F i n a l Report o f

Research Accompl i shed f o r the p e r i o d

June 15, 1982 through May 15, 1986 N87- 1 E S 7 2 (bASA-CR-180lS9) C f i l G l l A b C EVCLUTIOl OF

CSIIOBEGULATCBP EICBANISHS I b ELUE-GbEEN ALGAE ( C Y B H O b A C 3 E 3 I A ) AS B F U ; L C ' I I C N 01 RETAECLIC AND S I B U C ' I U E A L C C B E I E X J I Y : Uoclas L E E L E C T I C B S OF ( U a i v e r s i t y c*f S o u t h e r a 63/51 43610

Funded by the Grant e n t i t l e d :

O r i g i n and Evo lu t i on o f Osmoregulatory Mechanisms i n Blue-Green Algae (Cyanobacteria) as a Funct ion o f Metabol ic and S t r u c t u r a l Complexity: R e f l e c t i o n s

o f Precambrian Paleobio log ? [g ran t number: NAGW-344j

N a t i m a l Aeroneuti cs and Space A d m i n i s t r a t i o n

P r i nc i pal I n v e s t i g a t o r : John H. Yopp, Professor of Botany Bot any De pa rtmen t Southern I l l i n o i s U n i v e r s i t y Carbondale, I 1 1 i n o i s 62901

Co-Pr inc ipa l I n v e s t i g a t o r : Donald R. T i n d a l l , Professor o f Botany (June 15, 1982- Botany Department December 30, 1984) Southern I l l i n o i s U n i v e r s i t y

Carbondale, I 1 1 i n o i s 62901

Postdoctora l F e l l ow: Kenneth Pavl icek, Ph.D. Botany Department Southern I l l i n o i s U n i v e r s i t y Carbondale, I l l i n o i s 62901

https://ntrs.nasa.gov/search.jsp?R=19870009539 2020-04-08T11:07:13+00:00Z

Table of Contents o f F i n a l Report

Page

I . Abs t rac t (Summary) o f t he P ro jec t F i n a l Report ............ 11. D e s c r i p t i o n of t h e General Problem and ....................

J u s t i f i c a t i o n f o r and Statement o f S p e c i f i c Ob jec t i ves o f t h e Research Funded, 1982-1986

A. Statement o f S p e c i f i c Object ives f o r ................. Three-Year Per iod (June 15, 1982- June 14, 1985) and Time Course Proposed f o r Research Designed t o Achieve Them

B. Statement o f Object ives f o r F i n a l S i x ................ Month Per iod (December 15, 1984- June 14, 1985)

1. Object ives f o r F i n a l S i x Month .................. Per iod (December 15, 1984- June 14, 1985)

2 . Object ives f o r Proposed Extension ............... Per iod (June 15, 1985-May 15, 1986)

111. Progress i n Achieving the Object ives ...................... A . Previet ts Resul ts f rom our Laboratory ...*............. B. Progress i n Achieving the Ob jec t i ves ................. C. Presentat ions, A c t i v i t i e s and P u b l i c a t i o n s ...........

R e s u l t i n g f rom Research since 1985 Proposal and Progress Report

I V . L i t e r a t u r e C i ted .......................................... V. Appendices ................................................

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ABSTRACT

The Nat iona l Aeronaut ics and Space Admin i s t ra t i on (NASA) has, d u r i n g t h e pas t decade, funded research by our l a b o r a t o r y designed t o cha rac te r i ze t h e phys io log i ca l , biochemical and u l t r a s t r u c t u r a l features o f a h a l o p h i l i c b lue-green a lga t h a t enables i t t o surv ive i n a hypersa l ine environment o f ext remely 1 i m i t e d water a v a i l a b i 1 i t y . Th is research has l e d t o t h e c u l t u r a l and phys io log i ca l cha rac te r i za t i on , as w e l l as t h e research a v a i l a b i l i t y , o f one o f t h e most h a l o p h i l i c of t h e ea r th ' s oxygenic photosynthesizers, Aphanothece ha lophy t ica . I n add i t ion , t h i s research has l e d t o t h e r e a l i z a t i o n t h a t blue-green a lgae ( ' cyanobacter ia ' ) a re un ique ly s u i t e d t o research designed t o answer two of the most fundamental quest ions concerning t h e phys io logy o f water r e l a t i o n s i n photosynthe t ic organisms. The f i r s t i s t h e ques t ion o f how photosynthe t ic organisms o f d i ve rse s t r u c t u r a l and meta- b o l i c natures a d j u s t t h e i r i n t r a c e l l u l a r water p o t e n t i a l t o t h a t of a changing ex te rna l ( i .e. environmental ) water p o t e n t i a l . oxygenic photosynthesi zers osmoregul a te?

I n o t h e r words, how do

The second ques t ion concerns the t ime d u r i n g which metabol i c a l l y d i ve rse mechanisms o f osmoregulat ion i n oxygenic photosynthe t ic organisms had t h e i r o r i g i n and, subsequently evolved. Precambrian paleobio logy because oxygenic photosynthes is arose du r ing t h i s eon.

This ques t ion would necessa r i l y r e l a t e t o

The unique s u i t a b i l i t y of the blue-green a lgae as ob jec ts o f research on

Pa leob io log i s t s concerned w i t h t h e phylogeny o f these organisms

t h e second quest ion i s e a s i l y j u s t i f i e d . The f i r s t organisms t o evolve t h e oxygenic photosynthesis c h a r a c t e r i s t i c o f t h e h ighe r p l a n t s were t h e b lue- green algae. genera l l y b e l i e v e t h a t prokaryotes were s t r u c t u r a l l y and m e t a b o l i c a l l y com- p l e t e by t h e end o f t h e Precambrian. Indeed, t h e Precambrian eon has been termed " the age o f t h e blue-green algae." We w i l l f o r reasons g iven w i t h i n t h e t e x t r e f e r t o these organisms henceforth as cyanobacter ia. The cyano- b a c t e r i a a re a l s o unique i n t h a t they have n o t s i g n i f i c a n t l y changed morpho- l o g i c a l l y and m e t a b o l i c a l l y s ince the l a t e Pro terozo ic o r e a r l y Paleozoic eras. Therefore, i t i s assumed t h a t modern representa t ives of t h e f i v e major subgroups o f cyanobacter ia, which range f rom u n i c e l l u l a r through f i lamentous t o branched f i lamentous forms, possess the same metabo l ic ( n u t r i t i o n a l ) c a p a b i l i t i e s o f t h e i r Precambrian counterparts, now a p a r t of t h e f o s s i l record. These n u t r i t i o n a l types inc lude a v a i l a b l e s t r a i n s t h a t f u n c t i o n as o b l i g a t e photoautotrophs, f a c u l t a t i v e photoheterot rophs and f a c u l t a t i v e chemoheterotrophs. These types may i n some cases f u n c t i o n anaerob ica l l y as w e l l as a e r o b i c a l l y , and, even i n l i m i t e d numbers, as anoxygenic, hydrogen s u l f i d e - u t i l i z i n g photoautotrophs.

Recent research by prominent pa leob io log i s t s c l e a r l y i n d i c a t e s t h a t ecosystems changed d u r i n g the Precambrian f rom anaerobic- reduct ing t o aerobic- o x i d i z i n g types.

Furthermore, t h e geochemical evidence suggests t h a t t h e major s h i f t s i n t e c t o n i c mode d u r i n g t h e e a r l y Proterozoic l e d t o a g rea t increase i n favo r - ab1 e ecosystems f o r cyanobacter ia l p r o d u c t i v i t y and adequate environmental s t resses f o r e v o l u t i o n i n t h i s group. The i n v e s t i g a t o r s b e l i e v e t h a t t h e e v o l u t i o n o f t h e e a r l y biosphere must have inc luded t h e dominant cyanobacter ia responding t o changes i n t h e water p o t e n t i a l o f t h e i r d iverse, Precambrian

i

niches. p o s s i b l e w i t h i n t h e c o n s t r a i n t s o f s t r u c t u r a l complex i ty and t h e n u t r i t i o n a l parameters i nhe ren t i n anaerobic and aerobic photoautotrophy, photohetero- t rophy and chemoheterotrophy. The nature o f osmoregulatory mechanisms i n modern eukaryot ic , u n i c e l l u l a r and m u l t i c e l l u l a r photoautotrophs i s exceedingly compl icated by s t r u c t u r a l complexi ty and na tu re o f metabol ism i n v o l v e d i n t h e response. The i n v e s t i g a t o r s be l i eve t h a t such use of t h e s t r u c t u r a l and n u t r i t i o n a l s t r a i n s of cyanobacteria w i l l n o t on l y p rov ide impor tan t informa- t i o n concerning the na tu re of h igher p l a n t osmoregulat ion b u t w i l l h e l p dec ipher a major e v o l u t i o n a r y event i n Precambrian paleobio logy. today a considerable body of evidence t h a t t h e cyanobacter ia were t h e ances- t o r s of t he c h l o r o p l a s t s of h ighe r p lants .

Th i s response could i nvo l ve e v o l u t i o n o f osmoregulatory mechanisms

There e x i s t s

The unique s u i t a b i l i t y o f cyanobacteria as organisms f o r research on the f i r s t quest ion concerning t h e nature o f osmoregulat ion p e r se, i s l i k e w i s e j u s t i f i e d i n the s t r u c t u r a l and metabol ic c h a r a c t e r i s t i c s o f t h e cyanobacteria. The major unresolved aspects o f osmoregulat ion p e r se i n v o l v e t h e r e s o l u t i o n o f t h e f o l l o w i n g : (1) r e l a t i o n s h i p o f metabol ic c a p a b i l i t y t o type o f osmo- r e g u l a t o r y s o l u t e formed; ( 2 ) r o l e o f i n t r a c e l l u l a r compartmenta l izat ion i n i n t r a c e l l u l a r water p o t e n t i a l adjustment; ( 3 ) ex is tence o f a s i n g u l a r o r m u l t i p h a s i c sequence i n the process o f osmotic adjustment; and ( 4 ) t h e na tu re o f "compat ib le so l Utes" i n t h e metabol ism of osmotical ly-adapted-organisms.

Cyanobacteria a re prokaryotes and as such do n o t possess water vacuoles f o r compartmental izat ion of so lu tes. Cyanobacteria e x i s t i n a l l t h r e e major n u t r i t i o n a l modes, making poss ib le the establ ishment o f r e l a t i o n s h i p between na tu re of osmoregulatory s o l u t e and metabol i c capabi l i ty. Cyanobacteria have an extremely l i m i t e d and r e l a t i v e l y i n f l e x i b l e carbohydrate and organic a c i d metabolism t h a t w i l l f a c i l i t a t e the d i r e c t t r a c i n g of synthesized osmoregula- t o r y s o l Utes. Cyanobacteria genera l ly l a c k s imple c o n t r o l o f gene r e g u l a t i o n (feedback product and subs t ra te con t ro l o f enzyme syn thes i s ) . Th is a t t r i b u t e f a c i l i t a t e s the i n t e r p r e t a t i o n o f t h e sequence o f events i n a m u l t i p h a s i c osmoregulatory mechanism and the r o l e o f c e r t a i n chemicals as "compat ib le so lutes." syn thes i s o r uptake of almost a l l classes o f osmoregulatory so lu tes known f o r pho tosyn the t i c organisms.

F i n a l l y , cyanobacter ia possess the necessary pathways f o r t he

Another major type o f exper imentat ion d i r e c t e d toward t h e r e s o l u t i o n o f t he e v o l u t i o n a r y h i s t o r y o f t h e cyanobacteria has been t h e c a t a l o g i n g o f o l i g o n u c l e o t i d e sequences o f 16s ribosomal RNA. The use o f t h i s technique f o r measuring phy logenet ic r e l a t i o n s h i p s among prokaryotes has proven va luab le b u t has l e d t o some disagreement w i th i n t e r p r e t a t i o n o f such r e l a t i o n s h i p s f rom t h e f o s s i l record. The i n v e s t i g a t o r s f e l t t h a t , i n general, both l i n e s o f i n q u i r y support one another and t h a t d i f f e r e n c e s o f i n t e r p r e t a t i o n may a r i s e from d i f f e r e n c e s i n t ime o f e v o l u t i o n o f a p a r t i c u l a r genotype and t ime o f env i ronmental ly- induced-expressi on o f t h e phenotypes p o s s i b l e w i t h i n t h e genotype. and s t r u c t u r a l complexi ty. components o f t h e e v o l v i n g Precambrian biosphere e l i c i t e d d i f f e r e n t expressions o f an ever- increas ing genome. o f t h e nature proposed here (i .e. physiological-biochemical) w i l l p rov ide evidence f o r such d i f f e r e n t i a l expression i n the case o f osmoregulatory mechanisms w i t h i n n u t r i t i o n a l types o f t he cyanobacter ia.

It i s known t h a t t h e r e i s a d i r e c t r e l a t i o n s h i p between genome s i z e I t may be t h a t d i f ferences i n t h e environmental

The i n v e s t i g a t o r s f u r t h e r b e l i e v e t h a t a study

I n t h i s sense, t h i s

ii

study serves t o "b r i dge" the l i n e s of i n q u i r y i n v o l v i n g molecular genet ics and t h e f o s s i l record.

The o b j e c t i v e s of t he proposed study were t h e r e f o r e :

To a s c e r t a i n whether t h e r e e x i s t s any r e l a t i o n s h i p between mode o f n u t r i t i o n i n t h e cyanobacter ia ( i .e. photoautot rophic , photoheterot rophic o r h e t e r o t r o p h i c ) expressed under aerobic o r anaerobic c o n d i t i o n s and t h e na tu re o f t he so lu tes employed f o r adjustment o r i n t r a c e l l u l a r water p o t e n t i a l (osmoregulat ion) ;

To determine whether t h e r e e x i s t s any r e l a t i o n s h i p between s t r u c t u r a l complex i ty i n t h e cyanobacteria ( u n i c e l l u l a r , s imple f i lamentous o r m u l t i - s e r i a t e branched) and the na tu re of t h e s o l u t e s employed f o r adjustment o f i n t r a c e l l u l a r w a t e r p o t e n t i a l ;

To conduct t h e s t u d i e s on osmoregulation under environmental cond i t i ons t h a t correspond t o those o f the major stages i n the e v o l u t i o n of t h e Precambrian biosphere;

To i d e n t i f y and cha rac te r i ze the uptake and enzymatic mechanisms invo lved i n the p roduc t i on and accumulation of t he so lu tes employed i n adjustment o f i n t r a c e l l u l a r water p o t e n t i a l by r e p r e s e n t a t i v e n u t r i t i o n a l and s t r u c t u r a l types o f cyanobacteria, under t h e environmental cond i t i ons proposed i n the prev ious object ives;

To determine whether d i f f e rences i n metabolism u n d e r l i e t h e i n t r a c e l l u l a r adjustment t o lowered environmental water p o t e n t i a l achieved by t h e a d d i t i o n o f soditim c h l o r i d e o r nonion ic ( p e n e t r a t i n g o r non-penetrat ing) so fu t2s t o t h e g r w t h rnedium o f cyanbacter ia ;

To asce r ta in whether genome and o t h e r chemical c h a r a c t e r i s t i c s o f t he genet ic m a t e r i a l i n f l u e n c e the nature o f t he osmoregulatory response e l i c i t e d under va ry ing environmental c o n d i t i o n s i n t h e rep resen ta t i ve cyanobacteria;

To a s c e r t a i n whether adjustments o f i n t r a c e l l u l a r water p o t e n t i a l i n cyanobacter ia i s a mu1 t i p h a s i c process cu lm ina t i ng i n t h e accumulation o f a s o l u t e ( o r s o l u t e s ) compatible t o enzymatic f u n c t i o n i n g ; and t o de te r - mine whether t h e degree o f complexity i n any m u l t i p h a s i c process i s a f u n c t i o n o f metabol ic , genet ic o r s t r u c t u r a l complexi ty;

To determine t o what ex ten t the i no rgan ic and organic so lu tes accumulating d u r i n g osmoregulat ion account f o r ba lanc ing t h e ex te rna l water p o t e n t i a l ; and t o a s c e r t a i n whether t h e "sensing" o f changes i n environmental water i s by a t u r g o r - a c t i v a t e d mechanism o r not; and

To r e c o n s t r u c t a probable sequence o f evol u t i o n o f osmoregulatory mecha- nisms as a f u n c t i o n o f t h e adaptive i n t e r a c t i o n between t h e metabol ic capabi l i t i e s o f Precambrian cyanobacteria and f l u c t u a t i n g water p o t e n t i a l i n ecosystems o f a changing Precambrian biosphere.

The methodology proposed t o achieve the o b j e c t i v e s i nvo l ved the t r a c i n g o f t h e pathways o f format ion o f osmoregulatory s o l u t e s by t r a d i t i o n a l methods

iii

i n v o l v i n g C-14 l a b e l 1 ed substrates; gas chromatography; amino a c i d ana lys i s , X-ray a n a l y s i s us ing scanning transmission e l e c t r o n microscopy, and, most impor tan t l y , C-13 l a b e l l e d substrates, fo l lowed by Nuclear Magnetic Resonance (NMR) spectroscopy.

The t ime course proposed f o r achievement o f t h e o b j e c t i v e s was th ree years. 1986.

An extens ion was approved, due t o equipment f a i l u r e , u n t i l May 15, Work i s c o n t i n u i n g t o s a t i s f y t h e o b j e c t i v e s as o r i g i n a l l y proposed.

Major accomplishments under ly ing t h e bas i c understanding o f cyano- b a c t e r i a l r e s i s t a n c e t o s a l t to lerance and osmotic s t r e s s were made as a r e s u l t o f t h i s research.

(1)

I n summary, these are as f o l l o w s :

The cyanobacter ia, once termed metabol i c a l l y un i fo rm and evo lu t i ona ry , m e t a b o l i c a l l y conservat ive, employ a d i v e r s i t y o f organic , osmoregulatory so lu tes . These inc lude g lucosy lg l yce ro l i n many f reshwater and marine u n i c e l l u l a r forms; sucrose and t reha lose i n many f i lamentous and branched forms o f f r e s h and marine-water systems; and, most impor tan t l y , g l yc ine - be ta ine i n t h e extremely h a l o t o l e r a n t forms such as ou r o r i g i n a l i s o l a t e , Aphanothece halophyt ica.

(2 ) Osmore u l a t o r y s o l u t e s were found t o serve four f u n c t i o n s i n our research, i.e., 9 a) adjustment o f water a c t i v i t y ( Y T ) , ( b ) n o n - i n h i b i t i o n o f enzymes; ( c ) l ower ing K o f enzymes t o a l l o w f u n c t i o n i n g a t "normal" l e v e l s when i n t r a c e l l u l g r s a l t accumulates (a pre-adaptat ion) ; and (d ) extending t h e pH optimum o f enzymes as i n t r a c e l l u l a r pH r i s e s due t o proton-potassium i o n pump ac t i on d u r i n g osmoregulat ion. funct ions a re o r i g i n a l t o t h i s research.

The l a t t e r two

(3) Di f fe rences i n osmoregulatory S O ~ L J ~ P S i n cyanobacter ia may, b u t a re n o t always, be a t t r i b u t a b l e t o d i f f e rences i n n u t r i t i o n a l c a p a b i l i t i e s , i .e. photoautotrophy, photoheterotrophy and chemoheterotrophy.

(4)

(5)

The mechanism o f osmoregulation and concomitant sa l t to1 erance i n h a l o p h i l i c cyanobacter ia was elucidated. (which accumulates d u r i n g the i n i t i a l s t a e o f osmoregulat ion) a c t i v a t i o n o f t he enzyme S-adenosyl homocysteine (SAH 3 hydro1 ase t o cause t h e break- down o f SAH. me thy la t i on s tep o f g lyc ine-beta ine synthes is . A t t h e same t ime the reverse d i r e c t i o n o f t he enzymatic r e a c t i o n ( i . e . SAH syn thes i s ) i s i n h i b i t e d by t h e i n t r a c e l l u l a r potassium. When be ta ine i s synthesized t o a l e v e l t h a t p r o t e c t s t h e other s a l t - i n h i b i t e d enzymes, i n c l u d i n g SAH hydrolase i n t h e s y n t h e t i c d i r e c t i o n , be ta ine synthes is i s shut-down. Th is i s a simple, s e l f - r e g u l a t o r y mechanism, o r i g i n a l l y discovered d u r i n g t h i s research.

It invo lves t h e potassium i o n

SAH i s t h e key metabol ic i n h i b i t o r o f t h e e s s e n t i a l

Other o r i g i n a l d i scove r ies include: t h e Km o f potassium ion-inhibited-enzymes ( o n l y ) , thereby "pre-adapt ing enzymes t o i n h i b i t o r y s a l t concentrat ions a r i s i n g d u r i n g osmotic a d j u s t - ment; (b ) be ta ine p r o t e c t i o n of an enzyme i n h i b i t e d by potassium i o n i s i n v e r s e l y p r o p o r t i o n a l t o t h e degree o f s e n s i t i v i t y o f t h e enzyme t o such s a l t ; ( c ) S-Adenosyl homocysteine hydrolase, a key enzyme i n c o n t r o l o f c e l l u l a r methy lat ions was f i r s t d iscovered i n cyanobacter ia d u r i n g t h e course of t h i s research. This i s o f p a r t i c u l a r importance i n view o f t h e

( a ) t h a t be ta ine a c t s by l ower ing

i v

c u r r e n t hypothes is t h a t cyanobacter ia may be t h e ancestors of h ighe r p l a n t c h l o r o p l a s t s .

V

TITLE: O r i g i n and E v o l u t i o n o f Osmoregulatory Mechanisms i n Blue-Green Algae (Cyanobacter ia l ) as a Func t i on o f Me tabo l i c and S t r u c t u r a l Complexity: R e f l e c t i o n s o f Precambrian Paleobiology?

D e s c r i p t i o n of t he General Problem and J u s t i f i c a t i o n f o r and Statement o f S p e c i f i c Object ives

One o f t h e fundamental p r i n c i p l e s of t h e b i o l o g y o f l i f e forms on t h i s p l a n e t i s t h a t t h e i r phys io logy i s i n t r i n s i c a l l y r e l a t e d t o t h e physico- chemical p r o p e r t i e s o f water. Ea r th ' s l i f e forms have evolved i n a water m i l i e u ; t h e i r major c o n s t i t u e n t i s water; t h e i r metabolism i s p r i m a r i l y conducted i n a water m a t r i x ; and, t h e i r communication w i th , and a c q u i s i t i o n o f , t h e e s s e n t i a l chemicals of t he a b i o t i c environment i s through a water medium. Jus t as e a r t h ' s organisms have evolved i n a d i v e r s i t y o f environments va ry ing w i t h respect t o water a v a i l a b i l i t y , so too a r e the p h y s i o l o g i c a l a c t i v i t i e s o f these organisms adapted t o d i f f e r i n g degrees of water a v a i l a b i l - i t y ( i . e . water p o t e n t i a l ) . Therefore, one o f t he most impor tant aspects o f t h e phys io logy of a l l l i f e forms i s the mechanism(s) whereby they r e g u l a t e t h e i r i n t r a c e l l u l a r water p o t e n t i a l i n response t o a changing water p o t e n t i a l o f t h e i r environment. The movement o f water i n and o u t o f an organism i s by osmosis and t h e mechanisms regu la t i ng t h i s movement a re termed osmoregulatory mechanisms.

Water p o t e n t i a l ( Y ) i s the sum of t h e c o n t r i b u t i o n o f osmotic p o t e n t i a l (YIT) , which i s due t o s o l u t e concentrat ion (Yn=-miRT) ; pressure p o t e n t i a l (Yp), which i s due t o the t u r g o r pressure of c e l l w a l l r e s i s t a n c e t o volume increase; and m a t r i c p o t e n t i a l (Ym) , t h a t occurs as a r e s u l t of reduc t i on of water a c t i v i t y ~iperr i t s absorpt ion o f protop lasmic surfaces. I n t r a c e l l u l a r water p o t e n t i a l ( ' Y ) i s commonly decreased t o t h e l e v e l o f lower ex te rna l Y by uptake and/or synthes is o f so lu tes. L ikewise, i n t r a c e l l u l a r Y may be increased i n response t o a h ighe r external \Y by metabolism o f synthesized s o l u t e s and/or t he l o s s o f these so lutes t o the environment. of t h e osmotic p o t e n t i a l (YIT) component of a t o t a l Y i s termed osmore u l a t i o n . Adjustment o f i n t r a c e l l u l a r YJ i n response t o a suddenly lowered + termed upshock), o r g r a d u a l l y lowered, external Y may employ the r e g u l a t i o n of t h e o t h e r components o f water p o t e n t i a l ( i m and 'up) as w e l l . osmoregulatory mechanisms and mechanisms o f adjustment o f i n t r a c e l l u l a r \Y may n o t be synonymous.

The adjustment

I n t h i s sense

There have been e i g h t recen t reviews on t h e sub jec t o f osmoregulat ion i n pho tosyn the t i c algae and h ighe r p lan ts (9, 21, 22, 36, 43, 59, 75, 82). general conclusions may be drawn f r o m these ex tens i ve reviews. t h a t t h e r e a r e a t l e a s t s i x fundamental aspects o f osmoregulatory systems t h a t must be y e t e luc idated, a l l o f which a r e somehow r e l a t e d t o t h e organism's mode o f n u t r i t i o n and s t r u c t u r a l complexity. The second conclus ion i s t h a t l i t t l e i s known concerning the osmoregulatory mechanisms o f t he cyanobacter ia. The photoautot rophic prokaryotes were t h e f i r s t organisms on e a r t h t o possess the pho tosyn the t i c machinery ( i . e . photosystems I and 11) o f t h e euka ryo t i c a lgae and h ighe r p l a n t s (62,64). The e i g h t major books and rev iew a r t i c l e s on cyanobacter ia over t h e past decade have a l s o added on ly ve ry l i t t l e t o t h e s u b j e c t of osmoregulat ion i n cyanobacteria (14, 24, 28, 66, 70, 71, 75, 79). Walsby (75) has best reviewed the l i t e r a t u r e concerning t h i s t o p i c i n t h e

Two The f i r s t i s

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group. Our recent p u b l i c a t i o n on osmoregulat ion i n t h i s group, which was t h e f i r s t such study, and one o t h e r p u b l i c a t i o n represent an i n i t i a l view o f t h i s process i n e a r t h ' s e a r l i e s t oxygenic photosynthes izers (85).

The i n v e s t i g a t o r s b e l i e v e t h a t the cyanobacter ia represent organisms t h a t possess unique a t t r i b u t e s t h a t would f a c i l i t a t e t h e e l u c i d a t i o n o f t h e s i x fundamental aspects of osmoregulatory systems i n photosynthes iz ing p lan ts . The i n v e s t i g a t o r s a l s o b e l i e v e t h a t a s tudy o f t h e osmoregulatory mechanisms i n t h i s group w i l l y i e l d impor tant i n fo rma t ion on one o f t h e fundamental aspects o f Precambrian paleobiology. As s t a t e d by Schopf (64) , " t h e u l t i m a t e goal o f Precambrian paleobio logy i s t o decipher and document both the t i m i n g and n a t u r e o f major events i n t h e early h i s t o r y of l i f e . " T r u l y t h e e v o l u t i o n of osmoregulatory mechanisms i n t h e dominant organisms of t h e Precambrian i s one such major event.

The i n v e s t i g a t o r s p o s t u l a t e the f o l l o w i n g scenar io based upon the excel - (1) The cyanobacter ia evolved from

l e n t i n t e r p r e t a t i o n s o f t h e data on t h e e v o l u t i o n o f t h e e a r t h ' s e a r l i e s t ecosystems by Knol l (45) and Schopt (64): probable anaerobic , photosynthet ic bacter ia l -ancestors d u r i n g ( o r p o s s i b l y much p r i o r t o ) the l a t e Archean e ra (2900 t o 2500 m.y. ago) o f t he Precambrian eon (4500 t o 570 m.y. ago) (1, 61, 62, 63). photoautotrophs possessing a c h l o r p h y l l a based-photosynthesis t h a t i n c l u d e photosystems I and I 1 (66). The l a t t e r photosystem f u n c t i o n s t o u t i l i z e water as a source of hydrogen f o r t h e reduct ion o f CO and t h e oxygen l i b e r a t e d as a by-product. s t r u c t u r a l comFlexi ty o f present-day forms by t h e l a t e Pre-Cambrian (45, 62, 64). t he f o l l owing modes o f n u t r i t i o n (metabol i c capabi l i t i e s ) : anaerobic and ae rcb i c photoautotrophy; aerobic and anaerobic photoheterotrophy; aerobic chemoheterotrophy; and perhaps as evidenced by a few modern forms, anaerobic chemoheterotrophy (24, 64, 66). As such, modern rep resen ta t i ves o f these prokaryotes r e t a i n e d most o f t he major metabol ic innovat ions ( c a l l e d "bench- marks" by Schopf e t a1 , 64) t h a t character ized e a r t h ' s e a r l i e s t ecosystems. (3) P r i o r to , and du r ing the development of a s t a b l e aerobic hydrosphere and atmosphere d u r i n g the e a r l y Proterozoic (2500 and 1600 m.y. ago) as a conse- quence o f cyanobacter ia l photosynthet ic a c t i v i t y (18, 45, 63) , major geo log i c events occurred t h a t "chal lenged" the metabol ic machinery o f these prokaryotes t o develop osmoregulatory mechanisms. reco rd by Knol l (45), Cloud (18) and Schopf e t a1 (64) would mean, the re fo re , t h a t t h e e a r t h ' s e a r l i e s t ecosystems inc luded the dominant cyanobacter ia responding t o changes i n the water p o t e n t i a l o f t h e i r d iverse, Precambrian n iches by osmoregulatory mechanisms p o s s i b l e w i t h i n the c o n s t r a i n t s of anaerobic and aerobic photoautotrophy , anaerobic and aerobic photoheterotrophy and anaerobic and aerobic chemoheterotrophy.

These l i f e forms a r e aerobic

( 2 ) The cyanobacter ia evolved t o t i e f u l l range o f metabol ic and

During t h i s pe r iod o f evolut ion, cyanobacter ia developed and r e t a i n e d

The i n t e r p r e t a t i o n o f t h e geologic

It i s impor tant t o note a t t h i s p o i n t t h a t t h e r e has been d u r i n g the pas t decade an enormous amount o f research by molecular b i o l o g i s t s d i r e c t e d toward r e s o l v i n g t h e e v o l u t i o n o f t h e prokaryotes, i n c l u d i n g the cyanobacter ia (24, 29). P o i n t i n g o u t t he d i f f i c u l t i e s i n u t i 1 i z i n g s o l e l y morphological charac- t e r s t o assess phylogenet ic, as wel l as taxonomic r e l a t i o n s h i p s , Stackebrandt and Woese (68) r e c e n t l y reviewed the methodology of gene t i c research used t o e s t a b l i s h genealogies among the prokaryotes. f o r a new concept o f c e l l u l a r evo lu t i on (as presented by Woese and Fox, 78). Consequently, a new phylogenet ic s t r u c t u r e of t h e p r o k a r y o t i c domain (77) has

This research has been respons ib le

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l i k e w i s e r e s u l t e d as r e c e n t l y discussed by Fox e t a1 (29). Stackebrandt and Woese (68) , the cata log ing o f b l i g o n u c l e o t i d e sequences i n t h e e v o l u t i o n a r i l y conservat ive 16s ribosomal RNA i s t h e bes t method f o r e s t a b l i s h i n g genealogical r e l a t i o n s h i p s among prokaryotes. Th is method has been used t o study such r e l a t i o n s h i p s among t h e cyanobacter ia by D o o l i t t l e and Bonen and t h e i r assoc iates a t Dalhousie (5, 6, 24, 29). The assessment o f t he e x t e n t o f s i m i l a r i t i e s i n these 16s rRNA sequences by t h i s group has l e d t o some poss ib le disagreement w i t h the i n t e r p r e t a t i o n o f p h y l e t i c r e l a t i o n s h i p s rendered f rom the f o s s i l r eco rd (6, 24, 29, 77). agreement among i n t e r p r e t a t i o n s from e i t h e r method, e s p e c i a l l y w i t h respec t t o t h e t ime and sequence o f o r i g i n of the most s t r u c t u r a l l y complex cyanobacter ia (e.g. F i s h e r e l l a and Nostoc). Stackebrandt and Woese (68) p o i n t ou t i n t h i s rega rd t h a t these approaches represent "two measures o f evolut ion. . . . the phenotypic versus the genotypic. The phenotypic measure i s t h e c l a s s i c a l one; i t i s t h e one we use i n speaking o f e v o l u t i o n a r y progress ion ... p a r t i t r e f l e c t s e c o l o g i c a l change, o r t h e working o u t o f some ' e v o l u t i o n a r y p o t e n t i a l I i n a l i n e o f descent." The i n v e s t i g a t o r s r e a l i z e t h a t osmo- r e g u l a t o r y s t r a t e g i e s may r e f l e c t environmental ly induced-expression o f t h e same geneotype t h a t changed w i t h the e v o l u t i o n of t he Precambrian biosphere. However, we a re a l s o aware f rom the recen t work o f Herdman e t a1 (38, 39) and Rippka e t a1 (56) t h a t morphologica l ly i d e n t i c a l species ( o r s t r a i n s ) o f cyanobacter ia may have g r e a t l y d i f f e r i n g genome s i ze . t he re fo re , t h a t genome doubl ing (and f u r t h e r increases) provided the gene t i c m a t e r i a l f o r metabol ic and s t r u c t u r a l advancement d u r i n g t h e Precambrian. We propose t o t e s t these p o s s i b i l i t i e s and hope t o c o n t r i b u t e t o the r e c o n c i l i a - t i o n between the phenotypic and genetic approach by c a r e f u l s e l e c t i o n o f exper imental m a t e r i a l .

As noted by

However, t h e r e i s a l s o much

For the most

I t may w e l l be poss ib le ,

Perhaps the best summary statement o f j u s t i f i c a t i o n f o r t h i s aspect of oiur study i s g iven i n the most recent r e l e v a n t p u b l i c a t i o n by Schopf e t a1 (64) :

"Bearing such l i m i t a t i o n s ( ' i n t e r p r e t a t i o n of t h e f o s s i l r e c o r d ' ) i n mind, i t seems ev iden t t h a t questions regard ing the e a r l i e s t phases o f eco log i c development must be phrased b iochemica l l y and m e t a b o l i c a l l y ( r a t h e r than s o l e l y i n terms o f organismal morphology); t h a t t h e i r answers should be based on considerat ion of t o t a l i t y o f r e l e v a n t chemical, b i o l o g i c a l and geologica l evidence now a v a i l a b l e ( r a t h e r than on data f rom any s i n g l e d i s c i p l i n e alone); and t h a t a t present, any f r u i t f u l approach t o such questions, must, of necess i ty , be l a r g e l y model- dependent (determined t o a subs tan t i a l degree by t h e o r e t i c a l and i n d i r e c t cons iderat ion, r a t h e r than s o l e l y by d i r e c t observable features o f t h e f o s s i l r eco rd as now known.)"

I t seems c l e a r f rom the l i t e r a t u r e t h a t w h i l e the p a l e o b i o l o g i c a l and t h e mol ecul a r gene t i c approaches are we1 1 represented , the physi ol og i c a l - b iochemical approach i s s t i l l underrepresented.

I t i s r e l a t i v e l y easy then t o j u s t i f y a s tudy on an undoubtedly p r i m i t i v e p h y s i o l o g i c a l response i f i t s relevance t o e a r t h ' s e a r l i e s t ecosystems can be es tab l i shed . It i s somewhat more d i f f i c u l t t o j u s t i f y t h e use o f t h e cyano- b a c t e r i a t o s tudy the s i x bas ic problems y e t unresolved f o r osmoregulat ion i n pho tosyn the t i c eukaryotes. f o l l o w i n g : (1) - The i n f l u e n c e of the type ( i o n i c versus non- ion ic and

These problems i n v o l v e the e l u c i d a t i o n o f t h e

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penetrating versus non-penetrating) of solute used t o lower the external Y on the result ing internal solutes employed to lower the in t race l lu la r Y.

47, 76). on the quanTity and quality of osmoregulatory solutes formed. published l i t e r a tu re indicates tha t , depending upon the metabolic s ta tus of an organism, different intracel lular solutes may resu l t from imposition o f an external water s t ress (48, 85). (3) The existence of a possible sequence o f di f fe ren t intracel lular solutes accumulating as a consequence of changes i n metabolism e l i c i t ed by the i n i t i a l ionic or non-ionic solute accumulated has a lso been postulated (12 , 21, 43, 85 . photosynthetic and respiratory shock'just p r i o r t o deplasmolysis ( 2 , 3, 35,

Several studies have shown t h a t an influence of this type exis ts (11, 1 2 3 32, 33, 36,

( 2 ) The influence of metabolic capabi l i t ies of the upshocked organism Again the

This may explain the commonly observed

36, 58). compatible solutes remains to be fully resolved. solute derives from the f ac t t h a t the solute employed f o r lowering the intra- ce l lu la r YIT must allow enzymatic function as well. Ion ( K + and Na+) perform

( 4 ) The functional ro le o f the internal osmoregulatory solutes a s The concept o f compatible

t h i s role i n halophilic bacteria b u t are inhibitory ( i n v i t ro) t o the'same enzymes from eukaryotic photoautotrophs ( 7 , 26, 40, 8-s l a t t e r group employs a diversity of organic solutes (polyols, amino acids, carbohydrates) t o lower intracel lular YIT and s t i l l allow enzymatic ac t iv i ty (3 , 9 , 10, 85). However, recent studies (12 , 13, 31) indicate that some o f these eukaryotic photoautotrophs also accumulate high levels of ions ( a t l ea s t i n i t i a l l y ) t o aid i n lowering intracel lular YIT . protect enzymes from the s a l t inhibition as well as a i d in lowering intracel- l u l a r Y I T ( 7 , 9, 59, 60). The mechanism by which this protection i s afforded

In t h i s case the comparable solutes may

- .

i s one of much current. research. - enzyme( s ) responsible fo r production and/or uptake of the osmoregulatory solutes i n the well-studied systems remain largely unknown (22, 43, 44) . Only m s regulating the production of the organic solutes i n the f l age l l a t e Ochrzmcnas malhamensis as a funct ion n f external Y I T are well studied. This i s due to theexce l len t work of Kauss and his coworkers over the past decade (43). (6) The role of vacuolization and intracel lular colnprtmentalization of the sol u T e s a c c u m u l a t i n g r Z g u 1 atory recovery.

(5) T h e specif ic mechanism( s ) whereby the

The advantage o f using cyanobacteria t o study these aspects of the mechanism of regulation o f Y l i e s in the i r structural and metabolic charac- t e r i s t i c s . vacuo1 ar system. Therefore, there should be no structural compartmental i - zation of penetrating solutes from the lower external Y into vacuolar and cytoplasmic s i t e s . In addition, internal osmoregulatory sol Utes t h a t may accumulate sequentially will not be compartmentalized i n vacuolar and cyto- plasmic s i t e s , thereby complicating a time course study of deplasmolysis. Finally, the accumulating osmoregulatory solutes would be i n presumed contact w i t h lamellar and interlamellar enzymes rather than sequestered in a vacuole. In this sense a bet ter evaluation of the degree of compatibility of the solute coul d be obtained.

F i r s t , they are prokaryotes that possess low water content and no

Secondly, species of the cyanobacteria d i f f e r with respect t o structural complexity; such differences constitute tradit ional characters f o r taxonomic dis t inct ion. I t i s then possible t o study osmoregulation within a single nutri t ional mode (e.g. the photoheterotrophic) a s a function o f structural complexity. multi-seriate-branched filaments and as heterocyst-containing-simple and branched filaments.

Species of blue-green algae ex is t as unicells, simple filaments,

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T h i r d l y , t h e cyanobacter ia possess metabol ic c h a r a c t e r i s t i c s t h a t pe rm i t d i r e c t experimental t e s t i n g o f t he r e l a t i o n s h i p between metabol ic c a p a b i l i t y and na tu re of osmoregulatory so lu te (s ) employed f o r adjustment of i n t r a - c e l l u l a r YJ. These c h a r a c t e r i s t i c s are as f o l 1 ows :

Cyanobacteria c h a r a c t e r i s t i c a l l y e x i s t as ae rob ic o r micro- ae roph i l i c y oxygenic photosynthesizers (i .e. a r e photoautotrophs) b u t can e x i s t (o f ten t h e same s t r a i n o r i s o l a t e ) i n t h e o t h e r n u t r i t i o n a l modes (i .e. photoheterotrophy and chemoheterotrophy) (14, 66, 69, 70). Therefore, by man ipu la t i ng t h e environmental c o n d i t i o n s and/or us ing an i n h i b i t o r o f photosystem 11, such as DCMU (3 ' -3 , 4-dichlorophenyl ) 1'-1 dimethyl urea) the photoheterot rophic o r chemoheterotrophic (by dark maintenance) mode may be induced (55). Only a r e l a t i v e l y f e w organisms, o t h e r than t h e cyano- b a c t e r i a , may be so u t i l i z e d t o demonstrate the r e l a t i o n s h i p between n u t r i t i o n a l mode and nature o f osmoregulatory s o l u t e employed. Furthermore, some cyanobacter ia l i s o l a t e s a r e ob1 i g a t e photoauto- t rophs whi 1 e o t h e r c l o s e l y re1 a ted is01 ates a re facul t a t i v e photo- and even chemoheterotrophs (56). Th is permi ts y e t another i n s i g h t t o be made regard ing an o b l i g a t e o r f a c u l t a t i v e n u t r i t i o n a l r e l a t i o n - s h i p t o osmoregulation.

Use o f t h e photoheterot rophic cyanobacteria should p rov ide evidence o f t h e e f f e c t o f a photoass imi la ted organic s o l u t e on the c a p a b i l i t y f o r forma- t i o n of a p o t e n t i a l osmoregulatory solute. photoass imi la ted subst rates would, therefore, be formed i n the absence o f m e t a l l i c involvement f r o m photosynthesis. f u r t h e r degree o f metabol ic involvement i n osmoregulatory mechanisms i n l i g h t - t r e a t e d organisms.

Osmoregulatory so lu tes formed from

This w i l l a l l o w t h e assessment o f a

The use o f chemoheterotrophic cyanobacteria w i l l a l l o w t h e study o f osmoregulat ion i n organisms w i t h the p r i n c i p a l metabol ic machinery o f t h e o t h e r t w o n u t r i t i o n a l types, bu t i n the t o t a l absence of l i g h t . Supply ing these algae w i t h var ious (though s t i l l very l i m i t e d ) organic subs t ra tes may r e s u l t i n f u r t h e r i n s i g h t i n t o osmoregulation i n the absence o f even photo- system I o f photosynthesis.

Recent research has demonstrated t h a t c e r t a i n cyanobacter ia ( u n i c e l l s and f i lamentous types) may a l s o be induced t o use a p r i m i t i v e , anoxygenic photo- synthes is us ing H2S as a hydrogen donor (18, 30). Use o f a blue-green a lga i n t h i s n u t r i t i o n a l mode w i l l a l l o w the s tudy o f osmoregulat ion n o t o n l y under the cond i t i ons o f an extremely p r i m i t i v e atmosphere, b u t a1 so under c o n d i t i o n s i n which o n l y photosystem I o f photosynthesis i s ope ra t i ve (18).

(2) Cyanobacteria have an extremely l i m i t e d , and r e l a t i v e l y i n f l e x i b l e carbohydrate and organic ac id metabolism (15, 17, 24, 54, 66, 69). Indeed i t has been shown t h a t t h e Pentose Phosphate Pathway i s t h e major pathway o f carbohydrate metabolism f u n c t i o n i n g as such i n the photoautotroph i c , phot oh etero t r o ph i c , and c herno he t e r o t roph i c n u t r i - t i o n a l modes (66, 70, 79). Furthermore, t h e Krebs Cycle i s incom- p l e t e ( i n t e r r u p t e d by a lack o f a1 pha-ketogl u t a r a t e dehydrogenase) and serves on ly a b iosyn the t i c f u n c t i o n (53, 66). These metabol ic c h a r a c t e r i s t i c s a l l o w a much more d i r e c t assessment o f t h e r e l a t i o n - s h i p o f energy-y ie ld ing, metabol ic r e a c t i o n s t o osmoregulatory i o n

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f l u x e s o r synthes is o f osmoregulatory so lu tes . I n a d d i t i o n , a o f r e s p i r a t o r y breakdown o f c e r t a i n p h o t o h e t e r o t r o p h i c a l l y - o r chemoheterotrophically-assimilated, organic a c i d subst rates w i pe rm i t a more d i r e c t synthesis i n t o measureabl e osmoregulatory s o l Utes.

1 ack

1

Cyanobacteria a r e unique among t h e organisms possessing oxygen c photosynthesis w i t h respect t o a general l a c k a c o n t r o l o f enzyme synthes is (gene expression) by p o t e n t i a l subs t ra tes o r products o f t h e r e a c t i o n s mediated by t h e enzymes (17, 24, 66). As N.G. Carr notes "With o n l y one or two except ions t h e r a t e of synthes is o f enzymes o f both in termediary metabolism and of b iosyn thes i s have been shown t o be unal tered by the a v a i l a b i l i t y of subs t ra tes o r by t h e end product of t he b iosyn the t i c pathway r e s p e c t i v e l y " (17). While Carr i n t e r p r e t s t h i s as a poss ib le cause of photoautotrophy and D o o l i t t l e notes t h a t i t may i n f a c t be a consequence o f it, t h e r e i s l i t t l e doubt t h a t cyanobacter ia l metabolism i s charac- t e r i z e d by a de f i c iency (though n o t t o t a l l a c k ) i n ' 's imple" c o n t r o l s o f gene regu la t i on . o b l i g a t e photoautotroph represents an i d e a l system f o r t h e s tudy o f t h e na tu re and sequence o f appearance of osmoregulatory so lutes. Unl i ke photoautot rophic eukaryotes, which do respond ( t r a n s c r i p - t i o n a l systems present) t o added substrates, and which do possess end-product feedback contro ls , t he cyanobacter ia l metabol ic system should respond more d i r e c t l y t o ex te rna l water s t ress . Presumably organic so lu tes a r i s i n g i n t h e o b l i g a t e photoautotrophs as a r e s u l t o f lowered environmental YIT would n o t induce t h e enzymes o f t h e i r f u r t h e r d isseminat ion. Likewise p e n e t r a t i n g o rgan ic so lu tes used t o lower t h e environmental YS should n o t induce enzymes t h a t would i nsu re t he i r structural rearraflgement,

Whatever the explanat ion, t h e cyanobacter ia l -

Previous research, i n c l u d i n g a g r e a t deal o f ou r own over t h e pas t ten years , has demonstrated t h a t cyanobacter ia possess t h e b i o - s y n t h e t i c pathways needed t o produce the o rgan ic osmoregulatory so lu tes repo r ted f o r a l l o the r pho tosyn the t i c eukaryotes. There- fo re , t h e pathways, and i n many cases the i n d i v i d u a l enzymes, i nvo l ved i n the product ion o f poly01 s, monosaccharide e s t e r s w i t h g l y c e r o l , pro1 ine, glutamic ac id , sucrose, and o t h e r carbohydrates have been s tud ied i n cyanobacteria (16, 21, 28, 66, 79).

Not o n l y a r e t h e cyanobacteria l i n k e d t o t h e pho tosyn the t i c eukaryotes by t h e i r possession o f an i d e n t i c a l pho tosyn the t i c

g l y c e r o l nerr has been found t o f l u c t u a t e w i t h adjustment of i n t r a - c e l l u l a r Y both i n the blue-green and c e r t a i n p r i m i t i v e red algae (Rhodophyta) (4 ) . i n v e s t i g a t o r s t o c o n t a i n the photosynthet ic eukaryotes most c l o s e l y r e l a t e d t o cyanobacter ia (4, 6, 8, 29).

, b u t a type of organic s o l u t e (monosaccharide e s t e r o f

Th i s l a t t e r algae group i s be l i eved by many

(6) - The symbiosis hypothesis proposed by Margu l i s (49, 50) which s t a t e s t h a t t h e ancestor o f h igher p l a n t c h l o r o p l a s t s was a cyanobacter ia l c e l l t ype provides an important l i n k t o p h o t o s y n t h e t i c a l l y mediated osmoregulat ion i n crop plants.

6

These metabolic capabi l i t ies represent then a f inal jus t i f ica t ion f o r use of the cyanobacteria as subjects for the study of osmoregulation as a function of nutri t ional mode and structural complexity.

Pertinent Literature in Related Fields

A very detailed assessment of the pertinent l i t e r a t u r e for the purpose of establishing the background and significance of the objectives may be found in the original g r a n t proposal (NAGW-344) a1 ready approved for funding.

An update o f the pertinent l i t e ra ture was given i n the proposal fo r Phase I1 (the second eighteen months) submitted on February 23, 1983 and on f i l e .

Since early 1983, several publications have appeared tha t are relevant t o These are i n the area of solutes accumulated i n blue-green the present study.

algae (cyanobacteria) during osmotic adjustment. a r t i c l e on the osmotic significance of betaine has a lso appeared. review of these publications follows.

In addition, an additional A brief

Blumwald and Tel-Or (4a) reported t h a t a s a l t adapted Nostoc species accumulated sucrose as an osmoticum. reported t h a t certain unicellular marine blue-green algae synthesize glucosylglycerol as an osmoticum. tuation of trehalose w i t h s a l in i ty in the cyanobacterium Rivularia a t ra . Reed e t a1 . (54b) most recently reported on a survey of the osmoregulatory carbohy- drates in a large number of cyanobacterial isolates .

Erdmann (25b) and Mackay e t a l . (48a)

Reed and Stewart (54a) reported the fluc-

A summary and greatly simplified interpretation of these recent papers i s 6s E 1 1 *l.,C

181 iuw3:

a) Non-reducing carbohydrates play a major role in osmoregulation by cyanobacteria.

b ) The three major carbohydrates are gl ucosyl glycerol , trehalose and sucrose.

c ) Marine cyanobacteria appear t o general ly employ gl ucosyl glycerol , while fresh water forms employ sucrose.

d ) No c lear cut relationship between genus and osmolyte employed by cyanobacteria is now evident.

Our research reported above is supported by these findings. We have, however, found exceptions to many o f the conclusions drawn from the above papers.

In addition, an excellent review on the central importance of betaine in osmoregulation by Ruduber e t a1 (%a) has appeared in Science. further jus t i f ica t ion to our search fo r the f i r s t pathway of betaine synthesis.

This gives

7

The s p e c i f i c o b j e c t i v e s o f t h e proposed study a re as f o l l o w s :

(1) - To a s c e r t a i n whether there e x i s t s any r e l a t i o n s h i p s between mode o f n u t r i t i o n i n the cyanobacteria ( i .e. photoautot rophic , photohetero- t r o p h i c o r h e t e r o t r o p h i c ) expressed under aerobic o r anaerobic c o n d i t i o n s and t h e na tu re o f t h e so lu tes employed f o r adjustment o f i n t r a c e l l u l a r water p o t e n t i a l (osmoregulat ion);

( 2 ) - To determine whether there e x i s t s any r e l a t i o n s h i p s between s t r u c - t u r a l complex i ty i n the cyanobacteria ( u n i c e l l u l a r , s imple f i lamentous o r m u l t i - s e r i a t e branched) and the na tu re of t h e s o l u t e s employed f o r adjustment o f i n t r a c e l l u l a r water p o t e n t i a l ;

To conduct t he s tud ies on osmoregulat ion under environmental con- d i t i o n s t h a t correspond t o those of t h e major stages i n t h e evolu- t i o n o f t h e Precambrian biosphere;

(4 ) - To i d e n t i f y and character ize t h e uptake and enzymatic mechanisms invo lved i n the product ion and accumulation of t h e s o l u t e s employed i n adjustment o f i n t r a c e l l u l a r water p o t e n t i a l by r e p r e s e n t a t i v e n u t r i t i o n a l and s t r u c t u r a l types o f cyanobacter ia, under the env i ron- mental c o n d i t i o n s proposed i n t h e previous ob jec t i ves ;

To determine whether d i f f e rences i n metabolism under1 i e t h e i n t r a - c e l l u l a r adjustment t o lowered environmental water p o t e n t i a l achieved by the a d d i t i o n o f sodium c h l o r i d e o r non - ion i c ( p e n e t r a t i n g o r non-penetrat ing) s o l U t e s t o t h e growth medium o f cyanobacteria;

( 3 ) -

(5) -

(6) - To a s c e r t a i n whether genome s i z e and o t h e r chemical c h a r a c t e r i s t i c s n f t h e genet ic m a t e r i a l i n f l uence t h e na tu re o f t h e osmoregulatory response e l i c i t e d under varying environmental c o n d i t i o n s i n the r e p r e s e n t a t i v e cyanobacteria;

To a s c e r t a i n whether adjustment o f i n t r a c e l l u l a r water p o t e n t i a l i n cyanobacter ia i s a mu l t i phas i c process cu lm ina t i ng i n t h e accumu- l a t i o n o f a s o l u t e ( o r so lu tes) compat ib le t o enzymatic func t i on ing ; and t o determine whether the degree o f complex i ty i n any m u l t i p h a s i c process i s a f u n c t i o n o f metabolic, genet ic o r s t r u c t u r a l complexi ty;

(7) -

(8) - To determine t o what extent t he i no rgan ic o r organic so lu tes accu- mu la t i ng d u r i n g osmoregulation account f o r ba lanc ing the ex te rna l water p o t e n t i a l ; and t o asce r ta in whether the "sensing" of changes i n environmental water p o t e n t i a l i s by a t u r g o r a c t i v a t e d mechanism o r not; and

(9) - To r e c o n s t r u c t a probable sequence of e v o l u t i o n o f osmoregulatory mechanisms as a f u n c t i o n of t h e adapt ive i n t e r a c t i o n between t h e metabol ic c a p a b i l i t i e s o f Precambrian cyanobacter ia and f l u c t u a t i n g water p o t e n t i a l i n ecosystems of a changing Precambrian biosphere.

8

Time course proposed f o r research designed t o achieve the o b j e c t i v e s .

Year 1:

(1) - C u l t u r e of a l l s t r u c t u r a l and n u t r i t i o n a l types of cyanobacter ia proposed (see Table 2, o r i g i n a l proposal f o r f i r s t year, June 15, 1982 - June 15, 1983; t h i s o b j e c t i v e must be met before research on t h e o t h e r o b j e c t i v e s progresses);

C u l t u r e of t h e above s t r a i n s under c o n d i t i o n s t h a t w i l l e l i c i t ph o toa u t o t r o phy , phot ohe tero t r o phy and c hemohe t e r o t r o phy ; t h i s o b j e c t i v e w i l l con f i rm the ex i s tence of t h e approp r ia te n u t r i t i o n a l c a p a b i l i t i e s o f t he cyanobacteria, as i n d i c a t e d f rom the l i t e r a t u r e .

Determinat ion o f t he changes i n osmoregulatory s o l u t e s a f t e r upshock f o r approximately one- th i rd o f t h e s t r a i n s of cyanobacter ia i n c u l t u r e .

( 2 ) -

( 3 ) -

Year 2: ( A c t u a l l y t h e nex t eighteen months o f t h e t h r e e yea r s tudy)

(1) - Completion of t h e study of t h e na tu re of osmoregulatory so lu tes as a f u n c t i o n of s t r u c t u r a l complexity and n u t r i t i o n a l mode (photoauto- t rophy, photoheterotrophy and chemoheterotrophy) .

(2) - I d e n t i f i c a t i o n of t h e enzymatic mechanisms invo lved i n t h e produc- t i o n and accumulation o f the so lu tes employed i n adjustment o f i n t r a c e l 1 u l a r water p o t e n t i a l by r e p r e s e n t a t i v e n u t r i t i o n a l and s t r u c t u r a l types o f t he cyanobacteria cu l tu red .

To determfne t o what extent t he i n t r a c e l l u l a r s o l u t e s accumulating i n the cyanobacter ia studied account f o r ba lanc ing t h e ex te rna l water p o t e n t i a l .

(2)

( 4 ) - To determine whether solutes accumulating i n t h e se lec ted cyano- b a c t e r i a f o l l o w i n g upshock serve as compat ib le so lutes.

Year 3: ( A c t u a l l y t he f i n a l s i x months o f t h e o r i g i n a l t i m e t a b l e and the proposed s i x months extension)

(1) - To conduct t he s tud ies on osmoregulat ion under environmental con- d i t i o n s t h a t correspond t o those o f t he major stages i n t h e evolu- t i o n o f t h e Precambrian biosphere.

V e r i f i c a t i o n o f t he metabolic in termediates ( i .e. pathways) i n t h e synthes is o f t h e major osmoregulatory s o l u t e s i n se lec ted cyano- b a c t e r i a by 13C-NMR.

(2 ) -

The research on t h i s o b j e c t i v e has been g r e a t l y delayed due t o t h e breakdown o f our NMR instrument and l o s s o f our NMR operator . Th i s de lay was communicated t o D r . Donald DeVincenzi by the Univer- s i t y ' s D i r e c t o r o f t he O f f i c e o f Research Admin i s t ra t i on , D r . Michael R. Dingerson. This de lay has i n t u r n delayed research on c e r t a i n r e l a t e d object ives. It i s f o r t h i s reason t h a t t he

9

extens ion i s requested. There i s no request f o r a d d i t i o n a l funds f o r t h e extension.

( 3 ) - Cont inua t ion on the e l u c i d a t i o n o f t he pathways and enzymatic r e a c t i o n s i nvo l ved i n t h e p roduc t i on of osmoregulatory so lu tes i n t h e se lec ted cyanobacteria.

f i c a l l y , t h e fo l l ow ing exper imentat ion i s planned:

Determinat ion of t he changes i n osmoregulatory s o l u t e s i n t h e cyanobacteria, O s c i l l a t o r i a 1 ime t i ca and Aphanotece ha lophy t i ca when changed from cond i t i ons a l l o w i n g oxygenic photosynthesis t o environmental cond i t i ons e l i c i t i n g anoxygenic photosynthesis ( s u l f i d e u t i 1 i z a t i o n ) ;

Determinat ion of t h e s p e c i f i c pathway o f g l yc inebe ta ine synthe- s i s i n t h e cyanobacterium, Aphanothece halophyt ica; c u r r e n t l y t h ree poss ib le pathways have been proposed by two groups o f i n v e s t i g a t o r s ; i t i s impor tant t o a s c e r t a i n which o f these pathways i s operat ive i n e a r t h ' s e a r l i e s t , oxygenic photo- synthes izers;

Determinat ion o f t he e f f e c t o f s a l i n i t y (and s p e c i f i c a l l y , potassium i o n ) and beta ine on t h e synthes is o f be ta ine as the major compat ib le so lu te i n h a l o p h i l i c cyanobacteria; t h i s w i l l r e q u i r e t h e study o f t h e key, r e g u l a t o r y enzymes o f be ta ine synthes is and the e f f e c t o f potassium i o n and be ta ine on t h e i r a c t i v i t y ; and

13C-NMR s tud ies on t h e synthesis o f g l u c o s y l g l y c e r o l s betaine, t reha lose and sucrose i n se lec ted cyanobacter ia f o r c e d t o osmoregulate under c o n d i t i o n s i m u l a t i n g t h e Precambrian atmo- sphere.

Previous r e s u l t s f rom our l abo ra to ry r e l a t e d t o t h e proposed research.

Our l a b o r a t o r y has been engaged i n research t o determine the mechanisds) o f t o le rance f o r h y p e r s a l i n i t y and extremely low water p o t e n t i a l i n t h e h a l o p h i l i c cyanobacterium, Aphanothece ha lophy t i ca f o r t h e pas t f i f t e e n years. This research was supported by grants f rom the Na t iona l Aeronaut ics and Space A d m i n i s t r a t i o n (NASA-NGR-14-008-026). D e t a i l s o f t h e research a r e t h e sub jec t o f twelve publ i c a t i o n s , twenty-s ix publ ished a b s t r a c t s f rom n a t i o n a l meetings and ex tens i ve progress r e p o r t s t o NASA's D i v i s i o n o f P lanetary B io logy (19, 41, 42, 46, 51, 52a,b,c, 57, 72, 73, 74, 80, 82, 83-88).

I n a d d i t i o n , s p e c i f i c r e s u l t s from exper imentat ion conducted through support f rom t h e c u r r e n t g ran t (NAWA-344) have been repo r ted i n a previous progress r e p o r t (February 25, 1983) t o D r . Donald DeVincenzi, p a r t I 1 (second 18 months) o f t he t h r e e year study (February 25, 1983), and i n P a r t I11 f i n a l s i x month and s i x month extens ion (March 1985) t o D r . Donald DeVincenzi. These th ree prev ious progress repor ts and proposals a re g iven i n t h i s f i n a l r e p o r t as appendices I, 11, and 111, r e s p e c t i v e l y .

10

Progress i n ach iev ing t h e s t a t e d ob jec t i ves o f t h e g r a n t (NAGW-344).

t h e s t a r t o f t h e funded research. and t h e o b j e c t i v e s p a r t i a l l y achieved d u r i n g the course of t h e research:

Twenty-four (24) of t h e twenty-nine cyanobacter ia proposed f o r c u l t u r e ( i n Table 2 of f i r s t year proposal) have been s u c c e s s f u l l y cu l tu red . A l i s t of these i s prov ided as Table 1 of t h e present proposal .

E x c e l l e n t progress has been made on achievement o f most o b j e c t i v e s s ince The f o l l o w i n g i s a summary of t h i s progress

(1) -

It i s be l i eved t h a t the remaining f i v e cyanobacter ia (Synechococcus , ATCC# 29154, Synechococcus , ATCC# 29206) w i 11 be e v e n t u a l l y c u l t u r e d i n the remaining phase o f t h e grant .

The above c u l t u r e s are now i n a c t i v e l y growing cond i t i on , under a r e g u l a r t r a n s f e r protocol . Th i s research r e q u i r e d considerable exper imentat ion t o achieve t h e proper environmental c o n d i t i o n s o f l i g h t , temperature and physico-chemical c h a r a c t e r i s t i c s o f t h e c u l t u r e media.

Our group worked c l o s e l y w i th c e r t a i n s t a f f of t h e American Personal communication Type Cu l tu re C o l l e c t i o n (ATCC) f a c i l i t y .

w i t h D r . St jepko Golubic o f Boston U n i v e r s i t y d u r i n g t h e F i r s t Symposium on Chemical Evo lu t i on and t h e O r i g i n o f L i f e , organized by D r . Donald L. DeVincenzi, NASA Headquarters a l s o aided i n s o l v i n g some o f t he a n c i l l a r y problems.

Three a d d i t i o n a l blue-green a1 gae ( I cyanobacter i a I } prov ided by D r . Lynn M a v l l i s Y U ' o f Bostog University d u r i n g the s m e S y m p o s i ~ m on O r i g i n o f L i f e a t Ames Research Center, M o f f e t t F i e l d , C a l i f o r n i a were a1 so successful l y cu l tured.

I n a d d i t i o n our group i s o l a t e d a new h a l o t o l e r a t e d species o f Nostoc f rom eastern New Mexico i n 1983. F i n a l l y , D r . Y. Cohen has prov ided us w i th c u l t u r e s o f O s c i l l a t o r i a l i m n e t i c a f o r our s t u d i e s on osmoregulat ion d u r i n g anoxygenic photosynthesis.

These cyanobacteria, i n a c t i v e l y growing c u l t u r e s , a re now a v a i l a b l e f o r use by other s c i e n t i s t s working w i t h i n the same program funded by NASA. This represents, t he re fo re , a va luab le r e p o s i t o r y of t he "organisms o f t h e Precambrian" f o r immediate use by these s c i e n t i s t s . have a1 ready taken advantage of t h i s oppor tun i t y .

S i x s c i e n t i s t s i n u n i v e r s i t i e s i n t h i s country

A l i s t o f these cyanobacteria was r e c e n t l y prov ided t o scien- t i s t s a t NASA-Ames Research Laboratory M o f f e t t F i e l d , CA d u r i n g a week v i s i t , August 6-11, 1984.

(2) - One t h i r d o f t h e cyanobacteria (approx imate ly} from t h e group (group E of Table 1) o f f a c u l t a t i v e chemoheterotrophs a re now i n l a r g e sca le c u l t u r e f o r use i n exper imentat ion i n v o l v i n g osmoregulatory s o l u t e determinat ion and NMR s tud ies.

11

( 3 ) - The p r i n c i p a l osmoregul a tory s o l Ute (compat ib le so l Ute as we1 1 ) o f t h e extremely h a l o t o l erant Aphanothece ha1 ophyt ica , has been iden- t i f i e d as a be ta ine by NMR. concen t ra t i on o f f i v e t o f i f t e e n t imes those o f a l l of t h e s o l u b l e carbohydrates and f ree amino ac ids combined (Table 2).

Th is s o l u t e i s found i n c e l l u l a r

We have i d e n t i f i e d a g l u c o s y l g l y c e r o l t h a t a l s o f l u c t u a t e s w i t h changes i n ex te rna l (medium) osmo la r i t y . However, t h i s s o l u t e accumulates i n concentrat ions t o o low t o serve a major osmo- r e g u l a t o r y r o l e .

The be ta ine content o f A. ha lophy t i ca n o t o n l y increases dramat ica l l y w i t h increasing-NaC1 - m o l a r i t y o f t h e growth medi um (Table 2) b u t a l s o increases i n response t o r a p i d changes i n medium osmo la r i t y ( i .e. , upshock). Note t h a t be ta ine con ten t i s h i g h e r i n organisms grown or upshocked i n media c o n t a i n i n g g l y c y l g l y c i n e b u f f e r than i n media conta in ing MES b u f f e r . be ta ine content f a l l s w i t h downshock, b u t n o t t o i n i t i a l l e v e l s . There i s p r e l i m i n a r y evidence t h a t be ta ine i s metabol ized f o l l o w i n g downshock.

Beta ine synthes is i n A. ha lophyt ica r e q u i r e s l i g h t (87). organism i s an ob1 igaFe photoautotroph b u t does s low ly deplasmolyze i n t h e dark. However, growth w i l l n o t cont inue i n the dark.

Beta ine i s n o t merely a neutra l osmoregulatory s o l u t e b u t a l s o d i sp lays coun te rac t i ng e f fec ts on s a l t - i n h i b i t i o n o f enzyme a c t i v i t y . Glucose-6-phosphate dehydrogenase from A. h a l o h t i c a was assayed i n

i n h i b i t i x o f a c t i v i t y was obta ined between 0.3 and 0.4M KC1. concen t ra t i on has been shown t o occur i n A. ha lophy t i ca (see prev ious s t u d i e s ) . Next, t h i s enzyme wasassayed i n a medium c o n t a i n i n g 04.M KC1 p l u s e i t h e r g l y c e r o l , p r o l i n e o r beta ine. Although some r e l i e f f r o m s a l t i n h i b i t i o n was obtained w i t h p r o l i n e and g l y c e r o l , be ta ine c l e a r l y was the super io r coun te rac t i ng , compat ib le s o l u t e (52a).

We have a l s o found t h a t

Studies on t h i s quest ion a r e c u r r e n t l y i n progress.

(4) - Th is

(5 ) -

the presence o f increas ing concen t ra t i ons -+ o f KC1 pprox imate ly 50% This

F i n a l l y , t h e e f f e c t of t h e e x t e n t of me thy la t i on o f g l y c i n e on p r o t e c t i o n aga ins t s a l t i n h i b i t i o n o f glucose-6-phosphate dehydro- genase was examined. There was found a c l e a r r e l a t i o n s h i p between degree o f me thy la t i on and e x t e n t of s a l t coun te rac t i ng e f f e c t (52a).

The source (organism) o f the enzyme i s a f a c t o r i n t h e e x t e n t o f p r o t e c t i o n aga ins t s a l t i n h i b i t i o n a f f o r d e d by beta ine. Contrary t o r e p o r t s i n t h e l i t e r a t u r e (82) , a compatible, osmoregulatory s o l u t e w i l l n o t p r o t e c t a p a r t i c u l a r enzyme regard less o f source. We obta ined p u r i f i e d glucose-6-phosphate dehydrogenase from an euka ryo t i c organism, Torula yeas t and another p r o k a r y o t i c organism, Leuconostoc mesenteroides. The enzyme from t h e l a t t e r organism was s e v e r a l - f o l d more s a l t t o l e r a n t than t h e enzyme from t h e former organism. Betaine had d ramat i ca l l y d i f f e r e n t e f f e c t s on t h e degree o f s a l t p r o t e c t i o n f o r the enzyme from d i f f e r e n t sources. The i n h e r e n t l y more s a l t t o l e r a n t enzyme was a c t u a l l y i n h i b i t e d by be ta ine (52a).

(6) -

12

( 7 ) - Beta ine may e x e r t i t s "counteract ing ef fects ' ' on s a l t i n h i b i t i o n n o t by " r e s t o r i n g a c t i v i t y " o f s a l t i n h i b i t e d enzymes, b u t by l ower ing t h e Km o f t h e enzyme p e r se. We had repo r ted e a r l i e r t h a t KC1 i n h i b i t s enzyme a c t i v i t y i n h a l o t o l e r a n t blue-green algae by causing an increase i n Km. t h e presence of h i g h i n t e r n a l K by g r e a t l y i n c r e a s i n g t h e a f f i n i t y o f the enzyme f o r i t s subs t ra te - - fo r enzymes f rom c e r t a i n , b u t n o t a1 1 , organisms (52a, 52b) .

The changes, both q u a n t i t a t i v e and q u a l i t a t i v e , i n amino ac ids f o l lowing upshock of t h e ha1 o t o l e r a n t , Aphanothece ha1 ophyt ica, have been completely documented. made concerning these changes:

Betaine appsars t o ' 'a l low" enzyme f u n c t i o n i n

( 8 ) - The f o l l o w i n g major p o i n t s can now be

a) The predominant f r e e amino ac ids i n t h e pool a r e g l u t a m i o s e r i ne>gl yc ine>aspar t ic>al a n i ne. f r e e amino ac ids do n o t increase as t h e s a l i n i t y o f t h e growth medium increase (Table 2), an apparent increase occurs i n t h i s organism upon upshock i n medium c o n t a i n i n g g l y c y l g l y c i n e buffer. b a c t e r i a i n medium conta in ing MES b u f f e r . The increase under t h e former buffer cond i t i on appears i n t h e " l euc ine f r a c t i o n " o f t he ana lys i s obtained by i o n exchange w i t h i n t h e Beckman 19CL Amino Acid Analyzer. "amino ac id " t o be t h e b u f f e r g l y c y l g l y c i n e by us ing High Vol tage Paper Electrophoresis. t i d e g l y c y l g l y c i n e i t s e l f i s u t i l i z e d by t h e a lga d u r i n g upshock. There a re r e p o r t s o f o the r b a c t e r i a u t i l i z i n g t h e amino ac ids o f t h e i r growth media under upshock cond i t i ons . eve9 m ~ r e i n t e r e s t t o LIS i s t he consequence o f g l y c y l g l y c i n e accumulation d u r i n g upshock. The amino a c i d p r o l i n e accumu- l a t e s i n MES buffered-organisms f o l l o w i n g upshock. This i s a major d i f f e r e n c e i n v o l v i n g an amino a c i d known t o be i nvo l ved i n the osmoregulatory mechanism (perhaps as a compat ib le s o l u t e ) o f eukaryot ic and p r o k a r y o t i c organisms. noted a l s o t h a t g l y c y l g l y c i n e i s taken up from the upshock media under l i g h t , dark and dark anaerobic cond i t i ons . major d i f f e r e n c e i s t h a t l y s i n e g r e a t l y increases i n cyano- b a c t e r i a upshocked i n g l y c y l g l y c i n e , b u t n o t i n cyanobacter ia upshocked i n MES buffered-medium. i n 1983 (87).

Whereas t h e t o t a l

No s i m i l a r increase i s observed i n upshocked cyano-

However, we have now shown t h i s

It i s obvious t h a t t h e dipep-

Of

It should be

Another

These r e s u l t s were repo r ted

b) The amino ac ids changing t o the g rea tes t e x t e n t w i t h upshock a r e s e r i n e and glutamic ac id . ac ids o f t h e f r e e pool i n t h i s organism. always decreases t o a major e x t e n t and t h e s e r i n e concomi tant ly r i s e s . i n d i c a t e a metabol ic r e l a t i o n s h i p between t h e two i n which g lu tamic a c i d may serve as an amino donor f o r s e r i n e b i o - synthesis. I n add i t i on , i t should be noted t h a t t he r e l a t i o n - s h i p occurs independent o f type o f b u f f e r present i n t h e upshock media (87). We fee l t h a t these f i n d i n g s a r e o f p a r t i c - u l a r importance i n view of t h e f a c t t h a t s e r i n e i s t h e precur- so r t o be ta ine and t h a t t h i s amino a c i d i s " t r i g g e r e d " t o

These a re a l s o the major amino The g lu tamic a c i d

The r e l a t i o n s h i p i s be l i eved by t h e i n v e s t i g a t o r s t o

13

accumulate w i t h increas ing s a l i n i t y under a l l environmental cond i t i ons tested. The s i g n i f i c a n c e o f t h i s observat ion t o osmoregulat ion i n cyanobacteria and t o e v o l u t i o n o f osmo- r e g u l a t o r y mechanisms w i l l be discussed i n d e t a i l below.

(9) - The changes i n t o t a l so lub le carbohydrates, reducing carbohydrates, t o t a l amino ac ids and i n d i v i d u a l amino ac ids f o l l o w i n g upshock i n l i g h t and dark o r t h r e e d i f f e r e n t s t r u c t u r a l types o f cyanobacter ia have been determined. These were t h e u n i c e l l u l a r Synechocystis t h e f i 1 amentous, E; and the branched F i s h e r e l l a. The major f i ndings

t h i s exper imentat ion are as f o l l o w s :

- No be ta ine accumulated f o l l o w i n g upshock i n NaCl ( s u f f i c i e n t f o r p lasmolys is and recovery) i n any o f t he t h r e e s t r u c t u r a l types o f cyanobacteria. These r e s u l t s and severa l months of p r e l i m i n a r y exper imentat ion t o " f i nd " beta ines i n o t h e r cyano- bac te r ia , have l e d us t o t e n t a t i v e l y conclude t h a t be ta ine i s a compatible, osmoregulatory so l Ute formed i n ha1 o t o l e r a n t and h a l o p h i l i c oxygenic photoautotrophs, b u t n o t i n f r e s h water organisms of t h e same type. A l l t h r e e o f t h e s t r u c t u r a l types so t e s t e d were of fresh o r i g i n .

Q u a n t i t a t i v e l y speaking, t h e s o l u t e c l a s s i n c r e a s i n g t o the g r e a t e s t e x t e n t i n the Synechococcus, LPP ( O s c i l l a t o r i a type) and F i s c h e r e l l a was t h a t o f t h e non-reducing carbohydrates. Non-reducing carbohydrate amount i s obta ined by s u b t r a c t i n g reducing carbohydrates from t o t a l s o l u b l e carbohydrates i n our ana lys i s p ro toco l .

C1 e a r l y , t h e evnl lrti nnar i l y most advanced F i scherel 1 a %

conta ins t h e greatest amount o f non-reducing carbohydrates. a d d i t i o n , t h e non-reducing carbohydrate f r a c t i o n (we b e l i e v e one molecular type) more than doubles w i t h upshock a f t e r o n l y seven hours. Furthermore, t h i s increase occurs i n bo th l i g h t and dark. an immediate l i g h t requirement. i n reducing sugars f o l l o w i n g upshock i n the F i s c h e r e l l a .

I n

The carbohydrate( s ) synthes is then proceeds w i t h o u t There i s l i t t l e o r no increase

A much d i f f e r e n t e f f e c t o f upshock i s seen i n t h e l e s s e v o l u t i o n a r i l y advanced LPP. carbohydrates (again, we b e l i e v e one chemical species) more than doubles a f t e r s i x hours o f upshock, but, o n l y i n the l i g h t . Deplasmolysis i s a l s o very much slower i n t h e dark. Again, t h e r e i s no appreciable increase i n reducing sugars w i t h ups hock.

I n t h i s case, non-reducing

F i n a l l y , t he u n i c e l l u l a r cyanobacter ia Synechocystis behaves much t h e same way as the f i lamentous LPP w i t h respect t o t o t a l carbohydrate. However, under 1 i g h t c o n d i t i o n s only , t h e reducing carbohydrates ( b u t n o t non-reducing f r a c t i o n ) increase w i t h upshock.

14

It should be noted t h a t o n l y the F i s c h e r e l l a i s capable o f growing i n t h e dark on a carbohydrate source ( i .e . chemohetero- t r o p h i c a l l y ) .

c ) The changes i n f ree amino ac ids i n t h e t h r e e fresh-water cyanobacter ia d i d not c o r r e l a t e w i t h those found f o r t h e carbohydrates. Synechocystis and fi lamentous LPP. No increase i n t o t a l amino ac ids were observed f o r t h e F i sche re l 1 a.

Object ives concerned w i t h the de te rm ina t ion of r e l a t i o n s h i p between s t r u c t u r a l and n u t r i t i o n a l modes ( i .e. photoautotrophy, photohetero- t rophy and chemoheterotrophy) and t h e na tu re of t h e s o l u t e s employed f o r adjustment o f i n t r a c e l l u l a r water p o t e n t i a l i n cyanobacter ia ( o b j e c t i v e 1 and 2) a r e l a r g e l y achieved.

was grown i n a l l t h ree n u t r i t i o n a l modes and forced t o osmoregulate ( a f t e r upshock) i n each mode. Cal o t h r i x (ATCC #27914) was 1 i kewi se t r e a t e d i n t h e photoautot rophic n u t r i t i o n a l mode. The most advanced s t r u c t u r a l type ( i .e. branched), Chlorogl oeopsis (ATCC #27181) was forced t o osmoregul a t e i n a l l t h r e e n u t r i t i o n a l modes. Previous s tud ies (of growth alone) of cyanobacter ia i n a l l three n u t r i t i o n a l modes invo lved p r i o r growth ( e a s i e r and more rap id ) i n photoautot rophic c o n d i t i o n s fo l l owed by a few days t ransfer t o t h e o t h e r two modes p r i o r t o exper imentat ion. The present s tudy employed cyanobacter ia complete ly grown under th ree n u t r i t i o n a l modes.

The greatest changes occur i n t h e u n i c e l l u l a r

(10) -

The u n i c e l l u l a r s t r u c t u r a l type, Synechocystsis (ATCC #27178)

The f i lamentous s t r u c t u r a l type,

r-uh- bo1 uuhydrates rather than amins a c i d s a r e t h e principal solutes

(F igu re I ,

i nc reas ing d u r i n g osmotic adjustment i n S nechoc s t s i s under photo- au to t roph ic cond i t i ons (almost a 10-fold + d i f f e r e n c e Increases o f both so lu tes were g rea te r i n t h e l i g h t than i n t h e dark. Appendix 111) was fo l l owed by a r a p i d f a l l w i t h i n t h e nex t 24-hours. d i d n o t occur i n t h e carbohydrates. t h a t t h e carbohydrate f r a c t i o n i n c r e a s i n g was almost e n t i r e l y non-reducing disaccharides (see progress r e p o r t phase 11, f o r d e t a i l s ) .

Furthermore, t he r i s e i n amino ac ids a f t e r 24-hours Th is f a l l

Subsequent analyses revealed

SUC I11

The p r i n c i p a l carbohydrate i s g l u c o s y l g l y c e r o l , fo l l owed by .rose. Only sucrose accumulated i n t h e dark. (F igu re 2, Appendix )

t h i s a lga were g lu tamic acid and p r o l i n e (F igu re 3 o f Appendix 111). As i n t h e carbohydrate f r a c t i o n s , amino a c i d changes were more marked i n t h e l i g h t than i n the dark.

The p r i n c i p a l amino ac ids changing w i t h osmotic adjustment i n

Osmotic adjustment i n Synechocystis under photoheterot rophic c o n d i t i o n s d i f f e r e d somewhat from t h a t under photoautot rophic cond i t i ons . Both carbohydrates and amino ac ids increased i n t h e dark (a lmost as much as i n t h e l i g h t ) (F igu re 4, Appendix 111). Again, t he carbohydrates were q u a n t i t a t i v e l y the more impor tant so lu te . t he carbohydrate f r a c t i o n revealed t h a t both sucrose and

Gas chromatographic analyses (F igu re 5 o f Appendix 111) o f

15

g l u c o s y l g l y c e r o l increased w i th t h e former carbohydrate predominating. Likewise, di f ferences were noted i n the amino a c i d increases d u r i n g osmotic adjustment. Glutamic a c i d increased, as i t d i d i n t h e photoautot rophica l l y grown cyanobacteria, b u t p r o l i n e d i d n o t . Moreover, i n t h i s n u t r i t i o n a l mode, l y s i n e and v a l i n e d r a m a t i c a l l y increased f o l l o w i n g upshock. The much g r e a t e r ( t h a n i n photo- autrophy) increase i n carbohydrates i n dark upshocked c e l l s was seen t o be due t o sucrose, n o t g lucosy lg l yce ro l (F igures 3, 6 and 7 , Appendix 111).

Osmotic adjustment i n Synechocystis under chemoheterotrophic c o n d i t i o n s (dark o n l y ) was achieved b a s i c a l l y i n t h e same manner as i n t h e o the r two n u t r i t i o n a l modes. t he much g rea te r amino ac id con ten t o f t h e o s m o t i c a l l y ad jus t i ng , chemoheterotrophically-grown c e l l s (F igu re 8 o f Appendix 111). These l e v e l s were over twice those found i n c e l l s grown i n t h e o t h e r two modes. Sucrose was the p r i n c i p a l sugar. I n t h i s n u t r i t i o n a l mode, g lu tamic a c i d i s s t i l l t h e p r i n c i p a l amino ac id, b u t a l a n i n e and s e r i n e a re a l s o seen t o d r a m a t i c a l l y increase (F igu re 9 , Appen- d i x 111).

The s t r j k i n g d i f f e r e n c e was i n

Osmotic adjustment i n t h e f i lamentous s t r u c t u r a l form, C a l a t h r i x (ATCC #27914) i n t h e photoautot rophic mode i s s i m i l a r q u a n t i t a t i v e l y t o t h a t of Synechocystis. Carbohydrates (again non-reducing) a r e about 10 - fo ld g rea te r than amino ac ids i n c e l l s upshocked i n the l i g h t (Figure l o ) . Dark l e v e l s r a p i d l y decreased f o l l o w i n g an i n i t i a l , smal ler r i s e . T r i m e t h y l s i l y l d e r i v a t i v e s o f these carbohydrates revealed t h a t un l i ke Synechocystis , C a l o t h r i x employed (F igu re 11, Appendix I J I ) t h e non-reducing d isacchar ide, treha1~)se. Sucrose 31 so apprec iab ly increased. G1 utamate and a r g i n i n e account f o r most o f t h e amino a c i d increase d u r i n g osmotic adjustment (F igu re 12, Appendix 111).

Osmotic adjustment i n t h e most complex s t r u c t u r a l form o f t he cyanobacter ia (i .e. branched), Chlorogloeopsis (ATCC #27181) was l i k e t h e o t h e r two forms achieved p r i n c i p a l l y by carbohydrates under photoautot rophic condi t ions (F igu re 13 o f Appendix 111). cond i t i ons r e s u l t e d i n twice as much carbohydrate as i n dark con- d i t i o n s . The absolute l e v e l s o f carbohydrate was niuch h i g h e r i n t h i s s t r u c t u r a l form than i n t h e previous two forms. Amino a c i d l e v e l s were much lower than i n the Synechocystis and C a l a t h r i x . Gas chromatographic a n a l y s i s showed t h a t t h e p r i n c i p a l carbohydrate was t rehalose, b u t t h a t sucrose i s a l s o formed d u r i n g osmotic adjustment (F igu re 14 o f Appendix 111). amounts o f t reha lose were a l s o formed i n dark upshocked Chlorogloeopsis (F igu re 15 of Appendix 111). amino ac ids changing dur ing osmotic adjustment i n Chlorogloeopsis d i f f e r e n t f rom t h a t o f the o t h e r two s t r u c t u r a l forms. F igu re 16 shows t h a t g lu tamic a c i d f a l l s and two d e r i v a t i v e s o f g lu tamic ac id , p r o l i n e and a r g i n i n e r i s e d ramat i ca l l y .

cond i t i ons f o l l o w s b a s i c a l l y t h e same p lan ( q u a n t i t a t i v e l y ) as i n the photoautot rophic mode (F igu re 17 o f Appendix 111).

L i g h t

Unl i k e t h e case f o r C a l o t h r i x , l a r g e

The na tu re o f t h e

Osmotic adjustment i n Chlorogloeopsis under photoheterot rophic

S u r p r i s i n g l y ,

16

however, sucrose r a t h e r than t reha lose i s t h e predominant carbohy- d r a t e (F igu re 18). Trehalose i s never the less present. Again, as i n t h e photoautot rophic mode, pro1 i n e and a r g i n i n e r i s e d u r i n g osmotic adjustment, w h i l e glutamic a c i d f a l l s (F igu re 19 o f Appendix 111).

Osmotic adjustment i n Chloro l o e o s i s d u r i n g chemoheterotrophic c o n d i t i o n s ( o n l y i n t h e dark + f o l l o w s t h e same q u a n t i t a t i v e p l a n as i n t h e o t h e r two n u t r i t i o n a l modes. The amino a c i d l e v e l s remain low (F igu re 20 of Appendix 111). Trehalose and sucrose a r e a a i n t h e p r i n c i p a l carbohydrates formed d u r i n g osmotic adjustment ?Figure 21 o f Appendix 111).

A major change i n amino a c i d response was seen i n t h e chemo- h e t e r o t r o p h i c mode du r ing osmotic adjustment. Glutamic a c i d l e v e l rose and the p r o l i n e and a r g i n i n e l e v e l s remained almost constant (F igu re 22 o f Appendix 111). Chlorogloeopsis f o r t h e conversion o f g lu tamic a c i d t o p r o l i n e and a r g i n i n e .

Research on t h e pathways o f synthes is o f t h e osmoregulatory s o l u t e s has focused ma in l y on the pathway of be ta ine fo rma t ion i n t h e ha1 o p h i l i c (photoautotrophic mode, o n l y ) cyanobacterium, Aphanothece halophyt ica.

Apparent ly, l i g h t i s r e q u i r e d i n

(11) -

As Drev ious lv noted we have found t h a t t h e Drecursor o f t h e carbon 'Ibkeleton"" o f betaine, ser ine, increases i n - A. ha lophy t i ca d u r i n g osmotic adjustment, both i n l i g h t and dark. However, be ta ine synthes is i n th is-cyanobacter ium r e q u i r e s 1 i g h t . been d i r e c t e d toward t h e discovery o f t h e na tu re o f t h i s l i g h t requirement. Our f i r s t approach was t o determine the o r i g i n o f t he methyl groups o f beta ine. F igu re 1 prov ides our i n t e r p r e t a t i o n o f t he pathway o f be ta ine synthesis i n t h e s u b j e c t cyanobacterium. Note t h a t S-Adenosylmethionine (SAM) i s g iven i n t h i s f i g u r e as the probable methyl source. This compound i s w i d e l y considered t o be t h e u n i v e r s a l methyl donor i n t h e major b i o l o g i c a l me thy la t i ons (25a, 13a). L-methionine Methionine der ives i t s methyl group f rom methyl t e t r a - h y d r o f o l a t e ( thereby imp1 i c a t i n g f o l a t e metabol ism). f o l i c a c i d i s t h e c a r r i e r o f methyl groups i n t h i s reac t i on . receives t h e C - 1 (methyl) fragment i n i t i a l 1 quent reduc t i ons ) from formic a c i d ( formate 1 . h e l d (19a) t h a t one o f the p r i n c i p a l sources o f formate i n photo- syn thes i z ing p l a n t s i s the decarboxy lat ion o f g l y o x y l i c a c i d produced by pho to resp i ra t i on (bas i s o f t h e 1 i g h t requirement?). However, one problem remained. Cur ren t l y , t h e r e i s cons iderable support f o r t h e ex is tence of a d i f f e r e n t pho to resp i ra to ry pathway i n cyanobacter ia f rom t h a t i n h ighe r p lan ts . F igu re 2 d e p i c t s bo th pathways. I n eukaryotes g l y o s y l a t e i s transaminated t o g l y c i n e and g l y c i n e subsequently converted t o ser ine. I n cyanobacter ia, severa l prominent i n v e s t i g a t o r s (17a, 18a) pos tu la ted the ex i s tence of an a1 t e r n a t e pathway through t a r t r o n i c a c i d semial dehyde. based p a r t i a l l y on t h e lack o f e f f e c t o f t he i n h i b i t o r o f t h e enzyme c a t a l y z i n g t h e conversion o f g l y c i n e t o s e r i n e (h ighe r p l a n t pathway). Th is i n h i b i t o r i s I N H ( I s o n i c o t i n i c a c i d hydraz ide) . I f INH is a b l e

Our research has

I t s methyl group i s t h a t o f i t s c o n s t i t u e n t

Tetrahydro- It

(and be fo re two subse- It i s a l s o w i d e l y

Th is was

17

t o e x e r t i t s e f f e c t , and g l yc ine conversion t o s e r i n e i s blocked, g l y c i n e w i l l accumulate. We fo l l owed t h e e f f e c t s of t h e i n h i b i t o r I N H on g l y c i n e and be ta ine format ion. I t should n o t b lock be ta ine fo rma t ion i f the methyl group i s de r i ved from t h e C-2 atom o f g l y o x y l i c ac id . i t s e l f by t h e pho to resp i ra t i on methane a lpha-hydroxypyr id ine methane s u l f o n i c a c i d (HPMS) (see F igu re 2). t i o n o f g l y o x y l i c a c i d and the re fo re , f o r m i c a c i d p roduc t i on f rom i t s decarboxy lat ion. I n turn, be ta ine fo rma t ion should be d imin- ished due t o a l a c k of formate, which i s t h e source o f i t s methyl

The data from these experiments i s prov ided i n F igures 8?%”,f the l a s t progress r e p o r t (Appendix 111).

We consider the above data as c l e a r demonstrat ion o f a s t rong pho to resp i ra to ry pathway o f t h e t ype found i n h i g h e r p l a n t s .

The nex t quest ion concerned the metabol ic c o n t r o l of be ta ine fo rma t ion f o l l o w i n g upshock o f A. ha lophyt ica. mechanism was sought f rom the pFevious data. Note i n Table 2 t h a t t he be ta ine l e v e l s correspond t o t h e l e v e l s o f NaCl t h e cyano- bacter ium was grown i n . The h ighe r t h e NaCl t h e h i g h e r t h e i n t e r n a l betaine. We hav? a l s o shown (see r e f 51) t h a t i n t r a c e l l u l a r potassium i o n ( K ) increases w i t h i n c r e a s i n g e x t e r n a l s a l i n i t y . +It would appear then t h a t s a l i n i t y , perhaps through i n t r a c e l l u l a r K , was i n i t i a t i n g the synthesis o f t h e compatible, osmoregulatory s o l u t e betaine.

I n addi t ion, we blocked g l y c o l i c a c i d fo rma t ion

This should b lock t h e forma-

A c o n t r o l 1 i n g

A p r i n c i p a l regu lan t o f be ta ine synthes is would be t h e me thy la t i on o f ethanolamine (F igu re 23). t h a t t h e methylase enzymes emplnying A-Adennsyl methionine (SAM) as methyl donors a re regulated (feedback i n h i b i t i o n ) by t h e product o f t he reac t i on , S-Adenosylhomocysteine (SAH). 3 SAM + ethanolamine + 3 SAH + cho l i ne .

I t i s w i d e l y h e l d (13a)

The r e a c t i o n i s :

Chol ine i s ox id i zed t o beta ine. by the i n t r a c e l l u l a r SAMjSAH r a t i o s . f o re , g r e a t l y increases upon t h e enzymatic removal o f SAH.

Methy lat ions a r e then regu la ted Me thy la t i on a c t i v i t y , there-

I t i s c u r r e n t l y h e l d ( E a , 74a) t h a t prokaryotes and eukaryotes d i f f e r w i t h respect t o the mechanism o f SAH breakdown. eukaryotes the enzyme i s A-Adenosyl homocysteine hydro1 ase (SAH hydrolase). ( a c t u a l l y i n favor o f ) t o achieve t h e synthes is o f SAH f rom homocysteine and adenosine. o f SAH l e v e l s and consequently, b i o l o g i c a l methy lat ions. prokaryotes the enzyme ca ta l yz ing SAH removal i s S-Adenosyl homocysteine nucleosidase. i r r e v e r s i b l e.

I n

This enzyme a l so f u n c t i o n s i n the reverse d i r e c t i o n

I n t h i s sense i t i s a r e g u l a t o r y enzyme I n

The r e a c t i o n i s e s s e n t i a l l y

A potent, s p e c i f i c ( a s s p e c i f i c i t y goes) i n h i b i t o r o f t he euka ryo t i c enzyme SAH hydrolase i s t h e adenosine analog, 3-Deazaadenosine. 13a) t o i n h i b i t a l l b i o l o g i c a l methy lat ions, due t o i t s e f f e c t on p reven t ion o f SAH breakdown.

Consequently, i t i s c l i n i c a l l y w ide l y used (25a,

A f t e r numerous experiments t o

18

demonstrate SAH nucleosidase i n ou r cyanobacterium f a i l e d , we looked f o r t he enzyme be l i eved t o be present o n l y i n eukaryotes, SAH hydrolase. hydro lase p r e v i o u s l y be l ieved t o n o t be present i n prokaryotes. Figures 30-33 from the repo r t i n Appendix I11 show t h e data obta ined f rom these experiments. P re l im ina ry s tud ies ( n o t repo r ted here) u t i l i z e d an assay based upon t h e disappearance of L-homocysteine o c c u r r i n g upon measurement o f s y n t h e t i c a c t i v i t y . reveal ed good SAH hydro1 ase a c t i v i t y . based upon t h e more s p e c i f i c r a d i o l a b e l l e d adenosine i n c o r p o r a t i o n i n t o SAH ( s y n t h e t i c d i r e c t i o n ) . The data g iven i n t h i s r e p o r t a r e f rom t h i s l a t t e r assay.

We found t h a t a t l e a s t t h i s cyanobacterium has SAH

The assays However, 1 a t e r assays were

F igu re 30 o f Appendix I11 shows the e f f e c t o f c u l t u r e age on the a c t i v i t y o f SAH hydrolase. e f f e c t o f 0.4 M KC1 added t o t h e assay medium. s i s o f SAH (assay i n the syn the t i c r a t h e r than h y d r o l y t i c d i r e c t i o n ) i s i n h i b i t e d by the s a l t . F igu re 31 shows (another exper iment) t he assay o f SAH synthes is f rom t h e perspect ive of adenosine incorpo- r a t i o n i n t o SAH. Again 0.4M K C l i n h i b i t s t h i s i nco rpo ra t i on . Betaine, however, increases the a c t i v i t y of t h e enzyme even i n the absence o f KC1 s a l t . Furthermore, beta ine c l e a r l y r e l i e v e s t o a l a r g e e x t e n t t h e KC1- inh ib i t ion o f SAH synthesis. be ta ine accumulation would t h e o r e t i c a l l y be SAH accumulation, and, t h e r e f o r e shutdown o f f u r t h e r methy lat ion. I n t h i s manner be ta ine would r e g u l a t e i t s own synthesis.

A lso shown i n t h i s f i g u r e i s t h e C l e a r l y t h e synthe-

The n e t e f f e c t o f

F igu re 32 (Appendix 111) d e p i c t s t h e r e s u l t s o f an experiment on t h e e f f e c t o f t h e s p e c i f i c SAH hydrolase i n h i b i t o r , 3-Deazaadenssine nn cyaaohacter ia l SAH synthes is . I n h i b i t i o n was observed even a t l O u m i n h i b i t o r , thereby p r o v i d i n g a d d i t i o n a l evidence f o r the existence of t h i s enzyme i n cyanobacter ia.

hydro lase i n h i b i t o r , 3-Deazaadenosine on be ta ine synthes is i n the i n t a c t c e l l s f o l l o w i n g upshock. Betaine synthes is was completely i n h i b i t e d .

F i n a l l y , f i g u r e 33 (Appendix 111) shows t h e e f f e c t o f t h e SAH

(12) - Very extens ive s tud ies were performed t o determine t h e e f fec t o f be ta ine on the major k i n e t i c parameters of g l ucose-6- hosphate dehydrogenase from cyanobacter ia l species ( o r s t r a i n s P va ry ing i n s a l t t o le rance as w e l l as from a v a r i e t y o f o t h e r microorganisms whose p u r i f i e d enzymes d i sp lay va ry ing s a l t t o le rance . s tud ies have been t h e subject of two Master of Science theses and several presentat ions which were pub1 ished (91, 95, 96). Genome s i z e o f s t r a i n s o f t h e same species o f cyanobacter ia ( i . e . Synechocystis and LPP) was a l s o considered as a v a r i a b l e i n these enzymatic 3 tud ies m). (1.79 x 10 da l tons ) and h igh (3.5 x 10 da l tons ) DNA-content cyanobacter ia ( s t r a i n s 27184 and 29235, respecthvely, o f S nechoc t i s ) and enzymes f rom a low (2.5 x 10 d a l t o n s ) and h igh +hi-€P- da l tons ) DNA content LPP ( s t r a i n s 29126 and 29344) cyanobacteria, va ry ing amounts o n C 1 were needed f o r 50% i n h i b i t i o n (F igu re 3).

These

It was found t $ a t f o r enzymes f rom low

The lower DNA con ten t Synechocystis was much more

19

t o l e r a n t o f KC1 , as was i t s glucose-6-phosphate dehydrogenase , than the h ighe r DNA s t r a i n and i t s enzyme. when used as a probe, has much d i f f e r e n t e f fects on these enzymes f rom the two species of cyanobacteria. i d e n t i c a l species b u t d i f f e r e n t s t r a i n s showing v a r y i n g s a l t t o l e r - ance v a r i e d g r e a t l y w i t h respect t o pH.

F igures 4-8 show t h a t pH,

The enzymes even from

O f f a r g rea te r importance, however, i s t h e e f f e c t of g l y c i n e - be ta ine on enzymatic a c t i v i t y reduced 50% by assay KC1. F i ures 9 and 10 show t h a t t he greater t h e s e n s i t i v i t y o f t h e enzyme !from whatever source) t o KC1 the g r e a t e r w i l l be t h e e f f e c t of be ta ine on negat ing the KC1 i n h i b i t i o n . The converse i s a l s o t r u e (F igu re 10).

The s i g n i f i c a n c e o f t h i s can bes t be seen i n F igu re 11. Here a number o f glucose-6-phosphate dehydrogenases a r e assayed w i t h respect t o t h e i r s e n s i t i v i t y t o KC1 and t h e e f f e c t of be ta ine on t h i s KC1 i n h i b i t i o n . Again, t h e more i n h i b i t e d t h e enzyme by KC1 t h e g rea te r t he e f fec t o f be ta ine on " r e s t o r i n g n t h e a c t i v i t y . more KC1 t o l e r a n t enzymes a re n o t a f fec ted , o r a r e adverse ly a f f e c t e d by betaine. "Restorat ion" i s a c t u a l l y a misnomer because s tud ies from t h i s p r o j e c t have shown t h a t be ta ine independent o f s a l t , lowers Km, w h i l e h igh assay s a l t causes Km t o increase. adapt ive value of betaine. These f i gu res a re from reference 96.

Recent exper imentat ion i n our l a b o r a t o r y has a l s o demonstrated t h a t g l y c i n e be ta ine w i l l increase t h e to le rance of glucose-6-phosphate dehydrogenase t o increas ing pH (95). F igu re 12 shows t h i s dramat ic e f f e c t o f beta ine.

sect ion.

The

This i s t he

(13) -

The s i g n i f i c a n c e o f t h i s s u r p r i s i n g e f f e c t o f I...+, UCLai I i t 4 nrr t o t h e ~ s m r e g u l a t ~ r y mechanism will be disc~ussed i n a l a t e r

(14) - Our s tud ies on the r o l e o f t he enzyme S-Adenosylhomocysteine (SAH) hydrolase i n cyanobacter ia l osmoregulat ion and be ta ine synthes is have demonstrated t h a t the c o n t r o l l i n g f a c t o r i s i n t r a c e l l u l a r potassium i o n concentrat ion. Apparently, t h i s enzyme, which was f i r s t found i n cyanobacteria i n t h i s p r o j e c t , i s a "one-way" enzyme. This means t h a t as i n t r a c e l l u l a r potassium accumulates d u r i n g t h e i n i t i a l stage o f osmoregulat ion t o concentrat ions i n h i b i t o r y t o most enzymes, t h e SAH hydrolase s t i l l f u n c t i o n s u n i n h i b i t e d i n t h e h y d r o l y t i c d i r e c t i o n . It i s i n h i b i t e d , however, i n t h e favored (Keg.) synthes is d i r e c t i o n . As SAH i s a powerful i n h i b i t o r o f methylases which use S-Adenosylmethionine (SAM) t o methy late t h e precursor t o betaine, t h i s e f f e c t would a l l o w be ta ine synthes is t o cont inue t o a concentrat ion t h a t would a l l o w the SAH hydrolase t o func t i on i n the s y n t h e t i c d i r e c t i o n . SAH would then accumulate t o shut down be ta ine synthesis. Th is i s a p r i m i t i v e b u t e f f i c i e n t , adapt ive mechanism t h a t regulates be ta ine synthes is t o achieve s a l t to lerance. These f i n d i n g s have been repo r ted (90, 92) and accepted f o r pub1 i c a t i o n i n an i n t e r n a t i o n a l j o u r n a l (97).

Significance of the Research Accomplished During Phases I , I1 and 111

The specific f i n d i n g s reported above are s ignif icant w i t h respect t o qualification of certain conclusions concerning cyanobacteria and the nature of compatible solutes t h a t have been recently reported i n the l i t e r a tu re .

First, there ex is t s the problem of too closely g r o u p i n g the cyanobacteria

Gram-negative bacteria (not cyanobacteria however) show increases i n (o r blue-green algae) t o other gram-negative bacteria, as i s frequently done (17) . glutamic acid and/or prol ine and gamma aminobutyric acid (GABA) following upshock. In f ac t , the halo- tolerant cyanobacterium Aphanothece, always showed a deirease i n glutamic with upshock concomitant t o an increase in ser ine, even as K was increasing. Furthermore, we have been unable to f i n d any GABA, even a t the nanomole level i n this organism.

Obviously from our data this does not occur.

Secondly, our d a t a show t h a t proline and glycine, two solutes often found i n osmoregulating eukaryotes, do accumulate i n certain cyanobacteria under certain environmental conditions. prol i ne accumul ate.

In one case (Aphanothece) both betaine and

As previously reported we appear t o a lso f i n d , again i n certain cyano- bacteria, the glucosylglycerol involved i n osmoregulation. However, a recent review i n Science by Yancey, e t a1 (82) reports only this osmoregulatory solute (osmolyte) for the cyanobacteria. The major thesis of this particular a r t i c l e was the great uniformity (and lack of divers i ty) of chemicals evolved for the purpose of osmoregulation. We believe, from our i n i t i a l data, that there i s considerable diversity i n osmoregulatory solutes employed by the cyanobacteria from adaptive evoluticn t o water and sal ine s t ress .

Thirdly, and of considerable importance t o non-blue-green bacteria, amino acids i n the growth (cul ture) medium may be taken up (or diffuse i n ) d u r i n g upshock. These amino acids may, i f the case involving Aphanothece is repeated, influence the nature of the solutes formed d u r i n g osmoregulation. Early heterotrophic bacteria, which were probably the ea r th ' s f i r s t pro- karyotes, almost certainly had amino acids available to them. common of these was almost certainly glycine, and perhaps, the dipeptide, glycylglycine. These amino acids may have directed the pathways of osmo- regulatory sol Ute production.

One of the most

Fourth, recent research on the pathway of betaine synthesis i n higher p l a n t s by Andrew Hanson and his coworkers a t Michigan State (35a) confirms t h a t serine is the precursor o f betaine. The pathway i s as follows:

Serine + ethanolamine + N-methylethanolamine + dimethylethanolamine + choline + betaine aldehyde + betaine

However, these investigators have focused the i r attention t o the steps i n this pathway t h a t are responsive to increasing water s t r e s s o r sal ini ty--af ter the formation of serine. They fur ther note that betaine synthesis requires l i g h t , especially fo r the formation of one-carbon metabolite derivatives of formic acid. Our d a t a certainly agree w i t h those indicating a l i g h t requirement fo r betaine synthesis. However, we believe that our data show t h a t the decreased water potential and/or s a l i n i t y trigger the synthesis of serine. Serine

2 1

f o rma t ion i s t he l i m i t i n g s tep f o r beta ine synthes is and l a t e r stages i n t h e pathway may o r may n o t be s t imu la ted by decreasing water p o t e n t i a l o r i nc reas ing i n t e r n a l s a l i n i t y . Furthermore, we b e l i e v e t h a t t h e ser ine, w h i l e c e r t a i n l y n o t o f photorespi r a t o r y o r i g i n , probably comes from t h e non-phosphoryl ated, D -g l yce r i c pathway.

F i f t h , and f i n a l l y , our data a lso chal lenge, o r a t l e a s t q u a l i f y , t h e statement made by Yancey e t a1 (82, p . 1217) t h a t t h e "coun te rac t i ng e f f e c t s [ t o s a l t i n h i b i t i o n of enzymes] [of beta ine] a re independent o f t h e species source of p r o t e i n . Mammal s , t e l e o s t , amphibian and e l asmobranch p r o t e i n s respond s i m i l a r l y i n t h e presence of coun te rac t i ng so lutes, regard less o f whether they experience these solutes i n v ivo' ' and on p. 1221,

"Through t h e use o f compatible s o l u t e systems, p r o t e i n s a r e a b l e t o work i n t h e presence o f h igh o r v a r i a b l e s o l u t e concentrat ions, and t h e [gene t i c ] m o d i f i c a t i o n s of vast numbers of p r o t e i n s i s avoided."

We agree w i t h the importance o f t h e i r assessment of t he e v o l u t i o n a r y r o l e o f compat ib le so lutes, as t h i s gives even more j u s t i f i c a t i o n f o r t h e objec- t i v e s o f our proposed research. g l yc ine -be ta ine and glucose-phosphate dehydrogenase from d i f f e r e n t sources, leads us t o c a l l f o r more exper imentat ion on p r o t e i n m o d i f i c a t i o n i n organisms employing g r e a t l y d i f f e r e n t osmoregulatory systems.

However, our experience ( r e p o r t e d here) w i t h

S i x t h , we now know t h a t the cyanobacteria, once c a l l e d m e t a b o l i c a l l y uniform, employ a d i v e r s i t y of so lu tes t o achieve osmotic balance w i th an environment o f i n c r e a s i n g s a l i n i t y ( o r lowered water p o t e n t i a l ) . c e l l u l a r form, Synechocystis empl oyed a g l ucosyl g l y c e r o l . f i 1 amentous unbranced species employed t reha lose and sucrose. p r o w d t o be a miner component i n the tot.al solute change d u r i n g osmotic adjustment i n a l l s t r u c t u r a l forms. I n t e r e s t i n g l y , p r o l i n e (a common o s m o t i c a l l y impor tant s o l Ute) accumulated i n on l y t h e more advanced s t r u c t u r a l forms.

The un i -

Ami no ac ids The branched and

N u t r i t i o n a l modes o f t he cyanobacteria d i d n o t appear t o g r e a t l y i n f l u -

This i s s i g n i f i c a n t i n view o f t h e g r e a t e r a v a i l - ence t h e na tu re o f t h e so lu tes accumulating d u r i n g osmotic adjustment w i t h some p o s s i b l e except ions. a b i l i t y o f glucose i n photoheterot rophica l l y and chemoheterotrophical l y grown c e l l s .

L i g h t versus dark i ncuba t ion dur ing osmotic adjustment d i d p ro found ly a f f e c t t h e q u a n t i t a t i v e and q u a l i t a t i v e na tu re of t he s o l u t e response. C l e a r l y some o f t h e carbohydrates (e.g. g l u c o s y l g l y c e r o l ) and amino ac ids (e.g. p r o l i n e ) r e q u i r e 1 i g h t f o r t h e i r maximal synthesis.

Seventh, our demonstrat ion of a " t y p i c a l " h ighe r p l a n t c h l o r o p l a s t p h o t o r e s p i r a t o r y pathway i n the h a l o p h i l i c cyanobacterium i s s i g n i f i c a n t t o t h e o r i g i n o f t h e methyl groups of be ta ine f rom formate. doubt t h a t l a r g e amounts o f pho to resp i ra to ry carbon passes through g l y c i n e and s e r i n e i n t h i s cyanobacterium.

There i s l i t t l e

Eighth, i f we a r e c o r r e c t i n our hypothesis t h a t p h o t o r e s p i r a t o r y formate i s t h e source o f t he methyl groups o f be ta ine then t h i s pathway possesses a d e f i n i t e value t o s u r v i v a l o f cyanobacteria under c o n d i t i o n s o f extreme

22

s a l i n i t y . However, another quest ion a r i ses . P h o t o r e s p i r a t i o n r e q u i r e s e leva ted oxygen l e v e l s . t r i b u t e d formate t o be ta ine synthesis i n the Precambrian eon, an eon o f extremely low atmospheric oxygen. The p o s s i b i l i t y does e x i s t however, t h a t increased s a l i n i t y p e r se may e l i c i t phosphoglycol ic a c i d synthes is f rom r i b u l o s e 1, 5-bisphosphate. This p o s s i b i l i t y w i l l be tested. Another pos- s i b i l i t y i s t h e d i r e c t synthes is o f formic a c i d f rom carbon d iox ide . synthes is has been p r e v i o u s l y postulated.

It i s , therefore, u n l i k e l y t h a t t h i s pathway con-

Such a

Ninth, be ta ine synthes is i n a rep resen ta t i ve of e a r t h ' s e a r l i e s t oxygenic photoautotroph i s o f cons iderable evo lu t i ona ry importance. s i b i l i t y t h a t h i g h e r p l a n t ch lo rop las ts arose from cyanobacter ia (endosymbiot ical l y ) , and the recen t demonstrat ion t h a t be ta ine synthes is may occur w i t h i n p l a n t c h l o r o p l a s t s (35b) our research on t h i s pathway d i r e c t l y r e l a t e s t o t h e o r i g i n o f a very important pathway.

Given t h e pos-

O f even g r e a t e r importpnce i s the poss ib le mechanism of r e g u l a t i o n o f be ta ine synthes is through K and betaine l e v e l s . Th i s mechanism i s made p o s s i b l e by our demonstrat ion t h a t SAH hydro lase does i n f a c t e x i s t i n a prokaryote, t he cyanobacter ia. B r i e f l y our hypothesis i s :

a) With inq reas ing environmental s a l i n i t y t h e cyanobacterium accumu- l a t e s K ion; t h i s i o n st imulates SAH hydro lase i n t h e h y d r o l y t i c d i r e c t i o n thereby breaking down the methylase i n h i b i t o r SAH; increased methy la t i on r e s u l t s i n increased be ta ine synthes is ; be ta ine p r o t e c t s enzymes i n the presence o f t h e increased i n t r a c e l - 1 u l a r KC1 .

(b) I m r e a s e d K C l i n h i b i t s the SAH hydro lase i n t h e d i r e c t i o n o f SAH syn thes is ; t h i s i n h i h i t i o n r ~ s u l t s in t h e decreased synthes is o f t h e methylase i n h i b i t o r SAH; t h i s r e s u l t s i n more be ta ine synthesized.

( c ) As be ta ine accumulates i t p r o t e c t s SAH hydrolase from KC1 i n h i b i t i o n o f i t s synthes is d i r e c t i o n ; SAH w i l l accumulate once again and i n h i b i t methylase enzyme r e s u l t i n g i n a cessa t ion o f be ta ine synthesis.

Tenth, a recen t p u b l i c a t i o n i n Science (22a) deals w i t h the u n i v e r s a l a s s o c i a t i o n o f t reha lose w i t h p ro tec t i on aga ins t dehydrat ion o f b i o l o g i c a l membranes. had t h e same t r a n s i t i o n temperature as f u l l y hydrated membrane. We f e e l t h a t t h i s i s t he r o l e o f t reha lose i n osmoregulation by cyanobacter ia. Osmotic adjustment must a l s o r e q u i r e p r o t e c t i o n o f des iccated membranes.

I n t h e presence o f t rehalose membrane 1 i p i d s completely dehydrated

C u r r e n t l y we f e e l t h a t osmotic adjustment t o hypersa l i ne c o n d i t i o n s can o n l y be achieved i n those cyanobacteria capable of be ta ine synthes is . f u l f i l l s t h r e e r o l e s o f a compatible so lu te . a ) p rese rva t i on o f membranes from dehydrat ion i n j u r y dur ing osmotic adjustment; b) p rese rva t i on of enzyme f u n c t i o n i n g i n t h e presence o f h igh i n t r a c e l l u l a r s a l t ; and c ) balance o f t h e i n t r a c e l l u l a r water thermodynamical l y w i t h t h a t o f t h e e x t e r n a l environment.

Beta ine These are:

A l l o t h e r cyanobacter ia capable of osmotic adjustment t o much lower s a l i n i t i e s ( g e n e r a l l y up t o 1 M NaC1) employ compat ib le so lu tes t h a t merely a l l o w enzymatic f u n c t i o n i n g , n o t p ro tec t i on aga ins t h i g h s a l t . I n t h i s sense

23

betaine is more than a compatible solute, it is a protective solute or osmolyte. tection of membranes and allowance of enzymatic function.

The significance of the betaine effect on KC1-inhibited enzymes lies in the inherent salt sensitivity of the enzyme (in this case, glucose phosphate dehydrogenase). It would appear that there is an evolutionary bifurcation in adaptation to high intracell ul ar salt. (high) requiring enzymes, as did the halobacteria, or the metabolic pathways for the synthesis of betaine. One conclusion from this study is that without either structural salt tolerance (requirement) or betaine synthesis, a cyano- bacterium is greatly limited in its ability to tolerate salt.

Trehalose will serve two of the three functions, that is, pro-

Either the prokaryote evolved sal t

The significance of the betaine effect on shifting the pH optimum upward 1 ies in the probable increase in intracellular pH during osmoregulation. is the case in organisms possessing a proton (out)-potassium ion (in)-pump. Previous experiments in our 1 aboratory have shown that Aphanothece which accumulates potassium ion during osmoregulation also synthesizes betaine. Hence the connection.

This

Finally, perhaps the most significant of all of our findings involves that of the relationship between intracellular potassium accumulation to offset lowered water potential due to salt stress and the enzymatic synthesis of glycine-betaine. Our research has definitely shown that an important regulatory step in betaine synthesis is that of the methylation of phosphorylethanolamine using S-Adenosylmethionine as the methyl donor and a specific methylase to catalyze the reaction. The products o f the reaction, chol ine and eventual ly betaine, and S-Adenosylcysteine (SAH) are regulated by SAH which inhibits the methylase. High potassium ion concentration drives the h y d r s l y s i s i ~ f SA!-! by a l l ow ing the hydrnlytic d i r e c t i n n nf t h e SAH hydrolaw t o

inhibiting the synthetic step which leads to SAH formation. function while

This resu will ''protect" resulting will methylase.

It appear of sal t-induce

ts in increased betaine synthesis up to a concentration that SAH hydrolase in the synthetic direction. The increased SAH thereby shut down betaine synthesis by inhibiting the

that our project has elucidated the most primitive mechanism betaine synthesis in cyanobacteria. This may, if correct,

prove to be highly significant by virtue of the postulated (by some) cyanobacterial ancestry of the chloroplasts of higher plants.

C. Presentations , Activities and Publ ications Resulting from the Research Since 1985 P roposal and Progress Report

1. Publ ications and Abstracts:

Pavlicek, K.A. and J.H. Yo . 1985. Influence of KC1 and betaine on soluble and membrane N D(P)H dehydrogenases from the halophilic cyanobacterium, Aphanothece halophytica. Plant Physiol . 77:647-S.

Sibley, Marion H. and J.H. Yopp. 1985. Occurrence and osmoregulatory role of S-adevosyl homocysteine hydrolase in betaine synthesis by the

24

h a l o t o l e r a n t cyanobacterium, Aphanothece halophyt ica. P l a n t Phys io l . 77:771-5.

Myers, G.O. and J.H. Yopp. 1985. S a l t i n h i b i t i o n and be ta ine res to ra - t i o n o f a c t i v i t y of glucose-6-phosphate dehydrogenase from cyano- b a c t e r i a d i f f e r i n g i n genome s i ze . P l a n t Phys io l . 77:650-S.

S ib ley , Marion H. and John H. Yo . 1986. U n i d i r e c t i o n a l i n h i b i t i o n o f cyanobacter ia l S- A---Ee denosyl homocystei ne hydro1 ase by po tass i um c h l o r i d e : proposed r o l e i n osmoregulatory be ta ine synthes is . P l a n t Phvs io l . 80(4):699.

Hawkins, Lynda, D. R i t t e r and John H. Yopp. 1987. Proposed a d d i t i o n a l

p r o t e c t i o n aga ins t pH-induced l o s s o f r o l e f o r g l yc inebe ta ine i n t h e adaptat ion o f h a l o p h i l i c cyano- b a c t e r i a t o h y p e r s a l i n i t y : enzymatic a c t i v i t y . P lant Phys io l . I n press.

S ib ley, Marion and John H. Yopp. t h e h a l o p h i l i c cyanobacterium Aphanothece ha lophy t i ca : r o l e o f SAH hydrolase. Archives o f Microbio logy. Accepted f o r pub1 i c a t i o n , January.

1987. Regulat ion o f g l yc inebe ta ine i n proposed

Yopp, J.H., K.A. Pav l icek and Marion H. S ib ley . 1985. "Evo lu t i ona ry S i g n i f i c a n c e o f Osmoregulatory Mechanisms i n Cyanobacteria. Second Sympos. on Chem. Evol. and O r i g i n o f L i f e . Res. C t . , C a l i f .

Proc. 2:74, NASA,-

1985. "The r o l e o f s u l f u r i n osmoregulat ion and s a l i n i t y w a n c e i n cyanobacteria, a lgae and p lants . " I n : The Global

S u l f u r Cycle, Dor ion Sagan, (ed.); L i f e Sciences D i v i s i o n , NASA O f f i c e o f Space Science and App l i ca t i ons , Washington, D.C. , pp. 83-86.

2. Presentat ions:

Pavl icek, K.A. and J.H. Yo . 1985. In f luence o f KC1 and beta ine on s o l u b l e and membrane __qg N D(P)H dehvdroqenases from t h e h a l o p h i l i c cyanobacterium. American Soc ie ty o f P lan t Phys io log i s t s , Brown U n i v e r s i t y , P rov i -

Aphanothece ha lbphy t i cs Annual Meeting o f t h e

dence, Rhode I s land . June 23-28, 1985.

S ib ley , Marion H. and J.H. Yo . 1985. Occurrence and osmoregulatory r o l e o f S-adevosy + omocysteine hydrolase i n be ta ine synthes is by t h e h a l o t o l e r a n t cyanobacterium Aphanothece halophyt ica. Annual Meeting o f t he American Soc ie ty of P l a n t Phys io log i s t s , Brown U n i v e r s i t y , Providence , Rhode I s 1 and. June 23-28, 1985.

Myers, G.O. and J.H. Yopp. 1985. S a l t i n h i b i t i o n and be ta ine res to ra -

Annual Meeting o f t he American t i o n o f a c t i v i t y o f glucose-6-phosphate dehydrogenase from cyano- b a c t e r i a d i f f e r i n g i n genome s ize. Society o f P l a n t Phys io log is ts , Brown U n i v e r s i t y , Providence, Rhode I s land . June 23-28, 1985.

Yopp, J.H., K.A. Pav l icek and Marion H. S i b l e y . 1985. Evo lu t i ona ry s i g n i f i c a n c e o f osmoregulatory mechanisms i n cyanobacter ia. Second Symposium on Chemical Evolut ion and t h e O r i g i n o f L i f e . Nat ional Aeronaut ics and Space Admin i s t ra t i on , Ames Research Center. M o f f e t t F i e l d , Cal i f o r n i a. J u l y 23-26 , 1985.

S ib ley, Marion H. and John H. Yo . 1986. " U n i d i r e c t i o n a l i n h i b i t i o n o f

c h l o r i d e : Annual Meeting of t h e American Soc ie ty o f P l a n t P h y s i o l o g i s t s a t Louis iana S t a t e Un ive rs i t y , Baton Rouge, Louis iana. 1986.

cyanobacter ia l S- A-T-M denosy homocysteine hydrolase by potassium proposed r o l e i n osmoregulatory be ta ine synthesis."

June 8-12,

I n v i t a t i o n by coo rd ina to rs o f the NASA-San Jose S t a t e ( C a l i f o r n i a ) - - SPONSORED SUMMER RESEARCH PROGRAM (ANNUAL) , t o present two papers e n t i t l e d "The r o l e of oxygenic photoautotrophs i n the s u l f u r cyc le : f rom s u l f a t e a s s i m i l a t i o n t o d i m e t h y l s u l f i d e product ion," on August 3, 1984; and, "The r o l e of s u l f u r i n osmoregulat ion and s a l i n i t y t o le rance i n cyanobacteria, algae, and plants," on August 2, 1984; Drs. Lynn Margul is and E l l en Weaver, coord inators ; San Jose S t a t e U n i v e r s i t y .

3. Master o f Science Theses Supported by t h e NASA Grant--Completed Under D i r e c t i o n o f John H. Yopp:

Lynda Kaye Hawkins. "The E f fec ts o f Glycinebetaine, pH and Potassium Ch lo r ide on t h e Physico-Chemical P roper t i es and Funct ion o f Glucose-6-Phosphate Dehvdroqenase (E.C.1.1.1.49) f rom Two S t r a i n s o f Synechocystis kpp. D i f f i r i n g i n Genome Size and. S a l t Tolerance." I A i ! g ~ s t 'U , 1986.

Myers , Gerald 0. "P roper t i es o f G1 ucose-6-Phosphate Dehydrogenase (E.C.1.1.1.49) f r o m S t ra ins o f t h e Cyanobacteria Synechocystis spp. and LPP spp. D i f f e r i n g i n Genome Size." A p r i l , 1985.

4. Related Accomplishments from NASA-Supported Research (Grant NAG W - 344 )

Appointment t o the Society o f Sigma X i Lectureship. Midwest Lecture

(Lectures a t C i r c u i t (1984-1986) : Topic was NASA-sponsored research e n t i t l e d "Adaptat ions by P1 ants t o Extreme Environments. I' var ious u n i v e r s i t i e s , e.g. Augustana, U n i v e r s i t y o f L o u i s v i l l e . )

Appointed as a V i s i t i n g Scholar and Program Speaker i n the Oak Ridge Associated U n i v e r s i t i e s V i s i t i n g Schol a r s Program, 1986-1987, by D r . James Gumnick, Program D i r e c t o r . Topic: Phys io log i ca l and Biochemical Mechanisms o f Resistance t o H y p e r s a l i n i t y by Photosynthet ic Microorganisms. (NASA Research) 1971-1986.

I n v i t a t i o n t o present l e c t u r e e n t i t l e d , " S a l t Resistance Mechanisms i n Photosynthet ic Organisms and Ch lo rop las ts o f Higher Plants." Pedro Urena Nat ional Un ive rs i t y , Santo Domingo, Dominican Republic a t t h e I n v i t a t i o n o f Rector, Dr . Jaime Vinas Roman, August 15-21, 1986.

26

Presentat ion o f l e c t u r e and workshop e n t i t l e d "The Need t o I n t e g r a t e Chemistry, Physics, B io logy and Evo lu t i on i n t o a Treatment o f the O r i g i n o f the Universe and L i f e f o r High School Science Students" t o the Master B io logy Teachers of t he Honors I n s t i t u t e i n Microbio logy. Sponsored by the Nat ional Science Foundation ( g r a n t t o D r . Isaac Scheckmeister) , 1984.

Science p r o j e c t adv isor and i n s t r u c t o r f o r two Carbondale Community High School s tudents (Todd Mart in, M iche l l e Yee) t o a i d i n development o f NASA space s h u t t l e experiment, Spring, 1984.

Presenta t ion o f 2 l e c t u r e s and workshops e n t i t l e d : "The Need t o I n t e - g r a t e Chemistry, Physics, Biology, and Evo lu t i on i n t o a Treatment o f the O r i g i n o f t he Universe and L i f e f o r High School Science Students" and " I s o l a t i o n o f Extreme Organisms," t o the Master B io logy Teachers o f t he Honors I n s t i t u t e i n Microbio logy. Sponsored by the Nat iona l Science Foundation (g ran t t o D r . Isaac Schechmeister), January 26, 1985; March 1985; and tw ice i n 1986.

5. Other Major Accomplishments and Cont inu ing A c t i v i t i e s

Dar ry l R i t t e r , a c u r r e n t Ph.D. s tudent has now (1986-1987) i s o l a t e d DNA f rom Aphanothece halophyt ica and Lpp s t r a i n (ATCC) #29126 and made c lone banks us ing the Esher ichia c o l i p lasmid pBr 322. a re c u r r e n t l y being screened f o r t he expression o f key cyano- b a c t e r i a l enzymes w i t h subsequent c h a r a c t e r i z a t i o n o f gene and promoter-termi na to r sequences encoding f o r g l ucose-6-phosphate dehydrogenase.

These banks

P soutilerri I i i i no j s ~ . i i i i i e r s j t y 6t Carbondafe has ifow n i r r r h > c n A 2 ?nn M U 7 pul L. i iUJI -" u Y V " e . : , &

wide bore NMR t o f i n i s h the NMR C-13 s tud ies i n i t i a l l y proposed. Support w i l l be provided t o t a l l y by the u n i v e r s i t y .

m d d h N =k

V I- !- e .n d d

M 0 c\I m N =k

V I- I- Q

h 0 m h N =k

N =k

V V I- Q

m 0 h W =k

e n

V V I- e .n

co 0 -4- h =k

V V a

m 0 0 aJr-

L O S V c, 7 u S C V 5

V .C

m W =k

h =k

0 V a

V 0 a

V V a

h

.n aJ

.n

aJ 6 aJ

.n

aJ 5 aJ V 6 v u V o s v o 0 *C

Oc, L O TQ) Vv,

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T a J Ov,

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4

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c, S 6 T

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c3 a a -1

c3 a a 1

m

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x v w VI- Q=,

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29

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v)

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30

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M 0 l-4 h =ti=

0 0

.a

a

.n

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k n

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-r-

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31

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v) -0

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- al V

L aJ n

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(5, E

t t

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m

0

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m In h

*

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N

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C C t t C t

32

Ly

2 3 0 v)

Z

33

v) Ly c

s 0 a VI

6: V a + =

Ly VI

> x

p:

5

3 a z

U

N

o--O 27184 [1-13 29235 -29126 +-+ 29344

L c r

0.0 0.2 0.4 0.6 0.8 1 .o KCI (M)

FIGURE 3. KC1 i n h i b i t i o n o f Synechocystis and LPP spp. glucose-6-phosphate dehydrogenase.

35

.300<

.200

.loo

.ooo 0.0 1.0 2.0 4.0 6.0

G6P(mM)

10.0

FIGURE 4. Substrate s a t u r a t i o n curves f o r Synechocystis and LPP spp. glucose-6-phosphate dehydrogenase a t pH 6.4

36

.30@

.2004

.loo

.ooo

o-0 27184 e-0 29235 M 29126 P +-+ 29344

o i i 3 4 5 6 7 a 9 i o G6P(mM)

F I G U R E 5. Subs t ra te s a t u r a t i o n curves f o r Synechocyst is and LPP s p p . glucose-6-phosphate dehydrogenase a t pH 6 . 9

37

.400*

.300-

e

t .- Q) c 2 n

0 4 1 2 3 4 5 6 7 8 9 10

E t E

13 a

c

.- 0 t m

U

.loo*

.ooo

0-0 27184 0-U 29235

# 29344

A

I

G6P(mM)

FIGURE 6. Subs t r a t e s a t u r a t i o n curves f o r Synechocyst is and LPP spp . glucose-6-phosphate dehydrogenase a t pH 7 . 4

38

.400*

.3004

c

i .- Q,

e Q, 9

0 v)

c E

- = .2004 -

F - t E .- 0 * m

2 .loo a 4

.OOO,

0-0 27184 (3--Q 29235 - 29126 ++ 29344

A A/ A A t'

0.0 1.0 2.0 4.0 6.0 G6P(mM)

10.0

FIGURE 7 . Subs t r a t e s a t u r a t i o n curves f o r Synechocyst is and LPP spp . glucose-6-phosphate dehydrogenase a t pH 7.9

39

.4004

.3001

.200

.loo

.ooo

27184 M 29235 a 29126 H 29344

0.0 1.0 2.0 4.0 6.0

G6P(mM)

10.0

FJGURE 8. Subs t r a t e s a t u r a t i o n curves f o r Synechocys t i s and LPP s p p . glucose-6-phosphate dehydrogenase a t pH 8 . 4

40

400

300

200

100

0

o--o 27184 Q--c) 29235 H 29126

0.0 1 .o 210 Betaine (M)

3:O 4.0

FIGURE 9. The e f fec t of g l yc inebe ta ine on KC1 i n h glucose-6-phosphate dehydrogenase o f Synechocystis LPP spp. as a percent o f i n h i b i t e d c o n t r o l .

b i t e d and

41

170

1601

l a

120'

- c 9 100 C 0 U m aa

80 x .- z C t 3 0

.- - 60

s

40

20

0 I

3 1 .o 2:o 3 :O 470

Betaine(M)

FIGURE 10. The e f fec t o f g lyc inebeta ine o f KC1 i n h i b i t e d glucose-6-phosphate dehydrogenase o f Synechocystis and LPP spp. as a percent o f u n i n h i b i t e d c o n t r o l .

42

170-

160-

1 so= 140-

130-

Q 120- - %

' + OJ (3 110-

404

301 20

Sources of Glucose - 6 - PO, dehydrogenase 0

0 29235 Synechocystis spp.

0 Aphonothece holophyticu \ 4 29344 LPP spp.

\ Torula spp.

A 291 26 LPP spp.

0 271 04 Synechocystis spp.

X Leuconostoc spp.

\ r = 0.949

0 : I 1 1 I I I I I I 1 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

KCI(M) Needed for 50% Inhibition

FIGURE 11. The e f f e c t of 1M g l yc inebe ta ine on KC1 i n h i b i t e d glucose-6-phosphate dehydrogenase from severa l genera.

43

n I

I 8

n I

. m rn 1

I I

n I

0

44

LITERATURE

1. Barghoorn, E.X. and S.A. Ty le r . 1965. Microorganisms f rom t h e G u n f l i n t Chert . Science 147 : 563-577.

2. Ba t te r ton , J.C. and C. van Baalen. 1971. Growth responses o f b lue-green a lgae t o sodium c h l o r i d e concentrat ion. Arch M ik rob io l . 76:151-165.

3. Ben-Amotz, A. and M. Avron. 1975. The r o l e o f g l y c e r o l i n t h e osmotic r e g u l a t i o n o f t h e h a l o p h i l i c a lga D u n a l i e l l a parva. P l a n t Phys io l . 51~875-878.

4. B i s o n , M.A. and G.O. K i r s t . 1979. Osmotic adapta t ion i n t h e marine a lga G r i f f i t h s i a m o n i l i s (Rhodophyceae): o rgan ic compounds. Aust. J . P l a n t Phys io l . 6(4):523-538.

t h e r o l e o f ions and

4a. Blumwald, E. and E. Tel-Or. 1982. Osmoregulation and c e l l composi t ion i n sa l t -adap ta t i on o f Nostoc muscorum. Arch. M i c r o b i o l . 132:168-172.

5. Bonen, L. and W.F. D o o l i t t l e . 1978. Ribosomal RNA homoloaies and t h e evol u t i o n o f t h e f i lamentous b l ue-green bac te r ia . J. 4Mol. Evol . 10 : 283-291.

6. Bonen, L. 3nd W.F. D o o l i t t l e . 1979. Cyanobacter ia l evo lu t i on : r e s u l t s of 16 r i b o n u c l e i c a c i d sequence ana lys is . Can. J. Biochem. 57 :879-888.

7. Borowitzka, L.J. and A.D. Brown. 1974. The s a l t r e l a t i o n s o f marine and h a l o p h i l i c species o f u n i c e l l u l a r green alga, D u n a l i e l l a . M ic rob io l . 96:37-52.

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