The CLP and PREP protease systems coordinate maturation and degradation of the chloroplast proteome in Arabidopsis thaliana.

A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone-protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix. Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N-terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)). Strong synergistic interactions were observed between the CLP protease system (clpr1-2, clpr2-1, clpc1-1, clpt1, clpt2) and both PREP homologs (prep1, prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N-terminal proteomes showed that many nuclear encoded chloroplast proteins have alternatively processed N-termini in prep1prep2, clpt1clpt2 and prep1prep2clpt1clpt2. Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.

Autocatalytic Processing and Substrate Specificity of Arabidopsis Chloroplast Glutamyl Peptidase.

Chloroplast proteostasis is governed by a network of peptidases. As a part of this network, we show that Arabidopsis (Arabidopsis thaliana) chloroplast glutamyl peptidase (CGEP) is a homo-oligomeric stromal Ser-type (S9D) peptidase with both exo- and endo-peptidase activity. Arabidopsis CGEP null mutant alleles (cgep) had no visible phenotype but showed strong genetic interactions with stromal CLP protease system mutants, resulting in reduced growth. Loss of CGEP upregulated the chloroplast protein chaperone machinery and 70S ribosomal proteins, but other parts of the proteostasis network were unaffected. Both comparative proteomics and mRNA-based coexpression analyses strongly suggested that the function of CGEP is at least partly involved in starch metabolism regulation. Recombinant CGEP degraded peptides and proteins smaller than ∼25 kD. CGEP specifically cleaved substrates on the C-terminal side of Glu irrespective of neighboring residues, as shown using peptide libraries incubated with recombinant CGEP and mass spectrometry. CGEP was shown to undergo autocatalytic C-terminal cleavage at E946, removing 15 residues, both in vitro and in vivo. A conserved motif (A[S/T]GGG[N/G]PE946) immediately upstream of E946 was identified in dicotyledons, but not monocotyledons. Structural modeling suggested that C-terminal processing increases the upper substrate size limit by improving catalytic cavity access. In vivo complementation with catalytically inactive CGEP-S781R or a CGEP variant with an unprocessed C-terminus in a cgep clpr2-1 background was used to demonstrate the physiological importance of both CGEP peptidase activity and its autocatalytic processing. CGEP homologs of photosynthetic and nonphotosynthetic bacteria lack the C-terminal prosequence, suggesting it is a recent functional adaptation in plants.

The Plastoglobule-Localized Metallopeptidase PGM48 Is a Positive Regulator of Senescence in Arabidopsis thaliana.

Plastoglobuli (PG) are thylakoid-associated monolayer lipid particles with a specific proteome of ∼30 PG core proteins and isoprenoid and neutral lipids. During senescence, PGs increase in size, reflecting their role in dismantling thylakoid membranes. Here, we show that the only PG-localized peptidase PGM48 positively regulates leaf senescence. We discovered that PGM48 is a member of the M48 peptidase family with PGM48 homologs, forming a clade (M48D) only found in photosynthetic organisms. Unlike the M48A, B, and C clades, members of M48D have no transmembrane domains, consistent with their unique subcellular location in the PG. In vitro assays showed Zn-dependent proteolytic activity and substrate cleavage upstream of hydrophobic residues. Overexpression of PGM48 accelerated natural leaf senescence, whereas suppression delayed senescence. Quantitative proteomics of PG from senescing rosettes of PGM48 overexpression lines showed a dramatically reduced level of CAROTENOID CLEAVAGE ENZYME4 (CCD4) and significantly increased levels of the senescence-induced ABC1 KINASE7 (ABC1K7) and PHYTYL ESTER SYNTHASE1 (PES1). Yeast two-hybrid experiments identified PG core proteins ABC1K3, PES1, and CCD4 as PGM48 interactors, whereas several other PG-localized proteins and chlorophyll degradation enzymes did not interact. We discuss mechanisms through which PGM48 could possibly accelerate the senescence process.

The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

Loss of plastoglobule kinases ABC1K1 and ABC1K3 causes conditional degreening, modified prenyl-lipids, and recruitment of the jasmonic acid pathway.

Plastoglobules (PGs) are plastid lipid-protein particles. This study examines the function of PG-localized kinases ABC1K1 and ABC1K3 in Arabidopsis thaliana. Several lines of evidence suggested that ABC1K1 and ABC1K3 form a protein complex. Null mutants for both genes (abc1k1 and abc1k3) and the double mutant (k1 k3) displayed rapid chlorosis upon high light stress. Also, k1 k3 showed a slower, but irreversible, senescence-like phenotype during moderate light stress that was phenocopied by drought and nitrogen limitation, but not cold stress. This senescence-like phenotype involved degradation of the photosystem II core and upregulation of chlorophyll degradation. The senescence-like phenotype was independent of the EXECUTER pathway that mediates genetically controlled cell death from the chloroplast and correlated with increased levels of the singlet oxygen-derived carotenoid ß-cyclocitral, a retrograde plastid signal. Total PG volume increased during light stress in wild type and k1 k3 plants, but with different size distributions. Isolated PGs from k1 k3 showed a modified prenyl-lipid composition, suggesting reduced activity of PG-localized tocopherol cyclase (VTE1), and was consistent with loss of carotenoid cleavage dioxygenase 4. Plastid jasmonate biosynthesis enzymes were recruited to the k1 k3 PGs but not wild-type PGs, while pheophytinase, which is involved in chlorophyll degradation, was induced in k1 k3 and not wild-type plants and was localized to PGs. Thus, the ABC1K1/3 complex contributes to PG function in prenyl-lipid metabolism, stress response, and thylakoid remodeling.

Salicylic Acid Inhibits the Replication of Tomato bushy stunt virus by Directly Targeting a Host Component in the Replication Complex.

Although the plant hormone salicylic acid (SA) plays a central role in signaling resistance to viral infection, the underlying mechanisms are only partially understood. Identification and characterization of SA's direct targets have been shown to be an effective strategy for dissecting the complex SA-mediated defense signaling network. In search of additional SA targets, we previously developed two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SA-binding proteins (SABPs) from Arabidopsis. Using these approaches, we have now identified several members of the Arabidopsis glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein family, including two chloroplast-localized and two cytosolic isoforms, as SABPs. Cytosolic GAPDH is a well-known glycolytic enzyme; it also is an important host factor involved in the replication of Tomato bushy stunt virus (TBSV), a single-stranded RNA virus. Using a yeast cell-free extract, an in vivo yeast replication system, and plant protoplasts, we demonstrate that SA inhibits TBSV replication. SA does so by inhibiting the binding of cytosolic GAPDH to the negative (-)RNA strand of TBSV. Thus, this study reveals a novel molecular mechanism through which SA regulates virus replication.

New chemical and microbial perspectives on vitamin B1 and vitamer dynamics of a coastal system.

Vitamin B1 (thiamin, B1) is an essential micronutrient for cells, yet intriguingly in aquatic systems most bacterioplankton are unable to synthesize it de novo (auxotrophy), requiring an exogenous source. Cycling of this valuable metabolite in aquatic systems has not been fully investigated and vitamers (B1-related compounds) have only begun to be measured and incorporated into the B1 cycle. Here, we identify potential key producers and consumers of B1 and gain new insights into the dynamics of B1 cycling through measurements of B1 and vitamers (HMP: 4-amino-5-hydroxymethyl-2-methylpyrimidine, HET: 4-methyl-5-thiazoleethanol, FAMP: N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine) in the particulate and dissolved pool in a temperate coastal system. Dissolved B1 was not the primary limiting nutrient for bacterial production and was relatively stable across seasons with concentrations ranging from 74-117 pM, indicating a balance of supply and demand. However, vitamer concentration changed markedly with season as did transcripts related to vitamer salvage and transport suggesting use of vitamers by certain bacterioplankton, e.g. Pelagibacterales. Genomic and transcriptomic analyses showed that up to 78% of the bacterioplankton taxa were B1 auxotrophs. Notably, de novo B1 production was restricted to a few abundant bacterioplankton (e.g. Vulcanococcus, BACL14 (Burkholderiales), Verrucomicrobiales) across seasons. In summer, abundant picocyanobacteria were important putative B1 sources, based on transcriptional activity, leading to an increase in the B1 pool. Our results provide a new dynamic view of the players and processes involved in B1 cycling over time in coastal waters, and identify specific priority populations and processes for future study.

Nitrogen fixation in the widely distributed marine γ-proteobacterial diazotroph Candidatus Thalassolituus haligoni.

The high diversity and global distribution of heterotrophic bacterial diazotrophs (HBDs) in the ocean has recently become apparent. However, understanding the role these largely uncultured microorganisms play in marine N2 fixation poses a challenge due to their undefined growth requirements and the complex regulation of the nitrogenase enzyme. We isolated and characterized Candidatus Thalassolituus haligoni, a member of a widely distributed clade of HBD belonging to the Oceanospirillales. Analysis of its nifH gene via amplicon sequencing revealed the extensive distribution of Cand. T. haligoni across the Pacific, Atlantic, and Arctic Oceans. Pangenome analysis indicates that the isolate shares >99% identity with an uncultured metagenome-assembled genome called Arc-Gamma-03, recently recovered from the Arctic Ocean. Through combined genomic, proteomic, and physiological approaches, we confirmed that the isolate fixes N2 gas. However, the mechanisms governing nitrogenase regulation in Cand. T. haligoni remain unclear. We propose Cand. T. haligoni as a globally distributed, cultured HBD model species within this understudied clade of Oceanospirillales.

Use and detection of a vitamin B1 degradation product yields new views of the marine B1 cycle and plankton metabolite exchange.

Vitamin B1 (thiamin) is a vital nutrient for most cells in nature, including marine plankton. Early and recent experiments show that B1 degradation products instead of B1 can support the growth of marine bacterioplankton and phytoplankton. However, the use and occurrence of some degradation products remains uninvestigated, namely N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (FAMP), which has been a focus of plant oxidative stress research. We investigated the relevance of FAMP in the ocean. Experiments and global ocean meta-omic data indicate that eukaryotic phytoplankton, including picoeukaryotes and harmful algal bloom species, use FAMP while bacterioplankton appear more likely to use deformylated FAMP, 4-amino-5-aminomethyl-2-methylpyrimidine. Measurements of FAMP in seawater and biomass revealed that it occurs at picomolar concentrations in the surface ocean, heterotrophic bacterial cultures produce FAMP in the dark-indicating non-photodegradation of B1 by cells, and B1-requiring (auxotrophic) picoeukaryotic phytoplankton produce intracellular FAMP. Our results require an expansion of thinking about vitamin degradation in the sea, but also the marine B1 cycle where it is now crucial to consider a new B1-related compound pool (FAMP), as well as generation (dark degradation-likely via oxidation), turnover (plankton uptake), and exchange of the compound within the networks of plankton. IMPORTANCE Results of this collaborative study newly show that a vitamin B1 degradation product, N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (FAMP), can be used by diverse marine microbes (bacteria and phytoplankton) to meet their vitamin B1 demands instead of B1 and that FAMP occurs in the surface ocean. FAMP has not yet been accounted for in the ocean and its use likely enables cells to avoid B1 growth deficiency. Additionally, we show FAMP is formed in and out of cells without solar irradiance-a commonly considered route of vitamin degradation in the sea and nature. Altogether, the results expand thinking about oceanic vitamin degradation, but also the marine B1 cycle where it is now crucial to consider a new B1-related compound pool (FAMP), as well as its generation (dark degradation-likely via oxidation), turnover (plankton uptake), and exchange within networks of plankton.

Short-term acidification promotes diverse iron acquisition and conservation mechanisms in upwelling-associated phytoplankton.

Coastal upwelling regions are among the most productive marine ecosystems but may be threatened by amplified ocean acidification. Increased acidification is hypothesized to reduce iron bioavailability for phytoplankton thereby expanding iron limitation and impacting primary production. Here we show from community to molecular levels that phytoplankton in an upwelling region respond to short-term acidification exposure with iron uptake pathways and strategies that reduce cellular iron demand. A combined physiological and multi-omics approach was applied to trace metal clean incubations that introduced 1200 ppm CO2 for up to four days. Although variable, molecular-level responses indicate a prioritization of iron uptake pathways that are less hindered by acidification and reductions in iron utilization. Growth, nutrient uptake, and community compositions remained largely unaffected suggesting that these mechanisms may confer short-term resistance to acidification; however, we speculate that cellular iron demand is only temporarily satisfied, and longer-term acidification exposure without increased iron inputs may result in increased iron stress.

Intein-mediated cyclization of bacterial acyl carrier protein stabilizes its folded conformation but does not abolish function.

Bacterial acyl carrier protein (ACP) is essential for the synthesis of fatty acids and serves as the major acyl donor for the formation of phospholipids and other lipid products. Acyl-ACP encloses attached fatty acyl groups in a hydrophobic pocket within a four-helix bundle, but must at least partially unfold to present the acyl chain to the active sites of its multiple enzyme partners. To further examine the constraints of ACP structure and function, we have constructed a cyclic version of Vibrio harveyi ACP, using split-intein technology to covalently join its closely apposed N and C termini. Cyclization stabilized ACP in a folded helical conformation as indicated by gel electrophoresis, circular dichroism, fluorescence, and mass spectrometry. Molecular dynamics simulations also indicated overall decreased polypeptide chain mobility in cyclic ACP, although no major conformational rearrangements over a 10-ns period were noted. In vivo complementation assays revealed that cyclic ACP can functionally replace the linear wild-type protein and support growth of an Escherichia coli ACP-null mutant strain. Cyclization of a folding-deficient ACP mutant (F50A) both restored its ability to adopt a folded conformation and enhanced complementation of growth. Our results thus suggest that ACP must be able to adopt a folded conformation for biological activity, and that its function does not require complete unfolding of the protein.

Immune response and alveolar bone resorption in a mouse model of Treponema denticola infection.

Treponema denticola is considered to be an agent strongly associated with periodontal disease. The lack of an animal infection model has hampered the understanding of T. denticola pathogenesis and the host's immune response to infection. In this study, we have established an oral infection model in mice, demonstrating that infection by oral inoculation is feasible. The presence of T. denticola in the oral cavities of the animals was confirmed by PCR. Mice given T. denticola developed a specific immune response to the bacterium. The antibodies generated from the infection were mainly of the immunoglobulin G1 subclass, indicating a Th2-tilted response. The antibodies recognized 11 T. denticola proteins, of which a 62-kDa and a 53-kDa protein were deemed immunodominant. The two proteins were identified, respectively, as dentilisin and the major outer sheath protein by mass spectrometry. Splenocytes cultured from the infected mice no longer produced interleukin-10 and produced markedly reduced levels of gamma interferon relative to those produced by naïve splenocytes upon stimulation with T. denticola. Mandibles of infected mice showed significantly greater bone resorption (P < 0.01) than those of mock-infected controls.

Manganese and iron deficiency in Southern Ocean Phaeocystis antarctica populations revealed through taxon-specific protein indicators.

Iron and light are recognized as limiting factors controlling Southern Ocean phytoplankton growth. Recent field-based evidence suggests, however, that manganese availability may also play a role. Here we examine the influence of iron and manganese on protein expression and physiology in Phaeocystis antarctica, a key Antarctic primary producer. We provide taxon-specific proteomic evidence to show that in-situ Southern Ocean Phaeocystis populations regularly experience stress due to combined low manganese and iron availability. In culture, combined low iron and manganese induce large-scale changes in the Phaeocystis proteome and result in reorganization of the photosynthetic apparatus. Natural Phaeocystis populations produce protein signatures indicating late-season manganese and iron stress, consistent with concurrently observed stimulation of chlorophyll production upon additions of manganese or iron. These results implicate manganese as an important driver of Southern Ocean productivity and demonstrate the utility of peptide mass spectrometry for identifying drivers of incomplete macronutrient consumption.

Author Correction: Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis.

Almost all cells require thiamin, vitamin B1 (B1), which is synthesized via the coupling of thiazole and pyrimidine precursors. Here we demonstrate that 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cHET) is a useful in vivo B1 precursor for representatives of ubiquitous marine picoeukaryotic phytoplankton and Escherichia coli - drawing attention to cHET as a valuable exogenous micronutrient for microorganisms with ecological, industrial, and biomedical value. Comparative utilization experiments with the terrestrial plant Arabidopsis thaliana revealed that it can also use exogenous cHET, but notably, picoeukaryotic marine phytoplankton and E. coli were adapted to grow on low (picomolar) concentrations of exogenous cHET. Our results call for the modification of the conventional B1 biosynthesis model to incorporate cHET as a key precursor for B1 biosynthesis in two domains of life, and for consideration of cHET as a microbial micronutrient currency modulating marine primary productivity and community interactions in human gut-hosted microbiomes.

Electrospray ionization mass spectra of acyl carrier protein are insensitive to its solution phase conformation.

Electrospray ionization mass spectrometry (ESI-MS) can be used to monitor conformational changes of proteins in solution based on the charge state distribution (CSD) of the corresponding gas-phase ions, although relatively few studies of acidic proteins have been reported. Here, we have compared the CSD and solution structure of recombinant Vibrio harveyi acyl carrier protein (rACP), a small acidic protein whose secondary and tertiary structure can be manipulated by pH, fatty acylation, and site-directed mutagenesis. Circular dichroism and intrinsic fluorescence demonstrated that apo-rACP adopts a folded helical conformation in aqueous solution below pH 6 or in 50% acetonitrile/0.1% formic acid, but is unfolded at neutral and basic pH values. A rACP mutant, in which seven conserved acidic residues were replaced with their corresponding neutral amides, was folded over the entire pH range of 5 to 9. However, under the same solvent conditions, both wild type and mutant ACPs exhibited similar CSDs (6(+)-9(+) species) at all pH values. Covalent attachment of myristic acid to the phosphopantetheine prosthetic group of rACP, which is known to stabilize a folded conformation in solution, also had little influence on its CSD in either positive or negative ion modes. Overall, our results are consistent with ACP as a "natively unfolded" protein in a dynamic conformational equilibrium, which allows access to (de)protonation events during the electrospray process.

Qualitative and quantitative evaluation of protein extraction protocols for apple and strawberry fruit suitable for two-dimensional electrophoresis and mass spectrometry analysis.

A modified phenol-based protocol and a phenol-free protocol that involves hot SDS extraction followed by TCA precipitation in acetone were qualitatively and quantitatively compared and evaluated on apple peel and strawberry fruit. The phenol protocol resulted in significantly higher protein yields of 2.35 +/- 0.1 and 0.46 +/- 0.06 mg/g of FW from apple and strawberry fruit, respectively, compared to the SDS protocol, which produced 0.74 +/- 0.1 and 0.27 +/- 0.02 mg/g of FW, respectively. 2-DE analysis of apple protein extracts revealed 1422 protein spots associated with the phenol protocol and 849 spots associated with the SDS protocol. Of these, 761 were present only in phenol gels, whereas 23 were exclusive to SDS samples. For strawberry, SDS extraction produced poor-quality spots with a high degree of streaking, indicating possible contamination. The application of a cleanup procedure resulted in a purified protein extract with high-quality spots. 2-DE analysis of strawberry protein extracts revealed 1368 spots for the phenol protocol and 956 spots for the SDS protocol accompanied by the cleanup procedure. Of these, 599 spots were present only in phenol gels, whereas 109 were present only in SDS samples. Spots from each fruit tissue and extraction procedure were selected, and a total of 26 were identified by LC-MS/MS. Overall, this study demonstrates the complexity of protein extraction of fruit tissues and suggests that a phenol-based protein extraction protocol should be used as a standard procedure for recalcitrant fruit tissues, whereas a SDS protocol with or without a cleanup procedure may be used as an alternative protocol.