Sulfoquinovosyl diacylglycerol is required for dimerisation of the Rhodobacter sphaeroides reaction centre-light harvesting 1 core complex.

The reaction centre-light harvesting 1 (RC-LH1) core complex is indispensable for anoxygenic photosynthesis. In the purple bacterium Rhodobacter (Rba.) sphaeroides RC-LH1 is produced both as a monomer, in which 14 LH1 subunits form a C-shaped antenna around 1 RC, and as a dimer, where 28 LH1 subunits form an S-shaped antenna surrounding 2 RCs. Alongside the five RC and LH1 subunits, an additional polypeptide known as PufX provides an interface for dimerisation and also prevents LH1 ring closure, introducing a channel for quinone exchange that is essential for photoheterotrophic growth. Structures of Rba. sphaeroides RC-LH1 complexes revealed several new components; protein-Y, which helps to form the quinone channel; protein-Z, of unknown function and seemingly unique to dimers; and a tightly bound sulfoquinovosyl diacylglycerol (SQDG) lipid that interacts with two PufX arginine residues. This lipid lies at the dimer interface alongside weak density for a second molecule, previously proposed to be an ornithine lipid. In this work we have generated strains of Rba. sphaeroides lacking protein-Y, protein-Z, SQDG or ornithine lipids to assess the roles of these previously unknown components in the assembly and activity of RC-LH1. We show that whilst the removal of either protein-Y, protein-Z or ornithine lipids has only subtle effects, SQDG is essential for the formation of RC-LH1 dimers but its absence has no functional effect on the monomeric complex.

Exploiting Violet-Blue Light to Kill Campylobacter jejuni: Analysis of Global Responses, Modeling of Transcription Factor Activities, and Identification of Protein Targets.

Campylobacter jejuni is a microaerophilic foodborne zoonotic pathogen of worldwide concern as the leading cause of bacterial gastroenteritis. Many strains are increasingly antibiotic resistant and new methods of control are required to reduce food-chain contamination. One possibility is photodynamic inactivation (PDI) using violet-blue (VB) light, to which C. jejuni is highly susceptible. Here, we show that flavin and protoporphyrin IX are major endogenous photosensitizers and that exposure of cells to VB light increases intracellular reactive oxygen species (ROS) to high levels, as indicated by a dichlorodihydrofluorescein reporter. Unusually for an oxygen-respiring bacterium, C. jejuni employs several ROS-sensitive iron-sulfur cluster enzymes in central metabolic pathways; we show that VB light causes rapid inactivation of both pyruvate and 2-oxoglutarate oxidoreductases, thus interrupting the citric acid cycle. Cells exposed to VB light also lose heme from c-type cytochromes, restricting electron transport, likely due to irreversible oxidation of heme-ligating cysteine residues. Evaluation of global gene expression changes by RNAseq and probabilistic modeling showed a two-stage protein damage/oxidative stress response to VB light, driven by specific regulators, including HspR, PerR, Fur, and RacR. Deletion mutant analysis showed that superoxide dismutase and the cytochrome CccA were particularly important for VB light survival and that abolishing repression of chaperones and oxidative stress resistance genes by HcrA, HspR, or PerR increased tolerance to VB light. Our results explain the high innate sensitivity of C. jejuni to VB light and provide new insights that may be helpful in exploiting PDI for novel food-chain interventions to control this pathogen. IMPORTANCE Campylobacteriosis caused by C. jejuni is one of the most widespread zoonotic enteric diseases worldwide and represents an enormous human health and economic burden, compounded by the emergence of antibiotic-resistant strains. New interventions are urgently needed to reduce food-chain contamination. Although UV light is well known to be bactericidal, it is highly mutagenic and problematic for continuous exposure in food production facilities; in contrast, narrow spectrum violet-blue (VB) light is much safer. We confirmed that C. jejuni is highly susceptible to VB light and then identified some of the global regulatory networks involved in responding to photo-oxidative damage. The identification of damaged cellular components underpins efforts to develop commercial applications of VB light-based technologies.

Cysteine Biosynthesis in Campylobacter jejuni: Substrate Specificity of CysM and the Dualism of Sulfide.

Campylobacter jejuni is a highly successful enteric pathogen with a small, host-adapted genome (1.64 Mbp, ~1650 coding genes). As a result, C. jejuni has limited capacity in numerous metabolic pathways, including sulfur metabolism. Unable to utilise ionic sulfur, C. jejuni relies on the uptake of exogenous cysteine and its derivatives for its supply of this essential amino acid. Cysteine can also be synthesized de novo by the sole cysteine synthase, CysM. In this study, we explored the substrate specificity of purified C. jejuni CysM and define it as an O-acetyl-L-serine sulfhydrylase with an almost absolute preference for sulfide as sulfur donor. Sulfide is produced in abundance in the intestinal niche C. jejuni colonises, yet sulfide is generally viewed as highly toxic to bacteria. We conducted a series of growth experiments in sulfur-limited media and demonstrate that sulfide is an excellent sulfur source for C. jejuni at physiologically relevant concentrations, combating the view of sulfide as a purely deleterious compound to bacteria. Nonetheless, C. jejuni is indeed inhibited by elevated concentrations of sulfide and we sought to understand the targets involved. Surprisingly, we found that inactivation of the sulfide-sensitive primary terminal oxidase, the cbb3-type cytochrome c oxidase CcoNOPQ, did not explain the majority of growth inhibition by sulfide. Therefore, further work is required to reveal the cellular targets responsible for sulfide toxicity in C. jejuni.

FRET measurement of cytochrome bc1 and reaction centre complex proximity in live Rhodobacter sphaeroides cells.

In the model purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides, solar energy is converted via coupled electron and proton transfer reactions within the intracytoplasmic membranes (ICMs), infoldings of the cytoplasmic membrane that form spherical 'chromatophore' vesicles. These bacterial 'organelles' are ideal model systems for studying how the organisation of the photosynthetic complexes therein shape membrane architecture. In Rba. sphaeroides, light-harvesting 2 (LH2) complexes transfer absorbed excitation energy to dimeric reaction centre (RC)-LH1-PufX complexes. The PufX polypeptide creates a channel that allows the lipid soluble electron carrier quinol, produced by RC photochemistry, to diffuse to the cytochrome bc1 complex, where quinols are oxidised to quinones, with the liberated protons used to generate a transmembrane proton gradient and the electrons returned to the RC via cytochrome c2. Proximity between cytochrome bc1 and RC-LH1-PufX minimises quinone/quinol/cytochrome c2 diffusion distances within this protein-crowded membrane, however this distance has not yet been measured. Here, we tag the RC and cytochrome bc1 with yellow or cyan fluorescent proteins (YFP/CFP) and record the lifetimes of YFP/CFP Förster resonance energy transfer (FRET) pairs in whole cells. FRET analysis shows that that these complexes lie on average within 6 nm of each other. Complementary high-resolution atomic force microscopy (AFM) of intact, purified chromatophores verifies the close association of cytochrome bc1 complexes with RC-LH1-PufX dimers. Our results provide a structural basis for the close kinetic coupling between RC-LH1-PufX and cytochrome bc1 observed by spectroscopy, and explain how quinols/quinones and cytochrome c2 shuttle on a millisecond timescale between these complexes, sustaining efficient photosynthetic electron flow.

Cryo-EM structure of the dimeric Rhodobacter sphaeroides RC-LH1 core complex at 2.9†Å: the structural basis for dimerisation.

The dimeric reaction centre light-harvesting 1 (RC-LH1) core complex of Rhodobacter sphaeroides converts absorbed light energy to a charge separation, and then it reduces a quinone electron and proton acceptor to a quinol. The angle between the two monomers imposes a bent configuration on the dimer complex, which exerts a major influence on the curvature of the membrane vesicles, known as chromatophores, where the light-driven photosynthetic reactions take place. To investigate the dimerisation interface between two RC-LH1 monomers, we determined the cryogenic electron microscopy structure of the dimeric complex at 2.9 Šresolution. The structure shows that each monomer consists of a central RC partly enclosed by a 14-subunit LH1 ring held in an open state by PufX and protein-Y polypeptides, thus enabling quinones to enter and leave the complex. Two monomers are brought together through N-terminal interactions between PufX polypeptides on the cytoplasmic side of the complex, augmented by two novel transmembrane polypeptides, designated protein-Z, that bind to the outer faces of the two central LH1 ß polypeptides. The precise fit at the dimer interface, enabled by PufX and protein-Z, by C-terminal interactions between opposing LH1 αß subunits, and by a series of interactions with a bound sulfoquinovosyl diacylglycerol lipid, bring together each monomer creating an S-shaped array of 28 bacteriochlorophylls. The seamless join between the two sets of LH1 bacteriochlorophylls provides a path for excitation energy absorbed by one half of the complex to migrate across the dimer interface to the other half.

Cryo-EM structure of the monomeric Rhodobacter sphaeroides RC-LH1 core complex at 2.5†Å.

Reaction centre light-harvesting 1 (RC-LH1) complexes are the essential components of bacterial photosynthesis. The membrane-intrinsic LH1 complex absorbs light and the energy migrates to an enclosed RC where a succession of electron and proton transfers conserves the energy as a quinol, which is exported to the cytochrome bc1 complex. In some RC-LH1 variants quinols can diffuse through small pores in a fully circular, 16-subunit LH1 ring, while in others missing LH1 subunits create a gap for quinol export. We used cryogenic electron microscopy to obtain a 2.5 Šresolution structure of one such RC-LH1, a monomeric complex from Rhodobacter sphaeroides. The structure shows that the RC is partly enclosed by a 14-subunit LH1 ring in which each αß heterodimer binds two bacteriochlorophylls and, unusually for currently reported complexes, two carotenoids rather than one. Although the extra carotenoids confer an advantage in terms of photoprotection and light harvesting, they could impede passage of quinones through small, transient pores in the LH1 ring, necessitating a mechanism to create a dedicated quinone channel. The structure shows that two transmembrane proteins play a part in stabilising an open ring structure; one of these components, the PufX polypeptide, is augmented by a hitherto undescribed protein subunit we designate as protein-Y, which lies against the transmembrane regions of the thirteenth and fourteenth LH1α polypeptides. Protein-Y prevents LH1 subunits 11-14 adjacent to the RC QB site from bending inwards towards the RC and, with PufX preventing complete encirclement of the RC, this pair of polypeptides ensures unhindered quinone diffusion.

A Thermostable Protein Matrix for Spectroscopic Analysis of Organic Semiconductors.

Advances in protein design and engineering have yielded peptide assemblies with enhanced and non-native functionalities. Here, various molecular organic semiconductors (OSCs), with known excitonic up- and down-conversion properties, are attached to a de novo-designed protein, conferring entirely novel functions on the peptide scaffolds. The protein-OSC complexes form similarly sized, stable, water-soluble nanoparticles that are robust to cryogenic freezing and processing into the solid-state. The peptide matrix enables the formation of protein-OSC-trehalose glasses that fix the proteins in their folded states under oxygen-limited conditions. The encapsulation dramatically enhances the stability of protein-OSC complexes to photodamage, increasing the lifetime of the chromophores from several hours to more than 10 weeks under constant illumination. Comparison of the photophysical properties of astaxanthin aggregates in mixed-solvent systems and proteins shows that the peptide environment does not alter the underlying electronic processes of the incorporated materials, exemplified here by singlet exciton fission followed by separation into weakly bound, localized triplets. This adaptable protein-based approach lays the foundation for spectroscopic assessment of a broad range of molecular OSCs in aqueous solutions and the solid-state, circumventing the laborious procedure of identifying the experimental conditions necessary for aggregate generation or film formation. The non-native protein functions also raise the prospect of future biocompatible devices where peptide assemblies could complex with native and non-native systems to generate novel functional materials.

Quantifying the Interaction of Phosphite with ABC Transporters: MicroScale Thermophoresis and a Novel His-Tag Labeling Approach.

The combination of MicroScale Thermophoresis (MST) and near-native site-specific His-tag labeling enables simple, robust, and reliable determination of the binding affinity between proteins and ligands. To demonstrate its applicability for periplasmic proteins, we provide a detailed protocol for determination of the binding affinity of phosphite to three ABC transporter periplasmic-binding proteins from environmental microorganisms. ABC transporters are central to many important biomedical phenomena, including resistance of cancers and pathogenic microbes to drugs. The protocol described here can be used to quantify protein-ligand and protein-protein interactions for other soluble, membrane-associated and integral membrane proteins.

Atoms to Phenotypes: Molecular Design Principles of Cellular Energy Metabolism.

We report a 100-million atom-scale model of an entire cell organelle, a photosynthetic chromatophore vesicle from a purple bacterium, that reveals the cascade of energy conversion steps culminating in the generation of ATP from sunlight. Molecular dynamics simulations of this vesicle elucidate how the integral membrane complexes influence local curvature to tune photoexcitation of pigments. Brownian dynamics of small molecules within the chromatophore probe the mechanisms of directional charge transport under various pH and salinity conditions. Reproducing phenotypic properties from atomistic details, a kinetic model evinces that low-light adaptations of the bacterium emerge as a spontaneous outcome of optimizing the balance between the chromatophore's structural integrity and robust energy conversion. Parallels are drawn with the more universal mitochondrial bioenergetic machinery, from whence molecular-scale insights into the mechanism of cellular aging are inferred. Together, our integrative method and spectroscopic experiments pave the way to first-principles modeling of whole living cells.

Structural and functional characterization of IdiA/FutA (Tery_3377), an iron-binding protein from the ocean diazotroph Trichodesmium erythraeum.

Atmospheric nitrogen fixation by photosynthetic cyanobacteria (diazotrophs) strongly influences oceanic primary production and in turn affects global biogeochemical cycles. Species of the genus Trichodesmium are major contributors to marine diazotrophy, accounting for a significant proportion of the fixed nitrogen in tropical and subtropical oceans. However, Trichodesmium spp. are metabolically constrained by the availability of iron, an essential element for both the photosynthetic apparatus and the nitrogenase enzyme. Survival strategies in low-iron environments are typically poorly characterized at the molecular level, because these bacteria are recalcitrant to genetic manipulation. Here, we studied a homolog of the iron deficiency-induced A (IdiA)/ferric uptake transporter A (FutA) protein, Tery_3377, which has been used as an in situ iron-stress biomarker. IdiA/FutA has an ambiguous function in cyanobacteria, with its homologs hypothesized to be involved in distinct processes depending on their cellular localization. Using signal sequence fusions to GFP and heterologous expression in the model cyanobacterium Synechocystis sp. PCC 6803, we show that Tery_3377 is targeted to the periplasm by the twin-arginine translocase and can complement the deletion of the native Synechocystis ferric-iron ABC transporter periplasmic binding protein (FutA2). EPR spectroscopy revealed that purified recombinant Tery_3377 has specificity for iron in the Fe3+ state, and an X-ray crystallography-determined structure uncovered a functional iron substrate-binding domain, with Fe3+ pentacoordinated by protein and buffer ligands. Our results support assignment of Tery_3377 as a functional FutA subunit of an Fe3+ ABC transporter but do not rule out dual IdiA function.

Plant and algal chlorophyll synthases function in Synechocystis and interact with the YidC/Alb3 membrane insertase.

In the model cyanobacterium Synechocystis sp. PCC 6803, the terminal enzyme of chlorophyll biosynthesis, chlorophyll synthase (ChlG), forms a complex with high light-inducible proteins, the photosystem II assembly factor Ycf39 and the YidC/Alb3/OxaI membrane insertase, co-ordinating chlorophyll delivery with cotranslational insertion of nascent photosystem polypeptides into the membrane. To gain insight into the ubiquity of this assembly complex in higher photosynthetic organisms, we produced functional foreign chlorophyll synthases in a cyanobacterial host. Synthesis of algal and plant chlorophyll synthases allowed deletion of the otherwise essential native cyanobacterial gene. Analysis of purified protein complexes shows that the interaction with YidC is maintained for both eukaryotic enzymes, indicating that a ChlG-YidC/Alb3 complex may be evolutionarily conserved in algae and plants.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo-designed heme protein.

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo-designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b-induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Determination of Cell Doubling Times from the Return-on-Investment Time of Photosynthetic Vesicles Based on Atomic Detail Structural Models.

Cell doubling times of the purple bacterium Rhodobacter sphaeroides during photosynthetic growth are determined experimentally and computationally as a function of illumination. For this purpose, energy conversion processes in an intracytoplasmic membrane vesicle, the chromatophore, are described based on an atomic detail structural model. The cell doubling time and its illumination dependence are computed in terms of the return-on-investment (ROI) time of the chromatophore, determined computationally from the ATP production rate, and the mass ratio of chromatophores in the cell, determined experimentally from whole cell absorbance spectra. The ROI time is defined as the time it takes to produce enough ATP to pay for the construction of another chromatophore. The ROI time of the low light-growth chromatophore is 4.5-2.6 h for a typical illumination range of 10-100 µmol photons m-2 s-1, respectively, with corresponding cell doubling times of 8.2-3.9 h. When energy expenditure is considered as a currency, the benefit-to-cost ratio computed for the chromatophore as an energy harvesting device is 2-8 times greater than for photovoltaic and fossil fuel-based energy solutions and the corresponding ROI times are approximately 3-4 orders of magnitude shorter for the chromatophore than for synthetic systems.

Desert Dust as a Source of Iron to the Globally Important Diazotroph Trichodesmium.

The marine cyanobacterium Trichodesmium sp. accounts for approximately half of the annual 'new' nitrogen introduced to the global ocean but its biogeography and activity is often limited by the availability of iron (Fe). A major source of Fe to the open ocean is Aeolian dust deposition in which Fe is largely comprised of particles with reduced bioavailability over soluble forms of Fe. We report that Trichodesmium erythraeum IMS101 has improved growth rate and photosynthetic physiology and down-regulates Fe-stress biomarker genes when cells are grown in the direct vicinity of, rather than physically separated from, Saharan dust particles as the sole source of Fe. These findings suggest that availability of non-soluble forms of dust-associated Fe may depend on cell contact. Transcriptomic analysis further reveals unique profiles of gene expression in all tested conditions, implying that Trichodesmium has distinct molecular signatures related to acquisition of Fe from different sources. Trichodesmium thus appears to be capable of employing specific mechanisms to access Fe from complex sources in oceanic systems, helping to explain its role as a key microbe in global biogeochemical cycles.

Evidence for polyploidy in the globally important diazotroph Trichodesmium.

Polyploidy is a well-described trait in some prokaryotic organisms; however, it is unusual in marine microbes from oligotrophic environments, which typically display a tendency towards genome streamlining. The biogeochemically significant diazotrophic cyanobacterium Trichodesmium is a potential exception. With a relatively large genome and a comparatively high proportion of non-protein-coding DNA, Trichodesmium appears to allocate relatively more resources to genetic material than closely related organisms and microbes within the same environment. Through simultaneous analysis of gene abundance and direct cell counts, we show for the first time that Trichodesmium spp. can also be highly polyploid, containing as many as 100 genome copies per cell in field-collected samples and >600 copies per cell in laboratory cultures. These findings have implications for the widespread use of the abundance of the nifH gene (encoding a subunit of the N2-fixing enzyme nitrogenase) as an approach for quantifying the abundance and distribution of marine diazotrophs. Moreover, polyploidy may combine with the unusual genomic characteristics of this genus both in reflecting evolutionary dynamics and influencing phenotypic plasticity and ecological resilience.

Quantifying Integrated Proteomic Responses to Iron Stress in the Globally Important Marine Diazotroph Trichodesmium.

Trichodesmium is a biogeochemically important marine cyanobacterium, responsible for a significant proportion of the annual 'new' nitrogen introduced into the global ocean. These non-heterocystous filamentous diazotrophs employ a potentially unique strategy of near-concurrent nitrogen fixation and oxygenic photosynthesis, potentially burdening Trichodesmium with a particularly high iron requirement due to the iron-binding proteins involved in these processes. Iron availability may therefore have a significant influence on the biogeography of Trichodesmium. Previous investigations of molecular responses to iron stress in this keystone marine microbe have largely been targeted. Here a holistic approach was taken using a label-free quantitative proteomics technique (MSE) to reveal a sophisticated multi-faceted proteomic response of Trichodesmium erythraeum IMS101 to iron stress. Increased abundances of proteins known to be involved in acclimation to iron stress and proteins known or predicted to be involved in iron uptake were observed, alongside decreases in the abundances of iron-binding proteins involved in photosynthesis and nitrogen fixation. Preferential loss of proteins with a high iron content contributed to overall reductions of 55-60% in estimated proteomic iron requirements. Changes in the abundances of iron-binding proteins also suggested the potential importance of alternate photosynthetic pathways as Trichodesmium reallocates the limiting resource under iron stress. Trichodesmium therefore displays a significant and integrated proteomic response to iron availability that likely contributes to the ecological success of this species in the ocean.

Phosphite utilization by the globally important marine diazotroph Trichodesmium.

Species belonging to the filamentous cyanobacterial genus Trichodesmium are responsible for a significant fraction of oceanic nitrogen fixation. The availability of phosphorus (P) likely constrains the growth of Trichodesmium in certain regions of the ocean. Moreover, Trichodesmium species have recently been shown to play a role in an emerging oceanic phosphorus redox cycle, further highlighting the key role these microbes play in many biogeochemical processes in the contemporary ocean. Here, we show that Trichodesmium erythraeum IMS101 can grow on the reduced inorganic compound phosphite as its sole source of P. The components responsible for phosphite utilization are identified through heterologous expression of the T. erythraeum IMS101 Tery_0365-0368 genes, encoding a putative adenosine triphosphate (ATP)-binding cassette transporter and nicotinamide adenine dinucleotide (NAD)-dependent dehydrogenase, in the model cyanobacteria Synechocystis sp. PCC6803. We demonstrate that only combined expression of both the transporter and the dehydrogenase enables Synechocystis to utilize phosphite, confirming the function of Tery_0365-0367 as a phosphite uptake system (PtxABC) and Tery_0368 as a phosphite dehydrogenase (PtxD). Our findings suggest that reported uptake of phosphite by Trichodesmium consortia in the field likely reflects an active biological process by Trichodesmium. These results highlight the diversity of phosphorus sources available to Trichodesmium in a resource-limited ocean.

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent.

Campylobacter jejuni is a human pathogen of worldwide significance. It is commensal in the gut of many birds and mammals, where hydrogen is a readily available electron donor. The bacterium possesses a single membrane-bound, periplasmic-facing NiFe uptake hydrogenase that depends on the acquisition of environmental nickel for activity. The periplasmic binding protein Cj1584 (NikZ) of the ATP binding cassette (ABC) transporter encoded by the cj1584c-cj1580c (nikZYXWV) operon in C. jejuni strain NCTC 11168 was found to be nickel-repressed and to bind free nickel ions with a submicromolar K(d) value, as measured by fluorescence spectroscopy. Unlike the Escherichia coli NikA protein, NikZ did not bind EDTA-chelated nickel and lacks key conserved residues implicated in metallophore interaction. A C. jejuni cj1584c null mutant strain showed an approximately 22-fold decrease in intracellular nickel content compared with the wild-type strain and a decreased rate of uptake of (63)NiCl(2). The inhibition of residual nickel uptake at higher nickel concentrations in this mutant by hexa-ammine cobalt (III) chloride or magnesium ions suggests that low-affinity uptake occurs partly through the CorA magnesium transporter. Hydrogenase activity was completely abolished in the cj1584c mutant after growth in unsupplemented media, but was fully restored after growth with 0.5 mM nickel chloride. Mutation of the putative metallochaperone gene slyD (cj0115) had no effect on either intracellular nickel accumulation or hydrogenase activity. Our data reveal a strict dependence of hydrogenase activity in C. jejuni on high-affinity nickel uptake through an ABC transporter that has distinct properties compared with the E. coli Nik system.

Reduction of fumarate, mesaconate and crotonate by Mfr, a novel oxygen-regulated periplasmic reductase in Campylobacter jejuni.

Methylmenaquinol : fumarate reductase (Mfr) is a newly recognized type of fumarate reductase present in some epsilon-proteobacteria, where the active site subunit (MfrA) is localized in the periplasm, but for which a physiological role has not been identified. We show that the Campylobacter jejuni mfrABE operon is transcribed from a single promoter, with the mfrA gene preceded by a small open reading-frame (mfrX) encoding a C. jejuni-specific polypeptide of unknown function. The growth characteristics and enzyme activities of mutants in the mfrA and menaquinol : fumarate reductase A (frdA) genes show that the cytoplasmic facing Frd enzyme is the major fumarate reductase under oxygen limitation. The Mfr enzyme is shown to be necessary for maximal rates of growth by fumarate respiration and rates of fumarate reduction in intact cells measured by both viologen assays and 1H-NMR were slower in an mfrA mutant. As periplasmic fumarate reduction does not require fumarate/succinate antiport, Mfr may allow more efficient adaptation to fumarate-dependent growth. However, a further rationale for the periplasmic location of Mfr is suggested by the observation that the enzyme also reduces the fumarate analogues mesaconate and crotonate; fermentation products of anaerobes with which C. jejuni shares its gut environment, that are unable to be transported into the cell. Both MfrA and MfrB subunits were localized in the periplasm by immunoblotting and 2D-gel electrophoresis, but an mfrE mutant accumulated unprocessed MfrA in the cytoplasm, suggesting a preassembled MfrABE holoenzyme has to be recognized by the TAT system for translocation to occur. Gene expression studies in chemostat cultures following an aerobic-anaerobic shift showed that mfrA is highly upregulated by oxygen limitation, as would be experienced in vivo. Our results indicate that in addition to a role in fumarate respiration, Mfr allows C. jejuni to reduce analogous substrates specifically present in the host gut environment.

A Multicopper oxidase (Cj1516) and a CopA homologue (Cj1161) are major components of the copper homeostasis system of Campylobacter jejuni.

Metal ion homeostasis mechanisms in the food-borne human pathogen Campylobacter jejuni are poorly understood. The Cj1516 gene product is homologous to the multicopper oxidase CueO, which is known to contribute to copper tolerance in Escherichia coli. Here we show, by optical absorbance and electron paramagnetic resonance spectroscopy, that purified recombinant Cj1516 contains both T1 and trinuclear copper centers, which are characteristic of multicopper oxidases. Inductively coupled plasma mass spectrometry revealed that the protein contained approximately six copper atoms per polypeptide. The presence of an N-terminal "twin arginine" signal sequence suggested a periplasmic location for Cj1516, which was confirmed by the presence of p-phenylenediamine (p-PD) oxidase activity in periplasmic fractions of wild-type but not Cj1516 mutant cells. Kinetic studies showed that the pure protein exhibited p-PD, ferroxidase, and cuprous oxidase activities and was able to oxidize an analogue of the bacterial siderophore anthrachelin (3,4-dihydroxybenzoate), although no iron uptake impairment was observed in a Cj1516 mutant. However, this mutant was very sensitive to increased copper levels in minimal media, suggesting a role in copper tolerance. This was supported by increased expression of the Cj1516 gene in copper-rich media. A mutation in a second gene, the Cj1161c gene, encoding a putative CopA homologue, was also found to result in copper hypersensitivity, and a Cj1516 Cj1161c double mutant was found to be more copper sensitive than either single mutant. These observations and the apparent lack of alternative copper tolerance systems suggest that Cj1516 (CueO) and Cj1161 (CopA) are major proteins involved in copper homeostasis in C. jejuni.