Genetic control of rhizosheath formation in pearl millet.

The rhizosheath, the layer of soil that adheres strongly to roots, influences water and nutrients acquisition. Pearl millet is a cereal crop that plays a major role for food security in arid regions of sub-Saharan Africa and India. We previously showed that root-adhering soil mass is a heritable trait in pearl millet and that it correlates with changes in rhizosphere microbiota structure and functions. Here, we studied the correlation between root-adhering soil mass and root hair development, root architecture, and symbiosis with arbuscular mycorrhizal fungi and we analysed the genetic control of this trait using genome wide association (GWAS) combined with bulk segregant analysis and gene expression studies. Root-adhering soil mass was weakly correlated only to root hairs traits in pearl millet. Twelve QTLs for rhizosheath formation were identified by GWAS. Bulk segregant analysis on a biparental population validated five of these QTLs. Combining genetics with a comparison of global gene expression in the root tip of contrasted inbred lines revealed candidate genes that might control rhizosheath formation in pearl millet. Our study indicates that rhizosheath formation is under complex genetic control in pearl millet and suggests that it is mainly regulated by root exudation.

Haemodynamic changes during retroperitoneoscopic adrenalectomy for phaeochromocytoma.

Surgical removal of phaeochromocytoma may be accompanied by acute cardiovascular changes. We report the haemodynamic changes in seven patients with retroperitoneal laparoscopic adrenalectomy for phaeochromocytoma. Transient hypertension (systolic pressure (SBP) > 160 mm Hg) was observed in all patients during manipulation of the tumour, in two patients during pneumoretroperitoneum insufflation, and in one patient during intubation. Small doses of nicardipine were sufficient to control these episodes of hypertension. Transient hypotension (SBP < 100 mm Hg) was observed in two patients during exsufflation and in one patient during repositioning to the lateral position. Our observations suggest that this approach provides relative haemodynamic stability, especially during pneumoretroperitoneum insufflation.

Structural analysis of the exopolysaccharide from Burkholderia caribensis strain MWAP71.

Burkholderia caribensis strain MWAP71 was isolated from rhizosphere soil microaggregates in Martinique. The extracellular polysaccharide produced by this strain was found to be composed of D-glucose (D-Glc), 6-deoxy-L-talose (L-6dTal), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and an O-acetyl group in a molar ratio of 2:1:1:1. The primary structure of the polysaccharide was shown by sugar analysis, electrospray mass spectrometry, partial acid hydrolysis and 1-D and 2-D NMR spectroscopy to consist of a tetrasaccharide repeating unit having the following structure: [structure in text].

Multiple facets of bacterial porins.

Porins form channels allowing the transport of molecules across lipid bilayer membranes. Their structure, location and large number on the bacterial surface lend them multiple functions. Porin loops are potential targets for adhesion to other cells and binding of bactericidal compounds to the surface of Gram-negative bacteria. Variation of the loop structure as a mechanism to escape immune pressure, or modulation of the porin expression in response to the presence of antibiotics, are survival strategies developed by some pathogenic bacteria. Porins may play a significant role as pathogenesis effectors.

Phase-variable expression of an operon encoding extracellular alkaline protease, a serine protease homolog, and lipase in Pseudomonas brassicacearum.

The rhizobacterium Pseudomonas brassicacearum forms phenotypic variants which do not show extracellular protease and lipase activity. The operon encoding these enzymes, a serine protease homolog, and a type I secretion machinery was characterized. Transcriptional lacZ gene fusions revealed that the expression of the operon is under the control of phase variation.

Ecology and evolution of bacterial microdiversity.

Using high resolution molecular fingerprinting techniques like random amplification of polymorphic DNA, repetitive extragenic palindromic PCR and multilocus enzyme electrophoresis, a high bacterial diversity below the species and subspecies level (microdiversity) is revealed. It became apparent that bacteria of a certain species living in close association with different plants either as associated rhizosphere bacteria or as plant pathogens or symbiotic organisms, typically reflect this relationship in their genetic relatedness. The strain composition within a population of soil bacterial species at a given field site, which can be identified by these high resolution fingerprinting techniques, was markedly influenced by soil management and soil features. The observed bacterial microdiversity reflected the conditions of the habitat, which select for better adapted forms. In addition, influences of spatial separation on specific groupings of bacteria were found, which argue for the occurrence of isolated microevolution. In this review, examples are presented of bacterial microdiversity as influenced by different ecological factors, with the main emphasis on bacteria from the natural environment. In addition, information available from some of the first complete genome sequences of bacteria (Helicobacter pylori and Escherichia coli) was used to highlight possible mechanisms of molecular evolution through which mutations are created; these include mutator enzymes. Definitions of bacterial species and subspecies ranks are discussed in the light of detailed information from whole genome typing approaches.

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

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 microm 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 [(15)N]nitrate, we showed that inoculation made the use of fertilizer more effective by increasing nitrogen uptake by sunflower plantlets.

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.

Impact of crop management on intraspecific diversity of Pseudomonas corrugata in bulk soil.

The genetic structure of Pseudomonas corrugata populations was investigated in bulk soil to evaluate the impact of crop management on the intraspecific diversity of this bacterium stimulated in plant rhizosphere. As these bacteria are rare in bulk soil, an immunotrapping assay was developed to isolate them from soils located in Grignon (France), where adjacent plots with similar soil features were cultivated under maize/wheat crop rotation or continuous wheat cropping. Genomic fingerprinting of 291 isolates was performed using repetitive sequence-based polymerase chain reaction (ERIC-PCR). Most of these isolates (236 out of 291) were assigned to P. corrugata on the basis of an amplified ribosomal DNA restriction analysis (ARDRA) with 12 restriction enzymes. Data were analyzed by Pearson's chi-squared tests and genetic diversity was evaluated with specific indices for richness and evenness. Comparison of richness or evenness diversity indices (global diversity) showed no significant difference, whereas ERIC-PCR fingerprinting data (intraspecific diversity) showed that the genetic structure of P. corrugata was significantly affected by crop management.

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).

Disruption of the Paenibacillus polymyxa levansucrase gene impairs its ability to aggregate soil in the wheat rhizosphere.

Inoculation of wheat roots with Paenibacillus (formerly Bacillus) polymyxa CF43 increases the mass of root-adhering soil. We tested the role of levan, a fructosyl polymer produced by strain CF43, in the aggregation of soil adhering to wheat roots. The P. polymyxa gene homologous to the Bacillus subtilis sacB gene encoding levansucrase was cloned and sequenced. The corresponding gene product synthesises high molecular weight levan. A P. polymyxa mutant strain, SB03, whose sacB gene is disrupted, was constructed using heterogramic conjugation. Effects of wheat inoculation with the wild type and the mutant strain were compared using two different cultivated silt loam soils in four independent pot experiments. Roots of wheat plantlets inoculated with CF43 or SB03 were colonized after 7-14 days at the same level, and root and shoot masses were not significantly different from those of the non-inoculated control plants. The ratio of root-adhering soil dry mass to root tissue dry mass was significantly higher for plants inoculated with strain CF43 than for those inoculated with mutant strain SB03: +30% in Orgeval soil and +100% in Dieulouard soil. Thus the levan produced by P. polymyxa is implicated in the aggregation of root-adhering soil on wheat.

Bacteria in the Tatahouine meteorite: nanometric-scale life in rocks.

We present a study of the textural signature of terrestrial weathering and related biological activity in the Tatahouine meteorite. Scanning and transmission electron microscopy images obtained on the weathered samples of the Tatahouine meteorite and surrounding soil show two types of bacteria-like forms lying on mineral surfaces: (1) rod-shaped forms (RSF) about 70-80 nm wide and ranging from 100 nm to 600 nm in length; (2) ovoid forms (OVF) with diameters between 70 and 300 nm. They look like single cells surrounded by a cell wall. Only Na, K, C, O and N with traces of P and S are observed in the bulk of these objects. The chemical analyses and electron diffraction patterns confirm that the RSF and OVF cannot be magnetite or other iron oxides, iron hydroxides, silicates or carbonates. The sizes of the RSF and OVF are below those commonly observed for bacteria but are very similar to some bacteria-like forms described in the Martian meteorite ALH84001. All the previous observations strongly suggest that they are bacteria or their remnants. This conclusion is further supported by microbiological experiments in which pleomorphic bacteria with morphology similar to the OVF and RSF objects are obtained from biological culture of the soil surrounding the meteorite pieces. The present results show that bacteriomorphs of diameter less than 100 nm may in fact represent real bacteria or their remnants.

Comparative phylogeny of rrs and nifH genes in the Bacillaceae.

The rrs (16S rDNA) gene sequences of nitrogen-fixing endospore-forming bacilli isolated from the rhizosphere of wheat and maize were determined in order to infer their phylogenetic position in the Bacillaceae. These rhizosphere strains form a monophyletic cluster with Paenibacillus azotofixans, Paenibacillus polymyxa and Paenibacillus macerans. Two of them (RSA19 and TOD45) had previously been identified as Bacillus circulans (group 2) by phenotypic characterization (API 50CH). Evidence for nitrogen fixation by P. azotofixans, P. polymyxa, P. macerans and putative B. circulans strains RSA19 and TOD45 was provided by acetylene-reduction activity, and confirmed by amplifying and sequencing a nifH fragment (370 nt). The phylogenetic tree of nifH-derived amino acid sequences was compared to the phylogenetic tree of rrs sequences. All Paenibacillus nifH sequences formed a coherent cluster distinct from that of related nitrogen-fixing anaerobic clostridia and Gram-positive high-G+C-content frankiae. The nifH gene was neither detected in the B. circulans type strain (ATCC 4513T) nor in the type strains of Bacillus subtilis, Bacillus cereus, Bacillus alcalophilus, Bacillus simplex, Brevibacillus brevis and Paenibacillus validus. Accordingly, nitrogen fixation among aerobic endospore-forming Firmicutes seems to be restricted to a subset of species in the genus Paenibacillus.

Differentiation of Burkholderia species by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene and application to cystic fibrosis isolates.

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrociniaT were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes B. multivoransT) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepacia genomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.

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).

DNA extraction from activated sludges.

To optimize the cell lysis step for DNA extraction from activated sludge samples, two floc dispersion methods (sonication versus stirring with a cation exchange resin), and three cell lysis treatments (lysozyme + SDS, sonication in a water bath, and thermal shock) were tested. For dispersion, stirring with cation exchange resin was more efficient than sonication. The cell lysis procedures were applied in two sequences, and DNA was quantified after each cell lysis treatment. Lysozyme + SDS was the most effective step in the cell lysis procedures. The cell lysis treatment sequences giving the highest DNA yields were not the same for all the sludges. The differences in sludge microbial compositions and floc structures required specifically adapted cell lysis protocols. The proposed protocols were highly efficient for DNA extraction, yielding about 50 mg DNA g-1 volatile suspended solids, and allowed PCR amplification of 16S rDNA.

Colonization of wheat roots by an exopolysaccharide-producing pantoea agglomerans strain and its effect on rhizosphere soil aggregation

The effect of bacterial secretion of an exopolysaccharide (EPS) on rhizosphere soil physical properties was investigated by inoculating strain NAS206, which was isolated from the rhizosphere of wheat (Triticum durum L.) growing in a Moroccan vertisol and was identified as Pantoea aglomerans. Phenotypic identification of this strain with the Biotype-100 system was confirmed by amplified ribosomal DNA restriction analysis. After inoculation of wheat seedlings with strain NAS206, colonization increased at the rhizoplane and in root-adhering soil (RAS) but not in bulk soil. Colonization further increased under relatively dry conditions (20% soil water content; matric potential, -0.55 MPa). By means of genetic fingerprinting using enterobacterial repetitive intergenic consensus PCR, we were able to verify that colonies counted as strain NAS206 on agar plates descended from inoculated strain NAS206. The intense colonization of the wheat rhizosphere by these EPS-producing bacteria was associated with significant soil aggregation, as shown by increased ratios of RAS dry mass to root tissue (RT) dry mass (RAS/RT) and the improved water stability of adhering soil aggregates. The maximum effect of strain NAS206 on both the RAS/RT ratio and aggregate stability was measured at 24% average soil water content (matric potential, -0.20 MPa). Inoculated strain NAS206 improved RAS macroporosity (pore diameter, 10 to 30 &mgr;m) compared to the noninoculated control, particularly when the soil was nearly water saturated (matric potential, -0.05 MPa). Our results suggest that P. agglomerans NAS206 can play an important role in the regulation of the water content (excess or deficit) of the rhizosphere of wheat by improving soil aggregation.

Enzyme-linked immunofiltration assay To estimate attachment of thiobacilli to pyrite

An enzyme-linked immunofiltration assay (ELIFA) has been developed in order to estimate directly and specifically Thiobacillus ferrooxidans attachment on sulfide minerals. This method derives from the enzyme-linked immunosorbent assay but is performed on filtration membranes which allow the retention of mineral particles for a subsequent immunoenzymatic reaction in microtiter plates. The polyclonal antiserum used in this study was raised against T. ferrooxidans DSM 583 and recognized cell surface antigens present on bacteria belonging to the genus Thiobacillus. This antiserum and the ELIFA allowed the direct quantification of attached bacteria with high sensitivity (10(4) bacteria were detected per well of the microtiter plate). The mean value of bacterial attachment has been estimated to be about 10(5) bacteria mg-1 of pyrite at a particle size of 56 to 65 &mgr;m. The geometric coverage ratio of pyrite by T. ferrooxidans ranged from 0.25 to 2.25%. This suggests an attachment of T. ferrooxidans on the pyrite surface to well-defined limited sites with specific electrochemical or surface properties. ELIFA was shown to be compatible with the measurement of variable levels of adhesion. Therefore, this method may be used to establish adhesion isotherms of T. ferrooxidans on various sulfide minerals exhibiting different physicochemical properties in order to understand the mechanisms of bacterial interaction with mineral surfaces.

A major outer membrane protein of Rahnella aquatilis functions as a porin and root adhesin.

A 38-kDa major outer membrane protein (OMP) was isolated from the nitrogen-fixing enterobacterium Rahnella aquatilis CF3. This protein exists as a stable trimer in the presence of 2% sodium dodecyl sulfate at temperatures below 60 degrees C. Single channel experiments showed that this major OMP of R. aquatilis CF3 is able to form pores in the planar lipid membrane. Two oligonucleotides encoding the N-terminal portion of the 38-kDa OMP and C-terminal portion of OmpC were used to amplify the 38-kDa gene by PCR. The deduced amino acid sequence showed a strong homology with Escherichia coli, Klebsiella pneumoniae, Salmonella typhi, and Serratia marcescens OmpC sequences, except loops L6 and L7, which are postulated to be cell surface exposed. On the basis of the OmpF-PhoE three-dimensional structure, it seems likely that this 38-kDa organizes three 16-strand beta-barrel subunits. The relationship between the structure and the double functionality of this protein as porin and as a root adhesin is discussed.