- Volume 46, Issue 3, 1996

A novel proteolytic bacterium was isolated from rumen contents of New Zealand cattle grazing fresh forage and was designated strain B316T (T = type strain). Strain B316T cells were straight to slightly curved rods (width, 0.4 to 0.6 μm; length, 1.3 to 3.0 μm) that were gram-positive and possessed a single subterminal flagellum. This isolate did not produce catalase, indole, ammonia, lipase, or lecithinase or reduce nitrate, but it did produce a curd reaction with milk. Strain B316T was proteolytic, hydrolyzing casein and fraction I leaf protein. The crude proteinase was predominantly the serine type, but some cysteine proteinase and metallo-proteinase activities were also detected. The DNA base composition of strain B316T was 28 mol% G+C. A 16S ribosomal DNA sequence analysis of strain B316T indicated that it was most closely related to a member of clostridial cluster XIVa, viz., Clostridium aminophilum, an amino acid-fermenting organism isolated from the rumen; the similarity value was 92.2%. The results of the phenotypic characterization analysis, G+C content analysis, and phylogenetic analysis of the 16S ribosomal DNA sequence set strain B316T apart from all of the members of cluster XIVa. We propose that strain B316T should be designated a new species of the genus Clostridium, Clostridium proteoclasticum. Strain B316 is the type strain and has been deposited in the American Type Culture Collection as strain ATCC 51982.

Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahigh-temperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16S rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. megaterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16S rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. megaterium group, notably, B. firmus (30%), B. benzoevorans (28%), and B. circulans (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermodurans for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).

In order to determine the phylogenetic structure of the actinomycete family Micromonosporaceae, the 16S ribosomal DNA sequences of 17 type species of the genera Actinoplanes, Dactylosporangium, and Pilimelia were compared with the 16S ribosomal DNA sequences of species of the genera Catellatospora, Catenuloplanes, and Couchioplanes and with those of species of the genus Micromonospora and other actinomycete genera for which the sequences have been previously determined. All genera of the family, together with the genera Catellatospora, Catenuloplanes, and Couchioplanes, form a phylogenetically coherent cluster that is well separated from other families of the order Actinomycetales. Except for one species of the genus Catellatospora, all species cluster according to their genus affiliation. The inclusion of the genera Catellatospora, Catenuloplanes, and Couchioplanes into the family broadens the phenotypic and chemotaxonomic heterogeneity of this taxon. An emendation of the family Micromonosporaceae is given.

By using selective enrichment of polyhydroxyalkanoate-degrading bacteria and poly(3-hydroxyvalerate)-containing granules from Chromobacterium violaceum as the carbon source, 10 new Pseudomonas lemoignei strains were isolated; these strains were able to degrade poly(3-hydroxyvalerate), as well as poly(3-hydroxybutyrate), in vitro. The new isolates were characterized and identified by comparing them with P. lemoignei LMG 2207T (T = type strain). Like P. lemoignei LMG 2207T cells, the cells of the 10 new isolates contained mainly hexadecenoic, hexadecanoic, octadecenoic, and dodecanoic acids, as well as hydroxylated fatty acids, and exhibited respiration in the presence of methylpyruvate, 3-hydroxybutyrate, and 4-hydroxybutyrate, but not in the presence of the 92 other carbon sources included in Biolog GN microplates. The protein patterns of the new isolates were almost identical to each other and very similar to the protein pattern of P. lemoignei LMG 2207T. Some of the new isolates, but not P. lemoignei LMG 2207T, contained megaplasmids that were about 200 kbp long. The 16S ribosomal DNA genes of strain A62, a representative of the 10 new isolates, and of P. lemoignei LMG 2207T exhibited more than 0.99 sequence similarity. The DNA-DNA reassociation value for two representative strains was 100%, and the levels of DNA-DNA reassociation between these strains and the type strain were 60 and 61%. The taxonomy of P. lemoignei is briefly discussed.

Streptococcus dysgalactiae consists of at least five distinct subgroups on the basis of serogroups, biotypes, and hosts. A chemotaxonomic and phenotypic examination of 80 S. dysgalactiae strains representing the known diversity within this species and 49 reference strains representing all members of the streptococcal pyogenic species group revealed two subpopulations of strains within S. dysgalactiae. The name S. dysgalactiae subsp. dysgalactiae is proposed for strains of animal origin. These strains belong to Lancefield serogroups C and L, are alpha-, beta-, or nonhemolytic, and do not exhibit streptokinase activity on human plasminogen or proteolytic activity on human fibrin. The name S. dysgalactiae subsp. equisimilis is proposed for human isolates. These strains belong to Lancefield serogroups C and G, are beta-hemolytic, and exhibit streptokinase activity on human plasminogen and proteolytic activity on human fibrin.

A polyphasic approach was used to determine the relationships between well-characterized reference strains representing all seven Capnocytophaga species. One Centers for Disease Control (CDC) group DF-3 strain, a presumed relative of the genus Capnocytophaga, and 15 field isolates were included as well. Fourteen isolates were assigned to named Capnocytophaga species, all of which could be differentiated by means of whole-organism protein electrophoresis. A separate position was occupied by the CDC group DF-3 strain and by one field isolate representing a novel Capnocytophaga species. The phylogenetic position of each taxon was determined by means of 16S rRNA sequence analysis. A considerable genotypic heterogeneity within the genus Capnocytophaga was detected in spite of the minimal phenotypic differences. Comparative 16S rRNA sequence analysis revealed that CDC group DF-3 is not a close relative of the capnocytophagas but constitutes a separate genus that clusters together with Bacteroides forsythus and Bacteroides distasonis, two generically misclassified Bacteroides species. The degree of protein similarity correlated with our and published DNA-DNA binding values. Percentage 16S rRNA similarity values of greater than 97% did not guarantee conspecificity. All Capnocytophaga strains had very similar fatty acid contents characterized by significant amounts of 14:0, 15:0 iso (greater than 55%), 16:0, 16:0 30H, and 17:0 iso 30H. PCR-mediated DNA fingerprinting allowed discrimination of most species, although some strains could not be classified efficiently because of DNA polymorphisms.

A new coagulase-negative subspecies, Staphylococcus saprophyticus subsp. bovis, is described on the basis of a study of five strains isolated from the anterior nares of cows. This subspecies is differentiated from the other novobiocin-resistant staphylococci by its phenotypic properties, cell wall composition, and levels of genetic relatedness. The type strain of the new subspecies is KV 12 (=CCM 4410).

Spiroplasma sp. strain EA-1T (T = type strain) (subgroup VIII-1), which was isolated from the syrphid fly Eristalis arbustorum, was serologically distinct from other spiroplasma species, groups, and subgroups. The cells of this strain, as revealed by dark-field light microscopy, were short, helical, and motile. An electron microscopic examination revealed wall-less cells delimited by a single membrane. The unusually short cells passed through 220-nm filter pores with no reduction in titer. The organisms grew well in SM-1, MID, and SP-4 liquid media. Growth also occurred in conventional horse serum medium and 1% serum fraction medium. Strain EA-1T grew at temperatures between 10 and 41°C, and optimuum growth occurred at 32°C. The doubling time at the optimal temperature was 1.0 h. The strain catabolized glucose and hydrolyzed arginine but did not hydrolyze urea. The guanine-plus-cytosine content of the DNA was 30 ± 1 mol%. The genome size was about 1,230 kbp. Strain EA-1 (= ATCC 33826), which represents subgroup VIII-1, is designated the type strain of a new species, Spiroplasma syrphidicola.

[Pseudomonas] rubrisubalbicans. a mild plant pathogen, Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates. Our results show that they all belong to the genus Herbaspirillum. In addition to H. seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin. The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities. The type strain of H. rubrisubalbicans is NCPPB 1027 (=LMG 2286): Representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321). It has been confirmed that H. rubrisubalbicans is an endophytic diazotroph. It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae. Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine. Oligonucleotide probes based on partial sequences of the 23S rRNA of H. seropedicae and H. rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

Six strains of coryneform bacteria were isolated from blood samples obtained from healthy cows. Phenotypic and molecular genetic studies showed that these isolates represent a new species of the genus Sanguibacter, for which the name Sanguibacter inulinus is proposed. The type strain of S. inulinus is strain ST-50 (= NCFB 3024).

We describe two species of hyperacidophilic, thermophilic, heterotrophic, aerobic archaea that were isolated from solfataric hydrothermal areas in Hokkaido, Japan. These organisms, Picrophilus oshimae and Picrophilus torridus, represent a novel genus and a novel family, the Picrophilaceae, in the kingdom Euryarchaeota and the order Thermoplasmales. Both of these bacteria are more acidophilic than the genus Thermoplasma since they are able to grow at about pH 0.

The phylogenetic positions of six Vibrio costicola strains were determined by direct sequencing and analysis of their PCR-amplified 16S ribosomal DNAs. A comparative analysis of the sequence data revealed that the moderate halophile V. costicola forms a monophyletic branch that is distinct from other Vibrio species and from moderately halophilic species of other genera. These results complement phenotypic and genotypic data determined previously. The molecular evidence, together with several phenotypic differences, distinguishes V. costicola from species of the genus Vibrio and other species belonging to the gamma subclass of the Proteobacteria and indicates that V. costicola should be placed in a new and separate genus. The name Salinivibrio costicola gen. nov., comb. nov. is proposed for this bacterium. The guanine-plus-cytosine content of the DNA is 49.4 to 50.5 mol%. The type strain of S. costicola is strain NCIMB 701 (= ATCC 33508).

Several strains of a novel heterotrophic gas vacuolate bacterium were isolated from antarctic marine waters. The results of phylogenetic analyses in which 16S ribosomal DNA sequencing was used, coupled with pheno-typic tests, indicated that strain 34-PT (T = type strain) belongs to a new genus and species of the beta subgroup of the Proteobacteria, for which the name Polaromonas vacuolata is proposed. Although the other four strains studied probably belong to this new species, DNA-DNA hybridization tests were not conducted. The closest phylogenetic relatives of P. vacuolata are the photosynthetic nonsulfur purple bacterium Rhodoferax fermentans and the hydrogen autotroph Variovorax paradoxus.

Phylogenetic relationships among species of the genus Ureaplasma were elucidated by analyzing 16S rRNA sequence information. The 16S rRNA genes of six strains of the mammalian Ureaplasma species were amplified by PCR and were sequenced directly by a primer walking method. The phylogenetic tree based on the nucleotide sequence of the 16S rRNA genes corresponded to the evolutionary history of the host animal species.

Two type subspecies names created automatically under Rule 46 are presented.

A rule is proposed to replace Recommendation 30a in the International Code of Nomenclature of Bacteria; this rule makes mandatory the deposition of the type strains of cultivable bacterial species and subspecies in a permanent culture collection so that they are available for study.

The discrepancies between the current description of the CAMP test between Listeria monocytogenes and Rhodococcus equi in the latest edition of Bergey’s Manual of Determinative Bacteriology (L. monocytogenes is described as CAMP test negative with R. equi) and routine findings (positive reactions are usually described in many laboratories) make it advisable to review the current interpretation of the CAMP test to avoid confusion among people working in microbiological laboratories. Overall, 98.4% of the L. monocytogenes strains examined in this study, regardless of their source or the intensity of their hemolytic activity, displayed a synergic hemolytic reaction (CAMP phenomenon) with R. equi, indicating that L. monocytogenes can generally be considered CAMP positive with R. equi. We propose that L. monocytogenes, together with Listeria ivanovii, should be considered CAMP test positive with R. equi (circular or racket and semicircular or shovel shapes, respectively).

The Judicial Commission designated strain B213c (= DSM 20284) as the type strain of Pediococcus acidilactici in place of strain NCDO 1859, which seems to belong in the species Pediococcus pentosaceus.