A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitivenitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cellswere cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen.The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen.Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicatingthat alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogenlevels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. Theencystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine.Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. Bycontrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel blebdependentpolysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels. [IntMicrobiol 2013; 16(1):35-44

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

A novel bleb-dependent polysaccharide export system in nitrogen-fixing Azotobacter vinelandii subjected to low nitrogen gas levels

The alginate biofilm-producing bacterium Azotobacter vinelandii aerobically fixes nitrogen by oxygensensitivenitrogenases. Here we investigated the bacterial response to nitrogen/oxygen gas mixtures. A. vinelandii cellswere cultured in nitrogen-free minimal media containing gas mixtures differing in their ratios of nitrogen and oxygen.The bacteria did not grow at oxygen concentrations >75 % but grew well in the presence of 5 % nitrogen/25 % oxygen.Growth of wild-type and alginate-deficient strains when cultured with 50 % oxygen did not differ substantially, indicatingthat alginate is not required for the protection of nitrogenases from oxygen damage. In response to decreasing nitrogenlevels, A. vinelandii produced greater amounts of alginate, accompanied by the formation of blebs on the cell surface. Theencystment of vegetative cells occurred in tandem with the release of blebs and the development of a multilayered exine.Immunoelectron microscopy using anti alginate-antibody revealed that the blebs contained alginate molecules. Bycontrast, alginate-deficient mutants could not form blebs. Taken together, our data provide evidence for a novel blebdependentpolysaccharide export system in A. vinelandii that is activated in response to low nitrogen gas levels

Adenovirus Vector-Derived VA-RNA-Mediated Innate Immune Responses

The major limitation of the clinical use of replication-incompetent adenovirus (Ad) vectors is the interference by innate immune responses, including induction of inflammatory cytokines and interferons (IFN), following in vivo application of Ad vectors. Ad vector-induced production of inflammatory cytokines and IFNs also results in severe organ damage and efficient induction of acquired immune responses against Ad proteins and transgene products. Ad vector-induced innate immune responses are triggered by the recognition of Ad components by pattern recognition receptors (PRRs). In order to reduce the side effects by Ad vector-induced innate immune responses and to develop safer Ad vectors, it is crucial to clarify which PRRs and which Ad components are involved in Ad vector-induced innate immune responses. Our group previously demonstrated that myeloid differentiating factor 88 (MyD88) and toll-like receptor 9 (TLR9) play crucial roles in the Ad vector-induced inflammatory cytokine production in mouse bone marrow-derived dendritic cells. Furthermore, our group recently found that virus associated-RNAs (VA-RNAs), which are about 160 nucleotide-long non-coding small RNAs encoded in the Ad genome, are involved in IFN production through the IFN-β promoter stimulator-1 (IPS-1)-mediated signaling pathway following Ad vector transduction. The aim of this review is to highlight the Ad vector-induced innate immune responses following transduction, especially VA-RNA-mediated innate immune responses. Our findings on the mechanism of Ad vector-induced innate immune responses should make an important contribution to the development of safer Ad vectors, such as an Ad vector lacking expression of VA-RNAs