The bamA gene for anaerobic ring fission is widely distributed in the environment

Abstract

Benzoyl-CoA is the signature central metabolite associated with the anaerobic metabolism of a diverse range of compounds such as humic acid, lignin, amino acids, and industrial chemicals. Aromatic chemicals with different upstream degradation pathways all funnel into the downstream benzoyl-CoA pathway. Different genes encoding enzymes of the benzoyl-CoA pathway could be used as biomarkers for the anaerobic benzoyl-CoA pathway, however, the ring opening hydrolase, encoded by the bamA gene, is ideal because it is detected under a range of respiratory conditions, including under denitrifying, iron-reducing, sulfate-reducing, and fermentative conditions. This work evaluated DNA samples from six diverse environments for the presence of the bamA gene, and had positive results for every sample. Individual bamA gene clones from these sites were compared to published genome sequences. The clone sequences were distributed amongst the genome sequences, although there were clone sequences from two of the analyzed sites that formed a unique clade. Clone sequences were then grouped by site and analyzed with a functional operational taxonomic unit based clustering program to compare the bamA gene diversity of these sites to that of several locations reported in the literature. The results showed that the sequence diversity of the sites separated into two clusters, but there was no clear trend that could be related to the site characteristics. Interestingly, two pristine freshwater sites formed a subgroup within one of the larger clusters. Thus far the bamA gene has only been examined within the context of contaminated environments, however, this study demonstrates that the bamA gene is also detected in uncontaminated sites. The widespread presence of the bamA gene in diverse environments suggests that the anaerobic benzoyl-CoA pathway plays an important role in the global carbon cycle that has thus far been understudied.

Keywords: 6-oxocylcohex-1-ene-1-carbonyl-CoA hydrolase; anaerobic benzoyl-CoA pathway; anaerobic hydrocarbon biodegradation; bamA gene; monoaromatic degradation.

FIGURE 1

A comparison of the benzoyl-CoA downstream metabolic pathways. Benzoyl-CoA (I) is dearomatized to cyclohexa- 1,5-diene-1-carbonyl-CoA (II). For Thauera, Azoarcus, and Geobacter the metabolites include 6-hydroxycyclohex-1-ene-1-carbonyl-CoA (III), 6-oxocyclohex-1-ene-1-carbonyl-CoA (IV), and result in 3-hydroxypimelyl- CoA (V). The corresponding enzymes are indicated below the arrows in the pathway. The pathway is modified from Carmona et al. (2009).

FIGURE 2

BamA amino acid sequence from characterized isolates, genome sequences, and environmental clones. This is a neighbor-joining tree based on a 97 amino acid sequence alignment and rooted to Azoarcus sp. CIB methyl-6-ketocyclohex-1-ene-1-carbonyl-CoA hydrolase. Bootstrap values are given for 1000 replicate trees, and the values greater than 50% are indicated. Clones in blue indicate marine sediment DNA extracts (Eel Pond and Wild Harbor), green indicates groundwater samples (Crosswicks Creek and Six Mile Run), and orange represents methanogenic sites (anaerobic digester and peat bog). Clade-1 contains genome sequences from Geobacter, Magnetospirillum, and Thauera sp. Clade-2 is unique to this study and only contains sequences from Eel Pond and Wild Harbor. Clade-3 has representatives from the Syntrophus (Clade-3A) and Azoarcus (Clade-3B) isolates, along with a several sulfate-reducing microorganisms.

FIGURE 3

A functional OTU-based cluster analysis of environmental samples based on location. Amino acid sequences from all published culture-independent surveys were compared over an 81 amino acid region of the BamA hydrolase. Sites were compared using mothur software (Schloss et al., 2009) with a 95% cutoff.