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Günther Koraimann - One of the best experts on this subject based on the ideXlab platform.
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Silencing and activating type IV secretion Genes of the F-like conjugative resistance plasmid R1.
Microbiology (Reading England), 2013Co-Authors: Maria Anna Wagner, Karin Bischof, Dominiki Kati, Günther KoraimannAbstract:Expression of DNA Transfer (tra) Genes of F-type conjugative plasmids is required for the assembly of a functional type IV secretion machinery and subsequent plasmid DNA Transfer from donor to recipient cells. Transcription of tra Genes depends on the activation of a single promoter, designated PY, by the plasmid encoded TraJ protein. We here determine plasmid specificity of TraJ proteins from various subgroups of F-like plasmids and find that plasmid R1 conjugation and PY promoter activation can be achieved only by its cognate activator and by TraJ of the Salmonella plasmid pSLT and not by F or R100 TraJ proteins. In addition, we characterize the PY promoter of plasmid R1. We show that TraJ binds to PY DNA in vivo and that H-NS acts as a silencer of the PY promoter. In the natural plasmid context, H-NS silences Transfer Gene expression and horizontal plasmid DNA Transfer. In contrast to what was found for the F plasmid, lack of H-NS did not abolish the requirement for ArcA and TraJ to reach full tra Gene expression and DNA Transfer activity. We propose that, besides a passive de-silencing activity, both ArcA and TraJ play a direct role in synergistically stimulating tra operon transcription and subsequent DNA Transfer.
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GroEL Plays a Central Role in Stress-Induced Negative Regulation of Bacterial Conjugation by Promoting Proteolytic Degradation of the Activator Protein TraJ
Journal of bacteriology, 2007Co-Authors: Doris Zahrl, Andrea Wagner, Michael Tscherner, Günther KoraimannAbstract:Transcription of DNA Transfer Genes is a prerequisite for conjugative DNA Transfer of F-like plasmids. Transfer Gene expression is sensed by the donor cell and is regulated by a complex network of plasmid- and host-encoded factors. In this study we analyzed the effect of induction of the heat shock regulon on Transfer Gene expression and DNA Transfer in Escherichia coli. Raising the growth temperature from 22°C to 43°C transiently reduced Transfer Gene expression to undetectable levels and reduced conjugative Transfer by 2 to 3 orders of magnitude. In contrast, when host cells carried the temperature-sensitive groEL44 allele, heat shock-mediated repression was alleviated. These data implied that the chaperonin GroEL was involved in negative regulation after heat shock. Investigation of the role of GroEL in this regulatory process revealed that, in groEL(Ts) cells, TraJ, the plasmid-encoded master activator of type IV secretion (T4S) system Genes, was less susceptible to proteolysis and had a prolonged half-life compared to isogenic wild-type E. coli cells. This result suggested a direct role for GroEL in proteolysis of TraJ, down-regulation of T4S system Gene expression, and conjugation after heat shock. Strong support for this novel role for GroEL in regulation of bacterial conjugation was the finding that GroEL specifically interacted with TraJ in vivo. Our results further suggested that in wild-type cells this interaction was followed by rapid degradation of TraJ whereas in groEL(Ts) cells TraJ remained trapped in the temperature-sensitive GroEL protein and thus was not amenable to proteolysis.
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TraM of plasmid R1 controls Transfer Gene expression as an integrated control element in a complex regulatory network.
Molecular microbiology, 1997Co-Authors: Elisabeth Pölzleitner, Ellen L. Zechner, Wilfried Renner, Rainer Fratte, Bettina Jauk, Gregor Högenauer, Günther KoraimannAbstract:Site-directed mutaGenesis was used to investigate the functions of the traM Gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a Transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative Transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this Gene in conjugative Transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM Gene of plasmid R1 has at least two functions in conjugation: (i) positive control of Transfer Gene expression; and (ii) a function in a process distinct from Gene expression. Since expression of the negatively autoregulated traM Gene is itself affected positively by the expression of the Transfer operon Genes, this Gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the Genes necessary for horizontal DNA Transfer. Based on our studies, we present a novel model for the regulation of the Transfer Genes of plasmid R1 that might also be applicable to other IncF plasmids.
Gordon L. Archer - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation by TrsN of conjugative Transfer Genes on staphylococcal plasmid pGO1
Journal of bacteriology, 1994Co-Authors: Vijay K. Sharma, T M Morton, J L Johnston, Gordon L. ArcherAbstract:The major conjugative Transfer Gene cluster of staphylococcal plasmid pGO1 (trs) consists of 13 open reading frames (trsA to trsM) transcribed from one DNA strand and a single 189-bp open reading frame (trsN) within the first 348 bp of trs that is transcribed divergently. Promoter regions for trsN and trsA partially overlap. TrsN, a 7,181-Da protein, was purified as a fusion to glutathione S-Transferase and found to have DNA-binding activity. Increasing concentrations of the fusion protein progressively retarded the gel migration of PCR-Generated DNA fragments containing predicted promoters 5' to trsL, trsA, and trsN. The target sequences contained areas of identity, including regions of dyad symmetry, that were protected in DNase I footprinting studies. The binding of TrsN to its trsL target was required for this target DNA to be stably introduced into Staphylococcus aureus on a high-copy-number vector. Provision of excess TrsN from this high-copy-number vector in S. aureus decreased beta-galactosidase activity from a trsL-lacZ transcriptional fusion and decreased pGO1 conjugation frequency. Conversely, both transcription and conjugation increased in the presence of excess trsL target. We propose that TrsN negatively regulates the transcription of Genes essential for conjugative Transfer by binding to regions 5' to their translational start sites.
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DNA sequence and units of transcription of the conjugative Transfer Gene complex (trs) of Staphylococcus aureus plasmid pGO1.
Journal of bacteriology, 1993Co-Authors: T M Morton, D M Eaton, J L Johnston, Gordon L. ArcherAbstract:Abstract The conjugative Transfer Genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base Transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished Transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related Genes necessary for conjugative Transfer of plasmids in gram-negative species and 32% identity of trsC to a Gene required for conjugative mobilization of plasmid pC221 from staphylococci.
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Conjugative Transfer Genes in staphylococcal isolates from the United States.
Antimicrobial agents and chemotherapy, 1991Co-Authors: Gordon L. Archer, J ScottAbstract:Staphylococcus aureus and coagulase-negative staphylococcal isolates from various geographic areas in the United States were examined by using a conjugative Transfer Gene DNA probe in dot-blot hybridization assays. Of 175 S. aureus isolates, 47 (27%) hybridized with the probe, while 24 of 208 (11.5%) coagulase-negative staphylococci hybridized. However, among methicillin-resistant S. aureus 52% (45 of 89) were probe positive while only 2% (2 of 86) of methicillin-susceptible S. aureus were probe positive. In contrast, 12.5% (22 of 176) of methicillin-resistant and 6% (2 of 32) of methicillin-susceptible coagulase-negative staphylococci contained Transfer Genes. All but one of the staphylococci containing Transfer Genes were resistant to gentamicin; 91.5% of S. aureus and 65% of coagulase-negative staphylococci containing Transfer Genes Transferred gentamicin resistance to a S. aureus recipient. Of the 12 isolates that hybridized with the probe but did not Transfer resistance, 10 (6 coagulase-negative staphylococci and 4 S. aureus) carried both gentamicin resistance and conjugative Transfer Genes on the same plasmid. Of these 10, 6 contained plasmid target fragments of sizes different from that of the probe, suggesting additions or deletions of DNA essential for Transfer, while in 4 no such alterations could be detected. In two coagulase-negative staphylococci the entire Transfer region was apparently integrated into the chromosome. Thus, staphylococci carrying conjugative Transfer Genes are widely disseminated in the United States and are usually found in multiresistant isolates on plasmids that also encode gentamicin resistance.
Elisabeth Pölzleitner - One of the best experts on this subject based on the ideXlab platform.
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TraM of plasmid R1 controls Transfer Gene expression as an integrated control element in a complex regulatory network.
Molecular microbiology, 1997Co-Authors: Elisabeth Pölzleitner, Ellen L. Zechner, Wilfried Renner, Rainer Fratte, Bettina Jauk, Gregor Högenauer, Günther KoraimannAbstract:Site-directed mutaGenesis was used to investigate the functions of the traM Gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a Transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative Transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this Gene in conjugative Transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM Gene of plasmid R1 has at least two functions in conjugation: (i) positive control of Transfer Gene expression; and (ii) a function in a process distinct from Gene expression. Since expression of the negatively autoregulated traM Gene is itself affected positively by the expression of the Transfer operon Genes, this Gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the Genes necessary for horizontal DNA Transfer. Based on our studies, we present a novel model for the regulation of the Transfer Genes of plasmid R1 that might also be applicable to other IncF plasmids.
T M Morton - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional regulation by TrsN of conjugative Transfer Genes on staphylococcal plasmid pGO1
Journal of bacteriology, 1994Co-Authors: Vijay K. Sharma, T M Morton, J L Johnston, Gordon L. ArcherAbstract:The major conjugative Transfer Gene cluster of staphylococcal plasmid pGO1 (trs) consists of 13 open reading frames (trsA to trsM) transcribed from one DNA strand and a single 189-bp open reading frame (trsN) within the first 348 bp of trs that is transcribed divergently. Promoter regions for trsN and trsA partially overlap. TrsN, a 7,181-Da protein, was purified as a fusion to glutathione S-Transferase and found to have DNA-binding activity. Increasing concentrations of the fusion protein progressively retarded the gel migration of PCR-Generated DNA fragments containing predicted promoters 5' to trsL, trsA, and trsN. The target sequences contained areas of identity, including regions of dyad symmetry, that were protected in DNase I footprinting studies. The binding of TrsN to its trsL target was required for this target DNA to be stably introduced into Staphylococcus aureus on a high-copy-number vector. Provision of excess TrsN from this high-copy-number vector in S. aureus decreased beta-galactosidase activity from a trsL-lacZ transcriptional fusion and decreased pGO1 conjugation frequency. Conversely, both transcription and conjugation increased in the presence of excess trsL target. We propose that TrsN negatively regulates the transcription of Genes essential for conjugative Transfer by binding to regions 5' to their translational start sites.
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DNA sequence and units of transcription of the conjugative Transfer Gene complex (trs) of Staphylococcus aureus plasmid pGO1.
Journal of bacteriology, 1993Co-Authors: T M Morton, D M Eaton, J L Johnston, Gordon L. ArcherAbstract:Abstract The conjugative Transfer Genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base Transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished Transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related Genes necessary for conjugative Transfer of plasmids in gram-negative species and 32% identity of trsC to a Gene required for conjugative mobilization of plasmid pC221 from staphylococci.
Ellen L. Zechner - One of the best experts on this subject based on the ideXlab platform.
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TraM of plasmid R1 controls Transfer Gene expression as an integrated control element in a complex regulatory network.
Molecular microbiology, 1997Co-Authors: Elisabeth Pölzleitner, Ellen L. Zechner, Wilfried Renner, Rainer Fratte, Bettina Jauk, Gregor Högenauer, Günther KoraimannAbstract:Site-directed mutaGenesis was used to investigate the functions of the traM Gene in plasmid R1-mediated bacterial conjugation. Three mutant alleles, a null mutation, a sense mutation and a stop mutation, were recombined back into the R1-16 plasmid, a Transfer-derepressed (finO-) variant of plasmid R1. The frequency of conjugative Transfer of the traM null mutant derivative of R1-16 was 10(7)-fold lower than that of the isogenic parent plasmid, showing the absolute requirement for this Gene in conjugative Transfer of plasmid R1. Measurements of the abundance of plasmid specified traJ, traA and traM mRNAs, TraM protein levels, and complementation studies indicated that the traM Gene of plasmid R1 has at least two functions in conjugation: (i) positive control of Transfer Gene expression; and (ii) a function in a process distinct from Gene expression. Since expression of the negatively autoregulated traM Gene is itself affected positively by the expression of the Transfer operon Genes, this Gene constitutes a decisive element within a regulatory circuit that co-ordinates expression of the Genes necessary for horizontal DNA Transfer. Based on our studies, we present a novel model for the regulation of the Transfer Genes of plasmid R1 that might also be applicable to other IncF plasmids.