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4 Pure Cultures of  Exhibit Diverse Complex Patterns of Dechlorination of Commercial PCB Mixtures

4 Pure Cultures of  Exhibit Diverse Complex Patterns of Dechlorination of Commercial PCB Mixtures

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552



J. He and D.L. Bedard



Further experiments with single congeners proved that the dechlorination of

234-chlorophenyl rings was strongly influenced by the chlorine configuration on

the opposite ring; usually the doubly flanked meta chlorine was removed, but the

singly flanked para chlorine could also be removed. Hence 234-234-CB and 234245-CB were each dechlorinated by four distinct pathways comprising, respectively, six and seven different dechlorination reactions (Adrian et al. 2009). This

was a level of complexity not previously appreciated.



23.4.2 D. mccartyi Strain JNA

D. mccartyi strain JNA, which, like CBDB1, belongs to the Pinellas subgroup,

was recently isolated from the JN enrichment that metabolically dehalogenates

Aroclor 1260 (LaRoe et al. 2014; Bedard et al. 2007). It had not previously been

determined which of the congeners in Aroclor 1260 serve as respiratory electron

acceptors. LaRoe et al. (2014) tested multiple PCB congeners and found that 236236-CB was the best substrate for respiration. This was an unexpected finding

because 236-236-CB has four ortho chlorines and exists as two separate enantiomers. Despite this, strain JNA stoichiometrically dechlorinated 236-236-CB to 23626-CB and then 26-26-CB. Using 236-236-CB as sole electron acceptor, LaRoe

et al. (2014) carried out multiple serial transfers to extinction to isolate strain JNA.

Strain JNA almost exclusively removes flanked meta chlorines from PCB congeners with 3–8 chlorines. Eight chlorophenyl groups are confirmed substrates for

JNA: 34-, 234-, 235-, 236-, 245-, 2345-, 2346-, and 2356-CB (underscores indicate the chlorines removed). These chlorophenyl groups constitute 88.7 mol%

of the chlorophenyl groups in Aroclor 1260 (Frame et al. 1996), explaining

why this dechlorination is so extensive. The major dechlorination products are

24-24-CB, 24-26-CB, 24-25-CB, and 25-26-CB. This dechlorination matches

PCB Dechlorination Process N which occurs in the Housatonic River (Lenox,

Massachusetts, USA). LaRoe et al. (2014) determined that JNA carries out 85 distinct PCB dechlorination reactions and utilizes 56 different dechlorination pathways, demonstrating the complexity of dechlorination that this strain is capable

of. Genome sequencing is in progress, but it has already been determined that

this strain harbors at least 19 putative reductive dehalogenase genes (Fricker et al.

2014).



23.4.3 Optimizing Enrichment and Isolation of PCB

Dechlorinating D. mccartyi Strains

Like bacteria that reductively dechlorinate other halogenated compounds, PCB

dechlorinating D. mccartyi strains employ reductive dehalogenases to catalyze chlorine removal from biphenyl rings. Wang et al. set about isolating PCB



23  The Microbiology of Anaerobic PCB Dechlorination



CG-1



CG-4



CG-5



PCB-fed cultures



CG-1



CG-4



553



CG-5



PCE-fed cultures



Fig. 23.3  Microbial community profiling of the enrichment process in mineral salts medium

with lactate as the sole carbon source shows enrichment of Dehalococcoides mccartyi (Dhc, in

red) in Aroclor 1260 dechlorinating cultures via a single transfer with PCE as an alternative electron acceptor. The stacked bar graph depicts microbial community compositions (obtained by

Illumina sequencing of 16S rRNA genes) at the phylum level. Reprinted with permission from

PNAS (Wang et al. 2014)



dechlorinating strains from three of their sediment-free cultures with the specific

objective of identifying the functional genes responsible for PCB dechlorination.

One difficulty in obtaining a pure culture of PCB dechlorinators is that even after

enrichment with PCBs, the putative dechlorinators, in this case strains of D. mccartyi, represent a very small fraction of the total population. This is likely because

the solubility of PCBs is extremely low. Therefore, Wang et al. (2014) used PCE,

which is much more soluble than PCBs, as an alternative electron acceptor in

order to increase the proportion and total biomass of PCB dechlorinating bacteria. As seen in Fig. 23.3, this strategy substantially increased the proportion of D.

mccartyi cells and facilitated the isolation of D. mccartyi strains CG1, CG4, and

CG5.



23.4.4 D. mccartyi Strains CG1, CG4, and CG5

Phylogenetic analysis showed that strains CG1, CG4, and CG5 (see Sect. 23.4.3)

cluster into distinct D. mccartyi subgroups, Victoria, Cornell, and Pinellas

(Fig. 23.2), represented by previously sequenced strains VS, 195, and CBDB1,

respectively (Wang et 

al. 2014). Overall, each strain retained the PCB



J. He and D.L. Bedard



554

Abiotic control



CG4



CG1



Process N

CG5



Process H

CW-4



Fig. 23.4  Reductive dechlorination of Aroclor 1260 in three pure cultures and one sediment-free

enrichment after 6 months of incubation. Absolute difference in the PCB congener distribution of

Aroclor 1260 between the abiotic control and pure cultures CG1, CG4, and CG5, and mixed culture CW-4. Negative mol% indicates the amount of PCBs dechlorinated; positive numbers represent PCB congeners produced by dechlorination. Dechlorination Processes H and N are illustrated. The X-axes indicate the predominant PCB congeners in each peak; PCB congeners are

designated by IUPAC number. This figure is adapted from figures previously published in PNAS

and Plos One and is published here with permission (Wang et al. 2014; Wang and He 2013b)



dechlorinating specificity exhibited by its parent mixed culture (see Sect. 23.2.2)

(Fig. 23.4) (Wang et al. 2014; Wang and He 2013b). Strain CG1 removed the doubly flanked meta chlorines of 234-, 2345-, and 2346-chlorophenyl groups. We are

now able to describe the activity of strain CG4 in more detail and it appears to be

a novel dechlorination process. Strain CG4 preferentially removed doubly flanked

chlorines such as the meta chlorine of 234- and 2346-chlorophenyl groups, the

para chlorine of 2345-, and to a lesser extent, the meta chlorine of 2345-chlorophenyl groups. Strain CG4 also removed the flanked para chlorines of 2346-, and

to a much lesser extent, of 245-chlorophenyl groups. Strain CG4 removed nearly

equal numbers of meta and para chlorines from Aroclor 1260, 0.13 and 0.19 chlorines per biphenyl, respectively (Wang et al. 2014). Strain CG5 removed primarily flanked and doubly flanked meta chlorines from 245-, 234-, 236-, 2345-, and

2346-chlorophenyl groups.

When grown with Aroclor 1260, strain CG1 removed 9 % of the meta chlorines, strain CG4 removed 5 % of the meta chlorines and 14.4 % of the para



23  The Microbiology of Anaerobic PCB Dechlorination



555



chlorines, and strain CG5 removed 35.6 % of the meta chlorines and 6 % of the

para chlorines (Wang et al. 2014).

Continued growth with PCE increased the biomass of strains CG1, CG4, and

CG5 and enabled Wang et al. to sequence the genomes revealing that the three

strains have, respectively, 35, 15, and 32 putative RDase genes (Wang et al. 2014).



23.5 Identification and Characterization of Three PCB

Reductive Dehalogenases

23.5.1 Identification of PCB Reductive Dechlorinases

RDase genes in D. mccartyi strains are usually identified through a combination

of transcriptional analysis and enzymatic activity assays with purified RDases.

However, the transcriptional and proteomic descriptions required for positive

functional characterization require a high biomass of cells that cannot typically be

reached by PCB respiring isolates (Wang et al. 2014). A single identical RDase

gene dominated in both PCB- and PCE-fed cultures making the use of PCE to

increase the biomass of the cells possible, which was critical (Fig. 23.3) for harvesting enough biomass for subsequent enzymatic activity tests. This enabled

Wang and colleagues to use transcriptomic and in vitro enzyme activity assays

to identify three distinct PCB dechlorinating enzymes PcbA1, PcbA4 and PcbA5

from each of D. mccartyi strains CG1, CG4, and CG5 (Wang et al. 2014). Cellfree enzyme assays with the crude cell lysates confirmed that the dechlorination

specificity was the same as observed in the pure cultures. The three enzymes

encoded by these genes each exhibit different regiospecificities in catalyzing

dechlorination of a broad range of PCB congeners in Aroclor 1260.



23.5.2 Regiospecificity of the PCB RDases

Each PCB RDase gene was highly transcribed in pure culture amended with either

PCE or Aroclor 1260. Dechlorination activities on Aroclor 1260 and highly chlorinated congeners were measured in crude cell lysates. The cell lysate containing

PcbA1 from strain CG1 primarily removed doubly flanked meta chlorines from

2345-, 2346-, and 234-chlorophenyl rings. The cell lysate containing PcbA4 had

the same specificity as the pure culture, preferential removal of doubly flanked

chlorines and, to a much lesser extent, of the para chlorine of 245-chlorophenyl

groups. The cell lysate containing PcbA5 had the most extensive PCB dechlorination capability which is very similar to Process N, removing flanked and doubly

flanked meta chlorines from 2345-, 234-, 235-, 236-, and 245-chlorophenyl rings.



J. He and D.L. Bedard



556



DEHALGT0124-GT (ADC73492)

PcbA4-CG4 (AII58856)

DET1559-195 (AAW39215)

PcbA5-CG5 (AII60305)

RD11-JNA (AHZ58530)

MbrA-MB (ADF96893)

CbrA-CBDB1 (YP_307261)

PcbA1-CG1 (AII58466)

1350-GY50 (AHB14121)

DcpA-KS (AGS15112)

PceA-195 (YP_181066)

VcrA-VS (YP_003330719)

TceA-195 (YP_180831)

BvcA-BAV1 (AAT64888)

0.2



Fig. 23.5  Phylogenetic analysis of functionally characterized RDases in D. mccartyi including orthologs of the PCB reductive dehalogenases. DEHALGT0124, Det1559, and RD11 are

orthologs of PcbA4 and PcbA5 in D. mccartyi strains GT, 195, and JNA; they have not yet been

functionally characterized. Likewise, 1350-GY50 is an ortholog of Pcb1 in D. mccartyi strain

GY50 but has not been functionally characterized. The tree was constructed with MEGA 6

(Tamura et al. 2013) using the maximum likelihood method in the Jones-Taylor-Thornton (JTT)

model. Branch lengths indicate the number of substitutions per site



23.5.3 Phylogenetic Lineage of PCB RDases

Two of the PCB RDase enzymes, PcbA4 and PcbA5, which attack para- and meta

chlorines, respectively, are phylogenetically similar, sharing 97 % amino acid

sequence identity. The other, PcbA1, clusters in a distant clade and shares only 38 %

amino acid sequence identity with PcbA4 and PcbA5. No PCB RDase has been identified in strains 195 or JNA yet, but both of these strains have orthologs of PcbA4 and

PcbA5 that are potential candidates (Fig. 23.5) (Seshadri et al. 2005; Fricker et al.

2014). CBDB1 has no orthologs of either PcbA1 or the other two PCB RDases, so it

must use an enzyme of yet another lineage. These findings suggest the existence of at

least three different lineages of the PCB RDases in D. mccartyi. Moreover, the results

suggest considerable diversity of regiospecificity within lineages.



23.5.4 PCB Reductive Dechlorinases also Dechlorinate PCE

Transcriptome and enzyme activity assays prove that in addition to dechlorinating

PCBs, PcbA1, PcbA4, and PcbA5 dechlorinate PCE to TCE and then to cis- and



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