Project: Function and Importance of Marine Bacterial Transporters of Plankton Exometabolites

Project:

Observing expression of marine bacterial transporter systems with transcriptomic or proteomic tools can provide valuable information about the metabolomic environment. However, these ‘omics approaches are limited by the low rate of transporter gene annotation. Here, a barcoded, arrayed, mutant library of the marine bacterium Ruegeria pomeroyi DSS-3 is employed in high throughput screens to identify the target substrates of 13 transporter systems. A set of 156 isolated putative transporter mutants were screened for growth on minimal medium with 63 substrates, each as a sole carbon source. Mutants that demonstrated a growth defect on a specific substrate were selected for secondary, higher resolution, growth screening. Mutants that continued to demonstrate growth defect relative to the pooled-mutant library (pooled-BarSeq, used as an analog for wildtype) were screened for their ability to drawdown the target substate. Gene annotations were made when mutants of the given transporter demonstrated both growth and drawdown defects on the target substrate.

In addition to the isolated mutant screens, the pooled barcoded transposon mutant library (pooled-BarSeq) was grown on minimal medium with selected substrates, each as sole carbon source, such that the relative enrichment or depletion of each mutant could demonstrate its fitness cost associated with the loss of each disrupted gene when grown on each substrate. The results of pooled-BarSeq screens had mixed consistency with the isolated mutant screens, demonstrating the value of isolated mutants for transporter annotation.

Program:

The Center for Chemical Currencies of a Microbial Planet (C-CoMP) integrates research, education and knowledge transfer activities to develop a mechanistic understanding of surface ocean carbon flux within the context of a changing ocean and through increased participation in ocean sciences. C-CoMP supports science teams that merge biology, chemistry, modeling, and informatics to close long-standing knowledge gaps in the identities and dynamics of organic molecules that serve as the currencies of elemental transfer between the ocean and atmosphere.