Characterization of the promoter of the Rhizobium etli recA gene.

The promoter of the Rhizobium etli recA gene has been identified by primer extension and by making deletions affecting several regions located upstream of its coding region. A gel mobility shift assay carried out with crude extracts of cells of R. etli has been used to show that a DNA-protein complex is formed in the R. etli recA promoter region in vitro. Analysis of the minimal region of the recA promoter giving rise to this DNA-protein complex revealed the presence of an imperfect palindrome corresponding to the sequence TTGN11CAA. Site-directed mutation of both halves of this palindrome indicated that both motifs, TTG and CAA, are necessary for both normal DNA-protein complex formation in vitro and full DNA damage-mediated inducibility of the recA gene in vivo. However, the TTG motif seems to be more dispensable than the CAA one. The presence of this same palindrome upstream of the recA genes of Rhizobium meliloti and Agrobacterium tumefaciens, whose expression is also regulated in R. etli cells, suggests that this TTGN11CAA sequence may be the SOS box of at least these three members of the Rhizobiaceae.     

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

Abstract 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.     

Diverse rhizobia associated with Sophora alopecuroides grown in different regions of Loess Plateau in China

A total of seventy-five symbiotic bacterial strains isolated from root nodules of wild Sophora alopecuroides grown in different regions of China's Loess Plateau were characterized. Based on the combined RFLP patterns, thirty-five genotypes were defined among the rhizobia and they were classified into nine genomic species, including Mesorhizobium alhagi and M. gobiense as the main groups, as well as Agrobacterium tumefaciens, M. amorphae, Phyllobacterium trifolii, Rhizobium giardinii, R. indigoferae, Sinorhizobium fredii and S. meliloti as the minor groups according to the 16S rRNA and recA gene analyses. Five and three lineages of nodA and nifH were found, respectively, in these strains, implying that the symbiotic genes of the S. alopecuroides rhizobia had different origins or had divergently evolved. Results of correspondence analysis showed that there was a correlation between rhizobial genotypes and the geographic origins. Possible lateral transfer of the recA and 16S rRNA genes between the P. trifolii and A. tumefaciens strains, and that of symbiotic genes (nodA, nifH) between different genera, was shown by discrepancies of the phylogenetic relationships of the four gene loci. These results revealed diverse rhizobia associated with wild S. alopecuroides grown in different regions of China's Loess Plateau, and demonstrated for the first time the existence of symbiotic A. tumefaciens strains in root nodules of S. alopecuroides.     

Construction of an Agrobacterium tumefaciens C58 recA mutant.

Clones encoding the recA gene of Agrobacterium tumefaciens C58 were isolated from a cosmid bank by complementation of an Escherichia coli recA mutation. Subcloning and mutagenesis with the lacZ fusion transposon Tn3HoHo1 located the Agrobacterium recA gene to a 1.3-kilobase segment of DNA. beta-Galactosidase expression from the fusions established the direction in which the gene was transcribed. The gene restored homologous recombination as well as DNA repair functions in E. coli recA mutants. Similar complementation of DNA repair functions was observed in the UV-induced Rec- Agrobacterium mutant, LBA4301. The Agrobacterium recA gene was disrupted by insertion of a cassette encoding resistance to erythromycin, and the mutated gene was marker exchanged into the chromosome of strain NT-1. The resulting strain, called UIA143, was sensitive to UV irradiation and methanesulfonic acid methyl ester and unable to carry out homologous recombination functions. The mutation was stable and had no effect on other genetic properties of the Agrobacterium strain, including transformability and proficiency as a conjugal donor or recipient. Furthermore, strain UIA143 became tumorigenic upon introduction of a Ti plasmid, indicating that tumor induction is independent of recA functions. Sequence homology was detected between the recA genes of strain C58 and E. coli as well as with DNA isolated from agrobacteria representing the three major biochemically differentiated biovars of this genus. In some cases, biovar-specific restriction fragment length polymorphisms were apparent at the recA locus.     

Molecular characterization of regulatory elements controlling expression of the Bacillus subtilis recA+ gene.

Expression of the Bacillus subtilis recA gene is induced following DNA damage as well as during the development of the competent state. DNA damage-induction of the recA gene occurs by a RecA-dependent mechanism, whereas competence-induction occurs by a RecA-independent mechanism. To examine the molecular mechanisms that control the expression of the recA gene, a deletion analysis of the recA promoter region was performed. A regulatory region that is required for repression of recA expression was identified upstream of the recA promoter. Deletion of this regulatory region derepressed expression and abolished damage-induction of the recA promoter. Within this region are sequences similar to the consensus sequence that has been identified within DNA damage-inducible promoter regions of other B subtilis genes. Another regulatory region was identified that is required for the RecA-independent, competence-specific induction of the recA gene. Deletion of these sequences significantly reduced competence-induction of the recA promoter.     

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.     

Cloning and DNA sequence of a mycoplasmal recA gene.

Mycoplasmas are wall-less prokaryotes phylogenetically related to gram-positive bacteria. In order to investigate DNA recombination in these organisms, we have cloned the recA gene from the mycoplasma Acholeplasma laidlawii. DNA sequence data indicate extensive homology between the A. laidlawii recA gene and recA genes from other bacteria, particularly Bacillus subtilis. The recA sequences from three A. laidlawii strains (strains JA1, K2, and 8195) were compared, and surprisingly, the gene from A. laidlawii 8195 was found to contain a nonsense mutation that results in truncation of 36 amino acids from the carboxyl terminus of the RecA protein. By using sensitivity to UV irradiation as a measure of DNA repair, strain 8195 had an apparent RecA- phenotype. When carried on a multicopy plasmid, the wild-type A. laidlawii recA gene was detrimental to growth of Escherichia coli, perhaps because of improper regulation of the RecA protein.     

Application of recA and rpoB sequence analysis on phylogeny and molecular identification of Geobacillus species

Aims:  Some Geobacillus species have highly similar 16S rRNA gene sequences, making 16S rDNA sequence analysis-based identification problematic. To overcome this limitation, recA and rpoB sequence analysis was evaluated as an alternative for distinguishing Geobacillus species.
Methods and Results:  The phylogram of 16S rRNA gene sequences inferred from the neighbour-joining method showed that nine clusters of Geobacillus species were characterized with bootstrap values >90%. The recA and rpoB sequences of 10 reference strains in clusters V, VIb and VIc were amplified and sequenced using consensus primers. Alignment of recA sequences in clusters V, VIb and VIc revealed three types of recA genes, consistent with the putative amino acid sequences and in vivo recA splicing analysis. The phylogram constructed from rpoB sequences showed more divergence than that constructed from 16S rRNA gene sequences.
Conclusions:  recA and rpoB sequence analysis differentiated closely-related Geobacillus species and provided direct evidence for reclassifying some species dubiously categorized as Geobacilli . Additionally, this study revealed three types of recA genes in the different Geobacillus species.
Significance and Impact of the Study:  This study highlights the advantage of recA and rpoB sequence analysis to supplement 16S rRNA gene sequence analysis for efficient and convenient determination of Geobacillus species.     

Genotyping of Chromobacterium violaceum isolates by recA PCR-RFLP analysis

Intraspecies variation of Chromobacterium violaceum was examined by comparative sequence - and by restriction fragment length polymorphism analysis of the recombinase A gene (recA-PCR-RFLP). Primers deduced from the known recA gene sequence of the type strain C. violaceum ATCC 12472(T) allowed the specific amplification of a 1040bp recA fragment from each of the 13 C. violaceum strains investigated, whereas other closely related organisms tested negative. HindII-PstI-recA RFLP analysis generated from 13 representative C. violaceum strains enabled us to identify at least three different genospecies. In conclusion, analysis of the recA gene provides a rapid and robust nucleotide sequence-based approach to specifically identify and classify C. violaceum on genospecies level.     

Cloning of a chromosomal fragment from Lactococcus lactis subsp. lactis partially complementing Escherichia coli recA functions

A recA-like gene was isolated from a gene library of Lactococcus lactis subsp. lactis by intergeneric complementation of an E. coli recA mutant. A plasmid was obtained which fully complemented the RecA response to DNA damaging agents and UV inducibility of prophage, but not P1 plating efficiency in an E. coli recA mutant. The cloned DNA fragment also partially complemented the rec mutation in Lc. lactis MMS36. Hybridization studies showed that there was no detectable sequence homology between the recA gene of E. coli and Lc. lactis subsp. lactis chromosomal DNA.     

Isolation and characterization of the recA gene of Rhizobium meliloti.

Interspecific complementation of an Escherichia coli recA mutant with plasmids containing a gene bank of Rhizobium meliloti DNA was used to identify a clone which contains the recA gene of R. meliloti. The R. meliloti recA protein can function in recombination and in response to DNA damage when expressed in an E. coli recA host, and hybridization studies have shown that DNA sequence homology exists between the recA gene of E. coli and that of R. meliloti. The isolated R. meliloti recA DNA was used to construct a recA R. meliloti, and this bacterium was not deficient in its ability to carry out symbiotic nitrogen fixation.     

Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function

A recA deletion mutant of Mycobacterium smegmatis has been isolated by homologous recombination using a sacB counterselection strategy. Deletion of the recA gene from the chromosome was demonstrated by Southern hybridizations and by polymerase chain reaction (PCR). Western analysis using anti-RecA antibodies confirmed that the RecA protein was not made by the mutant strain. The recA deletion strain exhibited enhanced sensitivity to UV irradiation and failed to undergo homologous recombination. The results obtained from the recombination assays suggest that in wild-type M. smegmatis the majority of colonies arise from single cross-over homologous recombination events with only a very minor contribution from random integrations. The deficiencies in UV survival and recombination were complemented by introduction of the cloned M. smegmatis recA gene. Overexpression of RecA was found to be toxic in the absence of recX , which is found downstream of and co-transcribed with recA and is thus also affected by the deletion of recA . The M. smegmatis recA deletion strain was also complemented by the M. tuberculosis recA gene with or without its intein; most importantly, the frequency of double cross-over homologous recombination events was identical regardless of whether the M. tuberculosis recA gene contained or lacked the intein. Thus, the low frequency of homologous recombination observed in M. tuberculosis is not due to the presence of an intein-coding sequence in its recA gene per se .     

产生无标记农杆菌突变体方法的建立及优化

农杆菌已经用作许多生物过程研究的模型细菌,为了解析这些生物过程的分子机理,对农杆菌的某些基因进行突变就显得非常重要．以自杀性基因sacB作为反向可选择性标记基因,利用同源重组的原理,建立了一种可对农杆菌基因进行准确插入、删除和位点置换的突变方法,所获突变体不带任何不需要的外源DNA序列．通过详细研究同源序列的长度对农杆菌同源重组效率和突变体产生概率的影响,以及对农杆菌中的同源重组机理的分析,提出了优化该突变体产生方法的方案,即通过设计不等长的上下游同源序列和选择其中一种类型的单交换重组体来筛选二次交换重组体的方法,可以显著地提高理想突变体的产生概率．研究结果对如何提高突变体的产生概率和减少突变体筛选的工作量具重要的参考价值．利用该方法成功地获得了两个基因被同时删除而且不含抗性标记的农杆菌突变株．     

Isolation and characterization of the Rickettsia prowazekii recA gene.

The recA gene has been isolated from Rickettsia prowazekii, an obligate intracellular bacterium. Comparison of the amino acid sequence of R. prowazekii RecA with that of Escherichia coli RecA revealed that 62% of the residues were identical. The highest identity was found with RecA of Legionella pneumophila, in which 69% of the residues were identical. Amino acid residues of E. coli RecA associated with functional activities are conserved in rickettsial RecA, and the R. prowazekii recA gene complements E. coli recA mutants for UV light and methyl methanesulfonate sensitivities as well as recombinational deficiencies. The characterized region upstream of rickettsial recA did not contain a sequence homologous to an E. coli LexA binding site (SOS box), suggesting differences in the regulation of the R. prowazekii recA gene.     

Non-reciprocal regulation of Rhodobacter capsulatus and Rhodobacter sphaeroides recA genes expression

Abstract The Rhodobacter capsulatus recA gene has been isolated and sequenced. Its deduced amino acid sequence showed the closest identity with the Rhodobacter sphaeroides RecA protein (91% identity). However, the promoter regions of both R. capsulatus and R. sphaeroides recA genes are only 64% similar. An Escherichia coli -like LexA binding site was not present in the upstream region of the R. capsulatus recA gene. Nevertheless, the R. capsulatus recA gene is inducible by DNA damage in both hetero- and phototrophically growing conditions. The R. capsulatus recA gene is poorly induced when inserted into the chromosome of R. sphaeroides , indicating that the recA gene of both bacteria possess different control sequences despite their phylogenetically close relationship.