Characterization of genes involved in erythritol catabolism in Rhizobium leguminosarum bv. viciae

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2061-2074 ◽  
Author(s):  
Christopher K. Yost ◽  
Amber M. Rath ◽  
Tanya C. Noel ◽  
Michael F. Hynes
Keyword(s):  
Rhizobium Leguminosarum ◽  
Triosephosphate Isomerase ◽  
Genetic Locus ◽  
High Homology ◽  
Wild Type ◽  
High Sequence Identity ◽  
Catabolic Genes ◽  
Wide Range ◽  
Genomic Dna Sequence

A genetic locus encoding erythritol uptake and catabolism genes was identified in Rhizobium leguminosarum bv. viciae, and shown to be plasmid encoded in a wide range of R. leguminosarum strains. A Tn5-B22 mutant (19B-3) unable to grow on erythritol was isolated from a mutant library of R. leguminosarum strain VF39SM. The mutated gene eryF was cloned and partially sequenced, and determined to have a high homology to permease genes of ABC transporters. A cosmid complementing the mutation (pCos42) was identified and was shown to carry all the genes necessary to restore the ability to grow on erythritol to a VF39SM strain cured of pRleVF39f. In the genomic DNA sequence of strain 3841, the gene linked to the mutation in 19B-3 is flanked by a cluster of genes with high homology to the known erythritol catabolic genes from Brucella spp. Through mutagenesis studies, three distinct operons on pCos42 that are required for growth on erythritol were identified: an ABC-transporter operon (eryEFG), a catabolic operon (eryABCD) and an operon (deoR-tpiA2-rpiB) that encodes a gene with significant homology to triosephosphate isomerase (tpiA2). These genes all share high sequence identity to genes in the erythritol catabolism region of Brucella spp., and clustalw alignments suggest that horizontal transfer of the erythritol locus may have occurred between R. leguminosarum and Brucella. Transcription of the eryABCD operon is repressed by EryD and is induced by the presence of erythritol. Mutant 19B-3 was impaired in its ability to compete against wild-type for nodulation of pea plants but was still capable of forming nitrogen-fixing nodules.

Genetic characterization of Rhizobium sp. strain TAL1145 that nodulates tree legumes

1994 ◽  
Vol 40 (3) ◽  
pp. 208-215 ◽  
Author(s):  
M. L. C. George ◽  
J. P. W. Young ◽  
D. Borthakur
Keyword(s):  
Rhizobium Leguminosarum ◽  
Genetic Characterization ◽  
Rhizobium Meliloti ◽  
Wild Type ◽  
Tree Legumes ◽  
Wide Range ◽  
The Common ◽  
Rrna Sequences ◽  
Rhizobium Sp

Rhizobium sp. strain TALI 145 nodulates Leucaena ieucocephaia and Phaseolus vulgaris, in addition to a wide range of tropical tree legumes. Six overlapping clones that complemented nodulation defects in leucaena and bean rhizobia were isolated and a 40-kb map of the symbiosis region was constructed. The common nod and nifA genes were situated approximately 17 kb apart, with the nodlJ genes in between. These clones enabled a derivative of TAL1145 carrying a partially deleted pSym to form ineffective nodules on both leucaena and bean, and a similar derivative of Rhizobium etli TAL182 to form ineffective nodules on bean. When two representative clones, pUHR9 and pUHR114, were each transferred to wild-type rhizobial strains, they allowed ineffective nodulation by Rhizobium meliloti on both leucaena and bean and by Rhizobium leguminosarum bv. viciae on bean. Transconjugants of R. leguminosarum bv. trifolii formed effective nodules on leucaena and ineffective nodules on bean. Tn5 mutagenesis of the symbiosis region resulted in a variety of nodulation and fixation phenotypes on leucaena and bean. On the basis of 16S rRNA sequences, TAL1145 was found to be distinct from both R. tropici and NGR234, the two groups of leucaena symbionts that were previously described.Key words: Rhizobium, Leucaena leucocephala, nodulation, nitrogen fixation.

scholarly journals Regulation and characterization of mutants of fixABCX in Rhizobium leguminosarum.

2021 ◽  
Author(s):  
Isabel Webb ◽  
Jiabao Xu ◽  
Carmen Sanchez-Cañizares ◽  
Ramakrishnan Karunakaran ◽  
Vinoy Ramachandran ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Rhizobium Leguminosarum ◽  
Redox Balance ◽  
Rna Seq ◽  
Wild Type ◽  
Transcription Start Sites ◽  
Sym Plasmid ◽  
Microaerobic Conditions ◽  
Type Infection

Symbiosis between Rhizobium leguminosarum and Pisum sativum requires tight control of redox balance in order to maintain respiration under the microaerobic conditions required for nitrogenase, whilst still producing the eight electrons and sixteen molecules of ATP needed for nitrogen fixation. FixABCX, electron transfer flavoproteins essential for nitrogen fixation, are encoded on the Sym plasmid (pRL10), immediately upstream of nifA, which encodes the general transcriptional regulator of nitrogen fixation. There is a symbiotically-regulated NifA-dependent promoter upstream of fixA (PnifA1), as well as an additional basal constitutive promoter driving background expression of nifA (PnifA2). These were confirmed by 5’-end mapping of transcription start sites using differential (d) RNA-seq. Complementation of polar fixAB and fixX mutants (Fix- strains) confirmed expression of nifA from PnifA1 in symbiosis. Electron microscopy combined with single-cell Raman microspectroscopy characterization of fixAB mutants revealed previously unknown heterogeneity in bacteroid morphology within a single nodule. Two morphotypes of mutant fixAB bacteroids were observed. One was larger than wild-type bacteroids and contained high levels of polyhydroxy-3-butyrate, a complex energy/reductant storage product. A second bacteroid phenotype was morphologically and compositionally different and resembled wild-type infection thread cells. From these two characteristic fixAB mutant bacteroid morphotypes, inferences can be drawn on the metabolism of wild-type nitrogen-fixing bacteroids.

scholarly journals Cloning and Characterization of Four Genes of Rhizobium leguminosarum bv. trifolii Involved in Exopolysaccharide Production and Nodulation

1997 ◽  
Vol 10 (2) ◽  
pp. 290-301 ◽  
Author(s):  
Wilbert A. T. van Workum ◽  
Hayo C. J. Canter Cremers ◽  
André H. M. Wijfjes ◽  
Christa van der Kolk ◽  
Carel A. Wijffelman ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Structural Analysis ◽  
Rhizobium Leguminosarum ◽  
Second Step ◽  
Overlapping Genes ◽  
Wild Type ◽  
Exopolysaccharide Production ◽  
Polysaccharide Synthesis ◽  
Gene Mutant

Four different genes of Rhizobium leguminosarum bv. tri-folii strain RBL5599 involved in exopolysaccharide (EPS) production were identified by complementation of Tn5-induced EPS-deficient mutants (Exo mutants) with a cosmid bank. On one cosmid pssA was located, which was found to be almost identical to the pss4 gene from R. leguminosarum bv. viciae VF39 and highly homologous to a family of glycosyl transferases. Two pssA mutants, exo2 and exo4, were characterized and found to produce 19 and 1% of the wild-type amount of EPS, respectively. The three other genes were found to be closely linked on a different complementing cosmid. pssC revealed similarity to exoM and exoW of R. meliloti, both encoding glucosyl transferases involved in the synthesis of succinoglycan. A mutation in this gene (mutant exo50) did reduce EPS synthesis to 27% of the wild-type amount. We found an op-eron closely linked to pssC, consisting of two overlapping genes, pssD and pssE, that is essential for EPS production. Homology of pssD and pssE was found with cps14F and cps14G of Streptococcus pneumoniae, respectively: two genes responsible for the second step in capsule polysaccharide synthesis. Furthermore, pssD and pssE were homologous to the 5′ and 3′ parts, respectively, of spsK of Sphingomonas S88, which encodes a putative glycosyl transferase. Structural analysis of EPS produced by Exo mutants exo2, exo4, and exo50 showed it to be identical to that of the parental strain RBL5599, with the exception of acetyl groups esterified to one of the glucose residues being absent.

Characterization of some non-fixing mutants of common bean (Phaseolus vulgaris L.)

1993 ◽  
Vol 73 (4) ◽  
pp. 977-983 ◽  
Author(s):  
B. R. Buttery ◽  
S. J. Park
Keyword(s):  
Phaseolus Vulgaris ◽  
Nitrate Reductase ◽  
Common Bean ◽  
Rhizobium Leguminosarum ◽  
Variable Number ◽  
Wild Type ◽  
Parent Line ◽  
Rhizobium Strains ◽  
Wild Type Parent

With 18 strains of Rhizobium leguminosarum bv. phaseoli the bean mutants R99 and NOD125 remained essentially non-nodulating, while the mutant R69 produced a variable number of small white ineffective nodules, and the wild-type parent-line OAC Rico formed a variable number of pink effective nodules. Both R69 and R99 grew less vigorously than OAC Rico, but possessed similar levels of nitrate reductase in both roots and leaves, and responded in a normal way to increased supply of combined nitrogen. Reciprocal grafts between the non-nodulating R99 and NOD125, the ineffective R69, the wild-type parent line OAC Rico, and the supernodulating R32BS, demonstrated that the non-nodulating and ineffective characters were controlled by the root, and confirmed that the supernodulation character was controlled by the shoot. Key words: Common bean, nitrate reductase, non-fixing mutants, Phaseolus vulgaris, Rhizobium strains, supernodulation

scholarly journals Characterization of triosephosphate isomerase mutants with reduced enzyme activity in Mus musculus.

Genetics ◽  
1989 ◽  
Vol 123 (4) ◽  
pp. 837-844 ◽  
Author(s):  
S Merkle ◽  
W Pretsch
Keyword(s):  
Enzyme Activity ◽  
Triosephosphate Isomerase ◽  
Hematological Parameters ◽  
Glucose Consumption ◽  
Dominant Mode ◽  
Metabolic Energy ◽  
Wild Type ◽  
Total Lack ◽  
Mutant Lines

Abstract Four heterozygous triosephosphate isomerase (TPI) mutants with approximately 50% reduced activity in blood compared to wild type were detected in offspring of 1-ethyl-1-nitrosourea treated male mice. Breeding experiments displayed an autosomal, dominant mode of inheritance for the mutations. All mutations were found to be homozygous lethal at an early postimplantation stage of embryonic development, probably due to a total lack of TPI activity and consequently to the inability to utilize glucose as a source of metabolic energy. Although activity alteration was also found in liver, lung, kidney, spleen, heart, brain and muscle the TPI deficiency in heterozygotes has no influence on the following physiological traits: hematological parameters, plasma glucose, glucose consumption of blood cells, body weight and organo-somatic indices of liver, spleen, heart, kidney and lung. Biochemical investigations of TPI in the four mutant lines indicated no difference of physicochemical properties compared to the wild type. Results from immunoinactivation assays indicate that the decrease of enzyme activity corresponds to a decrease in the level of an immunologically active moiety. It is suggested that the mutations have affected the Tpi-1 structural locus and resulted in alleles which produce no detectable enzyme activity and no immunologically cross-reacting material. The study furthermore suggests one functional TPI gene per haploid genome in the erythrocyte and seven other tested organs of the mouse.

scholarly journals Characterization of Proteus mirabilisPrecocious Swarming Mutants: Identification of rsbA, Encoding a Regulator of Swarming Behavior

1998 ◽  
Vol 180 (23) ◽  
pp. 6126-6139 ◽  
Author(s):  
Robert Belas ◽  
Rachel Schneider ◽  
Michael Melch
Keyword(s):  
Genetic Locus ◽  
Regulatory Proteins ◽  
Wild Type ◽  
Histidine Kinases ◽  
Swarmer Cell ◽  
In Trans ◽  
Swarming Behavior ◽  
Bull's Eye

ABSTRACT Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull’s-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.

scholarly journals Novel Insights into Anthocyanin Metabolism and Molecular Characterization of Associated Genes in Sugarcane Rinds Using the Metabolome and Transcriptome

2021 ◽  
Vol 23 (1) ◽  
pp. 338
Author(s):  
Muhammad Junaid Rao ◽  
Mingzheng Duan ◽  
Mingchong Yang ◽  
Hongzeng Fan ◽  
Songhao Shen ◽  
...  
Keyword(s):  
Anthocyanin Biosynthesis ◽  
Saccharum Officinarum ◽  
Limited Information ◽  
Wild Type ◽  
Myb Gene ◽  
Wide Range ◽  
Transgenic Lines ◽  
Promising Source ◽  
First Time

Saccharum officinarum (sugarcane) is the fifth major cultivated crop around the world. Sugarcane rind is a promising source for anthocyanin pigments; however, limited information is available on the anthocyanin and its biosynthesis in sugarcane rinds. In this study, we have quantified 49 compounds including 6 flavonoids and 43 anthocyanins in the rind of 6 sugarcane cultivars by using LCMS/MS approach. Thirty of them were quantified for the first time in sugarcane. The 43 anthocyanins included 10 cyanidin (Cya), 11 pelargonidin (Pel), 9 peonidin (Peo), 5 malvidin (Mal), 4 delphinidin (Del), and 4 petunidin (Pet) metabolites. High contents of Cya derivatives were observed in the rind of YT71/210 (dark purple rind), such as cya-3-O-(6-O-malonyl)-glu 1283.3 µg/g and cya-3-O-glu 482.67 µg/g followed by ROC22 (red rind) 821.3 µg/g and 409 µg/g, respectively, whereas the YT93/159 (green rind) showed a minimum level of these compounds. Among six cultivars, ROC22 rind has high levels of Peo derivatives such as peo-3-O-glu (197 µg/g), peo-3-O-(6-O-malonyl)-glu (69 µg/g) and peo-3-O-(6-O-p-coumaryl)-glu (55.17 µg/g). The gene expression analysis revealed that some genes, including a MYB(t) gene, were highly associated with the color phenotype. Thus, we cloned and overexpressed the gene in Arabidopsis and found the pinkish brown color in the hypocotyl of all transgenic lines compared with the wild type. Hence, we have quantified a wide range of anthocyanins in major sugarcane cultivars, reported many new anthocyanins for the first time, and concluded that Cya and Peo derivatives are the major contributing factor of dissimilar colors in sugarcane. The finding and the verification of a novel MYB gene involved in anthocyanin biosynthesis have demonstrated that our study was very valuable for gene discovery and genetic improvement of sugarcane cultivars to harvest high anthocyanin contents.

scholarly journals The Marine Sponge Petrosia ficiformis Harbors Different Cyanobacteria Strains with Potential Biotechnological Application

2020 ◽  
Vol 8 (9) ◽  
pp. 638
Author(s):  
Patrizia Pagliara ◽  
Amilcare Barca ◽  
Tiziano Verri ◽  
Carmela Caroppo
Keyword(s):  
Cultured Cells ◽  
Biological Activities ◽  
Cell Type ◽  
High Sequence Identity ◽  
Toxic Activity ◽  
Bioactive Properties ◽  
Wide Range ◽  
Synechococcus Sp ◽  
Morphological And Molecular Characterization

Marine cyanobacteria are a source of bioactive natural compounds, with a wide range of biotechnological applications. However, information on sponge-associated cyanobacteria are relatively scarce to date. In this paper, we carried out the morphological and molecular characterization of eight cyanobacterial strains, previously isolated from the Mediterranean sponge Petrosia ficiformis, and evaluated their biological activities on epithelial- and neuron-like cultured cells of human and murine origin. The new analysis allowed maintaining the assignment of three strains (Cyanobium sp., Leptolyngbya ectocarpi, and Synechococcus sp.), while two strains previously identified as Synechococcus sp. and Leptolyngbya sp. were assigned to Pseudanabaena spp. One strain, i.e., ITAC104, and the ITAC101 strain corresponding to Halomicronema metazoicum, shared extremely high sequence identity, practically representing two clones of the same species. Finally, for only one strain, i.e., ITAC105, assignment to a specific genus was not possible. Concerning bioactivity analyses, incubation of cyanobacterial aqueous cell supernatants induced variable responses in cultured cells, depending on cell type, with some of them showing toxic activity on human epithelial-like cells and no toxic effects on human and rat neuron-like cells. Future investigations will allow to better define the bioactive properties of these cyanobacteria strains and to understand if they can be useful for (a) therapeutic purpose(s).

scholarly journals Genetic and biochemical characterization of the Na + /H + antiporters of Pseudomonas aeruginosa

2021 ◽  
Author(s):  
Sara Foreman ◽  
Kristina Ferrara ◽  
Teri Hreha ◽  
Ana Duran-Pinedo ◽  
Jorge Frias-Lopez ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biochemical Characterization ◽  
Physiological Role ◽  
Glycerol Metabolism ◽  
Kinetic Properties ◽  
Wild Type ◽  
Wide Range ◽  
Quadruple Mutant

Pseudomonas aeruginosa has four Na + /H + antiporters that interconvert and balance Na + and H + gradients across the membrane. These gradients are important for bioenergetics and ionic homeostasis. To understand these transporters, we have constructed four strains, each of which has only one antiporter: NhaB, NhaP, NhaP2, and Mrp. We also constructed a quadruple deletion mutant that has no Na + /H + antiporters. Although the antiporters of P. aeruginosa have previously been studied, the strains constructed here present the opportunity to characterize their kinetic properties in their native membranes and their roles in the physiology of P. aeruginosa . The strains expressing only NhaB or Mrp, the two electrogenic antiporters, are able to grow essentially as the wild type across a range of [Na + ] and pH. Strains with only NhaP or NhaP2, which are electroneutral, grow more poorly at increasing [Na + ], especially at high pH, with NhaP the most sensitive. The strain with no Na + /H + antiporters is extremely sensitive to [Na + ] and shows essentially no Na + (Li + )/H + antiporter activity but retains most K + /H + antiporter activity of the wild type at pH 7.5 and approximately half at pH 8.5. We also used the four strains that each express one of the four antiporters to characterize the kinetic properties of each transporter. RNA-seq analysis of the quadruple deletion strain showed widespread changes, including pyocyanin synthesis, biofilm formation, and nitrate and glycerol metabolism. Thus, the strains constructed for this study will open a new door to understanding the physiological role of these proteins and their activities in P. aeruginosa . Importance Pseudomonas aeruginosa has four Na + /H + antiporters that connect and interconvert its Na + and H + gradients. We have constructed four deletion mutants, each of which has only one of the four Na + /H + antiporters. These strains made it possible to study the properties and physiological roles of each antiporter independently in its native membrane. Mrp and NhaB are each able to sustain growth over a wide range of pH and [Na + ], whereas the two electroneutral antiporters, NhaP and NhaP2, are most effective at low pH. We also constructed a quadruple mutant, lacking all four antiporters in which the H + and Na + gradients are disconnected. This will make it possible to study the role of the two gradients independently.

scholarly journals l-Rhamnose Transport Is Sugar Kinase (RhaK) Dependent in Rhizobium leguminosarum bv. trifolii

2007 ◽  
Vol 189 (23) ◽  
pp. 8437-8446 ◽  
Author(s):  
Jason S. Richardson ◽  
Ivan J. Oresnik
Keyword(s):  
Abc Transporter ◽  
Rhizobium Leguminosarum ◽  
Nodule Occupancy ◽  
Vitro Assay ◽  
Catabolic Genes ◽  
Genes Encoding ◽  
Pentose Sugar ◽  
Sugar Kinase

ABSTRACT Strains of Rhizobium leguminosarum which are unable to catabolize l-rhamnose, a methyl-pentose sugar, are compromised in the ability to compete for nodule occupancy versus wild-type strains. Previous characterization of the 11-kb region necessary for the utilization of rhamnose identified a locus carrying catabolic genes and genes encoding the components of an ABC transporter. Genetic evidence suggested that the putative kinase RhaK carried out the first step in the catabolism of rhamnose. Characterization of this kinase led to the observation that strains carrying rhamnose kinase mutations were unable to transport rhamnose into the cell. The absence of a functional rhamnose kinase did not stop the transcription and translation of the ABC transporter components. By developing an in vitro assay for RhaK activity, we have been able to show that (i) RhaK activity is consistent with RhaK phosphorylating rhamnose and (ii) biochemical activity of RhaK is necessary for rhamnose transport.

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