Composition, Variation, and Dynamics of Major Osmotic Solutes in Methanohalophilus Strain FDF1

Methanohalophilus strain FDF1, a member of the halophilic genus of methanogens, can grow over a range of external NaCl concentrations from 1.2 to 2.9 M and utilize methanol, trimethylamine, and dimethyl sulfide as substrates for methanogenesis. It produces the osmolytes glycine betaine, beta-glutamine, and N-acetyl-beta-lysine with increasing external NaCl, but the relative ratio of these zwitterions depends primarily on the methanogenic substrate and less on the external osmolarity. When the cells are grown on methanol in defined medium, accumulation of glycine betaine predominates over the other zwitterionic solutes. The cells also synthesized a carbohydrate which was not detected in cells grown on trimethylamine. This negatively charged compound, identified as alpha-glucosylglycerate from the C and H chemical shifts, does not act as an osmoregulatory solute in the salt range 1.4 to 2.7 M in this methanogen as evidenced by its invariant intracellular concentration. CH(3)OH-pulse/CH(3)OH-chase experiments were used to determine half-lifes for these organic solute pools in the cells. l-alpha-Glutamate showed a rapid loss of heavy isotope, indicating that l-alpha-glutamate functions as a biosynthetic intermediate in these cells. Measurable turnover rates for both beta-glutamine, which acts as an osmolyte, and alpha-glucosylglycerate suggest that they function as metabolic intermediates as well. Molecules which function solely as osmolytes (glycine betaine and N-acetyl-beta-lysine) showed a slower turnover consistent with their roles as osmotic solutes in Methanohalophilus strain FDF1.     

Methanohalophilus profundi sp. nov., a methylotrophic halophilic piezophilic methanogen isolated from a deep hypersaline anoxic basin

A novel anaerobic methylotrophic halophilic methanogen strain SLHTYRO^T was isolated from a deep hypersaline anoxic basin called “Tyro” located in the Eastern Mediterranean Sea. Cells of SLHTYRO^T were motile cocci. The strain SLHTYRO^T grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L⁻¹ NaCl (optimum 135 g L⁻¹). Strain SLHTYRO^T was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYRO^T was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYRO^T was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYRO^T should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYRO^T (=DSM 108854 = JCM 32768 = UBOCC-M-3308).     

Glycine betaine and potassium ion are the major compatible solutes in the extremely halophilic methanogen Methanohalophilus strain Z7302.

Methanohalophilus strain Z7302 was previously isolated from a hypersaline environment and grows over a range of NaCl concentrations from 1.7 to 4.4 M. We examined the relationships between cell growth rate, cell volume, and intracellular solute concentrations with increasing salinity. This extremely halophilic methanogen synthesized three zwitterionic compounds, beta-glutamine, N epsilon-acetyl-beta-lysine, and glycine betaine, and also accumulated potassium ion as compatible solutes to balance the external and internal osmotic pressures. Potassium and glycine betaine were the predominant compatible solutes when Methanohalophilus strain Z7302 was grown at high external NaCl concentrations and approached intracellular levels of 3 and 4 M, respectively.     

Phylogenetic diversity of methyl-coenzyme M reductase (mcrA) gene and methanogenesis from trimethylamine in hypersaline environments

Methanogens have been reported in complex microbial communities from hypersaline environments, but little is known about their phylogenetic diversity. In this work, methane concentrations in environmental gas samples were determined while methane production rates were measured in microcosm experiments with competitive and non-competitive substrates. In addition, the phylogenetic diversity of methanogens in microbial mats from two geographical locations was analyzed: the well studied Guerrero Negro hypersaline ecosystem, and a site not previously investigated, namely Laguna San Ignacio, Baja California Sur, Mexico. Methanogenesis in these microbial mats was suspected based on the detection of methane (in the range of 0.00086 to 3.204 %) in environmental gas samples. Microcosm experiments confirmed methane production by the mats and demonstrated that it was promoted only by non-competitive substrates (trimethylamine and methanol), suggesting that methylotrophy is the main characteristic process by which these hypersaline microbial mats produce methane. Phylogenetic analysis of amino acid sequences of the methyl coenzyme-M reductase (mcrA) gene from natural and manipulated samples revealed various methylotrophic methanogens belonging exclusively to the family Methanosarcinaceae. Moderately halophilic microorganisms of the genus Methanohalophilus were predominant (>60 % of mcrA sequences retrieved). Slightly halophilic and marine microorganisms of the genera Methanococcoides and Methanolobus, respectively, were also identified, but in lower abundances.     

Isolation and characterization of Halomonas sp. strain IMPC, a p-coumaric acid-metabolizing bacterium that decarboxylates other cinnamic acids under hypersaline conditions

A moderately halophilic, mesophilic, Gram-negative, motile, nonsporulating bacterium, designated strain IMPC, was isolated from a table-olive fermentation rich in aromatic compounds, after enrichment on p-coumaric acid under halophilic conditions. Strain IMPC was able to degrade p-coumaric acid. p-hydroxybenzaldehyde and p-hydroxybenzoic acid were detected as breakdown products from p-coumaric acid. Protocatechuic acid was identified as the final aromatic product of p-coumaric acid catabolism before ring fission. Strain IMPC transformed various cinnamic acids with substituent H, OH, CH(3) or OCH(3) in the para- and/or meta-position of the aromatic ring to the corresponding benzoic acids, indicating a specific selection. A beta-oxidation pathway was proposed for these transformations. Phylogenetic analysis of the 16S rRNA gene revealed that this isolate was a member of the genus Halomonas. Strain IMPC was closely related to Halomonas elongata ATCC 33173(T)and Halomonas eurihalina ATCC 49336(T).     

Isolation and characterization of novel halotolerant and/or halophilic denitrifying bacteria with versatile metabolic pathways for the degradation of trimethylamine

Four denitrifying bacteria capable of degrading trimethylamine under both aerobic and denitrifying conditions were newly isolated from coastal sediments and wastewater contaminated by marine water. All strains were in alpha-Proteobacteria. Strain GP43 was classified as a member of genus Paracoccus, and strain PH32, PH34 and GRP21 were novel organisms with remote phylogenetic position from other genus alpha-Proteobacteria. Among these four strains were the halophilic strains PH32, PH34 and GRP21, which did not grow in the absence of sodium chloride in culture medium. Cells grown under denitrifying conditions possessed trimethylamine dehydrogenase while cells grown aerobically possessed two different enzymes for oxidation of trimethylamine, trimethylamine dehydrogenase and trimethylamine monooxygenase. The newly isolated strain PH32, PH34 and GRP21 may be the first halophilic bacteria to degrade trimethylamine under denitrifying conditions.     

Biosynthetic pathways of the osmolytes N epsilon-acetyl-beta-lysine, beta-glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses.

Methanohalophilus strain FDF1 synthesizes beta-glutamine, betaine, and N epsilon-acetyl-beta-lysine as osmoprotective agents when the cells are grown in high external concentrations of NaCl. Nuclear magnetic resonance spectroscopic analyses of 13CH3OH-12CO2 label incorporation by the cells provide information on the biosynthetic pathways of these organic osmolytes. The labeling studies indicate that Methanohalophilus strain FDF1 produces glutamate and beta-glutamine via a partial oxidative Krebs pathway. 13C labeling of betaine is consistent with methylation of glycine generated from serine (via serine hydroxymethyltransferase). The labeling pattern for N epsilon-acetyl-beta-lysine is consistent with the synthesis of its precursor alpha-lysine occurring by the diaminopimelate pathway in these cells.     

Influence of pH on Ammonia Accumulation and Toxicity in Halophilic, Methylotrophic Methanogens

We studied the effects of pH and ammonia concentration on the growth of three methanogens. These three halophilic, methylotrophic methanogens, Methanolobus bombayensis, Methanolobus taylorii, and Methanohalophilus zhilinaeae, grew at environmental pH ranges that overlapped with each other and spanned the pH range from 7.0 to 9.5. During growth they had reversed membrane pH gradients ((Delta)pH) at all pH values tested. The (Delta)pH was in the range of -0.4 to -0.9 pH units, with the cytosol being more acidic than the environmental pH. Methanohalophilus zhilinaeae had the most negative (Delta)pH (-0.9 pH units). These negative pH gradients resulted in the accumulation of ammonium (NH(inf4)(sup+)), and when grown at the highest external ammonia concentrations that allowed good growth, cells had cytosolic NH(inf4)(sup+) concentrations as high as 180 mM. The high concentrations of cytosolic NH(inf4)(sup+) were accompanied by greater (Delta)pH and lower concentrations of the major cytosolic cation K(sup+) (compared with cells grown in medium with only 5 mM ammonia). Methanolobus bombayensis and Methanolobus taylorii were more sensitive to total external ammonia at higher external pH values, but the inhibitory concentration of un-ionized ammonia that resulted in a 50% reduction of the growth rate was about 2 to 5 mM, regardless of the pH. This is consistent with growth inhibition by ammonia in other bacteria. However, Methanohalophilus zhilinaeae was more resistant to un-ionized ammonia than any other known organism. It had a 50% inhibitory concentration for un-ionized ammonia of 13 mM at pH 8.5 and 45 mM at pH 9.5. We examined the effects of pH on three ammonia-assimilating activities (glutamine synthetase, glutamate dehydrogenase, and alanine dehydrogenase) in cell lysates and found that the pH ranges were consistent with the observed ranges of intracellular pH.     

Anaerobic growth of halophilic archaeobacteria by reduction of dimethysulfoxide and trimethylamine N-oxide

Abstract Most representatives of the halophilic arachaeobacterial genera Halobacterium, Haloarcula and Haloferax tested were able to reduce dimethylsulfoxide (DMSO) to dimethylsulfide (DMS) and trimethylamine N -oxide (TMAO) to trimethylamine (TMA) under (semi)anaerobic conditions. In most cases the reduction of DMSO and TMAO was accompanied by an increase in cell yield. The ability to reduce DMSO or TMAO was not correlated to reduced DMSO or TMAO was not correlated with the ability to reduce nitrate to nitrite. Anaerobic respiration with DMSO and TMAO as electron acceptor supplies the halophilic archeobacteria with an additional mode of energy generation in the absence of molecular oxygen.     

Halobacterium saccharovorum sp. nov., a carbohydrate-metabolizing, extremely halophilic bacterium.

The previously described extremely halophilic bacterium, strain M6, metabolizes a variety of carbohydrates with the production of acid. In addition, the organism produces nitrite (but no gas) from nitrate, is motile, and grows most rapidly at about 50 degrees C. These characteristics distinguish it from all previously described halophilic bacteria in the genus Halobacterium. It is suggested that it be designated as a new species, Halobacterium saccharovorum.     

Phospholipid and fatty acid compositions of Alteromonas putrefaciens and A. haloplanktis

The phospholipid and fatty acid composition of Alteromonas putrefaciens S29 (non-halophilic type) and A. haloplanktis S5B (halophilic type) was determined. Major phospholipids of both strains were the same when they were grown in media containing optimum salt concentrations. However, the fatty acid composition of phos-pholipids in strain S29 was remarkably different from that of strain S5B. Strain S29 contained iso-C15: 0 and eicosapentaenoic acid (20: 5) as constituent fatty acids of phospholipids and also contained sterol ester and wax as neutral lipids. In contrast, strain S5B did not contain branched and polyunsaturated fatty acids, and neither sterol ester nor wax were detected.     

Alkalibacillus halophilus sp. nov., a new halophilic species isolated from hypersaline soil in Xin-Jiang province, China

A halophilic, Gram-positive, spore-forming motile Bacillus-like strain YIM 012(T), was isolated from one of the hypersaline soil samples collected in Xin-jiang province, China. Its optimum growth occurred at 10-20% of NaCl concentration (w/v), pH 7.0-8.0. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM 012(T) is a member of the genus of Alkalibacillus, which is well supported by its chemotaxonomic and molecular characteristics. Based on its phenotypic evidence and genotypic data, Alkalibacillus halophilus sp. nov. was proposed and strain YIM 012(T) (=DSM 17369(T)=KCTC 3990(T)) was assigned as the type strain of the novel species.     

<Emphasis Type="Italic">Streptomyces tritolerans</Emphasis> sp. nov., a novel actinomycete isolated from soil in Karnataka,India

A Gram-positive, nonmotile, moderately halophilic, alkali and thermotolerant strain designated DAS 165(T), was isolated from a dry land soil sample from the Gulbarga region, Karnataka province, India. The isolate produced yellow substrate mycelia and gray aerial mycelia on most tested media. Strain DAS 165(T) showed growth in the presence of 5 to 7% NaCl and at 45 degrees C. The DNA G + C content was 69.7%. 16S rRNA gene sequence analysis together with these characteristics consistently assigned strain DAS 165(T) to the genus Streptomyces. The 16S rRNA gene sequence analysis revealed that strain DAS 165(T) was most closely related to S. tendae ATCC 19812(T) (D 63873) with a sequence similarity of 99.6% (three nucleotide differences out of 1,517). Strain DAS 165(T) formed a distinct clade based on analysis of the almost complete sequence and 120-nucleotide variable gamma region of the 16S rRNA gene. Despite the high sequence similarity, strain DAS 165(T) was phenotypically different from S. tendae ATCC 19812(T). DNA-DNA hybridization between these strains was 47% showing that strain DAS 165(T) is a distinct genomic species. Phenetic and genetic results support the classification of strain DAS 165(T) as a new species, for which the name S. tritolerans is proposed, with strain DAS 165(T) as the type strain (=DSM 41899(T )= CCTCCAA 206013(T)).     

Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes

AIMS: The aim of this study was to determine the diversity of moderately halophilic bacteria with hydrolase activities. METHODS AND RESULTS: Screening bacteria from different hypersaline environments in South Spain led to the isolation of a total of 122 moderately halophilic bacteria able to produce different hydrolases (amylases, DNases, lipases, proteases and pullulanases). These bacteria are able to grow optimally in media with 5-15% salts and in most cases up to 20-25% salts. In contrast to strains belonging to previously described species, that showed very little hydrolase activities, environmental isolates produced a great variety of hydrolases. These strains were identified as members of the genera: Salinivibrio (55 strains), Halomonas (25 strains), Chromohalobacter (two strains), Bacillus-Salibacillus (29 strains), Salinicoccus (two strains) and Marinococcus (one strain), as well as eight non-identified isolates. CONCLUSIONS: Moderately halophilic bacteria are a source of hydrolytic enzymes such as amylases, DNases, lipases, proteases and pullulanases. SIGNIFICANCE AND IMPACT OF THE STUDY: Although most culture collection strains are not able to produce hydrolases, it has been shown that environmental isolates can produce these potentially biotechnological important enzymes.     

Glycine betaine transport in the obligate halophilic archaeon Methanohalophilus portucalensis

Transport of the osmoprotectant glycine betaine was investigated using the glycine betaine-synthesizing microbe Methanohalophilus portucalensis (strain FDF1), since solute uptake for this class of obligate halophilic methanogenic Archaea has not been examined. Betaine uptake followed a Michaelis-Menten relationship, with an observed K(t) of 23 microM and a V(max) of 8 nmol per min per mg of protein. The transport system was highly specific for betaine: choline, proline, and dimethylglycine did not significantly compete for [(14)C]betaine uptake. The proton-conducting uncoupler 2, 4-dinitrophenol and the ATPase inhibitor N, N-dicyclohexylcarbodiimide both inhibited glycine betaine uptake. Growth of cells in the presence of 500 microM betaine resulted in faster cell growth due to the suppression of the de novo synthesis of the other compatible solutes, alpha-glutamate, beta-glutamine, and N(epsilon)-acetyl-beta-lysine. These investigations demonstrate that this model halophilic methanogen, M. portucalensis strain FDF1, possesses a high-affinity and highly specific betaine transport system that allows it to accumulate this osmoprotectant from the environment in lieu of synthesizing this or other osmoprotectants under high-salt growth conditions.     

A novel denitrifying bacterial isolate that degrades trimethylamine both aerobically and anaerobically via two different pathways

The aerobic and anaerobic degradation of trimethylamine by a newly isolated denitrifying bacterium from an enrichment culture with trimethylamine inoculated with activated sludge was studied. Based on 16S rDNA analysis, this strain was identified as a Paracoccus sp. The isolate, strain T231, aerobically degraded trimethylamine, dimethylamine and methylamine and released a stoichiometric amount of ammonium ion into the culture fluid as a metabolic product, indicating that these methylated amines were completely degraded to formaldehyde and ammonia. The strain degraded trimethylamine also under denitrifying conditions and consumed a stoichiometric amount of nitrate, demonstrating that complete degradation of trimethylamine was coupled with nitrate reduction. Cell-free extract prepared from cells grown aerobically on trimethylamine exhibited activities of trimethylamine mono-oxygenase, trimethylamine N-oxide demethylase, dimethylamine mono-oxygenase, and methylamine mono-oxygenase. Cell-free extract from cells grown anaerobically on trimethylamine and nitrate exhibited activities of trimethylamine dehydrogenase and dimethylamine dehydrogenase. These results indicate that strain T231 had two different pathways for aerobic and anaerobic degradation of trimethylamine. This is a new feature for trimethylamine metabolism in denitrifying bacteria.     

Isolation and Characterization of a Halophilic Methanogen from Great Salt Lake

A halophilic methanogenic microorganism isolated from sediments collected from the southern arm of Great Salt Lake, Utah, is described. Cells were irregular, nonmotile cocci approximately 1.0 μm in diameter and stained gram negative. Colonies from anaerobic plates and roll tubes were foamy, circular, and cream-yellow. Methanol, methylamine, dimethylamine, and trimethylamine supported growth and methanogenesis. Hydrogen-carbon dioxide, formate, and acetate were not utilized. Sodium and magnesium were required for growth; the optimum NaCl concentration ranged between 1.0 and 2.0 M, with the minimum doubling time occurring at 2.0 M. The optimum growth temperature was 35°C, with maximum growth rate occurring at pH 7.5. The DNA base composition was 48.5 mol% guanine + cytosine. SLP is the type strain designation (= ATCC 35705).     

Gracilibacillus quinghaiensis sp. nov., isolated from salt-lake sediment in the Qaidam Basin, north-west China

A motile, Gram-positive, slightly halophilic, endospore-forming, catalase- and oxidase-positive, obligately aerobic, slender rod-shaped bacterium, strain YIM-C229T was isolated from the sediment of a salt lake in the Qaidam Basin, north-west China. Filamentous forms were present throughout the growth cycle. Strain YIM-C229T grew in the presence of 0.5-8% NaCl and at pH 6.0-8.5, with optimum growth at 1-3% NaCl and pH 7.0-7.5. It grew at 4-45 degrees C, with optimum growth at 37 degrees C. The major cellular fatty acids were anteiso-C15:0, C16:0, iso-C15:0, C16:1 omega11c and anteiso-C(17:0). The predominant respiratory quinone was MK-7, and diphosphatidylglycerol and phosphatidylglycerol were the polar lipids, with meso-diaminopimelic acid occurring in the cell-wall peptidoglycan. The genomic DNA G+C content was 40.9 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM-C229T was closely related to the type strains of the four recognized species of the genus Gracilibacillus: G. halotolerans NN T (sequence similarity 95.5%), G. dipsosauri DD1T (96.1%), G. orientalis XH-63T (96.8%) and G. boraciitolerans T-16X(T) (99.1%). The DNA-DNA relatedness between strain YIM-C229(T) and G. boraciitolerans DSM 17256(T) was 30.8%. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic differences and DNA-DNA hybridization data supported the view that this strain represented a novel species of the genus Gracilibacillus, for which the name Gracilibacillus quinghaiensis sp. nov. is proposed, with YIM-C229T (=DSM 17858T=CGMCC 1.6304T) as the type strain.     

Identification of moderately halophilic bacteria from Thai fermented fish ( pla-ra ) and proposal of Virgibacillus siamensis sp. nov

Forty-one isolates of moderately halophilic bacteria were isolated from fermented fish (pla-ra) in Thailand. On the basis of their phenotypic and chemotaxonomic characteristics, DNA-DNA relatedness and 16S rRNA gene sequences analyses, they were divided into six groups. The isolates in Group I to V were Gram-positive rod-shaped bacteria. They contained meso-diaminopimelic acid in the cell-wall peptidoglycan and menaquinone with seven isoprene units (MK-7). An isolate in Group VI was a Gram-negative rod-shaped bacterium. The DNA G+C contents of tested strains ranged from 36.5-63 mol%. Ten strains (Group I) were identified as Virgibacillus dokdonensis, 13 isolates (Group II) as V. halodenitrificans, 14 isolates (Group III) as V. marismortui, 1 isolate (Group IV) as Virgibacillus sp., 2 isolates (Group V) as Bacillus vietnamnensis, and 1 isolate (Group VI) as Chromohalobacter salexigens. Isolate MS3-4 in Group IV was closely related to V. carmonensis KCTC 3819(T) (95.9%). This strain contained anteiso-C(15:0) (55.8%) and anteiso-C(17:0) (17.7%) as major cellular fatty acids and had phosphatidylglycerol, diphosphatidylglycerol and an unidentified glycolipid as polar lipids. The DNA G+C content of MS3-4 was 38.0 mol%. The strain from Group IV is proposed as Virgibacillus siamensis sp. nov. and MS3-4(T) is the type strain (JCM 15395(T) =PCU 312(T) =TISTR 1957(T)).