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A G Shivakumar

Publications and source records attributed to A G Shivakumar.

13 recordsLinked to original sources
PubMed1995

Substrate requirements for ErmC' methyltransferase activity.

ErmC' is a methyltransferase that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics by catalyzing the methylation of 23S rRNA at a specific adenine residue (A-2085 in Bacillus subtilis; A-2058 in Escherichia coli). The gene for ErmC' was cloned and expressed to a high level in E. coli, and the protein was purified to virtual homogeneity. Studies of substrate requirements of ErmC' have shown that a 262-nucleotide RNA fragment within domain V of B. subtilis 23S rRNA can be utilized efficiently as a substrate for methylation at A-2085. Kinetic studies of the monomethylation reaction showed that the apparent Km of this 262-nucleotide RNA oligonucleotide was 26-fold greater than the value determined for full-size and domain V 23S rRNA. In addition, the Vmax for this fragment also rose sevenfold. A model of RNA-ErmC' interaction involving multiple binding sites is proposed from the kinetic data presented.

Bacillus subtilis↗
PubMed1989

Gene dosage effect on the expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium.

Significant differences in expression of the delta-endotoxin genes cryA1 and cryA2 of Bacillus thuringiensis subsp. kurstaki were observed in B. subtilis and B. megaterium. The cryA1 gene was expressed when present on a high-copy-number (hcn) vector in B. megaterium but not in B. subtilis. The cryA2 gene was expressed in both hosts, but at a higher level in B. megaterium. Expression of the cryA2 gene in B. megaterium was better from a hcn vector than from a low copy number vector. Inhibition of sporulation was observed when the toxin genes were present on hcn plasmids in B. subtilis while no such effect was evident in B. megaterium. In addition, there was a significant reduction in copy numbers in both B. subtilis and B. megaterium when delta-endotoxin genes or a spoVG promoter-containing fragment of DNA were cloned into hcn plasmids.

Bacillus↗
PubMed1986

Vegetative expression of the delta-endotoxin genes of Bacillus thuringiensis subsp. kurstaki in Bacillus subtilis.

Bacillus thuringiensis subsp. kurstaki total DNA was digested with BglII and cloned into the BamHI site of plasmid pUC9 in Escherichia coli. A recombinant plasmid, pHBHE, expressed a protein of 135,000 daltons that was toxic to caterpillars. A HincII-SmaI double digest of pHBHE was then ligated to BglII-cut plasmid pBD64 and introduced into Bacillus subtilis by transformation. The transformants were identified by colony hybridization and confirmed by Southern blot hybridization. A 135,000-dalton protein which bound to an antibody specific for the crystal protein of B. thuringiensis was detected from the B. subtilis clones containing the toxin gene insert in either orientation. A toxin gene insert cloned into a PvuII site distal from the two drug resistance genes of the pBD64 vector also expressed a 135,000-dalton protein. These results suggest that the toxin gene is transcribed from its own promoter. Western blotting of proteins expressed at various stages of growth revealed that the crystal protein expression in B. subtilis begins early in the vegetative phase, while in B. thuringiensis it is concomitant with the onset of sporulation. The cloned genes when transferred to a nonsporulating strain of B. subtilis also expressed a 135,000-dalton protein. These results suggest that toxin gene expression in B. subtilis is independent of sporulation. Another toxin gene encoding a 130,000- to 135,000-dalton protein was cloned in E. coli from a library of B. thuringiensis genes established in lambda 1059. This gene was then subcloned in B. subtilis. The cell extracts from both clones were toxic to caterpillars. Electron microscope studies revealed the presence of an irregular crystal inclusion in E. coli and a well-formed bipyramidal crystal in B. subtilis clones similar to the crystals found in B. thuringiensis.

Bacillus subtilis↗
PubMed1981

Characterization of a plasmid-specified ribosome methylase associated with macrolide resistance.

The ermC gene of plasmid pE194 specifies resistance to the macrolidelincosamide-streptogramin B antibiotics. This resistance, as well as synthesis of the 29,000 dalton protein product of ermC, has been shown to be induced by erythromycin. Weisblum and his colleagues have established that macrolide resistance is associated with a specific dimethylation of adenine in 23 S rRNA. We show that pE194 specifies an RNA methylase that can utilize either 50 S ribosomes or 23 S rRNA as substrates. Synthesis of this methylase is induced by low concentrations of erythromycin, and the enzyme is produced in elevated amounts by strains carrying a high copy number mutant of pE194. The methylase comigrates with the 29K ermC product on polyacrylamide gels. The purification and some properties of this methylase are described.

Amino Sugars↗
PubMed1981

Naturally occurring macrolide-lincosamide-streptogramin B resistance in Bacillus licheniformis.

Resistance to the macrolide-lincosamide-streptogramin B (MLS) group of antibiotics is widespread and of clinical importance. B. Weisblum and his coworkers have demonstrated that this resistance is associated with methylation of the 23S ribosomal ribonucleic acid of the large ribosomal subunit which results in a diminished affinity of this organelle for these antibiotics (Lai et al, J. Mol. Biol. 74:67-72, 1973). We report that 10 of 15 natural isolates of Bacillus licheniformis, a common soil organism, are resistant to the MLS antibiotics. The properties of this resistance (high level of tolerance for erythromycin, broad cross-resistance spectrum, and inducibility) suggest that resistance is conferred as described above. The resistance determinant from one of these strains was cloned onto a B. subtilis plasmid vector, and the resulting hybrid plasmid (pBD90) was used to prepare radioactive probe deoxyribonucleic acid for hybridization studies. All of the resistance B. licheniformis strains studied exhibited homology with the pBD90 insert. Plasmid pBD90 showed no homology to the following staphylococcal and streptococcal MLS-resistance plasmids: pE194, pE5, pAM77, pI258. Plasmids pE194 and pE5, on the other hand, carry homologous MLS genes but showed no detectable homology to one another in their replication genes. pBD90 specified a 35,000-dalton erythromycin-inducible protein, detectable in minicells, which therefore appears different from the 29,000-dalton inducible resistance protein specified by pE194. We conclude that there are at least three distinct MLS resistance determinants to be found among gram-positive bacteria.

Anti-Bacterial Agents↗
PubMed1980

Posttranscriptional regulation of an erythromycin resistance protein specified by plasmic pE194.

Induction of the synthesis of a plasmid-encoded polypeptide (E3) by erythromycin is known to be required for the inducible expression of resistance to the macrolide-lincosamide-streptogramin B group of antibiotics in Bacillus subtilis strains carrying the plasmid pE194. This resistance is mediated by a specific N6-dimethylation of adenine in the 23S rRNA of the large ribosomal subunit. We show in this report that E3 induction is regulated posttranscriptionally in the sense that it can occur when RNA synthesis is blocked and that induction is accompanied by an increase in the functional half-life of E3 mRNA but not of the mRNA species that code for the remaining four known pE194 polypeptides. The induction of E3 is subject to feedback regulation and involves the ribosome. Modification of the erythromycin binding site on the ribosome by methylation or by mutation interferes with induction.

Bacillus subtilis↗
PubMed1980

Characterization of chimeric plasmid cloning vehicles in Bacillus subtilis.

Restriction endonuclease cleavage maps of seven chimeric plasmids that may be used for molecular cloning in Bacillus subtilis are presented. These plasmids all carry multiple antibiotic resistance markers and were constructed by in vitro molecular cloning techniques. Several of the antibiotic resistance markers were shown to undergo insertional inactivation at specific restriction endonuclease sites. Kanamycin inactivation occurred at the BglII site of pUB110 derivatives, erythromycin inactivation occurred at the HpaI and BclI sites of pE194 derivatives, and streptomycin inactivation occurred at the HindIII site of pSA0501 derivatives. A stable mini-derivative of pBD12 was isolated and characterized. By using these plasmids, we identified proteins involved in plasmid-coded kanamycin and erythromycin resistance. The properties and uses of these chimeric plasmids in the further development of recombinant deoxyribonucleic acid technology in B. subtilis are discussed.

Bacillus subtilis↗
PubMed1978

Differential effect of hydroxyurea on the replication of plasmid and chromosomal DNA in Bacillus subtilis.

The replication in Bacillus subtilis of the staphylococcal R plasmids pE194, pBD15, pUB110, pSA0501, and pSA2100 has been studied in the presence of hydroxyurea. In all cases, an enrichment for covalently closed circular DNA compared with chromosomal DNA was observed. In this respect, hydroxyurea mimics the effect previously observed with pUB110, using strains carrying the conditional mutation dnaA13. This mutation has been reported to affect ribonucleotide reductase (G. W. Bazill and D. Karamata, Mol. Gen. Genet. 117:19-29, 1972). An explanation for these effects is offered, together with some supporting evidence.

Bacillus subtilis↗