RESEARCH ARTICLE

Structure and Evolution of Chlorate Reduction Composite Transposons

Iain C. Clark,a Ryan A. Melnyk,b Anna Engelbrektson,b John D. Coatesb Department of Civil and Environmental Engineeringa and Department of Plant and Microbial Biology,b University of California, Berkeley, California, USA

ABSTRACT The genes for chlorate reduction in six bacterial strains were analyzed in order to gain insight into the metabolism. A newly isolated chlorate-reducing bacterium (Shewanella algae ACDC) and three previously isolated strains ( dechlora- tans, Pseudomonas sp. strain PK, and Dechloromarinus chlorophilus NSS) were genome sequenced and compared to published sequences (Alicycliphilus denitrificans BC plasmid pALIDE01 and Pseudomonas chloritidismutans AW-1). De novo assembly of genomes failed to join regions adjacent to genes involved in chlorate reduction, suggesting the presence of repeat regions. Using a bioinformatics approach and finishing PCRs to connect fragmented contigs, we discovered that chlorate reduction genes are flanked by insertion sequences, forming composite transposons in all four newly sequenced strains. These insertion sequences delineate regions with the potential to move horizontally and define a set of genes that may be important for chlorate reduction. In addition to core metabolic components, we have highlighted several such genes through comparative analysis and visualiza- tion. Phylogenetic analysis places within a functionally diverse clade of type II dimethyl sulfoxide (DMSO) reductases, part of a larger family of with reactivity toward chlorate. Nucleotide-level forensics of regions surrounding (cld), as well as its phylogenetic clustering in a betaproteobacterial Cld clade, indicate that cld has been mobi- lized at least once from a reducer to build chlorate respiration. IMPORTANCE Genome sequencing has identified, for the first time, chlorate reduction composite transposons. These transposons are constructed with flanking insertion sequences that differ in type and orientation between organisms, indicating that this mo- bile element has formed multiple times and is important for dissemination. Apart from core metabolic enzymes, very little is known about the genetic factors involved in chlorate reduction. Comparative analysis has identified several genes that may also be important, but the relative absence of accessory genes suggests that this mobile metabolism relies on host systems for electron transport, regulation, and synthesis. Phylogenetic analysis of Cld and ClrA provides support for the hypothesis that chlorate reduction was built multiple times from type II dimethyl sulfoxide (DMSO) reductases and cld. In at least one case, cld has been coopted from a perchlorate reduction island for this purpose. This work is a significant step toward understanding the genetics and evolution of chlorate reduction.

Received 18 May 2013 Accepted 15 July 2013 Published 6 August 2013 Citation Clark IC, Melnyk RA, Engelbrektson A, Coates JD. 2013. Structure and evolution of chlorate reduction composite transposons. mBio 4(4):e00379-13. doi:10.1128/ mBio.00379-13. Invited Editor Ronald Oremland, U.S. Geological Survey Editor Douglas Capone, University of Southern California Copyright © 2013 Clark et al. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to John D. Coates, [email protected].

Ϫ Ϫ erchlorate (ClO4 ) and chlorate (ClO3 ) have natural and perchlorate and chlorate (9), while the chlorate reductase (Cl- Panthropogenic sources. While recent evidence suggests that rABC) can reduce only the latter (10, 11). Chlorite is an obligate these compounds are formed in the atmosphere (1, 2), contami- intermediate in both pathways and is detoxified by the chlorite nation of drinking water is often a result of human activity. Chlo- dismutase (Cld), which produces chloride and molecular oxygen rate has been used as an herbicide and defoliant and as a bleaching that is respired. The chlorate reductases of , agent in the paper industry; perchlorate is a solid oxidant found in Pseudomonas chloritidismutans AW-1, and Pseudomonas sp. strain ␣ ␤ ␥ flares, explosives, and propellants (3). Bacterial remediation of PDA have been purified as soluble heterotrimers ( 1 1 1) (10– contaminated water is a viable treatment option, which has 12). ClrABC in I. dechloratans and that in PDA are probably spurred both applied (4) and basic (5) science research. Perchlo- periplasmic, and while fractionation experiments support a cyto- rate and chlorate are respired by dissimilatory perchlorate- plasmic ClrABC in AW-1, a twin-arginine signal motif is pre- reducing (PRB) and chlorate-reducing bacteria (CRB), dicted (13), suggesting periplasmic localization. By comparison to respectively, almost all of which are (6), with a few structurally characterized enzymes EbdABC (14) and NarGHI exceptions (7, 8) (see Fig. S1 in the supplemental material). While (15), the ␣ subunit is predicted to contain a bis(molybdopterin all PRB isolated are also chlorate reducers, the reverse is not true. guanine dinucleotide)- cofactor and a [4Fe-4S] The distinction is at least partly a result of the specificity of the cluster coordinated by one histidine and three cysteines (10). The terminal reductase; the perchlorate reductase (PcrAB) can reduce ␤ subunit is predicted to contain four Fe-S clusters that form an

July/August 2013 Volume 4 Issue 4 e00379-13 ® mbio.asm.org 1 Clark et al. electron transfer pathway between a cytochrome b in the ␥ sub- Shewanella algae ACDC and Dechloromarinus chlorophilus unit (16) and the Fe-S cluster in the ␣ subunit. The ␦ subunit is NSS. ACDC was isolated from the same marine sediment enrich- homologous to NarJ and most likely participates in proper inser- ment from which NSS was previously isolated (6). Details of the tion of the molybdenum cofactor but is not part of the active physiology of ACDC will be published elsewhere. After improved (17, 18). assembly and annotation (Fig. 1A), two ISPpu12 insertion se- To date, three CRB (Ideonella dechloratans, P. chloritidismutans quences flanked clrABDC and cld (Fig. 1A). Five copies of this AW-1, and Alicycliphilus denitrificans BC) have had their genes for composite transposon were found in ACDC: two chromosomal chlorate reduction sequenced. As part of our continuing effort to insertions and one on a multicopy (~3) plasmid (Fig. 1D). The understand the genomics of chloroxyanion respiration by bacte- insertion sequences flanking the plasmid-borne copy are iso- ria, genome sequences were completed for four CRB: Ideonella forms, designated ISPpu12a and ISPpu12b, and contain 68 single dechloratans, Pseudomonas sp. strain PK, Dechloromarinus chloro- nucleotide polymorphisms in the transposase gene, as well as a philus NSS, and the newly isolated Shewanella algae ACDC. How- small deletion of 12 bp in czcD (Fig. 1B). The chromosomal com- ever, after de novo assembly, the genes for chlorate reduction were posite transposons contain matching flanking copies of ISPpu12b. found on small contigs, with no information about neighboring The reason for the discrepancy between plasmid and chromo- regions. Short reads from next-generation sequencing (NGS) somal insertion sequence isoforms is unknown but was the cause technologies often do not unambiguously connect regions sur- for negligible assembly of this region (Fig. 1C and D). Imperfect rounding repeats, and as a result, assemblers produce many con- 24-bp inverted repeats are located at the boundaries of both tigs instead of contiguous finished genomes. A fragmented ge- ISPpu12 isoforms and are identical to those reported for ISPpu12 nome may not be a research impediment if the genes of interest are in the Pseudomonas putida plasmid pWW0 (Fig. 1E). on a large contig. This was the case in our recent comparative Insertion of the composite transposon into the ACDC chro- analysis of genes for perchlorate reduction, in which conserved mosome occurred in the open reading frames of nrfA synteny and evidence of horizontal gene transfer led to the iden- (ammonium-forming nitrite reductase) and barA (hybrid sensor tification of a perchlorate reduction genomic island (PRI) that kinase). This finding is supported by conserved synteny surround- contained metabolic, regulatory, and electron transport chain ing nrfA and barA in closely related species, by the continuous components (19). coverage of mapped reads over the insertions, and by PCRs con- Using a bioinformatics approach, contigs containing genes for necting nrfA and barA to the interior of the composite transposon chlorate reduction (clrABDC and cld) were extended, connected (Fig. 1C). Presumably due to the insertion in nrfA, ACDC reduces to neighbors, and confirmed with finishing PCRs. In all four newly nitrate to nitrite but does not reduce nitrite to ammonium (I. C. sequenced chlorate-reducing bacteria, these genes are flanked by Clark, unpublished data). Duplications of 8 bp found surround- insertion sequences, forming composite transposons. Insertion ing composite transposon insertions in nrfA, barA, and the plas- sequences are small, autonomous, mobile genetic elements that mid (Fig. 1E) have been previously observed in other ISPpu12 encode a transposase and accessory genes enclosed by terminal elements following transposition (25). repeat sequences (20). Insertion sequences are widely recognized Assembly of the NSS genome produced contigs that matched as drivers of bacterial evolution; they are often present in large ACDC’s plasmid with 99.9% nucleotide identity (Fig. 1D). The numbers and serve as sites for homologous recombination and plasmid contains tra and trb gene clusters putatively involved in therefore chromosomal rearrangements, plasmid integration, and self-transmissibility. Unlike in ACDC, coverage of the composite deletions (21). As composite transposons, they construct and dis- transposon in NSS is consistent with it being only plasmid borne. seminate novel metabolic pathways, including those involved in However, NSS does have a full and partial copy of ISPpu12b in its the catabolic degradation of substituted aromatics and xenobiot- chromosome (Fig. 1F). This is not surprising, considering that ics (22, 23). The identification of chlorate reduction composite ISPpu12 transposes independently, in multiple copies (25), and transposons is an important step toward understanding the for- functions in alpha-, beta-, and gammaproteobacteria (26). mation and horizontal transfer of this metabolism. Pseudomonas chloritidismutans AW-1 and Pseudomonas sp. strain PK. P. chloritidismutans AW-1’s published sequence (Gen- Bank accession no. GQ919187) contains cld and a cytochrome c, RESULTS which are separated from clrABDC by an insertion sequence. Sim- After de novo assembly, genome sequences of PK, NSS, and ACDC ilar architecture is observed in PK, which contains an insertion contained cld on a contig with a gene cluster encoding a dimethyl sequence in the exact same location (Fig. 2C). However, despite sulfoxide (DMSO) reductase family type II enzyme. This was in- their identical positions, PK’s insertion sequence is ISPst12, while ferred to be the chlorate reductase based on its proximity to cld AW-1’s insertion sequence is ISPa16 (Fig. 2C). In both genomes, and phylogenetic clustering with other putative chlorate reducta- these insertion sequences have formed tandem duplications of ses (10, 11). Since no genetic system is available, the physiological TTAG. Insertion at CTAG has been previously observed for both chlorate reductase remains to be validated experimentally in all elements (27). In PK, ISPpu12 insertion sequences again flank the CRB. For newly sequenced chlorate reducers, the larger genomic genes for chlorate reduction (Fig. 2A). The ISPpu12 insertion se- context of the chlorate reductase was obscured by fragmented quences in PK are 99% identical to each other, but one assembly of NGS reads, which produced clrABDC and cld on con- (ISPpu12d) contains a notable 12-bp deletion in czcD. Compared tigs of less than 16 kb. To resolve neighboring genes, assemblies of to ACDC and NSS, PK’s ISPpu12 sequences are in the opposite I. dechloratans, PK, ACDC, and NSS were improved by mapping orientation with respect to cld and clrABDC, indicating that the reads to the ends of contigs, extending them computationally composite transposons found in these bacteria were formed inde- (24), and predicting links to other contigs (see Materials and pendently. Mapping coverage over this region (Fig. 2C) shows Methods). that the genes for chlorate reduction are present in one copy

2 ® mbio.asm.org July/August 2013 Volume 4 Issue 4 e00379-13 n Sp1b C opst rnpsnisrin in insertions transposon Composite (C) of ISPpu12b. PRI and the with synteny including NSS, and ACDC I 1 FIG uyAgs 03Vlm su e00379-13 4 Issue 4 Volume 2013 July/August n S r eryietcladcnanaclrt euto opst rnpsnflne yioom Sp1aadIPu2.TA n Mrepresent RM and T/AT ISPpu12b. and ISPpu12a isoforms ISSal by and NSS. ISPpu12a/b flanked in from transposon locations duplications and chromosomal composite repeats several inverted reduction in of chlorate Sequences inserted (E) a respectively. systems, contain modification restriction and and identical toxin/antitoxin nearly are NSS and C A D T/AT rpoN A. suillum

oprsno ee novdi hoaerdcini CCadNS A tutr n noaino hoaerdcincmoietasoosin transposons composite reduction chlorate of annotation and Structure (A) NSS. and ACDC in reduction chlorate in involved genes of Comparison RM ISPpu12a IRL -1 1 PS 0.89

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0.75 traO-C Lipoprotein signal Coverage(bp)

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ISPpu12b ISPpu12b klcA 123 glycosyl ltransferase

126

R 2IR2IL- IRR-1 IRL -1 IRR-2 IRL -2 repA 78 family protein 97 ISSal1

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NSS chromosomalISPpu12insertions lipoprotein signal peptide Duplication surroundingISSal1 123 126 heavy metal efflux

AAAATAGC -ISSal1 contig 22 - ISPpu12acontig51ISPu12b - ISPpu12bcontig51ISPu12b - ISPpu12bcontig51ISPu12b 78 contig 19 ISSal1 invertedrepeat 97 merR regulator hoaeRdcinCmoieTransposons Composite Reduction Chlorate P IS

51 ISPpu12b nrfA B contig 12 contig 8 ISPa38-like 123 ISPpu12b 126 ISPpu12a 78 ® 97 mbio.asm.org .()ISPpu12b (F) 1.

7 CAAAAATC... CTTTGATG... CAAATGGT... 3 Clark et al.

Pseudomonas sp. PK A IRL-1 IRR-1 IRL-2 IRR-2 IRL-1 IRR-1 B ISPpu12c ISPst12 ISPpu12d PK ISPpu12 inverted repeats IRL-1 GGGGTAAGCGGATTAAATGGTTGAT |||||| |||||| |||||||||| rev comp IRR-1 GGGGTATACGGATTTAATGGTTGAT PK ISPst12 inverted repeats IRL-2 CAGGCCGTTGAAAAA cld clrA clrB clrC clrD ||||||||||||||| rev comp IRR-2 CAGGCCGTTGAAAAA regulator regulator Transposase Transposase merR merR AW-1 ISPa16 inverted repeats

cytochrome c553 IRL GGATGGTCTGAAAAAGACTT Heavy metal efflux Heavy metal efflux glycosyl family protein

ATPase ParA/MinD ATPase ||| |||||||| || | | rev comp IRR GGAAGGTCTGAAGTAGCCCT Lipoprotein signal peptide Lipoprotein signal peptide

P. stutzeri ATCC 17588 C 10 kb D PK chromosomal insertions

P. stutzeri DSM 4166

P. chloritidismutans AW-1 fragment contig 3 contig 19 (GenBank: GQ919187)

P. chloritidismutans AW-1 (this study)

contig 7 contig 36 Pseudomonas sp. PK

300 PK coverage (bp) 274 200 contig 20 ISPpu12 contig 15 100 102 PK duplications surrounding ISPpu12 PK PCR reactions contig 3 - CGAATTTC - ISPpu12 - CGAATTTC - contig 19 PK NGS contigs contig 7 - ACGAAAAA - ISPpu12 - ACGAAAAA - contig 36 contig 20 - CAAAAAAG - ISPpu12 - CAAAAAAG - contig 15 28 6062 46 3460 62 46 18 FIG 2 Comparison of genes involved in chlorate reduction in AW-1 and PK. (A) Structure and annotation of the chlorate reduction composite transposon in PK. (B) Sequences of inverted repeats surrounding ISPpu12, ISPst12, and ISPa16. (C) Conserved synteny and genomic location of chlorate reduction genes in PK and AW-1. (D) Location of three ISPpu12 copies in PK’s chromosome and sequences of duplications created upon insertion.

(~chromosomal coverage) but that the ISPpu12d element is pres- tans presented here expands previous sequencing efforts, identi- ent in at least three other locations. We predicted these locations fying an arsR regulator, a partial methionine sulfoxide reductase and confirmed them with PCR. These insertions appear to have gene (msrA), and a cupin 2 domain gene, as well as an insertion generated duplications (Fig. 2D). In contrast, duplications at the sequence that we designate ISIde2 (Fig. 3A). outer edges of the composite transposon or surrounding individ- Most interesting, however, is the presence of matching inser- ual insertion sequences making up the composite transposon were tion sequences that flank this entire region in I. dechloratans and not found. form a composite transposon (Fig. 3A). The insertion sequences The left ISPpu12 insertion sequence was absent in the previ- are 99% identical to ISAav1 (27) from Acidovorax citrulli AAC00-1 ously published AW-1 sequence, but the chlorate reduction genes (GenBank accession no. AF086815), which forms a composite are in the same location in the chromosome and in the middle of transposon with s-triazine ring cleavage genes, and 99% identical a conserved region in other Pseudomonas sequences (Fig. 2C). to part of an insertion sequence on Pseudomonas sp. strain ADP’s This allowed PCR primers to be designed outward from clrC to a plasmid pADP-1 (RefSeq accession no. NC_004956), which con- conserved part of ompG in order to finish the rest of AW-1’s se- tains genes for atrazine degradation. The ISAav1 isoform in I. de- quence in this region. In contrast to PK, which contains two copies chloratans has perfect 26-bp inverted repeats (Fig. 3B). Due to the of ISPpu12, AW-1 contains only one copy and is therefore not a relatively poor quality of the assembled genome (see Table S1 in composite transposon (Fig. 2C). the supplemental material) and highly variable coverage (Fig. 3A), I. dechloratans and A. denitrificans BC. Ideonella dechlora- it was not possible to completely resolve the location(s) of the tans was one of the first characterized chlorate-reducing bacteria composite transposon or deduce copy number. However, the (28), and several fragments of genes putatively involved in chlor- presence of high-coverage areas (Ͼ200- versus 96-bp average cov- ate reduction have been sequenced, including cld and a conserved erage) suggests that it may exist in more than one copy. hypothetical gene (GenBank accession no. AJ296077.1) (29), a Alicycliphilus denitrificans BC was isolated as a benzene- cytochrome c and molybdopterin-guanine dinucleotide biosyn- degrading chlorate reducer and has been genome sequenced (31). thesis gene (GenBank accession no. EU768872.1) (30), and clr- Genes for chlorate reduction are located on a 119,718-bp plasmid, ABDC with the insertion sequence ISIde1 (GenBank accession no. pALIDE01 (GenBank accession no. CP002450). Apart from the AJ566363.1) (10) (Fig. 3A). The genome sequence of I. dechlora- absence of ISIde1, the region within the I. dechloratans composite

4 ® mbio.asm.org July/August 2013 Volume 4 Issue 4 e00379-13 Chlorate Reduction Composite Transposons

I. dechloratans IRLIRR IRL-2 IRR-2 IRL-1 IRR-1 IRL IRR ISIde1 duplications A ISAav1 ISIde2 ISIde1 ISAav1 B AAAATA - ISIde1 - AAAATA

ISAav1 inverted repeats IRL GTTGAATCACGAGCAAAACTGAGCCA IstB domain IstB domain Transposase Integrase family Cupin 2 domain msrA Hypothetical protein Transposase Cytochrome- MGD biosynthesis arsR Hypothetical protein IstB domain Transposase Bacteriophage protein Hypothetical protein Integrase family cld clrA clrB clrD clrC |||||||||||||||||||||||||| rev comp IRR GTTGAATCACGAGCAAAACTGAGCCA regulator framgment

ISIde1 inverted repeats c IRL-1 TGGCTCTATGCCGTTTCCAGTGGAAATTGACTTCTCAACAAACATGAC ||||||| |||||||||||||||||||||||||||||||||||||||| rev comp IRR-1 TGGCTCTGTGCCGTTTCCAGTGGAAATTGACTTCTCAACAAACATGAC Coverage (bp) ISIde2 inverted repeats IRL-2 TGCGTATTCCAGACCAAACTGGACACCGATTCCACGGCAAACTGGACAGT PCR reactions ||||||||||| ||||||||||| | | |||||||||||||||||||| NGS contigs 445 71 835 116 510 445 1025 rev comp IRR-2 TGCGTATTCCACG-CAAACTGGACAGCCAGTCCACGGCAAACTGGACAGT Previously sequenced AJ296077AJ566363 EU768872

ISAde1 inverted repeats ISIde1 clrA D IRL-4 CGGAATTTTCGGCGGTTCCGGCTTAGAACCCC C ||||| |||||||||||| || |||||||||| ISIde2 rev comp IRR-4 CGGAAATTTCGGCGGTTCTGGCTTAGAACCCC ISAav1 ISAav1

IRL-4Alide_4634 Alide_4607 Alide_4635 Alide_4606 Alide_4636 Alide_4605 Alide_4637 Alide_4604 Alide_4638 Alide_4639 Alide_4602 IRR-4 pALIDE01 (ISAde1)

qacF Delftia sp. pKV29 (JN648090)

Alide_461

A Alid Alide_4616 Alide_4614 Alide_4613 Alide_4613

I. dechloratans Alid Alide_4 Ali lide_ Al

BC Alide Alide_4610 TetR ide_4611 ide_4611 e_4617 Alide_46 de_4620 e_4619

4 _4621 6 612

1 IncP-1 beta pB8 (AJ863570)

5

8

09 A lide HopAF1 _46 2 IRL-4 A. denitrificans 2 ISA ISPsy30 de1 1 -4 _460 IRR e Alid Alide_4633 IS1071 inverted repeats IRL-5 GGGGTCCCCTCGTTTTCAGTGCAATAAGTGACGGTACG... IR R -4 |||||| ||||||||||||||||||||||||||||||| rev comp IRR-5 GGGGTCTCCTCGTTTTCAGTGCAATAAGTGACGGTACG...

IRL-5 IRR-5 ISAde1 IR L-4 IS1071 A lide_4641 Alide_4633/Alide_4601 A. denitrificans BC Alide_4622 pALIDE01 (119,718 bp) Alide_4641

FIG 3 Comparison of genes for chlorate reduction in I. dechloratans and A. denitrificans BC. (A) Structure, annotation, and finishing of the chlorate reduction composite transposon in I. dechloratans. (B) Sequences of inverted repeats and duplications associated with insertion sequences in I. dechloratans. (C) Compar- ison of syntenic regions in I. dechloratans and A. denitrificans BC pALIDE01. (D) Structure of the ISAde1 transposon containing a cld-like gene and cupin 2 domain gene. transposon is remarkably similar to a region on A. denitrificans BC into the evolution of chlorate reduction. Within the larger family pALIDE01, with just 14 single nucleotide polymorphisms in of type II DMSO reductases, ClrA resides in a functionally diverse 12,546 aligned bases. A 6-bp sequence (AAAATA) that was most clade that includes ethylbenzene dehydrogenase (EbdA), dimeth- likely duplicated during transposition of ISIde1 in I. dechloratans ylsulfide dehydrogenase (DdhA), and selenate reductase (SerA) is present only once on pALIDE01. The syntenic region (Fig. 3C, (Fig. 4A). ClrA proteins from I. dechloratans and A. denitrificans gray) includes everything carried within the I. dechloratans com- BC group with SerA from Thauera selenatis AX, while ClrA pro- posite transposon, except for the ArsR family regulator and a small teins from PK, AW-1, ACDC, and NSS group with DdhA from hypothetical gene. Rhodovulum sulfidophilum (Fig. 4B). In addition to the canonical The A. denitrificans plasmid pALIDE01 shows a history of ␣␤␦␥ organization of genes within this clade, two additional genes transposition and recombination in the region surrounding chlo- appear to be conserved in the subclade containing PK, AW-1, rate reduction genes (Fig. 3C). An insertion sequence, 99.8% iden- ACDC, and NSS. The first gene is annotated as an ATPase and tical to IS1071 after reconstruction, is fragmented and found in contains MipZ and ParA domains, and the second gene is anno- multiple copies (Fig. 3D). The plasmid contains two copies of an tated as a glycosyl transferase (Fig. 4B). The role of these genes in ISPsy30-like transposon, which have variable 5= regions with a relation to their neighboring reductase is unknown but worthy of conserved resolvase and transposase bound by inverted repeats further investigation. (Fig. 3D). This transposon, designated ISAde1, carries a toxin/ Cld proteins in ACDC, NSS, PK, and AW-1 group phyloge- antitoxin module, as well as the cld-like gene and a cupin 2 domain netically with a betaproteobacterial clade of Cld proteins from gene. ISAde1 flanks genes for chlorate reduction on pALIDE01 perchlorate reduction islands (Fig. 5A and C). In ACDC and NSS, (Fig. 3C), again forming a composite architecture, although the cld is surrounded by a napC fragment and a small 3= portion of ability of ISPsy30 transposons to mobilize large regions has not pcrD that matches the conserved gene organization of Dechlo- been demonstrated. romonas aromatica RCB and Azospira suillum PS (Fig. 1A) (19). In Phylogenetic analysis of ClrA and Cld. Four new genomes PK and AW-1, a small 3= portion of pcrD is present. This is highly expand the known sequences of ClrA and Cld and provide insight suggestive that the direction of horizontal gene transfer was from

July/August 2013 Volume 4 Issue 4 e00379-13 ® mbio.asm.org 5 Clark et al.

A NarG* (Q9P9I5)

54 NarG* (Q8ZSS1)

57

100 PcrA* (Q47CW6)

100 SerA* (Q9S1H0) 76 ClrA* (P60068) 100 100 DdhA 64 100 72 ClrA* (E3SES5)

100 EbdA Q2RH81 C7ML97 100 Q3AAD3

img:2513982058 S. selenatireducens AK4OH1 (2513258005) γ- proteobacteria 100 uniprot:Q9S1H0 SerA T. selenatis (CACR01001399) 77 β - proteobacteria B uniprot:C9E264 sp. CMS 78 img:2513981313 S. selenatireducens AK4OH1 (2513258005) γ- proteobacteria 100 uniprot:P60068 I. dechloratans 100 ClrA uniprot:E8U2K4 A. denitrificans BC (CP002450) ATPase β - proteobacteria uniprot:G2E459 T. drewsii AZ1 (PRJNA62879) 100 uniprot:Q8GPG4 DdhA (fragment) R. sulfidophilum (AF453479) 92 GH 100 uniprot:A3K6T1 αβδγ S. stellata E-37 (NZ_AAYA01000011) 100 α- proteobacteria uniprot:D0D3W5 Citreicella sp. SE45 (NZ_GG704599) 74 uniprot:E3SES5 P. chloritidismutans AW-1 (GQ919187) 70 to NarG img:2508531177 Pseudomonas sp. PK 45 100 ClrA γ- proteobacteria 100 img:2510563985 D. chlorophilus NSS 100 img:2510560151 S. algae ACDC uniprot:C0QP50 P. marina EX-H1 (NC_012440) 100 Aquificae Sulfurihydrogenibium azorense Az-Fu1 100 uniprot:C1DW86 αβγ uniprot:E4TYZ0 S. kujiense BAA-921 (NC_014762) ε-proteobacteria uniprot:Q5NZV2 A.aromaticum EbN1 (NC_006513) 100 EbdA β - proteobacteria uniprot:Q5P5I0 A.aromaticum EbN1 (NC_006513) αβγδ

FIG 4 Phylogenetic analysis of ClrA and closely related type II DMSO reductases. (A) Location of ClrA proteins within the larger type II DMSO reductase family. Asterisks signify that at least one enzyme in the clade has been shown to reduce chlorate in vitro or through genetic analysis. The black circle represents the ancestral state with proposed chlorate reductase activity. UniProt identifiers of the characterized protein are in parentheses. Conflicting biochemical data are reported in the literature for SerABC from Thauera selenatis (44, 74). (B) Phylogenetics of ClrA and closely related enzymes. Gene neighborhoods reveal two additional conserved genes. an RCB/PS-type PRI to a chlorate reduction composite trans- DISCUSSION poson. Despite a series of analogous biochemical reactions, genes for In A. denitrificans BC, I. dechloratans, and PK, an additional chlorate and perchlorate reduction have different genomic ar- cld-like gene was found unassociated with chlorate reduction chitectures and a distinct, yet intertwined, evolutionary his- genes. These encode Cld-like proteins that form a unique type II tory. Composite transposons containing chlorate reduction subclade (Fig. 5A), with exaggerated signatures of horizontal gene genes with different flanking insertion sequences (type and ori- transfer (Fig. 5B). In I. dechloratans, the cld-like gene is a passenger entation) have been identified in five chlorate-reducing bacte- on an ISPa38-like transposon, designated ISIde3. ISPa38 trans- ria. The transposon ISAde1 that surrounds cld and clr in posons contain a cupin 2 domain gene and are found in the ge- A. denitrificans BC (Fig. 3C) has not been reported to mobilize nomes of Aeromonas caviae (pFBAOT6), P. aeruginosa DK2, and genes as a composite and may not be relevant to horizontal NSS. In Burkholderia cepacia,acld-like gene appears to have re- transfer of chlorate reduction. However, ISPpu12 and ISAv1 placed a portion of the ISPa38 transposase (Fig. 5B). In A. denitri- ficans BC, the cld-like gene is colocated with a cupin 2 domain gene isoforms have been observed in a composite architecture pre- on the plasmid-borne transposon ISAde1. ISAde1 is found in Ral- viously, and the former can transpose as a composite (26). stonia pickettii 12D and 12J and in partial form in Cupriavi- Identification of 8-bp direct repeats at the outermost edges of dus metallidurans CH34 (Fig. 5B). The model tree species Populus the composite transposon in nrfA, barA, and the plasmid trichocarpa, which can reduce perchlorate and chlorate (32), con- (Fig. 1E) in ACDC strongly suggest that the entire element tains a cld-like gene that is 99.6% identical to the copy in ISAde1. transposed into these locations. These duplications probably The contig with the Populus trichocarpa cld also contains a small occurred when staggered cuts in the target site were filled in portion of the antitoxin gene from ISAde1, suggesting that it is following transposon insertion (20). This, together with the bacterial in origin (Fig. 5B). PK contains a cld-like gene and a fact that chlorate reduction composite transposons can be plas- cupin 2 domain gene that share synteny with a Pseudomo- mid borne, provides a conceivable mechanism by which the nas stutzeri A1501 region comprising phage-associated, UV resis- metabolism moves horizontally. Indeed, the 99.9% nucleotide tance genes (Fig. 5B). identity between plasmids in ACDC and NSS is indicative of

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refseq:ZP_01913751.1 Limnobacter sp. MED105 gb:CAH04648.1 Pseudomonas aeruginosa Gammaproteobacteria refseq:YP_004124080.1 Alicycliphilus denitrificans BC Betaproteobacteria A refseq:YP_002984396.1 Ralstonia pickettii 12D gb:ADD63334 Uncultured plasmid pAKD4 gb:EEE70332 Populus trichocarpa Streptophyta refseq:YP_001899051.1 Ralstonia pickettii 12J gb:ABF13199.1 Cupriavidus metallidurans CH34 refseq:YP_006965502.1 Burkholderia cepacia Betaproteobacteria img:2510552383 Ideonella dechloratans Anox B, ATCC 51718 refseq:ZP_16060981.1 Acinetobacter baumannii OIFC074 img:2508531231 Pseudomonas sp. PK refseq:YP_001173821.1 Pseudomonas stutzeri A1501 refseq:ZP_09161441.1 Marinobacter manganoxydans MnI7-9 Gammaproteobacteria gb:EGU94847 Escherichia coli MS 79-10 refseq:YP_001338852.1 Klebsiella pneumoniae MGH 78578 refseq:ZP_08308323.1 Klebsiella sp. MS 92-3 refseq:ZP_01125763.1 Nitrococcus mobilis Nb-231 refseq:ZP_10152326.1 Hydrogenophaga sp. PBC Betaproteobacteria refseq:ZP_10728314.1 Flavobacterium sp. CF136 Flavobacteriia refseq:ZP_10331624.1 Fibrisoma limi BUZ 3 Cytophagia refseq:ZP_11424577.1 Afipia clevelandensis ATCC 49720 refseq:ZP_08627255.1 Bradyrhizobiaceae bacterium SG-6C refseq:ZP_01046929.1 Nitrobacter sp. Nb-311A Alphaproteobacteria II refseq:YP_319047.1 Nitrobacter winogradskyi Nb-255 * refseq:ZP_10580941.1 Bradyrhizobium sp. YR681 refseq:YP_005606828.1 Bradyrhizobium japonicum USDA 6 img:2517556316 Methylosarcina fibrata AMLC10 Gammaproteobacteria refseq:YP_001352597.1 Janthinobacterium sp. Marseille Betaproteobacteria refseq:YP_003631388.1 Planctomyces limnophilus DSM 3776 refseq:YP_003369421.1 Pirellula staleyi DSM 6068 Planctomycetia refseq:ZP_11090713.1 Schlesneria paludicola DSM 18645 refseq:ZP_18299486.1 Rhizobium leguminosarum bv. viciae WSM1455 Alphaproteobacteria refseq:YP_001615619.1 Sorangium cellulosum So ce56 Deltaproteobacteria refseq:ZP_10722281.1 Herbaspirillum sp. CF444 Betaproteobacteria refseq:ZP_08504795.1 Methyloversatilis universalis FAM5 refseq:YP_001205457.1 Bradyrhizobium sp. ORS 278 Alphaproteobacteria refseq:NP_924112.1 Gloeobacter violaceus PCC 7421 Gloeobacteria refseq:YP_007060490.1 Synechococcus sp. PCC 6312 refseq:ZP_21052507.1 Gloeocapsa sp. PCC 73106 refseq:YP_007318361.1 Chamaesiphon minutus PCC 6605 Chroococcales refseq:YP_002482168.1 Cyanothece sp. PCC 7425 img:2509800733 Leptolyngbya sp. PCC 6306 refseq:YP_007157592.1 Anabaena cylindrica PCC 7122 Nostocales img:2513983006 Sedimenticola selenatireducens AK4OH1 Gammaproteobacteria gb:AAM92878.1 Dechloromonas agitata CKB Betaproteobacteria * img:2508503837 Dechlorobacter hydrogenophilus LT1 img:2506660333 Magnetospirillum bellicus VDY gb:AAT07043 Magnetospirillum sp. WD Alphaproteobacteria gb:AAT07045 Dechlorospirillum sp. DB gb:CAC14884.1 Ideonella dechloratans * refseq:YP_004124061.1 Alicycliphilus denitrificans BC refseq:YP_005026408.1 Azospira suillum PS Betaproteobacteria * gb:ACA21505.1 Dechloromonas hortensis refseq:YP_285781.1 Dechloromonas aromatica RCB I * gb:ADN79129.1 Dechloromonas sp. Miss R gb:ACA21503.1 Pseudomonas chloritidismutans AW-1 * img:2510560149 Shewanella algae ACDC img:2510563983 Dechloromarinus chlorophilus NSS Gammaproteobacteria gb:AAT07040.1 Pseudomonas sp. PK img:2013665516 Crenothrix polyspora refseq:YP_003797063.1 Candidatus Nitrospira defluvii Nitrospirales * refseq:NP_147071.2 Aeropyrum pernix K1 Thermoprotei refseq:YP_254735.1 Sulfolobus acidocaldarius DSM 639 Thermoprotei refseq:NP_393983.1 Thermoplasma acidophilum DSM 1728 Thermoplasmata

cld cupin IS1071 B YP_004124080.1 Alicycliphilus denitrificans BC YP_002984396.1 Ralstonia pickettii 12D ADD63334 Uncultured plasmid pAKD4 copB copH ISPsy30-like YP_001899051.1 Ralstonia pickettii 12J EEE70332 Populus trichocarpa merD merA merP merT merR IS1071 ABF13199.1 Cupriavidus metallidurans CH34 pMOL28 YP_006965502.1 Burkholderia cepacia ISPa38-like 2510552383 Ideonella dechloratans ZP_16060981.1 Acinetobacter baumannii OIFC074 IS4321 putative Tn 2508531231 impB rulA Pseudomonas sp. PK YP_001173821.1 Pseudomonas stutzeri A1501 ZP_09161441.1 Marinobacter manganoxydans MnI7-9 EGU94847 Escherichia coli MS 79-10 YP_001338852.1 Klebsiella pneumoniae MGH 78578 pKPN5 IS26 IS26 ZP_08308323.1 Klebsiella sp. MS 92-3

YP_005026408.1 Azospira suillum PS C ACA21505.1 Dechloromonas hortensis YP_285781.1 Dechloromonas aromatica RCB ADN79129.1 Dechloromonas sp. Miss R ACA21503.1 Pseudomonas chloritidismutans AW1 2510560149 Shewanella algae ACDC 2510563983 Dechloromarinus chlorophilus NSS AAT07040.1 Pseudomonas sp. PK

FIG 5 Phylogenetic analysis of Cld and Cld-like proteins. (A) Respiratory Cld proteins form a monophyletic clade (green) that is distinct from other Cld-like proteins found in many chlorate reducers (orange). Asterisks signify chlorite dismutase activity in vitro or through genetic analysis. Circles visually represent bootstrap support at nodes. (B) Superimposing gene neighborhoods highlights the association of cld-like genes with cupin domain genes and mobile genetic elements in a type II subclade. Cld is shown in blue at the center of the gene alignment, while the cupin 2 protein is green and usually located directly adjacent to Cld. (C) Cld proteins from chlorate-reducing strains ACDC, NSS, PK, and AW-1 group with betaproteobacterial Cld proteins from PS and RCB.

July/August 2013 Volume 4 Issue 4 e00379-13 ® mbio.asm.org 7 Clark et al. recent transfer from one to the other, which is plausible con- a hydrophobic may have precluded turnover of chlorate sidering that they were isolated from the same enrichment. (45). The most parsimonious hypothesis consistent with these The genomic architecture of chlorate reduction genes has im- phylogenetic data is that enzymes in this clade evolved from an plications for the study of this metabolism. In all of the chlorate ancestral enzyme capable of chlorate reduction. If this is correct, reducers examined, except for AW-1, which lacks the composite the key step in the evolution of chlorate reduction was the pres- structure, the location and copy number of chlorate reduction ence of a chlorite detoxification system. genes have the potential to change. The loss of the metabolism by Type I Cld proteins involved in respiratory perchlorate and homologous recombination of flanking insertion sequences is chlorate reduction are monophyletic (Fig. 5A) (46) but, based on possible and has been observed in other composite transposons incongruence between protein and species trees, are predicted to (22, 23, 33). This is expected at some frequency when cells are have undergone horizontal gene transfer (46, 47). We provide grown without chlorate and can dominate in a culture if loss of the evidence that cld found in four chlorate reduction composite metabolism is adventitious. For example, if no regulatory system transposons originated from a PRI, based on the transfer of small exists for controlling transcription under chlorate-free condi- PRI-specific regions surrounding cld, including part of pcrD tions, the presence of the composite transposon becomes a meta- (ACDC, NSS, PK, and AW-1) (Fig. 5C) and in some cases a napC bolic burden and provides a selective advantage to cells that have homolog (ACDC and NSS) (Fig. 1A). Given that a PRI cld has lost or silenced the metabolism. Composite transposons can also been coopted for chlorate reduction, why has an active type II increase in copy number (Fig. 1C) and confound transcriptional chlorite dismutase (46) (Fig. 5A) yet to be associated with a respi- studies that assume a single stable locus. ratory metabolism? One barrier is the lack of a signal sequence and Insertion sequences can activate transcription of neighboring therefore cytoplasmic localization, which would not protect cells genes by providing full or partial promoters (34). In ACDC, a from chlorite produced periplasmically. Another difference is Ϫ35/Ϫ10 promoter in ISPpu12 is located upstream of clrABDC. structural: the type II Cld from Nitrobacter winogradskyi is dimeric In PK, the promoter is on the opposite strand and could poten- (48) compared to the tetrameric (49, 50) or pentameric/hexam- tially drive cld transcription. It has been suggested that this pro- eric (51, 52) type I Cld proteins, but the implications of this dif- moter is active and constitutive in P. putida (25). Insertion se- ference are unknown. It is also possible that the diversity of respi- quences are also located between cld and clr in PK, AW-1, and ratory Cld has not been fully surveyed. I. dechloratans and could serve to change transcription of sur- In addition to type I Cld, several chlorate reducers contain rounding genes. While we cannot rule out the possibility that the Cld-like proteins located elsewhere on the chromosome that form repeated localization of insertion sequences between cld and clr is a subclade within the type II lineage (Fig. 5A, orange). This sub- random, it is suggestive either of a physiological role for these clade contains a set of cld-like genes that are part of transposons insertion sequences or of the historical mechanism of colocaliza- (ISPa38-like, ISPsy30-like, and IS26 composite), or associated tion of cld with clrABDC. with phage-related genes, and are therefore presumably highly With the exception of A. denitrificans BC, most chlorate reduc- mobile (Fig. 5B). In all but Acinetobacter baumannii OIFC074, a ers are unable to reduce nitrate (AW-1 [35], ASK-1 [36], PDA cupin 2 domain gene is colocated with cld; this occurs in multiple [37], I. dechloratans [28], and NSS [unpublished data]). I. dechlo- configurations and on different transposons (Fig. 5B). Colocaliza- ratans was reported to lose the ability to reduce nitrate after culti- tion of type I cld and cupin genes also occurs in some chlorate vation on chlorate (28), and AW-1 has been reported to regain the reduction composite transposons (Fig. 3A) and PRIs, and as a ability to denitrify after repeated aerobic subculturing in the pres- result, we hypothesize a functional connection between these ence of nitrate (38). We have identified insertions in the nitrite genes. reductase (nrfA) in ACDC and a putative nitrate-responsive his- Comparative analysis of composite transposons identified the tidine kinase (narX) in PK but have yet to find a genetic basis for core metabolic genes and several additional genes that may be NSS’s inability to reduce nitrate, given that it contains genes for a important for chlorate reduction. These are relatively scarce com- complete denitrification pathway (nar, nir, nor, and nos). The abil- pared to those in perchlorate reduction islands, suggesting that ity of the NarGHI to reduce chlorate to toxic this mobile metabolism often relies on endogenous systems for chlorite has been widely reported (39–41). Early experiments ex- electron transport, cofactor biosynthesis, and regulation. At the ploited this to isolate chlorate-resistant cells with mutations in least, a connection to the host’s electron transport chain must be parts of the nitrate reduction pathway. This identified the nitrate established. By analogy to the closely related enzymes SerABC (53) reductase, as well as genes for Mo cofactor biosynthesis, molyb- and DdhABC (54), and as previously proposed (55), it is possible date transport, and nitrate regulation (42). Given this, it is not that ClrABC connects to a quinol oxidase via a cytochrome c. surprising that parts of nitrate reduction pathways are inactivated ACDC, NSS, PK, and AW-1 all contain a cytochrome c next to cld in some chlorate reducers, but more work is needed to fully ap- that is a possible candidate. However, in I. dechloratans, the cyto- preciate the interplay between these metabolisms. chrome c in the chlorate reduction composite transposon does not Perchlorate, despite having a high redox potential, has a large donate electrons to ClrABC in vitro, suggesting that it does not activation energy that slows inadvertent reduction by metals and provide the necessary link to the electron transport chain (30). enzymes (43). In contrast, chlorate will react abiotically with Another cytochrome c in I. dechloratans has been shown to donate Fe(II) or Mn(II), and the evolution of an enzyme that overcomes electrons to ClrABC in vitro (56), and we identified its sequence the activation barrier for chlorate was conceivably less difficult (see Fig. S2 in the supplemental material) based on the fragment than for perchlorate. Many related enzymes, including PcrA (9), reported in the literature (55). A quick search (57) for similar NarG (39, 40), and SerA (44), can reduce chlorate in vitro (Fig. 4A; proteins gave NirM, the electron donor for nitrite reductase NirS starred clades contain at least one characterized enzyme with chlo- (58), as a top hit. rate reductase activity). In the case of DdhA, evolution to oxidize Regulation of chlorate reduction with respect to other electron

8 ® mbio.asm.org July/August 2013 Volume 4 Issue 4 e00379-13 Chlorate Reduction Composite Transposons acceptors is not well understood. Lack of regulatory genes in the img ϭ IMG GeneID. The alpha subunit of chlorate reductase (ClrA) from composite transposons of ACDC, NSS, PK, AW-1, and BC implies ACDC was used as the query to search IMG and NCBI (nr database) for that regulation may be absent or controlled by a chromosomal closely related proteins (BLAST-P). After duplicates were removed, the system. In I. dechloratans, an ArsR family regulator was identified top 100 sequences were added to prealigned DMSO reductase type II that may be involved in the observed increase of cld or clrA tran- family proteins (Pfam 00384 and 17524 sequences; http://pfam.sanger- .ac.uk/, accessed 4 December 2012). CD-HIT (67) was run with a cluster- scription under anaerobic conditions in the presence of chlorate ing threshold of 0.7, the reduced protein alignment was trimmed with (59). Although this family of regulators is usually associated with Gblocks (b1 ϭ N/2ϩ1 -b2 ϭ N/2ϩ1 -b3 ϭ N/2 -b4 ϭ 2 -b5 ϭ h, where N sensing metals, evidence exists for nonmetal signals (60). is the number of sequences) (68), and the protein phylogeny was recon- The discovery of multiple, independently formed, chlorate re- structed with FastTree (69) in order to obtain an outgroup basal to the duction composite transposons advances our understanding of clades of interest. Sequences exterior to this outgroup were discarded, and this metabolism at the gene level and supports the idea that chlo- the remaining sequences were realigned and retrimmed. The best amino rate reduction is a highly mobile metabolism. Flanking insertion acid substitution model was determined with ProtTest (70) to be MT- sequences help define a set of genes that move with clrABDC and MAM. Subsequent phylogenetic analysis was performed using RAxML cld, and although some of these genes may be passengers that were with 500 bootstraps (71). The same procedure was followed when build- captured as the composite transposons formed, others are likely to ing a phylogeny for the subclade containing ClrA. Chlorite dismutase (Cld) sequences were obtained with a PSI-BLAST against NCBI (three have roles in chlorate reduction. We have identified these genes by Ϫ iterations, e ϭ 10 3) using ACDC’s type I Cld as the starting query. These comparative analysis and hypothesized functions for several. The sequences were combined with the top 100 hits from a BLAST-P search of next step is to develop a genetic system in a chlorate reducer and both IMG and NCBI (nr) that used the A. denitrificans Cld-like protein as make clean deletions to test these predictions. a query. The same workflow as above was used to select an appropriate outgroup and reconstruct the phylogeny with RAxML. 16S rRNA gene MATERIALS AND METHODS sequences from perchlorate- and chlorate-reducing bacterial isolates were Genome sequencing. Whole-genome shotgun sequencing and initial as- obtained from NCBI and newly sequenced genomes. Three nearest- sembly of four chlorate-reducing bacteria, Ideonella dechloratans, Pseu- neighbor matches were found and aligned using Ribosomal Database domonas sp. strain PK, Dechloromarinus chlorophilus NSS, and Shewanella Project tools (72). Phylogenetic analysis was performed with MrBayes 3.2 algae ACDC, were completed by Eureka Genomics (Hercules, CA). A (73) until the average standard deviation of the split frequencies was less summary of this sequencing effort is provided in Table S1, and a summary than 0.01. Posterior probabilities were estimated after discarding the first of the assembly is provided in Table S2, both in the supplemental material. 25% of samples from the cold chain. Gene calling and annotation were performed using the IMG/ER server (61). Genomes are publically available through IMG (http://img.jgi.doe- SUPPLEMENTAL MATERIAL .gov/cgi-bin/w/main.cgi). A summary of genes internal to chlorate reduc- Supplemental material for this article may be found at http://mbio.asm.org tion composite transposons is provided in Table S3. Annotation of the /lookup/suppl/doi:10.1128/mBio.00379-13/-/DCSupplemental. manually assembled plasmid from ACDC and NSS was performed using Text S1, DOCX file, 0.1 MB. RAST (62). Figure S1, EPS file, 1 MB. Improving draft assemblies. Bowtie (63) was used to map reads Figure S2, DOCX file, 0.1 MB. (Bowtie -q -n 3 -l 60 -e 200 –best) to assembled genomes. Multiple hits Figure S3, EPS file, 1 MB. were not allowed in the Bowtie mapping stage. IMAGE (24) was used to Table S1, DOCX file, 0.1 MB. extend assembled contigs when possible, and extensions were blasted Table S2, DOCX file, 0.1 MB. against all other contigs to search for potential linkages (BLASTn Table S3, XLSX file, 0.1 MB. -word_size 10) (64). It should be noted that not all extensions were valid; Table S4, XLSX file, 0.1 MB. some were later determined to be erroneous after finishing PCRs were completed. Linkages were used to create a contig graph surrounding chlo- ACKNOWLEDGMENTS rate reduction genes. The graph was further simplified using coverage to We thank Itai Sharon for insights into bacterial genome finishing. understand the relative abundance of duplicated regions, which was im- I.C.C. gratefully acknowledges the support of an NSF Graduate Stu- portant for the identification of composite transposons. This workflow is dent Fellowship. Funding for research on perchlorate and chlorate reduc- further detailed in Text S1 and Fig. S3 in the supplemental material. PCR tion in the laboratory of J.D.C. has been provided through the Energy and sequencing were performed to confirm linkages. A list of finishing Biosciences Institute, University of California, Berkeley. primers is found in Table S4. To further support predicted in silico link- ages, reads were remapped to contig junctions and coverage was deter- REFERENCES mined with samtools mpileup (65). Gene visualization. Visualization of genes was performed using py- 1. Dasgupta PK, Martinelango PK, Jackson WA, Anderson TA, Tian K, Tock RW, Rajagopalan S. 2005. The origin of naturally occurring thon scripts and the GenomeDiagram package (66). Additional modifi- perchlorate: the role of atmospheric processes. Environ. Sci. Technol. 39: cations to diagrams were performed in Adobe Illustrator. ApE was used 1569–1575. for viewing and comparing nucleotide sequences and for manually anno- 2. Balaji Rao BR, Hatzinger PB, Böhlke JK, Sturchio NC, Andraski BJ, tating features. Mapped reads and sequenced PCR products were visual- Eckardt FD, Jackson W. 2010. Natural chlorate in the environment: ized by overlaying them on assembled contigs. Horizontal bars represent application of a new IC-ESI/MS/MS method with a Cl18O3-internal stan- PCR products, with black indicating the portion that was Sanger se- dard. Environ. Sci. Technol. 44:8429–8434. quenced. Inverted repeats are shown as black vertical lines at the outer 3. Aziz CE, Hatzinger PB. 2009. Perchlorate sources, source identification edges of insertion sequences. Syntenic regions of high nucleotide identity and analytical methods, p -55–78. In Stroo HF, Ward CH (ed), In situ are highlighted in gray. bioremediation of perchlorate in groundwater. Springer Verlag, New York, NY. Phylogenetics. Phylogenetics was performed using a compute cluster 4. Hatzinger PB. 2005. Perchlorate biodegradation for water treatment. En- running CentOS 5.6 at UC Berkeley’s QB3 Computational Genomics Re- viron. Sci. Technol. 39:239A–247A. source Laboratory. Proteins are labeled in figures with an identification 5. Coates JD, Achenbach LA. 2004. Microbial perchlorate reduction: code reflecting their source: uniprot ϭ UniProtID, refseq ϭ NCBI RefSeq rocket-fuelled metabolism. Nat. Rev. Microbiol. 2:569–580. ID, gi ϭ NCBI GI number, gb ϭ GenBank locus or accession number, and 6. Coates JD, Michaelidou U, Bruce RA, O’Connor SM, Crespi JN, Achen-

July/August 2013 Volume 4 Issue 4 e00379-13 ® mbio.asm.org 9 Clark et al.

bach LA. 1999. Ubiquity and diversity of dissimilatory (per)chlorate- capable of growing anaerobically with chlorate as an electron acceptor. reducing bacteria. Appl. Environ. Microbiol. 65:5234–5241. Syst. Appl. Microbiol. 17:58–64. 7. Balk M, Mehboob F, van Gelder AH, Rijpstra WI, Damsté JS, Stams AJ. 29. Thorell HD, Karlsson J, Portelius E, Nilsson T. 2002. Cloning, charac- 2010. (Per)chlorate reduction by an acetogenic bacterium, Sporomusa sp., terisation, and expression of a novel gene encoding chlorite dismutase isolated from an underground gas storage. Appl. Microbiol. Biotechnol. from Ideonella dechloratans. Biochim. Biophys. Acta 1577:445–451. 88:595–603. 30. Bohlin J, Bäcklund AS, Gustavsson N, Wahlberg S, Nilsson T. 2010. 8. Balk M, van Gelder T, Weelink SA, Stams AJ. 2008. (Per)chlorate Characterization of a cytochrome c gene located at the gene cluster for reduction by the thermophilic bacterium Moorella perchloratireducens sp. chlorate respiration in Ideonella dechloratans. Microbiol. Res. 165: nov., isolated from underground gas storage. Appl. Environ. Microbiol. 450–457. 74:403–409. 31. Oosterkamp MJ, Veuskens T, Plugge CM, Langenhoff AA, Gerritse J, 9. Kengen SW, Rikken GB, Hagen WR, van Ginkel CG, Stams AJ. 1999. Van Berkel WJ, Pieper DH, Junca H, Goodwin LA, Daligault HE, Bruce Purification and characterization of (per)chlorate reductase from the DC, Detter JC, Tapia R, Han CS, Land ML, Hauser LJ, Smidt H, Stams chlorate-respiring strain GR-1. J. Bacteriol. 181:6706–6711. AJ. 2011. Genome sequences of Alicycliphilus denitrificans strains BC and 10. Thorell HD, Stenklo K, Karlsson J, Nilsson T. 2003. A gene cluster for K601T. J. Bacteriol. 193:5028–5029. chlorate metabolism in Ideonella dechloratans. Appl. Environ. Microbiol. 32. Van Aken B, Schnoor JL. 2002. Evidence of perchlorate (ClO4-) reduc- 69:5585–5592. tion in plant tissues (poplar tree) using radio-labeled 36ClO4. Environ. 11. Wolterink AF, Schiltz E, Hagedoorn PL, Hagen WR, Kengen SW, Stams Sci. Technol. 36:2783–2788. AJ. 2003. Characterization of the chlorate reductase from Pseudomonas 33. Meulien P, Downing RG, Broda P. 1981. Excision of the 40kb segment of chloritidismutans. J. Bacteriol. 185:3210–3213. the TOL plasmid from Pseudomonas putida mt-2 involves direct repeats. 12. Steinberg LM, Trimble JJ, Logan BE. 2005. Enzymes responsible for Mol. Gen. Genet. 184:97–101. chlorate reduction by Pseudomonas sp. are different from those used for 34. Charlier D, Piette J, Glansdorff N. 1982. IS3 can function as a mobile perchlorate reduction by Azospira sp. FEMS Microbiol. Lett. 247:153–159. promoter in E. coli. Nucleic Acids Res. 10:5935–5948. 13. Bendtsen JD, Nielsen H, Widdick D, Palmer T, Brunak S. 2005. Pre- 35. Wolterink AF, Jonker AB, Kengen SW, Stams AJ. 2002. Pseudomonas diction of twin-arginine signal peptides. BMC Bioinformatics 6:167. doi: chloritidismutans sp. nov., A non-denitrifying, chlorate-reducing bacte- 10.1186/1471-2105-6-167. rium. Int. J. Syst. Evol. Microbiol. 52:2183–2190. 14. Kloer DP, Hagel C, Heider J, Schulz GE. 2006. Crystal structure of 36. Wolterink A, Kim S, Muusse M, Kim IS, Roholl PJ, van Ginkel CG, ethylbenzene dehydrogenase from Aromatoleum aromaticum. Structure Stams AJ, Kengen SW. 2005. Dechloromonas hortensis sp. nov. and strain 14:1377–1388. ASK-1, two novel (per)chlorate-reducing bacteria, and taxonomic de- 15. Bertero MG, Rothery RA, Palak M, Hou C, Lim D, Blasco F, Weiner JH, scription of strain GR-1. Int. J. Syst. Evol. Microbiol. 55:2063–2068. Strynadka NC. 2003. Insights into the respiratory electron transfer path- 37. Xu J, Trimble JJ, Steinberg L, Logan BE. 2004. Chlorate and nitrate way from the structure of nitrate reductase A. Nat. Struct. Biol. 10: reduction pathways are separately induced in the perchlorate-respiring 681–687. bacterium Dechlorosoma sp. KJ and the chlorate-respiring bacterium 16. Karlsson J, Nilsson T. 2005. The C subunit of Ideonella dechloratans Pseudomonas sp. PDA. Water Res. 38:673–680. chlorate reductase: expression, purification, refolding, and heme reconsti- 38. Cladera AM, García-Valdés E, Lalucat J. 2006. Genotype versus pheno- tution. Protein Expr. Purif. 41:306–312. type in the circumscription of bacterial species: the case of Pseudomonas 17. Blasco F, Dos Santos P, Magalon A, Frixon C, Guigliarelli B, Santini CL, stutzeri and Pseudomonas chloritidismutans. Arch. Microbiol. 184: Giordano G. 1998. NarJ is a specific chaperone required for molybdenum 353–361. cofactor assembly in nitrate reductase A of Escherichia coli. Mol. Micro- 39. Afshar S, Johnson E, de Vries S, Schröder I. 2001. Properties of a biol. 28:435–447. thermostable nitrate reductase from the hyperthermophilic archaeon Py- 18. Vergnes A, Pommier J, Toci R, Blasco F, Giordano G, Magalon A. 2006. robaculum aerophilum. J. Bacteriol. 183:5491–5495. NarJ chaperone binds on two distinct sites of the aponitrate reductase of 40. Yoshimatsu K, Sakurai T, Fujiwara T. 2000. Purification and character- Escherichia coli to coordinate molybdenum cofactor insertion and assem- ization of dissimilatory nitrate reductase from a denitrifying halophilic bly. J. Biol. Chem. 281:2170–2176. archaeon, Haloarcula marismortui. FEBS Lett. 470:216–220. 19. Melnyk RA, Engelbrektson A, Clark IC, Carlson HK, Byrne-Bailey K, 41. Bell LC, Richardson DJ, Ferguson SJ. 2001. Periplasmic and membrane- Coates JD. 2011. Identification of a perchlorate reduction genomic island bound respiratory nitrate reductases in Thiosphaera pantotropha. The with novel regulatory and metabolic genes. Appl. Environ. Microbiol. 77: periplasmic enzyme catalyzes the first step in aerobic denitrification. FEBS 7401–7404. Lett. 265:85–87. 20. Mahillon J, Chandler M. 1998. Insertion sequences. Microbiol. Mol. Biol. 42. Stewart V. 1988. Nitrate respiration in relation to facultative metabolism Rev. 62:725–774. in enterobacteria. Microbiol. Rev. 52:190–232. 21. Habermann P, Starlinger P. 1982. Bidirectional deletions associated with 43. Urbansky ET. 1998. Perchlorate chemistry: implications for analysis and IS4. Mol. Gen. Genet. 185:216–222. remediation. Bioremediat. J. 2:81–85. 22. Slater JH, Weightman AJ, Hall BG. 1985. Dehalogenase genes of Pseu- 44. Martinez-Espinosa RM, Dridge EJ, Bonete MJ, Butt JN, Butler CS, domonas putida PP3 on chromosomally located transposable elements. Sargent F, Richardson DJ. 2007. Look on the positive side! The orienta- Mol. Biol. Evol. 2:557–567. tion, identification and bioenergetics of archaeal membrane-bound ni- 23. Nakatsu C, Ng J, Singh R, Straus N, Wyndham C. 1991. Chlorobenzoate trate reductases. FEMS Microbiol. Lett. 276:129–139. catabolic transposon Tn5271 is a composite class I element with flanking 45. Hanlon SP, Toh TH, Solomon PS, Holt RA, McEwan AG. 2004. class II insertion sequences. Proc. Natl. Acad. Sci. USA 88:8312–8316. Dimethylsulfide: acceptor from Rhodobacter sulfidophilus. 24. Tsai IJ, Otto TD, Berriman M. 2010. Improving draft assemblies by Eur. J. Biochem. 239:391–396. iterative mapping and assembly of short reads to eliminate gaps. Genome 46. Maixner F, Wagner M, Lücker S, Pelletier E, Schmitz-Esser S, Hace K, Biol. 11:R41. doi: 10.1186/gb-2010-11-4-r41. Spieck E, Konrat R, Le Paslier D, Daims H. 2008. Environmental 25. Williams PA, Jones RM, Shaw LE. 2002. A third transposable element, genomics reveals a functional chlorite dismutase in the nitrite oxidizing ISPpu12, from the toluene-xylene catabolic plasmid pWW0 of Pseudomo- bacterium “Candidatus Nitrospira defluvii.” Environ. Microbiol. 10: nas putida mt-2. J. Bacteriol. 184:6572–6580. 3043–3056. 26. Weightman AJ, Topping AW, Hill KE, Lee LL, Sakai K, Slater JH, 47. Bender KS, Rice MR, Fugate WH, Coates JD, Achenbach LA. 2004. Thomas AW. 2002. Transposition of DEH, a broad-host-range trans- Metabolic primers for detection of (per)chlorate-reducing bacteria in the poson flanked by ISPpu12, in Pseudomonas putida is associated with environment and phylogenetic analysis of cld gene sequences. Appl. En- genomic rearrangements and dehalogenase gene silencing. J. Bacteriol. viron. Microbiol. 70:5651–5658. 184:6581–6591. 48. Mlynek G, Sjöblom B, Kostan J, Füreder S, Maixner F, Gysel K, 27. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. 2006. Furtmüller PG, Obinger C, Wagner M, Daims H, Djinovic´-Carugo K. ISfinder: the reference centre for bacterial insertion sequences. Nucleic 2011. Unexpected diversity of chlorite dismutases: a catalytically efficient Acids Res. 34:D32–D36. doi: 10.1093/nar/gkj014. dimeric enzyme from Nitrobacter winogradskyi. J. Bacteriol. 193: 28. Malmqvist Å, Welander T, Moore E, Ternström A, Molin G, Stenström 2408–2417. IM. 1994. Ideonella dechloratans gen. nov., sp. nov., a new bacterium 49. Goblirsch BR, Streit BR, Dubois JL, Wilmot CM. 2010. Structural

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features promoting dioxygen production by Dechloromonas aromatica I, Mavromatis K, Ivanova NN, Kyrpides NC. 2011. IMG: the integrated chlorite dismutase. J. Biol. Inorg. Chem. 15:879–888. microbial genomes database and comparative analysis system. Nucleic 50. Mehboob F, Wolterink AF, Vermeulen AJ, Jiang B, Hagedoorn PL, Acids Res. 40:D115–D122. doi: 10.1093/nar/gkr1044. Stams AJ, Kengen SW. 2009. Purification and characterization of a chlo- 62. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, rite dismutase from Pseudomonas chloritidismutans. FEMS Microbiol. Formsma K, Gerdes S, Glass EM, Kubal M, Meyer F, Olsen GJ, Olson Lett. 293:115–121. R, Osterman AL, Overbeek RA, McNeil LK, Paarmann D, Paczian T, 51. De Geus DC, Thomassen EA, Hagedoorn PL, Pannu NS, van Duijn E, Parrello B, Pusch GD, Reich C, Stevens R, Vassieva O, Vonstein V, Abrahams JP. 2009. Crystal structure of chlorite dismutase, a detoxifying Wilke A, Zagnitko O. 2008. The RAST server: rapid annotations using enzyme producing molecular oxygen. J. Mol. Biol. 387:192–206. subsystems technology. BMC Genomics 9:75. doi: 10.1186/1471-2164-9- 52. Kostan J, Sjöblom B, Maixner F, Mlynek G, Furtmüller PG, Obinger C, 75. Wagner M, Daims H, Djinovic´-Carugo K. 2010. Structural and func- 63. Langmead B, Trapnell C, Pop M, Salzberg SL. 2009. Ultrafast and tional characterisation of the chlorite dismutase from the nitrite-oxidizing memory-efficient alignment of short DNA sequences to the human ge- bacterium Candidatus Nitrospira defluvii identification of a catalytically nome. Genome Biol. 10:R25. doi: 10.1186/gb-2009-10-3-r25. important amino acid residue. J. Struct. Biol. 172:331–342. 64. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local 53. Lowe EC, Bydder S, Hartshorne RS, Tape HL, Dridge EJ, Debieux CM, alignment search tool. J. Mol. Biol. 215:403–410. Paszkiewicz K, Singleton I, Lewis RJ, Santini JM, Richardson DJ, Butler 65. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, CS. 2010. Quinol-cytochrome c oxidoreductase and cytochrome C4 me- Abecasis G, Durbin R, 1000 Genome Project Data Processing Sub- diate electron transfer during selenate respiration in Thauera selenatis.J. group. 2009. The sequence alignment/map format and SAMtools. Bioin- Biol. Chem. 285:18433–18442. formatics 25:2078–2079. 54. McDevitt CA, Hugenholtz P, Hanson GR, McEwan AG. 2002. Molec- 66. Pritchard L, White JA, Birch PR, Toth IK. 2006. GenomeDiagram: a ular analysis of dimethyl sulphide dehydrogenase from Rhodovulum python package for the visualization of large-scale genomic data. Bioin- sulfidophilum: its place in the dimethyl sulphoxide reductase family of formatics 22:616–617. microbial molybdopterin-containing enzymes. Mol. Microbiol. 44: 67. Fu L, Niu B, Zhu Z, Wu S, Li W. 2012. CD-HIT: accelerated for 1575–1587. clustering the next-generation sequencing data. Bioinformatics 28: 55. Bäcklund AS, Bohlin J, Gustavsson N, Nilsson T. 2009. Periplasmic c 3150–3152. cytochromes and chlorate reduction in Ideonella dechloratans. Appl. En- viron. Microbiol. 75:2439–2445. 68. Castresana J. 2000. Selection of conserved blocks from multiple align- 56. Bäcklund AS, Nilsson T. 2011. Purification and characterization of a ments for their use in phylogenetic analysis. Mol. Biol. Evol. 17:540–552. soluble cytochrome c capable of delivering electrons to chlorate reductase 69. Price MN, Dehal PS, Arkin AP. 2010. FastTree 2-approximately in Ideonella dechloratans. FEMS Microbiol. Lett. 321:115–120. maximum-likelihood trees for large alignments. PLoS One 5:e9490. doi: 57. Finn RD, Clements J, Eddy SR. 2011. HMMER web server: interactive 10.1371/journal.pone.0009490. sequence similarity searching. Nucleic Acids Res. 39:W29–W37. doi: 70. Abascal F, Zardoya R, Posada D. 2005. ProtTest: selection of best-fit 10.1093/nar/gkr367. models of protein evolution. Bioinformatics 21:2104–2105. 58. Hasegawa N, Arai H, Igarashi Y. 2001. Two c-type cytochromes, NirM 71. Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phy- and NirC, encoded in the nir gene cluster of Pseudomonas aeruginosa act as logenetic analyses with thousands of taxa and mixed models. Bioinformat- electron donors for nitrite reductase. Biochem. Biophys. Res. Commun. ics 22:2688–2690. 288:1223–1230. 72. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed- 59. Lindqvist MH, Johansson N, Nilsson T, Rova M. 2012. Expression of Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. 2009. chlorite dismutase and chlorate reductase in the presence of oxygen The Ribosomal Database Project: improved alignments and new tools for and/or chlorate as the terminal electron acceptor in Ideonella dechloratans. rRNA analysis. Nucleic Acids Res. 37:D141–D145. doi: 10.1093/nar/ Appl. Environ. Microbiol. 78:4380–4385. gkp353. 60. Gao CH, Yang M, He ZG. 2012. Characterization of a novel ArsR-like 73. Huelsenbeck JP, Ronquist F. 2001. MrBayes: bayesian inference of phy- regulator encoded by Rv2034 in Mycobacterium tuberculosis. PLoS One logenetic trees. Bioinformatics 17:754–755. 7:e36255. doi: 10.1371/journal.pone.0036255. 74. Schröder I, Rech S, Krafft T, Macy JM. 1997. Purification and charac- 61. Markowitz VM, Chen IMA, Palaniappan K, Chu K, Szeto E, Grechkin terization of the selenate reductase from Thauera selenatis. J. Biol. Chem. Y, Ratner A, Jacob B, Huang J, Williams P, Huntemann M, Anderson 272:23765–23768.

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