Pieter van Dillewijn

Proteomic Analysis Reveals the Participation of Energy- and Stress-Related Proteins in the Response of Pseudomonas putida DOT-T1E to Toluene

Pseudomonas putida DOT-T1E is tolerant to toluene and other toxic hydrocarbons through extrusion of the toxic compounds from the cell by means of three efflux pumps, TtgABC, TtgDEF, and TtgGHI. To identify other cellular factors that allow the growth of P. putida DOT-T1E in the presence of high concentrations of toluene, we performed two-dimensional gel analyses of proteins extracted from cultures grown on glucose in the presence and in the absence of the organic solvent. From a total of 531 spots, 134 proteins were observed to be toluene specific. In the absence of toluene, 525 spots were clearly separated and 117 proteins were only present in this condition. Moreover, 35 proteins were induced by at least twofold in the presence of toluene whereas 26 were repressed by at least twofold under these conditions. We reasoned that proteins that were highly induced could play a role in toluene tolerance. These proteins, identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry, were classified into four categories: 1, proteins involved in the catabolism of toluene; 2, proteins involved in the channeling of metabolic intermediates to the Krebs cycle and activation of purine biosynthesis; 3, proteins involved in sugar transport; 4, stress-related proteins. The set of proteins in groups 2 and 3 suggests that the high energy demand required for solvent tolerance is achieved via activation of cell metabolism. The role of chaperones that facilitate the proper folding of newly synthesized proteins under toluene stress conditions was analyzed in further detail. Knockout mutants revealed that CspA, XenA, and Tuf-1 play a role in solvent tolerance in Pseudomonas, although this role is probably not specific to toluene, as indicated by the fact that all mutants grew more slowly than the wild type without toluene.

Construction and Environmental Release of a Sinorhizobium meliloti Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation

ABSTRACT Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium melilotiPutA− mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492–498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. melilotistrains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of theputA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.

Bacterial diversity in the rhizosphere of maize and the surrounding carbonate‐rich bulk soil

Maize represents one of the main cultivar for food and energy and crop yields are influenced by soil physicochemical and climatic conditions. To study how maize plants influence soil microbes we have examined microbial communities that colonize maize plants grown in carbonate‐rich soil (pH 8.5) using culture‐independent, PCR‐based methods. We observed a low proportion of unclassified bacteria in this soil whether it was planted or unplanted. Our results indicate that a higher complexity of the bacterial community is present in bulk soil with microbes from nine phyla, while in the rhizosphere microbes from only six phyla were found. The predominant microbes in bulk soil were bacteria of the phyla Acidobacteria, Bacteroidetes and Proteobacteria, while Gammaproteobacteria of the genera Pseudomonas and Lysobacter were the predominant in the rhizosphere. As Gammaproteobacteria respond chemotactically to exudates and are efficient in the utilization of plants exudate products, microbial communities associated to the rhizosphere seem to be plant‐driven. It should be noted that Gammaproteobacteria made available inorganic nutrients to the plants favouring plant growth and then the benefit of the interaction is common.

Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

ABSTRACT The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.

Type II Hydride Transferases from Different Microorganisms Yield Nitrite and Diarylamines from Polynitroaromatic Compounds

ABSTRACT Homogenous preparations of XenB of Pseudomonas putida, pentaerythritol tetranitrate reductase of Enterobacter cloacae, and N-ethylmaleimide reductase of Escherichia coli, all type II hydride transferases of the Old Yellow Enzyme family of flavoproteins, are shown to reduce the polynitroaromatic compound 2,4,6-trinitrotoluene (TNT). The reduction of this compound yields hydroxylaminodinitrotoluenes and Meisenheimer dihydride complexes, which, upon condensation, yield stoichiometric amounts of nitrite and diarylamines, implying that type II hydride transferases are responsible for TNT denitration, a process with important environmental implications for TNT remediation.

Transcriptome profiling of a Sinorhizobium meliloti fadD mutant reveals the role of rhizobactin 1021 biosynthesis and regulation genes in the control of swarming

BackgroundSwarming is a multicellular phenomenom characterized by the coordinated and rapid movement of bacteria across semisolid surfaces. In Sinorhizobium meliloti this type of motility has been described in a fadD mutant. To gain insights into the mechanisms underlying the process of swarming in rhizobia, we compared the transcriptome of a S. meliloti fadD mutant grown under swarming inducing conditions (semisolid medium) to those of cells grown under non-swarming conditions (broth and solid medium).ResultsMore than a thousand genes were identified as differentially expressed in response to growth on agar surfaces including genes for several metabolic activities, iron uptake, chemotaxis, motility and stress-related genes. Under swarming-specific conditions, the most remarkable response was the up-regulation of iron-related genes. We demonstrate that the pSymA plasmid and specifically genes required for the biosynthesis of the siderophore rhizobactin 1021 are essential for swarming of a S. meliloti wild-type strain but not in a fadD mutant. Moreover, high iron conditions inhibit swarming of the wild-type strain but not in mutants lacking either the iron limitation response regulator RirA or FadD.ConclusionsThe present work represents the first transcriptomic study of rhizobium growth on surfaces including swarming inducing conditions. The results have revealed major changes in the physiology of S. meliloti cells grown on a surface relative to liquid cultures. Moreover, analysis of genes responding to swarming inducing conditions led to the demonstration that iron and genes involved in rhizobactin 1021 synthesis play a role in the surface motility shown by S. meliloti which can be circumvented in a fadD mutant. This work opens a way to the identification of new traits and regulatory networks involved in swarming by rhizobia.

Microbial responses to xenobiotic compounds. Identification of genes that allow Pseudomonas putida KT2440 to cope with 2,4,6-trinitrotoluene.

Pseudomonas putida KT2440 grows in M9 minimal medium with glucose in the presence of 2,4,6‐trinitrotoluene (TNT) at a similar rate than in the absence of TNT, although global transcriptional analysis using DNA microarrays revealed that TNT exerts some stress. Response to TNT stress is regulated at the transcriptional level, as significant changes in the level of expression of 65 genes were observed. Of these genes, 39 appeared upregulated, and 26 were downregulated. The identity of upregulated genes suggests that P. putida uses two kinds of strategies to overcome TNT toxicity: (i) induction of genes encoding nitroreductases and detoxification‐related enzymes (pnrA, xenD, acpD) and (ii) induction of multidrug efflux pump genes (mexEF/oprN) to reduce intracellular TNT concentrations. Mutants of 13 up‐ and 7 downregulated genes were analysed with regards to TNT toxicity revealing the role of the MexE/MexF/OprN pump and a putative isoquinoline 1‐oxidoreductase in tolerance to TNT. The ORF PP1232 whose transcriptional level did not change in response to TNT affected growth in the presence of nitroaromatic compounds and it was found in a screening of 4000 randomly generated mutants.

Exploring the rhizospheric and endophytic bacterial communities of Acer pseudoplatanus growing on a TNT-contaminated soil: towards the development of a rhizocompetent TNT-detoxifying plant growth promoting consortium

Background and aimsNumerous microorganisms have been isolated from trinitrotoluene (TNT)-contaminated soils, however TNT tends to persist, indicating that the microbial biomass or activity is insufficient for degradation. Deep-rooting trees at military sites have been found to take-up contaminants from groundwater, and the extensive root and endosphere provide ideal niches for microbial TNT-transformations.MethodsWe characterised the rhizosphere, root endosphere and endo-phyllosphere bacteria of Acer pseudoplatanus growing at a historically TNT-contaminated location, using 16S rRNA gene fingerprinting, bacteria isolation, oxidoreductase gene-cloning, in planta growth-promotion (PGP) tests, inoculation, plant physiology measurements and microscopy.ResultsBased on terminal-restriction-fragment-length-polymorphism analysis, bulk soil and rhizosphere samples were highly clustered. Proteo- and Actinobacteria dominated the rhizosphere and root endosphere, whereas Alphaproteobacteria were more abundant in shoots and Actinobacteria in leaves. We isolated multiple PGP-bacteria and cloned 5 flavin-oxidoreductases belonging to the Old Yellow Enzyme family involved in TNT-reduction from 3 Pseudomonas spp., the leaf symbiont Stenotrophomonas chelatiphaga and the root endophyte Variovorax ginsengisola.ConclusionsThe inoculation with a selection of these strains, consortium CAP9, which combines efficient TNT-transformation capabilities with beneficial PGP-properties, has the ability to detoxify TNT in the bent grass (Agrostis capillaris) rhizosphere, stimulate plant growth and improve plant health under TNT stress.

Subfunctionality of Hydride Transferases of the Old Yellow Enzyme Family of Flavoproteins of Pseudomonas putida

ABSTRACT To investigate potential complementary activities of multiple enzymes belonging to the same family within a single microorganism, we chose a set of Old Yellow Enzyme (OYE) homologs of Pseudomonas putida. The physiological function of these enzymes is not well established; however, an activity associated with OYE family members from different microorganisms is their ability to reduce nitroaromatic compounds. Using an in silico approach, we identified six OYE homologs in P. putida KT2440. Each gene was subcloned into an expression vector, and each corresponding gene product was purified to homogeneity prior to in vitro analysis for its catalytic activity against 2,4,6-trinitrotoluene (TNT). One of the enzymes, called XenD, lacked in vitro activity, whereas the other five enzymes demonstrated type I hydride transferase activity and reduced the nitro groups of TNT to hydroxylaminodinitrotoluene derivatives. XenB has the additional ability to reduce the aromatic ring of TNT to produce Meisenheimer complexes, defined as type II hydride transferase activity. The condensations of the primary products of type I and type II hydride transferases react with each other to yield diarylamines and nitrite; the latter can be further reduced to ammonium and serves as a nitrogen source for microorganisms in vivo.

Assessing Bacterial Diversity in the Rhizosphere of Thymus zygis Growing in the Sierra Nevada National Park (Spain) through Culture-Dependent and Independent Approaches.

Little is known of the bacterial communities associated with the rhizosphere of wild plant species found in natural settings. The rhizosphere bacterial community associated with wild thyme, Thymus zygis L., plants was analyzed using cultivation, the creation of a near-full length 16S rRNA gene clone library and 454 amplicon pyrosequencing. The bacterial community was dominated by Proteobacteria (mostly Alphaproteobacteria and Betaproteobacteria), Actinobacteria, Acidobacteria, and Gemmatimonadetes. Although each approach gave a different perspective of the bacterial community, all classes/subclasses detected in the clone library and the cultured bacteria could be found in the pyrosequencing datasets. However, an exception caused by inconclusive taxonomic identification as a consequence of the short read length of pyrotags together with the detection of singleton sequences which corresponded to bacterial strains cultivated from the same sample highlight limitations and considerations which should be taken into account when analysing and interpreting amplicon datasets. Amplicon pyrosequencing of replicate rhizosphere soil samples taken a year later permit the definition of the core microbiome associated with Thymus zygis plants. Abundant bacterial families and predicted functional profiles of the core microbiome suggest that the main drivers of the bacterial community in the Thymus zygis rhizosphere are related to the nutrients originating from the plant root and to their participation in biogeochemical cycles thereby creating an intricate relationship with this aromatic plant to allow for a feedback ecological benefit.