Pseudomonas aeruginosa is an opportunistic human pathogen causing devastating acute and chronic infections in individuals with compromised immune systems. Its highly notorious persistence in clinical settings is attributed to its ability to form antibiotic-resistant biofilms. Biofilm is an architecture built mostly by autogenic extracellular polymeric substances which function as a scaffold to encase the bacteria together on surfaces, and to protect them from environmental stresses, impedes phagocytosis and thereby conferring the capacity for colonization and long-term persistence. Here we review the current knowledge on P. aeruginosa biofilms, its development stages, and molecular mechanisms of invasion and persistence conferred by biofilms. Explosive cell lysis within bacterial biofilm to produce essential communal materials, and interspecies biofilms of P. aeruginosa and commensal Streptococcus which impedes P. aeruginosa virulence and possibly improves disease conditions will also be discussed. Recent research on diagnostics of P. aeruginosa infections will be investigated. Finally, therapeutic strategies for the treatment of P. aeruginosa biofilms along with their advantages and limitations will be compiled.

Alginate is an important polysaccharide used widely in the food, textile, printing and pharmaceutical industries for its viscosifying, and gelling properties. All commercially produced alginates are isolated from farmed brown seaweeds. These algal alginates suffer from heterogeneity in composition and material properties. Here, we will discuss alginates produced by bacteria; the molecular mechanisms involved in their biosynthesis; and the potential to utilize these bacterially produced or modified alginates for high-value applications where defined material properties are required.

SummaryA vast range of extracellular polysaccharides are produced by bacteria in order to adapt to and thrive in diverse environmental niches. Many of these polymers have attracted great attention due to their implication in biofilm formation, capsule formation, virulence, or for their potential medical and industrial uses. One important exopolysaccharide, alginate, is produced by various Pseudomonas spp. and Azotobacter vinelandii. Alginate is of particular interest due to its role in the pathogenesis of Pseudomonas aeruginosa lung infection in cystic fibrosis patients. Here, we will discuss the genetic organization and distribution of the genes involved in the biosynthesis of this significant polymer. The complex regulatory networks involved in the production of bacterial alginate will be reviewed, including transcriptional, posttranscriptional and posttranslational forms of regulation.

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Alginates are polysaccharides with many industrial and medical uses, from food additives to encapsulation agents in the emerging transplantation technologies. Alginate is composed of variable proportions of β-D-mannuronic acid and α-Lguluronic acid linked by 1-4 glycosidic bonds. Traditionally, commercial alginate has been produced by farmed brown seaweeds, but this alginate suffers from heterogeneity in composition and quality partly due to environmental variation. Two bacterial genera, Pseudomonas and Azotobacter, are also capable of producing alginate as an exopolysaccharide. These bacterial alginate producers can provide the means to produce alginates with defined monomer composition and possibly through genetic and protein engineering may allow for the production of 'tailor made' bacterial alginates. The paper discusses the mechanisms behind alginate production in bacteria and how they may be used in the commercial production of alginates.

Pseudomonas aeruginosa is an opportunistic pathogen of particular significance to cystic fibrosis patients. This bacterium produces the exopolysaccharide alginate, which is an indicator of poor prognosis for these patients. The proteins required for alginate polymerization and secretion are encoded by genes organized in a single operon; however, the existence of internal promoters has been reported. It has been proposed that these proteins form a multiprotein complex which extends from the inner to outer membrane. Here, experimental evidence supporting such a multiprotein complex was obtained via mutual stability analysis, pulldown assays, and coimmunoprecipitation. The impact of the absence of single proteins or subunits on this multiprotein complex, i.e., on the stability of potentially interacting proteins, as well as on alginate production was investigated. Deletion of algK in an alginate-overproducing strain, PDO300, interfered with the polymerization of alginate, suggesting that in the absence of AlgK, the polymerase and copolymerase subunits, Alg8 and Alg44, are destabilized. Based on mutual stability analysis, interactions between AlgE (outer membrane), AlgK (periplasm), AlgX (periplasm), Alg44 (inner membrane), Alg8 (inner membrane), and AlgG (periplasm) were proposed. Coimmunoprecipitation using a FLAG-tagged variant of AlgE further demonstrated its interaction with AlgK. Pulldown assays using histidine-tagged AlgK showed that AlgK interacts with AlgX, which in turn was also copurified with histidine-tagged Alg44. Detection of AlgG and AlgE in PAO1 supported the existence of internal promoters controlling expression of the respective genes. Overall experimental evidence was provided for the existence of a multiprotein complex required for alginate polymerization and secretion.

The activated intracellular precursor of alginate biosynthesis is GDPmannuronic acid. Carbon sources are oxidized to acetyl-CoA, which enters the citric acid cycle, providing the intermediate oxaloacetate. Oxaloacetate is converted via gluconeogenesis into fructose 6-phosphate. The central metabolite fructose 6-phosphate derived from gluconeogenesis is then converted to the activated alginate precursor GDP-mannuronic acid employing alginate-specific biosynthesis enzymes. This conversion requires four enzymatic steps catalysed by bifunctional phosphomannose isomerase:GDP-mannose pyrophosphorylase, phosphomannomutase and GDP-mannose dehydrogenase. GDP-mannuronic acid is polymerized to alginate by a membrane-anchored glycosyltransferase which is presumably represented by Alg8, which has been suggested to be a subunit of a multiprotein complex spanning the cytoplasmic membrane (Alg44), the periplasm (AlgX, AlgK, AlgG, AlgL) and the outer membrane (AlgE). These periplasmic proteins have been proposed to form

Enzymes are used as biocatalysts in a vast range of industrial applications. Immobilization of enzymes to solid supports or their self-assembly into insoluble particles enhances their applicability by strongly improving properties such as stability in changing environments, re-usability and applicability in continuous biocatalytic processes. The possibility of co-immobilizing various functionally related enzymes involved in multistep synthesis, conversion or degradation reactions enables the design of multifunctional biocatalyst with enhanced performance compared to their soluble counterparts. This review provides a brief overview of up-to-date in vitro immobilization strategies while focusing on recent advances in enzyme engineering towards in situ self-assembly into insoluble particles. In situ self-assembly approaches include the bioengineering of bacteria to abundantly form enzymatically active inclusion bodies such as enzyme inclusions or enzyme-coated polyhydroxyalkanoate granules. These one-step production strategies for immobilized enzymes avoid prefabrication of the carrier as well as chemical cross-linking or attachment to a support material while the controlled oriented display strongly enhances the fraction of accessible catalytic sites and hence functional enzymes.