Diversity of Benzylsuccinate Synthase-Like (bssA) Genes in Hydrocarbon-Polluted Marine Sediments Suggests Substrate-Dependent Clustering

Abstract

ABSTRACTThe potential of hydrocarbon biodegradation in marine sediments was determined through the detection of a functional biomarker, thebssAgene, coding for benzylsuccinate synthase, the key enzyme of anaerobic toluene degradation. EightbssAclone libraries (409 sequences) were constructed from polluted sediments affected by thePrestigeoil spill in the Atlantic Islands National Park and from hydrocarbon-amended sediment microcosms in Mallorca. The amplified products and database-derivedbssA-like sequences grouped into four major clusters, as determined by phylogenetic reconstruction, principal coordinate analysis (PCoA), and a subfamily prediction tool. In addition to the classicalbssAsequences that were targeted, we were able to detect sequences homologous to the naphthylmethylsuccinate synthase gene (nmsA) and the alkylsuccinate synthase gene (assA), thebssAhomologues for anaerobic 2-methylnaphthalene and alkane degradation, respectively. The detection ofbssA-like variants was determined by the persistence and level of pollution in the marine samples. The observed level of gene diversity was lower in the Mallorca sediments, which were dominated byassA-like sequences. In contrast, the Atlantic Islands samples, which were highly contaminated with methylnaphthalene-rich crude oil, showed a high proportion ofnmsA-like sequences. Some of the detected genes were phylogenetically related toDeltaproteobacteriacommunities, previously described as the predominant hydrocarbon degraders at these sites. Differences between all detectedbssA-like genes described to date indicate separation between marine and terrestrial sequences and further subgrouping according to taxonomic affiliation. Global analysis suggested thatbssAhomologues appeared to cluster according to substrate specificity. We observed undetected divergent gene lineages ofbssAhomologues, which evidence the existence of new degrader groups in these environments.