Lawrence Berkeley National Laboratory

The Environmental Stress Pathway Project (ESPP) aims to elucidate the molecular mechanisms by which microbial communities affect stress response and activity in the field and in the laboratory using sulfate reducing bacteria (SRB) as a model. To accomplish our goal of linking field observations to those in the laboratory, a systems-level understanding of SRB genome function is necessary. To meet this challenge, we are developing a mutagenesis and phenotyping strategy that is comprehensive across the genome and applicable to any microorganism amenable to transposon mutagenesis. Here we describe the application of our strategy to Shewanella oneidensis MR1 and the SRB Desulfovibrio desulfuricans G20. We have cloned and sequence-verified ~;;3000 barcode modules into a Gateway entry vector. Each module is a 175 base pair element containing two unique 20 base pair sequences, the UPTAG and DOWNTAG, flanked by common PCR priming sites. Each module can then be rapidly transferred in vitro to any DNA element, such as a transposon, that is made Gateway compatible. Transposon mutants marked by the modules will be sequenced to determine which of the ~;;3000 barcode modules was used and which gene was disrupted. Transposon mutants can be rapidly re-arrayed into a single pool containing ~;;3000 uniquely tagged, sequence-verified mutant strains. By sequencing saturating numbers of transposon mutants, we can identify and assay mutants in most nonessential genes in a given genome. The fitness of each mutant in the pool will be monitored in parallel by the hybridization of the barcodes to an Affymetrix microarray containing the barcode complements in a system identical to that used for the yeast deletion collection. Compared to other approaches for the parallel analysis of transposon mutants such as signature tagged mutagenesis, genetic footprinting, and transposon site hybridization, our approach offers much higher throughput, a single microarray design is universal for any organism, single mutational events are assayed, and mutant strains are archived for verification, distribution, and the systematic genetic interrogation of individual pathways. The successful completion of this project will enable the quantitative phenotypic analysis of thousands of mutants across a wide range of conditions. These data will be used to assign gene function on a global scale, aid in the identification of missing metabolic enzymes, and provide insight into the functional connectivity of different pathways. Our results in Desulfovibrio desulfuricans G20 will be extrapolated via comparative genomic analysis to other sequenced SRBs including Desulfovibrio vulgaris Hildenborough. Consequently, our findings will aid in the interpretation of both laboratory and field data collected by the ESPP.