Adaptive functions of extracellular autoregulators of microorganisms

Information about the functions of extracellular autoregulators, which adapt microorganisms to the stresses "scheduled" in the development cycle of microbial cultures (stresses of new medium, starvation, or space exhaustion (high cell density)) is summarized in the review. In a number of bacteria and yeasts, derivatives of alkylhydroxybenzenes (AHB), particularly of the class of alkyl resorcinols, act as autoregulators with adaptogenic functions. The chemical structure of AHB determines their amphiphility; capacity for physical and chemical interaction with membrane lipids, proteins, and DNA; properties as natural modifiers of biological membranes and enzymes; and the expression of antioxidant activity. Increase of AHB concentration up to the critical level (10(-5)-10(-4) M) results in cessation of cell division and in transition of the microbial culture to the stationary phase; further increase to 10(-4)-10(-3) M induces a transition of some of the cells of a post-stationary culture to the anabiotic state with the formation of cystlike resting cells (CRC), even in non-spore-forming bacteria. AHB participate in the regulation of the phenotypic variability of bacteria. The dynamics of extra- and intracellular concentrations of AHB in growing microbial cultures and the polymodality of their effect determine the adaptogenic functions of AHB as autoinhibitors of culture growth, autoinducers of anabiosis, and autoinhibitors of germination of resting forms. Manifestation of any given function depends on the concentration of AHB, the physiological state of the recipient cells, and on environmental factors. The species nonspecificity of AHB effects points to their significant role in the regulation of the development and functioning of microbial communities.     

Autoregulation of stress response in microorganisms

Examples are considered of the involvement of low-molecular-weight autoregulators in the development of resistance of proliferating microbial cultures to unfavorable environmental impacts of various intensity, including impacts programmed to occur in the developmental cycle (“new medium stress,” starvation stress) and nonprogrammed impacts. It was shown that extracellular adaptation factors control the reversible adhesion of cells in submerged cultures and the processes of cell reactivation in the poststress period and are involved in the stabilization of cellular biopolymers (proteins and DNA) and subcellular structures (membranes); the adaptogens of the phenolic type also act as efficient scavengers of reactive oxygen species. The protective effect of the adaptogenic autoregulators is manifested in the increase of resistance of microbial cells to stressors of various nature and in the preservation of the cell proliferative ability.     

Properties of the phenotypic variants of Pseudomonas aurantiaca and P. fluorescens

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 (μ_max) 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.     

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.     

The effect of extracellular metabolites on the frequency of thy+ revertants in Salmonella typhimurium populations

There is convincing evidence that adaptation and survival processes in bacterial populations depend on cell-to-cell interactions. Our studies showed that the frequency of stress-induced His⁺ reversions in an amino-acid-starved Salmonella typhimurium culture is inversely proportional to cell density in this culture. The effects of cell density and of different culture liquids prepared from cultures starved for histidine on the frequency of Thy⁺ revertants were also studied. It was found that the frequency of Thy⁺ revertants is inversely proportional (r = ?0.74) to the density of the bacterial culture starved of thymine. The culture liquid prepared from the culture starved of histidine exerted an inhibitory effect on the frequency of Thy⁺ reversions, indicating that mutations induced by different types of stress have a common mechanism. The study of the effect of the culture liquid prepared from a histidine-starved culture on the frequency of ethyl-methanesulfonate-induced His⁺ revertants showed that this liquid prevented the induction of His⁺ reversions.     

结合代谢组学和转录组学分析恶臭假单胞菌Y-9主动稳定胞内外pH的机制

【目的】揭示恶臭假单胞菌(Pseudomonas putida) Y-9在氨氧化过程中主动调节胞外和胞内pH稳态机制。【方法】在初始pH为7.19和9.40的硝化培养基中培养Y-9生长48 h,利用代谢组学对比分析Y-9氨氧化过程中的显著差异代谢产物并预测解离常数(pKa);结合转录组学对比分析Y-9氨氧化过程中的显著差异调控基因。【结果】Y-9在初始pH为7.19的相对酸性条件下,产生麦芽糖醇提高胞外pH;通过上调脱氨酶、脱亚胺酶和阳离子转运相关基因在相对酸性环境中的表达来维持细胞内pH稳定性。在初始pH为9.40的碱性条件下,产5-氨基戊酸3和草氨酸等有机酸及酸性物质降低胞外pH;通过调控NADH脱氢酶、细胞色素、ATP合酶和氨基酸转运相关基因的表达来维持细胞内酸度,应对碱性环境。【结论】本研究结果首先发现了Y-9具有稳定胞外pH的能力,探讨了其胞内pH稳态机制,拓展了对微生物与环境相互作用的认知,为进一步认识微生物脱氮过程中系统pH稳定机理提供了理论依据。     

The Role of Bacterial Growth Autoregulators (Alkyl Hydroxybenzenes) in the Response of Staphylococci to Stresses

The investigation of the response of a batch culture of Staphylococcus aureus to exogenous alkyl-substituted hydroxybenzenes (AHBs), chemical analogues of anabiosis autoinducers, showed that C₁-AHB at concentrations from 5 M to 1.5 mM did not influence the culture growth, whereas the more hydrophobic C₆-AHB inhibited it at concentrations of 0.5 mM and higher. Either of the AHBs drastically enhanced the phenotypic dissociation of staphylococcal cultures, which manifested itself in an increase in the fraction of cells producing small nonhemolyzing colonies of G type when plated on solid media with erythrocytes. In a submerged staphylococcal culture, the relative number of cells producing G-type colonies varied from 10 to 90%, depending on the concentration of the AHB added. The growth of S. aureus in the presence of AHBs also enhanced cell tolerance to heat shock (heating at 45 or 60°C for 10 min). The role of AHBs, which are structural modifiers of membranes and possess chaperone activity, in the mechanisms responsible for cell tolerance and phenotypic dissociation of microbial populations is discussed.     

电活性微生物胞外聚合物的特征与应用

电活性微生物是一类能够通过直接接触、导电菌毛或氧化还原介质与电极或者其他细胞进行胞外电子传递的微生物。而在这个过程中,胞外聚合物(extracellular polymeric substances, EPS)扮演着重要的角色。EPS是微生物生长过程中通过细胞裂解、水解分泌的高分子聚合物的混合物,主要由蛋白质、多糖和腐殖质等物质组成。来自电活性微生物的EPS的不同组成成分和特性会对EPS的电活性以及电活性微生物胞外电子传递产生一定的影响,同时在环境应用方面发挥重要作用。因此,为了更全面了解电活性微生物EPS的电活性及其对电活性微生物胞外电子传递的作用,本文总体介绍了电活性微生物EPS的电活性的直接表征方法,再从组成成分、化学性质、物理性质和空间分布4个方面综述了其对EPS电活性的影响及其在电子传递中的作用,介绍了当前电活性微生物EPS在染料废水脱色、重金属吸附、有机污染物的生物转化和渗滤液管理等方面的环境应用,并从表征方法、试验规模和互作机理研究等角度展望了未来的研究方向。     

Obtaining of Intrapopulational Dissociants of Some Bacilli and the Use of DIR-PCR for Their Identification

The paper is the first to suggest methods for rapid obtaining and genotypic identification of phenotypic (colonial–morphological) dissociants of bacterial cultures. For revealing the potential dissociation ability and obtaining dissociants, the use of bacterial cystlike refractile cells (CRC) is recommended. These cells are characterized by enhanced variability; upon their first passage, an abrupt increase in the dissociation index is observed as a result of the emergence of cells that form morphologically different types of colonies. The approaches elaborated were tested with Bacillus cereus, B. subtilis, and B. licheniformis, for which colonial–morphological dissociants of various types were obtained after the first passage of CRC (both of those formed in the developmental cycle of the bacteria and of those arising as a result of an artificial increase in the concentration of anabiosis autoinducers in the cultivation medium). The genomic distinctions between dissociants of B. cereus and B. subtilis were estimated using polymerase chain reaction with a primer system designed based on the analysis of nucleotide sequences of complete prokaryotic genomes available in the GenBank database (DIR-PCR). The application of the proposed method allowed distinctions to be revealed between the genomes of dissociants of Bacillus cereus and B. subtilis, which is in agreement with the hypothesis that assumes reversible intragenomic rearrangements to be the basis of bacterial dissociation into subpopulations.     

Diverse biological functions of extracellular collagen processing enzymes

Collagens are abundant proteins in higher organisms, and are formed by a complex biosynthetic pathway involving intracellular and extracellular post-translational modifications. Starting from simple soluble precursors, this interesting pathway produces insoluble functional fibrillar and non-fibrillar elements of the extracellular matrix. The present review highlights recent progress and new insights into biological regulation of extracellular procollagen processing, and some novel functions of byproducts of these extracellular enzymatic transformations. These findings underscore the notion that released propeptides and other proteolytic products of extracellular matrix proteins have important biological functions, and that structural proteins are multifunctional. An emerging concept is that a dynamic interplay exists between extracellular products and byproducts with cells that helps to maintain normal cellular phenotypes and tissue integrity.     

非生物因素对藻类胞外多聚糖含量影响

本文从研究铜绿微囊藻表型转换机制的需要出发,综述了非生物因素对藻类胞外多聚糖含量的影响.很多藻类的胞外多聚糖分泌量与环境中主要营养盐比例之间存在着一定的响应关系,在高碳氮比或高碳磷比条件下,即在氮不足或磷不足时,藻类光合作用固定的有机物质主要以不含氮磷的碳水化合物形式存在,胞内碳水化合物的过量累积导致其逐步向胞外转移释放,使得胞外多聚糖含量显著升高.在碳氮代谢不平衡的情况下,胞外多聚糖充当了接收过剩固定碳的汇.对于一些藻类,不同光谱、光强和光周期均可影响其胞外多聚糖的合成与分泌.温度对藻类胞外多聚糖的产生也有一定影响.由于胞外多聚糖在藻细胞相互粘结形成群体上发挥着重要作用,通过调控相关非生物因素使得铜绿微囊藻胞外多聚糖产量增加可能有助于在室内模拟铜绿微囊藻的群体形成.     

群感效应对电活性微生物胞外电子传递的影响

群感效应（quorum sensing,QS）是微生物之间以信号分子受体蛋白感知信号浓度变化,从而调控菌群的行为及功能,使其适应环境变化的信号通讯机制。电活性微生物（electroactive microorganisms,EAMs）能进行胞外电子传递,在可再生能源利用和环境修复方面具有广阔的应用前景。近年来,关于QS在EAMs胞外电子传递中的作用的研究日益增多。本文总结了QS对纯EAMs或混合产电菌群的直接或间接电子传递的影响效应及机制,阐述了基于QS的EAMs逻辑与门的构建及其应用前景,并从机制研究的角度展望其未来发展方向。     

Effects of extracellular matrices derived from different cell sources on chondrocyte functions

Cell-derived extracellular matrices (ECMs) are a key factor in regulating cell functions in tissue engineering and regenerative medicine. The fact that cells are surrounded by their specific ECM in vivo elicits the need to elucidate the effects of ECM derived from different cell sources on cell functions. Here, three types of ECM were prepared by decellularizing cultured chondrocytes, fibroblasts, and mesenchymal stem cells (MSC) and used for chondrocyte culture to compare their effects on chondrocyte adhesion, proliferation, and differentiation. Chondrocyte adhesion to the chondrocyte-derived ECM was greater than those to the fibroblast- and MSC-derived ECM. Chondrocyte proliferation on the chondrocyte-derived ECM was lower than those on the fibroblast- and MSC-derived ECM. The ECM showed no evident effect on chondrocyte differentiation. The effects of ECM on cell functions depended on the cell source used to prepare the ECM.     

Effects of extracellular matrix proteins in chondrocyte‐derived matrices on chondrocyte functions

Loss of cartilaginous phenotype during in vitro expansion culture of chondrocytes is a major barrier to the application of chondrocytes for tissue engineering. In previous study, we showed that dedifferentiation of chondrocytes during the passage culture was delayed by matrices formed by primary chondrocytes (P0‐ECM). In this study, we investigated bovine chondrocyte functions when being cultured on isolated extracellular matrix (ECM) protein‐coated substrata and P0‐ECM. Low chondrocyte attachment was observed on aggrecan‐coated substratum and P0‐ECM. Cell proliferation on aggrecan‐ and type II collagen/aggrecan‐coated substrata and P0‐ECM was lower than that on the other ECM protein (type I collagen and type II collagen)‐coated substrata. When chondrocytes were subcultured on aggrecan‐coated substratum, decline of cartilaginous gene expression was delayed, which was similar to the cells subcultured on P0‐ECM. These results indicate that aggrecan plays an important role in the regulation of chondrocyte functions and P0‐ECM may be a good experimental control for investigating the role of each ECM protein in cartilage ECM. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1331–1336, 2013     

Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.     

嗜麦芽寡养单胞菌胞外蛋白酶功能研究进展

嗜麦芽寡养单胞菌(Stenotrophomonas maltophilia)是广泛分布于自然界的革兰氏阴性杆菌。作为一种新型、与高死亡率相关的条件致病菌,嗜麦芽寡养单胞菌能够导致人类或其他生物感染多种疾病。近年来,越来越多的研究结果显示来自于细菌的胞外蛋白酶是导致宿主发病的关键蛋白质。因此,探究嗜麦芽寡养单胞菌胞外蛋白酶的组成成分和功能将不仅有助于阐明其致病机制,更为今后以其为靶点进行临床治疗奠定基础。本文试图对嗜麦芽寡养单胞菌胞外蛋白酶的性质、功能及其应用进行归纳总结。     

Activity-dependent formation and functions of chondroitin sulfate-rich extracellular matrix of perineuronal nets

Extracellular matrix molecules--including chondroitin sulfate proteoglycans, hyaluronan, and tenascin-R--are enriched in perineuronal nets (PNs) associated with subsets of neurons in the brain and spinal cord. In the present study, we show that similar cell type-dependent extracellular matrix aggregates are formed in dissociated cell cultures prepared from early postnatal mouse hippocampus. Starting from the 5th day in culture, accumulations of lattice-like extracellular structures labeled with Wisteria floribunda agglutinin were detected at the cell surface of parvalbumin-expressing interneurons, which developed after 2-3 weeks into conspicuous PNs localized around synaptic contacts at somata and proximal dendrites, as well as around axon initial segments. Physiological recording and intracellular labeling of PN-expressing neurons revealed that these are large fast-spiking interneurons with morphological characteristics of basket cells. To study mechanisms of activity-dependent formation of PNs, we performed pharmacological analysis and found that blockade of action potentials, transmitter release, Ca2+ permeable AMPA subtype of glutamate receptors or L-type Ca2+ voltage-gated channels strongly decreased the extracellular accumulation of PN components in cultured neurons. Thus, we suggest that Ca2+ influx via AMPA receptors and L-type channels is necessary for activity-dependent formation of PNs. To study functions of chondroitin sulfate-rich PNs, we treated cultures with chondroitinase ABC that resulted in a prominent reduction of several major PN components. Removal of PNs did not affect the number and distribution of perisomatic GABAergic contacts but increased the excitability of interneurons in cultures, implicating the extracellular matrix of PNs in regulation of interneuronal activity.     

Production of extracellular enzymes by isolates of Metarhizium anisopliae

Extracellular enzyme production in solid culture media was analyzed in order to determine the variability among different Metarhizium anisopliae isolates. Using specific substrates, amylase, lipase, chitinase, and protease production was tested in 11 isolates from different regions of Brazil. Enzyme production was determined by the formation of a halo around the colony, and the diameters of both halo and colony were measured. The enzymatic index was expressed by the colony diameter/halo diameter ratio. In general, the isolates from the same region had similar enzymatic indexes, although similar indexes were also found for isolates from geographically distinct regions. The different isolates were tentatively grouped according to index similarity.     

Structural and physiological diversity among cystlike resting cells of bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C₁₂-AHB, 10^?4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.     

Production of Resting Forms by the Gram-Negative Chemolithoautotrophic Bacteria Thioalkalivibrio versutusand Thioalkalimicrobium aerophilum

The haloalkaliphilic chemolithoautotrophic gram-negative bacteria Thioalkalivibrio versutus, strain AL2, and Thioalkalimicrobium aerophilum, strain AL3, were shown to possess the capacity to produce resting forms, namely cystlike refractile cells (CRC), whose production was controlled by the level of the d₁ extracellular factors exhibiting the function of anabiosis autoinducers. The conditions were elucidated that promote the formation of CRC in the developmental cycles of the cultures studied, in condensed cell suspensions undergoing autolysis, and under the action of exogenously introduced chemical analogues of anabiosis autoinducers (alkylhydroxybenzenes). The peculiarities of the fine structure of the resting cells obtained were studied. Distinctions were revealed (with respect to viability and thermotolerance) between the CRC formed under different conditions. The relationship between the growth strategy and survival strategy of extremophilic bacteria is discussed taking into account the effect of the d₁ autoregulatory factors. A new model of CRC formation is proposed: CRC production in the life cycle of bacteria developing under conditions of increased concentration of anabiosis autoinducers.