JOURNAL OF BACTERIOLOGY, Sept. 2010, p. 4720–4731
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 192, No. 18
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?¶
Choong-Soo Yun,1,2† Chiho Suzuki,1† Kunihiko Naito,1Toshiharu Takeda,1Yurika Takahashi,1
Fumiya Sai,1Tsuguno Terabayashi,1Masatoshi Miyakoshi,1‡ Masaki Shintani,1§
Hiromi Nishida,2Hisakazu Yamane,1and Hideaki Nojiri1,2*
Biotechnology Research Center1and Agricultural Bioinformatics Research Unit, Graduate School of Agricultural and
Life Sciences,2the University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Received 25 May 2010/Accepted 6 July 2010
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.
Nucleoid-associated proteins (NAPs) have architectural and
regulatory functions in bacterial cells. Bacterial chromosomal
DNA is folded into a compact nucleoid body by NAPs (9, 11).
Because of their DNA-binding ability, NAPs can also influence
the expression of genes (9, 11). Histone-like protein H1 (H-
NS), a NAP family member, is an oligomeric DNA-binding
protein identified in Escherichia coli because of its effect on
transcription in vitro (13, 16). H-NS acts as a global repressor
and binds to horizontally acquired DNA regions (28). Plasmid-
encoded H-NS can function as a “stealth” protein to switch off
gene expression on chromosomes or plasmids and to main-
tain host cell fitness (15). H-NS also interacts with paralo-
gous proteins, such as StpA and Hfp in E. coli, or other
NAPs (12, 16, 27).
Tendeng et al. (39) suggested that conserved MvaT proteins
from Pseudomonas bacteria belong to the H-NS family, despite
their limited sequence similarity with H-NS. Recently MvaT
and MvaU from Pseudomonas aeruginosa PAO1, functional
homologous H-NS proteins from Pseudomonas bacteria, were
shown to interact with each other (44). Castang et al. (5)
reported that these two H-NS family proteins bind to the same
chromosomal regions and that they function coordinately. In-
terestingly, P. putida KT2440 has five genes encoding H-NS
family proteins, and recently Renzi et al. (30) named them as
follows: PP_1366 (turA), PP_3765 (turB), PP_0017 (turC),
PP_3693 (turD), and PP_2947 (turE). TurA and TurB were
copurified as the TOL plasmid (pWW0) upper operon repres-
sors A and B, respectively, and both bound to the Pu promoter
(a ?54-dependent promoter of the operon encoding enzymes
for the upper pathway of toluene degradation in pWW0), sug-
gesting that these two proteins could interact with each other
(31). Renzi et al. (30) proposed that TurA and TurB belonged
to groups I and II, respectively, and that these groups con-
tained orthologous H-NS family proteins present in all Pseudo-
monadaceae species. Conversely, TurC, TurD, and TurE be-
longed to group III, which contained species-specific H-NS
family proteins (30).
The self-transmissible pCAR1, an IncP-7 archetypal plas-
mid, endows the host strain with carbazole-degrading ability
(23, 36, 38). pCAR1 carries the pmr gene, encoding the H-NS
family protein designated Pmr (plasmid-encoded MvaT-like
* Corresponding author. Mailing address: Biotechnology Research
Center, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657,
Japan. Phone: 81-3-5841-3064. Fax: 81-3-5841-8030. E-mail: anojiri
† C.-S.Y. and C.S. contributed equally to this work.
‡ Present address: Department of Environmental Life Sciences,
Graduate School of Life Sciences, Tohoku University, Sendai 980-
§ Present address: Japan Collection of Microorganisms, Microbe
Division, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama
¶ Supplemental material for this article may be found at http://jb
?Published ahead of print on 16 July 2010.
regulator) (25) and belonging to the above-mentioned group
III. The effect of plasmid carriage on host strains may change
in different hosts, and therefore, we performed transcriptome
comparisons between pCAR1-free and pCAR1-containing
KT2440 strains (25, 35). Based on the comparisons, pCAR1
carriage affected the iron acquisition system of the host
KT2440 strain, enhanced resistance to chloramphenicol by in-
ducing the mexEF-oprN operon, and induced the transcription
of PP_3700 (parI) (35). We also discovered that pmr was tran-
scribed in four distinct Pseudomonas host bacterial strains (26,
35). These data suggest that Pmr could interact with other
H-NS family proteins, such as TurA, TurB, TurC, TurD, and
TurE, encoded on the KT2440 chromosome.
In the present study, we assessed the in vivo transcriptional
profiles of genes encoding H-NS family proteins on both
pCAR1 and the KT2440 chromosome. Additionally, we inves-
tigated the in vitro interaction of Pmr with itself and with other
H-NS family proteins. Furthermore, we assessed the effect of
pmr disruption on the transcriptome of the host strain and
identified genome-wide Pmr-binding sites. Taken together, we
clarified the role of Pmr as a horizontally acquired H-NS family
MATERIALS AND METHODS
Bacterial strains and plasmids. The bacterial strains and plasmids used in this
study are listed in Table 1. E. coli strains for cloning and expression of genes were
grown in L broth (LB) (32) at 37°C or 25°C, and the Pseudomonas strains were
cultivated with LB at 30°C. Ampicillin (Ap) (50 ?g/ml), chloramphenicol (Cm)
(30 ?g/ml), kanamycin (Km) (50 ?g/ml), gentamicin (Gm) (120 ?g/ml), rifampin
(Rif) (250 ?g/ml), streptomycin (Sm) (450 ?g/ml), or tetracycline (Tc) (12.5
?g/ml) was added to the selective medium. For plate cultures, the above media
were solidified with 1.6% agar (wt/vol).
DNA manipulations. Plasmid DNA extraction from E. coli was performed
using the alkaline lysis method (32), and total DNA from Pseudomonas strains
was extracted using hexadecyltrimethylammonium bromide as described previ-
ously (1). Restriction enzymes (New England Biolabs, Ipswich, MA; Toyobo,
Tokyo, Japan) and the Ligation High reagent (Toyobo) were used according to
the manufacturers’ instructions. DNA fragments were extracted from agarose
gels using the Ezna gel extraction kit (Omega Bio-Tek, Norcross, GA) according
to the manufacturer’s instructions. PCR was performed with Ex Taq Hot Start
polymerase (Takara Bio, Shiga, Japan) according to the manufacturer’s instruc-
tions. All other experiments were performed according to standard methods
(32). All primers used are presented in Table S1 in the supplemental material.
RNA extraction. RNA extractions from strain KT2440, KT2440(pCAR1), or
KT2440(pCAR1?pmr) were performed as follows: an overnight culture of each
strain in LB was washed and transferred into 100 ml NMM-4 buffer (37) sup-
plemented with 0.1% succinate by adjusting the turbidity to 0.05 at 600 nm and
then incubated at 30°C in a rotating shaker at 120 rpm. At early log phase growth
(turbidity of 0.15 to 0.20 at 600 nm), we used the RNAprotect bacterial reagent
(Qiagen, Valencia, CA) to stabilize the total RNA in the bacterial cultures, and
subsequently, RNA extraction was performed using the RNeasy Midi kit (Qia-
gen) or Nucleospin RNA II (Macherey-Nagel GmbH & Co. KG, Du ¨ren, Ger-
many) according to the manufacturers’ instructions. The eluted RNA was treated
with RQ1 RNase-free DNase (Promega, Fitchburg, WI) at 37°C for 30 min.
Following inactivation of the DNase by the addition of the stop reagent and
subsequent incubation at 65°C for 10 min, RNA samples were repurified with the
TABLE 1. Bacterial strains and plasmids used in this study
Strain or plasmid Relevant characteristicsSource or reference
F??80dlacZ?M15 ?(lacZYA-argF)U169 endA1 recA1 hsdR17(rK
deoR thi-1,supE44 gyrA96 relA1 ??phoA
?) gal dcm ? (DE3)
KT2440 harboring pCAR1
KT2440 harboring pCAR1 carrying disrupted pmr gene by Gmrgene cassette
KT2440(pCAR1) carrying gene encoding His-tagged Pmr
Kmr, T7 promoter, lacI, pBR322 replicon
pET-26b(?), NdeI-XhoI fragment containing pmr
Apr, lacI, tac promoter expressing FLAG tag
pFLAG-CTC, NdeI-SalI fragment containing PP_0017
pFLAG-CTC, NdeI-SalI fragment containing PP_1366
pFLAG-CTC, NdeI-SalI fragment containing PP_2947
pFLAG-CTC, NdeI-SalI fragment containing PP_3693
pFLAG-CTC, NdeI-SalI fragment containing PP_3765
pFLAG-CTC, NdeI-SalI fragment containing pmr
Apr, ori1600 oriT(RP4), Flp recombinase expression vector
pFLP2, Kmrgene cassette inserted into its ScaI site
Kmr, oriT(RP4) sacB lacZ?, pMB1 replicon
pK19mobsacB containing gene encoding Pmr with 6? His at its C-terminal
end with Gmrgene cassette and FRT site at its 3?-terminal end
pK19mobsacB, containing 3.8-kb EcoRI-PstI fragment including Gmrgene
cassette inserted pmr flanking region (77486-77909 region of pCAR1 was
replaced by Gmrgene)
pBluescript II KS(?) with 0.7-kb SmaI fragment containing a nonpolar Gmr
Apr, lacZ?, T7 promoter, f1 origin, pUC/M13 priming sites
pT7Blue T-vector, Kmrgene cassette
Apr, lacZ?, pMB9 replicon
pUC18 containing 75211-80781 region of pCAR1
VOL. 192, 2010PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION4721
RNeasy Mini column (Qiagen) or Nucleospin RNA binding column (Macherey-
Nagel) according to each manufacturer’s RNA cleanup protocol.
Primer extension and pmr disruption. We identified the transcription start
point (tsp) of pmr by primer extension analysis, performed as described previ-
ously (25). We used the IRD800-labeled primer PMR-R (Aloka Co., Ltd.,
Tokyo, Japan) (see Table S1 in the supplemental material), which anneals to the
coding region of pmr from ?218 to ?237 (77782 to 77763 on pCAR1; see Table
S1). The extension reaction was performed with 4 ?l of 5? First Strand buffer
containing 10 ?g of total RNA, 2 pmol of the labeled primer, 100 U of Super-
Script III reverse transcriptase (Invitrogen, Carlsbad, CA), 40 U of RNaseOUT
(Invitrogen), 10 mM dithiothreitol (DTT), and 0.5 mM deoxynucleoside triphos-
phates (dNTPs) (Toyobo). After denaturation of the RNA and the labeled
primer at 65°C for 5 min, the remaining reagents were added, and then the
mixture was incubated at 50°C for 30 min. The extended product was purified by
phenol-chloroform extraction and ethanol precipitation and then dissolved in 2
?l of H2O and 1 ?l of IR2 stop solution (Li-Cor Inc., Lincoln, NE). The solution
was then denatured at 95°C for 2 min and subjected to electrophoresis using a
Li-Cor model 4200L-2 automated DNA sequencer (Li-Cor). A sequence ladder
was obtained using the same primer and the template plasmid pUB11 (Table 1).
pmr disruption in pCAR1 was designed by removing the region containing the tsp
(77486 to 77909). The 3.8-kb EcoRI-PstI fragment from 75681 to 79457 in pCAR1
(GenBank/EMBL/DDBJ accession number AB0088420) was inserted into
pK19mobsacB (33), and then the SmaI fragment containing the nonpolar Gm
resistance cassette of pSJ12 (21) was inserted into blunt-ended SalI-SacI sites from
77486 to 77909 in the opposite direction to yield pK19mobsacBpmrGm. Using a
method described previously (29), pK19mobsacBpmrGm was introduced into
KT2440(pCAR1) by filter mating with E. coli S17-1(?pir) transformants, and sub-
sequently, double-crossover recombinants were screened.
Quantitative RT-PCR. Quantitative reverse transcription-PCR (qRT-PCR)
was performed using the ABI 7300 real-time PCR system (Applied Biosystems,
Foster City, CA) as described previously (25). The primers used for qRT-PCR
are shown in Table S1 in the supplemental material, and all of the products were
between 100 and 150 bp in length. 16S rRNA was used as an internal normal-
ization standard. All of the reactions were carried out at least in triplicate, and
the data were normalized using the average of the internal standard.
Preparation of a KT2440(pCAR1) derivative containing a gene encoding the
His-tagged Pmr protein. The construction of the KT2440(pCAR1) derivative
strain expressing Pmr containing six histidine (His) residues at the C terminus
was performed using a homologous recombination-based gene replacement sys-
tem with suicide vectors, antibiotic resistance selection, and sucrose counters-
election (33). The preparation of the DNA region to replace the pmr gene with
a modified gene that expresses His-tagged Pmr was performed by overlap ex-
tension PCR as described by Choi and Schweizer (6). Briefly, the primers Pmr-
His01 and Pmr-His02 were used to amplify the His-tagged pmr gene. The prim-
ers Pmr-His03 and Pmr-His04 were used to amplify the downstream region of
the untagged pmr gene. Simultaneously, the primers Gm-F and Gm-R were used
to amplify the Gm cassette flanked by flippase recognition target (FRT) sites
from pPS856 (20). The primers used are listed in Table S1 in the supplemental
material. These three partially overlapping DNA fragments were amplified and
then spliced together by in vitro overlap extension PCR. The resulting DNA
fragment was cloned into the pT7Blue T vector. After verification of the inserted
sequence, the fragment was excised and then recombined into the suicide vector
pK19mobsacB to yield pK19mobsacBpmrHis. The pK19mobsacBpmrHis con-
struct was introduced into KT2440 by filter mating with E. coli S17-1(?pir)
transformants, and double-crossover recombinants were subsequently screened
by sucrose counterselection to yield the KT2440(pCAR1) derivative, replacing
the pmr gene with a gene encoding the His-tagged Pmr protein. Finally, the Gm
resistance gene was removed by site-specific recombination of FRT sites with Flp
recombinase supplied from E. coli S17-1(?pir) transformants containing
pFLP2Km. Then, pFLP2Km was constructed by insertion of the EcoRV frag-
ment of pTKm (47) containing the Km resistance gene cassette into the ScaI site
of pFLP2 (20). PCR analyses were performed to confirm the final construction
of the derivative strain.
Western blot analysis for growth phase-dependent expression of Pmr. Cell
lysates for Western blot analyses were prepared using the B-Per reagent (Pierce
Biotechnology, Inc., Rockford, IL) according to the manufacturer’s instructions.
The protein samples were quantified using the bicinchoninic acid (BCA) protein
assay reagent kit (Pierce), and 40 ?g of protein sample for Pmr or 5 ?g of protein
sample for the RNA polymerase ? subunit was loaded in each lane. Proteins
were separated on a 15% SDS-polyacrylamide gel and transferred to a Sequi-
Blot polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Foster City, CA).
Anti-His antibody (GE Healthcare Bio-Sciences, Piscataway, NJ) or anti-RNA
polymerase ? subunit (NeoClone, Madison, WI) was used as the primary anti-
body, and enhanced chemiluminescence (ECL) peroxidase-labeled anti-mouse
antibody (GE Healthcare Bio-Sciences) was used as the secondary antibody.
Proteins were detected using the Immobilon Western chemiluminescent horse-
radish peroxidase (HRP) substrate (Millipore, Billerica, MA), and LAS1000 plus
(Fujifilm, Tokyo, Japan) was used for imaging analyses.
Overexpression of Pmr and other H-NS family proteins in E. coli cells. To
construct the C-terminal-His-tagged Pmr expression plasmid, the pET-26b(?)
vector (Novagen, San Mateo, CA) was used. The insert was amplified by PCR
using the pCAR1-covered clone pUB11 as template DNA and the primer set
with artificial NdeI and XhoI sites at the 5? and 3? ends of the pmr gene. The
nucleotide sequence of the insert was confirmed, and the resultant expression
plasmid was designated pET-C-His-pmr. To express each C-terminal-FLAG-
tagged H-NS family protein (Pmr, PP_0017 [TurC], PP_1366 [TurA], PP_2947
[TurE], PP_3693 [TurD], PP_3765 [TurB]), pFLAG-CTC (Sigma-Aldrich, St.
Louis, MO) was used as a vector. Each insert was amplified by PCR using the
primer set with artificial NdeI and SalI sites at the 5? and 3? ends of each gene
and pUB11 (for pmr) or total DNA of the P. putida strain KT2440 (for others)
as a template. The resulting expression plasmids were designated pFLAGpmr,
pFLAG0017, pFLAG1366, pFLAG2947, pFLAG3693, pFLAG3765, and ex-
pressed FLAG-tagged forms of Pmr, PP_0017 (TurC), PP_1366 (TurA),
PP_2947 (TurE), PP_3693 (TurD), and PP_3765 (TurB), respectively. Trans-
formed E. coli BL21(DE3) harboring each expression plasmid of H-NS family
proteins was grown at 25°C to a cell turbidity at 600 nm of 0.6 to 0.8 and was
induced overnight by the addition of isopropyl ?-D-thiogalactoside (IPTG) at a
final concentration of 0.5 mM. The expression level of each protein was con-
firmed by Tricine-SDS-PAGE (34).
Pull-down assays. Pull-down assays were performed using the MagneHis pro-
tein purification system (Promega). Cells expressing His-tagged or FLAG-tagged
H-NS family proteins were harvested by centrifugation and washed twice with 25
mM Tris-HCl (pH 8.0, 4°C) containing 2 mM EDTA and 10% glycerol. Cells
were then resuspended in 700 ?l of MagneHis binding/wash buffer and broken by
ultrasonication, and crude extracts were obtained by centrifugation (17,000 ? g,
15 min, 4°C). Protein concentrations were estimated with the Bio-Rad protein
assay reagent (Bio-Rad) according to the manufacturer’s instructions. Crude
extract (200 ?g) containing His-tagged Pmr was mixed with the following
amounts of crude extract containing FLAG-tagged proteins, according to each
protein expression level: Pmr, 225 ?g; TurC, 900 ?g; TurA, 450 ?g; TurE, 225
?g; TurD, 1350 ?g; and TurB, 225 ?g. After the addition of 30 ?l of MagneHis
Ni particles (Promega), the protein mixture was incubated at 4°C and centrifuged
(10 rpm, 1 h). Elution of His-tagged Pmr was done according to the manufac-
turer’s instructions. Each protein sample was separated by Tricine-SDS-PAGE
and transferred to a PVDF membrane (iBlot gel transfer stack, PVDF, regular;
Invitrogen) using the iBlot gel transfer system (Invitrogen) according to the
manufacturer’s instructions. Anti-His antibody (GE Healthcare Bio-Sciences) or
monoclonal anti-FLAG M2 antibody (Sigma-Aldrich) was used as the primary
antibody, and ECL peroxidase-labeled anti-mouse antibody (GE Healthcare
Bio-Sciences) was used as the secondary antibody. Detection of the proteins was
performed similarly to that described above for Western blot analyses.
Phenotype MicroArray (PM) analyses. Phenotypic differences between
KT2440(pCAR1) and KT2440(pCAR1?pmr) in carbon metabolism were com-
pared for cell respiration of each strain using 96-well plate microarrays (Biolog
PM1 and PM2; Biolog, Hayward, CA) (4). Each plate well contained defined
medium with a unique carbon compound plus indicator dye for cell respiration,
and each medium was made at Biolog. Excluding carbon-free wells (negative
controls), the PM1 and PM2 Biolog assays can assess the ability to use 190
carbon compounds as the sole carbon source. Experiments were performed in
duplicate, according to the manufacturer’s instructions, except that the strains
were precultured on R2A plates (1.5% agar) and data collection was performed
manually using the Biolog MicroLog MicroStation system.
Tiling array transcriptome analyses of pCAR1 and the KT2440 chromosome.
Transcriptome analyses with our custom-made tiling arrays were performed as
described previously (26, 35). Briefly, total RNA was extracted in parallel from
samples of each host culture (1 ? 109cells from two exponential-phase cultures
[the turbidity of each culture was 0.15 to 0.20 at 600 nm] derived from two
independent precultures). cDNAs reverse transcribed from these RNAs were
hybridized individually with each microarray chip using the GeneChip hybrid-
ization oven 640 (Affymetrix, Inc., Santa Clara, CA) at 60 rpm and at 50°C for
16 h with the KT2440 chromosomal tiling array or at 45°C for 16 h with the
pCAR1 tiling array. After washing, staining, and scanning of the chips, the signal
intensities for each probe were computed using the Affymetrix Tiling Analysis
Software program, v.1.1 (TAS). We used the median signal intensities of the
probes located within each gene as an indicator of the expression level. Com-
parisons between two conditions were performed using each of the biologically
4722 YUN ET AL.J. BACTERIOL.
duplicated data, and we identified upregulated and downregulated open reading
frames (ORFs) with fold changes of ?1.5 in the four data comparisons (between
replicate 1 of KT2440(pCAR1) and replicate 1 of KT2440(pCAR1?pmr) and be-
tween replicate 1 of KT2440(pCAR1) and replicate 2 of KT2440(pCAR1?pmr);
see Tables S2 to S4 in the supplemental material). The data were visualized using
the IGB software package (Affymetrix). The fold change of pmr expression levels
between KT2440(pCAR1) and KT2440(pCAR1?pmr) was only ?4.5 to ?5.5
(see Table S2) because the Gm resistance gene introduced into the pmr gene was
transcribed in the counterdirection to the pmr gene, and the read through from
the Gm resistance gene was detected (see Fig. S2).
Chromatin affinity purification coupled with high density tiling chip (ChAP-
chip) analysis. An overnight culture of the KT2440(pCAR1) derivative express-
ing 6-His-tagged Pmr in LB at 30°C was inoculated into 200 ml NMM-4 supple-
mented with 0.1% (wt/vol) succinate to obtain an initial turbidity at 600 nm of
0.05 and then incubated at 30°C in a rotating shaker at 120 rpm for 4 h to a
turbidity at 600 nm of 0.20 to 0.30. The His-tagged Pmr and DNA in the cells
were in vivo cross-linked by the addition of formaldehyde to a final concentration
of 1% for 15 min with shaking at 30°C. The cross-linking reaction was quenched
by the addition of glycine to a final concentration of 125 mM for 5 min, and
then the cells were washed twice with chilled Tris-EDTA (TE) buffer (pH
8.0). The resulting harvested cells were disrupted by sonication on ice in 2.4
ml of QuickPick Imac wash buffer (Bio-Nobile, Turku, Finland). After cen-
trifugation (17,000 ? g, 20 min), the supernatant was affinity purified using
the QuickPick Imac metal affinity kit (Bio-Nobile) according to the manufac-
turer’s instructions to yield 6-His-tagged Pmr. Cross-links were dissociated by
heating at 65°C for 4 h, and the resulting DNA was purified using the Qiaquick
kit (Qiagen) according to the manufacturer’s instructions. Terminal labeling of
the purified DNA fragments and hybridization to the pCAR1 and KT2440
chromosomal tiling arrays were performed as described above. Signal intensities
of DNA hybridization on the arrays were computed to identify protein-binding
sites using TAS, which uses nonparametric quantile normalization and a Hodges-
Lehmann estimator for fold enrichment (Affymetrix Tiling Array Software v1.1
User’s Guide) with the biologically duplicated affinity-purified fractions (treat-
ment DNA) and those of DNA isolated from the biologically duplicated whole-
cell extract fractions before purification (control DNA).
Microarray data accession number. The array data reported in this article
have been deposited in the Gene Expression Omnibus (GEO) of the National
Center for Biotechnology Information (NCBI) (GEO; http://www.ncbi.nlm.nih
.gov/geo/) under the GEO Series accession no. GSE21968.
RESULTS AND DISCUSSION
Transcriptional profiles of pmr and other H-NS family
genes in P. putida KT2440. Transcriptional levels of H-NS
family proteins change in the presence or absence of other
H-NS homologous proteins, and they are not always tran-
scribed under the same growth condition (7, 27, 44). First, we
determined the transcription start point (tsp) of pmr to con-
struct a pmr disruptant strain by extinguishing its transcription
(see Materials and Methods). The tsp of pmr (?1) was located
69 bp upstream of the annotated start codon of Pmr (nucleo-
tide at 77571 of pCAR1; see Fig. S1 in the supplemental
material), corroborating our findings using a previous tiling
array analysis (26). To clarify the transcriptional profiles of the
H-NS family genes, qRT-PCR analyses were performed for
KT2440, KT2440(pCAR1), and KT2440(pCAR1?pmr), along
their growth curves. As demonstrated in Fig. 1A and B, the
transcriptional levels of turA (PP_1366), turC (PP_0017), and
turD (PP_3693) in early log-phase growth were higher than
those in the stationary phase, whereas turB (PP_3765) and turE
(PP_2947) were transcribed in the late log and stationary
growth phases, compared with the early log phase growth in
KT2440, confirming a previous report (48). In KT2440
(pCAR1), pmr was transcribed in early log phase growth (the
cell turbidity was about 0.18 at 600 nm), and its transcription
was reduced in the stationary phase (the cell turbidity was 0.56)
(Fig. 1A and B). The transcriptional profiles of other H-NS-
encoding genes did not change with pCAR1 carriage or with
pmr disruption (Fig. 1A and B). Similar results were also ob-
tained by transcriptome analysis using tiling arrays with these
three strains in early log phase growth: the signal intensities of
the H-NS family proteins did not change with pCAR1 carriage
or with pmr disruption (Fig. 1C). Taken together with the
results of the transcriptional profiles of pmr, turA, turB, turC,
turD, and turE, pmr and turA were the primary transcribed
genes in the early log phase growth, whereas turB was tran-
scribed in the late log and stationary growth phases in
KT2440(pCAR1) (Fig. 1B and C).
Translational profiles of Pmr in P. putida KT2440(pCAR1).
Because previous reports indicated that the translational pro-
files of some H-NS family proteins were different from their
transcriptional profiles (7, 44), we confirmed the translational
profiles of Pmr. Western blot analysis was performed with the
crude extract from KT2440(pCAR1) cells in the growth phase
that expressed C-terminal-6-His-tagged Pmr. Pmr signals in
KT2440(pCAR1) were detected throughout the growth phase,
and translational levels of Pmr were higher in the late log and
stationary growth phases than in early log phase growth (Fig.
1A and D). Notably, the translational profile of Pmr differed
from the transcriptional profile (Fig. 1B and D) and from those
of other previously reported H-NS family proteins (7, 44).
Currently, we could not explain the physiological meaning(s)
of the discrepancy between pmr transcription and translation.
Reciprocal transcription and translation of a gene encoding an
H-NS-like protein, Sfh of pSF-R27, have been investigated in
detail before (14). Those authors showed that a blockade of sfh
mRNA translation occurred in early exponential growth and
was relieved at the onset of stationary phase, responsible for
the expression pattern of Sfh (14). They proposed that con-
finement of Sfh expression may ensure that the conjugative
plasmid pSF-R27 carrying sfh minimizes the disruption on the
physiology of the host cell (14). It is therefore possible that
Pmr translation may have been regulated in a similar manner
to reduce effects on the host cell; however, further investiga-
tions are still necessary to clearly explain the Pmr translation
Pmr interacts with itself and with three other H-NS family
proteins. Many reports have indicated that H-NS family pro-
teins can interact with themselves and with paralogous pro-
teins, such as StpA, Hfp, or MvaU (7, 22, 27, 44). Thus, Pmr
may interact with itself or other H-NS family proteins ex-
pressed from the host chromosome. To assess this possibility,
we performed pull-down assays followed by Western blot anal-
yses to clarify whether Pmr interacted with itself and/or other
H-NS family proteins. As revealed in Fig. 2B (lane 1 of each
sample), we detected anti-FLAG signals from each crude ex-
tract, indicating that each H-NS family protein was expressed
in E. coli. Anti-His signals were also detected in each eluant
after the pull-down assays, indicating that His-tagged Pmr ex-
isted in each eluant (data not shown). In contrast, anti-FLAG
signals in the eluants were detected only in the mixtures of
His-tagged Pmr with FLAG-tagged Pmr, TurA, TurB, and
TurE, whereas those with FLAG-tagged TurC and TurD were
not detected (Fig. 2B, lane 2 of each sample). This result
indicates that the strength of the interactions between Pmr and
itself or between Pmr and TurA, TurB, or TurE is higher than
those between Pmr and TurC or TurD. One important feature
VOL. 192, 2010PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION 4723
of H-NS family proteins is their modular structure (10). Addi-
tionally, KT2440 proteins have putative structures similar to
that of H-NS: a well-conserved amino-terminal oligomeriza-
tion domain (see Fig. S3A, blue box, in the supplemental
material), a conserved carboxyl-terminal nucleic acid-binding
domain (see Fig. S3A, red box), and a poorly conserved flexible
linker that connects the two aforementioned domains (see Fig.
S3A). When the amino acid sequences of the H-NS family
proteins of KT2440 were aligned, their putative oligomeriza-
tion domains at the N-terminal regions were well conserved
(see Fig. S3A), although the identity between H-NS family
proteins of KT2440, including Pmr and the H-NS protein of E.
coli, was low (see Fig. S3B). Although it was difficult to predict
why Pmr could have heteromeric interactions with three H-NS
family proteins but not with two other H-NS family proteins,
some residues from the latter may be important for the inter-
action. Notably, the homologous proteins of TurA and TurB
are conserved in all Pseudomonadaceae species, but TurC,
TurD, and TurE are species-specific proteins (30) encoded in
the putative horizontally acquired DNA region (24). Taken
together with the result that turA and turB were transcribed
primarily in the early log and late log growth phases, respec-
tively, Pmr may primarily interact with TurA and TurB, al-
though the functional significance of TurE is presently unclear.
Considering the reciprocal transcription and translation of
Pmr (Fig. 1B and D), it is necessary to analyze the translational
levels of Tur proteins in vivo.
Phenotypic alteration by pmr disruption. To assess the ef-
fects of pmr disruption on the phenotypes of KT2440(pCAR1),
comparisons of the catabolic abilities of KT2440(pCAR1) and
KT2440(pCAR1?pmr) were performed using Biolog PM anal-
yses by measuring the absorbance of colored cultures derived
from a tetrazolium dye used as a reporter of cell respiration.
From the comparisons for each of the 190 substrates as a sole
carbon source, reproducible reductions of the maximum absor-
bance of the color were observed in the KT2440(pCAR1?pmr)
culture, compared with results for the KT2440(pCAR1) cul-
ture, with nine compounds (D-fructose, L-serine, L-valine, sac-
charic acid, D-malic acid, pyruvic acid, methyl pyruvate,
D-ribono-1,4-lactone, and inosine; see Fig. S4 in the supplemental
material). We did not detect any difference between the two
strains in the culture using the other carbon sources (for ex-
FIG. 1. Transcriptional profiles of the genes encoding H-NS family proteins and translational profiles of Pmr. (A) Growth curves of KT2440
(diamond), KT2440(pCAR1) (square), and KT2440(pCAR1?pmr) (triangle). The turbidity of each cell culture was measured at 600 nm. (B) The
mRNA levels of the genes encoding H-NS family proteins (Pmr, PP_0017 [TurC], PP_1366 [TurA], PP_2947 [TurE], PP_3693 [TurD], and
PP_3765 [TurB]) were measured by qRT-PCR. Means and standard deviations (error bars) of triplicate data are shown. (C) Signal intensities of
genes encoding H-NS family proteins in tiling array analyses with KT2440 (black), KT2440(pCAR1) (white), and KT2440(pCAR1?pmr) (gray).
Biologically duplicated data are shown. (D) The amount of Pmr-His protein was monitored at different points in the growth curve of
KT2440(pCAR1). Results of Western blot analysis using anti-His are shown in the upper panel, and those with use of an anti-RNA polymerase
? subunit as a control for sample loading are shown in the lower panel.
4724YUN ET AL.J. BACTERIOL.
ample, the result with D-glucose was shown in Fig. S4). These
results indicated that pmr disruption affected the catabolic abili-
ties of KT2440(pCAR1) with several carbon sources, suggesting
that Pmr may function as a global regulator of many genes.
Transcriptome alteration by pmr disruption. To confirm the
effects of pmr disruption on the host cells, we performed tran-
scriptome comparisons between KT2440(pCAR1) and KT2440
(pCAR1?pmr) using custom-made tiling arrays of genome se-
quences of pCAR1 and the KT2440 chromosome (26, 35). To
evaluate the transcriptional and translational profiles of pmr
(Fig. 1), transcriptome comparisons were performed for cells
in early log phase growth.
Overview. We found that the transcription of 31 genes on
pCAR1 and 159 genes on the KT2440 chromosome were al-
tered by pmr disruption, with a fold change of ?1.5 (see Ma-
terials and Methods; see also Tables S2 and S3 in the supple-
mental material). We identified 2 and 19 upregulated genes on
pCAR1 and the KT2440 chromosome, respectively, and 29 and
140 downregulated genes on pCAR1 and the KT2440 chromo-
some, respectively. Based on our previous study (35), we iden-
tified 112 genes altered by pCAR1 carriage with a fold change
of ?1.5 in both of the duplicate data (see Table S3). Notably,
the number of downregulated genes following pmr disruption
was larger than that with pCAR1 carriage (see Fig. S5), sug-
gesting that Pmr may play an important role in mediating the
transcription of the chromosomal genes of the host KT2440
by pCAR1 carriage. The comparison of the transcriptome
changes with pCAR1 carriage with those with pmr disruption
enabled us to classify 5,398 genes of KT2440 (after rRNA and
tRNA removal) based on their transcriptional patterns. First,
the transcription of 5,146 genes was not affected by pCAR1
carriage or by pmr disruption. Among the remaining 252 genes,
43 (group A) or 50 (group B) were upregulated or downregu-
lated by pCAR1 carriage, respectively, but neither of their
transcription levels was affected by pmr disruption (Fig. 3; see
also Table S4). Only one gene (group D) was downregulated
by both pCAR1 carriage and pmr disruption (no gene was
classified into group C) (Fig. 3 and Table 2; see also Table S4).
Seventeen genes (group E) were upregulated by pCAR1 car-
riage but downregulated by pmr disruption, and one gene
(group F) was the reverse (Fig. 3; see also Table S4). In total,
122 genes (group G) or 18 genes (group H) were upregulated
or downregulated by pmr disruption, respectively, but neither
group was affected by pCAR1 carriage (Fig. 3; see also Table
S4). Doyle et al. (15) proposed that these H-NS proteins en-
coded on plasmids have “stealth” functions to minimize the
effect on host strain fitness, comparing the number of genes
whose transcriptional levels were altered in the presence or
absence of the H-NS protein. Regarding KT2440(pCAR1), the
number of differentially transcribed genes with pmr disruption
(159 genes on the chromosome) was larger than that with
pCAR1 carriage (112 genes). Additionally, 88% of them (be-
longing to group G or H) were altered only by the absence of
Pmr, suggesting that Pmr had a “stealth” function, as mentioned
above. The transcription levels of only 12% of the differentially
transcribed genes with pmr disruption (18 genes) reverted to
FIG. 2. Identification of the interaction among H-NS family proteins by in vitro pull-down assays. (A) Tricine-SDS-PAGE profiles of each H-NS
family protein expressed in E. coli. “M” indicates protein marker (XL ladder Low, APRO Life Science Institute, Inc., Tokushima, Japan), and “1”
and “2” indicate the crude extract of cells expressing each FLAG-tagged H-NS family protein and the sample after the pull-down assay,
respectively. “His” indicates the crude extract of cells expressing His-tagged Pmr. (B) Western blot analysis using anti-FLAG and the same samples
in Tricine-SDS-PAGE shown in panel A.
VOL. 192, 2010 PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION4725
levels similar to those in pCAR1-free KT2440 (groups E and F in
Fig. 3; see also Table S4), suggesting that these genes were reg-
ulated primarily by Pmr itself, directly or indirectly. These results
suggest that Pmr is a key global regulator of many genes, both on
pCAR1 and on the host chromosome.
Martins dos Santos et al. (24) demonstrated that KT2440
had many putative horizontally acquired DNA regions. These
regions include 1,105 ORFs, corresponding to about 20% of
the total ORFs in KT2440. Because H-NS family proteins bind
to horizontally acquired DNA regions (16, 28), we calculated
the ratio of ORFs in the regions in the above differentially
transcribed genes (Fig. 3). Of the 112 genes (Fig. 3, groups A
to F) differentially transcribed by pCAR1 carriage, 23 (21%)
were located in the putative horizontally acquired DNA region
(Table 2; see also Table S4 in the supplemental material).
Conversely, 56 (35%) of 159 genes differentially transcribed by
pmr disruption (Fig. 3, groups C to H) were in this region
(Table 2; see also Table S4). Notably, the proportions of
groups B, G, and H were high: 28%, 39%, and 28%, respec-
tively (Table 2; see also Table S4). The average G?C content
of pCAR1 and the KT2440 chromosome is 56.3% and 61.6%,
respectively. We then calculated the G?C content in the 500
bp upstream of each ORF. The G?C content of pCAR1-borne
genes differentially transcribed by pmr disruption was signifi-
cantly below the average: for most upstream regions of 30
among 31 affected ORFs (Table 3), it was below 61.6%, and for
those of 27 ORFs, including the car or parAB genes (see Table
S2), it was even below 56.3% (Table 3). Concerning the ORFs
on the KT2440 chromosome, the G?C content of the up-
stream regions of 92 (58%) among 159 ORFs was below
61.6%, and that for 37 ORFs was below 56.3% (Table 3). As
revealed in Table 3, the ratio of these ORFs to the total
affected ORFs was higher (87% in pCAR1 and 23% in the
KT2440 chromosome) than the ratio of the ORFs whose up-
stream regions were low in G?C content (below 56.3%) to the
total ORFs (64% in pCAR1 and 17% in the KT2440 chro-
mosome). Notably, the ORFs with a ratio of ?56.3% in the
upstream region (16 ORFs) among the ORFs affected by
pCAR1 carriage (112 ORFs) was 14% (Table 3). Thus, some
ORFs with low-G?C regions may be specifically regulated
Downregulated genes on pCAR1 with pmr disruption. The
transcription levels of the genes on the car operon, involved in
carbazole degradation, were downregulated (Fig. 4A; see also
Table S2 in the supplemental material). When KT2440
(pCAR1) is grown with succinate, the car operon is constitu-
tively transcribed from the PcarAapromoter (26, 35), and it is
induced by anthranilate, an intermediate of the carbazole deg-
radation pathway, from the Pantpromoter, further upstream
(42). Thus, the constitutively expressed carbazole-degrading
enzymes will be required to produce anthranilate. This sug-
gests that the downregulation of the constitutive transcription
levels of car genes may have caused the growth delay with carba-
zole. In fact, the growth rate of KT2440(pCAR1?pmr) was de-
layed compared with that of KT2440(pCAR1) in NMM-4 buffer
with carbazole as a sole carbon source (data not shown). The
transcriptional levels of the parAB genes were also reduced in
the pmr disruptants (Fig. 4B; see also Table S2). The parAB
genes are required for the stable maintenance of pCAR1 in the
host strain (36), and thus, the downregulation of these genes
may cause instability of pCAR1. However, we did not detect
changes in the stability of pCAR1 or pCAR1?pmr in KT2440
cells (data not shown), suggesting that the effects of the down-
TABLE 2. Number and percentage of ORFs on the putative horizontally acquired DNA region in each group
No. (%) of ORFs
ORFs on putative horizontally-
acquired DNA region
5 (12) 14 (28)0 (0)0 (0) 4 (24)0 (0) 47 (39)5 (28) 1,105 (20)
435001 171 12218 5,398
FIG. 3. Classification of the differentially transcribed genes by
pCAR1 carriage and/or pmr disruption. Blue, red, and green bars
indicate the relative transcription levels in KT2440, KT2440(pCAR1),
and KT2440(pCAR1?pmr), respectively.
4726 YUN ET AL. J. BACTERIOL.
regulation of the parAB genes on plasmid stability may be
insignificant. It is also possible that the chromosomally en-
coded ParAB system (ParABKT2440) for the partition of the
KT2440 chromosome may have been involved in plasmid par-
tition; however, the transcriptional levels of these genes were
unaltered in the pmr disruptant (data not shown). Additionally,
the cis-acting centromere-like parS sequence is indispensable
for the function of ParABKT2440; however, the 16-nucleotide
(nt) parS sequence of P. putida KT2440 (5?-TGTTNCACGT
GAAACA-3?) (3, 18) was not found in the pCAR1 sequence
(data not shown). The reason pCAR1?pmr was stable in the
host strain was not clear. Notably, the transcriptional levels of
the car and parAB genes were altered in different host strains
(26, 35), and the transcription of these genes may be related to
the Pmr concentration.
pmr disruption alters chromosomal gene transcription that
is upregulated by pCAR1 carriage. The mexEF-oprN operon,
encoding the efflux pump, was upregulated in KT2440
(pCAR1) and downregulated in KT2440(pCAR1?pmr) (Fig.
5A; see also group E of Table S4 in the supplemental mate-
rial). In our previous study, these gene products enhanced the
chloramphenicol (Cm) resistance of the host strain; KT2440
(pCAR1) showed resistance to concentrations of Cm higher
than 300 ?g/ml, although KT2440 was not able to grow with
that concentration. (35). Therefore, we assessed the Cm resis-
tance (300 ?g/ml) of the pmr disruptants. Cm resistance re-
verted to the levels of pCAR1-free KT2440, indicating that the
downregulation of the mexEF-oprN operon may occur with the
loss of resistance at that concentration. Westfall et al. (45)
reported that the transcription of mexEF-oprN orthologous
genes in P. aeruginosa PAO1 (normally untranscribed) was
induced in the mvaT (PA4315) mutant on the PAO1 chromo-
some. Thus, H-NS family proteins may be involved in the
transcriptional regulation of these genes in PAO1. Although
our case contrasted with the PAO1 case, i.e., pmr disruption
caused the downregulation of the mexEF-oprN operon, Pmr
may have contributed to the transcriptional regulation of these
genes. Herrera et al. (19) recently reported that PhhR
(PP_4489), a transcriptional regulator of phenylalanine hy-
droxylase phhAB genes, modulates the level of expression of
mexEF-oprN together with MexT (PP_2826). Notably, the
transcriptional levels of both phhR and mexT were not changed
by pCAR1 carriage or by pmr disruption (data not shown),
suggesting that Pmr may be the third element for the regula-
tion of the mexEF-oprN operon.
The parI gene encodes a putative ParA-like ATPase con-
taining an N-terminal DNA-binding motif, and its transcrip-
tion was upregulated in KT2440(pCAR1) but downregulated
in KT2440(pCAR1?pmr) (Fig. 5B; see also group E of Table
S4 in the supplemental material). This corroborated our pre-
vious results that the parI promoter was activated in the pres-
ence of pCAR1 because of the parA product from pCAR1
(25). Therefore, the decrease in the parI transcriptional level in
KT2440(pCAR1?pmr) was caused by the reduced parA tran-
scription (Fig. 4B; see also Table S2), although the reasons for
the parA gene downregulation in KT2440(pCAR1?pmr) re-
Pmr preferentially binds to foreign DNA and low-G?C re-
gions of the host chromosome. Because the transcription of
many genes was affected by pCAR1 carriage and by pmr dis-
ruption, we identified genome-wide Pmr-binding DNA regions
on both pCAR1 and the KT2440 chromosome. We performed
ChAP-chip analyses to identify the Pmr-binding sites on the
KT2440 chromosome and in pCAR1 in early log phase grow-
ing cells, as well as transcriptome analyses, although the trans-
lational levels of Pmr were higher in the late log and stationary
growth phases than in early log phase growth (Fig. 1D).
Consequently, 241 and 26 Pmr-binding sites were detected
(with a P value of ?0.01) on the KT2440 chromosome and in
pCAR1, respectively (see Table S5 in the supplemental mate-
rial). First, we calculated the G?C content of the regions
identified. The average G?C content of the 241 Pmr-binding
regions in the KT2440 chromosome was significantly lower
(52.5%) than that of the entire KT2440 chromosome (61.6%).
The 26 Pmr-binding regions in pCAR1 also demonstrated an
average G?C content (52.5%) that was lower than that of the
entire pCAR1 plasmid (56.3%). Indeed, a high association was
found between the Pmr-binding sites on the KT2440 chromo-
some and the putative foreign DNA region (Fig. 6A). Notably,
73% of the Pmr-binding sites in the KT2440 chromosome were
located in foreign DNA regions.
Interestingly, many Pmr binding sites in pCAR1 overlapped
with the localization of the differentially transcribed genes with
pmr disruption (Fig. 6B). Similarly, many binding sites in the
KT2440 chromosome were also found near regions where the
differentially transcribed genes localized, although not every
gene near a binding-site region was affected by pmr disruption
(Fig. 6A). These data indicate that Pmr regulates the transcrip-
tion of many genes by binding to intergenic or intragenic re-
gions of target genes. To determine the relative positions of the
Pmr-binding sites to each intergenic or intragenic region of the
ORFs, distribution analyses were performed for the ChAP-
TABLE 3. Classification of KT2440 chromosomal and pCAR1-borne ORFs by G?C contents of their 500-bp
regions upstream of the putative start codon
% G?C content of 500-bp
upstream regions of ORFs
No. (%) of ORFs affected by:
No. (%) of total ORFs
pCAR1 carriage pmr disruption
pCAR1 KT2440 chromosomepCAR1KT2440 chromosome pCAR1KT2440 chromosome
TotalND112 31 1591925,398
aND, not determined.
VOL. 192, 2010PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION4727
chip analysis data (Fig. 7). The Pmr binding site number
peaked at around 200 bp upstream from the translational start
point and at around 300 bp downstream from the translational
endpoint (Fig. 7), which was similar in the ChAP-chip analysis
when different P-value thresholds were used (Fig. 7). This
analysis indicated that Pmr may bind preferentially to inter-
genic regions rather than to intragenic regions of ORFs, con-
firming many reports that H-NS family proteins regulate gene
expression by binding to target promoter regions (10, 16).
Our ChAP-chip analysis demonstrated that Pmr bound pref-
erentially to DNA with a low G?C content in KT2440
(pCAR1) and that Pmr bound to intergenic regions and reg-
ulated the transcription of genes in the flanking regions of the
We also performed ChAP-chip analysis to identify the bind-
ing sites of the TurA and TurB proteins, which are encoded on
the KT2440 chromosome. However, the detected TurA- and
TurB-binding sites were almost identical to those of Pmr, and
most of them were DNA regions with low G?C content (data
not shown). These results were similar to those observed with
E. coli or P. aeruginosa PAO1, in which the two H-NS family
proteins (H-NS and StpA or MvaT and MvaU) bound to the
same regions of the chromosome (5, 43). Recently Dillon et al.
(8) reported that the DNA binding sites of plasmid-encoded
Sfh of Salmonella overlapped with those of H-NS. Sfh does not
bind uniquely to any site, and the number of binding sites in
Sfh is smaller than that in H-NS. Although Sfh binding sites are
located within H-NS, the DNA binding sites greatly expand in
the absence of H-NS, suggesting that Sfh may play a “backup”
role for H-NS (8). These facts suggest that the three proteins
FIG. 4. RNA maps of downregulated genes on pCAR1 by pmr disruption of the flanking regions of the car operon (A) or parAB genes (B). The x
intensity of each probe: red bars represent RNA maps of pCAR1-containing KT2440, and green bars represent those of the pmr disruptants. The results
of two biological replicates (Replicate-1 and -2) are shown. Pentagons indicate the directions and locations of the annotated genes.
4728 YUN ET AL. J. BACTERIOL.
Pmr, TurA, and TurB may function coordinately as global
regulators in the cells and that Pmr may also perform “backup”
functions for the other proteins, different from those of H-NS
and Sfh, because the binding sites of Pmr, TurA, and TurB are
identical. However, previous genome-wide analyses of the
binding sites of H-NS family proteins, including ours, did not
necessarily take into account the in vivo protein-protein inter-
action(s). In other words, the detected sites are not necessarily
showing how they bind to the DNA sequences by forming the
homo- or heteromultimer of the H-NS family proteins in vivo.
Considering the coordinate functions for DNA binding and
transcriptional regulation by Pmr and TurA to TurE, analyses
from protein structure viewpoints will be necessary to under-
stand how they compose the homo- or heteromultimer in vivo
in the presence or absence of target DNA.
Conclusions. In this study, we demonstrated that the plas-
mid-encoded H-NS family protein Pmr forms homomeric and
heteromeric oligomers in vitro and that pmr, turA, and turB are
the primary transcribed genes at different growth phases. We
also revealed that pmr disruption affected the carbon catabo-
lism of KT2440(pCAR1) and that Pmr is a key factor that
regulates the transcription of genes on both pCAR1 and the
host chromosome in two ways: (i) Pmr may alter the transcrip-
tional levels of genes in group E or F, such as mexEF-oprN and
parI, and (ii) Pmr may minimize the effect of the transcription
of many genes in group G or H, such as those in the putative
FIG. 5. RNA maps of the flanking regions of the mexEF-oprN (A) or parI (B) gene on the KT2440 chromosome. Blue, red, and green bars
represent the signal intensities of each probe detected in the RNA maps of KT2440, KT2440(pCAR1), and KT2440(pCAR1?pmr) with the two
biological replicates (Replicate-1 and -2), respectively. Pentagons indicate the directions and locations of annotated genes.
FIG. 6. Distribution of Pmr-binding sites on the P. putida KT2440 chromosome (A) or pCAR1 (B). Genes or ORFs (gray) outside the circle
are coded clockwise, while those inside are coded counterclockwise. Genes upregulated or downregulated by pmr disruption are shown in purple
and magenta, respectively. Putative horizontally acquired DNA regions in the KT2440 chromosome (24) are shown in blue. The Pmr-binding
regions detected in this study are shown in red. The green line indicates the average G?C content of genomic DNA (KT2440, 1,000-nt span;
pCAR1, 100-nt span). The broken-line circle indicates the average G?C content of the entire KT2440 (61.6%) or pCAR1 (56.3%) genome.
VOL. 192, 2010PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION 4729
foreign DNA regions. The identification of genome-wide bind-
ing sites in Pmr by ChAP-chip analysis indicated that Pmr
binds to putative foreign DNA regions with low G?C content.
Additionally, Pmr binds preferentially to intergenic regions
and may regulate many genes in the flanking regions of the
binding sites. These findings indicate that Pmr is involved in
the regulation of the expression of many genes, directly or
indirectly, and that this regulation may be closely related to its
DNA-binding regions and its interaction with other H-NS fam-
ily proteins, primarily TurA and TurB. Recently three H-NS
family proteins in pathogenic E. coli, the endogenous hns and
stpA genes and the horizontally acquired hfp gene, were shown
to be differentially transcribed at distinct temperatures (27).
Thus, we must further analyze the transcriptional and transla-
tional levels of Pmr, TurA, TurB, TurC, TurD, and TurE under
conditions other than those we have used to date.
Notably, the pmr gene is conserved in another IncP-7 plas-
mid, pWW53 (46), indicating that Pmr is an important protein
for IncP-7 plasmids. Moreover, H-NS family proteins are ex-
pressed from other plasmids, such as H-NS from R27 (IncHI)
(17), Sfh from pSf-R27 (IncHI) (15), Orf4 from R446 (IncM)
(41), or an undeposited ORF from pQBR103 (IncP-3) (40). A
key function of H-NS expressed from mobile genetic elements
is maintaining host cell fitness (15). H-NS is a member of the
NAP family, and its coordinate functions with other NAPs are
also important for host cell fitness maintenance (9, 11). In the
case of the R27 studies, the Hha-like protein, a protein-protein
modulator of H-NS activity, is encoded on the plasmid (17);
however, no candidates for Pmr modulator-encoding genes are
found on pCAR1. Interestingly, pCAR1 harbors two other
genes encoding putative NAPs other than Pmr, although their
transcriptional levels were unaltered by pmr disruption. Anal-
yses of the function(s) of these gene products are necessary to
understand how these H-NS family proteins behave when
pCAR1 is introduced into the host cell by conjugative transfer.
Such information would help to explain the adaptive and evo-
lutionary mechanisms of bacteria acquiring foreign genes by
horizontal gene transfer.
We thank Akira Yokota of the Institute of Molecular and Cellular
Biosciences, the University of Tokyo, for use of his Biolog MicroLog
This study was supported by the Program for Promotion of Basic
Research Activities for Innovative Biosciences (PROBRAIN) in Ja-
pan. Y.T. was supported by research fellowships from the Japan So-
ciety for the Promotion of Science (JSPS) for Young Scientists.
1. Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A.
Smith, and K. Struhl (ed.). 1990. Current protocols in molecular biology.
John Wiley & Sons, Inc., New York, NY.
2. Bagdasarian, M., R. Lurz, B. Ruckert, F. C. H. Franklin, M. M. Bagdasar-
ian, J. Frey, and K. N. Timmis. 1981. Specific-purpose plasmid cloning
vectors. II. Broad host range, high copy number, RSF1010-derived vectors,
and a host-vector system for gene cloning in Pseudomonas. Gene 16:237–247.
3. Bartosik, A. A., K. Lasocki, J. Mierzejewska, C. M. Thomas, and G. Jagura-
Burdzy. 2004. ParB of Pseudomonas aeruginosa: interactions with its partner
ParA and its target parS and specific effects on bacterial growth. J. Bacteriol.
4. Bochner, B. R. 2009. Global phenotypic characterization of bacteria. FEMS
Microbiol. Rev. 33:191–205.
5. Castang, S., H. R. McManus, K. H. Turner, and S. L. Dove. 2008. H-NS
family members function coordinately in an opportunistic pathogen. Proc.
Natl. Acad. Sci. U. S. A. 105:18947–18952.
6. Choi, K. H., and H. P. Schweizer. 2005. An improved method for rapid
generation of unmarked Pseudomonas aeruginosa deletion mutants. BMC
7. Deighan, P., C. Beloin, and C. J. Dorman. 2003. Three-way interactions
among the Sfh, StpA and H-NS nucleoid-structuring proteins of Shigella
flexneri 2a strain 2457T. Mol. Microbiol. 48:1401–1416.
8. Dillon, S. C., A. D. Cameron, K. Hokamp, S. Lucchini, J. C. Hinton, and C. J.
Dorman. 2010. Genome-wide analysis of the H-NS and Sfh regulatory net-
works in Salmonella Typhimurium identifies a plasmid-encoded transcription
silencing mechanism. Mol. Microbiol. doi:10.1111/j.1365-2958.2010.07173.x.
9. Dillon, S. C., and C. J. Dorman. 2010. Bacterial nucleoid-associated proteins,
nucleoid structure and gene expression. Nat. Rev. Microbiol. 8:185–195.
10. Dorman, C. J. 2004. H-NS: a universal regulator for a dynamic genome. Nat.
Rev. Microbiol. 2:391–400.
11. Dorman, C. J. 2009. Nucleoid-associated proteins and bacterial physiology.
Adv. Appl. Microbiol. 67:47–64.
12. Dorman, C. J. 2010. Horizontally acquired homologues of the nucleoid-
associated protein H-NS: implications for gene regulation. Mol. Microbiol.
13. Dorman, C. J., and K. A. Kane. 2008. DNA bridging and antibridging: a role
for bacterial nucleoid-associated proteins in regulating the expression of
laterally acquired genes. FEMS Microbiol. Rev. 33:587–592.
14. Doyle, M., and C. J. Dorman. 2006. Reciprocal transcriptional and posttran-
scriptional growth-phase dependent expression of sfh, a gene that encodes a
paralogue of the nucleoid-associated protein H-NS. J. Bacteriol. 188:7581–
15. Doyle, M., M. Fookes, A. Ivens, M. W. Mangan, J. Wain, and C. J. Dorman.
2007. An H-NS-like stealth protein aids horizontal DNA transmission in
bacteria. Science 315:251–252.
16. Fang, F. C., and S. Rimsky. 2008. New insights into transcriptional regulation
by H-NS. Curr. Opin. Microbiol. 11:113–120.
17. Forns, N., R. C. Ban ˜os, C. Balsalobre, A. Jua ´rez, and C. Madrid. 2005.
Temperature-dependent conjugative transfer of R27: role of chromosome-
and plasmid-encoded Hha and H-NS proteins. J. Bacteriol. 187:3950–3959.
18. Godfrin-Estevenon, A. M., F. Pasta, and D. Lane. 2002. The parAB gene
products of Pseudomonas putida exhibit partition activity in both P. putida
and Escherichia coli. Mol. Microbiol. 43:39–49.
19. Herrera, M. C., E. Duque, J. J. Rodríguez-Herva, A. M. Ferna ´ndez-Escam-
illa, and J. L. Ramos. 2010. Identification and characterization of the PhhR
regulon in Pseudomonas putida. Environ. Microbiol. 12:1427–1438.
20. Hoang, T. T., R. R. Karkhoff-Schweizer, A. J. Kutchma, and H. P. Schweizer.
1998. A broad-host-range Flp-FRT recombination system for site-specific
excision of chromosomally-located DNA sequences: application for isolation
of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86.
21. Jain, S., and D. E. Ohman. 1998. Deletion of algK in mucoid Pseudomonas
aeruginosa blocks alginate polymer formation and results in uronic acid
secretion. J. Bacteriol. 180:634–641.
22. Johansson, J., S. Eriksson, B. Sonde ´n, S. N. Wai, and B. E. Uhlin. 2001.
Heteromeric interactions among nucleoid-associated bacterial proteins: lo-
calization of StpA-stabilizing regions in H-NS of Escherichia coli. J. Bacte-
23. Maeda, K., H. Nojiri, M. Shintani, T. Yoshida, H. Habe, and T. Omori. 2003.
Complete nucleotide sequence of carbazole/dioxin-degrading plasmid
FIG. 7. Distribution of Pmr-binding regions near the translation
start (A) or end (B) points. Pmr-binding regions located 500 bp up-
stream and downstream of the translation start (or end) points were
plotted. Pmr-binding numbers (y axis) were estimated for each nucle-
otide position in the range from 500 bp up- and downstream of the
translation start (end) points by vertical integration of the binding
regions around the translation start (end) points.
4730 YUN ET AL.J. BACTERIOL.
pCAR1 in Pseudomonas resinovorans strain CA10 indicates its mosaicity and
the presence of large catabolic transposon Tn4676. J. Mol. Biol. 326:21–33.
24. Martins dos Santos, V. A. P., K. N. Timmis, B. Tu ¨mmler, and C. Weinel.
2004. Genomic features of Pseudomonas putida strain KT2440, p. 77–112. In
J. L. Ramos (ed.), Pseudomonas, vol. 1. Kluwer Academic/Plenum Publish-
ers, New York, NY.
25. Miyakoshi, M., M. Shintani, T. Terabayashi, S. Kai, H. Yamane, and H.
Nojiri. 2007. Transcriptome analysis of Pseudomonas putida KT2440 harbor-
ing the completely sequenced IncP-7 plasmid pCAR1. J. Bacteriol. 189:
26. Miyakoshi, M., H. Nishida, M. Shintani, H. Yamane, and H. Nojiri. 2009.
High-resolution mapping of plasmid transcriptomes in different host bacte-
ria. BMC Genomics 10:12.
27. Mu ¨ller, C. M., G. Schneider, U. Dobrindt, L. Emo ¨dy, J. Hacker, and B. E.
Uhlin. 2010. Differential effects and interactions of endogenous and hori-
zontally acquired H-NS-like proteins in pathogenic Escherichia coli. Mol.
28. Navarre, W. W., S. Porwollik, Y. Wang, M. McClelland, H. Rosen, S. J.
Libby, and F. C. Fang. 2006. Selective silencing of foreign DNA with low GC
content by the H-NS protein in Salmonella. Science 313:236–238.
29. Pinyakong, O., H. Habe, A. Kouzuma, H. Nojiri, H. Yamane, and T. Omori.
2004. Isolation and characterization of genes encoding polycyclic aromatic
hydrocarbon dioxygenase from acenaphthene and acenaphthylene degrading
Sphingomonas sp. strain A4. FEMS Microbiol. Lett. 238:297–305.
30. Renzi, F., E. Rescalli, E. Galli, and G. Bertoni. 2010. Identification of genes
regulated by the MvaT-like paralogues TurA and TurB of Pseudomonas
putida KT2440. Environ. Microbiol. 12:254–263.
31. Rescalli, E., S. Saini, C. Bartocci, L. Rychlewski, V. De Lorenzo, and G.
Bertoni. 2004. Novel physiological modulation of the Pu promoter of TOL
plasmid: negative regulatory role of the TurA protein of Pseudomonas putida
in the response to suboptimal growth temperatures. J. Biol. Chem. 279:7777–
32. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory
manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Har-
33. Scha ¨fer, A., A. Tauch, W. Ja ¨ger, J. Kalinowski, G. Thierbach, and A. Pu ¨hler.
1994. Small mobilizable multi-purpose cloning vectors derived from the
Escherichia coli plasmids pK18 and pK19: selection of defined deletions in
the chromosome of Corynebacterium glutamicum. Gene 145:69–73.
34. Scha ¨gger, H. 2006. Tricine-SDS-PAGE. Nat. Protoc. 1:16–22.
35. Shintani, M., Y. Takahashi, H. Tokumaru, K. Kadota, H. Hara, M. Miya-
koshi, K. Naito, H. Yamane, H. Nishida, and H. Nojiri. 2010. Response of
the Pseudomonas host chromosomal transcriptome to carriage of the IncP-7
plasmid pCAR1. Environ. Microbiol. 12:1413–1426.
36. Shintani, M., H. Yano, H. Habe, T. Omori, H. Yamane, M. Tsuda, and H.
Nojiri. 2006. Characterization of the replication, maintenance, and transfer
features of the IncP-7 plasmid pCAR1, which carries genes involved in
carbazole and dioxin degradation. Appl. Environ. Microbiol. 72:3206–3216.
37. Shintani, M., T. Yoshida, H. Habe, T. Omori, and H. Nojiri. 2005. Large
plasmid pCAR2 and class II transposon Tn4676 are functional mobile ge-
netic elements to distribute the carbazole/dioxin-degradative car gene cluster
in different bacteria. Appl. Microbiol. Biotechnol. 67:370–382.
38. Takahashi, Y., M. Shintani, H. Yamane, and H. Nojiri. 2009. The complete
nucleotide sequence of pCAR2: pCAR2 and pCAR1 were structurally iden-
tical IncP-7 carbazole degradative plasmids. Biosci. Biotechnol. Biochem.
39. Tendeng, C., O. A. Soutourina, A. Danchin, and P. N. Bertin. 2003. MvaT
proteins in Pseudomonas spp.: a novel class of H-NS-like proteins. Microbi-
40. Tett, A., A. J. Spiers, L. C. Crossman, D. Ager, L. Ciric, J. M. Dow, J. C. Fry,
D. Harris, A. Lilley, A. Oliver, J. Parkhill, M. A. Quail, P. B. Rainey, N. J.
Saunders, K. Seeger, L. A. Snyder, R. Squares, C. M. Thomas, S. L. Turner,
X. X. Zhang, D. Field, and M. J. Bailey. 2007. Sequence-based analysis of
pQBR103; a representative of a unique, transfer-proficient mega plasmid
resident in the microbial community of sugar beet. ISME J. 1:331–340.
41. Tietze, E., and H. Tscha ¨pe. 1994. Temperature-dependent expression of
conjugation pili by IncM plasmid-harbouring bacteria: identification of plas-
mid-encoded regulatory functions. J. Basic Microbiol. 34:105–116.
42. Urata, M., M. Miyakoshi, S. Kai, K. Maeda, H. Habe, T. Omori, H. Yamane,
and H. Nojiri. 2004. Transcriptional regulation of the ant operon, encoding
two-component anthranilate 1,2-dioxygenase, on the carbazole-degradative
plasmid pCAR1 of Pseudomonas resinovorans strain CA10. J. Bacteriol.
43. Uyar, E., K. Kurokawa, M. Yoshimura, S. Ishikawa, N. Ogasawara, and T.
Oshima. 2009. Differential binding profiles of StpA in wild-type and h-ns
mutant cells: a comparative analysis of cooperative partners by chromatin
immunoprecipitation-microarray analysis. J. Bacteriol. 191:2388–2391.
44. Vallet-Gely, I., K. E. Donovan, R. Fang, J. K. Joung, and S. L. Dove. 2005.
Repression of phase-variable cup gene expression by H-NS-like proteins in
Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. U. S. A. 102:11082–11087.
45. Westfall, L. W., N. L. Carty, N. Layland, P. Kuan, J. A. Colmer-Hamood, and
A. N. Hamood. 2006. mvaT mutation modifies the expression of the Pseudo-
monas aeruginosa multidrug efflux operon mexEF-oprN. FEMS Microbiol.
46. Yano, H., C. E. Garruto, M. Sota, Y. Ohtsumo, Y. Nagata, G. Zylstra, P. A.
Williams, and M. Tsuda. 2007. Complete sequence determination combined
with analysis of transposition /site-specific recombination events to explain
genetic organization of IncP-7 TOL plasmid pWW53 and related mobile
genetic elements. J. Mol. Biol. 369:11–26.
47. Yoshida, T., Y. Ayabe, M. Yasunaga, Y. Usami, H. Habe, H. Nojiri, and T.
Omori. 2003. Genes involved in the synthesis of the exopolysaccharide meth-
anolan by the obligate methylotroph Methylobacillus sp. strain 12S. Micro-
48. Yuste, L., A. B. Herva ´s, I. Canosa, R. Tobes, J. L. Jime ´nez, J. Nogales, M. M.
Pe ´rez-Pe ´rez, E. Santero, E. Díaz, J. L. Ramos, V. De Lorenzo, and F. Rojo.
2006. Growth phase-dependent expression of the Pseudomonas putida
KT2440 transcriptional machinery analysed with a genome-wide DNA mi-
croarray. Environ. Microbiol. 8:165–177.
VOL. 192, 2010 PLASMID-BORNE H-NS PROTEIN ALTERS HOST FUNCTION4731