Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Genetic and chemical characterization of a mutant that disrupts synthesis of the lipopolysaccharide core tetrasaccharide in Rhizobium leguminosarum.

ABSTRACT: A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced. Two open reading frames (ORFs) were identified. The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis. ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases. Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-deoxy-D-manno-2-octulosonic acid (Kdo) residue which normally attaches the core tetrasaccharide to the O chain was missing, suggesting that IpcB may encode a CMP-Kdo:LPS Kdo transferase.

SUBMITTER: Allaway D

PROVIDER: S-EPMC178523 | biostudies-other | 1996 Nov

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Genetic and chemical characterization of a mutant that disrupts synthesis of the lipopolysaccharide core tetrasaccharide in Rhizobium leguminosarum.

Allaway D D Jeyaretnam B B Carlson R W RW Poole P S PS

Journal of bacteriology 19961101 21

A 2-kb region that complements the Tn5-derived lipopolysaccharide (LPS) rough mutant Rhizobium leguminosarum RU301 was sequenced. Two open reading frames (ORFs) were identified. The first ORF (lpcA) is homologous to a family of bacterial sugar transferases involved in LPS core tetrasaccharide biosynthesis. ORF2 (lpcB), in which Tn5 transposed, has no significant homology to any DNA in the GenBank-EMBL databases. Chemical characterization of LPS produced by strain RU301 demonstrated that the 3-de ...[more]

PMID: 8892852

Similar Datasets

Expression cloning of three Rhizobium leguminosarum lipopolysaccharide core galacturonosyltransferases.

Project description:The lipid A and core regions of the lipopolysaccharide in Rhizobium leguminosarum, a nitrogen-fixing plant endosymbiont, are strikingly different from those of Escherichia coli. In R. leguminosarum lipopolysaccharide, the inner core is modified with three galacturonic acid (GalA) moieties, two on the distal 3-deoxy-D-manno-octulosonic acid (Kdo) unit and one on the mannose residue. Here we describe the expression cloning of three novel GalA transferases from a 22-kb R. leguminosarum genomic DNA insert-containing cosmid (pSGAT). Two of these enzymes modify the substrate, Kdo2-[4'-(32)P]lipid IV(A) and its 1-dephosphorylated derivative on the distal Kdo residue, as indicated by mild acid hydrolysis. The third enzyme modifies the mannose unit of the substrate mannosyl-Kdo2-1-dephospho-[4'-(32)P]lipid IV(A). Sequencing of a 7-kb subclone derived from pSGAT revealed three putative membrane-bound glycosyltransferases, now designated RgtA, RgtB, and RgtC. Transfer by tri-parental mating of these genes into Sinorhizobium meliloti 1021, a strain that lacks these particular GalA residues, results in the heterologous expression of the GalA transferase activities seen in membranes of cells expressing pSGAT. Reconstitution experiments with the individual genes demonstrated that the activity of RgtA precedes and is necessary for the subsequent activity of RgtB, which is followed by the activity of RgtC. Electrospray ionization-tandem mass spectrometry and gas-liquid chromatography of the product generated in vitro by RgtA confirmed the presence of a GalA moiety. No in vitro activity was detected when RgtA was expressed in Escherichia coli unless Rhizobiaceae membranes were also included.

| S-EPMC2814240 | biostudies-literature

The genome of Rhizobium leguminosarum has recognizable core and accessory components.

Project description:Rhizobium leguminosarum is an alpha-proteobacterial N2-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.

| S-EPMC1557990 | biostudies-literature

Lipopolysaccharide O-chain core region required for cellular cohesion and compaction of in vitro and root biofilms developed by Rhizobium leguminosarum.

Project description:The formation of biofilms is an important survival strategy allowing rhizobia to live on soil particles and plant roots. Within the microcolonies of the biofilm developed by Rhizobium leguminosarum, rhizobial cells interact tightly through lateral and polar connections, forming organized and compact cell aggregates. These microcolonies are embedded in a biofilm matrix, whose main component is the acidic exopolysaccharide (EPS). Our work shows that the O-chain core region of the R. leguminosarum lipopolysaccharide (LPS) (which stretches out of the cell surface) strongly influences bacterial adhesive properties and cell-cell cohesion. Mutants defective in the O chain or O-chain core moiety developed premature microcolonies in which lateral bacterial contacts were greatly reduced. Furthermore, cell-cell interactions within the microcolonies of the LPS mutants were mediated mostly through their poles, resulting in a biofilm with an altered three-dimensional structure and increased thickness. In addition, on the root epidermis and on root hairs, O-antigen core-defective strains showed altered biofilm patterns with the typical microcolony compaction impaired. Taken together, these results indicate that the surface-exposed moiety of the LPS is crucial for proper cell-to-cell interactions and for the formation of robust biofilms on different surfaces.

| S-EPMC4292486 | biostudies-literature

Characterization of galacturonosyl transferase genes rgtA, rgtB, rgtC, rgtD, and rgtE responsible for lipopolysaccharide synthesis in nitrogen-fixing endosymbiont Rhizobium leguminosarum: lipopolysaccharide core and lipid galacturonosyl residues confer membrane stability.

Project description:Rhizobium lipopolysaccharide (LPS) contains four terminally linked galacturonic acid (GalA) residues; one attached to the lipid A and three attached to the core oligosaccharide moiety. Attachment of the GalA residues requires the lipid donor dodecaprenyl-phosphate GalA (Dod-P-GalA), which is synthesized by the GalA transferase RgtE reported here. The galacturonosyl transferases RgtA, -B, and -C utilize Dod-P-GalA to attach GalAs on the LPS core region, and RgtD attaches GalA to the lipid A 4' position. As reported here, the functions of the rgtD and rgtE genes were determined via insertion mutagenesis and structural characterization of the mutant lipid A. The rgtE(-) mutant lacked Dod-P-GalA as determined by mass spectrometry of total lipid extracts and the inability of rgtE(-) mutant membranes to provide the substrate for heterologously expressed RgtA activity. In addition, we created single mutations in each of the rgtA, -B, -C, -D, and -E genes to study the biological function of the GalA residues. The structures of the core oligosaccharide region from each of the rgt mutants were elucidated by glycosyl linkage analysis. Each mutant was assayed for its sensitivity to sodium deoxycholate and to the antimicrobial cationic peptide, polymyxin B (PmxB). The rgt mutants were more sensitive than the parent strain to deoxycholate by varying degrees. However, the rgtA, -B, and -C mutants were more resistant to PmxB, whereas the rgtD and E mutants were less resistant in comparison to the parent strain.

| S-EPMC3256847 | biostudies-other

Rhizobium gone native: unexpected plasmid stability of indigenous Rhizobium leguminosarum.

Project description:Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using chi2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

| S-EPMC24705 | biostudies-literature

Defining the Rhizobium leguminosarum Species Complex.

Project description:Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

| S-EPMC7831135 | biostudies-literature

Molecular basis of sulfolactate synthesis by sulfolactaldehyde dehydrogenase from <i>Rhizobium leguminosarum</i>.

Project description:Sulfolactate (SL) is a short-chain organosulfonate that is an important reservoir of sulfur in the biosphere. SL is produced by oxidation of sulfolactaldehyde (SLA), which in turn derives from sulfoglycolysis of the sulfosugar sulfoquinovose, or through oxidation of 2,3-dihydroxypropanesulfonate. Oxidation of SLA is catalyzed by SLA dehydrogenases belonging to the aldehyde dehydrogenase superfamily. We report that SLA dehydrogenase RlGabD from the sulfoglycolytic bacterium Rhizobium leguminsarum SRDI565 can use both NAD+ and NADP+ as cofactor to oxidize SLA, and indicatively operates through a rapid equilibrium ordered mechanism. We report the cryo-EM structure of RlGabD bound to NADH, revealing a tetrameric quaternary structure and supporting proposal of organosulfonate binding residues in the active site, and a catalytic mechanism. Sequence based homology searches identified SLA dehydrogenase homologs in a range of putative sulfoglycolytic gene clusters in bacteria predominantly from the phyla Actinobacteria, Firmicutes, and Proteobacteria. This work provides a structural and biochemical view of SLA dehydrogenases to complement our knowledge of SLA reductases, and provide detailed insights into a critical step in the organosulfur cycle.

| S-EPMC10599462 | biostudies-literature

A genetic locus necessary for rhamnose uptake and catabolism in Rhizobium leguminosarum bv. trifolii.

Project description:Rhizobium leguminosarum bv. trifolii mutants unable to catabolize the methyl-pentose rhamnose are unable to compete effectively for nodule occupancy. In this work we show that the locus responsible for the transport and catabolism of rhamnose spans 10,959 bp. Mutations in this region were generated by transposon mutagenesis, and representative mutants were characterized. The locus contains genes coding for an ABC-type transporter, a putative dehydrogenase, a probable isomerase, and a sugar kinase necessary for the transport and subsequent catabolism of rhamnose. The regulation of these genes, which are inducible by rhamnose, is carried out in part by a DeoR-type negative regulator (RhaR) that is encoded within the same transcript as the ABC-type transporter but is separated from the structural genes encoding the transporter by a terminator-like sequence. RNA dot blot analysis demonstrated that this terminator-like sequence is correlated with transcript attenuation only under noninducing conditions. Transport assays utilizing tritiated rhamnose demonstrated that uptake of rhamnose was inducible and dependent upon the presence of the ABC transporter at this locus. Phenotypic analyses of representative mutants from this locus provide genetic evidence that the catabolism of rhamnose differs from previously described methyl-pentose catabolic pathways.

| S-EPMC532407 | biostudies-literature

Genomic Diversity in the Endosymbiotic Bacterium Rhizobium leguminosarum.

Project description:Rhizobium leguminosarum bv. viciae is a soil α-proteobacterium that establishes a diazotrophic symbiosis with different legumes of the Fabeae tribe. The number of genome sequences from rhizobial strains available in public databases is constantly increasing, although complete, fully annotated genome structures from rhizobial genomes are scarce. In this work, we report and analyse the complete genome of R. leguminosarum bv. viciae UPM791. Whole genome sequencing can provide new insights into the genetic features contributing to symbiotically relevant processes such as bacterial adaptation to the rhizosphere, mechanisms for efficient competition with other bacteria, and the ability to establish a complex signalling dialogue with legumes, to enter the root without triggering plant defenses, and, ultimately, to fix nitrogen within the host. Comparison of the complete genome sequences of two strains of R. leguminosarum bv. viciae, 3841 and UPM791, highlights the existence of different symbiotic plasmids and a common core chromosome. Specific genomic traits, such as plasmid content or a distinctive regulation, define differential physiological capabilities of these endosymbionts. Among them, strain UPM791 presents unique adaptations for recycling the hydrogen generated in the nitrogen fixation process.

| S-EPMC5852556 | biostudies-literature

RhaU of Rhizobium leguminosarum is a rhamnose mutarotase.

Project description:Of the nine genes comprising the L-rhamnose operon of Rhizobium leguminosarum, rhaU has not been assigned a function. The construction of a Delta rhaU strain revealed a growth phenotype that was slower than that of the wild-type strain, although the ultimate cell yields were equivalent. The transport of L-rhamnose into the cell and the rate of its phosphorylation were unaffected by the mutation. RhaU exhibits weak sequence similarity to the formerly hypothetical protein YiiL of Escherichia coli that has recently been characterized as an L-rhamnose mutarotase. To characterize RhaU further, a His-tagged variant of the protein was prepared and subjected to mass spectrometry analysis, confirming the subunit size and demonstrating its dimeric structure. After crystallization, the structure was refined to a 1.6-A resolution to reveal a dimer in the asymmetric unit with a very similar structure to that of YiiL. Soaking a RhaU crystal with L-rhamnose resulted in the appearance of beta-L-rhamnose in the active site.

| S-EPMC2293258 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data