Biology subjects

Seow, D. C. S.

Publications and source records attributed to Seow, D. C. S..

3 recordsLinked to original sources

bioRxiv2022

Cost-effective hybrid long-short read assembly delineates alternative GC-rich Streptomyces chassis for natural product discovery

With the advent of rapid automated in silico identification of biosynthetic gene clusters (BGCs), genomics presents vast opportunities to accelerate natural product (NP) discovery. However, prolific NP producers, Streptomyces, are exceptionally GC-rich (>80%) and highly repetitive within BGCs. These pose challenges in sequencing and high-quality genome assembly which are currently circumvented via intensive sequencing. Here, we outline a more cost-effective workflow using multiplex Illumina and Oxford Nanopore sequencing with hybrid long-short read assembly algorithms to generate high quality genomes. Our protocol involves subjecting long read-derived assemblies to up to 4 rounds of polishing with short reads to yield accurate BGC predictions. We successfully sequenced and assembled 8 GC-rich Streptomyces genomes whose lengths range from 7.1 to 12.1 Mb at an average N50 of 5.9 Mb. Taxonomic analysis revealed previous misrepresentation among these strains and allowed us to propose a potentially new species, Streptomyces sydneybrenneri. Further comprehensive characterization of their biosynthetic, pan-genomic and antibiotic resistance features especially for molecules derived from type I polyketide synthase (PKS) BGCs reflected their potential as NP chassis. Thus, the genome assemblies and insights presented here are envisioned to serve as gateway for the scientific community to expand their avenues in NP discovery. Graphic abstractSchematic of hybrid long- and short read assembly workflow for genome sequencing of GC-rich Streptomyces. Boxes shaded blue and grey correspond to experimental and in silico workflows, respectively. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/519232v2_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1395af0org.highwire.dtl.DTLVardef@81798forg.highwire.dtl.DTLVardef@539c92org.highwire.dtl.DTLVardef@14c366f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIA cost-effective genome sequencing approach for GC-rich Streptomyces is presented C_LIO_LIHybrid assembly improves BGC annotation and identification C_LIO_LIA new species, Streptomyces sydneybrenneri, identified by taxonomic analysis C_LIO_LIGenomes of 8 Streptomyces species are reported and analysed in this study C_LI

genomics↗

bioRxiv2021

Application of Cas12j for Streptomyces editing and cluster activation

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are also oftentimes strain dependent, subsequently a variety of Cas proteins would allow for flexibility and enable genetic manipulation within a wider range of Streptomyces strains. In this work, we have further expanded the Cas toolbox by presenting the first example of Cas12j mediated editing in Streptomyces sp. A34053. In our study, we have also observed significantly improved editing efficiencies with Acidaminococcus sp. Cas12j compared to Cas12a, Francisella tularensis subsp. novicida U112s type V-A Cas (FnCpf1).

microbiology↗

bioRxiv2018

Biosynthetic intermediates of the enterobacterial common antigen overcome outer membrane lipid dyshomeostasis in Escherichia coli

The outer membrane (OM) is an essential component of the Gram-negative bacterial envelope that protects cells against external threats. To maintain a functional OM, cells require distinct mechanisms to ensure balance of proteins and lipids in the membrane. Mutations in OM biogenesis and/or homeostasis pathways often result in permeability defects, but how molecular changes in the OM affect barrier function is unclear. Here, we seek potential mechanism(s) that can alleviate permeability defects in Escherichia coli cells lacking the Tol-Pal complex, which accumulate excess PLs in the OM. We identify mutations in enterobacterial common antigen (ECA) biosynthesis that re-establish OM barrier function against large hydrophilic molecules, yet did not restore lipid homeostasis. Furthermore, we demonstrate that build-up of biosynthetic intermediates, but not loss of ECA itself, contributes to the rescue. This suppression of OM phenotypes is unrelated to known effects that accumulation of ECA intermediates have on the cell wall. Finally, we reveal that an unusual diacylglycerol pyrophosphoryl-linked lipid species also accumulates in ECA mutants, and might play a role in the rescue phenotype. Our work provides insights into how OM barrier function can be restored independent of lipid homeostasis, and highlights previously unappreciated effects of ECA-related species in OM biology.

microbiology↗