Journal of Environmental Biotechnology
Vol. 11, No. 1 · 2, 83–87, 2011

Note

Identification and Characterization of Methyl-Accepting Chemotaxis
Proteins for Trichloroethylene in Pseudomonas putida F1
Shota Oku, Haruki Kinoshita, Takahisa Tajima and Junichi Kato*
Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University,
Higashi-Hiroshima, Hiroshima 739-8530, Japan* TEL: +81–(0)82–424–7757 FAX: +81–(0)82–424–7047* E-mail: jun@hiroshima-u.ac.jp(Received; 31 October 2011/Accepted; 2 December, 2011)
Twenty seven methyl-accepting chemotaxis proteins (MCPs) from trichloroehylene (TCE)-degrading Pseudomonas putida
F1 were examined for their ability to detect TCE in Pseudomonas aeruginosa PCT2 and two proteins, CttA (Pput 2149)
and CttB (Pput 2348) were identified as MCPs for TCE. Chemotaxis assays revealed that CttA detected toluene as well as
TCE. Quantitative reverse-transcription PCR (qRT-PCR) demonstrated that both cttA and cttB were constitutively expressedin P. putida F1. Overexpression of cttA and cttB enhanced positive chemotaxis to TCE in P. putida F1.
Key words: chmoetaxis, trichloroethylene, Pseudomonas putida F1

Pseudomonas putida F1 is an aerobic motile bacterium
and is capable of utilizing toluene as a growth substrate via
the toluene oxygenase pathway 18). Toluene-induced cells of
P. putida F1 show the activity to degrade the priority environmental pollutant trichloroethylene (TCE) 6,15). Toluene
dioxygenase is responsible for TCE degradation in P. putida
F1. This TCE-degrading bacterium has been intensively
studied as bioremediation agents in chloroethylene-polluted
environments. One special feature of P. putida F1 is chemotaxis to TCE and toluene 8). When P. putida F1 is grown in
the presence of toluene, it shows chemotactic responses to
TCE and toluene. The migration of TCE-degrading bacteria toward TCE is expected to speed the biodegradation
process because it should bring the cells into contact with
TCE 9). Despite these fi ndings, very little is known about
the chemosensory protein mediating positive chemotaxis to
TCE and toluene in TCE-degrading bacteria. Chemotactic
ligands are detected by cell surface chemoreceptors called
methyl-accepting chemotaxis proteins (MCPs). Upon binding a chemotactic ligand, a MCP generates chemotaxis
signals that are communicated to the flagellar motor via aseries of chemotaxis proteins. In this study, we identified andcharacterized MCPs for TCE in P. putida F1.
For chemotaxis assays, P. putida F1, Pseudomonas
aeruginosa PCT2 (a pctA pctB pctC mutant of P. aeruginosa PAO1) 13), and their derivatives were grown overnightin 2x YT medium 10) with shaking. Five hundred microlitersof the culture broth was inoculated into T0 medium 17) in
a 50-ml screw-capped vial. When cells were exposed totoluene, minimum salts basal medium 2) supplemented with
50 mM pyruvate as a solo carbon source was used for

cultivation and 0.5 ml toluene was added to a small tube
inside the screw-capped vial. P. putida and P. aeruginosa
were cultivated at 28 and 37°C, respectively. Cells were
harvested by centrifugation, washed twice with chemotaxis
buﬀer A (10 mM HEPES [N-2-hydroxyethylpiperazine-N’2-ethanesulfonic acid], pH 7.0), and resuspended in thesame buﬀer. Chemotaxis was assayed by computer-assisted
capillary as described previously 7). Digital image processing
was used to count the number of bacteria around the mouthof a capillary containing a known concentration of a test
compound plus 1% agarose.
For quantitative reverse transcription-PCR (qRT-PCR)
analysis, P. putida F1 was cultivated as described above.
Cells were harvested by centrifugation at 4°C for 10 min.
Total RNA was extracted from bacterial cells using an
NucleoSpin® RNA II kit (MACHEREY-NAGEL, GmbH &Co., Düren, Germany) with a RNAprotect bacteria reagent(QIAGEN, Inc., Valencia, CA, USA) for the stabilization ofRNA. RNA samples were treated with a RNase-free DNase
set (MACHEREY-NAGEL) to eliminate contaminating
DNA. Complete removal of DNA was confirmed by PCR. A
One Step SYBR PrimeScript RT-PCR kit (Takara Bio, Inc.,
Shiga, Japan) was used for cDNA generation and quantitative RT-PCR. Quantitative RT-PCR was carried out usinga LightCycler system (Roche Diagnostics, Tokyo, Japan)
with an reverse transcription at 42°C for 15 s followed bycycles of denaturation at 95°C for 5 s, primer annealing at
57°C for 10 s, and extension at 72°C for 6 s. Standard procedures were used for plasmid DNA preparations, restriction
enzyme digestions, ligations, transformations and agarose
gel electrophoresis 10). Polymerase chain reactions (PCRs)

Oku et al.

84
Table 1. Oligonucleotides used in this study
Name
PPF02fSequence (5’ to 3’)
AATTGAGCTCTCAACGCATTCCGTTCGGTTAC

PPF02rTATGGGTACCAGGTCAGGCAGCGACGAGATAC

PPF04f

ACTTGCTCGAGCTGTCGATGCCACGACTTG

PPF04rGTAAACTGCAGAAAGACTGTGAGGCTATCGAACG

qPFgyrBf

CAGACTTACGTTCACGGTGTTCC

qPFgyrBr

GATGGCTTGAAGTGGATGTGG

qPFtodC2f AGCGTCCCTTCATCATCGTG
qPFtodC2r GACATTCACTACTTCATGCCCATTC
qPF02fACGTGTCTTCGGGCTTTATC

qPF02r

TCTGGCTCTTGTTGTTCAGC

qPF04f

CGCCTTGATGAAGTGGTTG

qPF04rTGGTATTCGCCACGGTTCKmRfGTAAGACATATGCGGGAAGATGCGTGATCTG

KmRr

ATGATGCATATGGGAAAGCCACGTTGTGTCTC

Ptaqf

ATCGATATCCAGCTTGTCTGTAAGCGGATG

Ptaqr

ATCGATATCCAGTAGTAGGTTGAGGCCGTTG

were carried out using KOD plus (Toyobo, Tokyo, Japan)
DNA polymerase according to the manufacturer’s instructions. Oligonucleotides used for PCR and qRT-PCR are
listed in Table 1. For P. aeruginosa, pUCP18 12) was used
as a plasmid vector. For P. putida F1, pUCPK18taq wasused as a plasmid vector. Kanamaycin resistant gene from
pUC4K 16) was introduced into pUCP18 and lac promoter of
pUCP18 was substituted with taq promoter from pHA10 1)
to construct pUCPK18taq.
Genome annotation has revealed that P. putida F1 possesses 27 putative MCP genes (locus tags: Pput 0339, Pput
0342, Pput 0601, Pput 0623, Pput 0804, Pput 1257, Pput
1390, Pput 1872, Pput 2091, Pput 2149, Pput 2217, Pput
2348, Pput 2731, Pput 2828, Pput 3459, Pput 3481, Pput
3489, Pput 3621, Pput 3628, Pput 3892, Pput 4234, Pput
4352, Pput 4520, Pput 4764, Pput 4863, Pput 4894, andPput 4895 [genome annotation data: NC 009512]). These
genes were PCR amplified from the P. putida F1 genomic
DNA and amplified products were cloned into pUCP18.
The resulting plasmids were introduced to P. aeruginosa
PCT2 and transformants were examined for their ability torespond to TCE. P. aeruginosa PCT2 is a pctA pctB pctC
triple mutant of P. aeruginosa PAO1 13). The pctA, pctB,
and pctC genes encode MCPs for negative chemotaxis to
TCE 11). P. aeruginosa PCT2 is a better host for screening a gene encoding MCP for TCE than P. aeruginosa
PAO1 because it shows much decreased repelled responses
to TCE. Chemotaxis assays revealed that two transformants harboring pHEC02 and pHEC04 were significantly
attracted by TCE (Fig. 1). Plasmids pHEC02 and pHEC04
contain genes encoding proteins Pput 2149 and Pput 2348,
respectively. Based on these results, the Pput 2149 and Pput
2348 genes were designated cttA and cttB (ctt stands forchemotactic transducer for TCE).
Typical MCPs are membrane-spanning homodimers
and their features are as follows: a positively charged N

Fig. 1. Chemotaxis responses to TCE (A) and toluene (B) by P.
aeruginosa strains. Digital image processing was used to countthe number of bacteria around the mouth of a capillary containing 0.8 mM TCE and 1% agarose (A) or 0.1 mM tolueneand 1% agarose (B). One videotape frame was analyzed ateach time point and the normalized cell number was calculated by dividing the number of bacteria at each time pointby that at the initiation of the observation. The chemotactic
response is presented as the normalized cell number. PCT2
(open squares), PCT2 [pHEC02] (closed circles), PCT2
[pHEC04] (closed triangles). Vertical bars represent the standard deviations of measurements done in triplicate experiments.

terminus followed by a hydrophobic membrane-spanning
region, a hydrophilic periplasmic domain, a second hydrophobic membrane-spanning region and a hydrophilic cytoplasmic domain 3). Chemotactic ligands bind to periplasmic
domains of MCPs and their binding initiates chemotaxis
signaling. The diverse ligand specificities among MCPs
reflect amino acid sequence diversities of periplasmic
domains of MCPs. The C-terminal cytoplasmic domains of
MCPs are relatively conserved. CttA has features of typical
MCP (Fig. 2) and Conserved Domain-Search (CD-Search)
program (National Center of Biotechnology Information)found HAMP and TarH signal domains, typical domains of
MCP, in its C-terminal region. Blastp search using aminoacid sequence of the putative periplasmic domain of CttA
(residues 38 to 189) as a query sequence revealed that P.
aeruginosa PAO1 does not possess a homologue of CttA.
CttB seems to be atypical MCP. Hydropathy plot foundno transmembrane region in CttB and PSORT program 5)
predicted that it is a cytoplasmic protein. CD-Search foundtwo PAS domains as well as MCP signal domain (TarH)
in CttB (Fig. 2). The PAS motif is known to comprise aIdentification and Characterization of Methyl-Accepting Chemotaxis Proteins for Trichloroethylene in Pseudomonas putida F1

85

Fig. 2. Domain organization of CttA and CttB from P. putida F1 and CttP from P. aeruginosa PAO1 2). TM1 and TM2, transmembraneregions 1 and 2; HAMP, conserved signal transduction domain in histidine kinase, adenylate cyclase, methyl-accepting chemotaxis proteins, and phosphatases; PAS, acronym formed from names of the proteins in which imperfectrepeat sequences wre first recognized (theDrosophia period clock protein [PER], the vertebrate aryl hydrocarbon receptor nucler translocator [ARNT], and the Drosophia singleminded protein [SIM]); TarH (TarH superfamily), methyl-accepting chemotaxis protein signaling domain; HCD, highly conserved signaling domain consisted of 44 amino acid residues in methyl-accepting chemotaxis protein 11).

Fig. 3. Expression levels of todC2, cttA and cttB in P. putida F1.Total RNA was extracted from P. putida F1 cells grown in the presence of
toluene (closed bars) and in the absence of toluene (open bars), and subjected to qRT-PCR. Relative expression was calculated based on
gyrB expression as internal control gene. Vertical bars represent the standard deviations of measurements done in triplicate experiments.

binding pocket for a prosthetic group 13). Blastp search using
N-terminal amino acid sequence of CttB (residues 30 to240) as a query reveled that CttB is 49% and 46% identicalto P. aeruginosa PAO1 MCPs BdlA (PA1423) and PA1930,
respectively, but it shows no significant similarity to CttP
(PA0180) which is MCP for positive chemotaxis to TCE in
P. aeruginosa PAO1 4).
Parales et al. reported that toluene induced chemotaxis to
TCE from P. putida F1 8). To investigate whether expressionof cttA and cttB is induced by toluene, RNA was extracted
from P. putida F1 cells grown in the presence and absenceof toluene and subjected to qRT-PCR analysis. Although
expression of toluene-inducible toluene dioxygenase gene
(todC2) 18) increased in cells grown in the presence of
toluene, cttA and cttB were constitutively expressed (Fig. 3).

It is unlikely that CttA and CttB are responsible for tolueneinducible chemotaxis to TCE in P. putida F1 although
they serve as MCPs for TCE. In this study, a MCP gene
responsible for toluene-inducible chemotaxis to TCE could
not be obtained probably because that gene was not functionally expressed in P. aeruginosa PCT2. Alternatively, an
additional factor may be required for the MCP for inducible
chemotaxis to TCE to detect TCE. For example, phosphate
specific transportation system is essential for P. aeruginosa
CtpL to act as a MCP for phosphate taxis 17).
Parales et al. demonstrated that toluene also induced
chemotaxis to toluene from P. putida F1 8). Although CttA
and CttB are not MCPs for inducible chemotaxis to TCE,
it is interesting to investigate whether these MCPs detect
toluene or not. So, we examined P. aeruginosa PCT2 har-86

Oku et al.

in the absence of showed repelled responses to 0.8–4 mM
TCE (Fig. 4). P. putida F1 [pMEF02] and P. putida F1[pMEF04] did not show repelled responses to TCE, rather
was attracted by 0.8 mM TCE. P. putida F1 wild-type cells
grown in the presence of toluene showed weak positive
chemotaxis to TCE, while P. putida [pHEF02] showed
increased responses to TCE. P. putida [pHEF04] did notshow any responses to TCE because this strain showedvery poor mobility in this cultivation condition. From theseresults, we can conclude that introduction of the cttA andcttB genes enhanced positive chemotaxis to TCE by P.
putida F1.
In conclusion, two MCPs for TCE, CttA and CttB, wereidentified. CttA detected toluene as well as TCE. Both cttAand cttB were constitutively expressed in P. putida F1. Overexpression of cttA and cttB enhanced positive chemotaxis to
TCE by P. putida F1. We are now searching MCP responsible for inducible chemotaxis to TCE in P. putida F1.

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Fig. 4. Chemotaxis responses toward TCE by P. putida F1 strains.
(A) chemotactic responses by P. putida F1 wild type strain
grown in the absence of toluene. H2O (negative control), open
squares; 0.8 mM TCE, closed diamonds; 2 mM TCE, open diamonds; 4 mM TCE, crosses. (B) chemotactic responses to
0.8 mM TCE by P. putida F1 (closed diamonds), F1 [pHEF02](open circles), and F1 [pMEF04] (open triangles) grown in theabsence of toluene. (C) chemotactic responses to 0.8 mM
TCE by P. putida F1 (closed diamonds), F1 [pHEF02] (opencircles), and F1 [pMEF04] (open triangles) grown in the presence of toluene. Vertical bars represent the standard deviationsof measurements done in triplicate experiments.

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