A synthetic <i>Escherichia coli</i> system identifies a conserved origin tethering factor in Actinobacteria

Donovan, Catriona; Sieger, Boris; Krämer, Reinhard; Bramkamp, Marc

doi:10.1111/j.1365-2958.2012.08011.x

Cited by 74 publications

(97 citation statements)

References 44 publications

(70 reference statements)

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“…Fitting with this, our experiments indicated that the C-terminal domain is the interaction module for RacA, which is a soluble cytoplasmic protein. It was recently reported that the interaction of C. glutamicum ParB, which is a chromosome-binding protein like RacA, with its cognate DivIVA requires central regions of the C-terminal domain as well (9). Our results thus confirm earlier speculations that the sporulation and the division functions of DivIVA can be separated.…”

Section: Discussionsupporting

confidence: 82%

“…Membrane binding and curvature sensitivity appear to be intrinsic features of DivIVA, as it was shown that DivIVA of Bacillus subtilis also localizes to curved membranes when expressed in other, nonrelated species, including yeast cells (4). DivIVA is used as a scaffold and recruits other proteins that function in cell division, cell wall biosynthesis, secretion, genetic competence, or chromosome segregation (5)(6)(7)(8)(9)(10)(11)(12)(13). The proteins that interact with DivIVA are therefore diverse and comprise both transmembrane and cytosolic proteins (14).…”

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confidence: 99%

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Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

Baarle

Celik

Kaval

et al. 2012

Journal of Bacteriology

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e DivIVA proteins are curvature-sensitive membrane binding proteins that recruit other proteins to the poles and the division septum. They consist of a conserved N-terminal lipid binding domain fused to a less conserved C-terminal domain. DivIVA homologues interact with different proteins involved in cell division, chromosome segregation, genetic competence, or cell wall synthesis. It is unknown how DivIVA interacts with these proteins, and we used the interaction of Bacillus subtilis DivIVA with MinJ and RacA to investigate this. MinJ is a transmembrane protein controlling division site selection, and the DNA-binding protein RacA is crucial for chromosome segregation during sporulation. Initial bacterial two-hybrid experiments revealed that the C terminus of DivIVA appears to be important for recruiting both proteins. However, the interpretation of these results is limited since it appeared that C-terminal truncations also interfere with DivIVA oligomerization. Therefore, a chimera approach was followed, making use of the fact that Listeria monocytogenes DivIVA shows normal polar localization but is not biologically active when expressed in B. subtilis. Complementation experiments with different chimeras of B. subtilis and L. monocytogenes DivIVA suggest that MinJ and RacA bind to separate DivIVA domains. Fluorescence microscopy of green fluorescent proteintagged RacA and MinJ corroborated this conclusion and suggests that MinJ recruitment operates via the N-terminal lipid binding domain, whereas RacA interacts with the C-terminal domain. We speculate that this difference is related to the cellular compartments in which MinJ and RacA are active: the cell membrane and the cytoplasm, respectively.

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Section: Discussionsupporting

confidence: 82%

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confidence: 99%

Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

Baarle

Celik

Kaval

et al. 2012

Journal of Bacteriology

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“…It is conceivable that the nucleation of ParA at the tip of the aerial hyphae depends on the TIPOC. Indeed, interaction between Streptomyces DivIVA and ParB has been demonstrated, albeit in the heterologous host E. coli (38). We have also shown that localization of FtsZ was compromised in the scy mutant (Fig.…”

Section: Discussionmentioning

confidence: 61%

Coiled-coil protein Scy is a key component of a multiprotein assembly controlling polarized growth inStreptomyces

Holmes¹,

Walshaw

Leggett³

et al. 2013

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

146

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Polarized growth in eukaryotes requires polar multiprotein complexes. Here, we establish that selection and maintenance of cell polarity for growth also requires a dedicated multiprotein assembly in the filamentous bacterium, Streptomyces coelicolor. We present evidence for a tip organizing center and confirm two of its main components: Scy (Streptomyces cytoskeletal element), a unique bacterial coiled-coil protein with an unusual repeat periodicity, and the known polarity determinant DivIVA. We also establish a link between the tip organizing center and the filamentforming protein FilP. Interestingly, both deletion and overproduction of Scy generated multiple polarity centers, suggesting a mechanism wherein Scy can both promote and limit the number of emerging polarity centers via the organization of the Scy-DivIVA assemblies. We propose that Scy is a molecular "assembler," which, by sequestering DivIVA, promotes the establishment of new polarity centers for de novo tip formation during branching, as well as supporting polarized growth at existing hyphal tips.bacterial polarized growth | polar multiprotein assembly | cell division | modified hendecad coiled coil H ow organisms, cells, or tissues establish polarity is one of the fundamental questions in developmental biology. In eukaryotes, from unicellular organisms to multicellular plants and animals, some of the core mechanisms for generating polarity are conserved (1). Sites for polarization must be selected using positional markers, followed by the recruitment of a complex assembly, which, in turn, orients cytoskeletal filaments that deliver vesicles to the particular sites. A specific example of polarity is polarized growth, during which cells select a single polarization site and generate elongated, cylindrical shapes, such as neuronal dendrites in animals, root hairs and pollen tubes in plants, hyphal growth of filamentous fungi, elongation of Schizosaccharomyces pombe, or the short period of polarized growth during budding in Saccharomyces cerevisiae. However, the presumed earliest examples of polarized growth can be found in bacteria. These include the actinomycete Streptomyces coelicolor, which is used as a model organism for studying morphological differentiation and filamentous growth.The complex life cycle of S. coelicolor begins with an ovoid spore that contains a single chromosome. During germination, long, multigenomic filaments (germ tubes) are formed, which branch regularly to generate a network of hyphal filaments. Branching is a necessity for exponential growth because the rate of tip elongation cannot exceed a certain maximum; hence, there is an exponential increase in the number of new tips. New tips develop on the lateral wall well behind the existing tip, a phenomenon also observed and described as apical dominance in eukaryotic filamentous fungi. When grown on semisolid agar medium, these hyphal filaments first grow across and into the solid medium, generating the vegetative mycelium, followed by the formation of an aerial mycelium by h...

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“…In B. subtilis, LCP proteins were found to interact with MreB, a cytoskeleton protein which is probably responsible for their localization (6). The localization of the LCP proteins in C. glutamicum suggests that, analogous to MreB/Tag-TUV in B. subtilis, they might be part of the elongasome, which is required for polar growth, and be spatially organized by DivIVA (17,(36)(37)(38). However, given that C. glutamicum lacks MreB and that the cytoplasmic part of LcpA is dispensable for function, the localization likely appears within the membrane or in the periplasm, and additional proteins would be required to connect it to the elongasome.…”

Section: Discussionmentioning

confidence: 99%

Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum

et al. 2016

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Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum. IMPORTANCEAbout one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics. The special cell wall architecture of Corynebacteriales is critical for treatments because it is either a direct target or a barrier that the drug has to cross. Here, we present the analysis of LcpA and LcpB of the closely related Corynebacterium glutamicum, the first of which is an essential protein involved in cell wall biogenesis. Our work provides a comprehensive characterization of the impact of LCP proteins on cell wall biogenesis in this medically and biotechnologically important class of bacteria. Special focus is set on the two periplasmic LcpA domains and their contributions to physiological function.

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A synthetic Escherichia coli system identifies a conserved origin tethering factor in Actinobacteria

Cited by 74 publications

References 44 publications

Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

Protein-Protein Interaction Domains of Bacillus subtilis DivIVA

Coiled-coil protein Scy is a key component of a multiprotein assembly controlling polarized growth inStreptomyces

Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum

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