Enhancing tellurite and selenite bioconversions by overexpressing a methyltransferase from Aromatoleum sp. CIB.

Pollution by metalloids, e.g., tellurite and selenite, is of serious environmental concern and, therefore, there is an increasing interest in searching for ecologically friendly solutions for their elimination. Some microorganisms are able to reduce toxic tellurite/selenite into less toxic elemental tellurium (Te) and selenium (Se). Here, we describe the use of the environmentally relevant ß-proteobacterium Aromatoleum sp. CIB as a platform for tellurite elimination. Aromatoleum sp. CIB was shown to tolerate 0.2 and 0.5 mM tellurite at aerobic and anaerobic conditions, respectively. Furthermore, the CIB strain was able to reduce tellurite into elemental Te producing rod-shaped Te nanoparticles (TeNPs) of around 200 nm length. A search in the genome of Aromatoleum sp. CIB revealed the presence of a gene, AzCIB_0135, which encodes a new methyltransferase that methylates tellurite and also selenite. AzCIB_0135 orthologs are widely distributed in bacterial genomes. The overexpression of the AzCIB_0135 gene both in Escherichia coli and Aromatoleum sp. CIB speeds up tellurite and selenite removal, and it enhances the production of rod-shaped TeNPs and spherical Se nanoparticles (SeNPs), respectively. Thus, the overexpression of a methylase becomes a new genetic strategy to optimize bacterial catalysts for tellurite/selenite bioremediation and for the programmed biosynthesis of metallic nanoparticles of biotechnological interest.

Motility, Adhesion and c-di-GMP Influence the Endophytic Colonization of Rice by Azoarcus sp. CIB.

Proficient crop production is needed to ensure the feeding of a growing global population. The association of bacteria with plants plays an important role in the health state of the plants contributing to the increase of agricultural production. Endophytic bacteria are ubiquitous in most plant species providing, in most cases, plant promotion properties. However, the knowledge on the genetic determinants involved in the colonization of plants by endophytic bacteria is still poorly understood. In this work we have used a genetic approach based on the construction of fliM, pilX and eps knockout mutants to show that the motility mediated by a functional flagellum and the pili type IV, and the adhesion modulated by exopolysaccarides are required for the efficient colonization of rice roots by the endophyte Azoarcus sp. CIB. Moreover, we have demonstrated that expression of an exogenous diguanylate cyclase or phophodiesterase, which causes either an increase or decrease of the intracellular levels of the second messenger cyclic di-GMP (c-di-GMP), respectively, leads to a reduction of the ability of Azoarcus sp. CIB to colonize rice plants. Here we present results demonstrating the unprecedented role of the universal second messenger cyclic-di-GMP in plant colonization by an endophytic bacterium, Azoarcus sp. CIB. These studies pave the way to further strategies to modulate the interaction of endophytes with their target plant hosts.

Enhancing the Rice Seedlings Growth Promotion Abilities of Azoarcus sp. CIB by Heterologous Expression of ACC Deaminase to Improve Performance of Plants Exposed to Cadmium Stress.

Environmental pollutants can generate stress in plants causing increased ethylene production that leads to the inhibition of plant growth. Ethylene production by the stressed plant may be lowered by Plant Growth-Promoting Bacteria (PGPB) that metabolizes the immediate precursor of ethylene 1-aminocyclopropane-1-carboxylate (ACC). Thus, engineering PGPB with ACC deaminase activity can be a promising alternative to mitigate the harmful effects of pollutants and thus enhance plant production. Here we show that the aromatics-degrading and metal-resistant Azoarcus sp. CIB behaves as a PGP-bacterium when colonizing rice as an endophyte, showing a 30% increment in plant weight compared to non-inoculated plants. The cloning and expression of an acdS gene led to a recombinant strain, Azoarcus sp. CIB (pSEVA237acdS), possessing significant ACC deaminase activity (6716 nmol mg-1 h-1), constituting the first PGPB of the Rhodocyclaceae family equipped with this PGP trait. The recombinant CIB strain acquired the ability to protect inoculated rice plants from the stress induced by cadmium (Cd) exposure and to increase the Cd concentration in rice seedlings. The observed decrease of the levels of reactive oxygen species levels in rice roots confirms such a protective effect. The broad-host-range pSEVA237acdS plasmid paves the way to engineer PGPB with ACC deaminase activity to improve the growth of plants that might face stress conditions.

ArxA From Azoarcus sp. CIB, an Anaerobic Arsenite Oxidase From an Obligate Heterotrophic and Mesophilic Bacterium.

Arsenic is a toxic element widely distributed in nature, but numerous bacteria are able to resist its toxicity mainly through the ars genes encoding an arsenate reductase and an arsenite efflux pump. Some "arsenotrophic" bacteria are also able to use arsenite as energy supplier during autotrophic growth by coupling anaerobic arsenite oxidation via the arx gene products to nitrate respiration or photosynthesis. Here, we have demonstrated that Azoarcus sp. CIB, a facultative anaerobic ß-proteobacterium, is able to resist arsenic oxyanions both under aerobic and anaerobic conditions. Genome mining, gene expression, and mutagenesis studies revealed the presence of a genomic island that harbors the ars and arx clusters involved in arsenic resistance in strain CIB. Orthologous ars clusters are widely distributed in the genomes of sequenced Azoarcus strains. Interestingly, genetic and metabolic approaches showed that the arx cluster of the CIB strain encodes an anaerobic arsenite oxidase also involved in the use of arsenite as energy source. Hence, Azoarcus sp. CIB represents the prototype of an obligate heterotrophic bacterium able to use arsenite as an extra-energy source for anaerobic cell growth. The arsenic island of strain CIB supports the notion that metabolic and energetic skills can be gained by genetic mobile elements.

Speeding up bioproduction of selenium nanoparticles by using Vibrio natriegens as microbial factory.

Selenium and selenium nanoparticles (SeNPs) are extensively used in biomedicine, electronics and some other industrial applications. The bioproduction of SeNPs is gaining interest as a green method to manufacture these biotechnologically relevant products. Several microorganisms have been used for the production of SeNPs either under aerobic or anaerobic conditions. Vibrio natriegens is a non-pathogenic fast-growing bacterium, easily cultured in different carbon sources and that has recently been engineered for easy genetic manipulation in the laboratory. Here we report that V. natriegens was able to perfectly grow aerobically in the presence of selenite concentrations up to 15 mM with a significant survival still observed at concentrations as high as 100 mM selenite. Electron microscopy and X-ray spectroscopy analyses demonstrate that V. natriegens cells growing aerobically in selenite-containing LB medium at 30 °C produced spherical electron-dense SeNPs whose size ranged from 100-400 nm. Selenite reduction just started at the beginning of the exponential growth phase and the release of SeNPs was observed after cell lysis. Remarkably, V. natriegens produced SeNPs faster than other described microorganisms that were proposed as model bioreactors for SeNPs production. Thus, the fast-growing V. natriegens bacterium becomes a suitable biocatalyst for bioremediation of selenite and for speeding-up the eco-friendly synthesis of SeNPs.

Biosynthesis of selenium nanoparticles by Azoarcus sp. CIB.

BACKGROUND: Different bacteria have been reported so far that link selenite resistance to the production of metallic selenium nanoparticles (SeNPs). Although SeNPs have many biotechnological applications in diverse areas, the molecular mechanisms involved in their microbial genesis are not fully understood. The Azoarcus genus is a physiologically versatile group of beta-proteobacteria of great environmental relevance. Azoarcus sp. CIB is a facultative anaerobe that combines the ability to degrade under aerobic and/or anaerobic conditions a wide range of aromatic compounds, including some toxic hydrocarbons such as toluene and m-xylene, with an endophytic life style in the root of rice. We unravel here an additional physiological feature of the strain CIB that is related to its resistance to selenium oxyanions and the formation of SeNPs. RESULTS: This work is the first report of a member of the Azoarcus genus that is able to anaerobically grow in the presence of selenite. Electron microscopy preparations and X-ray spectroscopy analyses demonstrate the reduction of selenite to spherical electron-dense SeNPs whose average size was 123 ± 35 nm of diameter. Our data suggest that the main molecular mechanism of selenite resistance resides on an energy-dependent selenite exporter. Azoarcus cells trigger the synthesis of SeNPs when they reach the stationary-phase of growth, and either the exhaustion of electron donor or acceptor, both of which lead to starvation conditions, produce the reduction of selenite to red elemental selenium. Azoarcus becomes a promising biocatalyst, either as whole cells or cellular extracts, for the anaerobic and/or aerobic green synthesis of SeNPs. CONCLUSIONS: Azoarcus turns out to be a new eco-friendly system to reduce selenite and produce spherical SeNPs. Moreover, this is the first report of a rice endophyte able to produce SeNPs. Since Azoarcus is also able to degrade both aerobically and anaerobically toxic aromatic compounds of great environmental concern, it becomes a suitable candidate for a more sustainable agricultural practice and for bioremediation strategies.