Methylotroph Quorum Sensing Signal Identification by Inverse Stable Isotopic Labeling.

Natural products are an essential source of bioactive compounds. Isotopic labeling is an effective way to identify natural products that incorporate a specific precursor; however, this approach is limited by the availability of isotopically enriched precursors. We used an inverse stable isotopic labeling approach to identify natural products by growing bacteria on a 13C-carbon source and then identifying 12C-precursor incorporation by mass spectrometry. We applied this approach to methylotrophs, ecologically important bacteria predicted to have significant yet underexplored biosynthetic potential. We demonstrate that this method identifies N-acyl homoserine lactone quorum sensing signals produced by diverse methylotrophs grown on three different one-carbon compounds. We then apply this approach to simultaneously detect five previously unidentified signals produced by a methylotroph and link these compounds to their synthases. We envision that this method can be used to identify other natural product classes synthesized by methylotrophs and other organisms that grow on relatively inexpensive 13C-carbon sources.

Halotolerance mechanisms of the methanotroph Methylomicrobium alcaliphilum.

Methylomicrobium alcaliphilum is an alkaliphilic and halotolerant methanotroph. The physiological responses of M. alcaliphilum to high NaCl concentrations, were studied using RNA sequencing and metabolic modeling. This study revealed that M. alcaliphilum possesses an unusual respiratory chain, in which complex I is replaced by a Na+ extruding NQR complex (highly upregulated under high salinity conditions) and a Na+ driven adenosine triphosphate (ATP) synthase coexists with a conventional H+ driven ATP synthase. A thermodynamic and metabolic model showing the interplay between these different components is presented. Ectoine is the main osmoprotector used by the cells. Ectoine synthesis is activated by the transcription of an ect operon that contains five genes, including the ectoine hydroxylase coding ectD gene. Enzymatic tests revealed that the product of ectD does not have catalytic activity. A new Genome Scale Metabolic Model for M. alcaliphilum revealed a higher flux in the oxidative branch of the pentose phosphate pathway leading to NADPH production and contributing to resistance to oxidative stress.

A Novel Cold-adapted Methylovulum species, with a High C16:1ω5c Content, Isolated from an Arctic Thermal Spring in Spitsbergen.

A novel cold-adapted methane-oxidizing bacterium, termed TFB, was isolated from the thermoglacial Arctic karst spring, Trollosen, located in the South Spitsbergen National Park (Norway). The source water is cold and extremely low in phosphate and nitrate. The isolate belongs to the Methylovulum genus of gammaproteobacterial methanotrophs, with the closest phylogenetic affiliation with Methylovulum miyakonense and Methylovulum psychrotolerans (96.2 and 96.1% 16S rRNA gene sequence similarities, respectively). TFB is a strict aerobe that only grows in the presence of methane or methanol. It fixes atmospheric nitrogen and contains Type I intracellular membranes. The growth temperature range was 2-22°C, with an optimum at 13-18°C. The functional genes pmoA, mxaF, and nifH were identified by PCR, whereas mmoX and cbbL were not. C16:1ω5c was identified as the major fatty acid constituent, at an amount (>49%) not previously found in any methanotrophs, and is likely to play a major role in cold adaptation. Strain TFB may be regarded as a new psychrotolerant or psychrophilic species within the genus Methylovulum. The recovery of this cold-adapted bacterium from a neutral Arctic thermal spring increases our knowledge of the diversity and adaptation of extremophilic gammaproteobacterial methanotrophs in the candidate family "Methylomonadaceae".

Repurposing a chemosensory macromolecular machine.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

A novel Type I methanotroph Methylolobus aquaticus gen. nov. sp. nov. isolated from a tropical wetland.

A novel gammaproteobacterial methanotroph; strain FWC3 was isolated from a tropical freshwater wetland sample collected near a beach in Western India. Strain FWC3 forms flesh pink/peach colored colonies, is non-motile, and the cells are present as diplococci, triads, tetracocci and aggregates. Strain FWC3 grows only on methane and methanol. As the 16S rRNA gene of strain FWC3 showed low similarities with other Type I methanotrophs (less than 94.3%), it was further investigated for its novelty and characterisation by a polyphasic approach. ANI indices and DDH values deduced from the draft genome of strain FWC3 (SEYW00000000.1) with the other nearest type strains (Methylocaldum marinum S8T and Methylococcus capsulatus BathT) were ~ 70% and ~ 15%, respectively. The low level similarities indicated that strain FWC3 can belong to a new genus and species. Additionally, strain FWC3 showed a unique fatty acid profile with the dominance of C16:1 ω7 and ω6c, C16:0 and C16:1 ω9c. During the characterisation of strain FWC3, a morphologically similar methanotroph, strain C50C1 was described (Ghashghavi et al. in mSphere 4:e00631-18, 2019) and named as 'Methylotetracoccus oryzae'. We found that strain FWC3 and strain C50C1 belonged to the same genus but could belong to different species based on the ANI indices and dDDH values (~ 94% and ~ 55%, respectively). However, strain C50C1 has not been deposited in two culture collections and not been validly described. Also, the 16S rRNA gene of strain C50C1 is neither available on the database nor can it be retrieved from the genome assembly. Based on the polyphasic characterisation and comparison to the other type strains of Methylococcaceae, we propose strain FWC3 (= JCM 33786T, = KCTC 72733T, = MCC 4198T) to be the type strain of a novel genus and species, for which the name Methylolobus aquaticus is proposed. Strain C50C1 (Ghashghavi et al. 2019) could represent another species ('Methylolobus oryzae').

[Moderate grazing increases the abundance of soil methane-oxidizing bacteria and CH4 uptake rate in a typical steppe of Inner Mongolia, China.] / é€‚åº¦æ”¾ç‰§å¢žåŠ å† è’™å¤å ¸åž‹è‰åŽŸç”²çƒ·æ°§åŒ–èŒä¸°åº¦å’Œç”²çƒ·å¸æ”¶.

Microbial oxidation is the only biological sink of atmospheric methane (CH4). It is essential to understand the variation of CH4 fluxes among different grassland use types for developing low-emission management system. Here, we measured the CH4 flux and the soil methane-oxidizing bacteria abundance in a typical steppe under grazing, mowing and fencing management in central Inner Mongolia, with the aims to determine the effects of these grassland use types on CH4 flux, and to test the hypothesis that pmoA functional gene abundance regulates CH4 fluxes. The measurements were conducted on the experimental grassland that had experienced four grassland use treatments over five years. The treatments were whole growing season grazing from May to September (T1), spring and summer grazing (twice in May and July)(T2), autumn mowing (T3) and enclosure (T0). We measured CH4 flux using static chamber method, and quantified the abundance of pmoA functional genes using molecular techniques. Moreover, we measured plant biomass and soil physicochemical properties. The results showed that moderate grazing significantly enhanced CH4 uptake rate and the methane-oxidizing bacteria abundance (i.e., the pmoA gene copy number per gram of dry soil). The pmoA gene copy number ranged from 6.9×104 to 3.9×105 per gram of dry soil in growing season. The CH4 uptake rate was (68.21±3.01) µg·m-2·h-1 under T1, which was 22.1%, 37.5% and 30.9% higher than that under T2, T3 or T0 , respectively. The CH4 uptake rate was positively correlated with abundance of CH4 oxidizing bacteria and soil sand content, but negatively correlated with soil silt content, soil moisture, NH4+-N and NO3--N content, and plant biomass. These results suggested that the steppe ecosystem is a CH4 sink under all land-use types in central Inner Mongolia, and that moderate grazing would enhance methane-oxidizing bacteria abundance and CH4 uptake by improving soil sand content, reducing soil mineral nitrogen content and plant production in the typical steppe ecosystem. These results were of significance for the development of low-emission grassland management system.

Methylotetracoccus oryzae Strain C50C1 Is a Novel Type Ib Gammaproteobacterial Methanotroph Adapted to Freshwater Environments.

Methane-oxidizing microorganisms perform an important role in reducing emissions of the greenhouse gas methane to the atmosphere. To date, known bacterial methanotrophs belong to the Proteobacteria, Verrucomicrobia, and NC10 phyla. Within the Proteobacteria phylum, they can be divided into type Ia, type Ib, and type II methanotrophs. Type Ia and type II are well represented by isolates. Contrastingly, the vast majority of type Ib methanotrophs have not been able to be cultivated so far. Here, we compared the distributions of type Ib lineages in different environments. Whereas the cultivated type Ib methanotrophs (Methylococcus and Methylocaldum) are found in landfill and upland soils, lineages that are not represented by isolates are mostly dominant in freshwater environments, such as paddy fields and lake sediments. Thus, we observed a clear niche differentiation within type Ib methanotrophs. Our subsequent isolation attempts resulted in obtaining a pure culture of a novel type Ib methanotroph, tentatively named "Methylotetracoccus oryzae" C50C1. Strain C50C1 was further characterized to be an obligate methanotroph, containing C16:1ω9c as the major membrane phospholipid fatty acid, which has not been found in other methanotrophs. Genome analysis of strain C50C1 showed the presence of two pmoCAB operon copies and XoxF5-type methanol dehydrogenase in addition to MxaFI. The genome also contained genes involved in nitrogen and sulfur cycling, but it remains to be demonstrated if and how these help this type Ib methanotroph to adapt to fluctuating environmental conditions in freshwater ecosystems.IMPORTANCE Most of the methane produced on our planet gets naturally oxidized by a group of methanotrophic microorganisms before it reaches the atmosphere. These microorganisms are able to oxidize methane, both aerobically and anaerobically, and use it as their sole energy source. Although methanotrophs have been studied for more than a century, there are still many unknown and uncultivated groups prevalent in various ecosystems. This study focused on the diversity and adaptation of aerobic methane-oxidizing bacteria in different environments by comparing their phenotypic and genotypic properties. We used lab-scale microcosms to create a countergradient of oxygen and methane for preenrichment, followed by classical isolation techniques to obtain methane-oxidizing bacteria from a freshwater environment. This resulted in the discovery and isolation of a novel methanotroph with interesting physiological and genomic properties that could possibly make this bacterium able to cope with fluctuating environmental conditions.

Methylococcaceae are the dominant active aerobic methanotrophs in a Chinese tidal marsh.

Although coastal marshes are net carbon sinks, they are net methane sources. Aerobic methanotrophs in coastal marsh soils are important methane consumers, but their activity and populations are poorly characterized. DNA stable-isotope probing followed by sequencing was used to determine how active methanotrophic populations differed in the main habitats of a Chinese coastal marsh. These habitats included mudflat, native plant-dominated, and alien plant-dominated habitats. Methylococcaceae was the most active methanotroph family across four habitats. Abundant methylotroph sequences, including methanotrophs and non-methane-oxidizing methylotrophs (Methylotenera and Methylophaga), constituted 50-70% of the 16S rRNA genes detected in the labeled native plant-dominated and mudflat soils. Methylotrophs were less abundant (~ 20%) in labeled alien plant-dominated soil, suggesting less methane assimilation into the target community or a different extent of carbon cross-feeding. Canonical correspondence analysis indicated a significant correlation between the active bacterial communities and soil properties (salinity, organic carbon, total nitrogen, pH, and available phosphorus). Importantly, these results highlight how changing vegetation or soil features in coastal marshes may change their resident active methanotrophic populations, which will further influence methane cycling.

Methanotroph-derived bacteriohopanepolyol signatures as a function of temperature related growth, survival, cell death and preservation in the geological record.

Interpretation of bacteriohopanepolyol (BHP) biomarkers tracing microbiological processes in modern and ancient sediments relies on understanding environmental controls of production and preservation. BHPs from methanotrophs (35-aminoBHPs) were studied in methane-amended aerobic river-sediment incubations at different temperatures. It was found that: (i) With increasing temperature (4°C-40°C) a 10-fold increase in aminopentol (associated with Crenothrix and Methylobacter spp. growth) occurred with only marginal increases in aminotriol and aminotetrol; (ii) A further increase in temperature (50°C) saw selection for the thermophile Methylocaldum and mixtures of aminopentol and C-3 methylated aminopentol, again, with no increase in aminotriol and aminotetrol. (iii) At 30°C, more aminopentol and an aminopentol isomer and unsaturated aminopentol were produced after methanotroph growth and the onset of substrate starvation/oxygen depletion. (iv) At 50°C, aminopentol and C-3 methylated aminopentol, only accumulated during growth but were clearly resistant to remineralization despite cell death. These results have profound implications for the interpretation of aminoBHP distributions and abundances in modern and past environments. For instance, a temperature regulation of aminopentol production but not aminotetrol or aminotriol is consistent with and, corroborative of, observed aminopentol sensitivity to climate warming recorded in a stratigraphic sequence deposited during the Paleocene-Eocene thermal maximum (PETM).

Quorum Sensing in a Methane-Oxidizing Bacterium.

Aerobic methanotrophic bacteria use methane as their sole source of carbon and energy and serve as a major sink for the potent greenhouse gas methane in freshwater ecosystems. Dissecting the molecular details of how these organisms interact in the environment may increase our understanding of how they perform this important ecological role. Many bacterial species use quorum sensing (QS) systems to regulate gene expression in a cell density-dependent manner. We have identified a QS system in the genome of Methylobacter tundripaludum, a dominant methane oxidizer in methane enrichments of sediment from Lake Washington (Seattle, WA). We determined that M. tundripaludum produces primarily N-3-hydroxydecanoyl-l-homoserine lactone (3-OH-C10-HSL) and that its production is governed by a positive feedback loop. We then further characterized this system by determining which genes are regulated by QS in this methane oxidizer using transcriptome sequencing (RNA-seq) and discovered that this system regulates the expression of a putative nonribosomal peptide synthetase biosynthetic gene cluster. Finally, we detected an extracellular factor that is produced by M. tundripaludum in a QS-dependent manner. These results identify and characterize a mode of cellular communication in an aerobic methane-oxidizing bacterium.IMPORTANCE Aerobic methanotrophs are critical for sequestering carbon from the potent greenhouse gas methane in the environment, yet the mechanistic details of chemical interactions in methane-oxidizing bacterial communities are not well understood. Understanding these interactions is important in order to maintain, and potentially optimize, the functional potential of the bacteria that perform this vital ecosystem function. In this work, we identify a quorum sensing system in the aerobic methanotroph Methylobacter tundripaludum and use both chemical and genetic methods to characterize this system at the molecular level.

Methane oxidation in a landfill cover soil reactor: Changing of kinetic parameters and microorganism community structure.

Changing of CH4 oxidation potential and biological characteristics with CH4 concentration was studied in a landfill cover soil reactor (LCSR). The maximum rate of CH4 oxidation reached 32.40 mol d-1 m-2 by providing sufficient O2 in the LCSR. The kinetic parameters of methane oxidation in landfill cover soil were obtained by fitting substrate diffusion and consumption model based on the concentration profile of CH4 and O2. The values of [Formula: see text] (0.93-2.29%) and [Formula: see text] (140-524 nmol kgsoil-DW-1·s-1) increased with CH4 concentration (9.25-20.30%), while the values of [Formula: see text] (312.9-2.6%) and [Formula: see text] (1.3 × 10-5 to 9.0 × 10-3 nmol mL-1 h-1) were just the opposite. MiSeq pyrosequencing data revealed that Methylobacter (the relative abundance was decreased with height of LCSR) and Methylococcales_unclassified (the relative abundance was increased expect in H 80) became the key players after incubation with increasing CH4 concentration. These findings provide information for assessing CH4 oxidation potential and changing of biological characteristics in landfill cover soil.

Multiple I-Type Lysozymes in the Hydrothermal Vent Mussel Bathymodiolus azoricus and Their Role in Symbiotic Plasticity.

The aim of this study was first to identify lysozymes paralogs in the deep sea mussel Bathymodiolus azoricus then to measure their relative expression or activity in different tissue or conditions. B. azoricus is a bivalve that lives close to hydrothermal chimney in the Mid-Atlantic Ridge (MAR). They harbour in specialized gill cells two types of endosymbiont (gram-bacteria): sulphide oxidizing bacteria (SOX) and methanotrophic bacteria (MOX). This association is thought to be ruled by specific mechanism or actors of regulation to deal with the presence of symbiont but these mechanisms are still poorly understood. Here, we focused on the implication of lysozyme, a bactericidal enzyme, in this endosymbiosis. The relative expression of Ba-lysozymes paralogs and the global anti-microbial activity, were measured in natural population (Lucky Strike--1700 m, Mid-Atlantic Ridge), and in in situ experimental conditions. B. azoricus individuals were moved away from the hydrothermal fluid to induce a loss of symbiont. Then after 6 days some mussels were brought back to the mussel bed to induce a re-acquisition of symbiotic bacteria. Results show the presence of 6 paralogs in B. azoricus. In absence of symbionts, 3 paralogs are up-regulated while others are not differentially expressed. Moreover the global activity of lysozyme is increasing with the loss of symbiont. All together these results suggest that lysozyme may play a crucial role in symbiont regulation.

Gammaproteobacterial methanotrophs dominate cold methane seeps in floodplains of West Siberian rivers.

A complex system of muddy fluid-discharging and methane (CH4)-releasing seeps was discovered in a valley of the river Mukhrinskaya, one of the small rivers of the Irtysh Basin, West Siberia. CH4 flux from most (90%) of these gas ebullition sites did not exceed 1.45 g CH4 h(-1), while some seeps emitted up to 5.54 g CH4 h(-1). The δ(13)C value of methane released from these seeps varied between -71.1 and -71.3‰, suggesting its biogenic origin. Although the seeps were characterized by low in situ temperatures (3.5 to 5°C), relatively high rates of methane oxidation (15.5 to 15.9 nmol CH4 ml(-1) day(-1)) were measured in mud samples. Fluorescence in situ hybridization detected 10(7) methanotrophic bacteria (MB) per g of mud (dry weight), which accounted for up to 20.5% of total bacterial cell counts. Most (95.8 to 99.3%) methanotroph cells were type I (gammaproteobacterial) MB. The diversity of methanotrophs in this habitat was further assessed by pyrosequencing of pmoA genes, encoding particulate methane monooxygenase. A total of 53,828 pmoA gene sequences of seep-inhabiting methanotrophs were retrieved and analyzed. Nearly all of these sequences affiliated with type I MB, including the Methylobacter-Methylovulum-Methylosoma group, lake cluster 2, and several as-yet-uncharacterized methanotroph clades. Apparently, microbial communities attenuating methane fluxes from these local but strong CH4 sources in floodplains of high-latitude rivers have a large proportion of potentially novel, psychrotolerant methanotrophs, thereby providing a challenge for future isolation studies.

Weak phylogenetic signal in physiological traits of methane-oxidizing bacteria.

The presence of phylogenetic signal is assumed to be ubiquitous. However, for microorganisms, this may not be true given that they display high physiological flexibility and have fast regeneration. This may result in fundamentally different patterns of resemblance, that is, in variable strength of phylogenetic signal. However, in microbiological inferences, trait similarities and therewith microbial interactions with its environment are mostly assumed to follow evolutionary relatedness. Here, we tested whether indeed a straightforward relationship between relatedness and physiological traits exists for aerobic methane-oxidizing bacteria (MOB). We generated a comprehensive data set that included 30 MOB strains with quantitative physiological trait information. Phylogenetic trees were built from the 16S rRNA gene, a common phylogenetic marker, and the pmoA gene which encodes a subunit of the key enzyme involved in the first step of methane oxidation. We used a Blomberg's K from comparative biology to quantify the strength of phylogenetic signal of physiological traits. Phylogenetic signal was strongest for physiological traits associated with optimal growth pH and temperature indicating that adaptations to habitat are very strongly conserved in MOB. However, those physiological traits that are associated with kinetics of methane oxidation had only weak phylogenetic signals and were more pronounced with the pmoA than with the 16S rRNA gene phylogeny. In conclusion, our results give evidence that approaches based solely on taxonomical information will not yield further advancement on microbial eco-evolutionary interactions with its environment. This is a novel insight on the connection between function and phylogeny within microbes and adds new understanding on the evolution of physiological traits across microbes, plants and animals.

Characterization and phylogeny of a novel methanotroph, Methyloglobulus morosus gen. nov., spec. nov.

A novel methanotrophic gammaproteobacterium, strain KoM1, was isolated from the profundal sediment of Lake Constance after initial enrichment in opposing gradients of methane and oxygen. Strain KoM1 grows on methane or methanol as its sole source of carbon and energy. It is a Gram-negative methanotroph, often expressing red pigmentation. Cells are short rods and occur sometimes in pairs or short chains. Strain KoM1 grows preferably at reduced oxygen concentrations (pO2=0.05-0.1bar). It can fix nitrogen, and grows at neutral pH and at temperatures between 4 and 30°C. Phylogenetically, the closest relatives are Methylovulum miyakonense and Methylosoma difficile showing 91% 16S rRNA gene sequence identity. The only respiratory quinone is ubiquinone Q8; the main polar lipids are phosphatidyl ethanolamine and phosphatidyl glycerol. The major cellular fatty acids are summed feature 3 (presumably C16:1ω7c) and C16:1ω5c, and the G+C content of the DNA is 47.7mol%. Strain KoM1 is described as the type strain of a novel species within a new genus, Methyloglobulus morosus gen. nov., sp. nov.

CorA is a copper repressible surface-associated copper(I)-binding protein produced in Methylomicrobium album BG8.

CorA is a copper repressible protein previously identified in the methanotrophic bacterium Methylomicrobium album BG8. In this work, we demonstrate that CorA is located on the cell surface and binds one copper ion per protein molecule, which, based on X-ray Absorption Near Edge Structure analysis, is in the reduced state (Cu(I)). The structure of endogenously expressed CorA was solved using X-ray crystallography. The 1.6 Å three-dimensional structure confirmed the binding of copper and revealed that the copper atom was coordinated in a mononuclear binding site defined by two histidines, one water molecule, and the tryptophan metabolite, kynurenine. This arrangement of the copper-binding site is similar to that of its homologous protein MopE* from Metylococcus capsulatus Bath, confirming the importance of kynurenine for copper binding in these proteins. Our findings show that CorA has an overall fold similar to MopE, including the unique copper(I)-binding site and most of the secondary structure elements. We suggest that CorA plays a role in the M. album BG8 copper acquisition.

Highly efficient methane biocatalysis revealed in a methanotrophic bacterium.

Methane is an essential component of the global carbon cycle and one of the most powerful greenhouse gases, yet it is also a promising alternative source of carbon for the biological production of value-added chemicals. Aerobic methane-consuming bacteria (methanotrophs) represent a potential biological platform for methane-based biocatalysis. Here we use a multi-pronged systems-level approach to reassess the metabolic functions for methane utilization in a promising bacterial biocatalyst. We demonstrate that methane assimilation is coupled with a highly efficient pyrophosphate-mediated glycolytic pathway, which under oxygen limitation participates in a novel form of fermentation-based methanotrophy. This surprising discovery suggests a novel mode of methane utilization in oxygen-limited environments, and opens new opportunities for a modular approach towards producing a variety of excreted chemical products using methane as a feedstock.

[Methanotrophic bacteria in cold seeps of the floodplains of northern rivers].

Small mud volcanoes (cold seeps), which are common in the floodplains of northern rivers, are a potentially important, although poorly studied sources of atmospheric methane. Field research on the cold seeps of the Mukhrina River (Khanty-Mansiysk Autonomous okrug, Russia) revealed methane fluxes from these structures to be orders of magnitude higher than from equivalent areas of the mid-taiga bogs. Microbial communities developing around the seeps were formed under conditions of high methane concentrations, low temperatures (3-5 degrees C), and near-neutral pH. Molecular identification of methane-oxidizing bacteria from this community by analysis of the pmoA gene encoding particulate methane monooxygenase revealed both type I and type II methanotrophs (classes Gammaproteobacteria and Alphaproteobacteria, respectively), with predomination of type I methanotrophs. Among the latter, microorganisms related to Methylobacterpsychrophilus and Methylobacter tundripaludum, Crenothrix polyspora (a stagnant water dweller), and a number of methanotrophs belonging to unknown taxa were detected. Growth characteristics of two isolates were determined. Methylobactersp. CMS7 exhibited active growth at 4-10 degrees C, while Methylocystis sp. SB12 grew better at 20 degrees C. Experimental results confirmed the major role ofmethanotrophic gammaproteobacteria in controlling the methane emission from cold river seeps.

Survival or revival: long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria.

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium.

Diversity and phylogeny of the ectoine biosynthesis genes in aerobic, moderately halophilic methylotrophic bacteria.

The genes of ectoine biosynthesis pathway were identified in six species of aerobic, slightly halophilic bacteria utilizing methane, methanol or methylamine. Two types of ectoine gene cluster organization were revealed in the methylotrophs. The gene cluster ectABC coding for diaminobutyric acid (DABA) acetyltransferase (EctA), DABA aminotransferase (EctB) and ectoine synthase (EctC) was found in methanotrophs Methylobacter marinus 7C and Methylomicrobium kenyense AMO1(T). In methanotroph Methylomicrobium alcaliphilum ML1, methanol-utilizers Methylophaga thalassica 33146(T) , Methylophaga alcalica M8 and methylamine-utilizer Methylarcula marina h1(T), the genes forming the ectABC-ask operon are preceded by ectR, encoding a putative transcriptional regulatory protein EctR. Phylogenetic relationships of the Ect proteins do not correlate with phylogenetic affiliation of the strains, thus implying that the ability of methylotrophs to produce ectoine is most likely the result of a horizontal transfer event.