The major targets for improvement of recombinant expression systems in microbial cells are gene dosage, transcriptional control machinery and, to some extent, translation. Here we show that optimization of fermentation conditions by applying statistically designed, multifactorial experiments offers an additional method for potential enhancement of gene expression systems. A chromosomally encoded fusion between the Bacillus subtilis aprE regulatory region and the E. coli lacZ gene carried by the B. subtilis host cells was used. The 2 x SG sporulation medium was used as a basal medium. Among the 11 fermentation factors we examined, the most significant variables influencing beta-galactosidase expression were statistically elucidated for optimization and included peptone, MgSO4.7H2O, and KCl. The optimum concentrations of these variables were predicted by using a second-order polynomial model fitted to the results obtained by applying the Box-Behnken design, a response surface method. Calculated optimum concentrations were predicted to confer a maximum yield of 2,423.5 beta-galactosidase specific activity units. A verification experiment performed under optimal conditions yielded 96% of the predicted specific activity value with an increase by a factor of almost 5 compared with the results obtained under basal conditions.

Urinary tract infection (UTI) is the most commonly acquired bacterial infection. Biofilm formation being the prime cause for antibiotic resistance by uropathogenes. The purpose of this study was to detect biofilm formation by uropathogenes isolated from UTIs, their antibiotic susceptibility pattern and detection of genes responsible for biofilm production in some isolates. The isolated bacteria were tested for biofilm production by tube adherence (TA), congo red agar (CRA) and micro titer plate (MtP) methods. Antibiotic susceptibility pattern of uropathogens was done by Kirby-Bauer disc diffusion method and detection of genes responsible for biofilm production in some isolates by PCR. Out of 320 cultures positive cases 146 (46.5 %) isolates were biofilm producers Ecoli was the commonest uropathogen followed by Staphylococcus aureus and Enterococcus feacalis. The antibiotic resistance was higher among biofilm producers to commonly used antibiotics as compared to non biofilm producers. The detection of icaD gene of Staph. areus and cup gene of pseudomonas by PCR was siginfiacntly related to its detection by congo red agar method, while there no significant relation between detection of ESP gene by PCR and the three used conventional methods. It was concluded that the ability of slime production, adherence and biofilm formation of uropathogenic strains had significant role in antibiotic resistance. Biofilm formation is the major virulence determinant of uropathogen, so it is necessary to screen all urinary isolates for biofilm production.

No abstract

A Bacillus subtilis wild type strain and a kinA (spoIIJ) isogenic mutant were compared as hosts for the expression of the Escherichia coli β‐galactosidase gene, lacZ, driven by the B. subtilis aprE promoter in a chromosomal system. The 2 × SG sporulation formula, with some modifications, was used as a basal medium. The specific activity values recorded by the mutant strain at the stationary phase were markedly higher than those achieved by the wild type host. Exposure of the cells to increasing levels of chloramphenicol resulted in significant amplifications of the lacZ region. Gene copy numbers of 19 and 11 were estimated in the amplified wild type and kinA strains, respectively, with high segregational stability records. The magnitude of β‐galactosidase over‐expression was dependent on, and roughly proportional to antibiotic resistance levels. Among five examined by‐products, a 2.3‐times diluted concentration of neutralized cheese whey was successfully used as a sole medium component for over‐expression of the recombinant β‐galactosidase gene in B. subtilis.