Functional role of Bradyrhizobium japonicum trehalose biosynthesis and metabolism genes during physiological stress and nodulation

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 DeltaotsA, DeltaotsA DeltatreS and DeltaotsA DeltatreY, and DeltaotsA DeltatreS DeltatreY 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.

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 cassette to create pK18mob-otsA::Ω. Arrows denote open reading frames. (b) treS (blr6767) and its flanking region. The Ω or aph cassette was inserted into the internal BamHI site of treS to create pK18mob-treS::Ω or pSUP-treS::aph, respectively. (c) treY (blr6771) and its flanking region. The tetA gene fragment was inserted into the internal EcoRI site of treY to create pK18mob-treY::tetA. B, BamHI; E, EcoRI; N, NruI.

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 values of trehalose synthesis genes in salinity-stressed cells. Black bars, nonstressed cells; gray bars, salt-stressed cells. (B) Absolute gene expression values of trehalose synthesis genes in desiccated cells. Black bars, nonstressed cells; gray bars, cells after 24 h incubation under desiccation stress; white bars, cells after 72 h incubation under desiccation stress. Error bars indicate standard deviations of data for three technical replicates.

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 cells. Error bars indicate standard deviations of data for three biological replicates.

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 ΔtreS (E), ΔotsA ΔtreY (F), ΔtreS ΔtreY (G), and ΔotsA ΔtreS ΔtreY (H) were inoculated onto AG agar medium and AG medium supplemented with 60 mM NaCl. Plates were photographed after 7 days of growth at 30°C.

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 (white bars) and after growth for 72 h (black bars). Error bars represent the standard deviation of data for three biological replicates.

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 the ΔotsA ΔtreS ΔtreY mutant. (C) Plants were inoculated with wild-type B. japonicum USDA 110, the ΔotsA ΔtreY double mutant, or the ΔotsA ΔtreS ΔtreY triple mutant and analyzed after 31 days. Bars, 0.2 mm.

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 synthase; and TreZ, malto-oligosyltrehalose trehalohydrolase.