Identification of residues essential for a two-step reaction by malonyl-CoA synthetase from Rhizobium trifolii.
(25/1949)
Malonyl-CoA synthetase (MCS) catalyses the formation of malonyl-CoA in a two-step reaction consisting of the adenylation of malonate with ATP followed by malonyl transfer from malonyl-AMP to CoA. In order to identify amino acid residues essential for each step of the enzyme, catalysis based on chemical modification and database analysis, Arg-168, Lys-170, and His-206 were selected for site-directed mutagenesis. Glutathione-S-transferase-fused enzyme (GST-MCS) was constructed and mutagenized to make R168G, K170M, R168G/K170M and H206L mutants, respectively. The MCS activity of soluble form GST-MCS was the same as that of wild-type MCS. Circular dichroism spectra for the four mutant enzymes were nearly identical to that for the GST-MCS, indicating that Arg-168, Lys-170 and His-206 are not important for conformation but presumably for substrate binding and/or catalysis. HPLC analysis of products revealed that the intermediate malonyl-AMP is not accumulated during MCS catalysis and that none of the mutant enzymes accumulated it either. Kinetic analysis of the mutants revealed that Lys-170 and His-206 play a critical role for ATP binding and the formation of malonyl-AMP, whereas Arg-168 is critical for formation of malonyl-CoA and specificity for malonyl-AMP. Molecular modelling based on the crystal structures of luciferase and gramicidin S synthetase 1 provided MCS structure which could fully explain all these biochemical data even though the MCS model was generated by comparative modelling. (+info)
Lethal effects of apidaecin on Escherichia coli involve sequential molecular interactions with diverse targets.
(26/1949)
Apidaecins, short proline-arginine-rich peptides from insects, are highly bactericidal through a mechanism that includes stereoselective elements but is completely devoid of any pore-forming activity. The spectrum of antibacterial activity, always limited to Gram-negatives, is further dependent on a small number of variable residues and can be manipulated. We show here that mutations in the evolutionary conserved regions result in a more general loss of function, and we have used such analogs to probe molecular interactions in Escherichia coli. First, an assay was developed to measure selectively chiral association with cellular targets. By using this method, we find that apidaecin uptake is energy-driven and irreversible and yet can be partially competed by proline in a stereospecific fashion, results upholding a model of a permease/transporter-mediated mechanism. This putative transporter is not the end point of apidaecin action, for failure of certain peptide analogs to kill cells after entering indicates the existence of another downstream target. Tetracycline-induced loss of bactericidal activity and dose-dependent in vivo inhibition of translation by apidaecin point at components of the protein synthesis machinery as likely candidates. These findings provide new insights into the antibacterial mechanism of a unique group of peptides and perhaps, by extension, for distant mammalian relatives such as PR-39. (+info)
Rhizobium etli bv. mimosae, a novel biovar isolated from Mimosa affinis.
(27/1949)
Fifty rhizobial isolates from root nodules of Mimosa affinis, a small leguminous plant native to Mexico, were identified as Rhizobium etli on the basis of the results of PCR-RFLP and RFLP analyses of small-subunit rRNA genes, multilocus enzyme electrophoresis and DNA-DNA homology. They are, however, a restricted group of lineages with low genetic diversity within the species. The isolates from M. affinis differed-from the R. etli strains that orginated from bean plants (Phaseolus vulgaris) in the size and replicator region of the symbiotic plasmid and in symbiotic-plasmid-borne traits such as nifH gene sequence and organization, melanin production and host specificity. A new biovar, bv. mimosae, is proposed within R. etli to encompass Rhizobium isolates obtained from M. affinis. The strains from common bean plants have been designated previously as R. etli bv. phaseoli. Strains of both R. etli biovars could nodulate P. vulgaris, but only those of bv. mimosae could form nitrogen-fixing nodules on Leucaena leucocephala. (+info)
Expression of the Bs2 pepper gene confers resistance to bacterial spot disease in tomato.
(28/1949)
The Bs2 resistance gene of pepper specifically recognizes and confers resistance to strains of Xanthomonas campestris pv. vesicatoria that contain the corresponding bacterial avirulence gene, avrBs2. The involvement of avrBs2 in pathogen fitness and its prevalence in many X. campestris pathovars suggests that the Bs2 gene may be durable in the field and provide resistance when introduced into other plant species. Employing a positional cloning strategy, the Bs2 locus was isolated and the gene was identified by coexpression with avrBs2 in an Agrobacterium-mediated transient assay. A single candidate gene, predicted to encode motifs characteristic of the nucleotide binding site-leucine-rich repeat class of resistance genes, was identified. This gene specifically controlled the hypersensitive response when transiently expressed in susceptible pepper and tomato lines and in a nonhost species, Nicotiana benthamiana, and was designated as Bs2. Functional expression of Bs2 in stable transgenic tomatoes supports its use as a source of resistance in other Solanaceous plant species. (+info)
Fix L, a haemoglobin that acts as an oxygen sensor: signalling mechanism and structural basis of its homology with PAS domains.
(29/1949)
Fix L, which contains a haemoglobin domain homologous to the PAS family and a histidine kinase domain, forms, with Fix J, a two-component signalling complex that regulates expression of nitrogenase genes in Rhizobium. Spin transitions of its haem iron trigger stereochemical changes in and around the haem that, together with steric effects, control the activity of the kinase. Homology with the PAS family is based on a common core of about 20 structurally equivalent sites from which polar residues are excluded. (+info)
Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate.
(30/1949)
Biological N(2) fixation represents the major source of N input in agricultural soils including those in arid regions. The major N(2)-fixing systems are the symbiotic systems, which can play a significant role in improving the fertility and productivity of low-N soils. The Rhizobium-legume symbioses have received most attention and have been examined extensively. The behavior of some N(2)-fixing systems under severe environmental conditions such as salt stress, drought stress, acidity, alkalinity, nutrient deficiency, fertilizers, heavy metals, and pesticides is reviewed. These major stress factors suppress the growth and symbiotic characteristics of most rhizobia; however, several strains, distributed among various species of rhizobia, are tolerant to stress effects. Some strains of rhizobia form effective (N(2)-fixing) symbioses with their host legumes under salt, heat, and acid stresses, and can sometimes do so under the effect of heavy metals. Reclamation and improvement of the fertility of arid lands by application of organic (manure and sewage sludge) and inorganic (synthetic) fertilizers are expensive and can be a source of pollution. The Rhizobium-legume (herb or tree) symbiosis is suggested to be the ideal solution to the improvement of soil fertility and the rehabilitation of arid lands and is an important direction for future research. (+info)
The replicator of the nopaline-type Ti plasmid pTiC58 is a member of the repABC family and is influenced by the TraR-dependent quorum-sensing regulatory system.
(31/1949)
The replicator (rep) of the nopaline-type Ti plasmid pTiC58 is located adjacent to the trb operon of this conjugal element. Previous genetic studies of this region (D. R. Gallie, M. Hagiya, and C. I. Kado, J. Bacteriol. 161:1034-1041, 1985) identified functions involved in partitioning, origin of replication and incompatibility, and copy number control. In this study, we determined the nucleotide sequence of a 6,146-bp segment that encompasses the rep locus of pTiC58. The region contained four full open reading frames (ORFs) and one partial ORF. The first three ORFs, oriented divergently from the traI-trb operon, are closely related to the repA, repB, and repC genes of the octopine-type Ti plasmid pTiB6S3 as well as to other repA, -B, and -C genes from the Ri plasmid pRiA4b and three large plasmids from Rhizobium spp. The fourth ORF and the partial ORF are similar to y4CG and y4CF, respectively, of the Sym plasmid pNGR234a. The 363-bp intergenic region between traI and repA contained two copies of the tra box which is the cis promoter recognition site for TraR, the quorum-sensing activator of Ti plasmid conjugal transfer. Expression of the traI-trb operon from the tra box II-associated promoter mediated by TraR and its acyl-homoserine lactone ligand, AAI, was negatively influenced by an intact tra box III. On the other hand, the region containing the two tra boxes was required for maximal expression of repA, and this expression was enhanced slightly by TraR and AAI. Copy number of a minimal rep plasmid increased five- to sevenfold in strains expressing traR but only when AAI also was provided. Consistent with this effect, constitutive expression of the quorum-sensing system resulted in an apparent increase in Ti plasmid copy number. We conclude that Ti plasmid copy number is influenced by the quorum-sensing system, suggesting a connection between conjugal transfer and vegetative replication of these virulence elements. (+info)
Characterization of two inducible phosphate transport systems in Rhizobium tropici.
(32/1949)
Rhizobium tropici forms nitrogen-fixing nodules on the roots of the common bean (Phaseolus vulgaris). Like other legume-Rhizobium symbioses, the bean-R. tropici association is sensitive to the availability of phosphate (P(i)). To better understand phosphorus movement between the bacteroid and the host plant, P(i) transport was characterized in R. tropici. We observed two P(i) transport systems, a high-affinity system and a low-affinity system. To facilitate the study of these transport systems, a Tn5B22 transposon mutant lacking expression of the high-affinity transport system was isolated and used to characterize the low-affinity transport system in the absence of the high-affinity system. The K(m) and V(max) values for the low-affinity system were estimated to be 34 +/- 3 microM P(i) and 118 +/- 8 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively, and the K(m) and V(max) values for the high-affinity system were 0.45 +/- 0.01 microM P(i) and 86 +/- 5 nmol of P(i) x min(-1) x mg (dry weight) of cells(-1), respectively. Both systems were inducible by P(i) starvation and were also shock sensitive, which indicated that there was a periplasmic binding-protein component. Neither transport system appeared to be sensitive to the proton motive force dissipator carbonyl cyanide m-chlorophenylhydrazone, but P(i) transport through both systems was eliminated by the ATPase inhibitor N,N'-dicyclohexylcarbodiimide; the P(i) transport rate was correlated with the intracellular ATP concentration. Also, P(i) movement through both systems appeared to be unidirectional, as no efflux or exchange was observed with either the wild-type strain or the mutant. These properties suggest that both P(i) transport systems are ABC type systems. Analysis of the transposon insertion site revealed that the interrupted gene exhibited a high level of homology with kdpE, which in several bacteria encodes a cytoplasmic response regulator that governs responses to low potassium contents and/or changes in medium osmolarity. (+info)