A total of thirty yeast strains were isolated from a whey beverage and screened for α-L-rhamnosidase enzyme production. Of these, only four isolates were capable of producing the α-L-rhamnosidase enzyme by hydrolyzing naringin. Scanning electron microscopy images showed that the morphology of the yeast isolate (isolate No. 84) producing the greatest enzyme, changed from oval to filamentous in the presence of naringin. On the basis of morphological and molecular characterization (ITS sequencing), these four isolates were identified as Clavispora lusitaniae-84, Clavispora lusitaniae-B82, Candida sp.-86 and Candida hyderabadensis-S82). Fermentation parameters and the biochemical characterization of the α-L-rhamnosidase-producing yeast isolates were studied based on carbon substrate utilization profiles using BIOLOG phenotype microarray plates. Intra-species genetic diversity among the isolates was evaluated by whole genome analysis with repetitive DNA sequences (ERIC, REP and BOX) based DNA fingerprinting. On the basis of these results, it was found that these isolates of yeast producing L-rhamnosidase have a great potential application for beverage quality enhancement, and can build a strong foundation of α-L-rhamnosidase-producing yeast strains in the debittering of citrus juice.
The discovery of plasmin-like microbial fibrinolytic enzymes having high specificity and negligible side effects is crucial for thrombolytic therapy. Herein, we report one such extra-cellular fibrinolytic enzyme producing Bacillus cereus SRM-001 isolated from the blood-laden soil of a chicken dump yard. The potency of the enzyme was established with fibrin plate assay and in-vitro blood clot lysis assay. The shake-flask operating parameters and media composition were optimized for maximizing the productivity of the enzyme. The operating parameters, pH 7, 37°C, 1% inoculum volume and 24 h inoculum age, were found to be the optimum. The levels of media components, corn flour (0.3% w/v), soyabean powder (1.9% w/v) and MnSO4 (11.5 mM) were optimized by statistical analysis using Box-Behnken design derived RSM. This resulted in an almost 1.8 fold increase in fibrinolytic enzyme productivity. The 3D response surface plots showed soyabean powder and MnSO4 to be the key ingredients for enhancing the enzyme productivity, whereas corn flour had a marginal effect. The in-vitro blood clot lysis assay conducted at near physiological pH 7 at 37°C showed the enzyme to be a potential therapeutic thrombolytic agent.
Bacterial strains capable of utilizing glyphosate as the sole carbon source were isolated from contaminated soil by the enrichment culture method and identified based on partial 16S rRNA gene sequence analysis. Pseudomonas spp. strains GA07, GA09 and GC04 demonstrated the best degradation capabilities towards glyphosate and were used for the laboratory experiments of glyphosate bioremediation. Inoculating glyphosate-treated soil samples with these three strains resulted in a 2–3 times higher rate of glyphosate removal than that in non-inoculated soil. The degradation kinetics was found to follow a first-order model with regression values greater than 0.96. Cell numbers of the introduced bacteria decreased from 4.4 × 106 CFU/g to 3.4–6.7 × 105 CFU/g dry soil within 18 days of inoculation. Due to the intense degradation of glyphosate, the total dehydrogenase activity of the soil microbial community increased by 21.2–25.6%. Analysis of glyphosate degradation products in cell-free extracts showed that glyphosate breakdown in strain GA09 was catalyzed both by C-P lyase and glyphosate oxidoreductase. Strains GA07 and GC04 degraded glyphosate only via glyphosate oxidoreductase, but no further metabolite was detected. These results highlight the potential of the isolated bacteria to be used in the bioremediation of GP-contaminated soils.
In this study, the picocyanobacterial species composition of Lake Miyagase was examined by analyzing the 16S rRNA gene in a clone library and by amplicon sequencing using a benchtop next-generation sequencer. Five separate samples were analyzed from different days over a ten-month period. In the picocyanobacterial lineage, 9 and 12 OTUs were identified from a clone library and by amplicon sequencing, respectively. Both analyses suggested that a picocyanobacterium related to Synechococcus sp. MW6B4 was dominant in Lake Miyagase. Our findings suggest that 16S rRNA gene amplicon sequencing enables detailed evaluation of picocyanobacteria composition. One OTU identified was found to be a novel cluster that does not group with any of the known freshwater picocyanobacteria.
Two-component signal transduction systems (TCSs) represent one of the primary means by which bacteria sense and respond to changes in their environment, both intra- and extracellular. The highly conserved WalK (histidine kinase)/WalR (response regulator) TCS is essential for cell wall metabolism of low G+C Gram-positive bacteria and acts as a master regulatory system in controlling and coordinating cell wall metabolism with cell division. Waldiomycin, a WalK inhibitor, has been discovered by screening metabolites from actinomycetes and belongs to the family of angucycline antibiotics. In the present study, we have shown that waldiomycin inhibited autophosphorylation of WalK histidine kinases in vitro from Bacillus subtilis, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans at half-maximal inhibitory concentrations of 10.2, 8.8, 9.2, and 25.8 μM, respectively. Quantitative RT-PCR studies of WalR regulon genes have suggested that waldiomycin repressed the WalK/WalR system in B. subtilis and S. aureus cells. Morphology of waldiomycin-treated S. aureus cells displayed increased aggregation instead of proper cellular dissemination. Furthermore, autolysis profiles of S. aureus cells revealed that waldiomycin-treated cells were highly resistant to Triton X-100- and lysostaphin-induced lysis. These phenotypes are consistent with those of cells starved for the WalK/WalR system, indicating that waldiomycin inhibited the autophosphorylation activity of WalK in cells. We have also confirmed that waldiomycin inhibits WalK autophosphorylation in vivo by actually observing the phosphorylated WalK ratio in cells using Phos-tag SDS-PAGE. The results of our current study strongly suggest that waldiomycin targets WalK histidine kinases and inhibits the WalR regulon genes expression, thereby affecting both cell wall metabolism and cell division.
The pollution of land and water by petroleum compounds is a matter of growing concern necessitating the development of methodologies, including microbial biodegradation, to minimize the impending impacts. It has been extensively reported that fungi from polluted habitats have the potential to degrade pollutants, including petroleum compounds. The Red Sea is used extensively for the transport of oil and is substantially polluted, due to leaks, spills, and occasional accidents. Tidal water, floating debris, and soil sediment were collected from mangrove stands on three polluted sites along the Red Sea coast of Saudi Arabia and forty-five fungal isolates belonging to 13 genera were recovered from these samples. The isolates were identified on the basis of a sequence analysis of the 18S rRNA gene fragment. Nine of these isolates were found to be able to grow in association with engine oil, as the sole carbon source, under in vitro conditions. These selected isolates and their consortium accumulated greater biomass, liberated more CO2, and produced higher levels of extracellular enzymes, during cultivation with engine oil as compared with the controls. These observations were authenticated by gas chromatography-mass spectrophotometry (GC-MS) analysis, which indicated that many high mass compounds present in the oil before treatment either disappeared or showed diminished levels.
Tamarix ramosissima is a tree species that is highly resistant to salt and drought. The Tamarix species survives in a broad range of environmental salt levels, and invades major river systems in southwestern United States. It may affect root-associated bacteria (RB) by increasing soil salts and nutrients. The effects of RB on host plants may vary even under saline conditions, and the relationship may be important for T. ramosissima. However, to the best of our knowledge, there have been no reports relating to T. ramosissima RB and its association with salinity and nutrient levels. In this study, we have examined this association and the effect of arbuscular mycorrhizal colonization of T. ramosissima on RB because a previous study has reported that colonization of arbuscular mycorrhizal fungi affected the rhizobacterial community (Marschner et al., 2001). T. ramosissima roots were collected from five locations with varying soil salinity and nutrient levels. RB community structures were examined by terminal restriction fragment (T-RF) length polymorphism, cloning, and sequencing analyses. The results suggest that RB richness, or the diversity of T. ramosissima, have significant negative relationships with electrical conductivity (EC), sodium concentration (Na), and the colonization of arbuscular mycorrhizal fungi, but have a significant positive relationship with phosphorus in the soil. However, at each T-RF level, positive correlations between the emergence of some T-RFs and EC or Na were observed. These results indicate that high salinity decreased the total number of RB species, but some saline-tolerant RB species multiplied with increasing salinity levels. The ordination scores of nonmetric multidimensional scale analysis of RB community composition show significant relationships with water content, calcium concentration, available phosphorus, and total nitrogen. These results indicate that the RB diversity and community composition of T. ramosissima are affected by soil salinity and nutrient levels. Sequence analysis detected one Bacteroidetes and eight Proteobacteria species. Most 16S rRNA gene sequences had high similarities with the bacteria isolated from saline conditions, indicating that at least a portion of the RB species observed in T. ramosissima was halotolerant.
We have previously demonstrated conjugation of Escherichia coli into vacuoles of the protozoal ciliate (Tetrahymena thermophila). This indicated a possible role of ciliates in evoking bacterial quorum sensing, directly connecting bacterial survival via accumulation in the ciliate vacuoles. We therefore assessed if ciliates promoted bacterial autoinducer (AI)-2 accumulation with vacuole formation, which controls quorum sensing. E. coli AI-2 accumulation was significantly enhanced in the supernatants of a mixed culture of ciliates and bacteria, likely depending on ciliate density rather than bacterial concentration. As expected, AI-2 production was significantly correlated with vacuole formation. The experiment with E. coli luxS mutants showed that ciliates failed to enhance bacterial AI-2 accumulation, denying a nonspecific phenomenon. Fluorescence microscopy revealed accumulation of fragmented bacteria in ciliate vacuoles, and, more importantly, expulsion of the vacuoles containing disrupted bacteria into the culture supernatant. There was no increase in the expression of luxS (encoding AI-2) or ydgG (a transporter for controlling bacterial export of AI-2). We conclude that ciliates promote bacterial AI-2 accumulation in a mixed culture, via accumulation of disrupted bacteria in ciliate vacuoles followed by expulsion of the vacuoles, independently of luxS or ydgG gene induction. This is believed to be the first demonstration of a relationship between E. coli AI-2 dynamics and ciliates. In the natural environment, ciliate biotopes may provide a survival advantage to bacteria inhabiting such biotopes, via evoking quorum sensing.