J. Biol. Macromol., 13(3), 86-91, 2013

Regular Article

Mutant deletions in Escherichia coli affect the cellular levels of undecaprenyl phosphate andundecaprenyl diphosphate
Yuichiro Saito, Tatsuya Ishikawa, Marie Murakami, Keisuke Suzuki
and Shingo Fujisaki*
Department of Biomolecular Science, Faculty of Science, Toho University, Miyama 2-2-1, Funabashi, Chiba
274-8510, Japan
Received November 12, 2013; Accepted December 10, 2013
Escherichia coli multiple-deletion mutants were constructed to examine the lipid phosphatase genes. Ratioof cellular levels of undecaprenyl phosphate (UP) and undecaprenyl diphosphate (UPP) was determinedusing cells labeled with 14C and measuring the radioactivities in these lipids. The labeled cells wereobtained through feeding [14C]isopentenyl diphosphate to an E. coli strain. The relative level of UP in thewild type strain W3110; in the YS1234 strain defective in ynbD, yeiU, ybjG, and pgpB; in the YS1235 strain
defective in ynbD, yeiU, ybjG, and bacA; and in the YS1245 strain defective in ynbD, yeiU, pgpB, and bacA
were 78.0% ± 3.5%, 74.7% ± 1.2%, 68.4% ± 6.8%, and 64.0% ± 7.5%, respectively. The quadruple mutant
strain that possessed the wild-type bacA showed almost identical UP level as the wild type strain W3110,
indicating that sufficient level of UPP phosphatse activity was conferred by a bacA product without the
products of ynbD, yeiU, ybjG, and pgpB. However, the mutants defective in bacA showed a slight but
significant (p < 0.01) decrease in the UP level.
Keywords: Undecaprenyl phosphate; Undecaprenyl diphosphate; isopentenyl diphosphate; lipid phosphatase;deletion mutant; lyophilized cell
Undecaprenyl phosphate (UP) is a sugar carrier in thedephosphorylate UPP. In the E. coli chromosome

biosyntheses of peptidoglycan and other cell surface

there are four genes that encode proteins with a PAP-2

polymers (1). UP is essential in forming undecaprenyl

type phosphatase motif (7) (Fig. 1). The chromosome

diphosphoryl sugar (UPP-sugar), which transports

also shows the presence of a bacA copy. Enzymatichydrophilic monomeric units of polysaccharides

activities

acrossthecellmembrane

to

the

of

BacA,

PgpB,

YbjG,

and

YeiU

external

dephosphorylate UPP to produce UP (5, 6, 8). The

polymerization sites (2). Undecaprenyl diphosphate

YeiU is also a phosphotransferase, which produces(UPP), the precursor of UP, is synthesized by thelipid A 1-diphosphate (10). As of now, there are nosequential condensation of isopentenyl diphosphsate

published reports that elucidate the function of YnbD.

(IPP) to farnesyl diphosphate (3, 4).

Lipid

El Ghachi et al. reported that an E. coli strain

phosphatases are integral membrane proteins that

deficient in bacA, pgpB, and ybjG was lethal, and that

dephosphorylate(5-8).

strains deficient in both bacA and pgpB or in both

BacA-type (5, 9) and the PAP-2 type (6, 7) lipid

bacA and ybjG could grow as efficiently as a wild type

phosphatases

arestrain (6). However, disruption of bacA decreased

membraneproteins

UPP

toproducedifferentinclassesEscherichia

UPofintegralcoli*To whom correspondence should be addressed,
fax: +81-47-472-1188
e-mail:sfujisak@biomol.sci.toho-u.ac.jp

that

UPP phosphatase activity by 75% in vitro

87

Cellular levels of undecaprenyl phosphate in E. coli mutants

Sequence
Protein
YbjG
YeiU
PgpB
YnbD
Phosphatase
motif

GHLFPHDRP (18)
PSDH (32)SRVYLGVHWPLD
KENAPGRRR (56)
PGDH (32)
PRVMIGAHWFTD
KDKVQEPRP (54)
PSGH (36)SRLLLGMHWPRD
PLKFSFIRP (23)
PSLH (32)
STLTTWQHHFIDKXXXXXXRP (12-54) PSGH (31-54) SRXXXXXHXXXD

Fig. 1. Consensus motif of the phosphatase domain and comparison with predicted lipid phosphatases. Bold letter
indicates the conserved amino acids. The numbers in parentheses indicate the number of residues separating eachmotif.
(5). To estimate the contribution of an each geneproduct to the in vivo UPP phosphatase activity, weconstructed multiple gene-deletion mutants forputative UPP phosphatases and measured the ratio of
UP and UPP in the cells. High performance liquid
chromatography (HPLC) can readily measure UP (4,11), however, direct measurement of UPP by HPLC isdifficult because UPP elutes in a crowded region (11).
Therefore, we specifically labeled UP and UPP with
14

C through feeding of [14C]IPP to an E. coli strain.

The ratio of cellular levels of UP and UPP was thendetermined by measuring the radioactivities in theselipids. Because IPP is impermeable to growing cells,
for labeling experiment, we prepared lyophilized cells
that had been confirmed to retain normal metabolic
activities (12).
Materials and Methods
Strains and plasmids – Strains used in this study
are described in Table 1. A recJ strain KM354 (13)and deletion mutants (14) JWK1270, JWK2162,
JWK3029, and JWK5112 were provided by Dr. Kenan
C. Murphy from University of Massachusetts and Dr.
Hirotada Mori from Nara Institute of Science andTechnology, respectively. Plasmid pCP20 (15) andplasmid pKRP10 (16) were provided by Dr. Hirotada
Mori and by Dr. Seiichi Yasuda from the National
Insitute of Genetics, respectively. A cloning vector
pCR-2.1TOPO was purchased from Invitrogen Co.
Chemicals – [14C]IPP (specific activity, 2.2 TBq
mol–1) and bacitracin (>40,000 u g–1) were acquired
from Amersham Bioscience and Wako Pure Chemical
Industries, Ltd., respectively.
Plasmid construction – The ynbD gene was

PCR-amplified from an E. coli W3110 chromosome
using oligonucleotides ynbDfw and ynbDrv (Table 2).
The 1.7-kb PCR fragment was cloned into thepCR-2.1TOPO vector plasmid to construct a
pCR-ynbD. pCR-ynbD was digested with SalI andligated with a chloramphenicol-resistant 0.9-kb SalI
fragment from pKRP10 to construct pCR-ynbD::CmR.
Strain construction – A strain carrying ynbD
disrupted with the chloramphenicol-resistant cassette
was constructed by transforming the recJ strain
KM354 with the 2.6-kb PCR fragment that was
amplifiedfrompCR-ynbD::CmR,
using
oligonucleotides ynbDfw and ynbDrv. The disrupted
ynbD was introduced into W3110 by P1 transduction.
Multiple-deletion mutants were generated bytransduction of kanamycin resistant markers withphage P1, followed by excision of the cassettes fromthe chromosome (15). P1 phages were prepared fromstrains of Keio collection (14), JW2162 (yeiU::Km),
JWK5112 (ybjG::Km), JWK1270 (pgpB::Km), andJWK3029 (bacA::Km). The disrupted gene wasintroduced into kanamycin-sensitive parental strains
derived from W3110 by P1 transduction.
Kanamycin-resistant clones were isolated and thedisruption of the yeiU, ybjG, pgpB, and bacA genes in
the chromosome was verified by PCR using
oligonucleotides yeiUfw and yeiUrv, ybjGfw andybjGrv, pgpBfw and pgpBrv, and bacAfw and bacArv,
respectively (Table 2).
Excision of thekanamycin-resistance genes from the chromosome of
the mutant was obtained by the transformation with
pCP20. Antibiotic-sensitive clones were isolated, andthe excision of the cassette was confirmed by PCR
analysis as described above.

Y. Saito, et al.

88

Cellular levels of undecaprenyl phosphate in E. coli mutants

89

Table 2. Oligonucleotides used in this study.
Oligonucleotide Sequence (5'-3')ynbDfw
AACTCACTCGCAGGTCTTGC
ynbDrvGCACAGTCTGACGCAAAAGC
yeiUfwGTGAAGTTTTCGGTGATGATCTG
yeiUrv
CCCTGATGATGTTAATTACTG
ybjGfwCTTGAGGGAAATAAGACGATG
ybjGrv
AAATGGCGTTGCTGGCGTGA
pgpBfw
ACAAGCGGACTTCATTGACG
pgpBrv
TATGGTCAACTTACCGCAAT
bacAfw
CTGCTCCCTTGCCACCGATA
bacArv
AGCGTCGCATCAGGCGTTGA

Preparation of lyophilized cells and 14C labeling ofcellular lipids – A previously described protocol (12)was slightly modified to prepare the lyophilized cells.
E. coli cells which were grown under shaking
conditions in 5 mL of L-broth (17) for 16 h at 37˚C
were harvested, washed once with 0.1 M potassium
phosphate buffer (pH 7.4), and then suspended in 200µL of 3% sodium glutamate. The suspensions in the
tubes were frozen in liquid nitrogen and lyophilized
under a pressure of less than 20 Pa for two hours. The
lyophilized cells were rehydrated with 0.1 mL of 45
mM potassium phosphate buffer (pH 7.5) containing
9,200 Bq of [14C]IPP and incubated for 20 min at
30˚C. The cells were washed with 0.1 M potassium
phosphate buffer and suspended in 0.85% NaCl for the
extraction of the labeled lipids.
Extraction and analysis of labeled lipids – Lipids
were extracted with chloroform–methanol (1:1, v/v)
according to the method of Bligh and Dyer (18), andanalyzed by thin-layer chromatography on silica gel
60 plate (Merck Co.) using diisobutyl ketone–acetic
acid–water (8:5:1, v/v/v) as a mobile phase (12).
Radioactivity on the plate was detected by BAS-1000(Fuji film Co.). For radioactivity quantification, thearea containing each product was scraped off of theplate into a counting vial containing scintillator.

containing bacA::KmR was unsuccessful. This result
confirmed the lethality of the inactivation of bacA,
ybjG, and pgpB genes (6). Every mutant strain thatwas obtained grew as efficiently as the parental strain
W3110.

The growth of the triple and quadruple

mutants defective in bacA, YS235, YS245, YS1235,
and YS1245 was slightly inhibited in the bacitracin
medium (120 u mL–1), whereas the growth of the wild
type strain in the same medium was not affected.
Determination

of

UP

and

UPP

level

by

measurement of radioactivity – In the labelingexperiment described in Materials and Methods, about
10% of radioactivity of added [14C]IPP was found in
the chloroform-methanol extract.

The 14C-labeled

lipids were separated on a silica gel plate and theradioactivities of the UP (Rf=0.5) and UPP (Rf=0.33)
spots were measured. The ratio of radioactivity in UP
to the sum of those in UP and UPP was calculated andconsidered to be a relative level of UP. Because therelative levels of UP in all single and double disruption
mutants (Table 1) were not significantly different fromthat in the parental strain, we constructed triple andquadruple mutants and measured the UP levels inthem. The relative level of UP in the wild type strain
W3110; in the YS1234 strain defective in ynbD, yeiU,
ybjG, and pgpB; in the YS1235 strain defective in
ynbD, yeiU, ybjG, and bacA; and in the YS1245 strain
defective in ynbD, yeiU, pgpB, and bacA were 78.0%± 3.5%, 74.7% ± 1.2%, 68.4% ± 6.8%, and 64.0% ±
7.5%, respectively (Table 3). The quadruple mutant
YS1234 that possessed the wild-type bacA showedalmost identical UP level as W3110, indicating that asufficient level of UPP phosphatase activity was

Results

conferred by a bacA product without the products ofConstruction of multiple deletion mutants – Strains

ynbD, yeiU, ybjG, and pgpB. However the quadruple

carrying multiple gene deletions were constructed bymutants

successive rounds of transduction of strains derived

wild-type pgpB and ybjG, respectively, showed slightfrom W3110 by phage P1 obtained from deletionbut significant (p < 0.01) decrease in the UP level,mutants of Keio collection containing integral

indicating that either a product of pgpB or ybjG alone

kanamycin resistance markers, followed by excision

could confer the UPP phosphatase activity slightlyof the markers from the chromosome.

Althoughlower than that in the wild type strain, which would

multiple-deletion mutants shown in Table 1 wereprove enough for its growth in an antibiotic-freeobtained without any difficulty, transduction of themedium.

ybjG and pgpB double-deficient mutant by phage P1

YS1235

and

YS1245that

possessed

Y. Saito, et al.

90

Table 3. Relative level of UP in parental strain and deletion mutants.
Relative level of UP (%)a
StrainRemaining gene for phosphatase
+
+
+
+
+
bacA , pgpB , ybjG , yeiU , ynbD
W3110
78.0 ± 3.5
bacA+
YS124374.7 ± 1.2
bacA+, yeiU+
YS143
80.0 ± 1.0
bacA+, ynbD+
YS234
82.7 ± 1.0
pgpB+
YS123568.4 ± 6.8 **pgpB+, yeiU+
YS135
67.8 ± 7.9*
pgpB+, ynbD+
YS235
74.6 ± 7.8
ybjG+
YS124564.0 ± 7.5 **ybjG+, yeiU+
YS145
66.8 ± 6.0 **ybjG+, ynbD+
YS245
67.5 ± 14.5
aValues shown represent averages and standard deviations of results obtained at least three assays.
t-test * P < 0.05; **p <0.01

small and probably deleterious to the cells (notDiscussion

shown).

We prepared lyophilized cells and labeled UP and
its derivatives in this study.

We had previously

Barreteau et al. (11) reported that 80% of the total
undecaprenol derivatives in growing cells of E. coli

confirmed that the labeled cells retained normal

were UPP.

metabolic activity (12). We had been able to grow thedifference of UP contents determined by HPLC before

labeled cells in L-broth again, and to trace theandmetabolism of the labeled UP derivatives (12, 19). In

measurement of UPP by HPLC was difficult.

the growing cells, radioactivity in UPP-sugar had been

Barreteau et al.’s estimated UPP value included that ofas high as that in UP and UPP, whereas before growing

UPP-sugar, which led to the over-estimation of UPP.

in L-broth, radioactivity in UPP-sugar had been much

They assumed that the UPP-sugar content was low;

lower than that in UP and UPP (12). Because thewhereas, our previous results suggested that thelabeled cells were not grown again in this study,

UPP-sugar content in growing cells was as high as thatradioactivity should primarily be in UP and UPP.

of UPP and UP (12). Thus, direct measurement ofafter

UPP was calculated by analyzing thealkalinehydrolysis

becausedirectThe UP level in multiple deletion mutants showed

UPP or UPP-sugar by liquid chromatography-massthat BacA plays an essential role in dephosphorylating

spectrometry analysis would solve the difference of

UPP.

the results of two groups.

UP levels were maintained in the triple orquadruple mutants that lacked both the ybjG and pgpB
genes. Although UP levels maintained by PgpB or

Acknowledgements

YjbG alone were sufficient for growth in an

We wish to thank Dr. Hirotada Mori from Nara

antibiotic-free medium, these levels were slightly

Institute of Science and Technology, Dr. Kenan C.

lower than that in the wild type strain. Thus, it isMurphy from University of Massachusetts, and Dr.

possible that this decrease in UP level causes

Seiichi Yasuda from National Institute of Genetics, forhypersensitivity of the deletion mutants in bacA toproviding the strains and plasmids. We also would

bacitracin,

like to thank Enago (www.enago.jp) for the English

theantibiotic

that

inhibits

UPP

dephosphorylation.

language review.

This is the first time that multiple-deletion mutants,
including the ynbD deletion, have been constructed.
The contribution of ynbD to UPP dephosphorylation
was small, but the negligible difference in the UP level
between YS1235 and YS235 may be due to thephosphatase activity of YnbD. Although we tried todetect the enzymatic activity of YnbD, we neverobtained this protein because its expression was very

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Communicated by Ishijima Sumio