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.

Comparative differential cuproproteomes of Rhodobacter capsulatus reveal novel copper homeostasis related proteins.

Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have developed sophisticated response mechanisms towards both the lack and the excess of Cu in their environments. In this study, we achieved a global view of Cu-responsive changes in the prokaryotic model organism Rhodobacter capsulatus using label-free quantitative differential proteomics. Semi-aerobically grown cells under heterotrophic conditions in minimal medium (∼0.3 µM Cu) were compared with cells supplemented with either 5 µM Cu or with 5 mM of the Cu-chelator bathocuproine sulfonate. Mass spectrometry based bottom-up proteomics of unfractionated cell lysates identified 2430 of the 3632 putative proteins encoded by the genome, producing a robust proteome dataset for R. capsulatus. Use of biological and technical replicates for each growth condition yielded high reproducibility and reliable quantification for 1926 of the identified proteins. Comparison of cells grown under Cu-excess or Cu-depleted conditions to those grown under minimal Cu-sufficient conditions revealed that 75 proteins exhibited statistically significant (p < 0.05) abundance changes, ranging from 2- to 300-fold. A subset of the highly Cu-responsive proteins was orthogonally probed using molecular genetics, validating that several of them were indeed involved in cellular Cu homeostasis.

NifA is the master regulator of both nitrogenase systems in Rhodobacter capsulatus.

Rhodobacter capsulatus fixes atmospheric nitrogen (N2 ) by a molybdenum (Mo)-nitrogenase and a Mo-free iron (Fe)-nitrogenase, whose production is induced or repressed by Mo, respectively. At low nanomolar Mo concentrations, both isoenzymes are synthesized and contribute to nitrogen fixation. Here we examined the regulatory interplay of the central transcriptional activators NifA and AnfA by proteome profiling. As expected from earlier studies, synthesis of the structural proteins of Mo-nitrogenase (NifHDK) and Fe-nitrogenase (AnfHDGK) required NifA and AnfA, respectively, both of which depend on the alternative sigma factor RpoN to activate expression of their target genes. Unexpectedly, NifA was found to be essential for the synthesis of Fe-nitrogenase, electron supply to both nitrogenases, biosynthesis of their cofactors, and production of RpoN. Apparently, RpoN is the only NifA-dependent factor required for target gene activation by AnfA, since plasmid-borne rpoN restored anfH transcription in a NifA-deficient strain. However, plasmid-borne rpoN did not restore Fe-nitrogenase activity in this strain. Taken together, NifA requirement for synthesis and activity of both nitrogenases suggests that Fe-nitrogenase functions as a complementary nitrogenase rather than an alternative isoenzyme in R. capsulatus.

Hydrogen production by hup(-) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors.

Photofermentative production of hydrogen is a promising and sustainable process; however, it should be coupled to dark fermentation to become cost effective. In order to integrate dark fermentation and photofermentation, the suitability of dark fermenter effluents for the photofermentative hydrogen production must be demonstrated. In this study, thermophilic dark fermenter effluent (DFE) of sugar beet thick juice was used as a substrate in photofermentation process to compare wild-type and uptake hydrogenase-deficient (hup (-)) mutant strains of Rhodobacter capsulatus by means of hydrogen production and biomass growth. The tests were conducted in small-scale (50 mL) batch and large-scale (4 L) continuous photobioreactors in indoor conditions under continuous illumination. In small scale batch conditions, maximum cell concentrations were 0.92 gdcw/L c and 1.50 gdcw/L c, hydrogen yields were 34 % and 31 %, hydrogen productivities were 0.49 mmol/(L c·h) and 0.26 mmol/(Lc·h), for hup (-) and wild-type cells, respectively. In large-scale continuous conditions, maximum cell concentrations were 1.44 gdcw/L c and 1.87 gdcw/L c, hydrogen yields were 48 and 46 %, and hydrogen productivities were 1.01 mmol/(L c·h) and 1.05 mmol/(L c·h), for hup (-) and wild-type cells, respectively. Our results showed that Rhodobacter capsulatus hup (-) cells reached to a lower maximum cell concentration but their hydrogen yield and productivity were in the same range or superior compared to the wild-type cells in both batch and continuous operating modes. The maximum biomass concentration, yield and productivity of hydrogen were higher in continuous mode compared to the batch mode with both bacterial strains.

Molecular mechanisms of superoxide production by complex III: a bacterial versus human mitochondrial comparative case study.

In this mini review, we briefly survey the molecular processes that lead to reactive oxygen species (ROS) production by the respiratory complex III (CIII or cytochrome bc1). In particular, we discuss the "forward" and "reverse" electron transfer pathways that lead to superoxide generation at the quinol oxidation (Qo) site of CIII, and the components that affect these reactions. We then describe and compare the properties of a bacterial (Rhodobacter capsulatus) mutant enzyme producing ROS with its mitochondrial (human cybrids) counterpart associated with a disease. The mutation under study is located at a highly conserved tyrosine residue of cytochrome b (Y302C in R. capsulatus and Y278C in human mitochondria) that is at the heart of the quinol oxidation (Qo) site of CIII. Similarities of the major findings of bacterial and human mitochondrial cases, including decreased catalytic activity of CIII, enhanced ROS production and ensuing cellular responses and damages, are remarkable. This case illustrates the usefulness of undertaking parallel and complementary studies using biologically different yet evolutionarily related systems, such as α-proteobacteria and human mitochondria. It progresses our understanding of CIII mechanism of function and ROS production, and underlines the possible importance of supra-molecular organization of bacterial and mitochondrial respiratory chains (i.e., respirasomes) and their potential disease-associated protective roles. This article is part of a Special Issue entitled: Respiratory complex III and related bc complexes.

Missense mutations in cytochrome c maturation genes provide new insights into Rhodobacter capsulatus cbb3-type cytochrome c oxidase biogenesis.

The Rhodobacter capsulatus cbb(3)-type cytochrome c oxidase (cbb(3)-Cox) belongs to the heme-copper oxidase superfamily, and its subunits are encoded by the ccoNOQP operon. Biosynthesis of this enzyme is complex and needs dedicated biogenesis genes (ccoGHIS). It also relies on the c-type cytochrome maturation (Ccm) process, which requires the ccmABCDEFGHI genes, because two of the cbb(3)-Cox subunits (CcoO and CcoP) are c-type cytochromes. Recently, we reported that mutants lacking CcoA, a major facilitator superfamily type transporter, produce very small amounts of cbb(3)-Cox unless the growth medium is supplemented with copper. In this work, we isolated "Cu-unresponsive" derivatives of a ccoA deletion strain that exhibited no cbb(3)-Cox activity even upon Cu supplementation. Molecular characterization of these mutants revealed missense mutations in the ccmA or ccmF gene, required for the Ccm process. As expected, Cu-unresponsive mutants lacked the CcoO and CcoP subunits due to Ccm defects, but remarkably, they contained the CcoN subunit of cbb(3)-Cox. Subsequent construction and examination of single ccm knockout mutants demonstrated that membrane insertion and stability of CcoN occurred in the absence of the Ccm process. Moreover, while the ccm knockout mutants were completely incompetent for photosynthesis, the Cu-unresponsive mutants grew photosynthetically at lower rates and produced smaller amounts of cytochromes c(1) and c(2) than did a wild-type strain due to their restricted Ccm capabilities. These findings demonstrate that different levels of Ccm efficiency are required for the production of various c-type cytochromes and reveal for the first time that maturation of the heme-Cu-containing subunit CcoN of R. capsulatus cbb(3)-Cox proceeds independently of that of the c-type cytochromes during the biogenesis of this enzyme.

Novel transporter required for biogenesis of cbb3-type cytochrome c oxidase in Rhodobacter capsulatus.

UNLABELLED: The acquisition, delivery, and incorporation of metals into their respective metalloproteins are important cellular processes. These processes are tightly controlled in order to prevent exposure of cells to free-metal concentrations that could yield oxidative damage. Copper (Cu) is one such metal that is required as a cofactor in a variety of proteins. However, when present in excessive amounts, Cu is toxic due to its oxidative capability. Cytochrome c oxidases (Coxs) are among the metalloproteins whose assembly and activity require the presence of Cu in their catalytic subunits. In this study, we focused on the acquisition of Cu for incorporation into the heme-Cu binuclear center of the cbb(3)-type Cox (cbb(3)-Cox) in the facultative phototroph Rhodobacter capsulatus. Genetic screens identified a cbb(3)-Cox defective mutant that requires Cu(2+) supplementation to produce an active cbb(3)-Cox. Complementation of this mutant using wild-type genomic libraries unveiled a novel gene (ccoA) required for cbb(3)-Cox biogenesis. In the absence of CcoA, the cellular Cu content decreases and cbb(3)-Cox assembly and activity become defective. CcoA shows homology to major facilitator superfamily (MFS)-type transporter proteins. Members of this family are known to transport small solutes or drugs, but so far, no MFS protein has been implicated in cbb(3)-Cox biogenesis. These findings provide novel insights into the maturation and assembly of membrane-integral metalloproteins and on a hitherto-unknown function(s) of MFS-type transporters in bacterial Cu acquisition. IMPORTANCE: Biogenesis of energy-transducing membrane-integral enzymes, like the heme copper-containing cytochrome c oxidases, and the acquisition of transition metals, like copper, as their catalytic cofactors are vital processes for all cells. These widespread and well-controlled processes are poorly understood in all organisms, including bacteria. Defects in these processes lead to severe mitochondrial diseases in humans and poor crop yields in plants. In this study, using the facultative phototroph Rhodobacter capsulatus as a model organism, we report on the discovery of a novel major facilitator superfamily (MFS)-type transporter (CcoA) that affects cellular copper content and cbb(3)-type cytochrome c oxidase production in bacteria.

Across membrane communication between the Q(o) and Q(i) active sites of cytochrome bc(1).

The ubihydroquinone:cytochrome c oxidoreductase (cyt bc(1)) contains two catalytically active domains, termed the hydroquinone oxidation (Q(o)) and quinone reduction (Q(i)) sites, which are distant from each other by over 30 A. Previously, we have reported that binding of inhibitors to the Q(i) site on one (n) side of the energy-transducing membrane changes the local environment of the iron-sulfur (Fe/S) protein subunit residing in the Q(o) site on the other (p) side of the lipid bilayer [Cooley, J. W., Ohnishi, T., and Daldal, F. (2005) Biochemistry 44, 10520-10532]. These findings best fit a model whereby the Q(o) and Q(i) sites of the cyt bc(1) are actively coupled in spite of their distant locations. Because the Fe/S protein of the cyt bc(1) undergoes a large-scale (macro) domain movement during catalysis, we examined various macromobility-defective Fe/S subunit mutants to assess the role of this motion on the coupling of the active sites and also during the multiple turnovers of the enzyme. By monitoring the changing environments of the Fe/S protein [2Fe-2S] cluster upon addition of Q(i) site inhibitors in selected mutants, we found that the Q(o)-Q(i) site interactions manifest differently depending on the ability of the Fe/S protein to move between the cytochrome b and cytochrome c(1) subunits of the enzyme. In the presence of antimycin A, an immobile Fe/S protein mutant exhibited no changes in its EPR spectra. In contrast, mobility-restricted mutants showed striking alterations in the EPR line shapes and revealed two discrete subpopulations in respect to the [2Fe-2S] cluster environments at the Q(o) site. These findings led us to conclude that the mobility of the Fe/S protein is involved in its response to the occupancy of the Q(i) site by different molecules. We propose that the heterogeneity seen might reflect the distinct responses of the two Fe/S proteins at the Q(o) sites of the dimeric enzyme upon the occupancy of the Q(i) sites and discuss it in terms of the function of the dimeric cyt bc(1) during its multiple turnovers.

Binding dynamics at the quinone reduction (Qi) site influence the equilibrium interactions of the iron sulfur protein and hydroquinone oxidation (Qo) site of the cytochrome bc1 complex.

Multiple instances of low-potential electron-transport pathway inhibitors that affect the structure of the cytochrome (cyt) bc(1) complex to varying degrees, ranging from changes in hydroquinone (QH(2)) oxidation and cyt c(1) reduction kinetics to proteolytic accessibility of the hinge region of the iron-sulfur-containing subunit (Fe/S protein), have been reported. However, no instance has been documented of any ensuing change on the environment(s) of the [2Fe-2S] cluster. In this work, this issue was addressed in detail by taking advantage of the increased spectral and spatial resolution obtainable with orientation-dependent electron paramagnetic resonance (EPR) spectroscopic analysis of ordered membrane preparations. For the first time, perturbation of the low-potential electron-transport pathway by Q(i)-site inhibitors or various mutations was shown to change the EPR spectra of both the cyt b hemes and the [2Fe-2S] cluster of the Fe/S protein. In particular, two interlinked effects of Q(i)-site modifications on the Fe/S subunit, one changing the local environment of its [2Fe-2S] cluster and a second affecting the mobility of this subunit, are revealed. Remarkably, different inhibitors and mutations at or near the Q(i) site induce these two effects differently, indicating that the events occurring at the Q(i) site affect the global structure of the cyt bc(1). Furthermore, occupancy of discrete Q(i)-site subdomains differently impede the location of the Fe/S protein at the Q(o) site. These findings led us to propose that antimycin A and HQNO mimic the presence of QH(2) and Q at the Q(i) site, respectively. Implications of these findings in respect to the Q(o)-Q(i) sites communications and to multiple turnovers of the cyt bc(1) are discussed.

FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.

A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus.

A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding-protein-dependent transporter was interrupted by Tn5 transposition. Cloning and sequence analysis of the wild-type operon revealed a four-gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP-binding proteins (BztD). To assess the function of this putative binding-protein-dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic-resistance marker. The deletion mutant grew more slowly than the wild type in NH4(+)-free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gln, and Asp. A complementing plasmid containing the wildtype copy of bztABCD was able to rescue all the mutant phenotypes. Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gln, Asp, and Asn. In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.

CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.

An albino mutant designated cla1-1 (for "cloroplastos alterados', or "altered chloroplasts') has been isolated from a T-DNA-generated library of Arabidopsis thaliana. In cla1-1 plants, chloroplast development is arrested at an early stage. cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.

cDNA cloning, expression during development, and genome mapping of PSY2, a second tomato gene encoding phytoene synthase.

The enzyme phytoene synthase (Psy) catalyzes the formation of phytoene, an intermediate in the carotenoid biosynthesis pathway. Expression of a previously described gene (PSY1) is induced by fruit ripening in tomato (Lycopersicon esculentum). We describe here the cloning of a partial cDNA for PSY2, a gene related to PSY1. A plasmid containing the PSY2 coding region under control of a bacterial promoter complements a Rhodobacter capsulatus phytoene synthase mutant, indicating that this gene has the capacity to encode an active enzyme. We used reverse transcriptase-polymerase chain reaction followed by digestion with a restriction enzyme to determine that both PSY1 and PSY2 are expressed during tomato development. PSY1 transcripts predominate in seedlings and in late stages of fruit ripening, whereas PSY2 transcripts are relatively more abundant in mature leaves. Both genes are expressed under photooxidative conditions induced by treatment with the carotenoid biosynthesis inhibitor Norflurazon. We used polymerase chain reaction-based restriction fragment length polymorphisms and alien addition lines to map PSY2 to chromosome 2. We conclude that PSY2 is a second tomato gene encoding phytoene synthase.

Purification and properties of a nif-specific flavodoxin from the photosynthetic bacterium Rhodobacter capsulatus.

A flavodoxin was isolated from iron-sufficient, nitrogen-limited cultures of the photosynthetic bacterium Rhodobacter capsulatus. Its molecular properties, molecular weight, UV-visible absorption spectrum, and amino acid composition suggest that it is similar to the nif-specific flavodoxin, NifF, of Klebsiella pneumoniae. The results of immunoblotting showed that R. capsulatus flavodoxin is nif specific, since it is absent from ammonia-replete cultures and is not synthesized by the mutant strain J61, which lacks a nif-specific regulator (NifR1). Growth of cultures under iron-deficient conditions causes a small amount of flavodoxin to be synthesized under ammonia-replete conditions and increases its synthesis under N2-fixing conditions, suggesting that its synthesis is under a dual system of control with respect to iron and fixed nitrogen availability. Here we show that flavodoxin, when supplemented with catalytic amounts of methyl viologen, is capable of efficiently reducing nitrogenase in an illuminated chloroplast system. Thus, this nif-specific flavodoxin is a potential in vivo electron carrier to nitrogenase; however, its role in the nitrogen fixation process remains to be established.