scholarly journals The PASTA domains of Bacillus subtilis PBP2B stabilize the interaction of PBP2B with DivIB

2019 ◽  
Author(s):  
Danae Morales Angeles ◽  
Alicia Macia-Valero ◽  
Laura C. Bohorquez ◽  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Bacterial Cell Division ◽  
Penicillin Binding Protein ◽  
Protein Protein Interactions ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Two Hybrid

AbstractBacterial cell division is mediated by a protein complex known as the divisome. Many protein-protein interactions in the divisome have been characterized. In this report, we analyse the role of the PASTA (Penicillin binding protein And Serine Threonine kinase Associated)-domains of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the PBP2B PASTA domains results in impaired cell division and a heat sensitive phenotype. This resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two hybrid and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is weakened when the PBP2B PASTA domains are removed. Combined, our results show that the PBP2B PASTA domains are required to stabilize the interaction between PBP2B and DivIB.

scholarly journals The PASTA domains of Bacillus subtilis PBP2B strengthen the interaction of PBP2B with DivIB

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 826-836 ◽  
Author(s):  
Danae Morales Angeles ◽  
Alicia Macia-Valero ◽  
Laura C. Bohorquez ◽  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Bacterial Cell Division ◽  
Penicillin Binding Protein ◽  
Protein Protein Interactions ◽  
Serine Threonine Kinase ◽  
Content Type ◽  
Threonine Kinase

Bacterial cell division is mediated by a protein complex known as the divisome. Many protein–protein interactions in the divisome have been characterized. In this report, we analyse the role of the PASTA (Penicillin-binding protein And Serine Threonine kinase Associated) domains of Bacillus subtilis PBP2B. PBP2B itself is essential and cannot be deleted, but removing the PBP2B PASTA domains results in impaired cell division and a heat-sensitive phenotype. This resembles the deletion of divIB, a known interaction partner of PBP2B. Bacterial two-hybrid and co-immunoprecipitation analyses show that the interaction between PBP2B and DivIB is weakened when the PBP2B PASTA domains are removed. Combined, our results show that the PBP2B PASTA domains are required to strengthen the interaction between PBP2B and DivIB.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

scholarly journals Multiple Interactions between the Transmembrane Division Proteins of Bacillus subtilis and the Role of FtsL Instability in Divisome Assembly

2006 ◽  
Vol 188 (21) ◽  
pp. 7396-7404 ◽  
Author(s):  
Richard A. Daniel ◽  
Marie-Françoise Noirot-Gros ◽  
Philippe Noirot ◽  
Jeff Errington
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Ring Structure ◽  
Protein Protein Interactions ◽  
Essential Proteins ◽  
Hybrid Approaches ◽  
Important Player ◽  
Multiple Interactions ◽  
Two Hybrid

ABSTRACT About 11 essential proteins assemble into a ring structure at the surface of the cell to bring about cytokinesis in bacteria. Several of these proteins have their major domains located outside the membrane, forming an assembly that we call the outer ring (OR). Previous work on division in Bacillus subtilis has shown that four of the OR proteins—FtsL, DivIC, DivIB, and PBP 2B—are interdependent for assembly. This contrasts with the mainly linear pathway for the equivalent proteins in Escherichia coli. Here we show that the interdependent nature of the B. subtilis pathway could be due to effects on FtsL and DivIC stability and that DivIB is an important player in regulating this turnover. Two-hybrid approaches suggest that a multiplicity of protein-protein interactions contribute to the assembly of the OR. DivIC is unusual in interacting strongly only with FtsL. We propose a model for the formation of the OR through the mutual association of the membrane proteins directed by the cytosolic inner-ring proteins.

scholarly journals Mammalian Hippo signalling: a kinase network regulated by protein–protein interactions

2012 ◽  
Vol 40 (1) ◽  
pp. 124-128 ◽  
Author(s):  
Alexander Hergovich
Keyword(s):  
Protein Interactions ◽  
Tumour Suppressor ◽  
Protein Protein Interactions ◽  
Large Tumour ◽  
Serine Threonine Kinase ◽  
Ww Domains ◽  
Threonine Kinase ◽  
Signalling Molecules ◽  
Kinase Cascade ◽  
Hippo Signalling

The Hippo signal transduction cascade controls cell growth, proliferation and death, all of which are frequently deregulated in tumour cells. Since initial studies in Drosophila melanogaster were instrumental in defining Hippo signalling, the machinery was named after the central Ste20-like kinase Hippo. Moreover, given that loss of Hippo signalling components Hippo, Warts, and Mats resulted in uncontrolled tissue overgrowth, Hippo signalling was defined as a tumour-suppressor cascade. Significantly, all of the core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in Drosophila. Furthermore, studies in Drosophila and mammalian cell systems showed that Hippo signalling represents a kinase cascade that is tightly regulated by PPIs (protein–protein interactions). Several Hippo signalling molecules contain SARAH (Salvador/RASSF1A/Hippo) domains that mediate specific PPIs, thereby influencing the activities of MST1/2 (mammalian Ste20-like serine/threonine kinase 1/2) kinases, the human Hippo orthologues. Moreover, WW domains are present in several Hippo factors, and these domains also serve as interaction surfaces for regulatory PPIs in Hippo signalling. Finally, the kinase activities of LATS1/2 (large tumour-suppressor kinase 1/2), the human counterparts of Warts, are controlled by binding to hMOB1 (human Mps one binder protein 1), the human Mats. Therefore Hippo signalling is regulated by PPIs on several levels. In the present paper, I review the current understanding of how these regulatory PPIs are regulated and contribute to the functionality of Hippo signalling.

scholarly journals YneA, an SOS-Induced Inhibitor of Cell Division in Bacillus subtilis, Is Regulated Posttranslationally and Requires the Transmembrane Region for Activity

2010 ◽  
Vol 192 (12) ◽  
pp. 3159-3173 ◽  
Author(s):  
Allison H. Mo ◽  
William F. Burkholder
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Signal Peptide ◽  
Protein Interactions ◽  
Signal Sequence ◽  
Protein Protein Interactions ◽  
Transmembrane Region ◽  
Extracellular Proteases ◽  
Peptide Cleavage ◽  
Daughter Cell Separation

ABSTRACT Cell viability depends on the stable transmission of genetic information to each successive generation. Therefore, in the event of intrinsic or extrinsic DNA damage, it is important that cell division be delayed until DNA repair has been completed. In Bacillus subtilis, this is accomplished in part by YneA, an inhibitor of division that is induced as part of the SOS response. We sought to gain insight into the mechanism by which YneA blocks cell division and the processes involved in shutting off YneA activity. Our data suggest that YneA is able to inhibit daughter cell separation as well as septum formation. YneA contains a LysM peptidoglycan binding domain and is predicted to be exported. We established that the YneA signal peptide is rapidly cleaved, resulting in secretion of YneA into the medium. Mutations within YneA affect both the rate of signal sequence cleavage and the activity of YneA. YneA does not stably associate with the cell wall and is rapidly degraded by extracellular proteases. Based on these results, we hypothesize that exported YneA is active prior to signal peptide cleavage and that proteolysis contributes to the inactivation of YneA. Finally, we identified mutations in the transmembrane segment of YneA that abolish the ability of YneA to inhibit cell division, while having little or no effect on YneA export or stability. These data suggest that protein-protein interactions mediated by the transmembrane region may be required for YneA activity.

scholarly journals Conserved Glycines in the C Terminus of MinC Proteins Are Implicated in Their Functionality as Cell Division Inhibitors

2004 ◽  
Vol 186 (9) ◽  
pp. 2841-2855 ◽  
Author(s):  
S. Ramirez-Arcos ◽  
V. Greco ◽  
H. Douglas ◽  
D. Tessier ◽  
D. Fan ◽  
...  
Keyword(s):  
Cell Division ◽  
Protein Interactions ◽  
Thermotoga Maritima ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Mutant Proteins ◽  
E Coli ◽  
C Terminus ◽  
Close Proximity ◽  
Two Hybrid

ABSTRACT Alignment of 36 MinC sequences revealed four completely conserved C-terminal glycines. As MinC inhibits cytokinesis in Neisseria gonorrhoeae and Escherichia coli, the functional importance of these glycines in N. gonorrhoeae MinC (MinCNg) and E. coli MinC (MinCEc) was investigated through amino acid substitution by using site-directed mutagenesis. Each mutant was evaluated for its ability to arrest cell division and to interact with itself and MinD. In contrast to overexpression of wild-type MinC, overexpression of mutant proteins in E. coli did not induce filamentation, indicating that they lost functionality. Yeast two-hybrid studies showed that MinCEc interacts with itself and MinDEc; however, no interactions involving MinCNg were detected. Therefore, a recombinant MinC protein, with the N terminus of MinCEc and the C terminus of MinCNg, was designed to test for a MinCNg-MinDNg interaction. Each MinC mutant interacted with either MinC or MinD but not both, indicating the specificity of glycine residues for particular protein-protein interactions. Each glycine was mapped on the C-terminal surfaces (A, B, and C) of the solved Thermotoga maritima MinC structure. We found that MinCEc G161, residing in close proximity to the A surface, is involved in homodimerization, which is essential for MinC function. Glycines corresponding to MinCEc G135, G154, and G171, located within or adjacent to the B-C surface junction, are critical for MinC-MinD interactions. Circular dichroism revealed no gross structural perturbations of the mutant proteins, although the contribution of glycines to protein flexibility and stability cannot be discounted. Using molecular modeling, we propose that exposed conserved MinC glycines interact with exposed residues of the α-7 helix of MinD.

The pneumococcal eukaryotic-type serine/threonine protein kinase StkP co-localizes with the cell division apparatus and interacts with FtsZ in vitro

Microbiology ◽  
2010 ◽  
Vol 156 (6) ◽  
pp. 1697-1707 ◽  
Author(s):  
Carmen Giefing ◽  
Kira E. Jelencsics ◽  
Dieter Gelbmann ◽  
Beatrice M. Senn ◽  
Eszter Nagy
Keyword(s):  
Cell Division ◽  
Cellular Localization ◽  
Binding Protein ◽  
Regulation Of Gene Expression ◽  
Pneumococcal Infection ◽  
Penicillin Binding Protein ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Ftsz Ring

The importance of serine/threonine phosphorylation in signalling and regulation of gene expression in prokaryotes has been widely recognized. Driven by our interest in StkP (the pneumococcal serine/threonine kinase homologue) for vaccine development, we studied its cellular localization. We found that the C-terminally located PASTA (penicillin-binding protein and serine/threonine kinase associated) domains, but not the N-terminal kinase domain of StkP, were located on the surface of live pneumococcal cells grown in vitro and were also accessible to antibodies during pneumococcal infection in mice and man. Most importantly, we discovered, by immunofluorescence microscopy, that StkP co-localized with the cell division apparatus. StkP and FtsZ, the prokaryotic tubulin homologue, co-localized at mid-cell in most cells. Formation and constriction of the ring-like structure of StkP followed the dynamic changes of FtsZ in dividing cells. This pattern resembles that of the ‘late’ divisome protein penicillin-binding protein 2X. The lack of StkP in gene deletion mutants did not disturb FtsZ ring formation, further suggesting that StkP joins the divisome after the FtsZ ring is assembled. We also present evidence that StkP binds and phosphorylates recombinant FtsZ in vitro; however, we could not detect changes in the phosphorylation of FtsZ in a stkP deletion strain relative to wild-type cells. Based on its cell-division-dependent localization and interaction with FtsZ, we propose that StkP plays a currently undefined role in cell division of pneumococcus.

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