Identification and Expression of Benzylsuccinate Synthase Genes in a Toluene-Degrading Methanogenic Consortium

Benzylsuccinate synthase (BSS) initiates anaerobic toluene biodegradation,and BSS genes have been found in several nitrate- and iron-reducingorganisms. Here, two new putative bssA genes were identifiedin a methanogenic toluene-degrading culture. Transcription wasupregulated with toluene but not with benzoate, consistent withthe proposed function. These are the first bss sequences froma methanogenic culture.

INTRODUCTION

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Introduction

Anaerobic toluene biodegradation can be initiated by benzylsuccinatesynthase (BSS) (3, 4, 6, 7, 13, 15, 16, 21), and the genes encodingBSS and related enzymes (bssDCABE) have to date been sequencedfrom six denitrifying bacteria (1, 9, 12, 14, 15, 19) and oneiron-reducing microorganism (13). No BSS gene sequences fromany sulfate-reducing or methanogenic cultures have yet beenpublished. This goal of this study was to identify BSS genesequences in a highly enriched toluene-degrading methanogenicconsortium. We have previously shown that benzylsuccinate isproduced by this culture (4) but were unable to amplify BSSgenes by the use of published primers (5).

Identification of putative bssA sequences.

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Identification of putative bssa...

The toluene-degrading methanogenic enrichment culture used inthis study (4, 10, 11) has been maintained for almost 15 yearswith toluene as the sole carbon source and electron donor. GenomicDNA was extracted from the culture by use of an UltraClean soilDNA extraction kit (Mo Bio Laboratories Inc., Solana Beach,CA), and PCR amplification of putative bssA sequences was performedusing a variety of primer sets designed from alignments of knownsequences and incorporating degeneracies (Table 1). A partialtranscriptional map of the bss operon and of the target regionslabeled TR1 to TR5, flanked by different forward and reverseprimer sets, is shown in Fig. 1. PCR amplifications were performedusing RediTaq (New England BioLabs, Mississauga, ON), and conditionswere as follows: initial denaturation at 94°C for 5 minfollowed by 30 cycles of denaturation at 94°C for 1 min,primer annealing for 1 min (for annealing temperatures, seeTable 1), and chain extension for 1.5 min (1 min for primerset 5) at 72°C followed by a final extension step at 72°Cfor 10 min. All PCRs were carried out in a PTC-200 DNA Enginethermocycler (MJ Research Inc., Waltham, MA). For each primerset, three PCRs were carried out in parallel, and the productswere pooled and run on a 1% agarose gel stained with ethidiumbromide. Where only one band of the expected length was observed,the PCR products were purified using a QIAquick PCR cleanupkit (QIAGEN, Valencia, CA). Where more than one band was observed,the band of the expected length was excised from the gel andpurified using a QIAGEN gel purification kit (QIAGEN, Valencia,CA). Amplicons were cloned using a TOPO TA cloning kit (Invitrogen,Carlsbad, CA), according to the manufacturer's instructions,and DNA from 10 to 15 clones was sequenced using vector primersT7f and M13r. To obtain complete sequences of putative bssAor bssABE clones, internal primers designed with Primer3 software(17) were used. Partial sequences of putative bssA or bssABEamplicons were aligned and assembled using ChromasPro 1.15 (TechnelysiumPty Ltd., Australia), and GeneMark (8) was used to predict startand/or stop codons of the putative bssA, bssB, and bssE sequencesidentified.

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TABLE 1. Degenerate PCR primers designed to amplify putative bssA or bssABE sequences in the methanogenic toluene-degrading culture

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FIG. 1. Partial transcriptional map of the bss operon. The target regions (TRs) for PCR amplification and sequencing are shown under the map. The primers used to amplify TR5 were 1985f and 2524r. The two dotted vertical lines shown in the bssA box illustrate the approximate positions of nucleotides coding for conserved cysteine (positions 1464 to 1467 in the Thauera aromatica K172 bssA sequence) and glycine (positions 2472 to 2475 in T. aromatica K172) residues in this gene.

One putative sequence was assembled from amplicons obtainedwith all of the primer sets listed in Table 1. This contiguoussequence (GenBank: EF134966) includes a putative bssA (referredto as bssA-1, encoding a protein with 747 amino acids), a putativebssB (85 amino acids) and a putative partial bssE sequence (113amino acids). A second putative bssA sequence, referred to asbssA-2, was only found using primers for TR1 and could not beassembled with any other sequences. BLAST queries of translatedbssA-1 and bssA-2 sequences against the NCBI non-redundant translateddatabase using default parameters (2) revealed 78-84% similarityto the seven previously known BssA sequences, and the expectationvalue for each hit was 0.0. The translated sequences of bssA-1and bssA-2 were 83% similar to each other. As with the previouslyknown BssA sequences, the C-terminal region of BssA-1 was highlyconserved, and a conserved glycine residue was present at aposition corresponding to residue 825 in the Thauera aromaticastrain K172 BssA sequence. The conserved glycine residue ischaracteristic of all glycyl radical enzymes (GREs) (13), andis a part of the glycyl radical fingerprint motif RVxG(FWY)x6-8(FL)x4Qx2(IV)x2Rthat is found in most GREs (18) and in all BssA sequences knownto date. In addition to the conserved glycine motif in BssA-1,both BssA-1 and BssA-2 contained a conserved cysteine residueat the position corresponding residue 489 of the T. aromaticaK172 BssA sequence. This conserved residue is also characteristicof GREs (18).

Differential transcription of bssA.

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Differential transcription of...

For this part of the study, 60-ml portions of the methanogenicculture were transferred anaerobically into each of six 125-mlglass bottles sealed with Mininert (VICI Precision Sampling,Baton Rouge, LA) caps. Three bottles were amended with toluene(42 µmol/bottle), while the other three were amended withbenzoate (49 µmol/bottle), a downstream metabolite inanaerobic toluene degradation. Toluene and methane concentrationswere analyzed as described previously (11). RNA was extractedfrom all bottles when 50% of the toluene or benzoate was degraded,and all bottles contained approximately the same concentrationof methane. Total RNA was extracted and purified from 45 mlof culture as previously described (20) except that culturesamples were centrifuged at 15,000 x g and 4°C for 30 min.Reverse transcription was performed using random hexamers andSuperscript III reverse transcriptase (RT) (Invitrogen, Carlsbad,CA). Each reaction mixture (25 µl total volume) contained5 µl of 5x First Strand buffer, 250 ng random hexamers(Invitrogen), 50 nmol of deoxynucleoside triphosphates, 2 µmolof dithiothreitol, and approximately 2 µg of RNA. RT reactionswere carried out in RNase-free PCR tubes, using a PTC-200 DNAEngine thermocycler (MJ Research). The program was as follows:incubation at 65°C for 5 min and then 25°C for 5 minfollowed by addition of 1 µl of RT to each sample, annealingat 25°C for 10 min, reverse transcription at 50°C for2 h, and finally inactivation of the RT at 70°C for 15 min.A no-RT control was run for each RNA sample to ensure that noDNA contamination was present.

PCRs were carried out on all reverse transcription products,including those from the no-RT control reactions. For thesePCR experiments, primers with no degenerate bases were designedto specifically target each of the bssA sequences found in thisstudy (Table 2). A primer set was also designed to target the16S rRNA gene for a specific microorganism referred to as Eub-1—likelya Desulfotomaculum sp.—found previously to be active inthe culture (11). Primer specificity was confirmed by amplifyingculture DNA and checking amplicon sequences. Positive or negativeamplification of the putative genes was assessed by runningPCR products on a 1% agarose gel stained with ethidium bromideand looking for bands of the expected length.

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TABLE 2. PCR primers used for amplifying specific genes in the toluene-degrading consortium

RT-PCRs conducted with RNA from toluene-degrading cultures resultedin the amplification of bssA-2, while no amplification was observedin benzoate-degrading cultures (Fig. 2A). Thus, bssA-2 transcriptionwas upregulated in the presence of toluene relative to benzoate,consistent with involvement of this gene product in the conversionof toluene to benzoate. Surprisingly, although the bssA-1 sequencewas readily amplified from culture DNA, transcription of bssA-1in the presence of either toluene or benzoate was not observed(data not shown). To ensure that all RNA extractions and RTreactions were comparable between treatments, cDNA was amplifiedwith 16S rRNA primers for Eub-1. In all cases, bands correspondingto amplicons of expected sizes and of similar intensities wereobserved by gel electrophoresis (Fig. 2B). No amplificationwas observed in any of the no-RT control reactions.

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FIG. 2. Agarose gels showing amplicons from RT-PCRs performed using cultures amended with toluene (T) or benzoate (B). Amplification was carried out using specific bssA-2 primers (panel A) and Eub-1 (16S rRNA gene) primers (panel B) listed in Table 2. Expected amplicon lengths were $~$ 300 bp for all reactions. Control reactions shown: "T-RT" and "B-RT," controls without reverse transcriptase; +, culture DNA; –, no sample.

The differential transcription of bssA-2, combined with thebioinformatic analysis of the sequence, provides strong evidencethat bssA-2 encodes an enzyme that shares the same catalyticfunction as those encoded by BssA sequences from the nitrate-and iron-reducing microorganisms discovered to date. Althoughthe transcription of bssA-1 was not demonstrated in this study,the strong similarity of BssA-1 sequence to other known BssAsequences and the presence of putative bssB and bssE sequenceson the same operon as bssA-1 support the proposed function ofthis gene. Further studies examining transcription of this geneat very high or low toluene concentrations or in the presenceof other alkylbenzenes may help to determine the nature of therole of bssA-1 in the culture.

The identification of new bssA sequences from the methanogenicculture, combined with the existing sequence data obtained fromnitrate-reducing and iron-reducing microorganisms, expands ourknowledge of the diversity of bssA, bssB, and bssE genes inthe environment. As more bssA sequences are discovered, particularlyfrom strict anaerobes such as those found in this study, morecomprehensive primers and probes can be designed to track thegrowth and activity of alkylbenzene-degrading organisms in theenvironment and confirm biodegradation of toluene and otheralkylbenzenes in contaminated soil and groundwater.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The putative bssA and bssABE sequences have been deposited inGenBank under accession numbers EF134965 and EF134966.

ACKNOWLEDGMENTS

We thank Monika Ficker for her previous work enriching the methanogenictoluene-degrading culture and Charles Whang for developing theRNA extraction procedure used.

The research was supported by the Natural Science and EngineeringResearch Council of Canada through a discovery grant awardedto E.A.E. and a postgraduate scholarship awarded to C.E.W.

FOOTNOTES

* Corresponding author. Mailing address: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College St., Toronto, Ontario, Canada M5S 3E5. Phone: (416) 946-3506. Fax: (416) 978-8605. E-mail: edwards{at}chem-eng.utoronto.ca.

Published ahead of print on 1 December 2006.

REFERENCES

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