FNR homologues constitute an important class of transcription factors that control a wide range of anaerobic physiological functions in a number of bacterial species. Since FNR homologues are some of the most pervasive transcription factors, an understanding of their involvement in regulating anaerobic gene expression in different species sheds light on evolutionary similarity and differences. To address this question, we used a combination of high throughput RNA-Seq and ChIP-Seq analysis to define the extent of the FnrL regulon in Rhodobacter capsulatus and related our results to that of FnrL in Rhodobacter sphaeroides and FNR in Escherichia coli.

Several Gram-negative species undergo development leading to the formation of metabolically dormant desiccation resistant cysts. Recent analysis of cyst development has revealed that ~20 % of the Rhodospirillum centenum transcriptome undergo temporal changes in expression as cells transition from vegetative to cyst forms. It has also been established that one trigger for cyst formation is the synthesis of the signaling nucleotide 3', 5'- cyclic guanosine monophosphate (cGMP) that is sensed by a homolog of the catabolite repressor protein called CgrA. CgrA in the presence of cGMP initiate a cascade of gene expression leading to the development of cysts.

The RegB/RegA two-component system from Rhodobacter capsulatus regulates global changes in gene expression in response to alterations in oxygen levels. Studies have shown that RegB/RegA controls many energy-generating and energy-utilizing systems such as photosynthesis, nitrogen fixation, carbon fixation, hydrogen utilization, respiration, electron transport and denitrification. In this report, we utilized RNA-seq and ChIP-seq to analyse the breadth of genes indirectly and directly regulated by RegA. A comparison of mRNA transcript levels in wild type cells relative to a RegA deletion strain shows that there are 257 differentially expressed genes under photosynthetic defined minimal growth medium conditions and 591 differentially expressed genes when grown photosynthetically in a complex rich medium. ChIP-seq analysis also identified 61 unique RegA binding sites with a well-conserved recognition sequence, 33 of which exhibit changes in neighbouring gene expression. These transcriptome results define new members of the RegA regulon including genes involved in iron transport and motility. These results also reveal that the set of genes that are regulated by RegA are growth medium specific. Similar analyses under dark aerobic conditions where RegA is thought not to be phosphorylated by RegB reveal 40 genes that are differentially expressed in minimal medium and 20 in rich medium. Finally, a comparison of the R. capsulatus RegA regulon with the orthologous PrrA regulon in Rhodobacter sphaeroides shows that the number of photosystem genes regulated by RegA and PrrA are similar but that the identity of genes regulated by RegA and PrrA beyond those involved in photosynthesis are quite distinct.

We recently described a new member of the CRP (cyclic AMP receptor protein)/FNR (fumarate and nitrate reductase regulatory protein) family called RedB, an acronym for redox brake, that functions to limit the production of ATP and NADH. This study shows that the RedB regulon significantly overlaps the FnrL regulon, with 199 genes being either directly or indirectly regulated by both of these global regulatory proteins. Among these 199 coregulated genes, 192 are divergently regulated, indicating that RedB functions as an antagonist of FnrL. Chromatin immunoprecipitation sequencing (ChIP-seq) analysis indicates that RedB and Fnr directly coregulate only 4 out of 199 genes. The primary mechanism for the divergent regulation of target genes thus involves indirect regulation by both RedB and FnrL (156 cases). Additional regulation involves direct binding by RedB and indirect regulation by FnrL (36 cases) or direct binding by FnrL and indirect regulation by RedB (3 cases). Analysis of physiological pathways under direct and indirect control by these global regulators demonstrates that RedB functions primarily to limit energy production, while FnrL functions to enhance energy production. This regulation includes glycolysis, gluconeogenesis, photosynthesis, hydrogen oxidation, electron transport, carbon fixation, lipid biosynthesis, and protein synthesis. Finally, we show that 75% of genomes from diverse species that code for RedB proteins also harbor genes coding for FNR homologs. This cooccurrence indicates that RedB likely has an important role in buffering FNR-mediated energy production in a broad range of species. IMPORTANCE The CRP/FNR family of regulatory proteins constitutes a large collection of related transcription factors, several of which globally regulate cellular energy production. A well-characterized example is FNR (called FnrL in Rhodobacter capsulatus), which is responsible for regulating the expression of numerous genes that promote maximal energy production and growth under anaerobic conditions. In a companion article (N. Ke, J. E. Kumka, M. Fang, B. Weaver, et al., Microbiol Spectr 10:e02353-22, 2022, https://doi.org/10.1128/Spectrum02353-22), we identified a new subgroup of the CRP/FNR family and demonstrated that a member of this new subgroup, called RedB, has a role in limiting cellular energy production. In this study, we show that numerous genes encompassing the RedB regulon significantly overlap genes that are members of the FnrL regulon. Furthermore, 97% of the genes that are members of both the RedB and FnrL regulons are divergently regulated by these two transcription factors. RedB thus functions as a buffer limiting the amount of energy production that is promoted by FnrL.

Purple nonsulfur bacteria adapt their physiology to a wide variety of environmental conditions often through the control of transcription. One of the main transcription factors involved in controlling expression of the Rhodobacter capsulatus photosystem is CrtJ, which functions as an aerobic repressor of photosystem genes. Recently, we reported that a vitamin B12 binding antirepressor of CrtJ called AerR is required for anaerobic expression of the photosystem. However, the mechanism whereby AerR regulates CrtJ activity is unclear. In this study, we used a combination of next-generation sequencing and biochemical methods to globally identify genes under control of CrtJ and the role of AerR in controlling this regulation. Our results indicate that CrtJ has a much larger regulon than previously known, with a surprising regulatory function under both aerobic and anaerobic photosynthetic growth conditions. A combination of in vivo chromatin immunoprecipitation-DNA sequencing (ChIP-seq) and ChIP-seq and exonuclease digestion (ChIP-exo) studies and in vitro biochemical studies demonstrate that AerR forms a 1:2 complex with CrtJ (AerR-CrtJ2) and that this complex binds to many promoters under photosynthetic conditions. The results of in vitro and in vivo DNA binding studies indicate that AerR-CrtJ2 anaerobically forms an extended interaction with the bacteriochlorophyll bchC promoter to relieve repression by CrtJ. This is contrasted by aerobic growth conditions where CrtJ alone functions as an aerobic repressor of bchC expression. These results indicate that the DNA binding activity of CrtJ is modified by interacting with AerR in a redox-regulated manner and that this interaction alters CrtJ's function.IMPORTANCE Photoreceptors control a wide range of physiology often by regulating downstream gene expression in response to light absorption via a bound chromophore. Different photoreceptors are known to utilize a number of different compounds for light absorption, including the use of such compounds as flavins, linearized tetrapyrroles (bilins), and carotenoids. Recently, a novel class of photoreceptors that use vitamin B12 (cobalamin) as a blue-light-absorbing chromophore have been described. In this study, we analyzed the mechanism by which the vitamin B12 binding photoreceptor AerR controls the DNA binding activity of the photosystem regulator CrtJ. This study shows that a direct interaction between the vitamin B12 binding photoreceptor AerR with CrtJ modulates CrtJ binding to DNA and importantly, the regulatory outcome of gene expression, as shown here with photosystem promoters.