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

Abstract

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.

Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea

“…The heat-stable (or histone-like) nucleoidstructural (H-NS) protein in Salmonella enterica binds DNA regions with low GC content rather than the remaining chromosomal DNA and inhibits expression of the genes contained in those regions, to which horizontally transferred DNA fragments locate (Lucchini et al, 2006;Navarre et al, 2006). Similar functions for nucleoid-associated proteins have been found in other bacteria (Castang et al, 2008;Gordon et al, 2010;Smits and Grossman, 2010;Yun et al, 2010). These gene-silencing systems depend on the fact that horizontally transferred DNAs have lower GC content than host chromosome DNAs do (Rocha and Danchin, 2002).…”

“…For example, in the Symbiobacterium thermophilus genome with high GC content (69%), transposase genes, markers of transposable genetic elements, are more frequently found in regions with lower GC content (less than 65% GC content) than in the remaining chromosomal DNA (Nishida and Yun, 2011). Interestingly, nucleoid-associated protein genes are distributed not only throughout bacterial chromosomes but also within plasmids, suggesting that plasmids have carried these genes (Yun et al, 2010;Takeda et al, 2011).…”

Structural and Molecular Genetic Analyses of the Bacterial Carbazole Degradation System

“…125) In addition, pull-down assays followed by Western blots indicated that the strength of the interactions between Pmr and itself and between Pmr and TurA, TurB, or TurE is higher than those between Pmr and TurC or TurD. 123) On the other hand, atomic force microscopic analysis revealed that Pmr has DNAbridging capacity. 125) Based on these results, we conclude that Pmr has features common to H-NS family proteins, and that it functions as a DNA-binding protein constituting homomeric and heteromeric oligomerizations with Pmr itself and at least two chromosomal P. putida KT2440, P. aeruginosa PAO1, and P. fluorescens Pf0-1.…”

“…Quantitative reverse transcription-PCR combined with RNA mapping analyses using tiling arrays suggested that pmr and turA are the primary transcribed H-NS family genes during early log-phase growth, whereas turB is transcribed in the late log and stationary growth phases in KT2440(pCAR1). 123) Gel filtration chromatography and protein-protein cross-linking analyses indicated that Pmr forms homo-oligomers consisting of its homodimers. 125) In addition, pull-down assays followed by Western blots indicated that the strength of the interactions between Pmr and itself and between Pmr and TurA, TurB, or TurE is higher than those between Pmr and TurC or TurD.…”

“…122) As the first step in clarifying the role of NAPs encoded on pCAR1, we assessed the function of Pmr in P. putida KT2440, which carries pCAR1. 123) KT2440 has five genes encoding H-NS family proteins, and recently Renzi et al 124) named them as follows: PP 1366 (turA), PP 3765 (turB), PP 0017 (turC), PP 3693 (turD), and PP 2947 (turE). Quantitative reverse transcription-PCR combined with RNA mapping analyses using tiling arrays suggested that pmr and turA are the primary transcribed H-NS family genes during early log-phase growth, whereas turB is transcribed in the late log and stationary growth phases in KT2440(pCAR1).…”