- Volume 48, Issue 3, 1998

As a continuation of an ongoing study of DNA relatedness among red-spored streptomycetes, the homology between the four species comprising the Streptomyces fulvissimus phenotypic cluster (S. fulvissimus, Streptomyces aureoverticillatus, Streptomyces longispororuber and Streptomyces spectabilis) and the four species comprising the Streptomyces griseoviridis phenotypic cluster (S. griseoviridis, Streptomyces chryseus, Streptomyces daghestonicus and Streptomyces murinus) was measured spectrophotometrically from C0t05 determinations. All strains were also compared to 12 strains representing previously determined DNA-relatedness clusters in the Streptomyces lavendulae phenotypic cluster, including the type strain NRRL B-1230T. The strains segregated into 15 cluster groups at DNA relatedness <80%, including 10 single-membered clusters. S. griseoviridis and S. daghestonicus were synonymous, as were S. chryseus and S. longispororuber. S. spectabilis exhibited species-level homology with strain NRRL B-2402, which had been received as S. lavendulae. S. aureoverticillatus exhibited significant DNA relatedness to Streptomyces flavotricini. None of the strains from the S. fulvissimus or S. griseoviridis clusters exhibited significant homology to the type strain of S. lavendulae.

An alkaliphilic, endospore-forming bacterium isolated from Brazilian soil was taxonomically studied and is proposed as a new Paenibacillus species. This organism (strain 324T) was particularly distinguishable from other Paenibacillus species by its ability to grow optimally at pH 10 and 40 °C. The DNA G+C content was 50-9 mol%. The diamino acid of the cell-wall peptidoglycan was meso-diaminopimelic acid. MK-7 was the predominant menaquinone and anteiso-C15:0 was the major fatty acid. Levels of 16S rDNA similarity between strain 324Tand other Paenibacillus species were 90-6-95-9%. Phylogenetically, strain 324Tformed an evolutionary lineage distinct from other species within the evolutionary radiation encompassing the genus Paenibacillus. Based on phenotypic and chemotaxonomic properties, and phylogenetic inference, it is proposed that strain 324Tshould be placed in the genus Paenibacillus as a new species, Paenibacillus campinasensis. The type strain of the new species is strain 324T(= KCTC 0364BPT).

Rickettsiae are classified in the order Rickettsiales and have been included in the α subclass of the class Proteobacteria on the basis of 16S rRNA gene sequence comparison. To estimate the evolutionary forces that have shaped the members of the spotted fever group (SFG) rickettsiae, the ompA gene (apart from the tandem repeat units), encoding an antigenic high-molecularmass membrane protein specific for the group, was amplified and sequenced from 21 isolates. The phylogenetic relationships between SFG rickettsiae were inferred from the comparison of both the gene and derived protein sequences, using the parsimony, neighbour-joining and maximum-likelihood methods. Three strongly supported phylogenetic sub-groups were distinguished: first, the Rickettsia conorii complex (R. conorii Malish, R. conorii M1, R. conorii Moroccan, R. conorii Indian tick typhus, Astrakhan fever rickettsia and Israeli tick typhus rickettsia); second, a cluster including Rickettsia africae, strain S, Rickettsia parkeri, Rickettsia sibirica and ‘Rickettsia mongolotimonae’; and, third, a cluster including Rickettsia aeschlimannii, Rickettsia rhipicephali, Rickettsia massiliae, Bar 29 and Rickettsia montanensis. Rickettsia rickettsii, Rickettsia japonica, Rickettsia slovaca and Thai tick typhus rickettsia did not cluster with any other Rickettsia species. To test whether positive selection was responsible for sequences diversity, rates of synonymous and nonsynonymous nucleotide substitutions were compared for Rickettsia ompA alleles and indicated that this gene is undergoing neutral evolution.

Pathogenic leptospires can be causative agents of reproductive problems in pigs. Cultures of uteri and kidneys from two pig herds in New South Wales and Victoria (Australia) yielded five strains identified as Leptospira on morphological and cultural grounds. Phenotypic characteristics (growth at 13 and 30°C, growth in the presence of 8-azaguanine) were intermediate between those of pathogenic and saprophytic leptospires. No cross-agglutination was observed with reference antisera representing the 24 pathogenic serogroups and the main saprophytic ones. Antiserum against one of the strains did not agglutinate reference strains representative of any serogroup. This provided evidence of a new serovar, designated hurstbridge. Genomic characterization of the five strains was achieved using five molecular approaches. Mapped restriction site polymorphisms in the rrs (16S rRNA) gene were not related to those of any reference strains. Arbitrarily primed PCR fingerprints suggested clonality of the five strains. The strains all showed an identical and unique PFGE profile. PCR, using primers specific for the rrs gene of pathogenic leptospires, amplified corresponding sequences from the strains. DNA-DNA hybridization (and reciprocal experiments) using the S1 nuclease/TCA method was performed between one of the strains and the reference strains of Leptospira species. The homology ranged from 0 to 36% (the latter being with Leptospira inadai) thus satisfying the criterion of a new species, Leptospira fainei (type strain BUT 67). Phylogenetic analysis of 16S rRNA sequences showed that L. fainei and L. inadai formed a clade separate from the previously recognized ‘prophyt’ and ‘pathoge’ clades.

Four species of the newly proposed genus Macrococcus, namely Macrococcus caseolyticus gen. nov., comb. nov. (formerly Staphylococcus caseolyticus Schleifer, Kilpper-Bälz, Fischer, Faller and Endl 1982, 19VP), Macrococcus equipercicus sp. nov., Macrococcus bovicus sp. nov. and Macrococcus carouselicus sp. nov., are described on the basis of a phylogenetic analysis comparing 16S rRNA sequences, DNA-DNA liquid hybridization, DNA base composition, normalized ribotype patterns, macrorestriction pattern analysis and estimation of genome size using PFGE, cell wall composition, phenotypic characteristics and plasmid profiles. Compared with their closest relatives, members of the genus Staphylococcus, these organisms demonstrated significantly lower 16S rRNA sequence similarities (93.4—95.3%), higher DNA G+C content (38—45 mol%), absence of cell wall teichoic acids (with the possible exception of M. caseolyticus), unique ribotype pattern types and macrorestriction patterns, smaller genome size (approx. 1500—1800 kb) and generally larger Gram-stained cell size (1.1—2.5 μm in diameter). Macrococci can be distinguished from most species of staphylococci (except Staphylococcus sciuri, Staphylococcus vitulus and Staphylococcus lentus) by their oxidase activity. The four Macrococcus species can be distinguished from one another on the basis of DNA-DNA hybridization, ribotype pattern types, macrorestriction patterns and their phenotypic properties, including colony morphology, cell morphology, haemolysins, Staph Latex agglutination, acid production from a variety of carbohydrates, acetoin production, nitrate reduction, aesculin hydrolysis, and DNase and urease activities. The type species is M. equipercicus. The type strains of M. equipercicus, M. caseolyticus, M. bovicus and M. carouselicus are ATCC 51831T(= DD 9350T), ATCC 13548T(= TDD 4508T) (Schleifer et al. 1982), ATCC 51825T(= DD 4516T) and ATCC 51828T(= DD 9348T), respectively.

Three strains of novel, extremely thermophilic, rod-shaped crenarchaeotes were isolated from acidic hot spring areas in Japan. Cells of the three strains were straight or slightly curved rods and occasionally branched out singly or extensively, or had spherical bodies protruding at the ends of the cells. They were heterotrophs that grew anaerobically or microaerobically. The presence of CO2 in the gas phase, archaeal cell-extracts and a vitamin mixture stimulated growth of the strains. Growth occurred at 45-82 °C and pH 2.6-5.9 and was optimal around 75 °C and pH 4.0. The strains utilized glycogen, starch, gelatin and various proteinaceous complex compounds as carbon sources. They required sulfur, thiosulfate or L-cystine as possible electron acceptors. The lipids mainly consisted of various cyclic glycerol-bisdiphytanyl-glycerol tetraethers. The G+C contents of the genomic DNAs were 52 mol%. Comparison of the 16S rDNA sequences indicated that they belonged to a separate lineage in the family Thermoproteaceae. The three strains were included in a single species due to high levels of DNA-DNA hybridization values. Based upon these results, the new isolates were assigned to a new genus and species in the family Thermoproteaceae, Thermocladium modestius gen. nov., sp. nov. The type strain is Thermocladium modestius IC-125T(= JCM 10088T).

Bacterial strain T1, originally isolated by P. J. Evans on the basis of its capacity for toluene degradation under denitrifying conditions, has been classified as Thauera aromatica. In a comprehensive study of strains of this species, it was found that the cells have a different type of flagellar insertion from that of cells of the type species of the genus, Thauera selenatis, suggesting the convenience of an emendation of the description of the genus Thauera. Further studies on a larger collection of strains with the above characteristics may serve in the future as the basis for the creation of a new generic designation.

For the rapid identification of Nocardioides strains, multiplex PCR, using 16S rDNA as target gene, was used and its value was evaluated. Forward primers specific for Nocardioides albus, Nocardioides jensenii, Nocardioides plantarum and Nocardioides simplex, among the five validly described Nocardioides species, were designed from the alignment of 16S rDNA sequences. Nocardioides luteus has been shown to be a member of the same species as N. albus by recent molecular systematic studies and preliminary DNA-DNA relatedness tests. Therefore, N. albus and N. luteus were considered as members of the same species in this study. Each primer was found to be species-specific by specificity testing. N. albus NSP01T, N. jensenii NSP19T, N. plantarum NSP21Tand N. simplex NSP22Tcould be clearly differentiated by PCR products characteristic for each species in the multiplex PCR assay. N. luteus gave an identical result to N. albus NSP01T. The additional 17 strains of N. albus and the additional four strains of N. simplex gave PCR products identical to those of N. albus NSP01Tand N. simplex NSP22T, respectively. Multiplex PCR was found to be rapid, species-specific and reproducible. The technique evaluated in this study proved to be effective for rapidly identifying Nocardioides strains to species level.

An actinomycete strain, ‘Nocardia flavorosea’ JCM 3332, was found to have properties consistent with its classification in the genus Nocardia. An almost complete gene sequence of the 16S rDNA of the strain was determined following cloning and sequencing of the amplified gene. The sequence was aligned with those available for nocardiae and phylogenetic trees were inferred using four tree-making algorithms. The organisms consistently formed a distinct clade with the type strain of Nocardia carnea. However, DNA relatedness experiments showed that the strain and N. carnea DSM 43397Tbelonged to two distinct genomic species. The organism was also distinguished from representatives of all of the validly described species of Nocardia using a combination of phenotypic properties. These genotypic and phenotypic data show that the strain merits recognition as a new species of the genus Nocardia. The name proposed for the new species is Nocardia flavorosea sp. nov. The type strain is JCM 3332T.

The taxonomic position of bacterial strain 141T, isolated from the mangrove rhizosphere, has been clarified by phenotypic, chemotaxonomic and phylogenetic studies. The strain possesses wall chemotype IV, MK-9(H2) as the predominant menaquinone, relatively long-chain mycolic acids (56-64 carbon atoms) and straight-chain saturated and monounsaturated fatty acids with a small amount of tuberculostearic acid. The G+C content of the DNA is 66.8 mol%. Similarity values for genes encoding 16S rRNA indicated that strain 141Trepresents a new species within the genus Gordonia for which the name Gordonia rhizosphera sp. nov. is proposed. The type strain of G. rhizosphera is 141T(= IFO 16068T).

An autotrophic, extremely thermophilic methanogen (MET) was isolated from a deep-sea hydrothermal chimney sample collected on the Mid-Atlantic Ridge at a depth of 3000 m. The heavily flagellated cells are motile and coccoid shaped. The new strain grows between 55 and 91 °C, with an optimum growth temperature at 85 °C. The optimum pH for growth is 6.5, and the optimum sea salt concentration for growth is around 25 g I-1. The organism uses H2 and CO2 as the only substrate for growth and methane production. Tungsten, selenium and yeast extract stimulate growth significantly. In the presence of CO2 and H2, the organism reduces elemental sulphur to hydrogen sulphide. The G+C content of the genomic DNA is 33 mol%. As determined by 16S gene sequence analysis, this organism is closely related to Methanococcus jannaschii strain JAL-1T. However, no significant homology was observed between them with DNA-DNA hybridization. It is proposed that this organism should be placed in a new species, Methanococcus infernus. The type strain is MET(-DSM 11812T.

Taxonomic studies were performed on eight strains of α-haemolytic streptococci that showed very low DNA-DNA hybridization similarity values with all established members of the mitis group of the genus Streptococcus. These strains were isolated from the tooth surface and pharynx of humans. 16S rRNA gene sequence analysis showed that these strains belonged to the mitis group, but that they fell into two new branches. DNA-DNA hybridization demonstrated two new similarity groups. From the results of the present study, the names Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov. are proposed for these new groups. The type strains are 0-66T(= GTC 848T= JCM 10158T) and 0-122T(= GTC 849T= JCM 10157T), respectively.

Phenotypic and phylogenetic studies were performed on a Gram-negative, rod-shaped bacterium isolated from three porpoises. Biochemical and physiological studies indicated that the bacterium was related to the family Pasteurellaceae. Comparative 16S rRNA gene sequencing studies confirmed these findings and demonstrated that the bacterium represents a hitherto unknown subline. The nearest phylogenetic relative of the unknown bacterium was Actinobacillus delphinicola, an organism also originating from sea mammals, although a sequence divergence of 3% demonstrated that the newly isolated bacterium is a distinct species. On the basis of the results of the phylogenetic analysis and phenotypic criteria, it is proposed that the bacterium should be classified as a new species, Actinobacillus scotiae sp. nov. The type strain of Actinobacillus scotiae sp. nov. is NCTC 12922T(= M2000/95/1T).

Two strains of Acetobacter sp., LTH 2460Tand LTH 2458T, have been isolated from running red wine and cider vinegar fermentations, respectively. Taxonomic characteristics of the isolates were investigated. Comparative analysis of the 16S rRNA sequences revealed < 99% similarity between strain LTH 2460Tand the type strains of the related species Acetobacter europaeus and Acetobacter xylinus and between strain LTH 2458Tand Acetobacter pasteurianus. On the other hand, low levels of DNA relatedness (> 34%) were determined in DNA-DNA similarity studies. This relatedness below the species level was consistent with specific physiological characteristics permitting clear identification of these strains within established species of acetic acid bacteria. Based on these results, the names Acetobacter oboediens sp. nov. and Acetobacter pomorum sp. nov. are proposed for strains LTH 2460Tand LTH 2458T, respectively. The phylogenetic positions of the new species are reflected by a 16S rRNA-based tree. Furthermore, a 16S rRNA-targeted oligonucleotide probe specific for A. oboediens was constructed.

DNA extracted from three papaya (Carica papaya L.) plants, individually affected by dieback, yellow crinkle or mosaic diseases, was subjected to PCR using phytoplasma-specific primers to amplify the 16S rRNA gene plus 16S-23S rRNA intergenic spacer region. Near-complete DNA sequences obtained for the three PCR amplimers were subjected to phylogenetic analyses and direct sequence comparison with other phytoplasma 16S rDNA and 16S-23S spacer region DNA sequences. The papaya yellow crinkle (PpYC) and papaya mosaic (PpM) sequences were identical to each other, but distinctly different from the papaya dieback (PpDB) sequence, showing 90.3% identity in the 16S rDNA and 87.8% identity in the 16S-23S spacer region DNA sequences. A phylogenetic tree based on 16S rDNA sequences was calculated, in which PpYC and PpM are most closely related to the tomato big bud phytoplasma (TBB; 99.7% 16S rDNA sequence identity) from Australia, within subclade iii. This subclade consists of strains only reported occurring in the Southern Asian region and Australia, which indicates an Asian/Australasian origin. PpDB is most closely related to the Phormium yellow leaf phytoplasma from New Zealand (PYL; 99.9% identity) and the Australian grapevine yellows phytoplasma (AGY; 99.7% identity). These three phytoplasma strains form a distinct clade within subclade xii, which also includes the European strains STOL and VK as another distinct clade. The origin of the closely related but geographically separated AGY-like strains and STOL-like strains of subclade xii is unclear. It is proposed that phytoplasma strains PpDB, PYL and AGY be included in the previously described taxon ‘Candidatus Phytoplasma australiense’, and that PpYC PpM and TBB be assigned to a new taxon, ‘Candidatus Phytoplasma australasia’.

A strictly anaerobic, nitrate-reducing bacterium, strain LuFRes1, was isolated using resorcinol as sole source of carbon and energy. The strain reduced nitrate to dinitrogen gas and was not able to use oxygen as an alternative electron acceptor. Cells were catalase-negative but superoxide-dismutase-positive. Resorcinol was completely oxidized to CO2.16S rRNA sequence analysis revealed a high similarity with sequences of Azoarcus evansii and Azoarcus tolulyticus. Strain LuFRes1T(= DSM 12081T) is described as a new species of the genus Azoarcus, Azoarcus anaerobius.

Complete nucleotide sequences of the 16S rDNAs were determined from Thiocapsa and Amoebobacter species, including all available type strains and some additional isolates. The distance-matrix analysis and the dendrogram for estimating the genetic relationships revealed that the investigated strains were found in two major clusters within the Chromatiaceae. One cluster comprises all Amoebobacter species, Thiocapsa roseopersicina and several isolates related to Thiocapsa roseopersicina. Representatives of the species Amoebobacter roseus, Amoebobacter pendens and Thiocapsa roseopersicina, the so called ‘Thiocapsa roseopersicina group’, are very closely related, justifying their inclusion into one genus, Thiocapsa, for which an emended description is presented. Amoebobacter purpureus and Amoebobacter pedioformis formed two separate lines of descent with less than 93% (89.6-92.9%) similarity to strains of the ‘Thiocapsa roseopersicina group’. Therefore, they will be considered as two separate genera. As a consequence, an emended description is presented for the genus Amoebobacter, with Amoebobacter purpureus as the new type species and A. pedioformis is transferred to Thiolamprovum pedioforme gen. nov., comb. nov. Two species, Thiocapsa pfennigii and Thiocapsa halophila, which have been classified with the genus Thiocapsa because of their morphological properties, were found within another major cluster of the Chromatiaceae and are only distantly phylogenetically related to the first cluster with 88.4-90.6% and 90.4-92.2% sequence similarity, respectively.

A new bacterial species belonging to the genus Shewanella is described on the basis of phenotypic characterization and sequence analysis of its 16S rRNA-encoding and gyrase B (gyrB) genes. This organism, isolated from shallowwater marine sediments derived from the Amazon River delta, is a Gramnegative, motile, polarly flagellated, facultatively anaerobic rod-shaped eubacterium and has a G+C content of 51.7 mol%. Strain SB2BTis exceptionally active in the anaerobic reduction of iron, manganese and sulfur compounds. SB2BTgrows optimally at 35 °C, with 1-3% NaCl and over a pH range of 7-8. Analysis of the 16S rDNA sequence revealed a clear affiliation between strain SB2BTand members of the gamma subclass of the class Proteobacteria. High similarity values were found with certain members of the genus Shewanella, especially with Shewanella putrefaciens, and this was supported by cellular fatty acid profiles and phenotypic characterization. DNA-DNA hybridization between strain SB2BTand its phylogenetically closest relatives revealed low similarity values (24.6-42.7%) which indicated species status for strain SB2BT. That SB2BTrepresents a distinct bacterial species within the genus Shewanella is also supported by gyrB sequence analysis. Considering the source of the isolate, the name Shewanella amazonensis sp. nov. is proposed and strain SB2BT(= ATCC 700329T) is designated as the type strain.

Reference strains, including two mis-named organisms, ‘Chromobacterium chocolatum’ and Flavobacterium marinotypicum. isolates from soil and clinical specimens, all previously recognized as Aureobacterium or Microbacterium, were characterized taxonomically. On the basis of morphological, physiological and chemotaxonomic characteristics, as well as DNA-DNA hybridization data, six new species and one new combination are proposed in the genus Microbacterium: Microbacterium ketosireducens sp. nov. (type strain IFO 14548T), Microbacterium chocolatum sp. nov. (type strain IFO 3758T), Microbacterium aurantiacum sp. nov. (type strain IFO 15234T), Microbacterium hominis sp. nov. (type strain IFO 15708T), Microbacterium thalassium sp. nov. (type strain IFO 16060T), Microbacterium halophilum sp. nov. (type strain IFO 16062T) and Microbacterium maritypicum comb. nov. (type strain IFO 15779T).