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RecA protein mediates homologous recombination repair in bacteria through assembly of long helical filaments on ssDNA in an ATP-dependent manner. RecX, an important negative regulator of RecA, is known to inhibit RecA activity by stimulating the disassembly of RecA nucleoprotein filaments. Here we use a single-molecule approach to address the regulation of ( Escherichia coli ) RecA-ssDNA filaments by RecX ( E. coli ) within the framework of distinct conformational states of RecA-ssDNA filament. Our findings revealed that RecX effectively binds the inactive conformation of RecA-ssDNA filaments and slows down the transition to the active state. Results of this work provide new mechanistic insights into the RecX-RecA interactions and highlight the importance of conformational transitions of RecA filaments as an additional level of regulation of its biological activity.

Bacteria often use multiple transcription factors to regulate specific biological processes. Biosynthesis of heat-stable antifungal factor (HSAF) is regulated by multiple factors in Lysobacter enzymogenes . However, the mechanism of HSAF biosynthesis regulation remains largely unknown. In this study, we screened a potential HSAF biosynthesis regulator, RecX, by a DNA pull-down assay. Deletion of recX resulted in a significant increase in the production of HSAF, and overexpression of recX significantly suppressed HSAF production. Importantly, our results showe that RecX directly binds to the promoter region of the lafB gene to inhibit its transcription and thus decreases HSAF production in L. enzymogenes . These findings reveal the novel mechanism of RecX regulation of antifungal antibiotic production in L. enzymogenes .

Male genitourinary tract (MGT) bacterial infections are considered responsible for 15% of male infertility, but the mechanisms underlying decreased semen quality are poorly known. We evaluated in vitro the effect of strains of Gram-negative uropathogenic species (two E.coli strains, three K. pneumoniae strains, P. aeruginosa and E. cloacae ) on motility, viability, mitochondrial oxidative status, DNA fragmentation and caspase activity of human spermatozoa. All strains, except P. aeruginosa , reduced significantly sperm motility, with variable effects. Sperm Immobilizing Factor (SIF) was largely responsible for deteriorating effects on sperm motility of E. coli strains since they were completely reverted by knockout of SIF coding recX gene. Sequence alignment for RecX showed the presence of high homologous sequences in K. pneumoniae and E. cloacae but not in P. aeruginosa. These results suggest that, in addition to E.coli , other common uropathogenic Gram-negative bacteria affect sperm motility through RecX products. In addition to sperm motility, the E. coli strain ATCC 35218 also affected sperm viability, and induced caspase activity, oxidative stress and DNA fragmentation suggesting an interspecies variability in the amount and/or type of the produced spermatotoxic factors. In general, our results highlight the need for a careful evaluation of semen infections in the diagnostic process of the infertile man.

ABSTRACT The RecA protein is a recombinase functioning in recombinational DNA repair in bacteria. RecA is regulated at many levels. The expression of the recA gene is regulated within the SOS response. The activity of the RecA protein itself is autoregulated by its own C-terminus. RecA is also regulated by the action of other proteins. To date, these include the RecF, RecO, RecR, DinI, RecX, RdgC, PsiB, and UvrD proteins. The SSB protein also indirectly affects RecA function by competing for ssDNA binding sites. The RecO and RecR, and possibly the RecF proteins, all facilitate RecA loading onto SSB-coated ssDNA. The RecX protein blocks RecA filament extension, and may have other effects on RecA activity. The DinI protein stabilizes RecA filaments. The RdgC protein binds to dsDNA and blocks RecA access to dsDNA. The PsiB protein, encoded by F plasmids, is uncharacterized, but may inhibit RecA in some manner. The UvrD helicase removes RecA filaments from RecA. All of these proteins function in a network that determines where and how RecA functions. Additional regulatory proteins may remain to be discovered. The elaborate regulatory pattern is likely to be reprised for RecA homologues in archaeans and eukaryotes.

The RecX protein has attracted considerable interest because the recX mutants exhibit multiple phenotypes associated with RecA functions. To further our understanding of the functional relationship between recA and recX , the effect of different stress treatments on their expression profiles, cell yield and viability were investigated. A significant correlation was found between the expression of Mycobacterium smegmatis recA and recX genes at different stages of growth, and in response to different stress treatments albeit recX exhibiting lower transcript and protein abundance at the mid-log and stationary phases of the bacterial growth cycle. To ascertain their roles in vivo , a targeted deletion of the recX and recArecX was performed in M . smegmatis . The growth kinetics of these mutant strains and their sensitivity patterns to different stress treatments were assessed relative to the wild-type strain. The deletion of recA affected normal cell growth and survival, while recX deletion showed no significant effect. Interestingly, deletion of both recX and recA genes results in a phenotype that is intermediate between the phenotypes of the ΔrecA mutant and the wild-type strain. Collectively, these results reveal a previously unrecognized role for M . smegmatis recX and support the notion that it may regulate a subset of the yet unknown genes involved in normal cell growth and DNA-damage repair.

Gonorrhea is a sexually transmitted infectious disease of the human genital and nasopharyngeal mucosa caused by the host-restricted bacterium Neisseria gonorrhoeae . The rise of antibiotic-resistant gonorrhea is an urgent global threat to public health. Pilus antigenic variation is a gene conversion process that allows N. gonorrhoeae to evade host immune surveillance, and a mechanistic understanding of this process is crucial to understanding N. gonorrhoeae pathogenesis. This report shows that we can adopt a digital PCR methodology to quickly and accurately measure pilin antigenic variation.

Multidrug-resistant Mycobacterium tuberculosis ( Mtb ) infection seriously endangers global human health, creating an urgent need for new treatment strategies. Efficient genome editing tools can facilitate identification of key genes and pathways involved in bacterial physiology, pathogenesis, and drug resistance mechanisms, and thus contribute to the development of novel treatments for drug-resistant tuberculosis . Here, we report a two-plasmid system, MtbCBE , used to inactivate genes and introduce point mutations in Mtb . In this system, the assistant plasmid pRecX-NucS E107A expresses RecX and NucS E107A to repress RecA-dependent and NucS-dependent DNA repair systems, and the base editor plasmid pCBE expresses a fusion protein combining cytidine deaminase APOBEC1, Cas9 nickase (nCas9), and uracil DNA glycosylase inhibitor (UGI). Together, the two plasmids enabled efficient G:C to A:T base pair conversion at desired sites in the Mtb genome. The successful development of a base editing system will facilitate elucidation of the molecular mechanisms underlying Mtb pathogenesis and drug resistance and provide critical inspiration for the development of base editing tools in other microbes.

Background To control the overpopulation and unintended pregnancies, vaginal contraceptives have gained recent surge of interest because of its topical application with possible avoidance of systemic effects. However non-specific cytotoxicity associated with detergent-based synthetic vaginal contraceptive agents limits their use and generates considerable interest in the development of vaginal contraceptives of biological origin for controlling reproduction and ultimately growing population. In this study, we have cloned, over-expressed an Escherichia coli gene encoding a sperm immobilizing factor (SIF) that inhibits sperm motility for the development of vaginal contraceptive from a biological source i.e. E. coli . The contraceptive efficacy of the Escherichia coli recombinant sperm immobilizing factor (r-SIF) was also determined. Methods Genomic DNA library of an E. coli strain isolated from semen sample of an infertile male was constructed for the identification and cloning of E. coli SIF coding gene. This gene was sub-cloned in pBADmycHisB for over-expression and the r-SIF was purified using Ni-NTA affinity chromatography. Effect of r-SIF on mouse sperm motility, viability and on morphology was evaluated. Binding of r-SIF to mouse sperm was demonstrated by fluorescent labeling. Contraceptive efficacy of r-SIF was checked in murine model. Results Genomic library resulted in five hundred transformants; five clones were found positive for sperm immobilizing activity. The protein product of the insert DNA sequence in one of the transformants showed maximum sperm immobilizing activity. Sequence analysis of ORFs in the insert revealed homology to recX on both nucleotide and protein level. 40 μg of the purified r-SIF showed immediate spermicidal activity in vitro for mouse sperm. Scanning electron micrograph of the r-SIF treated sperm showed intense morphological damage to sperm. FITC labeled r-SIF showed highest fluorescence at the head region of the sperm. 5 μg of purified r-SIF exhibited a complete contraceptive effect in mouse model. Conclusion r-SIF could be seen as potential target to be developed as potent and safe vaginal contraceptive in future.

Enterohemorrhagic Escherichia coli (EHEC) can cause severe diarrheic in humans. To improve therapy options, a better understanding of EHEC pathogenicity is essential. The genetic manipulation of EHEC with classical one-step methods, such as the transient overexpression of the phage lambda (λ) Red functions, is not very efficient. Here, we provide a robust and reliable method for increasing recombineering efficiency in EHEC based on the transient coexpression of recX together with gam, beta, and exo. We demonstrate that the genetic manipulation is 3–4 times more efficient in EHEC O157:H7 EDL933 Δstx1/2 with our method when compared to the overexpression of the λ Red functions alone. Both recombineering systems demonstrated similar efficiencies in Escherichia coli K-12 MG1655. Coexpression of recX did not enhance the Gam-mediated inhibition of sparfloxacin-mediated SOS response. Therefore, the additional inhibition of the RecFOR pathway rather than a stronger inhibition of the RecBCD pathway of SOS response induction might have resulted in the increased recombineering efficiency by indirectly blocking phage induction. Even though additional experiments are required to unravel the precise mechanistic details of the improved recombineering efficiency, we recommend the use of our method for the robust genetic manipulation of EHEC and other prophage-carrying E. coli isolates.

The RecA recombinase of Escherichia coli has not evolved to optimally promote DNA pairing and strand exchange, the key processes of recombinational DNA repair. Instead, the recombinase function of RecA protein represents an evolutionary compromise between necessary levels of recombinational DNA repair and the potentially deleterious consequences of RecA functionality. A RecA variant, RecA D112R, promotes conjugational recombination at substantially enhanced levels. However, expression of the D112R RecA protein in E. coli results in a reduction in cell growth rates. This report documents the consequences of the substantial selective pressure associated with the RecA-mediated hyperrec phenotype. With continuous growth, the deleterious effects of RecA D112R, along with the observed enhancements in conjugational recombination, are lost over the course of 70 cell generations. The suppression reflects a decline in RecA D112R expression, associated primarily with a deletion in the gene promoter or chromosomal mutations that decrease plasmid copy number. The deleterious effects of RecA D112R on cell growth can also be negated by over-expression of the RecX protein from Neisseria gonorrhoeae. The effects of the RecX proteins in vivo parallel the effects of the same proteins on RecA D112R filaments in vitro. The results indicate that the toxicity of RecA D112R is due to its persistent binding to duplex genomic DNA, creating barriers for other processes in DNA metabolism. A substantial selective pressure is generated to suppress the resulting barrier to growth.