Review on Bacterial Stress Topics

ANNA MARIA GIULIODORI,a CLAUDIO O. GUALERZI,a SARA SOTO,b

JORDI VILA,b AND MARIA M. TAVIOc

aLaboratory of Genetics, Department of Biology MCA, University of Camerino,Camerino (MC), 62032 ItalybMicrobiology Department, Hospital Clinic of Barcelona, 08036 SpaincFaculty of Health Sciences, University of Las Palmas de Gran Canaria, 35016Spain

ABSTRACT: A complex network of regulatory systems ensures a coor-dinated and effective response to different types of stress that can acton a bacterium. Bacterial stress response generates changes that influ-ence efflux system and virulence factor expression. Thus, partial or to-tal loss of pathogenicity islands in uropathogenic Escherichia coli canbe induced by SOS-dependent or SOS-independent pathways related toselection of quinolone-resistant mutants. Likewise, hyperosmolarity andsome chemicals, including fluoroquinolones, salicylate, nonantimicrobialmedicaments like diazepam and anti-inflammatory drugs are all able toinduce an increased active efflux, cyclohexane tolerance, loss of porins,and decreased susceptibility to multiple antimicrobials in enterobacterialstrains, suggesting that bacterial response to the stress caused by an in-crease in osmolarity might be linked to the development of the multidrug-resistant phenotypes. Finally, a sudden downshift of the growth temper-ature (cold-shock) triggers a drastic reprogramming of bacterial geneexpression to allow cell survival under the new unfavorable conditions.The strategy developed by E. coli to reach this goal consists in the induc-tion of a set of (cold-shock) genes whose expression is regulated at bothtranscriptional and posttranscriptional levels.

KEYWORDS: pathogenicity islands; quinolones; hyperosmotic stress;multidrug resistance; cold shock; transcriptional and posttranscrip-tional regulations

INTRODUCTION

Bacterial stress response can be defined as a cascade of alterations in geneexpression and protein activity for the purpose of surviving extreme and rapidly

Address for correspondence: Marıa M. Tavıo, Microbiology, Clinical Science Department, Facultyof Health Sciences, University of Las Palmas de G.C., Dr. Pasteur, 35016 Las Palmas de Gran Canaria,Spain. Voice: 34928453405; fax: 34928451416.

mtavio@dcc.ulpgc.es

Ann. N.Y. Acad. Sci. 1113: 95–104 (2007). C© 2007 New York Academy of Sciences.doi: 10.1196/annals.1391.008

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changing and potentially damaging conditions sensed by bacteria, and whichresults in the cells becoming broadly stress resistant or eliminating the stressagent and/or mediating repair of cell injury.1

�s, or RpoS, is a sigma subunit of RNA polymerase in Escherichia coliand the master regulator of the general stress response, which is triggered byvarious stressful conditions and, in turn, activates more than 70 �S-dependentgenes, resulting in resistance to hyperosmolarity, among other stresses.1

RpoS and other bacterial stress response regulators influence virulence andbiofilm formation, and most of them may contribute to the development ofmultidrug resistance, an issue of considerable clinical relevance.2

Furthermore, several studies have demonstrated that quinolone-resistanturopathogenic E. coli strains have fewer virulence factors than their susceptiblecounterparts,3 and quinolones may induce some bacterial stress responses, sug-gesting pathways evolved by bacteria to economize some molecular resourcesin order to accomplish a more effective survival after stress response.

Multidrug resistance is frequently associated with transcriptional regulatorsinvolved in bacterial stress response, such as marA, soxS, sdiA, etc.4 Suchregulators can activate the overexpression of efflux systems like AcrAB, whosenatural function might be the transport of quorum-sensing signals that wouldtrigger rpoS.5 The in vitro induction of multidrug resistance in enterobacteriastrains has been associated with inducer levels that are kept close to their ownminimal inhibitory concentrations, suggesting the involvement of mechanismsfor bacterial response to increasing medium osmolarity in the development ofmultidrug-resistant phenotypes.6 In fact, several sigma and quorum-sensingregulators, which seem to cooperate in the management of hyperosmotic stress,can promote the increased expression of multidrug transporters in E. coli.1,5

The most studied adaptive response to a temperature change is the heat-shockresponse while relatively less is known about the cellular response to the cold,a phenomenon that has been observed in many prokaryotes and eukaryotes.Unlike heat shock, which involves the activity of a sigma factor (�32) otherthan the vegetative one, the osmotic stress, and the control of virulence geneexpression, E. coli cells have evolved a �s-independent mechanism to copewith the effects caused by sudden lowering of the environmental temperature.The main features of the cold-shock (CS) response in E. coli, which is thebetter-characterized bacterium from this point of view, will be briefly describedhere.

The Cold-Shock Response in E. coliAnna M. Giuliodori & Claudio O. Gualerzi

When an exponential culture of the mesophilic bacterium E. coli is trans-ferred from 37◦C to a temperature below 20◦C, the cells transiently stop grow-ing and enter an acclimation phase in which the synthesis of a small set of CS

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proteins is induced, whereas that of most of the other gene products is repressed.At the end of the acclimation phase, which can last one to several hours de-pending on the temperature, the synthesis of CS proteins declines and cellulargrowth resumes at a rather slow rate.7 The list of the E. coli CS-induced proteinsso far identified, mainly by proteomic approaches, is essentially constitutedby nucleic acid–binding proteins involved in different cellular processes likeRNA degradation, transcription, DNA replication and supercoiling, transla-tion, and ribosome maturation, as well as by five members of the Csp familyof E. coli.7 CspA and its homologs are small proteins (65–70 aa) identifiedin all types of bacteria except for archaea and cyanobacteria. E. coli containsnine paralogues belonging to the csp family, probably generated as a resultof a number of gene duplication events. Among these proteins CspA, CspB,CspE, CspG, and CspI are cold-inducible, while CspC, CspD, CspF, and CspHare not. Notably, the nucleic acid–binding domain of the CS proteins, calledcold-shock domain (CSD), is the most evolutionary conserved nucleic acid–binding domain within prokaryotes and eukaryotes.8 The list of CS proteinsalso includes two protein chaperones, namely, TF and Hsc66. A recent globaltranscript profiling of E. coli during cold shock has revealed that the level ofother transcripts encoding molecular chaperones such as htpG, mopA, mopB,and ppiA (encoding HtpG, GroEL, GroES, and peptidyl-prolyl-cis-trans iso-merase, respectively) transiently increases after cold shock.9 In addition, thesame study has underlined the induction of many genes involved in sugar trans-port and metabolism, as well as in membrane synthesis and function. Therefore,the overall data seem to suggest that the CS response is intended for (a) dealingwith unfavorable secondary structures of nucleic acids induced/stabilized bythe cold, which are expected to hinder basic functions such as transcription,ribosome assembly, and translation; (b) opposing the CS-induced decreasein membrane fluidity; (c) accumulating sugars displaying a protective effectagainst the low temperature, such as trehalose; and (d) helping protein foldingat low temperatures.

The mechanisms that regulate the CS gene induction can be either tran-scriptional or posttranscriptional but, although most frequently both levels areinvolved, they do not seem to have the same relevance for all CS genes. Twomain mechanisms of posttranscriptional regulation have been identified: se-lective cold-shock–induced stabilization of the transcripts of CS genes and“translational bias” whereby at low temperature translation of the CS mR-NAs is preferentially favored while that of the non-CS mRNAs is disfavored.Unlike the heat-shock response, transcriptional induction was found to playa relevant role only for some of the genes under study.7 Examples of thesetypes of regulatory circuits can be found in the mechanisms underlying the CSinduction of infA and cspA. InfA, encoding the translation initiation factor IF1,is transcribed in E. coli from two promoters (P1 and P2) yielding two mRNAsthat differ for the length of their 5′-untranslated region (5′UTR). While undernormal growth conditions infA is predominantly transcribed from P2 promoter,

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after cold-shock infA is de novo transcribed preferentially from the otherwiseless used P1 promoter. A possible reason for this “promoter preference shift”is that transcription from P1 is stimulated by increasing concentrations of theCS protein CspA, which has essentially no effect on the P2 activity.10

As to the expression of cspA, its CS induction is mainly controlled at aposttranscriptional level since its mRNA, which is extremely unstable at 37◦C,is drastically stabilized immediately after cold shock. Furthermore, the stabilityof cspA mRNA during cold shock is modulated in such a way that the rateof decay of cspA mRNA increases again toward the end of the acclimationphase, when the accumulation of the CS proteins is no longer necessary. Thismechanism of regulation is not restricted to cspA mRNA but is active also forother CS transcripts.7

Finally, the CS expression of both cspA and infA is favored by the above-mentioned translational bias. Recent data indicate that this bias is partly due tointrinsic features of CS mRNAs, which make them prone to translation at lowtemperature. Preliminary results seem indeed to indicate that cspA mRNA un-dergoes conformational changes whereby the ribosome binding site becomesmore accessible at low temperature compared to that at 37◦C. Thus, cspAmRNA might behave as an RNA thermometer, whose conformation changesin response to temperature.11 Furthermore, translation of CS templates is selec-tively stimulated by trans-acting factors whose level increases on cold shock.The most important trans-acting factor involved in the CS translational biasis IF3, whose level with respect to ribosomes increases during cold shock to-gether with that of the other two initiation factors (IF1 and IF2).12 IF3 is able tostimulate selectively, at low temperature and in a dose-dependent manner, thetranslation of CS mRNAs, while translation of non-CS mRNAs displays littleor no IF3 dependence.12 In particular, at low temperature, IF3 stimulates therate of “30S initiation complexes” formation with CS mRNAs while inducingthe formation of nonproductive 70S initiation complexes with non-CS mR-NAs.13 Furthermore, it has been shown that a larger-than-normal amount ofIF1 and IF3 is needed at low temperature to overcome the increased tendencyof the ribosomal subunits to associate.10,13 These results suggest a mechanis-tic model that can explain the translational bias. On lowering the temperature,the formation of initiation complexes with non-CS mRNA becomes rate lim-iting, thus leading to a transient block of translation. This in turn triggers theCS induction of PY. This protein, whose likely function is that of “storing”the ribosomes as 70S monomers when the cell requires a reduced number oftranslating ribosomes would sequester a large proportion of 70S deriving frompolysome dissociation in a functionally inactive state.14 The transient increaseof the IFs level would counteract the tendency of the few pY-free 70S monomersto remain stably associated, thus maintaining a pool of free subunits capableof initiating the translation of CS mRNAs that are accumulating in the cellsowing to their increased stability. Furthermore, IF3 might reduce the transla-tion of non CS mRNAs. Finally, the synthesis of RNA chaperones, helicases,

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and protein chaperones would help the cell adapt to the low temperature andto resume growth at the end of the acclimation phase.

Quinolones and Loss of Virulence Factors in E. coliSara Soto & Jordi Vila

It is well known that resistance to quinolones in E. coli is an increasing prob-lem in Spain and other countries.15 Quinolone-resistant uropathogenic E. colistrains express fewer virulence factors than quinolone-susceptible strains,16

and this phenomenon might be particularly frequent among strains of theB2 phylotype.17 Among the virulence factors, the hemolysin and cytotoxicnecrotizing factor were more frequently found in quinolone-susceptible thanin quinolone-resistant E. coli isolates.16 These virulence factors are located inone or two pathogenicity islands, depending on the strain. Herein, we foundthat quinolones increase the frequency of loss of some pathogenicity island(PAI) in phylotype B2 E. coli strains. The loss of PAI-I was observed, whereasthe loss of PAI-IV was not. Spontaneous loss of hemolytic capacity was notobserved in any of the three tested strains in the absence of quinolone exposure.

Further studies are necessary to elucidate the mechanism involved in theinduction of the loss of PAIs by quinolones. The role of the SOS system wasanalyzed because it is well known that quinolones induce SOS system response(DNA repair mechanism) and this response could favor the splitting of bacte-riophages or related sequences from the chromosome.18 In this regard, Shaikhand Tarr demonstrated that quinolones promote the excision of stx2 bacterio-phages as well as complete and truncated stx1 bacteriophages.19 Nevertheless,in our study when recA was truncated, the knock-out mutant did not lose thePAI in presence of quinolones.

These findings suggest that quinolones induce the in vitro loss of pathogenic-ity islands in uropathogenic E. coli strains when they are under the effect ofsubinhibitory concentrations of these antimicrobial agents. This loss can bepartial or total depending on each strain, and, in some cases, the SOS sys-tem can be implicated. Nevertheless, the role of stress response regulatorsin response to quinolones, resulting in loss of virulence factors, must not beoverlooked. In this sense, quinolones induce not only SOS system responsebut also heat-shock response.20 Moreover, envelope stress regulators influencevirulence factor expression.21 Otherwise, the deletion processes may also playa role in the adaptation of uropathogenic E. coli strains during certain stagesof infection. Therefore, genetic flexibility of pathogenic microbes may createselective advantages over other, less flexible organisms, and may finally resultin proper replication in host organisms or other ecological niches. Althoughquinolone resistance is not necessary to lose PAIs, quinolone-resistant E. colistrains have likely been in previous contact with these antimicrobial agents,thus favoring the loss of the PAI.

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Osmotic Stress and Other Stressors Inducing Multidrug ResistanceMarıa M. Tavıo

Intrinsic multiple-antibiotic resistance in bacteria is a clinically importantproblem, since it plays the role of a fundamental stepping stone to higher levelsof resistance in clinical isolates. In this type of resistance, reduced penetrationof antibiotics as well as an increase in active efflux results in low-level, broadresistance to distinct classes of antimicrobial agents.22

Multidrug resistance in E. coli has frequently been linked to the expressionof transcriptional activators such as MarA, SoxS, or Rob, which belong to theXylS-AraC class of positive transcriptional regulators.4 Other transcriptionalregulators may also induce efflux pump expression, as in the case with SdiA,which is homologous to the LuxR family of quorum-sensing transcription fac-tors, and whose amplification has a global impact on several functions of bac-terial cell, such as cell septation (ftsQAZ cell division genes).23 This suppressorof division inhibition (SdiA) regulates cell division in a cell density–dependentor quorum-sensing manner, in which AcrAB seems to have the natural functionof pumping out quorum-sensing signals that do not diffuse easily on their own.AcrAB is an E. coli multidrug efflux transporter that allows passage of fluoro-quinolones. Interestingly, a 4-quinolone (2-heptyl-3-hydroxy-4-quinolone) isproduced by Pseudomonas aeruginosa and functions as a quorum-sensing sig-nal.23 Indeed, sublethal concentrations of fluoroquinolones and other antimi-crobials may induce both multidrug-resistant phenotypes and quorum-sensingregulator lux genes.5 Nevertheless, in vitro nonantimicrobial chemotherapeu-tic agents such as diazepam and anti-inflammatory drugs, among others, alsoinduce the multidrug resistance phenotypes in enterobacteria. The inducingeffect of such drugs is dose dependent in such a way that as they near the MIC,there is an increased cyclohexane tolerance, decreased susceptibility to distinctantimicrobials and loss of OmpF in the bacterial cell.6,22

Taking into account that osmotic stress due to increased osmotic pressureoutside the lipid bilayer surrounding the bacterial cell depends on the concen-tration of the solute but not on its identity, a possible involvement of hyperos-motic stress response regulators in the induction of multidrug resistance phe-notypes must not be overlooked. In this sense, high osmolarity of the mediumtriggers the expression of regulators like rpoS or cpxR that, in turn, activateefflux pump expression. Although, tolC upregulation (the outer-membranechannel that joins with the AcrAB or AcrEF efflux systems) is responsible forincreased cyclohexane tolerance in E. coli, this still has not been associatedwith rpoS or cpxR. However, TolC as well as AcrAB and AcrEF efflux sys-tems are overexpressed when sdiA upregulation is induced.24 Recently, we havefound sdiA overexpression in E. coli multidrug-resistant mutants in vitro se-lected with suprainhibitory concentrations of fluoroquinolones, ceftazidime,or diazepam.4 In E. coli, the amplification of sdiA, a positive activator of

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ftsQAZ genes that are essential for septation, results in mitomycin C resistanceas well as in a global impact on gene expression, including increased transcriptlevels of components of efflux systems such as acrD, acrE, acrF, acrA, acrB,and tolC.24 In fact, multiple data suggest that regulation of multidrug trans-porters has a strong relationship to (i) stress responses, (ii) the growth phase,and (iii) quorum sensing.

E. coli DNA mycroarrays have not identified micF or ompF as genes undersdiA regulation.24 On the other hand, both ompC and ompF genes are membersof the Cpx regulon, and CpxR activation results in a strong decrease in ompFexpression (not requiring micF) and an increase in ompC expression, and italso leads to an increased level of mutation owing at least partly to direct re-pression of the ung DNA repair gene.25 Perhaps suprainhibitory concentrationsof antimicrobials such as fluoroquinolones or ceftazidime, and chemicals likesalicylate or diazepam, might activate the Cpx regulon, resulting in ung generepression mediated by CpxR and consequently favoring in vitro selection ofmultidrug-resistant mutants.4 Otherwise, the sigma factors �S and �E (a co-operator of �S in the management of hyperosmotic stress) are also involved inthe regulation of porin expression in E. coli, although activation of �E resultsin decreased expression of both ompF and ompC.26

Therefore, a complex network of bacterial stress regulators would coop-erate during bacterial response to an increase in medium osmolarity causedby antimicrobial or nonantimicrobial agents, resulting in induced multidrug-resistant phenotypes. Trials aimed to elucidate the role of the bacterial stressresponse regulatory systems in the changes associated with the development ofintrinsic multiple-antimicrobial resistance might be a good starting point for afuture better understanding of the concerning problem of multidrug resistancein clinical or environmental bacterial isolates.

CONCLUSIONS AND PERSPECTIVES

Transcriptional and Posttranscriptional Controlof Cold-Shock Gene Expression

In spite of the failure in identifying the actual signal triggering the in-duction of the CS stimulon, and of the large number of questions that re-main open, the study of the CS response, probably one of the most complexadaptive responses known in bacteria, has shed light on novel mechanismsof gene regulation and produced results of considerable interest for theirpotential applications, mainly allowing the expression of recombinant pro-teins under conditions limiting the danger of the formation of inclusionbodies.

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Quinolones Induce the Loss of Virulence Factors in E. coli

Subinhibitory concentrations of quinolones might in vitro induce a bacterialstress response, resulting in the loss of virulence factors located in pathogenic-ity islands in uropathogenic E. coli strains.

Multidrug Resistance Induced by an Increasing Medium Osmolarity

The close relationship between high concentrations of inducers and selec-tion or induction of multidrug-resistant strains suggests the involvement ofhyperosmotic stress response regulators in such resistant phenotypes.

The role of such regulators in the appearance of multidrug-resistant strains inclinical isolates or in the environment, and the pathway by which they integrateinto the network of stress regulator systems, may be helpful in thoroughlyunderstanding how antimicrobial resistance occurs.

ACKNOWLEDGMENTS

The cold-shock response in E. coli: The costs of this investigation were par-tially supported by Italian MIUR grants (PRIN 2005 to Cynthia L. Pon andPRIN 2005 to COG). Quinolones and loss of virulence factors in uropathogenicE. coli: This research was supported by work contract from National HealthSystem CP05/00140. Osmotic stress and other stressors as inducers of mul-tidrug resistance: This work was partially supported by subsidy 2002/199awarded by Canary Government.

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