Robert F. Fisher

Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

Diverse Flavonoids Stimulate NodD1 Binding to nod Gene Promoters in Sinorhizobium meliloti

NodD1 is a member of the NodD family of LysR-type transcriptional regulators that mediates the expression of nodulation (nod) genes in the soil bacterium Sinorhizobium meliloti. Each species of rhizobia establishes a symbiosis with a limited set of leguminous plants. This host specificity results in part from a NodD-dependent upregulation of nod genes in response to a cocktail of flavonoids in the host plants root exudates. To demonstrate that NodD is a key determinant of host specificity, we expressed nodD genes from different species of rhizobia in a strain of S. meliloti lacking endogenous NodD activity. We observed that nod gene expression was initiated in response to distinct sets of flavonoid inducers depending on the source of NodD. To better understand the effects of flavonoids on NodD, we assayed the DNA binding activity of S. meliloti NodD1 treated with the flavonoid inducer luteolin. In the presence of luteolin, NodD1 exhibited increased binding to nod gene promoters compared to binding in the absence of luteolin. Surprisingly, although they do not stimulate nod gene expression in S. meliloti, the flavonoids naringenin, eriodictyol, and daidzein also stimulated an increase in the DNA binding affinity of NodD1 to nod gene promoters. In vivo competition assays demonstrate that noninducing flavonoids act as competitive inhibitors of luteolin, suggesting that both inducing and noninducing flavonoids are able to directly bind to NodD1 and mediate conformational changes at nod gene promoters but that only luteolin is capable of promoting the downstream changes necessary for nod gene induction.

ExoR is genetically coupled to the ExoS–ChvI two‐component system and located in the periplasm of Sinorhizobium meliloti

Sinorhizobium meliloti enters into a symbiotic relationship with legume host plants, providing fixed nitrogen in exchange for carbon and amino acids. In S. meliloti, exoR and the exoS–chvI two‐component system regulate the biosynthesis of succinoglycan, an exopolysaccharide important for host invasion. It was previously reported that a loss‐of‐function mutation in exoR and a gain‐of‐function mutation in exoS cause overproduction of succinoglycan and loss of motility, indicating that ExoR negatively regulates and ExoS–ChvI positively regulates downstream genes. However, a relationship between exoR and exoS–chvI has never been clearly established. By identification and detailed characterization of suppressor strains, we provide genetic evidence that exoR and exoS–chvI control many similar phenotypes. These include succinoglycan production, symbiosis, motility, and previously uncharacterized prototrophy and biofilm formation, all of which are co‐ordinately restored by suppressors. We further demonstrate that ExoR is located in the periplasm, suggesting that it functions to regulate downstream genes in a novel manner. In pathogenic bacteria closely related to S. meliloti, exoS–chvI homologues are required for virulence and the regulation of cell envelope composition. Our data suggest that periplasmically localized ExoR and ExoS–ChvI function together in a unique and critical regulatory system associated with both free‐living and symbiotic states of S. meliloti.

Predicting gene expression levels from codon biases in α-proteobacterial genomes

Predicted highly expressed (PHX) genes in five currently available high G+C complete α-proteobacterial genomes are analyzed. These include: the nitrogen-fixing plant symbionts Sinorhizobium meliloti (SINME) and Mesorhizobium loti (MESLO), the nonpathogenic aquatic bacterium Caulobacter crescentus (CAUCR), the plant pathogen Agrobacterium tumefaciens (AGRTU), and the mammalian pathogen Brucella melitensis (BRUME). Three of these genomes, SINME, AGRTU, and BRUME, contain multiple chromosomes or megaplasmids (>1 Mb length). PHX genes in these genomes are concentrated mainly in the major (largest) chromosome with few PHX genes found in the secondary chromosomes and megaplasmids. Tricarboxylic acid cycle and aerobic respiration genes are strongly PHX in all five genomes, whereas anaerobic pathways of glycolysis and fermentation are mostly not PHX. Only in MESLO (but not SINME) and BRUME are most glycolysis genes PHX. Many flagellar genes are PHX in MESLO and CAUCR, but mostly are not PHX in SINME and AGRTU. The nonmotile BRUME also carries many flagellar genes but these are generally not PHX and all but one are located in the second chromosome. CAUCR stands out among available prokaryotic genomes with 25 PHX TonB-dependent receptors. These are putatively involved in uptake of iron ions and other nonsoluble compounds.

Identification of Direct Transcriptional Target Genes of ExoS/ChvI Two-Component Signaling in Sinorhizobium meliloti

The Sinorhizobium meliloti ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between S. meliloti and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an exoS96::Tn5 mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with chvI gain-of-function and reduced-function strains. The chvI gain-of-function strain that we used contains a dominant gain-of-function chvI allele in addition to wild-type chvI. We identified genes that, relative to their expression level in the wild type, are both upregulated in the chvI gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of ropB1, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria.

Analysis of the requirements for transcription pausing in the tryptophan operon

RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide. Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon. We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro. A model is presented that accounts for many of the findings obtained in this and other pausing studies.

Rhizobium Meliloti Nodulation Gene Regulation and Molecular Signals

We have been studying the genes used by Rhizobium meliloti to form nodules on host alfalfa plants. In recent work, we have characterized the interaction of the NodD protein with upstream nod box promoters, and have determined the circuit of analysis for the nodD3 and syrM genes. We have found that a locus on the chromosome, mutated in strain B4, is required for full NodD activity and have discovered the B4 gene to be at least partly homologous to a family of chaperonin proteins. We have also used the homology of the nodP gene to E. coli as a means to trace the function of this gene, and have examined the response of single plant root hairs to bacterial signals by means of electrophysiological monitoring.

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.

The RNA Polymerase α Subunit from Sinorhizobium meliloti Can Assemble with RNA Polymerase Subunits from Escherichia coli and Function in Basal and Activated Transcription both In Vivo and In Vitro

Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the α subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (α)-rplQ (L17) found in the α-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti α supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti α and E. coli β, β′, and σ subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti α functions are conserved in heterologous host E. coli even though the two α subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

Genetic Analysis of Rhizobium-Plant Interactions

In approaching the question of plant-microbe recognition, we require studies at several levels. In addition, it is important to understand the many stages at which bacteria and plants interact. This is particularly relevant to the legume-Rhizobium symbiosis, which proceeds by a series of steps involving both the host plant and the bacterial symbiont. These steps, originally outlined by Vincent (1980), include attachment, root hair curling, meristem initiation, invasion of the plant, release of bacteria into plant cells, and successful expression of nitrogen fixation and other essential genes. At all of these stages, recognition events may occur.