Nitrate reductase (NaR) of a strain of Selenomonas ruminantium was purified, and the gene encoding NaR (nar) was sequenced. The 6.4 kbp nar gene consisted of narG, H, J, and I in this order. The deduced amino acid sequences of these subunits resembled those of membrane-bound nitrate reductase-A reported for Escherichia coli. It was shown that narG, H, J, and I are transcribed as a single polycistronic message (nar operon). The level of intracellular nar-mRNA was higher when S. ruminantium was grown with nitrate than when grown without nitrate, suggesting that nar transcription is enhanced by nitrate. The level of nar-mRNA, which was in parallel to the amount of NaR per cellular nitrogen, was suggested to be enhanced in response to the deficiency of energy and electron supply. Therefore, NaR synthesis in S. ruminantium appeared to be regulated at the transcriptional level in response to the availability of energy and electrons. S. ruminantium reduced nitrate and fumarate simultaneously with no significant effect of fumarate on nar transcription. Addition of fumarate stimulated nitrate reduction, which was caused by increased cell growth because of increased acquirement of ATP via electron transport phosphorylation coupled with fumarate reduction.
Three strains related to Cryptococcus flavus were isolated from plants collected in the Prioksko-terrasny biosphere reserve (Russia). Physiological characterization, mycocinotyping, sequencing of the D1/D2 domain of the 26S rDNA and the ITS region revealed their separate taxonomic position. The name Cryptococcus paraflavus is proposed to accommodate these isolates (type strain VKM Y-2923).
Whether fructose-1,6-bisphosphate (FBP) triggers the transcriptional regulation of the gene expression of lactate dehydrogenase (LDH) and pyruvate formate-lyase (PFL) in Streptococcus bovis was examined by constructing a recombinant strain that overexpresses FBP aldolase (FBA). When the recombinant strain was grown on glucose, intracellular FBP was much lower as compared to the parent strain, whereas dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (GAP) were slightly higher. Intracellular ATP and ADP were slightly lower, but the NADH/NAD+ ratio was not different. When glucose was replaced by lactose, a less readily utilized substrate, there was no great difference in FBP, DHAP, GAP, or adenine nucleotides. Overexpression of FBA decreased the level of LDH-mRNA, and increased the level of PFL-mRNA. Consequently, FBP concentration was positively related to the LDH-mRNA level and inversely related to the PFL-mRNA level. On the contrary, DHAP and GAP concentrations were positively related to the PFL-mRNA level and inversely related to the LDH-mRNA level. The levels of these mRNA were proportional to the amounts of corresponding enzymes in cells. As a result, the ratio of formate to lactate produced was increased by the overexpression of FBA. From these results, it could be presumed that FBP is involved in the transcriptional control of LDH and PFL synthesis in S. bovis.
Lactic acid bacteria (LAB) commonly used in food as starter cultures are known to produce antimicrobial substances such as bacteriocins and have great potential as food biopreservatives. LAB isolated from traditional fermented foods (appam batter and pickles) were screened for bacteriocin production. Two lactobacilli, LABB and LABP (one from each source) producing bacteriocins were characterized. Both the bacilli were homo-fermentative, catalase negative and micro-aerophilic in nature. LABB was found to be a thermobacterium growing at 45°C while LABP was a streptobacterium growing at 15°C. Both were able to grow at pH 4.5–8.6 but were intolerant to high salt concentration. They failed to produce gas from glucose as well as ammonia from arginine. Among the sugars examined they could not ferment arabinose, raffinose, rhamnose or xylose. Additionally, LABB could not ferment esculin, gluconate or mannose. LABB is identified as Lactobacillus acidophilus while LABP as Lb. casei. Their bacteriocins showed a broad inhibitory spectrum against the indicator organisms tested. They were active below pH 8.0 and after autoclaving as well. There was a complete loss of activity when treated with proteolytic enzymes such as trypsin indicating the proteinaceous nature of the active molecules. SDS-PAGE of partially purified bacteriocins indicated the molecular mass of the bacteriocin as 3.8 and 4.5 kDa for LABB and LABP respectively.
The aim of this study is to develop ecotoxicity assay for evaluating the influence of chemicals on a microbial ecosystem based on XTT reduction inhibition (XTT assay). XTT reduction method is used for quantification of the microbial respiratory activity. Since the XTT assay indicates the inhibition of microbial respiratory activity, it could evaluate the toxicity of chemicals. Suitable conditions for the XTT assay were determined to be 200 mg/L of particulate organic carbon as test microbe concentration and 15 min of assay time using activated sludge. Toxicities of several chemicals evaluated by activated sludge as test microbes were examined under these conditions. Sensitivity for the toxicity evaluated by the XTT assay using activated sludge microbes was almost the same value was that for the OECD activated sludge respiration inhibition test (ASRI test). XTT assay was also applied for evaluating the influence of chemicals on the soil microbial community and the XTT assay was used to evaluate a median effective concentration (EC50) value of 3,5-dichlorophenol (3,5-DCP). The EC50 value of 3,5-DCP was almost the same as the value using activated sludge as test microbes. These results suggest that the XTT assay using both mixed cultures of non-contaminated environments and chemical extracts from various contaminated environments could evaluate the influence on microbial ecosystems affected by toxic chemicals.