Metabolic analysis of the soil microbe Dechloromonas aromatica str. RCB: indications of a surprisingly complex life-style and cryptic anaerobic pathways for aromatic degradation
© Salinero et al; licensee BioMed Central Ltd. 2009
Received: 1 December 2008
Accepted: 3 August 2009
Published: 3 August 2009
Initial interest in Dechloromonas aromatica strain RCB arose from its ability to anaerobically degrade benzene. It is also able to reduce perchlorate and oxidize chlorobenzoate, toluene, and xylene, creating interest in using this organism for bioremediation. Little physiological data has been published for this microbe. It is considered to be a free-living organism.
The a priori prediction that the D. aromatica genome would contain previously characterized "central" enzymes to support anaerobic aromatic degradation of benzene proved to be false, suggesting the presence of novel anaerobic aromatic degradation pathways in this species. These missing pathways include the benzylsuccinate synthase (bss ABC) genes (responsible for fumarate addition to toluene) and the central benzoyl-CoA pathway for monoaromatics. In depth analyses using existing TIGRfam, COG, and InterPro models, and the creation of de novo HMM models, indicate a highly complex lifestyle with a large number of environmental sensors and signaling pathways, including a relatively large number of GGDEF domain signal receptors and multiple quorum sensors. A number of proteins indicate interactions with an as yet unknown host, as indicated by the presence of predicted cell host remodeling enzymes, effector enzymes, hemolysin-like proteins, adhesins, NO reductase, and both type III and type VI secretory complexes. Evidence of biofilm formation including a proposed exopolysaccharide complex and exosortase (epsH) are also present. Annotation described in this paper also reveals evidence for several metabolic pathways that have yet to be observed experimentally, including a sulphur oxidation (sox FCDYZAXB) gene cluster, Calvin cycle enzymes, and proteins involved in nitrogen fixation in other species (including RubisCo, ribulose-phosphate 3-epimerase, and nif gene families, respectively).
Analysis of the D. aromatica genome indicates there is much to be learned regarding the metabolic capabilities, and life-style, for this microbial species. Examples of recent gene duplication events in signaling as well as dioxygenase clusters are present, indicating selective gene family expansion as a relatively recent event in D. aromatica's evolutionary history. Gene families that constitute metabolic cycles presumed to create D. aromatica' s environmental 'foot-print' indicate a high level of diversification between its predicted capabilities and those of its close relatives, A. aromaticum str EbN1 and Azoarcus BH72.
D. aromatica strain RCB is a gram negative Betaproteobacterium found in soil environments . Other members of the Betaproteobacteria class are found in environmental samples (such as soil and sludge) or are pathogens (such as Ralstonia solanacearum in plants and Neisseria meningitidis in humans) and in general the genus Dechloromonas has been found to be ubiquitous in the environment.
A facultative anaerobe, D. aromatica was initially isolated from Potomac River sludge contaminated with BTEX compounds (benzene, toluene, ethylbenzene and xylene) based on its ability to anaerobically degrade chlorobenzoate . This microbe is capable of aromatic hydrocarbon degradation and perchlorate reduction, and can oxidize Fe(II) and H2S . Although several members of the Rhodocyclales group of Betaproteobacteria are of interest to the scientific community due to their ability to anaerobically degrade derivatives of benzene, D. aromatica is the first pure culture capable of anaerobic degradation of the stable underivitized benzene molecule to be isolated. This, along with its ability to reduce perchlorate (a teratogenic contaminant introduced into the environment by man) and inquiry into its use in biocells  has led to interest in using this organism for bioremediation and energy production. Since the isolation of D. aromatica, other species of Azoarcus have been found to possess the ability to anaerobically degrade benzene, but have not been genomically sequenced .
The pathway for anaerobic benzene degradation has been partially deduced , but the enzymes responsible for this process have yet to be identified, and remain elusive even after the intensive annotation efforts described here-in. Conversely, central anaerobic pathways for aromatic compounds described in various other species were not found to be present in this genome .
Three libraries (3 kb, 8 kb and 30 kb) were generated by controlled shearing (Hydroshear, Genomic Solutions, Ann Arbor, MI) of spooled genomic DNA isolated from D. aromatica strain RCB and inserted into pUC18, pCUGIblu21, and pcc1Fos vectors, respectively. Clonal DNA was amplified using rolling circular amplification http://www.jgi.doe.gov/ and sequenced on ABI 3700 capillary DNA sequencers (Applied Biosystems, Foster City, CA) using BigDye technology (Perkin Elmer Corporation, Waltham, MA). Paired end-reads  were used to aid in assembly, and proved particularly useful in areas of repeats.
The Phrap algorithm [8, 9] was used for initial assembly. Finishing and manual curation was conducted on CONSED v14 software , supplemented with a suite of finishing analysis tools provided by the Joint Genome Institute. In silico cross-over errors were corrected by manual creation of fake reads to guide the assembly by forcing the consensus to follow the correct path.
Gaps were closed through a combination of primer walks on the gap-spanning clones from the 3 and 8 kb libraries (identified by paired-end analysis in the CONSED software) as well as sequencing of mapped, unique PCR products from freshly prepared genomic DNA.
The final step required to create a finished single chromosomal sequence was to determine the number of tandem repeats for a 672 base DNA sequence of unknown length. This was done by creating the full tandem repeat insert from unique upstream and downstream primers using long-range PCR. We then determined the size of product (amplified DNA) between the unique sequences.
Protein sequence predictions/orfs
Annotation done at Oak Ridge National Laboratory consisted of gene calls using CRITICA , glimmer , and Generation http://compbio.ornl.gov/. Annotation at the Virtual Institute for Microbial Stress and Survival http://www.microbesonline.org used bidirectional best hits as well as recruitment to TIGRfam hidden Markov models (HMMs), as described in Alm et al. . Briefly, protein coding predictions derived from NCBI, or identified using CRITICA, with supplemental input from Glimmer, were analyzed for domain identities using the models deposited in the InterPro, UniProt, PRODOM, Pfam, PRINTS, SMART, PIR SuperFamily, SUPERFAMILY, and TIGRfam databases . Orthologs were identified using bidirectional unique best hits with greater than 75% coverage. RPS-BLAST against the NCBI COGs (Clusters of Orthologous Genes) in the CDD database were used to assign proteins to COG models when the best hit E-value was <1e-5 and coverage was >60%.
Each and every predicted protein in the VIMSS database http://www.microbesonline.org was assessed to compare insights obtained from recruitment to models from several databases (TIGRfams, COGs, EC and InterPro). Assignments that offered the most definitive functional assignment were captured in an excel spreadsheet with data entries for all proteins predicted in the VIMSS database. Extensive manual curation of the predicted protein set was carried out using a combination of tools including the VIMSS analysis tools, creation and assessment of HMMs, and phylogenomic analysis, as described [see Additional files 1, 2]. Changes in gene functional predictions and naming were captured in the excel spreadsheet, and predictions with strong phylogenetic evidence of function posted using the interactive VIMSS web-based annotation interface.
Phylogenomic analysis: Flower Power, SCI PHY and HMM scoring
Hidden Markov models were generated for a large subset of proteins of interest, as detailed [see Additional file 1], to predict functional classification with the highest confidence measures currently available. The HMMs allowed recruitment of proteins to phylogenetic tree alignments that most closely reflect evolutionary relatedness across species. The proteins were assembled within clades of proteins that are aligned along their full length (no missing functional domains), and that allow high confidence of shared function in each species.
Gene Family Expansion
A clustered set of paralogs [see Additional files 1, 2] was used to search for recent gene duplication events. After an initial assessment of the VIMSS gene information/homolog data, candidate proteins were used as seed sequences for Flower Power and internal tree-viewing tools or SCI-PHY analyses. These two approaches employed neighbor-joining trees using the Scoredist correction setting in the Belvu alignment editor, or the SCI-PHY utility and tree viewer. In either case resulting phylogenomic tree builds were reviewed, and contiguous protein alignments of two or more proteins from D. aromatica were considered to be candidates for a gene duplication event, either in the D. aromatica genome or in a predecessor species.
Resequencing to verify absence of plasmid structure
After finishing the D. aromatica genome, analysis of the annotated gene set revealed the notable absence of several anaerobic aromatic degradation pathways that were expected to be present, due to their presence in A. aromaticum EbN1 (an evolutionary near-neighbor, as determined by 16sRNA phylogeny). Because many catabolic pathways are encoded on plasmid DNA, we felt it was important to preclude this possibility. We re-isolated DNA from a clonal preparation of D. aromatica that experimentally supported anaerobic benzene degradation, using three different plasmid purification protocols, each based on different physical parameters. All three generated a single band of DNA. The protocol that generated the highest yield of DNA was used to create a complete, new library of 2 kb inserts, and the library was submitted to sequence analysis using the protocols previously cited.
Overview of Gene and Protein Features
The finished sequence for D. aromatica reveals a single circular, closed chromosome of 4,501,104 nucleotides created from 130,636 screened reads, with an average G+C content of 60% and an extremely high level of sequence coverage (average depth of 24 reads/base [see Additional file 3]). Specific probing for plasmids confirmed no plasmid structure was present in the clonal species sequenced, which supports anaerobic benzene degradation. It is noted however that the presence of two tra clusters (putative conjugal transfer genes; VIMSS582582-582597 and VIMSS582865-582880), as well as plasmid partitioning proteins, indicates this microbial species is likely to be transformationally competent and thus likely to be able to support plasmid DNA structures.
The Virtual Institute for Microbial Stress and Survival (VIMSS, http://www.microbesonline.org) and the Joint Genome Institute http://genome.jgi-psf.org/finished_microbes/decar/decar.home.html report 4170 and 4204 protein coding genes, respectively [see Additional file 3]. Cross-database comparisons were done to assure the highest probability of capturing candidate orfs for analysis. The majority of proteins are shared between data sets. Variations in N-termini start sites were noted, both between JGI and VIMSS datasets and between initial and later annotation runs (approximately 200 N-termini differences between four runs of orf predictions were noted for the initial two annotation runs, Joint Genome Institute's, done at Oak Ridge National Laboratories – ORNL, and VIMSS).
The most definitive functional classification, TIGRfams, initially defined approximately 10% of the proteins in this genome; as of this writing, 33% of predicted proteins in the D. aromatica genome are covered by TIGRfams, leaving 2802 genes with no TIGRfam classification [see Additional file 4]. Many proteins in the current and initial non-covered sets were investigated further using K. Sjölander's HMM building protocols (many of which are available at http://phylogenomics.berkeley.edu), to supplement TIGRfams. The Clusters of Orthologous Genes (COG) assignments were used for classification in the families of signaling proteins, but specific function predictions for these proteins also required further analyses. The metabolic and signaling pathways are discussed below, and the identity of orthologs within these pathways are based on analysis of phylogenomic profiles of clusters obtained by HMM analysis, with comparison to proteins having experimentally defined function.
Anaerobic aromatic degradation – absence of known enzymes indicates novel pathways
One of the more striking findings is the absence of known key enzymes for monoaromatic degradation under anaerobic conditions. One of the primary metabolic capabilities of interest for this microbe is anaerobic degradation of benzene. Fumarate addition to toluene via benzylsuccinate synthase (BssABCD) is recognized as the common mechanism for anaerobic degradation by a phylogenomically diverse population of microbes [14–16] and has been called "the paradigm of anaerobic hydrocarbon oxidation". Benzoyl CoA is likewise considered a central intermediate in anaerobic degradation, and is further catabolized via benzoyl CoA reductase (BcrAB) . Populated KEGG maps in the IMG and VIMSS databases, based on BLAST analyses, indicate the presence of some of the enzymes previously characterized as belonging to the Bss pathway in D. aromatica, yet more careful analysis shows the candidate enzymes to be members of a general family, rather than true orthologs of the enzymes in question. The majority of catabolic enzymes of interest for D. aromatica are not covered by TIGRfams or COGs families. For this reason Flower Power clustering, SCI-PHY subfamily clade analysis, and HMM scoring were used to ascertain the presence or absence of proteins of interest (for a detailed description, see Additional file 1). The most reliable prediction-of-function approaches for genomically sequenced protein orfs are obtained using the more computationally intensive HMM modelling and scoring utilities. This allows the protein in question to be assessed by phylogenetic alignment to protein families or sub-families with experimentally known function, providing much more accurate predictions [18, 19].
Anaerobic aromatic degradation enzymes in near-neighbor Aromatoleum aromaticum EbN1.
Proteins involved in the anaerobic aromatic pathways in Aromatoleum aromaticum str. EbN1
A. aromaticum EbN1 – representative protein used for HMM models
Azoarcus BH72 ortholog
D. aromatica RCB ortholog
VIMSS813888:pat (COG1448; EC 18.104.22.168)
3) benzyl alcohol/benzaldehyde
VIMSS816847:ald (COG1012; EC22.214.171.124)
VIMSS813733:pchC (EC: 126.96.36.199)
VIMSS813734:pchF (EC 188.8.131.52)
VIMSS816923:ppsA phenylphosphate synthase
VIMSS816681:hbcL1 4-hydroxybenzoate CoA ligase
Dch Had Oah
VIMSS813951:hbcL 3-hydroxybenzoate CoA ligase
For all pathways except the ubiquitous phenylacetic acid catabolic cluster, which is involved in the aerobic degradation of phenylalanine, and the PpcAB phenylphosphate carboxylase enzymes involved in phenol degradation via 4-hydroxybenzoate, all key anaerobic aromatic degradation proteins present in A. aromaticum EbN1 are missing from the D. aromatica genome (Table 1), and the majority are also not present in Azoarcus BH72. The lack of overlap for genes encoding anaerobic aromatic enzymes between these two species was completely unexpected, as both A. aromaticum EbN1 and D. aromatica are metabolically diverse degraders of aromatic compounds. In general Azoarcus BH72 appears to share many families of proteins with D. aromatica that are not present in A. aromaticum EbN1 (eg signaling proteins, noted below).
Anaerobic degradation of benzene occurs at relatively sluggish reaction rates, indicating that the pathways incumbent in D. aromatica for aromatic degradation under anaerobic conditions might serve in a detoxification role. Another intriguing possibility is that oxidation is dependent on intracellularly produced oxygen, which is likely to be a rate-limiting step. Alicycliphilus denitrificans strain BC couples benzene degradation under anoxic conditions with chlorate reduction, utilizing the oxygen produced by chlorite dismutase in conjunction with a monooxygenase and subsequent catechol degradation for benzene catabolism . A similar mechanism may account for anaerobic benzene oxidation coupled to perchlorate and chlorate reduction in D. aromatica. However, anaerobic benzene degradation coupled with nitrate reduction is also utilized by this organism, and remains enigmatic .
The extremely high divergence of encoded protein families in this functional grouping differs from the general population of central metabolic and housekeeping genes: Azoarcus BH72, Azoarcus aromaticum EbN1 and D. aromatica are evolutionarily near-neighbors within currently sequenced genomes, as defined both by the high level of protein similarity within house-keeping genes (defined by the COG J family of proteins), and 16sRNA sequence. Azoarcus BH72 and A. aromaticum EbN1 display the highest percent similarity between housekeeping proteins within this triad, with 138 of the 156 COG J proteins in A. aromaticum EbN1 displaying highest similarity to their BH72 counterparts. On average these two genomes display 83.5% amino acid identity across shared COG J proteins. D. aromatica is an outlier in the triad, with higher similarity to Azoarcus BH72 than A. aromaticum EbN1 (43 of D. aromatica's 169 COG J proteins are most homologous to A. aromaticum EbN1 orthologs with an average 71% identity, and 67 are most homologous to Azoarcus BH72 with an average 72% identity).
Comparative genomics have previously established that large amounts of DNA present in one species can be absent even from a different strain within the same species . In addition, the underestimation of the diversity of aromatic catabolic pathways (both aerobic and anaerobic) has been noted previously , and a high level of enzymatic diversity has been seen for pathways that have the same starting and end products, including anaerobic benzoate oxidation .
Aerobic aromatic degradation
The single predicted MhpA protein in D. aromatica (VIMSS584155), which is predicted to support an initial hydroxylation of a substituted phenol substrate, shares 64.4% identity to Rhodococcus OhpB 3-(2-hydroxyphenyl) propionate monooxygenase (GI:8926385) vs. 26.4% for Comamonas testosteroni (GI:5689247), yet the remainder of the ohp genes in the Rodococcus ohp clade do not share synteny with the D. aromatica mhp gene cluster.
Other aromatic oxygenases
Aromatic degradation in D. aromatica: Mono- and Di-oxygenases.
ferredoxin subunit of ring-hydroxylating dioxygenase
membrane protein; transport
cheY like protein
methyl-accepting chemotaxis protein
chemotaxis sensory transducer, membrane bound
EC184.108.40.206 Acetaldehyde dehydrogenase (acetylating)
2-hydroxymuconic semialdehyde dehydrogenase
NAD+-dependent dehydrogenase (EC220.127.116.11)
ring-cleaving extradiol dioxygenase
catechol 2,3 dioxygenase (18.104.22.168)
4-hydroxyphenylacetic acid catabolism pathway
S box domain
EAL domain containing protein (obsolete in current VIMSS database)
diguanylate phosphodiesterase; signaling
response regulator, tbuT family
activator of aromatic catabolism
2Fe-2S ferredoxin, iron-sulfur binding site
IPR000524: Bacterial regulatory protein GntR, HTH
ring-cleaving extradiol dioxygenase
catechol 2,3 dioxygenase (EC22.214.171.124)
2-hydroxymuconic semialdehyde dehydrogenase
NAD+-dependent dehydrogenase (EC126.96.36.199)
2-hydroxy-6-ketonona-2,4-dienedioic acid hydrolase
3-oxoacyl-[acyl-carrier-protein] reductase (EC188.8.131.52)
acetaldehyde dehydrogenase (acetylating; EC184.108.40.206)
One monooxygenase gene cluster, composed of VIMSS581514 to 581519 ('tbc2 homologs,' Fig. 2), is orthologous to the tbuA1UBVA2C/tmoAECDBF/touABCDEF/phlKLMNOP and tbc2ABCDEF gene families (from P. stutzeri, R. pickettii, and Burkholderia JS150). This gene cluster includes a transport protein that is orthologous to TbuX/TodX/XylN (VIMSS581520). Specificity for the initial monooxygenase is not established, but phylogenetic analysis places VIMSS581514 monooxygenase with near-neighbors TbhA , reported as a toluene and aliphatic carbohydrate monooxygenase (76.5% sequence identity), and BmoA , a benzene monooxygenase of low regiospecificity (79.6% sequence identity). The high level of similarity to the D. aromatica protein is notable. The region is also highly syntenic with, and homologous to, the tmo AECDBF (AY552601) gene cluster responsible for P. mendocina's ability to utilize toluene as a sole carbon and energy source .
Just downstream on the chromosome is a phc/dmp/phh/phe/aph-like cluster of genes, composed of the genes VIMSS812947 and VIMSS 581535 to 581540 ('tbc1 homologs,' Fig. 2). Overall, chromosomal organization is somewhat different for D. aromatica as compared to Ralstonia and Burkholderia. D. aromatica has a fourteen gene insert that encodes members of the mhp-like family of aromatic oxygenases between the tandem tbc 1 and 2-like oxygenase clusters (see Table 2), with an inversion of the second region compared to R. eutropha and Burkholderia. Clade analysis indicates a broad substrate phenol degradation pathway in this cluster, with high sequence identity to the TOM gene cluster of Bradyrhizobium, which has the ability to oxidize dichloroethylene, vinyl chlorides, and TCE [37, 38]. The VIMSS581522 response regulator gene that occurs between the two identified monooxygenase gene clusters shares 50.3% identity to the Thaurea aromatica tutB gene and 48.2% to the Pseudomonas sp. Y2 styrene response regulator (occupying the same clade in phylogenetic analysis). VIMSS581522 is likely to be involved in the chemotactic response in conjunction with VIMSS581521 (histidine kinase) and VIMSS581523 (methyl accepting chemotaxis protein), which would confer the ability to display a chemotactic response to aromatic compounds.
Overall, several mono- and di-oxygenases were found in the genome, indicating D. aromatica has diverse abilities in the aerobic oxidation of heterocyclic compounds.
There are several gene clusters indicative of benzoate transport and catabolism. All recognized pathways are aerobic. The benzoate dioxygenase cluster BenABCDR is encoded in VIMSS582483-582487, and is very similar to (and clades with) the xylene degradation (xyl XYZ) cluster of Pseudomonas.
There is also an hcaA oxygenase gene cluster, embedded in one of the mhp clusters (see cluster 5, Fig. 1). Specificity of the large subunit of the dioxygenase (VIMSS582049) appears to be most likely for a bicyclic aromatic compound, as it shows highest identity to dibenzothiophene and naphthalene dioxygenases.
Dechloromonas aromatica's sensitivity to the environment
Candidates for gene expansion in the D. aromatica genome.
Protein/protein family function
Number of duplicates
Number of triplicates
Signal transduction or regulatory – includes:
Nitrogen regulatory protein PII homolog
Hydrolase/transhydrogenase or hydratase
A relatively high level of diguanylate cyclase (GGDEF domain [40–42]) signaling capability is implied in D. aromatica by the presence of 57 proteins encoding a GGDEF domain (Interpro IPR000160 [see Additional file 5]) and an additional 10 with a GGDEF response regulator (COG1639) . E. coli, for comparison, encodes 19. This gene family also appears to have undergone recent expansion in this microbe's evolutionary history. Microbes having a large number of proteins or even a diverse array of COG T elements do not a priori encode a large number of GGDEF elements, as Stigmatella aurantiaca, Myxococcus, Xanthus DK1622 and Burkholderia pseudomallei, by contrast, have very large genomes with extensive COG T populations, yet each have 20 or fewer proteins identified as having GGDEF domains [see Additional file 5], and Prochloroccus spp. appear to have none. Conversely, Oceanospirillum has a relatively small genome, yet has 112 proteins identified as likely GGDEF domain/IPR000160 proteins. GGDEF/EAL domain response regulators have been implicated in root colonization in Pseudomonas putida (Matilla et al. 2007); in E. coli the GGDEF domain-containing YddV protein upregulates the transcription of a number of cell wall modification enzymes , and in point of fact, D. aromatica's VIMSS581804, a GGDEF domain containing homolog of the YddV E. coli protein, occurs upstream of a cluster of sixteen cell wall division proteins (encoded by VIMSS581805-581820).
Cellular interactions with community/environment – secretion
Fifteen transport clusters include a TolC-like outer membrane component, and recent gene family expansion is noted within several families of ABC transporters for this genome. TolC was originally identified in E. coli as the channel that exports hemolysin , and hemolysin-like proteins are encoded in this genome. Two groups of ABC transporters occur as a cluster of five transport genes; these five-component transporters have been implicated in the uptake of external macromolecules .
The presence of putative lytic factors, lipases, proteases, antimicrobials, invasins, hemolysins, RTXs and colicins near potential type I transport systems indicate that these might be effector molecules used by D. aromatica for interactions with host cells (eg. for cell wall remodeling). Iron acquisition is likely to be supported by a putative FeoAB protein cluster (VIMSS583997, 583998), as well as several siderophore-like receptors and a putative FhuE protein (outer membrane receptor for ferric iron uptake; VIMSS583312). Other effector-type proteins, likely to be involved in cell/host interactions (and which in some species have a role in pathogenicity ), are present in this genome. Adhesins, haemagglutinins, and oxidative stress neutralizers are relatively abundant in D. aromatica. A number of transporters occur near the six putative soluble lytic murein transglycosylases, indicating possible cell wall remodeling capabilities for host colonization in conjunction with the potential effector molecules noted above. Homologs of these transporters were shown to support invasin-type functions in other microbes . Interaction with a host is further implicated by: VIMSS581582, encoding a potential cell wall-associated hydrolase, VIMSS581622, encoding a predicted ATPase, and VIMSS3337824/formerly 581623, encoding a putative membrane-bound lytic transglycosylase.
Eleven tandem copies of a 672 nucleotide insert comprise a region of the chromosome that challenged the correct assembly of the genome, and finishing this region was the final step for the sequencing phase of this project (see Methods). Unexpectedly, analysis of this region revealed a potential open reading frame encoding a very large protein that has been variously predicted at 4854, 2519 or 2491 amino acids in size during sequential automated protein prediction analyses (VIMSS3337779/formerly 582095). This putative protein, even in its smallest configuration, contains a hemolysin-type calcium-binding region, a cadherin-like domain, and several RTX domains, which have been associated with adhesion and virulence. Internal repeats of up to 100 residues with multiple copies have also been found in proteins from V ibrio, C olwellia, B radyrhizobium, and S hewanella spp. (termed "VCBS" proteins as defined by TIGRfam1965).
Other potential effector proteins include: three hemolysin-like proteins adjacent to type I transporters, eight proteins with a predicted hemolysin-related function, including VIMSS583067, a hemolysin activation/secretion protein, VIMSS580979, hemolysin A, VIMSS583372, phospholipase/hemolysin, VIMSS581868, a homolog of hemolysin III, predicted by TIGRfam1065 to have cytolytic capability, VIMSS582079, a transport/hemolysin, and VIMSS581408, a general hemolysin. Five predicted proteins have possible LysM/invasin domains, including: VIMSS580547, 581221, 581781, 582766, and 583769. One gene, VIMSS583068, encodes a putative 2079 amino acid filamentous haemagglutinin, as well as a hasA-like domain, making it a candidate for hasA-like function (hasA is a hemophore that captures heme for iron acquisition ).
Type II secretion
Besides the constitutive Sec and Tat pathways, D. aromatica has several candidates for dedicated export secretons of unknown function, with 3–4 putative orthologs of PulDEFG interspersed with a lytic transglycosylase and a hemolysin (VIMSS582071-582085). The region from VIMSS581889 to VIMSS581897 includes pul DEFG type subunits and an exe A ATPase like protein. It is bracketed by signaling components comprised of a histidine kinase, adenylate cyclase, and a protein bearing similarity to the nitrogen response regulator gln G (VIMSS581898), which has been shown to be involved in NH3 assimilation in other species .
In addition, there is a nine-gene cluster that encodes several proteins related to toluene resistance (VIMSS581899 to 581906).
A pilus-like gene cluster (which can also be classified as type IV secretion) occurs in VIMSS580547-580553, encoding a putative lytic transglycosylase, ABC permease, cation transporter, pilin peptidase, pilin ATPase and PulF-type protein. This assembly resembles other pilin assemblies associated with attachment to a substrate, such as the pilus structure responsible for chitin/host colonization in Vibrio cholerae .
Another large pilus-like cluster (VIMSS584160-584173) occurs in close proximity to the mhp CEFDBAR oxygenase genes (see eg VIMSS584157, mhp R).
Type III secretion
D. aromatica has been shown to be chemotactic under various circumstances. The flagellar proteins (FliAEFGHIJKLMNOPQR, FlaABCDEFGHIJK) are followed by an additional cluster of 15 chemotaxis/signal transduction genes (VIMSS580462-580476), and homologs of FlhC and D regulatory elements required for the expression of flagellar proteins (VIMSS582640 and 582641) , identified by phylogenetic clustering, are also present. Since D. aromatica has a flagellum and displays chemotactic behavior, it is likely that the flagellar gene cluster is solely related to locomotion, though type III secretion systems can also encode dedicated protein translocation machineries that deliver bacterial pathogenicity proteins directly to the cytosol of eukaryotic host cells .
Type IV secretion
There are two copies of a twenty-one gene cluster that includes ten putative conjugal transfer (Tra) sex-pilus type genes in the D. aromatica genome (VIMSS582582-582601 and VIMSS582864-582884), indicating a typeIV secretion structure that is related to non-pathogenic cell-cell interactions .
Type VI secretion
A large cluster of transport proteins that is related to the virulence associated genetic locus HIS-1 of Pseudomonas aeruginosa and the VAS genes of V. cholerae [52, 53] includes homologs of hcp1, IcmF and clpV (as VIMSS583005, 582995 and 583009, respectively, in D. aromatica [see Additional file 6]). This IcmF-associated (IAHP) cluster has been associated with mediation of host interactions, via export of effector proteins that lack signal sequences . Further evidence for type VI secretion is found in the presence of three proteins containing a Vgr secretion motif modeled by TIGRfam3361, which is found only in genomes having type VI secretory apparatus. Though most bacteria that contain IcmF clusters are pathogenic agents that associate with eukaryotic cell hosts , it has been reported that the host interactions supported by this cluster are not restricted to pathogens .
The type IV pili systems might be involved in biofilm development, as interactions with biofilm surfaces are affected by force-generating motility structures, including type IV pili and flagella . Quorum sensing is a deciding input for biofilm formation, and the presence of an exopolysaccharide synthetic cluster lends further support for biofilm formation. Further, derivatives of nitrous oxide, which is an evident substrate for D. aromatica, are a key signal for biofilm formation vs cell dispersion in the microbe P. aeruginosa .
Cellular interactions with community – quorum sensing
Quorum sensing uses specific membrane bound receptors to detect autoinducers released into the environment. It is involved in both intra- and inter-species density detection [58, 59]. Cell density has been shown to regulate a number of cellular responses, including bioluminescence, swarming, expression of virulence factors, secretion, and motility (as reviewed in Withers et al. 2001 ).
D. aromatica encodes six histidine kinase receptor proteins that are similar to the quorum sensing protein QseC of E. coli (VIMSS580745, 582451, 582897, 583274, 3337577 (formerly 583538), and 583893), five of which co-occur on the chromosome with homologs of the CheY like QseB regulator, and two of which appear to be the product of a recent duplication event (VIMSS583893 & 3337577). Of the six QseC homologs, phylogenetic analysis indicates VIMSS582451 is most similar to QseC from E. coli, where the QseBC complex regulates motility via the FlhCD master flagellar regulators (VIMSS582640 and 582641). The presence of several qseC/B gene pairs indicates the possibility of specific responses that are dependant on different sensing strategies. In other species, expression of ABC exporters is regulated by quorum sensing systems ; gene family expansion is indicated in the ABC export gene pool as well as the qseC/B sensors in D. aromatica.
N-acyl-homoserine lactone is the autoinducer typical for gram negative bacteria , yet D. aromatica lacks any recognizable AHL synthesis genes. Ralstonia Betaproteobacteria likewise encode several proteins in the qse C gene family and display a diversity of candidate cell density signaling compounds other than AHL . The utility of having a diverse array of quorum sensing proteins remains to be determined, but appears likely to be associated with a complex, and possibly symbiotic, lifestyle for D. aromatica.
Cellular interactions with the environment – stress
Poly-hydroxyalkanoates (PHAs) store carbon energy, are synthesized from the catabolism of lipids, and constitute up to almost 90% of the dry weight of the Betaproteobacteria species Comamonas testosteroni . These lipid-like carbon/energy storage polymers are found in granular inclusions. PhaR candidate VIMSS583509 is likely to be a regulatory protein for PHA synthesis, and is found near other proteins associated with PHA granule biosynthesis and utilization in D. aromatica (VIMSS583511-513).
Phasins are relatively small proteins (180–200 aas) that have been shown to associate with PHA inclusions . There are six copies of phasin-type proteins, with indications of recent gene duplication for three of the phasin-type proteins (VIMSS581881, 582264, and 3337571 (formerly 583582)). There are also three homologs of the active subunit poly-B-hydroxybutyrate polymerase (PhaC orthologs) and two Pha reductase candidates present in a direct repeat, which is also found in Legionella pneumophila. Interestingly, one PhaC-like protein, VIMSS583511, is 70% identical to NodG of Azospirillum brasilense, a nodulation protein . No PhaA-like ketothiolase ortholog is present. The presence of an amplified gene pool for carbon storage granules in D. aromatica may confer the ability to survive under low nutrient conditions, and poly-3-hydroxybutyrate accumulation has recently been observed in A. aromaticum EbN1 cultures displaying reduced growth .
Inorganic polyphosphate storage appears likely, as both polyphosphate kinase (Ppk, VIMSS582444) and exopolyphosphatase (Ppx, VIMSS583870) are present. These genes are similar to those encoded in Pseudomonas aeruginosa, in that they are in disparate regions of the chromosome . Polyphosphate has been implicated in stress response due to low nutrients in the environment , and also in DNA uptake .
Phosphate transport appears to be encoded in a large cluster of genes (VIMSS581746-581752), and response to phosphate starvation is likely supported by the PhoH homolog VIMSS583854.
There is a large cluster of exopolysaccharide export (eps) associated genes, including a proposed exosortase (epsH, VIMSS582792). Presence of the eps family proteins (VIMSS582786, VIMSS582790-582801) indicates capsular exopolysaccharide production, associated with either host cell interactions (including root colonization ) or biofilm production in soil sediments . D. aromatica is one of a small number of species (19 out of 280 genomes assessed by Haft et al. ) that also encodes the PEP-CTERM export system. The PEP-CTERM signal, present in sixteen proteins in this genome, is proposed to be exported via a potential exportase, represented in this genome by epsH (VIMSS582792). Additionally, the presence of proteins encoding this putative exportase is seen only in genomes also encoding the eps genes.
D. aromatica closely reflects several metabolic pathways of R. capsulatus, which is present in the rhizosphere, and its assimilatory nitrate/nitrite reductase cluster is highly similar to the R. capsulatus cluster . Encoded nitrate response elements also indicate a possible plant association for this microbe, as nitrate can act as a terminal electron acceptor in the oxygen-limited rhizosphere. Alternatively, nitrous oxide (NO) reduction can indicate the ability to respond to anti-microbial NO production by a host (used by the host to mitigate infection ). Several gene families are present that indicate interactions with a eukaryotic host species, including response elements that potentially neutralize host defense molecules, in particular nitric oxide and other nitrogenous species.
Nitrate is imported into the cytosol by NasDEF in Klebsiella pneumoniae  and expression of nitrate and nitrite reductases is regulated by the nasT protein in Azotobacter vinelandii . A homologous set of these genes are encoded by the cluster VIMSS580377-580380 (NasDEFT), and a homolog of nar K is immediately downstream at VIMSS580384, and is likely involved in nitrite extrusion. Upstream, a putative nas A/nir BDC cluster (assimilatory nitrate and nitrite reduction) is encoded near the nar XL-like nitrate response element. VIMSS580393 encodes a nitrate reductase that is homologous to the NasA cytosolic nitrate reductase of Klebsiella pneumoniae . Community studies have correlated the presence of NasA-encoding bacteria with the ability to use nitrate as the sole source of nitrogen . The large and small subunits of nitrite reductase (VIMSS580391 nir B and VIMSS580390 nir D) are immediately adjacent to a transporter with a putative nitrite transport function (VIMSS580389 NirC-like protein). The NirB orf is also highly homologous to both NasB (nitrite reductase) and NasC (NADH reductase which passes electrons to NasA) of Klebsiella pneumoniae. HMMs created from alignments seeded by the NasB and NasC genes scored at 3.2e-193 and 4.0e-159, respectively, to the VIMSS580391 NirB protein. D. aromatica is similar to Methylococcus capsulatus, Ralstonia solanacearum, Polaromonas, and Rhodoferax ferrireducens for nas A, nir B and nir D gene clusters. However, the presence of the putative transporter nir C (VIMSS580389) shares unique similarity to the E. coli and Salmonella nir BCD clusters.
Putative periplasmic, dissimilatory nitrate reduction, which is a candidate for denitrification capability , is encoded by the nap DABC genes (VIMSS 3337807/581796-581799). A probable cytochrome c', implicated in nitric oxide binding as protection against potentially toxic excess NO generated during nitrite reduction , is encoded by VIMSS582015. Although most denitrifiers are free living, plant-associated denitrifiers do exist . There is no dissimilatory nitrate reductive complex nar GHIJ, but rather, NarG and NarH-like proteins are found in the evolutionarily-related perchlorate reductase alpha and beta subunits . These proteins are present in the pcr ABCDcld cluster, VIMSS582649-582652 and VIMSS584327, as previously reported for Dechloromonas species .
Ammonia incorporation appears to be metabolically feasible via a putative glu-ammonia ligase (VIMSS581081), an enzyme that incorporates free ammonia into the cell via ligation to a glutamic acid. An ammonium transporter and cognate regulator are likely encoded in the Amt and GlnK-like proteins VIMSS581101 and 581102.
Putative nitrogen fixation gene cluster in D. aromatica
FldA, flavodoxin typical for nitrogen fixation
NafY-1, nitrogenase accessory factor Y
NifB, nitrogenase cofactor biosynthesis protein
ferredoxin, nitric oxide synthase
Che-Y like receiver
UrtA urea transport
CynS cyanate lyase
S-box sensor, similar to oxygen sensor arcB
Protein of unknown function involved in nitrogen fixation
UrtB urea transport
UrtC urea transport
UrtD urea transport
UrtE urea transport
UreH urease accessory protein
Urea amidohydrolase gamma
Urea amidohydrolase beta
Urea amidohydrolase alpha/UreC urease accessory protein
UreE urease accessory protein
UreF urease accessory protein
UreG urease accessory protein
ferredoxin, subunit of nitrite reductase
NifH nitrogenase iron protein (EC220.127.116.11)
NifD nitrogenase molybdenum-iron protein alpha chain (EC18.104.22.168)
NifK nitrogenase molybdenum-iron protein beta chain (EC22.214.171.124)
NafY-2 nitrogenase accessory factor Y
NifW nitrogen fixation protein
Nitric oxide (NO) reductase
The chromosomal region around D. aromatica' s two nos Z homologs is notably different from near-neighbors A. aromaticum EbN1 and Ralstonia solanacearum which encode a nosRZDFYL cluster. D. aromatica's nosRZDFYL operon lacks the nosRFYL genes, and displays other notable differences with most nitrate reducing microbes. In D. aromatica, two identical nos Z reductase-like genes (annotated as nos Z1 and nos Z2, VIMSS583543 and VIMSS583547) are adjacent to two cytochrome c553s, a ferredoxin, and a transport accessory protein, and are uniquely embedded within a histidine kinase/response regulator cluster and include nosD and a napGH-like pair that potentially couples quinone oxidation to cytochrome c reduction. This indicates the NO response might be involved in cell signaling and as a possible general detoxification mechanism for nitric oxide.
The Epsilonproteobacteria Wolinella succinogenes is quite similar to D. aromatica for nitric oxide reductase genes (both have two nosZ genes, a nosD gene and a napGH pair in the same order and orientation ), but the W. succinogenes genome lacks the embedded signaling protein cluster. Further, nitric oxide reductase homologs NorDQEBC (VIMSS582097, 582100–582103), along with the cytochrome c' protein (VIMSS582015), which has been shown to bind nitric oxide (NO) prior to its reduction , are all present, and potentially act in detoxification roles. It has been shown that formation of anaerobic biofilms of P. aeruginosa (which cause chronic lung infections in cystic fibrosis) require NO reductase when quorum has been reached , so a role in signaling and complex cell behavior is possible.
W. succinogenes shares other genome features with D. aromatica. It encodes only 2042 orfs, yet has a large number of signaling proteins, histidine kinases, and GGDEF proteins relative to its genome size. It also encodes nif genes, several genes similar to virulence factors, and similarity in the nitrous oxide enzyme cluster noted above. W. succinogenes is evolutionarily related to two pathogenic species (Helicobacter pylori and Campylobacter jejuni), and displays eukaryotic host interactions, yet is not known to be pathogenic . The distinction between effector molecules causing a pathogenic interaction and a symbiotic one is unclear.
Nitrogen fixation capability in D. aromatica is indicated by a complex of nif-like genes (see Table 4), that include putative nitrogenase alpha (NifD, VIMSS583693) and beta (NifK, VIMSS583694) subunits of the molybdenum-iron protein, an ATP-binding iron-sulfur protein (NifH, VIMSS583692), and the regulatory protein NifL (VIMSS583623), that share significant sequence similarity and synteny to the free-living soil microbe Azotobacter vinelandii. D. aromatica further encodes a complex that is likely to transport electrons to the nitrogenase, by using a six subunit rnf ABCDGE-like cluster (VIMSS583616-583619, 583621 and 583622) that is phylogenomically related to the Rhodobacter capsulatus complex used for nitrogen fixation . There is a second rnf-like NADH oxidoreductase complex composed of VIMSS583911-583916, of unknown involvement (see Fig. 4). A. aromaticum EbN1 and Azoarcus BH72 each encode two rnf-like clusters as well.
Embedded in the putative nitrogen fixation cluster are two gene families involved in urea metabolism (Table 4). This includes the urea transport proteins (UrtABCDE) and urea hydrolase enzyme family (Ure protein family).
Hydrogenases associated with nitrogen fixation
Hydrogenase clusters associated with nitrogen fixation.
hydrogenase-1 small subunit
hydrogenase-1, nickel-dependent, large subunit
Ni/Fe-hydrogenase 1 b-type cytochrome subunit
hydrogenase expression/formation protein
hydrogenase assembly chaperone
nickel incorporation into hydrogenase-1 proteins
rubredoxin-type Fe(Cys)4 protein
HupJ/(similar to HoxT)
hydrogenase accessory protein
membrane-bound hydrogenase accessory protein
hydrogenase nickel insertion protein
hydrogenase accessory factor Ni(2+)-binding GTPase
hydrogenase maturation protein
periplasmic component, ABC transporter
signal transduction, GGDEF
hydrogenase-2 small subunit
Fe-S-cluster-containing hydrogenase component
cytochrome Ni/Fe component of hydrogenase-2
hydrogenase-2 large subunit
Ni, Fe-hydrogenase maturation factor
hydrogenase assembly chaperone
hydrogenase accessory protein
hydrogenase maturation protein
hydrogenase maturation protein
hydrogenase maturation protein
formation of active hydrogenase
response regulator with CheY domain (signal transduction)
regulatory [NiFe] Hydrogenase small subunit (sensor)
regulatory [NiFe] Hydrogenase large subunit (sensor)
histidine kinase with PAS domain sensor
VIMSS581384 encodes a homolog of the HoxA hydrogenase transcriptional regulator, which has been shown to be expressed only during symbiosis in some species . Regulation is indicated by homologs of NtrX (VIMSS581123) and NtrY (VIMSS581124); the NtrXY pathway comprises a two-component signaling system involved in the regulation of nitrogen fixation in Azorhizobium caulinodans ORS571 .
Carbon Fixation via the Calvin-Benson-Bassham cycle
The genes indicative of carbon fixation, using the Calvin cycle, are present in the D. aromatica genome. This includes Ribulose 1,5-bisphosphate carboxylase (RuBisCo, VIMSS581681), phosphoribulokinase (cbbP/PrkB, VIMSS581690), and a fructose bisphosphate (fba, VIMSS581693) of the Calvin cycle subtype. The RuBisCo cbbM gene is of the fairly rare type II form. D. aromatica CbbM displays a surprisingly high 77% amino acid identity to CbbM found in the deep-sea tube worm Riftia pachyptila symbiont . In a recent study of aquatic sediments, Rhodoferax fermentans, Rhodospirillum fulvum and R. rubrum were also found to possess the cbb M type II isoform of RuBisCo ; this sub-type is shared by a only a few microbial species.
Further putative Cbb proteins are encoded by VIMSS581680 & 581688, candidates for CbbR (regulator for the cbb operon) and CbbY (found downstream of RuBisCo in R. sphaeroides ), respectively.
The presence of the cbb M gene suggests the ability to carry out the energetically costly fixation of CO2, though such functionality has yet to be observed, and carbon dioxide fixation capability has been found in only a few members of the microbial community.
There is a potential glycolate salvage pathway indicated by the presence of two isoforms of phosphoglycolate phosphatase (gph, VIMSS583850 and 581830). In other organisms, phosphoglycolate results from the oxidase activity of RuBisCo in the Calvin cycle, when concentrations of carbon dioxide are low relative to oxygen. In Ralstonia (Alcaligenes) eutropha and Rhodobacter sphaeroides, the gph gene (cbbZ) is located on an operon along with other Calvin cycle enzymes, including RuBisCo. In D. aromatica, the gph candidates for this gene (VIMSS583850 and 581830), are removed from the other cbb genes on the chromosome in D. aromatica; however VIMSS581830 is adjacent to a homolog of Ribulose-phosphate 3-epimerase (VIMSS581829, rpe).
The cco SNOQP gene cluster codes for a cbb-type cytochrome oxidase that functions as the terminal electron donor to O2 in the aerobic respiration of Rhodobacter capsulatus . These genes are present in a cluster as VIMSS580484-580486 and VIMSS584273-584274; note that these genes are present in a large number of Betaproteobacteria.
Other carbon cycles, such at the reverse TCA cycle and the Wood-Ljungdahl pathways, are missing critical enzymes in this genome, and are not present as such.
Sulfate and thiosulfate transport appear to be encoded in the gene cluster composed of an OmpA type protein (VIMSS581631) followed by orthologs of a sulfate/thiosulfate specific binding protein Sbp (VIMSS581632), a CysU or T sulfate/thiosulfate transport system permease T protein (VIMSS581633), a CysW ABC-type sulfate transport system permease component (VIMSS581634), and a CysA ATP-binding component of sulfate permease (VIMSS581635).
In addition, candidates for the transcriptional regulator of sulfur assimilation from sulfate are present and include: CysB, CysH, and CysI (VIMSS582364, 582360 and 582362, respectively).
Conversely, the cytoplasmic SorAB complex  is not present in D. aromatica nor A. aromaticum EbN1, although it is found in several other Betaproteobacteria, including R. metallidurans, R. eutropha, R. solanacearum, C. violaceum, and B. japonicum.
Gene Family Expansion
To determine candidates for recent gene duplication events, extensive phylogenomic profile analyses were conducted for all sets of paralogs in the genome. Flower Power recruitment and clustering against the non-redundant Genbank protein set was done, and the resulting alignments were analyzed using the tree-building SCI-PHY or Belvu based neighbor-joining utilities. The alignment of two or more D. aromatica protein sequences in a clade such that they displayed higher % identity to each other than to orthologs present in other species was interpreted as an indication of a probable recent duplication event, either in the D. aromatica genome itself or in a progenitor species. Results of this analysis are shown in Table 3.
Potential gene family expansion is indicated in several functional groups, including the following: signaling proteins (including cAMP signaling, histidine kinases, and others), Mhp-like aromatic oxidation complexes, nitrogen metabolism proteins and transport proteins.
Most duplications indicate that a single gene, rather than sets of genes, were replicated. An exception is the Tra/Type IV transport cluster (VIMSS582581-582601 and VIMSS582864-582884) noted previously. In the protein sets for the histidine kinase/response regulator, duplication of histidine kinase appears to occur without duplication of the adjacent response regulator. The paralogs created by recent duplication events are typically found well-removed from one another on the chromosome, although some tandem repeats of single genes were noted. However, the highest percent identity was not found between pairs of genes in tandem repeats.
Discussion of results and analyses concerning aromatic degradation, various predicted metabolic cycles, secretion, signaling, quorum-sensing and gene family expansion are included in the relevant sections, above.
Dechloromonas aromatica strain RCB appears to support a highly complex lifestyle which might involve biofilm formation and interaction with a eukaryotic host. It lacks predicted enzyme families for anaerobic aromatic catabolism, though it supports degradation of several aromatic species in the absence of oxygen. The enzymes responsible for this metabolic function remain to be identified and characterized. It also encodes proteins suggestive of the ability to fix nitrogen and CO2, as well as thiosulfate oxidation. Converse to aromatic degradation, these enzymatic functionalities have yet to be experimentally demonstrated. In short, this genome was full of surprises.
The utility of TIGRfams and COGs families in these analyses cannot be overstated. New releases of TIGRfams during the course of this analysis provided new insights and identified new functionality (malonate degradation cluster, PEP-Cterm transport and the epsH putative translocon, and urea degradation all were identified in the TIGRfam 7.0 additions). The HMM model building and assessment utilized as the major annotation approach for this study was employed to cover those protein families of interest that are not currently covered by TIGRfams. We utilized K. Sjölander's modelling and analysis tools, which are highly similar to those used to produce TIGRfams models. Overall, the extensive use of HMMs during this analysis allowed high confidence in predicted protein function, as well as certainty that several families of previously characterized anaerobic degradation enzymes for aromatic compounds are not present (eg BssABCD and BcrAB).
KKS sincerely thanks Tanja Woyke for her very helpful suggestions and direction for creation of the tables and figures, Patrick Chain for helpful suggestions on the manuscript, Dan Kirshner for technical help on computational work, Nandini Krishnamurthy for building an internally clustered data-set of D. aromatica proteins as well as help with computational tools, Ching Shang for ideas regarding biochemical pathways, Paul Richardson for general support during the finishing phase and Frank W. Larimer for a cogent and extremely helpful critique of the manuscript. The genome finishing portion of this study was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Livermore National Laboratory under Contract No. W-7405-Eng-48, Lawrence Berkeley National Laboratory under Contract No. DE-AC02-05CH11231 and Los Alamos National Laboratory under Contract No. W-7405-ENG-36. The majority of the annotation was done as an independent project by KKS. Considerable intellectual support, computational and data analysis tools were provided by Adam Arkin, Katherine Huang, Morgan Price, Eric Alm, Dan Kirshner, and Kimmen Sjölander – sufficient gratitude cannot be expressed for their generous help.
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