recA Mutants Research Articles

High-Throughput Screening Method Using Escherichia coli Keio Mutants for Assessing Primary Damage Mechanism of Antimicrobials.

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Prevalent role of homologous recombination in the repair of specific double-strand breaks in Rhizobium etli.

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.

Rec-1 loss of function increases recombination in the central gene clusters at the expense of autosomal pairing centers.

Meiotic control of crossover (CO) number and position is critical for homologous chromosome segregation and organismal fertility, recombination of parental genotypes, and the generation of novel genetic combinations. We here characterize the recombination rate landscape of a rec-1 loss of function modifier of CO position in Caenorhabditis elegans, one of the first ever modifiers discovered. By averaging CO position across hermaphrodite and male meioses and by genotyping 203 single-nucleotide variants covering about 95% of the genome, we find that the characteristic chromosomal arm-center recombination rate domain structure is lost in the loss of function rec-1 mutant. The rec-1 loss of function mutant smooths the recombination rate landscape but is insufficient to eliminate the nonuniform position of CO. Lower recombination rates in the rec-1 mutant are particularly found in the autosomal arm domains containing the pairing centers. We further find that the rec-1 mutant is of little consequence for organismal fertility and egg viability and thus for rates of autosomal nondisjunction. It nonetheless increases X chromosome nondisjunction rates and thus male appearance. Our findings question the maintenance of recombination rate heritability and genetic diversity among C. elegans natural populations, and they further suggest that manipulating genetic modifiers of CO position will help find quantitative trait loci located in low-recombining genomic regions normally refractory to discovery.

Distinct Screening Approaches Uncover PA14_36820 and RecA as Negative Regulators of Biofilm Phenotypes in Pseudomonas aeruginosa PA14.

Pseudomonas aeruginosa commonly infects hospitalized patients and the lungs of individuals with cystic fibrosis. This species is known for forming biofilms, which are communities of bacterial cells held together and encapsulated by a self-produced extracellular matrix. The matrix provides extra protection to the constituent cells, making P. aeruginosa infections challenging to treat. We previously identified a gene, PA14_16550, which encodes a DNA-binding TetR-type repressor and whose deletion reduced biofilm formation. Here, we assessed the transcriptional impact of the 16550 deletion and found six differentially regulated genes. Among them, our results implicated PA14_36820 as a negative regulator of biofilm matrix production, while the remaining 5 had modest effects on swarming motility. We also screened a transposon library in a biofilm-impaired ΔamrZ Δ16550 strain for restoration of matrix production. Surprisingly, we found that disruption or deletion of recA increased biofilm matrix production, both in biofilm-impaired and wild-type strains. Because RecA functions both in recombination and in the DNA damage response, we asked which function of RecA is important with respect to biofilm formation by using point mutations in recA and lexA to specifically disable each function. Our results implied that loss of either function of RecA impacts biofilm formation, suggesting that enhanced biofilm formation may be one physiological response of P. aeruginosa cells to loss of either RecA function. IMPORTANCE Pseudomonas aeruginosa is a notorious human pathogen well known for forming biofilms, communities of bacteria that protect themselves within a self-secreted matrix. Here, we sought to find genetic determinants that impacted biofilm matrix production in P. aeruginosa strains. We identified a largely uncharacterized protein (PA14_36820) and, surprisingly, RecA, a widely conserved bacterial DNA recombination and repair protein, as negatively regulating biofilm matrix production. Because RecA has two main functions, we used specific mutations to isolate each function and found that both functions influenced matrix production. Identifying negative regulators of biofilm production may suggest future strategies to reduce the formation of treatment-resistant biofilms.

Genotoxic hazard and oxidative stress induced by wastewater irrigated soil with special reference to pesticides and heavy metal pollution

Due to enhancement of industrial growth and urbanization, soil contamination is increasing prominently. Therefore, it is important to examine possible adverse effects of industrial waste. Soil samples were might to be polluted with several heavy-metals and pesticides. Gas chromatographic results showed occurrence of high-level of organochlorine and organophosphate pesticides in studied soil samples. Genotoxicity of soil extracts was assessed using environmental-risk assessment models. Soil samples were extracted in hexane and dichloromethane solvents and were evaluated for genotoxic potential by prokaryotic (Ames test, plasmid nicking assay and E. coli K-12 DNA repair defective mutants) and eukaryotic (Allium cepa root chromosomal aberration and Vigna radiata seed-germination test) bioassays. Strain TA98 was found the most susceptible among soil extracts. The mutagenicity of hexane soil extract from wastewater irrigation was found to be higher than that of DCM samples in terms of mutagenic index, mutagenic potential, and induction factor for Ames strains. The damage in DNA repair defective mutants of hexane extracts were found higher compared to DCM extracts at dose of 20 μl/ml of culture. Survival in polA, lexA and recA mutants were 39%, 47% and 55% while treated with hexane extract. Allium cepa test, mitotic index was decreased in dose-dependent way and various kinds of chromosomal aberrations were found. Vigna radiata seeds germination and other parameters were also affected when treated with wastewater irrigated (WWI) soil. Oxidative stress in V. radiata roots were also showed under CLS microscope. Genotoxicity of WWI soil extract was also confirmed by plasmid nicking test. Our study provides possible explanation for the assessment of potential health and environmental hazards of the industrial region.

How do Streptomyces coordinate DNA repair and cell division following DNA damage?

DNA damage often results in a pause of cell division until damage is repaired. In bacteria, a widely conserved response to DNA damage is the SOS response which relies on two proteins: the multifunctional recombinase RecA and the transcriptional repressor LexA. Under DNA-damaging conditions, this response activates proteins involved in DNA repair and the inhibition of cell division which in most unicellular bacteria results in temporarily filamentous growth. However, it is unknown how naturally filamentous growing bacteria like Streptomyces cope with DNA damage and how DNA damage repair is coordinated with cell division. To identify novel regulators of cell division in Streptomyces that specifically function during DNA damaging growth conditions, we investigated the global response of Streptomyces venezuelae to several genotoxic agents, including mitomycin C, ciprofloxacin and methane methylsulfonate. To this end we have performed ChIP-seq experiments to identify the LexA regulon in Streptomycesandconducted RNA-seq experiments to determine the global response to DNA damaging agents in the wildtype and a recA mutant. The combined analysis of the available data sets has allowed us to obtain a comprehensive overview about the genes involved in the SOS response in Streptomycesand further analysis will enable us to understand how damage repair and cell division are coordinated in these bacteria.

Essential Role for an Isoform of Escherichia coli Translation Initiation Factor IF2 in Repair of Two-Ended DNA Double-Strand Breaks.

In Escherichia coli, three isoforms of the essential translation initiation factor IF2 (IF2-1, IF2-2, and IF2-3) are generated from separate in-frame initiation codons in infB. The isoforms have earlier been suggested to additionally participate in DNA damage repair and replication restart. It is also known that the proteins RecA and RecBCD are needed for repair of DNA double-strand breaks (DSBs) in E. coli. Here, we show that strains lacking IF2-1 are profoundly sensitive to two-ended DSBs in DNA generated by radiomimetic agents phleomycin or bleomycin, or by endonuclease I-SceI. However, these strains remained tolerant to other DSB-generating genotoxic agents or perturbations to which recA and recBC mutants remained sensitive, such as to mitomycin C, type-2 DNA topoisomerase inhibitors, or DSB caused by palindrome cleavage behind a replication fork. Data from genome-wide copy number analyses following I-SceI cleavage at a single chromosomal locus suggested that, in a strain lacking IF2-1, the magnitude of recombination-dependent replication through replication restart mechanisms is largely preserved but the extent of DNA resection around the DSB site is reduced. We propose that in the absence of IF2-1 it is the synapsis of a RecA nucleoprotein filament to its homologous target that is weakened, which in turn leads to a specific failure in assembly of Ter-to-oriC directed replisomes needed for consummation of two-ended DSB repair. IMPORTANCE Double-strand breaks (DSBs) in DNA are major threats to genome integrity. In Escherichia coli, DSBs are repaired by RecA- and RecBCD-mediated homologous recombination (HR). This study demonstrates a critical role for an isoform (IF2-1) of the translation initiation factor IF2 in the repair of two-ended DSBs in E. coli (that can be generated by ionizing radiation, certain DNA-damaging chemicals, or endonuclease action). It is proposed that IF2-1 acts to facilitate the function of RecA in the synapsis between a pair of DNA molecules during HR.

Study of the contribution of active defense mechanisms to ciprofloxacin tolerance in Escherichia coli growing at different rates

Using rpoS, tolC, ompF, and recA knockouts, we investigated their effect on the physiological response and lethality of ciprofloxacin in E. coli growing at different rates on glucose, succinate or acetate. We have shown that, regardless of the strain, the degree of changes in respiration, membrane potential, NAD+/NADH ratio, ATP and glutathione (GSH) strongly depends on the initial growth rate and the degree of its inhibition. The deletion of the regulator of the general stress response RpoS, although it influenced the expression of antioxidant genes, did not significantly affect the tolerance to ciprofloxacin at all growth rates. The mutant lacking TolC, which is a component of many E. coli efflux pumps, showed the same sensitivity to ciprofloxacin as the parent. The absence of porin OmpF slowed down the entry of ciprofloxacin into cells, prolonged growth and shifted the optimal bactericidal concentration towards higher values. Deficiency of RecA, a regulator of the SOS response, dramatically altered the late phase of the SOS response (SOS-dependent cell death), preventing respiratory inhibition and a drop in membrane potential. The recA mutation inverted GSH fluxes across the membrane and abolished ciprofloxacin-induced H2S production. All studied mutants showed an inverse linear relationship between logCFU ml-1 and the specific growth rate. Mutations shifted the plot of this dependence relative to the parental strain according to their significance for ciprofloxacin tolerance. The crucial role of the SOS system is confirmed by dramatic shift down of this plot in the recA mutant.

Potential genes associated with survival of Acinetobacter baumannii under ciprofloxacin stress

Acinetobacter baumannii is an opportunistic pathogen that has acquired resistance to all available drugs. The rise in multi-drug resistance in A. baumannii has been exacerbated by its ability to tolerate antibiotics due to the persister cells, which are phenotypic variants of normal cells that can survive various stress conditions, resulting in chronicity of infection. In the present study we observed that A. baumannii formed persister cells against lethal concentration of ciprofloxacin in exponential phase. The transcriptome of A. baumannii was analyzed after exposure to high concentration of ciprofloxacin (50X MIC) to determine the possible mechanisms of survival. Transcriptome analysis showed differential expression of 146 genes, of which 101 were up-regulated and 45 were down-regulated under ciprofloxacin stress. Differentially expressed genes that might be important for persistence against ciprofloxacin were involved in DNA repair, phenylacetic acid degradation, leucine catabolism, HicAB toxin-antitoxin system and ROS response (iron-sulfur clusters, hemerythrin-like metal binding and Kdp). recA, umuD and ddrR genes involved in SOS response were also up-regulated. Knockout of umuD showed significant decrease in persister cells formation while they were completely eradicated in recA mutant strain. The differentially expressed genes highlighted in the study merit further investigation as therapeutic targets for effective control of A. baumannii infections.

Cytotoxic and genotoxic assessment of agricultural soils from an industrial region.

Industrial effluents contain hazardous substances that can be a serious threat to the agriculture and human health. In the present study, the cytotoxic and genotoxic impacts of agricultural soil from the industrial area of Dera Bassi (Punjab, India) have been evaluated. Assays such as defects in DNA repair in K-12 mutants of Escherichia coli and chromosomal aberrations in Allium cepa were used to estimate the acute toxicity and chromosomal mutagenesis, respectively. Atomic absorption spectrometry and GC-MS analysis revealed contamination of the soil with high concentrations of heavy metals and organic compounds, respectively. Dichloromethane extract of site I soil sample caused maximum damage to 40 μL mL-1 DNA repair defective mutants and showed 38 and 49% survival in lexA and recA mutants, respectively, which was least among all the sites. In A. cepa test, an inverse relationship between soil extract concentration and the mitotic index was observed. Exposure of growing roots of A. cepa to soil extracts induced chromosomal abnormalities and alterations in mitotic phases in root tip cells. The study concludes that agricultural sites near the industrial area were contaminated with genotoxic and mutagenic compounds. Hence, adequate measures should be taken to reduce the toxicity of industrial effluents discharged onto the agricultural fields.

Characterization of gross genome rearrangements in Deinococcus radiodurans recA mutants

Genome stability in radioresistant bacterium Deinococcus radiodurans depends on RecA, the main bacterial recombinase. Without RecA, gross genome rearrangements occur during repair of DNA double-strand breaks. Long repeated (insertion) sequences have been identified as hot spots for ectopic recombination leading to genome rearrangements, and single-strand annealing (SSA) postulated to be the most likely mechanism involved in this process. Here, we have sequenced five isolates of D. radiodurans recA mutant carrying gross genome rearrangements to precisely characterize the rearrangements and to elucidate the underlying repair mechanism. The detected rearrangements consisted of large deletions in chromosome II in all the sequenced recA isolates. The mechanism behind these deletions clearly differs from the classical SSA; it utilized short (4–11 bp) repeats as opposed to insertion sequences or other long repeats. Moreover, it worked over larger linear DNA distances from those previously tested. Our data are most compatible with alternative end-joining, a recombination mechanism that operates in eukaryotes, but is also found in Escherichia coli. Additionally, despite the recA isolates being preselected for different rearrangement patterns, all identified deletions were found to overlap in a 35 kb genomic region. We weigh the evidence for mechanistic vs. adaptive reasons for this phenomenon.

Recombinational repair in the absence of holliday junction resolvases in E. coli.

Cells possess two major DNA damage tolerance pathways that allow them to duplicate their genomes despite the presence of replication blocking lesions: translesion synthesis (TLS) and daughter strand gap repair (DSGR). The TLS pathway involves specialized DNA polymerases that are able to synthesize past DNA lesions while DSGR relies on Recombinational Repair (RR). At least two mechanisms are associated with RR: Homologous Recombination (HR) and RecA Mediated Excision Repair (RAMER). While HR and RAMER both depend on RecFOR and RecA, only the HR mechanism should involve Holliday Junctions (HJs) resolvase reactions. In this study we investigated the role of HJ resolvases, RuvC, TopIII and RusA on the balance between RAMER and HR in E. coli MG1655 derivatives. Using UV survival measurements, we first clearly establish that, in this genetic background, topB and ruvC define two distinct pathways of HJ resolution. We observed that a recA mutant is much more sensitive to UV than the ruvC topB double mutant which is deficient in HR because of its failure to resolve HJs. This difference is independent of RAMER, the SOS system, RusA, and the three TLS DNA polymerases, and may be accounted for by Double Strand Break repair mechanisms such as Synthesis Dependent Strand Annealing, Single Strand Annealing, or Break Induced Replication, which are independent of HJ resolvases. We then used a plasmid-based assay, in which RR is triggered by a single blocking lesion present on a plasmid molecule, to establish that while HR requires topB, ruvC or rusA, RAMER is independent of these genes and, as expected, requires a functional UvrABC excinuclease. Surprisingly, analysis of the RR events in a strain devoid of HJ resolvases reveals that the UvrABC dependent repair of the single lesion present on the plasmid molecule can generate an excision track potentially extending to dozens of nucleotides.

Profiling of the assembly of RecA nucleofilaments implies a potential target for environmental factors to disturb DNA repair

Double-strand breaks (DSBs), one class of the most harmful DNA damage forms that bring elevated health risks, need to be repaired timely and effectively. However, an increasing number of environmental pollutants have been identified to impair DSB repair from various mechanisms. Our previous work indicated that the formation of unsaturated RecA nucleofilaments plays an essential role in homology recombination (HR) pathway which can accurately repair DSBs. In this study, by developing a benzonase cutting protection assay and combining it with traditional electrophoretic mobility shift assay (EMSA) analysis, we further investigated the assembly patterns of four RecA mutants that display differential DSB repair ability and ATPase activity. We observed that the mutants (G204S and S69G) possessing both ATP hydrolysis and DSB repair activities form unsaturated nucleofilaments similar to that formed by the wild type RecA, whereas the other two ATP hydrolysis-deficient mutants (K72R and E96D) that fail to mediate HR form more compacted nucleofilaments in the presence of ATP. These results establish a coupling of ATPase activity and effective DSB repair ability via the assembly status of RecA nucleofilaments. This linkage provides a potential target for environmental factors to disturb the essential HR pathway for DSB repair by suppressing the ATPase activity and altering the assembly pattern of nucleofilaments.

Plasmid DNA Production in Proteome-Reduced Escherichia coli.

The design of optimal cell factories requires engineering resource allocation for maximizing product synthesis. A recently developed method to maximize the saving in cell resources released 0.5% of the proteome of Escherichia coli by deleting only three transcription factors. We assessed the capacity for plasmid DNA (pDNA) production in the proteome-reduced strain in a mineral medium, lysogeny, and terrific broths. In all three cases, the pDNA yield from biomass was between 33 and 53% higher in the proteome-reduced than in its wild type strain. When cultured in fed-batch mode in shake-flask, the proteome-reduced strain produced 74.8 mg L−1 pDNA, which was four times greater than its wild-type strain. Nevertheless, the pDNA supercoiled fraction was less than 60% in all cases. Deletion of recA increased the pDNA yields in the wild type, but not in the proteome-reduced strain. Furthermore, recA mutants produced a higher fraction of supercoiled pDNA, compared to their parents. These results show that the novel proteome reduction approach is a promising starting point for the design of improved pDNA production hosts.

Rec A disruption unveils cross talk between DNA repair and membrane damage, efflux pump activity, biofilm formation in Mycobacterium smegmatis

Tuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) has emerged in recent decades as one of the leading causes of mortality worldwide. The burden of TB is alarmingly high, with one third affected global population as reported by WHO. Short-course treatment with an antibiotic is a powerful weapon to treat infection of susceptible MTB strain, however; MTB has developed resistance to anti-TB drugs, which is an escalating global health crisis. Thus there is urgent need to identify new drug targets. RecA is a 38 kilodalton protein required for the repair and maintenance of DNA and regulation of the SOS response. The objective of this study is to understand the effect of disruption of RecA gene (deletion mutant ΔdisA from previous study) in a surrogate model for MTB, Mycobacterium smegmatis. This study demonstrated that disruption of RecA causes enhanced susceptibility towards rifampicin and generation of ROS leading to lipid peroxidation and impaired membrane homeostasis as depicted by altered cell membrane permeability and efflux pump activity. Mass spectrometry based lipidomic analysis revealed decreased mycolic acid moieties, phosphatidylinositol mannosides (PIM), Phthiocerol dimycocerosate (DIM). Furthermore, biofilm formation was considerably reduced. Additionally, we have validated all the disrupted phenotypes by RT-PCR which showed a good correlation with the biochemical assays. Lastly, RecA mutant displayed reduced infectivity in Caenorhabditis elegans illustrating its vulnerability as antimycobacterial target. Together, present study establishes a link between DNA repair, drug efflux and biofilm formation and validates RecA as an effective drug target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

Engineering E. coli for improved microaerobic pDNA production.

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA-, which, together with W3110recA-vgb+, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

Distinct roles of Deinococcus radiodurans RecFOR and RecA in DNA transformation

RecFOR and RecA are key recombination factors in Deinococcus radiodurans, a bacterium that possesses robust DNA repair capability and is also naturally transformable. While RecFOR functioning as a RecA loader during DNA repair has been established, their relative roles in transformation need further exploration. Here, we constructed recFOR and recA deletion mutants of D. radiodurans, and investigated the effect of these mutations on DNA transformation. recA deletion causes defects in both plasmid and chromosomal transformation. However, it was found that recFOR is not involved in chromosomal transformation, and that only recO and recR mutations compromise plasmid transformation. How recO, recR and recA mutations influence plasmid transformation was further examined by complementation plasmids. Interestingly, the transformation process remains defective in the recA mutant, but gets restored in the recO and recR mutants. This indicates that unlike RecA, RecOR may not be essential for DNA uptake. Therefore, we provide evidence that RecFOR is dispensable for RecA to protect incoming exogenous DNA and to catalyze recombination during transformation. Instead, RecO and RecR are likely to promote later steps in plasmid transformation.

Tannic and gallic acids alter redox-parameters of the medium and modulate biofilm formation.

Tannic (TA) and gallic (GA) acids are known to have both anti- and prooxidant properties however recently they have been described as potential anti-biofilm agents although their mechanisms of action on bacterial cells remain obscure. The aim of our research was to elucidate the role of prooxidant actions of these plant phenolic compounds in bactericidal effects and biofilm formation. In our experiments, both compounds demonstrated strong oxidative properties that altered activity of stress regulons and contributed to decrease of CFU and ability of cells to maintain membrane potential. Stimulation of biofilm formation was observed in all the strains with the exception of the strains deficient in flagella synthesis. Both compounds demonstrated bactericidal effect which was weakened in biofilms. TA efficiently killed bacteria in the bioflms of pgaA mutant which pointed out an important role of poly-beta-1,6-N-acetyl-D-glucosamine (PGA) polysaccharide in matrix formation. Similar effects of TA in recA mutant indicate involvement of SOS-response into reaction towards exposure with TA. Gallic acid-induced killing was more pronounced in the biofilms of csgA mutant revealing role of curli in protection against GA toxicity.

Analysis of Pilin Antigenic Variation in Neisseria meningitidis by Next-Generation Sequencing.

Many pathogenic microbes evade host immune surveillance by varying the surface antigens, a process termed antigenic variation. While the process of pilin antigenic variation has been extensively studied in the human pathogen Neisseria gonorrhoeae (gonococcus [Gc]), relatively few studies of pilin antigenic variation have been conducted with Neisseria meningitidis (meningococcus [Mc]). Mc is usually a commensal organism that colonizes the human nasopharynx, but when it translocates to the bloodstream or meninges, it results in the severe and often deadly meningococcal disease. The type IV pili of Mc isolates play a critical role in host surface adherence, and its major pilin component (PilE) can undergo antigenic variation. In this study, Roche 454 pyrosequencing was used to examine the pilin antigenic variation of Mc strain 8013, as well as 8013 recA, recX, recQ, rep, and recJ mutants, Gc orthologues which have been shown to play a role in pilin antigenic variation. This study confirms that the Mc recA, rep, and recJ genes are essential for pilin antigenic variation. While the Mc recQ and recX gene products contribute to normal frequencies of antigenic variation, the loss of these factors does not alter the types of pilin variants produced. Overall, this study shows that the mechanisms of pilin antigenic variation are conserved between Gc and Mc.IMPORTANCE Antigenic variation is a strategy used by many pathogens to escape host immune surveillance and establish persistent infections. This study successfully applies next-generation sequencing to study pilin antigenic variation in the human pathogen Neisseria meningitidis This assay provides an affordable and efficient solution for quantifying antigenic variation frequency in mutant strains and for defining the recombination products of the process. We determined that there is a nonuniformity of silent donor copies used during meningococcus antigenic variation, and by the analysis of selected mutants deficient for specific recombination pathways, we show for the first time that the processes are conserved between N. meningitidis and Neisseria gonorrhoeae.