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.

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.

Quorum sensing (QS) is the process by which bacteria communicate with each other through small signaling molecules such as N-acylhomoserine lactones (AHLs). Certain bacteria can degrade AHL molecules by a process called quorum quenching (QQ); therefore, QQ can be used to control bacterial infections and biofilm formation. In this study, we aimed to identify new species of bacteria with QQ activity. Red Sea sediments were collected either from the close vicinity of seagrass or from areas with no vegetation. We isolated 72 bacterial strains, which were tested for their ability to degrade/inactivate AHL molecules. Chromobacterium violaceum CV026-based bioassay was used for the initial screening of isolates with QQ activity. QQ activity was further quantified using high-performance liquid chromatography-tandem mass spectrometry. We found that these isolates could degrade AHL molecules of different acyl chain lengths as well as modifications. 16S-rRNA sequencing of positive QQ isolates showed that they belonged to three different genera. Specifically, two isolates belonged to the genus Erythrobacter; four, Labrenzia; and one, Bacterioplanes. The genome of one representative isolate from each genus was sequenced, and potential QQ enzymes, namely, lactonases and acylases, were identified. The ability of these isolates to degrade the 3OXOC12-AHLs produced by Pseudomonas aeruginosa PAO1 and hence inhibit biofilm formation was investigated. Our results showed that the isolate VG12 (genus Labrenzia) is better than other isolates at controlling biofilm formation by PAO1 and degradation of different AHL molecules. Time-course experiments to study AHL degradation showed that VG1 (genus Erythrobacter) could degrade AHLs faster than other isolates. Thus, QQ bacteria or enzymes can be used in combination with an antibacterial to overcome antibiotic resistance.

The ubiquitous opportunistic human pathogen Pseudomonas aeruginosa secretes a viscous extracellular polysaccharide, called alginate, as a virulence factor during chronic infection of patients with cystic fibrosis. In the present study, it was demonstrated that the outer membrane protein AlgE is required for the production of alginate in P. aeruginosa. An isogenic marker-free algE deletion mutant was constructed. This strain was incapable of producing alginate but did secrete alginate degradation products, indicating that polymerization occurs but that the alginate chain is subsequently degraded during transit through the periplasm. Alginate production was restored by introducing the algE gene. The membrane topology of the outer membrane protein AlgE was assessed by site-specific insertions of FLAG epitopes into predicted extracellular loop regions.

An alarming increase in antibiotic-resistant bacterial strains is driving clinical demand for new antibacterial agents. One of the oldest antimicrobial agents is elementary silver (Ag), which has been used for thousands of years. Even today, elementary Ag is used for medical purposes such as treating burns, wounds, and microbial infections. In consideration of the effectiveness of elementary Ag, the present researchers generated effective antibacterial/antibiofilm agents by combining elementary Ag with biocompatible ultrashort peptide compounds. The innovative antibacterial agents comprised a hybrid peptide bound to Ag nanoparticles (IVFK/Ag NPs). These were generated by photoionizing a biocompatible ultrashort peptide, thus reducing Ag ions to form Ag NPs with a diameter of 6 nm. The IVFK/Ag NPs demonstrated promising antibacterial/antibiofilm activity against reference Gram-positive and Gram-negative bacteria compared with commercial Ag NPs. Through morphological changes in Escherichia coli and Staphylococcus aureus, we proposed that the mechanism of action for IVFK/Ag NPs derives from their ability to disrupt bacterial membranes. In terms of safety, the IVFK/Ag NPs demonstrated biocompatibility in the presence of human dermal fibroblast cells, and concentrations within the minimal inhibitory concentration had no significant effect on cell viability. These results demonstrated that hybrid peptide/Ag NPs hold promise as a biocompatible material with strong antibacterial/antibiofilm properties, allowing them to be applied across a wide range of applications in tissue engineering and regenerative medicine.

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