Monique Gillis

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.

Characterization of Azorhizobium caulinodans gen. nov. sp. nov., a Stem-Nodulating Nitrogen-Fixing Bacterium Isolated from Sesbania rostrata

Twenty stem- and root-nodulating bacterial strains isolated from stem nodules of Sesbania rostrata were compared by numerical analysis of 221 phenotypic features with nine strains which effectively nodulate only the roots of this plant and with representative strains from the genera Rhizobium and Bradyrhizobium. Representative organisms from the different clusters were investigated further, together with possibly related organisms, by performing comparative gel electrophoresis of whole-cell proteins and by performing deoxyribonucleic acid (DNA)-DNA and DNA-ribosomal ribonucleic acid (rRNA) hybridizations. 3H-labeled rRNA was prepared from Sesbania stem- and root-nodulating bacterial strain ORS 571T (T = type strain); [14C]rRNA from Bradyrhizobium japonicum NZP 5549T was also used. The following conclusions were drawn: (i) the Sesbania root-nodulating bacterial strains are genuine rhizobia; (ii) the Sesbania stem- and root-nodulating strains are quite different from Rhizobium and Bradyrhizobium, and thus they constitute a separate rRNA subbranch on the Rhodopseudomonas palustris rRNA branch in rRNA superfamily IV; and (iii) the closest relative of these organisms is Xanthobacter, but they are phenotypically and genotypically sufficiently different from the latter genus to deserve a separate generic rank. Because the feature of free-living nitrogen fixation is quite discriminative, a new genus, Azorhizobium, is proposed, with one species, Azorhizobium caulinodans. The type strain is strain ORS 571 (= LMG 6465).

Advantages of multilocus sequence analysis for taxonomic studies: a case study using 10 housekeeping genes in the genus Ensifer (including former Sinorhizobium)

There is a need for easy, practical, reliable and robust techniques for the identification and classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence analysis and DNA-DNA hybridization. Here, we demonstrate that multilocus sequence analysis (MLSA) of housekeeping genes is a valuable alternative technique. An MLSA study of 10 housekeeping genes (atpD, dnaK, gap, glnA, gltA, gyrB, pnp, recA, rpoB and thrC) was performed on 34 representatives of the genus Ensifer. Genetic analysis and comparison with 16S and 23S rRNA gene sequences demonstrated clear species boundaries and a higher discrimination potential for all housekeeping genes. Comparison of housekeeping gene sequence data with DNA-DNA reassociation data revealed good correlation at the intraspecies level, but indicated that housekeeping gene sequencing is superior to DNA-DNA hybridization for the assessment of genetic relatedness between Ensifer species. Our MLSA data, confirmed by DNA-DNA hybridizations, support the suggestion that Ensifer xinjiangensis is a later heterotypic synonym of Ensifer fredii.

Transfer of Rhizobium loti, Rhizobium huakuii, Rhizobium ciceri, Rhizobium mediterraneum, and Rhizobium tianshanense to Mesorhizobium gen. nov.

Reasons are advanced for removal of Rhizobium ciceri, Rhizobium huakuii, Rhizobium loti, Rhizobium mediterraneum, and Rhizobium tianshanense from the genus Rhizobium and for establishment of Mesorhizobium gen. nov. for these species. A description of the genus Mesorhizobium and amended descriptions of Mesorhizobium ciceri, Mesorhizobium huakuii, Mesorhizobium loti, Mesorhizobium mediterraneum, and Mezorhizobium tianshanense are provided.

DNA-DNA hybridization study of Bradyrhizobium strains.

DNA-DNA hybridizations were performed between Bradyrhizobium strains, isolated mainly from Faidherbia albida and Aeschynomene species, as well as Bradyrhizobium reference strains. Results indicated that the genus Bradyrhizobium consists of at least 11 genospecies, I to XI. The genospecies formed four subgeneric groups that were more closely related to each other (>40% DNA hybridization) than to other genospecies (<40% DNA hybridization): (i) genospecies I (Bradyrhizobium japonicum), III (Bradyrhizobium liaoningense), IV and V; (ii) genospecies VI and VIII; (iii) genospecies VII and IX; and (iv) genospecies II (Bradyrhizobium elkanii), X and XI. Photosynthetic Aeschynomene isolates were found to belong to at least two distinct genospecies in one subgeneric group. DNA-DNA hybridization data are compared with data from amplified fragment length polymorphism analysis and 165-23S rDNA spacer sequence analysis.

Emended description of Herbaspirillum; inclusion of [Pseudomonas] rubrisubalbicans, a milk plant pathogen, as Herbaspirillum rubrisubalbicans comb. nov.; and classification of a group of clinical isolates (EF group 1) as Herbaspirillum species 3.

[Pseudomonas] rubrisubalbicans, a mild plant pathogen. Herbaspirillum seropedicae, and EF group 1 strains (clustered by an immunological method) were investigated by a polyphasic approach with DNA-rRNA and DNA-DNA hybridizations and auxanography on 147 substrates. Our results show that they all belong to the genus Herbaspirillum. In addition to H. seropedicae, two other species are described: Herbaspirillum rubrisubalbicans and a new unnamed species, Herbaspirillum species 3, containing mainly strains of clinical origin. The three species can be differentiated on the basis of their auxanographic features and DNA-DNA similarities. The type strain of H. rubrisubalbicans is NCPPB 1027 (=LMG 2286); representative strains of the third Herbaspirillum species are strains CCUG 189 (=LMG 5523), CCUG 10263 (=LMG 5934), and CCUG 11060 (=LMG 5321). It has been confirmed that H. rubrisubalbicans is an endophytic diazotroph. It colonizes the roots, the stems, and predominantly the leaves of sugarcane (Saccharum spp.), while Herbaspirillum seropedicae colonizes in large numbers many different species of the Gramineae. Both diazotrophic Herbaspirillum species could be differentiated with meso-erythritol and N-acetylglucosamine. Oligonucleotide probes based on partial sequences of the 23S rRNA of H. seropedicae and H. rubrisubalbicans (HS and HR probes, respectively), were constructed and used as diagnostic probes.

Burkholderia ambifaria sp. nov., a novel member of the Burkholderia cepacia complex including biocontrol and cystic fibrosis-related isolates.

A polyphasic taxonomic study, including amplified fragment length polymorphism (AFLP) fingerprinting, DNA-DNA hybridizations, DNA base-ratio determinations, phylogenetic analysis, whole-cell fatty acid analyses and an extensive biochemical characterization, was performed on 19 Burkholderia cepacia-like isolates from the environment and cystic fibrosis (CF) patients. Several of the environmental isolates have attracted considerable interest due to their biocontrol properties. The polyphasic taxonomic data showed that the strains represent a new member of the B. cepacia complex, for which the name Burkholderia ambifaria sp. nov. is proposed. The type strain is strain LMG 19182T. B. ambifaria can be differentiated from the other members of the B. cepacia complex by means of AFLP fingerprinting, whole-cell fatty acid analysis, biochemical tests (including ornithine and lysine decarboxylase activity, acidification of sucrose and beta-haemolysis) and a newly developed recA gene-based PCR assay. 16S rDNA-based RFLP analysis and PCR tests allowed differentiation of B. ambifaria from Burkholderia multivorans, Burkholderia vietnamiensis and B. cepacia genomovar VI, but not from B. cepacia genomovars I and III and Burkholderia stabilis. The finding that this new taxon includes both strains isolated from CF patients and potentially useful biocontrol strains supports the general consensus that the large-scale use of biocontrol strains belonging to the B. cepacia complex would be ill-advised until more is known about their potential pathogenic mechanisms.

Photosynthetic Bradyrhizobia Are Natural Endophytes of the African Wild Rice Oryza breviligulata

ABSTRACT We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains ofBradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulatacould significantly enhance cultivated rice production.

Burkholderia fungorum sp. nov. and Burkholderia caledonica sp. nov., two new species isolated from the environment, animals and human clinical samples

A polyphasic taxonomic study that included DNA-DNA hybridizations, DNA base ratio determinations, 16S rDNA sequence analyses, whole-cell protein and fatty acid analyses and an extensive biochemical characterization was performed on 16 strains isolated from the environment, animals and human clinical samples. The isolates belonged to the genus Burkholderia, were phylogenetically closely related to Burkholderia graminis, Burkholderia caribensis and Burkholderia phenazinium and had G+C contents between 61.9 and 62.2 mol%. Seven strains isolated from the rhizosphere were assigned to Burkholderia caledonica sp. nov. [type strain LMG 19076T (= CCUG 42236T)]. Nine strains isolated from the environment, animals and human clinical samples were assigned to Burkholderia fungorum sp. nov. [type strain LMG 16225T (= CCUG 31961T)]. Differential tests for B. graminis, B. caribensis, B. phenazinium, B. caledonica and B. fungorum include the following: assimilation of trehalose, citrate, DL-norleucine, adipate and sucrose; nitrate reduction; growth in the presence of 0.5% NaCl; and beta-galactosidase activity.

Comparison of sequence analysis of 16S-23S rDNA spacer regions, AFLP analysis and DNA-DNA hybridizations in Bradyrhizobium

The sequences of the 16S-23S rDNA intergenic spacer region of 62 strains of Bradyrhizobium, including representatives of the three valid species, were determined. The majority of strains had a single rRNA operon type and all contained a tRNA(Ala) and a tRNA(Ile) gene. Analysis of the sequence data produced groupings in line with previously obtained AFLP data. DNA-DNA hybridizations were performed to determine to what extent spacer sequences and AFLP profiles reflected the overall genomic similarities. Strains belonging to the same AFLP group, and strains with spacer sequences diverging less than 4%, were found to belong to the same genospecies. More remote relationships (DNA homology levels of 40-60%) between species were reflected in the spacer sequence analysis, but not in the AFLP analysis. For the genus Bradyrhizobium, 16S-23S rDNA spacer sequence analysis provides taxonomic information similar, but not always identical to that obtained by DNA-DNA hybridizations. Our results indicate that this genus consists of a group of four highly related genospecies (Bradyrhizobium japonicum, Bradyrhizobium liaoningense and two other genospecies) and at least three other genospecies, one of which is Bradyrhizobium elkanii.