Complete Lipooligosaccharide Structure from Pseudoalteromonas nigrifaciens Sq02-Rifr and Study of Its Immunomodulatory Activity

Lipopolysaccharides (LPS) are surface glycoconjugates embedded in the external leaflet of the outer membrane (OM) of the Gram-negative bacteria. They consist of three regions: lipid A, core oligosaccharide (OS), and O-specific polysaccharide or O-antigen. Lipid A is the glycolipid endotoxin domain that anchors the LPS molecule to the OM, and therefore, its chemical structure is crucial in the maintenance of membrane integrity in the Gram-negative bacteria. In this paper, we reported the characterization of the lipid A and OS structures from Pseudoalteromonas nigrifaciens Sq02-Rifr, which is a psychrotrophic Gram-negative bacterium isolated from the intestine of Seriola quinqueradiata. The immunomodulatory activity of both LPS and lipid A was also examined.

Discovery of a novel glucuronan lyase system in Trichoderma parareesei

Glucuronan lyases (EC 4.2.2.14) catalyze depolymerization of linear β-(1, 4)-polyglucuronic acid (glucuronan). Only a few glucuronan lyases have been characterized until now, most of them originating from bacteria. Here we report the discovery, recombinant production, and functional characterization of the full complement of six glucuronan specific polysaccharide lyases in the necrotic mycoparasite Trichoderma parareesei . The enzymes belong to four different polysaccharide lyase families and have different reaction optima and glucuronan degradation profiles. Four of them showed endo-lytic action and two, TpPL8A and TpPL38A, displayed exo-lytic action. NMR revealed that the monomeric end-product from TpPL8A and TpPL38A underwent spontaneous rearrangements to tautomeric forms. Proteomic analysis of the secretomes from T. parareesei growing on pure glucuronan and lyophilized A. bisporus fruiting bodies, respectively, showed secretion of five of the glucuronan lyases and HPAEC-PAD analysis confirmed the presence of glucuronic acid in the A. bisporus fruiting bodies. By systematic genome annotation of more than 100 fungal genomes and subsequent phylogenetic analysis of the putative glucuronan lyases, we show that glucuronan lyases occur in several ecological and taxonomic groups in the fungal kingdom. Our findings suggest that a diverse repertoire of glucuronan lyases is a common trait among Hypocreales spp. with mycoparasitic and entomopathogenic lifestyles. IMPORTANCE This paper reports the discovery of a set of six complementary glucuronan lyase enzymes in the mycoparasite Tricoderma parareseei . Apart from the novelty of the discovery of these enzymes in T. parareesei, the key importance of the study is the finding that the majority of these lyases are induced when T. parareseei is inoculated on Basidiomycete cell walls that contain glucuronan. The study also reveals putative glucuronan lyase encoding genes in a wealth of other fungi that furthermore points at fungal cell wall glucuronan being a target C-source for many types of fungi. In a technical context, the findings may lead to controlled production of glucuronan oligomers for advanced pharmaceutical applications and pave the way for development of new fungal biocontrol agents.

Oral Bacteriophages

Bacteriophage or phage therapy involves using phages or their products as bio-agents for the treatment or prophylaxis of bacterial infections or diseases. Bacteriophages have the ability to regulate the oral microflora by lysing sensitive bacterial cells and releasing bacterial components with pro-inflammatory activity. Bacteriophages carry specific polysaccharide depolymerases that aid viral penetration and can disrupt the pathogenic process associated with biofilm and exopolysaccharide in the oral cavity. Oral diseases are mainly caused by biofilm forming microorganisms and phages are now being used for biocontrol of oral biofilms. Phages for Actinomyces species, Aggregatibacter actinomycetemcomitans, Enterococcus faecalis, Fusobacterium nucleatum, Lactobacillus species, Neisseria species, Streptococcus species, and Veillonella species have been isolated and characterized. Bacteriophages could be considered as potential therapeutic tools for the elimination of caries, periodontitis, and other diseases of the oral cavity.

Exopolysaccharides Producing Lactic Acid Bacteria in Wine and Other Fermented Beverages: For Better or for Worse?

Lactic acid bacteria (LAB) from fermented beverages such as wine, cider and beer produce a wide range of exopolysaccharides (EPS) through multiple biosynthetic pathways. These extracellular polysaccharides constitute key elements for bacterial species adaptation to such anthropic processes. In the food industry, LAB polysaccharides have been widely studied for their rheological, functional and nutritional properties; however, these have been poorly studied in wine, beer and cider until recently. In this review, we have gathered the information available on these specific polysaccharide structure and, biosynthetic pathways, as well as the physiology of their production. The genes associated with EPS synthesis are also presented and compared. Finally, the possible role of EPS for bacterial survival and spread, as well as the risks or possible benefits for the winemaker and the wine lover, are discussed.

Structure of the 4-O-[1-Carboxyethyl]-d-Mannose-Containing O-Specific Polysaccharide of a Halophilic Bacterium Salinivibrio sp. EG9S8QL

The moderately halophilic strain Salinivibrio sp. EG9S8QL was isolated among 11 halophilic strains from saline mud (Emisal Salt Company, Lake Qarun, Fayoum, Egypt). The lipopolysaccharide was extracted from dried cells of Salinivibrio sp. EG9S8QL by the phenol–water procedure. The OPS was obtained by mild acid hydrolysis of the lipopolysaccharide and was studied by sugar analysis along with 1H and 13C NMR spectroscopy, including 1H,1H COSY, TOCSY, ROESY, 1H,13C HSQC, and HMBC experiments. The OPS was found to be composed of linear tetrasaccharide repeating units of the following structure: →2)-β-Manp4Lac-(1→3)-α-ManpNAc-(1→3)-β-Rhap-(1→4)-α-GlcpNAc-(1→, where Manp4Lac is 4-O-[1-carboxyethyl]mannose.

Lipopolysaccharide of Pseudomonas mandelii, Isolated from Antarctica

Representatives of the Pseudomonas mandelii species are able to exist and multiply in places where the temperature is constantly low. The optimum growth temperature for P. mandelii is 25–30°C, although this bacterium can grow at 4°C but not at 37°C. Therefore, P. mandelii is an excellent example of psychrotolerant bacterium which like psychrophilic bacteria is characterized by a number of structural and functional adaptations that facilitate survival at low temperatures. To understand these microorganisms’ role in Antarctica the characterization of its biopolymers is vital. One of these biopolymers is lipopolysaccharide (LPS), composition and structure of which are diagnostically significant. This determines the aim of the work – to isolate lipopolysaccharides from the cells of Antarctic strain of P. mandelii, grown at different temperatures, to characterize them chemically, and to study their functional and biological activity. Methods. The object of the study was Pseudomonas sp. U1, isolated from moss on Galindez Island in Antarctica. Lipopolysaccharides were extracted from dried cells by 45% phenol water solution at 65–68°С by Westphal and Jann method. The amount of carbohydrates was determined by phenol-sulfuric method. Carbohydrate content was determined in accordance to the calibration curve, which was built using glucose as a standard. The content of nucleic acids was determined by Spirin, protein − by Lowry method. Serological activity of LPS was investigated by double immunodiffusion in agar using the method of Ouchterlony. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAAG electrophoresis) was performed according to Laemmli. Results. As a result of phylogenetic analysis (programs ClustalX 2.1, Tree view, Mega v. 6.00) it was shown that the Antarctic bacterial strain Pseudomonas sp. U1 associated with green moss has a 99.4% homology with the type strain from the GenBank database NR024902 P. mandelii CIP 105273T. According to these data and proximity to the corresponding cluster of species, the studied isolate can be identified as P. mandelii. A characteristic feature of LPS isolated from P. mandelii cells, grown at different temperatures (20°C and 4°C) is their heterogeneity. This is evidenced by the data of the monosaccharide composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. The R-form of LPS is also present, which does not contain an O-specific polysaccharide chains. Structural heterogeneity is also inherent in LPS lipid A. This is evidenced by the data of the fatty acid composition. In LPS grown at 4°C no unsaturated fatty acids were found, while such ones are synthesized in LPS of other bacteria grown in the cold, in response to a decrease in growth temperature. The study of the immunochemical properties of LPS was carried out using polyclonal O-antisera as antibodies, and LPS as antigens indicated that in homologous systems LPS exhibited serological activity. LPS obtained from P. mandelii U1 cells, grown at 20°C, had a complex antigenic composition and gave two clear lines of precipitation at a concentration of 1 mg/mL. LPS obtained from P. mandelii U1 cells, grown at 4°C, gave one line, which indicates their serological homogeneity. Conclusions. For the first time lipopolysaccharides were isolated from cells of P. mandelii U1, grown at 4°C and 20°С. A characteristic feature of these LPS is their heterogeneity. This is evidenced by the data of the monosaccharide and fatty acid composition, electrophoretic distribution, which showed that P. mandelii produces S- and SR-forms of LPS, differed in the length of the O-specific polysaccharide chains. LPS, obtained from cells, grown at different temperatures, are differed by serological activity.

Sift-PULs: A public repository for specific functional polysaccharide utilization loci

Background Polysaccharide utilization loci (PULs) were bacterial gene clusters encoding genes responsible for polysaccharide utilization process. PUL studies are blooming in recent years but the biochemical characterization speed is relative slow. There is a growing demand for PUL database with function annotations. Results Using signature genes corresponding for specific polysaccharide, 10422 PULs specific for 6 polysaccharides (agar, alginate, pectin, carrageenan, chitin and β-manan) from various bacterial phyla were predicted. Then online website of specific functional polysaccharide utilization loci (Sift-PULs) was constructed. Sift-PULs provides a repository where users could browse, search and download interested PULs without registration. Conclusions The key advantage of Sift-PULs is to assign a function annotation of each PUL, which is not available in existing PUL databases. PUL's functional annotation lays a foundation for studying novel enzymes, new pathways, PUL evolution or bioengineering. The website is available on http://sift-puls.org

Defining Polysaccharide-Specific Antibody Targets against Vibrio cholerae O139 in Humans following O139 Cholera and following Vaccination with a Commercial Bivalent Oral Cholera Vaccine, and Evaluation of Conjugate Vaccines Targeting O139

Cholera is a severe dehydrating illness of humans caused by Vibrio cholerae serogroup O1 or O139. Protection against cholera is serogroup specific, and serogroup specificity is defined by O-specific polysaccharide (OSP).