Thierry Heulin

Plant host habitat and root exudates shape soil bacterial community structure

The rhizosphere is active and dynamic in which newly generated carbon, derived from root exudates, and ancient carbon, in soil organic matter (SOM), are available for microbial growth. Stable isotope probing (SIP) was used to determine bacterial communities assimilating each carbon source in the rhizosphere of four plant species. Wheat, maize, rape and barrel clover (Medicago truncatula) were grown separately in the same soil under 13CO2 (99% of atom 13C) and DNA extracted from rhizosphere soil was fractionated by isopycnic centrifugation. Bacteria-assimilating root exudates were characterized by denaturing gradient gel electrophoresis (DGGE) analysis of 13C-DNA and root DNA, whereas those assimilating SOM were identified from 12C-DNA. Plant species root exudates significantly shaped rhizosphere bacterial community structure. Bacteria related to Sphingobacteriales and Myxococcus assimilated root exudates in colonizing roots of all four plants, whwereas bacteria related to Sphingomonadales utilized both carbon sources, and were identified in light, heavy and root compartment DNA. Sphingomonadales were specific to monocotyledons, whereas bacteria related to Enterobacter and Rhizobiales colonized all compartments of all four plants, used both fresh and ancient carbon and were considered as generalists. There was also evidence for an indirect important impact of root exudates, through stimulation of SOM assimilation by a diverse bacterial community.

Rhizosphere soil aggregation and plant growth promotion of sunflowers by an exopolysaccharide-producing Rhizobium sp. strain isolated from sunflower roots.

ABSTRACT Root-adhering soil (RAS) forms the immediate environment where plants take up water and nutrients for their growth. We report the effect of an exopolysaccharide (EPS)-producing rhizobacterium (strain YAS34) on the physical properties of sunflower (Helianthus annuus L.) RAS, associated with plant growth promotion, under both water stress and normal water supply conditions. Strain YAS34 was isolated as a major EPS-producing bacterium from the rhizoplane of sunflowers grown in a French dystric cambisol. Strain YAS34 was assigned to the Rhizobium genus by 16S ribosomal DNA gene sequencing. Inoculation of sunflower seeds and soil with strain YAS34 caused a significant increase in RAS per root dry mass (dm) (up to 100%) and a significant increase in soil macropore volume (12 to 60 μm in diameter). The effect of inoculation on sunflower shoot dm (up to +50%) and root dm (up to +70%) was significant under both normal and water stress conditions. Inoculation with strain YAS34 modified soil structure around the root system, counteracting the negative effect of water deficit on growth. Using [15N]nitrate, we showed that inoculation made the use of fertilizer more effective by increasing nitrogen uptake by sunflower plantlets.

Paenibacillus graminis sp. nov. and Paenibacillus odorifer sp. nov., isolated from plant roots, soil and food.

Sixteen gram-positive endospore-forming bacteria previously isolated from soil, plant rhizospheres, plant roots and pasteurized pureed vegetables were studied to determine their taxonomic positions. The isolates were formerly identified as Bacillus circulans based on their biochemical characters using API galleries. Two of these strains, RSA19T and TOD45T, were recently assigned to the genus Paenibacillus based on phylogenetic analysis of their 16S rRNA (rrs) gene sequence. In the present work, the sixteen isolates were assigned to two genomospecies using DNA-DNA hybridization, in agreement with rrs gene sequence analysis. These genomospecies can also be differentiated on the basis of their cultural and biochemical characters into two novel species, for which the names Paenibacillus graminis sp. nov. (type strain RSA19T = ATCC BAA-95T = LMG 19080T) and Paenibacillus odorifer sp. nov. (type strain TOD45T = ATCC BAA-93T = LMG 19079T) are proposed.

Alliance of proteomics and genomics to unravel the specificities of Sahara bacterium Deinococcus deserti.

To better understand adaptation to harsh conditions encountered in hot arid deserts, we report the first complete genome sequence and proteome analysis of a bacterium, Deinococcus deserti VCD115, isolated from Sahara surface sand. Its genome consists of a 2.8-Mb chromosome and three large plasmids of 324 kb, 314 kb, and 396 kb. Accurate primary genome annotation of its 3,455 genes was guided by extensive proteome shotgun analysis. From the large corpus of MS/MS spectra recorded, 1,348 proteins were uncovered and semiquantified by spectral counting. Among the highly detected proteins are several orphans and Deinococcus-specific proteins of unknown function. The alliance of proteomics and genomics high-throughput techniques allowed identification of 15 unpredicted genes and, surprisingly, reversal of incorrectly predicted orientation of 11 genes. Reversal of orientation of two Deinococcus-specific radiation-induced genes, ddrC and ddrH, and identification in D. deserti of supplementary genes involved in manganese import extend our knowledge of the radiotolerance toolbox of Deinococcaceae. Additional genes involved in nutrient import and in DNA repair (i.e., two extra recA, three translesion DNA polymerases, a photolyase) were also identified and found to be expressed under standard growth conditions, and, for these DNA repair genes, after exposure of the cells to UV. The supplementary nutrient import and DNA repair genes are likely important for survival and adaptation of D. deserti to its nutrient-poor, dry, and UV-exposed extreme environment.

Taxonomic characterization of Ochrobactrum sp. isolates from soil samples and wheat roots, and description of Ochrobactrum tritici sp. nov. and Ochrobactrum grignonense sp. nov

A large collection of bacterial strains, immunotrapped from soil and from the wheat rhizoplane, was subjected to polyphasic taxonomy by examining various pheno- and genotypic parameters. Strains were grouped on (inter) repetitive extragenic palindromic DNA (REP) PCR profiles at the intraspecies level. Pheno- and genotypic characters were assessed for representatives from 13 different REP groups. Strains of nine REP groups constituting two physiological BIOLOG clusters fell in the coherent DNA-DNA reassociation group of Ochrobactrum anthropi. Strains of two REP groups constituting a separate BIOLOG cluster fell in the coherent DNA-DNA reassociation group of Ochrobactrum intermedium. Additional phenotypic characters differentiating O. anthropi and O. intermedium were found. REP group K strains constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and a distinct phylogenetic lineage in 165 rDNA homology analysis, indicating that REP group K strains represent a new species. Diagnostic phenotypic characters were found. Closest relatives were Ochrobactrum species. The name Ochrobactrum grignonense sp. nov. is proposed (type strain OgA9aT = LMG 18954T = DSM 13338T). REP group J strains again constituted a different BIOLOG cluster, a separate DNA-DNA reassociation group and showed, as a biological particularity, a strict preference for the rhizoplane as habitat. Diagnostic phenotypic characters were found. This indicated that REP group J strains represent a further new species, although phylogenetic analyses using 16S rDNA homology were not able to separate the cluster of REP group J sequences significantly from 16S rDNA sequences of Ochrobactrum anthropi. The name Ochrobactrum tritici sp. nov. is proposed (type strain SCII24T = LMG 18957T = DSM 13340T).

Selenite and tellurite reduction by Shewanella oneidensis

ABSTRACT Shewanella oneidensis MR-1 reduces selenite and tellurite preferentially under anaerobic conditions. The Se(0) and Te(0) deposits are located extracellularly and intracellularly, respectively. This difference in localization and the distinct effect of some inhibitors and electron acceptors on these reduction processes are taken as evidence of two independent pathways.

Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Brassica napus and Arabidopsis thaliana

Bacteria isolates phenotypically related to Pseudomonas corrugata have frequently been isolated from the rhizosphere of Arabidopsis thaliana and Brassica napus grown on different soils. 16S rDNA (rrs) gene sequencing, DNA-DNA hybridization, biochemical characterization and siderophore typing showed that these isolates belong to two different species that are distinct from other species of the genus Pseudomonas, including P. corrugata. A description of properties of these two new species is given based on the study of 16 isolates. Proposed names are Pseudomonas brassicacearum (10 strains studied) and Pseudomonas thivervalensis (6 strains studied). The type strain of Pseudomonas brassicacearum is CFBP 11706T and that of Pseudomonas thivervalensis is CFBP 11261T.

Burkholderia caribensis sp. nov., an exopolysaccharide-producing bacterium isolated from vertisol microaggregates in Martinique.

Twenty-one exopolysaccharide-producing strains were isolated from the 5-20 microns fraction of a vertisol in the south-east of the island of Martinique in the French West Indies. Although these strains were phenotypically identified as Burkholderia cepacia or as Burkholderia glathei using BIOLOG microplates, they did not cluster genotypically by amplified rDNA restriction analysis (ARDRA) with any described Burkholderia species. A phylogenetic analysis revealed that the rrs (16S rDNA) sequences of three representative strains clustered in a single branch within the genus Burkholderia and distantly from all of the previously described species of Burkholderia for which rrs sequences were available. DNA-DNA hybridization data as well as phenotypic analyses indicated that the 21 isolates represented a single and new species for which the name Burkholderia caribensis sp. nov. is proposed (type strain MWAP64T = LMG 18531T).

Phenotypic Variation of Pseudomonas brassicacearum as a Plant Root-Colonization Strategy

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.

Heavy Metal Tolerance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an aerobic, non-fermentative Gram-negative bacterium widespread in the environment. S. maltophilia Sm777 exhibits innate resistance to multiple antimicrobial agents. Furthermore, this bacterium tolerates high levels (0.1 to 50 mM) of various toxic metals, such as Cd, Pb, Co, Zn, Hg, Ag, selenite, tellurite and uranyl. S. maltophilia Sm777 was able to grow in the presence of 50 mM selenite and 25 mM tellurite and to reduce them to elemental selenium (Se0) and tellurium (Te0) respectively. Transmission electron microscopy and energy dispersive X-ray analysis showed cytoplasmic nanometer-sized electron-dense Se0 granules and Te0 crystals. Moreover, this bacterium can withstand up to 2 mM CdCl2 and accumulate this metal up to 4% of its biomass. The analysis of soluble thiols in response to ten different metals showed eightfold increase of the intracellular pool of cysteine only in response to cadmium. Measurements by Cd K-edge EXAFS spectroscopy indicated the formation of Cd-S clusters in strain Sm777. Cysteine is likely to be involved in Cd tolerance and in CdS-clusters formation. Our data suggest that besides high tolerance to antibiotics by efflux mechanisms, S. maltophilia Sm777 has developed at least two different mechanisms to overcome metal toxicity, reduction of oxyanions to non-toxic elemental ions and detoxification of Cd into CdS.