Castellaniella gen. nov., to accommodate the phylogenetic lineage of Alcaligenes defragrans, and proposal of Castellaniella defragrans gen. nov., comb. nov. and Castellaniella denitrificans sp. nov.

Comparative 16S rRNA gene sequence analysis indicates that two distinct sublineages exist within the genus Alcaligenes: the Alcaligenes faecalis lineage, comprising Alcaligenes aquatilis and A. faecalis (with the three subspecies A. faecalis subsp. faecalis, A. faecalis subsp. parafaecalis and A. faecalis subsp. phenolicus), and the Alcaligenes defragrans lineage, comprising A. defragrans. This phylogenetic discrimination is supported by phenotypic and chemotaxonomic differences. It is proposed that the A. defragrans lineage constitutes a distinct genus, for which the name Castellaniella gen. nov. is proposed. The type strain for Castellaniella defragrans gen. nov., comb. nov. is 54PinT (=CCUG 39790T = CIP 105602T = DSM 12141T). Finally, on the basis of data from the literature and new DNA-DNA hybridization and phenotypic data, the novel species Castellaniella denitrificans sp. nov. (type strain NKNTAUT = DSM 11046T = CCUG 39541T) is proposed for two strains previously identified as strains of A. defragrans.

Thermomonas haemolytica gen. nov., sp. nov., a gamma-proteobacterium from kaolin slurry.

Four aerobic, gram-negative bacterial strains isolated from kaolin slurry used in the production of paper were subjected to a polyphasic analysis and characterization to determine their taxonomic position. Analysis of the 16S rDNA sequences of the four strains revealed that they represent a new lineage within the gamma-Proteobacteria, related to the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Luteimonas, Xylella and Rhodanobacter. Analysis of the quinone system, the polyamines, the fatty acids and the polar lipids revealed a combination of characteristics that is unique and not described for the phylogenetic relatives. The four strains contain a ubiquinone Q-8, spermidine as the major polyamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, and a fatty acid profile with predominantly iso-branched fatty acids. The G+C content of the genomic DNA was determined to be within the narrow range 67.1-68.7 mol%. Determination of DNA relatedness, as well as riboprint band patterns and amplified fragment length polymorphism profiles, clearly demonstrated that the four strains are members of a single species. Antibiotic-susceptibility patterns were identical for the four strains. Although showing a high degree of similarites in physiological and biochemical patterns, each of the four strains could be distinguished from the others on the basis of a few biochemical characteristics. On the basis of the estimates of phylogenetic relationships derived from the 16S rDNA sequence analyses, the observed chemotaxonomic characteristics and other phenotypic traits, a new genus, Thermomonas gen. nov., and species, Thermomonas haemolytica sp. nov., are proposed for the strains A50-7-3T (= DSM 13605T = LMG 19653T), B 50-7-1 (= DSM 13598 = LMG 19655), D50-7-1 (= DSM 13610 = LMG 19656) and B50-8-1 (= DSM 13599 = LMG 19654), with strain A50-7-3T as the type strain.

Detection of indigenous Halobacillus populations in damaged ancient wall paintings and building materials: molecular monitoring and cultivation.

Several moderately halophilic gram-positive, spore-forming bacteria have been isolated by conventional enrichment cultures from damaged medieval wall paintings and building materials. Enrichment and isolation were monitored by denaturing gradient gel electrophoresis and fluorescent in situ hybridization. 16S ribosomal DNA analysis showed that the bacteria are most closely related to Halobacillus litoralis. DNA-DNA reassociation experiments identified the isolates as a population of hitherto unknown Halobacillus species.

Alcaligenes faecalis subsp. parafaecalis subsp. nov., a bacterium accumulating poly-beta-hydroxybutyrate from acetone-butanol bioprocess residues.

The authors have previously isolated a solvent tolerant bacterium, strain G(T), (T = type strain) capable to convert acetone-butanol bioprocess residues into poly-beta-hydroxybutyrate. Strain G(T) was initially identified as Alcaligenes spp by standard bacteriological tests. In this study the taxonomic position of the bacterium was investigated in detail. The 165 rDNA sequence analysis, the G + C content of DNA (56 mol%) and the presence of ubiquinone Q-8 confirmed strain G(T) as a representative of the genus Alcaligenes. In the polyamine pattern of the bacterium putrescine and cadaverine were detected, but only trace amounts of 2-hydroxyputrescine. The extremely low content of 2-hydroxyputrescine is remarkable, since this unique diamine is a common marker for beta-proteobacteria. Phylogenetic analyses of 16S rDNA demonstrated that Alcaligenes sp. G(T) is most closely related to the species Alcaligenes faecalis (99.6% sequence similarity to A. faecalis HR4 and 98.7% sequence similarity to A. faecalis [ATCC 8750T = DSM 30030T]. On the basis of DNA-DNA relatedness (56% similarity), the unique polyamine pattern, the physiological and biochemical differences strain G(T) could be distinguished from the species A. faecalis. Therefore, a new subspecies for the species Alcaligenes faecalis is proposed; Alcaligenes faecalis subsp. parafaecalis subsp. nov.

Psychrobacter proteolyticus sp. nov., a psychrotrophic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting a cold-adapted metalloprotease.

An Antarctic marine bacterium (strain 116) excreting an extracellular cold-adapted metalloprotease was subjected to a detailed polyphasic taxonomic investigation. Strain 116 was previously isolated from the stomach of a specimen of the Antarctic krill Euphasia superba Dana and tentatively characterized as Sphingomonas paucimobilis 116. The 16S rDNA sequence analysis showed that the strain is in fact related to species of the genus Psychrobacter, next to Psychrobacter glacincola (97.4% similarity). Sequence similarities between strain 116 and other Psychrobacter species ranged from 96.9% (with P. urativorans) to 95.4% (with P. immobilis). Key phenotypic characteristics as well as chemotaxonomic features of the bacterium were congruent with the description of the genus Psychrobacter i.e. cells were strictly aerobic, strongly oxidase-positive, psychrotrophic, halotolerant, gram-negative non-motile coccobacilli, with ubiquinone-8 as the main respiratory lipoquinone and 18:1 cis 9, 16:1 cis and 17:1 (omega8c being the predominant cellular fatty acids. The G+C content of the DNA was 43.6 mol%. DNA-DNA hybridization studies showed that the relatedness between strain 116 and Psychrobacter glacinola is only 62.2%. Further differences were apparent in whole-cell SDS-PAGE protein pattern, cellular fatty acid profile and in a number of physiological and biochemical characteristics as well as in enzymatic activities. Tolerance to 5% bile salts, nitrate reduction, citrate utilization, acid production from carbohydrates, alkaline phosphatase, acid phosphatase, C4 esterase, C14 lipase and valine arylamidase were found to differentiate strain 116 from Psychrobacter glacincola. On the basis of this phenotypic and molecular evidences, strain 116, previously known as Sphingomonas paucimobilis 116, was recognized as a new species of the genus Psychrobacter for which the name Psychrobacter proteolyticus is proposed. Strain 116 has been deposited in the Collection de l'Institut Pasteur, France, as CIP106830T and in the Deutsche Sammlung von Mikroorganismen and Zellkulturen, as DSM13887.

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).

Reclassification of Pseudomonas echinoides Heumann 1962, 343AL, in the genus Sphingomonas as Sphingomonas echinoides comb. nov.

[Pseudomonas] echinoides DSM 1805T (= ATTC 14820T, DSM 50409T, ICBP 2835T, NCIB 9420T) has been reinvestigated to clarify its taxonomic position. 16S rDNA sequence comparisons demonstrated that this species clusters phylogenetically with species of the genus Sphingomonas. Investigation of fatty acid patterns, polar lipid profiles, polyamine patterns and quinone systems supported this delineation. Substrate utilization profiles and biochemical characteristics displayed no distinct overall similarity to any validly described species of the genus Sphingomonas. Therefore, the reclassification of [Pseudomonas] echinoides as Sphingomonas echinoides comb. nov. is proposed, based upon the estimated phylogenetic position derived from 16S rRNA gene sequence data, chemotaxonomic data and previously published genomic DNA G+C content data.

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.

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.

Classification of "Pseudomonas azotocolligans" Anderson 1955, 132, in the genus Sphingomonas as Sphingomonas trueperi sp. nov.

"Pseudomonas azotocolligans" ATCC 12417T (T = type strain), which was described as a diazotrophic bacterium, was reinvestigated to clarify its taxonomic position. 16S ribosomal DNA sequence comparisons demonstrated that this strain clusters phylogenetically with species of the genus Sphingomonas and represents a new species. The results of investigations of the fatty acid patterns, polar lipid profiles, and quinone system supported this placement. The substrate utilization profile and biochemical characteristics displayed no obvious similarity to the characteristics of any previously described species of the genus Sphingomonas. The new name Sphingomonas trueperi is proposed on the basis of these results and previously published data for the G + C content of the genomic DNA and the polyamine pattern.

Bacterial ghosts as multifunctional vaccine particles.

Expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex. Bacterial ghosts have been produced from a variety of bacteria including Escherichia coli. Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, and Helicobacter pylori. Such ghosts are used as non-living candidate vaccines and represent an alternative to heat or chemically inactivated bacteria. In recombinant ghosts, foreign proteins can be inserted into the inner membrane prior to E-mediated lysis via specific N-, or C-, or N- and C-terminal anchor sequences. The export of proteins into the periplasmic space or the expression of recombinant S-layer proteins vastly extents the capacity of ghosts or recombinant ghosts as carriers of foreign epitopes or proteins. Oral, aerogenic or parenteral applications of (recombinant) ghosts in experimental animals induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity. The most relevant advantage of ghosts and recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in the production of ghosts used as vaccines or as carriers of relevant antigens. The inserted target antigens into the inner membrane or into S-layer proteins are not limited in size.

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.