- Volume 45, Issue 4, 1995

Strains of Thermus silvanus sp. nov. and strains of Thermus chliarophilus sp. nov. were isolated from the hot spring at Vizela in northern Portugal and the hot spring at Alcafache in central Portugal, respectively. The strains of T. silvanus produce orange-red-pigmented colonies and have an optimum growth temperature of about 55°C, while the strains of T. chliarophilus produce yellow-pigmented colonies and have an optimum growth temperature of about 50°C. The strains of both species are catalase negative. These species can be distinguished from each other and from Thermus ruber by biochemical characteristics, fatty acid composition data, and 16S rRNA gene sequence data. Our phylogenetic analysis showed that strains VI-R2T (T = type strain) and ALT-8T belong to the T. ruber line of descent. The type strain of T. silvanus is strain VI-R2 (= DSM 9946), and the type strain of T. chliarophilus is strain ALT-8 (= DSM 9957).

The genetic diversity of chickpea strains was studied by using 30 isolates obtained from nodules on chickpeas growing in uninoculated fields over a wide geographic range. The following taxonomic approaches were used: DNA-DNA relatedness analysis, restriction fragment length polymorphism analysis of the amplified 16S ribosomal DNA (rDNA) intergenic spacer (IGS), and total 16S rRNA sequence analysis. The division of chickpea-infective strains into two major phylogenetic groups (groups A and B) that has been described previously was confirmed by the polymorphism of the 16S IGS rDNA. We identified a total of five genomic species, including the previously described species Rhizobium ciceri. All of the group B strains except one were homogeneous and belonged to a single genomic species corresponding to R. ciceri. Group A was heterogeneous, containing three genomic species and five strains that remained unclassified, and its members had very different PCR restriction fragment length polymorphism profiles. The complete 16S rRNA sequences of strains representing the two major groups. R. ciceri UPM-Ca7T (T = type strain) and genomic species 2 strain UPM-Ca36T, exhibited 19 mismatches. Both of these strains belonged to the Rhizobium loti-Rhizobium huakuii branch; R. ciceri UPM-Ca7T was closely related to R. loti, and strain UPM-Ca36T was clearly separated from R. ciceri and closely related to R. huakuii. Thus, genomic species 2 could be distinguished from R. ciceri by its 16S rRNA sequence, by DNA relatedness data, by the polymorphism of the 16S IGS rDNAs, and by previously described multilocus enzyme electrophoresis results and phenotypic characteristics. Therefore, we propose that strains belonging to genomic species 2 should be classified in a new species, Rhizobium mediterraneum, and that strain UPM-Ca36 should be the type strain.

The 16S rRNA gene sequences of eight Cellulomonas species, two Promicromonospora species, and Jonesia denitrificans were determined, and these sequences were compared with the sequences of about 50 representatives of the Arthrobacter line of descent in the order Actinomycetales. We found that in spite of its current assignment to the family Cellulomonadaceae, J. denitrificans branches outside the radiation of this taxon and cannot be considered a member of it. The two Promicromonospora species do not cluster separately from Cellulomonas species and are more closely related to Cellulomonas species than to each other.

The phylogenetic relationships among 30 mycolic acid-containing wall chemotype IV actinomycete strains and 12 strains belonging to allied taxa were examined by determining the amino acid sequences of the ribosomal AT-L30 proteins of these organisms. Sequencing 20 N-terminal amino acids of AT-L30 preparations revealed that the members of the genera containing mycolic acid-containing actinomycetes form two clusters; the first cluster contains the genera Nocardia, Rhodococcus, Gordona, and Tsukamurella, and the second cluster contains the genera Corynebacterium and Mycobacterium. The genus Nocardia was placed in a clade containing the genus Rhodococcus. The data showed that Tsukamurella paurometabolum is closely related phylogenetically to the genus Gordona. The phylogenetic clusters identified were entirely consistent with the proposal of Goodfellow that the family Nocardiaceae should encompass the mycolate-containing, cell wall type IV actinomycete genera Nocardia, Rhodococcus, Gordona, and Tsukamurella. The genera Actinomyces and Micrococcus exhibited AT-L30 amino acid sequence characteristics intermediate between those of actinomycetes and those of typical eubacteria. The genera Nocardia, Gordona, Mycobacterium, Actinoplanes, and Micromonospora were each a taxon that consisted of phylogenetically coherent species. In contrast, the genera Rhodococcus and Corynebacterium are taxa that consist of phylogenetically distantly related species. In general, my results are consistent with previous 16S rRNA sequencing results, but significant differences were also found. My data, together with previous AT-L30 sequencing data, show that phylogenetic relationships among taxa can be determined by using markers other than the ribosomal gene sequences.

A polyphasic study in which we performed an amplified ribosomal DNA restriction analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins, a gas chromatographic analysis of methylated fatty acids, pyrolysis mass spectrometry, a random amplified polymorphic DNA analysis, a phenotypic analysis, and an analysis of the levels of DNA binding of Paenibacillus gordonae and Paenibacillus validus strains (including both type strains) showed that these organisms form a homogeneous group and that the names P. gordonae and P. validus are therefore synonyms. P. validus has nomenclatural priority, and an emended description of this species is given; the type strain is strain LMG 11161 (= ATCC 43897).

A total of 17 Lactobacillus plantarum strains that originated from different environments and 24 reference strains were classified by performing a restriction endonuclease analysis of total chromosomal DNAs digested with Eco RI, Hind III, and Cla I, and the resulting patterns were visualized after the fragments were separated according to size by agarose gel electrophoresis. The patterns were analyzed by using the Pearson product moment correlation coefficient and the unweighted pair group algorithm with arithmetic averages. All but two L. plantarum strains formed a cluster that was separated from the reference strains at a similarity level of 29% (cluster 1). The two remaining L. plantarum strains (cluster 2) merged with cluster 1 at a level of similarity of 28%. The reference strains formed four additional clusters, and four reference strains were stragglers. Cluster 3 (three Lactobacillus pentosus strains) and cluster 4 (including Pediococcus acidilacti CCUG 32235T [T = type strain] and Lactobacillus fermentum ATCC 14931T) merged with cluster 1 at a level of similarity of 25%. Cluster 5 comprised and Lactobacillus reuteri strains, and cluster 6 contained the type strains of Lactobacillus amylovorus, Lactobacillus gasseri, and Lactobacillus vaginalis. Cluster 1 (L. plantarum) was divided into three subclusters, and this subdivision reflected to some extent certain phenotypic features of presumed ecological significance, including the ability to adhere to intestinal mucosa (subcluster 1c) and starch and glycogen degradation (subcluster la). A principal-component analysis significantly distinguished the strains belonging to different species. Also, the subcluster 1c strains could be separated from the rest of the L. plantarum strains. The results of restriction endonuclease analysis of total chromosomal DNA were found to be reproducible, and this method can be used to (i) differentiate between similar strains belonging to the same Lactobacillus species and group strains within a species, (ii) distinguish between strains of different Lactobacillus species, and (iii) place strains in specific Lactobacillus species.

Two strains of thermophilic photosynthetic bacteria, designated MD-66T (T = type strain) and YI-9, were isolated from bacterial mats in two separate hot springs in Japan. These new isolates were phenotypically similar to Chloroflexus aurantiacus in some respects. They were thermophilic filamentous photosynthetic bacteria that grew well at 55°C either anaerobically as photoheterotrophs or aerobically as chemoheterotrophs. They exhibited gliding motility, produced bacteriochlorophylls a and c, contained chlorosomes, and required thiamine and folic acid as growth factors. However, isolates MD-66T and YI-9 had the ability to rapidly form mat-like dense aggregates of filaments, an ability which has not been observed in any C. aurantiacus strain. Carbon source utilization tests revealed that unlike C. aurantiacus, the new isolates did not utilize acetate, citrate, ethanol, or glycylglycine. An analysis of the carotenoid components revealed that isolates MD-66T and YI-9 contained mainly γ-carotene and OH-γ-carotene glucoside fatty acid esters. These isolates also contained only trace amounts of β-carotene, which is a major carotenoid component (28.4% of the total carotenoids) in C. aurantiacus. The results of DNA hybridization studies suggested that the new strains were genetically distinct from C. aurantiacus (levels of similarity, 9 to 18%), and 16S rRNA sequence comparisons showed that strain MD-66T was related to C. aurantiacus at a similarity level of 92.8%. On the basis of our data, we propose that a new Chloroflexus species should be created for our new isolates; the name of this new species is Chloroflexus aggregans, and the type strain is strain MD-66 (= DSM 9485).

The results of a phylogenetic and chemotaxonomic analysis of the genus Micrococcus indicated that it is significantly heterogeneous. Except for Micrococcus lylae, no species groups phylogenetically with the type species of the genus, Micrococcus luteus. The other members of the genus form three separate phylogenetic lines which on the basis of chemotaxonomic properties can be assigned to four genera. These genera are the genus Kocuria gen. nov. for Micrococcus roseus, Micrococcus varians, and Micrococcus kristinae, described as Kocuria rosea comb. nov., Kocuria varians comb. nov., and Kocuria kristinae comb. nov., respectively; the genus Nesterenkonia gen. nov. for Micrococcus halobius, described as Nesterenkonia halobia comb. nov.; the genus Kytococcus gen. nov. for Micrococcus nishinomiyaensis, described as Kytococcus nishinomiyaensis comb. nov.; and the genus Dermacoccus gen. nov. for Micrococcus sedentarius, described as Dermacoccus sedentarius comb. nov. M. luteus and M. lylae, which are closely related phylogenetically but differ in some chemotaxonomic properties, are the only species that remain in the genus Micrococcus Cohn 1872. An emended description of the genus Micrococcus is given.

We performed a systematic study of 55 solvent-producing clostridial strains, the majority of which are currently classified as Clostridium acetobutylicum strains, by using a combination of biotyping and DNA fingerprint analysis. The biotyping procedures used included rifampin susceptibility testing, bacteriocin typing, and bacteriophage typing. The 55 strains examined exhibited a good correlation between their biotypes and DNA fingerprints, which allowed us to divide them into nine groups. The DNA fingerprints of the nine groups differed markedly, but within each group the DNA fingerprints exhibited a high level of similarity. To determine the phylogenetic relationships of the nine groups, we performed a 16S rRNA gene sequence analysis. The results of a comparative analysis of the partial sequence corresponding to positions 830 to 1383 (Escherichia coli numbering) of the 16S rRNA gene indicated that the nine biotype groups could be assembled into four taxonomic groups. The complete 16S rRNA sequences of strains representing these groups were determined. Our phylogenetic analysis revealed that the amylolytic type strain C. acetobutylicum ATCC 824 (taxonomic group I) was only distantly related to the saccharolytic strains belonging to taxonomic groups II, III, and IV (levels of sequence similarity, 90 to 90.5%). The strains belonging to taxonomic groups II, III, and IV, represented by C. acetobutylicum NCP 262, “Clostridium saccharoperbutylacetonicum” N1-4, and C. acetobutylicum NCIMB 8052T (T = type strain), respectively, were closely related (levels of sequence similarity, 98.2 to 98.9%). C. acetobutylicum NCIMB 8052T exhibited a level of similarity of 100% with the type strain of Clostridium beijerinckii. Reclassification of the saccharolytic solvent-producing strains is necessary, and possible names for the four taxonomic groups are discussed.

Seventeen strains of extra-slowly growing (ESG) soybean rhizobia isolated from root nodules of Glycine soja and Glycine max growing in five provinces (Liaoning, Heilongjiang, Shanxi, Hubei, and Anhui) in the People’s Republic of China were compared with 48 reference strains belonging to the genera Bradyrhizobium, Rhizobium, and Agrobacterium by performing a numerical analysis of 191 phenotypic features. Our results showed that all of the ESG strains examined clustered closely in the genus Bradyrhizobium but were separated from Bradyrhizobiumjaponicum at the species level and that they could be differentiated from Rhizobium and Agrobacterium species at the genus level. On the basis of the results of our numerical taxonomy analysis, a genomic DNA G+C content analysis, DNA-DNA hybridization experiments, a partial 16S rRNA sequence analysis, a serological analysis, an N and C content analysis, and an N/C ratio analysis of members of the three groups of soybean rhizobia, we propose the name Bradyrhizobium liaoningense sp. nov. for the ESG strains; the type strain of this species is strain 2281.

The phylogenetic positions of the moderately halophilic bacteria Chromohalobacter marismortui, Volcaniella eurihalina, and Deleya salina were determined by PCR amplification of rRNA genes and direct sequencing. The resulting data were compared with data for other bacteria obtained from 16S rRNA sequence databases. C. marismortui, V. eurihalina, and D. salina clustered phylogenetically within the gamma subclass of the Proteobacteria and are closely related to other species on the Halomonas-Deleya branch. C. marismortui belongs in the family Halomonadaceae and has the characteristic 16S rRNA signatures defined for this family, including the distinctive cytosine residue at position 486 found in all members of the Halomonadaceae. V. eurihalina is closely related to the type species of the genus Halomonas, Halomonas elongata, and we formally propose that V. eurihalina should be transferred to the genus Halomonas as Halomonas eurihalina comb. nov. The type strain of this species is strain F9-6 (= ATCC 49336). D. salina is not as closely related to other species belonging to the Halomonas-Deleya complex, but is more closely related to Halomonas elongata than to Deleya aquamarina, the type species of the genus Deleya. A polyphasic approach will be necessary to determine the natural taxonomic positions of the species belonging to the genera Halomonas and Deleya, as well as C. marismortui, V. eurihalina, Halovibrio variabilis, and Paracoccus halodenitrificans.

We describe a new species of the genus Actinomadura for four nitrite-producing strains which were isolated from experimental biofilters that had been supplied with ammonia. The whole cells of these strains contain meso-diaminopimelic acid, ribose, madurose, glucose, and galactose. The polar lipids include diphosphatidylglycerol, phosphatidylinositol, and phosphatidylinositol mannosides. The main menaquinone is MK-9(H6). The fatty acids include hexadecanoic acid and 10-methyloctadecanoic acid. The guanine-plus-cytosine content of the DNA is 74 mol%. The fatty acid composition, morphology, and physiological reaction profiles of these strains differentiate them from previously described species. The name proposed for the new species is Actinomadura nitritigenes.

The 16S rRNA gene sequences of 30 strains representing 23 validated Corynebacterium species and 7 currently non-valid Corynebacterium species were determined. These sequences were aligned with the sequences of other Corynebacterium species and related actinomycete taxa. A comparative sequence analysis revealed that there is considerable phylogenetic depth and internal structure in the genus Corynebacterium. Turicella otitidis and the amycolate species Corynebacterium amycolatum were located at the periphery of the genus Corynebacterium. It was evident that the species of the genus Corynebacterium form a monophyletic association and, together with other chemotype IV and mycolic acid-containing taxa (including the genera Dietzia, Gordona, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella), form a natural suprageneric group.

The genomes of various Spiroplasma citri strains were digested with several restriction enzymes, and the fragments were analyzed by pulsed-field gel electrophoresis. Polymorphism of the restriction patterns was observed. The genome sizes of the strains obtained when we added the restriction fragment sizes ranged from 1,650 to 1,910 kbp. Physical and genetic maps of 12 strains were constructed by using several DNA probes as genetic markers. The relative positions of mapped loci were conserved in most of the strains; the main differences were differences in the order and number of restriction sites and differences in the sizes of certain fragments. The distribution of viral sequences, which occur at multiple sites in the S. citri genome and are homologous to the sequences of S. citri virus SpV1 strains R8A2B and S102, was also studied.

Recently, Centers for Disease Control and Prevention coryneform group ANF-1 bacteria were described as Corynebacterium afermentans, and group ANF-1-like bacteria were described as Turicella otitidis. Over a 1.5-year period 10 strains of a previously undescribed, gram-positive, rod-shaped organism that was not partially acid fast and resembled ANF-1-like bacteria were isolated from different pediatric patients with ear infections. These previously undescribed coryneform bacteria exhibited a distinct colony morphology and consistency, had a carbon source utilization pattern distinct from the carbon source utilization patterns of C. afermentans and T. otitidis, had a cell wall based on meso-diaminopimelic acid, contained mycolic acids, and had DNA G+C contents of 68 to 74 mol%. A 16S rRNA gene sequence analysis revealed that these clinical isolates are members of the genus Corynebacterium and that they are distinct from C. afermentans and T. otitidis. On the basis of phenotypic and phylogenetic evidence we propose a new species, Corynebacterium auris, for these Centers for Disease Control and Prevention coryneform group ANF-1-like bacteria. The type strain is strain DSM 44122 (CCUG 33426).

We determined almost complete small-subunit ribosomal DNA sequences of 50 reference strains belonging to the genera Corynebacterium, Rhodococcus, and Gordona and compared these sequences with previously published sequences. Three phylogenetic methods (the neighbor-joining, maximum-likelihood, and maximum-parsimony methods), as well as a bootstrap analysis, were used to assess the robustness of each topology which we obtained. The results of comparative phylogenetic analyses confirmed that the genera Corynebacterium, Dietzia, Gordona, Mycobacterium, Nocardia, Tsukarmurella, and Turicella form a monophyletic taxon within the phylum containing the high-G+C-content gram-positive bacteria. The genus Corynebacterium appeared to be a monophyletic unit whose members could be divided into four major clusters. The validity of the genus Turicella is doubtful since members of this genus clearly belong to the genus Corynebacterium. The variability of chemotaxonomic characteristics within the genus Corynebacterium suggests that small-subunit ribosomal DNA sequence analysis is probably the most straightforward method for confirming that a bacterium belongs to this genus.

A novel extremely halophilic archaeon was isolated from the Dead Sea. This isolate is rod shaped and, like Halobacterium sodomense, requires a relatively low level of sodium ions for growth and a very high level of magnesium; optimal growth occurs in the presence of 0.6 to 1.0 M Mg2+. The new strain resembles members of the Halobacterium saccharovorum-Halobacterium sodomense-Halobacterium trapanicum group in many physiological properties. However, the polar lipid composition of this organism is characteristic of representatives of the genus Haloferax; a sulfated diglycosyl diether is present, and the glycerol diether analog of phosphatidylglycerosulfate is absent. The G+C content of the DNA is 70 mol%. We found that on the basis of 16S rRNA sequence data our new isolate occupies a position intermediate between the position of the Halobacterium saccharovorum group and the position of the genus Haloferax and is sufficiently different from the previously described members of the Halobacteriaceae to justify classification in a new species and a new genus. We propose the name Halobaculum gomorrense gen. nov., sp. nov. for this organism; the type strain is strain DSM 9297.

Small-subunit ribosomal DNA sequences were determined for 17 strains belonging to the genera Alteromonas, Shewanella, Vibrio, and Pseudomonas, and these sequences were analyzed by phylogenetic methods. The resulting data confirmed the existence of the genera Shewanella and Moritella, but suggested that the genus Alteromonas should be split into two genera. We propose that a new genus, the genus Pseudoalteromonas, should be created to accommodate 11 species that were previously Alteromonas species, including Pseudoalteromonas atlantica comb. nov., Pseudoalteromonas aurantia comb. nov., Pseudoalteromonas carrageenovora comb. nov., Pseudoalteromonas citrea comb. nov., Pseudoalteromonas denitrificans comb. nov., Pseudoalteromonas espejiana comb. nov., Pseudoalteromonas haloplanktis comb. nov. (with two subspecies, Pseudoalteromonas haloplanktis subsp. haloplanktis comb. nov. and Pseudoalteromonas haloplanktis subsp. tetraodonis comb. nov.), Pseudoalteromonas luteoviolacea comb. nov., Pseudoalteromonas nigrifaciens comb. nov., Pseudoalteromonas rubra comb. nov., and Pseudoalteromonas undina comb. nov., and one species that previously was placed in the genus Pseudomonas, Pseudoalteromonas piscicida comb. nov. We propose that P. haloplanktis (type strain, ATCC 14393) should be the type species of the genus Pseudoalteromonas. At this time the emended genus Alteromonas is restricted to a single species, Alteromonas macleodii.

The α-amylase-producing haloalkaliphilic archaeon Natronococcus sp. strain Ah-36T (T = type strain) was isolated previously from a Kenyan soda lake, Lake Magadi. Most cells of strain Ah-36T occurred in irregular clusters, and the colonies were orange-red. The polar lipids of this organism were composed of C20, C20 and C20, C25 derivatives of phosphatidylglycerol and phosphatidylglycerophosphate. Phosphatidylglycero-(cyclo-) phosphate, which is characteristic of Natronococcus occultus, was not detected. The complete nucleotide sequence of the 16S rRNA gene revealed that the closest relative of strain Ah-36T is N. occultus ATCC431011 (level of similarity, 96.4%), an extremely halophilic archaeon. However, strain Ah-36T did not exhibit a significant level of DNA homology to N. occultus ATCC43101T, which represents the only previously described species in the genus Natronococcus. We describe a new species for strain Ah-36T, for which we propose the name Natronococcus amylolyticus.