ndvB Mutant Research Articles

Bacterial cyclic β-(1,2)-glucans sequester iron to protect against iron-induced toxicity.

Cellular iron homeostasis is critical for survival and growth. Bacteria employ a variety of strategies to sequester iron from the environment and to store intracellular iron surplus that can be utilized in iron-restricted conditions while also limiting the potential for the production of iron-induced reactive oxygen species (ROS). Here, we report that membrane-derived oligosaccharide (mdo) glucan, an intrinsic component of Gram-negative bacteria, sequesters the ferrous form of iron. Iron-binding, uptake, and localization experiments indicated that both secreted and periplasmic β-(1,2)-glucans bind iron specifically and promote growth under iron-restricted conditions. Xanthomonas campestris and Escherichia coli mutants blocked in the production of β-(1,2)-glucan accumulate low amounts of intracellular iron under iron-restricted conditions, whereas they exhibit elevated ROS production and sensitivity under iron-replete conditions. Our results reveal a critical role of glucan in intracellular iron homeostasis conserved in Gram-negative bacteria.

High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: the ndvB gene is involved in the production of highly glycerol-phosphorylated -(1->3)-glucans, which bind aminoglycosides

Pseudomonas aeruginosa is an opportunistic pathogen that affects immunocompromised individuals and causes life-threatening infections in cystic fibrosis (CF) patients. Colonization of CF lung by P. aeruginosa involves a biofilm mode of growth, which is promoted by the production of exopolysaccharides. These polymers are essential components of the extracellular biofilm matrix. P. aeruginosa possesses several clusters contributing to the formation of the matrix, including the pel or psl genes. In the present study, we identified anionic cyclic glucans produced by P. aeruginosa, which are associated with the matrix of strains PAKDeltaretS and PA14. Their structure has been elucidated using chemical analysis, 1- and 2D nuclear magnetic resonance techniques and mass spectrometry. They belong to a family of cyclic beta-(1-->3)-linked glucans of 12-16 glucose residues with 30-50% of glucose units substituted by 1-phosphoglycerol at O-6. These glucans were also recovered in pel mutant strains, which indicated that their biosynthesis was pel independent. In an effort to understand the biogenesis of these glucans, we analyzed the matrix components of a previously characterized P. aeruginosa PA14 mutant, the PA14::ndvB mutant strain. The ndvB gene was predicted to be involved in the synthesis of perisplasmic glucans, capable of physically interacting with aminoglycoside antibiotics. We revealed that the highly glycerol-phosphorylated beta-(1-->3)-glucans are lacking in the ndvB mutant, and we showed that these glucans are capable of direct binding with the aminoglycoside antibiotic kanamycin. This observation fills a gap in our understanding of the relationship between biofilm, cyclic glucans and high-level antibiotic resistance.

Bacterial Cyclic β-(1,2)-Glucan Acts in Systemic Suppression of Plant Immune Responses

Although cyclic glucans have been shown to be important for a number of symbiotic and pathogenic bacterium-plant interactions, their precise roles are unclear. Here, we examined the role of cyclic beta-(1,2)-glucan in the virulence of the black rot pathogen Xanthomonas campestris pv campestris (Xcc). Disruption of the Xcc nodule development B (ndvB) gene, which encodes a glycosyltransferase required for cyclic glucan synthesis, generated a mutant that failed to synthesize extracellular cyclic beta-(1,2)-glucan and was compromised in virulence in the model plants Arabidopsis thaliana and Nicotiana benthamiana. Infection of the mutant bacterium in N. benthamiana was associated with enhanced callose deposition and earlier expression of the PATHOGENESIS-RELATED1 (PR-1) gene. Application of purified cyclic beta-(1,2)-glucan prior to inoculation of the ndvB mutant suppressed the accumulation of callose deposition and the expression of PR-1 in N. benthamiana and restored virulence in both N. benthamiana and Arabidopsis plants. These effects were seen when cyclic glucan and bacteria were applied either to the same or to different leaves. Cyclic beta-(1,2)-glucan-induced systemic suppression was associated with the transport of the molecule throughout the plant. Systemic suppression is a novel counterdefensive strategy that may facilitate pathogen spread in plants and may have important implications for the understanding of plant-pathogen coevolution and for the development of phytoprotection measures.

A motility revertant of the ndvB mutant of Bradyrhizobium japonicum.

A motility revertant of a Bradyrhizobium japonicum ndvB mutant was isolated and characterized. The ndvB mutants of B. japonicum have been reported to be osmotically sensitive, as well as defective in motility, periplasmic cyclic beta-(1-->3), (1-->6)-D-glucan synthesis, and symbiosis with soybean. The motility revertant was restored for osmotic tolerance but not for cyclic beta-glucan production or effective symbiosis. These results support our hypothesis that cyclic beta-glucans have an important role in symbiosis--the suppression of a plant defense response--in addition to their role in periplasmic osmoprotection.

Osmotic regulation of cyclic 1,2-beta-glucan synthesis.

In contrast to what happens in AGROBACTERIUM: tumefaciens and RHIZOBIUM: meliloti, synthesis of periplasmic cyclic 1,2-beta-glucan in BRUCELLA: spp. was not inhibited when bacteria were grown in media of high osmolarity. Studies performed with crude membrane preparations showed that cyclic 1,2-beta-glucan synthetase of BRUCELLA: spp. was not inhibited by 0.5 M KCl or potassium glutamate; concentrations that completely inhibit the osmosensitive enzymes of A. tumefaciens A348 or R. meliloti 102F34, respectively encoded by the chvB or ndvB genes. The BRUCELLA: abortus cyclic 1, 2-beta-glucan synthetase gene (cgs) was introduced into A. tumefaciens A1011 chvB and R. meliloti GRT21s ndvB mutants. Synthesis of cyclic 1,2-beta-glucan by the recombinant strains was not inhibited when grown in media of high osmolarity (0.25 M NaCl or 0.5 M mannitol). On the other hand, when the A. tumefaciens cyclic 1, 2-beta-glucan synthetase gene was introduced into the R. meliloti GRT21s ndvB mutant, the recombinant strain displayed marked inhibition of cyclic 1,2-beta-glucan synthesis when grown in high-osmolarity media. However, the same gene introduced into a B. abortus cgs mutant background resulted in no inhibition of glucan synthesis at high osmolarity. In vitro studies with crude membranes isolated from recombinant strains revealed that BRUCELLA: cyclic 1, 2-beta-glucan synthetase was not inhibited by high concentrations of KCl or potassium glutamate even when expressed in AGROBACTERIUM: or RHIZOBIUM: backgrounds. It was concluded that the lack of effect of high osmolarity on 1,2-beta-glucan synthesis in BRUCELLA: is due to two convergent mechanisms: a) the presence of a cyclic 1, 2-beta-glucan synthetase that is not affected by concentrations of solutes such as KCl or potassium glutamate and b) either the possible accumulation of compatible solutes that might protect the enzyme from the inhibition by potassium glutamate or the accumulation of other osmolytes that do not affect the 1, 2-beta-glucan synthetase.

Effects of Ionic and Osmotic Strength on the Glucosyltransferase of Rhizobium meliloti Responsible for Cyclic beta-(1,2)-Glucan Biosynthesis.

The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.

Cyclolaminarinose. A new biologically active β-(1 → 3) cyclic glucan

A unique glucan has been isolated from a recombinant strain of a Rhizobium meliloti TY7, a cyclic β-(1 → 2) glucan mutant carrying a locus specifying β-(1 → 3; 1 → 6) glucan synthesis from Bradyrhizobium japonicum USDA110. This compound, which appears to have considerable hydrophobic affinity, was separated from a perchloric acid cell extract by adsorption to a C-18 silica column. Unlike those cyclic glucans previously isolated from Rhizobium meliloti or Bradyrhizobium japonicum, this molecule contains neither phosphoglycerol nor phosphocholine substituents, respectively. 2D NMR, FAB mass spectrometric analysis and high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) confirmed that this glucan is a single, cyclic decasaccharide (cyclolaminarinose) in which one of the residues is substituted in its 6-position with β-laminarabiose. This structural assignment was confirmed by mass spectral and NMR analyses of the product obtained from two consecutive Smith degradations. Unlike the complex 13C spectrum of the unoxidized material, the spectrum of this product consisted of only six resonances due to rapid time averaging of its symmetrical structure on the relatively slow NMR timescale. Synthesis of this newly described cyclic β-glucan in the R. meliloti ndvB mutant restored the symbiotic and hypoosmotic adaptation characteristics of the R. meliloti wild type strain.

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.

Nodule development induced by mutants of Bradyrhizobium japonicum defective in cyclic B-glucan synthesis.

The soybean response to inoculation with B. japonicum mutants defective in cyclic beta-(1-->3), beta-(1-->6) glucan synthesis was examined by electron microscopy and by monitoring the expression of early and late nodulin genes. Two mutants were examined. Strain AB-14 is an ndvB mutant and is unable to synthesize beta-glucans. Strain AB-1 is an ndvC mutant and produces cyclic glucans containing 95 to 100% beta-(1-->3) glycosidic linkages. Nodules formed by either mutant were defective in nitrogen fixation activity. Soybean plants inoculated with strain AB-14 formed nodules roughly at the same rate as the wild-type strain USDA110, but nodulation by strain AB-1 was significantly delayed. Microscopy of nodules formed by strain AB-14 showed an overall ultrastructure similar to nodules formed by the wild type. However, in some nodules bacteroids were limited to only one part of the infected cells and in others the few bacteroids present showed signs of degradation. Nodulelike structures were formed by strain AB-1 that showed some signs of cellular differentiation. For example, clear parenchyma and sclerenchyma tissue could be seen. However, no infection threads or bacteria were evident in these structures. The expression of early (e.g., ENOD2 and ENOD55) and late (e.g., NOD26 and leghemoglobin) nodulins was examined. Nodules formed by strain AB-14 expressed all of the nodulins tested but at a reduced level. Expression of late nodulins was delayed in strain AB-14-induced nodules. Nodules formed by strain AB-1 were more strongly affected in nodulin expression. Although leghemoglobin synthesis was not detected, infection-specific nodulin transcripts of GmN93 and ENOD55 were detected. However, expression of the early nodulins ENOD2 and ENOD55 was considerably delayed and only apparent when assayed 21 days postinoculation. A low level of expression of intermediate nodulin GmN70 and late nodulin NOD26 could also be detected by 21 days postinoculation. The microscopy data show that tissue differentiation occurs in these nodules even in the absence of active bacterial invasion. These results demonstrate the importance of cyclic beta-(1-->3), beta-(1-->6) glucan synthesis to symbiotic development in soybean.

Isolation and characterization of an ndvB locus from Rhizobium fredii.

A gene (ndvB) in Rhizobium meliloti that is essential for nodule development in Medicago sativa (alfalfa), specifies synthesis of a large membrane protein. This protein appears to be an intermediate in beta-1,2-glucan synthesis by the microsymbiont. Southern hybridization analysis showed strong homology between an ndvB (chvB) probe and genomic DNA of R. fredii but not from Bradyrhizobium japonicum. A cosmid clone containing the putative ndvB locus was isolated from a Rhizobium fredii gene library. The cosmid clone which complemented R. meliloti ndvB mutants for synthesis of beta-1,2-glucans and effective nodulation of alfalfa was mapped and subcloned. Fragment-specific Tn5 mutagenesis followed by homologous recombination into the R. fredii genome indicated that the region was essential for beta-1,2-glucan synthesis and for formation of an effective symbiosis with Glycine max (soybean).

Suppression of the ndv mutant phenotype of Rhizobium meliloti by cloned exo genes

The ndvA and ndvB genes of Rhizobium meliloti are involved in the export and synthesis, respectively, of the small cyclic polysaccharide beta(1,2)glucan. We have previously shown that spontaneous symbiotic pseudorevertants of ndv mutants do not produce periplasmic beta(1,2)glucan. Here we show that the pseudorevertants also do not produce extracellular beta(1,2)glucan, but do show alterations in the amount of the major acidic exopolysaccharide produced. This exopolysaccharide is not detectably different from that produced by the wild type or by the ndv mutants. A cosmid which suppresses the symbiotic defect of both ndvA and ndvB mutants was isolated from a gene bank prepared from DNA of an ndvA pseudorevertant. This cosmid contains a number of exo genes, including exoH and exoF. Subcloning and Tn5 mutagenesis were used to show that the widely separated exoH and exoF genes are both involved in suppression of the ndv mutant phenotype and that the 3.5 kb DNA fragment which contains the exoH gene does not carry the mutation responsible for second site suppression.

An Osmorevertant of aRhizobium meliloti ndvBDeletion Mutant Forms Infection Threads but is Defective in Bacteroid Development

A defined deletion mutant in the ndvB locus of Rhizobium meliloti that was identified by its Inf- phenotype lacked the beta-1,2-glucans and showed osmotic instability. The Inf- phenotype was restored by a spontaneous mutation causing osmotic tolerance. This osmorevertant still lacked the beta-1,2-glucans but induced Fix- nodules containing infection threads. Release of bacteria from the infection thread was observed, but bacterial multiplication in the plant cell and development into bacteroids was blocked. In contrast to the original ndvB mutant, the osmorevertant was able to establish Fix+ nodules following mixed inoculation with a Nod- mutant. Thus, osmotic intolerance seems to be the reason for the Inf- phenotype of ndvB mutants. Reisolation experiments revealed that Fix+ nodules were occupied mainly by the Nod- mutant, indicating that the partially osmotic-tolerant osmorevertant still lacked functions that could not be complemented on the cellular level.

Expression of nodule-specific genes in alfalfa root nodules blocked at an early stage of development.

To help dissect the molecular basis of the Rhizobium-legume symbiosis, we used in vitro translation and Northern blot analysis of nodule RNA to examine alfalfa-specific genes (nodulins) expressed in two types of developmentally defective root nodules elicited by Rhizobium meliloti. Fix- nodules were elicited by R. meliloti nif mutants; these nodules were invaded by rhizobia and contained differentiated bacteroids. 'Empty' nodules were elicited by R. meliloti exo and ndv mutants and by Agrobacterium tumefaciens strains carrying the R. meliloti nod genes; these nodules contained a nodule meristem but lacked infection threads, intracellular bacteria, and bacteroids. Fix- nodules contained a spectrum of nodulins similar to wild-type nodules. In contrast, only two nodulins, Nms-30 and a nodulin homologous to ENOD2 of soybean, were detected in empty nodules. Although R. meliloti ndv and exo mutants elicited nodules with the same defective phenotype, ndv and exo mutants (except for exoC mutants) had distinct biochemical phenotypes. R. meliloti ndvA and ndvB mutants were deficient in cyclic glucan production but not the acidic exopolysaccharide; the converse was true for exoA, exoB, and exoF mutants. exoC mutants were defective in both exopolysaccharide and cyclic glucan biosynthesis. Our results support the model that the R. meliloti nod genes produce a signal that results in nodule meristem induction. Both the exopolysaccharide and cyclic glucan, however, appear to act at the next step in the developmental process and are involved in the production of a signal (or structure) that allows infection thread formation and invasion of the nodule.