Formation of virulence-associated type I pili in Escherichia coli should be considered as one of the most efficient models for investigating the mechanisms of regulating the heterogeneity of populations of genetically identical microbial cells. The present work focused on the role of alkylhydroxybenzenes (AHBs), density-dependent intercellular regulators, in controlling phase variations in type I pili formation (fimbriogenesis). The tested AHB homologue was C12-AHB; a genetically constructed strain E. сoli dsp250 containing the fimA-lacZ hybrid operon was used. In this operon, the fimA gene encodes the main subunit of the pili protein, and its expression results in β-galactosidase synthesis; pili-forming cells, therefore, become blue on the medium with the Х-gal substrate. Expression of fimA depends on the inversion of the fimS region that is located upstream of it. If the inversion is on, pili formation takes place, if it is off, no pili are formed. An increase in C12-AHB concentration (within the 5 × 10–5–2 × 10–4 M range) in the exponential-phase culture of strain dsp250 causes a dose-dependent change in the dominant phenotype that is displayed by up to 98–99% of the cells. Cells with this phenotype form colonies with a blue center and white edges. Up to 60% of the cells with this phenotype assume a metastable state and up to 11% and 44% of them transition to the alternative phenotypes of pili-forming and pili-less cells, respectively. The influence of C12-AHB on off-switching, i.e. the formation of the avirulent phenotype, was observed irrespective of the growth conditions of strain dsp250. Addition of glucose to the LB medium (5 or 10 mg/mL) resulted in catabolic repression via regulation by the cAMP-CNR complex and predictably induced pili formation in 49 and 75% of the cells, respectively. Against this background, C12-AHB caused a dose-dependent decrease in the share of pili-forming cells to 33–61% and an increase in the share of pili-less cells to 32–61%. If glucose was added in excess (2.5, 5 or 10 mg/mL) to the diluted LB/2 medium, pili formation was completely repressed, while C12-AHB still induced the off inversion to the pili-less phenotype in up to 30% of the cells. The conclusion can be drawn that C12-AHB is not involved in the pathway of fimbriogenesis regulation via cAMP. Since C12-AHB functions as an extracellular alarmon (activating the rpoS regulon and the SOS response as shown earlier, see Golod et al., 2009), its mechanism of action apparently involves stress signal transduction. It induces the synthesis of global regulators RpoS and H-NS and of intracellular alarmon (p) ppGpp; these factors are responsible for the on → off inversion and the proliferation of pili-less cells.

Survival of bacterial populations treated with lethal doses of antibiotics is ensured by very small numbers of persister cells. Unlike antibiotic-resistant cells, antibiotic tolerance of persisters is not inheritable and reversible. The present work provides evidence supporting the hypothesis on transformation (maturation) of persisters of an opportunistic pathogen Pseudomonas aeruginosa, revealed by ciprofloxacin (CF) treatment (25–100 μg/mL), into dormant cystlike cells (CLC) and nonculturable cells (NC), as was described previously for a number of non-spore-forming bacteria. Subpopulations of type 1 and type 2 persisters, which survived antibiotic treatment and developed into dormant forms, were heterogeneous in their capacity to form colonies or microcolonies upon germination as resistance to heating at 70°C and in cell morphology. Type 1 persisters, which were formed after 1-month incubation of the stationary-phase cultures grown in the medium with decreased C and N concentrations, developed in several types of surviving cells, including those similar to CLC in cell morphology. In the course of 1-month incubation of type 2 persisters, which were formed in exponentially growing cultures, other types of surviving cells developed: immature CLC and L-forms. Unlike P. aeruginosa CLC formed in the control post-stationary phase cultures without antibiotic treatment, most of 1-month persisters, especially type 2 ones, were characterized by the loss of colony-forming capacity, probably due to transition into an nonculturable state with relatively high numbers of live intact cells (Live/Dead test). Another survival strategy of P. aeruginosa populations was ensured by a minor subpopulation of CF-tolerant and CF-resistant cells able to grow in the form of microcolonies or regular colonies of decreased size in the presence of the antibiotic. The described P. aeruginosa dormant forms may be responsible for persistent forms in bacteria carriers and latent infections and, together with antibiotic-resistant cells, are important as components of test systems to assay the efficiency of potential pharmaceuticals against resistant infections.

Currently, limited information is available on the enzymatic activities in ancient paleosols and permafrost, which are recognized as unique depositories of extant microorganisms. In this work, samples from buried and modern top Kastanozems (Haplic Calcisol), modern tundra (Gleysol) soils, and permafrost were tested for amylase activity based on hydrolysis of starch to low-molecular sugars that are important energy sources for microorganisms. Our study demonstrated the preservation of amylase in buried paleosols (300 and 2000 years old) and permafrost (15,000-60,000 years old). The enzyme activity in buried soils was 5-7 times lower than in top soils and did not significantly (P > 0.05) differ from permafrost and tundra soils. Exogenously added allcylresorcinols, valine, and glycine influenced amylolytic activity in soil and permafrost samples in the dose-dependent manner. The amylase response to stimulating concentrations of alkylresorcinols and amino acids was greater in buried paleosols (4.3-11.5-fold) than in top soils (1.9-3.8-fold) over the enzymatic activities in the controls. Positive response of soil amylase even to little changes in concentrations of the low-molecular-weight compounds may be important during recovery of biological activity of microorganisms. Results of this study contribute to a more complete evaluation of hidden biological potential of ancient soils and permafrost and are relevant to paleomicrobiology and soil biology. (C) 2015 Elsevier Masson SAS. All rights reserved.

Effect of sublethal doses of physical and chemical stressors (heat shock for 2 h at 45°C and addition of C12-alkylhydroxybenzene, a microbial alarmone) on development of resistance to the subsequent lethal antibiotic attack and the role of the time interval between these treatments were studied on a submerged batch culture of Escherichia coli K12. The interval sufficient for the development of stress response provides for development of temporary adaptive resistance to the antibiotic attack, resulting in increased number of surviving persister cells. The interval below the time required for the stress response potentiates cell death and results in a decreased number of persisters. Heterogeneity of the fractions (10–4 to 10–2% of the initial CFU number) surviving lethal doses of an antibiotic (ampicillin or ciprofloxacin) was found. Apart from a low number of antibiotic-resistant cells (up to 0.005% of surviving cells), the fractions contained antibiotic-tolerant forms, such as temporarily resistant metabolically adapted cells, long-term surviving persisters, and the cells of slowly growing SCV variants with small colonies (d ≤ 1 mm). Persisters are hypothesized to act as precursors for cystlike dormant cells (CLC), in which the cell differentiation stage is completed and the processes of cell ametabolism (transition to the anabiotic state) are still incomplete.

Effect of the antibiotics kanamycin and ampicillin on the growth and phase variation of the populations of four strains of lactic acid bacteria (Lactobacillus sp. M76AT, L. casei MB, Enterococcus faecium M, and E. faecium M3185) was studied. The presence of antibiotics in the medium resulted in a dose-dependent decrease in viable cell numbers and in partial or complete substitution of the dominant S variant with the minor Sm and Sb variants. The variants differed in colony morphology, as well as in some physiological, biochemical, biotechnological, and probiotic characteristics. The Sm type variants of all strains exhibited the highest resistance to antibiotics. High production of exopolysaccharides was found in Sb variants of lactobacilli and in S variants of enterococci. The highest antibacterial activity was found in Sm variants of lactobacilli, especially in Lactobacillus sp. M76AT. The latter is biotechnologically the most promising strain, since all variants fermented milk yielding the products with uniformly pronounced functional and organoleptic properties. These patterns are of importance for the understanding of the mechanisms of antibiotic resistance and for selection of the variants with desired properties, as well as for quality control of the lactic acid bacteria starter cultures.

The effect of the extracellular peptide reactivating factor (RF) synthesized by Luteococcus casei on stress response of Escherichia coli cells subjected to UV irradiation was studied. For these studies, we constructed a test strain carrying the umuD-lacZ operon. The expression rate of this operon reflects the rate of SOS response. Protective effect of RF, defined as the number of cells retaining the colony-forming activity (CFU) after UV irradiation (49–1166 J/m2), was dose-dependent, species-nonspecific, and increasing with increase of the stress load. RF was demonstrated to possess the properties of a direct adaptogen: 15 min of preincubation with RF caused a 1.5–6-fold decrease in expression of the umuD SOS response gene in UV-treated cells, concurrently with a 1.2–7.5 times increase in the number of viable cells (those having retained their colony-forming activity). The probable mechanisms of the protective effect of RF are being discussed.

Cross protection of members of the domains Bacteria, Archaea, and lower Eukaryota from stress factors due to the action of extracellular low-molecular metabolites with adaptogenic functions was shown. The adaptogen produced by Luteococcus japonicus subsp. casei and described previously as a reactivating factor (RF) was shown to protect the yeasts Saccharomyces cerevisiae, archaea Haloarcula marismorti, and the cells of higher eukaryotes (HeLa) against weak stressor impacts. Production of an archaeal extracellular metabolite with a weak adaptogenic effect of the producer cells and capable of a threefold increase in survival of heat-inactivated yeast cells was discovered. Our results confirm the similarity of the compensatory adaptive reactions in prokaryotes (bacteria and archaea) and eukaryotes.

Изучено действие внеклеточного пептидного реактивирующего фактора (РФ), синтезируемого Luteococcus casei, на стрессовый ответ клеток Escherichia coli, подвергаемых УФ-облучению. В исследованиях использовали сконструированный тестерный штамм, несущий оперон umuD-lacZ, уровень экспрессии которого отражает степень активации SOS-ответа. Защитный эффект РФ, определяемый по численности клеток, сохраняющих колониеобразующую способность (КОЕ) после УФ-облучения (49–1166 Дж/м2), был дозозависим, видонеспецифичен и возрастал с увеличением стрессовой нагрузки. Было показано, что РФ обладает функциями прямого адаптогена: 15-минутная предынкубация с ним клеток обусловливала снижение в 1.5–6 раз экспрессии гена umuD SOS-ответа у УФ-облученных клеток параллельно с увеличением в 1.2–7.5 раз численности оставшихся жизнеспособными (сохранивших колониеобразующую способность) клеток. Возможные механизмы защитного действия РФ обсуждаются.

Представлены данные о перекрестной защите от стрессорных воздействий представителей доменов бактерий, архей и низших эукариот, обеспеченной действием внеклеточных низкомолекулярных метаболитов с функциями адаптогенов. Адаптоген, синтезируемый Luteococcus japonicus subsp. casei и описанный ранее как реактивирующий фактор (РФ), оказывал протекторное действие на клетки дрожжей Saccharomyces cerevisiae, галоархей Haloarcula marismortui, а также на клетки высших эукариот, HeLa, при слабых стрессорных воздействиях. Впервые показано образование археями внеклеточного метаболита, имеющего слабый адаптогенный эффект в отношении клеток продуцента и в 3 раза увеличивающего выживаемость дрожжей, подвергнутых термоинактивации. Полученные результаты являются подтверждением сходства компенсаторно-приспособительных стрессовых реакций прокариот (бактерий и архей) и эукариот.

The ability of the symbiotrophic rhizobium Sinorhizobium meliloti P221 to produce cells having all the properties of resting forms (RFs) during the development cycles of the culture or after addition of the threshold concentrations of anabiosis autoinducers was demonstrated. The numbers, properties, and ultra-structure of S. meliloti resting forms depended on the conditions of growth and poststationary-phase incubation. In the four-month poststationary-phase, cultures grown in media deficient in some nutrient elements and energy sources (nitrogen, phosphorus, or oxygen), numerous cells (24–76% of the number of CFUs in the stationary-phase cultures) exhibiting a high degree of heat resistance and reversibly inhibited metabolic activity (the absence of endogenous respiration) were detected. According to their ultrastructure, all the resting forms detected in starving cultures were divided into three groups: (1) cystlike resting cells (CRCs) with thick cell envelopes and compacted nucleoids, (2) CRCs containing numerous (up to three-quarters of their volumes) polyhydroxyalkanoate inclusions, and (3) RFs similar to Azotobacter cysts. The resting forms obtained in the culture grown at high concentrations (5 × 10−5 M) of C12-AHB, a chemical analogue of microbial anabiosis autoregulators, were incapable of endogenous respiration and retained the colony-forming ability. The CFU number after plating of these resting forms was twice as high as in the control culture; the heat resistance of these cells (55°C, 10 min) was an order of magnitude higher. The bacterial cells obtained from the resting forms either had a mixed (Swa+Gri+) type of motility in semisolid agar, typical of the dominant phenotype of the parent cells, or switched to the Gri+ type. Emergence of different motility phenotypes depended on the conditions of RF formation. More severe stress conditions of RF formation induced the emergence of the Gri+ type of cell motility. The results obtained can be used for development of a new generation of bacterial preparations based on bacterial CRCs which are able to preserve their viability for a long time and are highly resistant to stress impacts.

The adaptogenic effect of the chemical analogues of alkylhydroxybenzenes (AHBs), bacterial extracellular autoregulators (the individual compound C7-AHB and its technical preparation Sidovit), was demonstrated for two pseudomonad species, Pseudomonas aeruginosa and P. fluorescens . The protective effect of AHBs resulted in increased growth rate and biomass accumulation in bacteria grown under suboptimal conditions within the species tolerance range. The adaptogenic effect of AHBs (10–50 μmg/l) was more pronounced under more unfavorable growth conditions. In the case of P. fluorescens , the individual compound C7-AHB increased the biomass yield by 30% under alkaline conditions (pH 9.5), when the growth rate decreased by 80–90% compared to the optimum (pH 5.5–7.5). The Sidovit preparation, containing a mixture of natural AHBs with C7-AHB as the main component, increased the growth rate of P. aeruginosa by 40–60% at nonoptimal temperatures (45 and 10°C) or under enhanced salinity (1% NaCl). The action of AHBs as regulators of the rpoS and SOS response stress regulons was demonstrated to be among the mechanisms of their adaptogenic effect, as was demonstrated with the relevant reporter genes in the model strains E. coli C600 thi, thr, leu Δ (pro-lac) with the osmE-lacZ and umuD-lacZ hybrid operons, respectively. AHBs are technologically and economically acceptable as adaptogenic supplements for bacterial preparations used in soil bioremediation and oil spillage removal under conditions unfavorable for microbial growth, including increased salinity, extreme pH, and fluctuating sub- or supraoptimal temperatures.

The effect of hexylresorcinol (HR), a chemical analogue of microbial anabiosis autoinducers of the alkylhydroxybenzene (AHB) group, on the stability of biological membranes and monolamellar liposomes formed of egg phosphatidylcholine (ePC) was studied. According to spectrophotometry and electron microscopy studies of HR-loaded liposomes in the presence of a surfactant Tween 20, the critical ratio between HR and ePC for liposome preservation was found to be close to equimolar. The trends in HR influence on membrane structural organization and stability of liposomes were also confirmed in experiments on intact bacterial cells explaining non-species-specific effect of AHBs. The demonstrated high efficiency of AHB biocides may be used in material and equipment protection against biocorrosion.

In our study, we investigated the capacity of alkylhydroxybenzenes (AHB), which are microbial anabiosis autoinducers, for alteration of the enzymatic activity of the hen egg-white lysozyme, as well as the efficiency of hydrolysis of specific (peptidoglycan) and nonspecific (chitin) substrates catalyzed by lysozyme. AHB homologues (C7-AHB and C12-AHB), which differ in their hydrophobicity and effects in their interaction with lysozyme, were used as modifying agents. C7-AHB stimulated enzymatic activity within the whole range of concentrations used (10 −7 −10 −3 M). More hydrophobic C12-AHB exhibited this ability only at low concentrations and inhibited fermentative activity at high concentrations, acting as a mixed-type inhibitor. Both AHB homologues caused changes in the hydrophobicity of lysozyme molecules. An increase in the affinity level between the C7-AHB-modified enzyme and the nonspecific substrate (colloidal chitin or cell wall polymers of Saccharomyces sp.) was observed, which manifested itself in the enhancement of the hydrolysis rate by 200–500% (as compared to the native enzyme). A significant effect on the efficiency of the lysozyme-catalyzed modifications of the substrate (peptidoglycan, colloidal chitin) structure as a result of its complexation with AHB was demonstrated. A stabilizing effect of C7-AHB and C12-AHB was revealed, which ensured a high level of activity of the AHB-modified enzyme (as compared to the control) after heat treatment (functional stability), as well as at nonoptimal temperatures of catalysis (operational stability). The biological significance of lysozyme modification with AHB and the practical aspects of its application are discussed.

Different capacity for phenotypic variation of Pseudomonas aurantiaca and P. fluorescens in populations of cyst-like resting cells (CRC) during their germination on solid media, was shown to be a characteristic trait of biodiversity for the dormant forms of these bacteria. This biodiversity manifests itself as qualitative and quantitative differences in the spectra and emergence frequency of phenotype variants, obtained by plating of CRC, and depends on the conditions of CRC formation and storage time. In P. aurantiaca, the variation was associated with transition of the wild-type S-colonial phenotype into the R-type or the more pigmented P-type. These transitions were most pronounced for the CRC obtained under nitrogen depletion (a twofold N limitation), as well as under the influence of a chemical analogue of microbial anabiosis autoinducers, C12-AHB. In the latter case, the frequency of S➝R and S➝P transitions (up to 70% and 80%, respectively) depended on the C12-AHB concentration (1.0 × 10−4 M and 2.5 × 10−4 M) and on the storage time of CRC suspensions (from 3 days to 1.3 months). In the CRC populations grown in nitrogen-deficient media, R-type appeared with a frequency of up to 45% after at least four months of storage. In the case of P. fluorescens, S➝R transitions depended not only on the storage time of CRC and C12-AHB concentrations, but also on the composition of the solid medium used for plating. Differences were shown between the R-, P-, and S-variants of P. aurantiaca in such morphological, physiological, and biochemical characteristics as the growth rate (μmax) in a poor medium, biomass yield (Y max), resistance to streptomycin and tetracycline (LD50), and the productivity in extracellular proteases. The R-and S-variants of P. fluorescens differed in their growth characteristics, resistance to high salinity and oxidative stress, as well as in their sensitivity to exogenous introduction of chemical analogues of microbial autoregulators (C12-AHB and C7-AHB). Hence, both the formation of dormant forms of the various morphological types [1] and intrapopulation phenotypic variability observed during their germination are important for the survival strategy of pseudomonads under unfavorable environmental conditions.