Novel succinylated and large-sized osmoregulated periplasmic glucans of Pseudomonas syringae pv. syringae

Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the Gram-negative bacterial envelope and are important for bacterial-host interactions. The OPGs of Pseudomonas syringae pv. syringae have been known to be highly branched linear glucans ranging from 6 to 13 glucose residues devoid of any substituents, while having backbone structure similar to those of Escherichia coli and Erwinia chrysanthemi. Here, we report for the first time succinylated and large-sized OPGs from P. syringae pv. syringae. The glucans were isolated with trichloroacetic acid treatment and various chromatographic techniques. These were further characterized by thin-layer chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometer, and 1D ¹H nuclear magnetic resonance spectroscopy. The results demonstrate that novel anionic glucans with one succinyl residue at the C-6 position of the glucose unit as well as neutral glucans including large-sized glucans with up to 28 degrees of polymerization are produced in P. syringae pv. syringae. Furthermore, the succinylated and large-sized OPGs of P. syringae pv. syringae are necessary for hypoosmotic adaptation.     

Cell-associated glucans of Burkholderia solanacearum and Xanthomonas campestris pv. citri: a new family of periplasmic glucans.

The cell-associated glucans produced by Burkholderia solanacearum and Xanthomonas campestris pv. citri were isolated by trichloroacetic acid treatment and gel permeation chromatography. The compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry, and high-performance anion-exchange chromatography. B. solanacearum synthesizes only a neutral cyclic glucan containing 13 glucose residues, and X. campestris pv. citri synthesizes a neutral cyclic glucan containing 16 glucose residues. The two glucans were further purified by high-performance anion-exchange chromatography. Methylation analysis revealed that these glucans are linked by 1,2-glycosidic bonds and one 1,6-glycosidic bond. Our 600-MHz homonuclear and 1H-13C heteronuclear nuclear magnetic resonance experiments revealed the presence of a single alpha-1,6-glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The presence of this single alpha-1,6 linkage, however, induces such structural constraints in these cyclic glucans that all individual glucose residues could be distinguished. The different anomeric proton signals allowed complete sequence-specific assignment of both glucans. The structural characteristics of these glucans contrast with those of the previously described osmoregulated periplasmic glucans.     

Transfer of phosphoethanolamine residues from phosphatidylethanolamine to the membrane-derived oligosaccharides of Escherichia coli.

The membrane-derived oligosaccharides (MDO) of Escherichia coli are periplasmic constituents composed of glucose residues linked by beta-1,2 and beta-1,6 glycosidic bonds. MDO are substituted with phosphoglycerol, phosphoethanolamine, and succinic acid moieties. The phosphoglycerol residues present on MDO are derived from phosphatidylglycerol (B. J. Jackson and E. P. Kennedy, J. Biol. Chem. 258:2394-2398, 1983), but evidence as to the source of the phosphoethanolamine residues has been lacking. We now report that phosphatidylethanolamine, exogenously added to intact cells of E. coli, provides a source of phosphoethanolamine residues that are transferred to MDO. The biosynthesis of phosphoethanolamine-labeled MDO is osmotically regulated, with maximum synthesis occurring during growth in medium of low osmolarity.     

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.     

Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.     

Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan.

The periplasmic cyclic beta-1,2-glucan of Agrobacterium tumefaciens is believed to maintain high osmolarity in the periplasm during growth of the bacteria on low-osmotic-strength media. Strains with mutations in the chvA or chvB gene do not accumulate beta-1,2-glucan in their periplasm and exhibit pleiotropic phenotypes, including inability to form crown gall tumors on plants. We examined the effects of medium osmolarity to determine whether some or all of these phenotypes result from suboptimal periplasmic osmolarity. The mutants grew more slowly than wild-type cells and exhibited altered periplasmic and cytoplasmic protein content when cultured in low-osmotic-strength media, but not when cultured in high-osmotic-strength media. These observations support a role for periplasmic glucan in osmoadaptation. However, the mutants were avirulent and exhibited reduced motility regardless of the osmolarity of the medium. Therefore, beta-1,2-glucan may play roles in virulence and motility that are unrelated to its role in osmoadaptation.     

Identification of mdoD, an mdoG paralog which encodes a twin-arginine-dependent periplasmic protein that controls osmoregulated periplasmic glucan backbone structures

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic and highly branched oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. The glucan length, ranging from 5 to 12 glucose residues, is under strict control. Two genes that form an operon, mdoGH, govern glucose backbone synthesis. The new gene mdoD, which appears to be a paralog of mdoG, was characterized in this study. Cassette inactivation of mdoD resulted in production of OPGs with a higher degree of polymerization, indicating that OpgD, the mdoD product (according to the new nomenclature), controls the glucose backbone structures. OpgD secretion depends on the Tat secretory pathway. Orthologs of the mdoG and mdoD genes are found in various proteobacteria. Most of the OpgD orthologs exhibit a Tat-dependent secretion signal, while most of the OpgG orthologs are Sec dependent.     

Stimulation of exopolysaccharide production by fluorescent pseudomonads in sucrose media due to dehydration and increased osmolarity

Abstract Exopolysaccharides produced by plant pathogenic bacteria are thought to play an important role in both the general ecology and the virulence of the producing organism. The environmental factors affecting exopolysaccharide production in planta by Pseudomonas syringae pathovars are not known. We tested the effect of increased medium osmolarity and dehydration on exopolysaccharide production in a sucrose-containing medium by three P. syringae pathovars, one ( P. syringae pv. phaseolicola ) capable of levan and alginate production and two ( P. syringae pv. papulans and pv. savastanoi ) capable of only alginate production. Addition of NaCl and ethanol to the medium led to increased accumulation of alginate by all three pathovars as well as increased levan production by P. syringae pv. phaseolicola . Culture fluids of the two non-levan producers also contained increased amounts of neutral carbohydrate which was not levan. Based on sugar compostion this material may have originated from outer membrane lipopolysaccharide. In addition, the ratio of neutral material (levan or not) to alginate varied dependent on culture conditions.     

Characterisation of Mesorhizobium huakuii cyclic beta-glucan

Periplasmic and extracellular glucans of Mesorhizobium huakuii were isolated and characterized by compositional and MALDI-TOF analyses, as well as 1H and 13C NMR spectroscopy. It was shown that M. huakuii produces a cyclic beta-glucan composed entirely of nonbranched glucose chains and unmodified by nonsugar substituents. The degree of polymerisation of the cyclic oligosaccharides was estimated to be in the range from 17 to 28. The most abundant glucan molecules contained 22 glucose residues. Glucose residues within the glucan were connected by beta-(1,2) glycosidic linkages. The cyclic glucan produced by M. huakuii is quite similar to the periplasmic beta-(1,2) glucans synthesized by Agrobacterium and Sinorhizobium genera. The synthesis of beta-glucan in M. huakuii is osmoregulated and this glucan could function as an osmoprotectant in free living cells.     

The Tat pathway of the plant pathogen Pseudomonas syringae is required for optimal virulence

Pseudomonas syringae is a gram-negative bacterium that infects a number of agriculturally important plant species. The ability of the organism to deliver virulence factors across the plant cell wall is a key to its pathogenicity. Deletion mutants in the twin arginine translocation (Tat) pathway of two pathovars of P. syringae, pvs. tomato DC3000 and maculicola ES4326, displayed a range of pleiotropic phenotypic changes, such as defects in fluorescent siderophore production, a decrease in sodium dodecyl sulfate and copper resistance, and a significant loss in fitness using Arabidopsis thaliana or tomato as plant hosts. The genome sequence of P. syringae pv. tomato DC3000 encodes a number of potential virulence factors that are predicted to be translocated via the Tat pathway, including several proteins involved in iron scavenging (two siderophore receptors, PSPTO3474 and PSPTO3294, and an aminotransferase, PSPTO2155, involved in siderophore biosynthesis). Further candidates for Tat-dependent pathogenicity determinants include the homologs of a cell wall amidase (PSPTO5528), an enzyme involved in periplasmic glucans biosynthesis (PSPTO5542), and two putative phospholipases (PSPTO3648 and PSPTOB0005). Translocation of the putative amidase, aminotransferase, glucans biosynthetic enzyme, and the two phospholipases, but not the two siderophore receptors, is shown to be dependent on the Tat pathway. Strains deleted for the genes encoding the probable aminotransferase and amidase enzymes are significantly less infectious than the wild type. We conclude that the incremental effects due to the failure to correctly localize at least two, and possibly more, Tat substrates gives rise to the attenuated fitness phenotype of the P. syringae pv. tomato DC3000 tat strain.     

Involvement of the exopolysaccharide alginate in the virulence and epiphytic fitness of Pseudomonas syringae pv. syringae

Alginate, a co-polymer of O-acetylated beta-1,4-linked D-mannuronic acid and L-guluronic acid, has been reported to function in the virulence of Pseudomonas syringae, although genetic studies to test this hypothesis have not been undertaken previously. In the present study, we used a genetic approach to evaluate the role of alginate in the pathogenicity of P. syringae pv. syringae 3525, which causes bacterial brown spot on beans. Alginate biosynthesis in strain 3525 was disrupted by recombining Tn5 into algL, which encodes alginate lyase, resulting in 3525.L. Alginate production in 3525.L was restored by the introduction of pSK2 or pAD4033, which contain the alginate biosynthetic gene cluster from P. syringae pv. syringae FF5 or the algA gene from P. aeruginosa respectively. The role of alginate in the epiphytic fitness of strain 3525 was assessed by monitoring the populations of 3525 and 3525.L on tomato, which is not a host for this pathogen. The mutant 3525.L was significantly impaired in its ability to colonize tomato leaves compared with 3525, indicating that alginate functions in the survival of strain 3525 on leaf surfaces. The contribution of alginate to the virulence of strain 3525 was evaluated by comparing the population dynamics and symptom development of 3525 and 3525.L in bean leaves. Although 3525. L retained the ability to form lesions on bean leaves, symptoms were less severe, and the population was significantly reduced in comparison with 3525. These results indicate that alginate contributes to the virulence of P. syringae pv. syringae 3525, perhaps by facilitating colonization or dissemination of the bacterium in planta.     

Housekeeping gene sequencing and multilocus variable-number tandem-repeat analysis to identify subpopulations within Pseudomonas syringae pv. maculicola and Pseudomonas syringae pv. tomato that correlate with host specificity

Pseudomonas syringae pv. maculicola causes bacterial spot on Brassicaceae worldwide, and for the last 10 years severe outbreaks have been reported in the Loire Valley, France. P. syringae pv. maculicola resembles P. syringae pv. tomato in that it is also pathogenic for tomato and causes the same types of symptoms. We used a collection of 106 strains of P. syringae to characterize the relationships between P. syringae pv. maculicola and related pathovars, paying special attention to P. syringae pv. tomato. Phylogenetic analysis of gyrB and rpoD gene sequences showed that P. syringae pv. maculicola, which causes diseases in Brassicaceae, forms six genetic lineages within genomospecies 3 of P. syringae strains as defined by L. Gardan et al. (Int. J. Syst. Bacteriol. 49[Pt 2]:469-478, 1999), whereas P. syringae pv. tomato forms two distinct genetic lineages. A multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) conducted with eight minisatellite loci confirmed the genetic structure obtained with rpoD and gyrB sequence analyses. These results provide promising tools for fine-scale epidemiological studies on diseases caused by P. syringae pv. maculicola and P. syringae pv. tomato. The two pathovars had distinct host ranges; only P. syringae pv. maculicola strains were pathogenic for Brassicaceae. A subpopulation of P. syringae pv. maculicola strains that are pathogenic for Pto-expressing tomato plants were shown to lack avrPto1 and avrPtoB or to contain a disrupted avrPtoB homolog. Taking phylogenetic and pathological features into account, our data suggest that the DC3000 strain belongs to P. syringae pv. maculicola. This study shows that P. syringae pv. maculicola and P. syringae pv. tomato appear multiclonal, as they did not diverge from a single common ancestral group within the ancestral P. syringae genomospecies 3, and suggests that pathovar specificity within P. syringae may be due to independent genetic events.     

Pseudomonas syringae exchangeable effector loci: sequence diversity in representative pathovars and virulence function in P. syringae pv. syringae B728a

Pseudomonas syringae is a plant pathogen whose pathogenicity and host specificity are thought to be determined by Hop/Avr effector proteins injected into plant cells by a type III secretion system. P. syringae pv. syringae B728a, which causes brown spot of bean, is a particularly well-studied strain. The type III secretion system in P. syringae is encoded by hrp (hypersensitive response and pathogenicity) and hrc (hrp conserved) genes, which are clustered in a pathogenicity island with a tripartite structure such that the hrp/hrc genes are flanked by a conserved effector locus and an exchangeable effector locus (EEL). The EELs of P. syringae pv. syringae B728a, P. syringae strain 61, and P. syringae pv. tomato DC3000 differ in size and effector gene composition; the EEL of P. syringae pv. syringae B728a is the largest and most complex. The three putative effector proteins encoded by the P. syringae pv. syringae B728a EEL--HopPsyC, HopPsyE, and HopPsyV--were demonstrated to be secreted in an Hrp-dependent manner in culture. Heterologous expression of hopPsyC, hopPsyE, and hopPsyV in P. syringae pv. tabaci induced the hypersensitive response in tobacco leaves, demonstrating avirulence activity in a nonhost plant. Deletion of the P. syringae pv. syringae B728a EEL strongly reduced virulence in host bean leaves. EELs from nine additional strains representing nine P. syringae pathovars were isolated and sequenced. Homologs of avrPphE (e.g., hopPsyE) and hopPsyA were particularly common. Comparative analyses of these effector genes and hrpK (which flanks the EEL) suggest that the EEL effector genes were acquired by horizontal transfer after the acquisition of the hrp/hrc gene cluster but before the divergence of modern pathovars and that some EELs underwent transpositions yielding effector exchanges or point mutations producing effector pseudogenes after their acquisition.     

Analysis of the syrP gene, which regulates syringomycin synthesis by Pseudomonas syringae pv. syringae.

Syringomycin is a lipodepsinonapeptide phytotoxin synthesized by Pseudomonas syringae pv. syringae on multienzymatic peptide synthetases. Sequence analysis of the interval between the syrB and syrD genes of P. syringae pv. syringae strain B301D revealed a 1,059-bp open reading frame (ORF), designated syrP. The predicted product of this ORF was a 39.6-kDa protein consisting of 353 amino acid residues. Searches of protein sequence databases demonstrated that SyrP was most similar to histidine kinases such as the CheA regulatory protein of Escherichia coli. The predicted SyrP sequence was aligned with the N terminus of CheA, a region corresponding to the phosphotransfer and acceptor domains of CheA. The SyrP region that aligns with the phosphotransfer domain of CheA contained a His at position 101 which is flanked by a weak consensus sequence of the unorthodox sensory kinase subfamily of two-component regulatory systems. Strain B301D-31, obtained by site-directed insertional mutagenesis of the syrP gene, exhibited an unusual pleiotropic phenotype including a failure to produce syringomycin in liquid media in contrast to production of elevated levels of the toxin on agar media. The syrP mutant was relieved of the suppression of toxin production that accompanies inorganic phosphate concentrations of > 1 mM on agar media. Nevertheless, the syrP mutant was substantially less virulent than the wild-type strain in pathogenicity assays in cherry fruits. These results suggest that the syrP gene encodes a regulatory protein that participates in a phosphorylation cascade controlling syringomycin production and virulence in P. syringae pv. syringae.     

Synthesis of glycerophosphorylated cyclic beta-(1,2)-glucans by Rhizobium meliloti ndv mutants.

The periplasmic cyclic beta-(1,2)-glucans of Rhizobium spp. are believed to provide functions during hypoosmotic adaptation and legume nodulation. In Rhizobium meliloti, cyclic beta-(1,2)-glucans are synthesized at highest levels when cells are grown at low osmolarity, and a considerable fraction (> or = 35%) of these glucans may become substituted with phosphoglycerol moieties. Thus far, two chromosomally encoded proteins, NdvA and NdvB, have been shown to function during cyclic beta-(1,2)-glucan biosynthesis; however, the precise roles for these proteins remain unclear. In the present study, we show that R. meliloti mutants lacking up to one-third of the downstream region of ndvB synthesize cyclic beta-(1,2)-glucans similar to those produced by wild-type cells with respect to size and phosphoglycerol substituent profile. In contrast, no phosphoglycerol substituents were detected on the cyclic beta-(1,2)-glucans synthesized by an R. meliloti ndvA mutant.     

Phenotypic expression of the Pseudomonas syringae pv. syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin.

Plants, in general, appear to be able to detect the presence of incompatible Pseudomonas syringae strains by a hypothetical cell-cell recognition process to initiate inducible defense mechanisms that contribute to disease resistance. A 25-kb hrp/hrm gene cluster isolated from P. syringae pv. syringae 61(pHIR11) enables Escherichia coli to elicit a hypersensitive response (HR), a plant response generally considered to be a manifestation of recognition and resistance. To identify the nature of the HR-eliciting signal produced by E. coli cells carrying pHIR11, bacterial surface features were surveyed by immunological and biochemical procedures. No immunoreactive epitopes or outer membrane proteins were detected that were associated with expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100. Phenotypic expression of the P. syringae pv. syringae 61 hrp/hrm cluster in E. coli MC4100, however, was found to be dependent upon ompC and ompF, which control outer membrane permeability to hydrophilic solutes. The results suggest that deployment of the HR-eliciting signal occurs via outer membrane porins and imply that a low-molecular-weight, hydrophilic factor mediates signal exchange between the bacterium and the responding plant cell.     

Genetic Characterization of Pseudomonas syringae pv. syringae Strains from Stone Fruits in California

Strains of Pseudomonas syringae pv. syringae were isolated from healthy and diseased stone fruit tissues sampled from 43 orchard sites in California in 1995 and 1996. These strains, together with P. syringae strains from other hosts and pathovars, were tested for pathogenicity and the presence of the syrB and syrC genes and were genetically characterized by using enterobacterial repetitive intergenic consensus (ERIC) primers and PCR. All 89 strains of P. syringae pv. syringae tested were moderately to highly pathogenic on Lovell peach seedlings regardless of the host of origin, while strains of other pathovars exhibited low or no pathogenicity. The 19 strains of P. syringae pv. syringae examined by restriction fragment length polymorphism analysis contained the syrB and syrC genes, whereas no hybridization occurred with 4 strains of other P. syringae pathovars. The P. syringae pv. syringae strains from stone fruit, except for a strain from New Zealand, generated ERIC genomic fingerprints which shared four fragments of similar mobility. Of the P. syringae pv. syringae strains tested from other hosts, only strains from rose, kiwi, and pear generated genomic fingerprints that had the same four fragments as the stone fruit strains. Analysis of the ERIC fingerprints from P. syringae pv. syringae strains showed that the strains isolated from stone fruits formed a distinct cluster separate from most of the strains isolated from other hosts. These results provide evidence of host specialization within the diverse pathovar P. syringae pv. syringae.