Glycine betaine uptake after hyperosmotic shift in Corynebacterium glutamicum.

Osmoregulatory uptake of glycine betaine in whole cells of Corynebacterium glutamicum ATCC 13032 (wild type) was studied. The cells actively take up glycine betaine when they are osmotically shocked. The total accumulation and uptake rate were dependent on the osmotic strength of the medium. Kinetic analysis revealed a high-affinity transport system (Km, 8.6 +/- 0.4 microM) with high maximum velocity (110 nmol.min-1.mg [dry weight]-1). Glycine betaine functioned as a compatible solute when added to the medium and allowed growth at an otherwise inhibitory osmotic strength of 1.5 M NaCl. Proline and ectoine could also be used as osmoprotectants. Glycine betaine is neither synthesized nor metabolized by C. glutamicum. The glycine betaine transport system is constitutively expressed at a basal level of activity. It can be induced up to eightfold by osmotic stress and is strongly regulated at the level of activity. The transport system is highly specific and has its pH optimum in the slightly alkaline range at about pH 8. The uptake of the zwitterionic glycine betaine is mediated by a secondary symport system coupled to cotransport of at least two Na+ ions. It is thus driven both by the membrane potential and the Na+ gradient. An extremely high accumulation (internal/external) ratio of up to 4 x 10(6) was measured, which represents the highest accumulation ratio observed for any transport system.     

Response of marine microbial communities to anthropogenic stress

Marine microbial communities adapt rapidly to changingenvironmental conditions, including anthropogenicstress. Adaptation involves a wide range ofstrategies, including, (a) formation of resistant,dormant stages, (b) initiation of repair mechanisms, (c)immobilization of toxic chemicals, (d) active transportof chemicals out of the cell, (e) use of contaminantchemicals as carbon or energy sources, and (f)transformation of contaminants to less toxic or morevolatile forms.Adaptation responses are generally plasmid- orchromosomally-mediated and controlled throughinduction or derepression of a variety of biochemicalpathways. Characterization of microbial communityresponses at the molecular level provides biomarkersof contaminant exposure which in turn may be used toprovide an overall picture of ecosystem health. Thisreview will discuss the interactions betweenmicroorganisms and environmental contaminants and thepotential use of microbial biomarkers to assess thehealth of the microbial ecosystem.     

Role for glycine betaine transport in Vibrio cholerae osmoadaptation and biofilm formation within microbial communities

Vibrio cholerae is a halophilic facultative human pathogen found in marine and estuarine environments. Accumulation of compatible solutes is important for growth of V. cholerae at NaCl concentrations greater than 250 mM. We have identified and characterized two compatible solute transporters, OpuD and PutP, that are involved in uptake of glycine betaine and proline by V. cholerae. V. cholerae does not, however, possess the bet genes, suggesting that it is unable to synthesize glycine betaine. In contrast, many Vibrio species are able to synthesize glycine betaine from choline. It has been shown that many bacteria not only synthesize but also secrete glycine betaine. We hypothesized that sharing of compatible solutes might be a mechanism for cooperativity in microbial communities. In fact, we have demonstrated that, in high-osmolarity medium, V. cholerae growth and biofilm development are enhanced by supplementation with either glycine betaine or spent media from other bacterial species. Thus, we propose that compatible solutes provided by other microorganisms may contribute to survival of V. cholerae in the marine environment through facilitation of osmoadaptation and biofilm development.     

Quorum quenching in cultivable bacteria from dense marine coastal microbial communities

Acylhomoserine lactone (AHLs)-mediated quorum-sensing (QS) processes seem to be common in the marine environment and among marine pathogenic bacteria, but no data are available on the prevalence of bacteria capable of interfering with QS in the sea, a process that has been generally termed 'quorum quenching' (QQ). One hundred and sixty-six strains isolated from different marine dense microbial communities were screened for their ability to interfere with AHL activity. Twenty-four strains (14.4%) were able to eliminate or significantly reduce N-hexanoyl-l-homoserine lactone activity as detected by the biosensor strain Chromobacterium violaceum CV026, a much higher percentage than that reported for soil isolates, which reinforces the ecological role of QS and QQ in the marine environment. Among these, 15 strains were also able to inhibit N-decanoyl-l-homoserine lactone activity and all of them were confirmed to enzymatically inactivate the AHL signals by HPLC-MS. Active isolates belonged to nine different genera of prevalently or exclusively marine origin, including members of the Alpha- and Gammaproteobacteria (8), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (1). Whether the high frequency and diversity of cultivable bacteria with QQ activity found in near-shore marine isolates reflects their prevalence among pelagic marine bacterial communities deserves further investigation in order to understand the ecological importance of AHL-mediated QS and QQ processes in the marine environment.     

Design and testing of 'genome-proxy' microarrays to profile marine microbial communities

Microarrays are useful tools for detecting and quantifying specific functional and phylogenetic genes in natural microbial communities. In order to track uncultivated microbial genotypes and their close relatives in an environmental context, we designed and implemented a 'genome-proxy' microarray that targets microbial genome fragments recovered directly from the environment. Fragments consisted of sequenced clones from large-insert genomic libraries from microbial communities in Monterey Bay, the Hawaii Ocean Time-series station ALOHA, and Antarctic coastal waters. In a prototype array, we designed probe sets to 13 of the sequenced genome fragments and to genomic regions of the cultivated cyanobacterium Prochlorococcus MED4. Each probe set consisted of multiple 70-mers, each targeting an individual open reading frame, and distributed along each approximately 40-160 kbp contiguous genomic region. The targeted organisms or clones, and close relatives, were hybridized to the array both as pure DNA mixtures and as additions of cells to a background of coastal seawater. This prototype array correctly identified the presence or absence of the target organisms and their relatives in laboratory mixes, with negligible cross-hybridization to organisms having 相似文献   

Glycine betaine transmethylase mutant of Pseudomonas aeruginosa

The gene for glycine betaine transmethylase (gbt) was identified in Pseudomonas aeruginosa strain Fildes III by biochemical, physiological, and molecular approaches. Based on sequence analysis, the knockout gene corresponded to an open reading frame (ORF) named PA3082 in the genome of P. aeruginosa PAO1. The translated product of this ORF displayed similarity to transferases of different microorganisms. Mutation in gbt blocked the utilization of choline and glycine betaine as carbon and nitrogen sources.     

Glycine betaine as a cryoprotectant for prokaryotes

Osmoprotectants are low molecular weight, hydrophilic, nontoxic molecules that assist a cell under osmotic stress to stabilize its concentration of internal solutes. These properties are similar to compounds used as cryoprotectants for the preservation of prokaryotic cells during freezing. This study tested the ability of a common compatible solute, glycine betaine (GB), to act as a cryoprotectant. In a series of freeze-drying studies using a variety of prokaryotes, GB performed as well, or better than, two commonly used cryoprotectants, sucrose/bovine serum albumin (S/BSA) and trehalose/dextran (T/D). GB did especially well maintaining cell viability after long-term storage (simulated equivalent of 20 years) for microorganisms like Neisseria gonorrhoeae and Streptococcus pneumoniae. GB was tested for its ability to preserve members of the genus Acidothiobacillus, a difficult genus to preserve. For two strains of Acidithiobacillus ferrooxidans that were preserved using liquid drying, GB performed as well as S/BSA. Results were more mixed for two strains of Acidithiobacillus thiooxidans; one strain could be preserved with S/BSA but not GB, the other strain gave low recoveries with both cryoprotectants. GB also proved to be a useful cryoprotectant for liquid nitrogen preservation yielding equivalent results to the cryopreservative, glycerol for halophilic archaea, and neutrophilic Fe-oxidizing bacteria. These results indicate that GB is a simple and useful cryoprotectant that works for a wide range of prokaryotic organisms under different cryopreservation regimens.     

Metabolically active microbial communities in marine sediment under high-CO2 and low-pH extremes

Sediment-hosting hydrothermal systems in the Okinawa Trough maintain a large amount of liquid, supercritical and hydrate phases of CO₂ in the seabed. The emission of CO₂ may critically impact the geochemical, geophysical and ecological characteristics of the deep-sea sedimentary environment. So far it remains unclear whether microbial communities that have been detected in such high-CO₂ and low-pH habitats are metabolically active, and if so, what the biogeochemical and ecological consequences for the environment are. In this study, RNA-based molecular approaches and radioactive tracer-based respiration rate assays were combined to study the density, diversity and metabolic activity of microbial communities in CO₂-seep sediment at the Yonaguni Knoll IV hydrothermal field of the southern Okinawa Trough. In general, the number of microbes decreased sharply with increasing sediment depth and CO₂ concentration. Phylogenetic analyses of community structure using reverse-transcribed 16S ribosomal RNA showed that the active microbial community became less diverse with increasing sediment depth and CO₂ concentration, indicating that microbial activity and community structure are sensitive to CO₂ venting. Analyses of RNA-based pyrosequences and catalyzed reporter deposition-fluorescence in situ hybridization data revealed that members of the SEEP-SRB2 group within the Deltaproteobacteria and anaerobic methanotrophic archaea (ANME-2a and -2c) were confined to the top seafloor, and active archaea were not detected in deeper sediments (13–30 cm in depth) characterized by high CO₂. Measurement of the potential sulfate reduction rate at pH conditions of 3–9 with and without methane in the headspace indicated that acidophilic sulfate reduction possibly occurs in the presence of methane, even at very low pH of 3. These results suggest that some members of the anaerobic methanotrophs and sulfate reducers can adapt to the CO₂-seep sedimentary environment; however, CO₂ and pH in the deep-sea sediment were found to severely impact the activity and structure of the microbial community.     

嗜盐菌中甘氨酸甜菜碱的合成途径及其生物学功能

在嗜盐菌长期的盐适应或短期的盐胁迫过程中,甘氨酸甜菜碱(又名三甲基甘氨酸,N,N,N-trimethylglycine)发挥着极为重要的作用。甘氨酸甜菜碱在嗜盐菌中的生物合成有2种途径:胆碱氧化途径和甘氨酸甲基化途径。前者以胆碱为底物,由胆碱脱氢酶(cholinedehydrogenase,BetA)和甜菜碱乙醛脱氢酶(betaine aldehyde dehydrogenase,BetB)经2次氧化生成甜菜碱;后者以甘氨酸作为底物,由甘氨酸肌醇甲基转移酶(glycine sarcosine N-methyltransferase,GSMT)和肌氨酸二甲基甘氨酸甲基转移酶(sarcosine dimethylglycine N-methyltransferase,SDMT)经3次N-甲基化生成甜菜碱。目前在JGI-IMG和EZBioCloud数据库中公布了134株嗜盐菌标准菌株的全基因组序列。其中,约56.0%的嗜盐细菌和约39.6%的嗜盐古菌拥有胆碱氧化途径所需的2个基因;约9.7%的嗜盐细菌和约0.7%的嗜盐古菌携带甲基化途径所需的2个基因。其中,8株嗜盐细菌同时拥有胆碱氧化途径和甘氨酸甲基化途径所需的全部基因。甘氨酸甜菜碱生物合成基因在典型微生物菌株或经济作物中的表达可以提高其耐盐抗逆能力,这种独特的优势已经引起科学家们强烈的兴趣,相信未来,嗜盐菌中甘氨酸甜菜碱生物合成领域内的科学理论和技术应用会有重大的突破。     

Glycine betaine is the main organic osmotic solute in a stratified microbial community in a hypersaline evaporitic gypsum crust

We have examined the organic osmotic solutes content within the stratified microbial communities in an evaporitic gypsum crust found in an evaporation pond (~194 g/l total dissolved salts) of the salterns of the Israel Salt Company, Eilat. We extracted intracellular solutes from the upper three pigmented layers of the crust: a yellow-orange layer dominated by unicellular cyanobacteria, a green layer with filamentous cyanobacteria, and a layer colored red-purple by purple sulfur bacteria; dense communities of heterotrophic bacteria were present in all layers. The solutes were analyzed by Raman spectroscopy, ¹H and ¹³C nuclear magnetic resonance, and HPLC. All layers contained glycine betaine as the only detectable osmotic solute; ectoine and other solutes known to be produced by many halophilic and halotolerant prokaryotes were not found. In this first attempt to assess the osmotic solute content within complex natural communities of halophilic microorganisms, the predominant role of glycine betaine as an osmolyte was established. Most heterotrophic bacteria cannot produce glycine betaine but preferentially use it when it is supplied. Presence of glycine betaine produced by the photoautotrophic members of the community, therefore, may relieve the heterotrophs from the need to synthesize other compounds at a high-energy cost.     

Study of nitrogen fixation in microbial communities of oil-contaminated marine sediment microcosms

Aerobic microbial degradation of pollutant oil (petroleum) in aquatic environments is often severely limited by the availability of combined nitrogen. We therefore studied whether the microbial community enriched in marine sediment microcosms with an added oil layer and exposure to light harboured nitrogenase activity. The acetylene reduction (AR) assay indeed indicated active nitrogenase; however, similar activity was observed in oil-free control microcosms. In both microcosms, the AR rate was significantly reduced upon a dark shift, indicating that enriched cyanobacteria were the dominant diazotrophs. Analysis of structural dinitrogenase reductase genes (nifH) amplified from both microcosms indeed revealed NifH sequences related mostly to those of heterocystous cyanobacteria. NifH sequences typically affiliating with those of heterotrophic bacteria were more frequently retrieved from the oil-containing sediment. Expression analyses showed that mainly nifH genes similar to those of heterocystous cyanobacteria were expressed in the light. Upon a dark shift, nifH genes related to those of non-heterocystous cyanobacteria were expressed. Expression of nifH assignable to heterotrophs was apparently not significant. It is concluded that cyanobacteria are the main contributors of fixed nitrogen to oil-contaminated and pristine sediments if nitrogen is a limiting factor and if light is available. Hence, also the oil-degrading heterotrophic community may thus receive a significant part of combined nitrogen from cyanobacteria, even though oil vice versa apparently does not stimulate an additional nitrogen fixation in the enriched community.     

Analysis of marine microbial communities colonizing various metallic materials and rust layers

High-throughput sequencing was used to visualize microbial biocoenoses on different metallic surfaces and rust layers of highly corroded steels after immersion in coastal marine water for 30 months at Sanya, China. Distinct microbial community compositions were observed on these metallic surfaces. The dominant genus was the copper-tolerant, acid-producing Lactobacillus on copper alloys, the common aerobic surface colonizers Bacillus and Ruegeria on aluminum alloys, and aerobic biofilm-forming Pseudomonas on carbon steel. Most of these are copiotrophic microbes compared to planktonic microbes, which are oligotrophic. Additionally, sulfate-reducing prokaryotes (SRP) were detected in the rust layer, but the dominant genera changed from the outer layer to the inner part. The dominant genera detected in the outer, middle and inner rusts layers were Desulfotomaculum, Desulfonatronum (obligate anaerobe) and Desulfovibiro (electroactive), respectively. Further, the coexistence of methanogens with SRP suggests interspecies interactions.     

Unlocking the diversity and biotechnological potential of marine surface associated microbial communities

Marine sessile eukaryotic hosts provide a unique surface for microbial colonisation. Chemically mediated interactions between the host and colonising microorganisms, interactions between microorganisms in the biofilm community and surface-specific physical and chemical conditions impact differently on the diversity and function of surface-associated microbial assemblages compared with those in planktonic systems. Understanding the diversity and ecology of surface-associated microbial communities will greatly contribute to the discovery of next-generation, bioactive compounds. On the basis of recent conceptual and technological advances insights into the microbiology of marine living surfaces are improving and novel bioactives, including those previously ascribed as host derived, are now revealed to be produced by members of the surface-associated microbial community.     

环丙沙星对土壤微生物量碳和土壤微生物 群落碳代谢多样性的影响

采用氯仿熏蒸浸提法和Biolog法,分析环丙沙星作用下的土壤微生物量碳和微生物群落碳代谢多样性,以揭示环丙沙星在环境中残留对土壤微生物学性状的影响.结果表明,环丙沙星(wCIP≥0.1 μg/g)对土壤微生物量碳含量影响显著(P＜0.05),土壤中环丙沙星浓度愈高,微生物量碳含量愈低,100μg/g的环丙沙星处理使土壤微生物量碳含量下降58.69％.环丙沙星对土壤微生物群落碳代谢功能影响显著,环丙沙星降低了土壤微生物对碳水化合物、羧酸、氨基酸、聚合物、酚类和胺类的碳源利用率;环丙沙星(wCIP≥0.1 μg/g)显著影响了土壤微生物群落碳源代谢强度和代谢多样性,但不同浓度的环丙沙星对土壤微生物群落碳代谢功能的影响不同,0.1、1、10 μg/g的环丙沙星处理对土壤微生物群落碳代谢功能的影响主要表现在处理前期(用药第7天、21天),这种影响在处理后期(用药第35天)表现不明显,100μg/g的环丙沙星在用药的前期和后期均显著影响土壤微生物群落碳代谢功能,土壤中环丙沙星积累到该浓度可能对土壤微生物群落碳代谢功能产生难以逆转的长期影响.     

Shifts in coastal Antarctic marine microbial communities during and after melt water-related surface stratification

Antarctic coastal waters undergo major physical alterations during summer. Increased temperatures induce sea-ice melting and glacial melt water input, leading to strong stratification of the upper water column. We investigated the composition of micro-eukaryotic and bacterial communities in Ryder Bay, Antarctic Peninsula, during and after summertime melt water stratification, applying community fingerprinting (denaturing gradient gel electrophoresis) and sequencing analysis of partial 18S and 16S rRNA genes. Community fingerprinting of the eukaryotic community revealed two major patterns, coinciding with a period of melt water stratification, followed by a period characterized by regular wind-induced breakdown of surface stratification. During the first stratified period, we observed depth-related differences in eukaryotic fingerprints while differences in bacterial fingerprints were weak. Wind-induced breakdown of the melt water layer caused a shift in the eukaryotic community from an Actinocyclus sp.- to a Thalassiosira sp.-dominated community. In addition, a distinct transition in the bacterial community was found, but with a few days' delay, suggesting a response to the changes in the eukaryotic community rather than to the mixing event itself. Sequence analysis revealed a shift from an Alpha- and Gammaproteobacteria to a Cytophaga-Flavobacterium-Bacteroides-dominated community under mixed conditions. Our results show that melt water stratification and the transition to nonstabilized Antarctic surface waters may have an impact not only on micro-eukaryotic but also bacterial community composition.     

Glycine metabolism in anaerobes

Some strict anaerobic bacteria catalyze with glycine as substrate an internal Stickland reaction by which glycine serves as electron donor being oxidized by glycine-cleavage system or as electron acceptor being reduced by glycine reductase. In both cases, energy is conserved by substrate level phosphorylation. Except for the different substrate-activating proteins P _B , reduction of sarcosine or betaine to acetyl phosphate involves inEubacterium acidaminophilum the same set of proteins as observed for glycine, e.g. a unique thioredoxin system as electron donor and an acetyl phosphate-forming protein P _c interacting with the intermediarily formed Secarboxymethylselenoether bound to protein P _A .