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Elongation factor P (EF-P) is an essential protein that stimulates the formation of the first peptide bond in protein synthesis. Here we report the crystal structure of EF-P bound to the Thermus thermophilus 705 ribosome along with the initiator transfer RNA N-formyl-methionyl-tRNAi (fMet-tRNAifMET and a short piece of messenger RNA (mRNA) at a resolution of 3.5 angstroms EF-P hinds to a site located between the binding site fur the peptidyl tRNA (P site) and the exiting tRNA (E site), it spans both ribosomal subunits with its amino terminal domain positioned adjacent to the aminoacyl acceptor stem and its carboxyl terminal domain positioned next to the anticodon stem-loop of the P site--bound initiator tRNA. Domain II of EF-P interacts with the ribosomal protein L1, which results in the largest movement of the L1 stalk that has been observed in the absence of ratcheting of the ribosomal subunits. EF-P facilitates the proper positioning of the fMet-tRNAifMET for the formation of the first peptide bond during translation initiation. [ABSTRACT FROM AUTHOR]

Abstract: Plant viruses can be genetically modified to produce chimeric virus particles (CVPs) carrying heterologous peptides. The efficacy of plant-produced CVPs in inducing antibody responses specific to the displayed peptide has been extensively demonstrated. To determine if plants can be used to produce CVPs able to activate peptide-specific major histocompatibility complex (MHC) class I-restricted CD8+ T cells, potato virus X (PVX) has been engineered to display the H-2Db-restricted epitope ASNENMETM of influenza A virus nucleoprotein (NP). Engineering criteria were devised to comply not only with plant virus genetic stability and infectivity but also with antigen processing rules. The immunological properties of different doses of endotoxin-free preparations of CVPs or unmodified PVX have been evaluated by s.c. immunizing C57BL/6J mice and testing at different time intervals splenocyte responses by interferon γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay. These experiments demonstrated that CVPs activate ASNENMTEM-specific CD8+ T cells. Remarkably, the best response was achieved without adjuvant co-delivery. These results represent the proof of concept that well-designed plant virus carriers of epitopes produced in plant can reasonably be used into peptide vaccine formulations aimed to activate cell-mediated immune responses. [Copyright &y& Elsevier]

Background: Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters. Methodology/Principal Findings: Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides. Conclusions/Significance: In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to evaluate the antibody responses to different VlsE protein IR6 peptide variants and the synthetic C6 peptide in acute and convalescent (2–3 and 6 months) serum samples from Swedish patients with clinical erythema migrans (EM). Serum samples were prospectively collected from 148 patients with EM and compared to serum samples obtained from 200 healthy blood donors. The IgG responses to 3 IR6 peptide variants originating from Borrelia burgdorferi (B. burgdorferi) sensu stricto, B. garinii, and B. afzelii were measured by enzyme-linked immunosorbent assays (ELISAs) and compared to a commercial C6 peptide ELISA. Seropositivity rate in the IR6 or C6 peptide ELISAs ranged from 32% to 58% at presentation, 30–52% after 2–3 months, and 20–36% after 6 months. At presentation, positive antibodies in any of the 4 ELISAs were found in 66%. In 7/52 (13%), C6-negative EM cases, serological reaction was found to the B. burgdorferi sensu stricto-derived IR6 peptide. In patients reporting previous LB compared to those without previous LB, significantly higher seropositivity rates were noted for all IR6 peptides, but not for the C6 peptide. In the serology of EM in Europe, C6 ELISA does not seem to cover all cases. An ELISA using a mixture of B. burgdorferi sensu stricto IR6 peptide and the C6 peptide could be of value in the serodiagnosis of LB in Europe. Further studies on combinations of variant IR6 peptides and the C6 peptide in other manifestations of LB are needed to address this issue. [Copyright &y& Elsevier]

This study reports for the first time the presence of Anabaenopsis arnoldi blooms in Saudi freshwaters. This species has been investigated with high cell densities (3.8 × 103–264 × 103 cells mL−1) during June–November 2007 in Tendaha Lake, one of the major freshwater sources in Saudi Arabia. High temperature and conductivity, and a high concentration of phosphate, and low nitrate concentrations may have contributed to the formation of these blooms. The blooms were found to produce microcystins (MCYSTs) at concentrations up to 364 μg g−1 dry weight as detected by an enzyme-linked immunosorbent assay. MCYSTs were also detected in the raw and treated water of the lake at concentrations (1.6–8.3 and 0.33–1.6 μg L−1, respectively) exceeding the World Health Organization guideline level of 1 μg L−1 for these toxins. HPLC analysis revealed that the extracts of A. arnoldi blooms contained MCYST-RR, -YR and two unidentified MCYSTs, but a pure culture of A. arnoldi isolated from Tendaha Lake during the present study produced MCYST-RR and –YR only. This is the first study to report MCYST production by A. arnoldi. Therefore, this cyanobacterium should be taken into consideration during monitoring of toxic cyanobacterial blooms in drinking and recreational water sources in the world, particularly arid and semi-arid countries including Saudi Arabia. [ABSTRACT FROM AUTHOR]

Abstract: Enniatins are cyclic peptides isolated from bacteria, fungi, and plants, with numerous biological effects on animal systems. Recently, we have reported that certain enniatins (ENs), such as EN H and EN MK1688, have cytotoxic effects on several adenocarcinoma cell lines. In an effort to understand the mechanism behind their cytotoxicity, we investigated whether ENs can induce apoptosis in human colorectal carcinoma cells (HCT-15 cells). Treatment with the ENs H and MK1688 resulted in an alteration of cellular and nuclear morphology, leading to an increase in the number of the cells with apoptotic nuclei (seen as condensed or fragmented nuclei). Furthermore, it was observed that cellular DNA fragmentation increased in a dose-dependent manner in EN treated cells. These cells have elevated activity levels for caspase-3, the enzyme responsible for initiating cell death, compared with the untreated cells. Normal caspase-3 activity levels were observed when Z-VAD-FMK, a caspase inhibitor, was added simultaneously with the ENs. Based on our results, we propose that the new ENs H and MK1688 induce cytotoxicity via an apoptotic pathway. [Copyright &y& Elsevier]

Abstract: Adenosine triphosphate (ATP) is necessary in the enzymatic production of glutathione (GSH). Our aim was to improve GSH production by increasing the efficiency of ATP regeneration in a coupled system. Previous results suggested that low GSH production in the coupled system is due to the irreversible transformation of adenosine (Ado) to hypoxanthine (Hx) via inosine (Ino) pathway in Escherichia coli JM109 (pBV03). In this study, to block the transformation of Ado into Hx and enhance GSH production, a coupled ATP regeneration system was constructed with adenosine deaminase-deficient recombinant E. coli JW1615 (pBV03) and Saccharomyces cerevisiae WSH2. GSH production was improved (2.94-fold of the control), and ATP regeneration reaction was established in the coupled system. The results are applicable to ATP regeneration in other microbial processes. [Copyright &y& Elsevier]

Oligopeptide-binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4–35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open- and closed-liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino-acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline-rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline-rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino-acid composition rather than sequence. [ABSTRACT FROM AUTHOR]

The article focuses on the study of hetR and patS in Anabaena 7120. The hetR is served as the significant regulator of heterocyst development. The study reveals the key role of hetR proteins in heterocyst development. It recommends that heterocyst-forming cyanobacteria might appear between 2450 and Ma. It discovers that the peptide encoded by a gene next to hetR was similar to PatS functional pentapeptide among filamentous cyanobacteria. It concludes that the two genes, hetR and patS, could control heterocyst development.

Abstract: δ-hemolysin is a hemolytic peptide produced by Staphylococcus, and it has been studied for nearly 50 years. Therefore, it has become a model in the study of peptides interacting with membranes. In this review, we report some recent findings and compare them with previous works. δ-hemolysin is a 26 amino acid peptide, somewhat hydrophobic and presenting a zero net charge. Study of its structure has shown that δ-hemolysin is α-helical and amphipathic, such as many antimicrobial peptides (e.g. magainin and melittin). However, δ-hemolysin had not displayed any reported antimicrobial activity until a recent publication showed its high potency against Legionella. Its mode of action is based on direct interaction with target membranes. In accordance with its concentration, δ-hemolysin may slightly perturb a membrane or lead to cell lysis. Peptide charge plays an important role in its interaction with membranes, as is shown in the study of peptide variants. Some positively charged variants become highly hemolytic and even active against Escherichia coli and Staphylococcus aureus. Finally, it has recently been demonstrated that peptide preferentially binds to lipid-disordered domains. It has been postulated that as a result, enrichment in lipid-ordered domains might increase peptide concentration in lipid-disordered domains and thereby improve its activity. [Copyright &y& Elsevier]

Matrix effects on the microcystin-LR fluorescent immunoassay based on the evanescent wave all-fiber immunosensor (EWAI) and their elimination methods were studied. The results indicated that PBS and humic acid did not affect the monitoring of samples under the investigated conditions. When the pH was less than 6 or higher than 8, the fluorescence signals detected by immunosensor systems were obviously reduced with the decrease or increase of pH. When the pH ranged from 6 to 8, IC50 and the linear working range of MC-LR calculated from the detection curves were 1.01∼1.04 μg/L and 0.12∼10.5 μg/L, respectively, which was favourable for an MC-LR immunoassay. Low concentrations of Cu2+ rarely affected the detection performance of MC-LR. When the concentration of CuSO4 was higher than 5 mg/L, the fluorescence signal detected by EWAI clearly decreased, and when the concentration of CuSO4 was 10 mg/L, the fluorescence signal detected was reduced by 70%. The influence of Cu2+ on the immunoassay could effectively be compromised when chelating reagent EDTA was added to the pre-reaction mixture. [ABSTRACT FROM AUTHOR]

The article presents a clinical study on the potential medical use buforin II and rifampin on severe infections caused by acinetobacter baumannii sepsis using a rat model. It notes that acinetobacter baumannii is considered an important nosocomial pathogen and the increasing hospital oubreaks continues. It provides evidence for buforin II which shows considerable outcomes on sepsis survival.

Summary: Environmental factors account for 75% of the risk of developing multiple sclerosis (MS). Numerous infections have been suspected as environmental disease triggers, but none of them has consistently been incriminated, and it is unclear how so many different infections may play a role. We show that a microbial peptide, common to several major classes of bacteria, can induce MS-like disease in humanized mice by crossreacting with a T cell receptor (TCR) that also recognizes a peptide from myelin basic protein, a candidate MS autoantigen. Structural analysis demonstrates this crossreactivity is due to structural mimicry of a binding hotspot shared by self and microbial antigens, rather than to degenerate TCR recognition. Biophysical studies reveal that the autoreactive TCR binding affinity is markedly lower for the microbial (mimicry) peptide than for the autoantigenic peptide. Thus, these data suggest a possible explanation for the difficulty in incriminating individual infections in the development of MS. [Copyright &y& Elsevier]

Abstract: Streptococcus mutans (S. mutans) uses a quorum sensing (QS) signaling system, which is dependent on competence stimulating peptide (CSP), to regulate diverse physiological activities including bacteriocin production, genetic transformation, and biofilm formation. However, the mechanism of the QS system-induced biofilm formation remains unclear. Here, we demonstrated that the late-stage biofilm formation was increased by the addition of exogenous CSP in S. mutans. The numbers of dead cells in biofilms formed in presence of CSP was 64.5% higher than that without CSP after 12h (p <0.05) and 76.3% higher after 24h (p <0.05), the numbers of live cells in biofilms formed in presence of CSP were 89.3% higher than that without CSP after 24h (p <0.01). The expression of QS-associated genes was increased 3.4–5.3-fold by CSP in biofilms. Our results revealed that cell viability of S. mutans grown in biofilms is affected by the CSP-dependent QS system. [Copyright &y& Elsevier]

It is known that microcystin (MC) is subject to microbial degradation to provide three types of products, linearized MCLR (Adda-Glu-Mdha-Ala-Leu-MeAsp-Arg), tetrapeptide Adda-Glu-Mdha-Ala, and Adda. They can be readily detected by the usual HPLC, because they commonly have an Adda moiety with a diene and an absorption maximum at 238 nm as the chromophore. However, no other degradation products without such a chromophore have been isolated to date. In this study, cell preparation of a bacterium B-9 that can degrade MC and detection of the degradation products were devised. First, we regulated the B-9 hydrolytic activity by washing with sodium chloride solution to obtain a desired cell preparation, which permitted an additional intermediate and the final products of MCLR to be obtained. Second, the resulting products could be firmly identified using the advanced Marfey method with the aid of log D. As a result of these experiments, the following degradation products were further identified: a tetrapeptide, Adda-Glu-Mdha-Ala, tripeptides Adda-Glu-Mdha, Glu-Mdha-Ala, and Arg-MeAsp-Leu, a dipeptide, Glu-Mdha, and amino acids Adda, Arg, and methylamine derived from Mdha. The present study expands the hydrolytic activity of the B-9 strain, which can hydrolyze not only cyanobacterial cyclic peptides but also MC to the intermediates and final products. The established characterization method composed of the advanced Marfey method and log Dwould be a standard technique for the structural characterization of a mixture of amino acids and peptides. [ABSTRACT FROM AUTHOR]

This review reports on the effects of fermentation on the chemical constituents and antioxidant activity of plant-based food materials. Fermentation involves a series of reactions that modify the chemical components of the substrate. It could be considered a tool to increase the bioactive compounds and functional properties of food plant materials. Oxidative damage is key to the progression of many human diseases, and the production of antioxidant compounds by fermentation will be helpful to reduce the risk of these diseases. Fermentation also can improve antioxidant activity given its association with increased phytochemicals, antioxidant polysaccharides, and antioxidant peptides produced by microbial hydrolysis or biotransformation. Additionally, fermentation can encourage the breakdown of plant cell walls, which helps to liberate or produce various antioxidant compounds. Overall, results indicated that fermentation in many cases contributed to enhancing antioxidants' content and antioxidant capacity, supporting the fermentation use in the production of value-added functional food. This review provides an overview of the factors that impact the effects of fermentation on bioactive compound composition and antioxidant activity. The impacts of fermentation are summarized as a reference to its effects on food plant material. [ABSTRACT FROM AUTHOR]

Abstract: The twin-arginine translocation (Tat) system is a bacterial protein targeting pathway. Tat-targeted proteins display signal peptides containing a distinctive SRRxFLK ‘twin-arginine’ motif. The Escherichia coli trimethylamine N-oxide reductase (TorA) bears a bifunctional Tat signal peptide, which directs protein export and serves as a binding site for the TorD biosynthetic chaperone. Here, the physical interaction between TorD and the TorA signal peptide was investigated. A single substitution within the TorA signal peptide (L31Q) was sufficient to impair TorD binding. Screening of a random torD mutant library identified a variant TorD protein (Q7L) that displayed increased binding affinity for the TorA signal peptide. Structured summary: MINT-6796225, MINT-6796279, MINT-6796298, MINT-6796315, MINT-6796332, MINT-6796350, MINT-6796371, MINT-6796391, MINT-6796410, MINT-6796429, MINT-6796446, MINT-6796460: TorD (uniprotkb:P36662) physically interacts (MI:0218) with TorA (uniprotkb:P33225) by two-hybrid (MI:0018) MINT-6796515, MINT-6796563, MINT-6796589, MINT-6796624, MINT-6796648, MINT-6796666, MINT-6796770, MINT-6796750: TorA (uniprotkb:P33225) binds (MI:0407) to TorD (uniprotkb:P36662) by isothermal titration calorimetry (MI:0065) [Copyright &y& Elsevier]

Model class A amphipathic helical peptides mimic several properties of apolipoprotein A-I (apoA-I), the major protein component of high density lipoproteins. Previously, we reported the NMR structures of Ac-18A-NH2 (renamed as 2F because of two phenylalanines), the base-line model class A amphipathic helical peptide in the presence of lipid (Mishra, V. K., Anantharamaiah, G. M., Segrest, J. P., Palgunachari, M. N., Chaddha, M., Simon Sham, S. W., and Krishna, N. R. (2006) J Biol. Chem. 281, 6511-6519). Substitution of two Leu residues on the nonpolar face (Leu³ and Leu14) with Phe residues produced the peptide 4F (so named because of four phenylalanines), which has been extensively studied for its anti-inflammatory and antiatherogenic properties. Like 2F, 4F also forms discoidal nascent high density lipoprotein-like particles with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Since subtle structural changes in the peptide-lipid complexes have been shown to be responsible for their antiatherogenic properties, we undertook high resolution NMR studies to deduce detailed structure of 4F in 4F·DMPC discs. Like 2F, 4F adopts a well defined amphipathic α-helical structure in association with the lipid at a 1:1 peptide/lipid weight ratio. Nuclear Overhauser effect (NOE) spectroscopy revealed a number of intermolecular close contacts between the aromatic residues in the hydrophobic face of the helix and the lipid acyl chain protons. Similar to 2F, the pattern of observed peptide-lipid NOEs is consistent with a parallel orientation of the amphipathic α helix, with respect to the plane of the lipid bilayer, on the edge of the disc (the belt model). However, in contrast to 2F in 2F·DMPC, 4F in the 4F·DMPC complex is located closer to the lipid headgroup as evidenced by a number of NOEs between 4F and DMPC headgroup protons. These NOEs are absent in the 2F·DMPC complex. In addition, the conformation of the DMPC sn-3 chain in 4F·DMPC complex is different than in the 2F·DMPC complex as evidenced by the NOE between lipid 2.CH and βCH2 protons in 4F·MPC, but not in 2F·MPC, complex. Based on the results of this study, we infer that the antiatherogenic properties of 4F may result from its preferential interaction with lipid headgroups. [ABSTRACT FROM AUTHOR]

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids. [ABSTRACT FROM AUTHOR]

The microcystin (MC) content and cyanobacterial community structure of Antarctic microbial mat samples collected from 40 ponds, lakes, and hydroterrestrial environments were investigated. Samples were collected from Bratina Island and four of the Dry Valleys, Wright, Victoria, Miers, and Marshall. Enzyme-linked immunosorbent assays (ELISAs), liquid chromatography-mass spectrometry (LC-MS), and protein phosphatase 2A (PP-2A) inhibition assays resulted in the identification of low levels (1 to 16 mg/kg [dry weight]) of MCs in all samples. A plot of indicative potencies of MCs (PP-2A inhibition assay/ELISA ratio) versus total MCs (ELISA) showed a general decrease in potency, as total MC levels increased, and a clustering of values from discrete geographic locations. LC-tandem MS analysis on selected samples identified eight novel MC congeners. The low-energy collisional activation spectra were consistent with variants of [u-Asp3] MC-RR and ED-Asp3] MC-LR containing glycine [Gly'] rather than alanine and combinations of homoarginine [hAr2] or acetyldemethyl 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyl-4,6-decadienoic acid (acetyldemethyl ADDA) [ADMAdda'] substitutions. Nostoc sp. was identified as a MC producer using PCR amplification of a region of the 16S rRNA gene and the aminotransferase domain of the mcyE gene. Automated ribosomal intergenic spacer analysis (ARISA) was undertaken to enable a comparison of cyanobacterial mat community structure from distant geographical locations. Two-dimensional multidimensional scaling ordination analysis of the ARISA data showed that in general, samples from the same geographic location tended to cluster together. ARISA also enabled the putative identification of the MC-producing Nostoc sp. from multiple samples. [ABSTRACT FROM AUTHOR]

PEGylation of peptide drugs prolongs their circulating lifetimes in plasma. However, PEGylation can produce a decrease in the in vitro bioactivity. Longer poly(ethylene glycol) (PEG) chains are favourable for circulating lifetimes but unfavourable for in vitro bioactivities. In order to circumvent the conflicting effects of PEG length, a hydrophobic peptide, using an antimicrobial peptide as a model, was PEGylated with short PEG chains. The PEGylated peptides self-assembled in aqueous solution into micelles with PEG shell and peptide core. In these micelles, the core peptides were protected by the shell, thus reducing proteolytic degradation. Meanwhile, most of the in vitro antimicrobial activities still remained due to the short PEG chain attached. The stabilities of the PEGylated peptides were much higher than that of the unPEGylated peptides in the presence of chymotrypsin and serum. The antimicrobial activities of the PEGylated peptides in the presence of serum, an ex vivo assay, were much higher than that of the unPEGylated peptide. [ABSTRACT FROM PUBLISHER]

Mammalian ci-defensins all have a conserved triple-stranded α-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse α-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia co/i ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid! polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C1 A)-Crp4, native, β-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration. [ABSTRACT FROM AUTHOR]

Summary: The N-end rule targets specific proteins for destruction in prokaryotes and eukaryotes. Here, we report a crystal structure of a bacterial N-end rule adaptor, ClpS, bound to a peptide mimic of an N-end rule substrate. This structure, which was solved at a resolution of 1.15 Å, reveals specific recognition of the peptide α-amino group via hydrogen bonding and shows that the peptide''s N-terminal tyrosine side chain is buried in a deep hydrophobic cleft that pre-exists on the surface of ClpS. The adaptor side chains that contact the peptide''s N-terminal residue are highly conserved in orthologs and in E3 ubiquitin ligases that mediate eukaryotic N-end rule recognition. We show that mutation of critical ClpS contact residues abrogates substrate delivery to and degradation by the AAA+ protease ClpAP, demonstrate that modification of the hydrophobic pocket results in altered N-end rule specificity, and discuss functional implications for the mechanism of substrate delivery. [Copyright &y& Elsevier]

Microcystins (MCs) are a family of related cyclic hepatotoxic heptapeptides, of which more than 70 types have been identified. The chemically unique nature of the C20 β-amino acid, (2S,3S,8S,9S)-3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda), portion of the MCs has been exploited to develop a strategy to analyze the entirety. Oxidation of MCs causes the cleavage of MC Adda to form 2-methyl-3-methoxy-4-phenylbutanoic acid (MMPB). In the present study, we investigated the kinetics of MMPB produced by oxidation of the most-often-studied MC variant, MC-LR (L = leucine, R = arginine), with permanganate-periodate. This investigation allowed insight regarding the influence of the reaction conditions (concentration of the reactants, temperature, and pH) on the conversion rate. The results indicated that the reaction was second order overall and first order with respect to both permanganate and MC-LR. The second-order rate constant ranged from 0.66 to 1.35 M/s at temperatures from 10 to 30°C, and the activation energy was 24.44 kJ/mol. The rates of MMPB production can be accelerated through increasing reaction temperature and oxidant concentration, and sufficient periodate is necessary for the formation of MMPB. The initial reaction rate under alkaline and neutral conditions is higher than that under acidic conditions, but the former decreases faster than the latter except under weakly acidic conditions. These results provided new insight concerning selection of the permanganate-periodate concentration, pH, and temperature needed for the oxidation of MCs with a high and stable yield of MMPB. [ABSTRACT FROM AUTHOR]

Viral fusion proteins mediate the merger of host and viral membranes during cell entry for all enveloped viruses. Baculovirus glycoprotein gp64 (gp64) is unusual in promoting entry into both insect and mammalian cells and is distinct from established class I and class II fusion proteins. We report the crystal structure of its postfusion form, which explains a number of gp64′s biological properties including its cellular promiscuity, identifies the fusion peptides and shows it to be the third representative of a new class (III) of fusion proteins with unexpected structural homology with vesicular stomatitis virus G and herpes simplex virus type 1 gB proteins. We show that domains of class III proteins have counterparts in both class I and II proteins, suggesting that all these viral fusion machines are structurally more related than previously thought. [ABSTRACT FROM AUTHOR]

Abstract  This study evaluated the killing rate as well as the antimycoplasmal effect of surfactins isolated from Bacillus subtilis complex BC1212, either alone or in combination with various antibacterials against Mycoplasma hyopneumoniae. Prior to the killing rate and the combination effect studies of surfactins, the minimal inhibitory concentrations (MICs) of various antibacterials and surfactins (consisting of surfactins A, B, C, and D) against M. hyopneumoniae were investigated. The MIC of all surfactins was found to be 64 μg/ml. The MICs of colistin (COL), norfloxacin (NFX), oxytetracycline (OTC), streptomycin (SM) and tiamulin (TIA) were >256, 0.063, 0.025, 4, and 0.015 μg/ml, respectively. In the killing rate curve studies, surfactin C at 2× MIC and 4× MIC concentrations was found to reduce viability by >3 log10 c.c.u./ml within 2–4 h of incubation. Combination of surfactin C with other antibacterials showed additive interaction from the viewpoint of fractional inhibitory concentration (FIC) index of >0.5 but ≤2 as a borderline. Taking all results into consideration, surfactin C may be useful as a preventive or therapeutic adjuvant in the pathogenesis of mycoplasmal infection. [ABSTRACT FROM AUTHOR]

Cyanobacteria are capable of producing multiple microcystin variants simultaneously. The mechanisms that determine the composition of microcystin variants in cyanobacteria are still debated. [Asp3]microcystin-RR contains arginine at the position where the more toxic [Asp3]microcystin-LR incorporates leucine. We cultured the filamentous cyanobacterium Planktothrix agardhii strain 126/3 with and without external addition of leucine and arginine. Addition of leucine to the growth medium resulted in a strong increase of the [Asp3]microcystin LR/RR ratio, while addition of arginine resulted in a decrease. This demonstrates that amino acid availability plays a role in the synthesis of different microcystin variants. Environmental changes affecting cell metabolism may cause differences in the intracellular availability of leucine and arginine, which can thus affect the production of microcystin variants. Because leucine contains one nitrogen atom while arginine contains four nitrogen atoms, we hypothesized that low nitrogen availability might shift the amino acid composition in favor of leucine, which might explain seasonal increases in the [Asp3]microcystin LR/RR ratio in natural populations. However, when a continuous culture of P. agardhii was shifted from nitrogen-saturated to a nitrogen-limited mineral medium, leucine and arginine concentrations decreased, but the leucine/arginine ratio did not change. Accordingly, while the total microcystin concentration of the cells decreased, we did not observe changes in the [Asp3]microcystin LR/RR ratio in response to nitrogen limitation. [ABSTRACT FROM AUTHOR]

The article discusses findings of a study, which systematically map intracellular and extracellular microcystin concentrations in eutrophic portions of the Great Lakes region during a season when Microcystis was abundant. It also investigated the relationship between microcystin concentrations and both Microcystis colony abundance and the frequency of toxic genotypes in natural Microcystis populations. Several factors that may affect or influence Microcystis growth include nutrients, light, and shifts in community composition toward the predominance of toxic strains.

The article discusses findings of a study which examines the expression of protein phosphatases 2A (PP2A), CHOP, Bax, Bcl-2, and p53 proteins in microcystin-LR (MCLR)-induced apoptopsis in FL cells. Considered as the most frequently studied, MCLR is also the most toxic in the group of MC. Inhibiting the activity of protein phosphatases 1 (PP1) and 2A (PP2A) is one of the mechanisms of MC toxicity, and this leads to increase of protein phosphorylation which is directly related to their cytotoxic effects and tumor-promoting activity.

A certified reference material (CRM) for microcystins has been prepared by the National Institute for Environmental Studies (NIES). Microcystins are hepatotoxic cyclic peptides produced by cyanobacteria in eutrophic water bodies. At least seven microcystin variants were found by HPLC analysis of the NIES CRM, of which [Dha7]microcystin-RR and -LR were the major microcystins present. Because of the lack of available standards we determined the total microcystin concentration in the CRM by the MMPB method, and elucidated the structures of the main individual microcystin variants following their isolation. Analyses of NMR and MS spectra indicated that the remaining minor variants in the CRM were [ D-Asp3, Dha7]microcystin-RR and -LR, and [Dha7]microcystin-YR, -ThTyrR, and -HilR. The CRM is valuable not only as a standard material for the quantitation of total microcystins but also for the identification of individual [Dha7]microcystin variants. [ABSTRACT FROM AUTHOR]

During the summer of 2006, the western basin of Lake Erie experienced a bloom of the toxigenic cyanobacterium Microcystis aeruginosa. Across 11 sites, intracellular, particulate-bound microcystin levels in the seston increased to levels that exceeded World Health Organization guidelines for drinking water exposure (1 µg toxin·L–1). In contrast, toxin concentrations in yellow perch (Perca flavescens) muscle tissue (n = 68) declined from June to August, were negatively related to algal toxin levels, and never exceeded a conservative chronic exposure concentration estimated using proposed United States Environmental Protection Agency (US EPA) guidelines. Microcystin concentrations in yellow perch liver exceeded US EPA chronic exposure guidelines, were on average 125 times higher than muscle toxin concentrations per unit dry weight, and varied little throughout the summer. With current guidelines, humans do not appear to be at risk when consuming the muscle tissue of Lake Erie yellow perch collected during large-scale cyanobacterial blooms. However, this study highlights the need for a better understanding of the trophic transfer of cyanobacterial toxins through aquatic food webs in diverse ecosystems with an emphasis on understanding if these compounds could accumulate sufficiently to affect human health. [ABSTRACT FROM AUTHOR]

Immunity genes are activated in the Drosophila fat body by Rel and GATA transcription factors. Here, we present evidence that an additional regulatory factor, deformed epidermal autoregulatory factor-1 (DEAF-1), also contributes to the immune response and is specifically important for the induction of two genes encoding antimicrobial peptides, Metchnikowin (Mtk) and Drosomycin (Drs). The systematic mutagenesis of a minimal Mtk 5' enhancer identified a sequence motif essential for both a response to LPS preparations in 52 cells and activation in the larval fat body in response to bacterial infection. Using affinity chromatography coupled to multidimensional protein identification technology (MudPlT), we identified DEAF-1 as a candidate regulator. DEAF-1 activates the expression of Mtk and Drs promoter-luciferase fusion genes in S2 cells. SELEX assays and footprinting data indicate that DEAF-1 binds to and activates Mtk and Drs regulatory DNAs via a TTCGGBT motif. The insertion of this motif into the Diptericin (Dpt) regulatory region confers DEAF-1 responsiveness to this normally DEAF-1-independent enhancer. The coexpression of DEAF-1 with Dorsal, Dif, and Relish results in the synergistic activation of transcription. We propose that DEAF-1 is a regulator of Drosophila immunity. [ABSTRACT FROM AUTHOR]

Abstract  Cyanobacteria are able to produce a variety of secondary metabolites such as the microcystins. The ecological role of microcystins for the cyanobacteria themselves and in the aquatic ecosystem is not well understood. The aim of this study is to evaluate if microcystins might be used as a communication tool for interspecies cyanobacterial communication via the promotion of oxidative stress. Reactive oxygen species (ROS) are known to be used as plant signals. The main questions relate to the promotion of oxidative stress in Synechocystis sp. via exposure to the cyanobacterial toxins and the physiological effects. This study shows a few markers for oxidative stress, such as the antioxidative enzymes superoxide dismutase, peroxidases and catalases, and cell damage due to extensive generation of ROS leading to lipid peroxidation. End products of lipid peroxidation (malonaldehyde and 4-hydroxynonenal) are conjugated by glutathione S-transferase. [ABSTRACT FROM AUTHOR]

Occurrence of cyanobacteria and their potent toxins has imposed a risk to human health and raised concerns in drinking water treatment. In this study, the applicability of electrochemical oxidation on microcystin-LR produced by cyanobacteria was examined using Ti/RuO2 as anodes. The experiments focused on the variation and elimination of extracellular and intracellular microcystin-LR during the cyanobacterial inactivation by electrooxidation. The effects of applied current density, cell density, as well as electrolyte type on toxin degradation have been studied. The complete elimination of extracellular and intracellular microcystin-LR was achieved and 100% removal for total microcystin-LR was obtained at current density 5 mA/cm2. The results showed that when the cell densities decreased or the applied current densities increased, the efficiency of total microcystin-LR removal could be increased. More than 98% removal for total microcystin-LR was also achieved even if the cyanobacteria were inoculated in lake water or drinking water without adding any chemicals. It can be concluded that electro-oxidation of microcystin-LR is quite an efficient method, and can be applied to remove microcystin-LR from waters during the cyanobacterial inactivation. [ABSTRACT FROM AUTHOR]

The presence of cyanobacterial toxins, such as microcystins, in drinking water supplies is a major concern for water authorities worldwide due to possible adverse health effects associated with exposure to these compounds. Adsorption onto powdered activated carbon in the water treatment process offers an effective treatment option for the removal of these compounds. However, for the efficient use of powdered activated carbon, knowledge of the appropriate dose to apply is required. In this study the adsorption of two microcystin analogues, microcystin-LR and microcystin-LA, onto powdered activated carbon was studied for two waters. Analysis of batch kinetic and equilibrium adsorption data showed that the homogeneous surface diffusion model could successfully predict the adsorption kinetics of the two toxins. The application of the homogeneous surface diffusion model is extremely useful to water treatment plant operators as it can be used to predict the powdered activated carbon dose for the removal of algal toxins based on the analogue(s) present, influent toxin concentration and powdered activated carbon contact time. Adsorption was also found to be different for the two toxin analogues, and dependent on the water character. Microcystin-LR was much more easily removed than microcystin-LA in both waters, and adsorption of both analogues was lower in the water with a higher dissolved organic carbon concentration. [ABSTRACT FROM AUTHOR]

Apidaecins are heat-stable, nonhelical antibacterial peptides isolated from lymph fluid of the honeybee ( Apis mellifera). These peptides are active against a wide range of gram-negative bacteria and they are the most prominent components of the honeybee humoral defense against microbial invasion. In the present study, one isoform of apidaecin, apidaecin Ho, was expressed extracellularly in the food-grade bacterium Lactococcus lactis. Results showed that expression driven by the lactococcal nisA promoter and Usp45 signal peptide resulted in efficient secretion of apidaecin in L. lactis subsp. cremoris NZ9000. Recombinant apidaecin was purified by gel filtration and semipreparative RP-HPLC, and about 10 mg active recombinant apidaecin was obtained from 1,000 ml culture. This is the first report on the nisin-controlled extracellular production of active apidaecin in L. lacits. The expression and delivery of apidaecin in the food-grade L. lactis may provide a clue to facilitate the widespread application of apidaecin in the control and prevention of gram-negative bacteria infections of human and animals. [ABSTRACT FROM AUTHOR]

The article reports on the study of nonribosomal peptide synthetase (NRPS) and its role in the bassianolide synthesis in wood-rot fungus called Xylaria sp. BCC1067. It aims to identify the complete NRPS of the bassianolide biosynthesis through genetic approach. It isolates the biosynthetic gene of the bassianolide namely nrpsxy using the adenylation of NRPS. It uses gene disruption and functional analysis to identify the missing metabolite of the nrpsxy gene. The study also conducts chemical analysis and bioassay of bassianolide to reveal the metabolite's cyclooctadepsipeptide bassianolide.

The anti-microbial peptides β-defensins constitute a large family of innate immune effector molecules, conserved across a wide species range. In this paper, we describe a systematic search of the sequenced bovine genome to characterise this extensive gene family in Bos taurus, providing an insight into the pattern of conservation of β-defensin genes between species. We have sequenced a sub-set of these newly discovered bovine β-defensin genes and also report expression data for these genes across a range of tissues. We have synthesised the peptide product of one of these genes, bovine β-defensin 123, and found it to be a potent inhibitor of several pathogenic microbes, particularly Escherichia coli and Listeria monocytogenes. [ABSTRACT FROM AUTHOR]

Abstract: The persistence time and risk of microcystin-RR (MC-RR) in cropland via irrigation were investigated under laboratory conditions. In order to evaluate the efficiency of the potential adsorption and biodegradation of MC-RR in cropland and the persistence time of MC-RR for crop irrigation, high performance liquid chromatography (HPLC) was used to quantify the amount of MC-RR in solutions. Our study indicated that MC-RR could be adsorbed and biodegraded in cropland soils. MC-RR at 6.5mg/L could be completely degraded within 6 days with a lag phase of 1–2 days. In the presence of humic acid, the same amount of MC-RR could be degraded within 4 days without a lag phase. Accordingly, the persistence time of MC-RR in cropland soils should be about 6 days. This result also suggested the beneficial effects of the organic fertilizer utilization for the biodegradation of MC-RR in cropland soils. Our studies also demonstrated that MC-RR at low concentration (<10μg/L) could accelerate the growth of plants, while high concentration of MC-RR (>100μg/L) significantly inhibited the growth of plants. High sensitivity of the sprouting stage plants to MC-RR treatments as well as the strong inhibitory effects resulting from prolonged irrigation further indicated that this MC-RR growth-inhibition may vary with the duration of irrigation and life stage of the plants. [Copyright &y& Elsevier]

Background: Transgenic animals are widely used in biomedical research and biotechnology. Multicistronic constructs, in which several proteins are encoded by a single messenger RNA, are commonly used in genetically engineered animals. This is currently done by using an internal ribosomal entry site to separate the different coding regions. 2A peptides result in the cotranslational 'cleavage' of proteins and are an attractive alternative to the internal ribosomal entry site. They are more reliable than the internal ribosomal entry site and lead to expression of multiple cistrons at equimolar levels. They work in a wide variety of eukaryotic cells, but to date have not been demonstrated to function in transgenic mice in an inheritable manner. Results: To test 2A function in transgenic mice and uncover any possible toxicity of widespread expression of the 2A peptide, we made a bicistronic reporter construct containing the coding sequence for a membrane localised red fluorescent protein (Myr-TdTomato) and a nuclear localised green fluorescent protein (H2B-GFP), separated by a 2A sequence. When this reporter is transfected into HeLa cells, the two fluorescent proteins correctly localise to mutually exclusive cellular compartments, demonstrating that the bicistronic construct is a reliable readout of 2A function. The two fluorescent proteins also correctly localise when the reporter is electroporated into chick neural tube cells. We made two independent transgenic mouse lines that express the bicistronic reporter ubiquitously. For both lines, transgenic mice are born in Mendelian frequencies and are found to be healthy and fertile. Myr-TdTomato and H2B-GFP segregate to mutually exclusive cellular compartments in all tissues examined from a broad range of developmental stages, ranging from embryo to adult. One transgenic line shows X-linked inheritance of the transgene and mosaic expression in females but uniform expression in males, indicating that the transgene has integrated into the X chromosome in this line. Conclusion: The 2A peptide efficiently mediates co-translational cleavage in transgenic mice in which it has been inherited through the germ-line. Mice expressing it ubiquitously throughout development and into adulthood appear normal. It is therefore a viable tool for use in genetically engineered mice and represents a superior alternative to the widely used internal ribosomal entry site. [ABSTRACT FROM AUTHOR]

Internal ribosome entry site (IRES) RNAs initiate protein synthesis in eukaryotic cells by a noncanonical cap-independent mechanism. IRESes are critical for many pathogenic viruses, but efforts to understand their function are complicated by the diversity of IRES sequences as well as by limited high-resolution structural information. The intergenic region (IGR) IRESes of the Dicistroviridae viruses are powerful model systems to begin to understand IRES function. Here we present the crystal structure of a Dicistroviridae IGR IRES domain that interacts with the ribosome's decoding groove. We find that this RNA domain precisely mimics the transfer RNA anticodon–messenger RNA codon interaction, and its modeled orientation on the ribosome helps explain translocation without peptide bond formation. When combined with a previous structure, this work completes the first high-resolution description of an IRES RNA and provides insight into how RNAs can manipulate complex biological machines. [ABSTRACT FROM AUTHOR]

Bacteriophage P22 infects Salmonella enterica by injecting its genetic material through the cell envelope. During infection, a specialized tail needle, gp26, is injected into the host, likely piercing a hole in the host cell envelope. The 2.1-Å crystal structure of gp26 reveals a 240-Å elongated protein fiber formed by two trimeric coiled-coil domains interrupted by a triple β-helix. The N terminus of gp26 plugs the portal protein channel, retaining the genetic material inside the virion. The C-terminal tip of the fiber exposes β-hairpins with hydrophobic tips similar to those seen in class II fusion peptides. The α-helical core connecting these two functionally polarized tips presents four trimerization octads with consensus sequence IXXLXXXV. The slender conformation of the gp26 fiber minimizes the surface exposed to solvent, which is consistent with the idea that gp26 traverses the cell envelope lipid bilayers. [ABSTRACT FROM AUTHOR]

The component peptides of lacticin 3147 were degraded by α-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit. [ABSTRACT FROM AUTHOR]

Abstract: It is generally assumed that preprotein substrates must be presented in an unfolded state to the bacterial Sec-translocase in order to be translocated. Here, we have examined the ability of the Sec-translocase to translocate folded preproteins. Tightly folded human cardiac Ig-like domain I27 fused to the C terminus of proOmpA is translocated efficiently by the Sec-translocase and the translocation kinetics are determined by the extent of folding of the titin I27 domain. Accumulation of specific translocation intermediates around the fusion point that undergo translocation progress upon ATP binding suggests that the motor protein SecA plays an important and decisive role in promoting unfolding of the titin I27 domain. It is concluded that the bacterial Sec-translocase is capable of actively unfolding preproteins. [Copyright &y& Elsevier]

Objective. Anal fistulas are the result of chronic infection of an intersphincteric gland. Despite the passage through mesenchymal tissue, fistulas seldom lead to systemic infection. Antimicrobial peptides are secreted by a variety of epithelia, belonging to the innate immune system and are potential factors contributing to infection control. The aim of this study was to investigate whether epithelium is present in the fistulas and what the origin might be. Material and methods. Forty-seven chronic anal fistulas from patients, excluding Crohn's disease, were compared with healthy rectal and perianal control tissue. Expression of antimicrobial peptide mRNA was analysed by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry. Tissue was further studied by cytokine and cytokeratin staining. Results. Chronic anal fistulas express high levels of hBD-2 and hBD-3 and the newly identified antimicrobial peptides RNase7 and psoriasin compared to rectal mucosa from control patients. Perianal skin has almost identical levels of RNase7 and psoriasin expression to those in fistulas. IL-1b and IL-8 were the only cytokines detectable in fistulas. Fistulas are lined with squamous epithelium that expresses identical cytokeratines as skin. Conclusions. Epithelialization and local production of antimicrobial peptides in anal fistulas serve as defence mechanisms to prevent local and systemic infection by microbes from faeces passing through the fistula tract. [ABSTRACT FROM AUTHOR]

Infections can act as environmental triggers inducing or promoting autoimmune disease in genetically predisposed individuals. Identification of microbial peptides similar to self-tissues may by molecular mimicry, provide the inducing mechanism for an immune response. The aim of this study was to identify autoantibodies (autoAbs) in nonautoimmune individuals during acute bacterial, viral, or parasitic infections. Specific Abs or specific infections with an increased autoAb load may shed insight into the mechanisms of autoimmune disease. Sera from 88 patients with acute infections (41 bacterial, 23 viral, 17 parasitic, and 7 rickettsial) were tested by the ELISA method for antinuclear antibodies (ANA) 8 Pro, and Abs to thyroid peroxidase (TPO), thyroglobulin, phospholipids, annexin-V, laminin, anti– Saccharomyces cervisiae (ASCA), and prothrombin, along with 80 normal controls. Elevated titers of Abs to annexin-V and prothrombin were the most prevalent in viral, parasitic, and rickettsial infections and to laminin in viral and parasitic infections. Elevated titers of ASCA and ANA were found in viral and bacterial infections. Antiphospholipid Abs were found in parasitic and Q-fever infections. Thirty-four individuals harbored elevated titers of at least two Abs. An autoAb burden was detected in individuals with hepatitis A, hepatitis B, toxoplasma or Q-fever infections. In nonautoimmune individuals with various (bacterial, viral, parasitic, and rickettsial) infections, elevated titers of Abs to annexin-V, prothrombin, laminin, ASCA, ANA, and phospholipids were most frequently detected. [ABSTRACT FROM AUTHOR]

Burkholderia cepacia MBA4 is a bacterium that can utilize 2-haioacids as carbon and energy sources for growth. It has been proposed that dehalogenase-associated permease mediates the uptake of haloacid. In this paper, we report the first cloning and characterization of such a haloacid permease. The structural gene, designated deh4p, was found 353 bases downstream of the dehalogenase gene deh4a. Quantitative analysis of the expression of deh4p showed that it was induced by monochloroacetate (MCA), to a level similar to the MCA-induced level ofdeh4a. The nucleotide sequence ofdeh4p was determined, and an open reading frame of 1,656 bp encoding a putative peptide of 552 amino acids was identified. Deh4p has a putative molecular weight of 59,414 and an isoelectric point of 9.88. Deh4p has the signatures of sugar transport proteins and integral membrane proteins of the major facilitator superfamily. Uptake of [14C]MCA into the cell was Deh4p dependent. Deh4p has apparent Kms of 5.5 and 8.9 μM and Vmaxs of 9.1 and 23.1 nmol mg-1 min-1 for acetate and MCA, respectively. A mutant with a transposon-inactivated haloacid operon failed to grow on MCA even when deh4a was provided in trans. [ABSTRACT FROM AUTHOR]

The distribution and genotypic variation of potential microcystin (MC) producers along the southern and eastern shores of Lake Ontario in 2001 and 2003 were examined using a suite of PCR primers. Cyanobacterial, Microcystis sp., and Microcystis-specific toxin primer sets identified shoreline distribution of cyanobacterial DNA (in 97% of the stations) and MC synthetase genes (in 50% of the stations). Sequence analysis of a partial mcyA amplicon targeting Microcystis, Anabaena, and Planktothrix species indicated that the Microcystis sp. genotype was the dominant MC genotype present and revealed a novel Microcystis-like sequence containing a 6-bp insert. Analysis of the same samples with genus-specific mcyE primers confirmed that the Microcystis sp. genotype was the dominant potential MC producer. Genotype compositions within embayments were relatively homogenous compared to those for shoreline and tributary samples. MC concentrations along the shoreline exhibited both temporal and spatial differences as evidenced by the protein phosphatase inhibition assay, at times exceeding the World Health Organization guideline value for drinking water of 1.0 μg MC-LRcq liter-1. MC genotypes are widespread along the New York State shoreline of Lake Ontario, appear to originate nearshore, and can be carried through the lake via wind and surface water current patterns. [ABSTRACT FROM AUTHOR]

The reaction pathway of deformylation catalyzed by E. colipeptide deformylase (PDF) has been investigated by the density functional theory method of PBE1PBE on a small model and by a two-layer ONIOM method on a realistic protein model. The deformylation proceeds in sequential steps involving nucleophilic addition of metal-coordinated water/hydroxide to the carbonyl carbon of the formyl group, proton transfer, and cleavage of the C−N bond. The first step is rate-determining for the deformylation, which occurs through a pentacoordinated metal center. The estimated activation energies with the ONIOM method are about 23.0, 15.0, and 14.9 kcal/mol for Zn−, Ni−, and Fe−PDFs, respectively. These calculated barriers are in close agreement with experimental observations. Our results demonstrate that the preference for metal coordination geometry exerts a significant influence on the catalytic activity of PDFs by affecting the activation of the carbonyl group of the substrate, the deprotonation of the metal-coordinated water, and the stabilization of the transition state. This preference for coordination geometry is mainly determined by the ligand environment and the intrinsic electronic structures of the metal center in the active site of the PDFs [ABSTRACT FROM AUTHOR]

Bacterial peptide deformylase (PDF) represents a novel class of mononuclear iron peptidase, and has an intriguing metal preference different from most other metalloproteases. Using a hybrid density functional theory (B3LYP) QM/MM method, we have theoretically investigated its catalytic mechanism and metal specificity by studying both Fe2-PDF and Zn2-PDF. In both forms of PDF, the conserved Glu133 residue is protonated in the reactant complex, and acts as a general acid during the reaction. The initial reaction step is the nucleophilic attack of the metal-bound hydroxide on the carbonyl carbon of the substrate. Our calculations indicate that the metal ion in Fe2-PDF is always pentacoordinated during the reaction process, while that in Zn2-PDF is only tetrahedrally coordinated and not bound to the substrate in the reactant complex. This difference in their metal coordination is suggested to account for the lower activity of Zn2-PDF in comparison with Fe2-PDF. [ABSTRACT FROM AUTHOR]