ABSTRACT
Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium able to survive in diverse environments such as soil, plants, freshwater, and seawater. P. aeruginosa can be an opportunistic pathogen to humans when their immune system is deficient. Its pathogenicity may be linked to the production of virulence factors. We isolated P. aeruginosa strain RBS from the saltern of Sfax in Tunisia. In this study, we characterized the halotolerance, antibiotic susceptibility, and some virulence factors of strain RBS. High NaCl concentrations inhibited growth and motility. However, biofilm formation was enhanced to protect bacteria against salt stress. Among the 18 antibiotics tested, quinolones and tetracycline showed a significant inhibitory effect on growth, motility, and biofilm formation of strain RBS. ß-Lactams, however, did not have any inhibitory effect on neither bacterial growth nor motility. In some cases, resistance was due, in part, to biofilm formation. We also showed that RBS produces two proteases, LasB and AprA, which have been shown to be implicated in host infection. LasB was further characterized to study the role of metal ions in enzyme stability. It possesses two distinct metal ion-binding sites coordinating a calcium and a zinc ion. The effect of metal ion chelation was evaluated as well as substitutions of residues involved in metal ion binding. Impairing metal ion binding of LasB led to a loss of activity and a sharp decrease of stability. Our findings suggest that the binding of both metal ions is interdependent as the two metal ions' binding sites are linked via a hydrogen bond network.
Subject(s)
Ions/metabolism , Pseudomonas aeruginosa/metabolism , Pseudomonas aeruginosa/pathogenicity , Virulence Factors/metabolism , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/metabolism , Biofilms/drug effects , Humans , Peptide Hydrolases/metabolism , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , TunisiaABSTRACT
Enteric viruses contaminating the environment represent a danger for public health notably enteroviruses that are excreted in stools and can contaminate wastewater and shellfish. The ability of enteroviruses to grow in cell culture and the development of techniques of molecular biology applied to their detection make these viruses a good marker of viral pollution of aquatic environment. The aim of our study was to develop a rapid and sensitive RT-PCR technique, able to detect enterovirus RNA in wastewater and shellfish. From 26 samples of wastewater and 56 samples of shellfish, 50.0 and 42.8% were found positive by RT-PCR, respectively, whereas 38.4 and 28.5% were positive by culture, respectively (P=0.077 by chi square test). The two techniques were found concordant in 57.3% of the 82 combined samples, whereas 23 samples (28.0%) were positive only by RT-PCR and that 12 samples (14.6%) were positive only by culture. These discrepancies illustrate the fact that the two techniques are not equivalent: the molecular technique allows the detection of not cultivable samples but is sensitive to PCR inhibitors that are present in large amounts in environmental samples.
Subject(s)
Enterovirus/isolation & purification , Sewage/virology , Shellfish/virology , Animals , Enterovirus/genetics , Enterovirus/growth & development , Mollusca/virology , RNA, Viral/genetics , RNA, Viral/isolation & purification , Sensitivity and Specificity , Water Pollution/analysisABSTRACT
XylA gene of Streptomyces violaceoniger (Drocourt et coll. 1988) code for a D-xylose isomerase activity which is used as a D-glucose isomerase activity in large scale. This gene is a part of regulated region involved in xylose utilization (Marcel et coll. 1987). Sequence determination of this region enabled us to characterize xylB gene (Xylulose kinase activity) and xylX gene which is involved in xylA and xylB expression. In order to construct a new strain having a strong and constitutive glucose isomerase activity, a newly isolated strong streptomyces promoter (P1 promoter), has been cloned behind xylA gene. To avoid instability of plasmid and glucose-isomerase activity, the P1-xylA gene of S. violaceoniger has been integrated into the chromosome, using the integrative vector pTS55. The resultant CBS1 strain has four to five fold higher glucose-isomerase activity in absence of xylose compared to that of strain SV1 fully induced by xylose. In addition, specific glucose-isomerase activity of CBS1 strain increases in the secondary growth phase, in contrast to wild type and SV1 strains.
