Cloning, sequencing, and disruption of the Bacillus subtilis sigma 28 gene

Abstract

Bacillus subtilis contains multiple forms of RNA pol,ymerase holoenzyme, distinguished by the presence of different specificiy determinants known as (r factors.. The au factor was initially purified as a unique transcriptional activity in vegetatively growing B. subtilis cells. Purification of the a8 protein has allowed tryptic peptides to be prepared and sequenced. The sequence of one tryptic peptide fragment was used to prepare an oligonucleotide probe specific for the a8 -structural gene, and the gene was isolated from a B. subtilis subgenomic library. The complete nucleotide sequence of the a8 gene was determined, and the cloned 28 (F gene was used to construct a mutant strain which does not express the ('8 protein. This strain also failed to synthesize flageilin protein and grew as long filaments. The predicted (28 gene product is a 254-amino-acid polypeptide with a calculated molecular weight of 29,500. The d8 protein sequence was similar to that of other sequenced 'r factors and to theflbB gene product of Escherichia coli. Since theflbB gene product is a positive regulator of flagellar synthesis in E. coli, it is likely that cr8 functions to regulate flagellar synthesis in B. subtilis.Bacterial RNA polymerase is a multisubunit enzyme central to the process of gene expression. Although the catalytic activity resides in the core subunits of the enzyme, the promoter specificity of a particular holoenzyme is determined by the nature of the associated a factor (31). The majority of cellular transcription is dependent on the primary r factor, which exhibits a conserved promoter recognition specificity throughout the eubacteria (43). Many bacterial species also contain alternative a factors that are specific for transcription of distinct regulons of coordinately regulated genes. These alternative a factors normally recognize promoter sequences that are differett from those recognized by the primary cr factor. Examhples of alternative of factors in the enteric bacteria include a32 (15), specific for the transcription of heat shock genes, and (94 (19, 20), specific for transcription of fnitrogen-regulated genes. For Bacillus subtilis, at least six alternative factors have been described (26,31).In B. subtilis, alterations in cellular transcription, mediated at least in part by alternative cr factors, effect the precise temporal changes in gene expression necessary for endospore formation (26). The products of the spoOH (U30) and spoIIGB (r29) genes are sporulation-specific cr factors that have been characterized both genetically and biochemically (6,22,24,26,40,41). In addition, the spoIIAC gene product is homologous to other sequenced a factors (9) and may also function as a cf factor. The cr3 and a98 factors are found in vegetatively growing cells and are dispensable for sporulation, since disruption of these genes does not impair sporulation (3, 8; see below). To define the biological function of the B. subtilis ur28 factor, we have begun a genetic and structural analysis of the Cr28 structural gene (sigD) bas...

Characterization of the sigD transcription unit of Bacillus subtilis

“…1). To introduce the strong Ti and T2 transcription terminators from the rRNA operon of E. coli (8), we constructed a derivative of the integration plasmid pJM102 (19). The 480-bp HindlIl fragment from pKK5-1 (7), which contains the Ti and T2 terminators, was cloned into the Hindlll site of the pJM102 polylinker, generating pJM102T.…”

“…The che locus has therefore been renamed the fla/che region to more accurately represent the function of the proteins encoded therein (33). Strains containing transposon insertions within the fla/che region of DNA do not express flagellin (41), as is the case with strains lacking the CD factor (19,28). In fact the structural gene for aD, sigD, maps to the same area of the bacterial chromosome as the fla/che region does (28).…”

“…The a' factor is expressed during vegetative growth in B. subtilis (15) and has been shown to be required for flagellin and wild-type motility and/or chemotaxis functions (19,28). In fact, uD holoenzyme is directly responsible for transcription of the flagellin structural gene (30), the motility genes motA and motB (31), and the flgM regulatory gene (30a).…”

High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation

“…pJH6-2 DNA containing the upstream and 5' regions of the sigD gene (15) was digested with EcoRI, subjected to a fill-in reaction, and then further digested with Sall. The resulting 0.8-kb SalI-EcoRI (blunt-ended) fragment containing the 5' region of the sigD gene was recovered by using Gene Clean (Bio 101).…”

“…B. subtilis AC327 (purB his-i smo-1) (45) was used for RNA isolation in promoter-probing and primer extension analyses. B pUC18 plasmid into which a 1.5-kb HindIII fragment containing the upstream region and 5' region of the sigD gene have been inserted (15). pMC1871 (Pharmacia), a ,-galactosidase fusion vector, was used also for sigD-lacZ fusion.…”