Contribution of the metabolism of phenolic compounds to the virulence of Pseudomonas savastanoi in woody hosts

Most strains of the Pseudomonas syringae complex isolated from the trunks of woody plants and belonging to phylogroups (PGs) 1 and 3 share a genomic region, named WHOP (from woody host and Pseudomonas), which is partially conserved in some PG2 strains. Annotation of the 14 ORFs encoded within this region, which is absent in the genomes of P. syringae strains infecting herbaceous hosts, yielded functions possibly involved in the metabolism of phenolic compounds. The genetic organization of this region was analyzed by RT-PCR in Pseudomonas savastanoi pv. savastanoi (Psv), the causative agent of olive knot disease. The WHOP region is organized in four operons (antABC, catBCA, ipoABC and dhoAB) and three genes transcribed independently (antR, benR and PSA3335_3206). HPLC analyses confirmed that the antABC and catBCA operons mediate the catabolism of anthranilate and catechol, respectively, through the β-ketoadipate pathway. In addition, oxygenase activity on aromatic compounds, tested as the conversion of indole into indigo, was assigned to the ipoABC operon. Pathogenicity tests revealed that deletion of antABC, catBCA or ipoABC in Psv caused reduced symptoms in woody olive plants, while such phenotype was not observed in young micropropagated (non-woody) plants. Similarly, the catBCA and dhoAB operons and the PSA3335_3206 gene (encoding a putative aerotaxis receptor) are also required for full bacterial fitness exclusively in woody olive plants. At present, we are addressing the role in virulence of the orphan benR gene encoded in the WHOP region.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The tep1 gene of Sinorhizobium meliloti coding for a putative transmembrane efflux protein and N-acetyl glucosamine affect nod gene expression and nodulation of alfalfa plants

<p>Abstract</p> <p>Background</p> <p>Soil bacteria collectively known as <it>Rhizobium</it>, characterized by their ability to establish beneficial symbiosis with legumes, share several common characteristics with pathogenic bacteria when infecting the host plant. Recently, it was demonstrated that a <it>fadD </it>mutant of <it>Sinorhizobium meliloti </it>is altered in the control of swarming, a type of co-ordinated movement previously associated with pathogenicity, and is also impaired in nodulation efficiency on alfalfa roots. In the phytopathogen <it>Xanthomonas campestris</it>, a <it>fadD </it>homolog (<it>rpfB</it>) forms part of a cluster of genes involved in the <it>r</it>egulation of <it>p</it>athogenicity <it>f</it>actors. In this work, we have investigated the role in swarming and symbiosis of SMc02161, a <it>S. meliloti fadD</it>-linked gene.</p> <p>Results</p> <p>The SMc02161 locus in <it>S. meliloti </it>shows similarities with members of the Major Facilitator Superfamily (MFS) of transporters. A <it>S. meliloti </it>null-mutant shows increased sensitivity to chloramphenicol. This indication led us to rename the locus <it>tep1 </it>for <it>t</it>ransmembrane <it>e</it>fflux <it>p</it>rotein. The lack of <it>tep1 </it>does not affect the appearance of swarming motility. Interestingly, nodule formation efficiency on alfalfa plants is improved in the <it>tep1 </it>mutant during the first days of the interaction though <it>nod </it>gene expression is lower than in the wild type strain. Curiously, a <it>nodC </it>mutation or the addition of <it>N</it>-acetyl glucosamine to the wild type strain lead to similar reductions in <it>nod </it>gene expression as in the <it>tep1 </it>mutant. Moreover, aminosugar precursors of Nod factors inhibit nodulation.</p> <p>Conclusion</p> <p><it>tep1 </it>putatively encodes a transmembrane protein which can confer chloramphenicol resistance in <it>S. meliloti </it>by expelling the antibiotic outside the bacteria. The improved nodulation of alfalfa but reduced <it>nod </it>gene expression observed in the <it>tep1 </it>mutant suggests that Tep1 transports compounds which influence nodulation. In contrast to <it>Bradyrhizobium japonicum</it>, we show that in <it>S. meliloti </it>there is no feedback regulation of nodulation genes. Moreover, the Nod factor precursor, <it>N</it>-acetyl glucosamine reduces <it>nod </it>gene expression and nodulation efficiency when present at millimolar concentrations. A role for Tep1 in the efflux of Nod factor precursors could explain the phenotypes associated with <it>tep1 </it>inactivation.</p

Papel de una región cromosómica de Pseudomonas savastanoi pv. savastanoi NCPPB 3335 en la virulencia en plantas de olivo lignificadas

El genoma del patógeno de olivo Pseudomonas savastanoi pv. savastanoi (Psv) NCPPB 3335 (58.1% G+C) presenta una región cromosómica de aproximadamente 15 kb, denominada VR8 (60.4% G+C), ausente en los genomas de todos los patovares secuenciados del complejo Pseudomonas syringae que infectan plantas herbáceas, pero presente en los patovares patógenos de plantas leñosas. El análisis de esta región mediante retrotranscipción (RT)-PCR reveló la existencia de 4 posibles operones y el gen AER-1900 (posible receptor de aerotaxis). Con el objetivo de analizar la función y el papel en virulencia de estos genes se construyeron mutantes en los 4 operones y el gen AER-1900 mediante intercambio alélico. El consumo/acumulación de antranilato y catecol se cuantificó mediante HPLC en los mutantes de los operones antABC y catBCA, confirmando la participación de los mismos en el catabolismo de antranilato y catecol, respectivamente. Los otros dos operones, denominados AER-1901/2/3 y AER-1904/5, presentan homología con genes relacionados con la degradación de otros compuestos aromáticos. Hemos determinado que el operón AER-1901/2/3 está implicado en la modificación de indol hasta un compuesto azulado que podría ser índigo, mientras que el operón AER-1904/5 podría intervenir en el catabolismo de derivados clorados del catecol. Los síntomas generados por las cepas mutantes de los operones antABC y catBCA en plantas de olivo no lignificadas son similares a los generados por la cepa silvestre. Sin embargo, los síntomas producidos en plantas lignificadas por los mutantes en ambos operones y el operón AER-1901/2/3 son significativamente menores que los inducidos por la cepa silvestre, sugiriendo un posible papel de estos operones en la degradación de compuestos derivados de la lignina. En la actualidad, estamos analizando el papel en virulencia en plantas no lignificadas del gen AER-1900 y los operones AER-1901/2/3 y AER-1904/5 y en plantas lignificadas del gen AER-1900 y el operón AER-1904/5.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

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

58 pages, 7 figures, 3 tables, 3 additional files.-- Provisional PDF.[Background] Swarming 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).[Results] More 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.[Conclusions] The 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.JN was supported by a postdoctoral contract (Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía). This work was supported by a FPI fellowship from MICINN to CVA-G, and by grants BIO2007-62988 and CVI 03541 to MJS.Peer reviewe

Rhizobial Volatiles: Potential New Players in the Complex Interkingdom Signaling With Legumes

Bacteria release a wide range of volatile compounds that play important roles in intermicrobial and interkingdom communication. Volatile metabolites emitted by rhizobacteria can promote plant growth and increase plant resistance to both biotic and abiotic stresses. Rhizobia establish beneficial nitrogen-fixing symbiosis with legume plants in a process starting with a chemical dialog in the rhizosphere involving various diffusible compounds. Despite being one of the most studied plant-interacting microorganisms, very little is known about volatile compounds produced by rhizobia and their biological/ecological role. Evidence indicates that plants can perceive and respond to volatiles emitted by rhizobia. In this perspective, we present recent data that open the possibility that rhizobial volatile compounds have a role in symbiotic interactions with legumes and discuss future directions that could shed light onto this area of investigation

Physiological and transcriptomic characterization of a fliA mutant of Pseudomonas putida KT2440

Pseudomonas putida KT2440 encodes 23 alternative sigma factors. The fliA gene, which encodes σ28, is in a cluster with other genes involved in flagella biosynthesis and chemotaxis. Reverse transcriptase-PCR revealed that this cluster is comprised of four independent transcriptional units: flhAF, fleNfliA, cheYZA and cheBmotAB. We generated a nonpolar fliA mutant by homologous recombination and tested its motility, adhesion to biotic and abiotic surfaces, and responses to various stress conditions. The mutant strain was nonmotile and exhibited decreased capacity to bind to corn seeds, although its ability to colonize the rhizosphere of plants was unaffected. The mutant was also affected in binding to abiotic surfaces and its ability to form biofilms decreased by almost threefold. In the fliA mutant background expression of 25 genes was affected: two genes were upregulated and 23 genes were downregulated. In addition to a number of motility and chemotaxis genes, the fliA gene product is also necessary for the expression of some genes potentially involved in amino acid utilization or stress responses; however, we were unable to assign specific phenotypes linked to these genes since the fliA mutant used the same range of amino acids as the parental strain, and was as tolerant as the wild type to stress imposed by heat, antibiotics, NaCl, sodium dodecyl sulfate, H2O2 and benzoate. Based on the sequence alignment of promoters recognized by FliA and genome in silico analysis, we propose that P. putidaσ28 recognizes a TCAAG-t-N12-GCCGATA consensus sequence located between −34 and −8 and that this sequence is preferentially associated with an AT-rich upstream region

Taxonomic and functional metagenomic profiling of the microbial community in the anoxic sediment of a sub-saline Shallow Lake (Laguna de Carrizo, Central Spain)

The phylogenetic and functional structure of the microbial community residing in a Ca2+-rich anoxic sediment of a sub-saline shallow lake (Laguna de Carrizo, initially operated as a gypsum (CaSO4 × 2 H2O) mine) was estimated by analyzing the diversity of 16S rRNA amplicons and a 3.1 Mb of consensus metagenome sequence. The lake has about half the salinity of seawater and possesses an unusual relative concentration of ions, with Ca2+ and SO 4 2- being dominant. The 16S rRNA sequences revealed a diverse community with about 22% of the bacterial rRNAs being less than 94.5% similar to any rRNA currently deposited in GenBank. In addition to this, about 79% of the archaeal rRNA genes were mostly related to uncultured Euryarchaeota of the CCA47 group, which are often associated with marine and oxygen-depleted sites. Sequence analysis of assembled genes revealed that 23% of the open reading frames of the metagenome library had no hits in the database. Among annotated genes, functions related to (thio) sulfate and (thio) sulfonate-reduction and iron-oxidation, sulfur-oxidation, denitrification, synthrophism, and phototrophic sulfur metabolism were found as predominant. Phylogenetic and biochemical analyses indicate that the inherent physical–chemical characteristics of this habitat coupled with adaptation to anthropogenic activities have resulted in a highly efficient community for the assimilation of polysulfides, sulfoxides, and organosulfonates together with nitro-, nitrile-, and cyanide-substituted compounds. We discuss that the relevant microbial composition and metabolic capacities at Laguna de Carrizo, likely developed as an adaptation to thrive in the presence of moderate salinity conditions and potential toxic bio-molecules, in contrast with the properties of previously known anoxic sediments of shallow lakes.This research was supported by the Spanish CSD2007-00005 project and FEDER funds. M-E.G. thanks the CSIC for a JAE fellowship.Peer Reviewe

Bacterial ecotoxicity and shifts in bacterial communities associated with the removal of ibuprofen, diclofenac and triclosan in biopurification systems

The proliferation and possible adverse effects of emerging contaminants such as pharmaceutical and personal care products (PPCPs) in waters and the environment is a cause for increasing concern. We investigated the dissipation of three PPCPs: ibuprofen (IBP), diclofenac (DCF) and triclosan (TCS), separately and in mixtures, in the ppm range in biopurification system (BPS) microcosms, paying special attention to their effect on bacterial ecotoxicity, as well as bacterial community structure and composition. The results reveal that BPS microcosms efficiently dissipate IBP and DCF with 90% removed after 45 and 84 days of incubation, respectively. However, removal of TCS required a longer incubation period of 127 days for 90% removal. Furthermore, dissipation of the PPCPs was slower when a mixture of all three was applied to BPS microcosms. TCS had an initial negative effect on bacterial viability by a decrease of 34–43% as measured by live bacterial cell counts using LIVE/DEAD® microscopy; however, this effect was mitigated when the three PPCPs were present simultaneously. The bacterial communities in BPS microcosms were more affected by incubation time than by the PPCPs used. Nonetheless, the PPCPs differentially affected the composition and relative abundance of bacterial taxa. IBP and DCF initially increased bacterial diversity and richness, while exposure to TCS generally provoked an opposite effect without full recovery at the end of the incubation period. TCS, which negatively affected the relative abundance of Acidobacteria, Methylophilales, and Legionellales, had the largest impact on bacterial groups. Biomarker OTUs were identified in the BPS microcosms which were constrained to higher concentrations of the PPCPs and thus are likely to harbour degradation and/or detoxification mechanisms. This study reveals for the first time the effect of PPCPs on bacterial ecotoxicity and diversity in biopurification system microcosms and also facilitates the design of further applications of biomixtures to eliminate PPCPs.This study was funded by the Spanish Ministry of Economy and Competitiveness and the Spanish State Research Agency (AEI) (projects CTM2013-44271-R and CTM2017-86504-R), and co-financing from the European Regional Development Fund (ERDF). Inés Aguilar-Romero was supported by an FPI grant. Finally, we would like to thank Michael O'Shea for proofreading the manuscript

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

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

Árboles transgénicos resistentes a explosivos y que eliminan TNT

Árboles transgénicos resistentes a explosivos y que eliminan TNT. El objetivo de la presente patente es aportar una alternativa económica y respetuosa con el medio ambiente para eliminar compuestos nitroaromáticos, preferentemente 2,4,6-trinitrotolueno (TNT) de suelos y aguas subterráneas. Para ello se utilizan plantas transgénicas que eliminan estos compuestos. El método incluye el uso de chopos y abedules transgénicos portando los genes pnrA, xenB o nemA de Pseudomonas putida o el gen nemA de Escherichia coli expresados desde el promotor 35S del virus del mosaico de la coliflor (CaMV35S) que es constitutivo en plantas. La expresión de estos genes confiere a los árboles mayor resistencia a TNT e incrementa la capacidad para eliminar TNT de suelos y aguas subterráneas. La limpieza de los sitios contaminados pueden realizarla las plantas transgénicas cultivándolas en los suelos contaminados o en suelos próximos a acuíferos contaminados. Las plantas extraen TNT y lo detoxifican.Estas plantas transgénicas pueden restaurar sitios altamente contaminados, y así permitir la ecolonización del suelo limpio por plantas autóctonas que no verían inhibido su crecimiento debido a la eliminación del contaminante.Consejo Superior de Investigaciones Científicas (España)B1 Patente con informe sobre el estado de la ténic