ABSTRACT
Streptococcus pyogenes (group A streptococcus [GAS]), a multiple-amino-acid-auxotrophic human pathogen, may face starvation for essential amino acids during various stages of the infection process. Since the response of GAS to such conditions is likely to influence pathogenetic processes, we set out to identify by transcriptional analyses genes and operons that are responsive to amino acid starvation and examined whether functionally meaningful response patterns can be ascertained. We discovered that GAS are capable of mounting a relA-independent amino acid starvation response that involves transcriptional modulation of a wide array of housekeeping genes as well as accessory and dedicated virulence genes. Housekeeping genes that were upregulated during starvation of both wild-type and relA mutant strains included the newly identified T-box members of the aminoacyl-tRNA synthetase genes, the genes for components of the tmRNA-mediated peptide tagging and proteolysis system for abnormal proteins (ssrA, smpB, clpP, and clpC), and the operons for the dnaK and groE groups of molecular chaperones. In addition to upregulation of the genes for oligopeptide permease (opp), intracellular peptidase (pepB), and the two-component regulator covRS reported previously (K. Steiner and H. Malke, Mol. Microbiol. 38:1004-1016, 2000), amino acid starvation stimulated the transcription of the growth phase-associated, virulence-regulatory fas operon, the streptolysin S operon (sag), and the gene for autoinducer-2 production protein (luxS). A prominent feature of operons exhibiting internal transcriptional termination (opp, fas, and sag) was starvation-promoted full-length transcription, a mechanism that improves the efficacy of these systems by increasing the level of coordinate transcription of functionally related genes. Based on these results, a regulatory network with feedback mechanisms is proposed that counteracts the stringent response, links the levels of key rate-limiting enzymes to virulence gene expression, and enables the organism in a dynamic way to take advantage of protein-rich environments provided by its human host. As several of the affected target genes are controlled by more than one regulator, fine modulation may result in accordance with the demands imposed by ecologically different colonization sites upon the adaptive capacity of the pathogen.
Subject(s)
Amino Acids/deficiency , Bacterial Proteins , Homoserine/analogs & derivatives , Ligases/metabolism , Streptococcus pyogenes/physiology , Adenosine Triphosphatases/genetics , Amino Acyl-tRNA Synthetases/genetics , Endopeptidase Clp , Homoserine/biosynthesis , Lactones , Molecular Chaperones/genetics , Operon , Protein Synthesis Inhibitors/pharmacology , Puromycin/pharmacology , RNA, Bacterial/genetics , Serine Endopeptidases/genetics , Streptolysins/genetics , Transcription, Genetic/drug effectsABSTRACT
Considering that group A streptococci are multiple auxotrophs that may encounter shortage of amino acids during specific stages of the infectious process, we studied their adaptive response to amino acid deprivation. We found that, in addition to the (p)ppGpp-mediated stringent response characterized previously, Streptococcus pyogenes exhibits a relA-independent response comprising transcriptional modulation of a specific subset of genes involved in pathogenesis. Genes/operons transcriptionally upregulated during starvation of both wild type and relA mutants included the two-component signal transduction system covRS, the positive regulator (ropB) of the pyrogenic exotoxin B gene, speB, the oligopeptide (opp) and dipeptide (dpp) permease systems and the pepB gene putatively involved in the intracellular processing of oligopeptides. Upregulation of covRS was accompanied by downregulation of ska, one of the target genes of the negative CovR regulator, and the net effect of amino acid starvation also favoured repression of speB. A significant feature of upregulated opp expression was stimulated readthrough transcription of the operon-internal oppA terminator, leading to increased mRNA levels for synthesis of the translocator complex relative to the substrate-binding protein. Based on these and previous results, a stimulus-response network is proposed that counteracts the stringent response and may enable the pathogen to mount a dynamic response to the protein-rich environment provided by its human host.
Subject(s)
Amino Acids/metabolism , Ligases/metabolism , Streptococcus pyogenes/metabolism , Bacterial Proteins/genetics , Base Sequence , Culture Media , DNA Primers , Gene Expression Regulation, Bacterial , Operon , Streptococcus pyogenes/genetics , Transcription, GeneticABSTRACT
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.
Subject(s)
Gene Expression Regulation, Enzymologic , Streptococcus/enzymology , Streptococcus/genetics , Streptokinase/genetics , Base Sequence , Chromosome Mapping , DNA, Bacterial/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Promoter Regions, Genetic , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Recombinant Fusion Proteins/biosynthesis , Transcription, GeneticABSTRACT
The function of the streptococcal cytoplasmic membrane lipoprotein, LppC, was identified with isogenic Streptococcus equisimilis H46A and Escherichia coli JM109 strain pairs differing in whether they contained [H46A and JM109(pLPP2)] or lacked (H46A lppC::pLPP10 and JM109) the functional lppC gene for comparative phosphatase determinations under acidic conditions. lppC-directed acid phosphatase activity was demonstrated zymographically and by specific enzymatic activity assays, with whole cells or cell membrane preparations as enzyme sources. LppC acid phosphatase showed optimum activity at pH 5, and the enzyme activity was unaffected by Triton X-100, L-(+)-tartaric acid, or EDTA. Database searches revealed significant structural homology of LppC to the Streptococcus pyogenes LppA, Flavobacterium meningosepticum OplA, Helicobacter pylori HP1285, and Haemophilus influenzae Hel [e (P4)] proteins. These results suggest a possible function for these proteins and establish a novel function of streptococcal cell membrane lipoproteins.
Subject(s)
Acid Phosphatase/genetics , Bacterial Proteins/genetics , Lipoproteins/genetics , Streptococcus/genetics , Acid Phosphatase/physiology , Amino Acid Sequence , Bacterial Proteins/physiology , Cytoplasm/enzymology , Hydrogen-Ion Concentration , Membrane Proteins/genetics , Membrane Proteins/physiology , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Amino Acid , Streptococcus/enzymologyABSTRACT
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.
Subject(s)
Polysaccharide-Lyases/genetics , Streptococcus agalactiae/enzymology , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA, Bacterial , Escherichia coli , Gene Expression , Genes, Bacterial , Humans , Molecular Sequence Data , Plasmids , Streptococcus agalactiae/genetics , Transcription, GeneticABSTRACT
The region between the Streptococcus equisimilis streptokinase (skc) gene and the oppositely oriented rel-orf1 transcription unit contains only one termination site known to function bidirectionally in both the homologous host and in Escherichia coli. The terminator sequence is similar to other factor-independent terminators. Using two sets of point mutations that interrupt the hairpin-upstream oligo(dA) tract or the hairpin-downstream oligo(dT) tract, we examined the possible contribution of extended base pairing between the upstream rA and downstream rU residues to efficient termination and bidirectionality in both hosts, using terminator-cat reporter gene fusions in either polarity. The results show that interrupting the oligo(dA) tract preceding the hairpin has relatively little effect on terminator strength in either orientation in the homologous host, but abolishes termination in skc polarity in E. coli. Disruption of the hairpin-distal oligo(dT) tract inactivated the terminator in skc polarity in both hosts, had little effect on termination efficiency in rel-orf1 polarity in S. equisimilis, and also retained appreciable terminator activity in E. coli. In general, these alterations of the terminator sequence, together with additional mutations that reduce the spacing between the skc stop codon and the termination site or introduce a base substitution in the terminator stem, adversely affected the efficiency of termination to a greater extent in E. coli than in the homologous host. The disparity between the effects of certain mutations in the two hosts suggests that, in addition to thermodynamic properties, specific host factors, including RNA polymerase, contribute to terminator strength.
Subject(s)
Genes, Bacterial , Streptococcus/genetics , Streptokinase/genetics , Terminator Regions, Genetic , Base Sequence , Chloramphenicol O-Acetyltransferase/genetics , Codon, Terminator/genetics , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Genes, Reporter , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Polymerase Chain Reaction , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Streptococcus/enzymology , Transcription, GeneticABSTRACT
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.
Subject(s)
Bacterial Outer Membrane Proteins/genetics , Genes, Bacterial , Haemophilus influenzae/chemistry , Lipoproteins/genetics , Streptococcus/genetics , Amino Acid Sequence , Bacterial Outer Membrane Proteins/chemistry , Base Sequence , Hemin/pharmacology , Lipoproteins/chemistry , Molecular Sequence Data , Transcription, GeneticABSTRACT
The 739-codon rel(Seq) gene of Streptococcus equisimilis H46A is bifunctional, encoding a strong guanosine 3',5'-bis(diphosphate) 3'-pyrophosphohydrolase (ppGppase) and a weaker ribosome-independent ATP:GTP 3'-pyrophosphoryltransferase [(p)ppGpp synthetase]. To analyze the function of this gene, (p)ppGpp accumulation patterns as well as protein and RNA synthesis were compared during amino acid deprivation and glucose exhaustion between the wild type and an insertion mutant carrying a rel(Seq) gene disrupted at codon 216. We found that under normal conditions, both strains contained basal levels of (p)ppGpp. Amino acid deprivation imposed by pseudomonic acid or isoleucine hydroxamate triggered a rel(Seq)-dependent stringent response characterized by rapid (p)ppGpp accumulation at the expense of GTP and abrupt cessation of net RNA accumulation in the wild type but not in the mutant. Tetracycline added to block (p)ppGpp synthesis caused the accumulated (p)ppGpp to degrade rapidly, with a concomitant increase of the GTP pool (decay constant of ppGpp, approximately 0.7 min(-1)). Simultaneous addition of pseudomonic acid and tetracycline to mimic a relaxed response caused wild-type RNA synthesis to proceed at rates approximating those seen under either condition in the mutant. Glucose exhaustion provoked the (p)ppGpp accumulation response in both the wild type and the rel(Seq) insertion mutant, consistent with the block of net RNA accumulation in both strains. Although the source of (p)ppGpp synthesis during glucose exhaustion remains to be determined, these findings reinforce the idea entertained previously that rel(Seq) fulfils functions that reside separately in the paralogous reL4 and spoT genes of Escherichia coli. Analysis of (p)ppGpp accumulation patterns was complicated by finding an unknown phosphorylated compound that comigrated with ppGpp under two standard thin-layer chromatography conditions. Unlike ppGpp, this compound did not adsorb to charcoal and did not accumulate appreciably during isoleucine deprivation. Like ppGpp, the unknown compound did accumulate during energy source starvation.
Subject(s)
Guanosine Tetraphosphate/metabolism , Streptococcus/physiology , Amino Acids , Gene Expression Regulation, Bacterial/physiology , Glucose/physiology , Kinetics , Ligases/genetics , Ligases/physiology , Mupirocin/pharmacology , Mutation , Phenotype , Protein Synthesis Inhibitors/pharmacology , Pyrophosphatases/genetics , Pyrophosphatases/physiology , Streptococcus/enzymology , Streptococcus/genetics , Tetracycline/pharmacologySubject(s)
Guanosine Pentaphosphate/metabolism , Guanosine Tetraphosphate/metabolism , Streptococcus/metabolism , Chromosome Mapping , Escherichia coli/genetics , Genes, Bacterial , Genetic Vectors , Guanosine Pentaphosphate/genetics , Guanosine Tetraphosphate/genetics , Plasmids/genetics , Streptococcus/geneticsSubject(s)
Enterococcus/genetics , Streptococcus/genetics , Chromosome Mapping , DNA Transposable Elements/genetics , Evolution, Molecular , Gene Expression Regulation, Bacterial , Gene Transfer Techniques , Genome, Bacterial , Guanine Nucleotides/metabolism , Humans , Streptococcus/metabolism , Streptococcus/pathogenicity , Virulence/geneticsABSTRACT
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.
Subject(s)
DNA, Bacterial/genetics , DNA, Bacterial/ultrastructure , Genes, Bacterial/genetics , Streptococcus/enzymology , Streptokinase/genetics , Base Sequence , Molecular Sequence Data , Sequence Analysis, DNA , Streptococcus/geneticsABSTRACT
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.
Subject(s)
Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Plasminogen/metabolism , Streptococcus/genetics , Amino Acid Sequence , Base Sequence , Chromatography, Affinity , Chromatography, Gel , Cloning, Molecular , DNA, Recombinant , Electrophoresis, Polyacrylamide Gel , Fibrinolysin/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Glyceraldehyde-3-Phosphate Dehydrogenases/isolation & purification , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Kinetics , Molecular Sequence Data , Protein Binding , Sequence Homology, Amino AcidABSTRACT
The overlapping tandem promoters of the streptokinase gene, P1 and P2, identified previously by S1 nuclease transcript mapping were functionally dissected by mutagenesis of their -10 regions and fused transcriptionally with or without the 202-bp upstream region (USR) to the luciferase reporter gene (luc) from Photinus pyralis to analyze the contribution of the different sequence elements to promoter activity in Escherichia coli and the homologous Streptococcus equisimilis strain H46A. In E. coli, virtually the entire promoter activity derived from the upstream promoter P1. In S. equisimilis, luc expression increased in the following order of the involved sequence elements: P2 approximately equal to P2 + USR < P1 < P1 + P2 < P1 + USR < P1 + P2 + USR. This shows that (1) in the homologous system, P1 and P2 alone are extremely weak, (2) in the USR-less arrangement, only the combined core promoters have substantial activity, and (3) the USR stimulates only P1 and the combination of P1 + P2. Thus, the tandem promoters presumably function by mutual contributary action and their full activity strongly depends on the AT-rich and statically bent upstream region. The distinctive feature determining the strength of P1 in both hosts appears to be its extended -10 region which matches the consensus TRTGN established for strong S. pneumoniae and Bacillus subtilis promoters.
Subject(s)
Genes, Bacterial , Promoter Regions, Genetic/genetics , Streptococcus/genetics , Streptokinase/genetics , Base Sequence , Cloning, Molecular , Escherichia coli/genetics , Luciferases/analysis , Molecular Sequence Data , Plasmids , Sequence Analysis, DNAABSTRACT
We examined the functional attributes of a gene encountered by sequencing the streptokinase gene region of Streptococcus equisimilis H46A. This gene, originally called rel, here termed relS. equisimilis, is homologous to two related Escherichia coli genes, spoT and relA, that function in the metabolism of guanosine 5',3'-polyphosphates [(p)ppGpp]. Studies with a variety of E. coli mutants led us to deduce that the highly expressed rel S. equisimilis gene encodes a strong (p)ppGppase and a weaker (p)ppGpp synthetic activity, much like the spoT gene, with a net effect favoring degradation and no complementation of the absence of the relA gene. We verified that the Rel S. equisimilis protein, purified from an E. coli relA spoT double mutant, catalyzed a manganese-activated (p)ppGpp 3'-pyrophosphohydrolase reaction similar to that of the SpoT enzyme. This Rel S. equisimilis protein preparation also weakly catalyzed a ribosome-independent synthesis of (p)ppGpp by an ATP to GTP 3'-pyrophosphoryltransferase reaction when degradation was restricted by the absence of manganese ions. An analogous activity has been deduced for the SpoT protein from genetic evidence. In addition, the Rel S. equisimilis protein displays immunological cross-reactivity with polyclonal antibodies specific for SpoT but not for RelA. Despite assignment of rel S. equisimilis gene function in E. coli as being similar to that of the native spoT gene, disruptions of rel S. equisimilis in S. equisimilis abolish the parental (p)ppGpp accumulation response to amino acid starvation in a manner expected for relA mutants rather than spoT mutants.
Subject(s)
Genes, Bacterial , Guanosine Pentaphosphate/metabolism , Pyrophosphatases/genetics , Streptococcus/genetics , Cross Reactions , Gene Expression Regulation, Bacterial , Genetic Complementation Test , Guanosine Tetraphosphate/metabolism , Ligases/genetics , Ligases/immunology , Ligases/metabolism , Manganese/pharmacology , Pyrophosphatases/drug effects , Pyrophosphatases/immunology , Pyrophosphatases/metabolism , Streptococcus/enzymologyABSTRACT
Using ten gene-specific probes from the cloned and sequenced streptokinase gene (skc) region (8,931 bp) of Streptococcus equisimilis H46A, a human serogroup C strain, the conservation of these genes and their linkage relationships were studied by Southern hybridization in pathogenic streptococci differing taxonomically, serologically, in regard to their host range, and in the class of plasminogen activator produced. The results indicate that in S. pyogenes (strains A374, NZ131 and SF130/13) and a human group G strain (G19,908) both gene content and gene order as determined for H46A (dexB-abc-lrp-skc-orfl-rel) are preserved. The same is true of an equine S. equisimilis isolate (87-542-W), the streptokinase gene of which has been shown to hybridize detectably with skc, a result at variance with that obtained previously by others. In contrast, the chromosomal DNA of three S. uberis strains (0140J, C198, C216) of bovine origin, two of which produced a plasminogen activator different from streptokinase, hybridized only with dexB-, abc- and rel-specific probes, and the homologues of these genes appeared to lie close to each other. The maintenance of the organization of the streptokinase gene region in strains differing in overall chromosomal character suggests that this gene arrangement is of selective advantage.
Subject(s)
Bacterial Proteins/genetics , Streptococcus/genetics , Streptococcus/pathogenicity , Streptokinase/genetics , Blotting, Southern , Conserved Sequence , DNA Probes , Molecular Sequence Data , Restriction Mapping , Streptococcus/classification , Streptococcus pyogenes/genetics , Streptococcus pyogenes/pathogenicityABSTRACT
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.
Subject(s)
Bacterial Proteins/genetics , Gene Expression Regulation, Bacterial , Promoter Regions, Genetic , Streptococcus/genetics , Streptokinase/genetics , Base Sequence , Chromosome Mapping , DNA, Bacterial , Escherichia coli/genetics , Molecular Sequence Data , Mutation , Single-Strand Specific DNA and RNA Endonucleases/genetics , Streptococcus/enzymology , Transcription, GeneticABSTRACT
In Streptococcus equisimilis H46A, a hypersymmetrical transcription terminator with bidirectional activity was localized between the translational termination codons of the streptokinase gene, skc, and the rel-orf1 genes. These two transcription units are oriented towards each other, and under normal conditions the skc mRNA level exceeds that of the rel-orf1 genes by a factor of at least 1000. Reporter vectors based on the promoterless cat gene were constructed by transcriptional fusion of skc to cat, such that the region between the two genes contained the terminator in skc orientation or in rel-orf1 orientation. Additionally, skc and cat were fused directly, with deletion of the terminator. The reporter vectors were designed to be capable of being studied either as multicopy plasmids in Escherichia coli or in single copy following integration, via skc, into the S. equisimilis chromosome. Chloramphenicol acetyl transferase (CAT) activity assays in conjunction with determination of chloramphenicol resistance levels and Northern hybridization analysis showed that the terminator is active in either host and orientation. However, termination efficiency was host dependent, with high terminator strength being observed in the homologous streptococcal background and appreciable readthrough occurring in E. coli. The extent of transcriptional readthrough was dependent upon terminator orientation, with termination being more efficient in rel-orf1 polarity. The results suggest that, in S. equisimilis, transcription of both skc and rel-orf1 is efficiently terminated by a common signal, and that these genes are largely protected from convergent transcription, which otherwise would seem to be particularly detrimental to the weakly expressed rel-orf1 genes.
Subject(s)
Genes, Bacterial/genetics , Streptococcus/genetics , Streptokinase/genetics , Terminator Regions, Genetic/genetics , Base Sequence , Codon, Terminator/genetics , Escherichia coli/genetics , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Bacterial/biosynthesis , RNA, Bacterial/chemistry , RNA, Messenger/biosynthesis , RNA, Messenger/chemistry , Recombinant Fusion Proteins/biosynthesis , Species Specificity , Streptococcus/enzymology , Transcription, Genetic/geneticsABSTRACT
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.
Subject(s)
Genes, Bacterial , Streptococcus/enzymology , Streptococcus/genetics , Streptokinase/genetics , Alleles , Gene Expression Regulation, Bacterial , Genetic Variation , Humans , Phylogeny , Transcription, GeneticABSTRACT
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.
Subject(s)
Fibrinolysin/metabolism , Plasminogen/metabolism , Streptococcus/enzymology , Streptokinase/metabolism , Base Sequence , DNA, Bacterial , Humans , Molecular Sequence Data , Mutagenesis, Insertional , Receptors, Cell Surface/metabolism , Receptors, Urokinase Plasminogen Activator , Restriction Mapping , Streptococcal Infections/microbiology , Streptococcus/genetics , Streptokinase/geneticsABSTRACT
The complete nucleotide sequences of four genes and one open reading frame (ORF1) adjacent to the streptokinase gene, skc, from Streptococcus equisimilis H46A were determined. These genes are encoded on the opposite DNA strand to skc and are arranged as follows: dexB-abc-lrp-skc-ORF1-rel. The dexB gene, coding for an alpha-glucosidase (M(r) 61,733), and abc, encoding an ABC transporter (M(r) 42,080), are similar to the dexB and msmK genes, respectively, from the multiple sugar metabolism operon of S. mutans. The lrp gene specifies a leucine-rich protein (M(r) 32,302) that has a leucine-zipper motif at its C-terminus. The function of the Lrp protein is not known but appeared to be detrimental when overexpressed in Escherichia coli. Although lrp appears not to be an essential gene, as judged by plasmid insertion mutagenesis, it is conserved in all streptococcal strains carrying a streptokinase gene. The rel gene showed significant homology to the E. coli relA and spoT genes involved in the stringent response to amino acid deprivation. Multiple alignment of the amino acid sequences of Rel (M(r) 83,913), RelA and SpoT revealed 59.4% homology of the primary structures. Northern hybridization analyses of the genes in the skc region showed skc to be transcribed most abundantly. In addition to transcripts for skc, monocistronic mRNAs were detected for all three genes divergently transcribed from skc. Although there was also some read-through transcription from lrp into abc, and from abc into dexB, the transcription pattern suggests a high degree of transcriptional and functional independence not only of skc but also abc and dexB. Prominent structural features in intergenic regions included a static DNA bending locus located upstream and a putative bidirectional transcription terminator downstream of skc.