Kazuhiro Kutsukake

Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium

SummaryThrough genetic studies, the fliA gene product has been shown to regulate positively gene expression in late operons of the flagellar regulon in Salmonella typhimurium. In the present study, the fliA gene was cloned and sequenced. The fliA coding region consisted of 717 nucleotides beginning with the GTG initiation codon and the conserved sequence specific to promoters for flagellar operons was found to exist upstream of the coding region. The fliA gene product deduced from the nucleotide sequence was a protein with 239 amino acid residues and the calculated molecular mass was 27470 dalton. The deduced amino acid sequence was homologous with that of σ28, a flagellar specific sigma factor of Bacillus subtilis. The fliA gene product was identified as a protein of molecular mass 29 kDa in the in vitro transcription-translation system, while three proteins of 29 kDa, 31 kDa and 32 kDa were found in the products programmed by the fliA gene in minicells and in maxicells. The 29 kDa FHA protein was purified from the FliA overproducing strain which carried the ptac-fliA fusion. This protein activated the in vitro synthesis of flagellin, the fliC gene product. RNA polymerase containing the purified FliA protein was shown to transcribe the fliC gene. These results indicate that FliA protein functions as an alternative sigma factor specific for S. typhimurium flagellar operons.

A novel transcriptional regulation mechanism in the flagellar regulon of Salmonella typhimurium : an anti-sigma factor inhibits the activity of the flagellum-specific sigma factor, σF

We have studied the molecular mechanism of the negative regulation by flgM of the late operons of the flagellar regulon of Salmonella typhimurium. A 7.8 kDa protein that was identified as the flgM gene product was purified to homogeneity; its amino‐terminal sequence was identical to the deduced sequence except for the lack of the initiating methionine. The purified FlgM repressed transcription from the fliC promoter, one that is activated by the sigma factor, FliA (σF). No DNA‐binding activity was detected in FlgM. Chemical cross‐linking experiments showed that the purified FlgM bound to σF and disturbed Its ability to form a complex with RNA polymerase core enzyme. These results indicate that FlgM is a novel type of negative regulator that probably inactivates the flagellum‐specific sigma factor through direct interaction, i.e. it is an anti‐sigma factor.

Excretion of the anti-sigma factor through a flagellar substructure couples flagellar gene expression with flagellar assembly in Salmonella typhimurium.

More than 50 genes are required for flagellar formation and function in Salmonella typhimurium. According to the cascade model of the flagellar regulon, the flagellar operons are divided into three classes, 1, 2, and 3, with reference to their relative positions in the transcriptional hierarchy. This sequential transcription is coupled to the assembly process of the flagellar structure, that is, genes involved in formation of the hook-basal body complex belong to the class-2 operons, whereas those involved in formation of filament belong to the class-3 operons. The fliA gene encodes an alternative sigma factor specific for transcription of the class-3 operons. A negative regulatory gene, flgM, which is responsible for the coupling of expression of class-3 operons to flagellar assembly, encodes an anti-sigma factor that binds to FHA and prevents its association with RNA polymerase core enzyme. In the present study, we showed that the flgM gene is transcribed from two different promoters: one is its own class-3 promoter and the other is the class-2 promoter for the upstream gene, flgA. Furthermore, we showed that FlgM is excreted into culture medium from cells of the wild-type strain and of class-3 mutants that can produce complete hook-basal body structures. On the other hand, FlgM is not excreted from mutants defective in the hook-basal body genes. These results indicate that FIgM is excreted from the cells through the flagellar substructures that are formed by the function of the hook-basal body genes. We believe that this is the mechanism by which FlgM can monitor the assembly state of flagellar structure and couple flagellar gene expression to flagellar assembly.

Autogenous and global control of the flagellar master operon, flhD, in Salmonella typhimurium

Abstract Expression of the flagellar master operon, flhD, is known to be affected by growth conditions and by mutations in a variety of genes. In the present work, the transcriptional control of the Salmonella typhimurium flhD operon was investigated in various genetic backgrounds. First, we examined the effect of mutations in the global regulators cAMP-CRP, H-NS, OmpR and RpoS. Mutations in the cya, crp or hns gene reduced but did not eliminate flhD expression. However, expression was completely inhibited in the cya hns and crp hns double mutants. These results indicate that cAMP-CRP and H-NS independently activate the flhD operon and that maximal expression is attained in the presence of both regulators. On the other hand, the ompR and rpoS mutations did not affect either the motility phenotype or flhD expression. We next examined the expression of a chromosomal flhD-lac fusion gene in the presence of a plasmid carrying the wild-type flhD operon. It was found that under this condition the chromosomal flhD operon was repressed or activated, depending on the intracellular activity of FliA, an alternative sigma factor specific for late flagellar operons. In the absence of FliA or in the presence of both FliA and its cognate anti-sigma factor FlgM, the flhD operon was autogenously repressed, whereas in the flgM mutant background it was autogenously activated in the presence of FliA. This autoregulation was still observed in the crp or hns mutant background, indicating that the autogenous control is achieved by a mechanism that is independent of the cAMP-CRP and H-NS regulatory pathways.

FlgB, FlgC, FlgF and FlgG: A family of structurally related proteins in the flagellar basal body of Salmonella typhimurium☆

The flagellar basal body of Salmonella typhimurium consists of four rings surrounding a rod. The rod, which is believed to transmit motor rotation to the filament, is not well characterized in terms of its structure and composition. FlgG is known to lie within the distal portion of the rod, in the region where it is surrounded by the L and P rings, just before the rod-hook junction. The FlgC and FlgF proteins are also known to be flagellar basal-body components; by comparison of deduced and experimental N-terminal amino acid sequences we show here that FlgB is a basal-body protein. The flgB, flgC, flgF and flgG gene sequences and the deduced protein sequences are presented. The four proteins are clearly related to each other in primary sequence, especially toward the N and C termini, supporting the hypothesis (based on examination of basal-body subfractions) that FlgB, FlgC and FlgF are, like FlgG, rod proteins. From this and other information we suggest that the rod is the cell-proximal part of a segmented axial structure of the flagellum, with FlgB, FlgC and FlgF located (in unknown order) in successive segments of the proximal rod, followed by FlgG located in the distal rod; the axial structure then continues with the hook, HAPs and filament. Although the rod is external to the cell membrane, none of the four rod proteins contains a consensus signal sequence for the primary export pathway; comparison with the experimentally determined N-terminal amino acid sequence indicates that FlgB has had its N-terminal methionine removed, while the other three are not processed at all. This demonstrates that these proteins are not exported by the primary cellular pathway, and suggests that they are exported by the same flagellum-specific pathway as the flagellar filament protein flagellin. The observed sequence similarities among the rod proteins, especially a six-residue consensus motif about 30 residues in from the N terminus, may constitute a recognition signal for this pathway or they may reflect higher-order structural similarities within the rod.

Ribosome rescue by Escherichia coli ArfA (YhdL) in the absence of trans-translation system

Although SsrA(tmRNA)‐mediated trans‐translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in‐frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome‐rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA–arfA synthetic lethality was alleviated by SsrADD, an SsrA variant that adds a proteolysis‐refractory tag through trans‐translation, indicating that ArfA‐deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the ΔarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon‐independent mimic of aminoacyl‐tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non‐stop mRNA, which was otherwise SsrA‐tagged and degraded. In vitro, purified ArfA acted on a ribosome‐nascent chain complex to resolve the peptidyl‐tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3′ end of a non‐stop mRNA without involving trans‐translation.

Functional analysis of the flagellar genes in the fliD operon of Salmonella typhimurium

The fliD genes of Salmonella typhimurium and Escherichia coli encode the filament-cap protein of the flagellar apparatus, which facilitates the polymerization of endogenous flagellin at the tips of the growing filaments. Previous sequence analysis of this operon in both organisms has revealed that the fliD gene constitutes an operon together with two additional genes, fliS and fliT. Based on the gene-disruption experiment in E. coli, both the fliS and fliT genes have been postulated to be necessary for flagellation. In the present study, we constructed S. typhimurium mutants in which either fliS or fliT on the chromosome was specifically disrupted. Both mutants were found to produce functional flagella, indicating that these genes are dispensable for motility development in S. typhimurium. However, flagellar filaments produced by the fliS mutant were much shorter than those produced by the wild-type strain. This indicates that the fliS mutation affects the elongation step of filament assembly. The excretion efficiency of flagellin was examined in the fliD-mutant background, where the exported flagellin molecules cannot assemble onto the hooks, resulting in their excretion into the culture media. We found that the amount of flagellin excreted was much reduced by the fliS mutation. Based on these results, we conclude that FliS facilitates the export of flagellin through the flagellum-specific export pathway.

Escherichia coli YaeJ protein mediates a novel ribosome‐rescue pathway distinct from SsrA‐ and ArfA‐mediated pathways

Accumulation of stalled ribosomes at the 3′ end of mRNA without a stop codon (non‐stop mRNA) is supposed to be toxic to bacterial cells. Escherichia coli has at least two distinct systems to rescue such stalled ribosomes: SsrA‐dependent trans‐translation and ArfA‐dependent ribosome rescue. Combination of the ssrA and arfA mutations is synthetically lethal, suggesting the significance of ribosome rescue. In this study, we identified the E. coli yaeJ gene, encoding a peptide‐release factor homologue with GGQ motif, as a multicopy suppressor of the lethal phenotype of ssrA arfA double mutant. The YaeJ protein was shown to bind to ribosomes. Both in vivo and in vitro, YaeJ showed the ribosome‐rescue activity and promoted the hydrolysis of peptidyl‐tRNA residing in the stalled ribosome. Missense mutation in the GGQ motif or deletion of the C‐terminal unstructured tail abolished both the suppressor activity for ssrA arfA synthetic lethality and the ribosome‐rescue activity, suggesting the importance of these structural features. On the basis of these observations, we propose that YaeJ acts as a stop codon‐independent peptidyl‐tRNA hydrolysing factor through binding to ribosomes stalled at the 3′ end of non‐stop mRNAs. It was also suggested that ArfA and YaeJ rescue the stalled ribosomes by distinct mechanisms.

Genetic and molecular analyses of the interaction between the flagellum-specific sigma and anti-sigma factors in Salmonella typhimurium.

More than 50 genes are required for flagellar formation and function in Salmonella typhimurium. According to the cascade model of flagellar regulon, the flagellar operons are divided into three classes, 1, 2, and 3, with respect to transcriptional hierarchy. FliA is an alternative sigma factor specific for transcription of the class 3 operons, while FlgM is an anti‐sigma factor which binds to FliA and prevents its association with RNA polymerase core enzyme. In the present study, we isolated a number of fliA mutants in which the altered FliA proteins become insensitive to inhibition by FlgM. Sequence analysis of their mutation sites revealed that most of them caused the amino acid substitutions in region 4 of the conserved amino acid sequences of sigma factors which lies near the C‐terminal end of FliA. Using a set of fliA deletion mutants in a high‐expression plasmid, we demonstrated that polypeptides containing the C‐terminal portion of FliA could titrate the intracellular FlgM protein resulting in derepression of the class 3 operons. This result indicates that the C‐terminal region of FliA contains the FlgM‐binding domain. This was confirmed by a chemical cross‐linking experiment with FlgM and truncated FliA proteins.

FliT Acts as an Anti-FlhD2C2 Factor in the Transcriptional Control of the Flagellar Regulon in Salmonella enterica Serovar Typhimurium

Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(2)C(2) heterotetramer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. Class 2 expression is known to be enhanced by a disruption mutation in a flagellar gene, fliT. In this study, we purified FliT protein in a His-tagged form and showed that the protein prevented binding of FlhD(2)C(2) to the class 2 promoter and inhibited FlhD(2)C(2)-dependent transcription. Pull-down and far-Western blotting analyses revealed that the FliT protein was capable of binding to FlhD(2)C(2) and FlhC and not to FlhD alone. We conclude that FliT acts as an anti-FlhD(2)C(2) factor, which binds to FlhD(2)C(2) through interaction with the FlhC subunit and inhibits its binding to the class 2 promoter.