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Dehalogenimonas alkenigignens IP3-3T is a strictly anaerobic, mesophilic, Gram negative staining bacterium that grows by organohalide respiration, coupling the oxidation of H2 to the reductive dehalogenation of polychlorinated alkanes. Growth has not been observed with any non-polyhalogenated alkane electron acceptors. Here we describe the features of strain IP3-3T together with genome sequence information and its annotation. The 1,849,792 bp high-quality-draft genome contains 1936 predicted protein coding genes, 47 tRNA genes, a single large subunit rRNA (23S-5S) locus, and a single, orphan, small unit rRNA (16S) locus. The genome contains 29 predicted reductive dehalogenase genes, a large majority of which lack cognate genes encoding membrane anchoring proteins.
Strain IP3-3T (=JCM 17062, =NRRL B-59545) is the type strain of the species Dehalogenimonas alkenigignens . Currently, two pure cultures of D. alkenigignens have been described, namely, D. alkenigignens strains IP3-3T and SBP-1 . Both strains were isolated from chlorinated alkane- and alkene-contaminated groundwater collected at a Superfund Site near Baton Rouge, Louisiana (USA) . Construction of 16S rRNA gene libraries indicated that bacteria closely related or identical to D. alkenigignens were present at high relative abundance in the groundwater where strains IP3-3T and SBP-1 were first isolated .
Strains of D. alkenigignens possess the unique trait of growing via organohalide respiration, a process in which halogenated organic compounds are utilized as terminal electron acceptors. In particular, they are able to reductively dehalogenate a variety of polychlorinated alkanes that are of environmental concern on account of their potential to cause adverse health effects and their widespread occurrence as soil and groundwater pollutants [1–4]. In this report, we present a summary classification and a set of features for D. alkenigignens IP3-3T together with the description of the draft genomic sequence and annotation.
Dehalogenimonas alkenigignens is a member of the order Dehalococcoidales, class Dehalococcoidia, of the phylum Chloroflexi (Table 1). Based on 16S rRNA gene sequences, the closest related type strains are Dehalogenimonas lykanthroporepellens BL-DC-9T [1, 5] and Dehalococcoides mccartyi 195T , with sequence identities of 96.2 and 90.6 %, respectively .
Figure 1 shows the phylogenetic neighborhood of D. alkenigignens strain IP3-3T in a 16S rRNA gene based phylogenetic dendrogram. The sequence of the lone 16S rRNA gene copy in the draft genome is identical to the previously published 16S rRNA gene sequence (JQ994266).
The cells of D. alkenigignens IP3-3T are Gram negative staining, non-spore forming, irregular cocci to disk-shaped with a diameter of 0.4–1.1 μm  (Fig. 2). The strain was isolated in liquid medium using a dilution-to-extinction approach. Growth of the strain was not observed on agar plates even after long term (2 months) incubation . The temperature range for growth of strain IP3-3T is between 18 °C and 42 °C with an optimum between 30 °C and 34 °C . The pH range for growth is 6.0 to 8.0 with an optimum of 7.0 to 7.5 . The strain grows in the presence of <2 % (w/v) NaCl and is resistant to ampicillin and vancomycin at concentrations of 1.0 and 0.1 g/l, respectively .
D. alkenigignens IP3-3T is a strictly anaerobic chemotroph, coupling utilization of H2 as an electron donor and polychlorinated aliphatic alkanes as electron acceptors for growth. The chlorinated compounds known to be reductively dehalogenated include 1,2-dichloroethane, 1,2-dichloropropane, 1,1,2,2-tetrachloroethane, 1,1,2-trichloroethane, and 1,2,3-trichloropropane . In all of the reductive dechlorination reactions characterized to date, strain IP3-3T appears to exclusively utilize vicinally halogenated alkanes as electron acceptors via dihaloelimination reactions (i.e., simultaneous removal of two chlorine atoms from adjacent carbon atoms with concomitant formation of a carbon-carbon double bond) . Strain IP3-3T does not utilize carbon tetrachloride (tetrachloromethane), 1-chlorobenzene, chloroform, 1-chloropropane, 2-chloropropane, 1,2-dichlorobenzene, 1,1-dichloroethane, cis-1,2-dichloroethene, trans-1,2-dichloroethene, methylene chloride (dichloromethane), tetrachloroethene, 1,1,1-trichloroethane, or vinyl chloride as electron acceptors . Growth is not supported by acetate, butyrate, citrate, ethanol, fructose, fumarate, glucose, lactate, lactose, methanol, methyl ethyl ketone, propionate, pyruvate, succinate, or yeast extract in the absence of H2 .
Although sufficiently high chlorinated alkane concentrations were found to become inhibitory, D. alkenigignens IP3-3T was shown to reductively dehalogenate 1,2-dichloroethane, 1,2-dichloropropane, and 1,1,2-trichloroethane when present at initial aqueous-phase concentrations as high as 9.81±0.98, 5.05±.29, and 3.49±0.31 mM, respectively . When grown in the presence of mixtures of chlorinated alkanes, preferential dechlorination of 1,1,2-trichloroethane over both 1,2-dichloroethane and 1,2-dichloropropane was observed . 1,2-Dichloroethane in particular was not dechlorinated until 1,1,2-trichloroethane reached low concentrations. In contrast, D. alkenigignens IP3-3T concurrently dechlorinated 1,2-dichloroethane and 1,2-dichloropropane over a comparably large concentration range .
The major cellular fatty acids of D. alkenigignens IP3-3T are C18:1ω9c, C16:0, C14:0, and C16:1ω9c . The same fatty acids were also present in the closely related D. alkenigignens strain SBP-1 . Cellular fatty acids present in lower proportions include C18:0, C18:3ω6c(6,9,12), and unidentified fatty acids with equivalent chain lengths of 11.980, 13.768, 13.937, and 15.056 .
D. alkenigignens IP3-3T was chosen for genome sequencing because it is the type strain of the species and because of the importance of organohalide respiration in the field of environmental biotechnology and bioremediation. A summary of the project information is shown in Table 2. The D. alkenigignens strain IP3-3T genome project is deposited in the Genomes OnLine Database  and the genome sequence is available from GenBank.
D. alkenigignens strain IP3-3T (=JCM 17062, =NRRL B-59545) was cultured in liquid anaerobic basal medium  supplemented with 2 mM 1,2-dichloropropane. Cells were harvested from 9.9 L culture medium by centrifugation after at least 50 % of the starting 1,2-dichloropropane was dehalogenated. Total DNA was extracted using a GenElute Bacterial Genomic DNA kit (Sigma-Aldrich) following the manufacturer’s recommended protocol. Eluted DNA was concentrated using ethanol precipitation, air dried, and reconstituted in TE buffer (10 mM Tris–HCl, 0.5 mM EDTA, pH 9.0).
The genome of D. alkenigignens IP3-3T was sequenced using a combination of Illumina  and 454 technologies . A total of three libraries were constructed, a 454 Titanium standard library which generated 234,711 reads (42.35-fold coverage; 78.34 Mb), a 454 Titanium paired-end libraries with insert size of 8 kb which generated 238,686 reads (29.86-fold coverage; 55.23 Mb), and an Illumina paired-end library which generated 7,147,715 reads (read length 150 bp; 583.50-fold coverage; 1079.35 Mb). Libraries were prepared using 454 standard and paired-end protocols and the Illumina TruSeq DNA sample preparation protocol, as provided by each manufacturer.
The 454 Titanium standard data and the 454 paired-end data were assembled with gsAssembler ver. 2.6 (Roche). Illumina data were assembled with CLC Genomics Workbench ver. 6.5.1 (CLCbio). Each of the resulting scaffolds and contigs were integrated using CodonCode Aligner ver. 3.7.1 (CodonCode Corporation). Also, Illumina sequencing reads were mapped to the final contigs to correct misassembles and base errors. The final assembly generated one scaffold including two contigs representing 1,849,792 bp based on 655.71× coverage of 454 and Illumina sequencing data.
Genes were identified using Prodigal  as part of the JGI’s microbial annotation pipeline  followed by a round of manual curation using the JGI GenePRIMP pipeline . The predicted CDSs were translated and used to search the National Center for Biotechnology Information nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. These data sources were combined to assert a product description for each predicted protein. Non-coding genes and miscellaneous features were predicted using tRNAscan-SE , RNAMMer , Rfam , TMHMM , ARAGORN , bSECISearch , and signal . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes - Expert Review platform .
The draft genome of D. alkenigignens strain IP3-3T has a total length of 1,849,792 bp with 55.88 % G+C content (Table 3 and Fig. 3). Of the 1988 genes predicted, 1936 were protein-coding genes and 52 were RNAs. The majority of the protein-coding genes (74.9 %) were assigned a putative function, and the remaining were annotated as hypothetical proteins. The distribution of the predicted protein coding genes into COG functional categories is presented in Table 4.
The draft genome of D. alkenigignens IP3-3T is 163,282 bp larger than that of D. lykanthroporepellens BL-DC-9T (1,686,510 bp) and 380,072 bp larger than Dehalococcoides mccartyi 195T (1,469,720 bp). Although the genomes of D. alkenigignens IP3-3T, D. lykanthroporepellens BL-DC-9T , and Dehalococcoides mccartyi strains [22–24] contain similar number of rRNA and tRNA encoding genes, they lack overall synteny and differ in their GC content, gene density, and percentage of sequence that encodes proteins.
BLAST comparisons of protein sets of D. alkenigignens IP3-3T and D. lykanthroporepellens BL-DC-9T revealed that the two strains contain 1154 protein coding genes in common (bidirectional best hits, 20-95 % identity at the predicted protein level). Bidirectional best-hit comparisons indicated that D. alkenigignens IP3-3T contains 782 protein-coding genes with no homologs in D. lykanthroporepellens BL-DC-9T. The latter contained 566 protein-coding genes with no homologs in D. alkenigignens IP3-3T. Genome-specific genes identified in D. alkenigignens IP3-3T and D. lykanthroporepellens BL-DC-9T included those that encoded transposases, restriction endonucleases, acetyltransferases, permeases, reductases, hydrogenases, and dehalogenases. Some of these strain-specific genes were associated with IS elements.
Nine signature indels (insertions or deletions) specific for predicted proteins of the class Dehalococcoidia (which at present includes only the genera Dehalococcoides and Dehalogenimonas) were recently reported based on the results of comparative analyses of previously reported genomes . Of the nine proteins in which conserved signature indels were reported as specific for the class Dehalococcoidia , all were found to be present in the predicted proteins of D. alkenigignens IP3-3T, including those for a GTP binding protein LepA (DEALK_16110), F0F1-type ATP synthase alpha subunit (DEALK_14680), imidazoleglycerol-phosphate dehydratase (DEALK_15410), glycine/serine hydroxymethyltransferase (DEALK_18820), adenylate kinase (DEALK_03090), hydrogenase formation/expression protein HypD (DEALK_04300), DNA gyrase subunit A (DEALK_05640), excinuclease ABC subunit A (DEALK_13870), and ribulose-phosphate 3-epimerase (DEALK_13610). Of the conserved signature proteins (CSPs) that were reported previously to be specific for the class Dehalococcoidia , however, several did not have homologs in D. alkenigignens IP3-3T (DET0078, DET0236, DET0307, DET0767, DET1026, DET1283, and DET1511). Furthermore, four conserved signature proteins reported as specific for the genus Dehalococcoides  (DET0939, DET1011, DET1322, and DET1557) were found to have homologs in Dehalogenimonas alkenigignens IP3-3T (DEALK_12980, DEALK_11520, DEALK_01350, and DEALK_19030, respectively), indicating that these proteins are not as narrowly confined to the genus Dehalococcoides as once thought.
The genome of D. alkenigignens IP3-3T contains 47 tRNA genes, including those for all 20 standard amino acids as well as the less common amino acid selenocysteine. Consistent with the presence of a selC gene (DEALK_t00110) encoding a selenocysteine-inserting tRNA (tRNAsec), D. alkenigignens strain IP3-3T also contains genes that are putatively involved in synthesis of selenocysteine (DEALK_04960-04970) and a GTP-dependent selenocysteine-specific elongation factor (DEALK_04950) that forms a quaternary complex with selenocysteine-tRNAsec and the selenocysteine inserting sequence (SECIS), a hairpin loop found immediately downstream of the UGA codon in selenoprotein-encoding mRNA . This complex facilitates reading through the UGA codon and incorporation of selenocysteine instead of translation termination . Also consistent with the presence of the genes encoding the synthesis and incorporation of selenocysteine, D. alkenigignens strain IP3-3T contains multiple genes encoding putative selenocysteine-containing proteins including a selenophosphate synthase (DEALK_04975) and formate dehydrogenase (DEALK_19115) that have internal in-frame UGA stop codons followed by putative SECIS elements .
A number of microorganisms accumulate low molecular weight organic compounds commonly referred to as “compatible solutes” that help the microorganisms survive osmotic stress but do not interfere with metabolism . Ectoine is a compatible solute of many mesophilic bacteria capable of survival at high salt concentrations , while mannosylglycerate is a compatible solute accumulated by many thermophilic organisms . Homologs of a gene encoding a bifunctional mannosylglycerate synthase (mgsD) are found in Dehalococcoides mccartyi strains (e.g., DET1363) and D. lykanthroporepellens BL-DC-9T (Dehly_0877), an unusual occurrence for mesophilic bacteria [21, 29]. Comparative analysis revealed that D. alkenigignens IP3-3T contains a homologous gene (DEALK_12650, 55–70 % protein identity). This expands the range of mesophilic species containing genes putatively involved in the biosynthesis of mannosylglycerate. D. alkenigignens IP3-3T, however, lacks the operon (ectABC) encoding putative homologs of the enzymes involved in ectoine biosynthesis and regulation that were found to be present in D. lykanthroporepellens BL-DC-9T (Dehly_1306, Dehly_1307, Dehly_1308). The presence of these ectoine encoding genes in D. lykanthroporepellens BL-DC-9T but not D. alkenigignens IP3-3T may account for the ability of the former to reductively dechlorinate polychlorinated alkanes in the presence of higher NaCl concentrations than was observed for D. alkenigignens IP3-3T .
Genes encoding the enzymes characterized to date that are involved in catalyzing the reductive dehalogenation of chlorinated solvents are organized in rdhAB operons encoding a ~500 aa protein (RdhA) that functions as a reductive dehalogenase and a ~90 aa hydrophobic protein with transmembrane helices (RdhB) that is thought to anchor the RdhA to the cytoplasmic membrane [30–41]. D. alkenigignens IP3-3T contains several loci, accounting for 2.38 % of the genome, related to rdhA and/or rdhB genes scattered throughout the genome. The multiple rdhA and rdhB ORFs of D. alkenigignens IP3-3T have 32–97 % and 32–43 % identities at the predicted protein level, respectively. The closest homologs for most of the D. alkenigignens IP3-3TrdhA ORFs (Table 5) are found among Dehalogenimonas lykanthroporepellens BL-DC-9T, Dehalococcoides mccartyi strains, or uncultured bacteria. A twin-arginine motif followed by a stretch of hydrophobic amino acids, was identified in the N-terminus of a large majority (27 of 29) of the predicted RdhA sequences (Table 5). Consistent with the presence of the twin-arginine sequence in the N-terminus of most of its RdhA sequences, D. alkenigignens IP3-3T contains an operon encoding proteins that constitute a putative twin-arginine translocation (TAT) system (DEALK_04830-04860). This specialized system is involved in the secretion of folded proteins across the bacterial inner membrane into the periplasmic space [42, 43]. Dehalogenimonas lykanthroporepellens BL-DC-9T also contains an operon encoding an analogous TAT system that is related to the TAT system of D. alkenigignens IP3-3T (55–86 % protein identity).
Two conserved motifs each containing three or four cysteine residues, a feature associated with binding iron-sulfur clusters , were identified near the C-terminus of 28 of the 29 predicted RdhA sequences of D. alkenigignens IP3-3T. The first of these motifs had a consistent number of cysteine residues and consistent number of amino acids between the cysteine residues (CX2CX2CX3C), while the second motif was variable (Table 5). If a “full-length” rdhA is predicted to encode a protein containing a twin-arginine sequence in the N-terminus, two iron-sulfur cluster binding motifs in the C-terminus, and an intervening sequence of ~450 aa, then D. alkenigignens IP3-3T contains 27 such genes, a number appreciably larger than the 17 such genes found in Dehalogenimonas lykanthroporepellens BL-DC-9T . One of the non-full length rdhA genes (DEALK_17180) contains a predicted internal stop codon that putatively prevents complete translation of what would otherwise be a 458 aa protein containing two iron-sulfur binding clusters. rdhA genes with internal stop codons have been reported previously among the genomes of other organohalide respiring strains of the genera Dehalococcoides  and Dehalobacter [45, 46].
Within D. alkenigignens IP3-3T, only three of the rdhA ORFs (DEALK_11290, DEALK_17200, and DEALK_19050) have a cognate rdhB (Table 6). Two additional rdhB genes (DEALK_00250 and DEALK_05730) appear to be orphans with no cognate rdhA ORF. In at least one locus (DEALK_00250), it appears that transposon insertion has truncated the rdhA gene (annotated as pseudogene DEALK_00260). The predicted RdhB sequences of strain IP3-3T each contain two or three transmembrane helices (Table 6), similar to the features of the predicted RdhB sequences of Dehalogenimonas lykanthroporepellens BL-DC-9T and Dehalococcoides mccartyi strains [21, 22, 24, 47]. The predicted RdhB sequences of D. alkenigignens IP3-3T are most closely related to the RdhB of D. lykanthroporepellens strain BL-DC-9T, Dehalococcoides mccartyi strain GY, and an uncultured bacterium designated as Dehalogenimonas sp. WBC-2  (45-96 % identity at the protein level, Table 6). As was observed for D. lykanthroporepellens BL-DC-9T , genes putatively involved in the regulation of rdhAB operons in Dehalococcoides mccartyi strains (e.g., MarR-type or two-component transcriptional regulators [22, 24]) were not present in a majority of the rdhA loci of D. alkenigignens IP3-3T. Thus, it appears that regulation of rdh gene expression within the genus Dehalogenimonas may generally differ from that of the genus Dehalococcoides.
The predicted RdhA protein encoded by the rdhAB operon comprised of DEALK_17200-17210 shares 95 % identity with the 1,2-dichloropropane reductive dehalogenases (dcpAs) recently identified in Dehalococcoides mccartyi strains KS and RC and 92 % identity with the related dcpA in D. lykanthroporepellens BL-DC-9T . The putative membrane anchoring protein encoded by the rdhB (DEALK_17210) adjacent to the dcpA gene is also related (92–96 % identity at the protein level) to the RdhB cognate dcpA of D. lykanthroporepellens BL-DC-9T and Dehalococcoides mccartyi strains KS and RC . Interestingly, the putative dcpA gene present in D. alkenigignens IP3-3T had mismatches with all four primers/probes that were reported  for use in PCR or qPCR for detection and quantification of this gene (1 mismatch with dcpA-360 F, 3 mismatches with dcpA-1257 F, and two mismatches each with dcpA-1426R and dcpA-1449R).
The presence of insertion sequence elements adjacent to some rdhA/rdhB loci in D. alkenigignens IP3-3T indicates their acquisition from an unknown host. Previous studies of D. lykanthroporepellens BL-DC-9T and Dehalococcoides strains have also suggested horizontal transfer of reductive dehalogenase genes [21, 49, 50]. Additionally, the genomic region downstream of the ssrA gene (DEALK_tm00010) in D. alkenigignens IP3-3T shares some synteny with the mobile genetic elements reported for vinyl chloride reductases in Dehalococcoides strains . A 22 bp direct repeat of the 3’ end of the ssrA gene adjacent to one of the rdhA loci in D. alkenigignens IP3-3T (DEALK_11430) suggests that integration at the ssrA gene may have played a role in shaping the genome of D. alkenigignens IP3-3T.
It remains to be determined if D. alkenigignens IP3-3TrdhA genes lacking an rdhB ORF downstream encode functional reductive dehalogenases and whether or how they are membrane-bound. It is possible that a non-contiguous rdhB (e.g., the orphan DEALK_005730) could complement one or more of the strain IP3-3TrdhA genes lacking an rdhB ORF downstream. Alternatively, some of these genes may encode reductive dehalogenases that are not membrane bound or that are bound by an unknown mechanism. The finding that many of the D. lykanthroporepellens BL-DC-9TrdhA genes lacking cognate rdhB genes are simultaneously transcribed during the reductive dechlorination of 1,2-dichloroethane, 1,2-dichloropropane, and 1,2,3-trichloropropane  suggests that rdhA genes lacking a cognate rdhB may serve a purpose. An enzyme involved in the reductive dehalogenation of tetrachloroethene by Sulfurospirillum multivorans (basonym Dehalospirillum multivorans [52, 53]) was found in the cytoplasmic fraction , suggesting that some reductive dehalogenases are either loosely membrane-bound or soluble entities. The same may be the case for the majority of reductive dehalogenases of D. alkenigignens IP3-3T.
Genomic analysis of D. alkenigignens IP3-3T revealed the presence of components associated with synthesis of selenocysteine-containing proteins as well as numerous reductive dehalogenase homologous genes not previously studied. As with the related species D. lykanthroporepellens but in contrast to other dechlorinating genera, a large majority of the reductive dehalogenase homologous genes in D. alkenigignens IP3-3T lack apparent cognate genes encoding membrane anchoring components. The sequences of these diverse genes may aid future studies aimed at elucidating the strain’s mechanisms for transforming polychlorinated alkanes. The absence of genes encoding enzymes involved in ectoine biosynthesis in the genome of D. alkenigignens IP3-3T may account for the strain’s inability to dehalogenate chlorinated alkanes at higher NaCl concentrations that were observed for strains of the related species D. lykanthroporepellens.
This research was financially supported by a consortium of petrochemical companies. The work conducted by TK was supported in part by U.S. National Science Foundation grant DGE-1247192. The authors gratefully acknowledge Xiao Ying for assistance with scanning electron microscopy.
TK and KB carried out the microbial cultivation and genomic DNA isolation. YC and JC supervised and participated in sequencing and assembly. TK, DR, and WM participated in sequence alignment and conducted manual curation. TK, DR, KB, YC, JC, MC, FR, and WM all participated in drafting the manuscript. All authors read and approved the manuscript.
The authors declare that they have no competing interests.