- Volume 52, Issue 1, 2002

Prokaryotes constitute a single kingdom, Bacteria, here divided into two new subkingdoms: Negibacteria, with a cell envelope of two distinct genetic membranes, and Unibacteria, comprising the new phyla Archaebacteria and Posibacteria, with only one. Other new bacterial taxa are established in a revised higher-level classification that recognizes only eight phyla and 29 classes. Morphological, palaeontological and molecular data are integrated into a unified picture of large-scale bacterial cell evolution despite occasional lateral gene transfers. Archaebacteria and eukaryotes comprise the clade neomura, with many common characters, notably obligately co-translational secretion of N-linked glycoproteins, signal recognition particle with 7S RNA and translation-arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin, prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs), exosomes and similar replication, repair, transcription and translation machinery. Eubacteria (posibacteria and negibacteria) are paraphyletic, neomura having arisen from Posibacteria within the new subphylum Actinobacteria (possibly from the new class Arabobacteria, from which eukaryotic cholesterol biosynthesis probably came). Replacement of eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are the keys to neomuran origins. All 19 common neomuran character suites probably arose essentially simultaneously during the radical modification of an actinobacterium. At least 11 were arguably adaptations to thermophily. Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft of glycoprotein, not flagellin; DNA-binding protein lob; specially modified tRNA; absence of Hsp90) were subsequent secondary adaptations to hyperthermophily and/or hyperacidity. The insertional origin of protein-spliced tRNA introns and an insertion in proton-pumping ATPase also support the origin of neomura from eubacteria. Molecular co-evolution between histones and DNA-handling proteins, and in novel protein initiation and secretion machineries, caused quantum evolutionary shifts in their properties in stem neomura. Proteasomes probably arose in the immediate common ancestor of neomura and Actinobacteria. Major gene losses (e.g. peptidoglycan synthesis, hsp90, secA) and genomic reduction were central to the origin of archaebacteria. Ancestral archaebacteria were probably heterotrophic, anaerobic, sulphur-dependent hyperthermoacidophiles; methanogenesis and halophily are secondarily derived. Multiple lateral gene transfers from eubacteria helped secondary archaebacterial adaptations to mesophily and genome re-expansion. The origin from a drastically altered actinobacterium of neomura, and the immediately subsequent simultaneous origins of archaebacteria and eukaryotes, are the most extreme and important cases of quantum evolution since cells began. All three strikingly exemplify De Beer's principle of mosaic evolution: the fact that, during major evolutionary transformations, some organismal characters are highly innovative and change remarkably swiftly, whereas others are largely static, remaining conservatively ancestral in nature. This phenotypic mosaicism creates character distributions among taxa that are puzzling to those mistakenly expecting uniform evolutionary rates among characters and lineages. The mixture of novel (neomuran or archaebacterial) and ancestral eubacteria-like characters in archaebacteria primarily reflects such vertical mosaic evolution, not chimaeric evolution by lateral gene transfer. No symbiogenesis occurred. Quantum evolution of the basic neomuran characters, and between sister paralogues in gene duplication trees, makes many sequence trees exaggerate greatly the apparent age of archaebacteria. Fossil evidence is compelling for the extreme antiquity of eubacteria [over 3500 million years (My)] but, like their eukaryote sisters, archaebacteria probably arose only 850 My ago. Negibacteria are the most ancient, radiating rapidly into six phyla. Evidence from molecular sequences, ultrastructure, evolution of photosynthesis, envelope structure and chemistry and motility mechanisms fits the view that the cenancestral cell was a photosynthetic negibacterium, specifically an anaerobic green non-sulphur bacterium, and that the universal tree is rooted at the divergence between sulphur and non-sulphur green bacteria. The negibacterial outer membrane was lost once only in the history of life, when Posibacteria arose about 2800 My ago after their ancestors diverged from Cyanobacteria.

Two novel strains of the class Actinobacteria were isolated from a cave in China. Cells of both strains were gram-positive, non-motile, non-spore-forming and not acid-fast and exhibited a rod/coccus growth cycle. Both isolates grew well on complex organic media under aerobic conditions. Their cell wall peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The acyl type of the glycan chain of peptidoglycan was acetyl. The major respiratory quinone was MK-8(H4). The cellular fatty acid profile was characterized by the predominance of 13-methyltetradecanoic (i-C15:0), 15-methylhexadecanoic (i-C17:0), 14-methylpentadecanoic (i-C16:0) and 14-methylhexadecanoic (ai-C17:0) acids. The major polar lipids were phosphatidylethanolamine, phosphatidylinositol and diphosphatidylglycerol. Mycolic acids were absent. The DNA G+C composition was 68-69 mol%. 16S rDNA-based phylogenetic analysis revealed an intermediate phylogenetic position of the cave isolates between the genera Janibacter and Tetrasphaera, which did not permit their unambiguous affiliation to either genus. Differences in morphological, physiological and chemotaxonomic properties between the two isolates and their closest phylogenetic neighbours support the proposal of a new genus and two novel species, Knoellia sinensis gen. nov., sp. nov. and Knoellia subterranea sp. nov. The type and only strains of the species are respectively HKI 0119T (= DSM 12331T = CIP 106775T) and HKI 0120T (= DSM 12332T = CIP 106776T).

A novel, facultatively psychrophilic alkaliphile that grows on a chemically defined medium containing n-alkanes as the sole carbon source was isolated from a drain of a fish product-processing plant. The isolate was an aerobic, non-motile, gram-positive bacterium. The bacterium was catalase-positive and oxidase-negative. The cell wall contained meso-diaminopimelic acid, arabinose and galactose; the glycan moiety of the cell wall contained acetyl residues. The G+C content of the DNA was 69.6 mol %. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate was closely related to members of the genus Dietzia (96.1-96.8% similarity). Comparisons of phenotypic and chemotaxonomic characteristics between the isolate and the two known Dietzia species showed that they were very similar. However, the isolate differed from the two known Dietzia species in growth temperature range and certain physiological characteristics. DNA-DNA hybridization revealed that the isolate had 38.4 and 49.7% relatedness, respectively, to Dietzia maris and Dietzia natronolimnaea. On the basis of the physiological and biochemical characteristics, the phylogenetic position as determined by 16S rRNA gene analysis and DNA-DNA relatedness, it is concluded that the isolate should be designated as a novel species, for which the name Dietzia psychralcaliphila sp. nov. is proposed. The type strain is ILA-1T (= JCM 10987T = IAM14896T = NCIMB 13777T).

An extensive Fourier-transform infrared (FT-IR) spectroscopy database for the identification of bacteria from the two suborders Micrococcineae and Corynebacterineae (Actinomycetales, Actinobacteria) as well as other morphologically similar genera was established. The database consists of averaged IR spectra from 730 reference strains, covering 220 different species out of 46 genera. A total of 192 species are represented by type strains. The identity of 352 reference strains was determined by comparative 16S rDNA sequence analysis and, if necessary, strains were reclassified accordingly. FT-IR frequency ranges, weights and reproducibility levels were optimized for this section of high-G+C gram-positive bacteria. In an internal validation, 98.1% of 208 strains were correctly identified at the species level. A simulated external validation which was carried out using 544 strains from 54 species out of 16 genera resulted in a correct identification of 87.3% at the species level and 95.4% at the genus level. The performance of this identification system is well within the range of those having been reported in the literature for the identification of coryneform bacteria by phenotypical methods. Coryneform and related taxa display a certain degree of overlapping distribution of different taxonomical markers, leading to a limited differentiation capacity of non-genotypical identification methods in general. However, easy handling, rapid identification within 25 h starting from a single colony, a satisfactory differentiation capacity and low cost, render FT-IR technology clearly superior over other routine methods for the identification of coryneform bacteria and related taxa.

Four strains of aerobic, endospore-forming bacteria were isolated from the inner tissues of healthy cotton plants (Gossypium sp., Dushanbe, Tajikistan). The organisms had identical randomly amplified polymorphic DNA patterns that distinguished them from other bacilli that are commonly isolated from plant tissues, e.g. Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus and Bacillus subtilis. PCR amplification of 16S-23S rRNA spacer regions suggested that the four strains could be assigned to two highly related taxa, which correlated with differences in cell morphology. However, the cloned spacer region provided a simple and specific hybridization probe for all four strains. The virtually complete 16S rDNA sequences were prepared for representatives of the two groups (strains 2DT(T) and 12DX) and differed by only two bases, thus supporting classification of the four strains in a single taxon at the species level. Phylogenetic analyses indicated that strain 2DT(T) belonged to the genus Bacillus and was most closely related to Bacillus sporothermodurans DSM 10599T with a sequence similarity of 94.8%. It is concluded that the four strains belong to a novel species of Bacillus for which the name Bacillus endophyticus sp. nov. is proposed. The type strain is 2DT(T) (= UCM B-5715T = CIP 106778T).

A thermo-acidophilic gram-positive bacterium, strain CP-1T, which grows aerobically at 35-65 degrees C (optimum 55-60 degrees C) and at pH 3.5-6.0 (optimum pH 4.5-5.0), was isolated from a herbal tea made from the dried flowers of hibiscus. Phylogenetic analysis of the 16S rRNA gene sequences showed that this bacterium was clearly distinguishable from previously described species of the genera Alicyclobacillus and Sulfobacillus. Strain CP-1T had unique omega-cycloheptane fatty acids as the major membrane lipid component, a characteristic which is peculiar to Alicyclobacillus cycloheptanicus. However, phenotypic and chemotaxonomic characteristics of strain CP-1T were different from those of the type strain of A. cycloheptanicus. DNA-DNA hybridization between the type strains of Alicyclobacillus species and Sulfobacillus disulfidooxidans was <20%, indicating that strain CP-1T represents a distinct species. On the basis of these results, the name Alicyclobacillus herbarius is proposed for this organism. The type strain is strain CP-1T (= DSM 13609T = IAM 14883T = NRIC 0477T).

Novel isolates, strains HM-7, HM-6, HH-31, P9a-hT and UJB13-d, which were isolated from tongue plaque and necrotic dental pulp, were studied taxonomically and phylogenetically. These organisms were anaerobic, non-spore-forming, gram-positive, rod-shaped bacteria that were inert in most of the conventional biochemical tests and phenotypically resemble Mogibacterium species or asaccharolytic Eubacterium species. The G+C contents of the DNAs from the novel isolates ranged from 41 to 42 mol %. DNA-DNA hybridization studies demonstrated that these strains might be assigned to the genus Mogibacterium but not to the previously described species. It was also apparent that strain HM-7 belonged to the same species as strains HM-6 and HH-31, and that strains P9a-hT and UJB13-d belonged to a second species. The levels of DNA-DNA relatedness to asaccharolytic Eubacterium species, including Eubacterium brachy, Eubacterium nodatum, Eubacterium saphenum and the more recently proposed Eubacterium minutum and Eubacterium exiguum (reclassified as Slackia exigua), are less than 2%. The results of 16S rDNA sequence comparisons revealed that these organisms represent novel lineages distinct from all previously described species of gram-positive, rod-shaped bacteria. On the basis of phenotypic characteristics, DNA-DNA hybridization data and phylogenetic analysis with 16S rRNA gene sequence data, new species are proposed, namely Mogibacterium diversum (for strains HM-7, HM-6 and HH-31) and Mogibacterium neglectum (for strains P9a-hT and UJB13-d). HM-7T (= ATCC 700923T = JCM 11205T) is the type strain of the former and P9a-hT (= ATCC 700924T = JCM 11204T) is the type strain for the latter.

A gram-negative, moderately halophilic bacterial strain, YKJ-16T, which was isolated from jeotgal, a traditional Korean food, was considered to be a member of the genus Halomonas. Cells of strain YKJ-16T are non-motile and cocci or short rods, unlike most Halomonas species. However, chemotaxonomic and phylogenetic analyses demonstrated that strain YKJ-16T belongs to the genus Halomonas. The predominant isoprenoid quinone is ubiquinone-9. The major fatty acids are C18.1omega7c, C16:0, C19:0 cyclo omega8c and C16:1omega7c and/or iso C15:0 20H. The phylogenetic tree showed that strain YKJ-16T forms a distinct evolutionary lineage within the radiation comprising Halomonas species and forms a coherent cluster with Halomonas halodenitrificans, Halomonas cupida and Halomonas pacifica. Levels of 16S rDNA similarity between strain YKJ-16T and the type strains of other Halomonas species are 93.0-96.3%. Levels of DNA-DNA relatedness indicate a taxonomic status of strain YKJ-16T as a species different from the three species that form the coherent cluster mentioned above. Morphologically, strain YKJ-16T is also clearly differentiated from the type strains of H. cupida and H. pacifica. Accordingly, on the basis of the phenotypic characteristics, 16S rDNA sequence analysis and DNA relatedness data, strain YKJ-16T should be placed in the genus Halomonas as a novel species. The name Halomonas alimentaria sp. nov. is proposed with strain YKJ-16T (= KCCM 41042T = JCM 10888T) as the type strain.

Bacteria belonging to the genus Paenibacillus were isolated by enrichment from petroleum-hydrocarbon-contaminated sediment and salt marsh rhizosphere using either naphthalene or phenanthrene as the sole carbon source, and were characterized using phenotypic, morphological and molecular techniques. The isolates were grouped by their colony morphologies and polyaromatic hydrocarbon-degradation patterns. Phenanthrene-degrading isolates produced mottled colonies on solid media and were identified as P. validus by fatty acid methyl ester and 16S rRNA gene sequence analyses. In contrast, the naphthalene-degrading isolates with mucoid colony morphology were distantly related to Paenibacillus validus, according to fatty acid methyl ester and 16S rRNA gene sequence analyses. The predominant fatty acids of the mucoid isolates were 15:0 anteiso, 16:1omega11c, 16:0 and 17:0 anteiso, constituting, on average, 50.5, 12.0, 11.2 and 6.5% of the total, respectively. The G+C contents of their DNA ranged from 47 to 52 mol%. The 16S rDNA sequence analysis revealed the highest (< or = 94%) similarity to P. validus. In addition, phylogenetic analyses based on 16S rDNA sequences showed that the mucoid isolates formed a distinct cluster within Paenibacillus. DNA-DNA hybridization experiments showed only a 6% DNA similarity between the type strain of P. validus and mucoid strain PR-N1. On the basis of the morphological, phenotypic and molecular data, the naphthalene-degrading isolates merit classification as a new Paenibacillus species, for which the name Paenibacillus naphthalenovorans sp. nov. is proposed, with PR-N1T (= ATCC BAA-206T = DSM 14203T) as the type strain.

A taxonomic study was conducted to clarify the relationships of two bacterial populations belonging to the genus Weissella. A total of 39 strains originating mainly from Malaysian foods (22 strains) and clinical samples from humans (9 strains) and animals (6 strains) were analysed using a polyphasic taxonomic approach. The methods included classical phenotyping, whole-cell protein electrophoresis, 16S and 23S rDNA RFLP (ribotyping), determination of 16S rDNA sequence homologies and DNA-DNA reassociation levels. Based on the results, the strains were considered to represent two different species, Weissella confusa and a novel Weissella species, for which the name Weissella cibaria sp. nov. is proposed. Weisella confusa possessed the highest 16S rDNA sequence similarity to Weisella cibaria, but the DNA-DNA reassociation experiment showed hybridization levels below 49% between the strains studied. The numerical analyses of Weisella confusa and Weisella cibaria strains did not reveal any specific clustering with respect to the origin of the strains. Based on whole-cell protein electrophoresis, and ClaI and HindIII ribotyping patterns, food and clinical isolates were randomly located in the two species-specific clusters obtained.

A novel extremely halophilic archaeon was isolated from Lake Tebenquiche, situated in the northern part of the Atacama Saltern, Chile. The cells of these micro-organisms were mostly irregularly disc-shaped. They grew in medium containing saturated concentrations of NaCl and did not require magnesium for optimal growth. The polar lipid composition revealed the presence of mannosyl-2-sulfate-(1-4)-glycosyl-archaeol, the main glycolipid of the genus Halorubrum, and two new glycolipids. The G+C content of the DNA was 63.2 mol%. Phylogenetic analysis of the 16S rRNA gene placed strain ALT6-92T within the Halorubrum cluster. The low DNA-DNA hybridization value justified classification in a new species for which the name Halorubrum tebenquichense sp. nov. is proposed. The type strain is ALT6-92T (= CECT 5317T = DSM 14210T).

A novel anaerobic, moderately thermophilic, peptide-fermenting bacterium, strain NGA(T), was isolated from an anaerobic wool-scouring wastewater treatment lagoon. The cells were gram-negative, straight rods of 0.5-1.0 x 2.0-4.0 microm, motile by means of a single flagellum. The DNA G+C content was 51.5 mol%. The optimum pH and temperature range for growth were 6.6-7.3 and 55-60 degrees C, respectively. The optimum NaCl concentration was 0.08 g l(-1). The bacterium fermented organic acids (malate, tartrate, pyruvate, glycerol and fumarate), a few carbohydrates (starch, glucose, fructose and gluconate), Casamino acids, tryptone and yeast extract. Carbohydrates and organic acids were converted to acetate, hydrogen and CO2. The bacterium oxidized leucine to isovalerate with crotonate as an electron acceptor, but not in co-culture with Methanothermobacter thermoautotrophicus DSM 3720T. Thiosulfate, sulfur and cystine were reduced to sulfide and crotonate was reduced to butyrate with glucose and tryptone-yeast extract as electron donors. Phylogenetic analysis of the 16S rRNA gene indicated that strain NGA(T) was related to Anaerobaculum thermoterrenum (98% similarity), the only described species of the genus. The DNA-DNA hybridization value for strain NGA(T) and A. thermoterrenum ACM 5076T was 40.8%. On the basis of these results, strain NGA(T) is proposed as a novel species of the genus Anaerobaculum, namely Anaerobaculum mobile sp. nov. The type strain is NGA(T) (= DSM 13181T =ATCC BAA-54T).

Bartonella is a bacterial genus classified in the alpha-Proteobacteria on the basis of 165 rDNA sequence comparison. The highly conserved heat-shock chaperonin protein, GroEL, has proved to be a valuable resolving tool to classify ten Bartonella species. The groEL gene was amplified and sequenced from ten Bartonella isolates: Bartonella alsatica, Bartonella vinsonii subsp. arupensis, Bartonella taylorii, Bartonella tribocorum, Bartonella birtlesii, Bartonella henselae Marseille (URLLY8), B. henselae (90-615), B. henselae (Fizz), B. henselae (CAL-1) and B. henselae (SA-2). Then, phylogenetic relationships were inferred between our isolates and eight other species and subspecies from the comparison of both 16S rDNA and groEL sequences using parsimony, neighbour-joining and maximum-likelihood methods. By using groEL sequences, the first reliable classification of most known Bartonella species and subspecies was established. Four strongly supported subgroups were distinguished: firstly, the two human pathogens B. henselae and Bartonella quintana; secondly, a cluster including four rodent isolates, Bartonella elizabethae, B. tribocorum, Bartonella grahamii and B. taylorii; thirdly, a cluster including the B. vinsonii subspecies (B. vinsonii subsp. vinsonii, arupensis and berkhoffii); and lastly, B. birtlesii and 'Bartonella weissi'. 'Bartonella washoensis', B. alsatica, Bartonella doshiae, Bartonella bacilliformis and Bartonella clarridgeiae did not reliably cluster with any other Bartonella species. In addition, the groEL gene was shown to be useful in subtyping six B. henselae isolates into three variants: Houston, Marseille and Fizz.

The strictly anaerobic gram-negative bacterium strain Fru22T grows at 50 degrees C in media containing up to 75 g NaCl l(-1). Hexoses and pentoses are fermented to equal molar amounts of acetate, propionate and succinate, and no CO2 is formed. An orange-red pigment similar to flexirrubin is produced during stationary phase upon exposure to light for several days. Cells also produce a surface-active extracellular compound which lowers the surface tension of the medium. This tenside is heat-tolerant up to 70 degrees C and is destroyed by treatment with proteinase K or trypsin, but not by lipase. Comparative 16S rDNA sequence analysis confirmed a phylogenetic affiliation of strain Fru22T to the phylum Bacteroides (Cytophaga/Flavobacterium/Bacteroides), moderately related to the genus Marinilabilia. Therefore, on the basis of phylogenetic, phenotypic and physiological evidence, a new genus, Anaerophaga, is proposed to harbour strain Fru22T (DSM 12881T, OCM 798T) which is described as the type strain of a new species, Anaerophaga thermohalophila gen. nov., sp. nov.

A gram-negative, oxidase-positive, rod-shaped bacterium isolated from the heart of a cotton-topped tamarin was characterized by 16S rDNA sequence analysis, SDS-PAGE of whole-cell proteins, fatty acid analysis and biochemical tests. Outer-membrane proteins, iron-regulated outer-membrane proteins, lipopolysaccharides and siderophore production were studied. On the basis of the results, the organism belongs to the beta-Proteobacteria where it forms a separate line of descent, for which a novel genus and species are proposed, Brackiella oedipodis (LMG 19451T = DSM 13743T = NCIMB 13739T). Nearest phylogenetic neighbours of the new genus are Taylorella, Pelistega, Bordetella, Alcaligenes and Achromobacter.

A novel thermophilic, photosynthetic bacterium, designated strain HLO8T, was isolated from a bacterial mat in a Japanese hot spring. Morphologically, the isolate was an unbranched multicellular filament with a cell diameter of 0.8-1.0 microm. The bacterium was red to reddish-brown in colour and formed a distinct red bacterial mat in the natural environment. It was able to grow photoheterotrophically under anaerobic light conditions and also chemoheterotrophically under aerobic dark conditions. Optimal growth occurred at 50 degrees C and pH 7.5-8.0. The cells contained bacteriochlorophyll (Bchl) a and gamma-carotene derivatives as photosynthetic pigments, but lacked Bchl c and chlorosomes. Cellular fatty acids in the isolate were mainly C16:0, C14:0 and C15:0. The major quinone was menaquinone-11. The DNA G+C content was 62.0 mol% (by HPLC). Phylogenetic analysis based on 16S rDNA sequencing suggested that the isolate belonged to the anoxygenic filamentous phototrophic bacteria represented by Chloroflexus aurantiacus, but was clearly distant from all members in this group (the sequence similarities between the isolate and its relatives were less than 83.8%). Based on genotypic and phenotypic data, the name Roseiflexus castenholzii gen. nov., sp. nov. is proposed for this isolate; the type strain is HLO8T (= DSM 13941T = JCM 11240T).

Three strains of psychrophilic bacteria isolated from Antarctic coastal marine environments were studied to determine their taxonomic position. These bacteria were gram-negative rods, facultatively anaerobic and motile by means of a single polar flagellum. None of the bacterial isolates had an Na+ requirement. Only one of the strains was capable of producing H2S from thiosulfate. The DNA base content of these bacteria was 41-42 mol % G+C. DNA-DNA hybridization experiments showed that the isolates formed two related groups that exhibited about 70 and 24% DNA-DNA homology, respectively, with the type strain of Shewanella frigidimarina. The fatty acid profiles of the bacterial isolates were similar to the profiles of other Shewanella species. All the strains contained both ubiquinones and menaquinones, like Shewanella species. Methylmenaquinones were also found. 16S rRNA gene analysis confirmed that isolated strains belonged to the genus Shewanella and were phylogenetically related to the newly identified Shewanella frigidimarina. The results of the polyphasic taxonomic study assigned the three isolates to Shewanella and two of them specifically to Shewanella frigidimarina. The name Shewanella livingstonensis sp. nov. (type strain LMG 19866T) is proposed for the third organism.