Acid resistance and response to pH-induced stress in two Lactobacillus plantarum strains with probiotic potential.

Two new Lactobacillus plantarum strains, KR6-DSM 28780 and M5 isolated from sour turnip and traditional dried fresh cheese, respectively, were evaluated for species identity, antibiotic susceptibility, resistance to gastrointestinal conditions and adaptive response to low pH. Resistance mechanisms involved in the adaptation to acid-induced stress in these two strains were investigated by quantitative PCR of the atpA, cfa1, mleS and hisD genes. In addition to absence of antibiotic resistance, the two L. plantarum strains showed excellent survival rates at pH values as low as 2.4. Adaptive response to low pH was clearly observed in both strains; strain KR6 was superior to M5, as demonstrated by its ability to survive during 3 h incubation at pH 2.0 upon adaptation to moderately acidic conditions. In contrast, acid adaptation did not significantly affect the survival rate during simulated passage through the gastrointestinal tract. In both strains, induction of histidine biosynthesis (hisD) was upregulated during the acid adaptation response. In addition, significant upregulation of the cfa1 gene, involved in modulation of membrane fatty acid composition, was observed during the adaptation phase in strain KR6 but not in strain M5. Cells adapted to moderately acidic conditions also showed a significantly increased viability after the lyophilisation procedure, a cross-protection phenomenon providing additional advantage in probiotic application.

Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea.

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/PactI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.

Pro-oxidative vs antioxidative properties of ascorbic acid in chromium(VI)-induced damage: an in vivo and in vitro approach.

The effect of antioxidant ascorbic acid (vitamin C) pretreatment on chromium(VI)-induced damage was investigated using the yeast Saccharomyces cerevisiae as a model organism. The objective of this study was to pretreat yeast cells with the antioxidant ascorbic acid in an effort to increase cell tolerance against reactive chromium intermediates and reactive oxygen species formed during chromium(VI) reduction. Intracellular oxidation was estimated using the fluorescence indicators dihidro-2,7-dichlorofluorescein, dihydroethidium and dihydrorhodamine 123. The role of ascorbic acid pretreatment on chromium(VI) toxicity was determined by measuring mitotic gene conversion, reverse mutations, 8-OHdG, hydroxyl radical, superoxide anion and chromium(V) formation. The chromium content in the biomass was determined by flame atomic absorption spectrometry. In the absence of chromium, ascorbic acid effectively protected the cells against endogenous reactive oxygen species formed during normal cellular metabolism. In vitro measurements employing EPR and the results of supercoiled DNA cleavage revealed that the pro-oxidative action of ascorbic acid during Cr(VI) reduction was concentration-dependent and that harmful hydroxyl radical and Cr(V) had formed following Cr(VI) reduction. However, the in vivo results highlighted the important role of increased cytosol reduction capacity related to modification of Cr(V) formation, increased chromium accumulation, better scavenging ability of superoxide anions and hydrogen peroxide, and consequently decreased cytotoxicity and genotoxicity in ascorbic acid pretreated cells. Ascorbic acid influenced Cr(VI) toxicity both as a reducing agent, by decreasing Cr(V) persistence, and as an antioxidant, by decreasing intracellular superoxide anion and hydrogen peroxide formation and by quenching free radicals formed during Cr(VI) to Cr(III) reduction. Increased 8-OHdG and decreased reduced glutathione in ascorbic acid-treated cells might induce an endogenous antioxidant defense system and thus increase cell tolerance against subsequent Cr-induced stress.

The involvement of ATP sulfurylase in Se(VI) and Cr(VI) reduction processes in the fission yeast Schizosaccharomyces pombe.

The response of Schizosaccharomyces pombe towards the oxyanions selenate [Se(VI)] and dichromate [Cr(VI)] was investigated in order to establish the involvement of the yeast ATP sulfurylase in their reduction. An ATP sulfurylase-defective/selenate-resistant mutant of S. pombe (B-579 Se(R) -2) and an ATP sulfurylase-active/selenate-sensitive strain of S. pombe (B-579 Se(S)) were included in this study. The inhibitory effect of Se(VI) and Cr(VI) oxyanions on growth and bioaccumulation was measured. The sensitive strain showed natural sensitivity to selenate while the resistant mutant tolerated a 100-fold higher concentration of selenate. These results indicate that selenate toxicity to microorganisms is connected with the reduction of selenate to selenite. Both strains showed similar sensitivity to Cr(VI) and in this study there was no evidence that ATP sulfurylase participates in the reduction process of Cr(VI).