Formate-Dependent Autotrophic Growth in Sinorhizobium meliloti

Abstract

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...

Carbohydrate Kinase (RhaK)-Dependent ABC Transport of Rhamnose in Rhizobium leguminosarum Demonstrates Genetic Separation of Kinase and Transport Activities

“…An assay for rhamnose kinase activity was developed by adapting a fructose kinase assay that has been previously described (30,31). Briefly R. leguminosarum cells were grown, and cell-free lysates were prepared as previously described (32)(33)(34). The assay was initiated with the addition of rhamnose.…”

The Twin Arginine Transport System Appears To Be Essential for Viability in Sinorhizobium meliloti

“…To characterize the twin arginine transport pathway in S. meliloti, we set out to create a gene disruption using a Kan r cassette targeted to the tatC gene. The tatC gene was PCR amplified, cloned into pBluescript, disrupted with an antibiotic cassette, recloned into pRK7813, and used for an allelic exchange experiment, as previously described (47). Although putative recombinants were isolated, none of these was a bona fide double-crossover event; they appeared to be single-crossover events in which the vector was also integrated into the genome.…”

“…We note that the S. meliloti genome contains at least one other gene, smc04018, which is annotated to have the same putative function. Moreover, since S. meliloti can also live as a formate-dependent autotroph, it seems unlikely that a salvage pathway gene would be essential in S. meliloti (47). However, these prospective candidates will need to be functionally tested to determine whether they may, indeed, be essential for S. meliloti viability.…”

“…E. coli strains were routinely grown at 30°C or 37°C in Luria-Bertani (LB) medium. S. meliloti strains where routinely grown at 30°C in either LB medium, LB medium containing 2.5 mM magnesium and calcium (LB MC ), or Vincent's minimal medium (VMM), as previously described (47,51,56,61). For defined medium, carbon sources were filter sterilized and added to medium at a final concentration of 15 mM.…”

Inability To Catabolize Galactose Leads to Increased Ability To Compete for Nodule Occupancy in Sinorhizobium meliloti

“…Cells were grown in defined medium, cell-free lysates were prepared, and samples were separated by using nondenaturing polyacrylamide gel electrophoresis (PAGE) as previously described (48). Gels were developed by using a p-nitroblue tetrazolium-based dehydrogenase stain as previously described (37).…”