Comparative analysis of assembly algorithms to optimize biosynthetic gene cluster identification in novel marine actinomycete genomes

Many marine sponges harbor dense communities of microbes that aid in the chemical defense of these nonmotile hosts. Metabolites that comprise this chemical arsenal can have pharmaceutically-relevant activities such as antibacterial, antiviral, antifungal and anticancer properties. Previous investigation of the Caribbean giant barrel sponge Xestospongia muta revealed a microbial community including novel Actinobacteria, a phylum well known for its production of antibiotic compounds. This novel assemblage was investigated for its ability to produce compounds that inhibit M. tuberculosis by using a bioinformatics approach. Microbial extracts were tested for their ability to inhibit growth of M. tb and genomes of the 11 strains that showed anti-M. tb activity including Micrococcus (n=2), Micromonospora (n=4), Streptomyces (n=3), and Brevibacterium spp. (n=2) were sequenced by using Illumina MiSeq. Three assembly algorithms/pipelines (SPAdes, A5-miseq and Shovill) were compared for their ability to construct contigs with minimal gaps to maximize the probability of identifying complete biosynthetic gene clusters (BGCs) present in the genomes. Although A5-miseq and Shovill usually assembled raw reads into the fewest contigs, after necessary post-assembly filtering, SPAdes generally produced the most complete genomes with the fewest contigs. This study revealed the strengths and weaknesses of the different assemblers based on their ease of use and ability to be manipulated based on output format. None of the assembly methods handle contamination well and high-quality DNA is a prerequisite. BGCs of compounds with known anti-TB activity were identified in all Micromonospora and Streptomyces strains (genomes > 5 Mb), while no such BGCs were identified in Micrococcus or Brevibacterium strains (genomes < 5 Mb). The majority of the putative BGCs identified were located on contig edges, emphasizing the inability of short-read assemblers to resolve repeat regions and supporting the need for long-read sequencing to fully resolve BGCs

BIOPROSPECTING MARINE ACTINOMYCETES FOR NOVEL ANTI-TUBERCULOSIS DRUGS

Mycobacterium tuberculosis (M. tb), the causative agent of the infectious lung disease tuberculosis (TB), is estimated to infect approximately 1.7 billion people worldwide. This pathogen was responsible for more than 1.5 million deaths in 2020, and is likely to remain a global threat for many years to come due to the rising incidence of antibiotic resistance, as well as dramatic setbacks in treatment due to the ongoing COVID-19 pandemic. There is an urgent demand for novel therapeutics to treat the disease through unique mechanisms of action. In the search for these drugs, a novel collection of 101 marine actinomycetes previously isolated from a Caribbean giant barrel sponge Xestospongia muta was investigated for their ability to inhibit M. tb growth. Thirteen novel strains of Micrococcus, Micromonospora, Brevibacterium, and Streptomyces were identified as consistently producing extracts that inhibit M. tb in a dose-dependent manner. After sequencing the genomes of these strains, a comparative analysis between three assembly algorithms (SPAdes, A5-miseq, Shovill) was performed to determine which program yielded the best assembly from Illumina MiSeq data for biosynthetic gene cluster (BGC) mining. Upon characterizing the biosynthetic potential of each strain, two isolates generating highly potent extracts – Micrococcus sp. strain R8502A1 and Micromonospora sp. strain R45601 – were selected for further analysis through a dual genomics and chemistry-enabled approach. No compounds with obvious anti-TB activity were detected in the genome of Micrococcus sp. strain R8502A1, suggesting production of an elusive and novel anti-TB compound through a cryptic pathway. A comprehensive examination of all BGC-associated domains was conducted to evaluate possible biosynthetic pathways linked to the anti-TB activity observed. The active component of the Micrococcus extract was further isolated with high performance liquid chromatography (HPLC) and is under investigation with liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). In contrast, a BGC with 94% similarity to the selective and potent but poorly soluble anti-TB compound diazaquinomycin H/J was identified in the genome of Micromonospora sp. strain R45601, suggesting production of a chemical analog. LC-MS detected four peaks of interest, two of which are associated with mass-to-charge (m/z) values that do not correlate with any previously identified diazaquinomycin analogs. This analysis has identified at least two potentially novel anti-TB compounds, supporting continued investigation into sponge-associated marine actinomycetes for novel therapeutics

Effects of low-dose alcohol exposure in adolescence on subsequent alcohol drinking in adulthood in a rat model of depression

Objective: Adolescence drinking and subsequent development of alcohol use disorder (AUD) is a worldwide health concern. In particular, mood dysregulation or early alcohol exposure can be the cause of heavy drinking in some individuals or a consequence of heavy drinking in others. Methods: This study investigated the effects of voluntary alcohol intake during adolescence, i.e. continuous 10% alcohol access between postnatal days (PND) 29 to 43 and olfactory bulbectomy (OBX) model of depression (performed on PND 59) on alcohol drinking in Wistar rats during adulthood (PND 80–120, intermittent 20% alcohol access). In addition, the effect of NBQX, an AMPA/kainate receptor antagonist (5 mg/kg, IP) on spontaneous alcohol consumption was examined. Results: Rats exposed to 10% alcohol during adolescence exhibited a lower 20% alcohol intake in the intermittent paradigm during adulthood, while the OBX-induced phenotype did not exert a significant effect on the drinking behaviour. NBQX exerted a transient reduction on alcohol intake in the OBX rats. Conclusions: Our results indicate that exposure to alcohol during adolescence can affect alcohol drinking in adulthood and that further exploration of AMPA and/or kainate receptor antagonists in co-morbid alcoholism-depression is warranted