Tatjana P. Tourova

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov. — Novel Aerobic Facultatively Methylotrophic Bacteria Utilizing Dichloromethane

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).

Methylarcula marina gen. nov., sp. nov. and Methylarcula terricola sp. nov.: novel aerobic, moderately halophilic, facultatively methylotrophic bacteria from coastal saline environments.

A new genus, Methylarcula, with two new species, Methylarcula marina and Methylarcula terricola, are proposed for strains h1T and h37T of moderately halophilic facultatively methylotrophic bacteria isolated from the coastal saline habitats. These methylobacteria are aerobic, Gram-negative, asporogenous, non-motile, colourless rods that multiply by binary fission. Their cellular fatty acids profiles consist primarily of straight-chain unsaturated (C18:1; 70-80%), saturated (C18:0; 14-16%) and cyclopropane (C19:0; 5-6%) acids. The major ubiquinone is Q-10. The dominant phospholipids are phosphatidylethanolamine and phosphatidylcholine. Both strains could use methylamine, some sugars and organic acids as carbon and energy sources. They grew well under optimal conditions (29-35 degrees C, pH 7.5-8.5, 0.5-1.0 M NaCl) and accumulated intracellularly poly-beta-hydroxybutyrate and the compatible solute ectoine. The ectoine pool was found to increase upon increasing the external NaCl concentration and accounted for 18% of the dry cellular weight. Both strains oxidized methylamine by the N-methylglutamate (N-MG) pathway enzymes (gamma-glutamylmethylamide synthetase/lyase and N-MG synthetase/lyase) to formaldehyde and assimilated it via the icl- serine pathway. The DNA G+C content was 60-4 mol% for Methylarcula marina h1T and 57.1 mol% for Methylarcula terricola h37T. The DNA-DNA hybridization value between strains hl and h37 was 25-30%, although they had a low level of DNA relatedness (5-7%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Methylorhabdus and Methylopila. A comparative 16S rDNA sequence-based phylogenetic analysis placed the two species of Methylarcula into a separate branch of the alpha-3 subclass of the Proteobacteria. The type strains of the new species are Methylarcula marina h1T (= VKM B-2159T) and Methylarcula terricola h37T (= VKM B-2160T).

Sulfidogenesis under extremely haloalkaline conditions by Desulfonatronospira thiodismutans gen. nov., sp. nov., and Desulfonatronospira delicata sp. nov. - a novel lineage of Deltaproteobacteria from hypersaline soda lakes.

High rates of sulfidogenesis were observed in sediments from hypersaline soda lakes. Anaerobic enrichment cultures at 2 M Na(+) and pH 10 inoculated with sediment samples from these lakes produced sulfide most actively with sulfite and thiosulfate as electron acceptors, and resulted in the isolation of three pure cultures of extremely natronophilic sulfidogenic bacteria. Strain ASO3-1 was isolated using sulfite as a sole substrate, strain AHT 8 with thiosulfate and formate, and strain AHT 6 with thiosulfate and acetate. All strains grew in a mineral soda-based medium by dismutation of either sulfite or thiosulfate, as well as with sulfite, thiosulfate and sulfate as acceptors, and H(2) and simple organic compounds as electron donors. The acetyl-CoA pathway was identified as the pathway for inorganic carbon assimilation by strain ASO3-1. All strains were obligately alkaliphilic, with an optimum at pH 9.5-10, and grew in soda brines containing 1-4 M total Na(+) (optimum at 1.0-2.0 M). The cells accumulated high amounts of the organic osmolyte glycine betaine. They formed a new lineage within the family Desulfohalobiaceae (Deltaproteobacteria), for which the name Desulfonatronospira gen. nov. is proposed. Strains ASO3-1(T) and AHT 8 from Kulunda Steppe formed Desulfonatronospira thiodismutans sp. nov., and strain AHT 6(T) from Wadi al Natrun is suggested as Desulfonatronospira delicata sp. nov.

Methanosarcina lacustris sp. nov., a new psychrotolerant methanogenic archaeon from anoxic lake sediments.

A new psychrotolerant methanogenic archaeon strain ZS was isolated from anoxic lake sediments (Switzerland). The cells of the organism were non-motile cocci, 1.5-3.5 microm in diameter. The cells aggregated and formed pseudoparenchyma. The cell wall was Gram-positive. The organism utilized methanol, mono-, di-, trimethylamine and H2/CO2 with methane production. The temperature range for growth was 1-35 degrees C with an optimum at 25 degrees C. The DNA G+C content of the organism was 43.4. mol%. Analysis of the 16S rRNA gene sequence showed that strain ZS was phylogenetically closely related to members of the genus Methanosarcina, but clearly differed from all described species of this genus (95.6-97.6% of sequence similarity). The level of DNA-DNA hybridization of strain ZS with Methanosarcina barkeri and Methanosarcina mazei was 15 and 31%, respectively. Based on the results of physiological and phylogenetic studies strain ZS can be assigned to a new species of the genus Methanasarcina. The name Methanosarcina lacustris sp. nov. is proposed. The type strain is ZS (= DSM 13486T, VKM B-2268).

Proposal of Oscillochloridaceae fam. nov. on the basis of a phylogenetic analysis of the filamentous anoxygenic phototrophic bacteria, and emended description of Oscillochloris and Oscillochloris trichoides in comparison with further new isolates.

The nucleotide sequences of the genes of 16S rRNAs were determined for the type strain Oscillochloris trichoides DG-6T and three new strains of Oscillochloris-like mesophilic filamentous green bacteria. Two major clusters have been found within the family Chloroflexaceae by phylogenetic-analysis: one cluster includes thermophilic species of Chloroflexus and the second includes mesophilic strains of Oscillochloris. The degree of relatedness of these clusters was below an intergeneric level, having only 82.5-86.5% of 16S rDNA sequence similarity. These phylogenetic data correlate well with the significant physiological, biochemical and chemotaxonomical differences between members of both groups. Therefore, the Oscillochloris and Chloroflexus clusters should be considered as two separate families. The description of the new family, Oscillochloridaceae fam. nov., and emended descriptions of the genus Oscillochloris and the species Oscillochloris trichoides are presented.

Nitriliruptor alkaliphilus gen. nov., sp. nov., a deep-lineage haloalkaliphilic actinobacterium from soda lakes capable of growth on aliphatic nitriles, and proposal of Nitriliruptoraceae fam. nov. and Nitriliruptorales ord. nov.

A novel bacterial strain, designated ANL-iso2(T), was obtained from an enrichment culture inoculated with a mixture of soda lake sediments by using isobutyronitrile (iBN) as the carbon, energy and nitrogen source at pH 10. The enrichment resulted in a stable binary culture containing iBN-degrading Gram-positive rods and a satellite Gram-negative gammaproteobacterium Marinospirillum sp. strain (ANL-isoa) scavenging the products of nitrile hydrolysis. Cells of the iBN-degrading strain, ANL-iso2(T), were short, non-motile, non-spore-forming rods. Strain ANL-iso2(T) was capable of utilizing propionitrile (C(3)), butyronitrile (C(4)), isobutyronitrile (C(4)), valeronitrile (C(5)) and capronitrile (C(6)) as the only growth substrate. Growth on nitriles was biphasic with fast initial hydrolysis of nitriles to the corresponding amides, carboxylic acids and ammonia and slow further utilization of these products resulting in biomass growth. Cells of strain ANL-iso2(T) grown with iBN were capable of extremely active hydration of a wide range of nitriles into the corresponding amides and much slower hydrolysis of these amides to the corresponding carboxylic acids. This indicated the presence of the nitrile hydratase/amidase pathway of nitrile degradation in the novel bacterium. Strain ANL-iso2(T) showed obligately alkaliphilic growth on iBN within the pH range 8.4-10.6, with optimum growth at 9.0-9.5. It was moderately salt-tolerant, with a salt range for growth of 0.1-2.0 M Na(+) and an optimum salt concentration for growth of 0.2-0.3 M. The dominant fatty acids in the polar lipids were C(16 : 0), iso-C(14), C(14 : 0), iso-C(16) and C(16 : 1)omega7. The cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid. Phylogenetic analysis placed strain ANL-iso2(T) within the class Actinobacteria as an independent lineage with only uncultured bacteria from soda lakes as its nearest relatives. On the basis of its unique phenotype and distinct phylogeny, strain ANL-iso2(T) is considered to represent a novel species of a new genus, for which the name Nitriliruptor alkaliphilus gen. nov., sp. nov. is proposed. The type strain of the type species, Nitriliruptor alkaliphilus, is ANL-iso2(T) (=DSM 45188(T)=NCCB 100119(T)=UNIQEM U239(T)). Phylogenetic data suggest that the novel bacterium forms the basis of a new family Nitriliruptoraceae fam. nov. and a novel order Nitriliruptorales ord. nov. within the class Actinobacteria.

Methylopila capsulata gen. nov., sp. nov., a novel non-pigmented aerobic facultatively methylotrophic bacterium

A new genus, Methylopila, and one new species are described for a group of seven strains of facultatively methylotrophic bacteria with the serine pathway of C1 assimilation. These bacteria are aerobic, Gram-negative, non-spore--forming, motile, colourless rods that multiply by binary fission. Their DNA base content ranges from 66 to 70 mol % G + C. Their cellular fatty acid profile consists primarily of C18:1 omega 7 cis-vaccenic and C19:0 cyclopropane acids. The major hydroxy acid is 3-OH C14:0. The main ubiquinone is Q-10. The dominant cellular phospholipids are phosphatidylethanolamine and phosphatidylcholine. The new isolates have a low level of DNA-DNA homology (5-10%) with the type strains of the serine pathway methylobacteria belonging to the genera Methylobacterium, Aminobacter, Hyphomicrobium and Methylorhabdus. Another approach, involving 16S rRNA gene sequence analysis of strain IM1T, has shown that the new isolates represent a separate branch within the alpha-2 subclass of the Proteobacteria. The type species of the new genus is Methylopila capsulata sp. nov., with the type strain IM1T (= VKM B-1606T).

Sulfidogenesis under extremely haloalkaline conditions in soda lakes of Kulunda Steppe (Altai, Russia).

Sulfidogenic activity (SA) in anoxic sediments of several soda lakes with variable salinity in south Kulunda Steppe (Altai, Russia) has been investigated. The study included in situ measurements of sulfate reduction rates and laboratory experiments with sediment slurries in which sulfate, thiosulfate or elemental sulfur were used as electron acceptors. Despite the extreme conditions (high salt concentrations and high pH), the SA values were relatively high (ranging from 0.02 to 1.20 micromol HS(-) cm(-3) h(-1)), and only hampered under salt-saturated conditions. The highest SA was observed with elemental sulfur, followed by thiosulfate, while the lowest SA was determined in the presence of sulfate. Of all the electron donors tested, the addition of formate resulted in the highest SA with all three sulfur electron acceptors. Surprisingly, hydrogen as an electron donor had very little effect. Acetate was utilized as an electron donor only under sulfur-reducing conditions. Indigenous populations of sulfidogens in soda lake sediments showed an obligately alkaliphilic pH response of SA, showing a pattern that corresponded well to the in situ pH conditions. Sulfate reduction was much more susceptible to salt inhibition than thiosulfate and sulfur reduction. Microbiological investigations indicated that sulfate-reducing bacteria belonging to the orders Desulfovibrionales and Desulfobacterales could very likely be responsible for the SA with sulfate and thiosulfate as electron acceptors at moderate salt concentrations. Sulfur reduction at moderate salinity was carried out by a specialized group of haloalkaliphilic sulfur-reducing bacteria that utilize volatile fatty acids. In saturated soda brine, extremely natronophilic representatives of the order Halanaerobiales were responsible for the sulfur-dependent respiration.

Methylobacterium suomiense sp. nov. and Methylobacterium lusitanum sp. nov., aerobic, pink-pigmented, facultatively methylotrophic bacteria.

Two aerobic, pink-pigmented, facultatively methylotrophic bacteria, strains F20T and RXM(T), are described taxonomically. On the basis of their phenotypic and genotypic properties, the isolates are proposed as novel species of the genus Methylobacterium, Methylobacterium suomiense sp. nov. (type strain F20T = VKM B-2238T = NCIMB 13778T) and Methylobacterium lusitanum sp. nov. (type strain RXMT = VKM B-2239T = NCIMB 13779T).

Diversity of RuBisCO and ATP citrate lyase genes in soda lake sediments

Sediments from six soda lakes of the Kulunda Steppe (Altai, Russia) and from hypersaline alkaline lakes of Wadi Natrun (Egypt) were analyzed for the presence of cbb and aclB genes encoding key enzymes Ci assimilation (RuBisCO in Calvin-Benson and ATP citrate lyase in rTCA cycles, respectively). The cbbL gene (RuBisCO form I) was found in all samples and was most diverse, while the cbbM (RuBisCO form II) and aclB were detected only in few samples and with a much lower diversity. The cbbL libraries from hypersaline lakes were dominated by members of the extremely haloalkaliphilic sulfur-oxidizing Ectothiorhodospiraceae, i.e. the chemolithotrophic Thioalkalivibrio and the phototrophic Halorhodospira. In the less saline soda lakes from the Kulunda Steppe, the cbbL gene comprised up to ten phylotypes with a domination of members of a novel phototrophic Chromatiales lineage. The cbbM clone libraries consisted of two major unidentified lineages probably belonging to chemotrophic sulfur-oxidizing Gammaproteobacteria. One of them, dominating in the haloalkaline lakes from Wadi Natrun, was related to a cbbM phylotype detected previously in a hypersaline lake with a neutral pH, and another, dominating in lakes from the Kulunda Steppe, was only distantly related to the Thiomicrospira cluster. The aclB sequences detected in two samples from the Kulunda Steppe formed a single, deep branch in the Epsilonproteobacteria, distantly related to Arcobacter sulfidicus.