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Two novel Gram-strain-negative and rod-shaped bacteria, designated strain G1 T and G2 T , were isolated from sediment samples collected from the coast of Xiamen, PR China. The cells were motile by a single polar flagellum. Growth of strain G1 T occurred at 10-40 °C (optimum, 30 °C), at pH 6.0-9.0 (optimum, pH 7.5) and with 5-1530 mM NaCl (optimum, 510 mM), while the temperature, pH and NaCl concentration ranges for G2 T were 4-45 °C (optimum, 28 °C), pH 5.5-8.0 (optimum, pH 6.5) and 85-1530 mM NaCl (optimum, 340 mM). The two isolates were obligate chemolithoautotrophs capable of using thiosulfate, sulfide, elemental sulphur or tetrathionate as an energy source. Strain G1 T used molecular oxygen or nitrite as an electron acceptor, while strain G2 T used molecular oxygen as the sole electron acceptor. The dominant fatty acids of G1 T and G2 T were summed feature 3 (C 16:1 ω7 c and/or C 16:1 ω6 c ), C 16 : 0 and summed feature 8 (C 18:1 ω7 c and/or C 18:1 ω6 c ). The DNA G+C content of G1 T and G2 T were 45.1 and 48.3 mol%, respectively. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain G1 T and G2 T were members of the genus Thiomicrorhabdus , and most closely related to Thiomicrorhabdus hydrogeniphila MAS2 T (96.0 %) and Thiomicrorhabdus indica 13-15A T (95.4 %), respectively. The 16S rRNA gene sequence similarity between strains G1 T and G2 T was 95.8 %. Based on the phylogenetic, genomic and phenotypic data presented here, the isolate strains represent novel species of the genus Thiomicrorhabdus , for which the names Thiomicrorhabdus sediminis sp. nov. (type strain G1 T =MCCC 1A14511 T =KCTC 15841 T ) and Thiomicrorhabdus xiamenensis sp. nov. (type strain G2 T =MCCC 1A14512 T =KCTC 15842 T ) are proposed.

A new mesophilic bacterium, designated strain 13-15A T , was isolated from the deep-sea water from the Carlsberg Ridge, northwestern Indian Ocean. Cells were short rods and motile with a single polar flagellum. Growth was observed in the presence of 85-1700 mM NaCl (optimum 680 mM), at 10-45 °C (optimum, 28 °C) and pH 5.0-9.0 (optimum, pH 7.0). The isolate was an obligate chemolithoautotroph capable of growth using thiosulfate, sulfide, elemental sulfur or tetrathionate as the sole energy source, carbon dioxide as the sole carbon source, and molecular oxygen as the sole electron acceptor. Molecular hydrogen did not support growth. The major fatty acids were C 16 : 1 (45.0 %), C 18 : 1 (22.5 %) and C 16 : 0 (20.1 %). The G+C content of the genomic DNA was 41.6 mol%. The results of phylogenetic analysis based on 16S rRNA gene sequences showed that the novel isolate belonged to the genus Thiomicrorhabdus and was most closely related to Thiomicrorhabdus hydrogeniphila MAS2 T (94.8 % sequence similarity). On the basis of the taxonomic data obtained in this study, strain 13-15A T represents a novel species of the genus Thiomicrorhabdus , for which the name Thiomicrorhabdus indica sp. nov. is proposed, with the type strain 13-15A T (=MCCC 1A13986 T =KCTC 15750 T ).

Caligidosis and Piscirickettsiosis are currently the most important sanitary challenges for the Chilean salmon industry. Caligidosis is caused by the ectoparasite, Caligus rogercresseyi and Piscirickettsiosis is caused by the intracellular bacterium, Piscirickettsia salmonis. Both diseases are highly prevalent and widely distributed in farming areas in Chile. The co-occurrence of the two diseases is frequently reported on salmon farms. However, there is little epidemiological evidence as to whether these two diseases are associated and generate interactive effects. This study was undertaken to evaluate the potential effects of C. rogercresseyi infestation on P. salmonis-attributed mortalities in farmed salmonids in Chile. Using a linear regression model, the potential association between the mean abundance of adult C. rogercresseyi in a period of 10 weeks and Piscirickettsiosis cumulative mortalities observed in the following 10 weeks was evaluated, while controlling for important confounders. These two 10-week windows were set around the time-point at which Piscirickettsiosis weekly mortality exceeded 0.1% for the first time in a production cycle. We found that the mean abundance of adult C. rogercresseyi was significantly associated with the Piscirickettsiosis cumulative mortality, suggesting the two diseases have a synergistic relationship. This relationship was of the same intensity in Atlantic salmon and rainbow trout. Our findings highlight the importance of taking effective control measures for C. rogercresseyi as a part of the strategies in place to reduce P. salmonis-attributed mortalities on salmon farms in Chile., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Strain HaS4 T is an aerobic sulfur-oxidizing bacterium isolated from water of Lake Harutori in Japan. It was isolated and partially characterized in a previous study, but its taxonomic status has not been determined. The previous study revealed that the strain is an obligate chemolithoautotroph which grows at temperatures ranging from 0 to 25 °C (optimum, 22 °C) and pH from pH 6.2 to 8.8 (optimum, pH 6.6-7.4). In this study, further characterization of the strain was made to describe it as representative of a novel species. Cells of strain HaS4 T are rod-shaped, 1.6-2.5 µm long, 0.7-0.9 µm wide and Gram-stain-negative. Major cellular fatty acids were summed feature 3 (C 16 : 1 ω7 c and/or C 16 : 1 ω6 c ), summed feature 8 (C 18 : 1 ω7 c and/or C 18 : 1 ω6 c ) and C 16 : 0 . Phylogenetic analysis based on the 16S rRNA gene indicated that the strain is related to the genus Thiomicrorhabdus , but phylogenetically distinct from the type strains of existing species in the genus. On the basis of its phylogenetic and phenotypic properties, strain HaS4 T (=NBRC 112315 T =BCRC 81110 T ) is proposed as type strain of a new non-marine species of the genus Thiomicrorhabdus with the name Thiomicrorhabdus aquaedulcis sp. nov.

Members of the genera Hydrogenovibrio , Thiomicrospira , and Thiomicrorhabdus fix carbon at hydrothermal vents, coastal sediments, hypersaline lakes, and other sulfidic habitats. The genome sequences of these ubiquitous and prolific chemolithoautotrophs suggest a surprising diversity of mechanisms for the uptake and fixation of dissolved inorganic carbon (DIC); these mechanisms are verified here. Carboxysomes are apparent in the transmission electron micrographs of most of these organisms but are lacking in Thiomicrorhabdus sp. strain Milos-T2 and Thiomicrorhabdus arctica , and the inability of Thiomicrorhabdus sp. strain Milos-T2 to grow under low-DIC conditions is consistent with the absence of carboxysome loci in its genome. For the remaining organisms, genes encoding potential DIC transporters from four evolutionarily distinct families (Tcr_0853 and Tcr_0854, Chr, SbtA, and SulP) are located downstream of carboxysome loci. Transporter genes collocated with carboxysome loci, as well as some homologs located elsewhere on the chromosomes, had elevated transcript levels under low-DIC conditions, as assayed by reverse transcription-quantitative PCR (qRT-PCR). DIC uptake was measureable via silicone oil centrifugation when a representative of each of the four types of transporter was expressed in Escherichia coli The expression of these genes in the carbonic anhydrase-deficient E. coli strain EDCM636 enabled it to grow under low-DIC conditions, a result consistent with DIC transport by these proteins. The results from this study expand the range of DIC transporters within the SbtA and SulP transporter families, verify DIC uptake by transporters encoded by Tcr_0853 and Tcr_0854 and their homologs, and introduce DIC as a potential substrate for transporters from the Chr family. IMPORTANCE Autotrophic organisms take up and fix DIC, introducing carbon into the biological portion of the global carbon cycle. The mechanisms for DIC uptake and fixation by autotrophic Bacteria and Archaea are likely to be diverse but have been well characterized only for " Cyanobacteria " Based on genome sequences, members of the genera Hydrogenovibrio , Thiomicrospira , and Thiomicrorhabdus have a variety of mechanisms for DIC uptake and fixation. We verified that most of these organisms are capable of growing under low-DIC conditions, when they upregulate carboxysome loci and transporter genes collocated with these loci on their chromosomes. When these genes, which fall into four evolutionarily independent families of transporters, are expressed in E. coli , DIC transport is detected. This expansion in known DIC transporters across four families, from organisms from a variety of environments, provides insight into the ecophysiology of autotrophs, as well as a toolkit for engineering microorganisms for carbon-neutral biochemistries of industrial importance., (Copyright © 2019 American Society for Microbiology.)

Sulfide mineral precipitation occurs at mid-ocean ridge (MOR) spreading centers, both in the form of plume particles and seafloor massive sulfide structures. A common constituent of MOR is the iron-bearing sulfide mineral pyrrhotite, which was chosen as a substrate for in-situ incubation studies in shallow waters of Catalina Island, CA to investigate the colonization of iron-oxidizing bacteria. Microbial community datasets were obtained from in-situ incubated pyrrhotite, allowing for direct comparison to microbial communities of iron-sulfides from active and inactive chimneys in deep-sea environments. Unclassified Gammaproteobacteria and Alphaproteobacteria (Magnetovibrio) largely dominated the bacterial community on pyrrhotite samples incubated in the water column while samples incubated at the surface sediment showed more even dominance by Deltaproteobacteria (Desulfobulbus), Gammaproteobacteria (Piscirickettsiaceae), Alphaproteobacteria (Rhodobacteraceae), and Bacteroidetes (Flavobacteriia). Cultivations that originated from pyrrhotite samples resulted in the enrichment of both, sheath-forming and stalk-forming Zetaproteobacteria. Additionally, a putative novel species of Thiomicrospira was isolated and shown to grow autotrophically with iron, indicating a new biogeochemical role for this ubiquitous microorganism., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Atlantic salmon Salmo salar L. farmed in south-east Tasmania, Australia, are susceptible to infection by the Tasmanian Rickettsia-like organism (TRLO), a Gram-negative bacterium. Here, we report the first isolation of TRLO from south-east Tasmania in pure culture and show that the bacterium is culturable on both specialised enriched agar and in cell culture using the CHSE-214 cell line. In vitro cultured TRLO was used to reproducibly elicit disease in Atlantic salmon parr held in fresh water. In inoculated fish, TRLO was observed intracytoplasmically in peripheral blood leucocytes, suggesting that these cells are responsible for haematogenous dispersal of the bacterium within the host. Fish with experimentally induced disease presented with gross and histopathological changes similar to TRLO-infected fish at commercial marine farms. TRLO was also isolated in culture from farmed Atlantic salmon in the Tamar River and Macquarie Harbour production areas in Tasmania, both of which have no history of TRLO-associated disease. These TRLO isolates appear to be serologically distinct from each other as well as from isolates obtained from south-east Tasmania, linking each serotype to a specific geographical location within Tasmania. Despite the lack of clinical evidence of TRLO-linked disease in fish grown in the Tamar River and Macquarie Harbour, experimental infection trials demonstrably showed the pathogenic potential of these TRLO serovars. Together, these data provide evidence that TRLO is a fastidious, facultative intracellular bacterium and confirm TRLO as a pathogen of Atlantic salmon, causing a disease designated Tasmanian salmonid rickettsiosis.

A moderately psychrophilic, aerobic, hydrogen- and sulfur-oxidizing bacterium, designated strain MAS2T, was isolated from a tank containing coastal seawater from Tokyo Bay and a block of beef tallow added as organic material. Growth occurred under aerobic chemolithoautotrophic conditions in the presence of molecular hydrogen, thiosulfate, tetrathionate, elemental sulfur or sulfide as the sole energy source and bicarbonate as a carbon source. The isolate represented a Gram-staining-negative rod with a single polar flagellum and grew in artificial seawater medium with thiosulfate at 2-40 °C (optimum 30 °C). The isolate grew in media with thiosulfate at Na+ concentrations between 30 and 1380 mM (optimum 270 mM). MAS2T possessed C16 : 0, C16 : 1 and C18 : 1 as the major fatty acids. The G+C content of the genomic DNA was 39.6 mol%. The 16S rRNA gene sequence similarity analysis showed that the isolate represented a member of the genus Thiomicrospira within the class Gammaproteobacteria and was most closely related to Thiomicrospira frisia JB-A2T. On the basis of phenotypic and molecular properties, the isolate represents a novel species of the genus Thiomicrospira, for which the name Thiomicrospira hydrogeniphila sp. nov. is proposed (type strain, MAS2T=JCM 30760T=DSM 100274T).

Thiomicrospira species are ubiquitously found in various marine environments and appear particularly common in hydrothermal vent systems. Members of this lineage are commonly classified as sulfur-oxidizing chemolithoautotrophs. Although sequencing of Thiomicrospira crunogena's genome has revealed genes that encode enzymes for hydrogen uptake activity and for hydrogenase maturation and assembly, hydrogen uptake ability has so far not been reported for any Thiomicrospira species. We isolated a Thiomicrospira species (SP-41) from a deep sea hydrothermal vent and demonstrated that it can oxidize hydrogen. We show in vivo hydrogen consumption, hydrogen uptake activity in partially purified protein extracts and transcript abundance of hydrogenases during different growth stages. The ability of this strain to oxidize hydrogen opens up new perspectives with respect to the physiology of Thiomicrospira species that have been detected in hydrothermal vents and that have so far been exclusively associated with sulfur oxidation.

Two bacterial strains, designated JAM1(T) and JAM7(T), were isolated from a methanol-fed denitrification system treating seawater at the Montreal Biodome, Canada. They were affiliated within the genus Methylophaga of the Gammaproteobacteria by analysis of the 16S rRNA gene sequences. Strain JAM1(T) had the capacity to grow under denitrifying conditions by reducing nitrate into nitrite which is unique among the species of the genus Methylophaga. Major fatty acids were C16:1ω7c or ω6c, C16:0 and C18:1ω7c or ω6c. The major ubiquinone was Q8. Both strains required vitamin B12 and Na(+) ions for growth. The genomes of strains JAM1(T) and JAM7(T) have been completely sequenced and showed a DNA G+C content of 44.7 mol% and 47.8 mol%, respectively. Growth occurred at pH 6-11 and at 0.5-8% NaCl. Both genomes contained predicted ORFs encoding the key enzymes of the ribulose monophosphate pathway. Also, operons encoding two nitrate reductases (Nar), two nitric oxide reductases (Nor), one nitrous oxide reductase (Nos) and one truncated nitrite reductase (NirK) were clustered in a 67 kb chromosomal region in strain JAM1(T). No such operons were found in strain JAM7(T). These results supported the affiliation of the two strains as novel species within the genus Methylophaga. The names Methylophaga nitratireducenticrescens sp. nov. for type strain JAM1(T) (=DSM 25689(T)=ATCC BAA-2433(T)) and Methylophaga frappieri sp. nov. for type strain JAM7(T) (=DSM 25690(T)=ATCC BAA-2434(T)) are proposed.

A mesophilic, strictly microaerophilic, chemosynthetic bacterium, designated strain P2D(T), was isolated from the sediment of an active shallow-water hydrothermal vent in Paleochori Bay, on the Greek island of Milos. The cells were Gram-staining-negative rods that measured approximately 0.8-1.3 µm in length and 0.4-0.5 µm in width. Strain P2D(T) grew at 20-50 °C (optimum 35 °C), with 1.0-5.0% (w/v) NaCl (optimum 3.0%), and at pH 4.5-8.0 (optimum pH 5.5). The generation time under optimal conditions was 1.1 h. Growth occurred under chemolithoautotrophic conditions with S₂O₃²⁻ and CO(2) as the energy and carbon sources, respectively. Oxygen (5%) was used as sole terminal electron acceptor. No growth was observed in the presence of acetate, formate, lactate, tryptone or peptone. Chemolithoheterotrophic growth occurred when d-glucose or sucrose were present as carbon sources. None of the organic compounds tested was used as an electron donor. The genomic DNA G+C content of the novel strain was 44.9 mol%. In a phylogenetic analysis based on 16S rRNA gene sequences, strain P2D(T) was found to be most closely related to Thiomicrospira psychrophila DSM 13453(T) (92.8% sequence similarity). Based on the phylogenetic, physiological and chemotaxonomic evidence, strain P2D(T) represents a novel species of a new genus within the class Gammaproteobacteria of the family Piscirickettsiaceae, for which the name Galenea microaerophila gen. nov., sp. nov. is proposed. The type strain of the type species is P2D(T) ( = DSM 24963(T) = JCM 17795(T)).

Cycloclasticus sp. strain P1 was isolated from deep-sea sediments of the Pacific Ocean and characterized as a unique bacterium in the degradation of pyrene, a four-ring polycyclic aromatic hydrocarbon (PAH). Here we report the complete genome of P1 and genes associated with PAH degradation.

Methylophaga sp. strains JAM1 and JAM7 have been isolated from a denitrification system. Strain JAM1 was the first Methylophaga strain reported to be able to grow under denitrifying conditions. Here, we report the complete genome sequences of the two strains, which allowed prediction of gene clusters involved in denitrification in strain JAM1.

A moderately haloalkaliphilic methylotrophic bacterium possessing the ribulose monophosphate pathway for carbon assimilation, designated MPL(T), was isolated from Lonar Lake sediment microcosms that were oxidizing methane for two weeks. The isolate utilized methanol and was an aerobic, Gram-negative, asporogenous, motile, short rod that multiplied by binary fission. The isolate required NaHCO(3) or NaCl for growth and, although not auxotrophic for vitamin B(12), had enhanced growth with vitamin B(12). Optimal growth occurred with 0.5-2% (w/v) NaCl, at 28-30 °C and at pH 9.0-10.0. The cellular fatty acid profile consisted primarily of straight-chain saturated C(16:0) and unsaturated C(16:1)ω7c and C(18:1)ω7c. The major ubiquinone was Q-8. The dominant phospholipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol. Cells accumulated ectoine as the main compatible solute. The DNA G+C content was 50.0 mol%. The isolate exhibited 94.0-95.4% 16S rRNA gene sequence similarity with the type strains of methylotrophs belonging to the genus Methylophaga and 31% DNA-DNA relatedness with the reference strain, Methylophaga alcalica VKM B-2251(T). It is proposed that strain MPL(T) represents a novel species, Methylophaga lonarensis sp. nov. (type strain MPL(T)=VKM B-2684(T)=MCC 1002(T)).

Nitrate is a serious problem in closed-circuit public aquariums because its accumulation rapidly becomes toxic to many lifeforms. A moving bed biofilm denitrification reactor was installed at the Montreal Biodome to treat its 3,250-m(3) seawater system. Naturally occurring microorganisms from the seawater affluent colonized the reactor carriers to form a denitrifying biofilm. Here, we investigated the functional diversity of this biofilm by retrieving gene sequences related to narG, napA, nirK, nirS, cnorB, and nosZ. A total of 25 sequences related to these genes were retrieved from the biofilm. Among them, the corresponding napA1, nirK1, cnorB9, and nosZ3 sequences were identical to the corresponding genes found in Hyphomicrobium sp. NL23 while the narG1 and narG2 sequences were identical to the two corresponding narG genes found in Methylophaga sp. JAM1. These two bacterial strains were previously isolated from the denitrifying biofilm. To assess the abundance of denitrifiers and nitrate respirers in the biofilm, the gene copy number of all the narG, napA, nirS, and nirK sequences found in biofilm was determined by quantitative PCR. napA1, nirK1, narG1, and narG2, which were all associated with either Methylophaga sp. JAM1 or Hyphomicrobium sp. NL23, were the most abundant genes. The other genes were 10 to 10,000 times less abundant. nirK, cnorB, and nosZ but not napA transcripts from Hyphomicrobium sp. NL23 were detected in the biofilm, and only the narG1 transcripts from Methylophaga sp. JAM1 were detected in the biofilm. Among the 19 other genes, the transcripts of only two genes were detected in the biofilm. Our results show the predominance of Methylophaga sp. JAM1 and Hyphomicrobium sp. NL23 among the denitrifiers detected in the biofilm. The results suggest that Hyphomicrobium sp. NL23 could use the nitrite present in the biofilm generated by nitrate respirers such as Methylophaga sp. JAM1.

Elemental sulfur (S(0) ) is deposited each summer onto surface ice at Borup Fiord pass on Ellesmere Island, Canada, when high concentrations of aqueous H(2) S are discharged from a supraglacial spring system. 16S rRNA gene clone libraries generated from sulfur deposits were dominated by β-Proteobacteria, particularly Ralstonia sp. Sulfur-cycling micro-organisms such as Thiomicrospira sp., and ε-Proteobacteria such as Sulfuricurvales and Sulfurovumales spp. were also abundant. Concurrent cultivation experiments isolated psychrophilic, sulfide-oxidizing consortia, which produce S(0) in opposing gradients of Na(2) S and oxygen. 16S rRNA gene analyses of sulfur precipitated in gradient tubes show stable sulfur-biomineralizing consortia dominated by Marinobacter sp. in association with Shewanella, Loktanella, Rubrobacter, Flavobacterium, and Sphingomonas spp. Organisms closely related to cultivars appear in environmental 16S rRNA clone libraries; none currently known to oxidize sulfide. Once consortia were simplified to Marinobacter and Flavobacteria spp. through dilution-to-extinction and agar removal, sulfur biomineralization continued. Shewanella, Loktanella, Sphingomonas, and Devosia spp. were also isolated on heterotrophic media, but none produced S(0) alone when reintroduced to Na(2) S gradient tubes. Tubes inoculated with a Marinobacter and Shewanella spp. co-culture did show sulfur biomineralization, suggesting that Marinobacter may be the key sulfide oxidizer in laboratory experiments. Light, florescence and scanning electron microscopy of mineral aggregates produced in Marinobacter experiments revealed abundant cells, with filaments and sheaths variably mineralized with extracellular submicron sulfur grains; similar biomineralization was not observed in abiotic controls. Detailed characterization of mineral products associated with low temperature microbial sulfur-cycling may provide biosignatures relevant to future exploration of Europa and Mars., (© 2011 Blackwell Publishing Ltd.)

Methylophaga spp. are methylotrophs commonly associated with marine environments and have been defined as strict aerobic methylotrophs. They have been shown previously to represent 50-70% of the bacterial population in the biofilm of the methanol-fed denitrification reactor treating a large seawater aquarium at the Montreal Biodome. It was therefore surprising to find such a high concentration of Methylophaga spp. in anoxic conditions. In this study, we showed by cultivation-independent and -dependent approaches that one Methylophaga strain present in the anoxic biofilm is involved in the denitrification process. DNA stable-isotope probing (SIP) experiments in which the biofilm was cultured under denitrifying conditions with (13)C-methanol have revealed the enrichment of one particular taxon. By screening a 16S ribosomal RNA gene library derived from a (13)C-DNA fraction of the SIP gradients, 62% of the library was composed of one sequence affiliated with the genus Methylophaga. One strain, named JAM1, representing this Methylophaga species was isolated. It grows aerobically but also under denitrifying conditions by reducing nitrate into nitrite. The nitrate-reducing activity was correlated with the presence and the expression of two highly divergent narG genes (narG1 and narG2). narG1 showed a high percentage of identity with the corresponding part of narG found in Thiobacillus denitrificans, which suggests a recent acquisition of narG in strain JAM1 by horizontal gene transfer. This study provides the first direct evidence of the adaptation of a Methylophaga species to an oxygen-limited environment.

It is shown that neutrophilic methylobacteria Methylophaga thalassica and M. marina have higher rates of growth and ectoin accumulation compared to the haloalkaliphilic species M. alcalica and M. natronia and methanotrophs Methylomicrobium alcaliphilum and M. kenyense. The conditions of M. thalassica cultivation in methanol-containing medium were optimized. The yield of this process reached 60 g/l of absolutely dry biomass containing 15-19% (9-11 g/l) ectoine. The scheme of ectoin isolation from the biomass by extraction and subsequent purification, which allowed obtaining preparations of different degree of purity, was developed.

The perennial springs at Gypsum Hill (GH) and Colour Peak (CP), situated at nearly 80 degrees N on Axel Heiberg Island in the Canadian high Arctic, are one of the few known examples of cold springs in thick permafrost on Earth. The springs emanate from deep saline aquifers and discharge cold anoxic brines rich in both sulfide and sulfate. Grey-coloured microbial streamers form during the winter months in snow-covered regions of the GH spring run-off channels (-1.3 degrees C to 6.9 degrees C, approximately 7.5% NaCl, 0-20 p.p.m. dissolved sulfide, 1 p.p.m. dissolved oxygen) but disappear during the Arctic summer. Culture- and molecular-based analyses of the 16S rRNA gene (FISH, DGGE and clone libraries) indicated that the streamers were uniquely dominated by chemolithoautotrophic sulfur-oxidizing Thiomicrospira species. The streamers oxidized both sulfide and thiosulfate and fixed CO(2) under in situ conditions and a Thiomicrospira strain isolated from the streamers also actively oxidized sulfide and thiosulfate and fixed CO(2) under cold, saline conditions. Overall, the snow-covered spring channels appear to represent a unique polar saline microhabitat that protects and allows Thiomicrospira streamers to form and flourish via chemolithoautrophic, phototrophic-independent metabolism in a high Arctic winter environment characterized by air temperatures commonly below -40 degrees C and with an annual average air temperature of -15 degrees C. These results broaden our knowledge of the physical and chemical boundaries that define life on Earth and have astrobiological implications for the possibility of life existing under similar Martian conditions.

A novel restricted facultatively methylotrophic marine strain, MP(T), possessing the ribulose monophosphate pathway of C(1)-carbon compound assimilation was isolated from a seawater sample obtained from Mokpo, South Korea. The novel isolate is aerobic, Gram-negative, asporogenous and a non-motile short rod. It grows well on methanol, methylated amines, dimethylsulfide and DMSO. Optimal growth occurs with 3 % NaCl at 30 degrees C and pH 7.0. Fructose is utilized as a multicarbon source. Growth factors are not required and vitamin B(12) does not stimulate growth. The cellular fatty acid profile of the novel strain consists primarily of straight-chain saturated C(16 : 0) and unsaturated C(16 : 1) acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content is 44.9 mol% (T(m)). Based on 16S rRNA gene sequence analysis and DNA-DNA relatedness (25-41 %) with the type strains of marine methylotrophs belonging to the genus Methylophaga, it is suggested that isolate MP(T) represents a novel species, Methylophaga aminisulfidivorans sp. nov. (type strain MP(T)=KCTC 12909(T)=VKM B-2441(T)=JCM 14647(T)).

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with Methylobacter and Methylophaga species accounted for 56%+/-8% of total cells. In sediments below Beggiatoa mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the Desulfobulbus (DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94%+/-2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (Siboglinidae, formerly known as Pogonophora). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and Beggiatoa species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (mcrA) genes showed a strong dominance of a novel lineage, mcrA group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.

Dimethylsulfide (DMS)-degrading enrichment cultures were established from samples of coastal seawater, nonaxenic Emiliania huxleyi cultures, and mixed marine methyl halide-degrading enrichment cultures. Bacterial populations from a broad phylogenetic range were identified in the mixed DMS-degrading enrichment cultures by denaturing gradient gel electrophoresis (DGGE). Sequences of dominant DGGE bands were similar to those of members of the genera Methylophaga and Alcanivorax. Several closely related Methylophaga strains were obtained that were able to grow on DMS as the carbon and energy source. Roseobacter-related populations were detected in some of the enrichment cultures; however, none of the Roseobacter group isolates that were tested were able to grow on DMS. Oxidation of DMS by Methylophaga sp. strain DMS010 was not affected by addition of the inhibitor chloroform or methyl tert-butyl ether, suggesting that DMS metabolism may occur by a route different from those described for Thiobacillus species and other unidentified marine isolates. Addition of DMS and methanethiol to whole-cell suspensions of strain DMS010 induced oxygen uptake when strain DMS010 was grown on DMS but not in cells grown on methanol. The apparent K(m)s of strain DMS010 for DMS and for methanethiol were 2.1 and 4.6 microM, respectively, when grown on DMS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the biomass of strain DMS010 and analysis of peptide bands by mass spectrometry techniques and N-terminal sequencing provided the first insight into the identity of polypeptides induced during growth on DMS. These included XoxF, a homolog of the large subunit of methanol dehydrogenase for which a biological role has not been identified previously.

In 2004, cultured Nile tilapia Oreochromis niloticus in several Latin America farms began to succumb to a disease similar to the piscirickettsiosis-like syndrome previously reported in tilapia in Taiwan and the United States. Mortality increased during 2005; reductions in tilapia biomass ranged from 5% to 80% in individual ponds and averaged 50% overall. All ages of fish have been involved. Clinical signs include lethargy, loss of appetite, petechia, exophthalmia, and abnormal swimming behavior. Gross lesions have included splenomegaly, renomegaly, and numerous white nodules observed in the spleen, kidney, testes, heart, ovaries, and occasionally the liver. A previously unreported black granulomatous lesion was reported in up to 30% of the fillets. Histologically, granulomatous infiltrates were observed in the kidney, spleen, liver, testes, ovary, and choroid gland, and rarely in the brain and heart. A small pleomorphic bacterium was observed in Giemsa-stained blood smears and spleen imprints. The bacterium did not grow on standard microbiological media and has not been isolated in cell culture. We obtained a near-complete 16S ribosomal DNA sequence with high similarity to Francisella spp. sequences previously identified in tilapias Oreochromis spp. (Taiwan), Atlantic cod Gadus morhua (Norway), and three-line grunts Parapristipoma trilineatum (Japan).

Viable preparations of intact Piscirickettsia salmonis, purified from host cell material, are necessary for studying characteristics associated with this bacterium. However, purification of the organism is difficult due to its obligate intracellular nature. A simple and effective method for isolating whole P. salmonis, which is quick and easy to perform, but still maintains the viability and antigenicity of the bacterium is described. P. salmonis was purified by differential pelleting and density gradient centrifugation using 30%, 40%, or 50% (v/v) Percoll gradients. Following fractionation, a band with a density of 1.056-1.080 was found to be composed entirely of rickettsiae, confirmed by fluorescent antibody technique (IFAT). Purity of the P. salmonis from different stages of the purification process was assessed by light and transmission electron microscopy, and the viability of yields determined from a plaque assay and a tissue culture infective dose (TCID(50) ml(-1)). P. salmonis recovered from the 30% Percoll gradient appeared to retain their intracellular structure better than P. salmonis obtained from the 40% and 50% Percoll gradients, and appeared to have a greater viability. Differences were seen between P. salmonis-infected CHSE-214 cells and purified P. salmonis when compared by SDS-PAGE and Western blotting, and less host cell contamination was present in preparations obtained from the 30% Percoll gradient. Finally ten different P. salmonis isolates obtained from three different geographical locations and four different fish species, were purified using the 30% Percoll gradient. When the morphology of these was compared by transmission electron microscopy (TEM), they appeared similar in size and appearance, although isolate R980769 was highly pleomorphic and isolate R-29 was larger than the other isolates examined.

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function.

Denitrification is applied in the tertiary treatment of wastewater to reduce N-pollutants. Fluorescence in situ hybridisation (FISH), CARD (catalyzed reporter deposition)-FISH, cloning, and scanning electron microscopy (SEM) were applied to follow the evolution of the microbial composition and structure of granular sludge in autotrophic denitrifying bioreactors fed with nitrate and thiosulfate. With this goal, FISH oligonucleotide probes for the autotrophic denitrifiers, Thiobacillus denitrificans and Thiomicrospira denitrificans, were designed and their utility tested. CARD-FISH and cloning data showed that bacterial diversity changed with bioreactor operation time. After 110 days of operation, the abundance of Thiobacillus denitrificans cells increased considerably: from 1 to 35% of total DAPI-stained cells and from no isolated clones to 30% of the total positives clones. This fact strongly suggests that this microorganism played a dominant role in the autotrophic denitrification. The Archaeal diversity remained almost unchanged and it was mainly represented by Methanosaeta soehngenii. Scanning electron microscopy results indicated a considerable loss in the integrity of the sludge granules during the operation, with risk of sludge buoyancy.

Methylophaga sp. strain SK1 is a new restricted facultative methanol-oxidizing bacterium that was isolated from seawater. The aim of this study was to characterize the electron carriers involved in the methanol oxidation process in Methylophaga sp. strain SK1. The gene encoding cytochrome c(L) (mxaG) was cloned and the recombinant gene was expressed in Escherichia coli DH5 under strict anaerobic conditions. The recombinant cytochrome c(L) had the same molecular weight and absorption spectra as the wild-type cytochrome c(L) both in the reduced and oxidized forms. The electron flow rate from methanol dehydrogenase (MDH) to the recombinant cytochrome c(L) was similar to that from MDH to the wild-type cytochrome c(L). These results suggest that recombinant cytochrome c(L) acts as a physiological primary electron acceptor for MDH.

Two psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria were isolated from marine Arctic sediments sampled off the coast of Svalbard with thiosulfate as the electron donor and CO(2) as carbon source. Comparative analysis of 16S rRNA gene sequences suggested that the novel strains, designated SVAL-D(T) and SVAL-E(T), represent members of the genus Thiomicrospira. Further genotypic (DNA-DNA relatedness, DNA G+C content) and phenotypic characterization revealed that the strains represent members of two novel species. Both organisms are obligately autotrophic and strictly aerobic. Nitrate was not used as an electron acceptor. Chemolithoautotrophic growth was observed with thiosulfate, tetrathionate and sulfur. The temperature limits for growth of both strains were between -2 degrees C and 20.8 degrees C, with optima of 11.5-13.2 degrees C (SVAL-E(T)) and 14.6-15.4 degrees C (SVAL-D(T)), which is about 13-15 degrees C lower than the optima of all other recognized Thiomicrospira species. The maximum growth rate on thiosulfate at 14 degrees C was 0.14 h(-1) for strain SVAL-E(T) and 0.2 h(-1) for strain SVAL-D(T). Major fatty acids of SVAL-D(T) are C(16 : 1), C(18 : 0) and C(16 : 0), and those of SVAL-E(T) are C(16 : 1), C(18 : 1), C(16 : 0) and C(14 : 1). Cells of SVAL-D(T) and SVAL-E(T) are rods, like those of their closest relatives. To our knowledge the novel strains are the first psychrophilic, chemolithoautotrophic, sulfur-oxidizing bacteria so far described. The names Thiomicrospira arctica sp. nov. and Thiomicrospira psychrophila sp. nov. are proposed for SVAL-E(T) (=ATCC 700955(T)=DSM 13458(T)) and SVAL-D(T) (=ATCC 700954(T)=DSM 13453(T)), respectively.

A novel thermotolerant bacterium, designated strain I78(T), was isolated from a self-temperature-recording in situ colonization system deployed in a hydrothermal diffusing flow (maximal temperature 78 degrees C) at the TOTO caldera in the Mariana Arc, Western Pacific. Cells were highly motile curved rods with a single polar flagellum. Growth was observed at 15-55 degrees C (optimum 35-40 degrees C; 60 min doubling time) and pH 5.0-8.0 (optimum pH 6.0). The isolate was a microaerobic chemolithomixotroph capable of using thiosulfate, elemental sulfur or sulfide as the sole energy source, and molecular oxygen as the sole electron acceptor. The isolate was able to grow chemolithoautotrophically with carbon dioxide. Various organic substrates such as complex proteinaceous compounds, carbohydrates, organic acids, amino acids and sugars could also support growth as the carbon source instead of carbon dioxide with sulfur oxidation. The G+C content of the genomic DNA was 43.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Thiomicrospira and was most closely related to Thiomicrospira crunogena strain TH-55(T) and Thiomicrospira sp. strain L-12, while DNA-DNA hybridization demonstrated that the novel isolate could be genetically differentiated from previously described strains of Thiomicrospira. On the basis of its physiological and molecular properties the isolate is representative of a novel Thiomicrospira species, for which the name Thiomicrospira thermophila sp. nov. is proposed (type strain, I78(T)=JCM 12397(T)=DSM 16397(T)).

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the "Epsilonproteobacteria" related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 x 10(3) to 4.4 x 10(9) copies ml(-1) or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 x 10(1) to 2.2 x10(6) copies ml(-1) or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml(-1). The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.

Piscirickettsia salmonis is an obligate intracellular bacterial pathogen of salmonid fish and the etiological agent of the aggressive disease salmonid rickettsial syndrome. Today, this disease, also known as piscirickettsiosis, is the cause of high mortality in net pen-reared salmonids in southern Chile. Although the bacteria can be grown in tissue culture cells, genetic analysis of the organism has been hindered because of the difficulty in obtaining P. salmonis DNA free from contaminating host cell DNA. In this report, we describe a novel procedure to purify in vitro-grown bacteria with iodixanol as the substrate to run differential centrifugation gradients which, combined with DNase I digestion, yield enough pure bacteria to do DNA analysis. The efficiency of the purification procedure relies on two main issues: semiquantitative synchrony of the P. salmonis-infected Chinook salmon embryo (CHSE-214) tissue culture cells and low osmolarity of iodixanol to better resolve bacteria from the membranous structures of the host cell. This method resulted in the isolation of intact piscirickettsia organisms and removed salmon and mitochondrial DNA effectively, with only 1.0% contamination with the latter.

Methylotrophic bacteria are widespread in nature. They may play an important role in the cycling of carbon and in the metabolism of dimethylsulfide in a marine environment. Bacteria belonging to the genus Methylophaga are a unique group of aerobic, halophilic, non-methane-utilizing methylotrophs. Two 16S rRNA-targeted oligonucleotide probes were developed for the specific detection of Methylophaga species, marine methylobacteria, by fluorescence in situ hybridization. Probe MPH-730 was highly specific for all members of the genus Methylophaga while probe MPHm-994 targeted exclusively M. marina. The application of these probes were demonstrated by the detection of Methylophaga species in enrichment cultures from various marine sediments. All isolates recovered were visualized by using the genus specific probe MPH-730. The results were confirmed by 16S rDNA sequencing which demonstrated that all selected isolates belong to Methylophaga. Five isolates could be detected by the M. marina-specific probe MPHm-994 and were confirmed by rRNA gene restriction pattern (ribotyping). With the development of these specific probes, fluorescence in situ hybridization shows that the genus Methylophaga is widespread in marine samples.

A new, moderately haloalkaliphilic and restricted-facultatively methylotrophic bacterium (strain Bur2T) with the ribulose monophosphate pathway of carbon assimilation is described. The isolate, which utilizes methanol, methylamine and fructose, is an aerobic, Gram-negative, asporogenous, motile short rod multiplying by binary fission. It is auxotrophic for vitamin B12, and requires NaHCO3 or NaCl for growth in alkaline medium. Cellular fatty acids profile consists primarily of straight-chain saturated C16:0, unsaturated C16:1 and C18:1 acids. The major ubiquinone is Q-8. The dominant phospholipids are phosphatidylethanolamine and phosphatidylglycerol. Diphosphatidylglycerol is also present. Optimal growth conditions are 25-29 degrees C, pH 8.5-9.0 and 2-3% (w/v) NaCl. Cells accumulate ectoine and glutamate as the main osmoprotectants. The G + C content of the DNA is 45.0 mol%. Based on 16S rDNA sequence analysis and DNA-DNA relatedness (25-35%) with type strains of marine and soda lake methylobacteria belonging to the genus Methylophaga, the novel isolate was classified as a new species of this genus and named Methylophaga natronica (VKM B-2288T).

Piscirickettsia salmonis is the first Gram-negative, intracellular bacterial pathogen isolated from fish and is a significant cause of mortality in salmonid fish. Recent reports of P. salmonis or P. salmonis-like organisms from new fish hosts and geographic regions have increased the interest in the bacterium. In this review, the important characteristics of the bacterium including recent taxonomic changes, features of the disease caused by the bacterium including transmission, hosts, reservoirs, diagnostic procedures, and current approaches for prevention and treatment have been discussed. The reader is also directed to other reviews concerning the bacterium and the disease it causes (Fryer & Lannan 1994, 1996; Almendras & Fuentealba 1997; Lannan, Bartholomew & Fryer 1999; House & Fryer 2002; Mauel & Miller 2002).