RT Journal Article SR Electronic T1 Cosmid based mutagenesis causes genetic instability in Streptomyces coelicolor, as shown by targeting of the lipoprotein signal peptidase gene JF bioRxiv FD Cold Spring Harbor Laboratory SP 049320 DO 10.1101/049320 A1 John T Munnoch A1 David A. Widdick A1 Govind Chandra A1 Iain C. Sutcliffe A1 Tracy Palmer A1 Matthew I Hutchings YR 2016 UL http://biorxiv.org/content/early/2016/04/19/049320.abstract AB 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 Δlsp 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 St4A10Δlsp 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.