scholarly journals SpoVID Guides SafA to the Spore Coat inBacillus subtilis

2001 ◽  
Vol 183 (10) ◽  
pp. 3041-3049 ◽  
Author(s):  
Amanda J. Ozin ◽  
Craig S. Samford ◽  
Adriano O. Henriques ◽  
Charles P. Moran
Keyword(s):  
Direct Interaction ◽  
Spore Coat ◽  
Coat Proteins ◽  
Yeast Two Hybrid System ◽  
Spore Coat Proteins ◽  
Basement Layer ◽  
Two Hybrid ◽  
Spore Coat Protein

ABSTRACT Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encasesBacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number inPseudomonas aeruginosa

2001 ◽  
Vol 183 (22) ◽  
pp. 6636-6644 ◽  
Author(s):  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Binding Motif ◽  
Yeast Two Hybrid System ◽  
Gtp Binding ◽  
C Terminus ◽  
Enhancer Binding Protein ◽  
Two Hybrid

ABSTRACT Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption offleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

scholarly journals An XA21-Associated Kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors

2013 ◽  
Author(s):  
Xuewei Chen ◽  
Shimin Zuo ◽  
Benjamin Schwessinger ◽  
Mawsheng Chern ◽  
Patrick Canlas ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Immune Receptor ◽  
Immune Receptors ◽  
Yeast Two Hybrid System ◽  
Intracellular Domains ◽  
Activity Dependent

The rice XA21 immune receptor kinase and the structurally related XA3 receptor, confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast-two-hybrid system in a kinase activity dependent manner. OsSERK2 undergoes bidirectional trans-phosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. Taken together, these results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function.

scholarly journals Interaction of Vav with ENX-1, a putative transcriptional regulator of homeobox gene expression.

1996 ◽  
Vol 16 (6) ◽  
pp. 3066-3073 ◽  
Author(s):  
O Hobert ◽  
B Jallal ◽  
A Ullrich
Keyword(s):  
Gene Expression ◽  
Critical Role ◽  
Homeobox Gene ◽  
Transcriptional Regulators ◽  
Polycomb Group ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

The proto-oncogene product Vav plays a critical role in hematopoietic signal transduction. By using the yeast two-hybrid system, we identified a novel human protein, ENX-1, which interacts specifically with Vav both in vitro and in vivo. ENX-1 represents the human homolog of the Drosophila Enhancer of zeste gene, a member of the Polycomb group of genes, which are transcriptional regulators of homeobox gene expression. Interaction with ENX-1 suggests that Vav functions as an upstream element in the transcriptional regulation of homeobox genes, known to be important effectors in the hematopoietic system.

scholarly journals Role of Spore Coat Proteins in the Resistance of Bacillus subtilis Spores to Caenorhabditis elegans Predation

2008 ◽  
Vol 190 (18) ◽  
pp. 6197-6203 ◽  
Author(s):  
Maria-Halima Laaberki ◽  
Jonathan Dworkin
Keyword(s):  
Bacillus Subtilis ◽  
Caenorhabditis Elegans ◽  
Spore Coat ◽  
Coat Proteins ◽  
Host Interaction ◽  
Wide Range ◽  
In The Wild

ABSTRACT Bacterial spores are resistant to a wide range of chemical and physical insults that are normally lethal for the vegetative form of the bacterium. While the integrity of the protein coat of the spore is crucial for spore survival in vitro, far less is known about how the coat provides protection in vivo against predation by ecologically relevant hosts. In particular, assays had characterized the in vitro resistance of spores to peptidoglycan-hydrolyzing enzymes like lysozyme that are also important effectors of innate immunity in a wide variety of hosts. Here, we use the bacteriovorous nematode Caenorhabditis elegans, a likely predator of Bacillus spores in the wild, to characterize the role of the spore coat in an ecologically relevant spore-host interaction. We found that ingested wild-type Bacillus subtilis spores were resistant to worm digestion, whereas vegetative forms of the bacterium were efficiently digested by the nematode. Using B. subtilis strains carrying mutations in spore coat genes, we observed a correlation between the degree of alteration of the spore coat assembly and the susceptibility to the worm degradation. Surprisingly, we found that the spores that were resistant to lysozyme in vitro can be sensitive to C. elegans digestion depending on the extent of the spore coat structure modifications.

scholarly journals Rice Grassy Stunt Tenuivirus Nonstructural Protein p5 Interacts with Itself To Form Oligomeric Complexes In Vitro and In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 769-775 ◽  
Author(s):  
Pritsana Chomchan ◽  
Shi-Fang Li ◽  
Yukio Shirako
Keyword(s):  
Hybrid System ◽  
Nonstructural Protein ◽  
Transcription Activator ◽  
Yeast Two Hybrid ◽  
Western Blots ◽  
Yeast Two Hybrid System ◽  
Rice Tissue ◽  
Two Hybrid

ABSTRACT We investigated the interaction of Rice grassy stunt tenuivirus (RGSV) nonstructural protein p5, a protein of 22 kDa encoded on vRNA 5, with all 12 RGSV proteins by using a GAL4 transcription activator-based yeast two-hybrid system. The p5 protein interacted only with itself and not with any other viral protein; the interacting domains were localized within the N-terminal 96 amino acids of p5. The p5-p5 interaction was reproduced in an Sos recruitment-mediated yeast two-hybrid system as well in by far-Western blots. Native p5 protein extracted from RGSV-infected rice tissue was detected in a large complex with a molecular mass of approximately 260 kDa composed of 12 molecules of p5 or a p5 oligomer with an unidentified host factor(s).

scholarly journals Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

2003 ◽  
Vol 185 (8) ◽  
pp. 2393-2401 ◽  
Author(s):  
Yi-Ying Lee ◽  
Chiung-Fang Chang ◽  
Chueh-Ling Kuo ◽  
Meng-Ching Chen ◽  
Chien Hung Yu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hybrid System ◽  
Protein Interactions ◽  
Large Subunit ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
Two Hybrid

ABSTRACT The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpYΔI) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

scholarly journals The choice of anchoring protein influences interaction of recombinant Bacillus spores with the immune system

2017 ◽  
Vol 64 (2) ◽  
Author(s):  
Aurelia Piekarska ◽  
Paulina Pełka ◽  
Grażyna Peszyńska-Sularz ◽  
Alessandro Negri ◽  
Krzysztof Hinc ◽  
...  
Keyword(s):  
Immune System ◽  
Spore Coat ◽  
Coat Proteins ◽  
Heterologous Proteins ◽  
Protein Fusion ◽  
Mucosal Vaccination ◽  
Anchoring Protein ◽  
Bacillus Spores ◽  
Spore Coat Proteins ◽  
Spore Coat Protein

The technology of display of heterologous proteins on the surface of Bacillus subtilis spores enables use of these structures as carriers of antigens for mucosal vaccination. Currently there are no technical possibilities to predict, whether a designed fusion will be efficiently displayed on the spore surface and how such recombinant spores will interact with cells of the immune system. In this study we compared four variants of B. subtilis spores presenting a fragment of FliD protein of Clostridium difficile in fusion with CotB, CotC, CotG or CotZ spore coat proteins. We show that these spores promote their phagocytosis and activate both, J774 macrophages and JAWSII dendritic cells of murine cell lines. Moreover, we used these spores for mucosal immunization of mice. We conclude that observed effects vary with the type of displayed FliD-spore coat protein fusion and seem to be mostly independent on its abundance and localization in the spore coat structure.

scholarly journals Incorporation of protein into spore coats is not cell autonomous in Dictyostelium.

1992 ◽  
Vol 116 (5) ◽  
pp. 1291-1300 ◽  
Author(s):  
C M West ◽  
G W Erdos
Keyword(s):  
Flow Cytometry ◽  
Coat Protein ◽  
Viscous Fluid ◽  
Fluorescent Antibody ◽  
Fluid Phase ◽  
Spore Coat ◽  
Coat Proteins ◽  
Assembly Process ◽  
Spore Coat Proteins ◽  
Spore Coat Protein

At maturity, the spores of Dictyostelium are suspended in a viscous fluid droplet, with each spore being surrounded by its own spore coat. Certain glycoproteins characteristic of the spore coat are also dissolved in this fluid matrix after the spore coat is formed. To determine whether any proteins of the coat reside in this fluid phase earlier during the process of spore coat assembly, pairs of strains which differed in a spore coat protein carbohydrate marker were mixed and allowed to form spore coats in each other's presence. We reasoned that proteins belonging to an early, soluble, extracellular pool would be incorporated into the spore coats of both strains. To detect trans-incorporation, spores were labeled with a fluorescent antibody against the carbohydrate marker and each spore's fluorescence was analyzed by flow cytometry. Several proteins of both the outer and inner protein layers of the coat appeared to be faithfully and reciprocally trans-incorporated and hence judged to belong to a soluble, assembly-phase pool. Western blot analysis of sorted spores, and EM localization, confirmed this conclusion. In contrast, one outer-layer protein was not trans-incorporated, and was concluded to be insoluble at the time of secretion. Three classes of spore coat proteins can be described: (a) Insoluble from the time of secretion; (b) present in the early, soluble pool but not the late pool after spore coat formation; and (c) present in the soluble pool throughout spore coat assembly. These classes may, respectively: (a) Nucleate spore coat assembly; (b) comprise a scaffold defining the dimensions of the nascent spore coat; and (c) complete the assembly process by intercalation into the scaffold.

scholarly journals Hormone-Dependent Interaction between the Amino- and Carboxyl-Terminal Domains of Progesterone Receptor in Vitro and in Vivo

1999 ◽  
Vol 13 (6) ◽  
pp. 910-924 ◽  
Author(s):  
Marc J. Tetel ◽  
Paloma H. Giangrande ◽  
Susan A. Leonhardt ◽  
Donald P. McDonnell ◽  
Dean P. Edwards
Keyword(s):  
Progesterone Receptor ◽  
Transcriptional Activation ◽  
Hormone Receptors ◽  
Direct Interaction ◽  
Steroid Hormone Receptors ◽  
Creb Binding Protein ◽  
Two Hybrid ◽  
Dependent Interaction

Abstract Full transcriptional activation by steroid hormone receptors requires functional synergy between two transcriptional activation domains (AF) located in the amino (AF-1) and carboxyl (AF-2) terminal regions. One possible mechanism for achieving this functional synergy is a physical intramolecular association between amino (N-) and carboxyl (C-) domains of the receptor. Human progesterone receptor (PR) is expressed in two forms that have distinct functional activities: full-length PR-B and the amino-terminally truncated PR-A. PR-B is generally a stronger activator than PR-A, whereas under certain conditions PR-A can act as a repressor in trans of other steroid receptors. We have analyzed whether separately expressed N- (PR-A and PR-B) and C-domains [hinge plus ligand-binding domain (hLBD)] of PR can functionally interact within cells by mammalian two-hybrid assay and whether this involves direct protein contact as determined in vitro with purified expressed domains of PR. A hormone agonist-dependent interaction between N-domains and the hLBD was observed functionally by mammalian two-hybrid assay and by direct protein-protein interaction assay in vitro. With both experimental approaches, N-C domain interactions were not induced by the progestin antagonist RU486. However, in the presence of the progestin agonist R5020, the N-domain of PR-B interacted more efficiently with the hLBD than the N-domain of PR-A. Coexpression of steroid receptor coactivator-1 (SRC-1) and the CREB binding protein (CBP), enhanced functional interaction between N- and C-domains by mammalian two-hybrid assay. However, addition of SRC-1 and CBP in vitro had no influence on direct interaction between purified N- and C-domains. These results suggest that the interaction between N- and C-domains of PR is direct and requires a hormone agonist-induced conformational change in the LBD that is not allowed by antagonists. Additionally, coactivators are not required for physical association between the N- and C-domains but are capable of enhancing a functionally productive interaction. In addition, the more efficient interaction of the hLBD with the N-domain of PR-B, compared with that of PR-A, suggests that distinct interactions between N- and C-terminal regions contribute to functional differences between PR-A and PR-B.

Sign in / Sign up

Export Citation Format

Share Document