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:MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (KT2440)
Project description:MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (pCAR1)
Project description:Cooperative function of the three different kinds of nucleoid-associated proteins encoded on the IncP-7 plasmid pCAR1 [KT2440 chromosome chip]
Project description: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:Plasmids are one of the important mobile genetic elements in bacterial evolution. In this study, to evaluate the generality of the impact of plasmid carriage on host cell between different plasmids, we compared the response of Pseudomonas putida KT2440 to harboring three natural plasmids; RP4 (IncP-1, multidrug resistance, 60,099-bp), pCAR1 (IncP-7, carbazole-degradative, 200,231-bp) and NAH7 (IncP-9, naphthalene-degradative, 82,232-bp). We prepared two sets of plasmid-harboring strains from independent conjugation events to elucidate the reproducibility of the impact of the plasmid carriage. As results, the fitness was reduced by the carriage of RP4 and pCAR1 in liquid medium, while it was unaffected or even improved for NAH7-harboring strains. RP4-harboring KT2440 formed smaller colonies than the plasmid-free strain on solid medium (1.6% agar). The host cells were elongated by the carriage of the all plasmids, respectively. Copy number determination by quantitative PCR showed that the amount of each plasmid DNA in the host cell did not differed drastically. Whole genome resequencing showed that 13 SNPs (RP4), 24 SNPs (pCAR1) and 5 SNPs (NAH7) were the total differences between the two substrains for each plasmid-harboring strains. Transcriptome analyses showed that the impact of plasmid carriage was constantly larger in RP4-harboring strain than the other two plasmid-harboring strains. Genes involved in metal acquisition and metabolism were commonly affected by the carriage of the three plasmid. Indeed, plasmid-harboring strains showed greater growth inhibition than plasmid-free strains under iron-limiting condition. This feature could become future target to control plasmid spreading.
Project description:High-resolution mapping of the pCAR1 plasmid transcriptomes in the original host Pseudomonas resinovorans CA10 and the transconjugant Pseudomonas putida KT2440(pCAR1) While plasmids are replicated autonomously in their hosts, the transcription of plasmid genes can be switched through horizontal transfer by the change in the transcriptional networks. To examine whether and how the plasmid genome is differentially expressed, we analyzed the transcriptomes of the 199,035-bp IncP-7 carbazole catabolic and conjugative plasmid pCAR1 in the original host Pseudomonas resinovorans CA10 and the transconjugant Pseudomonas putida KT2440(pCAR1) during growth on carbazole or succinate using the high-resolution tiling array. The tiling array successfully detected the relatively large catabolic operons, for which transcription was induced during growth on carbazole regardless of the host. Compared between the hosts, nearly identical regions of pCAR1 were transcribed, but two hypothetical operons, i.e., ORF100-108 and ORF145-146, were transcribed at higher levels in KT2440(pCAR1) than in CA10. We verified the differential expression in heterologous hosts using quantitative RT-PCR. The tiling array analysis clearly revealed the transcription start sites, for which the positions and extents agreed with the primer extension experiments. Our data demonstrate that the transcriptome of the transmissible plasmid is altered through horizontal transfer, and we identified probable genes that are involved in plasmid functions in various hosts. This approach can be used to visualize flexible prokaryotic transcriptomes comprehensively. Keywords: high-resolution RNA mapping
Project description:Many bacteria harbor an incomplete quorum sensing system, wherein they possess LuxR homologues without the quorum sensing acyl-homoserine lactone (AHL) synthase, which is encoded by a luxI homolog. An artificial AHL-producing plasmid was constructed using a cviI gene encoding for C6-AHL (HHL) synthase from Chromobacterium violaceum and was introduced successfully into both wild-type and the ppoR (a luxR homolog) mutant. Our data provides evidence to suggest that the PpoR-HHL complex, but neither PpoR nor HHL alone, could attenuate growth, antibiotic resistance, and biofilm formation ability. In contrast, swimming motility, siderophore production, and indole degradation were enhanced by PpoR-HHL. The addition of exogenous indole increased biofilm formation and reduce swimming motility. Interestingly, indole proved ineffective in the presence of PpoR-HHL, thereby suggesting that the PpoR-HHL complex masks the effects of indole. Our data was supported by transcriptome analyses showing that the presence of the plasmid-encoded AHL synthase altered the expression of many genes on the chromosome in strain KT2440. Our results showed that heterologous luxI expression occurring via horizontal gene transfer can regulate a broad range of specific target genes, resulting in alterations of the phenotype and physiology of host cells.
Project description:We analyzed gene expression during conjugative transfer of plasmid RP4. Pairs of rifampicin-susceptible (RifS) and -resistance (RifR) strains of Pseudomonas putida KT2440 were conjugated for 10 minute on filter membrane in the presence of rifampicin to discriminate the expression changes in the donor and recipient cells.