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J. Biol. Chem., Vol. 276, Issue 9, 6093-6097, March 2, 2001
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From the
Received for publication, August 22, 2000, and in revised form, November 13, 2000
Coliphage There are at least two distinct mechanisms by which phage promote
destruction of the bacterial cell wall and subsequent cell lysis, the
choice of which appears to be determined by the phage genome size.
Bacteriophages with large genomes encode a holin-endolysin system. In
the prototypic Working from the premise that these phage proteins are targeting a host
protein to promote lysis, we took a genetic approach to isolate
lysis-resistant host mutants and identify the target genes. We recently
isolated dominant mutations in Escherichia coli mraY that
result in resistance to the bacteriophage The bacterial cell wall is composed of polysaccharide chains of the
repeating unit
N-acetyl-glucosamine-
The Lysis Protein E of
X174 Is a Specific Inhibitor of the
MraY-catalyzed Step in Peptidoglycan Synthesis*
,¶
Department of Biochemistry and Biophysics,
Texas A&M University and § Department of Medical
Biochemistry and Genetics, Texas A&M University System Health Science
Center, College Station, Texas 77843
ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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X174 encodes a single lysis protein,
E, a 91-amino acid membrane protein. Dominant mutations have been
isolated in the host gene mraY that confer E resistance.
mraY encodes translocase I, which catalyzes the formation
of the first lipid intermediate in bacterial cell wall synthesis,
suggesting a model in which E inhibits MraY and promotes cell lysis in
a manner analogous to cell wall synthesis inhibitors like penicillin.
To test this model biochemically, we monitored the effect of E on cell
wall synthesis in vivo and in vitro. We find
that expression of Emyc, encoding an epitope-tagged E
protein, from a multicopy plasmid inhibits the incorporation of
[3H]diaminopimelic acid into cell wall and leads to a
profile of labeled precursors consistent with MraY inhibition.
Moreover, we find that membranes isolated after Emyc
expression are drastically reduced in MraY activity, whereas the
activity of Rfe, an enzyme in the same superfamily, was unaffected. We
therefore conclude that E is indeed a cell wall synthesis inhibitor and
that this inhibition results from a specific block at the
MraY-catalyzed step in the pathway.
INTRODUCTION
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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model, the S holin protein accumulates in the cell
membrane and the R endolysin accumulates in the cytoplasm. At a
genetically programmed time, S forms a membrane lesion to release the R
endolysin into the periplasm where it can degrade the cell wall and
cause lysis (1). In contrast, bacteriophages with small genomes can
only afford to encode a single lysis protein. Three unrelated single
protein lysis systems are known: the E protein from X174 (ssDNA,
Microviridae), and the L and A2 proteins from
MS2 (ssRNA, group I) and Q
(ssRNA, group III) respectively (1-5).
The molecular mechanism of lysis caused by any of these proteins has
been elusive. No cell wall-degrading activity has been associated with
any of these phages, indicating that their lytic mechanism is distinct
from the holin-endolysin system of the larger phages.
X174 E lysis protein (6).
mraY encodes translocase I, which catalyzes the formation of
the first lipid intermediate in cell wall synthesis (7) and is a member
of the
UDP-GlcNAc/MurNAc1:polyisoprenyl-P
GlcNAc/MurNAc 1-P transferase family of enzymes, which we will
refer to as the GPT enzyme family (8). Inhibition of MraY by the
antibiotic mureidomycin results in lysis of Pseudomonas aeruginosa, and depletion of MraY activity from E. coli
also results in concomitant cell lysis (9, 10). These findings, coupled with the isolation of E-resistant mraY mutants, suggest a
model in which E inhibits MraY to elicit lysis by a mechanism similar to that of antibiotics like mureidomycin and penicillin that inhibit cell wall synthesis (see Fig.
1A) (11, 12).
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Fig. 1.
A, model for the mechanism of E-lysis.
Shown is a cartoon illustrating our current model for the mechanism of
E-lysis. E is drawn in the membrane according to its predicted
topology. The predicted topology of MraY is drawn according to the
results of Bouhss et al. (39). The positions of mutations
conferring E resistance are indicated with an x.
B, expression constructs. Shown is a linear representation
of the salient features of the expression constructs used in this
study. Both are derivatives of the expression vector pJF118EH (18).
Restriction sites are as follows: B, BamHI;
H, HindIII; R, EcoRI.
-1,4-N-acetyl-muramic acid (GlcNAc1,4MurNAc). Attached to the MurNAc sugar is a
pentapeptide side chain (in E. coli,
L-Ala-
-D-Glu-meso-DAP-D-Ala-D-Ala).
Adjacent polysaccharide strands are cross-linked by peptide bonds
between the free amino group of meso-diaminopimelic acid
(DAP) from one peptide chain and the carboxyl group of the penultimate
D-Ala of an adjacent peptide. The pathway for cell wall
synthesis can be divided into three phases: (i) cytoplasmic reactions,
(ii) membrane reactions, and (iii) periplasmic reactions (see Fig. 2) (13). In the cytoplasm, UDP-GlcNAc is
converted to UDP-MurNAc in two steps. In the first and committed step
in cell wall biosynthesis, an enolpyruvyl moiety from
phosphoenolpyruvate is added to C-3 by MurA. MurB catalyzes the
reduction of the enolpyruvyl moiety to a lactyl group using reducing
equivalents from NADPH. The pentapeptide is then extended from the
lactyl group of UDP-MurNAc, one or two amino acid residues at a time,
to generate UDP-MurNAc-pentapeptide. On the cytoplasmic face of the
inner membrane, MraY catalyzes the transfer of P-MurNAc-pentapeptide
from UMP to the lipid carrier undecaprenol-P, generating
undecaprenol-P-P-MurNAc-pentapeptide or lipid I. GlcNAc is then added
to lipid I from UDP-GlcNAc by MurG, generating lipid II. Lipid II is
flipped, by an as yet unidentified flippase, to expose the
disaccharide-pentapeptide monomer unit on the periplasmic face of the
membrane. Once exposed to the periplasm, the disaccharide-pentapeptide
is polymerized and cross-linked by multienzyme complexes, including the
penicillin-binding proteins, to form the mature cell wall (14).
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Fig. 2.
Overview of the cell wall synthesis
pathway. Shown is a simplified version of the cell wall synthesis
pathway adapted from Nanninga (13). The jagged line in the
membrane represents undecaprenol-P. G, GlcNAc; M,
MurNAc; pep3, tripeptide; pep5,
pentapeptide; PBPs, penicillin-binding proteins.
The purpose of this study is to test our model for the mechanism of
E-induced lysis (see Fig. 1A) biochemically by monitoring its effect on cell wall synthesis in vivo and in
vitro.
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Media, Chemicals, and Solvent Systems--
Bacterial cultures
were grown in Luria-Bertani (LB) broth (15) or minimal M9 media (16)
supplemented with 0.4% glucose and L-amino acids (20 µg/ml His and Tyr; 30 µg/ml Gly, Ile, Phe, Pro, Ser, and Val; 40 µg/ml Trp; 50 µg/ml Arg; 60 µg/ml Ala and Asp; 70 µg/ml Asn and
Lys; 80 µg/ml Glu; 90 µg/ml Gln; 100 µg/ml Met and Thr; 110 µg/ml Leu). Met and Thr were present at 100 µg/ml to reduce the
size of the intracellular DAP pool (17). All cultures were grown in the
presence of 40 µg/ml kanamycin (Kan). When indicated, bacterial
cultures were induced with
isopropyl--D-thiogalactopyranoside (IPTG) (Alexis, San
Diego, CA) at a concentration of 1 mM.
[3H]Racemic-DAP (1 mCi/ml; 45 Ci/mmol) was purchased from
American Radiolabeled Chemicals Inc. (St. Louis, MO), and
[3H]UMP (1 mCi/ml; 35 Ci/mmol) was purchased from Moravek
Biochemicals (Brea, CA). Unless otherwise indicated, all other
chemicals were purchased from Sigma Chemical Co. (St. Louis, MO).
Solvent systems used for chromatography were as follows: A, isobutyric
acid:1 M NH4OH (5:3; v/v); B, ethanol:1
M ammonium acetate (pH 7.5) (5:2); C, ethanol:1
M ammonium acetate (pH 3.8) (5:2).
Bacterial Strains and Plasmids-- E. coli K-12 strain ET505 (W3110 lysA::Tn10) was the host strain used in this study and was obtained from the E. coli Genetic Stock Center (New Haven, CT) on the web. The lysA mutation was required to prevent the conversion of added [3H]DAP to Lys, so that [3H]DAP is only incorporated into cell wall and its precursors. The plasmid pEmycZK has been described previously (6) and contains Emyc, encoding E with a C-terminal c-Myc epitope tag, cloned under control of the IPTG-inducible tac promoter (Fig. 1B). The control vector pJFlacZK is isogenic to pEmycZK, except that it does not contain Emyc (Fig. 1B). It was constructed by inserting the lacZ gene in the HindIII site of pJF118EH (18) and conversion to KanR as described previously for pEmycZK (6). All DNA manipulations were performed according to standard procedures (19).
Cell Wall Synthesis Measurements-- ET505 pEmycZK and ET505 pJFlacZK were grown in minimal M9 glucose media in 250-ml culture flasks at 37 °C to an A550 of ~0.3 when a portion of each culture was transferred to a small prewarmed 50-ml flask containing sufficient [3H]DAP to give a final activity of 5 µCi/ml. Constant aeration of all cultures was maintained throughout the experiment. After a 10-min prelabeling period, both labeled and unlabeled cultures were induced with IPTG. Culture growth was monitored from the unlabeled culture, and [3H]DAP incorporation into cell wall was monitored in the labeled culture as described previously (17) with minor modifications. Briefly, 0.2-ml aliquots were removed at the indicated times and added directly to 0.8 ml of boiling 5% SDS. The samples were boiled for 1 h and allowed to cool to room temperature before filtering through a 0.22-µm mixed cellulose-ester filter (type-GS, Millipore, Bedford, MA). The filters were washed with 30 ml of distilled H2O and allowed to dry completely, and the radioactivity associated with the cell wall was determined by counting the filters in a Beckman LS5000TD liquid scintillation counter using EcoscintA liquid scintillation fluid (National Diagnostics, Atlanta, GA). In control experiments, label incorporation into the cell wall was linear 10 min after addition of [3H]DAP, indicating that the precursor pools were in isotopic equilibrium.
Determination of Cell Wall Precursor Synthesis during Emyc Expression-- Cultures were grown as described above except that induction with IPTG was done at an A550 of 0.6. After 2 min, a portion of the culture was added to a prewarmed 50-ml flask containing sufficient [3H]DAP for a total activity of 35 µCi/ml. Constant aeration of all cultures was maintained throughout the experiment. After an 8-min pulse-labeling period, prior to any observable lysis, three 1-ml aliquots of labeled culture were removed and centrifuged for 10 min at 4 °C at maximum speed in a microcentrifuge. The cell pellets were washed with 1 ml of ice-cold media and resuspended in 10 µl of dH2O. The cell suspension was spotted on Whatman 3MM paper, and labeled cell wall precursors were separated by development with solvent system A for ~20 h as described (20). Each lane was cut into 1-cm strips and counted as described above. Cell wall, nucleotide, and lipid intermediate fractions ran at published RF values (0, 0.1, and 0.8, respectively).
Purification of UDP-MurNAc Peptides-- UDP-MurNAc-pentapeptide and -tripeptide were accumulated in Bacillus subtilis W23 in the presence of vancomycin and chloramphenicol or cycloserine and chloramphenicol, respectively, and purified through a Sephadex G-25 column (1.6 × 72 cm) as described previously (21). Fractions from the G-25 column were assayed for the presence of N-acetyl sugars as described previously (22). N-Acetyl sugar-containing fractions were pooled, lyophilized, and dissolved in a small volume of dH2O. The partially purified material was subjected to TLC analysis on Whatman Al-SIL-G plates using solvent systems A, B, and C and yielded a single UV-absorbing spot in each case. The UDP-MurNAc-pentapeptide and tripeptide were further purified on a 1-ml Hi-Trap Source Q anion exchange column (Amersham Pharmacia Biotech, Piscataway, NJ) using a 0-0.4 M gradient of NH4HCO3 over 20 column volumes and a flow rate of 1 ml/min. For the UDP-MurNAc-pentapeptide preparation, fractions corresponding to the major UV-absorbing peak at 254 nm were pooled, lyophilized, and dissolved in 200 µl of dH2O. Mass spectrometry at the Texas A&M Laboratory for Biological Mass Spectrometry and quantitative amino acid analysis at the Texas A&M Protein Chemistry Laboratory confirmed that the preparation contained highly purified UDP-MurNAc-pentapeptide (m/z = 1194 and a Ala:Glu:DAP ratio of 3:1:1). The UDP-MurNAc-tripeptide preparation was processed as described for the UDP-MurNAc-pentapeptide, and quantitative amino acid analysis confirmed its identity (Ala:Glu:DAP ratio of 1:1:1).
Accumulation of UDP-MurNAc Peptides during Emyc Expression-- ET505 pEmycZK and ET505 pJFlacZK were grown in 2 liters of LB-Kan at 37 °C to an A550 of ~0.7 and induced with IPTG. Just before induction, MgCl2 was added to 0.1 M to stabilize cells undergoing lysis (23). After 45 min of induction, the cells were harvested and UDP-N-acetyl sugars were purified and characterized as described above for the UDP-MurNAc-pentapeptide. For the preparation from ET505 pEmycZK, fractions corresponding to the major UV-absorbing peak in the anion exchange chromatogram were processed for amino acid analysis as described above. The retention time of the UDP-N-acetyl sugar that accumulated during Emyc expression was compared with those of authentic UDP-MurNAc-tripeptide and -pentapeptide standards using the anion exchange column described above with a 0-0.6 M gradient of NH4HCO3 over 20 column volumes and a flow rate of 1 ml/min.
Membrane Preparation--
200 ml cultures of ET505 pEmycZK and
ET505 pJFlacZK were grown in LB-Kan to an A550 of 0.6 and
induced with IPTG. After 9 min of induction, before any observable
lysis, the cells were harvested by centrifugation at 9000 × g at 4 °C for 15 min. Cell pellets were resuspended in 2 ml of French press buffer (50 mM Tris pH 8.0, 100 mM KCl, 1 mM dithiothreitol) and disrupted in a
French pressure cell (SLM Instruments Inc.) at 16000 psi. Membranes were pelleted by centrifugation at 100,000 × g for 60 min at 4 °C. Finally, the membrane pellets were resuspended in 300 µl of French press buffer and stored in small aliquots at 
80 °C.
Protein concentrations of the membrane preparations were determined on aliquots solubilized in 1% SDS with the Bio-Rad DC
detergent-compatible protein assay kit using bovine serum albumin as a
standard. Membranes isolated from ET505 pEmycZK and ET505 pJFlacZK
contained 17.4 and 21.2 mg/ml protein, respectively.
MraY Exchange Assay-- MraY activity was assayed in the membrane preparations using an exchange assay monitoring the exchange of [3H]UMP for the UMP moiety of UDP-MurNAc-pentapeptide (24). Reactions contained 1 µl of 0.5 M Tris-HCl (pH 8.0), 1 µl of 0.2 M MgCl2, 1 µl of 1 mM UMP, 1 µl of [3H]UMP (100,000 cpm), 1.5 µl of 4.8 mM UDP-MurNAc-pentapeptide, and 5-µl membranes. When indicated, 0.25 µl of a 5 mg/ml tunicamycin stock in methanol was added. Methanol alone did not affect the reaction. Reactions were incubated at 37 °C for 20 min and terminated by boiling for 3 min. After boiling, the membrane debris was pelleted by centrifugation at maximum speed for 5 min in a microcentrifuge. The supernatant was removed to a fresh tube, and the pellets were washed with 10 µl of distilled H2O and centrifuged as before. The wash was pooled with the previous supernatant, and 10 µl was spotted on a TLC plate (Whatman AL-SIL-G) and developed with solvent system C. UMP and UDP-MurNAc-pentapeptide (0.02 µmol each) were spotted in each lane as standards. The UV-absorbing spots corresponding to UMP (RF = 0.6) and UDP-MurNAc-pentapeptide (RF = 0.3) were scraped, and the radioactivity in each spot was determined by liquid scintillation counting as described above and normalized to total protein.
Rfe Exchange Assays--
Rfe activity was measured using an
exchange assay monitoring the exchange of [3H]UMP for the
UMP moiety of UDP-GlcNAc. Rfe reaction compositions were exactly as
those for MraY except that 1.5 µl of 5 mM UDP-GlcNAc was
added in place of UDP-MurNAc-pentapeptide, and 1 µl of
[3H]UMP (200,000 cpm) was used. The reactions were
terminated and processed as described above except that solvent system
A was used for TLC. UMP and UDP-GlcNAc (0.02 µmol each) were spotted in each lane as standards. The UV-absorbing spots corresponding to UMP
(RF = 0.3) and UDP-GlcNAc (RF = 0.2) were scraped, and the radioactivity in each spot was determined by
liquid scintillation counting as described above.
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E Is a Cell Wall Synthesis Inhibitor-- The isolation of lysis-resistant mraY mutants suggested that E is a cell wall synthesis inhibitor that blocks the MraY-catalyzed step in the pathway (Fig. 1A). To test this model, we monitored the effect of E expression on cell wall synthesis in vivo.
The mature cross-linked cell wall is the only cellular material that
remains insoluble when cells are boiled in 4% SDS (17). Therefore, a
convenient assay for monitoring cell wall synthesis is to measure the
incorporation of [3H]DAP, an amino acid unique to the
cell wall, into SDS-insoluble material (17). Using this assay we found
that [3H]DAP incorporation into the cross-linked cell
wall is inhibited 5 min after induction of pEmycZK well before any
detectable effect on growth (Fig. 3).
|
) and pJFlacZK (
) are
shown with arrows indicating the times that a portion
of culture was labeled with [3H]DAP (t = 0) and when IPTG (t = 10) was added to both labeled and
unlabeled cultures. Incorporation of [3H]DAP into 4%
SDS-insoluble material after a 10-min prelabeling period is shown for
ET505 pEmycZK (
) and pJFlacZK (
).
Cell Wall Precursor Synthesis during Emyc Expression--
To
determine the step in cell wall synthesis that is blocked by Emyc, we
monitored the synthesis of cell wall precursors during Emyc
expression from pEmycZK. After induction of Emyc, cells were pulse-labeled with [3H]DAP for 8 min (Fig.
4A), and the major cell wall
precursor fractions were separated by paper chromatography into cell
wall, nucleotide intermediates (UDP-MurNAc-tripeptide and
-pentapeptide), and lipid intermediates (lipids I and II) (20). As
shown in Fig. 4B, only the nucleotide intermediates are
labeled with [3H]DAP after Emyc induction,
whereas all three fractions are labeled in the control strain. Because
the ratio of the [3H]DAP-labeled UDP-MurNAc-tripeptide
and -pentapeptide species in the nucleotide fraction could not be
determined in this experiment, the data are consistent with an
Emyc-mediated block at either the MurF- or the MraY-catalyzed steps of
cell wall synthesis (Fig. 2).
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Accumulation of UDP-MurNAc-pentapeptide during Emyc
Expression--
For a more precise determination of which nucleotide
intermediates are synthesized following Emyc induction, we
purified the UDP-N-acetyl sugars that accumulate during
Emyc expression and compared them to those isolated from the
control strain (see "Experimental Procedures"). By
N-acetlylsugar assay (22) and A262
measurements, ~5 times more UDP-N-acetyl sugars were
isolated after Emyc expression than in the control (1.6 versus 0.3 µmol). Anion exchange chromatography of this
material revealed one major UV absorbing peak eluting between 25 and 30 ml (Fig. 5A), whereas the
control preparation was a complex mixture containing multiple peaks of
similar absorbance (Fig. 5B). Using a slightly different
elution gradient, authentic UDP-MurNAc-tripeptide and -pentapeptide
standards are clearly separable, and the intermediate that accumulates
during Emyc expression has an identical retention time to
UDP-MurNAc-pentapeptide. However, quantitative amino acid analysis
found a 2:1:1 Ala:DAP:Glu ratio for the peak fractions shown in Fig.
5B, which is consistent with an equimolar mixture of the
tripeptide and pentapeptide species. The reason for this discrepancy is
not known but may be due to contaminants found in the preparation from
E. coli that are absent in the standard preparations from
B. subtilis. Based on the chromatographic analysis, we
conclude that UDP-MurNAc-pentapeptide is synthesized and accumulates to
high levels after Emyc induction. Therefore, the step of
cell wall synthesis inhibited by Emyc must be the one catalyzed by
MraY.
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MraY Activity Is Dramatically Reduced in Membranes Isolated after Emyc Expression-- The in vivo experiments described above indicate that it is the MraY step that is blocked upon Emyc expression. To test this directly, we measured MraY activity in vitro using membranes isolated with and without prior expression of Emyc. MraY catalyzes the reaction,
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Rfe Activity Is Normal in Membranes Containing Emyc--
Besides
MraY, the only other known member of the GPT enzyme family in E. coli is Rfe (8, 26). Rfe catalyzes the formation of the first
lipid intermediate in enterobacterial common antigen synthesis,
undecaprenol-P-P-GlcNAc (26). To determine whether Emyc is a specific
inhibitor of MraY like the antibiotic mureidomycin (27) or a general
GPT enzyme inhibitor like tunicamycin (25, 26), we measured Rfe
activity in the membranes described above using an exchange reaction
similar to that catalyzed by MraY. Rfe activity was nearly identical in
membranes isolated with and without prior Emyc expression
(Fig. 6B). As expected, Rfe activity was completely
inhibited by the general inhibitor tunicamycin (Fig.
6B).
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In a previous report we described the isolation of dominant mutants in the host gene mraY that resulted in resistance to E-induced lysis (6). In addition, we found that overexpression of mraY from a medium-copy plasmid could also confer resistance (6). Given these results, we proposed a model in which E inhibits cell wall synthesis at the MraY-catalyzed step in the pathway to promote lysis (Fig. 1A). The results presented in this study provide compelling biochemical support for this model. By monitoring cell wall synthesis in vivo, we observed complete inhibition of [3H]DAP incorporation into cell wall after only 5 min of Emyc expression. We also found that the cytoplasmic nucleotide precursors are the last intermediates labeled with [3H]DAP (Fig. 4) and that UDP-MurNAc-pentapeptide accumulates to high levels during Emyc expression (Fig. 5). Taken together, the in vivo experiments indicate that it is the MraY step in the pathway that is blocked. This blockage was demonstrated unequivocally by the finding that in vitro MraY exchange activity is almost completely inhibited in membranes containing Emyc (Fig. 6A).
There are several antibiotics that block the MraY step, and they can be classified based on their mode of inhibition. They are either direct inhibitors of MraY, like mureidomycin and tunicamycin (25, 28), or indirect inhibitors like amphomycin, bacitracin, and colicin M (29-31), all of which are known to induce bacteriolysis (9, 29, 31-33). The direct inhibitors all resemble the nucleotide substrate with additional hydrophobic moieties attached. They can be further subdivided as specific MraY inhibitors, like mureidomycin (27), or general GPT enzyme inhibitors like tunicamycin (25, 26, 34). The indirect inhibitors limit the availability of the substrate undecaprenol-P by sequestering it or interfering with its recycling (30, 35). In addition to MraY, the general GPT enzyme inhibitors like tunicamycin inhibit the eukaryotic GPT enzyme involved in protein glycosylation, and the indirect inhibitors will bind its lipid substrate, dolichol-P (35, 36). Therefore, only specific MraY inhibitors are likely to have potential therapeutic applications in treating bacterial infections.
E is a 91-amino acid membrane protein. Protein fusion analysis has revealed that only the amino-terminal 29 amino acids of the molecule, encompassing its putative transmembrane domain (TMD), are required for lytic function (37, 38).2 With these observations in mind, E likely inhibits MraY in one of two ways, either by direct inhibition through a TMD-TMD interaction or by one of the indirect modes described above. To distinguish between these modes of inhibition, we monitored the effect of Emyc on the activity of the only other known E. coli GPT enzyme, Rfe, which catalyzes the formation of the first lipid intermediate in enterobacterial common antigen biosynthesis, undecaprenol-P-P-GlcNAc (26). We observed no inhibition of Rfe activity in membranes containing Emyc (Fig. 6B). If Emyc were a general or an indirect GPT inhibitor, it should have also inhibited Rfe. We therefore conclude that Emyc is a specific MraY inhibitor. This apparent specificity coupled with the small size of its lethal domain and its selectable lytic phenotype make E an attractive system to probe MraY function and inhibition. It may also help in the development of new specific MraY inhibitors with therapeutic applications. Coprecipitation and inhibition studies using a more purified system are underway to investigate E inhibition of MraY in more detail.
Now that there is both biochemical and genetic evidence that cell wall
synthesis inhibition is the mechanism of E-induced lysis, it is a
reasonable hypothesis that other single protein lysis systems also act
in the same way. We are currently taking similar genetic and
biochemical approaches to investigate whether the Q A2
and MS2 L lysis proteins are also cell wall synthesis inhibitors, and
if so, at which step they act.
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ACKNOWLEDGEMENTS |
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We thank all members of the Young laboratory for support and helpful discussions. We also thank Jinny Johnson and Larry Dangott of the Texas A&M Protein Chemistry Laboratory for technical assistance and for performing the quantitative amino acid analysis and Bill Russell of the Texas A&M Laboratory for Biological Mass Spectrometry for performing mass spectrometry. We are especially grateful to Hung Ton-That and Olaf Schneewind for helpful discussions and advice.
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grant GM27099 and by funds from the Robert A. Welch Foundation and the Texas Agricultural Experiment Station.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.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, 2128 TAMU, Texas A&M University, College Station, TX 77843-2128. Tel.: 979-845-2087; Fax: 979-862-4718; E-mail: ryland@tamu.edu.
Published, JBC Papers in Press, November 14, 2000, DOI 10.1074/jbc.M007638200
2 W. D. Roof and R. Young, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are:
MurNAc, N-acetylmuramic acid;
GlcNAc, N-acetylglucosamine;
Kan, kanamycin;
DAP, diaminopimelic
acid;
IPTG, isopropyl--D-thiogalactopyranoside;
undecaprenol-P, undecaprenol-phosphate;
GPT enzyme family, UDP-GlcNAc/MurNAc polyisoprenyl-P: GlcNAc/MurNAc 1-P transferase enzyme
family;
TMD, transmembrane domain.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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REFERENCES
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| 1. | Young, R., Wang, I.-N., and Roof, W. D. (2000) Trends Microbiol. 8, 120-128 |
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| 7. | Ikeda, M., Wachi, M., Jung, H., Ishino, F., and Matsuhashi, M. (1991) J. Bacteriol. 173, 1021-1026 |
| 8. | Lehrman, M. A. (1994) Glycobiology 4, 768-771 |
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| 10. | Boyle, D., and Donachie, W. (1998) J. Bacteriol. 180, 6429-6432 |
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