(Received for publication, September 8, 1995)
From the
The envA gene of Escherichia coli has been
shown previously to be essential for cell viability (Beall, B. and
Lutkenhaus, J.(1987) J. Bacteriol. 169, 5408-5415), yet
it encodes a protein of unknown function. Extracts of strains harboring
the mutant envA1 allele display 3.5-18-fold reductions
in UDP-3-O-acyl-N-acetylglucosamine deacetylase
specific activity. The deacetylase is the second enzymatic step of
lipid A biosynthesis. The structural gene coding for the deacetylase
has not been assigned. In order to determine if the envA gene
encodes the deacetylase, envA was cloned into an
isopropyl-1-thio--D-galactopyranoside-inducible T7-based
expression system. Upon induction, a protein of the size of envA was highly overproduced, as judged by SDS-PAGE. Direct deacetylase
assays of cell lysates revealed a concomitant
5,000-fold
overproduction of activity. Assays of the purified, overproduced EnvA
protein demonstrated a further
5-fold increase in specific
activity. N-terminal amino acid sequencing of the purified
protein showed that the first 20 amino acids matched the predicted envA nucleotide sequence. Contaminating species were present
at less than 1% of the level of the EnvA protein. Thus, envA is the structural gene for UDP-3-O-acyl-GlcNAc
deacetylase. Based on its function in lipid A biosynthesis, we propose
the new designation lpxC for this gene.
The outer membrane of Gram-negative bacteria provides a
formidable permeability barrier to the entry of large and hydrophobic
compounds, primarily because of the unique lipopolysaccharide (LPS) ()associated with the outer leaflet of the outer membrane
(Nakae, 1986; Nikaido, 1976; Nikaido and Vaara, 1985). Over the years,
a large number of mutant strains of Escherichia coli and other
Gram-negative bacteria that exhibit defects in this barrier have been
isolated and characterized (Weigand and Rothfield 1976; Roantree et
al., 1977; Scudamore et al., 1979; Coleman and Leive,
1979; Angus et al., 1982; Vaara, 1990; Vuorio et al.,
1991). For many of these mutants the molecular defect has not yet been
determined.
One well studied example of such mutations is envA1, originally described in the late 1960s by Normark and
colleagues (1969). The envA1 mutation was obtained in a study
of penicillin resistance by screening ethyl methanesulfonate-treated,
penicillinase-producing E. coli for increased ampicillin
sensitivity and smooth colony morphology. Strains harboring envA1 exhibit a complex phenotype, including permeability to a wide
variety of antibiotics and dyes, suggesting that their EDTA-sensitive
outer membrane surface layer is defective (Normark, 1970; Normark et al., 1969; Young and Silver, 1991). Mutants bearing envA1 also display a morphological defect in the completion of
cell septation, resulting in growth as short chains (Normark et
al., 1969; Normark et al., 1971). In addition, there is a
slight (30%) reduction in the content of apparently normal LPS
(Grundstrom et al., 1980).
Only the one original allele of the envA locus, the envA1 mutation, has been described (Normark et al., 1969). envA1 has since been used to locate precisely the mutated envA reading frame to minute 2 on the E. coli chromosome at the end of a large gene cluster required for cell division and cell wall biogenesis (Sullivan and Donachie, 1984). The sequence of the envA gene predicts a protein of 34 kDa. The gene itself has been demonstrated to be essential by insertional mutagenesis (Beall and Lutkenhaus, 1987), implying that the envA1 allele encodes a protein that retains some residual activity (Beall and Lutkenhaus, 1987). However, no further hints as to the function of envA have, as yet, been reported (Beall and Lutkenhaus, 1987), and no related sequences of known function from other organisms are available for comparative studies.
LPS is an essential component of the outer membrane and its associated barrier function (Morrison and Ryan, 1992; Rietschel, 1984). The lipid A component of LPS is the basic building block that comprises most of the outer leaflet (Raetz, 1990, 1993). The biosynthesis of lipid A in E. coli has been described (Raetz, 1990, 1993). It begins with the 3-O-acylation of UDP-GlcNAc with R-3-hydroxymyristate (Fig. 1), followed by deacetylation and reacylation at the glucosamine ring nitrogen with a second R-3-hydroxymyristate moiety (Raetz, 1990, 1993; Williamson et al., 1991). The genes encoding the UDP-GlcNAc acyltransferases of E. coli have been identified as lpxA and lpxD (firA), respectively (Coleman and Raetz, 1987; Kelly et al., 1993), but the structural gene encoding UDP-3-O-acyl-GlcNAc deacetylase (Anderson et al., 1988) has remained elusive. The deacetylase has been implicated as a point of regulation with respect to the biosynthesis of lipid A (Anderson et al., 1993; Mohan et al., 1994). Identification of the deacetylase structural gene could shed light on the nature of this regulation.
Figure 1: Relationship of key precursors of fatty acid, lipid A, and peptidoglycan biosynthesis. Three major cell envelope components arise from two key precursors of E. coli metabolism. UDP-GlcNAc serves as the glucosamine source in both peptidoglycan and lipid A biosynthesis. The lipid A pathway begins with the acylation at the 3-OH moiety of the glucosamine ring of UDP-GlcNAc with R-3-hydroxymyristate derived from R-3-hydroxymyristoyl acyl carrier protein. The latter is also the precursor of palmitate residues found in membrane glycerophospholipids. Since the equilibrium constant for UDP-GlcNAc acylation is unfavorable (Anderson et al., 1993), the second reaction of the lipid A pathway (the deacetylase) appears to function as the first committed step, and it may be regulated. U, uridine; ACP, acyl carrier protein; PtdEtn, phosphatidylethanolamine.
We now demonstrate that extracts of E. coli strains harboring the envA1 allele possess diminished levels of UDP-3-O-acyl-GlcNAc deacetylase activity, that T7-based overexpression of the envA reading frame causes a massive induction of deacetylase activity in cell extracts, and that UDP-3-O-acyl-GlcNAc deacetylase activity purifies with the EnvA protein. Thus, envA is an essential gene (Beall and Lutkenhaus, 1987), encoding the second enzymatic step unique to lipopolysaccharide biosynthesis. The intriguing questions relating to the molecular basis for the pleiotropic phenotypes associated with envA1 may now be studied at a biochemical level.
Cultures of MB5503 and MB5504,
inoculated from small overnight cultures into 125 ml of LB broth, were
grown at 37 °C to late log phase. MB5503 and MB5504 transformed
with the pENV15 plasmid were grown in parallel but under selection with
20 µg/ml streptomycin. Cells were harvested by centrifugation at
8000 g for 10 min, washed once in 1 volume of 10
mM sodium phosphate, pH 7.0, and resuspended in 2 ml (62-fold
concentration) of the same buffer.
For T7-based deacetylase activity
induction, log phase cultures of BL21(DE3)/pLysS alone or in
combination with pET11a or pET11a-EnvA, were grown with shaking in LB
broth at 37 °C. The overnight cultures used for inoculation had
been grown with appropriate selection (50 µg/ml ampicillin for the
pET vectors and 25 µg/ml chloramphenicol for pLysS). When A reached 0.15-0.2, the cells were induced
by the addition of IPTG to a final concentration of 1 mM. The
cells were incubated with shaking for another 2 h. Cells were harvested
by centrifugation at 6000
g for 10 min., washed in
volume of 10 mM sodium phosphate, pH 7.0, containing 20%
glycerol and 0.2 mM dithiothreitol (DTT), and resuspended in
this buffer (containing 0.5 mM DTT) to yield a 25-fold
concentrate relative to the original culture.
In each of the above
experiments, cell extracts were prepared by one passage of the washed,
concentrated cells through a French pressure cell at 18,000 p.s.i.
Unbroken cells and debris were removed by centrifugation at 8000
g for 10 min. Protein concentrations were determined
by the method of Bradford (Bio-Rad protein assay), using bovine serum
albumin as the standard (Bradford, 1976).
Figure 2: Cloning of the envA gene from E. coli and construction of pET11a-EnvA. Details of the construction strategy and selection and the sequence of primers used for PCR amplification are described under ``Experimental Procedures.'' For clarity, only restriction sites relevant to the construction are shown. Reading frames encoding resistance to antibiotics are placed as shown and are abbreviated as follows: Amp, ampicillin; Str/Spc, streptomycin/spectinomycin; Tet, tetracycline. The NdeI - BamHI restriction fragment resulting from digestion of pET11a-gyrB was removed prior to subsequent ligation, as described under ``Experimental Procedures.''
Briefly, this cloning was accomplished by preparing
a pool of 2.5-kilobase EcoRI fragments from the envAE. coli strain LS584. This pool
was ligated to EcoRI-digested and phosphatase-treated vector
pLL24, a hybrid of pBR322 and a pSC101 replicon, joined at their
respective EcoRI sites. This ligation mixture subsequently was
used to transform the envA
E. coli strain, LS583. Selection was for complementation of the envA1 allele (growth in the presence of rifampicin at 1 µg/ml) in
combination with either streptomycin or spectinomycin, each at 5
µg/ml (resistance associated with the pSC101 replicon) or
ampicillin at 10 µg/ml (resistance associated with the pBR322
replicon). A single, low copy, pSC101 origin-containing transformant
was obtained. This construct, conferring resistance to rifampicin in an envA
host as well as
streptomycin/spectinomycin resistance, and consisting of the
anticipated 2.5-kilobase fragment with the restriction sites expected
for the envA region (Sullivan, 1984), was named pENV15.
The NdeI-BamHI-cut PCR product was
ligated at room temperature (19 °C) overnight into similarly
cut and gel-purified pET11a (Studier et al., 1990). In order
to facilitate the complete digestion of this vector, properly digested
pET11a was actually obtained as the fragment released from NdeI and BamHI digestion of pET11a into which the
2415-base pair gyrB gene had been cloned. Upon gel
purification, this doubly digested vector was easily separated from any
incompletely digested products. This ligation mixture was used to
transform DH5
cells made competent by the method of Hanahan(1983),
and colonies resistant to ampicillin were selected. Plasmid was
isolated from a number of putative clones by the method of Holmes and
Quigley(1981) and analyzed for the presence of an insert of
approximately 900 base pairs. One resulting plasmid with such an insert
was designated pET11a-EnvA, and it was used to transform the envA
T7 polymerase-harboring host LS822
(BL21(DE3), envA1) to ampicillin resistance. The construct
pET11a-EnvA proved capable of complementing the phenotypic sensitivity
of this envA1 mutant to rifampicin at 1 µg/ml and
overproduced a protein band of appropriate molecular weight upon
induction with 1 mM IPTG. Sequencing of the envA reading frame harbored by this clone by the dideoxy
chain-termination method of Sanger(1977) verified the exact match of
the cloned sequence to that published previously by Beall and
Lutkenhaus(1987).
The cells were lysed and brought to an ammonium sulfate fraction in two batches. In each case, this was accomplished by thawing the cell paste on ice, followed by addition of Brij 58 to approximately 0.05% (w/v) and incubation in ice water until a viscous mixture was obtained as a result of autolysis mediated by the endogenous T7 lysozyme (encoded by the pLysS plasmid). This mixture was clarified to generate a cell-free extract by centrifugation in a Ti-45 rotor (Beckman) at 40,000 rpm for 60 min. Deacetylase activity was precipitated by the addition of 0.3 g of solid ammonium sulfate/ml of cell-free extract (approximately 50% saturation) over a 45-min period with a further incubation on ice with stirring for 20 min. The ammonium sulfate precipitate was collected by centrifugation in a Sorvall SS-34 rotor at 14,000 rpm for 20 min at 4 °C. The ammonium sulfate pellets were resuspended in buffer A (25 mM imidazole, pH 7.0, 20% glycerol, 2 mM DTT) and dialyzed against this buffer at 4 °C for 5 h, at which point the conductivity had decreased to a level appropriate for further chromatography. This fraction (40.5 ml, 2552 mg of protein) was rapidly frozen in two portions, which were further purified separately on a Q-Sepharose Fast Flow column.
A priori, these observations could be explained by a regulatory function for the EnvA protein. Alternatively, the EnvA protein could actually be the deacetylase. The deacetylase might be regulated in some manner to maintain a constant level of enzyme activity. The level of expression from each of the envA gene(s) in these complemented strains is not known, and such experiments do not allow us to distinguish between these two possibilities. However, the results do indicate that the level of deacetylase activity in these cells is, at a minimum, a function of the allelic state of the envA gene.
To verify the function of the reading frame in this construct, pET11a-EnvA was transformed into LS822 (BL21(DE3) envA1), a lysogenic strain containing the gene for T7 RNA polymerase under the control of tandem lac UV5 promoters and sensitive to low levels of rifampicin as a consequence of the envA1 allele. Transcription of T7 RNA polymerase in this strain is normally somewhat ``leaky,'' and the small amount of polymerase produced results in low basal transcription and translation of the gene(s) downstream of the T7 promotor. The pET11a-EnvA construct proved capable of complementing the envA1 permeability defect in this strain in the presence of IPTG as evidenced by the ability to grow on LB plates containing 1 µg/ml rifampicin.
The validated pET11a-EnvA construct was transformed into BL21(DE3)/pLysS. Cell-free extracts made from small cultures of BL21(DE3)/pLysS/pET11a-EnvA after 2-h induction with 1 mM IPTG were examined by SDS-PAGE. They showed strong overexpression of an approximately 34-kDa polypeptide (Fig. 3, lane 10), identical in size to the protein predicted from the inferred amino acid sequence of the envA gene. Further, this band was unique to these extracts and was not seen in induced extracts of either BL21(DE3)/pLysS (host) or BL21(DE3)/pLysS/pET11a (vector) (Fig. 3, lanes 4 and 7, respectively).
Figure 3:
Overexpression of the envA gene product under control of the T7
promoter. Portions of whole culture (lanes 2, 3, 5, 6, 8, and 9) and soluble extract
derived from these cultures (lanes 4, 7, and 10) were prepared for SDS-PAGE analysis, as described under
``Experimental Procedures.'' Lanes 1 and 11, protein molecular weight standards: phosphorylase b (97,400); bovine serum albumin (66,200); ovalbumin (45,000);
carbonic anhydrase (31,000); soybean trypsin inhibitor (21,500);
trypsin (14,400). Lane 2, strain BL21(DE3)/pLysS (host)
without IPTG induction. Lane 3, strain BL21(DE3)/pLysS after
IPTG induction. Lane 4, soluble extract derived from lane
3. Lane 5, strain BL21(DE3)/pLysS/pET11a (host/vector)
without induction. Lane 6, BL21(DE3)/pLysS/pET11a after IPTG
induction. Lane 7, soluble extract derived from lane
6. Lane 8, BL21(DE3)/pLysS/pET11-EnvA (host/vector with envA insert), without IPTG induction. Lane 9,
BL21(DE3)/pLysS/pET11-EnvA after IPTG induction. Lane 10,
soluble fraction derived from lane
9.
The soluble extracts of these induced host, vector, and pET11a-EnvA-bearing strains were assayed for UDP-3-O-acyl-GlcNAc deacetylase activity under standard conditions (Table 3, Experiment 2). The extract made from cells harboring the pET11a-EnvA clone was found to overexpress greatly the deacetylase activity, consistent with massive induction of the envA gene product. These results indicate that deacetylase activity parallels the apparent level of envA gene expression but formally does not rule out the alternative possibility that the expressed envA gene product directs expression of a similarly sized protein, which itself is the deacetylase.
From a 15-liter culture of BL21(DE3)/pLysS/pET11a-EnvA cells, grown and induced as described under ``Experimental Procedures,'' we obtained 73.5 g of cell paste. This material was conveniently lysed with a single freeze-thaw step and the subsequent addition of 0.05% (w/v) Brij detergent. The presence of T7 lysozyme in these cells, which aids in suppressing the expression of cloned protein during growth prior to induction, acts to autolyze the peptidoglycan layer upon freeze-thawing. Analysis of the high speed supernatant fraction by SDS-PAGE and deacetylase assay revealed the presence of a high level of a 34-kDa protein and of a correspondingly massive amount of enzymatic activity (Fig. 4, lane 1, and Table 4). We found that enzyme held at this stage of purification was unstable with a decay half-life of approximately 40 h (stability studies data not shown). This instability was essentially eliminated by the next step.
Figure 4: Analysis by SDS-PAGE of fractions obtained during purification of the EnvA protein. Samples from each step of purification were boiled for 5 min in SDS-PAGE buffer and analyzed by SDS-PAGE. Lane 4, protein molecular weight standards; phosphorylase b (97,400); bovine serum albumin (66,200); ovalbumin (45,000); carbonic anhydrase (31,000); soybean trypsin inhibitor (21,500); lysozyme (14,400). Lane 1, induced, soluble crude extract. Lane 2, dialyzed and filtered ammonium sulfate precipitate. Lane 3, Q-Sepharose chromatography peak fraction number 50.
The protein band and deacetylase activity co-precipitated in high yield in a 50% ammonium sulfate fraction (Fig. 4., lane 2, and Table 4). The latter was dialyzed, filtered, and processed by anion ion exchange chromatography. The 34-kDa protein band and deacetylase activity were both recovered at approximately 110 mM potassium chloride (Fig. 4, lane 3, and Fig. 5). The isolated protein was judged to be >98% pure by SDS-PAGE (Fig. 4, lane 3).
Figure 5: Co-chromatography of 34-kDa band and deacetylase activity. Equal volumes of representative fractions from the Q-Sepharose column were boiled for 5 min in SDS-PAGE buffer and analyzed by SDS-PAGE. Lane L, column load (lane 2, Fig. 4). Lane W, Q-Sepharose wash. Lanes 2-20, column fractions as indicated. Lane S, protein molecular weight standards; phosphorylase b (97,400); bovine serum albumin (66,200); ovalbumin (45,000); carbonic anhydrase (31,000); soybean trypsin inhibitor (21,500); and lysozyme (14,400).
A sample of similarly purified material was analyzed by Edman degradation in order to demonstrate that this isolated protein was indeed the expression product of the envA gene. The sequence obtained, Met-Ile-Lys-Gln-Arg-Thr-Leu-Lys-Arg-Ile-Val-Gln-Ala-Thr-Gly-Val-Gly-Leu-His, was exactly that predicted for the first 19 amino acids of the envA reading frame (Beall and Lutkenhaus, 1987). Further, the molar yield of phenylthiohydantoin-derivative products was consistent with the absence of measurable contaminating proteins, thus confirming the EnvA protein itself was the source of the comigrating deacetylase activity. We propose the designation lpxC for this gene.
Early studies of strains bearing the envA1 mutation indicated a slight reduction of lipopolysaccharide content compared with wild-type, possibly accounting for the antibiotic hypersensitivity associated with envA1 (Wolf-Watz and Normark, 1976; Grundstrom et al., 1980). We (Young and Silver, 1991) postulated that, in envA1 bearing mutants, increased entry of hydrophobic antibiotics might be due to the lowered LPS content but that increased entry of hydrophilic antibiotics and exit of periplasmic proteins could be due to breakage and rejoining of the outer membrane during inefficient separation at cell division. A number of pleiotropic mutants of E. coli and Salmonella exhibit similar ``leaky'' phenotypes with accompanying septal morphological defects (Weigand and Rothfield, 1976).
Wolf-Watz and Normark(1976)
found that N-acetylmuramyl-L-alanine amidase
activity, possibly involved in peptidoglycan remodeling at the septum,
was 6-fold decreased in an envA1 strain. Since a 20-fold
decrease of this amidase caused by a mutation at a different site in an envA background does not lead to increased
outer membrane permeability (Tomioka et al., 1983), envA is unlikely to be the structural gene for this amidase.
Recently, it was noted that point mutations in the lpxA and lpxB genes, encoding the first acyl transferase (Galloway and
Raetz, 1990) and the disaccharide synthase (Crowell et al.,
1987) of lipid A biosynthesis, respectively, display increased
antibiotic permeability (Vuorio and Vaara, 1992). ()In
addition, conditional mutants defective in lpxA (Galloway and
Raetz, 1990) leak periplasmic enzymes (
)under permissive
conditions. The fact that these mutations in lipid A biosynthesis
exhibit a phenotype similar to that associated with envA1 and
the finding that UDP-3-O-acyl-GlcNAc deacetylase activity is
decreased in envA1-bearing strains suggested that the envA gene product might also play a role in lipid A biosynthesis.
When assayed in the range of pH 5.5-6.5, we found that crude
extracts made from a number of common enterobacterial strains possess
deacetylase specific activities that are within an order of magnitude
of each other (Table 2). However, extracts of envA1-bearing strains of E. coli K12 were
3.5-18-fold less active than closely related constructs ( Table 2and Table 3). The depression of deacetylase activity
in strains harboring the envA1 mutation was especially
apparent in an isogenic pair of E. coli strains (Table 3). The deficit in deacetylase activity could be corrected
specifically, although not increased above normal levels, by
complementation in trans with the low copy vector pENV15 (envA).
Forced expression of the envA reading frame using the T7 system of
Studier(1987) resulted in massive overexpression of deacetylase
activity in broken cell preparations (Table 3). The demonstration
that essentially all the deacetylase activity in these extracts
purified with the expressed EnvA protein verified that the envA locus is indeed the structural gene encoding
UDP-3-O-acyl-GlcNAc deacetylase. We propose the designation lpxC to replace envA, given its function in
lipopolysaccharide biosynthesis.
In the lipid A pathway, the deacetylase functions between two acyltransferases, encoded by the lpxA and lpxD (firA) genes, respectively (Fig. 1). lpxA and lpxD map to a macromolecular synthesis operon at minute 4, containing genes involved in DNA, phospholipid, lipid A, and outer membrane protein biosynthesis (Fig. 6) (Kelly et al., 1993; Raetz, 1993). In contrast, the lpxC (envA) gene near minute 2 resides at the 3` end of a large cluster of murein and cell division genes, the regulation of which is complex and not fully understood (Donachie, 1993, Errington, 1993). LpxC (envA) appears to have its own promoter (Sullivan and Donachie, 1984), but transcripts arising from upstream promoters could also contribute to deacetylase expression under some conditions. Whether the up-regulation of the deacetylase (Anderson et al., 1993) under conditions of limited lipid A biosynthesis occurs at the level of transcription or by some other mechanism remains to be established. The association of lpxC with genes involved in peptidoglycan biosynthesis and cell division may reflect the operation of a novel, global regulatory network for envelope assembly.
Figure 6: Organization of the genes in the minute 2 and minute 4 regions of the E. coli chromosome. These regions have been completely sequenced, and the functions of most reading frames have been assigned as indicated (Errington, 1993; Tomasiewicz, 1987; Kelly et al., 1993). All genes shown at both minute 2 and minute 4 are transcribed from left to right (clockwise).
Genetic interruption of the early steps in LPS biosynthesis is bactericidal (Galloway and Raetz, 1993; Kelly et al., 1993). Mutants in these genes must be studied as conditional lethals (Raetz, 1990; Raetz, 1993). The previous demonstration by Beall and Lutkenhaus (Beall and Lutkenhaus, 1987) of lethality associated with insertional inactivation of the envA reading frame is consistent with these observations. We have confirmed that extracts of the envA1-bearing point mutant do indeed possess residual enzymatic function ( Table 2and Table 3), as postulated by Beall and Lutkenhaus(1987). The question of why the depletion of lipid A deacetylase activity has such pleiotropic effects on cell morphology and outer membrane permeability requires further examination.
The apparent instability of the lpxC/envA gene, when introduced into E.
coli on medium or high copy number plasmids (Sullivan and
Donachie, 1984), also requires further investigation. Overexpression of
the deacetylase might consume too much R-3-hydroxymyristoyl
acyl carrier protein (Fig. 1) resulting in depletion of membrane
glycerophospholipids.
The lpxC (envA) gene bears
no significant relationship to other reading frames in
GenBank. It contains no structural motifs that would
suggest to what family of amidase (i.e. metallo, serine, or
cysteine) it belongs. Mechanistic studies will be facilitated by the
availability of large amounts of deacetylase. Cloning of the lpxC gene from other bacterial species, like Pseudomonas,
would also serve to identify critical amino acid residues and might
shed light on the mechanism of the deacetylase.