Biomedical subjects

R Provvedi

Publications and source records attributed to R Provvedi.

6 recordsLinked to original sources

PubMed2001

NucA is required for DNA cleavage during transformation of Bacillus subtilis.

We have re-examined the roles of nucA and nin, in the transformation of Bacillus subtilis as conflicting accounts have been presented concerning the importance of these genes for transformation. The present report demonstrates that nucA deficiency lowers the rate of DNA transport and that NucA is needed for the double-strand cleavage of transforming DNA, probably acting directly as an endonuclease. A relative paucity of DNA termini, resulting from the absence of this endonuclease activity, most probably accounts for the decreased transport rate. NucA is a bitopic integral membrane protein, with its C-terminus external to the membrane where it is appropriately located to effect the cleavage of bound transforming DNA. We have also investigated the roles of the known competence genes in the DNA processing that accompanies transformation in B. subtilis. The genes that are required for DNA transport (comEA, comEC and comFA) are also required for the degradation of the non-transforming strand that accompanies internalization, but comEC and comFA are not needed for the double-strand cleavage that occurs external to the cell membrane.

Bacillus subtilis↗

PubMed1999

ComEA is a DNA receptor for transformation of competent Bacillus subtilis.

Competent cells of Bacillus subtilis efficiently bind and internalize DNA. ComEA and the seven proteins encoded by the comG operon are required in vivo for the binding step. We show here that ComEA, a bitopic membrane protein, is itself capable of high-affinity DNA binding. A domain necessary for DNA binding is located at the C-terminus of ComEA. Proteins with similar 60-80 amino acid residue domains are widespread among bacteria and higher organisms. ComEA shows a marked preference for double-stranded DNA and can bind to oligomers as small as 22 bp in length. DNA binding by ComEA exhibits no apparent base sequence specificity. Using a membrane vesicle DNA-binding assay system we show that in the absence of cell wall, ComEA is still required for DNA binding, whereas the requirement for the ComG proteins is bypassed. We conclude that the ComG proteins are needed in vivo to provide access of the binding domain of ComEA to exogenous DNA. Possible specific roles for the ComG proteins are discussed.

Amino Acid Sequence↗

PubMed1998

Insertion vectors for construction of recombinant conjugative transposons in Bacillus subtilis and Enterococcus faecalis.

The broad-host range of conjugal transfer and the chromosomal location make conjugative transposons (CT) attractive candidates as tools for genetic manipulation of a large variety of bacteria. In this paper we describe insertion vectors capable of integrating into Tn916, the prototype of CT in Gram-positive bacteria. The integration of vectors into a single chromosomal copy of Tn916 was studied both after natural transformation of Bacillus subtilis, and after electroporation in Enterococcus faecalis. Integration occurred either by double or by single crossover, and the integrated DNA segment was shown to be highly stable. All recombinant CT (rCT) were still able to excise from the chromosome to form circular intermediates, the first step of both transposition and conjugal transfer. All classes of rCT generated by insertion vector pSMB47 were capable of conjugal transfer, while using pVMB11 it was possible to generate non-conjugative rCT.

Bacillus subtilis↗

PubMed1996

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae.

Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252, to form the composite element Tn5253 of Streptococcus pneumoniae. We show that Tn5251 is identical in structure and size to Tn916. DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the two CT. Essentially all differences (66/73) were clustered in a 688-bp segment of tet(M), which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae. DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site (attB) of Tn5251 into Tn5252. Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10(6) chromosomes.

Base Sequence↗

PubMed1996

The tetracycline resistance gene tet(M) exhibits mosaic structure.

Tetracycline resistance genes of the M class, tet(M), are typically found on mobile genetic elements as the conjugative transposons of gram-positive bacteria. By comparing the sequences of eight different tet(M) genes (from Enterococcus faecalis, Streptococcus pneumoniae, Staphylococcus aureus, Ureaplasma urealyticum, and Neisseria), a mosaic structure was detected which could be traced to two distinct alleles. The two alleles displayed a divergence of 8% and a different G/C content. The block structure of these genes provides evidence for the contribution of homologous recombination to the evolution and the heterogeneity of the tet(M) locus. Unlike described cases of chromosomally located mosaic loci, tet(M) is a relatively recently acquired determinant in the species examined and it would appear that mosaic structure within tet(M) has evolved after acquisition of the gene by the mobile genetic elements upon which it is located.

Bacterial Proteins↗

PubMedDate not supplied

Internalizing DNA.

The steps involved in the transformation of Bacillus subtilis are reviewed. These include the initial binding, processing and passage of DNA across the cell wall and transport across the plasma membrane. Our understanding of the roles of the proteins known to be required for these steps is reviewed.

Bacillus subtilis↗