Karel Kersters

RECLASSIFICATION OF XANTHOMONAS.

A comprehensive DNA-DNA hybridization study was performed by using 183 strains of the genus Xanthomonas. This genus was shown to comprise 20 DNA homology groups which are considered genomic species. Four groups corresponded to the previously described species Xanthomonas albilineans, Xanthomonas fragariae, Xanthomonas oryzae, and Xanthomonas populi. The previously described species Xanthomonas campestris was heterogeneous and was divided into 16 DNA homology groups. One of these groups exhibited a high level of DNA homology with Xanthomonas axonopodis. The 62 pathovars represented in this study were allocated to appropriate species. Our results, together with previous taxonomic data, supported a comprehensive revision of the classification of the genus Xanthomonas. The species X. albilineans, X. fragariae, X. oryzae, and X. populi are not affected. The type species of the genus, X. campestris (Pammel 1895) Dowson 1939, is emended to include only the pathovars obtained from crucifers (i.e., X. campestris pv. aberrans, X. campestris pv. armoraciae, X. campestris pv. barbareae, X. campestris pv. campestris, X. campestris pv. incanae, and X. campestris pv. raphani). X. axonopodis Starr and Garces 1950 is emended to include 34 former X. campestris pathovars. The following species names are proposed: Xanthomonas arboricola sp. nov., including X. arboricola pv. corylina, X. arboricola pv. juglandis, X. arboricola pv. poinsettiicola (type C strains of the former X. campestris pathovar), X. arboricola pv. populi, and X. arboricola pv. pruni; Xanthomonas bromi sp. nov. for strains isolated from bromegrass; Xanthomonas cassavae (ex Wiehe and Dowson 1953) sp. nov., nom. rev.; Xanthomonas codiaei sp. nov., including type B strains of the former taxon X. campestris pv. poinsettiicola; Xanthomonas cucurbitae (ex Bryan 1926) sp. nov., nom. rev.; Xanthomonas hortorum sp. nov., including X. hortorum pv. hederae, X. hortorum pv. pelargonii, and X. hortorum pv. vitians; Xanthomonas hyacinthi (ex Wakker 1883) sp. nov., nom. rev.; Xanthomonas melonis sp. nov.; Xanthomonas pisi (ex Goto and Okabe 1958) sp. nov., nom. rev.; Xanthomonas sacchari sp. nov. for strains isolated from diseased sugarcane in Guadeloupe; Xanthomonas theicola sp. nov.; Xanthomonas translucens (ex Jones, Johnson, and Reddy 1917) sp. nov., nom. rev., including X. translucens pv. arrhenatheri, X. translucens pv. cerealis, X. translucens pv. graminis, X. translucens pv. hordei, X. translucens pv. phlei, X. translucens pv. phleipratensis, X. translucens pv. poae, X. translucens pv. secalis, X. translucens pv. translucens, and X. translucens pv. undulosa; Xanthomonas vasicola sp. nov., including X. vasicola pv. holcicola and X. vasicola pv. vasculorum (type B strains of the former taxon X. campestris pv. vasculorum); and Xanthomonas vesicatoria (ex Doidge 1920) sp. nov., nom. rev., which includes the type B strains of the former taxon X. campestris pv. vesicatoria. Differentiating characteristics were determined for the new species on the basis of metabolic activity on a range of carbon substrates by using the Biolog GN microplate system.

Cutting a gordian knot: Emended classification and description of the genus Flavobacterium, emended description of the family Flavobacteriaceae, and proposal of Flavobacterium hydatis nom nov (basonym, Cytophaga aquatilis Strohl and Tait 1978).

The phylogenetic positions and G+C contents of most species belonging to the genera Flavobacterium, Cytophaga, and Flexibacter and several related taxa were determined. Most of the strains included in this study belong to rRNA superfamily V, as shown by DNA-rRNA hybridization data, but the three main genera are highly polyphyletic. Several so-called Cytophaga and Flexibacter species isolated from soil and freshwater cluster with the type species of the genus Flavobacterium, Flavobacterium aquatile, and with Flavobacterium branchiophilum. The fatty acid and protein profiles of members of this group of organisms were determined. We provide an emended description of the genus Flavobacterium and propose new combinations for the following 7 of the 10 validly described species included in this genus: Flavobacterium columnare, Flavobacterium flevense, Flavobacterium johnsoniae (we also correct the specific epithet of this taxon), Flavobacterium pectinovarum, Flavobacterium psychrophilum, Flavobacterium saccharophilum, and Flavobacterium succinicans. A new name, Flavobacterium hydatis, is proposed for [Cytophaga] aquatilis Strohl and Tait 1978. The emended genus Flavobacterium contains bacteria that have the following main characteristics: gram-negative rods that are motile by gliding, produce yellow colonies on agar, are chemoorganotrophs and aerobes, decompose several polysaccharides but not cellulose, and are widely distributed in soil and freshwater habitats. Three Flavobacterium species are pathogenic for fish. The G+C contents of Flavobacterium DNAs range from 32 to 37 mol%. An emended description of the family Flavobacteriaceae is also provided.

Evaluation of the DNA fingerprinting method AFLP as a new tool in bacterial taxonomy

We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In totals, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xanthomonas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aeromonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specific adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5% (w/v) polyacrylamide/8.3 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 30-50 visualized PCR products in the size range 80-550 bp, were captured by a high-resolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecific level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies.

Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov.

We performed an integrated genotypic and phenotypic analysis of 128 strains of the genera Burkholderia, Ralstonia, and Pseudomonas in order to study the taxonomic structure of Burkholderia cepacia and its relationships with other Burkholderia species. Our data show that presumed B. cepacia strains isolated from cystic fibrosis patients belong to at least five distinct genomic species, one of which was identified as Burkholderia vietnamiensis. This group of five phenotypically similar species is referred to as the B. cepacia complex. The name Burkholderia multivorans is proposed for one of these genomic species, which was formerly referred to as B. cepacia genomovar II; the remaining B. cepacia groups are referred to as genomovars I, III, and IV, pending additional differential phenotypic tests. The role and pathogenic potential of each of these taxa, particularly in view of the potentially fatal infections in cystic fibrosis patients, need further evaluation. The data presented also demonstrate that Pseudomonas glathei and Pseudomonas pyrrocinia should be reclassified as Burkholderia species.

New perspectives in the classification of the flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen-nov, and Empedobacter nom-rev.

Our present knowledge concerning the genotypic, chemotaxonomic, and phenotypic characteristics of members of the genus Flavobacterium and some related genera, including the genus Weeksella, was used to revise the classification of these organisms. The generically misclassified organisms Flavobacterium balustinum, Flavobacterium gleum, Flavobacterium indologenes, Flavobacterium indoltheticum, Flavobacterium meningosepticum, and Flavobacterium scophthalmum are included in a new genus, Chryseobacterium, with Chryseobacterium gleum as the type species. The generically misclassified organism Flavobacterium breve is included in the revived genus Empedobacter as Empedobacter brevis, whereas the generically misclassified organism Weeksella zoohelcum is included in the new genus Bergeyella as Bergeyella zoohelcum.

Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp. nov.

A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analysis, whole-cell protein and fatty acid analyses, AFLP (amplified fragment length polymorphism) fingerprinting and an extensive biochemical characterization was performed on 10 strains provisionally identified as Alcaligenes faecalis-like bacteria. The six environmental and four human isolates belonged to the genus Ralstonia and were assigned to a new species for which the name Ralstonia gilardii sp. nov. is proposed. The type strain is LMG 5886T.

Transfer of Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972 to Pantoea gen. nov. as Pantoea agglomerans comb. nov. and description of Pantoea dispersa sp. nov.

Deoxyribonucleic acid (DNA)-DNA hybridization was performed with 10 strains belonging to the “Erwinia herbicola-Enterobacter agglomerans complex” by using the competition method on nitrocellulose filters. These strains exhibited more than 75% DNA binding to Erwinia herbicola ATCC 14589T (T = type strain) and constitute DNA hybridization group 14589 (including strains ATCC 14589T and CDC 1429-71 from DNA hybridization group III [D. J. Brenner, G. R. Fanning, J. K. Leete Knutson, A. G. Steigerwalt, and M. J. Krichevsky, Int. J. Syst. Bacteriol. 34:45-55, 1984]). The high level of genomic relatedness of these strains was confirmed by the similarities observed in their electrophoretic protein patterns. On the basis of our data, DNA hybridization group 14589 constitutes a discrete species within the family Enterobacteriaceae. Its closest relative is DNA hybridization group 27155 (41 to 53% DNA relatedness), which was previously defined and includes the type strains, among others, of Enterobacter agglomerans, Erwinia herbicola, and Erwinia milletiae (A. Beji, J. Mergaert, F. Gavini, D. Izard. K. Kersters, H. Leclerc, and J. De Ley, Int. J. Syst. Bacteriol. 38:77-88, 1988). We propose to unite DNA hybridization groups 14589 and 27155 in a single genus, Pantoea gen. nov. Pantoea agglomerans (Beijerinck 1888) comb. nov. is proposed to contain most strains of DNA hybridization group 27155 (including DNA hybridization group XIII of Brenner et al.), and its type strain is strain ATCC 27155 (= NCTC 9381 = LMG 1286). Pantoea dispersa sp. nov. is proposed to contain DNA hybridization group 14589, and its type strain is strain ATCC 14589 (= LMG 2603). Descriptions of the genus and its two species are given.

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.

High-Resolution Genotypic Analysis of the Genus Aeromonas by AFLP Fingerprinting

We investigated the ability of a recently developed genomic fingerprinting technique, named AFLP, to differentiate the 14 currently defined DNA hybridization groups (HGs) in the genus Aeromonas. We also determined the taxonomic positions of the phenospecies Aeromonas allosaccharophila, Aeromonas encheleia, Aeromonas enteropelogenes, and Aeromonas ichthiosmia, which have not been assigned to HGs yet. A total of 98 Aeromonas type and reference strains were included in this study. For the AFLP analysis, the total genomic DNA of each strain was digested with restriction endonucleases ApaI and TaqI. Subsequently, restriction fragments were selectively amplified under high-stringency PCR conditions. The amplification products were electrophoretically separated on a polyacrylamide gel and visualized by autoradiography. Following high-resolution densitometric scanning of the resulting band patterns, AFLP data were further processed for a computer-assisted comparison. A numerical analysis of the digitized fingerprints revealed 13 AFLP clusters which, in general, clearly supported the current Aeromonas taxonomy derived from DNA homology data. In addition, our results indicated that there is significant genotypic heterogeneity in Aeromonas eucrenophila (HG6), which may lead to a further subdivision of this species. A. allosaccharophila and A. encheleia did not represent a separate AFLP cluster but were found to be genotypically related to HG8/10 and HG6, respectively. In addition, the results of the AFLP analysis also confirmed the phylogenetic findings that A. enteropelogenes and A. ichthiosmia are in fact identical to Aeromonas trota (HG13) and Aeromonas veronii (HG8/10), respectively. The results of this study clearly show that the AFLP technique is a valuable new high-resolution genotypic tool for classification of Aeromonas species and also emphasize that this powerful DNA fingerprinting method is important for bacterial taxonomy in general.

Acetobacter diazotrophicus sp. nov., a Nitrogen-Fixing Acetic Acid Bacterium Associated with Sugarcane

Results of deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid and DNA-DNA hybridizations, together with a phenotypic and chemotaxonomic analysis, revealed that nitrogen-fixing bacteria isolated from roots and stems of sugarcane belong to a new species in the genus Acetobacter, for which the name Acetobacter diazotrophicus sp. nov. is proposed. Strain LMG 7603 (= Dobereiner PAI 5 = ATCC 49037) is the type strain.