Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing.
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Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%. (+info)
Transformation of sulfur compounds by an abundant lineage of marine bacteria in the alpha-subclass of the class Proteobacteria.
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Members of a group of marine bacteria that is numerically important in coastal seawater and sediments were characterized with respect to their ability to transform organic and inorganic sulfur compounds. Fifteen strains representing the Roseobacter group (a phylogenetic cluster of marine bacteria in the alpha-subclass of the class Proteobacteria) were isolated from seawater, primarily from the southeastern United States. Although more than one-half of the isolates were obtained without any selection for sulfur metabolism, all of the isolates were able to degrade the sulfur-containing osmolyte dimethyl sulfoniopropionate (DMSP) with production of dimethyl sulfide (DMS). Five isolates also degraded DMSP with production of methanethiol, indicating that both cleavage and demethylation pathways for DMSP occurred in the same organism, which is unusual. Five isolates were able to reduce dimethyl sulfoxide to DMS, and several isolates also degraded DMS and methanethiol. Sulfite oxygenase activity and methanesulfonic acid oxygenase activity were also present in some of the isolates. The ability to incorporate the reduced sulfur in DMSP and methanethiol into cellular material was studied with one of the isolates. A group-specific 16S rRNA probe indicated that the relative abundance of uncultured bacteria in the Roseobacter group increased in seawater enriched with DMSP or DMS. Because this group typically accounts for >10% of the 16S ribosomal DNA pool in coastal seawater and sediments of the southern United States, clues about its potential biogeochemical role are of particular interest. Studies of culturable representatives suggested that the group could mediate a number of steps in the cycling of both organic and inorganic forms of sulfur in marine environments. (+info)
Purification and characterization of the soluble methane monooxygenase of the type II methanotrophic bacterium Methylocystis sp. strain WI 14.
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Methane monooxygenase (MMO) catalyzes the oxidation of methane to methanol as the first step of methane degradation. A soluble NAD(P)H-dependent methane monooxygenase (sMMO) from the type II methanotrophic bacterium WI 14 was purified to homogeneity. Sequencing of the 16S rDNA and comparison with that of other known methanotrophic bacteria confirmed that strain WI 14 is very close to the genus Methylocystis. The sMMO is expressed only during growth under copper limitation (<0.1 microM) and with ammonium or nitrate ions as the nitrogen source. The enzyme exhibits a low substrate specificity and is able to oxidize several alkanes and alkenes, cyclic hydrocarbons, aromatics, and halogenic aromatics. It has three components, hydroxylase, reductase and protein B, which is involved in enzyme regulation and increases sMMO activity about 10-fold. The relative molecular masses of the native components were estimated to be 229, 41, and 18 kDa, respectively. The hydroxylase contains three subunits with relative molecular masses of 57, 43, and 23 kDa, which are present in stoichiometric amounts, suggesting that the native protein has an alpha(2)beta(2)gamma(2) structure. We detected 3.6 mol of iron per mol of hydroxylase by atomic absorption spectrometry. sMMO is strongly inhibited by Hg(2+) ions (with a total loss of enzyme activity at 0.01 mM Hg(2+)) and Cu(2+), Zn(2+), and Ni(2+) ions (95, 80, and 40% loss of activity at 1 mM ions). The complete sMMO gene sequence has been determined. sMMO genes from strain WI 14 are clustered on the chromosome and show a high degree of homology (at both the nucleotide and amino acid levels) to the corresponding genes from Methylosinus trichosporium OB3b, Methylocystis sp. strain M, and Methylococcus capsulatus (Bath). (+info)
Poly(aspartic acid) degradation by a Sphingomonas sp. isolated from freshwater.
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A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (<5 kDa), while the cell extract hydrolyzed high-molecular-mass poly(aspartic acid)s of 5 to 150 kDa to yield aspartic acid monomer. (+info)
Characterization of a separate small domain derived from the 5' end of 23S rRNA of an alpha-proteobacterium.
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We demonstrate the presence of a separate processed domain derived from the 5' end of 23S rRNA in ribosomes of Rhodopseudomonas palustris, a member of the alpha-++proteobacteria. Previous sequencing studies predicted intervening sequences (IVS) at homologous positions within the 23S rRNA genes of several alpha-proteobacteria, including R.palustris, and we find a processed 23S rRNA 5' domain in unfractionated RNA from several species. 5.8S rRNA from eukaryotic cytoplasmic large subunit ribosomes and the bacterial processed 23S rRNA 5' domain share homology, possess similar structures and are both derived by processing of large precursors. However, the internal transcribed spacer regions or IVSs separating them from the main large subunit rRNAs are evolutionarily unrelated. Consistent with the difference in sequence, we find that the site and mechanism of IVS processing also differs. Rhodopseudomonas palustris IVS-containing RNA precursors are cleaved in vitro by Escherichia coli RNase III or a similar activity present in R.palustris extracts at a processing site distinct from that found in eukaryotic systems and this results in only partial processing of the IVS. Surprisingly, in a reaction unlike characterized cases of eubacterial IVS processing, an RNA segment larger than the corresponding DNA insertion is removed which contains conserved sequences. These sequences, by analogy, serve to link the 23S rRNA 5' rRNA domains or 5.8S rRNAs to the main portion of other prokaryotic 23S rRNAs or to eukaryotic 28S rRNAs, respectively. (+info)
Bartonella vinsonii subsp. berkhoffii and related members of the alpha subdivision of the Proteobacteria in dogs with cardiac arrhythmias, endocarditis, or myocarditis.
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Cardiac arrhythmias, endocarditis, or myocarditis was identified in 12 dogs, of which 11 were seroreactive to Bartonella vinsonii subspecies berkhoffii antigens. Historical abnormalities were highly variable but frequently included substantial weight loss, syncope, collapse, or sudden death. Fever was an infrequently detected abnormality. Cardiac disease was diagnosed following an illness of short duration in most dogs, but a protracted illness of at least 6 months' duration was reported for four dogs. Valvular endocarditis was diagnosed echocardiographically or histologically in eight dogs, two of which also had moderate to severe multifocal myocarditis. Four dogs lacking definitive evidence of endocarditis were included because of seroreactivity to B. vinsonii antigens and uncharacterized heart murmurs and/or arrhythmias. Alpha proteobacteria were not isolated from the blood by either conventional or lysis centrifugation blood culture techniques. Using PCR amplification and DNA sequencing of a portion of the 16S rRNA gene, B. vinsonii was identified in the blood or heart valves of three dogs. DNA sequence alignment of PCR amplicons derived from blood or tissue samples from seven dogs clustered among members of the alpha subdivision of the Proteobacteria and suggested the possibility of involvement of one or more alpha proteobacteria; however, because of the limited quantity of sequence, the genus could not be identified. Serologic or molecular evidence of coinfection with tick-transmitted pathogens, including Ehrlichia canis, Babesia canis, Babesia gibsonii, or spotted fever group rickettsiae, was obtained for seven dogs. We conclude that B. vinsonii subsp. berkhoffii and closely related species of alpha proteobacteria are an important, previously unrecognized cause of arrhythmias, endocarditis, myocarditis, syncope, and sudden death in dogs. (+info)
Oxidation of methyl halides by the facultative methylotroph strain IMB-1.
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Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils. (+info)
Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.
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Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes. (+info)