Functional Redundancy in the Hydroxycinnamate Catabolism Pathways of the Salt Marsh Bacterium Sagittula stellata E-37

Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata. We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors. ABSTRACT The hydroxycinnamates (HCAs) ferulate and p-coumarate are among the most abundant constituents of lignin, and their degradation by bacteria is an essential step in the remineralization of vascular plant material. Here, we investigate the catabolism of these two HCAs by the marine bacterium Sagittula stellata E-37, a member of the roseobacter lineage with lignolytic potential. Bacterial degradation of HCAs is often initiated by the activity of a hydroxycinnamoyl-coenzyme A (hydroxycinnamoyl-CoA) synthase. Genome analysis of S. stellata revealed the presence of two feruloyl-CoA (fcs) synthase homologs, an unusual occurrence among characterized HCA degraders. In order to elucidate the role of these homologs in HCA catabolism, fcs-1 and fcs-2 were disrupted using insertional mutagenesis, yielding both single and double fcs mutants. Growth on p-coumarate was abolished in the fcs double mutant, whereas maximum cell yield on ferulate was only 2% of that of the wild type. Interestingly, the single mutants demonstrated opposing phenotypes, where the fcs-1 mutant showed impaired growth (extended lag and ∼60% of wild-type rate) on p-coumarate, and the fcs-2 mutant showed impaired growth (extended lag and ∼20% of wild-type rate) on ferulate, pointing to distinct but overlapping roles of the encoded fcs homologs, with fcs-1 primarily dedicated to p-coumarate utilization and fcs-2 playing a dominant role in ferulate utilization. Finally, a tripartite ATP-independent periplasmic (TRAP) family transporter was found to be required for growth on both HCAs. These findings provide evidence for functional redundancy in the degradation of HCAs in S. stellata E-37 and offer important insight into the genetic complexity of aromatic compound degradation in bacteria. IMPORTANCE Hydroxycinnamates (HCAs) are essential components of lignin and are involved in various plant functions, including defense. In nature, microbial degradation of HCAs is influential to global carbon cycling. HCA degradation pathways are also of industrial relevance, as microbial transformation of the HCA, ferulate, can generate vanillin, a valuable flavoring compound. Yet, surprisingly little is known of the genetics underlying bacterial HCA degradation. Here, we make comparisons to previously characterized bacterial HCA degraders and use a genetic approach to characterize genes involved in catabolism and uptake of HCAs in the environmentally relevant marine bacterium Sagittula stellata. We provide evidence of overlapping substrate specificity between HCA degradation pathways and uptake proteins. We conclude that S. stellata is uniquely poised to utilize HCAs found in the complex mixtures of plant-derived compounds in nature. This strategy may be common among marine bacteria residing in lignin-rich coastal waters and has potential relevance to biotechnology sectors.