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Appl Environ Microbiol. Feb 2010; 76(4): 1071–1081.
Published online Dec 18, 2009. doi:  10.1128/AEM.02483-09
PMCID: PMC2820964

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation [down-pointing small open triangle]

Abstract

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.

Rhizobia induce the formation of nodules on the roots of legume plants, in which atmospheric nitrogen is fixed and supplied to the host plant, thereby enhancing growth under nitrogen-limiting conditions. The symbiotic interaction between rhizobia and their cognate leguminous plants is important for agricultural productivity, especially in less developed countries. However, physiological stresses, such as desiccation and salinity, negatively affect these symbiotic interactions by limiting nitrogen fixation (44). The osmotic environment within the rhizosphere may affect root colonization, infection thread development, nodule development, and the formation of effective N2-fixing nodules (21). Moreover, when legume seeds are inoculated with appropriate rhizobial strains prior to planting in the field, the vast majority of nodules produced are often not formed by the inoculant bacteria but rather by indigenous strains in the soil (36). This is in part due to the death of inoculant strains from rapid seed coat-mediated desiccation. Therefore, improvement of the survival of rhizobia under conditions of physiological stresses may promote biological nitrogen fixation and enhance plant growth.

Rhizobia synthesize and accumulate compatible solutes, including trehalose, in response to desiccation and solute-mediated physiological stresses (5, 21, 42). Trehalose, a nonreducing disaccharide with an α,α-1,1 linkage between the two glucose molecules, has been shown to protect cell membranes and proteins from stress-induced inactivation and denaturation (8, 23, 24). The relationship between trehalose accumulation and symbiotic phenotype is dependent on rhizobial species and host genotype. Suarez et al. (39) reported an increase in root nodule number and nitrogen fixation by Phaseolus vulgaris inoculated with a trehalose-6-phosphate synthase-overexpressing strain of Rhizobium etli. In contrast, trehalose accumulation in Rhizobium leguminosarum and Sinorhizobium meliloti cells did not result in an increase in nitrogen-fixing nodules but led to enhancement of competitiveness on clover and on certain alfalfa genotypes, respectively (1, 16, 20).

Four trehalose biosynthetic pathways, mediated by OtsAB, TreS, TreYZ, and TreT, have been reported thus far for prokaryotes (8, 25). The OtsAB pathway results in the condensation of glucose-6-phosphate with UDP-glucose by trehalose-6-phosphate synthase (OtsA) to form trehalose-6-phosphate. Trehalose is subsequently formed from trehalose-6-phosphate by the action of trehalose-6-phosphate phosphatase (OtsB). The TreS pathway involves a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with α,α-1,4 linkage between the two glucose molecules, to trehalose. The third pathway, mediated by TreYZ, involves the conversion of maltodextrins into trehalose. The terminal α-1,1-glycosylic bond at the end of the maltodextrin polymer is hydrolyzed by maltooligosyltrehalose synthase (TreY), and trehalose is subsequently released from the end of the polymer via hydrolysis by maltooligosyltrehalose trehalohydrolase (TreZ). More recently, a trehalose glycosyltransferring synthase (TreT) was shown to catalyze the reversible formation of trehalose from ADP-glucose and glucose (25).

In addition to biosynthesis, Gram-negative bacteria have also been reported to have trehalose degradation systems. Typically, trehalose is hydrolyzed into two glucose moieties by periplasmic and cytoplasmic trehalase enzymes, TreA and TreF, respectively (13, 15). However, Sinorhizobium meliloti also uses ThuA and ThuB for trehalose utilization (16).

Bradyrhizobium japonicum, the root nodule symbiont of soybeans, accumulates trehalose in cultured cells and bacteroids (34, 35). Biochemical studies indicated that B. japonicum has three independent trehalose biosynthetic pathways involving trehalose synthase (TreS), maltooligosyltrehalose synthase (TreYZ), and trehalose-6-phosphate synthetase (OtsAB) (38). Sequence analysis of the B. japonicum USDA 110 genome identified the genes that encode these biosynthetic pathways: otsAB (bll0322 to bll0323), two homologs of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771), but not treT (17). Orthologous gene sequences to the trehalose degradation genes treA, treF, and thuAB have not been found in the genome of B. japonicum USDA 110. Cytryn et al. (6) reported that expression of otsA, treS (blr6767), and treY genes were highly induced by desiccation stress. Moreover, the concentrations of these three enzymes increased when B. japonicum was cultured in the presence of salt (38). Trehalose concentration in B. japonicum has been reported to increase due to desiccation stress (6), and this sugar is purported to act as an osmoprotectant. The addition of exogenously supplied trehalose has been reported to enhance the survival of B. japonicum in response to desiccation and salinity stresses (9, 37). Despite this information, little is known about how the various trehalose biosynthetic pathways modulate stress tolerance and symbiotic performance in B. japonicum.

The purpose of this study was to examine the functional role(s) of the B. japonicum trehalose biosynthetic pathways on stress survival by constructing single, double, and triple mutants and by producing strains that overexpress the trehalose biosynthesis enzymes. Here we report on the relationship between trehalose accumulation and physiological responses to salinity and desiccation stresses in mutant and overexpression strains and that mutations in the trehalose biosynthesis pathways altered the symbiotic performance of B. japonicum USDA 110 on soybeans. Results of these studies indicate that trehalose accumulation in B. japonicum plays a prominent role in the saprophytic and symbiotic competence of this agriculturally important soil bacterium.

MATERIALS AND METHODS

Bacterial strains, plasmids, and growth conditions.

Bacterial strains and plasmids used in this study are shown in Table Table1.1. The B. japonicum strains were grown at 30°C in arabinose-gluconate (AG) medium (26), and the Escherichia coli strains were grown at 37°C in LB medium (29). Media were supplemented with antibiotics, as required, at the following concentrations (per ml): for B. japonicum, kanamycin at 100 μg, streptomycin at 100 μg, spectinomycin at 100 μg, tetracycline at 100 μg, and polymyxin B at 50 μg; and for E. coli, ampicillin at 50 μg, kanamycin at 50 μg, tetracycline at 12.5 μg, streptomycin at 25 μg, and spectinomycin at 25 μg.

TABLE 1.
Bacterial strains and plasmids used in this study

DNA manipulations.

The isolation of plasmid DNA, restriction enzyme digestions, DNA ligations, and the transformation of E. coli were done as described by Sambrook and Russell (29).

Construction of B. japonicum USDA 110 mutants in trehalose synthesis genes.

A mobilizable otsA inactivation plasmid was constructed as follows: the 1.0-kb otsA (bll0322) coding region from B. japonicum USDA 110 was amplified by PCR using genomic DNA as the template and the oligonucleotide primers tre6PSFN and tre6PR (see Table S1 in the supplemental material). The PCR product was ligated into the pGEM-T Easy vector (Promega, Madison, WI), to create pGEM-otsA. The Ω cassette, which encodes resistance to spectinomycin and streptomycin (via aadA), was digested from pHP45Ω (10) and inserted into the NruI site of pGEM-otsA, resulting in pGEM-otsA::Ω. Plasmid pGEM-otsA::Ω was digested with EcoRI, and the 3.0-kb fragment containing otsA::Ω was ligated into suicide vector pK18mob (30) to create pK18mob-otsA::Ω (Fig. (Fig.1).1). For inactivation of the treS gene, the 1.7-kb treS (blr6767) coding region from B. japonicum USDA 110 was amplified by PCR using genomic DNA as the template and the oligonucleotide primers treSF and treS2R (see Table S1). The PCR product was ligated into the pGEM-T Easy vector, yielding pGEM-treS. The Ω cassette, or the aminoglycoside 3-phosphotransferase (aph) cassette from pUC4-K (41), was inserted into the BamHI site of pGEM-treS, to create pGEM-treS::Ω and pGEM-treS::aph, respectively. Plasmid pGEM-treS::Ω was digested with EcoRI, and the resulting 3.7-kb fragment containing treS::Ω was ligated into suicide vector pK18mob to create pK18mob-treS::Ω (Fig. (Fig.1).1). Plasmid pGEM-treS::aph was digested with BamHI, and the 2.9-kb fragment containing treS::aph was ligated into suicide vector pSUP202 (31), resulting in pSUP-treS::aph (Fig. (Fig.1).1). For inactivation of the treY gene, the 1.5-kb treY (blr6771) coding region from B. japonicum USDA 110 was amplified by PCR using genomic DNA as the template and the oligonucleotide primers treY-Fbam and treY-Rbam (see Table S1) and ligated into pGEM-T vector (Promega, Madison, WI), yielding pGEM-treY. The tetA gene, which confers tetracycline resistance, was amplified by PCR from plasmid pALTER-1 (Promega, Madison, WI) by using the oligonucleotide primers tetA-Feco and tetA-Reco (see Table S1). The tetA fragment was subsequently digested with EcoRI and inserted into the EcoRI site of pGEM-treY, resulting in pGEM-treY::tetA. Plasmid pGEM-treY::tetA was digested with BamHI, and the 3.1-kb fragment containing treY::tetA was ligated into suicide vector pK18mob to create pK18mob-treY::tetA (Fig. (Fig.1).1). Plasmids containing pK18mob-otsA::Ω (for the otsA mutation), pK18mob-treS::Ω (for the treS mutation), pSUP-treS::aph (for the treS mutation), and pK18mob-treY::tetA (for the treY mutation) were introduced into B. japonicum USDA 110 by triparental mating, using pRK2013 as a helper plasmid (11). Mutated strains were selected on AG agar plates containing 50 μg of polymyxin B per ml (to eliminate E. coli) and other appropriate antibiotics as required. Gene replacement, double crossover mutants were verified by antibiotic resistance phenotypes and by PCR using primers for the inserted gene and primers that spanned the insertion sites.

FIG. 1.
Physical maps of the otsA, treS, and treY genes (and their flanking regions) used in the construction of B. japonicum USDA 110 mutants. (a) otsA (bll0322) and its flanking region. The internal NruI fragment in otsA was removed and replaced with the Ω ...

Construction of strains overexpressing trehalose synthesis genes.

To overexpress OtsA, the 1.5-kb full-length otsA gene, including its Shine-Dalgarno (SD) sequence, from B. japonicum USDA 110 was amplified by PCR using genomic DNA as the template and the oligonucleotide primers otsAFpst and otsARbam (see Table S1 in the supplemental material). The PCR product was ligated into the pGEM-T Easy vector to create pGEM-otsAEX. The pGEM-otsAEX was subsequently digested with PstI and BamHI, and the 1.5-kb fragment containing otsA was inserted into the PstI and BamHI sites, downstream of the trp promoter, of the broad-host-range vector pTE3 (7), yielding plasmid pTE3-otsA. Expression of genes inserted into this vector are constitutively expressed and under the control of the trp promoter. For overexpression and complementation of treS, the 3.3-kb full-length treS gene, including its SD sequence, was amplified by PCR using genomic DNA as the template and the oligonucleotide primers treSF-pst3 and treSR-pst3 (see Table S1). The PCR product was digested with PstI and ligated into pBluescript II SK(+) vector (Invitrogen Co., Carlsbad, CA) to create pBS-treSEX. The pBS-treSEX was digested with PstI to obtain the 3.3-kb treS-containing fragment and ligated into the PstI site of pTE3, resulting in pTE3-treS. For overexpression of treY, the 3.0-kb full-length treY gene, including its SD sequence, from B. japonicum USDA 110 was amplified by PCR using genomic DNA as the template and the oligonucleotide primers treY-FpstEX2 and treY-RbamEX (see Table S1). The PCR product was digested with PstI and BamHI and ligated into the pBluescript II SK(+) vector to create pBS-treYEX. The pBS-treYEX vector was digested with PstI and BamHI, and the 3.0-kb fragment containing treY was inserted into the PstI site of pTE3, resulting in pTE3-treS.

The pTE3-otsA, pTE3-treS, or pTE3-treY plasmid construct was introduced into wild-type B. japonicum USDA 110 by triparental mating as described above. Transconjugant strains were selected on AG agar plates containing 50 μg of polymyxin B per ml and 100 μg of tetracycline per ml. For complementation analyses, pTE3-treS was introduced into the treS mutant by triparental mating, and transconjugants were selected on AG agar plates containing 50 μg of polymyxin B per ml and 100 μg of tetracycline, spectinomycin, and streptomycin per ml.

Determination of trehalose and maltose contents in Bradyrhizobium cells.

Cell extracts of wild-type and mutant strains were prepared by using a hot ethanol extraction method. The B. japonicum cells were cultured in 25 ml AG medium or AG medium supplemented with 60 mM NaCl, until an optical density at 600 nm (OD600) of 0.5 to 0.6 was reached. Cells were collected by centrifugation at 10,000 × g for 10 min, resuspended in 500 μl 80% ethanol, and incubated at 85°C for 15 min. The cell suspension was centrifuged at 15,000 × g for 2 min, and the supernatant was transferred to a 1.5-ml microcentrifuge tube. This procedure was repeated, and the approximately 1.0 ml of cell extract was evaporated by using a Spin-Vap centrifugal concentrator. The dried sample was suspended in 100 μl distilled H2O and stored at −20°C until used for trehalose and maltose quantification analyses.

Trehalose and maltose in cell extracts were detected following enzymatic conversion to glucose by reaction with trehalase (Sigma, St. Louis, MO) and maltase (Sigma), respectively. Reaction mixtures (50 μl) using trehalase contained 30 μl of 135 mM citric acid buffer, pH 5.7; 10 μl of cell extract; and 10 μl of trehalase enzyme solution, which was prepared by dissolving 0.2 units of enzyme per ml in cold 135 mM citric acid buffer, pH 5.7. Reactions were incubated for 1 h at 37°C and terminated by addition of 50 μl of 500 mM Tris-HCl, pH 7.5. Reaction mixtures using maltase contained 20 μl of 50 mM potassium phosphate buffer, pH 6.0; 25 μl of cell extract; and 5 μl of maltase enzyme solution, which was prepared by dissolving 1.0 unit of enzyme per ml in cold 50 mM potassium phosphate buffer, pH 6.0. Reactions were incubated for 1 h at 25°C. The amount of glucose produced was measured by using a glucose assay kit (Sigma), according to the manufacturer's instruction. The concentration of trehalose or maltose in samples was calculated from the difference in the concentration of glucose in samples with and without the addition of trehalase or maltase, respectively.

RNA extraction, cDNA synthesis, and quantitative RT-PCR.

A 25-ml culture of B. japonicum was grown in AG medium or AG medium supplemented with 60 mM NaCl to an optical density at 600 nm of 0.5 to 0.7. Cultures were immediately transferred to centrifuge tubes containing 2.5 ml of cold stop solution (5% H2O-saturated phenol, pH 4.3, in 95% ethanol). The suspension was centrifuged at 4°C for 10 min at 10,000 × g, and cell pellets were frozen immediately in liquid nitrogen and stored at −80°C until used for RNA isolation. Desiccated cell samples for RNA extraction were prepared as described by Cytryn et al. (6). RNA isolation from B. japonicum cells and DNA digestions were done as described by Chang et al. (4). The cDNA was synthesized from 5 μg of total RNA template. Reaction mixtures contained 250 ng of random hexamers (Invitrogen), 1 μl of 10 mM dNTP mix, and 11 μl of RNA solution. The mixture was heated for 5 min at 65°C and chilled on ice for 1 min, and 4 μl of 5× first-strand buffer, 1 μl of 0.1 M dithiothreitol, 1 μl of RNaseOUT (Invitrogen), and 200 units of Superscript III reverse transcriptase (RT) (Invitrogen) were added to the solution. The solution was incubated at 50°C for 1 h and placed in a 70°C heating block for 15 min. The resulting cDNA sample was kept at −20°C until used in quantitative RT-PCR (qRT-PCR) experiments.

For qRT-PCR, each 25-μl reaction mixture contained the cDNA synthesized from 0.25 μg RNA, 12.5 μl iTaq SYBR green Supermix with ROX (Bio-Rad, Hercules, CA), 11.25 μl nuclease-free water, and 0.5 μM (each) forward and reverse gene-specific primers (see Table S1 in the supplemental material). The primer sets used in the PCR to amplify products from the transcript were as follows: bll0322-F2 and bll0322-R2 (for otsA), blr6767-F2 and blr6767-R2 (for treS), bll0902-F and bll0902-R (for bll0902), blr6771-F and blr6771-R (for treY), and bll0631-F and bll0631-R (for parA). The qRT-PCRs were run on an Applied Biosystems (Foster City, CA) 7500 real-time PCR system using Sequence Detection System software, version 1.3. The PCR program consisted of an initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation at 95°C for 15 s, and annealing and extension at 60°C for 1 min. Expression values for three biological replicates for each treatment were normalized to the expression level of parA (bll0631), which is a housekeeping gene in the B. japonicum USDA 110 genome.

Measurement of viable cell numbers after desiccation stress.

Desiccation stress assays were done as described by Cytryn et al. (6). The relative humidity (RH) in the desiccators was adjusted to 50% by using a saturated solution of potassium acetate. The viability of desiccated cells was determined by using plate count assays.

Nodulation studies.

Wild-type B. japonicum USDA 110 and the ΔotsA ΔtreY, ΔotsA ΔtreS ΔtreY, and ΔtreS mutant strains were grown in AG medium supplemented with the appropriate antibiotics, collected by centrifugation at 8,000 × g for 10 min, and washed twice with sterile distilled water. These resultant cultures were diluted to 107 cells per ml, and 1-ml aliquots of these dilutions were inoculated onto surface-sterilized Glycine max cv. Lambert seeds. Plant assays were done in sterile Leonard jar assemblies containing a 3:1 mixture of vermiculite and perlite, as previously described (26). Seeds of G. max cv. Lambert were surface sterilized by immersion in 100% ethanol for 5 s and treated with 1.0% sodium hypochlorite for 5 min. Seeds were washed 10 times with sterile distilled water prior to planting. After inoculation, seeds were planted in a 3:1 mixture of vermiculite and perlite and covered with a 1-cm layer of sterilized paraffin-coated sand. Plants were watered with nitrogen-free plant nutrient solution (18) and incubated in a plant growth chamber at 25°C with a photoperiod of 16 h. The number of mature and immature nodules and nodule and plant dry weights (wt) were determined 31 days after inoculation.

RESULTS

Expression of trehalose biosynthetic genes under stress conditions.

otsAB (bll0322 to bll0323), two copies of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771) were previously annotated as trehalose biosynthetic genes in B. japonicum strain USDA 110 (17). In this study, real-time qRT-PCR analyses were done to examine expression of these genes under conditions of salinity and desiccation stress, relative to that seen with nonstressed cells. Results depicted in Fig. Fig.22 show that the expression of otsA (bll0322), the two homologs of treS (blr6767 and bll0902), and treY (blr6771) were induced by stress imparted by supplementation of the growth medium with 60 mM NaCl and by growth under desiccating conditions. The absolute gene expression value of otsA was greater than those seen for the other trehalose synthesis genes. In contrast, expression of bll0902, coding for the TreS homolog, was the lowest of all the tested genes. These results suggested that the OtsAB pathway may play a major role in stress-induced trehalose biosynthesis in B. japonicum.

FIG. 2.
qRT-PCR analysis of differentially expressed trehalose synthesis genes (otsA, treS, and treY) in salinity- and desiccation-stressed B. japonicum USDA 110 cells. Values are normalized to those of the housekeeping gene parA. (A) Absolute gene expression ...

Construction of trehalose biosynthesis mutants and overexpressing strains.

In order to investigate the roles that the different trehalose biosynthesis pathways play in stress survival of B. japonicum USDA 110, antibiotic resistance cassettes were used to create insertions in genes coding for OtsA, TreS (blr6767), and TreY (Fig. (Fig.1).1). Single, double, and triple mutations in these three genes were constructed by homologous recombination. The treS (bll0902) gene was eliminated as a target in this study, since the expression of this gene under stress conditions was lower than that of other genes (Fig. (Fig.22).

Trehalose accumulation in wild-type cells increased about fourfold in response to salinity stress (4.7 ± 0.21 μmol/g fresh wt) compared to cells grown under nonstressed conditions (1.2 ± 0.02 μmol/g fresh wt). This result correlated well with gene expression data (Fig. (Fig.2A).2A). In contrast, strains growing under salt stress conditions and carrying the otsA mutation, the ΔotsA, ΔotsA ΔtreS, ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, accumulated smaller amounts of trehalose, relative to the wild-type strain (Fig. (Fig.3A).3A). In particular, the two strains lacking the OtsAB and TreYZ pathways, the ΔotsA ΔtreY and ΔotsA ΔtreS ΔtreY mutants, accumulated extremely low levels of trehalose. Surprisingly, the treS mutant (ΔtreS) strain accumulated a larger amount of trehalose than that seen with salt-grown wild-type cells. The strain containing the treY single mutation and the wild-type strain accumulated approximately the same amount of trehalose, suggesting that the maltooligosyltrehalose synthase does not play a major role in trehalose synthesis in B. japonicum under the tested growth conditions.

FIG. 3.
Trehalose and maltose accumulation by wild-type B. japonicum USDA 110, trehalose synthesis mutants, and overexpressing strains grown in AG medium supplemented with 60 mM NaCl. (A) Trehalose contents in Bradyrhizobium cells. (B) Maltose contents in Bradyrhizobium ...

Wild-type USDA 110 strains overexpressing OtsA or TreY accumulated slightly greater amounts of trehalose than those of the wild-type strain carrying the empty plasmid vector pTE3. In contrast, trehalose accumulation in wild-type cells overexpressing TreS was less than that found with wild-type USDA 110(pTE3) cells. This result suggested that TreS was involved in trehalose degradation in B. japonicum USDA 110. In addition, the ΔtreS mutant strain complemented with the treS gene, ΔtreS (pTE3-treS), accumulated less trehalose than the parental control ΔtreS (pTE3) strain. Taken together, these results suggested that trehalose biosynthesis in B. japonicum is mediated mainly by the OtsAB pathway and marginally by TreYZ pathways and that the TreS protein encoded by blr6767 is likely involved in trehalose degradation or repression of trehalose biosynthesis. Moreover, since wild-type USDA 110 cells overexpressing OtsA accumulated levels of trehalose similar to those of the control strain containing the empty vector, our results suggest that the concentration of trehalose in salt-stressed Bradyrhizobium cells may be controlled by the combined activities of trehalose biosynthesis and degradation systems.

The B. japonicum treS gene is involved in degradation of trehalose to maltose.

In order to investigate whether TreS catalyzes the degradation of trehalose in B. japonicum, maltose accumulation levels in wild-type, mutant, and overexpressing strains were determined. The accumulation of maltose by all of the tested strains is shown in Fig. Fig.3B.3B. The strains carrying a mutation in treStreS, ΔotsA ΔtreS, ΔtreS ΔtreY, and ΔotsA ΔtreS ΔtreY) accumulated amounts of maltose smaller than those of the wild-type strain, and the strain overexpressing TreS resulted in an increase in the intracellular concentration of maltose. In addition, the mutant ΔtreS strain overexpressing TreS (via pTE3-treS) accumulated an amount of maltose significantly larger than that of the ΔtreS control strain (pTE3). The single and double otsA mutant strains, the ΔotsA and ΔotsA ΔtreY mutants, respectively, which accumulated relatively low levels of trehalose, also produced relatively less maltose than did the wild-type strain. These results indicated that TreS is likely involved in the degradation of trehalose to maltose in B. japonicum strain USDA 110.

Influence of trehalose on expression of otsA and treS.

Since the OtsA-overexpressing strain did not accumulate much trehalose (Fig. (Fig.3A),3A), our results strongly suggested that the intracellular concentration of trehalose in Bradyrhizobium cells is controlled by the combined action of the trehalose biosynthesis and degradation systems. To determine if genes controlling trehalose synthesis and degradation are regulated by trehalose, real-time qRT-PCR analyses were done using primers specific for otsA and treS transcripts. The absolute expression level of the otsA and treS genes was initially examined with the wild-type B. japonicum strain cultured in AG medium supplemented with 0.1, 1, or 10 mM filter-sterilized trehalose. However, the expression levels of both genes were not different relative to cells grown without trehalose supplementation (data not shown). In contrast, there were significant differences in the expression levels of otsA and treS in strains overexpressing the otsA and treS genes. The expression of otsA in USDA 110(pTE3-otsA) was 119-fold greater than that of the control wild-type strain containing the empty vector, and treS gene expression in wild-type cells overexpressing OtsA (pTE3-otsA) was 1.9-fold greater than that seen with wild-type cells containing pTE3 (Table (Table2).2). In addition, treS gene expression in USDA 110(pTE3-treS) was 300-fold greater than that seen with the control strain, USDA 110(pTE3), and otsA gene expression in wild-type cells overexpressing TreS (pTE3-treS) was 2.8-fold greater than that seen with vector control cells (Table (Table2).2). Results of these studies suggested that expression of the trehalose synthesis gene (otsA) and degradation gene (treS) is likely modulated by the intracellular concentration of trehalose in B. japonicum.

TABLE 2.
qRT-PCR analysis of otsA and treS genes in B. japonicum strains overexpressing trehalose-6-phosphate synthetase and trehalose synthase

Effects of trehalose accumulation on salinity stress survival.

To determine if trehalose accumulation influenced growth of salt-stressed cells, the various trehalose synthesis mutants were grown on agar plates containing AG medium or AG medium containing 60 mM NaCl. While all the tested strains grew well on AG medium without added salt, the single, double, and triple mutant strains lacking the OtsAB pathway (the ΔotsA, ΔotsA ΔtreS or ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants) were inhibited for growth on 60 mM NaCl (Fig. (Fig.4).4). The ΔotsA ΔtreY double mutant and the ΔotsA ΔtreS ΔtreY triple mutant, which lack both the OtsAB and TreYZ pathways, failed to grow on salt-containing medium (Fig. (Fig.4).4). These growth effects were correlated with trehalose accumulation levels (Fig. (Fig.3A)3A) but were not correlated with maltose accumulation levels (Fig. (Fig.3B).3B). These results indicated that trehalose in B. japonicum plays a role as an osmoprotectant for growth under conditions of salt-induced osmotic stress.

FIG. 4.
Influence of salt stress on growth of wild-type B. japonicum USDA 110 and mutants with insertions in trehalose synthesis genes. Wild-type B. japonicum USDA 110 (A) and mutants ΔotsA (B), ΔtreS (C), ΔtreY (D), ΔotsA Δ ...

Survival phenotypes of trehalose biosynthetic gene mutants under desiccation stress.

To examine the effects of trehalose biosynthesis on desiccation stress, Bradyrhizobium cells were filtered onto polycarbonate filters and incubated under conditions of 50% RH. Results depicted in Fig. Fig.55 show that the viable cell numbers of the ΔotsA and ΔotsA ΔtreY mutants, which are low trehalose accumulators, were not different than that found for the wild-type strain. In contrast, the mutants lacking the TreS pathway (the ΔtreS, ΔotsA ΔtreS, ΔtreS ΔtreY, and ΔotsA ΔtreS ΔtreY mutants) survived poorly under desiccation stress conditions relative to the wild-type strain, although the initial cell numbers were not different. The survival of the TreS overexpressing strain, USDA110 (pTE3-treS), was slightly greater than that of the wild-type control strain containing pTE3. Moreover, the decreased survival caused by a lack of the TreS pathway was recovered by complementing the mutant strain with treS. It should be noted, however, that the growth responses of mutant and wild-type strains to desiccation stress were not correlated with intracellular accumulation levels of trehalose or maltose (Fig. (Fig.3).3). Thus, trehalose and maltose accumulation in B. japonicum did not enhance survival against desiccation stress, and trehalose metabolism via the TreS pathway is likely important for survival under desiccating conditions.

FIG. 5.
Viable cell numbers of trehalose synthesis mutants, treS-overexpressing strains, and wild-type B. japonicum after incubation under desiccating condition (50% RH). Plate count data are expressed as log10 CFU/ml for desiccated cells at time of inoculation ...

Symbiotic phenotypes of trehalose synthesis mutants on soybeans.

To determine if trehalose accumulation in B. japonicum influences its ability to form symbiotic interactions with its host legume, soybean (G. max cv. Lambert) seeds were inoculated independently with the ΔotsA ΔtreY, ΔotsA ΔtreS ΔtreY, and ΔtreS mutants and the wild-type strain. Results represented in Table Table33 show that the number of mature nodules produced by the ΔotsA ΔtreY or ΔotsA ΔtreS ΔtreY mutant strains was less than that seen with soybean seeds inoculated with the wild-type strain. However, the mutant strains induced a large number of immature, ineffective nodules on the roots of soybean (Fig. 6A and B; Table Table3).3). The dry masses of soybean plants and nodules produced when plants were inoculated with the ΔotsA ΔtreY and ΔotsA ΔtreS ΔtreY mutants were less than those seen with plants inoculated with the wild-type USDA 110 strain. Moreover, the leaves of soybean plants inoculated with the mutants were chlorotic and looked similar to the uninoculated control plants (Fig. (Fig.6C).6C). Thus, the nitrogen fixation ability of nodules induced by the ΔotsA ΔtreY and ΔotsA ΔtreS ΔtreY mutants was likely severely impaired relative to that found with the wild-type strain. In contrast, the nodulation and nitrogen fixation phenotype of the ΔtreS mutant strain was not different from that of the wild-type strain (Table (Table3).3). These results indicated that trehalose accumulation in B. japonicum is linked to the development of soybean nodules and that TreS-mediated trehalose degradation has a limited role in the symbiotic interaction of this bacterium with its legume host.

FIG. 6.
Symbiotic phenotypes of wild-type and mutant B. japonicum strains inoculated onto G. max cv. Lambert plants. (A) Nodules formed on soybean roots inoculated with the wild-type strain. (B) Mature and immature nodules formed on soybean roots inoculated with ...
TABLE 3.
Nodulation phenotype of Glycine max cv. Lambert plants inoculated with wild-type and trehalose synthesis and degradation mutants of B. japonicum USDA 110a

DISCUSSION

While four trehalose biosynthetic pathways (OtsAB, TreS, TreYZ, and TreT) have thus far been described for prokaryotes, the combination of pathways possessed by a single bacterium appears to be bacterial species dependent. In B. japonicum strain USDA 110, three independent trehalose pathways (OtsAB, TreS, and TreYZ) were detected by enzymatic assays (38), and the otsAB, treS, and treYZ genes have been identified by genome sequence analyses (17). In this study, the presence and role of the putative trehalose biosynthesis genes in B. japonicum USDA 110 were determined using genetic and molecular approaches.

Results of the studies presented here show that disruption of otsA caused a decrease in trehalose accumulation levels in cells, especially those of the otsA treY double mutant, which accumulated extremely low levels of trehalose when grown under conditions of salinity stress. In contrast the single treY mutation had little effect on trehalose accumulation. Thus, trehalose biosynthesis in B. japonicum appears to be mediated mainly by the OtsAB pathway and only marginally by the TreYZ pathway (Fig. (Fig.7).7). Our results also indicated that despite construction of double (ΔotsA ΔtreY) and triple (ΔotsA ΔtreS ΔtreY) mutations in the trehalose biosynthetic genes, low levels of trehalose were still detected with these strains. This is likely due to the existence of other yet-defined or -annotated genes. For example, bll0902 is annotated as treS-like and may play a role in trehalose synthesis in B. japonicum.

FIG. 7.
Proposed trehalose biosynthetic and metabolic pathways in B. japonicum. AglA, alpha-glucosidase; MalQ, 4-alpha-glucanotransferase; OtsA, trehalose-6-phosphate synthase; OtsB, trehalose-phosphatase; TreS, trehalose synthase; TreY, malto-oligosyltrehalose ...

The trehalose biosynthesis and degradation systems present in B. japonicum are similar to those reported for Rhodobacter sphaeroides (19), a bacterium taxonomically related to B. japonicum. Thus, the alphaproteobacteria likely contain evolutionarily conserved systems controlling trehalose levels in cells. In contrast, osmoregulated trehalose biosynthesis in the Gram-positive bacterium Corynebacterium glutamicum is mediated by the TreYZ pathway and not by the OtsAB pathway (43).

The functional redundancy of multiple genes and pathways for trehalose biosynthesis in B. japonicum likely points to the overall importance of this sugar for the survival of this bacterium growing in soils and plants. This redundancy, however, is lacking in several tested fast-growing Rhizobium strains, and only trehalose-6-phosphate activity has been reported for S. meliloti and Mesorhizobium loti (38). However, like B. japonicum, an otsA mutant of R. leguminosarum accumulated relatively less trehalose than did a treY mutant (20). The OtsAB pathway has also been shown to be essential for trehalose biosynthesis in other Gram-negative bacteria, including Escherichia coli and Rhodobacter sphaeroides (14, 19). This suggests that the OtsAB pathway may be a dominant trehalose biosynthetic pathway in Gram-negative bacteria, including rhizobia.

Our studies suggested that the TreS pathway is involved in trehalose catabolism in B. japonicum. The maltose which is produced by the TreS protein in B. japonicum might be further metabolized to glucose by 4-alpha-glucanotransferase (MalQ) and alpha-glucosidase (AglA), which are enzymes catalyzing maltose degradation (Fig. (Fig.7)7) (2, 3). Therefore, it would seem logical that trehalose can be used to support growth of this bacterium. However, earlier studies have shown that B. japonicum cannot utilize trehalose as a sole source of carbon for growth (34), despite the fact that B. japonicum bacteroids have also been reported to have trehalase, an enzyme catalyzing degradation of trehalose into two molecules of glucose (27, 28, 33). Interestingly, strain USDA 110 contains malQ (bll6765) and aglA (blr0901), and these genes, as well as the trehalose biosynthetic genes, were highly induced by desiccation stress (6). However, genome sequence analyses indicated that the B. japonicum USDA 110 genome did not contain homologs of treA and treF, coding for trehalase, and thuAB, which codes for trehalose utilization enzymes. Thus, other studies are needed to fully understand trehalose and maltose metabolism in this bacterium.

Gene expression studies reported here indicate that the trehalose biosynthesis genes otsA, treS, and treY were induced by salinity and desiccation stresses. This correlates well with previous whole-genome microarray data of desiccation-stressed cells (6). However, Chang et al. (4) reported that expression of the trehalose synthesis genes in B. japonicum were not induced by salt stress. This discrepancy is likely due to the fact that different concentrations of NaCl were used in both studies. Our studies also suggested that expression of the otsA and treS genes were likely modulated by the intracellular concentration of trehalose (Table (Table2;2; Fig. Fig.7).7). Although the OtsA-overexpressing strain did not accumulate much trehalose (Fig. (Fig.3A),3A), the expression of the trehalose degradation gene (treS) was induced due to overexpression of the otsA gene. In contrast, overexpression of the treS gene led to repression of the trehalose biosynthetic gene otsA. Interestingly, while our results suggested that trehalose accumulation in the triple mutant should be more than that seen with the double otsA treY mutant, this was not observed (Fig. (Fig.3A).3A). Since TreS utilizes trehalose as its substrate, this may be due in part to a lack of effectiveness of the TreS degradation pathway with the lower levels of trehalose present in the otsA treY mutant strain. Thus, it seems logical that B. japonicum controls trehalose accumulation by modulating gene expression levels. Recently, the PhyR/EcfG regulon was found to be a stress response regulator in B. japonicum USDA 110 (12). The authors of that recent study reported that expression of otsA and treS was downregulated in phyR and ecfG mutants, and this involves an ECF-type sigma factor. A conserved σecfG-type promoter region is located upstream of otsA in USDA 110. Thus, the PhyR/EcfG regulon may also be involved in regulation of trehalose synthase genes in B. japonicum cells growing under physiological stress conditions. In contrast, otsA expression in E. coli is under the control of rpoS, which is the stationary-phase sigma factor (14). Moreover, a deletion mutant of another stationary-phase sigma factor, sigD, in Myxococcus xanthus did not accumulate trehalose in cells and was more sensitive to salinity stress compared to the wild-type strain (40). Since the B. japonicum USDA 110 strain has two copies of sigD, but no rpoS, it is possible that sigD may also modulate expression of the trehalose synthesis genes in this bacterium.

The accumulation of trehalose is a common response to salinity- and desiccation-induced stress in several bacteria (5, 21, 42). Our results also indicate that trehalose accumulation in B. japonicum affects tolerance of salinity stress. Surprisingly, cells of the mutants lacking the trehalose degradation pathway mediated by TreS survived poorly under desiccation stress conditions relative to the wild-type strain. Similarly, mutant strains of Saccharomyces cerevisiae and Corynebacterium glutamicum lacking trehalose degradation activity were severely impaired in their ability to recover from physiological stresses (22, 43). Singer and Lindquist postulated that high concentrations of trehalose inhibited the refolding and reactivation of denatured proteins by molecular chaperones (32). Thus, TreS-mediated trehalose degradation in B. japonicum cells subjected to desiccation stress may improve refolding and reactivation of denatured proteins and play an important role in the bacterium's recovery from dehydrating conditions.

Trehalose accumulation in B. japonicum also positively influenced nodulation of G. max cv. Lambert, since nodulation studies indicated that mutants lacking both the OtsAB and TreYZ pathways induced only a small number of mature nodules on soybean plants (Table (Table3).3). This is similar to what has been reported for the Rhizobium etli-Phaseolus vulgaris symbiotic interaction (39). However, the effect of trehalose accumulation by rhizobial cells on their symbiotic phenotype appears to be dependent on the rhizobial species and host genotype examined and is not consistent across symbiotic systems. For example, R. leguminosarum mutants lacking the OtsAB and TreYZ pathways were reported to form effective nitrogen-fixing nodules on Trifolium repens, although the mutants had enhanced competitiveness relative to the wild-type strain (20). In contrast, thuB mutants, which lacked trehalose catabolism, showed an enhanced competitiveness phenotype only on certain host genotypes in the Sinorhizobium-Medicago symbiotic interaction (1). Similarly, trehalose concentration in bacteroids has been shown to vary in different host genotypes that have been inoculated by Bradyrhizobium strains (34, 38). Taken together, our results indicate that trehalose accumulation in Bradyrhizobium cells influences its symbiosis with soybeans, perhaps by acting as a compatible solute to overcome host-induced osmotic stress that occurs during the nodulation possess.

Supplementary Material

[Supplemental material]

Acknowledgments

This study was supported, in part, by grant 2004-35604-14708 from the USDA/CSREES/NRI and grant 0543238 from the National Science Foundation and by the University of Minnesota Agricultural Experiment Station.

Footnotes

[down-pointing small open triangle]Published ahead of print on 18 December 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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