Microbiology

The genome of the obligately methanotrophic bacterium Methyloferula stellata AR4 encodes thirty glycoside hydrolases. The closest homologues for most of these proteins belong to other members of the class Alphaproteobacteria. Two exceptions are represented by the genes encoding glycoside hydrolases of the families GH39 and GH65, which presumably appeared in M. stellata AR4 due to the lateral transfer from distantly related bacteria. This work was devoted to the study of the evolutionary history of these two genes. In the case of a member of the GH65 family of glycoside hydrolases, this scenario was not confirmed. Kojibiose phosphorylase encoded by this gene is common among Alphaproteobacteria. The suggested lateral transfer of the corresponding gene had an opposite direction, into one of the evolutionary lineages of the class Betaproteobacteria. The potential glycoside hydrolase of the GH39 family was shown to be the only one which gene is not of proteobacterial origin. The role of lateral transfers in the evolution of glycoside hydrolases and their homologues in methanotrophs and other bacteria is discussed.

Inactivation of one of the genes (CDS AZOBR_p60123) of the ABC transporter ATP-binding protein Wzt in the p60123::aphAI mutant of Azospirillum baldaniorum Sp245 and the introduction of an additional copy of this gene into the parent or mutant strain affected the chain-length heterogeneity of O polysaccharides (OPSs) and lipopolysaccharides (LPSs), the OPS and LPS immunochemical characteristics, and the strain properties related to polysaccharide production (biofilm construction). Biofilms of the p60123::aphAI mutant Sp245.4-1-1 accumulated two times less biomass than those of Sp245. Introduction of pRK415-p60123 into the cells of Sp245.4-1-1 or Sp245 resulted in a respective increase or decrease in the ability of the resulting derivative strains to accumulate biofilm biomass, as compared with the original strains.

Strain K-11 was isolated from the highly saline brown semi-desert soil of the Astrakhan region. Based on analysis of the 16S rRNA gene sequence, this strain was identified as Streptomyces carpaticus K-11 RCAM04697 (SCPM-O-B-9993). Whole genome sequencing of the strain was performed. Phytotoxicity, antiviral, antioxidant, antifungal, and insecticidal activities of the strain were studied. All extracts and suspensions of S. carpaticus strain RCAM04697 had plant-stimulating activity. Antiviral properties was exhibited as suppression of development and propagation of viral pathogens in laboratory conditions: Tomato Mosaic Virus (ToMV) – 26.3%, Cucumber Mosaic Virus (CMV) – 33.8%, Y-Potato Virus (YVK) (Potato Y potyvirus, PVY) – 51.3%, Potato X-Virus (PVX) (Potato X potyvirus, PVX) – 41.3%. The highest antioxidant activity was shown by a suspension of S. carpaticus strain RCAM04697 (88.8%) and its aqueous-alcoholic (20 : 80) extract (76.0%). The strain inhibited growth of the phytopathogenic fungus Fusarium sporotrichioides to varying degrees. The insecticidal activity against Aphis fabae after 6 h of treatment was 100% in the variants with suspension treatment, water-alcohol (80 : 20, 50 : 50), methanol, and hexane extracts. The metabolites of the S. carpaticus RCAM04697 strain included flavonoids, alkaloids, glycosides, organic acids (isocitric, acetic, fumaric, lactic, pyruvic, and malic), alcohols, aldehydes, hydrocarbons, ethers, sulfur-containing compounds, and other groups of low-molecular weight organic compounds.

Research on the microorganisms inhabiting deep aquifers is based on sampling the water released from deep wells and is seldom concerned with the physicochemical processes of the water-rock system. The issue of metabolism of aerobic prokaryotes revealed in deep habitats by molecular techniques remains unclear. Cultivation is required for direct determination of relation of prokaryotes to oxygen. In the present work, aerobic and anaerobic bacteria, which were revealed in thermal radon baths of the Belokurikha resort by molecular techniques, were isolated. Profiling by the 16S rRNA gene revealed predominance of members of the Deionococcus-Thermus group belonging to the genus Meiothermus (17.6% reads) and considered strictly aerobic. Anaerobic sulfate-reducing Thermodesulfovibrio were also present in the sample. The habitat was characterized by reductive, alkaline conditions. Target-oriented cultivation revealed aerobically growing Meiothermus sp. 1165, which was closely related to Meiothermus cerbereus. An alkaliphilic anaerobic sulfate reducer Thermodesulfovibrio sp. 1176 was also isolated. The rate of sulfate reduction measured in the Belokurikha water using yielded the value of 41.4 ± 1.06 µm S_red L^–1 day^–1, or 1.29 nmol S mL^–1 day^–1. Analysis of the genome of strain 1176 revealed the presence of various mechanisms responsible for its relative resistance to oxygen and oxidative stress, which included superoxide reductase, rubredoxin, a Fe-Mn family superoxide dismutase, a KatG catalase-peroxidase, and a cytochrome bd ubiquinol oxidase. The low redox potential and intense anaerobic sulfate reduction provide evidence for the generally reduced conditions in the Belokurikha deep horizons. Spatial separation of aerobes and anaerobes in the water-rock system, similar to the one occurring in the terrestrial microbial mats, may be hypothesized, as well as occurring of aerobic processes in microniches.

—Microorganisms associated with algae and able to utilize complex substrates (e.g., plant heteropolysaccharides) may be important producers of hydrolytic enzymes. The microbial communities of the red algae Corallina sp. and Phyllophora sp. sampled in the Kandalaksha Gulf basin of the White Sea were analyzed using high-throughput sequencing of the V4-variable region of the 16S rRNA gene. The dominant phyla in microbiomes of both samples were Pseudomonadota and Bacteroidota (GTDB classification, https://gtdb.ecogenomic.org/). For the Corallina sp. sample, dominance of the Vibrio, Agarivorans, and Photobacterium genera was shown, while Granulosicoccus and Aliivibrio dominated in the Phyllophora sp. sample. The analyzed red macroalgae with associated microbiota were used as an inocula to obtain microbial enrichment cultures growing on β-1,4-xylan or β-1,3-glucan (сurdlan). It was shown that, similar to environmental samples Pseudomonadota and Bacteroidota phyla representatives were prevalent in all enrichment cultures. However, unlike the environmental samples, in the enrichment cultures the dominant genera were Marinomonas, Reinekea, Polaribacter, and Pseudoalteromonas. The latter, as well as the representatives of Vibrio sp., were isolated in pure cultures for which the xylanolytic activity was shown.

Plasma membrane H⁺-ATPase (PMA1), the key enzyme of yeast metabolism, undergoes multiple phosphorylation during biogenesis and functioning. In the course of this process, the ATPase is getting activated (glucose effect). We have employed site-directed mutagenesis to determine the functional role of potentially phosphorylable amino acid residues located in the extracytosolic L9-10 loop (846-SENWTD). The mutant enzyme forms were expressed at the plasma membrane to examine the effect of substitutions on biogenesis and ATPase activity. Immunobloting revealed that the mutant ATPase expression was not significantly impaired. In the absence of glucose, basal activity of the mutant enzymes differed insignificantly from that of the wild type. At the same time, regulation of the mutant E847A, T850A, and D851A enzymes has been impaired; the level of enzyme activation by glucose was lower by 2.0−2.5-fold. On the contrary, the S846A mutant displayed elevated basal activity, maintaining the ability to undergo further activation. These data indicate that these residues (especially, Ser-846, Thr-850, and Asp-851) are essential for the normal functioning of the PMA1 and its regulation by glucose.