JOURNAL OF BACTERIOLOGY, July 2011, p. 3304–3312
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 13
Pantethine Rescues Phosphopantothenoylcysteine Synthetase and
Phosphopantothenoylcysteine Decarboxylase Deficiency in
Escherichia coli but Not in Pseudomonas aeruginosa?†
Carl J. Balibar,* Micah F. Hollis-Symynkywicz, and Jianshi Tao
Department of Infectious Diseases, Novartis Institutes for BioMedical Research, 500 Technology Square,
Cambridge, Massachusetts 02139
Received 9 March 2011/Accepted 26 April 2011
Coenzyme A (CoA) plays a central and essential role in all living organisms. The pathway leading to CoA
biosynthesis has been considered an attractive target for developing new antimicrobial agents with novel
mechanisms of action. By using an arabinose-regulated expression system, the essentiality of coaBC, a single
gene encoding a bifunctional protein catalyzing two consecutive steps in the CoA pathway converting 4?-
phosphopantothenate to 4?-phosphopantetheine, was confirmed in Escherichia coli. Utilizing this regulated
coaBC strain, it was further demonstrated that E. coli can effectively metabolize pantethine to bypass the
requirement for coaBC. Interestingly, pantethine cannot be used by Pseudomonas aeruginosa to obviate coaBC.
Through reciprocal complementation studies in combination with biochemical characterization, it was dem-
onstrated that the differential characteristics of pantethine utilization in these two microorganisms are due to
the different substrate specificities associated with endogenous pantothenate kinase, the first enzyme in the
CoA biosynthetic pathway encoded by coaA in E. coli and coaX in P. aeruginosa.
Coenzyme A (CoA) is a ubiquitous and essential cofactor in
all living organisms, where it functions as a carrier for activated
acyl groups in numerous central metabolic processes. Perhaps
most notably, CoA provides the indispensable phospho-
pantetheine prosthetic group posttranslationally appended to
acyl carrier proteins upon which fatty acids are biosynthesized;
however, CoA participates in many other biochemical pro-
cesses from the tricarboxylic acid (TCA) cycle to amino acid
degradation (30). The five-step biochemical pathway for con-
version of vitamin B5 pantothenate to CoA is conserved across
all taxa and begins with formation of 4?-phosphopantothenate,
catalyzed by pantothenate kinase (PanK). 4?-Phosphopanto-
thenate is subsequently condensed with cysteine, which is in
turn decarboxylated to form 4?-phosphopantetheine by the en-
zymes phosphopantothenoylcysteine synthetase (PPCS) and
phosphopantothenoylcysteine decarboxylase (PPCDC), re-
spectively. Finally, 4?-phosphopantetheine is converted to de-
phospho-CoA by the addition of an AMP moiety catalyzed by
4?-phosphopantetheine adenylyltransferase (PPAT) and then
the 3?-hydroxyl of the AMP ribose is phosphorylated by de-
phospho-CoA kinase (DPCK) to form CoA (8) (Fig. 1).
In bacteria, CoA plays a vital role in biogenesis of the cell
envelope. Membrane lipid biogenesis utilizes fatty acid precur-
sors that are biosynthesized using the iterative condensation of
acetyl-CoA precursors (20). The peptidoglycan, an essential
bacterial structure that maintains the mechanical integrity of
the cell and participates in several key processes, including cell
division and virulence (9), is biosynthesized from the primary
precursor UDP-N-acetylglucosamine. GlmU, the final enzyme
required to convert D-fructose-6-phosphate to UDP-N-acetyl-
glucosamine, utilizes acetyl-CoA for the acetylation of gluco-
samine-1-phosphate (26). Furthermore, UDP-N-acetylgluco-
samine is a precursor to teichoic acid(s) in Gram-positive
bacteria (19) and lipid A in Gram-negative bacteria (4), both of
which are surface-exposed molecules that impact the permea-
bility of the bacterial cell. Finally, the majority of Gram-posi-
tive pathogens, including Staphylococcus aureus, Enterococcus
faecalis, and Streptococcus pneumoniae, utilize the mevalonate
pathway for isoprenoid biosynthesis. In the mevalonate path-
way, 3 units of acetyl-CoA are condensed to form isopentenyl
pyrophosphate (IPP), the building block for the cell wall car-
rier lipid undecaprenyl pyrophosphate (UPP) (7).
Despite its significance in bacteria, the complete biosyn-
thetic machinery for synthesis of CoA has been elucidated only
relatively recently, culminating in identification of the Esche-
richia coli coaBC gene, which encodes a single bifunctional
PPCS/PPCDC protein (43). The existence of coaBC as a single
gene encoding one protein with 2 functional domains in bac-
teria is in stark contrast to the pattern seen with eukaryotes
such as plants and mammals, where PPCS and PPCDC are
individual enzymes encoded by separate coaB and coaC genes,
respectively (1–3, 13, 28). Given the paucity of information
concerning coaBC in bacteria and its potential as a target for
antimicrobial intervention, we sought to investigate the effect
on E. coli of disruption of coaBC. Interestingly, despite the
validated essentiality of coaBC (13, 15) and reports that pan-
tothenate is the most advanced precursor to CoA that has been
characterized as being actively transported into bacteria (8,
30), we find that chemical complementation with pantethine
* Corresponding author. Mailing address: Department of Infectious
Diseases, Novartis Institutes for BioMedical Research, 500 Technol-
ogy Square, Cambridge, MA 02139. Phone: (617) 871-5745. Fax: (617)
871-5791. E-mail: email@example.com.
† Supplemental material for this article may be found at http://jb
?Published ahead of print on 6 May 2011.
renders coaBC nonessential in E. coli but not in Pseudomonas
aeruginosa. This bypass mechanism is dependent on the pres-
ence of pantothenate kinase.
MATERIALS AND METHODS
General materials and procedures. Standard DNA, molecular cloning, and
microbiological procedures were performed as described previously (39). Plas-
mids, genomic DNA, PCR fragments run on agarose gels, and restriction digests
were purified using a QIAprep spin miniprep kit, DNeasy blood and tissue kit,
QIAquick gel extraction kit, and QIAquick PCR purification kit, respectively
(Qiagen). Primers and ultramers were purchased from Integrated DNA Tech-
nologies. Phusion high-fidelity DNA polymerase, Quick ligase, and restriction
enzymes were from NEB. Electroporation was performed on a MicroPulser
Electroporator (Bio-Rad). One Shot TOP10 and PIR1 E. coli were from Invi-
trogen. DNA sequencing was performed by Beckman Coulter Genomics. LB, LB
agar, and Pseudomonas isolation agar (PIA) were from Difco. NZYM media and
sucrose were from MP Biomedicals. ATP, NADH, kanamycin, chloramphenicol,
gentamicin, ampicillin, pantethine, CoA, dephospho-CoA, L-arabinose, lactate
dehydrogenase, pyruvate kinase, and phosphoenolpyruvate (PEP) were from
Sigma. Pantothenate, IPTG (isopropyl-?-D-thiogalactopyranoside), and imid-
azole were from Acros Organics. Carbenicillin was from Fisher Scientific, dithio-
threitol (DTT) was from Promega, TCEP [tris(2-carboxyethyl)phosphine] was
from Thermo Scientific, Complete protease inhibitor cocktail was from Roche,
and cobalt Talon metal affinity resin was from Clontech. Fast protein liquid
chromatography (FPLC) purification of proteins was performed using a HiLoad
16/60 Superdex 200 prep grade column, an A ¨KTA FPLC system, and Unicorn 5.0
software (GE Healthcare).
Construction of coaBC- and coaD-regulated E. coli strains. A new vector was
created that allowed the one-step ? Red recombinase knockout method devel-
oped by Datsenko and Wanner (12) to be adapted to perform one-step incor-
poration of the PBADpromoter in front of any gene. The araC-PBADregion from
the pBAD18 plasmid (17) was amplified by PCR using primers P1 and P2 (all
primers and ultramers are listed in Table S1 in the supplemental material), and
the majority of pKD4 (12) was amplified by PCR using primers P3 and P4. The
PCR fragments were digested with ClaI/XhoI, ligated, and transformed into
PIR1 chemically competent cells. The resulting plasmid, pKD4-PBAD, was used
as a template in subsequent PBADpromoter integrations.
The PBADpromoter was integrated 15 bp upstream of the coaBC start
codon by the use of the linear PCR product generated by amplification from a
template with the P5 and P6 ultramers, generating the
BW25113PBADcoaBC strain (all strains are listed in Table S2 in the supplemen-
tal material). The PBADpromoter was integrated 25 bp upstream of the coaD
start codon by the use of the linear PCR product generated by amplification from
template with the P7 and P8 ultramers, generating the
BW25113PBADcoaD strain. Transformation and selection were performed as
described previously (6). Briefly, overnight cultures of BW25113 cells harboring
pKD46 (6) were diluted 100-fold into fresh LB containing 100 ?g/ml ampicillin
and 0.2% (wt/vol) arabinose, grown at 30°C until an optical density at 600 nm
(OD600) of 0.5 was reached, washed twice with an equal volume of cold water,
washed three times with 1 ml 10% glycerol, and finally resuspended in 10%
glycerol using 1/250 of the initial culture volume. Competent cells (50 ?l) were
mixed with 100 to 300 ng (not exceeding 5 ?l) of gel-purified PCR product to be
inserted into the chromosome, electroporated in a 0.2-cm-gap cuvette using an
EC2 setting (2.5 kV, 5 ms), recovered in 1 ml SOC medium (Invitrogen) con-
taining 0.2% (wt/vol) arabinose at 37°C for 2 h, and finally plated on LB agar
containing 30 ?g/ml kanamycin and 1 mM arabinose. Colonies that grew the next
day were replica plated to confirm loss of pKD46 and acquisition of arabinose-
dependent growth. All integrants were confirmed by PCR and sequencing.
Deletion of panF and replacement of coaA from E. coli with coaX from P.
aeruginosa in BW25113PBADcoaBC. The panF gene was deleted, leaving only the
first and last six codons, by using the linear PCR product generated by amplifi-
cation from pKD3 (12) with the P9 and P10 ultramers, generating strain
BW25113PBADcoaBC?panF::cat. For replacement of the coaA gene from E. coli
(EccoaA), splicing by overlap extension PCR (21) was used to fuse a chloram-
phenicol resistance cassette downstream of the coaX gene from P. aeruginosa
(PacoaX) to facilitate selection. In the first round of PCR, ultramer P11 and
primer P12 were used to amplify PacoaX from P. aeruginosa genomic DNA, and
primer P13 and ultramer P14 were used to amplify the cat gene from pKD3. In
the second round of PCR, products from the first round were mixed and further
amplified using P11 and P14. This second-round complete PCR product was
used to replace the EccoaA gene from the start to the stop codon, generating
FIG. 1. The CoA biosynthetic pathway.
VOL. 193, 2011 PANTETHINE BYPASS OF coaBC3305
25. Jackowski, S., and C. O. Rock. 1984. Metabolism of 4?-phosphopantetheine
in Escherichia coli. J. Bacteriol. 158:115–120.
26. Kotnik, M., P. S. Anderluh, and A. Prezelj. 2007. Development of novel
inhibitors targeting intracellular steps of peptidoglycan biosynthesis. Curr.
Pharm. Des. 13:2283–2309.
27. Kropinski, A. M., J. Kuzio, B. L. Angus, and R. E. Hancock. 1982. Chemical
and chromatographic analysis of lipopolysaccharide from an antibiotic-su-
persusceptible mutant of Pseudomonas aeruginosa. Antimicrob. Agents Che-
28. Kupke, T., P. Hernandez-Acosta, and F. A. Culianez-Macia. 2003. 4?-Phos-
phopantetheine and coenzyme A biosynthesis in plants. J. Biol. Chem. 278:
29. Leonardi, R., et al. 2005. A pantothenate kinase from Staphylococcus aureus
refractory to feedback regulation by coenzyme A. J. Biol. Chem. 280:3314–
30. Leonardi, R., Y. M. Zhang, C. O. Rock, and S. Jackowski. 2005. Coenzyme
A: back in action. Prog. Lipid Res. 44:125–153.
31. Levintow, L., and G. Novelli. 1954. The synthesis of coenzyme A from
pantetheine: preparation and properties of pantetheine kinase. J. Biol.
32. Meier, J. L., A. C. Mercer, H. J. Rivera, and M. D. Burkart. 2006. Synthesis
and evaluation of bioorthogonal pantetheine analogues for in vivo protein
modification. J. Am. Chem. Soc. 128:12174–12184.
33. Mendelson, M. H., et al. 1994. Pseudomonas aeruginosa bacteremia in pa-
tients with AIDS. Clin. Infect. Dis. 18:886–895.
34. Mercer, A. C., J. L. Meier, J. W. Torpey, and M. D. Burkart. 2009. In vivo
modification of native carrier protein domains. Chembiochem 10:1091–1100.
35. Poole, K. 2005. Efflux-mediated antimicrobial resistance. J. Antimicrob. Che-
36. Poole, K. 2004. Efflux-mediated multiresistance in Gram-negative bacteria.
Clin. Microbiol. Infect. 10:12–26.
37. Rana, A., et al. 2010. Pantethine rescues a Drosophila model for pantothe-
nate kinase-associated neurodegeneration. Proc. Natl. Acad. Sci. U. S. A.
38. Rubio, S., et al. 2006. An Arabidopsis mutant impaired in coenzyme A
biosynthesis is sugar dependent for seedling establishment. Plant Physiol.
39. Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratory
manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Har-
40. Schweizer, H. D. 1993. Small broad-host-range gentamycin resistance gene
cassettes for site-specific insertion and deletion mutagenesis. Biotechniques
41. Simon, R., U. Priefer, and A. Puehler. 1983. A broad host range mobilization
system for in vivo genetic engineering: transposon mutagenesis in Gram-
negative bacteria. Nat. Biotechnol. 1:784–791.
42. Strauss, E., and T. P. Begley. 2002. The antibiotic activity of N-pentylpan-
tothenamide results from its conversion to ethyldethia-coenzyme A, a coen-
zyme A antimetabolite. J. Biol. Chem. 277:48205–48209.
43. Strauss, E., C. Kinsland, Y. Ge, F. W. McLafferty, and T. P. Begley. 2001.
Phosphopantothenoylcysteine synthetase from Escherichia coli. Identifica-
tion and characterization of the last unidentified coenzyme A biosynthetic
enzyme in bacteria. J. Biol. Chem. 276:13513–13516.
44. Thomas, J., and J. E. Cronan. 2010. Antibacterial activity of N-pentylpan-
tothenamide is due to inhibition of coenzyme A synthesis. Antimicrob.
Agents Chemother. 54:1374–1377.
45. Vallari, D. S., and C. O. Rock. 1985. Isolation and characterization of
Escherichia coli pantothenate permease (panF) mutants. J. Bacteriol. 164:
46. Vallari, D. S., and C. O. Rock. 1987. Isolation and characterization of
temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia
coli. J. Bacteriol. 169:5795–5800.
47. Vallari, D. S., and C. O. Rock. 1985. Pantothenate transport in Escherichia
coli. J. Bacteriol. 162:1156–1161.
48. Wu, Z., C. Li, S. Lv, and B. Zhou. 2009. Pantothenate kinase-associated
neurodegeneration: insights from a Drosophila model. Hum. Mol. Genet.
49. Zhang, Y. M., et al. 2004. Acyl carrier protein is a cellular target for the
antibacterial action of the pantothenamide class of pantothenate anti-
metabolites. J. Biol. Chem. 279:50969–50975.
50. Zhou, B., et al. 2001. A novel pantothenate kinase gene (PANK2) is defec-
tive in Hallervorden-Spatz syndrome. Nat. Genet. 28:345–349.
51. Zimmermann, W. 1979. Penetration through the gram-negative cell wall: a
co-determinant of the efficacy of beta-lactam antibiotics. Int. J. Clin. Phar-
macol. Biopharm. 17:131–134.
3312 BALIBAR ET AL.J. BACTERIOL.