Three well differentiated processes must be separated in the
general metabolism of sulfur: synthesis of the sulfur-containing
amino acids (cysteine and methionine), together with that of
the sulfur-containing coenzymes or prosthetic groups; catabolism
and equilibration of the pool of sulfur containing molecules;
and methionine recycling (a topic in itself, be it only because
of the role of methionine as the first residue of all proteins).
Several databases devoted to metabolism allow one to retrieve
extant data relevant to sulfur metabolism such as: the Kyoto
Encyclopedia of Genes and Genomes (KEGG),
the WIT (What is
there?) database of the US Department of Energy at Argonne, BRENDA and
the database of SRI, EcoCyc.
Specialized microbial genome databases, Colibri and SubtiList provide
direct information about the genes and genomes of the model
organisms Escherichia coli and Bacillus subtilis.
Other databases maintained within the GenoChore suite
allow one to have access to further information on a variety of bacteria.
Cell compartmentalization is therefore a prerequisite
to sulfur assimilation and this is reflected in the organization
of sulfur metabolism genes into clusters in the chromosome
et al., 2000).
Sulfate is first transported into the cell, as we shall
detail later on, then it is assimilated in the form of
adenosine phosphosulfate (APS), by an ATP sulfurylase (coded
by genes cysDandcysN). This reaction, which
yields APS and pyrophosphate from ATP and sulfate, is thermodynamically
strongly shifted towards ATP synthesis (and, therefore,
not towards sulfate incorporation), because the DG0'
of hydrolysis of the APS phosphate-sulfate bond (-19 kcal/mol)
is considerably higher than that of hydrolysis of the a,b
bond of ATP (-10,7 kcal/mol, Liu, 1998). It is therefore
necessary that an important activity keeps efficiently
the pyrophosphate concentration to a low level (through
hydrolysis into inorganic phosphate) to pull the reaction
towards the anabolic direction. This is assumed by one
or several pyrophosphatases, whose essential role is to
pull the pyrophosphate-producing macromolecular biosynthesis
reactions towards the direction of anabolism (figure 1).
However, as witnessed by the rather high intracellular
pyrophosphate concentration in E. coli, it
seems that this reaction is far from equilibrium. It cannot
therefore be sufficient to pull the reaction toward synthesis
of APS (Liu, 1998). This is why the synthesis of this latter
molecule is also linked with hydrolysis of the b,g bond
of GTP, which favors the reaction of sulfate incorporation
(105-fold with respect to the reaction in absence
of GTP). The coupling of the synthesis of APS with GTP
hydrolysis displaces the equilibrium of the reaction towards
sulfate assimilation (DG0' = -6,8 kcal/mol) (Liu, 1998).
However, this implies an extremely high cost of incorporation
of sulfur in sulfur-containing molecules. This drives a
strong selection pressure for the recovery of molecules
which contain sulfur in reduced state, a fact that has
to be taken into consideration when analyzing sulfur metabolism.
A further energy-consuming step sometimes shifts further
the reactions towards sulfur assimilation. In E. coli APS
is the substrate of a kinase (CysC), in a reaction that
utilises a second ATP molecule to phosphorylate the 3OH
positionofAPS, which is meatbolized into 3phosphoadenosine
phosphosulfate (PAPS). The raison d'être of
this reaction, which from ATP produces PAPS and ADP, seems
to be a supplementary means to pull the reaction towards
the anabolic direction. The sulfate activation pathway
is essential for the assimilation of sulfate and, in many
bacteria, is comprised of three reactions: the synthesis
of APS (adenosine 5' phosphosulfate), the hydrolysis of
GTP, and the 3' phosphorylation of APS to produce PAPS,
whose sulfuryl group is reduced or transferred to other
metabolites. The entire sulfate activation pathway is organized
into a single complex in Mycobacterium tuberculosis (Sun
et al., 2004). The catalytic efficiency of PAPS synthesis
is considerably higher than that of APS synthesis, and
PAPS is formed extremely fast.
drives the overall pathway towards PAPS synthesis.
In fact, not only the 3' phosphate of PAPS has no specific
function, but it must be metabolized into a by-product
of the reaction, PAP (3'-5'ADP), with no known function
in metabolism (figure 1). In many bacteria (but we did
not find the corresponding enzymes in E. coli or B. subtilis),
the plants and the animals, PAPS is the necessary precursor
for the sulfatation of various molecules (carbohydrates
in particular) by sulfotransferases (Kusche, 1991; Suiko,
1992; Varin, 1992). In M. tuberculosis PAPS
is involved in sulfatation of a lipid important for protecting
the baceria against the host defenses, and hence releavant
to pathogenicity (Mougous
et al. 2004).
The enzyme involved in the reduction of sulfate, phosphoadenosine
phosphosulfate reductase (EC 184.108.40.206) (thioredoxin-dependent
PAPS reductase) (PADOPS reductase) is coded by gene cysH.
The sulfite ion is reduced by NADPH-sulfite reductase
(EC 220.127.116.11), an enzyme comprising two subunits, coded
by operon cysJIH. Subunit a (coded by gene cysJ)
involves FAD, whereas subunit b (coded by gene cysI)
involves an iron-sulfur center and a siroheme prosthetic
group (analogous to siroheme-dependent nitrite reductases).
This implies a strong link between iron and sulfur metabolims,
as well as with heme or vitamin B12 biosynthesis. The sulfhydryl
ion, HS-, is quite reactive and toxic to the
cell if it is concentrated. The synthesis of siroheme is
catalysed by a SAM-dependent uroporphyrinogene III methylase
(EC 18.104.22.168), encoded by gene cysG that forms
an operon with genes nirBDC coding for the nitrite
reductase and possibly with genes yhfLMNOPQR, coding
for the metabolism of fructoselysine. Siroheme is present
in two enzymes, sulfite reductase and nitrite reductase.
In addition, it is a precursor of the B12 coenzyme (that
neither E. coli K12 nor B. subtilis 168
can entirely synthesize).
Many kinds of intermediary sulfur oxidation states exist:
sulfite, thiosulfate, etc. Complex oxido-reduction systems
permit to reach the ultimate reduction state, that of hydrogen
sulfide, H2S. These systems are often poorly known.
However, one knows that, for example, thiosulfate can be
an excellent sulfur source in E. coli Sirko,
1995). With respect to organic sulfur forms, things are also
poorly known. The situation in Bacillus subtilis is
being unravelled but not yet completely understood. It is
significantly different from its counterpart in E. coli.
Biosynthesis of methionine originates from the homoserine
pathway (which branches to lysine via diaminopimelate,
an essential component of mureine
to threonine, and to isoleucine), starting from the
synthesis of an activated derivative, O-succinylhomoserine.
This activated homoserine condenses directly with cysteine,
giving cystathionine. A S-lyase, belonging to a large
family of enzymes that allow the cleavage of molecules of the
X-CH2-S-CH2-Y, or X-CH2-S-S-CH2-Z type,
from either side of the sulfur atom, liberates homocysteine
and serine (which is cleaved into pyruvate and ammonium). Homocysteine
is the precursor of methionine, whose methyl group comes from
the one-carbon metabolism (figure 3). In E. coli,
two enzymes which catalyze this methylation exist. One of them,
the product of the metH gene (EC 22.214.171.124), utilises
vitamin B12 as a cofactor, and the methyl group can be taken
from 5-methyltetrahydrofolate or from its polyglutamyl derivative.
The second one, the product of gene metE (EC 126.96.36.199),
which is not coenzyme B12-dependent, catalyses the methylation
with 5-methyltetrahydropteroyltri-L-glutamate as the methyl
groupe donor Kung, 1972). In both cases the original carbon
derives from serine.
One can wonder why there exist two different genes for this
metabolic step. In fact, one of them utilises coenzyme B12
(that necessitates at least 26 steps for its synthesis, starting
from uroporphyrinogene III (Michal, 1999) and consumes
seven molecules of S-adenosylmethionine. E. coli does
not synthesize coenzyme B12, but possesses a transport system
(btuBCDE and btuR) highly specific and efficient
for this coenzyme (Colibri).
The reaction catalyzed by protein MetH with coenzyme B12 is
more than one hundred-fold faster than that catalyzed by the
B12-independent enzyme, MetE (EC 188.8.131.52) Greene, 1996). It
follows that the availability in methyl groups (we shall see
below how important they are), via methionine, is provided
much more easily in the presence than in the absence of coenzyme
There exists finally a pathway for recycling of methyl groups,
recently found, that utilises S-methylmethionine as
a methyl donor. This molecule, synthesized by plants, is scavenged
by the product of gene mmuM (formerly yagD, with
a strong similarity with metH), S-methylmethionine:homocysteine
methyltransferase, which transfers methyl group directy onto
homocysteine to give methionine (Thanbichler, 1999, Table 2).
There certainly exist permeases for the sulfur-containing
amino-acid: cysteine, its oxidation product cystine, homocysteine
and homocystine, and methionine. They have however rarely
been characterised without ambiguity. One may think that
transport of methionine is carried out by branched-chain
amino-acid permeases (liv in E. coli, azlCD and braB
brnQ in B. subtilis), but one must wonder
about the possible existence of specific permeases Greene,
1996). Indeed, there exists a permease for S-methylmethionine,
encoded by gene mmuP (formerly yfkD), in
an operon with gene mmuM (Table 2). In the case
of B. subtilis methionine is transported by several permeases
but the most efficient one is the metNPQ (yusCBA)
operon that encodes an ABC permease transporting methionine
sulfoxide, D- and L-methionine (Hullo
et al., 2004).
Independently from their transport, the presence of reduced
amino acids (cysteine and homocysteine) must pose a problem
to the cell, since they are very reactive (reducers), and this
must be taken into account when one explores the way in which
they permeate the cell. In order to understand their possible
effects, it is useful to remark that the distribution of cysteine-containing
proteins is not random in general. In the cytoplasm, one often
finds proteins containing a metal, and in particular the diverse
types of iron-sulfur centers (often with clustered cysteine
residues), isolated cysteines (often involved in the catalytic
mechanism), and cysteines burried in the hydrophobic regions
of proteins. In contrast, the periplasm possesses a strongly
oxidizing character (in the presence of oxygen), necessary
for the formation of tertiary and quaternary structures of
all kinds of proteins through disulfide bridges. The intrusion
of strongly reducing molecules may therefore have a deleterious
Probably, the problem of cysteine toxicity in its reduced
form does not occur frequently, because it is not present in
nature in significant amount, but is saved perhaps in environments
devoid of oxygen. Cystine is likely to be more common, and
it is probable that there exists a system permetting its transport,
and its rapid metabolism (a b-lyase for example). This seems
more necessary because cystine is structurally similar to diaminopimelic
acid (figure 4), an essential component of many eubacteria
cell wall, E. coli in particular. As in the case
of every molecular mimic, it may therefore take the corresponding
place, and interfere with the synthesis of mureine, leading
to cell lysis Richaud, 1993).
There exist many proteases and peptidases in bacteria Miller,
1996), and the externl medium (the gut in particular) such
as those faced by E. coli often contain peptides.
Several transport systems allow their salvage (Dpp, Tpp and
Opp Oliver, 1996). A specially important case, despite the
fact that it has hardly been studied, is that of peptides from
the amino-terminal ends of proteins. They often carry a N-formyl-methionine
group, specific for translation initiation. It has indeed been
discovered that in the eucaryote hosts that live in the presence
of commensal or pathogenic bacteria, there exist several recognition
and transport systems for these peptides Schiffmann, 1975;
Prossnitz, 1999). This indicates that these molecules are produced
and secreted in significant quantity. It seems likely that
bacteria also possess transport systems allowing their salvage.
Nobody knows, at this time, if permeases Dpp, Tpp or Opp are
adapted to this transport, or if there exist other similar
permeases. In the same way, the pathways for the degradation
of these peptides allowing methionine salvage, have not been
studied. This would be more interesting because these molecules
can easily be visualized as participating in a new process
allowing "quorum sensing".
In the metabolic pathways that have just been described,
cystathionine plays a central role since, by its very
construction, it allows to go indiscriminately from cysteine
to homocysteine and vice versa, as long as there exist
b-lyase and g-lyase, together with b-synthase and g-synthase
activities (figure 5). Therefore the organisms carrying
these activities should grow equally well with cysteine
or homocysteine as sulfur sources. Growth on methionine
requires a methyl group transfer reaction. But as S-adenosylmethionine
is involved in a large number of activities that produces
homocysteine, this should be easily achieved. Furthermore,
the reaction catalyzed by MmuM, shows that the transfer
of a methyl group from a sulfonium group is not difficult
to achieve Thanbichler, 1999). Finally, the reaction
homologous to that leading from serine to cysteine must
easily result from the evolution of the corresponding
proteins and allow the use of homoserine as a substrate
in the place of serine (O-acylhomoserine sulfhydrylase,
or even homoserine phosphate sulfhydrylase; Michal, 1999).
One can therefore easily appreciate the versatility in
the utilisation of sulfur sources in different organisms,
take the case of Pseudomonas putida for example
Vermeij, 1999). All depends on the selection pressure
of the biotope in which they strive to survive.
In these conditions, it is remarkable that E. coli,
that can grow on cysteine, cannot grow on methionine Kredich,
1996). This inability is certainly due to the fact that the
anaerobic medium in which E. coli generally lives
is a sulfur-rich medium. The problem that is posed to the
bacteria is therefore how to eliminate the sulfur excess
rather than its elaborate utilization. In such a case, there
is no bypass from methionine (or homocysteine) to cysteine. E. coli metabolizes
cystathionine only by the action of a b-lyase (MetC), the
g-lyase being apparently absent from its genome. Interestingly,
this lack of continuity in sulfur metabolism allows E. coli to
use selenomethionine in the place of methionine, as was shown
by Georges Cohen (Cohen, 1957; Cowie, 1957). We have indeed
verified that E. coli was not able to grow with
methionine as a sulfur source. It follows that E. coli is
an organism of choice for testing by heterologous complementation
the existence of sulfur equilibrating pathways in other microorganisms.
Methionine is the object of several cycles, particulary
important when sulfur is limiting (in plants for example).
The first of these cycles concerns the process of translation
of messenger RNAs into proteins. Indeed, all the proteins
of the cell start with a methionine residue, which implies
an importance of this amino acid. This methionine is however
modified by a formyl group, and all neosynthesized proteins
start with a N-formyl-methionine.
In eubacteria translation begins in an original way. A particular
transfer RNA, tRNAFmet is first charged by methionine as
its homologous tRNAMmet, by the action of methionine tRNA
synthetase, encoded by gene metG in E. coli.
(metS in B. subtilis). The remarkable
feature comes now. This Met-tRNAFmet is formylated by a transformylase
(identified both in E. coli and B. subtilis, fmt),
that utilises 10-formyltetrahydrofolate as donor of the formyl
group (cf. Danchin, 1973). It is this FMet-tRNAFmet that,
in the presence of translation initiation factor IF2 (encoded
by gene infB) and GTP, will correctly position this
charged tRNA at the translation initiation site of a messenger
RNA (at codons AUG, UUG or GUG). In the presence of a transfer
RNA corresponding to the subsequent codon, charged with its
cognate amino acid and loaded on factor EF-Tu in the presence
of GTP and then factor EF-G bound to GTP, the first peptidic
link is formed. Let us note here a second oddity: the formation
of this first peptidic link, except if it links FMet to an
aromatic amino-acid, necessitates another factor, named EFP
(enoded by gene efp) Aoki, 1997). The precise role
of protein EFP, which is an essential protein, remains to
be determined. Interestingly, we must remark that factor
EFP is homologous to the eucaryotic factor eIF5-A (a family
of highly conserved essential proteins), that carries a residue
derived from spermidine, hypusine. The need for its presence
suggests that it operates at a "fragile" step of
translation, and that at this stage this process can be spontaneously
interrupted. Also, the role of factor EFP in the liberation
of short peptidyl-tRNA must be taken into account when one
wishes to count the pool of methionine available in the cell.
The synthesis of the protein then goes on normally.
One observes therefore that, as a function of the efficiency
of this step (and more generally of every first steps of
translation), one will obtain either a complete protein,
carrying FMet at its extremity, or a transfer RNA carrying
a peptide that will be hydrolyzed by peptidyl-tRNA hydrolase
(EC 184.108.40.206), an essential protein encoded by gene pth Heurgue-Hamard,
1998). The need for the step of dissociation of peptidyl-tRNA
from the ribosome is apparently necessary to ensure the accuracy
of translation Heurgue-Hamard, 1998).
A general assessment of translation indicates that 10% at
least of initiated translations are abortive Heurgue-Hamard,
1998). This shows that the effect of formation of the first
peptidic link on the utilisation of sulfur is far from negligible.
As a consequence, there must exist a recycling process permitting
methionine salvage from peptidyl FMet-aan. An alternative
to recycling is excretion of formylated peptides, but this
leads to sulfur leakage, in the form of methionine. We have
already indicated that these peptides are recognized by receptors
in eucaryotes Prossnitz, 1999), showing that this mechanism
is most probably significant. It may also be involved in "quorum
A second series of reactions associated to translation plays
a general role. The neosynthesized protein must be deformylated
by a deformylase (specified by gene def, in operon
with fmt, in E. coli and B. subtilis).
The sequence of these two steps
formylation and deformylation must have an important
role, since it is conserved throughout evolution in all Bacteria
(and even in mitochondria and chloroplasts). But, oddly enough,
it does not appear to be absolutely necessary since E. coli can
grow without formylation, in particular when ribosomal protein
S12 is altered to become streptomycin resistant (Danchin,
1973; Harvey, 1973; Petersen
et al., 1978; Mazel et al., 1994). Subsequently, methionine
is cleaved by an original amino-peptidase, encoded by gene map with
a zinc cofactor, but also, in many organisms, the cobalt
Cobalt is a relatively rare ion. Its concentration in soil
varies from 10 to 15 mg/g (mg of cobalt by g of soil) Kucera,
1998). Living organisms have therefore developped very efficient
transport systems for this indispensable ion. Note that amino-peptidase
plays a particularly important role in all organisms because
it is an essential activity for the cell. It has been found
that the antibiotic fumagilline (synthesized by the fungus Aspergillus
fumigatus) is a strong inhibitor of this enzyme in eucaryotes
Liu, 1998). In B. subtilis two genes, map (mapA)
and yflG (mapB) code for a methionine aminopeptidase.
While MapB (YflG) analogs are found in Gram positive cocci,
they do not possess an direct counterpart of the Bacillus MapA.
The successive action of these two enzymes (deformylase
and methionine aminopeptidase) recycles methionine. This
liberates the amino-terminal extremity of the protein, which
may thus be submitted to the degradative action of diverse
aminopeptidases, allowing fine regulation of the concentration
of the corresponding protein in the cell (see Varshavsky's
"N-end rule" Bachmair, 1986). It is essential to
remark that the cleavage of the N-terminal methionine can
only happen after deformylation Solbiati, 1999). The activity
of deformylase is controlled by an essential ferrous ion,
which makes it particularly sensitive to the presence of
oxygen Rajagopalan, 1998). Taken together, these observations
show that these steps probably play a crucial role in the
regulation of gene expression, in particular in the presence
The E. coli biotope is not likely to be
limited in sulfur. Many observations substantiate this
contention: cysteines are present in large quantity in
its proteins and its metabolism does not seem to care
for sulfur availability. This is illustrated by the following
example. Spermidine biosynthesis produces a sulfur-containing
methylthioadenosine (MTA) (figure 6 and see below).
In a close relative of E. coli, Klebsiella
pneumoniae, this molecule is transformed into methylthioribose-1-phosphate
and recycled (see below). In E. coli however,
it is excreted Schroeder, 1973), which implies an enormous
loss of sulfur, because for each molecule of spermidine
a molecule of methylthioribose (MTR) is produced and
excreted (and we shall see that other reactions involving
AdoMet produce also MTA)!
What do we know more generally about sulfur sources for E. coli?
In its mammalian hosts, apart from quite varied sources (depending
on the diet), they derive probably from two pathways of cysteine
degradation: cysteine-sulfinate (sulfinoalanine)-dependent,
and cysteine-sulfinate independent, because methionine is
recycled (and its sulfur lost as MTR). The cysteine-sulfinate-dependent
pathway arises by oxidation of cysteine by the cysteine dioxygenase
(CDO, EC 220.127.116.11), which produces cysteine-sulfinate (sulfinoalanine).
The latter can be catabolized in two different ways. Either
it is transaminated by an aspartate aminotransferase (AAT,
EC 18.104.22.168) with production of pyruvate and sulfate, or it
is decarboxylated by cysteine sulfinate decarboxylase (sulfinoalanine
decarboxylase, CSAD, EC 22.214.171.124) into hypotaurine. Hypotaurine
is probably oxidized non-enzymatically into taurine Bella,
1996). Neither animals, nor plants (where it is rarer) can
metabolize taurine. The excess of taurine produced by animals
is excreted either directly in urine, or in bile in the form
of taurocholate. This molecule is therefore abundant in E. coli's
diet. Taurine is an important metabolite, involved in aging,
in homeostasis or in the defense against free radicals or
alterations in the concentration of calcium.
Cysteine, in the presence of oxygen, can be oxidized spontaneously
into cysteic acid (frequent oxidation in aging proteins).
Cysteic acid is decarboxylated directly into taurine by sulfinoalanine
decarboxylase (CSAD, also named sulfoalanine decarboxylase,
an enzyme specific of two similar substrates, cysteine-sulfinate
and cysteate (see Swiss-Prot: EC 126.96.36.199 and KEGG: MAP00430)).
Alternatively it is transformed in the presence of H2S into
cysteine and sulfite by a cysteine lyase (EC 188.8.131.52). However,
most of these reactions are unknown (and, in fact, a cysteine
sulfinate sulfinase has just been described Mihara, 1997
#280), and these reactions are just presented as hypotheses
reflecting our ignorance
lyase: L-cysteine + sulfite <=> L-cysteate + H2S
can use a second molecule of cysteine (producing lanthionine)
or other alkyl thiols as replacing agents). As an illustration
of this metabolism in bacteria (it does not appear to exist
in E. coli), one finds in B. subtilis a
gene (yubC) that looks like a cysteine dioxygenase,
enzyme known to regulate the intracellular level of cysteine,
of methionine and of glutathione in mammals (SubtiList) Eppler,
1998 #253. This, together with the fact that B. subtilis possesses
a very small number of cysteines in its proteins, may indicate
a physiological role particular to this amino acid.
- Repair of oxidized sulfur amino-acids
Disulfide bridges are very important structural elements
of many proteins. They are extremely rare in cytoplasmic
proteins because of the reducing nature of the intracellular
medium. In contrast, the periplasm possesse a strongly
oxidizing character (in the presence of oxygen). An environment
with this character is necessary to the formation of
tertiary and quaternary structures of all kinds of proteins,
via the formation of disulfide bridges. These covalent
bonds are essential to the stability and activity of
many extracellular proteins. This has been demonstrated
in pathogenic bacteria in the case of some toxins, secreted
cellulases and pectate lyases Missiakas, 1995 #213. The
corresponding oxidation mechanism (2 Cys > Cys-S-S-Cys
+ 2 H+ + 2 e-), can be spontaneous in the presence of
oxygen, but in E. coli the proteins of the
family Dsb (DiSulfide Bond formation) that catalyse the
formation of appropriate disulfide bridges, have an essential
function. This family comprises proteins DsbA, DsbB,
DsbC and DsbD Missiakas, 1995 #213 and proteins DsbE
and DsbF that have been recently discovered Metheringham,
1996 #227. Among these proteins, DsbA and DsbC that are
located in the periplasm have a strong oxidizing activity
on cysteines. The membrane protein DsbB is essential
for the recycling of the active site (active thiol) of
DsbA, but the mechanism of this reaction is not yet known.
Protein DsbD counteracts the action of the two oxidizing
forms (A and C) by reduction of disulfide bridges in
an equilibrium between the thiols and the disulfide bonds
in the periplasm. The role of Dsb proteins in the shaping
of proteins is fundamental, and one can therefore wonder
about the nature of their function: are they enzymes,
or molecular chaperones? The case of protein DnaJ illustrates
well the question. Identified as a chaperone, it possesses
an enzymatic activity capable of reducing, oxidizing,
or isomerizing disulfide bridges Wang, 1998 #307.
Bader et al. reconstituted the oxidative folding
system using purified DsbA and DsbB. They identified the
sources of oxidative power for protein folding and showed
how disulfide bond formation is linked to cellular metabolism:
disulfide bond formation is directly coupled to the electron
transport chain. DsbB uses quinones as electron acceptors,
allowing various choices for electron transport to support
disulfide bond formation. Electrons flow via cytochrome bo
oxidase to oxygen under aerobic conditions or via cytochrome
bd oxidase under partially anaerobic conditions. Under anaerobic
conditions, menaquinone shuttles electrons to alternate final
electron acceptors such as nitrate or fumarate Bader, 1999).
Intracellular proteins are in majority present in the thiol
form, with a low representation of cysteines (1.6%), whereas
extracellular proteins contain disulfide bridges and are
rich in cysteines (4.1%) Fahey, 1977). This absence of disulfide
bridges inside the cell, as we have seen, is linked to the
strongly reducing character of the cytoplasm (production
of NAD(P)H by the catabolic processes of respiration and
glycolysis). In E. coli, many factors participate
in the maintenance of the balance between thiols and disulfide
bridges in the cytoplasm (disulfide bridges can be formed
by the action of free radicals, active oxygen or cosmic rays,
which lead to inactivation of the proteins sensitive to their
action). In the cytoplasm the main system maintaining the
correct ratio between thiols and disulfide bridges is constituted
by a tripeptide containing cysteine, glutathione (see also
below). Glutathione is present in E. coli at
a high concentration (about 5 mM), and it is maintained almost
entirely in the reduced form (redox potential of -230 mV).
the ratio between reduced and oxidized glutathione is between
50:1 and 200:1 Prinz, 1997).
E. coli possesses at least four thiol-disulfide
oxidoreductases that permit reduction of the disulfide bridges
of cytoplasmic proteins: a thioredoxin (encoded by gene trxA)
and three glutaredoxins (1, 2 and 3, encoded by genes grxABC).
Thioredoxin is a reducing protein more efficient (redox potential
of -270 mV) than glutaredoxins (redox potential of -233 mV
to -198 mV). After reducing the disulfide bridges, the thiol-disulfide
oxidoreductases are oxidized. A system based on the transfer
of protons from NADPH to these proteins allow their reduction,
a sine qua non step to keep them functional. Thioredoxins
are reduced directly by a thioredoxin reductase (FAD enzyme,
EC 184.108.40.206, encoded by gene trxB), whereras glutaredoxins
are reduced by glutathione, that in its turn is reduced by
glutathione reductase (FAD enzyme, EC 220.127.116.11, encoded by
gene gor). In E. coli, these two systems:
thioredoxin reductase and thioredoxin, and glutaredoxin reductase,
glutathione and glutaredoxin, participate in the reduction
of disulfide bridges of essential cytoplasmic enzymes that
need this step of reduction to accomplish their catalytic
function (for example ribonucleotide reductase, PAPS reductase
or peptidyl methionine reductase).
One mechanism to regulate the activity of redox proteins
in the cell is through reversible formation of disulfide
bridges. This is often achieved with the help of glutathione,
glutaredoxin, or thioredoxin. Thus, the activity of key redox
regulatory proteins is responsive to the intracellular thiol-disulfide
redox status. For example, OxyR, the prototypic redox-regulated
transcription factor in E. coli, is activated
through the formation of a disulfide bond using cysteine
sulfenic acid as an intermediate, and is deactivated by enzymatic
reduction of this disulfide bond with glutaredoxin 1 Zheng,
1998 #363; Aslund, 1999 #362. Likewise, the catalytic mechanism
by which peroxiredoxins scavenge hydrogen peroxide or alkyl
hydroperoxide also involves reduction and re-formation of
disulfide bonds with a cysteine sulfenic acid as intermediate
(reviewed in Jin, 1999 #367). Known peroxiredoxins in E. coli include
AhpC and scavengase p20/Tpx Chae, 1994 #366; Wan, 1997 #364;
The oxidation of methionine is a case of damage that can
happen to proteins through action of endogenous or environmental
oxidizing agents. Although in some cases oxidation does not
have a large effect on the activity of proteins, in the majority
of cases it abolishes their catalytic function. The cell
has two ways to take into account proteins containing oxidized
methionine residues (Met(O)). Oxidized proteins are either
degraded, or an enzyme can reduce the modified residues.
This latter function is performed by peptidyl-methionine
sulfoxide reductase MsrA (encoded by gene msrA) that
reduces oxidized residues in the protein, restoring its function
Moskovitz, 1995). This enzyme, apart from its capacity to
repair oxidized residues in proteins, can also reduce methionine
sulfoxide in its free amino-acid form. It permits therefore
utilisation of methionine sulfoxide as a source of sulfur
An interesting observation established recently a relationship
between the capacity of repair of oxidized methionines and
virulence in enterobacteria pathogenic for plants, Erwinia
chrysanthemi Hassouni, 1999). Alteration of virulence
(inability to cause systemic invasion) is associated with
enhancement of sensitivity to oxidative stress and with the
decrease of motility on a solid substrate. It involves the msrA gene,
coding for peptidyl methionine sulfoxide reductase. Indeed,
the mutants of this gene can no longer repair their proteins
altered by the defensive action of plants (a process that
utilises active oxygen and free radicals). Moreover, motility
is an essential factor of virulence, in particular motility
on solid surface is affected in mutant msrA. Curiously,
the msrA mutants remain motile in liquid medium. This
shows that the affected mechanisms do not involve flagellae.
It is possible that the target process is not the process
of movement creation but, for example, chemotaxis or adhesion.
Combining these data, one can imagine that MsrA has the double
function of a general repair system and a regulator of the
production of extracellular appendages. The production of
these appendages may be under the control of a regulator
extremely sensitive to oxidative stress, normally repaired
by MsrA. In any event, peptidyl methionine sulfoxide reductase
is a particulary important protein since it seems to be present
in all extant organisms: it belongs to the class of genes
that Eugene Koonin named "the minimal gene set"
Mushegian, 1996) and that we named the paleome.
S-adenosylmethionine and its metabolism
Methionine is also an essential element in a large number
of methylation processes it is a universal methylating
agent (and it was even proposed that it could act in the
absence of enzymes, but this has not been found to be the
case Posnick, 1999 #373
via S-adenosylmethionine (SAM or AdoMet). Some of
these methylations are essential to cell functioning, and
SAM synthetase is therefore an essential enzyme. The exact
role of these methylations is often poorly known, but it
is a type of chemical modification so frequent that it probably
indicates a metabolic need linked to the availability in
one-carbon residues ("one-carbon pool"). Sulfur
in its sulfonium form is an ideal atom as an intermediate,
being both a receptor and donor of alkyl groups. As a case
in point, it appears now that another molecule comprising
this group, S-methylmethionine, also plays an important
(but poorly known) role both in the storage and in the exchange
of methyl groups Thanbichler, 1999).
Biosynthesis of S-adenosylmethionine
AdoMet is synthesized from methionine and ATP by SAM synthase
(EC 18.104.22.168), the product of gene metK. AdoMet is
an essential molecule. This is due to the frequent (and poorly
understood) involvement of AdoMet in the regulation of gene
expression (cf. Thomas, 1991; Chen, 1999) and this accounts
well for the ambiguous results of the genetics of metK.
SAM synthase being essential, the metK gene can never
be totally inactive. If one obtains, among ethionine resistant
mutants, mutants that make little AdoMet, they are nevertheless
not entirely deprived of it Saint-Girons, 1988). One should
not forget this fact, that has long made scientists think
that there existed two AdoMet synthetases in E. coli (which
is true in S. cerevisiae, but not in E. coli)
Thomas, 1987; Greene, 1996).
AdoMet synthetase condenses methionine with ATP by hydrolyzing
the latter into phosphate and pyrophosphate. In turn, pyrophosphate
is cleaved by pyrophosphatase, pulling the reaction toward
the biosynthesis direction:
ATP + methionine > AdoMet + Pi + PPi
This is therefore a very energy costly reaction since it
consumes three energy-rich bonds, while creating only one,
that of the sulfonium group. The sulfur atom of methionine
is, in the molecule of AdoMet, positively charged. It is
coordinated to three groups that it can theoretically donate
in a more or less equivalent way: a methyl- group, an aminobutyryl-
(or, more aptly, 3-amino-3-carboxylpropyl-) group and an
adenosyl- group. It is generally the methyl- group which
is donated, but the other reactions are possible (a variant
is seen in the synthesis of spermidine, with transfer of
the aminopropyl- group from dAdoMet). For example, some transfer
RNA molecules (tRNAPhe) can be modified with the 3-amino-3-carboxylpropyl-
group Nishimura, 1974). A last type of transfer (transfer
of the ribosyl- group) is the basis of another complicated
modified base of tRNA, queuosine Slany, 1993).
Because of its reactivity this sulfonium group can be spontaneously
hydrolyzed, and it is the masking from water molecules that
makes it reactive towards other acceptor substrates. In the
presence of water it spontaneously yields methionine and
adenosine, methylthioribose and homoserine, or methanol and
adenosylhomocysteine. It must therefore exist a strict control
of the pool of AdoMet in the cell.
The main product of the transmethylation reaction is S-adenosylhomocysteine.
In E. coli, this molecule seems to be recycled
in the following way. A nucleosidase (EC 22.214.171.124), encoded
by gene pfs (see below) hydrolyses this molecule into
adenine and S-ribosylhomocysteine, that hydrolyses
spontaneously into homocysteine and ribose Della Ragione,
1985). Homocysteine is converted into methionine, and ribose
and adenine must be recycled (but we did not find a general
assessment of these reactions). In plants and animals, there
exists an adenosylhomocysteinase (EC 126.96.36.199) that produces
adenosine and homocysteine directly. This seems more economical
and produces adenosine, an important mediator of some cascades
of regulation Cohen, 1998 #10. It seems clear that it would
be useful to characterize more explicitly the outcome of
these pathways in bacteria.
The reactions of the AdoMet sulfonium group
Methylation reactions are very numerous. The best known,
because they are better understood, are those which correspond
to methylation of DNA, in particular to protect DNA against
restriction enzymes (in general the genes are in an operon
coding for a DNA methylase, followed by the restriction enzyme:
a case in point is the hsdRM operon in E. coli K12).
They can also be involved in covalent modifications leading
to processes of epigenetic heredity (as sexual imprint in
vertebrates, or controlling the form of flowers Cubas, 1999).
Methylation of nucleic acids
Most often, methylated sites are located at position N6
of adenine, or C5 of cytosine, but all kinds of reactions
are possible: for example, positions 2' or 3' of ribose can
also be methylated, as well as the amino group of adenine.
In E. coli, two major sites of methylation of
DNA are observed: on the one hand the adenine present at
site GATC, is methylated by Dam methylase, and on the other
hand the second cytosine sites recognized by enzyme EcoRII
CC(A/T)GG, by methylase Dcm Urieli-Shoval, 1983). GATC methylation
sites are involved in at least three fondamental processes:
(i) the replication proof-reading system ("long patch
mismatch repair"), where protein MutH cleaves the unmethylated
strand of sequence GATC, and hydrolyses the daughter strand
when it interacts with a mismatch base pair recognized by
the MutS MutL complex, followed by the reparation of this
strand by DNA polymerase I Laengle-Rouault, 1986); (ii) the
fixation of protein DnaA, to permit correct initiation of
replication of DNA at the Ori region Campbell, 1990 #242;
(iii) the control, possibly epigenetic in part, of the transcription
of genes involved in pili formation Blyn, 1990). Finally,
an analysis of the genome sequence suggested a particular
role of GATC sequences, perhaps hemimethylated, in the control
of the transition of growth from anaerobiosis to growth in
the presence of oxygen Hénaut, 1996). A recent work
on the formation of E. coli biofilms controlled
by the regulator OxyR that binds to regions containing several
GATC substantiates experimentally this hypothesis Hasman,
1999). The case of DNA regions methylated by protein Dcm
corresponds mostly to the correction of very local errors
("very short mismatch repair") Lieb, 1986 #245.
RNA molecules are also often methylated: it is the case
of many positions in transfer RNA Bjork, 1996), in particular
of the thymine residue in the TYCG loop (gene trmA).
Some positions in ribosomal RNA may be methylated permanently
Bjork, 1996 #254, or in the presence of genes confering resistance
to some antibiotics (such as erythromycine Thakker-Varia,
1985 #255). The role of these methylations is poorly known,
and inactivatiing the corresponding genes does not give a
simple phenotype (apart from a variation in the accurracy
of translation, Bjork, 1999).
In the case of ribosomal RNA eubacteria differ from eucaryotes
and archaebacteria in that it is much less methylated, and
probably methylated by methylases specific for each methylated
region. In contrast, in the latter cases, it seems that a
small set of methylases recognize structures made of ribosomal
RNA associated to guide RNAs, that can methylate several
regions of the rRNA.
Many proteins may also be methylated: ribosomal proteins
Chen, 1977 #258; Mardones, 1980 #259; chemotaxis control
proteins (methylation of a glutamate residue in the chemotaxis
receptors by methyltransferase CheR and demethylation by
methylesterase CheB Simms, 1987 #257); N-methylation
of the amino-terminal residue of some proteins Stock, 1987
#260; or methylation of isoaspartate residues in aging proteins.
In this latter case, methylation on the aspartate group creates
an unstable methyl bond, that is corrected following spontaneous
hydrolysis Visick, 1998).
The sites of these methylations are varied: it is often
the e-terminal group of lysines, but one also finds histidine
Chen, 1988 #262, aspartate, glutamate, arginine Rawal, 1995),
glycine Ogawa, 1998 residues, or the N-terminal extremity
of proteins. In this latter case, the modifications concern
methionine, phenylalanine, alanine or proline residues, which
leads to the formation of monomethylmethionine, monomethylphenylalanine,
monomethylalanine, trimethylalanine and dimethylproline Stock,
1987 #260. Little is known concerning methylation of proteins
in bacteria, and it is probable that the study of the proteome
will lead to suprising discoveries in this domain.
Methylation of metabolites
AdoMet is involved in the synthesis of a large number of
metabolites, and in particular of essential coenzymes such
as ubiquinone, menaquinone, siroheme and vitamin B12. One
also finds detoxification systems (methyl-selenocysteine),
antibiotics and all kinds of molecules involved in the cell's
achitecture (terpene derivatives, sterols, lignin) or metabolites
involved in the regulation of osmotic pressure (betaïne,
choline). In plants, one finds derivatives of methionine,
such as S-methylmethionine (SMM) James, 1995 #265
or S-methylcysteine Chow, 1972 #266. The role of SMM
is not clear but it is used by plants as an osmoprotectant.
It has recently been implicated in the metabolism of selenium.
Its methyl group can be transfered to homocysteine to give
two molecules of methionine (role of storage for methyl groups).
In plants, SMM can also be cleaved into dimethylmercaptan
and homoserine or, finally, decarboxylated in the presence
of pyridoxal-phosphate and metal ions. This decarboxylation
product (dimethylsulfonium propylamino) is an element active
in the anticancer molecule, bleomycine A2 Cohen, 1998 #10.
Some of these metabolites exist in diverse strains of E. coli,
and it is likely that among the unknown genes of pathogenicity
islands one finds genes involved in some of these methylations.
The aminobutyryl group and its avatars
The second group which may be donated by AdoMet is the aminobutyryl
group. It has been found recently that the aminobutyryl group
is utilized by the cells in several reactions.
Bacteria are often considered as isolated individuals, incapable
of the organized behaviors observed in multicellular organisms.
However, W. Hastings, who studied marine bacteria forming
luminescent colonies, discovered twenty years ago that their
capacity to emit light was entirely determined by their relative
number Wilson, 1998 #271. These bacteria had to be concentrated
enough to produce light. Everything went as if they could
measure their number to induce expression of the genes necessary
for light production. This supposed, as in general assemblies,
that a "quorum"
was reached, hence the name of "quorum sensing" given
to this phenomenon. "Quorum sensing" is a mechanism
of communication between bacteria that leads them to display
an organized collective behavior.
The following questions were asked: what are the molecules
secreted by the bacteria, that permitted them to trigger
this phenomenon, how these molecules were synthesized, and
what did they control?
Among the many molecules that carry this information (autoinducers)
one may distinguish at least two classes. Gram negative bacteria
produce N-acyl homoserine lactones (N-acyl
HSL), small molecules composed of homoserine cyclised in
lactone and an aliphatic chain. Other bacteria, such as Staphylococcus
aureus or B. subtilis, produce peptidic autoinducers.
In the case of Gram bacteria, the paradigm of which
is Vibrio fisheri or V. harveyi, a protein
belonging to the family LuxI catalyses the formation of N-acyl
HSL from a molecule of S-adenosylmethionine and acyl-ACP
(acyl-Acyl Carrier Protein) Val, 1998 #268; Parsek, 1999
#267. AdoMet is used in this reaction as a donor of the aminobutyryl
group (it participates in the creation of the lactone cycle),
which produces a molecule of methylthioadenosine (MTA). The
aliphatic chain (in general short chained) comes from the
biosynthesis of fatty acids, and it is transported by ACP.
The fact to carry an aliphatic chain of a certain length
(but not too long) permits free diffusion of the molecule
through the cell membrane, even in the absence of permease.
This leads to a rapid equilibration of its concentration
between the external medium and the inside of the cell.
When this concentration reaches a threshold value the regulator
effects of "quorum sensing" begin to control a
large number of processes, such as: luminescence (V. fisheri,
or V. harveyi), conjugative transfer of plasmids
(Agrobacterium tumefaciens), collective movement of
bacteria (swarming) (Serratia licfaciens), synthesis
of some reserve molecules (e.g. poly-3-hydroxybutyrate, V. harveyi),
or finally virulence Val, 1998).
The synthesis of ethylene
The gas ethylene (CH2=CH2) is the first multifonctional
gaseous hormone that has been discovered (in 1934, in plants),
well before nitrogen monoxide, curiously now much more famous
Cohen, 1998 #10. Ethylene participates, among other processes,
in fruit ripening, plant aging and/or in the formation of
roots or flowers. The biosynthesis of ethylene derives from
methionine, or more precisely of AdoMet. Ethylene is also
synthesized in bacteria such as E. coli, Pseudomonas
syringae or the fungus Cryptococcus albidus. The
existence of microbes producing ethylene indicates a possible
interference with the plant metabolism during infection of
In microorganisms, there are at least two known pathways
for ethylene biosynthesis. A first one starts with methionine
(as in plants) and the second one uses a-ketoglutarate as
a precursor. In Penicillium digitatum, there exists
yet another precursor, 2-keto-4-methylthiobutyrate (KMBA),
produced by the transamination of methionine (or, as we shall
see, the product of recycling of methylthioribose in K.
pneumoniae). KMBA can be directly converted into ethylene.
The main pathway using AdoMet as a precursor, is composed
of two enzymes. The first one, ACC synthase (pyridoxal 5-phosphate
enzyme, EC 188.8.131.52), converts AdoMet into 1-aminocyclopropane-1-carboxylic
acid (ACC) yielding methylthioadenosine. Subsequently, ACC
oxidase converts ACC into ethylene (figure 7) Cohen, 1998).
Cyanobacteria also produce ethylene, but its role is not
well understood. For example, in Fremyella displosiphon,
a photoreceptor protein which belongs to the family of two-component
regulators is homologous to a gene governing the reponse
to ethylene in Arabidopsis thaliana Kehoe, 1996 #176.
The authors speculate that this protein may be involved in
the control of cellular processes regulated by light. It
is possible that the receptors of ethylene and the phytochromes
(red/infrared receptors) evolved from a common ancestor that
was sensitive to the regulation by two stimuli: light and
ethylene. We cannot exclude their possible role as mediators
of differentiation in higher cyanobacteria, especially because
there exists in these organisms an ethylene receptor binding
protein homologous to that of Arabidopsis thaliana that
seems to be functional (ethylene response sensor protein
(ETR1) Rodriguez, 1999).
Synthesis of biotin
A step of biotin synthesis (bioA) uses AdoMet not
as a source of methyl group, but as a source of amino group,
yielding as a by-product of the reaction S-adenosyl-4-methylthio-2-oxobutanoate.
This molecule, that is similar to a precursor of ethylene
synthesis, must therefore be recycled. This step is not known.
Modification of tRNA
Transfer RNAs are known to have a large number of modifications
in their bases, some of which as frozen traces of ancestral
1989). The most recent works have identified 79 modifications
in enterobacteria Bjork, 1999 #246. One of these modifications
results from transfer of the 3-amino-3-carboxypropyl group
of S-adenosylmethionine Nishimura, 1974). This modification
concerns phenylalanine tRNA and is on the uridine of the
supplementary loop m7G-X-C, where X represents 3-(3-amino-3-carboxypropyl)-uridine.
As a product of the reaction, apart from the modified nucleoside,
one obtains methylthioadenosine.
The synthesis of spermidine
The last point that we shall consider here is the transfer
of the aminopropyl group of S-adenosylmethionine in
the synthesis of polyamines. This reaction is of significant
biological importance, despite the fact that, from a purely
quantitative point of view, transmethylations are more significant.
However, in the case of methylations, sulfur is immediaty
recycled as homocysteine, which is not the case in polyamine
biosynthesis. In this case, AdoMet is first decarboxyled
by S-adenosylmethionine decarboxylase (EC 184.108.40.206)
Cohen, 1998 #10. Subsequently, its aminopropyl group is transfered
by spermidine synthase (aminopropyltransferase, EC 220.127.116.11)
on putrescine to give a molecule of spermidine and a molecule
of methylthioadenosine (see figure 6). This reaction, that
traps sulfur in a little studied molecule, is a universal
reaction, present in all living organisms, with the exception
of a few halophilic microorganisms that do not possess spermidine.
In eucaryotes and some procaryotes, this aminopropyl group
can be subsequently transfered onto spermidine to give spermine.
This reaction is catalysed by an enzyme very similar to spermidine
synthases (spermine synthase, EC 18.104.22.168), with production
of a molecule of methylthioadenosine, the fate of which will
be considered below. Because of its availability, one may
wonder about the possible existence of aminopropyl group
transfers to other molecules: in principle one may expect
that all the substrates of methylation, and in particular
numerous proteins could be modified by this group. This may
explain that analysis of bacterial proteome displays modified
polypeptides in proportions much larger than expected.
Transfer of the ribosyl group
Queuosine (Q) [7-(((4,5-ciS-dihydroxy-2-cyclopenten-1-yl)amino)methyl)-7-deaza-guanosine],
is a nucleoside that takes the place of guanosine at position
34 of some transfer RNAs (those of asparagine, aspartate,
histidine and tyrosine) in Bacteria and in Eukarya, with
the exception of archaebacteria and yeast. Bacteria synthesize
the base queuine de novo, whereas eucaryotes
must insert this base directly into the tRNA from a precursor
obtained from food. For a few years only, one knows that
this nucleoside is a derivative of S-adenosylmethionine
Slany, 1993 #238. It is an extremely interesting case because
AdoMet acts here as a donor of the ribosyl group. This is,
as yet, the only known example of this reaction. Only two
steps of the biosynthesis pathway of queuosine are well characterized.
This pathway starts with GTP, that is converted by a unknown
mechanism, requiring iron, into 7-(aminomethyl)7-deaza-guanine
(preQ1). PreQ1 is then inserted at position 1 of the anticodon
of some tRNAs in exchange of guanine by a tRNA-guanine transglycosylase
(EC 22.214.171.124, encoded by gene tgt). The preQ1 present
in these tRNAs is converted into epoxyqueuosine (oQ) by attachement
of the epoxycyclopentanediol group. This reaction is catalysed
by S-adenosylmethionine:tRNA ribosyltransferase-isomerase
(EC 5.-.-.-, encoded by gene queA). The cyclopentane
group is the isomerized derivative of ribose coming from
AdoMet. Nobody knows the outcome of adenine during this reaction.
In fact, because we do not have AdoMet labeled on ribose,
it is difficult to determine whether it is the ribosyl group
alone or the adenosyl group that is used. The last step of
reduction of epoxyqueuosine into queuosine is catalysed by
an unidentified enzyme using vitamin B12 as a cofactor (when
present). Finally, for reasons that are not quite understood,
queuosine or its derivatives is excreted at high levels in
the medium. This may be used for quorum sensing and/or for
interaction with the hosts.
It seems interesting to stress that all the types of activated
groups in AdoMet are used to provide modifications of transfer
RNA. The methyl group is most often transfered, for example
to form ribothymidine. The 3-amino-3-carboxypropyl group
is used to give 3-(3-amino-3-carboxypropyl)-uridine and the
ribosyl (or possibly adenosyl) group are involved in the
formation of queuosine.
AdoMet also acts as a transient donor of the oxyadenosyl
radical in the synthesis of L-b-lysine from L-lysine (reaction
catalysed by a lysine 2,3-aminomutase) in anaerobic conditions
in Closthree-dium subterminale Lieder, 1998 #270.
In the same conditions, AdoMet plays a role in E. coli in
the synthesis of deoxyribonucleotides, reaction catalysed
by a ribonucleotide reductase (EC 126.96.36.199, coded by genes nrdD and nrdG)
Ollagnier, 1997 #274. One sees therefore that the metabolism
of this molecule is quite complicated
Recycling of methylthioadenosine: the methionine salvage
AdoMet can also be decarboxyled as dAdoMet, the precursor
of spermidine. During this reaction methylthioadenosine (MTA)
is produced (figure 8). Many bacteria [13, 33], yeast
, plants , rat  and human  utilize the methionine
salvage pathway recycling MTA to methioine. Organic sulfur
being often limiting, this molecule must be recycled in most
cases. Several studies in bacteria related to E. coli, K. pneumoniae,
suggest the metabolic pathway indicated
The pathway has now been completely unravelled in B.
subtilis, and in several other bacteria, including Pseudomonas
aeruginosa. It remains however not completely understood
in several organisms, such as Thermoanaerobacter tengcongensis.
In a first step in E. coli, MTA is hydrolysed
by a nucleosidase (EC 188.8.131.52, coded by gene pfs,
now mtnN), yielding methylthioribose (MTR) and adenine
Cornell, 1996 #273; Cornell, 1998 #272. In B. subtilis the
corresponding gene yrrU, is present in a complex operon yrrTmtnNyrhABC,
that appears to metabolise sulfur containing molecules Sekowska,
1999). In contrast, in animals and their parasites, MTA is
phosphorolysed, giving methylthioribose-1-phosphate and adenine.
In the case where sulfur is not limiting it happens that
MTR is the final product excreted into the medium. This seems
to be the case in E. coli (Schroeder, 1973).
In order to harmonize nomencalture pfs and yrrU should
perhaps be renamed mtnN, for methylthioadenosine nucleosidase
(reserving mtnP for the phosphorylase present in several
Bacteria, Archaea and Eukarya).
However, MTR is usually recycled (figure
9). In the presence of oxygen, the pathway
deciphered in K. pneumoniae begins with phosphorylation
of MTR by a MTR kinase, that uses ATP as phosphate donor
and produces MTR-1-phosphate (Wray, 1995). MTR-1-P is
transformed into methylthioribulose-1-phosphate (MTRu-1-P)
by an aldose-ketose isomerase. MTRu-1-P is subsequently
dehydrated (and converted into a ketone) by a not yet
identified dehydratase, to give 2,3-diketo-5-methylthiopentyl-1-phosphate
(DK-MTP-1-P). This latter molecule is first converted
into phosphoene-diol (DHK-MTPene) via the intermediate,
to give 1,2-dihydroxy-3-keto-5-methylthiopentene (aci-reductone).
These two steps are catalysed by a bi-functional enzyme,
enolase-phosphatase E-1. In K. pneumoniae,
1,2-dihydroxy-3-keto-5-methylthiopentene can be processed
in two different ways, by hte action of a dioxygenase
E-2 (aci-reductone oxidase) either into formate, 3-methylthiopropionate
and CO (Wray, 1993), or, depending on the nature of the
transition ion bound to the enzyme (either nickel or
iron), into formate and a-keto-4-methylthiobutyrate (KMTB),
the direct ketoacid precursor of methionine. Only the
second pathway permits the direct way back to methionine
thanks to the transamination of KMTB by an original tyrosine
aminotransferase (TyrAT), that utilises as an amino group
donor glutamate or aromatic amino-acids Heilbronn, 1999)
9). It must be stressed that the carbon
atoms 2, 3, 4 and 5 of ribose are found again in the
methionine backbone, and that the methyl group carried
by sulfur is not exchanged: this recycling process therefore
does not require a source of one-carbon residues, but,
in contrast, it produces one (in the form of formaldehyde
or formate). This fact is quite remarkable (figure
9). Indeed, this production of one-carbon
residues in the form of a chain reaction is typically
explosive. One expects therefore that methionine metabolism
will be particularly stringently regulated to control
these explosive reactions. In this context, the storage
in the form of S-methylmethionine may be a way
to modify rapidy the availabilty of methyl groups.
At this stage, experimental work was needed to identify
all the biochemical steps of the pathway. A remarkable work
by Yokota and his colleagues established the following, in B.
subtilis (). The mtnW (ykrW) gene
coding for a RuBisCO-like Protein (RLP) is the first gene
in the mtnWXBD operon, located close to the mtnKA operon
(Sekowska and Danchin, 2002). These operons have S-box riboswitches
that regulate the expression of the genes involved in sulfur
metabolism in B. subtilis and other A+T-rich Gram
positives. MtnD is highly homologous to the 1,2-dihydroxy-3-keto-5-methylthiopentene
dioxygenase and MtnK was identified as the methylthioribose
kinase (Sekowsha et al., 2001). This suggested that both
operons were functioning in the methionine salvage pathway
9). Therefore Yokota et al. first predicted
and proved that B. subtilis MtnA, rather than coding
for a translation initiation factor, coded for the first
step of MTA recycling; RLP would catalyze a reaction step
somewhere in this pathway. Each step of this pathway has
been predicted in Klebsiella sp. by analysis of
metabolic intermediates but two enzymes, MTR-1-P isomerase
and MTRu-1-P dehydratase, were unknown. Moreover, each reaction
step in this pathway was uncharacterized in B. subtilis,
except for that catalyzed by MTA nucleosidase, MTR kinase
and KMTB aminotransferase encoded by mtnN, mtnK and mtnE,
respectively. This fact suggested to them that RLP functions
as a DK-MTP-1-P enolase in the methionine salvage pathway
in Bacillus species. One must remark here the special role
played by dioxygenase E-2 (that gives an interesting idea
of the general possible function of dioxygenases). Indeed,
this enzyme directly consumes dioxygen, O2, and may therefore
be utilised to prevent the toxic effects of oxygen (more
numerous than we can summarize here, see for example Gardner,
1998). In such a case, the production of MTA (MTR) may be
considered as a means (perhaps existing very early in evolution,
before the evolving of respiration) to protect the cell against
the toxic effects of oxygen. This may indirectly account
for the antioxidizing properties of polyamines coupled with
their synthesis Lovaas, 1991; Pavlovic, 1992).
Special metabolism of sulfur
We just broadly outlined the general metabolism of sulfur,
but it still exists a series of more or less unknown metabolic
pathways where sulfur is involved in some way. Sulfur is
an element present in molecules as glutathione. It is also
a constituent of iron-sulfur centers or of essential coenzymes
such as thiamine, biotin, molybdopterin, pantothenate and
lipoic acid. It also belongs to the many modifications of
the nucleotides that constitute transfer RNA, via reactions
that we just begin to understand.
We begin with this cofactor because it is directy associated
to cysteine metabolism. Its transfer onto spermidine probably
leads to storage of the latter in stationary growth phase
(Cohen, 1998). In E. coli, gene gsp is
involved in this transfer. The corresponding protein, GSP,
is a bifunctional protein that catalyses the formation of
glutathionyl-spermidine (glutathionyl-spermidine synthase,
EC 184.108.40.206), but also its hydrolysis (glutathionyl-spermidine
amidohydrolase, EC 220.127.116.11). The two catalytic activities
lie in two distinct parts of the protein, the amidase function
is located in the amino-terminal part, whereas the synthetase
function is located in the carboxy-terminal part (Swiss-Prot:
P43675). There exists also another protein, glutathione S-transferase
or GST (EC 18.104.22.168, encoded by gene gst), that tranfers
glutathione to several kinds of molecules.
Glutathione is an isotripeptide, very frequently found in
cells. It is derived from the condensation of cysteine on
the g-terminal COOH of glutamate. A glycine residue is subsequently
added by formation of an usual peptidic bond. Glutathione
is used to recycle the sulfide groups that can be spontaneously
oxidized, following electron transfer due to catalysis or
free radicals, or other oxidation phenomena. In the course
of this process, two molecules of glutathione form a disulfide
bridge that is thus reduced by glutathione oxidoreductase.
In E. coli, the genes involved are the following:
• gene gor codes for glutathione oxidoreductase;
it is in operon with an unknown gene yhiR.
• gene gshA codes for g-glutamyl-cysteine
synthetase; it lies in a likely operon yqaBAgshAygaG (YqaB
and YqaA may be phosphatases, similar to the phosphatase
of the HPr protein of B. subtilis).
• gene gshB codes for glutathione
synthetase; it lies in a likely operon gshByqgEyqgF (YqgE
and YqgF display some analogy with phosphotransferases).
These unexpected associations suggest an interesting track
to explore: is the biosynthesis of glutathione controled
by protein phosphorylation?
The iron-sulfur centers
Sulfur is also an element of iron-sulfur centers (minute
analogues of iron pyrite) that are involved in a large number
of oxido-reductions, and constitute the core of ferredoxins.
In the history of the origin of life, hypotheses become more
and more convincing that the electron transfers between Fe2+
iron and Fe3+ iron and the numerous possible oxidation states
of sulfur play a role of prime importance (Granick, 1957;
Wächtershäuser, 1988; Danchin,
1989). It is therefore
interesting to wonder how the iron-sulfur centers are formed
in the cell.
One knows very little about the formation of elemental sulfur,
apart from conditions where it acts in the electron transfers
allowing anaerobic respiration (Ehrlich, 1996). It is very
unlikely that it is sulfur in this oxidation state that is
incorporated in the diverse types of centers (Fe-S)n. A possible
start point seems to be cystine, possibly by directly coupling
a cysteine to a cysteine present in the protein that carries
the iron-sulfur center, using a hydrogenase. A beta-lyase,
analogous to cystathionine b-lyase may then provide the thiocysteine
that serves as donor of sulfur atom. But it is the study
of protein NifS in Azotobacter vinelandii that brings
most information on a possible mechanism. This protein is
a cysteine sulfurylase that, from thiocysteine, liberates
sulfur, yielding cysteine (Zheng, 1998). Several enzymes
homologous to the product of gene nifS of A. vinelandi existent
in E. coli. One of those seems to code for a
cysteine sulfinate sulfinase (Mihara, 1997). The existence
of these reactions may provide tracks to assign a function
to some genes labeled "y".
Much work in years 2000-2010 has unraveled the pathway of
formation of iron-sulfur clusters, starting from a cysteine
desulfurase (IscS and related enzymes) that extracts the
sulfur atom of cysteine to make alanine and put it on a carrier
cysteine sulfur, making a persulfide). "Several
IscS-interacting partners including IscU, a scaffold for
Fe-S cluster assembly; TusA, the first member of a sulfur
to sulfur incorporation into the wobble uridine of several
tRNAs; ThiI, involved in tRNA modification and thiamine
biosynthesis; and rhodanese RhdA are sulfur acceptors. Other
proteins, such as CyaY/frataxin and IscX, also bind to IscS,
their functional roles are not directly related to sulfur
transfer" (Shi et al, 2010).
Thiamine is an essential cofactor comprising a sulfur heterocycle.
Its biosynthesis needs two separated pathways. The first
synthesizes the thiazole group (5-methyl-4-(-hydroxyethyl)thiazole),
and the second a pyrimidine (4-amino-5-hydroxymethylpyrimidine
pyrophosphate) (figure 10). These two molecules are combined
to yield thiamine pyrophosphate. The pyrimidine moiety is
derived from 5-aminoimidazole ribotide. Thiazole is derived
from tyrosine, cysteine, and 1-oxy-D-xylulose-5-phosphate.
The enzymatic mechanisms involved in the formation of thiazole
and pyrimidine remain poorly understood. In particular a
synthesis pathway for pyrimidines, related to that of purines,
but that does not stem from normal ammonium transfer on PrPP,
seems to exist. This pathway involves several genes of unknown
function such as apbA, apbE or ybgF (the
homologous gene in B. subtilis is yabC).
Their inactivation makes the mutants particularly sensitive
An operon of five genes (thiCEFGH) involved in the
biosynthesis of thiamine has been cloned and characterized
(Table 2 and figure 10).
Gene thiC complements mutants auxotrophic for 4-amino-5-hydroxymethylpyrimidine,
genes thiFGH complement mutants auxotrophic for thiazole,
and gene thiE codes for thiamine phosphate synthase.
Another gene (thiI), necessary for the biosynthesis
of thiazole in Salmonella typhimurium has recently
been identified (Webb, 1997). A gene homologous to thiI of S. typhimurium is
located at 9.5 min in the chromosome of E. coli and
complements a mutation nuvC. NuvC is necessary for
the synthesis of the thiouridine present in tRNA and of the
thiazole moiety of thiamine. This suggests that ThiI plays
a role in the chemistry of sulfur transfer in the biosynthesis
A new observation recently completed this pathway. When
the product of gene thiG, purified from a strain that
expressed it, has been analysed by mass spectrometry, it
appeared that it was in fact made of two proteins. A subunit,
now named ThiS, had a mass of 7310.74 Da. The second
subunit, for which one keeps the name ThiG, had a mass of
26896.5 Da. In fact, although the mass anomaly of ThiG
had been noted before, thiS had escaped attention
during the identification of CDSs in the E. coli genome.
This was caused in part by the short length of the corresponding
CDS, and by the presence of sequence errors (quoted in Taylor,
1998). To this difficulties we may add that ThiS is poorly
coloured with Coomassie blue and migrates with the front
in the polyacrylamide gels. ThiS contains the -Gly-Gly sequence
found at the carboxy-terminal end of ubiquitinylated human
red cells. This similarity in sequence was more remarkable
because ThiF is similar to an enzyme that activates ubiquitine.
The similarity includes the presence of an ATP binding site.
This suggested that ThiF may well catalyse adenylation of
ThiS, and that the adenylate derivative ThiS-CO-AMP
may react with cysteine (or a sulfur donor derived from cysteine)
to yield the thiocarboxylate derivative ThiS-COSH
(figure 11 B). Furthermore, the double role of ThiI
in the biosynthesis of thiazole and 4-thiouridine indicated
that ThiI may play a role in the reaction of sulfur transfer.
This is what has been proposed by Taylor and his colleagues
(figure 11 Taylor, 1998).
The enzymatic mechanisms of sulfur transfer in the biosynthesis
of thiamine, molybdopterin, biotin, and lipoic acid remain
however poorly known.
Thiamine is involved in the anabolic processes of decarboxylation
(linked to dehydrogenation), where two cofactors operate
successively, thiamine pyrophosphate and lipoic acid. Lipoate
is synthesized by insertion of sulfur coming from cysteine
into the hydrocarbon chain of octanoic acid (an intermediary
in the biosynthesis of fatty acids) at position C-6 and C-8.
This reaction is probably performed by a mechanism involving
a radical Michal, 1999 #166. The sources of sulfur used for
the formation of biotin and lipoic acid are unknown, and
the mechanism of insertion of sulfur into the protein has
not yet been established. At the end of biosynthesis, a molecule
of lipoate activated by ATP is linked to the e-amino group
of a lysine of the corresponding enzyme to yield the functional
cofactor. Lipoic acid is a cofactor linked covalently to
many dehydrogenases, such as pyruvate dehydrogenase (EC 3.3.1.)
or a-ketoglutarate dehydrogenase (EC 3.8.1.). Most living
organisms synthesize lipoic acid, that must be produced in
situ to be functional, and it seems difficult to recover
from the environment. However, in some bacteria, lipoate
is a growth factor Michal, 1999 #166.
Three genes of the biosynthesis pathway of lipoate have
been identified: lipA (lipoate synthase, Swiss-Prot
P25846), and lplA and lipB (lipoyl-protein
ligases A and B, EC 6.-.-.-, Swiss-Prot P32099 and P30976,
respectively). But it is clear that for understanding the
whole pathway a lot of data are still missing. One possibly
has in E. coli an operon comprising dacA (D-alanine
carboxypeptidase), ybeD (similar to yitC of B. subtilis,
similar to an oxo-acyl CoA carrier protein synthase) and lipB (LipB
links lipoate to the e-amino group of a lysine in a receptor
protein, a reaction performed from a complex and not from
free lipoate), an operon comprising ybeF (YbeF is
an activator of the LysR family, whose effector is unknown)
and lipA whose product may be involved in insertion
of sulfur during lipoate biosynthesis. There exist two proteins
similar to LipA in B. subtilis, YutB (54% identity)
and YqeV (23% identity). YqeV is remarkable because it is
very conserved in all bacteria, but without known function,
with a likely role in the synthesis of lipoic acid (Table
The modifications of transfer RNA
Among the 79 types of transfer RNA modifications that have
been identified, at least 50 of which being present in E.
coli, three or four involve sulfur-containing molecules
In E. coli and related bacteria, the nucleotide
in position 8 in transfer RNA is a modified uridine, 4-thiouridine.
This nucleotide seems to have as its principal role a protective
function against violent irradiations by near ultraviolet
light Mueller, 1998). Indeed, this nucleotide absorbs radiation
at this wavelength and therefore protects the cell. In the
presence of sufficient irradiation at 365 nm, this nucleotide
reacts with a neighbor cytosine producing a covalent bond.
The tRNA molecule thus modified can no longer be charged
by the corresponding amino acid, in most cases. This triggers
the stringent response, whose signal is the ppGpp molecule,
coupling translation to transcription. The macromolecular
syntheses stop and bacteria are shifted to a state that permit
them to better resist this situation. One does not yet know
how the atom of sulfur is incorporated into the nucleotide.
Although mutants controlling this process have been isolated
more than 25 years ago (nuvA and nuvC Lipsett,
1978), the thiI gene involved in this reaction has
only been characterised recently Mueller, 1998). Furthermore,
this gene is involved in the synthesis of thiamine.
There exist several modifications at position 34 (first
position of anticodon) in E. coli. In the case
of tRNAs specific for glutamate, lysine and glutamine, the
modified nucleoside is 5-methylaminomethyl-2-thiouridine
(mnm5s2U34). The reaction of uridine 34 modification comprises
several steps, some of which are poorly known. The first
step of thiolation at position two of uridine (s2U34) is
catalysed by the product of gene mnmA (also known
in the litterature as trmU or asuE). The product
of gene mnmE (trmE) catalyses the first step
of the modification at position five, but one does not know
the steps that lead to the modification into 5-carbonylmethylaminomethyl-2-thiouridine
(cmnm5s2U34). The two modifications at position two and five
are synthesized independently of each other. The product
of gene mnmC (trmC), that catalyses the following
step of the modification at position five, possesses two
enzymatic activities that catalyse successively the reactions
to liberate the acetate group of cmnm5s2U34 (which leads
to nm5s2U34, 5-methylamino-2-thiouridine), and to methylate
the nm5s2U34 in mnm5s2U34 (AdoMet-dependent reaction) Hagervall,
The modification at position 34 participates probably in
the discrimination between codons of the same family that,
varying at their third position, code for different amino
acids (for example lysine is coded by two triplets AAA and
AAG, which can lead to the confusion with asparagine codons
AAU and AAC). However, experience shows that this role is
perhaps not direct, because the mutants partially modified
at position 34 of tRNA seem to be more accurate in translation
(there is less confusion between lysine and asparagine) than
the wild type, with a tRNA containing normally modified uridine.
The authors propose that this modification has a role in
the interactions with the ribosome or with the translation
elongation or termination factors, rather than in the direct
codon discrimination Hagervall, 1998). In this case, the
thiol group present in these modified bases stabilises the
structure of transfer RNA, which is centrally important to
the reconnaissance of tRNA correspondings by their synthetases
and to their charge with their amino acids Kruger, 1998).
At position 32 of some tRNAs of eubacteria, one finds another
modification, 2-thiocytidine, but little is known about the
genes involved in its synthesis and about the biological
signification of this modification Bjork, 1996).
Another modification (2-methylthio-N6-isopentenyl
adenosine (ms2i6A), adds a S-methyl group at position
2 of adenine at position 37 of transfer RNAs specific for
codons beginning with U, with the exception of tRNASer I
and V). It may result from the transfer of the methyl group
of AdoMet, whereas sulfur atom would come from cysteine.
Two genes, miaB and miaC, are necessary for
this reaction. Gene miaA catalyses the first step
of conversion of adenosine into 6-isopentenyl-adenosine Esberg,
1995). The position of these genes in the chromosome of E. coli was
not clearly established until recently. The miaB gene
has been shown to be identical to gene yleA. MiaB
contains a cysteine cluster reminiscent of iron-binding sites.
It is therefore assumed that it participates in the thiolation
step of tRNA Esberg, 1999). In S. typhimurium,
a fourth gene, miaE, oxidizes 2-methylthio-N6-(isopentenyl)-adenosine
(ms2io6A) Persson, 1998).
Some modifications of tRNA are important for cellular metabolism
(in S. typhimurium), or virulence (in Shigella)
Bjork, 1999 #246. Among others, these modifications concern
position 37, such as the modification of adenosine just described
(ms2i6A), or the modification of guanosine catalysed by tRNA(m1G37)methyltransferase,
encoded by gene trmD (1-methyl-guanosine (m1G37)),
or another modification of guanosine at position 34 in queuosine.
The molybdenum coenzyme (molybdopterin)
With the exception of nitrogenase, in all molybdoenzymes,
the atom of molybdenum belongs to an organometallic structure
named the molybdenum cofactor (MoCo). In this structure,
molybdenum is linked to its organic ligand by a dithiolene
group located in the 6-alkyl chain of molybdopterin (MPT).
In E. coli, MoCo is present in the slightly different
form of molybdopterin guanine dinucleotide (the conversion
of MoCo in dinucleotide is placed just before incorporation
of the cofactor in the protein). The biosynthesis of MPT
starts with GTP as a precursor (figure 12). There are several
steps, that comprise the opening of the GTP cycle, lead to
the direct precursor of MPT, precursor Z Wuebbens, 1995).
This latter molecule is a 6-alkylpterine with a phosphate
group at the extremity of the aliphatic chain. This phosphate
group leads to the formation of a six atom heterocycle by
using two carbons of the chain (C-2' and C-4') (figure 12).
Precursor Z contains none of the sulfur atoms present in
MPT. The conversion of precursor Z into molybdopterin necessitates
ring opening of the heterocycle and transfer of sulfur to
create the dithiolene group needed for the chelation of molybdenum.
The transfer of sulfur in the biosynthesis of thiamine and
molybdopterin follows the same course. A system of three
enzymes (MoeB, MoaD, and MoaE) permitting the transfer of
sulfur to precursor Z of molybdopterin has been reconstituted
in E. coli Pitterle, 1993; Pitterle, 1993) and
in Aspergillus nidulans (figure 12) Appleyard, 1998).
The first enzyme, MoeB (also known as ChlN), molybdopterin
synthase sulfurylase, is responsible for the activation of
MoaD by adenylation, as well as the transfer of sulfur to
activated MoaD Appleyard, 1998). MoaD (small subunit) with
MoaE (large subunit) form molybdopterin synthase (known in
the litterature as "converting factor"). This enzyme
possesses two catalytic activities: opening of the cycle
created by the phosphate group and sulfur transfer on precursor
Z. Protein MoeB is very similar to ThiF; MoaD and ThiS comprise
the same Gly-Gly dipeptide at their carboxyl-terminal end.
One can therefore think that sulfur is transfered in the
same way by adenylation followed by formation of thiocarboxylate,
as in the case of the biosynthesis of thiamine Taylor, 1998
(see figure 11).
In E. coli the genes involved in the biosynthesis
of MPT are situated in two operons: moeA (function
unknown) moeB, and moaABCDE. moaABC have
an unknown function (Table 2).
Apart from the metabolism of energy, transamination and
decarboxylation are the two most central steps of intermediary
metabolism. In general, transamination is involved in anabolism,
while decarboxylation is most often a first step of catabolism
(cf. Danchin, 1990 #49). A particular coenzyme, biotin, plays
a central role in many decarboxylation reactions (but also
carboxylations and transcarboxylations). Although generally
essential, this coenzyme is rare in nature. For this reason,
living organisms have developped many systems to capture
and transport biotin. These systems are so powerful that
they have been used as probes in many processes used in molecular
biology. The biotin/(strept)avidine couple is often more
efficient and more specific than the antigen/antibody couples.
One can use biotin and streptavidine to the coupling of a
reaction (molecular hybridization for example) with an enzymatic
amplification system Prescott, 1999).
Biotin is synthesized by bacteria, yeast and plants essentially
in the same way. However, this biosynthesis is not completely
understood, and in particular one does not yet know how sulfur
is incorporated into biotin. It begins probably by the condensation
of three molecules of malonyl-CoA into pimeloyl-CoA with
liberation of two molecules of carbon dioxide. Subsequently,
the condensation of pimeloyl-CoA with alanine (pyridoxal-phosphate-dependent)
leads to the formation of a molecule of 8-amino-7-oxopelargonate
(KAPA) with the liberation of CO2 and CoA-SH. KAPA is then
converted into 7,8-diaminepelargonate (DAPA) by a transamination
reaction. This reaction involves S-adenosylmethionine
as an amino group donor. An ATP-dependent carboxylation leads
to the closing of the imidazolidone cycle and to the formation
of dethiobiotin. The last step, that places sulfur in the
molecule (is still an unidentified mechanism, because it
can neither be sulfur S0, nor an iron-sulfur center, but
probably of sulfur coming from a protein with an iron-sulfur
center, perhaps biotin synthase Bui, 1998 #339, yields biotin
by the closure of the thiophane cycle Michal, 1999). Because
biotin (vitamin H) is an important food supply, its
production is of industrial interest.
In E. coli, we know that the synthesis of biotin
is controled by genes linked to locus galETK (lambda
phage deletions often lead to auxotrophy for biotin), but
all the steps are not yet known. Two operons containing genes
involved in the synthesis of biotin are lying next to each
other in a divergent way. One has therefore possibly an operon
of three genes: bioA (acid diaminepelargonic synthetase), ybhB (unknown
gene that is similar to yxkA of B. subtilis (31%
identity), itself similar to ... bioA) and ybhC (weakly
similar to YdgB of B. subtilis (29% identity),
without known function). The divergent operon comprises four
genes: bioB (coding for biotin synthase), bioF (coding
for 7-keto-8-aminopelargonic acid synthase), bioC (function
unknown, the mutant of this gene is blocked in the formation
of pimeloyl-CoA) and bioD (coding for dethiobiotin
synthase). Gene bioH is located elsewhere in the chromosome,
and its function is not known but the corresponding mutant
is blocked in the formation of pimeloyl-CoA.
Pantothenic acid and coenzyme A
The presence of membranes in the form of a lipid bilayer
is a universal feature of life. Lipids, and especially aliphatic
long chain fatty acids, are ubiquitous constituents of membranes.
To know their biosynthesis is therefore crucial to understand
the metabolism of living organisms. This biosynthesis is
based on the synthesis and degradation of a thioester bond,
which have an ancient origin and probably started before
the origin of life. The mobile arm in these reactions catalysed
by ACP (Acyl Carrier Protein), is 4-phosphopantetheine. In
some reactions this arm is linked to a nucleotide, and forms
coenzymeA (CoA, or "acyl carrier group"). The biosynthetic
mechanism of CoA synthesis is not entirely known, although
the main enzymatic steps have been identified (figure 13).
Thee precursor of panthothenate biosynthesis is 2-oxoisovalerate,
the valine transamination product. 2-oxoisovalerate is methylated
by 5,10-methylene-THF, giving 2-hydropantoate, which is subsequently
reduced into pantoate. An ATP-dependent condensation of the
latter with b-alanine (derived from aspartate a-decarboxylation,
or from pyrimidine degradation) yields (R)-pantothenate.
This latter product, which is strongly excreted by E. coli,
is source of this vitamin for mammals Michal, 1999).
Coenzyme A is synthesized by all organisms from (R)-pantothenate
that is first phosphorylated in (R)-4'-P-N-pantothenate.
Its condensation with cysteine in presence of CTP produces
pyrophosphate, CMP and (R)-4'-P-N-pantothenoyl-cysteine.
Following decarboxylation, one obtains 4'-P-pantetheine.
Phosphopantetheine adenylyltransferase (PPAT), produces dephosphoCoA
Izard, 1999), that is subsequently phosphorylated by an unknown
dephospho-CoA kinase to give the final product, CoA.
The 4-phosphopantetheine group of CoA is transfered on a
serine residue of ACP (apo-ACP), the core protein of the
fatty acids biosynthesis enzymes and also of peptide antibiotics.
The same mechanism operates for the formation of an active
protein EntF, necessary for the syntehsis of siderophores
Gehring, 1998). At this step, there is liberation of 3'5'adenosine
diphosphate (PAP), the same molecule that is liberated during
assimilation of sulfate, and that is hydrolysed in 5'AMP
by the product of gene cysQ. It is therefore important
to notice that PAP creates a possible link between sulfur
assimilation, lipid biosynthesis, iron transport and antibiotic
In E. coli, the few genes involved in the biosynthesis
of pantothenate and of CoA are distributed in several operons
dispersed in the chromosome (Table 2). They are indicated
in figure 13. We have operon panBC where panB codes
for ketopantoate hydroxymethyltransferase, EC 22.214.171.124 and panC coding
for pantothenate synthetase "panthoate activating enzyme",
EC 126.96.36.199. Gene panD codes for aspartate 1-decarboxylase,
EC 188.8.131.52 and coaA codes for pantothenate kinase,
EC 184.108.40.206. The operon kdtAB, where kdtB has
been wrongly annotated in Swiss-Prot, codes for phosphopantetheine
adenyltransferase Izard, 1999). Finally, operon rnc era
recO pdxJ acpS contains acpS that codes for holo-ACP
synthase (EC 220.127.116.11).
Some elements of regulation
Such a complex metabolism requires an important coordination.
Indeed, there exist, in E. coli, at least four
general regulators (without counting specific regulators)
that coordinate sulfur utilisation both in the anabolic and
in the catabolic direction. Three of these regulators, CysB,
MetR and Cbl, are LysR-type activators Greene, 1996; Kredich,
1996; van der Ploeg, 1997). The regulators of this family
(more than one hundred are known) are similar to each other
in a region of about 280 residues, comprising in its amino-terminal
end a DNA binding site, the"helix-turn-helix"
motif Schell, 1993). To these regulators we can add protein
MetJ, that has a totally different structure, with almost
no equivalent among the proteins known to bind DNA Greene,
CysB is a tetramer composed of identical subunits (Mr =
36 kDa), that controls expression of genes involved in the
biosynthesis of cysteine in Gram negative bacteria. This
system comprises many genes associated to sulfate transport,
to its reduction into sulfide and to the formation of cysteine
from serine and acetyl-CoA Kredich, 1996). CysB is a transcriptional
regulator that acts as an activator of transcription. It
is also the repressor of its own synthesis (it is generally
the case of LysR type regulators). The activity of CysB is
modulated by a cofactor, N-acetylserine. As we have
seen, O-acetylserine (the direct precursor of the
synthesis of cysteine) is not stable. By internal cyclisation
and transfer of the acetyl group, O-acetylserine leads
to a more stable molecule, N-acetylserine. It is this
molecule that plays the role of probe for the available level
of O-acetylserine in the cell, and regulates the cysteine
As in the case of many promotors functioning in the presence
of an activator, the promotors of genes under the control
of CysB possess a "-35" region that differs from
the consensus sequence of sigma factor 70 (TTGACA). The activation
promotors of this type necessitate the fixation of the activator
upstream from region
"-35", which facilitates the formation of transcription
initiation complexe Kredich, 1992). It is difficult to predict
the interaction sites of LysR type regulators. Their experimental
identification Schell, 1993 leads several authors to describe
a consensus binding sequence for CysB to DNA Hryniewicz,
1995). But because of its length and its small number of
conserved residues TTANTNcNNtTNNNNNTNN and NNATNNNNAaNCNNTNNNT
a consensus of this type is probably without much significance.
One must also remark that, curiously, the authors have never
made a statistical study of the corresponding sequence. As
we can see, the usual search for consensus sequences (generally
disputable Hénaut, 1996) seems therefore particulary
ill suited to proteins of this type and to their operators.
The underlying molecular mechanisms were not really understood
until recently. One often remarked that the binding sites
of these activator proteins apparently occur in strongly
curved regions of DNA. Indeed, one finds CysB in the control
of other responses where the supercoiling and the curvature
of DNA have been noticed, such as the adaptation to acid
Rowbury, 1997). The analysis in electron microscopy of the
structure of sigma 70 promotors, during transcription initiation,
leads to a remarkable observation. It seems established that
DNA circles completely the molecule of RNA polymerase, forming
a superhelix with a very small diameter (10 nm), which involves
a strong DNA curvature at the promotor, placing the -70 region
and the +24 region of promotor in close vicinity Rivetti,
1999). One therefore understands that the stabilisation of
curved structures could help transcription initiation. One
must also stress that there exists a phenomenon of winding
of the double helix, that can play an important role. This
is illustrated by the promotors controlled in B. subtilis by
factor Mta (Multidrug transporter activation), a member of
the family of transcriptional regulators MerR, which have
regions -10 and -35 spaced by 19 base pairs instead of 17
et al., 1999). One does not know yet, in the case of
CysB, the relative contributions of the bending and torsion
of the promotors that it regulates.
In the same way as we do not know their operators, it is
difficult to identify the effector molecules for LysR type
regulators. One has identified in vitro an inducer
of CysB, but sulfide and thiosulfate inverse the effect of N-acetylserine
on its binding with the promotors of regulon cys.
They inhibit transcription initiation by exerting an anti-inducer
role on CysB. The absence of physico-chemical kinship between
these diverse molecules pose the question of identification
of molecules that bind to CysB.
The CysB protein has been crystallised, and its three-dimensional
structure is known. The crystal has been obtained from a
solution saturated in ammonium sulfate, then passed through
several steps of sieving and purification in solutions containing
no sulfate. However, in the crystal, a sulfate ion rests
at the core of the protein. The authors of this work did
not expect to find this ion there, although one could naturally
expect its presence as regulator of this metabolic pathway
Tyrrell, 1997). The sulfate ion is likely to be placed at
the binding site for N-acetylserine, that seems be
the true inducer of the pathway Lynch, 1994). One can therefore
wonder whether sulfate does not play an antagonist role to N-acetylserine,
for example to repress the pathway for sulfate transport.
Another possibility is that the binding site of sulfate is
the natural place of thiosulfate, that although structurally
similar to sulfate, is an anti-inducer of regulon cys Tyrrell,
1997). Finally, this observation of the presence of sulfate
in the inducer binding cavity is very interesting because
of the very strong similarity of the three-dimensional structure
of proteins of the LysR family (CysB in particular) with
periplasmic proteins binding sulfate Tyrrell, 1997). It remains
however to be proven that this is really a biologically significant
phenomenon. This is the more so if one remarks that CysB
comprises a sequence (YVRLGLGVGVIASMAVD) which is remarkably
similar to that of the consensus sequence of several AdoMet
binding proteins [AMLIVF] [AMLIVF] [DE] [AMLIVF] G X G X
G X [AMLIVF] X X X [AMLIVF] [AMLIVF] [DENQRKHST] (Wu, 1992, Sekowska
et al. 2000), suggesting that AdoMet could also modulate
its activity. In B. subtilis the corresponding regulation
is indeed performed by AdoMet, but on S-box riboswitches
The regulator Cbl is an activator of operon tauABCD and
other genes belonging to the SSI class "sulfate
in cooperation with CysB van der Ploeg, 1997). One knows
little on the operators recognized or on the effectors involved.
As for the pathway of cysteine biosynthesis, the pathway
of methionine biosynthesis possesses also its regulators.
MetR is an activator of expression of genes glyA, metE and metH and
probably also of gene metF Cowan, 1993). The activation
by MetR necessitates the presence of homocysteine as
coactivator in the case of glyA and metE or
as co-repressor (in the case of metH) Plamann,
1989), the mechanisms of activation by this activator
are not understood.
It remains finally an original regulator, MetJ, that controls
the synthesis of methionine in reponse to AdoMet (for a review
see Greene, 1996). MetJ belongs to the small class of transcriptional
regulators that bind DNA by a motif "ribbon-helix-helix"
(RHH) and not with "helix-turn-helix" as most regulators
do in bacteria. This family contains, in addition to MetJ,
the two repressors Arc and Mnt of bacteriophage P22 of S. typhimurium He,
1992). The regulators of the HU family of B. stearothermophilus also
belong to this class Vis, 1994). Protein MetJ is a dimer
composed of two identical subunits (Mr = 12 kDa), that regulate
expression of all genes involved in the biosynthesis of methionine
(with the exception of metH) and of gene metK coding
for SAM-synthase Greene, 1996). It also regulates its own
expression Saint-Girons, 1984). MetJ binds upstream sequences
of genes that have two to five repeats of the eight nucleotides
'AGACGTCT', the "methionine box". Each methionine
box acts as a recognition site for a molecule of repressor.
However, one needs at least two boxes to bind a molecule
of repressor. The number of repeats (and their similarity
with the consensus) determined the number of repressor molecules
that will bind, and therefore the level of repression. MetJ
is an aporepressor, that for its activity needs a co-repressor,
AdoMet, whose affinity for the aporepressor is weak (Kd =
200 mM). The dissociation constant of the repressor-operator
complex is of 1 nM in the presence of AdoMet and 10-fold
less in its absence Saint-Girons, 1986).
As we can see, the regulation of sulfur utilisation and
the corresponding enzyme activities involve various and complicated
mechanisms that are not always well understood. This regulation
is linked to the methylation potential of the cell (it is
the role of AdoMet) and possibly also to its oxido-reduction
potential. The very nature of sulfur leads us to investigate
the processes that permit the cell to manage its many oxidation
The situation in many organisms, and in particular in E. coli,
is complicated because the metabolism of sulfur is associated
to that of another atom, with a particularly enigmatic role,
Selenium and sulfur
Selenium is an element both indispensible and very toxic
to living organisms. Its "normal" intracellular
concentration is only ten-fold lower than the concentration
for which selenium becomes toxic. This implies that the cell
must manage the transport and the mechanisms of detoxification
of this element. Selenium, that is very similar to sulfur,
is the element immediately below sulfur in the same series
of Mendeleieff table. It can therefore very easily take its
place inside biological molecules. Selenium's atomic radius
is however larger than that of sulfur, which means that the
bonds involving selenium atoms are much longer and weaker.
Replacement of sulfur by selenium therefore changes the form
of molecules or the distances between the atoms. Moreover,
the chemical properties of selenocysteine and cysteine are
very different. As a consequence, at physiological pH, the
selenol- group (SeH) of selenocysteine is principally
ionized, whereas the sulfhydryl group(SH) of cysteine
is principally protonated.
It follows that incorporation of selenocysteine in proteins
has a very strong effect on their tertiary structure, and
alters the catalytic activity of residues comprising an atom
of selenium. The chemical differences between selenomethionine
and methionine are not that important, but the sensitivity
to oxygen of the former is however higher. The toxic effects
of selenium are therefore due to the substitution of sulfur,
leading mainly to the formation of selenocysteine.
Selenium is also more easily oxidized than sulfur. As sulfur,
elementary selenium is not soluble. Its oxidized forms (selenate
and selenite) are very soluble and constitute the source
of selenium for the cell. Selenium oxides play in biological
systems the role of a strong oxidizing centre. Selenite is
an extremely toxic compound because it reacts with the sulfhydryl
groups of glutathione (GSH) or of cysteine, producing molecules
of the type RSSeSR or RSSR and Se0. The reaction
of selenite with thiols produces also H2O2 and O2-, participating
to its toxicity and giving it a mutagenic role Kramer, 1988).
Because of the significant chemical similarity between sulfur
and selenium, many organisms do not know how to discriminate
between these two elements. Selenium enters in the pathway
of sulfur assimilation, and it is non-specifically integrated
in various molecules, that normally contain sulfur. The proportion
of sulfur replacement by selenium depends on the intracellular
concentration of these two elements, but also on the affinity
of enzymes involved in these sulfur assimilation pathways,
vis-a-vis substrates containing sulfur or selenium.
However, there exist systems permitting biological discrimination
between sulfur and selenium. A first example is specific
insertion of selenocysteine in some proteins, in the presence
of codon UGA in the corresponding messenger RNA. Thus, selenocysteine
plays the role of twenty first amino acid of the genetic
code. The synthesis of selenocysteine is separated from sulfur
metabolism. It begins with a transfer RNA carrying an activated
serine. In this case, selenium stems from monoselenophosphate,
a molecule synthesized by selenophosphate synthase (product
of gene selD) from selenide (HSe-) and ATP Lacourciere,
1998). Monoselenophosphate is also the selenium donor for
the conversion of 2-thiouridine into 2-selenouridine in some
tRNAs. The selenoproteins have an important role in the anaerobic
metabolism of E. coli (for example the formate
hydrogenase Axley, 1990).
A second example of biological discrimination between sulfur
and selenium exists in plants. It is useful to understand
what happens in bacteria. It is manifested by greater tolerance
to selenium, which is thus accumulated in a large number
of organic molecules (Se-methylselenocysteine, g-glutamyl-Se-methylselenocysteine
or selenocystathionine). Most plants resistant to selenium
belong to the genus Astragalus (Fabaceae).
They are characterised by: (i) a strong accumulation of selenium
in the form of Se-methylselenocysteine, (ii) enhancement
of selenium tolerance, and (iii) a strong reduction of selenium
incorporation into proteins Neuhierl, 1999). Until very recently,
the mechanisms of resistance of plants to selenium, were
not clearly established. One supposed that this resistance
was linked to the presence of enzymes that, in recruiting
storage metabolites containing sulfur, they transformed them
into selenium derivatives that could no longer be incorporated
into proteins. Indeed, a methyltransferase specific for selenocysteine
(selenocysteine methyltransferase) that uses S-methylmethionine
as donor of methyl group has been purified in Astragalus
bisulcatus (species tolerant to selenium) Neuhierl, 1999).
In A. bisulcatus, selenium is metabolized by
the sulfur assimilation pathway, which leads to the formation
of selenocysteine, as primary compound. Selenocysteine is
subsequently methylated very efficiently by selenocysteine
methyltransferase, which prevents incorporation of selenocysteine
in these proteins or others molecules containing sulfur.
It is probable that this compound is subsequently discarded
and/or stored in vacuoles, but this remains to be seen.
In E. coli, there exists a homologue of selenocysteine
methyltransferase, homocysteine methyltransferase encoded
by gene mmuA. This enzyme presents only a slight preference
for selenohomocysteine as compared to homocysteine, whereas
the enzyme from A. bisulcatus is specific for
selenohomocysteine or selenocysteine. It is very likely that
the two enzymes are related, and that the enzyme involved
in detoxification has evolved from an ancestral protein which
did not discriminate between sulfur and selenium.
The specificity for S-methylmethionine (and probably
also for the S(+) isomer of S-adenosylmethionine)
is unusual for methyltransferases. However, the two methyltransferases
described above use S-methylmethionine as donor of
methyl group, this product being abundant in plants. Furthermore,
both enzymes can use the non-physiological stereoisomer of
AdoMet (a spontaneous product of the racemisation of groups
coordinated to a sulfur atom). In addition to the role of
these enzymes in the detoxification and catabolism of S-methylmethionine,
they may thus have a function in the scavenging of these
molecules Neuhierl, 1999).
Sulfur is an essential component of cells. It has been associated
to life from its very early steps, and it still plays a role
of major importance in life. In E. coli, among
the ca 4,500 genes in the genome, more than one hundred genes
are directly involved in some step of sulfur metabolism.
Analysis of Table 2 suggests that many more genes are probably
related to sulfur in a way or another. Curiously enough,
not much work has been devoted not only to the identification
of many metabolic steps involving sulfur, but also to the
regulation of sulfur availability, disposal or to the metabolic
steps controlled by sulfur containing molecules. We hope
that the present work will be an incentive for further exploration
of this enigmatic domain of gene functions, especially in
the context of genetics of genomes.
We wish to thank Dr Dong-Yan Jin for careful reading and
constructive comments on the manuscript, Drs Philippe Bertin,
Nicolas Glansdorff, Philippe Glaser, Isabelle Martin-Verstraete
and Marc Salomé
for their constructive comments in preliminary stages of
this review, and Dr Chun-Kwun Wun for his constant encouragement.
Pr Art Aronson pointed to us that sulfur metabolism in Gram
positive bacteria has to be investigated in depth, since
it appears that bacteria such as Bacillus subtilis lack
the ubiquitous peptide glutathione.
Figure 1. Sulfate assimilation pathway and cysteine
biosynthesis in Escherichia coli.
Figure 2. Methionine biosynthesis in Escherichia coli.
Figure 3. One-carbon residues cycle. On a souligne in the
figure the cycle leading to thymine synthesis, in which tetrahydrofolate
is not simply recycled as a coenzyme, but used as a substrate
Figure 4. Comparison of cystine and diaminopimelate structure.
Figure 5. Methionine recycling. In the figure are represented
known pathways, presumed pathways, and unknown but likely
pathways. There certainly exist other pathways that we were
not able to imagine. 1 : O-acetylserine sulfhydrylase,
2 : cystathionine g-synthase, 3 : cystathionine
b-lyase, 4 : methionine synthase, 5 : O-acylhomoserine
sulfhydrylase, 6 : SAM synthase, 7 : SAM methyltransferase,
8 : adenosylhomocysteine nucleosidase, 9 : cystathionine
g-lyase, 11 : S-methylmethionine:homocysteine methyltransferase,
12 : enzyme yielding MTA (e.g. spermidine synthase),
13 : pathway for MTA recycling (see figure 9), 14 :
methionine aminotransferase, 15 : KMBA decarboxylase,
16 : 3-methylpropionate lyase, 17 : methanethiol
dioxygenase, 18 : methionine g-lyase (this enzyme donne,
in addition to methanethiol, ammonium and 2-ketobutyrate),
19 : sulfate assimilation pathway (see figure 1).
Figure 6. Polyamines biosynthesis in Escherichia coli.
Figure 7. Biosynthesis pathway for ethylene in plants.
Figure 8. Structure of the S-adenosylmethionine derivatives.
Figure 9.Methylthioribose recycling in Klebsiella pneumoniae.
Figure 10 Taylor, 1998 #207: Pathway generally admitted
for the synthesis of thiamine in E. coli.
Figure 11 Taylor, 1998 #207: Activation of ubiquitin ThiS
and mechanism of formation of ThiS-COSH. A. Reactions
catalysed by enzyme activation of ubiquitin. One shows only
the COOH-terminal end of the protein and the thiol group
of cysteine in the active site of the enzyme activating ubiquitin.
B. Sulfur transfer in the biosynthesis of thiazole.
Figure 12 Appleyard, 1998 #288: Structure of molecules and
metabolic pathway for the synthesis of the molybdenum cofactor
in Aspergillus nidulans.
At the beginning of the metabolic pathway, a derivative
of guanosine is converted into precursor Z. Sulfur is subsequently
added to precursor Z to form the thiolene active group.
Figure 13. Coenzyme A and Acyl Carrier Protein biosynthesis
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