BIOCHEMICAL SOCIETY TRANSACTIONS
994
prise a tight complex or small number of complexes containing only a few polypeptide chains to which the undecaprenyl phosphate is firmly associated (Leaver et al., 1981).
Taking this into account, a simple interpretation of the
experiments with protoplasts and autolysed cells would
be that the complex, together with its bound prenyl phosphate, occupies a trans-membrane location and either
rotates or re-orients itself such that active sites are first
exposed on the inner surface where successive interactions
with nucleotide precursors occur, thereby transferring
residues to the prenyl phosphate. As loading of the lipid
occurs the complex either rotates or re-arranges so that
growing polymer attached to its lipid emerges from the
outer surface. At this point active sites on the enzymes
become temporarily exposed on the outer surface and it
is this that enables synthesis to be demonstrated on the
outer surface or protoplasts (Fig. 1). In the illustration
the model comprises a single rotating complex; two or
more adjacent complexes could be envisaged, however,
and re-arrangement within the complex, rather than rotation, is a possibility.
A major question posed by such a model is the amount
and source of energy required t o bring about translocation.
The nature of the environment within the membrane in the
immediate vicinity of the enzyme complex is not known
and so it is not possible to assess the forces exerted between
complex and membrane. Nevertheless, it seems unlikely
that the bond energy changes during synthesis of the
polymers from precursors would be able to provide the
necessary driving force. We have therefore examined the
possibility that the electrochemical proton gradient of
the membrane might provide the energy (Harrington &
Baddiley, 1984).
In the glycerol auxotroph, Bacillus subtilis 61360,
peptidoglycan synthesis was inhibited by the uncouplers
dinitrophenol and carbonylcyanide rn-chlorophenylhydrazone, with the accumulation of nucleotide precursors.
These uncouplers also affect the transport of N-acetylglucosamine and have been reported to initiate autolytic activity,
so the study was extended t o teichoic acid synthesis in vivo
and the effect of the ionophore valinomycin. It was found
that whereas valinomycin caused little or no inhibition of
glycerol incorporation into the cell, or into the nucleotide
precursor CDP-glycerol within the cell, under certain
conditions marked inhibition of wall teichoic acid synthesis
was observed. Similarly, inhibition of peptidoglycan synthesis was noted, accompanied by the accumulation of
'
Teichoic acid
Wall
Membrane
ChP-ribitol
UDP-GlcNAc
CDP-glycerol
Cell
Fig. 1. M o d e l f o r t e i c h o i c acid synthesis a n d translocation
across t h e cell m e m b r a n e
UDP-MurNAc-peptides. In contrast, valinomycin did not
inhibit the synthesis of wall teichoic acid by membrane
preparations, nor did it inhibit lipoteichoic acid synthesis
in vivo. From the action of valinomycin at different pH
values and ionic concentrations, it was concluded that the
electrochemical proton gradient, rather than either one of
its components (A$ and ApH), is essential for wall synthesis in the living cell. This dependence of wall synthesis
on the integrity of the electrochemical proton gradient is
consistent with our model.
I should like to close by acknowledgement of my
appreciation of the skills, ingenuity and devoted efforts
of my many co-workers over the years. They are too
numerous to name individually but I have had the greatest
pleasure in our association and friendship.
Bertram, K., Hancock, 1. C. & Baddiley, J. (1981) J. Bacreriol.
148,406-412
Harrington, C. R. & Baddiley, J. (1983) J. Bacreriol. 155, 776-792
Harrington, C. R. & Baddiley, I. (1984)J. Bacteriol. 159, 925-933
Leaver, I., Hancock, I. C. & Baddiley, J. (1981) J. Bacreriol. 146,
847-852
Kaya, S., Yokoyama, K., Araki, Y. & Ito, E. (1984) J. Bacreriol.
158,990-996
Neuhaus, F . C . (1985) Biochem. SOC. Trans. 13,987-990
The regulation of synthesis of wall polymers and of wall assembly in Bacillus
I. C. HANCOCK
Department of Microbiology, University of Newcastle
upon Tyne, The Medical School, Frarnlington Place,
Newcastle upon Tyne NE2 4HH, U.K.
The cell walls of Bacillus subtilis and Bacillus licheniformis
consist mainly of peptidoglycan, teichoic acids and teichuronic acid. In the work described here we have used
mainly B. subtilis W23, in which the teichoic acid is
poly(fl-glucosy1 ribitolphosphate) attached to a muramic
acid residue of peptidoglycan by a 'linkage unit' consisting
of three glycerol phosphate residues, and an N-acetyl
glucosamine I-phosphate residue that is phosphodiesterlinked to the muramic acid (Coley et al., 1978). Recent
Abbreviations used: lipid-LU, lipid-bound linkage unit; LU-TA,
linkage unit-bound teichoic acid.
work by I~o's group indicates that an additional N-acetylmannosamine residue may intervene between N-acetylglucosamine and the first glycerol phosphate linkage unit
(Kojima et al., 1985) although it is not clear whether this
is essential for biosynthesis in vifro. The linkage unit is
synthesized from UDP-N-acetylglucosamine and CDPglycerol and is assembled on a polyisoprenylphosphatecarrier lipid. Subsequently the main poly(ribito1phosphate)
chain is assembled on the lipid-bound linkage unit (lipidLU) by sequential additions of ribitolphosphate units from
CDP-ribitol. All these reactions are catalysed by membranebound enzymes (Hancock, 198 1). Teichuronic acid, which
only appears in the walls of B. subtilis at low phosphate
concentrations but is synthesized constitutively by B.
lichenijiormis, is a polymer of equimolar amounts of glucuronic acid and N-acetylgalactosamine, probably with a
disaccharide repeating unit structure. Ward & Curtis (1982)
1985
995
BIRMINGHAM MEETING
have shown that in B. lichenifomis the repeating unit is
synthesized on a polyisoprenylphosphate-carrier lipid, and
then polymerized by successive transfers of repeating unit
to the lipid-bound end of the growing polymer chain.
Using isolated membrane preparations and whole cells
made permeable to exogenous nucleotide precursors by
treatment with toluene, we have been able to assay several
stages in these biosynthetic pathways (Fig. 1). Synthesis of
lipid I, lipid-LU and linkage unit-bound teichoic acid
(LU-TA) can be measured in the presence of UDP-Nacetylglucosamine, CDP-glycerol and CDP-ribitol. With
isolated membranes the activity of the polymerase alone
can be measured taking advantage of the fact that artifactually alanine-free lipoteichoic acid in the membrane
can act as an artificial acceptor (Fischer et al., 1980). The
activities of the membrane-bound reactions of teichuronic
acid synthesis and of UDP-glucose dehydrogenase, catalysing the formation of the precursor UDP-glucuronic acid,
can be measured using the appropriate radioactive nucleotides and permeabilized cells (McGettrick et al., 1982;
Hancock, 1983).
The most striking change in wall composition due to a
change in growth conditions occurs when bacteria become
phosphate-limited in a chemostat. Teichoic acid synthesis
ceases and the polymer is lost from the wall by turnover,
being replaced by teichuronic acid (Ellwood & Tempest,
1972). Investigations of synthesis of teichoic acid and
teichuronic acid using radioactive labelling in vivo by
Rosenberger (1976) and by Glaser & Loewy (1979) showed
that protein synthesis was essential for acquisition of
teichuronic acid synthesis, or for full recovery of teichoic
acid synthesis on re-addition of phosphate, although some
teichoic acid was synthesized even when protein synthesis
was inhibited (Glaser & Loewy, 1979). We have examined
the enzymic basis of this phenomenon by using chemostatgrown bacteria in a technique that minimizes stress on the
bacteria and enables us to measure polymer synthesis from
exogenous nucleotide precursors in small samples, at rates
of the same order of magnitude as those found in vivo
(Hancock, 1983).
Using these techniques we have distinguished three
groups of enzymes according to their regulatory response
to changes in phosphate concentration in the chemostat
medium (Table 1). A particularly interesting feature of the
enzymes of classes A and B is that they are inactivated
when polymer synthesis ceases. In the case of class B
enzymes the inactivation is irreversible and re-activation
requires synthesis of new enzyme protein, but the membrane-bound enzymes of linkage unit synthesis are reversibly inactivated by a mechanism that must involve reorganization of the enzyme complex or covalent enzyme
modification. Although the regulatory response of the
membrane-bound enzymes of linkage unit synthesis to
phosphate did not require protein synthesis, full recovery
of teichoic acid synthesis in vivo on re-addition of phosphate to phosphate-limited bacteria did. The explanation
for this has been found t o lie in the behaviour of the
enzyme CDP-glycerol pyrophosphorylase (Fig. 1). This
enzyme was rapidly and irreversibly inactivated at the
onset of phosphate depletion (Table 1) and since synthesis
of new enzyme was completely repressed under phosphatelimitation (Cheah et al., 1981) protein synthesis was
required for induction of activity when phosphate was
restored. Since CDP-glycerol is required for synthesis of
linkage unit in B. subtilis W23 (Fig. l), teichoic acid synthesis becomes rate-limited by low levels of the enzyme
(Hancock, 1983).
In order to determine which steps in the biosynthetic
pathway are responsible for regulating the rate of teichoic
acid synthesis we took advantage of the observation of
Anderson et al. (1978) that in the chemostat at concentrations of Pi slightly higher than those causing growth-
UDPGlcNAc
Tunicamyctn
Lipld I
Prenol P
Prenol PPGlcNAc-PG
Pepiidoglycan
-
~
.
ncMA
LU-Lipid
Prenol PPGlcNAcPGPG-PG-IP-ribtiol),
LU-TA-
- .-
Prenol PPGlcNAcPG-PG-
~
r
r
c
e
r
o
t
CMP
nCDP-ribitol
(Prenol PPGlcNAcPGPG-PGI
CTP+RP
Fig. 1. Biosynthesis of teichoic acid (polyribitolphosphate) and its linkage t o
peptidogly can
All the reactions are catalysed by membrane-bound enzymes except reaction 1
(CDP-ribitol pyrophosphorylase) and reaction 2 (CDP-glycerol pyrophosphorylase).
Abbreviations: GlcNAc, N-acetylglucosamine; RP, ribitol phosphate; PG, glycerol
phosphate.
Vol. 13
BIOCHEMICAL SOCIETY TRANSACTIONS
996
Table 1 . Regulation of’ activity of e n z y m e s of teichoic acid and teichuronic acid
synthesis in Bacillus subtilis W23
Type of regulation
Enzymes
Group A
Membrane-bound enzymes
of LU-TA synthesis
Group B
CDP-glycerol pyrophosphorylase,
poly(ribito1phosphate) polymerase, teichuronic acid
polymerase
Group C
CDP-ribitol pyrophosphorylase,
UDP-glucose dehydrogenase
Inactivation
Activation
Reversible, at low
Pi concentrations
At high Pi concentrations,
no protein synthesis
required
Change in Pi concentration,
irreversible inactivation
Change in Pi concentration,
induction of enzyme
synthesis
Change in Pi concentration,
synthesis repressed, no
inactivation
Induction of new enzyme
synthesis
limitation the relative proportions of teichoic acid and
teichuronic acid in the wall are dependent on the Pi concentration in the medium. We therefore compared the
activities of several stages of teichoic acid synthesis (Fig. 1)
with the amount of teichoic acid incorporated into the
cell wall at various steady-state phosphate concentrations,
and looked for correlations between changes in enzyme
activities and changes in wall teichoic acid content (Cheah
et aL, 1982; Hancock, 1983). The clear conclusion drawn
from the results was that over the range of non-limiting
phosphate concentrations which modulated the amount of
teichoic acid in the wall, changes in the activities of the
enzymes catalysing the synthesizing of lipid-LU, together
with CDP-glycerol pyrophosphorylase, were responsible
for the observed changes in teichoic acid levels in the wall.
The activities of the enzymes associated with synthesis of
the main polymer chain (CDP-ribitol pyrophosphorylase,
polymerase, glucosyl-transferase) did not change over this
range of phosphate concentrations and decreased only
when phosphate became growth-limiting. Since in B.
subtilis W23 the only role for CDP-glycerol is in linkage
unit synthesis it is clear that the site of regulation of
teichoic acid synthesis, except under phosphate-limitation
(or starvation), is linkage unit synthesis.
It was pointed out by Baddiley’s group several years ago
that competition between pathways for a shared pool of
polyisoprenylphosphate-carrier lipid could provide a
mechanism for regulating wall composition. Evioence for
such competition has come from several sources (Watkinson
ef al., 1971; Anderson et al,, 1972; Rundell & Shuster,
1975). The more recent recognition that carrier lipids
serve not only as acceptors for the assembly of polymer
repeating units but also as membrane-bound carriers of
nascent polymer chains before their incorporation into the
wall leads to the idea that the rate of transfer of polymers
into the wall could have a feed-back effect on the activity
of biosynthetic sites where long occupancy of carrier
lipid molecules by nascent polymer chains would reduce
the number of sites for synthesis of lipid-bound precursors
and initiation of new polymer chains. There is evidence for
interaction between the synthetic pathways of different
wall polymers that could be explained in this way. We
found (Hancock, 1983) that initiation of new teichoic acid
synthesis on reactivation in previously phosphate-limited
bacteria was faster if the cells had been prevented from
synthesizing teichuronic acid during phosphate limitation.
In unpublished work we have also seen evidence for interaction of the peptidoglycan and teichoic acid synthetic
pathways. Brief treatment ( 5 min) of growing bacteria with
bacitracin, which sequesters polyisoprenylpyrophosphate
generated during peptidoglycan synthesis, almost completely prevented subsequent teichoic acid synthesis in
permeabilized cells, although the antibiotic was not inhibitory t o teichoic acid synthesis per s e . Recently Fischer &
Tomasz (1984) have found that peptidoglycan synthesis in
Streptococcus pneurnoniae is inhibited when teichoic acid
synthesis is blocked by deprivation of an exogenous precursor in viuo.
There is thus increasing evidence that wall composition
is regulated by the interaction of individual metabolic
pathways at the level of enzymic reactions that involve
polyisoprenylphosphate-carrier lipid. The details of the
regulatory mechanisms are, however, unknown and it is
only by determining what these mechanisms are that we
can expect to obtain a thorough understanding of wall
assembly.
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1985