Phage Display Selection of Cyclic Peptides That Inhibit Andes Virus Infection

Specific therapy is not available for hantavirus cardiopulmonary syndrome caused by Andes virus (ANDV). Peptides capable of blocking ANDV infection in vitro were identified using antibodies against ANDV surface glycoproteins Gn and Gc to competitively elute a cyclic nonapeptide-bearing phage display library from purified ANDV particles. Phage was examined for ANDV infection inhibition in vitro, and nonapeptides were synthesized based on the most-potent phage sequences. Three peptides showed levels of viral inhibition which were significantly increased by combination treatment with anti-Gn- and anti-Gc-targeting peptides. These peptides will be valuable tools for further development of both peptide and nonpeptide therapeutic agents.Andes virus (ANDV), an NIAID category A agent linked to hantavirus cardiopulmonary syndrome (HCPS), belongs to the family Bunyaviridae and the genus Hantavirus and is carried by Oligoryzomys longicaudatus rodents (11). HCPS is characterized by pulmonary edema caused by capillary leak, with death often resulting from cardiogenic shock (9, 16). ANDV HCPS has a case fatality rate approaching 40%, and ANDV is the only hantavirus demonstrated to be capable of direct person-to-person transmission (15, 21). There is currently no specific therapy available for treatment of ANDV infection and HCPS.Peptide ligands that target a specific protein surface can have broad applications as therapeutics by blocking specific protein-protein interactions, such as preventing viral engagement of host cell receptors and thus preventing infection. Phage display libraries provide a powerful and inexpensive tool to identify such peptides. Here, we used selection of a cyclic nonapeptide-bearing phage library to identify peptides capable of binding the transmembrane surface glycoproteins of ANDV, Gn and Gc, and blocking infection in vitro.To identify peptide sequences capable of recognizing ANDV, we panned a cysteine-constrained cyclic nonapeptide-bearing phage display library (New England Biolabs) against density gradient-purified, UV-treated ANDV strain CHI-7913 (a gift from Hector Galeno, Santiago, Chile) (17, 18). To increase the specificity of the peptides identified, we eluted phage by using monoclonal antibodies (Austral Biologicals) prepared against recombinant fragments of ANDV Gn (residues 1 to 353) or Gc (residues 182 to 491) glycoproteins (antibodies 6B9/F5 and 6C5/D12, respectively). Peptide sequences were determined for phage from iterative rounds of panning, and the ability of phage to inhibit ANDV infection of Vero E6 cells was determined by immunofluorescent assay (IFA) (7). Primary IFA detection antibodies were rabbit polyclonal anti-Sin Nombre hantavirus (SNV) nucleoprotein (N) antibodies which exhibit potent cross-reactivity against other hantavirus N antigens (3). ReoPro, a commercially available Fab fragment which partially blocks infection of hantaviruses in vitro by binding the entry receptor integrin β₃ (5), was used as a positive control (80 μg/ml) along with the original antibody used for phage elution (5 μg/ml). As the maximum effectiveness of ReoPro in inhibiting hantavirus entry approaches 80%, we set this as a threshold for maximal expected efficacy for normalization. The most-potent phage identified by elution with the anti-Gn antibody 6B9/F5 bore the peptide CPSNVNNIC and inhibited hantavirus entry by greater than 60% (61%) (Table ​(Table1).1). From phage eluted with the anti-Gc antibody 6C5/D12, those bearing peptides CPMSQNPTC and CPKLHPGGC also inhibited entry by greater than 60% (66% and 72%, respectively).

TABLE 1.

Peptide-bearing phage eluted from ANDV

Zinc-Independent Folate Biosynthesis: Genetic,Biochemical, and Structural Investigations Reveal New Metal Dependence for GTP Cyclohydrolase IB

GTP cyclohydrolase I (GCYH-I) is an essential Zn²⁺-dependent enzyme that catalyzes the first step of the de novo folate biosynthetic pathway in bacteria and plants, the 7-deazapurine biosynthetic pathway in Bacteria and Archaea, and the biopterin pathway in mammals. We recently reported the discovery of a new prokaryotic-specific GCYH-I (GCYH-IB) that displays no sequence identity to the canonical enzyme and is present in ∼25% of bacteria, the majority of which lack the canonical GCYH-I (renamed GCYH-IA). Genomic and genetic analyses indicate that in those organisms possessing both enzymes, e.g., Bacillus subtilis, GCYH-IA and -IB are functionally redundant, but differentially expressed. Whereas GCYH-IA is constitutively expressed, GCYH-IB is expressed only under Zn²⁺-limiting conditions. These observations are consistent with the hypothesis that GCYH-IB functions to allow folate biosynthesis during Zn²⁺ starvation. Here, we present biochemical and structural data showing that bacterial GCYH-IB, like GCYH-IA, belongs to the tunneling-fold (T-fold) superfamily. However, the GCYH-IA and -IB enzymes exhibit significant differences in global structure and active-site architecture. While GCYH-IA is a unimodular, homodecameric, Zn²⁺-dependent enzyme, GCYH-IB is a bimodular, homotetrameric enzyme activated by a variety of divalent cations. The structure of GCYH-IB and the broad metal dependence exhibited by this enzyme further underscore the mechanistic plasticity that is emerging for the T-fold superfamily. Notably, while humans possess the canonical GCYH-IA enzyme, many clinically important human pathogens possess only the GCYH-IB enzyme, suggesting that this enzyme is a potential new molecular target for antibacterial development.The Zn²⁺-dependent enzyme GTP cyclohydrolase I (GCYH-I; EC 3.5.4.16) is the first enzyme of the de novo tetrahydrofolate (THF) biosynthesis pathway (Fig. ​(Fig.1)1) (38). THF is an essential cofactor in one-carbon transfer reactions in the synthesis of purines, thymidylate, pantothenate, glycine, serine, and methionine in all kingdoms of life (38), and formylmethionyl-tRNA in bacteria (7). Recently, it has also been shown that GCYH-I is required for the biosynthesis of the 7-deazaguanosine-modified tRNA nucleosides queuosine and archaeosine produced in Bacteria and Archaea (44), respectively, as well as the 7-deazaadenosine metabolites produced in some Streptomyces species (33). GCYH-I is encoded in Escherichia coli by the folE gene (28) and catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (55), a complex reaction that begins with hydrolytic opening of the purine ring at C-8 of GTP to generate an N-formyl intermediate, followed by deformylation and subsequent rearrangement and cyclization of the ribosyl moiety to generate the pterin ring in THF (Fig. ​(Fig.1).1). Notably, the enzyme is dependent on an essential active-site Zn²⁺ that serves to activate a water molecule for nucleophilic attack at C-8 in the first step of the reaction (2).Open in a separate windowFIG. 1.Reaction catalyzed by GCYH-I, and metabolic fate of 7,8-dihydroneopterin triphosphate.A homologous GCYH-I is found in mammals and other higher eukaryotes, where it catalyzes the first step of the biopterin (BH₄) pathway (Fig. ​(Fig.1),1), an essential cofactor in the biosynthesis of tyrosine and neurotransmitters, such as serotonin and l-3,4-dihydroxyphenylalanine (3, 52). Recently, a distinct class of GCYH-I enzymes, GCYH-IB (encoded by the folE2 gene), was discovered in microbes (26% of sequenced Bacteria and most Archaea) (12), including several clinically important human pathogens, e.g., Neisseria and Staphylococcus species. Notably, GCYH-IB is absent in eukaryotes.The distribution of folE (gene product renamed GCYH-IA) and folE2 (GCYH-IB) in bacteria is diverse (12). The majority of organisms possess either a folE (65%; e.g., Escherichia coli) or a folE2 (14%; e.g., Neisseria gonorrhoeae) gene. A significant number (12%; e.g., B. subtilis) possess both genes (a subset of 50 bacterial species is shown in Table ​Table1),1), and 9% lack both genes, although members of the latter group are mainly intracellular or symbiotic bacteria that rely on external sources of folate. The majority of Archaea possess only a folE2 gene, and the encoded GCYH-IB appears to be necessary only for the biosynthesis of the modified tRNA nucleoside archaeosine (44) except in the few halophilic Archaea that are known to synthesize folates, such as Haloferax volcanii, where GCYH-IB is involved in both archaeosine and folate formation (13, 44).

TABLE 1.

Distribution and candidate Zur-dependent regulation of alternative GCYH-I genes in bacteria^a

The Diagnosis and Management of Hereditary Haemochromatosis

Hereditary haemochromatosis (HH) is a common genetic disorder of iron metabolism in individuals of Northern European ancestry which leads to inappropriate iron absorption from the intestine and iron overload in susceptible individuals. Iron overload is suggested by elevations in serum ferritin and transferrin saturation. The majority of patients with clinically significant iron overload are homozygous for the C282Y mutation of the HFE gene, however only a minority of C282Y homozygotes fully express the disease clinically. Those with a high serum ferritin (>1000 μg/L) and additional hepatic insults from cofactors are more likely to develop cirrhosis and its complications. The mainstay of treatment is venesection. Those without cirrhosis who undergo appropriate venesection have a normal life expectancy. Family screening is recommended for all first degree relatives of an individual with the disease.HH refers to a group of inherited disorders that result in progressive iron overload. Mutations of the HFE gene are responsible for the majority of cases of HH,¹ although disease expression is highly variable.² The ready availability of testing for the two clinically relevant mutations: C282Y and H63D, has substantially altered the approach to suspected iron overload in clinical practice. A number of rare but important forms of non-HFE related HH have also been described.³ The other main causes of iron overload are outlined in the

Rethinking Guard Cell Metabolism

Stomata control gaseous fluxes between the internal leaf air spaces and the external atmosphere and, therefore, play a pivotal role in regulating CO₂ uptake for photosynthesis as well as water loss through transpiration. Guard cells, which flank the stomata, undergo adjustments in volume, resulting in changes in pore aperture. Stomatal opening is mediated by the complex regulation of ion transport and solute biosynthesis. Ion transport is exceptionally well understood, whereas our knowledge of guard cell metabolism remains limited, despite several decades of research. In this review, we evaluate the current literature on metabolism in guard cells, particularly the roles of starch, sucrose, and malate. We explore the possible origins of sucrose, including guard cell photosynthesis, and discuss new evidence that points to multiple processes and plasticity in guard cell metabolism that enable these cells to function effectively to maintain optimal stomatal aperture. We also discuss the new tools, techniques, and approaches available for further exploring and potentially manipulating guard cell metabolism to improve plant water use and productivity.Stomata are microscopic, adjustable pores on the leaf surface. The evolution of stomata more than 400 million years ago (Edwards et al., 1986, 1992, 1998) helped facilitate the adaptation of plants to a terrestrial environment, where water is typically a limiting resource. Each stoma is composed of two kidney- or dumbbell-shaped guard cells, whose volume changes to adjust pore aperture, allowing plants to simultaneously regulate CO₂ uptake and water loss. This facilitation of gas exchange by stomatal opening is one of the most essential processes in plant photosynthesis and transpiration, affecting plant water use efficiency and agricultural crop yields (Lawson and Blatt, 2014).Plant physiologists have a long history of investigating the behavior of these fascinating structures, reaching back more than a century to the pioneering work of Sir Francis Darwin (Darwin, 1916) and the American botanist Francis Ernest Lloyd (Lloyd, 1908). Major contributions to stomatal research arose from inventing and improving equipment and methods for quantitatively measuring the effects of environmental factors on stomatal pore aperture. After Darwin’s work, it became clear that the stomatal aperture actively responds to changes in the environment and regulates leaf transpiration rates (Meidner, 1987). Over the past century, much has been learned about their structure, development, and physiology.Despite the anatomical simplicity of the stomatal valve, the surrounding guard cells are highly specialized. Guard cells are morphologically distinct from general epidermal cells and possess complex signal transduction networks, elevated membrane ion transport capacity, and modified metabolic pathways. These features allow rapid modulations in guard cell turgor in response to endogenous and environmental signals, promoting the opening and closure of the stomatal pore in time scales of seconds to hours (Assmann and Wang, 2001). A variety of osmotically active solutes contribute to the buildup of stomatal turgor. Potassium (K⁺) and chloride (Cl⁻) act as the main inorganic ions, and malate²⁻ and sucrose (Suc) function as the main organic solutes. Whereas K⁺ and Cl⁻ are taken up from the apoplast, Suc and malate²⁻ can be imported or synthesized internally using carbon skeletons deriving from starch degradation and/or CO₂ fixation in the guard cell chloroplast (Roelfsema and Hedrich, 2005; Vavasseur and Raghavendra, 2005; Lawson, 2009; Kollist et al., 2014). The accumulation of these osmotica lowers the water potential, promoting the inflow of water, the swelling of guard cells, and the opening of the stomatal pore. Most of the ions taken up, or synthesized by guard cells, are sequestered into the vacuole (Barbier-Brygoo et al., 2011). As a result, the guard cell vacuoles undergo dynamic changes in volume and structure, which are crucial for achieving the full amplitude of stomatal movements (Gao et al., 2005; Tanaka et al., 2007; Andrés et al., 2014). During stomatal closure, guard cells reduce their volume through the release of ions into the cell wall and the consequent efflux of water.The transport of osmolytes across the plasma and tonoplast guard cell membranes is energized by H⁺-ATPase activity, which generates a proton motive force by translocating H⁺ ions against their concentration gradient (Blatt, 1987a, 1987b; Thiel et al., 1992; Roelfsema and Hedrich, 2005; Gaxiola et al., 2007). After the pioneering work of Fischer demonstrated the importance of K⁺ uptake in stomatal opening (Fischer, 1968; Fischer and Hsiao, 1968), K⁺ transport became of central interest and has long been considered the essence of stomatal movement regulation. The development of the voltage clamp technique, along with the relative easy acquisition of knockout mutants and transgenics in the model plant Arabidopsis (Arabidopsis thaliana), helped to uncover the precise mechanism and function of K⁺ fluxes in guard cells. It is well established that changes in membrane potential in response to several stimuli (e.g. light/darkness, CO₂, and abscisic acid [ABA]) alter the direction of K⁺ transport (Thiel et al., 1992; Blatt, 2000; Roelfsema et al., 2001, 2002, 2004). During stomatal opening, the activation of the proton pump generates a sufficiently negative electric potential to cause the uptake of K⁺ through the inward-rectifying K⁺ channels (K⁺_in; Fig. 1). During stomatal closure, K⁺ outflow from outward-rectifying K⁺ channels (K⁺_out) results from membrane depolarization (Fig. 2; Blatt, 1988; Schroeder, 1988; Anderson et al., 1992; Sentenac et al., 1992). Besides being gated by opposing changes in voltage, the activation of (K⁺_out) channels is dependent on the extracellular K⁺ concentration, while that of K⁺_in is not (Blatt, 1988, 1992; Roelfsema and Prins, 1997; Dreyer and Blatt, 2009). There is also strong evidence for H⁺-coupled K⁺ symport in guard cells, which could account for up to 50% of total K⁺ uptake during stomatal opening (Blatt and Clint, 1989; Clint and Blatt, 1989; Hills et al., 2012). At least for K⁺_in, the loss of a single-channel gene in Arabidopsis has little or no impact on stomatal movement (Szyroki et al., 2001), showing the redundancy among the different K⁺_in isoforms and of K⁺ transport in general.Open in a separate windowFigure 1.Integration of guard cell carbohydrate metabolism with membrane ion transport during stomatal opening. Sugars in guard cells can be imported from the apoplast, derive from starch breakdown, or be synthesized in the Calvin cycle. These sugars then can be stored as osmotically active solutes in the vacuole or metabolized in the cytosol to yield energy, reducing equivalents, and phosphoenolpyruvate (PEP). PEP can be further metabolized to pyruvate in the mitochondrial tricarboxylic acid (CAC) cycle or used as carbon skeletons for the biosynthesis of malate via PEP carboxylase (PEPC) and NAD-dependent malate dehydrogenase (NAD-MDH). Malate (which also can be imported from the apoplast) and the inorganic ions K⁺ and Cl⁻ accumulate in the vacuole, lowering the guard cell osmotic potential, thereby promoting stomatal opening. ABCB14, ATP-binding cassette transporter B14; AcetylCoA, acetyl-CoA; ALMT, aluminum-activated malate transporter; ATP-PFK, ATP-dependent phosphofructokinase; AttDT, dicarboxylate transporter; cINV, cytosolic invertase; cwINV, cell wall invertase; Fru6P, Fru-6-P; Fru1,6P₂, fructose 1,6-bisphosphate; Gl6P, Glc-6-P; G3P, glyceraldehyde 3-phosphate; iPGAM, phosphoglycerate mutase isoforms; NRGA1, negative regulator of guard cell ABA signaling1; OAA, oxaloacetate; 2-PGA, 2-phosphoglycerate; 3-PGA, 3-phosphoglycerate; PPi-PFK, PPi-dependent Fru-6-P phosphotransferase; STP, monosaccharide/H⁺ cotransporter; SUC, Suc/H⁺ cotransporter; SuSy, Suc synthase; TPT, triose phosphate/phosphate translocator. Compartments are not to scale. The dotted line indicates multiple metabolic steps.Open in a separate windowFigure 2.Proposed pathways of osmolyte dissipation during stomatal closure. While the removal of Cl⁻ and K⁺ is well described in the literature, the fate of Suc and malate during stomatal closure is unclear. Suc can be cleaved by cytosolic invertase (cINV), and the resulting hexoses can be imported into the chloroplast in the form of Glc-6-P (Glc6P). Glc6P is used subsequently for starch biosynthesis. Malate can be removed from the cell via decarboxylation to pyruvate by malic enzyme (ME) and the subsequent complete oxidation in the mitochondrial tricarboxylic acid (CAC) cycle. Alternatively, malate can be converted to PEP via NAD⁺-dependent malate dehydrogenase (NAD-MDH) and PEP carboxykinase (PEPCK). Gluconeogenic conversion of PEP to Glc6P establishes a possible link between malate removal and starch synthesis. Compartments are not to scale. PEP, Phosphoenolpyruvate; OAA; oxaloacetate; STP, monosaccharide/H⁺ cotransporter; SUC, Suc/H⁺ cotransporter; SuSy, Suc synthase; cINV, cytosolic invertase; NRGA1, negative regulator of guard cell ABA signaling1; ALMT, aluminum-activated malate transporter; GPT, Glc-6-P/Pi translocator; cwINV, cell wall invertase; HK, hexokinase; QUAC1, quickly activating anion channel1.Despite the undisputed importance of K⁺ uptake in stomatal opening, the accumulation of K⁺ ions alone cannot account for the increase in osmotic pressure necessary to explain stomatal aperture. Studies from the 1980s by MacRobbie and Fischer demonstrated that Vicia faba guard cells take up approximately 2 pmol of K⁺ during stomatal opening. Assuming that K⁺ uptake is balanced by the accumulation of similar amounts of counter ions (Cl⁻ and/or malate²⁻), the expected increase in stomatal turgor to approximately 3 MPa is less than the 4.5 MPa expected for fully open stomata (Fischer, 1972; MacRobbie and Lettau, 1980a, 1980b; Chen et al., 2012). The realization that other solutes must accumulate in addition to K⁺ salts was one of the major paradigm shifts in stomatal physiology research in the last decades, equal to the discovery of ion channels. Suc was put forward as the most likely candidate for the additional osmoticum to support stomatal opening (MacRobbie, 1987; Tallman and Zeiger, 1988; Talbott and Zeiger, 1993, 1998). Nonetheless, this research area subsequently failed to attract notice commensurate with its importance.In the last few years, the metabolism of starch, sugars and, organic acids in guard cells has seen a rebirth, making this the perfect time to review the developments in this field. In this review, we focus on photosynthetic carbon assimilation and respiratory metabolism in guard cells and provide a historical overview of the subject that highlights the most up-to-date and novel discoveries in guard cell research. We describe the various metabolic pathways separately, but as metabolism is an integrated network, we also discuss their reciprocal and beneficial interactions. Finally, we highlight their connection with the metabolism in the subjacent mesophyll cells and how they integrate with guard cell signal transduction networks and membrane ion transport to regulate stomatal movements. The enzymes and transporters discussed in this review are listed in Arabidopsis Genome Initiative CodeGeneProteinFunctionMalate transport AT1G28010ABCB14ATP-binding cassette transporter B14Import of apoplastic malate AT5G47560tDTDicarboxylate transporterTransport of carboxylates into the vacuole AT3G18440ALMT9Aluminum-activated malate transporter9Transport of Cl⁻/malate²⁻ into the vacuole AT2G17470ALMT6Aluminum-activated malate transporter6Transport of malate²⁻ into the vacuole AT4G17970ALMT12/QUAC1Aluminum-activated malate transporter12Export of cytosolic Cl⁻/malate²⁻ to the apoplastMalate metabolism –PEPCPhosphoenolpyruvate carboxylaseβ-Carboxylation of PEP to OAA –NAD-MDHNAD⁺-dependent malate dehydrogenaseReduction of OAA to malate –MEMalic enzymeOxidative decarboxylation of malate to pyruvate AT4G37870PEPCK1PEP carboxykinase1Conversion of OAA to PEP –PPDKPyruvate, orthophosphate dikinaseConversion of pyruvate to PEPOther carboxylates –TPTTriose phosphate/phosphate translocatorExport of triose phosphate from the chloroplast to the cytosol AT4G05590NRGA1Negative regulator of guard cell ABA signaling1Putative mitochondrial pyruvate carrier –SDH2Succinate dehydrogenase2Oxidation of succinate to fumarate AT2G47510FUM1Fumarase1Hydration of fumarate to malate –iPGAMPhosphoglycerate mutaseInterconversion of 3-PGA to 2-PGA –PPi-PFKPPi-dependent Fru-6-P phosphotransferasePhosphorylation of Fru-6-P to Fru-1,6-bisphosphate –ATP-PFKATP-dependent phosphofructokinasePhosphorylation of Fru-6-P to Fru-1,6-bisphosphateCalvin cycle –RubiscoRubiscoCarboxylation of ribulose 1,5-bisphosphate AT3G55800SBPaseSedoheptulose-bisphosphataseDephosphorylation of sedoheptulose-1,7-bisphosphate to sedoheptulose-7-phosphateSugar metabolism AT4G29130HK1Hexokinase1Phosphorylation of Glc to Glc-6-P AT4G02280SuSySuc synthase3Interconversion of Suc to Fru and UDP-Glc –cINVCytosolic invertaseHydrolysis of Suc to Fru and Glc –cwINVCell wall invertaseHydrolysis of Suc to Fru and GlcSugar transport AT1G11260STP1Monosaccharide/H⁺ cotransporter1Import of apoplastic hexose sugars AT3G19930STP4Monosaccharide/H⁺ cotransporter4Import of apoplastic hexose sugars AT1G71880SUC1Suc/H⁺ cotransporter1Import of apoplastic Suc AT2G02860SUC3Suc/H⁺ cotransporter3Import of apoplastic SucStarch degradation AT3G23920BAM1β-Amylase1Hydrolysis of α-1,4 external glucoside linkages in starch AT1G69830AMY3α-Amylase3Hydrolysis of α-1,4 internal glucoside linkages in starchStarch synthesis –GPTGlc-6-P/Pi translocatorUptake of cytosolic Glc-6-P into the chloroplast AT4G24620PGIPhosphoglucose isomeraseConversion of Fru-6-P to Glc-6-P AT5G51820PGM1Phosphoglucomutase1Conversion of Glc-6-P to Glc-1-P AT5G48300APS1ADPGlc pyrophosphorylase small subunitConversion of Glc-1-P to ADPGlc, catalytic subunit –APLADPGlc pyrophosphorylase large subunitConversion of Glc-1-P to ADPGlc, regulatory subunitVarious AT3G45780PHOT1Phototropin1Blue light photoreceptor AT5G58140PHOT2Phototropin2Blue light photoreceptor AT4G14480BLUS1Blue light signaling1Protein kinase, regulator of blue light-induced stomatal opening –PP1Protein phosphatase1Regulator of blue light-induced stomatal opening AT3G01500CA1Carbonic anhydrase1Interconversion of CO₂ and water into H₂CO₃ AT1G70410CA4Carbonic anhydrase4Interconversion of CO₂ and water into H₂CO₃ AT1G62400HT1High leaf temperature1Protein kinase, regulator of CO₂-induced stomatal closureOpen in a separate window     

Alleviation of Proteolytic Sensitivity To Enhance Recombinant Lipase Production in Escherichia coli

Two amino acids, Leu149 and Val223, were identified as proteolytically sensitive when Pseudozyma antarctica lipase (PalB) was heterologously expressed in Escherichia coli. The functional expression was enhanced using the double mutant for cultivation. However, the recombinant protein production was still limited by PalB misfolding, which was resolved by DsbA coexpression.Though Escherichia coli retains popularity as a host for recombinant protein production, it has technical limitations for expressing eukaryotic proteins. Host/protein incompatibility is critical, since eukaryotic proteins heterologously expressed in E. coli are recognized as a foreign object that can induce heat shock responses and drive protease-mediated degradation. E. coli contains several proteases situated in various intracellular compartments, including the cytoplasm, periplasm, inner membrane, and outer membrane (7, 9). While these proteases play a pivotal role in maintaining cell physiology by clearing up abnormal proteins (4), they can potentially degrade recombinant proteins (6). Nevertheless, the mechanisms associated with protein degradation and protein substrate specificity are incompletely understood.Since the proteolysis of recombinant proteins is a common issue, approaches to overcome this limitation have been extensively explored. These include using protease-deficient mutants as an expression host (10, 18), lowering the cultivation temperature (3), coexpressing folding factors (19, 20), applying protein fusion technology (2, 8), eliminating protease cleavage sites (5, 15), and modifying target protein hydrophobicity (11). While genetic elimination of proteolytically sensitive sequences without impacting the target protein''s bioactivity appears to be technically attractive, there is no effective prediction or identification of potential protease cleavage sites. Proteolytically sensitive sequences might not be specific to certain proteases. Various factors affecting the folding, solubility, steric hindrance, and binding of the substrate protein, e.g., neighboring domains or amino acids, can contribute to the sensitivity of proteolytic sites, making sequence manipulation for proteolysis alleviation more difficult.Herein, the recombinant protein''s sensitivity toward intracellular proteolysis was genetically alleviated to enhance the functional expression of PalB, an industrial enzyme with justified applications (1), in E. coli and to understand the relationship between proteolytic specificity and substrate protein sequence. Since there are three intramolecular disulfide bonds whose formation can potentially affect PalB structure and bioactivity, PalB expression in the oxidative periplasm of E. coli was explored (20, 22). However, the expressed PalB protein was extremely sensitive to intracellular proteolysis, resulting in complete degradation. To overcome this problem, PalB mutant variants that were stable against proteolysis were derived (Table ​(Table11).

TABLE 1.

Strains, plasmids, and oligonucleotides

Variation in Adult Plant Phenotypes and Partitioning among Seed and Stem-Borne Roots across Brachypodium distachyon Accessions to Exploit in Breeding Cereals for Well-Watered and Drought Environments

Seedling roots enable plant establishment. Their small phenotypes are measured routinely. Adult root systems are relevant to yield and efficiency, but phenotyping is challenging. Root length exceeds the volume of most pots. Field studies measure partial adult root systems through coring or use seedling roots as adult surrogates. Here, we phenotyped 79 diverse lines of the small grass model Brachypodium distachyon to adults in 50-cm-long tubes of soil with irrigation; a subset of 16 lines was droughted. Variation was large (total biomass, ×8; total root length [TRL], ×10; and root mass ratio, ×6), repeatable, and attributable to genetic factors (heritabilities ranged from approximately 50% for root growth to 82% for partitioning phenotypes). Lines were dissected into seed-borne tissues (stem and primary seminal axile roots) and stem-borne tissues (tillers and coleoptile and leaf node axile roots) plus branch roots. All lines developed one seminal root that varied, with branch roots, from 31% to 90% of TRL in the well-watered condition. With drought, 100% of TRL was seminal, regardless of line because nodal roots were almost always inhibited in drying topsoil. Irrigation stimulated nodal roots depending on genotype. Shoot size and tillers correlated positively with roots with irrigation, but partitioning depended on genotype and was plastic with drought. Adult root systems of B. distachyon have genetic variation to exploit to increase cereal yields through genes associated with partitioning among roots and their responsiveness to irrigation. Whole-plant phenotypes could enhance gain for droughted environments because root and shoot traits are coselected.Adult plant root systems are relevant to the size and efficiency of seed yield. They supply water and nutrients for the plant to acquire biomass, which is positively correlated to the harvest index (allocation to seed grain), and the stages of flowering and grain development. Modeling in wheat (Triticum aestivum) suggested that an extra 10 mm of water absorbed by such adult root systems during grain filling resulted in an increase of approximately 500 kg grain ha⁻¹ (Manschadi et al., 2006). This was 25% above the average annual yield of wheat in rain-fed environments of Australia. This number was remarkably close to experimental data obtained in the field in Australia (Kirkegaard et al., 2007). Together, these modeling and field experiments have shown that adult root systems are critical for water absorption and grain yield in cereals, such as wheat, emphasizing the importance of characterizing adult root systems to identify phenotypes for productivity improvements.Most root phenotypes, however, have been described for seedling roots. Seedling roots are essential for plant establishment, and hence, the plant’s potential to set seed. For technical reasons, seedlings are more often screened than adult plants because of the ease of handling smaller plants and the high throughput. Seedling-stage phenotyping may also improve overall reproducibility of results because often, growth media are soil free. Seedling soil-free root phenotyping conditions are well suited to dissecting fine and sensitive mechanisms, such as lateral root initiation (Casimiro et al., 2003; Péret et al., 2009a, 2009b). A number of genes underlying root processes have been identified or characterized using seedlings, notably with the dicotyledonous models Arabidopsis (Arabidopsis thaliana; Mouchel et al., 2004; Fitz Gerald et al., 2006; Yokawa et al., 2013) and Medicago truncatula (Laffont et al., 2010) and the cereals maize (Zea mays; Hochholdinger et al., 2001) and rice (Oryza sativa; Inukai et al., 2005; Kitomi et al., 2008).Extrapolation from seedling to adult root systems presents major questions (Hochholdinger and Zimmermann, 2008; Chochois et al., 2012; Rich and Watt, 2013). Are phenotypes in seedling roots present in adult roots given developmental events associated with aging? Is expression of phenotypes correlated in seedling and adult roots if time compounds effects of growth rates and growth conditions on roots? Watt et al. (2013) showed in wheat seedlings that root traits in the laboratory and field correlated positively but that neither correlated with adult root traits in the field. Factors between seedling and adult roots seemed to be differences in developmental stage and the time that growing roots experience the environment.Seedling and adult root differences may be larger in grasses than dicotyledons. Grass root systems have two developmental components: seed-borne (seminal) roots, of which a number emerge at germination and continue to grow and branch throughout the plant life, and stem-borne (nodal or adventitious) roots, which emerge from around the three-leaf stage and continue to emerge, grow, and branch throughout the plant life. Phenotypes and traits of adult root systems of grasses, which include the major cereal crops wheat, rice, and maize, are difficult to predict in seedling screens and ideally identified from adult root systems first (Gamuyao et al., 2012).Phenotyping of adult roots is possible in the field using trenches (Maeght et al., 2013) or coring (Wasson et al., 2014). A portion of the root system is captured with these methods. Alternatively, entire adult root systems can be contained within pots dug into the ground before sowing. These need to be large; field wheat roots, for example, can reach depths greater than 1.5 m depending on genotype and environment. This method prevents root-root interactions that occur under normal field sowing of a plant canopy and is also a compromise.A solution to the problem of phenotyping adult cereal root systems is a model for monocotyledon grasses: Brachypodium distachyon. B. distachyon is a small-stature grass with a small genome that is fully sequenced (Vogel et al., 2010). It has molecular tools equivalent to those available in Arabidopsis (Draper et al., 2001; Brkljacic et al., 2011; Mur et al., 2011). The root system of B. distachyon reference line Bd21 is more similar to wheat than other model and crop grasses (Watt et al., 2009). It has a seed-borne primary seminal root (PSR) that emerges from the embryo at seed germination and multiple stem-borne coleoptile node axile roots (CNRs) and leaf node axile roots (LNRs), also known as crown roots or adventitious roots, that emerge at about three leaves through to grain development. Branch roots emerge from all root types. There are no known anatomical differences between root types of wheat and B. distachyon (Watt et al., 2009). In a recent study, we report postflowering root growth in B. distachyon line Bd21-3, showing that this model can be used to answer questions relevant to the adult root systems of grasses (Chochois et al., 2012).In this study, we used B. distachyon to identify adult plant phenotypes related to the partitioning among seed-borne and stem-borne shoots and roots for the genetic improvement of well-watered and droughted cereals (Fig. 1; Krassovsky, 1926; Navara et al., 1994), nitrogen, phosphorus (Tennant, 1976; Brady et al., 1995), oxygen (Wiengweera and Greenway, 2004), soil hardness (Acuna et al., 2007), and microorganisms (Sivasithamparam et al., 1978). Of note is the study by Krassovsky (1926), which was the first, to our knowledge, to show differences in function related to water. Krassovsky (1926) showed that seminal roots of wheat absorbed almost 2 times the water as nodal roots per unit dry weight but that nodal roots absorbed a more diluted nutrient solution than seminal roots. Krassovsky (1926) also showed by removing seminal or nodal roots as they emerged that “seminal roots serve the main stem, while nodal roots serve the tillers” (Krassovsky, 1926). Volkmar (1997) showed, more recently, in wheat that nodal and seminal roots may sense and respond to drought differently. In millet (Pennisetum glaucum) and sorghum (Sorghum bicolor), Rostamza et al. (2013) found that millet was able to grow nodal roots in a dryer soil than sorghum, possibly because of shoot and root vigor.Open in a separate windowFigure 1.B. distachyon plant scanned at the fourth leaf stage, with the root and shoot phenotypes studied indicated. Supplemental Table S1.

Characterization of a Thermostable Short-Chain Alcohol Dehydrogenase from the Hyperthermophilic Archaeon Thermococcus sibiricus

Short-chain alcohol dehydrogenase, encoded by the gene Tsib_0319 from the hyperthermophilic archaeon Thermococcus sibiricus, was expressed in Escherichia coli, purified and characterized as an NADPH-dependent enantioselective oxidoreductase with broad substrate specificity. The enzyme exhibits extremely high thermophilicity, thermostability, and tolerance to organic solvents and salts.Alcohol dehydrogenases (ADHs; EC 1.1.1.1.) catalyze the interconversion of alcohols to their corresponding aldehydes or ketones by using different redox-mediating cofactors. NAD(P)-dependent ADHs, due to their broad substrate specificity and enantioselectivity, have attracted particular attention as catalysts in industrial processes (5). However, mesophilic ADHs are unstable at high temperatures, sensitive to organic solvents, and often lose activity during immobilization. In this relation, there is a considerable interest in ADHs from extremophilic microorganisms; among them, Archaea are of great interest. The representatives of all groups of NAD(P)-dependent ADHs have been detected in genomes of Archaea (11, 12); however, only a few enzymes have been characterized, and the great majority of them belong to medium-chain (3, 4, 14, 16, 19) or long-chain iron-activated ADHs (1, 8, 9). Up to now, a single short-chain archaeal ADH from Pyrococcus furiosus (10, 18) and only one archaeal aldo-keto reductase also from P. furiosus (11) have been characterized.Thermococcus sibiricus is a hyperthermophilic anaerobic archaeon isolated from a high-temperature oil reservoir capable of growth on complex organic substrates (15). The complete genome sequence of T. sibiricus has been recently determined and annotated (13). Several ADHs are encoded by the T. sibiricus genome, including three short-chain ADHs (Tsib_0319, Tsib_0703, and Tsib_1998) (13). In this report, we describe the cloning and expression of the Tsib_0319 gene from T. sibiricus and the purification and the biochemical characterization of its product, the thermostable short-chain ADH (TsAdh319).The Tsib_0319 gene encodes a protein with a size of 234 amino acids and the calculated molecular mass of 26.2 kDa. TsAdh319 has an 85% degree of sequence identity with short-chain ADH from P. furiosus (AdhA; PF_0074) (18). Besides AdhA, close homologs of TsAdh319 were found among different bacterial ADHs, but not archaeal ADHs. The gene flanked by the XhoI and BamHI sites was PCR amplified using two primers (sense primer, 5′-GTTCTCGAGATGAAGGTTGCTGTGATAACAGGG-3′, and antisense primer, 5′-GCTGGATCCTCAGTATTCTGGTCTCTGGTAGACGG-3′) and cloned into the pET-15b vector. TsAdh319 was overexpressed, with an N-terminal His₆ tag in Escherichia coli Rosetta-gami (DE3) and purified to homogeneity by metallochelating chromatography (Hi-Trap chelating HP column; GE Healthcare) followed by gel filtration on Superdex 200 10/300 GL column (GE Healthcare) equilibrated in 50 mM Tris-HCl (pH 7.5) with 200 mM NaCl. The homogeneity and the correspondence to the calculated molecular mass of 28.7 kDa were verified by SDS-PAGE (7). The molecular mass of native TsAdh319 was 56 to 60 kDa, which confirmed the dimeric structure in solution.The standard ADH activity measurement was made spectrophotometrically at the optimal pH by following either the reduction of NADP (in 50 mM Gly-NaOH buffer; pH 10.5) or the oxidation of NADPH (in 0.1 M sodium phosphate buffer; pH 7.5) at 340 nm at 60°C. The enzyme exhibited a strong preference for NADP(H) and broad substrate specificity (Table ​(Table1).1). The highest oxidation rates were found with pentoses d-arabinose (2.0 U mg⁻¹) and d-xylose (2.46 U mg⁻¹), and the highest reduction rates were found with dimethylglyoxal (5.9 U mg⁻¹) and pyruvaldehyde (2.2 U mg⁻¹). The enzyme did not reduce sugars which were good substrates for the oxidation reaction. The kinetic parameters of TsAdh319 determined for the preferred substrates are shown in Table ​Table2.2. The enantioselectivity of the enzyme was estimated by measuring the conversion rates of 2-butanol enantiomers. TsAdh319 showed an evident preference, >2-fold, for (S)-2-butanol over (RS)-2-butanol. The enzyme stereoselectivity is confirmed by the preferred oxidation of d-arabinose over l-arabinose (Table ​(Table1).1). The fact that TsAdh319 is metal independent was supported by the absence of a significant effect of TsAdh319 preincubation with 10 mM Me²⁺ for 30 min before measuring the activity in the presence of 1 mM Me²⁺ or EDTA (Table ​(Table3).3). TsAdh319 also exhibited a halophilic property, so the enzyme activity increased in the presence of NaCl and KCl and the activation was maintained even at concentration of 4 M and 3 M, respectively (Table ​(Table33).

TABLE 1.

Substrate specificity of TsAdh319

Analysis of the Clonal Relationship of Serotype O26:H11 Enterohemorrhagic Escherichia coli Isolates from Cattle

Twelve cluster groups of Escherichia coli O26 isolates found in three cattle farms were monitored in space and time. Cluster analysis suggests that only some O26:H11 strains had the potential for long-term persistence in hosts and farms. As judged by their virulence markers, bovine enterohemorrhagic O26:H11 isolates may represent a considerable risk for human infection.Shiga toxin (Stx)-producing Escherichia coli (STEC) strains comprise a group of zoonotic enteric pathogens (42). In humans, infections with some STEC serotypes result in hemorrhagic or nonhemorrhagic diarrhea, which can be complicated by hemolytic-uremic syndrome (HUS) (49). These STEC strains are also designated “enterohemorrhagic E. coli” (EHEC). Consequently, EHEC strains represent a subgroup of STEC with a high pathogenic potential for humans. Strains of the E. coli serogroup O26 were originally classified as enteropathogenic E. coli due to their association with outbreaks of infantile diarrhea in the 1940s. In 1977, Konowalchuk et al. (37) recognized that these bacteria produced Stx, and 10 years later, the Stx-producing E. coli O26:H11/H− strains were classified as EHEC. EHEC O26 strains constitute the most common non-O157 EHEC group associated with diarrhea and HUS in Europe (12, 21, 23, 24, 26, 27, 55, 60). Reports on an association between EHEC O26 and HUS or diarrhea from North America including the United States (15, 30, 33), South America (51, 57), Australia (22), and Asia (31, 32) provide further evidence for the worldwide spread of these organisms. Studies in Germany and Austria (26, 27) on sporadic HUS cases between 1996 and 2003 found that EHEC O26 accounted for 14% of all EHEC strains and for ∼40% of non-O157 EHEC strains obtained from these patients. A proportion of 11% EHEC O26 strains was detected in a case-control study in Germany (59) between 2001 and 2003. In the age group <3 years, the number of EHEC O26 cases was nearly equal to that of EHEC O157 cases, although the incidence of EHEC O26-associated disease is probably underestimated because of diagnostic limitations in comparison to the diagnosis of O157:H7/H− (18, 34). Moreover, EHEC O26 has spread globally (35). Beutin (6) described EHEC O26:H11/H−, among O103:H2, O111:H, O145:H28/H−, and O157:H7/H−, as the well-known pathogenic “gang of five,” and Bettelheim (5) warned that we ignore the non-O157 STEC strains at our peril.EHEC O26 strains produce Stx1, Stx2, or both (15, 63). Moreover, these strains contain the intimin-encoding eae gene (11, 63), a characteristic feature of EHEC (44). In addition, EHEC strains possess other markers associated with virulence, such as a large plasmid that carries further potential virulence genes, e.g., genes coding for EHEC hemolysin (EHEC-hlyA), a catalase-peroxidase (katP), and an extracellular serine protease (espP) (17, 52). The efa1 (E. coli factor for adherence 1) gene was identified as an intestinal colonization factor in EHEC (43). EHEC O26 represents a highly dynamic group of organisms that rapidly generate new pathogenic clones (7, 8, 63).Ruminants, especially cattle, are considered the primary reservoir for human infections with EHEC. Therefore, the aim of this study was the molecular characterization of bovine E. coli field isolates of serogroup O26 using a panel of typical virulence markers. The epidemiological situation in the beef herds from which the isolates were obtained and the spatial and temporal behavior of the clonal distribution of E. coli serogroup O26 were analyzed during the observation period. The potential risk of the isolates inducing disease in humans was assessed.In our study, 56 bovine E. coli O26:H11 isolates and one bovine O26:H32 isolate were analyzed for EHEC virulence-associated factors. The isolates had been obtained from three different beef farms during a long-term study. They were detected in eight different cattle in farm A over a period of 15 months (detected on 10 sampling days), in 3 different animals in farm C over a period of 8 months (detected on 3 sampling days), and in one cow on one sampling day in farm D (Table ​(Table1)1) (28).

TABLE 1.

Typing of E. coli O26 isolates

Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Focus on Metabolism: Posttranslational Protein Modifications in Plant Metabolism

Posttranslational modifications (PTMs) of proteins greatly expand proteome diversity, increase functionality, and allow for rapid responses, all at relatively low costs for the cell. PTMs play key roles in plants through their impact on signaling, gene expression, protein stability and interactions, and enzyme kinetics. Following a brief discussion of the experimental and bioinformatics challenges of PTM identification, localization, and quantification (occupancy), a concise overview is provided of the major PTMs and their (potential) functional consequences in plants, with emphasis on plant metabolism. Classic examples that illustrate the regulation of plant metabolic enzymes and pathways by PTMs and their cross talk are summarized. Recent large-scale proteomics studies mapped many PTMs to a wide range of metabolic functions. Unraveling of the PTM code, i.e. a predictive understanding of the (combinatorial) consequences of PTMs, is needed to convert this growing wealth of data into an understanding of plant metabolic regulation.The primary amino acid sequence of proteins is defined by the translated mRNA, often followed by N- or C-terminal cleavages for preprocessing, maturation, and/or activation. Proteins can undergo further reversible or irreversible posttranslational modifications (PTMs) of specific amino acid residues. Proteins are directly responsible for the production of plant metabolites because they act as enzymes or as regulators of enzymes. Ultimately, most proteins in a plant cell can affect plant metabolism (e.g. through effects on plant gene expression, cell fate and development, structural support, transport, etc.). Many metabolic enzymes and their regulators undergo a variety of PTMs, possibly resulting in changes in oligomeric state, stabilization/degradation, and (de)activation (Huber and Hardin, 2004), and PTMs can facilitate the optimization of metabolic flux. However, the direct in vivo consequence of a PTM on a metabolic enzyme or pathway is frequently not very clear, in part because it requires measurements of input and output of the reactions, including flux through the enzyme or pathway. This Update will start out with a short overview on the major PTMs observed for each amino acid residue (PTMs, including determination of the localization within proteins (i.e. the specific residues) and occupancy. Challenges in dealing with multiple PTMs per protein and cross talk between PTMs will be briefly outlined. We then describe the major physiological PTMs observed in plants as well as PTMs that are nonenzymatically induced during sample preparation (PTMs, in particular for enzymes in primary metabolism (Calvin cycle, glycolysis, and respiration) and the C4 shuttle accommodating photosynthesis in C4 plants (PTMs observed in plants