Various aerobic culturable bacteria (1,133 isolates) were isolated from the gut of Apostichopus japonicus (black adult, green adult, black small, green small, black juvenile, and green juvenile sea cucumbers) and from the sea sediment and the seawater using different culture conditions and without enrichment culture. By molecular analysis of partial 16S rRNA gene sequences of 231 isolates, they were tentatively affiliated with 53 described species in the phyla Firmicutes (42 species), Proteobacteria (9 species) and Actinobacteria (2 species). Eighteen species were often found among the intestines and the sea sediment. High diversity was observed in the genus Bacillus (20 species), Oceanobacillus and Virgibaillus but there were no isolates affiliated to members of the genus Vibrio, well-known sea pathogens. There were no clear differences in the bacterial communities among the hosts varied in size and color. Most isolates showed various polysaccharide degradation activities, suggesting their possible contributions in the digestion of organic matters in the gut.
A highly sensitive and specific multiplex PCR assay has been developed to detect the presence of Escherichia coli O157:H7 from naturally contaminated raw milk samples within 10 h. The primers explored in the assay were targeted against the uidR gene specific for all types of E. coli and the fliCH7 gene specific for the h7 flagellar antigen of E. coli O157:H7. The multiplex PCR assay developed was found to be highly specific as it produced PCR products of 152 bp (E. coli specific) and 625 bp (E. coli O157:H7 specific). The assay was tested for its specificity against different serotypes of E. coli as well as other pathogenic strains like Salmonella, Shigella, Klebsiella, Enterobacter, Staphylococcus aureus, Lactobacillus and Lactococcus etc. When this multiplex PCR assay was directly applied to 24 raw milk samples collected from different sources, E. coli O157:H7 could be detected in one of the milk samples without 4 h enrichment in CT-SMAC broth and three samples after 4 h enrichment in CT-SMAC broth. However, all the pasteurized milk samples gave a negative signal for this organism.
A soil metagenomic library was constructed and two functionally diverse lipase genes, SMlipB and SMlipD, were screened by a function-driven approach and characterized. The optimal temperature for enzyme activity of SMlipB and SMlipD was 50°C and 30°C, respectively, and optimal pH was determined to be 7.0 and 9.0, respectively. Both enzymes exhibited broad substrate specificity and showed enhanced activity in the presence of SDS and Tween 20. The SMlipB enzyme was highly resistant to many organic solvents, especially isopropanol, ethanediol, DMSO, methanol and xylene, whereas SMlipD activity was inhibited in all the solvents except xylene. Sequence analysis revealed SMlipB consisted of an open reading frame of 1,212 bp and encoded for 404 amino acids. It contained the GXXGXD motifs, which are supposed to be involved in Ca2+ binding in proteases and lipases, and an extreme C-terminal motif consisting of a negatively charged amino acid followed by four hydrophobic residues, essential for the secretion of metalloprotease, and belongs to lipase subfamily I.3. SMlipD contained 1,071 bp ORF and encoded for 357 amino acids. It contains Ca2+ ion binding sites extending from amino acid 282 to 294 and two Cystein residues (218,308), proven necessary for forming a disulfide bridge and belongs to lipase subfamily1.2.
A novel actinobacterial strain, designated YM16-381T, was isolated from sediments of Lake Nakaumi in Shimane Prefecture, Japan. The cell of the strain was motile, non-spore-forming and Gram-positive. The colony was gray-pink and circular on marine agar 2216 medium. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain belongs to the family Dermatophilaceae of the suborder Micrococcineae. The highest sequence similarity value of the isolate was 96.2% against Kineosphaera limosa. The diaminopimelic acid in the cell wall was meso-A2pm. The major menaquinone was MK-8(H4). The DNA G+C contents were 70.5 mol%. The major cellular fatty acids were C17:1ω8c, C16:0, C15:0 and C18:1ω9c. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol and ninhydrin-positive phosphoglycolipid. On the basis of polyphasic taxonomic studies, strain YM16-381T represents a novel species of the genus Kineosphaera within the family Dermatophilaceae, for which the name Kineosphaera nakaumiensis sp. nov. is proposed. The type strain is YM16-381T (=KCTC 29138T=NBRC 109121T).
The mechanism of bacterial resistance to tributyltin (TBT) is still unclear. The results herein presented contribute to clarify that mechanism in the TBT-resistant bacterium Aeromonas molluscorum Av27. We have identified and cloned a new gene that is involved in TBT resistance in this strain. The gene is highly homologous (84%) to the Aeromonas hydrophila-sugE gene belonging to the small multidrug resistance gene family (SMR), which includes genes involved in the transport of lipophilic drugs. In Av27, expression of the Av27-sugE was observed at the early logarithmic growth phase in the presence of a high TBT concentration (500 μM), thus suggesting the contribution of this gene for TBT resistance. E. coli cells transformed with Av27-sugE become resistant to ethidium bromide (EtBr), chloramphenicol (CP) and tetracycline (TE), besides TBT. According to the Moriguchi logP (miLogP) values, EtBr, CP and TE have similar properties and are substrates for the sugE-efflux system. Despite the different miLogP of TBT, E. coli cells transformed with Av27-sugE become resistant to this compound. So it seems that TBT is also a substrate for the SugE protein. The modelling studies performed also support this hypothesis. The data herein presented clearly indicate that sugE is involved in TBT resistance of this bacterium.
A polyphasic molecular approach was used in order to characterize and taxonomically assign Debaryomyces yeast isolates of different origins. Actin 1 (ACT1) gene sequences coupled with AFLP markers showed that the investigated yeasts belonged to the recently reinstated species D.hansenii, D.fabryi and D.tyrocola. The strain HA1179 was supposed to be a D.hansenii strain with introgressed D.fabryi DNA segments. This strain acquired ribosomal RNA encoding genes (rDNA) and the ACT1 gene from the species D.fabryi and D.hansenii respectively. Comparative sequence analysis of the ACT1 gene, ITS1-5.8S-ITS2 (5.8S-ITSs) and D1/D2 regions, suggested that five strains isolated from a municipal wastewater treatment plant could represent a new taxon of the genus, for which the name Debaryomycesvindobonensis was proposed. The calculated degree of similarity between the AFLP patterns indicated that the strains of D.vindobonensis and the closely related species were separated by the values ＜0.5. New yeast isolates showed very similar morphological and physiological properties to related Debaryomyces species. They differed notably only by the assimilation of rhamnose and growth at 50% glucose. In contrast to the other species, D.vindobonensis was unable to assimilate starch.
Klebsiella oxytoca Rs-5 isolated with ACC (1-aminocyclopropane-1-carboxylate) deaminase activity as the sole nitrogen source could obviously promote cotton seedling growth under salt stress and produce phytohormone indole-3-acetic acid (IAA). The amount of IAA produced by the strain Rs-5 was measured, and the effect of IAA on cotton growth under salt stress was studied. Different treatments were set to treat cotton seeds with fermentation broth containing strain Rs-5 (FB), strain Rs-5, fermentation broth with bacteria removed (FB-NB), fermentation broth without bacteria or IAA (FB-NB-NI) and single IAA solutions (SI) according to the IAA concentration after strain Rs-5 culturing of 48, 72 and 120 h. The germination rate, dry weight, plant height, root length and malondialdehyde (MDA), proline and endogenous IAA content in roots were determined. The results showed that both IAA produced by strain Rs-5 and the strain were effective in promoting cotton growth under salt stress. The growth and ability to resist salt stress of cotton seedlings were increased with the enhancement of IAA concentration. The treatment of FB containing bacteria and IAA at 120 h obtained the best state of cotton growth, when the IAA content was the highest in the fermentation broth (42.14 μg·L–1). The germination rate, dry weight, plant height and root length were increased by 29.4%, 24.3%, 27.2% and 27.2% , respectively, compared to the saline control. The strain Rs-5 and/or IAA could obviously reduce the MDA and proline content and increase the endogenous IAA content in cotton seedlings. However, the efficacy of other components in the fermentation broth was inconspicuous.
Taxonomic analysis of budding yeast strains isolated from flowers of Wisteria sinensis (Fabales, Fabaceae) abundantly visited by flying insects, mainly bees in city parks of Baku is described. The isolates forming slightly pink colonies and propagating by budding represent a hitherto unknown yeast species for which the name Starmerella caucasica is proposed. The sequences of the D1/D2 domains of the large subunit rRNA genes and the ITS1-5.8S-ITS2 regions were highly similar in the isolates and indicated a close relationship with Candida kuoi and Starmerella bombicola in the phylogenetic analysis. S. caucasica can be separated from these species by its growth on glucosamine and D-tryptophan, in vitamin-free medium and at 37°C, and its inability to grow on citrate, ethylamine, cadaverine and in media supplemented with 0.01% of cycloheximide. The type strain is 11-1071.1T. It has been deposited in Centralbureau voor Schimmelcultures (Utrecht, the Netherlands) as CBS 12650T, the National Collection of Agricultural and Industrial Microorganisms (Budapest, Hungary) as NCAIM Y.02030T and the Culture Collection of Yeasts (Bratislava, Slovakia) as CCY 90-1-1T. The GenBank accession numbers for nucleotide sequences of S. caucasica are JX112043 (D1/D2 domain of the 26S rRNA gene) and JX112044 (ITS1-5.8S-ITS2). Mycobank: MB 800536.
Genetic immobilization of the yeast RNase Rny1p was performed by creating a hybrid protein containing the signal sequence of the S. cerevisiae cell wall protein Ccw12p followed by the catalytic part of the Rny1p (amino acids 19 to 293) and additionally 73 amino acids of the Ccw12p including the GPI-anchoring signal. The construct was expressed in S. cerevisiae VMY5678 and the hybrid protein was secreted through the plasma membrane and incorporated into the cell wall through GPI-anchoring in the same way as the Ccw12p. Thus, it could be released from the wall by β-1,3-glucanase. It retained RNase activity with the optimal pH of about 9 and the optimal temperature at 60°C. It was significantly more stable than the wild type enzyme and retained activity at 50°C for at least 6 hours; at 60°C it maintained full activity for at least 4 h, and at 70°C it lost activity in about 2 h. No DNase activity of the Rny1/Ccw12p was detected. Yeast cells expressing the hybrid protein were successfully used instead of RNase A in a standard procedure for yeast chromosomal DNA preparation with the advantage of quick and easy quantitative removal of the RNase activity from the reaction mixture.
In view of antibiotic resistance among pathogens, the present study is to address the toxicity of Ag2O nanoparticles against the Gram-positive and Gram-negative bacteria through in vitro assays. The preliminary screening by agar diffusion assay confirms the antibacterial activity of Ag2O nanoparticles against all the test bacteria. Comparative antibacterial activity of Ag2O nanoparticles and respective antibiotics reveals their broad range of activity and lower inhibitory dose against the used bacterial strains. Further, they can inhibit E.coli with an effective dose of 0.036 mg/ml within 1 h of exposure time as determined by luciferin based ATP assay. Moreover, the Ag2O nanoparticles exhibit higher antibacterial efficacy against Gram-negative bacteria than Gram-positive bacteria, as revealed by their MIC & MBC values. Therefore, Ag2O nanoparticles pave the way for a new generation of antibacterial agents against the emerging multidrug resistant pathogens.