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.     

Degenerate oligonucleotide primers for enzymatic amplification of recA sequences from gram-positive bacteria and mycoplasmas.

RecA protein in gram-negative bacteria, especially in Escherichia coli, has been extensively studied, but little is known about this key enzyme in other procaryotes. Described here are degenerate oligonucleotide primers that have been used to amplify by the polymerase chain reaction (PCR) recA sequences from several gram-positive bacteria and mycoplasmas. The DNA sequences of recA PCR products from Streptococcus pyogenes, Streptococcus mutans, Enterococcus faecalis, and Mycoplasma pulmonis were determined and compared. These data indicate that the M. pulmonis recA gene has diverged significantly from recA genes of other eubacteria. It should be possible to use cloned recA PCR products to construct recA mutants, thereby providing the means of elucidating homologous genetic recombination and DNA repair activities in these organisms.     

Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment.

Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria. This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii. An internal fragment of the recA gene from A. laidlawii was cloned into a plasmid that does not replicate in this organism. When this plasmid construct was used to transform A. laidlawii, it inserted into the chromosome, disrupting the recA gene. The phenotype of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene. As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.     

Multilocus sequence typing of Lactobacillus casei reveals a clonal population structure with low levels of homologous recombination

Robust genotyping methods for Lactobacillus casei are needed for strain tracking and collection management, as well as for population biology research. A collection of 52 strains initially labeled L. casei or Lactobacillus paracasei was first subjected to rplB gene sequencing together with reference strains of Lactobacillus zeae, Lactobacillus rhamnosus, and other species. Phylogenetic analysis showed that all 52 strains belonged to a single compact L. casei-L. paracasei sequence cluster, together with strain CIP107868 (= ATCC 334) but clearly distinct from L. rhamnosus and from a cluster with L. zeae and CIP103137(T) (= ATCC 393(T)). The strains were genotyped using amplified fragment length polymorphism, multilocus sequence typing based on internal portions of the seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA, and recG, and tandem repeat variation (multilocus variable-number tandem repeats analysis [MLVA] using nine loci). Very high concordance was found between the three methods. Although amounts of nucleotide variation were low for the seven genes (pi ranging from 0.0038 to 0.0109), 3 to 12 alleles were distinguished, resulting in 31 sequence types. One sequence type (ST1) was frequent (17 strains), but most others were represented by a single strain. Attempts to subtype ST1 strains by MLVA, ribotyping, clustered regularly interspaced short palindromic repeat characterization, and single nucleotide repeat variation were unsuccessful. We found clear evidence for homologous recombination during the diversification of L. casei clones, including a putative intragenic import of DNA into one strain. Nucleotides were estimated to change four times more frequently by recombination than by mutation. However, statistical congruence between individual gene trees was retained, indicating that recombination is not frequent enough to disrupt the phylogenetic signal. The developed multilocus sequence typing scheme should be useful for future studies of L. casei strain diversity and evolution.     

Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24

Abstract An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers. Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain. A 4.4 kb Bam HI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24. Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp. The highest similarity (∼78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E. coli promoter the S. lividans recA gene could partially complement an Escherichia coli recA mutant.     

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. Significance and Impact of the Study: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.     

Detection of cultured and uncultured Burkholderia cepacia complex bacteria naturally occurring in the maize rhizosphere

The species composition of a Burkholderia cepacia complex population naturally occurring in the maize rhizosphere was investigated by using both culture-dependent and culture-independent methods. B. cepacia complex isolates were recovered from maize root slurry on the two selective media Pseudomonas cepacia azelaic acid tryptamine (PCAT) and trypan blue tetracycline (TB-T) and subjected to identification by a combination of restriction fragment length polymorphism (RFLP) analysis and species-specific polymerase chain reaction (PCR) tests of the recA gene. DNA extracted directly from root slurry was examined by means of nested PCR to amplify recA gene with species-specific B. cepacia complex primers and to obtain a library of PCR amplified recA genes. Using the culture-dependent method the species Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia ambifaria and Burkholderia pyrrocinia were identified, whereas using the culture-independent method also the species Burkholderia vietnamiensis was detected. The latter method also allowed us to highlight a higher diversity within the B. cenocepacia species. In fact, by using the culture-independent method the species B. cenocepacia recA lineages IIIA and IIID besides B. cenocepacia recA lineage IIIB were detected. Moreover, higher heterogeneity of recA RFLP patterns was observed among clones assigned to the species B. cenocepacia than among B. cenocepacia isolates from selective media.     

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Intraspecies discrimination of Lactobacillus paraplantarum by PCR

Lactobacillus paraplantarum is a species phenotypically close to Lactobacillus plantarum. Several PCR methods were evaluated to discriminate L. paraplantarum strains and among them, a PCR using an enterobacterial repetitive intergenic consensus (ERIC) sequence differentiated L. paraplantarum from other Lactobacillus species. In addition, a combination of ERIC and random amplified polymorphic DNA (RAPD) analysis distinguished among seven strains of L. paraplantarum tested. ERIC-PCR profiles showed several strain-specific DNA fragments in L. paraplantarum, among them, a 2.2-kb ERIC marker, termed LpF1, found to be specific to strain FBA1, which improved the skin integrity in an animal model. The LpF1 encodes three proteins similar to Lactobacillus fermentum AroA, TyrA, and AroK, which are involved in the shikimate pathway. A primer pair specific to FBA1 based on the internal sequence of LpF1 amplified a 950-bp FBA1-specific fragment LpF2. Southern blot analysis of Dra I-digested genomic DNA of L. paraplantarum strains using LpF2 as a probe showed that LpF2 is distinctive of strain FBA1 among 16 L. paraplantarum strains. Because both ERIC- and RAPD-PCR are fast and technically simple methods, they are useful for the rapid discrimination of L. paraplantarum strains and for the development of new strain-specific DNA markers for identifying industrially important strains.     

Genomic species typing of acinetobacters by polymerase chain reaction amplification of the recA gene

Abstract The recA gene has been used as a target in screening for the presence of acinetobacters on the genospecies level and differentiation of relevant acinetobacter species from one another by PCR. Primers deduced from known recA gene sequences of Acinetobacter calcoaceticus and Neisseria gonorrhoeae allowed the amplification of DNAs from all Acinetobacter genospecies. The size of the amplified DNA fragment from all genospecies tested was approximately 435–500 bp relative to DNA size markers. The amplified products were examined further by restriction fragment length polymorphism (RFLP) analysis. Restriction analysis with only two enzymes, Mbo I and Hin fI, enabled us to identify all known genospecies. Since this method uses conserved recA gene sequences for primers, it is expected to be applicable for the identification of most bacterial species.     

High-throughput PCR screening of genes for three-component regulatory system putatively involved in quorum sensing from low-G + C gram-positive bacteria

Quorum sensing of gram-positive bacteria is often regulated by three-component regulatory system composed of autoinducing peptide, sensor kinase and response regulator. We used PCR to study a gene cassette encoding this three-component regulatory system. Degenerate primers were designed from consensus amino acid sequences in the HPK10 subfamily, mostly involved in quorum sensing. Products amplified from genomic DNA of Lactobacillus, Enterococcus, and Clostridium species were cloned and sequenced; their deduced amino acid sequences were similar to those of members of the HPK10 subfamily. Complete genes for the putative gene cassette were cloned by inverse PCR from L. paracasei E93490 and L. plantarum WCFS6. Phylogenetic analysis grouped the cloned putative HPKs into the HPK10 subfamily. These results indicated the usefulness of this high-throughput gene screening and suggested that the three-component regulatory gene cassette are widely present.     

pPSY: a vector for the stable cloning and expression of streptomycete single gene phenotypes in Escherichia coli

pPSY is a 12kb cloning vector derived from the IncW plasmid R388, which provides a rapid and easy way to stably clone phenotypes encoded in DNA segments <10kb. In the present study three different genes were amplified by PCR, cloned into pGEM-T Easy and sub-cloned into the EcoRI site of pPSY. The first gene, vioA, is a FAD-dependent l-tryptophan amino acid oxygenase from the high G+C Gram-negative bacterium Chromobacterium violaceum. VioA is involved in the synthesis of the indolocarbazole antitumour antibiotic violacein. It was found that vioA was strongly expressed in Escherichia coli from its native promoter. Two other genes encoding recombinase A (recA) and an amylase (amyA), derived from the high G+C Gram-positive streptomycete, Streptomyces lividans, were also tested. Despite recA lacking its native promoter sequence, it was strongly expressed in E. coli using the lac promoter of pGEM-T Easy. Similar to vioA, S. lividansamyA was strongly expressed in E. coli from its native promoter. Unlike pGEM-T Easy, pPSY stably maintained all three genes without the requirement for antibiotic selection. These results demonstrate the applicability of pPSY as a stable amplicon cloning vector for the expression of heterologous genes in E. coli.     

PCR amplification and direct sequencing of gyrB genes with universal primers and their application to the detection and taxonomic analysis of Pseudomonas putida strains.

Degenerate PCR primers, UP-1 and UP-2r, for the amplification of DNA gyrase subunit B genes (gyrB) were designed by using consensus amino acid sequences of gyrases from Escherichia coli, Pseudomonas putida, and Bacillus subtilis. In addition to the degenerate sequences, these primers have sequences at the 5' end which allow direct sequencing of amplified PCR products. With these primers, DNA segments of the predicted size were amplified from a variety of gram-negative and gram-positive genera. The nucleotide sequences of the amplified gyrB DNA from three P. putida strains were determined directly from the amplified fragments. The base substitution frequency of gyrB between the strains of P. putida was much higher than that of the 16S rRNA gene. With a specific set of PCR primers, it was possible to amplify gyrB fragments selectively from P. putida or its subgroups. The direct sequencing method of gyrB developed in this study provides a rapid and convenient system for bacterial identification, taxonomic analysis, and monitoring of bacteria in the natural environment.     

Mobilization transfer of the pUB110 plasmid between gram-positive bacteria]

The three factor crosses between the donor strain Bacillus subtilis 168 harbouring the plasmid pUB102-4, Bacillus thuringiensis strain carrying the mobilizing plasmid pAM beta 1 and recipient strain Lactobacillus fermenti were conducted in order to elaborate the optimal conditions of the plasmid pUB102-4 mobilization for transfer into gram-positive microorganisms and to elucidate the possible expression of endogluconase genes in a lactobacillus strain. The Lactobacillus fermenti transconjugants carrying the pUB102-4 plasmid were obtained in the three factor reciprocal crosses with the streptococcus recipient strain and Bacillus subtilis recipients. The presence of the plasmids in transconjugants was confirmed by colony hybridization with the [32P]-labelled plasmid DNA and KMC-ase activity in transconjugant cells. The proposed system of crosses using the high copy number plasmid derivatives of pUB110 mobilized with high frequency by the pAM beta 1 plasmid demonstrates the possibility to increase the circle of gram-positive host bacteria avoiding time and labour consuming operations.     

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.     

Differentiation of Lactobacillus plantarum, L. pentosus, and L. paraplantarum by recA gene sequence analysis and multiplex PCR assay with recA gene-derived primers

In this study, we succeeded in differentiating Lactobacillus plantarum, Lactobacillus pentosus, and Lactobacillus paraplantarum by means of recA gene sequence comparison. Short homologous regions of about 360 bp were amplified by PCR with degenerate consensus primers, sequenced, and analyzed, and 322 bp were considered for the inference of phylogenetic trees. Phylograms, obtained by parsimony, maximum likelihood, and analysis of data matrices with the neighbor-joining model, were coherent and clearly separated the three species. The validity of the recA gene and RecA protein as phylogenetic markers is discussed. Based on the same sequences, species-specific primers were designed, and a multiplex PCR protocol for the simultaneous distinction of these bacteria was optimized. The sizes of the amplicons were 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum. This strategy permitted the unambiguous identification of strains belonging to L. plantarum, L. pentosus, and L. paraplantarum in a single reaction, indicating its applicability to the speciation of isolates of the L. plantarum group.     

The majority of inducible DNA repair genes in Mycobacterium tuberculosis are induced independently of RecA

In many species of bacteria most inducible DNA repair genes are regulated by LexA homologues and are dependent on RecA for induction. We have shown previously by analysing the induction of recA that two mechanisms for the induction of gene expression following DNA damage exist in Mycobacterium tuberculosis. Whereas one of these depends on RecA and LexA in the classical way, the other mechanism is independent of both of these proteins and induction occurs in the absence of RecA. Here we investigate the generality of each of these mechanisms by analysing the global response to DNA damage in both wild-type M. tuberculosis and a recA deletion strain of M. tuberculosis using microarrays. This revealed that the majority of the genes that were induced remained inducible in the recA mutant stain. Of particular note most of the inducible genes with known or predicted functions in DNA repair did not depend on recA for induction. Amongst these are genes involved in nucleotide excision repair, base excision repair, damage reversal and recombination. Thus, it appears that this novel mechanism of gene regulation is important for DNA repair in M. tuberculosis.     

DNA probe and PCR-specific reaction for Lactobacillus plantarum

A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus , albeit more weakly. Two internal primers of this probe were selected (LbPl1 and LbPl2) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum .     

Two recA genes in Myxococcus xanthus.

Two recA genes, recA1 and recA2, in Myxococcus xanthus were cloned by using the recA gene of Escherichia coli, and their DNA sequences were determined. On the basis of deduced amino acid sequences, RecA1 and RecA2 have 67.0% identity to each other and 60.5 and 60.9% identities to E. coli RecA, respectively. Expression of recA2 was detected in both vegetative and developmental cells by Northern blot (RNA) analysis, and a threefold induction was observed when cells were treated with nalidixic acid. Repeated attempts to isolate a recA2 disruption mutant have failed, while a recA1 disruption mutant was readily isolated. Both the recA1 and recA2 genes expressed in E. coli complement the UV sensitivity of an E. coli recA strain.