A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component.

Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.

Profiling the interactome of oligonucleotide drugs by proximity biotinylation.

Drug-ID is a novel method applying proximity biotinylation to identify drug-protein interactions inside living cells. The covalent conjugation of a drug with a biotin ligase enables targeted biotinylation and identification of the drug-bound proteome. We established Drug-ID for two small-molecule drugs, JQ1 and SAHA, and applied it for RNaseH-recruiting antisense oligonucleotides (ASOs). Drug-ID profiles the drug-protein interactome de novo under native conditions, directly inside living cells and at pharmacologically effective drug concentrations. It requires minimal amounts of cell material and might even become applicable in vivo. We studied the dose-dependent aggregation of ASOs and the effect of different wing chemistries (locked nucleic acid, 2'-methoxyethyl and 2'-Fluoro) and ASO lengths on the interactome. Finally, we demonstrate the detection of stress-induced, intracellular interactome changes (actinomycin D treatment) with an in situ variant of the approach, which uses a recombinant biotin ligase and does not require genetic manipulation of the target cell.

Integrative proteomics highlight presynaptic alterations and c-Jun misactivation as convergent pathomechanisms in ALS.

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease mainly affecting upper and lower motoneurons. Several functionally heterogeneous genes have been associated with the familial form of this disorder (fALS), depicting an extremely complex pathogenic landscape. This heterogeneity has limited the identification of an effective therapy, and this bleak prognosis will only improve with a greater understanding of convergent disease mechanisms. Recent evidence from human post-mortem material and diverse model systems has highlighted the synapse as a crucial structure actively involved in disease progression, suggesting that synaptic aberrations might represent a shared pathological feature across the ALS spectrum. To test this hypothesis, we performed the first comprehensive analysis of the synaptic proteome from post-mortem spinal cord and human iPSC-derived motoneurons carrying mutations in the major ALS genes. This integrated approach highlighted perturbations in the molecular machinery controlling vesicle release as a shared pathomechanism in ALS. Mechanistically, phosphoproteomic analysis linked the presynaptic vesicular phenotype to an accumulation of cytotoxic protein aggregates and to the pro-apoptotic activation of the transcription factor c-Jun, providing detailed insights into the shared pathobiochemistry in ALS. Notably, sub-chronic treatment of our iPSC-derived motoneurons with the fatty acid docosahexaenoic acid exerted a neuroprotective effect by efficiently rescuing the alterations revealed by our multidisciplinary approach. Together, this study provides strong evidence for the central and convergent role played by the synaptic microenvironment within the ALS spinal cord and highlights a potential therapeutic target that counteracts degeneration in a heterogeneous cohort of human motoneuron cultures.

An Unprecedented Tolerance to Deletion of the Periplasmic Chaperones SurA, Skp, and DegP in the Nosocomial Pathogen Acinetobacter baumannii.

The outer membrane (OM) of Gram-negative bacteria efficiently protects from harmful environmental stresses such as antibiotics, disinfectants, or dryness. The main constituents of the OM are integral OM ß-barrel proteins (OMPs). In Gram-negative bacteria such as Escherichia coli, Yersinia enterocolitica, and Pseudomonas aeruginosa, the insertion of OMPs depends on a sophisticated biogenesis pathway. This comprises the SecYEG translocon, which enables inner membrane (IM) passage; the chaperones SurA, Skp, and DegP, which facilitate the passage of ß-barrel OMPs through the periplasm; and the ß-barrel assembly machinery (BAM), which facilitates insertion into the OM. In E. coli, Y. enterocolitica, and P. aeruginosa, the deletion of SurA is particularly detrimental and leads to a loss of OM integrity, sensitization to antibiotic treatment, and reduced virulence. In search of targets that could be exploited to develop compounds that interfere with OM integrity in Acinetobacter baumannii, we employed the multidrug-resistant strain AB5075 to generate single gene knockout strains lacking individual periplasmic chaperones. In contrast to E. coli, Y. enterocolitica, and P. aeruginosa, AB5075 tolerates the lack of SurA, Skp, or DegP with only weak mutant phenotypes. While the double knockout strains ΔsurAΔskp and ΔsurAΔdegP are conditionally lethal in E. coli, all double deletions were well tolerated by AB5075. Strikingly, even a triple-knockout strain of AB5075, lacking surA, skp, and degP, was viable. IMPORTANCE Acinetobacter baumannii is a major threat to human health due to its ability to persist in the hospital environment, resistance to antibiotic treatment, and ability to deploy multiple and redundant virulence factors. In a rising number of cases, infections with multidrug-resistant A. baumannii end up fatally, because all antibiotic treatment options fail. Thus, novel targets have to be identified and alternative therapeutics have to be developed. The knockout of periplasmic chaperones has previously proven to significantly reduce virulence and even break antibiotic resistance in other Gram-negative pathogens. Our study in A. baumannii demonstrates how variable the importance of the periplasmic chaperones SurA, Skp, and DegP can be and suggests the existence of mechanisms allowing A. baumannii to cope with the lack of the three periplasmic chaperones.

Nemertean, Brachiopod, and Phoronid Neuropeptidomics Reveals Ancestral Spiralian Signaling Systems.

Neuropeptides are diverse signaling molecules in animals commonly acting through G-protein coupled receptors (GPCRs). Neuropeptides and their receptors underwent extensive diversification in bilaterians and the relationships of many peptide-receptor systems have been clarified. However, we lack a detailed picture of neuropeptide evolution in lophotrochozoans as in-depth studies only exist for mollusks and annelids. Here, we analyze peptidergic systems in Nemertea, Brachiopoda, and Phoronida. We screened transcriptomes from 13 nemertean, 6 brachiopod, and 4 phoronid species for proneuropeptides and neuropeptide GPCRs. With mass spectrometry from the nemertean Lineus longissimus, we validated several predicted peptides and identified novel ones. Molecular phylogeny combined with peptide-sequence and gene-structure comparisons allowed us to comprehensively map spiralian neuropeptide evolution. We found most mollusk and annelid peptidergic systems also in nemerteans, brachiopods, and phoronids. We uncovered previously hidden relationships including the orthologies of spiralian CCWamides to arthropod agatoxin-like peptides and of mollusk APGWamides to RGWamides from annelids, with ortholog systems in nemerteans, brachiopods, and phoronids. We found that pleurin neuropeptides previously only found in mollusks are also present in nemerteans and brachiopods. We also identified cases of gene family duplications and losses. These include a protostome-specific expansion of RFamide/Wamide signaling, a spiralian expansion of GnRH-related peptides, and duplications of vasopressin/oxytocin before the divergence of brachiopods, phoronids, and nemerteans. This analysis expands our knowledge of peptidergic signaling in spiralians and other protostomes. Our annotated data set of nearly 1,300 proneuropeptide sequences and 600 GPCRs presents a useful resource for further studies of neuropeptide signaling.

ß-Actin mRNA interactome mapping by proximity biotinylation.

The molecular function and fate of mRNAs are controlled by RNA-binding proteins (RBPs). Identification of the interacting proteome of a specific mRNA in vivo remains very challenging, however. Based on the widely used technique of RNA tagging with MS2 aptamers for RNA visualization, we developed a RNA proximity biotinylation (RNA-BioID) technique by tethering biotin ligase (BirA*) via MS2 coat protein at the 3' UTR of endogenous MS2-tagged ß-actin mRNA in mouse embryonic fibroblasts. We demonstrate the dynamics of the ß-actin mRNA interactome by characterizing its changes on serum-induced localization of the mRNA. Apart from the previously known interactors, we identified more than 60 additional ß-actin-associated RBPs by RNA-BioID. Among these, the KH domain-containing protein FUBP3/MARTA2 has been shown to be required for ß-actin mRNA localization. We found that FUBP3 binds to the 3' UTR of ß-actin mRNA and is essential for ß-actin mRNA localization, but does not interact with the characterized ß-actin zipcode element. RNA-BioID provides a tool for identifying new mRNA interactors and studying the dynamic view of the interacting proteome of endogenous mRNAs in space and time.

Import of extracellular ATP in yeast and man modulates AMPK and TORC1 signalling.

AMP-activated kinase (AMPK) and target of rapamycin (TOR) signalling coordinate cell growth, proliferation, metabolism and cell survival with the nutrient environment of cells. The poor vasculature and nutritional stress experienced by cells in solid tumours raises the question: how do they assimilate sufficient nutrients to survive? Here, we show that human and fission yeast cells import ATP and AMP from their external environment to regulate AMPK and TOR signalling. Exposure of fission yeast (Schizosaccharomyces pombe) and human cells to external AMP impeded cell growth; however, in yeast this restraining impact required AMPK. In contrast, external ATP rescued the growth defect of yeast mutants with reduced TORC1 signalling; furthermore, exogenous ATP transiently enhanced TORC1 signalling in both yeast and human cell lines. Addition of the PANX1 channel inhibitor probenecid blocked ATP import into human cell lines suggesting that this channel may be responsible for both ATP release and uptake in mammals. In light of these findings, it is possible that the higher extracellular ATP concentration reported in solid tumours is both scavenged and recognized as an additional energy source beneficial for cell growth.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry.

Ubiquitination controls a plethora of cellular processes. Modifications by linear polyubiquitin have so far been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (OTULIN) and a very few linear polyubiquitinated substrates have been identified. Current methods for studying lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present an approach to discovering linear polyubiquitin-modified substrates by combining a lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-REx HEK293T cells and validated several newly identified linear polyubiquitin targets. We demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for NFκB signaling.

A flagellum-specific chaperone facilitates assembly of the core type III export apparatus of the bacterial flagellum.

Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP-FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

MapZ marks the division sites and positions FtsZ rings in Streptococcus pneumoniae.

In every living organism, cell division requires accurate identification of the division site and placement of the division machinery. In bacteria, this process is traditionally considered to begin with the polymerization of the highly conserved tubulin-like protein FtsZ into a ring that locates precisely at mid-cell. Over the past decades, several systems have been reported to regulate the spatiotemporal assembly and placement of the FtsZ ring. However, the human pathogen Streptococcus pneumoniae, in common with many other organisms, is devoid of these canonical systems and the mechanisms of positioning the division machinery remain unknown. Here we characterize a novel factor that locates at the division site before FtsZ and guides septum positioning in pneumococcus. Mid-cell-anchored protein Z (MapZ) forms ring structures at the cell equator and moves apart as the cell elongates, therefore behaving as a permanent beacon of division sites. MapZ then positions the FtsZ ring through direct protein-protein interactions. MapZ-mediated control differs from previously described systems mostly on the basis of negative regulation of FtsZ assembly. Furthermore, MapZ is an endogenous target of the Ser/Thr kinase StkP, which was recently shown to have a central role in cytokinesis and morphogenesis of S. pneumoniae. We show that both phosphorylated and non-phosphorylated forms of MapZ are required for proper Z-ring formation and dynamics. Altogether, this work uncovers a new mechanism for bacterial cell division that is regulated by phosphorylation and illustrates that nature has evolved a diversity of cell division mechanisms adapted to the different bacterial clades.

Extracellular Matrix-Specific Platelet Activation Leads to a Differential Translational Response and Protein De Novo Synthesis in Human Platelets.

Platelets are exposed to extracellular matrix (ECM) proteins like collagen and laminin and to fibrinogen during acute vascular events. However, beyond hemostasis, platelets have the important capacity to migrate on ECM surfaces, but the translational response of platelets to different extracellular matrix stimuli is still not fully characterized. Using 2D-gel electrophoresis, confocal microscopy, polysome analysis and protein sequencing by mass spectrometry, we demonstrate that platelets show a differential expression profile of newly synthesized proteins on laminin, collagen or fibrinogen. In this context, we observed a characteristic, ECM-dependent translocation phenotype of translation initiation factor eIF4E to the ribosomal site. eIF4E accumulated in polysomes with increased binding of mRNA and co-localization with vinculin, leading to de novo synthesis of important cytoskeletal regulator proteins. As the first study, we included a proteome analysis of laminin-adherent platelets and interestingly identified upregulation of essentially important proteins that mediate cytoskeletal regulation and mobility in platelets, such as filamin A, talin, vinculin, gelsolin, coronin or kindlin-3. In summary, we demonstrate that platelet activation with extracellular matrix proteins results in a distinct stimulus-specific translational response of platelets that will help to improve our understanding of the regulation of platelet mobility and migration.

TORC1 and TORC2 converge to regulate the SAGA co-activator in response to nutrient availability.

Gene expression regulation is essential for cells to adapt to changes in their environment. Co-activator complexes have well-established roles in transcriptional regulation, but less is known about how they sense and respond to signaling cues. We have previously shown that, in fission yeast, one such co-activator, the SAGA complex, controls gene expression and the switch from proliferation to differentiation in response to nutrient availability. Here, using a combination of genetic, biochemical, and proteomic approaches, we show that SAGA responds to nutrients through the differential phosphorylation of its Taf12 component, downstream of both the TORC1 and TORC2 pathways. Taf12 phosphorylation increases early upon starvation and is controlled by the opposing activities of the PP2A phosphatase, which is activated by TORC1, and the TORC2-activated Gad8AKT kinase. Mutational analyses suggest that Taf12 phosphorylation prevents cells from committing to differentiation until starvation reaches a critical level. Overall, our work reveals that SAGA is a direct target of nutrient-sensing pathways and has uncovered a mechanism by which TORC1 and TORC2 converge to control gene expression and cell fate decisions.

The Integrin Activating Protein Kindlin-3 Is Cleaved in Human Platelets during ST-Elevation Myocardial Infarction.

Kindlins are important proteins for integrin signaling and regulation of the cytoskeleton, but we know little about their precise function and regulation in platelets during acute ischemic events. In this work, we investigated kindlin-3 protein levels in platelets isolated from patients with ST-elevation myocardial infarction (STEMI) compared to patients with non-ischemic chest pain. Platelets from twelve patients with STEMI and twelve patients with non-ischemic chest pain were isolated and analyzed for kindlin-3 protein levels and intracellular localization by immunoblotting and two-dimensional gel electrophoresis. Platelet proteome analysis by two-dimensional gel electrophoresis and protein sequencing identified kindlin-3 as a protein that is cleaved in platelets from patients with myocardial infarction. Kindlin-3 full-length protein was significantly decreased in patients with STEMI compared to patients with non-ischemic chest pain (1.0 ± 0.2 versus 0.28 ± 0.2, p < 0.05) by immunoblotting. Kindlin-3 showed a differential distribution and was primarily cleaved in the cytosolic and membrane compartment of platelets in myocardial infarction. Platelet activation with thrombin alone did not affect kindlin-3 protein levels. The present study demonstrates that kindlin-3 protein levels become significantly reduced in platelets of patients with myocardial infarction compared to controls. The results suggest that kindlin-3 cleavage in platelets is associated with the ischemic event of myocardial infarction.

Heat-Shock Protein 27 (HSPB1) Is Upregulated and Phosphorylated in Human Platelets during ST-Elevation Myocardial Infarction.

Heat-shock proteins are a family of proteins which are upregulated in response to stress stimuli including inflammation, oxidative stress, or ischemia. Protective functions of heat-shock proteins have been studied in vascular disease models, and malfunction of heat-shock proteins is associated with vascular disease development. Heat-shock proteins however have not been investigated in human platelets during acute myocardial infarction ex vivo. Using two-dimensional electrophoresis and immunoblotting, we observed that heat-shock protein 27 (HSPB1) levels and phosphorylation are significantly increased in platelets of twelve patients with myocardial infarction compared to patients with nonischemic chest pain (6.4 ± 1.0-fold versus 1.0 ± 0.9-fold and 5.9 ± 1.8-fold versus 1.0 ± 0.8-fold; p < 0.05). HSP27 (HSPB1) showed a distinct and characteristic intracellular translocation from the cytoskeletal fraction into the membrane fraction of platelets during acute myocardial infarction that did not occur in the control group. In this study, we could demonstrate for the first time that HSP27 (HSPB1) is upregulated and phosphorylated in human platelets during myocardial infarction on a cellular level ex vivo with a characteristic intracellular translocation pattern. This HSP27 (HSPB1) phenotype in platelets could thus represent a measurable stress response in myocardial infarction and potentially other acute ischemic events.

The polarity protein Scribble positions DLC3 at adherens junctions to regulate Rho signaling.

The spatial regulation of cellular Rho signaling by GAP proteins is still poorly understood. By performing mass spectrometry, we here identify the polarity protein Scribble as a scaffold for the RhoGAP protein DLC3 (also known as StarD8) at cell-cell adhesions. This mutually dependent interaction is mediated by the PDZ domains of Scribble and a PDZ ligand (PDZL) motif in DLC3. Both Scribble depletion and PDZL deletion abrogated DLC3 junctional localization. Using a RhoA biosensor and a targeted GAP domain, we demonstrate that DLC3 activity locally regulates RhoA-ROCK signaling at and Scribble localization to adherens junctions, and is required for their functional integrity. In a 3D model of cyst development, we furthermore show that DLC3 depletion impairs polarized morphogenesis, phenocopying the effects observed upon Scribble knockdown. We thus propose a new function for Scribble in Rho regulation that entails positioning of DLC3 GAP activity at cell junctions in polarized epithelial cells.

TOR complex 2 localises to the cytokinetic actomyosin ring and controls the fidelity of cytokinesis.

The timing of cell division is controlled by the coupled regulation of growth and division. The target of rapamycin (TOR) signalling network synchronises these processes with the environmental setting. Here, we describe a novel interaction of the fission yeast TOR complex 2 (TORC2) with the cytokinetic actomyosin ring (CAR), and a novel role for TORC2 in regulating the timing and fidelity of cytokinesis. Disruption of TORC2 or its localisation results in defects in CAR morphology and constriction. We provide evidence that the myosin II protein Myp2 and the myosin V protein Myo51 play roles in recruiting TORC2 to the CAR. We show that Myp2 and TORC2 are co-dependent upon each other for their normal localisation to the cytokinetic machinery. We go on to show that TORC2-dependent phosphorylation of actin-capping protein 1 (Acp1, a known regulator of cytokinesis) controls CAR stability, modulates Acp1-Acp2 (the equivalent of the mammalian CAPZA-CAPZB) heterodimer formation and is essential for survival upon stress. Thus, TORC2 localisation to the CAR, and TORC2-dependent Acp1 phosphorylation contributes to timely control and the fidelity of cytokinesis and cell division.

Structural and Functional Characterization of the Bacterial Type III Secretion Export Apparatus.

Bacterial type III protein secretion systems inject effector proteins into eukaryotic host cells in order to promote survival and colonization of Gram-negative pathogens and symbionts. Secretion across the bacterial cell envelope and injection into host cells is facilitated by a so-called injectisome. Its small hydrophobic export apparatus components SpaP and SpaR were shown to nucleate assembly of the needle complex and to form the central "cup" substructure of a Salmonella Typhimurium secretion system. However, the in vivo placement of these components in the needle complex and their function during the secretion process remained poorly defined. Here we present evidence that a SpaP pentamer forms a 15 Å wide pore and provide a detailed map of SpaP interactions with the export apparatus components SpaQ, SpaR, and SpaS. We further refine the current view of export apparatus assembly, consolidate transmembrane topology models for SpaP and SpaR, and present intimate interactions of the periplasmic domains of SpaP and SpaR with the inner rod protein PrgJ, indicating how export apparatus and needle filament are connected to create a continuous conduit for substrate translocation.

Proteome Response of a Metabolically Flexible Anoxygenic Phototroph to Fe(II) Oxidation.

The oxidation of Fe(II) by anoxygenic photosynthetic bacteria was likely a key contributor to Earth's biosphere prior to the evolution of oxygenic photosynthesis and is still found in a diverse range of modern environments. All known phototrophic Fe(II) oxidizers can utilize a wide range of substrates, thus making them very metabolically flexible. However, the underlying adaptations required to oxidize Fe(II), a potential stressor, are not completely understood. We used a combination of quantitative proteomics and cryogenic transmission electron microscopy (cryo-TEM) to compare cells of Rhodopseudomonas palustris TIE-1 grown photoautotrophically with Fe(II) or H2 and photoheterotrophically with acetate. We observed unique proteome profiles for each condition, with differences primarily driven by carbon source. However, these differences were not related to carbon fixation but to growth and light harvesting processes, such as pigment synthesis. Cryo-TEM showed stunted development of photosynthetic membranes in photoautotrophic cultures. Growth on Fe(II) was characterized by a response typical of iron homeostasis, which included an increased abundance of proteins required for metal efflux (particularly copper) and decreased abundance of iron import proteins, including siderophore receptors, with no evidence of further stressors, such as oxidative damage. This study suggests that the main challenge facing anoxygenic phototrophic Fe(II) oxidizers comes from growth limitations imposed by autotrophy, and, once this challenge is overcome, iron stress can be mitigated using iron management mechanisms common to diverse bacteria (e.g., by control of iron influx and efflux).IMPORTANCE The cycling of iron between redox states leads to the precipitation and dissolution of minerals, which can in turn impact other major biogeochemical cycles, such as those of carbon, nitrogen, phosphorus and sulfur. Anoxygenic phototrophs are one of the few drivers of Fe(II) oxidation in anoxic environments and are thought to contribute significantly to iron cycling in both modern and ancient environments. These organisms thrive at high Fe(II) concentrations, yet the adaptations required to tolerate the stresses associated with this are unclear. Despite the general consensus that high Fe(II) concentrations pose numerous stresses on these organisms, our study of the large-scale proteome response of a model anoxygenic phototroph to Fe(II) oxidation demonstrates that common iron homeostasis strategies are adequate to manage this. The bulk of the proteome response is not driven by adaptations to Fe(II) stress but to adaptations required to utilize an inorganic carbon source. Such a global overview of the adaptation of these organisms to Fe(II) oxidation provides valuable insights into the physiology of these biogeochemically important organisms and suggests that Fe(II) oxidation may not pose as many challenges to anoxygenic phototrophs as previously thought.