Effects of Three Different Nucleoid-Associated Proteins Encoded on IncP-7 Plasmid pCAR1 on Host Pseudomonas putida KT2440

Abstract

e Nucleoid-associated proteins (NAPs), which fold bacterial DNA and influence gene transcription, are considered to be global transcriptional regulators of genes on both plasmids and the host chromosome. Incompatibility P-7 group plasmid pCAR1 carries genes encoding three NAPs: H-NS family protein Pmr, NdpA-like protein Pnd, and HU-like protein Phu. In this study, the effects of single or double disruption of pmr, pnd, and phu were assessed in host Pseudomonas putida KT2440. When pmr and pnd or pmr and phu were simultaneously disrupted, both the segregational stability and the structural stability of pCAR1 were markedly decreased, suggesting that Pmr, Pnd, and Phu act as plasmid-stabilizing factors in addition to their established roles in replication and partition systems. The transfer frequency of pCAR1 was significantly decreased in these double mutants. The segregational and structural instability of pCAR1 in the double mutants was recovered by complementation of pmr, whereas no recovery of transfer deficiency was observed. Comprehensive phenotype comparisons showed that the host metabolism of carbon compounds, which was reduced by pCAR1 carriage, was restored by disruption of the NAP gene(s). Transcriptome analyses of mutants indicated that transcription of genes for energy production, conversion, inorganic ion transport, and metabolism were commonly affected; however, how their products altered the phenotypes of mutants was not clear. The findings of this study indicated that Pmr, Pnd, and Phu act synergistically to affect pCAR1 replication, maintenance, and transfer, as well as to alter the host metabolic phenotype.

MvaT Family Proteins Encoded on IncP-7 Plasmid pCAR1 and the Host Chromosome Regulate the Host Transcriptome Cooperatively but Differently

“…A markerless deletion mutant of pmr was constructed previously (30). In the present study, each single-gene deletion mutant of turA and turB was prepared This study similarly from KT2440(pCAR1) by using a homologous recombinationbased unmarked gene replacement system (27) as described previously.…”

Reconsidering plasmid maintenance factors for computational plasmid design

“…In contrast to smaller or nontransmissible plasmids, larger and transferable plasmids carried multiple NAP genes [ 89 , 90 ]. Three different NAPs encoded on plasmid pCAR1 are involved in plasmid stability and its conjugation in the host cells [ 91 ]. Therefore, minimizing unnecessary transcription may be important for minimizing the cost imposed by plasmids.…”

Proteome and acylome analyses of the functional interaction network between the carbazole‐degradative plasmid pCAR1 and host Pseudomonas putida KT2440

“…Pmr binds numerous sites not only on pCAR1 but also on the chromosome, and disruption of pmr resulted in significant reshaping of the transcriptome of the host (Yun et al, 2010). Furthermore, comprehensive transcriptome and phenotypic analyses of single gene mutants and double gene mutants of the three NAPs demonstrated that these proteins are key determinants of the transcriptional networks in pCAR1-harbouring host cells (Suzuki-Minakuchi et al, 2015). The proteome data in this study showed that several NAPs encoded on the chromosome, including the HU proteins HupA (PP_5313) and HupN (PP_0975), and NdpA (PP_0973), were downregulated in the pCAR1-harbouring strain, suggesting that carriage of pCAR1 might result in additional modifications in the composition of the nucleoid in the host.…”

“…Carriage of pCAR1 reduced fitness, swimming motility, resistance to osmotic and pH stresses and altered the primary metabolic capabilities of the cells, suggesting modifications in the metabolic background of the hosts . Furthermore, comprehensive phenotypic and transcriptome analyses revealed that three pCAR1encoded nucleoid-associated proteins (NAPs), Pmr, Pnd and Phu, play an important role in the plasmid-host cross-talk (Yun et al, 2010;Suzuki-Minakuchi et al, 2015;Yun et al, 2016).…”