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A clonal variant of serotype M1 group A streptococcus, strain 90-131, disseminated to several continents, where it was associated with severe systemic infections and toxic shock. Although this strain harbours the speA gene and is efficiently internalized by human epithelial cells, clinical isolates often fail to express the erythrogenic toxin under laboratory growth conditions. Cultures of strain 90-131 were observed to phase vary between small, dry, compact and larger, more mucoid colonies. The former were shown to be poorly internalized by epithelial cells. Analysis of RNA by Northern hybridization demonstrated that the emml, hasA and speA genes were weakly transcribed in cultures derived from the small colonies and highly transcribed in those derived from the large colonies. An insertion mutation in mga (the multigene activator) downregulated the invasion of epithelial cells and the transcription of emm1 and hasA, but had little impact on the transcription of speA. These are the first data to suggest the existence of a common regulatory circuit linking intracellular invasion, M protein, hyaluronic acid capsule and erythrogenic toxin expression by group A streptococcus. Moreover, the genetic instability of toxin expression exhibited by this serotype may impact on laboratory studies that attempt to associate toxin production with toxic shock.

RegA is a transcriptional activator that controls exotoxin A (ETA) production in Pseudomonas aeruginosa. To date, functional assays performed with the purified protein have not clearly defined the molecular mechanism of action of RegA. In this study, we sought to identify important coding regions of regA by analysing the sequences around linker insertion mutations in regA that affected toxA transcription. First, we constructed a strain with the regAB locus deleted from the chromosome, PA103 delta regAB::Gm. toxA transcription was obliterated in strain PA103 delta regAB::Gm, demonstrating that the regAB locus is essential for ETA production. Next, we constructed a series of 6 bp linker insertion mutations distributed throughout regA. These regA linker insertion mutants were sequenced and screened in PA103 delta regAB::Gm for their effects on regulation of ETA production. Six linker insertion mutations occurring between amino acids (aa) 53 and 163 of RegA were isolated that resulted in depression of toxA transcription to varying levels relative to the parental regAB locus. One of these linker insertion mutations (pTR53), resulted in a lack of iron-regulated ETA production and occurred directly upstream from a predicted transmembrane alpha-helix. The other five linker mutations (pTR88, pTR124, pTR132, pTR132-2 and pTR163) occurred within or flanked a region of RegA between aa 87-142 with similarity to the transcriptional activation domains of ToxR, VirG and OmpR. These data suggest the presence of a previously unidentified transcriptional activation domain in RegA between aa 87-142 and implicate the predicted transmembrane alpha-helix in the N-terminus as being involved in sensory transduction.

Because the ferric uptake regulator (fur) appears to be an essential gene in Pseudomonas aeruginosa, resistance to manganese was used as an enrichment to isolate strains carrying point mutations in the fur gene in order to assess its role in the co-ordinate expression of siderophores and exotoxin A (ETA). This report describes a detailed molecular and phenotypic characterization of four mutants and one revertant, which carry point mutations in the fur gene. Two parental strains were used in this study. Three mutants were isolated from the widely used strain, PAO1. One of these, CS (cold sensitive), has a mutation in the 5' non-coding region of the fur gene while the two other mutants derived from this parent have mutations resulting in the following deduced changes in Fur: mutant A2, H86-->R; mutant A4, H86-->Y. The other mutant (C6) and its revertant (C6Rv) were derived from PAO6261, a mutant of PAO1 with a deletion in the anr gene (anaerobic regulation of arginine deiminase and nitrate reduction) that controls anaerobic respiration in P. aeruginosa. Fur from the C6 mutant has an A10-->G mutation while in the C6Rv spontaneous revertant the mutant Gly residue has been changed to Ser at this position. All mutants were examined for alterations in the iron-regulated expression of siderophores and ETA. The A2 and A4 mutants expressed higher levels of siderophores in iron-deficient media and in iron-replete media. The CS mutant constitutively expressed siderophores at 25 degrees C. At 42 degrees C siderophore biosynthesis was iron repressed as in the parental strain PAO1. The deletion of anr in PAO6261 had no apparent effect on the iron-mediated regulation of siderophore synthesis, but the C6 mutant derived from this strain produces siderophores constitutively. The iron-regulated production of siderophores by C6Rv was similar to the parental strain PAO6261 and PAO1. Because one of the parental strains used in this study is an Anr mutant, regulation of ETA production was assessed under aerobic and microaerobic conditions. Iron-dependent repression of ETA synthesis in both parental strains and A2 and A4 mutants was found to be 50-100-fold under aerobic and microaerobic conditions, as assayed by quantitative Western dot-blot assays. By contrast in the CS and C6 mutants, while iron-dependent repression os ETA synthesis was similar to both parental strains under aerobic conditions, ETA production in these mutants was constitutive in a microaerobic environment. RNase protection analysis of toxA and regAB transcription in PAO1, PAO6261 and the C6 mutant corroborated the results of quantitative dot-blot assays of ETA. The mutant Fur proteins were purified and examined for their ability to bind to the promoter of a gene (pvdS) that positively regulates the expression of siderophores and ETA. Fur from the A2 and A4 mutants and from the C6Rv revertant was able to bind to the target DNA, but with reduced affinity by comparison to wild-type Fur. Fur from the C6 mutant in DNase I footprint experiments failed to protect the promoter region of the pvdS gene, but it retained some weak binding activity in gel mobility shift assays. The data presented in this study not only furnish some additional insights into the structure-function relationships of Fur, but also afford novel perspectives of virulence factors in P. aeruginosa under environmental conditions that have not previously been considered.

Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron-deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four-to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb Kpnl-Bg/II fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino-terminus region of PtxR contains a putative helix-turn-helix DNA-binding motif. Specific ptxR-deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA-lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.

Phaseolotoxin, a phytotoxin of Pseudomonas syringae pv. phaseolicola, is produced at 18 degrees C but not at 28 degrees C. Here we report that a fragment (24.4 kb) cloned from the wild-type strain, which does not harbour a gene(s) involved in phaseolotoxin biosynthesis, abolishes this thermoregulation in the wild type and suppresses a Tox- mutant at both temperatures. A subclone harbouring a 485 bp fragment contains motifs that are characteristic of DNA-binding sites. In mobility shift assays we have detected a protein(s) from the wild-type and the mutant strains, grown at appropriate temperatures, that specifically binds to the fragment containing the DNA-binding motifs. We propose that the binding protein is a repressor which is 'titrated' by this fragment when it is present in the cell on a multiple copy plasmid, thus allowing expression of phaseolotoxin genes.

We have studied the expression of the gene fragments encoding the enzymatically active portion of three bacterial cytotoxins: exotoxin A (ETA) of Pseudomonas aeruginosa, and pertussis toxin (PT) and adenylate cyclase toxin (CYA) of Bordetella pertussis, in sensitive mammalian target cells. Expression of active ETA and CYA was lethal to the producing cells and stable transfectants of Cos-1 cells containing the corresponding genes could not be obtained. The expression of the PTS1 subunit was tolerated by the producing mammalian cells. Since PT is cytotoxic because of ADP-ribosylation of G-proteins, we assume that the endogenously expressed PTS1 may not find the cellular target G proteins or PTS1 alone may not be sufficient for ADP-ribosylation of these proteins in vivo.

A multicopy plasmid containing the Escherichia coli fur gene was introduced into Pseudomonas aeruginosa strain PA103C. This strain contains a toxA-lacZ fusion integrated into its chromosome at the toxA locus. Beta-galactosidase synthesis in this strain is regulated by iron, as is seen for exotoxin A production. Beta-galactosidase synthesis and exotoxin A production in PA103C containing multiple copies of E. coli fur was still repressed in low iron conditions. The transcription of regA, a positive regulator of toxA, was also found to be inhibited by multiple copies of the E. coli fur gene. In addition, the ability of PA103C containing multiple copies of E. coli fur to produce protease was greatly reduced relative to PA103C containing a vector control. A polyclonal rabbit serum containing antibodies that recognize E. coli Fur was used to screen whole-cell extracts from Vibrio cholerae, Shigella flexneri, Salmonella typhimurium and Pseudomonas aeruginosa. All strains tested expressed a protein that was specifically recognized by the anti-Fur serum. These results and those described above suggest that Fur structure and function are conserved in a variety of distinct bacterial genera and that at least some of these different genera use this regulatory protein to control genes encoding virulence factors.

The yield of exotoxin A from Pseudomonas aeruginosa has been shown to be strain-dependent. Exotoxin A production requires the presence of the positive regulatory gene, regA. We cloned the regA genetic locus from the prototypical P. aeruginosa strain PAO1 and examined its ability to influence exotoxin A yields compared to the same region cloned from the hypertoxin-producing strain, PA103. The P. aeruginosa regA mutant strain, PA103-29, containing the PAO1 regA locus in trans produced approximately five to seven times less extracellular exotoxin A than PA103-29 containing the regA locus cloned from the hypertoxigenic strain, PA103. Nucleotide sequence analysis of the PAO1 regA locus revealed several differences, the most striking of which was the absence of a second open reading frame that was present in the analogous PA103 DNA. In addition, an amino acid substitution was found at position 144 of RegA (Thr in PAO1 and Ala in PA103). Recombinant molecules were constructed to test the contribution of each of these changes in nucleotide sequence on extracellular exotoxin A yields. The amino acid substitution in the PAO1 RegA protein was found not to affect overall exotoxin A yields. In contrast, the presence of the second open reading frame immediately downstream of the PA103 regA gene was found to influence extracellular exotoxin A yields. This open reading frame encodes a gene which we call regB. Nucleotide sequence analysis indicates that regB is 228 nucleotides in length and encodes a protein of 7527 Daltons. Our data suggest that regB is required for optimal exotoxin A production and its absence in strain PAO1 partially accounts for the difference in yield of extracellular exotoxin A between P. aeruginosa strains PAO1 and PA103.

A mobilizable plasmid which carries the promoter for the exotoxin A (ETA) structural gene fused to lacZ was integrated into the chromosome of wild-type and mutant strains of Pseudomonas aeruginosa at the toxA locus by homologous recombination. beta-galactosidase synthesis in the strains (cointegrates) carrying the toxA-lacZ fusions was regulated like ETA synthesis is in P. aeruginosa. Two multicopy plasmids carrying a positive regulatory gene designated toxR were constructed which are identical except with respect to the orientation of toxR to the lacZ promoter on the plasmid. These plasmids were then introduced into P. aeruginosa cointegrate strains. When toxR was using its own promoter, synthesis of beta-galactosidase in the cointegrate strains was increased but the pattern of iron regulation was not altered. In contrast, when the lacZ promoter was directing synthesis of the toxR product in the cointegrate strains, iron regulation of beta-galactosidase and ETA synthesis were abolished.