Disulfide bond formation catalyzed by disulfide oxidoreductases occurs in the periplasm and plays a major role in the properfolding and integrity of many proteins . In this study, we wereinterested in elucidating factors that influence the regulationof dsbA, a gene coding for the primary disulfide oxidoreductasefound in Salmonella enterica serovar Typhimurium . Strains withmutations created by transposon mutagenesis were screened forstrains with altered expression of dsbA . A mutant [NLM2173]was found where maximal expression of a dsbA::lacZ transcriptional fusion occurred in the exponential growth phase in contrastto that observed in the wild type where maximal expression occursin stationary phase . Sequence analysis of NLM2173 demonstratedthat the transposon had inserted upstream of the gene encodingH-NS . Western immunoblot analysis using H-NS and StpA antibodiesshowed decreased amounts of H-NS protein in NLM2173, and thisreduction in H-NS correlated with an increase of StpA protein.Northern blot analysis with a dsbA-specific probe showed anincrease in dsbA transcript during exponential phase of growth.Direct binding of H-NS to the dsbA promoter region was verifiedusing purified H-NS in electrophoretic mobility shift assays.Thus, a reduction in H-NS protein is correlated with a derepressionof dsbA in NLM2173, suggesting that H-NS normally plays a rolein suppressing the expression of dsbA during exponential phasegrowth.

 
Salmonella enterica serovar Typhimurium is a major cause of gastroenteritis or food poisoning in humans [34] . This gram-negative,facultative, intracellular pathogen has evolved a number ofdistinct strategies to survive and propagate in a wide varietyof cell types in the host . Many of these strategies involve proteins that are exported from the cytoplasm to either the periplasm or outer membrane or secreted out of the cell [15]. Some proteins are transported or assembled by means of specialized secretory systems, but many of these proteins pass through the periplasm, where they undergo some degree of folding into their native conformation . Disulfide bonds usually contribute to the stabilization of a folded protein conformation [2, 35] . In gram-negativebacteria, disulfide bond formation is mediated by the foldaseDsbA, which is part of a disulfide oxidoreductase system thatincludes other Dsb proteins, such as DsbB, DsbC, and DsbD [2,24, 35] . DsbA, a soluble periplasmic disulfide oxidoreductase, was first discovered in Escherichia coli [4] and has also beencharacterized from a number of gram-negative bacteria, includingS . enterica serovar Typhimurium [49] . Disulfide bonding is anessential step for the proper folding and hence, function, ofa number of disulfide bond-containing proteins that are bacterialvirulence factors, such as exotoxins, fimbriae, and adhesins[52] . Although DsbA is not essential for growth under laboratoryconditions, lack of disulfide oxidoreductase activity in serovarTyphimurium renders cells nonmotile and slows growth in definedminimal medium [49] . Interestingly, in contrast to the observationsmade in E . coli [6], DsbA is growth phase regulated in S . enterica serovar Typhimurium, with expression levels increasing during late exponential phase of growth and remaining elevated forat least 72 h in liquid culture [16] . This stationary-phase regulation is not dependent upon RpoS [16], a common stationary-phasesigma factor [27, 36] or SlyA, a serovar Typhimurium stationary-phase transcriptional regulator [8].

This study details the investigation of a new facet of DsbA regulation involving the global regulator H-NS . By characterizing mutants that were derepressed for expression of dsbA from a plasmid-encoded dsbA::lacZ construct in S . enterica serovar Typhimurium during exponential phase growth, it was determined that H-NS was involved in the growth phase-dependent regulationof dsbA . H-NS is a major protein of the bacterial nucleoid andis involved in the regulation of both housekeeping and virulencegenes in E . coli [10, 21] . H-NS is a small, abundant proteinthat has affinity for all types of nucleic acids but binds preferentiallyto curved DNA substrates [37, 47] . A number of hns mutant alleleshave been shown to cause slow growth, reduce motility, and confermucoid appearance on the mutant strain [5, 19] . H-NS has beenshown to negatively or positively regulate more than 200 genesin E . coli [21] . Many of the target genes that are affectedby H-NS are also regulated by other global transcription factors,such as LRP, VirF, CfaD, RpoS, and the DNA-binding protein FIS[1, 41] . Hence, the effect of H-NS on many target genes is not straightforward . In this study, we demonstrate that H-NS bindsto the dsbA promoter region and that a reduction in the amountof H-NS protein derepresses dsbA expression early in the growth cycle, suggesting that H-NS normally represses dsbA until late log or early stationary phase.

 
Bacterial strains, media, and culture conditions. The bacterial strains and plasmids used in this study are listedin Table 1 . In general, bacteria were grown overnight at 30°C in Luria-Bertani [LB] medium [39] with the appropriate antibioticselection . When required, antibiotics were used at the followingconcentrations: chloramphenicol [30 µg ml^-1], tetracycline[10 µg ml^-1], and ampicillin [100 µg ml^-1] . Whenscreening for blue or white colonies, 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside [X-Gal] was used at a concentration of 40 µg ml^-1.

 
P22 transduction. P22 transduction was performed by the method of Maloy [32].An aliquot of the lysate containing a pool of random Tn10d[T-POPII]insertions in S . enterica serovar Typhimurium LT2 was kindlyprovided by John Roth [38] . Tn10d[T-POPII] is a Tn10 derivativethat has been modified such that its insertion between any geneor operon and its promoter causes the expression of the geneor downstream gene to become tetracycline dependent . StrainNLM2275 was created by transducing a hns null allele [osmZ]from strain S4105 into a SL1344 background.

Motility assays. Salmonella strains were grown overnight at 30°C with appropriateantibiotics . The following morning, all the strains were standardizedto an A₆₀₀ of 0.04 . A flat-ended sterile toothpick was dippedinto standardized bacterial culture and stabbed into semisolid0.3% agar LB plates [19] . The plates were incubated for up to16 h at 30°C, and the swarming behavior of each strain wasmeasured as an indicator of motility.

ß-Galactosidase assays. Transcription of plasmid-borne dsbA-lacZ fusions were monitoredby ß-galactosidase assays of cells cultured to mid-exponentialphase and stationary phase by the method of Miller [33] . Assayswere performed in triplicate, and experiments were done at leastthree times.

RNA protocols. Total RNA was extracted from 3-ml samples of cells at the appropriategrowth phase by the Trizol method [Gibco BRL] . Diethyl pyrocarbonate-treatedwater or formamide was used to resuspend the RNA pellets . Theconcentrations and purity of the RNA samples were determinedspectrophotometrically and by visual inspection of formaldehyde-agarosegels [see below].

For Northern blotting, samples of RNA [30 µg] were denaturedat 65°C, loaded onto 1.5% formaldehyde-agarose gels, electrophoresed within buffer containing 20 mM MOPS [3[N-morpholino] propanesulfonic acid], 5 mM sodium acetate, and 1 mM EDTA at 80 V for 2 to 3h, and transferred to Hybond-N nylon membranes [Amersham] . Themembranes were washed, denatured, neutralized, air dried, andcross-linked following established protocols [40] . The templates used for the DNA probe were a PCR fragment amplified from the sequence of the dsbA gene using NLM88 [5'-CTGGCGAACCCCAGGTACTG-3'] and NLM78 [5'CGCATCAACGAACACTTTACGG-3'] or an amplicon specific for hns using primers NM93 [5'-ATAAGCTCTTTTTTGTGCGGTG-3'] and NM94 [5'-TATTTTTTTCGCGGCCTAAATG-3'] . The DNA fragment was labeled with digoxigenin, and prehybridization and hybridization were performed as recommended by the manufacturer [Amersham] . Chemiluminescence detection as described by the Genius guide was used for probe detection.

For reverse transcriptase PCR [RT-PCR], RNA isolated from S. enterica serovar Typhimurium strains at various phases of growth was subjected to DNase treatment [RQ1 RNase-free DNase; Promega]and subsequent purification using RNeasy columns [RNeasy Minikit; Qiagen] . Reverse transcription reactions [Retroscript kit;Ambion] using 100 pmol of primer NM112 hns primer [5'-GCAGTTTACGAGTGCGTTCTTCC-3'] were performed on approximately 3 µg of purified RNA [Trizol method] . Reverse transcription negative-control reactions were performed simultaneously where water was added instead of theRT enzyme . PCR amplification was performed using forward NM111 [5'-TAGCGACAGACGGTGAGTATCC-3'] and reverse NM112 [5'-GCAGTTTACGAGTGCGTTCTTCC-3'] hns-specific primers . PCRs [50 µl] were performed using 2.5 U of Taq DNA polymerase [Gibco BRL], 5 µl of reverse transcription reaction mixture sample as the template, 50 pmolof each PCR primer pair, 1x PCR buffer, 0.2 mM [each] deoxynucleosidetriphosphate, and 1.5 mM MgCl₂ . Template cDNA was denaturedfor 2 min at 94°C before Taq DNA polymerase was added . Twentycycles of PCR were performed, with 1 cycle consisting of denaturation[45 s at 4°C], annealing [30 s at 60°C], and extension[1 min at 72°C] . The final extension step was 7 min at 72°C.To aid in qualitative analysis, we normalized the RT-PCR productto an established endogenous internal control [tsf encodingthe elongation factor EF-Tsf] [20] . After the PCR, 5 µlof PCR product was visualized by agarose gel electrophoresis.

Gel electrophoresis and Western blotting. Proteins were separated by the method of Laemmli [26] usinga sodium dodecyl sulfate-12% polyacrylamide gel . Cell cultureswere centrifuged at a specific optical density, and the cellpellet was resuspended in loading buffer and boiled before loading.The amount of protein in each whole-cell lysate was determined,and equal amounts of protein [2 x 10⁷ cells] were loaded ineach lane . The expression of StpA and H-NS proteins in S . entericaserovar Typhimurium was determined using anti-StpA polyclonalantibody [S100] that does not cross-react with H-NS and anti-H-NSmonoclonal antibody [H113] that does not cross-react with StpA[45].

Electrophoretic mobility shift assay. Purified H-NS protein from S . enterica serovar Typhimurium waskindly provided by John Ladbury [Department of Biochemistryand Molecular Biology, University College London] . The bandshift reaction contained various concentrations of H-NS proteinin the picomolar range and 10 ng of radiolabeled probe DNA inbinding buffer [10 mM Tris-HCl [pH 7.5], 15 mM KCl, 0.1 mM EDTA,2 mM spermidine, 15% glycerol] and was performed as describedpreviously [23] . The template used for the DNA probe was a PCRfragment amplified from the promoter region of the dsbA geneusing primer pair NLM22 [5'-ACAAGATCTATTAATACATTGGCGTT-3'] andNLM24 [5'-CCCCTCGAGAAGCTTATCAAGAAGTT-3'] and primer pair NM111 [5'-TAGCGACAGACGGTGAGTATCC-3'] and NM112 [5'-GCAGTTTACGAGTGCGTTCTTCC-3'] for the promoter region of hns . The reaction mixture was incubated at room temperature for 30 min, and the samples were loaded onto a 5% polyacrylamide gel in Tris-acetate-EDTA and electrophoresed at 35 mA for 2 h . After electrophoresis, the gel was dried,and radiolabeled DNA was detected by autoradiography.

 
Transposon mutagenesis strategy for the isolation of dsbA regulatory mutants. A pool of random Tn10d[T-POPII] [38] insertions from S . entericaserovar Typhimurium LT2 was obtained as a P22 phage lysate andused to transduce S . enterica serovar Typhimurium SL1344 carryinga dsbA::lacZ transcriptional fusion on a low-copy-number plasmid,pMEG2 [strain NLM2160] . This dsbA::lacZ fusion is normally regulated by growth phase, with maximal induction of expression occurringupon entry into the stationary phase of growth [16] . The pool of mutants resulting from T-POPII insertion mutations in strain NLM2160 were screened for those altered in dsbA regulation by monitoring ß-galactosidase activity of isolated coloniesusing X-Gal . White colonies were chosen, as these colonies wereconsidered potential regulatory mutants . Of several white coloniesisolated, the phenotypes of three of these colonies was causedby a single transposon insertion, as confirmed by 100% linkageof the dsbA::lacZ phenotype with the tetracycline resistancemarker upon transduction into a fresh strain of SL1344 [NLM2160].Two of these insertion mutants contained the T-POPII insertionin the same location, and this mutant was designated NLM2173,while the third remains to be characterized . The mutant phenotypeof NLM2173 was somewhat unstable at temperatures higher than30°C where secondary mutations led to renewed expressionof ß-galactosidase . These suppressor mutants were visible as blue sectors in the colonies on agar plates containing X-Gal . This suppressor phenotype, as exemplified by strain NLM2174, was able to grow in the presence of tetracycline, confirming the maintenance of the transposon . The suppressor phenotypewas not, however, cotransduced with the tetracycline resistancemarker, confirming that the secondary mutations were not closelylinked to the transposon insertion site . The ratio of the appearanceof suppressor mutants to the original mutant was observed tobe much lower at 30°C [0.55%] than at 37°C [24%].

On solid LB agar plates, the mutant NLM2173 produced coloniesthat were generally smaller in size than strain SL1344 or thesuppressor mutant, NLM2174 . When grown in LB broth at 30°C,the growth rate of strain NLM2173 was lower than that of theparental strain [Fig. 1A] . In addition to the difference inthe growth rate of strain NLM2173, a decrease in motility wasalso observed . The parental strain NLM2160 was fully motile[diameter of the motility zone, 5.0 ± 0.4 cm], whereasstrain NLM2173 had reduced motility [diameter of the motilityzone, 2.8 ± 0.2 cm].

 
Expression of dsbA::lacZ transcriptional fusion in S . enterica serovar Typhimurium strains. In order to measure the effects of the transposon insertionon dsbA promoter activity, the ß-galactosidase activityfrom strain NLM2173 containing the dsbA::lacZ transcriptionalfusion was measured throughout the growth cycle [Fig . 1B] . Asexpected, maximal expression of the dsbA::lacZ fusion occurredat the onset of stationary phase in wild-type strain NLM2160.In contrast, strain NLM2173, in either the absence or presenceof tetracycline, showed a shift in the activation of the dsbApromoter to earlier in the growth cycle, from stationary phaseto log phase . If the Tn10d[T-POPII] transposon were to disrupta promoter, then the expression of any downstream gene couldbecome tetracycline dependent [38] . This was not the case, however, for NLM2173 . In addition, stationary-phase levels of dsbA promoter activity in NLM2173 are lower than in the wild type . The dsbA::lacZ expression was also assayed in the suppressor strain NLM2174, where no shift in the induction of dsbA::lacZ expression was observed and the stationary-phase ß-galactosidaselevels were the same as those of the wild type [Fig . 1B] . When the Tn10d[T-POPII] interrupted locus was transduced into a dsbA null strain, the overall level of dsbA promoter induction was even higher than in the wild type but still occurred prior to the onset of stationary-phase growth [Fig . 1C] . Previous studieshave shown that the dsbA::lacZ activity from pMEG2 is higherin a dsbA null background than in a wild-type background [16].These observations led to the hypothesis that a feedback loopexists for the expression of DsbA in S . enterica serovar Typhimurium,whereby the absence of DsbA activity signals the cell to producemore DsbA [16], and this mechanism of autoregulation appearsto be independent of the effect generated by the T-POPII insertion.

Localization and identification of the site of transposon insertion. Genomic DNA from strain NLM2173 was digested with SacI, SalI, and HindIII, shotgun cloned into pBluescript, and selected by screening for clones that conferred tetracycline resistance. Using outward facing primers specific to the Tn10d[T-POPII] transposon, the regions flanking the transposon were sequencedand localized to a 3,629-bp contig [B-STM1107] from the S . enterica serovar Typhimurium sequencing project . The transposon insertion occurred 580 bp upstream of the hns coding region and 116 bp upstream of the tdk gene on the opposite strand encoding a thymidinekinase . Two other open reading frames could be recognized onthis genomic DNA fragment; downstream of hns, a putative galUgene was detected, and upstream of hns, a putative adhE genewas also found [Fig . 2] . Mutations at the hns locus are highlypleiotropic [5, 19, 53] . In general, hns mutant strains growmore slowly than wild-type strains, show reduced motility, andare mucoid in nature [5] . These phenotypic characteristics ofan hns mutant were also shared by the mutant strain NLM2173,consistent with hns being the locus that affected dsbA regulation.

 
It was not immediately apparent how the T-POPII insertion 580bp upstream of the hns coding region, produced an hns phenotype. However, Hinton et al . [19] and Hulton et al . [22] have demonstratedthat S . enterica serovar Typhimurium strain CH1794, which containsa Tn10 insertion 377 bp upstream of the translational initiationcodon of hns, produced reduced levels of H-NS protein than otherhns mutants and the wild type . Unlike mutations within the hnsstructural gene, this hns-106::Tn10d insertion caused only alow level of derepression of the proU locus and had no detectedeffect on DNA supercoiling [19] . This hns mutant strain wasalso included in this study for comparison.

hns mutations differentially affect the levels of dsbA mRNA at mid-logarithmic growth. To examine the effects of hns mutations on dsbA transcription,Northern blot analysis was performed on total RNA extractedfrom several strains using a probe complementary to the transcribeddsbA gene . Goecke et al . [16] previously showed that two transcriptswere consistently detected for dsbA and that the amount of thesetwo transcripts varied with growth conditions . In the presentstudy, this transcription pattern was observed in the wild-typestrain NLM2160 and the suppressor mutant NLM2174 . However, therewas a substantial increase in the amount of the shorter dsbA-specifictranscripts compared to the larger transcript in strains NLM2173and CH1794 relative to the wild type or the suppressor strain[Fig . 3] . This increase in the amount of dsbA transcript duringlog phase growth correlated with the increase in expressionof the dsbA::lacZ fusion, suggesting that dsbA promoter activityis elevated in strain NLM2173.

 
Comparison of expression from the dsbA promoter in hns mutant strains. The effect on dsbA transcription of the Tn10 insertion 377 bpupstream of the translational initiation codon of hns [strainCH1794] was compared to that of the T-POPII insertion mutantNLM2173 under log phase growth conditions [Fig . 4] . Both NLM2173and CH1794 showed increased dsbA promoter activity at mid- andlate log phase, suggesting that although the transposons areinserted 200 bases apart from each other, their effect on hnsis similar . An hns null strain [NLM2275] was also tested andhad even higher levels of ß-galactosidase activitythan NLM2173 . NLM2174 was also included in this comparison, and although the dsbA promoter activity is closer to the wild type in this suppressor strain, it is not identical to thatof the wild type.

 
H-NS binds to the dsbA promoter region. In order to determine whether H-NS could interact directly withthe dsbA promoter region, band shift assays were performed.As H-NS had previously been shown to bind to its own promoterwith high affinity [11, 51], the hns promoter region was usedas a control for binding specificity . The results show that the dsbA promoter fragment begins to shift at an H-NS concentration equivalent to that required to demonstrate binding to the hns promoter [Fig . 5] . This binding is specific, as demonstratedby the fact that a mobility shift does not occur in the 112-bpdigested hns fragment but does occur in the 267-bp fragmentpreviously shown to contain the H-NS binding domain [Fig. 5].

 
H-NS expression is altered in strains NLM2173 and CH1794. As the results indicated that H-NS was affecting the dsbA promoter activity, Western immunoblotting using a H-NS-specific monoclonal antibody was undertaken to monitor steady-state H-NS levels.As previous work had demonstrated that both H-NS and the homologous protein, StpA, are implicated in a global regulatory systemand that the stpA gene is derepressed in hns mutants of E . coli [12, 44, 4], StpA protein levels were also monitored using anStpA polyclonal antibody . If a reduction in H-NS protein wasoccurring in NLM2173 and CH1794, increased expression of StpAwould be expected . The amount of H-NS protein produced was lowerin strain NLM2173 than in wild-type strain NLM2160, and thereverse pattern was observed when the same samples were probedwith the StpA-specific antibody [Fig . 6] . Thus, H-NS proteinproduction was decreased in the transposon mutant, and thisreduction in H-NS protein was associated with an increase in expression of StpA . A similar decrease in H-NS and increasein StpA levels were also observed in CH1794 [data not shown].

 
hns transcription and growth phase. Although the connection between decreased H-NS protein and increaseddsbA transcription was established, it was not clear how transposons inserted either 377 or 580 bases upstream of the hns coding region could cause a decrease in H-NS protein levels . Transcriptional analysis of hns was undertaken to assess the effects of these transposons on hns transcript abundance . RT-PCR was used to assess the amount of hns transcript produced at different time points during the growth cycle [Fig . 7] . For each strain, an hns-specific transcript was detected at all growth phases tested. In the wild-type strain [NLM2160], different amounts of the RT-PCR product were produced in the different growth phases,with more abundant transcript being detected earlier in thegrowth cycle, as expected . However, the relative intensitiesof the RT-PCR product in strains NLM2173 and CH1794 were higherin mid-log phase than those of the wild-type strain, NLM2160,and the suppressor strain, NLM2174 . These RT-PCR results weresurprising, as they did not correlate with the data showinga reduction in the steady-state H-NS protein levels, but theseresults were confirmed when Northern blotting or multiplex RT-PCR[17] were used to assess the hns transcript [data not shown].

 
The growth phase-regulated expression of dsbA in S . enterica serovar Typhimurium has been shown to be independent of the stationary-phase sigma factor, RpoS, and the transcriptional activator, SlyA [16] . This study was thus initiated to determinethe factors that may be involved in influencing the levels ofdsbA expression in the cell . Screening a T-POPII mutant libraryusing a dsbA::lacZ transcriptional fusion led to the isolationof strain NLM2173 that exhibited alterations in its abilityto regulate dsbA . Cloning and sequencing of the transposon-containingDNA fragments from strain NLM2173 revealed that the zde-5A1t::Tn10d[T-POPII]locus is around 38.4 min on the chromosome of S . enterica serovarTyphimurium, 116 bp upstream of the thymidine kinase gene [tdk]and 580 bp upstream of the hns gene . Several phenotypic characteristics of the mutant, such as increased mucoidy, decreased growth rate,and decreased motility, suggested that modification of expressionof the hns locus was being affected . The zde-5A1t::T-POPII mutation was later designated hns-112::Tn10d[T-POPII] . NLM2173 was ableto grow on plates containing thymidine kinase [25], confirmingthat the tdk locus was not affected by hns-112::Tn10d [datanot shown] . The motility of strain NLM2173 is about 56% that of the wild-type strain, suggesting that the transposon insertion does not completely eliminate the expression of H-NS but changes the level of H-NS expressed in the cell . Motility is reducedto different levels by several hns alleles, and it was previously shown in strain CH1794 that the insertion 377 bp upstream ofthe translation initiation codon of hns also resulted in a partial loss of motility [55% of the wild type], while a strain withan hns null allele lacks flagella [19] . In E . coli, the presenceof an hns mutation decreased the transcription of flhD and fliAgenes required for the synthesis of flagella [7, 46], a ratherrare example of H-NS acting as a positive regulator . DsbA isalso involved in flagellar biosynthesis . Work done by Bardwellet al . [3] showed that DsbA is essential for flagellar assemblyand function in E . coli, and Turcot et al . [49] demonstrated that an S . enterica serovar Typhimurium dsbA null strain was also immotile . Thus, the decrease in motility observed in the transposon mutant strains in the present study is unlikely tobe related to reduced expression of the wild-type dsbA gene, since the expression of dsbA is derepressed in NLM2173.

The phenomenon of spontaneous second-site mutations arisingin hns mutants has been observed previously [5, 18, 30] . Inaddition, Barth et al . [5] found that hns suppressor strains had lost the increased mucoidy characteristic for hns mutants and grew faster, exhibiting shorter doubling times, than theparental strains, as was seen with the present study . Barthet al . [5] found that some of their suppressor mutants carriedalterations at the rpoS locus, raising the possibility thatthe suppressor mutation in strain NLM2174 was in the rpoS gene.Qualitative assays of catalase activity showed an increase inNLM2173 relative to the wild type, but catalase activity inNLM2174 was similar to NLM2173 [data not shown] . This increasein catalase activity in the hns mutant probably resulted fromincreased rpoS transcription [5] and, because activity was unchanged in NLM2174, suggests that the suppressor mutation in NLM2174is not located in the rpoS gene.

Figure 1B clearly shows that expression of the dsbA::lacZ transcriptionalfusion in strain NLM2173 occurs earlier in the growth cycle,with a twofold derepression of dsbA expression in mid- and latelog phase [Fig . 4] . This twofold derepression of dsbA was alsoseen for the expression of the dsbA::lacZ transcriptional fusionin another hns mutant strain [CH1794] that also contains a Tn10insertion upstream of the hns coding region, while analysisof the hns null mutant NLM2275 showed even higher levels ofdsbA promoter activity [Fig . 4] . Taken together, these results imply that the level of H-NS in the cell influences the transcription of dsbA in S . enterica serovar Typhimurium . H-NS is known to act as a transcriptional repressor by binding to DNA in the promoter region [51] and shows a preference for binding to intrinsicallycurved DNA [9] . Sequence analysis of the region upstream ofthe dsbA translation start site revealed the presence of a regionpredicted to bend [data not shown] . Band shift assays with purifiedH-NS demonstrated high-affinity binding to the dsbA promoterregion, suggesting that normally dsbA expression is directlyrepressed by H-NS [Fig. 5] . By surveying the literature, Atlungand Ingmer [1] determined that H-NS has a larger effect on target gene expression in scenarios where expression is not also mediated by positive transcription factors and noted that any repression by H-NS is virtually eliminated when positive transcriptionfactors are artificially induced . We see an increase in dsbApromoter activity in NLM2173 during exponential phase of onlytwo- to threefold, the magnitude of which could be influencedby a positive regulator; it also could be due to the fact thatH-NS is not completely abolished.

H-NS is autoregulated [11, 12, 50], positively regulated atthe transcriptional level [41], and posttranscriptionally regulatedby DsrA RNA [28] . Free and Dorman [12] found that the hns transcriptis virtually absent in stationary-phase cells but is presentat high levels within 1 h of subculturing a stationary-phaseculture . Additionally, Dorman et al . [10] showed that the ratioof H-NS synthesis to DNA synthesis was constant, which couldexplain why hns expression is reduced in stationary phase whenDNA synthesis slows . In the present study, steady-state levelsof H-NS in the wild-type strain were seen to be slightly higherin exponential phase growth than in stationary phase . The levelsof H-NS were decreased in NLM2173 than in the wild type, correlatingwith the observed derepression of the dsbA promoter in exponentialphase.

The steady-state levels of StpA were also examined . StpA isa paralogue of H-NS that shows 52% identity at the amino acidlevel and has a DNA-binding affinity that is comparable to thatof H-NS [45] . H-NS and StpA can act cooperatively to repressmany H-NS-regulated genes [13, 14] . Furthermore, it has been demonstrated that the expression of stpA is derepressed in an hns mutant strain [14, 44] . This increase in StpA expressionin an hns mutant strain appears to compensate for the lack ofH-NS and allows repression of many H-NS-regulated genes [45],although not all H-NS-repressed loci can be regulated by StpA[10, 54] . In this study, StpA levels were higher in strain NLM2173 than in the wild type [NLM2160], showing that the hns-112::Tn10d mutation caused a sufficient decrease in H-NS levels to exerta biologically relevant effect . However, the increase in StpAprotein levels in NLM2173 did not allow StpA to substitute forH-NS in the repression of the dsbA promoter.

It was not clear how transposon insertions significantly upstream of the hns coding region caused a decrease in H-NS levels, especially since the transposons were inserted further from the promoter region than any previously described regulatory regions . Transcriptional autorepression occurs as a result of H-NS binding to extended regions of DNA 150 nucleotides upstream of its coding region[11, 48] . In the present study, it was hypothesized that the T-POPII and Tn10 transposons somehow affect the hns promoter region, lowering the transcription of the hns locus . Using RT-PCR [Fig . 7], it was shown that the level of hns transcript is abundantearly in the growth cycle and lower when the cells reach stationaryphase in the wild-type strain . However, there was a marked increasein the amount of hns transcript in strains NLM2173 and CH1794at mid-log phase compared to that of the wild type, suggestingthat the transposon insertions enhanced hns transcription . BothNorthern blotting and multiplex primer extension approachesto measuring the hns transcript abundance also showed increasedtranscription in NLM2173 and CH1794 than in the wild type [datanot shown] . The data clearly establish enhanced hns transcription,suggesting that the transposons have affected a previously uncharacterizedregulatory element upstream of hns . The data also suggest thatautoregulation of hns transcription has been disrupted, as theresults are similar to that seen in an hns deletion strain wherebasal hns transcription levels are more than twofold higherthan the level in the wild type [29].

With hns transcription increased, the observed decrease in H-NS protein levels still requires an explanation . It is hypothesized that DsrA, a small RNA, is involved in the repression of H-NS in these mutants . DsrA is an untranslated, regulatory RNA thatis involved in the expression of RpoS [31, 43] and H-NS [29,31, 42] . It is thought that DsrA and hns mRNA interact and thatthis interaction enhances the turnover of hns mRNA, resultingin the production of less H-NS protein [28] . In experimentsperformed by Lease et al . [29], DsrA expression decreased H-NSprotein levels in a wild-type background and had no effect onthe level of hns transcript . Lease et al . [29] also showed thatStpA is still produced when DsrA is overexpressed, and it appearsthat the DsrA-mediated reduction in H-NS actually leads to anincrease in StpA levels [29] . A direct connection between increased hns transcription and increased DsrA activity remains to be established in future experiments.

In this study, we have demonstrated that a reduction of H-NS protein correlates with a derepression of dsbA expression in log phase . Since the regulation of DsbA is growth phase dependent, the involvement of H-NS, a protein abundant in log phase, fitswith the expression profile of this disulfide oxidoreductase.There must also be as yet unidentified positively regulatingfactors involved in dsbA transcription to account for the increasein stationary-phase expression . DsbA appears to facilitate protein folding in stationary phase rather than exponential growth phase where it is expected that protein secretion would require foldasesin order to be rapid and efficient . Given the involvement ofH-NS in the expression of genes related to cell survival understressful growth conditions, DsbA expression may reflect theneed for foldase activity in the context of environmental factorscausing stress to the bacterial cell.

 
We are grateful to John Roth who provided us with an aliquotof the lysate containing a pool of random Tn10d[T-POPII] inS . enterica serovar Typhimurium LT2 . Purified H-NS was kindly provided by John Ladbury, Paul McDermott provided protocolsfor Western immunoblotting, and Martin Goldberg provided themultiplex primer extension protocol . We thank D . Low for strainCH1794 [DL3157 [his strain designation]].

This work was supported by a Canadian Institutes of Health Research [CIHR] grant to N . L . Martin.

 
* Corresponding author . Mailing address: Department of Microbiology and Immunology, Queen's University, Kingston, Ontario, Canada K7L 3N6 . Phone: [613] 533-2460 . Fax: [613] 533-6796 . E-mail: nlm@post.queensu.ca .

C.V.G . and T.P . contributed equally to the work presented inthis report.

1. Atlung, T., and H . Ingmer. 1997 . H-NS: a modulator of environmentally regulated gene expression . Mol . Microbiol . 24:7-17.
2. Bardwell, J . C . A., and J . Beckwith. 1993 . The bonds that tie: catalyzed disulfide bond formation . Cell 74:769-771.
3. Bardwell, J . C . A., J . O . Lee, G . Jander, N . Martin, D . Belin, and J . Beckwith. 1993 . A pathway for disulfide bond formation in vivo . Proc . Natl . Acad . Sci . USA 90:1038-1042.
4. Bardwell, J . C . A., K . McGovern, and J . Beckwith. 1991 . Identification of a protein required for disulfide bond formation in vivo . Cell 67:581-589.
5. Barth, M., C . Marschall, A . Muffler, D . Fischer, and R . Hengge-Aronis. 1995 . Role for the histone-like protein H-NS in growth phase-dependent and osmotic regulation of sigma S and many sigma S-dependent genes in Escherichia coli . J . Bacteriol . 177:3455-3464.
6. Belin, P., and P . L . Boquet. 1994 . The Escherichia coli dsbA gene is partly transcribed from the promoter of a weakly expressed upstream gene . Microbiology 140:3337-3348.
7. Bertin, P., E . Terao, E . H . Lee, P . Lejeune, C . Colson, A . Danchin, and E . Collatz. 1994 . The H-NS protein is involved in the biogenesis of flagella in Escherichia coli . J . Bacteriol . 176:5537-5540.
8. Buchmeier, N., S . Bossie, C . Y . Chen, F . C . Fang, D . G . Guiney, and S . J . Libby. 1997 . SlyA, a transcriptional regulator of Salmonella typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages . Infect . Immun. 65:3725-3730.
9. Dame, R . T., C . Wyman, and N . Goosen. 2001 . Structural basis for preferential binding of H-NS to curved DNA . Biochimie 83:231-234.
10. Dorman, C . J., J . C . Hinton, and A . Free. 1999 . Domain organization and oligomerization among H-NS-like nucleoid-associated proteins in bacteria . Trends Microbiol . 7:124-128.
11. Falconi, M., N . P . Higgins, R . Spurio, C . L . Pon, and C . O . Gualerzi. 1993 . Expression of the gene encoding the major bacterial nucleotide protein H-NS is subject to transcriptional auto-repression . Mol . Microbiol . 10:273-282.
12. Free, A., and C . J . Dorman. 1995 . Coupling of Escherichia coli hns mRNA levels to DNA synthesis by autoregulation: implications for growth phase control . Mol . Microbiol . 18:101-113.
13. Free, A., M . E . Porter, P . Deighan, and C . J . Dorman. 2001 . Requirement for the molecular adapter function of StpA at the Escherichia coli bgl promoter depends upon the level of truncated H-NS protein . Mol . Microbiol . 42:903-917.
14. Free, A., R . M . Williams, and C . J . Dorman. 1998 . The StpA protein functions as a molecular adapter to mediate repression of the bgl operon by truncated H-NS in Escherichia coli . J . Bacteriol . 180:994-997 .
15. Galan, J . E., and A . Collmer. 1999 . Type III secretion machines: bacterial devices for protein delivery into host cells . Science 284:1322-1328 .
16. Goecke, M., C . Gallant, P . Suntharalingam, and N . L . Martin. 2002 . Salmonella typhimurium DsbA is growth phase regulated . FEMS Lett. 206:229-234.
17. Goldberg, M . D., M . Johnson, J . C . Hinton, and P . H . Williams. 2001 . Role of the nucleoid-associated protein Fis in the regulation of virulence properties of enteropathogenic Escherichia coli . Mol . Microbiol . 41:549-559.
18. Harrison, J . A., D . Pickard, C . F . Higgins, A . Khan, S . N . Chatfield, T . Ali, C . J . Dorman, C . E . Hormaeche, and G . Dougan. 1994 . Role of hns in the virulence phenotype of pathogenic salmonellae . Mol . Microbiol . 13:133-140.
19. Hinton, J . C., D . S . Santos, A . Seirafi, C . S . Hulton, G . D . Pavitt, and C . F . Higgins. 1992 . Expression and mutational analysis of the nucleoid-associated protein H-NS of Salmonella typhimurium . Mol . Microbiol . 6:2327-2337.
20. Holmstrom, K., T . Toker-Nielsen, and S . Molin. 1999 . Physiological states of individual Salmonella typhimurium cells monitored by in situ reverse transcription-PCR . J . Bacteriol . 181:1733-1738 .
21. Hommais, F., E . Krin, C . Laurent-Winter, O . Soutourina, A . Malpertuy, J . P . Le Caer, A . Danchin, and P . Bertin. 2001 . Large-scale monitoring of pleiotropic regulation of gene expression by the prokaryotic nucleoid-associated protein, H-NS . Mol . Microbiol . 40:20-36.
22. Hulton, C . S., A . Seirafi, J . C . Hinton, J . M . Sidebotham, L . Waddell, G . D . Pavitt, T . Owen-Hughes, A . Spassky, H . Buc, and C . F . Higgins. 1990 . Histone-like protein H1 [H-NS], DNA supercoiling, and gene expression in bacteria . Cell 63:631-642.
23. Jordi, B . J., A . E . Fielder, C . M . Burns, J . C . Hinton, N . Dover, D . W . Ussery, and C . F . Higgins. 1997 . DNA binding is not sufficient for H-NS-mediated repression of proU expression . J . Biol . Chem . 272:12083-12090 .
24. Kadokura, H., F . Katzen, and J . Beckwith. 2003 . Protein disulfide bond formation in prokaryotes . Annu . Rev . Biochem . 72:111-135.
25. Kaehler, R., M . Strauss, and U . Kiessling. 1984 . A well transformable E . coli tdk-strain—suitable for direct rescue of tk gene plasmids from mammalian cells . Biomed . Biochim . Acta 43:K25-K29.
26. Laemmli, U . K. 1970 . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature 227:680-685.
27. Lange, R., and R . Hengge-Aronis. 1991 . Identification of a central regulator of stationary-phase gene expression in Escherichia coli . Mol . Microbiol . 5:49-59.
28. Lease, R . A., and M . Belfort. 2000 . Riboregulation by DsrA RNA: trans-actions for global economy . Mol . Microbiol . 38:667-672.
29. Lease, R . A., M . E . Cusick, and M . Belfort. 1998 . Riboregulation in Escherichia coli: DsrA RNA acts by RNA:RNA interactions at multiple loci . Proc . Natl . Acad . Sci . USA 95:12456-12461 .
30. Lejeune, P., and A . Danchin. 1990 . Mutations in the bglY gene increase the frequency of spontaneous deletions in Escherichia coli K-12 . Proc . Natl . Acad . Sci . USA 87:360-363.
31. Majdalani, N., C . Cunning, D . Sledjeski, T . Elliott, and S . Gottesman. 1998 . DsrA RNA regulates translation of RpoS message by an anti-antisense mechanism, independent of its action as an antisilencer of transcription . Proc . Natl . Acad . Sci . USA 95:12462-12467 .
32. Maloy, S . R. 1990 . Experimental techniques in bacterial genetics . Jones and Bartlett Publishers, Inc., Boston, Mass.
33. Miller, J . H. 1972 . Experiments in molecular genetics . Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
34. Miller, S . I., E . Hohmann, and D . Pegues. 1995 . Salmonella [including Salmonella typhi] . Churchill Livingstone, New York, N.Y.
35. Missiakas, D., and S . Raina. 1997 . Protein folding in the bacterial periplasm . J . Bacteriol . 179:2465-2471.
36. Nguyen, L . H., D . B . Jensen, N . E . Thompson, D . R . Gentry, and R . R . Burgess. 1993 . In vitro functional characterization of overproduced Escherichia coli katF/rpoS gene product . Biochemistry 32:11112-11117.
37. Owen-Hughes, T . A., G . D . Pavitt, D . S . Santos, J . M . Sidebotham, C . S . Hulton, J . C . Hinton, and C . F . Higgins. 1992 . The chromatin-associated protein H-NS interacts with curved DNA to influence DNA topology and gene expression . Cell 71:255-265.
38. Rappleye, C . A., and J . R . Roth. 1997 . A Tn10 derivative [T-POP] for isolation of insertions with conditional [tetracycline-dependent] phenotypes . J . Bacteriol . 179:5827-5834.
39. Roth, R . J. 1970 . Genetic techniques in studies of bacterial metabolism . Methods Enzymol . 17A:3-35.
40. 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.
41. Schroder, O., and R . Wagner. 2002 . The bacterial regulatory protein H-NS—a versatile modulator of nucleic acid structures . Biol . Chem . 383:945-960.
42. Sledjeski, D., and S . Gottesman. 1995 . A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli . Proc . Natl . Acad . Sci . USA 92:2003-2007.
43. Sledjeski, D . D., A . Gupta, and S . Gottesman. 1996 . The small RNA, DsrA, is essential for the low temperature expression of RpoS during exponential growth in Escherichia coli . EMBO J . 15:3993-4000.
44. Sonden, B., and B . E . Uhlin. 1996 . Coordinated and differential expression of histone-like proteins in Escherichia coli: regulation and function of the H-NS analog StpA . EMBO J . 15:4970-4980.
45. Sonnenfield, J . M., C . M . Burns, C . F . Higgins, and J . C . Hinton. 2001 . The nucleoid-associated protein StpA binds curved DNA, has a greater DNA-binding affinity than H-NS and is present in significant levels in hns mutants . Biochimie 83:243-249.
46. Soutourina, O., A . Kolb, E . Krin, C . Laurent-Winter, S . Rimsky, A . Danchin, and P . Bertin. 1999 . Multiple control of flagellum biosynthesis in Escherichia coli: role of H-NS protein and the cyclic AMP-catabolite activator protein complex in transcription of the flhDC master operon . J . Bacteriol . 181:7500-7508 .
46. Spaink, H . P., R . J . H . Okker, C . A . Wijffelman, E . Pees, and B . J . J . Lugtenberg. 1987 . Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1J1 . Plant Mol . Biol . 9:27-39.
47. Spurio, R., M . Durrenberger, M . Falconi, A . La Teana, C . L . Pon, and C . O . Gualerzi. 1992 . Lethal overproduction of the Escherichia coli nucleoid protein H-NS: ultramicroscopic and molecular autopsy . Mol . Gen . Genet . 231:201-211.
48. Spurio, R., M . Falconi, A . Brandi, C . L . Pon, and C . O . Gualerzi. 1997 . The oligomeric structure of nucleoid protein H-NS is necessary for recognition of intrinsically curved DNA and for DNA bending . EMBO J . 16:1795-1805 .
49. Turcot, I., T . V . Ponnampalam, C . W . Bouwman, and N . L . Martin. 2001 . Isolation and characterization of a chromosomally encoded disulphide oxidoreductase from Salmonella enterica serovar Typhimurium . Can . J . Microbiol . 47:711-721.
50. Ueguchi, C., M . Kakeda, and T . Mizuno. 1993 . Autoregulatory expression of the Escherichia coli hns gene encoding a nucleoid protein: H-NS functions as a repressor of its own transcription . Mol . Gen . Genet . 236:171-178.
51. Ueguchi, C., and T . Mizuno. 1993 . The Escherichia coli nucleoid protein H-NS functions directly as a transcriptional repressor . EMBO J . 12:1039-1046.
51. Wray, C., and W . J . Sojka. 1978 . Experimental Salmonella typhimurium in calves . Res . Vet . Sci . 25:139-143.
52. Wulfing, C., and A . Pluckthun. 1994 . Protein folding in the periplasm of Escherichia coli . Mol . Microbiol . 12:685-692.
53. Yamashino, T., C . Ueguchi, and T . Mizuno. 1995 . Quantitative control of the stationary phase-specific sigma factor, sigma S, in Escherichia coli: involvement of the nucleoid protein H-NS . EMBO J. 14:594-602.
54. Zhang, A., S . Rimsky, M . E . Reaban, H . Buc, and M . Belfort. 1996 . Escherichia coli protein analogs StpA and H-NS: regulatory loops, similar and disparate effects on nucleic acid dynamics . EMBO J . 15:1340-1349.

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