- Volume 58, Issue 6, 2008

Seventeen strains of lespedeza rhizobia, a previously defined novel group, were further characterized by comparison with phylogenetically related species using a polyphasic approach, including SDS-PAGE of whole-cell proteins, PCR-based restriction fragment length polymorphism analysis of ribosomal intergenic spacer sequences, determination of DNA G+C contents, DNA–DNA hybridization and analysis of phenotypic features. Based on data obtained in the present and previous studies, a novel species, Rhizobium miluonense sp. nov., is proposed for these strains. The type strain of R. miluonense is CCBAU 41251T (=LMG 24208T=HAMBI 2971T). Strain CCBAU 41251T could form ineffective nodules on Phaseolus vulgaris in addition to its original host Lespedeza chinensis.

A novel ovoid, phototrophic, purple non-sulfur bacterium was isolated in pure culture from a sample of sediment from an industrially polluted pond (Noor Mohammad Kunta) in Hyderabad, India. Strain JA234T was found to be Gram-negative and non-motile and grew photoheterotrophically with a number of organic compounds serving as carbon source/electron donor. Photo-organoheterotrophic and chemo-organoheterotrophic growth were demonstrated. Biotin and thiamine were required for growth of strain JA234T. Phylogenetic analysis on the basis of 16S rRNA gene sequences showed that strain JA234T is most closely related to Rhodobacter azotoformans (97.3 % sequence similarity) and clusters with other species of the genus Rhodobacter of the family Rhodobacteraceae. However, DNA–DNA hybridization with Rba. azotoformans JCM 9340T showed a relatedness of only 31.3 % with respect to strain JA234T. On the basis of 16S rRNA gene sequence analysis, DNA–DNA hybridization data and morphological and physiological characteristics, strain JA234T represents a novel species of the genus Rhodobacter, for which the name Rhodobacter ovatus sp. nov. is proposed. The type strain is JA234T (=JCM 14779T=CCUG 55049T).

A Gram-negative, psychrophilic, motile, rod-shaped bacterium, designated strain 20188T, was isolated from marine samples collected from the Arctic (7 ° 00′ 24″ N 16 ° 59′ 37″ W), and was identified taxonomically by means of a polyphasic study. On the basis of 16S rRNA gene sequence similarity, strain 20188T was closely related to members of the genera Phaeobacter. 16S rRNA gene sequence similarities between strain 20188T and the type strains of Phaeobacter inhibens, Phaeobacter gallaeciensis and Phaeobacter daeponensis were 97.0, 96.8 and 96.2 %, respectively. The temperature range for growth was 0–25 °C, with optimum growth occurring at 19–20 °C and at approximately pH 6.0–9.0. Strain 20188T had ubiquinone-10 as the major respiratory quinone and C18 : 1 ω7c and 11-methyl C18 : 1 ω7c as major fatty acids. The genomic DNA G+C content was 59.6 mol%. On the basis of phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data, strain 20188T is considered to represent a novel species of the genus Phaeobacter, for which the name Phaeobacter arcticus sp. nov. is proposed. The type strain is 20188T (=CGMCC 1.6500T=JCM 14644T).

A Gram-negative bacterium, strain MDA0585T, isolated from a sputum culture, was characterized by a polyphasic approach. The 16S rRNA gene and a conserved portion of the DNA gyrase A gene were sequenced and analysed phylogenetically. Strain MDA0585T showed the closest relationships with Chromobacterium violaceum ATCC 12472T and Chromobacterium subtsugae PRAA4-1T (96.1 % and 96.3 % 16S rRNA gene sequence similarity, respectively). The cellular fatty acids of strain MDA0585T consisted mainly of C16 : 0, C16 : 1 ω7c and C16 : 1 ω6c (summed feature 3) and C18 : 1 ω7c and C18 : 1 ω6c (summed feature 8), a profile that was similar to, but distinguishable from, those of C. violaceum ATCC 12472T and C. subtsugae PRAA4-1T. In culture, strain MDA0585T differed from C. violaceum and C. subtsugae in several ways: lack of violet pigmentation, the ability to haemolyse sheep blood, differences in several biochemical reactions and higher resistance to antibiotics. The culture supernatant of strain MDA0585T also caused remarkable haemolysis of human erythrocytes. These results suggest that strain MDA0585T represents a novel species within the genus Chromobacterium, for which the name Chromobacterium haemolyticum sp. nov. is proposed. The type strain is MDA0585T (=CCUG 53230T=JCM 14163T=DSM 19808T).

A Gram-negative, rod-shaped, non-motile, non-spore-forming bacterium, designated strain HR2T, was isolated from a soil sample from the Taklimaken Desert in Xinjiang Province, China. Strain HR2T grew optimally at pH 7.0–8.0 and 30–37 °C in the presence of 0–1 % (w/v) NaCl. An analysis of 16S rRNA gene sequences revealed that strain HR2T fell within the radiation of the genus Pseudomonas, the highest level of similarity being found with respect to Pseudomonas luteola IAM 13000T (97.5 %); the levels of sequence similarity with respect to other recognized Pseudomonas species were <96.4 %. DNA–DNA hybridization showed that the genetic relatedness between strain HR2T and P. luteola IAM 13000T was 53.2 %. The G+C content of the genomic DNA of strain HR2T was 55.2 mol%. The major fatty acids were 18 : 1, summed feature 3 and 16 : 0. The hydroxylated fatty acids 10 : 0 3-OH, 12 : 0 3-OH and 12 : 0 2-OH were also present. The data obtained in this polyphasic study indicated that this isolate represents a novel species of the genus Pseudomonas, for which the name Pseudomonas duriflava sp. nov. is proposed. The type strain is HR2T (=KCTC 22129T=CGMCC 1.6858T).

Bacterial strain CCBAU 25048T was isolated from root nodules of Kummerowia stipulacea grown in Shandong province of China. Cells of the strain were Gram-negative, strictly aerobic, non-spore-forming, motile short rods. Phylogeny of 16S rRNA gene sequences revealed that the strain belonged to the genus Shinella, a member of family Rhizobiaceae. Its closest phylogenetic relatives were Shinella granuli Ch06T and Shinella zoogloeoides IAM 12669T, respectively showing 98.3 and 98.9 % 16S rRNA gene sequence similarity. Strain CCBAU 25048T had DNA–DNA relatedness of 43.5 and 34.8 %, respectively, with S. zoogloeoides JCM 20728T and S. granuli JCM 13254T. In addition, in TP-RAPD analysis, different patterns were obtained for these three strains and some rhizobial strains. The nifH, nodC and nodD sequences of CCBAU 25048T were identical or very similar to those of bean-nodulating Rhizobium tropici strains. Several phenotypic characteristics, including the use of citrate and d-ribose as carbon sources and growth at pH 11.0, as well as the fatty acid composition, could differentiate CCBAU 25048T from the two defined Shinella species. Therefore, a novel species Shinella kummerowiae sp. nov. is proposed, with strain CCBAU 25048T (=JCM 14778T =LMG 24136T) as the type strain.

A novel aerobic, Gram-negative, rod-shaped, motile bacterium, designated strain 5516S-11T, was isolated from air samples collected in the Suwon region of the Republic of Korea. Analysis of the 16S rRNA gene sequence indicated that the organism belongs to the genus Massilia; the highest sequence similarity (97.2 %) was found with respect to Massilia aurea DSM 18055T. Cells of strain 5516S-11T contained ubiquinone Q-8 as the predominant isoprenoid quinone and possessed summed feature 3 (C16 : 1 ω7c/iso-C15 : 0 2-OH; 35.2 %), C16 : 0 (30.6 %) and C18 : 1 ω7c (11.7 %) as the major fatty acids. DNA–DNA hybridization revealed 32 % relatedness between strain 5516S-11T and M. aurea DSM 18055T. The G+C content of the DNA of strain 5516S-11T was 68.9 mol%. It is clear from the genotypic and phenotypic data presented that strain 5516S-11T represents a novel species of the genus Massilia, for which the name Massilia aerilata sp. nov. is proposed. The type strain is 5516S-11T (=KACC 12505T =DSM 19289T).

Two Gram-negative, rod-shaped, non-spore-forming bacteria, PR17T and DSM 7216T, isolated from the potato rhizosphere and an industrial environment, respectively, were studied for their taxonomic allocation. By rrs (16S rRNA) gene sequencing, these strains were shown to belong to the Alphaproteobacteria, most closely related to Ochrobactrum pseudogrignonense (98.4 and 99.3 % similarity to the type strain, respectively). Chemotaxonomic data (major ubiquinone Q-10; major polyamines spermidine, sym-homospermidine and putrescine; major polar lipids phosphatidylethanolamine, phosphatidylmonomethylethanolamine, phosphatidylglycerol and phosphatidylcholine and the Ochrobactrum-specific unidentified aminolipid AL2; major fatty acids C18 : 1 ω7c and C19 : 0 cyclo ω8c) supported the genus affiliation. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of the isolates from all hitherto-described Ochrobactrum species. Hence, both isolates represent novel species of the genus Ochrobactrum, for which the names Ochrobactrum rhizosphaerae sp. nov. (type strain PR17T =CCUG 55411T =CCM 7493T =DSM 19824T) and Ochrobactrum thiophenivorans sp. nov. (type strain DSM 7216T =CCUG 55412T =CCM 7492T) are proposed.

[Enterobacter] sakazakii is an opportunistic pathogen that can cause infections in neonates. This study further clarifies the taxonomy of isolates described as [E.] sakazakii and completes the formal description of the proposed reclassification of these organisms as novel species and subspecies within a proposed novel genus, Cronobacter gen. nov. [E.] sakazakii was first defined in 1980, however recent polyphasic taxonomic analysis has determined that this group of organisms consists of several genomospecies. In this study, the phenotypic descriptions of the proposed novel species are expanded using Biotype 100 and Biolog Phenotype MicroArray data. Further DNA–DNA hybridization experiments showed that malonate-positive strains within the [E.] sakazakii genomospecies represent a distinct species, not a subspecies. DNA–DNA hybridizations also determined that phenotypically different strains within the proposed species, Cronobacter dublinensis sp. nov., belong to the same species and can be considered as novel subspecies. Based on these analyses, the following alternative classifications are proposed: Cronobacter sakazakii gen. nov., comb. nov. [type strain ATCC 29544T (=NCTC 11467T)]; Cronobacter malonaticus sp. nov. [type strain CDC 1058-77T (=LMG 23826T=DSM 18702T)]; Cronobacter turicensis sp. nov. [type strain z3032T (=LMG 23827T=DSM 18703T)]; Cronobacter muytjensii sp. nov. [type strain ATCC 51329T (=CIP 103581T)]; Cronobacter dublinensis sp. nov. [type strain DES187T (=LMG 23823T=DSM 18705T)]; Cronobacter dublinensis subsp. dublinensis subsp. nov. [type strain DES187T (=LMG 23823T=DSM 18705T)]; Cronobacter dublinensis subsp. lausannensis subsp. nov. [type strain E515T (=LMG 23824=DSM 18706T)], and Cronobacter dublinensis subsp. lactaridi subsp. nov. [type strain E464T (=LMG 23825T=DSM 18707T)].

Strain 20041T was isolated from sea-ice of the Canadian Basin (7 ° 23′ 14″ N 14 ° 06′ 55″ W). Phylogenetic analysis based on 16S rRNA gene homology showed that strain 20041T was related to members of the genus Marinobacter and had highest 16S rRNA gene sequence similarity with the type strain of Marinobacter maritimus. Cells were Gram-negative, rod-shaped, psychrophilic and motile. The temperature range for growth was 0–22 °C, with optimum growth occurring at 16–18 °C and at approximately pH 6.0–9.0. Strain 20041T had ubiquinone-9 as the major respiratory quinone and iso-C15 : 0 2OH and/or C16 : 1 ω7c, C16 : 0, C18 : 1 ω9c and C12 : 0 3OH as major fatty acids. The genomic DNA G+C content was 55.4 mol%. On the basis of the phenotypic characteristics, phylogenetic analysis and DNA–DNA relatedness data, strain 20041T is considered to represent a novel species, for which the name Marinobacter psychrophilus sp. nov. is proposed. The type strain is 20041T (=CGMCC 1.6499T=JCM 14643T).

A polyphasic study was carried out to clarify the taxonomic position of a pink-coloured, Gram-negative bacterium isolated from air in the Suwon region of Korea. The novel strain, 5420S-30T, grew in the temperature range of 5–35 °C (optimum 30 °C) and pH range of 5–9 (optimum pH 6–7). Growth did not occur in the presence of as little as 1 % (w/v) NaCl. It contained Q-10 as the predominant ubiquinone. The major fatty acids were C18 : 1 ω7c, summed feature 3 (C16 : 1 ω7c/iso-C15 : 0 2-OH) and C16 : 0 (together representing 77.7 % of the total). The DNA G+C content was 72.1 mol%. The 16S rRNA gene sequence similarities between strain 5420S-30T and members of the genus Roseomonas were in the range 91.6–95.1 %, with the highest sequence similarities (95.1 and 94.5 %, respectively) to Roseomonas aquatica TR53T and Roseomonas gilardii ATCC 49956T. On the basis of its phenotypic and genotypic characteristics, strain 5420S-30T represents a novel species of the genus Roseomonas, for which the name Roseomonas aerilata sp. nov. is proposed, with 5420S-30T (=KACC 12521T =DSM 19363T) as the type strain.

Three methanol-utilizing yeast strains were isolated from basidiocarps of Ganoderma sp. collected from a tree trunk in Mangshan Mountain, Hunan Province, southern China. These strains formed hat-shaped ascospores in unconjugated and deliquescent asci. Sequence analysis of the large-subunit rRNA gene D1/D2 domain and internal transcribed spacer (ITS) region, electrophoretic karyotype comparison and phenotypic characterization demonstrated that the three strains represent a novel species of the genus Ogataea, which is described as Ogataea ganodermae sp. nov. (type strain SHS 2.1T =CGMCC AS 2.3435T =CBS 10646T). Phylogenetically, the novel species was closely related to Ogataea pini and Ogataea henricii. The latter two taxa with similar D1/D2 sequences were confirmed to represent separate species by ITS sequence and electrophoretic karyotype comparisons.

A novel strictly anaerobic, vancomycin-resistant, Gram-positive coccus (strain CCRI-16110T) was isolated from a human faecal specimen. This strain was characterized using morphological, biochemical and molecular taxonomic methods. The organism was unable to hydrolyse aesculin and failed to produce acid from cellobiose, d-lactose and α-raffinose. Acetic acid was the sole product of glucose fermentation by the organism. On the basis of 16S rRNA and tuf gene sequence comparison, strain CCRI-16110T was most closely related to species of the genus Ruminococcus and formed a hitherto unknown sublineage within the Clostridium coccoides rRNA cluster of organisms (cluster XIVa). Based on phenotypic and phylogenetic evidence, a novel species, Ruminococcus gauvreauii sp. nov., is proposed. The type strain is CCRI-16110T (=NML 060141T =CCUG 54292T =JCM 14987T).

A novel obligately anaerobic, alkalithermophilic, chemo-organotrophic bacterium was isolated from a small and very shallow geothermally heated pool at Pushino (Kamchatka, Far East Russia). The bacterium, designated strain JW/VK-KS5YT, was a Gram staining negative, Gram type positive rod. The cells were sometimes branched, with a tendency to grow in long chains, and were non-sporulating and non-motile. The shortest observed doubling time was 28 min when the novel strain was grown at 54–60 °C in 120 mM sodium carbonate-containing medium at pH25 °C 8.5–9.0. The novel bacterium grew on yeast extract and soytone as sole carbon and energy sources but could also use fumarate, thiosulfate and sulfur as electron acceptors. The DNA G+C content was 32.5 mol%. Based on phylogenetic, DNA–DNA hybridization and phenotypic data, it was concluded that isolate JW/VK-KS5YT (=VKM B-2436T=DSM 18970T) represents the type strain of a novel species, Anaerobranca zavarzinii sp. nov.

Five novel strains (2002T, 2902, 2006, 108T and 117) of cellulose-degrading, anaerobic, thermophilic bacteria were isolated from terrestrial hot springs of Kamchatka (Far East, Russia). Strains 2002T and 108T were non-spore-forming bacteria with a Gram-positive type cell wall and peritrichous flagella. Optimum growth of strains 2002T and 108T occurred at pH 7.0 and at temperatures of 70 and 65 °C, respectively. The G+C contents of the DNA of strains 2002T and 108T were 35.1 and 36.4 mol%, respectively. Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to the genus Caldicellulosiruptor. However, DNA–DNA hybridization experiments indicated that the levels of relatedness between strains 2002T and 108T and those of recognized members of the genus Caldicellulosiruptor ranged between 32 and 54 %. Based on both phenotypic and genomic differences, strains 2002T and 108T are considered to represent two novel species of the genus Caldicellulosiruptor. The names proposed for these organisms are Caldicellulosiruptor kronotskyensis sp. nov. (type strain 2002T=DSM 18902T=VKM B-2412T) and Caldicellulosiruptor hydrothermalis sp. nov. (type strain 108T=DSM 18901T=VKM B-2411T).

A Gram-positive, aerobic, rod-shaped bacterium (strain Eur1 9.5T) was isolated from a 9-m-deep permafrost sample from the Canadian high Arctic. Strain Eur1 9.5T could not be cultivated in liquid medium and grew over the temperature range 5–37 °C; no growth was observed at 42 °C and only slow growth was observed at 5 °C following 1 month of incubation. Eur1 9.5T grew over the pH range 5.5–8.9 and tolerated NaCl concentrations of 0–0.5 % (w/v). Eur1 9.5T grew heterotrophically on complex carbon substrates and chemolithoautotrophically on inorganic sulfur compounds, as demonstrated by growth on sodium thiosulfate and sulfite as sole electron donors. Eur1 9.5T contained iso-C15 : 0 as the major cellular fatty acid and menaquinone 7 (MK-7) as the major respiratory quinone. The cell-wall peptidoglycan was of type A1γ. The DNA G+C content was 53.1 mol%. The 16S rRNA gene sequence of strain Eur1 9.5T was only distantly related (≤87 % sequence similarity over 1407 bp) to any recognized bacterial species. Based on physiological and phylogenetic analyses, strain Eur1 9.5T is suggested to represent a novel species of a new genus, for which the name Tumebacillus permanentifrigoris gen. nov., sp. nov. is proposed. The type strain of Tumebacillus permanentifrigoris is Eur1 9.5T (=DSM 18773T =JCM 14557T).

The rplKAJL–rpoBC operon or β operon is a classic bacterial gene cluster, which codes for proteins K, A, J and L of the large ribosomal subunit, as well as proteins B (β subunit) and C (β′ subunit) of RNA polymerase. In the early 1990s, the operon was obtained as a 2.6 kbp DNA fragment (In-2.6) by random cloning of DNA from periwinkle plants infected with the Poona (India) strain of the huanglongbing agent, later named ‘Candidatus (Ca.) Liberibacter asiaticus’. DNA from periwinkle plants infected with the Nelspruit strain (South Africa) of ‘Ca. L. africanus’ was amplified with a primer pair designed from In-2.6 and yielded, after cloning and sequencing, a 1.7 kbp DNA fragment (AS-1.7) of the β operon of ‘Ca. L. africanus’. The β operon of the American liberibacter, as well as the three upstream genes (tufB, secE, nusG), have now also been obtained by the technique of chromosome walking and extend over 4673 bp, comprising the following genes: tufB, secE, nusG, rplK, rplA, rplJ, rplL and rpoB. The sequence of the β operon was also determined for a Brazilian strain of ‘Ca. L. asiaticus’, from nusG to rpoB (3025 bp), and was found to share 99 % identity with the corresponding β operon sequences of an Indian and a Japanese strain. Finally, the β operon sequence of ‘Ca. L. africanus’ was extended from 1673 bp (rplA to rpoB) to 3013 bp (nusG to rpoB), making it possible to compare the β operon sequences of the African, Asian and American liberibacters over a length of ∼3000 bp, from nusG to rpoB. While ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’ shared 81.2 % sequence identity, the percentage for ‘Ca. L. americanus’ and ‘Ca. L. africanus’ was only 72.2 %, and identity for ‘Ca. L. americanus’ and ‘Ca. L. asiaticus’ was only 71.4 %. The ∼3000 bp nusG–rpoB sequence was also used to construct a phylogenetic tree, and this tree was found to be identical to the known 16S rRNA gene sequence-based tree. These results confirm earlier findings that ‘Ca. L. americanus’ is a distinct liberibacter, more distantly related to ‘Ca. L. africanus’ and ‘Ca. L. asiaticus’ than ‘Ca. L. africanus’ is to ‘Ca. L. asiaticus’. The dates of speciation have also been estimated.

This is the first study that estimates mycobacterial phylogeny using the maximum-likelihood method (PhyML-aLRT) on a seven-gene concatenate (hsp65, rpoB, 16S rRNA, smpB, sodA, tmRNA and tuf) and the super distance matrix (SDM) supertree method. Two sets of sequences were studied: a complete seven gene sequence set (set R, type strains of 87 species) and an incomplete set (set W, 132 species) with some missing data. Congruencies were computed by using the consense program (phylip package). The evolution rate of each gene was determined, as was the evolution rate of each strain for a given gene. Maximum-likelihood trees resulting from concatenation of the R and W sets resulted in a similar phylogeny, usually showing an early separation between slow-growing (SG) and rapidly growing (RG) mycobacteria. The SDM tree for the W set resulted in a different phylogeny. The separation of SG and RG was still evident, but it was located later in the nodes. The SG were therefore positioned as a subgroup of RG. Maximum-likelihood phylogenetic reconstruction was less affected by increasing the number of strains (with incomplete data), but did seem to cushion the variability of the evolution rate (ER), whereas the SDM method seemed to be more accurate and took into account both the differing ER values and the incomplete data. With regard to ER, it was observed that the 16S rRNA gene was the gene that displayed the slowest evolution, whereas smpB was the most rapidly evolving gene. Surprisingly, these two genes alone accurately separated the SG from the RG on the basis of their ER values. This study focused on the differences in ER between genes and in some cases linked the ER to the phenotypic classification of the mycobacteria.