Expression and regulation of the streptokinase gene.

Recent research in various areas has appreciably expanded our knowledge of streptokinase, a plasminogen activator produced by all human group A (GAS), group C (GCS), and group G (GGS) streptococci. Several molecular genetic approaches are described here to study the expression of the streptokinase gene, skn. Southern hybridization analysis demonstrated homology of synteny of ska, skc, and skg in the genomes of the above serogroups. S1 nuclease mapping, the use of transcriptional fusions to beta-galactosidase and luciferase reporter genes, in conjunction with site-directed mutagenesis, led to the localization of the core promoter region of skc and the identification of a cis-active upstream region required for full promoter activity. Circular permutation analysis of the promoter upstream region identified an intrinsic DNA bending locus as the pivotal DNA element stimulating the activity of the core promoter. The detection of skn allele-specific expression phenotypes, which proved not to be due to different skn mRNA half-lives, prompted allele swap experiments, showing that promoter activity is dictated by the host genetic background, rather than the sequence of the regulatory region. These findings suggest the involvement in skn expression of an as yet unidentified transcriptional activator that contacts the bent DNA region. Transcription termination of skc is directed by a bidirectional terminator whose structural requirements for termination efficiency were determined with base substitution mutants fused to a chloramphenicol acetyl transferase reporter. Finally, mutagenic plasmids are described for insertion-duplication and allele replacement mutagenesis of the skn locus.

Identification, cloning, and expression of the CAMP factor gene (cfa) of group A streptococci.

The CAMP reaction is a synergistic lysis of erythrocytes by the interaction of an extracellular protein (CAMP factor) produced by some streptococcal species with the Staphylococcus aureus sphingomyelinase C (beta-toxin). Group A streptococci (GAS [Streptococcus pyogenes]) have been long considered CAMP negative, and this reaction commonly has been used to distinguish GAS from Streptococcus agalactiae. We here provide evidence that GAS possess this gene and produce an extracellular CAMP factor capable of participating in a positive CAMP reaction. The S. pyogenes CAMP factor is specified by a 774-bp open reading frame homologous to the CAMP factor genes from S. agalactiae and Streptococcus uberis. This gene, designated cfa, was isolated on a 1,256-bp fragment and cloned in Escherichia coli. Recombinant clones of E. coli expressing cfa secreted an active CAMP factor. The deduced 28.5-kDa protein encoded by cfa consists of 257 amino acids, with a predicted 28-amino-acid signal peptide. The cfa gene is widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U. The cfa gene was cloned and actively expressed in Escherichia coli and gene fusions were made, placing the beta-galactosidase gene (lacZ) under control of the cfa promoter. These cfa promoter-lacZ fusions were introduced into S. pyogenes via a bacteriophage-derived site-specific integration vector where they showed that the cfa gene has a strong promoter that may be subject to as-yet-unidentified regulatory factors. The results presented here, along with previous reports, indicate that the CAMP factor gene is fairly widespread among streptococci, being present at least in groups A, B, C, G, M, P, R, and U.

The Streptococcus agalactiae hylB gene encoding hyaluronate lyase: completion of the sequence and expression analysis.

We report the cloning, sequencing and expression analysis of the Streptococcus agalactiae strain 4755 hylB4755 allele, the first chromosomally-encoded streptococcal hyaluronate lyase gene to be cloned and sequenced completely. This gene lies in a region homologous to that found in S. mutans, between the mutX and rmlB genes, a region involved in the synthesis of the serotype c-specific polysaccharide antigen of this organism. Sequencing of hylB4755 revealed a 3216-bp open reading frame that encodes a 121.2-kDa polypeptide possessing a 30-amino acid signal sequence which was theoretically predicted and experimentally confirmed. A recombinant plasmid, pHYB100, containing hylB4755 together with its promoter and terminator was constructed and used to analyze the expression of the gene in Escherichia coli. In Northern hybridization experiments, hylB4755 was found to be transcribed as 3.3-kb monocistronic mRNA from its own promoter which exhibits an extended, sigma70-like 10 consensus sequence. Transcript mapping by primer extension analysis placed the major transcription initiation site leading to the longest transcript 38 bp upstream of the translational initiation codon, ATG. E. coli TG1(pHYB100) efficiently synthesized hyaluronan-cleaving enzyme activity at approximately 7000 working units/109 cells, with lyase activity detectable in all principle cellular locations. Zymography and Western analysis identified functional activity in TG1(pHYB100) to be associated with approximately 118, 110 and 94-kDa polypeptides, with the two low molecular weight species constituting the major components of the enzyme purified from the culture supernatant fluid of S. agalactiae 4755. The 118-kDa form was shown to represent the undegraded mature enzyme, whereas the smaller species are likely to arise from proteolytic cleavage in the N-terminal part of the mature protein. The HylB4755 protein showed extensive sequence identity to the homologous enzymes from S. agalactiae 3502 and S. pneumoniae characterized by others but sequence comparisons clearly show that incomplete genes truncated at their 5' ends had been isolated from these two organisms.

The lppC gene of Streptococcus equisimilis encodes a lipoprotein that is homologous to the e (P4) outer membrane protein from Haemophilus influenzae.

We report the cloning, sequencing, and analysis of a novel chromosomal gene of Streptococcus equisimilis strain H46A that codes for a membrane lipoprotein, designated LppC. The lppC gene is located 3' adjacent to, and co-oriented with, the unrelated gapC gene that encodes the previously characterized glyceraldehyde-3-phosphate dehydrogenase. Sequencing of lppC revealed an 855-bp open reading frame that predicted a 32.4-kDa polypeptide possessing a potential lipoprotein signal sequence and modification site (VTGC). Signal sequence processing of LppC synthesized in the homologous host or expressed from plasmid pLPP2 in Escherichia coli was sensitive to globomycin, a selective inhibitor of lipoprotein-specific signal peptidase II. Subcellular localization of LppC using polyclonal antibodies raised to the hexahistidyl-tagged protein proved LppC to be tightly associated with the cytoplasmic membrane of S. equisimilis and with the outer membrane of E. coli JM109 (pLPP2). Southern, Northern and Western analyses indicated that lpp was conserved in S. pyogenes, and transcribed independently of gap as monocistronic 0.9-kb mRNA from a sigma 70-like consensus promoter. Database searches found homology of LppC to the hel gene-encoded outer membrane protein e (P4) from Haemophilus influenzae to which it exhibits 58% sequence similarity. However, unlike the hel gene, lppC was unable to complement hemA mutants of E. coli for growth on hemin as sole porphyrin source in aerobic conditions. Furthermore, neither the wild type nor an lppC insertion mutant of S. equisimilis could grow on hemin in iron-limited medium. These results, together with findings indicating that S. equisimilis H46A had no absolute requirement for iron, led us to conclude that lppC, in contrast to hel, is not involved in hemin utilization and has yet to be assigned a function.

Localization of the sequence-determined DNA bending center upstream of the streptokinase gene skc.

DNA sequences upstream of the core promoter region of the streptokinase gene (skc) from Streptococcus equisimilis H46A increase skc transcription more than tenfold in vivo. This promoter upstream region contains a segment of intrinsically bent DNA, the precise location of which was determined experimentally by circular permutation analysis and theoretically by computer prediction. Electrophoretic analysis of circularly permuted upstream DNA fragments placed the bend center approximately at position -100 with respect to the major transcription initiation site of skc. This position was in excellent agreement with the center of maximum curvature predicted theoretically. Knowledge of the precise location of the bend center will be useful for future studies of the possible effect of DNA bending on skc transcription.

Cloning, sequencing and functional overexpression of the Streptococcus equisimilis H46A gapC gene encoding a glyceraldehyde-3-phosphate dehydrogenase that also functions as a plasmin(ogen)-binding protein. Purification and biochemical characterization of the protein.

We previously identified DNA sequences involved in the function of the complex promoter of the streptokinase gene from Streptococcus equisimilis H46A, a human serogroup C strain known to express this gene at a high level. As a prerequisite to understanding possible mechanisms that control the balance between the plasminogen activating and plasmin(ogen) binding capacities of H46A, we describe here its gapC gene encoding glyceraldehyde-3-phosphate dehydrogenase (GraP-DH, EC 1.2.1.12), a glycolytic enzyme apparently transported to the cell surface where it functions as a plasmin(ogen).binding protein. The gapC gene was cloned and sequenced and found to code for a 336-amino-acid polypeptide (approximately 35.9 kDa) exhibiting 94.9% sequence identity to the Plr protein from Streptococcus pyogenes shown by others to be capable of plasmin binding [Lottenberg, R., Broder, C. C., Boyle, M. D., Kain, S. J., Schroeder, B. L. & Curtiss, R. III (1992) J. Bacteriol. 174, 5204-5210]. To study the properties of the GapC protein, its gene was inducibly overexpressed in Escherichia coli from QIAexpress expression plasmids to yield the authentic GapC or (His)6GapC carrying a hexahistidyl N-terminus to permit affinity purification. Both proteins were functionally active, exhibiting specific GraP-DH activities of about 80 kat/mol (approximately 130 U/mg) after purification. Their binding parameters [association (ka) and dissociation (kd) rate constants, and equlibrium dissociation constants (Kd = kd/ka)] for the interaction with human Gluplasminogen and plasmin were determined by real-time biospecific interaction analysis using the Pharmacia BIAcore instrument. For comparative purposes, the commercial GraP-DH from Bacillus stearothermophilus (BstGraP-DH), a nonpathogenic organism, was included in these experiments. The Kd values for binding of plasminogen to GapC, (His)6GapC and BstGraP-DH were 220 nM, 260 nM and 520 nM, respectively, as compared to 25 nM, 17 nM and 98 nM, respectively, for the binding to plasmin. These data show that both the zymogen and active enzyme possess low-affinity binding sites for the gapC gene product and that the hexahistidyl terminus does not affect its function. Prior limited treatment with plasmin enhanced the subsequent plasminogen binding capacity of all three GraP-DHs, presumably by the exposure of new C-terminal lysine residues for binding to the zymogen.

Complex transcriptional control of the streptokinase gene of Streptococcus equisimilis H46A.

On the Streptococcus equisimilis H46A chromosome, the divergent coding sequences of the genes for the plasminogen activator streptokinase (skc) and a leucine-rich protein (lrp), the function of which is unknown, are separated by a 328 bp intrinsically bent DNA region rich in AT tracts. To begin to understand the expression control of these two genes, we mapped their transcriptional initiation sites by S1 nuclease analysis and studied the influence of the bent intergenic region on promoter strength, using promoter-reporter gene fusions of skc' and lrp' to 'lacZ from Escherichia coli. The major transcriptional start sites, in both S. equisimilis and E. coli, mapped 22 bases upstream of the ATG start site of lrp (G), and 24 and 32 bases upstream of the translational initiation codon of skc (A and G, respectively), indicating the existence of two overlapping canonical skc promoters arranged in tandem on opposite faces of the helix. The reporter gene fusions were cloned in E. coli on a vector containing a 1.1 kb fragment of the S. equisimilis dexB gene, thus allowing promoter strength to be measured in multiple plasmid-form copies in the heterologous host and in single-copy genomic form following integration into the skc region of the homologous host. In S. equisimilis, skc'-'lacZ was expressed about 200-fold more strongly than the corresponding lrp'-'lacZ fusion. In contrast, in E. coli, the corresponding levels of expression differed by only about 11-fold. Deletion of the 202 bp bent region upstream of the skc and lrp core promoters caused a 13-fold decrease in skc promoter activity in S. equisimilis but did not alter lrp promoter strength in this host. In contrast, when studied in E. coli, this deletion did not alter the strength of the skc-double promoter and even increased by 2.4- to 3-fold the activity of the lrp promoter. This comparative promoter analysis shows that skc has a complex promoter structure, the activity of which in the homologous genomic environment specifically depends on sequences upstream of the two core promoters. Thus, the skc promoter structure resembles that of an array of promoters involved in a transcriptional switch; however, the nature of the potential switch factor(s) remains unknown.

The streptokinase gene: allelic variation, genomic environment and expression control.

The genes for streptokinase, the most important prokaryotic plasmingoen activator, exhibit allelic variation predominantly due to the polymorphism of an internal 220-base pair fragment that divides the phylogenetic tree of their products into two primary branches. Current molecular genetic research seeks functional correlates of the allelic variation, aims at analyzing the genomic environment of the streptokinase gene, skc, and focuses on understanding its expression. Of the six genes cloned and sequenced in the skc region of Streptococcus equisimilis H46A, skc is expressed most abundantly in a fashion that involves two overlapping core promoters and upstream sequences rich of AT tracts. Transcription of skc is terminated at a hypersymmetrical site that functions bidirectionally and prevents convergent transcription of the oppositely oriented skc and rel-orf1 genes whose mRNA abundance differs by a factor of at least three orders of magnitude.

Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen.

The streptokinase gene of Streptococcus equisimilis H46 was inactivated by plasmid insertion mutagenesis to study the relationship between elaboration of streptokinase and acquisition of cell-associated plasmin activity after incubation of wild-type and mutant cells in media containing plasminogen or plasmin. The results showed that H46A binds both the zymogen and active enzyme, generates surface-associated plasmin activity in the presence of plasminogen when producing streptokinase, and expresses its plasmin(ogen) receptor(s) independently of a functional streptokinase gene. At least part of the plasmin(ogen) binding capacity may be due to the glyceraldehyde-3-phosphate dehydrogenase type of receptor molecule, as judged by the detection of the corresponding gene.

Expression and subcellular location of native and mutant hTNF alpha proteins in Escherichia coli.

Several mutant hTNF alpha genes were constructed by deletion and stepwise reconstitution of regions coding for C-terminal sequences. The mutant hTNF alpha proteins behaved differently from native hTNF alpha when expressed in Escherichia coli. They were either sensitive to proteolytic degradation or formed insoluble aggregates depending on the strains and conditions used for expression. By contrast, native hTNF alpha was always present in a soluble form and had a tendency to associate with the cytoplasmic membrane. It was even transported to the periplasmic space in E. coli as shown by both cell fractionation and immunoelectron microscopy. The different behaviour of mutant hTNF alpha proteins probably results from a disturbance of protein folding.

Immunogold labelling of human IFN alpha 2 and an IFN alpha 1/ alpha 2 hybrid produced by recombinant Escherichia coli.

In recombinant Escherichia coli strains the subcellular location of human interferon (IFN) alpha 2 and a hybrid IFN alpha 1/alpha 2 was investigated by immunogold labelling techniques. The gold label was scattered throughout the cytoplasm in cells containing the gene for mature IFN alpha 2 under the control of heterologous staphylokinase sak42D transcription and translation initiation signals. In contrast, in cells containing in addition the sak42D signal peptide coding region in front of the IFN gene, the gold label was found mainly near the cell membrane and in the periplasmic space. Inclusion bodies were identified in cells accumulating IFN in the cytoplasm.

Synergistic cytotoxic effect of human recombinant tumor necrosis factor alpha combined with dihydroambazone.

Dihydroambazone (DHA) is a water-soluble derivative of the experimental anticancer drug ambazone. In vitro, a combination of DHA and human recombinant tumor necrosis factor alpha (TNF) exerted a strong synergism of cytotoxicity against both mouse melanoma B16K cells and the TNF-sensitive mouse fibroblast line L-M (S). Furthermore, in a colony-forming assay with B16K cells a combination of TNF and DHA inhibited colony-formation much more severely than either drug alone. An increased antiproliferative efficiency was also confirmed in vivo against established subcutaneous melanoma B16 tumors of C57BL/6 mice.

Critical role of the C-terminus in the biological activities of human tumour necrosis factor-alpha.

Alterations of the C-terminal amino acid sequence of human tumour necrosis factor-alpha (hTNF-alpha) caused significant changes in its biological activity. Thus shortening of the C-terminus by removal of two or three amino acids led to a very marked loss of cytotoxic activity. Other, more subtle changes introduced by site-directed mutagenesis resulted in a less drastic reduction in cytotoxicity. The mitogenic activity towards human fibroblasts of the hTNF-alpha was reduced in parallel with the loss of cytotoxicity. These results suggest that the C-terminal amino acids of hTNF-alpha are critical for its biological actions and that they may be part of the receptor-binding site.

Intracellular expression of hIFN alpha genes in Escherichia coli and Bacillus subtilis directed by staphylokinase signals.

Portable expression units for intracellular formation of heterologous proteins in Escherichia coli and Bacillus subtilis were constructed by inserting the transcription and translation initiation signals of the staphylokinase sak42D gene into the polylinker of plasmid pUC18. Fusions with ATG-gene cassettes coding for mature human interferons (hIFN) alpha 1 and alpha 2 resulted in intracellular expression of both proteins in E. coli. The 20 fold lower yield of hIFN alpha 2 was not due to unfavorable mRNA secondary structure formation as ruled out by constructing a hybrid hIFN alpha 1/alpha 2 gene. Intracellular expression of IFN alpha 1 in B. subtilis reached 6 x 10(4) IU/ml. Nuclease S1 mapping of transcriptional start sites revealed differential promoter usage in E. coli and B. subtilis. In E. coli transcription from the sak42D promoter was drastically reduced by by transcription initiating from upstream lac and tet promoters. In contrast, in B. subtilis transcription proceeded exclusively from the sak42D promoter.