Dehalogenimonas lykanthroporepellens gen. nov., sp. nov., a reductively dehalogenating bacterium isolated from chlorinated solvent-contaminated groundwater

The phylum Chloroflexi is a deep branching lineage within the domain Bacteria. The phylum contains at least six major lineages, five of which, at the time of writing, have cultured representatives. These lineages include the classes Anaerolinea (Yamada et al., 2006), Caldilinea (Yamada et al., 2006), ‘Chloroflexi’ (Castenholz, 2001) and Thermomicrobia (Hugenholtz & Stackebrandt, 2004). Also included in the phylum Chloroflexi is a lineage for which the class ‘Dehalococcoidetes’ was informally proposed by Hugenholtz & Stackebrandt (2004) to accommodate the tetrachloroethene-respiring coccus ‘Dehalococcoides ethenogenes’ (Maymó-Gatell et al., 1997).

Two novel anaerobic bacterial strains that cluster within the phylum Chloroflexi, designated BL-DC-8 and BL-DC-9^T, were recently demonstrated to be able to reductively dehalogenate a variety of polychlorinated alkanes (Yan et al., 2009). These represent the first strains isolated in pure culture to be able anaerobically to reductively dehalogenate 1,2,3-trichloropropane. For each of these strains, 1,2,3-trichloropropane was transformed to the intermediate allyl chloride (3-chloro-1-propene). Allyl chloride is unstable and underwent abiotic hydrolysis to form allyl alcohol. Allyl chloride also underwent abiotic reactions with sulfide and cysteine, reducing agents which were present in the growth medium, to form various other compounds, including diallyl sulfide, diallyl disulfide, allyl mercaptan and allyl methyl sulfide. These final products are compounds that contribute to the recognizable odour associated with garlic (Laakso et al., 1989; Lawson, 1996). The two strains were isolated from groundwater collected from a waste recovery well at the PetroProcessors of Louisiana, Inc. Superfund Site, near Baton Rouge, LA, USA, an area heavily contaminated with chlorinated solvents (Bowman et al., 2006). Data from the present study show that strains BL-DC-9^T and BL-DC-8 represent a single novel species of a new genus in the phylum Chloroflexi.

The genomic DNA G+C content of strains BL-DC-8 and BL-DC-9^T was determined by HPLC as described by Mesbah et al. (1989) following DNA isolation by using an UltraClean microbial DNA isolation kit (MoBio Laboratories Inc.).

Cells for analysis of cellular fatty acids were grown in anaerobic basal medium prepared as described by Yan et al. (2009) except that the sodium sulfide solution was replaced with titanium citrate solution (Zehnder & Wuhrmann, 1976) as a reducing agent at a final concentration of 1.0 mM Ti(III) and 2.0 mM citrate. The medium was supplemented with 5 mM sodium acetate and 0.5 mM 1,1,2-trichloroethane, and provided with hydrogen gas (10 %, v/v) in the headspace. Cells were harvested via centrifugation (10 000 g) following static incubation in the dark at 30 °C for 2 weeks. Cellular fatty acids were extracted, saponified and methylated according to the protocol of the Sherlock Microbial Identification System (MIDI). The fatty acids were analysed by using a gas chromatograph equipped with the Microbial Identification software package (Sasser, 1990).

Scanning electron microscopy was performed as described by Yan et al. (2009). For transmission electron microscopy, cells were collected and fixed in the same manner as for scanning electron microscopy. Preparations were rinsed with 0.02 M glycine in 0.1 M cacodylate buffer, post-fixed in 2 % osmium tetroxide and stained with 0.5 % uranyl acetate. Preparations were dehydrated with a graded ethanol series. The membrane was infiltrated with 1 : 1 ethanol/LR white embedding resin and infiltrated and embedded with 100 % resin. Samples were sectioned with a DuPont MT5000 ultramicrotome and stained with Reynolds' lead citrate. Cross-sections were imaged with a JEOL 100CX transmission electron microscope.

Genomic DNA extraction, PCR amplification and 16S rRNA gene sequencing were performed as described by Yan et al. (2009). Phylogenetic analyses were performed by using the arb program (Ludwig et al., 2004). The neighbour-joining algorithm was used to build the phylogenetic tree, with Jukes–Cantor correction (Jukes & Cantor, 1969) followed by bootstrap analysis with phylip 3.62 (Felsenstein, 1985) based on 1000 replications.

The genomic DNA G+C contents of strains BL-DC-8 and BL-DC-9^T were 54.0 and 53.8 mol%, respectively. The major cellular fatty acids included C_18 : 1ω9c, C_16 : 1ω9c, C_16 : 0 and C_14 : 0 (Table 1⇓). Apart from the presence of summed feature 8 (comprising C_18 : 1ω7c and/or C_18 : 1ω6c) as 4.7 % of the total in strain BL-DC-8 and its absence from strain BL-DC-9^T, there were only minor differences in the cellular fatty acid contents of the two strains. Cells of the two strains were non-spore-forming, irregular cocci, 0.3–0.6 μm in diameter (see Supplementary Fig. S1 in IJSEM Online). Additional phenotypic characteristics were described previously (Yan et al., 2009) and are included in the species description. The two strains shared nearly identical phenotypic properties, exhibiting identical substrate utilization profiles and differing only slightly in temperature range for growth (20–34 °C for strain BL-DC-8 and 20–37 °C for strain BL-DC-9^T). The two strains exhibited reductive dechlorination of 1,2-dichloropropane, a growth-related process, in the pH range 6.0–8.0 but not at pH ≤5.5 or ≥8.5 during a 2-month incubation period (Yan et al., 2009). As the groundwater at the location from which the strains were isolated had a pH of 5.1 (Bowman et al., 2006), the habitat of these strains probably extends to pH levels somewhat lower than determined in the laboratory test conditions employed by Yan et al. (2009).

Phylogenetically, strains BL-DC-8 and BL-DC-9^T were closely related, sharing 99.93 % 16S rRNA gene sequence similarity (1 nt difference). In the neighbour-joining tree, the novel strains were placed distantly from recognized genera within a deep branch of the Chloroflexi (Fig. 1⇓). Their closest cultured relatives were ‘Dehalococcoides’ strains, but with only 90 % 16S rRNA gene sequence similarity. The clustering of strains BL-DC-8 and BL-DC-9^T with the ‘Dehalococcoides’ was supported by a bootstrap value of 94 %.

Maymó-Gatell et al. (1997) informally proposed the name ‘Dehalococcoides ethenogenes’ for a bacterial strain (strain 195) isolated from an enrichment culture derived from anaerobically digested wastewater sludge. This bacterium was the first strain isolated in pure culture that was shown to reductively dehalogenate tetrachloroethene to ethene. Additional strains with close phylogenetic relatedness to ‘Dehalococcoides ethenogenes’ 195 (>99 % 16S rRNA gene sequence similarity) have been reported subsequently in the literature as ‘Dehalococcoides’ sp. (He et al., 2003, 2005; Kube et al., 2005; Sung et al., 2006). There has been intense interest in this group of bacteria because of their ability to reductively dehalogenate a variety of chlorinated compounds that are widespread pollutants of environmental importance (Hendrickson et al., 2002; Bowman et al., 2006). At the time of writing, however, the genus ‘Dehalococcoides’ has not been formally described. The strain designated in the literature as ‘Dehalococcoides ethenogenes’ 195 (Maymó-Gatell et al., 1997) and the related ‘Dehalococcoides’ strains BAV1, CBDB1, FL2 and GT (Adrian et al., 2000; He et al., 2003, 2005; Sung et al., 2006) have not been deposited in public culture collections as required for valid publication under the Bacteriological Code (Lapage et al., 1992), and no formal taxonomic descriptions for these exist. The above genus and species names, however, are widely used in the literature to the extent that ‘Dehalococcoides’ has been carried to the class level with the informal proposal of Hugenholtz & Stackebrandt (2004) of the class ‘Dehalococcoidetes’. In order to move the taxonomy of this lineage forward and to establish an anchor point for future higher-level taxa, we propose the genus Dehalogenimonas gen. nov. to accommodate strains BL-DC-8 and BL-DC-9^T, this genus being related most closely to but distinct from the ‘Dehalococcoides’ cluster.

Although strains BL-DC-8 and BL-DC-9^T share several common phenotypic features with the ‘Dehalococcoides’ strains reported to date, including strictly anaerobic respiration, requirement for H₂ as an electron donor, apparently exclusive use of halogenated compounds as electron acceptors, coupling of cell growth with reductive dehalogenation, Gram-negative staining, small irregular cell morphology and resistance to the antibiotics ampicillin and vancomycin (Adrian et al., 2000; He et al., 2003, 2005; Sung et al., 2006; Yan et al., 2009), they can be differentiated from members of the ‘Dehalococcoides’ cluster based on genotypic, chemotaxonomic and phenotypic properties. Based on 16S rRNA gene sequence comparisons, strains BL-DC-8 and BL-DC-9^T branch with but represent a distinct lineage at the 90 % similarity level from ‘Dehalococcoides’ strains (Fig. 1⇑). Such a low level of 16S rRNA gene sequence similarity is indicative of a taxon distinct to at least the genus level. The genomic DNA G+C contents of strains BL-DC-8 and BL-DC-9^T (54.0 and 53.8 mol%, respectively) are appreciably higher than those for ‘Dehalococcoides ethenogenes’ 195 (48.9 mol%; Seshadri et al., 2005), ‘Dehalococcoides’ sp. CBDB1 (47.0 mol%; Kube et al., 2005) and ‘Dehalococcoides’ sp. BAV1 (47.1 mol%; ).

The cellular fatty acids determined for strains BL-DC-8 and BL-DC-9^T (Table 1⇑) differ from those reported for ‘Dehalococcoides’ strains BAV1 and FL2, the only ‘Dehalococcoides’ strains characterized to date in this regard. Strains BL-DC-8 and BL-DC-9^T contained large proportions of C_16 : 1ω9c (23.0 and 23.2 %, respectively), significant concentrations of summed feature 5 (one or more of C_18 : 2ω6c, C_18 : 2ω9c and anteiso-C_18 : 0) (7.9 and 6.5 %, respectively) and summed feature 3 (one or more of C_16 : 1ω7c, C_16 : 1ω6c and iso-C_15 : 0 3-OH), while these were not detected in strain BAV1 or FL2 (White et al., 2005). 10-Methyl C_16 : 0 made up a large proportion of the cellular fatty acids in ‘Dehalococcoides’ sp. BAV1 and FL2 (21.3 and 24.4 %, respectively; White et al., 2005) but was not detected in strain BL-DC-8 or BL-DC-9^T. C_18 : 1ω9c comprised a major fraction of the cellular fatty acids in strains BL-DC-8 and BL-DC-9^T (21.8 and 23.4 %, respectively) but was present in only trace amounts in ‘Dehalococcoides’ strains BAV1 and FL2 (0.4 and 0.5 %, respectively; White et al., 2005).

Additionally, strains BL-DC-8 and BL-DC-9^T were shown to reductively dehalogenate polychlorinated alkanes (e.g. 1,2-dichloropropane, 1,2-dichloroethane, 1,1,2-trichloroethane, 1,1,2,2-tetrachloroethane, 1,2,3-trichloropropane), but not monochlorinated alkanes (e.g. 1-chloropropane, 2-chloropropane), chlorinated alkenes (e.g. tetrachloroethene, trichloroethene, cis-dichloroethene, trans-dichloroethene, vinyl chloride) or chlorinated benzenes (1-chlorobenzene, 1,2-dichlorobenzene) (Yan et al., 2009). All dechlorination reactions determined to date appear to involve exclusively a dichlorelimination reaction mechanism (Yan et al., 2009). This range of compounds serving as electron acceptors and the presence of apparently exclusive dichlorelimination (involving simultaneous removal of two chlorines from adjacent carbon atoms and formation of a carbon–carbon double bond) for strains BL-DC-8 and BL-DC-9^T differs from the ‘Dehalococcoides’ strains reported to date, which can reductively dehalogenate chlorinated alkenes and chlorinated benzenes and employ a reaction mechanism involving sequential removal of one chlorine atom at a time (Maymó-Gatell et al., 1997, 1999, 2001; Adrian et al., 2000; He et al., 2003, 2005; Sung et al., 2006).

Based on these characteristics, strains BL-DC-8 and BL-DC-9^T are considered to represent a new genus distinct from the informally proposed genus ‘Dehalococcoides’. It should be noted that any future valid publication of the name ‘Dehalococcoides’ would not be impacted by, nor would it impact, the genus Dehalogenimonas proposed herein.

Levels of 16S rRNA gene sequence similarity between strains BL-DC-8 and BL-DC-9^T and members of recognized genera in the phylum Chloroflexi were extremely low, and phylogenetic distance readily served as a differentiating property (Fig. 1⇑). Phenotypic characteristics also differentiated strains BL-DC-8 and BL-DC-9^T from recognized genera in the phylum Chloroflexi (Supplementary Table S1). Within the class Anaerolinea, members of the genera Anaerolinea (Sekiguchi et al., 2003; Yamada et al., 2006), Bellilinea (Yamada et al., 2007), Leptolinea (Yamada et al., 2006), Levilinea (Yamada et al., 2006) and Longilinea (Yamada et al., 2007) are filamentous, whereas cells of strains BL-DC-8 and BL-DC-9^T are irregular cocci. Additionally, members of the genera Anaerolinea and Bellilinea are thermophilic, while strains BL-DC-8 and BL-DC-9^T are mesophilic. At the time of writing, the class Caldilineae comprises a single genus with a single species, Caldilinea aerophila (Sekiguchi et al., 2003; Yamada et al., 2006), which can be differentiated from strains BL-DC-8 and BL-DC-9^T based on the fact that it is filamentous, thermophilic and facultatively aerobic (Supplementary Table S1).

Phenotypically, strains BL-DC-8 and BL-DC-9^T could be differentiated from the filamentous anoxygenic phototrophs of the genera Chloroflexus, Chloronema, Heliothrix, Oscillochloris and Roseiflexus on the basis of cell morphology (Supplementary Table S1). In addition, the type strains of species of the genera Chloroflexus, Heliothrix and Roseiflexus are thermophilic (Pierson & Castenholz, 1974; Pierson et al., 1985; Hanada et al., 1995, 2002) whereas strains BL-DC-8 and BL-DC-9^T are mesophilic (Yan et al., 2009). Strains BL-DC-8 and BL-DC-9^T could be differentiated from the chemoheterotrophic genus Herpetosiphon on the basis that the latter has lower DNA G+C content and filamentous cell morphology and exhibits aerobic metabolism (Holt & Lewin, 1968; Lewin, 1970; Holt & Castenholz, 2001).

Strains BL-DC-8 and BL-DC-9^T may be differentiated from members of the class Thermomicrobia on the basis that the species assigned to this class, namely Thermomicrobium roseum (Perry, 2001; Hugenholtz & Stackebrandt, 2004) and Sphaerobacter thermophilus (Demharter et al., 1989; Hugenholtz & Stackebrandt, 2004), are thermophilic, aerobic, rod-shaped and have appreciably higher DNA G+C contents. In addition, Sphaerobacter thermophilus is Gram-positive (Demharter et al., 1989) whereas strains BL-DC-8 and BL-DC-9^T stain Gram-negative.

On the basis of phylogenetic, chemotaxonomic and phenotypic data, strains BL-DC-8 and BL-DC-9^T are clearly distinct from other genera in the phylum Chloroflexi. We suggest that strains BL-DC-8 and BL-DC-9^T represent a novel species of a new genus, for which the name Dehalogenimonas lykanthroporepellens gen. nov., sp. nov. is proposed.