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

By John T Munnoch, David A. Widdick, Govind Chandra, Iain C. Sutcliffe, Tracy Palmer, Matthew I. Hutchings

Posted 19 Apr 2016
bioRxiv DOI: 10.1101/049320 (published DOI: 10.1038/srep29495)

Bacterial lipoproteins are a class of 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 the lsp. We report resequencing of the genomes of the wild-type M145 and the cis-complemented ∆lsp mutant (BJT1004), mapping and identifying secondary mutations, including an insertion into a novel putative small RNA, scr6809. Disruption of scr6809 led to a range of developmental phenotypes. However, these secondary mutations do not increase the efficiency of disrupting lsp suggesting they are not lsp specific suppressors. Instead we suggest that these were induced by introducing the cosmid St4A10∆lsp as part of the Redirect mutagenesis protocol, which transiently duplicates a number of important cell division genes. Disruption of lsp using no gene duplication resulted in the previously observed phenotype. We conclude that lsp is not essential in S. coelicolor but loss of lsp does lead to developmental defects due to the loss of lipoproteins from the cell. Significantly, our results indicate the use of cosmid libraries for the genetic manipulation of bacteria can lead to unexpected phenotypes not necessarily linked to the gene or pathway of interest.

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