Draft Genome Sequence of Amycolatopsis lurida NRRL 2430, Producer of the Glycopeptide Family Antibiotic Ristocetin.

We report here the first draft genome sequence for Amycolatopsis lurida NRRL 2430, the producer of the glycopeptide antibiotic ristocetin. The 9-Mbp genome is predicted to harbor 8,143 genes, including those belonging to the ristocetin biosynthesis cluster and 31 additional predicted secondary metabolite gene clusters.This work was supported by the grants from the Royal Society (516002.K5877/ROG) and the Medical Research Council (G0700141).This paper was originally published in Genome Announcements (Kwun MJ, Hong H-J, Genome Announcements 2014, 2(5):e01050-14. doi:10.1128/genomeA.01050-14)

The activity of glycopeptide antibiotics against resistant bacteria correlates with their ability to induce the resistance system.

Glycopeptide antibiotics containing a hydrophobic substituent display the best activity against vancomycin-resistant enterococci, and they have been assumed to be poor inducers of the resistance system. Using a panel of 26 glycopeptide derivatives and the model resistance system in Streptomyces coelicolor, we confirmed this hypothesis at the level of transcription. Identification of the structural glycopeptide features associated with inducing the expression of resistance genes has important implications in the search for more effective antibiotic structures.This work was supported by the Royal Society (516002.K5877/ROG) and the Medical Research Council (G0700141).This is the accepted manuscript version. The final version is available from ASM at http://aac.asm.org/content/early/2014/07/30/AAC.03668-14.abstract

Genome Sequence of Streptomyces toyocaensis NRRL 15009, Producer of the Glycopeptide Antibiotic A47934.

Here we report the draft genome sequence of Streptomyces toyocaensis strain NRRL 15009 which is the producer of the glycopeptide antibiotic A47934. The genome sequence is predicted to harbor a total of 26 secondary metabolite biosynthetic gene clusters including the A47934 cluster.This work was supported by grants from the Royal Society (516002.K5877/ ROG) and the Medical Research Council (G0700141).This is the final published version, also available from ASM at http://genomea.asm.org/content/2/4/e00749-14

High-Resolution Mass Spectrometry Based Proteomic Analysis of the Response to Vancomycin-Induced Cell Wall Stress in Streptomyces coelicolor A3(2).

Understanding how bacteria survive periods of cell wall stress is of fundamental interest and can help generate ideas for improved antibacterial treatments. In this study we use tandem mass tagging to characterize the proteomic response of vancomycin resistant Streptomyces coelicolor to the exposure to sublethal levels of the antibiotic. A common set of 804 proteins were identified in triplicate experiments. Contrasting changes in the abundance of proteins closely associated with the cytoplasmic membrane with those taking place in the cytosol identified aspects of protein spatial localization that are associated with the response to vancomycin. Enzymes for peptidoglycan precursor, mycothiol, ectoine and menaquinone biosynthesis together with a multisubunit nitrate reductase were recruited to the membrane following vancomycin treatment. Many proteins with regulatory functions (including sensor protein kinases) also exhibited significant changes in abundance exclusively in the membrane-associated protein fraction. Several enzymes predicted to be involved in extracellular peptidoglycan crossbridge formation became significantly depleted from the membrane. A comparison with data previously acquired on the changes in gene transcription following vancomycin treatment identified a common high-confidence set of changes in gene expression. Generalized changes in protein abundance indicate roles for proteolysis, the pentose phosphate pathway and a reorganization of amino acid biosynthesis in the stress response.HJH was supported by the Royal Society (516002.K5877/ROG) and by grant number GO700141 from the Medical Research Council.This is the final version of the article. It first appeared from ACS Publications via http://dx.doi.org/10.1021/acs.jproteome.5b0024

In Vivo Characterization of the Activation and Interaction of the VanR-VanS Two-Component Regulatory System Controlling Glycopeptide Antibiotic Resistance in Two Related Streptomyces Species.

This is the author accepted manuscript. The final version is available from the American Society for Microbiology via http://dx.doi.org/10.1128/AAC.01367-15The VanR-VanS two-component system is responsible for inducing resistance to glycopeptide antibiotics in various bacteria. We have performed a comparative study of the VanR-VanS systems from two streptomyces strains, Streptomyces coelicolor and Streptomyces toyocaensis, to characterize how the two proteins cooperate to signal the presence of antibiotics and to define the functional nature of each protein in each strain background. The results indicate that the glycopeptide antibiotic inducer specificity is determined solely by the differences between the amino acid sequences of the VanR-VanS two-component systems present in each strain rather than by any inherent differences in general cell properties, including cell wall structure and biosynthesis. VanR of S. coelicolor (VanRsc) functioned with either sensor kinase partner, while VanR of S. toyocaensis (VanRst) functioned only with its cognate partner, S. toyocaensis VanS (VanSst). In contrast to VanRsc, which is known to be capable of phosphorylation by acetylphosphate, VanRst could not be activated in vivo independently of a VanS sensor kinase. A series of amino acid sequence modifications changing residues in the N-terminal receiver (REC) domain of VanRst to the corresponding residues present in VanRsc failed to create a protein capable of being activated by VanS of S. coelicolor (VanSsc), which suggests that interaction of the response regulator with its cognate sensor kinase may require a region more extended than the REC domain. A T69S amino acid substitution in the REC domain of VanRst produced a strain exhibiting weak constitutive resistance, indicating that this particular amino acid may play a key role for VanS-independent phosphorylation in the response regulator protein.This work was supported by funding from the Medical Research Council, UK (G0700141) and the Royal Society, UK (516002.K5877/ROG). the American Society for Microbiology

Genome-wide dynamics of a bacterial response to antibiotics that target the cell envelope.

BACKGROUND: A decline in the discovery of new antibacterial drugs, coupled with a persistent rise in the occurrence of drug-resistant bacteria, has highlighted antibiotics as a diminishing resource. The future development of new drugs with novel antibacterial activities requires a detailed understanding of adaptive responses to existing compounds. This study uses Streptomyces coelicolor A3(2) as a model system to determine the genome-wide transcriptional response following exposure to three antibiotics (vancomycin, moenomycin A and bacitracin) that target distinct stages of cell wall biosynthesis. RESULTS: A generalised response to all three antibiotics was identified which involves activation of transcription of the cell envelope stress sigma factor σ(E), together with elements of the stringent response, and of the heat, osmotic and oxidative stress regulons. Attenuation of this system by deletion of genes encoding the osmotic stress sigma factor σ(B) or the ppGpp synthetase RelA reduced resistance to both vancomycin and bacitracin. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. Sensitivity studies using mutants constructed on the basis of the transcriptome profiling confirmed a role for several such genes in antibiotic resistance, validating the usefulness of the approach. CONCLUSIONS: Antibiotic inhibition of bacterial cell wall biosynthesis induces both common and compound-specific transcriptional responses. Both can be exploited to increase antibiotic susceptibility. Regulatory networks known to govern responses to environmental and nutritional stresses are also at the core of the common antibiotic response, and likely help cells survive until any specific resistance mechanisms are fully functional.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Antibiotic resistance mechanisms inform discovery: identification and characterization of a novel amycolatopsis strain producing ristocetin.

Discovering new antibiotics is a major scientific challenge, made increasingly urgent by the continued development of resistance in bacterial pathogens. A fundamental understanding of the mechanisms of bacterial antibiotic resistance will be vital for the future discovery or design of new, more effective antibiotics. We have exploited our intimate knowledge of the molecular mechanism of glycopeptide antibiotic resistance in the harmless bacterium Streptomyces coelicolor to develop a new two-step cell wall bioactivity screen, which efficiently identified a new actinomycete strain containing a previously uncharacterized glycopeptide biosynthetic gene cluster. The screen first identifies natural product extracts capable of triggering a generalized cell wall stress response and then specifically selects for glycopeptide antibacterials by assaying for the induction of glycopeptide resistance genes. In this study, we established a diverse natural product extract library from actinomycete strains isolated from locations with widely varying climates and ecologies, and we screened them using the novel two-step bioassay system. The bioassay ultimately identified a single strain harboring the previously unidentified biosynthetic gene cluster for the glycopeptide ristocetin, providing a proof of principle for the effectiveness of the screen. This is the first report of the ristocetin biosynthetic gene cluster, which is predicted to include some interesting and previously uncharacterized enzymes. By focusing on screening libraries of microbial extracts, this strategy provides the certainty that identified producer strains are competent for growth and biosynthesis of the detected glycopeptide under laboratory conditions.This work was supported by funding from the Royal Society, UK (516002.K5877/ROG), the Medical Research council, UK (G0700141) and St. John’s College, University of CambridgeThis the the author accepted manuscript. The final version is available from ASM at http://aac.asm.org/content/early/2014/07/09/AAC.03349-14.abstract

Chemotranscriptomic profiling defines drug-specific signatures of the glycopeptide antibiotics dalbavancin, vancomycin and chlorobiphenyl-vancomycin in a VanB-type-resistant streptomycete

Dalbavancin, vancomycin and chlorobiphenyl-vancomycin share a high degree of structural similarity and the same primary mode of drug action. All inhibit bacterial cell wall biosynthesis through complexation with intermediates in peptidoglycan biosynthesis mediated via interaction with peptidyl-d-alanyl–d-alanine (d-Ala–d-Ala) residues present at the termini of the intermediates. VanB-type glycopeptide resistance in bacteria encodes an inducible reprogramming of bacterial cell wall biosynthesis that generates precursors terminating with d-alanyl–d-lactate (d-Ala–d-Lac). This system in Streptomyces coelicolor confers protection against the natural product vancomycin but not dalbavancin or chlorobiphenyl-vancomycin, which are semi-synthetic derivatives and fail to sufficiently activate the inducible VanB-type sensory response. We used transcriptome profiling by RNAseq to identify the gene expression signatures elucidated in S. coelicolor in response to the three different glycopeptide compounds. An integrated comparison of the results defines both the contribution of the VanB resistance system to the control of changes in gene transcription and the impact at the transcriptional level of the structural diversity present in the glycopeptide antibiotics used. Dalbavancin induces markedly more extensive changes in the expression of genes required for transport processes, RNA methylation, haem biosynthesis and the biosynthesis of the amino acids arginine and glutamine. Chlorobiphenyl-vancomycin exhibits specific effects on tryptophan and calcium-dependent antibiotic biosynthesis and has a stronger repressive effect on translation. Vancomycin predictably has a uniquely strong effect on the genes controlled by the VanB resistance system and also impacts metal ion homeostasis and leucine biosynthesis. Leaderless gene transcription is disfavoured in the core transcriptional up- and down-regulation taking place in response to all the glycopeptide antibiotics, while HrdB-dependent transcripts are favoured in the down-regulated group. This study illustrates the biological impact of peripheral changes to glycopeptide antibiotic structure and could inform the design of future semi-synthetic glycopeptide derivatives

A Study of Parameters Related to the Etch Rate for a Dry Etch Process Using NF 3

The characteristics of the dry etching of SiNx:H thin films for display devices using SF6/O2 and NF3/O2 were investigated using a dual-frequency capacitively coupled plasma reactive ion etching (CCP-RIE) system. The investigation was carried out by varying the RF power ratio (13.56 MHz/2 MHz), pressure, and gas flow ratio. For the SiNx:H film, the etch rates obtained using NF3/O2 were higher than those obtained using SF6/O2 under various process conditions. The relationships between the etch rates and the usual monitoring parameters—the optical emission spectroscopy (OES) intensity of atomic fluorine (685.1 nm and 702.89 nm) and the voltages VH and VL—were investigated. The OES intensity data indicated a correlation between the bulk plasma density and the atomic fluorine density. The etch rate was proportional to the product of the OES intensity of atomic fluorine (I(F)) and the square root of the voltages (Vh+Vl) on the assumption that the velocity of the reactive fluorine was proportional to the square root of the voltages

Microsatellite Analysis of the Genetic Diversity and Population Structure in Dairy Goats in Thailand

The genetic relationships between different populations and breeds of exotic dairy goats in Thailand were studied using 12 microsatellite markers. Blood samples were obtained from 211 goats from Department of Livestock Development breeding and research farms: 29 Anglonubian (AN), 21 Alpine (AP), 23 Jamunapari (JAM), 50 Saanen (SN), and 88 Toggenburg (TG). Five of the 12 microsatellite markers were found to be polymorphic. A mean of 7.40 alleles per locus was found, with a range from 5 (SPS115 and ETH225) to 11 (TGLA122). We found 24, 27, 19, 32, and 24 alleles in the AN, AP, JAM, SN, and TG breeds, respectively; 37 alleles were present in all breeds. The mean number of alleles in each population ranged from 3.2 (ETH225 locus) to 7.6 (TGLA122 locus). Genetic variability within the breeds was moderate as evidenced by the mean expected heterozygosity of 0.539. The average observed heterozygosity across the 5 markers in all breeds was 0.529 with the maximum observed at the BM1818 locus (0.772) and the minimum at the ETH225 locus (0.248). The observed and expected heterozygosity for all breeds for the 5 microsatellite markers ranged from 0.419 to 0.772 and 0.227 to 0.792, respectively. On the basis of their means, the TGLA122 and BM1818 loci were the most suitable markers for distinguishing genetic diversity among the goats. The estimated average Fis value for the breeds ranged from −0.044 (ETH225) to 0.180 (SPS115), while the estimated average Fst value ranged from 0.021 (SPS115) to 0.104 (ETH10). These results indicated that TGLA122 and BM1818 markers are suitable to be used for aiding conservation and breeding improvement strategies of dairy