Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803

Although the biogeochemistry of the two environmentally hazardous compounds arsenic and sulfide has been extensively investigated, the biological interference of these two toxic but potentially energy-rich compounds has only been hypothesized and indirectly proven. Here we provide direct evidence for the first time that in the photosynthetic model organism Synechocystis sp. strain PCC6803 the two metabolic pathways are linked by coregulated genes that are involved in arsenic transport, sulfide oxidation, and probably in sulfide-based alternative photosynthesis. Although Synechocystis sp. strain PCC6803 is an obligate photoautotrophic cyanobacterium that grows via oxygenic photosynthesis, we discovered that specific genes are activated in the presence of sulfide or arsenite to exploit the energy potentials of these chemicals. These genes form an operon that we termed suoRSCT, located on a transposable element of type IS4 on the plasmid pSYSM of the cyanobacterium. suoS (sll5036) encodes a light-dependent, type I sulfide:quinone oxidoreductase. The suoR (sll5035) gene downstream of suoS encodes a regulatory protein that belongs to the ArsR-type repressors that are normally involved in arsenic resistance. We found that this repressor has dual specificity, resulting in 200-fold induction of the operon upon either arsenite or sulfide exposure. The suoT gene encodes a transmembrane protein similar to chromate transporters but in fact functioning as an arsenite importer at permissive concentrations. We propose that the proteins encoded by the suoRSCT operon might have played an important role under anaerobic, reducing conditions on primordial Earth and that the operon was acquired by the cyanobacterium via horizontal gene transfer

Heterologous Expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] Hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H2 evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria

Effect of inactivation of genes involved in ammonium regulation on the biohydrogen production of Rhodobacter capsulatus

Hydrogen production by nitrogenase is an energetically expensive process for the cell, hence strictly controlled at different levels. Ammonium is one of the substances regulating nitrogenase activity. The key proteins in the regulation of nitrogenase by ammonium are two regulatory proteins; GlnB and GlnK. In order to increase hydrogen production of Rhodobacter capsulatus DSM1710 (wild type strain) grown on agricultural materials/wastes, ammonium inhibition of nitrogenase enzyme has to be eliminated. In this study, GlnB and GlnK were targeted to be inactivated by in frame site-directed mutagenesis. The glnB mutant R. capsulatus (GP1 strain) was obtained at the end of mutagenesis studies. In the case of glnK, the suicide vector was constructed and delivered into the cells. However, glnK mutant could not be obtained

Evaluation of hydrogen production by Rhodobacter sphaeroides OU001 and its hupSL deficient mutant using acetate and malate as carbon sources

Rhodobacter sphaeroides O.U.001 is one of the candidates for photobiological hydrogen production among purple non-sulfur bacteria. Hydrogen is produced by Mo-nitrogenase from organic acids such as malate or lactate. A hupSL in frame deletion mutant strain was constructed without using any antibiotic resistance gene. The hydrogen production potential of the R. sphaeroides O.U.001 and its newly constructed hupSL deleted mutant strain in acetate media was evaluated and compared with malate containing media. The hupSL(-) R. sphaeroides produced 2.42 l H-2/l culture and 0.25 l H-2/l culture in 15 mM malate and 30 mM acetate containing media, respectively, as compared to the wild type cells which evolved 1.97 l H-2/l culture and 0.21 l H-2/l culture in malate and acetate containing media, correspondingly. According to the results, hupSL- R. sphaeroides is a better hydrogen producer but acetate alone does not seem to be an efficient carbon source for photo-heterotrophic H-2 production by R. sphaeroides. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved

Phages from Genus Bruynoghevirus and Phage Therapy: Pseudomonas Phage Delta Case

The applicability and safety of bacteriophage Delta as a potential anti-Pseudomonas aeruginosa agent belonging to genus Bruynoghevirus (family Podoviridae) was characterised. Phage Delta belongs to the species Pseudomonas virus PaP3, which has been described as a temperate, with cos sites at the end of the genome. The phage Delta possesses a genome of 45,970 bp that encodes tRNA for proline (Pro), aspartic acid (Asp) and asparagine (Asn) and does not encode any known protein involved in lysogeny formation or persistence. Analysis showed that phage Delta has 182 bp direct terminal repeats at the end of genome and lysogeny was confirmed, neither upon infection at low nor at high multiplicity of infection (MOI). The turbid plaques that appear on certain host lawns can result from bacteriophage insensitive mutants that occur with higher frequency (10−4). In silico analysis showed that the genome of Delta phage does not encode any known bacterial toxin or virulence factor, determinants of antibiotic resistance and known human allergens. Based on the broad host range and high lytic activity against planktonic and biofilm cells, phage Delta represents a promising candidate for phage therapy

Improved hydrogen production by uptake hydrogenase deficient mutant strain of Rhodobacter sphaeroides OU001

Rhodobacter sphaeroides O.U.001 is a purple non-sulfur bacterium producing hydrogen under photoheterotrophic conditions. Hydrogen is produced by Mo-nitrogenase enzyme and substantial amount of H-2 is reoxidized by a membrane-bound uptake hydrogenase in the wild type strain. To improve the hydrogen producing capacity of the cells, a suicide vector containing a gentamicin cassette in the hupSL genes was introduced into R. sphaeroiodes O.U.001 and the uptake hydrogenase genes were destroyed by site directed mutagenesis. The correct integration of the construct was confirmed by uptake hydrogenase activity measurement, PCR and subsequent sequence analysis. The wild type and the mutant cells showed similar growth patterns but the total volume of hydrogen gas evolved by the mutant was 20% higher than that of the wild type strain. This result demonstrated that the hydrogen produced by the nitrogenase was not consumed by uptake hydrogenase leading to higher hydrogen production. (c) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved

Are Bordetella bronchiseptica Siphoviruses (Genus Vojvodinavirus) Appropriate for Phage Therapy—Bacterial Allies or Foes?

Bordetella bronchiseptica is a respiratory animal pathogen that shows growing resistance to commonly used antibiotics, which has necessitated the examination of new antimicrobials, including bacteriophages. In this study, we examined the previously isolated and partially characterized B. bronchiseptica siphoviruses of the genus Vojvodinavirus (LK3, CN1, CN2, FP1 and MW2) for their ability to inhibit bacterial growth and biofilm, and we examined other therapeutically important properties through genomic analysis and lysogeny experiments. The phages inhibited bacterial growth at a low multiplicity of infection (MOI = 0.001) of up to 85% and at MOI = 1 for >99%. Similarly, depending on the phages and MOIs, biofilm formation inhibition ranged from 65 to 95%. The removal of biofilm by the phages was less efficient but still considerably high (40–75%). Complete genomic sequencing of Bordetella phage LK3 (59,831 bp; G + C 64.01%; 79 ORFs) showed integrase and repressor protein presence, indicating phage potential to lysogenize bacteria. Lysogeny experiments confirmed the presence of phage DNA in bacterial DNA upon infection using PCR, which showed that the LK3 phage forms more or less stable lysogens depending on the bacterial host. Bacterial infection with the LK3 phage enhanced biofilm production, sheep blood hemolysis, flagellar motility, and beta-lactam resistance. The examined phages showed considerable anti-B. bronchiseptica activity, but they are inappropriate for therapy because of their temperate nature and lysogenic conversion of the host bacterium