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.

Cloning and characterization of the metE gene encoding S-adenosylmethionine synthetase from Bacillus subtilis.

The metE gene, encoding S-adenosylmethionine synthetase (EC 2.5.1.6) from Bacillus subtilis, was cloned in two steps by normal and inverse PCR. The DNA sequence of the metE gene contains an open reading frame which encodes a 400-amino-acid sequence that is homologous to other known S-adenosylmethionine synthetases. The cloned gene complements the metE1 mutation and integrates at or near the chromosomal site of metE1. Expression of S-adenosylmethionine synthetase is reduced by only a factor of about 2 by exogenous methioinine. Overproduction of S-adenosylmethionine synthetase from a strong constitutive promoter leads to methionine auxotrophy in B. subtilis, suggesting that S-adenosylmethionine is a corepressor of methionine biosynthesis in B. subtilis, as others have already shown for Escherichia coli.

Cloning, sequencing, and characterization of the Bacillus subtilis biotin biosynthetic operon.

A 10-kb region of the Bacillus subtilis genome that contains genes involved in biotin-biosynthesis was cloned and sequenced. DNA sequence analysis indicated that B. subtilis contains homologs of the Escherichia coli and Bacillus sphaericus bioA, bioB, bioD, and bioF genes. These four genes and a homolog of the B. sphaericus bioW gene are arranged in a single operon in the order bioWAFDR and are followed by two additional genes, bioI and orf2. bioI and orf2 show no similarity to any other known biotin biosynthetic genes. The bioI gene encodes a protein with similarity to cytochrome P-450s and was able to complement mutations in either bioC or bioH of E. coli. Mutations in bioI caused B. subtilis to grow poorly in the absence of biotin. The bradytroph phenotype of bioI mutants was overcome by pimelic acid, suggesting that the product of bioI functions at a step prior to pimelic acid synthesis. The B. subtilis bio operon is preceded by a putative vegetative promoter sequence and contains just downstream a region of dyad symmetry with homology to the bio regulatory region of B. sphaericus. Analysis of a bioW-lacZ translational fusion indicated that expression of the biotin operon is regulated by biotin and the B. subtilis birA gene.

Cloning and characterization of the Bacillus subtilis birA gene encoding a repressor of the biotin operon.

The Bacillus subtilis birA gene, which regulates biotin biosynthesis, has been cloned and characterized. The birA gene maps at 202 degrees on the B. subtilis chromosome and encodes a 36,200-Da protein that is 27% identical to Escherichia coli BirA protein. Three independent mutations in birA that lead to deregulation of biotin synthesis alter single amino acids in the amino-terminal end of the protein. The amino-terminal region that is affected by these three birA mutations shows sequence similarity to the helix-turn-helix DNA binding motif previously identified in E. coli BirA protein. B. subtilis BirA protein also possesses biotin-protein ligase activity, as judged by its ability to complement a conditional lethal birA mutant of E. coli.

Streptomyces genes involved in biosynthesis of the peptide antibiotic valinomycin.

We have identified genes from Streptomyces levoris A-9 involved in the biosynthesis of the peptide antibiotic valinomycin. Two segments of chromosomal DNA were recovered from genomic libraries, constructed by using the low-copy-number plasmid pIJ922, by complementation of valinomycin-deficient (vlm) mutants of S. levoris A-9. One set of plasmids restored valinomycin production to only one mutant, that carrying vlm-1, whereas a second set of plasmids restored productivity to seven vlm mutants, those carrying vlm-2 through vlm-8. Additional complementation studies using subcloned restriction enzyme fragments showed that the vlm-1+ gene was contained within a 2.5-kilobase (kb) DNA region, whereas alleles vlm-2+ through vlm-8+ were contained in a 12-kb region, representing at least three genes. Physical mapping experiments based on the isolation of cosmid clones showed that the two vlm loci were 50 to 70 kb apart. Southern hybridization experiments demonstrated that the vlm-2+ gene cluster was highly conserved among other valinomycin-producing Streptomyces strains, whereas the vlm-1+ gene was ubiquitous among Streptomyces species tested. Increasing the copy number of the vlm-2+ gene cluster in S. levoris A-9 by the introduction of low-copy-number recombinant plasmids resulted in a concomitant increase in the level of valinomycin production.

Second-site rho mutation: genetic linkage and polyC-dependent ATPase.

Rho has been purified to homogeneity from Escherichia coli double mutant rho-115 sur-38 cells, and from rho6 and rho-115 cells. The sur-38 mutation suppresses the original rho-115 phenotype. We observe that the polyC-dependent ATPases of these three rho preparations have the same specific activities. However, the ATPase of rho from the double rho-115 sur-38 mutant is extremely heat labile, while that from rho-115 shows a heat lability intermediate between the wild type and the double mutant. Transduction analysis suggests that sur-38 is closely linked to rho-115 in the order ilv--sur-38--rho-115--metE. These data are consistent with the model that the sur-38 mutation affects the structural gene for rho.

Rho and ribosome mutation interaction: lethality of rho-15 in rpsL or rpsE strains, and rho-15 methionine auxotrophy in rps+ strains of Escherichia coli.

The phenotype of Escherichia coli K-12 carrying rho-15 in the genetic background DW319 ilv lacZ::IS1 is described. Seventy-eight percent (70/90) of Ilv+ transductants acquired the following phenotype: temperature-sensitive growth on minimal salts medium, Ts+ growth on complex medium and suppression of the lac polar mutation. At 42 degrees on minimal medium, the rho-15 transductants were cross-fed by a substance diffusing from Rho+ transductants or controls. The requirement for this substance was satisfied by methionine or cystathionine, but not by any other single amino acid or combination of amino acids, by spermidine, or by mono- or divalent cationic salts.--Transduction of rho-15 into four other Ilv- recipients revealed two phenotypic patterns. Recipients with rpsL or rpsE ribosomes yielded rho-15 transductants that were Ts on all media, or Ts on minimal medium whether or not methionine was present. The effect of the ribosome on expression of rho-15 was confirmed by transduction of appropriate rps alleles into DW319, followed by co-transduction of rho-15 with Ilv+. The growth rate of double rho-15 rpsL or rho-15 rpsE strains was severely reduced at 42 degrees in comparison with strains carrying any of these single mutations. Models for rho and ribosome interaction are presented.

Rifampicin supersensitivity of rho strains of E. coli, and suppression by sur mutation.

Escherichia coli strains with mutations rho-115, rho-ts15, rho-101 (psu-1) or rho-102 (psu-2) are more sensitive ("supersensitive") to rifampicin than isogenic parent strains, as measured by growth rate in broth and colony forming efficiency on solid media with 5, 10, or 20 microgram of rifampicin per ml. There is no change in sensitivity of rho mutants to the antibiotics penicillin, erythromycin, chloramphenicol, or the detergent desoxycholate. The rho-101 or rho-102 mutations confer rifampicin supersensitivity at 32 degrees C but not 42 degrees C. Mutants of a rho-115 strain that have lost polarity suppression can be isolated by selection for rifampicin resistance. This phenotype, Sur, is not due to reversion of the original rho gene mutation but to a second mutation perhaps in the gene for rho protein or the gene for the beta subunit of RNA polymerase. One class of Sur mutation, occurring in rho-115 cells isolated as resistant to 20 microgram of rifampicin per ml, is co-transducible with the marker ilv, and the gene order is rbs-ilv-sur-38. A model suggested by this map position is that the mutations rho-115 and sur-38 define the domain of rho protein which interacts with the beta subunit of RNA polymerase.