|
|
|
|||||||
|
|||||||||
(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 (
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
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
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
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
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 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 units 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
units
Volume 271,
Number 1,
Issue of January 5, 1996 pp. 32-39
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)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.
Bacterial Strains and Plasmids
E. coli strain JM83 (ara 
[lac-proAB] rpsL thi 80
dlacZ
M15) (19) was used as the host strain for
plasmids and for the purification of the glmM gene product.
Strain W1485 pgm::tet and plasmid pML14 carrying
the E. coli pgm (phosphoglucomutase) gene (20) were
kindly provided by S. Slater. pJP900, a pUC9-derived plasmid carrying
the 2.4-kb XhoII fragment of bacteriophage with the p
promoter inserted into its BamHI site,
was obtained from J. Plá (CSIC, Universidad
Autonoma de Madrid, Spain). Cloning vector pUC18 and the Kan
cartridge originating from the pUC4K plasmid were purchased from
Pharmacia Biotech Inc. Plasmid pMAK705 bearing a thermosensitive
replicon was obtained from S. R. Kushner(21) .Growth Conditions
Unless otherwise noted, 2YT was
used as a rich medium for growing cells(22) . Growth was
monitored at 600 nm with a spectrophotometer (model 240, Gilford
Instrument Laboratories, Inc., Oberlin, Ohio). For strains carrying
drug resistance genes, antibiotics were used at the following
concentrations (in µg
ml): ampicillin
(100), kanamycin (30), and chloramphenicol (25).
General DNA Techniques and E. coli Cell
Transformation
Small- and large-scale plasmid isolations were
carried out by the alkaline lysis method(23) . Standard
procedures for endonuclease digestions, ligation, filling-in of
5`-protruding ends by using the Klenow fragment of DNA polymerase I,
and agarose electrophoresis were used(23, 24) . E.
coli cells were made competent for transformation with plasmid DNA
by the method of Dagert and Ehrlich(25) .Construction of Plasmids
The 17-kb chromosomal
fragment from E. coli which is carried by phage 14F11 (clone
521) from the bank of Kohara et al.(26) was used as
the starting material for the construction of the different plasmids
described in Fig. 2. The pMLD90 plasmid was constructed by
inserting the 3.1-kb BamHI fragment carrying the glmM gene (yhbF) into the BamHI site of the pUC18
vector (with glmM in the opposite orientation as compared to
the lac promoter from the vector). The pMLD96 plasmid was
constructed by inserting the 2.1-kb PstI fragment from pMLD90
into the corresponding site of the pUC18 vector (with glmM and lac promoter in the same orientation). An internal AccI-ClaI deletion in pMLD96 produced the plasmid
pMLD99 (Fig. 2). For the expression of glmM under the
control of the p
promoter, plasmid pMLD100
was constructed by inserting the 2.4-kb XhoII fragment
(carrying the structural gene cI857 encoding a thermosensitive
form of the
cI repressor, the strong p
promoter, and the ribosome-binding site of cro) (27) into the unique BamHI site of
pMLD99. The disruption of the glmM gene was obtained by
inserting the 1.28-kb HincII kan cartridge
originating from pUC4K into the unique BstEII site of plasmid
pMLD90 lying within the glmM gene coding sequence, generating
plasmid pMLD94 (Fig. 2). The pGMMkan plasmid was
constructed by inserting the 4.5-kb BamHI fragment from pMLD94
(harboring the disrupted glmM gene) into the BamHI
site of pMAK705(21) . The pGMM plasmid was isolated as
described below during the excision process that followed integration
of the pGMMkan plasmid at the chromosomal glmM locus.
Disruption of the Chromosomal glmM Gene
The
wild-type chromosomal copy of glmM was replaced by a disrupted
one by following the procedure of Hamilton et al.(21) which uses pMAK705, a plasmid bearing a
thermosensitive replicon. pGMMkan, a pMAK705 derivative
carrying the disrupted glmM gene, was transformed into JM83.
Integration of the plasmid into the chromosome was selected for by
plating cells at 44 °C on 2YT-chloramphenicol. Several clones were
picked up, and the integration of pGMMkan at the glmM locus was verified by Southern analysis. Excision of the plasmid
from the chromosome was then achieved as follows. Cointegrants were
grown at 30 °C in 2YT-chloramphenicol for at least 30 doubling
times and subsequently plated at 30 °C. Individual clones were then
screened for sensitivity to chloramphenicol at 44 °C, which is
indicative of plasmid excision. The structures of the excised plasmids
from several clones were determined by DNA restriction analysis. One
clone containing a plasmid bearing the wild-type glmM gene
(called pGMM) was chosen. Finally, the replacement in that strain
(named GPM83) of the chromosomal glmM copy by the inactivated
one was verified by Southern analysis (data not shown).Pool Levels of Peptidoglycan Precursors
Cells of
GPM83 (1-liter cultures) were grown exponentially at 30 °C in 2YT
medium. At a cell density of 5 
10
ml, the temperature of the
culture was either maintained at 30 °C or increased to 43 °C.
Incubation was continued until the optical density of the culture at 43
°C reached a plateau value of 0.5-0.7 about 5 to 6 h later.
At this time, cells were rapidly chilled to 0 °C and harvested in
the cold. Control cultures were made with strain JM83 which carries an
intact chromosomal copy of glmM. The extraction of
peptidoglycan nucleotide precursors as well as the analytical procedure
used for their quantitation were as described
previously(6, 7) .
Isolation of Sacculi and Quantitation of
Peptidoglycan
Exponential-phase cells of JM83 and GPM83 were
grown at 30 °C or first at 30 °C and then 43 °C as
described above. Harvested cells were washed with a cold 0.85% NaCl
solution and centrifuged again. Bacteria were then rapidly suspended by
vigorous stirring in 40 ml of a hot (95 to 100 °C) aqueous 4% SDS
solution for 30 min. After standing overnight at room temperature, the
suspensions were centrifuged for 30 min at 200,000 g,
and the pellets were washed several times with water. Final suspensions
made in 5 ml of water were homogenized by brief sonication. Aliquots
were hydrolyzed and analyzed as described previously, and the
peptidoglycan content of the sacculi was expressed in terms of its DAP
content(6, 28) .Preparation of Crude Enzyme
Cells of JM83
harboring the different plasmids described above (excepting pMLD100)
were grown exponentially at 37 °C in 2YT-ampicillin medium (1-liter
cultures). Cells were harvested in the cold when the optical density of
the cultures reached 0.7. A different protocol was used with strain
JM83(pMLD100): cells were grown first at 30 °C and at an absorbance
of 0.01 (5 10
cellsml),
cultures were shifted to 42 °C for 3 h during which time the
absorbance reached a value of 0.7. Strains JM83 and GPM83 were grown at
30 °C or 43 °C as described above for the extraction of
peptidoglycan precursors. In all cases, harvested cells were washed
with 40 ml of cold 0.02 M potassium phosphate buffer (pH 7)
containing 1 mM
-mercaptoethanol. The wet cell pellet was
suspended in 9 ml of the same buffer and disrupted by sonication
(Sonicator 150; T. S. Ultrasons, Annemasse, France) for 10 min with
cooling. The resulting suspension was centrifuged at 4 °C for 30
min at 200,000 g. The supernatant was dialyzed
overnight at 4 °C against 100 volumes of the same phosphate buffer,
and the resulting solution (10 ml, 10 to 12 mg of
proteinml) designated as crude enzyme was
stored at -20 °C. SDS-PAGE analysis of proteins was performed
as described previously using 13% polyacrylamide gels(29) .
Protein concentrations were determined by the method of Lowry, using
bovine serum albumin as standard(30) .
Assay for Phosphoglucosamine Mutase Activity
A
coupled assay was routinely used in which the GlcN-1-P synthesized from
GlcN-6-P by the mutase was quantitatively converted to UDP-GlcNAc in
the presence of purified bifunctional GlmU enzyme(12) . The
standard assay mixture contained 50 mM Tris-HCl buffer (pH
8.0), 3 mM MgCl
, 1 mM GlcN-6-P, 0.4
mM [
C]acetyl-CoA (700 Bq), 10 mM UTP, 0.7 mM Glc-1,6-diP, pure GlmU enzyme (1 µg), and
enzyme (0.1 to 10 µg of protein, depending on overexpression or
purification factors) in a final volume of 100 µl. Mixtures were
incubated at 37 °C for 30 min, and reactions were terminated by the
addition of 10 µl of acetic acid. Reaction products were separated
by high-voltage electrophoresis on Whatman No. 3MM filter paper in 2%
formic acid (pH 1.9) for 90 min at 40 V/cm using an LT36 apparatus
(Savant Instruments, Hicksville, NY). The only two radioactive spots
(acetyl-CoA and UDP-GlcNAc) were located by overnight autoradiography
using type R2 films (3 M, St. Paul, MN) or with a
radioactivity scanner (model Multi-Tracermaster LB285, Berthold France,
Elancourt, France). They were cut out and counted in an Intertechnique
SL 30 liquid scintillation spectrophotometer with a solvent system
consisting of 2 ml of water and 13 ml of Aqualyte mixture (J. T. Baker
Chemicals, Deventer, Netherlands). 1 unit of enzyme activity was
defined as the amount which catalyzed the synthesis of 1 µmol of
GlcN-1-P in 1 min.Purification of GlmM from Strain JM83
A large
culture (3 18 liters) of strain JM83 was performed at 37 °C
in a fermenter. Cells were harvested at the end of the exponential
phase and disrupted by sonication as described above. The resulting
soluble protein extract (5 g of protein, 240 units of
phosphoglucosamine mutase activity, 198 ml) was loaded onto a column (5
25 cm) of DEAE-Trisacryl-M (BioSepra, Villeneuve-la-Garenne,
France) pre-equilibrated with buffer A (20 mM potassium
phosphate buffer, pH 7.4, containing 5 mM MgCl,
2.5 mM -mercaptoethanol, and 5% (v/v) glycerol). The
elution was run at a flow rate of 2 mlmin,
first with 120 ml of buffer A, and then with a linear gradient (1
liter) of NaCl (0 to 160 mM) in buffer A. Fractions (12 ml)
were collected and assayed for phosphoglucosamine mutase activity. The
most active fractions which eluted in 70 mM NaCl were pooled
(96 units, 420 mg of protein) and concentrated to 30 ml by
ultrafiltration through PM10 membranes (Amicon). The buffer was
exchanged with buffer B (5 mM potassium phosphate buffer, pH
7.4, containing the same additives as buffer A) by repeated
concentration and dilution using PM10 membranes. The protein solution
was loaded onto a column (5
17 cm) of hydroxylapatite-Ultrogel
(BioSepra) pre-equilibrated with buffer B. The elution was run at a
flow rate of 2 mlmin, first with 150 ml of
buffer B, and then with a linear gradient (800 ml) of potassium
phosphate (5 mM to 200 mM). 10-ml fractions were
collected and assayed for phosphoglucosamine mutase activity. The most
active fractions which eluted in 150 mM potassium phosphate
were pooled (50 units, 41 mg of protein) and concentrated as above to
about 5 ml. This solution was loaded onto a column (1
50 cm) of
Ultrogel-AcA 44 (BioSepra) equilibrated with buffer C (as buffer A but
100 mM in potassium phosphate), and the elution was run at a
flow rate of 0.1 mlmin. Active fractions (48
units, 32 mg of protein) were pooled, concentrated as above to 5 ml,
and dialyzed against buffer A. This solution was loaded onto a column
(2.5
13 cm) of carboxymethyl-cellulose CM32 equilibrated with
buffer D (buffer A at pH 6.15). The elution was run at a flow rate of 1
mlmin, first with 60 ml buffer D, and then
with a linear gradient (400 ml) of NaCl (0 to 200 mM) in
buffer D. Under these conditions, the phosphoglucosamine mutase eluted
in 10 mM NaCl, slightly later than the remaining contaminant
proteins. Most active fractions were pooled (20 units, 7.7 mg of
protein) and dialyzed against buffer A.
Amino Acid Composition and Peptide
Sequencing
Quantitative amino acid analyses were performed with
an analyzer (model LC 2000; Biotronik, Frankfurt, FRG) after hydrolysis
of samples in 6 M HCl at 95 °C for 16 h.-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.Chemicals
[C]acetyl-CoA
(1.85 GBqmmol) was purchased from ICN.
GlcN-1-P, GlcN-6-P, Glc-1,6-diP, UTP, and UDP-GlcNAc were from Sigma.
Purification and Amino Acid Sequencing of
Phosphoglucosamine Mutase
Starting from a 54-liter culture of
wild-type strain JM83 which yielded 5 g of total soluble proteins, the
phosphoglucosamine mutase activity was purified approximately 150-fold
to a final specific activity of 7.2
µmolminmg (Table 1). The purest fractions eluted from the last column
contained a major protein species migrating after denaturating
polyacrylamide gel electrophoresis as a protein of 45-50 kDa (Fig. 3). It was the only one coeluting perfectly in all
purification steps with the phosphoglucosamine mutase activity. The
N-terminal amino acid sequence of this protein species was thus
determined after electroblotting on Immobilon-P and appeared as
follows: SNRKYFGTXXI.
Search for the glmM Gene Using Data Bases
The
alignment of this sequence with those of proteins from the Swissprot
and Genpro libraries revealed that it matched perfectly with the
N-terminal sequence MSNRKYFGTDGI predicted for the product of an E.
coli open reading frame of unknown function named yhbF(31) or mrsA (GenBank accession number U01376)
which was located near folP at 69.5 min on the chromosome.
Several discrepancies were observed when comparing the two reported
sequences of this gene. Using plasmid pMLD96 (Fig. 2) as the
template and appropriate synthetic oligonucleotides, the complete
1335-bp coding sequence was thus verified again and appeared similar to
that published first (yhbF). Taking into account the
post-translational removal of the N-terminal methionine (revealed by
protein sequencing), the mature product of this gene is theoretically a
444-amino acid protein with a molecular mass of 47,380.(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.Levels of Phosphoglucosamine Mutase in E. coli
Cells
The specific activity of phosphoglucosamine mutase in
strains overexpressing the yhbF gene was estimated. A
significant overproduction (up to 18-fold) in cells carrying plasmids
with the yhbF gene expressed under the control of the lac promoter was observed (Table 2). As shown in Fig. 4,
this was accompanied by the accumulation in the cell content of a
protein species of about 47 kDa, a value in agreement with that
(47,380) calculated from the DNA sequence(31) . A more
efficient overproducing plasmid pMLD100 with the yhbF gene
expressed under the control of the strong p
promoter was constructed. When cells of JM83(pMLD100) were grown
exponentially at 30 °C and then shifted to 42 °C, an initial
increase in the growth rate was observed, but growth progressively
slowed down and the culture finally reached a plateau value. As soon as
1 h after the temperature shift, a large accumulation of the 47K
protein was observed, which further increased to finally account for
more than 20-30% of the cell proteins after a 3-h incubation
period (Fig. 4). It comigrated with the protein overproduced
from the other plasmid construction as well as with the
phosphoglucosamine mutase purified from wild-type strain JM83 (Fig. 4). A typical fractionation procedure of cell extracts
showed that the highly overproduced protein was mainly found in the
soluble fraction, but that significant amounts (10 to 20%) remained
associated with the particulate fraction. This finding was certainly
due to the formation of aggregates at this high level of protein
expression, as confirmed by the presence of multiple inclusion bodies
in induced cells when observed by optical microscopy. Finally, we
showed that after 3 h of growth at 42 °C, JM83(pMLD100) cells
contained about 50-fold more phosphoglucosamine mutase activity than
cells carrying the pUC18 control vector (Table 2). All these
different results taken together supported the initial proposal that
the yhbF gene coded for the phosphoglucosamine mutase. This
gene was thus named glmM (for glucosamine mutase) following
the nomenclature previously adopted for the glmS and glmU genes from the same pathway (11, 17) .
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.
Hexose 1,6-Diphosphate-dependent Phosphorylation of
Phosphoglucosamine Mutase
By similarity with the reaction
mechanism of other well characterized hexose-phosphate mutases (33, 34, 35, 36, 37) , the
phosphoglucosamine mutase was assumed to be active only in a
phosphorylated form. As explained before, the serine residue at
position 102 was the putative phosphorylated site and GlcN-1,6-diP was
expected to be the phosphorylating agent. Since the latter compound was
not commercially available, we used instead Glc-1,6-diP in our
enzymatic assays, as generally was done with hexosephosphate mutases
other than the phosphoglucomutase species(35, 37) .
The apparent GlmM activity that could be detected in the extract from
wild-type strain JM83 was very low when Glc-1,6-diP was omitted from
the reaction mixture (Table 3). This basal level was greatly
enhanced (up to 20-fold) by increasing the concentration of this
compound (Table 3), according to a hyperbolic saturation curve
from which a constant of 150 µM could be calculated (data
not shown). Interestingly, the overproduction of the enzyme activity
was no longer detected in crude extracts from strains JM83(pMLD96) and
JM83(pMLD100) when assays were performed in the absence of Glc-1,6-diP.
Under these conditions, the apparent activity of GlmM was similar or
curiously lower in these extracts than that detected in the wild-type
extract (Table 3). The addition of a saturating concentration of
Glc-1,6-diP was accompanied by a very large increase (500- or
2000-fold) of the activity, which was consistent with the relative
amounts of GlmM known to be present in these various extracts (see
above). These different results first indicated that only a small part
of the enzyme extracted from the cell content was present in the active
phosphorylated form (5%, as judged from results obtained with the
extract of strain JM83(pUC18)). Furthermore, the total amount of
phosphorylated enzyme was clearly not increased in strains
overproducing this protein to high levels, suggesting that the level of
enzyme phosphorylation could be tightly regulated.
Inactivation of the Chromosomal glmM Gene and Its Effect
on Bacterial Growth
To inactivate the glmM gene on the E. coli chromosome, we used the procedure described by
Hamilton et al.(21) that is particularly well adapted
for the disruption of essential genes, as recently shown for the
construction of mutants altered in the glmU and murI genes which are essential for peptidoglycan synthesis (11, 38) . First, the glmM gene coding
sequence carried by the pMLD90 plasmid was disrupted by inserting at
the unique BstEII site the 1.28-kb kanamycin resistance gene
from pUC4K, generating plasmid pMLD94 (Fig. 2). The complete
insert from the pMLD94 plasmid was inserted into the pMAK705 vector
which bears a thermosensitive replicon. The resulting plasmid,
pGMMkan, was used to construct strain GPM83 (JM83 glmM::kan [pGMM]) having the inactivated glmM gene on the chromosome and the wild-type allele on the
pMAK705 vector. At the restrictive temperature for plasmid replication
(43 to 44 °C), GPM83 failed to grow on 2YT plates, indicating that
this strain with a disrupted chromosomal copy of the glmM gene
was viable only in the presence of a plasmid carrying the wild-type
gene. The failure to transduce by phage P1 the glmM::kan marker from GPM83 to other E. coli K12 strains further
confirmed that glmM was an essential gene.
10ml), 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.
Biochemical Effects of the glmM Mutation
The
effects of the mutation on bacterial growth were clearly associated
with the depletion of the phosphoglucosamine mutase activity. GPM83
cells effectively contained 50 fold less phosphoglucosamine mutase
activity than the parental strain after growth for 5 to 6 h at the
restrictive temperature (Table 2). As shown in Table 4, an
arrest in the de novo synthesis of functional GlmM enzyme
molecules rapidly led to the depletion of the pools of the two main
nucleotide precursors UDP-GlcNAc and UDP-MurNAc-pentapeptide (Fig. 1). The pools of the other intermediates from
UDP-GlcNAc-enolpyruvate to UDP-MurNAc-tripeptide, which are always
detected at a much lower intracellular concentration(28) , were
also depleted (data not shown). This finding confirmed that the
mutational block was in one of the early steps located upstream from
UDP-GlcNAc which are required for both peptidoglycan and
lipopolysaccharide biosyntheses (Fig. 1). As a result, the
synthesis of both cell-envelope components was inhibited and GPM83
cells finally were lysed when their peptidoglycan content was decreased
to a critical value representing about 60% of its normal level (Table 4). The highly reduced peptidoglycan content determined in
the mutant cells most probably represented the lowest physiological
value compatible with cell integrity.
Capability of Different Plasmids to Complement the
glmM::kan Mutation
The different pUC18-derived plasmids which
carry the glmM gene were tested for their capability to
complement the thermosensitivity of strain GPM83. Interestingly, only
those carrying inserts with glmM in the same orientation as
the lac vector promoter (pMLD96, pMLD99) could restore the
growth of GPM83 at 43 °C. The pMLD90 plasmid carrying the BamHI fragment where glmM and most (800 bp) of the
preceding folP gene were inserted in the orientation opposite
to that of the lac promoter (Fig. 2) failed to
complement the mutation. As described in Table 2, no
overproduction of phosphoglucosamine mutase could be detected in cells
carrying the pMLD90 plasmid. These results implied that transcription
of the chromosomal glmM gene may occur from a promoter located
far upstream from its initiation codon, suggesting that glmM is cotranscribed with the proximal folP gene.::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.
mg 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.
mg 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.
)-D-Glu-meso-DAP-D-Ala-D-Ala;
GlcN-6-P, glucosamine 6-phosphate; Glc-1,6-diP, glucose1,6-diphosphate;
PAGE, polyacrylamide gel electrophoresis; kb, kilobase pair(s); bp,
base pair(s).
We wish to thank J. Plumbridge and B. Badet for
helpful discussions and critical reading of the manuscript, and N.
Kleckner, A. Labigne, Y. Kohara, Y. Mechulam, and S. Slater for the
generous gift of phages, plasmids, and bacterial strains. M. Nicaise
and C. Ghelis are greatly acknowledged for the sequencing of the
purified GlmM enzyme.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  I. C Schoenhofen, E. Vinogradov, D. M Whitfield, J.-R. Brisson, and S. M Logan The CMP-legionaminic acid pathway in Campylobacter: Biosynthesis involving novel GDP-linked precursors Glycobiology, July 1, 2009; 19(7): 715 - 725. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. T. Park and T. Uehara How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan) Microbiol. Mol. Biol. Rev., June 1, 2008; 72(2): 211 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Saskova, L. Novakova, M. Basler, and P. Branny Eukaryotic-Type Serine/Threonine Protein Kinase StkP Is a Global Regulator of Gene Expression in Streptococcus pneumoniae J. Bacteriol., June 1, 2007; 189(11): 4168 - 4179. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. M. Cunneen and P. R. Reeves The Yersinia kristensenii O11 O-Antigen Gene Cluster was Acquired by Lateral Gene Transfer and Incorporated at a Novel Chromosomal Locus Mol. Biol. Evol., June 1, 2007; 24(6): 1355 - 1365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Joyce, J. L. Reed, A. White, R. Edwards, A. Osterman, T. Baba, H. Mori, S. A. Lesely, B. O. Palsson, and S. Agarwalla Experimental and Computational Assessment of Conditionally Essential Genes in Escherichia coli J. Bacteriol., December 1, 2006; 188(23): 8259 - 8271. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Uehara and J. T. Park The N-Acetyl-D-Glucosamine Kinase of Escherichia coli and Its Role in Murein Recycling J. Bacteriol., November 1, 2004; 186(21): 7273 - 7279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. R. Vimr, K. A. Kalivoda, E. L. Deszo, and S. M. Steenbergen Diversity of Microbial Sialic Acid Metabolism Microbiol. Mol. Biol. Rev., March 1, 2004; 68(1): 132 - 153. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shao, J. Zhang, P. Kowal, and P. G. Wang Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose Appl. Envir. Microbiol., November 1, 2002; 68(11): 5634 - 5640. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. Park Identification of a Dedicated Recycling Pathway for Anhydro-N-Acetylmuramic Acid and N-Acetylglucosamine Derived from Escherichia coli Cell Wall Murein J. Bacteriol., July 1, 2001; 183(13): 3842 - 3847. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. M. Tavares, L. Jolly, F. Pompeo, J. H. Leitão, A. M. Fialho, I. Sá-Correia, and D. Mengin-Lecreulx Identification of the Pseudomonas aeruginosa glmM Gene, Encoding Phosphoglucosamine Mutase J. Bacteriol., August 15, 2000; 182(16): 4453 - 4457. [Abstract] [Full Text]
|
|
|
|
 |
|
 J. S. Edwards and B. O. Palsson The Escherichia coli MG1655 in silico metabolic genotype: Its definition, characteristics, and capabilities PNAS, May 9, 2000; 97(10): 5528 - 5533. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. M. Pooley and D. Karamata Incorporation of [2-3H]glycerol into cell surface components of Bacillus subtilis 168 and thermosensitive mutants affected in wall teichoic acid synthesis: effect of tunicamycin Microbiology, April 1, 2000; 146(4): 797 - 805. [Abstract] [Full Text]
|
|
|
|
|
|
L. Jolly, F. Pompeo, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx Autophosphorylation of Phosphoglucosamine Mutase from Escherichia coli J. Bacteriol., March 1, 2000; 182(5): 1280 - 1285. [Abstract] [Full Text]
|
|
|
|
|
|
C. Y. Loo, D. A. Corliss, and N. Ganeshkumar Streptococcus gordonii Biofilm Formation: Identification of Genes that Code for Biofilm Phenotypes J. Bacteriol., March 1, 2000; 182(5): 1374 - 1382. [Abstract] [Full Text]
|
|
|
|
 |
|
 P. Glanzmann, J. Gustafson, H. Komatsuzawa, K. Ohta, and B. Berger-Bächi glmM Operon and Methicillin-Resistant glmM Suppressor Mutants in Staphylococcus aureus Antimicrob. Agents Chemother., February 1, 1999; 43(2): 240 - 245. [Abstract] [Full Text]
|
|
|
|
|
|
J. Plumbridge and E. Vimr Convergent Pathways for Utilization of the Amino Sugars N-Acetylglucosamine, N-Acetylmannosamine, and N-Acetylneuraminic Acid by Escherichia coli J. Bacteriol., January 1, 1999; 181(1): 47 - 54. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Mio, T. Yamada-Okabe, M. Arisawa, and H. Yamada-Okabe Saccharomyces cerevisiae GNA1, an Essential Gene Encoding a Novel Acetyltransferase Involved in UDP-N-acetylglucosamine Synthesis J. Biol. Chem., January 1, 1999; 274(1): 424 - 429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Pompeo, J. van Heijenoort, and D. Mengin-Lecreulx Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from Escherichia coli: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes J. Bacteriol., September 15, 1998; 180(18): 4799 - 4803. [Abstract] [Full Text]
|
|
|
|
|
|
M. K. B. Berlyn Linkage Map of Escherichia coli K-12, Edition 10: The Traditional Map Microbiol. Mol. Biol. Rev., September 1, 1998; 62(3): 814 - 984. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Pompeo, Y. Bourne, J. van Heijenoort, F. Fassy, and D. Mengin-Lecreulx Dissection of the Bifunctional Escherichia coli N-Acetylglucosamine-1-phosphate Uridyltransferase Enzyme into Autonomously Functional Domains and Evidence That Trimerization Is Absolutely Required for Glucosamine-1-phosphate Acetyltransferase Activity and Cell Growth J. Biol. Chem., February 2, 2001; 276(6): 3833 - 3839. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |