A high performance Trichoderma reesei strain that reveals the importance of xylanase III in cellulosic biomass conversion.

The ability of the Trichoderma reesei X3AB1strain enzyme preparations to convert cellulosic biomass into fermentable sugars is enhanced by the replacement of xyn3 by Aspergillus aculeatus β-glucosidase 1 gene (aabg1), as shown in our previous study. However, subsequent experiments using T. reesei extracts supplemented with the glycoside hydrolase (GH) family 10 xylanase III (XYN III) and GH Family 11 XYN II showed increased conversion of alkaline treated cellulosic biomass, which is rich in xylan, underscoring the importance of XYN III.

In vitro catalytic activity of N-terminal and C-terminal domains in NukM, the post-translational modification enzyme of nukacin ISK-1.

Lantibiotics are antibacterial peptides containing unique thioether cross-links termed lanthionine and methyllanthionine. NukM, the modifying enzyme of nukacin ISK-1, which is produced by Staphylococcus warneri ISK-1, catalyzes the dehydration of specific Ser/Thr residues in a precursor peptide, followed by conjugative addition of intramolecular Cys to dehydrated residues to generate a cyclic structure. By contrast, the precursor peptide of nisin is modified by 2 enzymes, NisB and NisC, which mediate dehydration and cyclization, respectively.

Genome-wide identification of small RNAs in the opportunistic pathogen Enterococcus faecalis V583.

Small RNA molecules (sRNAs) are key mediators of virulence and stress inducible gene expressions in some pathogens. In this work we identify sRNAs in the gram positive opportunistic pathogen Enterococcus faecalis. We characterized 11 sRNAs by tiling microarray analysis, 5' and 3' RACE-PCR, and Northern blot analysis.

Engineering unusual amino acids into peptides using lantibiotic synthetase.

Alteration of protein structure and function by introducing unusual amino acids has great potential to develop new biological tool and to produce novel therapeutic agents. Lantibiotics produced by Gram-positive bacteria are ribosomally synthesized and post-translationally modified antimicrobial peptides. The modification enzyme involved in lantibiotic biosynthesis can catalyze the formation of unusual amino acids in the nascent lantibiotic prepeptide.

The sigma factor AlgU plays a key role in formation of robust biofilms by nonmucoid Pseudomonas aeruginosa.

The extracytoplasmic function sigma factor AlgU of Pseudomonas aeruginosa is responsible for alginate overproduction, leading to mucoidy and chronic infections of cystic fibrosis patients. We investigated here the role of AlgU in the formation of nonmucoid biofilms. The algU mutant of P.

Effect of a negatively charged lipid on membrane-lacticin Q interaction and resulting pore formation.

Lacticin Q is an antimicrobial peptide that forms pores on membranes. We investigated effects of negatively charged lipids on the binding and pore formation of lacticin Q with liposomes by surface plasmon resonance analysis and fluorescence dye leakage experiments respectively. Negatively charged lipids accelerated the binding of lacticin Q on the membranes and the resulting pore formation.

Characterization of modification enzyme NukM and engineering of a novel thioether bridge in lantibiotic nukacin ISK-1.

The lantibiotic nukacin ISK-1 is an antimicrobial peptide containing unusual amino acids such as lanthionine and dehydrobutyrine. The nukacin ISK-1 prepeptide (NukA) undergoes posttranslational modifications, such as the dehydration and cyclization reactions required to form the unusual amino acids by the modification enzyme NukM. We have previously constructed a system for the introduction of unusual amino acids into NukA by coexpression of NukM in Escherichia coli.

ATP-dependent leader peptide cleavage by NukT, a bifunctional ABC transporter, during lantibiotic biosynthesis.

NukT, a possible ABC transporter maturation and secretion (AMS) protein, may contribute to the cleavage of the leader peptide of NukA, which is the prepeptide of the lantibiotic nukacin ISK-1, and to nukacin ISK-1 transport. In this study, we reconstituted in vitro peptidase activity of the full-length NukT overexpressed in inside-out membrane vesicles of Staphylococcus carnosus TM300. We found that the presence of unusual amino acids in NukA is required for leader peptide cleavage.

Binding specificity of the lantibiotic-binding immunity protein NukH.

NukH is a lantibiotic-binding immunity protein that shows strong binding activity against type A(II) lantibiotics. In this study, the binding specificity of NukH was analyzed by using derivatives of nukacin ISK-1, which is a type A(II) lantibiotic produced by Staphylococcus warneri ISK-1. Interactions between cells of Lactococcus lactis transformants expressing nukH and nukacin ISK-1 derivatives were analyzed by using a quantitative peptide-binding assay.

Lantibiotic engineering: molecular characterization and exploitation of lantibiotic-synthesizing enzymes for peptide engineering.

Lanthionine-containing peptide antibiotics called lantibiotics are produced by a large number of Gram-positive bacteria. Nukacin ISK-1 produced by Staphylococcus warneri ISK-1 is type-A(II) lantibiotic. Ribosomally synthesized nukacin ISK-1 prepeptide (NukA) consists of an N-terminal leader peptide followed by a C-terminal propeptide moiety that undergoes several post-translational modification events including unusual amino acid formation by the modification enzyme NukM, cleavage of leader peptide and export by the dual functional ABC transporter NukT, finally yielding a biologically active peptide.

Lanthionine introduction into nukacin ISK-1 prepeptide by co-expression with modification enzyme NukM in Escherichia coli.

We demonstrated lanthionine introduction into hexa-histidine-tagged (His-tagged) nukacin ISK-1 prepeptide NukA by modification enzyme NukM in Escherichia coli. Co-expression of nukA and nukM, purification of the resulting His-tagged prepeptide by affinity chromatography, and subsequent mass spectrometry analysis showed that the prepeptide was converted into a postulated peptide with decrease in mass of 72Da which resulted from dehydration of four amino acids. Characterization of the resultant prepeptide indicated the presence of unusual amino acids, such as dehydrated amino acid, lanthionine or 3-methyllanthionine, in its C-terminal propeptide moiety.

A novel type of immunity protein, NukH, for the lantibiotic nukacin ISK-1 produced by Staphylococcus warneri ISK-1.

Staphylococcus warneri ISK-1 produces a lantibiotic, nukacin ISK-1. The nukacin ISK-1 gene cluster consists of at least six genes, nukA, -M, -T, -F, -E, and -G, and two open reading frames, ORF1 and ORF7 (designated nukH). Sequence comparisons suggested that NukF, -E, -G, and -H contribute to immunity to nukacin ISK-1.

Localization and interaction of the biosynthetic proteins for the lantibiotic, Nukacin ISK-1.

Nukacin ISK-1 is a type-A(II) lantibiotic produced by Staphylococcus warneri ISK-1. In this study, we characterized NukM and NukT, which are predicted to be involved in modification of prepeptide (NukA) and cleavage of leader peptide and subsequent secretion respectively. Localization analysis of NukM and NukT in the wild-type strain indicated that both proteins were located at the cytoplasm membrane.

Characterization of functional domains of lantibiotic-binding immunity protein, NukH, from Staphylococcus warneri ISK-1.

The immunity to a lantibiotic, nukacin ISK-1, is conferred by NukFEG (ABC transporter) and NukH (lantibiotic-binding protein) cooperatively. The present study identifies the functional domains of NukH. The topological analysis indicated that NukH possesses two external loops and three transmembrane helices.