Whole Genome Sequencing and Metabolomic Study of Cave Streptomyces Isolates ICC1 and ICC4

The terrestrial subsurface microbiome has gained considerable amount of interests in the recent years because of its rich potential resource for biomining novel genes coding for metabolites possessing antimicrobial activities. In our previous study, we identified two Streptomyces isolates, designated as ICC1 and ICC4, from the Iron Curtain Cave, Chilliwack, Canada that exhibited antagonistic activities against the multidrug resistant strains of Escherichia coli. In this study, the genomes of these two isolates were sequenced by Illumina MiSeq, assembled and annotated. The genes associated with secondary metabolite production were identified and annotated using the bioinformatics platforms antiSMASH and BAGEL. ICC1 and ICC4 were then cultivated and ICC1 metabolome characterized by UHPLC-ESI-HRMS. The Global Natural Products Social Molecular Networking was used to identify metabolites based on the MS/MS spectral data. ICC1 and ICC4 showed a high level of sequence identity with the terrestrial bacteria Streptomyces lavendulae; however, they possess a greater secondary metabolite potential as estimated by the total number of identified biosynthetic gene clusters (BGCs). In particular, ICC1 and ICC4 had a greater number of polyketide and non-ribosomal peptide BGCs. The most frequently detected BGCs were those predicted to generate terpenes, small and low complexity dipeptides and lipids. Spectral analysis clearly identified a number of diketopiperazine products through matched reference spectra for cyclo (Leu-Pro), cyclo (Pro-Val) and cyclo [(4-hydroxyPro)-Leu]. One of the terpenes gene clusters predicted by antiSMASH possesses a seven-gene pathway consistent with diazepinomicin biosynthesis. This molecule contains a very rare core structure and its BGC, to date, has only been identified from a single bacterial genome. The tetrapeptide siderophore coelichelin BGC was unambiguously identified in the genome, however, the metabolite could not be identified from the culture extracts. Two type III polyketides, 2′, 5′ – dimethoxyflavone and nordentatin, were identified from the UHPLC-HRMS data of the aqueous and n-butanolic fractions of Streptomyces sp. ICC1, respectively. A BGC likely encoding these metabolites was predicted in both genomes. The predicted similarities in molecule production and genome shared by these two strains could be an indicative of a cooperative mode of living in extreme habitats instead of a competitive one. This secondary metabolite potential may contribute to the fitness of ICC1 and ICC4 in the Iron Curtain Cave

Transcriptomic and Exometabolomic Profiling Reveals Antagonistic and Defensive Modes of Clonostachys rosea Action Against Fusarium graminearum

The mycoparasite Clonostachys rosea ACM941 is under development as a biocontrol organism against Fusarium graminearum, the causative agent of Fusarium head blight in cereals. To identify molecular factors associated with this interaction, the transcriptomic and exometabolomic profiles of C. rosea and F. graminearum GZ3639 were compared during coculture. Prior to physical contact, the antagonistic activity of C. rosea correlated with a response heavily dominated by upregulation of polyketide synthase gene clusters, consistent with the detected accumulation of corresponding secondary metabolite products. Similarly, prior to contact, trichothecene gene clusters were upregulated in F. graminearum, while those responsible for fusarielin and fusarin biosynthesis were downregulated, correlating with an accumulation of trichothecene products in the interaction zone over time. A concomitant increase in 15-acetyl deoxynivalenol-3-glucoside in the interaction zone was also detected, with C. rosea established as the source of this detoxified mycotoxin. After hyphal contact, C. rosea was found to predominantly transcribe genes encoding cell wall–degradation enzymes, major facilitator superfamily sugar transporters, anion:cation symporters, as well as alternative carbon source utilization pathways, together indicative of a transition to necrotropism at this stage. F. graminearum notably activated the transcription of phosphate starvation pathway signature genes at this time. Overall, a number of signature molecular mechanisms likely contributing to antagonistic activity by C. rosea against F. graminearum, as well as its mycotoxin tolerance, are identified in this report, yielding several new testable hypotheses toward understanding the basis of C. rosea as a biocontrol agent for continued agronomic development and application

Discovery of a New Natural Product and a Deactivation of a Quorum Sensing System by Culturing a “Producer” Bacterium With a Heat-Killed “Inducer” Culture

Herein we describe a modified bacterial culture methodology as a tool to discover new natural products via supplementing actinomycete fermentation media with autoclaved cultures of “inducer” microbes. Using seven actinomycetes and four inducer microbes, we detected 28 metabolites that were induced in UHPLC-HRESIMS-based analysis of bacterial fermentations. Metabolomic analysis indicated that each inducer elicited a unique response from the actinomycetes and that some chemical responses were specific to each inducer-producer combination. Among these 28 metabolites, hydrazidomycin D, a new hydrazide-containing natural product was isolated from the pair Streptomyces sp. RKBH-B178 and Mycobacterium smegmatis. This result validated the effectiveness of the strategy in discovering new natural products. From the same set of induced metabolites, an in-depth investigation of a fermentation of Streptomyces sp. RKBH-B178 and autoclaved Pseudomonas aeruginosa led to the discovery of a glucuronidated analog of the pseudomonas quinolone signal (PQS). We demonstrated that RKBH-B178 is able to biotransform the P. aeruginosa quorum sensing molecules, 2-heptyl-4-quinolone (HHQ), and PQS to form PQS-GlcA. Further, PQS-GlcA was shown to have poor binding affinity to PqsR, the innate receptor of HHQ and PQS

A season-long study of questioning behaviours by an international rugby coach

AIM To understand how an international rugby coach used questioning behaviours in on-pitch activities during training and competition. METHODS The coach’s behavioural intentions and “coaching philosophy” were investigated through semi-structured interviews. On-pitch coach behaviour was then systematically observed using the Rugby Coach Activities and Behaviours Instrument (RCABI). The RCABI includes behaviour and activity classifications enabling actions to be understood within their micro-contexts. RESULTS Overall, questioning accounted for only 6.34% of on-pitch behaviours; yet, this was the coach’s most frequent direct interaction with players. Time spent questioning varied between activity contexts during training, but was never used during match play. The impacts of intentions, contexts and constraints on questioning behaviour are discussed. CONCLUSION The nuances of questioning behaviour need to be examined in greater depth and with more rugby coaches. It is essential to understand how and why questioning is used in on-pitch and off-pitch contexts. Future studies need to investigate the content of questions (i.e., convergent, divergent types), their intended purpose, how they are influenced by contextual factors, and the perceptions of recipients (i.e., how they impact player outcomes)

Doing research with high-performing coaches: reflection on a case study in international rugby union

Introduction Criticism has been levelled at research for its lack of impact on the coaching process. The origins of this theory-practice gap lie, in part, in the way coaching research has traditionally been undertaken – the coach has been treated as the “other” to be studied by the coaching “scientist”. Consequently, methods that maintain an “objective” distance between researcher and participant have been popular. In addition, high-performance sport is often off limits to researchers, as coaches remain secretive about what they do in order to protect a perceived competitive advantage. Thus, when trying to advance a more authentic understanding of the complexities of real-world, high-performance coaching through, for example, ethnographic methods (where the researcher becomes part of the coaching context) various philosophical and practical challenges must be overcome. The purpose of this presentation is to explore these challenges. Method Following a mixed-methods, season-long, ethnographic case study of the coaching process in international rugby union, the researcher and coach engaged in a series of collaboratively reflexive interviews to reflect upon their experiences together. These were audio recorded and transcribed verbatim. As part of the broader study, the data analysis was informed by the principles of grounded theory, which included inductive and deductive reasoning. Findings The findings highlight challenges and opportunities inherent in the coming together of coach and researcher in the coaching context, which serve to identify how the coaching process and research process were interrelated. Key themes to be discussed include continually negotiating access to the high performance context, trying to blend in as an “outsider”, remaining flexible and influencing the data as a researcher. This presentation will take a pragmatic approach with the intention of highlighting implications for other researchers planning and undertaking of similar approaches. It is hoped that by reporting findings generated through collaboration and reflexivity the coach and researcher might be reframed as reciprocal allies to address the theory-practice gap

A Holistic Model of the Coaching Process: Conceptualising the Challenge of Effectiveness in Practice

This chapter introduces a holistic model of the coaching process, which was developed during a mixed-methods, longitudinal case study in international rugby union. Fundamentally, it shows that the coaching process is constituted of the interactions of a coach and their associates within a coaching context. Each of these personal, social and contextual influences on coaching practice are described and explored using practical examples. Numerous constraints and opportunities experienced by coaches in their complex work are highlighted and key considerations for practitioners in different contexts are raised. The holistic model is presented in order to help coaches from diverse backgrounds to think critically about the intricacies of their own coaching practice. More specifically and against the backdrop of this book’s title, it is hoped that the holistic model will act as a framework for reflective practice and awareness building for readers who wish to be effective coaches. Moreover, we hope to stimulate further discussion about the coaching process as a sophisticated, holistic and contingent project, one imbued with interconnected idiosyncrasies, possibilities and challenges, as well as significant breadth and depth

Doing hybrid management work in elite sport: The case of a head coach in top-level Rugby Union

This paper examines a head coach’s enactment of their hybrid role (i.e., it was comprised of leadership, management, and coaching responsibilities) in an elite level sport organisation. A methodological bricolage comprising a) participant observation and reflexive field notes, b) ethnographic film, and c) semi-structured and stimulated recall interviews was used to generate data during 20 months of ethnographic fieldwork with an international rugby union team. Data were iteratively analysed using Crossley’s (2011) relational theorising. The head coach was found to strategically manage nuanced relations with, and between, more and less trusted collaborators in the organisation. This positioned her to maintain oversight and to influence the support, capital, resources and information flowing through relational networks towards the organisation’s key mission. Strategic interaction that responded to others’ thoughts, feelings and interests was used to generate buy-in, space and time to carry out the manager’s agendas. The findings highlight the importance for researchers, policy makers, sport organisations, educators and practitioners of further engaging with management as an ongoing exercise in collective behaviour, which includes micropolitical struggles and exchange

Terrosamycins A and B, Bioactive Polyether Ionophores from Streptomyces sp. RKND004 from Prince Edward Island Sediment

Terrosamycins A (1) and B (2), two polycyclic polyether natural products, were purified from the fermentation broth of Streptomyces sp. RKND004 isolated from Prince Edward Island sediment. The one strain-many compounds (OSMAC) approach coupled with UPLC-HRMS-based metabolomics screening led to the identification of these compounds. The structure of 1 was determined from analysis of NMR, HRMS, and X-ray diffraction data. NMR experiments performed on 2 revealed the presence of two methoxy groups replacing two hydroxy groups in 1. Like other polyether ionophores, 1 and 2 exhibited excellent antibiotic activity against Gram-positive pathogens. Interestingly, the terrosamycins also exhibited activity against two breast cancer cell lines

Evolution of the Ergot Alkaloid Biosynthetic Gene Cluster Results in Divergent Mycotoxin Profiles in <i>Claviceps purpurea</i> Sclerotia

Research into ergot alkaloid production in major cereal cash crops is crucial for furthering our understanding of the potential toxicological impacts of Claviceps purpurea upon Canadian agriculture and to ensure consumer safety. An untargeted metabolomics approach profiling extracts of C. purpurea sclerotia from four different grain crops separated the C. purpurea strains into two distinct metabolomic classes based on ergot alkaloid content. Variances in C. purpurea alkaloid profiles were correlated to genetic differences within the lpsA gene of the ergot alkaloid biosynthetic gene cluster from previously published genomes and from newly sequenced, long-read genome assemblies of Canadian strains. Based on gene cluster composition and unique polymorphisms, we hypothesize that the alkaloid content of C. purpurea sclerotia is currently undergoing adaptation. The patterns of lpsA gene diversity described in this small subset of Canadian strains provides a remarkable framework for understanding accelerated evolution of ergot alkaloid production in Claviceps purpurea