Significance of wall structure, macromolecular composition, and surface polymers to the survival and transport of Cryptosporidium parvum oocysts.

The structure and composition of the oocyst wall are primary factors determining the survival and hydrologic transport of Cryptosporidium parvum oocysts outside the host. Microscopic and biochemical analyses of whole oocysts and purified oocyst walls were undertaken to better understand the inactivation kinetics and hydrologic transport of oocysts in terrestrial and aquatic environments. Results of microscopy showed an outer electron-dense layer, a translucent middle layer, two inner electron-dense layers, and a suture structure embedded in the inner electron-dense layers. Freeze-substitution showed an expanded glycocalyx layer external to the outer bilayer, and Alcian Blue staining confirmed its presence on some but not all oocysts. Biochemical analyses of purified oocyst walls revealed carbohydrate components, medium- and long-chain fatty acids, and aliphatic hydrocarbons. Purified walls contained 7.5% total protein (by the Lowry assay), with five major bands in SDS-PAGE gels. Staining of purified oocyst walls with magnesium anilinonaphthalene-8-sulfonic acid indicated the presence of hydrophobic proteins. These structural and biochemical analyses support a model of the oocyst wall that is variably impermeable and resistant to many environmental pressures. The strength and flexibility of oocyst walls appear to depend on an inner layer of glycoprotein. The temperature-dependent permeability of oocyst walls may be associated with waxy hydrocarbons in the electron-translucent layer. The complex chemistry of these layers may explain the known acid-fast staining properties of oocysts, as well as some of the survival characteristics of oocysts in terrestrial and aquatic environments. The outer glycocalyx surface layer provides immunogenicity and attachment possibilities, and its ephemeral nature may explain the variable surface properties noted in oocyst hydrologic transport studies.

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene.

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.

Bacterial expression system with tightly regulated gene expression and plasmid copy number.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

Tightly regulated vectors for the cloning and expression of toxic genes.

A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

Studying the salt dependence of the binding of sigma70 and sigma32 to core RNA polymerase using luminescence resonance energy transfer.

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of sigma70 with thebeta' coiled-coil fragment (amino acids 100-309). Here we describe an LRET binding assay used to monitor the interaction of E. coli sigma70 and sigma32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of sigma70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the sigma32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of sigma32 with core RNAP is stronger than sigma70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E.coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.

High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.

BACKGROUND: Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. METHODOLOGY/ PRINCIPAL FINDINGS: By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. CONCLUSIONS/ SIGNIFICANCE: Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

Conformational flexibility in sigma70 region 2 during transcription initiation.

Prokaryotic RNA polymerase holoenzyme is composed of core subunits (alpha(2)betabeta'omega) plus a sigma factor that confers promoter specificity allowing for regulation of gene expression. Holoenzyme is known to undergo several conformational changes during the multiple steps of transcription initiation. However, the effects of these changes on the functions of specific regions have not been well characterized. In this work, we addressed the role of possible conformational change in region 2 of Escherichia coli sigma(70) by engineering disulfide bonds that "lock" region 2.1 with region 2.2 and region 2.2 with region 2.3. When these mutant holoenzymes were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond did not result in a fundamental block, indicating that the disulfide-containing holoenzymes are active. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. Our results suggest that conformational flexibility within sigma(70) region 2 facilitates open complex formation and transcription initiation.

Using disulfide bond engineering to study conformational changes in the beta'260-309 coiled-coil region of Escherichia coli RNA polymerase during sigma(70) binding.

RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the beta' subunit (beta'260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with sigma(70) both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to sigma(70). In this work, we engineered a disulfide bond within beta'240-309 that locks the beta' coiled-coil region in the coiled-coil conformation, and we show that this "locked" peptide is able to bind to sigma(70). We also show that the locked coiled-coil is capable of inducing a conformational change within sigma(70) that allows recognition of the -10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding sigma(70) or upon recognition of the -10 nontemplate strand of DNA.

On-column tris(2-carboxyethyl)phosphine reduction and IC5-maleimide labeling during purification of a RpoC fragment on a nickel-nitrilotriacetic acid Column.

Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the beta(')-subunit from Escherichia coli RNA polymerase (RNAP) to sigma70, we synthesized a fluorescent-labeled beta(')-fragment (residues 100-309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5-maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The beta(')-fragment with a N-terminal His(6)-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni-NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni-NTA chemistry. The labeled beta(')-fragment was tested with sigma70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes.