Biomedical subjects
Publications and source records attributed to V Scarlato.
The complete nucleotide sequence of bacteriophage T4D gene 28 has been determined. Gene 28 product is a structural component of the viral baseplate for which an enzymatic activity has also been proposed.
Expression of virulence factors of Bordetella pertussis is coordinately regulated by the products of the bvg locus, which codes for a sensory protein (BvgS) and a positive regulator of transcription (BvgA), a pair in the family of bacterial 'two-component' regulators. Transcription of the bvg-regulated promoters is repressed by modulating environmental factors such as 50 mM MgSO4, 10 mM nicotinic acid (NA) or low temperature (25 degrees C). We have isolated a spontaneous mutant (SK170) which expresses virulence genes at either 25 degrees C, or in the presence of 1-5 mM NA, or 10-50 mM MgSO4. Virulence factors in strain SK170 are still repressed by higher concentrations of NA (10 mM), or by a combination of low temperature (25 degrees C) and one of the other modulating agents. From this strain, we have isolated a second mutant (SK180) that showed constitutive synthesis of the virulence factors under any growth regime. Nucleotide (nt) and deduced amino acid (aa) sequence analysis showed that SK170 contains a substitution at aa570 of BvgS and SK180 contains an additional substitution at aa680. These substitutions are confined to a 161-aa sequence that links the transmembrane (TM) and kinase domains of BvgS. These mutations also alter the transcriptional autoregulation of the P1 and P2 promoters of the bvg locus. P1, which in the wild-type (wt) strain is repressed by modulating agents, is constitutively active in the mutant strains. On the contrary, P2, which is normally induced by all three modulating agents, is active in strain SK170 only in the presence of MgSO4 or NA, while in strain SK180 this promoter is repressed by modulating agents. The mutants exhibit elevated levels of the BvgA regulatory protein and have a virulent phenotype also in the presence of modulating agents.
Bordetella pertussis, the human pathogen of whooping cough, when grown at 22 degrees C is nonvirulent and unable to bind eukaryotic cells. In response to a temperature shift to 37 degrees C, the bacterium acquires the ability to bind eukaryotic cells in a time-dependent fashion. By studying in vitro the temperature-induced transition, from the nonvirulent to the virulent state, we found that binding to CHO cells is mediated by the Arg-Gly-Asp-containing domain of filamentous hemagglutinin (FHA), a protein with multiple binding specificities. This protein is synthesized as a 367-kDa polypeptide within 10 min after temperature shift, but requires 2 hr before it is detected on the bacterial cell surface and starts to bind CHO cells. Mutations affecting the cell surface export of FHA abolish bacterial adhesion to CHO cells, while mutations in the outer membrane protein pertactin strongly reduce binding. This suggests that multiple chaperon proteins are required for a correct function of FHA. Finally, several hours after maximum binding efficiency is achieved, the N-terminal 220-kDa portion of FHA that contains the binding regions is cleaved off, possibly to release the bacteria from the bound cells and facilitate spreading. The different forms of FHA may play different roles during bacterial infection.
Many bacteria respond in a coordinate manner to environmental changes. External stimuli, sensed by receptors, are transduced to regulatory proteins which participate in well defined pathways of gene expression by varying their structure and mode of action. The network of environmental signal transduction is responsible for a fine and continuous communication between the host and the pathogenic bacteria. As a result, the gene expression machinery of the pathogen is modified continuously, in order to establish the optimal conditions for bacterial survival and multiplication.
We analysed transcription of the DNA region immediately downstream of the origin of replication in the chlamydial plasmid pCT. This region comprises two convergent open reading frames (ORF7, ORF8), encoding putative polypeptides that are homologous to each other and with C-terminal domains typical of the phage integrase family of proteins. Northern blot and RNA 5' end mapping analyses indicated that both ORFs were transcribed in the late phase of the chlamydial replicative cycle. RNA mapping showed the presence of a transcript starting 31 nucleotides (nt) before the ATG start codon of ORF7, and two temporally regulated transcripts starting 59 and 89 nt upstream of the ATG start codon of ORF8. Two abundant RNA species of 225 and 415 nt were also identified as overlapping anti-sense transcripts (AS-RNAs), complementary to the 3' end of ORF8 mRNA, with identical 5' ends but different 3' ends. In vitro and in vivo experiments in Escherichia coli showed that the sigma 70-RNA polymerase complex was capable of initiating RNA synthesis at the same sites as observed in Chlamydia trachomatis for ORF7 and AS-RNA transcripts, but was not able to transcribe ORF8. In accord with this, sequences at -10 and -35 nt upstream of the RNA 5' ends resemble sigma 70 consensus promoters in the case of ORF7 and AS, but not in the case of the two ORF8 transcripts. Therefore, transcription of ORF7 and ORF8 is controlled by different types of promoters.(ABSTRACT TRUNCATED AT 250 WORDS)
Plasmid pCT is present in essentially all isolates of Chlamydia trachomatis and may encode factors important for survival in the natural environment. However, no pCT-associated phenotype has been described so far. With the purpose of investigating the possibility of a role of pCT in C. trachomatis pathogenicity we examined the expression of an ORF (ORF3), potentially encoding a 28 kDa polypeptide (pgp3). Analysis of RNA extracted from chlamydia-infected Vero cells detected ORF3-specific transcripts, from 20 h post-infection onwards, mainly as discrete RNA species of 1390 nucleotides comprising the downstream ORF4 sequence. ORF3 DNA was cloned and expressed in Escherichia coli as a 39 kDa fusion protein (MS2/pgp3). Antibodies raised against purified MS2/pgp3, specifically recognized a 28 kDa protein on Western blots of protein from purified chlamydial elementary bodies (EBs). The same antibodies detected chlamydial inclusions in methanol-fixed infected cells by immunofluorescence. Western blot analysis of EBs extracted with 2% Sarkosyl, showed that a large proportion of the 28 kDa antigen is associated with the detergent-insoluble ('membrane') fraction. Antibodies recognizing pgp3 epitopes were detected in sera from patients with chlamydial infections, but not in sero-negative control sera. The finding support the hypothesis that pCT may provide a function related to chlamydial cell physiology.
The bvg locus of Bordetella pertussis encodes an environmentally inducible operon essential for the expression of virulence genes. We show that in Escherichia coli, the PTOX promoter cloned in cis of the bvg locus is activated and environmentally regulated. Cotransformation of E. coli with the bvg locus cloned in a low-copy-number plasmid and with the PTOX promoter cloned in a high-copy-number plasmid can give rise to two different results. If the PTOX promoter is cloned in the pGem-3 vector, transcription is absent. If the PTOX promoter is cloned in the plasmid pKK232, containing the PTOX promoter between two ribosomal gene terminators of transcription, transcription occurs, although regulation of transcription is abolished. Under these conditions, the intracellular amount of RNA transcripts is increased by adding to the culture medium novobiocin, an inhibitor of bacterial gyrases. In vitro, the transcription of the PTOX promoter is activated on E. coli RNA polymerase supplemented with cell extracts from wild-type B. pertussis. Addition of DNA gyrase to the mixture dramatically reduces the amount of RNA synthesized. Our data show that the products of the bvg locus, BvgA and BvgS, are directly involved in the regulation of the PTOX promoter in E. coli and that DNA topology may play a role in the induction of transcription.
The bacteriophage SPO1 DNA polymerase-encoding gene, which contains a self-splicing intron, has been sequenced and its amino acid (aa) sequence has been deduced. The aa sequence of SPO1 DNA polymerase shows a high degree of similarity with that of DNA polymerase I from Escherichia coli (Po1I). Alignment with the sequences of Po1I, and the phi 29 and SPO1 DNA polymerases indicate that the aa residues that have been implicated in 3'----5' exonuclease activities are conserved.
The bacteriophage SP01 gene 30, whose function is essential for DNA synthesis, has been analyzed for its primary structural features. Conditionally lethal mutations in the gene 30 locus have been mapped and sequenced, and the wild-type amino acid (aa) sequence has been deduced along with that of a co-transcribed and possibly co-translated upstream unidentified reading frame (URF). The aa sequence deduced for gene 30 shares partial similarity with protein P of bacteriophage lambda, which participated in lambda DNA replication, and also with the exonuclease, gp46, of bacteriophage T4. A lysine-rich region of the hypothetical product of the URF shares similarity with both the T4 DNA topoisomerase and the phi 29 gene 3-encoded protein; the latter codes for a terminal protein which participates in the priming of DNA elongation.
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Bacterial pathogens undergo profound physiological changes when they infect their host and require co-ordinated regulation of gene expression in response to the stress encountered during infection. In Bordetella pertussis, the human pathogen which causes whooping cough, virulence factors are synthesized in response to environmental signals under the control of the bvg regulatory locus. Here we demonstrate that the bvg locus is responsible for two events of gene activation. In the first step the bvg locus transactivates its own autoregulated promoter (P1) and the promoter of the adherence factor filamentous haemagglutinin (PFHA). The second step occurs several hours later and consists of the transactivation of adenylate cyclase and pertussis toxin genes. We provide evidence that the second step of transactivation requires overexpression of regulatory proteins. Our results imply that bacterial adhesion and tissue colonization--intoxication are two separate steps at the molecular level.
Phage SPO1 middle transcripts are known to fall into two classes, m and m1l. Class m1l transcripts continue to be made late in the viral infection, while the synthesis of class m transcripts ceases soon after the onset of replication and late transcription. The experiments that are reported here deal with the regulatory nature of this diversity. The accumulation of transcripts associated with eight middle promoters was analyzed by S1 nuclease mapping. DNA sequence surrounding these middle promoters was determined or redetermined, and the stability of RNA associated with most of these promoters was also analyzed. Class m1l transcription was shown to be associated with SPO1 middle promoters that remain active at late stages of viral development, when middle promoters of class m are repressed. The consensus sequences of class m and m1l middle promoters were found to be indistinguishable and the search for sequences consensual with late promoters yielded only divergent candidates. No other consensus sequence that is specific and exclusive to either class of middle promoters was detected within a hundred base pairs upstream or downstream of these promoters. Considerable variations in the stabilities of SPO1 middle transcripts were found. Two promoters that are only 71 base pairs apart yielded transcripts that had substantially different stabilities. The 5'-flanking segment of the transcript associated with the upstream promoter apparently conferred a high degree of stability on this RNA.
The bvg locus contains two genes, bvgA and bvgS, which control the expression of the virulence-associated genes in Bordetella species by a system similar to the two-component systems used by a variety of bacterial species to respond to environmental stimuli. We determined the nucleotide sequence of the bvg loci of Bordetella parapertussis and Bordetella bronchiseptica and compared them with the previously determined sequence of Bordetella pertussis. The nucleotide and amino acid sequences of the bvg loci of these species are well conserved in those regions coding for the protein domains which have putative kinase and DNA-binding activities. In marked contrast, the region of BvgS that codes for the protein domain with putative sensor activity shows a high degree of variability. In total, we find 198 base-pair changes in the bvg loci of B. parapertussis and B. bronchiseptica relative to the bvg locus of B. pertussis. One hundred and seventy-three of these base-pair changes are identical in B. parapertussis and B. bronchiseptica. This confirms our previous observation that B. parapertussis and B. bronchiseptica are more related to each other than to B. pertussis. We have mapped the mutations that cause phase changes in B. bronchiseptica and we have found that in three cases these are due to spontaneous deletions in the bvgS gene. The wild-type bvg locus present on a multicopy plasmid cannot complement avirulent derivatives of B. bronchiseptica to wild-type levels, but it can do so when the bvgA gene on the plasmid is inactivated. This suggests that hyperexpression of bvgA down-regulates the bvg system.
The expression of the virulence-associated genes in Bordetella species is co-ordinately regulated by the gene products encoded by the bvg locus. In Bordetella pertussis the expression of this locus is regulated by the P1, P2, P3 and P4 promoters which are located in a 350 bp DNA fragment also containing the PFHA promoter. Here we report the transcriptional regulation of the bvg locus and the fha gene in Bordetella parapertussis and a sequence analysis of the bvg-regulated promoters. The Pp1, Pp2, Pp4 and PpFHA promoters are indistinguishable, both in transcription initiation sites and environmental regulation, from the corresponding promoters of B. pertussis, while the Pp3 promoter is not active. Sequence homologies from nine bvg-regulated promoters show a conserved dinucleotide, 5'-TG-3', at approximately one turn of helix upstream of the -10 5'-A.AaTat-3' region, and a 5'-TTTCC-3' sequence in the -90 region. Since the nucleotide sequence of the inactive Pp3 promoter shows several base substitutions with respect to the found sequence homologies, it is likely that some of these bases play an essential role in promoter activity.
Magnesium sulfate is known to repress the expression of the virulence factors of Bordetella pertussis that are coordinately regulated by the bvg locus. We have tested the time required by MgSO4 to repress the synthesis of several bvg-regulated mRNA species and found that the promoters of the virulence genes (pertussis toxin, adenylate cyclase, and filamentous hemagglutinin) are repressed in 6 min, while the autogenously regulated promoters of the bvg locus (P1, P3, and P4) are repressed only several hours later. These data show a differential behavior between regulated and autoregulated genes of the bvg regulon.
We report a self-splicing intron in bacteriophage SPO1, whose host is the gram-positive Bacillus subtilis. The intron contains all the conserved features of primary sequence and secondary structure previously described for the group IA introns of eukaryotic organelles and the gram-negative bacteriophage T4. The SPO1 intron contains an open reading frame of 522 nucleotides. As in the T4 introns, this open reading frame begins in a region that is looped out of the secondary structure, but ends in a highly conserved region of the intron core. The exons encode SPO1 DNA polymerase, which is highly similar to E. coli DNA polymerase I. The demonstration of self-splicing introns in viruses of both gram-positive and gram-negative eubacteria lends further evidence for their early origin in evolution.
The complete E7 protein-encoding open reading frame of human papillomavirus type 16 (HPV-16) was expressed in the fission yeast Schizosaccharomyces pombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in S. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in Escherichia coli). The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in S. pombe is identical to that of the E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed S. pombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity.
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