S. N. Filippova

Non-species-specific effects of unacylated homoserine lactone and hexylresorcinol, low molecular weight autoregulators, on the growth and development of bacteria

We conducted a comparative study of the effects of α-amino-γ-butyrolactone, the common structural element of extracellular microbial regulators of the homoserine lactone (HSL) group, and of 4-n-hexylresorcinol, an autoregulator of the alkylhydroxybenzene (AHB) group, on the growth and development of grampositive and gram-negative bacteria. We revealed non-species-specific effects of HSL and AHB and characterized their concentration dependencies. The addition of 10−5−10−3 M HSL or 10−5−10−4 M AHB during the exponential growth phase of the cultures grown on balanced media resulted in cell division arrest and accelerated the transition to the stationary phase that culminated in endospore formation in Bacillus cereus, Alicyclobacillus tolerans, and Sulfobacillus thermosulfidooxidans. When bacilli grew under the cultivation conditions that resulted in a low-zero spore percentage, 10−4−10−3 M HSL cancelled the inhibition of spore formation. In the gram-negative bacteria Pseudomonas aurantiaca and Azotobacter vinelandii, AHB at concentrations of 10−4 to (1.5−2.5)×10−4 M induced the formation of dormant cells. Studies with the actinobacterium Streptomyces avermitilis revealed that the HSL effect varied depending on the age of the test cultures. The addition of 10−4 M HSL during the lag phase of a submerged streptomycete culture accelerated its transition to the stationary phase and induced the formation of endospores, the dormant cells that are regarded as alternatives to exospores (conidia). If HSL (3.64 and 4.55 mg per 1 cm2 disc) was locally added to a surface S. avermitilis culture, the growing mycelium formed rings that differed in their density, in the extent of the development of aerial mycelium, and in the presence/absence of exospores. Ring-shaped growth of streptomycete mycelia was also induced by 0.075–0.75 mg of AHB; however, unlike HSL, AHB repressed exospore formation. The data on non-species-specific effects of HSL and AHB suggest that they may perform regulatory functions at the microbial community level.

Long-term storage of collection cultures of actinobacteria

A total of 553 collection cultures of actinobacteria, including 453 reference ISP strains, were studied after long-term storage in a lyophilized state, as soil cultures, and under mineral oil. It was established that their viability reached a near-critical level. A number of methodological approaches to optimization of activation and proliferation of actinobacteria made it possible to restore the viability and major cultural and morphological properties of 65% of actinobacteria stored in a lyophilized state or under mineral oil. The actinobacteria stored as soil cultures almost completely lost their viability. Resuscitated actinobacteria exhibited a high level of genetic instability, which resulted in the emergence of more than three phenotypically different types of colonies. The population spectrum shifted towards an increase in the content of minor phenotypes with low spore-forming capacity. Significant changes in the cultural and morphological properties of a number of resuscitated strains were observed. Desirability of the application of antioxidants or growth-stimulating compounds in order to restore the viability of Actinobacteria cultures and to stabilize them after long-term storage was demonstrated.

Endospore formation by Streptomyces avermitilis in submerged culture

The ability of streptomycetes to form endospores during their life cycle was studied in submerged cultures of Streptomyces avermitilis. Submerged S. avermitilis spores were most intensely formed (1) during the culture development cycle on synthetic medium CP1 with glucose under phosphate limitation and (2) in autolysing cell suspensions of high density obtained by tenfold concentration in phosphate buffer (pH 7.2) with 0.2% CaCl2 of stationary-phase cells grown in synthetic medium. Endospores of S. avermitilis formed in submerged cultures shared the major characteristics of specialized microbial resting forms: heat resistance, resistance to lysozyme, ability to retain the main species-specific features, and ultrastructural organization characteristic of endospores. They can be considered a resting form of streptomycetes alternative to the spores formed exogenously on aerial mycelium in surface cultures.

Detection of Phage Infection in the Bacterial Population of Lake Untersee (Antarctica)

ISSN 0026!2617, Microbiology, 2013, Vol. 82, No. 3, pp. 383–386.

A New Regulatory Function of A-Factor: Stimulation of the Germination of Streptomycete Spores

Spore germination in streptomycetes was shown to be stimulated by exogenously added A-factor. Agar medium either containing or not containing A-factor was inoculated with spore suspensions of three strains differing in their ability to produce regulators of the A-factor group: Streptomyces griseus 773, which produces A-factor and two its lower homologs; S. coelicolor A3(2), which forms six Acl-factors (A-factor analogues); and S. avermitilis JCM5070, which fails to form regulators of this group. A count of the grown colonies showed that exogenous A-factor stimulated spore germination in strains that were themselves able to synthesize regulators of the A-factor group. In S. griseus 773, the number of germinated spores increased by 67% on average after the addition of A-factor to the medium in an amount of 10 μg/ml. In strain S. coelicolor A3 (2), the number of germinated spores increased by 75% after the addition of 1 μg/ml of A-factor. During germination of the S. avermitilis JCM5070 spores, no changes in the CFU number was observed after the addition of A-factor.

Programmed cell death as one of the stages of streptomycete differentiation

Programmed cell death (PCD), i.e., active, genetically determined cell death controlled by special intracellular programs, is a necessary part of development of living organisms. PCD of streptomycetes, a widespread and biotechnologically important group of mycelial bacteria, is poorly known, in contrast to an immense amount of data on their growth processes. This review deals with the results of PCD studies in streptomycetes as one of the stages of their development, considered as a part of analysis of growth and differentiation of this bacterial group. PCD events in streptomycetes are considered together with their other feautres, which support analogies with multicellular organisms. The results of investigation of PCD in streptomycetes are required for development of new approaches to optimization of the yield of their biosynthetic products. Basic PCD research is of medical and pharmacological importance for development of fundamentally new approaches to counteracting microbial pathogens.

Diversity of Bacterial Forms in Ice Wedge of the Mamontova Gora Glacial Complex (Central Yakutiya)

Electron microscopic investigation of four samples of ancient ice wedge from the Pleistocene glacial complex of Mamontova Gora (Yakutiya, Russia) revealed high diversity of bacteriomorphic particles. Their structural features included the presence of electron-transparent zones, presumably inclusions containing storage compounds, and microenvironments (capsules or external sheaths). These features may be a result of adaptive strategies providing for microbial survival under permafrost conditions. Predominance of rod-shaped forms morphologically resembling coryneform actinobacteria was found. X-ray microanalysis revealed organic origin of bacteriomorphic particles. Some particles were characterized by incomplete spectra of the major biogenic elements, resulting probably from low-temperature damage to the cellular structures. Total numbers of aerobic heterotrophic bacteria determined by plating on nutrient media were comparable to the values obtained for permafrost soils and Arctic ice. Predominance of coryneform actinobacteria was observed. Abundance of these evolutionarily early groups of actinobacteria may indicate the ancient origin of the microflora of the relic frozen rocks.

Bacteriophages in Arctic and Antarctic low-temperature systems

Comparative analysis of the presence of bacteriophages was carried out for the water column of a permanently ice-covered, extremely oligotrophic Lake Untersee (East Antarctica) and the ancient ice wedge of the Mamontova Gora outcrop (Aldan River, Central Yakutia). Microscopy revealed bacteriophages in the Mamontova Gora ice samples and in the lysates of the pure cultures of phage-sensitive bacteria isolated from the same samples. Bacteriophages isolated from these cultures were filamentous and interacted with bacteria as moderate (lysogenic) phages. A similar filamentous bacteriophage was isolated from the Lake Untersee water column. The highest morphological diversity of bacteriophages was revealed by microscopy in the oxic Lake Untersee water column in the chemocline zone (70–76 m) and in the sulfide layer (85 m). Detection of similar filamentous bacteriophages in a relic ice sample and in the samples from Antarctic Lake Untersee indicate wide occurrence of bacteriophages and lysogeny in microbial communities of low-temperature ecosystems.

Growth induction and stabilization of population composition in Saccharopolyspora erythraea by catecholamine compounds

Dopamine and epinephrine, hormone compounds belonging to the catecholamine group, produced a polymodal effect on the culture of the erythromycin-producing actinobacterium Saccharopolyspora erythraea. They stimulated spore germination and stabilized the composition of its population. Plating of monospore suspensions of strains RIA 1387 and RIA 120 in the presence of catecholamines increased the colony number by 2- and 2.3-fold, respectively (with epinephrine), and 3- and 4-fold, respectively (with dopamine). The optimum effect of both catecholamines was attained at a concentration of 10−5 M. The influence of exogenous catecholamines resulted in a significant shift in the population structure of the tested strains with an increased share of the colonies of the dominant morphotype, which is the most efficient antibiotic producer. Considerable differences in the biological activity of the catecholamines were revealed after exposure to ultralow temperatures both during medium-term storage (for three months) and after short-term freezing (for 10 min) in liquid nitrogen. An appreciable effect was produced by exogenous dopamine introduced at the resuscitation stage, resulting in a two- to three-fold increase in the viability of actinobacterial spores. Almost 100% of the resulting populations were composed of colonies of the dominant morphotype. In contrast to dopamine, epinephrine failed to produce a growth-stimulating effect when added to spore suspensions exposed to ultralow temperatures.

Modeling of dissemination of microbial cells and phages from the sites of permafrost thawing

A method is proposed for integral assessment of the propagation of microbial cells and phage particles during seasonal thawing of relic ice wedge layers. The results of field and laboratory investigation carried out in the upper part of permafrost exposure at Mamontova Gora (Yakutiya, Russia) are presented. Suspensions of yeast, bacteria, and two coliphages were introduced as biomarkers directly on the surface of thawing ice and in the meltwater flow. Microorganisms and phages were shown (a) to possess particular parameters of dissemination in the meltwater flow and (b) were able to move 132 m in 25–35 min with the stream water flow.