Molecular cloning and nucleotide sequence of the Rhizobium phaseoli recA gene

Summary A recombinant phage carrying the recA gene of Rhizobium phaseoli was isolated from a R. phaseoli genomic library by complementation of the Fec^– phenotype of the recombinant phage in Escherichia coli. When expressed in E. coli, the cloned recA gene was shown to restore resistance to both UV irradiation and the DNA alkylating agent methyl methanesulphonate (MMS). The R. phaseoli recA gene also promoted homologous recombination in E. coli. The cloned recA gene was only weakly inducible in E. coli recA strains by DNA damaging agents. The DNA sequence of the R. phaseoli recA gene was determined and compared with published recA sequences. No LexA-binding site was detected in the R. phaseoli recA upstream region.     

Complementation of Bacteriophage Induction and Recombination Defects in <Emphasis Type="Italic">Escherichia coli</Emphasis> RecA<Superscript>−</Superscript> Mutants by Expression of the Cloned T4 Bacteriophage <Emphasis Type="Italic">uvsX</Emphasis> Gene

Previous workers reported that the T4 bacteriophage UvsX protein could promote neither RecA-LexA-mediated DNA repair nor induction of lysogenized bacteriophage, only recombination. Reexamination of these phenotypes demonstrated that, in contrast to these prior studies, when this gene was cloned into a medium but not a low-copy-number vector, it stimulated both a high frequency of spontaneous induction and mitomycin C-stimulated bacteriophage induction in a strain containing a recA13 mutation, but not a recA1 defect. The gene when cloned into a low- or medium- copy-number vector also promoted a low frequency of recombination of two duplicated genes in Escherichia coli in a strain with a complete recA gene deletion. These results suggest that a narrow concentration range of T4 UvsX protein is required to promote both high-frequency spontaneous and mitomycin C-stimulated bacteriophage induction in a recA13 gene mutant, but it facilitates recombination of duplicated genes at only a very low frequency in E. coli RecA⁻ mutants with a complete recA deletion. These results also suggest that the different UvsX phenotypes are affected differentially by the concentration of UvsX protein present. Received: 11 February 2002 / Accepted: 12 April 2002     

Cloning and characterization of the recA gene of Aquaspirillum magnetotacticum

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence.This paper is affectionately dedicated to Prof. John L. Ingraham     

Investigation of a phage resistantSerratia entomophila strain (BC4B), establishment of generalised transduction and construction ofS. entomophila RecA mutants

ArecA clone was isolated from a cosmid library ofSerratia entomophila constructed in theEscherichia coli strain HB101. Subcloning and transposon mutagenesis were used to identify a 1.36 kb fragment containing therecA gene. A clonedrecA mutation, generated by transposon mutagenesis and the replacement of a portion of therecA gene with an antibiotic resistance cassette, was introduced into the chromosome via a marker exchange technique. TherecA strains created were deficient in DNA repair, homologous recombination and both the spontaneous and UV induction of prophages.S. entomophila recA strains showed continued pathogenicity towards the New Zealand grass grub,Costelytra zealandica. Simple procedures for further construction ofS. entomophila recA strains have been demonstrated.     

Construction of an Azospirillum brasilense Sp7 recA mutant

Summary Cosmid clones encoding the recA gene of Azospirillum brasilense were isolated by intergeneric complementation of an Escherichia coli recA mutant. Site-directed Tn5 mutagenesis and subcloning of one complementing cosmid clone allowed us to localize the A. brasilense recA gene on a 1.2 kb DNA fragment. One Tn5 insertion that inactivates the cloned recA gene was crossed into the chromosome of A. brasilense by marker exchange. The resulting A. brasilense recA mutant showed increased sensitivity to the DNA methylating agent methyl methanesulfonate and to ultraviolet light and had at least one hundredfold reduced recombinational activity compared to the parent strain.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Expression and Complementation Analyses of a Chloroplast-Localized Homolog of Bacterial RecA in the Moss Physcomitrella patens

RecA protein is widespread in bacteria, and it plays a crucial role in homologous recombination. We have identified two bacterial-type recA gene homologs (PprecA1, PprecA2) in the cDNA library of the moss Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA2 to the green fluorescent protein (GFP) caused a targeting of PpRecA2 to the chloroplasts. Mutational analysis showed that the first AUG codon acts as initiation codon. Fusion of the full-length PpRecA2 to GFP caused the formation of foci that were colocalized with chloroplast nucleoids. The amounts of PprecA2 mRNA and protein in the cells were increased by treatment with DNA damaging agents. PprecA2 partially complemented the recA mutation in Escherichia coli. These results suggest the involvement of PpRecA2 in the repair of chloroplast DNA.     

RecA mediates MgpB and MgpC phase and antigenic variation in Mycoplasma genitalium, but plays a minor role in DNA repair

Mycoplasma genitalium, a sexually transmitted human pathogen, encodes MgpB and MgpC adhesins that undergo phase and antigenic variation through recombination with archived ‘MgPar’ donor sequences. The mechanism and molecular factors required for this genetic variation are poorly understood. In this study, we estimate that sequence variation at the mgpB/C locus occurs in vitro at a frequency of > 1.25 × 10^?4 events per genome per generation using a quantitative anchored PCR assay. This rate was dramatically reduced in a recA deletion mutant and increased in a complemented strain overexpressing RecA. Similarly, the frequency of haemadsorption‐deficient phase variants was reduced in the recA mutant, but restored by complementation. Unlike Escherichia coli, inactivation of recA in M. genitalium had a minimal effect on survival after exposure to mitomycin C or UV irradiation. In contrast, a deletion mutant for the predicted nucleotide excision repair uvrC gene showed growth defects and was exquisitely sensitive to DNA damage. We conclude that M. genitalium RecA has a primary role in mgpB/C–MgPar recombination leading to antigenic and phase variation, yet plays a minor role in DNA repair. Our results also suggest that M. genitalium possesses an active nucleotide excision repair system, possibly representing the main DNA repair pathway in this minimal bacterium.     

The role of DNA polymerase I and the rec system in survival of bacteria and bacteriophages damaged by the photodynamic action of acridine orange

Summary The effect of acridine orange (AO)-sensitized photodynamic treatment (PD) was studied in various repair-deficient mutants of Salmonella typhimurium and Escherichia coli. Bacteria of either species carrying mutations in the polA gene and hence deficient in the enzyme DNA polymerase I were significantly more sensitive to PD-killing than polA ⁺ parent bacteria or phenotypically POL⁺ revertants of the polA strains (selected on the basis of resistance to methyl methanesulphonate). It therefore appears that DNA polymerase I plays an important role in cellular recovery from PD treatment. E. coli carrying a mutation in the recA gene was also more sensitive to PD-treatment than its parent strain, as was S. typhimurium carrying a mutation of the recA type. In S. typhimurium the rec mutant was somewhat less sensitive to PD-killing than the pol mutant even although it is much more sensitive to ultraviolet killing. E. coli strains with mutations in the recB and recC genes were intermediate in PD sensitivity between the recA and the parent strain. S. typhimurium and E. coli bacteria with mutations in the polA and recA genes showed reduced ability to host-cell reactivate PD-damaged bacteriophages ES 18 and c1, indicating that the polA ⁺ and recA ⁺ gene products also contribute to repair of bacteriophages damaged by PD treatment. It is suggested that the recombinational repair process is less important for recovery from PD than for recovery from UV, and that the primary contribution of the rec genes to recovery from PD may be in repair of single-strand gaps by repair resynthesis.     

Cloning of theHelicobacter pylori recA gene and functional characterization of its product

The RecA protein is a key enzyme involved in DNA recombination in bacteria. Using a polymerase chain reaction (PCR) amplification we cloned arecA homolog fromHelicobacter pylori. The gene revealed an open reading frame (ORF) encoding a putative protein of 37.6 kDa showing closest homology to theCampylobacter jejuni RecA (75.5% identity). A putative ribosome binding site and a near-consensus σ⁷⁰ promoter sequence was found upstream ofrec A. A second ORF, encoding a putative protein with N-terminal sequence homology to prokaryotic and eukaryotic enolases, is located directly downstream ofrecA. Compared to the wild-type strains, isogenicH. pylori recA deletion mutants of strains 69A and NCTC11637 displayed increased sensitivity to ultraviolet light and abolished general homologous recombination. The recombinantH. pylori RecA protein produced inEscherichia coli strain GC6 (recA ⁻) was 38 kDa in size but inactive in DNA repair, whereas the corresponding protein inH. pylori 69A migrated at the greater apparent molecular weight of approx. 40 kDa in SDS-polyacrylamide gels. However, complementation of theH. pylori mutant using the clonedrecA gene on a shuttle vector resulted in a RecA protein of the original size and fully restored the general functions of the enzyme. These data can be best explained by a modification of RecA inH. pylori which is crucial for its function. The potential modification seems not to occur when the protein is produced inE. coli, giving rise to a smaller but inactive protein.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Transposition of the kanamycin-resistance transposon Tn903

Summary The insertion of the kanamycin-resistance transposon, Tn903, into the Escherichia coli chromosome was studied. Tn903 is similar in structure to the well known transposons Tn5 and Tn10 in that it has a unique central sequence flanked by inverted repeat sequences extending more than a thousand base pairs. However, the central region of Tn903 has enough single-frame coding capacity only for the drug modifying enzyme, whereas Tn5 and Tn10 carry multigenic unique sequences. In this paper we demonstrate that two different classes of insertion event occur: (1) the first class is a complex event in which all or part of the genome of the bacteriophage lambda vector is co-inserted near the purE locus on the E. coli chromosome (11.7 min); (2) the second class appears to be a simple transposition event in which the transposon alone is inserted at relatively nonspecific sites in the chromosome, as has been described for Tn5 and Tn10. Furthermore both classes show dependency on homology-requiring recombination systems. We suggest that Tn903 transposes infrequently because it must utilize a recA-controlled host function, whereas Tn5 and Tn10 are recA-independent and encode similar but more active functions on the transposon DNA.     

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 pheno-type 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.     

Quantitative evaluation of recA gene expression in Escherichia coli

Summary A recA::lac operon fusion was constructed using the phage Mu d(Ap, lac) in Escherichia coli to obtain precise measurements of the level of recA gene expression in various genetic backgrounds. The RecA protein normally represents 0.02% of total protein. This value is known to increase dramatically after treatments interrupting DNA synthesis; kinetic experiments showed that the rate of recA expression increases 17-fold within 10 min after UV irradiation or thymine starvation. In mutants affected in SOS regulation or repair the following observations were made: (i) the tif-1 mutation in the recA gene does not alter the basal level of recA expression, suggesting that it improves the protease activity of RecA; (ii) the lexA3 mutation does not create a super-repressor of recA; (iii) the tsl-1 mutation in the lexA gene makes the LexA protein a poor repressor of recA at 30°C (2.5-fold derepression) and a poor substrate for RecA protease (3-fold stimulation of recA expression by UV); (iv) the spr-55 amber mutation in the lexA gene causes a 30-fold increase in recA expression, higher than all inducing treatments, and this level cannot be further increased by nalidixic acid; (v) the zab-53 mutation at the recA locus, known to abolish tsl-mediated induction of recA expression, is trans-recessive and thus probably affects a regulatory site on the DNA; (vi) uvrA, B and C, recB and recF mutations do not increase the basal level of recA expression, suggesting that there are not sufficient spontaneous lesions to cause induction even when any one of these three repair pathways is inoperative.Abbreviations Ap ampicillin - Km kanamycin - Cm chloramphenicol - Tc terracycline - Sm streptomycin - Ts thermosensitive - Tr thermoresistant - Nal nalidixic acid - X-Gal 5-bromo-4-chloro-3-indolyl--D-galactoside - mito C mitomycin C - LFT low frequency transducing - HFT high frequency transducing     

Conferral of transposable properties to a chromosomal gene in Escherichia coli

A normally stable gene of Escherichia coli was converted into a transposable element. A bacterial strain was constructed in which the malK gene was flanked on each side by the transposable element Tn5. The resulting Tn5-malK⁺-Tn5 structure (Tn651) became a transposable element with properties very similar to those of Tn5 itself. Tn651 transposes into regions of both the E. coli chromosome and bacteriophage lambda and is able to induce mutations. Transposition of Tn651 does not require the product of recA. Based on a physical analysis of lambda Tn651 DNA it is shown that the two Tn5s flanking the malK gene are in inverted orientation. In these experiments a new derivative of bacteriophage lambda is used that can accept a 14 kilobase insertion in vivo and still yield a plaque-forming transducing particle.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

Gene sequence of recA+ and construction of recA mutants of Vibrio cholerae

The recA ⁺ gene of Vibrio cholerae O1 has been cloned, its nucleotide sequence determined and the product characterized. A deletion mutation was constructed in the recA gene and mutants showed the typical sensitivity to UV and to DNA-damaging agents, as well as an inability to mediate homologous DNA recombination. The chromosomal recA deletion mutants in V. cholerae do not show altered virulence in the infant mouse cholera model and are thus ideal strains for use in complementation studies.