Osmoregulated periplasmic glucans of Erwinia chrysanthemi

We report the initial characterization of the osmoregulated periplasmic glucans (OPGs) of Erwinia chrysanthemi. OPGs are intrinsic components of the bacterial envelope necessary to the pathogenicity of this phytopathogenic enterobacterium (F. Page, S. Altabe, N. Hugouvieux-Cotte-Pattat, J.-M. Lacroix, J. Robert-Baudouy and J.-P. Bohin, J. Bacteriol. 183:0000-0000, 2001 [companion in this issue]). OPGs were isolated by trichloracetic acid treatment and gel permeation chromatography. The synthesis of these compounds appeared to be osmoregulated, since lower amounts of OPGs were produced when bacteria were grown in media of higher osmolarities. However, a large fraction of these OPGs were recovered in the culture medium. Then, these compounds were characterized by compositional analysis, high-performance anion-exchange chromatography, matrix-assisted laser desorption mass spectrometry, and (1)H and (13)C nuclear magnetic resonance analyses. OPGs produced by E. chrysanthemi are very heterogeneous at the level of both backbone structure and substitution of these structures. The degree of polymerization of the glucose units ranges from 5 to 12. The structures are branched, with a linear backbone consisting of beta-1,2-linked glucose units to which a variable number of branches, composed of one glucose residue, are attached by beta-1,6 linkages in a random way. This glucan backbone may be substituted by O-acetyl and O-succinyl ester-linked residues.     

Osmoregulated periplasmic glucans synthesis gene family of Shigella flexneri

Osmoregulated periplasmic glucans (OPGs) of food- and water-borne enteropathogen Shigella flexneri were characterized. OPGs were composed of 100% glucose with 2-linked glucose as the most abundant residue with terminal glucose, 2-linked and 2,6-linked glucose also present in high quantities. Most dominant backbone polymer chain length was seven glucose residues. Individual genes from the opg gene family comprising of a bicistronic operon opgGH, opgB, opgC and opgD were mutagenized to study their effect on OPGs synthesis, growth in hypo-osmotic media and ability to invade HeLa cells. Mutation in opgG and opgH abolished OPGs biosynthesis, and mutants experienced longer lag time to initiate growth in hypo-osmotic media. Longer lag times to initiate growth in hypo-osmotic media were also observed for opgC and opgD mutants but not for opgB mutant. All opg mutants were able to infect HeLa cells, and abolition of OPGs synthesis did not affect actin polymerization or plaque formation. Ability to synthesize OPGs was beneficial to bacteria in order to initiate growth under low osmolarity conditions, in vitro mammalian cell invasion assays, however, could not discriminate whether OPGs were required for basic aspect of Shigella virulence.     

Osmoregulated periplasmic glucans of the free-living photosynthetic bacterium Rhodobacter sphaeroides.

The osmoregulated periplasmic glucans (OPGs) produced by Rhodobacter sphaeroides, a free-living organism, were isolated by trichloracetic acid treatment and gel permeation chromatography. Compounds obtained were characterized by compositional analysis, matrix-assisted laser desorption ionization mass spectrometry and nuclear magnetic resonance. R. sphaeroides predominantly synthesizes a cyclic glucan containing 18 glucose residues that can be substituted by one to seven succinyl esters residues at the C6 position of some of the glucose residues, and by one or two acetyl residues. The glucans were subjected to a mild alkaline treatment in order to remove the succinyl and acetyl substituents, analyzed by MALDI mass spectrometry and purified by high-performance anion-exchange chromatography. Methylation analysis revealed that this glucan is linked by 17 1,2 glycosidic bonds and one 1,6 glycosidic bond. Homonuclear and (1)H/(13)C heteronuclear NMR experiments revealed the presence of a single alpha-1,6 glycosidic linkage, whereas all other glucose residues are beta-1,2 linked. The different anomeric proton signals allowed a complete sequence-specific assignment of the glucan. The structural characteristics of this glucan are very similar to the previously described OPGs of Ralstonia solanacearum and Xanthomonas campestris, except for its different size and the presence of substituents. Therefore, similar OPGs are synthesized by phytopathogenic as well as free-living bacteria, suggesting these compounds are intrinsic components of the Gram-negative bacterial envelope.     

Osmoregulated periplasmic glucans are needed for competitive growth and biofilm formation by Salmonella enterica serovar Typhimurium in leafy-green vegetable wash waters and colonization in mice

Osmoregulated periplasmic glucans (OPGs) are major periplasmic constituents of Gram-negative bacteria. The role of OPGs has been postulated in symbiotic as well as pathogenic host–microorganism interactions. Here, we report the role of OPGs from Salmonella enterica serovar Typhimurium during growth and biofilm formation in leafy-green vegetable wash water. The opgGH mutant strain, which was defective in OPG biosynthesis, initiated the growth at a slower rate in wash waters obtained from spinach, lettuce and green collard and severely impaired biofilm formation. The lack of OPG synthesis did not influence biofilm formation by the opgGH mutant in low-nutrient low-osmolarity laboratory media. In coculture experiments initiated with equal proportions of cells, the opgGH mutant was outnumbered by the wild-type strain under the planktonic as well as the biofilm growth conditions. The opgGH mutant strain poorly colonized mouse organs when introduced orally along with the wild-type strain. This is the first report demonstrating the role of OPGs of Salmonella in competitive colonization of biofilms, planktonic cultures and mouse organs.     

Osmoregulated periplasmic glucan synthesis is required for Erwinia chrysanthemi pathogenicity

Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.     

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.     

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.     

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica serovar Typhimurium SL1344 in mice virulence

opgB gene of Salmonella enterica serovar Typhimurium was identified earlier in a genome-wide screen for mice virulence (Valentine et al. in Infect Immun 66:3378-3383, 1998). Although mutation in opgB resulted in avirulent Salmonella strain, how this gene contributes to pathogenesis remains unclear. Based on DNA homology, opgB is predicted to be responsible for adding phosphoglycerate residues to osmoregulated periplasmic glucans (OPGs) giving them anionic characteristics. In Escherichia coli, yet another gene, opgC, is also reported to contribute to anionic characteristics of OPGs by adding succinic acid residues. We constructed opgB, opgC, and opgBC double mutants of S. enterica serovar Typhimurium strain SL1344. As predicted opgBC mutant synthesized neutral OPGs that were devoid of any anionic substituents. However, opgB, opgC, and opgBC mutations had no significant impact on mice virulence as well as on competitive organ colonization. In low osmotic conditions, opgB, opgC, and opgBC mutants exhibited delay in growth initiation in the presence of sodium deoxycholate. Anionic substituents of OPGs from Salmonella although appear to be needed to overcome resistance of deoxycholate in hypoosmotic growth media, no evidence was found for their role in mice virulence.     

Structural analysis of Escherichia coli OpgG, a protein required for the biosynthesis of osmoregulated periplasmic glucans

Osmoregulated periplasmic glucans (OPGs) G protein (OpgG) is required for OPGs biosynthesis. OPGs from Escherichia coli are branched glucans, with a backbone of beta-1,2 glucose units and with branches attached by beta-1,6 linkages. In Proteobacteria, OPGs are involved in osmoprotection, biofilm formation, virulence and resistance to antibiotics. Despite their important biological implications, enzymes synthesizing OPGs are poorly characterized. Here, we report the 2.5 A crystal structure of OpgG from E.coli. The structure was solved using a selenemethionine derivative of OpgG and the multiple anomalous diffraction method (MAD). The protein is composed of two beta-sandwich domains connected by one turn of 3(10) helix. The N-terminal domain (residues 22-388) displays a 25-stranded beta-sandwich fold found in several carbohydrate-related proteins. It exhibits a large cleft comprising many aromatic and acidic residues. This putative binding site shares some similarities with enzymes such as galactose mutarotase and glucodextranase, suggesting a potential catalytic role for this domain in OPG synthesis. On the other hand, the C-terminal domain (residues 401-512) has a seven-stranded immunoglobulin-like beta-sandwich fold, found in many proteins where it is mainly implicated in interactions with other molecules. The structural data suggest that OpgG is an OPG branching enzyme in which the catalytic activity is located in the large N-terminal domain and controlled via the smaller C-terminal domain.     

Four PCR primers necessary for the detection of periplasmic nitrate reductase genes in all groups of Proteobacteria and in environmental DNA

Generic primers are available for detecting bacterial genes required for almost every reaction of the biological nitrogen cycle, the one notable exception being napA (gene for the molybdoprotein of the periplasmic nitrate reductase) encoding periplasmic nitrate reductases. Using an iterative approach, we report the first successful design of three forward oligonucleotide primers and one reverse primer that, in three separate PCRs, can amplify napA DNA from all five groups of Proteobacteria. All 140 napA sequences currently listed in the NCBI (National Center for Biotechnology Information) database are predicted to be amplified by one or more of these primer pairs. We demonstrate that two pairs of these primers also amplify PCR products of the predicted sizes from DNA isolated from human faeces, confirming their ability to direct the amplification of napA fragments from mixed populations. Analysis of the resulting amplicons by high-throughput sequencing will enable a good estimate to be made of both the range and relative abundance of nitrate-reducing bacteria in any community, subject only to any unavoidable bias inherent in a PCR approach to molecular characterization of a highly diverse target.     

Osmoregulated trehalose-derived oligosaccharides in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium accumulating glutamate, N-acetylglutaminyl glutamine amide and trehalose in hyperosmolarity. Besides these compatible solutes, we highlighted several compounds in S. meliloti Rm1021 wild-type strain. The purification and the structural characterization based on liquid chromatography evaporative light scattering detector, electrospray ionization high resolution mass spectrometry and nuclear magnetic resonance techniques showed they were four linear oligosaccharides composed of 3, 4, 5 and 6 glucose units all linked by α-(1 → 2) linkages except a terminal α-(1 ↔ 1) linkage. These oligosaccharides were cytoplasmic and were observed in several wild-type strains suggesting they were common features in S. meliloti strains grown in hyperosmolarity.     

Electrophoresis of algal glucans

The ability of reactive dyes based on chloro-s-triazine to reactwith the primary and secondary hydroxyl groups of glucose, formingcovalent bonds, has been used to dye the storage polyglucosidesof the algae, Cytmidium caldarium, Oscillatoria princeps, Rhodymeniapertusa and Spirogyra setiformis, prior to electrophoresis oncellulose polyacetate strips in borate buffer. The techniqueresolves mixtures of glucans and defines some of the parametersof the internal structure of these glucans. The similarity ofthe glucans from Cyanidium caldarium and Oscillatoria princepsis evident using this method. The two components of the "starch"from Spirogyra setiformis have been studied and identified bytheir reactions with the dye and the mobilities of the dyedcomponents during electrophoresis. Inferences as to the biphyleticevolutionary pathways leading from the Cyanophyceae to the Rhodophyceaeand the Chlorophyceae can be made based on the data. (Received June 27, 1973; )     

Changes in the glucans of ripening apples

The noncellulosic glucose content, like the xylose content, of cell walls of cortex tissue of apples showed little change as the fruit ripened and the cellulosic glucose also remained constant. There was a considerable loss of galactose residues from the walls, however, whilst only a small change in arabinose was observed. The starch was rapidly hydrolysed.     

The dif/Xer Recombination Systems in Proteobacteria

In E. coli, 10 to 15% of growing bacteria produce dimeric chromosomes during DNA replication. These dimers are resolved by XerC and XerD, two tyrosine recombinases that target the 28-nucleotide motif (dif) associated with the chromosome''s replication terminus. In streptococci and lactococci, an alternative system is composed of a unique, Xer-like recombinase (XerS) genetically linked to a dif-like motif (dif _SL) located at the replication terminus. Preliminary observations have suggested that the dif/Xer system is commonly found in bacteria with circular chromosomes but that assumption has not been confirmed in an exhaustive analysis. The aim of the present study was to extensively characterize the dif/Xer system in the proteobacteria, since this taxon accounts for the majority of genomes sequenced to date. To that end, we analyzed 234 chromosomes from 156 proteobacterial species and showed that most species (87.8%) harbor XerC and XerD-like recombinases and a dif-related sequence which (i) is located in non-coding sequences, (ii) is close to the replication terminus (as defined by the cumulative GC skew) (iii) has a palindromic structure, (iv) is encoded by a low G+C content and (v) contains a highly conserved XerD binding site. However, not all proteobacteria display this dif/XerCD system. Indeed, a sub-group of pathogenic ε-proteobacteria (including Helicobacter sp and Campylobacter sp) harbors a different recombination system, composed of a single recombinase (XerH) which is phylogenetically distinct from the other Xer recombinases and a motif (dif _H) sharing homologies with dif _SL. Furthermore, no homologs to dif or Xer recombinases could be detected in small endosymbiont genomes or in certain bacteria with larger chromosomes like the Legionellales. This raises the question of the presence of other chromosomal deconcatenation systems in these species. Our study highlights the complexity of dif/Xer recombinase systems in proteobacteria and paves the way for systematic detection of these components in prokaryotes.     

CHDL: a cadherin-like domain in Proteobacteria and Cyanobacteria

We identified a cadherin-like domain (CHDL) using computational analysis. The CHDL domain is mostly distributed in Proteobacteria and Cyanobacteria, although it is also found in some eukaryotic proteins. Prediction of three-dimensional protein folding indicated that the CHDL domain has an immunoglobulin beta-sandwich fold and belongs to the cadherin superfamily. The CHDL domain does not have LDRE and DxNDN motifs, which are conserved in the cadherin domain, but has three other motifs: PxAxxD, DxDxD and YT-V/I-S/T-D, which might contribute to forming a calcium-binding site. The identification of this cadherin-like domain indicates that the cadherin superfamily may exhibit wider sequence and structural diversity than previously appreciated. Domain architecture analysis revealed that the CHDL domain is also associated with other adhesion domains as well as enzyme domains. Based on computational analysis and previous experimental data, we predict that the CHDL domain has calcium-binding and also carbohydrate-binding activity.     

Profiling the secretomes of plant pathogenic Proteobacteria

Secreted proteins are central to the success of plant pathogenic bacteria. They are used by plant pathogens to adhere to and degrade plant cell walls, to suppress plant defence responses, and to deliver bacterial DNA and proteins into the cytoplasm of plant cells. However, experimental investigations into the identity and role of secreted proteins in plant pathogenesis have been hindered by the fact that many of these proteins are only expressed or secreted in planta, that knockout mutations of individual proteins frequently have little or no obvious phenotype, and that some obligate and fastidious plant pathogens remain recalcitrant to genetic manipulation. The availability of genome sequence data for a large number of agriculturally and scientifically important plant pathogens enables us to predict and compare the complete secretomes of these bacteria. In this paper we outline strategies that are currently being used to identify secretion systems and secreted proteins in Proteobacterial plant pathogens and discuss the implications of these analyses for future investigations into the molecular mechanisms of plant pathogenesis.