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  • 1
    Electronic Resource
    Electronic Resource
    Specific binding of the Listeria monocytogenes transcriptional regulator PrfA to target sequences requires additional factor(s) and is influenced by iron (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 22 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The PrfA protein, which is a member of the Crp/Fnr family of prokaryotic transcription activators, regulates the virulence genes of Listeria monocytogenes. In this work, specific binding of PrfA to its target DNA was determined by electrophoretic mobility-shift assays (EMSAs) using cell-free extracts from the two L. monocytogenes strains EGD and NCTC 7973. PrfA-specific binding differs between the two strains, even when the concentration of PrfA was adjusted to similar levels. Both strains exhibited increased PrfA-specific binding after a shift into minimal essential medium (MEM) without showing a significant change in the amount of PrfA protein, relative to extracts from bacteria grown in brain–heart infusion (BHI). The purified PrfA protein from strain EGD produced in Escherichia coli did not exhibit specific binding to the target DNA but did so upon addition of PrfA-free extracts from various Listeria species and Bacillus subtilis. The observed activation of PrfA seems to be caused by a PrfA-activating factor (Paf), which is probably a protein since elevated temperature, but not RNase treatment, destroyed the activation potential of such PrfA-free extracts. Moreover, fractionation of these extracts by sucrose gradient centrifugation yielded the Paf activity in a fraction sedimenting at 3.2 S. Specific binding of PrfA-containing extracts from strain EGD to the hly and actA promoter sequences was strongly inhibited by iron, whereas that of extracts from strain NCTC 7973 was only slightly reduced. The iron effect seems to be mediated by Paf rather than by PrfA itself.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.d01-1722.x
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  • 2
    Electronic Resource
    Electronic Resource
    A new PrfA-regulated gene of Listeria monocytogenes encoding a small, secreted protein which belongs to the family of internalins (1996)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 21 (1996), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: A mutant of Listeria monocytogenes EGD was constructed that carries an extended deletion removing the entire PrfA-regulated gene cluster from plcA to plcB and a second deletion inactivating the inlA gene. Upon supplementation of this mutant with multiple gene copies of prfA, a protein of 30 kDa was detected in the supernatant of the mutant strain. The gene encoding this protein was obtained by direct and inverse polymerase chain reaction using oligonucleotide primers that were deduced from partial amino acid sequences of the purified 30 kDa protein. The amino acid sequence of the gene product revealed a protein of 297 amino acids that carried eight repeat units with high homology to those of the two known internalin proteins A and B. This secretory protein, termed internalin C, is much smaller than InlA or InlB and its complete sequence is related to the two known internalins. The gene inlC is transcribed into a monocistronic mRNA from a single promoter which shows a typical consensus sequence for PrfA-binding at the position −40. In contrast to the transcription of the inlAB operon, which is downregulated after shift of an L. monocytogenes EGD culture from brain–heart infusion into minimum essential medium (MEM), transcription of inlC is induced in MEM like most of the other known PrfA-regulated virulence genes. In addition, inlC is strongly transcribed in the cytoplasm of phagocytic J774 cells whereas inlA is poorly transcribed under these conditions, suggesting that internalin C may play a role in a late stage of L. monocytogenes infection rather than in the uptake of L. monocytogenes by non-professional phagocytic cells. An inlC deletion mutant shows reduced virulence when tested in an intravenous mouse model, but intracellular replication of the mutant in Caco-2 and J774 cells appears to be comparable with that of the wild-type strain.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1996.541414.x
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  • 3
    Electronic Resource
    Electronic Resource
    Supportive and inhibitory elements of a putative PrfA-dependent promoter in Listeria monocytogenes (2005)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 55 (2005), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Elements essential for PrfA-dependent transcription were analysed on two promoters of Listeria monocytogenes, the PrfA-dependent promoter of the phospholipase gene plcA (PplcA) and a putative promoter of the aroA gene (ParoA2) which contains a similar PrfA-binding site and a similar −10 box as PplcA but does not function as PrfA-dependent promoter. We constructed a series of hybrid plcA-aroA promoters by exchanging corresponding sequence elements of these two ‘promoters’. The results showed that the two critical elements of PrfA-dependent promoters, the PrfA-box and the −10 box, can be functionally exchanged as long as the distance in between is maintained to 22 or 23 bp. However, the interspace sequence and the sequence downstream of the −10 box of ParoA2 were strongly inhibitory for PrfA-dependent transcription. A detailed analysis of these two sequences revealed that the RNA polymerase binding site being part of the actual in vivo and in vitro used aroA promoter (ParoA1) and a sequence immediately downstream of the putative −10 site, possibly blocking the formation of the open complex, were responsible for the inhibition of PrfA-dependent transcription from ParoA2. Taking into consideration the lessons learned from this study we were able to construct a functional PrfA-dependent aroA promoter.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2005.04417.x
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  • 4
    Electronic Resource
    Electronic Resource
    PrfA mediates specific binding of RNA polymerase of Listeria monocytogenes to PrfA-dependent virulence gene promoters resulting in a transcriptionally active complex (2000)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 36 (2000), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: There is accumulating evidence that the coordinate transcription of the virulence genes in Listeria monocytogenes constitutes a very complex regulation mechanism which might require other factors in addition to PrfA. We previously described an unknown proteinaceous component from crude bacterial cell extracts, which, together with PrfA, formed a specific complex (CI) in electrophoretic mobility shift assays (EMSA) with an hly promoter probe. Here we identify the RNA polymerase (RNAP) of L. monocytogenes as an essential component of the CI complex. Addition of purified RNAP plus PrfA to the hly promoter probe allowed reconstitution of a complex migrating at the same height as CI. By using EMSA and DNaseI footprint experiments it could be shown that PrfA leads to an enhanced and specific binding of RNAP. Transcriptional activity of RNAP in vitro, using the actA promoter, was strictly dependent on PrfA.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.2000.01868.x
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  • 5
    Electronic Resource
    Electronic Resource
    New Listeria monocytogenes prfA* mutants, transcriptional properties of PrfA* proteins and structure–function of the virulence regulator PrfA (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 52 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: PrfA, a transcription factor structurally related to Crp/Fnr, activates Listeria monocytogenes virulence genes during intracellular infection. We report two new PrfA* mutations causing the constitutive overexpression of the PrfA regulon. Leu-140Phe lies in αD adjacent to the DNA-binding motif in the C-terminal domain, like a previously characterized PrfA* mutation (Gly-145Ser). Ile-45Ser, in contrast, maps to the N-terminal β-roll, a structure similar to that of the Crp cAMP binding site. The in vitro transcriptional properties of recombinant PrfA*I45S and PrfA*G145S were compared to those of PrfAWT at two differentially regulated PrfA-dependent promoters, PplcA and PactA. The two PrfA* mutations increased the affinity for the target DNA to a different extent, and the differences in DNA binding (PrfA*G145S 〉 PrfA*I45S 〉〉〉 PrfAWT) correlated with proportional differences in transcriptional activity. The use of the PrfA* proteins revealed that PplcA had a greater affinity for, and was more sensitive to, PrfA than PactA. RNA polymerase (RNAP) initiated transcription independently of PrfA at PplcA, but not at PactA, consistent with bandshift experiments suggesting that PplcA has a greater affinity for RNAP than PactA. Thus, differences in affinity for both PrfA and RNAP appear to determine the different expression pattern of PrfA-regulated promoters. Modelling of the PrfA* mutations in the crystal structure of PrfA and comparison with structure–function analyses of Crp, in which similar mutations lead to constitutively active (cAMP-independent) Crp* proteins, suggested that PrfA shares with Crp an analogous mechanism of cofactor-mediated allosteric shift. Our data support a regulatory model in which changes in PrfA-dependent gene expression are primarily accounted for by changes in PrfA activity.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2004.04052.x
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  • 6
    Electronic Resource
    Electronic Resource
    In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and -independent promoters that are differentially activated by GTP (2004)
    Oxford, UK : Blackwell Science Ltd
    Molecular microbiology 52 (2004), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA. Using our recently established in vitro transcription system, we have studied the PrfA-dependent promoter (PinlC) regulating the expression of the small, secreted internalin C. PrfA-dependent and PrfA-independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same −10 box. Although the PrfA-dependent transcription requires, as expected, the PrfA-box, PrfA-independent transcription depends on a −35 box located directly downstream of the PrfA-box. PrfA-independent transcription starts at A, 7 bp downstream of the common −10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the −35 box. PrfA-dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the −10 box can also be used. The −35 box of the PrfA-independent promoter can be functionally inactivated without affecting PrfA-dependent transcription as long as the distance between the PrfA-box and the −10 box remains fixed to 22 (or 23) bp. Vice versa, the PrfA-box can be deleted without affecting PrfA-independent transcription from PinlC, which is no longer inhibited by PrfA. The PrfA-dependent transcription initiation needs, in contrast to the PrfA-independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP. Overlapping PrfA-dependent and PrfA-independent promoter activity was also demonstrated for the mpl promoter (Pmpl). Again, PrfA-dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA-independent transcription at low GTP. Here too, the PrfA-dependent and the PrfA-independent promoters share the same −10 box characteristic of SigA-loaded RNA polymerase. High GTP concentration also appears to be necessary for transcription initiation at other PrfA-dependent promoters (Phly, PactA) but not at the PrfA-independent promoter PinlC-m8.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1365-2958.2003.03960.x
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  • 7
    Electronic Resource
    Electronic Resource
    Analysis of the SlyA-controlled expression, subcellular localization and pore-forming activity of a 34 kDa haemolysin (ClyA) from Escherichia coli K-12 (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
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    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Escherichia coli K-12 harbours a chromosomal gene, clyA (sheA, hlyE ), that encodes a haemolytic 34 kDa protein. Recombinant E. coli overexpressing the cloned clyA gene accumulated this haemolysin in the periplasm and released only very small amounts of it into the external medium. The secretion of ClyA was confined to the log phase and paralleled by the partial release of several other periplasmic proteins. Sequencing of ClyA revealed the translational start point of the clyA gene and demonstrated that the clyA gene product is not N-terminally processed during transport. The transcription of clyA from its native promoter region was positively controlled by SlyA, a regulatory protein found in E. coli, Salmonella typhimurium and other Enterobacteriaceae. SlyA-controlled transcription started predominantly 72 bp upstream from clyAas shown by primer extension. The corresponding putative promoter contains an unusual −10 sequence (TATGAAT) that is separated from a conventional −35 sequence by a GC-rich spacer. Site-directed deletion of the G in the −10 sequence abrogated the SlyA requirement for strong ClyA production, whereas a reduction in the G+C content of the spacer diminished the capability of SlyA to activate the clyA expression. Osmotic protection assays and lipid bilayer experiments suggested that ClyA forms stable, moderately cation-selective transmembrane pores that have a diameter of about 2.5–3 nm.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01196.x
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  • 8
    Electronic Resource
    Electronic Resource
    Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin (1999)
    Oxford BSL : Blackwell Science Ltd
    Molecular microbiology 31 (1999), S. 0 
    Details
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartate-rich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat region and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1365-2958.1999.01183.x
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