ABSTRACTaJ37 is a minor species of RNA polymerase cr factor found in the Gram-positive bacterium Bacillus subtilis. 37 governs the transcription in vitro of genes that are turned on at an early stage in spore formation, as well as other genes that are switched on at the end of the exponential phase of growth but that are not under sporulation control. To study the role of O37 in B. subtilis gene expression, we have cloned the gene for this minor species of (F factor in Escherichia coli by using as a hybridization probe a synthetic oligonucleotide that was designed on the basis of the NH2-terminal amino acid sequence of o'37 protein. We determined the nucleotide sequence of the entire o.37 gene, which was found to encode a 262-amino acid residue polypeptide of 29.9 kDa. The predicted amino acid sequence of a37 showed significant homology to that of other (r proteins in a region that has been proposed to be the site ofbinding of these factors to core RNA polymerase. holoenzyme forms in vitro, is induced at the end of the exponential phase of growth but under nutritional conditions that do not support spore formation (38). Unlike spoVG transcription, ctc RNA synthesis is not dependent on the products ofspoO genes but is instead stimulated by mutations in these regulatory genes. Studies on the interaction of E&37 with the ctc and spoVG promoters indicate that promoter recognition by this minor holoenzyme form is determined by contacts with conserved bases in the "-35" and "-10" regions of its cognate promoters (refs. 19 and 21-23; P. Zuber, J. Healy, and R.L., unpublished results).Although much has been learned about the interaction of Eoa37 with spoVG and ctc from in vitro studies, it has not yet been possible to assess the contribution of this minor holoenzyme form to the expression and differential regulation of these genes in vivo or to investigate the general role of Eo-37 in the overall pattern of B. subtilis gene expression.As a way of approaching this problem, we have cloned in Escherichia coli the structural gene for a37 and we have determined the location of its coding sequence on the B. subtilis chromosome. We show that the predicted product of the 37 gene, which we designate sigB, contains a region of significant homology to other B. subtilis and E. coli a factors and is evidently not essential for growth or sporulation, as judged by the construction of a sigB insertion mutation. MATERIALS AND METHODSBacterial Strains and Plasmids. Cloning of DNA from B. subtilis strain 168 (trpC2) was carried out in E. coli strains MM294 and HB101 using the plasmid vectors pBR322 and pJH101 (24). Mapping experiments were carried out in B. subtilis strain QB928 (aroI, purB33, dal, and trpC2; see ref. 25).Abbreviations: kb, kilobase(s); bp, base pair(s). 5943The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The anhydrotetracycline (ATC) oxygenase enzyme which carries out the conversion of ATC to dehydrotetracycline was purified and the N-terminal amino acid sequence was determined. The sequence displays a significant similarity to that of the p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. This is consistent with the activity of the oxygenase, i.e., addition of a hydroxyl moiety to an aromatic ring structure.Oligonucleotide probes were designed and used to clone the corresponding fragment of chromosomal DNA from Streptomyces rimosus. This DNA fragment was used to screen a cosmid library, allowing the isolation of flanking DNA sequences. Surprisingly, the gene was located within the previously cloned cluster of genes involved in the synthesis of the biosynthetic intermediate ATC and not as had been expected (P. M. Rhodes, N. Winskill, E. J. Friend, and M. Warren, J. Gen. Microbiol. 124:329-338, 1981) at a separate locus on the other side of the chromosome. Subcloning of an appropriate DNA fragment from one of the cosmid clones onto pIJ916 produced Streptomyces lividans transformants which synthesized oxytetracycline.Oxytetracyline (OTC) is a commercially important broadspectrum antibiotic produced by Streptomyces rimosus. OTC is a member of the polyketide class of antibiotics which includes, among others, avermectin, monensin, erythromycin, and other tetracyclines such as 7-chlorotetracycline.The OTC biosynthetic pathway of S. rimosus has been the subject of both biochemical and genetic analysis (15). Figure 1 indicates the proposed biosynthetic pathway from malonyl coenzyme A to the final product. The isolation and characterization of mutants blocked at various points in the pathway indicated that the genes involved in OTC biosynthesis are clustered into two distinct positions mapping at diametrically opposite locations on the circular map of S. rimosius.The loci thought to be responsible for all pathway steps leading to the production of anhydrotetracycline (ATC) were assigned to the "4 o'clock" locus, whereas the genes encoding enzymes for the final part of the pathway, including those responsible for the production of a flavinlike cofactor (cosynthetic factor 1 [CSF1]) were assigned to a 10 o'clock location.The isolation (3; M. J. Butler, E. J. Friend, I. S. Hunter, F. S. Kaczmarek, D. A. Sugden, and M. Warren, Mol. Gen. Genet., in press) of two genes, otrA and otrB, involved in host resistance to OTC and the analysis of flanking DNA indicated that the 4 o'clock cluster was organized as a single group of biosynthetic genes flanked by the two resistance loci (Fig. 2). However, the genes responsible for the conversion of ATC to OTC remained unidentified. This paper describes the isolation of the gene (otcC) encoding the ATC oxygenase, the enzyme catalyzing the hydroxylation of ATC to dehydrotetracycline. otcC (7) except that S. lividans mycelium was grown for 3 days instead of 2 days in YEME medium containing glycine and MgCl2 at 0.5% and 5 mM, respectively. S. rimosus protoplast formation, regener...

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A strain of Streptomyces lividans 66 deleted for a major tripeptidyl aminopeptidase (Tap) was used as a host to screen an S. lividans genomic library for clones overexpressing activity against the chromogenic substrate Ala-Pro-Ala-␤-naphthylamide. In addition to reisolation of the tap gene, clones representing another locus, slpD, were uncovered. slpD was analyzed by deletion subcloning to localize its functional sequence. Nucleotide sequence determination revealed an open reading frame encoding a 55-kDa protein exhibiting significant amino acid sequence homology to Tap, particularly around the putative active-site serine residue. No secreted protein was observed for strains harboring the slpD clone, but inspection of the predicted protein sequence revealed a putative lipoprotein signal peptide (signal peptidase II type), suggesting a mycelial location for the SlpD proteinase. In an attempt to isolate an endoprotease known to be active against some heterologous proteins, a second clone was isolated by using a longer substrate (t-butyloxycarbonyl [Boc]-APARSPA-␤-naphthylamide) containing a chemical blocking group at the amino terminus to prevent aminopeptidase cleavage. This locus, slpE, appeared to also encode a 55-kDa mycelium-associated (lipoprotein) proteinase, whose predicted protein sequences showed significant amino acid homology to Tap and SlpD, particularly around the putative active-site serine residues. Chromosomal integration and deletion analysis in both the wild-type and Tap-deficient backgrounds appeared to indicate that SlpD was essential for viability and SlpE was required for growth on minimal media.