Mapping of a Protein Interaction Network Required for Enterobactin Biosynthesis in Escherichia coli

Protein complexes are essential components of many biological processes. Therefore, protein-protein interactions are crucial for many essential cellular functions and are considered good targets for the development of novel therapeutics. Siderophore biosynthesis is one of the biological processes that has an absolute requirement for protein-protein interactions. Siderophores are small iron-scavenging molecules that are synthesized and secreted by iron-starved bacteria to chelate ferric iron (Fe3+) from the environment. Ferric iron, which is essential for survival and growth of most bacteria, is insoluble at neutral pH, or is bound to host iron storage proteins such as transferrin. By taking up Fe3+-siderophore complexes, such bacteria can survive and proliferate in low-iron environments. Enterobactin is a catecholate type siderophore of E. coli that is synthesized in its cytoplasm by seven enzymes, EntA-F and EntH. These sequentially-related enzymes function together to produce enterobactin, which is a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine. Enterobactin biosynthetic enzymes are organized in two functional modules: the DHB module (EntCBA) and the non-ribosomal peptide synthesis (NRPS) module (EntBDEF). Interactions between EntBDEF in the NRPS module have been previously reported. Our research group has since reported in vitro evidence of an interaction between EntA and EntE, the enzymes at the interface of the DHB and NRPS modules. The research presented here is focused on the identification of novel protein-protein interactions in the DHB module as well as the study of subunit orientation in the Ent complexes. The first research chapter is centered on the subunit orientation in the intracellular EntA-EntE complex. In this study Chrome Azurol S (CAS) assays and bacterial adenylate cyclase two-hybrid (BACTH) assays were employed to study the EntA-EntE complexation in vivo. CAS assays were used to validate the functionality of EntA and EntE BACTH constructs. BACTH experiments were then performed to identify the intracellular complexation of EntA and EntE and to determine the orientation of EntA relative to EntE in the complex. BACTH results were further validated by automated docking simulations. The second research chapter focuses on the construction of two Fur-controlled bidirectional protein expression vectors. Ferric Uptake Regulator (Fur) is a protein involved in iron homeostasis in E. coli. When intracellular iron is abundant, Fur forms a complex with Fe2+. This complex binds to the Fur box and inhibits the transcription of iron responsive genes such as ent genes. The Fur box is the consensus sequence that is located near or within the promoter region of iron responsive genes. The novel expression vectors are derivatives of low copy number plasmids pACYC184 and pBR322 and contain a bidirectional promoter, FLAG or HA tags, TEV cleavage site and a multiple cloning site (MCS) compatible with the MCS of BACTH vectors. The third research chapter involves the identification of a novel protein-protein interaction between two enzymes in the DHB module, EntA and EntB. Furthermore, ternary complex formation between EntA, EntB and EntE was investigated in this chapter. BACTH was employed as the primary method for the detection of protein-protein interactions between EntA and EntB. Functionality of all the constructs used in the BACTH was confirmed using the CAS assay and growth studies. Automated docking simulations were also used to generate a model for an EntA-EntB-EntE ternary complex. The EntE-EntB interaction interface in the generated model was in accordance with the published crystal structure for the EntE-EntB complex and therefore supported our experimental results

Subunit Orientation in the Escherichia coli Enterobactin Biosynthetic EntA-EntE Complex Revealed by a Two-Hybrid Approach

The siderophore enterobactin is synthesized by the enzymes EntA-F and EntH in the E. coli cytoplasm. We previously reported in vitro evidence of an interaction between tetrameric EntA and monomeric EntE. Here we used bacterial adenylate cyclase two-hybrid (BACTH) assays to demonstrate that the E. coli EntA-EntE interaction occurs intracellularly. Furthermore, to obtain information on subunit orientation in the EntA-EntE complex, we fused BACTH reporter fragments T18 and T25 to EntA and EntE in both N-terminal and C-terminal orientations. To validate functionality of our fusion proteins, we performed Chrome Azurol S (CAS) assays using E. coli entE- and entA- knockout strains transformed with our BACTH constructs. We found that transformants expressing N-terminal and C-terminal T18/T25 fusions to EntE exhibited CAS signals, indicating that these constructs could rescue the entE- phenotype. While expression of EntA with N-terminal T18/T25 fusions exhibited CAS signals, C-terminal fusions did not, presumably due to disruption of the EntA tetramer in vivo. Bacterial growth assays supported our CAS findings. Co-transformation of functional T18/T25 fusions into cya- E. coli BTH101 cells resulted in positive BACTH signals only when T18/T25 fragments were fused to the N-termini of both EntA and EntE. Co-expression of N-terminally fused EntA with C-terminally fused EntE resulted in no detectable BACTH signal. Analysis of protein expression by Western blotting confirmed that the loss of BACTH signal was not due to impaired expression of fusion proteins. Based on our results, we propose that the N-termini of EntA and EntE are proximal in the intracellular complex, while the EntA N-terminus and EntE C-terminus are distal. A protein-protein docking simulation using SwarmDock was in agreement with our experimental observations

A novel set of vectors for Fur-controlled protein expression under iron deprivation in Escherichia coli

Background In the presence of sufficient iron, the Escherichia coli protein Fur (Ferric Uptake Regulator) represses genes controlled by the Fur box, a consensus sequence near or within promoters of target genes. De-repression of Fur-controlled genes occurs upon iron deprivation. In the E. coli chromosome, there is a bidirectional intercistronic promoter region with two non-overlapping Fur boxes. This region controls Fur-regulated expression of entCEBAH in the clockwise direction and fepB in the anticlockwise direction. Results We cloned the E. coli bidirectional fepB/entC promoter region into low-copy-number plasmid backbones (pACYC184 and pBR322) along with downstream sequences encoding epitope tags and a multiple cloning site (MCS) compatible with the bacterial adenylate cyclase two-hybrid (BACTH) system. The vector pFCF1 allows for iron-controlled expression of FLAG-tagged proteins, whereas the pFBH1 vector allows for iron-controlled expression of HA-tagged proteins. We showed that E. coli knockout strains transformed with pFCF1-entA, pFCF1-entE and pFBH1-entB express corresponding proteins with appropriate epitope tags when grown under iron restriction. Furthermore, transformants exhibited positive chrome azurol S (CAS) assay signals under iron deprivation, indicating that the transformants were functional for siderophore biosynthesis. Western blotting and growth studies in rich and iron-depleted media demonstrated that protein expression from these plasmids was under iron control. Finally, we produced the vector pFCF2, a pFCF1 derivative in which a kanamycin resistance (KanR) gene was engineered in the direction opposite of the MCS. The entA ORF was then subcloned into the pFCF2 MCS. Bidirectional protein expression in an iron-deprived pFCF2-entA transformant was confirmed using antibiotic selection, CAS assays and growth studies. Conclusions The vectors pFCF1, pFCF2, and pFBH1 have been shown to use the fepB/entC promoter region to control bidirectional in trans expression of epitope-tagged proteins in iron-depleted transformants. In the presence of intracellular iron, protein expression from these constructs was abrogated due to Fur repression. The compatibility of the pFCF1 and pFBH1 backbones allows for iron-controlled expression of multiple epitope-tagged proteins from a single co-transformant

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研究紀要

An Anillin-Ect2 Complex Stabilizes Central Spindle Microtubules at the Cortex during Cytokinesis

Cytokinesis occurs due to the RhoA-dependent ingression of an actomyosin ring. During anaphase, the Rho GEF (guanine nucleotide exchange factor) Ect2 is recruited to the central spindle via its interaction with MgcRacGAP/Cyk-4, and activates RhoA in the central plane of the cell. Ect2 also localizes to the cortex, where it has access to RhoA. The N-terminus of Ect2 binds to Cyk-4, and the C-terminus contains conserved DH (Dbl homologous) and PH (Pleckstrin Homology) domains with GEF activity. The PH domain is required for Ect2's cortical localization, but its molecular function is not known. In cultured human cells, we found that the PH domain interacts with anillin, a contractile ring protein that scaffolds actin and myosin and interacts with RhoA. The anillin-Ect2 interaction may require Ect2's association with lipids, since a novel mutation in the PH domain, which disrupts phospholipid association, weakens their interaction. An anillin-RacGAP50C (homologue of Cyk-4) complex was previously described in Drosophila, which may crosslink the central spindle to the cortex to stabilize the position of the contractile ring. Our data supports an analogous function for the anillin-Ect2 complex in human cells and one hypothesis is that this complex has functionally replaced the Drosophila anillin-RacGAP50C complex. Complexes between central spindle proteins and cortical proteins could regulate the position of the contractile ring by stabilizing microtubule-cortical interactions at the division plane to ensure the generation of active RhoA in a discrete zone

Intracellular co-localization of the Escherichia coli enterobactin biosynthetic enzymes EntA, EntB, and EntE

Bacteria utilize small-molecule iron chelators called siderophores to support growth in low-iron environments. The Escherichia coli catecholate siderophore enterobactin is synthesized in the cytoplasm upon iron starvation. Seven enzymes are required for enterobactin biosynthesis: EntA-F, H. Given that EntB-EntE and EntA-EntE interactions have been reported, we investigated a possible EntA-EntB-EntE interaction in E. coli cells. We subcloned the E. coli entA and entB genes into bacterial adenylate cylase two-hybrid (BACTH) vectors allowing for co-expression of EntA and EntB with N-terminal fusions to the adenylate cyclase fragments T18 or T25. BACTH constructs were functionally validated using the CAS assay and growth studies. Co-transformants expressing T18/T25-EntA and T25/T18-EntB exhibited positive two-hybrid signals indicative of an intracellular EntA-EntB interaction. To gain further insights into the interaction interface, we performed computational docking in which an experimentally validated EntA-EntE model was docked to the EntB crystal structure. The resulting model of the EntA-EntB-EntE ternary complex predicted that the IC domain of EntB forms direct contacts with both EntA and EntE. BACTH constructs that expressed the isolated EntB IC domain fused to T18/T25 were prepared in order to investigate interactions with T25/T18-EntA and T25/T18-EntE. CAS assays and growth studies demonstrated that T25-IC co-expressed with the EntB ArCP domain could complement the E. coli entB- phenotype. In agreement with the ternary complex model, BACTH assays demonstrated that the EntB IC domain interacts with both EntA and EntE

Anillin interacts with Ect2 during cytokinesis.

<p>A) Western blots show the interaction of endogenous Ect2 (and Cyk-4) with the AHD of anillin. The top western blot shows Hela lysates from cells treated with nocodazole, purvalanol and/or Ect2 RNAi pulled down with MBP or MBP:AHD and probed for endogenous Ect2. The bottom western blot shows Hela lysates from cells treated with nocodazole, purvalanol and/or Cyk-4 RNAi and/or Ect2 RNAi pulled down with MBP:AHD and probed for endogenous Ect2 (top panel) or Cyk-4 (bottom panel). B) Z-stack projections of MeOH-fixed Hela cells transfected with Myc:Ect2 and co-stained for Myc (red) and anillin (green). Both a lateral view and an end-on view are shown. C) Z-stack projections of MeOH-fixed Hela cells+/−anillin RNAi treated with 100 µM Blebbistatin to inhibit oscillations and stained for Plk1 (red) and anillin (green). Scale bar is 10 µm.</p

Anillin interacts with an Ect2 complex at the cortex.

<p>A) Single plane images of fixed Hela cells transfected with Myc:Ect2 C-terminal constructs+/−Ect2 siRNAs co-stained for DNA (DAPI) and Myc. Inverted images show Myc localization for D668G and D668G; Ras Tail. Arrows point to the cortex. Scale bar is 10 µm. B) A western blot of active RhoA pulled down with GST:RBD using lysates from HEK-293 cells expressing Myc:Ect2 C-terminal constructs. The fold-change of RhoA binding vs. control is indicated for each lane. A ponceau stain of the blot is shown below. C) Western blots of PIP strips incubated with lysates from HEK-293 cells expressing GFP, GFP:RhoA, GFP:anillin (C-term), Myc:Ect2 (C-term wt or D668G), and GFP:Ect2 (C-term; Ras Tail or D668G; Ras Tail). D) A western blot of lysates from HEK-293 cells transfected with Myc:Ect2 (E3 wt, Ras Tail, D668G or D668G; Ras Tail) pulled down with GST:AHD of anillin and stained for Myc.</p

The PH region of Ect2 interacts with the AHD of anillin.

<p>A) Structures of Ect2 (BRCT: BRCA1 C terminus domain, DH: Dbl homology, PH: Pleckstrin Homology, C: C-region) and anillin (My: Myosin, Ac: Actin, AHD: Anillin Homology Domain, PH: Pleckstrin Homology). B) Coomassie-stained gels show the various MBP and GST-tagged recombinant anillin proteins used in this study. The MBP-tagged proteins are shown in the gel on the left, and the GST-tagged proteins are shown in the gel on the right. C) A western blot of lysates from HEK-293 cells transfected with Myc: Ect2 FL, E1 (N-term), E2 (BRCT) and E3 (C-term) pulled down with MBP or MBP:AHD (A2) of anillin and stained for Myc. D) Western blots of lysates from HEK-293 cells transfected with Myc-tagged Ect2 constructs (FL, E3–E8) pulled down with MBP or MBP:AHD of anillin stained for Myc. E) Western blots of lysates from Hela cells expressing Myc:Ect2 (E3) treated with nocodazole, purvalanol A, and Cyk-4 or RhoA RNAi, and pulled down with MBP or MBP:AHD of anillin, stained for Myc, Cyk-4 and RhoA. F) Coomassie-stained gels show recombinant, cleaved Ect2 fragments [E6 (DH domain) or E4 (DH+PH domains)] pulled down with MBP or MBP:AHD. Boxes outline E6 or E4, and are shown below. G) A western blot of lysates from HEK-293 cells transfected with Myc:Ect2 (E5) pulled down with anillin fragments (A1, A2, A3 and A4) tagged with MBP and stained for Myc.</p