Advances in heterologous gene expression by Streptomyces.

During the past year, noteworthy advances have been made in our ability to express foreign genes in Streptomyces. These advances were due, in part, to a detailed examination of the critical parameters that limit expression by Streptomyces of soluble forms of the human T-cell receptor CD4. Significant progress has also been made in our understanding of transcriptional regulation and protease gene expression. Application of this knowledge to expression vector design and the construction of alternative expression hosts should improve our ability to easily and routinely express foreign genes in Streptomyces.

Streptomyces: a host for heterologous gene expression.

Streptomyces species offer many potential advantages as hosts for the expression and secretion of eukaryotic gene products. In this review we discuss the expression and localization signals that have been used to direct heterologous gene expression and the applications of these signals. Finally, we discuss future strategies aimed at increasing the capacity of this host for the high level production of biologically active eukaryotic gene products.

Cloning and functional characterization of a novel human CC chemokine that binds to the CCR3 receptor and activates human eosinophils.

Eotaxin has been found to bind exclusively to a single chemokine receptor, CCR3. Using expression sequence tag screening of an activated monocyte library, a second chemokine has been identified; it was expressed and purified from a Drosophila cell culture system and appears to only activate CCR3. Eotaxin-2, MPIF-2, or CKbeta-6, is a human CC chemokine with low amino acid sequence identity to other chemokines. Eotaxin-2 promotes chemotaxis and Ca2+ mobilization in human eosinophils but not in neutrophils or monocytes. Cross-desensitization calcium mobilization experiments using purified eosinophils indicate that eotaxin and MCP-4, but not RANTES, MIP-1alpha, or MCP-3, can completely cross-desensitize the calcium response to eotaxin-2 on these cells, indicating that eotaxin-2 shares the same receptor used by eotaxin and MCP-4. Eotaxin-2 was the most potent eosinophil chemoattractant of all the chemokines tested. Eotaxin-2 also displaced 125I-eotaxin bound to the cloned CCR3 stably expressed in CHO cells (CHO-CCR3) and to freshly isolated human eosinophils with affinities similar to eotaxin and MCP-4. 125I-Eotaxin-2 binds with high affinity to eosinophils and both eotaxin and cold eotaxin-2 displace the ligand with equal affinity. Eotaxin and eotaxin-2 promote a Ca2+ transient in RBL-2H3 cells stably transfected with CCR3 (RBL-2H3-CCR3) and both ligands cross-desensitized the response of the other but not the response to LTD4. The data indicate that eotaxin-2 is a potent eosinophil chemotactic chemokine exerting its activity solely through the CCR3 receptor.

Stroke genomics: approaches to identify, validate, and understand ischemic stroke gene expression.

Sequencing of the human genome is nearing completion and biologists, molecular biologists, and bioinformatics specialists have teamed up to develop global genomic technologies to help decipher the complex nature of pathophysiologic gene function. This review will focus on differential gene expression in ischemic stroke. It will discuss inheritance in the broader stroke population, how experimental models of spontaneous stroke might be applied to humans to identify chromosomal loci of increased risk and ischemic sensitivity, and also how the gene expression induced by stroke is related to the poststroke processes of brain injury, repair, and recovery. In addition, we discuss and summarise the literature of experimental stroke genomics and compare several approaches of differential gene expression analyzes. These include a comparison of representational difference analysis we have provided using an experimental stroke model that is representative of stroke evolution observed most often in man, and a summary of available data on stroke differential gene expression. Issues regarding validation of potential genes as stroke targets, the verification of message translation to protein products, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, and the emerging problems of handling novel genes that may be discovered during differential gene expression analyses will also be addressed.

Characterization of Streptomyces promoter sequences using the Escherichia coli galactokinase gene.

A gene fusion system that uses the Escherichia coli galK gene has been developed to characterize Streptomyces transcriptional regulatory sequences. The system consists of galK-deficient Streptomyces lividans mutants and plasmids containing the E. coli galK gene with its natural ribosome-binding site and sites upstream of galK for insertion of transcription signals. Expression of the E. coli galK gene in S. lividans can be quantitated by either an enzymatic or immunoblot assay or detected by genetic complementation of an S. lividans galK- mutant. The utility of the plasmid to select, detect and assess promoter function was examined using the S. lividans XP55 and S. fradiae aph gene promoters. The potential use of the galK fusion system to isolate and characterize Streptomyces transcription signals is discussed.

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Molecular and pharmacological characterization of the murine tachykinin NK(3) receptor.

Starting with a partial sequence from Genbank, polymerase chain reaction (PCR) was utilized to isolate the full-length cDNA for NK(3) receptor from mouse brain. The murine NK(3) receptor has a predicted sequence of 452 amino acids, sharing 96% and 86% identity to the rat and human NK(3) receptors, respectively. Binding affinities and functional potencies of tachykinin receptor agonists were similar in HEK (human embryonic kidney) 293 cells expressing murine NK(3) receptor and human NK(3) receptor, although substance P and neurokinin A were more potent stimulators of Ca(2+) mobilization in murine NK(3) receptor cells. NK(3) receptor-selective antagonists from two structural classes, had 10- to 100-fold lower binding affinities for murine NK(3) receptor compared to human NK(3) receptor, and about 5- to 10-fold reduced potency in the murine NK(3) receptor functional assay. The results demonstrate species differences in the potencies of tachykinin receptor antagonists in murine and human NK(3) receptors, and the lower potencies in the former should be taken into consideration when using murine disease models.

Soluble forms of the human T cell receptor CD4 are efficiently expressed by Streptomyces lividans.

We have developed a new gene expression and secretion system for Streptomyces lividans and used it to produce soluble forms of a human T-cell receptor CD4 at levels greater than 300 mg/l. The system uses the transcription, translation and secretion signals of the serine protease inhibitor gene STI-II which is naturally produced by S. longisporus. Using these signals, soluble derivatives of CD4 were secreted directly into the culture supernatant as correctly processed soluble, biologically active proteins. High level expression of the CD4 proteins depended on the transcription initiation signal, the amino acid sequence surrounding the signal peptide cleavage site and temporally controlled protease activities. We discuss these results in the context of the potential of this system for producing other eukaryotic proteins in Streptomyces.

Validation and long term performance characteristics of a quantitative enzyme linked immunosorbent assay (ELISA) for human anti-PA IgG.

Accurate, reliable and standardized quantification of anti-protective antigen (PA) IgG antibody levels is essential for comparative analyses of anti-toxin immune responses in anthrax cases, recipients of PA-based anthrax vaccines and for evaluation of anti-PA based immunotherapies. We have previously reported the early performance characteristics and application of a quantitative anti-PA IgG enzyme linked immunosorbent assay. The principal application of this assay was in a Phase 4 human clinical trial of anthrax vaccine adsorbed (AVA, BioThrax), the central component of the CDC Anthrax Vaccine Research Program (AVRP) and in humans following bioterrorism associated Bacillus anthracis infection (Quinn et al., 2002; Quinn et al., 2004; Marano et al., 2008). The objective of the AVRP was to determine the feasibility of reducing the number of priming series and booster doses of the licensed Anthrax Vaccine Adsorbed (AVA) (BioThrax®; Emergent BioSolutions, Lansing, MI) and changing the route of administration from subcutaneous (SC) to intramuscular (IM) (Marano et al., 2008). In this paper we report the validation and long term performance characteristics of the assay during its six year application in the AVRP (2002-2008). The critical features are 1) extensive validation of the assay using two standard reference sera; 2) long term stability and 3) consistency of the data for quantitative analysis of human long term anti-PA IgG responses. The reportable value (RV) of the assay was expressed as anti-PA IgG concentration (µg/ml). Accuracy of the assay was high with a percent error (%ER) range of 1.6-11.4%. Overall intra-operator and intermediate precision were high with Coefficients of Variation (%CVs) of 2.5-15.4% and 6.3-13.2%, respectively. The assay demonstrated excellent dilutional linearity for human sera using log(10) transformed data with the slope=0.95 to 0.99, intercept=0.02 to 0.06 and r(2)=0.980-0.987. The assay was robust, tolerating changes in serum incubation temperatures from 35 to 39°C, serum incubation times from 55 to 65min and changes in key reagents. The long-term assay stability over 6years using consecutive reference sera AVR414 and AVR801 demonstrated sustained high accuracy and precision for the assay, confirming its suitability for long term studies of PA protein-based anthrax vaccines.

Two transcribing activities are involved in expression of the Streptomyces galactose operon.

The Streptomyces galactose operon is transcribed from two independently regulated promoters: galP1, located at the 5' end of the operon and responsible for galactose-dependent transcription of the operon, and galP2, an internal constitutive promoter. We identified and partially separated two distinct transcribing activities involved in expression of this operon. Using RNA polymerase from Streptomyces lividans and Streptomyces coelicolor partially purified by chromatography on heparin-agarose and DNA-cellulose, we detected activities capable of initiating transcription in vitro specifically from either galP1 or galP2. Circumstantial evidence suggests that the activity for galP2 transcription is a holoenzyme species associated with the previously described sigma 28 protein (referred to here as sigma C). The galP1-transcribing activity is more difficult to evaluate. This activity may correspond to a holoenzyme species associated with sigma A (formerly sigma 35), although other possibilities are discussed. This would be the second reported example of a catabolite-controlled gene in Streptomyces species expressed from multiple promoters recognized by different holoenzyme forms. This may indicate that the involvement of RNA polymerase heterogeneity in gene expression in Streptomyces species is a more general strategy for regulation than the specialized gene expression seen in Escherichia coli.

Native complex formation between apolipoprotein E isoforms and the Alzheimer's disease peptide A beta.

To explore whether the genetic linkage between apolipoprotein E (ApoE) alleles and susceptibility to Alzheimer's disease might be attributable to a direct molecular interaction between ApoE and the amyloid peptide A beta, we have produced ApoE variants in Escherichia coli and studied their interactions with A beta under native conditions. When incubated with A beta at 20-40 microM concentrations, all three isoforms of ApoE (2, 3, and 4) readily form complexes with A beta which can be isolated by gel filtration in native buffer. Freshly mixed ApoE and A beta generate a complex that co-migrates in gel filtration with the main A280 peak, which migrates identically to the ApoE tetramer alone. After several hours incubation, an additional, high molecular weight, soluble aggregate appears which also contains both ApoE and A beta. Neither ApoE nor A beta incubated by themselves produces high molecular weight aggregates under these conditions. Incubation of A beta with control proteins bovine serum albumin and immunoglobulin generates negligable binding in the gel filtration assay. Similar results were obtained whether A beta (1-40) or A beta (1-42) was used, and plasma-derived ApoE gave similar results to E. coli-produced material. The data are consistent with a role for ApoE-A beta interactions in modulating the development of AD. Since no major differences were observed in the behavior of the three ApoE isotypes, however, the molecular basis of the genetic trend between ApoE alleles and AD cannot be attributed to specific activity differences between the molecular forms of ApoE characterized in this study.

xylE functions as an efficient reporter gene in Streptomyces spp.: use for the study of galP1, a catabolite-controlled promoter.

We describe the development of a convenient and sensitive reporter gene system for Streptomyces spp. based on the use of a promoterless copy of the xylE gene of Pseudomonas putida. The xylE gene product is a catechol dioxygenase, which converts the colorless substrate catechol to an intensely yellow hydroxymuconic semialdehyde. A promoterless copy of xylE was placed under the transcriptional control of galP1, a glucose-repressed and galactose-induced promoter from Streptomyces lividans, and its expression was examined in bacterial colonies on agar plates or in liquid cultures grown in the presence of glucose or galactose as the sole carbon source. On plates, colonies of bacteria grown on galactose turned bright yellow within a few minutes of being sprayed with a solution of catechol, whereas colonies on glucose-containing plates remained white or only slightly colored, even after extensive incubation. Activity of galP1-xylE fusions was conveniently measured in crude cell extracts with a simple colorimetric assay and was shown to faithfully reflect intracellular RNA levels, as determined by quantitative dot blots. Moreover, differences in expression levels of xylE fusions driven by mutant galP1 promoters were readily apparent in color reactions on plates. The properties of xylE as a reporter gene thus make it suitable not only for quantitatively monitoring expression of regulated promoters in Streptomyces spp. but also for recovering mutations that alter the expression levels of promoters of interest.

Identification of a complex operator for galP1, the glucose-sensitive, galactose-dependent promoter of the Streptomyces galactose operon.

The galP1 promoter is responsible for galactose-dependent, glucose-sensitive transcription of the galactose utilization operon of Streptomyces coelicolor and Streptomyces lividans. We describe the characterization of mutations that were positioned directly upstream of the apparent transcription start site of galP1 and that resulted in deregulated expression. Certain combinations of base changes within a series of hexamers that lie within two pairs of direct repeat sequences resulted in significant expression from galP1 in the absence of inducer. These motifs are further implicated in regulation by the observation that DNA fragments containing the hexamers and direct repeat sequences resulted in increased transcription from the chromosomal copy of galP1 on multicopy plasmids in the absence of galactose. We suggest that these hexamers and direct repeat sequences constitute an operator for the negative regulation of the Streptomyces gal operon.

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.

Two promoters, one inducible and one constitutive, control transcription of the Streptomyces lividans galactose operon.

Galactose utilization in Streptomyces lividans was shown to be controlled by an operon that is induced in the presence of galactose and repressed by glucose. Two promoters, galP1 and galP2, which direct transcription of two distinct polycistronic transcripts, have been identified. galP1 is located immediately upstream of the operon and is induced in the presence of galactose. This promoter directs transcription of the galT, galE, and galK genes. The second promoter, galP2, is located within the operon just upstream of the galE gene. This promoter is responsible for constitutive transcription of the galE and galK genes. Comparison of the S. lividans gal operon to the Escherichia coli gal operon indicates the presence of a constitutive promoter positioned upstream of galE in both operons. We suggest that coupling the operon's constitutive promoter to the galE gene fulfills a physiological requirement for constitutive UDPgalactose 4-epimerase expression in Streptomyces.

Identification of a small-molecule, nonpeptide macrophage scavenger receptor antagonist.

Class A scavenger receptor (SR-A) antagonists may prevent the initiation of atherosclerosis, because a recent report found that SR-A/apolipoprotein E (apoE) double-knockout mice had 60% smaller lesions than apoE single-knockout littermates. We transfected human embryonic kidney (HEK) 293 cells with SR-A type I or II receptors to find small-molecule antagonists. Uptake of 1,1'-dioctadecyl-3,3,3', 3'-tetramethylindocarbocyanine perchlorate-labeled acetylated low-density lipoprotein (DiI-AcLDL) showed that among common polyanionic ligands, polyinosine was the most potent, with an IC50 of 0.74 microgram/ml, whereas the novel compound (E)-methyl 4-chloro-alpha-[4-(4-chlorophenyl)-1, 5-dihydro-3-hydroxy-5-oxo-1-(2-thiazolyl)-2H-pyrrol-2-ylidene]benzene acetate gave an IC50 of 6.1 microgram/ml (13 microM). The novel antagonist also inhibited DiI-AcLDL uptake in cultured human peripheral and rat peritoneal macrophages with IC50 values of 21 microM and 17 microM, respectively. With [125I]AcLDL as ligand for transfected HEK 293 cells, binding/uptake and degradation at 37 degrees C for 5 h was saturable and selective. In a comparison of both types of receptor, we found no difference between the capacity of SR-AI or SR-AII for either binding or degradation. Polyinosine competed both [125I]AcLDL binding and degradation with a Ki of 1 microgram/ml, whereas the novel antagonist competed with a Ki of 19 microgram/ml (40 microM) and 8.6 microgram/ml (18 microM), respectively, for binding and degradation. Saturation binding in the presence of the ionophore monensin indicated that the novel compound behaved as a noncompetitive antagonist and perhaps as an allosteric effector. This is the first report to describe a small-molecule macrophage scavenger receptor antagonist. Utilization of this permanently transfected HEK 293 cell line will allow the identification of more potent macrophage scavenger receptor antagonists, so that their utility as therapeutics for atherosclerosis can be determined.

The Streptomyces galP1 promoter has a novel RNA polymerase recognition sequence and is transcribed by a new form of RNA polymerase in vitro.

We report the identification of DNA sequences that determine the activity of the Streptomyces galP1 promoter and a new form of RNA polymerase holoenzyme that recognizes these sequences in vitro. Base substitutions were introduced throughout the galP1 promoter region, and bases at positions -34, -36, and -11 with respect to the transcription start site were shown to be required for promoter function. These bases correspond in their positions to regions known to be important for RNA polymerase binding in several classes of eubacterial promoters, but the sequences themselves are not similar to those previously described. The -35 region of the galP1 promoter consists of six G residues, and base changes in this G hexamer had a dramatic effect on promoter activity. By using galP1-containing DNA template, a new RNA polymerase activity was purified from Streptomyces. Holoenzyme reconstitution experiments identified a new sigma factor that directs galP1 transcription in vitro. DNase I protection experiments identified a binding site for this new holoenzyme immediately upstream of the galP1 transcription start site.

Interferon action: transcriptional control of a gene specifying a 56,000-Da protein in Ehrlich ascites tumor cells.

The exposure of cells to interferons enhances the accumulation of particular mRNAs and of the corresponding proteins. A cDNA clone (clone 202) complementary to an mRNA (202 mRNA) whose level is enhanced over 12-fold in mouse Ehrlich ascites tumor cells upon exposure to beta-interferon for 10 hr has previously been isolated. The level of this mRNA was also increased in other beta-interferon-responsive mouse cell lines (i.e., L929, L1210S) but not in a line (L1210R) which is not responsive to beta-interferon. The extent of induction in Ehrlich ascites tumor cells depended on the beta-interferon concentration and reached its maximal level between 300 and 1000 units of interferon/ml. Nuclei isolated from Ehrlich ascites tumor cells which had been exposed to beta-interferon produced in vitro more 202 specific RNA than nuclei from control Ehrlich ascites tumor cells: an increase in this production was detectable 2 hr after beginning the exposure of the cells to 1000 units/ml of beta-interferon and the increase reached its maximal level, around 18-fold, after 18 hr exposure. Much, if not all of this increase, appeared to be due to an increase in the rate of synthesis of the RNA and not to a decrease in its rate of turnover. The 202 mRNA was translated in a reticulocyte lysate into a 56,000-Da protein.

Secretion of interleukin-1 beta and Escherichia coli galactokinase by Streptomyces lividans.

The functionality of the Streptomyces lividans beta-galactosidase signal peptide to direct heterologous protein export was examined. The signal peptide plus eight amino acids of mature protein were sufficient to export not only a naturally exported protein, interleukin-1 beta, but also a naturally occurring cytoplasmic protein, Escherichia coli galactokinase. Interestingly, cells which expressed yet exported galactokinase were phenotypically Gal-. The potential use of the exported galactokinase system for the isolation and characterization of mutations within signal peptides and the export machinery of the host is discussed.