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Exopolysaccharide (EPS) is a general term for the forms of bacterial polysaccharides found outside the cell wall of bacteria. The industrial microbiologist has become interested in exploiting microbial exopolymer production due to an increasing interest and need for novel polysaccharides. The use of bacterial EPS in acid milk curd (AMC) production accounts for a quality improvement of the milk curd in terms of stability during the normal operations of storage and transportation. On the other hand, EPS have been used as prebiotic because of its health-promoting effects in human beings. The objective of this study was to evaluate the rheological properties (RP) and their statistical correlations in acid milk curd (AMC) samples added with bacterial EPS. Rheometric properties are suitable parameters to evaluate food quality such as AMC. Some of the RP normally used in this evaluation include elastic modulus (EM), hardness (HR), breaking energy (BE), viscosity (VIS) and stress. Correlation between pairs of these parameters are important to understand in order to predict statistically other parameters in case they cannot be easily determined. Acid milk curd samples were formulated using 10, 15, and 20 % of reconstituted skim milk (RSM), to which 0, 0.2, 0.4, 0.6, 0.8, and 1 % level of EPS were added, respectively. Samples were pasteurized at 60 [degrees] C for 30 min, and at 85 [degrees]C for 30s. Afterwards, AMC samples were tempered to 40 [degrees]C in[degrees]Culated with Lactobacillus delbrueckii subsp. bulgaricus B-5b, and incubated at 37 [degrees]C for 16 h. Results indicated that viscosity and hardness showed the strongest and highest correlation among all the parameters evaluated (r=0.9272) and it was comparable to that between viscosity and breaking energy (r=0.8349) and breaking energy and hardness (r=0.7694). However, elastic modulus and viscosity showed a rather low correlation (r=0.5394) and very low correlation (r=0.1830), respectively, suggesting that estimation of elastic modulus from viscosity values would be inaccurate. In general, rheometric properties of acid milk curd increased with higher EPS concentrations until 1 % level of EPS addition. Therefore, it is recommended to use BE and HR values, and BE and VIS values to predict accurate measurements of other RP values. Key words: Exopolysaccharide (EPS), acid milk curd (AMC), rheometric properties (RP), statistical correlation (SC), INTRODUCTION The term 'exopolysaccharides' (EPS) was proposed by Sutherland in 1972 and provides a general term for all forms of bacterial polysaccharides found outside of the cell wall [1,2,3,4,5,6,7]. The [...]

Sequence analysis of the large virulence plasmid pLVPK in Klebsiella pneumoniae CG43 revealed the presence of another mucoid factor encoding gene rmpA besides rmpA2. Promoter activity measurement indicated that the deletion of rmpA reduced K2 capsular polysaccharide (CPS) biosynthesis, resulting in decreased colony mucoidy and virulence in mice. Introduction of a multicopy plasmid carrying rmpA restored CPS production in the rmpA or rmpA2 mutant but not in the rcsB mutant. Transformation of the rmpA deletion mutant with an rcsB-carrying plasmid also failed to enhance CPS production, suggesting that a cooperation of RmpA with RcsB is required for regulatory activity. This was further corroborated by the demonstration of in vivo interaction between RmpA and RcsB using two-hybrid analysis and coimmunoprecipitation analysis. A putative Fur binding box was only found at the 5' noncoding region of rmpA. The promoter activity analysis indicated that the deletion of fur increased the rmpA promoter activity. Using electrophoretic mobility shift assay, we further demonstrated that Fur exerts its regulatory activity by binding directly to the promoter. As a result, the fur deletion mutant exhibited an increase in colony mucoidy, CPS production, and virulence in mice. In summary, our results suggested that RmpA activates CPS biosynthesis in K. pneumoniae CG43 via an RcsB-dependent manner. The expression of rmpA is regulated by the availability of iron and is negatively controlled by Fur. doi: 10.1128/JB.00031-10

Streptococcus mutans is a key contributor to the formation of the extracellular polysaccharide (EPS) matrix in dental biofilms. The exopolysaccharides, which are mostly glucans synthesized by streptococcal glucosyl-transferases (Gtfs), provide binding sites that promote accumulation of microorganisms on the tooth surface and further establishment of pathogenic biofilms. This study explored (i) the role of S. mutans Gtfs in the development of the EPS matrix and microcolonies in biofilms, (ii) the influence of exopolysaccharides on formation of microcolonies, and (iii) establishment of S. mutans in a multispecies biofilm in vitro using a novel fluorescence labeling technique. Our data show that the ability of S. mutans strains defective in the gtfB gene or the gtfB and gtfC genes to form microcolonies on saliva-coated hydroxyapatite surfaces was markedly disrupted. However, deletion of both gtfB (associated with insoluble glucan synthesis) and gtfC (associated with insoluble and soluble glucan synthesis) is required for the maximum reduction in EPS matrix and biofilm formation. S. mutans grown with sucrose in the presence of Streptococcus oralis and Actinomyces naeslundii steadily formed exopolysaccharides, which allowed the initial clustering of bacterial cells and further development into highly structured microcolonies. Concomitantly, S. mutans became the major species in the mature biofilm. Neither the EPS matrix nor microcolonies were formed in the presence of glucose in the multispecies biofilm. Our data show that GtfB and GtfC are essential for establishment of the EPS matrix, but GtfB appears to be responsible for formation of microcolonies by S. mutans; these Gtf-mediated processes may enhance the competitiveness of S. mutans in the multispecies environment in biofilms on tooth surfaces. doi: 10.1128/JB.01649-09

The effect of four sugars (glucose, galactose, lactose and fructose) on exopolysaccharide (EPS) production by Bifidobacterium longum subsp, longum CRC 002 was evaluated. More EPS was produced when CRC 002 was grown on lactose in the absence of pH control, with a production of 1080 [+ or -] 120 mg EPS [I.sup-1] (P DOI 10.1099/mic.0.033720-0

Sinorhizobium meliloti is a soil bacterium that elicits the formation of root organs called nodules on its host plant, Medicago sativa. Inside these structures, the bacteria are able to convert atmospheric nitrogen into ammonia, which is then used by the plant as a nitrogen source. The synthesis by S. meliloti of at least one exopolysaccharide, succinoglycan or EPS II, is essential for a successful symbiosis. While exopolysaccharide-deficient mutants induce the formation of nodules, they fail to invade them, and as a result, no nitrogen fixation occurs. Interestingly, the low-molecular-weight fractions of these exopolysaccharides are the symbiotically active forms, and it has been suggested that they act as signals to the host plant to initiate infection thread formation. In this work, we explored the role of these rhizobial exopolysaccharides in biofilm formation and their importance in the symbiotic relationship with the host. We showed that the ExpR/Sin quorum-sensing system controls biofilm formation in S. meliloti through the production of EPS II, which provides the matrix for the development of structured and highly organized biofilms. Moreover, the presence of the low-molecular-weight fraction of EPS II is vital for biofilm formation, both in vitro and in vivo. This is the first report where the symbiotically active fraction of EPS II is shown to be a critical factor for biofilm formation and root colonization. Thus, the ability of S. meliloti to properly attach to root surfaces and form biofilms conferred by the synthesis of exopolysaccharides may embody the main function of these symbiotically essential molecules. doi: 10.1128/JB.01063-09

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of gram-negative bacteria that serves as a barrier against harmful molecules, including antibiotics. The waaYZ locus that encodes the LPS core biosynthetic function in Escherichia coli was found to be induced strongly by superoxide generators but not by [H.sub.2][O.sub.2], ethanol, or heat shock. This induction was dependent on SoxRS, a superoxide and nitric oxide sensing system, through a soxbox in the waaY promoter that binds SoxS. A [DELTA]waaYZ mutant became more sensitive to some superoxide generators, and the activation of SoxR by these drugs became more sensitized in the mutant. Through phenotypic microarray analysis, we found that the mutant became sensitive to a wide variety of chemicals not restricted to oxidizing agents. We found that the mutant is under envelope stress and is altered in LPS composition, as monitored by the level of [[sigma].sup.E] activation and changes in the electrophoretic mobility of LPS, respectively, waaY expression was also regulated by MarA (multiple-antibiotic resistance regulator), which shares a binding site (soxbox) with SoxS, and was induced by salicylate, a nonoxidative compound. These results demonstrate a novel way of protecting gram-negative bacteria against various compounds by modifying LPS, possibly through phosphorylation. Since either oxidant or nonoxidant compounds elicit resistance toward themselves and other toxic drugs, this mechanism could serve as an efficient way for pathogenic bacteria to enhance survival during antibiotic treatment within an oxidant-rich host immune environment.

The periodontopathic bacterium Porphyromonas gingivalis forms pigmented colonies when incubated on blood agar plates as a result of accumulation of [mu]-oxo haem dimer on the cell surface. Gingipain--adhesin complexes are responsible for production of [mu]-oxo haem dimer from haemoglobin. Non-pigmented mutants (Tn6-5, Tn7-1, Tn7-3 and Tn10-4) were isolated from P. gingivalis by Tn4351 transposon mutagenesis [Hoover & Yoshimura (1994), FEMS Microbiol Lett 124, 43-48]. In this study, we found that the Tn6-5, Tn7-1 and Tn7-3 mutants carried Tn4351 DNA in a gene homologous to the ugdA gene encoding UDP-glucose 6-dehydrogenase, a gene encoding a putative group 1 family glycosyltransferase and a gene homologous to the rfa gene encoding ADP heptose-LPS heptosyltransferase, respectively. The Tn10-4 mutant carried Tn4351 DNA at the same position as that for Tn7-1. Gingipain activities associated with cells of the Tn7-3 mutant (rfa) were very weak, whereas gingipain activities were detected in the culture supernatants. Immunoblot and mass spectrometry analyses also revealed that gingipains, including their precursor forms, were present in the culture supernatants. A lipopolysaccharide (LPS) fraction of the rfa deletion mutant did not show the ladder pattern that was usually seen for the LPS of the wild-type P. gingivalis. A recombinant chimera gingipain was able to bind to an LPS fraction of the wild-type P. gingivalis in a dose-dependent manner. These results suggest that the rfa gene product is associated with biosynthesis of LPS and/or cell-surface polysaccharides that can function as an anchorage for gingipain--adhesin complexes.

The obligate anaerobe Bacteroides fragilis is a normal resident of the human gastrointestinal tract. The clinically derived B. fragilis strain NCTC 9343 produces an extensive array of extracellular polysaccharides (EPS), including antigenically distinct large, small and micro- capsules. The genome of NCTC 9343 encodes multiple gene clusters potentially involved in the biosynthesis of EPS, eight of which are implicated in production of the antigenically variable micro-capsule. We have developed a rapid and robust method for generating marked and markerless deletions, together with efficient electroporation using unmodified plasmid DNA to enable complementation of mutations. We show that deletion of a putative wzz homologue prevents production of high-molecular-mass polysaccharides (HMMPS), which form the micro-capsule. This observation suggests that micro-capsule HMMPS constitute the distal component of LPS in B. fragilis. The long chain length of this polysaccharide is strikingly different from classical enteric O-antigen, which consists of short-chain polysaccharides. We also demonstrate that deletion of a putative wbaP homologue prevents expression of the phase-variable large capsule and that expression can be restored by complementation. This suggests that synthesis of the large capsule is mechanistically equivalent to production of Escherichia coil group 1 and 4 capsules.

In Escherichia coli, FtsH (HflB) is a membrane-bound, ATP-dependent metalloendoprotease belonging to the AAA family (ATPases associated with diverse cellular activities). FtsH has a limited spectrum of known substrates, including the transcriptional activator [[sigma].sup.32]. FtsH is the only known E. coli protease that is essential, as it regulates the concentration of LpxC, which carries out the first committed step in the synthesis of lipid A. Here we identify a new FtsH substrate--3-deoxy-o-manno-octulosonate (KDO) transferase--which carries out the attachment of two KDO residues to the lipid A precursor (lipid [IV.sub.A]) to form the minimal essential structure of the lipopolysaccharide (LPS) ([KDO.sub.2]-lipid A). Thus, FtsH regulates the concentration of the lipid moiety of LPS (lipid A) as well as the sugar moiety (KDO-based core oligosaccharides), ensuring a balanced synthesis of LPS.

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the Bacillus subtilis teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations. In this work we explored the acceptor substrate promiscuity and polymer length disregulation that appear to be characteristic of TagF activity away from biological membranes. Using isotope labeling, steady-state kinetics, and chemical lability studies, we demonstrated that the enzyme can synthesize poly(glycerol phosphate) teichoic acid using the elongation substrate CDP-glycerol as an acceptor. This suggests that substrate specificity is relaxed in the region distal to the glycerol phosphate moiety in the acceptor molecule under these conditions. Polymer synthesis proceeded at a rate (27 [min.sup.-1]) comparable to that in the reconstituted membrane system after a distinct lag period which likely represented slower initiation on the unnatural CDP-glycerol acceptor. We confirmed that polymer length became disregulated in the soluble system as the polymers synthesized on CDP-glycerol acceptors were much larger than the polymers synthesized on the membrane or previously found attached to bacterial cell walls. Finally, polymer synthesis on protease-treated membranes suggested that proper length regulation is retained in the absence of accessory proteins and provided evidence that such regulation is conferred through proper association of the polymerase with the membrane.

The Rhizobium leguminosarum biovar viciae genome contains several genes predicted to determine surface polysaccharides. Mutants predicted to affect the initial steps of polysaccharide synthesis were identified and characterized. In addition to the known cellulose (cel) and acidic exopolysaccharide (EPS) (pss) genes, we mutated three other loci; one of these loci (gmsA) determines glucomannan synthesis and one (gelA) determines a gel-forming polysaccharide, but the role of the other locus (an exoY-like gene) was not identified. Mutants were tested for attachment and biofilm formation in vitro and on root hairs; the mutant lacking the EPS was defective for both of these characteristics, but mutation of gelA or the exoY-like gene had no effect on either type of attachment. The cellulose (celA) mutant attached and formed normal biofilms in vitro, but it did not form a biofilm on root hairs, although attachment did occur. The cellulose-dependent biofilm on root hairs appears not to be critical for nodulation, because the celA mutant competed with the wild-type for nodule infection. The glucomannan (gmsA) mutant attached and formed normal biofilms in vitro, but it was defective for attachment and biofilm formation on root hairs. Although this mutant formed nodules on peas, it was very strongly outcompeted by the wild type in mixed inoculations, showing that glucomannan is critical for competitive nodulation. The polysaccharide synthesis genes around gmsA are highly conserved among other rhizobia and agrobacteria but are absent from closely related bacteria (such as Brucella spp.) that are not normally plant associated, suggesting that these genes may play a wide role in bacterium-plant interactions.

Lipopolysaccharide (LPS) is an essential component of the outer membrane (OM) in most gram-negative bacteria, and its structure and biosynthetic pathway are well known. Nevertheless, the mechanisms of transport and assembly of this molecule at the cell surface are poorly understood. The inner membrane (IM) transport protein MsbA is responsible for flipping LPS across the IM. Additional components of the LPS transport machinery downstream of MsbA have been identified, including the OM protein complex LptD/LptE (formerly Imp/RlpB), the periplasmic LptA protein, the IM-associated cytoplasmic ATP binding cassette protein LptB, and LptC (formerly YrbK), an essential IM component of the LPS transport machinery characterized in this work. Here we show that depletion of any of the proteins mentioned above leads to common phenotypes, including (i) the presence of abnormal membrane structures in the periplasm, (ii) accumulation of de novo-synthesized LPS in two membrane fractions with lower density than the OM, and (iii) accumulation of a modified LPS, which is ligated to repeating units of colanic acid in the outer leaflet of the IM. Our results suggest that LptA, LptB, LptC, LptD, and LptE operate in the LPS assembly pathway and, together with other as-yet-unidentified components, could be part of a complex devoted to the transport of LPS from the periplasmic surface of the IM to the OM. Moreover, the location of at least one of these five proteins in every cellular compartment suggests a model for how the LPS assembly pathway is organized and ordered in space.

The Wzz proteins are important for determining the length of the ()-antigen side chain attached to lipopolysaecharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wildtype PA 103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzzl protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.

A study presents evidence that binding of surfactant protein D (SP-D) to R-LPS (rough lipopolysaccharides) is mediated by interactions between the carbohydrate binding site of the carbohydrate recognition domain and the inner core oligosaccharides of the lipopolysaccharide (LPS). Interactions of the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.

ATP-binding-cassette (ABC) transporters are responsible for the export of a wide variety of cell-surface glycoconjugates in both Gram-positive and Gram-negative bacteria. These include the O-antigenic polysaccharide (O-PS) portion of lipopolysaccharide, a crucial virulence determinant in Gram-negative pathogens. O-PSs are synthesized by one of two fundamentally different pathways. Escherichia coli O serotypes O8 and O9a provide the prototype systems for studying O-PS export via ABC transporters. The transporter is composed of the transmembrane component Wzm and the nucleotide-binding component Wzt. Although the N-terminal domain of Wzt is a conventional ABC protein, the C-terminal domain of Wzt (C-Wzt) is a unique structural element that determines the specificity of the transporter for either the O8 or O9a O-PS. We show here that the two domains of Wzt can function when expressed as separate polypeptides; both are essential for export. In vitro, C-Wzt binds its cognate O-PS by recognizing a residue located at the nonreducing end of the polymer. The crystal structure of C-[Wzt.sub.O9a] is reported here and reveals a [beta] sandwich with an immunoglobulin-like topology that contains the O-PS-binding pocket. Substrate interactions with nucleotide-binding domains have been demonstrated in an ABC exporter previously. However, to our knowledge substrate binding by a discrete, cytoplasmic accessory domain in an extended nucleotide-binding domain polypeptide has not previously been demonstrated. Elucidation of the substrate-recognition system involved in O-PS export provides insight into the mechanism that coordinates polymer biosynthesis, termination, and export. lipopolysaccharide | O-antigen biosynthesis | outer membrane | polymer export | polymer recognition

Bordetellae are respiratory pathogens that infect both humans and animals. Bordetella bronchiseptica establishes asymptomatic and long-term to life-long infections of animal nasopharynges. While the human pathogen Bordetella pertussis is the etiological agent of the acute disease whooping cough in infants and young children, it is now being increasingly isolated from the nasopharynges of vaccinated adolescents and adults who sometimes show milder symptoms, such as prolonged cough illness. Although it has been shown that Bordetella can form biofilms in vitro, nothing is known about its biofilm mode of existence in mammalian hosts. Using indirect immunofluorescenee and scanning electron microscopy, we examined nasal tissues from mice infected with B. bronchiseptiea. Our results demonstrate that a wild-type strain formed robust biofilms that were adherent to the nasal epithelium and displayed architectural attributes characteristic of a number of bacterial biofilms formed on inert surfaces. We have previously shown that the Bordetella Bps polysaceharide encoded by the bpsABCD locus is critical for the stability and maintenance of three-dimensional structures of biofilms. We show here that Bps is essential for the formation of efficient nasal biofilms and is required for the colonization of the nose. Our results document a biofilm lifestyle for Bordetella in mammalian respiratory tracts and highlight the essential role of the Bps polysaccharide in this process and in persistence of the nares.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.01.046 Byline: Young-Sun Kang (1), Yoonkyung Do (1), Hae-Kyung Lee (1), Sung Ho Park (1), Cheolho Cheong (1), Rebecca M. Lynch (1), Jutta M. Loeffler (2), Ralph M. Steinman (1), Chae Gyu Park (1) Abstract: The intricate system of serum complement proteins provides resistance to infection. A pivotal step in the complement pathway is the assembly of a C3 convertase, which digests the C3 complement component to form microbial binding C3 fragments recognized by leukocytes. The spleen and C3 provide resistance against blood-borne S. pneumoniae infection. To better understand the mechanisms involved, we studied SIGN-R1, a lectin that captures microbial polysaccharides in spleen. Surprisingly, conditional SIGN-R1 knockout mice developed deficits in C3 catabolism when given S. pneumoniae or its capsular polysaccharide intravenously. There were marked reductions in proteolysis of serum C3, deposition of C3 on organisms within SIGN-R1.sup.+ spleen macrophages, and formation of C3 ligands. We found that SIGN-R1 directly bound the complement C1 subcomponent, C1q, and assembled a C3 convertase, but without the traditional requirement for either antibody or factor B. The transmembrane lectin SIGN-R1 therefore contributes to innate resistance by an unusual C3 activation pathway. Author Affiliation: (1) Laboratory of Cellular Physiology and Immunology and Chris Browne Center for Immunology and Immune Diseases, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA (2) Laboratory of Bacterial Pathogenesis, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA Article History: Received 11 October 2005; Revised 26 December 2005; Accepted 17 January 2006 Article Note: (miscellaneous) Published: April 6, 2006

CpsA, CpsB, CpsC, and CpsD are part of a tyrosine phosphorylation regulatory system involved in modulation of capsule synthesis in Streptococcus pneumoniae and many other gram-positive and gram-negative bacteria. Using an immunoblotting technique, we observed distinct laddering patterns of S. pneumoniae capsular polysaccharides of various serotypes and found that transfer of the polymer from the membrane to the cell wall was independent of size. Deletion of cps2A, cps2B, cps2C, or cps2D in the serotype 2 strain D39 did not affect the ability to transfer capsule to the cell wall. Deletion of cps2C or cps2D, which encode two domains of an autophosphorylating tyrosine kinase, resulted in the production of only short-chain polymers. The function of Cps2A is unknown, and the polymer laddering pattern of the cps2A deletion mutants appeared similar to that of the parent, although the total amount of capsule was decreased. Loss of Cps2B, a tyrosine phosphatase and a kinase inhibitor, resulted in an increase in capsule amount and a normal ladder pattern. However, Cps2B mutants exhibited reduced virulence following intravenous inoculation of mice and were unable to colonize the nasopharynx, suggesting a diminished capacity to sense or respond to these environments. In D39 and its isogenic mutants, the amounts of capsule and tyrosine-phosphorylated Cps2D (Cps2D[tilde]P) correlated directly. In contrast, restoration of type 2 capsule production followed by deletion of cps2B in Rxl, a laboratory passaged D39 derivative containing multiple uncharacterized mutations, resulted in decreased capsule amounts but no alteration in Cps2D[tilde]P levels. Thus, a factor outside the capsule locus, which is either missing or defective in the Rxl background, is important in the control of capsule synthesis.

A new glycoside hydrolase (GH) family 11 xylanase, Xyn11B, is identified in Pseudomonas cellulosa. The genes of the Xyn11B and other GH11 family xylanases may show differential temporal expression but they are all induced by the same polysaccharides. The Xyn11A and Xyn11B are the two major extracellular xylanases..

Research points out that all three of the genes, algI, algJ, and algF are involved in adding O-acetyl groups to alginate by O acetylation. Further, the encoded proteins AlgI, AlgJ, and AlgF appear to form a complex in the membrane where O acetylation reaction occurs.

With the goal of identifying and characterizing traits of Enterococcus faecalis that play key roles in human disease, we identified an operon specifying synthesis of a capsular carbohydrate of the type most commonly expressed by clinical isolates. This surface-exposed carbohydrate consists of glycerol phosphate, glucose, and galactose residues, and its biosynthesis is encoded by a determinant that includes 11 ORFs. Insertional inactivation of genes in this pathway yielded mutants with enhanced susceptibility to phagocytic killing in vitro and compromised in the ability to persist in regional lymph nodes in vivo.

Mutational studies indicate that the homologous polysaccharide biosynthetic protein WbjA adds the terminal glucose to O-antigen unit of the Pseudomonas aeruginosa serogroup O11 lipopolysaccharide.

The gene CaVrg4 is a key regulator of mannan synthesis in Candida albicans and encodes GDP-mannose transporter involved in the formation of Golgi apparatus. Data indicate that a conditional loss of the gene results in lethality, reduction in cell wall mannan, and morphological abnormalities.

The review discusses plant cell wall polysaccharide degrading enzymes by Aspergillus in terms of the genes coding for the enzymes and their genetic regulation. Data show that bulk of these enzymes are highly glycosylated proteins. Industrial applications of these enzymes are also presented.

Results indicate that carbon dioxide reduces the expression of capsular polysaccharide type 5 at the transcriptional level in Staphylococcus aureus. Regulation of capsular polysaccharide type 8 expression is strain dependent, suggesting that genetic background determines the carbon dioxide response.

Research examines the process of septal murein synthesis and segregation in Escherichia coli. Results demonstrate that impairment of penicillin-binding protein 2 and RodA function results in inactivation of sacculus extension and activation of the septal murein biosynthetic process at perioding intervals during each cell cycle event.

Results show that the colanic acid deficient mutant of enterohemorrhagic Escherichia coli serotype O157:H7 W6-13 under environmental stress conditons is less tolerant to acid pH and heat indicating that colanic acid confers protection. Data indicate W6-13 cells tolerate stress and produce large amounts of exopolysaccharide composed of colanic acid.

Results reveal that Erwinia chrysanthemi osmoregulated periplasmic glucans exhibit size heterogeneity and structural similarities to Escherichia coli and Pseudomonas syringae osmoregulated periplasmic glucans. Data indicate that the E. chrysanthemi glucan backbones can be substituted with O-acetyl and O-succinyl ester-linked residues.

Study of the structure of the cell envelope of corynebacteria is discussed with attention to the importance of the noncovalently bound lipids in the formation of the cell wall fracture plane and permeability barrier. Ultrastructural appearance, molecular compositional analysis, and freeze-etch electron microscopy study were used. Most corynomycolate-containing strains had low-molecular-mass porins. The cell wall-permeability barrier in corynebacteria likely involve both covalently linked corynomycolates and noncovalently bound lipids of their cell envelopes.

Results demonstrate that the gene vpsR, which regulates the vps biosynthetic gene cluster, is homologous to response element of two-component regulatory systems involved in external stimuli response. Data further show that the vpsR influences colony morphology, exopolysaccharide synthesis, and biofilm formation.

Research shows that family of carbohydrate-binding modules, comprising complex hydrolytic extracellular bacterial enzymes, possess different polysaccharide-binding specificities. Two modules flank Laminarinase Lam16A from Thermotoga nepolitana, which bind to different glucan molecules such as soluble laminarin and barley glucan at the N-terminus and curdlan, pustulan and yeast glucan at the C-terminus.

Research on Lactococus lactis has produced clues for methods to increase exopolysaccharide (EPS) production via genetic manipulation. Nuclear magnetic resonance was used to determine phosphorylated intermediate concentrations in cell wall and EPS biosynthetic pathways; metabolic targets include an increase in pool size of the lipid carrier and an enhancement of EPS precursor production flux.

Research reveals that the cps gene cluster encodes multiple proteins that are involved in various functions such as polymerization and export of the polysaccharide, sugar transport, and EPS biosynthesis.

Research reveals in Azotobacter vinelandii nitrogenase protection is mediated by alginate. Results demonstrate depending on the oxygen tension in the culture medium, alginate composition varies and an alginate capsule on the cell surface functions as a barrier for oxygen diffusion into the cell.

Research demonstrates that transposon insertion into the ampDE operon enhances the transcription of algD leading to increased production of alginate in Azotobacter vinelandii. Data further suggest that alginate production and bacterial encystment are influenced by cell wall recycling.

Research demonstrates that Sphingomonas grown in the presence of alginate produce oligoalginate lyase which degrades alginate oligosaccharides to monosaccharides. The enzyme is a monomer of 84 kilodalton, cleaves glycosidic bonds, and is involved in total depolymerization of alginate reaction.

Results show that cpsIaH and cpsIIIH genes encode Ia and III capsular polysaccharide polymerases respectively, leading to the synthesis of type-specific capsular polysaccharides. Data point out that these polysaccharides aid in host immune defence mechanism in Streptococcus agalactiae.

Results demonstrate that low-molecular weight polysaccharides are critical for establishing symbiosis with alafalfa by Sinorhizobium meliloti bacterium. Data also indicate that the efficiency of nodulation is dependent on the chemical nature of the polysaccharides with succinoglycans being efficient both in the infection and post-infection processes.

Results indicate that Lon protease in Sinorhizobium meliloti regulates the turnover of constitutively expressed proteins. Data also suggest that such a regulation is essential as otherwise nodule formation and growth of the bacteria are affected.

Results demonstrate that a reduced type 3 spontaneous mutant is involved in the capsule production. Data further point out that most of the phosphoglucomutase activity responsible for type 3 capsule production is derived from the cellular phosphoglucomutase.

Results indicate that glycanases exhibit carboxymethyl cellulose degrading activity and they are released from the Rhizobium cell surface in an inactive form. Data reveal that upon contact with the bacteria the glycanases are activated by an exopolysaccharide-related component.

Results demonstrate the nucleotide sequence of the capsular gene cluster contisting of 12 open reading frames and a 14.7-kb fragment that can transform an unencapsulated type 3 Streptococcus pneumoniae strain to type 8 capsule strain.

Results describe sequencing and analysis of the capsular polysaccharide biosynthetic locus PS B2 in Bacteroides fragilis 638R and NCTC 9343 strains. Data show that two genes of the region wcgD and wcgC encode a UDP-N-acetylglucosamine 2-epimerase and a UDP-N-acetylmannosamine dehydrogenase, respectively.

Initial velocity, product inhibition and dead-end inhibition methods have been used in studying the steady-state dynamics of cytidine 5'-monophosphate N-acetylneuraminic acid (CMP-NeuAc) synthetase. The latter is an activating enzyme that binds sialic acid to the lipooligosaccharide of Haemophilus ducreyi. Results indicate that the reaction proceeds through an ordered bi-bi kinetic mechanism in which CTP binds first and CMP-NeuAc is the last to dissociate.

A study was conducted to examine the mechanism of the adipose tissue inducible nitric oxide synthase (iNOS) in endotoxemia. Male Sprague-dawley rats were injected with bacterial endotoxin lipopolysaccharide (LPS) to increase the iNOS messenger RNA and protein levels of the white and brown adipose tissues. Results suggest that LPS treatment has significantly increased iNOS activity and expression in both the white and brow adipose tissues.

Byline: Kesheng Zhao (1), Irena Kirman (1), Ingomar Tschepen (1), Rise Schwab (1), Marc E. Weksler (1) Abstract: The contribution of peritoneal cells to lipopolysaccharide (LPS)-induced elevation of serum TNF-[alpha] and IL-6 levels and mortality has been studied. Peritoneal lavage performed before LPS administration reduced serum cytokine levels by approximately 50% and mortality from 50 to 100%. The effect of peritoneal lavage is due to the removal of peritoneal cells as reinjection of peritoneal cells eliminated the protective effect of lavage on LPS-induced mortality. A special role of peritoneal macrophages in the systemic response to LPS was suggested by the finding that LPS-induced an increase in intracellular TNF-[alpha] and IL-6 in peritoneal macrophages but in neither splenic nor bone marrow macrophages. Intraperitoneal injection of thioglycollate broth 4 days prior to lavage increased the number of peritoneal cells removed by lavage and increased protection from LPS mortality. Peritoneal lavage performed 30 to 120 minutes after the LPS administration completely protected all mice from LPS-induced mortality, suggesting the possibility that such treatment may offer a novel therapeutic approach to septic shock. Author Affiliation: (1) Division of Geriatrics and Gerontology, Cornell University Medical College, New York, New York, 10021 Article History: Registration Date: 04/09/2004

Escherichia coli lipopolysaccharide (LPS) inhibits the synthesis of nitric oxide (NO) and impairs the activity of nitric oxide synthase (NOS) gene expression in vitro and in vivo. Repeated administration of endotoxins in cell cultures and in whole organisms reduced the abundance of intracellular NOS messenger ribonucleic acid. An endotoxin refractory state for NOS was also detected 12 hours after administration with LPS.

The capsule-specific coliphage K5 of Escherichia coli contains an enzyme that degrades the capsular K5 polysaccharide into many oligosaccharides through beta-elimination. The polysaccharides are capsule phage receptors and the enzyme is a K5 polysaccharide lyase. The major components of the reaction are hexa-, octa- and decasaccharides. A 4,5-unsaturated glucuronic acid is present at the products nonreducing end.