Lysiane Hauben

Phylogenetic position of phytopathogens within the Enterobacteriaceae

The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae. The species of the genus Erwinia may be divided into three phylogenetic groups. Cluster I represents the true erwinias and comprises E. amylovora, E. mallotivora, E. persicinus, E. psidii, E. rhapontici and E. tracheiphila. We propose to unite the species of cluster II, E. carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, E. carotovora subsp. carotovora, E. carotovora subsp. odorifera, E. carotovora subsp. wasabiae, E. cacticida, E. chrysanthemi and E. cypripedii in the genus Pectobacterium respectively as P. carotovorum subsp. atrosepticum comb. nov., P. carotovorum subsp. betavasculorum comb. nov., P. carotovorum subsp. carotovorum comb. nov., P. carotovorum subsp. odoriferum comb. nov., P. carotovorum subsp. wasabiae comb. nov., P. cacticidum comb. nov., P. chrysanthemi and P. cypripedii. The species E. alni, E. nigrifluens, E. paradisiaca, E. quercina, E. rubrifaciens and E. salicis, comprising cluster III, are being classified into a new genus Brenneria gen. nov. respectively as B. alni comb. nov., B. nigrifluens comb. nov., B. paradisiaca comb. nov., B. quercina comb. nov., B. rubrifaciens comb. nov. and B. salicis comb. nov. The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

Comparison of 16s Ribosomal DNA Sequences of All Xanthomonas Species

The phylogenetic relationships of all validly described species of the genus Xanthomonas and the type strain of Stenotrophomonas maltophilia were analyzed by sequencing and comparing 16S ribosomal DNAs (rDNAs). The two genera exhibited a mean sequence similarity value of 96.6%, corresponding to differences at 50 nucleotide positions on average. The species of the genus Xanthomonas exhibited relatively high levels of overall sequence similarity; the mean similarity value was 98.2%, which corresponds to an average of 14 mutual nucleotide differences. Within the genus Xanthomonas, a group containing Xanthomonas albilineans, Xanthomonas hyacinthi, Xanthomonas theicola, and Xanthomonas translucens clustered apart from the main Xanthomonas core, whereas Xanthomonas sacchari formed a third phylogenetic lineage. Due to the very restricted variability in 16S rDNA sequences within the genus Xanthomonas, rDNA signatures that have possible diagnostic value for differentiating the Xanthomonas species could not be determined with certainty. When sequence similarities were compared with DNA-DNA pairing data determined previously, there was only a limited correlation. This illustrates the different resolving powers of the techniques for determining phylogenetic hierarchies and for species delineation.

Genomic diversity of the genus Stenotrophomonas

The clinical and environmental importance of Stenotrophomonas bacteria requires thorough, molecular studies on their epidemiology and taxonomy. In order to obtain a complete genomic profile of this genus, over 100 Stenotrophomonas maltophilia strains from various origins were investigated by AFLP fingerprinting. A subset of these strains was analysed by DNA hybridization and 16S rDNA sequencing. In contrast to their high phenotypic homogeneity, the strains were found to be very heterogeneous genotypically by AFLP fingerprinting. Nevertheless, ten cores of highly similar strains representing ten genomic groups were observed. The same groups could be retrieved by DNA hybridizations and also, partly, by 16S rDNA sequence analysis. The intergroup DNA similarities were too high to create confident species delineations, neither could the genomic groups be characterized by phenotypic features.

Reclassification of non-pigmented Erwinia herbicola strains from trees as Erwinia billingiae sp. nov.

Twenty-two Erwinia-like strains, isolated from trees since the late fifties and belonging to a distinct phenotypic group with resemblance to Pantoea agglomerans, were further characterized by conventional biochemical tests, the BIOLOG metabolic fingerprinting system and fatty acid analysis. Their phylogenetic positions were determined by comparing the 16S rRNA gene sequence of a representative strain to available sequences of Erwinia, Pantoea, Pectobacterium and Brenneria species. The strains were shown to belong to the genus Erwinia, with Erwinia rhapontici and Erwinia persicina as the closest phylogenetic relatives. The name Erwinia billingiae sp. nov. is proposed (type strain LMG 2613T) and a description of the species is given.

Isolation and Identification of Poly(3-Hydroxyvalerate)-Degrading Strains of Pseudomonas lemoignei

By using selective enrichment of polyhydroxyalkanoate-degrading bacteria and poly(3-hydroxyvalerate)-containing granules from Chromobacterium violaceum as the carbon source, 10 new Pseudomonas lemoignei strains were isolated; these strains were able to degrade poly(3-hydroxyvalerate), as well as poly(3-hydroxybutyrate), in vitro. The new isolates were characterized and identified by comparing them with P. lemoignei LMG 2207(T) (T = type strain). Like P. lemoignei LMG 2207(T) cells, the cells of the 10 new isolates contained mainly hexadecenoic, hexadecanoic, octadecenoic, and dodecanoic acids, as well as hydroxylated fatty acids, and exhibited respiration in the presence of methylpyruvate, 3-hydroxybutyrate, and 4-hydroxybutyrate, but not in the presence of the 92 other carbon sources included in Biolog GN microplates. The protein patterns of the new isolates were almost identical to each other and very similar to the protein pattern of P. lemoignei LMG 2207(T). Some of the new isolates, but not P. lemoignei LMG 2207(T), contained megaplasmids that were about 200 kbp long. The 16S ribosomal DNA genes of strain A62, a representative of the 10 new isolates, and of P. lemoignei LMG 2207(T) exhibited more than 0.99 sequence similarity. The DNA-DNA reassociation value for two representative strains was 100%, and the levels of DNA-DNA reassociation between these strains and the type strain were 60 and 61%. The taxonomy of P. lemoignei is briefly discussed.

Application of AFLP for taxonomic and epidemiological studies of Photobacterium damselae subsp. piscicida

A collection of 106 Photobacterium damselae subsp. piscicida strains and 19 Photobacterium damselae subsp. damselae strains, including reference and type strains, were genetically characterized using AFLP. The total genomic DNA of each bacterial strain was digested using restriction endonucleases HindIII and TaqI. Using numerical analysis, six clusters were recognized. The largest cluster (n = 106) contained the majority of the strains tested and consisted exclusively of Photobacterium damselae subsp. piscicida. The Photobacterium damselae subsp. damselae strains fell outside this cluster. DNA-DNA hybridization experiments showed 77% DNA binding between the two subspecies, indicating a close genetic relationship. This clearly demonstrates the applicability of AFLP in studying the taxonomic position of Photobacterium damselae subsp. piscicida. In addition, AFLP proved to be a useful genotypic technique for epidemiological surveys of the pathogen, since it was able to discriminate between Mediterranean and Japanese Photobacterium damselae subsp. piscicida isolates.

Biodegradation of Poly (3 -hYdroxYalkanoates in Anaerobic Sludge and Characterization of a Poly (3 -hYdroxYalkanoates Degrading Anaerobic Bacterium

Summary The degradation of samples of poly (3-hydroxybutyrate) [P(3HB)], copolymers of 3-hydroxybutyrate and 10 and 20% 3-hydroxyvalerate [P(3HB-co-3HV)], and poly (3-hydroxyoctanoate-co-3-hydroxydecanoate) [P(3H0-co-3HD)] in anaerobic sludge was investigated by gravimetry, changes in molecular weight and mechanical properties. After 123 days, the mass of P(3HB) decreased by 15%. P(3HB-co-3HV) showed 7 and 11% mass loss, while P(3H0-co-3HD) showed no change in mass. The average molecular weight of all samples decreased with incubation time but this decrease was not correlated to the loss of mass, and is to be attributed to abiotic hydrolysis. The tensile strength of the P(3HB) and P(3HB-co-3HV) samples decreased with incubation time, but elongation remained practically unchanged. By enumeration, it was shown that anaerobic P(3HB) degrading bacteria were enriched during the incubation period. An anaerobic bacterium LMG 16094, able to degrade P(3HB) and P(3HB-co-19% 3HV) in pure culture, but not P(3HB-co-97%3HV), P(3H0-co-3HD) or poly (e-caprolactone), was isolated and characterized by fatty acid analysis. Its cells contained mainly straight chain, saturated and unsaturated fatty acids, with a high portion of fatty acids with 16 carbons, and did not contain hydroxylated or branched fatty acids. Its 16S rDNA gene sequence was found to be related to those of members of Clostridium group I ( Collins et al., 1994). In a simple in vitro test using strain LMG 16094 in pure culture, an extruded P(3HB-co-12% 3HV) film lost 10% of its initial mass within 21 days of anaerobic incubation.