scholarly journals Physical Interaction between Coat Morphogenetic Proteins SpoVID and CotE Is Necessary for Spore Encasement in Bacillus subtilis

2012 ◽  
Vol 194 (18) ◽  
pp. 4941-4950 ◽  
Author(s):  
Melissa de Francesco ◽  
Jake Z. Jacobs ◽  
Filipa Nunes ◽  
Mónica Serrano ◽  
Peter T. McKenney ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Mutational Analysis ◽  
Physical Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Protein Protein Interactions ◽  
Content Type ◽  
Morphogenetic Protein ◽  
Physical Interactions

ABSTRACTEndospore formation byBacillus subtilisis a complex and dynamic process. One of the major challenges of sporulation is the assembly of a protective, multilayered, proteinaceous spore coat, composed of at least 70 different proteins. Spore coat formation can be divided into two distinct stages. The first is the recruitment of proteins to the spore surface, dependent on the morphogenetic protein SpoIVA. The second step, known as encasement, involves the migration of the coat proteins around the circumference of the spore in successive waves, a process dependent on the morphogenetic protein SpoVID and the transcriptional regulation of individual coat genes. We provide genetic and biochemical evidence supporting the hypothesis that SpoVID promotes encasement of the spore by establishing direct protein-protein interactions with other coat morphogenetic proteins. It was previously demonstrated that SpoVID directly interacts with SpoIVA and the inner coat morphogenetic protein, SafA. Here, we show by yeast two-hybrid and pulldown assays that SpoVID also interacts directly with the outer coat morphogenetic protein, CotE. Furthermore, by mutational analysis, we identified a specific residue in the N-terminal domain of SpoVID that is essential for the interaction with CotE but dispensable for the interaction with SafA. We propose an updated model of coat assembly and spore encasement that incorporates several physical interactions between the principal coat morphogenetic proteins.

scholarly journals Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Kurt M. Dahlstrom ◽  
Krista M. Giglio ◽  
Alan J. Collins ◽  
Holger Sondermann ◽  
George A. O’Toole
Keyword(s):  
Protein Interactions ◽  
Second Messenger ◽  
Target Protein ◽  
Physical Interaction ◽  
Content Type ◽  
Signaling Specificity ◽  
Cellular Processes ◽  
Diguanylate Cyclases ◽  
Cellular Behaviors ◽  
Physical Interactions

ABSTRACT Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such “local signaling” is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network. IMPORTANCE An important question in microbiology is how bacteria make decisions using a signaling network made up of proteins that make, break, and bind the second messenger c-di-GMP, which is responsible for controlling many cellular behaviors. Previous work has shown that a given DGC enzyme will signal for specific cellular outputs, despite making the same diffusible molecule as its sibling DGCs in the unpartitioned space of the bacterial cell. Understanding how one DGC differentiates its output from the dozens of other such enzymes in the cell is synonymous with understanding a large component of the bacterial decision-making machinery. We present evidence for a helix on a DGC used to physically associate with its target protein, which is necessary to achieve maximal signaling.

scholarly journals Functional and Physical Interactions between AML1 Proteins and an ETS Protein, MEF: Implications for the Pathogenesis of t(8;21)-Positive Leukemias

1999 ◽  
Vol 19 (5) ◽  
pp. 3635-3644 ◽  
Author(s):  
Shifeng Mao ◽  
Richard C. Frank ◽  
Jin Zhang ◽  
Yasushi Miyazaki ◽  
Stephen D. Nimer
Keyword(s):  
Dna Binding ◽  
Protein Interactions ◽  
Myeloid Leukemia ◽  
Mutational Analysis ◽  
Myeloid Differentiation ◽  
Protein Protein Interactions ◽  
Ets Proteins ◽  
Ets Family ◽  
Physical Interactions ◽  
Definitive Hematopoiesis

ABSTRACT The AML1 and ETS families of transcription factors play critical roles in hematopoiesis; AML1, and its non-DNA-binding heterodimer partner CBFβ, are essential for the development of definitive hematopoiesis in mice, whereas the absence of certain ETS proteins creates specific defects in lymphopoiesis or myelopoiesis. The promoter activities of numerous genes expressed in hematopoietic cells are regulated by AML1 proteins or ETS proteins. MEF (for myeloid ELF-1-like factor) is a recently cloned ETS family member that, like AML1B, can strongly transactivate several of these promoters, which led us to examine whether MEF functionally or physically interacts with AML1 proteins. In this study, we demonstrate direct interactions between MEF and AML1 proteins, including the AML1/ETO fusion protein, in t(8;21)-positive acute myeloid leukemia (AML) cells. Using mutational analysis, we identified a novel ETS-interacting subdomain (EID) in the C-terminal portion of the Runt homology domain (RHD) in AML1 proteins and determined that the N-terminal region of MEF was responsible for its interaction with AML1. MEF and AML1B synergistically transactivated an interleukin 3 promoter reporter gene construct, yet the activating activity of MEF was abolished when MEF was coexpressed with AML1/ETO. The repression by AML1/ETO was independent of DNA binding but depended on its ability to interact with MEF, suggesting that AML1/ETO can repress genes not normally regulated by AML1 via protein-protein interactions. Interference with MEF function by AML1/ETO may lead to dysregulation of genes important for myeloid differentiation, thereby contributing to the pathogenesis of t(8;21) AML.

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.

Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions

1991 ◽  
Vol 11 (3) ◽  
pp. 1578-1589
Author(s):  
L D Fresco ◽  
D S Harper ◽  
J D Keene
Keyword(s):  
Protein Interactions ◽  
Leucine Zipper ◽  
Tandem Repeats ◽  
Mutational Analysis ◽  
Particle Density ◽  
Protein Protein Interactions ◽  
Small Nuclear Ribonucleoprotein ◽  
Cell Extracts ◽  
Associated Proteins ◽  
Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

scholarly journals In Vivo Phosphorylation of Partner Switching Regulators Correlates with Stress Transmission in the Environmental Signaling Pathway of Bacillus subtilis

2004 ◽  
Vol 186 (18) ◽  
pp. 6124-6132 ◽  
Author(s):  
Tae-Jong Kim ◽  
Tatiana A. Gaidenko ◽  
Chester W. Price
Keyword(s):  
Bacillus Subtilis ◽  
Protein Interactions ◽  
Current Model ◽  
Ethanol Stress ◽  
Protein Protein Interactions ◽  
General Stress Response ◽  
Switching Regulators ◽  
Stress Transmission ◽  
Stress Signals

ABSTRACT Exposure of bacteria to diverse growth-limiting stresses induces the synthesis of a common set of proteins which provide broad protection against future, potentially lethal stresses. Among Bacillus subtilis and its relatives, this general stress response is controlled by the σB transcription factor. Signals of environmental and energy stress activate σB through a multicomponent network that functions via a partner switching mechanism, in which protein-protein interactions are governed by serine and threonine phosphorylation. Here, we tested a central prediction of the current model for the environmental signaling branch of this network. We used isoelectric focusing and immunoblotting experiments to determine the in vivo phosphorylation states of the RsbRA and RsbS regulators, which act in concert to negatively control the RsbU environmental signaling phosphatase. As predicted by the model, the ratio of the phosphorylated to unphosphorylated forms of both RsbRA and RsbS increased in response to salt or ethanol stress. However, these two regulators differed substantially with regard to the extent of their phosphorylation under both steady-state and stress conditions, with RsbRA always the more highly modified. Mutant analysis showed that the RsbT kinase, which is required for environmental signaling, was also required for the in vivo phosphorylation of RsbRA and RsbS. Moreover, the T171A alteration of RsbRA, which blocks environmental signaling, also blocked in vivo phosphorylation of RsbRA and impeded phosphorylation of RsbS. These in vivo results corroborate previous genetic analyses and link the phosphorylated forms of RsbRA and RsbS to the active transmission of environmental stress signals.

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