Redox Brake Regulator RedB and FnrL Function as Yin-Yang Regulators of Anaerobic-Aerobic Metabolism in Rhodobacter capsulatus.

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.

Iterative mutagenesis induced by atmospheric and room temperature plasma treatment under multiple selection pressures for the improvement of coenzyme Q10 production by Rhodobacter sphaeroides.

CoQ10, which has been widely applied in medicine by dietary supplement, possesses important functions in antioxidant process and bioenergy generation. Iterative mutagenesis introduced by atmospheric and room temperature plasma (ARTP) treatment was studied to improve the coenzyme Q10 (CoQ10) production of Rhodobacter sphaeroides (R. sphaeroides), and multiple selection pressures including vitamin K3 (VK3), Na2S and benzoic acid (BA) were adopted for the first time. After two rounds of mutation and screening, a mutant strain R.S 17 was obtained, and the product titer was increased by 80.37%. The CoQ10 titer and cell density reached 236.7 mg L-1 and 57.09 g L-1, respectively, in the fed-batch fermentation, and the CoQ10 content was 22.1% higher than that of the parent strain. In addition, the spectral scanning results indicated the metabolic flux improvement contributing to the CoQ10 production in R.S 17, and the genetic stability was validated. Based on the iterative mutagenesis introduced by ARTP under multiple selection pressures, the promotion of CoQ10 production by R. sphaeroides was achieved. The significant improvement in fermentation performances and the good genetic stability of R.S 17 indicate a potential way for the efficient biosynthesis of CoQ10.

TRAP transporter TakP: a key player in the resistance against selenite-induced oxidative stress in Rhodobacter sphaeroides.

Almost one-third of all proteins require metal ions as an essential component in key biological processes and approximately half of all enzymes are associated with one or more metal ions. The naturally occurring selenium is very toxic at higher levels, but few bacteria can reduce it into the less toxic insoluble elemental selenium. Selenium is required for the synthesis of selenocysteine, an essential residue involved in the active sites of various enzymes. The purple non-sulphur bacteria, Rhodobacter sphaeroidesis demonstrated for its selenite reduction capacity. The exact mechanism of selenite toxicity is unknown but it reacts with glutathione to form selenodiglutathione, producing the highly toxic compounds namely, H2O2and O2-. A R. sphaeroidesstrain with mutated takP gene, a member of the TRAP (tripartite ATP-independent periplasmic) family of transporter, was reported to be showing more resistance towards selenite in the growth medium but the reason for the resistance is unknown. TRAP transporters are the best-studied family of substrate-binding protein and in our previous study it was confirmed that the gene takP in R. sphaeroides is down-regulated by a small non-coding RNA SorY, providing more resistance to the bacterium against the oxidative stress. By comparative growth analysis and sensitivity assays in the presence of 2 mM selenite, it was observed that the SorY knockout strain is more sensitive to selenite while overexpression of the sRNA conferred more resistance to the bacterium like the takP mutant strain. TakP is involved in the import of malate into the cell, which under oxidative stress needs to be down-regulated to limit malate flux into the cell. Limited malate flux leads to metabolic rearrangements in the cell to avoid excessive generation of prooxidant NADH and facilitate constant generation of antioxidant NADPH. In the presence and absence of selenite, a drastic increase in the NADPH and decrease in the NADH levels are reported respectively. Accumulation of metallic selenium in the cytoplasm was detected via atomic absorption spectrophotometer and our analysis clearly demonstrated the presence of more selenium in the electron micrographs of the SorY knockout strain compared to the takP mutant grown under dark semi-aerobic growth conditions in the presence of selenite. Hence based on our analysis, it is confirmed that lack of TakP transporter led to reduced selenite influx into the cytoplasm, relieving cells with limited generation of ROS, eventually exhibiting more resistance against selenite-induced oxidative stress.

Polychromatic solar energy conversion in pigment-protein chimeras that unite the two kingdoms of (bacterio)chlorophyll-based photosynthesis.

Natural photosynthesis can be divided between the chlorophyll-containing plants, algae and cyanobacteria that make up the oxygenic phototrophs and a diversity of bacteriochlorophyll-containing bacteria that make up the anoxygenic phototrophs. Photosynthetic light harvesting and reaction centre proteins from both kingdoms have been exploited for solar energy conversion, solar fuel synthesis and sensing technologies, but the energy harvesting abilities of these devices are limited by each protein's individual palette of pigments. In this work we demonstrate a range of genetically-encoded, self-assembling photosystems in which recombinant plant light harvesting complexes are covalently locked with reaction centres from a purple photosynthetic bacterium, producing macromolecular chimeras that display mechanisms of polychromatic solar energy harvesting and conversion. Our findings illustrate the power of a synthetic biology approach in which bottom-up construction of photosystems using naturally diverse but mechanistically complementary components can be achieved in a predictable fashion through the encoding of adaptable, plug-and-play covalent interfaces.

Elevated ß-Carotene Synthesis by the Engineered Rhodobacter sphaeroides with Enhanced CrtY Expression.

ß-Carotene is a precursor of vitamin A and a dietary supplement for its antioxidant property. Producing ß-carotene by microbial fermentation has attracted much attention owing to consumers' preference for the natural product. In this study, an engineered photosynthetic Rhodobacter sphaeroides producing ß-carotene was constructed by the following strategies: (1) five promoters of different strengths were used to investigate the effect of the expression level of crtY on ß-carotene content. It was found that PrrnB increased the ß-carotene content by 109%. (2) blocking of the branched pentose phosphate pathway by zwf deletion, and (3) overexpressing dxs could restore the transcriptional levels of crtE and crtB. Finally, the engineered RS-C3 has the highest ß-carotene content of 14.93 mg/g dry cell weight (DCW) among all of the reported photosynthetic bacteria and the ß-carotene content reached 3.34 mg/g DCW under light conditions. Our results will be available for industrial use to supply a large quantity of natural ß-carotene.

Mutagenesis of Rhodobacter sphaeroides using atmospheric and room temperature plasma treatment for efficient production of coenzyme Q10.

Coenzyme Q10 (CoQ10) plays an important role in the human respiratory chain and is widely used as medicine and dietary supplement. To improve the fermentation efficiency of CoQ10, a modified version of atmospheric and room temperature plasma (ARTP) treatment was used to mutate Rhodobacter sphaeroides. Meanwhile, Vitamin K3, a structural analog of CoQ10, was used as an inhibitor for mutant selection. In the first round of screening in 24-well plates, three mutants were obtained, with the production of CoQ10 at 311 mg/L, 307 mg/L, and 309 mg/L, which were increased from the parent's production at 265 mg/L. Furthermore, a second round of mutation and screening was performed based on the mutant strain with the highest production in the first round, leading to the identification of a mutant AR01 with the production of CoQ10 at ∼330 mg/L. Finally, 590 mg/L CoQ10 was obtained for AR01 after 100 h fermentation, which was ∼25.5% higher than that of the original parent strain. It is the first report of ARTP treatment usage for the selection of CoQ10 producing bacteria and the results show that plasma jet, driven by helium-based ARTP, can be a feasible strategy for mutation feeding.

Modifying the endogenous electron fluxes of Rhodobacter sphaeroides 2.4.1 for improved electricity generation.

The purple bacteria Rhodobacter sphaeroides serve as a promising biocatalyst in the photo-microbial fuel cell system (photo-MFC). This gram-negative species performs highly efficient anoxygenic photosynthesis that ensures an anaerobic environment in the anode compartment. Previous studies incorporating R. sphaeroides into photo-MFC were conducted using platinum as the anode electrode. In this study, we detected a steady current generation of R. sphaeroides in a bioelectrochemical system where glassy carbon was the working electrode and a typical growth medium was the electrolyte. The bioelectricity generation synchronized with the supplementation of reduced carbon source and showed immediate response to illumination, which strongly indicated the correlation between the observed current and the cytoplasmic quinone activity. Modifications of the endogenous electron flows mediated by quinone pool are shown to have significantly enhanced the bioelectricity generation. We anticipate that the findings in this study would advance future optimization of R. sphaeroides as an anode strain, as well as facilitate the study of bioenergetics in photosynthetic bacteria.

Tackling codon usage bias for heterologous expression in Rhodobacter sphaeroides by supplementation of rare tRNAs.

The photosynthetic Rhodobacter species are promising alternative expression hosts in bioproduction and biorefinery due to their unique metabolic capacities. With prominent inner membrane areas and efficient endogenous translocation machineries, they are especially attractive for membrane protein expression. However, codon usage bias could be a limitation in the engineering of Rhodobacter species and has seldom been investigated. In this study, we tackled the codon bias of Rhodobacter by functionally expressing 8 rare tRNAs of Rhodobacter sphaeroides with a multi-copy vector. The impact of tRNA supplementation was evaluated through monitoring expression levels of two heterologous proteins with different phylogenetic origins, a membrane subunit of the riboflavin transporter, RibU, from Lactobacillus acidophilus La-14 and a decaheme cytochrome, MtrA, from Shewanella oneidensis. Our results showed that the performances were closely related to medium composition and rare codon percentages of raw DNA sequences. Provision of rare tRNAs has increased RibU production by 7.7-folds and 2.86-fold in minimal medium and rich medium, respectively, while MtrA levels were increased by 1-fold in minimal medium. The present study confirms the presence of codon bias in R. sphaeroides and offers a facile tool for improving heterologous expression of rare-codon containing genes. We anticipate that this tRNA supplementation system can be further extended to other species of Rhodobacter, and thus will facilitate the engineering of purple bacteria for interesting applications in microbial technology.

An integrated approach to reconstructing genome-scale transcriptional regulatory networks.

Transcriptional regulatory networks (TRNs) program cells to dynamically alter their gene expression in response to changing internal or environmental conditions. In this study, we develop a novel workflow for generating large-scale TRN models that integrates comparative genomics data, global gene expression analyses, and intrinsic properties of transcription factors (TFs). An assessment of this workflow using benchmark datasets for the well-studied γ-proteobacterium Escherichia coli showed that it outperforms expression-based inference approaches, having a significantly larger area under the precision-recall curve. Further analysis indicated that this integrated workflow captures different aspects of the E. coli TRN than expression-based approaches, potentially making them highly complementary. We leveraged this new workflow and observations to build a large-scale TRN model for the α-Proteobacterium Rhodobacter sphaeroides that comprises 120 gene clusters, 1211 genes (including 93 TFs), 1858 predicted protein-DNA interactions and 76 DNA binding motifs. We found that ~67% of the predicted gene clusters in this TRN are enriched for functions ranging from photosynthesis or central carbon metabolism to environmental stress responses. We also found that members of many of the predicted gene clusters were consistent with prior knowledge in R. sphaeroides and/or other bacteria. Experimental validation of predictions from this R. sphaeroides TRN model showed that high precision and recall was also obtained for TFs involved in photosynthesis (PpsR), carbon metabolism (RSP_0489) and iron homeostasis (RSP_3341). In addition, this integrative approach enabled generation of TRNs with increased information content relative to R. sphaeroides TRN models built via other approaches. We also show how this approach can be used to simultaneously produce TRN models for each related organism used in the comparative genomics analysis. Our results highlight the advantages of integrating comparative genomics of closely related organisms with gene expression data to assemble large-scale TRN models with high-quality predictions.

Enhanced production of coenzyme Q10 by self-regulating the engineered MEP pathway in Rhodobacter sphaeroides.

Fine-tuning the expression level of an engineered pathway is crucial for the metabolic engineering of a host toward a desired phenotype. However, most engineered hosts suffer from nonfunctional protein expression, metabolic imbalance, cellular burden or toxicity from intermediates when an engineered pathway is first introduced, which can decrease production of the desired product. To circumvent these obstacles, we developed a self-regulation system utilizing the trc/tac promoter, LacI(q) protein and ribosomal binding sites (RBS). With the purpose of improving coenzyme Q10 (CoQ10 ) production by increasing the decaprenyl diphosphate supplement, enzymes DXS, DXR, IDI, and IspD were constitutively overexpressed under the control of the trc promoter in Rhodobacter sphaeroides. Then, a self-regulation system combining a set of RBSs for adjusting the expression of the LacI(q) protein was applied to tune the expression of the four genes, resulting in improved CoQ10 production. Finally, another copy of the tac promoter with the UbiG gene (involved in the ubiquinone pathway of CoQ10 biosynthesis) was introduced into the engineered pathway. By optimizing the expression level of both the upstream and downstream pathway, CoQ10 production in the mutants was improved up to 93.34 mg/L (7.16 mg/g DCW), about twofold of the wild-type (48.25 mg/L, 3.24 mg/g DCW).

Role of the Irr protein in the regulation of iron metabolism in Rhodobacter sphaeroides.

In Rhizobia the Irr protein is an important regulator for iron-dependent gene expression. We studied the role of the Irr homolog RSP_3179 in the photosynthetic alpha-proteobacterium Rhodobacter sphaeroides. While Irr had little effect on growth under iron-limiting or non-limiting conditions its deletion resulted in increased resistance to hydrogen peroxide and singlet oxygen. This correlates with an elevated expression of katE for catalase in the Irr mutant compared to the wild type under non-stress conditions. Transcriptome studies revealed that Irr affects the expression of genes for iron metabolism, but also has some influence on genes involved in stress response, citric acid cycle, oxidative phosphorylation, transport, and photosynthesis. Most genes showed higher expression levels in the wild type than in the mutant under normal growth conditions indicating an activator function of Irr. Irr was however not required to activate genes of the iron metabolism in response to iron limitation, which showed even stronger induction in the absence of Irr. This was also true for genes mbfA and ccpA, which were verified as direct targets for Irr. Our results suggest that in R. sphaeroides Irr diminishes the strong induction of genes for iron metabolism under iron starvation.

Metabolic engineering to improve 5-aminolevulinic acid production.

5-Aminolevulinic acid (ALA) has recently attracted significant attentions due to its potential applications in many diverse fields. The majority of engineered ALA producers are based on the whole cell catalysis, supplemented with succinate and glycine as precursors. Recently, we succeeded in producing ALA directly from inexpensive glucose, through re-constructing the native C5 pathway of ALA synthesis in Escherichia coli. Herein, we further discuss ALA production by manipulating the C5 and C4 pathways in Escherichia coli through the strategy of metabolic engineering.

Hydrogen bonds between nitrogen donors and the semiquinone in the Qi-site of the bc1 complex.

The ubisemiquinone stabilized at the Qi-site of the bc1 complex of Rhodobacter sphaeroides forms a hydrogen bond with a nitrogen from the local protein environment, tentatively identified as ring N from His-217. The interactions of 14N and 15N have been studied by X-band (approximately 9.7 GHz) and S-band (3.4 GHz) pulsed EPR spectroscopy. The application of S-band spectroscopy has allowed us to determine the complete nuclear quadrupole tensor of the 14N involved in H-bond formation and to assign it unambiguously to the Nepsilon of His-217. This tensor has distinct characteristics in comparison with H-bonds between semiquinones and Ndelta in other quinone-processing sites. The experiments with 15N showed that the Nepsilon of His-217 was the only nitrogen carrying any considerable unpaired spin density in the ubiquinone environment, and allowed calculation of the isotropic and anisotropic couplings with the Nepsilon of His-217. From these data, we could estimate the unpaired spin density transferred onto 2s and 2p orbitals of nitrogen and the distance from the nitrogen to the carbonyl oxygen of 2.38+/-0.13A. The hyperfine coupling of other protein nitrogens with semiquinone is <0.1 MHz. This did not exclude the nitrogen of the Asn-221 as a possible hydrogen bond donor to the methoxy oxygen of the semiquinone. A mechanistic role for this residue is supported by kinetic experiments with mutant strains N221T, N221H, N221I, N221S, N221P, and N221D, all of which showed some inhibition but retained partial turnover.

Multiple chromosomes in bacteria. The yin and yang of trp gene localization in Rhodobacter sphaeroides 2.4.1.

The existence of multiple chromosomes in bacteria has been known for some time. Yet the extent of functional solidarity between different chromosomes remains unknown. To examine this question, we have surveyed the well-described genes of the tryptophan biosynthetic pathway in the multichromosomal photosynthetic eubacterium Rhodobacter sphaeroides 2.4.1. The genome of this organism was mutagenized using Tn5, and strains that were auxotrophic for tryptophan (Trp(-)) were isolated. Pulsed-field gel mapping indicated that Tn5 insertions in both the large (3 Mb CI) and the small (0.9 Mb CII) chromosomes created a Trp(-) phenotype. Sequencing the DNA flanking the sites of the Tn5 insertions indicated that the genes trpE-yibQ-trpGDC were at a locus on CI, while genes trpF-aroR-trpB were at locus on CII. Unexpectedly, trpA was not found downstream of trpB. Instead, it was placed on the CI physical map at a locus 1.23 Mb away from trpE-yibQ-trpGDC. To relate the context of the R. sphaeroides trp genes to those of other bacteria, the DNA regions surrounding the trp genes on both chromosomes were sequenced. Of particular significance was the finding that rpsA1, which encodes ribosomal protein S1, and cmkA, which encodes cytidylate monophosphate kinase, were on CII. These genes are considered essential for translation and chromosome replication, respectively. Southern blotting suggested that the trp genes and rpsA1 exist in single copy within the genome. To date, this topological organization of the trp "operon" is unique within a bacterial genome. When taken with the finding that CII encodes essential housekeeping functions, the overall impression is one of close regulatory and functional integration between these chromosomes.

Identification and characterization of a GroEL homologue in Rhodobacter sphaeroides.

A protein closely related to the Escherichia coli GroEL protein has been isolated from Rhodobacter sphaeroides. Native and SDS-polyacrylamide gel electrophoresis of this protein have shown that it is present in the cell as a multimeric complex of Mr 670,000 which is composed of a monomer of Mr 58,000. Antisera raised against the Mr 58,000 polypeptide have been shown to cross-react with GroEL and the alpha subunit of the pea plastid chaperonin. The N-terminal amino acid sequence of the Mr 58,000 polypeptide is identical to that of GroEL at 15 of 19 residues and is also closely related to the alpha subunit of the pea plastid chaperonin, though less so to the beta subunit.