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The PufX protein is found in the photosynthetic membranes of several purple bacteria and is involved in ubiquinol-ubiquinone exchange at the Q B site of the reaction center. We have studied quinone pool reduction in chromatophores from PufX + and PufX - strains of Rhodobacter capsulatus by time-resolved FTIR difference spectroscopy under and after continuous illumination. To our knowledge, it is the first time that quinone pool reduction has been directly followed in real time in Rba. capsulatus membranes. Thanks to the availability in the literature of IR marker bands for protein conformational changes, ubiquinone consumption, ubiquinol production, Q---QH 2 quinhydrone complex formation, as well as for RC-bound Q A - and Q B - semiquinone species, it is possible to follow all the molecular events associated with light-induced quinone pool reduction. In Rba. capsulatus PufX  +  chromatophores, these events resemble the ones found in Rba. sphaeroides wild-type membranes. In PufX - chromatophores the situation is different. Spectra recorded during 22.7 s of illumination showed a much smaller amount of photoreduced quinol, consistent with previous observations that PufX is required for efficient QH 2 / Q exchange at the Q B site of the RC. Q consumption and QH 2 formation are rapidly associated with Q A - formation, showing that the structure of the RC-LH1 complex in PufX - membranes does not provide efficient access to the Q B site of the RC to a large fraction of the quinone pool, evidently because the LH1 ring increases in size to impair access to the RC. The presence of a positive band at 1560 cm -1 suggests also the transient formation, in a fraction of chromatophores or of RC-LH1 complexes, of a Q---QH 2 quinhydrone complex. Experiments carried out after 2-flash and 10-flash sequences make it possible to estimate that the size of the quinone pool with access to the Q B site in PufX - membranes is ≥ 5 ubiquinone molecules per RC. The results are discussed in the framework of the current knowledge of protein organization and quinone pool reduction in bacterial photosynthetic membranes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Prokaryotes contribute to the global sulfur cycle by using diverse sulfur compounds as sulfur sources or electron acceptors. In this study, we report that a nitrogenase-like enzyme (NFL) and a radical SAM enzyme (RSE) are involved in the novel anaerobic assimilation pathway of a sulfonate, isethionate, in the photosynthetic bacterium Rhodobacter capsulatus . The nflHDK genes for NFL are localized at a locus containing genes for known sulfonate metabolism in the genome. A gene nflB encoding an RSE is present just upstream of nflH , forming a small gene cluster nflBHDK . Mutants lacking any nflBHDK genes are incapable of growing with isethionate as the sole sulfur source under anaerobic photosynthetic conditions, indicating that all four NflBHDK proteins are essential for the isethionate assimilation pathway. Heterologous expression of the islAB genes encoding a known isethionate lyase that degrades isethionate to sulfite and acetaldehyde restored the isethionate-dependent growth of a mutant lacking nflDK , indicating that the enzyme encoding nflBHDK is involved in an isethionate assimilation reaction to release sulfite. Furthermore, the heterologous expression of nflBHDK and ssuCAB encoding an isethionate transporter in the closely related species R. sphaeroides , which does not have nflBHDK and cannot grow with isethionate as the sole sulfur source, conferred isethionate-dependent growth ability to this species. We propose to rename nflBHDK as isrBHDK ( is ethionate r eductase). The isrBHDK genes are widely distributed among various prokaryote phyla. Discovery of the isethionate assimilation pathway by IsrBHDK provides a missing piece for the anaerobic sulfur cycle and for understanding the evolution of ancient sulfur metabolism.IMPORTANCENitrogenase is an important enzyme found in prokaryotes that reduces atmospheric nitrogen to ammonia and plays a fundamental role in the global nitrogen cycle. It has been noted that nitrogenase-like enzymes (NFLs), which share an evolutionary origin with nitrogenase, have evolved to catalyze diverse reactions such as chlorophyll biosynthesis (photosynthesis), coenzyme F 430 biosynthesis (methanogenesis), and methionine biosynthesis. In this study, we discovered that an NFL with unknown function in the photosynthetic bacterium Rhodobacter capsulatus is a novel isethionate reductase (Isr), which catalyzes the assimilatory degradation of isethionate, a sulfonate, releasing sulfite used as the sulfur source under anaerobic conditions. Isr is widely distributed among various bacterial phyla, including intestinal bacteria, and is presumed to play an important role in sulfur metabolism in anaerobic environments such as animal guts and microbial mats. This finding provides a clue for understanding ancient metabolism that evolved under anaerobic environments at the dawn of life., Competing Interests: The authors declare no conflict of interest.

Gene transfer agents (GTAs) are phage-like entities that package and transfer random host genome fragments between prokaryotes. RcGTA, produced by Rhodobacter capsulatus , is hypothesized to originate from a prophage ancestor. Most of the evidence supporting this hypothesis came from the finding of RcGTA-like genes in phages. More than 75% of the RcGTA genes have a phage homolog. However, only a few RcGTA homologs have been identified in a (pro)phage genome, leaving the hypothesis that GTAs evolved from prophages through gene loss with only weak evidence. We herein report the discovery of an inducible prophage (vB_MseS-P1) from a Mesorhizobium sediminum strain that contains the largest number (12) of RcGTA homologs found in a phage genome to date. We also identified three putative prophages and two prophage remnants harboring 12-14 RcGTA homologs in a Methylobacterium nodulans strain. The protein remote homology detection also revealed more RcGTA homologs from other phages than we previously thought. Moreover, the head-tail gene architecture of these newly discovered prophage-related elements closely resembles that of RcGTA. Furthermore, vB_MseS-P1 virions have structural proteins similar to RcGTA particles. Close phylogenetic relationships between certain prophage genes and RcGTA-like genes in Alphaproteobacteria further support the shared ancestry between RcGTA and prophages. Our findings provide new relatively direct evidence of the origin of RcGTA from a prophage progenitor.IMPORTANCEGTAs are important genetic elements in certain groups of bacteria and contribute to the genetic diversification, evolution, and ecological adaptation of bacteria. RcGTA, a common type of GTA, is known to package and transfer random fragments of the bacterial genome to recipient cells. However, the origin of RcGTA is still elusive. It has been hypothesized that RcGTA evolved from a prophage ancestor through gene loss. However, the few RcGTA homologs identified in a (pro)phage genome leave the hypothesis lacking direct evidence. This study uncovers the presence of a large number of RcGTA homologs in an inducible prophage and several putative prophages. The similar head-tail gene architecture and structural protein compositions of these newly discovered prophage-related elements and RcGTA further demonstrate an unprecedentedly observed close evolutionary relationship between prophages and RcGTA. Together, our findings provide more direct evidence supporting the origin of RcGTA from prophage., Competing Interests: The authors declare no conflict of interest.

Protein engineering is an established method for tailoring enzymatic reactivity. A commonly used method is directed evolution, where the mutagenesis and natural selection process is mimicked and accelerated in the laboratory. Here, we describe a reliable method for generating saturation mutagenesis libraries by Golden Gate cloning in a broad host range plasmid containing the pBBR1 replicon. The applicability is demonstrated by generating a mutant library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter capsulatus, which is subsequently screened for the improved formation of molecular hydrogen., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The rates, yields, mechanisms and directionality of electron transfer (ET) are explored in twelve pairs of Rhodobacter (R.) sphaeroides and R. capsulatus mutant RCs designed to defeat ET from the excited primary donor (P*) to the A-side cofactors and re-direct ET to the normally inactive mirror-image B-side cofactors. In general, the R. sphaeroides variants have larger P + H B - yields (up to ∼90%) than their R. capsulatus analogs (up to ∼60%), where H B is the B-side bacteriopheophytin. Substitution of Tyr for Phe at L-polypeptide position L181 near B B primarily increases the contribution of fast P* → P + B B -  → P + H B - two-step ET, where B B is the "bridging" B-side bacteriochlorophyll. The second step (∼6-8 ps) is slower than the first (∼3-4 ps), unlike A-side two-step ET (P* → P + B A -  → P + H A - ) where the second step (∼1 ps) is faster than the first (∼3-4 ps) in the native RC. Substitutions near H B , at L185 (Leu, Trp or Arg) and at M-polypeptide site M133/131 (Thr, Val or Glu), strongly affect the contribution of slower (20-50 ps) P* → P + H B - one-step superexchange ET. Both ET mechanisms are effective in directing electrons "the wrong way" to H B and both compete with internal conversion of P* to the ground state (∼200 ps) and ET to the A-side cofactors. Collectively, the work demonstrates cooperative amino-acid control of rates, yields and mechanisms of ET in bacterial RCs and how A- vs. B-side charge separation can be tuned in both species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Membrane proteins carrying redox cofactors are key subunits of respiratory chain complexes, yet the exact path of their folding and maturation remains poorly understood. Here, using cryo-EM and structure prediction via Alphafold2, we generated models of early assembly intermediates of cytochrome b (Cytb), a central subunit of complex III. The predicted structure of the first assembly intermediate suggests how the binding of Cytb to the assembly factor Cbp3-Cbp6 imposes an open configuration to facilitate the acquisition of its heme cofactors. Moreover, structure predictions of the second intermediate indicate how hemes get stabilized by binding of the assembly factor Cbp4, with a concomitant weakening of the contact between Cbp3-Cbp6 and Cytb, preparing for the release of the fully hemylated protein from the assembly factors., (© 2024 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Genetic engineering enables the forced expression of desired products in bacteria, which can then be used for a variety of applications, including functional analysis and pharmaceuticals. Here, we describe a method for tuning translation in bacteria, including Escherichia coli and Rhodobacter capsulatus, based on a phenomenon known as TED (translation enhancement by a Dictyostelium gene sequence). This method promotes translation of mRNA encoded by downstream genes by inserting a short nucleotide sequence into the 5' untranslated region between the promoter and the Shine-Dalgarno (SD) sequence. Various expression levels can be observed depending on the inserted sequence and its length, even with an identical promoter., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Formate dehydrogenases catalyze the reversible oxidation of formate to carbon dioxide. These enzymes play an important role in CO 2 reduction and serve as nicotinamide cofactor recycling enzymes. More recently, the CO 2 -reducing activity of formate dehydrogenases, especially metal-containing formate dehydrogenases, has been further explored for efficient atmospheric CO 2 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 Rc FDH, 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 (Lys 157 ) in the β-subunit of the enzyme is essential for the binding of NAD + . RcFDH variants that had Glu 259 exchanged for either a positively charged or uncharged amino acid had additional activity with NADP + . The FdsB L279R and FdsB K276A variants also showed activity with NADP + . Kinetic parameters for all the variants were determined and tested for activity in CO 2 reduction. The variants were able to reduce CO 2 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.

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., (© 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

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 β-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., (open access.)

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 (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 αβ-subunits. Four αβ-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., (© 2023. The Author(s).)

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.

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.

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.

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.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (© 2022. The Author(s).)

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Purple non-sulfur photosynthetic bacteria (PNSB) such as Rhodobacter capsulatus serve as a versatile platform for fundamental studies and various biotechnological applications. In this study, we sought to develop the class II RNA-guided CRISPR/Cas12a system from Francisella novicida for genome editing and transcriptional regulation in R. capsulatus. Template-free disruption method mediated by CRISPR/Cas12a reached ˜ 90% editing efficiency when targeting ccoO or nifH gene. When both genes were simultaneously edited, the multiplex editing efficiency reached > 63%. In addition, CRISPR interference (CRISPRi) using deactivated Cas12a was also evaluated using reporter genes egfp and lacZ, and the transcriptional repression efficiency reached ˜ 80%. In summary, our work represents the first report to develop CRISPR/Cas12a-mediated genome editing and transcriptional regulation in R. capsulatus, which would greatly accelerate PNSB-related researches., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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 β-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 β-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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

CcoA belongs to the widely distributed bacterial copper (Cu) importer subfamily CalT ( C co A - l ike T ransporters) of the Major Facilitator Superfamily (MFS) and provides cytoplasmic Cu needed for cbb 3 -type cytochrome c oxidase ( cbb 3 -Cox) biogenesis. Earlier studies have supported a 12-transmembrane helix (TMH) topology of CcoA with the well-conserved Met 233 xxxMet 237 and His 261 xxxMet 265 motifs in its TMH7 and TMH8, respectively. Of these residues, Met 233 and His 261 are essential for Cu uptake and cbb 3 -Cox production, whereas Met 237 and Met 265 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 Met 237 and Met 265 completely abolishes Cu uptake and cbb 3 -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 Cys 49 located at the end of TMH2, the Cys 247 on a surface loop between TMH7 and THM8, and the C 367 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 cbb 3 -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.

Energy crisis and global climate change have driven an increased effort toward biofuel synthesis from renewable feedstocks. Herein, purple nonsulfur photosynthetic bacterium (PNSB) of Rhodobacter capsulatus was explored as a platform for high-titer production of a terpene-based advanced biofuel-bisabolene. A multilevel engineering strategy such as promoter screening, improving the NADPH availability, strengthening the precursor supply, suppressing the side pathways, and introducing a heterologous mevalonate pathway, was used to improve the bisabolene titer in R. capsulatus . The above strategies enabled a 35-fold higher titer of bisabolene than that of the starting strain, reaching 1089.7 mg/L from glucose in a shake flask. The engineered strain produced 9.8 g/L bisabolene with a yield of >0.196 g/g-glucose under the two-phase fed-batch fermentation, which corresponds to >78% of theoretical maximum. Taken together, our work represents one of the pioneering studies to demonstrate PNSB as a promising platform for terpene-based advanced biofuel production.

Reactive sulfur species (RSS) are involved in bioactive regulation via persulfidation of proteins. However, how cells regulate RSS-based signaling and RSS metabolism is poorly understood, despite the importance of universal regulation systems in biology. We previously showed that the persulfide-responsive transcriptional factor SqrR acts as a master regulator of sulfide-dependent photosynthesis in proteobacteria. Here, we demonstrated that SqrR also binds heme at a near one-to-one ratio with a binding constant similar to other heme-binding proteins. Heme does not change the DNA-binding pattern of SqrR to the target gene promoter region; however, DNA-binding affinity of SqrR is reduced by the binding of heme, altering its regulatory activity. Circular dichroism spectroscopy clearly showed secondary structural changes in SqrR by the heme binding. Incremental change in the intracellular heme concentration is associated with small, but significant reduction in the transcriptional repression by SqrR. Overall, these results indicate that SqrR has an ability to bind heme to modulate its DNA-binding activity, which may be important for the precise regulation of RSS metabolism in vivo., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc 1 (Complex III, CIII 2 ), and may have specific cbb 3 -type cyt c oxidases (Complex IV, CIV) instead of the canonical aa 3 -type CIV. Electron transfer between these complexes is mediated by soluble (c 2 ) and membrane-anchored (c y ) cyts. Here, we report the structure of an engineered bc 1 -cbb 3 type SC (CIII 2 CIV, 5.2 Å resolution) and three conformers of native CIII 2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt c y and the assembly factor CcoH. The cyt c y is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c 2 . The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

Protein phosphorylation is a universal mechanism for transducing cellular signals in prokaryotes and eukaryotes. The histidine kinase CckA, the histidine phosphotransferase ChpT, and the response regulator CtrA are conserved throughout the alphaproteobacteria. In Rhodobacter capsulatus , these proteins are key regulators of the gene transfer agent (RcGTA), which is present in several alphaproteobacteria. Using purified recombinant R. capsulatus proteins, we show in vitro autophosphorylation of CckA protein, and phosphotransfer to ChpT and thence to CtrA, to demonstrate biochemically that they form a phosphorelay. The secondary messenger cyclic di-GMP changed CckA from a kinase to a phosphatase, resulting in reversal of the phosphotransfer flow in the relay. The substitutions of two residues in CckA greatly affected the kinase or phosphatase activity of the protein in vitro , and production of mutant CckA proteins in vivo confirmed the importance of kinase but not phosphatase activity for the lytic release of RcGTA. However, phosphatase activity was needed to produce functional RcGTA particles. The binding of cyclic di-GMP to the wild-type and mutant CckA proteins was evaluated directly using a pulldown assay based on biotinylated cyclic di-GMP and streptavidin-linked beads. IMPORTANCE The CckA, ChpT, and CtrA phosphorelay proteins are widespread in the alphaproteobacteria, and there are two groups of organisms that differ in terms of whether this pathway is essential for cell viability. Little is known about the biochemical function of these proteins in organisms where the pathway is not essential, a group that includes Rhodobacter capsulatus This work demonstrates biochemically that CckA, ChpT, and CtrA also form a functional phosphorelay in the latter group and that the direction of phosphotransfer is reversed by cyclic di-GMP. It is important to improve understanding of more representatives of this pathway in order to obtain deeper insight into the function, composition, and evolutionary significance of a wider range of bacterial regulatory networks., (Copyright © 2021 American Society for Microbiology.)

Screening of high temperature tolerant strains is important for photo-fermentative hydrogen production in natural conditions which exhibit wide temperature variations. Hence, a temperature-tolerant strain of Rhodobacter capsulatus was isolated by transposon mutagenesis. The mutant strain Rhodobacter capsulatus MX01 could convert cornstalk hydrolysate into hydrogen successfully, and exhibited better hydrogen production performance at higher culture temperature (33 °C and 37 °C) and light intensity (5000 lx and 7000 lx) than the wild type strain. At 33 °C and 5000 lx, the total hydrogen production yield and rate of MX01 from cornstalk hydrolysate were 3.64 ± 0.18 mol-H 2 /g-cornstalk and 40.07 ± 1.70 mmol-H 2 /(h·g-cornstalk), respectively. The energy conversion efficiency of cornstalk hydrolysate to hydrogen for the mutant strain MX01 was 10.6%. This higher temperature- and light intensity-tolerant mutant MX01 could carry out photo-fermentation at outdoor settings, which is important for eco-friendly, low-cost and energy-saving practical application of bio-hydrogen production., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important intracellular signaling molecule that affects diverse physiological processes in bacteria. The intracellular levels of c-di-GMP are controlled by proteins acting as diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that synthesize and degrade c-di-GMP, respectively. In the alphaproteobacterium Rhodobacter capsulatus, flagellar motility and gene exchange via production of the gene transfer agent RcGTA are regulated by c-di-GMP. One of the R. capsulatus proteins involved in this regulation is Rcc00620, which contains an N-terminal two-component system response regulator receiver (REC) domain and C-terminal DGC and PDE domains. We demonstrate that the enzymatic activity of Rcc00620 is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. In addition, we discovered that the rcc00620 and rcc00621 genes are present in only one lineage within the genus Rhodobacter and were acquired via horizontal gene transfer from a distantly related alphaproteobacterium in the order Sphingomonadales. Therefore, a horizontally acquired regulatory system regulates gene transfer in the recipient organism., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

The photosynthetic α-proteobacterium Rhodobacter capsulatus reduces and thereby fixes atmospheric dinitrogen (N 2 ) by a molybdenum (Mo)-nitrogenase and an iron-only (Fe)-nitrogenase. Differential expression of the structural genes of Mo-nitrogenase (nifHDK) and Fe-nitrogenase (anfHDGK) is strictly controlled and activated by NifA and AnfA, respectively. In contrast to NifA-binding sites, AnfA-binding sites are poorly defined. Here, we identified two highly similar AnfA-binding sites in the R. capsulatus anfH promoter by studying the effects of promoter mutations on in vivo anfH expression and in vitro promoter binding by AnfA. Comparison of the experimentally determined R. capsulatus AnfA-binding sites and presumed AnfA-binding sites from other α-proteobacteria revealed a consensus sequence of dyad symmetry, TAC-N 6 -GTA, suggesting that AnfA proteins bind their target promoters as dimers. Chromosomal replacement of the anfH promoter by the nifH promoter restored anfHDGK expression and Fe-nitrogenase activity in an R. capsulatus strain lacking AnfA suggesting that AnfA is required for AnfHDGK production, but dispensable for biosynthesis of the iron-only cofactor and electron delivery to Fe-nitrogenase, pathways activated by NifA. These observations strengthen our model, in which the Fe-nitrogenase system in R. capsulatus is largely integrated into the Mo-nitrogenase system., (© 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

5-Aminolevulinic acid (ALA) is an important metabolic intermediate compound with high value and has recently been used in agriculture and medicine. In this study, we have constructed six recombinant Escherichia coli (E. coli) strains that are involved in pET system under the regulation of the T7 promoter and LacI to express codon-optimized hemA gene from Rhodobacter capsulatus (RchemA) for ALA production via the C4 pathway. Due to codon optimization, hemA has a high transcriptional level; however, most RcHemA proteins were formed as inclusion body. To improve expression in soluble form, the vector with TrxA fusion tag was successfully used and co-expressed with partner GroELS as chaperone in another vector. As a result, ALA production increased significantly from 1.21 to 3.67 g/L. In addition, optimal ALA production was developed through adjustment of induction time and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, as well as substrate addition conditions. By adopting a two-stage induction strategy, the highest ALA reached 5.66 g/L when 0.1 mM of IPTG was added at early exponential phase (i.e., OD 600 was equal to 0.7 to 0.8), while 6 g/L of glycine, 2 g/L of succinate, and 0.03 mM of pyridoxal 5'-phosphate (PLP) were provided in the mid-exponential phase in fermentation.

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.

Rhodobacter capsulatus fixes atmospheric nitrogen (N 2 ) 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., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Genetic exchange mediated by viruses of bacteria (bacteriophages) is the primary driver of rapid bacterial evolution. The priority of viruses is usually to propagate themselves. Most bacteriophages use the small terminase protein to identify their own genome and direct its inclusion into phage capsids. Gene transfer agents (GTAs) are descended from bacteriophages, but they instead package fragments of the entire bacterial genome without preference for their own genes. GTAs do not selectively target specific DNA, and no GTA small terminases are known. Here, we identified the small terminase from the model Rhodobacter capsulatus GTA, which then allowed prediction of analogues in other species. We examined the role of the small terminase in GTA production and propose a structural basis for random DNA packaging. IMPORTANCE Random transfer of any and all genes between bacteria could be influential in the spread of virulence or antimicrobial resistance genes. Discovery of the true prevalence of GTAs in sequenced genomes is hampered by their apparent similarity to bacteriophages. Our data allowed the prediction of small terminases in diverse GTA producer species, and defining the characteristics of a "GTA-type" terminase could be an important step toward novel GTA identification. Importantly, the GTA small terminase shares many features with its phage counterpart. We propose that the GTA terminase complex could become a streamlined model system to answer fundamental questions about double-stranded DNA (dsDNA) packaging by viruses that have not been forthcoming to date., (Copyright © 2019 Sherlock et al.)

Bacteriophage-like gene transfer agents (GTAs) have been discovered in both of the prokaryotic branches of the three-domain phylogenetic tree of life. The production of a GTA (RcGTA) by the phototrophic alphaproteobacterium Rhodobacter capsulatus is regulated by quorum sensing and a phosphorelay homologous to systems in other species that control essential functions such as the initiation of chromosome replication and cell division. In wild-type strains, RcGTA is produced in <3% of cells in laboratory cultures. Mutants of R. capsulatus that exhibit greatly elevated production of RcGTA were created decades ago by chemical mutagenesis, but the nature and molecular consequences of the mutation were unknown. We show that the number of cells in a population that go on to express RcGTA genes is controlled by a stochastic process, in contrast to a genetic process. We used transposon mutagenesis along with a fluorescent protein reporter system and genome sequence data to identify a gene, rcc00280 , that encodes an RTX family calcium-binding protein homologue. The Rc280 protein acts as an extracellular repressor of RcGTA gene expression by decreasing the percentage of cells that induce the production of RcGTA. IMPORTANCE GTAs catalyze horizontal gene transfer (HGT), which is important for genomic evolution because the majority of genes found in bacterial genomes have undergone HGT at some point in their evolution. Therefore, it is important to determine how the production of GTAs is regulated to understand the factors that modulate the frequency of gene transfer and thereby specify the tempo of evolution. This work describes a new type of genetic regulation in which an extracellular calcium-binding protein homologue represses the induction of the Rhodobacter capsulatus GTA, RcGTA., (Copyright © 2019 American Society for Microbiology.)

Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis.

In bacterial reaction centers (RCs), photon-induced initial charge separation uses an A-side bacteriochlorophyll (BChl, B A ) and bacteriopheophytin (BPh, H A ), while the near-mirror image B-side B B and H B cofactors are inactive. Two new sets of Rhodobacter capsulatus RC mutants were designed, both bearing substitution of all amino acids for the native histidine M180 (M-polypeptide residue 180) ligand to the core Mg ion of B B . Residues are identified that largely result in retention of a BChl in the B B site (Asp, Ser, Pro, Gln, Asn, Gly, Cys, Lys, and Thr), ones that largely harbor the Mg-free BPh in the B B site (Leu and Ile), and ones for which isolated RCs are comprised of a substantial mixture of these two RC types (Ala, Glu, Val, Met and, in one set, Arg). No protein was isolated when M180 is Trp, Tyr, Phe, or (in one set) Arg. These findings are corroborated by ground state spectra, pigment extractions, ultrafast transient absorption studies, and the yields of B-side transmembrane charge separation. The changes in coordination chemistries did not reveal an RC with sufficiently precise poising of the redox properties of the B B -site cofactor to result in a high yield of B-side electron transfer to H B . Insights are gleaned into the amino acid properties that support BChl in the B B site and into the widely observed multi-exponential decay of the excited state of the primary electron donor. The results also have direct implications for tuning free energies of the charge-separated intermediates in RCs and mimetic systems.

This study investigated the effect of different nitrogen sources, namely, ammonium chloride and glutamate, on photoheterotrophic metabolism of Rhodobacter capsulatus grown on acetate as the carbon source. Genes that were significantly differentially expressed according to Affymetrix microarray data were categorized into Clusters of Orthologous Groups functional categories and those in acetate assimilation, hydrogen production, and photosynthetic electron transport pathways were analyzed in detail. Genes related to hydrogen production metabolism were significantly downregulated in cultures grown on ammonium chloride when compared to those grown on glutamate. In contrast, photosynthetic electron transport and acetate assimilation pathway genes were upregulated. In detail, aceA encoding isocitrate lyase, a unique enzyme of the glyoxylate cycle and ccrA encoding the rate limiting crotonyl-CoA carboxylase/reductase enzyme of ethylmalonyl-coA pathway were significantly upregulated. Our findings indicate for the first time that R. capsulatus can operate both glyoxylate and ethylmalonyl-coA cycles for acetate assimilation.

Bacterial microcompartments (BMCs) are large (∼100-nm) protein shells that encapsulate enzymes, their substrates, and cofactors for the purposes of increasing metabolic reaction efficiency and protecting cells from toxic intermediates. The best-studied microcompartment is the carbon-fixing carboxysome that encapsulates ribulose-1,5-bisphosphate carboxylase and carbonic anhydrase. Other well-known BMCs include the Pdu and Eut BMCs, which metabolize 1,2-propanediol and ethanolamine, respectively, with vitamin B 12 -dependent diol dehydratase enzymes. Recent bioinformatic analyses identified a new prevalent type of BMC, hypothesized to utilize vitamin B 12 -independent glycyl radical enzymes to metabolize substrates. Here we use genetic and metabolic analyses to undertake in vivo characterization of the newly identified glycyl radical enzyme microcompartment 3 (GRM3) class of microcompartment clusters. Transcriptome sequencing analyses showed that the microcompartment gene cluster in the genome of the purple photosynthetic bacterium Rhodobacter capsulatus was expressed under dark anaerobic respiratory conditions in the presence of 1,2-propanediol. High-performance liquid chromatography and gas chromatography-mass spectrometry analyses showed that enzymes coded by this cluster metabolized 1,2-propanediol into propionaldehyde, propanol, and propionate. Surprisingly, the microcompartment pathway did not protect these cells from toxic propionaldehyde under the conditions used in this study, with buildup of this intermediate contributing to arrest of cell growth. We further show that expression of microcompartment genes is regulated by a two-component system located downstream of the microcompartment cluster. IMPORTANCE BMCs are protein shells that are designed to compartmentalize enzymatic reactions that require either sequestration of a substrate or the sequestration of toxic intermediates. Due to their ability to compartmentalize reactions, BMCs have also become attractive targets for bioengineering novel enzymatic reactions. Despite these useful features, little is known about the biochemistry of newly identified classes of BMCs. In this study, we have undertaken genetic and in vivo metabolic analyses of the newly identified GRM3 gene cluster., (Copyright © 2019 American Society for Microbiology.)

Gene transfer agents (GTAs) are thought to be ancient bacteriophages that have been co-opted into serving their host and can now transfer any gene between bacteria. Production of GTAs is controlled by several global regulators through unclear mechanisms. In Rhodobacter capsulatus, gene rcc01865 encodes a putative regulatory protein that is essential for GTA production. Here, I show that rcc01865 (hereafter gafA) encodes a transcriptional regulator that binds to the GTA promoter to initiate production of structural and DNA packaging components. Expression of gafA is in turn controlled by the pleiotropic regulator protein CtrA and the quorum-sensing regulator GtaR. GafA and CtrA work together to promote GTA maturation and eventual release through cell lysis. Identification of GafA as a direct GTA regulator allows the first integrated regulatory model to be proposed and paves the way for discovery of GTAs in other species that possess gafA homologues.

Comparative genomic studies of the bacterial MFS-type copper importer CcoA, required for cbb 3 -type cytochrome c oxidase (cbb 3 -Cox) biogenesis, revealed a widespread CcoA-like transporters (CalT) family, containing the conserved CcoA Cu-binding MxxxM and HxxxM motifs. Surprisingly, this family also included the RfnT-like proteins, earlier suggested to transport riboflavin. However, presence of the Cu-binding motifs in these proteins raised the possibility that they might be Cu transporters. To test this hypothesis, the genomic context of the corresponding genes was examined, and three of such genes from Ochrobactrum anthropi, Rhodopseudomonas palustris and Agrobacterium tumefaciens were expressed in Escherichia coli (ΔribB) and Rhodobacter capsulatus (ΔccoA) mutants. Copper and riboflavin uptake abilities of these strains were compared with those expressing R. capsulatus CcoA and Rhizobium leguminosarum RibN as bona fide copper and riboflavin importers, respectively. Overall data demonstrated that the "RfnT-like" CalT proteins are unable to efficiently transport riboflavin, but they import copper like CcoA. Nevertheless, even though expressed and membrane-localized in a R. capsulatus mutant lacking CcoA, these transporters were unable to accumulate Cu or complement for cbb 3 -Cox defect. This lack of functional exchangeability between the different subfamilies of CalT homologs suggests that MFS-type bacterial copper importers might be species-specific.

The ubihydroquinone:cytochrome (cyt) c oxidoreductase (or cyt bc 1 ) is an important enzyme for photosynthesis and respiration. In bacteria like Rhodobacter capsulatus, this membrane complex has three subunits, the iron‑sulfur protein (ISP) with its Fe 2 S 2 cluster, cyt c 1 and cyt b, forming two catalytic domains, the Q o (hydroquinone (QH 2 ) oxidation) and Q i (quinone (Q) reduction) sites. At the Q o site, the electron transfer pathways originating from QH 2 oxidation are known, but their associated proton release routes are less well defined. Earlier, we demonstrated that the His291 of cyt b is important for this latter process. In this work, using the bacterial cyt bc 1 and site directed mutagenesis, we show that Lys329 of cyt b is also critical for electron and proton transfer at the Q o site. Of the mutants examined, Lys329Arg was photosynthesis proficient and had quasi-wild type cyt bc 1 activity. In contrast, the Lys329Ala and Lys329Asp were photosynthesis-impaired and contained defective but assembled cyt bc 1 . In particular, the bifurcated electron transfer and associated proton(s) release reactions occurring during QH 2 oxidation were drastically impaired in Lys329Asp mutant. Furthermore, in silico docking studies showed that in this mutant the location and the H-bonding network around the Fe 2 S 2 cluster of ISP on cyt b surface was different than the wild type enzyme. Based on these experimental findings and theoretical considerations, we propose that the presence of a positive charge at position 329 of cyt b is critical for efficient electron transfer and proton release for QH 2 oxidation at the Q o site of cyt bc 1 ., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Catalytic reactions of quinol oxidoreductases may lead to the generation of superoxide due to electron leaks from unstable semiquinone intermediates (SQ). For cytochrome bc 1 , the mechanism of suppression of superoxide generation remains unknown. We analyzed conditions of formation of a spin-spin-coupled state between SQ and the Rieske cluster (SQ-FeS) associated with catalysis of the quinol oxidation site of cytochrome bc 1 . We reveal that mutants that preclude direct interaction between SQ and the Rieske cluster do not form SQ-FeS and release enhanced superoxide. In the enzymes generating SQ-FeS, little or no superoxide is detected. We propose that SQ-FeS suppresses superoxide generation, becoming an element modulating superoxide release under physiologically relevant conditions slowing electron flow through the enzyme., (© 2018 Federation of European Biochemical Societies.)

The maturation of bacterial molybdoenzymes is a complex process leading to the insertion of the bulky bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor into the apo-enzyme. Most molybdoenzymes were shown to contain a specific chaperone for the insertion of the bis-MGD cofactor. Formate dehydrogenases (FDH) together with their molecular chaperone partner seem to display an exception to this specificity rule, since the chaperone FdhD has been proven to be involved in the maturation of all three FDH enzymes present in Escherichia coli. Multiple roles have been suggested for FdhD-like chaperones in the past, including the involvement in a sulfur transfer reaction from the l-cysteine desulfurase IscS to bis-MGD by the action of two cysteine residues present in a conserved CXXC motif of the chaperones. However, in this study we show by phylogenetic analyses that the CXXC motif is not conserved among FdhD-like chaperones. We compared in detail the FdhD-like homologues from Rhodobacter capsulatus and E. coli and show that their roles in the maturation of FDH enzymes from different subgroups can be exchanged. We reveal that bis-MGD-binding is a common characteristic of FdhD-like proteins and that the cofactor is bound with a sulfido-ligand at the molybdenum atom to the chaperone. Generally, we reveal that the cysteine residues in the motif CXXC of the chaperone are not essential for the production of active FDH enzymes., Competing Interests: The authors have declared that no competing interests exist.

Phototrophic microorganisms adjust photosystem synthesis in response to changes in light intensity and wavelength. A variety of different photoreceptors regulate this process. Purple photosynthetic bacteria synthesize a novel photoreceptor AerR that uses cobalamin (B 12 ) as a blue-light absorbing chromophore to control photosystem synthesis. AerR directly interacts with the redox responding transcription factor CrtJ, affecting CrtJ's interaction with photosystem promoters. In this study, we show that AerR is translated as two isoforms that differ by 41 amino acids at the amino terminus. The ratio of these isoforms was affected by light and cell growth phase with the long variant predominating during photosynthetic exponential growth and the short variant predominating in dark conditions and/or stationary phase. Pigmentation and transcriptomic analyses show that the short AerR variant represses, while long variant activates, photosynthesis genes. The long form of AerR also activates many genes involved in cellular metabolism and motility., Competing Interests: HY, MF, VD, CB No competing interests declared, (© 2018, Yamamoto et al.)

Several of the enzymes involved in the conversion of adenosylcobyric acid (AdoCby) to adenosylcobamide (AdoCba) are yet to be identified and characterized in some cobamide (Cba)-producing prokaryotes. Using a bioinformatics approach, we identified the bluE gene (locus tag RSP&#95;0788) of Rhodobacter sphaeroides 2.4.1 as a putative functional homolog of the L-threonine kinase enzyme (PduX, EC 2.7.1.177) of S. enterica. In AdoCba, (R)-1-aminopropan-2-ol O-phosphate (AP-P) links the nucleotide loop to the corrin ring; most known AdoCba producers derive AP-P from L-Thr-O-3-phosphate (L-Thr-P). Here, we show that RsBluE has L-Thr-independent ATPase activity in vivo and in vitro. We used 31 P-NMR spectroscopy to show that RsBluE generates L-Thr-P at the expense of ATP and is unable to use L-Ser as a substrate. BluE from R. sphaeroides or Rhodobacter capsulatus restored AdoCba biosynthesis in S. enterica ΕpduX and R. sphaeroides ΕbluE mutant strains. R. sphaeroides ΕbluE strains exhibited a decreased pigment phenotype that was restored by complementation with BluE. Finally, phylogenetic analyses revealed that bluE was restricted to the genomes of a few Rhodobacterales that appear to have a preference for a specific form of Cba, namely Coᴽ-(ᴽ-5,6-dimethylbenzimidazolyl-Coᵦ-adenosylcobamide (a.k.a. adenosylcobalamin, AdoCbl; coenzyme B 12 , CoB 12 )., (© 2018 John Wiley & Sons Ltd.)

Several members of the Rhodobacterales ( Alphaproteobacteria ) produce a conserved horizontal gene transfer vector, called the gene transfer agent (GTA), that appears to have evolved from a bacteriophage. The model system used to study GTA biology is the Rhodobacter capsulatus GTA (RcGTA), a small, tailed bacteriophage-like particle produced by a subset of the cells in a culture. The response regulator CtrA is conserved in the Alphaproteobacteria and is an essential regulator of RcGTA production: it controls the production and maturation of the RcGTA particle and RcGTA release from cells. CtrA also controls the natural transformation-like system required for cells to receive RcGTA-donated DNA. Here, we report that dysregulation of the CckA-ChpT-CtrA phosphorelay either by the loss of the PAS domain protein DivL or by substitution of the autophosphorylation residue of the hybrid histidine kinase CckA decreased CtrA phosphorylation and greatly increased RcGTA protein production in R. capsulatus We show that the loss of the ClpXP protease or the three C-terminal residues of CtrA results in increased CtrA levels in R. capsulatus and identify ClpX(P) to be essential for the maturation of RcGTA particles. Furthermore, we show that CtrA phosphorylation is important for head spike production. Our results provide novel insight into the regulation of CtrA and GTAs in the Rhodobacterales IMPORTANCE Members of the Rhodobacterales are abundant in ocean and freshwater environments. The conserved GTA produced by many Rhodobacterales may have an important role in horizontal gene transfer (HGT) in aquatic environments and provide a significant contribution to their adaptation. GTA production is controlled by bacterial regulatory systems, including the conserved CckA-ChpT-CtrA phosphorelay; however, several questions about GTA regulation remain. Our identification that a short DivL homologue and ClpXP regulate CtrA in R. capsulatus extends the model of CtrA regulation from Caulobacter crescentus to a member of the Rhodobacterales We found that the magnitude of RcGTA production greatly depends on DivL and CckA kinase activity, adding yet another layer of regulatory complexity to RcGTA. RcGTA is known to undergo CckA-dependent maturation, and we extend the understanding of this process by showing that the ClpX chaperone is required for formation of tailed, DNA-containing particles., (Copyright © 2018 American Society for Microbiology.)

Heme biosynthesis, a complex, multistage, and tightly controlled process, starts with 5-aminolevulinate (ALA) production, which, in metazoa and certain bacteria, is a reaction catalyzed by 5-aminolevulinate synthase (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. Functional aberrations in ALAS are associated with several human diseases. ALAS can adopt open and closed conformations, with segmental rearrangements of a C-terminal, 16-amino acid loop and an α-helix regulating accessibility to the ALAS active site. Of the murine erythroid ALAS (mALAS2) forms previously engineered to assess the role of the flexible C-terminal loop versus mALAS2 function one stood out due to its impressive gain in catalytic power. To elucidate how the simultaneously introduced seven mutations of this activity-enhanced variant affected structural and dynamic properties of mALAS2, we conducted extensive molecular dynamics simulation analysis of the dimeric forms of wild-type mALAS2, hepta-variant and Rhodobacter capsulatus ALAS (aka R. capsulatus HemA). This analysis revealed that the seven simultaneous mutations in the C-terminal loop, which extends over the active site of the enzyme, caused the bacterial and murine proteins to adopt different conformations. Specifically, a new β-strand in the mutated 'loop' led to interaction with two preexisting β-strands and formation of an anti-parallel three-stranded β-sheet, which likely endowed the murine hepta-variant a more 'stable' open conformation than that of wild-type mALAS2, consistent with a kinetic mechanism involving a faster closed-to-open conformation transition and product release for the mutated than wild-type enzyme. Further, the dynamic behavior of the mALAS2 protomers was strikingly different in the two dimeric forms.