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Journal of Bacteriology, March 2008, p. 1658-1670, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01649-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

The LysR-Type Transcriptional Regulator LeuO Controls Expression of Several Genes in Salmonella enterica Serovar Typhi ^,

I. Hernández-Lucas,^1, A. L. Gallego-Hernández,^1, S. Encarnación,² M. Fernández-Mora,¹ A. G. Martínez-Batallar,² H. Salgado,² R. Oropeza,¹ and E. Calva^1*

Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, México,¹ Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62210, México²

Received 11 October 2007/ Accepted 11 December 2007

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
LeuO is a LysR-type transcriptional regulator that has been implicated in the bacterial stringent response and in the virulence of Salmonella. A genomic analysis with Salmonella enterica serovar Typhi revealed that LeuO is a positive regulator of OmpS1, OmpS2, AssT, and STY3070. In contrast, LeuO down-regulated the expression of OmpX, Tpx, and STY1978. Transcriptional fusions supported the positive and negative LeuO regulation. Expression of ompS1, assT, and STY3070 was induced in an hns mutant, consistent with the notion that H-NS represses these genes; transcriptional activity was lower for tpx and STY1978 in an hns background, suggesting that this global regulatory protein has a positive effect. In contrast, ompS2 and ompX expression appeared to be H-NS independent. LeuO specifically bound to the 5' intergenic regions of ompS2, assT, STY3070, ompX, and tpx, while it was not observed to bind to the promoter region of STY1978, suggesting that LeuO regulates in direct and indirect ways. In this work, a novel set of genes belonging to the LeuO regulon are described; interestingly, these genes are involved in a variety of biological processes, suggesting that LeuO is a global regulator in Salmonella.

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Salmonella enterica serovar Typhi is a gram-negative facultative intracellular pathogen that causes typhoid fever in humans. The complete genomic sequence of Salmonella serovar Typhi is composed of a 4.8-Mb chromosome and two small replicons (218 and 106 kb). The genome includes 250 genes involved in transcriptional regulatory functions (41). LeuO belongs to the LysR family of transcription regulators present in Enterobacteriaceae such as Yersinia, Shigella, Escherichia coli, and Salmonella; these regulators typically are 300 amino acids long and have an N-terminal DNA binding domain and a C-terminal sensing domain. They can function as activators or repressors and regulate genes with promoters that are divergent from their own promoter. The family of LysR transcriptional factors is involved in microbe-plant and microbe-animal interactions (48). The LeuO regulator has been reported to be a virulence factor in Caenorhabditis elegans and in the mouse model with Salmonella (26, 46, 52). LeuO expression is enhanced in stationary phase and by phosphorous restriction (10, 56); it is also involved in Vibrio cholerae biofilm formation and in the stringent response (30, 35). LeuO regulates the bgl and cadAB operons, as well as the ompS1, ompS2, dsrA, and rovA genes (5, 12, 27, 45, 50, 55), and has been shown to be part of a promoter relay mechanism that explains the coordinate expression of the ilvIH-leuO-leuABCD gene cluster (9). In this paper, a novel set of proteins that was induced by the LeuO regulator in Salmonella serovar Typhi is described. Transcriptional fusions validated the proteomic data and showed that the genes regulated positively by LeuO are strictly LeuO dependent. Expression profiles of the LeuO-regulated genes showed that some members of the LeuO regulon described here are either negatively or positively regulated by the global regulatory protein H-NS. Based on primer extension and footprinting experiments, the transcription initiation sites and the LeuO regions protected from DNase I were determined for new members of the LeuO regulon. Interestingly, bioinformatics analyses showed that LeuO did not recognize a specific motif in the promoter region of the LeuO-dependent genes.

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Bacterial strains and culture conditions. The bacterial strains and plasmids used are listed in Table S1 in the supplemental material. Salmonella serovar Typhi was grown in LB medium (10 g of tryptone per liter, 5 g of yeast extract per liter, 10 g of NaCl per liter) or in liquid MA medium (7 g of nutrient broth per liter, 1 g of yeast extract per liter, 2 g of glycerol per liter, 3.75 g of K₂HPO₄ per liter, 1.3 g of KH₂PO₄ per liter) (21). When required, the following antibiotics were added: kanamycin (30 µg/ml), tetracycline (12 µg/ml), and ampicillin (100 µg/ml). Salmonella serovar Typhi and E. coli strains were grown aerobically at 37°C.

DNA manipulation. Plasmid isolation and genomic DNA isolation were performed using previously described protocols (47). Primers for PCR amplification were provided by the oligonucleotide synthesis facility at our institute (see Table S2 in the supplemental material). Restriction enzymes, ligase, kinases, nucleotides, and polymerases were obtained from New England Biolabs or Gibco BRL. For sequencing, double-stranded DNA was purified with a High Pure plasmid isolation kit (Boehringer, Mannheim, Germany), and sequencing was performed with an automatic Perkin Elmer/Applied Biosystems 377-18 system.

Preparation of crude cell extracts. Salmonella serovar Typhi harboring plasmid pFMTrc12 (strain IMSS-II) (12) or plasmid pFMTrcleuO-50 (strain IMSS-III) (5) and Salmonella serovar Typhi leuO harboring plasmid pFMTrc12 (strain IMSS-32) were grown in MA medium supplemented with ampicillin and isopropyl-β-D-thiogalactopyranoside (IPTG) (50 µM) to an optical density at 595 nm (OD₅₉₅) of 0.6. Salmonella cultures (100 ml) were pelleted and washed with 1x phosphate-buffered saline. Cellular proteins were obtained by sonication at 24 kHz for 1 min in the on position and 1 min in the off position for five cycles at 4°C using a Vibra Cell (Sonics, United States) in the presence of a protease inhibitor (Complete tablets; Roche Diagnostics GmbH, Mannheim, Germany). To further limit proteolysis, protein isolation was performed using phenol extraction (17). To solubilize proteins and to obtain completely denatured and reduced proteins, pellets were dried and resuspended as previously reported (6). Prior to electrophoresis samples were mixed with 7 M urea, 2 M thiourea, 4% 3-[(3-choloamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) (Roche Diagnostics GmbH, Germany), 2 mM tributhyl phosphine, 2% ampholytes, and 60 mM dithiothreitol.

2-DGE. The methods used for sample preparation, analytical two-dimensional gel electrophoresis (2-DGE), image analysis, and preparative 2-DGE have been described previously (7), and pH gradients were determined using a two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis standard (Sigma, United States). For isoelectric focusing 500 µg of total protein was loaded. All gel experiments were performed more than three times.

In-gel digestion and mass spectrometry (MS)-based identification of proteins. Selected spots from Coomassie blue-stained preparative two-dimensional gels were excised manually and frozen at –70°C until they were used. Samples were prepared for mass spectrum analysis by using a slight modification of a previously described procedure (6, 7, 49). Protein spots were destained, reduced, alkylated, and digested with trypsin (Promega, Madison, WI). Before the mass spectra of the peptide mixtures were obtained, the mixtures were desalted using a C₁₈ Zip Tip (Millipore, Bedford, MA) according to the manufacturer's recommendations.

Mass spectra were obtained using a Bruker Daltonics Autoflex (Bruker Daltonics, Billerica, MA) operated in the delayed extraction and reflectron mode. Spectra were externally calibrated using a peptide calibration standard (Bruker Daltonics 206095). Peptide mixtures were analyzed using a saturated solution of alpha-cyano-4-hydroxycinnamic acid in 50% acetonitrile-0.1% trifluoroacetic acid. Peak lists of the tryptic peptide masses were generated and searched against the NCBI nr databases using the Mascot search program (Matrix Science, Ltd., London, United Kingdom; http://www.matrixscience.com). Proteins not identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis were excised from preparative gels and analyzed by liquid chromatography-MS/MS using a nanoflow chromatograph (Agilent 1100 LC NanoPump; Agilent, Waldbronn, Germany) coupled to a hybrid triple quadrupole linear ion trap (3200 Q TRAP system; Applied Biosystems/MDS Sciex, Ontario, Canada) equipped with a Nanospray II source and using information-dependent acquisition. Digested proteins were separated using a 60-min acetonitrile-0.1% formic acid elution gradient (Mallinckrodt Baker, United States) and a flow rate of 0.4 µl/min with a Zorbax C₁₈ column (75 µm by 150 mm; 3.5 µm). Precursor ions were determined using an enhanced MS scan over a mass range from m/z 400 to 1,500 at 4,000 atomic mass units/s (with no trapping in Qo and a LIT fill time of 20.00 ms) with an ion spray voltage of 2,400 V applied to a Picotip (FS360-75-15; New Objective, Woburn, MA) with ion spray gas (nitrogen). Precursors ions were collided in Q2 using rolling collision energy (maximum allowed collision energy, CE-80). Enhanced product ion scans (MS/MS) were performed over a mass range from 50 to 1,700 m/z at 4,000 atomic mass units/s, and collision voltages were determined dynamically. The entire precursor ion mass/charge ratio was confirmed with an Enhance Resolution scan. Proteins were identified by using the Mascot algorithm (available at http://www.matrixscience.com) (59).

Determination of LeuO cellular concentration. To evaluate the LeuO concentration used in this work, Salmonella serovar Typhi IMSS-III was grown in MA medium (supplemented with 50 µM IPTG) to an OD₅₉₅ of 0.6; a total-extract sample was collected, boiled, and loaded on a 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. Proteins were transferred to nitrocellulose membranes using the Trans-Blot SD system (Bio-Rad). Membranes were probed using rabbit anti-LeuO His-tagged polyclonal antibody. Protein concentrations were determined with the Bradford protein assay reagent using bovine serum albumin as a standard (Bio-Rad). To calculate the LeuO concentration, a comparative analysis of the LeuO blot signals from the total extract and from various amounts of the LeuO purified protein was performed.

Construction of transcriptional reporter fusions. Oligonucleotides (see Table S2 in the supplemental material) were designed to PCR amplify the complete 5' intergenic regions of the genes regulated by LeuO. PCR fragments were double digested with BamHI-KpnI or BamHI-HindIII and ligated into pKK232-8 or pKK232-9 (see Table S1 in the supplemental material), which contained the promoterless cat gene. Fusions were sequenced in order to verify the DNA sequences of the PCR fragments.

CAT assays. The chloramphenicol acetyltransferase (CAT) assays were performed as follows (31). Salmonella serovar Typhi strains were grown in MA medium supplemented with ampicillin or kanamycin and with IPTG (50 µM) to OD₅₉₅ of 0.4, 0.6, 0.8, and 1 and for 12 h. Salmonella serovar Typhi STYhns99 was also grown in MA medium. Portions (1.5 ml) of bacterial cultures were collected by centrifugation and washed with 0.8 ml of TDTT buffer (50 mM Tris-HCl [pH 7.8], 30 µM DL-dithiothreitol). Bacterial cells were resuspended in 0.6 ml of TDTT buffer and sonicated on ice using 10-s bursts with 10-s rest periods until the extract was clear. The homogenate was centrifuged, and the supernatant was used to measure activity. For CAT assays 5 µl of each extract was added in duplicate to a 96-well enzyme-linked immunosorbent assay plate, which was followed by addition of 0.2 ml of a reaction mixture that contained 1 mM 5,5'-dithiobis(2-nitrobenzoic acid), 0.1 mM acetyl coenzyme A, and 0.1 mM chloramphenicol in 0.1 M Tris-HCl (pH 7.8). The absorbance at 412 nm was measured every 5 s for 5 min using a scanning autoreader and Ceres 900 microplate workstation. The protein concentrations of the cell extracts used in the CAT assays were determined using the bicinchoninic acid protein assay reagent (Pierce). The protein values and the mean rate of product formation by cat were used to determine the CAT specific activity, which was expressed in micromoles per minute per milligram of protein. The results presented below are the means of three independent experiments.

RNA isolation and primer extension analysis. Salmonella serovar Typhi strains were grown at 37°C in MA medium for 12 h. Bacterial cells (5 ml) were collected, and total RNA was isolated using a commercial kit (RNeasy; Qiagen). The concentration of RNA was determined by measuring the absorbance at 260 nm. The integrity of RNA was determined by using a 1.5% agarose gel. Five to 20 µg of total RNA was denatured at 95°C for 3 min and then slowly cooled to 45°C. The RNA was annealed with [-³²P]ATP-labeled primers. Primers were extended with reverse transcriptase at 42°C for 1 h, and then the extended products were purified by ethanol precipitation and analyzed by electrophoresis in 8% polyacrylamide-8 M urea gels alongside sequencing ladders (39). Sequencing ladders were generated from plasmids that contained the regulatory regions of the LeuO-controlled genes.

DNase I footprinting assays. DNA fragments encompassing the promoter regions of the LeuO-controlled genes were PCR amplified using labeled primers. LeuO was purified as previously described (12). Binding of the recombinant LeuO protein to the labeled promoter was performed at room temperature for 30 min in a reaction buffer containing 20 mM HEPES (pH 7.9), 100 mM KCl, 2 mM MgCl₂, 0.1 mM EDTA, 0.1 mM dithiothreitol, 20% glycerol, and 1.0 µg of poly(dI-dC) (Boehringer)/ml. The reaction mixture was treated without DNase I and with 0.4 U of DNase I. After precipitation with ethanol, the digested DNA fragments were separated by electrophoresis in 8% polyacrylamide-8 M urea gels alongside sequencing ladders. Sequencing ladders were generated, with the labeled primer used for PCR amplification, from plasmids containing the regulatory regions of the LeuO-controlled genes.

Bioinformatic analysis. To determine whether LeuO recognizes a specific DNA sequence, two bioinformatics approaches were used: pattern matching and pattern discovery. In both approaches, we used 400 bp upstream and 50 bp downstream of the putative ATG translational start codon. In the first method, the experimental LeuO regions protected from DNase I were used as a training set to run the consensus program (16); thus, we obtained alignments and weight matrices of binding sites that were 8 to 12 bp long. To assign match scores, we used an "alphabet" based on the frequency of occurrence of each base in the regulatory regions analyzed. The search was conducted with a single strand. The weight matrix generated (highest information content) was used to search binding sites. Using patser, we searched for the highest-scoring sequence in each region of the training set. To evaluate the quality of the matrix, we evaluated different sets, including upstream regions of LeuO-regulated genes, 1,000 groups of six randomly selected regions, and non-LeuO-regulated genes. The mid-value of the scores was used as a threshold because negative scores were obtained with the training set. The second method, pattern discovery, assumed that LeuO binding sites are unknown; thus, the regulated LeuO sequences together with the Oligo and dyad analysis programs were used to identify a motif in the LeuO interacting sites (57). To evaluate this approach, we counted the number of times that we could locate a known binding site in a collection of upstream regions. The dyad and Oligo analysis programs were designed to find overrepresented "small words" (57). Overrepresented "words" would be expected to occur at the binding sites, and thus we determined whether the resulting Oligo or dyad analysis was in agreement with the LeuO sites detected experimentally. The parameters used to determine the LeuO consensus sequence are available at http://www.ccg.unam.mx/Computational_Genomics/tools/LeuO/.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Detection and identification of LeuO-regulated proteins in Salmonella serovar Typhi. In order to define the LeuO-regulated proteins, Salmonella serovar Typhi harboring plasmid pFMTrc12 (strain IMSS-II) (12) or pFMTrcleuO-50 (strain IMSS-III) (5) and Salmonella serovar Typhi leuO containing plasmid pFMTrc12 (strain IMSS-32) were grown to an OD₅₉₅ of 0.6 in MA medium supplemented with IPTG (50 µM). These conditions were the conditions determined previously for simultaneous expression of the two minor porins OmpS1 and OmpS2, as the activation of the corresponding genes is dependent on the level of induction of LeuO (5). Whole-cell proteins were obtained and separated by 2-DGE; about 600 proteins with pI values ranging from 4 to 8 and with molecular masses ranging from 20 to 90 kDa were resolved in each gel. The protein patterns of IMSS-II and IMSS-32 were similar. However, a comparison of the proteomic profiles of IMSS-III and IMSS-32 showed that overexpression of LeuO quantitatively up-regulated and down-regulated 10 and 3 protein spots, respectively (Fig. 1). To identify the LeuO-regulated proteins, spots of interest were excised, trypsin digested, and analyzed by MALDI-TOF MS. These analysis showed that LeuO activated the expression of OmpS1 (five isoforms), OmpS2, AssT (two isoforms), and STY3068, the third gene of the STY3070-STY3064 operon. In contrast, LeuO repressed the expression of OmpX, STY1978, and Tpx. As expected, recombinant LeuO was also detected in the 2-DGE gel (Table 1).

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FIG. 1. Proteomic profiles of Salmonella serovar Typhi total protein extracts. (A) Salmonella serovar Typhi leuO containing pFMTrc12 (IMSS-32). (B) Salmonella serovar Typhi containing pFMTrcleuO-50 (IMSS-III). Cells were grown in MA broth to an OD595 of 0.6. Cultures were supplemented with 50 µM IPTG. The labeled spots were excised and identified using MALDI-TOF MS. The identities of circled proteins are shown in Table 1. More than three independent experiments were performed, and representative 2-DGE gels are shown.

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TABLE 1. LeuO-regulated proteins in Salmonella serovar Typhi identified by MALDI-TOF MSa

The sequence coverage of the LeuO-dependent proteins ranged from 7.2 to 54%, and the Mascot database search algorithm revealed that the proteins identified are identical to the corresponding proteins of Salmonella serovar Typhi CT18 (41) (Table 1). OmpS1 and AssT were detected in five and two isoforms, respectively. The OmpS1 pI values were 4.44, 4.25, 5.09, 5.06, and 6.12, and the AssT pI values were 6.71 and 6.51. The variation in pI values was not accompanied by a substantial change in molecular weight (Table 1), suggesting that the isoforms may involve modifications such as phosphorylation, methylation, or deamidation rather than introduction of high-molecular-weight groups. Previous studies with prokaryotes showed that posttranslational modifications, such as methylation, are relevant in bacterial pathogenesis (42, 54). Multiple protein isoforms have been found previously in Salmonella, including isoforms involved in carbon metabolism in S. enterica serovar Typhimurium and S. enterica serovar Pullorum (8). Previously, other loci regulated by LeuO, such as bgl, rovA, dsrA, cadC, and leuO, have been reported (9, 27,45, 50, 55); however, they were not detected in our proteomic analysis. The bgl and rovA genes are not present in Salmonella serovar Typhi, and dsrA codes for a small RNA. CadC and LeuO were not found, perhaps due to mRNA instability or low expression, because they overlapped with other proteins on the 2-DGE, or because the LeuO concentration was not the optimal concentration for induction of these genes.

The cellular LeuO concentration in IMSS-III harboring pFMTrcleuO-50 with 50 µM IPTG was calculated by Western blot analysis of total proteins and comparison to purified LeuO (data not shown). The data obtained indicated that LeuO was present at a level of 6 x 10⁵ molecules per cell (or 0.4 µg/100 µg of total cellular protein).

The proteins identified in this study thus corresponded to outer membrane proteins, such as OmpS1, OmpS2, and OmpX (11, 12, 33); to proteins involved in detoxification, such as Tpx and AssT (20, 22, 24); and to the hypothetical STY1978 and STY3068 proteins (41). Previously, we described isolation, characterization, and regulation analysis of the quiescent porins OmpS1 and OmpS2; furthermore, we showed that the genes encoding these proteins are LeuO targets, and thus we considered these proteins relevant controls for proteomic analysis.

Transcriptional analysis of the LeuO-regulated Salmonella serovar Typhi genes. To define the role of LeuO in the regulation of the identified genes, transcriptional fusions were constructed with each of the LeuO-regulated genes. PCR fragments containing the putative promoter sequence of these genes were obtained and cloned into pKK232-9, which contained the cat reporter gene. The plasmids harboring the fusions (see Table S1 in the supplemental material) were transformed independently into Salmonella serovar Typhi IMSS-III and Salmonella serovar Typhi IMSS-II, which contained pFMTrcleuO-50 and pFMTrc12, respectively. To measure CAT specific activity, Salmonella cells were grown to different optical densities in MA medium supplemented with 50 µM IPTG when necessary.

The effect of LeuO on the transcription of the STY3068 gene, the third gene of a putative large operon consisting of seven open reading frames, was analyzed by fusing the STY3068 upstream region to the cat reporter. Interestingly, we were unable to detect expression (data not shown), indicating that a LeuO-dependent promoter is not present in the closest 5' region (602 bp) of STY3068. Thus, a gene reporter fusion was constructed with the complete 5' intergenic region of the first gene (STY3070) of the operon, showing that this gene was positively regulated by LeuO, since we found CAT activity at all ODs tested (Fig. 2a). Low transcriptional activity of STY3070 was also detected at 12 h without IPTG, suggesting that the basal accumulation of LeuO was enough for induction. This indicates that LeuO positively regulates the putative STY3070-STY3064 operon.

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FIG. 2. Transcriptional profiles of the LeuO-dependent genes fused to the cat reporter gene. Bacterial strains IMSS-II (Salmonella serovar Typhi IMSS-1 harboring plasmid pFMTrc12) and IMSS-III (Salmonella serovar Typhi IMSS-1 harboring plasmid pFMTrcleuO-50) were independently transformed with fusions of each of the LeuO-regulated genes (pKK232-9 [STY3070], pKK232-9 [assT], pKK232-9 [ompS1], pKK232-9 [ompS2], pKK232-9 [tpx], pKK232-9 [ompX], and pKK232-9 [STY1978]) to generate individual strains containing the LeuO regulator, as well as the promoter region of the LeuO-dependent gene. To evaluate the effect of H-NS in the regulation of each of the LeuO-regulated genes, individual fusions were transformed into Salmonella serovar Typhi hns mutant STYhns99. The CAT specific activity of each strain was determined using samples collected at OD595 of 0.4, 0.6, 0.8, and 1 and at 12 h. For strains IMSS-II and IMSS-III 50 µM IPTG was used as an inducer. The bars indicate the means of three independent experiments performed in duplicate.

In the case of assT and ompS1, no transcriptional activity was detected when LeuO was absent, but upon induction with IPTG, high transcriptional activity of the assT and ompS1 fusions was detected (Fig. 2b and c). For ompS2, we found that in the absence of IPTG considerable transcriptional activity was detected only at 12 h, showing that, as observed for STY3070, the basal expression from the recombinant LeuO plasmid was enough for induction of the quiescent porin and the putative operon. Expression from the ompS2 fusion was also detected in the presence of IPTG at different ODs (Fig. 2d); however, the activities obtained at different ODs with IPTG were three times lower than the activity detected without IPTG at 12 h, in accordance with the previous observation that the amount of LeuO determines the expression level of ompS2 (5). It is relevant that in the case of STY3070, assT, ompS1, and ompS2, transcriptional activity was not detected with pFMTrc12 (Fig. 2a to d), indicating that these genes are strictly LeuO dependent.

The proteomic data mentioned above also revealed that LeuO represses the expression of tpx, ompX, and STY1978. The results obtained with the fusions showed that in the absence of LeuO these three loci are constitutively expressed, but when LeuO was induced with 50 µM IPTG, strong repression was observed for tpx (Fig. 2e). In the case of ompX and STY1978, down-regulation by LeuO was also observed at different ODs (Fig. 2f and g).

To demonstrate the specificity of the LeuO-mediated regulation, a transcriptional fusion containing the complete 5' intergenic region of the ompC gene was evaluated. As we expected, no regulation of ompC by LeuO was observed since no differences in CAT activity were detected in the presence or absence of LeuO (data not shown). The results obtained with the transcriptional fusions support the proteomic data in demonstrating the positive and negative regulatory roles of LeuO in Salmonella serovar Typhi.

Identification of the transcription start site of the genes regulated by LeuO. To determine the promoters associated with each of the LeuO-dependent genes, primer extension experiments were performed. Salmonella serovar Typhi IMSS-II and Salmonella serovar Typhi IMSS-III, transformed with pKK232-9 derivatives harboring the 5' regions of the LeuO-dependent genes (see Table S1 in the supplemental material), were grown in MA medium to stationary phase. Using total RNA isolated from MA medium-grown cells, the transcription start sites of STY3070, assT, ompX, tpx, and STY1978 were determined (Fig. 3). The transcription initiation sites of ompS1 and ompS2 were reported previously (12, 39). The primer extension results showed that STY3070 contains two transcription start sites (Fig. 3a, panel A), whereas assT, ompX, tpx, and STY1978 each had a single transcription initiation site (Fig. 3b, panel A; Fig. 3d, panel A; Fig. 3e, panel A; and Fig. 3f, panel A, respectively). Our results also show that in the case of STY3070 (Fig. 3a, panel A) and assT (Fig. 3b, panel A), the initiation sites were detected only in IMSS-III (containing pFMTrcleuO-50), while no signals were detected in the absence of LeuO (pFMTrc12). In contrast, for ompX, tpx, and STY1978 the initiation sites were detected in the presence of pFMTrc12, but diminished signals were observed with pFMTrcleuO-50 (Fig. 3d, panel A; Fig. 3e, panel A; and Fig. 3f, panel A, respectively). This observation supports the hypothesis that there is strict LeuO dependence of STY3070 and assT, as well as the hypothesis that down-regulation of ompX, tpx, and STY1978 is exerted by the LeuO regulator. In Fig. 3, the nucleotide sequences corresponding to the region containing the transcription start sites of STY3070, assT, ompX, tpx, and STY1978 are shown.

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FIG. 3. Transcriptional start sites and LeuO protected regions of genes belonging to the LeuO regulon. Primer extension and DNase I protection assays for the STY3070, assT ompS2, ompX, and tpx genes were performed. For STY1978, only the transcriptional initiation results are shown. (A) Transcription initiation in the presence (+) or absence (–) of LeuO. (B) LeuO sites protected from DNase I. The LeuO concentrations used are indicated at the top. D– and D+ indicate without and with DNase I, respectively. DHR, DNase I hypersensitive regions. (C) Schematic diagram of the regulatory region of the LeuO-dependent genes. The nucleotides corresponding to the transcription initiation site () the LeuO regions protected from DNase I (I and II), and the translation initiation sites (ATG) are indicated.

To further support the experimentally determined transcription start sites, we used neural network promoter prediction (38). The in silico promoter prediction results were similar to the experimental data, since for STY3070 the experimental data showed that there were two initiation sites at 85 and 90 bp upstream of the ATG codon, whereas the in silico analysis predicted that there was a single promoter at 85 bp upstream of the ATG codon. For ompX, tpx, assT, and STY1978 the experimental results showed that the initiation sites were 240, 33, 165, and 11 bp upstream of the ATG codon, respectively, and the in silico results predicted that the transcription initiation sites were located 240, 34, 135, and 29 bp upstream of the ATG codon, respectively. Hence, the locations of two LeuO-dependent promoters (STY3070 and ompX) were in agreement as determined by the experimental and in silico approaches.

LeuO binds to the regulatory regions of the LeuO-controlled genes. The proteomic data, the gene fusions, and the primer extension experiments described above indicated that LeuO is a genetic component involved in the regulation of STY3070, assT, ompS1, ompS2, tpx, ompX, and STY1978. In order to elucidate whether LeuO directly interacted with the 5' upstream regulatory region of these LeuO-dependent genes, footprinting experiments were performed. A previous report showed that LeuO bound to the promoter region of ompS2 at –93 to –109 and –139 to –152 bp with respect to the transcription start site (12). In this work the same footprinting profile was obtained (Fig. 3c). For ompS1, two LeuO binding sites were recently detected (5). Interestingly, for the novel LeuO-dependent genes, two regions protected from DNase I by LeuO were also detected. In the 5' intergenic region of STY3070, the sites were identified at nucleotides –65 to –105 and –110 to –140 (Fig. 3a, panel B). In the case of the regulatory region of assT, the first protected sequence was located at nucleotides –112 to –122 and the second was located at nucleotides –132 to –165 (Fig. 3b, panel B). For the 5' region of ompX, the LeuO DNase I-protected sites were detected at nucleotides 1 to 9 and 28 to 60 (Fig. 3d, panel B). The promoter region of tpx had two LeuO binding regions located between nucleotides –22 to –46 and between nucleotides –51 to –59 (Fig. 3e, panel B). In the footprinting experiments (Fig. 3), a LeuO concentration of 150 nM was high enough for protection of ompS2 (12), whereas a higher concentration was required for STY3070, assT, ompX, and tpx.

Interestingly, in the 5' intergenic region (41 bp) of STY1978, no LeuO protected sites were detected, suggesting that LeuO indirectly regulates the expression of this gene. Furthermore, we evaluated 131 bp upstream and 38 bp downstream of the 5' intergenic region of STY1978 in order to see whether LeuO bound at a greater distance, but protection from DNase I was not detected. Alternatively, binding of LeuO to the 5' regulatory region of STY1978 might require an accessory molecule, as reported for other LysR family members (48), or even could occur outside the boundaries of the region studied.

Hence, the footprinting results showed that LeuO directly interacts with the regulatory regions of STY3070, assT, ompX, and tpx and with ompS2, as previously shown (12). Likewise, LeuO interacts directly with the ompS1 regulatory region, as has been recently illustrated (5). In addition, LeuO probably indirectly regulates STY1978. Figure 3 shows the DNA sequences recognized by LeuO in each member of the LeuO regulon.

To determine a consensus sequence for LeuO interaction, the LeuO sites determined in this study were analyzed by using two bioinformatics approaches. The results showed that the matrix generated with the pattern-matching method identified binding sites in the non-LeuO-regulated genes and in the random sequences and detected only one-half of the experimental DNA binding sites reported in this work. In this set of genes false DNA binding sites were also detected (see http://www.ccg.unam.mx/Computational_Genomics/tools/LeuO/), indicating that the interacting sites are not sequence specific. In addition, the lack of either significant oligonucleotides or dyads in the upstream regulatory region of the LeuO-dependent genes supported the notion that the LeuO binding sites are not sequence specific and may not be easily represented with a consensus. The results of the complete analysis are available at http://www.ccg.unam.mx/Computational_Genomics/tools/LeuO/.

Effect of the nucleoid-associated protein H-NS on regulation of the LeuO-dependent genes. Since the proposed role of LeuO has been as an antagonist of H-NS action (2, 5, 55), the functional effect of the global regulatory protein H-NS on the genetic expression of the LeuO-regulated genes was evaluated. To this end, transcriptional fusions of each member of the LeuO regulon were transformed independently into Salmonella serovar Typhi STYhns99 and CAT specific activity was measured in MA medium at different ODs. The transcriptional profile obtained was consistent with the hypothesis that H-NS represses the expression of ompS1 (5) (Fig. 2c), assT (Fig. 2b), and STY307O (Fig. 2a), suggesting that the STY3070-STY3064 operon also belongs to the H-NS regulon. For ompS2 and ompX expression, no change in the transcriptional pattern was observed in the hns mutant (Fig. 2d and f). In the case of tpx and STY1978, the CAT activity decreased 60% in each case in the absence of H-NS, suggesting that H-NS enhances positive regulation (Fig. 2e and g). These data are in agreement with the notion that LeuO and H-NS regulate the same genetic loci (2, 5, 27, 45, 50, 55).

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In this study, a proteomic approach was used to elucidate the genes regulated by LeuO in Salmonella under a particular set of conditions. We found that LeuO regulates the expression of seven proteins with different biological functions. For instance, the OmpS1 porin of Salmonella serovar Typhimurium is proposed to be involved in swarming, motility, and biofilm formation (34, 53, 58), and expression profiles of Salmonella serovar Typhi and Salmonella serovar Typhimurium showed that the ompS1 gene is induced with hydrogen peroxide (43). Interestingly, the microarray results showed that the ompS2 gene was also expressed with hydrogen peroxide, although significant induction was detected only in Salmonella serovar Typhi (43). The assT gene encodes a putative arylsulfate sulfotransferase that converts toxic phenolic compounds into nontoxic compounds and is proposed to be involved in a detoxification process (20, 24). The STY3070 operon encodes uncharacterized proteins predicted to be involved in DNA repair, replication, and recombination (18).

The STY3070 to STY3064 open reading frames are also associated with short palindromic repeats (CRISPR) that are widely distributed in prokaryotes (15) and have been proposed to represent a form of mobile genetic elements which move via horizontal gene transfer (14). Another LeuO-dependent gene reported in this work is ompX, encoding an outer membrane protein which increases E^E activity when multiple copies are present (32, 33) and which also appears to be expressed under different pH conditions (51). Inactivation of ompX increased tolerance to sodium dodecyl sulfate and antibiotic compounds, suggesting that OmpX affects transport of hydrophobic compounds across the membrane (40). The tpx gene was also negatively regulated by LeuO; this gene is proposed to code for a periplasmic antioxidant enzyme that is induced under several pH conditions (51), in the exponential growth phase, and during biofilm formation (22). STY1978 is another LeuO-dependent gene, and it encodes a hypothetical protein with no assigned functional role (41).

Recent data have shown that LeuO is involved in the regulation of mechanosensitive channels that allow bacteria to survive during a rapid increase in turgor pressure (28). Interestingly, Wu and Fang have commented that LeuO affects the expression of at least 27 genes either positive or negatively (60). Together, all these data indicate that LeuO regulates proteins involved in several biological processes and may be a global regulator in Enterobacteriaceae.

To further support the proteomic data, transcriptional gene fusions were constructed, and similar results were obtained. Interestingly, the transcriptional results showed that LeuO positively induced STY3070, assT, and ompS1 at different points of the growth curve, and the highest activity was observed in stationary phase (Fig. 2a to c). In order to verify that this effect was dependent on the LeuO concentration, growth curves were obtained with different amounts of IPTG (25, 50, 100, and 200 µM). The gene reporter results with different IPTG concentrations suggest that for assT and STY3070 induction is dependent on the LeuO concentration (data not shown). In the case of ompS2, LeuO also acted positively when it was present at low levels, but if the amount of LeuO increased, ompS2 expression diminished (Fig. 2d), supporting the observation that an ompS2 gene fusion is expressed at higher levels at an OD of 0.6 upon induction of LeuO with 20 µM IPTG from pFMTrcleuO-50 (5).

In the case of tpx, LeuO was a strong repressor since low transcription levels were detected at any OD (Fig. 2e); for ompX and STY1978, transcriptional down-regulation was also observed (Fig. 2f and g). These results suggest that LeuO has the ability to differentially repress gene expression.

To elucidate whether LeuO directly regulates the LeuO-dependent genes, footprinting experiments were performed. The results showed that two LeuO protected sites were found in the LeuO-dependent genes reported in this work, and a DNase I hypersensitivity region was observed when increasing amounts of LeuO were used, suggesting that there was a DNA conformational change upon binding of LeuO (Fig. 3), which is consistent with our previous observations for the ompS2 and ompS1 genes (5, 12). For the positively regulated LeuO-dependent genes, the LeuO regions protected from DNase I are far from the transcription initiation site, suggesting that additional regulatory inputs might be involved in the regulation of these genes. For the repressed genes, the LeuO sites overlap with the initiation site and with the putative promoter region; thus, the down-regulation exerted by LeuO could be explained by competition or hindrance with RNA polymerase. Further work is necessary to validate these hypotheses.

In previous work (2), three LeuO binding sites were found in the ilvIH-leuO-leuABCD cluster. Two of these sites (AT7 and AT3) were mapped upstream of the leuO promoter, whereas the third site was located downstream of the leuO gene, and it was suggested that H-NS represses leuO expression by interacting with an AT8 region located between LeuO binding sites AT7 and AT3. However, the LeuO protein interacts with the AT7 region and the LeuO binding site located downstream of leuO, making a DNA loop to counteract repression by H-NS (2, 3). Recently, our group reported a novel ompS1 regulatory mechanism in which LeuO acts as an antirepressor by displacing the negative regulator H-NS (5). Therefore, different LeuO regulatory mechanisms are present in Enterobacteriaceae; hence, the two LeuO sites detected in every member of the LeuO regulon described here will be the subject of future studies to explain the mechanism of LeuO regulation in detail.

Previous reports showed that LeuO reduces the expression of the dsrA gene (45) and that the nucleotide sequences necessary for this effect have a high A+T content. Additional reports (2, 3, 4) on gene expression of the ilvIH-leuO-leuABCD gene cluster in Salmonella serovar Typhimurium and E. coli showed that the AT7 and the EAT16 regions have high A+T contents. However, at present, we believe that it is difficult to define a consensus DNA binding motif for the LeuO-dependent genes reported in this work. Thus, each of the LeuO protected regions should be evaluated by site-directed mutagenesis to determine the specific LeuO interacting motifs.

LeuO and H-NS are involved in regulation of leuO and ompS1 (2, 5), in addition to bgl, cadC, dsrA, and rovA (27, 45, 50, 55). In order to determine whether the LeuO-dependent genes described in this work were regulated by this nucleoid protein, transcriptional expression of the LeuO-dependent genes was evaluated in a Salmonella serovar Typhi hns mutant. The results of our experiments concur with the concept that H-NS represses the expression of STY3070, assT, and ompS1 in Salmonella serovar Typhi (Fig. 2a to c). Previous studies also showed that assT and ompS1 are repressed by H-NS in Salmonella serovar Typhimurium and Salmonella serovar Typhi, respectively (13, 37). Moreover, microarrays for the STY3070-STY3064 operon indicated that in three of the genes, repression was exerted by H-NS, although ChIP on-chip analysis suggested that there was indirect regulation since no coimmunoprecipitation was reported (29, 37). Interestingly, the results presented here indicate that H-NS is a positive regulator of tpx and STY1978; one possibility is that in an hns mutant the levels of LeuO increase since leuO is repressed by H-NS. The microarray experiments with Salmonella serovar Typhimurium (37) also showed that H-NS positively induces the expression of STY1978; however, in contrast to our data, the global microarray analysis indicated that H-NS represses tpx expression.

In E. coli, a positive role of H-NS has been described for MalT, lamB, and malE (19), and a proteomic analysis has also revealed that H-NS has the ability to act as a positive regulator (25, 36, 61). Previous reports claimed that H-NS plays an indirect role in the flhDC operon involved in flagellation synthesis via HdfR (1, 23).

The data obtained in this work suggest that tpx and STY1978 belong to the repertoire of genes that are positively regulated by H-NS; however, additional work is necessary to determine if this regulation is direct or if other proteins are involved in this process. The transcription experiments also revealed no evident changes in the expression of the ompS2 and ompX genes in the hns mutant. In both cases our results are opposite those of a Salmonella serovar Typhimurium microarray analysis, which indicated that H-NS represses the expression of these two genes (37). This suggests that there is differential regulation by H-NS even in highly related bacterial serovars.

Altogether, these data support the hypothesis that most of the LeuO-regulated genes described in this work are dependent on H-NS (Table 2). Under the conditions used in this work, a discrete number of LeuO-dependent genes were identified; however, the use of other approaches, such as microarray and ChIP on-chip analyses, could increase the number of LeuO-regulated genes detected in Salmonella.

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TABLE 2. LeuO- and H-NS-regulated genesa

 
We thank S. Contreras, R. Noguez, M. A. De la Cruz, C. Guadarrama, A. Vazquez, F. J. Santana, J. A. Ramirez-Trujillo, M. Wiesner, E. Villa, P. Jarillo, J. Tellez, E. Lopez, S. Becerra, J. Yañez, P. Gaytan, S. C. Janga, A. Medina-Rivera, and L. Medina for technical help and useful scientific opinions that improved this work.

This research was supported in part by grants to E.C. from CONACyT, Mexico (grant 46115Q) and DGAPA/UNAM (grant IN201407) and to I.H.-L. from DGAPA/UNAM (grant IN206705).

 
* Corresponding author. Mailing address: Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos 62210, México. Phone: (52) (777) 329-1645. Fax: (52) (777) 313-8673. E-mail: ecalva{at}ibt.unam.mx

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://jb.asm.org/.

I.H.-L. and A.L.G.-H. contributed equally to this work.

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Journal of Bacteriology, March 2008, p. 1658-1670, Vol. 190, No. 5
0021-9193/08/$08.00+0     doi:10.1128/JB.01649-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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