a genomics based approach identifies a thioviridamide like compound with selective anticancer activity

Thioviridamide is a structurally novel ribosomally synthesized and post-translational modified peptide (RiPP) produced by Streptomyces olivoviridis NA005001. It is characterized by a structure that features a series of thioamide groups and possesses potent antiproliferative activity in cancer cell lines. Its unusual structure allied to its promise as an anticancer compound led us to investigate the diversity of thioviridamide-like pathways across sequenced bacterial genomes. We have isolated and characterized three diverse members of this family of natural products. This characterization is supported by transformation-associated recombination cloning and heterologous expression of one of these compounds, thiostreptamide S4. Our work provides an insight into the diversity of this rare class of compound and indicates that the unusual N-terminus of thioviridamide is not introduced biosynthetically but is instead introduced during acetone extraction. A detailed analysis of the biological activity of one of th..

Expressed in the yeast Saccharomyces cerevisiae, human ERK5 is a client of the Hsp90 chaperone that complements loss of the Slt2p (Mpk1p) cell integrity stress-activated protein kinase

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2Delta Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2Delta yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5

Bacteriophages limit the existence conditions for conjugative plasmids

Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome

Bottromycins - biosynthesis, synthesis and activity

Covering: 1950s up to the end of 2020Bottromycins are a class of macrocyclic peptide natural products that are produced by several Streptomyces species and possess promising antibacterial activity against clinically relevant multidrug-resistant pathogens. They belong to the ribosomally synthesised and post-translationally modified peptide (RiPP) superfamily of natural products. The structure contains a unique four-amino acid macrocycle formed via a rare amidine linkage, C-methylation and a d-amino acid. This review covers all aspects of bottromycin research with a focus on recent years (2009-2020), in which major advances in total synthesis and understanding of bottromycin biosynthesis were achieved

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

Nurses’ decision-making around gastric residual volume measurement in adult intensive care: a four-centre survey

Background: Despite increasing evidence of the potential inaccuracy and unwarranted practice of regular gastric residual volume (GRV) measurement in critically in adults, this practice persists within the United Kingdom.Aim: To explore adult intensive care nurses’ decision-making around the practice of gastric residual volume measurement to guide enteral feeding.Methods: A cross sectional 16 item electronic survey in four adult intensive care units in England and Wales.Results: Two hundred and seventy-three responses were obtained across four intensive care units with acceptable response rates for most [Unit 1 74 /127 = 58.2%; Unit 2 87/129 =67.4%; Unit 3 77/120= 64.1%; Unit 4 35/168 = 20.8%]. Most (243/273 (89%) reported measuring GRV 4-6 hourly, with most (223/273 82%) reporting that the main reason was to assess feed tolerance or intolerance and 37/273 (13.5%) saying their unit protocol required it. In terms of factors affecting decision-making, volume obtained was the most important factor, followed by the condition of the patient, with aspirate colour and appearance less important. When asked how they would feel about not measuring gastric residual volume routinely, the majority (78.2%) of nurses felt worried (140/273 = 51.2%) or very worried (74/273 = 27%).Conclusions: Factors affecting the nurses’ decision making around gastric residual volume were based on largely on fear of risk (around vomiting and pulmonary aspiration) and compliance with unit protocols. Relevance to clinical practice: Despite increasing evidence suggesting it is unnecessary, nurses’ beliefs around the value of this practice persist and it continues to be embedded into unit protocols around feeding.<br/

CDK-dependent Hsp70 phosphorylation controls G1 cyclin abundance and cell-cycle progression

In budding yeast, the essential functions of Hsp70 chaperones Ssa1-4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

CDK-Dependent Hsp70 Phosphorylation Controls G1 Cyclin Abundance and Cell-Cycle Progression

In budding yeast, the essential functions of Hsp70 chaperones Ssa1–4 are regulated through expression level, isoform specificity, and cochaperone activity. Suggesting a novel regulatory paradigm, we find that phosphorylation of Ssa1 T36 within a cyclin-dependent kinase (CDK) consensus site conserved among Hsp70 proteins alters cochaperone and client interactions. T36 phosphorylation triggers displacement of Ydj1, allowing Ssa1 to bind the G1 cyclin Cln3 and promote its degradation. The stress CDK Pho85 phosphorylates T36 upon nitrogen starvation or pheromone stimulation, destabilizing Cln3 to delay onset of S phase. In turn, the mitotic CDK Cdk1 phosphorylates T36 to block Cln3 accumulation in G2/M. Suggesting broad conservation from yeast to human, CDK-dependent phosphorylation of Hsc70 T38 similarly regulates Cyclin D1 binding and stability. These results establish an active role for Hsp70 chaperones as signal transducers mediating growth control of G1 cyclin abundance and activity

Understanding the biosynthesis, metabolic regulation, and anti-phytopathogen activity of 3,7-dihydroxytropolone in Pseudomonas spp.

The genus Pseudomonas is a prolific source of specialized metabolites with significant biological activities, including siderophores, antibiotics, and plant hormones. These molecules play pivotal roles in environmental interactions, influencing pathogenicity, inhibiting microorganisms, responding to nutrient limitation and abiotic challenges, and regulating plant growth. These properties mean that pseudomonads are suitable candidates as biological control agents against plant pathogens. Multiple transposon-based screens have identified a Pseudomonas biosynthetic gene cluster (BGC) associated with potent antibacterial and antifungal activities, which produces 7-hydroxytropolone (7-HT). In this study, we show that this BGC also makes 3,7-dihydroxytropolone (3,7-dHT), which has strong antimicrobial activity toward Streptomyces scabies, a potato pathogen. Through metabolomics and reporter assays, we unveil the involvement of cluster-situated genes in generating phenylacetyl-coenzyme A, a key precursor for tropolone biosynthesis via the phenylacetic acid catabolon. The clustering of these phenylacetic acid genes within tropolone BGCs is unusual in other Gram-negative bacteria. Our findings support the interception of phenylacetic acid catabolism via an enoyl-CoA dehydratase encoded in the BGC, as well as highlighting an essential role for a conserved thioesterase in biosynthesis. Biochemical assays were used to show that this thioesterase functions after a dehydrogenation–epoxidation step catalyzed by a flavoprotein. We use this information to identify diverse uncharacterized BGCs that encode proteins with homology to flavoproteins and thioesterases involved in tropolone biosynthesis. This study provides insights into tropolone biosynthesis in Pseudomonas, laying the foundation for further investigations into the ecological role of tropolone production

Activation of Secondary Metabolite Gene Clusters in Streptomyces clavuligerus by the PimM Regulator of Streptomyces natalensis

Expression of non-native transcriptional activators may be a powerful general method to activate secondary metabolites biosynthetic pathways. PAS-LuxR regulators, whose archetype is PimM, activate the biosynthesis of polyene macrolide antifungals and other antibiotics, and have been shown to be functionally preserved across multiple Streptomyces strains. In this work we show that constitutive expression of pimM in Streptomyces clavuligerus ATCC 27064 significantly affected its transcriptome and modifies secondary metabolism. Almost all genes in three secondary metabolite clusters were overexpressed, including the clusters responsible for the biosynthesis of the clinically important clavulanic acid and cephamycin C. In comparison to a control strain, this resulted in 10- and 7-fold higher production levels of these metabolites, respectively. Metabolomic and bioactivity studies of S. clavuligerus::pimM also revealed deep metabolic changes. Antifungal activity absent in the control strain was detected in S. clavuligerus::pimM, and determined to be the result of a fivefold increase in the production of the tunicamycin complex