Summary
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 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.
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References
Adler HI, Fisher WD, Cohen A, Hardigree AA (1967) Miniature Escherichia coli cells deficient in DNA. Proc Natl Acad Sci USA 57:321–326
Amann E, Ochs B, Abel K-J (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69:301–315
Arnosti DN, Chamberlin MJ (1989) Secondary σ factor controls transcription of flagellar and chemotaxis genes in Escherichia coli. Proc Natl Acad Sci USA 86:830–834
Bahl H, Echols D, Straus DB, Court D, Crowl R, Georgopoulos CP (1987) Induction of the heat shock response of E. coli through stabilization of σ32 by the phage λ cIII protein. Genes Dev 1:57–64
Bartlett DH, Frantz BB, Marsumura P (1988) Flagellar transcription activators FlbB and FlaI: Gene sequences and 5′ consensus sequences of operons under FlbB and Flal control. J Bacteriol 170:1575–1581
Boyd DH, Porter LM, Young BS, Wright A (1979) The in vitro detection of defects in temperature sensitive RNA polymerases from mutants of Salmonella typhimurium. Mol Gen Genet 173:279–287
Brosius J, Holy A (1984) Regulation of ribosomal RNA promoters with a synthetic lac operator. Proc Natl Acad Sci USA 81:6929–6933
Brosius J, Dull TJ, Sleeter DD (1981) Gene organization and primary structure of a ribosomal RNA operon from Escherichia coli. J Mol Biol 148:107–127
Burgess RR, Jendrisak JJ (1975) A procedure for the rapid, largescale purification of Escherichia coli DNA-dependent RNA polymerase involving Polymin P precipitation and DNA-cellulose chromatography. Biochemistry 14:4634–4638
Burton Z, Burgess RR, Lin J, Moore D, Holder S, Gross CA (1981) The nucleotide sequence of the cloned rpoD gene for the RNA polymerase sigma subunit from E. coli K12. Nucleic Acids Res 9:2889–2903
Erickson BD, Burton ZF, Watanabe KK, Burgess RR (1985) Nucleotide sequence of the rpsU-dnaG-rpoD operon from Salmonella typhimurium and comparison of the sequence with the homologous operon of Escherichia coli. Gene 40:67–78
Gitt MA, Wang L-F, Doi RH (1985) A strong sequence homology exists between the major RNA polymerase sigma factors of Bacillus subtilis and Escherichia coli. J Biol Chem 260:7178–7185
Gribskov M, Burgess RR (1986) Sigma factors from E. coli B. subtilis, phage SPO1, and phage T4 are homologous proteins. Nucleic Acids Res 14:6745–6763
Grossman AD, Erickson JW, Gross CA (1984) The htpR gene product of E. coli is a sigma factor for heat shock promoters. Cell 38:383–390
Hara H, Nishimura Y, Kato J, Suzuki H, Nagasawa H, Suzuki A, Hirota Y (1989) Genetic analyses of processing involving C-terminal cleavage in penicillin-binding protein 3 of Escherichia coli. J Bacteriol 171:5882–5889
Helmann JD, Chamberlin MJ (1987) DNA sequence analysis suggests that expression of flagellar and chemotaxis genes in Escherichia coli and Salmonella typhimurium is controlled by an alternative a factor. Proc Natl Acad Sci USA 84:6422–6424
Helmann JD, Chamberlin MJ (1988) Structure and function of bacterial sigma factors. Annu Rev Biochem 57:839–872
Helmann JD, Marquez LM, Chamberlin MJ (1988) Cloning, sequencing, and disruption of the Bacillus subtilis σ28 gene. J Bacteriol 170:1568–1574
Hirschman J, Wong PK, Sei K, Keener J, Kustu S (1985) Products of nitrogen regulatory genes ntrA and ntrC of enteric bacteria activate glnA transcription in vitro: evidence that the ntrA product is a σ factor. Proc Natl Acad Sci USA 82:7525–7529
Iino T, Komeda Y, Kutsukake K, Macnab RM, Matsumura P, Parkinson JS, Simon MI, Yamaguchi S (1988) New unified nomenclature for the flagellar genes of Escherichia coli and Salmonella typhimurium. Microbiol Rev 52:533–535
Inoue YH, Kutsukake K, Iino T, Yamaguchi S (1989) Sequence analysis of operator mutants of phase-1 flagellan-encoding gene, fliC, in Salmonella typhimurium. Gene 85:221–226
Jo Y-L, Nara F, Ichihara S, Mizuno T, Mizushima S (1986) Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem 261:15252–15256
Kajitani M, Ishihama A (1983) Determination of the promoter strength in the mixed transcription system: promoters of lactose, tryptophan and ribosomal protein L10 operons from Escherichia coli. Nucleic Acids Res 11:671–686
Komeda Y (1982) Fusions of flagellar operons to lactose genes on a Mu lac bacteriophage. J Bacteriol 150:16–26
Komeda Y (1986) Transcriptional control of flagellar genes in Escherichia coli K-12. J Bacteriol 168:1315–1318
Komeda Y, Kutsukake K, Iino T (1980) Definition of additional flagellar genes in Escherichia coli K12. Genetics 94:277–290
Kutsukake K, Nakao T, Iino T (1985) A gene for DNA invertase and an invertible DNA in Escherichia coli K-12. Gene 34:343–350
Kutsukake K, Ohya Y, Iino T (1990) Transcriptional analysis of the flagellar regulon of Salmonella typhimurium. J Bacteriol 172:741–747
Kutsukake K, Ohya Y, Yamaguchi S, Iino T (1988) Operon structure of flagellar genes in Salmonella typhimurium. Mol Gen Gene 214:11–15
Lowe PA, Hager DA, Burgess RR (1979) Purification and properties of the σ subunit of Escherichia coli DNA-dependent RNA polymerase. Biochemistry 18:1344–1352
Macnab RM (1987) Flagella. In: Neidhardt FC (ed) Escherichia coli and Salmonella typhimurium. American Society for Microbiology, Washington DC, pp 70–83
Makino K, Shinagawa H, Amemura M, Kimura S, Nakata A, Ishihama A (1988) Regulation of the phosphate regulon of Escherichia coli. Activation of pstS transcription by PhoB protein in vitro. J Mol Biol 203:85–95
Matsudaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membrane. J Biol Chem 262:10035–10038
Mirel DB, Chamberlin MJ (1989) The Bacillus subtilis, flagellin gene (hag) is transcribed by the σ28 form of RNA polymerase. J Bacteriol 171:3095–3101
Reiner AM (1969) Isolation and mapping of polynucleotide phosphorylate mutants of Escherichia coli. J Bacteriol 97:1431–1436
Richet E, Raibaud O (1987) Purification and properties of the MalT protein, the transcription activator of the Escherichia coli maltose regulon. J Biol Chem 262:12647–12653
Roozen KJ, Fenwick RG Jr, Curtiss RIII (1971) Synthesis of ribonucleic acid and protein in plasmid-containing minicells of Escherichia coli K-12. J Bacteriol 107:21–33
Sancar A, Hack AM, Rupp WD (1979) Simple method for identification of plasmid-coded proteins. J Bacteriol 137:692–693
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Straus DB, Walter WA, Gross CA (1987) The heat shock response of E. coli is regulated by changes in the concentration of σ32. Nature 329:348–351
Tilly K, Spence J, Georgopoulos C (1989) Modulation of stability of Escherichia coli heat shock regulatory factor σ32. J Bacteriol 171:1585–1589
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268
Yamaguchi S, Fujita H, Taira T, Kutsukake K, Homma M, Iino T (1984) Genetic analysis of three additional fla genes in Salmonella typhimurium. J Gen Microbiol 130:3339–3342
Yura T, Tobe T, Ito K, Osawa T (1984) Heat shock regulatory gene (htpR) of Escherichia coli is required for growth at high temperature but is dispensable at low temperature. Proc Natl Acad Sci USA 81:6803–6807
Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7:267–287
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Ohnishi, K., Kutsukake, K., Suzuki, H. et al. Gene fliA encodes an alternative sigma factor specific for flagellar operons in Salmonella typhimurium . Molec. Gen. Genet. 221, 139–147 (1990). https://doi.org/10.1007/BF00261713
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DOI: https://doi.org/10.1007/BF00261713