Although pressure is an important environmental parameter in microbial niches such as the deep sea and is furthermore usedin food preservation to inactivate microorganisms, the fundamental understanding of its effects on bacteria remains fragmentary.Our group recently initiated differential fluorescence induction screening to search for pressure-induced Escherichia coli promoters and has already reported induction of the heat shock regulon. Here the screening was continued, and we report for the firsttime that pressure induces a bona fide SOS response in E . coli, characterized by the RecA and LexA-dependent expression of uvrA, recA, and sulA . Moreover, it was shown that pressure is capableof triggering lambda prophage induction in E . coli lysogens.The remnant lambdoid e14 element, however, could not be inducedby pressure, as opposed to UV irradiation, indicating subtle differences between the pressure-induced and the classical SOS response . Furthermore, the pressure-induced SOS response seemsnot to be initiated by DNA damage, since recA and lexA1 [Ind^–]mutants, which are intrinsically hypersensitive to DNA damage,were not sensitized or were only very slightly sensitized forpressure-mediated killing and since pressure treatment was notfound to be mutagenic . In light of these findings, the currentknowledge of pressure-mediated effects on bacteria is discussed.

 
Pressure is an environmental parameter that varies only between narrow limits and thus has little or no influence in most commonly studied microbial niches . However, in some specific niches and situations, the life and death of microorganisms are strongly affected by pressure . This is the case for piezophilic and piezotolerant microorganisms [respectively requiring or tolerating high pressure during growth] living in the deep sea and the deep subsurface[40, 54] and also for nonpiezophiles that are subjected to pascalization,an emerging process for preserving foods by treatment with ultrahighpressure [100 to 1,000 MPa] [15] . Although pressure, like temperature,is a thermodynamically well-known physical parameter, the effectsof high pressure on microorganisms remain poorly characterized,unlike those of heat . Some effects of pressure on biomoleculesand biological systems that have been well studied in vitroand explained on the basis of thermodynamic principles are proteindenaturation and phase transition in membranes [5] . Therefore,most pressure effects on microorganisms observed in vivo, suchas inhibition of key enzymes [48] and processes [20, 41] anddisruption of cellular structures [35] and membranes [22, 42],are believed to stem from these primary events.

Microorganisms that are adapted to normal atmospheric pressure [0.1 MPa], such as Escherichia coli, can often grow at pressures up to a few tens of megapascals, but only at a strongly reduced rate . The deep sea, on the other hand, with pressures rangingfrom 30 up to 100 MPa, constitutes a reservoir of piezophilicand piezotolerant microorganisms, which have become a modelfor studying piezophysiology and to gain insight into cellularadaptation strategies for coping with high-pressure stress.Although these organisms remain difficult to study, severalresearch groups have revealed specific piezoprotective alterationsin their membrane and protein components, such as an increasedproportion of unsaturated fatty acids and a decreased occurrenceof helix-destabilizing amino acid residues, respectively [reviewedin references 1 and 8].

Pressures in the range of 100 to 1,000 MPa kill most microorganisms and are being used in high-pressure food preservation, because they leave most of the sensorial and nutritional propertiesof the food intact, as opposed to thermal treatments [15] . Detailed pressure inactivation studies, however, revealed significant differences in pressure sensitivities among vegetative bacterial species and even between strains within a single species [3, 9] . Moreover, several groups have reported the selection ofpressure-resistant mutants of E . coli [23, 28] and Listeriamonocytogenes [31] . Although not affecting pressure growth limits,the resistance in E . coli was extended by an extraordinary 500MPa [28] . Molecular characterization of such pressure-resistantmutants of E . coli and L . monocytogenes revealed in both casesthe abundance of heat shock proteins [2, 32], stressing proteinmanagement as an important feature for withstanding extremelyhigh pressures.

In order to understand the piezoprotective adaptations in dedicated deep-sea bacteria as well as in surface-dwelling bacteria that have mutated to become resistant to high-pressure inactivation,a better insight into cellular awareness of pressure is necessary.One way to achieve this is by characterizing the bacterial high-pressure response . To date, only a few groups have embarked on dissectionof the genetic response of piezotolerant or piezosensitive microorganisms to pressure . For the deep-sea bacterium Photobacterium profundum SS9, Bartlett and colleagues discovered the expression of outer membrane proteins to be pressure dependent and to be regulatedat the molecular level by homologues of the Vibrio choleraeToxR and E . coli RpoE proteins [7, 16, 53] . Using two-dimensionalsodium dodecyl sulfate-polyacrylamide gel electrophoresis andgene arrays, two other studies revealed a link between the pressureresponse and the heat shock or freeze-thaw response of E . coliand Saccharomyces cerevisiae, respectively [30, 52] . Using adifferential fluorescence induction [DFI] screening [50], ourgroup recently was able to demonstrate induction of several heat shock promoters after pressure treatment in E . coli, further substantiating the link between the genetic heat shock and pressure responses [2].

The characterization of the genetic response of microorganisms upon pressure treatment, however, remains fragmentary and isfar from finished . In this paper, based on further DFI screening,we describe the unexpected discovery of a pressure-induced SOSresponse for E . coli . The SOS response is typically inducedupon DNA damage, for example, after UV irradiation or mitomycinC treatment, resulting in stalling of the DNA replication forkand disassembly of the replication apparatus . This then resultsin the exposure of single-stranded DNA, which is rapidly sensedand stabilized by the RecA protein, generating a nucleoproteinfilament that activates the autoproteolytic activity of LexA.Intact LexA acts as a repressor of the SOS response, controllingmore than 40 genes involved in stabilization of single-strandedDNA, base or nucleotide excision repair, recombinational repair,translesion synthesis, and control of cell division [21, 36, 37] . Pressure induction of the SOS response is surprising and has never been reported earlier, but it provides an explanation for some as yet unexplained pressure-related phenomena in bacteria.

 
Strains and growth conditions. The effects of high-pressure treatment were studied with E.coli strain MG1655 [13] . Derivatives of MG1655 carrying either recA, lexA1 [Ind^–], or e14::Tn10 were constructed by P1 transduction [46], using QC2411 [recA srl::Tn10] [19], AM121[lexA1 [Ind^–] malF3089::Tn10] [4], and CH1494 [e14-1272::Tn10][14], respectively, as donor strains . Tetracycline-resistanttransductants were isolated and, in the case of recA and lexA1[Ind^–] strains, checked for UV sensitivity . Lysogens ofMG1655 wild-type and recA and lexA1 strains were constructedusing [P3rpoH::lacZ] isolated from ADA600 [10] and selectedon medium containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [20 µg/ml] . Finally, C600 was used as a host for countingPFU.

Overnight cultures were obtained by growth in Luria Bertanibroth [LB] [46] for 21 h at 37°C under well-aerated conditions. Antibiotics [Applichem, Darmstadt, Germany] were added when necessary to obtain the following concentrations: 100 µgof ampicillin/ml and 20 µg of tetracycline/ml.

Construction and screening of promoter trap library of MG1655. A promoter trap library of MG1655, consisting of ca . 15,000 independent clones, each containing 1- to 3-kb fragments ofa partial Sau3AI digest of MG1655 chromosomal DNA inserted upstreamof the promoterless gfp gene of pFPV25 [50], was constructed earlier [2] . Screening of the promoter trap library was basedon the DFI technique described by Valdivia and Falkow [50] andslightly customized for isolating pressure-induced promoters as recently described [2].

Pressure treatment. Overnight cultures were diluted 1/100 in fresh prewarmed LBwith 100 µg of ampicillin/ml and further incubated untillate exponential phase [optical density at 600 nm [OD₆₀₀] =0.6] . Portions [4 ml] of this culture were then pelleted by centrifugation [5 min at 6,000 x g] and resuspended in the samevolume of fresh prewarmed LB . For pressure induction, a 500-µlsample was sealed without air bubbles in a polyethylene bagand pressurized for 15 min in an 8-ml pressure vessel, maintainedat 20°C with an external cooling circuit [Resato, Roden,The Netherlands] . It should be noted that pressurization causedsome adiabatic heating of the sample; however, this was less than 3°C at 100 MPa, which was the maximum inducing pressure used in this study . For inactivation experiments, pressuresup to 300 MPa were used . After treatment, cultures were maintainedat 37°C and used for the measurement of gfp induction, phageinduction, or determination of viability.

Green fluorescent protein [GFP] fluorescence measurements. After induction, 300-µl samples were transferred to microplatewells and placed in a fluorescence reader [Fluoroscan AscentFL; Thermolabsystems, Brussels, Belgium] . Fluorescence at 520nm was then measured at 30-min intervals with intermittent shaking[every 5 min] at 37°C, using an excitation wavelength of480 nm . At the same time, OD₆₀₀ was measured and fluorescencewas expressed per unit of OD₆₀₀ . Alternatively, 3 h after induction,cultures were analyzed by fluorescence-activated cell sorting[FACS] analysis using a FACSCalibur apparatus [Becton Dickinson,Erembodegem, Belgium] fitted with an argon laser emitting at488 nm . Fluorescence data shown are representative results fromat least four independent experiments . To determine the foldinduction after pressure treatment, population means of fluorescencecalculated by the FACSCalibur software were used.

Determination of viability. Serial dilutions from pressurized and nonpressurized sampleswere plated on Tryptone Soy Agar [Oxoid, Basingstoke, UnitedKingdom] with a spiral plater [Spiral Systems Inc., Cincinnati,Ohio] . Twenty-four hours later, colonies on the plates werecounted, and reduction factors [RF] were determined as follows:RF = [no . of CFU/ml [nonpressurized sample]]/[no . of CFU/ml[pressurized sample]].

Construction of plasmids. Specific GFP transcriptional fusions were constructed in pFPV25with the promoter regions of recA [P_recA] and sulA [P_sulA], which were obtained by PCR [Platinum Pfx DNA polymerase; Invitrogen,Merelbeke, Belgium], using the primer pairs 5'-TACGTCTAGATTATACTCCTGTCATGCCGGG-3'and 5'-TAGCGGATCCTGTCTATTAGTGGTATCGCC-3' for P_recA and 5'-GCATTCTAGATTAACGATGTGCATAGCCTC-3'and 5'-GCATGGATCCCCCGAAGATACAACTCACC-3' for P_sulA . Both PCR products and pFPV25 were cut with BamHI and XbaI to allow directional cloning of the promoters upstream of gfp . Subsequently, these constructs were transformed to wild-type, recA and lexA1 [Ind^–]backgrounds of MG1655 and confirmed by sequencing the promoterfragment and the gfp 5' end . All restriction enzymes were purchasedfrom Roche Diagnostics Belgium [Vilvoorde, Belgium].

Sequencing. Inserts in the pFPV25 plasmid were sequenced by MWG BiotechAG [Ebersberg, Germany], using 5'-GACAAGTGTTGGCCATGGAACAGGTAG-3'in the 5' region of gfp as a sequencing primer.

Phage induction. Late exponential cultures [OD₆₀₀ = 0.6] of the lysogenizedstrains were pressure treated as described above and subsequentlyincubated at 37°C . At different time points after pressuretreatment, 500-µl portions of pressure treated and untreated control cells were centrifuged [5 min at 24,000 x g], and thesupernatant was sterilized by adding 30 µl of chloroformand vortexing . Afterwards, dilutions of this supernatant wereadded to 1 ml of stationary-phase C600 cells, which are a moreefficient plating host than MG1655 . Subsequently, 3 ml of TBMMtop agar [34] was added to this culture, and the mixture waspoured on a LB plate . After 24 h, the number of PFU was countedand recalculated to number of PFU/milliliter of the originalculture.

UV treatment. The UV oven used in this work [Bio-Link, Vilber Lourmat, France]was equipped with five fluorescent lamps of 8 W each, emittingfrom 180 to 280 nm with a peak at 254 nm . UV doses were programmedand are controlled by a radiometer that constantly monitorsthe UV light emission.

For measuring induction of promoters by UV, 1-ml portions of late-exponential-phase cultures [OD₆₀₀ = 0.6] of strains containing promoter-gfp fusions or carrying a prophage were poured ina petri dish and irradiated in the UV oven [0.1 kJ/m²] . Afterwards,the cultures were used for measurement of production of GFPor phage particles . For measuring UV inactivation or e14::Tn10 excision, a dilution series of cells was plated on LB agar and directly irradiated in the UV oven [0 to 0.1 kJ/m²] . The distance between the lamps and the plates was 14 cm . After 24 h, colonies were counted . It should be noted that irradiating a 1-ml culture in a petri dish resulted in far less inactivation than direct irradiation of plated bacteria [about 1 log cycle for the wildtype and the lexA1 mutant and 1.5 log cycles for the recA mutant].

 
uvrA induction by pressure, detected by DFI. DFI screening of a random promoter probe library of E . coli MG1655, constructed in pFPV25, led to the isolation of several plasmids containing putative pressure-inducible promoters . Sequence analysis of the cloned fragments revealed in one particularclone the presence of the uvrA promoter [P_uvrA] with its LexA binding box upstream of gfp . This clone showed a ca . fivefold increase in fluorescence 3 h after sublethal pressurizationat 100 MPa for 15 min [Fig . 1], whereas the vast majority of clones containing random promoter fragments did not show pressure-induced GFP expression [data not shown] . The functionality of P_uvrA in the checked fragment was further confirmed by a dose-response curve showing UV induction [data not shown].

 
Induction of a bona fide SOS response by pressure. Since uvrA is part of the SOS regulon, two additional gfp transcription fusions were constructed with the promoters of recA [P_recA] and sulA [P_sulA] . Like P_uvrA, these promoters also harbor LexAbinding boxes, but the LexA dissociation constant for each promoteris different [in order of increasing LexA affinity: P_uvrA, P_recA, and P_sulA] . Both P_recA and P_sulA proved to be responsive towardspressure treatment, showing ca . 18-fold and ca . 20-fold fluorescenceinduction at 100 MPa, respectively [Fig . 1].

Since pressure can cause protein denaturation [5], one possibleexplanation for the observed induction of the SOS response wasdenaturation of the LexA repressor, resulting in clearance of the LexA boxes and in a short-cut mechanism of induction ofthe SOS response . Alternatively, the SOS response could be theresult of a bona fide physiological response, dependent on thekey regulators RecA and LexA . To distinguish between the twopossibilities, we investigated the effect of a RecA deletion[recA] and a cleavage-resistant LexA protein [lexA1 Ind^–] on pressure induction of P_uvrA, P_recA, and P_sulA . Figure 2 clearlyshows that both defects abolish pressure induction of all threepromoters at 100 MPa, similar to what they do for their inductionby UV . Since recA strains were slightly more inactivated at100 MPa than wild-type and lexA1 [Ind^–] strains [see Fig.4A], the same experiment was repeated at 75 MPa, where the differencesin inactivation between all three strains were negligible andloss of responsiveness in the recA background could not be attributedto loss of viability . Although the induction at 75 MPa was slightlylower, a similar pattern was observed [data not shown] . Fromthis we can conclude that a genuine, RecA and LexA-dependentSOS response is elicited by sublethal pressures.

 
Induction of the lytic development of lysogenic by pressure. Since the activated RecA nucleoprotein fragment stimulates autocleavagenot only of LexA but also of the phage CI repressor, responsiblefor maintaining lysogeny [36], we anticipated that pressurewould cause induction of prophage from lysogenized MG1655.Figure 3A shows that an almost 10⁴-fold induction of prophagesindeed occurred 3 h after pressure treatment . Since this inductionwas not accompanied by visible lysis of the culture, we presumethat only a subpopulation of cells was induced . In addition, pressure-mediated prophage induction was also shown to be LexA dependent [Fig . 3B], since lysogens displayed severely diminishedinduction in a lexA1 background . Finally, in a recA background,no phage induction could be observed [Fig . 3B] . A similar patternof prophage induction by UV irradiation [Fig . 3B] or mitomycinC treatment [data not shown] was observed.

 
Stability of the e14 element is not affected by pressure treatment. The e14 element is a 15.4-kb lambdoid bacteriophage remnantknown to excise itself from the E . coli genome upon SOS inductionby UV treatment [26] . The use of an antibiotic resistance marker positioned in e14 [see "Strains and growth conditions"] allows direct demonstration of the presence or absence of this element[14] . Loss of e14::Tn10 was assayed after pressure or UV treatment by streaking ca . 400 survivors on LB with 20 µg of tetracycline/ml. While ca . 60% of UV-treated [0.02 kJ/m²] cells showed loss of e14, no loss was observed for any of the pressures applied [Table 1].

 
recA and lexA1 mutants are not sensitized against pressure treatment. MG1655 recA and lexA1 mutants displayed no sensitivity or onlya very slightly increased sensitivity towards pressure treatmentsthat are sublethal [0 to 150 MPa] or lethal [150 to 300 MPa]to MG1655 [Fig. 4A], although both mutants were, as expected, extremely sensitive towards UV irradiation [Fig . 4B] . Together with the stability of the e14 element, unaffected by pressure, these observations provide a remarkable difference in the behaviorof E . coli towards pressure versus other that toward SOS-inducing treatments described so far.

 
The bacterial SOS response is typically induced by DNA-damaging treatments, such as UV irradiation or exposure to mitomycinC [21, 36, 37]; however, its induction by pressure as shownin this work [Fig . 1] was previously unreported . Although pressureis known to denature proteins, it has been shown to stabilizeDNA helices in vitro by promoting hydrogen bonds and enhancingstacking of the hydrophobic bases, resulting in compact DNA[6] . The induction of the heat shock response by high pressure,as has been reported previously [2], therefore seems more logicalthan activation of the SOS regulon . However, our results provethat the pressure-induced SOS response is not a consequenceof LexA repressor denaturation by pressure but genuinely dependson the physiologically active RecA and LexA proteins, sincemutant strains with a RecA deletion or producing the autocleavage-resistantLexA1 variant did not exhibit SOS induction [Fig . 2.].

In addition to the transcriptional activation of uvrA, recA, and sulA, prophage induction by pressure provided a secondindependent manifestation of the pressure-mediated SOS responsethat was also RecA and LexA dependent [Fig . 3A and B] . Whereas lysogens in wild-type E . coli after pressure induction produced an almost 10⁴-fold-increased level of phage particles, only about a 10-fold induction was observed in a lexA1 background. This residual 10-fold induction is probably due to activation of RecA protein that is formed by leaky expression of its LexA-dependent promoter . While pressure-induced expression of the gfp fusions is silenced in a lexA1 background because of the inability of LexA1 to cleave itself, the limited amount of RecA protein after activation by pressure could mediate some cleavage of the CIrepressor . In wild-type lysogens, however, LexA also becomes cleaved, resulting in derepression of the recA promoter, production of additional RecA protein, and enhanced CI autocleavage stimulation. In lysogens in a recA background, the noninduced productionof phage particles was strongly reduced [ca . 5 PFU/ml, comparedto ca . 10³ to 10⁴ PFU/ml in the wild type], and no inductionby pressure was observed [Fig . 3B] because the CI repressoris not cleaved in the absence of RecA . Similar patterns of prophageinduction were obtained with UV irradiation, but UV inductionresulted in higher titers of phage than pressure induction,indicating that the applied pressure treatments induced a smallerfraction of lysogens [Fig. 3B] . Thus, although Fig . 1 shows a uniform induction of the SOS response throughout the population, cleavage of the CI repressor seems to occur only in a relatively small fraction of the pressurized cells . Interestingly, duringthe preparation of this report, our attention was drawn to a40-year-old study of Rutberg [45] [hard-copy citations from 1953 to 1965 were added online to Medline on 30 September 2003],who demonstrated unexplainable similarities between UV and pressure induction of in E . coli K12 . Therefore, in hindsight, our resultsprovide a molecular description for this cryptic phenotype observed40 years ago.

Some lambdoid prophages confer virulence properties to theirhost upon lysogenization [51] . A well-known example is the Shiga toxin-converting [Stx] bacteriophages, which carry the genes for production of Shiga toxins in Stx-producing E . coli strains [47] . In fact, we recently found Stx lysogens to be inducibleby high-pressure treatment [A . Aertsen, D . Faster, and C . W.Michiels, submitted for publication] . In food preservation, therefore, pressure treatment could potentially enhance the spread of these phages and lead to the emergence of new pathogensor pathogens with increased virulence . The probability of sucha scenario is difficult to estimate, but recently, Toth et al.[49] demonstrated that Stx bacteriophages could indeed readilylysogenize enteropathogenic E . coli strains in porcine ligatedileal loops.

Interestingly, the e14 element, which is a lambdoid prophage remnant, remained unaffected by pressure treatment of differ-ent intensities, while an SOS response evoked by UV irradiationcaused its excision in ca . 60% of the survivors [Table 1] . Possibly, this difference can be attributed to the observation that the pressure-mediated SOS response induces the excision only ina smaller fraction of cells than with UV treatment, and we wereunable to detect it accordingly . Indeed, it could be that noneof the pressures used supports the sustained levels of activatedRecA necessary to induce detectable e14 excision . A similarobservation was made for prophage induction [see above] . Anadditional distinguishing feature with classical SOS-inducingtreatments is that recA and lexA1 mutants, which are hypersensitivetowards DNA-damaging treatments, exhibited almost wild-typetolerance to pressure [Fig . 4A] . Although recA strains wereslightly more inactivated by pressure treatment than the wildtype and lexA1 mutants, this difference was small compared tothe difference in UV sensitivity between these strains [Fig.4B] . This feature may reflect a lack of DNA damage inductionby high pressure . Indeed, MG1655 cultures showed no increasedincidence of rifampin resistance after high-pressure treatment[data not shown] . Moreover, in the literature, pressure has never been associated with mutagenesis . Although two early studies reported an increased occurrence of petite mutants in a pressurized S . cerevisiae culture [44] or pressure-induced color mutationin Euglena gracilis [27], both phenotypes might be related toloss of mitochondria or chloroplasts, respectively, rather thanto mutagenesis . In the absence of DNA lesions after pressuretreatment, the causal trigger of this SOS response remains enigmatic.

Nevertheless, the presence of an activated RecA nucleoprotein filament can reasonably be assumed, since cleavage of both LexAand CI seems to occur . This nucleoprotein complex originateswhen RecA binds to single-stranded DNA [ssDNA] in the cell [36], implicating the formation or exposure of ssDNA during or after pressure treatment . While the DNA helix itself is stabilizedunder pressure, the DNA replication machinery performs one ofthe most pressure-sensitive essential cellular processes inE . coli [8] . In fact, all multisubunit protein complexes arehighly susceptible to dissociation upon pressurization, andtherefore, we believe pressure to cause disassembly of the replicationfork . Without the requirement for DNA lesions to stall the replicationfork, this direct denaturation of the replication complex canthen result in exposure of ssDNA, which in turn can activatethe RecA protein and trigger SOS induction.

Interestingly, in recent years several microbial responses to stresses seemingly unrelated to DNA damage, such as starvation, aging, and translational stress, have been shown to evoke orrely on members of the SOS regulon [12, 29] . It has been hypothesizedthat the increased mutation and recombination rate, empoweredby these SOS proteins, might generate genetic diversity in timesof stress [43] . When the specific physiological stress responsesfall short, relaxation of the functions involved in safeguardingthe integrity of the DNA might well be a last resort for a populationto survive a high level of stress by increasing its geneticdiversity . Future research should determine whether pressurecan trigger adaptive mutations and assess its possible implicationsfor microorganisms exposed to high pressure in the deep sea,the deep subsurface, or during high-pressure food preservation.

We have reason to believe that our findings present a new paradigm in explaining several pressure-related phenomena . The first pressure-responsive genes, discovered in Photobacterium profundum SS9 by the group of Bartlett [7], comprise ompH and ompL . Bothencode outer membrane proteins, of which OmpL is predominantunder ambient pressures and is replaced by OmpH under high pressures.Since the absence of these genes did not influence growth orsurvival of SS9 at high or ambient pressures, it was hypothesizedthat in the deep sea, characterized by high pressure and nutrientscarcity, OmpL was replaced by OmpH to facilitate nutrient uptake.Also in E . coli, reduced expression of OmpC, OmpF, and OmpXwas observed upon growth at sublethal pressures [39] . This repressionseemed to occur independently from the normal EnvZ-OmpR signaltransduction cascade . Interestingly, Garvey et al . [24] demonstratedearlier that OmpC, OmpF, and OmpA disappeared in E . coli uponthe induction of the SOS response by nalidixic acid . Althoughthis awaits further confirmation, we therefore propose that pressure might well influence outer membrane protein expression in the above-mentioned cases by inducing an SOS response.

Another typical pressure-related phenomenon is cell filamentation in piezosensitive bacteria growing at permissive high pressures[50 MPa] [38, 55] . Interestingly, we found pressure-inducedcell elongation also to occur in the first hours after sublethalhigh-pressure treatment [unpublished data] . Moreover, duringthe preparation of this report, an independent study reported a similar effect in E . coli [33] after short high-pressure treatment.In many cases, cell elongation is typically the result of anSOS response and, more specifically, of SulA-mediated inhibitionof FtsZ ring formation, which constitutes an early and crucialstep in the cell division process . However, our observationswere consistent with those presented by Kawarai et al . [33], showing no elimination of cell elongation in a sulA or recA mutant background after sublethal pressure shock . In addition, no effect of the absence of SulA or RecA on cell filamentation induced by growth at permissive pressures was observed [unpublished data] . Although both Kawarai et al . [33] and Molina-Hoppner et al . [38] suggested that elongation results from direct high-pressureinhibition of FtsZ ring formation, we believe that high-pressure-inducedcell elongation in this respect resembles the "transient filamentation"phenotype observed by Gottesman et al . [25] after UV treatment,which was also shown to be independent of SulA . Also the recentfinding of Bidle and Bartlett [11] that recD of the piezotolerant deep-sea isolate SS9 allows E . coli to grow at high pressures without filamentation is more compatible with a model in which pressure-induced filamentation is mediated by the SOS responsethan with the model assuming a direct effect of pressure onFtsZ, because RecD is involved in DNA recombination and repair.The exact mechanism by which RecD affects pressure-induced filamentationin E . coli, however, awaits further clarification.

Finally, Chilukuri et al . [17] have identified piezoadaptiveamino acid substitutions in the ssDNA binding proteins of marineShewanella strains living at elevated pressures that are essentialfor the functionality of these proteins at high pressure, whileWelch et al . [52] observed increased rates of synthesis of coldshock proteins, RecA, and some other proteins in cultures ofE . coli growing at 55 MPa . The idea was then formulated thatpressure might mimic cold shock and that elevated levels ofDNA binding proteins would be necessary to compensate for decreasedDNA binding at elevated pressures [18] . Our data place thesedistinct observations in a broader framework of a pressure-inducedSOS response.

In conclusion, we have demonstrated the high-pressure inductionin E . coli of a genuine SOS response that nevertheless showssome peculiar differences from the typical SOS response as inducedby DNA-damaging treatments . This SOS response provides a plausible explanation for several previous observations made with bacteria under high pressure and at the same time raises some intriguingnew questions on how high pressure triggers a response thatis normally initiated by DNA damage . In both ways, this workcontributes to a better understanding of the effects of pressureon cells and cellular processes.

 
We acknowledge financial support by fellowships to authors A.A.and R.V.H . from the Flemish Institute for the Promotion of Scientific Technical Research [IWT] and from the Fund for Scientific Research Flanders [F.W.O . Vlaanderen], respectively, and by researchgrants OT/01/35 from the K.U.Leuven Research Fund and G.0195.02from F.W.O . Vlaanderen.

We thank J . Vanderleyden [Centre for Microbial and Plant Genetics, K.U.Leuven] for providing access to his FACS facilities.

 
* Corresponding author . Mailing address: Laboratory of Food Microbiology, K.U.Leuven, Kasteelpark Arenberg 22, B-3001 Heverlee, Belgium . Phone: 32-[0]16-32 15 78 . Fax: 32-[0]16-32 19 60 . E-mail: chris.michiels@agr.kuleuven.ac.be .

1. Abe, F., C . Kato, and K . Horikoshi. 1999 . Pressure-regulated metabolism in microorganisms . Trends Microbiol . 7:447-453.
2. Aertsen, A., K . Vanoirbeek, P . De Spiegeleer, J . Sermon, K . Hauben, A . Farewell, T . Nyström, and C . W . Michiels. 2004 . Heat shock protein-mediated resistance towards high hydrostatic pressure in Escherichia coli . Appl . Environ . Microbiol . 70:2660-2666 .
3. Alpas, H., N . Kalchayanand, F . Bozoglu, A . Sikes, C . P . Dunne, and B . Ray. 1999 . Variation in resistance to hydrostatic pressure among strains of food-borne pathogens . Appl . Environ . Microbiol . 65:4248-4251 .
4. Balashov, S., and M . Z . Humayun. 2002 . Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells . J . Mol . Biol . 315:513-527.
5. Balny, C., P . Masson, and K . Heremans. 2002 . High pressure effects on biological macromolecules: from structural changes to alteration of cellular processes . Biochim . Biophys . Acta 1595:3-10.
6. Barciszewski, J., J . Jurczak, S . Porowski, T . Specht, and V . A . Erdmann. 1999 . The role of water structure in conformational changes of nucleic acids in ambient and high-pressure conditions . Eur . J . Biochem . 260:293-307 .
7. Bartlett, D., M . Wright, A . A . Yayanos, and M . Silverman. 1989 . Isolation of a gene regulated by hydrostatic pressure in a deep-sea bacterium . Nature 342:572-574.
8. Bartlett, D . H. 2002 . Pressure effects on in vivo microbial processes . Biochim . Biophys . Acta 1595:367-381.
9. Benito, A., G . Ventoura, M . Casadei, T . Robinson, and B . Mackey. 1999 . Variation in resistance of natural isolates of Escherichia coli O157 to high hydrostatic pressure, mild heat, and other stresses . Appl . Environ . Microbiol . 65:1564-1569 .
10. Bianchi, A . A., and F . Baneyx. 1999 . Hyperosmotic shock induces the sigma32 and sigmaE stress regulons of Escherichia coli . Mol . Microbiol . 34:1029-1038.
11. Bidle, K . A., and D . H . Bartlett. 1999 . RecD function is required for high-pressure growth of a deep-sea bacterium . J . Bacteriol . 181:2330-2337 .
12. Bjedov, I., O . Tenaillon, B . Gerard, V . Souza, E . Denamur, M . Radman, F . Taddei, and I . Matic. 2003 . Stress-induced mutagenesis in bacteria . Science 300:1404-1409 .
13. Blattner, F . R., G . Plunkett III, C . A . Bloch, N . T . Perna, V . Burland, M . Riley, J . Collado-Vides, J . D . Glasner, C . K . Rode, G . F . Mayhew, J . Gregor, N . W . Davis, H . A . Kirkpatrick, M . A . Goeden, D . J . Rose, B . Mau, and Y . Shao. 1997 . The complete genome sequence of Escherichia coli K-12 . Science 277:1453-1474 .
14. Brody, H., A . Greener, and C . W . Hill. 1985 . Excision and reintegration of the Escherichia coli K-12 chromosomal element e14 . J . Bacteriol . 161:1112-1117.
15. Cheftel, J . C. 1995 . Review: high-pressure, microbial inactivation and food preservation . Food Sci . Technol . Int . 1:75-90.
16. Chi, E., and D . H . Bartlett. 1995 . An rpoE-like locus controls outer membrane protein synthesis and growth at cold temperatures and high pressures in the deep-sea bacterium Photobacterium sp . strain SS9 . Mol . Microbiol . 17:713-726.
17. Chilukuri, L . N., D . H . Bartlett, and P . A . Fortes. 2002 . Comparison of high pressure-induced dissociation of single-stranded DNA-binding protein [SSB] from high pressure-sensitive and high pressure-adapted marine Shewanella species . Extremophiles 6:377-383.
18. Chilukuri, L . N., P . A . Fortes, and D . H . Bartlett. 1997 . High pressure modulation of Escherichia coli DNA gyrase activity . Biochem . Biophys . Res . Commun . 239:552-556.
19. Dukan, S., and T . Nystrom. 1999 . Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells . J . Biol . Chem . 274:26027-26032 .
20. Erijman, L., and R . M . Clegg. 1998 . Reversible stalling of transcription elongation complexes by high pressure . Biophys . J . 75:453-462 .
21. Friedberg, E . C., G . C . Walker, and W . Siede. 1995 . DNA repair and mutagenesis . ASM Press, Washington, D.C.
22. Ganzle, M . G., and R . F . Vogel. 2001 . On-line fluorescence determination of pressure mediated outer membrane damage in Escherichia coli . Syst . Appl . Microbiol . 24:477-485.
23. Gao, X., J . Li, and K . C . Ruan. 2001 . Barotolerant Escherichia coli induced by high hydrostatic pressure . Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao [Shanghai] 33:77-81.
24. Garvey, N., A . C . St John, and E . M . Witkin. 1985 . Evidence for RecA protein association with the cell membrane and for changes in the levels of major outer membrane proteins in SOS-induced Escherichia coli cells . J . Bacteriol . 163:870-876.
25. Gottesman, S., E . Halpern, and P . Trisler. 1981 . Role of sulA and sulB in filamentation by Lon mutants of Escherichia coli K-12 . J . Bacteriol . 148:265-273.
26. Greener, A., and C . W . Hill. 1980 . Identification of a novel genetic element in Escherichia coli K-12 . J . Bacteriol . 144:312-321.
27. Gross, J . A. 1965 . Pressure-induced color mutation of Euglena gracilis . Science 147:741-742.
28. Hauben, K . J., D . H . Bartlett, C . C . Soontjens, K . Cornelis, E . Y . Wuytack, and C . W . Michiels. 1997 . Escherichia coli mutants resistant to inactivation by high hydrostatic pressure . Appl . Environ . Microbiol . 63:945-950.
29. Humayun, M . Z. 1998 . SOS and Mayday: multiple inducible mutagenic pathways in Escherichia coli . Mol . Microbiol . 30:905-910.
30. Iwahashi, H., H . Shimizu, M . Odani, and Y . Komatsu. 2003 . Piezophysiology of genome wide gene expression levels in the yeast Saccharomyces cerevisiae . Extremophiles 7:291-298.
31. Karatzas, K . A., and M . H . Bennik. 2002 . Characterization of a Listeria monocytogenes Scott A isolate with high tolerance towards high hydrostatic pressure . Appl . Environ . Microbiol . 68:3183-3189 .
32. Karatzas, K . A., J . A . Wouters, C . G . Gahan, C . Hill, T . Abee, and M . H . Bennik. 2003 . The CtsR regulator of Listeria monocytogenes contains a variant glycine repeat region that affects piezotolerance, stress resistance, motility and virulence . Mol . Microbiol . 49:1227-1238.
33. Kawarai, T., M . Wachi, H . Ogino, S . Furukawa, K . Suzuki, H . Ogihara, and M . Yamasaki. 2004 . SulA-independent filamentation of Escherichia coli during growth after release from high hydrostatic pressure treatment . Appl . Microbiol . Biotechnol . 64:255-262.
34. Kleckner, N., J . Bender, and S . Gottesman. 1991 . Uses of transposons with emphasis on Tn10 . Methods Enzymol . 204:139-180.
35. Kobori, H., M . Sato, A . Tameike, K . Hamada, S . Shimada, and M . Osumi. 1995 . Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections . FEMS Microbiol . Lett . 132:253-258.
36. Kuzminov, A. 1999 . Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda . Microbiol . Mol . Biol . Rev . 63:751-813 .
37. Little, J . W. 1993 . LexA cleavage and other self-processing reactions . J . Bacteriol . 175:4943-4950.
38. Molina-Hoppner, A., T . Sato, C . Kato, M . G . Ganzle, and R . F . Vogel. 2003 . Effects of pressure on cell morphology and cell division of lactic acid bacteria . Extremophiles 7:511-516.
39. Nakashima, K., K . Horikoshi, and T . Mizuno. 1995 . Effect of hydrostatic pressure on the synthesis of outer membrane proteins in Escherichia coli . Biosci . Biotechnol . Biochem . 59:130-132.
40. Newman, D . K., and J . F . Banfield. 2002 . Geomicrobiology: how molecular-scale interactions underpin biogeochemical systems . Science 296:1071-1077 .
41. Niven, G . W., C . A . Miles, and B . M . Mackey. 1999 . The effects of hydrostatic pressure on ribosome conformation in Escherichia coli: an in vivo study using differential scanning calorimetry . Microbiology 145:419-425.
42. Pagan, R., and B . Mackey. 2000 . Relationship between membrane damage and cell death in pressure-treated Escherichia coli cells: differences between exponential- and stationary-phase cells and variation among strains . Appl . Environ . Microbiol . 66:2829-2834 .
43. Radman, M. 2001 . Fidelity and infidelity . Nature 413:115.
44. Rosin, M . P., and A . M . Zimmerman. 1977 . The induction of cytoplasmic petite mutants of Saccharomyces cerevisiae by hydrostatic pressure . J . Cell Sci . 26:373-385.
45. Rutberg, L. 1964 . On the effects of high hydrostatic pressure on bacteria and bacteriophage . 3 . Induction with high hydrostatic pressure of Escherichia coli K lysogenic for bacteriophage lambda . Acta Pathol . Microbiol . Scand . 61:98-105.
46. Sambrook, J., E . F . Fritsch, and T . Maniatis. 1989 . Molecular cloning: a laboratory manual, 2nd ed . Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
47. Schmidt, H. 2001 . Shiga-toxin-converting bacteriophages . Res . Microbiol . 152:687-695.
48. Simpson, R . K., and A . Gilmour. 1997 . The effect of high hydrostatic pressure on the activity of intracellular enzymes of Listeria monocytogenes . Lett . Appl . Microbiol . 25:48-53.
49. Toth, I., H . Schmidt, M . Dow, A . Malik, E . Oswald, and B . Nagy. 2003 . Transduction of porcine enteropathogenic Escherichia coli with a derivative of a shiga toxin 2-encoding bacteriophage in a porcine ligated ileal loop system . Appl . Environ . Microbiol . 69:7242-7247 .
50. Valdivia, R . H., and S . Falkow. 1996 . Bacterial genetics by flow cytometry: rapid isolation of Salmonella typhimurium acid-inducible promoters by differential fluorescence induction . Mol . Microbiol . 22:367-378.
51. Wagner, P . L., and M . K . Waldor. 2002 . Bacteriophage control of bacterial virulence . Infect . Immun . 70:3985-3993.
52. Welch, T . J., A . Farewell, F . C . Neidhardt, and D . H . Bartlett. 1993 . Stress response of Escherichia coli to elevated hydrostatic pressure . J . Bacteriol . 175:7170-7177.
53. Welch, T . J., and D . H . Bartlett. 1998 . Identification of a regulatory protein required for pressure-responsive gene expression in the deep-sea bacterium Photobacterium species strain SS9 . Mol . Microbiol . 27:977-985.
54. Yayanos, A . A. 1995 . Microbiology to 10,500 meters in the deep sea . Annu . Rev . Microbiol . 49:777-805.
55. ZoBell, C . E., and A . B . Cobet. 1964 . Filament formation by Escherichia coli at increased hydrostatic pressures . J . Bacteriol. 87:710-719.

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