environmental stress responses in lactococcus lactis · the stress response mechanisms of l. lactis...
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ELSEVIER FEMS Microbiology Reviews 23 (l999À 483-501
Environmental stress responses in Lactococcus lactis
Jan Willern Sanders I,*, Gerard Venerna, Jan Kok
Abstract
Bacteria can encounter a variet y of physical conditions during their life. Bacterial cells are able to survive these (oftenadverse) conditions by the induction of specific or general protection mechanisms. The lactic acid bacteriUffi Lactococcus lactisis widely used for the production of cheese. Before and during this process as weIl as in its natural habitats, it is subjected toseveral stressful conditions. Such conditions include oxidation, heating and cooling, acid, high osmolarity/dehydration andstarvation. In many environments combinations of these parameters occur. Understanding the stress response behaviour of L.lactis is important to optimize its application in industrial fermentations and is of fundamental interest as L. lactis is a non-differentiating Gram-positive bacterium. The stress response mechanisms of L. lactis have drawn increasing attention in recentyears. The presence in L. lactis of a nUffiber ofthe conserved systems (e.g. the heat shock proteins) has been confirmed. Some ofthe regulatory mechanisms responding to an environmental stress condition are related to those found in other Gram-positivebacteria. Other stress response systems are conserved at the protein level but are under control of mechanisms unique for L.lactis. In a nuffiber of cases exposure to a single type of stress provides resistance to other adverse conditions. The unravellingof the underlying regulatory systems gives insight into the development of such cross resistance. Taken together, L. lactis has aunique set of stress response mechanisms, most of which have been identified on the basis of homology with proteins knownfrom other bacteria. A nuffiber of the regulatory elements may provide attractive ~ols for the development of food gradeinducible gene expression systems. Here an overview of the growth limits of L. lactis iind the molecular characterization of itsstress resistance mechanisms is presented. @ 1999 Federation of European Microbiological Societies. Published by ElsevierScience B. V. All rights reserved.
Keywords: Lactococcus lactis; Lactic acid bacterium; Physical stress condition; Stress resistance; Gene regulation
Contents
484484484
lntroduction 1.1. Stress conditions
1.2. L.lactis
* Correspondin~ author. Tel.: +31 (10) 4606256; Fax: +31 (10) 4605383; E-mail: [email protected]
1 Present address: Unilever Research Laboratorium Vlaardingen, 01ivier van Noortlaan 120, 3133 AT Vlaardingen, The Neth
erlands.
0168-6445/99/$20.00 ((J 1999 Federation of European Microbiological Societies. Published by EIsevier Science B.V. AII rights reserved.
PIl: SO 168-6445(99)00017-0
MICROBIOLOGVREVIEWS
Department of Genetics, Groningen Riomolecular Sciences and Riotechnology Institute, University of Groningen, Kerklaan 30,
9751 NN Haren, The Netherlands
Received 9 December 1998; revised 31 March 1999; accepted 3 April 1999
484 J. w Sanders el al.1 FEMS Microbiology Reviews 23 ( 1999) 483-501
485485488490491492494494495495496498
2. Stress. 2.1. Heatshock 2.2. Low temperature 2.3. Osmoticstress 2.4. Oxidative stress and DNA damage 2.5. Low pH 2.6. Starvation. 2.7. Cross protection and global regulation .
3. Development of expression systems for L. lactis
3.1. Stress inducible gene expression systems
4. Concluding remarks References
Introduction making grass a likely source of inoculation of rawmilk [I]. Both on plant material and in industrialprocesses L. lactis faces a wide range of differentconditions such as extremes in temperature, pR, orosmotic pressure. The organism is subject to rela-tively high temperatures in soil or during 'cooking'in cheddar cheese production, in which the temper-ature is increased to the growth limiting levelof40°C. Low temperatures occur during storage of fro-zen starter cultures and during cheese ripening (8-16°C). Osmotic pressure can vary from very low inrain water to high in pressed cheeses containing 0.56M NaCI. Many of these different stress conditionswilloften coincide. This is, for example, the casewith carbohydrate starvation and acid stress, as sug-ar fermentation results in high levels of lactic acid.Dried cells in Iyophilized or spray dried starter cul-tures suffer from both osmotic and oxidative stress.Cells are exposed to both high temperatures and uvradiation in sunlight. Optimal conditions for growthare rare and require specific metabolic adaptationsand can, therefore, also be seen as a stress condition[2]. An increasing number of studies show that L.lactis, when confronted with dangerous environmen-tal conditions, can survive by the activation of spe-cific protection mechanisms.
Lactococcus lactis has been associated with foodproduction and preservation since ancient times.Nowadays, defined starter cultures of L. lactis areof great economic importance in the bulk productionof cheese. This organism plays a key role in the for-mation of flavour and texture of cheese and in itspreservation. From an industrial point of view it isimportant to select strains that perform well in fer-mentation and that resist adverse conditions qccur-ring during the fermentation process. In addition,such strains should survive storage and handlingprocedures that are cheap and convenient. In somefood products (e.g. fresh milk) the same L. lactis isregarded as a spoilage organism. The presence of L.lactis in food systems can be controlled by choosingspecific conditions, either to promo te lactococcalproliferation when desired or to prevent spoilage inproducts that need no lactococcal fermentation. Anumber of (physical) parameters can be manipulatedto controllactococci without changing the safety andnutritive value of the final product. Therefore, it isimportant to know which conditions are favourableand which are detrimental for the life of this organ-ism and which mechanisms are essential for its sur-vival under such conditions.
1.2. L. lactis
1.1. Stress conditions
L. lactis strains have for long been maintained in
milk and, therefore, have adapted to survival in that
environment. With the establishment of the dairy
industry the use of selected and defined cultures be-
came practice. Strains have been selected for rapid
acidification, proteolytic activity and resistance
A first insight into the stress resistance of Lacto-coccus is given by its 'natural' habitats and the ex-treilles of the conditions encountered. L. lactis isCoillillonly found in illilk, which is a nitrogen andcarbon rich substrate, and in various plant illaterials,
J. w: Sanders el af. / FEMS Microbiology Reviews 23 ( 1999) 483-501 485
reasons. Scientific interest is fuelled by the fact thatL. lactis is a mesophilic Gram-positive microorgan-ism with a relatively small genome (2.5 Mbp) [6] thatis unable to differentiate (by sporulation) in responseto stress conditions. Stress-induced gene expressioncan be very complex as regulation can take place atthe levelof transcription, translation or mRNAstability. In addition, regulation by post-translation-al modifications is possible. For example, the activityof a gene product may be influenced by its phospho-rylation state. Regulation can also take place by dif-ferential degradation of a protein. Key examples ofstress proteins which are controlled at these variouslevels are as [7] and CspA [8,9] in Escherichia coli.
The influence of stress conditions on L. lactis hasbeen studied globally by analyzing their effects ongrowth and total protein synthesis (see Table I)and by genetic analyses of known stress-related genes(summarized in Table 2). In the next subsectionsboth global effects and genetic studies will be dis-cussed for a number of stress conditions. Compari-son of the different studies in this field is hamperedby differences in the analysis techniques and by dif -
ferences in challenge conditions and incubation timesthat have been used to stress cells. Careful interpre-tation of studies that aimed at subjecting cells to asingle stress condition in a single experiment isneeded as the conditions used may have posed acombination of stresses to the cells. For example, asingle stress may be deleterious under standardgrowth conditions but may not be so at lower tem-peratures or in an anaerobic environment.
against bacteriophages and bacteriocins. The rela-tively small set of strains that is currently used is,thus, not specifically selected for stress resistancebut this may have been selected for indirectly. Thevarious studies discussed in this review either de-scribe L. lactis subsp. lactis or L. lactis subsp. cre-moris. The two subspecies have different stress resist-ance properties but these differences have onlyrecently been studied in more detail. Kim et al. [3]showed that the subsp. lactis is, in general, morerobust than subsp. cremoris with respect to acid re-sistance, bile salt resistance and freezing resistance.The fact that most subsp. lactis strains can grow at40°C and in the presence of 4% NaCl, whereas mostsubsp. cremoris strains do not, is used to phenotypi-cally distinguish the two [4]. Genotypic allalysis ofisolates from both industrial and natural sourceshave revealed that subsp. cremoris strains isolatedfrom nature can have a stress resistance phenotypethat is similar to that of the subsp. lactis [5]. Indus-trial subsp. cremoris strains probably have lost someproperties that are essential to survive in nature butnot or even disadvantageous for continuous subcul-turing in a dairy environment.
The aim of this review is to describe the currentinsight into the environmental stress response of L.lactis, and some other lactic acid bacteria. In addi-tion, some potential applications of this knowledgewill be discussed.
2. Stress
2.1. Heat shockThe stress responses of L. lactis have gained inter-
est in recent years not only because of the industrial
relevance of the organism but also for basic scientific When an L. lactis culture was shifted from its
Table 1Global response to environmental stress in L. lactis
Stress Conditions ReferenceNUffiber of proteins with
Enhanced expression Reduced expression
most17 (at 16°C)
majority>45most30
[10]
[30]
[28]
[83]
[12]
[27]
42°C, 5-25 min8°C, 1-10 hpH 5.5, 30 min3 h galactose and arginine exhaustiol) in CDMa2.5% NaCI, 10--40 min254 nm, 100 J/ml
Heat shockLow temperatureLow pHStarvationSaltUV light
acDM, chemically defined medium.
486 J w Sanders el af. / FEMS Microbiology Reviews 23 ( 1999) 483~501
Table 2Gelles induced by environmental stress in L. lactis
Gene Stress conditionFunction of protein lllductioll Allalysisfactor metbod
Reference
groESL
groES
chaperone
chaperone
heat shockheat shocklow pHUV lightheat shockheat shocklow pHheat shockheat shockheat shock
43°C, 15 min in GSAa42°C, 30 min in GM17apH 5.5, 30 min 1actic acid254 nm, 100 J/m237°C, 60 min in GM1742°C, 30 min in GM17pH 5.5, 30 min 1actic acid42°C, 10 min in wpa43°C, 15 min in GSA43°C, 15 min in GSA
10
12
3.8
2.3
3
4.9
2.4
3 ta 4
10
5
RNA
protein
protein
protein
protein
protein
proteinRNA
RNA
RNA
[14]
[29]
[29]
[29]
[23]
[29]
[29]
[15]
[14]
[14]
groEL ;haperone
dnaJ chaperone
ORFllhrcA llegative regulator
of heat shock gelles
grpE chaperolle heat shockheat shockheat shockheat shocklow pHheat shocklow pHsaltheat shock
43°C, 10 min in GSA37°C,60 min in GM1743°C, 15 min in GSA37°C,60 min in GM17pR 5.5, 30 min 1actic acid43°C, 15 min in GSApR 5.1, 10 min RC12.5% NaC1, 10 min43°C, 10 min in GSA
RNA
proteinRNA
protein
proteinRNA
RNA
proteinRNA
5
3.7
100
3.4
2.1
10
3
4
+b
[14]
[23]
[14]
[23]
[28]
[18]
[18]
[12]
[14]
chaperone
c/pP protease
ftsH/hfiB regulator of heat shock
responseheat shock 37°C,60 min in GMI7low pH pH 5.1, 15 minDNA damage 0.01% methylmethanesulfonate, or
I /.lg mi-l mitomycin Coxidation aerated cultureDNA damage 0.01% methylmethanesulfonate, or
I /.lg mi-l mitomycin Coxidation aerated cultureoxidation aerated culturecold shock 10°C, I h in GMI7cold shock 10°C,4 h in GMI7cold shock 10°C,2 h in GMI7cold shock 10°C,4 h in GMl7low pH growth at pH 5.2 vs. pH 7.0low temperature growth at 15°C vs. 30°Cchloride growth with 0.5 M NaCl vs. no NaCIlow pH growth with 0.3 M NaCl, in non-
buffered vs. buffered M17, pH 4.2 vs.pH 5.5
aGSA, glucose SA medium [102]; GMI7, glucose MI7 broth; WP, whey permeate.bUninduced expression level below detection limit.c+ , not quantified.
dDetermined from a lacZ transcriptional fusion.
5.2
5 ta 6
3 ta 5
[23]
[86]
r571
protein
RNA
RNArecA SOS regulator
DNA repairsas regulator
RNARNA
[57]
[57]fpg
+c
3 ta 5
DNA repair
02 scavengingRNA stabilization
RNA stabilization
RNA stabilization
RNA stabilization
RNA
reporterdRNA
RNA
RNA
RNA
reporter
reporter
reporter
reporter
[57]
[51]
[41]
[41]
[41]
[41]
[81]
[81]
[49]
f491
+c
2
10
40
8
30
50
7.5
1000
10
sodA
cspA
cs pB
cs pC
cspD
Pl70
gadCB acid stress resistance
optimal growth temperature of 30°C to 42°C cellsstopped growing [10]. Growth resumed af ter ashift-down to 30°C with the growth rate reachingthe preshock level af ter 1 h at 30°C. Cells werehardly capable to recover from exposure to 50°C
for 30 min. In this case growth only resumed af ter48 h. The number of viabIe cells was slightly reducedaf ter a 30 min heat shock at 42°C but was more than1000-fold reduced by incubation at 50°C. However,pretreatment at 42°C for 10 min resulted in survival
w Sanders el al.1 FEMS Microbiology Reviews 23 ( 1999) 483-501 487
of more than 10% upon heat shock at 50°C [10].Increased expression of 12 to 17 proteins was ob-served upon exposure of L. lactis to 42°C [10-12].This phenomenon has been observed in organisms asdiverse as plants, anilIlals and bacteria. Detailed im-munological and genetic analyses have confirmed thepresence of the conserved heat shock proteins(HSPs) DnaK, DnaJ, GroEL, GroES and GrpE inL. lactis. These HSPs are protein chaperones thatfunction in folding and maturation of new or dena-tured proteins [13]. The organization of the genesencoding these proteins in L. lactis is partially differ-ent from other bacteria. Whereas in most cases dnaKand dnaJ form one operon, lactococcal dnaJ is tran-scribed independent I y from dnaK [14,15]. dnak ispart of a gene cluster that is also found in otherGram-positive bacteria and consists of ORF1(hr-cA)-grpE-dnaK [16], foliowed by a putative transcrip-tion terminator and ORF4 that is not induced athigh temperature [14]. A dnaK mutant (dnaK~l,lacking 174 out of 607 amino acids from the C-ter-minus of the protein) shows a clear temperature sen-sitivity phenotype [17]. It grows at a reduced rate at30°C, whereas it is unable to grow at 33°C or highertemperatures. The mutant strain is still able to ac-quire thermotolerance by preincubation at 40°C,although less efficiently than the wild-type.
Arnau et al. [14] integrated data from severalgroups by analyzing the expression of seven genesat the RNA level in response to a standardizedheat shock in a single strain. They showed that af ter15 min at 42°C dnaK-specific mRNA levels in L.lactis MG1363 had increased 100-fold and that itrepresented the single dnaK transcript. hrcA- andgrpE-specific mRNAs were of similar size and thelevels of both were 5-fold higher af ter 10 min ofheat shock, indicating that these genes are tran-scribed together. On the basis of the nucleotide se-quence, a promoter could be identified upstream ofhrcA (see below) but not upstream of dnaK [16]. Thediscrepancy between this observation and the ob-served lengths of the mRNAs may be explained byefficient processing of a single messenger co veringhrcA-grpE-dnaK [14] and differential stability of theprocessing products. Indeed, larger mRNAs could bedetected both with grpE and dnaK probes. A groELprobe hybridized with a 2.2 kb transcript that was10-fold induced af ter 15 min. This transcript may~
also include groES. The mRNA levels of dnaJ,dnaK, ORFl and grpE had decreased significantly20 min af ter the onset of the heat shock. Only thelevelof groEL transcript was lower at this time thanits normallevel at 30°C. The expression of heatshock gelles upon induction in rich medium was gen-erally lower and faster than in a defined medium[14]. The temporal induction of the HSPs af ter heatshock was monitored by pulse labelling of stressedcells and separation by 2D-PAGE [12]. DnaK pro-duction is induced 35-fold in the first 10 min of heatshock and production declilles thereafter, whereasGroEL and GroES are produced at levels 45- and35-fold over unstressed levels, respectively, in thefirst 10 min and synthesis contillues at a high levelin the following 30 min. Five other unidentified pro-teins were induced more than 10-fold during the first10 min of heat shock. A second group of nine un-identified HSPs was induced to lower levels: theirexpression levels continued to increase during thefirst 30 min of heat shock at 43°C to levels rangingfrom 2- to 8-fold higher [12]. One of these has beenidentified as the protease ClpP [18]. ClpP is, mostlikely, involved in the degradation of misfolded pro-teins, as a clpP mutant has a reduced capacity for thedegradation of puromycyl-containing polypeptides.The mutant strain expressed higher levels of theheat shock chaperones when grown at 30°C, whichis an indication for the accumulation of unfoldedproteins due to reduced proteolytic activity. TheclpP mutant is unable to form colonies at 37°C.clpP mRNA levels increase up to 10-fold within5 min af ter a shift to 43°C. A number of putativegelles induced at 43°C were found using a probemade by RNA subtractive hybridization. These in-cluded transposase gelles and putative L. lactishomologs of the cell division gene ftsZ, the heatshock gene hsp86, and the gene asp23 encoding analkaline shock protein, as well as a gene of which theproduct showed homology to transcriptional repress-ors of the deoR-like family [19].
No central regulator for the heat shock responsehas been identified so far. Most attention has beenfocussed on the regulation of the expression of thechaperone operons. The gelles hrcA, dnaJ andgroESL are preceded by vegetative promoters andhighly conserved repetitive sequence elements, con-sisting of two 9 b p inverted repeats separated by a
488 J w Sanders el af. / FEMS Microbiology Reviews 23 ( 1999) 483-50.
9 b p loop. These CIRCE (controlling inverted repeatof chaperone expression) elements are present up-stream of heat shock gelles in 27 different bacterialspecies [20]. They have a regulatory role asnegatively acting cis-elements, as deletion of theCIRCE upstream of the L. lactis dnaJ promoterresulted in temperature-independent expression of adnaJ: :amyS fusion [15]. From studies in B. subtilis itis known that HrcA is a negative regulator of dnaKand groE and that HrcA can bind to CIRCE ele-ments [20]. Experiments in B. subtilis support a reg-ulatory mechanism in which the levels of activeHrcA are modulated by the chaperone GroEL.GroEL is, thus, a negative regulator for chaperoneexpression and functions as the molecular thermom-eter [21]. Heat shock and ethanol lead to increasedlevels of non-native proteins that titrate the levels ofGroEL, thereby reducing the capacity of GroEL toactivate the repressor protein HrcA [22]. DnaK of B.subtilis plays no role in the modulation of chaperoneexpression, unlike E. coli DnaK. However, recentexperiments show that in L. lactis the levels of theheat shock mRNAs hrcA, dnaK, and dnaJ and thelevels of the heat shock proteins GroEL, GroES,Hsp84, Hsp85 and Hsp100 are higher in the dnaKL\lmutant than in the wild-type strain [17]. Levels ofhfiB mRNA (encoding the HflB protease, see below)were not elevated in this mutant. These data suggestthat DnaK may be the central sensor for denaturedproteins in L. lactis and can modulate chaperoneexpression via HrcA. Additional factors are likelyto act on HrcA to exp1ain the remaining capacityof the dnaK~l mutant to develop thermotoleranceby preadaptation and to elicit a low level heat shockresponse. Alternatively, these properties may also beascribed to the N-terminal part of the DnaK proteinthat is still expressed in this mutant strain.
Another regulator of heat shock gelles in L. lactismay be RecA. A culture of a recA mutant shifted to37°C showed a lag of 8 h before growth resumed at alow rate [23]. Two- to 3-fold reduced levels of DnaK,GroEL and GrpE were observed in the recA strainand induction of these proteins by heat shock wasdelayed. In E. coli, the expression of most heat shockgelles is controlled by the transcription factor 0"32.The levelof 0"32 is negatively regulated by HflB,which is also an HSP. An analogue of HflB (alsocalled FtsH) has been identified in L. lactis [24]~
and its expression is induced at high temperature[23]. An L. lactis hflB mutant was unable to growat elevated temperature (38°C) [24]. RflB levels were3-fold higher in the recA strain at both 30°C and37°C. The reduced levels of the RSPs in the recAmutant may be caused by the higher levelof RflBin this strain. In a model based on these observa-tions, RecA is proposed to regulate the heat shockresponse via RflB that, in turn, may govern thestability of an unknown positive factor which regu-lates RSP expression, as is the case in E. coli [23,25].Rowever, at present there are no indications of theexistence of such a positive factor, either or not asigma factor, in L. lactis. Therefore, it is still unclearwhat other control circuits regulate RSP expressionin L. lactis and what the exact roles of RecA andRflB are. Some genes that may be involved in thecontrol of heat shock resistance were identified byscreening for heat resistant mutants of a recA strain[26]. Among these were deoB, guaA, hpt, pnpA, pstB,tktA, and trmA. Mutation of deoB, guaA, hpt, andtktA also conferred heat resistance in a wild-typebackground. These four genes play a role in nucleo-tide synthesis or uptake. The expression of the RSPsis also induced under other stress conditions such aslow pR, salt, and uv radiation (Table 2) [12,27-29],but not by phage infection [10]. Induction of RSPsby uv light (see below), together with the data dis-cussed above, point to the relevance of recA in RSP
expression.
2.2. Low temperature
Whereas growth at high temperatures is deleteri-ous to a cell, growth at low temperatures merelyslows down biological processes. The doubling timeof a lactococcal culture increased from 48 min at30°C to 3! h, 57 h and 7 days at 16°C, 8°C, and4°C, respectively [30]. No lag period was observedaf ter a temperature downshift. The survival of L.lactis increased at low temperature. Survival of bac-teria first grown at 30°C to stationary phase andsubsequently incubated at 4°C for 28 days was30%, whereas 0.03% of the culture survived whenheld at 30°C throughout the experiment [30]. Simi-larly, survival from a freezing-thawing cycle was bet-ter (95%) af ter preincubation at 8°C for 48 h thanwithout such an adaptive treatment (75%) [31]. Pre-
w Sanders el al.1 FEMS Microbiology Reviews 23 ( 1999) 483-501 489
detectab!e phenotype and was shown to be compen-sated by the enhanced expression of the remainingcsp genes [39]. Doub!e mutants showed a severe re-duction in ce!!u!ar growth (both at 37°C and at15°C), stationary phase surviva! and a deregu!atedprotein synthesis. A minimum of one csp gene isessentia! for viabi!ity of B. subtilis.
Recent!y, members of the CSP fami!y were identi-fied in L. lactis [32,40,4!]. A set of primers designedon the basis of conserved regions in the known CSPswas used to amp!ify an interna! fragment of a !acto-cocca! csp gene by P CR. This c!oned P CR fragmenthybridized with four chromosoma! HindIIl restric-tion fragments [4!]. Three of these were c!oned andrevea!ed the presence of five csp genes, named cspA,cs pB, cs pC, cspD and cspE. cspA and cs pB werepresent on one fragment and were separated by360 bp. cs pC and cspD were a!so c!ose!y !inked.The !inked csp genes are transcribed in the samedirection. A simi!ar organization was found for E.coli cs pB and cs pG, which are divergent!y transcribedfrom the same genomic region as cs pF and cspH,respective!y [36]. The deduced amino acid sequencesof the five L. lactis genes show 45 to 65% identity toE. coli CspA and B. subtilis Cs pB. The !actococca!CSPs have 52 to 85% identica! amino acid residues.Expression of cspA is about 10-fo!d higher af ter 1 hof incubation at 10°C whereas cs pB expression is 40-fold induced af ter 4 h at 10°C, as was shown byNorthern hybridization. Expression of cs pC is 8-fold higher af ter 2 h at !ow temperature and cspDexpression reaches an optimum of 30-fold induction4 h af ter a shift to !ow temperature (10°C). cspEexpression appeared to be temperature independent[41]. Al! transcripts were about 300 nucleotides (nt)in length, indicating that al! genes, including cs pBand cs pC, are monocistronic. The start points ofthe csp mRNAs have been mapped. The nucleotidesequences of the approximately 87 nt non-translatedleaders of the messengers of cspA, cs pB, cs pC andcspD are high!y conserved. The leader of the non-cold-induced cspE shows much more differences.This may point to a regu!atory function of the !ead-er, as is the case for the 5'-end untranslated region ofthe E. coli cspA messenger [42]. The simi!arity in theprimary structure and the pIs of CspA and Cs pCcompared to those of Cs pB and CspD as wel! astheir simi!arity in !evels and time spans of expression~
incubation at 16°C or 4°C did not increase freezing-thawing survival significantly. Survival from freez-ing-thawing cycles was lower when cells were incu-bated in a 0.85% NaCl solution instead of culturemedium [32]. Log phase cells are more resistant tofreeze-thaw than cells in the stationary growth phase[32]. L. lactis subspecies lactis strains were shown toacquire freezing resistance by preadaptation at lo°Cwhereas strains of the subspecies cremoris are equallyfreezing sensitive with or without preadaptation [3].Surprisingly, preincubation at 8°C for 48 h improvedsurvival of a 30 min challenge at 52°C 60-fold com-pared to a non-adapted culture [31]. Incubation atlow temperature resulted in the induction of a spe-cific set of 12 proteins in L. lactis [30]. The levelofinduction depended on the incubation time and tem-perature. The maximum observed overexpression ofone of the proteins was 10-fold after 1 h at 8°C whencompared with 30°C.
After a cold shock treatment, E. coli and B. sub-tilis are known to overexpress several proteins (re-viewed by Jones [33], Graumann [34,35] and Yama-naka [36]). Research on cold shock responses wasfocused on a family of small (7 kDa) highly con-served cold shock proteins (CSPs), three of whichwere identified in B. subtilis and nine in E. coli[34]. All three B. subtilis CSPs are cold inducedwhereas this is only true for four of the nine CSPsof E. coli. Some of the other E. coli CSPs are in-duced upon starvation (CspD) or are expressed con-stitutively (CspC and CspE). Expression of one ofthe cold-induced proteins, CspA from E. coli, wasshown to be induced 200-fold at low temperatureby a combination of increased transcription, stabilityand translation efficiency of cspA mRNA [8,9]. CspAand its relatives are thought to have a role as RNAchaperones. CspA was shown to bind RNA andssDNA with a broad sequence specificity and in-creases the susceptibility of RNA to RNases [37].Therefore, CspA was suggested to prevent the for-mation of secondary structures in RNA molecules atlow temperatures and in that way to stimulate trans-lation efficiency. The first csp deletion mutant de-scribed, cspB of B. subtilis, showed a defective coldshock response and a reduced induction at low tem-perature of 15 proteins, suggesting a regulatory func-tion for cspB in the cold shock response [38]. Dele-tion of B. subtilis cspC or cspD did not lead to a~
490 J. w Sanders el at I FEMS Microbiology Reviews 23 (1999 ) 483-501
in response to cold shock suggest that CspA andCspC may have a similar role in the cell. Possiblythey are involved in the induction of cspB and cspD,their respective neighbours on the chromosome [41].On the basis of amino acid sequence similarity andstructural conservation it is likely that the L. lactisCSPs function as RNA chaperones in a way similarto E. coli CspA. PCR studies using degenerate prim-ers suggest the presence of csp homologs in Lacto-bacillus helveticus, Pediococcus pentosaceus andStreptococcus thermophilus [32]. From another lacticacid bacterium, Lactobacillus plantarum, the two cspgenes cspL and cspP have been cloned and se-quenced [43]. The 66 amino acid proteins encodedby these genes differ by only eight amino acids andshow about 66% identity to E. coli CspA. Two dis-tinct transcripts of 330 and 760 nt were detected witha cspL-specific probe, whereas a single 330 nt RNAwas detected with a cspP-specific probe. A 3- to 5-fold increase of all three transcripts over the basallevel at 37°C was observed after 1 h at lo°C. Mes-senger RNA levels declined to preshock levels within1 h after return to 37°C.
2.3. Osmotic stress
Bacterial cell envelopes are perrneable to water .Therefore, an increase in the osmolarity of thegrowth medium would result in rapid efflux of waterfrom the cytoplasm. To retain water in the cell and,thus, to maintain turgor pressure, bacteria have sys-tems to accumulate specific solutes that do not inter-fere with cell physiology [44]. Such compatible sol-utes are either taken up from the environment ornewly synthesized in the cytoplasm; the uptakemechanism is found in most lactic acid bacteria.One of the compatible solutes mostly used by bac-teria is glycine betaine Cbetaine). Growth of L. lactiswas shown to be inhibited at high salt concentra-tions: 2.5% NaCl Ca level close to that in somecheese types) reduces the growth rate to 25 to 50%of the unstressed rate [12]. Growth under these con-ditions was stimulated considerably by the presenceof betaine in the medium [45]. Cells grown in C DMin the presence of 0.5 M KCl accumulated high levelsof proline and, to a lesser extent, aspartate. In thesame medium in the presence of betaine, cells accu-mulated aspartate, glutamate and betaine but no
proline. L. lactis has a high affinity uptake systemfor betaine that is constitutively expressed. In addi-tion, a low affinity proline uptake system was alsoactive but only so in a chemically defined medium(C DM) of high osmolarity and not in rich media orin the absence of KCl. Proline transport was inhib-ited by betaine and exchange of proline for betainewas also observed, suggesting that the proline trans-port system may also transport betaine. This wouldexplain the observed absence of proline in the cYto-plasm when betaine is present in the medium. Up-take by both transport systems is energy dependentbut, most likely, not driven by the proton motiveforce [45]. Information on the regulation or the ge-netic determinants of these systems is not available.Growth of L. plantarum at high osmotic pressure isstimulated by betaine, which is the preferred osmo-lyte of this organism [46]. Levels of alanine, gluta-mate, proline, and glycine increased 3-, 6-, 35-, and48-fold, respectively, upon growth in C DM with 0.8M KCl compared to low osmolarity medium, withglutamate and proline being the most abundantlyaccumulated amino acids. The four amino acids ac-cumulated to lower levels when betaine was presentin the high osmolarity medium. Betaine transportwas increased 3-fold in cells cultured in high osmo-larity medium, suggesting enhanced expression of tht;transport system. Uptake rates also increased whenthe osmolarity of the assay buffer was raised, whichpoints towards activation of the transport protein.Effiux of betaine upon osmotic downshock dependedon the post-shock osmolarity of the medium. Sepa-rate transport systems were postulated for the uptakeand effiux of betaine [47].
Analysis of protein production by 2D-PAGE re-vealed that total protein synthesis dropped to about50% of the preshift level in cells stressed with 2.5%NaCl [12]. The synthesis of at least 12 proteins isinduced, their levelof induction varying from 2- to9-fold. The expression of one protein, labelled Ssp21,gradually increased 35-fold during 40 min af ter stressinduction. Most of the salt-induced proteins identi-fied were also induced by heat shock. The heat shockproteins DnaK, GroEL and GroES were induced 5-to 9-fold within 10 min at 2.5% NaCl, resemblingtheir induction by heat shock, although at a lowerlevel. ClpP was induced 4-fold af ter 10 min at 2.5%NaCl [12].
I. w Sanders el al. / FEMS Microbiology Reviews 23 ( 1999) 483-501 491
In a search for environmentally regulated genes inL. lactis an NaCl inducible promoter structure wasidentified that was independent from the mediumosmolarity but required chloride for induction [48].The two genes transcribed from this promoter func-tion in low pR survival. One of these, gadC, maycode for a glutamate-y-aminobutyrate antiporterbut the involvement of the genes in osmoregulation,if any, is still unclear [49].
An L. lactis hjlB mutant is unable to grow in M17medium in the presence of 4% NaCl and grows veryslowly in l% NaCl whereas growth of the wild-typeis only reduced at the high NaCl concentration [24].Interestingly, the pattern of membrane-associatedproteins in the hjlB mutant is different from that ofthe wild-type, suggesting improper assembly of mem-brane proteins, some of which may be essential forsalt tolerance. hjlB expression is not induced at high
osmolarity [24].
2.4. Oxidative stress and DNA damage
L. lactis is an obligatory fermenting bacterium. It
can tolerate oxygen as its growth is not affected by
aeration [50,51]. Cells are able to use oxygen in the
presence of a carbon source by a closely coupled
NADH oxidase/NADH peroxidase system. This is
an alternative way to regenerate NAD+, next to
the conversion of pyruvate to lactate or ethanol.
pyruvate is then available for conversion to acetate,
y ielding extra ATP. Therefore, aerobic fermentation
produces different (amounts ot) end products as
compared to anaerobic fermentation [50,52]. The ac-
tivity of NADH oxidase/NADH peroxidase is about
5-fold higher in galactose grown aerated cells than in
non-aerated cells. This enzyme activity may generate
the highly toxic oxygen intermediate superoxide
(02). Two-fold higher levels of superoxide dismutase
(which removes 02) were found in aerated cultures
[51,52]. The toxicity of 02 is illustrated by the mark-
edly reduced growth rate of a superoxide dismutase
(sodA) mutant during aerobic growth [51]. The re-
maining systems can still cope with 02 to a certain
extent. Alternative reducing capacity may be pro-
vided by glutathione of which a relatively high level
is present in L. lactis [53]. A number of L. lactis
strains were reported to accumulate glutathione
[54]. Accumulation is medium dependent [54] andrelies Dn transport froll the environment [55].
Bath NADH oxidase and superoxide dislliutaseproduce the toxic compound H2O2. L. lactis hasno catalase and depends solely Dn NADH peroxi-dase ta keep H202 levels at subinhibitory concentra-tions. At sublethal H2O2 levels, L. lactis develops anadaptive response against Iethal concentrations ofH2O2 [56]. The L. lactis recA gene clearly plays arole in oxidative stress. Expression of recA was in-duced in aerated cultures and a recA mutant ishighly sensitive ta aeration, as evidenced by a lowergrowth rate and reduced viability during stationaryphase [57]. The doubling time of an aerated recAculture is restored ta that of a non-aerated cultureby the presence of catalase Dr the Fe2+ chelator fer-rozine in the medium, while catalase also illprovessurvival. This observation points ta the involvementof hydroxyl radical (O OH), the most reactive oxygenspecies, because this is formed in the H2O2- andFe2+-dependent Fenton re action (H2O2+Fe2++H+ ---'; °OH+H2O+Fe3+) [57]. °OH may be the causeof the observed higher rate of DNA dallage in aer-ated cells as compared ta standing L. lactis cultures[23]. In E. coli, 02 caused an increase in the intra-cellular paolof free iron, which promoted the rate atwhich H2O2 caused DNA dallage [58].
RecA is the key protein in the SOS response taDNA dallage and in holliologous recombination inE. coli. In analogy , recombination in L. lactis recAwas more than 104-fold lower than in the wild-typeand the mutant was sensitive ta DNA dallage in-duced by uv light, mitolliycin C ar methyl methallesulphonate [23]. The latter compounds also inducean increase in the levelof recA mRNA {57], but anincrease in the RecA protein level does nat occurupon introduction of DNA dallage [59]. recA in L.lactis is cotranscribed with a gene encoding the DNArepair enzyme formallidopyrillidine DNA glycosyl-ase ((pg) [57]. Like its E. coli counterpart, F pg frollL. lactis has DNA glycosylase activity and can nickDNA at abasic sites. Furthermore, it suppresses anE. coli mutator phenotype [60].
A nulliber of gelles that are in same way involvedin the DNA dallage response were found by ISSlmutagenesis. Mutants defective in the rexAB gelleswere identified by their lack of exonuclease activity;
492 J w Sanders el al I FEMS Microbiology Reviews 23 ( 1999) 483-501
these are more UV sensitive than wild-type cells andare recombination deficient [61]. This points to a rolefor rexAB (encoding the lactococcal recBCD-likeexonuclease) in resisting DNA damage. Other UVand mitomycin C sensitive mutants contained lesionsin genes (identified on the basis of homology) in-volved in DNA metabolism (hexB, polA, anddeoB), cell envelope formation (gerC and dltD) andvarious metabolic pathways [62]. hexB, polA, anddeoB mutants were sensitive to both low and highdoses of UV (10 and 50 J/m2 at 260 nm, respec-tively), whereas the other mutants only showed sen-sitivity to high doses. The strain lacking the putativemismatch repair system HexB permitted recombina-tion between DNA molecules with mismatches andthe occurrence of spontaneous mutations at a higherfrequency than the wild-type. polA encodes the bac-terial DNA polymerase I. Consequently the L. lactispolA strain could not support replication of a thetareplicating plasmid. DeoB is part of a salvage path-way for purines and pyrimidines. The UV sensitivityof the deoB mutant was relieved by the presence ofnucleotides in the culture medium. Although thefunction of the other genes in uv resistance remainsto be elucidated, this set of mutants strongly suggeststhat UV resistance, apart from DNA repair, is medi-ated by other mechanisms.
The lactococcal lacX and lacN genes were foundto complement an E. coli recA strain sensitive forsome mutagens. LacX and LacN may be a sensorand response regulator of a two component regula-tory system with a function in the SOS response, buttheir function in L. lactis is unknown [63].
2.5. Low pH
Lactic acid bacteria produce lactic acid duringsugar fennentation. This implies that they are fre-quently confronted with acid stress. It is importantto note that lactic acid is a weak organic acid that isnot charged at low pH and can easily pass the cellmembrane in the protonated form. At cytoplasmicpH, it dissociates and, thus, poses a stronger stressto cells at a given extracellular pH than for examplehydrochloric acid [64]. The intracellular pH of L.lactis cells in suspension was slightly reduced (from7.0 to 6.0) when the extracellular pH was reducedwith HCl (from 6.75 to 5.0) [65,66] but decreased
linearly (from 7.0 to 5.25) with the extracellular pHwhen that was adjusted with lactic acid [65]. Whenmeasured in growing cultures, the intracellular pHdecreased with the extracellular pH. In this way aconstant t1pH of 0.7 units was maintained [65]. L.lactis can resist pH 4.5 (with HC1) in minimal me-dium, but its viability rapidly decreases by incuba-tion at pH 4.0 [59]. Kim et al. [3] reported that L.lactis subsp. lactis strains can survive a pH as low as2.5 (in M17 with HC1) and that the subsp. cremoriscan resist a pH of 3.0. Cells can survive a low pHwhen adapted to a sublethal pH (5.5) for only 15min, both in a defined medium adjusted with HCl[59] or in M 17 medium with lactic acid [28]. In an-other study [3], three exponentially growing and pre-adapted L. lactis subsp. lactis strains were shown toresist exposure to a lethal pH (2.5) whereas a largepercentage of cells of three subsp. cremoris strainsdid not survive a lethal pH of 3.0. Stationary phasecells of both subspecies survived a lethal pH equallywell. Acid adaptation was shown to be chloramphen-icol sensitive in L. lactis subsp. cremoris MG 1363[59] but chloramphenicol independent in L. lactissubsp. lactis IL1403 [28]. Therefore, the latter strainseems to be acid resistant without the need for denovo protein synthesis. Incubation at pH 5.5 for 30min triggered the synthesis of 33 proteins, amongwhich DnaK, GroEL [28] and ClpP [18].
A number of mechanisms have been shown toconfer acid resistance. The primary mechanism forcontrol of intracellular pH is the F oF 1 A TPase thattranslocates protons to the environment at the ex-pense of A TP .Both the expression level and theactivity of this protein complex are increased atlow pH [67,68]. An acid sensitive L. lactis mutantwas isolated that is unable to maintain a neutralintracellular pH in an acidic environment [69]. Theacid sensitivity of this mutant was shown to becaused by a mutation in the A TPase structuralgene resulting in a reduced enzyme activity. ThemRNA levels and F 113 protein levels in this mutantwere elevated at low pH, as is the case in the wild-type strain.
A second mechanism for pH homeostasis is thearginine deiminase (ADI) pathway. This pathway al-lows L. lactis to neutralize its environment by NH3production [70] and the concomitant A TP generationenables extrusion of cytoplasmic protons by the
J. w Sanders el al.1 FEMS Microbiology Reviews 23 ( 1999) 483-501 493
FoFl ATPase. The AD! pathway consists of threecytoplasmic enzymes: arginine deiminase, ornithinecarbamoyltranferase and carbamate kinase, thatcatalyse the reaction: arginine+R2O+ADP+Pi ~ornithine+CO2+NR3+ATP. These enzymes areactive at low pR (2 to 3) in several Streptococcusspecies [71]. Arginine and ornithine are exchangedwithout the need for metabolic energy by a mem-brane located antiporter. The activity of the pathwayis induced 3- to 5-fold in the presence of arginine[72]. The presence of the AD! pathway is a uniquephenotypic property of L. lactis subsp. lactis [4], as itis only rarely observed in the subsp. cremoris. Arelationship between the presence of the AD! path-way and higher salt and temperature tolerance in thesubsp. lactis is likely as AD!+ transductants of thesubsp. cremoris showed increased stress tolerance[73]. Recently, genetic analysis of the AD! pathwayin Lactobacillus sake revealed the presence of fivegenes (arcAECTD) encoding the four componentsof the pathway and a putative transaminase gene(arcT) [74]. Transcription of this pathway is inducedby arginine and repressed by glucose.
L. lactis expresses a glutamate-dependent acid re-sistance mechanism in the presence of chloride[48,49]. The systern is encoded ' by an operon consist-
ing of two genes, gadC and gadE, which specify aputative glutamate-y-aminobutyrate antiporter and aglutamate decarboxylase, respectively. The combinedaction of these two proteins may confer acid resist-ance by the removal of a proton frorn the cytoplasmand the export of y-aminobutyrate which is morebasic than the imported glutamate. Alternatively, itmay also result in the formation of a proton motiveforce as was shown for a Lactobacillus sp. that couldgenerate ATP in the presence of glutamate [=75]. Ex-pression of L. lactis gadCE in cultures that wereallowed to acidify further during growth, by theomission of buffer frorn the medium, was higherthan in buffered medium and gadCE expressionmay also depend on glutamate [49]. Similar mecha-nisms, based on the combination of amino acid anti-port and amino acid decarboxylation have been de-scribed for lactobacilli. An aspartate-alanineantiporter that could stimulate A TP formation wasdescribed in Lactobacillus subsp. M3 [76]. Lactoba-cillus buchneri is able to generate a proton motiveforce by histidine decarboxylation and electrogenic~
histidine-histamine antiport [77]. Interestingly, a his-tidine decarboxylase mutant of Lactobacil!us 30a wasunable to alkalinize its environment in the presenceof histidine [78], suggesting that these systems maysupport acid survival by restoring the pR to levelswhich permit growth.
Another acid stress response mechanism was re-cently described in L. lactis subsp. lactis biovar. di-acetylactis. In this organism the transcription of citP,encoding the citrate-lactate antiporter CitP, is in-duced at low pR [79]. The strain could grow to ahigh cell density when inoculated in growth mediumat pR 4.5 containing both glucose and citrate, where-as proliferation was very poor in the presence of onlyglucose or citrate, or in the absence of CitP. Thismay be explained on the basis of studies on citrolac-tic fermentation in Leuconostoc mesenteroides de-scribing the alkalinization of the growth medium inthe presence of citrate and the generation of energyby the electrogenic precursor/product antiport of cit-rate and lactate [80]. In conclusion, co-metabolism ofglucose and citrate provides selective advantage atacidic pR to cells expressing CitP .
In a study using random transposon insertion,clones were selected that expressed the transposonencoded lacZ reporter at a higher level in responseto low pR [81]. One of these, PA170, showed 50-foldhigher lacZ expression in a chemostat maintained atpR 5.2 than at pR 7.0. Expression was only observedin the stationary phase and was also induced 7-foldat 15°C compared to 30°C. Genetic analysis of thispromoter revealed that a 50 b p region is sufficient fortranscription initiation in response to all three induc-ing conditions [82]. The function of the gene tran-scribed from the Pl70 promoter is not known.
To study regulation mechanisms that control acidresistance, acid resistant ISSl insertion mutants in L.lactis were selected [59]. One of the genes identifiedin this way shows homology with ahrC, a regulatorof arginine synthesis and catabolism in B. subtilis,and may be involved in control of the arginine de-iminase pathway. A number of the other acid resist-ant mutants contained insertions in genes that haveroles in nucleotide metabolism and may act in thebiosynthesis of (p )ppGpp, the stringent response reg-ulator. This result points to the possible involvementof the stringent response in pR regulation in L. lactis
[59].
494 J. w: Sanders el af. / FEMS Microbiology Reviews 23 (1999 ) 483-501
2.6. Starvation ing conditions. This is of relevance for cells inenvironments where they can be exposed to combi-nations of stress conditions. The best example ofsuch a multiple stress resistance is that acquired inthe stationary phase. Also, exposure to uv light (100J/m2) conferred cross protection against a treatmentwith heat, acid or ethanol and, to a lesser extent,against R2O2 [27]. Another aspect of the same phe-nomenon was seen by total protein analysis in 20gels. uv challenge induced 14 proteins in L. lactis ofwhich four were also induced by other stress condi-tions. Similarly, nine of the 33 proteins that wereinduced at low pB were also induced by heat shock[28]. This is suggestive of an overlap between thecontrol circuits regulating responses to these stresses.Other evidence for the existence of such interaction isthe involvement of recA in both oxidative stress, andheat shock and the acid inducibility of hfiB [86],which is also implicated in heat shock response. In-terestingly, in a selection procedure for thermoresist-ant mutants of the thermosensitive L. lactis recAstrain, a number of the disrupted genes appearedto be the same as those found in selection for acidresistant mutants [59,29]. One of these (deoB) wasalso found by selection for UV resistant mutants[62]. These observations suggest a link between theheat shock response and acid resistance in L. lactis.A number of the genes found in these mutagenesisstudies are involved in metabolic pathways for gua-nine and phosphate. As these are related to(p)ppGpp synthesis, the stringent response may beinvolved. Rowever, there is no evidence for directcontrol of heat shock genes or regulators and acidresistance genes by the stringent response.
Another open question as to the control of stressresponses in L. lactis is the involvement of sigmafactors. Oespite several attempts to identify alterna-tive sigma factors, thus far only the vegetative sigmafactor has been described [87,88]. Control mecha-nisms in L. lactis are likely to resembie those in theGram-positive bacterium B. subtilis more than thoseof the Gram-negative E. coli. Indeed, remarkablesimilarities in the control of heat shock genes, includ-ing the CIRCE element and hrcA, have been foundbetween L. lactis and B. subtilis (see above). Anothergroup of B. subtilis genes involved in general stressresistance is controlled by (JB [20]. It would be inter-esting to elucidate whether such general stress pro-
Exhaustion of an essential nutrient limits growthof a culture which then enters the stationary growthphase. It is generally accepted that stationary phaseis the most common state of bacterial cells in nature.Glycolytic and arginine deiminase pathway activitiesdecline rapidly upon entry of L. lactis in the station-ary phase [83]. These activities could be restored bythe addition of sugar. Resumption of glycolytic ac-tivity was slower af ter a longer period in stationaryphase and was chloramphenicol sensitive. In con-trast, arginine deiminase pathway activity raised tothe prestarvation rate independent from the durationof starvation and required no protein synthesis. To-tal cell protein is degraded during exponentialgrowth and in the first 30-90 min of stationaryphase, but proteins were stably maintained after-wards [83]. Cells react to starvation by increasingthe levelof 14 proteins, despite the lack of metabolicenergy. Major changes were also observed in theprotein composition of the cytoplasmic membrane,but amino acid transport capacity was hardly af -
fected in the stationary phase [83]. Although L. lactisis unable to react to starvation by, for instance, thedevelopment of competence or the formation ofspores, it does develop multiple stress resistance.The first sign of such an adaptation was the obser-vation that lactose starved cells survived longer whencultured at a lower imposed growth rate before star-vation [84]. L. lactis appeared to be highly resistant,already at the onset of the stationary phase, to in-cubation at 52°C or pR 4.0, or to the presence of20% ethanol, 3.5 M NaCl or 15 mM R202 [85].Analysis of acid resistance of cells during growthrevealed that it is acquired during the late exponen-tial phase. The latter is not surprising as growth ofL. lactis always results in acid formation.
Genetic studies on starvation response have onlyjust started.
2.7. Cross protection and global regulation
Subjection to a mild stress makes cells resistant toa Iethal challenge with the same stress condition, ashas been discussed above for a number of stress con-ditions. Moreover, preadaptation to one stress con-dition can render cells resistant to other stress impos-
J. w Sanders et alt FEMS Microbiology Reviews 23 (1999 ) 483-501
teins and their regulator ()"B are present in L. lactis.The striking properties of recA in relation to heatshock response and oxidative stress suggest that con-trol of stress responses in L. lactis is markedly differ-ent from other bacteria, as has been noted for thecontrol of other cellul ar functions [89].
3. Development of expression systems for L. lactis
Another reason to study stress response mecha-nisms of L. lactis is related to its biotechnologicalpotential. As L. lactis has the GRAS status (gener-ally regarded as safe), it is an interesting tool for theproduction of new fermented food products or for insitu modification of foods. Strains with new traits,different from the ones currently known may be de-sirable. These can be obtained by the introduction ofgenes from non-lactococcal sources, thereby openingthe way for specific new applications. Genetically L.lactis is the best characterized species of the lacticacid bacteria. Sophisticated techniques for the genet-ic modification of L. lactis have been developed inthe last 15 years [90,91]. These include cloning vec-tors, integration systems, expression signals, and se-lection markers entirely based on lactococcal DNA.
The gene expression systems developed thus fardirect, with a few exceptions, constitutive gene ex-pression. However, for the expression oflethal geneproducts, inducible gene expression systems are in-dispensabIe. Such systems are also preferred in manyother cases in order, for instance, to obtain highyields of non-lethal proteins. The induction of cer-tain activities during an (industrial) process requiresexpression signals that allow tight control. The in-ducing signals should be food compatible andshould, thus, be either a safe food additive or achange in an environmental condition that occursnaturally or during the fermentation process, orcan be easily incorporated in the process. In otherwords, regulatory systems that respond to changes inthe environment that normally control stress adapta-tion mechanisms hold promise for use in food gradeinducible gene expression systems.
is regulated (reviewed in [89]). Only a few of thesedepend on stress conditions. The regulatory elementsinvolved that have been used for the controlled ex-pression of other genes will be discussed here.
Regulation signals of lactococcal heat shock geneshave been used for heterologous gene expression.Rowever, the levels of induction from these pro-moters upon heat shock were rather low [92,93]. Afusion of the dnaJ promoter with usp45: :amySshowed a 4-fold higher levelof secreted Bacillusstearothermophilus a-amylase activity 30 min af tera shift from 30°C to 42°C [15].
Pgod, the promoter of the lactococcal gadCB oper-on [49], can be induced more than 1000-fold bygrowth in the presence of 0.5 M NaCI. In addition,expression is induced at low pR. A positive regulatorencoded by gadR is indispensable for the chloride-dependent expression of gadCB. The P god promoterwas amplified together with gadR and this expressioncassette was inserted upstream of the lysis cassettelytPR of the lactococcal bacteriophage rIt and up-stream of acmA of L. lactis, the gene specifying thecell wall hydrolase. Induction of L. lactis carryingthese fusions with NaCI resulted in overexpressionof the cell wall degrading enzymes and in cell lysis,evidenced by the release of cytoplasmic enzyme ac-tivity [94]. The basal expression levelof the fusionswas very low and did not harm the cells.
Another way of obtaining a stress inducible pro-moter is by mutagenesis of a regulated promoterand/or its repressor that normally respond to a dif-ferent stimulus. This approach was chosen for theexploitation of the genetic switch element of the tem-perate lactococcal bacteriophage r 1 t. This element isnormally activated by DNA damage caused by uvradiation or by chemicals like mitomycin C and re-sults in a change in the life cycle of the phage fromthe lysogenic state to the to lytic phase. The switchelement consists of two divergently transcribedgenes, rro, encoding the phage repressor, and tec.Expression of the genes (including tec) of the lyticgrowth phase downstream of promoter P2 is re-pressed by Rro. When E. coli lacZ was placed on aplasmid downstream of P2, j3-galactosidase expres-sion was induced about 70-fold af ter addition ofI ~g mI-l mitomycin C [95]. Rro was shown tobind specifically to an operator sequence presenttwice in the promoter region and once in tec. In
Stress inducible gene expression systems
The expression of a nulliber of lactococcal operons
496 J. w Sanders el af. / FEMS Microbiology Reviews 23 (1999 ) 483-501
this way it shuts off expression of the P2 driven
genes. Recently, a temperature sensitive repressor
mutant (RroI2) was constructed by site directed
mutagenesis on the basis of comparative molecular
modelling. Promoter P2 driven expression of lacZ is
efliciently repressed by Rro12 at 24°C and is 600-fold
higher at 40°C [96].
4. Concluding remarks
The increasing number of studies on stress in L.lactis allows an overview of its stress response behav-iour and an opportunit y for comparison with otherorganisms. Various stress conditions and the cellularresponses to these conditions are summarized in Fig.I. Best studied is the response to heat shock. Inparticular, the insight into the mechanisms that con-trol the expression of the chaperone proteins in re-sponse to various conditions is increasing. In con-trast, almost nothing is known of the regulatorymechanisms controlling the responses to other typesof stress, including the induction of non-chaperoneheat shock proteins. The number of induced proteinsobserved in 2D-PAGE analyses indicates that muchmore gelles and proteins are involved in stress re-sponses than currently known and indicated in Fig.
Obviously, growth studies confirm that L. lactis isa mesophile. It is, however, able to survive moreharsh conditions, in particular when pretreatedwith a mild stress condition. It is questionablewhether the laboratory conditions and culture mediaare representative for survival in a natural situation,either in a milk ( derived) environment or on plantmaterial. The results of, for example, an acid resist-ance test differ considerably depending on the use forthe low pH challenge of either a buffer, a definedmedium, or a complex medium ( our unpublished ob-servations). Natural situations may confront L. lactisnot only with physical stress but also with toxic com-pounds. Interestingly, L. lactis appears to harbourspecialized multidrug transporters for the removalof certain classes of (putative toxic) drugs or metab-olites from its cytoplasm, as reviewed elsewhere [97].
It is no surprise that the systems employed by L.lactis for protection against various environmentalstress conditions are similar to those found in other
bacterial species and higher organisros. Several ofsuch mechanisros are not present in more closelyrelated Grall-positive species but were found inmore distant Grall-negative bacteria. Whereas thestress resistance systells found thus far are weIl con-served at the protein level, the underlying molecularmechanisros responsible for the timing and fine tun-ing of the expression of these proteins in response toenvironmental changes are in most cases rather dif-ferent and unique for L. lactis. A striking exallple isthe regulation of the chaperone protein expression,which depends on HrcA and DnaK and in whichalso RecA is involved. The latter was thus far seenas a key protein in horoologous recombination andDNA repair.
The mechanisros for sensing environmental condi-tions and the coupled cellular signalling routes arestill undefined. Regulation could be organized eitherglobally or be stress specific. There are no signs thusfar for the presence of multiple sigma factors such ashas been described for E. coli and, in particular, forB. subtilis. Only the gene for the vegetative sigmafactor, rpoD, has been found [87,88] and no deviat-ing promoter sequences for certain classes of stressgenes have been reported. Many sensors in otherspecies that monitor environmental conditions be-long to the class of two component regulatory sys-tells. These usually comprise a merobrane locatedhistidine protein kinase that senses a specific signaland transfers this by phosphorylation to a responseregulator. This protein, when phosphorylated, canactivate cellular processes, often at the levelof tran-scription. The two component regulatory systells inlactic acid bacteria characterized thus far regulatethe expression of bacteriocins by sensing the concen-tration of bacteriocin or a bacteriocin-like peptide intheir environment (reviewed in [98]). However, recentdata show that genes for other horoologs of histidineprotein kinases and response regulators are presenton the chromosome of L. lactis. The cellular proc-esses controlled by these systells and the conditionsthey respond to are still unknown [99].
The present knowledge on the growth lillits of L.lactis supports the insights gained froro centuries ofpractical use of the organisro in dairying. This formsa basis for the systematic evaluation of combinationsof different strategies to obtain optillal food preser-vation. The available data can be combined with the
498 J. w. Sanders el al. I FEMS Microbiology Reviews 23 '999) 483-50
underlying molecular mechanisms may provide tar-gets for specific manipulation to promote or preventlactococcal growth. For example starter cultures thatare stored frozen will have a higher viability whenincubated at 10°C prior to freezing. An alternativestorage procedure is to dry starter cultures. Survivalof the osmotic stress imposed by this treatment willbe promoted by preculturing the starter in the pres-ence of osmolytes. A number of studies [70-72,79,80]have made clear that the ability to ferment citrateand/or arginine has two advantages: it provides cellswith an alternative source of energy and gives pro-tection against low pR. This notion can be used forthe selection of strains and for the design of culturemedia. Stress-induced proteins are clear molecularmarkers for the fitness of starter cultures. These pro-teins indicate that a culture has been subjected to astress and, therefore, that the cells may not optimallyperform in a following large fermentation. On theother hand, these markers could also be used as pos-itive indicators for a culture that is fully adapted toresist an upcoming stress condition. Mutants affectedin a stress resistance mechanism, e.g. acid resistance,may proliferate only partially under certain condi-tions. These may be useful to control fermentation,for example to limit acidification of a culture. Other-wise, mutants with improved stress resistance proper-ties, for in stance due to a break in a negative controlcircuit, may survive adverse conditions that occur inindustrial processes.
Of particular interest for future work will be theprocesses operating in cells that enter the stationarygrowth phase in a low pR environment, as this is acondition commonly encountered by and unique forlactic acid bacteria.
References
[1] Sandine, W.E., Radich, P.C. and Elliker, P.R. (1972) Ecologyof the lactic streptococci. A review. J. Milk Food Technol. 35,176-184.
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