Structural Insights into the Mode of Action of the Peptide Antibiotic Copsin.

Defensins make up a class of cysteine-rich antimicrobial peptides, expressed by virtually all eukaryotes as part of their innate immune response. Because of their unique mode of action and rapid killing of pathogenic microbes, defensins are considered promising alternatives to clinically applied antibiotics. Copsin is a defensin-like peptide, previously identified in the mushroom Coprinopsis cinerea. It exerts its activity against a range of Gram-positive bacteria by binding to the peptidoglycan precursor lipid II and prevention of proper cell wall formation. In this study, we present a new workflow for the generation, production, and activity-driven selection of copsin derivatives, based on their expression in Pichia pastoris. One hundred fifty-two single-amino acid mutants and combinations thereof allowed the identification of k-copsin, a peptide variant exhibiting significantly enhanced activity against Bacillus subtilis and Staphylococcus aureus. Furthermore, we performed in silico characterizations of membrane interactions of copsin and k-copsin, in the presence and absence of lipid II. The molecular dynamics data highlighted a high variability in lipid II binding, with a preference for the MurNAc moiety with 47 and 35% of the total contacts for copsin and k-copsin, respectively. Mutated amino acids were located in loop regions of k-copsin and shown to be crucial in the perturbation of the bacterial membrane. These structural studies provide a better understanding of how defensins can be developed toward antibacterial therapies less prone to resistance issues.

Copsin, a novel peptide-based fungal antibiotic interfering with the peptidoglycan synthesis.

Fungi and bacteria compete with an arsenal of secreted molecules for their ecological niche. This repertoire represents a rich and inexhaustible source for antibiotics and fungicides. Antimicrobial peptides are an emerging class of fungal defense molecules that are promising candidates for pharmaceutical applications. Based on a co-cultivation system, we studied the interaction of the coprophilous basidiomycete Coprinopsis cinerea with different bacterial species and identified a novel defensin, copsin. The polypeptide was recombinantly produced in Pichia pastoris, and the three-dimensional structure was solved by NMR. The cysteine stabilized α/ß-fold with a unique disulfide connectivity, and an N-terminal pyroglutamate rendered copsin extremely stable against high temperatures and protease digestion. Copsin was bactericidal against a diversity of Gram-positive bacteria, including human pathogens such as Enterococcus faecium and Listeria monocytogenes. Characterization of the antibacterial activity revealed that copsin bound specifically to the peptidoglycan precursor lipid II and therefore interfered with the cell wall biosynthesis. In particular, and unlike lantibiotics and other defensins, the third position of the lipid II pentapeptide is essential for effective copsin binding. The unique structural properties of copsin make it a possible scaffold for new antibiotics.

The responses of Vitreoscilla hemoglobin-expressing hybrid aspen (Populus tremula × tremuloides) exposed to 24-h herbivory: expression of hemoglobin and stress-related genes in exposed and nonorthostichous leaves.

The responses of transcriptome and phenolic compounds were determined with Populus tremula L. × Populus tremuloides Michx. expressing the hemoglobin (Hb) of Vitreoscilla (VHb) and non-transformant (wt) line. After 24-h exposure of leaves to Conistra vaccinii L., the transcript levels of endogenous non-symbiotic class 1 Hb (PttHb1) and truncated Hb (PttTrHb) genes were modestly reduced and increased, respectively, in both wt and VHb-expressing line. Besides the herbivory exposed leaves showing the most significant transcriptome changes, alterations were also detected in the transcriptome of nonorthostichous leaves positioned directly above the exposed leaves. Both wt and VHb-expressing line displayed similar herbivory-induced effects on gene expression, although the extent of responses was more pronounced in the wt than in the VHb-expressing line. The contents of phenolic compounds were not altered due to herbivory and they were alike in the wt and VHb-expressing line. In addition, we determined the relative growth rates (RGRs) of Orthosia gothica L., Ectropis crepuscularia Denis & Schiff. and Orgyia antiqua L. larvae, and found no variation in the RGRs between the lines. Thus, VHb-expressing P. tremula × tremuloides lines showed to be comparable with wt in regards to the food quality of leaves.

Expression of rat beta(1,4)-N-acetylglucosaminyltransferase III in Nicotiana tabacum remodels the plant-specific N-glycosylation.

Plant N-linked glycans differ substantially from their mammalian counterparts, mainly with respect to modifications of the core glycan, which typically contains a beta(1,2)-xylose and an alpha(1,3)-fucose. The addition of a bisecting N-acetylglucosamine residue by beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII) is known to control the processing of N-linked glycans in mammals, for example by preventing alpha(1,6)-fucosylation of the core glycan. In order to outcompete plant-specific beta(1,2)-xylose and alpha(1,3)-fucose modifications, rat GnTIII was expressed either with its native localization domain (GnTIII) or with the cytoplasmic tail, transmembrane domain and stem region (CTS) of Arabidopsis thaliana mannosidase II (ManII) (GnTIII(A.th.)). Both CTSs targeted enhanced yellow fluorescent protein (eYFP) to a brefeldin A-sensitive compartment, indicative of Golgi localization. GnTIII expression increased the fraction of N-glycans devoid of xylose and fucose from 13% +/- 7% in wild-type plants to 60% +/- 8% in plants expressing GnTIII(A.th.). N-Glycans of plants expressing rat GnTIII contained three major glycan structures of complex bisected, complex, or hybrid bisected type, accounting for 70%-85% of the total N-glycans. On expression of GnTIII(A.th.), N-glycans displayed a higher heterogeneity and were of hybrid type. Co-expression of A. thaliana ManII significantly increased the amount of complex bisected structures relative to the plants expressing GnTIII or GnTIII(A.th.), whereas co-expression of human ManII did not redirect the pool of hybrid structures towards complex-type structures. The method described offers the advantage that it can be implemented in any desired plant system for effective removal of beta(1,2)-xylose and alpha(1,3)-fucose from the N-glycan.

Intrinsic non-symbiotic and truncated haemoglobins and heterologous Vitreoscilla haemoglobin expression in plants.

To date, haemoglobins (Hbs) have been shown to exist in all kingdoms of life. The least studied and understood groups are plant non-symbiotic haemoglobins (nsHbs) and the recently found plant truncated Hbs (trHbs). From a biotechnological point of view, the best characterized and almost exclusively applied Hb is the bacterial Vitreoscilla haemoglobin (VHb). In this review, the present state of knowledge of structural features and ligand binding kinetics of plant nsHbs and trHbs and their proposed roles as oxygen carriers, oxygen sensors, and for oxygen storage, in nitric oxide (NO) detoxification, and in peroxidase activity are described. Furthermore, in order to predict the functioning of plant Hbs, their characteristics will be compared with those of the better known bacterial globins. In this context, the effects of heterologous applications of VHb on plants are reviewed. Finally, the challenging future of plant Hb research is discussed.

A small-scale method for the preparation of plant N-linked glycans from soluble proteins for analysis by MALDI-TOF mass spectrometry.

The use of plants as production hosts for recombinant glycoproteins, which is rapidly developing, requires methods for fast and reliable analysis of plant N-linked glycans. This study describes a simple small-scale method for the preparation of N-linked glycans from soluble plant protein and analysis thereof by matrix assisted laser desorption ionisation time of flight mass spectrometry (MALDI-TOF MS). Concentration and protease digestion of plant protein as well as deglycosylation is carried out in a single concentrator unit without the need for intermittent purification to minimize adsorptive loss and to facilitate handling. Plant protein is concentrated in a unit with a 5kDa cutoff, and after buffer exchange, pepsin (EC 3.4.23.1) digestion is carried out in the concentrator overnight to obtain peptides as substrates for deglycosylation. Deglycosylation is carried out with peptide-N-glycosidase A (PNGase A; EC 3.5.1.52) for 24h. Released N-glycans are purified using reverse-phase and cation exchange chromatography micro-columns for removal of peptides and desalting. N-Glycans are directly analyzed by MALDI-TOF MS without derivatization. The method for isolation of N-glycans is compatible with secreted proteins from cell culture supernatant as well as with soluble protein extracts from leaf tissue. As little as 5mug of plant glycoprotein is sufficient for N-glycan preparation for MALDI-TOF MS analysis using this method.

Induction of Pseudomonas aeruginosa fhp and fhpR by reactive oxygen species.

In Pseudomonas aeruginosa, flavohemoglobin (Fhp) and its cognate regulator FhpR (PA2665) form a protective regulatory circuit, which responds to reactive nitrogen species and is also capable of protecting cells against nitrosative stress. Recently, it has been shown that the expression of the fhp promoter is regulated not only by FhpR, but also by two new regulators, PA0779 and PA3697. It has also been suggested that the bacterial flavohemoglobins (flavoHbs) could play a crucial role in the protection of cells against reactive oxygen species (ROS). Therefore, the role and function of the Fhp/FhpR system during oxidative stress were studied by assessing the viability and membrane integrity of P. aeruginosa cells and by analyzing the promoter activities of fhp and fhpR upon exposure to paraquat, hydrogen peroxide, and tert-butyl hydroperoxide, under both aerobic and low-oxygen conditions. The results showed that under aerobic conditions, both fhp and fhpR promoters are induced by ROS generated by the stressors. Thus, the Fhp/FhpR system is implicated in the oxidative stress response. ROS-induced fhp promoter activity was dependent on FhpR, PA0779, and PA3697 regulators. Tert-butyl hydroperoxide-induced fhpR promoter activity was found to be highly repressed by PA0779, and FhpR showed negative autoregulation of its own promoter. Under low-oxygen conditions, the activity of the fhp promoter was not inducible by ROS, but fhpR promoter activity was induced by paraquat, and hydrogen peroxide was repressed in both cases by the regulators PA0779 and PA3697.

Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria.

Bacteria are the main nutritional competitors of saprophytic fungi during colonization of their ecological niches. This competition involves the mutual secretion of antimicrobials that kill or inhibit the growth of the competitor. Over the last years it has been demonstrated that fungi respond to the presence of bacteria with changes of their transcriptome, but the significance of these changes with respect to competition for nutrients is not clear as functional proof of the antibacterial activity of the induced gene products is often lacking. Here, we report the genome-wide transcriptional response of the coprophilous mushroom Coprinopsis cinerea to the bacteria Bacillus subtilis and Escherichia coli. The genes induced upon co-cultivation with each bacterium were highly overlapping, suggesting that the fungus uses a similar arsenal of effectors against Gram-positive and -negative bacteria. Intriguingly, the induced genes appeare to encode predominantly secreted peptides and proteins with predicted antibacterial activities, which was validated by comparative proteomics of the C. cinerea secretome. Induced members of two putative antibacterial peptide and protein families in C. cinerea, the cysteine-stabilized αß-defensins (Csαß-defensins) and the GH24-type lysozymes, were purified, and their antibacterial activity was confirmed. These results provide compelling evidence that fungi are able to recognize the presence of bacteria and respond with the expression of an arsenal of secreted antibacterial peptides and proteins.

Assessment of biotechnologically relevant characteristics of heterologous hemoglobins in E. coli.

The use of the heterologous bacterial hemoglobin (VHb) from Vitreoscilla to enhance growth and productivity of Escherichia coli under conditions of oxygen limitation has been one of the foremost examples of metabolic engineering. Although VHb has earned its merits during the last two decades by providing enhanced physiological enhancements to organisms from all kingdoms of life, it has been the candidate of choice primarily for historical reasons. Findings made during the last years, however, suggest that hemoglobin and flavohemoglobin proteins from bacterial species other than Vitreoscilla or artificially generated mutant proteins or fusion variants of hemoglobins and flavohemoglobins may be better suited for use in biotechnological processes. This account provides guidelines for the assessment of biotechnologically relevant characteristics conferred by such novel heterologous hemoglobins and flavohemoglobins in E. coli.

Endogenous PttHb1 and PttTrHb, and heterologous Vitreoscilla vhb haemoglobin gene expression in hybrid aspen roots with ectomycorrhizal interaction.

Present knowledge on plant non-symbiotic class-1 (Hb1) and truncated (TrHb) haemoglobin genes is almost entirely based on herbaceous species while the corresponding tree haemoglobin genes are not well known. The function of these genes has recently been linked with endosymbioses between plants and microbes. In this work, the coding sequences of hybrid aspen (Populus tremulaxtremuloides) PttHb1 and PttTrHb were characterized, indicating that the key residues of haem and ligand binding of both genes were conserved in the deduced amino acid sequences. The expression of PttHb1 and PttTrHb was examined in parallel with that of the heterologous Vitreoscilla haemoglobin gene (vhb) during ectomycorrhiza/ectomycorrhizal (ECM) interaction. Both ECM fungi studied, Leccinum populinum and Xerocomus subtomentosus, enhanced root formation and subsequent growth of roots of all hybrid aspen lines, but only L. populinum was able to form mycorrhizas. Real-time PCR results show that the dual culture with the ECM fungus, with or without emergence of symbiotic structures, increased the expression of both PttHb1 and PttTrHb in the roots of non-transgenic hybrid aspens. PttHb1 and PttTrHb had expression peaks 5 h and 2 d after inoculation, respectively, pointing to different functions for these genes during interaction with root growth-improving fungi. In contrast, ECM fungi were not able to enhance the expression of hybrid aspen endogenous haemoglobin genes in the VHb lines, which may be a consequence of the compensating action of heterologous haemoglobin.

Analysis of the contribution of the globin and reductase domains to the ligand-binding properties of bacterial haemoglobins.

Bacterial Hbs (haemoglobins), like VHb (Vitreoscilla sp. Hb), and flavoHbs (flavohaemoglobins), such as FHP (Ralstonia eutropha flavoHb), have different autoxidation and ligand-binding rates. To determine the influence of each domain of flavoHbs on ligand binding, we have studied the kinetic ligand-binding properties of oxygen, carbon monoxide and nitric oxide to the chimaeric proteins, FHPg (truncated form of FHP comprising the globin domain alone) and VHb-Red (fusion protein between VHb and the C-terminal reductase domain of FHP) and compared them with those of their natural counterparts, FHP and VHb. Moreover, we also analysed polarity and solvent accessibility to the haem pocket of these proteins. The rate constants for the engineered proteins, VHb-Red and FHPg, do not differ significantly from those of their natural counterparts, VHb and FHP respectively. Our results suggest that the globin domain structure controls the reactivity towards oxygen, carbon monoxide and nitric oxide. The presence or absence of a reductase domain does not affect the affinity to these ligands.

Impact of the small RNA RyhB on growth, physiology and heterologous protein expression in Escherichia coli.

The small noncoding RNA RyhB is a regulator of iron homeostasis in Escherichia coli. During iron limitation, it downregulates the expression of a number of iron-containing proteins, including enzymes of the tricarboxylic acid cycle and the respiratory chain. Because this infers a potential for RyhB to limit energy metabolism and biosynthetic capacity, the effect of knocking out ryhB on the physiology and heterologous protein productivity of E. coli has been analyzed. During iron limitation, induced either through insufficient extracellular supply or through overexpression of an iron-containing protein, ryhB mutants showed unaltered growth and substrate consumption. They did, however, exhibit significantly lowered acetate production rates. Plasmid-based expression of green fluorescent protein and the heterologous Vitreoscilla hemoglobin VHb was negatively affected by the ryhB knock-out.

Globin-expression postpones onset of stationary phase specific gene expression in Escherichia coli.

We have analyzed gene expression of Escherichia coli MG1655 expressing native and engineered bacterial globin proteins, in order to identify the molecular mechanisms leading to the improved phenotypical traits relative to control cells under oxygen-limited conditions. Regulated expression of hemoglobin and flavohemoglobin proteins postponed the onset of rpoS expression relative to plasmid bearing control cells. This change in expression pattern coincided with the expression pattern of stationary-phase specific genes including sigma(S)-dependent and sigma(S)-independent genes. Furthermore, several genes known to affect rpoS transcription, rpoS mRNA stability and sigma(S) turnover were regulated in such a manner as to ultimately lower the cellular level of sigma(S) in all globin-expressing strains. In a strain harboring an rpoS-lacZ fusion, lacZ expression correlated with acetate accumulation, a metabolite that is known to activate rpoS transcription, but not with growth. Therefore, we hypothesize that reduced excretion of acetate in globin expressing cells prevents induction of stationary phase specific genes. Additionally, several genes responding to carbon starvation (e.g. csrAB, cstA, sspA) were expressed at lower levels in globin-expressing cells. These findings are in good agreement with previous reports showing a more efficient energy household, i.e. also reduced glucose consumption, in hemoglobin- and flavohemoglobin-expressing cells relative to controls.

Characterization of heterologous hemoglobin and flavohemoglobin promoter regulation in Escherichia coli.

Bacterial hemoglobins and flavohemoglobins have been used to improve cell growth and productivity in biotechnological applications. The expression of globin genes can be induced by reducing the oxygen supply or applying external stressors, which provide a simple and inexpensive mechanism for induction of heterologous protein production. It is in the interest of the biotechnological industry to seek new promoters, which are non-patented, cheap and simple to induce. Therefore, new globin gene promoters have been isolated from Campylobacter jejuni, Bacillus subtilis, Deinococcus radiodurans, Streptomyces coelicolor, and Salmonella typhi. The goal was to obtain insights about the regulation mechanisms of these promoters in Escherichia coli using in silico and experimental methods. The recognition of these promoters by the E. coli transcriptional machinery was first analyzed by computational methods. Computer analysis revealed that all the promoters, except the promoter of S. coelicolor, should be functional in E. coli and most of them also contain putative binding sites for ArcA, CRP, and FNR global regulators. Furthermore, the expression profiles of the promoters fused to the chloramphenicol acetyl transferase gene were analyzed under various conditions using E. coli mutants devoid of regulatory molecules. In vivo regulation studies of globin promoters mainly verified the in silico predictions.

Heterologous expression of Vitreoscilla hemoglobin (VHb) and cultivation conditions affect the alkaloid profile of Hyoscyamus muticus hairy roots.

Fast-growing hairy root cultures of Hyoscyamus muticus induced by Agrobacterium rhizogenes offer a potential production system for tropane alkaloids. Oxygen deficiency has been shown to limit growth and biomass accumulation of hairy roots, whereas little experimental data is available on the effect of oxygen on alkaloid production. We have investigated the effect of Vitreoscilla hemoglobin (VHb) expression and cultivation conditions on the complete alkaloid profile of H. muticus hairy roots in shake flasks and in a laboratory scale bioreactor. We optimized the growth medium composition and studied the effects of sucrose, ammonium, nitrate, and phosphate on growth and alkaloid production. Maximum biomass accumulation was achieved with the highest and maximum hyoscyamine content with the lowest sucrose concentration. The optimum nitrate concentration for growth was higher for the VHb line than the control. Neither VHb expression nor aeration improved the hyoscyamine content significantly, thus suggesting that hyoscyamine biosynthesis is not limited by oxygen availability. Interestingly, the effect of VHb expression on the alkaloid profile was slightly different from that of aeration. VHb expression did not affect the concentrations of cuscohygrine, which was increased by aeration. Therefore, the effect of VHb is probably not related only to its ability to increase the intracellular effective oxygen concentration.

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology.

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.

Dual targeted poplar ferredoxin NADP(+) oxidoreductase interacts with hemoglobin 1.

Previous reports have connected non-symbiotic and truncated hemoglobins (Hbs) to metabolism of nitric oxide (NO), an important signalling molecule involved in wood formation. We have studied the capability of poplar (Populus tremula × tremuloides) Hbs PttHb1 and PttTrHb proteins alone or with a flavin-protein reductase to relieve NO cytotoxicity in living cells. Complementation tests in a Hb-deficient, NO-sensitive yeast (Saccharomyces cerevisiae) Δyhb1 mutant showed that neither PttHb1 nor PttTrHb alone protected cells against NO. To study the ability of Hbs to interact with a reductase, ferredoxin NADP(+) oxidoreductase PtthFNR was characterized by sequencing and proteomics. To date, by far the greatest number of the known dual-targeted plant proteins are directed to chloroplasts and mitochondria. We discovered a novel variant of hFNR that lacks the plastid presequence and resides in cytosol. The coexpression of PttHb1 and PtthFNR partially restored NO resistance of the yeast Δyhb1 mutant, whereas PttTrHb coexpressed with PtthFNR failed to rescue growth. YFP fusion proteins confirmed the interaction between PttHb1 and PtthFNR in plant cells. The structural modelling results indicate that PttHb1 and PtthFNR are able to interact as NO dioxygenase. This is the first report on dual targeting of central plant enzyme FNR to plastids and cytosol.

Nitric oxide detoxification--a new era for bacterial globins in biotechnology?

For more than a decade, the expression of Vitreoscilla hemoglobin (VHb) has been used to improve the growth and/or productivity of various organisms that are important for the production of valuable metabolites and recombinant proteins by biotechnological processes. Extensive experimental data have shown that VHb enhances the energy status of the cell under oxygen-limited conditions, presumably by improving the supply of intracellular oxygen. Recently, bacterial globin proteins have gained more attention in research because of their ability to detoxify nitric oxide (NO) in vivo. These new results have increased our knowledge, encouraging us to reconsider the role of VHb in vivo. The expression of heterologous globins might improve cellular protection against nitrosative stress under oxygen-limited conditions.

Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro.

Targeted expression of Vitreoscilla haemoglobin (VHb) has been analysed in Nicotiana tabacum plants and suspension cultures under various growth and stress conditions. VHb localization to different cell compartments (cytoplasm, chloroplast and mitochondria) was successful, as judged by signal peptide cleavage. The presence of VHb in subcellular compartments did not result in phenotypical differences between these plant lines. In contrast with previous reports, we were unable to discern any significant changes in growth and other phenotypical characteristics between VHb-expressing and transformed control plants under standard growth conditions. When exposed to nitrosative stress, growth of VHb-expressing cultures was less affected relative to transformed controls. Furthermore, a diminished inactivation of the NO-sensitive enzyme aconitase was observed in the presence of VHb. In contrast, no protective effect of VHb expression against oxidative stress could be detected.

Expression of Vitreoscilla haemoglobin in hybrid aspen (Populus tremula x tremuloides).

We describe the first ever expression of Vitreoscilla haemoglobin (VHb) in an economically important boreal woody plant hybrid aspen (Populus tremula x tremuloides). VHb has mainly been expressed in biotechnologically important unicellular organisms of both prokaryotic and eukaryotic origin. VHb expression, in this study, was analysed under different greenhouse cultivation conditions and under elevated UV-B illumination. Microscope analyses of leaves grown under optimized conditions revealed significant differences both in cell structure and size when the transgenic VHb lines were compared with the control lines. VHb lines displayed a higher relative volume of mitochondria and a significantly enhanced accumulation of starch in chloroplasts, all of which pointed towards changes in cellular energy production. Under elevated UV-B illumination, the differences between VHb lines became evident. Some specific VHb lines had elevated levels of total flavonoids, individual quercetin, kaempferol- and myricetin-derivatives relative to controls and other transgenic lines. This observation may reflect the availability of extra energy resources for secondary metabolite production and possibly an enhanced protection ability of these transgenic lines against UV-B illumination. Thus, all these findings point to changes in the energy metabolism of VHb lines. In the cultivation conditions tested this observation did not, however, result in a general improvement of elongation growth.