Development of a Functional Genomics Platform for
            <i>Sinorhizobium meliloti</i>
            : Construction of an ORFeome

Schroeder, Brenda K.; House, Brent; Mortimer, Michael W.; Yurgel, Svetlana N.; Maloney, Scott C.; Ward, Kristel L.; Kahn, Michael L.

doi:10.1128/aem.71.10.5858-5864.2005

Cited by 41 publications

(40 citation statements)

References 24 publications

(37 reference statements)

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“…Plasmid pK18acnA was constructed by inserting a 1.35-kb HindIII/PstI fragment of the acnA open reading frame (corresponding to positions 3512129 to 3513478 in the strain 1021 genome) from pESMc03846 (34) into the HindIII/PstI sites in pK18mobsacB (33). Plasmids pFL237, pFL3738, and pFL2240 (5) were kindly provided by T. Finan.…”

Section: Methodsmentioning

confidence: 99%

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

et al. 2009

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The symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti 1021 encodes only one predicted aconitase (AcnA) in its genome. AcnA has a significant degree of similarity with other bacterial aconitases that behave as dual proteins: enzymes and posttranscriptional regulators of gene expression. Similar to the case with these bacterial aconitases, AcnA activity was reversibly labile and was regained upon reconstitution with reduced iron. The aconitase promoter was active in root nodules. acnA mutants grew very poorly, had secondary mutations, and were quickly outgrown by pseudorevertants. The acnA gene was stably interrupted in a citrate synthase (gltA) null background, indicating that the intracellular accumulation of citrate may be deleterious for survival of strain 1021. No aconitase activity was detected in this mutant, suggesting that the acnA gene encodes the only functional aconitase of strain 1021. To uncover a function of AcnA beyond its catalytic role in the tricarboxylic acid cycle pathway, the gltA acnA double mutant was compared with the gltA single mutant for differences in motility, resistance to oxidative stress, nodulation, and growth on different substrates. However, no differences in any of these characteristics were found.Aconitases (EC 4.2.1.3) are key enzymes in the tricarboxylic acid (TCA) cycle in both prokaryotes and eukaryotes, catalyzing the reversible isomerization of citrate into isocitrate via the intermediate cis-aconitate. The active site contains a [4Fe-4S] cluster that is required for enzymatic activity.

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Section: Methodsmentioning

confidence: 99%

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

et al. 2009

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“…To carry this out, we utilized the S. meliloti ORFeome platform that contains each of the annotated open reading frames from S. meliloti Rm1021 in a Gateway-compatible vector (26,45). The rhaK open reading frame was recombined into the destination vector pCO37 (25), yielding pDR35.…”

Section: Rivers and Oresnikmentioning

confidence: 99%

The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

Rivers

Oresnik

2015

J Bacteriol

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Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N-and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are the carbohydrate uptake transporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport. C ells are separated from the external environment by a selectively permeable membrane. A means to transport physiologically relevant substrates across these membranes is required (1-3). The largest and most widely distributed family of transporters is the ATP binding cassette (ABC) transporters (1). ABC transporters are responsible for the transport of a very diverse set of substrates and can be broken into two main classes, importers and exporters (1, 3-5). Much of our understanding of the structure, mechanism, and kinetics of these systems has been derived from the study of relatively few model transporters (6).Gram-negative ABC importers typically consist of a permease component that is made up of two membrane-spanning proteins (encoded by either one or two genes), a periplasm...

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“…As a result of the whole-genome sequence data, greater effort has been made to address the functionality of many of the unknown open reading frames. Systems that have utilized the construction of either genetic deletions or genetic fusions of reporter genes have been developed for this purpose (15,28,40,52).Earlier efforts to uncover functionality associated with the megaplasmids in S. meliloti relied on the creation of precise deletions or plasmid-curing experiments (12, 45). These works showed that a number of carbon-catabolic loci, as well as transport systems, were associated with these plasmids (5, 12, 13, 45).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Formate-Dependent Autotrophic Growth in Sinorhizobium meliloti

Pickering

Oresnik

2008

J Bacteriol

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It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.Sinorhizobium meliloti is a gram-negative rhizobium that is capable of forming nodules on alfalfa, sweet clover, and trigonella. Bacterial infection occurs at the root hairs of the host plant, involving a signal exchange between the rhizobia and the plant (6, 55). Root hair curling encapsulates the bacteria, which subsequently travel through an infection thread and are finally released into plant cells, where they differentiate into bacteroids (24). In this environment, the bacteria reduce atmospheric nitrogen in exchange for nutrients and carbon compounds (37).From the sequencing of its genome, it has become apparent that S. meliloti retains an array of genes that are capable of encoding enzymes for many metabolic pathways that allow the organism to scavenge energy from many different substrates (7,23,25). As a result of the whole-genome sequence data, greater effort has been made to address the functionality of many of the unknown open reading frames. Systems that have utilized the construction of either genetic deletions or genetic fusions of reporter genes have been developed for this purpose (15,28,40,52).Earlier efforts to uncover functionality associated with the megaplasmids in S. meliloti relied on the creation of precise deletions or plasmid-curing experiments (12, 45). These works showed that a number of carbon-catabolic loci, as well as transport systems, were associated with these plasmids (5, 12, 13, 45). It was also observed that an S. meliloti strain cured of pSymA had a reduced ability to respire formate (I. J. Oresnik, unpublished observation).Formate is a ubiquitous compound in the environment. Many plants and bacterial species produce and excrete formate into their envi...

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Development of a Functional Genomics Platform for Sinorhizobium meliloti : Construction of an ORFeome

Cited by 41 publications

References 24 publications

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

Deletion of Citrate Synthase Restores Growth of Sinorhizobium meliloti 1021 Aconitase Mutants

The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

Formate-Dependent Autotrophic Growth in Sinorhizobium meliloti

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