Characterization of Rhizobium leguminosarum Exopolysaccharide Glycanases That Are Secreted via a Type I Exporter and Have a Novel Heptapeptide Repeat Motif

The prsDE genes encode a type I protein secretion system required for the secretion of the nodulation protein NodO and at least three other proteins from Rhizobium leguminosarum bv. viciae. At least one of these proteins was predicted to be a glycanase involved in processing of bacterial exopolysaccharide (EPS). Two strongly homologous genes (plyA and plyB) were identified as encoding secreted proteins with polysaccharide degradation activity. Both PlyA and PlyB degrade EPS and carboxymethyl cellulose (CMC), and these extracellular activities are absent in a prsD (protein secretion) mutant. The plyA gene is upstream of prsD but appears to be expressed at a very low level (if at all) in cultured bacteria. A plyB::Tn5 mutant has a very large reduction in degradation of EPS and CMC. Cultures of plyB mutants contained an increased ratio of EPS repeat units to reducing ends, indicating that the EPS was present in a longer-chain form, and this correlated with a significant increase in culture viscosity. Thus, PlyB may play a role in processing of EPS. Analysis of the symbiotic properties of a plyA plyB double mutant revealed that these genes are not required for symbiotic nitrogen fixation and that nodulation was not significantly affected. PlyA and PlyB are similar to bacterial and fungal polysaccharide lyases; they contain 10 copies of what we propose as a novel heptapeptide repeat motif that may constitute a fold similar to that found in the family of extracellular pectate lyases. PlyA and PlyB lack the Ca²⁺-binding RTX nonapeptide repeat motifs usually found in proteins secreted via type I systems. We propose that PlyA and PlyB are members of a new family of proteins secreted via type I secretion systems and that they are involved in processing of EPS.     

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.     

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.     

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.     

Cloning and characterization of a FAD-monooxygenase gene ( cadA) involved in degradation of chloranilic acid (2,5-dichloro-3,6-dihydroxybenzo-1,4-quinone) in Pseudomonas putida TQ07

A bacterium culture was isolated on the basis of its ability to degrade chloranilic acid, and was later identified as Pseudomonas putida (TQ07). Several transposon insertion mutants unable to degrade chloranilic acid were selected. The characterization of the site of insertion of one of these mutants led to the identification of the cadA gene encoding an enzyme with significant homology with FAD-monooxygenases involved in the degradation of aromatic and chloroaromatic compounds. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of "halo" formation (a zone of clearing color on agar plates around TQ07 colonies that degrade chloranilic acid) and degradation of chloranilic acid, unequivocally assigned cadA a function in the metabolism of this compound. We also found that most of the transposon insertion mutants unable to degrade chloranilic acid are clustered in a 10-kb region of the P. putida genome that is encoded in a megaplasmid or in an unstable chromosomal region. Electronic Publication     

Solid media containing carboxymethylcellulose to detect CX cellulose activity of micro-organisms.

Solid media containing carboxymethyl cellulose (CMC) were developed to detect CX cellulose-producing micro-organisms. Hydrolysis of CMC was seen as a clear zone around colonies after flooding plates with 1% aqueous hexadecyltrimethyl-ammonium bromide. Tests with ten bacterial and four fungal species showed that the degree of substitution (DS) of the CMC affects both growth and enzyme production. Most of the organisms produced more CX cellulase on CMC with a DS of 0-9, but CMC with a DS of 0-4 was better for one fungus. A qualitative measure of cellulase production may be obtained by calculating the ratio of zone size to colony diameter. Solid media containing CMC provided a more rapid assay of CX cellulose production than a medium containing native cellulose.     

Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Degradation of cellulose by the major endoglucanase produced from the brown-rot fungus Fomitopsis pinicola

An endoglucanase that is able to degrade both crystalline and amorphous cellulose was purified from the culture filtrates of the brown-rot fungus Fomitopsis pinicola grown on cellulose. An apparent molecular weight of the purified enzyme was approximately 32 kDa by SDS-PAGE analysis. The enzyme was purified 11-fold with a specific activity of 944 U/mg protein against CMC. The partial amino acid sequences of the purified endoglucanase had high homology with endo-beta-1,4-glucanase of glycosyl hydrolase family 5 from other fungi. The K(m) and K(cat)values for CMC were 12 mg CMC/ml and 670/s, respectively. The purified EG hydrolyzed both cellotetraose (G4) and cellopentaose (G5), but did not degrade either cellobiose (G2) or cellotriose (G3).     

Polysaccharide synthesis in relation to nodulation behavior of Rhizobium leguminosarum.

In this study, we characterized four Tn5 mutants derived from Rhizobium leguminosarum RBL5515 with respect to synthesis and secretion of cellulose fibrils, extracellular polysaccharides (EPS), capsular polysaccharides, and cyclic beta-(1,2)-glucans. One mutant, strain RBL5515 exo-344::Tn5, synthesizes residual amounts of EPS, the repeating unit of which lacks the terminal galactose molecule and the substituents attached to it. On basis of the polysaccharide production pattern of strain RBL5515 exo-344::Tn5, the structural features of the polysaccharides synthesized, and the results of an analysis of the enzyme activities involved, we hypothesize that this strain is affected in a galactose transferase involved in the synthesis of EPS only. All four mutants failed to nodulate plants belonging to the pea cross-inoculation group; on Vicia sativa they induced root hair deformation and rare abortive infection threads. All of the mutants appeared to be pleiotropic, since in addition to defects in the synthesis of EPS, lipopolysaccharide, and/or capsular polysaccharides significant increases in the synthesis and secretion of cyclic beta-(1,2)-glucans were observed. We concluded that it is impossible to correlate a defect in the synthesis of a particular polysaccharide with nodulation characteristics.     

Regulation of colony morphology and biofilm formation in Shewanella algae

Bacterial colony morphology can reflect different physiological stages such as virulence or biofilm formation. In this work we used transposon mutagenesis to identify genes that alter colony morphology and cause differential Congo Red (CR) and Brilliant Blue G (BBG) binding in Shewanella algae, a marine indigenous bacterium and occasional human pathogen. Microscopic analysis of colonies formed by the wild-type strain S. algae CECT 5071 and three transposon integration mutants representing the diversity of colony morphotypes showed production of biofilm extracellular polymeric substances (EPS) and distinctive morphological alterations. Electrophoretic and chemical analyses of extracted EPS showed differential patterns between strains, although the targets of CR and BBG binding remain to be identified. Galactose and galactosamine were the preponderant sugars in the colony biofilm EPS of S. algae. Surface-associated biofilm formation of transposon integration mutants was not directly correlated with a distinct colony morphotype. The hybrid sensor histidine kinase BarA abrogated surface-associated biofilm formation. Ectopic expression of the kinase and mutants in the phosphorelay cascade partially recovered biofilm formation. Altogether, this work provides the basic analysis to subsequently address the complex and intertwined networks regulating colony morphology and biofilm formation in this poorly understood species.     

A Confocal microscopic study on colony morphology and sporulation of Bacillus sp.

Hexavalent chromium Cr(VI) is one of the dominant oxidation states of chromium that exist in the environment and is highly toxic to all forms of life. In the present study, we employ a confocal laser scanning microscope (CLSM) and investigate the effect of Cr(VI) on colony morphology of a Bacillus sp. isolated from soil exposed to tannery effluent. The colonies grown at chromium concentrations, control and 100 ppm are found to be opaque and beyond 200 ppm the colonies were translucent thus exhibiting phase variation. CLSM studies on colonies grown on control plates showed significant increase in height and in biovolume as a function of time whereas, the translucent colonies showed very little change in height and biovolume corresponding to the colony growth. Exopolymeric substance (EPS) content of translucent colonies was lesser than that of opaque colonies, indicating that EPS also plays a role in the observed phenomenon of phase variation. Studies on the effect of Cr(VI) on spore formation showed that Cr(VI) concentrations up to 100 ppm favored spore formation, while concentrations beyond 100 ppm showed a steady decline in spore formation.     

The Rhizobium meliloti exoK gene and prsD/prsE/exsH genes are components of independent degradative pathways which contribute to production of low-molecular-weight succinoglycan

When grown on medium supplemented with the succinoglycan-binding dye, Calcofluor, and visualized under UV light, colonies of Rhizobium meliloti (Sinorhizobium meliloti) exoK mutants produce a fluorescent halo with a delayed onset relative to wild-type colonies. By conducting transposon mutagenesis of exoK mutants of R. meliloti and screening for colonies with even more severe delays in production of these fluorescent halos, we identified three genes, designated prsD, prsE, and exsH, which are required for the eventual production of fluorescent halos by exoK colonies. Nucleotide sequence indicates that the prsD and prsE genes encode homologues of ABC transporters and membrane fusion proteins of Type I secretion systems, respectively, whereas exsH encodes a homologue of endo-1,3-1,4-beta-glycanases with glycine-rich nonameric repeats typical of proteins secreted by Type I secretion systems. The exoK gene and the prsD/prsE/exsH genes were shown to be components of independent pathways for production of extracellular succinoglycan degrading activities and for production of low-molecular-weight succinoglycan by R. meliloti. Based on these results, we propose that ExsH is a succinoglycan depolymerase secreted by a Type I secretion system composed of PrsD and PrsE, and that the ExsH and ExoK glycanases contribute to production of low-molecular-weight succinoglycan.     

Variation of colony morphology and chromosomal rearrangement in Candida tropicalis pK233

UV irradiation treatment of the asexual yeast Candida tropicalis gave rise to morphological mutants exhibiting at least four different types of abnormal colonies on glucose-containing solid medium. These mutants were named according to their colony morphologies: 'doughnut', 'frilly', 'echinoid' and 'walnut' mutants. The doughnut mutant produced a wrinkled colony with a hollow in its central region that was rich in filamentous pseudohyphal cells. With increased incubation time, the colony gradually changed to a reticulate shape. The parent strain, which normally produced smooth colonies, gave similar colonies to those of the doughnut mutant when grown in medium containing oleic acid as carbon source. Both the frilly and the walnut mutants produced pseudohyphal cells in a similar fashion to the doughnut mutant. The echinoid mutant produced an echinulate colony morphology with aerial hyphae and contained true hyphal cells as well as pseudohyphal ones. Pulsed-field gel electrophoresis showed that the echinoid and frilly mutants had different karyotypes from that of their parent strain, suggesting the occurrence of chromosomal rearrangements associated with these morphological mutations.     

Detection, with the dye phloxine B, of yeast mutants unable to utilize nitrogenous substances as the sole nitrogen source.

Yeast mutants unable to degrade certain nitrogen compounds produce characteristic small red colonies on an agar medium containing the red dye phloxine B, galactose, the test nitrogen compound, and a small amount of ammonium chloride.     

Conformational Changes during Pore Formation by the Perforin-Related Protein Pleurotolysin

Membrane attack complex/perforin-like (MACPF) proteins comprise the largest superfamily of pore-forming proteins, playing crucial roles in immunity and pathogenesis. Soluble monomers assemble into large transmembrane pores via conformational transitions that remain to be structurally and mechanistically characterised. Here we present an 11 Å resolution cryo-electron microscopy (cryo-EM) structure of the two-part, fungal toxin Pleurotolysin (Ply), together with crystal structures of both components (the lipid binding PlyA protein and the pore-forming MACPF component PlyB). These data reveal a 13-fold pore 80 Å in diameter and 100 Å in height, with each subunit comprised of a PlyB molecule atop a membrane bound dimer of PlyA. The resolution of the EM map, together with biophysical and computational experiments, allowed confident assignment of subdomains in a MACPF pore assembly. The major conformational changes in PlyB are a ∼70° opening of the bent and distorted central β-sheet of the MACPF domain, accompanied by extrusion and refolding of two α-helical regions into transmembrane β-hairpins (TMH1 and TMH2). We determined the structures of three different disulphide bond-trapped prepore intermediates. Analysis of these data by molecular modelling and flexible fitting allows us to generate a potential trajectory of β-sheet unbending. The results suggest that MACPF conformational change is triggered through disruption of the interface between a conserved helix-turn-helix motif and the top of TMH2. Following their release we propose that the transmembrane regions assemble into β-hairpins via top down zippering of backbone hydrogen bonds to form the membrane-inserted β-barrel. The intermediate structures of the MACPF domain during refolding into the β-barrel pore establish a structural paradigm for the transition from soluble monomer to pore, which may be conserved across the whole superfamily. The TMH2 region is critical for the release of both TMH clusters, suggesting why this region is targeted by endogenous inhibitors of MACPF function.     

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.     

Cellulose-degrading potentials and phylogenetic classification of carboxymethyl-cellulose decomposing bacteria isolated from soil

In a previous study, culturable carboxymethyl-cellulose (CMC) decomposing soil bacteria isolated from different sampling positions across an agricultural encatchment have been classified into 31 pattern groups by digestion of amplified 16S rDNA using a single restriction enzyme (Ulrich and Wirth: Microb. Ecol. 37, 238-247, 1999). In order to reveal relationships between phylogenetic diversity and phenotypic functions, a further differentiation of two selected site-specific pattern groups (I and H) was performed, resulting in a sub-classification of four and three ARDRA groups, respectively. Based on sequencing a representative isolate of each ARDRA group, the isolates were assigned to the genus Streptomyces. The ARDRA groups were dispersed across various clades of the genus with a direct affiliation to species known for cellulolytic activity in one group, only. The isolates differed in potentials to degrade colloidal, native or highly crystalline cellulose derivatives. Out of 39 isolates, 11 were capable of degrading all substrates, 17 were restricted to degrade CMC only, and 11 were active decomposers of exclusively both CMC and colloidal cellulose. In most cases, the genetic classification of the isolates corresponded with groupings based on cellulose degrading capabilities. Thus, isolates of four ARDRA groups were restricted to the degradation of CMC, while two further isolates which efficiently degraded all cellulose derivatives formed two separate ARDRA groups. The major ARDRA group, however; displayed a high variability of degradation capabilities. The study of additional phenotypic features revealed a broad potential to decompose a set of various carbon substrates, which matched the phylogenetic classification in several cases.     

Screening for distinct xylan degrading enzymes in complex shake flask fermentation supernatants

The efficient degradation of complex xylans needs collaboration of many xylan degrading enzymes. Assays for xylan degrading activities based on reducing sugars or PNP substrates are not indicative for the presence of enzymes able to degrade complex xylans: They do not provide insight into the possible presence of xylanase-accessory enzymes within enzyme mixtures. A new screening method is described, by which specific xylan modifying enzymes can be detected.Fermentation supernatants of 78 different fungal soil isolates grown on wheat straw were analyzed by HPLC and MS. This strategy is powerful in recognizing xylanases, arabinoxylan hydrolases, acetyl xylan esterases and glucuronidases.No fungus produced all enzymes necessary to totally degrade the substrates tested. Some fungi produce high levels of xylanase active against linear xylan, but are unable to degrade complex xylans. Other fungi producing relative low levels of xylanase secrete many useful accessory enzyme component(s).     

Biofilm growth and detachment of Actinobacillus actinomycetemcomitans

The gram-negative, oral bacterium Actinobacillus actinomycetemcomitans has been implicated as the causative agent of several forms of periodontal disease in humans. When cultured in broth, fresh clinical isolates of A. actinomycetemcomitans form tenacious biofilms on surfaces such as glass, plastic, and saliva-coated hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize the oral cavity and cause disease. We examined the morphology of A. actinomycetemcomitans biofilm colonies grown on glass slides and in polystyrene petri dishes by using light microscopy and scanning and transmission electron microscopy. We found that A. actinomycetemcomitans developed asymmetric, lobed biofilm colonies that displayed complex architectural features, including a layer of densely packed cells on the outside of the colony and nonaggregated cells and large, transparent cavities on the inside of the colony. Mature biofilm colonies released single cells or small clusters of cells into the medium. These released cells adhered to the surface of the culture vessel and formed new colonies, enabling the biofilm to spread. We isolated three transposon insertion mutants which produced biofilm colonies that lacked internal, nonaggregated cells and were unable to release cells into the medium. All three transposon insertions mapped to genes required for the synthesis of the O polysaccharide (O-PS) component of lipopolysaccharide. Plasmids carrying the complementary wild-type genes restored the ability of mutant strains to synthesize O-PS and release cells into the medium. Our findings suggest that A. actinomycetemcomitans biofilm growth and detachment are discrete processes and that biofilm cell detachment evidently involves the formation of nonaggregated cells inside the biofilm colony that are destined for release from the colony.     

Succinate transport in Rhizobium leguminosarum.

The transport of succinate was studied in an effective streptomycin-resistant strain of Rhizobium leguminosarum. High levels of succinate transport occurred when cells were grown on succinate, fumarate, or malate, whereas low activity was found when cells were grown on glucose, sucrose, arabinose, or pyruvate as the sole carbon source. Because of the rapid metabolism of succinate after transport into the cells, a succinate dehydrogenase-deficient mutant was isolated in which intracellular succinate accumulated to over 400 times the external concentration. Succinate transport was completely abolished in the presence of metabolic uncouplers but was relatively insensitive to sodium arsenate. Succinate transport was a saturable function of the succinate concentration, and the apparent Km and Vmax values for transport were determined in both the parent and the succinate dehydrogenase mutant. Malate and fumarate competitively inhibited succinate transport, whereas citrate and malonate had no effect. Succinate transport mutants were isolated by transposon (Tn5) mutagenesis. These mutants were unable to transport succinate or malate and were unable to grow on succinate, malate, or fumarate as the sole carbon source. The mutants grew normally on pyruvate, oxaloacetate, citrate, or arabinose, and revertants isolated on succinate minimal medium had regained the ability to grow on malate and fumarate. From these data, we conclude that R. leguminosarum possesses a C4-dicarboxylic acid transport system which is inducible and mediates the active transport of succinate, fumarate, and malate into the cell.