Rapid and accurate species and genomic species identification and exhaustive population diversity assessment of Agrobacterium spp. using recA-based PCR

Agrobacteria are common soil bacteria that interact with plants as commensals, plant growth promoting rhizobacteria or alternatively as pathogens. Indigenous agrobacterial populations are composites, generally with several species and/or genomic species and several strains per species. We thus developed a recA-based PCR approach to accurately identify and specifically detect agrobacteria at various taxonomic levels. Specific primers were designed for all species and/or genomic species of Agrobacterium presently known, including 11 genomic species of the Agrobacterium tumefaciens complex (G1-G9, G13 and G14, among which only G2, G4, G8 and G14 still received a Latin epithet: pusense, radiobacter, fabrum and nepotum, respectively), A. larrymoorei, A. rubi, R. skierniewicense, A. sp. 1650, and A. vitis, and for the close relative Allorhizobium undicola. Specific primers were also designed for superior taxa, Agrobacterium spp. and Rhizobiaceace. Primer specificities were assessed with target and non-target pure culture DNAs as well as with DNAs extracted from composite agrobacterial communities. In addition, we showed that the amplicon cloning-sequencing approach used with Agrobacterium-specific or Rhizobiaceae-specific primers is a way to assess the agrobacterial diversity of an indigenous agrobacterial population. Hence, the agrobacterium-specific primers designed in the present study enabled the first accurate and rapid identification of all species and/or genomic species of Agrobacterium, as well as their direct detection in environmental samples.     

Phase variation and genomic architecture changes in Azospirillum

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V(I), which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V(I) differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V(I) genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.     

Presence of Hydrogenophilus thermoluteolus DNA in accretion ice in the subglacial Lake Vostok, Antarctica, assessed using rrs, cbb and hox

The 3561 m Vostok ice core sample originating from the subglacial Lake Vostok accretion (frozen lake water) ice with sediment inclusions was thoroughly studied by various means to confirm the presence of the thermophile bacterium Hydrogenophilus thermoluteolus reported earlier in the 3607 m accretion ice sample. PCR and molecular-phylogenetic analyses performed in two independent laboratories were made using different 16S rRNA gene (rrs) targeted primers. As a result, rrs-targeted PCR permitted to recover several very closely related clones with a small genetic distance to Hydrogenophilus thermoluteolus (< 1%). In addition, RubisCO (cbbL or rbcL) and NiFe-Hydrogenase (hoxV or hupL) targeted PCR have also allowed to recover sequences highly related to Hydrogenophilus thermoluteolus. All these results point to the presence of thermophilic chemoautotrophic microorganisms in Lake Vostok accretion ice. They presumably originate from deep faults in the bedrock cavity containing the lake in which episodes of seismotectonic activity would release debris along with microbial cells.     

Progress on the genetics of the N2-fixing actinorhizal symbiont Frankia

The actinomycete Frankia is of fundamental and ecological interests for several reasons including its wide distribution, its ability to fix nitrogen, differentiate into sporangium and vesicle (specialized cell for nitrogen-fixation), and to nodulate plants from about 24 genera. Here, we present a review on the genetics performed so far on Frankia. At the end of July 2001, 293 kbp of Frankia DNA sequences were found in the databases. Thirty five percent of these sequences corresponded to full gene or gene cluster sequences. These genes could be divided according to their role into 6 key activities: gene translation (rrnA and tRNA ^pro gene), proteolysis (pcr genes), assimilation of ammonium (glnA and glnII), protection against superoxide ions (sodF), nitrogen fixation (nif cluster), and plasmid replication. We present a review of these genetic islands; their function, expression, localization and particular properties are discussed. A comparative analysis of Frankia nif genes from various strains and species is presented. An improved nomenclature for some of these genes is suggested to avoid conflicts. Frankia plasmids DNA sequences are also presented. The novel trends in Frankia genetics are described.     

Analysis of Hydroxycinnamic Acid Degradation in Agrobacterium fabrum Reveals a Coenzyme A-Dependent,Beta-Oxidative Deacetylation Pathway

The soil- and rhizosphere-inhabiting bacterium Agrobacterium fabrum (genomospecies G8 of the Agrobacterium tumefaciens species complex) is known to have species-specific genes involved in ferulic acid degradation. Here, we characterized, by genetic and analytical means, intermediates of degradation as feruloyl coenzyme A (feruloyl-CoA), 4-hydroxy-3-methoxyphenyl-β-hydroxypropionyl–CoA, 4-hydroxy-3-methoxyphenyl-β-ketopropionyl–CoA, vanillic acid, and protocatechuic acid. The genes atu1416, atu1417, and atu1420 have been experimentally shown to be necessary for the degradation of ferulic acid. Moreover, the genes atu1415 and atu1421 have been experimentally demonstrated to be essential for this degradation and are proposed to encode a phenylhydroxypropionyl-CoA dehydrogenase and a 4-hydroxy-3-methoxyphenyl-β-ketopropionic acid (HMPKP)–CoA β-keto-thiolase, respectively. We thus demonstrated that the A. fabrum hydroxycinnamic degradation pathway is an original coenzyme A-dependent β-oxidative deacetylation that could also transform p-coumaric and caffeic acids. Finally, we showed that this pathway enables the metabolism of toxic compounds from plants and their use for growth, likely providing the species an ecological advantage in hydroxycinnamic-rich environments, such as plant roots or decaying plant materials.     

Rapid and Efficient Identification of Agrobacterium Species by recA Allele Analysis

The analysis of housekeeping recA gene sequences from 138 strains from 13 species or genomic species of Agrobacterium, nine being biovar 1 genomospecies, and the others Agrobacterium larrymoorei, Agrobacterium rubi, Agrobacterium sp. NCPPB 1650, and Agrobacterium vitis and one “former” Agrobacterium species, Rhizobium rhizogenes, led to the identification of 50 different recA alleles and to a clear delineation of the 14 species or genomospecies entirely consistent with that obtained by amplified fragment length polymorphism (AFLP) analysis. The relevance of a recA sequencing approach for epidemiological analyses was next assessed on agrobacterial Tunisian isolates. All Tunisian isolates were found to belong to the Agrobacterium tumefaciens/biovar 1 species complex by both biochemical tests and rrs sequencing. recA sequence analysis further permitted their unambiguous assignment to A. tumefaciens genomospecies G4, G6, G7, and G8 in total agreement with the results of an AFLP-based analysis. At subspecific level, several Tunisian recA alleles were novel, indicating the power and accuracy of recA-based typing for studies of Agrobacterium spp.