The structure of pyogenecin immunity protein, a novel bacteriocin-like immunity protein from Streptococcus pyogenes

<p>Abstract</p> <p>Background</p> <p>Many Gram-positive lactic acid bacteria (LAB) produce anti-bacterial peptides and small proteins called bacteriocins, which enable them to compete against other bacteria in the environment. These peptides fall structurally into three different classes, I, II, III, with class IIa being pediocin-like single entities and class IIb being two-peptide bacteriocins. Self-protective cognate immunity proteins are usually co-transcribed with these toxins. Several examples of cognates for IIa have already been solved structurally. <it>Streptococcus pyogenes</it>, closely related to LAB, is one of the most common human pathogens, so knowledge of how it competes against other LAB species is likely to prove invaluable.</p> <p>Results</p> <p>We have solved the crystal structure of the gene-product of locus Spy_2152 from <it>S. pyogenes</it>, (PDB:<ext-link ext-link-id="2fu2" ext-link-type="pdb">2fu2</ext-link>), and found it to comprise an anti-parallel four-helix bundle that is structurally similar to other bacteriocin immunity proteins. Sequence analyses indicate this protein to be a possible immunity protein protective against class IIa or IIb bacteriocins. However, given that <it>S. pyogenes </it>appears to lack any IIa pediocin-like proteins but does possess class IIb bacteriocins, we suggest this protein confers immunity to IIb-like peptides.</p> <p>Conclusions</p> <p>Combined structural, genomic and proteomic analyses have allowed the identification and <it>in silico </it>characterization of a new putative immunity protein from <it>S. pyogenes</it>, possibly the first structure of an immunity protein protective against potential class IIb two-peptide bacteriocins. We have named the two pairs of putative bacteriocins found in <it>S. pyogenes </it>pyogenecin 1, 2, 3 and 4.</p

Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 Å, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein–ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics

Fixed-target serial crystallography at the Structural Biology Center

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two β-lactamases: a class D serine β-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-β-lactamase from Stenotrophomonas maltophilia K279a

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis

Crystal Structure of an Integron Gene Cassette-Associated Protein from Vibrio cholerae Identifies a Cationic Drug-Binding Module

Background The direct isolation of integron gene cassettes from cultivated and environmental microbial sources allows an assessment of the impact of the integron/gene cassette system on the emergence of new phenotypes, such as drug resistance or virulence. A structural approach is being exploited to investigate the modularity and function of novel integron gene cassettes. Methodology/Principal Findings We report the 1.8 A crystal structure of Cass2, an integron-associated protein derived from an environmental V. cholerae. The structure defines a monomeric beta-barrel protein with a fold related to the effector-binding portion of AraC/XylS transcription activators. The closest homologs of Cass2 are multi-drug binding proteins, such as BmrR. Consistent with this, a binding pocket made up of hydrophobic residues and a single glutamate side chain is evident in Cass2, occupied in the crystal form by polyethylene glycol. Fluorescence assays demonstrate that Cass2 is capable of binding cationic drug compounds with submicromolar affinity. The Cass2 module possesses a protein interaction surface proximal to its drug-binding cavity with features homologous to those seen in multi-domain transcriptional regulators. Conclusions/Significance Genetic analysis identifies Cass2 to be representative of a larger family of independent effector-binding proteins associated with lateral gene transfer within Vibrio and closely-related species. We propose that the Cass2 family not only has capacity to form functional transcription regulator complexes, but represents possible evolutionary precursors to multi-domain regulators associated with cationic drug compounds.National Health and Medical Research Council (Australia) (NHMRC grant 488502)National Institutes of Health (U.S.) (Grant GM62414-0 )Ontario. Ministry of Revenue (Challenge Fund

Isolation and identification of thiocyanate utilizing chemolithotrophs from gold mine soils

A mixed bacterial culture capable of growing in potassium-thiocyanate containing medium (200 mg KSCN) has been isolated from bacterial suspensions of soil samples collected near gold mines in Kumjung (Korea). The isolates were initially characterized by metabolic profile analysis and were identified as Bacillus thermoglucosidasius, Bacillus cereus, Bacillus licheniformis, Bacillus mycoides, Brevibacterium epidermidis, Brevibacterium otitidis, and Corynebacterium nitrilophilus. One of the seven isolates was initially characterized as Brevibacterium epidermidis, which is not known to degrade thiocyanate. However, using 16S rDNA sequencing, this strain was identified as a member of Klebsiella. The strain shows high similarity values (95.8 to 96.4%) with Klebsiella species, and the closest known relative was found to be K. ornithinolytica ATCC 31898. The result indicates that species of the genus Klebsiella were the closest phylogenetic relatives of the investigated strain. This is the first known report of a member of Klebsiella that is capable of utilizing thiocyanate as sole source of carbon and nitrogen.close71