the identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide...

Molecular Microbiology (1996) 19(1), 37-52

The identification, cloning and mutagenesis of a genetic locus required for lipopolysacchar ide biosynthesis in Bordetella per tussis

Andrew Allen and Duncan Maskell* Department of 6iochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London SW7 ZAY, UK.

Summary

Bordetella pertussis lipopolysaccharide (LPS) is biologically active, being both toxic and immunogenic. Using transposon mutagenesis we have identified a genetic locus required for the biosynthesis of LPS in B. pertussis, which has been cloned and sequenced. We have also identified equivalent loci in Bordetella bronchiseptica and Bordetella parapertussis and cloned part of it from B. parapertussis. The amino acid sequences derived from most of the genes present in the sequenced B. pertussis locus are similar to proteins required for the biosynthesis of LPS and other complex polysaccharides from a variety of bacteria. The genes are in a unique arrangement in the locus. Several of the genes identified are similar to genes previously shown to play specific roles in LPS 0-antigen biosynthesis. In particular, the amino acid sequence derived from one of the genes is similar to the enzyme encoded by rfbP from Salmonella enterica, which catalyses the transfer of galactose to the undecaprenol phosphate antigen carrier lipid as the first step in building oligosaccharide 0-antigen units, which are subsequently assembled to form polymer- ized 0-antigen structures. Defined mutation of this gene in the B. pertussis chromosome results in the inability to express band A LPS, possibly suggesting that the trisaccharide comprising band A is a single 0-antigen-like structure and that 6. pertussis LPS is similar to semi-rough LPS seen in some mutants of enteric bacteria.

Introduction

Bordetella pertussis is a Gram-negative bacterium that causes whooping cough in children. The surge of molecular biological research into the virulence and vaccinology

Received 10 February, 1995; revised 11 August, 1995; accepted 25 August, 1995. *For correspondence. E-mail D.MASKELL@ IC.AC.UK; Tel. (0171) 594 5247; Fax (0171) 594 5255.

1996 Blackwell Science Ltd

of B. pertussis over the last few years has concentrated almost exclusively on proteins (Rappuoli et a/., 1992; Cherry, 1992) and little work has been done on non-protein structures that may be involved in the infectious process.

One of the most biologically active and important cell- surface components of all Gram-negative bacteria is the lipopolysaccharide (LPS), yet little is known about the role of LPS in infections caused by Bordetella spp. 6. pertussis LPS is highly immunogenic and displays the properties expected of an endotoxin (Chaby and Caroff, 1988; Amano et a/., 1990; Watanabe et a/., 1990). It shares a general common architecture with LPS molecules from other non-enteric bacteria, in that it has an endotoxic lipid A domain linked via keto-deoxyoctulosonic acid (KDO) to a branched-chain oligosaccharide domain containing hep- toses and hexoses (Caroff et a/., 1990; Lasfargues et a/., 1993; Lebbar et al., 1994). B. pertussis is usually described as lacking a repetitive 0-antigen structure, whereas Bordetella parapertussis and some strains of Bordetella bronchiseptica have an 0-antigen-like structure consisting of a homopolymer of 2,3-dideoxyQ,3-di-N-acetylgalactos- aminuronic acid (2,3-diNAcGalA) (Di Fabio et a/., 1992). The sugars in 6. pertussis core oligosaccharide include galactosaminuronic acid (GalNAcA), glucuronic acid and glucosamine, all of which are charged, and all of which are not commonly found as constituents of other LPS core molecules.

On silver-stained SDS-PAGE, 6. pertussis LPS appears as two bands: band A and band B (Peppler, 1984). Band B consists of the core oligosaccharide, whereas band A cor- responds to the core oligosaccharide plus a terminal trisaccharide. The terminal trisaccharide consists of three charged sugars: N-acetylglucosamine (GlcNAc), 2,3-dideoxy-2,3-di-N-acetylmannosaminuronic acid (2,3-diNAc- ManA), and N-acetyl-N-methylfucosamine (FucNAcMe). These sugars are somewhat similar to those found in enterobacterial common antigen (ECA) (Dell et a/., 1984). The expression of band A LPS has been suggested as being important for full virulence. Monoclonal antibodies (mAbs) specific for band A LPS are able to inhibit the invasion of HeLa cells by 6. pertussis (Ewanowich etal., 1989) and to adoptively transfer immunity to infection in an infant mouse lung infection model (Shahin et a/., 1994).

While evidence concerning the role of LPS in 6. pertus- sisvirulence is not abundant, even less is known about the

38 A. Allen and D. Maskell

metabolic pathways and enzymes required to synthesize an LPS molecule containing some fairly unusual sugars. In addition, nothing is known of the genetics and molecular biology of this important and interesting molecule, despite the major advances being made in the field of LPS genetics in other bacteria, both enteric and non-enteric (Reeves, 1994). A knowledge of 6. pertussis LPS genetics would allow the construction of mutants with defined, non- reverting LPS phenotypes, which could be used to investi- gate the role of LPS in infection. It might also elucidate and unravel some of the metabolic pathways required to synthesize the uncommon sugars present in the LPS and might thus assist a deeper understanding of sugar metabolism in bacteria. To this end, we report in this paper the identification, cloning and mutagenesis of genes required for LPS biosynthesis in 6. pertussis.

Results

Identification of LPS genes

To identify regions of the 6. pertussis genome encoding enzymes required for LPS biosynthesis, we mutagenized wild-type B. pertussis BP536 with TnphoA and then screened the 180 mutants generated with the mAb BL-2, specific for band A LPS (Archambault et a/., 1991 ; Martin et a/., 1992) (kindly provided by Drs Bernard Brodeur and Denis Martin, Ottawa, Canada). Two of the transposon mutants (numbers 5 and 103) did not react with the antibody and were used in further analysis.

The LPS phenotypes of mutants 5 and 103 were assessed by SDS-PAGE followed by silver staining and Western blotting. On the silver-stained gel, the LPS samples from both mutants ran as two bands which stained with approximately equal intensity (Fig. 1, panel 1, columns B and C). In the wild-type control, the faster migrating band (band 8) stained much less intensely than the slower migrating band (band A) (Fig. 1, panel 1, column A), as has been observed previously (Peppler, 1984). On Wes- tern blotting, all the LPS molecules bound the band B-specific mAb BL-8 (Fig. 1, panel 3) whereas only the wild-type LPS bound the band A-specific mAb BL-2 (Fig. 1, panel 2), confirming that transposon mutants 5 and 103 had lost structures defining band A LPS.

Cloning of LPS genes

Chromosomal DNA from mutants 5 and 103 was analysed by Southern hybridization using an oligonucleotide (oligo) specific for IS50 (which flanks TnphoA). Each of the mutants contained two chromosomal copies of IS50, indicating that each contained one transposon insert (data not shown). In mutant 5, a 2.8 kb Pstl and a 3 kb Sall restriction fragment hybridized with the oligo. These

fragments contained some DNA from IS50 and some from 6. pertussis, and were cloned and sequenced as described in the Experimental procedures. The sequence of the DNA from this region was then used to construct oligonucleotides specific for the B. pertussis DNA flanking the transposon insertion site. The polymerase chain reaction (PCR) using these oligonucleotides and wild-type B. pertussis DNA as template gave a band of the expected size and confirmed that the two sequences were indeed flanking the site of transposon insertion. The PCR product was sequenced, found to contain an EcoRl site and subsequently was used to probe a chromosomal Southern blot of 6. pertussis DNA in order to identify larger restriction fragments containing more of the locus. This identified a 2.6 kb EcoRl fragment and a 4.7 kb EcoRI-Clal fragment. Both were cloned as described in the Experimental procedures.

After preliminary sequence analysis of these restriction fragments, it was clear that they did not encompass the whole locus. Consequently a large fragment was sought that would be likely to cover all the genes involved in LPS biosynthesis from this locus. Pulsed-field gel electrophoresis-grade DNA isolated in agarose blocks from BP536 was digested with a variety of enzmes and electro- phoresed in a pulsed-field gel, which was then Southern blotted. Using the 2.6 kb EcoRl fragment as a probe, several large fragments hybridized, and an approximately 33 kb Asp7181 fragment was chosen for cloning. This fragment was purified from a preparative pulsed-field gel and ligated with a cosmid vector digested with Asp7181. The vector was constructed by cloning the polylinker from pBluescript on a Pvull fragment into the cosmid vector pHC79 (see the Experimental procedures). The ligation was packaged into h particles and this was infected into

Fig. 1. SDS-PAGE and Western blots of purified LPS from wild- type and mutant B. pertussis. LPS was purified from wild-type B. pertussis (column A), B. pertussis TnphoA mutant 5 (column B), and 103 (column C), and the allelic replacement insertion mutants in bplH (column D), and bplG (column E). Panel 1. Silver-stained SDS-PAGE of~the LPS. Panels 2 and 3. Corresponding Western blots using the band A-specific mAb BL-2 (mAb BL-2) and the band B-specific mAb BL-8 (mAb BL-8), respectively.

0 1996 Blackwell Science Ltd, Molecular Microbiohgy, 19, 37-52

Bordetella pertussis lipopolysaccharide genes 39

the amino acid sequences of the 6. pertussis, H. influenzae Rd (Fleischmann ef al., 1995) and f. coli (Clement2 and Raetz, 1991) KDO transferase proteins, as well as those from Chlamydia spp., shows that there is extensive homology across the full length of all these proteins (Fig. 3). We are currently expressing the 6. pertussis and H. influenzae kdtA genes with a view to assaying the activity of these enzymes, and whether they can add one or two KDO residues to the lipid A precursor.

fscherichia coli. Resultant cosmids were isolated from ampicillin-resistant colonies and were digested with various restriction enzymes to confirm that the LPS locus had been cloned.

DNA sequence analysis

The DNA sequence of 15 389 bp from this cosmid, deter- mined on both strands, is presented here and is deposited with the GenBanWEMBL database with the accession number X90711. The overall GC content of the sequence is 66.3%. DNA from the transposon mutants was also sequenced to identify the sites of transposon insertion. Insertion of IS50 generates a 9 bp repeat flanking the insert. For mutant 5 this covers bases 10709 to 10717 inclusive and for mutant 103 bases 9966 to 9974 inclusive (Fig. 2). These insertion sites are, therefore, both within one of the putative open reading frames (ORFs) (see below).

Analysis of the translated DNA sequence revealed the presence of a number of ORFs. These ORFs all give high scores when compared for codon usage with the housekeeping enzyme encoded by the 8. pertussis aroA gene (Maskell et a/., 1988) using the method of Staden (1 984), providing supportive evidence that they are genuine B. pertussis genes. Each of these genes is described in turn, with their position given in base pairs starting from the beginning of the GenBank entry. The direction of transcription/translation is with respect to the orientation of the sequence in Fig. 2.

baf: base pairs 1 + 91. At the left-hand end (Fig. 2), and pointing rightwards, the DNA sequence is a 100% match to the baf gene (Deshazer eta/., 1995), which is required for the expression of pertussis toxin in a bvgdependent fashion in f. coli. There is no evidence that baf plays any role in LPS biosynthesis, so we are provisionally using this to define one end of the LPS locus.

kdtA: base pairs 289 t 1575. Next to baf but pointing leftwards is an ORF that, when translated, is similar to kdfA. kdtA encodes KDO transferase, responsible in E. coli for adding KDO to the lipid A precursor, lipid IVA (Raetz, 1993). This intriguing enzyme can catalyse the addition of two KDO residues in quick succession in E. coli, to the extent that the individual transferase activites cannot be distinguished in in vifro assays (Raetz, 1993). From structural analysis of the LPS, 6. pertussis apparently contains only one KDO residue in its LPS molecule, suggesting that the 6. pertussis KDO transferase is only able to catalyse the addition of one of the residues. Haemophilus influenzae also contains only one KDO in its LPS and, similarly, might be expected to have a KDO transferase able to add only a single KDO. Comparison of

&) 1996 Blackwell Science Ltd, Molecular Microbiology, 19, 37-52

rfaC: base pairs1577 c 2584. The next gene, upstream of kdtA and also pointing to the left, when translated has a high degree of similarity with the products of rfaC from a number of other bacteria (Sirisena eta/, 1992; Zhou eta/., 1994). rfaC encodes the glycosyltransferase that catalyses the transfer of the first heptose residue in the core oligosaccharide to KDO in E. coli and Salmonella enterica serovar fyphimurium (Sirisena eta/., 1992). rfaC and kdtA are separated here by a single base pair, and we suggest that the two genes are in an operon and may be translationally coupled. kdtA also is downstream of rfaC in this arrangement and this is in keeping with the observation that the genes for sequential steps in LPS biosynthesis, when found together, are usually arranged such that the gene for the second step in the pathway would be transcribed before the gene for the first step (Reeves, 1994). It is also interesting to note here that rfaC and kdfA are separated by approximately 11 kb in the rfa locus of S. enterica and E. coli and are not in the same operon, while Neisseria gonorrhoeae rfaC is transcribed from its own promoter and appears not to be in an operon (Zhou et a/., 1994). The arrangement of rfaC-kdfA in 6. pertussis, therefore, is at present unique in the literature.

lntergenic region. Between the start of rfaC and the start of the next gene, which points rightwards, is an intergenic region of 129 bp with a GC content of 49.6%. Within this region may be divergent promoters for rfaC and the rightward-pointing genes.

Rightward-pointing genes. A series of 12 coding sequences extends to the Clal site that marks the right-hand end of the double-stranded sequence reported here. The putative function of most of these is suggested by similarity at the amino acid level with other proteins in the pub- lic databases. Each of these is dealt with in turn below. For ease of identification we have used the gene symbol bpl (for ,B. pertussis LPS). These functions are, in the main, only suggested b; similarity searching and clearly should be confirmed by enzymological and mutagenesis analysis where possible.

bplA: base pairs 271 4 -+ 3766. The protein encoded by bp/A is similar to a number of proteins in the database. The

Tn

Tn

No.

103

No.5

P 0

?

j;

I

> m

>o- -00-

P

L-

f-

;

77

B

'i 1'

B B

F ia'

?

ua

V

3

3 4

w M

tA

cw bp

lA

bplB

bp

lC

bPD

bp

IE

bplF

bp

lC

bpW

bp

11

bpU

bp

lK

bplL

IS

D

-1

i5 kn

n 0

lh

b

V kon

-

Fig.

2.

Dia

gram

mat

ic re

pres

enta

tion

of t

he B

. per

tuss

is L

PS

locu

s. T

he b

pl g

enes

are

sho

wn

with

thei

r di

rect

ion

of tr

ansc

riptio

n/tra

nsla

tion.

The

site

s of

ins

ertio

n of

the

tra

nspo

son

mut

ants

ar

e sh

own

by a

rrow

s la

belle

d Tn

No.

5 a

nd T

n N

o. 1

03. T

he s

ites

of in

serti

on o

f th

e de

fined

alle

lic r

epla

cem

ent m

utan

ts a

re s

how

n by

arr

ows

labe

lled

kan.

B, B

amH

I; C

, Cla

l E,

Eco

RI;

S, Sa

ll. T

he C

lal s

ite in

the

mid

dle

of t

he lo

cus

in b

plE

doe

s no

t dig

est i

n w

ild-ty

pe E

. col

i bec

ause

of

over

lapp

ing

dam

met

hyla

tion.

!&

cpsa

Cpn

Ctr

Cpsb

Hin

Eco

Cons.

Bpe

Cpsa

Ctr

Cpsb

Hin

Cpn

ECO

Bpe

Cons.

Cpsa

Cpn

ctr

Cpsb

Hin

Eco

Cons.

Bpe

Cpsa

Ctr

Cpsb

Hin

ECO

BPe

Cons.

Cpn

1

60

MMLRGIHRIF KCFYDVVLVC AFVIALPKLL Y

KMLW

GKYK

KSLAVRFGLK K......PHV

MMLRGVHRIF KCFYDVVLVC AFVIALPKLL YKMLVYGKYK KSLAVRFGLK K..

....

PHV

MIRRWLT SRLYDAFLVC AFFVSAPRIF YKVFFHGKYI DSWKIRFGVQ K......PFV

MVGLPRIL YKRFVHGKYT KSLGIRFGFK K......PEV

MWRFFYTS LLLICQPLIL CFIGLLSVKS PRYRQRLA.E RYGFYG.NAS

MLELLYTA LLYLIQPLIW IRLWVRGRKA PAYi7KRWG.E RYGFYR.HP.

MGRGWTL ALRGLAPLIW LWMWRRARRA GGQWELFAPA RFGRAGARAP

M YT

L ELI

RG

61

120

PGEGPLVWFH GASVGEVRLL LPVLEXFCEE FPGWRCLVTS CTELGVQVAS QVFIPMGATV

PGEGPLVWFH GASVGEVRLL LPVLEKFCEE FPGWRCLVTS CTELGVQVAS QVFIPMGATV

KGEGPLVWFH GASVGEVSLL APLLNFWREE FPEWRFVVTT CSEAGVHTAR RLYESLGATV

PGTGPVAWFH GASVGETALL LPLLKRFMKE YPEWRCWTS CTESGHENAH RLFGPLGVTT

CPPPQGIFIH AASVGEVIAA TPLVRQLQQD YPHLSITFTT FTPTGSERVK ATFGDSV ..

. .LKPGGIMLH SVSVGETLAA IPLVRALRHR YPDLPITVTT MTPTGSERVQ SAFGKDV ..

. APLAAPVWVH AVSLGETRAA QPLVQALLER ..GLPVLLTH 'M'ATGRAEGE RLFGAAIGRG

H SGEAAELVL

L T

TT

G

FG

121

180

SI ..

.. LPLD FSIIIKSWA KLRPSLVVFS EGDCWLNFIE EAKTVQVQTY ISVNQWRISI

SI ..

.. LPLD FSIIIKSWA KLRPSLVVFS EGDCWLNFIE EAKRIGATTL VING..RISI

FV.... LPLD LSCIIKSWR KLAPDIVIFS EGDCWLHFLT ESKRLGAKAF LING..KLSE

FI ..

.. LPLD LSIIIKPVVR AISPSLLVFS EGDCWLNFIE EAKRLGATAV IING..KLSA

..FHYYLPLD LPFSIHRFIN FVQPKLCIVM ETELWPNLIH QLFLRNIPFV IANA..RLSA

..QHVYLPYD LPDALNRFLN KVDPKLVLIM ETELWPNLIA ALHKRKIPLV IANA..RLSA

QLQQAWLPYD FPGATRRFLA RHAPRCGLLM EREVWPNLLA AARAQGVPMA LVSA..RFSA

A R SA

181

240

DSSKRFKFLK RLGKTYFSPV DKIDFYYRQV KTAFLSLDPS

....

....

.. .

....

....

. DSSKRFKFLK RLGKNYFSPV DGFLLQDEVQ KQRFLSLGIP EHKLQVENI KTWAAQTAL

HSCKRFSFLK RLGRNYFAPL DLLILQDELY KQRFMQIGIS SDKIHVTGNM KTFIESSLAT

NSCKRFTILK RFGRNYFSPV DGFLLQDEQH KARFLQLGVD KEKIQVTGNI KTYTETLSEN

RSAHRYGKIK AHLQTMWSQI SLIAAQDNIS GKRYATLGYP KEKLNITGNI KYDLNTNDEL

RSAAGYAKLG KFVRRLLRRI TLIAAQNEED GARFVALGAK NNQVTVTGSL KFDISVTPQL

SSLRQAGWLG QALREALAGL DRVLAQTDED GARLCQAGA.

.NAYTVTCSL KFDVALPEAQ

P L

EI2

P

RF

E ME ENPNL

s AQ

GR

G TG

KD

Cpsa

Cpn

ctr

Cpsb

Hin

Eco

BPe

Cons.

Cpsa

Ctr

Cpsb

Hin

Eco

Cons.

Cpn

Bpe

Cpsa

Ctr

Cpsb

Hin

Eco

Cons.

Cpn

Bpe

Cpsa

Ctr

Cpsb

Hin

Eco

Cons.

Cpn

Bpe

241

300

.LPLQAWRDR LRLPTDSKLV VLGSMHRSDA G

KWLPWQKL IKE. ..

.. GV SVLWVPRHVE

HLERETWRDR LRLPTDSKLV ILGSMHRSDA GKWLPWQKL IKE. ....GV SVLWPRHVE

N.RRDFWRAK LQISSQDRLI VLGSVHPKDV FXWAEVVSHF HNS. ..

.. ST KILWVPRHLE

N.QRDYWREK LQLAQDTELL VLGSVHPKDV EVWLPVVREL R.R. ....NL KVLWVPRHIE

LRKIDSLRTL W..KQDRPIW

IAASTHNGED EIILKSHRAL LAK...YPNL LLLLVPRHPE

AAKAVTLRRQ W..APHRPVW IATSTHEGEE SWIAAHQAL LQQ

... FPN

L LLILVPRHPE

LRVGHAWAG. ..

. ATGRPVI ALASTREGED AMFIEAIGAL QAHRAATPRP LILLIPRHPQ

301

360

KTKDVEESLH RCTFLMGCGA VVHQFSYTSV .V

WDEN

WLV LKQLYVAGDL AFVGGTFDPK

KTKDVEESLH RLHIPYGLWS RGANFSYVPV .V

WDEI

GL. LKQLYVAGDL AFVGGTFDPK

KLKEHAKLLE KAGILFGLWS QGASFRQYNS .LIMDAMGV. LKDIYSAADI AFVGGTFDPS

RSKELEALLS KENISYGLWS KEATFAQHDA .IIVDAIGW. LKQLYSAADL AFVGGTFDDR

RFNWADLLK KEKFQFIRRS TN.ELPNENT QVILGDSMGE LMLMYGISDI AFVGGSL.VK

RFPDAINLVR QAGLSYITRS SG.WPSTST QWVGmMGE LMLLYGIADL AFVGGSL.VE

RFDEAAAQLQ AAGLAYARRS AGSGEFGPHI DVLLGDTLGE MPFYYAAADV A1V

GGSF

.W

RP

ST GE

AL

L L

ap

RF

RS

P V

GD GE

Y R

AY

GE

S

361

420

IGGHNLLEPL QCEVPLIFGP HITSQSDVAQ RLLLSGAGLC LDEIEPIID. TVSFLLNNQE

IGGHNLLEPL QCEVPLIFGP HITSQSELAQ RLLLSGAGLC LDEIEPIID. TVSFLLNNQE

VGGHNLLEPL QKEVPLMFGP YIYSQSVLAE KLREKEAGLS VNK.ETLLD. W

TDLLQNEX

IGGHNLLEPL QCGVPLIFGP HIQSQSDLAE RLLSMGAGCC LDK.TNIVK. VITFLLDHPE

HGGHNPLEPL A

FKMP

VIEK

HTFNFPEIFR MLVEVQGVLE VNSTADALER

AVEALLNSKE

RGGHNPLEM AHAIPVLMGP HTFNFKDICA RLEQASGLIT V.TDATTLAK EVSSLLTDAD

LGGQNLIEAC AAGTPVIVGP HTFNFKDAAR D

AIMGAALR APDARTALDW

ALQ.LLAEPA

GG

E

A PV E

HTFNF

L LL

421

457

MREAYVQKGK VFLKAETASF DR'IWRALKSY IPLYKNS*

VREAYVQKGK VFVKAETASF DRTWRALKSY IPLYKNS*

NRQAYIEKGK SFLKQEENSF QQTWEILKSQ ITCMKI*

ERAAYIQKGA MFLHEEKVAF DRTWESFKRY IPCVKI*

SRERLGNAGY W

NRGAL QRLLDLLKPY LERNV*

YRSFYGRHAV EVLYQNQGAL QRLLQLLEPY LPPKTH'

RRQAMSEAAR AWTAAHAGAT RRTLDALEDW LG

* B

GA

RL L

L

Fig.

3. P

WJ

P a

naly

sis

of K

DO

tran

sfer

ase

sequ

ence

s fro

m tw

o di

ffere

nt C

hlam

ydia

psi

ttaci

gen

es (

Cps

a an

d C

psb)

, C

hlam

ydia

pne

umon

iae

(Cpn

), C

hlam

ydia

trac

hom

atis

(Ctr)

, H. i

nflu

enza

e (H

in),

E. C

ali (

Eco

) and

B. p

ertu

ssis

(Bpe

). C

ons.

sig

nifie

s a

cons

ensu

s se

quen

ce b

ased

on

iden

titie

s be

twee

n th

e B

pe,

Eco

and

Hin

seq

uenc

es. T

here

are

a c

onsi

dera

ble

num

ber o

f co

nser

vativ

e su

bstit

utio

ns, b

ut th

ese

are

not r

efle

cted

in th

e co

nsen

sus

sequ

ence

. Whe

re a

ll se

ven

sequ

ence

s in

the

alig

nmen

t are

iden

tical

, the

con

sens

us a

min

o ac

id is

und

erlin

ed. N

ote

that

th

e se

quen

ces

are

sim

ilar o

ver

thei

r fu

ll le

nath

s. A

dot

sia

nifie

s a

aap

aene

rate

d bv

the

com

pute

r ana

lysi

s.


context, it is noteworthy that the B. pertussis protein is also homologous to the CDP-4-keto-6-deoxy-o-glucose 3-dehydrase from both Yersinia pseudotuberculosis and S. enterica (encoded by rfbH) (Kessler et a/., 1993; Reeves, 1994; Thorson eta/., 1994), the putative perosa- mine synthase from Vibrio cholerae (encoded by rfbE) (Manning et a/,, 1994) and an ORF from the rfff-rff7 intergenic region in f. coli, (encoded by 0299) (Daniels et a/., 1992), involved in ECA biosynthesis. We, therefore, speculate that this ' ORF is involved in the biosynthetic pathway to the 2,bdideoxy amino sugar FucNAcMe or the 2,3-dideoxy diamino sugar 2,3-diNAcManA.

most similar include 4,5-dihydro-4,5-dihydroxyphthalate dehydrogenase (PHT4), which is a phthalate degradation enzyme from Pseudomonas putida (Nomura et a/., 1992), a glucose-fructose oxidoreductase from Zymomonas mobilis (Kanagasundaram and Scopes, 1992), my@ inositol 2-dehydrogenase from Bacillus subtilis (Fujita et a/., 1991), and the strl gene from Sfreptomyces griseus, a dehydrogenase required for streptomycin biosynthesis (Mansouri and Piepersberg, 1991). We, therefore, propose that BplA is a dehydrogenase. The B. pertussis LPS contains three sugars where a dehydrogenase might act in the biosynthetic pathway: glucuronic acid, GalNAcA and 2,3-diNAcManA. Because of the context in which bplA is found (see below) we propose that its most likely function is in the biosynthesis of 2,3-diNAcManA found in the band A trisaccharide.

bp/B: base pairs 3770 -+ 4345. The BplB protein is again similar to several proteins in the databases. These divide into two families. The first family includes the acyltrans- ferases encoded by lpxA and /pxD (Raetz, 1993). These are enzymes that transfer fatty acyl chains from acyl carrier protein to glucosamine in the biosynthesis of lipid A. Whether the B. pertussis gene found here is a genuine Ipxgene is under investigation, but it is somewhat unlikely since Ipxgenes are usually found in complex operons with other genes involved in lipid A biosynthesis and fatty acid metabolism (Raetz, 1993). The second family encompas- ses the acetyl transferases encoded by cysf. These transfer acetyl groups from acetyl-CoA to serine in the biosynthesis of cysteine (Wigley et a/., 1990, Gagnon et a/., 1994, Lai and Baumann, 1992). We propose that BplB is an acetyl transferase involved in the transfer of an acetyl group to one of the N-acetyl sugars, probably 2,3- diNAcManA, present in 6. pertussis LPS.

bplC: base pairs 4349 --* 5449. The BplC protein is most similar to the protein encoded by degT in Bacillus stearothermophilus (Takagi et a/., 1990) and the product of the SpsC gene in €3. subtilis (Glaser eta/., 1993) (Fig. 4). It is also homologous to the /mbS gene product required for lincomycin biosynthesis (Peschke eta/., 1995) as well as to several similar proteins involved in the biosynthesis of other sugar-containing antibiotics (e.g. streptomycin, erythromycin, daunorubicin, tylosin) (Dhillon et a/., 1989; Pissowotzki et a/., 1991 ; Stutzman-Engwall et a/., 1992; Merson-Davies and Cundliffe, 1994). DegT protein and its homologues were previously thought to function as protein kinase sensor elements in two-component regulatory systems (Takagi et a/., 1990). However, more recently they have been proposed as enzymes required as part of the pathway to biosynthesize 2,6-, 3,6- and 4,6-dideoxyhexoses, and as enzymes required for transaminations leading to amino sugars (Thorson ef a/. , 1993). In this

0 1996 Blackwell Science Ltd, Molecular Microbiology, 19, 37-52

bp/D: base pairs 5457 -+ 6545. The fourth gene encodes a protein which is similar to those encoded by 0389 in f. coli, required for ECA biosynthesis (Daniels eta/., 1992), epsC in Pseudomonas solanacearum (Huang and Schell, 1995), required for exopolysaccharide biosynthesis, and rfbC, required for 054 biosynthesis in S. enterica serovar Borreze. This rfbC gene encodes a putative UDP-GlcNAc 2-epimerase (W.J. Keenleyside and C. Whitfield, manu- script in preparation). We, therefore, propose that BplD is the enzyme catalysing the epimerization of UDP-GlcNAc to UDP-N-acetylmannosamine (UDP-ManNAc) as the putative first step in biosynthesis of 2,3-diNAcManA.

A putative pathway from UDP-GlcNAc to UDP-2,3- diNAcManA. bp/A-bp/D may constitute a group of genes involved in sequential steps in the biosynthesis of UDP- 2,3-diNAcManA from UDP-GlcNAc. It should be noted that each of these genes is separated from the next by only three to seven base pairs, and, therefore, may be translationally coupled. The bplD gene is separated from the next gene in the locus, bp/€, by 70 bp. The putative biosynthetic pathway is shown in Fig. 5. Again it is noteworthy that the enzymes for the proposed steps in this pathway are encoded by the genes in the reverse order to that in which they appear in the locus (Reeves, 1 994).

bp/€: base pairs 6616 -+ 7827. The BplE protein is similar to a hypothetical protein (ORF2.4) upstream of the cpsB gene in the E. coli cps gene cluster (Aoyama eta/., 1994), Yersinia enterocolitica trsf (Skurnik et a/., 1995) and Streptomyces lincolnensis /mbT (Peschke et a/., 1995). These proteins all have a low degree of homology with glycosyltransferases. We suggest that BplE is the glycosyltransferase responsible for adding either 2,3- diNAcManA or FucNAcMe to the band A trisaccharide.

bp/F base pairs 7890 + 9077. The BplF protein is another member of the family of proteins to which BplC belongs (Fig. 4). The BplC and BplF proteins are only 58% identical and, therefore, almost certainly perform different

YpsRf bH

StyRfbH

BsuSpsC

SliLmbS

Bs t DegT

SerErbS

SpeDnrJ

VchRfbE

Ec002 9 9

Boe

BD

lF

Boe

Bol

C

YpsRfbH

StyRfbH

BsuSpsC

SliLmbS

BDeBolC

Bs t DegT

SerErbS

SpeDnrJ

VchRfbE

EcoO2 9 9

BD

eBol

F

YpsRfbH

StyRfbH

BsuSpsC

SliL

mbS

BDeBDlC

BstDegT

SerErbS

SpeDnrJ

VchRfbE

Eco0299

Bae

BD

lF

YpsRfbH

StyRfbH

BD

sBol

F BsuSpsC

SliLmbS

BDeBDlC

BstDegT

SerErbS

SpeDnrJ

VchRfbE

Eco0299

1 60

MSQEELRQQI AELVAQYAET AMAPKPFEAG KSWPPSGKV IG..TKELQL MVEASLDGWL

MTANNLREQI SQLVAQYANE ALSPKPFVAG TSWPPSGKV IG..AKELQL MVEASLDGWL

M SDFLPFALPD IG..EAEIQA VTESMRSGWL

MVQKR NHFLPYSLPL IG..KEEIQE VTETLESGWL

M SDYIPFAAPC FD..TAEEEA VLRVVRSGWV

MQFIDLKTQY QALRDTINPR IQAVLDHGQF

MN

VPMLDLSEQY EQLKPEIMRV LDEVMRSSRF

MD VPFLDLQAAY LELRSDIDQA CRRVLGSGW

MS TYVWQYLNEY REERADILDA VETVFESGQL

MIPVYEPS LD..GNERKY LNDCIDSGWV

MIPFNAP. .PWGTELDY MQSAMGSGKL

61

12

0

..TTGRFNDA FEKKLGEYLG VP.YVLTTTS GSSANLLALT ALTSPKLGVR ALKPGDEVIT

..TTGRFNDA FEKKLGEFIG VP.HVLTTTS GSSANLLALT ALTSPKLGER ALKPGDEVIT

.TTG.PNARE FEREFAAYIG ADVEAVAVNS ATAGLHLALE A1 ..

....

.. GVGPGDEVIT

.SKG.PKVQQ FEKEFAAFVG AK.HAVAVNS CTAALFLALK AK ..

....

.. GI

GPGDEVIT

.STG.AEAQS FEEEFAAYIG VA.HAVALTS CTAALHVALK AY ..

....

.. GIGPGDEVIV

.IMG.PEVKE LEAALCAYTG AK.HCITVAS GTEALLISLM AL ..

....

.. G

VKAGDEVIT

.LHG.PENEA FEAEFAAYCE NA.HCVTVGS GCDALELSLV AL .

....

... GVGQGDEVIV

.ILG.TSVRS FEEEFAAYHG LP.YCTGVDN GTNALVLGLR AL ..

....

.. GIGPGDEWT

SSRG.KYIDR FETEFAEFLK VK.HATTVSN GTVALHLAMS AL ..

....

.. GITQGDEVIV

CGIX.GFTRR CQQWLEQRFG SA.KVLLTPS CTASLEMAAL LL ..

....

.. DIQPGDEVIM

.ILG.DYVKK LEADIAAYSR AK.HGIGCGN GSDAIHIALQ AA ..

....

.. GVGPGDEVIT

121

180

VAAGFPTTVN PTIQNGLIPV FVDVDIPTYN VNASLIEAAV SDKTKAIMIA HTLGNLFDLA

VAAGFPTTVN PAIQNGLIPV FVDVDIPTYN IDASLIEAAV TEKSKAIMIA HTLGNAFNLS

TTHTFTASAE VARYLGAEPV LVDIDPATLC ISPAAIERAI TPRTRAIVPV HYGGLSCDMD

SPLTFSSTAN TIIHEATPV FADIDENTLN IDPVKLEAAV TPRTKAWPV HFGGQSCDMD

PTMTFVATAT SWHAGAAPV LADVGPEHLT FDPDQVKSLI TERTKAWPV HLFGRMAAME

TSFTFVATAE VIALLGAKPV FVDVEPDTCN IKVSEIEAKI TPRTKAIIPV SLYGQCGDMD

TAFTFFATAG SIARAGAKPV FVDIDPVTFN IDPAQVEAAV TEKTKAIIPV HLYGQMADME

PSHTFIATWL GVP.VGAVPV PVEPEGVSHT LDPALVEQAI TPRTAAILPV HLYGHPADLD

VSNTAAPTW AIDAVGATPV FVDVHEENYL MDTGRLRSVI GPRTRCLLPV HLYGQSVDMT

PTFTYVASVN TIVQCGALPV FAEIEGESLQ VSVEDVKRXI NKKTKAVMAV HIYGQACDIQ

PSYTFVSTAN AFVLRGAKIV FVDVRPDTMN IDETLIEAAI TDKTRVIVPV HYAGVACEMD

181

240

EVRRVADKYN LWLIEDCCDA LGSTYDGKMA GTF.GDIGTV SFYPAHHIT. MGEGGAVFTQ

EVRRIADKYN LWLIEDCCDA LGTTYEGQMV GTF.GDIGTV SFYPAHHIT. MGEGGAVFTK

SILEIARKHG LKVIEDAAHA LPASWQGRRI GSLESDLTVY SFYATKTLAT .GEGGMVJTR

AILAVAQNHG LFVLEDAAHA WTTYKQRMI GSI.GDA?AF

SFYATKNLAT .GEGGMLTTD

PLRELCDSHG LTLLEDAAHT LPA.RDGDAV AGRAGDASAF SFFATKPITT .AEGGMLCTD

EVNAVAARHG LPVIEDAAQS FGATYKGRKS CNL.STIGCT SFFPSKPLGC YGDGGALFTN

AIAAIAKRHG LWIEDAAQA IGAKYNGKCV GEL.GT?ATY SFFPTKNLGA YGIXGMIITN

ALRAIADRHG LALVEDVAQA VGARHRGHRV GAG.SNAAAF SFYPGKNLGA LGDGGAWTT

PVLELAAEHD LKVLEDCAQA HGARRHGRLV GTQ.GHAAAF SFYPTKVLGA YGDGGAWTP

SLRDLCDEHG LYLIEDCAEA IGTAVNGKKV GTF.GDVSTF SFFGNKTITS .GEGGMVVSN

TIMALAKKHN LWEDAAQG VMSTYKGRAL GTI.GHIGCF SFHETKNYTA GGEGGATLIN

YpsRfbH

StyRfbH

Boe

BD

lF

BsuSpsC

SliLmbS

BstDegT

SerErbS

SpeDnrJ

VchRfbE

Eco02 9 9

BDeB

olC

YpsRfbH

StyRfbH

BsuSpsC

SliLrnbS

Boe

Bol

C

BstDegT

SerErbS

SpeDnrJ

VchRfbE

EcoO2 9 9

BDeBalF

YpsRfbH

StyRfbH

B s u S p s C

SliLrnbS

BstDegT

SerErbS

SpeDnrJ

VchRfbE

Eco0299

BDeBDlF

EiLx

.&&

YpsRfbH

S t yR

f bH

BsuSpsC

SliLmbS

BstDegT

S e r

Er bS

SpeDnrJ

VchR f bE

BoeBDlF

EiLx

.&&

241

SAELKSIIES FRDWGRDCYC APGCDNTCKK RFGQQLGSLP

SGELKKIIES FRDWGRDCYC APGCDNTCGK RFGQQLGSLP

DPALAKRCRV MRLHGIDRDA FDRFTSKKPA W

Y ..

....

..

DEELADKIRV LSLHGMSKAA WNRYSS.NGS W

Y ..

....

..

DARVADEARR WSLHGLSRGA VNRYRPGHSA .A. ..

....

. DDELAQAMRE IRVHGQS ..

....

....

.. G RY ..

....

..

DDELAEKCRV IRVHGSK ..

....

....

.. P KY ..

....

..

DPALAERIRL LRNYGSK...

....

....

. Q KY ..

....

..

DAEVDRRLRR LRYYGMG ..

....

....

.. E RY ..

....

..

SDIIIDKCLR LKNQGW ..

....

....

. AG KR ..

....

..

DKALIERAEI IREKG..TNR SQFFRGQVDK YT ..

....

..

300

FGYDHKYTYS HLGYNLKITD

FGYDHKYTYS HLGYNLKITD

.. YE1

....

V APGFKYNMTD

.. YEV....E SPGYKMNMFD

.. YDV ..

.. D RPGHKYNMSD

.. Y.H ..

.. A RIGVGGRMDT

. .Y .H .

... H VLGYNSRLDE

.. V.H ..

.. E VRGTNARLDE

..Y

W..

.. D TPGHNSRLDE

.. YWH....D LVAYNYRMTN

.. W .

....

. R DIGSSYLMSD

30

1

360

MQAACGLAQL ERIEEFVEKR KANFKYLKDA LQSCADFL..

....

....

EL PEATENSDPS

MQAACGLAQL ERVEEFVEQR KANFSYLKQG LQSCTEFL..

....

....

EL PEATEKSDPS

TAAAMGRVQL QRVQQMRDRR AQIAAnYDQA FADLPLTLPP GPGRTPGVER VAHRDDDEHS

LQAALGLHQL KRLDDMQKRR EEIAGRYQTA FQQIPGLITP FV ..

....

....

. HDDGRHA

LAAALGRAQL AKAGRLHARR TAIAEWLRE LAGLDRLELP

....

....

.. AADTATNRSS

LQCAWLGKL ERFDWEIAQR IKIGARYQQL LADLPGG.AC TV ..

....

.. .

TVRPDRDSV

MQAAILSVKF PHLDRWTEQR RKHAATYTRL LEEAVGDLW TP ..

....

.. .KEVDGRYHV

LQAAVLRVKL RHLDDWNARR TTLAQHYQTE LKDVPGITLP ET ..

....

.. .

HPWAD..SA

VQAEILRRKL RRLDAYVEGR RAVARRYEEG LGDLDGLVLP TI ..

....

.. .A..EGNDHV

LCAAIGVAQL ERVDKIIKAK RDIAEIYRSE LAGLPMQV..

....

....

.. HKESNGTFHS

LQAAYLWAQL E ..

....

....

....

....

....

....

AAD RINQQRLALW QNY ..

....

.

401

WFGFPITL.. KEDSGVSRID LVKFLDEAKV GTRLLFAGNL TRQPYFHDVK

WFGFPITL.. KETSGVNRVE LVKFLDEAKI GTRLLFAGNL IRQPYFANVK

WHLYAIR1.H PQAPLKCDDF 1VRMTEN.GI GCSVHYV.PL HLQPYWRD..

WHLYVLQVDE KKAGVTRSEM ITALKDEYNI GTSVHFI.PV HIHPYYQK..

WYLFPVRVHG HRRDAFRQRL

....

. HALGV GTSVHFE.PL HRFTWLRD..

WAQFTVMVPN REA. ..

....

VIAQLKEAGI PTAVHYPRPI HAQPAY.E..

FHQYTIRAPK RDE ..

....

. LQAFLKEQGI ATMWYPLPL HLQPVF.A..

WHLFVLRCEN RDH. ..

....

LQRHLTDAGV QTLIHYPTPV HLSPAY.A..

YYVYWRHPE RDR.. .

....

ILEALTAYDI HLNISYPWPV HTMSGF.A..

YWLTSIILD. .QEFEVHREG LMTFLENNDI ESRPFF.YPA HTLPMY.E..

... YDALRLW RKPGVSSCRR FPMAACRTRI CSTLNCGY*

421

462

ELTNTDRIMN QTFWIGIYPG LTHDHLDYW SKFEEFFGLN F*

ELTNTDRIMN QTFWIGIYPG LTTEHLDYW SKFEEFFGLN F'

MYPHSQAAFE GMASLPIYSR MTDADVQRVI ASVRQLLRP'

DFPNAMNYYK RTLSLPLYPS MSDDDVDDVI EAVRDIVKGA D*

GFPVADAMD TLVSLPVFPA MDDAAVSRW AAVRRAHDEA GR*

ATPVSDDLAA RVMSLPMHPD LDEATQDKIV AALRQALN*

QLPEAEKAAK EALSLPMFPE LKEEQQQYW EKIAEFYRHF A*

SFPVAESLAG EVLSLPIGPH LSREAADHVI ATLKAGA*

DLPVTERLAG EIFSLPMYPS LRPDAQEKVI DAVREWGSL*

.FPLSNSYSH RGINLPSWPG LCDDQVKEIC NCIKNYFNCI*

420

YRV ..

... VG

YRV ..

... VG

... RYGLTPD

... QFGYKEA

.. .H

WRTGQ

.. .QYAEGAG

... SLGYKEG

... DLGLPPG

... HLGYGPG

... HLAEKTA

Fig.

4.

PIL

EU

P a

naly

sis

of B

plC

and

Bpl

F w

ith d

ideo

xy s

ugar

bio

synt

hesi

s en

zym

es a

nd n

ucle

otid

e-su

gar

amin

otra

nsfe

rase

s. Y

psR

fbH

: R

fbH

pro

tein

from

Y. p

seud

ofub

ercu

losi

s re

quire

d fo

r 0-

antig

en b

iosy

nthe

sis.

Sty

Rfb

H:

Rfb

H p

rote

in fr

om S

. en

teric

a se

rova

r fy

phim

uriu

m re

quire

d fo

r 0-

antig

en b

iosy

nthe

sis.

Bsu

Sps

C: S

psC

pro

tein

invo

lved

in s

pore

coa

t ass

embl

y fro

m

B. s

ubtil

is. T

his

gene

was

pre

viou

sly

desi

gnat

ed ip

a-65

d. S

liLm

bS:

LmbS

pro

tein

invo

lved

in li

ncom

ycin

bio

synt

hesi

s in

Stre

pfom

yces

linco

lnen

sis.

Bst

Deg

T: D

egT

prot

ein

from

B

. ste

arot

herm

ophi

lus.

Ser

Erb

S: E

rbS

pro

tein

from

Sac

char

opol

yspo

ra e

ryth

raea

, inv

olve

d in

ery

thro

myc

in b

iosy

nthe

sis.

Spe

Dnr

J: D

nrJ

prot

ein

from

Stre

pfom

yces

peu

cefiu

s in

volv

ed in

da

unor

ubic

in b

iosy

nthe

sis.

Vch

Rfb

E:

V. c

hole

rae

pero

sam

ine

synt

heta

se in

volv

ed in

LP

S b

iosy

nthe

sis.

Eco

0299

: 02

99

pro

tein

from

E. c

oli r

equi

red

for

EC

A b

iosy

nthe

sis.


agalactiae (Rubens eta/., 1993) and ORF14 from the cps locus of Klebsiella pneumoniae (Arakawa ef a/., 1995), which are all required for capsule biosynthesis, as well as to AmsG from Erwinia amy/ovora (Bugert and Geider, 1995) and Xanthomonas campestris GumD (GenBank entry XCU22511). It is also similar to the C-terminal part of RfbP from S. enferica and a homologue of this gene found during the H. influenzae genome sequencing project (Fig. 5) (Jiang et a/., 1991; Wang and Reeves, 1994; Fleischmann eta/., 1995). The ExoY proteins and the 6. pertussis BplG protein all start at about 250 to 300 amino acids into the other sequences. The S. agalactiae CpsD protein starts at about 140 amino acids into the other proteins (Fig. 6).

bplH: base pairs 9863 -+ 1 1035. The precise definition of the start of the next orf is somewhat problematic since the first ATG only appears at position 10325. There is, however, a l T G codon at position 9863, in frame with a long orf. Codon usage analysis strongly suggests that this off is genuine. In addition, transposon mutants 5 and 103 map within this off. Indeed the insertion site for mutant 103 is only 104 bp downstream of the proposed I T G start codon and is upstream of the first available ATG codon. The translated amino acid sequence from this region is similar over its entire length to a number of glycosyltransferase genes. This lends further weight to the identification of the l T G as the start codon (Robison et a/., 1994). Similar proteins include the RfbF galactosyltransferase from Serratia marscecens LPS biosynthesis (Szabo et a/., 1995) as well as the galactosyltransferase encoded by the r fp6 gene in Shigella dysenteriae (Gohmann eta/., 1994). Others include the glycosyltransferase sucrose synthase from a variety of plant species, as well as other glycosyltransferases involved in LPS biosynthesis from other bacteria. However, the strongest homology is with the protein encoded by the capM gene of Staphylococcus aureus M, which is part of the locus required for type 1 capsule biosynthesis in this organism (Lin eta/., 1994). Type 1 capsule in s. aureus M consists of Kacetyl fucosamine (Fuc- NAc) linked to GalNAcA. Other capsule types in S. aureus contain other sugars (e.g. Kacetyl-mannosaminuronic acid (Man NAcA) and Kacetyl-glucosaminuronic acid (GlcNAcA)) and some of the genes from the type 5 cap region hybridize with regions of the type 1 cap locus in Southern blots (Lee et a/., 1994). capM is at the extreme right-hand end of the type 1 cap locus and, therefore, may be common to type 1 and type 5 loci. We speculate that the 6. pertussis BplH protein is a glycosyltransferase involved in transferring a sugar of the terminal trisaccharide of 6. pertussis LPS.

bpll, bplJ, bplK: base pairs 1 1032 -+ 1 1736; 1 1828 -+

12394; 12454 --f 13035. The protein products of the next three genes are only minimally similar to any proteins in

roH TO"

0-UDP OH NAc epimerisation

HO

kAc UDP-ManNAc

UDP-GlCNAC B I + amination

roH

acetylation =i roH

d e h y d r e s e I * coon

Fig. 5. Proposed pathway for conversion of UDP-GlcNAc to UDP- 2,3-diNAcManA, with the products of bplA-bplD acting in reverse order.

functions. BplF is more similar to RfbH proteins and SpsC from 6. subtilis whereas BplC is more similar to DegT from 6. sfearofhermophilus and LmbS from S. lincohensis. Again it is likely that BplF is involved in the biosynthesis of an amino sugar. If the proposed pathway for the biosynthesis of 2,3-diNAcManA is correct, it is possible that BplF is required for FucNAcMe biosynthesis. The stop codon of bplF overlaps with the start codon of the next ORF.

bplG: base pairs 9074 + 9667. The BplG protein is similar to a family of proteins, from a variety of bacteria, required for polysaccharide biosynthesis (Fig. 6). The greatest similarities were with ExoY proteins (previously also called Pss) from various Rhizobiurn spp. (Muller et a/., 1993). ExoY is essential for the biosynthesis of the succinoglycan exopolysaccharide. It is probably the enzyme catalysing the transfer of galactose to an antigen carrier lipid (ACL), which is the first step in exopolysaccharide biosynthesis (Reuber and Walker, 1993). BplG was also similar to CpsE from two different serotypes of Streptococcus pneumoniae (Guidolin et a/., 1994; Gen- Bank entry SPCPS14E), CpsD from Streptococcus


t a 1

60

301

360

KpnOr f 14

MTISQHRFRS NANASIISML QRFSDILIIF LGIYFSCFIN DYFFNLHYVL

KpnOrfl4

IIVSSLILIL ISPILLVIAT AVKTTSKGPV IFRQVRYGMD GKPIKVWKFR SM.TVMENDD

XcaGumD

MLLADLSSAT YTTSSPRLLS KYSAAADLVL RVFDLTMWA SGLIAYRIVF GWVPMPYR

RlePss

IFSSLSALLV LAPFLLFVAL LIKLDSFGPV LFKQTRWGKN CKAIKVYKFR SMRTDLCDVS

EamAmsG

StyRfbP

HinRfbP

SpnCpsl4E

SpnCpsl9E

MREIEF TFKGLLIRLS LALSDLIFFN IALALAIVLI NGFPGEILTG IPQHELDLKI

XcaGumD

KILAVIALMG LWPLMLAIAV GVKMSSPGPV FFRQRRHGLG GREFYMFKFR SMRVHDDHGT

?

MDNIDN KYNPQLCKIF LAISDLIFFN LALWFSLGCV YFIFDQVQRF IPQDQLDTRV

BoeBDlG

WCSGLGLLA LLPLLALIAI AIKLDSPGPV FFRQERVGKD GVPFRIHKLR SMSVRQDPQA

ia'

StyRfbP

IVCSIMILII ASPLMIYLWY KVT.RDGGPA IYGHQRVGRH GKLFPCYKFR SMVMNSQEVL

MDEKGLK IFLAVLQSII VILLVYFLSF VRETELERSS

HinRfbP

IWGSLAIII FSPVLLYLYF AVK.KDZGNA IYGHPRIGRN GKTFNCLKFR TMAVNSKEVL

5 RmeEXOY

VLAASVALLL FSPLFLLIMA LVKFSDGGSV FYGHRRIGHN GQSFKCLKFR TMMEKGDEVL

p

61

120

SpnCps14E

ICGATIGLIL FAIASLVLVP LIR.KDGGPA IFAQTRIGKN GRHFTFYKFR SMRIDAEVIK

KpnOrfl4

MALVALWFQ MIG.GITDFY RSWRGVEFSV ELILILKNWS LSFLLTLGFV TLFSDFDLTF

SpnCpsl9E

ICGATIGLIL FAIASLVLVP LIR .KDGGPA IFAQTRIGTN GRHFTFYKFR SMRIDAEAIK

$ XcaGumD

VAIAWLLYS VICFALFPLY RSWRGRGLLS ELVVLGGAFG GVFALFAVHA LIVQVGEQVS

SagCpsD

ITGAIIGLLI CGIVAIFLVP QIR .KDGGPA IFSQNRVGRN GRIFRFYKFR SMRVDAEQIK

EamAmsG

ATHILLSVIC VGWFWVRLRH YTYRKPFWFE LKEVFRTILI FSIVDLSVSA LSKWELSRWI

StyRfbP

ITHFILSWC VGWFWIRLRH YTIRKPFWYE LKEIFRTIVI FAIFDLALIA F

TKWQFSRW

361

420 '

HinRfbP

YIHAVLAGIC VGWFAIRLRH YTYRKPFWFE LKEIFRTLII FAIFELAIVA FPKLYFSRYL

KpnOrf 14

....

.. .

... KVIQAT KNDIRVTKVG KFLRSTSLDE LPQFFNVLFG QMSWGPRPH

SpnCpsl4E

MVILYLLHFF V.FYFSSYGN KFFKRGYLVE FNSTIRYIFF FAIAISVLNF FIAERFSISR

RlePss

....

.. .

... GVAQTV KNDPRITRIG AILRRTNVDE LPQLLNVLLG H

MSWGPRCH

SpnCpsl9E

MVILYLLHFF V.FYFSSYGN NFFKRGYLVE FNSTIRYIFF FAIAISVLNG FIAERFSISR

XcdGumD

....

....

.. .

... TIQQAT KNDTRITRFG SFLRRSSLDE LPQIFNVLGG SMSIVGPRPH

BoeBDlG

....

....

.. .

... G.QITV GADPRITRVG KWIRKWKLDE LVQLIDVFTG SMSLVGPRPE

121

180

EamAmsG

EEVLRTDPVA RAEWDEDFKL KNDPRITRIG HFIRKTSLDE LPQLWNWRG EMSLVGPRPV

KpnOrfl4

RTFI.FWYLA VCAGFWTRP LIRALAGFFR RIGYNKRRVA FAGSLPAGIS LLETFRKQPW

StyRfbP

KELLANDPIA RAEWEKDFKL KNDPRITAVG RFIRKTSLDE LPQLFNVLKG DMSLVGPRPI

XcaGumC

RGWVGLWFVG GLVSLVAART LLRGFLNHLR TQGVDVQRW WGLRHPVMK ISHYLSRNPW

HinRfbP

DELLRTDPEA RAEWEKDFKL KNDPRITKIG AFIRKTSLDE LPQLFNVLKG EMSLVGPRPI

EamAmsG

WILTWLLSM. ..

... AMVPF GRACVKRLLN RKKLWKKQSI IIGSGKNAQE AWQALQSEEM

RmeExoY

EEFFRINPOA YEEWRATRKL QNDPRVTWG AVLRKLSLDE LPQLLNIIRG EMSWGPRPV

StyRfbP

WVFCWTFAL. ..

... ILVPF FRALTKHLLN KLGIWKKKTI ILGSGQNARG AYSALQSEEM

SpnCpSl4E

EQLMDQNTM. .

.. RGGMFKM DNDPRVTKIG RFIRKTSLDE LPQFWNVFIG DMSLVGTRPP

HinRfbP

WALTWGITF. ..

... LLFPL ARVLVKKFLI KSGWFLRDTI MIGSGDNAFD WNALRDEPY

SpnCpsl9E

EQLMDQNTM. ..

. QGGMFKM DNDPRVTKIG RFIRKTSLDE LPQFWNVFIG DMSLVGTRPP

SpnCpsldE

RGMVYFLTLE GISLYLLNFL VKKYWKHVFF NPK.NSKKIL LLTVTENIEK VLDKLLESDE

SagCpsD

KDLLVHNQM. ..

. TGLMFKL EDDPRITKIG KFIRKTSIDE LPQFYNVLKG DMSLAGTASH

SpnCpsl9E

RGMWLLTLE GISLYLLNFL VKKYWKHVFF NLK.NSKKIL LLTVTKNMEK VLDKLLESDE

SagCpsD

MNSF LKYYRXYSYA KFS.RDTKW LITNKDS ..

. .LSKMTFRNK

42 1

480

KpnOrfl4

AVSHNEQ ..

. YRSLIQGYML RHKVKPGITG LAQINGWRGE TDTL EK....

181

240

RlePss

AIGMRAGGML YEELVPEYHQ RHAMRPGMTG LAQMRGLRGP TDRP AK....

KpnOrfl4

LGFEVKGIYE DSFSGTYDLE

....

. LYAGK ISDLINEARK GTIDRIYIAM HMRDEVAIKN

XcaGumD

MQH "H

... YEKLINHYMQ RHYVKPGITG WAQVNGFRGE TPELRT ..

.. .

....

...M

K

XcaGumD

VGMNMVGYFR TPYDLAVAEQ RQGL.PCLGD PDELIEYLKN NQVEQVWISL PLGERDHIKQ

BD

eBol

G

VPRY ...W

YPDALRDLVL S..VRPGITD PASIR.FRNE NEXLGQSSDP ERTYREIILP

EamAmsG

MGFDVIAFYD VDGSQT ..

. A LELFGVPVLK EEQQLWSL.V DSDTQFIVAV EYEQSQSRDR

EamAmSG

IE AE...... LERYAGDVDY YFMAKPGMTG LWQVSGRNDV

.S..

....

..

....

....

YE

StyRfbP

MGFDVIAFFD TDASDA ..

. E INML..PVIK DTEIIWDLNR TGDVHYILAY EYTELEKTHF

. LERYCDDVDY YLMAKPGTE LWQVSGRNDV .D........

....

....

YD

HinRfbP

LGFQVTHFIS VSNISN ..

. N VKELNIPILN SMSSWTSVTK KTD.QFIIAL EDDEEVDR"

HinRfbP

VIDE ..

....

LERYEENVDY YLMARPGMX LWQVSGR"1

.D........

....

....

YN

SpnCpsl4E

LSWKLVAVSV LDKS ..

... D FQHDKIWIE KEKIIEFATH EVVDEVFVDL PGE ..

. SYDI

RmeExoY

VEDE ..

....

LELYDSAAVF YLRSRPGLTG LWQISGRNDV

.S........

....

....

YA

MNRLFFSKIA LWLLDFLTFN ISFLLSLFVI SYYHNGYEKY LPIYEIDDRT

EamAmSG

LVGALSIITL LLPALVILIF RVS .RDGGAP IYGHERVGRD GRKFKCLKLR SMWNSKEVL

MDKKGLE IFLAVLQSII VILLWFLSF VRETELERSS

g SpnCpsl9E

LSWKLVAVSV LDKS ..

... D FQHDKIWIE KEKIIEFATH

FWD

EV

FVN

L PGE ..

. SYDI

SpnCpsl4E

TV

DE

....

..

YDQYTPEQKR RLSFKPGITG LWQVSGRSKI TD ..

....

.. .

..._

0)

SagCpsD

YDHNYIAVCI LDSSEKDCYD LKHNSLRIIN KDALTSELTC LTVDQAFINI PIELFGKYQI

SpnCpsl9E

TVDE ..

....

YVQYTSEQKR RLSFKPGITG LWQVSGRSKI TD

m

SagCpsD

S'

b 241

300

2 KpnOrf 14

MVSQLTDTTC .SVLYIPDVF TFNILQSRTE EINGVPWPL FDSPLNGIN.

. .MVFKRLED

481

516

'D -

RlePss

MD

. . .LVLKRAFD

KpnOrfl4

KRIEYDLLYI RGWSIWLDLK IIFLTVFKGF INKSAY*

LLQRLDRYPI .NVKLVPDLF DFGLLNQSAE QIGSVPVINL RQGGVDRDNY

.FWAKALQD

RlePss

ARIASDLYYV GNFSIVMDMR IIFGTWSEL TRGKGF*

MIKRLFD

XcaGumD

KRIQYDLDYI RRWSLWLDIR

IIVLTAVRVL GQKTAY*

$$ XcaGumD

2-

2

EamAmsG

WLKNLATHNC RSVSVIPSLR GVPL.YGTDM AYIFSHEVMI LRVSNNLAKH SSRFLKRTFD

StyRfbP

WLRELSKHHC R

SVTWPSFR GLPL.YNTDM SFIFSHEVML LRIQNNLAKR SSRFLKRTFD

EamAmsG

TRWFDSWW KNWSLWNDIA ILFKTIGWL KRLXAY'

.a HinRfbP

WLRYFSTNGY RSVSVIPTLR GLPL .YNTDM SFMFSHEIML LQMNNNLAKL SSRILKRTMD

StyRfbP

TRWFDSWYV KNWTLWNDIA ILFKTAKWL RRDGAY*

RmeExoY

MKSAT RSASS ..

...

.._

_..

.._

. PFFIPEETGA VRPIGGMAKR

S.......FD

HinRfbP

TRWFDSWYV KNWSLWNDIA 1LFK"VVL NRDGAY*

RmeExoY

TRVAFDTQYV QNWSLFADLV IVFKTIPAVC LSRGSY*

SpnCpsl9E

.GEIISRFET MGIDVTVNLK AFDKNLGRNX QIHEMVGLNV VTFSTNFYKT SHVISKRILD

SpnCpsl4E

DVVKLDVAYI DNWTIWKDIE ILLKTVKWF MRNGAK*

SdgCpsD

.QDIINDIEA MGVIVNVNVE ALSFDNIGEK RIQTFEGYSV ITYSMKFYKY SHLIAKRFLD

SpnCpsl9E

DVVKLDVAYI DNWTIhKDIE ILLKTVKWF MRDGAK*

BgzS

QLG

EKLRIQAEYV QTRTFLGDLK IIAHTLLAVA R*

3 9 8 u,

$$

Fig.

6. P

ILE

UP

ana

lysi

s of

Bpl

G w

ith m

embe

rs o

f the

Rfb

P fa

mily

of

prot

eins

. Kpn

OR

F14:

OR

F14

from

the

Kle

bsie

lla p

neum

onia

e C

hedi

d ca

psul

e lo

cus.

Xca

Gum

D:

Gum

D p

rote

in fr

om

Xan

thom

onas

cam

pest

ris re

quire

d fo

r xan

than

gum

bio

synt

hesi

s. E

amA

msG

: Am

sG p

rote

in fr

om E

rwin

ia a

myl

ovor

a re

quire

d fo

r ca

psul

e bi

osyn

thes

is. S

tyR

fbP

: Rfb

P fr

om S

. ent

eric

a se

rova

r W

phim

uriu

m re

quire

d fo

r 0-

antig

en b

iosy

nthe

sis.

Hin

Rfb

P: R

fbP

pro

tein

hom

olog

ue fr

om H

. inf

luen

zae

Rd

geno

me

sequ

enci

ng p

roje

ct. S

pnC

psl4

E a

nd S

pnC

psl9

E: C

psE

pro

tein

s fro

m th

e ca

psul

e bi

osyn

thes

is lo

ci o

f S

trept

ococ

cus

pneu

mon

iae

type

s 14

and

19,

resp

ectiv

ely.

Sag

Cps

D:

Cps

D p

rote

in fr

om th

e ca

psul

e bi

osyn

thes

is lo

cus

of S

trept

ococ

cus

agal

actia

e. R

leP

ss:

Rhi

zobi

um le

gum

jnos

arum

Pss

prot

ein

requ

ired

for e

xopo

lysa

ccha

ride

bios

ynth

esis

. Rm

eExo

Y: E

xoY

pro

tein

from

Rbi

zobi

um m

elilo

ti req

uire

d fo

r ex

opol

ysac

char

ide

bios

ynth

esis

. Bpe

Bpl

G:

6. pe

rtus

sis

Bpl

G p

rote

in. N

ote

that

the

Sag

Cps

D s

eque

nce

star

ts a

t pos

ition

137

in th

e di

agra

m, R

meE

xoY

at p

ositi

on 2

46,

Rle

Pss

at p

ositi

on 2

87 a

nd th

e B

plG

seq

uenc

e at

pos

ition

294

. I IG


described in the Experimental procedures. Two mutants were constructed: an insertion in the BamHl site in bplG and an insertion in the EcoRl site in bplH (Fig. 2). The mutants were checked by Southern hybridization and found to have the expected chromosomal rearrangements (data not shown). LPS from both mutants was isolated and assessed by SDS-PAGE and Western blotting.

Only band B was visible in SDS-PAGE of the LPS from the bplG mutant (Fig. 1, panel 1, column E), and on Wes- tern blotting it reacted with band B-specific mAb BL-8 (Fig. 1, panel 3, column E) but not with band A-specific mAb BL-2 (Fig. 1, panel 2, column E). This is consistent with the entire trisaccharide constituting band A being absent, as would be expected if BplG is a functional homologue of RfbP.

The bplH mutant displayed a different phenotype. On SDS-PAGE, band B was present, but in addition there was a faster-migrating form of band A (Fig. 1, panel 1, column D). These two bands were present in approximately a 1: l ratio, whereas in wild-type 6. pertussis, band A accounts for the vast majority of the stained mate- rial visible on the gel. On Western blotting, once again BL- 8 bound to the LPS (Fig. 1, panel 3, column D) whereas BL-2 did not (Fig. 1, panel 2, column D). This is clear evidence that band A has been modified. The phenotype of this mutant is, as expected, identical to the phenotype of the transposon mutants 5 and 103 (Fig. 1, columns B and C). It is likely that bplH encodes a protein required for the transfer of one of the sugars in the trisaccharide that constitutes band A, such that the bplH mutant synthe- sizes a truncated version of the trisaccharide which is then transferred to band B with reduced efficiency. Reduced efficiency transfer of truncated O-antigen subunits to core LPS was previously described in S. dysenteriae. Here it was suggested that the O-antigen ligase has a strong pre- ference for complete O-units but will allow the transfer of a single truncated unit (Reeves, 1994; Klena and Schnait- man, 1993). This observation further supports the idea that the €3. pertussis band A trisaccharide is a single 0- antigen unit.

The LPS molecules from both these mutants are currently being analysed to assess their chemical structures. These studies will give a much clearer indication of the precise role of the mutated genes in LPS biosynthesis. In addition, we are analysing the proteins described here enzymologically in an attempt to provide unequivocal evidence regarding their functions, though this is difficult given the lack of commercially available sugar substrates for such assays.

the databases. The stop codon of bplH overlaps a double ATG at the start of bpll. Bpll is intriguingly similar at a low level to the 6. pertussis BvgS protein involved in virulence gene regulation. BplJ is slightly similar to O r f l l from the K. pneumoniae cps locus, involved in capsule biosynthesis. The best similarity for BplK is the beta subunit of fatty acid synthase from Saccharomyces cerevisiae.

bplL: base pairs 13084 + 14958. bpll is the last gene in the locus and encodes a protein that is highly similar to a number of proteins involved in modification of nucleotide sugars (Fig. 7). It is most similar to Y. enterocolitica TrsG (Skurnik eta/., 1995) over its entire length. It is also similar to CapD from S. aureus, involved in type 1 capsule biosynthesis (Lin et a/., 1994) and to the partial ORF downstream of the rfb locus in V. cholerae 0 1 (Manning et a/., 1994). Starting at approximately 300 amino acids into the BplL protein are some strong similarities to a number of dTDP-glucose 4,6-dehydratases (Bechthold et a/., 1995; Linton et a/., 1995) (Fig. 7). These enzymes are usually involved in synthesizing 6-deoxy and dideoxy sugars. It seems likely, then, that the C-terminal domain of BplL is required for biosynthesis of the 2,6-dideoxy- galactose derivative FucNAcMe.

lnsertion sequence: base pairs 14955 t end. Overlap- ping the stop codon of bpll and transcribedhranslated on the opposite strand of the DNA is the gene for the transposase of an insertion sequence (IS) previously described in 6. pertussis (McLafferty eta/., 1988). The stop codons of the transposase and bplL genes actually overlap precisely. The sequence from position 14955 to 14982 inclusive is a perfect match for the 28 bp terminal inverted repeat of the IS. The Clal site marking the end of the sequence pub- lished here is equivalent to the Clal site in the transposase coding sequence. We have sequenced beyond this site, but the sequence is not double stranded and contains compression artefacts. However, it is clear that the transposase is present in its entirety and that a second 28 bp repeat is present at an appropriate distance, indicating that a complete IS is present.

Signal recognition particle. Beyond the IS lies a sequence which, when translated, gives a high degree of similarity with the signal recognition particle, or ‘fifty-four homologue’, of E. coli (Miller et a/., 1994). This protein is known to function in protein targeting to the cytoplasmic membrane protein translocation machinery. It is intriguing that this protein should be located next to a locus required for LPS biosynthesis.

Mutagenesis and phenotypic analysis of mutants

To confirm that the genes in the locus are required for LPS biosynthesis, defined allelic replacement mutants were constructed in the B. pertussis chromosome, using techniques


Presence of genes in other Bordetella spp.

The cloned 2.6 kb EcoRl restriction fragment from B. pertussis was used as a probe in Southern hybridization

YeTrsG

SaCapD

BBBnsL

YeTrsG

BBBnsL

SaCapD

YeTrsG

SaCapD

BBBnsL

YeTrsG

SaCapD

EEEL

QlL

YeTrsG

SaCapD

SeGdh

SfTylA2

SvGraE

SgStrE

BB

Bnl

L

YeTrsG

SaCapD

SeGdh

SfTylA2

SvGraE

SgStrE

BnB

plL

YeTrsG

SaCapD

SeGdh

SfTylA2

SvGraE

SgStrE

BREP

LL

1 60

MFLVF ..

... LLSLPRPVKR TIMLLLDTIL IALAYWGAFW VRLDVDSPFT SIEQWVALAA

MTLPYAIRRL FVDLPRPFKQ MLAIVLDAVI LLGAFHLALW LR

FEL ..

. FF LTDQYLFLSL

MTSISAKLRF LILIIIDSFI VTFSVFLGYA 1.LEPYFKGY SIDLLVLSSV

61

120

.I.IPPTLFA YIKLGLYRTV LRWSAKIVS I

VLVGWLSS GL.LVLGSYF LGV.YLPRTV

.LACAGGIAA LAAFGVYLYI LRYMSERVLA AILGGIWSV MV.VTAG"F

LQLATISRGV

ILLVSHHIFA YV.FNLYHRA WEYASVSELM SVLKAVTSSI WTLLLVSLL ISESPFLRLY

121

iao

SVMFFIFSLV LICGSRLFFR MLL ..

.. NYG VRGQIPWIY GAGASGRQLL PALMQASEYF

LVLYAALALV GLIGVRLIAR KLLFPADHHM ADPRTPVLIY

GAGGAGSQLA MALRTGPHYR

F1TW.MMHLL LIGGSRLFWR WRRYFIDNA VEKKATLW. GAGQGGSVLI REMLRSQDMR

181

240

..PIAFVDDN PKLHKAVIHG

... W

PSE KLEYLIGRYG IKKVLLAMPS VSQSQRRAW

..PVAMLDDD KRKHRLVVNG

... LRVYPPE QLPKLIDRHN IRQLLIAMPS APPKQIRSIV

MQPVLAVDDD KNKQKMTITE RVKVQGW.E DIPELVKKFR IKKIIIAIPT LSQKRLNEIN

241

300

NKLENLSCEV LSIPGMSDLV EGRAQISSLK KVSIEELLGR DPWPDEKLL AKNITGKVVM

EAAEPYRLRI RLVPSMRELI DPTNGV.RLR DVQVEDLLGR DPVAPIDTLL GRCVTDRVVM

KZCNIEGVEL FKMPNIEDVL SGELEVNNLK KV-DLLGR

DPVELDMALI SRELTNKTIL

MRV

L MRVL

MRLL

MTTHLL

301

360

VTGAGGSIGS ELCRQIIV.. .

.. EKPSLLI LFDISEFSLY SIENEM.AA1 CKKNKIETEF

VTGAGGSIGS ELCRQILA.. ..

. LRPRKLV LFEIAEPALY AIEQDLRQRI GE

RN ..

. IEI

VTGAGGSIGS EICRQVSK..

... FDPQKII LLGHGENSIY

SIHQELSKW GNR....IEF

VTGGAGFIGS HYVRQLLGGA YPAFAGADW VLDKLT. ..Y AGNEENLRPV A..DDPRFRF

VTGGAGFIGS HFTGQLLTGA YPDLGATRTV VLDKLT. ..Y AGNPANLEHV A..GHPDLEF

VTGAAGFIGS HYVREILAGS YPESDDVHVT WDRLT. ..Y AGRRDNLP.. E..HHERLDF

VTGAAGFIGS QYVRTLLGPG GP..PDVWT ALDALT ..

. Y AGNPDNLAAV R..GHPRYRF

361

420

VALLGSVQSE KRLVQIMSNF HVNTVYHAAA YKHVPLVENN VIEGVRNNIF GTLYCAKAAI

AGVLGSVRDA AHCLAQLQEH GVQTIYHAAA YKHVPIVEHN VSEGIRTNAF GTLNMAETAI

VPVIADVQNK TRILEVMNEF KPYAVYHAAA HKHVPLMEYN PHEAIRNNIL GTKNVAESAK

VR..GDICEW D

WSEVMRE. .VDVWHFAA ETHVDRSILG ASDFWTNW GTNTLLQGAL

VR..GDIADH GWWRRLMEG. .VGLWHFAA ESHVDRSIES SEAFVRTNVE GTRVLLQAAV

VH..GDICDR DLLDRVLPG. .HDAWHFAA ESHVDRSLTG PGEFVRTNVM GTQQLLDAAL

ER..GDICD. APGRRVMAG. .QDQWHLAA ESHVDRSLLD ASVFVRTNVH GTQTLLDAAT

YeTrsG

SaCapD

SeGdh

SfTylA2

SvGraE

SgStrE

LQBR

lL

YeTrsG

SaCapD

SeGdh

SFTY

IA?.

SvGraE

SgStrE

BBBe

lL

YeTrsG

SaCapD

SeGdh

SfTy1.42

SvGraE

SgSt rE

BnBplL

YeTrsG

SaCapD

SeGdh

SfTylA2

SvGraE

SgStrE

BBBelL

YeTrsG

SaCaDD

BBBB

lL

421

48

0

KSGVEKFVLI STDK ..

....

...

....

. AV RPTNTMGATK RMAELVLQAL STEQNKTKFC

QAGVLDFVLI STDK. ..

...

....

....

AV RPTNVMGASK RLAELILQAH AQIQDKTRFS

EGEVSKFVMI STDK. ..

...

....

....

AV NPSNVMGATK RIAEMVIQSL NEDNSKTSfV

AANVSKFVHV STDEWGTIE HGSWPEDHLL EPNSPYSAAK AGSDLIARAY HRTHG.LPVC

DAGVGRFVHI STDEWGSIA EGSWPEDHPV APNSPYAATK AASDLLALAY HRTYG.LDVR

HAGVDRVLHV STDEWGSLD SG'WTEDSPL LPNSPYAASK ASTTWSAAPT TVRHG.LDVR

RHGVASFVQV STDEVYGSLE HGSWTEDEPL RPNSPYSASK ASGDLLALAH HVSHG.LDVR

481

540

MVRFGNVLGS ..

. SGSWPL FKKQIAEGGP 1TL.THKDII RYFMTIPEAA QLVIQAGAMG

MVRFGNVLGS ..

. SGSWPL FRRQILEGGP 1TL.THPEIT RYFMTIPEAA QLVLQAGAMG

AVRFGNVLGS ..

. RGSVIPL FKNQIESGGP VTV.THPEMT RYFMTIPEAS RLVLQAGALA

ITRCSNNYGP YQFPEKVLPL FITNLMDGRR VPLYGDGLNV RDWLHVTDHC RGIQLVAESG

VTRCSNNYGP RQYPEKAVPL FTTNLLDGLP VPLYGDGGNT REWLHVDDHC RGVALVGAGG

ITRCSNNYGP RQHPEKLIPN FVTRLLTGRQ VPLYGDGRNV REWLHVDDHC RALQLVLTKG

VTRCSNNYGP RQFPEKLIPR FITLLNDGHR VPL

YGDG

LNV-

REWL

HVDD

HV RGIEAVRTRG

541

600

QGGDVFVLDM GDPVKIIDLA KRMINLSGLS IKSEENLDGD IAIEISGLRP GEKLYEELLI

ESGSVFVLDM GEPVLIRELA ERMVRLYGLT VKNSDQPDGD

IEIRITGLRP GEKLYEELLI

QGGEVFVLDM GKPVKIVDLA KNLIRLSG.. .KKEE....D IGIEFSGIRP GEKLYEELLN

RAGEIYNIGG GTELTNKELT ERVLELMGQD WSMVQPVTDR KGHDRRYSW HTKISEELGY

RPGVIYNIGG GTELTNAELT DRILELCGAD RSALRRVADR PGHDRRYSVD TTKIREELGY

RAGEIYNIGG GSGMSNREMT ARLLDLLGAD WDMVRHVEDR LGHDFRYAID DSKIREELGY

RAGRWNIGG GATLSNKELV GLLLEAAGAD WGSVEYVEDR KGHDRRYAVD STRIQRELGF

601

660

GDSVQ.HTYH PRIMTA ..

.. .

.. TEIMLEW DDLNILLNKI ETACNDFNYE CIRSLLLEAP

GEDSR.ETLH PRI MRA....

... TE

YSLPY ETLMGQLRML D. ..

....

.. .

.RSLQMCSP

KNEIHPQQVY EKIYRGKVDH YIKTEVDLIV EDLINNFSK.

....

....

_. ..EKLLKIAN

EPWPFERGL AETIEWYRDN RAWWEPLKSA PEGGK'

APRTGITEGL AGTVAWYRDN RAWWEFLKRS PGGRELERA*

APRWSIESGL GAVVUWYRDH PD FW....RA PAS'

APAVDLADGL AATVAWYHKH RSWWEPLVPA GSLPA'

661

68

8

TGFQPTDGIC DVVWQKTHSE NAKNVIVH*

R..QAAELLG QIVREYASVT YA*

R*

Fig.

7.

PILE

UP

anal

ysis

of B

plL

with

hom

olog

ous

prot

eins

. YeT

rsG

: Tr

sG p

rote

in fr

om Y

. ent

eroc

oliti

ca re

quire

d fo

r 0

3 b

iosy

nthe

sis.

BpB

plL:

6. p

ertu

ssis

Bpl

L pr

otei

n. S

aCap

D: S.

aure

us C

apD

pr

otei

n, re

quire

d fo

r ty

pe 1

cap

sule

bio

synt

hesi

s. S

eGdh

: S

acch

arop

olys

pora

eiy

fhra

ea d

TDP

-glu

cose

4,6

-deh

ydra

tase

invo

lved

in e

ryth

rom

ycin

bio

synt

hesi

s. S

fTyl

A2:

Str

epto

myc

es fr

adia

e dT

DP

-glu

cose

4,6

-deh

ydra

tase

invo

lved

in ty

losi

n bi

osyn

thes

is. S

vGra

E:

Str

epto

myc

es v

iola

ceor

uber

Tu2

2 dT

DP

-glu

cose

4,6

-deh

ydra

tase

invo

lved

in g

rani

ticin

bio

synt

hesi

s. S

gStr

E: S.

gris

eus

dTD

P-g

luco

se 4

,6-d

ehyd

rata

se in

volv

ed in

stre

ptom

ycin

bio

synt

hesi

s. N

ote

that

the

four

dTD

P-g

luco

se 4

,6-d

ehyd

rata

ses

are

only

hom

olog

ous

with

the

C-te

rmin

us o

f th

e B

plLl

TrsG

I C

apD

pro

tein

s.

P

cn b

b

5

a, P a P s m h


to co-ordinately regulate the first two steps in LPS core biosynthesis. The presence of these genes next to the bpl genes is intriguing and may also suggest some co- ordinate regulation of inner core biosynthesis with band A LPS biosynthesis. We are currently investigating the transcriptional regulation of the genes in this locus.

The arrangement of the bplgenes is also worthy of note. These genes are somewhat similar to an rfa or rfb locus from an enteric bacterium, or a capsule biosynthesis locus, in that genes for modification and biosynthesis of nucleotide sugars are present next to genes for glycosyltransferases. Although we have no direct enzymological evidence to confirm the nature of the reactions catalysed by the bpl gene products, we have made several reasonable speculations as to their functions. The products of the first four genes may act, in reverse order, to convert UDP-GlcNAc to UDPQ,3-diNAcManA (Fig. 5). The action of genes in reverse order to the order that they appear in a locus has been previously commented upon (Reeves, 1994). Once generated, UDP-2,3-diNAcManA must be transferred to the band A trisaccharide. It seems plausible that this is achieved by the BplE protein.

The BplF protein is similar to BplC and must also be required for biosynthesis of an amino sugar (Thorson et a/., 1993; 1994). It is possible that BplF is responsible for one of the steps in FucNAcMe biosynthesis.

A completely unexpected finding was the homology of the BplG protein with a family of proteins that catalyse the first step in building polysaccharide units for export and assembly. The most studied of these is RfbP from S. enterica. RfbP catalyses the transfer of galactose to an ACL, in this case undecaprenol phosphate (Wang and Reeves, 1994), this being the first step in biosynthesis of 0-antigen. Subsequently, the biosynthesis of the 0-unit is completed on the ACL before being incorporated into growing 0-antigen polymers. This mechanism is also used by other bacteria for biosynthesis of capsular polysaccharides (Muller et a/., 1993; Reuber and Walker, 1993; Rubens et a/., 1993; Guidolin et a/., 1994; Lin et a/., 1994; Arakawa et a/., 1995; Huang and Schell, 1995), and it is, therefore, not surprising that BplG is also homologous with these proteins. BplG appears to be homologous with the C-terminal domain only of S. enterica RfbP. This is consistent with the view that S. enterica RfbP is a two-domain protein, with the C-terminal domain being the galactosyltransferase and the N-terminal domain possibly being the ‘flippase’ that transports the 0 units across the cytoplasmic membrane (Wang and Reeves, 1994). It seems reasonable to suggest that BplG is transferring a sugar to an ACL, but the identification of that sugar will be difficult, given the non-availability of the nucleotide-sugar substrates required for definitive enzy- mology studies. We propose that FucNAcMe is the sugar that is being transferred, and that the band A trisaccharide

experiments using chromosomal DNA isolated from €3. bronchseptica and B. parapertussis Under conditions of high stringency, the probe bound to a single 2.6 kb EcoRl band in both DNA samples tested. This indicated that at least part of the LPS locus found in B. pertussis is also present in the other two Bordetella spp. We have recently cloned the B. parapertussis DNA and partial sequence analysis shows that, in this region, the loci are extremely highly conserved. This is somewhat surprising given that the B. pertussis and 6. parapertussis LPS molecules appear to differ considerably, the latter having an O-antigen-like structure consisting of repeats of 2,3-diNAcGalA and a core that appears not to share structures in common with 6. pertussis LPS (Di Fabio eta/., 1992). It is possible that certain genes are inactive in one or other of the bordetellae, or that other genes are present in the other Bordetella spp. leading to the biosynthesis of alternative LPS molecules. It is also possible that 6. perfussis is able to synthesize an LPS with an 0-antigen similar to the other bordetellae but that under the conditions that must be used to grow the organism it is not expressed. Alternatively, the other bordetellae might make a structure similar to band A but again it is masked or not synthesized when these organisms are grown in vitro. A precedent for this exists in that when Chlamydia spp. are grown under different conditions from those usually used to grow these bacteria, they are able to synthesize 0-antigen-like structures (Lukacova et a/., 1994). Pre- viously it was thought that this organism was unable to make 0-antigen.

Discussion

In this paper, we describe the identification, cloning, sequencing and mutagenesis of a genetic locus from 6. pertussis required for LPS biosynthesis. To our knowledge, no other genes for LPS biosynthesis have been reported from Bordetella spp. Similarity searching has allowed hypoth- eses to be formed regarding the functions of the products of some of the genes within the locus. It has been suggested that the arrangement in a single locus of a number of genes involved in the biosynthesis of a polysaccharide has commonly occurred in the evolution of bacteria (Reeves, 1994). The arrangement of genes found in the 6. pertussis locus is, however, unique amongst all those currently discovered, in that genes for deep inner core biosynthesis (kdtA and rfaC) are next to genes proposed to play a role in distal polysaccharide biosynthesis (bplA- L). kdfA and rfaC are found as part of the rfa locus in S. enterica and E. cob, but they are not next to each other and are found in different transcriptional units (Reeves, 1994). The evolutionary pressure for 6. pertussis to place rfaC and kdtA together, apparently in an operon, is unclear, but this arrangement does provide the potential

0 1996 Blackwell Science Ltd. Molecular Microbiology, 19, 37-52


is built like a single 0-antigen unit. The mutant that we generated in BplG in 6. pertussis supports this conjecture since it is no longer able to make band A LPS, while band B appears to be synthesized normally. This in turn implies that band A LPS is equivalent to semi-rough LPS found in enteric bacteria (Reeves, 1994), an idea that has been suggested previously (Caroff eta/., 1990).

From similarity analysis, BplH is probably a glycosyltransferase. The fact that the BplH mutant has a band A LPS of lower molecular weight implies that BplH transfers one of the sugars of the terminal trisaccharide. The size of the mutant band suggests that it transfers GlcNAc and we are currently testing this idea enzymologically. BplH, Bpll and BplK are of unknown function and are being investi- gated by mutagenesis with subsequent phenotypic analysis of the LPS.

BplL is highly similar over its entire length with CapD from S. aureus (Lin et al., 1994) and TrsG from Y. enterocolitica (Skurnik et al., 1995). It is also similar over the C- terminal domain with dTDP-glucose 4,6-dehydratases, mainly isolated from sugar-containing antibiotic biosynthesis pathways. These enzymes are involved in the biosynthesis of 6-deoxy sugars, so we propose that in 6. pertussis the C-terminal domain of BplL is required for the biosynthesis of FucNAcMe, since fucose is the 6- deoxy derivative of galactose. The possible function of the N-terminal domain remains unclear.

The presence of the 6. pertussis IS in intimate associa- tion with the bpl locus is intriguing. This IS element is not seen in 5. parapertussis or 6. bronchiseptica (McLafferty et a/., 1988). We have shown by hybridization analysis that these bacteria possess DNA homologous to the bpl locus and have recently cloned and partially sequenced the EcoRl restriction fragment from 6. parapertussis that contains the bpF, bplG and bplH genes. However, the Clal site at the right-hand end of the locus in 6. pertussis, provided by the IS, is absent in 6. parapertussis and 6. bronchiseptica, in accordance with the fact that this IS has not been observed in these bacteria. Since IS elements are suggested as playing an important evolutionary role in mediating chromosomal rearrangements, it seems likely that the bpl locus has been altered by the presence of the IS in 6. pertussis. This might contribute to the expression of the structurally different LPS molecules by the different Bordetella species (Di Fabio ef a/., 1992). Comparison of the sequences at this end of the locus between the different bordetellae will provide useful information in addressing this issue.

Cloning of the bpl locus has enabled us for the first time to generate defined non-reverting mutants of 6. pertussis with defined LPS structures. These mutants will be invalu- able tools in studying whether LPS plays a role in the pathogenicity of infections with these bacteria and will also provide information regarding the function@) of defined

LPS structures in the biological activities associated with 6. pertussis LPS.

Experimental procedures

Bacterial strains and plasmids

B. pertussis BP536 (Relman et a/., 1990) was used as the wild-type strain in all experiments. As a source of chromosomal DNA for Southern blot experiments, 6. parapertussis CN 2591 and B.bronchiseptica CN 7635E were used. For cloning experiments and maintaining plasmids E. coli XL1- Blue (Stratagene) was used. All cloning and DNA sequencing experiments used the pUC or the pBluescript series of plasmids. The vector used in conjugation experiments for the gen- eration of mutants in B. pertussis was pRTP1 (Stibitz et a/., 1986). This vector is a ColEl replicon and, therefore, cannot replicate in B. pertussis. It also has an s72 allele conferring streptomycin sensitivity on streptomycin-resistant bacteria, allowing selection against maintenance of vector sequences via single crossover events. It contains an oriT mobilizable by pNJ5000 (Grintner, 1983). For conjugation experiments involving pRTPl , E. coliCC118 was used to maintain the plasmid and E. coli S17-1 (pNJ5000) was used to mobilize it. The cosmid vector used was pHC79 (Hohn and Collins, 1980) into which a Pvull fragment containing the polylinker from pBlue- script was cloned. This provided a cosmid vector with an Asp7181 site for the cloning of the Asp71 81 fragment containing the locus.

Media, chemicals and reagents

8. pertussis was routinely cultured on Cohen-Wheeler medium supplemented by 10% (vh) horse blood (TCS Biologicals). E. coli was cultured on Luria broth or agar (Sarn- brook et a/., 1989). Media were purchased from Difco or Oxoid. Antibiotics were used where appropriate. For 6. pertussis nalidixic acid was used at 50pgml-', kanamycin at 40 pg ml-' and streptomycin at 200 pg rnl-'. For E. coli, kanamycin was used at 40pgml-' and ampicillin at lOOpgml-'. All antibiotics and routine chemicals were purchased from Sigma Chemical Company. Restriction and modifying enzymes were purchased from Boehringer Mannheim. DNA ligase was purchased from Gibco-BRL. Sequenase sequencing kits were purchased from United States Biochemical through Amersham International.

Jransposon mutagenesis

Transposon TnphoA (Boquet et a/., 1987) was used to muta- genize 6. pertussis. The transposon was conjugated into B. pertussis on the suicide vector pRT733 from E. coli SMlO hpiras donor. NalR, SmR, KmR transconjugants were selected on Cohen-Wheeler medium and then streaked out in dupli- cate on Cohen-Wheeler plates containing antibiotics. Bac- teria from one of these plates were lifted on to nitrocellulose discs (Shleicher and Schuell). These discs were subsequently screened with anti-LPS mAbs. These antibodies were kindly provided by Drs Denis Martin and Bernard Brodeur, National Laboratory for Immunology, Ottawa, Canada.



DNA flanking either side of the transposon insertion site were used to generate oligonucleotides which were subsequently used in a PCR on wild-type B. pertussis DNA. This generated an approximately 500 bp band that was confirmed as equivalent to the DNA flanking the transposon insertion by DNA sequence analysis and contained an EcoRl restriction site. This PCR product was used in high-stringency Southern blots to identify a 2.6 kb EcoRl and 4.7 kb EcoRI- Clal restriction fragments in B. pertussis chromosomal DNA. These fragments were subsequently cloned as follows. B. pertussis chromosomal DNA (1 0 pg) was digested with the appropriate restriction enzymes. After electrophoresis in a 0.8% preparative agarose gel, DNA of the required size was excised from the gel and purified. The DNA was then ligated into the appropriate restriction site(s) in pBluescript SK - and transformed into the E. coli strain XL-1 Blue. Colonies were screened with the radiolabelled 500 bp PCR product.

To clone a large restriction fragment containing the whole locus, a CHEF gel was run on a BioRad apparatus, using various digests of B. pertussis DNA prepared in agarose blocks (Sambrook eta/., 1989). This gel was Southern transferred and hybridized with DNA from within the locus. This identified a number of bands and we chose an approximately 33 kb Asp7181 band for cloning. To clone this band, an Asp7181 digest of B. pertussis DNA was run on a 0.5% low-melting- point agarose gel in the cold. The region of high-molecular- weight DNA was cut from this gel and DNA was extracted by melting the agarose followed by phenol extraction at 37", chloroform extraction and ethanol precipitation of the DNA. This was then ligated with the cosmid vector and packaged in h particles using Gigapack Gold packaging mixes (Strata- gene). The packaged cosmid was then used to infect €. cdi XL1 -Blue, with subsequent selection on ampicillin. This generated thousands of colonies. Twenty four of these were screened using PCR with locus-specific oligonucleotides as primers. This identified one cosmid carrying the locus. This was confirmed using restriction digest analysis and DNA sequence analysis.

Monoclonal antibody screening

Nitrocellulose discs with colony lifts were first blocked using phosphate-buffered saline (PBS), 0.02% (v/v) Tween 20, 3% (w/v) bovine serum albumin (BSA) for 30min at room temperature. They were then incubated with a 1:lOOO dilution (in PBS, 3% (w/v) skimmed milk) of mAb BL-2 (specific for band A LPS), for 30 min at room temperature. Unbound pri- mary antibody was then removed by three 10 minute washes in PBS-Tween 20 (0.02%, v/v). After this, the discs were incubated with rabbit anti-mouse IgG (whole molecule) alkaline phosphatase-conjugated second antibody (Sigma) (1 :lo00 dilution, in PBS-Tween 20) for 30min at room temperature. The discs were then washed as above. Substrate for the second antibody, consisting of BCIP/NBT (Sigma), was then added to the washed disks. The reaction was left to develop until there were clear differences between those colonies reacting and those not. Non-reactive colonies were picked and plated on Cohen-Wheeler medium for further analysis.

LPS preparation, SDS-PAGE and Western blots

LPS was purified using a modification of the method of Hitch- cock and Brown (1983). Briefly, B. pertussis was grown on Cohen-Wheeler plates for 2 to 3days then harvested into PBS. Resuspended bacteria were lysed by addition of a one-third volume of lysis solution (0.1875 M Tris-HCI pH 6.8, 3% (w/v) SDS, 30% (v/v) glycerol), followed by incubation at 100°C for 10min. After cooling, Proteinase K was added to a final concentration of 50pgml-'. This was incubated at 55°C for 60 min. SDS-PAGE was run in a tricine buffer sys- tem according to the method of Lesse et a/. (1 990). Western blots were done according to Towbin eta/. (1 979). After transfer, Western blot filters were processed by the same method as the colony immunoblots.

Southern hybridizations

Southern hybridizations were performed according to Sam- brook et a/. (1989) with probes labelled with [32P]-dCTP or [32P]-dATP using a random primed labelling kit (Stratagene).

Cloning of LPS genes

Chromosomal DNA was isolated from the bordetellae as described previously (Maskell et a/., 1988). Restriction fragments corresponding to the sites of insertion of the transposon in BL-2-non-reactive mutants were identified by Southern hybridization using oligonucleotides specific for IS50 sequences. The fragments chosen for further study were a 3.0 kb Sall fragment and a 2.8 kb Pstl fragment. To clone this DNA, restriction fragments from around the correct size range were eluted from a preparative agarose gel and ligated with pBluescript vector II SK- digested with the appropriate restriction enzyme. The resulting ligation mixture was then used as a template for PCR, using the IS50-specific oligonucleotide and forward sequencing primer as primers. This generated the expected band in both cases, which was then subcloned. Subsequent DNA sequence analysis identified the junction between the transposon and the B. pertussis DNA in these fragments. The sequences of the B. pertussis

0 1996 Blackwell Science Lid, Molecular Microbiology, 19, 37-52

DNA sequencing

Plasmid DNA was sequenced using Sequenase 2. Because DNA from Bordetella spp. has a high GC content, deaza-G was used to prevent compressions and terminal transferase was used to remove stalls. Particularly difficult compressions were resolved using dlTP in the mixes. Sequences were assembled and analysed using the GCG package (Devereux eta/., 1984) or the Staden package (Staden, 1984) running on the Oxford University molecular biology VAX.

Mutagenesis of B. pertussis

The suicide plasmid pRT733 carrying transposon TnphoA was conjugated into 6. pertussis strain BP536, which is SmR and NalR. Transposon mutants of B. perfussis were selected on kanamycin, with streptomycin and nalidixic acid in the medium to select against the E. coli conjugative donor. B. pertussis mutants were then transferred to nitrocellulose discs and screened as described above. For allelic replacement mutagenesis, the gene to be mutated was inser- tionally inactivated using a KmR gene cassette and then this


was cloned onto pRTPl . The mutated allele was introduced into B. pertussis via a triparental plate conjugation using S17-l(pNJ5000) to mobilize the pRTP1 construct into B. pertussis, as described previously (Roberts eta/., 1990).

Acknowledgements

We would like to thank Bernard Brodeur and Denis Martin of the Laboratory Center for Disease Control, Ottawa, Canada for the gift of monoclonal antibodies. We also thank Sunil Sarda for help with the artwork This work was funded by The Wellcome Trust with grant number 036991/z/92/z.

References

Amano, K.-I., Fukushi, K., and Watanabe, M. (1990) Bio- chemical and immunological comparison of lipopolysaccharides from Bordetella species. J Gen Microbioi 136: 481 -487.

Aoyama, K., Haase, A.M., and Reeves, P.R. (1994) Evidence for effect of random genetic drift on G+C content after lateral transfer of fucose pathway genes to Escherichia coli

Arakawa, Y., Wacharotayankun, R., Nagatsuka, T., Ito, H., Kato, N., and Ohta, M. (1995) Genomic organization of the Klebsiella pneumoniae cps region responsible for serotype K2 capsular polysaccharide synthesis in the virulent strain Chedid. J Bacterioll77: 1788-1 796.

Archambault, D., Rondeau, P., Martin, D., and Brodeur, B.R. (1 991) Characterization and comparative bactericidal activity of monoclonal antibodies to Bordetella pertussis lipooligosaccharide A. J Gen Microbiol 137: 905-91 1.

Bechthold, A., Sohng, J., Smith, T.M., Chu, X., and Floss, H.G. ( 1 995) Identification of Streptomyces violaceoruber Tu22 genes involved in the biosynthesis of granaticin. Mol Gen Genet 248: 610-620.

Boquet, P.L., Manoil, C., and Beckwith, J. (1987) Use of TnphoA to detect genes for exported proteins in Escher- ichia coli : identification of the plasmid-encoded gene for a periplasmic phosphatase. J Bacteriol 169: 1663-1 669.

Bugert, P., and Geider, K. (1995) Molecular analysis of the ams operon required for exopolysaccharide synthesis of Erwinia amylovora. Mol Microbiol 15: 917-933.

Caroff, M., Chaby, R., Karibian, D., Perry, J., Deprun, C., and Szabo, L. (1990) Variations in the carbohydrate regions of Bordetella pertussis lipopolysaccharides: electrophoretic, serological and structural features. J Bacterioll72: 1121- 1128.

Chaby, R., and Caroff, M. (1988) Lipopolysaccharide of Bordetella pertussis endotoxin. In Pathogenesis and Immunity in Pertussis. Wardlaw, A.C., and Parton R. (eds). New York: John Wiley, pp. 247-271.

Cherry, J.D. (1992) Pertussis: the trials and tribulations of old and new pertussis vaccines. Vaccine 10: 1033-1 038.

Clement2 T., and Raetz, C.R.H. (1991) A gene coding for 3- deoxy-D-manno-octulosonic acid transferase in fscherichia coli - identification, mapping, cloning, and sequencing. J Biol Chem 266: 9687-9696.

Daniels, D.L., Plunkett 111, G., Burland, V., and Blattner, F.R. (1992) Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 min. Science 257:

K-12. MOl B id EVO~ 11 : 829-838.

771 -778.

Dell, A,, Oates, J., Lugowski, C., Romanowska, E., Kenne, L., and Lindberg, B. (1 984) The enterobacterial common- antigen, a cyclic polysaccharide. Carbohydr Res 133: 95- 104.

Deshazer, D., Wood, G.E., and Friedman, R.L. (1995) Identification of a Bordetella pertussis regulatory factor required for transcription of the pertussis toxin operon in Escherichia coli. J Bacterioll77: 3801 -3807.

Devereux, J., Haeberli, P., and Smithies, 0. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12: 387-395.

Dhillon, N., Hale, R.S., Cortes, J., and Leadlay, P.F. (1989) Molecular characterization of a gene from Saccharopoly- spora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis. Mol Microbiol 3:

Di Fabio, J.L., Caroff, M., Karibian, D., Richards, J.C., and Perry, M.B. (1992) Characterization of the common antigenic lipopolysaccharide 0-chains produced by Borde- tella bronchiseptica and Bordetella parapertussis. FEMS Microbiol Letts 97: 275-282.

Ewanowich, C.A., Melton, A.R., Weiss, A.A., Sherburne, R.K., and Peppler, M.S. (1989) Invasion of HeLa 299 cells by virulent Bordetella pertussis. lnfect lmmun 57: 2698- 2704.

Fleischmann, R.D. et a/. (1 995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269: 496-512.

Fujita, Y., Shindo, K., Miwa, Y., and Yoshida, K.4. (1991) Bacillus subtilis inositol dehydrogenase-encoding gene (idh): sequence and expression in fscherichia coli. Gene

Gagnon, Y., Breton, R., Putzer, H., Pelchat, M., Grunberg- Manago, M., and Lapointe, J. (1994) Clustering and co- transcription of the Bacillus subtilis genes encoding the aminoacyl-tRNA synthetases specific for glutamate and for cysteine and the first enzyme for cysteine biosynthesis. J Biol Chem 269: 7473-7482.

Glaser, P., Kunst, F., Arnaud, M., Coudart, M.P., Gonzales, W., Hullo, M.F., lonescu, M., Lubochinsky, B., Marcelino, L., Moszer, I., Presecan, E., Santana, M., Schneider, E., Schweizer, J., Vertes, A., Rapoport, G., and Danchin, A. (1 993) Bacillus subtilis genome project: cloning and sequencing of the 97 kb region from 325 (degrees) to 333 (degrees). Mol Microbiol 10: 371 -384.

Gohmann, S., Manning, P.A., Alpert, C.-A,, Walker, M.J., and Timmis, K.N. (1 994) Lipopolysaccharide 0-antigen biosynthesis in Shigelia dysenteriae serotype 1 : analysis of the plasmid-carried rfp determinant. Microbial Pathogen

Grintner, N.J. (1983) A broad host range cloning vector transposable to various replicons. Gene 21: 133-143.

Guidolin, A,, Morona, J.K., Morona, R., Hansman, D., and Paton, J.C. (1 994) Nucleotide sequence analysis of genes essential for capsular polysaccharide biosynthesis in Streptococcus pneumoniae type 19F. lnfect lmmun 62:

Hitchcock, P.J., and Brown T.M. (1 983) Morphological heterogeneity among Salmonella lipopolysaccharide che- motypes in silver-stained polyacrylamide gels. J Bacteriol

1405-1414.

108: 121-125.

16: 53-64.

5384-5396.

154: 269-77.

0 1996 Blackwell Science Ltd, Molecular Microbio/ogy, 19, 37-52


Mansouri, K., and Piepersberg, W. (1991) Genetics of streptomycin production in Streptomyces griseus: nucleotide sequence of five genes, strFGHIK, including a phosphatase gene. Mol Gen Genet 228: 459-469.

Martin, D., Peppler, M.S., and Brodeur, B.R. (1992) Immu- nological characterization of the lipooligosaccharide B band of Bordetella pertussis. Infect lmmun 60: 271 8-2725.

Maskell, D.J., Morrisey, P., and Dougan, G. (1988) Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis. J Bacterioll70: 2467-2471 .

McLafferty, M.A., Harcus, D.R., and Hewlett, E.L. (1988) Nucleotide sequence and characterization of a repetitive DNA element from the genome of Bordetella pertussis with characteristics of an insertion sequence. J Gen Microbiol

Merson-Davies, L.A., and Cundliffe, E. (1994) Analysis of five tylosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome. Mol Microbioll3: 349-355.

Miller, J.D., Bernstein, H.D., and Walter, P. (1994) Interaction of Escherichia coli ffh14.5 s ribonucleoprotein and ftsY mimics that of mammalian signal recognition particle and its receptor. Nature 367: 657-659.

Muller, P., Keller, M., Weng, W.M., Quandt, J., Arnold, W., and Puhler, A. (1993) Genetic analysis of the Rhizobium meliloti exoYFQ operon: ExoY is homologous to sugar transferases and ExoQ represents a transmembrane protein. Mol Plant-Microb Interact 6: 55-65.

Nomura, Y., Nakagawa, M., Ogawa, N., Harashima, S., and Oshima, Y. (1992) Genes in PHT plasmid encoding the initial degradation pathway of phthalate in Pseudornonas putida. J Ferment Bioeng 74: 333-344.

Peppler, M.S. (1 984) Two physically and serologically distinct lipopolysaccharide profiles in strains of Bordetella pertussis and their phenotype variants. Infect lmmun 43: 224-232.

Peschke, U., Schmidt, H., Zhang, H.-Z., and Piepersberg, W. (1 995) Molecular characterization of the lincomycin-production gene cluster of Streptomyces lincolnensis 78-1 1. Mol Microbiol 16: 1 137-1 156.

Pissowotzki, K., Mansouri, K., and Piepersberg, W. (1991) Genetics of streptomycin production in Sfreptomyces griseus: molecular structure and putative function of genes strELMB2N. Mol Gen Genet 231 : 1 13-1 23.

Raetz, C.R.H. (1993) The enzymatic synthesis of lipid A. In Bacterial Endotoxin: Recognition and Effector Mech- anisms, Endotoxin Research Series, Vol. 2. Levin, J., Alving, C.R., Munford, R.S., and Stutz, P.L. (eds). Amsterdam: Elsevier Science, pp. 39-48.

Rappuoli, R., Pizza, M., Covacci, A,, Bartoloni, A., Nencioni, L., Podda, A., and de Magistris, M.T. (1992) Recombinant acellular pertussis vaccine - from the laboratory to the clinic: improving the quality of the immune response. FEMS Microbiol lmmunollO5: 161 -1 70.

Reeves, P. (1 994) Biosynthesis and assembly of lipopolysaccharide. In Bacterial Cell Wall. Ghuysen, J.-M., and Hakenbeck, R. (eds). Amsterdam: Elsevier Science, pp.

Relman, D., Tuomanen, E., Falkow, S., Golenbock, D.T., Saukkonen, K., and Wright, S.D. (1993) Recognition of a bacterial adhesin by an integrin: macrophage CR3 (ciMpP, CDl 1 b/CD18) binds filamentous hemagglutinin of Borde- tella pertussis. Cell 61 : 1375-1 382.

134: 2297-2306.

281-317.

Hohn, B., and Collins, J. (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11 : 291 -298.

Huang, J., and Schell, M. (1 995) Molecular characterization of the eps gene cluster of Pseudomonas nacearum and its transcriptional regulation at a single promoter. Mol Microbioll6: 977-989.

Jiang, X.-J., Neal, B., Santiago, F., Lee, S.J., Romana, L.K., and Reeves, P.R. (1991) Structure and sequence of the rfb (0 antigen) gene cluster of Salmonella serovar typhimurium (strain LT2). Mol Microbiol5: 695-71 3.

Kanagasundaram, V., and Scopes, R.K. (1 992) Cloning, sequence analysis, and expression of the structural gene encoding glucose-fructose oxidoreductase from Zymomo- nas mobilis. J Bacterioll74: 1439- 1447.

Kessler, A.C., Haase, A., and Reeves, P.R. (1993) Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup HA. J Bacteriol

Klena, J.D., and Schnaitman, C.A. (1993) Function of the rfb gene cluster and the rfe gene in the synthesis of 0 antigen by Shigella dysenteriae 1. Mol Microbiol9: 393-402.

Lai, C.-Y., and Baumann, P. (1992) Sequence analysis of a DNA fragment from Buchnera aphidicola (an endosymbiont of aphids) containing genes homologous to dnaG, rpoD, cysE, and secB. Gene 11 9: 11 3-1 18.

Lasfargues, A,, Caroff, M., and Chaby, R. (1993) Structural features involved in the mitogenic activity of Bordetella pertussis lipopolysaccharides for spleen cells of C3H/HeJ mice. FEMS lmmunol Med Microbiol7: 1 19-1 30.

Lebbar, S., Caroff, M., Szabo, L., Merienne, C., and Szilogyi, L. (1994) Structure of a hexasaccharide proximal to the hydrophobic region of lipopolysaccharides present in Bordetella pertussis endotoxin preparations. Carbohydr Res 259: 257-275.

Lee, J.C., Xu, S., Albus, A., and Livolsi, P.J. (1994) Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus. J Bacterioll76: 4883-4889.

Lesse, A.J., Campagnari, A.A., Bittner, W.E., and Apicella, M.A. (1 990) Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulphate-polyacrylamide gel-electrophoresis. J lmmunol Meth 126: 109-1 11.

Lin, W.S., Cunneen, T., and Lee, C.Y. (1994) Sequence analysis and molecular characterization of genes required for the biosynthesis of type 1 capsular polysaccharide in Staphylococcus aureus. J Bacterioll76: 7005-701 6.

Linton, K.J., Jarvis, B.W., and Hutchinson, C.R. (1995) Cloning of the genes encoding thymidine diphospho- glucose 4,6-dehydratase and thymidine diphospho-4-keto- 6-deoxyglucose 3,5-epimerase from the erythromycin-pro- ducing Saccharopolyspora erythraea. Gene 153: 33-40.

Lukacova, M., Baumann, M., Brade, L., Mamat, U., and Brade, H. (1 994) Lipopolysaccharide smooth-rough phase variation in bacteria of the genus Chlamydia. lnfect lmmun

Manning, P.A., Stroeher, U.H., and Morona, R. (1994) Molecular basis for 0-antigen biosynthesis in Vibrio cholerae 0 1 : Ogawa-lnaba switching. In Vibrio cholerae and Cholera: Molecular to global perspectives. Wachs- muth, I.K., Blake, P.A., and Olsvik, 0. (eds). Washington DC: American Society for Microbiology.

175: 1412-1422.

62: 2270-2276.

0 1496 Blackwell Science Ltd, Molecular Microbiofogy, 19, 37-52


Reuber, T.L., and Walker, G.C. (1993) Biosynthesis of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti. Cell 74: 269-280.

Roberts, M., Maskell, D., Novotny, P., and Dougan, G. (1990) Construction and characterization in vivo of Bordetella pertussis aroA mutants. lnfect lmmun 58: 732-739.

Robison, K., Gilbert, W., and Church, G.M. (1994) Large scale bacterial gene discovery by similarity search. Nature Genet 7 : 205-214.

Rubens, C.E., Heggen, L.M., Haft, R.F., and Wessels, M.R. (1993) Identification of cpsD, a gene essential for type 111 capsule expression in group B streptococci. Mol Microbiol

Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbour Laboratory Press.

Shahin, R.D., Hamel, J., Leef, M.F., and Brodeur, B.R. (1994) Analysis of protective and non-protective monoclonal antibodies specific for Bordetella pertussis lipooligosaccharide. lnfect lmmun 62: 722-725.

Sirisena, D.M., Brozek, K.A., Maclachlan, P.R., Sanderson, K.E., and Raetz, C.R.H. (1992) The rfaC gene of Salmonella typhimurium-cloning, sequencing, and enzymatic function in heptose transfer to lipopolysaccharide. J Biol Chem 267: 1 8874- 18884.

Skurnik, M., Venho, R., Toivanen, P., and Al-Hendy, A. (1 995) A novel locus of Yersinia enterocolifica serotype 0:3 involved in lipopolysaccharide core biosynthesis. Mol Microbiol 17: 575-594.

Staden, R. (1984) Graphic methods to determine function of nucleic acid sequences. Nucl Acids Res 12: 521 -538.

Stibitz, S., Black, W., and Falkow, S. (1986) The construction of a cloning vector designed for gene replacement in Bordetella pertussis. Gene 50: 133-1 40.

Stutzman-Engwall, K.J., Otten, S.L., and Hutchinson, C.R. (1 992) Regulation of secondary metabolism in Strepto- myces spp. and overproduction of daunorubicin in Strepto- myces peucetius. J Bacterioll74: 144-1 54.

8: 843-855.

Szabo, M., Bronner, D., and Whitfield, C. (1995) Relation- ships between rfb gene clusters required for biosynthesis of identical D-galactose-containing 0 antigens in Klebsiella pneumoniae serotype-01 and Serratia marcescens serotype 0-1 6. J Bacterioll77: 1544-1 553.

Takagi, M., Takada, H., and Imanaka, T. (1990) Nucleotide- sequence and cloning in Bacillus subfilis of the Bacillus stearothermophilus pleiotropic regulatory gene degT. J Bacterioll72: 41 1-418.

Thorson, J.S., Lo, S.F., and Liu, H.-W. (1993) Biosynthesis of 3,6-dideoxyhexoses: new mechanistic reflections upon 2,6- dideoxy, 4,6-dideoxy, and amino sugar construction. J Am Chem Soc 115: 6993-6994.

Thorson, J.S., Lo, S.F., Ploux, O., He, X., and Liu, H.-W. (1 994) Studies of the biosynthesis of 3,6-dideoxyhexoses: molecular cloning and characterization of the asc (ascar- ylose) region from Yersinia pseudotuberculosis serogroup VA. J Bacterioll76: 5483-5493.

Towbin, H., Staehelin, T., and Gordon, J. (1979) Electro- phoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nafl Acad Sci USA 76: 4350-4354.

Wang, L., and Reeves, P.R. (1994) Involvement of the galactosyl-1 -phosphate transferase encoded by the Sal- monella enterica rfbP gene in 0-antigen subunit pro- cessing. J Bacteriol 176: 4348-4356.

Watanabe, M., Takimoto, H., Kumazawa, Y., and Amano, K.I. (1 990) Biological properties of lipopolysaccharides from Bordetella species. J Gen Microbiof 136: 489-493.

Wigley, D.B., Derrick, J.P., and Shaw, W.V. (1990) The serine acetyltransferase from Escherichia coli. Over- expression, purification and preliminary crystallographic analysis. FEBS Letts 277: 267-271.

Zhou, D., Lee, N.-G., and Apicella, M.A. (1994) Lipo- oligosaccharide biosynthesis in Neisseria gonorrhoeae: cloning, identification and characterization of the crl,5- heptosyltransferase I gene (rfaC). Mol Microbioll4: 609- 618.


the identification, cloning and mutagenesis of a genetic locus required for lipopolysaccharide...

Documents