Effect of RecA inactivation and detoxification systems on the evolution of ciprofloxacin resistance in Escherichia coli.

BACKGROUND: Suppression of SOS response and overproduction of reactive oxygen species (ROS) through detoxification system suppression enhance the activity of fluoroquinolones. OBJECTIVES: To evaluate the role of both systems in the evolution of resistance to ciprofloxacin in an isogenic model of Escherichia coli. METHODS: Single-gene deletion mutants of E. coli BW25113 (wild-type) (ΔrecA, ΔkatG, ΔkatE, ΔsodA, ΔsodB), double-gene (ΔrecA-ΔkatG, ΔrecA-ΔkatE, ΔrecA-ΔsodA, ΔrecA-ΔsodB, ΔkatG-ΔkatE, ΔsodB-ΔsodA) and triple-gene (ΔrecA-ΔkatG-ΔkatE) mutants were included. The response to sudden high ciprofloxacin pressure was evaluated by mutant prevention concentration (MPC). The gradual antimicrobial pressure response was evaluated through experimental evolution and antibiotic resistance assays. RESULTS: For E. coli BW25113 strain, ΔkatE, ΔsodB and ΔsodB/ΔsodA mutants, MPC values were 0.25 mg/L. The ΔkatG, ΔsodA, ΔkatG/katE and ΔrecA mutants showed 2-fold reductions (0.125 mg/L). The ΔkatG/ΔrecA, ΔkatE/ΔrecA, ΔsodA/ΔrecA, ΔsodB/ΔrecA and ΔkatG/ΔkatE/ΔrecA strains showed 4-8-fold reductions (0.03-0.06 mg/L) relative to the wild-type. Gradual antimicrobial pressure increased growth capacity for ΔsodA and ΔsodB and ΔsodB/ΔsodA mutants (no growth in 4 mg/L) compared with the wild-type (no growth in the range of 0.5-2 mg/L). Accordingly, increased growth was observed with the mutants ΔrecA/ΔkatG (no growth in 2 mg/L), ΔrecA/ΔkatE (no growth in 2 mg/L), ΔrecA/ΔsodA (no growth in 0.06 mg/L), ΔrecA/ΔsodB (no growth in 0.25 mg/L) and ΔrecA/ΔkatG/ΔkatE (no growth in 0.5 mg/L) compared with ΔrecA (no growth in the range of 0.002-0.015 mg/L). CONCLUSIONS: After RecA inactivation, gradual exposure to ciprofloxacin reduces the evolution of resistance. After suppression of RecA and detoxification systems, sudden high exposure to ciprofloxacin reduces the evolution of resistance in E. coli.

No effect of recombinase-mediated DNA transfer on production efficiency of transgenic rats.

It was reported that recombinase-A protein (RecA)-coated exogenous DNA was more likely to be integrated into mouse, goat and pig genomes. The objective of this study was to investigate whether integration of exogenous DNA into the rat genome is improved by the recombinase-mediated DNA transfer. Pronuclear microinjection of RecA-coated EGFP or OAMB DNA resulted in a production efficiency of transgenic rats of 1.4-2.9%, comparable with 0.9-2.6% when non-coated control DNA was used. Intracytoplasmic injection of the sperm heads exposed to RecA-coated EGFP DNA did not produce any transgenic rats (0 vs. 0-2.8% in control groups). Thus, the recombinase-mediated DNA transfer contributed very little to the production of transgenic rats by means of pronuclear microinjection and intracytoplasmic sperm injection.

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair.

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated lambda red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA(+) or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD(+) allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged lambda DNA and that its anti-recombinogenic activity is operative at elevated levels only.

Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage.

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

DNA strand exchange mediated by the Escherichia coli RecA protein initiates in the minor groove of double-stranded DNA.

The Escherichia coli RecA protein can recognize sequence homology between a single-stranded DNA (ssDNA) and homologous double-stranded DNA (dsDNA). One model for the homology recognition invokes a DNA triplex intermediate in which specific hydrogen bonds connect the ssDNA with groups in the major groove of dsDNA. Using photo-cross-linking methods, we have analyzed the arrangement of DNA strands after the local strand exchange. The results showed that the displaced strand sits in the major groove of the hybrid duplex product. This arrangement indicates that the ssDNA invades the minor groove of dsDNA and hence argues against the involvement of triplex intermediates. The results support an alternative model for the homology recognition that invokes melting of the dsDNA and annealing of the one strand to the invading ssDNA.

Conformations of three-stranded DNA structures formed in presence and in absence of the RecA protein.

Using FTIR and UV spectroscopies, we have studied the structures of three-stranded DNA complexes (TSC) having two identical strands, containing all four bases, in parallel orientation. In the first system, an intermolecular TSC is formed by the addition of the third strand (ssDNA) previously coated with RecA protein to an hairpin duplex (dsDNA), in presence of ATP gamma S. In the second one, the formation of an intramolecular triplex is forced by folding back twice on itself an oligonucleotide. The sequences of the three strands are the same in both systems. The formation of the RecA-TSC, which accommodates all four bases, is evidenced by gel retardation assay, and by its biphasic melting profile observed by UV spectroscopy. Using FTIR spectroscopy, N-type sugars are detected in this structure. This shows that in the RecA-TSC studied in presence of the protein, the nucleic acid part adopts an extended form, in agreement with the model proposed by Zhurkin et al. (1,2) and electron microscopy observations (3-6). In contrast, the RecA-free intramolecular triplex in a non extended form has S-type sugars.

Inhibition of chloroplast DNA recombination and repair by dominant negative mutants of Escherichia coli RecA.

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involved or their biological significance. The endosymbiotic origin of plastids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, suggest that the plastid recombination system is related to its eubacterial counterpart. Therefore, we examined whether dominant negative mutants of the E. coli RecA protein can interfere with the activity of their putative homolog in the chloroplast of the unicellular green alga Chlamydomonas reinhardtii. Transformants expressing these mutant RecA proteins showed reduced survival rates when exposed to DNA-damaging agents, deficient repair of chloroplast DNA, and diminished plastid DNA recombination. These results strongly support the existence of a RecA-mediated recombination system in chloroplasts. We also found that the wild-type E. coli RecA protein enhances the frequency of plastid DNA recombination over 15-fold, although it has no effect on DNA repair or cell survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather than by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothesis could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of their genomes.

Membrane permeability and sensitivity to lethal heat are affected by lexA and recA mutations in Escherichia coli K12

Membrane permeability was evaluated in several SOS-deficient strains. Great heat sensitivity was observed in all the lexA (Ind-) strains, which was associated to an increase in membrane permeability (up to 120 per cent increase above the wild-type control), as assayed by the crystal violet (CV) growth inhibition. After irradiation with a single UV dose (75 J.m-2 delivered to wild-type and 2 J.m-2 to the lex A3 strain), survival was followed by plating cells in both nutrient and membrane permeability-selective (nutrient + CV) media and a great lethality due to CV was observed in a lexA mutant, which appeared to be about 100 times more sensitive to CV compared to its wild-type parent stain. The decreased membrane integrity found in the lex A-deficient strains suggests that LexA protein and/or LexA-repressed genes may interact with the bacterial membrane, which could be the location of SOS events

The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein.

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 delta(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

RecA protein-facilitated DNA strand breaks. A mechanism for bypassing DNA structural barriers during strand exchange.

RecA protein promotes an unexpectedly efficient DNA strand exchange between circular single-stranded DNA and duplex DNAs containing short (50-400-base pair) heterologous sequences at the 5' (initiating) end. The major mechanism by which this topological barrier is bypassed involves DNA strand breakage. Breakage is both strand and position specific, occurring almost exclusively in the displaced (+) strand of the duplex within a 15-base pair region of the heterology/homology junction. Breakage also requires recA protein, ATP hydrolysis, and homologous sequences 3' to the heterology. Although the location of the breaks and the observed requirements clearly indicate a major role for recA protein in this phenomenon, the molecular mechanism is not yet clear. The breakage may reflect a DNA structure and/or some form of structural stress within the DNA during recA protein-mediated DNA pairing which either exposes the DNA at this precise position to the action of a contaminating nuclease or induces a direct mechanical break. We also find that when heterology is located at the 3' end of the linear duplex, strand exchange is halted (without DNA breakage) about 500 base pairs from the homology/heterology junction.

RecA protein-promoted homologous pairing between duplex molecules: functional role of duplex regions of gapped duplex DNA.

RecA protein promotes homologous pairing and symmetrical strand exchange between partially single-stranded duplex DNA and fully duplex molecules. We constructed circular gapped DNA with a defined gap length and studied the pairing reaction between the gapped substrate and fully duplex DNA. RecA protein polymerizes onto the single-stranded and duplex regions of the gapped DNA to form a nucleoprotein filament. The formation of such filaments requires a stoichiometric amount of RecA protein. Both the rate and yield of joint molecule formation were reduced when the pairing reaction was carried out in the presence of a sub-saturating amount of RecA protein. The amount of RecA protein required for optimal pairing corresponds to the binding site size of RecA protein at saturation on duplex DNA. The result suggests that in the 4-stranded system the single-stranded as well as the duplex regions are involved in pairing. By using fully duplex DNA that shares different lengths and regions of homology with the gapped molecule, we directly showed that the duplex region of the gapped DNA increased both the rate and yield of joint molecule formation. The present study indicates that even though strand exchange in the 4-stranded system must require the presence of a single-stranded region, the pairing that occurs in duplex regions between DNA molecules is functionally significant and contributes to the overall activity of the gapped DNA.

Caffeine-induced reduction of the survival of gamma-irradiated HeLa cells and the reversal of the caffeine effect by Escherichia coli RecA protein.

It is confirmed that survival of gamma-irradiated HeLa cells is decreased by post-treatment with caffeine. The caffeine effect is believed to be the result of an inhibition of the repair of gamma-ray-induced DNA damage. In this work we show that the caffeine-induced reduction of the survival of gamma-irradiated HeLa cells is reversed when Escherichia coli RecA protein is introduced into the cells with the aid of liposomes.

[The restoration by Escherichia coli RecA protein of the survival of gamma-irradiated HeLa cells reduced by the DNA repair inhibitors caffeine and 3-aminobenzamide]. / Vosstanovlenie RecA-belkom Escherichia coli vyzhivaemosti gamma-obluchennykh kletok HeLa, snizhennoi ingibitorami reparatsii DNK kofeinom i 3-aminobenzamidom.

It is confirmed that inhibitors of DNA repair caffeine and 3-aminobenzamide decrease the survival of gamma-irradiated HeLa cells. It is shown that the decreased survival of irradiated cells is reversed when Escherichia coli RecA protein is introduced into cell nucleases with the aid of liposomes. This effect is more expressed in caffeine-treated (before or after irradiation) than in 3-aminobenzamide-treated (before irradiation) cells. It is suggested that E. coli 38 kD RecA protein may compensate the function of HeLa RecA-like protein, inhibited by DNA repair inhibitors, which is necessary for the repair of single-strand breaks and double-strand breaks of DNA.

RecA protein promoted homologous pairing in vitro. Pairing between linear duplex DNA bound to HU Protein (nucleosome cores) and nucleoprotein filaments of recA protein-single-stranded DNA.

RecA protein promotes two distinct types of synaptic structures between circular single strands and duplex DNA; paranemic joints, where true intertwining of paired strands is prohibited and the classically intertwined plectonemic form of heteroduplex DNA. Paranemic joints are less stable than plectonemic joints and are believed to be the precursors for the formation of plectonemic joints. We present evidence that under strand exchange conditions the binding of HU protein, from Escherichia coli, to duplex DNA differentially affects homologous pairing in vitro. This conclusion is based on the observation that the formation of paranemic joint molecules was not affected, whereas the formation of plectonemic joint molecules was inhibited from the start of the reaction. Furthermore, introduction of HU protein into an ongoing reaction stalls further increase in the rate of the reaction. By contrast, binding of HU protein to circular single strands has neither stimulatory nor inhibitory effect. Since the formation of paranemic joint molecules is believed to generate positive supercoiling in the duplex DNA, we have examined the ability of positive superhelical DNA to serve as a template in the formation of paranemic joint molecules. The inert positively supercoiled DNA could be converted into an active substrate, in situ, by the action of wheat germ topoisomerase I. Taken collectively, these results indicate that the structural features of the bacterial chromosome which include DNA supercoiling and organization of DNA into nucleosome-like structures by HU protein modulate homologous pairing promoted by the nucleoprotein filaments of recA protein single-stranded DNA.

Homologous pairing and the formation of nascent synaptic intermediates between regions of duplex DNA by RecA protein.

The RecA protein from E. coli gains access to duplex DNA, by nucleation from a short single-stranded gap, to form a spiral nucleoprotein filament that is capable of interaction with homologous duplex DNA. The observations described here demonstrate that any part of the nucleoprotein filament, whether it contains single- or double-stranded DNA, is capable of pairing with homologous duplex DNA. Homologous contacts between regions of duplex DNA lead to an increase in the initial rate and final extent of joint molecule formation. The experiments indicate that pairing is facilitated by the formation of nascent synaptic intermediates between duplex DNA sequences. Using chimeric form I DNA, which is incapable of forming an inter-wound or plectonemic joint with the gapped DNA due to the presence of flanking heterologous sequences, we show that these duplex-duplex pairing reactions involve extensive underwinding of the double helix.

Tn10 transposition promotes RecA-dependent induction of a lambda prophage.

We present evidence that Tn10 transposition, or a closely correlated event, induces expression of bacterial SOS functions. We have found that lambda prophage induction is increased in Escherichia coli lambda lysogens containing increased Tn10 transposase function plus single or multiple copies of an appropriate pair of transposon ends. This increase occurs by the normal pathway for prophage induction, which involves RecA-mediated cleavage of the phage lambda repressor protein. We also present evidence that Tn10 promotes induction of expression of the E. coli sfiA gene. Tn10 transposes by a nonreplicative mechanism. We propose that the signal for RecA protease activation and SOS induction is generated by degradation of the transposon donor molecule and suggest that SOS induction is biologically important in helping a cell undergoing transposition to repair and/or recover from damage to the transposon donor chromosome.

UmuD mutagenesis protein of Escherichia coli: overproduction, purification, and cleavage by RecA.

The mutation rate of Escherichia coli increases approximately 100-fold after treatment with replication-inhibiting agents such as UV light. This enhanced mutation rate requires the action of the UmuD and UmuC proteins, which are induced as part of the SOS response to DNA damage. To initiate a biochemical characterization of the role of these proteins, we have developed a plasmid system that gives efficient expression of the umuD and umuC genes. The umuD and umuC genes were placed under the control of a regulated phage lambda PL promoter and a synthetic ribosome-binding site, and the distance to the UmuD start was adjusted to maximize gene expression. Starting from this overproduction system, we have purified the UmuD protein and studied its interaction with RecA. The SOS response is turned on by the capacity of RecA protein to mediate cleavage of the LexA repressor for SOS-controlled operons. Others have shown that UmuD exhibits sequence homology to LexA around the cleavage site, suggesting a possible cleavage reaction for UmuD. We show that RecA mediates cleavage of UmuD, probably at this site. As with LexA, UmuD also undergoes a self-cleavage reaction. We infer that RecA-mediated cleavage of UmuD is another role for RecA in SOS mutagenesis, probably activating UmuD for its mutagenic function.

RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.

rec-A protein-promoted recombination reaction consists of two independent processes, homologous matching and processive unwinding. A study involving an anti-rec-A protein-monoclonal IgG.

recA protein promotes the formation and processing of joint molecules of homologous double-stranded DNA and single-stranded DNA. We studied the effects of an anti-recA protein monoclonal IgG (ARM193) on two processes carried out by the recA protein. The homologous matching, i.e. pairing of double-stranded DNA and single-stranded DNA by forming intermolecular base-pairing at homologous regions was found to occur even in the presence of an excess amount of antibody ARM193. On the other hand, processive unwinding, i.e. the propagation of the unwinding of double-stranded DNA through a processive reaction of recA protein, which occurs even in the absence of single-stranded DNA, was found to be very sensitive to the inhibition by antibody ARM193. Therefore, we conclude that homologous matching and processive unwinding are independent of each other. Analysis of the effect of antibody ARM193 on the various activities of recA protein suggests that the entire reaction of the formation of joint molecules and their processing can be rationalized in terms of these two underlying processes, homologous matching and processive unwinding. This analysis also suggests that homologous matching seems to require only the binding itself of active units of recA protein to single-stranded DNA but not necessarily either the cooperativity of the protein or unwinding.