A Red Fluorescent Protein Reporter System Developed for Measuring Gene Expression in Photosynthetic Bacteria under Anaerobic Conditions

Abstract

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.

Fluorescence Microscopy Study of the Intracellular Sulfur Globule Protein SgpD in the Purple Sulfur Bacterium Allochromatium vinosum

“…Figure 1 B shows that the cells were fully and homogeneously fluorescent after 70 to 80 min of exposure to air. Our results are fully consistent with experiments in the anoxygenic phototroph R. palustris , in which mCherry also became fluorescent when switched from anoxic to oxic conditions [ 32 ]. The spectroscopic properties of mCherry are the same irrespective of whether it is originally produced under aerobic or anaerobic conditions [ 32 ].…”

“…In the light, A. vinosum forms sulfur globules only under anaerobic conditions [ 20 ], while the red fluorescence protein mCherry needs molecular oxygen for the maturation of its chromophore. As pointed out by Jiang and coworkers [ 32 ], oxygen access into the fully folded barrel protein is of great importance, but may also trigger irreversible photobleaching and reduced photostability of a fluorescent protein [ 66 ]. In our experiments, the fluorescence signal of A. vinosum was fully recovered after exposure to oxygen for 60 min and was stably maintained at the maximum fluorescence intensity for at least 30 min.…”

“…Fluorescent reporter proteins, such as green fluorescent protein (GFP), are valuable non-invasive molecular tools for real-time in vivo imaging of living specimens and have the greatest potential to address these questions. One limitation of fluorescent proteins in pigmented phototrophic bacteria is signal quenching when they emit light at a wavelength absorbed by the pigments, as discussed for the anoxygenic phototrophic Alphaprotebacteria Rhodobacter capsulatus and Rhodopseudomonas palustris [ 31 , 32 ]. GFP is an example of a fluorescent protein that is incompatible with bacteriochlorophyll a and carotenoids, the pigments that drive photosynthesis not only in R. capsulatus and R. palustris but also in A. vinosum .…”

The Role of Glutamine Synthetase in Regulating Ammonium Assimilation and Iron-Only Nitrogenase Expression in a Photosynthetic Diazotroph

“…Given that glutamine is a key signal of the intracellular nitrogen status that regulates the expression of nitrogenase, GlnA1 could play an important role in nitrogenase regulation by modulating the glutamine biosynthesis in R. palustris . To examine if the deletion of glnA genes will induce the expression of nitrogenase in the presence of ammonium, a condition that inhibits the nitrogenase expression of the wild-type R. palustris , an RFP reporter system was firstly used to determine the levels of expression of the nitrogenase genes ( 13 ). When R. palustris Δ glnA1 mutant was grown in PM supplemented with molybdenum, the deletion of glnA1 induced the expression of Mo nitrogenase (NifHDK) ( Fig.…”

“…A 200 μL resuspended culture was then transferred to the 96-well clear and black plates for the measurement of OD 660 nm and relative fluorescence intensity by a microplate reader (Synergy TMH4, BioTek Instruments), respectively. When measuring the relative fluorescence of RFP, the excitation and emission wavelengths were set to 587 nm and 610 nm, respectively ( 13 ).…”