Evaluation of the Glucuronic Acid Production and Antibacterial Properties of Kombucha Black Tea.

BACKGROUND: Kombucha beverage is considered as a dietary supplement and drinking it strengthens the body's immune system which prevents diseases. OBJECTIVE: The purpose of this study was to determine the amount of glucuronic acid and antibacterial activity of Kombucha black tea drink during its production at different storage temperature. METHODS: The extent of glucuronic acid at temperatures of 20°C and 30°C was explored by the use of the HPLC system for 21 days. To analyse the antibacterial property, the influence of Kombucha black tea supernatant on the growth of Salmonella typhimurium, Staphylococcus aureus, and Lactobacillus rhamnosus bacteria was examined via the two procedures of the disc and agar well diffusion. RESULTS: The production of glucuronic acid underwent a variation at 20°C from 17.0 mg/L on day 1 to roughly 27.2 mg/L on day 21, and the difference was significant. Furthermore, the quantity of this acid at 30°C increased from 42.2 mg/L on day 1 to 48.0 mg/L on day 21. The amount of glucuronic acid produced at 30°C was significantly greater than that at 20°C (p<0.05). This study indicated that the Kombucha black tea has antibacterial activity against Salmonella typhimurium and Staphylococcus aureus, but not against Lactobacillus rhamnosus. However, there are no statistical differences in antibacterial activity of Kombucha between incubation at 20oC and 30oC (P>0.05). CONCLUSION: This study offers a perspective on glucuronic acid production (especially in 30°C rather than 20°C) and antibacterial activity of Kombucha black tea beverage.

Outer-membrane protein LptD (PA0595) plays a role in the regulation of alginate synthesis in Pseudomonas aeruginosa.

PURPOSE: The presence of alginate-overproducing (Alg+) strains of Pseudomonas aeruginosa in cystic fibrosis patients is indicative of chronic infection. The Alg+ phenotype is generally due to a mutation in the mucA gene, encoding an innermembrane protein that sequesters AlgT/U, the alginate-specific sigma factor. AlgT/U release from the anti-sigma factor MucA is orchestrated via a complex cascade called regulated intramembrane proteolysis. The goal of this study is to identify new players involved in the regulation of alginate production. METHODOLOGY: Previously, a mutant with a second-site suppressor of alginate production (sap), sap27, was isolated from the constitutively Alg+ PDO300 that harbours the mucA22 allele. A cosmid from a P. aeruginosa minimum tiling path library was identified via en masse complementation of sap27. The cosmid was transposon mutagenized to map the contributing gene involved in the alginate production. The identified gene was sequenced in sap27 along with algT/U, mucA, algO and mucP. The role of the novel gene was explored using precise in-frame algO and algW deletion mutants of PAO1 and PDO300.Results/Key findings. The gene responsible for restoring the mucoid phenotype was mapped to lptD encoding an outer-membrane protein. However, the sequencing of sap27 revealed a mutation in algO, but not in lptD. In addition, we demonstrate that lipopolysaccharide transport protein D (LptD)-dependent alginate production requires AlgW in PAO1 and AlgO in PDO300. CONCLUSION: LptD plays a specific role in alginate production. Our findings suggest that there are two pathways for the production of alginate in P. aeruginosa, one involving AlgW in the wild-type, and one involving AlgO in the mucA22 mutant.

Seasonal biomass and alginate stock assessment of three abundant genera of brown macroalgae using multispectral high resolution satellite remote sensing: A case study at Ekas Bay (Lombok, Indonesia).

The potential of Indonesian bays as alginate producers was assessed by determining the stock of wild brown algae and exploring their biomass as alginophytes at the scale of entire bay, using a combination of field observations, remote sensing high resolution data and GIS tools. Ekas Bay in Lombok Island presented a stock of brown macroalgae which varied with season and species: for Padina the biomass reached 97.85±12.63 and 79.54±2.53tons in May/June and November respectively; for Sargassaceae species, it reached 669.70±109.64 and 147.70±77.97tons in May/June and November respectively. The best alginate yields occurred during the May/June period: Padina could produce 9.10±0.06tons DW of alginates. Interestingly, Sargassum/Turbinaria together allow 207.61±0.42tons DW of alginates. This study suggests that wild Sargassaceae represent an interesting stock in terms of biomass, alginate yield and M/G ratio.

Synergistic Activity of Berberine with Azithromycin against Pseudomonas Aeruginosa Isolated from Patients with Cystic Fibrosis of Lung In Vitro and In Vivo.

BACKGROUND/AIMS: Pseudomonas aeruginosa (PA) is one of the major opportunistic pathogens which can cause chronic lung infection of cystic fibrosis (CF). The formation of PA biofilm promotes CF development and restricts the antimicrobial efficacies of current antibiotics. METHODS: The antimicrobial effects of azithromycin (AZM) and berberine (BER) alone and in combination were evaluated using microdilution method, checkerboard assay, time-kill test, qRT-PCR analysis and absorption method. The treatments of AZM and/or BER were further evaluated in an animal lung infection model via observing survival rate, bacterial burden and histopathology of lung, the levels of pro-/anti-inflammatory cytokines. RESULTS: AZM-BER were demonstrated to be synergistic against ten clinical PA isolates as well as the standard reference PA ATCC27853, in which PA03 was the most susceptible isolate to AZM-BER with FICI of 0.13 and chosen for subsequent experiments. The synergism of AZM-BER was further confirmed against PA03 in time-kill test and scanning electron microscope (SEM) at their concentrations showing synergism. In PA03, we found that AZM-BER could significantly attenuate productions of a series of virulence factors including alginate, LasA protease, LasB protease, pyoverdin, pyocyanin, chitinase as well as extracellular DNA, and remarkably inhibit the levels of quorum sensing (QS) molecules and the expressions of lasI, lasR, rhlI, rhlR at 1/2×MIC, 1×MIC and 2×MIC. In the infection model, the mice survival were increased markedly, the inflammations of infected lungs were improved greatly along with reduced IL-6, IL-8 and ascended IL-10 at 0.8 mg/kg of AZM combined with 3.2 mg/kg of BER. CONCLUSION: BER might be a promising synergist to enhance the antimicrobial activity of AZM in vitro and in vivo.

Two-component system CbrA/CbrB controls alginate production in Azotobacter vinelandii.

Azotobacter vinelandii, belonging to the Pseudomonadaceae family, is a free-living bacterium that has been considered to be a good source for the production of bacterial polymers such as alginate. In A. vinelandii the synthesis of this polymer is regulated by the Gac/Rsm post-transcriptional regulatory system, in which the RsmA protein binds to the mRNA of the biosynthetic algD gene, inhibiting translation. In several Pseudomonas spp. the two-component system CbrA/CbrB has been described to control a variety of metabolic and behavioural traits needed for adaptation to changing environmental conditions. In this work, we show that the A. vinelandii CbrA/CbrB two-component system negatively affects alginate synthesis, a function that has not been described in Pseudomonas aeruginosa or any other Pseudomonas species. CbrA/CbrB was found to control the expression of some alginate biosynthetic genes, mainly algD translation. In agreement with this result, the CbrA/CbrB system was necessary for optimal rsmA expression levels. CbrA/CbrB was also required for maximum accumulation of the sigma factor RpoS. This last effect could explain the positive effect of CbrA/CbrB on rsmA expression, as we also showed that one of the promoters driving rsmA transcription was RpoS-dependent. However, although inactivation of rpoS increased alginate production by almost 100 %, a cbrA mutation increased the synthesis of this polymer by up to 500 %, implying the existence of additional CbrA/CbrB regulatory pathways for the control of alginate production. The control exerted by CbrA/CbrB on the expression of the RsmA protein indicates the central role of this system in regulating carbon metabolism in A. vinelandii.

Different responses in the expression of alginases, alginate polymerase and acetylation genes during alginate production by Azotobacter vinelandii under oxygen-controlled conditions.

Alginate production and gene expression of genes involved in alginate biosynthesis were evaluated in continuous cultures under dissolved oxygen tension (DOT) controlled conditions. Chemostat at 8% DOT showed an increase in the specific oxygen uptake rate [Formula: see text] from 10.9 to 45.3 mmol g-1 h-1 by changes in the dilution rate (D) from 0.06 to 0.10 h-1, whereas under 1% DOT the [Formula: see text] was not affected. Alginate molecular weight was not affected by DOT. However, chemostat at 1% DOT showed a downregulation up to 20-fold in genes encoding both the alginate polymerase (alg8, alg44), alginate acetylases (algV, algI) and alginate lyase AlgL. alyA1 and algE7 lyases gene expressions presented an opposite behavior by changing the DOT, suggesting that A. vinelandii can use specific depolymerases depending on the oxygen level. Overall, the DOT level have a differential effect on genes involved in alginate synthesis, thus a gene expression equilibrium determines the production of alginates of similar molecular weight under DOT controlled.

Influence of the alginate production on cell-to-cell communication in Pseudomonas aeruginosa PAO1.

Many bacteria communicate with each other through signalling molecules, a process known as cell-to-cell communication. During this process, it is important for the signalling molecules to: (1) reach the target cells; and (2) to be received by the cognate receptor. Barriers such as the presence of extracellular matrix may prevent signals from reaching their targets; however, the influence of the extracellular matrix on cell-to-cell communication has scarcely been studied. Here, we demonstrate that the overproduction of an extracellular matrix, alginate, in a Pseudomonas aeruginosa mucoid variant, alters cell-to-cell communication by interfering with the response to quinolone signals while having no effect on N-acyl-L-homoserine lactones. The inhibition of quinolone signalling by alginate is limited to the alginate overproducer and has no effect on neighbour cells that do not produce alginate. Our study indicates that alginate overproduction affects the cell-to-cell communication of the mucoid variant, which may results in different downstream behaviours when it emerges in the presence of the wild-type (WT).

Identification of genes affecting alginate biosynthesis in Pseudomonas fluorescens by screening a transposon insertion library.

BACKGROUND: Polysaccharides often are necessary components of bacterial biofilms and capsules. Production of these biopolymers constitutes a drain on key components in the central carbon metabolism, but so far little is known concerning if and how the cells divide their resources between cell growth and production of exopolysaccharides. Alginate is an industrially important linear polysaccharide synthesized from fructose 6-phosphate by several bacterial species. The aim of this study was to identify genes that are necessary for obtaining a normal level of alginate production in alginate-producing Pseudomonas fluorescens. RESULTS: Polysaccharide biosynthesis is costly, since it utilizes nucleotide sugars and sequesters carbon. Consequently, transcription of the genes necessary for polysaccharide biosynthesis is usually tightly regulated. In this study we used an engineered P. fluorescens SBW25 derivative where all genes encoding the proteins needed for biosynthesis of alginate from fructose 6-phosphate and export of the polymer are expressed from inducible Pm promoters. In this way we would avoid identification of genes merely involved in regulating the expression of the alginate biosynthetic genes. The engineered strain was subjected to random transposon mutagenesis and a library of about 11500 mutants was screened for strains with altered alginate production. Identified inactivated genes were mainly found to encode proteins involved in metabolic pathways related to uptake and utilization of carbon, nitrogen and phosphor sources, biosynthesis of purine and tryptophan and peptidoglycan recycling. CONCLUSIONS: The majority of the identified mutants resulted in diminished alginate biosynthesis while cell yield in most cases were less affected. In some cases, however, a higher final cell yield were measured. The data indicate that when the supplies of fructose 6-phosphate or GTP are diminished, less alginate is produced. This should be taken into account when bacterial strains are designed for industrial polysaccharide production.

New insights into Pseudomonas fluorescens alginate biosynthesis relevant for the establishment of an efficient production process for microbial alginates.

Alginate denotes a family of linear polysaccharides with a wide range of industrial and pharmaceutical applications. Presently, all commercially available alginates are manufactured from brown algae. However, bacterial alginates have advantages with regard to compositional homogeneity and reproducibility. In order to be able to design bacterial strains that are better suited for industrial alginate production, defining limiting factors for alginate biosynthesis is of vital importance. Our group has been studying alginate biosynthesis in Pseudomonas fluorescens using several complementary approaches. Alginate is synthesised and transported out of the cell by a multiprotein complex spanning from the inner to the outer membrane. We have developed an immunogold labelling procedure in which the porin AlgE, as a part of this alginate factory, could be detected by transmission electron microscopy. No time-dependent correlation between the number of such factories on the cell surface and alginate production level was found in alginate-producing strains. Alginate biosynthesis competes with the central carbon metabolism for the key metabolite fructose 6-phosphate. In P. fluorescens, glucose, fructose and glycerol, are metabolised via the Entner-Doudoroff and pentose phosphate pathways. Mutational analysis revealed that disruption of the glucose 6-phosphate dehydrogenase gene zwf-1 resulted in increased alginate production when glycerol was used as carbon source. Furthermore, alginate-producing P. fluorescens strains cultivated on glucose experience acid stress due to the simultaneous production of alginate and gluconate. The combined results from our studies strongly indicate that the availability of fructose 6-phosphate and energy requires more attention in further research aimed at the development of an optimised alginate production process.

Responses of Pseudomonas putida to Zinc Excess Determined at the Proteome Level: Pathways Dependent and Independent of ColRS.

Zinc is an important micronutrient for bacteria, but its excess is toxic. Recently, the ColRS two-component system was shown to detect and respond to zinc excess and to contribute to zinc tolerance of Pseudomonas putida. Here, we applied a label-free whole-cell proteome analysis to compare the zinc-induced responses of P. putida and colR knockout. We identified dozens of proteins that responded to zinc in a ColR-independent manner, among others, known metal efflux systems CzcCBA1, CzcCBA2, CadA2 and CzcD. Nine proteins were affected in a ColR-dependent manner, and besides known ColR targets, four new candidates for ColR regulon were identified. Despite the relatively modest ColR-dependent changes of wild-type, colR deficiency resulted in drastic proteome alterations, with 122 proteins up- and 62 down-regulated by zinc. This zinc-promoted response had remarkable overlap with the alternative sigma factor AlgU-controlled regulon in P. aeruginosa. The most prominent hallmark was a high induction of alginate biosynthesis proteins and regulators. This response likely alleviates the zinc stress, as the AlgU-regulated alginate regulator AmrZ was shown to contribute to zinc tolerance of colR knockout. Thus, the ColRS system is important for zinc homeostasis, and in its absence, alternative stress response pathways are activated to support the zinc tolerance.

Pseudomonas aeruginosa AmrZ Binds to Four Sites in the algD Promoter, Inducing DNA-AmrZ Complex Formation and Transcriptional Activation.

During late stages of cystic fibrosis pulmonary infections, Pseudomonas aeruginosa often overproduces the exopolysaccharide alginate, protecting the bacterial community from host immunity and antimicrobials. The transcription of the alginate biosynthesis operon is under tight control by a number of factors, including AmrZ, the focus of this study. Interestingly, multiple transcription factors interact with the far-upstream region of this promoter (PalgD), in which one AmrZ binding site has been identified previously. The mechanisms of AmrZ binding and subsequent activation remain unclear and require more-detailed investigation. In this study, in-depth examinations elucidated four AmrZ binding sites, and their disruption eliminated AmrZ binding and promoter activation. Furthermore, our in vitro fluorescence resonance energy transfer experiments suggest that AmrZ holds together multiple binding sites in PalgD and thereafter induces the formation of higher-order DNA-AmrZ complexes. To determine the importance of interactions between those AmrZ oligomers in the cell, a DNA phasing experiment was performed. PalgD transcription was significantly impaired when the relative phase between AmrZ binding sites was reversed (5 bp), while a full-DNA-turn insertion (10 bp) restored promoter activity. Taken together, the investigations presented here provide a deeper mechanistic understanding of AmrZ-mediated binding to PalgD IMPORTANCE: Overproduction of the exopolysaccharide alginate provides protection to Pseudomonas aeruginosa against antimicrobial treatments and is associated with chronic P. aeruginosa infections in the lungs of cystic fibrosis patients. In this study, we combined a variety of microbiological, genetic, biochemical, and biophysical approaches to investigate the activation of the alginate biosynthesis operon promoter by a key transcription factor named AmrZ. This study has provided important new information on the mechanism of activation of this extremely complex promoter.

Analysis of respiratory activity and carbon usage of a mutant of Azotobacter vinelandii impaired in poly-ß-hydroxybutyrate synthesis.

In this study, the respiratory activity and carbon usage of the mutant strain of A. vinelandii AT6, impaired in poly-ß-hydroxybutyrate (PHB) production, and their relationship with the synthesis of alginate were evaluated. The alginate yield and the specific oxygen uptake rate were higher (2.5-fold and 62 %, respectively) for the AT6 strain, compared to the control strain (ATCC 9046), both in shake flasks cultures and in bioreactor, under fixed dissolved oxygen tension (1 %). In contrast, the degree of acetylation was similar in both strains. These results, together with the analysis of carbon usage (% C-mol), suggest that in the case of the AT6 strain, the flux of acetyl-CoA (precursor molecule for PHB biosynthesis and alginate acetylation) was diverted to the respiratory chain passing through the tricarboxylic acids cycle, and an important % C-mol was directed through alginate biosynthesis, up to 25.9 % and to a lesser extent, to biomass production (19.7 %).

Efficient production of polyhydroxyalkanoates (PHAs) from Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) as the sole carbon source.

Conditions for the optimal production of polyhydroxyalkanoate (PHA) by Pseudomonas mendocina PSU using a biodiesel liquid waste (BLW) were determined by response surface methodology. These were an initial carbon to nitrogen ratio (C/N) of 40 (mole/mole), an initial pH of 7.0, and a temperature of 35 °C. A biomass and PHA concentration of 3.65 g/L and about 2.6 g/L (77% DCW), respectively, were achieved in a growth associated process using 20 g/L glycerol in the BLW after 36 h of exponential growth. The PHA monomer compositions were 3HB (3-hydroxybutyrate), a short-chain-length-PHA, and the medium-chain-length-PHA e.g. 3-hydroxyoctanoate and 3-hydroxydecanoate. Both the phbC and phaC genes were characterized. The phbC enzyme had not been previously detected in a Pseudomonas mendocina species. A 2.15 g/L of an exopolysaccharide, alginate, was also produced with a similar composition to that of other Pseudomonas species.

Oxygen-dependent regulation of c-di-GMP synthesis by SadC controls alginate production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces increased levels of alginate in response to oxygen-deprived conditions. The regulatory pathway(s) that links oxygen limitation to increased synthesis of alginate has remained elusive. In the present study, using immunofluorescence microscopy, we show that anaerobiosis-induced alginate production by planktonic PAO1 requires the diguanylate cyclase (DGC) SadC, previously identified as a regulator of surface-associated lifestyles. Furthermore, we found that the gene products of PA4330 and PA4331, located in a predicted operon with sadC, have a major impact on alginate production: deletion of PA4330 (odaA, for oxygen-dependent alginate synthesis activator) caused an alginate production defect under anaerobic conditions, whereas a PA4331 (odaI, for oxygen-dependent alginate synthesis inhibitor) deletion mutant produced alginate also in the presence of oxygen, which would normally inhibit alginate synthesis. Based on their sequence, OdaA and OdaI have predicted hydratase and dioxygenase reductase activities, respectively. Enzymatic assays using purified protein showed that unlike OdaA, which did not significantly affect DGC activity of SadC, OdaI inhibited c-di-GMP production by SadC. Our data indicate that SadC, OdaA and OdaI are components of a novel response pathway of P. aeruginosa that regulates alginate synthesis in an oxygen-dependent manner.

Alginate Biosynthesis Factories in Pseudomonas fluorescens: Localization and Correlation with Alginate Production Level.

Pseudomonas fluorescens is able to produce the medically and industrially important exopolysaccharide alginate. The proteins involved in alginate biosynthesis and secretion form a multiprotein complex spanning the inner and outer membranes. In the present study, we developed a method by which the porin AlgE was detected by immunogold labeling and transmission electron microscopy. Localization of the AlgE protein was found to depend on the presence of other proteins in the multiprotein complex. No correlation was found between the number of alginate factories and the alginate production level, nor were the numbers of these factories affected in an algC mutant that is unable to produce the precursor needed for alginate biosynthesis. Precursor availability and growth phase thus seem to be the main determinants for the alginate production rate in our strain. Clustering analysis demonstrated that the alginate multiprotein complexes were not distributed randomly over the entire outer cell membrane surface.

Effect of Oxygen Tension and Medium Components on Monomer Distribution of Alginate.

Alginate is a natural biopolymer composed of mannuronic and guluronic acid monomers. It is produced by algae and some species of Azotobacter and Pseudomonas. This study aims to investigate the effect of dissolved oxygen tension (DOT) and growth medium substrate and calcium concentrations on the monomeric composition of alginate produced by Azotobacter vinelandii ATCC® 9046 in a fermenter. Results showed that alginate production increased with increasing DOT from 1 to 5 %. The highest alginate production was obtained as 4.51 g/L under 20 g/L of sucrose and 50 mg/L of calcium at 5 % DOT. At these conditions, alginate was rich in mannuronic acid (up to 61 %) and it was particularly high at low calcium concentration. On the other hand, at extreme conditions such as high DOT level (10 % DOT) and low sucrose concentration (10 g/L), guluronic acid was dominant (ranging between 65 and 100 %).

Production of polyhydroxybutyrate and alginate from glycerol by Azotobacter vinelandii under nitrogen-free conditions.

Glycerol is an interesting feedstock for biomaterials such as biofuels and bioplastics because of its abundance as a by-product during biodiesel production. Here we demonstrate glycerol metabolism in the nitrogen-fixing species Azotobacter vinelandii through metabolomics and nitrogen-free bacterial production of biopolymers, such as poly-d-3-hydroxybutyrate (PHB) and alginate, from glycerol. Glycerol-3-phosphate was accumulated in A. vinelandii cells grown on glycerol to the exponential phase, and its level drastically decreased in the cells grown to the stationary growth phase. A. vinelandii also overexpressed the glycerol-3-phosphate dehydrogenase gene when it was grown on glycerol. These results indicate that glycerol was first converted to glycerol-3-phosphate by glycerol kinase. Other molecules with industrial interests, such as lactic acid and amino acids including γ-aminobutyric acid, have also been accumulated in the bacterial cells grown on glycerol. Transmission electron microscopy revealed that glycerol-grown A. vinelandii stored PHB within the cells. The PHB production level reached 33% per dry cell weight in nitrogen-free glycerol medium. When grown on glycerol, alginate-overproducing mutants generated through chemical mutagenesis produced 2-fold the amount of alginate from glycerol than the parental wild-type strain. To the best of our knowledge, this is the first report on bacterial production of biopolymers from glycerol without addition of any nitrogen source.

Mutational Analyses of Glucose Dehydrogenase and Glucose-6-Phosphate Dehydrogenase Genes in Pseudomonas fluorescens Reveal Their Effects on Growth and Alginate Production.

The biosynthesis of alginate has been studied extensively due to the importance of this polymer in medicine and industry. Alginate is synthesized from fructose-6-phosphate and thus competes with the central carbon metabolism for this metabolite. The alginate-producing bacterium Pseudomonas fluorescens relies on the Entner-Doudoroff and pentose phosphate pathways for glucose metabolism, and these pathways are also important for the metabolism of fructose and glycerol. In the present study, the impact of key carbohydrate metabolism enzymes on growth and alginate synthesis was investigated in P. fluorescens. Mutants defective in glucose-6-phosphate dehydrogenase isoenzymes (Zwf-1 and Zwf-2) or glucose dehydrogenase (Gcd) were evaluated using media containing glucose, fructose, or glycerol. Zwf-1 was shown to be the most important glucose-6-phosphate dehydrogenase for catabolism. Both Zwf enzymes preferred NADP as a coenzyme, although NAD was also accepted. Only Zwf-2 was active in the presence of 3 mM ATP, and then only with NADP as a coenzyme, indicating an anabolic role for this isoenzyme. Disruption of zwf-1 resulted in increased alginate production when glycerol was used as the carbon source, possibly due to decreased flux through the Entner-Doudoroff pathway rendering more fructose-6-phosphate available for alginate biosynthesis. In alginate-producing cells grown on glucose, disruption of gcd increased both cell numbers and alginate production levels, while this mutation had no positive effect on growth in a non-alginate-producing strain. A possible explanation is that alginate synthesis might function as a sink for surplus hexose phosphates that could otherwise be detrimental to the cell.

Biotechnologically produced microbial alginate dressings show enhanced gel forming capacity compared to commercial alginate dressings of marine origin.

Marine alginate fibre dressings are well established in wound management. Alginate fibres can absorb plenty of wound exudate due to their gel forming abilities and ion exchange. Alginates from bacteria have never been studied for medical applications so far, although the microbial polymer raises expectations for improved gelling capacity due to its unique O-acetylation. To prove the gelling capacity of bacterial alginate, we extracted the co-polymer from fermentation of the soil bacterium Azotobacter vinelandii ATCC 9046, cultivated on crude glycerol as an alternative carbon source. Bacterial alginate was isolated in high purity and extruded by a wet spinning method. Fibre structure and properties were characterised by infrared spectroscopy, NMR, GPC, scanning electron microscopy and tensile testing. The fibres could be processed into biocompatible needle web dressings, which showed more than twice the gel formation in saline compared to commercial dressings made of marine alginates. Gelled dressings of bacterial alginate formed stable hydrogels of sufficient shape and strength for wound healing applications. This work suggests that the increased gel formation of bacterial alginate from A. vinelandii may be optimal for the preparation of novel wound dressings.

The capability of Pseudomonas aeruginosa to recruit zinc under conditions of limited metal availability is affected by inactivation of the ZnuABC transporter.

The ability of a large number of bacterial pathogens to multiply in the infected host and cause disease is dependent on their ability to express high affinity zinc importers. In many bacteria, ZnuABC, a transporter of the ABC family, plays a central role in the process of zinc uptake in zinc poor environments, including the tissues of the infected host. To initiate an investigation into the relevance of the zinc uptake apparatus for Pseudomonas aeruginosa pathogenicity, we have generated a znuA mutant in the PA14 strain. We have found that this mutant strain displays a limited growth defect in zinc depleted media. The znuA mutant strain is more sensitive than the wild type strain to calprotectin-mediated growth inhibition, but both the strains are highly resistant to this zinc sequestering antimicrobial protein. Moreover, intracellular zinc content is not evidently affected by inactivation of the ZnuABC transporter. These findings suggest that P. aeruginosa is equipped with redundant mechanisms for the acquisition of zinc that might favor P. aeruginosa colonization of environments containing low levels of this metal. Nonetheless, deletion of znuA affects alginate production, reduces the activity of extracellular zinc-containing proteases, including LasA, LasB and protease IV, and decreases the ability of P. aeruginosa to disseminate during systemic infections. These results indicate that efficient zinc acquisition is critical for the expression of various virulence features typical of P. aeruginosa and that ZnuABC also plays an important role in zinc homeostasis in this microorganism.