Douglas Fabiano Gomes

Proteomic profiling of Rhizobium tropici PRF 81: identification of conserved and specific responses to heat stress

BackgroundRhizobium tropici strain PRF 81 (= SEMIA 4080) has been used in commercial inoculants for application to common-bean crops in Brazil since 1998, due to its high efficiency in fixing nitrogen, competitiveness against indigenous rhizobial populations and capacity to adapt to stressful tropical conditions, representing a key alternative to application of N-fertilizers. The objective of our study was to obtain an overview of adaptive responses to heat stress of strain PRF 81, by analyzing differentially expressed proteins when the bacterium is grown at 28°C and 35°C.ResultsTwo-dimensional gel electrophoresis (2DE) revealed up-regulation of fifty-nine spots that were identified by MALDI-TOF/TOF-TOF. Differentially expressed proteins were associated with the functional COG categories of metabolism, cellular processes and signaling, information storage and processing. Among the up-regulated proteins, we found some related to conserved heat responses, such as molecular chaperones DnaK and GroEL, and other related proteins, such as translation factors EF-Tu, EF-G, EF-Ts and IF2. Interestingly, several oxidative stress-responsive proteins were also up-regulated, and these results reveal the diversity of adaptation mechanisms presented by this thermotolerant strain, suggesting a cross-talk between heat and oxidative stresses.ConclusionsOur data provide valuable protein-expression information relevant to the ongoing genome sequencing of strain PRF 81, and contributes to our still-poor knowledge of the molecular determinants of the thermotolerance exhibited by R. tropici species.

Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation.

BackgroundNodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes—with an emphasis on common bean (Phaseolus vulgaris)—and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated.ResultsPhenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes.ConclusionOur proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis.

Opening the “black box” of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899

BackgroundTranscription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants.MethodsThe three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.).ResultsPhenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.ConclusionsWe report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.

Two-dimensional proteome reference map of Rhizobium tropici PRF 81 reveals several symbiotic determinants and strong resemblance with agrobacteria.

Rhizobium tropici strain PRF 81 is used in commercial inoculants for common‐bean crops in Brazil because of its high efficiency in nitrogen fixation and, as in other strains belonging to this species, its tolerance of environmental stresses, representing a useful biological alternative to chemical nitrogen fertilizers. In this study, a proteomic reference map of PRF 81 was obtained by two‐dimensional gel electrophoresis and MALDI‐TOF/TOF‐TOF mass spectrometry. In total, 115 spots representing 109 different proteins were successfully identified, contributing to a better understanding of the rhizobia‐legume symbiosis and supporting, at proteomics level, a strong resemblance with agrobacteria.

NrcR, a New Transcriptional Regulator of Rhizobium tropici CIAT 899 Involved in the Legume Root-Nodule Symbiosis.

The establishment of nitrogen-fixing rhizobium-legume symbioses requires a highly complex cascade of events. In this molecular dialogue the bacterial NodD transcriptional regulators in conjunction with plant inducers, mostly flavonoids, are responsible for the biosynthesis and secretion of Nod factors which are key molecules for successful nodulation. Other transcriptional regulators related to the symbiotic process have been identified in rhizobial genomes, including negative regulators such as NolR. Rhizobium tropici CIAT 899 is an important symbiont of common bean (Phaseolus vulgaris L.), and its genome encompasses intriguing features such as five copies of nodD genes, as well as other possible transcriptional regulators including the NolR protein. Here we describe and characterize a new regulatory gene located in the non-symbiotic plasmid pRtrCIAT899c, that shows homology (46% identity) with the nolR gene located in the chromosome of CIAT 899. The mutation of this gene, named nrcR (nolR-like plasmid c Regulator), enhanced motility and exopolysaccharide production in comparison to the wild-type strain. Interestingly, the number and decoration of Nod Factors produced by this mutant were higher than those detected in the wild-type strain, especially under salinity stress. The nrcR mutant showed delayed nodulation and reduced competitiveness with P. vulgaris, and reduction in nodule number and shoot dry weight in both P. vulgaris and Leucaena leucocephala. Moreover, the mutant exhibited reduced capacity to induce the nodC gene in comparison to the wild-type CIAT 899. The finding of a new nod-gene regulator located in a non-symbiotic plasmid may reveal the existence of even more complex mechanisms of regulation of nodulation genes in R. tropici CIAT 899 that may be applicable to other rhizobial species.

Proteomic analysis of free-living Bradyrhizobium diazoefficiens: highlighting potential determinants of a successful symbiosis

BackgroundStrain CPAC 7 (=SEMIA 5080) was recently reclassified into the new species Bradyrhizobium diazoefficiens; due to its outstanding efficiency in fixing nitrogen, it has been used in commercial inoculants for application to crops of soybean [Glycine max (L.) Merr.] in Brazil and other South American countries. Although the efficiency of B. diazoefficiens inoculant strains is well recognized, few data on their protein expression are available.ResultsWe provided a two-dimensional proteomic reference map of CPAC 7 obtained under free-living conditions, with the successful identification of 115 spots, representing 95 different proteins. The results highlighted the expression of molecular determinants potentially related to symbiosis establishment (e.g. inositol monophosphatase, IMPase), fixation of atmospheric nitrogen (N2) (e.g. NifH) and defenses against stresses (e.g. chaperones). By using bioinformatic tools, it was possible to attribute probable functions to ten hypothetical proteins. For another ten proteins classified as “NO related COG” group, we analyzed by RT-qPCR the relative expression of their coding-genes in response to the nodulation-gene inducer genistein. Six of these genes were up-regulated, including blr0227, which may be related to polyhydroxybutyrate (PHB) biosynthesis and competitiveness for nodulation.ConclusionsThe proteomic map contributed to the identification of several proteins of B. diazoefficiens under free-living conditions and our approach—combining bioinformatics and gene-expression assays—resulted in new information about unknown genes that might play important roles in the establishment of the symbiosis with soybean.

Genome of Rhizobium leucaenae strains CFN 299 T and CPAO 29.8: searching for genes related to a successful symbiotic performance under stressful conditions

BackgroundCommon bean (Phaseolus vulgaris L.) is the most important legume cropped worldwide for food production and its agronomic performance can be greatly improved if the benefits from symbiotic nitrogen fixation are maximized. The legume is known for its high promiscuity in nodulating with several Rhizobium species, but those belonging to the Rhizobium tropici “group” are the most successful and efficient in fixing nitrogen in tropical acid soils. Rhizobium leucaenae belongs to this group, which is abundant in the Brazilian “Cerrados” soils and frequently submitted to several environmental stresses. Here we present the first high-quality genome drafts of R. leucaenae, including the type strain CFN 299T and the very efficient strain CPAO 29.8. Our main objective was to identify features that explain the successful capacity of R. leucaenae in nodulating common bean under stressful environmental conditions.ResultsThe genomes of R. leucaenae strains CFN 299T and CPAO 29.8 were estimated at 6.7–6.8 Mbp; 7015 and 6899 coding sequences (CDS) were predicted, respectively, 6264 of which are common to both strains. The genomes of both strains present a large number of CDS that may confer tolerance of high temperatures, acid soils, salinity and water deficiency. Types I, II, IV-pili, IV and V secretion systems were present in both strains and might help soil and host colonization as well as the symbiotic performance under stressful conditions. The symbiotic plasmid of CPAO 29.8 is highly similar to already described tropici pSyms, including five copies of nodD and three of nodA genes. R. leucaenae CFN 299T is capable of synthesizing Nod factors in the absence of flavonoids when submitted to osmotic stress, indicating that under abiotic stress the regulation of nod genes might be different.ConclusionA detailed study of the genes putatively related to stress tolerance in R. leucaenae highlighted an intricate pattern comprising a variety of mechanisms that are probably orchestrated to tolerate the stressful conditions to which the strains are submitted on a daily basis. The capacity to synthesize Nod factors under abiotic stress might follow the same regulatory pathways as in CIAT 899T and may help both to improve bacterial survival and to expand host range to guarantee the perpetuation of the symbiosis.

Proteomic Analysis of Soybean (Glycine max (L.) Merrill) Roots Inoculated with Bradyrhizobium japonicum Strain CPAC 15

This research intended to analyze the expression pattern of proteins in roots of the Brazilian soybean cultivar Conquista when inoculated with Bradyrhizobium japonicum CPAC 15, a strain broadly used in commercial inoculants in Brazil. At ten days after bacterial inoculation, whole-cell proteins were extracted from roots and separated by 2-D gel electrophoresis. Comparative analysis revealed significant changes in the intensity of 37 spots due to the inoculation (17 up-regulated and 20 down-regulated proteins), identified by MALDI-TOF/TOF-TOF. Identified proteins were associated with COG functional categories of information storage and processing, cellular processes and signaling, metabolism, and also in the “poorly characterized” and “not in COG” categories. Among the up-regulated proteins, we identified sucrose synthase (nodulin-100), β-tubulin, rubisco activase, glutathione-S-transferase, a putative heat-shock 70-kDa protein, pyridine nucleotide-disulphideoxidoreductase and a putative transposase. Proteomic analysis allowed for the identification of some putative symbiotic functions and confirmed the main biological processes triggered in the nitrogen-fixing symbiosis with soybean.

Genome Sequence of Rhizobium ecuadorense Strain CNPSo 671T, an Indigenous N2-Fixing Symbiont of the Ecuadorian Common Bean (Phaseolus vulgaris L.) Genetic Pool

ABSTRACT Rhizobium ecuadorense CNPSo 671T was isolated from a common bean nodule in Ecuador. The draft genome brings novelty about indigenous rhizobial species in centers of genetic diversity of the legume.

Draft Genome Sequence of Pseudomonas fluorescens Strain ET76, Isolated from Rice Rhizosphere in Northwestern Morocco

ABSTRACT Pseudomonas fluorescens ET76 was isolated from rice rhizosphere in northwestern Morocco. Its draft genome was estimated to be 6,681,652 bp with 5,789 coding sequences (CDSs). Genes encoding for type I to VI secretion systems, PvdQ, proteases, siderophores, hydrogen cyanide synthase, ACC-deaminase, among others, highlight its potential use in biological control of plant pathogens.