PcrA is an essential DNA helicase of Bacillus subtilis fulfilling functions both in repair and rolling‐circle replication

Abstract

SummaryThe only DNA helicase essential for Escherichia coli viability is DnaB, the chromosome replication fork helicase. In contrast, in Bacillus subtilis, in addition to the DnaB counterpart called DnaC, we have found a second essential DNA helicase, called PcrA. It is 40% identical to the Rep and UvrD DNA helicases of E. coli and 61% identical to the PcrA helicase of Staphylococcus aureus. This gene is located at 55Њ on the chromosome and belongs to a putative operon together with a ligase gene (lig ) and two unknown genes named pcrB and yerH. As PcrA was essential for cell viability, conditional mutants were constructed. In such mutants, chromosomal DNA synthesis was slightly decreased upon PcrA depletion, and rolling-circle replication of the plasmid pT181 was inhibited. Analysis of the replication intermediates showed that leading-strand synthesis of pT181 was prevented upon PcrA depletion. To compare PcrA with Rep and UvrD directly, the protein was produced in rep and uvrD mutants of E. coli. PcrA suppressed the UV sensitivity defect of a uvrD mutant but not its mutator phenotype. Furthermore, it conferred a Rep ¹ phenotype on E. coli. Altogether, these results show that PcrA is an helicase used for plasmid rolling-circle replication and suggest that it is also involved in UV repair.

The Crystal Structure of (S)-3-O-Geranylgeranylglyceryl Phosphate Synthase Reveals an Ancient Fold for an Ancient Enzyme

“…2A) and a protein of unknown function that is conserved in some Gram-positive bacteria, including the human pathogens B. anthracis, S. aureus, and L. monocytogenes (8,12,15). Based on the similarity between AfGGGPS and BsPcrB crystal structures (1.3 Å root mean square deviation over 209 C␣ atoms; Fig.…”

“…Second, GGGPS belongs to a class of prenyltransferases that catalyzes the transfer of isoprenoid groups onto nonisoprenoid acceptors; this class includes protein prenyltransferases, DMAPP-tRNA transferase, DMAPP-AMP transferase, dimethylallyltryptophan synthase, and the recently characterized aromatic prenyltransferases (11,14). Finally, GGGPS is a homologue of PcrB, a protein of unknown function that is conserved in some Gram-positive bacteria, including the human pathogens Bacillus anthracis, Staphylococcus aureus, and Listeria monocytogenes (8,12,15).…”

5′ End-independent RNase J1 Endonuclease Cleavage of Bacillus subtilis Model RNA

“…For experiments with reduced RNase J1 or RNase Y expression from the IPTG-inducible p spac promoter, BG626 was transformed to erythromycin resistance using chromosomal DNA from the RNase J1- (20) or RNase Y-conditional mutant strains (19). The conditional mutant strains also contained plasmid pMAP65, which carries extra copies of the lacI gene for stronger repression (21). For determination of trp leader RNA half-life in the absence of RNase J2, an rnjB deletion strain (20) was transformed with pGD5 (see below).…”

Characterization of tRNACys processing in a conditional Bacillus subtilis CCase mutant reveals the participation of RNase R in its quality control

“…The Cm r cassette in pX : : birA was later exchanged for a Tet r cassette using plasmid pCm : : Tc (Steinmetz & Richter, 1994). To obtain higher repression of Pspac : : cca this strain was then transformed with plasmid pMAP65, which carries lacI q (Petit et al, 1998;Yansura & Henner, 1984).…”