- Volume 45, Issue 4, 1995

A new bacterial strain, strain CR-43T (T = type strain), which was isolated from tropical soil and was previously shown to have antinematodal and antibiotic properties, is described. The name Streptomyces costaricanus is proposed for this organism. The generic placement of strain CR-43T was based on its typical morphology, its production of ll-diaminopimelic acid, and its fatty acid composition. To clarify the taxonomic position of strain CR-43T, it was compared with the type strains of similar Streptomyces species. The results of a number of biochemical tests and a profile analysis of the hydrolyzable fatty acids indicated that CR-43T differs from previously described species. Strain CR-43 (= ATCC 55274 = NRRL B-16897) is the type strain of S. costaricanus sp. nov.

Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. “Candidatus Arthromitus” is proposed as the provisional generic name for this group of organisms.

A strictly anaerobic, thermophilic, gram-positive, spore-forming eubacterium designated strain SEBR 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92°C. The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 μm) with peritrichous flagella. The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomorphic and filamentous (length, 15 μm) in old cultures. Growth occurred at temperatures of 40 to 75°C, and optimum growth occurred at temperatures between 55 and 60°C. The fermentable substrates included glucose, fructose, galac-tose, mannose, cellobiose, maltose, sucrose, lactose, d-xylose, d-ribose, mannitol, pyruvate, and starch. The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2. The DNA base composition was 35 mol% G+C. The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacterfinnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99%. The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of >70% were obtained, these organisms belong to the same species. Therefore, we propose that T. finnii should be reclassified as a subspecies of T. brockii, Thermoanaerobacter brockii subsp. finnii comb. nov. This automatically creates Thermoanaerobacter brockii subsp. brockii. We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T. brockii, Thermoanaerobacter brockii subsp. lactiethylicus. The latter differs from T. brockii subsp. brockii and T. brockii subsp. finnii by its 16S rRNA sequence, DNA sequence diversity, lower temperature optimum, G+C content, and carbohydrate utilization spectrum. Strain SEBR 5268T has been deposited in the Deutsche Sammlung von Mikroorganismen as strain DSM 9801T.

A new extremely halophilic chemoorganotrophic bacterium (strain H200T [T = type strain]) was isolated from the hypersaline sediments of Retba Lake in Senegal. This organism was a sluggishly motile, rod-shaped, non-spore-forming, gram-negative, obligate anaerobe that grew optimally at 40°C in the presence of 180 to 200 g of NaCl per liter. The DNA base composition was 32 mol% guanine plus cytosine. The fermentation products from glucose were ethanol, acetate, H2, and CO2. Yeast extract was required for growth. The fermentable substrates included d-fructose, galactose, d-xylose, cellobiose, lactose, maltose, sucrose, starch, d-mannitol, glycerol, and Casamino Acids. On the basis of the results of a 16S rRNA sequence analysis, strain H200T was found to be related to Haloanaerobium species. The 16S rRNA sequence of strain H200T differed from the sequences of the three previously described Haloanaerobium species, and strain H200T also differed from these organisms in its NaCl range for growth (60 to 340 g/liter); strain H200T grew in the presence of the highest NaCl concentration recorded for any halophilic anaerobic organism, including the three previously described Haloanaerobium species. We propose that strain H200T (= DSM 10165) belongs to a new Haloanaerobium species, Haloanaerobium lacusroseus.

We performed this study to determine the 16S rRNA sequences of the type strains of Streptococcus adjacens and Streptococcus defectivus and to calculate the phylogenetic distances between these two nutritionally variant streptococci (NVS) and other members of the genus Streptococcus. S. adjacens and S. defectivus belonged to one cluster, but this cluster was not closely related to other streptococcal species. A comparative analysis of the sequences of these organisms and other low-G+C-content gram-positive bacteria revealed that the two NVS species formed a distinct cluster and were only remotely related to the Aerococcus and Carnobacterium clusters. The highest level of homology (93.7%) was found between S. adjacens and Carnobacterium divergens. Carnobacterium species have meso-diaminopimelic acid in their cell walls, but S. adjacens and S. defectivus have l-lysine as the diamino acid at position 3 in their peptidoglycan tetrapeptides. On the basis of our findings and the results of previous phenotypic studies, we propose that the NVS species should be placed in a new genus, the genus Abiotrophia, as Abiotrophia adiacens comb. nov. and Abiotrophia defectiva comb. nov.

The ixodid tick Ixodes Persulcatus is the most important vector of Lyme disease in Japan. Most spirochete isolates obtained from I. persulcatus ticks have been classified as Borrelia burgdorferi sensu lato because of their genetic, biological, and immunological characteristics. However, we found that a small number of isolates obtained from I. persulcatus contained a smaller 38-kDa endoflagellar protein and single 23S-5S rRNA gene unit. Representative isolate HT31T (T = type strain) had the same 23S rRNA gene physical map as Borrelia turicatae. The DNA base composition of strain HT31T was 28.6 mol% G+C. DNA-DNA hybridization experiments revealed that strain HT31T exhibited moderate levels of DNA relatedness (24 to 51%) with Borrelia hermsii, B. turicatae, Borrelia parkeri, and Borrelia coriaceae. However, the levels of DNA reassociation with the previously described Lyme disease borreliae (B. burgdorferi, Borrelia garinii, and Borrelia afzelii) were only 8 to 13%. None of the previously described species examined exhibited a high level of DNA relatedness with strain HT31T. In addition, the 16S rRNA gene sequence (length, 1,368 nucleotides) of strain HT31T was determined and aligned with the 16S rRNA sequences of other Borrelia species. Distance matrix analyses were performed, and a phylogenetic tree was constructed. The results showed that isolate HT31T is only distantly related to both previously described Lyme disease borreliae and relapsing fever borreliae. Thus, the spirochetes isolated from I. persulcatus and closely related isolates should be classified as members of a new Borrelia species. We propose the name Borrelia miyamotoi sp. nov. for this spirochete; strain HT31 is the type strain.

We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium. We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus. A 70°C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol%. The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with α-33P-labeled primers, again with extension at 70°C. Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase. Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material. Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup. Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously. Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed.

A novel obligately intracellular bacterium, ileal symbiont intracellularis, which was obtained from the intestines of pigs with proliferative enteropathy disease, was grown in pure cocultures with tissue cultures of rat cells. An examination of the 16S ribosomal DNA gene sequence revealed that the isolates which we obtained are members of the delta subdivision of the Proteobacteria and that the sequences of these organisms exhibit a level of similarity of 91% with the sequence of Desulfovibrio desulfuricans ATCC 27774. These isolates were homogeneous and differed in cellular morphology, acid fastness, phenotype, electrophoretic protein profile, and habitat from Desulfovibrio species. On the basis of the results of an integrated study of the phenotype and genotype of a consistent morphological entity found in particular porcine cells and associated with a well-defined clinical condition, we concluded that these bacteria belong to a previously undescribed genus and species, for which we propose the name Lawsonia intracellularis gen. nov., sp. nov. A species-specific recombinant DNA probe was cloned previously, and this probe was used to identify the bacterium in tissue culture cells and in the ileal epithelia of pigs with proliferative enteropathy disease. Coculture of the organism with a rat enterocyte cell line allowed us to designate strain NCTC 12656 the type strain and to describe the new genus and species. The organism which we cultured is pathogenic for pigs and causes proliferative enteropathy lesions in their ilea and colons, and Koch’s postulates were fulfilled for this organism.

Because of similarities in the cellular fatty acid compositions of Hallella seregens and Mitsuokella dentalis, we determined the 16S rRNA gene sequences of the type strains of these species to assess their relationship. A very high level of sequence relatedness (approximately 99.8%) was found between H. seregens and M. dentalis, indicating that these species are genealogically closely related. A comparative sequence analysis revealed that these two species are members of the genus Prevotella and are phylogenetically remote from Mitsuokella multiacidus (the type species of the genus Mitsuokella), which was found to be a member of the Sporomusa subbranch of the Clostridium subphylum of the gram-positive bacteria. On the basis of our phylogenetic findings, we propose that M. dentalis should be reclassified as Prevotella dentalis comb. nov.

Phylogenetic evidence derived from a 16S ribosomal DNA analysis indicated that the type strain of Micrococcus agilis, DSM 20550 (= ATCC 966 = CCM 2390), is less closely related to the type species of the genus Micrococcus, Micrococcus luteus, than to the type species of the genus Arthrobacter, Arthrobacter globiformis, and related Arthrobacter species. The phylogenetic position of M. agilis is supported by the presence of peptidoglycan variation A3α and by the presence of MK-9(H2) as the major isoprenolog, a characteristic also found in strains of A. globiformis, Arthrobacter crystallopoietes, Arthrobacter atrocyaneus, Arthrobacter citreus, Arthrobacter aurescens, Arthrobacter ilicis, Arthrobacter ureafaciens, Arthrobacter oxydans, Arthrobacter histidinolovorans, and Arthrobacter nicotinovorans. The last six species and M. agilis are characterized by the presence of threonine in the interpeptide bridge of the peptidoglycan. Threonine has not been found in the peptidogiycans of other Arthrobacter species or in members of the genus Micrococcus. Despite the fact that a morphological life cycle is not known, these data support the proposal that M. agilis should be transferred to the genus Arthrobacter as Arthrobacter agilis comb. nov.

Streptococcus shiloi strains, including the type strain, which were isolated in Israel and the United States, and Streptococcus iniae ATCC 29178T (T = type strain) are phenotypically identical (as determined with API 20 STREP and API 50CH kits; beta-hemolytic on sheep blood agar). DNA-DNA hybridization experiments revealed levels of homology of 77 to 100%. Thus, S. shiloi should be considered a junior synonym of S. iniae. This bacterium is a major fish pathogen that is distributed worldwide.

One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and gas-liquid chromatography of cellular fatty acids were used to determine chemotaxonomic characteristics of strains belonging to Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica. Species-specific protein patterns and fatty acid profiles were found. Only B. bronchiseptica strains contained 12:0 2OH, 18:1 to7c, and 19:0 cyclo, and the presence of 10:0 3OH was a unique feature of B. pertussis and B. parapertussis strains. The latter two species were differentiated by the absence of 17:0 cyclo in B. pertussis strains. These chemotaxonomic data and the multiple differences in the classical phenotypes of these three species do not correlate with the high DNA homology values. Our findings support the present status of the three taxa as different species and emphasize the importance of a polyphasic approach in bacterial classification.

The primary endosymbionts (P-endosymbionts) of tsetse flies (Diptera: Glossinidae) are harbored inside specialized cells (mycetocytes) in the anterior region of the gut, and these specialized cells form a white, U-shaped organelle called mycetome. The P-endosymbionts of five tsetse fly species belonging to the Glossinidae have been characterized morphologically, and their 16S ribosomal DNA sequences have been determined for phylogenetic analysis. These organisms were found to belong to a distinct lineage related to the family Enterobacteriaceae in they subdivision of Proteobacteria, which includes the secondary endosymbionts of various insects and Escherichia coli. These bacteria are also related to the P-endosymbionts of aphids, Buchnera aphidicola. Signature sequences in the 16S ribosomal DNA and genomic organizational differences which distinguish the tsetse fly P-endosymbionts from members of the Enterobacteriaceae and from the genus Buchnera are described in this paper. I propose that the P-endosymbionts of tsetse flies should be classified in a new genus, the genus Wigglesworthia, and a new species, Wigglesworthia glossinidia. The P-endosymbiont found in the mycetocytes of Glossina morsitans morsitans is designated the type strain of this species.

A procedure for preparing actinomycete DNA samples for pulsed-field gel electrophoresis is described. When this method was used, DNAs from strains belonging to the genera Streptomyces, Kutzneria, Dactylosporangium, Microtetraspora, Actinoplanes, Saccharothrix, and Micromonospora were successfully restriction digested with infrequently cutting enzymes, such as Dral and Asel.

Comparative 16S rRNA analysis was used to determine the phylogenetic positions of Catonella morbi and Johnsonella ignava, which are members of two monospecific genera of gram-negative anaerobic bacilli isolated from human gingival crevices. Both of these genera were found to belong to cluster XIVa (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812–826, 1994) of the Clostridium subphylum of gram-positive bacteria. Within this cluster, which contains several Clostridium, Coprococcus, Eubacterium, and Ruminococcus species, C. morbi and J. ignava formed two distinct lines that were separate from all other taxa. Our findings support the separate generic status of the genera Catonella and Johnsonella and show that these genera do not belong to the family Bacteroidaceae but instead belong to the gram-positive Clostridium subphylum.

On the basis of DNA-DNA hybridization data and a dendrogram based on quantitative fatty acid data, we suggested previously (A. Yokota, T. Tamura, T. Hasegawa, and L. H. Huang, Int. J. Syst. Bacteriol. 43:805–812, 1993) that 13 strains belonging to the genus Catenuloplanes could be divided into six groups, including one group represented by Catenuloplanes japonicus N381–16T (T = type strain). The results of a subsequent study of phenotypic characteristics supported this division. In this paper we propose the following five new Catenuloplanes species for the remaining 12 strains: Catenuloplanes niger for strain N406–14T (= IFO 14177T); Catenuloplanes indicus for strains RA327, RA328T (= IFO 15575T), and RA329; Catenuloplanes atrovinosus for strains RA330, RA331, and RA332T (= IFO 15579T); Catenuloplanes castaneus for strains RA336, RA337, RA338, and RA344T (= IFO 15584T); and Catenuloplanes nepalensis for strain RA343T (= IFO 15583T).

We describe a new group (type 3) of the recently proposed species Mycobacterium celatum isolated from eight patients with AIDS in London, England. Sequences of genes coding for 16S rRNA (EMBL accession no. Z46664) showed a divergence of 17 bases from M. celatum type 2 reference isolates and a divergence of 7 bases from M. celatum type 1 reference isolates. A reference strain is available (NCTC 12882).

Newly isolated Xanthobacter strains were characterized. In addition, the fatty acid compositions and quinone systems of gram-negative, yellow-pigmented, hydrogen-oxidizing bacteria belonging to the genera Xanthobacter, Hydrogenophaga, and Variovorax and related species were studied. Xanthobacter strains are nitrogen-fixing organisms that have a Q-10 ubiquinone system; the cellular fatty acids of these organisms include high levels of C18:1 acid, and their hydroxy fatty acids include high levels of 3-OH C16:0 acid. Hydrogenophaga strains are polarly flagellated organisms that have a Q-8 ubiquinone system. These bacteria can be divided into two groups on the basis of cellular fatty acid and hydroxy fatty acid compositions. Variovorax strains are peritrichously flagellated, non-nitrogen-fixing organisms that have a Q-8 ubiquinone system; the cellular fatty acids of these strains include high levels of C16:0, C16:1 and C18:1 acids, and their hydroxy fatty acids include 3-OH C10:0 and 2-OH C14:0 acids. Xanthobacter, Hydrogenophaga, and Variovorax strains can be clearly distinguished from each other on the basis of their quinone systems and cellular fatty acid compositions.