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,
Lorenz Adrian,
and
Stephen H. Zinder*
Department of Microbiology, Wing Hall, Cornell University, Ithaca, New York 14853
Received 26 January 2007/ Accepted 15 May 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The genome sequence of strain 195 (30) revealed 17 RD-homologous genes in addition to tceA (designated DET0079) and pceA (designated DET0318; J. Magnuson, personal communication). Common features of RDs include the presence of a putative twin-arginine transport signal sequence used for transport into the periplasm of folded proteins that can contain prosthetic groups, iron-sulfur cluster-binding motifs, and an adjacent "B" RD gene predicted to encode a small hydrophobic protein proposed to serve as a membrane anchor. Two of the RD-homologous genes may not be functional. One (DET0162) contains a verified TGA stop codon that would truncate the predicted gene product from 488 to 59 amino acids and a shorter corresponding "B" gene (DET0163). DET0088 encodes a protein predicted to be 153 amino acids long, corresponding to the C termini of other RDs, and lacks a corresponding "B" gene. Sixteen of the 19 RD genes in D. ethenogenes have transcriptional regulator genes in close proximity, including pceA, suggesting they are transcriptionally regulated. The genome sequence of Dehalococcoides strain CBDB1 revealed 32 potential RD genes (15), 12 of which share up to 95.4% amino acid sequence identity with RDs from strain 195, suggesting a partial overlap of substrates. Multiple RD genes have been identified in Dehalococcoides strains FL-2, KB1 (mixed culture), and BAV1 (14, 14, and 10, respectively) (10, 35). Although sequenced Dehalococcoides genomes share high sequence similarity and synteny among "housekeeping" genes, isolates harbor different suites of RD genes and exhibit different dehalogenation spectra. Identifying which RD genes are expressed during dehalogenation of different substrates can provide insights into reductive dehalogenase function and their potential activities at contaminated sites.
Here we describe D. ethenogenes RD genes expressed during growth and the reductive dehalogenation of PCE, TCE, and 2,3-DCP by comparing RD gene expression and corresponding dehalogenation activities for strain 195. Expression and activity results indicated that tceA was under transcriptional control, and liquid chromatography-tandem mass spectrometry (LC/MS/MS) proteomic approaches identified TceA protein fragments only in the presence of PCE and TCE but not 2,3-DCP. pceA was the only intact RD gene expressed in cells grown with 2,3-DCP and is most likely the 2,3-DCP reductive dehalogenase, which suggests that PceA has broad substrate specificity.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Growth conditions.
D. ethenogenes strain 195 was cultured with PCE, TCE, or 2,3-dichlorophenol as previously described (1, 20). Briefly, culture inoculum sizes were 2% (vol/vol) in either 27-ml culture tubes, 120-ml serum vials, or 1,000-ml incubation containers containing 10, 50, or 500 ml of growth medium, respectively. Basal salts medium was amended with 2 mM acetate, a vitamin solution containing 0.05 mg of vitamin B12 per liter, 10% (vol/vol) filter-sterilized anaerobic digestor sludge supernatant, and 1% (vol/vol) mixed butyrate-PCE culture extract (20). Doses of PCE, TCE, and H2 were added as previously described (20), and a filter-sterilized stock solution of 2,3-DCP was added with a syringe at increasing doses of 30, 50, and 75 µM. Culture tubes were sealed with Teflon-coated butyl rubber stoppers and incubated at 35°C. Reductive dechlorination of ethenes was monitored using a Perkin-Elmer 8500 gas chromatograph with a flame ionization detector (8). Chlorophenols were analyzed using a high-pressure liquid chromatograph (Beckman Coulter, Fullerton, CA) equipped with an Alltima C8 3-µM-bead-diameter column (length, 53 mM; inside diameter, 7 mm; Alltech, Deerfield, IL) at ambient temperature. The solvent for isocratic elution was acetonitrile:water:glacial acetic acid (50:50:0.1) at 1.5 ml/min, and chlorophenols were detected by their absorbance at 220 nm. To monitor growth, cells were fixed with 25% formaldehyde, filtered onto 0.2-µm GTBP Isopore membrane filters (Millipore, Billerica, MA), and stained with 5 µg/ml 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride. All resting-cell assays and RNA and protein extractions were performed at near-maximum growth and dechlorination rates.
Nucleic acid extraction.
Prior to extraction, 40 ml of PCE-fed or 200 ml of 2,3-DCP-fed cultures were placed on ice for 30 min, centrifuged at 12,000 x g for 10 min at 4°C, and resuspended in 500 µl of sterile ultrapure RNase-free water. DNA extractions were performed according to the method of Fennell et al. (6) except for the elimination of a glass bead homogenization step. In short, cell pellets for RNA extractions were processed using the RNeasy minikit according to the manufacturer's instructions (QIAGEN, Valencia, CA). RNA was eluted with 50 µl RNase-free water and quantified by measuring absorbances at 260 and 280 nm with a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE). RNA samples were treated twice with RNase-free DNase 1 (Fisher Scientific, Rockville, MD) to eliminate contaminating DNA.
Reverse transcription and quantitative PCR.
First-strand cDNA synthesis reactions were performed with random hexamer primers using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Reactions were performed in 20-µl solutions containing 10 ng of RNA incubated at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min in a DNA Engine PTC-200 thermocycler (MJ Research, Hercules, CA). Quantitative PCR amplifications were performed on triplicate samples using an ABI 7000 Real Time PCR machine (Applied Biosystems, Foster City, CA). Individual reactions contained iQ SYBR Green Super Mix (Bio-Rad, Hercules, CA) with 1 ng of cDNA template and 200 nM of primer targeting one of the 19 RD genes or the gene encoding the RNA polymerase beta subunit (rpoB) (see Table S1 in the supplemental material). Primers were designed to target RD genes and not to amplify any other sequences in the D. ethenogenes genome. Specificity of each primer set was tested by PCR amplification and sequencing of amplified DNA. All primers were tested to amplify at a minimum of 97% reaction efficiency for each experiment. PCR amplifications were carried out with the following parameters: 95°C for 10 min and 30 cycles of 95°C for 15 s and 60°C for 1 min. Melting-curve analysis and amplicon sequencing were used to screen for primer dimers, and RNA samples incubated without reverse transcriptase did not lead to a PCR product, showing that no DNA was present. cDNA target amplifications were compared to DNA standards obtained by serial dilution of genomic DNA. RD expression levels were calculated from DNA standard curves generated during each run and with each primer pair and related to corresponding rpoB expression levels. In preliminary experiments, we found that using rpoB expression as a standard provided the most uniform and reproducible results compared to other potential standards, including atpA and the 16S rRNA gene. Rahm et al. (29) found that rpoB was highly expressed in mixed cultures containing D. ethenogenes that were actively reductively dechlorinating chloroethenes. Values presented and their standard deviations were taken from triplicate samples from at least two different cultures.
Mapping transcriptional start sites.
The transcription start sites (TSS) of genes tceA, pceA, infA (DET0497; initiation factor IF-1), and DET1407 were identified using the 5' rapid amplification of cDNA ends (5'-RACE) system according to the manufacturer's instructions (Invitrogen, Carlsbad, CA). A series of two or three nested primers was designed starting 20 bp downstream from the translational start codon (see Table S1 in the supplemental material). The PCR product was sequenced and aligned with the upstream region of the corresponding gene.
Proteomic analyses.
PCE- and 2,3-DCP-grown cell pellets were prepared from 500 ml of culture by centrifugation at 4°C for 30 min. The supernatant was discarded, and cell pellets were stored at –20°C. Crude pellet fractions were prepared by resuspension in 20 mM Tris, pH 7.4, passing of the cells through a French pressure cell at 55 MPa, and centrifugation for 10 min at 10,000 x g. Membrane-enriched material was obtained by transferring 1.5 ml of soluble fraction to new microcentrifuge tubes, and purified membrane material was pelleted at 104,000 x g using a tabletop ultracentrifuge. Membrane-enriched proteins were solubilized in 0.1% dodecylmaltoside in 20 mM Tris (pH 7.4), and approximately 5 to 10 µg of protein from PCE-grown cells and <1 µg of protein from 2,3-DCP grown cells was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel lanes in the regions estimated to be between 20 to 120 kDa were then divided into four sections, which were stored at –20°C for subsequent in-gel digestion and manual extraction. In-gel digestion and tryptic peptide extractions were performed following a protocol modified from the method of Shevchenko et al. (31).
Peptide and identification by nano-LC/MS/MS.
Samples were reconstituted in 15 µl of 0.1% formic acid with 2% acetonitrile prior to MS analysis. The nano-LC was carried out by using an LC Packings Ultimate integrated capillary high-performance LC system equipped with a Switchos valve switching unit (Dionex, Sunnyvale, CA). The gel-extracted peptides were injected using a Famous autosampler on a C18 µ-precolumn cartridge for on-line desalting and then separated on a PepMap C-18 RP nanocolumn, eluted in a 60-min gradient of 5% to 45% acetonitrile in 0.1% formic acid at 250 nl/min. The nano-LC was connected in-line to a hybrid triple quadrupole linear ion trap mass spectrometer, 4000 Q Trap, from ABI/MDS Sciex (Framingham, MA), equipped with a Micro Ion Spray Head ion source.
Data acquisition on the MS was performed using Analyst 1.4.1 software (Applied Biosystems) in the positive-ion mode for information-dependent acquisition (IDA) analysis. In IDA analysis, after each survey scan for m/z 400 to m/z 1550 and an enhanced-resolution scan, the three highest-intensity ions with multiple charge states were selected for MS/MS with rolling collision energy applied for detected ions based on different charge states and m/z values.
MS/MS data generated from nano-LC/electron spray ionization-based IDA analysis were interrogated using the ProID 1.4 search engine (Applied Biosystems) for database searching against the Dehalococcoides strain 195 database. One trypsin miscleavage, the carbobamidomethyl modification of cysteine, and a methionine oxidation were used for all searches. Initial protein identification was limited to peptide hits with >95% confidence. Subsequent identification was limited to at least one peptide with a Pro Group confidence score of >95 and at least one additional peptide with a Pro Group confidence score of >20.
Resting-cell assay of reductive dehalogenase activity.
Reductive dechlorination resting-cell assays were performed as described by Magnuson et al. and Nijenhuis et al. (18, 27) with the following modifications. Cells grown on PCE or DCP were concentrated 10- or 100-fold relative to their original volumes, respectively, and were prepared with Ti(III) citrate as a reducing agent. Assays were set up in 8-ml crimp-top vials with either 1 ml of cells for PCE and TCE reductive dechlorination assays or 2 ml of cells for the 2,3-DCP reductive dechlorination assay, along with 8.3 mM Ti(III) citrate, 57% hydrogen in the head space, and 0.5 to 1 mM chlorinated ethene or 20 to 50 µM 2,3-DCP. Dechlorination was monitored as described above for chlorinated ethenes and phenols.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transcription start sites for tceA and pceA.
We used 5'-RACE to determine TSSs for tceA, pceA, and two "housekeeping" genes not involved in reductive dehalogenation that are likely to be highly expressed. DET0497 is annotated as encoding translation initiation factor 1 (infA) and is highly expressed in growing cells of Escherichia coli (3), whereas DET1407 was found to be one of the most abundant membrane-associated proteins in a proteomic survey of strain 195 and was hypothesized to encode part of the S-layer cell wall (25). The TSSs of these genes were determined to be 86 to 156 bp upstream of the predicted translational start codons (Fig. 2A). Attempts to determine TSSs for DET0162 and rpoB were not successful.
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70 promoter site is TTGACa-(16 to 19 bp)-TAtAaT-(5 to 9 bp)-TSS, in which capitalized bases are present more than 50% of the time, and these two conserved hexamers are called –35 and –10 regions, respectively (16). All four upstream regions had acceptable –10 hexamers, all beginning with TA and ending with T. Moreover, the infA and DET1407 upstream regions had TGTG motifs one base upstream of the predicted –10 region, known to obviate the need for a strong –35 region in Bacillus subtilis (34), and tceA had a TG in the same position, which can play a similar role in E. coli (23). The only gene we examined with a suitable –35 region was infA, which had the important TTG as the first three bases. DET1407 had essentially no match near –35 but curiously had a TTGACA sequence located only 10 bp upstream of the –10 region. The potential –35 region for tceA had a 4/6-bp match but lacked a beginning T, and only a 2/6 match could be found for the pceA –35 region. Matches to other potential sigma factor binding sites were not apparent.
We also examined the leader regions between the TSS and the translation start sites for homology with other potential leader regions. BLAST analyses revealed the upstream region of tceA from strain 195 to be nearly 100% identical with upstream regions of close tceA homologues sequenced from diverse Dehalococcoides enrichments and from strain FL2 (14) (data not presented). Unfortunately, these sequences began downstream of the predicted –10 and –35 regions, so that these regions could not be compared. Comparison of the upstream regions of tceA and the vcrA vinyl chloride RD of Dehalococcoides strain VS (26) revealed 43% sequence identity overall, including low identity in the predicted promoter region (Fig. 2A), which contains a canonical –35 hexamer. Interestingly, there is a conserved stretch of 15 nucleotides with a single T-to-C transition mutation in approximately the same location within the two leader regions (Fig. 2B). The region upstream of the predicted translation start site of pceA is ca. 80% identical over 200 bases with that of CbdbA1588 from Dehalococcoides strain CBDB1, with only five nucleotide differences found in the promoter region (data not shown). The nucleotide and predicted amino acid sequences of the two genes are 86.1% and 93.7% identical, respectively.
Detection of RDs by LC/MS/MS proteomics.
Proteomic analyses were performed on PCE- and 2,3-DCP-grown cells to detect RD polypeptides (24). Similar to previous results (24, 25), TceA and PceA RDs were detected with high peptide coverage in membrane-enriched fractions from PCE-grown cells and were among the top five polypeptides in terms of peptide coverage, along with the products of three genes annotated as cochaperonin GroEL (DET1428), BNR/Asp box repeat domain protein (DET1407), and formate dehydrogenase (DET0187) (Fig. 3). Twenty-three unique peptides corresponding to TceA (54% coverage) and 25 unique peptides corresponding to PceA (55% coverage) were identified in PCE-grown membrane-enriched protein fractions, and no peptides from other RDs were detected. The same dominant proteins were identified in membrane-enriched fractions from 2,3-DCP-grown cells, with the notable exception of TceA. Although fewer proteins were detected and overall peptide coverage was lower in samples from 2,3-DCP-grown cells, which contained less protein than those from PCE-grown cells because the cultures reached lower cell densities, 19 unique peptides corresponding to PceA (51% coverage) were identified, whereas no peptides from other RDs were detected.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transcript levels for pceA were higher relative to those for rpoB in 2,3-DCP-grown cells than they were in PCE-grown cells. Moreover, PceA was the only RD with significant peptide coverage in proteomic analyses of 2,3-DCP-grown cells. In contrast, tceA transcript levels were more than 2 orders of magnitude lower than those of rpoB, peptides from TceA were not detected in 2,3-DCP-grown cells, and 2,3-DCP-grown cells did not have detectable TCE RD activity, indicating that tceA is not expressed in 2,3-DCP-grown cells.
It is not surprising that evidence for regulation of expression of RD-homologous genes in Dehalococcoides spp. is beginning to accumulate, since many are located adjacent to genes predicted to encode transcriptional regulators (15, 30). Johnson et al. (13), in their studies of a TCE-grown enrichment culture from Alameda Naval Air Station that contained Dehalococcoides spp., reported increased levels of tceA mRNA in starved cells given TCE, cis-DCE, trans-DCE, or 1,1-DCE but not PCE or VC. These findings suggest that the molecular mechanism of control over tceA is finely tuned to recognize specific halogenated compounds. This culture did not use PCE and therefore could not produce TCE potentially needed to induce tceA. In strain 195, tceA was induced in cells growing on TCE or PCE (which is metabolized to TCE) but not in cells growing on 2,3-DCP. Curiously, unlike most other RD genes, there are no genes with strong resemblances to transcription regulators adjacent to tceA. Of the adjacent genes, DET0080 is annotated as having unknown function (www.tigr.org/tdb/mdb) but has similarity to genes encoding the ArsR family of regulators below the noise cutoff in a hidden Markov model search. While this gene product is a possible candidate for regulating tceA expression, it is also possible that regulatory circuits encoded elsewhere in the chromosome play a role in regulation.
These results also suggest that the PceA RD is responsible for dechlorination of 2,3-DCP. It has the highest transcript levels in 2,3-DCP-grown cells, and it was detected with high peptide coverage, whereas no other potential RD was detected. Further bolstering this proposition is the finding that PCE-grown cells dechlorinated 2,3-DCP without a lag (Fig. 4), indicating the appropriate reductive dehalogenase was already present. In an analogous experiment, 2,3-DCP-grown cells dechlorinated PCE to TCE without a lag but did not dechlorinate TCE. In Desulfitobacterium strain PCE1, a member of the Firmicutes, 2-chlorophenol and PCE dechlorination are carried out by distinct RD enzymes with little cross-reactivity towards the other substrate (33). However, the 2-chlorophenol RD from this organism does not require adjacent chlorines, as are present 2,3-DCP and PCE, so that it clearly has different substrate specificity from the 2,3-DCP-dechlorinating RD in strain 195, and its gene sequence is phylogenetically distinct from pceA. In light of the large number of potential RD genes present in the D. ethenogenes genome (30) with unknown function, it is surprising that PceA rather than one of the other RDs uses 2,3-DCP, but this finding does suggest an evolutionary route for Dehalococcoides spp. to take from utilizing chlorophenols, considered to be naturally occurring substrates, especially in soils (4), to utilizing PCE, considered a xenobiotic.
Dehalococcoides strain CBDB1 is adept at dehalogenating chlorinated aromatics (2), including chlorophenols (1). Similar to strain 195, strain CBDB1 reductively dehalogenates 2,3-DCP from the ortho position and reductively dehalogenates PCE to TCE and trans-dichloroethene (L. Adrian, personal observation). Strain CBDB1 can also reductively dehalogenate higher chlorinated chlorophenols, such as 2,3,4-trichlorophenol, from the meta position. In strain 195, the translated sequence of pceA has 93.7% amino acid identity with CbdbA1588 from strain CBDB1 (15). Downstream of cbdbA1588 are predicted histidine kinase (CbdbA1590) and response regulator (CbdbA1589) genes of a two-component regulatory system that have 92.1% and 91.1% amino acid identities to their homologues associated with pceA in strain 195. Morris et al. (24) recently found that in cultures of strain CBDB1 grown on 2,3-DCP, CbdbA1588 peptides were detected with 31% coverage, while in contrast to strain 195, CbdbA080, homologous to DET1559, was detected with 13% peptide coverage and CbdbA088, with no homologues in strain 195, was detected with 10% peptide coverage. This result suggests that the Cbdb1588 serves as a 2,3-DCP reductive dehalogenase in strain CBDB1, but with the detection of multiple RDs, the situation may be more complicated in this organism.
Since little is known about transcription initiation and regulation in Dehalococcoides spp., we determined the TSSs and examined upstream regions of two expressed RD genes, tceA and pceA, and two "housekeeping" genes expected to be highly expressed, infA and DET1407. The genome of strain 195 contains a gene predicted to encode a 70 homologue (DET0551) (17), as well as two smaller genes (DET0169 and DET1348) predicted to encode W and extracellular sigma factor homologues, respectively, which usually regulate accessory functions. The predicted amino acid sequence of DET0551 contains the conserved residues within its region 4.2 that are involved in nucleotide contact with the –10 and –35 hexamers in other 70 homologues (5, 9, 22), suggesting that its recognition sequences should resemble the canonical ones. Thus, it is reasonable to expect some housekeeping genes and other genes to show 70 consensus binding sites. In all four genes, there was a reasonably good match to the consensus –10 70 binding site, but only infA had a close match to the –35 consensus sequence, as does the vcrA gene from Dehalococcoides strain VS (Fig. 2A). Both infA and DET1407 have potential extended –10 regions with a TGTG motif (34), and it is likely that this allows DET1407 to be transcribed in the absence of an acceptable –35 region. The region upstream of the tceA TSS has a TG motif extending its –10 region, which may allow transcription despite the moderate match of its –35 region (23), whereas the pceA gene has no TG element and has a poor –35 match. For both RD genes, binding by an activator may be needed for transcription, in the case of pceA, perhaps the response regulator of the two-component system predicted to be encoded by DET0315 and DET0316 adjacent to it. Finally, the conserved 15-bp sequence in the leader regions of tceA from strain 195 and vcrA from strain VS may bind homologous regulatory proteins.
Since DET0162 was not considered to encode a functional RD, it was surprising that it showed high transcript levels in cells grown in PCE, TCE, or 2,3-DCP; however, peptides from its translation product were not detected in any cells in this or a previous (25) study, suggesting either that it is not translated or that the translation product is unstable, and it is therefore a nonfunctional pseudogene in the process of degradation. It was recently suggested that pseudogenes are a common feature of microbial genomes (28). While we were unable to identify a transcription start site for DET0162 using 5'-RACE, there is an acceptable –10 region 108 bases upstream of the predicted translation start site, in similarity to other genes (Fig. 2), with TTGACA, a perfect –35 region match, 16 bases upstream of that, making its transcription possible. Why this presumably nonfunctional gene showed such high transcript levels is unclear.
The list of potential RD genes with unknown functions in Dehalococcoides spp. has increased rapidly over the past several years, while the list of halogenated substrates has increased at a slower pace. Dehalococcoides spp. grow to low densities, making traditional protein purification techniques and genetic analyses of RDs difficult, but PCR-based measurements of transcripts and sensitive proteomic techniques allowed us to identify RD genes expressed in D. ethenogenes cells using different electron acceptors, and these techniques were supplemented by measuring RD enzymatic activity in cells. This approach should continue to be useful in identifying RDs involved in use of other substrates by this microbial group important to bioremediation.
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ACKNOWLEDGMENTS |
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Helpful comments by J. Helmann and help with proteomic analyses from S. Zhang are gratefully acknowledged.
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FOOTNOTES |
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Published ahead of print on 18 May 2007.
Supplemental material for this article may be found at http://aem.asm.org/.
Present address: Ecology, Evolution, and Marine Biology, University of California, Santa Barbara, CA 93106.
Present address: FG Technische Biochemie, Technical University of Berlin, Berlin, Germany.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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70 family: sequence conservation and evolutionary relationships. J. Bacteriol. 174:3843-3849.This article has been cited by other articles:
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