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Journal of Bacteriology, November 2006, p. 7581-7591, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00685-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Reciprocal Transcriptional and Posttranscriptional Growth-Phase-Dependent Expression of sfh, a Gene That Encodes a Paralogue of the Nucleoid-Associated Protein H-NS

Marie Doyle and Charles J. Dorman^*

Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Ireland

Received 15 May 2006/ Accepted 16 August 2006

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IncHI1 self-transmissible plasmid pSf-R27 from Shigella flexneri 2a strain 2457T harbors sfh, a gene that codes for a protein with strong amino acid sequence homology to the global transcription regulator and nucleoid-associated protein H-NS and to its paralogue, StpA. Previously, we discovered that the expression of sfh mRNA is growth phase dependent such that in cultures growing in Lennox broth at 37°C, the transcript is readily detectable in the early stages of exponential growth but is not detectable at the onset of stationary phase. In contrast, the Sfh protein is poorly expressed in early-exponential growth when sfh mRNA is abundant whereas it is expressed to a high level in early stationary phase, when sfh transcript expression is low (P. Deighan, C. Beloin, and C. J. Dorman, Mol. Microbiol. 48:1401-1416, 2003). This unusual pattern of reciprocal mRNA and protein expression is not due to growth phase-dependent effects on either mRNA or protein stability, nor is it due to the known abilities of the Sfh, StpA, and H-NS proteins to influence sfh gene expression. Instead, our data point to a blockade of sfh mRNA translation in early-exponential growth that is relieved as the culture enters the stationary phase of growth. Replacing the 5' end and translation initiation signals of the sfh mRNA with heterologous sequences did not alter the growth phase-dependent expression of the Sfh protein, suggesting that growth phase control of translation is intrinsic to another component of the message.

Top Abstract Introduction Materials and Methods Results Discussion References

 
DNA supercoiling and the nucleoid-associated proteins collaborate to organize the structure of the bacterial nucleoid. These factors also influence transcription on a global level and modulate other major DNA transactions, such as replication, recombination (general and site specific), and transposition (17, 24, 47). H-NS is a prominent member of the nucleoid-associated protein family. It can constrain DNA supercoiling both in vitro and in vivo, and it influences transcription throughout the cell, usually by acting as a repressor (10, 11, 15, 41). H-NS has been identified as a repressor of virulence gene transcription in several gram-negative pathogens, including Shigella flexneri (4, 15, 16, 39). Here, it binds to the promoters of the major virulence gene operons located on the 230-kb virulence plasmid (4), where the transcription activator VirF or VirB counteracts its gene silencing activity (3, 6, 35, 39, 46). Virulence gene activation occurs under growth conditions that approximate those found in the lower human gut and involves a combination of environmental signals, such as temperature, osmolarity, and pH (16, 37). The molecular mechanism of gene activation operates through disruption of the H-NS-imposed silencing of the virulence gene promoters. The model that currently applies in the case of the S. flexneri virulence genes envisions an environmentally wrought change in DNA structure accompanied by a disruption of the H-NS-DNA complex leading to transcription derepression (16, 39).

S. flexneri 2a strain 2457T has been used extensively in studies of Shigella virulence gene regulation. It harbors an IncHI1 plasmid that is closely related to R27 but lacks genes for resistance to antibiotics or other antimicrobials (4, 50). This plasmid has not been found in other S. flexneri isolates, although the R27 prototype plasmid is widely distributed among epidemic strains of Salmonella enterica serovar Typhi from South and Southeast Asia (48, 49) and was discovered originally in S. enterica serovar Typhimurium (42).

The plasmid from strain 2457T, pSf-R27, encodes an H-NS-like protein called Sfh. This brings to three the number of H-NS-like proteins found in this bacterium: H-NS, StpA, and Sfh. Each protein can repress the transcription of its own gene and the genes coding for the other two paralogues (12). In addition, each protein can form a heteromeric complex with either of the other two, presumably owing to the strong amino acid sequence conservation seen in their oligomerization domains (12). There has been considerable speculation that the homomeric and heteromeric forms of H-NS-like proteins might have distinct biological activities (18, 27, 43, 51). Knockout mutants lacking Sfh do not have obvious phenotypes (12). This can be explained by the abilities of the H-NS, StpA, and Sfh proteins to substitute for one another functionally. For example, classic phenotypes of hns mutants, such as loss of Escherichia coli motility (44), derepression of the cryptic bgl locus (14, 26), low-osmolarity expression of the osmotic upshock-inducible proU locus (26, 40, 43), and an abnormal outer membrane porin protein expression profile (13), can all be complemented by genes coding for Sfh or StpA (5). Furthermore, combining sfh and hns knockout mutations in the same S. flexneri strain results in an enhancement of the already-derepressed virulence gene expression pattern normally seen in an hns single mutant (5). The three proteins can bind to the same DNA sequences and all share the well-documented preference of H-NS for binding to intrinsically curved DNA sequences (5, 12). This DNA binding preference contributes to the abilities of the three proteins to auto-repress transcription of their own genes and to cross-regulate negatively one another's genes. Preliminary analysis has shown that the three proteins have distinct expression patterns: H-NS is present at an approximately constant level throughout growth, StpA is expressed principally in early exponential phase, and Sfh is seen predominantly in late-logarithmic growth (12, 20, 43). The expression pattern of the Sfh protein is unusual in being the reciprocal of that of the sfh mRNA. The protein is abundant at the onset of stationary phase, whereas the mRNA is plentiful in early exponential phase and becomes difficult to detect by the onset of stationary phase (12). Here, we investigated the expression pattern of the sfh gene in order to elucidate its molecular biology in more detail.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains and growth conditions. All bacterial strains were derivatives of S. flexneri 2a strain 2457T or Escherichia coli K-12 strain MC4100 and are listed in Table 1. E. coli strain MD11 (MC4100/pSf-R27Gen^r) was constructed by conjugating plasmid pSf-R27Gen^r (19) into strain MC4100 as previously described (45). Exconjugants were selected for and twice single-colony purified on L-Str⁵⁰Gen¹⁵ plates, and the presence of pSf-R27Gen^r was confirmed by Southern blot and PCR analyses (data not shown). Bacteria were cultured routinely in Lennox (L) broth or on L agar plates at 37°C. Antibiotics were used at the following concentrations: carbenicillin, 50 µg ml^–1; streptomycin, 50 µg ml^–1; gentamicin, 15 µg ml^–1; kanamycin, 50 µg ml^–1; tetracycline, 15 µg ml^–1; chloramphenicol, 25 µg ml^–1; spectinomycin, 200 µg ml^–1; and rifampin, 250 µg ml^–1.

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TABLE 1. Strains and plasmids used in this study

RNA isolation and analyses by RT-PCR and Northern blotting. Total RNA extracts were prepared by harvesting 6 optical density at 600 nm (OD₆₀₀) units of bacteria and RNA isolated using TRI reagent (Sigma-Aldrich Ltd.) according to the manufacturer's guidelines. Total RNA was then DNase I treated using a DNA-free kit (Ambion Inc.) to ensure no DNA contamination. Reverse transcription (RT)-PCR analyses were carried out using a OneStep RT-PCR kit (QIAGEN), 1.2 µg DNA-free RNA as a template, and gene-specific primer pairs to yield amplicons of 200 to 250 bp in size (Table 2). The RT-PCR program was used according to the manufacturer's guidelines. Reaction mixtures were subject to one cycle at 50°C for 30 min and 95°C for 15 min, followed by 20 cycles at 94°C for 30 s, 50°C for 30 s, and 72°C for 1 min, followed by a final extension at 72°C for 10 min. The RT-PCR products were electrophoresed through 2% (wt/vol) Tris-acetate-EDTA agarose gels and stained with ethidium bromide and then quantified by densitometry using Quantity-One software (Bio-Rad, Hercules, Calif.). For Northern blot analyses, samples of total RNA (5 µg) were denatured for 10 min at 70°C in 50% formamide, loaded onto 1.25% Reliant agarose gels (Flowgen), electrophoresed in MOPS (morpholinepropanesulfonic acid) buffer, and capillary transferred to Biodyne B nylon membrane (PALL). Digoxigenin-labeled riboprobes were produced from plasmids p18sfh and p18rrnA as described previously (5). Overnight hybridization was carried out at 68°C, and after stringency washes, the bound digoxigenin-labeled probes were detected using the chemiluminescence substrate CDP-Star (Roche). Transcript levels were quantified by densitometry using Quantity-One software (Bio-Rad, Hercules, Calif.). To correct for possible differences in RNA integrity and loading in each lane, the analysis of a reference transcript, the 16S rrnA rRNA, was included. All experiments were performed on at least three independent occasions.

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TABLE 2. Primers

Western immunoblotting. Whole-cell lysates were prepared by harvesting 2 OD₆₀₀ units of bacteria and resuspending them in 50 µl B-PER reagent (Pierce) supplemented with lysozyme (500 µg ml^–1) and DNase I (100 U ml^–1). The protein concentrations of the lysates were determined using the Bradford assay (8). Total protein extracts were resolved using 12% sodium dodecyl sulfate-polyacrylamide gels, and proteins were electroblotted to Protran nitrocellulose membranes (Schleicher and Schuell). Nitrocellulose membranes were stained with Ponceau (0.2% Ponceau dye, 3% trichloroacetic acid) to confirm consistent transfer before being blocked with 5% dried skim milk in phosphate-buffered saline. Membranes were probed with primary anti-Sfh antiserum (1:1,000) diluted in blocking solution. Membranes were then washed in phosphate-buffered saline and incubated with a secondary goat anti-rabbit horseradish peroxidase-conjugated antiserum. Membranes were developed using a chemiluminescent Pierce West Pico Super Signal kit. Protein levels were quantified by densitometry using Quantity-One software (Bio-Rad, Hercules, Calif.).

Determination of protein stability. The stability of Sfh protein was monitored using a method described previously (21, 27, 28). Bacterial cells were cultured to exponential phase (OD₆₀₀ = 0.3) or early stationary phase (OD₆₀₀ = 1.5) and treated with spectinomycin (200 µg ml^–1) to inhibit translation. Protein samples were then isolated in a time course experiment, and determination of Sfh protein stability was followed by Western blotting. Experiments were performed on at least three independent occasions.

Determination of mRNA stability. The stability of sfh mRNA was monitored using a method described previously (13). Bacterial cells were cultured to exponential phase (OD₆₀₀ = 0.2) or early stationary phase (OD₆₀₀ = 1.5) and treated with rifampin (250 µg ml^–1) to inhibit transcription. Total RNA samples were then isolated in a time course experiment, and determination of sfh mRNA stability was followed by RT-PCR using the primer pair sfh-FRT and sfh-RRT (Table 2). As a control, hns mRNA stability was monitored using the primer pair hns-RTF and hns-RTR (Table 2), as its mRNA stability does not change significantly with growth phase. Experiments were performed on at least three independent occasions.

Plasmid construction. To assay sfh promoter activity, the sfh regulatory region was cloned upstream of the promoterless gfp reporter gene of plasmid pZep08 (Table 1). Primers sfh-FG and sfh-RG (Table 2) were used to amplify by PCR a 587-bp product encompassing the sfh regulatory region (nucleotides [nt] –497 to +90 with respect to the translation start site of sfh). The amplimer and vector pZep08 were then both digested with XbaI and SmaI so that following ligation, the sfh promoter read into the gfp gene. The structure of the new plasmid was verified by DNA sequencing and designated psfh-gfp (Table 1).

For controlled expression of the Sfh protein, the sfh gene was cloned without its own promoter downstream of the arabinose-inducible P_BAD promoter in plasmids pBAD18 and pBAD24 (Table 1). To construct plasmid pBAD18sfh, PCR primers pBAD18-F and sfh+780 (Table 2) were used to amplify an 820-bp product encompassing the 5' untranslated region (UTR) and open reading frame (ORF) of sfh (nt –40 to +780 with respect to the translation start site of sfh). The amplimer and vector pBAD18 were then both digested with NheI and HindIII so that following ligation, the P_BAD promoter read into the sfh gene. The structure of the plasmid was verified by DNA sequencing and designated pBAD18sfh (Table 1).

Plasmid pBAD24sfh was constructed in a manner similar to that for pBAD18sfh except that PCR primers pBAD24-F and sfh+780 (Table 2) amplified only the sfh ORF (nt +1 to +780 with respect to the translation start site of sfh) and not its 5' UTR and translation initiation signals. The sfh ORF was cloned behind the pBAD24 ribosome binding site. The amplified sfh ORF DNA fragment was extended with a HindIII site at the 3' end. After digestion with HindIII, the amplimer was cloned into pBAD24 that had been linearized with HindIII and NcoI. Following NcoI digestion and prior to HindIII digestion, the Klenow fragment of DNA polymerase was employed to create a blunt end at the NcoI-cut sites. The relative locations of the blunt end and HindIII-cut end of pBAD24 and the amplimer ensured that following ligation, the P_BAD promoter read into the sfh gene. The structure of the plasmid was verified by DNA sequencing and designated pBAD24sfh (Table 1). All PCRs were performed using Pfu polymerase (Promega) and S. flexneri BS184 genomic DNA as the template. GATC Biotech performed custom automated DNA sequencing, and MWG Biotech supplied all the oligonucleotides.

Controlled expression of Sfh. The plasmids pBAD18sfh and pBAD24sfh (Table 1) were transformed separately into the E. coli K-12 strain MC4100 (Table 1). Bacterial cells were cultured to early exponential phase (OD₆₀₀ = 0.2), and arabinose was added at time zero to the cultures at a final concentration of 0.2% to induce expression of Sfh from the P_BAD promoter. Protein samples were then isolated in a time course experiment, and Sfh expression was monitored by Western blotting.

Bioinformatic analysis. The secondary structure of sfh mRNA was predicted by the folding program Mfold (32, 52) (http://www.bioinfo.rpi.edu/applications/mfold/rna/form1.cgi).

Electrophoretic mobility shift assay. The association of recombinant StpA and H-NS proteins (5) with the sfh promoter was investigated using a gel retardation assay (7). A 438-bp PCR amplimer of the sfh promoter region (corresponding to nt –327 to +111 with respect to the sfh transcription start site) was amplified using Pfu polymerase (Promega) and the primer pair sfh-BSF and sfh-BSR (Table 2). The PCR probe was incubated with pBR322 digested with TaqI and SspI restriction enzymes and purified protein in a reaction buffer containing 40 mM HEPES (pH 8), 100 mM potassium glutamate, 10 mM magnesium aspartate, 0.022% NP-40, 0.1 µg ml^–1 bovine serum albumin, and 10% glycerol. The reactions were incubated at room temperature for 15 min and then electrophoresed through 3% molecular screening agarose (Roche). After migration, the gels were stained with ethidium bromide. Experiments were performed on at least two independent occasions.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sfh promoter is growth phase regulated. A gfp transcriptional fusion to the sfh promoter was monitored in S. flexneri 2a strain BS184 and its sfh knockout mutant derivative in cultures grown in L broth at 37°C. The patterns of expression were similar in both strains, although the level of gfp expression was higher in the absence of a functional sfh gene (Fig. 1). This was consistent with the known ability of Sfh to repress the sfh promoter (12). In both bacterial strains, sfh-gfp expression showed a marked growth phase dependency, with transcription being maximal in early exponential phase and declining steadily thereafter. Both S. flexneri strains contained the pSf-R27 plasmid. To investigate the possibility that growth phase-dependent expression was imposed by a factor or factors encoded by this plasmid, the sfh-gfp fusion was moved to E. coli K-12 strain MC4100. Here, a growth phase-dependent expression pattern similar to that observed in S. flexneri was seen, with maximal transcription occurring in the early exponential phase of growth (data not shown). Adding the pSf-R27 plasmid did not alter this expression pattern (data not shown). This ruled out a role for other pSf-R27 genes in the growth phase regulation of sfh transcription while showing that the pattern seen in S. flexneri was conserved when the sfh gene was transferred to E. coli K-12.

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FIG. 1. Expression of the sfh gene as a function of growth phase. Expression of an sfh-gfp fusion in S. flexneri 2a strain 2457T (filled squares) and its sfh derivative (filled diamonds) in L broth at 37°C as a function of time is shown. Expression of the gfp plasmid vector without the sfh promoter insert (filled ovals) is also shown. The inset is the growth curve of the S. flexneri 2457T culture. The other two strains exhibited identical growth curves (not shown).

Cross-regulation as a function of growth phase. Previously, genetic evidence established roles for the H-NS and StpA proteins as repressors of sfh transcription (12). Electrophoretic mobility shift assays showed that H-NS and StpA bind to the sfh promoter region (Fig. 2). This was consistent with the previously demonstrated binding of Sfh to the same DNA fragment (12). We wished to investigate the possibility that the H-NS or StpA proteins might influence the growth phase regulation of sfh. Therefore, we examined sfh transcription by Northern blotting in wild-type, hns, stpA, and hns stpA strains at different stages of growth. In all four strains, the same growth phase-dependent expression pattern of sfh was detected (Fig. 3). However, the loss of the paralogous proteins affected the level of sfh expression at each stage of growth. Inactivation of stpA resulted in increased sfh transcription at early exponential phase but had no effect thereafter. Loss of the hns gene expression caused sfh to be transcribed at a higher level at all three stages of growth. This effect was enhanced when the stpA and hns mutations were combined in the same strain (Fig. 3). These data showed that while StpA and H-NS repress transcription of sfh at all stages of growth, they are not responsible for the growth phase-dependent pattern of sfh transcription.

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FIG. 2. Binding of the H-NS and StpA proteins to the sfh promoter. Results for competitive electrophoretic mobility shift assays showing binding of the H-NS (left) and StpA (right) proteins to the sfh promoter are shown. Plasmid pBR322 DNA was digested with TaqI and SspI, and the resulting DNA fragments were mixed with a 438-bp PCR amplimer of the sfh promoter region. The DNA mixture was incubated with H-NS or StpA in the 0 to 2 µM range. An arrow and an asterisk indicate the positions of the sfh and the pBR322 bla promoter fragments, respectively. The curved DNA bla fragment is a positive control that is known to bind H-NS-like proteins (4). The positions of the 400-, 500-, and 600-bp molecular size markers are also indicated.

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FIG. 3. Expression of sfh mRNA as a function of growth phase in the wild type (wt) and mutants deficient in H-NS, StpA, or both. (A) Northern blotting with a specific sfh riboprobe was used to monitor sfh mRNA levels at the early, mid-, and late exponential phases of growth in the wild type and its stpA, hns, and hns stpA derivatives. The data indicate that the sfh transcript is approximately 500 nucleotides in length, which is believed to be a more accurate estimate than our previous one of 650 nucleotides (12). As a control for RNA integrity and loading, levels of rrnA rRNA were measured in the same samples with an rrnA-specific riboprobe. Samples were isolated at OD600s of 0.1 (early exponential phase) (lanes a), 0.6 (mid-exponential phase) (lanes b), and 1.5 (early stationary phase) (lanes c). (B) The Northern blots were scanned and the data used to generate histograms. The transcript levels are expressed as percentages of the sfh mRNA content of wild-type BS184 cells at an OD600 of 0.1 (early exponential phase), the value of which was set at 100%. The data are averages for three independent experiments, and a representative blot is shown.

We used Western blotting to monitor Sfh protein expression in the wild type and the hns, stpA, and hns stpA mutants. The results showed that each mutant expressed Sfh protein to a higher level than the wild type, in the following order: hns stpA > hns > stpA > the wild type (Fig. 4). These data supported those from the Northern blot analysis (Fig. 3), where H-NS and StpA were confirmed as negative influences on sfh gene expression but were found not to be determinants of growth phase-dependent expression.

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FIG. 4. Expression of Sfh protein as a function of growth phase in the wild type (wt) and mutants deficient in H-NS, StpA, or both. (A) Western blot analysis of Sfh protein levels in the BS184 wild type and its hns, stpA, and hns stpA mutant derivatives at fixed OD600 values during growth in L broth at 37°C. Samples were isolated at OD600s of 0.1 (early exponential phase) (lanes a), 0.6 (mid-exponential phase) (lanes b), and 1.5 (early stationary phase) (lanes c). An arrow indicates the Sfh protein band. (B) The Western blots were scanned and the data used to generate histograms. The protein levels are expressed as percentages of the Sfh protein content of wild-type BS184 cells at an OD600 of 1.5 (early stationary phase), the value of which was set at 100%. The data are averages for three independent experiments, and a representative blot is shown.

Stability of the sfh mRNA as a function of growth. The previously described finding that sfh mRNA and Sfh protein each reach their maxima at distinct phases of growth (12) suggested that sfh gene expression was regulated posttranscriptionally, at the level of either the sfh mRNA or the Sfh protein. Therefore, we decided to measure the stability of each of these molecules. Cultures of wild-type strain BS184, grown at 37°C in L broth to early exponential and early stationary phases, were treated with rifampin to stop all de novo transcription. The sfh transcript was then detected by RT-PCR at 5-min intervals over a 25-min period (Fig. 5). The experiment was also performed with hns mRNA. No significant difference in hns mRNA stability was detectable when results for early exponential and stationary phases of growth were compared. The rates of decay for the sfh mRNA, while similar, did suggest that the message was less stable in stationary-phase than in exponential-phase cultures. These results showed that if anything, the sfh mRNA was less stable at the later stage of growth, a period when the level of the Sfh protein was maximal. These results showed that the differential expression of Sfh protein at exponential and stationary phases of growth was not due to enhanced sfh mRNA stability in stationary-phase cultures.

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FIG. 5. Stability of sfh mRNA in exponential and stationary phases. Rifampin treatment was used to arrest transcription in wild-type BS184 cells growing in L broth at 37°C in exponential phase (OD600 = 0.2) or stationary phase (OD600 = 1.5). Total RNA was extracted at 5-min intervals over a 25-min period. The 0-min sample was taken just prior to addition of rifampin. RT-PCR analysis was used to assess mRNA stability by using primers specific for sfh and for a control transcript, hns. The data for sfh mRNA were scanned densitometrically and plotted as percentages of sfh mRNA remaining as a function of time. Similar decay rates were found for exponential-phase (filled diamonds) and stationary-phase (filled squares) cultures. The experiment was performed on three occasions, and representative blots are shown.

Sfh protein stability as a function of growth. The high level of sfh mRNA and the correspondingly very low level of Sfh protein at the same stage in growth were consistent with Sfh protein instability, possibly due to the rapid turnover of the protein. The wild-type strain BS184 was grown in L broth at 37°C to early exponential or early stationary phase. In each case, all protein synthesis was blocked by the addition of 200 µg ml^–1 spectinomycin to the cultures. Protein samples were removed at the time of spectinomycin addition (time zero) and at intervals of 15, 30, and 45 min thereafter. The Sfh protein was then detected by Western blotting. The results showed that the Sfh protein was equally stable during early exponential growth (Fig. 6A) and at entry into stationary phase (Fig. 6B). These data did not support a model in which Sfh protein is differentially unstable at different stages of culture growth. Instead, they pointed to a regulatory regime in which abundant sfh mRNA in early-exponential-phase cultures was poorly translated while low levels of sfh transcript in early-stationary-phase cultures were translated very efficiently.

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FIG. 6. Sfh protein stability. Treatment with spectinomycin was used to arrest translation in exponential (OD600 = 0.3) (A)- and stationary (OD600 = 1.5) (B)-phase cultures of wild-type BS184 cells and the hns, stpA, and hns stpA mutant derivatives growing at 37°C in L broth. Total protein was isolated for 45 min at 15-min intervals following translation inhibition, and Sfh protein was detected by Western blotting using a specific anti-Sfh antibody. The data from the Western blots were scanned densitometrically, and percentages of Sfh protein remaining were plotted as a function of time. The experiment was performed on three occasions, and representative blots are shown.

Sfh protein stability is dependent on H-NS. We had established that although the H-NS and StpA proteins were repressors of sfh transcription, growth phase-dependent expression of the sfh mRNA and Sfh protein was independent of these proteins (Fig. 3 and 4). We next examined the influence of knockout mutations in the hns and stpA genes, separately and in combination, on the stability of the Sfh protein. The results showed that the absence of H-NS (but not StpA) resulted in the reduced stability of the Sfh protein (Fig. 6). However, the same effect was seen in both exponential-phase (Fig. 6A) and stationary-phase (Fig. 6B) cultures. This ruled out a role for H-NS or StpA as a factor involved in growth phase-dependent control of Sfh protein stability.

Growth phase control is imposed after translation initiation. Our present and previous (12) data pointed to a scenario in which sfh mRNA was expressed in abundance in early exponential phase but not translated until the onset of stationary phase. The most straightforward explanation for these observations was that a translational blockade was imposed until an appropriate point in the growth of the culture was reached. Translational control might involve interference with the translational signals of the sfh gene, especially the ribosome binding site and the translation initiation codon. The Mfold mRNA secondary structure prediction algorithm (52) suggested that the native sfh mRNA could indeed adopt a secondary structure in which the translation initiation signals were sequestered in a region of base-paired RNA (Fig. 7). It was reasoned that if signal sequestration contributed to growth phase control of sfh mRNA translation, then the replacement of the native translation initiation signals with those from an unrelated gene would alter the pattern of growth phase regulation. To test this hypothesis, the sfh ORF was cloned with or without its native translation initiation signals into pBAD18 or pBAD24, respectively (see Materials and Methods). In each case, production of sfh mRNA was controlled by inducing the P_BAD promoter in each plasmid with arabinose. Following induction with arabinose at time zero, the expression of Sfh protein was monitored in the culture by Western blotting and compared with that of Sfh expressed from the native pSf-R27 plasmid (Fig. 8). The expression patterns of the Sfh protein were remarkably similar in all three cases, reaching a maximum 4 hours after induction of transcription. In each case, there was a strong correlation between the appearance of high levels of Sfh protein and the entry of the culture into the stationary phase of growth. These data showed that placing the sfh open reading frame under the control of foreign translation initiation signals did not disrupt its characteristic growth phase-dependent pattern of expression. This suggests that the point at which growth phase control is exerted lies at a later stage in the process of translation.

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FIG. 7. Proposed secondary structure of sfh mRNA. (A) The Mfold RNA folding program (52) was used to predict the secondary structure of sfh mRNA (GenBank accession number NP_457198). The 5' region of the transcript is magnified to show that the Shine-Dalgarno sequence (5'-gagg, with the bases within ovals) and translation initiation codon (5'-AUG, with the bases within squares) are sequestered within regions of base pairing. (B) The complete ribonucleotide sequence of sfh mRNA. The regions within the dashed boxes correspond to the magnified portion of the sfh mRNA secondary structure shown in panel A. The Shine-Dalgarno sequence is within the oval and the translation initiation codon within a rectangle. The bases in lowercase lettering form the 5' untranslated part of the message.

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FIG. 8. Effect of altering translation signals on Sfh protein expression. Bacterial strains containing the native sfh gene in pSf-R27 (filled triangles), the sfh gene with heterologous translation initiation signals in plasmid pBAD24sfh (filled squares), or the sfh gene with its own translation initiation signals in plasmid pBAD18sfh (filled diamonds) were grown to early exponential phase (OD600s of 0.15 to 0.18) in LB at 37°C. The PBAD promoter in plasmids pBAD18sfh and pBAD24sfh was induced by the addition of arabinose to a final concentration of 0.2% (wt/vol) at time zero. Samples were removed at the indicated time points, and Western blotting was used to monitor the expression of the Sfh protein. In each case, the maximum expression of the Sfh protein correlated with the onset of stationary phase.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The discovery of the Sfh protein brings to three the number of H-NS-like proteins expressed in S. flexneri 2a strain 2457T. Because H-NS has such wide-ranging effects on the global gene expression patterns of the bacterium (7, 15, 40), it is important to understand the potential for Sfh to influence these patterns in bacteria that acquire the sfh gene following the horizontal transfer of the plasmid that harbors it. As a first step in increasing our understanding of sfh biology, we investigated the expression of this gene. The sfh promoter was found to be at its most active in early-exponential growth, declining to a low level of activity as the culture approached late exponential phase (Fig. 1). In addition, clear evidence of growth phase dependency was found for expression of both sfh mRNA and Sfh protein. However, we previously noted a marked and surprising mismatch between the expression patterns of the sfh gene at the levels of mRNA and protein: the expression patterns at the transcriptional and posttranscriptional levels were reciprocal, with sfh mRNA being abundant in early-exponential growth but scarce later in growth, while the Sfh protein had the opposite pattern of expression (12).

Previous work has shown that the H-NS, StpA, and Sfh proteins each repress the sfh promoter (12). In the present study, we show that H-NS and StpA bind to the same sfh promoter DNA fragment to which Sfh had been shown previously to bind (Fig. 2). However, we could find no evidence that the growth phase-dependent expression pattern of sfh mRNA was influenced by these proteins. The removal of H-NS resulted simply in an overall increase in transcription, while the removal of StpA had little effect (Fig. 3).

The Sfh protein is expressed poorly in early-exponential growth (Fig. 4). This was not due to the enhanced turnover of the protein compared with that in stationary-phase cultures. Sfh was found to be equally stable in exponential- and stationary-phase cultures following inhibition of de novo protein synthesis (Fig. 6). We could find no evidence that the presence or absence of the H-NS or StpA proteins was responsible for the differences in Sfh protein stability in exponential- and stationary-phase cultures. Removal of StpA had no effect on Sfh stability at either phase of growth, while removal of H-NS resulted in the enhanced turnover of the protein in both exponential and stationary phases of growth (Fig. 6). The enhanced instability of Sfh that was seen in the hns mutant was reminiscent of the previously reported destabilization of StpA that is seen in the absence of H-NS (26, 27). This is due to the protection of StpA from protease turnover through the formation of heteromeric H-NS-StpA complexes. It is likely that Sfh enjoys a similar protection through interaction with H-NS, and this is consistent with previous data showing that Sfh and H-NS can form heteromers (12). Presumably, in the absence of H-NS, the complex that the StpA protein is known to form with Sfh (12) is not protective because Sfh-StpA heteromers are vulnerable to proteolytic cleavage or because there is insufficient intact StpA to act protectively due to rapid StpA turnover.

Abundant sfh mRNA in exponentially growing bacteria is not accompanied by a high level of Sfh protein, whereas small amounts of sfh mRNA in stationary phase correlate with enhanced expression of Sfh protein (12). We have ruled out differential growth phase-dependent mRNA stability or Sfh protein stability as a cause of the observed reciprocal transcript and protein expression patterns. Our data are consistent with a blockade of translation that is active in early-exponential growth and relieved at the onset of stationary phase. Negative regulation of translation can be imposed in cis, in trans, or through a combination of cis- and trans-acting processes. Mechanisms acting in cis usually involve the formation of a secondary structure in the message that precludes translation (1, 2, 29). The sfh mRNA is predicted by Mfold analysis (52) to form a secondary structure in which the likely translation initiation signals are sequestered within a base-paired region (Fig. 7). An attractive hypothesis envisions that this secondary structure is modified by a trans-acting factor at the onset of stationary phase, allowing the message to be translated. Alternatively, the translational blockade may be imposed by a trans-acting factor that sequesters the translation initiation signals by directly binding to them in early exponential phase. In the first case, the trans-acting factor is a positive regulator, and in the second, it is a negative regulator of translation. The putative trans-acting factor could be a protein or another RNA molecule, such as one of the many small RNA molecules that are known to influence translation in gram-negative bacteria (13, 22, 28, 30, 31). When this hypothesis was tested by replacing the native sfh translation initiation signals and 5' UTR with foreign sequences from the E. coli araB gene, it was discovered that the characteristic growth phase-dependent expression of the Sfh protein was retained (Fig. 8). Thus, it seems likely that growth phase control is exerted at a stage in the translation process later than initiation. This could still involve a role for mRNA secondary structure and a trans-acting regulatory factor. The identity of this trans-acting regulator remains elusive at the present time, but our data indicate that it is encoded by the chromosomes of both S. flexneri 2a strain 2457T and E. coli K-12 and not the pSf-R27 plasmid that harbors the sfh gene.

Why does the Sfh protein exhibit its particular pattern of expression? These bacteria already express two H-NS-like proteins (H-NS itself and StpA) from genes on the chromosome, and it is possible that the presence of a third protein may not be tolerated well under all physiological conditions. Consistent with this proposal is the observation that the bacterial culture harboring the pBAD18sfh plasmid in which Sfh was expressed from its natural translation signals entered stationary phase at a low optical density (OD₆₀₀ = 0.39) following the induction of the P_BAD promoter (Fig. 8). The culture carrying the pBAD24sfh plasmid in which sfh utilized foreign translation initiation signals also ceased growing earlier than the control following transcriptional induction. Control cultures harboring just the pBAD18 and pBAD24 plasmids do not exhibit this behavior following arabinose induction (data not shown). By confining Sfh protein expression to stationary phase, the sfh gene may ensure that the conjugative plasmid that harbors it imposes minimal disruption on the physiology of the bacteria that acquire it by horizontal transfer.

 
We thank Padraig Deighan and Christophe Beloin for very helpful discussions.

This work was supported by a grant from Science Foundation Ireland.

 
* Corresponding author. Mailing address: Department of Microbiology, Moyne Institute of Preventive Medicine, University of Dublin, Trinity College, Dublin 2, Ireland. Phone: 353 1 896 2013. Fax: 353 1 679 9294. E-mail: cjdorman{at}tcd.ie.

Published ahead of print on 25 August 2006.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Bacteriology, November 2006, p. 7581-7591, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00685-06
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