Sequence and properties of pIM13, a macrolide-lincosamide-streptogramin B resistance plasmid from Bacillus subtilis.

We initiated a study of pIM13, a multicopy, macrolide-lincosamide-streptogramin B (MLS) plasmid first isolated from a strain of Bacillus subtilis and described by Mahler and Halvorson (J. Gen. Microbiol. 120:259-263, 1980). The copy number of this plasmid was about 200 in B. subtilis and 30 in Staphylococcus aureus. The MLS resistance determinant of pIM13 was shown to be highly homologous to ermC, an inducible element on the S. aureus plasmid pE194. The product of the pIM13 determinant was similar in size to that of ermC and immunologically cross-reactive with it. The MLS resistance of pIM13 was expressed constitutively. The complete base sequence of pIM13 is presented. The plasmid consisted of 2,246 base pairs and contained two open reading frames that specified products identified in minicell extracts. One was a protein of 16,000 molecular weight, possibly required for replication. The second was the 29,000-molecular-weight MLS resistance methylase. The regulatory region responsible for ermC inducibility was missing from pIM13, explaining its constitutivity. The remainder of the pIM13 MLS determinant was nearly identical to ermC. The ends of the region of homology between pIM13 and pE194 were associated with hyphenated dyad symmetries. A segment partially homologous to one of these termini on pIM13 and also associated with a dyad was found in pUB110 near the end of a region of homology between that plasmid and pBC16. The entire sequence of pIM13 was highly homologous to that of pE5, an inducible MLS resistance plasmid from S. aureus that differs from pIM13 in copy control.     

Characterization of yhcN,a new forespore-specific gene of Bacillus subtilis

A new Bacillus subtilis sporulation-specific gene, yhcN, has been identified, the expression of which is dependent on the forespore-specific sigma factor σ^G and to a much lesser extent on σ^F. A translational yhcN-lacZ fusion is expressed at a very high level in the forespore, and the protein encoded by yhcN was detected in the inner spore membrane. A yhcN mutant sporulates normally and yhcN spores have identical resistance properties to wild-type spores. However, the outgrowth of yhcN spores is slower than that of wild-type spores.     

A quantitative study of gene regulatory pathways in Bacillus subtilis for virulence and competence phenotype by quorum sensing

Quorum sensing (QS) is a process which allows a population of bacteria to coordinately regulate gene expression of their entire community. Bacillus subtilis is a soil organism which uses QS to alternate between competence for DNA uptake and sporulation. We propose a model to describe the components involved in QS and to analyze reaction species involved in the regulation of QS machinery. We targeted only those QS phenotypes for which the genetic organization and molecular characterization of the components are fully elucidated. We have analyzed simulations for concentration of different species involved in competence as well as sporulation pathways at diverse time period using quantitative methods. It was observed that there is possibility of achieving different measurement from reactions taken place between species by applying irreversible Michaelis–Menten kinetic law. We obtain variation in measurement on changing parameters such as concentrations ranging from 0.3 to 50 μM in stepwise manner by setting end time in the range of 0.1–100 ms. Additionally we observe covariance between different reaction species involved in QS by fluctuating their quantities in real-time simulations. Our model mimics correctly the phenotype for competence and virulence. We concluded that time factor play major role to determine rate kinetics of diverse reaction species as compared to their concentrations and support the hypothesis of getting genetic stability while colonies are in synchronization.     

Sequence of the Bacillus subtilis glutamine synthetase gene region

The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features. An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation. Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site. One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites. The derived amino acid (aa) sequence of B. subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum. The B. subtilis and C. acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations. The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B. subtilis GS despite its lack of adenylylation.