Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from Man3GlcNAc2-PA to Man9GlcNAc2-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.
Acinetobacter baumannii has been well recognized as a problematic human pathogen and several reports has shown the incidence of multidrug and pandrug-resistant A. baumannii strains in infirmary infections. A. baumannii grows only on an air-liquid interface and does not form a contiguous biofilm. Extracellular matrices (ECM) and slanted glass coverslips are (SGC) used as biofilm substrates and biofilms have been investigated by SEM, confocal and crystal violet staining. ECM has shown enhanced biofilm formation under dynamic conditions rather than static conditions. SGC biofilm yield assay has shown higher levels of continuous layers and packed thicker biofilm formation with glass coverslip inserts, up to 1.7 to 3 times higher biofilm formation, than when compared with no glass coverslip inserts. A media immersed ECM study revealed that biofilm grown on extracellular matrixes formed thread-like pili structures, and that these structures had contact with the ECM and also showed excellent cell-to-cell interaction. In summary, A. baumannii showed higher biofilm formation capacities with ECM, while the prominent results were directly related with the biofilm formation capacity of A. baumannii. For the initial step of biofilm formation, adherence is an important factor and, consequently, strains with a comparatively high capability to adhere to extracellular matrices and slanted glass coverslips provide a new method of enhanced biofilm growth for in vitro assays. ECM can be used as a substrate for immersed biofilm formation studies and the SGC method for air-liquid interface exposed biofilm formation studies, and these substrates can provide better biofilm growth and easy handling for in vitro adherence and biofilm assays.
Alkyl hydroperoxidase reductase AhpD, which is functionally equivalent to the bacterial flavin-containing disulfide reductase AhpF, acts as a proton donor for the organic peroxide-scavenging alkyl hydroperoxidase AhpC. Although AhpD has long been demonstrated in Mycobacterium tuberculosis, its physiological and biochemical functions remain largely unknown in other actinobacteria, including Corynebacterium glutamicum, Streptomyces, and Mycobacterium smegmatis. Here, we report that C. glutamicum AhpD contributed to regenerate a variety of thiol-dependent peroxidase in the decomposition of peroxide by linking a dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide succinyltransferase (SucB)/NADH system through the cyclization of their own active site dithiol to the oxidized disulphide. The CXXC motif of AhpD was essential to maintain the peroxides reduction activity of thiol-dependent peroxidase. ΔahpD1ΔahpD2 mutants exhibited significantly decreased resistance to adverse stress conditions and obviously increased the accumulation of reactive oxygen species (ROS). The physiological roles of AhpD in resistance to adverse stresses, were corroborated by their induced expression under various stresses and their direct regulation under the stress-responsive ECF-sigma factor SigH. C. glutamicum AhpDs were disulfide oxidoreductases behaving like thioredoxin (Trx) in regenerating thiol-dependent peroxidase for stress response, which provides the theoretical basis for an in-depth study of the reduction system in ahpC-lacking bacteria.
The yeast Lipomyces accumulates triacylglycerols (TAGs) as intracellular fat globules, and these TAGs can be used as source materials for biodiesel production. In this study, we aimed to use this yeast to produce lipids from renewable resources. Using plate culture and micrograph methods, strains with a high lipid-accumulation ability were screened from 15,408 types of systems combining renewable resources, strains, and culture temperatures. The lipid-accumulation ability of the strains was estimated from the fat globule volume, which was calculated using a micrograph. The reliability of this method was examined, and strains with a high lipid-accumulation ability were identified for each renewable resource. Seventy-seven Lipomyces strains (7 deposit, 68 wild-type, 2 mutants) with a high lipid-accumulation ability were selected. A few strains possessed the ability to accumulate large amounts of TAGs from more than four different renewable resources. We found that strains with a high lipid-accumulation ability could efficiently convert consumed carbon sources into TAGs, which could be easily recovered from the fat globules of these strains through physical disruption.
Numerous microbes reside in the rhizosphere having plant growth promoting activity, and enhancing the property by increasing plant yield. Plant growth promoting rhizobacteria (PGPR) has gradually increased in agriculture and offers an attractive way to replace chemical fertilizers, pesticides and supplements. Soil was collected from the rhizosphere of an agricultural farm and the psychrotrophic bacterial strains STA3 (KY888133) and RM2 (KY888134) were successfully isolated, and screened on the basis of phosphate solubilization. Further characterization was carried out by morphological, biochemical, and 16S rDNA characterization methods. The unique nature of psychrotrophic Pentoea ananatis and a suitable combination with Pseudomonas fluorescens regarding plant growth promotion activity has not been studied before to our knowledge. An assessment of various parameters of plant growth promoting activity, such as IAA, phosphate solubilization, bio-control activity, HCN and siderophore production, has been carried out. Both strains were found to be positive in various parameters except HCN and Biocontrol activity, which were positive only for the strain RM2. Also, shelf life and their efficacy was determined before and after formulation. A great consistency was observed in all the cultures, even after 70 days of storage under bio-formulation at room temperature, while in the case of the co-culture CPP-2, the cfu ml-1 was greater, followed by RM2 and STA3. Moreover, the growth indices of the pea plant were found to be better in the co-culture CPP-2 compared with individual strains, followed by RM2 and STA3. Thus, the study suggests that the co-culture CPP-2 has a great potential for plant growth promotion as compared with individual strains followed by RM2 and STA3.
Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.
The prokaryotic and eukaryotic microbial communities of activated sludge in a chemical plant wastewater treatment facility, processing relatively oligotrophic wastewater containing aromatic compounds and high-strength bromide ions, were characterized by high-throughput sequencing of rRNA genes based on DNA and RNA extracts. The microbial community structure was distinct from those previously reported from domestic wastewater treatment plants. Several abundant OTUs in the RNA-based prokaryotic community were related to aromatic compound-degrading bacteria, which most likely contributed to the removal of recalcitrant chemicals from the wastewater. Furthermore, both prokaryotic and eukaryotic predators were highly abundant. These might promote stabilization of the microbial food chain and affect biomass in the activated sludge, maintaining the waste-removal function of the microbial community.