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Microbiology (2003), 149, 695–705
DOI 10.1099/mic.0.25875-0
Characterization of AcmB, an
N-acetylglucosaminidase autolysin from
Lactococcus lactis
Carine Huard,13 Guy Miranda,1 Françoise Wessner,1 Alexander Bolotin,2
Jonathan Hansen,3 Simon J. Foster3 and Marie-Pierre Chapot-Chartier1
1,2
Marie-Pierre Chapot-Chartier
Unité de Biochimie et Structure des Protéines1 et Unité de Génétique Microbienne2, INRA,
Domaine de Vilvert, 78352 Jouy-en-Josas Cedex, France
[email protected]
3
Correspondence
Received 12 July 2002
Revised 24 October 2002
Accepted 28 November 2002
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court,
Western Bank, Sheffield S10 2TN, UK
A gene encoding a putative peptidoglycan hydrolase, named acmB, which is a paralogue of the
major autolysin acmA gene, was identified in the Lactococcus lactis genome sequence. The acmB
gene is transcribed in L. lactis MG1363 and its expression is modulated during cellular growth. The
encoded AcmB protein has a modular structure with three domains: an N-terminal domain,
especially rich in Ser, Thr, Pro and Asn residues, resembling a cell-wall-associated domain; a
central domain homologous to the Enterococcus hirae muramidase catalytic domain; and a Cterminal domain of unknown function. A recombinant AcmB derivative, devoid of its N-terminal
domain, was expressed in Escherichia coli. It exhibited hydrolysing activity on the peptidoglycan of
several Gram-positive bacteria, including L. lactis. Though showing sequence similarity with
enterococcal muramidase, AcmB has N-acetylglucosaminidase specificity. The acmB gene was
inactivated in order to evaluate the role of the enzyme. AcmB does not appear to be involved in cell
separation but contributes to cellular autolysis.
INTRODUCTION
Peptidoglycan, a polymer of amino sugars cross-linked by
short peptides, is the major component of Gram-positive
bacterial cell walls and ensures cell wall integrity and rigidity. Bacteria synthesize peptidoglycan hydrolases capable
of hydrolysing their own peptidoglycan (Shockman &
Höltje, 1994). Although threatening cell integrity, peptidoglycan hydrolases are synthesized during cellular growth.
They are involved in a number of cellular functions which
require cell wall remodelling, such as cell separation after
division, cell wall turnover and cell wall expansion (Smith
et al., 2000). According to the chemical bond cleaved inside
the peptidoglycan molecule, four different specificities are
defined: N-acetylmuramidase, N-acetylglucosaminidase,
N-acetylmuramyl-L-alanine amidase and endopeptidase.
Lactococcus lactis is a lactic acid bacterium widely used in
starters for cheese making. During cheese ripening, the
bacterial intracellular enzyme content contributes to the
3Present address: AFSSA, LERPRA, Les Templiers, 105 route des
Chappes, BP111, 06902 Sophia-Antipolis Cedex, France.
Abbreviations: MALDI-TOF, matrix-assisted laser desorption ionization
time-of-flight; PI, propidium iodide; TFA, trifluoroacetic acid.
The EMBL/GenBank accession number for the sequence reported in
this paper is AJ414526.
0002-5875 G 2003 SGM
development of organoleptic properties (Chapot-Chartier,
1996; Crow et al., 1995). The cell envelope appears as a
physical barrier for the bacterial enzymes to reach the
extracellular substrates. Bacterial autolysis was previously
shown to enhance the contribution of enzymes to cheese
flavour formation. Especially, autolysis leads to the release
of the intracellular peptidases into the cheese curd, and as
a result more free amino acids, which are aroma precursors,
are produced and hydrophobic bitter peptides are degraded
(Chapot-Chartier et al., 1994; Wilkinson et al., 1994).
Besides this traditional use in dairy fermentation, L. lactis
has been proposed as a vaccine vehicle or delivery vector
for use in human medicine (Drouault et al., 1999; Wells
et al., 1996). For these applications, L. lactis autolysis in the
gastro-intestinal tract is also a critical parameter to obtain
an optimal response. In order to get insight into the autolysis mechanism and to control it, it is necessary to identify
and characterize the peptidoglycan hydrolases involved.
Up to now, only the major autolysin, named AcmA, has
been characterized at the molecular level in L. lactis (Buist
et al., 1995). Like most bacterial peptidoglycan hydrolases,
AcmA has a modular structure with two domains. The
N-terminal domain exhibits sequence similarity with the
catalytic domain of Enterococcus hirae muramidase Mur-2,
and the C-terminal one contains three amino acid repeats
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C. Huard and others
involved in cell wall binding (Buist, 1997). As regards its
enzymic specificity, AcmA was recently shown to be an
N-acetylglucosaminidase (Steen et al., 2001), rather than
an N-acetylmuramidase as predicted by sequence similarity.
AcmA is required for proper separation of cells after cell
division and is involved in cellular autolysis in stationary
phase in synthetic culture medium (Buist et al., 1997).
However, other factors than AcmA contribute to determining the autolysis rate when cells are in the cheese matrix
environment (Pillidge et al., 1998). This prompted us to
search for new peptidoglycan hydrolases and evaluate their
contribution to cellular autolysis in L. lactis.
In the genome sequence of L. lactis subsp. lactis IL1403
(Bolotin et al., 2001), we could identify four putative
peptidoglycan hydrolases, in addition to AcmA, on the basis
of a sequence similarity search. In this study, we have
investigated the functionality, hydrolytic bond specificity
and the role of one of these peptidoglycan hydrolases,
named AcmB.
METHODS
L. lactis, 150 mg ml21 for E. coli), chloramphenicol (5 mg ml21 for
L. lactis; 20 mg ml21 for E. coli) and ampicillin (100 mg ml21 for
E. coli).
General recombinant DNA techniques. Molecular cloning tech-
niques were performed using standard procedures (Sambrook et al.,
1989). Restriction enzymes (Eurogentec or Roche Molecular
Biochemicals), T4 DNA ligase (Epicentre), Taq DNA polymerase
(Appligene Oncor) and calf intestinal alkaline phosphatase (New
England Biolabs) were used according to the suppliers’ recommendations. The oligonucleotides were purchased from Invitrogen.
L. lactis DNA was isolated as described previously (Pospiech &
Neumann, 1995). PCR amplifications were carried out in a
GeneAmp PCR System 2400 (Perkin Elmer). pGEMT easy vector
(Promega Corporation) was used to clone PCR products in E. coli.
DNA sequences were determined on an Applied Biosystems 370A
automated DNA sequencer using the ABI PRISM Dye Terminator
Cycle Sequencing Kit and the Dye Primer Cycle Sequencing Kit
(Perkin Elmer). DNA and protein sequences were assembled and
analysed with the Genetic Computer Group sequence analysis package (GCG, Madison, WI, USA) and the tools available on the
Expasy (Expert Protein Analysis System) Biology Server of the Swiss
Institute of Bioinformatics (SIB) (http://www.expasy.org). Electroporation of L. lactis was performed as described by Holo & Nes
(1995) and transformants were plated on M17+glucose agar plates
containing the required antibiotic.
Bacterial strains, plasmids and growth conditions. The bacter-
Cloning of the acmB gene from L. lactis MG1363. With
ial strains and plasmids used in this study are listed in Table 1.
Escherichia coli strains were grown at 37 ˚C in Luria–Bertani (LB)
medium with shaking. L. lactis strains were grown in M17 medium
(Difco) supplemented with 0?5 % (w/v) glucose at 30 ˚C. Growth
was monitored by measuring OD650 with a spectrophotometer
(Uvicon 931, Biotek Kontron). The following antibiotics were added
as selective agents when appropriate: erythromycin (5 mg ml21 for
the primers AU4 (59-GATTATCTTTATCTCGTACAC-39 and AU11
(59-CAACTGCAGCACAAATTCC-39) selected from the IL1403
sequence genome (Bolotin et al., 2001), a 1?6 kb DNA fragment was
amplified by PCR from L. lactis subsp. cremoris MG1363 total DNA.
This fragment contained only part of the MG1363 acmB gene. PCR
cloning of the 59-end failed, although several pairs of primers were
tested. The sequence of the 1077 bp region upstream of the 1674 bp
Table 1. Bacterial strains and plasmids used in this study
Strain or plasmid
Strains
E. coli TG1
E. coli TG1repA+
E. coli M15(pREP4)
L.
L.
L.
B.
L.
lactis IL1403
lactis MG1363
lactis MG1363acmAD1
subtilis HR
lactis MG1363acmB
L. lactis MG1363acmAD1acmB
Plasmids
pQE60
pG+host9
pTIL328
pGEMT-easy
Relevant genotype/phenotype
supE hsdD5 thi D(lac–proAB) F9[traD36 proAB+ lacIq lacZDM15]
TG1 derivative with repA gene integrated into the chromosome,
allowing replication of L. lactis plasmids
Derivative of E. coli K-12, containing pREP4 plasmid ensuring the
production of high levels of lac repressor protein; Kanr
Plasmid-free strain
Plasmid-free strain
Derivative of MG1363 carrying a 701 bp deletion in acmA
Derivative of B. subtilis 168, trpC2
Derivative of MG1363 carrying a 414 bp deletion
in acmB and a Cmr cassette inserted; Cmr
Derivative of MG1363 carrying deletions in both acmA and acmB; Cmr
Expression vector for C-terminal hexa-His-tag fusion; Apr
Thermosensitive plasmid, for gene replacement
by double-crossover integration; Eryr
pG+host9 with 414 bp deleted acmB gene and a Cmr cassette inserted
Cloning vector for PCR products; Apr
Source/reference
Gibson (1984)
P. Renault*
Qiagen
Chopin et al. (1984)
Gasson (1983)
Buist et al. (1995)
S. J. Foster3
This study
This study
Qiagen
Biswas et al. (1993)
This study
Promega
*INRA, Jouy-en-Josas, France.
3University of Sheffield, UK.
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Microbiology 149
Peptidoglycan hydrolase AcmB from L. lactis
amplified region was obtained from a long-range 10 kb DNA
fragment amplified by Multiplex Long accurate PCR from MG1363
total DNA (Bolotin et al., 2002). This 10 kb fragment was amplified with two primers derived from the IL1403 sequence: mc91.pri
(59-ATCCGTACGGCCGAAGTGCCT-39) and mb95.pri (59-GGTATCGGTAACATCCACGGTC-39), corresponding to metK and fbaA
MG1363 sequencing tags. Finally, we obtained the sequence of a
2751 bp DNA fragment encompassing the acmB gene, which was
located in the central region of the fragment.
Inactivation of acmB by double crossing-over integration. A
2?4 kb fragment encompassing the acmB gene was amplified by PCR
with the two primers: AU57 (59-TCCCCCCGGGAATGGAAACCCAAAGTATAGG-39; SmaI restriction site underlined) and AU58
(59-GGGGTACCTTTTTCATCCGAAGTTTCTG; KpnI restriction
site underlined) with MG1363 DNA as template. The PCR product
was cloned into the pGEMT-easy vector. The insert was recovered
with SmaI and KpnI digestion and was then ligated in the corresponding sites of pG+host9 plasmid. The ligation mixture was used
to transform E. coli TG1repA+competent cells. In the resulting
recombinant plasmid, an internal 414 bp fragment of the acmB gene
was deleted by digestion with EcoRI and PstI. A chloramphenicolresistance cassette with a size of 0?9 kb (Trieu-Cuot et al., 1992) was
then ligated into the PstI and EcoRI sites. The final plasmid pTIL328
was electroporated into L. lactis MG1363 and MG1363acmAD1.
Integration of pTIL328 into the chromosome and subsequent excision was achieved according to the previously developed protocol
(Biswas et al., 1993). Mutant strains were screened first on their
resistance to chloramphenicol and second on the size of the fragment amplified by PCR with primers AU57 and AU58. The presence
of a correct insertional event was further verified by Southern
blotting. When the properties of the acmB mutants were analysed,
chloramphenicol was omitted from the culture.
Northern blotting. Total RNA fractions were extracted from L. lactis
MG1363 grown at 30 ˚C as previously described (Anba et al., 1995), at
different stages of growth, corresponding to OD650 0?1, 0?5, 1?0, 1?5,
2?0, 2?5 and 3?0. Northern blot hybridization was performed according to a standard protocol (Sambrook et al., 1989). Twenty micrograms of total RNA were electrophoresed in a 1 % (w/v) agarose gel
(Seakem GTG Agarose, BMA Bioproducts), transferred to HybondN+ nylon membrane (Amersham Pharmacia Biotech) and fixed by
heat treatment (80 ˚C, 2 h). The membrane was probed with a 1?5 kb
DNA fragment corresponding to the entire acmB gene, and labelled
with [a-32P]dCTP with the Random Primed DNA Labelling Kit
(Roche Molecular Biochemicals). Hybridization was performed under
high-stringency conditions (50 % formamide). The membrane was
then dehybridized by immersion in boiling 16 SSC/0?2 % SDS and
subsequent incubation for 1 h at room temperature. It was rehybridized with a 16S rRNA specific probe obtained by PCR amplification
as described by Cibik et al. (2000). Radioactivity was quantified with
a PhosphorImager (Molecular Dynamics) with the ImageQuaNT program. The relative amounts of acmB transcript were standardized by
hybridization with a L. lactis 16S rRNA specific probe.
Expression and purification of His-tagged proteins in E. coli.
The proteins were overexpressed in E. coli M15(pREP4) as C-terminal
hexa-His-tagged proteins, with the expression vector pQE60
(Qiagen). Two truncated derivatives of the acmB gene encoding
polypeptides corresponding to the AcmB catalytic domain alone,
and to the catalytic domain plus the C-terminal domain of AcmB,
were amplified by PCR from L. lactis MG1363 total DNA. The
DNA fragments were amplified respectively with the primers AU65
(59-CATGCCATGGTATATATGGGGCCTGTATT-39; NcoI restriction
site underlined) and AU68 (59-GAAGATCTACTGCGACATTATCATAAAAA-39; BglII restriction site underlined), and AU65 and
AU70 (59-GAAGATCTTTTAGGTTGGATATAAGTTGC-39; BglII
restriction site underlined). PCR fragments were digested by NcoI
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and BglII introduced with the primers and cloned in-frame
upstream of the hexa-His box sequence in the pQE60 vector, precut
by the same enzymes. E. coli M15(pREP4) competent cells were
transformed with the resulting plasmids. IPTG was added at a final
concentration of 1 mM to the culture at an OD650 of 0?5 to induce
the expression of the hexa-His-tagged proteins. Bacteria were grown
at 37 ˚C until IPTG addition and were then transferred at 28 ˚C
during the expression time (4 h) to avoid the formation of inclusion
bodies. The cells were harvested by centrifugation and broken by
one passage at a pressure of 1600 bar with a Constant Cell Disruption
System (Constant System, Warwickshire, UK). The soluble fraction
containing the recombinant protein was collected by centrifugation
at 15 000 g for 15 min at 4 ˚C. The hexa-His-tagged proteins were
purified by affinity chromatography on Ni2+-nitrilotriacetic acid
(Ni-NTA) spin columns (Qiagen), according to the manufacturer’s
instructions.
Preparation of cell wall peptidoglycan. Peptidoglycan from
Bacillus subtilis HR vegetative cells was prepared as described previously (Atrih et al., 1999). Peptidoglycan from L. lactis MG1363
was prepared from an exponentially growing culture (OD650 0?7)
according to the protocol of Mainardi et al. (2000). Briefly, cells
were boiled in 4 % (w/v) SDS for 30 min. Cell walls were recovered
by centrifugation for 90 min at 140 000 g and washed three times
with water to eliminate SDS. To remove proteins, the cell wall pellet
was treated with Pronase (200 mg ml21) for 16 h at 37 ˚C, then
by trypsin (200 mg ml21) for 16 h at 37 ˚C. The pellet containing
peptidoglycan was washed twice with water and stored at 220 ˚C.
SDS-PAGE and renaturing SDS-PAGE. SDS-PAGE was per-
formed as described by Laemmli (1970) with 15 % (w/v) polyacrylamide separating gels. Renaturing SDS-PAGE was performed as
previously described (Lepeuple et al., 1998). The polyacrylamide gels
contained 0?2 % (w/v) Micrococcus luteus ATCC 4698 (Sigma) or
0?4 % (w/v) L. lactis autoclaved cells, or 0?16 % (w/v) MG1363
peptidoglycan as enzyme substrate. Gels were washed for 1 h in
deionized H2O at room temperature and then incubated in buffer
containing 1 % (v/v) Triton X-100, overnight at 37 ˚C. The following
incubation buffers were used: 25 mM sodium citrate/50 mM
sodium phosphate at pH 3?0, 4?0 or 5?0; 50 mM MES at pH 6?0;
and 50 mM Tris/HCl at pH 7?0 and 8?0. The gels were subsequently
washed for 1 h in deionized H2O and when required, with 10 mM
Tris/HCl, pH 7?0, containing 0?1 % SDS to remove precipitated
proteins from the gels. The gels were then stained with 0?1 % methylene blue in 0?01 % (w/v) KOH for 2 h at room temperature and
destained in deionized H2O. Gel images were generated with a
Duoscan T1200 scanner (Agfa-Gevaert) customized for proper gel
handling and controlled by the Agfa Photolook 3.0 software.
Determination of hydrolytic bond specificity. The hydrolytic
bond specificity of AcmB was analysed using peptidoglycan from
B. subtilis HR vegetative cells as substrate. Peptidoglycan (5 mg) was
incubated overnight at 37 ˚C, with 500 mg purified hexa-His-tagged
recombinant AcmB[C+Z]-His in a final volume of 500 ml buffer
(25 mM sodium citrate/50 mM sodium phosphate, pH 4?0). Samples
were boiled for 3 min to stop the reaction. Insoluble material was
removed by centrifugation at 14 000 g. Half of the soluble muropeptide fraction was further digested with Cellosyl (250 mg ml21). The
soluble muropeptides obtained after digestion with AcmB[C+Z]-His
or AcmB[C+Z]-His plus Cellosyl were reduced with sodium borohydride (8 mg ml21) as described previously (Atrih et al., 1999). The
reduced muropeptides were separated by RP-HPLC with a Biocad
Sprint system (Perkin Elmer) equipped with a variable-wavelength
double detector and an automatic collector (Advantec) using a
Hypersyl PEP100 C18 column (25064?6 mm, 5 mm, 100 Å) (Thermo
Finnigan). Elution was carried out at a flow rate of 1 ml min21 for
10 min with 100 % solvent A (TFA/H2O; 1?15 : 1000, v/v) and subsequently with a 120 min linear gradient (0 to 20 %) of solvent B
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C. Huard and others
(TFA/CH3CN; 1 : 1000, v/v). Absorbance was monitored at both 220
and 280 nm and the eluate collected in 1 ml volume fractions.
Muropeptide-containing fractions were dried and then resuspended
in 10 ml 0?15 % TFA in water. They were subsequently analysed by
matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry using a Voyager DE STR mass spectrometer (Perseptive Biosystem, Framingham, USA). The precision in
the mass determination was 0?01 %. One microlitre of the sample
was mixed with 1 ml a-cyano-4-hydroxycinnamic acid. A 1?2 ml drop
of the mixture was deposited on the steel plate and was allowed to
air dry. The muropeptides were desorbed and ionized by a N2 laser
in the positive- and/or negative-ion mode. Each mass spectrum was
a mean of 200 scans. For all experiments, the accelerating potential
was held at 20 kV and laser power was set to the minimum level
necessary to generate a reasonable signal. An external mass calibration standard containing bradikinin fragment 1–5, angiotensin I,
neurotensin and melittin was employed for all the analyses.
Autolysis in buffer solution. L. lactis strains were grown in M17
medium to mid-exponential phase (OD650 1?0). Cells were harvested
by centrifugation at 5000 g for 15 min at 4 ˚C and washed once with
sterile deionized water. They were resuspended in 50 mM potassium
phosphate buffer pH 7?0 at OD650 0?8 and incubated at 30 ˚C.
Autolysis was monitored by measuring the OD650 of the cell suspension. The extent of autolysis was expressed as the percentage
decrease in OD650. The intracellular X-prolyldipeptidyl aminopeptidase (PepX) was chosen as a marker for cell autolysis (Lepeuple
et al., 1998). Its release into the culture supernatant was monitored
by measuring enzymic activity with Ala-Pro-p-nitroanilide (Bachem)
chromogenic substrate. Enzymic activity was expressed in katals
(1 katal is the quantity of enzyme releasing 1 mol p-nitroaniline
s21) per ml of culture.
Determination of the proportion of cells with a damaged
membrane. The membrane integrity of the cells incubated in buffer
solution as described above was investigated by the fluorescence
labelling procedure described by Niven & Mulholland (1998). The
fraction of permeable cells in the whole L. lactis cell population was
estimated by labelling with propidium iodide (PI), a fluorescent dye,
which stained only bacteria with a damaged membrane. The whole
population of cells was estimated by PI-induced fluorescence after
treatment with cetyltrimethylammonium bromide (CTAB) (Sigma),
a cationic surfactant used to permeabilize bacterial cytoplasmic
membranes. Bacterial suspension (1 ml) was incubated with 30 mM
PI (Molecular Probes) with or without 200 mM CTAB for 30 min
at 25 ˚C in the dark. Labelled cell suspensions were kept on ice
until fluorescence measurement (less than 15 min). Fluorescence
was measured in a 1 cm cuvette with an excitation wavelength of
500 nm and emission wavelength of 600 nm with a Kontron
Instrument SFM25 spectrofluorimeter. The fraction of permeable
cells in the cell population was calculated with the following formula
(cellsPI 2 cellsalone 2 bufferPI)/(cellsPI+CTAB 2 cellsalone 2 bufferPI+CTAB)
as described by Walker & Klaenhammer (2001).
study acmB from L. lactis subsp. cremoris MG1363, a strain
cured of its inducible prophage (Gasson, 1983). The
homologous acmB gene from MG1363 total DNA was
first cloned. The nucleotide sequence of a 2751 bp DNA
fragment encompassing acmB was determined (accession
number AJ414526). acmB contains 1616 bp and is preceded by a putative ribosome-binding site. Two putative
promoters as identified by 235 and 210 boxes are present
upstream of the start codon and a putative transcription
terminator is present downstream of the stop codon.
AcmB, a peptidoglycan hydrolase with an
uncommon three-domain modular structure
The acmB gene encodes a 538-residue protein with calculated molecular mass of 57?1 kDa and pI of 4?72. The first
33 N-terminal residues of AcmB possess the characteristics
of a signal peptide (Nielsen et al., 1997). They could also
constitute an N-terminal signal anchor sequence since a
transmembrane helix is predicted in positions 14–33
(Tusnady & Simon, 2001). According to protein sequence
similarities, the AcmB protein has a three-domain modular
organization (Fig. 1A).
The central domain (residues 212–390) of AcmB exhibits
sequence similarity with the catalytic domain of Ent. hirae
muramidase-2 (39?9 % identity) (Chu et al., 1992) and
numerous related proteins present in the databases. In
particular, sequence similarity was found with peptidoglycan hydrolases from lactic acid bacteria – L. lactis major
autolysin AcmA (41?5 %) (Buist et al., 1995), Streptococcus
thermophilus Mur1 (44?5 %) (Husson-Kao et al., 2000),
Leuconostoc citreum Mur (38 %) (Cibik et al., 2001) – and
with two other L. lactis paralogue proteins (Bolotin et al.,
2001) (Fig. 2). The catalytically important acidic residues
identified in the enterococcal muramidase family (Joris
et al., 1992) are conserved in L. lactis AcmB (Glu at position
299 and Asp at position 319).
The C-terminal domain (residues 390–538) shares sequence
similarity with several cell-wall-bound proteins such as the
putative transfer protein TraG of Staphylococcus aureus
RESULTS
Cloning of acmB from L. lactis subsp. cremoris
MG1363
From the sequence similarity search, a gene named acmB,
encoding a putative peptidoglycan hydrolase, was identified
in the complete genome sequence of L. lactis subsp. lactis
IL1403 (Bolotin et al., 2001). Since the IL1403 genome
contains several inducible prophages, and the induction
can cause cellular lysis (Chopin et al., 2001), we chose to
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Fig. 1. Schematic representation of the domain structure of
AcmB (A) and the His-tagged AcmB derivatives (B). SP, signal
peptide; [A], putative cell-wall-associated domain; [C], putative
catalytic domain; [Z], C-terminal domain containing a putative
zinc-binding motif. Black boxes indicate the hexa-His tag added
at the C-terminus of AcmB derivatives expressed in E. coli.
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Microbiology 149
Peptidoglycan hydrolase AcmB from L. lactis
Fig. 2. Alignment of amino acid sequences
of AcmA, AcmB, AcmC and AcmD of
L. lactis, muramidase-2 of Ent. hirae (Mur2),
Mur1 of Strep. thermophilus and Mur of Ln.
citreum. Alignment was done with the
ClustalW program. (*), identical amino acids
in the seven proteins; (:), similar amino acids.
Glu and Asp residues conserved in the
active centre of the enzymes are shaded.
involved in conjugative transfer of plasmids (Morton et al.,
1993), the immunogenic secreted protein (Isp) of group
A streptococci (McIver et al., 1996) and the major
secreted L. lactis protein Usp45 with unknown function
(van Asseldonk et al., 1990). The precise role of this
domain in the proteins is not known. Interestingly, the
AcmB C-terminal domain contains a 33-amino-acid
sequence Tyr-X-His-X7-Tyr-X13-Gly-X7-His resembling a
zinc-binding motif (Ramadurai et al., 1999).
The N-terminal domain (residues 34–211) has a remarkably
high content of the amino acids Ser (29 %), Thr (15 %), Asn
(10 %) and Pro (8 %). This characteristic is encountered in
protein domains associated with the cell wall in Grampositive bacteria (Fischetti et al., 1991). A very similar Ser-,
Thr-, Gly-, Pro-rich domain was previously described in the
cell-bound fructosyltransferase of Streptococcus salivarius
(Rathsam et al., 1993) and this domain was shown to play
a role in cell surface attachment of the protein (Rathsam &
Jacques, 1998).
A putative protein similar to AcmB and composed of three
homologous domains with 39 % sequence similarity was
found only in the Staph. aureus genome sequence (Kuroda
et al., 2001).
acmB expression is regulated during cellular
growth
Transcriptional analysis of acmB during cellular growth
was studied by Northern blot analysis. Total RNA was
isolated from cells harvested at different stages of growth at
30 ˚C in M17 medium. Using an acmB probe, we detected
a single 1?6 kb transcript (Fig. 3A). This size corresponds
to a monocistronic organization of acmB. Quantitative
mRNA analysis revealed that the relative abundance of
the transcript varied during growth (Fig. 3B). Earlyexponential-phase cells (OD650 0?1) contained a substantial
amount of acmB mRNA, which rapidly declined to become
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Fig. 3. Northern blot analysis of acmB expression during cellular growth. Total RNA was prepared from L. lactis MG1363
cells, grown in M17 medium, at different times of the culture
and analysed by Northern blotting. (A) Autoradiogram of the
membrane hybridized successively with radioactive acmB and
16S rRNA probes. (B) Quantitative analysis of the acmB transcript during cellular growth. Growth was followed by OD650
(e) measurement. Hybridization signals were quantified with a
PhosphorImager. The relative amount of acmB transcript (&) is
expressed with respect to the highest level of transcript (measured at OD650 0?1). The relative amount of acmB transcript
was standardized by hybridization with a L. lactis 16S rRNA
specific probe.
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C. Huard and others
3?5 times less abundant at an OD650 of 0?5. Then, the
amount of acmB mRNA increased slightly during the
exponential growth phase, followed by a slow decline until
stationary phase. These results indicate that acmB is
transcribed in L. lactis MG1363 and its transcription is
regulated during cell growth.
AcmB has peptidoglycan-hydrolysing activity
No activity band which could correspond to AcmB was
detected by renaturing SDS-PAGE with M. luteus or L. lactis
cells as substrate, in cell extracts from L. lactis MG1363 or
from the acmA mutant, although this latter strain lacks the
activity bands of AcmA and its degradation products (data
not shown). Therefore, in order to examine AcmB
peptidoglycan-hydrolysing activity, the enzyme was overproduced in E. coli. The full-length protein without its
putative signal sequence and with a C-terminal hexa-His tag
could not be produced in E. coli despite several attempts
with different expression vectors and recipient strains.
Conversely, two truncated derivatives of AcmB were
obtained in large amounts with the pQE60 expression
system. These derivatives correspond to the catalytic
domain plus the C-terminal domain of AcmB (AcmB
[C+Z]-His) and to the catalytic domain of AcmB alone
(AcmB[C]-His), which were expressed without signal
peptide and with a C-terminal hexa-His tag (Fig. 1B).
The His-tagged derivatives of AcmB were purified by nickel
affinity chromatography under native conditions (Fig. 4A).
Their peptidoglycan-hydrolysing activity was examined by
renaturing SDS-PAGE with M. luteus autoclaved cells as
substrate (Fig. 4B). The two proteins gave a clear hydrolysis
band with incubation buffer at pH 4?0. The detection of
AcmB[C]-His activity indicates that the [C] domain retains
peptidoglycan hydrolase activity and confirms its assignment as the catalytic domain. In addition, these results
indicate that cell wall binding is not an absolute requirement for enzyme activity. It is worth noting that a rather
high amount of the purified His-tagged proteins was
loaded on the gel for activity to be detected. Thus, the
absence of detection of AcmB activity in L. lactis cellular
extracts results most probably from too low an amount of
protein expressed in the cells.
The activity of AcmB[C+Z]-His was also examined by
renaturing SDS-PAGE on L. lactis cell walls with incubation
buffer at pH 4?0. A clear hydrolysis band was observed
(data not shown). Since the N-terminal [A] domain is most
probably not involved in specific recognition of the cell
wall, we can conclude that AcmB is an autolysin.
The influence of the incubation buffer pH on activity
detection was tested on M. luteus cell substrate. AcmB
[C+Z]-His activity was detected at pH 4?0 and 5?0 but
not at pH 6?0 and higher, nor at pH 3?0. AcmB[C+Z]His has a low pI (5?03) and interestingly, its activity was
detected only at acidic pH in the renaturing electrophoresis test. This result suggests that interaction with
the negatively charged cell walls is favoured by positive
or neutral charge of the protein.
AcmB is an N-acetylglucosaminidase
The purified recombinant N-terminal-domain-truncated
AcmB (AcmB[C+Z]-His) was found by renaturing SDSPAGE to hydrolyse B. subtilis vegetative cell walls (data not
shown). Therefore, the hydrolytic bond specificity of AcmB
was investigated on B. subtilis vegetative peptidoglycan,
whose fine structure has been previously studied in detail
(Atrih et al., 1999).
Peptidoglycan extracted from B. subtilis vegetative cells was
incubated with purified recombinant AcmB[C+Z]-His
overnight at 37 ˚C in phosphate/citrate buffer at pH 4?0.
A slight reduction of OD450 was observed (less than 5 %).
The soluble fraction was recovered and analysed by RPHPLC (Fig. 5A). Peaks 1 and 2 were products of AcmB
digestion since they were absent from the control consisting
of cell walls incubated without enzyme (data not shown).
When analysed by MALDI-TOF mass spectrometry, they
gave molecular ions with m/z values of 892?36 and 1814?70
respectively. According to the data of Atrih et al. (1999),
these m/z values correspond to peak 1 containing the
disaccharide tripeptide muropeptide, and peak 2 containing
Fig. 4. SDS-PAGE and renaturing SDSPAGE analysis of purified AcmB[C+Z]-His
(lane 1) and AcmB[C]-His (lane 2). The
purified proteins were migrated on a 15 %
polyacrylamide gel. (A) Coomassie blue
staining; (B) renaturing SDS-PAGE in gel
containing 0?2 % (w/v) M. luteus cells.
Renaturation was done in citrate/phosphate
buffer at pH 4?0 with 1 % Triton X-100. The
molecular masses (in kDa) of the standard
proteins are indicated on the left.
700
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Microbiology 149
Peptidoglycan hydrolase AcmB from L. lactis
Properties of an acmB mutant
To investigate the role of AcmB, a single acmB mutant
and a double acmA acmB mutant were constructed by
gene replacement with the pG+host-9 thermosensitive
vector in the MG1363 strain. The chromosomal copy of
acmB was replaced by a mutant copy obtained by deletion
of a 414 bp fragment inside the region encoding the
catalytic domain of AcmB, followed by insertion of a
chloramphenicol-resistance cassette.
Fig. 5. Analysis by RP-HPLC of the soluble muropeptides
released from B. subtilis vegetative cell walls after incubation
with AcmB (A) or AcmB and Cellosyl (B). The numbers indicate the peaks analysed by MALDI-TOF mass spectrometry.
the disaccharide tripeptide disaccharide tetrapeptide muropeptide (Table 2, Fig. 6). The AcmB-soluble muropeptides
were subsequently incubated with Cellosyl, which is an
N-acetylmuramidase. New major peaks appeared on the
RP-HPLC profile (Fig. 5B), whereas the peaks 1 and 2
present in the AcmB digest became minor peaks. By
MALDI-TOF analysis, peaks 3, 4 and 5 gave molecular ions
with m/z values of 689?11, 1386?66 and 1611?96 respectively.
These values correspond to peak 3 containing disaccharide
tripeptide missing one N-acetylglucosamine, and peaks 4
and 5 containing disaccharide tripeptide disaccharide
tetrapeptide missing two and one N-acetylglucosamine
respectively (Table 2, Fig. 6). These results indicate that
the muropeptides generated by AcmB hydrolysis can be
cleaved by Cellosyl and thus that N-acetylglucosamine is
present on the reducing end of the disaccharide of these
muropeptides. AcmB hydrolyses the peptidoglycan bonds
between N-acetylglucosamine and N-acetylmuramic acid
and thus AcmB is an N-acetylglucosaminidase.
The acmB mutant was not impaired in cellular growth nor
in cell separation compared to the wild-type strain. No
change in the cellular morphology of the mutants was
observed by phase-contrast microscopy. The double acmA
acmB mutant was not affected in its growth rate and
formed long chains of cells compared to MG1363, as
previously described for the acmA single mutant (Buist
et al., 1995).
Autolysis of the acmB mutant was measured in 50 mM
potassium phosphate buffer solution at pH 7?0, and compared to that of the wild-type MG1363. As shown in
Fig. 7, the acmB mutant autolysed at a slightly but
significantly lower rate than the wild-type strain. Also the
final extent of autolysis was reduced in the mutant strain
(61 %) compared with the wild-type strain (78 %). The
OD650 decrease was accompanied by a concomitant release
of intracellular peptidase PepX into the culture supernatant
(Fig. 7), which confirms that the OD650 decrease reflected
cellular autolysis and release of intracellular content. The
acmB mutant released a lower amount of PepX activity
than the wild-type MG1363, confirming the differences
revealed by optical density measurement.
The membrane integrity of the cells incubated in buffer
solution was investigated by labelling with PI. PI is a
fluorescent marker for double-stranded DNA, which
cannot cross an intact cell membrane and which thus
selectively labels cells with a damaged membrane. For both
Table 2. Calculated and observed m/z values for protonated, sodiated or deprotonated molecular ions of muropeptides
obtained after hydrolysis of B. subtilis peptidoglycan by AcmB (A) or by AcmB followed by Cellosyl (B) and purification by
RP-HPLC
Peak
Ion
m/z
Observed
Peak
Peak
Peak
Peak
1
2
3
4
(A)
(A)
(B)
(B)
Peak 5 (B)
(M+Na)+
(M+Na)+
(M+Na)+
(M+H)+
(M+Na)+
(M+Na)+
(M2H)2
892?12
1814?70
689?11
1386?66
1408?65
1611?96
1588?04
Dm (Da)*
Muropeptide identification3
Calculated
893?36
1816?76
893?76
1794?80
1816?76
1816?76
1792?76
21?24
22?06
2204?65
2408?14
2408?11
2204?80
2204?76
ds
ds
ds
ds
tri
tri–ds tetra
tri missing 1 GlcNAc
tri–ds tetra missing 2 GlcNAc
ds tri–ds tetra missing 1 GlcNAc
*Dm, difference between the calculated m/z and the observed m/z.
3ds, disaccharide (MurNAc-GlcNAc); di, dipeptide; tri, tripeptide; tetra, tetrapeptide.
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701
C. Huard and others
Fig. 6. Structure of the muropeptides from B. subtilis peptidoglycan obtained after AcmB digestion (1 and 2) or AcmB and
Cellosyl digestion (3, 4 and 5). Numbers refer to the peaks on the chromatograms presented in Fig. 5.
the wild-type and acmB mutant strains, the percentage of
labelled cells at the beginning of incubation in the buffer
solution at pH 7?0 was close to zero. After 6 h incubation,
this percentage reached 36 % and 30 % for wild-type
MG1363 and the acmB mutant, respectively. After 24 h
incubation, 100 % of the MG1363 cells were labelled,
whereas only 78 % of the acmB mutant cells were labelled.
Similar results were obtained in three independent experiments (data not shown). As a conclusion, acmB inactivation leads to a slower rate of membrane integrity loss
under starvation conditions in buffer solution.
For the double MG1363 acmA acmB mutant, no difference
in autolytic properties in buffer solution was observed
compared with the single MG1363 acmA mutant. Both of
them exhibited a lower autolysis rate and lower autolysis
extent compared with the wild-type strain MG1363 (Fig. 7).
Thus, AcmB does not appear to be responsible for the
residual autolytic activity detected in the MG1363 acmA
mutant. The fact that acmB inactivation leads to a reduction of the autolysis rate and extent in wild-type MG1363,
but not in the acmA mutant, suggests that AcmB could act
only in concert with AcmA and that AcmA could potentiate
the hydrolytic action of AcmB.
Fig. 7. Autolysis of L. lactis MG1363 and its derivative mutants
in 50 mM potassium phosphate, pH 7?0, at 30 ˚C. Cells were
grown in M17 medium up to an OD650 of 1?0, washed and
resuspended in the buffer at an OD650 of 0?8. Lysis of wildtype MG1363 (X), acmB mutant ($), acmA mutant (%) and
acmA acmB mutant (n) was monitored by measuring the
OD650 decrease of the cell suspension (continuous lines) and
the PepX activity released into the buffer (dashed lines). PepX
activity was measured with Ala-Pro-p-nitroanilide as substrate.
Similar results were obtained in three independent experiments.
702
DISCUSSION
In this work, we have characterized at the molecular level a
second peptidoglycan hydrolase of L. lactis named AcmB.
The acmB gene was identified on the basis of sequence
similarity in the complete genome sequence of L. lactis
IL1403. Our results show that acmB is expressed during
growth in L. lactis MG1363 and that it specifies an active
autolysin with N-acetylglucosaminidase specificity. Like
most of the previously described bacterial peptidoglycan
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Microbiology 149
Peptidoglycan hydrolase AcmB from L. lactis
hydrolases, AcmB protein has a modular structure. It has
an uncommon structure with three domains.
(4?65) and is active at acidic pH, may be active during
cellular growth.
AcmB exhibits sequence similarity with the Ent. hirae
muramidase-2 catalytic domain (Joris et al., 1992). However,
like the major L. lactis autolysin AcmA (Steen et al., 2001)
and B subtilis peptidoglycan hydrolase LytG (Horsburgh,
2001), which are also homologous to the Ent. hirae
muramidase-2, AcmB exhibits N-acetylglucosaminidase
hydrolytic specificity. Thus, it appears that the enterococcal
muramidase family defined by Smith et al. (2000) on the
basis of sequence similarity contains glycosidases with the
two types of specificities: muramidase or glucosaminidase.
Hitherto, one muramidase, Mur-2 of Ent. hirae, and three
glucosaminidases, LytG, AcmA and AcmB, had been
identified, whereas the hydrolytic specificity of the other
homologous enzymes was not determined experimentally.
In this study, we have shown that AcmB contributes to
cellular autolysis but to a lesser extent than AcmA. In
addition, AcmB action appears to be potentiated by that
of AcmA. This feature is consistent with AcmB being an
exoenzyme. It has been demonstrated previously that the
amino acid repeats present in Staph. aureus Atl autolysin
direct the enzyme to cell division sites (Baba & Schneewind,
1998). AcmB is devoid of such repeats and thus could have a
uniform localization around the cell surface. In contrast to
AcmA, which plays a role in cell separation, AcmB could be
more likely involved in other cellular processes such as cell
enlargement and insertion of new peptidoglycan units. The
fact that inactivation of AcmB did not impair cellular
growth does not exclude such a role for this protein. Indeed,
functional redundancy was previously observed among
B. subtilis autolysins (Smith et al., 2000). Thus, other L. lactis
peptidoglycan hydrolases could compensate for its absence.
Besides AcmA and AcmB, at least three other putative
peptidoglycan hydrolases are present in the L. lactis IL1403
genome sequence.
B. subtilis LytG was proposed to have exoglucosaminidase
specificity (Horsburgh, 2001). The fact that AcmB leads to
only limited hydrolysis of B. subtilis peptidoglycan (less
than 5 % optical density decrease), and that in these
conditions only small muropeptides were released, suggests
that AcmB could also be an exoglucosaminidase.
AcmB is devoid of sequence repeats termed LysM domains
(Bateman & Bycroft, 2000) previously described as cell-walltargeting and peptidoglycan-binding sequences. In contrast, three such LysM repeats are found in L. lactis AcmA
major autolysin. Also, no LPXTG motif, enabling covalent
binding to peptidoglycan (Fischetti et al., 1990), could be
found close to the C-terminus. However, AcmB contains
a domain with a high content of Ser, Thr, Asn and Pro
residues, which could be involved in cell wall interaction.
Such a domain was previously described in Strep. salivarius
fructosyltransferase, and was shown to be required in
combination with a C-terminal transmembrane anchor,
for cell wall attachment of the enzyme (Rathsam et al.,
1993). By analogy, in AcmB, this domain plus the
N-terminal putative transmembrane sequence could be
involved in cell wall attachment of the protein. Like other
Pro-, Ser-, Thr-rich domains (Fischetti et al., 1991), the
AcmB N-terminal domain is likely to have an extended
conformation, and could serve as a cell wall spacer allowing
exposure of the catalytic domain of AcmB at the cellular
surface. Another remarkable feature of this domain is the
presence of several consensus sequences (Asn-X-Ser/Thr)
for N-glycosylation and the high amount of Ser and Thr
residues, which could be sites for O-glycosylation. Glycosylation of the protein could protect it from proteolytic
degradation in the cell wall (Moens & Vanderleyden, 1997).
Recent studies provided evidence that actively metabolizing
B. subtilis cells secrete protons, which bind to anionic sites
in the cell wall (Calamita et al., 2001). It was proposed that
acidification of the cell wall during growth may be one
means for autolysin inhibition (Kemper et al., 1993). We
can speculate that in contrast AcmB, which has a low pI
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ACKNOWLEDGEMENTS
We are grateful to A. Sorokin and D. Ehrlich (Génétique Microbienne,
INRA) for making available the IL1403 sequence before publication.
We thank J.-C. Huet for helpful discussions on muropeptide mass
determination, C. Beauvallet for some help in mass spectrometry
experiments and V. Monnet for critical reading of the manuscript.
C. H. was recipient of a short-term EMBO fellowship. This work was
carried out with financial support of the Commission of the European
Communities (FAIR contract CT98-4396, LAB-lysis).
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