Ewald B. M. Denner

Marinobacter aquaeolei sp. nov., a halophilic bacterium isolated from a Vietnamese oil- producing well

Several strains of moderately halophilic and mesophilic bacteria were isolated at the head of an oil-producing well on an offshore platform in southern Vietnam. Cells were Gram-negative, non-spore-forming, rod-shaped and motile by means of a polar flagellum. Growth occurred at NaCl concentrations between 0 and 20%; the optimum was 5% NaCl. One strain, which was designated VT8T, could degrade n-hexadecane, pristane and some crude oil components. It grew anaerobically in the presence of nitrate on succinate, citrate or acetate, but not on glucose. Several organic acids and amino acids were utilized as sole carbon and energy sources. The major components of its cellular fatty acids were C12:0 3-OH, C16:1, omega 9c, C16:0 and C18:1 omega 9c. The DNA G + C content was 55.7 mol%. 16S rDNA sequence analysis indicated that strain VT8T was closely related to Marinobacter sp. strain CAB (99.8% similarity) and Marinobaster hydrocarbonoclasticus (99.4% similarity). Its antibiotic resistance, isoprenoid quinones and fatty acids were similar to those of Marinobacter hydrocarbonoclasticus and Pseudomonas nautica. However, the whole-cell protein pattern of VT8T differed from that of other halophilic marine isolates, including P. nautica. DNA-DNA hybridization indicated that the level of relatedness to Marinobacter hydrocarbonoclasticus was 65% and that to P. nautica was 75%. Further differences were apparent in Fourier-transformed IR spectra of cells and lipopolysaccharide composition. It is proposed that VT8T should be the type strain of a new species and should be named Marinobacter aquaeolei. P. nautica may have been misclassified, as suggested previously, and may also belong to the genus Marinobacter.

Classification and identification of bacteria: current approaches to an old problem. Overview of methods used in bacterial systematics

Most of the bacterial species are still unknown. Consequently, our knowledge about bacterial ecology is poor and expectations about specialized species with novel enzymatic functions or new products are high. Thus, bacterial identification is a growing field of interest within microbiology. In this review, suitability of developments for identification based on miniaturized biochemical and physiological investigations of bacteria are evaluated. Special emphasis is given to chemotaxonomic methods such as analysis of quinone system, fatty acid profiles, polar lipid patterns, polyamine patterns, whole cell sugars, peptidoglycan diaminoacids, as well as analytical fingerprinting methods and cellular protein patterning. 16S rDNA sequencing introduced to investigate the phylogenetic relationships of bacteria, nucleic acids hybridization techniques and G + C content determination are discussed as well as restriction fragment length polymorphism (RFLP), macrorestriction analysis and random amplified polymorphic DNA (RAPD). The importance of the different approaches in classification and identification of bacteria according to phylogenetic relationships are demonstrated on selected examples.

Chemotaxonomic characterisation of Sphingomonas.

Based on published results and investigations done for this study, chemotaxonomic characteristics of all validly described species of the genus Sphingomonas, as well as unnamed strains of this genus and related genera such as Rhizomonas and Blastomonas, are presented. All representatives of this group, here designated as sphingomonads, contain ubiquinone (Q-10). The two different polyamine patterns characterized either by the predominant polyamine sym-homospermidine or spermidine separate the sphingomonads into two major groups. Complex polar lipid profiles were found in sphingomonads in addition to the characteristic compound sphingoglycolipid. Identical profiles were found only in a few phylogenetically highly related species. Common to all sphingomonads is a fatty acid composition with 2-hydroxy fatty acids (14:0 2OH in all currently recognized species) and the lack of 3-hydroxy acids, which distinguishes them from taxa outside this group. Qualitative and quantitative differences in the fatty acid compositions, in several cases, were also suitable for identification at the species level. Thus, the differences in the chemotaxonomic characteristics demonstrate that the analyses of these low molecular weight cell compounds are suitable for classification of sphingomonads.

Halococcus salifodinae sp. nov., an Archaeal Isolate from an Austrian Salt Mine

A novel extremely halophilic archaeon (archaebacterium) was isolated from rock salt obtained from an Austrian salt mine. The deposition of the salt is thought to have occurred during the Permian period (225 × 106 to 280 × 106 years ago). This organism grew over a pH range of 6.8 to 9.5. Electron microscopy revealed cocci in tetrads or larger clusters. The partial 16S rRNA sequences, polar lipid composition, and menaquinone content suggested that this organism was related to members of the genus Halococcus, while the whole-cell protein patterns, the presence of several unknown lipids, and the presence of pink pigmentation indicated that it was different from previously described coccoid halophiles. We propose that this isolate should be recognized as a new species and should be named Halococcus salifodinae. The type strain is Blp (= ATCC 51437 = DSM 8989). A chemotaxonomically similar microorganism was isolated from a British salt mine.

Classification of three airborne bacteria and proposal of Hymenobacter aerophilus sp. nov.

Three aerobic, gram-negative, rod-shaped, non-spore-forming, red-pigmented, airborne bacteria (I/26-Cor1T, I/32A-Cor1 and I/74-Cor2) collected in the Museo Correr (Venice, Italy) were investigated to determine their taxonomic status by analysing their biochemical, physiological and chemotaxonomic features and the G+C content of genomic DNA and by comparing their genomic fingerprints. Additionally, the almost complete 16S rRNA gene sequence of strain I/26-Cor1T was analysed. The three strains were nearly identical in their morphological, physiological, biochemical and chemotaxonomic properties. The strains contained a menaquinone system with the predominant menaquinone MK-7 and a fatty acid profile with C15:0 anteiso, C15:0 iso and C16:1 predominant. Phosphatidylethanolamine and several unidentified lipids were detected in the polar lipid profiles. The polyamine pattern consisted of sym-homospermidine as the major compound. meso-Diaminopimelic acid was found as the characteristic cell-wall diamino acid. The DNA base composition of the three strains ranged from 60 to 63 mol% G+C. Phylogenetically, strain I/26-Cor1T was most closely related to Hymenobacter actinosclerus (95.8% 16S rRNA gene sequence similarity). Physiological and genomic characteristics indicated that the two strains I/26-Cor1T and I/32A-Cor1 are representatives of the same species. The phylogenetic distance to any validly described taxon as indicated by 16S rRNA gene sequence similarities demonstrates that I/26-Cor1T and I/32A-Cor1 represent a novel species, for which the name Hymenobacter aerophilus sp. nov. is proposed, with the type strain I/26-Cor1T (= DSM 13606T = LMG 19657T). I/32A-Cor1 (= DSM 13607 = LMG 19658) is another strain of the species Hymenobacter aerophilus. Since the taxonomic status of strain I/74-Cor2 within the genus Hymenobacter was not determined unambiguously, it is designated Hymenobacter sp. I/74-Cor2 (= DSM 13611 = LMG 19659).

Very similar strains of Halococcus salifodinae are found in geographically separated permo-triassic salt deposits.

The authors have previously isolated a novel extremely halophilic archaeon, Halococcus salifodinae Blp, from Austrian rock salt deposited about 250 million years ago. In this study they compared strain Blp with two other halococci isolated independently from geographically distant salt deposits of similar age, and with two recent isolates (N1 and H2) from the same site as strain Blp. Strain BG2/2 was from a salt mine in Germany and strain Br3 from a halite deposit in England; both resembled Hc. salifodinae Blp in cellular and colonial morphology. Strains Blp, BG2/2 and Br3 had identical 16S rRNA sequences, very similar whole-cell protein patterns, which were different from those of other halococci, similar G+C contents and identical sequences in a 108-base insertion in their 5S rRNA gene. Other similarities included composition and relative abundances of polar lipids, antibiotic susceptibility, enzymic activities and Fourier-transform infrared spectra. Strains N1 and H2 showed similar morphology, whole-cell protein patterns and biochemical characteristics as strains Blp, Br3 and BG2/2. Their partial 16S rRNA sequences (682 and 641 bases, respectively) were indistinguishable from those of strains Blp, Br3 and BG2/2. Therefore strains N1 and H2 can be considered as reisolates of Hc. salifodinae which were obtained 8 years after the first samples were taken from that mine. The results presented suggest that viable halophilic archaea, which belong to the same species, occur in widely separated evaporite locations of similar geological age, and support the notion that these halophilic isolates from subterranean salt deposits may be the remnants of populations which inhabited ancient hypersaline seas.

Erythrobacter citreus sp. nov., a yellow-pigmented bacterium that lacks bacteriochlorophyll a, isolated from the western Mediterranean Sea.

Two facultatively oligotrophic, intensely yellow-pigmented bacterial strains, RE35F/1T and RE10F/45, have been previously isolated from the western Mediterranean Sea (Bay of Calvi, Corsica, France) by 0.2 microm membrane filtration. The organisms were gram-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped and non-motile. Their respiratory lipoquinone profiles consisted exclusively of ubiquinone-10 (Q-10) and the G+C contents of their DNAs were 62.0 and 62.4 mol%, respectively. Among the cellular fatty acids, octadecenoic acid (18:1omega7c) was the major component. Both isolates also contained hydroxy fatty acids (14:0 2-OH, 18:1 2-OH and 16:0 iso 3-OH) and branched fatty acids (15:0 anteiso, 16:0 anteiso and 17:0 anteiso). Polar lipid fingerprints were characterized by the presence of a sphingoglycolipid. Comparative analyses of their 16S rRNA gene sequences indicated that both isolates were phylogenetically closely related (sequence similarity of 99.9%) and formed a coherent cluster with aerobic bacteriochlorophyll a-containing species of the Erythrobacter/Porphyrobacter/Erythromicrobium cluster within the family Sphingomonadaceae. The closest relative was Erythrobacter litoralis DSM 8509T (97.4 and 97.5% 16S rRNA gene sequence similarity between this strain and RE35F/1T and RE10F/45, respectively). DNA-DNA reassociation studies confirmed that strains RE35F/1T and RE10F/45 represent a single species (79.6% DNA homology), but also demonstrated that they do not belong to the species Erythrobacter litoralis (25.2 and 34.2% DNA homology, respectively). Notably, both RE35F/1T and RE10F/45 lacked bacteriochlorophyll a. Based upon phenotypic and molecular evidence, a novel species of the genus Erythrobacter, Erythrobacter citreus sp. nov., is proposed. Strain RE35F/1T (= CIP 107092T = DSM 14432T) is the type strain.

Emended descriptions of the genus micrococcus, Micrococcus luteus (Cohn 1872) and Micrococcus lylae (Kloos et al. 1974)

Nine yellow-pigmented, spherical bacterial strains isolated from a medieval wall painting (strain D7), from indoor air (strains 3, 6, 7, 13C2, 38, 83 and 118) and from an activated-sludge plant (strain Ballarat) were classified by a polyphasic approach. Analyses of the 16S rRNA gene sequences of three representatives (strains D7, 118 and Ballarat) indicated that they all belong to the genus Micrococcus. The three isolates shared the highest sequence similarities with Micrococcus luteus DSM 20030T (97.9-98%), Micrococcus antarcticus AS 1.2372T (97.9-98.3%) and Micrococcus lylae DSM 20315T (97.5-97.9%). DNA-DNA reassociation studies clearly demonstrated that all nine isolates belong to the species M. luteus. However, neither their chemotaxonomic features nor their physiological and biochemical properties were consistent with those of M. luteus DSM 20030T. In contrast to M. luteus DSM 20030T, all isolates investigated possessed MK-8(H2) as the major respiratory quinone, and strain Ballarat had an A4alpha peptidoglycan type. On the basis of analyses of their Fourier transform-infrared spectroscopy spectra, isolates D7, 3, 6, 7, 13C2, 38, 83 and 118 could be grouped into a single cluster separate from M. luteus DSM 20030T, strain Ballarat and M. lylae DSM 20315T. In addition, all these isolates could be distinguished from M. luteus DSM 20030T by their ability to assimilate D-maltose, D-trehalose, DL-3-hydroxybutyrate, DL-lactate, pyruvate and L-histidine and to hydrolyse casein. Strains D7, 3, 6, 7, 13C2, 38, 83 and 118 differed from both M. luteus DSM 20030T and strain Ballarat by their ability to assimilate acetate, L-phenylalanine, L-serine and phenylacetate. Furthermore, REP-PCR fingerprinting yielded one common band for these strains, whereas this band was not observed for M. luteus DSM 20030T, strain Ballarat or M. lylae DSM 20315T. On the basis of these data, the species M. luteus can be divided into three biovars that are distinguished by several chemotaxonomic and biochemical traits: biovar I, represented by M. luteus DSM 20030T; biovar II, represented by strains D7 (= DSM 14234 = CCM 4959), 3, 6, 7, 13C2, 38, 83 and 118; and biovar III, represented by strain Ballarat (= DSM 14235 = CCM 4960). On the basis of the results generated in this study, emended descriptions of the genus Micrococcus and the species M. luteus and M. lylae are given.

Sphingomonas pituitosa sp. nov., an exopolysaccharide-producing bacterium that secretes an unusual type of sphingan

Strain EDIVT, an exopolysaccharide-producing bacterium, was subjected to polyphasic characterization. The bacterium produced copious amounts of an extracellular polysaccharide, forming slimy, viscous, intensely yellow-pigmented colonies on Czapek-Dox (CZD) agar. The culture fluids of the liquid version of CZD medium were highly viscous after cultivation for 5 d. Cells of strain EDIVT were Gram-negative, catalase-positive, oxidase-negative, nonspore-forming, rod-shaped and motile. Comparisons of 16S rDNA gene sequences demonstrated that EDIVT clusters phylogenetically with the species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (64.5 mol%), the presence of ubiquinone Q-10, the presence of 2-hydroxymyristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the detection of sym-homospermidine as the major component in the polyamine pattern, together with the presence of sphingoglycolipid, supported this delineation. 16S rDNA sequence analysis indicated that strain EDIVT is most closely related (99.4% similarity) to Sphingomonas trueperi LMG 2142T. DNA-DNA hybridization showed that the level of relatedness to S. trueperi is only 45.5%. Further differences were apparent in the cellular fatty acid profile, the polar lipid pattern, the Fourier-transform infrared spectrum and whole-cell proteins and in a number of biochemical characteristics. On the basis of the estimated phylogenetic position derived from 16S rDNA sequence data, DNA-DNA reassociation and phenotypic differences, strain EDIVT (= CIP 106154T = DSM 13101T) was recognized as a new species of Sphingomonas, for which the name Sphingomonas pituitosa sp. nov. is proposed. A component analysis of the exopolysaccharide (named PS-EDIV) suggested that it represents a novel type of sphingan composed of glucose, rhamnose and an unidentified sugar. Glucuronic acid, which is commonly found in sphingans, was absent. The mean molecular mass of PS-EDIV was approximately 3 x 10(6) Da.

Description of Sphingomonas xenophaga sp. nov. for strains BN6T and N,N which degrade xenobiotic aromatic compounds.

The taxonomic position of two bacterial strains, BN6T and N,N, with the ability to degrade xenobiotic aromatic compounds (naphthalenesulfonates or N,N-dimethylaniline) was investigated. The 165 rRNA gene sequence, the G+C content of the DNA (62-63 mol%) and the detection of ubiquinone Q-10, 2-hydroxymyristic acid and the sphingoglycolipid present clearly placed the two strains into the genus Sphingomonas. Both strains are representatives of one species according to the level of DNA relatedness (70.7%). The strains could be separated from all validly described taxa of the genus Sphingomonas, according to the 16S rRNA gene sequence (the highest sequence similarity observed was 96 % to Sphingomonas yanoikuyae), the pattern of the polar lipids and physiological characteristics. Therefore, the new species Sphingomonas xenophaga is proposed to accommodate strains BN6T (= DSM 6383T) and N,N (= DSM 8566).