A CRISPR interference system for the nitrogen-fixing bacterium Azotobacter vinelandii

Abstract

A grand challenge for the next century can be found in mitigating the effects of changing climate through bioengineering solutions. Biological nitrogen fixation, the globally consequential, nitrogenase-catalyzed reduction of atmospheric nitrogen to bioavailable ammonia, is a particularly vital area of focus. Nitrogen fixation engineering relies upon extensive understanding of underlying genetics in microbial models, including the broadly utilized gammaproteobacterium, Azotobacter vinelandii (A. vinelandii). Here we report the first CRISPR interference (CRISPRi) system for targeted gene silencing in A. vinelandii that integrates genomically via site-specific transposon insertion. We demonstrate that CRISPRi can repress transcription of an essential nitrogen fixation gene by [~]60%. Further, we show that nitrogenase genes are suitably expressed from the transposon insertion site, indicating that CRISPRi and engineered nitrogen fixation genes can be co-integrated for combinatorial studies of gene expression and engineering. Our established CRISPRi system extends the utility of A. vinelandii and will aid efforts to engineer microbial nitrogen fixation for desired purposes. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/565814v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@128b75forg.highwire.dtl.DTLVardef@1ecdf4org.highwire.dtl.DTLVardef@6363c6org.highwire.dtl.DTLVardef@12b8358_HPS_FORMAT_FIGEXP M_FIG C_FIG