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J Bacteriol. Mar 2006; 188(6): 2233–2243.
PMCID: PMC1428135

Identification of New Flagellar Genes of Salmonella enterica Serovar Typhimurium

Abstract

RNA levels of flagellar genes in eight different genetic backgrounds were compared to that of the wild type by DNA microarray analysis. Cluster analysis identified new, potential flagellar genes, three putative methyl-accepting chemotaxis proteins, STM3138 (McpA), STM3152 (McpB), and STM3216(McpC), and a CheV homolog, STM2314, in Salmonella, that are not found in Escherichia coli. Isolation and characterization of Mud-lac insertions in cheV, mcpB, mcpC, and the previously uncharacterized aer locus of S. enterica serovar Typhimurium revealed them to be controlled by σ28-dependent flagellar class 3 promoters. In addition, the srfABC operon previously isolated as an SsrB-regulated operon clustered with the flagellar class 2 operon and was determined to be under FlhDC control. The previously unclassified fliB gene, encoding flagellin methylase, clustered as a class 2 gene, which was verified using reporter fusions, and the fliB transcriptional start site was identified by primer extension analysis. RNA levels of all flagellar genes were elevated in flgM or fliT null strains. RNA levels of class 3 flagellar genes were elevated in a fliS null strain, while deletion of the fliY, fliZ, or flk gene did not affect flagellar RNA levels relative to those of the wild type. The cafA (RNase G) and yhjH genes clustered with flagellar class 3 transcribed genes. Null alleles in cheV, mcpA, mcpB, mcpC, and srfB did not affect motility, while deletion of yhjH did result in reduced motility compared to that of the wild type.

What is a flagellar gene? Loss of motility when a gene is disrupted might seem a good definition of a flagellar gene. However, the disruption of many genes not considered flagellar genes results in a motility-defective phenotype. These include genes such as hns, crp, cya, dnaJ and dnaK, pss, and psd (6). These are global regulators that affect gene expression in a number of regulons, including the flagellar regulon. There are a number of genes that are directly responsible for the structure and assembly of the bacterial flagellum and its normal function in chemotaxis. These originally defined the flagellar genes, and their characterization has led to the discovery that the flagellar regulon is a hierarchy of coordinately transcribed genes (30). Structural genes are assigned to three assembly classes of early, middle, and late depending on when their products are needed for assembly and function (7). The promoters responsible for flagellar gene expression are also assigned to three classes, class 1, class 2, and class 3, according to their temporal expression after induction of the flagellar regulon (25). A number of new flagellar genes have been identified, not by their effect on motility but by the fact that they are transcribed from flagellar promoters in operons with known flagellar genes. Some of these, such as flhE, fliY, fliZ, and fliT, have little or no effect on motility (37), but in the case of fliZ and fliT they have positive and negative effects on transcription of other flagellar genes (29).

The flagellar regulon of Salmonella enterica serovar Typhimurium includes over 60 genes. Our current understanding of this process is diagrammed in Fig. Fig.11 (reviewed in references 6 and 34). At the top of the transcriptional hierarchy lies the flagellar master operon, flhDC, where the fundamental decision to produce flagella is controlled. The flhDC operon is expressed from what is defined as the class 1 promoter. As mentioned above, a number of global regulatory signals influence transcription of the flhDC promoter, which has been shown to contain at least six different transcriptional start sites (52). The FlhD and FlhC proteins form a heteromultimeric complex (FlhD2C2) that acts as a transcriptional activator to promote σ70-dependent transcription from the class 2 flagellar promoters (32, 33). The FlhDC complex also acts to autorepress transcription of the class 1 promoter (26).

FIG. 1.
Flagellar regulon expression coupled to flagellum assembly. The flagellar genes are organized into a transcriptional hierarchy that is coupled to assembly of the flagellar organelle as described in the text. The σ28 structural gene, fliA, is labeled ...

The class 2 promoters direct transcription of the flagellar middle assembly genes needed for the structure and assembly of the hook-basal body (HBB) structure. Also transcribed from class 2 promoters are three late assembly genes, flgK, flgL, and fliD, whose products are assembled immediately after HBB completion (27). In addition to structural genes, a number of regulatory genes are also transcribed from class 2 promoters, including the fliA and flgM genes (17, 23). The fliA gene encodes the flagellar-specific transcription factor, σ28, which directs transcription from class 3 promoters (38). It is held inactive prior to HBB completion by the anti-σ28 factor, FlgM (25, 39). Upon completion of the HBB, there is a switch in secretion specificity of the flagellar type III secretion apparatus from HBB-type (middle gene assembly) substrates to late secretion substrates that include FlgK, FlgL, FliD, FlgM, and flagellin (either FliC or FljB) (14, 19). These late secretion substrates all have corresponding type III secretion chaperones (TTSCs), which facilitate substrate secretion either directly or indirectly by preventing proteolytic degradation of substrates in the cytoplasm prior to secretion (4, 12). The TTSCs also have roles as regulators of flagellar gene expression. The TTSC for FlgK and FlgL is FlgN (13). Loss of FlgN results in reduced intracellular FlgM levels due to a decrease in flgM gene translation (24). The TTSC for FliD is FliT (13). Loss of FliT results in an increase in class 2 transcription (29). The TTSC for FlgM is σ28 (K. Hughes and P. Aldridge, unpublished results). Loss of σ28 results in no class 3 transcription (38). The TTSC for flagellin is FliS (3). This study demonstrates that the loss of FliS results in an increase in class 3 transcription.

A set of genes that includes the late secretion substrates and their secretion chaperones and important regulatory proteins are transcribed from both class 2 and class 3 promoters. These are the flgM, flgN, flgK, flgL, fliA, fliD, fliS, and fliT genes (15, 23, 27). The flgM and flgN genes are in the class 2 flgAMN and class 3 flgMN transcripts. The flgK and flgL genes are in the class 2 flgBCDEFGHIJKL and class 3 flgKL operons. The fliAZY operon is transcribed from a class 2 promoter, and the shorter fliAZ operon is from a separate class 3 promoter (23). The role that transcription from multiple promoters plays in the coupling of flagellar gene expression to flagellar assembly has yet to be determined.

In the work presented here, we compared levels of RNA from strains of S. enterica serovar Typhimurium LT2 defective in different flagellar regulatory genes to the levels of RNA expressed in the wild-type strain. This was done by DNA microarrays prepared from PCR-amplified open reading frames (ORFs) derived from the annotated sequence of the S. enterica serovar Typhimurium LT2 genome. Using a clustering analysis of eight mutant backgrounds, the flagellar genes were grouped to the known transcriptional classes (class 2 or class 3) to assign previously uncharacterized flagellar genes and the genes expressed from both class 2 and class 3 promoters. In addition, novel Salmonella flagellar genes were identified.

MATERIALS AND METHODS

Bacterial strains and plasmids.

Bacterial strains used in this study and their origins are listed in Table Table1.1. Plasmid pRP1 was constructed as follows. Strain TH3234 carrying a MudJ insertion in the fliA gene (fliA::MudJ) was converted to a Mud-P22 insertion (fliA::MudP) (54). The fliA::MudP strain was induced with mitomycin C, and DNA was isolated from the induced phage lysate (the DNA packaged from the induced fliA::MudP is enriched for chromosomal DNA flanking the 5′ end of the fliA locus [54]; the DNA was digested with EcoRI [New England Biolabs]). A 3.5-kbp fragment that includes the fliB+ and fliC+ genes and portions of the adjacent IS200 and fliD sequences was ligated (with T4 DNA ligase [New England Biolabs]) into the pBluescript II KS vector (Stratagene) digested with EcoRI to create pRP1.

TABLE 1.
List of bacterial strains

Media and standard genetic manipulations.

Media, growth conditions, transductional methods, and motility assays were as described previously (15, 16). The generalized transducing phage of S. enterica serovar Typhimurium P22 HT105/1 int-201 was used in all transductional crosses (9). Transductions involving tetracycline-sensitive (Tcs) selection was done as follows: 0.1 ml of a 10-fold dilution from a standard phage prep was mixed with 0.1 ml of an overnight culture (yielding a final mixture of 108 to 109 phage and 2 × 108 cells). As a control for spontaneous tetracycline sensitive mutants, 0.1 ml of 0.85% saline solution was mixed with 0.1 ml of the same overnight culture. Both solutions were allowed to stand at room temperature for about 30 min and then diluted 10- and 100-fold prior to plating on tetracycline-sensitive selection plates and incubated at 42°C (35). It takes 2 days for Tcs colonies to appear. If the transduction works well, there should be at least 100 times the number of Tcs colonies on the experimental plates as there is on the no-phage control plates.

Isolation of targeted flagellar gene deletions.

S. enterica serovar Typhimurium chromosomal gene deletion and replacement with drug resistance cassette FRT-Kan-FRT (FKF) or FRT-Chlor-FRT (FCF) were performed by the lambda Red phage mediated recombination system (8). The oligonucleotide sequences below in uppercase are the sites of recombination in the chromosome. The lowercase sequences are sites of P1 or P2 for PCR amplification of pKD4 (FKF) or pKD3 (FCF) (8). TH4911 ΔflgN5626::FKF (deletion of entire coding region) 1frtflgN, 5′-ATTCGCCAGGCGCAGAGCTACTTACAGAGTAAATAAGCGTgtgtaggctggagctgctcc-3′; 2frtflgN, 5′-CAGGCCGGAAAGGCGCAACGTCGCCATCCGGCAATGATTAcatatgaatatcctccttag-3′; TH4912 Δflk-5627::FKF (deletion of entire coding region) 1FRTFLK, 5′-GCATATATTGCTCAGATTTATGGTTAAAGGATAATTAATTgtgtaggctggagctgcttc-3′; 2FRTFLK, 5′-GGTTGGCATCCTGGTGACGATATTATCGTTCCGGCGCGGTcatatgaatatcctccttag-3′; TH5071 ΔflgM5628::FKF (deletion from codon 5 to 97 and the intervening region between flgM and flgN) FlgMP1, 5′-AACGTAACCCTCGATGAGGATAAATAAATGAGCATTGACCGgtgtaggctgcagctgcttc-3′; FlgNATGP2, 5′-ACGGTGGTCATCTGGTCAAGTATTTCTGACAAACGAGTCATcatatgaatatcctcctta-3′; TH5360 ΔfliA5467::FCF (deletion from codon 9 to 218) fliAP1, 5′-GTGTAATGGATAAACACTCGCTGTGGCAGCGTTATGTACCgtgtaggctggagctgcttc-3′; fliAP2, 5′-AATCGTTTGATGGCCTGACTATGCAACTGGCTGACCCGCGcatatgaatatcctccttag-3′; TH5711 ΔfliZ5737::FKF (deletion of entire coding region) fliZF1, 5′-CGAAAAGTGCCGCACAACGTATAGACTACCAGGAGTTCTCgtgtaggctggagctgcttc-3′; fliZF2, 5′-GTTTCACCAACACGACTCTGCTACATCTTATGCTTTTTAAcatatgaatatcctccttag-3′; TH5737 ΔfliS5720::FKF (deletion from codon 2 to 135) 5′fliS-FRT, 5′-AATTTACAGCTATGAACAAGTCCTGATAACAGAGGTCACCATgtgtaggctggagctgcttcg-3′; 3′fliS-FRT, 5′-ACGCTGCCAACGGTTGATAAACTCCACGGTTGAGGTCATTAcatatgaatatcctccttag-3′.

Isolation of a Tn10dCm insertion in the IS200 element near the fliB gene.

To target Mud insertions to specific regions within the flagellar fli region, a number of Tn10dCm insertions were isolated that were linked to a fliC::Tn10 allele, which was transduced into strain LT2 (to create TH453) from strain MH111 (Table (Table1).1). P22 was grown on strain TT10604, which caries a Tn10dCm insertion in the F plasmid, and used to transduce strain TT10427 to chloramphenicol resistance. TT10427 carries plasmid pNK972 that constitutively expresses Tn10 transposase (50). Because there was no F plasmid in strain TT10427, it was not possible to inherit the Tn10dCm transposon by homologous recombination. The chloramphenicol-resistant (Cmr) transductants were inherited by transposition into the recipient chromosome. Greater than 50,000 Cmr transductants were pooled together, and a P22 lysate was prepared on the pooled cells carrying Tn10dCm insertions located at positions throughout the chromosome. To isolate Tn10dCm insertions in the fliC region, the pooled lysate was used to transduce TH453 (fliC::Tn10) to Cmr on L-Cm plates, and the colonies were screened for loss of the fliC::Tn10 allele by replica printing onto L-Cm-Tc plates and screening for those that became Tcs upon transduction to Cmr. This allows for the isolation of Tn10dCm insertions near fliC, because the recipient does not carry Tn10 transposase, so the Tn10dCm elements from the pooled donor lysate are inherited by homologous recombination. Those close to fliC can inherit Tn10dCm-encoded Cmr, and if they also coinherit the fliC+ allele by cotransduction, they become Tcs. A total of 19 Cmr Tcs transductants were kept for further analysis. Of these 19, 10 were found to be nonmotile, indicating that they had inserted into one of the flagellar genes in the fli region, 1 was nonmotile in a fljB null strain only, indicating that it was inserted in the fliC gene, and 8 were motile. Flair complementation analysis was performed on the 10 nonmotile fli::Tn10dCm insertions. Of the 10, 5 were located on the fliD gene, 1 was located in fliF and 4 were located in fliQ. Linkage analysis of the 8 motile insertions revealed that four were linked to the fliR locus (located counterclockwise of fliR on the standard S. enterica serovar Typhimurium linkage map), two were located between the fliT and fliE genes, one was located between the fliC and fliD genes, and one was between the fliC and fliA genes. DNA sequence analysis was performed on the insertion between the fliC and fliA genes, named fli-5448::Tn10dCm. The fli-5448::Tn10dCm transposon had inserted counterclockwise after base pair 25 of the presumed ATG start codon of the transposase gene of the IS200 element (IS200IV) located between the fliA and fliB loci.

Isolation of the fliB5968::MudJ and fliB5740::MudK insertions.

Strain TH3730 carries a Tn10dTc[del-25] (T-POP) insertion located between the class 1 flhDC promoter and the flhDC structural genes. This insertion places the flhDC operon and thus the entire flagellar regulon under control of the inducible tetA promoter (25). The MudJ transposon was introduced into strain TH1059 (fli-5448::Tn10dCm) carrying the Tn10dCm insertion in the IS200IV element next to the fliB gene by the method of transitory cis complementation (20). Greater than 50,000 MudJ-encoding kanamycin-resistant (Kmr) transductants were pooled, and P22 transducing phage was grown on the pooled cells. The lysate was used to transduce strain TH3730 to Cmr on L-Cm plates, and these plates were then replica printed to L-Cm-Km plates to identify Cmr Kmr recipients that had also inherited a MudJ insertion along with the fli-5448::Tn10dCm allele, presumably by cotransduction. The Cmr Kmr transductants were screened for motility and expression of the lac genes in the presence of Tc (flhDC-inducing condition). Putative fliB::MudJ isolates were defined as being greater than 85% linked to the fli-5448::Tn10dCm allele by P22 transduction, exhibited a Tc-inducible Lac+ phenotype in the presence of the PflhDC::T-POP insertion, and were motile (loss of fliB does not affect motility). Subsequent PCR and DNA sequence analysis identified the fliB5968::MudJ transposon to be inserted after base 38 of the fliB coding region (fliB is transcribed counterclockwise on the standard S. enterica serovar Typhimurium linkage map).

The fliB5740::MudK insertion was isolated in a hunt for MudK insertions in the fliC gene (5). MudK insertions greater that 85% linked to the fli-5448::Tn10dCm allele that were motile were analyzed by PCR and DNA sequence analysis. One, fliB5740::MudK, was found to be inserted 813 nucleotides into the fliB coding region, resulting in a translational fusion of the first 271 amino acids of FliB to LacZ, and it was used for further studies.

Isolation of MudJ insertions in the cheV, mcpB, mcpC, and aer loci.

To isolate insertions of MudJ in cheV, we obtained a linked ΔpmrD::Cm allele from Eduardo Groisman. About 50,000 random MudJ insertion mutants in a ΔpmrD::Cm-containing strain were pooled, and P22-transducing phage was used to transduce this pool into strain TH3730, which has the flhDC operon under control of the tetA promoter. Four MudJ insertions that showed Tc-dependent Lac expression were tested by PCR using a primer that hybridized just outside the 3′ end of cheV and a primer that hybridizes to the left end of Mu, which reads out of the MudJ element. All four putative cheV::MudJ insertions gave PCR products of sizes expected for insertions in cheV.

To isolate insertions of MudJ in the mcpC and aer genes, we utilized a Tn10dCm insertion already present in the strain collection, which by DNA sequence analysis revealed it to be at the very 3′ end of the mcpB gene (data not shown). About 50,000 random MudJ insertions mutants in the mcpC::Tn10dCm-containing strain were pooled, and P22 transducing phage was used to transduce this pool into strain TH3730, which has the flhDC operon under control of the tetA promoter. Six MudJ insertions that showed Tc-dependent Lac expression were separated from the mcpC::Tn10dCm insertion by P22 transduction. They were then tested by PCR using either a primer that hybridized just outside the 3′ end of mcpC or a primer that hybridized just outside the 3′ end of aer as well as a primer that hybridizes to the left end of Mu, which reads out of the MudJ element. Two MudJ insertions were located on the mcpC gene, and four were located on the aer gene.

The MudJ pools isolated above were used with strain TH9670 that was defective in the mcpB-linked metC gene and has the flhDC operon under control of the tetA promoter. Three MudJ insertions that showed Tc-dependent Lac expression were tested by PCR using a primer that hybridized just outside the 3′ end of mcpB and a primer that hybridizes to the left end of Mu, which reads out of the MudJ element. All three putative mcpB::MudJ insertions gave PCR products of sizes expected for insertions in mcpB.

β-Galactosidase assays.

β-Galactosidase assays were performed as described by Maloy (35). Cells were grown to an optical density at 600 nm of 0.6 to 0.8. Each data point was the sum of three independently grown bacterial cultures each assayed twice, and the values were recorded as β-galactosidase units (nanomoles per minute per unit of optical density at 650 nm per milliliter).

Primer extension.

RNA was isolated as described previously (47). RNase inhibitor (Boehringer Mannheim) and DNase (Bethesda Research Laboratories) were added to the RNA in RNase-free water (sterile irrigation water; Baxter Healthcare Corp.). 32P labeling of primer FliBPE (5′-GGGTGACAAAGGCAGGTTCAGTGACGGTGA-3′) was done with T4 polynucleotide kinase and [γ-32P]ATP (New England Nuclear) (2). Primer extensions were carried out (2) with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) and buffer supplied by the manufacturer at 42°C for 30 min.

Microarray design and manufacture.

The S. enterica serovar Typhimurium LT2 microarray was constructed from specific PCR products for each gene. These were amplified from LT2 genomic DNA, and whenever possible these encompassed the whole ORF. The initial array set was used in all studies, except for two of the comparisons of the ΔflhDC and LT2 strains. For these, the microarray contained 4,442 whole ORF PCR products, representing 96.6% of the Salmonella enterica serovar Typhimurium LT2 genome and the pSLT plasmid. For the latter two experiments, the overall S. enterica serovar Typhimurium genome coverage for the array was 99.4% (4,466 genes). The arrays also contained PCR products representing the genes found on the LT2 virulence plasmid pSLT. The DNAs were spotted onto Ultra-GAPS glass slides (Corning Inc., Corning, N.Y.) in 50% dimethyl sulfoxide and were hybridized by the method of Brown et al. (http://cmgm.stanford.edu/brown/protocols/4_Ecoli_RNA.txt). Details of the construction of the Salmonella array were described previously (41).

RNA labeling and hybridization.

cDNA probes were labeled with Cy3- and Cy5-dye-linked dUTP by direct incorporation during reverse transcription from total RNA to cDNA, following the method described by Pat Brown (http://cmgm.stanford.edu/pbrown/protocols/4_Ecoli_RNA.txt), with the following modifications. Fifty micrograms of total RNA and 2.4 μg of random hexamers were resuspended in 30 μl of water, and subsequently the amounts and volumes of all components were doubled compared to those of the Brown protocol. Two microliters of RNAsin (F. Hoffmann-La Roche Ltd., Basel, Switzerland) was added to the reverse transcription, and the reaction was incubated at 42°C for 2 h. After the first hour of incubation, an additional 2 μl of Superscript II reverse transcriptase was added. Probes were purified using the QIAquick PCR purification kit (QIAGEN, Valencia, CA) and eluted in 1 mM Tris-HCl, pH 8. 0.

Hybridization and data acquisition.

Probes were hybridized to the Salmonella array overnight in 25% formamide, 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate at 42°C using a hybridization chamber (Corning) submerged in a water bath. Protocols suggested by the manufacturer for hybridizations in formamide buffer (http://www.corning.com/Lifesciences/technical_information/techDocs/gaps_ii_manual_protocol_5_02_cls_gaps_005.pdf) were applied for prehybridization, hybridization, and posthybridization processing. The cDNA probes from wild-type and mutant strains were hybridized to three arrays, and then dyes were switched and samples were hybridized to three additional arrays. Scans were performed on a ScanArray 5000 Laser scanner (Packard BioChip Technologies, Billerica, MA) using ScanArray 2.1 software.

Data analysis.

Signal intensities were quantified using the QuantArray 3.0 software package (Packard BioChip Technologies, Billerica, MA). Spots were analyzed by adaptive quantitation, and local background was subsequently subtracted from the recorded spot intensities. Ratios of the contribution of each spot to total signal in each channel were calculated (data normalization). Negative values (i.e., local background intensities higher than spot signal) were considered no data. The expression ratio of each gene was then calculated as the median of the six ratios from the six hybridizations. RNA measurements were analyzed by calculating ratios and standard deviations between the mutant versus wild-type strains. Genes with signals less than two standard deviations above background in both conditions were considered not detected and were removed prior to any analysis.

RESULTS

Effect of flagellar regulatory mutations on flagellar gene expression.

Two DNA microarray experiments were performed to identify novel flagellar genes of Salmonella enterica serovar Typhimurium. In one experiment, genomic RNA samples isolated from different flagellar mutant backgrounds were compared to RNA samples isolated from the wild-type strain LT2 (see Materials and Methods). In this experiment, each comparison was done between one sample from each of eight different strains and LT2. A cluster analysis program was then used to identify new genes that clustered with known flagellar genes. In a second experiment, four independent comparisons of RNA levels from a strain deleted for the flagellar master operon, flhDC, were compared to RNA levels from wild-type strain LT2.

Cluster analysis.

The expression levels of whole cellular RNAs from the eight different flagellar mutant strains relative to LT2 were subject to cluster analysis using the J-Express program (MolMine AS, Bergen, Norway). These strains included TH3358 (ΔflgM5301), TH4912 (Δflk-5627), TH5071 (ΔflgM5628), TH5360 (ΔfliA5647), TH5720 (flhC2035), TH6554 (ΔflgM5628 ΔfliA5647), TH6555 (ΔflgM5628 ΔflhDC2039), and TH6572 (ΔflgM5628 ΔfliA5647 ΔflhDC2039). Two clusters were found to include predominantly class 2 or class 3 flagellar genes and are presented in Fig. Fig.22.

FIG.2.
Cluster analysis of flagellar genes using J-Express. A. Cluster [1,1] included structural genes of the hook-basal body (fliF-N and flgA-J), the flagellin methylase (fliB), secretion chaperones fliS and fliT, a putative methyl-accepting chemotaxis gene, ...

In the class 2 flagellar cluster (Fig. (Fig.2A),2A), we observed many of the genes required for hook-basal body (HBB) formation. The late secretion substrate genes, flgK and flgL, and late secretion chaperone genes, fliS and fliT, which are transcribed from both class 2 and class 3 flagellar promoters, also clustered with the class 2 genes. In addition, the flhC gene clustered with class 2 genes, which probably reflects the fact that the flhDC operon is under autogenous control. The fliB gene, whose promoter has not been characterized, also clustered with the class 2 genes, which suggested that fliB was independently transcribed from the upstream class 3 fliC gene. In results presented below, we show that this is the case.

In addition to known flagellar genes, three genes from a presumed virulence gene operon, srfABC, and an uncharacterized gene, STM3152, annotated as a methyl-accepting chemotaxis protein clustered with the class 2 genes. We show below that the srfABC operon is under FlhDC control but not σ28 control, indicating that this operon is a flagellar class 2 operon. STM3152 was found to be a σ28-dependent class 3 gene, although it did not cluster with the class 3 genes. A large number of other flagellar genes were not present in the class 2 cluster. While the entire flgB-L operon and the flgA gene were in the class 2 cluster, the flhBAE operon and flhD, fliE, fliJ, and fliOPQR genes were not. It is possible that the flhBAE and fliE operons have different expression patterns in the different flagellar mutant strains compared to the other class 2 genes, but other genes cotranscribed with flhD, fliE, fliJ and fliOPQR did cluster as class 2 genes. It is likely that the low level of expression of these genes did not produce the significant changes in expression needed in the various mutant backgrounds to cluster with the other class 2 genes. Genes at the 3′ end of a transcript could be at low RNA levels due to mRNA degradation. Also, in E. coli it was shown that σ28 regulates expression of some class 2 operons (such as fliL-R) but not others (such as flhBAE) (33). This could account for lower levels of the flhBAE operon genes. The fliK gene was not present in the array slides used for these studies.

In the class 3 flagellar cluster (Fig. (Fig.2B)2B) we observed primarily late assembly genes, the motor force generator genes motAB, and the genes of the chemotaxis system. Unlike flgKL and fliST, the other late secretion substrate gene, fliD clustered with the class 3 genes (as did the fliAZ operon), which are expressed from both class 2 and class 3 promoters. In addition to known flagellar genes, the cafA gene, encoding RNase G, clustered as a class 3 gene, but data below will show this to be an error of the particular set of array chips used early in this study. In addition to known flagellar genes, a putative dicyclic GMP phosphodiesterase gene, yhjH, identified previously to have a class 3 flagellar promoter clustered with the flagellar class 3 genes as did three uncharacterized genes, STM2314, STM3138, and STM3152, annotated to be cheV (a cheW-cheY hybrid gene known from Bacillus subtilis) and two methyl-accepting chemotaxis proteins, respectively. We did not observe the flgMN operon in either cluster, and the tar (cheM) gene was not in the array slides.

FlhDC-dependent flagellar genes.

In a second set of experiments, we looked at genes dependent on the flhDC master operon for expression. Using one set of microarray chips, two independent RNA isolates from a ΔflhDC strain were compared for reduced expression from two independent RNA isolates from strain LT2 (see Materials and Methods). This was repeated with an independently constructed set of microarray chips, and the top 90 FlhDC-dependent genes from all four comparisons are shown in Table Table2.2. As expected, flagellar gene expression was reduced compared to that of pathogenesis genes. The flagellar regulatory system was shown to affect the regulation of many virulence-associated genes (10, 46, 48). The list of FlhDC-dependent genes includes those identified by the cluster analysis as novel flagellar genes, yhjH and STM2314 (cheV), as well as putative MCP genes STM3138 (mcpA), STM3152 (mcpB), and STM3216 (mcpC). We have renamed the STM3138, STM3152, and STM3216 loci mcpA, mcpB, and mcpC due to their homology to known methyl-accepting chemotaxis protein genes. The srfABC operon that clustered with flagellar class 2 genes ranked highest of all the virulence-associated genes on the FlhDC-dependent list, which was consistent with its clustering with flagellar genes. The flagellar genes that did not cluster (flgMN, flhBAE, fliE, fliJ, fliOPQR, and fliY) were less affected by the loss of flhDC than the genes that did. The fliY gene is expressed by a σ70-dependent promoter in addition to flagellar promoters (23), which would explain the reduced sensitivity to the loss of flhDC. Surprisingly, two flagellar class 2 genes were, in fact, not significantly affected by the absence of the flhDC operon. The fliR gene averaged only 10% lower RNA levels in the absence of the flhDC operon, while the fliQ gene was unaffected. This likely reflects low levels of fliQR mRNA in the presence of flhDC. Finally, the cafA gene, which clustered with flagellar class 3 genes, showed FlhDC dependence using the first set of array slides (ΔflhDC/LT2 ratio, 0.17) but not in the second independently constructed set of array slides (ΔflhDC/LT2 ratio, 1.3), suggesting that the initial clustering with flagellar genes was due to some error in array slide construction. Also, cafA appears to be part of a large operon that includes the mreBCD genes, which were not FlhDC dependent on either set of array slides.

TABLE 2.
FlhDC-dependent Salmonella genesa

Isolation and characterization of Mud-lac operon fusions to new flagellar genes.

To verify the microarray results for the identification of novel flagellar genes, we took advantage of the fact that a Mud-lac reporter fusion to the srfB gene had been isolated and generously provided to us by Fred Heffron. We also took advantage of the close linkage of STM3152 to the metC gene and the fact that we already had an insertion of Tn10dCm at the very 3′ end of the STM3216 gene to isolate Mud-lac insertions in STM3152, STM3216, and the aer locus (see Materials and Methods) as well as the close linkage of the cheV homolog gene, STM2314, to a ΔpmrD::Cm insertion mutation generously provided to us by Eduardo Groisman.

Insertions of the lac transcriptional reporter vector MudJ in STM2314, STM3152, STM3216, and aer loci were isolated (see Materials and Methods). These, along with the srfB::MudJ insertion kindly provided by Fred Heffron, were assayed for effects of deletions in flhDC and fliA28) on expression of these fusions to determine if these genes were transcribed from flagellar class 2 or class 3 promoters.

Expression of the srfB::MudJ insertion in strains carrying null alleles of flhDC or fliA confirmed that the srfABC operon was a flagellar class 2 operon. Transcription was 30-fold reduced in the absence of FlhDC but was not significantly affected by the fliA null allele (Table (Table33).

TABLE 3.
β-Galactosidase assays using the MudJ lac operon fusion vector as transcriptional reportera

MudJ insertions in the previously uncharacterized putative Salmonella cheV (STM2314) and mcp genes, STM2314 (mcpA), STM3152 (mcpB), STM3216 (mcpC), and aer, were isolated and assayed in strains carrying null alleles of flhDC or fliA and in strains locked in either the fliCON or fljBON orientations of the flagellar phase variation system to determine if any were under control of flagellar phase variation (Table (Table3).3). The MudJ insertion in STM2314 was σ28 dependent and not affected by flagellar phase variation. As with the cheV::MudJ insertion, MudJ insertions in the STM3152 (mcpB), STM3216 (mcpC), and aer loci were σ28-dependent class 3 flagellar genes and were not affected by flagellar phase variation. The cheV::MudJ, mcpB::MudJ, mcpC::MudJ, and aer::MudJ fusions were not affect by flagellar phase variation. Only mcpB::MudJ showed a somewhat reduced level in the fliCOFF fljBON (Dhin-5718::FCF) orientation, but the level observed was still within the error range of the β-galactosidase assays. While we did not obtain a MudJ insertion in STM3138, we presumed that its clustering with the flagellar class 3 genes, its dependence of FlhDC for expression (RNA levels were 67-fold lower in ΔflhDC compared to LT2 [Table [Table2]),2]), its amino acid homology to MCP proteins, and the presence of a σ28 consensus promoter sequence upstream (the consensus −10 sequence is 80 bases upstream of the ATG start codon) was sufficient evidence to include it with the new Salmonella flagellar class 3 genes.

We included the characterization of recently isolated flgK::MudJ in these studies (1). The flgKL operon is transcribed from both class 2 and class 3 promoters (27), and it clustered with the class 2 flagellar genes (Fig. (Fig.2).2). We tested the expression of the flgK-lac transcriptional fusion in wild-type and ΔflhDC and fliA mutant strains (Table (Table3).3). The loss of σ28-dependent class 3 transcription resulted in a fourfold reduction in flgK-lac transcription, and transcription was abolished in the ΔflhDC strain background. This is consistent with the flgKL operon being transcribed from both class 2 and class 3 promoters.

Finally, we also isolated and characterized fliB::Mud-lac reporter constructs. The fliB gene was shown over 40 years ago to encode a protein that methylates the lysine residues of flagellin (45). Prior to this array analysis, we presumed that the fliB gene was transcribed in an operon with the fliC gene flagellin. However, it was not determined if the fliC and fliB genes were cotranscribed in an operon, and it had not been reported where the fliB gene resided within the flagellar regulatory hierarchy. Given its role as a flagellin methylase, it was expected to be transcribed from a class 3 promoter. However, the DNA array data showed that fliB transcription was primarily dependent on FlhDC and not on σ28, indicating that it was transcribed in a class 2 transcript (Fig. (Fig.2A).2A). Transcriptional and translational lac reporter fusions to the fliB reporter were constructed using the Mud-lac transposons MudJ and MudK (see Materials and Methods) (18). The effect of FlhDC and σ28 on the fliB::Mud-lac fusion constructs was determined and is presented in Table Table3.3. Loss of σ28 resulted in a twofold reduction in fliB transcription using the fliB::MudJ (lac operon) transcriptional fusion vector, consistent with other class 2 genes (28). Loss of FlhDC resulted in complete loss of β-galactosidase activity for the fliB::MudJ fusion. These results support the DNA array data and indicate that transcription of the fliB gene was from a class 2 promoter. Low levels of β-galactosidase were obtained with the fliB::MudK construct, suggesting that there may be additional regulation of fliB gene expression at the posttranscriptional level.

Primer extension assay of the fliB gene.

The classification of fliB as a class 2 transcript predicted the presence of a promoter between the 3′ end of the class 3 fliC transcript and the beginning of the fliB open reading frame. Flagellar class 3 promoters have −10 (GCCGATAA) and −35 (TAAAGTTT) regions that are well conserved (21), but the class 2 promoters lack a conserved −35 region, and even the −10 sequences are not well conserved, although 4 out of 10 class 2 promoters have an identical −10 sequence (GATAAT) (22). The closest sequence upstream of the fliB start codon that corresponded to this sequence was CACAAT, 44 bases upstream of the fliB coding region. We performed primer extension assays on RNA samples isolated from isogenic wild-type (LT2), ΔflhDC (TH2231), and pfliB+/WT (pRP1/LT2) strains to locate a transcriptional start site for the fliB gene. The results of the primer extension analysis are shown in Fig. Fig.3.3. The observed FlhDC-dependent start site maps to a guanine position (marked by an asterisk) that was located 9 bases downstream from the putative class 2 CACAAT (−10) sequence mentioned above. In addition, a polar Tn10 insertion in fliC had no effect on fliB-lac expression (data not shown). These results confirm that fliB was transcribed independently from the fliC promoter from its own class 2 flagellar promoter.

FIG. 3.
A. Transcriptional start site mapping for the S. enterica serovar Typhimurium fliB gene. Lanes G, A, C, and T are the sequencing ladder. Primer extensions were measured in isogenic wild-type (WT), ΔflhDC, and pfliB+ wild-type (pfliB+ ...

Motility phenotypes of strains defective in the srfB, yhjH, cafA, and fliB loci.

In the original array, the original slides had cafA clustered with the flagellar genes. It was also identified by another group using the original Salmonella array slides as a gene coregulated with the flagellar genes (48). Using the most recent set of Salmonella array slides, cafA did not show FlhDC-dependent regulation. The location of the cafA gene in an operon of other nonflagellar genes and lack of any potential flagellar promoter sequences supports that cafA is not a flagellar gene. We deleted the cafA and yhjH loci, and the DcafA and DyhjH mutant strains were tested for motility along with strains containing Mud-lac null alleles of srfB or fliB (Fig. (Fig.4).4). The DcafA strain was unaffected in motility. However, a polar, null allele in srfB was also unaffected in motility, even though it was verified to be dependent on an FlhDC-dependent class 2 promoter for its expression. Loss of fliB was also not affected for motility. This is consistent with the idea that flagellin methylation by FliB is required for Salmonella virulence, not for flagellin function (K. Hughes and R. Curtiss III, unpublished data). The deletion of yhjH caused a motility defect, as had been reported previously (43). The yhjH gene product was annotated as a dicyclic GMP phosphodiesterase. The effect of yhjH on motility has yet to be determined.

FIG. 4.
Motility assays of novel flagellar genes. Null alleles in fliB (flagellin methylase), cafA (RNase G), yhjH (unknown), and srfB (unknown) were tested and compared to the wild-type strain LT2 (positive control) and a strain deleted for hook-basal body structural ...

DISCUSSION

In this study, we screened the Salmonella genome for novel genes that were coregulated with known flagellar genes using microarrays. The Salmonella flagellar genes clustered primarily according to whether they were expressed from class 2 or class 3 promoters. One additional operon, the srfABC operon, was also included in the flagellar class 2 gene cluster. The srfC locus encodes a homolog to an ADP ribosyltransferase that is secreted by the virulence-associated Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000 (40). The srfABC operon includes 3 of over 20 genes controlled by SsrAB, a two-component system located within Salmonella pathogenecity island 2 (49). Thus, the srfABC operon is thought to be associated with Salmonella virulence. The operon showed the strongest regulation of all virulence genes by FlhDC. A srfB::MudJ insertion exhibited normal motility on a soft agar plate, indicating that the srf operon is not required for flagellar construction.

We were surprised that several flagellar structural genes, the flhBAE genes, and the fliE, fliJ, and fliOPQR genes did not cluster with class 2 genes, even though they are known flagellar class 2 genes. It is noteworthy that all flagellar genes that showed the least dependence on FlhDC were at the 3′ end of operons (Table (Table2).2). This leads us to suspect that the mRNA of these operons was degraded from the 3′ end so the levels of 3′ gene mRNA was low. We are intrigued by the possibility that microarrays, if set up to be quantitative, can be used to study mRNA degradation pathways. This would be an important use for DNA microarrays in analyzing operon expression.

We were also surprised a little by the overlap between the FlhDC-dependent genes of Salmonella and those of E. coli. In E. coli, six nonflagellar operons were identified that require FlhDC for expression not picked up in this study of Salmonella FlhDC-dependent genes. These were genes required for cytoskelatin genes, mreBCD, and genes required for anaerobic respiration, glpABC, napFAGHBC, nrfABCDEFG, dmsABC, and hydNhypF (42). It is not clear why these were not picked up as FlhDC-dependent genes in Salmonella and may reflect the difference in lifestyles of the two organisms.

There are four new class 3 chemotaxis genes present in Salmonella that are not present in E. coli. These genes, formerly named STM2314, STM3138, STM3152, and STM3216, are here renamed cheV, mcpA, mcpB, and mcpC, respectively. These genes clustered with the known class 3 flagellar genes. The structural motifs of these new genes are shown in Fig. Fig.55 along with Aer and the well-characterized Tar and Tsr MCP receptors. The CheV protein is a fusion of CheW to a CheY receiver domain. McpA, McpB, McpC, and Aer all have a methyl-accepting signaling domain (MA domain [chemotaxis sensory transducer]) common to all MCP proteins. Aer of Salmonella is essentially the same as Aer from E. coli (data not shown). McpB and McpC are similar to Tar and Tsr, with a putative periplasmic ligand-binding domain flanked by transmembrane domains followed by a HAMP domain (histidine kinases, adenylyl cyclases, methyl binding proteins, and phosphatases) and the MA domain. McpA is unusual. McpA appears to have just a cytoplasmic MA domain followed by a coiled-coil region (green). Perhaps McpA interacts with the C-terminal domains of other Mcp receptors to influence their signaling. Expression analysis of Mud-lac insertions in cheV, mcpB, and mcpC confirmed that they are under the control of σ28-dependent promoters. The mcpC gene is in an apparent operon downstream of the Salmonella mcp gene required for aerotaxis, the aer gene. We also obtained Mud-lac insertions in the previously uncharacterized Salmonella aer gene and verified that it was also expressed from a σ28-dependent promoter.

FIG. 5.
Analysis of functional domains in new Salmonella flagellar genes cheV, mcpA, mcpB, mcpC, and aer. The functional domains were determined using the SMART domain-based sequence annotation resource (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1 ...

The three novel MCPs not present in E. coli cluster to the same region of the Salmonella chromosome in the vicinity of the metC locus. This is one of the largest regions of uncharacterized genes in the S. enterica serovar Typhimurium genome (44), with a high density of S. enterica serovar Typhimurium-specific (STM) genes (36). The mcpA (STM3138) and mcpB (STM3152) loci are located only 12.6 kbp from each other, and mcpC (STM3216) is another 64.8 kbp clockwise of mcpB on the S. enterica serovar Typhimurium chromosome (36). Curiously, mcpC (STM3216) is in an operon with the aerotaxis MCP gene aer, which is present in both S. enterica serovar Typhimurium and E. coli. The location of mcpA immediately adjacent to 22 STM genes is suggestive that it may be part of a uncharacterized STM pathogenecity island, and future studies on a possible role of cheV and mcpA, mcpB, and mcpC in S. enterica serovar Typhimurium pathogenesis or survival as a commensal organism in S. enterica serovar Typhimurium hosts are warranted.

The fliB gene was identified as a class 2 gene, and primer extension analysis revealed the presence of an FlhDC-dependent promoter located between fliC and fliB. There is apparent overlap between the 3′-untranslated region of the fliC transcript and the promoter region of fliB (Fig. (Fig.3).3). The fliB transcriptional start site is located within the apparent Rho-independent termination signal for fliC, suggesting that transcription of fliC might interfere with that of fliB. However, a polar Tn10 insertion in fliC did not affect expression of an fliB-lac reporter construct (data not shown). Expression of fliB from a class 2 promoter suggests that the FliB methylase must be temporally expressed before the fliC or fljB filament gene product on which it acts is produced. The exact role in flagellin methylation on Salmonella virulence has yet to be determined, but methylation of exposed lysine residues would remove the reactive positive charge of the lysine side group that could aid in avoiding the host immune response.

Acknowledgments

This work was supported by Public Health Service (PHS) grant GM56141 from the National Institutes of Health (NIH), awarded to K.T.H., and by PHS NIH grants AI034829, AI052237, and AI022933 to M.M. H.R.B. was a recipient of a PHS National Research Service Award (T32 GM07270) from the National Institute for General Medical Sciences.

We thank Andy Lee for technical assistance with the primer extension assays, Angela Fung for help with the fliB::MudK isolation, Ryan Peterson for construction of plasmid pRP1, Lewis Weil for technical assistance with β-galactosidase assays, Cyndy Baker, Syed Tanveer Haider, and Ka Ye for help with the array analysis, Felisa Blackmer for technical assistance, our colleague Steffen Porwollik for invaluable assistance in array construction and data interpretation, and Eduardo Groisman for his generous gift of the ΔpmrD::Cm allele.

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