The photosynthetic bacterium Rhodopseudomonas palustris converts nitrogen gas (N2) to fertilizer ammonia (NH3) and also produces clean energy hydrogen gas (H2) from protons (H+) when it is grown anaerobically in nitrogen fixing medium with illumination, a condition that promotes the expression of active nitrogenase. Compared with quantitative real-time PCR (qRT-PCR) and the lacZ reporter system, two methods commonly used for in vivo study of nitrogenase regulation in photosynthetic bacteria, the fluorescent protein reporter system has advantages in terms of its simplicity and sensitivity. However, little is known concerning if the fluorescent protein reporter system can be used in bacterial cells that need to grow anaerobically. Here, we developed an RFP-based method to measure the nitrogenase gene expression in photosynthetic bacteria grown anaerobically. This method was able to determine the levels of both the genome-based and the plasmid-based nitrogenase expression under anaerobic conditions, providing a better method for in vivo study of gene expression affected by oxygen. The RFP reporter system developed here will promote a better understanding of the molecular mechanism of nitrogenase regulation and will be used on other genes of interest in a wider range of anaerobic bacteria.
Keywords: Rhodopseudomonas palustris; anaerobic reporter system; gene regulation; nitrogenase; photosynthetic bacteria; red fluorescent protein.