LraI from Lactococcus raffinolactis BGTRK10-1, an Isoschizomer of EcoRI, Exhibits Ion Concentration-Dependent Specific Star Activity

Restriction enzymes are the main defence system against foreign DNA, in charge of preserving genome integrity. Lactococcus raffinolactis BGTRK10-1 expresses LraI Type II restriction-modification enzyme, whose activity is similar to that shown for EcoRI; LraI methyltransferase protects DNA from EcoRI cleavage. The gene encoding LraI endonuclease was cloned and overexpressed in E. coli. Purified enzyme showed the highest specific activity at lower temperatures (between 13 degrees C and 37 degrees C) and was stable after storage at -20 degrees C in 50% glycerol. The concentration of monovalent ions in the reaction buffer required for optimal activity of LraI restriction enzyme was 100 mM or higher. The recognition and cleavage sequence for LraI restriction enzyme was determined as 5'-G/AATTC-3', indicating that LraI restriction enzyme is an isoschizomer of EcoRI. In the reaction buffer with a lower salt concentration, LraI exhibits star activity and specifically recognizes and cuts another alternative sequence 5'-A/AATTC-3', leaving the same sticky ends on fragments as EcoRI, which makes them clonable into a linearized vector. Phylogenetic analysis based on sequence alignment pointed out the common origin of LraI restriction-modification system with previously described EcoRI-like restriction-modification systems

Virulence traits associated with Burkholderia cenocepacia ST856 epidemic strain isolated from cystic fibrosis patients

Background: Burkholderia cenocepacia is considered one of the most problematic cystic fibrosis (CF) pathogens. Colonization prevalence in the Serbian CF population is high and virtually exclusively limited to a single highly transmissible clone of B. cenocepacia ST856 which is positive for both the B. cepacia epidemic strain marker (BCESM) and cable pilin, and is closely related to the epidemic strain CZ1 (ST32). Methods: Biofilm formation for 182 isolates, and adhesion to components of the host extracellular matrix, proteolytic activity, mucoidy and motility of selected ST856 representatives, as well as B. cenocepacia ST858 and ST859, and B. stabilis ST857, novel STs isolated from Serbian CF patients, were investigated in this study. The presence of the cepI, cepR, fliG, llpE, wbiI, and bcscV genes was analyzed. Results: Biofilm-formation ability of analyzed strains was poor under standard laboratory conditions, but changed in stress conditions (cold stress) and conditions that mimic CF milieu (increased CO2). All strains expressed ability to bind to collagen and fibronectin albeit with different intensity. Representatives of ST856 exhibited gelatinase activity. ST858, ST859 and 9/11 of ST856 genotypes were positive for swimming and twitching motility whereas ST857 was non-motile. Mucoidy was demonstrated in all ST856 genotypes, ST857 was semi-mucoid, and ST858 and ST859 were non-mucoid. Molecular analysis for major virulence factors revealed that ST856 and ST857 carried the six analyzed genes, while ST858 and ST859 were negative for the llpE gene. Conclusion: Variations in virulence phenotypes in different genotypes of epidemic B. cenocepacia ST856 clone, in vitro, could be a consequence of diversification driven by pathoadaptation. Diversity of epidemic clone genotypes virulence, could be challenging for accurate diagnosis and treatment, as well as for infection control

PsrA Regulator Connects Cell Physiology and Class 1 Integron Integrase Gene Expression Through the Regulation of lexA Gene Expression in Pseudomonas spp.

Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, rpoS and psrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in rpoS and psrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status

Bacterial Diversity among the Sediments of Glacial Lakes in the Western Balkans: Exploring the Impact of Human Population

16S rRNA gene-based metagenomic approach was used to assess the biodiversity of bacterial communities in the sediments of selected glacial lakes in the Western Balkans and to assess the impact of human population on these microbial communities. Sediment samples were collected from three glacial lakes, viz., Plav Lake (in a zone of the highest impact of human population), Black Lake (a zone of medium impact of human population), and Donje Bare Lake (a remote lake with minimal impact of human population). Canonical correlation analysis analysis indicated correlation between the distance of the lake from urbanized population and bacterial diversity in Donje Bare Lake sediment. Bacterial diversity of Black Lake sediment was correlated with high content of phosphorous and pH value. Chemical compounds exhibiting the most prominent correlation with bacterial diversity of Plav Lake were NH4-N, K2O, CaCo3, and total nitrogen . Additionally, CCA analysis indicated that population density was correlated with biodiversity of bacterial communities in Plav Lake sediment, which is the most exposed to human population. Multivariate regression revealed the highest correlation between the presence of Proteobacteria classes and population density and levels of NH4-N. The influence of human population was observed to be important for shaping the sediment communities in addition to biological and chemical factors

Metagenomic Analysis of Bacterial Community and Isolation of Representative Strains from Vranjska Banja Hot Spring, Serbia

The hot spring Vranjska Banja is the hottest spring on the Balkan Peninsula with a water temperature of 63–95 °C and a pH value of 7.1, in situ. According to the physicochemical analysis, Vranjska Banja hot spring belongs to the bicarbonated and sulfated hyperthermal waters. The structures of microbial community of this geothermal spring are still largely unexplored. In order to determine and monitor the diversity of microbiota of the Vranjska Banja hot spring, a comprehensive culture-independent metagenomic analysis was conducted in parallel with a culture-dependent approach for the first time. Microbial profiling using amplicon sequencing analysis revealed the presence of phylogenetically novel taxa, ranging from species to phyla. Cultivation-based methods resulted in the isolation of 17 strains belonging to the genera Anoxybacillus, Bacillus, Geobacillus, and Hydrogenophillus. Whole-genome sequencing of five representative strains was then performed. The genomic characterization and OrthoANI analysis revealed that the Vranjska Banja hot spring harbors phylogenetically novel species of the genus Anoxybacillus, proving its uniqueness. Moreover, these isolates contain stress response genes that enable them to survive in the harsh conditions of the hot springs. The results of the in silico analysis show that most of the sequenced strains have the potential to produce thermostable enzymes (proteases, lipases, amylases, phytase, chitinase, and glucanase) and various antimicrobial molecules that can be of great importance for industrial, agricultural, and biotechnological applications. Finally, this study provides a basis for further research and understanding of the metabolic potential of these microorganisms

Fluoroquinolone-resistant Achromobacter xylosoxidans clinical isolates from Serbia: high prevalence of the aac-(6)-Ib-cr gene among resistant isolates

The aim of this study was to evaluate the contribution of plasmid-mediated genes and efflux to fluoroquinolone resistance in collection of Achromobacter spp. gathered during a 3-year period. Susceptibility to ciprofloxacin and levofloxacin was tested by disk diffusion and microdilution tests for a collection of 98 Achromobacter spp. clinical isolates. Identification of fluoroquinolone-resistant isolates was performed by sequencing and phylogenetic analyses of the nrdA gene. Genetic relatedness among resistant isolates was determined by pulsed-field gel electrophoresis (PFGE) analysis. The influence of an H+ conductor cyanide m-chlorophenyl hydrazone (CCCP) and a resistance-nodulation-division-type efflux pump inhibitor phenylalanine-arginine beta-naphthylamide (PAN) on minimal inhibitory concentration (MIC) value was evaluated by broth microdilution. The presence of the plasmid-mediated qnrA, qnrB, qnrC, qnrS, and aac-(6)-Ib-cr genes was investigated by PCR and sequencing. Achromobacter spp. isolates that were resistant or intermediately resistant to fluoroquinolones in disk diffusion tests (44/98) were subjected to microdilution. As a result, 20/98 isolates were confirmed to be resistant to ciprofloxacin while 10/98 was resistant to levofloxacin. CCCP decreased twofold MIC value for ciprofloxacin in six isolates and more than 16 times in one isolate, while MIC value for levofloxacin was decreased in all isolates (twofold to more than eightfold). Fluoroquinolone-resistant isolates were identified as A. xylosoxidans with the nrdA gene sequencing. PFGE revealed that resistant isolates belonged to seven different genotypes. Ten isolates belonging to four genotypes were positive for the aac-(6)-Ib-cr gene. Although resistance to fluoroquinolones was not widespread among analyzed isolates, detected contribution of efflux pumps and the presence of the aac-(6)-Ib-cr gene present a platform for emergence of more resistant strains