Vol 88, No 2 (2019)

The chemical structure of the extracellular reactivating factor (RF) from Luteococcus japonicus subsp. casei was determined; this factor promotes survival of a small subpopulation of the producer cells under lethal stress impact. For the isolation and purification of this RF, the previously developed method for RF isolation from Saccharomyces cerevisiae was optimized. A total of 15 fractions were obtained from the culture liquid of Luteococcus casei, two of which (I and IV) exhibited reactivation activity against the cells subjected to a lethal stress impact (UV irradiation). The method included solid-phase extraction of the peptides on a hydrophobic sorbent with the C₈ phase and subsequent multistage separation using RP-HPLC. Mass spectral analysis (MALDI-TOF) was used to determine the molecular characteristics of fraction IV. Efficient ionization was not achieved for fraction I. Mass charges for fraction IV were 773.394 and 788.102 Da. Edman automatic sequencing was used to identify these components as peptides: Ala-Pro-Asn-Glu-Asn-Gln-Gly and Ala-Pro-Asn-Glu-Glu-Gln-Gly. No similarity to any known full-size functional peptide molecules in the databases on polypeptide primary structures was revealed. Formation of biologically active peptides by L. casei may be associated with non-template synthesis and probably involves proteolysis of a large protein.

Phototrophic communities forming in the bottom of the Berikei highly mineralized mesothermal sulfide springs (Kayakent region, Dagestan, Russia) were investigated. The Berikei springs are an interesting example of combined effect of such factors as temperature, salinity, pH, and sulfide of occurrence and structure of phototrophic microbial communities. The water was of the sodium chloride type with salinity of 48‒97 g/L, near-neutral brine pH, and sulfide concentration of ~1 mM. The temperature at the stream bottom was as high as 60°C. Elevated temperature and high salinity limited the development of phototrophic communities. Formation of cyanobacterial mats occurred at temperatures below 54°C. Phormidium-like cyanobacteria and unicellular Synechocystis sp. predominated in the mats. The number of cyanobacterial species increased at desalination to 48‒57 g/L with emergence of the species morphologically resembling Spirulina sp., Leptolyngbya sp., and Oscillatoria sp. Among anoxygenic phototrophic bacteria, halophilic purple bacteria Ectothiorhodospira sp., Marichromatium sp., and Rhodovulum sp., green sulfur bacteria Prosthecochloris sp., and unidentified Chloroflexi were present. Oxygenic photosynthesis in the mats was not inhibited by sulfide. Production of cyanobacterial mats was up to 4.7‒53.8 µg/(cm² h). The contribution of anoxygenic phototrophic bacteria to photosynthetic production varied from 0 to 100%. The composition of this microbial community was compared to those of the phototrophic microbial communities of the mesothermal springs of the Dead Sea coast (Israel), the Washington warm lake (United States), and the Paoha Island hot springs (Mono Lake, United States.

Biofilm formation by monocultures and mixed cultures of nitrile-hydrolyzing bacteria Alcaligenes faecalis 2 and Rhodococcus ruber gt 1 was studied. Biofilm formation was assessed by staining with Crystal violet, by the energy state of the cells, and by the output of the polymer matrix. A quantitative method for assessment of the output of the polymer matrix by the fluorescence of konA-tetramethylrhodamine was developed. The medium used for obtaining mixed biofilms of Al. faecalis 2 and R. ruber gt 1 contained acetamide and glucose as carbon sources. In mixed biofilms Al. faecalis 2 was shown to act as a primary colonizer, while R. ruber gt 1 was a satellite microorganism. Compared to monoculture biofilms, mixed biofilms exhibited higher output of the polymer matrix and increased biofilm formation by Al. faecalis 2 and R. ruber gt 1 on addition of the culture liquid of the second strain. Daily addition of the toxic substrate acetonitrile to planktonic and biofilm cultures resulted in bacterial adaptation, as was indicated by higher numbers of surviving cells compared to the variants with acetonitrile addition every 10 min, mainly due to nitrile hydratase activity of R. ruber gt 1, which transformed acetonitrile into nontoxic acetamide. Our results show that binary biofilms of amidase-containing Al. faecalis 2 and R. ruber gt 1, a strain with high nitrile hydratase activity, are promising as biocatalysts for acrylic acid production and as the basis for a biofilter for nitrile removal from wastewater.

Anabiotic dormant forms (DF) of gram-negative bacterial epibionts of the smooth clawed frog Xenopus laevis—Stenotrophomonas sp. strain FM3 and Morganella morganii subsp. sibonii strain FF1, were obtained and characterized. The ultrastructural features of the dormant cells of strains FM3 and FF1 (large periplasmic space, dense heterogeneous cytoplasm, and ordered reorganization of the compacted nucleoid) were typical of the cystlike cells (CLC) of the previously studied other taxa of gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, P. fluorescens, Azospirillum brasilense , etc.). Preservation of potentially active enzymes of the antioxidant defense systems was not previously reported for anabiotic dormant forms. They are probably responsible for DF survival at early stages of their germination, providing for resistance to new medium stress and other stressors in the absence of de novo enzyme synthesis. Our results are of interest for both basic microbiology and biomedicine, since strains FM3 and FF1 are phylogenetically related to bacterial agents of nosocomial clinical infections and emergence of their dormant forms may explain resistance of nosocomial infections to sanitary treatment.

Iron is an essential micronutrient for all living organisms. The mechanisms of iron transport and homeostasis have been studied in detail in Saccharomyces cerevisiae yeasts, and iron metabolism in the strains isolated from different ecological niches was found to vary. In the present work, comparative analysis of iron homeostasis in the wine and flor yeast strains was carried out. Flor yeasts are characterized by their ability to grow as a biofilms on the surface of fortified wine materials, where they carry out oxidative metabolism. We have found that flor strains are more sensitive to elevated iron concentrations as compared to wine strains and accumulate iron more efficiently when grown in iron-limited media. These features correlate with the Q648X mutation in the Aft1p transcription factor, resulting in emergence of its truncated form and with the chromosomal rearrangement resulting in the deletion of the FRE-FIT cluster containing the Fre3p and Fre5p iron reductase genes and of the Fit2p and Fit3p membrane proteins responsible for accumulation of siderophore-bound iron in the cell wall. Our results indicate a significant adaptive role of these variations in the iron homeostasis genes for the physiology of flor yeasts, since selection of these organisms was accompanied by adaptation to growth on materials with low iron content, such as fortified fermented wort obtained from grapes grown in traditional areas of sherry wine-making, which is important for the preservation of specific properties of sherry wines.

Intertidal sponges, during tidal flux and filter feeding cycle, often get exposed to an influx of water of varying salinities containing halophilic microbes and suspended agricultural detritus. Microbes get established in the lumen of the host during its filter feeding process and might participate in the degradation of incoming particulate matter. We successfully isolated two haloarchaeal bionts, strains GUMFAS1 and GUMFAS7, producing cellulase-free xylanases from one such sponge, identified as Cinachyrella cavernosa inhabiting the intertidal rocky region of Anjuna, Goa-India. The bionts exhibited 4.791 and 8.714 U/mg of cellulase-free xylanase activity, respectively. Xylanase from GUMFAS7 (XS7) showed maximum activity at 37°C, pH 5 and 20% NaCl concentration. Zymographic analysis of XS7 revealed a presence of three bands with xylanase activity of molecular weight 238, 60, and 18 kDa, respectively. The strains were identified by 16S rRNA gene sequencing as Halococcus thailandensis (GUMFAS7) and Halorubrum saccharovorum (GUMFAS1). The latter was identical to another isolate obtained from the waters surrounding the host. These findings reflect a unique type of interaction between haloarchaea and sponge. An in-depth study on this cellulase-free xylanase is underway and expected to be of significance in the field of extremozymes.