Analysis of a chemotaxis operon in Rhizobium meliloti*

Genes controlling chemotaxis towards L-amino acids and d -mannitol in Rhizobium meliloti have been identified by Tn5 insertions that lead to chemotaxis-deficient mutants. The tagged genes span an 8.7 kbp region that has been sequenced. These genes are part of a large operon containing three novel open reading frames, orf1, orf2 and orf9, and six familiar chemotaxis (che) genes, cheY1-cheA-cheW-cheR-cheB-cheY2, that have been assigned by their similarity to known Escherichia coli genes. The second copy of cheY may be part of a second signalling chain; orf1 and orf2 encode sequence motifs that resemble the signalling domain of E. coli MCPs (methyl-accepting chemotaxis proteins), while the product of orf9 may contain a transmembrane domain. No protein methylation has been observed in Rhizobium meliloti in response to l -amino acids. However, the presence of cheR (methyltransferase gene) and cheB (methyl-esterase gene) suggested that MCPs are likely components of the chemotactic response in R. meliloti. Therefore, it is postulated that two chemotaxis pathways are functional in R meliloti: one responds to l -amino acids via ORF1-ORF2, whereas the other (probably responding to specific plant exudates) acts via MCP-like receptors, and both interact with the central components CheW-CheA-CheY1 and/or CheY2.     

Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti

An unusual regulatory mechanism involving two response regulators, CheY1 and CheY2, but no CheZ phosphatase, operates in the chemotactic signalling chain of Sinorhizobium meliloti . Active CheY2-P, phosphorylated by the cognate histidine kinase, CheA, is responsible for flagellar motor control. In the absence of any CheZ phosphatase activity, the level of CheY2-P is quickly reset by a phospho-transfer from CheY2-P first back to CheA, and then to CheY1, which acts as a phosphate sink. In studying the mechanism of this phosphate shuttle, we have used GFP fusions to show that CheY2, but not CheY1, associates with CheA at a cell pole. Cross-linking experiments with the purified proteins revealed that both CheY2 and CheY2-P bind to an isolated P2 ligand-binding domain of CheA, but CheY1 does not. The dissociation constants of CheA–CheY2 and CheA–CheY2-P indicated that both ligands bind with similar affinity to CheA. Based on the NMR structures of CheY2 and CheY2-P, their interactions with the purified P2 domain were analysed. The interacting surface of CheY2 comprises its C-terminal β4-α4-β5-α5 structural elements, whereas the interacting surface of CheY2-P is shifted towards the loop connecting β5 and α5. We propose that the distinct CheY2 and CheY2-P surfaces interact with two overlapping sites in the P2 domain that selectively bind either CheY2 or CheY2-P, depending on whether CheA is active or inactive.     

Sinorhizobium meliloti CheA complexed with CheS exhibits enhanced binding to CheY1, resulting in accelerated CheY1 dephosphorylation

Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain is a widespread mechanism for efficient dephosphorylation of the activated response regulator. First discovered in Sinorhizobium meliloti, the main response regulator CheY2-P shuttles its phosphoryl group back to CheA, while a second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA-P. We have identified a new component in this phospho-relay system, a small 97-amino-acid protein named CheS. CheS has no counterpart in enteric bacteria but revealed distinct similarities to proteins of unknown function in other members of the α subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In vitro binding, as well as coexpression and copurification studies, gave evidence of CheA/CheS complex formation. Using limited proteolysis coupled with mass spectrometric analyses, we defined CheA(163-256) to be the CheS binding domain, which overlaps with the N-terminal part of the CheY2 binding domain (CheA(174-316)). Phosphotransfer experiments using isolated CheA-P showed that dephosphorylation of CheY1-P but not CheY2-P is increased in the presence of CheS. As determined by surface plasmon resonance spectroscopy, CheY1 binds ～100-fold more strongly to CheA/CheS than to CheA. We propose that CheS facilitates signal termination by enhancing the interaction of CheY1 and CheA, thereby promoting CheY1-P dephosphorylation, which results in a more efficient drainage of the phosphate sink.     

Physiology of behavioral mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway

Wild-type and nonchemotactic mutant strains of Rhizobium meliloti were tested for attraction to localized sites on alfalfa roots and for attraction to numerous small molecules, including sugars, amino acids, and two fractions derived from alfalfa root extracts. Four strains (carrying mutations che-6, che-11, che-12, and che-26) lost all responses and were classified as generally nonchemotactic mutants. One strain (carrying mutation che-7) lost responses to a group of structurally unrelated amino acids but retained all other responses and was classified as a putative sensory transducer mutant. Two strains (carrying mutations che-1 and che-3) lost responses to all the amino acids and sugars tested but retained normal responses to localized sites on roots and to the root fractions. These two mutant strains could not be classified according to the generally accepted model for a sensory pathway, derived from studies of enteric bacteria, and provided evidence for a dual chemotaxis pathway in R. meliloti.     

Conserved Nodulation Genes in Rhizobium meliloti and Rhizobium trifolii

Plasmids which contained wild-type or mutated Rhizobium meliloti nodulation (nod) genes were introduced into Nod⁻R. trifolii mutants ANU453 and ANU851 and tested for their ability to nodulate clover. Cloned wild-type and mutated R. meliloti nod gene segments restored ANU851 to Nod⁺, with the exception of nodD mutants. Similarly, wild-type and mutant R. meliloti nod genes complemented ANU453 to Nod⁺, except for nodCII mutants. Thus, ANU851 identifies the equivalent of the R. meliloti nodD genes, and ANU453 specifies the equivalent of the R. meliloti nodCII genes. In addition, cloned wild-type R. trifolii nod genes were introduced into seven R. meliloti Nod⁻ mutants. All seven mutants were restored to Nod⁺ on alfalfa. Our results indicate that these genes represent common nodulation functions and argue for an allelic relationship between nod genes in R. meliloti and R. trifolii.     

Mechanism of phosphatase activity in the chemotaxis response regulator CheY

Response regulator proteins are phosphorylated on a conserved aspartate to activate responses to environmental signals. An intrinsic autophosphatase activity limits the duration of the phosphorylated state. We have previously hypothesized that dephosphorylation might proceed through an intramolecular attack, leading to succinimide formation, and such an intramolecular dephosphorylation event is seen for CheY and OmpR during mass spectrometric analysis [Napper, S., Wolanin, P. M., Webre, D. J., Kindrachuk, J., Waygood, B., and Stock, J. B. (2003) FEBS Lett 538, 77-80]. Succinimide formation is usually associated with the spontaneous deamidation of Asn residues. We show here that an Asp57 to Asn mutant of the CheY chemotaxis response regulator undergoes an unusually rapid deamidation back to the wild-type Asp57, supporting the hypothesis that the active site of CheY is poised for succinimide formation. In contrast, we also show that the major route of phosphoaspartate hydrolysis in CheY occurs through water attack on the phosphorus both during autophosphatase activity and during CheZ-mediated dephosphorylation. Thus, CheY dephosphorylation does not usually proceed via a succinimide or any other intramolecular attack.     

Ammonium assimilation in Rhizobium meliloti

We have characterized a mutant of Rhizobium meliloti strain 2011 which cannot use ammonium as a nitrogen source. This mutant, RTm2620, was found to have significantly altered glutamate synthase activity. Both the mutant and the wild-type strains had glutamate dehydrogenase activity, which, although stimulated in the presence of glutamate and ammonium, was apparently insufficient to allow ammonium assimmilation. We conclude that the glutamine synthetase-glutamate synthase pathway may be the normal mode of ammonium assimilation by this strain in the free-living state. Independent revertants of Rm2620 were isolated and fell into two classes. Class I revertants regained partial glutamate synthase activity and had the same levels of glutamate dehydrogenase activity as Rm2620. Class II revertants retained the altered glutamate synthase activity but acquired a very high level of assimilatory glutamate dehydrogenase activity. Both classes were found to be altered in their symbiotic properties, although the original Rm2620 mutant was normal in this regard.     

General transduction in Rhizobium meliloti

General transduction by phage phi M12 in Rhizobium meliloti SU47 and its derivatives is described. Cotransduction and selection for Tn5 insertions which are closely linked to specific loci were demonstrated. A derivative of SU47 carrying the recA::Tn5 allele of R. meliloti 102F34 could be transduced for plasmid R68.45 but not for chromosomally located alleles. Phage phi M12 is morphologically similar to Escherichia coli phage T4, and restriction endonuclease analysis indicated that the phage DNA was ca. 160 kilobases in size.     

Glutamate catabolism in Rhizobium meliloti

The pathway by which glutamate is degraded as a carbon source has not previously been elucidated, but enzymatic analysis of Rhizobium meliloti CMF1 indicated that both glutamate dehydrogenase (GDH) and gamma-aminobutyrate (GABA) bypass activities were present in free living cells. However, when similar studies were performed on R. meliloti CMF1 bacteroids, isolated from alfalfa nodules, only GABA bypass activities were detectable. Both GDH and GABA bypass activities were influenced by the carbon source provided, with maximum activities being detected when glutamate was present as sole carbon and nitrogen source. Addition of a second carbon source, such as succinate, to the growth medium did not influence GDH activity but substantially decreased levels of the first enzyme of the GABA bypass, glutamate decarboxylase (GDC). Cyclic adenosine 3′5′-monophosphate (cAMP) failed to increase GDC activities in R. meliloti CMF1 cells grown in the presence of an additional carbon source. It is proposed that the GABA bypass is a major mechanism of glutamate carbon degradation in R. meliloti CMF1, a system whose enzymatic activities are influenced by the nature of the carbon source present in the growth environment.     

Rhizobium meliloti competitiveness and the alfalfa agglutinin

We have isolated two types of isolates having identical colony morphologies from stock cultures of two different Rhizobium meliloti strains. One isolate was agglutinated at a high-dilution titer (HA, highly agglutinable) of the alfalfa agglutinin and was sensitive to phage F20, and the other was agglutinated at a lower agglutinin titer (LA) and was sensitive to phage 16B. All LA isolates from the original slant produced nodules on alfalfa earlier than did HA strains from the original slant. When these HA and LA strains were mixed and used as the inoculum in both vermiculite and field soil in the laboratory, LA strains were always the predominant strains recovered from the nodules. LA strains were obtained from HA cells by selection for resistance to phage F20, and HA strains were obtained from LA cells by selection for resistance to phage 16B. All of the strains with the HA phenotype that were derived from LA strains by phage selection had the nodulation properties of the HA strains from the original slant. Two classes of strains with the LA phenotype were obtained from HA cells by phage selection. One was identical to the original LA strains from the slant, and the other had the nodulation properties of the HA strains. Thus, we have shown that some cell surface properties change the nodulation abilities of R. meliloti strains and, furthermore, that specific phages can be used to enrich for more competitive rhizobia.     

Comparison between Rhizobium galegae and Rhizobium meliloti plasmid contents

Plasmid profiles of two strains of a newly classified rhizobial species- Rhizobium galegae -were compared with the profiles of several strains of another fast-growing Rhizobium species- Rhizobium meliloti .
The existence of a plasmid DNA band with a lower electrophoretic mobility than the R. meliloti megaplasmid band was demonstrated in the two R. galegae strains by a modified horizontal Eckhardt method. Thus R. galegae species contain giant plasmid(s) larger than the R. meliloti 1000 MD megaplasmids, previously considered to be the largest plasmids in the Rhizobiaceae family.
In one of the R. galegae strains an additional middle-size plasmid only a little smaller than 140 MD pRme41a of R. meliloti 41 was observed.     

CheZ-mediated dephosphorylation of the Escherichia coli chemotaxis response regulator CheY: role for CheY glutamate 89

The swimming behavior of Escherichia coli at any moment is dictated by the intracellular concentration of the phosphorylated form of the chemotaxis response regulator CheY, which binds to the base of the flagellar motor. CheY is phosphorylated on Asp57 by the sensor kinase CheA and dephosphorylated by CheZ. Previous work (Silversmith et al., J. Biol. Chem. 276:18478, 2001) demonstrated that replacement of CheY Asn59 with arginine resulted in extreme resistance to dephosphorylation by CheZ despite proficient binding to CheZ. Here we present the X-ray crystal structure of CheYN59R in a complex with Mn(2+) and the stable phosphoryl analogue BeF(3)(-). The overall folding and active site architecture are nearly identical to those of the analogous complex containing wild-type CheY. The notable exception is the introduction of a salt bridge between Arg59 (on the beta3alpha3 loop) and Glu89 (on the beta4alpha4 loop). Modeling this structure into the (CheY-BeF(3)(-)-Mg(2+))(2)CheZ(2) structure demonstrated that the conformation of Arg59 should not obstruct entry of the CheZ catalytic residue Gln147 into the active site of CheY, eliminating steric interference as a mechanism for CheZ resistance. However, both CheYE89A and CheYE89Q, like CheYN59R, conferred clockwise flagellar rotation phenotypes in strains which lacked wild-type CheY and displayed considerable (approximately 40-fold) resistance to dephosphorylation by CheZ. CheYE89A and CheYE89Q had autophosphorylation and autodephosphorylation properties similar to those of wild-type CheY and could bind to CheZ with wild-type affinity. Therefore, removal of Glu89 resulted specifically in CheZ resistance, suggesting that CheY Glu89 plays a role in CheZ-mediated dephosphorylation. The CheZ resistance of CheYN59R can thus be largely explained by the formation of the salt bridge between Arg59 and Glu89, which prevents Glu89 from executing its role in catalysis.     

Host Restriction and Transduction in Rhizobium meliloti

A host restriction difference exists between Rhizobium meliloti Rm41 and SU47 exists as indicated by the reduce plating efficiency of transducing phage ΦM12h1. Restriction can be attenuated by incubating cells at 42°C for 3 h; this procedure overcomes a block to transduction from SU47 to Rm41.