Role Of An FtsK Homolog In Genetic Stability In Streptomyces Coelicolor A3(2)

Streptomyces coelicolor A3(2) does not have a canonical cell division cycle duringmost of its complex life cycle, yet it contains a gene (ftsK_SC) encoding a protein similar toFtsK, which couples the completion of cell division and chromosome segregation inunicellular bacteria such as Escherichia coli. This work is to investigate the role of ftsK_SCduring the development of Streptomyces.Two ftsK_SC null mutants were constructed: WL1, containing an aac(3)â…£genereplacement, and WL11, an in-frame deletion mutant. The observation and complementexperiments demonstrated that the ftsK_SC null mutation resulted in genetic instability inStreptomyces.Bioinformatic analysis indicated that, like other FtsK/Spoâ…¢E family members, FtsK_SCconsists of three main parts: an N-terminal trans-membrane domain was separated from aC-terminal conserved ATP-dependent DNA translocase domain by a linker region. Thecomplement experiments of truncated FtsK_SC versions suggested that the three parts areall important for the maintenance of genetic stability.The germination experiment and the increased frequency of thechloramphenicol-sensitive and arginine auxotrophs mutation indicated that the ftsK_SCdeletion might be associated with genomic instability. The results of 5 PCR experimentsshowed that most of abnormal colonies had lost one or both of the chromosome terminalfragments and some of these deletions removed more than 841 kb.The observation of the FtsK_SC-EGFP fusion protein located in Streptomyces hyphaeindicated a role for FtsK_SC in chromosome segregation during sporulation. It alsosuggested that the cytoplasmic location of FtsK_SC is required for its function.Use of a fluorescent reporter showed that in ftsK_SC mutans several spore compartmentsin most spore chains failed to express the late sporulation-specific sigma factor gene sigF,even though they contained chromosomal DNA. The observation of these sporecompartments suggested that sigF expression is autonomously activated in each sporecompartment in response to completion of chromosome transfer which would thus be apreviously unknown checkpoint for late sporulation-specific gene expression.The location of the FtsZ_SC-EGFP fusion protein in the fisK_SC null mutant showed that inS. coelicolor the ftsK_SC deletion didn't affect the location of the division protein FtsZ_SCduring sporulation. None of the mutant cumulative effect was been observed in the parAB and ftsK_SCdouble mutants implying that the roles of ParAB and FtsK_SC in chromosome segregationare substantive.S1 nuclease protection revealed that there were at least two promoters to transcriptftsK_SC: one was over 400 bp upstream the start coden GTG; the other was upstream theosaB gene implying co-transcription. More than one promoter for ftsK_SC implies that theexpression of ftsK_SC may be regulated in a strict and complicated way.Another ftsK-related gene SCO4508 in S. coelicolor was disrupted. No distinctphenotype was observed compared with the parent strains respectively. Bioinformaticsanalysis of functional domains suggested that the SCO4508 product may be a YukA-liketransporter protein, a crucial component of the Gram-positive typeâ…£secretion system.