In Salmonella typhimurium, nearly 50 genes are involved in flagellar formation and function and constitute at least 13 different operons. In this study, we examined the transcriptional interaction among the flagellar operons by combined use of Mu dl(Apr Lac) cts62 and TnlO insertion mutants in the flagellar genes. The results showed that the flagellar operons can be divided into three classes: class I contains only theflhD operon, which is controlled by the cAMP-CAP complex and is required for expression of all of the other flagellar operons; class U contains seven operons, flgA, flgB, flhB, fliA, fliE, fliF, and fliL, which are under control of class I and are required for the expression of class Ill; class III contains five operons,flgK,fliDfliC, motA, and tar. This ordered cascade of transcription closely paralels the assembly of the flagellar structure. In addition, we found that the fliD defect enhanced expression of the class III operons. This suggests that the fliD gene product may be responsible for repression of the class Ill operons in the mutants in the class II genes. These results are compared with the cascade model of the flagellar regulon ofEscherichia coli proposed previously (Y. Komeda, J. Bacteriol. 170:1575-1581, 1982.The bacterial flagellum is composed of three structural components, a basal body, a hook, and a filament. The filament extends into the extracellular space and is connected by the hook to the basal body embedded in the cell membrane. Genetic analysis of flagellar mutants have revealed that there are nearly 50 genes involved in flagellar formation and function in both Salmonella typhimurium and Escherichia coli (24,25). Intergeneric complementation analysis revealed functional homology in the flagellar genes between these two organisms (8,21,39). Genes responsible for flagellar formation are calledflg,flh,fli, orflj. Except for a few genes, mutants defective in these genes are nonflagellate and some of them produce presumptive precursor structures of the flagellum (35, 36). There are three kinds of genes responsible for flagellar function, including flagellar rotation (mot), chemotaxis (che), and transmembrane signal transduction of chemotactic stimuli (tar, trg, tsr, etc.) Mutants defective in these genes produce flagellar structures indistinguishable from those of the wild-type strain. Most of the flagellar genes are clustered in three regions of the bacterial chromosome, termed regions I, II, and III. These clustered genes constitute 14 and 13 different operons in E. coli (16, 32) and S. typhimurium (22), respectively. Flagellar genes of S. typhimurium are summarized in Fig. 1.In E. coli, Komeda (14) constructed fusions of most of the flagellar operons to the lac genes by using Mu dl(Apr Lac) bacteriophage developed by Casadaban.and Cohen (5). This phage contains the lac genes with no promoter, and its integration in a gene can result in rescue of expression of the lac genes due to the promoter of that gene. By using these operon fusions, he examined the transcriptional interac...

SummaryAlthough 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 SsrA DD , 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 DarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codonindependent 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 ribosomenascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3Ј end of a non-stop mRNA without involving trans-translation.

Through 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 27,470 dalton. The deduced amino acid sequence was homologous with that of sigma 28, 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 FliA 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.