Value of remote sensing in forest surveys

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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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Human papillomavirus in amniotic fluid

BACKGROUND: There is evidence to suggest that human papillomavirus (HPV) can cross the placenta resulting in in-utero transmission. The goal of this study was to determine if HPV can be detected in amniotic fluid from women with intact amniotic membranes. METHODS: Residual amniotic fluid and cultured cell pellets from amniocentesis performed for prenatal diagnosis were used. PGMY09/11 L1 consensus primers and GP5+/GP6+ primers were used in a nested polymerase chain reaction assay for HPV. RESULTS: There were 146 paired samples from 142 women representing 139 singleton pregnancies, 2 twin pregnancies, and 1 triplet pregnancy. The women were 78% Caucasian, 5% African American, 14% Asian, and 2% Hispanic. The average age was 35.2 years with a range of 23–55 years. All samples were β-globin positive. HPV was not detected in any of the paired samples. CONCLUSION: Given the age range, race, and ethnicity of the study population, one would anticipate some evidence of HPV if it could easily cross the placenta, but there was none

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