Bacteriophage SPO1 genes 33 and 34. Location and primary structure of genes encoding regulatory subunits of Bacillus subtilis RNA polymerase.

Bacteriophage SPO1 gene 33 and 34 products are required for SPO1 late gene transcription. Both proteins bind to the core RNA polymerase of the Bacillus subtilis host to direct the recognition of SPO1 late gene promoters, whose sequences differ from those of SPO1 early and middle gene promoters. We have located and cloned the genes for these two regulatory proteins, and have engineered their expression in Escherichia coli by placing them under the control of the bacteriophage lambda PL promoter. Nucleotide sequence analysis indicated that genes 33 and 34 overlap by 4 base-pairs and encode highly charged, slightly basic proteins of molecular weight 11,902 and 23,677, respectively.

Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus.

A chitinase (Cht)-encoding gene from Streptomyces plicatus was previously cloned and expressed in Escherichia coli [Robbins et al., J. Biol. Chem., 263 (1988) 442-447]. We have sequenced this gene, compared its sequence with other genes encoding Cht and have explored its expression and regulation when reintroduced into Streptomyces lividans on multicopy plasmids. We have also cloned two other Streptomyces Cht-encoding genes and a beta-hexosaminidase-encoding gene in E. coli by expression in the lambda ZAP-Bluescript vector. The hexosaminidase and one of the Chts were expressed directly from the genomic library in E. coli at a high level as chimeric fusions with the beta-galactosidase alpha-complementing peptide encoded by the vector. Direct cloning and high-level expression of such chimeric proteins, which overcomes the difficulties associated with expressing Streptomyces genes in E. coli, should generally be possible wherever large numbers of transformants can be conveniently screened.

Structure of a Bacillus subtilis bacteriophage SPO1 gene encoding RNA polymerase sigma factor.

Gene 28 of Bacillus subtilis bacteriophage SPO1 codes for a regulatory protein, a sigma factor known as sigma gp28, that binds to the bacterial core RNA polymerase to direct the recognition of phage middle gene promoters. middle promoters exhibit distinctive and conserved nucleotide sequences in two regions centered about 10 and 35 base pairs upstream from the start point of mRNA synthesis. Here we report the cloning of gene 28 and its complete nucleotide sequence. We infer that sigma gp28 is a 25,707-dalton protein of 220 amino acids. Neither the nucleotide sequence of gene 28 nor the inferred amino acid sequence of sigma gp28 exhibits extensive homology to the gene or protein sequence of Escherichia coli sigma factor.

Distinctive nucleotide sequences of promoters recognized by RNA polymerase containing a phage-coded "sigma-like" protein.

We report the nucleotide sequences of two promoters for bacteriophage SP01 "middle" genes. These promoters are recognized by a modified form of Bacillus subtilis RNA polymerase that contains a phage-coded "sigma-like" regulatory protein (gp28) in place of the bacterial sigma factor. Both promoters shared the identical hexanucleotide 5'A-G-G-A-G-A at about 35 base pairs preceding the start point of transcription and the identical heptanucleotide 5'-T-T-T-A-T-T-T (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA) located about 10 base pairs preceding the transcriptional start point. The significance of these sequences in comparison with nucleotide sequences of promoters recognized by sigma-containing RNA polymerases is discussed.

Promoter recognition by phage SP01-modified RNA polymerase.

A modified form of Bacillus subtilis RNA polymerase containing a phage SP01-coded regulatory protein (the gene 28 product) selectively transcribes "middle" genes of the phage genome in vitro. In this paper, we identify a subset of restriction endonuclease fragments of SP01 DNA that promote specific transcription by the phage-modified polymerase. In the absence of nucleoside triphosphates, RNA polymerase containing the gene 28 protein selectively binds to these DNA fragments thereby forming stable binary complexes that can be isolated on nitrocellulose filters. In contrast, unmodified RNA polymerase containing sigma factor selectively binds to and transcribes a subset of phage DNA fragments that contain "early" sequences and that are in large part distinct from the fragments recognized by the phage-modified transcriptase. Our results strongly suggest that phage "early" and "middle" genes are transcribed from distinct promoters and that the RNA polymerase containing the gene 28 protein binds to sites that are located at or near promoters for SP01 "middle" genes.

Bacillus subtilis RNA polymerase and its modification in sporulating and phage-infected bacteria.

Bacillus subtilis RNA polymerase holoenzyme consists of the subunits beta', beta, sigma, alpha, delta, and omega. In sporulating bacteria and in bacteria infected with phages SP01 and SP82, this enzyme undergoes changes in subunit composition and transcriptional specificity that could play a regulatory role in gene transcription. Sporulating bacteria may contain a specific component that inhibits the activity of the sigma subunit of polymerase probably by interfering with the binding of sigma-polypeptide to core enzyme. The hypothetical inhibitor may be metabolically unstable, since its activity is rapidly depleted from sporulating cells in the presence of chloramphenicol. Inhibition of sigma-polypeptide activity may restrict the transcription of phage DNA an infected sporulating cells. Although lacking the sigma-subunit, RNA polymerase purified from sporulating cells contains sporulation-specific subunits of 85,000 and 27,000 daltons. In SP01-infected bacteria, the sigma-subunit is replaced by phage-induced subunits. Purified enzyme containing the protein product of SP01 regulatory gene 28 directs the transcription of phage middle genes in vitro, while enzyme containing phage-induced polypeptides V and VI preferentially copies late genes. Accurate transcription of middle and late genes in vitro requires the host delta-subunit of polymerase (or high ionic strength) but not sigma-subunit. Phage PBS2 induces an entirely new multisubunit RNA polymerase that specifically transcribes PBS2 DNA in vitro. This enzyme is synthesized de novo after infection and does not arise by modification of the B. subtilis holoenzyme.

Overproduction and purification of a bacteriophage SPO1-encoded RNA polymerase sigma factor.

Gene 28 of bacteriophage SPO1 encodes an RNA polymerase sigma factor sigma gp28, which replaces the host Bacillus subtilis sigma subunit sigma 55, to alter the promoter recognition specificity of RNA polymerase. A fragment of SPO1 DNA containing gene 28 was placed under the control of the PL promoter of bacteriophage lambda in an Escherichia coli expression vector. When transcription of gene 28 was induced by derepression of PL, the sigma gp28 synthesized constituted several per cent of total cellular protein. Sigma gp28 purified from these cells was able to confer specificity for SPO1 middle gene promoters upon B. subtilis core RNA polymerase, and also enabled E. coli core RNA polymerase to recognize and initiate transcription from an SPO1 middle gene promoter.

Nucleotide sequence of a promoter recognized by Bacillus subtilis RNA polymerase.

We report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the "-35 region" and the "Pribnow box" were 5'TTGACT and 5'CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA Polymerase as well as by B. subtilis RNA polymerase.

New phage-SPO1-induced polypeptides associated with Bacillus subtilis RNA polymerase.

RNA polymerase was precipitated from extracts of Bacillus subtilis infected with phage SP01 by antiserum prepared against core RNA polymerase. As shown by sodium dodecyl sulfate gel electrophoresis, the precipitates contained at least five new polypeptides not present in uninfected bacteria, in addition to the known subunits of RNA polymerase. The molecular weights of these polypeptides are (1) 85,000; (II) 40,000; (III) 28,000; (IV) 25,000; and (V) 23,000. Four of the polypeptides (I, III, IV, and V) co-purified with RNA polymerase through gel filtration and phosphocellulose chromatography. A pulse-chase experiment indicated that all five polypeptides are synthesized de novo after infection. The synthesis of polypeptides II, III, and IV commences almost immediately after infection, while polypeptides I and V first appear several minutes later. A sus mutant blocked early in transcription, susF21 [Fujita, et al. (1971) J. Mol. Biol. 57, 301-317] failed to induce polypeptides I, IV, and V, while two other mutants, susF4 and susF14, blocked late in transcription both failed only to induce polypeptide V.

Gene encoding a minor extracellular protease in Bacillus subtilis.

The gene for a minor, extracellular protease has been identified in Bacillus subtilis. The gene (epr) encoded a primary product of 645 amino acids that was partially homologous to both subtilisin (Apr) and the major internal serine protease (ISP-1) of B. subtilis. Deletion analysis indicated that the C-terminal 240 amino acids of Epr were not necessary for activity. This C-terminal region exhibited several unusual features, including a high abundance of lysine residues and the presence of a partially homologous sequence of 44 amino acids that was directly repeated five times. The epr gene mapped near sacA and was not required for growth or sporulation.

Bacteriophage SPO1 gene 27: location and nucleotide sequence.

Bacteriophage SPO1 gene 27, whose product is required for late gene transcription and DNA replication, has been cloned in Escherichia coli, and its complete nucleotide sequence has been determined. We infer that the product of gene 27 is a highly basic 17,518-dalton protein of 155 amino acids. The gene for this regulatory protein is transcribed from two promoters: an early promoter situated before the adjacent upstream gene 28 and a middle promoter located between genes 28 and 27.

Restriction cleavage map of SP01 DNA: general location of early, middle, and late genes.

A detailed restriction endonuclease map for the genome of Bacillus subtilis phage SP01 is presented. Sites of cleavage for the restriction enzymes BglII, EcoRI, HaeIII, and SalI were determined. This physical map showed that SP01 DNA was 140 kilobases in length and contained a repeated sequence of 12.4 kilobases at its termini. Combined with previously published information, we were also able to identify the general locations of genes expressed at early, middle, or late times in the phage lytic cycle. In particular, early genes were largely clustered in the terminal repeats, whereas a major cluster of late genes was located in the left-central portion of the genome.

In vitro synthesis of ribosomal RNA by Bacillus subtilis RNA polymerase.

Two kinds of hybridization competition experiments show that Bacillus subtilis RNA polymerase synthesizes ribosomal RNA (rRNA) in vitro with B. subtilis DNA as a template. First, RNA synthesized in vitro competes with the hybridization of [(32)P]rRNA synthesized in vivo to the heavy strand of B. subtilis DNA. Second, unlabeled rRNA synthesized in vivo competes with the hybridization of [(3)H]RNA synthesized in vitro to denatured DNA or heavy-strand DNA, but not to light-strand DNA. The ability of RNA polymerase holoenzyme to synthesize rRNA in vitro is not lost after extensive purification. RNA polymerase core enzyme, however, which is missing the sigma factor, synthesizes little rRNA in vitro.RNA polymerase purified from wild-type sporulating cells synthesizes little rRNA in vitro, while the in vitro synthesis of rRNA by RNA polymerase from stationary phase cells of the sporulation-defective mutant rfr 10 is apparently unimpaired.

Chloramphenicol restores sigma factor activity to sporulating Bacillus subtilis.

The sigma subunit of RNA polymerase from sporulating Bacillus subtilis is markedly inhibited in its ability to direct active transcription of phage varphie DNA in vitro. Treatment of sporulating bacteria with chloramphenicol rapidly restores sigma activity, suggesting that sporulating cells contain an inhibitor of sigma that is physiologically unstable or that becomes unstable after drug treatment. The hypothetical inhibitor is depleted exponentially with an apparent half-life of 11 min at 37 degrees .

Highly asymmetric transcription by RNA polymerase containing phage-SP01-induced polypeptides and a new host protein.

An RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) has been purified from phage-SP01-infected Bacillus subtilis that copies RNA almost exclusively from the heavy strand of native SP01 DNA, the DNA strand from which "middle" and "late" classes of RNA are copied in vivo. Hybridization-competition established that this RNA polymerase termed enzyme A, preferentially synthesizes middle RNA in vitro. Enzyme A contains beta',beta, alpha, and two newly identified host polypeptides, variation of (21,500 daltons) and omega (11,000 daltons). All of these polypeptides are associated with highly purified RNA polymerase from uninfected bacteria. In addition, enzyme A contains phage-induced subunits of 26,000, 24,000, and 13,500 daltons. Enzyme A lacks sigma polypeptide, and strand-selective transcription by this enzyme is resistant to anti-sigma antibody. A reconstitution experiment strongly suggests that the host variation of protein is required in addition to a phage-induced subunit(s) (or an unidentified phage-induced modification) for strand-selective transcription of SP01 middle genes in vitro.

Purification and comparative properties of the delta and sigma subunits of RNA polymerase from Bacillus subtilis.

Bacillus subtilis delta protein is a 21 500-Mr polypeptide that can be isolated in association with RNA polymerase holoenzyme from uninfected bacteria and with modified forms of RNA polymerase from cells infected with phage SP01 [Pero, J., Nelson, J. and Fox, T. (1975) Proc. Natl Acad. Sci. U.S.A. 72,1589]. Although no function has been assigned to delta protein in uninfected cells, this host polypeptide enhances the specificity of transcription by phage-modified forms of RNA polymerase that contain SP01-coded regulatory subunits. This report describes the purification of delta and sigma proteins from uninfected B. subtilis and examines the comparative effects of these polypeptides on transcription by core RNA polymerase. Purified sigma polypeptide was found to stimulate the transcription of phage DNA while having little effect on RNA synthesis with the synthetic DNA poly(dA-dT) as template. In contrast, purified delta protein markedly depressed the transcription of poly(dA-dT) while having little effect on enzyme activity with phage DNA as template. The inhibitory effect of delta protein on poly (dA-dT) transcription was strongly dependent on the presence of KC1 in the RNA synthesis reaction mixture.

Identification and characterization of transcripts from the biotin biosynthetic operon of Bacillus subtilis.

Northern (RNA) blot analysis of the Bacillus subtilis biotin operon, bioWAFDBIorf2, detected at least two steady-state polycistronic transcripts initiated from a putative vegetative (Pbio) promoter that precedes the operon, i.e., a full-length 7.2-kb transcript covering the entire operon and a more abundant 5.1-kb transcript covering just the first five genes of the operon. Biotin and the B. subtilis birA gene product regulated synthesis of the transcripts. Moreover, replacing the putative Pbio promoter and regulatory sequence with a constitutive SP01 phage promoter resulted in higher-level constitutive synthesis. Removal of a rho-independent terminator-like sequence located between the fifth (bioB) and sixth (bioI) genes prevented accumulation of the 5.1-kb transcript, suggesting that the putative terminator functions to limit expression of bioI, which is thought to be involved in an early step in biotin synthesis.