The sequencing and annotation of the genome of “Dehalococcoides ethenogenes
” 195, the first Dehalococcoides
bacterium sequenced, provided insights into its unique physiology (33
). The subsequent sequencing of the genome of Dehalococcoides
sp. strain CBDB1, which has different dechlorination abilities than strain 195 (1
), allowed a genetic comparison of two functionally different Dehalococcoides
). That study revealed a high degree of gene sequence conservation and synteny within central metabolism and information-processing systems (“housekeeping” genes). However, these genomes appear to be punctuated by regions of high plasticity, many of which contained putative reductive dehalogenase (RD) genes and integrated genetic elements (IEs), suggesting a high degree of evolutionary variability (23
). Many coding regions found in strain 195 are absent in strain CBDB1, including all nine predicted IEs in strain 195 and six putative RD genes, along with their predicted regulatory systems (23
). Other strains of Dehalococcoides
, specifically Dehalococcoides
sp. strain BAV1 (15
) (U.S. Department of Energy Joint Genome Institute [DOE JGI], completed genome), Dehalococcoides
sp. strain VS (6
) (DOE JGI, draft), and Dehalococcoides
sp. strain GT (35
) (DOE JGI, draft), have been isolated and selected for genome sequencing to further improve our understanding of the physiology and evolution of this unique group of microorganisms.
In addition to pure-culture studies, Dehalococcoides
-organism-containing enrichment cultures have also received significant attention (7
strains were found to be dominant members in a number of anaerobic TCE-dehalogenating enrichment cultures, including ANAS, an anaerobic microbial consortium enrichment culture derived from contaminated sediments taken from Alameda Navel Air Station in California that dehalogenates TCE to ethene (9
). ANAS contains two different strains of Dehalococcoides
, one that dechlorinates TCE and DCE to vinyl chloride (VC) and another that dechlorinates TCE, DCE, and VC to ethene (18
). The Dehalococcoides
strains in ANAS are functionally distinct from strain 195 in that they lack the ability to degrade PCE (31
). Because enrichment cultures can reflect the communities and microbial interactions occurring in the environment more closely than pure cultures, the study of enrichment cultures capable of complete reductive dechlorination, such as ANAS, may help to elucidate the natural microbial interactions required to support the degradation of chlorinated solvents in the environment. However, the complex nature of mixed microbial communities impedes the application of typical genetic and genomic-scale studies that might reveal crucial physiological processes.
Microarrays have been successfully applied to analyze intraspecies genome mutations in yeasts and bacterial pathogens (5
) but have not been widely used for genomic comparisons of microorganisms involved in environmental biodegradation. These studies revealed fundamental strain-specific differences. For example, genome rearrangements and deletions in strains of Yersinia pestis
and Yersinia pseudotuberculosis
are often a result of the recombination of insertion sequences and the acquisition of phage-related sequences (16
). The strain 195 genome, likewise, contains several IEs and phage-related genes (33
), making strain 195 an interesting candidate for comparative genomics studies. With an analogous framework, this study applies a microarray designed to query the whole genome of strain 195 to characterize the genomic content of the Dehalococcoides
-organism-containing ANAS enrichment. The results provide further insights into the diversity and evolution of this environmentally important microorganism.
MATERIALS AND METHODS
195 and Dehalobacter restrictus
PER-K23 (DSMZ strain number 9455) were grown in 100-ml-batch pure cultures with a defined mineral salts medium containing 5 mM acetate as the carbon source, H2
as the electron donor (added as H2
gas [80:20, vol/vol]), and 78 μmol TCE as the electron acceptor, as previously described (14
ANAS was enriched from contaminated soil obtained from Alameda Naval Air Station and has been functionally stable for over seven years, with the ability to reductively dechlorinate TCE completely to ethene. Previous clone library studies have shown that Dehalococcoides
bacteria comprise approximately 30% of the ANAS culture (31
). Lactate (25 mM final concentration) is supplied as both the carbon source and electron donor, while TCE (0.1 mM final concentration) is supplied as the terminal electron acceptor. The semibatch growth and maintenance procedures have been previously described (24
A 50-ml sample of the ANAS culture was collected after one 100 μM dose of TCE was degraded to VC and ethene. Cells were collected by centrifugation (12,000 × g for 3 min at 4°C), the supernatants were discarded, and the cell pellets were stored at −80°C until processing. ANAS genomic DNA (gDNA) was isolated from frozen cell pellets by using an UltraClean Mega Prep soil DNA kit (Mo Bio Laboratories, Carlsbad, CA) according to the manufacturer's instructions. DNA was stored at −80°C prior to further use.
D. restrictus and strain 195 cells were harvested from 10 100-ml batches per culture to ensure that a sufficient quantity of DNA could be extracted for application to microarrays. Cells were collected by vacuum filtration with hydrophilic Durapore membrane filters (0.22-μm pore size, 47-mm diameter; Millipores, Billerica, MA), and the filters were stored in 2-ml microcentrifuge tubes at −80°C until further processing. gDNA was isolated from the filters by using a phenol extraction method as follows. The 2-ml microcentrifuge tubes containing frozen filters were amended with 250 μl lysis buffer (200 mM Tris [pH 8.0], 50 mM EDTA, and 200 mM NaCl), 100 μl 10% sodium dodecyl sulfate, 1 g 100-μm-diameter zirconia-silica beads (Biospec Products, Bartlesville, OK), and 1.0 ml buffer-equilibrated phenol (pH 8.0) (Sigma-Aldrich, St. Louis, MO). The cells were lysed by heating the tubes to 65°C for 2 min, bead beating with a mini bead beater (Biospec Products) for 2 min, incubating for 8 min at 65°C, and bead beating again for an additional 2 min. Cellular debris was collected by centrifugation (12,000 × g for 5 min at 4°C), and the aqueous lysate was transferred to a new, DNase-free microcentrifuge tube. The aqueous lysate was extracted twice with 1 volume of phenol (pH 8.0)-chloroform-isoamylalcohol (125:24:1, vol/vol) and once with 1 volume of chloroform-isoamylalcohol (24:1, vol/vol) (Sigma-Aldrich). DNA was precipitated by adding 0.1 volume of 3 M ammonium acetate (pH 5.2) and 1 volume of 100% isopropanol. The precipitate was collected by centrifugation, washed once with 80% ethanol, and resuspended in 40 μl of nuclease-free water. Contaminating RNA was removed by RNase digestion with DNase-free RNase according to the manufacturer's instructions (Roche Applied Science, Indianapolis, IN). The purified DNA was stored at −80°C prior to further use.
The mass of DNA per volume was quantified by using a fluorometer (model TD-700; Turner Designs, Sunnyvale, CA) and a Quant-iT PicoGreen dsDNA assay kit (Invitrogen Molecular Probes, Carlsbad, CA) according to the manufacturer's instructions.
The microarrays targeting genes within the genome of Dehalococcoides ethenogenes
195 were produced by Affymetrix (Santa Clara, CA) as prokaryotic midi format (format 100) photolithographic microarray chips. Each target sequence is represented on the array as a collection of 11 probe pairs (22 total probes) consisting of 25-mer oligonucleotide probes which are distributed along the length of the respective gene and are collectively referred to as a probe set. Each chip contains 1,624 probe sets. To control for nonspecific hybridization, each probe pair consists of a perfect-match probe and a corresponding single-mismatch probe in which the 13th nucleotide consists of a base that would mismatch the target. While the perfect-match probe provides measurable fluorescence when sample binds to it, the paired mismatch probe is used to detect and eliminate any false or contaminating fluorescence within that measurement resulting from nonspecific hybridization (2
). Probe sets were designed to target 1,560 nonredundant genes from the genome of strain 195, including 48 of 51 structural-RNA-coding sequences and 1,512 of 1,514 of the predicted protein-coding sequences identified by Seshadri et al. (33
). The number of probe sets on the array is less than the total number of genes in the strain 195 genome because the genome contains a 33-gene duplication and a 22-gene triplication. In addition, 45 probe sets, 24 as positive controls and 21 as negative controls, were designed on the array to facilitate calibration and to resolve background signals. The positive-control probe sets targeted Bacillus subtilis
, and dap
. To generate control DNA, plasmids containing lys
, and dap
were purified from strains ATCC 87482, ATCC 87483, ATCC 87484, and ATCC 87486, respectively. The negative-control probe sets were designed to hybridize with specific Escherichia coli
and bacteriophage genes that were not expected to be present in the experimental cultures.
Sample preparation for microarray.
gDNA was prepared for application to the microarrays according to the protocols outlined in section 3 of the Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA), with minimal modifications. Briefly, purified gDNA (1 μg per array) was mixed with the positive-control plasmids (7.6 nM lys, 15.2 nM phe, 30.4 nM thr, and 114.0 nM dap) described above and fragmented to an average size of 50 to 200 bp by enzymatic digestion with amplification grade DNase I (Invitrogen Life Technologies, Carlsbad, CA). Each 50-μl fragmentation reaction consisted of gDNA and 0.02 units DNase I per μg DNA diluted in 1× One-Phor-All buffer (Amersham Biosciences). The mixture was incubated for 20 min at 25°C, followed by 10 min at 98°C to inactivate the DNase I. The fragmentation products were visualized on a 4% MetaPhor agarose (Cambrex Bio Science Rockland, Inc., Rockland, ME) electrophoresis gel run at 70 V for 2 h. A 25-μl amount of the fragmentation product was then used for further processing. The fragmentation products were biotin end labeled by adding 25 μl of fragmentation product to 10 μl of 5× reaction buffer (Affymetrix, Santa Clara, CA), 2 μl 7.5 mM GeneChip DNA-labeling reagent (Affymetrix), 2 μl terminal deoxynucleotidyl transferase (Promega), and 11 μl nuclease-free water, incubating at 37°C for 60 min, and adding 2 μl 0.5 M EDTA (pH 8.0) (Invitrogen Life Technologies, Carlsbad, CA) to stop the reaction. Microarray chips were prehybridized with 1× hybridization buffer (100 mM morpholineethanesulfonic acid [MES; Sigma-Aldrich], 1 M Na+ [Sigma-Aldrich], 20 mM EDTA [Sigma-Aldrich], 0.01% Surfact-Amps Tween 20 [Pierce Chemical]) in a chip-rotisserie oven for at least 20 min at 60 rpm and 45°C. The samples were denatured by being heated to 99°C for 5 min. Hybridization buffer was removed from each microarray chip and replaced with an equal volume of sample hybridization mixture (1× MES hybridization buffer, 7.8% dimethyl sulfoxide [Sigma-Aldrich], 0.1 mg/ml herring sperm DNA [Promega Corporation], 0.5 mg/ml bovine serum albumin [Invitrogen Life Technologies, Carlsbad, CA], 50 pM B2 control oligonucleotide [Affymetrix], and 1 μg fragmented, labeled DNA). The hybridization was carried out by incubation in a chip-rotisserie oven (Affymetrix) for 16 h at 45°C and 60 rpm. Following hybridization, the hybridization mixture was removed, and the microarrays were processed and scanned according to standard Affymetrix protocols.
gDNA from each culture was divided into replicate samples which were independently fragmented, labeled, and hybridized to the arrays. Two microarrays were processed for the positive control (strain 195), two for the negative control (D. restrictus), and five for ANAS (two analyses from one biological sample followed 1 year later by three analyses of a second biological sample).
The hybridization signal intensity for perfect match and mismatches in each probe set from each scan was computed by using Affymetrix GeneChip software and the MAS5 algorithm (3
). The data set of each microarray was normalized by scaling the signal intensities of the added positive controls to a target signal intensity of 2,500, thus allowing for comparisons between microarray chips. The GeneChip MAS5 software was used to implement the Wilcoxon signed-rank-based algorithm to evaluate the “presence” or “absence” of individual target sequences and return a statistical P
). A gene was considered “present” in a sample if each replicate probe set had a signal intensity greater than the highest signal intensity measured for the negative controls and a P
value of less than 0.05. Due to the stringency of the P
value criterion, only 8 genes total were designated absent due solely to the signal intensity criterion.
PCR confirmation of microarray results.
PCR primer sets were designed to target several selected protein-coding genes of strain 195. These primer sets were then used to test for the presence of these genes in the ANAS enrichment. PCR amplification was performed in reaction mixtures containing 20 to 100 ng of gDNA and (as final concentrations) 1× PCR buffer II (Perkin Elmer), 2.5 mM MgCl2, a 200 μM concentration of each deoxynucleoside triphosphate, a 300 nM concentration of each forward and reverse primer, and 0.025 U of AmpliTaq Gold (Perkin Elmer) per μl of reaction mix. The reaction mixtures were incubated in an Eppendorf Mastercycler gradient thermocycler (Eppendorf, Westbury, NY) at 94°C for 12 min (for initial denaturation and activation of AmpliTaq Gold), followed by 30 cycles at 94°C for 1 min, 50°C for 45 s, and 72°C for 2 min and then by a final extension period of 12 min at 72°C. Amplified DNA was processed by using an Agilent 2100 bioanalyzer. Visual detection of a band corresponding to a DNA fragment of expected size indicated the presence of a gene. The absence of a band of the expected fragment size indicated the absence of the gene. The PCR results were compared with the microarray probe hybridization results to support determinations of presence/absence.
Microarray data accession number.
This study represents the first application of microarrays to query the whole genome of Dehalococcoides bacteria, both in a pure and a mixed culture. The technology was validated with the successful detection of every gene targeted by the array when 1 μg of strain 195 gDNA was applied. The detection of high numbers of genes when 1 μg of gDNA of the multiple-Dehalococcoides-organism-containing mixed culture ANAS was applied to the array extends the usefulness of the array to mixed enrichment cultures and, potentially, environmental samples. The results obtained with traditional PCR techniques targeting more than 20 genes supported the microarray data. However, for a comprehensive analysis of the genetic capacity of Dehalococcoides organism-containing mixed cultures, PCR-based approaches are inadequate. Conversely, microarray technology provides a high-throughput method, requiring only small quantities of nucleic acids, to simultaneously examine every gene in these unique bacteria.
The challenges associated with applying DNA from microbial communities to microarrays targeting a single strain must be considered when analyzing the data in this study. For example, it is not possible to determine whether the probe signal intensities are the result of genes carried by individual or multiple strains within the community. In addition, lower overall signal intensities were observed for ANAS DNA than for strain 195 DNA. This result is the expected reflection of both the small portion of total DNA that is derived from Dehalococcoides
bacteria in ANAS (approximately 10% of total microbial cells) and nucleotide polymorphisms that cause imperfect hybridization to the probes (13
). These phenomena likely account for the large range of signal intensities observed for ANAS DNA in comparison to the range observed for strain 195. Also, when using strain-specific microarrays to query complex microbial communities, it is important to note that although genes present in both the isolate and the community can be detected, and genes present in the isolate that are not found in the community can be identified, genes that are present in the community but not in the isolate (such as vcrA
) will not be detected by this method.
The high degree of hybridization of gDNA from the ANAS enrichment culture to microarrays targeting the whole genome of strain 195 demonstrates the close evolutionary relationship between at least one of the strains within the ANAS enrichment culture and strain 195. Both cultures are able to dechlorinate TCE to ethene, with the distinction that VC and ethene are approximately equal end products for strain 195 while ANAS generates primarily ethene. The cultures share genetic sequences for most housekeeping and metabolic genes other than RD genes, as similarly found in a study comparing the genomes of strain 195 and strain CBDB1 (23
). The conservation of many of the sequences of various genes throughout different Dehalococcoides
strains facilitates the creation of Dehalococcoides
-specific probes. Such probes can be utilized in both laboratory and field applications to investigate the presence of Dehalococcoides
organisms but should not be used to infer the dehalogenation ability of Dehalococcoides
-organism-containing microbial communities, since different strains are known to have diverse RD contents (19
). The detection of genes encoding nitrogenase in ANAS is interesting since these genes are not present in the genomes of strains CBDB1 (23
) or BAV1, both of which are more genetically distant from strain 195 than are the Dehalococcoides
strains in ANAS. It is unclear whether the ancestor of these strains possessed nitrogenase and the branch with CBDB1 and BAV1 lost it or whether these genes were transferred to an ancestor of strain 195 and the Dehalococcoides
organisms in ANAS.
The results of this study identify significant genetic differences between the Dehalococcoides
strains in ANAS and strain 195 that may be responsible for functional differences in dehalogenation, such as the inability of ANAS to degrade PCE and the dissimilar degradation end products of the two cultures. Similar to comparisons with the genomes of strains CBDB1 (23
) and BAV1, the prominent genetic differences between strain 195 and Dehalococcoides
organisms in ANAS involve RD genes and associated transcriptional regulators, most of which exist in “islands” near the proposed origin of replication, and the absence of several MGEs identified as transposons or bacteriophages. Only 6 of the 19 genes in the strain 195 genome annotated to potentially encode A subunits of RDs are present in ANAS. As previously described (20
(DET0079) was detected. Interestingly, DET0088, a gene located near the tceA
gene that encodes only the C-terminal end of an RD and is missing a cognate B subunit anchoring gene and, therefore, is presumed to be nonfunctional, was detected in ANAS. This gene is not expressed in TCE-grown cells of strain 195 (10
) nor found in its proteome (29
), and its presence suggests that the truncated RD gene may occur in some Dehalococcoides
genomes because of its proximity to the highly functional tceA
gene. The pceA
gene, the product of which reduces PCE to TCE, is not useful for organisms in the TCE-grown ANAS culture and was not detected. Interestingly, 52% of strain 195 IE I is absent from the ANAS culture, yet two of the six RD genes detected in ANAS are located in this IE, suggesting that RD genes may be transferred in or out of a genome independently of IE carriers. RD genes and RD regulator genes, both with and without proximal MGEs, comprise a group of genes present in strain 195 that are not found in the Dehalococcoides
strains in ANAS. The high degree of conservation of housekeeping genes and the lack of conservation of RD genes and regulators between strain 195 and ANAS strains is consistent with the hypothesis that the RDs in Dehalococcoides
strains are commonly exchanged (19
), thus transferring the dechlorination abilities of the strains within an evolutionarily short time frame.
An unexpected result of the genomic comparison of strain 195 and the ANAS enrichment culture is found in amino acid transport and metabolism genes. Several genes of the tryptophan operon were not detected in the Dehalococcoides
strains in ANAS, although these genes were identified among the genes that are most highly conserved between strain 195 and strain CBDB1 (23
). These genes, however, are in a genetically active region of the genome proximal to RD genes and IE region VIII of Seshadri et al. (33
) and within atypical region F of Regeard et al. (30
), and a genetic rearrangement may have facilitated their loss. Indeed, several genes from this region are missing in the ANAS strains (Fig. 3
). It is possible that other microorganisms in ANAS provide tryptophan, obviating the need for the Dehalococcoides
strains to biosynthesize this amino acid and facilitating the evolutionary loss of these genes. The absence of genes in the tryptophan operon has not been described for any other Dehalococcoides
strains to date. Further investigation of potential tryptophan pathways in ANAS is needed for clarification.
This study also extends the use of whole-genome microarrays in environmental microbiology, demonstrating a tool to compare the genomes of isolates with enrichment cultures containing closely related strains. The ability to query complex microbial communities against a known genome to comprehensively identify homology between strains without a priori selection of individual sequences is a valuable tool. This technology has promise to provide insights into the physiology of environmental bacteria that are essential to the degradation of environmental contaminants in natural and engineered bioremediation systems.