Histone-like protein H1 (H-NS) family proteins are nucleoid-associated proteins (NAPs) conserved among many bacterial species. The IncP-7 plasmid pCAR1 is transmissible among various Pseudomonas strains and carries a gene encoding the H-NS family protein, Pmr. Pseudomonas putida KT2440 is a host of pCAR1, which harbors five genes encoding the H-NS family proteins PP_1366 (TurA), PP_3765 (TurB), PP_0017 (TurC), PP_3693 (TurD), and PP_2947 (TurE). Quantitative reverse transcription-PCR (qRT-PCR) demonstrated that the presence of pCAR1 does not affect the transcription of these five genes and that only pmr, turA, and turB were primarily transcribed in KT2440(pCAR1). In vitro pull-down assays revealed that Pmr strongly interacted with itself and with TurA, TurB, and TurE. Transcriptome comparisons of the pmr disruptant, KT2440, and KT2440(pCAR1) strains indicated that pmr disruption had greater effects on the host transcriptome than did pCAR1 carriage. The transcriptional levels of some genes that increased with pCAR1 carriage, such as the mexEF-oprN efflux pump genes and parI, reverted with pmr disruption to levels in pCAR1-free KT2440. Transcriptional levels of putative horizontally acquired host genes were not altered by pCAR1 carriage but were altered by pmr disruption. Identification of genome-wide Pmr binding sites by ChAP-chip (chromatin affinity purification coupled with high-density tiling chip) analysis demonstrated that Pmr preferentially binds to horizontally acquired DNA regions. The Pmr binding sites overlapped well with the location of the genes differentially transcribed following pmr disruption on both the plasmid and the chromosome. Our findings indicate that Pmr is a key factor in optimizing gene transcription on pCAR1 and the host chromosome.

The IncP-7 plasmid pCAR1 of Pseudomonas resinovorans CA10 confers the ability to degrade carbazole upon transfer to the recipient strain P. putida KT2440. We designed a customized whole-genome oligonucleotide microarray to study the coordinated expression of pCAR1 and the chromosome in the transconjugant strain KT2440(pCAR1). First, the transcriptome of KT2440(pCAR1) during growth with carbazole as the sole carbon source was compared to that during growth with succinate. The carbazole catabolic car and ant operons were induced, along with the chromosomal cat and pca genes involved in the catechol branch of the ␤-ketoadipate pathway. Additionally, the regulatory gene antR encoding the AraC/XylS family transcriptional activator specific for car and ant operons was upregulated. The characterization of the antR promoter revealed that antR is transcribed from an RpoN-dependent promoter, suggesting that the successful expression of the carbazole catabolic operons depends on whether the chromosome contains the specific RpoN-dependent activator. Next, to analyze whether the horizontal transfer of a plasmid alters the transcription network of its host chromosome, we compared the chromosomal transcriptomes of KT2440(pCAR1) and KT2440 under the same growth conditions. Only subtle changes were caused by the transfer of pCAR1, except for the significant induction of the hypothetical gene PP3700, designated parI, which encodes a putative ParA-like ATPase with an N-terminal Xre-type DNA-binding motif. Further transcriptional analyses showed that the parI promoter was positively regulated by ParI itself and the pCAR1-encoded protein ParA.Many catabolic plasmids can be transferred horizontally between different bacteria and play an important role in the distribution of the ability to degrade and utilize recalcitrant chemical compounds (15,90). Several catabolic plasmids within members of the genus Pseudomonas have been identified and have been classified mostly into incompatibility groups IncP-1, IncP-2, IncP-7, and IncP-9. Recently, the complete genome sequences of several IncP-1, IncP-7, and IncP-9 catabolic plasmids were determined (16,28,44,45,47,76,77,83,86,93). The 199,035-bp catabolic plasmid pCAR1 was originally discovered in Pseudomonas resinovorans CA10, which is able to utilize carbazole as its sole source of carbon, nitrogen, and energy (53, 56), and was the first IncP-7 plasmid to be completely sequenced (45). pCAR1 carries the car and ant operons, which encode the upper and meta pathway enzymes and the anthranilate 1,2-dioxygenase, respectively (see Fig. 1A). The operons are transcribed from two identical anthranilate-inducible promoters, P ant , under the control of the AraC/XylS family activator AntR (85), and the constitutive promoter P carAa also originates the transcription of the car operon (50). Carbazole catabolism begins with the upper pathway to yield anthranilate and 2-hydroxypenta-2,4-dienoate, and then 2-hydroxypenta-2,4-dienoate is mineralized into pyruvate and acetyl coenzyme A (acetylCoA) by the met...

Pmr, a histone-like protein H1 (H-NS) family protein encoded on plasmid pCAR1, is a key factor in optimizing gene transcription on both pCAR1 and the host chromosome. To clarify the mode of function of Pmr, we performed gel filtration chromatography analysis and protein-protein cross-linking, and found that Pmr forms homo-oligomers, consisting of its homodimers. We also found, by atomic force microscopy, that Pmr has DNA-bridging capacity. From these results, Pmr was deduced to have features common to H-NS family proteins. Additionally, evaluating protein-DNA affinity is important to clarify the mode of function of Pmr, and hence we performed an electrophoretic mobility shift assay. Though Pmr formed high-order protein-DNA complexes and did not show preference for nucleic acid sequences, the C-terminal region of Pmr did, suggesting that the DNA-binding affinity of Pmr can be evaluated by using its C-terminal region.

Pseudomonas putida KT2440 is an ideal soil bacterium for expanding the range of degradable compounds via the recruitment of various catabolic plasmids. In the course of our investigation of the host range of IncP-7 catabolic plasmids pCAR1, pDK1 and pWW53, we found that the IncP-7 miniplasmids composed of replication and partition loci were exceptionally unstable in KT2440, which is the authentic host of the archetypal IncP-9 plasmid pWW0. This study identified ParI, a homologue of ParA family of plasmid partitioning proteins encoded on the KT2440-specific cryptic genomic island, as a negative host factor for the maintenance of IncP-7 plasmids. The miniplasmids were destabilized by ectopic expression of ParI, and the loss rate correlated with the copy number of ParB binding sites in the centromeric parS region. Mutations in the conserved ATPase domains of ParI abolished destabilization of miniplasmids. Furthermore, ParI destabilized miniplasmid derivatives carrying the partition-deficient parA mutations but failed to impact the stability of miniplasmid derivatives with parB mutations in the putative arginine finger. Altogether, these results indicate that ParI interferes with the IncP-7 plasmid partition system. This study extends canonical partition-mediated incompatibility of plasmids beyond heterogeneous mobile genetic elements, namely incompatibility between plasmid and genomic island.