(Received for publication, July 26, 1995; and in revised form, October 10, 1995)
From the
Two different approaches to identify the gene encoding the phosphoglucosamine mutase in Escherichia coli were used: (i) the purification to near homogeneity of this enzyme from a wild type strain and the determination of its N-terminal amino acid sequence; (ii) the search in data bases of an E. coli protein of unknown function showing sequence similarities with other hexosephosphate mutase activities. Both investigations revealed the same open reading frame named yhbF located within the leuU-dacB region at 69.5 min on the chromosome (Dallas, W. S., Dev, I. K., and Ray, P. H.(1993) J. Bacteriol. 175, 7743-7744). The predicted 445-residue protein with a calculated mass of 47.5 kDa contained in particular a short region GIVISASHNP with high similarity to the putative active site of hexosephosphate mutases. In vitro assays showed that the overexpression of this gene in E. coli cells led to a significant overproduction (from 15- to 50-fold) of phosphoglucosamine mutase activity. A hexose 1,6-diphosphate-dependent phosphorylation of the enzyme, which probably involves the serine residue at position 102, is apparently required for its catalytic action. As expected, the inactivation of this gene, which is essential for bacterial growth, led to the progressive depletion of the pools of precursors located downstream from glucosamine 1-phosphate in the pathway for peptidoglycan synthesis. This was followed by various alterations of cell shape and finally cells were lysed when their peptidoglycan content decreased to a critical value corresponding to about 60% of its normal level. The gene for this enzyme, which is essential for peptidoglycan and lipopolysaccharide biosyntheses, has been designated glmM.
UDP-GlcNAc ()is one of the main cytoplasmic
precursors of bacterial cell-wall
peptidoglycan(1, 2) . In Escherichia coli and
other Gram-negative bacteria, it is also the precursor for outer
membrane lipopolysaccharide, as well as for the synthesis of the
enterobacterial common antigen(3, 4) . The genes and
enzymes involved in the steps located downstream from this branchpoint
(shown schematically in Fig. 1) in the different pathways have
in most cases been characterized and studied in
detail(3, 4, 5, 6, 7, 8, 9, 10) .
In comparison, the metabolic route leading to the formation of
UDP-GlcNAc has been poorly investigated. Considering that it was a
potential site for the regulation of the flow of metabolites going
through the peptidoglycan and lipopolysaccharide pathways, the latter
reaction sequence has recently been investigated in more detail (11, 12) . Four successive steps are required for the
synthesis of UDP-GlcNAc from fructose 6-phosphate (13, 14) (Fig. 1). The first of these reactions
is catalyzed by the L-glutamine:D-fructose-6-phosphate amidotransferase
(also named glucosamine-6-P synthase)(15) . Mutants altered in
this activity are characterized by an auxotrophy for GlcN or
GlcNAc(16, 17) , and the corresponding glmS gene has been located at 84 min on the E. coli map(15, 18) . The subsequent steps from GlcN-6-P
to UDP-GlcNAc are via GlcN-1-P (Fig. 1). We have previously
shown that the gene of unknown function preceding glmS on the E. coli chromosome codes in fact for the GlcNAc-1-P
uridyltransferase, and the gene for this final step leading to
UDP-GlcNAc (Fig. 1) was named glmU(11) . More
recently, we demonstrated that the glmU gene product also
catalyzes the preceding step of acetylation of GlcN-1-P and thus
appears as a bifunctional enzyme catalyzing two subsequent steps in the
same pathway (12) (Fig. 1). Here we describe the partial
purification and some properties of the phosphoglucosamine mutase which
catalyzes the interconversion of GlcN-6-P and GlcN-1-P isomers, as well
as the identification of the corresponding glmM gene on the
chromosome of E. coli.
Figure 1: Biosynthesis and cellular utilization of UDP-N-acetylglucosamine in E. coli.
Figure 2: Localization of the glmM gene at 69.5 min on the E. coli chromosome. Locations of some other genes present on the DNA fragment carried by phage clone 14F11 are indicated at the top, and their orientation relative to the chromosome is indicated by an arrow. Bacterial DNA present in plasmid inserts is shown below. The glmM gene is represented as a hatched region, and the position of the lac promoter relative to the insert in each pUC18-derived plasmid is indicated by an arrow. Positions of cleavage sites are shown for BamHI (B), BstEII (Bs), ClaI (C) EcoRI (E), KpnI (K), HindIII (H), and PstI (P).
For
NH-terminal amino acid analysis, 25 µg of partially
purified phosphoglucosamine mutase was resolved by SDS-PAGE and
electroblotted to an Immobilon-P membrane (Millipore). The membrane was
stained with Ponceau Red, and the major protein band (corresponding to
the enzyme) was excised. Microsequencing was performed with a model
473A protein sequencer (Applied Biosystems) under standard conditions
involving the detection of phenylthiohydantoin-derivatives.
Figure 3: Purification of the phosphoglucosamine mutase from E. coli strain JM83. SDS-PAGE analysis of active fractions recovered after each purification step. Lane a, crude extract. Lanes b-d, pools of active fractions recovered after elution from the columns of DEAE-Trisacryl, hydroxylapatite-Ultrogel, and Ultrogel-AcA 44, respectively. Lane e, purest fraction eluted from the last column of carboxymethyl-cellulose which was used for electroblotting and protein sequencing. MW, molecular mass standards (kilodaltons) indicated on the left are as follows: phosphorylase b (94), bovine serum albumin (67), ovalbumin (43), and carbonic anhydrase (30).
In fact,
this gene was identified at the same time by searching in data bases
for a protein exhibiting sequence similarities with other known
hexosephosphate mutases. The sequence of the phosphomannomutase from E. coli (the cpsG gene product) was used in the
search which yielded all phosphoglucomutase and phosphomannomutase
sequences characterized to date, as well as the YhbF putative peptide
(26% identity in 433 overlaps). The alignment of YhbF with the same
libraries gave as best scores two other proteins of unknown function
containing 445 and 463 amino acids, respectively: UreC from Helicobacter pylori (44.1% identity on 425 residues) (32) and a putative protein from Mycobacterium leprae (Genpro accession number U00020.PE23, 44% identity on 440
residues). All these sequences contain in particular a short region
previously characterized as the putative active site of hexosephosphate
mutases. This motif which is described as (GA)(LIVM)X (LIVM)(ST)(PGA)S*HXPX(GN) in the PROSITE data
base (PS00710) appeared as GIVISAS*HNPFYDNG in YhbF, where S*
represents the active-site serine residue. It was generally assumed
that the serine residue was phosphorylated during the catalytic action
of the hexosephosphate mutases, a reaction requiring the presence of
the corresponding hexose
1,6-diphosphate(33, 34, 35) . This serine
residue was located at position 102 in the sequence of YhbF. All these
data clearly showed that the yhbF gene encoded the
phosphoglucosamine mutase.
Figure 4:
Overproduction of phosphoglucosamine
mutase in E. coli cells. Crude extracts from strains carrying
plasmids with the glmM gene expressed under the control of lac or p
promoters were analyzed by
SDS-PAGE. Lanes a-c, soluble fraction extracted from cells of
JM83(pUC18), JM83(pMLD96), and JM83(pMLD99), respectively. Lanes
d-f, soluble fraction extracted from cells of JM83(pMLD100)
grown at 30 °C (d) or first at 30 °C and then for 1 h (e) or 3 h (f) at 42 °C. Lane g,
purified GlmM enzyme. MW, molecular mass standards
(kilodaltons) indicated on the left are those indicated in the
legend from Fig. 3.
Since the pGMM plasmid bears a thermosensitive replicon, the effects of the specific inactivation of the glmM gene were observed by shifting exponentially growing cells of GPM83 and JM83 from 30 °C to 43 °C. Both strains showed an identical growth rate and cell morphology when grown at 30 °C. However, after 5 to 6 h at 43 °C, the growth rate of GPM83 rapidly slowed down and cells apparently entered a stationary phase at a lower cell mass (Fig. 5). In addition, GPM83 cells progressively changed from rods to greatly enlarged ovoids when observed by phase-contrast microscopy, whereas the morphology of the parental strain was unaltered (data not shown). Cells finally lysed after prolonged incubation at the restrictive temperature, as judged by a progressive decrease of turbidity of the culture (Fig. 5) and the presence of many ghosts within the cell population. This was consistent with the involvement of the glmM gene product in the biosynthesis of a cell-envelope component. The fact that these different effects were observed only after a few hours was explained by the time required for the progressive dilution or inactivation of the low-copy number pGMM plasmid and of the functional GlmM enzyme molecules present at the time of the temperature shift. As previously observed with a glmU mutant, these morphological changes were amplified when using a growth medium deprived of NaCl, and the precocious stationary phase which characterized GPM83 cells grown at 43 °C was no longer observed when either 2% NaCl or 20% sucrose was added to the growth medium.
Figure 5:
Effect of the inactivation of the glmM gene on bacterial growth. GPM83 and JM83 cells were grown
exponentially at 30 °C in 2YT medium. At the time indicated by the
arrow (cell density = 5
10
ml
), the temperature of the
culture was either maintained at 30 °C or shifted to 43 °C.
Optical density (O.D.) values below 0.01 correspond to values
theoretically obtained after the appropriate dilutions of the cultures.
Symbols:
, GPM83 at 30 °C;
, JM83 at 30 °C;
, GPM83 at 43 °C;
, JM83 at 43
°C.
We failed to detect any significant accumulation of GlcN-6-P (the substrate of GlmM) in GPM83 cells grown at the restrictive temperature (data not shown). This unexpected finding probably resulted from its permanent and rapid conversion by the GlcN-6-P deaminase (the nagB gene product) back to fructose 6-phosphate (Fig. 1) (14, 39) .
Owing to
the fact that the phosphoglucomutase and phosphoglucosamine mutase
activities catalyzed similar reactions and used substrates which only
differ by the presence of the amino group at position 2 of the sugar, a
plasmid pML14 (20) carrying the pgm gene from E.
coli was also tested but it failed to restore growth of GPM83
cells at 43 °C. Reciprocally, the pMLD96 plasmid was assayed for
complementation of a strain deficient in phosphoglucomutase activity.
W1485 pgm::tet(20) appeared as pink
colonies of good size on Mac Conkey-galactose plates, when transformed
with this plasmid. As compared to the large red colonies obtained with
the pML14 plasmid and to the very small white colonies obtained with
the control vector pUC18, this result could be interpreted as a partial
complementation. It suggested that GlmM could catalyze at least to some
extent the interconversion of the glucose-phosphate isomers. This side
activity of phosphoglucosamine mutase was not investigated further.
As described above, two proteins of unknown function from Helicobacter pylori and Mycobacterium leprae showing more than 40% sequence identity with GlmM were found in data bases. It was tempting to speculate that these proteins were also phosphoglucosamine mutases. The observation that a plasmid carrying the ureC gene from H. pylori (pILL594 in (32) ) fully complemented the GPM83 mutant apparently confirmed this hypothesis.
Recently, we showed that both glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities from E. coli were carried by the glmU gene product which thus acted as a bifunctional enzyme catalyzing two contiguous steps in this pathway(11, 12) . The actual characterization of a phosphoglucosamine mutase activity in crude extracts of E. coli and the demonstration that it is essential for growth is a final confirmation that the steps leading from GlcN-6-P to UDP-GlcNAc are via GlcN-1-P in bacteria (Fig. 1). This finding is consistent with the previous demonstration that exogenously supplied GlcNAc had to be deacetylated before it could be incorporated into cell walls(13) . It suggests that any isomerase converting GlcNAc-6-P to GlcNAc-1-P has insignificant activity and that the only (or major) flux goes via GlcN-1-P which has to be reacetylated before formation of the nucleotide. Interestingly, a different reaction sequence is encountered in yeast where synthesis of GlcNAc-1-P from GlcN-6-P occurs by the successive actions of glucosamine-6-P acetylase and N-acetylglucosamine-phosphate mutase activities(37, 40) .
The glmM gene encoding phosphoglucosamine mutase was the last gene of the pathway for UDP-GlcNAc synthesis to be identified. Evidence is here provided that it corresponded to the previously sequenced open reading frame yhbF located at 69.5 min on the E. coli map. glmM is thus not linked to the related glmS and glmU genes previously identified at 84 min (11, 18) and which are probably cotranscribed(39, 41) . The presence of other genes involved in peptidoglycan metabolism in the vicinity of glmM is noteworthy: in particular, the dacB and murZ genes encoding penicillin-binding protein 4 (42) and phosphoenolpyruvate:UDP-GlcNAc enoylpyruvyl transferase(43) , respectively (Fig. 2). However, it is clear that these genes of related function do not belong to a cluster of tightly packed genes as observed for the mur genes in the 2-min region(8) . The experiments of complementation described in this work and the lack of an obvious promoter sequence on the DNA upstream of glmM(31, 44) suggested that this gene could be cotranscribed with the proximal folP gene. This is quite surprising when considering that the function of the folP gene product (dihydropteroate synthase involved in tetrahydrofolic acid synthesis) has no apparent relationship with cell-envelope metabolism (44) . In fact, the same was observed in H. pylori where the gene for phosphoglucosamine mutase appears inserted within the urease operon, the reason for its initial designation as ureC(32) . The transcription in this chromosomal region and a possible regulation of the glmM gene expression now has to be examined.
The effects of inactivating the glmM gene on the growth and cell morphology of E. coli were reminiscent of those observed with a glmU mutant altered in the next step from the same pathway(11, 12) . Cells progressively lost their rod shape to become greatly enlarged ovoids and growth stopped early at a lower cell density. Interestingly, this was not followed by an abrupt decrease of culture absorbance indicative of cell lysis, as generally observed with mutants defective in peptidoglycan synthesis(8, 9, 38, 45) . This was most probably due to the fact that the glmM mutation not only affects peptidoglycan but also lipopolysaccharide synthesis. Effectively, thermosensitive mutants altered in the lipopolysaccharide pathway and in particular in the essential steps leading from UDP-GlcNAc to lipid A are characterized by an arrest of growth at the restrictive temperature(4, 5) . The effects observed here with the glmM mutation are probably those expected from a simultaneous depletion of both cell-envelope components.
In the
present paper, the first characterization and purification to
near-homogeneity of a bacterial phosphoglucosamine mutase is described.
The final preparation had a specific activity of 7.2 unitsmg of
protein
whereas that of the crude extract from a
wild-type strain was approximately 0.05 (Table 1). Assuming that
there was no loss of activity in this crude extract from 4
10
cells (5 g of protein) and that the purified enzyme
contained only active GlmM molecules, a copy number of about 10,000 per
cell could be estimated for this enzyme in a plasmid-free parental
strain. This significant cellular abundance (0.5 to 1% of cell
proteins) has certainly facilitated the successful purification in
milligram quantities of this protein from a wild-type strain. It also
explains why a 50-fold overproduction factor is enough to make it
represent more than 20-30% of total cell proteins.
The amino acid sequence of the phosphoglucosamine mutase contains the characteristic signature of hexosephosphate mutases. This motif includes the putative serine residue (S102) whose phosphorylation is a prerequisite for enzyme activity. By similarity with other mutase activities and in particular the well-characterized phosphoglucomutase species(33, 34, 35) , the reaction catalyzed by GlmM is thought to proceed in two subsequent steps as follows:
GlcN-1,6-diP which appears as an intermediate in the catalytic process could be also considered as the compound required for the initial activation (phosphorylation) of the enzyme. However, this is not yet clearly established as Glc-1,6-diP itself could efficiently phosphorylate GlmM. An extension of this work will be to characterize a putative enzyme involved in the specific synthesis of GlcN-1,6-diP in E. coli.
The fact that the phosphoglucosamine mutase is
active only in a phosphorylated form is of great interest when
considering the regulation of the flow of metabolites in this pathway.
When assayed in the absence of added hexose-1,6-diphosphate, the
apparent GlmM activity that could be measured after cell extraction
theoretically reflects the total amount of phosphorylated enzyme
present in E. coli growing cells. As described in this work,
this basal activity in a wild-type strain was enhanced about 20-fold in
the presence of saturating concentrations of Glc-1,6-diP, suggesting
that most of the GlmM molecules were dephosphorylated and thus inactive in vivo. From the specific activity of 7.2
unitsmg
determined for the purified enzyme in
the presence of a saturating concentration of Glc-1,6-diP and assuming
a molecular weight of 47,380, a turnover number of 340 min
could be calculated. Under the in vitro conditions used,
the phosphoglucosamine mutase can catalyze the formation of
approximately 10
molecules of GlcN-1-P in each cell during
a 30-min generation time (10,000
340
30). Even if a 50%
turnover of peptidoglycan material is taken into
consideration(46) , this value is much higher than that
required for the formation of the average peptidoglycan content of
exponentially growing cells previously estimated in the range from 3.5
to 5.5
10
, depending on growth
conditions(6, 28) . The requirements for GlcN-1-P
(UDP-GlcNAc) molecules of the lipopolysaccharide pathway have not been
precisely determined but seem to be more or less
equivalent(4, 47) . This relative excess of enzyme is
consistent with the observation that only a small number of enzyme
molecules are apparently phosphorylated and thus active in
vivo. It was also noteworthy that this basal activity was clearly
not increased in strains overproducing as much as 50-fold the GlmM
protein, a result indicating that the total amount of phosphorylated
enzyme present in cells was unchanged and thus probably tightly
regulated. Any specific regulation of the activity of this enzyme which
catalyzes the first step in this reaction sequence could adjust in some
way the synthesis of GlcN-1-P molecules to the specific requirements of
the peptidoglycan and lipopolysaccharide pathways. The extent of enzyme
phosphorylation could therefore be an important factor in the control
of enzyme activity which should be investigated in detail now. Taking
advantage of the plasmids constructed in this work, the enzyme is now
being purified in large amounts for more precise investigations of its
kinetic parameters and structure.