A protease and a lipoprotein jointly modulate the conserved ExoR-ExoS-ChvI signaling pathway critical in Sinorhizobium meliloti for symbiosis with legume hosts.

Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S. meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S. meliloti with Medicago sativa and S. medicae with M. truncatula. Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli. Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.

Identification of a Novel Pyruvyltransferase Using 13C Solid-State Nuclear Magnetic Resonance To Analyze Rhizobial Exopolysaccharides.

The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPSs), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts and, thus, for the formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation and can facilitate invasion in the absence of succinoglycan on some host plants. The biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to identify putative pyruvyltransferase and acetyltransferase genes, construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan, and analyze EPS from mutant bacterial strains. EPS samples were examined by 13C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enables the detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains with deletions in five candidate acyl/acetyltransferase genes (exoZ, exoH, SMb20810, SMb21188, and SMa1016) and a putative pyruvyltransferase (wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through the ectopic expression of plasmid-borne wgaE. Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides. IMPORTANCE Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPSs) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. The biosynthesis of galactoglucan is not as well characterized as that of succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild-type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into the biosynthesis of an important S. meliloti EPS and expands the knowledge of enzymes that modify polysaccharides.

A high-throughput system to identify inhibitors of Candidatus Liberibacter asiaticus transcription regulators.

Citrus greening disease, also known as huanglongbing (HLB), is the most devastating disease of Citrus worldwide. This incurable disease is caused primarily by the bacterium Candidatus Liberibacter asiaticus and spread by feeding of the Asian Citrus Psyllid, Diaphorina citriCa L. asiaticus cannot be cultured; its growth is restricted to citrus phloem and the psyllid insect. Management of infected trees includes use of broad-spectrum antibiotics, which have disadvantages. Recent work has sought to identify small molecules that inhibit Ca L. asiaticus transcription regulators, based on a premise that at least some regulators control expression of genes necessary for virulence. We describe a synthetic, high-throughput screening system to identify compounds that inhibit activity of Ca L. asiaticus transcription activators LdtR, RpoH, and VisNR. Our system uses the closely related model bacterium, Sinorhizobium meliloti, as a heterologous host for expression of a Ca L. asiaticus transcription activator, the activity of which is detected through expression of an enhanced green fluorescent protein (EGFP) gene fused to a target promoter. We used this system to screen more than 120,000 compounds for compounds that inhibited regulator activity, but not growth. Our screen identified several dozen compounds that inhibit regulator activity in our assay. This work shows that, in addition to providing a means of characterizing Ca L. asiaticus regulators, an S. meliloti host can be used for preliminary identification of candidate inhibitory molecules.

Symbiotic Performance of Sinorhizobium meliloti Lacking ppGpp Depends on the Medicago Host Species.

Host specificity in the root-nodule symbiosis between legumes and rhizobia is crucial for the establishment of a successful interaction and ammonia provision to the plant. The specificity is mediated by plant-bacterial signal exchange during early stages of interaction. We observed that a Sinorhizobium meliloti mutant ∆relA, which is deficient in initiating the bacterial stringent response, fails to nodulate Medicago sativa (alfalfa) but successfully infects Medicago truncatula. We used biochemical, histological, transcriptomic, and imaging approaches to compare the behavior of the S. meliloti ∆relA mutant and wild type (WT) on the two plant hosts. ∆relA performed almost WT-like on M. truncatula, except for reduced nitrogen-fixation capacity and a disorganized positioning of bacteroids within nodule cells. In contrast, ∆relA showed impaired root colonization on alfalfa and failed to infect nodule primordia. Global transcriptome analyses of ∆relA cells treated with the alfalfa flavonoid luteolin and of mature nodules induced by the mutant on M. truncatula revealed normal nod gene expression but overexpression of exopolysaccharide biosynthesis genes and a slight suppression of plant defense-like reactions. Many RelA-dependent transcripts overlap with the hypo-osmolarity-related FeuP regulon or are characteristic of stress responses. Based on our findings, we suggest that RelA is not essential until the late stages of symbiosis with M. truncatula, in which it may be involved in processes that optimize nitrogen fixation.

Most Sinorhizobium meliloti Extracytoplasmic Function Sigma Factors Control Accessory Functions.

Bacteria must sense alterations in their environment and respond with changes in function and/or structure in order to cope. Extracytoplasmic function sigma factors (ECF σs) modulate transcription in response to cellular and environmental signals. The symbiotic nitrogen-fixing alphaproteobacterium Sinorhizobium meliloti carries genes for 11 ECF-like σs (RpoE1 to -E10 and FecI). We hypothesized that some of these play a role in mediating the interaction between the bacterium and its plant symbiotic partner. The bacterium senses changes in its immediate environment as it establishes contact with the plant root, initiates invasion of the plant as the root nodule is formed, traverses several root cell layers, and enters plant cortical cells via endocytosis. We used genetics, transcriptomics, and functionality to characterize the entire S. meliloti cohort of ECF σs. We discovered new targets for individual σs, confirmed others by overexpressing individual ECF σs, and identified or confirmed putative promoter motifs for nine of them. We constructed precise deletions of each ECF σ gene and its demonstrated or putative anti-σ gene and also a strain in which all 11 ECF σ and anti-σ genes were deleted. This all-ECF σ deletion strain showed no major defects in free-living growth, in Biolog Phenotype MicroArray assays, or in response to multiple stresses. None of the ECF σs were required for symbiosis on the host plants Medicago sativa and Medicago truncatula: the strain deleted for all ECF σ and anti-σ genes was symbiotically normal.IMPORTANCE Fixed (reduced) soil nitrogen plays a critical role in soil fertility and successful food growth. Much soil fertility relies on symbiotic nitrogen fixation: the bacterial partner infects the host plant roots and reduces atmospheric dinitrogen in exchange for host metabolic fuel, a process that involves complex interactions between the partners mediated by changes in gene expression in each partner. Here we test the roles of a family of 11 extracytoplasmic function (ECF) gene regulatory proteins (sigma factors [σs]) that interact with RNA polymerase to determine if they play a significant role in establishing a nitrogen-fixing symbiosis or in responding to various stresses, including cell envelope stress. We discovered that symbiotic nitrogen fixation occurs even when all 11 of these regulatory genes are deleted, that most ECF sigma factors control accessory functions, and that none of the ECF sigma factors are required to survive envelope stress.

Genome-wide identification of genes directly regulated by ChvI and a consensus sequence for ChvI binding in Sinorhizobium meliloti.

ExoS/ChvI two-component signaling in the nitrogen-fixing α-proteobacterium Sinorhizobium meliloti is required for symbiosis and regulates exopolysaccharide production, motility, cell envelope integrity and nutrient utilization in free-living bacteria. However, identification of many ExoS/ChvI direct transcriptional target genes has remained elusive. Here, we performed chromatin immunoprecipitation followed by microarray analysis (chIP-chip) to globally identify DNA regions bound by ChvI protein in S. meliloti. We then performed qRT-PCR with chvI mutant strains to test ChvI-dependent expression of genes downstream of the ChvI-bound DNA regions. We identified 64 direct target genes of ChvI, including exoY, rem and chvI itself. We also identified ChvI direct target candidates, like exoR, that are likely controlled by additional regulators. Analysis of upstream sequences from the 64 ChvI direct target genes identified a 15 bp-long consensus sequence. Using electrophoretic mobility shift assays and transcriptional fusions with exoY, SMb21440, SMc00084, SMc01580, chvI, and ropB1, we demonstrated this consensus sequence is important for ChvI binding to DNA and transcription of ChvI direct target genes. Thus, we have comprehensively identified ChvI regulon genes and a 'ChvI box' bound by ChvI. Many ChvI direct target genes may influence the cell envelope, consistent with the critical role of ExoS/ChvI in growth and microbe-host interactions.

Corrigendum: Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

[This corrects the article on p. 76 in vol. 9, PMID: 29467773.].

Characterization of Novel Plant Symbiosis Mutants Using a New Multiple Gene-Expression Reporter Sinorhizobium meliloti Strain.

The formation of nitrogen fixing root nodules by Medicago truncatula and Sinorhizobium meliloti requires communication between both organisms and coordinated differentiation of plant and bacterial cells. After an initial signal exchange, the bacteria invade the tissue of the growing nodule via plant-derived tubular structures, called infection threads. The bacteria are released from the infection threads into invasion-competent plant cells, where they differentiate into nitrogen-fixing bacteroids. Both organisms undergo dramatic transcriptional, metabolic and morphological changes during nodule development. To identify plant processes that are essential for the formation of nitrogen fixing nodules after nodule development has been initiated, large scale mutageneses have been conducted to discover underlying plant symbiosis genes. Such screens yield numerous uncharacterized plant lines with nitrogen fixation deficient nodules. In this study, we report construction of a S. meliloti strain carrying four distinct reporter constructs to reveal stages of root nodule development. The strain contains a constitutively expressed lacZ reporter construct; a PexoY-mTFP fusion that is expressed in infection threads but not in differentiated bacteroids; a PbacA-mcherry construct that is expressed in infection threads and during bacteroid differentiation; and a PnifH-uidA construct that is expressed during nitrogen fixation. We used this strain together with fluorescence microscopy to study nodule development over time in wild type nodules and to characterize eight plant mutants from a fast neutron bombardment screen. Based on the signal intensity and the localization patterns of the reporter genes, we grouped mutants with similar phenotypes and placed them in a developmental context.

OxyR-Dependent Transcription Response of Sinorhizobium meliloti to Oxidative Stress.

Reactive oxygen species such as peroxides play an important role in plant development, cell wall maturation, and defense responses. During nodulation with the host plant Medicago sativa, Sinorhizobium meliloti cells are exposed to H2O2 in infection threads and developing nodules (R. Santos, D. Hérouart, S. Sigaud, D. Touati, and A. Puppo, Mol Plant Microbe Interact 14:86-89, 2001, https://doi.org/10.1094/MPMI.2001.14.1.86). S. meliloti cells likely also experience oxidative stress, from both internal and external sources, during life in the soil. Here, we present microarray transcription data for S. meliloti wild-type cells compared to a mutant deficient in the key oxidative regulatory protein OxyR, each in response to H2O2 treatment. Several alternative sigma factor genes are upregulated in the response to H2O2; the stress sigma gene rpoE2 shows OxyR-dependent induction by H2O2, while rpoH1 expression is induced by H2O2 irrespective of the oxyR genotype. The activity of the RpoE2 sigma factor in turn causes increased expression of two more sigma factor genes, rpoE5 and rpoH2 Strains with deletions of rpoH1 showed improved survival in H2O2 as well as increased levels of oxyR and total catalase expression. These results imply that ΔrpoH1 strains are primed to deal with oxidative stress. This work presents a global view of S. meliloti gene expression changes, and of regulation of those changes, in response to H2O2IMPORTANCE Like all aerobic organisms, the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti experiences oxidative stress throughout its complex life cycle. This report describes the global transcriptional changes that S. meliloti makes in response to H2O2 and the roles of the OxyR transcriptional regulator and the RpoH1 sigma factor in regulating those changes. By understanding the complex regulatory response of S. meliloti to oxidative stress, we may further understand the role that reactive oxygen species play as both stressors and potential signals during symbiosis.

Novel Genes and Regulators That Influence Production of Cell Surface Exopolysaccharides in Sinorhizobium meliloti.

Sinorhizobium meliloti is a soil-dwelling alphaproteobacterium that engages in a nitrogen-fixing root nodule symbiosis with leguminous plants. Cell surface polysaccharides are important both for adapting to stresses in the soil and for the development of an effective symbiotic interaction. Among the polysaccharides characterized to date, the acidic exopolysaccharides I (EPS-I; succinoglycan) and II (EPS-II; galactoglucan) are particularly important for protection from abiotic stresses, biofilm formation, root colonization, and infection of plant roots. Previous genetic screens discovered mutants with impaired EPS production, allowing the delineation of EPS biosynthetic pathways. Here we report on a genetic screen to isolate mutants with mucoid colonial morphologies that suggest EPS overproduction. Screening with Tn5-110, which allows the recovery of both null and upregulation mutants, yielded 47 mucoid mutants, most of which overproduce EPS-I; among the 30 unique genes and intergenic regions identified, 14 have not been associated with EPS production previously. We identified a new protein-coding gene, emmD, which may be involved in the regulation of EPS-I production as part of the EmmABC three-component regulatory circuit. We also identified a mutant defective in EPS-I production, motility, and symbiosis, where Tn5-110 was not responsible for the mutant phenotypes; these phenotypes result from a missense mutation in rpoA corresponding to the domain of the RNA polymerase alpha subunit known to interact with transcription regulators.IMPORTANCE The alphaproteobacterium Sinorhizobium meliloti converts dinitrogen to ammonium while inhabiting specialized plant organs termed root nodules. The transformation of S. meliloti from a free-living soil bacterium to a nitrogen-fixing plant symbiont is a complex developmental process requiring close interaction between the two partners. As the interface between the bacterium and its environment, the S. meliloti cell surface plays a critical role in adaptation to varied soil environments and in interaction with plant hosts. We isolated and characterized S. meliloti mutants with increased production of exopolysaccharides, key cell surface components. Our diverse set of mutants suggests roles for exopolysaccharide production in growth, metabolism, cell division, envelope homeostasis, biofilm formation, stress response, motility, and symbiosis.

SnapShot: Signaling in Symbiosis.

The nitrogen-fixing Rhizobium-legume partnership is presently the best understood of all host-microbe symbioses. Bacterial and plant partners signal across developmental time and space.

LDSS-P: an advanced algorithm to extract functional short motifs associated with coordinated gene expression.

Identifying functional elements in promoter sequences is a major goal in computational and experimental genome biology. Here, we describe an algorithm, Local Distribution of Short Sequences for Prokaryotes (LDSS-P), to identify conserved short motifs located at specific positions in the promoters of co-expressed prokaryotic genes. As a test case, we applied this algorithm to a symbiotic nitrogen-fixing bacterium, Sinorhizobium meliloti The LDSS-P profiles that overlap with the 5' section of the extracytoplasmic function RNA polymerase sigma factor RpoE2 consensus sequences displayed a sharp peak between -34 and -32 from TSS positions. The corresponding genes overlap significantly with RpoE2 targets identified from previous experiments. We further identified several groups of genes that are co-regulated with characterized marker genes. Our data indicate that in S. meliloti, and possibly in other Rhizobiaceae species, the master cell cycle regulator CtrA may recognize an expanded motif (AACCAT), which is positionally shifted from the previously reported CtrA consensus sequence in Caulobacter crescentus Bacterial one-hybrid experiments showed that base substitution in the expanded motif either increase or decrease the binding by CtrA. These results show the effectiveness of LDSS-P as a method to delineate functional promoter elements.

Contributions of Sinorhizobium meliloti Transcriptional Regulator DksA to Bacterial Growth and Efficient Symbiosis with Medicago sativa.

UNLABELLED: The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis. IMPORTANCE: The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system.

Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules.

The bacterium Sinorhizobium meliloti interacts symbiotically with legume plant hosts such as Medicago truncatula to form nitrogen-fixing root nodules. During symbiosis, plant and bacterial cells differentiate in a coordinated manner, resulting in specialized plant cells that contain nitrogen-fixing bacteroids. Both plant and bacterial genes are required at each developmental stage of symbiosis. We analyzed gene expression in nodules formed by wild-type bacteria on six plant mutants with defects in nitrogen fixation. We observed differential expression of 482 S. meliloti genes with functions in cell envelope homeostasis, cell division, stress response, energy metabolism, and nitrogen fixation. We simultaneously analyzed gene expression in M. truncatula and observed differential regulation of host processes that may trigger bacteroid differentiation and control bacterial infection. Our analyses of developmentally arrested plant mutants indicate that plants use distinct means to control bacterial infection during early and late symbiotic stages.

The Sinorhizobium meliloti SyrM regulon: effects on global gene expression are mediated by syrA and nodD3.

UNLABELLED: In Sinorhizobium meliloti, three NodD transcriptional regulators activate bacterial nodulation (nod) gene expression. NodD1 and NodD2 require plant compounds to activate nod genes. The NodD3 protein does not require exogenous compounds to activate nod gene expression; instead, another transcriptional regulator, SyrM, activates nodD3 expression. In addition, NodD3 can activate syrM expression. SyrM also activates expression of another gene, syrA, which when overexpressed causes a dramatic increase in exopolysaccharide production. In a previous study, we identified more than 200 genes with altered expression in a strain overexpressing nodD3. In this work, we define the transcriptomes of strains overexpressing syrM or syrA. The syrM, nodD3, and syrA overexpression transcriptomes share similar gene expression changes; analyses imply that nodD3 and syrA are the only targets directly activated by SyrM. We propose that most of the gene expression changes observed when nodD3 is overexpressed are due to NodD3 activation of syrM expression, which in turn stimulates SyrM activation of syrA expression. The subsequent increase in SyrA abundance results in broad changes in gene expression, most likely mediated by the ChvI-ExoS-ExoR regulatory circuit. IMPORTANCE: Symbioses with bacteria are prevalent across the animal and plant kingdoms. Our system of study, the rhizobium-legume symbiosis (Sinorhizobium meliloti and Medicago spp.), involves specific host-microbe signaling, differentiation in both partners, and metabolic exchange of bacterial fixed nitrogen for host photosynthate. During this complex developmental process, both bacteria and plants undergo profound changes in gene expression. The S. meliloti SyrM-NodD3-SyrA and ChvI-ExoS-ExoR regulatory circuits affect gene expression and are important for optimal symbiosis. In this study, we defined the transcriptomes of S. meliloti overexpressing SyrM or SyrA. In addition to identifying new targets of the SyrM-NodD3-SyrA regulatory circuit, our work further suggests how it is linked to the ChvI-ExoS-ExoR regulatory circuit.

Exopolysaccharides from Sinorhizobium meliloti can protect against H2O2-dependent damage.

Sinorhizobium meliloti requires exopolysaccharides in order to form a successful nitrogen-fixing symbiosis with Medicago species. Additionally, during early stages of symbiosis, S. meliloti is presented with an oxidative burst that must be overcome. Levels of production of the exopolysaccharides succinoglycan (EPS-I) and galactoglucan (EPS-II) were found to correlate positively with survival in hydrogen peroxide (H2O2). H2O2 damage is dependent on the presence of iron and is mitigated when EPS-I and EPS-II mutants are cocultured with cells expressing either exopolysaccharide. Purified EPS-I is able to decrease in vitro levels of H2O2, and this activity is specific to the symbiotically active low-molecular-weight form of EPS-I. This suggests a potential protective function of exopolysaccharides against H2O2 during early symbiosis.

Isolation and characterization of mutant Sinorhizobium meliloti NodD1 proteins with altered responses to luteolin.

NodD1, a member of the NodD family of LysR-type transcriptional regulators (LTTRs), mediates nodulation (nod) gene expression in the soil bacterium Sinorhizobium meliloti in response to the plant-secreted flavonoid luteolin. We used genetic screens and targeted approaches to identify NodD1 residues that show altered responses to luteolin during the activation of nod gene transcription. Here we report four types of NodD1 mutants. Type I (NodD1 L69F, S104L, D134N, and M193I mutants) displays reduced or no activation of nod gene expression. Type II (NodD1 K205N) is constitutively active but repressed by luteolin. Type III (NodD1 L280F) demonstrates enhanced activity with luteolin compared to that of wild-type NodD1. Type IV (NodD1 D284N) shows moderate constitutive activity yet can still be induced by luteolin. In the absence of luteolin, many mutants display a low binding affinity for nod gene promoter DNA in vitro. Several mutants also show, as does wild-type NodD1, increased affinity for nod gene promoters with added luteolin. All of the NodD1 mutant proteins can homodimerize and heterodimerize with wild-type NodD1. Based on these data and the crystal structures of several LTTRs, we present a structural model of wild-type NodD1, identifying residues important for inducer binding, protein multimerization, and interaction with RNA polymerase at nod gene promoters.

Global mapping of transcription start sites and promoter motifs in the symbiotic α-proteobacterium Sinorhizobium meliloti 1021.

BACKGROUND: Sinorhizobium meliloti is a soil-dwelling α-proteobacterium that possesses a large, tripartite genome and engages in a nitrogen fixing symbiosis with its plant hosts. Although much is known about this important model organism, global characterization of genetic regulatory circuits has been hampered by a lack of information about transcription and promoters. RESULTS: Using an RNAseq approach and RNA populations representing 16 different growth and stress conditions, we comprehensively mapped S. meliloti transcription start sites (TSS). Our work identified 17,001 TSS that we grouped into six categories based on the genomic context of their transcripts: mRNA (4,430 TSS assigned to 2,657 protein-coding genes), leaderless mRNAs (171), putative mRNAs (425), internal sense transcripts (7,650), antisense RNA (3,720), and trans-encoded sRNAs (605). We used this TSS information to identify transcription factor binding sites and putative promoter sequences recognized by seven of the 15 known S. meliloti σ factors σ70, σ54, σH1, σH2, σE1, σE2, and σE9). Altogether, we predicted 2,770 new promoter sequences, including 1,302 located upstream of protein coding genes and 722 located upstream of antisense RNA or trans-encoded sRNA genes. To validate promoter predictions for targets of the general stress response σ factor, RpoE2 (σE2), we identified rpoE2-dependent genes using microarrays and confirmed TSS for a subset of these by 5' RACE mapping. CONCLUSIONS: By identifying TSS and promoters on a global scale, our work provides a firm foundation for the continued study of S. meliloti gene expression with relation to gene organization, σ factors and other transcription factors, and regulatory RNAs.

Development of tools for the biochemical characterization of the symbiotic receptor-like kinase DMI2.

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.