- Volume 45, Issue 2, 1995

Neorickettsia helminthoeca (tribe Ehrlichieae, family Rickettsiaceae) is the agent of salmon poisoning disease, which affects members of the family Canidae. This bacterium is unusual in that it is the only known obligately intracellular bacterium that is transmitted via a helminth vector. The nucleotide sequence of the N. helminthoeca 16S rRNA gene was determined and compared with the sequences of intracellular bacteria belonging to the alpha subgroup of the Proteobacteria. The N. helminthoeca sequence was most similar to the sequences of two Ehrlichia species, Ehrlichia risticii and Ehrlichia sennetsu (levels of sequence similarity, >95%). All other members of the tribe Ehrlichieae, including members of the other Ehrlichia species, and the related species Cowdria ruminantium and Anaplasma marginale, were only distantly related phylogenetically (levels of sequence similarity, 84 to 86%). Our results corroborate the results of previous ultrastructural and Western blot (immunoblot) comparisons of N. helminthoeca with other ehrlichial species. The genus Ehrlichia is phylogenetically incoherent and can be separated into three identifiable clusters of species. Each cluster is closely associated with a species classified in another non-Ehrlichia bacterial genus. The close relationships among N. helminthoeca, E. risticii, and E. sennetsu and the striking differences between these organisms and other members of the tribe Ehrlichieae suggest that in the future, these organisms should be treated as members of a new bacterial genus separate from the genus Ehrlichia.

Random amplified polymorphic DNA fingerprinting was used to examine 31 mosquito-pathogenic and 14 nonpathogenic strains of Bacillus sphaericus. We verified that DNA bands that migrated the same distance in an agarose gel were homologous by using PCR-generated probes made from the random amplified polymorphic DNA bands. The band patterns obtained with eight primers were analyzed by using the Jaccard coefficient and unweighted pair group with arithmetic average clustering. Pathogenic strains belonging to DNA homology group IIA were similar to strains belonging to nonpathogenic homology groups at an average level of similarity of 6.3%. Individual serotypes were clearly identified among the pathogenic strains. This suggests that there is overall genetic homogeneity among strains within serotypes. It is also consistent with the uniform toxicity pattern found for each serotype (unlike the toxin diversity found in Bacillus thuringiensis serotypes). These results, together with DNA homology data, support the proposal that a new species should be described for the pathogenic strains.

Strain MLFT (T = type strain), a new thermophilic, spore-forming sulfate-reducing bacterium, was characterized and was found to be phenotypically, genotypically, and phylogenetically related to the genus Desulfotomaculum. This organism was isolated from a butyrate enrichment culture that had been inoculated with a mixed compost containing rice hulls and peanut shells. The optimum temperature for growth was 50°C. The G+C content of the DNA was 51.2 mol%. Strain MLFT incompletely oxidized pyruvate, butyrate, and butanol to acetate and presumably CO2. It used long-chain fatty acids and propanediols. We observed phenotypic and phylogenetic differences between strain MLFT and other thermophilic Desulfotomaculum species that also oxidize long-chain fatty acids. On the basis of our results, we propose that strain MLFT is a member of a new species, Desulfotomaculum thermosapovorans.

Nine streptomycete strains that cause deep-pitted scab of potato were characterized by performing 138 physiological and morphological tests. A numerical analysis revealed that the deep-pitted scab-inducing organisms were related to strains belonging to cluster 1 of Williams et al. (Streptomyces albidoflavus). The levels of similarity between the deep-pitted scab-inducing strains and strains of Streptomyces scabies and Streptomyces acidiscabies were low (52 and 54%, respectively). The fatty acid profiles of the deep-pitted scab-inducing organisms differed considerably from those of S. scabies and S. acidiscabies. The deep-pitted scab-inducing bacteria were characterized by the predominance in their profiles of the 15:0 anteiso, 15:0 iso, 16:0 iso, 16:0, and 17:0 anteiso acids. The results of physiological characterization experiments, a fatty acid analysis, and whole-cell protein electrophoresis showed that the deep-pitted scab-inducing strains characterized in this study formed a relatively homogeneous group. These pathogenic strains could also be differentiated from S. scabies by their high cellulolytic and proteolytic activities.

Strains of a previously undescribed species of purple nonsulfur phototrophic bacteria were isolated from coastal seawater in Japan. These new isolates were gram-negative, motile, budding rods that contained lamellar intracytoplasmic membranes and produced pink to red cultures. Cell extracts of photosynthetic cultures exhibited absorption maxima at 377, 468, 500, 530, 591, 802, and 870 nm, indicating that bacterio-chlorophyll a and carotenoids of the spirilloxanthin series were present. The new isolates were halophilic, facultatively aerobic photoheterotrophs that grew anaerobically in the light or aerobically in the dark. Maximum growth occurred in the presence of 4 to 5% NaCl. Anaerobic growth in the dark with nitrate as a terminal electron acceptor also occurred. Various organic compounds were used as photosynthetic electron donors and carbon sources. Sulfate was used as a sulfur source. Both menaquinone 10 and ubiquinone 10 were produced; these quinones were the major quinones. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain MB312T(T = type strain), a representative of the new phototrophs, was a member of a lineage that was distinct from members of the genus Rhodopseudomonas; Rhodopseudomonas marina was the closest relative. On the basis of the data described above, we propose the name Rhodobium orientis gen. nov., sp. nov. for the new isolates. We also propose that Rhodopseudomonas marina Imhoff 1983 should be transferred to the genus Rhodobium as Rhodobium marinum comb. nov.

Isolates belonging to the “Streptococcus milleri” species group that appear to exhibit a gliding type of motility, which is expressed as spreading growth on certain types of agar media, are described. These strains resembled a biotype of “S. milleri” that is usually isolated from genitourinary sources and is notable for its ability to ferment a wide array of carbohydrates. This biotype, which is currently included in the species Streptococcus anginosus, has been implicated in cases of neonatal infection. The “S. milleri” isolates which we studied lacked any observable organelles of motility and gave negative results when they were tested in conventional motility test medium stab cultures. Colonies growing on certain agar media, however, spread over the surfaces of plates and increased in area with increasing time of incubation. Chocolate agar supported maximum spreading, while this characteristic was barely discernible on blood agar. Electron microscopy studies revealed that there was more production of extracellular glycocalyx by motile strains than by a nonmotile isolate having a similar biotype. The results of an analysis of 16S rRNA gene sequences suggested that the motile strains are closely related to S. anginosus and represent a distinct rRNA population within the “S. milleri” species complex.

Partial sequences of the 16S rRNA genes of the type strains of nine species of the genus Nocardia were determined following the isolation and cloning of the amplified genes. These sequences were aligned with the sequences of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus, and Tsukamurella, and phylogenetic trees were inferred by using the Fitch-Margoliash and neighbor-joining methods. The genus Nocardia formed a distinct clade that was most closely associated with the genus Rhodococcus. The average level of sequence similarity found among the type strains of the Nocardia species was 97.2 ± 0.7%. Two sublines were recognized within the Nocardia clade; one encompassed Nocardia asteroides and related species, and the other encompassed Nocardia otitidiscaviarum and allied taxa. Separation of the two sublines is based on differences in helix 37-1. The results of isoprenoid quinone analyses provided evidence that nocardiae can be distinguished from all other actinomycete taxa on the basis of their characteristic menaquinone profiles. Nocardiae typically contain hexahydrogenated menaquinones with eight isoprene units in which the two end units are cyclized.

Members of the genera Bacteroides, Prevotella, and Porphyromonas were investigated for their glucose 6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) activities by using spectrophotometric (SP) and alloenzyme elecrophoresis (AE) detection methods. When the SP and AE methods were compared, the AE method failed to detect activity in two of the six strains which exhibited G6PDH and 6PGDH activities as determined by the SP detection method. On the basis of the results of SP detection, Bacteroides levii ATCC 29147T(T = type strain) (G6PDH and 6PGDH negative) is not a member of the genus Bacteroides as currently defined, which reflects recent 16S rRNA placement, nor do Prevotella heparinolytica ATCC 35895T, Prevotella zoogleoformans ATCC 33285T, Porphyromonas canoris 12835T, and Porphyromonas salivosa NCTC 11632T(all G6PDH and 6PGDH positive) conform to their respective genus descriptions. When these organisms were grown in prereduced peptone-yeast extract broth containing 10% (wt/vol) glucose, the amounts of glucose remaining after 5 days were less than the amounts present initially for members of the genus Bacteroides (Bacteroides fragilis ATCC 25285T and B. levii) and members of the genus Prevotella (Prevotella melaninogenica ATCC 25845T, Prevotella buccae ATCC 33574T, Prevotella heparinolytica, and Prevotella zoogleoformans). In addition, the glucose levels were lower after 5 days of incubation in broth media containing Porphyromonas asaccharolytica ATCC 25845Tand Porphyromonas salivosa, but not in media containing the other members of the genus Porphyromonas tested (Porphyromonas canoris, Porphyromonas circumdentaria NCTC 12469T, Porphyromonas endodontalis ATCC 35406T, and Porphyromonas gingivalis ATCC 33277T). The reductions in glucose levels were not directly related to the final pH values. Our results suggest that the descriptions of the genera Bacteroides, Prevotella, and Porphyromonas may require emendation to reflect variability in G6PDH and 6PGDH activities and glucose utilization.

The 16S rRNA gene of a new infectious agent, strain AS145T(T = type strain), which was isolated from a wild mouse in Japan, was amplified by using the PCR. The amplimers were directly sequenced by dideoxynucleotide methods with Taq DNA polymerase. Sequence comparisons with other members of the tribe Ehrlichieae and related species revealed that the infectious agent isolated from the mouse is a new species of the genus Ehrlichia that is most closely related to Ehrlichia chaffeensis (level of sequence similarity, 97.9%), an agent of human ehrlichiosis in the United States. This result was consistent with the results of an immunoblot analysis performed with immune sera against different ehrlichiosis agents. On the basis of these findings and other morphological, biological, and serological characteristics of the organism, we propose that ehrlichiae with these properties belong to a new species, Ehrlichia muris.

We investigated expression of acid phosphatases containing low-molecular-mass (25 to 27-kDa) polypeptides (Lmmp-APs) similar to those described previously for Salmonella enterica serovar typhimurium and Morganella morganii in strains that were representatives of 43 different enterobacterial species by using a zymogram technique developed for detection of Lmmp-AP activities and for analysis of some of the properties of these enzymes. Under conditions that were suitable for detection of the previously described Lmmp-APs, production of similar enzymes appeared to be widespread but not universal among enteric bacteria, and heterogeneous patterns of expression were found among strains belonging to different genera and, in some cases, among strains belonging to different species of the same genus. We found that class A Lmmp-APs (i.e., Lmmp-Aps similar to the Morganella morganii PhoC and Salmonella enterica serovar typhimurium PhoN acid phosphatases) were also expressed in Cedecea spp., Enterobacter aerogenes, Hafitia alvei, Klebsiella spp., Providencia stuartii, Serratia plymuthica, and Yokenella regensburgei strains and that class B Lmmp-APs (i.e., Lmmp-APs similar to the Morganella morganii NapA and Salmonella enterica serovar typhimurium NapII acid phosphatases) were also expressed in strains of Citrobacter spp., Escherichia coli, Escherichia fergusonii, Hafnia alvei, Proteus mirabilis, Providencia spp., Salmonella enterica serovar typhi, ShigeUa dysenteriae, and Shigella flexneri. No Lmmp-AP activity was detected in strains of Enterobacter spp. other than Enterobacter aerogenes, Escherichia hermanii, Kluyvera ascorbata, Leclercia adecarboxylata, Leminorella grimontii, Moellerella wisconsensis, Proteus vulgaris, Proteus penneri, Serratia spp. other than Serratia plymuthica, and Yersinia spp. Because of the heterogeneous patterns of expression of Lmmp-APs, analysis of these enzymes could be useful for evolutionary studies of the enterobacterial genome and for precise phylogenetic positioning of enteric bacteria.

The Mycobacterium chelonae-like organism (MCLO) is a recently described member of the Mycobacterium fortuitum complex which causes posttraumatic skin infections and catheter sepsis. This taxon is a distinct group biochemically and has a unique mycolic acid profile as determined by high-performance liquid chromatography. Its phylogenetic relationships to other mycobacteria, however, have not been studied previously. We sequenced 1,062 bp of the 16S rRNA genes from three MCLO strains obtained from the American Type Culture Collection and compared our results with the sequences of previously described taxa of rapidly growing and slowly growing mycobacteria. Two biochemically typical strains (ATCC 49650T[T = type strain] and ATCC 49651) had identical sequences, while the sequence of a biochemically atypical strain (ATCC 49649) differed by 4 bp from the sequence of the two typical strains. The Hamming distances between these MCLO strains and related rapidly growing mycobacteria are comparable to the Hamming distances among taxa of rapidly growing mycobacteria established as species by DNA-DNA hybridization. We propose the name Mycobacterium mucogenicum sp. nov. for this new taxon because of the highly mucoid nature of most isolates on solid media.

I analyzed various families of ribosomal proteins obtained from selected species belonging to the genus Pseudomonas sensu stricto and allied organisms which were previously classified in the genus Pseudomonas. Partial amino acid sequencing of L30 preparations revealed that the strains which I examined could be divided into three clusters. The first cluster, which was assigned to the genus Pseudomonas sensu stricto, included Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas mendocina, and Pseudomonas fluorescens. The second cluster included Burkholderia pickettii and Burkholderia plantarii. The third cluster, which was a deeply branching cluster in the stem of gram-negative bacteria, included Brevundimonas diminuta and Brevundimonas vesicularis. Despite the different levels of conservation of the N-terminal sequences of ribosomal protein families (the highest level of similarity was 74% for L27 proteins and the lowest level of similarity was 42% for L30 proteins), similar phylogenetic trees were constructed by using data obtained from sequence analyses of various ribosomal protein families, including the S20, S21, L27, L29, L31, L32, and L33 protein families. Thus, I demonstrated the efficacy of ribosomal protein analysis in bacterial taxonomy.

The taxonomic position of nitrogen-fixing strains that were isolated from rhizosphere macerates of rice cultivated in the Binh Thanh region of Vietnam was determined by using polyphasic taxonomy. We determined the phylogenetic relationships of these organisms by performing DNA-rRNA hybridization experiments with a labeled rRNA probe from the type strain of Burkholderia cepacia, and we found that they belong to a single rRNA complex. Other members of this rRNA complex were also studied, and the N2-fixing strains were found to be closely related to B. cepacia. In addition, all members of the rRNA complex containing B. cepacia were studied by performing auxanographic and DNA-DNA hybridization experiments. Phenotypically and genotypically, the N2-fixing isolates constitute a single cluster together with two strains of clinical origin. These organisms constitute a new Burkholderia species, for which the name Burkholderia vietnamiensis is proposed; the type strain of this species is TVV75 (= LMG 10929). All members of this species can fix nitrogen. On the basis of our polyphasic taxonomy results and previously published data we concluded that the genus Burkholderia should be restricted to the following species: B. cepacia (the type species), Burkholderia mallei, Burkholderia pseudomallei, B. vietnamiensis, Burkholderia gladioli, Burkholderia caryophylli, Burkholderia plantarii, Burkholderia glumae, Burkholderia vandii, Burkholderia cocovenenans comb. nov., and Burkholderia andropogonis comb. nov. On the basis of genotypic and phenotypic results [Alcaligenes] eutrophus, [Burkholderia] solanacearum, and [Burkholderia] pickettii belong to two other clusters whose internal structures must be studied further.

We describe a new species on the basis of phenotypic characteristics and the results of an analysis of small-subunit rRNA sequences. Three strains of this organism were isolated from a culture of the toxin-producing dinoflagellate Prorocentrum lima. These bacteria are gram-negative, strictly aerobic, ovoid organisms that are motile by means of one or two subpolar flagella. They grow at temperatures ranging from 10 to 37°C and in the presence of NaCl concentrations ranging from 0.1 to 2 M and have an absolute requirement for sodium ions. They are strictly aerobic with a nonfermentative type of metabolism and are not able to grow anaerobically in presence or absence of nitrate. They do not denitrify. They exhibit oxidase, catalase, gelatinase, esculinase, β-galactosidase, and (to a lesser extent) amylase activities. The three strains which we examined require thiamine and biotin for growth. They grow only when glucose, trehalose, saccharose, fructose, maltose, pyruvate, malate, citrate, esculin, 2-ketoglutarate, 5-ketogluconate, glutamate, or shikimate is present as a sole carbon source. The three strains have identical small-subunit rRNA sequences. A phylogenetic analysis of these sequences revealed that these bacteria belong to the alpha subdivision of the Proteobacteria and that they form a distinct and robust monophyletic group with Roseobacter denitrificans and Roseobacter litoralis. This result and the general phenotypic characteristics of the organisms place them in the genus Roseobacter, although they do not produce bacteriochlorophyll a, in contrast to previously described Roseobacter species. On the basis of the phenotypic and genetic similarities of these strains, we assigned them to a single species, for which the name Roseobacter algicola is proposed. The type strain is R. algicola FF3 (= ATCC 51440).

A gram-negative, anaerobic, nonmotile, non-spore-forming, rod-shaped bacterium that fermented succinate quantitatively to propionate was isolated from a high dilution of rumen ingesta obtained from a dairy cow fed a production diet containing grass silage as the main roughage source. This organism did not grow on any of the following energy sources: 12 carbohydrates, pyruvate, lactate, 7 dicarboxylic acids, aspartate, citrate, and trans-aconitate. Both rumen fluid and yeast extract were necessary for good growth on succinate. The organism was negative for the following characteristics: production of propionate from threonine, protein digestion, sulfide production, nitrate reduction, catalase activity, and urease activity. There was no growth at 22°C and reduced growth at 45°C compared with growth at 39°C. The DNA base composition was 52 mol% G+C. The complete 16S rRNA sequence (EMBL accession number, X81137) was obtained, and the phylogenetic relationships of the organism were determined. The most closely related genera were the genera Acidaminococcus and Phascolarctobacterium. The name proposed for this bacterium is Succiniclasticum ruminis gen. nov., sp. nov.; the type strain is strain SE10 (= DSM 9236). Additional isolation attempts revealed that S. ruminis is a common inhabitant of the rumina of cows that are fed production diets and of cows on pasture.

A strictly anaerobic, moderately halophilic, gram-negative, rod-shaped bacterium was isolated from Great Salt Lake, Utah, sediments and designated GSLST(T = type strain). Strain GSLSTgrew optimally at pH 6.7 to 7.0 but had a very broad pH range for growth (pH 5.8 to 10.0). The optimum temperature for growth was 37°C, and no growth occurred at 15 or 55°C. The optimum salt concentration for growth was 10%. Strain GSLSTrequired yeast extract and Trypticase peptone to ferment carbohydrates, pyruvate, and glycine betaine. Strain GSLSTwas resistant to penicillin, d-cycloserine, tetracycline, and streptomycin. The G+C content of this isolate was 31 mol%. The fermentation products from glucose utilization were acetate, butyrate, lactate, CO2, and H2, and in addition strain GSLSTfermented glycine betaine to acetate and trimethylamine. All of these traits distinguish this organism from all previously described halophilic anaerobes. The 16S rRNA gene sequence of strain GSLSTwas found to be similar to, but also significantly different from, the 16S rRNA sequences of Haloanaerobium salsugo and Haloanaerobium praevalens. Therefore, strain GSLST(= DSM 8275T) is described as a new species, Haloanaerobium alcaliphilum.

A thermophilic, glucose-fermenting, strictly anaerobic, rod-shaped bacterium, strain SEBR 6459T(T = type strain), was isolated from an African oil-producing well. This organism was identified as a member of the genus Thermotoga on the basis of the presence of the typical outer sheath-like structure (toga) and 16S rRNA signature sequences and its ability to grow on carbohydrates (glucose, arabinose, fructose, lactose, maltose, and xylose). Major differences in its 16S rRNA gene sequence, its lower optimum temperature for growth (66°C), its sodium chloride range for growth (0 to 2.8%), its lack of lactate as an end product from glucose fermentation, and its peritrichous flagella indicate that strain SEBR 6459Tis not similar to the three previously described Thermotoga species. Furthermore, this organism does not belong to any of the other genera related to the order Thermotogales that have been described. On the basis of these findings, we propose that this strain should be described as a new species, Thermotoga elfii. The type strain of T. elfii is SEBR 6459 (= DSM 9442).

Ehrlichia risticii is the causative agent of Potomac horse fever. Variations among the major antigens of different local E. risticii strains have been detected previously. To further assess genetic variability in this species or species complex, the sequences of the 16S rRNA genes of several isolates obtained from sick horses diagnosed as having Potomac horse fever were determined. The sequences of six isolates obtained from Ohio and three isolates obtained from Kentucky were amplified by PCR. Three groups of sequences were identified. The sequences of five of the Ohio isolates were identical to the sequence of the type strain of E. risticii, the Illinois strain. The sequence of one Ohio isolate, isolate 081, was unique; this sequence differed in 10 nucleotides from the sequence of the type strain (level of similarity, 99.3%). The sequences of the three Kentucky isolates were identical to each other, but differed by five bases from the sequence of the type strain (level of similarity, 99.6%). The levels of sequence similarity of isolate 081, the Kentucky isolates, and the type strain to the next most closely related Ehrlichia sp., Ehrlichia sennetsu, were 99.3, 99.2, and 99.2%, respectively. On the basis of the distinct antigenic profiles and the levels of 16S rRNA sequence divergence, isolate 081 is as divergent from the type strain of E. risticii as E. sennetsu is. Therefore, we suggest that strain 081 and the Kentucky isolates may represent two new distinct Ehrlichia species.

Several strains of phototrophic purple nonsulfur bacteria were isolated from colored blooms occurring in tidal and seawater pools in Japan. All of these isolates had ovoid to rod-shaped cells that were motile by means of single polar flagella and contained vesicular intracytoplasmic membranes together with bacteriochlorophyll a and carotenoids of the spheroidene series. They produced ubiquinone 10 as the major quinone and contained straight-chain fatty acids, with C18:1predominating. They were mesophilic, halophilic, and photoheterotrophic, utilized sulfide and thiosulfate as electron donors for phototrophic growth, and photoassimilated a wide variety of organic compounds as carbon sources. Our results suggested that all of these isolates are members of the recently described genus Rhodovulum. The isolates were classified into four groups (designated groups I through IV) on the basis of phenotypic and genotypic data. The group I isolates, which were the most abundant purple nonsulfur bacteria recovered from the blooms, grew in the presence of NaCl concentrations ranging from 0.5 to 3.0% (optimum NaCl concentration, 0.8%) and at pH values ranging from 7.5 to 9.0 (optimum pH, 8.0 to 8.5). On the basis of these unique physiological traits, together with genotypic and phylogenetic data, we propose that the group I isolates should be classified as members of a new species, Rhodovulum strictum. The group II isolates were identified definitely as Rhodovulum sulfidophilum, and the group III and IV isolates were phenotypically most similar to R. sulfidophilum and Rhodovulum adriaticum, respectively, but could be differentiated from these specks by DNA-DNA pairing data.