The essential role of <scp>SepF</scp> in mycobacterial division

Gola, Susanne; Münder, Thomas; Casonato, Stefano; Manganelli, Riccardo; Vicente, Miguel

doi:10.1111/mmi.13050

Cited by 60 publications

(75 citation statements)

References 101 publications

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“…S. venezuelae lacks homologs of ZipA and FtsA, the FtsZstabilizing proteins that anchor the Z-ring to the membrane in many other bacteria, but it encodes the actinomycete-specific proteins SsgA and SsgB, involved in FtsZ-ring positioning (16) and three SepF-like proteins: Sven1372, Sven1734, and Sven5776. Sven1734 is encoded in the division and cell wall (dcw) gene cluster, like the single SepF protein found in B. subtilis and Mycobacterium tuberculosis (23,32). Based on this synteny, we named Sven1734 SepF and the additional Streptomyces SepF-like proteins SepF2 (Sven5776) and SepF3 (Sven1372).…”

Section: Resultsmentioning

confidence: 99%

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Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Schlimpert

Wasserström

Chandra

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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During sporulation, the filamentous bacteria Streptomyces undergo a massive cell division event in which the synthesis of ladders of sporulation septa convert multigenomic hyphae into chains of unigenomic spores. This process requires cytokinetic Z-rings formed by the bacterial tubulin homolog FtsZ, and the stabilization of the newly formed Z-rings is crucial for completion of septum synthesis. Here we show that two dynamin-like proteins, DynA and DynB, play critical roles in this process. Dynamins are a family of large, multidomain GTPases involved in key cellular processes in eukaryotes, including vesicle trafficking and organelle division. Many bacterial genomes encode dynamin-like proteins, but the biological function of these proteins has remained largely enigmatic. Using a cell biological approach, we show that the two Streptomyces dynamins specifically localize to sporulation septa in an FtsZ-dependent manner. Moreover, dynamin mutants have a cell division defect due to the decreased stability of sporulation-specific Z-rings, as demonstrated by kymographs derived from time-lapse images of FtsZ ladder formation. This defect causes the premature disassembly of individual Z-rings, leading to the frequent abortion of septum synthesis, which in turn results in the production of long spore-like compartments with multiple chromosomes. Two-hybrid analysis revealed that the dynamins are part of the cell division machinery and that they mediate their effects on Z-ring stability during developmentally controlled cell division via a network of protein-protein interactions involving DynA, DynB, FtsZ, SepF, SepF2, and the FtsZ-positioning protein SsgB.he active organization and remodeling of cellular membranes is a fundamental process for all organisms. Many of these remodeling events involve the reshaping, fission, or fusion of lipid bilayers to generate new organelles, release transport vesicles, or stabilize specific membrane structures. In eukaryotes, key cellular processes, such as the fission and fusion of mitochondria, the division of chloroplasts, endocytosis, and viral resistance, are mediated by members of the dynamin superfamily (1). Dynamins and dynamin-like proteins are mechanochemical GTPases that polymerize into helical scaffolds at the surface of membranes. GTP hydrolysis is coupled to a radical conformational change in the protein structure that forces the underlying lipid layer into an energetically unstable conformation that promotes membrane rearrangements, probably via a hemifusion intermediate (2, 3).Dynamin-like proteins are found in many bacterial species, yet the precise roles of these proteins are still largely unknown. They share a conserved domain architecture with the canonical human Dynamin 1, including the N-terminal GTPase domain, a neck domain involved in dynamin dimerization, and a trunk domain involved in stimulation of GTPase activity (4, 5). In contrast, bacterial dynamins lack the pleckstrin homology motif and proline-rich sequences found in classical dynamins and contain instead o...

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Section: Resultsmentioning

confidence: 99%

“…Instead, in other bacterial systems FtsZ filaments are tethered to the membrane through interaction with membrane-anchoring proteins such as FtsA, ZipA, and SepF (20)(21)(22)(23)(24). Additional factors, such as the ZapC and ZapD, are critically involved in the stabilization of preformed Z-rings to ensure normal cell division (25)(26)(27).…”

mentioning

confidence: 99%

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Schlimpert

Wasserström

Chandra

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…Since then, the TetR/Pip‐OFF system has been successfully used by several groups to study essential genes and validate drug targets in vitro and in vivo in different mycobacterial species (Serafini et al ., 2009, 2013; Cortes et al ., 2011; Di Luca et al ., 2012; Mondino et al ., 2013; Ventura et al ., 2013; Ahmed et al ., 2014, 2016; Bazet Lyonnet et al ., 2014; Bhowmick et al ., 2014; Boldrin et al ., 2014; Kolly et al ., 2014a,b; Pandey and Rodriguez, 2014; Verma and Chatterji, 2014; Gola et al ., 2015; Gupta et al ., 2015; Mori et al ., 2015; Hu et al ., 2016; Degiacomi et al ., 2017). This broad experience taught us that the main drawback of this otherwise very succesful system was the strength of the P ptr promoter, which causes overexpression of the gene of interest, when this is physiologically expressed at low level, with resulting accumulation of its product.…”

Section: Introductionmentioning

confidence: 99%

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Boldrin

Degiacomi

Serafini

et al. 2017

Microbial Biotechnology

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SummaryA range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip‐OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr, a strong Pip‐repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild‐type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip‐OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole‐cell screening assay to identify inhibitors of this enzyme.

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“…Cell division is initiated by the assembly of the protein FtsZ into a ring-like structure at midcell. FtsZ assembles concomitantly with FtsA, which anchors FtsZ to the membrane together with SepF (Bi and Lutkenhaus, 1991; Jensen et al, 2005; Duman et al, 2013; Gola et al, 2015; Gupta et al, 2015), and ZapA that promotes higher order assemblies of FtsZ protofilaments (Gueiros-Filho and Losick, 2002). In the Gram-positive bacterium Bacillus subtilis , the membrane protein EzrA is also one of the earliest proteins that is recruited to the Z-ring, and is involved in the regulation of FtsZ polymerization and the coordination of peptidoglycan synthesis of the division septum (Levin et al, 1999; Claessen et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Cooperative Recruitment of FtsW to the Division Site of Bacillus subtilis

2016

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Five essential proteins are known to assemble at the division site of Bacillus subtilis. However, the recruitment of the FtsW homolog is still unclear. Here, we take advantage of spore germination to facilitate the depletion of essential proteins and to study the divisome assembly in the absence of previous division events. We show that, unlike what has been shown for the Escherichia coli divisome, the assembly of FtsW is interdependent with the localization of PBP 2B and FtsL, which are key components of the membrane bound division complex. Interestingly, the Z-ring appeared to disassemble upon prolonged depletion of late division proteins. Nevertheless, we could restore Z-ring formation and constriction by re-inducing FtsW, which suggests that the stability of the Z-ring is stimulated by the assembly of a functional division complex.

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The essential role of SepF in mycobacterial division

Cited by 60 publications

References 101 publications

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Two dynamin-like proteins stabilize FtsZ rings during Streptomyces sporulation

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Cooperative Recruitment of FtsW to the Division Site of Bacillus subtilis

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