Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals: Streptomyces strains and phages for antibiotic discovery

Abstract

17 The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of 18 healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the 19 world's antibiotics are derived from natural products produced by Streptomyces encoded 20 biosynthetic gene clusters. Thus, in order to identify novel gene clusters, we sequenced the 21 genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and 22 used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the 23 mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 24 101 biosynthetic gene clusters. Some of these clusters were predicted to produce previously 25 studied antibiotics; however, the known mechanisms of these molecules could not fully account 26 for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode 27 antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, 28 three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus 29 aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial 30 strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, 31 and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 32 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a 33 useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas 34 systems and specific spacer sequences that allowed us to predict phage host ranges.35 Ultimately, this study identified Streptomyces strains with the potential to produce novel 36 chemical matter as well as integrase-encoding phages that could potentially be used to 37 manipulate these strains.38 Introduction 39 Antibiotic discovery is an international priority requiring immediate action. 1 The increasing 40 prevalence of multi-drug resistant (MDR) bacterial pathogens has resulted in an increased use 41 of last-resort antibiotics. [1][2][3] Microbes that produce natural products are the most prolific source 42 of clinically approved antibiotics. 4 In particular, soil dwelling Actinobacteria, notably 43 Streptomyces, account for two-thirds of the antibiotics currently on the market. 5-7 Despite 44 intensive studies, however, the full potential of microbes to produce natural products has not 45 been realized. 8 Genome mining studies have shown that microbes encode many biosynthetic 46 gene clusters (BGCs) that have not yet been characterized. 8 It is widely assumed that many of 47 these clusters produce novel natural products and that further characterization of Streptomyces 48 bacteria increases the probability of identifying molecules with unique chemical structures and 49 new mechanisms of action. 9 50 51 In addition to identifying Streptomyces strains containing potentially novel BG...