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Metal reduction pathways important for arsenic mobilization
Iron Hello my name is Chad Saltikov and I thank you for inviting me to talk with you about my research on how bacteria respire arsenate, that is how microbes utilize arsenate as a terminal electron acceptor during anaerobic respiration. Prior to the isolation of the first As(V) respiring microbe in the mid 90’s, it had been know for quite sometime that bacteria could detoxify arsenicals by reduction/oxidation and efflux. When I first read that microbes could use As(V) for respiration, I became fascinated yet puzzled as to how this toxic compound with such an infamous past could be used beneficially by organisms. Since that early report, numerous As(V) respiring strains have been isolated, however little is still known about this potentially important microbial process. Therefore, the goal of my research has been to simply determine how microbes respire arsenate. Chad W. Saltikov Assistant Professor UC, Santa Cruz Microbiology and Environmental Toxicology
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Pore water concentrations of As and Fe
MCL Suvasis Dixit and Janet Hering Caltech
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Metal Reduction and Arsenic Mobilization
As(III) Oxidizers e- As(III) As(V) Reducers As(V) iron oxide As(V) e- Fe(III) Reducers As(V) Fe(II)
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Metal-Reduction in Shewanella
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Toxicity, Fate, and Transport of Arsenic
Environmental impact of metal-reduction Electron transport chains for ARR/Mtr Regulation of metal reduction pathways Fe(III) As(V) As(III) HPO32- NO3- O2
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Shewanella sp. strain ANA-3: our model
Substrate Growth Arsenate + Nitrate Fumarate TMAO Fe(OH)3 MnO2 Oxygen Thiosulfate DMSO - Saltikov et al. AEM 2003
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Arsenate Respiratory Reductase in Bacteria
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The 23 kb “Arsenic Island” in Shewanella species
As(V) respiration Arsenic detoxification Gsh: glutathione synthetase; not found in MR-1 genome or on any bacterial genome. Similar to eukaryotic gsh however ANA-3’s has a putative tat signal. Perm: permease; top hit is in MR-1 genome but it’s labeled as a conserved hypothetical protein. New sequence data downstream of the arrAB operon reveals another cluster of ars genes however they are duplications yet divergent in the DNA sequence. Protein analysis by BLAST: ArsR1: ArsR regulator, 95% similarity to MR-1 ArsC1: Low molecular weight phosphatatse family; arsC homolog; 64% similarity P. aeruginosa Protein-tyrosine-phosphatase ArsR2: ArsR regulator, 81% similarity to MR-1 ArsC2: ArsC, 67% similarity MR-1 Pairwise comparison of amino acid sequence: ArsR1 vs ArsR2 50% similarity ArsC1 vs ArsC2 47% similarity UCSC Genome Browser
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Metal reduction genes in Shewanella
mtrD mtrF mtrC mtrA mtrB mtrE omcA UCSC Genome Browser
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Impacts of metal-reducing bacteria on arsenic contamination and water quality
Fe(III) vs. As(V) reduction Arsenic Iron
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Fe(III) vs. As(V) reduction and As mobilization
Strain Genotype Fe(III) Reduction As(V) Reduction ANA-3 wt + ARM1 ∆arrA, ∆arsC - FERM1 ∆mtrDEF ∆omcA ∆mtrCAB FARM1 ∆arrA, ∆arsC, ∆mtr/omc As:HFO= 0.015 500 mg HFO-As(V) 1 mM As(V) total Lactate 20 mM 0.5 mM Phosphate 10 mM HEPES pH 7 Basalt salts medium
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Iron(III) and Arsenate Reduction In batch cultures
Dissolved Iron Dissolve Arsenate
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End-point solid phase arsenic chemistry
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Arsenic mobilization from ferrihydrite coated sand: advective flow
263 mg As/kg sand 5500 mg Fe/kg sand 24 μL/min (no Fe reduction) (no As reduction) Iron oxide coated sand Picture from Herbal and Fendorf
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Comparison of Fe and As Elution under advective conditions
Dissolved Fe Dissolved As WT FERM ARM WT FERM no cells no cells ARM 3 mM lactate As(V) on ferrihydrite (3.5 mmol Kg-1) 25 % of the adsorption maximum at pH 7.1
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Preliminary Conclusions
ArrA reduces solid phase arsenate reduction ∆arrA strain has a problem reducing Fe(III)-oxide As(III) mobilization highest in ∆mtr/omc strain How does ArrA access solid-phase As(V)? Is the “Fe(III) reductase” blocked by As(V)? Why does uncoupling Fe-reduction increase As release?
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Characterizing the metabolic pathways essential for arsenic mobilization
CymA and ArrAB and Mtr/OmC Regulation of arrA Regulation of mtr/omc
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CymA and As(V) and Fe(III) reduction
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∆cymA causes pleiotropic effects on respiration pathways
ANA-3 CN-32 MR-1 Substrate cymA wt Arsenate - + Fumarate DMSO nd Nitrate TMAO +/- O2 Thiosulfate Fe(III) Mn(IV) - no growth + growth Murphy and Saltikov, 2007
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Does CymA interact with Menaquinone?
Fe(III) Fe(II) Mtr/Omc ArrAB As(V) As(III) c-heme CymA Mo 4X[FeS] [FeS] Q QH2 NADH2 NAD+
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Predicted structure of CymA
NrfH CymA hemes D97 D89 K96 K82 K91 K90 Lys (K) to Gln (Q) (basic to neutral) Asp (D) (acidic)
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Over-expression of CymA in E. coli
Western (anti-V5) Cytoplasmic Membranes Heme Stain Ladder No pCYMA CymA - ccm CymA + ccm K90Q + ccm CymA - ccm CymA + ccm K90Q + ccm
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MQH2-mediated reduction of CymA
DT Relative Abs DMNH2 nm nm nm Dithionite DMNH2 0.3 mg/ml protein ~4% CymA ~600 nM NaBH4 DMN DMNH2 2,3 dimethyl 1,4 naphthoquinone(ol)
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Does CymA interact with menaquionols?
CymA + ccm CymA-K90Q + ccm No CymA Relative Fluorescence Calculated disassociation Kd is ~90 nM HOQNO
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CymA-MQH2 effects on ARR
(Gln, neutral) (Met, neutral) (Arg, basic)
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Next steps for CymA Quinone substrate range? ArrAB reduction by CymA
Mtr/Omc reduction by CymA Multasking nature of CymA D97 K96 K91 K90
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How does arrA and mtr/omc gene expression respond to different environmental conditions?
Oxygen should repress both pathways Arsenate should induce arrA Fe should induce mtr/omc.
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Expression of mtr/omc in ANA-3
mtrD mtrE mtrC omcA mtrA mtrB mtrF Note: Beliaev et al showed 2-8 fold decrease in MR-1
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arrA expression with other substrates?
20 mM concentrations of each TEA. 10 mM As(V) was added prior to inoculating with an aerobically grown overnight of ANA-3 to a final dilution of 1/100 in 10 ml. 1 mM As(III) was added prior to inoculation with ANA-3. Cells were harvested at OD600 = 0.1 similar to other experiments. Expression is the normalized expression of arrA or arsC to gyrB. Induction refers to the expression value for the TEA+As divided by the expression value for the TEA without As. Blue box shows that nitrate represses expression. Red boxes just points out that As(III) induces almost as well as As(V). Saltikov et al. J. Bact. 2005
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Possible regulators of arr and mtr/omc
Environmental Condition Transcription Factor/Sensor Arsenite ArsR √ Arsenate ? Anaerobic Fnr , ArcA Aerobic cAMP CRP, Cya Nitrate/Nitrite NarP, NarQ
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Arsenite regulators: arr ars ArsR Six arsR like genes in ANA-3 ArsR
helix turn helix, ~12 kDa DNA binding Binds As(III), Sb(III), PAO Six arsR like genes in ANA-3 Quantitative Gene Expression ars arr ArsR As(III) As(III)
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Effects of arsR2 on arrA/arsC gene expression
1 kb
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ArsR binds the arr/ars intergenic region
Parr Pars ars arr ArsR2 ~350 bp 5’-Cy3 ArsR2 Binding Free DNA Protein+DNA
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As(V) respiration and global regulators
Growth on 10 mM Arsenate (fnr)
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CRP and arrA gene expression
A. arrA Expression Growth O2 Shift -O2 + As(V) Sample: 0hr, 8 hr B. 16S rRNA Expression
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Arsenate reduced after 8 hours
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ArsR and CRP binding @ arr promoter
Parr ars arr ArsR2 B Shift (ArsR/CRP) Shift (CRP) Shift (ArsR) Free DNA
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Model for arr and mtr/omc regulation
NO3- NO2- low O2, ? As(V) Low O2 ArrS ? Cya NarQ Hpt NarP-P ArrR ArrR-P NarP cAMP ATP ArcA ArcA-P CRP cAMP genes arrAB mtrDEF omcA mtrCAB +As(III) high redox ArsR Fnr (EtrA) -As(III) low redox
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Thank you! Acknowledgments: UCSC: Stanford: Funding:
Julie Murphy, Carolina Reyes, Kamrun Zargar Stanford: Prof. Scott Fendorf, Kate Tufano Funding: NSF, UC Toxic Substance, CRCC, MBRS, Cota Robles ~Santa Cruz, California, USA~
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