otsA Genes Research Articles

Bacterial Endophytes from Legumes Native to Arid Environments Are Promising Tools to Improve Mesorhizobium-Chickpea Symbiosis under Salinity.

Symbiotic nitrogen fixation is a major contributor of N in agricultural ecosystems, but the establishment of legume-rhizobium symbiosis is highly affected by soil salinity. Our interest is focused on the use of non-rhizobial endophytes to assist the symbiosis between chickpea and its microsymbiont under salinity to avoid loss of production and fertility. Our aims were (1) to investigate the impact of salinity on both symbiotic partners; including on early events of the Mesorhizobium-chickpea symbiosis, and (2) to evaluate the potential of four non-rhizobial endophytes isolated from legumes native to arid regions (Phyllobacterium salinisoli, P. ifriqiyense, Xanthomonas translucens, and Cupriavidus respiraculi) to promote chickpea growth and nodulation under salinity. Our results show a significant reduction in chickpea seed germination rate and in the microsymbiont Mesorhizobium ciceri LMS-1 growth under different levels of salinity. The composition of phenolic compounds in chickpea root exudates significantly changed when the plants were subjected to salinity, which in turn affected the nod genes expression in LMS-1. Furthermore, the LMS-1 response to root exudate stimuli was suppressed by the presence of salinity (250 mM NaCl). On the contrary, a significant upregulation of exoY and otsA genes, which are involved in exopolysaccharide and trehalose biosynthesis, respectively, was registered in salt-stressed LMS-1 cells. In addition, chickpea co-inoculation with LMS-1 along with the consortium containing two non-rhizobial bacterial endophytes, P. salinisoli and X. translucens, resulted in significant improvement of the chickpea growth and the symbiotic performance of LMS-1 under salinity. These results indicate that this non-rhizobial endophytic consortium may be an appropriate ecological and safe tool to improve chickpea growth and its adaptation to salt-degraded soils.

Increased trehalose biosynthesis improves Mesorhizobium ciceri growth and symbiosis establishment in saline conditions

Cicer arietinum (chickpea) is a legume very sensitive to salinity, and so are most of its rhizobial symbionts belonging to the species Mesorhizobium ciceri. We observed that exogenous trehalose (i.e., added to the growth medium) can significantly improve growth of M. ciceri strain Rch125 under moderate salinity. In order to test if endogenous trehalose (i.e., synthesized by the cell) could also enhance salt tolerance, strain Rch125 was genetically modified with various trehalose biosynthesis genes from Sinorhizobium meliloti 1021 (otsA, treS, treY) and Mesorhizobium loti MAFF 303099 (otsAB). We found that overexpression of otsA or otsAB, but not treS or treY, significantly improved M. ciceri Rch125 growth in saline media. This growth improvement correlated with enhanced trehalose accumulation in otsA- and otsAB-modified cells, suggesting that increased trehalose synthesis via trehalose-6-phosphate can enhance bacterial salt tolerance. Chickpea plants inoculated with M. ciceri Rch125 derivatives carrying extra otsAB or otsA genes formed more nodules and accumulated more shoot biomass than wild type inoculated plants when grown in the presence of NaCl. These results support the notion that improved salt tolerance of the bacterial symbiont can alleviate the negative effects of salinity on chickpeas, and that such improvement in M. ciceri can be achieved by manipulating trehalose metabolism.

The dual nature of trehalose in citrus canker disease: a virulence factor for Xanthomonas citri subsp. citri and a trigger for plant defence responses.

Xanthomonas citri subsp. citri (Xcc) is a bacterial pathogen that causes citrus canker in susceptible Citrus spp. The Xcc genome contains genes encoding enzymes from three separate pathways of trehalose biosynthesis. Expression of genes encoding trehalose-6-phosphate synthase (otsA) and trehalose phosphatase (otsB) was highly induced during canker development, suggesting that the two-step pathway of trehalose biosynthesis via trehalose-6-phosphate has a function in pathogenesis. This pathway was eliminated from the bacterium by deletion of the otsA gene. The resulting XccΔotsA mutant produced less trehalose than the wild-type strain, was less resistant to salt and oxidative stresses, and was less able to colonize plant tissues. Gene expression and proteomic analyses of infected leaves showed that infection with XccΔotsA triggered only weak defence responses in the plant compared with infection with Xcc, and had less impact on the host plant's metabolism than the wild-type strain. These results suggested that trehalose of bacterial origin, synthesized via the otsA-otsB pathway, in Xcc, plays a role in modifying the host plant's metabolism to its own advantage but is also perceived by the plant as a sign of pathogen attack. Thus, trehalose biosynthesis has both positive and negative consequences for Xcc. On the one hand, it enables this bacterial pathogen to survive in the inhospitable environment of the leaf surface before infection and exploit the host plant's resources after infection, but on the other hand, it is a tell-tale sign of the pathogen's presence that triggers the plant to defend itself against infection.

Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli

BackgroundThe compatible solute trehalose is involved in the osmostress response of Rhizobium etli, the microsymbiont of Phaseolus vulgaris. In this work, we reconstructed trehalose metabolism in R. etli, and investigated its role in cellular adaptation and survival to heat and desiccation stress under free living conditions.ResultsBesides trehalose as major compatible solute, R. etli CE3 also accumulated glutamate and, if present in the medium, mannitol. Putative genes for trehalose synthesis (otsAB/treS/treZY), uptake (aglEFGK/thuEFGK) and degradation (thuAB/treC) were scattered among the chromosome and plasmids p42a, p42c, p42e, and p42f, and in some instances found redundant. Two copies of the otsA gene, encoding trehalose-6-P-synthase, were located in the chromosome (otsAch) and plasmid p42a (otsAa), and the latter seemed to be acquired by horizontal transfer. High temperature alone did not influence growth of R. etli, but a combination of high temperature and osmotic stress was more deleterious for growth than osmotic stress alone. Although high temperature induced some trehalose synthesis by R. etli, trehalose biosynthesis was mainly triggered by osmotic stress. However, an otsAch mutant, unable to synthesize trehalose in minimal medium, showed impaired growth at high temperature, suggesting that trehalose plays a role in thermoprotection of R. etli. Desiccation tolerance by R. etli wild type cells was dependent of high trehalose production by osmotic pre-conditioned cells. Cells of the mutant strain otsAch showed ca. 3-fold lower survival levels than the wild type strain after drying, and a null viability after 4 days storage.ConclusionsOur findings suggest a beneficial effect of osmotic stress in R. etli tolerance to desiccation, and an important role of trehalose on the response of R. etli to high temperature and desiccation stress.

Identification at the species and symbiovar levels of strains nodulating Phaseolus vulgaris in saline soils of the Marrakech region (Morocco) and analysis of the otsA gene putatively involved in osmotolerance

Salinity is an increasing problem in Africa affecting rhizobia-legume symbioses. In Morocco, Phaseolus vulgaris is cultivated in saline soils and its symbiosis with rhizobia depends on the presence of osmotolerant strains in these soils. In this study, 32 osmotolerant rhizobial strains nodulating P. vulgaris were identified at the species and symbiovar levels by analysing core and symbiotic genes, respectively. The most abundant strains were closely related to Rhizobium etli and R. phaseoli and belonged to symbiovar phaseoli. A second group of strains was identified as R. gallicum sv gallicum. The remaining strains, identified as R. tropici, belonged to the CIAT 899TnodC group, which has not yet been described as a symbiovar. In representative strains, the otsA gene involved in the accumulation of trehalose and putatively in osmotolerance was analysed. The results showed that the phylogeny of this gene was not completely congruent with those of other core genes, since the genus Ensifer was more closely related to some Rhizobium species than others. Although the role of the otsA gene in osmotolerance is not well established, it can be a useful protein-coding gene for phylogenetic studies in the genus Rhizobium, since the phylogenies of otsA and other core genes are coincident at the species level.

The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5'-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields

BackgroundAssociated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T.ResultsThe most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the β-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined.ConclusionsThe soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

Characterization ofEscherichia coliOtsA, a trehalose-6-phosphate synthase from glycosyltransferase family 20

The Ots gene cluster of Escherichia coli encodes the synthetic apparatus for the formation of alpha,alpha-1,1-trehalose, a non-reducing glucose disaccharide. The otsA gene encodes a trehalose-6-phosphate synthase, a glycosyltransferase which catalyses the synthesis of alpha,alpha-1,1-trehalose-6-phosphate from glucose-6-phosphate using a UDP-glucose donor. It has been classified into glycosyltransferase family GT-20 based upon amino-acid sequence similarities. The otsA gene has been cloned and recombinant protein overexpressed using a pET-based system in E. coli BL21 cells. The recombinant protein (MW approximately 54.7 kDa) is active and has been crystallized in two forms suitable for X-ray diffraction analysis. The first is orthorhombic, P2(1)2(1)2(1), with unit-cell parameters a = 104.1, b = 127.8, c = 179.9 A. Data for this form have been collected to 3.0 A resolution at the CLRC Daresbury Synchrotron Radiation Source. The second form has unit-cell parameters a = b = 141.9, c = 317.8 A and displays the apparent space group P4(2). These crystals diffract beyond 2 A resolution, but display merohedral twinning.

Expression of Escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis

The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.

Analysis of the otsBA operon for osmoregulatory trehalose synthesis in Escherichia coli and homology of the OtsA and OtsB proteins to the yeast trehalose-6-phosphate synthase/phosphatase complex.

The Escherichia coli otsBA operon, located at min 42, was sequenced and shown to encode a 29.1-kDa trehalose-6-phosphate phosphatase (OtsB) and a 53.6-kDa trehalose-6-phosphate synthase (OtsA). Both proteins display sequence homology with subunits of the Saccharomyces cerevisiae trehalose-6-phosphate synthase/phosphatase complex, which is made up of the subunits TPS1, TPS2 and TPS3 (TSL1). OtsA has homology to the full-length TPS1, the N-terminal part of TPS2 and an internal region of TPS3 (TSL1). OtsB has homology to the C-terminal part of TPS2, but no homology to the other subunits. Primer extension analysis showed only one transcription start point upstream from otsB and one upstream from otsA, regardless of the growth conditions tested. The start codons of the otsB and otsA genes were established by N-terminal sequence determination of the proteins. The 3' end of the otsB coding region overlaps the 5' end of the otsA coding region by 23 nucleotides. The araH gene is located directly upstream from otsBA, and otsB may be identical to pexA.

A yeast gene for trehalose-6-phosphate synthase and its complementation of an Escherichia coli otsA mutant

A Saccharomyces cerevisiae gene for trehalose-6-phosphate synthase (TPS1) was sequenced. The gene appeared to code for a protein of 495 amino acid residues, giving the protein a molecular mass of 56 kDa. The TPS1 gene was able to restore both osmotolerance and trehalose accumulation during salt stress in an Escherichia coli strain mutated in the otsA gene encoding trehalose-6-phosphate synthase. Complementation studies with E. coli galU mutants showed that the TPS1-encoded trehalose-6-phosphate synthase is UDP-glucose-dependent. Sequence analysis and data base searches showed that TPS1 is allelic to GGS1, byp1, cif1 and fdp1. A possible gene for trehalose-6-phosphate synthase in Methanobacterium thermoautotrophicum was identified.