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J. Biol. Chem., Vol. 275, Issue 36, 27768-27774, September 8, 2000
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§¶,§
, and**
From the Laboratory of Microbiology, The Rockefeller
University, New York, New York 10021 and the § Molecular
Genetics Unit, Instituto de Tecnologia Química e
Biológica, Universidade Nova de Lisboa, 2780 Oeiras, Portugal
Received for publication, May 30, 2000, and in revised form, June 22, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The murMN operon, recently identified
in the genome of Streptococcus pneumoniae, encodes for
enzymes involved in the synthesis of branched structured muropeptides
in the pneumococcal peptidoglycan; inactivation of murMN
causes production of a peptidoglycan composed exclusively of linear
muropeptides and a virtually complete loss of resistance in
penicillin-resistant strains (Filipe, S. R., and Tomasz, A. (2000)
Proc. Natl. Acad. Sci. U. S. A. 97, 4891-4896). The
experiments described in this paper follow up these observations. Primer extension analysis was used to identify the putative promoter region of the murMN operon in penicillin-susceptible and
-resistant strains. Selective inactivation of the murN gene
in the penicillin-resistant strain Pen6 caused production of an unusual
peptidoglycan that contained only single amino acid residues in the
muropeptide branches, indicating that the product of murN
was involved with the addition of the second amino acid and the product
of murM was involved with the addition of the first amino
acid (alanine or serine) to the peptidoglycan cross-bridge. Allelic
replacement of the mosaic murM gene of strain Pen6 with
murM of the penicillin-susceptible laboratory strain caused
enrichment of the peptidoglycan in linear muropeptides. The findings
suggest that the genetic determinant primarily controlling the
synthesis of branched muropeptides in the pneumococcal peptidoglycan is
murM.
The cell wall of Streptococcus pneumoniae is a
heterogeneous polymer composed of peptidoglycan and polysaccharides of
different nature (capsular polysaccharides and teichoic acids) and
proteins. The peptidoglycan itself is a highly complex molecule
composed of a glycan (polymers of N-acetylated and
nonacetylated (1) glucosamine and N-acetylmuramic acid
residues) with short stem peptides attached to the glycan chains. These
peptides, when cross-linked by penicillin-binding proteins
(PBPs),1 interconnect
different glycan chains enabling the bacteria to resist high osmotic
pressures. The complexity of the peptidoglycan structure has been fully
recognized after the introduction of a high resolution analytical
technique, high pressure liquid chromatography (HPLC) (2). Use of this
technique for the analysis of stem peptide composition of the
peptidoglycan of clinical and laboratory strains of S. pneumoniae showed a species-specific peptidoglycan characterized
by highly conserved molar ratios of 18 different muropeptides (3). A
peculiar feature of the pneumococcal peptidoglycan is the simultaneous
presence of both directly and indirectly cross-linked (branched)
components. In the latter, alanyl-serine or alanyl-alanine dipeptides
form the cross-bridge between neighboring muropeptides (3, 4). In the
species-specific peptidoglycan, the percentage of these branched
peptides, although detectable, is very small (3-5). Cell wall analysis
by HPLC of the first high level penicillin-resistant clinical isolates,
from South Africa and Hungary, revealed that in the peptidoglycan of
these strains the proportion of branched muropeptides was greatly
increased, and this abnormality of wall structure was to a significant
degree co-transferred with resistance to penicillin during genetic
transformation (6). It has been suggested that the abnormally high
proportion of branched muropeptides in the cell wall of the resistant
strains may provide the bacteria with a set of cell wall precursors,
the branched structure of which has a better "fit" into the
altered, low affinity active site(s) (7) of the remodeled PBPs of the
resistant pneumococcus (6). However, examination of a larger number of
penicillin-resistant isolates showed that the abnormally high
proportion of branched wall peptides was not always associated with
resistance to penicillin. In fact, the abnormality of wall composition
detected in several isolates appeared to be related to the particular
genetic lineage rather than being an obligatory correlate of resistance
itself (4, 5).
The molecular mechanism of penicillin resistance in S. pneumoniae involves remodeling of the Here we report the identification of the sites of action of the MurM
and MurN proteins in the assembly of the dipeptide branches and
describe the impacts of selective inactivation of murN and allelic replacement of murM on the composition of peptidoglycan.
Strains and Growth Conditions--
All strains and plasmids used
in this study are listed in Table I.
S. pneumoniae strains were grown in a casein-based
semisynthetic medium (C + Y) at 37 °C without aeration, as described
previously (6). S. pneumoniae and Escherichia
coli strains containing pJDC9 or its derivatives were grown in the
presence of 1 µg/ml and 1 mg/ml erythromycin (Sigma), respectively.
Growth rates of the insertionally inactivated mutants of Pen6 were
determined with cultures first grown in C + Y containing
erythromycin at 1 µg/ml and then diluted 100-fold in fresh C + Y. The OD was then measured at 590 nm over the time.
DNA and RNA Techniques--
All routine DNA manipulations were
performed using standard methods (16, 17). The chromosomal DNA from
S. pneumoniae was isolated as described previously (18).
Plasmids were isolated using the Wizard Plus Minipreps DNA Purification
System (Promega), and polymerase chain reaction products were purified
using the Wizard PCR Preps DNA Purification System (Promega).
Oligonucleotides were purchased from Life Technologies, Inc. Nucleotide
and derived amino acid sequences were analyzed using DNASTAR software.
RNA was prepared from exponentially growing cultures at
A590 of 0.5 and was extracted by using
the FastRNA isolation kit (Bio101) according to the recommendations of
the manufacturer.
Primer Extension Analysis--
Primer extension analysis was
performed by using primer ZOO36 (5'-TGTTCTTTGACAAACTGATC-3') (Fig. 1),
which was end-labeled with [ Transformation and Population Analysis Profiles--
S.
pneumoniae strains were transformed according to published
procedures (5). To induce competence, synthetic CSP Inactivation of the murN Gene--
For gene disruption
experiments by insertion-duplication mutagenesis, an internal fragment
of murN was amplified by polymerase chain reaction using as
template chromosomal DNA from R36A and cloned into pJDC9, a plasmid
that does not replicate in S. pneumoniae (15). The following
primers were used-ZOO1KP (5'-TATGGTACCGGCCGATTTATACCCAACAAG-3') and
ZOO2BM (5'-TATGGATCCAGTCTCGCGCTTCTGCTTTTC-3'), giving origin to the
plasmid pZOO3. Plasmid pZOO3 was used to inactivate the murN
gene in Pen6 by transforming competent cells.
Allelic Replacement of the murM Gene--
The abnormal
murM gene from penicillin-resistant Pen6 strain was replaced
by the murM gene from R36A laboratory strain by transforming
competent cells from Pen6 with chromosomal DNA from R36AmurMN (R36A with inactivated murMN).
Transformants were selected with erythromycin (1 µg/ml), and their
penicillin susceptibility was confirmed. Revertants from these
transformants that result from the loss of the plasmid inactivating
murMN were selected by their penicillin resistance and
erythromycin susceptibility. The excision of the plasmid carrying the
erythromycin resistance marker allowed the reconstruction of active
murM allele from R36A. The allelic replacement of the
murM gene was confirmed by polymerase chain reaction
amplification using primers ZOO7 (5'-CATAGCGCTGGAACTCAC-3') and ZOO30
(5'-ATATTCTCTACGTTCAGAGG-3') followed by restriction with
PstI and HindIII, two enzymes that cut the Pen6
allele but not the R36A allele.
Cell Wall Preparation--
Pneumococcal cell walls were prepared
by a previously published method (4, 19) except for the process of
breaking the cells, which was done by shaking with acid-washed glass
beads with the help of FastPrep FP120 (Bio 101).
Enzymatic Digestion of Cell Walls--
Cell wall material (2 mg)
was suspended in 25 mM sodium phosphate buffer, pH 7.4, and
treated with affinity-purified pneumococcal amidase (5 µg) at
37 °C for 18-24 h with constant stirring. The products were dried,
the precipitate was washed with acetone, and the peptides were
extracted with acetonitrile/isopropyl alcohol/water (25:25:50)
containing 0.1% trifluoroacetic acid as already described (4, 19, 20).
After removal of the solvents by evaporation in a SpeedVac, the
peptides were dissolved in 0.1% trifluoroacetic acid.
Separation and Analysis of the Cell Wall Stem
Peptides--
Peptides were separated with a Shimadzu LC-10AVP HPLC
system, as described previously (4). The column used was a Vydac 218TP54 (The Separations Group). The peptides were eluted with an
80-min linear gradient from 0 to 15% acetonitrile (Fisher) in 0.1%
trifluoroacetic acid (Pierce) pumped at a flow rate of 0.5 ml/min. The
eluted fractions were detected and quantified by determination of their
ultraviolet absorption at 210 nm (A210).
Characterization of Stem Peptides--
The stem peptides
generated by enzymatic hydrolysis of cell walls of mutants
Pen6murN and Pen6murMN were recovered from the HPLC column and dried in the SpeedVac. The amino acid composition (21)
and the peptides' molecular mass were determined at the Rockefeller
University Protein/DNA Technology Center. Approximately 1.1 nmol of
sample from the stem peptides 3a, Ia, and 7a were used to obtain the
sequence of the part of the stem peptide amenable to Edman degradation.
The procedure was performed as recommended by the manufacturer's
program in a Hewlett-Packard G-1000A protein sequencer using chemistry
3.5, and the phenylthiohydantoin-derivatives were identified by
on-line HPLC at the Rockefeller University Protein/DNA Technology Center.
Determination of the transcription initiation site. Primer
extension analysis was performed to determine the transcription start
site using the primer ZOO36 that hybridizes with the murMN transcript of Pen6 and R6Hex (Fig. 1).
Based on this analysis, it was determined that the transcript that
includes murM can initiate at two different adenine residues
(Fig. 2A) located 25 and 26 base pairs upstream of the murM start codon in the case of
Pen6 (Fig. 1). In R6Hex, the transcript can initiate at an adenine or a
thymine residue located 26 and 27 base pairs, respectively, upstream of
the murM start codon (Figs. 1 and 2B). These
results indicate that the same region contains the promoter in both the penicillin-resistant strain Pen6 and in the susceptible strain R6Hex
(Fig. 1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-lactam target enzymes,
the PBPs, in such a way that their affinity toward the antibiotic molecule is greatly reduced (7). This is achieved by the construction of pbp mosaic genes that are believed to be the result of
heterologous recombinational events in the case of clinical isolates
(8-10) or mutations in the pbp genes in the case of
laboratory mutants (11). The recent identification of the genetic
determinants of the cell wall branching system murMN (12)
has allowed a reexamination of the relationship between muropeptide
structure and penicillin resistance. It was shown that inactivation of
the murMN operon resulted in the production of a
peptidoglycan, both in penicillin-sensitive and penicillin-resistant
strains, from which all branched muropeptide components were missing,
and, concomitantly, there was a complete loss of penicillin resistance
in each one of several penicillin-resistant strains examined (12). The
mechanistic connection between the functioning of murMN and
the expression of the penicillin-resistant phenotype remains to be elucidated.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Relevant properties of the strains and plasmids used in this study
-32P]ATP and purified with
the AGTC Gel Filtration Cartridges (Edge BioSystems). RNA from Pen6,
R6Hex, and R36A (50 µg) was hybridized with the primer at 65 °C
for 90 min and slowly cooled to room temperature. Reverse transcription
was carried out by using SuperScript RT (Life Technologies, Inc.) at
42 °C for 90 min, and the reaction mixture was heated at 65 °C
for 10 min to inactivate the enzyme. The reaction product was incubated
with RNase H (3 units) at 37 °C for 30 min,
ethanol-precipitated, resuspended in 10 µl of Sequenase stop
solution, denatured, and applied to a 6% sequencing gel. Sequencing
reaction mixtures prepared by using the T7 Sequenase Kit version 2.0 (Amersham Pharmacia Biotech) primed by ZOO36 were also applied to the gel.
was added to the
medium at a concentration of 250 ng/ml. The competent cells were then
incubated for 30 min at 30 °C in the presence of plasmid DNA
followed by the addition of 2 ml of C + Y and a 2-h incubation
at 37 °C. Transformants were selected on blood agar plates (tryptic
soy agar plus 3% (v/v) sheep blood) containing 1 µg/ml
erythromycin. Population analysis profiles were determined by plating
serial dilutions of early stationary phase cultures on plates of
tryptic soy agar containing 5% (v/v) of sheep blood (Micropure Medical
Inc., White Bear Lake, MN) and different concentrations of
penicillin G (Sigma) (0, 0.01, 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2, 4, 8, 16 µg/ml). The population analysis profiles were done with
and without the presence of erythromycin 1 µg/ml in the medium. Plates were incubated at 37 °C in a 5% CO2 in air
atmosphere for 24 h, and the number of bacteria capable of forming
colonies in the presence of various penicillin concentrations was
plotted against the concentration of penicillin in the agar medium.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (20K):
[in a new window]
Fig. 1.
Nucleotide sequence of the region upstream of
murM (GenBankTM data base accession
numbers AJ250766 (Pen6) and AJ250764 (R36A)). The putative
promoter region is highlighted by boxed sequences
and labeled 
10 and 35. The promoter is
designated PmurMN. The putative ribosome-binding
site is underlined and labeled SD. The 5'-ends of
the RNA determined by primer extension are labeled +1. The
start codon is in boldface type and
double underlined. The primer ZOO36 is indicated
by an arrow. Part of the deduced amino acid sequence of
murM from R36A or Pen are aligned under the DNA
sequence.
View larger version (58K):
[in a new window]
Fig. 2.
Mapping of the 5'-end of the murMN
transcript by primer extension from Pen6 (A) and
R6Hex (B). The sequencing encompassing the
transcription start site (marked by asterisks) is
enlarged.
Gene Disruption and Characterization of the murMN and murN
Mutants--
Inactivation of the murMN operon by insertion
duplication mutagenesis in the penicillin-resistant strain Pen6 did not
cause any significant change in growth rate of the cultures; the mass doubling times of the parental strain Pen6 and its Pen6murMN
mutant were 34.0 ± 0.5 and 32.5 ± 0.3 min, respectively.
However, when murN alone was inactivated the doubling time
of the mutant increased to 48.5 ± 0.9 min. The rates of autolysis
in the stationary phase of growth were the same for
Pen6murMN and for the parental strain Pen6 (1.7 ± 0.3 × 10
3 and 2.3 ± 0.5 × 103 min1,
respectively), although the mutant culture started lysing sooner than
the parental strain Pen6.
While inactivation of murMN caused a virtually complete
block in the expression of penicillin resistance (12), inactivation of
murN resulted only in a modest (2-fold) decrease of the MIC to penicillin (Fig. 3) despite the major
impact of the inactivation on the composition of the peptidoglycan (see
below).
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Composition of the Peptidoglycan in the Pen6murMN and Pen6murN
Strains--
The cell wall of the Pen6murMN and
Pen6murN was analyzed by HPLC (Fig.
4, A and B). The
stem peptide composition of the strains shown as a percentage of the
total peptides of the peptidoglycan is presented in Table
II, and the corresponding chemical
structures are shown in Fig. 6. As was shown before (12), disruption of the murMN operon led to the disappearance of all branched
muropeptide monomers and dimers accompanied by a parallel increase in
the percentage of linear structured stem peptides and in the appearance of novel peptide structures (peptides 10 and 11, Fig.
4A).
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HPLC analysis of the cell walls of strain Pen6 in which the
murN gene was inactivated showed major changes in the
elution profile of stem peptides (Fig.
5); the branched components found in the
peptidoglycan of Pen6 disappeared, but in this case there was no
increase of the linear peptides. Instead, inactivation of the
murN led to the appearance of novel peptide components that
were not seen before in pneumococcal cell wall preparations. Similarly,
these novel stem peptides with anomalous retention times were also
detected in the cell walls of the penicillin-susceptible strain R6Hex
with inactivated murN (data not shown). Nevertheless, there
was no change in the degree of cross-linking of the peptidoglycan in
the Pen6murMN and Pen6murN mutants (as seen from
the unchanged percentage of monomers relative to the total of peptides
in Table II).
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Characterization of the Novel Stem Peptides in the murN and murMN
Mutants--
Analysis of the peptide composition of the peptidoglycan
from the murN mutant of Pen6 showed an accumulation of
two novel peptides (3a and Ia; see Fig.
6). Based on the results of
molecular weight determination and amino acid composition, peptides 3a
and Ia are proposed to be monomers with only one serine or one alanine, respectively, attached to the 
-NH2-terminal of the
stem peptide lysine residue (Table
III). Components 7a, IV/Va, and
VIa appear to be dimeric peptides that would result from the
transpeptidation reaction of peptides 3a and Ia.
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The structures of the peptides 3a, Ia, and 7a were confirmed by Edman degradation. According to the proposed structures (Table III), the only sources of free amino termini in stem peptide 3a would be two alanine residues, and in stem peptide Ia one alanine and one serine. The results of Edman degradation confirmed these predictions; only one alanine residue was released from peptide 3a, and similar amounts of alanine and serine were released from peptide Ia. Edman degradation of peptide 7a resulted in the release of the same number of serine residues as in peptide Ia but twice the amount of alanine, consistent with the proposed structure.
The two new components detected in the peptidoglycan of cells with inactivated murMN operon (peptides 10 and 11) were tentatively identified (on the basis of amino acid composition and molecular mass) as trimers and tetramers composed of linear peptide units (Table III).
Allelic Replacement of murM-- The murM alleles from R36A and Pen6 encode proteins that differ by 15% at the amino acid level (12). To determine if the different peptidoglycan types of these two strains were related to the two different MurM proteins, we introduced the R36A murM allele into the Pen6 background by genetic transformation, in order to generate the construct Pen6murMR36A. Confirmation of efficient allelic replacement was obtained by sequencing the murMN operon of this mutant (data not shown).
The cell wall composition of the mutant Pen6murMR36A was analyzed by HPLC (Fig. 5). The introduction of the murM allele from strain R36A caused a large increase in the representation of the monomeric linear peptide 1 (from 2.5% in the parental strain Pen6 to 17.5% in the allelic replacement mutant) and an increased proportion of the directly cross-linked tri-tetra dimer (from 3.1 to 10.5%). The percentage of branched peptide 3 was decreased from 13.8% (in Pen6) to around 8.1%, a value similar to that found in R6Hex (10.3%). (This value was considerably higher than the value found in strain R36A, 2.5%.) There was a considerable reduction in the proportion of the branched peptide I (from 13.9 to 3.6%) more pronounced than the reduction in peptide 3.
Despite the extensive variation in the ratio of branched to linear
peptides (from 8.0 in Pen6 to 1.4 in the mutant
Pen6murMR36A) the level of cross-linking, determined by the
percentage of all cross-linked muropeptide species, was similar in all
mutants and in their parental strains (62% in the
Pen6murMR36A and 63% in Pen6).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The enzymes involved in the addition of lateral peptides to the
-amino group of lysine have been identified in the Staphylococci family (22-24) and recently in S. pneumoniae (12). In the
case of Staphylococcus aureus, the synthesis of the
pentaglycine bridge involves three enzymes: FmhB, responsible for the
addition of the first glycine to the linear pentapeptide precursor
(25), and FemA and FemB, involved in the addition of the glycyl
residues 2 and 3 (26-27) and glycyl residues 4 and 5 (28),
respectively. In S. aureus, the fmhB gene product
was shown to be essential, which may be related to the fact that dimers
of linear muropeptides are extremely rare in this bacterial species. In
contrast to S. aureus, S. pneumoniae appears to
be able to use either linear or branched cell wall precursors as
substrates for the PBPs in the cross-linking reaction of peptidoglycan.
The disruption of the murMN operon did not impair growth,
indicating that the murMN genes are not essential in
S. pneumoniae. However, the growth rate of the mutant
Pen6murN was significantly reduced, suggesting that the
semibranched peptides of this mutant may not be used as efficiently for
some growth-limiting function in S. pneumoniae.
A combination of genetic and biochemical studies described in this communication has allowed the clarification of the roles of the murM and murN gene products in the assembly of the branched muropeptides.
Inactivation of the murMN operon in S. pneumoniae
abolished the addition of any amino acid residue to the -amino group
of the stem peptide lysine, resulting in the production of a
peptidoglycan that completely lacked muropeptides of branched structure
(12). Selective inactivation of murN caused the formation of
an unusual peptidoglycan in which all dipeptide branches were replaced
by branches composed of only one seryl or alanyl residue. These results suggest that the MurN protein is involved with the addition of the
second amino acid residue (alanine) and that the MurM protein is
involved with the addition of the first amino acid (alanine or serine)
to the cross-bridge.
Interestingly, replacement of the dipeptide bridges by branches composed of single amino acids did not alter the overwhelmingly branched muropeptide composition of strain Pen6; the proportion of branched peptides was 89% in Pen6 and 90% in Pen6murN (see Table II). Furthermore, if one considers all branched muropeptides irrespective of whether they contain one or two amino acid residues, the characteristic proportions of the various specific branched muropeptide species also appeared to be retained in the Pen6murN mutant. These observations suggest that the amount of branched muropeptides in the pneumococcal peptidoglycan is primarily determined by the activity of the murM gene product.
Additional evidence for the dominant role of murM is
provided by the changes observed in the peptidoglycan composition of the penicillin-resistant strain Pen6 in which the mosaic
murM of this strain (12) was replaced by murM of
the penicillin-susceptible strain R36A. The murM alleles
from R36A and Pen6 differ by 15% at the amino acid level (12).
Comparison of the peptidoglycan of the two strains shows that Pen6 has
a much higher percentage of branched peptides and can add an alanine or
a serine as the first amino acid of the cross-bridge, whereas
R36A only adds a serine residue efficiently. These differences may be
related to the observed 15% difference at the amino acid level of the
MurM. In order to determine if there were in fact any differences in the activity of the MurM from Pen6 and R36A, we transformed the R36A
murM allele into Pen6. This mutant Pen6murMR36A
only diverges from the parental strain in the sequence of
murM allele. Data in Table II show that the
introduction of the R36A allele of murM shifted the
peptidoglycan composition in the direction characteristic of the
penicillin-susceptible strain: the proportion of total branched
muropeptides was reduced from 89% (in Pen6) to 58% (in Pen6murMR36A), which is close to the proportion of branched
peptides (55%) in strain R36A. The ratio of branched to linear
peptides also changed from 8.0 (in Pen6) to 1.4 (in
Pen6murMR36A), which is comparable with the ratio (1.3) seen
in strain R36A. These results suggest that the MurM from R36A is not as
efficient as the one from Pen6 in the addition of the first amino acid
to the -NH2 group of the lysine residue.
It is conceivable that these differences are related to different rates of transcription of the two kinds of murM genes. Based on the result obtained by primer extension analysis for the determination of the transcription initiation sites, we proposed a virtually identical promoter region for the murM genes in strains Pen6 and R36A/R6Hex. The fact that R6Hex has a murM allele identical to R36A but has a higher percentage of branched peptides indicates that additional factors may also contribute to the regulation of the amount of branched peptides in the peptidoglycan. However, when the murM allele from R36A is introduced in the background of Pen6 and therefore subject to the same regulation, the observed difference in the peptidoglycan composition should be due only to the differences in the protein. Therefore, compositional differences between the peptidoglycans of resistant and susceptible strains most likely reflect differences in the specific activities of the two types of MurM proteins. This possibility is currently under investigation.
Analysis of the composition of the peptidoglycan from Pen6 and its
murN and murMN mutants showed similar proportions
of cross-linked species in the peptidoglycan, suggesting that the PBPs
from strain Pen6 are not very specific regarding their substrates. In
the absence of antibiotic, PBPs seem to be able to use either linear, branched or semibranched peptides as substrates for the
transpeptidation reaction in the synthesis of peptidoglycan. On the
other hand, when a -lactam antibiotic is present in the medium, the
bacteria seem to depend on the availability of branched or at least
semibranched peptides for the expression of penicillin resistance. It
should be noticed that in strain R6Hex 74% of all stem peptides have a
branched structure, yet this strain is not resistant to penicillin, indicating that the presence of branched peptides alone is not sufficient for the expression of penicillin resistance.
The fact that PBPs can efficiently cross-link either linear, branched,
or semibranched peptides taken together with the fact that substituting
the murM allele of Pen6 by the allele from R36A results in
drastic changes in the peptidoglycan composition indicates that the
primary determinants of the type of peptidoglycan stem peptide
structure (linear versus branched peptides) must be MurM and MurN.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. Sandford Lacks for plasmid pJDC9. We also thank Mario Ramirez for stimulating and helpful discussions.
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Addendum |
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Confirmatory evidence for murM and
murN and the role(s) of these genes in peptidoglycan
structure and expression of -lactam resistance, first described in
Ref. 12, has appeared recently by Weber et al. (29), who
identified the same genes but named them fibA (same as
murM) and fibB (same as murN).
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These studies were supported, in part, by National Institutes of Health Grant RO1 AI37275 and by the Irene Diamond Foundation.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ250766 (Pen6) and AJ250764 (R36A).
¶ Supported by Fundação para a Ciência e Tecnologia PRAXIS XXI Grant BD/9071/96.
Supported by Fundação para a Ciência e
Tecnologia PRAXIS XXI Grant BD/9079/96.
** To whom correspondence should be addressed: Laboratory of Microbiology, The Rockefeller University, 1230 York Ave., New York, NY 10021. Tel.: 212-327-8278; Fax: 212-327-8688; E-mail: tomasz@rockvax.rockefeller.edu.
Published, JBC Papers in Press, June 26, 2000, DOI 10.1074/jbc.M004675200
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ABBREVIATIONS |
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The abbreviations used are: PBP, penicillin-binding protein; HPLC, high pressure liquid chromatography.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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),
Pen6 (
), mutant Pen6murMN (×), and mutant
Pen6murN (
) were plated at different cell concentrations
on blood agar plates containing different concentrations of penicillin,
and the number of bacterial colonies were counted, as described for
population analysis under "Experimental Procedures."
