The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

Abstract

The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

Full text

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The MurC Ligase Essential for Peptidoglycan Biosynthesis Is
Regulated by the Serine/Threonine Protein Kinase PknA in
Corynebacteriumglutamicum*
Receivedforpublication,September16,2008,andinrevisedform,October28,2008 Published,JBCPapersinPress,October29,2008,DOI10.1074/jbc.M807175200
Maria Fiuza‡1, Marc J. Canova§, Delphine Patin¶, Michal Letek‡, Isabelle Zanella-Cle ´on§, Michel Becchi§,
Luís M. Mateos‡, Dominique Mengin-Lecreulx¶?, Virginie Molle§2, and Jose ´ A. Gil‡3
Fromthe‡DepartamentodeBiologíaMolecular,A´readeMicrobiología,FacultaddeBiología,UniversidaddeLeo ´n,Leo ´n24071,Spain,
the§InstitutdeBiologieetChimiedesProte ´ines,UMR5086,CNRS,Universite ´Lyon1,IFR128BioSciences,Lyon-Gerland,7passagedu
Vercors,Lyon69367,Cedex07,France,the¶UniversityParis-Sud,InstitutdeBiochimieetBiophysiqueMole ´culaireetCellulaire,UMR8619,
OrsayF-91405,France,and?CNRS,LaboratoiredesEnveloppesBacte ´riennesetAntibiotiques,UMR8619,OrsayF-91405,France
The Mur ligases play an essential role in the biosynthesis of
bacterial cell-wall peptidoglycan and thus represent attractive
targets for the design of novel antibacterials. These enzymes
catalyze the stepwise formation of the peptide moiety of the
peptidoglycan disaccharide peptide monomer unit. MurC is
responsible of the addition of the first residue (L-alanine) onto
the nucleotide precursor UDP-MurNAc. Phosphorylation of
proteins by Ser/Thr protein kinases has recently emerged as a
major physiological mechanism of regulation in prokaryotes.
Herein,thehypothesisofaphosphorylation-dependentmecha-
nism of regulation of the MurC activity was investigated in
Corynebacterium glutamicum. We showed that MurC was
phosphorylatedinvitrobythePknAproteinkinase.Ananalysis
of the phosphoamino acid content indicated that phosphoryla-
tionexclusivelyoccurredonthreonineresidues.Sixphosphoac-
ceptor residues were identified by mass spectrometry analysis,
and we confirmed that mutagenesis to alanine residues totally
abolished PknA-dependent phosphorylation of MurC. In vitro
and in vivo ligase activity assays showed that the catalytic activ-
ityofMurCwasimpairedfollowingmutationofthesethreonine
residues. Further in vitro assays revealed that the activity of the
MurC-phosphorylated isoform was severely decreased com-
pared with the non-phosphorylated protein. To our knowledge,
thisisthefirstdemonstrationofaMurCligasephosphorylation
in vitro. The finding that phosphorylation is correlated with a
decreaseinMurCenzymaticactivitycouldhavesignificantcon-
sequences in the regulation of peptidoglycan biosynthesis.
Due to the increasing number of antibiotic-resistant strains
and the emergence of new pathogenic microorganisms, one of
the biggest challenges for modern biomedical research is the
continuous development of new antimicrobial drugs targeting
bacterial essential mechanisms such as cell division or pepti-
doglycan (PG)4biosynthesis (1). The bacterial cell wall PG is a
giant molecule that sustains the shape of the bacterial cell and
contains the outward forces generated in maintaining an
osmotic pressure gradient against the environment. Without
thisPGlayerthecellintegritywouldberuptured,andthiscould
lead to cell death. Therefore the PG biosynthesis machinery
represents a promising source of putative targets for antibacte-
rial chemotherapy (2, 3).
The biosynthesis of bacterial PG is a complex two-stage
process (4). The first stage involves the assembly of the disac-
charide peptide monomer unit by enzymes located in the cyto-
plasm or at the inner surface of the cytoplasmic membrane (3,
5). The peptide moiety of the monomer unit is assembled step-
wise by the successive additions of L-alanine, D-glutamic acid,
meso-diaminopimelic acid or L-lysine, and D-alanyl-D-alanine
to UDP-N-acetylmuramic acid (UDP-MurNAc). These steps
arecatalyzedbyspecificpeptidesynthetases(ligases),whichare
designated as MurC, MurD, MurE, and MurF, respectively, all
participating in non-ribosomal peptide bond formation with
the concomitant hydrolysis of ATP. The MurNAc-pentapep-
tide motif of the resulting nucleotide precursor is then trans-
ferred by the MraY translocase onto the undecaprenyl phos-
phatecarriermolecule,generatingthelipidintermediateI.The
subsequentadditionoftheN-acetylglucosaminemotifofUDP-
GlcNAc onto lipid I generates lipid II in a reaction catalyzed by
MurG (6). The second stage of the PG biosynthesis consists in
the polymerization by transglycosylation and transpeptidation
reactions of the disaccharide pentapeptide monomers, a reac-
tion taking place in the periplasmic space and that is catalyzed
by the penicillin-binding proteins.
The PG biosynthesis pathway enzymes, which are essential
and specific for bacteria, represent important potential targets
for screening novel antibacterial compounds. Due to the grow-
ing emergence of bacterial multiresistance to currently used
antibiotics, the discovery of new therapeutic compounds has
* This work was supported in part by grants from the Re ´gion Rho ˆne-Alpes (to
M.C.);bytheCNRS,theUniversityofLyon(France),andtheNationalResearch
Agency (ANR-06-MIME-027-01 to V.M.); by the Junta de Castilla y Leo ´n (Ref.
LE040A07toJ.A.G.);bytheMinisteriodeCienciayTecnología(Spain)(Grants
BIO2008-00519 and BIO2005-02723 to J.A.G. and L.M.M.); and by the CNRS
andUniversite ´ Paris-SudXI(GrantUMR8619toD.M.L.).Thecostsofpublica-
tionofthisarticleweredefrayedinpartbythepaymentofpagecharges.This
article must therefore be hereby marked “advertisement” in accordance with
18U.S.C.Section1734solelytoindicatethisfact.
1A beneficiary of a fellowship from the Ministerio de Educacio ´n y Ciencia.
2To whom correspondence may be addressed. Tel.: 33-4-72-72-26-79; Fax:
33-4-72-72-26-41; E-mail: vmolle@ibcp.fr.
3To whom correspondence may be addressed. Tel.: 34-987-29-15-03; Fax:
34-987-29-14-09; E-mail: jagils@unileon.es.
4The abbreviations used are: PG, peptidoglycan; MurNAc, N-acetylmuramic
acid;STPK,Ser/Thrproteinkinase;GST,glutathioneS-transferase;MS,mass
spectrometry;MS/MS,tandemMS;LC,liquidchromatography;ESI,electro-
spray ionization; TEV, tobacco etch virus.
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indeed become a necessity. In recent years, an extensive search
for specific inhibitors interfering with the cytoplasmic steps of
this pathway, and in particular with the four steps catalyzed by
the Mur ligases, has been developed (2, 7, 8). The UDP-Mur-
NAc:L-alanine ligase (MurC), encoded by the murC gene, rep-
resents such an interesting candidate for drug development (9,
10). Recently, different phosphinic acid derivatives and sub-
strate analogues have been identified as Mur ligase inhibitors
(11, 12).
Corynebacterium glutamicum is a rod-shaped non-patho-
genicGram-positiveactinomycetewidelyusedintheindustrial
production of amino acids such as L-lysine and L-glutamic acid
(13). C. glutamicum has been extensively studied as a model
microorganism due to the strategies employed by this actino-
mycetetoachievearod-shapedmorphology.Infact,themech-
anisms taking place in C. glutamicum happened to be com-
pletelydifferentfromthatofEscherichiacoliorBacillussubtilis
(14, 15), whereas the number of genes involved in cell division
and PG biosynthesis in C. glutamicum is lower (16).
Interestingly, an earlier work on the phosphoproteome of C.
glutamicum (17) identified MurC has being phosphorylated in
vivo, suggesting that protein phosphorylation plays a much
broader function in C. glutamicum than was previously
expected. Recently, we described the characterization of the
four STPKs from C. glutamicum ATCC 13869 and highlighted
their role in cell division (18). Moreover, Thakur and
Chakraborti (19) showed that MurD from Mycobacterium
tuberculosis was phosphorylated by the Ser/Thr protein kinase
(STPK) PknA, although no further characterization of the role
ofthephosphorylationontheMurDenzymeactivitywasinves-
tigated.Therefore,itwastemptingtospeculatethatMurCinC.
glutamicum could also be regulated by STPK phosphorylation.
ThefocusofthisworkistostudytheregulationofMurCinC.
glutamicum via phosphorylation. As a first step in deciphering
thepotentialrole/participationofthecorynebacterialSTPKsin
theregulationofMurCactivity,weconfirmeditsspecificphos-
phorylation by the PknA kinase through a combination of in
vitro phosphorylation assays and mass spectrometric identifi-
cation of the different MurC phosphorylation sites. Moreover,
we demonstrated that the murein ligase activity of MurC was
negatively regulated upon its phosphorylation. To our knowl-
edge, this work represents the first evidence of a Mur enzyme
regulated by phosphorylation.
EXPERIMENTAL PROCEDURES
Bacterial Strains and Growth Conditions—Bacterial strains
and plasmids are described in Table 1. Strains used for cloning
and expression of recombinant proteins were E. coli TOP10
(Invitrogen) and E. coli BL21(DE3)Star (Stratagene), respec-
tively. E. coli cells were grown and maintained at 37 °C in LB
medium supplemented with 100 ?g/ml ampicillin and/or 50
?g/mlkanamycin,whenrequired.ThemurCtemperature-sen-
sitive E. coli strain H1119 was grown at 30 °C in 2YT (1.6%
Bactotrypton, 1.0% Bactoyeast extract, 0.5% NaCl, pH 7.0)
medium and was used for genetic complementation experi-
ments with plasmids carrying wild-type or mutated copies of
themurCgene.C.glutamicumcellsweregrownat30 °CinTSB
(Trypticasesoybroth,Oxoid)orTSA(TSBcontaining2%agar)
medium supplemented with 12.5 ?g/ml kanamycin. Plasmids
to be transferred by conjugation from E. coli to corynebacteria
wereintroducedbytransformationintothedonorstrainE. coli
S17-1. Mobilization of plasmids from E. coli S17-1 to C. glu-
tamicum R31 was accomplished as described previously (20).
Cloning, Expression, and Purification of MurC Proteins—
First, the murC gene was cloned to generate a recombinant
MurC protein expressed in E. coli. Therefore, the murC gene
was amplified by PCR using C. glutamicum ATCC 13869
genomic DNA as a template and the primers pair murC1/
murC2 (Table 2), containing NdeI and NheI restriction sites,
respectively. The 1461-bp amplified product was digested by
NdeI and NheI and ligated to the pETTev vector (Table 1) gen-
erating the pTEVmurC plasmid. E. coli BL21(DE3)Star cells
transformed with this construction were used for expression
and purification of His6-tagged MurC, as previously described
(21). Finally, the purified His6-tagged MurC was treated with
TEV protease according to the manufacturer’s instructions
(Invitrogen). Secondly, overexpression and purification of
MurC from C. glutamicum cultures was performed using
standard PCR strategies. The murC gene from C. glutamicum
was amplified using the primers pair murC1/murHisNdeI2
(Table 2). The PCR product carrying a His tag at its C-terminal
endwasdigestedwithNdeIandsubsequentlyclonedunderthe
controlofthePdivpromoterintoplasmidpEDiv(Table1).The
resulting expression vector, named pEDivmurHis, was intro-
duced by conjugation into C. glutamicum R31. Purification of
thesolubleHis6-taggedMurCproteinfromC.glutamicumwas
performed as described previously (21).
In Vitro Kinase Assays—In vitro phosphorylation was per-
formedwith2?gofMurCin20?lofbufferP(25mMTris-HCl,
pH7.0,1mMdithiothreitol,5mMMgCl2,1mMEDTA)with200
?Ci/ml [?-33P]ATP corresponding to 65 nM (PerkinElmer Life
Sciences, 3000 Ci/mmol), and 0.5 ?g of kinase. Plasmids
pGEXA, pGEXB, pGEXL, and pTEVGfull (Table 1) were used
for the expression and purification in E. coli of the four recom-
binantSTPKsfromC.glutamicumaspreviouslydescribed(18).
After 15-min incubation, the reaction was stopped by adding
sample buffer and heating the mixture at 100 °C for 5 min. The
reaction mixtures were analyzed by SDS-PAGE. After electro-
phoresis, gels were soaked in 20% trichloroacetic acid for 10
min at 90 °C, stained with Coomassie Blue, and dried. Radioac-
tive proteins were visualized by autoradiography using direct
exposure films.
Analysis of the Phosphoamino Acid Content of Proteins—
MurCsample(5?g)phosphorylatedinvitrobytheGST-tagged
PknA, and unreacted [?-33P]ATP were separated by one-di-
mensional gel electrophoresis and electroblotted onto an
Immobilon polyvinylidene difluoride membrane. The33P-la-
beled protein bands were detected by autoradiography and
excisedfromtheImmobilonblotandhydrolyzedin6 MHClfor
1 h at 110 °C. The acid-stable phosphoamino acids released
were separated by electrophoresis in the first dimension at pH
1.9 (800 V/h) in 7.8% acetic acid, 2.5% formic acid, followed
by ascending chromatography in the second dimension in
2-methyl-1-propanol/formic acid/water (8:3:4, v/v). After
migration, radioactive compounds were detected by autora-
diography. Authentic phosphoserine, phosphothreonine and
MurCPhosphorylationinC.glutamicum
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phosphotyrosinewereruninparallelandvisualizedbystaining
with ninhydrin.
Cloning and Purification of PknA Mutant Proteins—Site-di-
rectedmutagenesiswasdirectlyperformedonthepGEXAexpres-
sion plasmid (Table 1) using inverse-PCR amplification with the
following self-complementary primers (Table 2): N-pknAT179A/
C-pknAT179A, N-pknAT181A/C-pknAT181A, N-pknAT179A-
T181A/C-pknAT179A-T181A, and N-pknAK49M/C-pknAK49M
to generate pGEXpknAT179A, pGEXpknAT181A, pGEXpkn-
AT179A/T181A, and pGEXpknAK49M, respectively (Table 1).
All constructs were verified by DNA sequencing. The different
GST-tagged recombinant fusion proteins were overexpressed
and purified as reported earlier (18).
Site-directedMutagenesis—ThesixthreonineresiduesfromC.
glutamicum MurC identified by mass spectrometry after in vitro
phosphorylationwithGST-taggedPknAwerereplacedbyalanine
residues by site-directed mutagenesis using inverse-PCR amplifi-
cation.AfirstPCRwascarriedoutusingpTEVmurC(Table1)asa
TABLE1
Bacterial strains and plasmids used in this study
Strains or plasmids
E. coli TOP10
Genotype or descriptionSource or reference
Invitrogen
F?mcrA ?(mrr-hsdRMS-mcrBC) ?80lacZ?M15 ?lacX74 deoR recA1
araD139 ?(ara-leu)7697 galU galK rpsL endA1 nupG; used for general
cloning
F2 ompT hsdSB(rB2 mB2) gal dcm (DE3); used to express recombinant
proteins in E. coli
murC temperature-sensitive mutant
Mobilizing donor strain, pro recA, which possesses an RP4 derivative
integrated into the chromosome
Wild-type control strain
C. glutamicum ATCC 13869; derivative used as recipient in conjugation
experiments
pET15b (Novagen) derivative including the replacement of the thrombin site
coding sequence with a tobacco etch virus (TEV) protease site
pTEV derivative used to express His-tagged fusion of MurC
E. coli vector designed to make GST gene fusions
pGEX4T-3 derivative used to express GST fusion of PknA cytoplasmic
domain
pGEX4T-3 derivative used to express GST fusion of PknB cytoplasmic domain
pGEX4T-3 derivative used to express GST fusion of PknL cytoplasmic domain
pETTev derivative used to express His-tagged PknG
Mobilizable plasmid able to replicate in E. coli and C. glutamicum; kan and cat
resistance genes
pEDiv derivative used to express His-tagged fusion of MurC in C. glutamicum
pGEXA derivative used to express GST fusion of PknA cytoplasmic domain
carrying the mutation T179A
pGEXA derivative used to express GST fusion of PknA cytoplasmic domain
carrying the mutation T181A
pGEXA derivative used to express GST fusion of PknA cytoplasmic domain
carrying the mutations T179A and T181A
pGEXA derivative used to express GST fusion of PknA cytoplasmic domain
carrying the mutation K49A
pTEVmurC derivative used to express His-tagged fusion of MurC1T carrying
the mutation T362A
pTEVmurC1T derivative used to express His-tagged fusion of MurC2T
carrying the mutation T362A/T365A
pTEVmurC2T derivative used to express His-tagged fusion of MurC3T
carrying the mutation T362A/T365A/T51A
pTEVmurC3T derivative used to express His-tagged fusion of MurC4T
carrying the mutation T362A/T365A/T51A/T120A
pTEVmurC4T derivative used to express His-tagged fusion of MurC5T
carrying the mutation T362A/T365A/T51A/T120A/T167A
pTEVmurC5T derivative used to express His-tagged fusion of MurC6T
carrying the mutation T362A/T365A/T51A/T120A/T167A/T133A
pTEVmurC derivative used to express His-tagged fusion of MurCT51A
carrying the mutation T51A
pTEVmurC derivative used to express His-tagged fusion of MurCT120A
carrying the mutation T120A
pTEVmurC derivative used to express His-tagged fusion of MurCT133A
carrying the mutation T133A
pTEVmurC derivative used to express His-tagged fusion of MurCT167A
carrying the mutation T167A
pTEVmurC derivative used to express His-tagged fusion of MurCT362A
carrying the mutation T362A
pTEVmurC derivative used to express His-tagged fusion of MurCT365A
carrying the mutation T365A
E. coli vector allowing high level expression under the IPTG inducible trc
promoter
pTrc99A derivative carrying murC
pTrc99A derivative carrying murC1T (T362A)
pTrc99A derivative carrying murC2T (T362A/T365A)
pTrc99A derivative carrying murC3T(T362A/T365A/T51A)
pTrc99A derivative carrying murC4T(T362A/T365A/T51A/T120A)
pTrc99A derivative carrying murC5T(T362A/T365A/T51A/T120A/T167A)
pTrc99A derivative carrying murC6T(T362A/T365A/T51A/T120A/T167A/
T133A)
E. coli BL21(DE3)Star Stratagene
E. coli H1119
E. coli S17-1
(23)
(38)
C. glutamicum 13869
C. glutamicum R31
ATCC
(39)
pETTev(40)
pTEVmurC
pGEX4T-3
pGEXA
This work
GE Healthcare
(18)
pGEXB
pGEXL
pTEVGfull
pEDiv
(18)
(18)
(18)
A. Ramos (unpublished)
pEDivmurHis
pGEXpknAT179A
This work
This work
pGEXpknAT181AThis work
pGEXpknAT179/181AThis work
pGEXpknAK49AThis work
pTEVmurC1TThis work
pTEVmurC2TThis work
pTEVmurC3T This work
pTEVmurC4TThis work
pTEVmurC5TThis work
pTEVmurC6T This work
pTEVmurCT51A This work
pTEVmurCT120AThis work
pTEVmurCT133AThis work
pTEVmurCT167AThis work
pTEVmurCT362A This work
pTEVmurCT365AThis work
pTrc99APharmacia
pTrc99murC
pTrc99murC1T
pTrc99murC2T
pTrc99murC3T
pTrc99murC4T
pTrc99murC5T
pTrc99murC6T
This work
This work
This work
This work
This work
This work
This work
MurCPhosphorylationinC.glutamicum
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template with the primers pair N-murC362 and C-murC362
(Table2)togeneratepTEVmurC1T(T362A).AsecondPCRwas
carried out using pTEVmurC (Table 1) as a template with the
primers pair N-murC362/365 and C-murC362/365 (Table 2) to
generate pTEVmurC2T (T362A/T365A). This mutant and the
subsequent additional mutants were used as templates in subse-
quentPCRreactionsusingthefollowingprimerspairs:N-murC51
and C-murC51, N-murC120 and C-murC120, N-murC167 and
C-murC167, and N-murC133 and C-murC133 (see Table 2) to
generate pTEVmurC3T (T362A/T365A/T51A), pTEVmurC4T
(T362A/T365A/T51A/T120A), pTEVmurC5T (T362A/T365A/
T51A/T120A/T167A),andpTEVmurC6T
T51A/T120A/T167A/T133A), respectively. Individual mutants
were generated using pTEVmurC (Table 1) as a template
with the primers pairs N-murC51/C-murC51, N-murC120/
C-murC120,N-murC133/C-murC133, N-murC167/C-mur-
C167, N-murC362/C-murC362, and N-murC365/C-murC365
(Table 2) to generate pTEVmurCT51A, pTEVmurCT120A,
pTEVmurCT133A, pTEVmurCT167A, pTEVmurCT362A, and
pTEVmurC T365A, respectively (Table 1). All the resulting con-
structs were verified by DNA sequencing. The different His6-
tagged mutant proteins were overexpressed and purified, as
describedabove.
MS Analysis—Purified wild-type and mutant MurC proteins
were subjected to in vitro phosphorylation by GST-tagged
PknA as described above, excepted that [?-33P]ATP was
replaced with 5 mM ATP. Subsequent analyses using NanoLC/
nanospray/tandem mass spectrometry (LC-ESI/MS/MS) were
performed as previously described (18).
Immunoblotting—Corynebacterial MurC purified either
from E. coli or C. glutamicum was loaded on a 10% polyacryl-
amide gel, electrophoresed, blotted on polyvinylidene difluo-
ride, and detected using either monoclonal mouse anti-phos-
pho-threonine, -serine, -tyrosine, or polyclonal rabbit anti-His
(T362A/T365A/
antibodies used at 1:100 and 1:10,000 dilution, respectively.
Alkaline phosphatase-conjugated anti-mouse or anti-rabbit
was used as a secondary antibody at a 1:5,000 dilution.
Complementation with C. glutamicum MurC—Plasmids
pTEVmurC, pTEVmurC1T, pTEVmurC2T, pTEVmurC3T,
pTEVmurC4T, pTEVmurC5T, and pTEVmurC6T were used
as templates for PCR amplification of the murC gene with the
primers pair murH1119-1/murH1119-2 (Table 2) containing a
BspHI and BglII restriction site, respectively. The resulting
1461-bp products, carrying the wild-type and mutated gene
copies, respectively, were digested with BspHI and BglII, and
cloned into the plasmid vector pTrc99A (22) between the
compatible NcoI and BamHI sites, generating pTrcmurC,
pTrcmurC1T, pTrcmurC2T, pTrcmurC3T, pTrcmurC4T,
pTrcmurC5T, and pTrcmurC6T, respectively. These plasmids
allowing high level expression of the C. glutamicum MurC and
MurC mutants under the control of the strong isopropyl
1-thio-?-D-galactopyranoside-inducible trc promoter, were
transformed in the E. coli MurC temperature-sensitive mutant
strainH1119(23).Transformantsweregrownatthepermissive
temperature (30 °C) before being shifted to the restrictive tem-
perature(42 °C).Complementationwasjudgedbytheabilityof
the mutant to grow at the restrictive temperature.
UDP-MurNAc L-alanine Ligase Assay—The L-alanine-add-
ingactivityofwild-typeandmutantMurCproteinswasassayed
according to Liger et al. (24) by following the formation of
UDP-MurNAc-L-[14C]alaninein40?lofreactionmixturecon-
taining100mMTris-HCl,pH8.6,20mMMgCl2,20mMammo-
nium sulfate, 0.5 mM L-[14C]alanine (0.6 KBq, 5.5 Gbq/mmol,
Amersham), 1 mM UDP-MurNAc, 5 mM ATP, and enzyme (25
?l of an appropriate dilution in buffer A: 20 mM potassium
phosphate, pH 7.2, containing 1 mM dithiothreitol and 10%
glycerol). The mixture was incubated at 37 °C for 30 min and
the reaction was stopped by addition of 8 ?l of acetic acid,
TABLE2
Primers used in this study
PrimerGene
murC
murC
murC
pknA
pknA
pknA
pknA
pknA
pknA
pknA
pknA
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
murC
5? to 3? Sequencea,b
murC1
murC2
murHisNdeI
N-pknAK49M
C-pknAK49M
N-pknAT179A
C-pknAT179A
N-pknAT181A
C-pknAT181A
N-pknAT179A/T181A
C-pknAT179A/T181A
N-murC362
C-murC362
N-murC362/365
C-murC362/365
N-murC365
C-murC365
N-murC51
C-murC51
N-murC120
C-murC120
N-murC167
C-murC167
N-murC133
C-murC133
murH1119-1
murH1119-2
aRestriction sites are underlined and specified by brackets.
bMutagenized codons are shown in bold.
GGAATTCCATATGGTGACCACTCCACAC (NdeI)
TGGAATTCGCTAGCCTAATTGTTTTGCAGCTGATCC (NheI)
GGAATTCCATATGCTAATGATGATGATGATGATGATTGTTTTGCAGC(NdeI)
GATCGCGAAGTAGCCATCATGGTACTGCGCCCGGAATTTTCC
GGAAAATTCCGGGCGCAGTACCATGATGGCTACTTCGCGATC
GCCGCTGCTGTGCCTTTGGCCCGCACCGGCATGGTGGTG
CACCACCATGCCGGTGCGGGCCAAAGGCACAGCAGCGGC
GCTGTGCCTTTGACCCGCGCCGGCATGGTGGTGGGTACT
AGTACCCACCACCATGCCGGCGCGGGTCAAAGGCACAGC
GCCGCTGCTGTGCCTTTGGCCCGCGCCGGCATGGTGGTGGGT
ACCCACCACCATGCCGGCGCGGGCCAAAGGCACAGCAGCGGC
GATTACGCACACCACCCAGCGGAAGTAACTGCAGTGCTC
GAGCACTGCAGTTACTTCCGCTGGGTGGTGTGCGTAATC
GCACACCACCCAGCGGAAGTAGCTGCAGTGCTCAGCGCTGCG
CGCAGCGCTGAGCACTGCAGCTACTTCCGCTGGGTGGTGTGC
CACCACCCAACGGAAGTAGCTGCAGTGCTCAGCGCGGCG
CGCAGCGCTGAGCACTGCAGCTACTTCCGTTGGGTGGTG
GATGCCAAAGATTCCCGCGCCTTGCTTCCACTCCGCGCC
GGCGCGGAGTGGAAGCAAGGCGCGGGAATCTTTGGCATC
GAATTGCTGGAAGGCTCCGCCCAGGTCTTGATCGCGGGT
ACCCGCGATCAAGACCTGGGCGGAGCCTTCCAGCAATTC
ACCAATGCGCACCATGGAGCTGGTGAGGTCTTTATCGCT
AGCGATAAAGACCTCACCAGCTCCATGGTGCGCATTGGT
ACCCACGGTAAGACCTCCGCCACCTCTATGTCTGTGGTA
TACCACAGACATAGAGGTGGCGGAGGTCTTACCGTGGGT
TTTAATCATGACCACTCCACACTTGG (BspHI)
CTTACAGATCTCTAATTGTTTTGCAGCTG (BglII)
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followedbylyophilization.Totesttheeffectofthephosphoryl-
ation of MurC on its enzyme activity, the assay was performed
as described above excepted that the MurC enzyme (20 ?g/ml)
was preincubated overnight at 37 °C with or without wild-type
PknA (16 ?g/ml), or with PknA_K49M mutant (30 ?g/ml) in
buffer A supplemented with 2 mM ATP and 5 mM MgCl2. The
radioactive product UDP-MurNAc-L-[14C]alanine and sub-
strate L-[14C]alanine were separated by reversed-phase high-
performance liquid chromatography on a Nucleosil 5C18 col-
umn (4.6 ? 150 mm, Alltech France) using 50 mM ammonium
formate, pH 3.9, as the eluent, at a flow rate of 0.6 ml/min.
Detection was performed with a radioactive flow detector
(model LB506-C1, Berthold France, La Garenne-Colombes,
France) using the Quicksafe Flow 2 scintillator (Zinsser Ana-
lytic,Maidenhead,UK)at0.6ml/min.Quantitationwascarried
out with the Radiostar software (Berthold).
RESULTS AND DISCUSSION
MurC Is a Substrate of the C. glutamicum PknA—The pep-
tide moiety of the PG monomer unit is assembled stepwise in
the cytoplasm by the successive actions of four Mur ligases
designated as MurC, MurD, MurE, and MurF. These enzymes
share limited sequence identity but have several highly con-
served regions that map primarily to the active site. Each of
them comprises three structural domains: an N-terminal
domain with a Rossmann-type fold primarily responsible for
binding of the UDP-MurNAc(-peptide) substrate, a large cen-
tral ATP-binding (ATPase) domain, and a C-terminal domain
associated with binding of the amino acid substrate (25). Based
on protein sequence alignments and on the three-dimensional
structure of the E. coli MurC protein (26), these three domains
in the C. glutamicum MurC may extend between Met1–Gly118,
Ser119–Arg334, and Arg335–Asn486, respectively (Figs. 1 and 4).
By comparing the amino acid sequences of the MurC, -D, -E,
and-Fligasesfromdifferentbacterialgenera,severalconserved
residues were identified. This analysis led to the characteriza-
tion of the ATP-binding motif located in the conserved
sequence (GXXGK(T/S)), and therefore named the GKT motif
(Fig. 1). There are also a number of common conserved amino
acids residues present in the different Mur ligases. Taking
E. coli as a model, it was established that Asp50, Lys130, Glu174,
and Asp351were essential for the catalytic process of these
ligases, whereas residues His199, Asn293, Asn296, and Arg327
wereinvolvedinthestructureoftheactivesite.Thesequenceof
severalMurCproteinsfromrepresentativespeciesofmycobac-
teria and corynebacteria were aligned using the ClustalW and
Espript programs (Fig. 1). The invariant residues Asp50, Lys130,
Glu174, Asn296, and Arg327appeared conserved in all the
sequences aligned, thus confirming that MurC from C. glu-
tamicum harbors most of the characteristics of a functional
Mur ligase enzyme. Therefore, the recombinant MurC protein
wasexpressedandpurifiedfromE. coliBL21(DE3)Starharbor-
ing the pTEVmurC. The protein contained an N-terminal His
FIGURE1.MultiplesequencealignmentofMurCorthologproteinsfromcorynebacteria,mycobacteria,andE. coli.Thealignmentwasperformedusing
ClustalWandEspriptprograms(C_glu,C.glutamicum;C_eff,C.efficiens;C_dip,C.diphtheriae;C_jei,C.jeikeium;M_tub,M.tuberculosis;M_sme,M.smegmatis;and
E_col, E. coli). The conserved GKT motif involved in ATP binding is represented by a shaded oval. Triangles represent PknA-dependent phosphorylation sites
(Thr51, Thr120, Thr133, Thr167, Thr362, and Thr365) of C. glutamicum MurC. Residues indicated by an asterisk in the E. coli MurC sequence correspond to residues
thatwerepreviouslyidentifiedasinvariantwithinthewholeMurligasefamily(3,25):theAsp50,Lys130(GKTmotif),andGlu174residuesinvolvedinthecatalytic
process,andtheAsn296andArg327residuesinvolvedinthestructuralorganizationoftheactivesite.NumberingofaminoacidscorrespondstotheMurCprotein
fromC.glutamicum.
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tag, which was subsequently removed after cleavage with the
TEV protease. Analysis of the purified recombinant protein by
SDS-PAGErevealedthattheproteinwasexpressedinasoluble
form migrating slightly above its predicted molecular mass of
52 kDa (Fig. 2A).
Previous work on the phosphoproteome of C. glutamicum
(17) identified MurC as being phosphorylated in vivo. More-
over,wehaverecentlyreportedthecharacterizationofthefour
STPKsfromC.glutamicumATCC13869andhighlightedtheir
role in cell division (18). These results prompted us to investi-
gate whether MurC would also represent a substrate for
corynebacterial STPKs. A systematic approach was used to
investigate whether STPKs of C. glutamicum (PknA, PknB,
PknG, or PknL) could phosphorylate MurC. All these STPKs
wereexpressedandpurifiedfromE. coliasdescribedpreviously
(18). The different STPKs migrate as diffuse bands reflecting
the different levels of phosphorylation for each isoform, and
this aberrant profile of migration of STPKs kinases has already
been reported in earlier studies (27–29). Interestingly, when
STPKs were incubated in the presence of recombinant MurC
and [?-33P]ATP, phosphorylation of MurC was specifically
observedwiththePknAkinase,whereasPknBandPknL,which
display autophosphorylation activity in vitro, did not phospho-
rylateMurC(Fig.2A).ThePknGkinaseneedstobeactivatedby
the PknA kinase to trigger its autophosphorylation activity,
therefore PknG was phosphorylated by PknA as previously
described
transphosphorylate MurC (18). As
shown on Fig. 2A, MurC did not
show any radioactive band, thus
indicating that PknG, even if acti-
vated by its cognate kinase PknA,
could not phosphorylate MurC in
vitro. Moreover, MurC alone is
unable to incorporate [?-33P]ATP,
confirming that MurC is a substrate
of PknA (Fig. 2A).
We next determined the nature of
theaminoacidresiduesphosphoryla-
ted in MurC by analyzing the phos-
phoamino acid content of PknA-
phosphorylated MurC. The MurC
protein (5 ?g) was labeled with
[?-33P]ATP in vitro, separated by
SDS-PAGE,excised,andsubjectedto
acid hydrolysis. Fig. 2B shows that
MurC was exclusively phosphoryla-
tedonthreonineresidues.
Together, these data suggest a
specificityofsubstraterecognition
by PknA toward MurC. Although
a specific interaction between
MurC and PknA may exist, it
remains to be established whether
thisspecificpartneringalsooccurs
in vivo.
The Activation Loop Thr179and
Thr181ResiduesAreEssentialforthe
andthenusedto
AutophosphorylationActivityofC.glutamicumPknA—Diverse
mechanisms of eukaryotic protein kinase regulation have been
described. The transition between active and inactive forms
may occur via control of access to the catalytic site and/or the
substrate-binding site. These regulatory mechanisms involve
phosphorylation/dephosphorylation
mechanism or the action of other kinases and phosphatases.
Theactivationloopappearstobeamajorcontrolelementofan
active/inactive conformational switch in numerous kinases
(30),andtheconformationoftheactivationloopoftendepends
onthephosphorylationstate(31).Basedonstructuralstudies,it
is thought that the activation loop controls both the accessibil-
ity to the catalytic site and the binding of the substrate.
Recently,Canovaetal.(27)demonstratedthatthephosphoryl-
ated residues Thr173and Thr175present in the activation loop
were essential for the autophosphorylation activity of the M.
tuberculosis PknL kinase, and that phosphorylation of the
Thr173residue was also required for optimal PknL-mediated
phosphorylation of its substrate Rv2175c. Interestingly, the
corresponding threonine residues Thr179and Thr181found in
the C. glutamicum PknA were recently identified as phospho-
rylation sites (18). The presence of such residues in PknA from
C. glutamicum supports the concept that phosphorylation of
the activation loop could play a regulatory role, as recently
described for other mycobacterial STPKs (27, 32, 33).
viaanautocatalytic
FIGURE 2. In vitro phosphorylation of C. glutamicum MurC. A, in vitro phosphorylation of MurC by coryne-
bacterial STPKs. The four recombinant STPKs (PknA, PknB, PknG, and PknL) were expressed and purified as
previously described by Fiuza et al. (18). Recombinant MurC was treated with the TEV protease to remove the
N-terminal His tag and then incubated with [?-33P]ATP and the different kinases. Samples were separated by
SDS-PAGE(upperpanel)andvisualizedbyautoradiography(lowerpanel).Upperbandsillustratetheautokinase
activityofeachSTPK,whereaslowerbandsreflectphosphorylationofMurC.B,phosphoaminoacidcontentof
MurC. MurC was phosphorylated in vitro in presence of PknA and [?-33P]ATP, analyzed by SDS-PAGE, electro-
blotted onto an Immobilon polyvinylidene difluoride membrane, excised, and hydrolyzed in acid. The phos-
phoamino acids thus liberated were separated by electrophoresis in the first dimension (1D) and ascending
chromatographyintheseconddimension(2D).Aftermigration,radioactivemoleculesweredetectedbyauto-
radiography (lower panel). Authentic phosphoserine (P-Ser), phosphothreonine (P-Thr), and phosphotyrosine
(P-Tyr) were run in parallel as internal standard controls, and visualized by ninhydrin staining (upper panel).
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To determine a possible link between Thr179and Thr181and
the autophosphorylation activity of PknA, the two residues
were individually substituted by Ala to generate PknA_T179A
and PknA_T181A, respectively. In addition, a double mutant
was also created and designated PknA_T179A/T181A. These
PknA mutants were expressed and purified from E. coli as pre-
viously described (18). The electrophoretic migration profile
showed that the different PknA mutants migrated differently
fromthewild-typeprotein(Fig.3A,upperpanel).Wereasoned
that, because these proteins displayed different electrophoretic
mobilityproperties,theymustdifferintheirintrinsicphospho-
rylation states. This hypothesis was confirmed by labeling the
mutant proteins in vitro with [?-33P]ATP. PknA_T179A gave
rise to a lower radioactive signal than the wild-type enzyme
(Fig. 3A, lower panel). Quantification of the signal intensity
indicated an autophosphorylation activity of 47% with respect
to the activity of the wild-type protein, which was arbitrarily
placedat100%.ThisresultarguesthatThr179isakeyphospho-
rylation site of the activation loop, necessary for PknA auto-
phosphorylation activity. The signal intensity generated by
PknA_T181A was intermediate between those of the wild-type
andThr179mutantproteins(Fig.3A,lowerpanel),representing
81% of residual activity with respect to the wild-type protein
activity. Therefore, this result confirms the requirement of
Thr181
for optimal PknA autophosphorylation activity,
although this residue appears less important than Thr179.
When both Thr residues were mutated, the intensity of the
signal was similar to the one of the single Thr179mutant (Fig.
3A, lower panel). Together, these results confirm that double
phosphorylation of the activation loop residues Thr179and
Thr181is necessary for full kinase activity and unambiguously
demonstrate the role of both phosphothreonines.
To exclude the possibility of ex-
ogenous contamination that could
explain labeling of PknA with
[?-33P]ATP, Lys49was mutated to
Met. Purified PknA_K49M, was
incubated inthe
[?-33P]ATP, and as expected, no
signal could be detected as Lys49is
essential for catalyzing the phos-
phorylation reaction, in agreement
withpreviousreports(Fig.3A,lower
panel) (34, 35). This confirms that
the radioactive signals observed for
the different isoforms of the PknA
kinase are only representing the
autophosphorylation
PknAandnotacontaminationfrom
the purification procedure.
Phosphorylation
Dependent on the PknA Activati-
on Loop Thr179Residue—Several
recent publications indicated that
phosphorylation of the threonine
residuespresentintheSTPKactiva-
tion loop is not only necessary for
controlling the kinase activity but is
presenceof
activityof
of MurCIs
also required for recruitment and phosphorylation of its sub-
strate(27,36).Theseresultspromptedustoanalyzethecontri-
bution of the activation loop key residues of PknA (Thr179and
Thr181) in the transphosphorylation mechanism between the
kinase and its substrate MurC. This was achieved by incubat-
ing recombinant MurC with PknA_K49M, PknA_T179A,
PknA_T181A, or PknA_T179A/T181A in the presence of
[?-33P]ATP (Fig. 3B). PknA_K49M was unable to phosphoryl-
ate C. glutamicum MurC, indicating that phosphorylation of
PknA is a prerequisite to the transphosphorylation reaction
(Fig. 3B, lower panel). More importantly, whereas the T179A
mutation completely abolished the transphosphorylation reac-
tion, replacement of Thr181by Ala did not alter PknA-depend-
ent phosphorylation of MurC (Fig. 3B, lower panel). Further-
more, MurC could not be phosphorylated by PknA_T179A/
T181A,clearlydemonstratingthatphosphorylationofMurCis
dependent on Thr179. The finding that MurC interacts only
withthephosphorylatedformofPknAandthatthisinteraction
is abolished by the T179A substitution suggests that this phos-
phopeptide recognition motif is involved in protein-protein
interaction between the two corynebacterial partners, as previ-
ouslydescribedinitsrelatedactinomyceteM.tuberculosis(27).
MurC Is Phosphorylated on Multiple Threonine Residues—
The role of post-translational mechanisms like phosphoryla-
tion in regulatory processes or the effect of phosphorylation on
a given substrate in the physiology and/or cell division repre-
sent key events to our understanding of the signaling mecha-
nisms through serine/threonine phosphorylation. This usually
requires the identification of the phosphorylated sites, which
often remains very challenging. To identify which of the 31
threonine residues of the C. glutamicum MurC corresponded
to the phosphorylated site(s), a mass spectrometry approach
FIGURE 3. In vitro phosphorylation of MurC by PknA and the PknA activation loop mutants. A, in vitro
phosphorylation of the different PknA activation loop mutants. All proteins were overproduced in E. coli and
purified as GST fusions. The following proteins were incubated with [?-33P]ATP: PknA_WT, PknA_K49M,
PknA_T179A, PknA_T181A, and PknA_T179A/T181A. Proteins were separated by SDS-PAGE and stained with
CoomassieBlue(upperpanel),andtheradioactivebandswererevealedbyautoradiography(lowerpanel).B,in
vitro phosphorylation of MurC by PknA and the different PknA activation loop mutants. Recombinant MurC
was treated with the TEV protease to remove the N-terminal His tag and then used in phosphorylation assays
in the presence of [?-33P]ATP and PknA_WT, PknA_K49M, PknA_T179A, PknA_T181A, or PknA_T179A/T181A.
Proteins were separated by SDS-PAGE and stained with Coomassie Blue (upper panel), and the radioactive
bands were revealed by autoradiography (lower panel).
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was used. This technique has recently been proven to be a
method of choice for characterizing post-translational modifi-
cations such as phosphorylation (18, 27, 37). LC-ESI/MS/MS
was applied for the identification of phosphorylated peptides
andforthelocalizationofphosphorylationsitesinMurC.Puri-
fied MurC was subjected to in vitro phosphorylation by PknA
with non-radioactive ATP, prior to resolving on SDS-PAGE,
andin-geldigestionwitheithertrypsinorchymotrypsin.Phos-
phorylated amino acid residues were assigned by peptide frag-
mentation in MS/MS: y and b daughter ions containing one
phosphothreonine were associated with a neutral loss of phos-
phoricacid(?H3PO4,i.e.?98Da).AsdetailedinTable3,anal-
ysis of tryptic and chymotryptic digests allowed the character-
ization of six phosphorylation sites in MurC corresponding to
Thr51,Thr120,Thr133,Thr167,Thr362,andThr365(Figs.1and4).
These residues were found in the three structural domains of
FIGURE4.StructuralorganizationofMurCandphosphorylationofMurCmutantsbyPknA.A,schematicrepresentationofMurCfromC.glutamicum.The
MurC protein comprises three structural domains, an N-terminal domain responsible for the binding of the UDP-MurNAc substrate, a central ATP-binding
(ATPase)domain,andaC-terminaldomaininvolvedinthebindingoftheaminoacidsubstrate L-alanine.Thesedomainsareshownbyshadedcirclesandboxes.
The phosphorylation sites identified in MurC are indicated by a “P.” B, in vitro phosphorylation of MurC mutants by PknA. The different MurC mutants were
treatedwiththeTEVproteasetoremovetheN-terminalHistagandthenusedinphosphorylationassaysinequalamountsinthepresenceof[?-33P]ATPand
PknA. The MurC_1T, MurC_2T, MurC_3T, MurC_4T, MurC_5T, and MurC_6T mutant proteins corresponding to MurC_T362A, MurC_T362A/T365A,
MurC_T362A/T365A/T51A,MurC_T362A/T365A/T51A/T120A,MurC_T362A/T365A/T51A/T120A/T167A,andMurC_T362A/T365A/T51A/T120A/T167A/T133A,
respectively,wereseparatedbySDS-PAGEandstainedwithCoomassieBlue(upperpanel),andtheradioactivebandswererevealedbyautoradiography(lower
panel).
TABLE3
Phosphorylation status of recombinant MurC as determined by mass spectrometry
Phosphorylated tryptic and chymotryptic peptide sequenceNumber of detected phosphate groups
LC-ESI/MS/MS
1
1
2
1
1
1
1
1
1
1
Phosphorylated residue(s)
?348–372? FNGAAITDDYAHHPTEVTAVLSAAR
?348–372? FNGAAITDDYAHHPTEVTAVLSAAR
?348–372? FNGAAITDDYAHHPTEVTAVLSAAR
?40–56?
TVTGSDAKDSRTLLPLR
?48–56?
DSRTLLPLR
?108–130? RSDLLGELLEGSTQVLIAGTHGK
?109–130? SDLLGELLEGSTQVLIAGTHGK
?159–184? AGTNAHHGTGEVFIAEADESDASLLR
?131–158? TSTTSMSVVAMQAAGMDPSFAIGGQLNK
?124–141? IAGTHGKTSTTSMSVVAM
Thr362
Thr365
Thr362and Thr365
Thr51
Thr51
Thr120
Thr120
Thr167
Thr133
Thr133
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the MurC protein, two of them, namely Thr120and Thr133,
being located very close to the ATP-binding site of the protein
(129GKT131). Thus, MS profiling coupled to in vitro kinase
assays unambiguously demonstrated the phosphorylation of
MurC by the PknA kinase. The fact that threonines but not
serine residues were identified was consistent with our phos-
phoamino analysis (Fig. 2B).
Definitive identification and localization of the six threonine
residues identified by mass spectrometry was achieved by site-
directed mutagenesis by introducing mutations that prevent
their specific phosphorylation. Thus, all the six threonine resi-
dues were sequentially replaced by alanine, yielding the
MurC_1T to MurC_6T mutants. These mutant proteins were
expressed, purified as His-tagged proteins in E. coli BL21(DE3)
Star harboring the different mutant constructs (Table 1), and
used in an in vitro kinase assay. After TEV protease cleavage of
the His tag, the recombinant MurC phosphorylation site
mutants were incubated with [?-33P]ATP and PknA. The pro-
teins were separated by SDS-PAGE and analyzed by autora-
diography.AsshowninFig.4B(upperpanel),equalamountsof
MurC_WT or MurC mutants were used. Phosphorylation of
the MurC_6T protein in which all six threonine residues were
replaced by alanine appeared completely abolished, compared
with phosphorylation of the intermediate mutants, as evi-
denced by the absence of a specific radioactive band (Fig. 4B,
lower panel). Thus, these results unambiguously demonstrated
that MurC without its six threonine phosphorylation sites has
lost its ability to be phosphorylated by PknA, thus confirming
the identification of all the sites of phosphorylation. An addi-
tional round of mass spectrometry analysis was also performed
directly on the MurC_6T protein, which failed to identify any
additional phosphate group that could eventually have arisen
from a compensatory mechanism to the loss of the specific
phosphorylation sites.
This type of analysis provided us the essential groundwork
for mechanistic/functional studies of MurC regulation and
demonstrated the efficiency of combining genetics and mass
spectrometry analyses, with precise identification of phos-
phoacceptors, a prerequisite for a further understanding of the
MurC mode of action. In particular, strains with defined muta-
tionswithinthephosphorylationsiteswillbeextremelyhelpful
to establish the role of MurC phosphorylation-dependent
regulation in corynebacterial growth and physiology.
In Vivo Phosphorylation of MurC in C. glutamicum—To
assess the relevance of in vitro phosphorylation, the in vivo
phosphorylation of MurC was also investigated in C. glutami-
cum. Therefore, to determine whether phosphorylation of
MurC occurs on threonine residues in vivo, Western blot anal-
ysiswasperformedusingspecificanti-phosphothreonine,anti-
phosphoserine, anti-phosphotyrosine, or anti-His antibodies.
To overproduce and purify phosphorylated MurC, an His tag
was attached at the C terminus of the MurC protein under the
control of the C. glutamicum divIVA promoter (15) in plasmid
pEDiv(Table1),andtherecombinantproteinwasexpressedin
C. glutamicum. Cultures of E. coli_pTEVmurC or C. glutami-
cum_pEDivmurHis overexpressing the His-tagged MurC pro-
tein were collected and lysed, and the soluble MurC was then
purified to homogeneity as previously described (21). It was
found that MurC purified from the C. glutamicum strain was
only phosphorylated on threonine residues (Fig. 5). This was
consistent with our phosphoamino acid content analyses and
the in vitro identification of the MurC phosphorylation sites
when phosphorylated by PknA. It clearly establishes that the
murein ligase MurC is highly phosphorylated in vivo on
threonines. In contrast, the anti-phosphothreonine antibodies
did not react with recombinant MurC protein purified from
E. coli thus confirming that phosphorylation of the C. glutami-
cum MurC did not occur following heterologous expression in
E. coli, which thus confirmed its specific phosphorylation in C.
glutamicum (Fig. 5).
MurC Phosphorylation Sites Are Critical for in Vitro and in
VivoActivity—Theimportanceofthephosphorylationsitesfor
MurC ligase activity was investigated in vitro by site-directed
mutagenesis. The six threonine residues were replaced by ala-
nine,andthecatalyticactivityofthewild-typeandmutantpuri-
fied MurC proteins was assayed by following the incorporation
of L-[14C]alanineintothePGnucleotideprecursor,asdescribed
under “Experimental Procedures.” As shown in Fig. 6A, the
activity of mutants MurC_1T, MurC_2T, and MurC_3T, cor-
responding to successive mutagenesis of Thr362, Thr365, and
Thr51residues, was reduced by ?30–40% as compared with
the wild-type level. Remarkably, the activity of the MurC_4T
(T362A/T365A/T51A/T120A),
T51A/T120A/T167A), and MurC_6T (T362A/T365A/T51A/
T120A/T167A/T133A) mutants appeared much more drasti-
cally altered and almost completely abolished in the six-
threoninemutant(Fig.6A).Moreover,todiscernwhethersome
threonines may be more important to murC activity than oth-
ersindividualmutagenesiswasperformed.Theligaseactivityof
each individual mutant corresponding to MurC_T51A,
MurC_T120A, MurC_T133A, MurC_T167A, MurC_T362A,
andMurC_T365A,wasof72%,29%,44%,79%,26%,and95%as
compared with the wild-type level. Therefore, these results
confirmed that Thr120, Thr133, and Thr362represent critical
residues for MurC ligase activity, as already observed with the
MurC_1T to MurC_6T mutants proteins (Fig. 6A).
These results confirmed that the overall activity of MurC
relies on the presence of these threonine residues. In fact, one
could imagine that replacing these critical residues present in
allthreecharacteristicdomainsofMurCcouldhaveadramatic
effect on the enzymatic activity of the ligase, and especially the
MurC_5T(T362A/T365A/
FIGURE5.InvivophosphorylationofMurCinC.glutamicum.Recombinant
MurC purified from either E. coli or C. glutamicum strains were analyzed by
SDS-PAGEanddetectedbyimmunoblottingusingantibodiesagainstHistag,
phosphothreonine, phosphoserine, or phosphotyrosine residues.
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mutagenesis of Thr133, a key residue closed to the GKT motif
necessary for the binding of ATP.
The activity of the MurC proteins was also tested in vivo
using a functional assay based on complementation of the
E. coli temperature-sensitive murC mutant strain H1119. This
strain, which grows normally at 30 °C but lyses when shifted at
the non-permissive temperature of 42 °C, was transformed
with the pTrc99A plasmid, or the pTrc99murC and
pTrc99murC1T, 2T, 3T, 4T, 5T, and 6T derivatives (Table 1)
expressing wild-type or mutated forms of MurC. As shown in
Fig. 6B, all types of transformants grew normally at the permis-
sive temperature of 30 °C, but only those expressing the wild-
type protein or the 1T, 2T, and 3T mutated proteins restored
growth of the E. coli mutant at the temperature of 42 °C. No
complementation by the plasmids expressing the 4T, 5T, and
6T mutants was observed (Fig. 6B).
A perfect correlation was thus observed between the in vitro
and in vivo data, i.e. between the catalytic activity of these pro-
teins and their ability to complement the murC temperature-
sensitive E. coli mutant. Combined together, the results
obtainedwiththedifferentphosphorylationsitemutantsstress
the importance of these threonine residues for MurC activity.
These data not only suggest that these residues are important
for a fully expressed activity of the MurC protein, due probably
to their localization in critical regions of the enzyme, but also
indicate,ifweputforwardtheanalysis,thattheintroductionof
anegativechargeduetothephosphorylationofthreoninesmay
have an impact on the regulation of the MurC enzyme activity.
Phosphorylation Negatively Modulates MurC Ligase Activity—
Therefore, we assessed whether phosphorylation by PknA
could have a direct effect on the regulation of the ligase activity
of MurC. The C. glutamicum MurC protein purified from
E. coli was pre-treated with either the PknA_WT kinase or the
PknA_K49M kinase, yielding the phosphorylated and non-
phosphorylated isoforms of MurC, respectively, which were
subsequently tested for activity (Fig. 7). The PknA_K49M
kinase,whichisunabletoautophosphorylate(Fig.3)andthere-
fore transphosphorylate its substrate, represented here an
appropriate control, because one could imagine that the kinase
itselfcouldberesponsiblefortheeffectonMurCactivity.Inter-
estingly,wefoundthattheligaseactivityofthephosphorylated
isoform of MurC was severely inhibited, representing ?30% of
the wild-type activity, whereas no decrease of activity was
observed following treatment by the PknA_K49M kinase (Fig.
7). An apparent increase of the MurC activity was observed in
thelattercase,suggestingthatthepresenceofthemutantPknA
protein, although inactive, stabilized in some way the MurC
protein (protein-protein interactions) and reduced the slight
loss of activity observed for MurC during the preincubation
period. These results were consistent with the in vitro and in
vivoanalysesperformedwiththeMurCmutantsindicatingthat
specific threonine residues were important in terms of enzy-
matic activity. Therefore, the PknA-mediated phosphorylation
of MurC seems to represent a key mechanism for regulating/
controlling its activity. Phosphorylation may rather modulate
MurC activity on a fine-tuned level rather than a strict on/off
mechanism. However, further work is needed to understand at
a molecular level whether and how phosphorylation of MurC
modifies the overall structure of this protein and negatively
regulatesthebindingof L-alaninetoUDP-MurNActogenerate
the peptide moiety of the PG disaccharide peptide monomer
unit.
We have previously shown that C. glutamicum PknA and
PknB are key players in signal transduction pathways for the
regulationofthecellshapeandarebothessentialforsustaining
corynebacterialgrowth(18).Inthisstudy,weextendedourpre-
vious findings and demonstrated for the first time that PknA is
able to specifically phosphorylate the essential murein ligase
FIGURE 6. Effect of MurC phosphorylation site mutagenesis on in vitro
and in vivo activity. A, in vitro assays of the wild-type and phosphorylation
sitemutant(1Tto6T)MurCproteins.PurifiedMurCproteinswereassayedas
described under “Experimental Procedures.” Three independent experi-
ments were performed, yielding similar results. The activity of the wild-type
protein (here represented as 100%) was 2650 nmol/min/mg of protein, and
those of the 1T, 2T, 3T, 4T, 5T, and 6T MurC mutants were 875, 800, 1050, 75,
20,and10nmol/min/mgofprotein,respectively.B,invivofunctionalcomple-
mentation assays using an E. coli temperature-sensitive murC mutant strain.
The pTrc99A plasmid vector and derivative plasmids expressing wild-type or
mutated versions of the C. glutamicum MurC protein were transformed into
the E. coli thermosensitive murC mutant strain H1119. Functional comple-
mentation was assayed by following the growth of the transformants at the
permissivetemperatureof30 °C(leftpanel),oratthenon-permissivetemper-
ature of 42 °C (right panel).
FIGURE7.ComparativeenzymaticactivitiesofMurCphosphorylatedver-
sus non-phosphorylated. Purified wild-type MurC protein was phosphoryl-
atedeitherwithPknAorPknA_K49M,andMurCligaseactivitywasassayedas
describedunder“ExperimentalProcedures.”Twoindependentproteinprep-
arations were assayed in triplicate, yielding similar results.
MurCPhosphorylationinC.glutamicum
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enzyme MurC, both in vitro and in vivo, and that post-transla-
tional modifications appear to be a way to regulate its ligase
activity. Moreover, due to the fact that corynebacteria are
extensively used for industrial production of amino acids such
asglutamate,theseresultsmayopenthewaytomanipulationof
the cell wall chemical structure to increase amino acid secre-
tion. Furthermore, these findings could be useful to extend our
presentunderstandingofthedcw(division-cellwallbiosynthe-
sis) cluster in Gram-positive bacteria toward designing of new
antimicrobial drugs targeting the cell wall metabolism pro-
cesses like the one involving the Mur enzymes. Moreover, if
theseinhibitorsarecapableofinterferingwiththeseregulatory
processes through selective inhibition of PknA or MurC phos-
phorylation, it may lead to the design of novel classes of inhib-
itors. Indeed, the development of STPK inhibitors and the
expertise in designing such inhibitors for eukaryotic STPKs
represent an intense research area and could be exploited for
the development of new drugs, especially toward the patho-
genic corynebacteria Corynebacterium diphtheriae.
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