1. The Polysaccharide Peptidoglycan and How it is Influenced by (Antibiotic) Stress

Ute Bertsche

The peptidoglycan or murein sacculus is the stress-bearing structure of bacterial cells. It consists of glycan strands cross-linked by peptide bridges. Even though studies on murein have a very long tradition, it is not known how the glycan strands are actually arranged. However, the chemical fine structure and the muropeptide composition of different Gram-negative and Gram-positive bacteria have been investigated in detail. This chapter discusses Escherichia coli and Staphylococcus aureus as representatives for both Gram forms. During cell growth the stress-bearing structure has to be elongated and/or divided by the insertion of new and elimination of old material without loosing its strength. Therefore multienzyme complexes containing both murein synthases and murein hydrolases have been postulated. Peptidoglycan biosynthesis is the target for many antibiotics such as β-lactams, D-cycloserine and glycopeptide-antibiotics like vancomycin. The last part of this chapter gives an overview of different bacterial strategies of bacteria for coping with antibiotic and osmotic stress.

2. Genetics and Regulation of Bacterial Lipopolysaccharide Synthesis

Mikael Skurnik and José Antonio Bengoechea

Lipopolysaccharide (LPS) is the major component of the outer leaflet of the outer membrane of Gram-negative bacteria. The LPS molecule is composed of two biosynthetic entities: the lipid A - core and the O-polysaccharide (O-antigen). Most biological effects of LPS are due to the lipid A part, however, there is an increasing body of evidence indicating that O-antigen (O-ag) plays an important role in effective colonization of host tissues, resistance to complement-mediated killing and in the resistance to cationic antimicrobial peptides that are key elements of the innate immune system. Recently, data has started to accumulate on the intricacies in the genetic regulation of the structural components of this molecule and this is highly relevant to the biological function of the molecule. In this review we discuss the regulation, mainly using Yersiniae as model organisms but also discussing other bacteria where relevant.

3. Mycobacterial Cell Wall Arabinogalactan: A Detailed Perspective on Structure, Biosynthesis, Functions and Drug Targeting

Suresh Bhamidi, Michael S. Scherman and Michael R. McNeil

Mycobacteria have an intricate cell wall core structure consisting of a hydrophobic mycolate layer and a peptidoglycan layer tethered together by a polysaccharide, arabinogalactan. The biosynthetic pathways of all the three major cell wall core components have been studied in great deal for their role as potential drug targets for tuberculosis. This chapter specifically deals with the structural details of the arabinogalactan and its biosynthetic pathways, starting from the precursors to its polymerization, and subsequent attachment to peptidoglycan and the mycolic acids. Further, various aspects of drug targeting are discussed in detail for each of these enzymes. The available enzyme assays and the difficulties associated with others will be discussed. Finally, future aspects of tuberculosis research with respect to the arabinogalactan will be presented.

4. Genetics and Regulation of Bacterial Polysaccharide Expression in Human Pathogenic Bacteria

David Corbett and Ian S. Roberts

The presentation of a polysaccharide capsule on the cell surface is a common feature of many bacteria. The presence of a polysaccharide capsule mediates interactions between the bacterium and its immediate environment. As such, regulation of capsule expression will be vital in the survival of bacteria in new environments, as will moderating capsule expression in response to local changes in the environment as a consequence of bacterial growth. This ability to regulate capsule expression is particularly important for pathogenic bacteria where the local environment will change as a consequence of the host response to infection and where more or less capsule expression will dictate the likely survival of the bacteria in a hostile environment. In this chapter we discuss the regulation of capsule gene expression in a number of pathogenic bacteria and describe the regulation of Group 2 capsule gene expression in Escherichia coli in detail.

5. Therapies Directed at Pseudomonas aeruginosa Polysaccharides

Joanna B. Goldberg

Given the critical importance of Pseudomonas aeruginosa as an opportunistic pathogen, it is necessary to consider novel targets for therapeutic development. This is especially true as this bacterium is naturally resistant to many antimicrobials and with the over use of antibiotics has become resistant to those it was once sensitive. Thus, there is a real need for new drugs and approaches to combat the myriad of diseases caused by this pathogen. Polysaccharides would appear to be suitable candidates in this regard. They are generally the outermost molecules on the surface of P. aeruginosa. Some have roles in adherence and/or biofilm formation. Other polysaccharides are considered virulence factors and are immunodominant antigens. Thus, this group of molecules would seem ideal for the development of therapeutic interventions. Unfortunately the identification of reagents to specifically inhibit polysaccharides has lagged far behind the obvious need for such drugs.

6. Immune Responses to Microbial Polysaccharides

Darran J. Wigelsworth, Erin B. Troy and Dennis L. Kasper

The immune system is capable of recognizing almost any biological polymer (including proteins and glycolipids) and presenting it to T cells via major histocompatability proteins. These adaptive immune responses have long been considered the territory of antigenic proteins, whereas carbohydrates are considered T-cell-independent antigens and are not recognized by the complete adaptive machinery. However, recent work reveals a larger role for sugars in immune recognition. The zwitterionic polysaccharide PSA from the commensal organism Bacteroides fragilis has recently been added to the list of antigens presented to T cells. This molecule has been shown not only to be presented on MHCII but also to play a role in immunomodulation and suppression of inflammatory diseases such as colitis. Bacteria have developed polysaccharide capsules to take advantage of the fact that polysaccharides are generally not recognized by the human immune system. However, the recent finding that sugars play a role in immune recognition makes them more attractive targets in the search for antigenic epitopes. This chapter will discuss in detail the immune response to foreign antigens, the function of zwitterionic polysaccharides in immune recognition, and finally the use of carbohydrates in glycoconjugated vaccines, undoubtedly one of the biggest scientific breakthroughs of the past century.

7. Polysaccharides of Gram-negative Periodontopathic Bacteria

Ryoma Nakao

Periodontitis is caused by periodontal biofilms, complex communities of microorganisms, and is one of the most common oral diseases of adults. In this chapter, recent reports concerning lipopolysaccharides, capusular polysaccharides, and glycoproteins of periodontopathic bacteria, mainly Porphyromonas gingivalis, are reviewed in the context of the their particular roles in attachment and biofilm formation. Understanding these roles of the bacterial polysaccharides provides helpful insights in bacteria-bacteria and bacteria-host interactions, which are crucial for the progress of periodontitis.

8. Bacterial Polysaccharides in Dental Plaque

Roy R. B. Russell

Dental plaque is a complex biofilm containing several hundred different species of bacteria. While these are normally harmless commensals, shifts in the population structure can lead to the plaque-related diseases such as dental caries and periodontal disease. Surface polysaccharides are important in coaggregation reactions that bind particular species together and aid colonisation and metabolic interaction while sucrose-derived glucans and fructans have a significant effect on plaque properties. Fructans serve as an extracellular energy store while glucans contribute to adhesion, modify the permeability of plaque and alter ion-binding capacity and thus have a powerful influence on the creation of conditions in plaque that can lead to dental caries.

9. Composition and Functional Role of Polysaccharides and Extracellular Polymeric Substances in Gram-positive Biofilm Infections

Christian Theilacker and Johannes Hübner

Biofilms seem to be the default mode of growth for most if not all bacterial species and this phenomenon has profound consequences in numerous clinical settings. For Gram-positive bacteria several mechanisms involving surface proteins and carbohydrate-containing structures have been identified. Poly-N-acetylglucosamine (PIA/PNAG) has been first described in staphylococci but recently has been shown to be present also in a large number of different bacterial species. Mutants in the gene locus responsible for the synthesis of this molecule lead to a biofilm-negative phenotype. Teichoic acids are polyanionic molecules found in the cell walls of Gram-positive bacteria with low GC content. Teichoic acids are either attached covalently to the peptidoglycan (wall teichoic acids) or inserted into the cell membrane (lipoteichoic acids) and both types have been associated with biofilm formation and adhesion to eukaryotic cells. Recent evidence indicates that extracellular DNA may also be involved in biofilm formation and primary attachment of many Gram-positve organisms and this fact may explain previous observations that autolysin-defective mutants are also impaired in biofilm formation. A more detailed understanding of the molecules involved in biofilm formation is needed to device novel treatment and preventive approaches for Gram-positive infections.

10. Poly-N-acetyl-glucosamine as a Mediator of Bacterial Biofilm Formation

Kimberly K. Jefferson

A number of evolutionarily disparate bacterial species synthesize and secrete a β-(1,6)-linked polymer of N-acetylglucosamine. This polysaccharide is known as polysaccharide intercellular adhesin (PIA), PNAG, or PGA depending on the species that produces it, but all are biochemically similar. The conservation of this polysaccharide is a tribute to its success as a virulence factor and it plays dual roles in this capacity. It is an essential scaffold in bacterial biofilms and also plays a role in immune evasion by interfering with opsonophagocytosis. It is loosely associated with the bacterial cell surface and is therefore not considered a capsular polysaccharide. In addition, PIA/PNAG/PGA is more structurally simplistic than most capsular polysaccharides. Nonetheless, the role of this polysaccharide in virulence in such species as Staphylococcus aureus, S. epidermidis, Escherichia coli, and Actinobacillus actinomycetemcomitans is clear, and advances in our understanding of its synthesis and structure may lead to its use as a therapeutic or vaccine target.

11. Surface Polysaccharides as Fitness factors of Rhizospheric Nitrogen-fixing Bacteria

Elizaveta Krol and Anke Becker

Nitrogen-fixing plant-associated bacteria include rhizospheric bacteria, endophytic diazotrophs and rhizobia, the symbiotic root nodule bacteria. Surface polysaccharides produced by bacteria contribute to their survival in the rhizosphere by improvement of the soil structure, enhancement of cell aggregation, and protection against harmful substances. Attachment of bacteria to plant roots and soil particles, which is important for colonisation of rhizosphere and roots and for infection of the plant, can be mediated by exopolysaccharides. Symbiotic interactions between root nodule bacteria and leguminous plants include infection of root tissues by bacteria and formation of nitrogen-fixing nodules, where bacteria differentiate into intracellular bacteroids. During early stages of the symbiotic interaction, exopolysaccharides, capsular polysaccharides or lipopolysaccharides can be involved in recognition by the plant and suppression of the immune response. At the stage of intracellular infection, lipopolysaccharides play an important role in maintenance of the bacteroids. Biosynthesis of surface polysaccharides is tightly regulated during both free-living growth and symbiotic development. Two-component regulatory systems, general stress response mechanisms, quorum sensing and symbiotic regulators constitute a flexible network for fine-tuning of surface polysaccharide production. In this chapter, we will focus on the structure of surface polysaccharides, produced by nodule bacteria, mechanisms of genetic regulation of their biosynthesis and their functions as fitness factors for free-living survival and symbiosis with a plant.

12. Levansucrase and Levan Formation in Pseudomonas syringae and Related Organisms

Abhishek Srivastava, Daria Zhurina and Matthias S. Ullrich

Bacterial levan is formed wherever bacteria encounter moderate to high sucrose concentrations, i.e. in the plant environment, in the mammalian oral cavity, or during the fermentation of plant-borne substrates, i.e. for bio-ethanol production. Consequently, the transcriptional and translational regulation, synthesis, secretion, and enzymatic properties of levan-forming enzymes such as levansucrase (Lsc) from various bacterial species have raised substantial interest among researchers in plant pathology, dental medicine, food manufacturing, and renewable energy technologies. Experimental work with Lsc was accelerated by three major features, i.e. its intrinsically high protein stability as an extra-cellular enzyme, no need for reaction-catalyzing co-factors, and an enormous ease of enzymatic detection due to its glucose releasing activity. Aside of many possible applied aspects, Lsc enzymatic activity as well as its regulation and secretion are exciting biochemical and molecular model systems for the in-depth understanding of global carbon utilization, protein transport processes, and biofilm formation.

13. Structure, Biosynthesis, and Regulation of Capsular Exopolysaccharide of Erwinia amylovora and Other Erwinia Species and Role in Pathogenicity

Klaus Geider

The fire blight pathogen E. amylovora and related bacteria synthesize complex EPS with galactose, glucuronic acid and eventually glucose residues. For E. amylovora, the capsules of amylovoran have been investigated by staining techniques and visualized by light and electron microscopy. They protect the pathogen against plant defense recognition, provide a moist environment against desiccation and bind ions and nutrients to support the cell metabolism. Amylovoran consists of repeating units forming chains up to 5 MDa and can be detached from the cells by mild treatments. EPS deficient mutants of E. amylovora and E. pyrifoliae are non-pathogenic. Synthesis requires at least twelve genes for assembly of the repeating units, transport and polymerization. Similar genes were found for other Erwinia species that produce EPS related to amylovoran. Expression of the EPS encoding gene clusters depends on specific or global regulators including siRNAs, such as RcsA, B, C or Hns and RsmA/rsmB, which may adjust the EPS level to actual needs of the bacteria. Degradation or absence of EPS capsules results in pathogen recognition by plant defense mechanisms and provides a strategy to control fire blight.

14. Osmoregulated Periplasmic Glucans (OPGs), Alginate, and Biofilm Formation in Pseudomonas syringae

Alejandro Penaloza-Vazquez, Christina M. Baker and Carol L. Bender

In recent years it became increasingly clear that the cell envelope, loosly attached, or tightly fixed exopolymeric substances might play an important role for plant pathogenic bacteria such as Pseudomonas syringae. Herein, the role and synthesis of the osmoregulated periplasic glucans (OPGs) in P. syringae are described in detail. OPGs are produced by many proteobacteria, and are important for the bacterial envelope and bacterial-host interactions. Mutants defective in production of OPGs in P. syringae exhibit a pleiotropic phenotype including the overproduction of the exopolysaccharide (EPS) alginate, decrease in cell viability under low osmolarity, low epiphytic fitness on leaves, and are unable to form biofilms on plant leaves. Results for the plant pathogen are compared to those for the opportunistic human pathogen, P. aeruginosa. In addition, we discussed the possibility of molecular interactions between OPGs production, the physical integrity of the membrane, and the associated EPS regulatory systems.

15. Ecology of Exopolysaccharide Formation by Lactic Acid Bacteria: Sucrose Utilisation, Stress Tolerance, and Biofilm Formation

Michael Gänzle and Clarissa Schwab

Lactic acid bacteria (LAB) synthesise a wide variety of exopolysaccharides (EPS); these polysaccharides are synthesised extracellularly from sucrose by glycansucrases, or intracellularly by glycosyltransferases from sugar nucleotide precursors. This chapter provides an overview on emerging concepts related to the ecological significance of EPS production by LAB. Biofilm formation, stress resistance and sucrose utilisation are clearly linked to the formation of EPS in individual species of LAB. The high frequency of homopolysacharide (HoPS) and heteropolysaccharide (HePS) producing LAB in the oral cavity and intestinal ecosystems argues in favour for an important role of EPS formation for the persistence of LAB in these habitats. The intricate regulatory network controlling the expression of glycansucrases in oral streptococci is in keeping with the contribution of HoPS and extracellular glycansucrases to biofilm formation and persistence in the oral cavity. EPS production by intestinal lactobacilli may play a comparable role. Glycansucrases in Lb. reuteri of HoPS and FOS production are regulated in response to stress sensed by the cytoplasmic membrane. The products of glycansucrases improve survival of lactobacilli in a scenario characterised by strong fluctuations in water activity, temperature, pH, and nutrient supply, and the presence of natural inhibitors. Because the expression of glycansucrases in many strains of lactobacilli and Leuconostoc species is induced by sucrose, the contribution of glycansucrases to sucrose catabolism may be their main ecological role in some strains.

16. Biosynthesis and Chemical Composition of Exopolysaccharides Produced by Lactic Acid Bacteria

Patricia Ruas-Madiedo, Nuria Salazar, and Clara G. de los Reyes-Gavilán

Exopolysaccharides (EPS) produced by lactic acid bacteria (LAB) can be classified according to the chemical composition and biosynthesis mechanism as homopolysaccharides (HoPS) and heteropolysaccharides (HePS). HoPS are composed of a single monosaccharide type, either glucose or fructose, and they are polymerised extracellularly by means of glycansucrases, sucrose being the donor of the corresponding sugar moiety. These α- or β-D-glucans and β-fructans are polymers of high molar mass (≥ 1 x106 Da) which can have different degrees of branching and they are usually produced in amounts higher than 1 g/L, although this value varies according to the strain and the production conditions. The glucansucrase and fructansucrase enzymes present a conserved functional structure in which four domains can be recognized, although enzymes produced by different strains do not share similar sequences. The HePS are built up of repeating unit structures of two or more types of monosaccharides, substituted monosaccharides and other organic and inorganic molecules. To date, around 42 different unique structures comprising from di- to octasaccharides have been described. Galactose, glucose, rhamnose and to a lower extent N-acetyl-glucosamine and N-acetyl-galactosamine are present in their composition. The HePS biosynthesis is a complex process not fully understood, that involves the activity of several specific, as well as housekeeping, enzymes which also participate in the metabolism of carbohydrates and in the synthesis of some components of the cell-wall. The genes involved in HePS synthesis are organised in eps clusters that share a common structural organization in which genes involved in regulation, export, polymerisation and chain length determination, as well as glycosyltransferases responsible for the intracellular assembly of the repeated units, can be recognised. This report summarises an overview of the chemical composition, structure and physical properties, as well the genetics and biosynthesis of EPS produced by LAB and some bifidobacteria and propionibacteria strains.

17. Commercial Exploitation of Homo-exopolysaccharides in Non-dairy Food Systems

Florian Waldherr and Rudi F. Vogel

Lactic acid bacteria play a crucial role in various food fermentations. Several strains can produce long chain sugar polymers called exopolysaccharides (EPS). These can be classified due to different criteria e.g. the composition of different or just one kind of sugar monomer in hetero- and homopolysaccharides. While heteropolysaccharides are intensively used as additives in milk products, homopolysaccharides can be introduced in sourdough products influencing structural quality, baking ability and reducing bread staling factors. Additionally, beneficial effects on enteral health are discussed. An example for industrial use of EPS in bakery products is the application of dextran in panettone and other breads. Also the addition of non-bacterial hydrocolloids is well established in industrial baking. Several investigations concerning the replacement of these additives by bacterial EPS have been made and provide data of dough and bread parameters such as textural factors, water retention and moisture and specific bread volume. Practically no information is available on the effects of bacterial EPS in other fermented foods as fermented meat products, sauerkraut or vinegar. EPSs can be as defined additives or alternatively via in situ production by starter cultures. The addition of purified EPS has to be labelled on the end product, which is a disadvantage since consumers demand for fewer additives in foods. On the other hand, in situ production appears to be less effective in traditional wheat and rye dough systems due to strain-dependent acid formation, which may be required but counteracts positive EPS effects. Forthcoming chances of EPS applications may therefore lie within special applications as in gluten free breads where both, reduced pH and EPS should have synergistic positive effects on structure.

18. Exploitation of Exopolysaccharides from Lactic Acid Bacteria: Nutritional and Functional Benefits

Alan D. Welman

The quest to find food ingredients with valuable bioactive properties has encouraged interest in exopolysaccharides (EPSs) from lactic acid bacteria (LAB). Functional food products that offer health and sensory benefits beyond their nutritional composition are becoming progressively more important in the food supply chain. Informed consumers are becoming increasingly more health conscious and more aware that disease prevention can be effected through dietary means and, more specifically, by particular components of foods. Although the sensory benefits of EPSs are now well established, more knowledge to fully understand how these polysaccharides can impact on human health and nutrition is needed. Because of the wide variation in molecular structures of EPSs, and the complexity of the mechanisms by which physical changes in foods and bioactive effects are elicited, the future commercial development of EPS-producing cultures and ingredients will depend heavily upon this platform of knowledge. In this chapter, evidence for the health properties that are attributable to LAB EPSs is reviewed, linking some of the very first findings in this area to current discoveries. Because the sensory attributes of foods are critical to their acceptance, the basis for the texture-imparting properties of EPSs is also reviewed; this section describes what is known about the basis of the structure_function relationships of EPSs, and their applications in a dairy food context. It is hoped that this chapter will stimulate interest in an area in which far more fundamental biochemical and clinical understanding is required.

19. Synthesis of Bacterial Polysaccharides as a Limiting Factor for Biofuel Production

K. Geetha, K. N. Rajnish, J. Rajendhran and P. Gunasekaran

Bio-ethanol has been considered as a potential alternative energy source to the conventional petroleum based fuel. Zymomonas mobilis is an efficient ethanol producing bacterium and it is advantageous over Saccharomyces cerevisiae with respect to ethanol productivity and tolerance. Z. mobilis possesses nearly 100% theoretical ethanol conversion rate on glucose-based media, while it has only about 70% theoretical yield from sucrose. The reduced ethanol yield has been attributed to the formation of by-products such as levan, fructooligosaccharides and sorbitol. The efficiency of ethanol production by Z. mobilis from sucrose-based substrates can be improved by limiting the formation of by-products either by optimizing the fermentation conditions or genetic improvements of Z. mobilis . Recent strategies developed for the improved production of ethanol, while limiting the formation of levan and the vice versa are discussed.