Phylogenetic relationships ofThiomicrospira species and their identification in deep-sea hydrothermal vent samples by denaturing gradient gel electrophoresis of 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA fragments was used to explore the genetic diversity of hydrothermal vent microbial communities, specifically to determine the importance of sulfur-oxidizing bacteria therein. DGGE analysis of two different hydrothermal vent samples revealed one PCR band for one sample and three PCR bands for the other sample, which probably correspond to the dominant bacterial populations in these communities. Three of the four 16S rDNA fragments were sequenced. By comparison with 16S rRNA sequences of the Ribosomal Database Project, two of the DGGE-separated fragments were assigned to the genusThiomicrospira. To identify these ‘phylotypes’ in more detail, a phylogenetic framework was created by determining the nearly complete 16S rRNA gene sequence (approx. 1500 nucleotides) from three describedThiomicrospira species, viz.,Tms. crunogena, Tms. pelophila, Tms. denitrificans, and from a new isolate,Thiomicrospira sp. strain MA2-6. AllThiomicrospira species exceptTms. denitrificans formed a monophyletic group within the gamma subdivision of the Proteobacteria.Tms. denitrificans was assigned as a member of the epsilon subdivision and was distantly affiliated withThiovulum, another sulfur-oxidizing bacterium. Sequences of two dominant 16S rDNA fragments obtained by DGGE analysis fell into the gamma subdivisionThiomicrospira. The sequence of one fragment was in all comparable positions identical to the 16S rRNA sequence ofTms. crunogena. Identifying a dominant molecular isolate asTms. crunogena indicates that this species is a dominant community member of hydrothermal vent sites. Another ‘phylotype’ represented a newThiomicrospira species, phylogenetically in an intermediate position betweenTms. crunogena andTms. pelophila. The third ‘phylotype’ was identified as aDesulfovibrio, indicating that sulfate-reducing bacteria, as sources of sulfide, may complement sulfur- and sulfide-oxidizing bacteria ecologically in these sulfide-producing hydrothermal vents.     

Competition for inorganic substrates among chemoorganotrophic and chemolithotrophic bacteria

In aerobic enrichment experiments with a chemostat, using phosphate-limited lactate medium, aSpirillum sp. predominated at the lower range of dilution rates. At the higher dilution rates an (chemoorganotrophic) unidentified rod-shaped bacterium came to the fore. The same result was obtained in competition experiments with pure cultures of the two bacteria. Growth parameters were: Rod, _max=0.48 hr^–1,k _s(PO₄ ^3–)=6.6×10^–N M;Spirillum, _max=0.24 hr^–1· k_s(PO₄ ^3–) =2.7×10^–8 M. TheSpirillum grew faster than the rod at low dilution rates, not only under phosphate-limitation but also in K⁺-,Mg²⁺-, NH₄ ⁺-, aspartate-, succinate-, and lactate-limited cultures. Both organisms showed little substrate specificity and could utilize a similar range of carbon and energy sources. The results support the view that part of the diversity among bacteria in the natural environment is based on selection toward substrate concentration. Another set of competition experiments was carried out with pure cultures of two marine obligately chemolithotrophic colorless sulfur bacteria,Thiobacillus thioparus andThiomicrospira pelophila. Tms. pelophila outgrewT. thioparus at low dilution rates under iron limitation, while the reverse was true at high dilution rates. It is concluded that the relatively fast growth ofTms. pelophila at low iron concentration may explain its higher sulfide tolerance. Organisms showing a selection advantage at very low concentrations of limiting substrates appear to have a relatively high surface to volume ratio.     

Growth and pigment production byArthrobacter pyridinolis n. sp.

A new bacterium capable of growing on 2-hydroxypyridine as sole source of carbon and nitrogen was isolated from soil. During its growth on solid medium, approximately 50% of this substrate was converted to a brilliant blue crystalline pigment which was deposited extracellularly in the colony mass. The pigment was identical to that produced byArthrobacter crystallopoietes during its growth on 2-hydroxypyridine. The new isolate exhibited the typical cycle of morphogenesis characteristic of the genusArthrobacter. The organism is different from all other reported species ofArthrobacter. It is proposed that the organism be namedArthrobacter pyridinolis n. sp.List of Abbreviations MSP mineral salts phosphate basal culture medium containing 2-hydroxypyridine, yeast extract and trace salts - 2-HP 2-hydroxypyridine - PFU plaque forming units - G+C guanine+cytosine - T _m midpoint of thermal denaturation     

Taxonomic relationships ofThiobacillus halophilus,T. aquaesulis,and other species ofThiobacillus,as determined using 16S rDNA sequencing

Total base sequences of the 16S rRNA genes ofThiobacillus halophilus andThiobacillus aquaesulis show that these bacteria fall into the gamma- and beta-subdivisions, respectively of the Proteobacteria. The closest relative ofT. halophilus isThiobacillus hydrothermalis (with 98.7% similarity), and the closest relative ofT. aquaesulis isThiobacillus thioparus (93.2% similarity). Physiological properties and mol% G+C content of their DNA serve to confirm that these four organisms are all distinct species. It is reiterated that the species currently assigned to the genusThiobacillus are clearly so diverse that they need reclassification into several genera. The type species,T. thioparus, is unequivocally placed in the beta-subdivision of the Proteobacteria, thus requiring that the use of the genus nameThiobacillus be restricted to the chemolithoautotrophic species falling into that group.T. aquaesulis andT. thioparus may thus be regarded as true species ofThiobacillus. The relatively large number of obligately chemolithoautotrophicThiobacillus species falling in the gamma-subdivision of the Proteobacteria need further study in order to assess the case for reclassification into one or more new or different genera.     

Desulforhopalus vacuolatus gen. nov., sp. nov., a new moderately psychrophilic sulfate-reducing bacterium with gas vacuoles isolated from a temperate estuary

A new type of gas-vacuolated, sulfate-reducing bacterium was isolated at 10° C from reduced mud (E₀ < 0) obtained from a temperate estuary with thiosulfate and lactate as substrates. The strain was moderately psychrophilic with optimum growth at 18–19° C and a maximum growth temperature of 24° C. Propionate, lactate, and alcohols served as electron donors and carbon sources. The organism grew heterotrophically only with hydrogen as electron donor. Propionate and lactate were incompletely oxidized to acetate; traces of lactate were fermented to propionate, CO₂, and possibly acetate in the presence of sulfate. Pyruvate was utilized both with and without an electron acceptor present. The strain did not contain desulfoviridin. The G+C content was 48.4 mol%. The differences in the 16S rRNA sequence of the isolate compared with that of its closest phylogenetic neighbors, bacteria of the genus Desulfobulbus, support the assignment of the isolate to a new genus. The isolate is described as the type strain of the new species and genus, Desulforhopalus vacuolatus. Received: 4 March 1996 / Accepted: 17 June 1996     

Induction of a Sensitive Response to Helminthosporium maydis Race T Toxin in Resistant Mitochondria of Corn (Zea mays L.) by Removal of the Outer Mitochondrial Membrane

Mitochondria isolated from Texas cytoplasmically male sterile (Tms) and normal (N) versions of corn (Zea mays L.) exhibit differential sensitivity to toxin(s) produced by Helminthosporium maydis race T, the causal organism of southern corn leaf blight. Malate dehydrogenase was inhibited by toxin(s) in intact Tms mitochondria but was unaffected in N mitochondria. Removal or rupture of the outer mitochondrial membrane resulted in retention of sensitivity of malate dehy-drogenase in Tms mitochondria to toxin(s), and induction of a sensitive response in normally toxin-insensitive N mitochondria. This suggests that a permeability difference in the respective outer membranes of N and Tms mitochondria may affect the passage of toxin(s) to a mitochondrial site of action. Mitochondrial bioassays indicate that more toxin was bound by Tms mitochondria than by N mitochondria; the greatest toxin binding was associated with the inner membrane of Tms mitochondria.     

Polycyclovorans algicola gen. nov., sp. nov., an Aromatic-Hydrocarbon-Degrading Marine Bacterium Found Associated with Laboratory Cultures of Marine Phytoplankton

A strictly aerobic, halotolerant, rod-shaped bacterium, designated strain TG408, was isolated from a laboratory culture of the marine diatom Skeletonema costatum (CCAP1077/1C) by enrichment with polycyclic aromatic hydrocarbons (PAHs) as the sole carbon source. 16S rRNA gene sequence analysis placed this organism within the order Xanthomonadales of the class Gammaproteobacteria. Its closest relatives included representatives of the Hydrocarboniphaga-Nevskia-Sinobacter clade (<92% sequence similarity) in the family Sinobacteraceae. The strain exhibited a narrow nutritional spectrum, preferring to utilize aliphatic and aromatic hydrocarbon compounds and small organic acids. Notably, it displayed versatility in degrading two- and three-ring PAHs. Moreover, catechol 2,3-dioxygenase activity was detected in lysates, indicating that this strain utilizes the meta-cleavage pathway for aromatic compound degradation. Cells produced surface blebs and contained a single polar flagellum. The predominant isoprenoid quinone of strain TG408 was Q-8, and the dominant fatty acids were C_16:0, C_16:1 ω7c, and C_18:1 ω7c. The G+C content of the isolate''s DNA was 64.3 mol% ± 0.34 mol%. On the basis of distinct phenotypic and genotypic characteristics, strain TG408 represents a novel genus and species in the class Gammaproteobacteria for which the name Polycyclovorans algicola gen. nov., sp. nov., is proposed. Quantitative PCR primers targeting the 16S rRNA gene of this strain were developed and used to show that this organism is found associated with other species of marine phytoplankton. Phytoplankton may be a natural biotope in the ocean where new species of hydrocarbon-degrading bacteria await discovery and which contribute significantly to natural remediation processes.     

Peptostreptococcus canis sp. nov., isolated from subgingival plaque from canine oral cavity

A polyphasic taxonomic study was performed on two strains of an unknown Gram-positive, asaccharolytic, nonspore-forming, obligately anaerobic coccus-shaped bacterium isolated from oral subgingival plaque of Labrador retriever dogs. Comparative 16S rRNA gene sequencing confirmed that these isolates were highly related to each other and formed a hitherto unknown linage within the clostridial rRNA XI cluster of organisms. Pairwise analysis demonstrated that the novel organism to be most closely related to members of the genus Peptostreptococcus with 16S rDNA gene sequence similarity values between 92.8% and 96.7%, respectively. The G + C DNA base composition was 30.8 mol% and the major cellular fatty acids included iso-C_14:0, iso-C_16:0, and iso-C_{16:0 DMA}. Based on biochemical, chemotaxonomic, and phylogenetic evidence it is proposed that the unknown bacterium be classified as a new species, Peptostreptococcus canis sp. nov. The type strain is CCUG 57081^T.     

Paramoeba aparasomata n. sp., a symbiont-free species,and its relative Paramoeba karteshi n. sp. (Amoebozoa,Dactylopodida)

Two brackish water amoebae have been isolated and studied from the benthic biotopes of the Chupa Inlet (Kandalaksha Bay, northwestern Russia). Both strains can be identified as new species of the genus Paramoeba (Amoebozoa, Dactylopodida, Paramoebidae) based on light microscopical characters, structure of microscales on the cell surface and molecular evidence based on the analyses of two genes, nuclear SSU rRNA and mitochondrial cytochrome c oxidase subunit 1 (COI). Paramoeba aparasomata n. sp. is of particular interest because this amoeba is permanently lacking a symbiotic Perkinsela-like organism (PLO) present in other species of Paramoeba and Neoparamoeba. The results obtained show that scaly dactylopodial amoebae lacking PLO are not necessarily members of Korotnevella. In particular, we suggest that Korotnevella nivo Smirnov, 1997, with microscales very similar to those of Paramoeba eilhardi and the species studied here in structure, may be in fact a member of Paramoeba. Molecular data on K. nivo have to be obtained and analysed to test this hypothesis. Based on our new results we emend the diagnosis of the genus Paramoeba to make it more fit to the current phylogenetic conception.     

Dysgonomonas hofstadii sp. nov., isolated from a human clinical source

A Gram-negative staining, facultative anaerobic, cocco-bacillus-shaped organism was isolated from a post-operative abdominal wound. Based on morphological and biochemical criteria, strain MX 1040 ( = CCUG 54731^T) was tentatively identified as Bacteroidaceae but did not correspond to any recognized species of this family. Comparative 16S rRNA gene sequencing analysis demonstrated the organism to be related to species of the genus Dysgonomonas, although sequence divergence values of >5% with the other members of this genus demonstrated the organism to represent a novel species. Phylogenetic analysis revealed the novel organism to be most closely related to Dysgonomonas gadei. The major long-chain cellular fatty acids of the novel species consisted of iso-C_14:0, anteiso-C_15:0, C_16:0, and iso-C_16:0. Based on the phenotypic criteria and phylogenetic considerations, it is proposed that strain MX 1040 from a human clinical source represents a new species of the genus Dysgonomonas, as Dysgonomonas hofstadii sp. nov. The type strain of D. hofstadii is CCUG 54731^T ( = CCM 7606^T).     

Grellamoeba robusta gen. n., sp. n., a possible member of the family Acramoebidae Smirnov,Nassonova et Cavalier-Smith, 2008

A strain of naked amoeba isolated from pikeperch (Sander lucioperca (L.)) kidney tissue has been characterized using light- and transmission electron microscopy. Sequencing of SSU rDNA and phylogenetic analysis based on a broad dataset of sequences completed our study. All data obtained suggest that this strain belongs to a species that has not been described before. As none of the existing genera of amoebae is applicable to this organism, the new genus Grellamoeba is established and the type species Grellamoeba robusta is described. Although the phylogenetic position of the SSU rDNA sequence of the type strain of G. robusta is sensitive to the method of analysis applied, a tendency to group with Acramoeba dendroida Smirnov, Nassonova et Cavalier-Smith, 2008 is evident.     

Isolation and characteristics ofTreponema zuelzerae nov. spec., an anaerobic,free-living spirochete

Summary An anaerobic, free-living spirochete was isolated from mud. The organism can be cultivated in ordinary nutrient media, e.g. yeast extract-glucose. End products of glucose fermentation are: lactic, acetic, and succinic acids, CO₂, and H₂. In cultures of this organism spheroid bodies are formed, especially during the stationary growth phase. Studies of slide cultures showed that these bodies, when inoculated in fresh medium, do not give rise to spiral cells whereas a rapid multiplication of normal cells, also present in the inoculum, was observed. Since the organism is serologically related toTreponema pallidum, it has been assigned to the genusTreponema, and is here described asTreponema zuelzerae nov. spec. Part of this work was carried out at the Hopkins Marine Station of Stanford University, Pacific Grove, U.S.A., under a Rockefeller Foundation fellowship. Present address: Laboratorium voor Microbiologie, Wageningen, the Netherlands.     

Tachypteron franzeni n. gen., n. sp., earliest emballonurid bat from the Middle Eocene of Messel (Mammalia, Chiroptera)

A new genus and species of emballonurid microchiropteran,Tachypteron franzeni, is described from the early Middle Eocene (MP 11) of Grube Messel, near Darmstadt, Germany. The holotype is extraordinarily well-preserved. It is the first unequivocal representative of an extant clade among Messel bats and the oldest unequivocal record of Emballonuridae. The flight apparatus ofT. franzeni is highly specialized for a rapid and constant flight style. The proportions of the strikingly narrow wing, the outline of the flight membranes and external ear, the morphology of the postcranial skeleton, and the relative cochlea size ofT. franzeni and extantTaphozous species are almost identical, while the dentition ofT. franzeni is distinguished by more plesiomorphic features.Tachypteron and the fairly diverse Paleogene record of emballonurids from France, consisting mainly of isolated teeth, document the earliest radiation known from an extant bat family and suggest a rather intense pre-Middle Eocene diversification of emballonurids.     

Description ofAnaphes lemae n. sp. and notes onPatasson declinata Soyka [Hym., Mymaridae]

The autor describes a new species,Anaphes lemae, reared fromOulema (Lema) spp in France, Yugoslavia, and Poland and shows a misidentification ofPatasson valkenburgica Soyka. He points out the existence ofPatasson declinata Soyka in Poland and the Netherlands.     

Chemotactic responses ofThiobacillus thioparus

A capillary assay was employed to quantify chemotactic responses in the chemoautotrophic bacterium,Thiobacillus thioparus. NH₄Cl, KNO₃, and Na₂S₂O₃ were strong attractants. The minimum concentration of each of these inorganic chemicals needed to elicit an observable response was approximately 10^–4 M.T. thioparus did not respond to carbohydrates or amino acids.     

Enhanced removal efficiency of malodorous gases in a pilot-scale peat biofilter inoculated with Thiobacillus thioparus DW44

A newly isolated autotrophic bacterium, Thiobacillus thioparus DW44, which is capable of degrading sulfur-containing gases, was inoculated into a pilot-scale peat biofilter to treat the exhaust gas from a night soil treatment plant. Hydrogen sulfide (H₂S), methanethiol (MT), dimethyl sulfide (DMS) and dimethyl disulfide (DMDS) in the exhaust gas were efficiently removed for six months. Average removal ratios were 99.8% for H₂S, 99.0% for MT, 89.5% for DMS and 98.1% for DMDS at a space velocity of 46 h⁻¹ during the period of operation. No acclimation period was needed to reach such a high efficiency in the removal of the gases, indicating that the ability of this bacterium to remove these gases was occurred immediately after its inoculation to the peat. Ammonia (NH₃) in the exhaust gas was neutralized with SO₄²⁻, which is the final product of the oxidation of H₂S, MT, DMS and DMDS by the bacterium. No remarkable decline of pH, which often causes a deterioration in bacterial activity, was observed, mainly because of the reaction of SO₄²⁻ with NH₃. This study is the first report on the application of an isolated microorganism to a practical deodorizing system. The inoculation of T. thioparus DW44 into the pilot-scale peat biofilter could overcome such disadvantages of the conventional peat biofilter as a long acclimation period to reach a constant gas removability and the low removability of DMS, and resulted in enhanced removal efficiency of malodorous gases.     

A novel milky disease organism from Australian scarabaeids: Ultrastructure

The unusual ultrastructure of a milky disease bacterium isolated from Aphodius tasmaniae is described and compared with a similar bacterium from Tipula paludosa. Both organisms possess an elongate sporangium with a small central spore, a multilayered exosporium, and longitudinally arranged extrasporal fibrils which arise from plates attached to the ends of the spore. The morphogenesis of the sporangium is described and illustrated for the organism from A. tasmaniae. It is suggested this organism and that from T. paludosa are closely related. Their taxonomic position remains obscure.     

Isolation of Highly Radioresistant Bacterium,Arthrobacter radiotolerans nov. sp.

A highly γ-ray resistant bacterium, which has not been described hitherto, has been isolated from water containing mud, fur and moss at a radioactive hot spring, Misasa, Tottori Prefecture, Japan, This bacterium was Gram-positive, non-sporulating, pink to red colored and pleomorphic rod at young stage and predominantly coccoid or small short rod at old. The radiosensitivity of this bacterium was lower than that of well-known bacterium Micrococcus radio- durans. When its exponentially growing cells were irradiated in buffer solution aerated sufficiently at room temperature, shoulder dose and D₀ were calculated to be 6 × 10⁵ and 1 × 10⁶ rads, respectively. The morphological, cultural and physiological characteristics of the bacterium have been studied. These attributes suggested that the organism was a new species, and the name Arthrobacter radiotolerans has been assigned with respect to its high radioresistance.     

Pelovirga terrestris gen. nov., sp. nov., anaerobic,alkaliphilic, fumarate-, arsenate-, Fe(III)- and sulfur-reducing bacterium isolated from a terrestrial mud volcano

A novel anaerobic, mesophilic, alkaliphilic, chemoorganotrophic bacterium (strain M08fum^T) was isolated from a salsa lake of a terrestrial mud volcano (Taman Peninsula, Russia). Cell of strain M08fum^T were Gram-stain-negative, rod-shaped, non-spore forming motile rods. The temperature range for growth was 10–45 °C (optimum at 30 °C). The pH range for growth was 7.0–11.0, with an optimum at pH 8.5–9.0. The isolate was capable of organic acids fermentation and anaerobic respiration with elemental sulfur, Fe(III) and arsenate. The end products of fumarate fermentation were succinate, acetate and CO₂. The closest phylogenetic relatives of strain M08fum^T were members of the family Geopsychrobacteraceae, class Desulfuromonadia. The genome of strain M08fum^T had a size of 3.10 Mb with a DNA G + C content of 53.1% (WGS). Genome analysis revealed the presence of genes involved in fumarate fermentation, arsenate reduction and resistance, sulfur respiration and Fe (III) reduction. Based on the phenotypic, genotypic and phylogenetic characteristics we propose to assign strain M08fum^T to a new species of a novel genus Pelovirga terrestris gen. nov., sp. nov. within the family Geopsychrobacteraceae. The type strain of Pelovirga terrestris is M08fum^T (=KCTC 15919^T = VKM B-3407^T). This is the first representative of the class Desulfuromonadia, isolated in pure culture from a mud volcano and the first alkaliphile in the family Geopsychrobacteraceae.