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Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene

Lookup NU author(s): Professor Tracy Palmer FRS FRSE FMedSciORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


Bacterial lipoproteins are extracellular proteins tethered to cell membranes by covalently attached lipids. Deleting the lipoprotein signal peptidase (lsp) gene in Streptomyces coelicolor results in growth and developmental defects that cannot be restored by reintroducing lsp. This led us to hypothesise that lsp is essential and that the lsp mutant we isolated previously had acquired compensatory secondary mutations. Here we report resequencing of the genomes of wild-type M145 and the cis-complemented Δlsp mutant (BJT1004) to map and identify these secondary mutations but we show that they do not increase the efficiency of disrupting lsp and are not lsp suppressors. We provide evidence that they are induced by introducing the cosmid St4A10Δ;lsp, as part of ReDirect PCR mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using a suicide vector (which does not result in gene duplication) still results in growth and developmental delays and we conclude that loss of Lsp function results in developmental defects due to the loss of all lipoproteins from the cell membrane. Significantly, our results also indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to phenotypes not necessarily linked to the gene(s) of interest.

Publication metadata

Author(s): Munnoch JT, Widdick DA, Chandra G, Sutcliffe IC, Palmer T, Hutchings MI

Publication type: Article

Publication status: Published

Journal: Scientific Reports

Year: 2016

Volume: 6

Online publication date: 12/07/2016

Acceptance date: 20/06/2016

Date deposited: 14/02/2019

ISSN (electronic): 2045-2322

Publisher: Nature Publishing Group


DOI: 10.1038/srep29495

PubMed id: 27404047


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Funder referenceFunder name
BBSRC grants BB/F009429/1 and BB/F009224/1 to MIH and TP, respectively.