Microbiology of Reductive Dehalogenation: Something Old, Something New

Steve Zinder MBL, June 19, 2013 Chlorinated organic compounds PCE TCE cis-DCE

trans-DCE 1,1-DCE VC Aerobic Degradation of Chloroethenes

H H mineralization C=!C CO2 H Cl non specific H H oxygenases C=!C CO2, etc. Cl Cl mineralization (Strain JS666)

Cl H Cl O H C=!C C–C! Cl Cl Cl Cl

Cl Cl toluene C=!C ?? monooxygenase Cl Cl of PCE

Cl Cl 2H HCl Cl H 2H HCl H H 2H HCl H H 2H HCl H H C C C C C C C C C C Cl Cl Cl Cl Cl Cl H Cl H H PCE TCE DCEs VC ETH

James Gossett

Known human carcinogen PCE conversion to ETH by a MeOH/PCE enrichment from the Ithaca Sewage Plant

Hours

Envir Sci Technol 28:973, 1994 H+/H 2 -0.4

- -0.3 Organochloride CO2/Acetate CO2/CH4 2- -0.2 respiration SO4 /H2S (halorespiration -0.1 dehalorespiration, 0.0 +0.1 respiratory reductive Fe(OH) /Fe(CO ) 3 3 +0.2 dechlorination) +0.3 cDCE/VC - - +0.4 NO3 /NO2 VC/ETH +0.5 TCE/cDCE PCE/TCE +0.6

- +0.7 NO3 /N2 +0.8 O2/H2O +0.9 3-Chlorobenzoate dehalogenating community

Desulfomonile tiedjei

Syntrophus

Methanospirillum Methanosaeta Organisms reducing PCE and TCE to cDCE MeOH/PCE-dechlorinating culture

Phase contrast Fluorescence, Xavier Maymó-Gatell “ ethenogenes” strain 195 (Science, 276:1568, 1997) EM of Dhc strain 195

S-layer cell wall No peptido- glycan Methanosarcina Sulfolobus Planctomyces Gloeobacter Nostoc Cyanobacteria! Mycoplasma Clostridium restrictus Desulfitobacterium.dehalogens Desulfitobacterium.chlororespirans Desulfitobacterium.frappieri Desulfitobacterium.hafniens Desulfitobacterium StrainDCE Actinomyces Streptomyces Actinobacteria Desulfovibrio Dehalospirillum multivorans Desulfuromonas chloroethenica Proteobacteria Agrobacterium Enterobacter MS1 Escherichia coli Chloroflexus Sphaerobacter SJA15 TCB consortium OPB12 Hot Spring WCHB1.50Chl. solvent aquifer GCA112 WCHA1.69 Chl. solvent aquifer OPB9 Hot spring SJA170 TCB consortium Chloroflexi! MUG9 Anaerobic bioreactors TUG8 H12f Hanford, WA reactor “GNS ” SAR202 Sargasso sea RFLP17 PCB consortium Dehalococcoides ethenogenes 16S rRNA gene vadinBA26 Wine waste reactor ACE22 Antarctic lake H3.93 t0.8.f Hanford, WA aquifer Phylogeny-2000 H1.4.f MT61 0.1 Dehalococcoides and relatives Dhc FL2, AF357918 Dhc GT, AY914178 Dhc CBDB1, AJ965256 Strain KB1-VC, AY146779 Pinellas Clone JN18_V35_B, EF059529 Dhc BAV1, AY165308 Dehalogenating Strain KB1-PCE, AY146780 All > 97% 16S ID Chloroflexi Dhc VS, AY323233 Victoria Dhc 195, AF004928 Strain TM-EtOH, AY882433 Cornell

Dehalogenimonas lykanthroporepellens EU679418 Clone LaC15H20, EF667695 Dehalobium chlorocoercia Strain DF-1, AF393781

Clone SAR202, U20797 Clone H1.2.f,, AF005747 Marine Chloroflexi Clone FTL276, AF529110

Anaerolinea thermophila UNI-1, AB046413 Anaerolinea Caldilinea aerolinea, AB067647

Chloroflexus aurantiacus, M34116 “Classical” Chloroflexi Dehalococcoides mccartyi

Perry McCarty with Tyler Prize for Environmental Achievement The real world: Site 1381 Cape Canaveral Air Station (CCAS)

Amy Carroll detected Dhc with PCR

Pump lactate as electron donor  H2 Biostimulaton With Electron Donors Butyrate, Ethanol, Benzoate, Lactate, or Vegetable Oil PCE ETH

H2 4 HCl Dechlorinators

CO2 Methanogens Methane

Acetate CO2 Bioremediation at Site 1381: fate of chloroethenes in a central well after adding lactate M ic h ig a n R iv e rs Red Cedar River Ki t c h n e r Père Marquertte River It h a c a Ke n t New a r k Au Sable River Niagara Falls Ft. Lewis Neeco Park (Niagara Falls) Textron (Niagara Falls) Alameda NAS P om p to n La k e s Oa k l e y Dover AFB LF 13 Lo r e n z Dover AFB Pilot S ac ra m en to Ja c k s o n v i l l e Wi n f i e l d Cape D o rd re c h t Kelly AFB LF Canaveral Am s te rd a m Kelly AFB Pilot Vi c t o r i a De Li s l e C ent ra l Holland Be a u m o n t Pi n e l l a s Berlin, GR C he s h ir e , Cornell Labs Tiedje’s Lab N. England RTDFR Sites* Geosyntec* Stuttgart, GR Du Pont’s Sites* German Labs Compiled by Edwin R. Hendrickson, DuPont Company,CRD/CCER & CRG * Data Contributed by Edwin R. Hendrickson, DuPont Co. Commercial Dehalococcoides cultures for bioaugmentation

Shaw Group SDC-9

+ Bioremediation Consulting International (BCI) Labs, and Bioaug LLC + Zinder Lab, Summer 2001

Ivonne Nijenhuis Amy Carroll

Tim Anguish! Dehalococcoides ethenogenes genome • Sequenced by the Institute for Genomic Research (JCVI) (funded by DOE)- • John Heidelberg, Rekha Seshadri, and Derrick Fouts main collaborators 1 • Size: 1,470,272 bp ≤ /3 that of E. coli • %G+C = 48.9 • Predicted protein-encoding ORFs: 1592 • Similar to known function: 904 (56.7%) • Similar to unknown function: 386 (24.3%) • No match: 302 (19%) • Seshadri et al. Science 307:105, 2005 TCE reductive dehalogenase (RD)

A Corrinoid “Periplasm” 3-4Fe4S 4Fe4S

Membrane TAT B

RR PC C Twin arginine export signal Three FeS proline motif centers

PCE RDH: PCE + 2H  TCE + HCl TCE RDH: TCE + 4H  VC + 2HCl ( ETH) Magnuson et al. AEM 66: 5141 (2000) Dehalococcoides! ethenogenes ! genome map- RDs DET0318 pceA 2C Regs

%GC Trinucs GC MarR skew = Mobile genetic elements ≈ 13.6% of genome

Phage?

17 complete + 2 defective RDHs Most have adjacent two-component or MarR transcriptional regulators RDH homologues detected in PceA Dehalococcoides Chloro- A large (96) and phenols phylogenetically diverse family

Only know functions for 5 TceA homologous groups BvcA-VC VcrA-VC CbrA - Chlorobenzenes

RDHs from Firmicutes ACT comparison of genomes 17 RDHs

36 RDHs

32 RDHs

HPR = high plasticity regions RDH genes in black RDH homologues in yellow Homologues transcribed in same direction in red, opposite direction in blue D. ethenogenes temperate phage • Heather Fullerton has succeeded in growing Det in defined medium • Genes (DET 1067-1104 encoding an ostensibly complete phage are found in the DET genome – related to G+ phages – no att site • Phage particles can be detected by fluorescence and electron microscopy and appear to be 10-fold induced by mitomycin C Dehalococcoides: lessons learned • Dehalococcoides “Born to dechlorinate” • RDHs are a large phylogenetically deep gene family • RDH genes usually found in “islands” of high genetic plasticity – Housekeeping genes are syntenic and highly conserved between strains – Fits core genome/pan genome model for species • Reductive dehalogenation past DCE at a contaminated sites apparently requires the presence Dehalococcoides • RDHs are better biomarkers for dehalogenation than 16S rRNA Chlorobenzenes • Chlorobenzenes (CBs) used as chemical precursors, solvents, and pesticides • Tons of monochlorobenzene (MCB) in groundwater at Montrose Chemical Plant in Fullerton CA – made DDT • Diverse aerobes like Pseudomonas and Burkholderia can degrade DCBs and MCB – Use well known oxygenase based aromatic degradation pathways

1,4-DCB in moth balls and CBs in car wax MCB used to Chlorobenzenes: anaerobic degradation • CBs can form dense nonaqueous phase liquids (DNAPLs) that migrate to anaerobic zones • Dehalococcoides CBDB1 and 195 reductively dehalogenate CBs with ≥3 Cl • Dhc stop at TCBs and DCBs • In ~2005 DuPont asked our lab to investigate reductive dehalogenation of DCBs and MCB • Much less was known about anaerobic degradation of DCBs and MCB Model for anaerobic DCB dechlorination - 2005

CH4 , CO Trace amounts 2H HCl during DCB to MCB Methanogenic conditions Cl 2H HCl ortho)-dichlorobenzene 2- 3+ SO4 , Fe or - benzene NO3 -reducing conditions

Cl Cl CO2 meta)-dichlorobenzene monochlorobenzene 2- 3+ Cl SO4 , Fe or - NO3 -reducing CO2 Microcosms conditions slow rates

Cl Solid arrows = known reactions Two early eras: Brian Weisenstein  Jennifer Fung DuPont Chambers Works • On Delaware River next to Memorial Bridge • Dye factory in early 1900s - chloroanilines and CBs • Samples came from a drainage ditch (DD) at the site • Received on Dec 5, 2005 • Brown mud with no detectable organic pollutants

• Dec 15- preliminary microcosm experiment – • 20 g mud, • 50 ml anoxic water, • 3 DCB isomers DD • yeast extract as electron donor • GC readings at 1 day and at 20 days (after break) The Xmas miracle

100 90 80 70 12DCB 60 13DCB 14DCB 50 MCB umoles 40 Tol 30 Benz 20 10 0 0 10 20 30 40 50 60 Days CW microcosms: all three DCB isomers

Fung et al. ES&T 43:2302 (2009) CW microcosms: individual CBs

1,2-DCB 1,3-DCB 1,4-DCB

MCB DCBs: Culture experiments • Jen Fung developed three enrichment cultures, one for each DCB isomer • The 1,2-DCB culture was the fastest, so we studied it • We expected Dehalococcoides to be the dehalogenator • However, vancomycin inhibited 1,2-DCB dehalogenation– Dhc is resistant • We couldn’t detect Dhc with specific 16S primers • Made a 16S rRNA gene clone library with universal bacterial primers (Hinsby Cadillo-Quiroz) • No Dhc, but > 25% of clones Dehalobacter Dehalobacter

Firmicutes (Gram +) • PER-K23 PCE/ TCE to cis-DCE – Requires B Vitamins, Arg, His, Thr for growth • Dehalobacter strain TCA1 1,1,1-TriChloroethAne/1,1-DCA to CA • Dehalobacter-containing mixed cultures – 1,2-DCA to ethene – Tetrachlorophthalide – Hexachlorocyclohexane (Lindane) • Specialists

– Only H2 or sometimes formate as electron donor – Only chlorinated electron acceptors Optimizing the 1,2-DCB Enrichment • Project was taken over by Jenny Nelson • Goal is to make Dhb most numerous organism in culture • Organic nutrients cut down to 2 mM acetate (C source), vitamins, and 20 mg/L CAA to minimize heterotrophs • Methanogens removed with BES – Acetogens removed using butyrate as an electron donor

– Poises H2 too low for acetogens -6 – Followed by 10 dilution into H2-DCB medium • Add DCB dissolved in hexadecane – Can add at much higher nominal concentrations (~1 mM)

• Replace Na2S or Ti (III) as reducing agent with FeS precipitate – faster growth – – But FeS chews up DNA Purified 1,2-DCB enrichment Purified 1,2-DCB enrichment Bacterial 16S rRNA gene clone library of purified 1,2-DCB enrichment • 80/82 clones were Dhb • 31 had a 100 bp insert at 5’ end • Two other sequences – Sedimentibacter (amino acid fermenting Firmicutes found in other dehalogenating enrichments) – Desulfovibrio Dv?

Sb? Isolation of 1.2-DCB utilizer Association with FeS particles • Culture was isolated by 10-8 dilution with Sedimentibacter extract • After isolation, the cells no longer needed the extracts and grew in defined medium

Acridine Orange Fluorescence Phase Contrast The 1,3-DCB culture

• The 1,3-DCB culture was studied by undergraduate Michael Schwarz • It also initially required Sedimentibacter extracts to grow in 10-8 dilutions • Like the 1,2-DCB culture, transfers no longer required extracts The1,4-DCB culture • The 1,4-DCB culture was by far the slowest and most difficult to grow • It was studied by Dr. Jiandong Jiang • Found that it could grow without CAAs thereby greatly reducing the numbers of contaminants • Growth was obtained from a 10-6 dilution MCB_June_1 Phylogeny of MCB_June_4 MCB_June_3 the three Dhb FTH1 AB294742 Dhb_clone MDAF14 EU214535 strains MCB_June_2 D154 AF422677 1,3-DCB_Early 2 SHA-67 AJ249096 SJA-19 AJ009454 Dhb_sp.MS DQ663785 MCB_Dec_1 MCB_Dec_2 Dhb_Sp_SHD-11 AJ278164 1,3-DCB_Early 1 1,3-DCB_Late Strain 13DCB1 Dhb_sp.WL DQ250129 D. restrictus Y10164 Dhb_sp.E3 AY673992 1,2- & 1,3-DCB Dhb_sp.1,1-DCA1 DQ777749 1,2-DCB* Strain 12DCB1 1,4-DCB Strain 14DCB1 FTH2 AB294743 1,2-DCB Strain 12DCB1 Desulfitobacterium_hafniense Y51 AP008230 0.0070 Physiology: Dehalobacter is a specialist

Electron acceptors and Required for growth donors that did not support growth Acetate – carbon source Hydrogen/formate – Fumarate Ethanol Methanol electron donor Glycine Glycerol – Variable activity in formate/12DCB1 transfers Sulfate Succinate Chlorinated electron Sulfite Glucose acceptor Nitrate Butyrate Nitrite Acetate B Vitamins including B12 Fumarate Oxygen Pyruvate Dehalogenation occurred Lactate between 15°C-37°C, not at 10°C Substrate utilization DCBs and PCE/TCE

1,2-DCB 1,3-DCB 1,4-DCB Cl Cl Cl Cl X X Cl Cl

MCB Cl Benzene

Cl! Cl! 2H! HCl!Cl! H! 2H! HCl! H! C! C! C! C! C! C! Cl! Cl! Cl! Cl! Cl! PCE! TCE! c-DCE! Utilization of 1,2,3 TCB

1,2,3-TCB 1,2,3-TCB Cl Cl Dehalobacter 12DCB1 Cl Cl (single-flanked Cl)

Cl Cl ? X Cl Cl 1,3-DCB Cl Cl [ ] Cl Cl

Cl Dehalococcoides CBDB1 (double-flanked Cl) Tetra to hexachlorobenzene

1,2,3,4-TeCB PeCB HCB Cl Cl Cl Cl Cl Cl Cl

Cl Cl X Cl Cl Cl Cl Cl Cl

No single flanked chlorines

Cl Cl 1,3-DCB culture 1,2-DCB 1,3-DCB 1,4-DCB Cl Cl Cl 1,3,5-TCB 1,2,3,4-TeCB Cl Cl Cl X Cl Cl Cl Cl Cl Cl Cl Cl! 2H! HCl!Cl! H! 2H! HCl! H! H! C! C! C! C! C! C! Cl! Cl! Cl! Cl! Cl! PCE! TCE! c-DCE! Cl Cl

Cl Cl 1,4-DCB culture – highly enriched

1,2-DCB 1,3-DCB 1,4-DCB 1,2,4-TCB Cl Cl Cl Cl Cl Cl X Cl Cl Cl

Cl Cl Cl Cl

Cl Chlorobenzene Model

1,2,3,4-TeCB 1,2,3-TCB 1,2-DCB Cl Cl Cl Cl Cl Cl

Cl Cl Cl HCB QCB 1,2,4,5-TeCB 1,2,4-TCB 1,3-DCB MCB Cl Cl Cl Cl Cl Benzene Cl Cl Cl Cl

Cl Cl Cl Cl Cl Cl Cl Cl 1,2,3,5-TeCB 1,3,5-TCB 1,4-DCB Strain 12DCB1 Cl Cl Cl Cl Strain 13DCB1

Strain 14DCB1 Cl Cl Cl Cl Cl

Also some chlorotoluenes Chlorobenzene dehalogenation

CH , CO Need 4 172 2H HCl Methanogenic conditions Cl Dhb 2H HCl ortho)-dichlorobenzene 6 2- 3+ SO4 , Fe or Dhb - Dhb benzene NO3 -reducing 203 104 conditions

Cl Dhb Cl CO2 meta)-dichlorobenzene monochlorobenzene 2- 3+ Cl SO4 , Fe or - NO3 -reducing CO2 conditions 159 Microcosms

Cl Solid arrows = known reactions Chocolate and peanut butter experiment

ES&T 47:2378 (2013) Dhb draft genomes (Heather Fullerton) CLC Assembly Geneious Assembly Paired-End Lengths Longest Contig Longest Contig Genome (bp) Reads Contig # K-mer Contig # 12DCB1 130-350 13,065,516 680,274 78 22 680,510 62 13DCB1 110-275 13,319,332 173,044 121 22 229,964 79 14DCB1 100-330 13,021,098 630,194 70 22 630,448 54 Dhb genomes

Genome 12DCB1 13DCB1 14DCB1

Genome Size (bp) 2,885,128 2,994,781 3,062,036 DNA G+C content 44.3% 44.7% 43.9% Number of Replicons 1 1 1 tRNA genes 54 53 54 rRNA Operons 3 3 3 Coding Sequences 2854 2956 3028 rdhAB Genes 39 28 39 Rdh operon 13DCB1_2417-2420

A B Fnr/Crp Septum Formation sigE, 1065, 1340, 1295 sigF, 0361, 0532, 0560 spoIIAB, 0362, 0533, 0561 spoIIGA, 1066, 1341, 1296 spoIIE, 0175, 0111, 0251 gerA*, 2128, 2338, 2211 E F spoIIR, 2529, 2512, 2698 σ σ gerB*, 2021, 2165, 2115 cwlJ, 2619, 2604, 2805 spoIID, 2743, 2747, 2930 sleB, 2781, 2782, 2968 spoIIM, 0352, 0532, 0550 ypeB, 2780, 2781, 2967 spoIIP, 2435, 2882, 2552 gerQ, no homologues EngulfmentspoIIIA, 0319, 0491, 0518 12DCB1 spoIIID, 2738, 2744, 2925 13DCB1 Germination & spoIIIJ, 2853, 2958, 3040 14DCB1 Cortex Hydrolysis

σE σF Mature Spore sigG, 1064, 1339, 1294 Cortex sigK, 0299, 0462, 0498 cwlC, 1495, 1498, 1730 spoIVA, 0832, 0959, 1027 cwlH, 2292, 1170, 2323 Formation spoVA*, 0364, 0535, 0569 spoIVB, 0348, 0519, 0546 nucB, 0414, 2164, 0619 spoIVFA, no homologuesa spoVB, 0637, 0735, 0837 spoIVFB, 2469, 2916, 2585 spoVD, 1082, 1357, 1312 Mother Cell spoVE, 1077, 1352, 1307 Lysis Spore spoVF, 1823, 1970, 1920 spoVR, no homologues σK σG Coat ACT comparison

13DCB1

14DCB1 COG comparison

Dehalococcoides Dehalobacter Desulfitobacterium Super specialist Metabolic specialist Generalist Conclusions/next steps

• Dhb are highly adapted to reductive dehalogenation like Dhc • Their genomes are 2x larger with greater physiological adaptation (sporulation, motility and chemotaxis) to their environments • Like Dhc, 16S sequence doesn’t predict dehalogenation spectrum (pangenome) • We are presently using proteomics to define which Rdhs are expressed on different substrates • We are also trying to grow cultures on MCB Acknowledgements • Mixed culture • Pure culture Dehalococcoides Dehalococcoides • Prof. Jim Gossett (Cornell) • Ivonne Nijenhuis • Amy Carroll • Dave Freedman • Rekha Seshadri (TIGR) • Tom Distefano • Donna Fennell • Prof Lorenz Adrian (Berlin) • Jennifer Fung • Valter Tandoi • Tim Anguish • Bob Morris • Heather Fullerton • Xavier Maymó-Gattell • Prof. Ruth Richardson (Cornell) • Chlorobenzenes • Prof. Lisa Alvarez-Cohen (Berkeley) • Brian Weisenstein • Support: • Jennifer Fung • Xu Cheng • US Air Force and SERDP/ESTCP • Cornell Biotechnology Institute • Jenny Nelson • NSF Metabolic Biochemistry • Hinsby Cadillo-Quiroz • DOE Genomes to Life • Erin Mack • Cornell Biogeochemistry and • George Dang Biocomplexity Program • Michael Schwartz • DuPont