J. Biol. Macromol., 18(3), 51-58 (2018)
Regular Article

Construction of Staphylococcus aureus Mutant Deficient in the Undecaprenol Kinase Gene, dgkA, andElucidation of the Role of Undecaprenol Kinase in the Metabolism of Undecaprenyl Phosphate
Naoki Kawakami1, Kasumi Shimada1, Takaaki Fujita1, Izumo Kanesaka1,2, Intetsu Kobayashi2, and Shingo Fujisaki1*
1

Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba 274-8510, Japan2

Department of Infection Control and Prevention Nursing, Faculty of Nursing, Toho University, Ohta-ku, Tokyo 143-0015,

Japan
Received January 17, 2018; Accepted February 21, 2018
A dgkA homolog was cloned from the Staphylococcus aureus genome and expressed in Escherichia coli. The purified
DgkA homolog encoded by the cloned gene revealed undecaprenol (UOH) kinase activity. A deletion mutant of dgkA
homolog was constructed to examine the undecaprenyl phosphate (UP) metabolism in S. aureus. Cellular levels of
UOH and UP were determined using cells grown in the medium containing 14C-labeled and 3H-labeled
mevalonolactone by measuring the radioactivities in UOH and UP that were extracted from cells and separated bythin-layer chromatography. The relative UOH level in the deletion mutant was significantly higher than that in theparental strain both in the exponential growth phase and in the stationary phase. Conversion of UOH accumulated in
the stationary phase to UP in the recultivated cells occurred not only in the parental strain but also in the deletionmutant.
Keywords: undecaprenyl phosphate, undecaprenyl diphosphate phosphatase, undecaprenol kinase, Staphylococcus aureus

Undecaprenyl phosphate (UP) is an essential compound in
the biosynthesis of bacterial cell surface polysaccharides
such as cell wall peptidoglycan [1] (Fig. 1). UP is called asugar carrier lipid because it is bound to a hydrophilic sugar
moiety to form a lipid intermediate that is translocated fromthe inner side to the outer side of the cytoplasmic membranein the polysaccharide biosynthesis pathway [2]. Then, thesugar unit is incorporated into the growing polysaccharide
chain by the action of the specific polymerase, andundecaprenyl diphosphate (UPP) is released at the outer sideof the cytoplasmic membrane [3]. UP is produced in both denovo
synthesis
andrecycling
pathwaysbydephosphorylation of UPP [2,4–6]. In the de novo synthesis
pathway, UPP is synthesized by sequential condensations ofisopentenyl diphosphate to farnesyl diphosphate by theaction of UPP synthase [7–9] at the inner side of thecytoplasmic membrane. In the recycling pathway, UPP isreleased from the lipid intermediate at the outer side of themembrane as described above. Dephosphorylation of UPP
is catalyzed by one of three enzymes, BacA and two lipid
phosphatases belonging to the type 2 phophatidic acidphosphatase family, in E. coli at*Corresponding author. Fax: 81 47 472 1188
Email: sfujisak@biomol.sci.toho-u.ac.jp (S. Fujisaki).

the periplasmic space [10–12]. Although MurJ is theflippase that translocate sugar lipid intermediate for
peptidoglycan biosynthesis form the inner side of thecytoplasmic membrane to the outer side [13], the flippasethat enhances transbilayer movement of UP and/or UPP is
not yet known.
Gram-positive bacteria contain a remarkable amountof undecaprenol (UOH) [14–16], which is phosphorylated
to UP, although UOH has not been found in Gram-negative
bacteria [17,18]. The DgkA protein in Bacillus subtilis [19]and Streptococcus mutans [20], which is homologous todiacylglycerol kinase in Escherichia coli [21], catalyzes thephosphorylation of UOH. It is possible that phosphorylationof UOH is a third pathway to produce UP at least in Grampositive bacteria [2,4–6]. To investigate the role of a DgkA
homolog in UP metabolism in Staphylococcus aureus, wecloned the dgkA homolog of S. aureus, and constructed thedeletion mutant. The expression of the cloned gene in E. coli
and the characterization of enzyme activity of the purified
DgkA homolog were carried out. Cellular levels of UOH and
UP in the mutant and parental strain were estimated by tracer
experiment using cells grown in the medium containing
radiolabeled mevalonolactone, the precursor for isoprenoid
biosynthesis [22].

Construction and characterization of the dgkA mutant of S. aureus

52

Fig. 1. Undecaprenyl phosphate (UP) metabolism in cell wall biosynthesis. The short black bar represents the undecaprenyl group, andthe circled P represents the phosphate group. Undecaprenyl diphosphate (UPP) is synthesized by the sequential condensation of isopentenyl
diphosphate (IPP) to farnesyl diphosphate (FPP) by the action of UPP synthase (IspU/Rth) [7–9]. Dephosphorylation of UPP is catalyzed
by BacA and lipid phosphatases belonging to the type 2 phophatidic acid phosphatase (PAP2) family [10–12]. UPP disaccharide
pentapeptide (lipid II) was synthesized by the sequential actions of MraY [31] and MurG [32] at the inner side of cytoplasmic membrane.
The hydrophilic disaccharide pentapeptide moiety of lipid II is translocated from the inner side of the membrane to the outer side by MurJ
[13]. Then the disaccharide pentapetide is incorporated into the growing chain of peptidoglycans by the action of penicillin-binding proteins(PBPs) [33], and UPP is released at the outer side of the cytoplasmic membrane [1–3]. The released UPP is dephosphorylated by BacA [10]or PAP2s [11,12]. The flippase that enhances transbilayer movement of UP and/or UPP is not yet known. A remarkable amount of
undecaprenol (UOH) exists in Gram-positive bacteria [14–16]. DgkA protein, which is homologous to diacylglycerol kinase of Escherichia
coli, catalyzes the phosphorylation of UOH [19,20].

Materials and methods
Bacterial strains and plasmids. The E. coli and S.aureus
strains used are listed in Table 1. E. coli DH5 was used

for plasmid construction and expression of the cloned dgkA
homolog. S. aureus ATCC29213 was used for preparation of
genomic DNA. Genomic DNA of ATCC29213 wasextracted using RBC Genomic DNA

Table 1. Bacterial strains and plasmids.
Strain or plasmid

Relevant characteristics or derivation

Source or reference

Host strain for construction of recombinant plasmids and recombinant gene expression

Toyobo Co.

Escherichia coli
DH5

Staphylococcus aureus
ATCC29213

Wild type strain

Laboratory stock

RN4220Highly transformable strain[23]

RN4220DdgkA

Constructed by allelic exchangeThis work

pColdIE. col i cloning vector for gene expression

Takara bio Co.

pCold-dgkA

Plasmid for dgkA expression

This work

pKOR1

Vector for allelic exchange[24]

pKOR-DdgkA

Plasmid harboring disrupted dgkA

This workKawakami et al.

53

Table 2. Oligonucleotides used in this study.
aOligonucleotide

Sequence (5'-3')dgkA_Fw1

CCACCCATATGAAAAGGTTTAAATATGCACTTG

dgkA_Rv1

TGGGGATCCGTGCTTTTCTAACTTCTTG

attB1_DdgkA_Fw

GGGGACAAGTTTGTACAAAAAAGCAGGCTATTGTATTAACAAGACCTGC

attB1_DdgkA_Rv

CATCCGCGGATTTTCAGCCCATCAAGTGC

attB2_DdgkA_Fw

CATCCGCGGATAGCGTTATTTTAGGGAGG

attB2_DdgkA_Rv

GGGGACCACTTTGTACAAGAAAGCTGGGT AGGTCCTTCGGGTAAATACG

dgkA_Fw2

ATGAAAAGGTTTAAATATGCACTTG

dgkA_Rv2

GTGCTTTTCTAACTTCTTG

a

Sequences of restriction sites are underlined. Sequences of specific recombination sites are double underlined.

Extraction Kit (RBC Bioscience Co.). pColdI (Takara BioCo.) was used as a vector for expression of dgkA homolog
in E. coli. The primers used are listed in Table 2. The primers
dgkA_Fw1 and dgkA_Rv1 were used for amplification of acoding region of dgkA homologue from a genomic DNA of
ATCC29123 by PCR (Fig. 2A). The amplified DNA
fragment and pColdI were digested with NdeI and BamHI
and ligated. The resulting plasmid pCold-dgkA was used forexpression of the fusion gene to produce hexahistidinetagged DgkA homolog. S. aureus

RN4220 [23] was used as a parental strain for constructionof dgkA disruptant RN4220DdgkA. pKOR1 is a vector for
allelic exchange [24]. Primers attB1_DdgkA_Fw andattB1_DdgkA_Rv were used for amplification of an
upstream and an amino terminal region, and primers
attB2_DdgkA_Fw and attB2_DdgkA_Rv were used for
amplification of a downstream and a carboxyl terminal
region of dgkA homolog in the S. aureus genome. The PCR
amplified fragments were digested with SacII and ligated toconstruct a truncated dgkA that lacked 82% of coding region.
The ligated fragment was amplified again by PCR using
primers attB1_DdgkA_Fw and attB2_DdgkA_Rv, and theamplified fragment was reacted with pKOR1 by BP clonase(Thermo Fisher Co.) [25] to construct pKOR-DdgkA.
Media and growth conditions. E. coli strains weregrown in L-broth [26]. Tryptic soy broth (TSB) for
cultivation of S. aureus was a product of Becton Dickinson
Co. L-agar and TSB agar (TSA) were prepared by adding
1.5% of agar to L-broth and TSB, respectively. For theselection of E. coli transformant, antibiotics were added to
50 µg/mL for ampicillin and to 20 µg/mL for
chloramphenicol (Cm). For the selection of S. aureus
transformant, Cm was added to 10 µg/mL.

Fig. 2. Construction of the dgkA deletion mutant. (A) A thickline including a large arrow denotes a dgkA region in thechromosome of S. aureus, and a large arrow denotes the open
reading frame of dgkA. Small arrows denote the primer bindingsites for PCR. A sequence of the cloned fragment was deposited to
GenBank/EMBL/DDBJ, and its accession number is LC365784.
(B) PCR analysis of the chromosomes of the dgkA disruptant andthe parental strain was carried out. DNA fragments of the dgkA
region were amplified from chromosomal preparations of
RN4220DdgkA and RN4220 on PCR. The resulting fragments wereseparated by electrophoresis in a 0.85% agarose gel. Lanes: 1,
RN4220; 2, RN4220DdgkA, 3, molecular weight markers (100 bp
ladder).

Chemicals. [-32P]ATP (specific activity 111
TBq/mmol) was a product of Perkin Elmer Co. RS-[53H(N)]mevalonolactone (specific activity 1.26 TBq/mmol)and RS-[2-14C]mevalonolactone (specific activity 2.00
GBq/mmol) were products of Perkin Elmer Co. C95-dolichol
and C95-polyprenol were provided by Collection of
Polyprenols of the Institute of Biochemistry and Biophysics,
the Polish Academy of Science. UOH, solanesol (all-Enonaprenol), all-E-farnesol, and dipalmitoyl glycerol werepurchased from Sigma Chemical Co. Geraniol waspurchased from Nacalai Tesque Co. Nerolidol and nerol
were purchased from Wako Pure Chemicals Co. The thin-

54

Construction and characterization of the dgkA mutant of S. aureus

layer chromatography (TLC) plates (silica gel 60 F254 and
LKC-18) were products of Merck Co. and Whatman Co.,
respectively.
Expression and purification of hexahistidine- tagged
DgkA homolog. E. coli DH5 cells harboring pCold-dgkA
were grown in L-broth medium deprived of glucose andcontaining ampicillin at 37°C. At the point in which A600
reached 0.5, the culture was chilled and kept at 15°C for 30
min. After addition of isopropyl -D-thiogalactopyranoside(final concentration 0.2 mM), cells were grown for 24 h at
15°C. Cells from 100 mL of medium were collected,
suspended in 2.5 mL of ice-cold 50 mM sodium phosphatebuffer (pH 8.0) containing 300 mM NaCl and 10 mM
imidazole (buffer A), and disrupted by sonication. The
mixture was centrifuged at 21,000×g for 15 min andremoved supernatant. The pellet was suspended in 2 mL ofbuffer A containing 0.3% of Triton X-100, and thesuspension was centrifuged at 21,000×g for 15 min. One
milliliter of the supernatant containing solubilized DgkA
homolog was collected and mixed with 0.25 mL of Ninitrilotriacetic acid agarose (Qiagen Co.). Purification of thehexahistidine-tagged DgkA homolog was carried out per the
manufacturer’s protocol with buffer containing 0.1% of
Triton X-100. The eluted fraction from the Ni-nitrilotriacetic
acid agarose column was dialyzed against 0.1 M Tris-HCl(pH 8.5) containing 0.1% of Triton X-100.
UOH kinase activity. The activities of purified DgkA
homolog were measured as described previously [27] withslight modifications. The reaction mixture for thephosphotransfer contained, in a final volume of 100 µL, 10µmol of MgCl2, 0.3% (w/v) Triton X-100, 10% (w/v)
dimethylsulfoxide, 5 µmol of Tris-HCl buffer (pH 8.5), theindicated amount of a lipid, the indicated amount of [32
P]ATP as described in a legent to Figure 4, and 50 ng of
the purified enzyme. After incubation for 10 min at 25°C, 50µL of 0.1 M EDTA trisodium salt (pH 8.0) was added to stopthe enzyme reaction. The products of the reaction wereextracted with chloroform-methanol or 1-butanol, and theradioactivity in the extract was measured after the extract
was washed with chloroform-methanol-water (3:48:47
v/v/v) or water saturated with 1-butanol, respectively.
Construction of the dgkA deletion mutant of S.
aureus. S. aureus RN4220 was transformed with pKORDdgkA by electroporation [28], and transformants wereselected on TSA containing 10 µg/mL of Cm at 30°C. A
colony on the selection plate was picked up and streaked on
the TSA containing Cm, and the plate was incubated overnight at 43°C (integration process). The resulting colony
was inoculated in TSB and grown over night at 30°C
(resolution process). The culture was diluted 10 5 times with

TSB and inoculated on TSA containing 1 µg/mL ofanhydrotetracycline. The plate was incubated over night at
37°C (counter selection). The resulting colonies were
inoculated in TSB and grown overnight. The genomic DNA
was prepared from grown cells to examine allelicreplacement at the dgkA site. The primers, dgkA_Fw2 anddgkA_Rv2 were used for PCR to amplify the fragment
including dgkA coding region.
Radiolabeling of S. aureus strains. S. aureus RN4220
and RN4220DdgkA were inoculated in 1 mL of TSB, grownfor 1 h at 37°C followed by addition of 37 kBq of RS-[214C]mevalonolactone, and grown for 24 h at 37°C. Cells inthe stationary phase were collected by centrifugation andsuspended in 1 mL of fresh TSB. The suspensions weredivided into two 0.5 mL of aliquots. Then the first aliquots
were centrifuged, and the collected cells were used for the
extraction of radiolabeled products. The remaining aliquots
were added to 10 mL of TSB, grown for 1 h followed by theaddition of 185 kBq of RS-[5-3H(N)]mevalonolactone, andgrown for 2 h at 37°C. Cells grown to the exponential
growth phase were collected, and used for the extraction ofradiolabeled products.
Extraction and analysis of radiolabeled polyprenols
and polyprenyl phosphates.
The radiolabeled cells were suspended in 2 mL of methanol0.3% NaCl (10:1 v/v), and 1 mL of 60% (w/w) KOH wasadded to the mixture and the mixture was heated in boilingwater for 1 h. Polyprenols were extracted twice with 2 mLof hexane and then once with 1 mL of hexane. The hexane
layer was rinsed with 1 mL of methanol-0.3% NaCl (10:1v/v), and 100 µL of hexane layer was used for measuring
radioactivity by liquid scintillation counter, Tri-Carb 2810
TR (Perkin Elmer Co.), using the single label 14C DPM
measurement mode or the double label DPM measurement
mode. The residual solution was dried under N 2, dissolved
with 20 µL of hexane, and analyzed by normal phase andreversed phase TLC. Normal phase TLC was carried out ona silica gel 60 plate with benzene-ethyl acetate (4:1, v/v).
Reversed-phase TLC was carried out on an LKC-18 platewith acetone-water (19:1 v/v). Radioactivity on the plate
was detected by FLA-7000 (Fuji Film Co.). For
radioactivity quantification, the area containing eachproduct was scraped off of the plate, and radioactive
compounds were dissolved in methanol for measurement byliquid scintillation counter. Polyprenyl phosphates wereextracted twice with 2 mL of diethyl ether and once with 3
mL of diethyl ether from the residual suspension in alkaline
methanol-water layer after the extraction with hexane. The
diethyl ether extract was washed with 5% acetic acid anddried under N2. The residue was dissolved with chloroformmethanol (2:1 v/v) and 100 µL of solution was used forKawakami et al.

measuring radioactivity. Residual polyprenyl phosphates
were treated with acid phosphatase as described previously[29]. The resulting polyprenols were extracted with hexane
and analyzed as described above.
Results55

phosphorylation of solanesol and all-E-farnesol were both
6% of that of undecaprenol. Phosphorylation of nerolidol,
geraniol, nerol, and dipalmitoyl glycerol was not detected.
To examine whether the various nucleotide triphosphates
were substrates for the DgkA homolog, competition assay
was carried out (Fig. 4B). Only cold ATP competed with theincorporation of radioactivity into lipid fraction from

Properties of hexahistidine-tagged DgkA homolog of S.
aureusE. coli cells expressing DgkA homolog were disrupted
by sonication in the buffer without a detergent at first. SDSpolyacrylamide gel electrophoresis (PAGE) analysis
revealed a specific 15-kDa band in both supernatant andpellet fraction in centrifugation. Hexahistidine-tagged DgkA
homolog could be solubilized and purified by Ninitrilotriacetic acid agarose chromatography using the buffer
containing Triton X-100 (Fig. 3). The analysis of fractions
eluted with the buffer containing 250 mM of imidazole in
SDS-PAGE revealed a major 15-kDa band and a minor 20kDa band. The second and third fractions

Fig. 3. SDS-polyacrileamide gel electrophoresis of DgkA
homolog. Fractions in the purification of the hexahistidine-tagged
DgkA homolog were analyzed on 12.5% acrylamide gel. Lane M,
moleculare weight marker: mixture of phosphorylase b (97,200),
bovine-serum albumin (66,400), ovalbumin (45,000), carbonic
anhydrase (29,000), trypsin inhibitor (20,100), and lysozyme(14,300). The solubilized fraction was applied to a Ni-nitriloacetic
acid agarose column as described in Materials and Methods. Lane1, flow through; lanes 2–4, fractions of 2.5 mL each eluted with 50
mM sodium phosphate buffer (pH 8.0) containing 300 mM NaCl,
0.1% (w/v) Triton X-100, and 20 mM imidazole; lanes 5–10,
fractions of 0.25 mL each eluted with 50 mM sodium phosphatebuffer (pH 8.0) containing 300 mM NaCl, 0.1% (w/v) Triton X100, and 250 mM imidazole.

of this buffer (lanes 6 and 7 in Fig. 3) were combined andused for characterization of lipid kinase activity.
Approximately 42 µg of protein was obtained in thesefractions. The most effective lipid substrate was Z,E-mixed
UOH (Fig. 4A). The activities of phosphorylation of Z,Emixed C95-polyprenol and C95-dolichol were 45% and 12%of that of UOH, respectively. The activities ofFig. 4. Substrate specificity of the DgkA homolog. (A) Enzyme
reactions with 150 µM ATP and various alcohols were carried out,
and radioactivities in reaction products extracted with 1-butanol
were measured. UOH, undecaprenol; 95PLE, C95-polyprenol;
95DOL, C95-dolicohol; SOH, solanesol (all-E-nonaprenol); FOH,
all-E-farnesol; NeOH, nerol; GOH, geraniol; NdOH, nerolidol; DG,
dipalmitoyl glycerol. C95-polyprenol and C95-dolicohol were added
to 75 µM, and the other alcohols were added to 150 µM. (B)Enzyme reactions with 150 µM UOH, 30 µM [-32P]ATP , andvarious nucleoside triphosphate were carried out, andradioactivities in a reaction product extracted with chloroformmethanol were measured. basic, without additional nucleoside
triphosphate; +ATP, 300 µM of ATP was added; +UTP,
300 µM of UTP was added; +GTP, 300 µM of GTP was added;+CTP, 300 µM of CTP was added.

[-32P]ATP, and the other nucleotides UTP, GTP, and CTP
did not affect the incorporation of radioactivity. Thus the
enzyme was shown to be specific to ATP. The Km value for
UOH that was obtained while holding the concentration of
ATP constant (150 µM) was 41 µM. The Km value for ATP
that was obtained while holding the concentration of UOH
constant (150 µM) was 56 µM.

56Construction and characterization of the dgkA mutant of S. aureus

Construction of dgkA disruptant
After the integration and resolution process, counterselection was carried out as described in Materials andMethods. Four colonies were selected to examine whether
the disrupted dgkA homolog (DdgkA) was introduced into achromosome. PCR analysis showed that three colonies gave
a 0.1-kb band resulting from DdgkA, and that one colony
gave a 0.4-kb band resulting from wild type dgkA (Fig. 2B).
Thus, we were able to obtain gene disruptant RN4220DdgkA.
The growth rate of the disruptant was same as that of theparental strain. The doubling times in TSB at 37°C of bothstrains were 30 min.

to be UOH, as it showed lower Rf value (0.32) than that ofsolanesol in reversed phase TLC (Fig. 5D). A spot thatshowed highest Rf value (0.67) was considered to bemenaquinone-8. Diethylether extract treated with acidphosphatase also contained octaprenol and UOH (Fig. 5B),
showing that diethyether extract contained octaprenyl
phosphate and UP.
Radioactivities in UOH and UP are shown in Figure 6.

Quantification of UOH and UP derivatives in S. aureus
strain by tracer experiment
To investigate UP metabolism in RN4220 and
RN4220Ddgk, a tracer experiment was carried out using
radiolabeled precursor for isoprenoids. 14C-labeled
compounds extracted from cells in the stationary phase wereanalyzed by TLC (Fig. 5). Analysis of hexane extract

Fig. 5. Autoradiograms of radiolabeled products from S.
aureus cells grown in the presence of 14C-labeled
mevalonolactone. Radiolabeling of cells of S. aureus and analysisof radioactive products were carried out as described in Materials
and Methods. Ori., Origin; SF, solvent front; C45, solanesol. (A)The hexane extract from the stationary cells of RN4220 was
analyzed by normal phase TLC. (B) The diethylether extract fromthe stationary cells of RN4220 was treated with acid phosphatase,and resulting products were analyzed by normal phase TLC. (C)The compound in the spot of Rf= 0.44 in the normal phase TLC (A)was eluted from the silica gel and analyzed by reversed phase TLC.
(D) The compound in the spot of Rf= 0.54 in the normal phase TLC(A) was eluted from the silica gel and analyzed by reversed phaseTLC.

in normal phase TLC revealed three radioactive spots (Fig.
5A). A spot that had a slightly lower Rf value (0.44) than thatof solanesol (Rf =0.45) was considered to be octaprenol, as
it had a higer Rf value (0.45) than that of solanesol (Rf =0.38)
in reversed phase TLC (Fig. 5C). A spot that had a modestly
higher Rf value (0.54) than that of solanesol was considered

Fig. 6. Radioactivity of undecaprenol and undecaprenyl
phosphate in the cells of S. aureus RN4220 and RN4220DdgkA
grown in the presence of 14C-labeled mevalonolactone and/or
3H-labeled mevalonolactone. Radiolabeling of S. aureus RN4220(A) and RN4220DdgkA (B) and analysis of radioactive products
were carried out as described in Materials and Methods. Bar graphs
show averages of three assays. Error bars show standard deviations.
White bar, 14C radioactivity in the stationary phase cells; black bar,
14C radioactivity in the recultivated cells; hatched bar, 3H
radioactivity in the recultivated cells.

Radioactivities of 14C in the compound extracted from cells
in the stationary phase are considered to reflect the levels ofthese compounds in the cells in the stationary phase, andradioactivities of 3H in the compound extracted from
regrown cells are considered to reflect the levels of thesecompounds in the cells in the exponential growth phase.
The relative UOH level to the sum of UOH level andKawakami et al.
UP level in the stationary phase calculated from 14C
radioactivities was 85.3%±2.2% in the dgkA deletion mutant
and was significantly higher (p<0.01) than that in theparental strain (56.4% ±3.7%). The relative UOH level inthe exponential growth phase calculated from 3H
radioactivities was 42.2%±5.3% in the dgkA deletion mutant
and was also significantly higher (p<0.01) than that in theparental strain (13.8%±6.6%). Radioactivities of 14C in thecompounds extracted from regrown cells are considered toreflect the result of conversion of radioactive compounds
that had accumulated in the stationary phase during
recultivation. The relative UOH level significantly
decreased (p<0.01) during recultivation of cells that hadreached the stationary phase (from 56.4% ±3.7% to26.1%±7.5%) in the parental strain, indicating that theconversion of UOH to UP occurred. The relative UOH levelin the deletion mutant also decreased (p<0.01) during the
regrowth of cells (from 85.3%±2.2% to 54.8%±9.7%),
indicating that the conversion of UOH to UP occurred also
in the deletion mutant.
Discussion
In the early study of cell wall biosynthesis, UOH
kinase was purified from S. aureus. It is an extremelyhydrophobic protein with a molecular mass of 17 kDa in
SDS-PAGE that catalyzes the phosphorylation of UOH with
ATP in the presence of magnesium ion [27]. The DgkA
homolog purified in this study showed similar properties tothat of previous purified UOH kinase. DgkA homolog
located mainly in precipitate fraction was solubilized with
Triton X-100 and gave a 15-kDa band in SDS-PAGE. Km
values of DgkA homolog (56 µM for ATP and 41 µM for
UOH) were almost the same as that of previous purified
UOH kinase (57 µM for ATP and 57 µM for UOH). Thus,
dgkA of S. aureus was confirmed to be the gene for UOH
kinase as that of B. subtilis[19] and S. mutans[20].
We constructed a dgkA deletion mutant using aplasmid for allelic exchange. A tracer experiment showedthat the relative UOH levels in the mutant were higher bothin the exponential growth phase and in the stationary phasethan that in the parental strain. These results indicate that the
DgkA homolog has a role in maintaining the cellular UP
level by phosphorylation of UOH both in the exponential
growth phase and in the stationary phase. Recently, Radeck
et al. constructed a dgkA deletion mutant of B. subtilis [30].
The upregulation of the expression of UPP phosphatase genein the deletion mutant was observed, though the mutant did
not show remarkable growth or morphological phenotype.
This result suggests that lack of UOH kinase causes modest
shortage of UP also in the cell of B. subtilis as well.
Relative UOH levels in the stationary phase were57

higher than in the exponential growth phase, both in thedeletion mutant and in the parental strain. By therecultivation of the 14C-labeled stationary phase cells,
metabolism of accumulated 14C-labeled lipids was examined.
Surprisingly conversion of UOH to UP was observed in the
dgkA deletion mutant as well as in the wild type parental
strain. Although DgkA homologue is UOH kinase thatmaintain cellular UP level, there should be another enzyme
to phosphorylate UOH in S. aureus. The possible candidateof phosphate donor is not only ATP but also the othercompounds such as phospholipids.
Because all known UPP phosphatases examined formembrane topology have substrate binding sites at the outerside of the membrane [10–12], there should be a flippase totranslocate UPP from the inner side of the membrane to theouter side or a novel UPP phosphatase that act at the innerside of the membrane for the de novo UP synthesis (Fig. 1).
However both of them have not been found yet. Sincetransbilayer movement of UOH is much faster than that of
UP and UPP, UOH can move across the membrane without
any flippase. If the transfer of phosphate from UPP to UOH
occurred at the cytoplasmic side of the membrane, it would
provide a means of de novo synthesis of UP from UPP. The
enzyme that catalyzes phosphotransfer to UOH in the dgkA
mutant should be investigated in future studies.
Acknowledgements
We thank Dr. Longzhu Cui (Jichi Medical University) for
generously providing strain RN4220 and plasmid pKOR1.
References[1] Bouhss A, Trunkfield AE, Bugg TD, et al. The
biosynthesis of peptidoglycan lipid-linked intermediates.
FEMS Microbiol Rev. 2008;32:208–233.
[2] Manat G, Roure S, Auger R, et al. Deciphering themetabolism of undecaprenyl-phosphate: the bacterial cellwall unit carrier at the membrane frontier. Microb Drug
Resit. 2014;20:199–214.
[3] van Heijenoort Y, Gomez M, Derrien M, et al.
Membrane intermediate in the peptidoglycan metabolism ofEscherichia coli: possible roles of PBP 1b and PBP 3. J
Bacteriol. 1992;174:3549–3557.
[4] Hartley MD, Imperiali B. At the membrane frontier: aprospectus on the remarkable evolutionary conservation ofpolyprenols and polyprenyl-phosphates. Arch Bichem
Biophys. 2012;517:83–97.
[5] Coker OO, Palittapongarnpim P. Current understandingof de novo synthesis of bacterial lipid carrier (undecaprenyl
phosphate): more enzymes to be discovered. Afr J Microbiol
Res. 2011;5:2555–2565.
[6] Kawakami N, Fujisaki S. Undecaprenyl phosphatemetabolism in Gram-negative and Gram-positive bacteria.
Biosci
Biotechnol
Biochem.
2017;
doi.org/10.1089/09168451.2017.1401915[7] Shimizu N, Koyama T, Ogura K. Molecular cloning,
expression, and purification of undecaprenyl diphosphatesynthase. No sequence similarity between E- and Z-prenyl
diphosphate synthases. J Biol Chem. 1998 273:19476–

58

Construction and characterization of the dgkA mutant of S. aureus

19481.
[8] Apfel CM, Takacs B, Fountoulakis M, et al. Use of
genomics to identify bacterial undecaprenyl pyrophosphatesynthetase: Cloning, expression, and characterization of theessential uppS gene. J. Bacteriol. 1999;181: 483–492.
[9] Kato J, Fujisaki S, Nakajima K, et al. The Escherichia
coli homologue of yeast Rer2, a key enzyme of dolichol
synthesis, is essential for the carrier lipid formation inbacterial cell wall synthesis. J. Bacteriol. 1999;181:2733–
2738.
[10] Manat G, El Ghachi M, Auger R, et al. Membrane
topology and biochemical characterization of the
Escherichia coli BacA undecaprenyl-pyrophosphate
phosphatase. PLoS One. 2015;10:e0142870.
[11] Tatar LD, Marolda CL, Polischuk AN, et al. An
Escherichia coli undecaprenyl- pyrophosphate phosphatase
implicated in undecaprenyl phosphate recycling.
Microbiology. 2007;153:2518–2529.
[12] Touzé T, Blanot D, Mengin-Lecreulx D. Substratespecificity and membrane topology of Escherichia coli
PgpB, an undecaprenyl pyrophosphate phosphatase. J Biol
Chem. 2008; 283:16573–16583.
[13] Sham LT, Butler EK, Lebar MD, et al. Bacterial cellwall. MurJ is the flippase of lipid-linked precursors for
peptidoglycan biogenesis. Science. 2014;345:220–222.
[14]
HigashiY, Strominger
JL, Sweeley
CC.
Biosynthesis of the peptidoglycan of bacterial cell walls.
XXI. Isolation of free C55-isoprenoid alcohol of lipid in
peptidoglycan synthesis from Staphylococcus aureus. J Biol
Chem. 1970;245:3697–3702.
[15] Gough DP, Kirby AL, Richards JB, et al. The
characterization of undecaprenol of Lactobacillus
plantarum. Biochem J. 1970;118:167–170.
[16] Thorne KJ. Identification of prenol intermediates ofwall biosynthesis in growing cells of Lactobacillus
plantarum. J Bacteriol. 1973;116:235–244.
[17] Fujisaki S, Nishino T, Katsuki H. Biosynthesis of
isoprenoids in intact cells of Escherichia coli. J. Biochem.
1986;99:1137–1146.
[18] Barreteau H, Magnet S, El Ghachi M, et al. Quantitative
high-performance liquid chromatography analysis of thepool levels of undecaprenyl phosphate and its derivatives inbacterial membranes. J Chromatogr B Analyt Technol
Biomed Life Sci. 2009;877:213–220.
[19] Jerga A, Lu YJ, Schujman GE, et al. Identification of asoluble diacylglycerol kinase required for lipoteichoic acidproduction in Bacillus subtilis. J Biol Chem.
2007;282:21738–21745.
[20] Lis M, Kuramitsu HK. The Stress-responsive dgk genefrom Streptococcus mutans encodes a putative undecaprenol
kinase activity. Infect Immun. 2003;71:1938–1943.
[21] Van Horn WD, Sanders CR. Prokaryotic
Diacylglycerol kinase and undecaprenol kinase. Annu Rev
Biophys. 2012;41:81–101.
[22] Matsumoto Y, Yasukawa J, Ishii M, et al. A critical roleof mevalonate for peptidoglycan synthesis in
Staphylococcus aureus. Sci Rep. 2016;6:22894.
doi:10.1038/srep22894.
[23] Kreiswirth BN, Lofdahl S, Betly MJ, et al. The toxic
shock syndrome exotoxin structural gene is not detectablytransmitted by a prophage. Nature. 1983;305:709–712.
[24] Bae T, Schneewind O. Allelic replacement in
Staphylococcus aureus with inducible counter-selection.

Plasmid. 2006;55:58–63.
[25] Hartley JL, Temple GF, Brash MA. DNA cloning using
in vitro site-specific recombinations. Genome Res.
2000;10:1788–1795.
[26] Lennox ES. Transduction of linked genetic charactersof the host by bacteriophage P1. Virology. 1955;1:190–206.
[27] Sandermann H Jr, Strominger JL. Purification andproperties of C55-isoprenoid alcohol phosphokinase from
Staphylococcus aureus. J Biol Chem. 1972;247:5123–5131.
[28] Monk IR, Shah IM, Xu M, et al. Trnsforming theuntransformable: application of direct transformation to
manipulate genetically Staphylococcus aureus andStaphylococcus epidermis. mBio. 2012;3:e00277-11.
doi:10.1128/mBio.00277-11.
[29] Fujii H, Koyama T, Ogura K. Efficient enzymatichydrolysis of polyprenyl pyrophosphate. Biochim Biophys
Acta. 1982;712:716–718.
[30] Radeck J, Lautenschläger N, Mascher T. The essential
UPP phosphatase pair BcrC and UppP connects cell wall
homeostasis during growth and sporulation with cellenvelope stress response in Bacillus subtilis. Front
Microbiol. 2017;8:2403. doi:10.3389/fmib.2017.02403[31] Bugg TD, Lloyd AJ, Roper DI. Phospho-MurNAcpnetapeptide translocase (MraY) as a target for antibacterial
agents and antibacterial proteins. Infect Disord Drug Target.
2006;6:85–106.
[32] Mengin-Lecreulx D, Texier L, Rousseau M, et al. The
murG gene of Escherichia coli codes for the UDP-Nacetylmuramyl-(pentapeptide)pyrophosphorylundecaprenol N-acetylglucosamine transferase involved in
the membrane steps of peptidoglycan synthesis. J Bacteriol.
1991;173:4625–4636.
[33] Suzuki H, van Heijenoort Y, Tamura T, et al. In vitro
peptidoglycan polymerization catalyzed by penicillin
binding protein 1b of Escherichia coli K-12. FEBS Lett.
1980; 110:245-249.

Communiated by Ishijima Sumio