Synthetic metabolism for biohalogenation.

The pressing need for novel bioproduction approaches faces a limitation in the number and type of molecules accessed through synthetic biology. Halogenation is widely used for tuning physicochemical properties of molecules and polymers, but traditional halogenation chemistry often lacks specificity and generates harmful by-products. Here, we pose that deploying synthetic metabolism tailored for biohalogenation represents an unique opportunity towards economically attractive and environmentally friendly organohalide production. On this background, we discuss growth-coupled selection of functional metabolic modules that harness the rich repertoire of biosynthetic and biodegradation capabilities of environmental bacteria for in vivo biohalogenation. By rationally combining these approaches, the chemical landscape of living cells can accommodate bioproduction of added-value organohalides which, as of today, are obtained by traditional chemistry.

Contribution of mechanosensitive channels to osmoadaptation and ectoine excretion in Halomonas elongata.

For osmoadaptation the halophilic bacterium Halomonas elongata synthesizes as its main compatible solute the aspartate derivative ectoine. H. elongata does not rely entirely on synthesis but can accumulate ectoine by uptake from the surrounding environment with the help of the osmoregulated transporter TeaABC. Disruption of the TeaABC-mediated ectoine uptake creates a strain that is constantly losing ectoine to the medium. However, the efflux mechanism of ectoine in H. elongata is not yet understood. H. elongata possesses four genes encoding mechanosensitive channels all of which belong to the small conductance type (MscS). Analysis by qRT-PCR revealed a reduction in transcription of the mscS genes with increasing salinity. The response of H. elongata to hypo- and hyperosmotic shock never resulted in up-regulation but rather in down-regulation of mscS transcription. Deletion of all four mscS genes created a mutant that was unable to cope with hypoosmotic shock. However, the knockout mutant grew significantly faster than the wildtype at high salinity of 2 M NaCl, and most importantly, still exported 80% of the ectoine compared to the wildtype. We thus conclude that a yet unknown system, which is independent of mechanosensitive channels, is the major export route for ectoine in H. elongata.

Revision and reannotation of the Halomonas elongata DSM 2581T genome.

The genome of the Halomonas elongata type strain DSM 2581, an industrial producer, was reevaluated using the Illumina HiSeq2500 technology. To resolve duplication-associated ambiguities, PCR products were generated and sequenced. Outside of duplications, 72 sequence corrections were required, of which 24 were point mutations and 48 were indels of one or few bases. Most of these were associated with polynucleotide stretches (poly-T stretch overestimated in 19 cases, poly-C underestimated in 15 cases). These problems may be attributed to using 454 technology for original genome sequencing. On average, the original genome sequence had only one error in 56 kb. There were 23 frameshift error corrections in the 29 protein-coding genes affected by sequence revision. The genome has been subjected to major reannotation in order to substantially increase the annotation quality.

Tetra-Antimony(III)-Bridged 18-Tungsto-2-Arsenates(V), [(LSb(III))4(A-α-As(V)W9O34)2](10-) (L = Ph, OH): Turning Bioactivity On and Off by Ligand Substitution.

Two tetra-antimony(III)-bridged, sandwich-type 18-tungsto-2-arsenates(V), [(LSb(III))4(A-α-As(V)W9O34)2](10-) (L = Ph (1), OH (2)), were prepared and fully characterized in the solid state and in solution. Both polyanions are stable in aqueous physiological medium for at least 24 h (at concentrations ≥2.5 × 10(-6) M). Despite the presence of an isostructural tetra-antimony(III) motif in 1 and 2, distinctly different antibacterial activity was observed for both polyanions. The minimum inhibitory concentrations (MIC) of 1 (7.8-62.5 µg/mL) is lower than for any other organoantimony(III)-containing polyoxometalate reported to date.

19-Tungstodiarsenate(III) Functionalized by Organoantimony(III) Groups: Tuning the Structure-Bioactivity Relationship.

A family of three discrete organoantimony(III)-functionalized heteropolyanions-[Na{2-(Me2HN(+)CH2)C6H4Sb(III)}As(III)2W19O67(H2O)](10-) (1), [{2-(Me2HN(+)CH2)C6H4Sb(III)}2As(III)2W19O67(H2O)](8-) (2), and [{2-(Me2HN(+)CH2)C6H4Sb(III)}{WO2(H2O)}{WO(H2O)}2(B-ß-As(III)W8O30)(B-α-As(III)W9O33)2](14-) (3)-have been prepared by one-pot reactions of the 19-tungstodiarsenate(III) precursor [As(III)2W19O67(H2O)](14-) with 2-(Me2NCH2)C6H4SbCl2. The three novel polyanions crystallized as the hydrated mixed-alkali salts Cs3KNa6[Na{2-(Me2HN(+)CH2)C6H4Sb(III)}As(III)2W19O67(H2O)]·43H2O (CsKNa-1), Rb2.5K5.5[{2-(Me2HN(+)CH2)C6H4Sb(III)}2As(III)2W19O67(H2O)]·18H2O·Me2NCH2C6H5 (RbK-2), and Rb2.5K11.5[{2-(Me2HN(+)CH2)C6H4Sb(III)}{WO2(H2O)}{WO(H2O)}2(B-ß-As(III)W8O30)(B-α-As(III)W9O33)2]·52H2O (RbK-3), respectively. The number of incorporated {2-(Me2HN(+)CH2)C6H4Sb(III)} units could be tuned by careful control of the experimental parameters. Polyanions 1 and 2 possess a dimeric sandwich-type topology, whereas 3 features a trimeric, wheel-shaped structure, representing the largest organoantimony-containing polyanion. All three compounds were fully characterized in the solid state via single-crystal X-ray diffraction (XRD), infrared (IR) spectroscopy, and thermogravimetric analysis, and their aqueous solution stability was validated by ultraviolet-visible light (UV-vis) and multinuclear ((1)H, (13)C, and (183)W) nuclear magnetic resonance (NMR) spectroscopy. Effective inhibition against six different types of bacteria was observed for 1 and 2, and we could extract a structure-bioactivity relationship for these polyanions.

Organoantimony(III)-Containing Tungstoarsenates(III): From Controlled Assembly to Biological Activity.

A family of three sandwich-type, phenylantimony(III)-containing tungstoarsenates(III), [(PhSb(III) ){Na(H2 O)}As(III) 2 W19 O67 (H2 O)](11-) (1), [(PhSb(III) )2 As(III) 2 W19 O67 (H2 O)](10-) (2), and [(PhSb(III) )3 (B-α-As(III) W9 O33 )2 ](12-) (3), have been synthesized by one-pot procedures and isolated as hydrated alkali metal salts, Cs3 K3.5 Na4.5 [(PhSb(III) ){Na(H2 O)}As(III) 2 W19 O67 (H2 O)]⋅41H2 O (CsKNa-1), Cs4.5 K5.5 [(PhSb(III) )2 As(III) 2 W19 O67 (H2 O)]⋅35H2 O (CsK-2), and Cs4.5 Na7.5 [(PhSb(III) )3 (B-α-As(III) W9 O33 )2 ]⋅42H2 O (CsNa-3). The number of incorporated {PhSb(III) } units could be selectively tuned from one to three by careful control of the reaction parameters. The three compounds were characterized in the solid state by single-crystal XRD, IR spectroscopy, and thermogravimetric analysis. The aqueous solution stability of sandwich polyanions 1-3 was also studied by multinuclear ((1) H, (13) C, (183) W) NMR spectroscopy. Effective inhibitory activity against six different kinds of bacteria was identified for all three polyanions, for which the activity increased with the number of incorporated {PhSb(III) } groups.

Reduction of cytotoxicity of benzalkonium chloride and octenidine by Brilliant Blue G.

The irritative effects of preservatives found in ophthalmologic solution, or of antiseptics used for skin disinfection is a consistent problem for the patients. The reduction of the toxic effects of these compounds is desired. Brilliant Blue G (BBG) has shown to meet the expected effect in presence of benzalkonium chloride (BAK), a well known preservative in ophthalmic solutions, and octenidine dihydrochloride (Oct), used as antiseptic in skin and wound disinfection. BBG shows a significant protective effect on human corneal epithelial (HCE) cells against BAK and Oct toxicity, increasing the cell survival up to 51 % at the highest BAK or Oct concentration tested, which is 0.01 %, both at 30 min incubation. Although BBG is described as a P2x7 receptor antagonist, other selective P2x7 receptor antagonists, OxATP (adenosine 5'-triphosphate-2',3'-dialdehyde) and DPPH (N'-(3,5-dichloropyridin-4-yl)-3-phenylpropanehydrazide), did not reduce the cytotoxicity of neither BAK nor Oct. Therefore we assume that the protective effect of BBG is not due to its action on the P2x7 receptor. Brilliant Blue R (BBR), a dye similar to BBG, was also tested for protective effect on BAK and Oct toxicity. In presence of BAK no significant protective effect was observed. Instead, with Oct a comparable protective effect was seen with that of BBG. To assure that the bacteriostatic effect is not affected by the combinations of BAK/BBG, Oct/BBG and Oct/BBR, bacterial growth inhibition was analyzed on different Gram-negative and Gram-positive bacteria. All combinations of BAK or Oct with BBG hinder growth of Gram-positive bacteria. The combinations of 0.001 % Oct and BBR above 0.025 % do not hinder the growth of B. subtilis. For Gram-negative bacteria, BBG and BBR reduce, but do not abolish, the antimicrobial effect of BAK nor of Oct. In conclusion, the addition of BBG at bacterial inhibitory concentrations is suggested in the ready-to-use ophthalmic preparations and antiseptic solutions.

Bacterial N2-fixation in mangrove ecosystems: insights from a diazotroph-mangrove interaction.

Mangrove forests are highly productive ecosystems but represent low nutrient environments. Nitrogen availability is one of the main factors limiting mangrove growth. Diazotrophs have been identified as key organisms that provide nitrogen to these environments. N2-fixation by such organisms was found to be higher in the mangrove roots than in surrounding rhizosphere. Moreover, previous studies showed that mangroves grew better in the presence of N2-fixers indicating a potentially mutualistic relationship. However, the molecular signals and mechanisms that govern these interactions are still poorly understood. Here we present novel insights in the interaction of a diazotroph with a mangrove species to improve our understanding of the molecular and ecophysiological relationship between these two organisms under controlled conditions. Our results showed that Marinobacterium mangrovicola is a versatile organism capable of competing with other organisms to survive for long periods in mangrove soils. N2-fixation by this bacterium was up-regulated in the presence of mangrove roots, indicating a possible beneficial interaction. The increase in N2-fixation was limited to cells of the exponential growth phase suggesting that N2-fixation differs over the bacterial growth cycle. Bacterial transformants harboring a transcriptional nifH::gusA fusion showed that M. mangrovicola successfully colonized mangrove roots and simultaneously conducted N2-fixation. The colonization process was stimulated by the lack of an external carbon source suggesting a possible mutualistic relationship. M. mangrovicola represents an interesting genetically accessible diazotroph, which colonize mangrove roots and exhibit higher N2-fixation in the presence of mangrove roots. Consequently, we propose this microorganism as a tool to study molecular interactions between N2-fixers and mangrove plants and to better understand how changes in the environment could impact these important and relatively unknown interactions.

Expression of extra-cellular levansucrase in Pseudomonas syringae is controlled by the in planta fitness-promoting metabolic repressor HexR.

BACKGROUND: Pseudomonas syringae pv. glycinea PG4180 causes bacterial blight on soybean plants and enters the leaf tissue through stomata or open wounds, where it encounters a sucrose-rich milieu. Sucrose is utilized by invading bacteria via the secreted enzyme, levansucrase (Lsc), liberating glucose and forming the polyfructan levan. P. syringae PG4180 possesses two functional lsc alleles transcribed at virulence-promoting low temperatures. RESULTS: We hypothesized that transcription of lsc is controlled by the hexose metabolism repressor, HexR, since potential HexR binding sites were identified upstream of both lsc genes. A hexR mutant of PG4180 was significantly growth-impaired when incubated with sucrose or glucose as sole carbon source, but exhibited wild type growth when arabinose was provided. Analyses of lsc expression resulted in higher transcript and protein levels in the hexR mutant as compared to the wild type. The hexR mutant's ability to multiply in planta was reduced. HexR did not seem to impact hrp gene expression as evidenced by the hexR mutant's unaltered hypersensitive response in tobacco and its unmodified protein secretion pattern as compared to the wild type under hrp-inducing conditions. CONCLUSIONS: Our data suggested a co-regulation of genes involved in extra-cellular sugar acquisition with those involved in intra-cellular energy-providing metabolic pathways in P. syringae.

Marinobacterium mangrovicola sp. nov., a marine nitrogen-fixing bacterium isolated from mangrove roots of Rhizophora mangle.

A nitrogen-fixing marine bacterium, designated strain Gal22(T), was isolated from mangrove roots of Rhizophora mangle. Cells were Gram-stain-negative rods, motile with a single polar flagellum. Growth was observed at 4-42 °C, pH 5.5 to 10 and with 0-18 % (w/v) NaCl. Strain Gal22(T) was positive for catalase and oxidase. Q-8 was the predominant lipoquinone. The DNA G+C content was 57.0 mol%. Based on phylogenetic analysis of 16S rRNA gene, strain Gal22(T) belongs to the genus Marinobacterium. The closely related strains were shown to be Marinobacterium lutimaris DSM 22012(T) and Marinobacterium litorale IMCC1877(T) with 99 % and 96 % 16S rRNA gene sequence similarity, respectively. DNA-DNA relatedness analysis indicated that strain Gal22(T) was different from M. lutimaris DSM 22012(T). On the basis of genotypic, morphological and biochemical characteristics, a novel species, Marinobacterium mangrovicola sp. nov. (type strain, Gal22(T) = DSM 27697(T) = CIP 110653(T)), is proposed.

Synthesis and biological activity of organoantimony(III)-containing heteropolytungstates.

Three discrete organoantimony(III)-containing heteropolytungstates [(PhSb(III))(4)(A-α-Ge(IV)W(9)O(34))(2)](12-) (1), [(PhSb(III))(4)(A-α-P(V)W(9)O(34))(2)](10-) (2), and [{2-(Me(2)NCH(2)C(6)H(4))Sb(III)}(3)(B-α-As(III)W(9)O(33))](3-) (3) have been synthesized in one-pot reactions in aqueous medium using the appropriate lacunary heteropolyanion precursor and organoantimony(III) source. Polyanions 1-3 were isolated as hydrated salts, (NH(4))(12)[(PhSb(III))(4)(A-α-Ge(IV)W(9)O(34))(2)]·20H(2)O (1a), Rb(9)Na[(PhSb(III))(4)(A-α-P(V)W(9)O(34))(2)]·20H(2)O (2a), and Rb(3)[{2-(Me(2)NCH(2)C(6)H(4))Sb(III)}(3)(B-α-As(III)W(9)O(33))]·7H(2)O (3a). The compounds 1a-3a were fully characterized in the solid state using infrared (IR) spectroscopy, single-crystal XRD, and thermogravimetric and elemental analyses. The stability of 1-3 in aqueous solution was confirmed by multinuclear NMR ((1)H, (13)C, (31)P, and (183)W) spectroscopy. Preliminary studies on the biological activity of 1-3 showed that all three compounds might act as potent antimicrobial agents.