ABSTRACT: Pmr, a histone-like protein H1 (H-NS) family protein encoded by the IncP-7 plasmid pCAR1, is a key global regulator that alters host function
Project description:MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (pCAR1)
Project description:MvaT proteins are recognized as a member of H-NS family proteins in pseudomonads. IncP-7 conjugative plasmid pCAR1, which was originally found in Pseudomonas resinovorans, carries an mvaT homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, it was previously reported that pmr and chromosomally-encoded homologous genes, turA and turB, are majorly transcribed, and that Pmr interacts with TurA and TurB in vitro (Yun et al. J. Bacteriol. 192:4720-4731, 2010). In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Modified ChIP-chip analyses suggested that the binding sites of Pmr, TurA, and TurB in P. putida KT2440(pCAR1) genome were almost identical; nevertheless, transcriptome analyses using deletion mutants of each MvaT protein gene at the log and early-stationary growth phases clearly suggested that their regulons were different with one another. Especially, significant dissimilarity of regulons was found between Pmr and other two proteins. Transcriptions of the larger number of genes were affected by Pmr deletion at the early-stationary phase than at the log phase, suggesting that Pmr minimizes the pCAR1 effects on host fitness more effectively at the early-stationary phase. On the other hand, similarity found between the regulons of TurA and TurB implied that they may have complementary roles as global transcriptional regulators in response to the plasmid carriage. ChIP-chip: Pseudomonas putida KT2440 harboring plasmid pCAR1 cells were ChIPed with His-tag (C-terminus of each MvaT homologs) and compared with input control. Transcriptome analysis: pCAR1 RNA maps of mvaT deletion mutants were compared with those of wild-type cells.
Project description:MvaT proteins are recognized as a member of H-NS family proteins in pseudomonads. IncP-7 conjugative plasmid pCAR1, which was originally found in Pseudomonas resinovorans, carries an mvaT homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, it was previously reported that pmr and chromosomally-encoded homologous genes, turA and turB, are majorly transcribed, and that Pmr interacts with TurA and TurB in vitro (Yun et al. J. Bacteriol. 192:4720-4731, 2010). In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Modified ChIP-chip analyses suggested that the binding sites of Pmr, TurA, and TurB in P. putida KT2440(pCAR1) genome were almost identical; nevertheless, transcriptome analyses using deletion mutants of each MvaT protein gene at the log and early-stationary growth phases clearly suggested that their regulons were different with one another. Especially, significant dissimilarity of regulons was found between Pmr and other two proteins. Transcriptions of the larger number of genes were affected by Pmr deletion at the early-stationary phase than at the log phase, suggesting that Pmr minimizes the pCAR1 effects on host fitness more effectively at the early-stationary phase. On the other hand, similarity found between the regulons of TurA and TurB implied that they may have complementary roles as global transcriptional regulators in response to the plasmid carriage.
Project description:MvaT proteins are recognized as a member of H-NS family proteins in pseudomonads. IncP-7 conjugative plasmid pCAR1, which was originally found in Pseudomonas resinovorans, carries an mvaT homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, it was previously reported that pmr and chromosomally-encoded homologous genes, turA and turB, are majorly transcribed, and that Pmr interacts with TurA and TurB in vitro (Yun et al. J. Bacteriol. 192:4720-4731, 2010). In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Modified ChIP-chip analyses suggested that the binding sites of Pmr, TurA, and TurB in P. putida KT2440(pCAR1) genome were almost identical; nevertheless, transcriptome analyses using deletion mutants of each MvaT protein gene at the log and early-stationary growth phases clearly suggested that their regulons were different with one another. Especially, significant dissimilarity of regulons was found between Pmr and other two proteins. Transcriptions of the larger number of genes were affected by Pmr deletion at the early-stationary phase than at the log phase, suggesting that Pmr minimizes the pCAR1 effects on host fitness more effectively at the early-stationary phase. On the other hand, similarity found between the regulons of TurA and TurB implied that they may have complementary roles as global transcriptional regulators in response to the plasmid carriage.
Project description:MvaT proteins are recognized as a member of H-NS family proteins in pseudomonads. IncP-7 conjugative plasmid pCAR1, which was originally found in Pseudomonas resinovorans, carries an mvaT homologous gene, pmr. In Pseudomonas putida KT2440 bearing pCAR1, it was previously reported that pmr and chromosomally-encoded homologous genes, turA and turB, are majorly transcribed, and that Pmr interacts with TurA and TurB in vitro (Yun et al. J. Bacteriol. 192:4720-4731, 2010). In the present study, we clarified how the three MvaT proteins regulate the transcriptome of P. putida KT2440(pCAR1). Modified ChIP-chip analyses suggested that the binding sites of Pmr, TurA, and TurB in P. putida KT2440(pCAR1) genome were almost identical; nevertheless, transcriptome analyses using deletion mutants of each MvaT protein gene at the log and early-stationary growth phases clearly suggested that their regulons were different with one another. Especially, significant dissimilarity of regulons was found between Pmr and other two proteins. Transcriptions of the larger number of genes were affected by Pmr deletion at the early-stationary phase than at the log phase, suggesting that Pmr minimizes the pCAR1 effects on host fitness more effectively at the early-stationary phase. On the other hand, similarity found between the regulons of TurA and TurB implied that they may have complementary roles as global transcriptional regulators in response to the plasmid carriage. ChIP-chip: Pseudomonas putida KT2440 harboring plasmid pCAR1 cells were ChIPed with His-tag (C-terminus of each MvaT homologs) and compared with input control. Transcriptome analysis: Chromosomal RNA maps of mvaT deletion mutants were compared with those of wild-type cells.
Project description:Nucleoid-associated proteins (NAPs) are known to fold bacterial DNA and influence global transcription. Incompatibility P-7 plasmid pCAR1 carries three genes encoding NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. Because previous reports about plasmid-encoded NAPs mainly focused on H-NS homologs, functions and importance of different kinds of NAPs encoded on a plasmid remained unknown. Here, we assessed the effects of single or double disruption of pmr, pnd, and phu in a host P. putida KT2440. When pmr and pnd or pmr and phu were disrupted simultaneously, stability and conjugation frequency of pCAR1 decreased significantly. In the comprehensive phenotypes comparisons, host availabilities of some compounds, which were reduced by pCAR1carriage, were restored by NAP-gene(s)-disruption. Transcriptome analyses showed that Pmr and Pnd have different regulons, whereas Phu mainly supports their gene regulation. These cooperative functions of the three NAPs were not simply due to protein-protein interactions because hetero-oligomers of them were not detected in pull-down assays. Our present study is the first report about the cooperative function of plasmid-encoded different kinds of NAPs, which show no homology with each other.
Project description:Nucleoid-associated proteins (NAPs) are known to fold bacterial DNA and influence global transcription. Incompatibility P-7 plasmid pCAR1 carries three genes encoding NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. Because previous reports about plasmid-encoded NAPs mainly focused on H-NS homologs, functions and importance of different kinds of NAPs encoded on a plasmid remained unknown. Here, we assessed the effects of single or double disruption of pmr, pnd, and phu in a host P. putida KT2440. When pmr and pnd or pmr and phu were disrupted simultaneously, stability and conjugation frequency of pCAR1 decreased significantly. In the comprehensive phenotypes comparisons, host availabilities of some compounds, which were reduced by pCAR1carriage, were restored by NAP-gene(s)-disruption. Transcriptome analyses showed that Pmr and Pnd have different regulons, whereas Phu mainly supports their gene regulation. These cooperative functions of the three NAPs were not simply due to protein-protein interactions because hetero-oligomers of them were not detected in pull-down assays. Our present study is the first report about the cooperative function of plasmid-encoded different kinds of NAPs, which show no homology with each other.
Project description:Cooperative function of the three different kinds of nucleoid-associated proteins encoded on the IncP-7 plasmid pCAR1 [pCAR1 plasmid chip]
Project description:Nucleoid-associated proteins (NAPs) are known to fold bacterial DNA and influence global transcription. Incompatibility P-7 plasmid pCAR1 carries three genes encoding NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. Because previous reports about plasmid-encoded NAPs mainly focused on H-NS homologs, functions and importance of different kinds of NAPs encoded on a plasmid remained unknown. Here, we assessed the effects of single or double disruption of pmr, pnd, and phu in a host P. putida KT2440. When pmr and pnd or pmr and phu were disrupted simultaneously, stability and conjugation frequency of pCAR1 decreased significantly. In the comprehensive phenotypes comparisons, host availabilities of some compounds, which were reduced by pCAR1carriage, were restored by NAP-gene(s)-disruption. Transcriptome analyses showed that Pmr and Pnd have different regulons, whereas Phu mainly supports their gene regulation. These cooperative functions of the three NAPs were not simply due to protein-protein interactions because hetero-oligomers of them were not detected in pull-down assays. Our present study is the first report about the cooperative function of plasmid-encoded different kinds of NAPs, which show no homology with each other. The NAPs-dependent change of chromosomal RNA maps in early exponential phases.
Project description:Nucleoid-associated proteins (NAPs) are known to fold bacterial DNA and influence global transcription. Incompatibility P-7 plasmid pCAR1 carries three genes encoding NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. Because previous reports about plasmid-encoded NAPs mainly focused on H-NS homologs, functions and importance of different kinds of NAPs encoded on a plasmid remained unknown. Here, we assessed the effects of single or double disruption of pmr, pnd, and phu in a host P. putida KT2440. When pmr and pnd or pmr and phu were disrupted simultaneously, stability and conjugation frequency of pCAR1 decreased significantly. In the comprehensive phenotypes comparisons, host availabilities of some compounds, which were reduced by pCAR1carriage, were restored by NAP-gene(s)-disruption. Transcriptome analyses showed that Pmr and Pnd have different regulons, whereas Phu mainly supports their gene regulation. These cooperative functions of the three NAPs were not simply due to protein-protein interactions because hetero-oligomers of them were not detected in pull-down assays. Our present study is the first report about the cooperative function of plasmid-encoded different kinds of NAPs, which show no homology with each other. The NAPs-dependent change of RNA maps in early exponential phases.