RAD51 supports DMC1 by inhibiting the SMC5/6 complex during meiosis.

Meiosis is a fundamental process for sexual reproduction in most eukaryotes and the evolutionarily conserved recombinases RADiation sensitive51 (RAD51) and Disrupted Meiotic cDNA1 (DMC1) are essential for meiosis and thus fertility. The mitotic function of RAD51 is clear, but the meiotic function of RAD51 remains largely unknown. Here we show that RAD51 functions as an interacting protein to restrain the Structural Maintenance of Chromosomes5/6 (SMC5/6) complex from inhibiting DMC1. We unexpectedly found that loss of the SMC5/6 partially suppresses the rad51 knockout mutant in terms of sterility, pollen inviability, and meiotic chromosome fragmentation in a DMC1-dependent manner in Arabidopsis thaliana. Biochemical and cytological studies revealed that the DMC1 localization in meiotic chromosomes is inhibited by the SMC5/6 complex, which is attenuated by RAD51 through physical interactions. This study not only identified the long-sought-after function of RAD51 in meiosis but also discovered the inhibition of SMC5/6 on DMC1 as a control mechanism during meiotic recombination.

Changes in lactic acid bacteria and components of Awa-bancha by anaerobic fermentation.

Awa-bancha is a post-fermented tea produced in Naka and Kamikatsu, Tokushima, Japan. We investigated the lactic acid bacteria in each stage of production of Awa-bancha and evaluated the relationships with the components. Lactic acid bacteria were isolated from tea leaves cultured with de Man, Rogosa, and Sharpe (MRS) agar plates, and the species were identified by homology of the 16 S rRNA gene and multiplex polymerase chain reaction (PCR) of the recA gene to distinguish the Lactobacillus plantarum group. As a result, a variety of species were isolated from the raw tea leaves, and Lactobacillus pentosus was isolated most frequently after anaerobic fermentation. Regarding the tea leaf components, organic acids, such as lactic acid, increased, free amino acids decreased, and catechins changed owing to anaerobic fermentation. Our results suggest that the microbial flora mainly composed of L. pentosus is important in the anaerobic fermentation process for flavor formation of Awa-bancha.

p-Coumaric acid inhibits the Listeria monocytogenes RecA protein functions and SOS response: An antimicrobial target.

Bacterial RecA plays an important role in the evaluation of antibiotic resistance via stress-induced DNA repair mechanism; SOS response. Accordingly, RecA became an important therapeutic target against antimicrobial resistance. Small molecule inhibitors of RecA may prevent adaptation of antibiotic resistance mutations and the emergence of antimicrobial resistance. In our study, we observed that phenolic compound p-Coumaric acid as potent RecA inhibitor. It inhibited RecA driven biochemical activities in vitro such as ssDNA binding, strand exchange, ATP hydrolysis and RecA coprotease activity of E. coli and L. monocytogenes RecA proteins. The mechanism underlying such inhibitory action of p-Coumaric acid involves its ability to interfere with the DNA binding domain of RecA protein. p-Coumaric acid also potentiates the activity of ciprofloxacin by inhibiting drastic cell survival of L. monocytogenes as well as filamentation process; the bacteria defensive mechanism in response to DNA damage. Additionally, it also blocked the ciprofloxacin induced RecA expression leading to suppression of SOS response in L. monocytogenes. These findings revealed that p-Coumaric acid is a potent RecA inhibitor, and can be used as an adjuvant to the existing antibiotics which not only enhance the shelf-life but also slow down the emergence of antibiotic resistance in bacteria.

Association of virulence gene expression with colistin-resistance in Acinetobacter baumannii: analysis of genotype, antimicrobial susceptibility, and biofilm formation.

BACKGROUND: Acinetobacter baumannii causes difficult-to-treat nosocomial infections, which often lead to morbidity due to the development of antimicrobial drug resistance and expression of virulence genes. Data regarding the association of resistance to colistin, a last treatment option, and the virulence gene expression of A. baumannii is scarce. METHODS: We evaluated the MLVA genotype, antimicrobial resistance, and biofilm formation of 100 A. baumannii isolates from burn patients, and further compared the in vitro and in vivo expression of four virulence genes among five colistin-resistant A. baumannii (Cst-R-AB) isolates. Five Cst-R-AB isolates were tested; one from the present study, and four isolated previously. RESULTS: Our results showed that reduced expression of recA, along with increased in vivo expression of lpsB, dnaK, and blsA; are associated with colistin resistance among Cst-R-AB isolates. Differences in virulence gene expressions among Cst-R-AB isolates, may in part explain common discrepant in vitro vs. in vivo susceptibility data during treatment of infections caused by Cst-R-AB. CONCLUSIONS: Our findings highlight the intricate relationship between colistin-resistance and virulence among A. baumannii isolates, and underscore the importance of examining the interactions between virulence and antimicrobial resistance toward efforts to control the spread of multidrug-resistant A. baumannii (MDR-AB) isolates, and also to reduce disease severity in burn patients with MDR-AB infection.

Pararhizobium polonicum sp. nov. isolated from tumors on stone fruit rootstocks.

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different stone fruit rootstocks in Poland: strains F5.1(T) and F5.3 from Prunus avium F12/1, strains CP3.5 and CP17.2.1 from Prunus avium and strain AL5.1.8 from Prunus cerasifera. On the basis of 16S rDNA phylogeny, the strains cluster together and belong to the genus Pararhizobium with type strain of Pararhizobium herbae (99.6-99.8%) as their closest relative. Phylogenetic analysis of the novel strains using housekeeping genes atpD, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to P. herbae. DNA-DNA hybridization of strains F5.1(T), with the type strain of P. herbae LMG 25718(T) and Pararhizobium giardinii R-4385(T) revealed 28.3% and 27.9% of DNA-DNA relatedness, respectively. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Pararhizobium, for which the name Pararhizobium polonicum sp. nov. (type strain F5.1(T)=LMG 28610(T)=CFBP 8359(T)) is proposed.

Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism.

Bacterial cell envelope is generally accepted as the primary target for a photo-induced oxidative stress. It is plausible that DNA damage occurs during the antimicrobial photoinactivation. Here we investigate the correlation between DNA damage and photoinactivation by evaluating the level of RecA-based DNA repair system in Staphylococcus aureus. By using exogenous photosensitizers (new methylene blue (NMB), toluidine blue O (TBO), 5,10,15,20-tetrakis(1-methyl-4-pyridinio)porphyrin tetra(p-toluenesulfonate) (TMPyP), zinc phthalocyanine (ZnPc), Rose Bengal (RB)) and ALA-induced endogenous porphyrin-dependent blue light (405 nm), several outcomes were observed: (i) an increase of DNA damage (from gel electrophoresis in DNA damage assay), (ii) an increase of recA expression (luminescence assay in recA-lux strain), and (iii) an increase of RecA protein level (Western blotting). When recA expression was repressed by novobiocin, or abolished by deleting the gene, S. aureus susceptibility towards photoinactivation was increased at approximately a hundred-fold. The absence of RecA increases DNA damage to yield bactericidal effect. In novobiocin-resistant mutant (gyrB), as opposed to wild type, neither RecA protein level nor cell's susceptibility was affected by photoinactivation (when novobiocin is present). This is to suggest that GyrB-dependent inhibition mediated recA repression. Therefore, we have established the role of RecA in DNA damage during photoinactivation. With the use of rifampicin mutation frequency and Ames tests, we demonstrated that photoinactivation did not increase S. aureus mutagenesis and potentially is not mutagenic toward eukaryotic cells. The results suggest that the treatment is considered safe. In conclusion, we provide an evidence that recA inhibitor may serve as therapeutic adjuvant for antimicrobial photoinactivation. Clinical relevance of our findings warrants further investigations.

[Role of double strand DNA break repair for quinolone sensitivity in Escherichia coli: therapeutic implications]. / El papel de la reparación de roturas de doble cadena de ADN en Escherichia coli en la sensibilidad a quinolonas: implicaciones terapéuticas.

INTRODUCTION: Quinolones are one of the types of antibiotics with higher resistance rates in the last years. At molecular level, quinolones block type II topoisomerases producing double strand breaks (DSBs). These DSBs could play a double role, as inductors of the quinolone bactericidal effects but also as mediators of the resistance and tolerance mechanisms. MATERIAL AND METHODS: In this work we have studied the molecular pathways responsible for DSBs repair in the quinolone susceptibility: the stalled replication fork reversal (recombination-dependent) (RFR), the SOS response induction, the translesional DNA synthesis (TLS) and the nucleotide excision repair mechanisms (NER). For this reason, at the European University in Madrid, we analysed the minimal inhibitory concentration (MIC) to three different quinolones in Escherichia coli mutant strains coming from different type culture collections. RESULTS: recA, recBC, priA and lexA mutants showed a significant reduction on the MIC values for all quinolones tested. No significant changes were observed on mutant strains for TLS and NER. DISCUSSION: These data indicate that in the presence of quinolones, RFR mechanisms and the SOS response could be involved in the quinolone susceptibility.

Graft-transmissible movement of inverted-repeat-induced siRNA signals into flowers.

In plants, small interfering RNAs (siRNA) and microRNAs move to distant tissues where they control numerous developmental and physiological processes such as morphogenesis and stress responses. Grafting techniques and transient expression systems have been employed to show that sequence-specific siRNAs with a size of 21-24 nucleotides traffic to distant organs. We used inverted-repeat constructs producing siRNA targeting the meiosis factor DISRUPTED MEIOTIC cDNA 1 (DMC1) and GFP to test whether silencing signals move into meiotically active tissues. In grafted Nicotiana tabacum, a transgenic DMC1 siRNA signal made in source tissues preferably entered the anthers formed in the first flowers. Here, the DMC1 siRNA interfered with meiotic progression and, consequently, the flowers were at least partially sterile. In agro-infiltrated N. benthamiana plants, a GFP siRNA signal produced in leaves was allocated and active in most flower tissues including anthers. In hypocotyl-grafted Arabidopsis thaliana plants, the DMC1 silencing signal consistently appeared in leaves, petioles, and stem, and only a small number of plants displayed DMC1 siRNA signals in flowers. In all three tested plant species the systemic silencing signal penetrated male sporogenic tissues suggesting that plants harbour an endogenous long-distance small RNA transport pathway facilitating siRNA signalling into meiotically active cells.

Hybrid recreation by reverse breeding in Arabidopsis thaliana.

Hybrid crop varieties are traditionally produced by selecting and crossing parental lines to evaluate hybrid performance. Reverse breeding allows doing the opposite: selecting uncharacterized heterozygotes and generating parental lines from them. With these, the selected heterozygotes can be recreated as F1 hybrids, greatly increasing the number of hybrids that can be screened in breeding programs. Key to reverse breeding is the suppression of meiotic crossovers in a hybrid plant to ensure the transmission of nonrecombinant chromosomes to haploid gametes. These gametes are subsequently regenerated as doubled-haploid (DH) offspring. Each DH carries combinations of its parental chromosomes, and complementing pairs can be crossed to reconstitute the initial hybrid. Achiasmatic meiosis and haploid generation result in uncommon phenotypes among offspring owing to chromosome number variation. We describe how these features can be dealt with during a reverse-breeding experiment, which can be completed in six generations (∼1 year).

Arabidopsis meiotic crossover hot spots overlap with H2A.Z nucleosomes at gene promoters.

PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot-enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates.

Haploid meiosis in Arabidopsis: double-strand breaks are formed and repaired but without synapsis and crossovers.

Two hallmark features of meiosis are i) the formation of crossovers (COs) between homologs and ii) the production of genetically-unique haploid spores that will fuse to restore the somatic ploidy level upon fertilization. In this study we analysed meiosis in haploid Arabidopsis thaliana plants and a range of haploid mutants to understand how meiosis progresses without a homolog. Extremely low chiasma frequency and very limited synapsis occurred in wild-type haploids. The resulting univalents segregated in two uneven groups at the first division, and sister chromatids segregated to opposite poles at the second division, leading to the production of unbalanced spores. DNA double-strand breaks that initiate meiotic recombination were formed, but in half the number compared to diploid meiosis. They were repaired in a RAD51- and REC8-dependent manner, but independently of DMC1, presumably using the sister chromatid as a template. Additionally, turning meiosis into mitosis (MiMe genotype) in haploids resulted in the production of balanced haploid gametes and restoration of fertility. The variability of the effect on meiosis of the absence of homologous chromosomes in different organisms is then discussed.

Honeybees and beehives are rich sources for fructophilic lactic acid bacteria.

Fructophilic lactic acid bacteria (FLAB) are a specific group of lactic acid bacteria (LAB) characterized and described only recently. They prefer fructose as growth substrate and inhabit only fructose-rich niches. Honeybees are high-fructose-consuming insects and important pollinators in nature, but reported to be decreasing in the wild. In the present study, we analyzed FLAB microbiota in honeybees, larvae, fresh honey and bee pollen. A total of 66 strains of LAB were isolated from samples using a selective isolation technique for FLAB. Surprisingly, all strains showed fructophilic characteristics. The 66 strains and ten FLAB strains isolated from flowers in a separate study were genotypically separated into six groups, four of which being identified as Lactobacillus kunkeei and two as Fructobacillus fructosus. One of the L. kunkeei isolates showed antibacterial activity against Melissococcus plutonius, a causative pathogen of European foulbrood, this protection being attributable to production of an antibacterial peptide or protein. Culture-independent analysis suggested that bee products and larvae contained simple Lactobacillus-group microbiota, dominated by L. kunkeei, although adult bees carried a more complex microbiota. The findings clearly demonstrate that honeybees and their products are rich sources of FLAB, and FLAB are potential candidates for future bee probiotics.

Microbial diversity analysis of fermented mung beans (Lu-Doh-Huang) by using pyrosequencing and culture methods.

In Taiwanese alternative medicine Lu-doh-huang (also called Pracparatum mungo), mung beans are mixed with various herbal medicines and undergo a 4-stage process of anaerobic fermentation. Here we used high-throughput sequencing of the 16S rRNA gene to profile the bacterial community structure of Lu-doh-huang samples. Pyrosequencing of samples obtained at 7 points during fermentation revealed 9 phyla, 264 genera, and 586 species of bacteria. While mung beans were inside bamboo sections (stages 1 and 2 of the fermentation process), family Lactobacillaceae and genus Lactobacillus emerged in highest abundance; Lactobacillus plantarum was broadly distributed among these samples. During stage 3, the bacterial distribution shifted to family Porphyromonadaceae, and Butyricimonas virosa became the predominant microbial component. Thereafter, bacterial counts decreased dramatically, and organisms were too few to be detected during stage 4. In addition, the microbial compositions of the liquids used for soaking bamboo sections were dramatically different: Exiguobacterium mexicanum predominated in the fermented soybean solution whereas B. virosa was predominant in running spring water. Furthermore, our results from pyrosequencing paralleled those we obtained by using the traditional culture method, which targets lactic acid bacteria. In conclusion, the microbial communities during Lu-doh-huang fermentation were markedly diverse, and pyrosequencing revealed a complete picture of the microbial consortium.

Low-level infrared laser effect on plasmid DNA.

Low-level laser therapy is used in the treatment of many diseases based on its biostimulative effect. However, the photobiological basis for its mechanism of action and adverse effects are not well understood. The aim of this study, using experimental models, was to evaluate the effects of laser on bacterial plasmids in alkaline agarose gel electrophoresis and Escherichia coli cultures. The electrophoretic profile of bacterial plasmids in alkaline agarose gels were used for studying lesions in DNA exposed to infrared laser. Transformation efficiency and survival of Escherichia coli AB1157 (wild-type), BH20 (fpg/mutM(-)), BW9091 (xth(-)), and DH5αF'Iq (recA(-)) cells harboring pBSK plasmids were used as experimental models to assess the effect of laser on plasmid DNA outside and inside of cells. Data indicate low-level laser: (1) altered the electrophoretic profile of plasmids in alkaline gels at 2,500-Hz pulsed-emission mode but did not alter at continuous wave, 2.5- and 250-Hz pulsed-emission mode; (2) altered the transformation efficiency of plasmids in wild-type and fpg/mutM(-) E. coli cells; (3) altered the survival fpg/mutM(-), xthA(-) and recA(-) E. coli cultures harboring pBSK plasmids. Low-level infrared laser with therapeutic fluencies at high frequency in pulsed-emission modes have effects on bacterial plasmids. Infrared laser action can differently affect the survival of plasmids in E. coli cells proficient and deficient in DNA repair mechanisms, therefore, laser therapy protocol should take into account fluencies, frequencies and wavelength of laser, as well as tissue conditions and genetic characteristics of cells before beginning treatment.

RecA proteins from Deinococcus geothermalis and Deinococcus murrayi--cloning, purification and biochemical characterisation.

BACKGROUND: Escherichia coli RecA plays a crucial role in recombinational processes, the induction of SOS responses and mutagenic lesion bypasses. It has also been demonstrated that RecA protein is indispensable when it comes to the reassembly of shattered chromosomes in γ-irradiated Deinococcus radiodurans, one of the most radiation-resistant organisms known. Moreover, some functional differences between E. coli and D. radiodurans RecA proteins have also been shown. RESULTS: In this study, recA genes from Deinococcus geothermalis and Deinococcus murrayi, bacteria that are slightly thermophilic and extremely γ-radiation resistant, were isolated, cloned and expressed in E. coli. After production and purification, the biochemical properties of DgeRecA and DmuRecA proteins were determined. Both proteins continued to exist in the solutions as heterogenous populations of oligomeric forms. The DNA binding by DgeRecA and DmuRecA proteins is stimulated by Mg2+ ions. Furthermore, both proteins bind more readily to ssDNA when ssDNA and dsDNA are in the same reaction mixture. Both proteins are slightly thermostable and were completely inactivated in 10 s at 80°C. Both proteins hydrolyze ATP and dATP in the presence of ssDNA or complementary ssDNA and dsDNA, but not in the absence of DNA or in the presence of dsDNA only, and dATP was hydrolyzed more rapidly than ATP. They were also able to promote DNA strand exchange reactions by a pathway common for other RecA proteins. However, we did not obtain DNA strand exchange products when reactions were performed on an inverse pathway, characteristic for RecA of D. radiodurans. CONCLUSIONS: The characterization of DgeRecA and DmuRecA proteins made in this study indicates that the unique properties of D. radiodurans RecA are probably not common among RecA proteins from Deinococcus sp.

Cisplatin inhibits protein splicing, suggesting inteins as therapeutic targets in mycobacteria.

Mycobacterium tuberculosis harbors three protein splicing elements, called inteins, in critical genes and their protein products. Post-translational removal of the inteins occurs autocatalytically and is required for function of the respective M. tuberculosis proteins. Inteins are therefore potential targets for antimycobacterial agents. In this work, we report that the splicing activity of the intein present in the RecA recombinase of M. tuberculosis is potently inhibited by the anticancer drug cisplatin (cis-diamminedichloro-platinum(II)). This previously unrecognized activity of cisplatin was established using both an in vitro intein splicing assay, which yielded an IC(50) of ∼2 µM, and a genetic reporter for intein splicing in Escherichia coli. Testing of related platinum(II) complexes indicated that the inhibition activity is highly structure-dependent, with cisplatin exhibiting the best inhibitory effect. Finally, we report that cisplatin is toxic toward M. tuberculosis with a minimum inhibitory concentration of ∼40 µM, and in genetic experiments conducted with the related Mycobacterium bovis bacillus Calmette-Guérrin (BCG) strain, we show that cisplatin toxicity can be mitigated by intein overexpression. We propose that cisplatin inhibits intein activity by modifying at least one conserved cysteine residue that is required for splicing. Together these results identify a novel active site inhibitor of inteins and validate inteins as viable targets for small molecule inhibition in mycobacteria.

Fructobacillus tropaeoli sp. nov., a fructophilic lactic acid bacterium isolated from a flower.

A fructophilic lactic acid bacterium, designated strain F214-1(T), was isolated from a flower of Tropaeolum majus in South Africa. Based on phylogenetic analysis of 16S rRNA gene sequences, the strain formed a subcluster with Fructobacillus ficulneus and Fructobacillus pseudoficulneus and, based on recA gene sequences, the strain formed a subcluster with F. ficulneus. DNA-DNA hybridization studies showed that strain F214-1(T) was phylogenetically distinct from its closest relatives. Acid was produced from the fermentation of d-glucose, d-fructose and d-mannitol only. d-Fructose was the preferred sole carbon and energy source and was fermented more rapidly than d-glucose. Growth of the strain on d-glucose under anaerobic conditions was very weak but external electron acceptors such as oxygen and pyruvate enhanced growth on d-glucose. Lactic acid and acetic acid were produced from d-glucose in equimolar amounts. Ethanol was produced at very low levels, despite the strain's obligately heterofermentative metabolism. Based on these data, strain F214-1(T) represents a novel species of fructophilic bacteria in the genus Fructobacillus, for which the name Fructobacillus tropaeoli sp. nov. is proposed. The type strain is F214-1(T) ( = JCM 16675(T)  = DSM 23246(T)).

Rhizobium phenanthrenilyticum sp. nov., a novel phenanthrene-degrading bacterium isolated from a petroleum residue treatment system.

Strain F11(T), a phenanthrene-degrading bacterium, was isolated from a petroleum residue treatment system, and classified under the genus Rhizobium based on the similarity analysis of its 16S rRNA and recA gene sequences. Strain F11(T) falls into the same phylogenetic clade with Rhizobium oryzae Alt 505(T) (96.8% 16S rRNA gene sequence similarity) and Rhizobium pseudoryzae J34A-127(T) (96.2%). Major cellular fatty acids of strain F11(T) are C(16:0) (6.24%) and summed feature 8 (C(18:1ω7c) and/or C(18:1ω6c), 76.59%), which are also the major fatty acids of R. oryzae Alt 505(T) and R. pseudoryzae J34A-127(T). The DNA G+C content of strain F11(T) was 59.3±0.4 mol%. Based on the phylogenetic analysis as well as biochemical and physiological characteristics, strain F11(T) could be separated from all recognized Rhizobium species. Strain F11(T) (=DSM 21882(T) =CCTCC AB 209029(T)) was considered to be representative of a novel species of Rhizobium, for which the name Rhizobium phenanthrenilyticum sp. nov. is proposed.

Characterization of strains unlike Mesorhizobium loti that nodulate lotus spp. in saline soils of Granada, Spain.

Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.

Characterization of Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease, reveals a unique mode of DNA binding, helical distortion, and cleavage compared with a canonical LAGLIDADG homing endonuclease.

Mycobacterium leprae, which has undergone reductive evolution leaving behind a minimal set of essential genes, has retained intervening sequences in four of its genes implicating a vital role for them in the survival of the leprosy bacillus. A single in-frame intervening sequence has been found embedded within its recA gene. Comparison of the M. leprae recA intervening sequence with the known intervening sequences indicated that it has the consensus amino acid sequence necessary for being a LAGLIDADG-type homing endonuclease. In light of massive gene decay and function loss in the leprosy bacillus, we sought to investigate whether its recA intervening sequence encodes a catalytically active homing endonuclease. Here we show that the purified M. leprae RecA intein (PI-MleI) binds to cognate DNA and displays endonuclease activity in the presence of alternative divalent cations, Mg2+ or Mn2+. A combination of approaches, including four complementary footprinting assays such as DNase I, copper-phenanthroline, methylation protection, and KMnO4, enhancement of 2-aminopurine fluorescence, and mapping of the cleavage site revealed that PI-MleI binds to cognate DNA flanking its insertion site, induces helical distortion at the cleavage site, and generates two staggered double strand breaks. Taken together, these results implicate that PI-MleI possesses a modular structure with separate domains for DNA target recognition and cleavage, each with distinct sequence preferences. From a biological standpoint, it is tempting to speculate that our findings have implications for understanding the evolution of the LAGLIDADG family of homing endonucleases.