Genetic diversity of Lactobacillus paracasei isolated from in situ human oral biofilms.

AIM: To determine the genetic diversity and possible origin of Lactobacillus paracasei found in the oral biofilm. METHODS AND RESULTS: Lactobacilli were isolated from a biofilm model, formed in situ prior to and during a period of exposure to 20% sucrose solution (28 days), using Rogosa Agar. The lactobacillus colonies were randomly selected (n = 222) and subcultured. The isolates were identified using pheS or rpoA gene sequence analysis. Lactobacilli identified as Lact. paracasei (n = 75) were subjected to multilocus sequencing typing (MLST) analysis by determining partial sequences of seven housekeeping genes fusA, ileS, lepA, leuS, pyrG, recA and recG. An increase recovery of lactobacilli after sucrose phase compared with nonsucrose period was observed (31 prior to and 191 following a sucrose exposure period). Seven subjects harboured Lact. paracasei and these represented 14 sequence types (ST). Comparison of the STs showed that unrelated subjects may harbour the same ST and that individuals harbour multiple STs. Three subjects harboured STs previously isolated from dairy products. CONCLUSION: The present data supports the hypothesis that oral lactobacilli may be of exogenous origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The study allow us to gain insight into the genetic diversity of Lact. paracasei in oral biofilm.

Evaluation of antimicrobial activity in Paenibacillus spp. strains isolated from natural environment.

AIMS: Paenibacillus isolates were selected to test antimicrobial activity against bacteria, filamentous fungi and yeasts isolates, with the purpose of finding new bacterium species for microbiological control. METHODS AND RESULTS: Fifty-five strains belonging to 15 species of Paenibacillus were inoculated on trypticase soya agar, potato dextrose agar and sabouraud agar plates in order to evaluate their antimicrobial activity against 16 indicator bacteria, 14 filamentous fungi and six yeasts isolates, both reference and field strains. After these screening, culture supernatant of 17 isolates was prepared. Twenty-five Paenibacillus isolates presented antimicrobial activity, where seven species (Paenibacillus chibensis; P. koreensis; P. illinoiensis; P. validu; P. pabuli; P. brasilensis and P. peoriae) stood out inhibiting at least 13 of the 16 indicator bacteria. Only 14 of the 55 isolates exhibited antifungal activity. P. peoriae inhibited 13 among the 14 filamentous fungi and all yeasts indicator strains. Fourteen isolates produced culture supernatant with antimicrobial activity. CONCLUSIONS: Among the 55 isolates analysed, 25 exhibited a broad inhibition spectrum against bacteria and pathogenic fungi. P. validus, P. chibensis, P. koreensis and P. peoriae isolates proved to be the subject matter for studies on the production of antimicrobial agents. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study revealed other two species with antimicrobial activity: P. validus and P. chibensis, and it contributed to enhance Paenibacillus biocontrolling potential, proving that it exhibit a broad action spectrum.

Lateral diffusion of human CD2 wild type and mutants with large deletions in the transmembrane domain.

Integral membrane proteins anchor to the cell surface and span the lipid bilayer by an alpha-helix of 17-30 amino acids, the transmembrane segment. However, little is known about the association of this alpha-helix and the lipid bilayer. In the present study human CD2 molecule was chosen as a model for an integral membrane protein. Truncate forms with transmembrane segments 14 and 12 amino acids long were created by oligonucleotide site-directed mutagenesis. Lateral diffusion revealed that even large deletions in the membrane domain of CD2 do not interfere with its lateral mobility. On the other hand, the fraction of free molecules for diffusion was higher in CD2 protein with transmembrane region 12 amino acids long. These results suggest that deletions in the transmembrane domain can interfere with the stability of the protein within the lipid bilayer.

Variation in the expression of human 3 beta-hydroxysteroid dehydrogenase.

Polymorphic genetic variation shows that the genes for human 3 beta-hydroxysteroid dehydrogenases (3 beta-HSD) types I and II are closely linked. The type II mutations A82T, S100N and L173R are associated with male pseudohermaphroditism and A82T is associated, with variable penetrance, with female premature puberty. When expressed in vitro A82T showed less than 5% of normal activity and L173R showed a 30-50% reduction in activity. PCR experiments and direct genomic cloning show that there is a larger family of 3 beta-HSD sequences which require to be tested for expression. The phenomenon of epitopic heterogeneity of 3 beta-HSD is discussed and is now shown to apply to testicular Leydig cells as well as extrauterine trophoblast. RT-PCR analyses indicate that the phenomenon is most likely to be due to post-translational modification affecting the carboxytermini 3 beta-HSD types I and II. This phenomenon may reflect a further level at which enzyme activity is regulated.