Co-opting bacterial viruses for DNA exchange: structure and regulation of gene transfer agents.

Horizontal gene transfer occurs via a range of mechanisms, including transformation, conjugation and bacteriophage transduction. Gene transfer agents (GTAs) are an alternative, less-studied route for interbacterial DNA exchange. Encoded within bacterial or archaeal genomes, GTAs assemble into phage-like particles that selflessly package and transmit host DNA to recipient bacteria. Several unique features distinguish GTAs from canonical phages such as an inability to self-replicate, thus producing non-infectious particles. GTAs are also deeply integrated into the physiology of the host cell and are maintained under tight host-regulatory control. Recent advances in understanding the structure and regulation of GTAs have provided further insights into a DNA transfer mechanism that is proving increasingly widespread across the bacterial tree of life.

Two distinct ferredoxins are essential for nitrogen fixation by the iron nitrogenase in Rhodobacter capsulatus.

Nitrogenases are the only enzymes able to fix gaseous nitrogen into bioavailable ammonia and hence are essential for sustaining life. Catalysis by nitrogenases requires both a large amount of ATP and electrons donated by strongly reducing ferredoxins or flavodoxins. Our knowledge about the mechanisms of electron transfer to nitrogenase enzymes is limited: The electron transport to the iron (Fe)-nitrogenase has hardly been investigated. Here, we characterized the electron transfer pathway to the Fe-nitrogenase in Rhodobacter capsulatus via proteome analyses, genetic deletions, complementation studies, and phylogenetics. Proteome analyses revealed an upregulation of four ferredoxins under nitrogen-fixing conditions reliant on the Fe-nitrogenase in a molybdenum nitrogenase knockout strain, compared to non-nitrogen-fixing conditions. Based on these findings, R. capsulatus strains with deletions of ferredoxin (fdx) and flavodoxin (fld, nifF) genes were constructed to investigate their roles in nitrogen fixation by the Fe-nitrogenase. R. capsulatus deletion strains were characterized by monitoring diazotrophic growth and Fe-nitrogenase activity in vivo. Only deletions of fdxC or fdxN resulted in slower growth and reduced Fe-nitrogenase activity, whereas the double deletion of both fdxC and fdxN abolished diazotrophic growth. Differences in the proteomes of ∆fdxC and ∆fdxN strains, in conjunction with differing plasmid complementation behaviors of fdxC and fdxN, indicate that the two Fds likely possess different roles and functions. These findings will guide future engineering of the electron transport systems to nitrogenase enzymes, with the aim of increased electron flux and product formation.IMPORTANCENitrogenases are essential for biological nitrogen fixation, converting atmospheric nitrogen gas to bioavailable ammonia. The production of ammonia by diazotrophic organisms, harboring nitrogenases, is essential for sustaining plant growth. Hence, there is a large scientific interest in understanding the cellular mechanisms for nitrogen fixation via nitrogenases. Nitrogenases rely on highly reduced electrons to power catalysis, although we lack knowledge as to which proteins shuttle the electrons to nitrogenases within cells. Here, we characterized the electron transport to the iron (Fe)-nitrogenase in the model diazotroph Rhodobacter capsulatus, showing that two distinct ferredoxins are very important for nitrogen fixation despite having different redox centers. In addition, our research expands upon the debate on whether ferredoxins have functional redundancy or perform distinct roles within cells. Here, we observe that both essential ferredoxins likely have distinct roles based on differential proteome shifts of deletion strains and different complementation behaviors.

Changing the Electron Acceptor Specificity of Rhodobacter capsulatus Formate Dehydrogenase from NAD+ to NADP.

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO2-reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO2 capture. Here, we investigate the nicotinamide binding site of formate dehydrogenase from Rhodobacter capsulatus for its specificity toward NAD+ vs. NADP+ reduction. Starting from the NAD+-specific wild-type RcFDH, key residues were exchanged to enable NADP+ binding on the basis of the NAD+-bound cryo-EM structure (PDB-ID: 6TG9). It has been observed that the lysine at position 157 (Lys157) in the ß-subunit of the enzyme is essential for the binding of NAD+. RcFDH variants that had Glu259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP+. The FdsBL279R and FdsBK276A variants also showed activity with NADP+. Kinetic parameters for all the variants were determined and tested for activity in CO2 reduction. The variants were able to reduce CO2 using NADPH as an electron donor in a coupled assay with phosphite dehydrogenase (PTDH), which regenerates NADPH. This makes the enzyme suitable for applications where it can be coupled with other enzymes that use NADPH.

The structure of AcrIC9 revealing the putative inhibitory mechanism of AcrIC9 against the type IC CRISPR-Cas system.

CRISPR-Cas systems are known to be part of the bacterial adaptive immune system that provides resistance against intruders such as viruses, phages and other mobile genetic elements. To combat this bacterial defense mechanism, phages encode inhibitors called Acrs (anti-CRISPR proteins) that can suppress them. AcrIC9 is the most recently identified member of the AcrIC family that inhibits the type IC CRISPR-Cas system. Here, the crystal structure of AcrIC9 from Rhodobacter capsulatus is reported, which comprises a novel fold made of three central antiparallel ß-strands surrounded by three α-helixes, a structure that has not been detected before. It is also shown that AcrIC9 can form a dimer via disulfide bonds generated by the Cys69 residue. Finally, it is revealed that AcrIC9 directly binds to the type IC cascade. Analysis and comparison of its structure with structural homologs indicate that AcrIC9 belongs to DNA-mimic Acrs that directly bind to the cascade complex and hinder the target DNA from binding to the cascade.

UbiN, a novel Rhodobacter capsulatus decarboxylative hydroxylase involved in aerobic ubiquinone biosynthesis.

Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ-biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ-containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin-containing UQ-biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3-decaprenylphenol, which is a UQ-biosynthetic intermediate. In addition, 3-decaprenyl-4-hydroxybenzoic acid, which is the substrate of UQ-biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD-X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild-type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.

Extracellular Polysaccharide Receptor and Receptor-Binding Proteins of the Rhodobacter capsulatus Bacteriophage-like Gene Transfer Agent RcGTA.

A variety of prokaryotes produce a bacteriophage-like gene transfer agent (GTA), and the alphaproteobacterial Rhodobacter capsulatus RcGTA is a model GTA. Some environmental isolates of R. capsulatus lack the ability to acquire genes transferred by the RcGTA (recipient capability). In this work, we investigated the reason why R. capsulatus strain 37b4 lacks recipient capability. The RcGTA head spike fiber and tail fiber proteins have been proposed to bind extracellular oligosaccharide receptors, and strain 37b4 lacks a capsular polysaccharide (CPS). The reason why strain 37b4 lacks a CPS was unknown, as was whether the provision of a CPS to 37b4 would result in recipient capability. To address these questions, we sequenced and annotated the strain 37b4 genome and used BLAST interrogations of this genome sequence to search for homologs of genes known to be needed for R. capsulatus recipient capability. We also created a cosmid-borne genome library from a wild-type strain, mobilized the library into 37b4, and used the cosmid-complemented strain 37b4 to identify genes needed for a gain of function, allowing for the acquisition of RcGTA-borne genes. The relative presence of CPS around a wild-type strain, 37b4, and cosmid-complemented 37b4 cells was visualized using light microscopy of stained cells. Fluorescently tagged head spike fiber and tail fiber proteins of the RcGTA particle were created and used to measure the relative binding to wild-type and 37b4 cells. We found that strain 37b4 lacks recipient capability because of an inability to bind RcGTA; the reason it is incapable of binding is that it lacks CPS, and the absence of CPS is due to the absence of genes previously shown to be needed for CPS production in another strain. In addition to the head spike fiber, we found that the tail fiber protein also binds to the CPS.

Rhodobacter capsulatus forms a compact crescent-shaped LH1-RC photocomplex.

Rhodobacter (Rba.) capsulatus has been a favored model for studies of all aspects of bacterial photosynthesis. This purple phototroph contains PufX, a polypeptide crucial for dimerization of the light-harvesting 1-reaction center (LH1-RC) complex, but lacks protein-U, a U-shaped polypeptide in the LH1-RC of its close relative Rba. sphaeroides. Here we present a cryo-EM structure of the Rba. capsulatus LH1-RC purified by DEAE chromatography. The crescent-shaped LH1-RC exhibits a compact structure containing only 10 LH1 αß-subunits. Four αß-subunits corresponding to those adjacent to protein-U in Rba. sphaeroides were absent. PufX in Rba. capsulatus exhibits a unique conformation in its N-terminus that self-associates with amino acids in its own transmembrane domain and interacts with nearby polypeptides, preventing it from interacting with proteins in other complexes and forming dimeric structures. These features are discussed in relation to the minimal requirements for the formation of LH1-RC monomers and dimers, the spectroscopic behavior of both the LH1 and RC, and the bioenergetics of energy transfer from LH1 to the RC.

Cryo-EM structure of a monomeric RC-LH1-PufX supercomplex with high-carotenoid content from Rhodobacter capsulatus.

In purple photosynthetic bacteria, the photochemical reaction center (RC) and light-harvesting complex 1 (LH1) assemble to form monomeric or dimeric RC-LH1 membrane complexes, essential for bacterial photosynthesis. Here, we report a 2.59-Å resolution cryoelectron microscopy (cryo-EM) structure of the RC-LH1 supercomplex from Rhodobacter capsulatus. We show that Rba. capsulatus RC-LH1 complexes are exclusively monomers in which the RC is surrounded by a 15-subunit LH1 ring. Incorporation of a transmembrane polypeptide PufX leads to a large opening within the LH1 ring. Each LH1 subunit associates two carotenoids and two bacteriochlorophylls, which is similar to Rba. sphaeroides RC-LH1 but more than one carotenoid per LH1 in Rba. veldkampii RC-LH1 monomer. Collectively, the unique Rba. capsulatus RC-LH1-PufX represents an intermediate structure between Rba. sphaeroides and Rba. veldkampii RC-LH1-PufX. Comparison of PufX from the three Rhodobacter species indicates the important residues involved in dimerization of RC-LH1.

Shared properties of gene transfer agent and core genes revealed by comparative genomics of Alphaproteobacteria.

Gene transfer agents (GTAs) are phage-like particles that transfer pieces of cellular genomic DNA to other cells. Homologues of the Rhodobacter capsulatus GTA (RcGTA) structural genes are widely distributed in the Alphaproteobacteria and particularly well conserved in the order Rhodobacterales. Possible reasons for their widespread conservation are still being discussed. It has been suggested that these alphaproteobacterial elements originate from a prophage that was present in an ancestral bacterium and subsequently evolved into a GTA that is now widely maintained in extant descendant lineages. Here, we analysed genomic properties that might relate to the conservation of these alphaproteobacterial GTAs. This revealed that the chromosomal locations of the GTA gene clusters are biased. They primarily occur on the leading strand of DNA replication, at large distances from long repetitive elements, and thus are in regions of lower plasticity, and in areas of extreme GC skew, which also accumulate core genes. These extreme GC skew regions arise from the preferential use of codons with an excess of G over C, a distinct phenomenon from the elevated GC content that has previously been found to be associated with GTA genes. The observed properties, along with their high level of conservation, show that GTA genes share multiple features with core genes in the examined lineages of the Alphaproteobacteria.

DNA Gyrase Inhibitors Increase the Frequency of Bacteriophage-like RcGTA-Mediated Gene Transfer in Rhodobacter capsulatus.

Rhodobacter capsulatus produces a bacteriophage-like particle called the gene transfer agent (RcGTA) that mediates horizontal gene transfer. RcGTA particles transfer random ~4.5-kb fragments of genomic DNA that integrate into recipient genomes by allelic replacement. This work addresses the effect of sub-inhibitory concentrations of antibiotics on gene transfer by RcGTA. A transduction assay was developed to test the effects of various substances on gene transfer. Using this assay, low concentrations of DNA gyrase inhibitors were found to increase the frequency of gene transfer. Novobiocin was studied in more detail, and it was found that this antibiotic did not influence the production or release of RcGTA but instead appeared to act on the recipient cells. The target of novobiocin in other species has been shown to be the GyrB subunit of DNA gyrase (a heterotetramer of 2GyrA and 2GyrB). R. capsulatus encodes GyrA and GyrB homologues, and a GyrB overexpression plasmid was created and found to confer resistance to novobiocin. The presence of the overexpression plasmid in recipient cells greatly diminished the novobiocin-mediated increase in gene transfer, confirming that this effect is due to the binding of novobiocin by GyrB. The results of this work show that antibiotics affect gene transfer in R. capsulatus and may be relevant to microbial genetic exchange in natural ecosystems.

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.

Loss of the Rhodobacter capsulatus Serine Acetyl Transferase Gene, cysE1, Impairs Gene Transfer by Gene Transfer Agents and Biofilm Phenotypes.

Biofilms are widespread in the environment, where they allow bacterial species to survive adverse conditions. Cells in biofilms are densely packed, and this proximity is likely to increase the frequency of horizontal gene transfer. Gene transfer agents (GTAs) are domesticated viruses with the potential to spread any gene between bacteria. GTA production is normally restricted to a small subpopulation of bacteria, and regulation of GTA loci is highly coordinated, but the environmental conditions that favor GTA production are poorly understood. Here, we identified a serine acetyltransferase gene, cysE1, in Rhodobacter capsulatus that is required for optimal receipt of GTA DNA, accumulation of extracellular polysaccharide, and biofilm formation. The cysE1 gene is directly downstream of the core Rhodobacter-like GTA (RcGTA) structural gene cluster and upregulated in an RcGTA overproducer strain, although it is expressed on a separate transcript. The data we present suggest that GTA production and biofilm are coregulated, which could have important implications for the study of rapid bacterial evolution and understanding the full impact of GTAs in the environment. IMPORTANCE Direct exchange of genes between bacteria leads to rapid evolution and is the major factor underlying the spread of antibiotic resistance. Gene transfer agents (GTAs) are an unusual but understudied mechanism for genetic exchange that are capable of transferring any gene from one bacterium to another, and therefore, GTAs are likely to be important factors in genome plasticity in the environment. Despite the potential impact of GTAs, our knowledge of their regulation is incomplete. In this paper, we present evidence that elements of the cysteine biosynthesis pathway are involved in coregulation of various phenotypes required for optimal biofilm formation by Rhodobacter capsulatus and successful infection by the archetypal RcGTA. Establishing the regulatory mechanisms controlling GTA-mediated gene transfer is a key stepping stone to allow a full understanding of their role in the environment and wider impact.

The archetypal gene transfer agent RcGTA is regulated via direct interaction with the enigmatic RNA polymerase omega subunit.

Gene transfer agents (GTAs) are small virus-like particles that indiscriminately package and transfer any DNA present in their host cell, with clear implications for bacterial evolution. The first transcriptional regulator that directly controls GTA expression, GafA, was recently discovered, but its mechanism of action has remained elusive. Here, we demonstrate that GafA controls GTA gene expression via direct interaction with the RNA polymerase omega subunit (Rpo-ω) and also positively autoregulates its own expression by an Rpo-ω-independent mechanism. We show that GafA is a modular protein with distinct DNA and protein binding domains. The functional domains we observe in Rhodobacter GafA also correspond to two-gene operons in Hyphomicrobiales pathogens. These data allow us to produce the most complete regulatory model for a GTA and point toward an atypical mechanism for RNA polymerase recruitment and specific transcriptional activation in the Alphaproteobacteria.

Heterologous Production of Plant Terpenes in the Photosynthetic Bacterium Rhodobacter capsulatus.

Terpenes are one of the largest classes of secondary metabolites that occur in all kingdoms of life and offer diverse valuable properties for food and pharma industry including pleasant odor or taste as well as antimicrobial or anticancer activities. A multitude of terpene biosynthesis pathways are known, but their efficient biotechnological exploitation requires an adequate microorganism as host which can ideally provide an optimal supply with biosynthetic isoprene precursors. Rhodobacter capsulatus, a Gram-negative, facultative anaerobic, photosynthetic non-sulfur purple bacterium belonging to the α-proteobacteria represents such a host particularly suitable for terpene production. Here, we describe methods for the expression of terpene biosynthetic enzymes in R. capsulatus and the extraction of products for analysis. At the same time, we summarize the current strategies to adjust the biosynthetic precursor supply via isoprenoid biosynthetic pathways.

Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus.

Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.

Genomic diversity of bacteriophages infecting Rhodobacter capsulatus and their relatedness to its gene transfer agent RcGTA.

The diversity of bacteriophages is likely unparalleled in the biome due to the immense variety of hosts and the multitude of viruses that infect them. Recent efforts have led to description at the genomic level of numerous bacteriophages that infect the Actinobacteria, but relatively little is known about those infecting other prokaryotic phyla, such as the purple non-sulfur photosynthetic α-proteobacterium Rhodobacter capsulatus. This species is a common inhabitant of freshwater ecosystems and has been an important model system for the study of photosynthesis. Additionally, it is notable for its utilization of a unique form of horizontal gene transfer via a bacteriophage-like element known as the gene transfer agent (RcGTA). Only three bacteriophages of R. capsulatus had been sequenced prior to this report. Isolation and characterization at the genomic level of 26 new bacteriophages infecting this host advances the understanding of bacteriophage diversity and the origins of RcGTA. These newly discovered isolates can be grouped along with three that were previously sequenced to form six clusters with four remaining as single representatives. These bacteriophages share genes with RcGTA that seem to be related to host recognition. One isolate was found to cause lysis of a marine bacterium when exposed to high-titer lysate. Although some clusters are more highly represented in the sequenced genomes, it is evident that many more bacteriophage types that infect R. capsulatus are likely to be found in the future.

Hydrogen bonding rearrangement by a mitochondrial disease mutation in cytochrome bc1 perturbs heme bH redox potential and spin state.

Hemes are common elements of biological redox cofactor chains involved in rapid electron transfer. While the redox properties of hemes and the stability of the spin state are recognized as key determinants of their function, understanding the molecular basis of control of these properties is challenging. Here, benefiting from the effects of one mitochondrial disease-related point mutation in cytochrome b, we identify a dual role of hydrogen bonding (H-bond) to the propionate group of heme bH of cytochrome bc1, a common component of energy-conserving systems. We found that replacing conserved glycine with serine in the vicinity of heme bH caused stabilization of this bond, which not only increased the redox potential of the heme but also induced structural and energetic changes in interactions between Fe ion and axial histidine ligands. The latter led to a reversible spin conversion of the oxidized Fe from 1/2 to 5/2, an effect that potentially reduces the electron transfer rate between the heme and its redox partners. We thus propose that H-bond to the propionate group and heme-protein packing contribute to the fine-tuning of the redox potential of heme and maintaining its proper spin state. A subtle balance is needed between these two contributions: While increasing the H-bond stability raises the heme potential, the extent of increase must be limited to maintain the low spin and diamagnetic form of heme. This principle might apply to other native heme proteins and can be exploited in engineering of artificial heme-containing protein maquettes.

A unique photochromic UV-A sensor protein, Rc-PYP, interacting with the PYP-binding protein.

Photoactive yellow protein (PYP) is one of the typical light sensor proteins. Although its photoreaction has been extensively studied, no downstream partner protein has been identified to date. In this study, the intermolecular interaction dynamics observed between PYP from Rhodobacter capsulatus (Rc-PYP) and a possible downstream protein, PYP-binding protein (PBP), were investigated. It was found that UV light induced a long-lived product (pUV*), which interacts with PBP to form a stable hetero-hexamer (Complex-2). The reaction scheme for this interaction was revealed using transient absorption and transient grating methods. Time-resolved diffusion detection showed that a hetero-trimer (Complex-1) is formed transiently, which produced Complex-2 via a second-order reaction. Any other intermediates, including those from pBL, do not interact with PBP. The reaction scheme and kinetics are determined. Interestingly, long-lived Complex-2 dissociates upon excitation with blue light. These results demonstrate that Rc-PYP is a photochromic and new type of UV sensor to sense the relative intensities of UV-A and blue light.

Production of C20, C30 and C40 terpenes in the engineered phototrophic bacterium Rhodobacter capsulatus.

Terpenes constitute one of the largest groups of secondary metabolites that are used, for example, as food-additives, fragrances or pharmaceuticals. Due to the formation of an intracytoplasmic membrane system and an efficient intrinsic tetraterpene pathway, the phototrophic α-proteobacterium Rhodobacter capsulatus offers favorable properties for the production of hydrophobic terpenes. However, research efforts have largely focused on sesquiterpene production. Recently, we have developed modular tools allowing to engineer the biosynthesis of terpene precursors. These tools were now applied to boost the biosynthesis of the diterpene casbene, the triterpene squalene and the tetraterpene ß-carotene in R. capsulatus SB1003. Selected enzymes of the intrinsic isoprenoid pathway and the heterologous mevalonate (MVA) pathway were co-expressed together with the respective terpene synthases in various combinations. Remarkably, co-expression of genes ispA, idi and dxs enhanced the synthesis of casbene and ß-carotene. In contrast, co-expression of precursor biosynthetic genes with the squalene synthase from Arabidopsis thaliana reduced squalene titers. Therefore, we further employed four alternative pro- and eukaryotic squalene synthases. Here, the synthase from Methylococcus capsulatus enabled highest product levels of 90 mg/L squalene upon co-expression with ispA. In summary, we demonstrate the applicability of R. capsulatus for the heterologous production of diverse terpene classes and provide relevant insights for further development of such platforms.

Cysteine Mutants of the Major Facilitator Superfamily-Type Transporter CcoA Provide Insight into Copper Import.

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT (CcoA-like Transporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb3-type cytochrome c oxidase (cbb3-Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met233xxxMet237 and His261xxxMet265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met233 and His261 are essential for Cu uptake and cbb3-Cox production, whereas Met237 and Met265 contribute partly to these processes. CcoA also contains five Cys residues of unknown role and, remarkably, its structural models predict that three of these are exposed to the highly oxidizing periplasm. Here, we first demonstrate that elimination of both Met237 and Met265 completely abolishes Cu uptake and cbb3-Cox production, indicating that CcoA requires at least one of these two Met residues for activity. Second, using scanning mutagenesis to probe plausible metal-interacting Met, His, and Cys residues of CcoA, we found that the periplasm-exposed Cys49 located at the end of TMH2, the Cys247 on a surface loop between TMH7 and THM8, and the C367 located at the end of TMH11 are important for CcoA function. Analyses of the single and double Cys mutants revealed the occurrence of a disulfide bond in CcoA in vivo, possibly related to conformational changes it undergoes during Cu import as MFS-type transporter. Our overall findings suggest a model linking Cu import for cbb3-Cox biogenesis with a thiol:disulfide oxidoreduction step, advancing our understanding of the mechanisms of CcoA function. IMPORTANCE Copper (Cu) is a redox-active micronutrient that is both essential and toxic. Its cellular homeostasis is critical for supporting cuproprotein maturation while avoiding excessive oxidative stress. The Cu importer CcoA is the prototype of the widespread CalT subfamily of the MFS-type transporters. Hence, understanding its molecular mechanism of function is significant. Here, we show that CcoA undergoes a thiol:disulfide oxidoreduction cycle, which is important for its Cu import activity.