Source: UNIVERSITY OF MISSOURI submitted to NRP
GENETIC APPROACHES TO MAPPING ENERGY CONVERSION PATHWAYS IN SULFATEREDUCING ANAEROBES OF THE GENUS DESULFOVIBRIO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1006863
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Jul 1, 2015
Project End Date
Feb 14, 2020
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Biochemistry
Non Technical Summary
Microbes use the cellular membrane as an insulated wire where electrons move from power sourcesto outlets that provide power to do work. At times, the electron flow is short-circuited by compoundsthat can cause oxidative damage. The majority of cells maintain a cache of electrons in a pool of thesmall molecule named glutathione that is readily mobilized to repair the damage. The strictlyanaerobicbacteria that use sulfate as the normal "outlet" do not have glutathione as a capacitor forholding electrons but they do have small proteins called thioredoxins that might serve that role.Currently little is known about the source and sinks of the electrons in the thioredoxin cache andthese proteins do not appear in the wiring diagram of the bacteria. It is our goal to determine wherethese proteins fit in the pattern of electron flow. We will use genetic and biochemical approaches toexplore the role(s) of the thioredoxins and report our findings in appropriate scholarly journals.The anaerobic bacteria that are the focus of this project have also been shown to deliver electrons to anumber of toxic metals and, thereby, alter the properties of the metals to be less toxic in severalcases. This capacity has been suggested to be of benefit to efforts of remediation of sitescontaminated with toxic metals. As an extension to our exploration of the wiring diagram of themodel anaerobic bacterium, we will determine whether the thioredoxins are involved in the deliveryof electrons to two metals of concern, mercury and uranium.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1334099100035%
1337010104040%
1334099110012%
1337010110013%
Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
4099 - Microorganisms, general/other; 7010 - Biological Cell Systems;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology; 1000 - Biochemistry and biophysics;
Goals / Objectives
A) Energy conversion in DvH:Thioredoxins are small redox proteins, transferring electrons through dithiol/disulfide exchanges.These proteins interact with Flavin-dependent reductases that maintain these proteins in a reductionstate ready to respond to oxidative stresses (Sarin and Sharma 2006). In a number of microbialgenera in which the oxidative stress protection system of glutathiones is absent, thioredoxins areconsidered to fill this role and to be essential for DNA replication and repair (Reott et al., 2009, Luet al., 2013).The roles of thioredoxins have not been clearly elucidated for the anaerobic bacteria of the genusDesulfovibrio. No glutathione system is present to maintain redox balance; therefore, thethioredoxins of Desulfovibrio vulgaris Hildenborough (encoded in DVU0378 and DVU1839) havebeen suggested to be critical for protection against oxidative damage (Pieulle et al., 2011).Interestingly, in sulfate-reducing growth conditions were sulfide is abundant, it is not clear thatthioredoxins would be solely responsible for protection to oxidative stress.Because members of the Desulfovibrio genus are present in the Oak Ridge FRC that is impacted byboth uranium and nitrate contamination (Ramsay et al., 2015), a potential role of the thioredoxins inprotection against both of these contaminants would be relevant to remediation capacities.Surprisingly, recent research has implemented thioredoxins as necessary for U(VI) reduction to theinsoluble U(IV) by Desulfovibrio alaskensis G20 (Li and Krumholz 2009, Li et al., 2014). TheWall laboratory has genetic tools that can be applied for examining the roles of the thioredoxins inthe model strain, D. vulgaris Hildenborough (DvH). Therefore, we will explore the function ofthese thioredoxins by the construction of deletion mutations in the genes for thioredoxins and theirreductases. The genome of DvH is annotated to encode two putative thioredoxins and at least threethioredoxin reductases (Pieulle et al., 2011). While the anaerobic lifestyle of this model bacteriumhas been the focus of energy budget research within the last decade, thioredoxins do not appear inany general models for energy conversion.We propose the following aims for thioredoxins: a) To determine whether thioredoxins and their reductases are essential for DvH,mutants lacking each of the thioredoxin encodinggenes will be generated, if each isdispensable, along with a mutant lacking both thioredoxin genes and a mutant lackinggenes for all three reductases. b) To explore conditional lethal phenotypes of mutants constructed in a), growthcharacteristics will be elucidated under sulfate-, thiosulfate- and non-sulfate-reducing(fermentative) conditions. c) To explore the roles of thioredoxins (if deletions cannot be obtained), resistance tothe bacterial thioredoxin reductase inhibitor ebselen will be determined and thegrowth conditions under which this inhibitor may be effective will be revealed. d) To elucidate changes in redox balance in the mutants altered in thioredoxin levels oractivity, intracellular levels of reduced thioredoxins and pyridine nucleotides will bedetermined in each mutant and/or after the addition of the inhibitor ebselen. e) To determine the substrates interacting with the thioredoxins, the DVU1839thioredoxin (more dramatically increased in expressionunder stress conditions thanDVU0378) will be isolated and binding affinities for substrates and interactingproteins will be determined.B.) Interactions of DvH thioredoxins with toxic metals, mercury and uranium:SRM have been shown capable of reducing both the toxic metals mercury and uranium (Lovleyand Phillips 1992, Lovley et al., 1993). The two electron reductions of Hg(II) to Hg0 (Kritee etal., 2008) and U(VI) to U(IV) (Gorby and Lovley 1992) are considered potential bioremediationapproaches since Hg0 is volatile and can be diluted in the atmosphere; whereas, the U(IV) existsas an insoluble mineral uraninite with reduced bioavailability. Knowing the electron donors forthe reductions might allow redirection of the electron flow to favor metal reductions. Potentialelectron donors are hydrogenases, thioredoxins and cytochromes. Evidence that proteins in eachof these categories can interact with metals has been reported in the past (Payne et al., 2002,Vignais and Billoud 2007, Li and Krumholz 2009). a) Test the possibility that sub-inhibitory concentrations Hg(II) or U(VI) might serve asterminal electron acceptors for SRM. b) Determine whether thioredoxins are components of the electron transfer pathway usedto deliver electrons to Hg(II) or U(VI). c) Establish whether reduced thioredoxin (DVU1839) can directly deliver electrons toHg(II) or U(VI).
Project Methods
A) Energy conversion in DvH:Bioinformatic analyses have revealed that members of five phyla are able to couple sulfaterespirationwith ATP formation, Firmicutes, Nitrospirae, Deltaproteobacteria, Euryarchaeotaand Crenarchaeota (Barton and Fauque 2009). This vast heterogeneity underscores the benefitsof this respiratory mode and demands caution for extrapolation of physiological characteristics ofone sulfate-reducing microbe to another. The model SRMs, Desulfovibrio strains that aremembers of the Deltaproteobacteria, were selected for experimentation because of the relativeease of their cultivation and rapid rate of growth. The Wall lab has been involved in thecharacterization of gene regulation and genomics of Desulfovibrio strains, with a particular focuson D. vulgaris Hildenborough (DvH) and much of the information is publically accessible athttp://www.microbesonline.org/ . Recent efforts have elucidated different aspects oftranscriptional changes in response to stresses (He et al., 2006, Mukhopadhyay et al., 2006,Mukhopadhyay et al., 2007, Zhou et al., 2011); better identified and annotated key genes in thesulfate reduction pathway (Li et al., 2009, Zane et al., 2010, Keller and Wall 2011, Keller et al.,2014); and shed light on thecontroversy surrounding the "Hydrogen Cycling Hypothesis"(Odom and Peck Jr. 1981, Odom and Peck 1984, Keller et al., 2014), a mechanism to augmentthe energy derived from oxidation of organic acids with sulfate reduction.Genetic tools have been developed in the Wall lab for manipulation of DvH genes that includemarker exchange deletions (Chhabra et al., 2011, Keller et al., 2011), marker exchange-evictiontechniques that allow the construction of in-frame unmarked mutations (Keller et al., 2009),fluorescently tagged proteins (Gorur et al., 2010) and site directed mutations (Smith et al., 2015).Table 1 illustrates the capacity of the tools to generate unmarked mutations of the genesencoding the hydrogenase isozymes in DvH. Surprisingly all isozymes were dispensable whenDvH was grown by sulfate respiration with lactate.B. Hg (II) and U(VI) interactions with DvHA major driver for study of the electron flow pathways in the SRM has been their rich metalmetabolism for their potential application to toxic metal bioremediation. Not only are the SRMbelieved to be the primary culprits in anaerobic corrosion of ferrous metals through oxidativeprocesses (Hamilton 2003) (Enning and Garrelfs 2014), but the low redox potential of theirmetabolism allows various electron carriers to deliver electrons to a number of heavy metals andmetalloids [summarized in (Barton and Fauque 2009)]. In the case of uranium present in theenvironment as U(VI), addition of two electrons generating U(IV) produces the uraninite mineralcausing precipitation from groundwater (Gorby and Lovley 1992). This observation has stimulatedmuch research into the application of SRMs for bioremediation of uranium contaminatedenvironments (Williams et al., 2013).

Progress 07/01/15 to 02/14/20

Outputs
Target Audience:Audience was collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. Changes/Problems:Changes in approach have occurred because I officially retired July 1, 2018 and officially closed my laboratory in September of 2019. Without equipment, funds or personnel, it is difficult to make progress. What opportunities for training and professional development has the project provided?Postdoctoral fellow, Erica Majumder, learned genetic approaches to research questions and an undergraduate, Sharien Fitriasari, helped make the mutants. Sharien became a graduate student at the Stowers Institute for Medical Research. Erica is now an Assistant Professor at UW at Madison. How have the results been disseminated to communities of interest?Audience was collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? i) Genes encoding two thioredoxins were identified in D. vulgaris by gene homology to bona fide thioredoxins, trx1 and trx3 at gene locus numbers DVU1839 and DVU0378, respectively. ii) Genes putatively encoding four thioredoxin reductases were identified: trxR1 (locus no. DVU1838) trxR3 (DVU0377) trxRi (DVU1457) ahpF (DVU0283) iii) Transposon insertions into the genes encoding each were obtained as individual mutants from the library of whole genome random transposon available in the Wall lab from previous research. Their study showed that single mutations of trx3 and trxR1 were impaired in growth but not were not lethal. Interruptions in trx1 were not recovered suggesting that those mutations were not viable and that Thioredoxin1 was essential for the cells under the standard growth conditions. Other transposon mutants in possible thioredoxin reductase genes were without effect on growth kinetics. iv) In-frame deletions of the reductases and Trx3 were constructed to avoid complications of polarity in operons. The process of construction employed marker exchange mutagenesis. Mutagenesis of the trx1 gene was again not successful which confirmed the earlier deduction that Trx1 protein is likely essential for growth in our standard protocols. Single gene mutants of all other thioredoxin associated genes were obtained without difficulty. v) Double and triple mutants of thioredoxins and reductases, respectively, were not constructed. That leaves open the possibility that any one of the reductases might be sufficient for maintaining the redox status. Double thioredoxins could not be made since Thioredoxin 1 appears to be essential and not complemented by Thioredoxin 3. No differences in the ability of the constructed mutants of D. vulgaris to grow with sulfate, thiosulfate or sulfite as terminal electron acceptor were observed when compared with the non-mutated parent strain. Experiments with the reductase inhibitor ebselen were not performed because of the lack of availability of the inhibitor from commercial sources. Not accomplished. To determine the substrates interacting with Thioredoxin 1, one of the two cysteines forming the site at which the electrons are delivered to oxidized substrates was mutated to an alanine in trx1. That mutated version of the trx1 gene was modified by the addition of an affinity tag and the modified, mutated version of trx1 was transferred into a non-mutated D. vulgaris. Other researchers have shown that removal of one of the two thiol functional groups (cysteines) short circuits the reduction of substrates producing a disulfide bond between the remaining cysteine and the substrate. Purification of the mutated Thioredoxin 1 by the affinity tag should also produce a sampling of the substrates covalently attached. This was achieved and a list of potential substrates (and "sticky" proteins) has been obtained. B) Interactions of DvH thioredoxins with toxic metals, mercury and uranium. a) Hg(II) or U(VI) as terminal electron acceptors was not examined. b) We did assay the U(VI) reduction capacity of the DvH mutants lacking individual thioredoxin reductases and that lacking Trx3.No differences were observed in the ability of any mutant of the thioredoxin-associated genes to form U(IV) when compared with the reduction ability of the parental strain in which these genes were functional. c) No in vitro experiments were performed to test the direct delivery of electrons.

Publications


    Progress 10/01/19 to 02/14/20

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? i) Genes encoding two thioredoxins were identified in D. vulgaris by gene homology to bona fide thioredoxins, trx1 and trx3 at gene locus numbers DVU1839 and DVU0378, respectively. ii) Genes putatively encoding four thioredoxin reductases were identified: trxR1 (locus no. DVU1838) trxR3 (DVU0377) trxRi (DVU1457) ahpF (DVU0283) iii) Transposon insertions into the genes encoding each were obtained as individual mutants from the library of whole genome random transposon available in the Wall lab from previous research. Their study showed that single mutations of trx3 and trxR1 were impaired in growth but not were not lethal. Interruptions in trx1 were not recovered suggesting that those mutations were not viable and that Thioredoxin1 was essential for the cells under the standard growth conditions. Other transposon mutants in possible thioredoxin reductase genes were without effect on growth kinetics. iv) In-frame deletions of the reductases and Trx3 were constructed to avoid complications of polarity in operons. The process of construction employed marker exchange mutagenesis. Mutagenesis of the trx1 gene was again not successful which confirmed the earlier deduction that Trx1 protein is likely essential for growth in our standard protocols. Single gene mutants of all other thioredoxin associated genes were obtained without difficulty. v) Double and triple mutants of thioredoxins and reductases, respectively, were not constructed. That leaves open the possibility that any one of the reductases might be sufficient for maintaining the redox status. Double thioredoxins could not be made since Thioredoxin 1 appears to be essential and not complemented by Thioredoxin 3. No differences in the ability of the constructed mutants of D. vulgaris to grow with sulfate, thiosulfate or sulfite as terminal electron acceptor were observed when compared with the non-mutated parent strain. Experiments with the reductase inhibitor ebselen were not performed because of the lack of availability of the inhibitor from commercial sources. Not accomplished. To determine the substrates interacting with Thioredoxin 1, one of the two cysteines forming the site at which the electrons are delivered to oxidized substrates was mutated to an alanine in trx1. That mutated version of the trx1 gene was modified by the addition of an affinity tag and the modified, mutated version of trx1 was transferred into a non-mutated D. vulgaris. Other researchers have shown that removal of one of the two thiol functional groups (cysteines) short circuits the reduction of substrates producing a disulfide bond between the remaining cysteine and the substrate. Purification of the mutated Thioredoxin 1 by the affinity tag should also produce a sampling of the substrates covalently attached. This was achieved and a list of potential substrates (and "sticky" proteins) has been obtained. B) Interactions of DvH thioredoxins with toxic metals, mercury and uranium. a) Hg(II) or U(VI) as terminal electron acceptors was not examined. b) We did assay the U(VI) reduction capacity of the DvH mutants lacking individual thioredoxin reductases and that lacking Trx3.No differences were observed in the ability of any mutant of the thioredoxin-associated genes to form U(IV) when compared with the reduction ability of the parental strain in which these genes were functional. c) No in vitro experiments were performed to test the direct delivery of electrons.

    Publications


      Progress 10/01/18 to 09/30/19

      Outputs
      Target Audience:Audiences were collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Continued training of Dr. Kara De Leon, postdoctoral fellow, learning how to close a lab and how to start her own in Oklahoma! Advising Leslie Day on applications to graduate school and her choices. How have the results been disseminated to communities of interest?Audience was collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. What do you plan to do during the next reporting period to accomplish the goals?Complete publications in progress.

      Impacts
      What was accomplished under these goals? B) Interactions of DvH thioredoxins with toxic metals, mercury and uranium. a) Hg(II) or U(VI) as terminal electron acceptors was not examined. b) We did assay the U(VI) reduction capacity of the DvH mutants lacking individual thioredoxin reductases and that lacking Trx3.No differences were observed in the ability of any mutant of the thioredoxin-associated genes to form U(IV) when compared with the reduction ability of the parental strain in which these genes were functional. Thus these proteins are not uniquely needed for transfer of electrons to this metal. c) No in vitro experiments were performed to test the direct delivery of electrons.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2018 Citation: Ge X, Vaccaro BJ, Thorgersen MP, Poole FL 2nd, Majumder EL, Zane GM, De Le�n KB, Lancaster WA, Moon JW, Paradis CJ, von Netzer F, Stahl DA, Adams PD, Arkin AP, Wall JD, Hazen TC, Adams MWW. (2018) Iron- and aluminum-induced depletion of molybdenum in acidic environments impedes the nitrogen cycle. Environ Microbiol. 2018 Oct 5. doi: 10.1111/1462-2920.14435
      • Type: Journal Articles Status: Published Year Published: 2018 Citation: Bertran E, Leavitt WD, Pellerin A, Zane GM, Wall JD, Halevy I, Wing BA, Johnston DT. (2018) Deconstructing the dissimilatory sulfate reduction pathway: isotope fractionation of a mutant unable of growth on sulfate. Front Microbiol. 9:3110. doi: 10.3389/fmicb.2018.03110.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Rajeev L, Garber ME, Zane GM, Price MN, Dubchak I, Wall JD, Novichkov PS, Mukhopadhyay A, Kazakov AE. (2019) A new family of transcriptional regulators of tungstoenzymes and molybdate/tungstate transport. Environ Microbiol. 2019 Feb;21(2):784-799. doi: 10.1111/1462-2920.14500.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: 1. Rajeev L, Luning EG, Zane GM, Juba TR, Kazakov AE, Novichkov PS, Wall JD, Mukhopadhyay A. (2019) LurR is a regulator of the central lactate oxidation pathway in sulfate-reducing Desulfovibrio species. PLoS One. 14(4):e0214960. doi: 10.1371/journal.pone.0214960.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: Date SS, Parks JM, Rush KW, Wall JD, Ragsdale SW, Johs A. (2019) Kinetics of enzymatic mercury methylation at nanomolar concentrations catalyzed by HgcAB. Appl Environ Microbiol. 2019 Apr 26. pii: AEM.00438-19. doi: 10.1128/AEM.00438-19.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: 3. Christensen GA, Gionfriddo CM, King AJ, Moberly JG, Miller CL, Somenahally AC, Callister SJ, Brewer H, Podar M, Brown SD, Palumbo AV, Brandt CC, Wymore AM, Brooks SC, Hwang C, Fields MW, Wall JD, Gilmour CC, Elias DA. (2019) Determining the reliability of measuring mercury cycling gene abundance with correlations with mercury and methylmercury concentrations. Environ Sci Technol. 53(15):8649-8663. doi:10.1021/acs.est.8b06389.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: 4. Day LA, De Le�n KB, Kempher ML, Zhou J, Wall JD. (2019) Complete genome sequence of Desulfovibrio desulfuricans IC1, a sulfonate-respiring anaerobe. Microbiol Resour Announc. 8(31):e00456-19. doi: 10.1128/MRA.00456-19.
      • Type: Journal Articles Status: Published Year Published: 2019 Citation: 5. Mehta-Kolte MG, Stoeva MK, Mehra A, Redford SA, Youngblut MD, Zane G, Gr�goire P, Carlson HK, Wall J, Coates JD. (2019) Adaptation of Desulfovibrio alaskensis G20 to perchlorate, a specific inhibitor of sulfate reduction. Environ Microbiol. 21(4):1395-1406. doi: 10.1111/1462-2920.14570.
      • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Kara B. De Le�n, Thomas R. Juba, J.E. Graham. Grant M. Zane, D. Ning, Aifen Zhou, C.S. Raman, Judy D. Wall. 2019. Tandem gene duplications are frequent in microbial genomes but go undetected in genome assemblies. Annual meeting of the American Society for Microbiology, Microbe 2019, San Francisco, MBP-1 Poster.


      Progress 10/01/17 to 09/30/18

      Outputs
      Target Audience:Audience was collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. Changes/Problems:Changes in approach occurred because I officially retired July 1, 2018. What opportunities for training and professional development has the project provided?Postdoctoral fellow, Erica Majumder, learned genetic approaches to research questions and an undergraduate, Sharien Fitriasari, helped make the mutants. Sharien became a graduate student at the Stowers Institute for Medical Research. Erica is now an Assistant Professor at UW at Madison. How have the results been disseminated to communities of interest?Audiences were collaborators, researchers, students and interested laypersons. Scholarly journals (Appl. Environ. Microbiol., mBio, etc.) and professional meetings (annual meeting of the American Society for Microbiology, annual retreat for ENIGMA, annual meeting of principal investigators of DOE BER, etc.) provided the platforms for information delivery. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

      Impacts
      What was accomplished under these goals? Under objective A: i) Genes encoding two thioredoxins, trx1 and trx3, were identified in D. vulgaris by gene homology to bona fide thioredoxins, at gene locus numbers DVU1839 and DVU0378, respectively. ii) Genes putatively encoding four thioredoxin reductases were identified: trxR1 (locus no. DVU1838) trxR3 (DVU0377) trxRi (DVU1457) ahpF (DVU0283) iii) Transposon insertions into the genes encoding each were obtained as individual mutants from the library of whole genome random transposon available in the Wall lab from previous research. Their study showed that single mutations of trx3 and trxR1 were impaired in growth but not were not lethal. Interruptions in trx1 were not recovered suggesting that those mutations were not viable and that Thioredoxin1 was essential for the cells under the standard growth conditions. Other transposon mutants in possible thioredoxin reductase genes were without effect on growth kinetics. iv) In-frame deletions of the genes encoding reductases and Trx3 were constructed to avoid complications of polarity in operons. The process of construction employed marker exchange mutagenesis. Mutagenesis of the trx1 gene was again not successful which confirmed the earlier deduction that Trx1 protein is likely essential for growth in our standard protocols. Single gene mutants of all other thioredoxin associated genes were obtained without difficulty. v) Double and triple mutants of thioredoxins and reductases, respectively, were not constructed. That leaves open the possibility that any one of the reductases might be sufficient for maintaining the redox status. Double thioredoxins could not be made since Thioredoxin 1 appears to be essential and not complemented by Thioredoxin 3. No differences in the ability of the constructed mutants of D. vulgaris to grow with sulfate, thiosulfate or sulfite as terminal electron acceptor were observed when compared with the non-mutated parent strain. Experiments with the reductase inhibitor ebselen were not performed because of the lack of availability of the inhibitor from commercial sources. Not accomplished. To determine the substrates interacting with Thioredoxin 1, one of the two cysteines forming the site at which the electrons are delivered to oxidized substrates was mutated to an alanine in trx1. That mutated version of the trx1 gene was modified by the addition of an affinity tag and the modified, mutated version of trx1 was transferred into a non-mutated D. vulgaris. Other researchers have shown that removal of one of the two thiol functional groups (cysteines) short circuits the reduction of substrates producing a disulfide bond between the remaining cysteine and the substrate. Purification of the mutated Thioredoxin 1 by the affinity tag should also produce a sampling of the substrates covalently attached. This was achieved and a list of potential substrates (and "sticky" proteins) has been obtained. Under objective B: Not tested. Not determined. Not established.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2017 Citation: Huan T, Forsberg EM, Rinehart D, Johnson CH, Ivanisevic J, Benton HP, Fang M, Aisporna A, Hilmers B, Poole FL, Thorgersen MP, Adams MWW, Krantz G, Fields MW, Robins PD, Niedernhofer LJ, Ideker T, Majumder EL, Wall JD, Rattray NJW, Goodacre R, Lairson LL, Siuzdak G (2017) Systems biology guided by XCMS online metabolomics. Nat Methods 14, 461-462. https://doi.org/10.1038/nmeth.4260
      • Type: Journal Articles Status: Published Year Published: 2017 Citation: De Le�n KB, Zane GM, Trotter VV, Krantz GP, Arkin AP, Butland GP, Walian PJ, Fields MW, Wall JD. (2017) Unintended laboratory-driven evolution reveals genetic requirements for biofilm formation by Desulfovibrio vulgaris Hildenborough. MBio. 8(5). pii: e01696-17. doi: 10.1128/mBio.01696-17.
      • Type: Journal Articles Status: Published Year Published: 2018 Citation: Christensen GA, Somenahally AC, Moberly JG, Miller CM, King AJ, Gilmour CC, Brown SD, Podar M, Brandt CC, Brooks SC, Palumbo AV, Wall JD, Elias DA. (2018) Carbon amendments alter microbial community structure and net mercury methylation potential in sediments. Appl Environ Microbiol. 84(3). pii: e01049-17. doi: 10.1128/AEM.01049-17


      Progress 10/01/16 to 09/30/17

      Outputs
      Target Audience:Environmental scientists from around the world are the target audience. These results may also be pertinent to the petroleum industry and to navies of all countries. The U.S. Department of Energy is also an audience as this department has responsibility for clean up of toxic metal wastes generated from weapons production. Target Audience includes collaborators and scientists in the Department of Energy Lawence Berkeley National LaboratoryScience Focus Area ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies). Further distribution of our findings are made available through attendance at national and international meetings. Changes/Problems:I will be retirng this summer. What opportunities for training and professional development has the project provided?Erica Majumder, postdoctoral fellow, has learned and now taught molecular biology skills with the anaerobic bacteria. She has also gained first-hand knowledge of proteomics and metabolomics. Erica has also taught lab skills to two undergraduates, Sharien Fitriasari and Fawn Whittle. An additional undergraduate has worked to improve our assay for uranium reduction by DvH. How have the results been disseminated to communities of interest?" Presentations at focused meetings such as our collaboration Annual Retreat with colleagues at Berkeley, Montana State, Washington Univ at Seattle and others have also been a major communication opportunity. " Campus poster sessions for undergraduate researchers also allow for benefits to our presenters and to those on the campus who learn about the research. " Much of the research will be published in scholarly journals along with being presented at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?I will be retiring in three months. Much of the research will be published after this date but will recognize our support.

      Impacts
      What was accomplished under these goals? A) Energy conversion: a) & b) Thioredoxin role(s) in DvH energetics. Mutants (marker-exchange deletions) were constructed successfully for three thioredoxin reductases, a putative regulator for a thioredoxin operon and the thioredoxin 3 isozyme. Physiological tests with sulfate, sulfite, thiosulfate respiration or under fermentation conditions indicated that none of the above proteins were needed for growth on these substrates. No conditional lethal phenotypes were observed. We interpret that to mean that there is likely compensatory activity when only one deletion has been constructed in a given strain. Multiple mutations are now being targeted. Thioredoxin 1 was unique. This protein appears to be essential to the growth of DvH in any of the conditions listed since we were unable to make an inactivating mutation of the gene encoding this protein. c) No experiments with ebselen have been conducted. d) Thioredoxin-related deletion mutants are being tested to determine whether responses to oxidative stresses have been affected by the removal of the genes encoding these functions. Initial experiments with peroxide treatments have revealed the sensitivity of the parental strain to oxidative environments. Targeted metabolomics experiments are being designed to determine reduced pyridine nucleotide levels before and after oxidative stress in the parental and mutant strains. The results from these experiments should provide a mechanism for changes in oxidative responses. e) Protein substrates for the thioredoxin 1 and thioredoxin 3 proteins have been explored though protein pulldown reactions with thioredoxins mutated to form a covalent bond to substrate proteins. The interacting proteins have been identified through proteomics analyses and data are being analyzed. B) Interactions of thioredoxins with mercury or uranium. Only one significant question has been answered with the mutants in hand. Thioredoxin 3 is not involved in the reduction of U(VI) in DvH.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2017 Citation: Majumder, E.L-W and J.D. Wall. (2017). Uranium bio-transformations: chemical or biological processes? Open J. Inorg. Chem.: Actinide Special Issue. [doi]:4236/ojic.2017.72003
      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Martins M., F.O. Mourato C, Morais-Silva, C. Rodrigues-Pousada, G. Voordouw, J.D. Wall, I.A. Pereira. 2016. Electron transfer pathways of formate-driven H2 production in Desulfovibrio. Appl Microbiol Biotechnol. 100(18):8135-46. doi: 10.1007/s00253-016-7649-7. PMID: 27270746
      • Type: Journal Articles Status: Published Year Published: 2017 Citation: Turkarslan S, Raman AV, Thompson AW, Arens CE, Gillespie MA, von Netzer F, Hillesland KL, Stolyar S, L�pez Garc�a de Lomana A, Reiss DJ, Gorman-Lewis D, Zane GM, Ranish JA, Wall JD, Stahl DA, Baliga NS. (2017) Mechanism for microbial population collapse in a fluctuating resource environment. Mol Syst Biol. 13(3):919. doi: 10.15252/msb.20167058. PMID: 28320772
      • Type: Conference Papers and Presentations Status: Accepted Year Published: 2017 Citation: Majumder, E.L.W., Forsberg, E.M., Trotter, V.V., Butland, G.P., Siuzdak, G., Wall, J.D. (2017) Global stable isotope metabolomics of sulfate-reducing bacteria reveals unique sulfur metabolome. Symposium presentation: Microbial Sulfur Cycling, American Society for Microbiology (ASM) Microbe 2017, June 1-5, New Orleans.


      Progress 10/01/15 to 09/30/16

      Outputs
      Target Audience:The results were disseminated to our large research collaboration, ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies), funded as a subcontract from the Science Focus Area of the Lawrence Berkeley National Laboratory during our monthly teleconference and posters presented at focussed meetings such as the ENIGMA collaboration Annual Retreat. Here at MU, campus poster sessions for undergraduate researchers and postdoctoral fellows disseminated our results. Dr. Majumder presented her findings at the Gordon Reesarch Conference for Metals in Biology and the Bioinorganic Chemistry Grodon Research Seminar, Jan, 24-29, 2016, in Ventura, CA, and at the American Society for Microbiology at Boston, MA, June 16-20, 2016. Plans are in place for publication of a manuscript describing these results. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Dr. Majumder joined the laboratory and successfully constructed her first mutations in a bacterium. She has also pioneered a clever approach of identifying the substrates reduced by thioredoxins in DvH. This identification of the thioredoxin interactome is in progress though application of proteomics analyses. An undergraduate Biochemistry student, Sharien Fitriasari from Indonesia who had no research laboratory experience, began working with Dr. Majumder in the fall of 2015. She actively carried out genetic constructs in the anaerobic bacteria and characterized the resulting mutants. This experience influenced Sharien to pursue her Ph.D. in graduate school. Recently she was accepted for graduate studies in the Stowers Institute for Medical Research located in Kansas City. How have the results been disseminated to communities of interest? Presentations at focussed meetings such as the ENIGMA collaboration Annual Retreat have been a major communication opportunity for Dr. Majumder as well as presentation at the 2016 Gordon Research Conference on Metals in Biology held in conjunction with the "Bioinorganic Chemistry" Gordon Research Seminar, in Ventura, CA. Dr. Majumder presented most of this work at the 2016 American Society for Microbiology meeting in Boston, MA. Campus poster sessions for the Life Science Week engaged both postdoctoral fellows and our undergraduate researchers. This event allows for benefits to our presenters and to those on the campus. Much of the research will be published in scholarly journals along with being presented at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? The thioredoxin interactome study will be completed. The sulfur metabolites of DvH will be determined to query whether this sulfate-reducing bacterium has unique metabolites. 34-sulfate versus 32S-cysteine will be used to explore the relative source of sulfur in the metabollites. Determine the role of the thioredoxins in metal reduction, in particular, uranium. Establish a colorimetric assay for uranium reduction. Determine whether thioredoxin mutants of the anaerobe DvH are more sensitive to exposure to oxygen and other oxidants than the parental strain.

      Impacts
      What was accomplished under these goals? Bioinformatic searches carried out by Dr. Erica Majumder showed that only about 50% of bacteria (aerobes or anaerobes) have the low molecular weight thiol glutathione that contributes to redox homeostasis in the cell. All organisms -- prokaryotes, eukaryotes or archaea -- have thioredoxins, small thiol proteins (ca. 12 kDa). Desulfovibrio vulgaris Hildenborough (DvH) has no glutatione but does have two paralogs of thioredoxin, Trx1 and Trx3. Trx1 (gene locus DVU1839) is an essential gene in DvH. Deletion of the encoding gene was not accomplished. Trx3 (gene locus DVU0378) is not essential and cannot replace Trx1 for function in DvH. There are four thioredoxin reductase (trxR) orthologs in DvH each of which was deleted as single mutants. Mutants lacking both TrxR1 (DVU1838) and TrxR3 (DVU0377) were not viable.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Kurczy, M., Forsberg, E., Thorgersen, M., Poole, F., Benton, H. P., Ivanisevic, J., Tran, M., Wall, J., Elias, D., Adams, M., Siuzdak, G. (2016) Global isotope metabolomics reveals adaptive strategies for nitrogen assimilation in Pseudomonas. ACS Chemical Biology 11(6):1677-85. DOI: 10.1021/acschembio.6b00082. PMID: 27045776
      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shatsky M., S. Allen, B.L. Gold, N.L. Liu, T.R. Juba, S.A. Reveco, D.A. Elias, R. Prathapam, J. He, W. Yang, E.D. Szakal, H. Liu, M.E. Singer, J.T. Geller, B.R. Lam, A. Saini, V.V. Trotter, S.C. Hall, S.J. Fisher, S.E. Brenner, S.R. Chhabra, T.C. Hazen, J.D. Wall, H.E. Witkowska, M.D. Biggin, J.M. Chandonia, G. Butland. 2016. Bacterial interactomes: interacting protein partners share similar function and are validated in independent assays more frequently than previously reported. Mol Cell Proteomics. 15(5):1539-55. doi: 10.1074/mcp.M115.054692. PMID: 26873250
      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Shen Q., J.D. Wall, Z. Hu. 2016. Solids retention time dependent phototrophic growth and population changes in chemostat cultivation using wastewater. Water Environ Res. 88(1):5-12. doi:10.2175/106143014X13975035526103. PMID: 26803021
      • Type: Journal Articles Status: Published Year Published: 2016 Citation: Martins M., F.O. Mourato C, Morais-Silva, C. Rodrigues-Pousada, G. Voordouw, J.D. Wall, I.A. Pereira. 2016. Electron transfer pathways of formate-driven H2 production in Desulfovibrio. Appl Microbiol Biotechnol. 100(18):8135-46. doi: 10.1007/s00253-016-7649-7. PMID: 27270746
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: De Le�n K.B., S.M. Utturkar, L.B. Camilleri, D.A. Elias, A.P. Arkin, M.W. Fields, S.D. Brown, J.D. Wall. 2015. Complete genome sequence of Pelosinus fermentans JBW45, a member of a remarkably competitive group of Negativicutes in the Firmicutes phylum. Genome Announc. 3(5). pii: e01090-15. doi: 10.1128/genomeA.01090-15. PMID: 26404608
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Zhou A, Hillesland KL, He Z, Schackwitz W, Tu Q, Zane GM, Ma Q, Qu Y, Stahl DA, Wall JD, Hazen TC, Fields MW, Arkin AP, Zhou J. (2015) Rapid selective sweep of pre-existing polymorphisms and slow fixation of new mutations in experimental evolution of Desulfovibrio vulgaris. ISME J. 9(11):2360-72. doi: 10.1038/ismej.2015.45. PMID: 25848870.


      Progress 07/01/15 to 09/30/15

      Outputs
      Target Audience:Target Audience included collaborators and scientists in the Department of Energy Lawence Berkeley National Laboratory Science Focus Area ENIGMA (Ecosystems and Networks Integrated with Genes and Molecular Assemblies). This includes scientists at UC Berkeley; University of Montana; University of Washington, Seattle; University of Oklahoma; Massachusetts Institute of Technology; University of Tennessee; and Oak Ridge National Laboratory. Further distribution of our findings are made available through attendance at national and international meetings but not during the three months of this report. Dr. Erica Majumder joined my laboratory as a Postdoctoral Fellow at the end of August, 2015. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Erica Majumder, postdoctoral fellow, obtained her PhD in Chemistry and is now expanding her skillset to include microbial genetics. She is also developing an intimate relationship with the Microscale Thermophoresis technology. We have four undergraduates working in the laboratory and two graduate students who are benefiting through our weekly lab group meeting exchanges. How have the results been disseminated to communities of interest?Dissemination has been through individual training of visiting scientists in the research techniques used frequently during the construction of targeted mutations. Presentations at focussed meetings such as the ENIGMA collaboration Annual Retreat have also been a major communication opportunity. Campus poster sessions for undergraduate researchers also allow for benefits to our presenters and to those on the campus who learn about the research. Much of the research will be published in scholarly journals along with being presented at national and international meetings. What do you plan to do during the next reporting period to accomplish the goals?A a) The constructed plasmids will be used to attempt to delete each of the thioredoxins and thioredoxin reductases. Those genes that can be deleted will indicate the non-essential genes. Those that are recalcitrant to deletion are likely to be essential. Thus we will get and early view of the more important players in the cell's metabolism. These deletions will be constructed in a host strain so that the antibiotic resistance used to select the deletion can be removed leaving an inframe, markerless mutation. Therefore, multiple mutations will be constructed in the same strain. A b) Growth characteristics will be determined on an array of substrates to determine whether distinguishable phenotypes have been revealed that indicate the role of the proteins in metabolism. A c) Inhibitors of thioredoxin that are accessible will be explored for their affects on the parental strain not mutated for thioredoxins or the reductases and the mutants obtained. Phenotypes of inhibited cells will be compared to those of the deletion strains. A d) If redox levels are changed in the various deletion mutants, the cells may show differential sensitivity to oxidants or stressors. We will determine whether sensitivity levels to oxygen, nitrate, nitrite, uranium, chromate, and mercury have been changed through the deletion of the targeted genes. A e) Using the Microscale Thermophoresis that should be assembled within the next year, we will determine whether metals interact with the thioredoxins or whether sulfite binds to these proteins. Proteins identified as potentially interacting with the reductases or thioredoxins will be purified through the attached tandem tags and used in the MST for affinity measurements. B a) Batch fed reactors will be used to determine whether the redox active metals mercury and uranium can act as terminal electron acceptors if maintained at sub-inhibitory concentrations. This may also provide information on the site of metal interaction wtih the cells. B b) Rates of metal reduction will be tested in the various mutants obtained and/or when the mutants are grown on substrates that show a growth difference from the parental strains. B c) Determination of a role for thioredoxins in metal reduction will likely require an in vivo assay. In the in vitro setting, the reactions could be driven simply by reduction potential and proximity. Such data would not speak to the ability of metals to reach these strongly reducing proteins in the cells.

      Impacts
      What was accomplished under these goals? Notable results obtained during the three months of this report were limited. A. a) To determine whether thioredoxins and their reductases are essential for DvH,mutants lacking each of the thioredoxin encodinggenes will be generated. a) Plasmids for marker-exchange mutagenesis of each of the annotated thioredoxin genes have been constructed. A b) Growthcharacteristics of mutants will be elucidated under sulfate-, thiosulfate- and non-sulfate-reducing(fermentative) conditions. b) Mutants were not constructed at this time. A c) Resistance tothe bacterial thioredoxin reductase inhibitor ebselen will be determined. c) Ebselen not explored since mutant construction will precede the experiments with inhibitors. A d) Iintracellular levels of reduced thioredoxins and pyridine nucleotides will bedetermined in each mutant. d) To explore redox changes, mutant sensitivity to oxygen exposure or peroxide will be examined before the more difficult measurements of redox status are pursued. A e) To determine the substrates interacting with the thioredoxins, the thioredoxin proteins will be isolated and binding affinities for substrates and interactingproteins will be determined. e) The tandem tag, Strep-TEV-FLAG tag, for affinity purification to determine interacting proteins is available in the laboratory. Earlier resarch in the laboratory generated three tagged versions of the target proteins that can be used in the current study. B a) Test the possibility that sub-inhibitory concentrations Hg(II) or U(VI) might serve asterminal electron acceptors for SRM. a) Not completed. B b) Determine whether thioredoxins are components of the electron transfer pathway usedto deliver electrons to Hg(II) or U(VI). b) Mutants that are generated will be tested. The Kinetic Phosphorescence Analyzer used to determine U(VI) has been troublesome even though a new laser and new Uraplex needed for the assay have been obtained. B c) Establish whether reduced thioredoxin (DVU1839) can directly deliver electrons toHg(II) or U(VI). c) This approach will take advantage of the newly described instrument, the Microscale Thermophoresis instrument. Upon arrival, my new postdoctoral fellow, Erica contacted my colleague who had volunteered to construct a "homemade" version of this instrument in his lab. Erica has been involved in this process from her arrival. With purified fluorescent protein, we should be able to begin to examine the affinitiies of metals with the thioredoxins.

      Publications

      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Ramos, A.R., F. Grein, G.P. Oliveira, S.S. Venceslau, K.L. Keller, J.D. Wall, and I.A. Pereira. 2015. The FlxABCD-HdrABC proteins correspond to a novel NADH dehydrogenase/heterodisulfide reductase widespread in anaerobic bacteria and involved in ethanol metabolism in Desulfovibrio vulgaris Hildenborough. Environ Microbiol. 17(7):2288-305 doi: 10.1111/1462-2920.12689
      • Type: Journal Articles Status: Published Year Published: 2015 Citation: Christensen, G.A., G.M. Zane, A.E. Kazakov, X. Li, D.A. Rodionov, P.S. Novichkov, I. Dubchak, A.P. Arkin, and J.D. Wall. 2015. Rex (encoded by DVU0916) in Desulfovibrio vulgaris Hildenborough is a repressor of sulfate adenylyl transferase and Is regulated by NADH. J Bacteriol. 197:29-39. doi: 10.1128/JB.02083-14
      • Type: Book Chapters Status: Published Year Published: 2015 Citation: Rabus, R., Venceslau, S.S., W�hlbrand, L., Voordouw, G., Wall, J.D., and Pereira, I.A.C. 2015. A post-genomic view of the ecophysiology, catabolism and biotechnological relevance of sulphate-reducing prokaryotes. R.K. Poole (Series Ed.) Advances in Microbial Physiology, Academic Press an imprint of Elsevier, London, Oxford, and San Diego. Adv Microb Physiol. 66:55-321. PMID: 26210106