Ulrike Kappler

University of Queensland, Brisbane, Queensland, Australia

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Publications (82)234.15 Total impact

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    ABSTRACT: Mononuclear molybdenum enzymes of the dimethylsulfoxide (DMSO) reductase family occur exclusively in prokaryotes, and a loss of some these enzymes has been linked to a loss of bacterial virulence in several cases. The MobA protein catalyzes the final step in the synthesis of the molybdenum guanine dinucleotide (MGD) cofactor that is exclusive to enzymes of the DMSO reductase family. MobA has been proposed as a potential target for control of virulence since its inhibition would affect the activities of all molybdoenzymes dependent upon MGD. Here, we have studied the phenotype of a mobA mutant of the host-adapted human pathogen Haemophilus influenzae. H. influenzae causes and contributes to a variety of acute and chronic diseases of the respiratory tract, and several enzymes of the DMSO reductase family are conserved and highly expressed in this bacterium. The mobA mutation caused a significant decrease in the activities of all Mo-enzymes present, and also resulted in a small defect in anaerobic growth. However, we did not detect a defect in in vitro biofilm formation nor in invasion and adherence to human epithelial cells in tissue culture compared to the wild-type. In a murine in vivo model, the mobA mutant showed only a mild attenuation compared to the wild-type. In summary, our data show that MobA is essential for the activities of molybdenum enzymes, but does not appear to affect the fitness of H. influenzae. These results suggest that MobA is unlikely to be a useful target for antimicrobials, at least for the purpose of treating H. influenzae infections.
    Full-text · Article · Nov 2015 · Frontiers in Microbiology
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    ABSTRACT: Interprotein electron transfer underpins the essential processes of life and relies on the formation of specific, yet transient protein-protein interactions. In biological systems, the detoxification of sulfite is catalyzed by the sulfite-oxidizing enzymes (SOEs), which interact with an electron acceptor for catalytic turnover. Here, we report the structural and functional analyses of the SOE SorT from Sinorhizobium meliloti and its cognate electron acceptor SorU. Kinetic and thermodynamic analyses of the SorT/SorU interaction showed the complex is dynamic in solution, and that the proteins interact with K d = 13.5 {plus minus} 0.8 βM. The crystal structures of the oxidized SorT and SorU both in isolation and in complex, reveal the interface to be remarkably electrostatic, with an unusually large number of direct hydrogen bonding interactions. The assembly of the complex is accompanied by an adjustment in the structure of SorU and conformational sampling provides a mechanism for dissociation of the SorT/SorU assembly.
    Full-text · Article · Nov 2015 · eLife Sciences
  • Ulrike Kappler · Hendrik Schäfer
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    ABSTRACT: Dimethylsulfide (DMS) is a naturally occurring chemical that is part of the biogeochemical sulfur cycle and has been implicated in climate-relevant atmospheric processes. In addition, DMS occurs in soil environments as well as in food stuff as a flavor compound and it can also be associated with disease states such as halitosis. A major environmental source of DMS is the marine algal osmoprotectant dimethylsulfoniopropionate (DMSP). A variety of bacterial enzyme systems lead either to the production of DMS from DMSP or dimethylsulfoxide (DMSO) or its oxidation to, e.g., DMSO. The interconversion of DMS and DMSO is catalyzed by molybdenum-containing metalloenzymes that have been very well studied, and recently another enzyme system, an NADH-dependent, flavin-containing monooxygenase, that produces formaldehyde and methanethiol from DMS has also been described. DMS conversions are not limited to a specialized group of bacteria – evidence for DMS-based metabolism exists for heterotrophic, autotrophic and phototrophic bacteria and there is also evidence for the occurrence of this type of sulfur compound conversion in Archaea.
    No preview · Article · Nov 2014 · Metal ions in life sciences
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    ABSTRACT: The respiratory DMSO reductase from Rhodobacter capsulatus catalyzes the reduction of dimethyl sulfoxide to dimethyl sulfide. Herein, we have utilized this Mo enzyme as an enantioselective catalyst to generate optically pure sulfoxides (methyl p-tolyl sulfoxide, methyl phenyl sulfoxide and phenyl vinyl sulfoxide) from racemic starting materials. A hexaaminecobalt coordination compound in its divalent oxidation state was employed as the mediator of electron transfer between the working electrode and DMSO reductase to continually reactivate the enzyme after turnover. In all cases, chiral HPLC analysis of the reaction mixture revealed that the S-sulfoxide was reduced more rapidly leading to enrichment or isolation of the R isomer.
    No preview · Article · Nov 2014 · JBIC Journal of Biological Inorganic Chemistry
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    Ulrike Kappler · John H Enemark
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    ABSTRACT: Sulfite-oxidizing enzymes (SOEs) are molybdenum enzymes that exist in almost all forms of life where they carry out important functions in protecting cells and organisms against sulfite-induced damage. Due to their nearly ubiquitous presence in living cells, these enzymes can be assumed to be evolutionarily ancient, and this is reflected in the fact that the basic domain architecture and fold structure of all sulfite-oxidizing enzymes studied so far are similar. The Mo centers of all SOEs have five-coordinate square pyramidal coordination geometry, which incorporates a pyranopterin dithiolene cofactor. However, significant differences exist in the quaternary structure of the enzymes, as well as in the kinetic properties and the nature of the electron acceptors used. In addition, some SOEs also contain an integral heme group that participates in the overall catalytic cycle. Catalytic turnover involves the paramagnetic Mo(V) oxidation state, and EPR spectroscopy, especially high-resolution pulsed EPR spectroscopy, provides detailed information about the molecular and electronic structure of the Mo center and the Mo-based sulfite oxidation reaction.
    Preview · Article · Sep 2014 · JBIC Journal of Biological Inorganic Chemistry
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    ABSTRACT: The Ralstonia solanacearum species complex has long been recognized as a group of phenotypically diverse strains that can be subdivided into four phylotypes. Using a polyphasic taxonomic approach on an extensive set of strains, this study provides evidence for a taxonomic and nomenclatural revision of members of this complex. Data obtained from phylogenetic analysis of 16S rRNA gene sequences, 16S-23S rRNA intergenic spacer (ITS) region sequences, partial endoglucanase (egl) gene sequences and DNA-DNA hybridizations demonstrate that the Ralstonia solanacearum species complex is comprised of three genospecies. One of these includes the type strain of R. solanacearum and consists of R. solanacearum phylotype II strains only. The second genospecies includes the type strain of R. syzygii and contains only phylotype IV strains. This genospecies is subdivided into three distinct groups, namely R. syzygii, the causal agent of Sumatra disease on clove trees in Indonesia, R. solanacearum phylotype IV strains isolated from different host plants mostly from Indonesia, and strains of the blood disease bacterium (BDB), the causal agent of the Banana Blood Disease, a bacterial wilt disease in Indonesia affecting bananas and plantains. The last genospecies is composed of R. solanacearum strains belonging to phylotypes I and III. As these genospecies are also supported by phenotypic data that allow the differentiation of the three genospecies, the following taxonomic proposals are made: emendation of the descriptions of R. solanacearum and R. syzygii, descriptions of Ralstonia syzygii subsp. syzygii (R 001T = LMG 10661T = DSM 7385T) for the current R. syzygii strains, Ralstonia syzygii subsp. indonesiensis subsp. nov. (UQRS 464T = LMG 27703T = DSM 27478T) for the current R. solanacearum phylotype IV strains, Ralstonia syzygii subsp. celebesensis subsp. nov. (UQRS 627T = LMG 27706T = DSM 27477T) for the BDB strains and Ralstonia pseudosolanacearum sp. nov. (UQRS 461T = LMG 9673T = NCPPB 1029T) for the R. solanacearum phylotype I and III strains.
    Full-text · Article · Jun 2014 · International Journal of Systematic and Evolutionary Microbiology
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    ABSTRACT: Sulfite dehydrogenase from the soil bacterium Sinorhizobium meliloti (SorT) is a periplasmic, homodimeric molybdoenzyme with a molecular mass of 78 kDa. It differs from most other well studied sulfite oxidizing enzymes as it bears no heme cofactor. SorT does not readily reduce ferrous horse heart cytochrome c which is the preferred electron acceptor for vertebrate sulfite oxidases. In the present study, ferrocene methanol (FM) (in its oxidized ferrocenium form) was utilised as an artificial electron acceptor for the catalytic SorT sulfite oxidation reaction. Cyclic voltammetry of FM was used to generate the active form of the mediator at the electrode surface. The FM-mediated catalytic sulfite oxidation by SorT was investigated by two different voltammetric methods namely (i) SorT freely diffusing in solution and (ii) SorT confined to a thin layer at the electrode surface by a semi-permeable dialysis membrane. A single set of rate and equilibrium constants was used to simulate the catalytic voltammograms performed under different sweep rates and with various concentrations of sulfite and FM which provides new insights into the kinetics of SorT catalytic mechanism. Further, we were able to model the role of the dialysis membrane in the kinetics of the overall catalytic system.
    No preview · Article · Jun 2014 · The Journal of Physical Chemistry B
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    ABSTRACT: Haemophilus influenzae is a host adapted human pathogen known to contribute to a variety of acute and chronic diseases of the upper and lower respiratory tract as well as the middle ear. At the sites of infection as well as during growth as a commensal the environmental conditions encountered by H. influenzae will vary significantly, especially in terms of oxygen availability, however, the mechanisms by which the bacteria can adapt their metabolism to cope with such changes have not been studied in detail. Using targeted metabolomics the spectrum of metabolites produced during growth of H. influenzae on glucose in RPMI-based medium was found to change from acetate as the main product during aerobic growth to formate as the major product during anaerobic growth. This is likely caused by a switch in the major pyruvate degrading route. Neither lactate nor succinate or fumarate were major products of H. influenzae growth under any condition studied Gene expression studies and enzyme activity data revealed that despite an identical genetic makeup and very similar metabolite production profiles, H. influenzae strain Rd appeared to favour glucose degradation via the PPP, while strain 2019, a clinical isolate, showed higher expression of enzymes involved in glycolysis. Components of the respiratory chain were most highly expressed during microaerophilic and anaerobic growth in both strains, but again clear differences existed in the expression of genes associated e.g. with NADH oxidation, nitrate and nitrite reduction in the two strains studied.Together our results indicate that H. influenzae uses a specialized type of metabolism that could be termed ‘respiration assisted fermentation’ where the respiratory chain likely serves to alleviate redox imbalances caused by incomplete glucose oxidation, and at the same time provides a means of converting a variety of compounds including nitrite and nitrate that arise as part of the host defence mechanisms.
    Preview · Article · Mar 2014 · Frontiers in Microbiology
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    Ulrike Kappler · Amanda S Nouwens
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    ABSTRACT: The highly diverse and metabolically versatile microbial communities found in soil environments are major contributors to the global carbon, nitrogen, and sulfur cycles. We have used a combination of genome -based pathway analysis with proteomics and gene expression studies to investigate metabolic adaptation in a representative of these bacteria, Starkeya novella, which was originally isolated from agricultural soil. This bacterium was the first facultative sulfur chemolithoautotroph that was isolated and it is also able to grow with methanol and on over 39 substrates as a heterotroph. However, using glucose, fructose, methanol, thiosulfate as well as combinations of the carbon compounds with thiosulfate as growth substrates we have demonstrated here that contrary to the previous classification, S. novella is not a facultative sulfur chemolitho- and methylotroph, as the enzyme systems required for these two growth modes are always expressed at high levels. This is typical for key metabolic pathways. In addition enzymes for various pathways of carbon dioxide fixation were always expressed at high levels, even during heterotrophic growth on glucose or fructose, which suggests a role for these pathways beyond the generation of reduced carbon units for cell growth, possibly in redox balancing of metabolism. Our results then indicate that S. novella, a representative of the Xanthobacteraceae family of methylotrophic soil and freshwater dwelling bacteria, employs a mixotrophic growth strategy under all conditions tested here. As a result the contribution of this bacterium to either carbon sequestration or the release of climate active substances could vary very quickly, which has direct implications for the modeling of such processes if mixotrophy proves to be the main growth strategy for large populations of soil bacteria.
    Full-text · Article · Oct 2013 · Frontiers in Microbiology
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    ABSTRACT: The arsenite oxidase (Aio) from the facultative autotrophic Alphaproteobacterium Rhizobium sp. NT-26 is a bioenergetic enzyme involved in the oxidation of arsenite to arsenate. The enzyme from the distantly related heterotroph, Alcaligenes faecalis, which is thought to oxidise arsenite for detoxification, consists of a large α subunit (AioA) with bis-molybdopterin guanine dinucleotide at its active site and a 3Fe-4S cluster, and a small β subunit (AioB) which contains a Rieske 2Fe-2S cluster. The successful heterologous expression of the NT-26 Aio in Escherichia coli has resulted in the solution of its crystal structure. The NT-26 Aio, a heterotetramer, shares high overall similarity to the heterodimeric arsenite oxidase from A. faecalis but there are striking differences in the structure surrounding the Rieske 2Fe-2S cluster which we demonstrate explains the difference in the observed redox potentials (+225 mV vs. +130/160 mV, respectively). A combination of site-directed mutagenesis and electron paramagnetic resonance was used to explore the differences observed in the structure and redox properties of the Rieske cluster. In the NT-26 AioB the substitution of a serine (S126 in NT-26) for a threonine as in the A. faecalis AioB explains a -20 mV decrease in redox potential. The disulphide bridge in the A. faecalis AioB which is conserved in other betaproteobacterial AioB subunits and the Rieske subunit of the cytochrome bc 1 complex is absent in the NT-26 AioB subunit. The introduction of a disulphide bridge had no effect on Aio activity or protein stability but resulted in a decrease in the redox potential of the cluster. These results are in conflict with previous data on the betaproteobacterial AioB subunit and the Rieske of the bc 1 complex where removal of the disulphide bridge had no effect on the redox potential of the former but a decrease in cluster stability was observed in the latter.
    Full-text · Article · Aug 2013 · PLoS ONE
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    ABSTRACT: We describe the catalytic voltammetry of the periplasmic arsenite oxidase (Aio) from the chemolithoautotrophic bacterium Rhizobium sp. str. NT-26 that oxidizes arsenite to arsenate. Electrochemistry of the enzyme was accomplished using its native electron transfer partner, cytochrome c552 (cyt c552), as a mediator. The protein cyt c552 adsorbed on a mercaptoundecanoic acid (MUA) modified Au electrode exhibited a stable, reversible one-electron voltammetric response at +275mV vs NHE (pH7). In the presence of arsenite and Aio the voltammetry of cyt c552 is transformed from a transient response to an amplified sigmoidal (steady state) wave consistent with an electro-catalytic system. Digital simulation was performed using single set of enzyme dependent parameters for all catalytic voltammetry obtained at different sweep rates and various substrate concentrations. The obtained kinetic constants from digital simulation provide new insight into the kinetics of the NT-26 Aio catalytic mechanism.
    Full-text · Article · Jul 2013 · Biochimica et Biophysica Acta
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    ABSTRACT: NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.
    Full-text · Dataset · Apr 2013
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    ABSTRACT: NtrYX is a sensor-histidine kinase/response regulator two-component system that has had limited characterization in a small number of Alphaproteobacteria. Phylogenetic analysis of the response regulator NtrX showed that this two-component system is extensively distributed across the bacterial domain, and it is present in a variety of Betaproteobacteria, including the human pathogen Neisseria gonorrhoeae. Microarray analysis revealed that the expression of several components of the respiratory chain was reduced in an N. gonorrhoeae ntrX mutant compared to that in the isogenic wild-type (WT) strain 1291. These included the cytochrome c oxidase subunit (ccoP), nitrite reductase (aniA), and nitric oxide reductase (norB). Enzyme activity assays showed decreased cytochrome oxidase and nitrite reductase activities in the ntrX mutant, consistent with microarray data. N. gonorrhoeae ntrX mutants had reduced capacity to survive inside primary cervical cells compared to the wild type, and although they retained the ability to form a biofilm, they exhibited reduced survival within the biofilm compared to wild-type cells, as indicated by LIVE/DEAD staining. Analyses of an ntrX mutant in a representative alphaproteobacterium, Rhodobacter capsulatus, showed that cytochrome oxidase activity was also reduced compared to that in the wild-type strain SB1003. Taken together, these data provide evidence that the NtrYX two-component system may be a key regulator in the expression of respiratory enzymes and, in particular, cytochrome c oxidase, across a wide range of proteobacteria, including a variety of bacterial pathogens.
    No preview · Article · Apr 2013 · Journal of bacteriology
  • Ulrike Kappler · Amanda S Nouwens
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    ABSTRACT: Molybdenum enzymes are known to underpin key reactions in the biological carbon, nitrogen and sulfur cycles, however, the diversity of these enzymes and the reactions they catalyze especially in bacteria is much greater than currently known. We have analysed the molybdoproteome of the soil bacterium Starkeya novella as a function of growth mode and identified a complete pathway for Mo-PPT synthesis, a Mo transporter and 18 gene loci encoding mononuclear Mo-enzymes of the Xanthine Oxidase, Sulfite Oxidase and DMSO Reductase enzyme families. This relatively high number of Mo enzymes may be a specific property of the taxonomic group (Xanthobacteraceae) to which S. novella belongs. About 70% of S. novella Mo enzymes have no characterized close relatives, and two thirds of them are expressed under the conditions analysed, which included heterotrophy, methylotrophy, chemolithotrophy and mixotrophy. Many enzymes were clearly regulated in response to either the type of carbon source present in the growth medium or the presence of thiosulfate, and two, in particular, including an uncharacterized enzyme of the XO family (Snov_3370) were highly abundant under all growth conditions tested. We also uncovered novel enzymes with links to growth in the presence of thiosulfate, such as a PaoABC-type aldehyde oxidoreductase and an uncharacterized group 1 sulfite oxidase family enzyme, although the function of these enzymes during sulfur oxidation is unclear at present. Clearly, further work is needed to uncover the significance of these enzymes for cell metabolism.
    No preview · Article · Jan 2013 · Metallomics
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    Dataset: Table S4
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    ABSTRACT: Crystallographic data collection and refinement statistics. (DOC)
    Preview · Dataset · Oct 2012
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    Dataset: Figure S6
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    ABSTRACT: Growth of all IMPDH mutants on MPA. Serial dilution spotting assays of all IMPDH mutants on YNB plus 5 µg/mL MPA. The juxtaposed images depict which portions of the IMPDH allele are present, with yellow representing CnIMD1 and blue representing CgIMD1, split into three thirds with the vertical bars depicting the six unique residues. Two proteins are depicted for the H99 background where the wild-type allele is also present. (A) Transformation of IMPDH variants (point mutants, double mutants, sextuple mutants, chimeras plus the two wild-type alleles) into the imd1Δ deletion background. (B) Transformation of IMPDH variants (point mutants, double mutants, sextuple mutants, chimeras plus the two wild-type alleles) into the wild-type H99 background, which possesses one copy of the MPA-sensitive allele. (TIF)
    Preview · Dataset · Oct 2012
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    Dataset: Figure S2
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    ABSTRACT: Quantification of melanin production. Melanin production was measured in liquid l-DOPA medium from culture supernatant at OD475. (A) imd1Δ at 30°C. (B) imd1Δ at 37°C. (C) hpt1Δ at 30°C. (D) hpt1Δ at 37°C. Bars represent mean OD475 from three replicates with standard error shown. No significant differences were found between strains. (TIF)
    Preview · Dataset · Oct 2012
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    Dataset: Table S1
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    ABSTRACT: Primers used in this study. (DOC)
    Preview · Dataset · Oct 2012
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    Dataset: Figure S8
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    ABSTRACT: Inhibitor kinetics of Cryptococcus IMPDH. Inhibition by MPA was investigated at fixed concentration of IMP (850 µM for CnImd1 and 1,800 µM for CgImd1) and varying NAD+ (62.5 µM–2,000 µM) and indicated MPA concentrations. Initial velocity data was best described by the uncompetitive tight-binding inhibition equation versus NAD+, while initial velocity data of MPA versus IMP were best fit by a noncompetitive/mixed tight-binding model. (A) CnImd1. (B) CgImd1. (TIF)
    Preview · Dataset · Oct 2012
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    Dataset: Table S2
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    ABSTRACT: Wild-type strains used in this study. (DOC)
    Preview · Dataset · Oct 2012

Publication Stats

1k Citations
234.15 Total Impact Points

Institutions

  • 2001-2015
    • University of Queensland
      • • School of Chemistry and Molecular Biosciences
      • • School of Information Technology and Electrical Engineering
      Brisbane, Queensland, Australia
  • 1998-2001
    • University of Bonn
      • Institut für Mikrobiologie und Biotechnologie
      Bonn, North Rhine-Westphalia, Germany