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Cooperative function of the three different kinds of nucleoid-associated proteins encoded on the IncP-7 plasmid pCAR1 [KT2440 chromosome chip]

ABSTRACT: 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.

ORGANISM(S): Pseudomonas putida KT2440 Pseudomonas putida

PROVIDER: GSE53067 | GEO | 2013/12/07

SECONDARY ACCESSION(S): PRJNA230794

REPOSITORIES: GEO

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Cooperative function of the three different kinds of nucleoid-associated proteins encoded on the IncP-7 plasmid pCAR1 [KT2440 chromosome 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.

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Cooperative function of the three different kinds of nucleoid-associated proteins encoded on the IncP-7 plasmid pCAR1 [pCAR1 plasmid chip]

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Effects of carbazole-degradative plasmid pCAR1 on biofilm morphology in three Pseudomonas strains.

Project description:To elucidate the impact of the carriage of carbazole-degradative plasmid pCAR1 on biofilm formation by host bacteria, we compared biofilm morphology of pCAR1-free and pCAR1-carrying three Pseudomonas hosts: P. putida KT2440, P. aeruginosa PAO1 and P. fluorescens Pf0-1 using confocal laser scanning microscopy. Although pCAR1 carriage had no significant influence on biofilm formed by PAO1 and Pf0-1, pCAR1-carrying KT2440 became filamentous and formed flat biofilm, whereas pCAR1-free KT2440 formed mushroom-like biofilm. pCAR1 carries three genes encoding nucleoid-associated proteins (NAPs); Pmr, Pnd, and Phu. Because intensive filamentous morphology was observed in two double mutants (DpmrDpnd and DpmrDphu), these NAPs cooperatively suppress cell filamentation and flat biofilm formation in KT2440(pCAR1) cells. Based on the results of transcriptome analyses of these double mutants, we could find 32 candidate genes which may be involved in the filamentation of KT2440(pCAR1). Overproduction in KT2440 and KT2440(pCAR1) of three of candidate genes (PP_2193, PP_0308, or PP_0309) induced temporal filamentation of the cells, suggesting that pCAR1 induced the abnormal filamentous morphology of KT2440 cells by overexpression of plural genes including PP_2193, PP_0308, and PP_0309. In addition, pCAR1-borne NAPs suppress the morphological changes probably by decreasing the transcriptome change by pCAR1 carriage. The pCAR1 carriage impact of chromosomal RNA maps in biofilm formation.

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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:H-NS family proteins are known as a member of nucleoid associated proteins (NAPs) conserved in genus Pseudomonas bacteria. Incompatibility group (Inc) P-7 archetype plasmid pCAR1 is transmissible among various Pseudomonas strains and carries genes encoding H-NS family protein, Pmr. P. putida KT2440 is one of pCAR1 hosts, which possesses five genes encoding H-NS family proteins, PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE) on its chromosome. Quantitative RT-PCR showed that the presence of Pmr did not affect on the transcriptions of genes encoding H-NS family proteins, and only Pmr, TurA and TurB were majorly transcribed. In vitro Pull down assay revealed that Pmr strongly interacted with Pmr itself and TurA, TurB, and TurE. Transcriptome comparisons of pmr disruptant, with KT2440 and KT2440(pCAR1) showed that the pmr disruption had larger effects on the host transcriptome than those by pCAR1 carriage. The transcriptional levels of some genes increased by pCAR1 carriage were reverted to those of pCAR1-free KT2440 by disruption of pmr, such as mexEFoprN genes for efflux pump, and parI. Transcriptional levels of many genes on the putative foreign DNA regions were not altered by carriage of pCAR1 but by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip analysis showed that Pmr preferentially binds to the foreign DNA region. Pmr binding sites were well overlapping with the location of the differentially transcribed genes by disruption of pmr. Our findings indicate that Pmr is a key factor to optimize the gene transcription onpCAR1 and host chromosome.

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MvaT family proteins encoded on IncP-7 plasmid pCAR1 and the host chromosome regulate host transcriptome cooperatively but differently (KT2440)

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.

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