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.

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.

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.

Molecular characterization of the Pseudomonas syringae pv. pisi plasmid-borne avirulence gene avrPpiB which matches the R3 resistance locus in pea.

An avirulence gene (designated avrPpiB) from race 3 of Pseudomonas syringae pv. pisi was cloned and sequenced. The gene corresponded to a single open reading frame of 831 nt identified by transposon mutagenesis and subcloning. This ORF encodes a predicted hydrophilic protein of 276 amino acids (MW 31,300). It effects the expression of a resistance mechanism governed by a single genetic locus in pea. Cosegregation of resistance at the R3 locus of pea was observed towards race 3 and a transconjugant carrying the cloned avrPpiB gene according to the predicted 3:1 ratio of resistant:susceptible F2 progeny from a cross between Jade (R3 R3) and Kelvedon Wonder (rr) cultivars. DNA hybridization studies showed avrPpiB to be plasmid-borne in race 3 and suggested the presence of other alleles on one of the endogenous plasmids of races 1 and 7. Disruption of the avrPpiB allele of race 1 and its complementation confirmed its behavior towards pea cultivars expressing the R3 locus. Homologs of avrPpiB were detected in P. syringae pv. phaseolicola, P. syringae pv. maculicola, and P. syringae pv. tomato. The presence of avrPpiB homologs in P. syringae pv. phaseolicola does not match any gene-for-gene pattern of interaction with bean cultivars.

Nucleotide sequence of nifD from Frankia alni strain ArI3: phylogenetic inferences.

The complete nucleotide sequence of the nifD gene encoding the alpha subunit of component I of nitrogenase from Frankia alni strain ArI3 was determined. The coding region is 1,458 bp in length and encodes a polypeptide of 486 residues with a predicted molecular weight of 53,500. Phylogenetic inferences with 12 complete published nifD sequences were drawn using a variety of approaches. Frankia nifD clusters with proteobacteria rather than with Clostridium pasteurianum, the other Gram-positive bacterium studied. Extant eubacterial nif genes seem to have at least three distinct evolutionary origins as a result of ancient gene duplications. Within the Gram-positive bacterial phylum, functional nif genes descend from different duplicates.