Clay beads as artificial trapping matrices for monitoring bacterial distribution among urban stormwater infiltration systems and their connected aquifers.

Stormwater infiltration systems (SIS) have been developed to limit surface runoff and flooding in urban areas. The impacts of such practices on the ecological and biological quality of groundwater ecosystems remain poorly studied due to the lack of efficient methodologies to assess microbiological quality of aquifers. In the present study, a monitoring method based on the incubation of artificial matrices (clay beads) is presented to evaluate microbial biomass, microbial activities, and bacterial community structure. Four microbial variables (biomass, dehydrogenase and hydrolytic activities, bacterial community structures) were measured on clay beads incubated in three urban water types (stormwater surface runoffs, SIS-impacted and non-impacted groundwaters) for six SIS. Analyses based on next-generation sequencing (NGS) of partial rrs (16S rRNA) PCR products (V5-V6) were used to compare bacterial community structures of biofilms on clay beads after 10 days of incubation with those of waters collected from the same sampling points at three occasions. Biofilm biomass and activities on clay beads were indicative of nutrient transfers from surface to SIS-impacted groundwaters. Biofilms allowed impacts of SIS on groundwater bacterial community structures to be determined. Although bacterial communities on clay beads did not perfectly match those of waters, clay beads captured the most abundant bacterial taxa. They also captured bacterial taxa that were not detected in waters collected at three occasions during the incubation, demonstrating the integrative character of this approach. Monitoring biofilms on clay beads also allowed the tracking of bacterial genera containing species representing health concerns.

Accumulated sediments in a detention basin: chemical and microbial hazard assessment linked to hydrological processes.

Accumulated sediments in a 32,000-m(3) detention basin linked to a separate stormwater system were characterized in order to infer their health hazards. A sampling scheme of 15 points was defined according to the hydrological behaviour of the basin. Physical parameters (particle size and volatile organic matter content) were in the range of those previously reported for stormwater sediments. Chemical analyses on hydrocarbons, PAHs, PCBs and heavy metals showed high pollutant concentrations. Microbiological analyses of these points highlighted the presence of faecal indicator bacteria (Escherichia coli and intestinal enterococci) and actinomycetes of the genus Nocardia. These are indicative of the presence of human pathogens. E. coli and enterococcal numbers in the sediments were higher at the proximity of the low-flow gutter receiving waters from the catchment. These bacteria appeared to persist over time among urban sediments. Samples highly contaminated by hydrocarbons were also shown to be heavily contaminated by these bacteria. These results demonstrated for the first time the presence of Nocardial actinomycetes in such an urban context with concentrations as high as 11,400 cfu g(-1).

Detection and enumeration of Pseudomonas aeruginosa in soil and manure assessed by an ecfX qPCR assay.

AIMS: To develop a qPCR approach for the detection of Pseudomonas aeruginosa in soil and manure and explore its efficacy and limitations compared with that of a classical culture-dependent approach. METHODS AND RESULTS: A Ps. aeruginosa ecfX qPCR assay was developed. This assay was optimized for soils of contrasting physico-chemical properties and evidenced a three-log dynamic range of detection [5 × 10(4)  - 5 × 10(6) cells (g drywt soil)(-1) ] in inoculated microcosms. Sensitivity was determined to be around 5 × 10(4)  cells (g drywt soil)(-1) . In parallel, the minimum detection limit was estimated in the range of 10-100 CFU (g drywt soil)(-1) using a culture-dependent approach based on the use of a selective medium (cetrimide agar base medium supplemented with nalidixic acid), coupled to ecfX gene amplification to confirm isolate identity. These soil samples led to the growth of abundant non-Ps. aeruginosa colonies mainly belonging to other Pseudomonas species but also some beta-Proteobacteria. These bacteria strongly impacted the detection threshold of this approach. Efficacy of these approaches was compared for Ps. aeruginosa enumeration among manure and agricultural soil samples from various sites in France, Tunisia and Burkina Faso. CONCLUSIONS: The developed qPCR assay enabled a specific detection of Ps. aeruginosa in soil and manure samples. The culture-based approach was usually found more sensitive than the qPCR assay. However, abundance of non-Ps. aeruginosa species among the indigenous communities able to grow on the selective medium affected the sensitivity of this latter approach. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the first specific and sensitive qPCR assay for the detection and enumeration of Ps. aeruginosa in soil and manure and shows its complementarity with a culture-based approach.

Identification of Stenotrophomonas maltophilia strains isolated from environmental and clinical samples: a rapid and efficient procedure.

AIMS: Aim of the study is to identify accurately Stenotrophomonas maltophilia isolates recovered from environmental and clinical samples. METHODS AND RESULTS: Recovery of Sten. maltophilia-like isolates from soil samples using the vancomycin, imipenem, amphotericin B (VIA) selective agar medium enabled distinction of various morphotype colonies. A set of soil and clinical isolates was tested for species identification using different methods. 16S rDNA analyses showed the dark green with a blue halo morphotype to be typical Sten. maltophilia strains. The API-20NE, Vitek-2 and Biolog phenotypic analyses typically used for the identification of clinical isolates did not perform well on these soil isolates. The species-specific PCR screening targeting Sten. maltophilia 23S rDNA and the multiplex smeD/ggpS PCR, differentiating Sten. maltophilia from Stenotrophomonas rhizophila, were tested for improvement of these identification schemes. The latter multiplex PCR identified all isolates tested in this study, whatever be their origin. CONCLUSIONS: Isolation on VIA medium and confirmation of Sten. maltophilia species membership by smeD PCR is proposed to identify environmental and clinical isolates of Sten. maltophilia. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed approach enables isolation and identification of Sten. maltophilia from different environments in an easy and rapid way. This approach will be useful to accurately manage studies on the abundance and distribution of Sten. maltophilia in hospital and nonhospital environments.

Reliability of Pseudomonas aeruginosa semi-automated rep-PCR genotyping in various epidemiological situations.

The purpose of this study was to evaluate the possibility of using a semi-automated repetitive DNA sequences-based polymerase chain reaction (rep-PCR) for typing Pseudomonas aeruginosa isolates. rep-PCR profiles obtained by the DiversiLab system of 84 P. aeruginosa isolates from distinct epidemiological situations were obtained. rep-PCR groupings were in good agreement with the origin of these isolates. Linked rep-PCR profiles were observed for isolates recovered from a same family of cystic fibrosis (CF) patients, for the etiological agents of clustered cases of nosocomial infections, and for some isolates recovered from a same hospital room. rep-PCR and pulsed-field gel electrophoresis SpeI restricted genomic DNA (PFGE-SpeI) profiles were compared. In a few instances, rep-PCR revealed genetic divergences among isolates of a same group of PFGE-SpeI profiles. These divergences could reflect genetic drifts among closely related isolates, as illustrated by those observed between clinical and environmental isolates of a same group of PFGE-SpeI profiles. The interpretation of such differences will require further studies, but the rep-PCR analysis of P. aeruginosa diversity appeared to be an appropriate method to investigate infra-specific genetic relatedness.

RISA-HPLC analysis of lung bacterial colonizers of cystic fibrosis children.

Microbiological analysis of sputum samples, from children affected by cystic fibrosis (CF) and showing signs of acute or chronic infections, is routinely performed by culture-dependent approaches involving selective media and biochemical tests. These identification schemes are time-consuming, and may lead to false negative results. The aim of this work was to evaluate the efficacy of a Ribosomal Intergenic Spacer Analysis (RISA) coupled to high performance liquid chromatography (HPLC) for the detection and monitoring of CF lung microbial colonizers including Staphylococcus aureus, Haemophilus influenzae, Pseudomonas aeruginosa, the Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans. These RISA-HPLC analyses were performed over a 10-months period on infants (below 18 months) and children that were or were not yet known to be colonised by P. aeruginosa. The RISA-HPLC profiles were found specific of the patients' microbial communities. A specific P. aeruginosa RISA-HPLC peak corresponding to 550 bp PCR products was recorded, and used to investigate P. aeruginosa persistence through time and after various therapeutic treatments. The RISA-HPLC profiles showed the CF children to be colonized by few bacterial species, and sometimes revealed peaks corresponding to bacterial species that were not detected by the selective plating approaches. Significant RISA-HPLC infra-specific variations were observed for most bacterial colonizers of CF lungs except P. aeruginosa. These species could yield as much as 5 distinct RISA-HPLC peaks, with some of these profiles being strain-specific. RISA-HPLC shows a great potential for revealing colonization by novel emerging pathogens, and for evaluating the efficacy of therapeutic treatments on the global bacterial community of CF lungs.

Spatio-temporal analysis of infra-specific genetic variations among a Pseudomonas aeruginosa water network hospital population: invasion and selection of clonal complexes.

AIMS: To investigate infra-specific spatio-temporal dynamics of a hospital water network Pseudomonas aeruginosa population. To infer the origin of water network isolates and assess their potential health hazard. METHODS AND RESULTS: 168 P. aeruginosa strains were isolated from tap waters and swabs of tap nozzle aerators of a hospital unit, over 2 years, and from rectal swabs and nosocomial infections. Genetic diversity among this collection was assessed by pulsed field gel electrophoresis of SpeI restricted genomic DNA. Virulence gene sets, biofilm properties, and hypochlorite resistance were analysed. Exactly 68% of the water samples and 74% of the tap nozzle aerators harboured P. aeruginosa. The strains were divided into 22 clonal lineages, with one dominant clone shown to have been involved in a nosocomial infection. CONCLUSIONS: An important turnover among the P. aeruginosa hospital population was observed. Some clonal lineages were found to persist, spread in the unit, and diversify into clonal complexes. Rectal carriage appeared an important source of contamination of the water network. SIGNIFICANCE AND IMPACT OF THE STUDY: High P. aeruginosa infra-specific population diversity suggested a broad ability in colonizing water networks but persistence analysis indicated a strong selection leading to the emergence of dominant clones.

Improved reliability of Pseudomonas aeruginosa PCR detection by the use of the species-specific ecfX gene target.

Reliability of the most widely used PCR screenings for the human opportunistic pathogen Pseudomonas aeruginosa was evaluated. Specificity analyses showed the gyrB, toxA, and 16S-23S rDNA internal transcribed spacer (ITS) but not the 16S rDNA, oprI, oprL, and fliC PCR screenings to discriminate P. aeruginosa cells from a collection of fifteen Pseudomonas species. Sensitivity analyses showed all these PCR except the toxA one to be reliable for 100% of the P. aeruginosa strains tested in this study. Specificity of the ITS and gyrB PCR screenings were further investigated on 9 soils and 29 freshwater DNA extracts of different origins, and on DNA extracted from 3 horse manures. The ITS PCR showed the highest efficacy on water and soil DNA extracts but only the gyrB one detected P. aeruginosa DNA in horse manure. DNA sequence analyses of ITS and gyrB PCR products revealed uncertainties and false positive results in these P. aeruginosa identification schemes. A novel PCR screening, targeting the ecfX gene, was thus developed. ecfX encodes an ECF (extracytoplasmic function) sigma factor which is restricted to P. aeruginosa, and might play a role in haem-uptake and virulence. Specificity and sensitivity analyses showed the ecfX PCR screening to be highly reliable, giving PCR products of the expected size for all P. aeruginosa strains tested and not amplifying DNA from any of the other Pseudomonas species tested. The ecfX PCR screening was validated on environmental DNA extracts. DNA sequence analyses of the ecfX PCR products confirmed their identity and allocation to P. aeruginosa. These investigations suggest a preferential colonization of water rather than soil environments by P. aeruginosa. Detection limits of P. aeruginosa in environmental samples were improved by the ecfX PCR screening.

Distribution and genetic diversity of bacterial thiopurine methyltransferases in soils emitting dimethyl selenide.

Dimethyl selenide (DMSe) and dimethyl diselenide (DMDSe) emissions by soil samples spiked with selenite or (methyl)selenocysteine, with or without a supplement of nutrient broth and glucose were measured. DMSe was the main form of volatile Se produced, and was observed for both Se-substrates. DMDSe was only emitted from soils spiked with (methyl)selenocysteine. Two bacterial thiopurine methyltransferases (TPMTs), TPMT-I and TPMT-E, have been reported to be involved in DMSe and DMDSe emissions [J. Bacteriol. 184 (2002) 3146; Appl. Environ. Microbiol. 69 (2003) 3784]. To establish if these TPMTs or other members of their gene family could have contributed to the DMSe emissions observed, the diversity of bTPMT gene (tpm) sequences among the soils of this study was investigated. Total DNAs from these soils were extracted and screened using the tpm PTCF2-PTCR2 consensus primers defined to PCR amplify this gene family. The PCR products obtained from two soils were cloned, analysed by PCR-RFLP, and sequenced. Their analysis showed an important diversity of tpm lineages (around 12) in soils. Phylogenetic analysis of the deduced TPMT sequences of these soils revealed lineages not previously recorded in the databases, sequences closely related or identical to freshwater TPMTs, or sequences encoding TPMTs closely related to those of Pseudomonas fragi TPMT-K, Pseudomonas Hsa.28 TPMT-I, or Colwellia psychrerythraea TPMT-Z. Nested PCRs, allowing detection of about 13 distinct tpm soil and freshwater lineages by PTCF2-PTCR2 PCR screenings, were performed on the soil total DNAs. These PCRs confirmed the sequencing data, and allowed to recover lineages not detected by the cloning strategy. These results indicate that soils, like the freshwater samples, harbour TPMT-I gene sequences but may also have distinct tpm lineages. This study further supports our hypothesis that TPMTs contribute to DMSe soil emissions.

Freshwater selenium-methylating bacterial thiopurine methyltransferases: diversity and molecular phylogeny.

The diversity of bacterial thiopurine methyltransferases (bTPMT) among five natural Se-methylating freshwaters was investigated by polymerase chain reaction (PCR) screenings and sequencings. DNA sequence analyses confirmed the cloned products' identity and revealed a broad diversity of freshwater TPMTs. Neighbour-joining (NJ) phylogenetic analyses combining these sequences, all GenBank entries closely related to these sequences and deduced TPMTs obtained in this work from selected gamma-proteobacteria showed TPMTs to form a distinct radiation, closely related to UbiG methyltransferases. Inside the TPMT phylogenetic cluster, eukaryote sequences diverged early from the bacterial ones, and all the bacterial database entries belonged to a subgroup of gamma-proteobacteria, with an apparent lateral transfer of a particular allele to beta-proteobacteria of Bordetella. The NJ phylogenetic tree revealed 22 bTPMT lineages, 10 of which harboured freshwater sequences. All lineages showed deep and long branches indicative of major genetic drifts outside regions encoding highly conserved domains. Selected residues among these highly variable domains could reflect adaptations for particular ecological niches. PCR lineage-specific primers differentiated Se-methylating freshwaters according to their 'tpm lineage' signatures. Most freshwater tpm alleles were found to be distinct from those available in the databases, but a group of tpm was found encoding TPMTs identical to an Aeromonas veronii TPMT characterized in this work.

Radiation and functional specialization of the family-3 glycoside hydrolases.

A phylogenetic analysis of the glycoside hydrolases of family 3 (GH3s) was conducted in order to infer particular trends in its evolution: functional specialization, gene transfer events, gene duplications and paralogous evolution, and gene deletions. The phylogenetic analysis of GH3s revealed six clusters, i.e., A, B, C, D, E, and F that could fit the definition of 3 sub-families, i.e., AB, AB' and AB". While the sub-families AB' and AB" contain a single cluster, F and E, respectively, the AB sub-family is sub-divided into four clusters. Global analysis of the GH3 phylogenetic tree suggests a primary burst of amplification of the GH3s that might have led to these sub-families. Specializations, gene transfers, and gene duplications among each of these sub-families and phylogenetic clusters might then have occurred and have been inferred. The fine comparison of the enzyme properties and phylogenetic relationships of GH3s allowed to detect common functional groups that belong to the same cluster (D, E or F), or sub-cluster (A1, A2 or B2). The prokaryotic and eukaryotic beta-xylosidases and beta-glucosidases belong to the AB and AB' sub-families, and the N-acetylglucosaminidases are in sub-family AB" (in cluster E). In some instances (B1, B2, C1, C2, and C3), the lack of data and/or the high heterogeneity of the hydrolytic properties did not allow to infer a particular link between an enzyme functional group and a phylogenetic cluster, suggesting the emergence of some highly specialized GH3s.

Characterization of a Pinus pinaster cDNA encoding an auxin up-regulated putative peroxidase in roots.

As part of a study to identify host plant genes regulated by fungal auxin during ectomycorrhiza formation, we differentially screened a cDNA library constructed from roots of auxin-treated Pinus pinaster (Ait.) Sol. seedlings. We identified three cDNAs up-regulated by auxin. Sequence analysis of one of these cDNAs, PpPrx75, revealed the presence of an open reading frame of 216 amino acids with the characteristic consensus sequences of plant peroxidases. The deduced amino acid sequence showed homology with Arabidopsis thaliana (L.) Heynh., Arachis hypogaea L. and Stylosanthes humilis HBK cationic peroxidases. Amino acid sequence identities in the conserved domains of plant peroxidases ranged from 60 to 100%. In PpPrx75, there are five cysteine residues and one histidine residue that are found at conserved positions among other peroxidases. A potential glycosylation site (NTS) is present in the deduced sequence. Phylogenetic analysis showed that PpPrx75 is closely related to two A. thaliana peroxidases. The PpPrx75 cDNA was induced by active auxins, ethylene, abscisic acid and quercetin, a flavonoid possibly involved in plant-microorganism interactions. Transcript accumulation was detected within 3 h following root induction by auxin, and the amount of mRNA increased over the following 24 h. The protein synthesis inhibitor cycloheximide did not inhibit indole-3-acetic acid-induced transcript accumulation, suggesting that PpPrx75 induction is a primary (direct) response to auxin. This cDNA can be used to study expression of an auxin-regulated peroxidase during ectomycorrhiza formation.

Modification of the protein expression pattern induced in the nitrogen-fixing actinomycete Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host Alnus glutinosa and cloning of the sodF gene.

Two-dimensional (2-D) polyacrylamide gel electrophoresis was used to detect proteins induced in Frankia sp. strain ACN14a-tsr by root exudates of its symbiotic host, Alnus glutinosa. The 5 most prominent proteins were purified from 2-D gels and characterized by N-terminal sequencing. All of these proteins had a high percentage of similarity with known stress proteins. One protein match was the Fe superoxide dismutase (Fe-SOD), another was a tellurite resistance protein (Ter), the third was a bacterioferritin comigratory protein (Bcp); and two matches, differing only by their isoelectric point, were the same small heat shock protein (Hsp), a major immune reactive protein found in mycobacteria. This suggests that the symbiotic microorganism Frankia, first responds with a normal stress response to toxic root products of its symbiotic host plant. To confirm its identity, the gene corresponding to the Fe-SOD protein, sodF was isolated from a genomic library by a PCR-approach and sequenced. It is the first stress response gene characterized in Frankia.

Analysis of pFQ31, a 8551-bp cryptic plasmid from the symbiotic nitrogen-fixing actinomycete Frankia.

The actinomycete Frankia has never been transformed genetically. To favour the development of Frankia cloning vectors, we have fully sequenced the Frankia alni pFQ31 cryptic plasmid and performed analyses to characterise its coding and non-coding regions. This plasmid is 8551 bp-long and contains 72% G+C. Computer-assisted analyses identified 18 open reading frames (ORFs). These ORFs show a synonymous codon usage different from the one of Frankia chromosomal genes, suggesting an evolutionary bias linked to the nature of the replicon or a horizontal transfer. Three ORFs were found to encode genes likely to be involved in plasmid replication and stability: parFA (partition protein), ptrFA (transcriptional repressor of the GntR family) and repFA (initiation of replication). DNA signatures of a replication origin were identified in the ptrFA-repFA intergenic region. These structural motifs are similar to those observed among origins of iteron-containing plasmids replicating via a θ mode.

Burkholderia cepacia genomovar III Is a common plant-associated bacterium.

A polyphasic taxonomic study involving DNA-DNA hybridization, whole-cell protein electrophoresis, and 16S ribosomal DNA sequence analysis revealed that a group of Burkholderia cepacia-like organisms isolated from the rhizosphere or tissues of maize, wheat, and lupine belong to B. cepacia genomovar III, a genomic species associated with "cepacia syndrome" in cystic fibrosis patients. The present study also revealed considerable protein electrophoretic heterogeneity within this species and demonstrated that the B. cepacia complex consists of two independent phylogenetic lineages.

Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2.

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium. The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10(3) to 10(4) agrobacteria. g of dry soil(-1) in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.

Characterization of the 20S proteasome from the actinomycete Frankia.

Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non-leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non-existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts-r. It is composed of one alpha-subunit and one beta-subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin-like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the beta-subunit is encoded with a 52-amino-acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active.

Analysis of Frankia evolutionary radiation using glnII sequences.

Using a glnII (encoding glutamine synthetase II) PCR selective screening, a Frankia ACN14a gene library clone was isolated. A derived glnII-hybridising 2.7-kb HindIII subclone was characterised. Identities of 95% and 93% were observed, respectively, with the corresponding Frankia CpI1 glnI and glnII regions. A variable segment of the glnII region was selected, PCR amplified from various Frankia genomes, sequenced, and used to investigate phylogenetic relationships within the genus. glnII phylogenetic inferences are well-resolved and allowed us to deduce evolutionary trends among Frankia. Frankia radiation seems to begin with a diversification according to the ability or not to infect actinorhizal plants. The infective strains are divided into two clusters matching plant-colonising specificities.

Genetic complementation of rhizobial nod mutants with Frankia DNA: artifact or reality?

Two divergent reports have been published on the genetic complementation of rhizobial nod mutants using Frankia DNA. In 1991 putative Frankia cosmid library clones were reported to restore normal nodulation properties to Rhizobium leguminosarum biovar viciae nodD::Tn5, but no supporting sequence data were published. In 1992 a second group reported a failure to find any evidence of functional complementation of various rhizobial nod mutants by Frankia DNA (nodA, nodB and nodC). Complementation tests of nine NodR. leguminosarum bv. viciae or Sinorhizobium meliloti Tn5 mutants (nodA-, nodB-, nodC-, nodD-, nodF-, nodL-, nodH-) were thus performed using a Frankia gene library in pLAFR3 to clarify this situation. Rhizobial transconjugants obtained by tri-parental matings were screened for restoration of the nodulation phenotype on their host plants, Vicia sativa subsp. nigra or Medicago sativa. Nodulation was observed on plants inoculated with transconjugants of the R. leguminosarum bv. viciae nodC::Tn5 mutant. The Nod+ rhizobial transconjugants were isolated and analysed. The Nod+ phenotype of these transconjugants was found to be due to Tn5 excision/transposition. No functional complementation was found with any of the mutants used, suggesting that rhizobial complementation of nod mutants with Frankia DNA is unlikely to occur.

Burkholderia graminis sp. nov., a rhizospheric Burkholderia species, and reassessment of [Pseudomonas] phenazinium, [Pseudomonas] pyrrocinia and [Pseudomonas] glathei as Burkholderia.

In a survey of soil and wheat or maize rhizoplane bacteria isolated using a medium containing azelaic acid and tryptamine as sole carbon and nitrogen sources, respectively, a large proportion of Burkholderia-like bacteria were found. Among them, a homogeneous group of strains was identifiable based on phenotypic properties, fatty acid composition, DNA-DNA hybridizations and 16S rDNA sequences. According to molecular data, this group belongs to the genus Burkholderia but its weak similarity to previously described species suggests that it belongs to a novel species. Closest 16S rDNA phylogenetic neighbours of this species are Burkholderia caryophylli and two previously named Pseudomonas species which clearly appear to be part of the Burkholderia genus and were thus named Burkholderia glathei comb. nov. and Burkholderia phenazinium comb. nov. Strains of the new species are oxidase- and catalase-positive, produce indole and gelatinase, and use L-xylose, lactose, rhamnose, trehalose, D-lyxose, L-arabitol, xylitol and D-raffinose as sole carbon source. This novel taxon is named Burkholderia graminis. In the course of this study, [Pseudomonas] pyrrocinia also proved to be a member of the Burkholderia genus.