![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
INSERM E0004-LRMA, UFR Broussais-Hôtel Dieu, Université Paris VI, 75270 Paris,1 Département Régulations, Développement et Diversité Moléculaire, Museum National d'Histoire Naturel, USM0502-CNRS UMR8041, 75005 Paris,France,4 University of Massachusetts Dartmouth, North Dartmouth, Massachusetts 02747,2 Department of Emergency Medicine, Somerville Hospital, Harvard Medical School, Somerville, Massachusetts 021433
Received 30 May 2003/ Accepted 17 September 2003
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
L-Ala-L-Ala-Lys3
peptidoglycan cross-bridge. Thus, the croRS mutant was
susceptible to ceftriaxone despite the production of PBP5 and the
synthesis of wild-type peptidoglycan precursors. These observations
constitute the first description of regulatory genes essential for
PBP5-mediated ß-lactam resistance in
enterococci. |
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Enterococci are resistant to the newer cephalosporins which have been developed to treat infections due to gram-negative bacteria producing ß-lactamases. Treatment with cephalosporins is one of the risk factors for colonization and infection by multidrug-resistant enterococci. Cephalosporin resistance is mediated by a specific class B penicillin-binding protein (PBP) commonly referred to as low-affinity PBP5 (7, 27). Production of PBP5 also confers moderate-level resistance to ampicillin (MIC, 2 to 16 µg/ml). Acquisition of higher levels of ampicillin resistance, seen mainly in E. faecium, results from overproduction of PBP5 (11, 14), amino acid substitutions that further decrease interaction of PBP5 with ß-lactams (26, 32), and modification of as-yet-unidentified non-PBP factors (18, 29).
In this report, we show that a two-component regulatory system (designated CroRS [for "ceftriaxone resistance"]) is essential for intrinsic ß-lactam resistance in E. faecalis. This system, designated RR05-HK05 in the classification of Hancock and Perego (15), was initially chosen because of sequence similarity with two-component systems that control acquired enterococcal resistance to the glycopeptide antibiotics vancomycin and teicoplanin (2, 15). We report the resistance phenotype associated with deletions from the croRS locus, purification of the CroR response regulator and of a soluble fragment of the CroS histidine protein kinase to test their activity, and transcriptional analysis of the croRS locus. Since defects in the assembly of peptidoglycan precursors are associated with impaired expression of methicillin resistance in S. aureus (10, 25), we also analyzed the impact of the croRS deletion on the assembly of cytoplasmic precursors and on peptidoglycan cross-bridge formation.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Deletion
of the croR and croS genes.
Deletions were made from the
chromosome of E. faecalis JH2-2 by homologous recombination
using derivatives of the suicide vector pHS1, which is thermosensitive
for replication and confers gentamicin resistance (A. Arbeloa and M.
Arthur, unpublished data). Briefly, DNA fragments (H1A, H1B, and H2)
flanking the sequences targeted for deletion were amplified with
primers (for H1A, primers
5'-ATTGATTTCTGAATCGC-3' and
5'-AGATCTATCTGGTGTTGTGTGC-3';
for H1B, primers
5'-ATTGATTTCTGAATCGC-3' and
5'-TTTTAGATCTTTAACGAGCATCGATCTTAT-3';
and for H2, primers
5'-AGATCTGAGTTAATTGACATCCC-3'
and 5'-GCAGACACATCATTCCG-3')
containing BglII restriction sites (underlined) to
facilitate subsequent cloning steps. The fragments were cloned (with or
without an intervening BglII erythromycin resistance cassette
[erm]) into pHS1 to generate the inserts
(H1A-erm-H2, H1A-H2, and H1B-H2) as shown in the insets in
Fig.
1. To replace croRS by erm
(
croRS/erm), the derivative of plasmid pHS1
carrying H1A-erm-H2 (pHS1
H1A-erm-H2) was
introduced by electrotransformation into E. faecalis JH2-2.
Replacement of croRS by erm was selected on agar
containing erythromycin (10 µg/ml) at the nonpermissive
temperature (42°C) for plasmid replication (Fig.
1B). One clone resistant
to erythromycin and susceptible to gentamicin was identified by replica
plating on BHI agar containing gentamicin (128 µg/ml) and was
designated JH2-2croRS/erm.
|
croRS/erm
was obtained in two steps using a derivative of pHS1 carrying H1A
directly fused to H2 (H1A-H2; Fig.
1C). In the first step,
integration of plasmid pHS1H1A-H2 by homologous recombination
was selected at 42°C on agar containing gentamicin (128
µg/ml), generating a partial duplication of the locus, since
the sequence of the pHS1 vector was flanked by the H1A-H2 and
H1A-erm-H2 alleles. Serial subcultures at the permissive
(28°C) and nonpermissive (42°C) temperatures in the
absence of antibiotic were used to stimulate the excision and loss of
pHS1H1A-erm-H2, leaving the H1A-H2 allele in the
chromosome. One clone (designated JH2-2croRS) was
obtained by screening for gentamicin and erythromycin
susceptibility.
Replacement of the erm cassette of
JH2-2croRS/erm by the croR open
reading frame was obtained by the same two-step procedure with a
derivative of pHS1 carrying croR as a part of the H1B-H2
insert (Fig. 1D). The
resulting clone, JH2-2croS, lacked the precise
croS open reading frame. PCR and Southern blot hybridization
were used to confirm that the expected deletions from and gene
replacements in JH2-2croRS/erm,
JH2-2croRS, and JH2-2croS had taken
place.
Shuttle plasmids for croS, croRS, serS, and pbp5 expression. The croS open reading frame was amplified with primers P80 and P87. Primer P80 (5'-ATCGAGGTACCGGATCCTAAAATATCGGAGGGTTTATTATGCTCGTTAAACCTAAAAA-3') contained a KpnI restriction site (underlined), an artificial ribosome binding site (italicized), and 20 bases complementary to the 5' end of croS that included the translation initiation codon (italicized). Primer P87 (5'-ATCGATCTAGAAGATCTTTAACTCTCTGATTTCTTGT-3') contained an XbaI restriction site (underlined) and 20 bases complementary to the 3' end of croS that included the stop codon (italicized). The croS open reading frame was cloned under the control of the aphA-3p promoter (1) in the shuttle vector pAT18 (30) to generate pRQ12(croS). The serS open reading frame was amplified with primers DS (5'-AAGAGCTCTCATTTCGTCCCAAGAATATT-3') and ES (5'-TTTTGGTACCTTATTTAATAACTG-3'), digested with SacI and KpnI (underlined), and cloned under the control of the aphA-3p promoter to generate pYC5(serS). A DNA fragment containing the rrnC-croR intergenic region, croR, and croS (coordinates 1191 to 3315) was amplified with primer P25 (5'-AGTTCGGTACCTAAGACATGTAATAATATACCAA-3') and P87 (described above) and cloned into pAT18 using BamHI (underlined) and XbaI to generate pRQ13(croRS). Plasmid pAA15 (Arbeloa and Arthur, unpublished) contains the PBP5 open reading frame cloned downstream from aphA-3p. DNA sequencing was performed for all recombinant plasmids used in this study to check the accuracy of the PCRs.
Purification and analysis of mRNA. Strains of E. faecalis were grown in 6 ml of BHI broth to an optical density at 600 nm (OD600) of 0.6. Bacteria were collected by centrifugation (12,000 x g for 30 s at 4°C) and treated with lysozyme and lysostaphin (GramCracker kit; Ambion Inc., Austin, Tex.), and total RNA was extracted with phenol and chloroform (RNAwiz; Ambion Inc.). RNA concentration and purity were determined by the absorbance at 260 nm (A260) and the A260/A280 ratio, respectively.
Mapping of the 5' extremity of mRNA
isolated in vivo was performed by primer extension with
oligonucleotides P49
(5'-AATACTCAATAGTTCTACAATTTC-3'),
P51
(5'-CACGCCACGGGTTTGTAGCTTTGC-3'),
and P76
(5'-GACTTCTTTATAGATGAATGTTT-3').
Primers (10 pmol) were end labeled with
[
-32P]ATP (Perkin Elmer Life Sciences
Inc., Boston, Mass.) (3,000 Ci/mmol) by using 10 U of polynucleotide
kinase (primer extension system; Promega Corp., Madison, Wis.) and
annealed to 30 µg of total RNA for 20 min at 58°C
followed by 10 min at 20°C. Avian myeloblastosis virus reverse
transcriptase (primer extension system; Promega) (1 U) was added, and
incubation was continued for 30 min at 42°C. The reverse
transcription products were analyzed in 6% denaturing
polyacrylamide gels. DNA sequencing reactions were performed with the
same primers (Sequenase version 2.0 DNA; USB Corp., Cleveland,
Ohio).
Northern blot hybridization was performed with commercial
denaturing and running buffers (NorthernMax; Ambion Inc.). Briefly,
total RNA (30 µg) was denatured in formaldehyde loading dye for
15 min at 65°C. Electrophoresis was performed in formaldehyde
denaturing gel at 5 V/cm for 3 h, and RNA was transferred by
vacuum onto a nylon membrane (BrightStar-Plus; Ambion Inc.). RNA was
cross-linked, using UV light at 254 nm (Stratalinker UV Cross-linker
1800; Stratagene, La Jolla, Calif.) (120,000
microjoules/cm2), to the membrane. Prehybridization was
performed overnight at 68°C (ULTRAhyb solution; Ambion Inc.).
Double-stranded DNA fragments used as probes (50 ng) were labeled with
[
-32P]dCTP (Ready-to-Go DNA labeling
beads; Amersham Pharmacia Biotech, Piscataway, N.J.) (3,000 Ci/mmol),
denatured at 90°C for 10 min, and added to the prehybridization
solution. Hybridization was performed overnight at 42°C. The
membranes were washed (using washing solutions from a NorthernMax kit
[Ambion Inc.]) twice with a low-stringency solution for 5 min
at room temperature and twice with a high-stringency solution for 15
min at 90°C.
Purification of
CroRH.
The
croR open reading frame of E. faecalis JH2-2 was
amplified with primers 1R61
(5'-TCATGAAAATTTTAGTTGC-3')
and 2R61His
(5'-AGATCTACGAGCATCGATCTTAT-3')
containing BspHI and BglII restriction
sites (underlined), respectively. The BspHI-BglII
fragment was cloned into the expression vector pTRCHis60
(23) digested with
NcoI and BglII. For protein production,
Escherichia coli JM83 harboring plasmid
pTRCHis60croRH was grown at 37°C
to an OD600 of 0.7 in 1 liter of BHI broth containing
ampicillin (100 µg/ml).
Isopropyl-D-thiogalactopyranoside (IPTG) was added to a
final concentration of 1 mM, and incubation was continued for
3 h at 30°C. Bacteria were harvested by
centrifugation (8,000 x g for 20 min at 4°C),
washed in 180 ml of 50 mM Tris-HCl (pH 7.5), and resuspended in 10 ml
of the same buffer containing 100 mM NaCl. Bacteria were disrupted by
sonication for 2 min with cooling, the extract was centrifuged at
12,000 x g for 30 min at 4°C, and the
supernatant was mixed with 4 ml of
Ni2+-nitrilotriacetate-agarose resin (Amersham
Pharmacia Biotech, Saclay, France) previously equilibrated with 50 mM
Tris-HCl (pH 7.8). After 1 h of incubation at 4°C,
the resin was recovered by centrifugation and washed with 50 mM
Tris-HCl (pH 7.8) containing increasing concentrations of imidazole
(20, 25, 40, 100, and 250 mM). The protein fraction eluting at 250 mM
was dialyzed with 50 mM Tris-HCl (pH 7.4). Gel filtration was performed
on a Superdex HR10/30 column (Amersham Pharmacia Biotech) equilibrated
with 50 mM Tris-HCl (pH 7.5) containing 300 mM NaCl at a flow rate of
0.5 ml/min.
Purification of
CroSS.
A portion
of the croS open reading frame of E. faecalis JH2-2
was amplified with primers S61int4
(5'-GGTGGTCATATGCTGGTGGATAGTACTGTCG-3')
and S61int3
(5'-GGTGGTTGCTCTTCCGCAACTCTCTGATTTCTTGTTG-3'),
and the PCR product was digested with NdeI and
SapI (underlined) and cloned into pTYB1 (New England Biolabs,
Frankfurt am Main, Germany) digested with the same enzymes. The
resulting plasmid, pTYB1croSS, encoded a
fusion protein consisting of a methionine specified by the ATG
initiation codon of pTYB1, residues 145 to 393 of croS, and
the self-cleavable C-terminal intein tag. E. coli ER2566 (New
England Biolabs) harboring pTYB1croSS was
grown at 37°C to an OD650 of 0.5 in 3 liters of BHI
broth containing ampicillin (300 µg/ml). IPTG was added to
achieve a final concentration of 0.5 mM, and incubation was continued
for 17 h at 16°C. CroSS was purified from
a clarified lysate by affinity chromatography on chitin beads followed
by cleavage of the fusion protein with 2-mercaptoethanol (50 mM) for
18 h at 20°C (IMPACT-CN kit; New England Biolabs).
Gel filtration was performed as described above for
CroRH.
Protein
phosphorylation assays.
The
kinetics of CroSS autophosphorylation was tested at
20°C in a total volume of 64 µl containing the purified
protein (30 µM), [-32P]ATP
triethylammonium salt (Amersham Pharmacia Biotech) (3.4 µM;
0.37 TBq/mmol), and buffer A (50 mM Tris-HCl, 25 mM KCl, 0.5 mM
MgCl2, pH 7.4). Samples (12 µl) were taken at 0, 5,
10, 30, and 60 min, and the reaction was quenched by the addition of 5
µl of a solution containing 125 mM Tris-HCl (pH 6.8),
2.5% sodium dodecyl sulfate (SDS), 2 mM EDTA, 0.025%
bromophenol blue, and 25% glycerol. Samples were applied
directly to SDS-13.5% polyacrylamide gels.
Gels were dried and subjected to autoradiography without Coomassie blue
staining.
To test the transfer of the phosphate group from
CroSS to CroRH, phosphorylated CroSS
(phospho-CroSS) was prepared by incubating the protein (24
µM) with [-32P]ATP for 60 min in
a total volume of 64 µl as described above.
Phospho-CroSS was separated from
[-32P]ATP by ultrafiltration (Microcon
YM10; Millipore Corporation, Bedford, Mass.). CroRH (24
µM) was incubated with phospho-CroSS in buffer A (64
µl), and samples (15 µl) were analyzed by
SDS-polyacrylamide gel electrophoresis
(PAGE).
Assay for in vivo promoter activity. DNA fragments were cloned upstream from the promoterless lacZ reporter gene of the promoter probing vector pTCV-lac (24). Strains of E. faecalis harboring derivatives of pTCV-lac were grown to an OD600 of 0.55 in broth containing erythromycin (10 µg/ml) in addition to the drug tested for induction. Mueller-Hinton broth (Bio-Rad, Marnes-la-Coquettes, France) was used for trimethoprim, and BHI broth was used for all other drugs. Bacteria were collected by centrifugation and permeabilized with toluene. The ß-galactosidase activity was expressed in arbitrary units calculated according to the equation 103 x {(the OD420 value of the reaction mixture) - (1.75 x the OD550 value)/[the time of the reaction (in minutes) x the OD600 value of the quantity of cells used in the assay]}, as described previously (24).
Analysis of PBPs. The technique used for the analysis of PBPs of the different strains was employed as previously described (31) except that labeling was performed with 40 µg of benzyl[14C]penicillin potassium (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, England)/ml (2.11 GBq/mmol).
Peptidoglycan structure analysis. Preparation and structure assignment of muropeptides by mass spectrometry were performed as previously described (6). Briefly, bacteria were grown at 37°C in BHI broth to an optical density of 0.8. Peptidoglycan was extracted with 8% SDS at 100°C, treated with pronase (200 µg/ml) and trypsin (200 µg/ml), and digested with lysozyme (200 µg/ml) and mutanolysin (200 µg/ml). Muropeptides were reduced with sodium borohydrate and separated by reverse-phase high-performance liquid chromatography (rpHPLC) on a C18 column (Interchrom, Monluçon, France) (3 µm; 4.6 by 250 mm) at a flow rate of 0.5 ml/min with a 0 to 20% gradient applied at between 10 and 90 min (buffer A, 0.05% trifluoroacetic acid in water; buffer B, 0.035% trifluoroacetic acid in acetonitrile [per volume]). The relative abundance of muropeptides was estimated according to the percentage of the integrate area of peaks detected by the absorbance at 210 nm. Mass spectral data were collected with an electrospray time-of-flight mass spectrometer operating in the positive mode (Qstar Pulsar I; Applied Biosystems, Courtaboeuf, France) directly connected to the C18 column (flow rate, 0.5 ml/min). The data were acquired with a capillary voltage of 5,200 V and a declustering potential of 20 V. The mass scan range was from m/z 400 to m/z 2,500, and the scan cycle was 1 s.
Preparation and analysis of the cytoplasmic peptidoglycan precursors. Bacteria were grown to an OD650 of 0.7 and treated with vancomycin (100 µg/ml) for 15 min. Peptidoglycan precursors were extracted with formic acid (1.1 M) as previously described (4) and analyzed by rpHPLC with a µ-Bondapak C18 column (Waters, Milford, Mass.) (3.0 by 250 mm) at a flow rate of 0.5 ml/min with 50 mM ammonium formiate (pH 3.8). A methanol gradient (0 to 20%) was applied between 24 and 44 min, and elution with 20% methanol was continued for 10 min. The relative abundance of the UDP-MurNAc-peptide was estimated according to the percentage of the integrate area of peaks detected with the absorbance at 262 nm. For mass spectral analysis, products isolated by rpHPLC were lyophilized and dissolved in a solution containing acetonitrile (49.5%) and formic acid (0.5%). The samples were desalted by rpHPLC with isocratic elution (50% acetonitrile) at a flow rate of 10 µl/min. Tandem mass spectrometry analyzes were performed with a cone voltage of 55V and with argon as the collision gas at a pressure of 15 lb/in2 (energy, 20 to 50 eV) as previously described (6).
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
croRS/erm (Fig.
1B), the sequence encoding
a large C-terminal portion of CroR and the entire croS open
reading frame was replaced by an erm erythromycin resistance
cassette. The mutant retained the first 45 codons of croR.
Deletion of a larger portion of croR by homologous
recombination was not attempted, since this would have required the use
of a significant portion of rrnC which is repeated in the
three other rRNA clusters. JH2-2croRS was obtained by
removing the erm cassette from the chromosome of
JH2-2croRS/erm (Fig.
1C). Replacement of the
cassette by the croR open reading frame generated
JH2-2croS, which differed from wild-type JH2-2 by a
precise deletion of the croS gene (Fig.
1D).
The
croRS and croS deletions led to a 4,000-fold decrease
in the MIC of ceftriaxone and a 4-fold decrease in the MIC of
ampicillin (Table
1). Deletion of croRS also led to similarly large
(>100-fold) decreases in the MICs of
expanded-spectrum cephalosporins (e.g., cefuroxime and
cefepime) and moderate (2- to 8-fold) decreases in the MICs of other
ß-lactams (e.g., cephalothin, imipenem, amdinocillin, and
oxacillin). A similar phenotype was observed following deletion of the
pbp5 gene from the chromosome of JH2-2
(JH2-2pbp5; Arbeloa and Arthur, unpublished). A
trans-complementation of the croRS deletion was
obtained with a DNA fragment containing the rrnC-croR
intergenic region, croR, and croS cloned into the
replicative vector pAT18 (Table
1). Expression of
croS alone under the control of a heterologous promoter
(aphA-3p) complemented the croS deletion. Thus, the
croR and croS genes were both required for intrinsic
ß-lactam resistance.
|
croRS (Table
1) despite overproduction
of PBP5. These results show that low-affinity PBP5 was produced but
could not mediate ceftriaxone resistance in
JH2-2croRS. Expression of the serS seryl
tRNA synthetase gene under the control of aphA-3p did not
restore ceftriaxone resistance in JH2-2croRS (Table
1). The role of the latter
gene was investigated in the present study, since two-component
regulatory systems frequently regulate adjacent
genes. Phosphotransfer reactions catalyzed by purified CroSS and CroRH. A soluble fragment of CroS lacking the two putative trans-membrane segments of the protein was produced in E. coli as a translational fusion containing a C-terminal intein tag. Following affinity purification on chitin beads and cleavage of the tag, the soluble fragment of the sensor kinase (designated CroSS) was expected to differ from CroS by the absence of the first 144 amino acid residues and the presence of an additional methionine introduced for translation initiation. Analysis of purified CroSS (28,183 Da, 250 residues) by SDS-PAGE showed a 31-kDa protein band estimated to be 95% pure (3 mg of protein per liter of culture). Gel filtration under the conditions described in Materials and Methods revealed a protein peak with an estimated mass of 60 kDa, indicating that CroSS eluted as a dimer. No protein was detected at the elution volume expected for the monomer.
Full-length CroR fused to a C-terminal six-histidine tag (Ser-Arg-His6) was purified by affinity chromatography on a nickel column. The protein (designated CroRH) was judged using SDS-PAGE to be more than 95% pure and had an estimated mass of 27 kDa (30 mg per liter of culture). CroRH eluted as a monomer in the gel filtration column.
Autophosphorylation of
CroSS was assayed by incubating the purified protein with
[32-P]ATP (Fig.
2). A radioactive protein band corresponding to phospho-CroSS
was detectable after 5 min of incubation and increased up to 60 min
(Fig. 2A). The
phospho-CroSS adduct was sufficiently stable to allow for
removal of [-32P]ATP by ultrafiltration
(Fig. 2B, lane 1). Upon
addition of purified CroRH, the radiolabeled phosphate group
was entirely transferred from CroSS to CroRH in
less than 2 min. Upon further incubation, the intensity of the
phospho-CroRH protein band slowly decreased due to
dephosphorylation of the protein.
|
|
croRS, and JH2-2croS was
performed with probes generated from internal fragments of
croR, croS, and serS (Fig.
3B). The croR and
croS probes detected the same ca. 2,400-base band in RNA
preparations from JH2-2, indicating that the two genes were
cotranscribed (lanes 1 and 5). Growth in the presence of ceftriaxone
(1,000 µg/ml) led to the same hybridization pattern (lanes 2
and 6). As expected, the 1,161-bp croS deletion resulted in a
decrease in the size of the RNA band detected by the croR
probe in JH2-2croS (lane 3). A unique 1,500-base band
was detected by the serS probe in RNA preparations from the
three strains (lanes 9 to 12), indicating that croRS and
serS were transcribed
independently.
In vivo activity of the
croRp and serSp promoters.
DNA fragments carrying the
croR and serS promoters (Fig.
3A) were cloned upstream
from the lacZ reporter gene of plasmid pTCV-lac and
introduced into E. faecalis JH2-2,
JH2-2croRS, and JH2-2croS. Control
experiments were also performed with the vector alone and the
heterologous aphA-3p promoter previously characterized in this
system (24).
Determination of ß-galactosidase activity (Table
2) showed that the croRp promoter in JH2-2 was inducible by
ceftriaxone. The basal level of expression of the
croRp-lacZ transcriptional fusion appeared reduced in
JH2-2croRS (fivefold) and in
JH2-2croS (threefold) in comparison to the level seen
with the JH2-2 host. The serSp promoter in JH2-2 did not
respond to ceftriaxone, and similar ß-galactosidase activity
was detected in the croS and croRS
mutants.
|
|
croRS and JH2-2croS (data not
shown). The role of CroR and CroS in induction could not be fully
investigated with this drug, since the concentrations required in JH2-2
for induction were inhibitory for the mutants. For this reason, the
glycosyltransferase inhibitor moenomycin was also tested, revealing
induction in JH2-2 but not in JH2-2croRS or
JH2-2croS (data not
shown).
Structure of cytoplasmic
peptidoglycan precursors and of muropeptides.
UDP-MurNAc-peptide precursors from
E. faecalis JH2-2 and JH2-2croRS were
compared by rpHPLC and mass spectrometry (Table
4). Deletion of the croRS locus was not associated with any defect
in the assembly of the nucleotide UDP-MurNAc-pentapeptide, since
precursors containing incomplete peptide stems were present in similar
low amounts in both strains (UDP-MurNAc-tripeptide) or detected in
neither strain (UDP-MurNAc-L-Ala and UDP-MurNAc-L-Ala-D-Glu). Addition
of L-Ala to the 
-amino group of the pentapeptide
stem of the nucleotide by the BppA1 transferase
(5) resulted in similar
amounts of UDP-MurNAc-hexapeptide in the croRS mutant
and in the parental strain. As expected, the pools of
UDP-MurNAc-hexapeptide were small, since the BppA1 transferase
preferentially uses lipid intermediates as substrates
(5).
|
croRS were also similar (Fig.
4 and data not shown). The predominant muropeptides contained two
D-alanyl residues at the free C-terminal end and two
L-alanyl residues both in the cross-bridge and at the free
N-terminal end. Muropeptides of lesser abundance differed from the
above structures by combinations of MurNAc O-acetylation and
loss of the C-terminal D-Ala residues, as previously
described (6). These
results indicate that deletion of the croRS locus did not
affect the extent or mode of peptidoglycan cross-linking in E.
faecalis JH2-2.
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The croR and
croS genes were cotranscribed from a promoter, croRp,
which was inducible by inhibitors of peptidoglycan synthesis in E.
faecalis JH2-2 (Table
2). The reporter gene was
expressed at a constitutive low level in JH2-2croRS
and JH2-2croS. Thus, the adaptive response elicited
by CroR and CroS involved increased transcription of the regulatory
genes. This autoregulation mechanism is common to other two-component
regulatory systems (16).
The sensor kinases encoded by glycopeptide resistance gene clusters
appear to specifically respond to vancomycin (VanB-type resistance) or
to inhibitors of the transglycosylation reaction (VanA-type resistance)
(2). In contrast, the
CroRS system did not respond to inhibition of a specific step of
peptidoglycan synthesis, since the croRp-lacZ transcriptional
fusion was inducible by all peptidoglycan synthesis inhibitors that
were tested, including compounds acting on early cytoplasmic steps, the
metabolism of the lipid intermediates, transglycosylation, and
transpeptidation (Table
3). A recent study of the
role of two-component regulatory systems of E. faecalis in the
response to environmental stresses suggests that the CroS sensor kinase
responds to an even broader spectrum of signals
(19).
Sequences
flanking the croRS locus were independently transcribed, since
the rrnC rRNA operon was in a divergent orientation and
Northern blot hybridization revealed a distinct mRNA for the downstream
serS seryl-tRNA synthetase gene. The serSp promoter
was not inducible by peptidoglycan synthesis inhibitors and was
similarly active in JH2-2, JH2-2croRS, and
JH2-2croS (Table
2). Expression of
serS under the control of the heterologous aphA-3p
promoter did not restore ceftriaxone resistance in the
JH2-2croRS mutant. Thus, there is apparently no
functional link between the croRS locus and the flanking
serS locus. Of note, this region of the chromosome does not
seem to undergo frequent recombination events, since the relative
positions of croRS and serS are conserved in E.
faecalis and in E. faecium (data not
shown).
Deletion of croRS and pbp5 had the same
impact on the MICs of ß-lactam antibiotics (Table
1). The patterns of PBPs
labeled with benzyl[14C]penicillin were similar
for JH2-2, JH2-2croRS, and
JH2-2croS (data not shown). Introduction of plasmid
pAA15, harboring pbp5 under the control of aphA-3p,
led to overproduction of PBP5 in JH2-2croRS, but the
MIC of ceftriaxone was only marginally increased (Table
1). These results indicate
that susceptibility to ceftriaxone caused by the croRS
deletion cannot be attributed to the lack of PBP5
production.
Gram-positive cocci produce branched peptidoglycan
precursors containing a side chain consisting of two
L-alanyl residues in E. faecalis (Fig.
4A), five glycyl residues
in S. aureus, and the sequence
L-Ser-L-Ala or L-Ala-L-Ala
in S. pneumoniae
(28). The genes encoding
the transferases for synthesis of the pentaglycine side chain in S.
aureus were initially identified as factors essential for
methicillin resistance (fem) after random mutagenesis of the
S. aureus chromosome
(3,
25). More recently,
production of incomplete side chains was also reported to lead to
impaired expression of acquired resistance to ß-lactam
antibiotics in S. pneumoniae
(13) and of intrinsic
resistance to ceftriaxone in E. faecalis
(6). The screening for
impaired expression of methicillin resistance in S. aureus
also identified mutations in genes encoding enzymes for the assembly of
the nucleotide precursor UDP-MurNAc-pentapeptide
(17,
21). In the latter case,
the mutations could affect the amount of precursor produced rather than
its structure (10). Since
(despite the production of PBP5) deletion of the croRS locus
was associated with ceftriaxone susceptibility, the structures of
cytoplasmic precursors and muropeptides were analyzed to screen for
defects in the production of the substrate of PBP5. Deletion of
croRS was not associated with accumulation of nucleotide
precursors containing incomplete stem peptides (Table
4). Further, synthesis of
the L-alanyl-L-alanine side chain was not
impaired since the mutant produced wild-type levels of
UDP-MurNAc-hexapeptide (Table
4) and two
L-alanyl residues were present both in the cross-bridge and
in the free N-terminal end of the muropeptides (Fig.
4). Finally, the relative
proportions of monomer, dimer, trimer, and tetramer were similar in
JH2-2croRS and in the parental strain. These results
indicate that PBP5 did not mediate ceftriaxone resistance in
JH2-2croRS, despite delivery of an apparently
unaltered supply of disaccharide-peptide subunits to the peptidoglycan
polymerization complexes.
|
ACKNOWLEDGMENTS |
|---|
E. faecalis genome sequence data were kindly provided by The Institute for Genomic Research as publicly released at www.tigr.org.
|
FOOTNOTES |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
gene for conjugal transfer of DNA from
Escherichia coli to gram-positive bacteria.Gene
102:99-104.[CrossRef][Medline]
This article has been cited by other articles:
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| Appl. Environ. Microbiol. | Infect. Immun. | Eukaryot. Cell |
|---|---|---|
| Mol. Cell. Biol. | J. Virol. | Microbiol. Mol. Biol. Rev. |
| ALL ASM JOURNALS |
