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PubMed1995

The recA gene of Streptococcus pneumoniae is part of a competence-induced operon and controls lysogenic induction.

The recently identified recA gene of the naturally transformable bacterium Streptococcus pneumoniae has been further characterized by constructing a recA null mutation and by investigating its regulation. The recA mutation has been shown to confer both DNA repair (as judged from sensitivity to u.v. and methyl methane sulphonate) and recombination deficiencies. Plasmid transformation into the recA mutant was also drastically reduced. Western blotting established that recA gene expression is increased several fold at the onset of competence for genetic transformation. Increased expression was associated with the appearance of a recA-specific transcript, approximately 5.7 kb long. This transcript indicated that recA is part of a competence-inducible (cin) operon. The major (about 4.3 kb) transcript detected from non-competent cells did not include cinA, the first gene in the operon, suggesting that this gene could be specifically required at some stage in the transformation process. Detection of small amounts of the 5.7 kb polycistronic mRNA in cells treated with mitomycin C suggested that the operon could also be damage inducible. In addition, mitomycin C treatment of a recA- lysogenic strain did not lead to prophage induction and cell lysis. This is unlike the situation of a recA+ lysogen. Together these results demonstrate that RecA controls lysogenic induction and suggest the existence of a SOS repair system in S. pneumoniae.

Bacterial Proteins↗
PubMed2004

Chromosomal fragmentation in dUTPase-deficient mutants of Escherichia coli and its recombinational repair.

Recent findings suggest that DNA nicks stimulate homologous recombination by being converted into double-strand breaks, which are mended by RecA-catalysed recombinational repair and are lethal if not repaired. Hyper-rec mutants, in which DNA nicks become detectable, are synthetic-lethal with recA inactivation, substantiating the idea. Escherichia coli dut mutants are the only known hyper-recs in which presumed nicks in DNA do not cause inviability with recA, suggesting that nicks stimulate homologous recombination directly. Here, we show that dut recA mutants are synthetic-lethal; specifically, dut mutants depend on the RecBC-RuvABC recombinational repair pathway that mends double-strand DNA breaks. Although induced for SOS, dut mutants are not rescued by full SOS induction if RecA is not available, suggesting that recombinational rather than regulatory functions of RecA are needed for their viability. We also detected chromosomal fragmentation in dut rec mutants, indicating double-strand DNA breaks. Both the synthetic lethality and chromosomal fragmentation of dut rec mutants are suppressed by preventing uracil excision via inactivation of uracil DNA-glycosylase or by preventing dUTP production via inactivation of dCTP deaminase. We suggest that nicks become substrates for recombinational repair after being converted into double-strand DNA breaks.

Amino Acid Sequence↗
PubMed2005

RecN protein and transcription factor DksA combine to promote faithful recombinational repair of DNA double-strand breaks.

In rapidly dividing bacterial cells, the machinery for repair of DNA double-strand breaks has to contend not only with the forces driving replication and transmission of the DNA but also its transcription. By exploiting I-SceI homing endonuclease to break the Escherichia coli chromosome at one or more defined locations, we have been able to investigate how these processes are co-ordinated and repair is accomplished. When breaks are induced at a single site, the SOS-inducible RecN protein and the transcription factor DksA combine to promote efficient repair. When induced at two or more, distantly located sites, RecN becomes almost indispensable. Many cells that do survive have extensive deletions of sequences flanking the break, with end points often coinciding with imperfect repeat elements. These findings herald a much greater complexity for chromosome repair than suggested by current mechanistic models and reveal a role for RecN in protecting the chromosome from break-induced chromosome rearrangements.

Bacterial Proteins↗
PubMed1995

The recA gene of Chlamydia trachomatis: cloning, sequence, and characterization in Escherichia coli.

The recA gene of Chlamydia trachomatis was isolated by complementation of an Escherichia coli recA mutant. The cloned gene restored resistance to methyl methanesulfonate in E. coli recA mutants. The DNA sequence of the chlamydial gene was determined and the deduced protein sequence compared with other RecA proteins. In E. coli recA deletion mutants, the cloned gene conferred moderate recombinational activity as assayed by Hfr matings. The chlamydial recA gene was efficient in repairing alkylated DNA but less so in repairing of UV damage when compared with the E. coli homologue. As detected by an SOS gene fusion, a small but measurable amount of LexA co-cleavage was indicated.

Chlamydia trachomatis↗
PubMed1999

Mechanisms of mutation in nondividing cells. Insights from the study of adaptive mutation in Escherichia coli.

When populations of cells are subjected to nonlethal selection, mutations arise in the absence of cell division, a phenomenon that has been called "adaptive mutation." In a strain of Escherichia coli that cannot metabolize lactose (Lac-) but that reverts to lactose utilization (Lac+) when lactose is its sole energy and carbon source, the mutational process consists of two components. (1) A highly efficient, recombination-dependent mechanism giving rise to mutations on the F' episome that carries the Lac- allele; and (2) a less efficient, unknown mechanism giving rise to mutations elsewhere in the genome. Both selected and nonselected mutations arise in the Lac- population, but nonselected mutations are enriched in Lac+ mutants, suggesting that some Lac+ cells have passed though a transient period of increased mutation. These results have several evolutionary implications. (1) DNA synthesis initiated by recombination could be an important source of spontaneous mutation, particularly in cells that are not undergoing genomic replication. (2) The highly active mutational mechanism on the episome could be important in the horizontal transfer of variant alleles among species that carry and exchange conjugal plasmids. (3) A sub-population of cells in a state of transient mutation could be a source of multiple variant alleles and could provide a mechanism for rapid adaptive evolution under adverse conditions.

Adaptation, Biological↗
PubMed1987

Characterization of the Pseudomonas aeruginosa recA analog and its protein product: rec-102 is a mutant allele of the P. aeruginosa PAO recA gene.

We cloned a 2.3-kilobase-pair fragment of the Pseudomonas aeruginosa PAO chromosome which is capable of complementing recA mutations of Escherichia coli. The recA-complementing activity was further localized to a 1.5-kilobase-pair PvuII-HindIII fragment. Southern blot analysis under conditions of high stringency indicated that DNA sequence homology is shared by the E. coli recA gene and the P. aeruginosa recA analog. The cloned recA analog was shown to restore resistance to methyl methanesulfonate, nitrofurantoin, and UV irradiation to E. coli recA mutants. Upon introduction of the cloned P. aeruginosa gene, these mutants regained recombination proficiency in HfrH-mediated conjugation and the ability to induce lambda prophages and SOS functions (din gene transcription) after exposure to DNA-damaging agents. Lambda prophage carrying a cI ind mutation was not inducible, suggesting that the mechanism of induction of these SOS functions by the P. aeruginosa RecA analog is similar to that by the activated E. coli RecA protein. The product of the recA analog was identified in minicells as a protein of approximately 47,000 daltons. Western blot analysis using anti-E. coli RecA antibody demonstrated that this protein is antigenically cross-reactive with the E. coli recA protein. The recA-containing fragment was cloned into the broad-host-range vector pCP13 and introduced into Rec- strains of P. aeruginosa containing the rec-102 allele. The plasmid was shown to restore recombination proficiency in FP5-mediated conjugations and to restore resistance to UV irradiation and methyl methanesulfonate to these Rec- mutants. It was shown that a wild-type allele of rec-102 is necessary for UV-mediated induction of D3 and F116 prophages. The cloned recA analog restored the UV inducibility of these prophages in rec-102 mutants. These data indicate that rec-102 is a mutant allele of the P. aeruginosa recA gene and suggest that there has been considerable conservation of the recA gene in the evolution of the gram-negative bacteria.

Alleles↗
PubMed1987

Regulation of the Escherichia coli uvrD gene in vivo.

The roles of two putative promoter sequences, P1 and P2, and a potential antiterminator sequence found in the uvrD control region were examined in vivo. Constitutive and SOS-induced levels of uvrD mRNA were determined by S1 mapping, and it was shown that the majority of uvrD transcripts are from P1, while P2 plays only a minor role. A series of increasing deletions from the 5' end of the uvrD gene was used to assay transcription in the promoterless vector pKO-1. Loss of just the -35 region of P1 was sufficient to switch off detectable transcription from both P1 and P2. Disruption of the antiterminator by site-specific mutagenesis had no effect on constitutive levels of transcription, but led to a significant increase over wild-type levels following SOS induction. This suggests that the attenuator comes into play following DNA damage to moderate the increase in UvrD protein synthesis.

Adenosine Triphosphatases↗
PubMed1987

Escherichia coli K-12 mutants in which viability is dependent on recA function.

A gene required for growth and viability in recA mutants of Escherichia coli K-12 was identified. This gene, rdgB (for Rec-dependent growth), mapped near 64 min on the E. coli genetic map. In a strain carrying a temperature-sensitive recA allele, recA200, and an rdgB mutation, DNA synthesis but not protein synthesis ceased after 80 min of incubation at 42 degrees C, and there was extensive DNA degradation. The rdgB mutation alone had no apparent effect on DNA synthesis or growth; however, mutant strains did show enhanced intrachromosomal recombination and induction of the SOS regulon. The rdgB gene was cloned and its-gene product identified through the construction and analysis of deletion and insertion mutations of rdgB-containing plasmids. The ability of a plasmid to complement an rdgB recA mutant was correlated with its ability to produce a 25-kilodalton polypeptide as detected by the maxicell technique.

Alleles↗
PubMed1988

SOS-like induction in Bacillus subtilis: induction of the RecA protein analog and a damage-inducible operon by DNA damage in Rec+ and DNA repair-deficient strains.

We quantitated the induction of the Bacillus subtilis Rec protein (the analog of Escherichia coli RecA protein) and the B. subtilis din-22 operon (representative of a set of DNA damage-inducible operons in B. subtilis) following DNA damage in Rec+ and DNA repair-deficient strains. After exposure to mitomycin C or UV irradiation, each of four distinct rec (recA1, recB2, recE4, and recM13) mutations reduced to the same extent the rates of both Rec protein induction (determined by densitometric scanning of immunoblot transfers) and din-22 operon induction (determined by assaying beta-galactosidase activity in din-22::Tn917-lacZ fusion strains). The induction deficiencies in recA1 and recE4 strains were partially complemented by the E. coli RecA protein, which was expressed on a plasmid in B. subtilis; the E. coli RecA protein had no effect on either induction event in Rec+, recB2, or recM13 strains. These results suggest that (i) the expression of both the B. subtilis Rec protein and the din-22 operon share a common regulatory component, (ii) the recA1 and recE4 mutations affect the regulation and/or activity of the B. subtilis Rec protein, and (iii) an SOS regulatory system like the E. coli system is highly conserved in B. subtilis. We also showed that the basal level of B. subtilis Rec protein is about 4,500 molecules per cell and that maximum induction by DNA damage causes an approximately fivefold increase in the rate of Rec protein accumulation.

Bacillus subtilis↗
PubMed1988

Attachment site of the genetic element e14.

The Escherichia coli K-12 genetic element, e14, contains a 216-base-pair region that is homologous to a portion of the host chromosome. This region serves as the integration site for the element. The 216-base-pair homology is interrupted by 28 mismatches distributed through the sequence. The actual integrative crossover occurs within the first 11 base pairs from one end of the region. To test factors which affect e14 site-specific recombination, we cloned the attachment sites of free e14 and the host chromosome into the same plasmid. The cloned attachment sites recombined intramolecularly in a process that required the presence of a chromosomal copy of e14 in the host cell as well as the induction of SOS. Recombination events that mimicked both integration and excision occurred under the same conditions and to roughly the same extent.

Attachment Sites, Microbiological↗
PubMed1988

Characterization of recombination-deficient mutants of Bacillus subtilis.

An isogenic set of "prophage-free," DNA repair-proficient and -deficient strains of Bacillus subtilis were characterized phenotypically. The mutant strains were provisionally classified into four categories on the basis of their sensitivity to DNA-damaging agents, their ability to release phage after lysogenization followed by damage to chromosomal DNA, and their impairment in genetic exchange. The properties of double Rec- mutants showed that recF and addA belong to different epistatic groups, whereas recF, recL, and recH fall into the same group. More than one pathway for genetic exchange might be operative in B. subtilis.

Bacillus subtilis↗
PubMed1992

New mutations in and around the L2 disordered loop of the RecA protein modulate recombination and/or coprotease activity.

The RecA protein plays a key role in Escherichia coli recombination and DNA repair. We have created new recA mutants with mutations in the vicinity of the recA430 mutation (Gly-204----Ser) which is known to affect RecA coprotease activity. Mutants carrying recA659 or recA611, located 3 and 7 amino acids downstream of residue 204, respectively, lose all RecA activities, while the mutant carrying recA616, which is located at 12 amino acids from this residue, keeps the coprotease activity but is unable to promote recombination. Complementation experiments show that both mutations recA611 and recA659 are dominant over the wild-type or recA430 allele while recA616 seems to be recessive to recA+ and dominant over recA430. It is suggested that these mutations are located in RecA domains which direct conformational modifications.

Alleles↗
PubMed1993

Genetic analysis of double-strand break repair in Escherichia coli.

We had reported that a double-strand gap (ca. 300 bp long) in a duplex DNA is repaired through gene conversion copying a homologous duplex in a recB21 recC22 sbcA23 strain of Escherichia coli, as predicted on the basis of the double-strand break repair models. We have now examined various mutants for this repair capacity. (i) The recE159 mutation abolishes the reaction in the recB21C22 sbcA23 background. This result is consistent with the hypothesis that exonuclease VIII exposes a 3'-ended single strand from a double-strand break. (ii) Two recA alleles, including a complete deletion, fail to block the repair in this recBC sbcA background. (iii) Mutations in two more SOS-inducible genes, recN and recQ, do not decrease the repair. In addition, a lexA (Ind-) mutation, which blocks SOS induction, does not block the reaction. (iv) The recJ, recF, recO, and recR gene functions are nonessential in this background. (v) The RecBCD enzyme does not abolish the gap repair. We then examined genetic backgrounds other than recBC sbcA, in which the RecE pathway is not active. We failed to detect the double-strand gap repair in a rec+, a recA1, or a recB21 C22 strain, nor did we find the gap repair activity in a recD mutant or in a recB21 C22 sbcB15 sbcC201 mutant. We also failed to detect conservative repair of a simple double-strand break, which was made by restriction cleavage of an inserted linker oligonucleotide, in these backgrounds. We conclude that the RecBCD, RecBCD-, and RecF pathways cannot promote conservative double-strand break repair as the RecE and lambda Red pathways can.

Bacterial Proteins↗
PubMed1994

The RecF pathway of homologous recombination can mediate the initiation of DNA damage-inducible replication of the Escherichia coli chromosome.

DNA damage-inducible DNA replication in SOS-induced Escherichia coli cells, termed inducible stable DNA replication (iSDR), has previously been shown to require either the RecBCD or the RecE pathway of homologous recombination for initiation. Here, we demonstrate that recB recC sbcC quadruple mutant cells are capable of iSDR induction and that a mutation in the recJ gene abolishes the inducibility. These results indicate that the RecF pathway of homologous recombination can also catalyze iSDR initiation.

Bacterial Proteins↗
PubMed1995

Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.

To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.

Chromosomes, Bacterial↗
PubMed1998

Only the N-terminal domain of FtsK functions in cell division.

Deletion of ftsK results in the inhibition of cell division, but this inhibition can be reversed by a plasmid carrying only the first approximately 17% of ftsK. The division block can be suppressed in most mutants by deletion of dacA, which codes for the D-alanine:D-alanine carboxypeptidase PBP5, or in all mutants by overexpression of ftsN. Overexpression of ftsK inhibits cell division and the formation of FtsZ rings. This division block is not due to the induction of either the SOS or the heat shock regulons.

Bacterial Proteins↗