Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Related Publications
• Parameters
• Instruments
• Project Information
• Program Information
• Funding

Each column heading provides the treatment condition and replicate. The first 4 letters are substrate abbreviation (provided below) followed by the bacterial identifier. The bacterial identity is either "WT" indicating the wildtype analog (pooled-RBTnSeq library) or four numbers indicating the locus tag of the disrupted gene (e.g. "1234" represents a mutant of "SPO1234").  Finally, the underscore and lowercase letter ("_a",  "_b",  "_c",  "_d") indicates the biological replicate. 

Values are optical density at 600 nm.

Methods & Sampling

Mutant cultures were pre-grown overnight in ½ YTSS medium with 50 mg ml-1 kanamycin. Screens were performed in L1 minimal medium (Guillard and Hargraves 1993) (dx.doi.org/10.17504/protocols.io.jvccn2w) modified to a salinity of 20 ppt and amended with ammonium (3 mM), kanamycin (50 mg ml-1), and phosphorus as PO43- at 36 mM. Overnight cultures of individual mutants were washed 3 times in substrate free medium. Four replicate 200 µl mutant cultures were prepared by inoculating 2 ml of washed  overnight culture into 96 well plates containing 198 ml modified L1 medium and a single substrate at 8 mM carbon. As a positive control, four wells with the same medium were inoculated with washed overnight cultures of the pooled-RB-TnSeq library, used as a proxy for wild-type R. pomeroyi growth but harboring a transposon/kanamycin resistance gene insertion. Cultures were grown at 25oC in a Synergy H1 plate reader (BioTek, Winooski, VT, USA) shaking at 425 rpm for 68-72 h. OD600 readings were collected once each hour.

Data Processing Notes from Submitter:

The data were imported into Excel and compiled on a single sheet. Values are OD600, they have not been pathlength corrected, they have not been blank corrected. We recommend blanking each sample to its first time point.

BCO-DMO Processing Notes:

• Replaced spaces and special characters in column header names with underscores ("_")

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.

Functions carried out by microscopic inhabitants of the surface ocean affect every aspect of life on our planet, regardless of distance from the coast. Ocean phytoplankton are responsible for half of the photosynthesis on Earth, the first step in a complex system that annually withdraws 50 billion metric tons of carbon from the atmosphere to sustain their growth. Of this, 25 billion metric tons participate in a rapid cycle in which biologically reactive material is released into seawater and converted back into carbon dioxide by marine bacteria within hours to days. The chemical-microbe network at the heart of this fast cycle remains poorly constrained; consequently, its primary currencies and controls remain elusive; its sensitivities to changing ocean conditions are unknown; and its responses to future climate scenarios are not predictable. 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. C-CoMP fosters education, outreach, and knowledge transfer activities that engage students of all ages, broaden participation in the next generation of ocean scientists, and extend novel open-science approaches into complementary academic and industrial communities. The Center framework is critical to this mission, uniquely facilitating an open exchange of experimental and computational science, methodological and conceptual challenges, and collaborations that establish integrated science and education partnerships. With expanded participation in ocean science research and ocean literacy across the US society, the next generation of ocean scientists will better reflect the diverse US population.

Climate-carbon feedbacks on the marine carbon reservoir are major uncertainties for future climate projections, and the trajectory and rate of ocean changes depend directly on microbial responses to temperature increases, ocean acidification, and other perturbations driven by climate change. C-CoMP research closes an urgent knowledge gap in the mechanisms driving carbon flow between ocean and atmosphere, with global implications for predictive climate models. The Center supports interdisciplinary science teams following open and reproducible science practices to address: (1) the chemical currencies of surface ocean carbon flux; (2) the structure and regulation of the chemical-microbe network that mediates this flux; and (3) sensitivity of the network and its feedbacks on climate. C-CoMP leverages emerging tools and technologies to tackle critical challenges in these themes, in synergy with existing ocean programs and consistent with NSF’s Big Ideas. C-CoMP education and outreach activities seek to overcome barriers to ocean literacy and diversify participation in ocean research. The Center is developing (1) initiatives to expand ocean literacy in K-12 and the broader public, (2) ocean sciences undergraduate curricula and research opportunities that provide multiple entry points into research experiences, (3) post-baccalaureate programs to transition undergraduates into graduate education and careers in ocean science, and (4) interdisciplinary graduate student and postdoctoral programs that prepare the next generation of ocean scientists. The C-CoMP team includes education faculty who evaluate the impacts of education and outreach activities and export successful STEM initiatives to the education community. C-CoMP is revolutionizing the technologies for studying chemical transformations in microbial systems to build understanding of the outsized impact of microbes on elemental cycles. Open science, cross-disciplinary collaborations, community engagement, and inclusive practices foster strategic advances in critical science problems and STEM initiatives. C-CoMP science, education, and knowledge-transfer themes are efficiently addressed through a sustained network of scientists addressing critical research challenges while broadening the workforce that will tackle multi-disciplinary problems with academic, industrial and policy partners.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The Program's Data Management Plan (DMP) is available as a PDF document.