- Volume 50, Issue 5, 2000

Seventy phytoplasma isolates, including 10 previously characterized reference strains, of the aster yellows group were examined by RFLP analysis of PCR-amplified rDNA and RFLP and sequence analysis of the tuf gene. On the basis of rDNA restriction profiles, seven previously proposed 16S rDNA subgroups (16SrI-A, -B, -C, -D, -E, -F and -K) were recognized in the material examined. In addition, three new subgroups that differ in the RFLP profiles were identified and designated 16SrI-L, 16SrI-M and 16SrI-N. Of the two types of rDNA sequences used, an 1800 bp fragment comprising the entire 16S rRNA gene and the 16S-23S rDNA spacer region proved more suitable for AY-group phytoplasma differentiation than a 1240 bp fragment of the 16S rRNA gene. Many differences in the rDNA profiles between the subgroups could be explained by sequence heterogeneity of the two phytoplasmal rRNA operons. The subgroups delineated by RFLP analysis of a 940 bp tuf gene fragment are consistent with subgroups defined on the basis of rDNA sequences. However, subgroups 16SrI-D, -L and -M showed the same tuf gene restriction profiles as subgroup 16SrI-B. This result was confirmed by sequence analysis in which these subgroups differed slightly in their tuf gene sequence, when compared with members of subgroup 16SrI-B. On the basis of combined analyses of rDNA and tuf gene sequences and in view of pathological aspects, the taxonomic distinction of AY-subgroups 16SrI-A, -B, -C, -D, -E, -F, -K and -N appears to be substantial.

The mesophilic round-spored bacteria embrace four species, namely Bacillus sphaericus, Bacillus fusiformis, Bacillus silvestris and Bacillus pasteurii. Although not displayed by all strains, mosquito pathogenicity is a noteworthy characteristic of B. sphaericus sensu lato. Phylogenetic analysis based on 16S rDNA sequences from 58 strains identified as B. sphaericus was used to examine the genetic heterogeneity of the taxon. Results from sequence analysis were compared with whole-cell fatty acid profiles and other phenotypic determinations. The B. sphaericus-like strains segregated into seven distinct clusters in a phylogenetic tree generated from 16S sequences. One cluster represented B. sphaericus and another B. fusiformis. A third cluster containing all of the pathogenic strains was closely related to, or was possibly part of, the B. fusiformis group. The remaining four groups were distinct and represented unnamed taxa that were more closely related to B. sphaericus and B. fusiformis than to the psychrophilic round-spored species, Bacillus globisporus and Bacillus psychrophilus. Groups based on phenotypic analysis corresponded to the 16S rDNA phylogenetic clusters. Data showed that B. sphaericus was genetically and phenotypically a highly heterogeneous taxon including at least seven genetically distinct taxa. The pathogenic strains were members of a distinct group and not of the species B. sphaericus sensu stricto. This heterogeneity partially accounts for the apparent variability of mosquito pathogenicity among B. sphaericus strains.

A mesophilic hydrogenotrophic methanogen, strain MHT-1T, was isolated from the leachate of a sea-based site for solid waste disposal (the port of Osaka, Japan). Strain MHT-1T was found to be an irregular coccus and was able to use H2/CO2 and formate as energy sources. Acetate was required for growth. The optimum temperature and pH for growth were 35 degrees C and 6.5-7.5, respectively. Strain MHT-1T was resistant to high concentrations of several heavy metals such as CdCl2 and CuSO4. The G+C content of the DNA was 51.9 mol%. Analysis of the 16S rRNA gene revealed that the isolate was a member of the genus Methanocalculus but distinct from its nearest neighbour, Methanocalculus halotolerans, there being a sequence similarity of 98.9%. DNA-DNA hybridization analysis revealed 51% relatedness with the DNA of M. halotolerans strain SEBR 4845T. The optimum NaCl concentration was 1.0%, whereas the optimum in M. halotolerans was 5.0%. A new species, Methanocalculus pumilus, is proposed for strain MHT-1T. The type strain is MHT-1T (= DSM 12632T = JCM 10627T).

Psychrophilic actinobacterial isolates from permanently cold groundwater in Finland were characterized using a polyphasic approach. Growth on agar plates was observed at temperatures down to -2 degrees C, with an optimum at 15-17 degrees C, but no growth was observed at 30 degrees C. The peptidoglycan type was B2y and the characteristic diamino acid was diaminobutyric acid. The cell wall sugars of strain K265T were rhamnose, ribose, xylose and mannose and those of strain K300T were glucose, rhamnose and xylose. The polar lipids included phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid and two glycolipids. The main whole-cell fatty acids were 12-methyltetradecanoic acid, 14-methylpentadecanoic acid and 14-methylhexadecanoic acid. Large amounts of anteiso-1,1-dimethoxy-pentadecane and also iso-1,1-dimethoxyhexadecane were present as diagnostic markers. The predominant menaquinones were MK-9 and MK-10. The G+C content of the DNA of strains K265T and K300T was 64.4 and 67.8 mol%, respectively. Comparison of 16S rRNA gene sequences revealed that strains K265T and K300T represent a new lineage among the type-B-peptidoglycan actinomycetes. The closest relatives were Clavibacter michiganensis, Frigoribacterium faeni and Rathayibacter rathayi. On the basis of 16S rDNA sequence, G+C content and chemotaxonomical and physiological characteristics, K265T and K300T clearly represent a new genus. The genus Subtercola gen. nov. is described, together with two species, namely Subtercola boreus sp. nov. (type strain K300T = DSM 13056T = CCUG 43135T) and Subtercola frigoramans sp. nov (type strain K265T = DSM 13057T = CCUG 43136T).

Aerobic endospore-forming bacteria were isolated from soils taken from active fumaroles on Mount Rittmann and Mount Melbourne in northern Victoria Land, Antarctica, and from active and inactive fumaroles on Candlemas Island, South Sandwich archipelago. The Mt Rittmann and Mt Melbourne soils yielded a dominant, moderately thermophilic and acidophilic, aerobic endospore-former growing at pH 5.5 and 50 degrees C, and further strains of the same organism were isolated from a cold, dead fumarole at Clinker Gulch, Candlemas Island. Amplified rDNA restriction analysis, SDS-PAGE and routine phenotypic tests show that the Candlemas Island isolates are not distinguishable from the Mt Rittmann strains, although the two sites are 5600 km apart, and 16S rDNA sequence comparisons and DNA relatedness data support the proposal of a new species, Bacillus fumarioli, the type strain of which is LMG 17489T.

Two strains of a Gram-positive, catalase-negative, facultatively anaerobic, rod-shaped bacterium isolated from common seals were characterized using phenotypic and molecular taxonomic methods. The two strains closely resembled each other based on their biochemical characteristics, and PAGE analysis of whole-cell protein patterns confirmed their close phenotypic affinity. 16S rRNA gene sequencing showed that the two strains were genetically highly related (99.8% sequence similarity) and that they constitute a new line of descent within the lactic acid group of bacteria. The nearest phylogenetic neighbours of the unknown bacterium were Granulicatella spp., with related taxa such as enterococci, carnobacteria, Desemzia incerta, Lactosphaera pasteurii, Melissococcus plutonius, tetragenococci and vagococci more distantly related. Based on phylogenetic and phenotypic evidence it is proposed that the unknown bacterium from seals be classified in a new genus as Atopobacter phocae gen. nov., sp. nov. The type strain of Atopobacter phocae is CCUG 42358T (= CIP 106392T).

The dnaK operon in gram-positive bacteria includes grpE, dnaJ and, in some members, hrcA as well. Both DnaK and DnaJ have been utilized for constructing phylogenetic relationships among various organisms. Multiple copies exist for dnaK and dnaJ genes in some bacterial genera, as opposed to a single gene copy for grpE and for hrcA, according to the currently available data. Here, we present a partial protein-based phylogenetic tree for gram-positive bacteria, derived by using the amino acid sequence identity of GrpE; the results are compared with the phylogenetic trees generated from 5S rRNA, 16S rRNA, dnaK and dnaJ sequences. Our results indicate three main groupings: two are within low-G+C DNA gram-positive bacteria comprising Bacillus species and Staphylococcus aureus on the one hand and Streptococcus species/Lactococcus lactis/Enterococcus faecalis/Lactobacillus sakei on the other hand; the Mycobacterium species and Streptomyces coelicolor, belonging to the high-G+C DNA gram-positive bacteria, form the third cluster. This hierarchical arrangement is in close agreement with that obtained with 16S rRNA and DnaK sequences but not DnaJ-based phylogeny.

Collinsella aerofaciens-like strains isolated from human faeces were characterized by biochemical tests, cell wall murein analysis and 16S rDNA analysis. The results indicated that these strains are phylogenetically a member of the family Coriobacteriaceae and close to the genus Collinsella. Their phenotypic characters resembled those of Collinsella aerofaciens. Determination of DNA-DNA relatedness showed that these strains could be divided into two groups (groups 1 and 2). Collinsella aerofaciens and both new groups have A4-type cell wall murein. Based on their phenotypic and phylogenetic characters, two new species of the genus Collinsella are proposed for the isolated strains: Collinsella stercoris for group 1 and Collinsella intestinalis for group 2. Species-specific PCR primer sets for these two species were also constructed. Using these primer sets, Collinsella stercoris and Collinsella intestinalis can be identified easily and rapidly.

The genes for rickettsial outer-membrane protein A (rOmpA), a distinguishing feature of spotted fever group (SFG) rickettsiae, and rOmpB, a genus-specific protein, were identified and sequenced in Rickettsia australis. The amino acid sequences of domains I, III and IV of the R. australis rOmpA share close homology with those of rOmpA of other SFG rickettsiae, but the repeat region (domain II) is dramatically different from that of other known SFG rOmpA. R. australis rOmpB is more similar to rOmpB of other SFG rickettsiae than to that of typhus group rickettsiae.

'Flexispira rappini' is a provisional name given to gram-negative, microaerophilic, motile, spindle-shaped micro-organisms with spiral periplasmic fibres and bipolar tufts of sheathed flagella. Several investigators, including Kirkbride, Romero, and Archer isolated strains possessing this morphology. Previously, the phylogenetic position of three 'Flexispira rappini' strains was determined by 165 rRNA sequencing, which indicated that flexispira were members of the genus Helicobacter. As more organisms with 'F. rappini' morphology were isolated, it became apparent that there were multiple Helicobacter taxa with this distinctive morphology. The purpose of this study was to examine a collection of 36 'F. rappini' strains from diverse habitats by using 165 rRNA sequence analysis. The strains fell into 10 taxa, each possibly representing a novel Helicobacter species. Two of these flexispira taxa were previously named, by us, Helicobacter bilis and Helicobacter trogontum. Currently, none of the flexispira taxa contains enough phenotypically and genotypically characterized strains to be formally named 'Helicobacter rappinii'.

A bacteriocin-producing lactic acid bacterium, which was isolated from the Korean fermented-vegetable food kimchi, was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods. This organism (MT-1077T) has phenotypic properties that are consistent with the description characterizing the genus Lactobacillus. Phylogenetic analysis based on 16S rDNA sequences showed clearly that strain MT-1077T is a member of the genus Lactobacillus. The closest phylogenetic relatives are Lactobacillus alimentarius KCTC 3593T and Lactobacillus farciminis LMG 9200T, with levels of 16S rDNA similarity of 98.4 and 98.2%, respectively. Levels of 16S rDNA similarity between strain MT-1077T and other Lactobacillus species were less than 93.0%. Differences in some phenotypic characteristics and DNA-DNA relatedness data indicated that strain MT-1077T should be distinguished from L. alimentarius KCTC 3593T and L. farciminis LMG 9200T. On the basis of the data presented, it is proposed that strain MT-1077T should be placed in the genus Lactobacillus as a new species, Lactobacillus kimchii sp. nov. The type strain of the new species is strain MT-1077T (= KCTC 8903PT = JCM 10707T).

Thiobacillus novellus is a facultatively chemolithoautotrophic and methylotrophic, Gram-negative, rod-shaped sulfur bacterium, shown by 16S rRNA gene sequence analysis to be a member of the alpha-2 subclass of the Proteobacteria. As such, it must be excluded from the genus Thiobacillus, whose species are members of the beta-Proteobacteria. It closest phylogenetic neighbour appears to be Ancylobacter, from which it is distinct morphologically and in some physiological characteristics. It is distinct physiologically and biochemically in a number of diagnostic features from Paracoccus versutus, in the alpha-3 subclass of the Proteobacteria and does not appear to be sufficiently closely related to any other genus of the alpha-Proteobacteria to be reassigned to a known genus. The new genus and species name Starkeya novella is proposed for T. novellus. The type strain is ATCC 8093T (= NCIMB 10456T = NCIMB 9113T = DSM 506T = IAM 12100T = IFO 12443T = CCM 1077T).

Five strains of simmondsin-degrading, lactic-acid-producing bacteria were isolated from fermented jojoba meal. These isolates were facultatively anaerobic, gram-positive, non-motile, non-spore-forming, homofermentative, rod-shaped organisms. They grew singly and in short chains, produced lactic acid but no gas from glucose, and did not exhibit catalase activity. Growth occurred at 15 and 45 degrees C. All strains fermented cellobiose, D-fructose, D-galactose, D-glucose, lactose, maltose, D-mannitol, D-mannose, melibiose, D-ribose, salicin, D-sorbitol, sucrose and trehalose. Some strains fermented L-(-)-arabinose and L-rhamnose. D-Xylose was not fermented and starch was not hydrolysed. The mean G+C content of the DNA was 48 mol%. Phylogenetic analyses of 16S rDNA established that the isolates were members of the genus Lactobacillus. DNA reassociation of 45% or less was obtained between the new isolates and the reference strains of species with G+C contents of about 48 mol%. The isolates were differentiated from other homofermentative Lactobacillus spp. on the basis of 16S rDNA sequence divergence, DNA relatedness, stereoisomerism of the lactic acid produced, growth temperature and carbohydrate fermentation. The data support the conclusion that these organisms represent strains of a new species, for which the name Lactobacillus arizonensis is proposed. The type strain of L. arizonensis is NRRL B-14768T (= DSM 13273T).

A previously uncharacterized, slowly growing, scotochromogenic Mycobacterium species was detected by HPLC analysis of the cell-wall-bound mycolic acids. The mycolic acid pattern standard was shown to be a late-eluting, contiguous peak cluster occurring at approximately 8-9 min. The mycolic acid pattern was noted to be most similar in number of peaks and range of elution to that reported previously for Mycobacterium asiaticum. However, the relative distribution of peaks within the elution range demonstrated a pattern with prominent peaks that started to emerge later than the characteristic M. asiaticum pattern. Standard biochemical identification test results were similar to those of the photochromogenic species M. asiaticum. Comparative 16S rRNA gene sequence analysis confirmed the genetic uniqueness of the strains and demonstrated the unclassified mycobacteria to be in a unique, intermediate position between slow and rapid growers in the phylogenetic tree of Mycobacterium. The name Mycobacterium kubicae sp. nov. is proposed for this taxon. The type strain is CDC 941078T (= ATCC 700732T = CIP 106428T).

A strain isolated from a lung abscess in an elephant that died from chronic respiratory disease was found to have properties consistent with its classification in the genus Mycobacterium. An almost complete sequence of the 165 rDNA of the strain was determined following the cloning and sequencing of the amplified gene. The sequence was aligned with those available on mycobacteria and phylogenetic trees inferred by using three tree-making algorithms. The organism, which formed a distinct phyletic line within the evolutionary radiation occupied by rapidly growing mycobacteria, was readily distinguished from members of validly described species of rapidly growing mycobacteria on the basis of its mycolic acid pattern and by a number of other phenotypic features, notably its ability to grow at higher temperatures. The type strain is Mycobacterium elephantis DSM 44368T.

A bacterial strain, CS12T, which was isolated from soil around a wastewater treatment plant, was subjected to a polyphasic taxonomic study using phenotypic characterizations and genetic methods. The cell wall of strain CS12T contains meso-diaminopimelic acid as the diamino acid but no arabinose and galactose. The predominant menaquinone is MK-8(H4). Mycolic acids are absent. Strain CS12T has a cellular fatty acid profile containing saturated, unsaturated, branched and 10-methyl fatty acids. The major fatty acids are iso-C16:0, C18:1 omega9c and anteiso-C17:0, The G+C content is 69 mol%. A phylogenetic tree based on 16S rDNA sequences showed that strain CS12T forms an evolutionary lineage within the radiation enclosing the members of the family Intrasporangiaceae and, in particular, a coherent cluster with Janibacter limosus DSM 11140T. The level of 16S rDNA similarity between strain CS12T and J. limosus DSM 11140T is 98.7%. The phenotypic characteristics and DNA-DNA relatedness data indicate that strain CS12T should be distinguished from J. limosus DSM 11140T. Therefore, on the basis of the data presented, a new species of the genus Janibacter, Janibacter terrae, is proposed. The type strain of the new species is strain CS12T (= KCCM 80001T = JCM 10705T).

A Gram-negative and tryptophanase-positive thermophile, whose growth is dependent on co-culture with an associating Bacillus strain, had been reported and tentatively named Symbiobacterium thermophilum strain T(T). Axenic culture of strain T(T) was recently established by dialysing cultures with the supporting bacterial strains or adding their culture broth. Phylogenetic analysis of strain T(T), based on the 16S rDNA sequence, was conducted for the validation of S. thermophilum. The sequence of strain T(T) was located at the outermost position in the high-G+C Gram-positive group distinctly isolated from any other branches hitherto known. Ten sequences identical to that of strain T(T), and one sequence closely related to it, were identified for the first time from soil and compost samples. The outer membrane of strain T(T) had a three-layered structure, outside the cytoplasmic membrane, which is similar to the S-layer in the cells of members of the Bacillaceae. Chemical analysis of the cells revealed that menaquinone-6 is a major component of the quinone system. According to these results, along with several previous observations (i.e. a G+C DNA content of 65 mol% and the identification of iso-C15:0 and iso-C17:0 acids as major cellular fatty acids), the new taxon Symbiobacterium thermophilum gen. nov., sp. nov. is proposed. The type strain is S. thermophilum strain T(T) (= IAM 14863T).

The formal description of Prochlorococcus marinus Chisholm et al. 1992, 299 was based on the non-axenic nomenclatural type, strain CCMP 1375T. The purification and properties of the axenic strain PCC 9511, derived from the same primary culture (SARG) as the type species, are reported here. Prochlorococcus PCC 9511 differs from the latter in possessing horseshoe-shaped thylakoids, exhibiting a low chlorophyll b2 content and lacking phycoerythrin, but shares these phenotypic properties with Prochlorococcus strain CCMP 1378. This relationship was confirmed by 16S rRNA sequence analyses, which clearly demonstrated that the axenic isolate is not co-identic with the nomenclatural type. Strain PCC 9511 has a low mean DNA base composition (32 mol% G+C) and harbours the smallest genome of all known oxyphotobacteria (genome complexity 1.3 GDa = 2 Mbp). Urea and ammonia are the preferred sources of nitrogen for growth, whereas nitrate is not utilized. Several different organic phosphorus compounds efficiently replace phosphate in the culture medium, indicative of ecto-phosphohydrolase activity. In order to distinguish strain PCC 9511 from the nomenclatural type, a new subspecies is proposed, Prochlorococcus marinus Chisholm et al. 1992 subsp. pastoris subsp. nov.