P. De Vos

Proposed minimal standards for describing new taxa of aerobic, endospore-forming bacteria

Minimal standards for describing new taxa within the aerobic endospore-forming bacteria are proposed, following Recommendation 30b of the Bacteriological Code (1990 Revision). These minimal standards are recommended as guidelines to assist authors in the preparation of descriptions for novel taxa. They encourage broad polyphasic characterization and the construction of descriptions that are practically useful in routine diagnostic laboratories. The proposals have been endorsed by the Subcommittee on the Taxonomy of the Genus Bacillus and Related Organisms of the International Committee on Systematics of Prokaryotes.

Applicability of combined amplified ribosomal DNA restriction analysis (ARDRA) patterns in bacterial phylogeny and taxonomy

Abstract A standardized method for amplified ribosomal DNA restriction analysis (ARDRA) is described. The first step involves selection of five tetracutter restriction enzymes on the basis of theoretical digestions of known 16S rDNA (rRNA) sequences. In the second step, the experimentally obtained restriction patterns are normalized and combined by means of the pattern recognition and analysis software GelCompar. Finally, numerical analysis allows the strains to be grouped according to the similarities in their combined ARDRA patterns. Results obtained with representatives of two phylogenetic lineages, the genera Alcaligenes and Bordetella and the genera Bacillus and Paenibacillus , are presented. In general, the clustering of the strains corresponded well with known species delineations and topology of phylogenetic groupings except for the discrepant position of the Bacillus lautus type strain, which can probably be explained by non-authenticity of this strain in one of the analyses. The effect of using less than five restriction enzymes on the clustering was evaluated. The frequent occurrence of interoperon variability of the 16S rRNA gene in Bacillus and Paenibacillus was also demonstrated. Because ARDRA detects interspecies and interstrain as well as interoperon variability and enables a relatively fast multiple strain analysis per taxon, this technique is appropriate to obtain indicative phylogenetic and taxonomic information. This information can be used to select strains for further detailed taxonomic studies. ARDRA fingerprinting also allows the construction of a database for indentification purposes.

Intra- and Intergeneric Similarities of Pseudomonas and Xanthomonas Ribosomal Ribonucleic Acid Cistrons

We hybridized 23s 2- 14C-labeled ribosomal ribonucleic acids (rRNAs) from type strains Pseudomonas fluorescens ATCC 13525, Pseudomonas acidovorans ATCC 15668, Pseudomonas solanacearum NCPPB 325, and Xanthomonas campestris NCPPB 528 with deoxyribonucleic acids (DNAs) from 65 Pseudomonus strains, 23 Xanthomonas strains, and 148 mostly gram-negative strains belonging to 43 genera and 93 species and subspecies including more than 60 type strains. Our findings confirm and extend the findings derived from ribonucleic acid hybridizations by the Berkeley group, but differed in some respects from the groupings of Pseudomonas in Bergey s Manual of Determinative Bacteriology, 8th ed. The genus Pseudomonas Migula 1894, 237 was divided into three large, distinct groups. The PseudomonasJIuorescens rRNA branch contains Pseudomonus aeruginosa, Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas aureofaciens, Pseudomonas syringae, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas mendocina, Pseudomonas cichorii, Pseudomonas alcaligenes, and Pseudomonas pseudoalcaligenes. The Pseudomonas acidovorans rRNA branch contains Pseudomonas acidovorans, Pseudomonas testosteroni, Pseudomonas delajieldii, Pseudomonas facilis, Pseudomonas palleronii, Pseudomonas saccharophila, and Pseudomonas flava. The third rRNA branch contains Pseudomonas solanacearum, Pseudomonas cepacia, Pseudomonas marginata, Pseudomonas caryophylli, and Pseudomonas lemoignei. Each of these rRNA branches is as heterogeneous as a genus. The Pseudomonas solanacearum and Pseudomonas acidovorans rRNA branches are about as far removed from each other as they are from the genera Janthinobacterium and Derxia and the authentic genus Alcaligenes. These branches are members of the third rRNA superfamily. The Pseudomonas fluorescens rRNA branch is quite different, as it is a member of the second rRNA superfamily, which also contains Azotobacter, Azomonas, Xanthomonas, and some other genera. Along with data from rRNA hybridizations involving. many different gram-negative taxa, these results show clearly that the three Pseudomonas rRNA branches differ at least at the genus level. The genus Xanthomonas is separate in its own right. It constitutes a very tight cluster consisting of Xanthomonas campestris, Xanthomonas fragariae, Xanthomonas axonopodis, and Xanthomonas albilineans (Xanthomonas campestris covers older species names no longer in use). Xanthomonas (Aplanobacter) populi has rRNA cistrons that are indistinguishable from the rRNA cistrons of the xanthomonads mentioned above. There are a number of misnamed taxa. Pseudomonas maltophilia is a somewhat unusual member of Xanthomonas; likewise, Pseudomonas diminuta and Pseudomonas vesicularis are not members of the genus Pseudomonas, and Xanthomonas ampelina is definitely not a member of the genus Xanthomonas. The exact taxonomic positions of the latter three species are unknown. A quantitative comparison showed that fine differentiation of strains by means of DNA-DNA hybridization under stringent conditions at TOR (temperature of optimal renaturation) was meaningful only in the top 7 to 8°C Tm(c) (thermal elution temperature range, 73 to 81°C) of our DNA-rRNA similarity maps and dendrograms (a difference of 1°C in thermal elution temperature Tm(e) from ribosomal DNA similarity corresponded to roughly 14% DNA homology).

Virgibacillus : a new genus to accommodate Bacillus pantothenticus (Proom and Knight 1950). Emended description of Virgibacillus pantothenticus

Twelve strains named Bacillus pantothenticus, at least 29 Bacillus strains representing 16 species belonging to rRNA groups 1 and 2, one Bacillus dipsosauri strain, and 38 strains of Amphibacillus, Aneurinibacillus, Brevibacillus, Halobacillus, Paenibacillus, Sporosarcina and Marinococcus, were characterized genotypically using amplified rDNA restriction analysis (ARDRA), and phenotypically using routine diagnostic characters comprising 61 biochemical tests in the API System and 15 observations of vegetative cell and sporangial morphology. The B. pantothenticus strains were also characterized by fatty acid methyl ester analysis and SDS-PAGE of whole-cell proteins. ARDRA revealed that strains of B. pantothenticus formed a cluster quite separate from other species in rRNA group 1, supporting the recognition of the former as a separate genus, for which the name Virgibacillus is proposed. The polyphasic data also indicate the presence of an as yet undescribed new species within this genus. The species Virgibacillus pantothenticus and related organisms comprising this new genus can be distinguished from members of Bacillus rRNA group 1 (Bacillus sensu stricto), and from members of Paenibacillus and other aerobic endospore-forming bacteria by routine phenotypic tests.

Transfer of Pseudomonas maltophilia Hugh 1981 to the Genus Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov.

Genotypic and phenotypic data (results of deoxyribonucleic acid-ribosomal ribonucleic acid and deoxyribonucleic acid-deoxyribonucleic acid hybridizations, guanine-plus-cytosine content, comparative enzymology, type of ubiquinones, cellular fatty acid composition, growth, niches, and several other characteristics) showed that Pseudomonas maltophilia (Hugh 1981) is generically misnamed and should be transferred to Xanthomonas as Xanthomonas maltophilia (Hugh 1981) comb. nov.

Aerobic Denitrification in Various Heterotrophic Nitrifiers

Various heterotrophic nitrifiers have been tested and found to also be aerobic denitrifiers. The simultaneous use of two electron acceptors (oxygen and nitrate) permits these organisms to grow more rapidly than on either single electron acceptor, but generally results in a lower yield than is obtained on oxygen, alone. One strain, formerly known as “Pseudomonas denitrificans”, was grown in the chemostat and shown to achieve nitrification rates of up to 44 nmol NH3 min−1 mg protein−1 and denitrification rates up to 69 nmol NOinf3sup−1min−1 mg protein−1.Unlike Thiosphaera pantotropha, this strain needed to induce its nitrate reductase. However, the remainder of the denitrifying pathway was constitutive and, like T. pantotropha, “Ps. denitrificans” probably possesses the copper nitrite reductase.

Classification of metal-resistant bacteria from industrial biotopes as Ralstonia campinensis sp. nov., Ralstonia metallidurans sp. nov. and Ralstonia basilensis Steinle et al. 1998 emend.

Thirty-one heavy-metal-resistant bacteria isolated from industrial biotopes were subjected to polyphasic characterization, including 16S rDNA sequence analysis, DNA-DNA hybridizations, biochemical tests, whole-cell protein and fatty-acid analyses. All strains were shown to belong to the Ralstonia branch of the beta-Proteobacteria. Whole-cell protein profiles and DNA-DNA hybridizations revealed two clearly distinct groups, showing low similarity to known Ralstonia species. These two groups, of 8 and 17 isolates, were assigned to two new species, for which the names Ralstonia campinensis sp. nov. and Ralstonia metallidurans sp. nov. are proposed. The type strains are WS2T (= LMG 19282T = CCUG 44526T) and CH34T (= LMG 1195T = DSM 2839T), respectively. Six isolates were allocated to Ralstonia basilensis, which presently contains only the type strain; an emendation of the latter species description is therefore proposed.

Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas island, south Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov.

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.

Bordetella trematum sp. nov., isolated from wounds and ear infections in humans, and reassessment of Alcaligenes denitrificans Rüger and Tan 1983.

Ten strains recognized on the basis of a computer-assisted numerical comparison of whole-cell protein patterns as members of a novel species belonging to the family Alcaligenaceae were examined by using an integrated phenotypic and genotypic approach. This species, for which we propose the name Bordetella trematum sp. nov., was more closely related to the type species of the genus Bordetella (Bordetella pertussis) than to the type species of the genus Alcaligenes (Alcaligenes faecalis) and had the general characteristics of members of this family (i.e., a DNA base ratio in the range from 57 to 70 mol%, a fatty acid profile characterized by high percentages of 16:0, 17:0 cyclo, and 14:0 3OH, nonsaccharolytic metabolism, and several classical biochemical characteristics, including aerobic and microaerobic growth, catalase activity, assimilation of citrate, an absence of anaerobic growth, and an absence of acetylmethylcarbinol and indole production, gelatin liquefaction, and esculin hydrolysis). A reevaluation of the criteria used to classify Alcaligenes denitrificans Rüger and Tan 1983 and Achromobacter xylosoxidans Yabuuchi and Ohyama 1971 as subspecies of Alcaligenes xylosoxidans and additional evidence provided in recent studies revealed that, consistent with present standards, it is appropriate to consider these two taxa distinct species of the genus Alcaligenes.

Genotypic relationships and taxonomic localization of unclassified Pseudomonas and Pseudomonas-like strains by deoxyribonucleic acid: ribosomal ribonucleic acid hybridizations

The deoxyribonucleic acid (DNA):ribosomal ribonucleic acid (rRNA) hybridization technique was used to reveal the relationships and taxonomic positions of an additional 83 strains belonging to 43 saprophytic or pathogenic Pseudomonas species and 29 named and unnamed Pseudomonas-like strains. The DNA:rRNA hybrids were characterized by the following two parameters: (i) the temperature at which one-half of the hybrid was eluted and (ii) the percentage of rRNA binding (the amount of rRNA bound per 100 g of filter-fixed DNA). We also used, for a limited number of strains, numerical analysis of carbon assimilation tests to delineate the finer taxonomic relationships of organisms. Of the 83 strains examined, 78 could be definitely assigned either to an rRNA branch or to an rRNA superfamily within the Proteobacteria. Only 25 of our strains belong in the genus Pseudomonas sensu stricto (our Pseudomonas fluorescens rRNA branch). In general, about two-thirds of the named Pseudomonas species have been misclassified and are distributed over at least seven genera all through the Proteobacteria. These organisms need to be reclassified and generically renamed according to their phylogenetic relationships. However, more detailed phenotypic and genotypic studies are necessary before definite nomenclatural proposals can be made. A comprehensive list of the phylogenetic affiliations of the Pseudomonas species is included.