Journal of General Microbiology (1989), 135, 1499-1 506. Printed in Great Britain
1499
Determination of Murein Precursors during the Cell Cycle of
Escherichia coli
By U T Z K O H L R A U S C H , ' F R A N S B. W I E N T J E S 2 A N D
JOACHIM-VOLKER HOLTJE1*
Max-Planck-Institut fur Entwicklungsbiologie, Abteilung Biochemie, Spemannstrasse 35,
0-7400 Tiibingen, FRG
Universiteit van Amsterdam, Vakgroep Moleculaire Cellbiologie, Sectie uoor Moleculaire
Cytologie, Plantage Muidergracht 14, 1018 T I / Amsterdam, The Netherlands
t
(Received 4 October 1988; revised 12 January 1989; accepted 22 February 1989)
A convenient and reliable method has been established that allows a quantitative
determination of m-diamino[3H]pimelic acid-labelled murein precursors in 1 ml culture samples
of Escherichia coli. Prior to separation by reversed-phase high-pressure liquid chromatography
the lipid-linked intermediates were hydrolysed to release the muropeptides. Tl-e accuracy for the
measurement of UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide) was
& 1.9% (sD), for undecaprenyl-P-P-MurNAc-pentapeptide (lipid I) & 10% (SD) and for
undecaprenyl-P-P-(GlcNAc-pl-t4)MurNA~-pentapeptide
(lipid 11) f 5 % (sD). The ratio of
UDP-MurNAc-pentapeptide :lipid I :lipid I1 was about 300 : 1 : 3 for E. coli MC4100. The
relative cellular concentrations of all three precursor molecules were found not to vary
throughout the cell cycle. It is concluded that elongation and division of the murein sacculus is
not controlled by oscillations in the concentrations of these late murein precursors.
INTRODUCTION
Propagation of bacteria is a cyclic process in which enlargement of cell mass and volume is
interrupted by cell division. Numerous metabolic processes have to be co-ordinated in a precise
sequence during the bacterial cell cycle. In particular, cell division has to be synchronized with
DNA replication. In addition, septation is also coupled to growth rate. Cells growing in rich
medium are larger than those growing in poor medium (Helmstetter et al., 1968). It has,
therefore, been proposed that cell division may also be regulated by changes in crucial
metabolite pool sizes (Kubitscheck & Pai, 1988; R. D'Ari, personal communication).
Regulation of enzymic reactions by substrate and/or product concentrations is quite common.
Feedback control in multi-step biosynthetic pathways is only one example of such control
circuits.
The most important structural event during cell division is the formation of a cross-wall of
murein, which after completion of cell septation forms the new polar caps of the two daughter
cells (Nanninga & Woldringh, 1985). Within the cell cycle, synthesis of a cross-wall alternates
with synthesis of cylindrical murein during cell elongation. Both processes have been suggested
to be catalysed by different enzyme systems (Spratt, 1975). Indeed, the structure of the murein
synthesized (Olijhoek et al., 1982; Kraus & Holtje, 1987), the sensitivity towards p-lactam
antibiotics (Schwarz et al., 1969)and the rate of synthesis (Hoffmann et al., 1972; Olijhoek et al.,
1982) have been shown to differ during cell elongation and division. Septum formation has been
found to be controlled and executed by quite a number of proteins (Donachie et al., 1984). The
leading enzyme involved in septation is the penicillin-binding protein (PBP) 3 (Spratt, 1979,
Abbreviations : A2pm, diaminopimelic acid; GlcNAc, N-acetylglucosamine; MurNAc, N-acetylmuramic acid.
0001-5172 0 1989 SGM
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u. KOHLRAUSCH,
F . B. W I E N T J E S A N D J . - V . H O L T J E
which is present in the cell in constant amounts throughout the cell cycle (Wientjes et al., 1983).
Nevertheless, the activity(ies) of at least one of the enzymic reactions involved in cell division is
expected to oscillate during the cycle. However, the regulatory signals are still unknown.
Murein biosynthesis takes place in different compartments of the cell (Holtje & Schwarz,
1985), commencing with the synthesis of the nucleotide precursors UDP-N-acetylglucosamine
(UDP-GlcNAc) and UDP-N-acetylmuramyl-L-Ala-D-Glu-m-A,pm-D-Ala-D-Ala (UDP-MurNAc-pentapeptide) in the cytoplasm. The activated amino-sugar derivatives are subsequently
translocated onto a lipid carrier (bactoprenol) residing in the cytoplasmic membrane. First,
undecaprenyl-P-P-MurN Ac-pentapeptide (lipid I) and finally the undecaprenyl-P-P-(GlcNAc/?1+4)MurNA~-pentapeptide (lipid 11) are formed. In a still unknown manner, the lipid-linked
disaccharide pentapeptide is then transferred to the outer leaflet of the cytoplasmic membrane
and inserted into the pre-existing murein sacculus. In E. coli this takes place in the periplasmic
space.
The undecaprenylphosphate carrier lipid is present in the cell in limiting amounts (Rothfield
& Romeo, 1971) and is re-used. It also participates in the synthesis of other wall polymers such as
lipopolysaccharides and capsular polysaccharides (Rogers et al., 1980). As a branch point in the
biosynthetic pathways of various wall components it is an ideal site to co-ordinate the synthesis
of these cell wall structures (Taschner, 1988). In particular, murein synthesis could be regulated
by the actual concentrations of the ultimate precursors. It was of major interest to gain
information on the concentration of the intermediates of the lipid cycle during the cell cycle.
Therefore, we established a method to determine the relative cellular concentration of UDPmuramylpentapeptide, lipid I and lipid I1 in synchronized cells of E. coli.
METHODS
Bacterial strain and growth conditions. E. coli K12 MC4100 lysA ref+ was grown at 28 "C with agitation in
minimal citrate medium (Vogel & Bonner, 1956) supplemented with lysine, methionine, threonine (50 pg ml-I
each), thiamin (4 pg ml-l) and 0.4% (w/v) glucose (Wientjes et al., 1985). The exponential mass-doubling time was
about 80 min.
Synchronization procedure. An exponentially growing culture (400 ml) was harvested by centrifugation at an
optical density at 450 nm of 0.45, which corresponds to about 2.4 x lo8 cells ml-l. The cells were synchronized by
the centrifugal elutriation technique of Figdor et al. (1981). The elutriation and synchronous growth were
monitored by Coulter counter volume distribution and cell number measurements. To minimize metabolic
disturbances, elutriation and collection of the newborn cells were done at 10 "C (instead of the usual 4 "C). The
time point of septum formation was deduced from the number of constricting cells, which was determined by
electron microscopy from at least 200 cells per sample.
Labelling of murein precursors and murein. Small cells (3.5 x los cells ml-l), obtained by centrifugal elutriation at
10 "C, were shifted to 28 "C to start synchronous growth. [3H]Diaminopimelic acid (A2pm) (meso-2,6diamin0[3,4,5-~H]pirnelicacid; 0.85 TBq mmol-' ; CEA) was added to a final concentration of 0-96 MBq ml-l.
After 50 min, samples (1 ml) of the synchronously growing culture were taken at intervals of 8 min and added to
2.5 ml of icecold extraction solvent (n-butanol/6 M-pyridinium acetate pH 4; 4 : 1, v/v) to isolate the murein
precursors. After vigorous shaking with 0.5 g of glass beads (0.17-0.18 mm diameter; Braun) in a vibrator for
30 min at 4 "C, the samples were centrifuged for 10 min at 2500 g and portions from both phases were withdrawn
for quantification of the murein precursors.
Incorporation of [3H]A2pm into TCA-precipitable material was measured as previously described (Wientjes
et al., 1985).
Quantijication of UDP-MurNAc-pentapepride. Portions (0.1 ml) of the aqueous phase were centrifuged to remove
insoluble material, freeze-dried to remove traces of organic solvents and dissolved in 0.1 ml H 2 0 . Samples were
separated by reversed-phase high-pressure liquid chromatography (HPLC) on a column (125 by 4.6 mm)
prepacked with Hypersil ODS-C18,5 pm (Bischoff). The column was eluted at 35 "C at a flow rate of 1 ml min-l
with 50 mM-NaOH adjusted to pH 4-0 with phosphoric acid. The radioactivity in the fractions (217 yl) was
determined (TriCarb 1500 liquid scintillation analyser; Canberra Packard) in 10 volumes of scintillator cocktail
(Hydroluma, J. T. Baker Chemicals).
Quant$cation ofthe murein lipid intermediates. Portions (1.7 ml) of the organic extraction phase were washed
twice with 0.5 ml H 2 0 and dried under vacuum. The material was hydrolysed with 0.1 M - H C for
~ 15 min in a
boiling water-bath. After neutralization with 0.1 M-NaOH the samples were freeze-dried, dissolved in 80 yl water
and analysed by HPLC. Prior tochromatography the muramyl residues of the muropeptides were reduced to their
muramitol derivatives (Glauner et al., 1988). Accordingly, the samples (80 pl) were diluted with an equal volume
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Murein precursors in the cell cycle of E. coli
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of 0.5 M-sodium borate buffer, pH 9, and incubated in the presence of 1 mg sodium borohydride ml-l for 30 min at
room temperature. The reaction was stopped by adjusting the pH to 4-5 with 4 M-phosphoric acid. HPLCfractionation was done as described above except that the pH of the eluent was 4.5 and the fraction size 300 p1.
R ESU L TS
Labelling of murein in synchronized cells of E. coli MC4100
The centrifugal elutriation method has yielded physiologically only slightly disturbed cells
with good synchrony, especially when the synchronization was done at 10°C (Figdor et al.,
1982; Olijhoek et al., 1982; Wientjes et al., 1983). In synchronously growing cultures of E. coli
MC4100 the cell number doubled over a period of about 0-62 times the generation time (as
calculated from Fig. 4a). The percentage of constricting cells fluctuated between 1 and 60%
(Fig. 4a).
E. coli MC4100, which is Dap+, was used instead of E. coli W7, which is Dap-, because the
latter strain incorporates exogenously supplied A2pm in a biphasic manner (Wientjes et al.,
1985); this would interfere with the pool size determinations during the cell cycle. Incorporation
of [3H]A2pmin MC4100 was found to be already in steady state 20-30 min after the addition of
the amino acid to the medium (Fig. 1).
Method for the determination of murein precursors
A precise and reliable procedure was established for the extraction of both the water-soluble
and the lipophilic murein precursors. By shaking the cells with glass beads in a vibrator in the
presence of the extraction solvent, reproducible extraction of the murein precursors was
achieved ; these were separated and quantified by high-pressure liquid chromatography.
The determination of the water-soluble precursors was greatly affected by traces of organic
solvent in the aqueous phase after the extraction step. Therefore, the samples were routinely
dried under vacuum and redissolved in water before HPLC separation (UDP-muramylpentapeptide was shown to be stable under these conditions). Fig. 2 shows the separation of UDPMurNAc-pentapeptide from other components of the aqueous extract.
For the analysis of the lipid-linked precursors the extracted material was hydrolysed to the
free muropeptide residues. The lipid I precursor yields muramylpentapeptide which is identical
to the product of hydrolysis of UDP-muramylpentapeptide. Therefore, the water-soluble
nucleotide precursors had to be completely removed from the organic solvent phase containing
the lipid precursors. This was done by washing the sample twice with water. The first wash
contained less than 0.1 % of UDP-muramylpentapeptide, which was no longer detectable in the
X
*I
2
0
50
100
Time (rnin)
150
Fig. 1. Incorporation of [3H]A2pminto TCA-precipitable material. A synchronously growing culture
of E. coli MC4100 in minimal-citrate medium at 28 "C was labelled at t = 0 min with 0.96 MBq
[ 3H]A2pmml-1 . The radioactivity incorporated intq TCA-precipitable material was determined in
culture samples (25 pl) at the indicated time points.
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u.
KOHLRAUSCH, F . B . WIENTJES AND J . - V . HOLTJE
O P ' "
h
ci
g 10000
h
..->
c)
4 4
0
.'D
5000
2
3.6
Retention time (min)
14.4
Fig. 2. Separation of UDP-MurNAc-pentapeptide from other components of the aqueous extract by
reversed-phase HPLC. Chromatography was on a 5 pm Hypersil ODS column (125 x 4.6 mm), using
isocratic elution conditions with 50 mM-sodium hydroxide adjusted to pH 4.0 with phosphoric acid
(flow rate, 1.0 ml min-' ; column temperature, 35 "C).(a) UV-absorbance at 205 nm. (b) Radioactivity
of the fractions (21 7 PI). Peak A represents UDP-MurNAc-pentapeptide (identified by running an
authentic standard).
0.1
p:
0.05
0
5
10
Retention time (min)
Fig. 3. Separation of MurNAc-pentapeptide and disaccharide-pentapeptide after hydrolysis and
reduction of the extracted lipophilic compounds (see Methods) by reversed-phase HPLC.
Chromatographic conditions were as described in Fig. 2 except that the pH was 4.5. (a) UV-absorbance
at 205 nm. (b)Radioactivity of the fractions (300 pl). Peak A represents MurNAc-pentapeptide; peak B
represents GlcNAc(/?I+4)MurNA~-pentapeptide (identified by running authentic standards).
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Murein precursors in the cell cycle of E. coli
0
1503
50
I00
150
Time (min)
Fig. 4. Cellular concentration of murein precursors during synchronous growth of E. coli. A
synchronized culture of E . coii MC4100 was labelled at t = 0 min with 0-96 MBq [3H]A2pm ml-I. (a)
Increase in cell number (a) and percentages of cells showing a visible constriction (0)
were
determined as described in Methods. Samples (1 ml) were withdrawn at the indicated time points and
analysed for the relative concentration of (b)UDP-MurNAc-pentapeptide; (c) lipid I and (d)lipid I1 as
described in Methods.
second wash. The separation of MurNAc-pentapeptide and GlcNAc-MurNAc-pentapeptide
after reduction is shown in Fig. 3.
The extraction procedure has been described by Anderson et al. (1967) to be complete for the
lipid-linked precursors. For the water-soluble nucleotide precursors we found that at least 99 %
of a known amount of UDP-MurNAc-pentapeptide added to a sample was recovered. Samples
of 1 ml of bacterial culture at a density of about 4 x lo8 cells ml-l were sufficient for the analysis.
The 'accuracy of the analytical method was determined by running fourfold samples; the
standard deviation was 1-9% for the determination of UDP-MurNAc-pentapeptide, 10% for
lipid I and 5% for lipid 11.
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KOHLRAUSCH, F . B. WIENTJES AND J . - V . HOLTJE
Pool sizes of murein precursors in the cell cycle of E. coli
The concentration of the water-soluble UDP-MurNAc-pentapeptide and the undecaprenylphosphate-linked precursors, lipid I and lipid 11, were determined every 8 min in a
synchronously growing culture of E. coli MC4100 during a period of 96 min covering a complete
cell cycle with the septum formation (maximum of constricting cells) in the mid-point of the time
span analysed (Fig. 4a). The amount of labelled UDP-MurNAc-pentapeptide decreased
exponentially (Fig. 4b). This is easily explained by the dilution kinetics of the labelled A,pm,
which is the result of two processes going on during cell growth. First, the specific radioactivity
of the [3H]A2pmpool decreases due to the endogenous synthesis of A,pm; second, the amino
acid is used up by its incorporation into murein. Taking this into consideration the results
indicate that the intracellular amount of UDP-MurNAc-pentapeptide does not vary throughout
the cell cycle. The same holds true for the two lipid-linked precursors (Fig. 4c, d). This means
that the concentrations of the murein precursors UDP-MurNAc-pentapeptide, lipid I and lipid
I1 are kept constant during the cell cycle.
Determination of the relative amounts of murein precursors in E. coli
Our results directly specify the relative amounts of the different precursor molecules. A
calculation of the ratios of the murein precursors at different time points in the cell cycle yielded
constant values throughout. Since we found that the precursors do not vary within the cell cycle
the specific radioactivity of the different compounds at each time point must be the same.
Therefore, the proportional numbers reflect the relative pool sizes of the various murein
precursors. Under the growth conditions employed in this study the following ratios were
determined (mean values of all 13 time points f.standard deviation) : UDP-MurNAcpentapeptide exceeds lipid I by a factor of 277-6 & 41.2, and lipid I1 by 89.7 & 15.97. The ratio of
lipid I1 to lipid I was 3-13 f.0.35.
DISCUSSION
Several control mechanisms of murein biosynthesis influence the cellular concentrations of
the nucleotide murein precursors (UDP-MurNAc-peptides and UDP-GlcNAc) and the lipid
carrier-linked intermediates. Studying the mode of action of penicillin, Park (1952) observed an
accumulation of UDP-derivatives (formerly called Park-nucleotides) in the presence of
p-lactams. Later it was proposed that UDP-MurNAc-pentapeptide exerts a feedback inhibition
on the first enzyme of the biosynthetic pathway of UDP-muramic acid, the phosphoenolpyruvate :UDP-GlcNAc enolpyruvyl transferase (Venkateswaran et al., 1973). Also, the
stringent control mechanism regulates the cellular concentration of UDP-MurNAc-pentapeptide. It has been shown that the reIA gene product prevents an accumulation of nucleotide
murein precursors during amino acid starvation (Ishiguro & Ramey, 1978).
Unfortunately, a detailed analysis of the concentrations of both the cytoplasmic nucleotide
precursors and the lipid-linked intermediates in the cytoplasmic membrane has only partially
been accomplished (Mengin-Lecreulx et al., 1982, 1983). In particular, determination of lipid
precursors was problematic because of the low intracellular levels and incomplete recoveries
during isolation and separation of these compounds.
We succeeded in establishing a convenient and rapid method that allows a reliable and precise
measurement of the relative concentration of the UDP-MurNAc-pentapeptide and the lipid
intermediates. Thereby, the relative amount of the lipid I precursor could be determined with
increased accuracy as compared with earlier results by others (Ramey & Ishiguro, 1978; Dai &
Ishiguro, 1988). It was found to be only one-third of the lipid I1 precursor.
Our method enabled us to determine the relative cellular concentrations of the murein
precursors within the cell cycle of E. coli. This was of interest because the pools of murein
intermediates may regulate murein synthesis, which is known to vary during the growth cycle of
E. coli (Olijhoek et al., 1982). The pools of both the cytoplasmic UDP-MurNAc-pentapeptide
and the two undecaprenylphosphate-linked intermediates were found to show no oscillation
throughout the cycle. Therefore, these intermediates cannot govern murein synthesis. The
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molecular signal(s) that initiate(s) the synthesis of the septum, a prerequisite for cell division,
remains unknown.
The suggestion that the known coupling of cell division to growth rate may be achieved by
changes in the internal pool sizes of certain metabolites (R. D’Ari, personal communication)
remains very attractive. Other promising candidates for such regulatory compounds may be
found among the numerous nucleotide derivatives which have been called alarmones (Stephens
et af.,1975) and which include such important control factors as cyclic AMP and ppGpp. Only
the concentration of cyclic AMP has been analysed during the cell cycle of E. coli. It was found
not to oscillate, excluding this nucleotide as a regulatory element in cell-cycle-dependent
processes (Holtje & Nanninga, 1984). A complete survey of the concentrations of the whole list
of nucleotide derivatives during the cell cycle would be of utmost importance for the
understanding of the regulation of the bacterial cell cycle. Convenient (two-dimensional thinlayer and HPLC) methods have been described (Bochner & Ames, 1982) which are capable of
analysing a great number of different nucleotides in one assay.
We thank Nanne Nanninga and Uli Schwarz for their interest and generous support throughout this
investigation.
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