Characterization and genomic analysis of chromate resistant and reducing Bacillus cereus strain SJ1

<p>Abstract</p> <p>Background</p> <p>Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by <it>Bacillus cereus </it>SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence.</p> <p>Results</p> <p><it>Bacillus cereus </it>SJ1 isolated from chromium-contaminated wastewater of a metal electroplating factory displayed high Cr(VI) resistance with a minimal inhibitory concentration (MIC) of 30 mM when induced with Cr(VI). A complete bacterial reduction of 1 mM Cr(VI) was achieved within 57 h. By genome sequence analysis, a putative chromate transport operon, <it>chrIA</it>1, and two additional <it>chrA </it>genes encoding putative chromate transporters that likely confer chromate resistance were identified. Furthermore, we also found an azoreductase gene <it>azoR </it>and four nitroreductase genes <it>nitR </it>possibly involved in chromate reduction. Using reverse transcription PCR (RT-PCR) technology, it was shown that expression of adjacent genes <it>chrA</it>1 and <it>chrI </it>was induced in response to Cr(VI) but expression of the other two chromate transporter genes <it>chrA</it>2 and <it>chrA</it>3 was constitutive. In contrast, chromate reduction was constitutive in both phenotypic and gene expression analyses. The presence of a resolvase gene upstream of <it>chrIA</it>1, an arsenic resistance operon and a gene encoding Tn7-like transposition proteins ABBCCCD downstream of <it>chrIA</it>1 in <it>B. cereus </it>SJ1 implied the possibility of recent horizontal gene transfer.</p> <p>Conclusion</p> <p>Our results indicate that expression of the chromate transporter gene <it>chrA</it>1 was inducible by Cr(VI) and most likely regulated by the putative transcriptional regulator ChrI. The bacterial Cr(VI)-resistant level was also inducible. The presence of an adjacent arsenic resistance gene cluster nearby the <it>chrIA</it>1 suggested that strong selective pressure by chromium and arsenic could cause bacterial horizontal gene transfer. Such events may favor the survival and increase the resistance level of <it>B. cereus </it>SJ1.</p

Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence - by controlling for phylogenetic structure - for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01

<p>Abstract</p> <p>Background</p> <p>Many strains of <it>Thermus </it>have been isolated from hot environments around the world. <it>Thermus scotoductus </it>SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different <it>Thermus thermophilus </it>strains have been completed. This paper represents the completed genome from a second <it>Thermus </it>species - <it>T. scotoductus</it>.</p> <p>Results</p> <p>The genome of <it>Thermus scotoductus </it>SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the <it>Thermus thermophilus </it>genomes. The <it>T. thermophilus </it>megaplasmid genes are part of the <it>T. scotoductus </it>chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of <it>T. scotoductus and T. thermophilus</it>. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from <it>Meiothermus ruber</it>. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of <it>Thermus scotoductus </it>was confirmed experimentally as expected as most of the proteins of the natural transformation system of <it>Thermus thermophilus </it>are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle.</p> <p>Conclusions</p> <p>The genome of <it>Thermus scotoductus </it>SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to <it>Thermus thermophilus</it>. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of <it>Thermus scotoductus </it>illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.</p

Characterisation of a Desmosterol Reductase Involved in Phytosterol Dealkylation in the Silkworm, Bombyx mori

Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C29 and C28) yielding cholesterol (C27). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD- dependent reaction

Photobiocatalytic chemistry of oxidoreductases using water as the electron donor

[EN] To date, water has been poorly studied as the sacrificial electron donor for biocatalytic redox reactions using isolated enzymes. Here we demonstrate that water can also be turned into a sacrificial electron donor to promote biocatalytic redox reactions. The thermodynamic driving force required for water oxidation is obtained from UV and visible light by means of simple titanium dioxide-based photocatalysts. The electrons liberated in this process are delivered to an oxidoreductase by simple flavin redox mediators. Overall, the feasibility of photobiocatalytic, water-driven bioredox reactions is demonstrated.Financial support from the Spanish Science and Innovation Ministry (Consolider Ingenio 2010-MULTICAT CSD 2009-00050, Subprograma de apoyo a Centros y Universidades de Excelencia Severo Ochoa SEV 2012 0267). M. M. acknowledges the Spanish Science and Innovation Ministry for a 'Juan de la Cierva' postdoctoral contract. S. G. acknowledges the European Union Marie Curie Programme (ITN 'Biotrains', Grant Agreement No. 238531).Mifsud Grau, M.; Gargiulo, S.; Iborra Chornet, S.; Arends, IWCE.; Hollmann, F.; Corma Canós, A. (2014). Photobiocatalytic chemistry of oxidoreductases using water as the electron donor. Nature Communications. 5:1-6. https://doi.org/10.1038/ncomms4145S165Bornscheuer, U. T. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).Breuer, M. et al. Industrial methods for the production of optically active intermediates. Angew. Chem. Int. Ed. 43, 788–824 (2004).Pollard, D. J. & Woodley, J. M. Biocatalysis for pharmaceutical intermediates: the future is now. Trends Biotechnol. 25, 66–73 (2007).Ran, N., Zhao, L., Chen, Z. & Tao, J. Recent applications of biocatalysis in developing green chemistry for chemical synthesis at the industrial scale. Green. Chem. 10, 361–372 (2008).Schmid, A. et al. Industrial biocatalysis today and tomorrow. Nature 409, 258–268 (2001).Schmid, A., Hollmann, F., Park, J. B. & Bühler, B. The use of enzymes in the chemical industry in Europe. Curr. Opin. Biotechnol. 13, 359–366 (2002).Schoemaker, H. E., Mink, D. & Wubbolts, M. G. Dispelling the myths-biocatalysis in industrial synthesis. Science 299, 1694–1697 (2003).Turner, N. J. & O’Reilly, E. Biocatalytic retrosynthesis. Nat. Chem. Biol. 9, 285–288 (2013).Drauz K., Gröger H., May O. (eds)Enzyme Catalysis in Organic Synthesis Wiley-VCH: Weinheim, (2012).Weckbecker, A., Gröger, H. & Hummel, W. Regeneration of nicotinamide coenzymes: principles and applications for the synthesis of chiral compounds. inBiosystems Engineering I: Creating Superior Biocatalysts pp195–242Springer: Berlin, (2010).Van der Donk, W. A. & Zhao, H. Recent developments in pyridine nucleotide regeneration. Curr. Opin. Biotechnol. 14, 421–426 (2003).Wu, H. et al. Methods for the regeneration of nicotinamide coenzymes. Green. Chem. 15, 1773–1789 (2013).Rodriguez, C., Lavandera, I. & Gotor, V. Recent advances in cofactor regeneration systems applied to biocatalyzed oxidative processes. Curr. Org. Chem. 16, 2525–2541 (2012).Reipa, V., Mayhew, M. P. & Vilker, V. L. A direct electrode-driven P450 cycle for biocatalysis. Proc. Natl Acad. Sci. USA 94, 13554–13558 (1997).Bernard, J., van Heerden, E., Arends, I. W. C. E., Opperman, D. J. & Hollmann, F. Chemoenzymatic reduction of conjugated C=C double bonds. Chem. Cat. Chem. 4, 196–199 (2012).Hollmann, F., Arends, I. W. C. E. & Bühler, K. Biocatalytic redox reactions for organic synthesis: nonconventional regeneration methods. Chem. Cat. Chem. 2, 762–782 (2010).Hollmann, F., Hofstetter, K., Habicher, T., Hauer, B. & Schmid, A. Direct electrochemical regeneration of monooxygenase subunits for biocatalytic asymmetric epoxidation. J. Am. Chem. Soc. 127, 6540–6541 (2005).Hollmann, F., Lin, P.-C., Witholt, B. & Schmid, A. Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a fad-dependent monooxygenase for catalysis. J. Am. Chem. Soc. 125, 8209–8217 (2003).Hollmann, F. & Schmid, A. Towards [Cp*Rh(bpy)(H2O)]2+-promoted P450 catalysis: direct regeneration of CytC. J. Inorg. Biochem. 103, 313–315 (2009).Hollmann, F., Taglieber, A., Schulz, F. & Reetz, M. T. A light-driven stereoselective biocatalytic oxidation. Angew. Chem. Int. Ed. 46, 2903–2906 (2007).Mifsud Grau, M. et al. Photoenzymatic reduction of C=C double bonds. Adv. Synth. Catal. 351, 3279–3286 (2009).Ruinatscha, R., Dusny, C., Buehler, K. & Schmid, A. Productive asymmetric styrene epoxidation based on a next generation electroenzymatic methodology. Adv. Synth. Catal. 351, 2505–2515 (2009).Schwaneberg, U., Appel, D., Schmitt, J. & Schmid, R. D. P450 in biotechnology: zinc driven ω-hydroxylation of p-nitrophenoxydodecanoic acid using P450 BM-3 F87A as a catalyst. J. Biotechnol. 84, 249–257 (2000).Taglieber, A., Schulz, F., Hollmann, F., Rusek, M. & Reetz, M. T. Light-Driven Biocatalytic Oxidation and Reduction Reactions: Scope and Limitations. Chem. Bio. Chem. 9, 565–572 (2008).Udit, A. K., Arnold, F. H. & Gray, H. B. Cobaltocene-mediated catalytic monooxygenation using holo and heme domain cytochrome P450 BM3. J. Inorg. Biochem. 98, 1547–1550 (2004).Udit, A. K., Hill, M. G., Bittner, V. G., Arnold, F. H. & Gray, H. B. Reduction of dioxygen catalyzed by pyrene-wired heme domain cytochrome p450 bm3 electrodes. J. Am. Chem. Soc. 126, 10218–10219 (2004).Unversucht, S., Hollmann, F., Schmid, A. & van Pée, K.-H. FADH2-Dependence of Tryptophan 7-Halogenase. Adv. Synth. Catal. 347, 1163–1167 (2005).Zilly, F. E., Taglieber, A., Schulz, F., Hollmann, F. & Reetz, M. T. Deazaflavins as mediators in light-driven cytochrome P450 catalyzed hydroxylations. Chem. Commun. 7152–7154 (2009).Yehezkeli, O. et al. Integrated photosystem II-based photo-bioelectrochemical cells. Nat. Commun. 3, 742 (2012).Duan, L. et al. A molecular ruthenium catalyst with water-oxidation activity comparable to that of photosystem II. Nat. Chem. 4, 418–423 (2012).Dau, H., Zaharieva, I. & Haumann, M. Recent developments in research on water oxidation by photosystem II. Curr. Opin. Chem. Biol. 16, 3–10 (2012).Qu, Y. & Duan, X. Progress, challenge and perspective of heterogeneous photocatalysts. Chem. Soc. Rev. 42, 2568–2580 (2013).Takanabe, K. & Domen, K. Preparation of inorganic photocatalytic materials for overall water splitting. Chem. Cat. Chem. 4, 1485–1497 (2012).Wee, T.-L. et al. Photochemical synthesis of a water oxidation catalyst based on cobalt nanostructures. J. Am. Chem. Soc. 133, 16742–16745 (2011).Cargnello, M. & Fornasiero, P. Photocatalysis by nanostructured TiO2 based semiconductors. inHandbook of Green Chemistry, Green Nanoscience (eds Selva M., Perosa A. Wiley-VCH: Weinheim, (2010).Liu, S. Q. & Chen, A. C. Coadsorption of horseradish peroxidase with thionine on TiO2: Nanotubes for biosensing. Langmuir 21, 8409–8413 (2005).Zhang, Y., He, P. L. & Hu, N. F. Horseradish peroxidase immobilized in TiO2 nanoparticle films on pyrolytic graphite electrodes: direct electrochemistry and bioelectrocatalysis. Electrochim. Acta 49, 1981–1988 (2004).Chen, D., Zhang, H., Li, X. & Li, J. H. Biofunctional titania nanotubes for visible-light-activated photoelectrochemical biosensing. Anal. Chem. 82, 2253–2261 (2010).Gomes Silva, C. U., Juárez, R., Marino, T., Molinari, R. & García, H. Influence of excitation wavelength (UV or visible light) on the photocatalytic activity of titania containing gold nanoparticles for the generation of hydrogen or oxygen from water. J. Am. Chem. Soc. 133, 595–602 (2010).Opperman, D. J., Piater, L. A. & van Heerden, E. A novel chromate reductase from Thermus scotoductus SA-01 related to old yellow enzyme. J. Bacteriol. 190, 3076–3082 (2008).Opperman, D. J. et al. Crystal structure of a thermostable old yellow enzyme from Thermus scotoductus SA-01. Biochem. Biophys. Res. Commun. 393, 426–431 (2010).Choi, S. H. et al. The influence of non-stoichiometric species of V/TiO2 catalysts on selective catalytic reduction at low temperature. J. Mol. Catal. A: Chem. 304, 166–173 (2009)

Introgression of Ivermectin Resistance Genes into a Susceptible Haemonchus contortus Strain by Multiple Backcrossing

Anthelmintic drug resistance in livestock parasites is already widespread and in recent years there has been an increasing level of anthelmintic drug selection pressure applied to parasitic nematode populations in humans leading to concerns regarding the emergence of resistance. However, most parasitic nematodes, particularly those of humans, are difficult experimental subjects making mechanistic studies of drug resistance extremely difficult. The small ruminant parasitic nematode Haemonchus contortus is a more amenable model system to study many aspects of parasite biology and investigate the basic mechanisms and genetics of anthelmintic drug resistance. Here we report the successful introgression of ivermectin resistance genes from two independent ivermectin resistant strains, MHco4(WRS) and MHco10(CAVR), into the susceptible genome reference strain MHco3(ISE) using a backcrossing approach. A panel of microsatellite markers were used to monitor the procedure. We demonstrated that after four rounds of backcrossing, worms that were phenotypically resistant to ivermectin had a similar genetic background to the susceptible reference strain based on the bulk genotyping with 18 microsatellite loci and individual genotyping with a sub-panel of 9 microsatellite loci. In addition, a single marker, Hcms8a20, showed evidence of genetic linkage to an ivermectin resistance-conferring locus providing a starting point for more detailed studies of this genomic region to identify the causal mutation(s). This work presents a novel genetic approach to study anthelmintic resistance and provides a “proof-of-concept” of the use of forward genetics in an important model strongylid parasite of relevance to human hookworms. The resulting strains provide valuable resources for candidate gene studies, whole genome approaches and for further genetic analysis to identify ivermectin resistance loci

The map-1 Gene Family in Root-Knot Nematodes, Meloidogyne spp.: A Set of Taxonomically Restricted Genes Specific to Clonal Species

Taxonomically restricted genes (TRGs), i.e., genes that are restricted to a limited subset of phylogenetically related organisms, may be important in adaptation. In parasitic organisms, TRG-encoded proteins are possible determinants of the specificity of host-parasite interactions. In the root-knot nematode (RKN) Meloidogyne incognita, the map-1 gene family encodes expansin-like proteins that are secreted into plant tissues during parasitism, thought to act as effectors to promote successful root infection. MAP-1 proteins exhibit a modular architecture, with variable number and arrangement of 58 and 13-aa domains in their central part. Here, we address the evolutionary origins of this gene family using a combination of bioinformatics and molecular biology approaches. Map-1 genes were solely identified in one single member of the phylum Nematoda, i.e., the genus Meloidogyne, and not detected in any other nematode, thus indicating that the map-1 gene family is indeed a TRG family. A phylogenetic analysis of the distribution of map-1 genes in RKNs further showed that these genes are specifically present in species that reproduce by mitotic parthenogenesis, with the exception of M. floridensis, and could not be detected in RKNs reproducing by either meiotic parthenogenesis or amphimixis. These results highlight the divergence between mitotic and meiotic RKN species as a critical transition in the evolutionary history of these parasites. Analysis of the sequence conservation and organization of repeated domains in map-1 genes suggests that gene duplication(s) together with domain loss/duplication have contributed to the evolution of the map-1 family, and that some strong selection mechanism may be acting upon these genes to maintain their functional role(s) in the specificity of the plant-RKN interactions

Genome-wide survey and analysis of microsatellites in nematodes, with a focus on the plant-parasitic species Meloidogyne incognita

<p>Abstract</p> <p>Background</p> <p>Microsatellites are the most popular source of molecular markers for studying population genetic variation in eukaryotes. However, few data are currently available about their genomic distribution and abundance across the phylum Nematoda. The recent completion of the genomes of several nematode species, including <it>Meloidogyne incognita</it>, a major agricultural pest worldwide, now opens the way for a comparative survey and analysis of microsatellites in these organisms.</p> <p>Results</p> <p>Using MsatFinder, the total numbers of 1-6 bp perfect microsatellites detected in the complete genomes of five nematode species (<it>Brugia malayi</it>, <it>Caenorhabditis elegans</it>, <it>M. hapla</it>, <it>M. incognita</it>, <it>Pristionchus pacificus</it>) ranged from 2,842 to 61,547, and covered from 0.09 to 1.20% of the nematode genomes. Under our search criteria, the most common repeat motifs for each length class varied according to the different nematode species considered, with no obvious relation to the AT-richness of their genomes. Overall, (AT)_<it>n</it>, (AG)_{<it>n </it>}and (CT)_{<it>n </it>}were the three most frequent dinucleotide microsatellite motifs found in the five genomes considered. Except for two motifs in <it>P. pacificus</it>, all the most frequent trinucleotide motifs were AT-rich, with (AAT)_{<it>n </it>}and (ATT)_{<it>n </it>}being the only common to the five nematode species. A particular attention was paid to the microsatellite content of the plant-parasitic species <it>M. incognita</it>. In this species, a repertoire of 4,880 microsatellite loci was identified, from which 2,183 appeared suitable to design markers for population genetic studies. Interestingly, 1,094 microsatellites were identified in 801 predicted protein-coding regions, 99% of them being trinucleotides. When compared against the InterPro domain database, 497 of these CDS were successfully annotated, and further assigned to Gene Ontology terms.</p> <p>Conclusions</p> <p>Contrasted patterns of microsatellite abundance and diversity were characterized in five nematode genomes, even in the case of two closely related <it>Meloidogyne </it>species. 2,245 di- to hexanucleotide loci were identified in the genome of <it>M. incognita</it>, providing adequate material for the future development of a wide range of microsatellite markers in this major plant parasite.</p

Tm1: A Mutator/Foldback Transposable Element Family in Root-Knot Nematodes

Three closely related parthenogenetic species of root-knot nematodes, collectively termed the Meloidogyne incognita-group, are economically significant pathogens of diverse crop species. Remarkably, these asexual root-knot nematodes are capable of acquiring heritable changes in virulence even though they lack sexual reproduction and meiotic recombination. Characterization of a near isogenic pair of M. javanica strains differing in response to tomato with the nematode resistance gene Mi-1 showed that the virulent strain carried a deletion spanning a gene called Cg-1. Herein, we present evidence that the Cg-1 gene lies within a member of a novel transposable element family (Tm1; Transposon in Meloidogyne-1). This element family is defined by composite terminal inverted repeats of variable lengths similar to those of Foldback (FB) transposable elements and by 9 bp target site duplications. In M. incognita, Tm1 elements can be classified into three general groups: 1) histone-hairpin motif elements; 2) MITE-like elements; 3) elements encoding a putative transposase. The predicted transposase shows highest similarity to gene products encoded by aphids and mosquitoes and resembles those of the Phantom subclass of the Mutator transposon superfamily. Interestingly, the meiotic, sexually-reproducing root-knot nematode species M. hapla has Tm1 elements with similar inverted repeat termini, but lacks elements with histone hairpin motifs and contains no elements encoding an intact transposase. These Tm1 elements may have impacts on root-knot nematode genomes and contribute to genetic diversity of the asexual species