Proteomic and transcriptomic analyses of Cutibacterium acnes biofilms and planktonic cultures in presence of epinephrine.

Transcriptomic and proteomic analysis were performed on 72 h biofilms of the acneic strain Cutibacterium acnes and planktonic cultures in the presence of epinephrine. Epinephrine predominantly downregulated genes associated with various transporter proteins. No correlation was found between proteomic and transcriptomic profiles. In control samples, the expression of 51 proteins differed between planktonic cultures and biofilms. Addition of 5 nM epinephrine reduced this number, and in the presence of 5 µM epinephrine, the difference in proteomic profiles between planktonic cultures and biofilms disappeared. According to the proteomic profiling, epinephrine itself was more effective in the case of C. acnes biofilms and potentially affected the tricarboxylic acid cycle (as well as alpha-ketoglutarate decarboxylase Kgd), biotin synthesis, cell division, and transport of different compounds in C. acnes cells. These findings are consistent with recent research on Micrococcus luteus, suggesting that the effects of epinephrine on actinobacteria may be universal.

Epinephrine affects gene expression levels and has a complex effect on biofilm formation in M icrococcus luteus strain C01 isolated from human skin.

In this study, the effect of epinephrine on the biofilm formation of Micrococcus luteus C01 isolated from human skin was investigated in depth for the first time. This hormone has a complex effect on biofilms in various systems. In a system with polytetrafluoroethylene (PTFE) cubes, treatment with epinephrine at a physiological concentration of 4.9 × 10-9 M increased the total amount of 72-h biofilm biomass stained with crystal violet and increased the metabolic activity of biofilms, but at higher and lower concentrations, the treatment had no significant effect. On glass fiber filters, treatment with the hormone decreased the number of colony forming units (CFUs) and changed the aggregation but did not affect the metabolic activity of biofilm cells. In glass bottom plates examined by confocal microscopy, epinephrine notably inhibited the growth of biofilms. RNA-seq analysis and RT-PCR demonstrated reproducible upregulation of genes encoding Fe-S cluster assembly factors and cyanide detoxification sulfurtransferase, whereas genes encoding the co-chaperone GroES, the LysE superfamily of lysine exporters, short-chain alcohol dehydrogenase and the potential c-di-GMP phosphotransferase were downregulated. Our results suggest that epinephrine may stimulate matrix synthesis in M. luteus biofilms, thereby increasing the activity of NAD(H) oxidoreductases. Potential c-di-GMP pathway proteins are essential in these processes.

[Regulation of Biofilm Formation by Pseudomonas chlororaphis in an vitro System].

The mutants of Pseudomonas chlororaphis 449 with completely or partially suppressed accumulation of N-acyl homoserine lactones exhibited the absence or a pronounced decrease of their capacity for stimulation of biofilm growth in the presence of azithromycin. Biofilms of the wild type strain preformed in the presence of the stimulatory azithromycin concentrations exhibited more intense staining with a polysaccharide-specific dye 1,9-dimethyl methylene blue (DMMB) and were more resistant to heat shock. These findings indicate accumulation of the structural matrix polysaccharides, which play a protective role under the conditions of thermal shock. Extremely low azithromycin concentrations (0.001-0.01 µg/mL) inhibit biofilm formation by P. chlororaphis 449 and P. chlororaphis 66 with suppression of the synthesis of DMMB-staining polysaccharides.

[Activation of formation of bacterial biofilms by azithromycin and prevention of this effect].

Growth of members of most of the studied genera of gram-positive (Dietzia, Kocuria, and Rhodo- coccus) and gram-negative bacteria (Pseudomonas and Chromobacterium) in biofilms exhibited higher resistance to an translation inhibitor, azithromycin compared to the growth of planktonic cultures of the same strains. Low concentrations of azithromycin were found to stimulate biofilm formation by the studied saprotrophic strains. The rate of synthesis of the polysaccharide matrix component exceeded the rate of cell growth, indicating implementation of the biofilm phenotype under these conditions. It was found that an alkylhydroxybenzene (AHB) compound 4-hexylresorcinol was capable of almost uniform suppression of growth of both planktonic cultures and biofilms of the saprotrophic strains under study. In some cases, combined action ofazithromycin and AHB resulted in an additive inhibitory effect and prevented the stimulation of biofilm growth by subinhibitory azithromycin concentrations. Thus, AHB may be considered a promising antibiofilm agent.

[Multi-Species Biofilms in Ecology, Medicine, and Biotechnology].

The structure, composition, and developmental patterns of multi-species biofilms are analyzed, as well as the mechanisms of interaction of their microbial components. The main methodological approaches used for analysis of multi-species biofilms, including omics technologies, are characterized. Environmental communities (cyanobacterial mats and methanotrophic communities), as well as typical multi-species communities of medical importance (oral cavity, skin, and gut microbiomes) are described. A special section deals with the role of multi-species biofilms in such biotechnological processes as wastewater treatment, heavy metal removal, corrosion control, and environmental bioremediation.

[Antimicrobic features of phenolic lipids].

The connection between the efficiency of phenolic lipids (PL) and their hydrophobic property (solubility) and hydrophobic property of microorganisms' cell structure is shown. The mixture of amphiphilic di(oxiphenil)-phenil-methanes, which act bacteriostatically under 15 mg/l, possesses maximal efficiency against Staphylococcus aureus. Against Mycobacterium smegmatis with hydrophobic cell wall, hydrophobic 2,4-dialkylocibenzol 70 mg/l was the most effective. Hexylresorcin (HR) stops the development of gram-positive bacteria in concentrations 20-50 mg/l, that of gram-negative bacteria in concentration 65 mg/l, that of M. smegmatis at 70 mg/l, and that of yeast and fungi at 300 mg/l. HR prevails bacteria spores germination in the concentration 25-100 mg/l. The dependence of antibacterial action of isomers and homologues of alkylresorcins on their structure--number, position, and length of alkyl substituents--is studied.

[Resistance of the petroleum-oxidizing microorganism Dietzia sp. to hyperosmotic shock in reconstituted biofilms].

A number of halotolerant and halophilic bacterial strains were isolated from the Romashkinskoe oil field (Tatarstan) stratal waters having a salinity of up to 100 g/l. The isolation of pure cultures involved biofilm reconstitution on M9 medium with paraffins. The associations obtained were dispersed and reinoculated onto solid media that contained either peptone and yeast extract (PY) or paraffins. It was shown that such associations included both oil-oxidizing bacteria and accompanying chemoheterotrophic bacteria incapable of oil oxidation. The pure cultures that were isolated were used for creating binary biofilms. In these biofilms, interactions between halophilic and nonhalophilic bacteria under hypo- and hyperosmotic shocks were investigated. We conducted a detailed study of a biofilm obtained from an oil-oxidizing halotolerant species (with an upper growth limit of 10-12% NaCl) identified as Dietzia sp. and an extremely halophilic gram-negative bacterium (growing within the 5-20% NaCl concentration range) of the genus Chromohalobacter that did not oxidize paraffins. If these microorganisms were grown in a mixed suspension (planktonic) culture that was not supplemented with an additional amount of NaCl, no viable cells of the halophilic microorganism were detected after reinoculation. In contrast, only halophilic cells were detected at a NaCl concentration of 15%. Thus, no mutual protective influence of the microorganisms manifested itself in suspension culture, either under hypo- or under hyperosmotic shock. Neither could the halophile cells be detected after reinoculating a biofilm obtained on a peptone medium without addition of NaCl. However, biofilms produced at a NaCl concentration of 15% contained approximately equal numbers of cells of the halophilic and halotolerant organisms. Thus, the halophile in biofilms sustaining a hyperosmotic shock exerts a protective influence on the halotolerant microorganism. Preliminary data suggest that this effect is due to release by the halophile of osmoprotective substances (ectoine and glutamate), which are taken up by the halotolerant species. Such substances are diluted by a large medium volume in suspension cultures, whereas, in biofilms, their diffusion into the medium is apparently hampered by their interaction with the intercellular polymer matrix.

[Changes in physicochemical properties of proteins, caused by modification with alkylhydroxybenzenes].

Kinetic characteristics of model enzymes and physicochemical properties of globular proteins modified by chemical analogues of low-molecular-weight microbial autoregulators (alkylhydroxybenzenes, AHBs) have been studied. C7 and C12 AHB homologues were used, differing in the length of the alkyl radical and the capacity for weak physicochemical interactions. Both homologues affected the degree of protein swelling, viscosity, and the degree of hydrophobicity. The effects depended on the structure of AHBs, their concentration, and pH of the solution, which likely reflects changes in the charge of the protein globule and its solvate cover. Variations of hydrophobicity indices of AHB-modified enzymes (trypsin and lysozyme) were coupled to changes in the catalytic activity. The values of K(M), measured for the enzymes within both AHB complexes, did not change, whereas V(max) increased (in the case of C7 complexes) or decreased (C12 complexes). Possible molecular mechanisms of changes in the physicochemical and catalytic parameters of enzymatically active proteins, induced by modification with structurally distinct AHBs, are described, with emphasis on targeted regulation of functional activity.

[Effect of bacterial satellites on Chlamydomonas reinhardtii growth in an algo-bacterial community].

The growth characteristics of an algo-bacterial community (Chlamydomonas reinhardtii and bacterial satellites) were studied, as well as the mechanism and patterns of bacterial effect on algae. Four strains of predominant bacteria were isolated and partially characterized. They were assigned to the following taxa: Rhodococcus terrea, Micrococcus roseus, and Bacillus spp. A pure culture of the alga under study was obtained by plating serial dilutions on agarized media with ampicillin. Within the algo-bacterial association, the alga had a higher growth rate (0.76 day(-1)) and yield (60 microg chlorophyll/ml culture) than in pure cultures (0.4 day(-1) and 10 microg chlorophyll/ml culture, respectively). The viability of the algal cells within the association was retained longer than in pure culture. Among the isolated bacterial satellites, strains B1 and Y1, assigned to the species Rhodococcus terrae, had the highest stimulatory effect on algal growth. The culture liquid of bacteria incubated under the conditions not permitting growth stimulated algal growth; the culture liquid of actively growing bacteria had an opposite effect.

[Biofilm--"City of microbes" or an analogue of multicellular organisms?].

The definition of the term "biofilm" and the validity of the analogy between these structured microbial communities and multicellular organisms are discussed in the review. The mechanisms of biofilm formation, the types of interrelations of the components of biofilms, and the reasons for biofilm resistance to biocides and stress factors are considered in detail. The role of biofilms in microbial ecology and in biotechnology is discussed.

["Oxygen regulation" of the respiratory chain composition in the yeast Debaryomyces hansenii under multiple stress].

It was shown that two stress factors, hypoxia and hyperosmotic shock, if applied simultaneously to the yeast Debaryomyces hansenii, display an antagonistic mode of interaction, which results in an increased degree of halophily of this microorganism under microaerobic conditions. Studies of the effects of respiration inhibitors (sodium azide and salicyl hydroxamic acid, SHA) and of the pattern of changes in the composition of the respiratory chain of Debaryomyces hansenii under the stated stress conditions led to the suggestion of three (or four) chains of electron transfer functioning simultaneously in the cell: the classical respiratory chain involving cytochrome-c oxidase, an alternative respiratory chain involving a cyanide- and azide-resistant oxidase, and additional respiratory chains involving oxidases resistant to salt, azide and SHA. Thus, the antagonistic mode of interaction between hypoxia and hyperosmotic shock results from the redirection of the electron flow from the salt-susceptible respiratory systems to the salt-unsusceptible ones encoded by "the hypoxia genes" and activated (induced) under microaerobic conditions.

[Influence of the degree of aeration on halotolerance of yeasts of the genera Candida, Rhodotorula, and Malassezia]. / Vliianie stepeni aératsii sredy na galotolerantnost' drozhzhei rodov Candida, Rhodotorula i Malassezia.

The biochemical mechanisms were studied that determine different reactions of yeasts of different genera to two simultaneously imposed stressors, hypoxia and osmotic shock. For Candida lipolytica, these two stressors were antagonistic, which resulted in stimulation of yeast growth by NaCl (in a wide range of concentrations) under microaerobic conditions. The reaction of Malassezia sp. was different: the degree of halotolerance of this microorganism was lower under microaerobic conditions. An intervening reaction pattern was characteristic of Rhodotorula aurantiaca. These differences were found to be determined, above all, by the induction of a salt-resistant respiratory system (oxidase) in Candida lipolytica, which could not be induced in Malassezia sp. In addition, the synthesis of catalase was enhanced in Candida lipolytica, which provided for neutralization of the active forms of oxygen accumulating as a result of inhibition of other protective enzymes by salt.

[Antagonistic interactions between stress factors during the growth of microorganisms under conditions simulating the parameters of their natural ecotopes]. / Antagonistitseskoe vzaimodeistvie stressornykh faktorov pri roste mikroorganizmov v usloviiakh, imitiruiushchikh parametry ikh prirodnykh ékotopov.

Two stress factors, hypoxia (microaerobic conditions) and a high salt concentration, if applied simultaneously to aerobic microorganisms, display an antagonistic mode of interaction. As a result, the NaCl level that is usually optimal for moderate halophiles (5-6%) becomes optimal for the growth of weak halophiles (Rhodococcus erythropolis and Shewanella sp. CN32); the halotolerant yeast Yarrowia lypolytica acquires halophilic properties (with a growth optimum at a NaCl concentration of 10%), and the growth rate of the extremely halophilic Halobacterium salinarum increases at supraoptimal salt concentrations (25-34%). This phenomenon is apparently due to multiple changes in metabolic reactions. In particular, high salt concentrations suppress respiration and the formation of enzymes (superoxide dismutase and catalase) that protect the cell from toxic oxygen species. Therefore, establishment of microaerobic conditions compensates for the loss of these protective mechanisms and enables cell growth at higher salt concentrations than under aerobic conditions. Of some importance can also be the increase in the intracellular concentrations of osmoprotectants caused by the suppression of their intracellular oxidation. The implications of this phenomenon for the ecophysiology of microorganisms (including oiloxidizing species) and for the classification of weak and moderate halophiles are discussed.