Extracellular glycanases of Rhizobium leguminosarum are activated on the cell surface by an exopolysaccharide-related component

Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS(+) strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn of R. leguminosarum. This indicates that the surface of A. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.     

Proteins exported via the PrsD-PrsE type I secretion system and the acidic exopolysaccharide are involved in biofilm formation by Rhizobium leguminosarum

The type I protein secretion system of Rhizobium leguminosarum bv. viciae encoded by the prsD and prsE genes is responsible for secretion of the exopolysaccharide (EPS)-glycanases PlyA and PlyB. The formation of a ring of biofilm on the surface of the glass in shaken cultures by both the prsD and prsE secretion mutants was greatly affected. Confocal laser scanning microscopy analysis of green-fluorescent-protein-labeled bacteria showed that during growth in minimal medium, R. leguminosarum wild type developed microcolonies, which progress to a characteristic three-dimensional biofilm structure. However, the prsD and prsE secretion mutants were able to form only an immature biofilm structure. A mutant disrupted in the EPS-glycanase plyB gene showed altered timing of biofilm formation, and its structure was atypical. A mutation in an essential gene for EPS synthesis (pssA) or deletion of several other pss genes involved in EPS synthesis completely abolished the ability of R. leguminosarum to develop a biofilm. Extracellular complementation studies of mixed bacterial cultures confirmed the role of the EPS and the modulation of the biofilm structure by the PrsD-PrsE secreted proteins. Protein analysis identified several additional proteins secreted by the PrsD-PrsE secretion system, and N-terminal sequencing revealed peptides homologous to the N termini of proteins from the Rap family (Rhizobium adhering proteins), which could have roles in cellular adhesion in R. leguminosarum. We propose a model for R. leguminosarum in which synthesis of the EPS leads the formation of a biofilm and several PrsD-PrsE secreted proteins are involved in different aspects of biofilm maturation, such as modulation of the EPS length or mediating attachment between bacteria.     

PssP2 Is a Polysaccharide Co-Polymerase Involved in Exopolysaccharide Chain-Length Determination in Rhizobium leguminosarum

Production of extracellular polysaccharides is a complex process engaging proteins localized in different subcellular compartments, yet communicating with each other or even directly interacting in multicomponent complexes. Proteins involved in polymerization and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum are encoded within the chromosomal Pss-I cluster. However, genes implicated in polysaccharide synthesis are common in rhizobia, with several homologues of pss genes identified in other regions of the R. leguminosarum genome. One such region is chromosomally located Pss-II encoding proteins homologous to known components of the Wzx/Wzy-dependent polysaccharide synthesis and transport systems. The pssP2 gene encodes a protein similar to polysaccharide co-polymerases involved in determination of the length of polysaccharide chains in capsule and O-antigen biosynthesis. In this work, a mutant with a disrupted pssP2 gene was constructed and its capabilities to produce EPS and enter into a symbiotic relationship with clover were studied. The pssP2 mutant, while not altered in lipopolysaccharide (LPS), displayed changes in molecular mass distribution profile of EPS. Lack of the full-length PssP2 protein resulted in a reduction of high molecular weight EPS, yet polymerized to a longer length than in the RtTA1 wild type. The mutant strain was also more efficient in symbiotic performance. The functional interrelation between PssP2 and proteins encoded within the Pss-I region was further supported by data from bacterial two-hybrid assays providing evidence for PssP2 interactions with PssT polymerase, as well as glycosyltransferase PssC. A possible role for PssP2 in a complex involved in EPS chain-length determination is discussed.     

Membrane cholesterol and sphingomyelin,and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein,pleurotolysin B

The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.     

The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification

NodO is a secreted protein from Rhizobium leguminosarum bv. viciae with a role in signalling during legume nodulation. A Tn5-induced mutant was identified that was defective in NodO secretion. As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated. This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules. In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics. Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype. The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids. The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length. The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity. DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa. An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R. leguminosarum bv. viciae in a prsD-dependent manner. We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.     

psi, a plasmid-linked Rhizobium phaseoli gene that inhibits exopolysaccharide production and which is required for symbiotic nitrogen fixation

Summary A strain of R. phaseoli cured of its symbiotic plasmid, pRP2JI, retained the ability to make exopolysaccharide (EPS). However, a region of pRP2JI, when cloned at an increased copy number in wide host-range vectors and transferred to this and other strains of Rhizobium, inhibited EPS synthesis. The gene responsible was termed psi (polysaccharide inhibition) and was located in a region of the symbiotic plasmid close to nodulation and nitrogen fixation genes. psi is important in the symbiosis since a wild-type strain containing psi cloned on a multicopy plasmid failed to form Phaseolus nodules, and mutant strains containing psi::Tn5 mutations failed to fix nitrogen in Phaseolus nodules. It is proposed that the function of psi may be to repress in the bacteriod the expression of genes such as those for EPS synthesis which are normally expressed in free-living culture.     

NopC Is a Rhizobium-Specific Type 3 Secretion System Effector Secreted by Sinorhizobium (Ensifer) fredii HH103

Sinorhizobium (Ensifer) fredii HH103 is a broad host-range nitrogen-fixing bacterium able to nodulate many legumes, including soybean. In several rhizobia, root nodulation is influenced by proteins secreted through the type 3 secretion system (T3SS). This specialized secretion apparatus is a common virulence mechanism of many plant and animal pathogenic bacteria that delivers proteins, called effectors, directly into the eukaryotic host cells where they interfere with signal transduction pathways and promote infection by suppressing host defenses. In rhizobia, secreted proteins, called nodulation outer proteins (Nops), are involved in host-range determination and symbiotic efficiency. S. fredii HH103 secretes at least eight Nops through the T3SS. Interestingly, there are Rhizobium-specific Nops, such as NopC, which do not have homologues in pathogenic bacteria. In this work we studied the S. fredii HH103 nopC gene and confirmed that its expression was regulated in a flavonoid-, NodD1- and TtsI-dependent manner. Besides, in vivo bioluminescent studies indicated that the S. fredii HH103 T3SS was expressed in young soybean nodules and adenylate cyclase assays confirmed that NopC was delivered directly into soybean root cells by means of the T3SS machinery. Finally, nodulation assays showed that NopC exerted a positive effect on symbiosis with Glycine max cv. Williams 82 and Vigna unguiculata. All these results indicate that NopC can be considered a Rhizobium-specific effector secreted by S. fredii HH103.     

Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation

 The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycinemax [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5–6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation. Received: 6 October 1999 / Accepted: 18 November 1999     

Expression in Myxococcus xanthus of foreign genes coding for secreted pectate lyases of Erwinia chrysanthemi

Structural genes for the five major pectate lyases of Erwinia chrysanthemi, pelA, pelB, pelC, pelD and pelE, have been transferred and expressed in Myxococcus xanthus. These proteins, which are secreted by their original host, are also produced, mainly into the medium, by Myxococcus. These results are discussed in the context of current knowledge about secretion in bacteria.     

Exopolysaccharide biosynthesis is important for <Emphasis Type="Italic">Mesorhizobium tianshanense</Emphasis>: plant host interaction

Mesorhizobium tianshanense is a nitrogen-fixing bacterium that can establish symbiotic associations with Glycyrrhiza uralensis in the form of root nodules. Nodule formation in rhizobia often requires various secreted carbohydrates. To investigate exopolysaccharide (EPS) production and function in M. tianshanense, we performed a genome-wide screen using transposon mutagenesis to identify genes involved in EPS production. We identified seven mutants that produced significantly lower amounts of EPS as well as a two-component sensor kinase/response regulator system that is involved in the activation of EPS synthesis. EPS mutants formed significantly less biofilm and displayed severely reduced nodulation capacity than wild type bacteria, suggesting that EPS synthesis can play important roles in the symbiosis process. Peng Wang, Zengtao Zhong and Jing Zhou have contributed equally to this work.     

Mini-TnhlyAs: a new tool for the construction of secreted fusion proteins

A simple and efficient procedure for the construction of secreted fusion proteins inEscherichia coli is described that uses a new minitransposon, termed TnhlyAs, carrying the secretion signal (HlyAs) ofE. coli hemolysin (HlyA). This transposon permits the generation of random gene fusions encoding proteins that carry the HlyAs at their C-termini. For the construction of model gene fusions we usedlacZ, encoding the cytoplasmicβ-galactosidase (β-Gal), andphoA, encoding the periplasmic alkaline phosphatase, as target genes. Our data suggest that allβ-Gal-HlyAs fusion proteins generated are secreted, albeit with varying efficiencies, by the HlyB/HlyD/TolC hemolysin secretion machinery under Sec-proficient conditions. In contrast, the PhoA-HlyAs fusion proteins are efficiently secreted in asecA mutant strain only under SecA-deficient conditions.     

Binding of a pleurotolysin ortholog from Pleurotus eryngii to sphingomyelin and cholesterol-rich membrane domains

A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus, pleurotolysin (Ply)A, from the extract of edible mushroom Pleurotus eryngii, named PlyA2. Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.     

Characterization of NopP, a type III secreted effector of Rhizobium sp. strain NGR234

The type three secretion system (TTSS) encoded by pNGR234a, the symbiotic plasmid of Rhizobium sp. strain NGR234, is responsible for the flavonoid- and NodD1-dependent secretion of nodulation outer proteins (Nops). Abolition of secretion of all or specific Nops significantly alters the nodulation ability of NGR234 on many of its hosts. In the closely related strain Rhizobium fredii USDA257, inactivation of the TTSS modifies the host range of the mutant so that it includes the improved Glycine max variety McCall. To assess the impact of individual TTSS-secreted proteins on symbioses with legumes, various attempts were made to identify nop genes. Amino-terminal sequencing of peptides purified from gels was used to characterize NopA, NopL, and NopX, but it failed to identify SR3, a TTSS-dependent product of USDA257. By using phage display and antibodies that recognize SR3, the corresponding protein of NGR234 was identified as NopP. NopP, like NopL, is an effector secreted by the TTSS of NGR234, and depending on the legume host, it may have a deleterious or beneficial effect on nodulation or it may have little effect.     

PssO, a unique extracellular protein important for exopolysaccharide synthesis in Rhizobium leguminosarum bv. trifolii

Synthesis and secretion of polysaccharides by Gram-negative bacteria are a result of a concerted action of enzymatic and channel-forming proteins localized in different compartments of the cell. The presented work comprises functional characterization of PssO protein encoded within the previously identified, chromosomal exopolysaccharide (EPS) biosynthesis region (Pss-I) of symbiotic bacterium Rhizobium leguminosarum bv. trifolii TA1 (RtTA1). pssO gene localization between pssN and pssP genes encoding proteins engaged in exopolysaccharide synthesis and transport, suggested its role in EPS synthesis and/or secretion. RtTA1 pssO deletion mutant and the PssO protein overproducing strains were constructed. The mutant strain was EPS-deficient, however, this mutation was not complemented. The PssO-overproducing strain was characterized by increase in EPS secretion. Subcellular fractionation, pssO-phoA/lacZ translational fusion analyses and immunolocalisation of PssO on RtTA1 cell surface by electron microscopy demonstrated that PssO is secreted to the extracellular medium and remains attached to the cell. Western blotting analysis revealed the presence of immunologically related proteins within the species R. leguminosarum bv. trifolii, bv. viciae and Rhizobium etli. The secondary structure of PssO-His(6), as determined by FTIR spectroscopy, consists of at least 32% alpha-helical and 12% beta-sheet structures. A putative function of PssO in EPS synthesis and/or transport is discussed in the context of its cellular localization and the phenotypes of the deletion mutant and pssO-overexpressing strain.     

Functional Analysis of NopM, a Novel E3 Ubiquitin Ligase (NEL) Domain Effector of Rhizobium sp. Strain NGR234

Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant''s defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.     

Disruption of PMR1, Encoding a Ca2+-ATPase Homolog in Yarrowia lipolytica, Affects Secretion and Processing of Homologous and Heterologous Proteins

The Yarrowia lipolytica PMR1 gene (YlPMR1) is a Saccharomyces cerevisiae PMR1 homolog which encodes a putative secretory pathway Ca²⁺-ATPase. In this study, we investigated the effects of a YlPMR1 disruption on the processing and secretion of native and foreign proteins in Y. lipolytica and found variable responses by the YlPMR1-disrupted mutant depending on the protein. The secretion of 32-kDa mature alkaline extracellular protease (AEP) was dramatically decreased, and incompletely processed precursors were observed in the YlPMR1-disrupted mutant. A 36- and a 52-kDa premature AEP were secreted, and an intracellular 52-kDa premature AEP was also detected. The acid extracellular protease activity of the YlPMR1-disrupted mutant was increased by 60% compared to that of the wild-type strain. The inhibitory effect of mutations in secretory pathway Ca²⁺-ATPase genes on the secretion of rice α-amylase was also observed in the Y. lipolytica and S. cerevisiae PMR1-disrupted mutants. Unlike rice α-amylase, the secretion of Trichoderma reesei endoglucanase I (EGI) was not influenced by the YlPMR1 disruption. However, the secreted EGI from the YlPMR1-disrupted mutant had different characteristics than that of the control. While wild-type cells secreted the hyperglycosylated form of EGI, hyperglycosylation was completely absent in the YlPMR1-disrupted mutant. Our results indicate that the effects of the YlPMR1 disruption as manifested by the phenotypic response depend on the characteristics of the reporter protein in the recombinant yeast strain evaluated.     

Exopolysaccharide (EPS) synthesis in Bradyrhizobium japonicum : sequence, operon structure and mutational analysis of an exo gene cluster

The nucleotide sequence of a 8330-bp DNA fragment from Bradyrhizobium japonicum 110spc4 was determined. Sequence analysis revealed that six ORFs were present and the deduced amino acid sequences were homologous to enzymes involved in exopolysaccharide (EPS) biosynthesis. The genes appear to be organized into at least four different operons. One gene was found to be homologous to exoB, which encodes a UDP-galactose 4′-epimerase. Other ORFs were homologous to UDP-hexose transferases and one ORF showed similarity to Sinorhizobium (Rhizobium) meliloti ExoP, which has been suggested to be involved in EPS chain-length determination. A set of deletion and insertion mutants was constructed and the resulting B. japonicum strains were tested for their symbiotic traits. Deletion mutant ΔP22, which lacks the C-terminal part of ExoP, the UDP-hexose transferase ExoT and the N-terminal part of ExoB, shows a delayed nodulation phenotype and induces symptoms of plant defense reactions; its EPS does not contain galactose and no high molecular weight fraction is synthesized. In contrast, insertion mutant EH3, which expresses an exoP gene product that is truncated in its putative periplasmic domain, produced an EPS containing both HMW and LMW fractions. However, the interaction of EH3 with soybeans was severely perturbed. As a rule, only the initial steps of nodule formation were observed.