Transcriptomic Studies Reveal that the Rhizobium leguminosarum Serine/Threonine Protein Phosphatase PssZ has a Role in the Synthesis of Cell-Surface Components, Nutrient Utilization, and Other Cellular Processes

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing symbiotic associations with clover plants (Trifolium spp.). Surface polysaccharides, transport systems, and extracellular components synthesized by this bacterium are required for both the adaptation to changing environmental conditions and successful infection of host plant roots. The pssZ gene located in the Pss-I region, which is involved in the synthesis of extracellular polysaccharide, encodes a protein belonging to the group of serine/threonine protein phosphatases. In this study, a comparative transcriptomic analysis of R. leguminosarum bv. trifolii wild-type strain Rt24.2 and its derivative Rt297 carrying a pssZ mutation was performed. RNA-Seq data identified a large number of genes differentially expressed in these two backgrounds. Transcriptome profiling of the pssZ mutant revealed a role of the PssZ protein in several cellular processes, including cell signalling, transcription regulation, synthesis of cell-surface polysaccharides and components, and bacterial metabolism. In addition, we show that inactivation of pssZ affects the rhizobial ability to grow in the presence of different sugars and at various temperatures, as well as the production of different surface polysaccharides. In conclusion, our results identified a set of genes whose expression was affected by PssZ and confirmed the important role of this protein in the rhizobial regulatory networ

Hanks-Type Serine/Threonine Protein Kinases and Phosphatases in Bacteria: Roles in Signaling and Adaptation to Various Environments

Reversible phosphorylation is a key mechanism that regulates many cellular processes in prokaryotes and eukaryotes. In prokaryotes, signal transduction includes two-component signaling systems, which involve a membrane sensor histidine kinase and a cognate DNA-binding response regulator. Several recent studies indicate that alternative regulatory pathways controlled by Hanks-type serine/threonine kinases (STKs) and serine/threonine phosphatases (STPs) also play an essential role in regulation of many different processes in bacteria, such as growth and cell division, cell wall biosynthesis, sporulation, biofilm formation, stress response, metabolic and developmental processes, as well as interactions (either pathogenic or symbiotic) with higher host organisms. Since these enzymes are not DNA-binding proteins, they exert the regulatory role via post-translational modifications of their protein targets. In this review, we summarize the current knowledge of STKs and STPs, and discuss how these enzymes mediate gene expression in prokaryotes. Many studies indicate that regulatory systems based on Hanks-type STKs and STPs play an essential role in the regulation of various cellular processes, by reversibly phosphorylating many protein targets, among them several regulatory proteins of other signaling cascades. These data show high complexity of bacterial regulatory network, in which the crosstalk between STK/STP signaling enzymes, components of TCSs, and the translational machinery occurs. In this regulation, the STK/STP systems have been proved to play important roles

Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.Polish National Science Centre grant no. DEC-2012/07/B/NZ1/0009

Deciphering the Symbiotic Significance of Quorum Sensing Systems of Sinorhizobium fredii HH103

Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.Spanish Ministerio de Economía y Competitividad (MINECO) BIO2016-78409-

Regulation of Protein Secretion Systems Mediated by Cyclic Diguanylate in Plant-Interacting Bacteria

The ubiquitous second messenger cyclic diguanylate (c-di-GMP) is involved in the regulation of different processes in bacteria. In phytopathogens, intracellular fluctuations in the concentration of this molecule contribute to the lifestyle switching from a motile and virulent stage to a sessile and biofilm-forming phase. Among the virulence mechanisms used by bacterial pathogens, different specific type secretion systems (TSSs) and the effector proteins that they translocate are included. Some of these TSS are conceived to suppress host immune responses during bacterial colonization. The modulation of the expression of secretion systems components and/or effector proteins can be influenced by c-di-GMP levels at transcriptional, translational, or post-translational levels and can take place directly by binding to specific or global regulators, or via transducer proteins. Different genera of plant-interacting bacteria have been analyzed to shed some light in the implications of c-di-GMP in the regulation of host plant colonization through protein secretion systems. Expression of (1) adhesins secreted by Type 1 secretion systems to bind the host plant in Pectobacterium (formerly Erwinia) and some beneficial Pseudomonas strains; (2) catalytic exoproteins delivered by Type 2 secretion systems to break plant cell wall in Dickeya; (3) effectors secreted by Type 3 secretion systems to suppress plant immunity in Xanthomonas; or (4) the activity of Type 6 secretion systems to export an ATPase in Pseudomonas, are finely tuned by c-di-GMP levels. In this minireview, we summarize the knowledge available about the implications of c-di-GMP in the regulation of protein secretion in different plant-interacting bacteria.Spanish Ministry of Economy, Industry and Competitivity BIO2016-78409-

Regulatory elements located in the upstream region of the Rhizobium leguminosarum rosR global regulator are essential for its transcription and mRNA stability

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a symbiotic relationship with clover (Trifolium spp.). Previously, the rosR gene, encoding a global regulatory protein involved in motility, synthesis of cell-surface components, and other cellular processes was identified and characterized in this bacterium. This gene possesses a long upstream region that contains several regulatory motifs, including inverted repeats (IRs) of different lengths. So far, the role of these motifs in the regulation of rosR transcription has not been elucidated in detail. In this study, we performed a functional analysis of these motifs using a set of transcriptional rosR-lacZ fusions that contain mutations in these regions. The levels of rosR transcription for different mutant variants were evaluated in R. leguminosarum using both quantitative real-time PCR and β-galactosidase activity assays. Moreover, the stability of wild type rosR transcripts and those with mutations in the regulatory motifs was determined using an RNA decay assay and plasmids with mutations in different IRs located in the 5′ -untranslated region of the gene. The results show that transcription of rosR undergoes complex regulation, in which several regulatory elements located in the upstream region and some regulatory proteins are engaged. These include an upstream regulatory element, an extension of the -10 element containing three nucleotides TGn (TGn-extended -10 element), several IRs, and PraR repressor related to quorum sensing.Polish National Science Centre DEC-2012/07/B/NZ1/0009

Sinorhizobium fredii HH103 RirA is required for oxidative stress resistance and efficient symbiosis with Soybean

Members of Rhizobiaceae contain a homologue of the iron-responsive regulatory protein RirA. In different bacteria, RirA acts as a repressor of iron uptake systems under iron-replete conditions and contributes to ameliorate cell damage during oxidative stress. In Rhizobium leguminosarum and Sinorhizobium meliloti, mutations in rirA do not impair symbiotic nitrogen fixation. In this study, a rirA mutant of broad host range S. fredii HH103 has been constructed (SVQ780) and its free-living and symbiotic phenotypes evaluated. No production of siderophores could be detected in either the wild-type or SVQ780. The rirA mutant exhibited a growth advantage under iron-deficient conditions and hypersensitivity to hydrogen peroxide in iron-rich medium. Transcription of rirA in HH103 is subject to autoregulation and inactivation of the gene upregulates fbpA, a gene putatively involved in iron transport. The S. fredii rirA mutant was able to nodulate soybean plants, but symbiotic nitrogen fixation was impaired. Nodules induced by the mutant were poorly infected compared to those induced by the wild-type. Genetic complementation reversed the mutant’s hypersensitivity to H2O2, expression of fbpA, and symbiotic deficiency in soybean plants. This is the first report that demonstrates a role for RirA in the Rhizobium-legume symbiosis.Andalucian Government Grant No. P11-CVI-7500Spanish Government Grant Nos. BIO2013-42801-P and BIO2016-78409-REuropean Regional Development Funds (ERDF)VPPI (V Plan Propio de Investigación) of University of Seville

Sinorhizobium fredii Strains HH103 and NGR234 Form Nitrogen Fixing Nodules With Diverse Wild Soybeans (Glycine soja) From Central China but Are Ineffective on Northern China Accessions

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals.España, Junta de Andalucía P11-CVI-7500España Ministerio de Economía y Competitividad BIO2016-78409-

Mortalidad y duración potencial de las uniones

Estadística Española Volumen 58, número 191 / 2016, pp. 301-312La mejora de la mortalidad modifica no sólo las edades que la experimentan, sino todo el transcurso de vida posterior. Se analiza el efecto de la mortalidad sobre la potencial duración de las uniones conyugales, en ausencia de otras causas de disolución, mediante un modelo de supervivencia de las uniones que combina las tablas de mortalidad de ambos sexos con distintos supuestos de edad media a la unión. El resultado evidencia cambios históricos en la duración posible de las uniones, y otras consecuencias sobre la viudedad, una profunda reestructuración de los hogares y un escenario de cambios sustanciales en los roles de género y edad.Peer reviewe

Sustainability assessment of green ammonia production to promote industrial decarbonization in Spain

This article investigates the economic and environmental implications of implementing green ammonia production plants in Spain. To this end, one business-as-usual scenario for gray ammonia production was compared with three green ammonia scenarios powered with different renewable energy sources (i.e., solar photovoltaic (PV), wind, and a combination of solar PV and wind). The results illustrated that green ammonia scenarios reduced the environmental impacts in global warming, stratospheric ozone depletion, and fossil resource scarcity when compared with conventional gray ammonia scenario. Conversely, green ammonia implementation increased the environmental impacts in the categories of land use, mineral resource scarcity, freshwater eutrophication, and terrestrial acidification. The techno-economic analysis revealed that the conventional gray ammonia scenario featured lower costs than green ammonia scenarios when considering a moderate natural gas cost. However, green ammonia implementation became the most economically favorable option when the natural gas cost and carbon prices increased. Finally, the results showed that developing efficient ammonia-fueled systems is important to make green ammonia a relevant energy vector when considering the entire supply chain (production/transportation). Overall, the results of this research demonstrate that green ammonia could play an important role in future decarbonization scenarios.This study has been supported by the Research Spanish Agency (AEI) through the projects REEMEs (PID2021-127028OB-I00) and SUSTDESALT (TED2021-131708B-C21). Additionally, the authors acknowledge the Open Innovation - Research Translation and Applied Knowledge Exchange in Practice through University–Industry Cooperation (OpenInnoTrain) with the grant agreement number (GAN) 823971 (H2020-MSCA-RISE-2018-823971), and the Catalan government (2021-SGR-GRC-00596). J. L. Cortina received support for the research through the “ICREA Academia” recognition for excellence in research funded by the Generalitat de Catalunya.Peer ReviewedPostprint (published version