Search

The exposure of methyl viologen (a bipyridine salt) can lead to the production of reactive oxygen species, causing oxidative stress to organisms. ABC transporters have been reported to be involved in multi-drug resistance and have a role in MV detoxification. Here, we performed a protein structure simulation of the Slr0982 protein encoding ABC transporters, and confirmed that the region from Phe57 to Gln257 was the ABC transporter-type domain of the Slr0982 protein. The results of protein sequence alignment showed that Slr0982 protein was similar to Slr2108 protein (polysialic acid transport ATP-binding protein) and Slr0354 protein (ABC transporter). We reported that the mutation of slr0982 reduced the tolerance of Synechocystis sp. PCC 6803 to oxidative stress induced by methyl viologen. The deletion of slr0982 reduced the ability of cells to resist oxidative stress. Our data confirmed that the deletion of slr0982 could affect the concentration of exopolysaccharide and the expression of some genes related to carbohydrate metabolism, thus decreasing polysaccharide transport. Importantly, the exogenous addition of exopolysaccharide extracted from wild type can effectively reduce the oxidative damage to Δslr0982 by methyl viologen. This study expands the role of ABC transporters in MV-induced oxidative stress and provides an insight into the further analysis of the response of cyanobacteria to oxidative stress.

Summary Gut-dwelling Prevotella copri (P. copri), the most prevalent Prevotella species in the human gut, have been associated with diet and disease. However, our understanding of their diversity and function remains rudimentary because studies have been limited to 16S and metagenomic surveys and experiments using a single type strain. Here, we describe the genomic diversity of 83 P. copri isolates from 11 human donors. We demonstrate that genomically distinct isolates, which can be categorized into different P. copri complex clades, utilize defined sets of polysaccharides. These differences are exemplified by variations in susC genes involved in polysaccharide transport as well as polysaccharide utilization loci (PULs) that were predicted in part from genomic and metagenomic data. Functional validation of these PULs showed that P. copri isolates utilize distinct sets of polysaccharides from dietary plant, but not animal, sources. These findings reveal both genomic and functional differences in polysaccharide utilization across human intestinal P. copri strains.

Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.

Haemophilus influenzae type b is a common cause of invasive bacterial disease, especially among children in underdeveloped countries. The type b polysaccharide capsule is a polymer of ribose and ribitol-5-phosphate and is a critical determinant of virulence. Expression of the type b capsule is dependent upon the cap b locus, which consists of three functionally distinct regions, designated regions 1 to 3. Region 3 contains the hcsA and hcsB genes, which share significant homology with genes that have been implicated in encapsulation in other pathogenic bacteria but have unclear functions. In this study, we inactivated hcsA alone, hcsB alone, and both hcsA and hcsB together and examined the effects of these mutations on polysaccharide transport and bacterial virulence properties. Inactivation of hcsA alone resulted in accumulation of polysaccharide in the periplasm and a partial decrease in surface-associated polysaccharide, whereas inactivation of hcsB alone or of both hcsA and hcsB together resulted in accumulation of polysaccharide in the periplasm and complete loss of surface-associated polysaccharide. All mutations eliminated serum resistance and abrogated bacteremia and mortality in neonatal rats. These results indicate that the hcsA and hcsB gene products have complementary functions involved in the transport of polysaccharide across the outer membrane and are essential for virulence.

KpsM is an integral membrane protein involved in the translocation of the polysialic acid capsule of Escherichia coli K1. The kpsMG93E allele is a point mutation in the first cytoplasmic loop (Cl) of KpsM which partially disrupts translocation of the capsule. While producing polymer of wild-type length, strains harboring the G93E allele exhibit a decreased production of capsular polymer and a reduced rate of polymer translocation to the cell surface.

SUMMARY: Biosynthesis of the Escherichis coli K5 polysaccharide requires the Kf iA, Kf iB, KfiC and KfiD proteins. The subsequent transport of the polysaccharide onto the cell surface requires the KpsC, KpsD, KpsE, KpsM, KpsS and KpsT proteins, which are conserved between different group II capsular polysaccharides. The KfiA and KfiC, together with the KpsC, KpsS and KpsT proteins, were purified and polyclonal antisera to each protein generated. These antisera, together with one previously generated (by others) against the purified KfiD protein, were used in Western blot analysis to locate the corresponding proteins within the cell. Analysis of membrane fractions revealed that KfiA (involved in initiation of polysaccharide synthesis), Kf iC (K5 glycosyl transferase) and the Kf iD protein (UDP-glucose dehydrogenase) were associated with the inner membrane. The KpsC, KpsS and KpsT proteins involved in polysaccharide transport were associated with the inner membrane and this membrane association occurred in the absence of any other capsule-related proteins. The effect of mutations in individual kps genes on the localization of each protein was determined. Mutations in the kpC# kpsM, kpsS and kpsT genes resulted in a loss of membrane targeting for KfiA and KfiC, suggesting some form of hetero-oligomeric membrane-bound biosynthetic complex. Osmotic shock caused the release of KfiA, KfiC, KpsC and KpsS from the inner membrane into the periplasm, suggesting that the polysaccharide biosynthetic complex may be associated with sites of adhesion between the inner and outer membrane.

Streptococcus suis serotype 16 can infect pigs and humans. We describe the identification and the characterization of the capsular polysaccharides synthesis locus of S. suis serotype 16. Using PCR primers flanking the capsular polysaccharides synthesis locus, a 30,101-bp fragment was amplified. Twenty-nine open reading frames related to transcriptional regulation, glycosyl transfer, oligosaccharide repeat unit polymerization, polysaccharide transport, sialic acid synthesis and modification were identified. The data suggests that the serotype 16 capsule is synthesized by a Wzy-dependent pathway. So far, no rapid and sensitive diagnostic method is available for detection of serotype 16 isolates. A serotype specific PCR test for the rapid and sensitive detection of S. suis serotype 16 was developed. Cross hybridization experiments of individual cps genes with chromosomal DNAs of 33 serotypes showed that the cps16G and cps16K genes hybridized with serotype 16 only. Primers based on cps16G were used to develop a serotype 16 specific PCR. The PCR assay was successfully used to identify S. suis serotype 16 in the 99 Chinese S. suis clinical isolates and 8 European isolates.

The gene cluster of the capsular K5 polysaccharide, a representative of group II capsular antigens of Escherichia coli, has been cloned previously, and three regions responsible for polymerization and surface expression have been defined (I.S. Roberts, R. Mountford, R. Hodge, K. B. Jann, and G. J. Boulnois, J. Bacteriol. 170:1305-1330, 1988). Region 1 has now been sequenced, and five open reading frames (kpsEDUCS) have been defined (C. Pazzani, C. Rosenow, G. J. Boulnois, D. Bronner, K. Jann, and I. S. Roberts, J. Bacteriol. 175:5978-5983, 1993). In this study, we characterized region 1 mutants by immunoelectron microscopy, membrane-associated polymerization activity, cytoplasmic CMP-2-keto-3-deoxyoctonate (KDO) synthetase activity, and chemical analysis of their K5 polysaccharides. Certain mutations within region 1 not only effected polysaccharide transport (lack of region 1 gene products) but also impaired the polymerization capacity of the respective membranes, reflected in reduced amounts of polysaccharide but not in its chain length. KDO and phosphatidic acid (phosphatidyl-KDO) substitution was found with extracellular and periplasmic polysaccharide and not with cytoplasmic polysaccharide. This and the fact that the K5 polysaccharide is formed in a kpsU mutant (defective in capsule-specific K-CMP-KDO synthetase) showed that CMP-KDO is engaged not in initiation of polymerization but in translocation of the polysaccharide.

The moderately halophilic strainHalomonas mauraS-30 produces a high-molecular-mass acidic polymer (4·7×106 Da) composed of repeating units of mannose, galactose, glucose and glucuronic acid. This exopolysaccharide (EPS), known as mauran, has interesting functional properties that make it suitable for use in many industrial fields. Analysis of the flanking regions of a mini-Tn5insertion site in an EPS-deficient mutant ofH. maura, strain TK71, led to the identification of five ORFs (epsABCDJ), which form part of a gene cluster (eps) with the same structural organization as others involved in the biosynthesis of group 1 capsules and some EPSs. Conserved genetic features were found such as JUMPstart andopselements, which are characteristically located preceding the gene clusters for bacterial polysaccharides. On the basis of their amino-acid-sequence homologies, their putative hydropathy profiles and the effect of their mutations, it is predicted that EpsA (an exporter-protein homologue belonging to the OMA family) and EpsC (a chain-length-regulator homologue belonging to the PCP family) play a role in the assembly, polymerization and translocation of mauran. The possibility that mauran might be synthesized via a Wzy-like biosynthesis system, just as it is for many other polysaccharides, is also discussed. This hypothesis is supported by the fact that EpsJ is homologous with some members of the PST-exporter-protein family, which seems to function together with each OMA–PCP pair in polysaccharide transport in Gram-negative bacteria, transferring the assembled lipid-linked repeating units from the cytoplasmic membrane to the periplasmic space. Maximum induction of theepsgenes is reached during stationary phase in the presence of 5 % (w/v) marine salts.

The pssT gene was identified as the fourth gene located upstream of the pssNOP gene cluster possibly involved in the biosynthesis, polymerization, and transport of exopolysaccharide (EPS) in Rhizobium leguminosarum bv. trifolii strain TA1. The hydropathy profile and homology searches indicated that PssT belongs to the polysaccharide-specific transport family of proteins, a component of the type I system of the polysaccharide transport. The predicted membrane topology of the PssT protein was examined with a series of PssT-PhoA fusion proteins and a complementary set of PssT-LacZ fusions. The results generally support a predicted topological model for PssT consisting of 12 transmembrane segments, with amino and carboxyl termini located in the cytoplasm. A mutant lacking the C-terminal part of PssT produced increased amounts of total EPS with an altered distribution of high- and low-molecular-weight forms in comparison to the wild-type RtTA1 strain. The PssT mutant produced an increased number of nitrogen fixing nodules on clover.

The capsular polysaccharides (CPS) play a major role in pathogenicity of Actinobacillus pleuroIpneumoniae , the causative agent of porcine pleuropneumonia. The purpose of the present study was to isolate a mutant in CPS biosynthesis by using a mini-Tn 10 transposon mutagenesis system and evaluate its adherence to host cells. One mutant apparently did not possess CPS as it did not react with a monoclonal antibody against A. pleuropneumoniae serotype 1 capsular antigen. Absence of capsule was confirmed by flow cytometry and also by transmission electron microscopy after polycationic ferritin labelling. The site of insertion of the mini-Tn 10 was determined and found to be in the cpxC gene. Its gene product, CpxC, is a protein involved in polysaccharide transport across the cytoplasmic membrane during CPS biosynthesis. Use of piglet tracheal frozen sections indicated that the CPS mutant adhered significantly ( P =0.0001) more than the parent strain. The non-capsular mutant was less virulent in pigs compared to the parent strain and showed no mortality in experimentally infected pigs. The CPS mutant was however resistant to pig serum. This CPS mutant is the first A. pleuropneumoniae mutant in a CPS transport gene. It is also the first time that adherence of a CPS mutant of A. pleuropneumoniae is evaluated. Our observations indicate that capsular polysaccharides of A. pleuropneumoniae serotype 1 are not involved in adherence to piglet tracheal frozen sections but rather mask, at least in part, the adhesive functions.

A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf ) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O -acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

Analysis of the Escherichia coli K10 capsule gene cluster identified two regions, regions 1 and 3, conserved between different group III capsule gene clusters. Region 1 encodes homologues of KpsD, KpsM, KpsT, and KpsE proteins, and region 3 encodes homologues of the KpsC and KpsS proteins. An rfaH mutation abolished K10 capsule production, suggesting that expression of the K10 capsule was regulated by RfaH in a manner analogous to group II capsule gene clusters. An IS 3 element and a φR73-like prophage, both of which may have played a role in the acquisition of group III capsule gene clusters, were detected flanking the K10 capsule genes.

Background Synergistic action of different enzymes is required to complete the degradation of plant biomass in order to release sugars which are useful for biorefining. However, the use of single strains is often not efficient, as crucial parts of the required enzymatic machinery can be absent. The use of microbial consortia bred on plant biomass is a way to overcome this hurdle. In these, secreted proteins constitute sources of relevant enzyme cocktails. Extensive analyses of the proteins secreted by effective microbial consortia will contribute to a better understanding of the mechanism of lignocellulose degradation. Results Here, we report an analysis of the proteins secreted by a microbial consortium (metasecretome) that was grown on either wheat straw (RWS), xylose or xylan as the carbon sources. Liquid chromatography–tandem mass spectrometry was used to analyze the proteins in the supernatants. Totals of 768 (RWS), 477 (xylose) and 103 (xylan) proteins were identified and taxonomically and functionally classified. In RWS, the proteins were mostly affiliated with Sphingobacterium-like consortium members (~50 %). Specific abundant protein clusters were predicted to be involved in polysaccharide transport and/or sensing (TonB-dependent receptors). In addition, proteins predicted to degrade plant biomass, i.e. endo-1,4-beta-xylanases, alpha-l-arabinofuranosidases and alpha-l-fucosidases, were prominent. In the xylose-driven consortium, most secreted proteins were affiliated with those from Enterobacteriales (mostly Klebsiella species), whereas in the xylan-driven one, they were related to Flavobacterium-like ones. Notably, the metasecretomes of the consortia growing on xylose and xylan contained proteins involved in diverse metabolic functions (e.g. membrane proteins, isomerases, dehydrogenases and oxidoreductases). Conclusions An analysis of the metasecretomes of microbial consortia originating from the same source consortium and subsequently bred on three different carbon sources indicated that the major active microorganisms in the three final consortia differed. Importantly, diverse glycosyl hydrolases, predicted to be involved in (hemi)cellulose degradation (e.g. of CAZy families GH3, GH10, GH43, GH51, GH67 and GH95), were identified in the RWS metasecretome. Based on these results, we catalogued the RWS consortium as a true microbial enzyme factory that constitute an excellent source for the production of an efficient enzyme cocktail for the pretreatment of plant biomass. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0387-8) contains supplementary material, which is available to authorized users.