I call any geometrical figure or group of point chiral, and say that it has chirality if its image in a plane mirror idealy realised, cannot be brought to coincide with itself

Conference on molecular dynamics, 1884

Table of Contents

The arsenic failure

Metabolic studies
European collaborations

Sulfur metabolism in Bacteria, with emphasis on Escherichia coli and Bacillus subtilis


An earlier presentation of the views proposed here has been published in year 2000 in Hong Kong and should be used as a reference for work published previous to that year.

Sulfur is an ubiquitous element of the Earth crust where it is mostly present as sulfate salts. Far from dioxygen it may be present in reduced metal-binding forms, such a pyrite (fool's gold) in rocks. It is an essential component of life. Most recent scenarios depicting the Origin of Life place sulfur at a crucial position. In biological processes the role of sulfur is limited to a series of highly specific objects, in particular involved in electron transfers. The presence of gaseous dioxygen has further enhanced the versatility of the processes where this atom is involved. This is probably due to the fact that it is a very reactive atom, and that many chemical reactions involving sulfur consume a large quantity of energy. It is therefore of prime importance to understand sulfur metabolism in model organisms and then extend the corresponding knowledge to other ecological niches. The various inorganic states of sulfur have been well studied, and the corresponding knowledge is fundamental for understanding mineralogy and soil biology (for a review see Ehrlich, 1996). In contrast, the organic cycle of sulfur is much less well known, and metabolism of sulfur, in spite of its central importance (but perhaps because of its difficult chemistry), has been relatively neglected by investigators. For this reason, many steps in sulfur metabolism have been mistakenly ascribed wrong functions that still plague genome annotations.

In 2010 was published a surprizing article that proposed arsenic as replacing phosphorus in the backbone of nucleic acids. A straightforward analysis of the chemical situation, based on reasoning that is useful to understand the core or sulfur metabolism, would have told scientists and educated persons that this is certainly not possible. The arsenic nightmare tells us that we should be very careful to base our knowledge of the previous knowledge accumulated over the years by scientists all over the world, and not to reinvent a crippled wheel...

A. General sulfur metabolism

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 provided direct information about the genes and genomes of the model organisms Escherichia coli and Bacillus subtilis. Other databases maintained within the GenoChore suite allowed one to have access to further information on a variety of bacteria.


Sulfur distribution On average, the sulfur concentration at the surface of the Earth is estimated to be of the order of 520 ppm. It varies in rocks between 270 and 2400 ppm. In fresh water, it is 3.7 ppm on average. In sea water, it reaches 905 ppm. In temperate regions, it varies between 100 and 1500 ppm in soil (Ehrlich, 1996). However, its concentration in plants is usually low. This element is present essentially in the form of the amino acids cysteine and methionine, their oxidation products, as well as molecules of reserve or osmoprotectants, such as S-methylmethionine (Kocsis et al., 1998) or various types of sulfonates (Cook & Denger, 2001; Kertesz & Wietek, 2001). It is also found in many derivatives of secondary metabolism (in particular in garlic-related plants, Atmaca, 2004), where these sulfur-containing metabolites play a very efficient antimicrobial role), and as sulfated carbohydrates or aminoglycosides (Chai et al. 2004; Nazarenko et al., 2003).

Oxido-reduction and assimilation of sulfur In the presence of oxygen, sulfur metabolism is particularly energy costly. As sulfate, it must first permeate the cell, usually against the intracellular electric potential which is usually strongly negative (-70 mV), then change from a highly oxidized state to a reduced state. This requires a significant consumption of energy, as well as the maintenance of a very low oxido-reduction potential, a proces that seems difficult to achieve with the simultaneous presence of oxygen molecules (Table 1- translated from A. Sekowska PhD thesis).

Table 1. Electron transfers in the absence of photosynthesis

Reducing agent
Redox couplea
E0' [mV]b
DG0' [kJ*mol-1]c

Carbon monoxide



'Carboxidobacteria' e.g. Pseudomonas


2 H+/H2


'Knallgas' bacteria




Thiobacillus, Beggiatoa, Wolinella succinogenes




Thiobacillus, Sulfolobus





















Fe2+(pH 2)



Thiobacillus ferrooxidans, Sulfolobus





a. The reaction procedes from the reduced state to the oxidized state, but convention asks that it is represented in the order: product/substrate.
b. Redox reactions are reactions where a substrate is reduced (electron acceptor) and another one is oxidized (electron donor). DE is the difference potentiel between the end and the start of the reaction. DE0' is the redox potentiel difference (that can for example generate a protonmotrice force) of a biochemical reaction in standard conditions: 298 K (25°C), pH 7.0 and where the concentration of each reagent is 1 mol/liter except for water (normal concentration 55.55 mol/liter) and gases (pressure of 101.3 kPa = 1 atm). The reaction occurs spontaneously only when the value of DE is negative, i.e. when the potential evolves towards more negative values. The redox potential is usually represented with the negative values above, and the spontaneous reactions procede from top to bottom.
c. For each component of the system one attributes a quantity of free energy 'G', composed of an enthalpy 'H' (internal energy plus pressure multiplied by the volume) and of an entropy 'S' (measuring the degrees of freedom of the system, in terms of positions and energy levels available to its different components). In the cases where the absolute values are not large, the changes in G (DG) are decisive for the chemical reaction. The reaction occurs spontaneously only when the value of DG is negative. DG0' is the DG of the biochemical reaction in standard conditions as defined above.

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 (Rocha et al., 2000).

In Escherichia coli, where the pathway has been best established, the genes involved in these reduction steps are organized as several operons (Kredich, 1996). Operon cysDNC codes for subunit 2 of sulfate adenylyltransferase, subunit 1 of ATP sulfurylase (ATP:sulfate adenylyltransferase) and adenylylsulfate kinase. Genes cysZ and cysK may not be co-transcribed, although they are neighbours in the chromosome. CysZ is probably a membrane protein, but its function is unknown. CysK is O-acetylserine sulfhydrylase, phylogenetically related to tryptophane synthase (Lévy & Danchin, 1988, see also minerals and the origin of life). E. coli ATP sulfurylase forms a tight, catalytically-coupled complex with the last enzyme in the cysteine biosynthetic pathway, O-acetylserine sulfhydrylase (Wei et al., 2002). Operon cysPUWAM codes for a thiosulfate (and sulfate) periplasmic binding protein, an ABC-type membrane permease, and a minor O-acetylserine sulfhydrylase, specific for thiosulfate. Finally cysE codes for serine O-acetyl-transferase. It may lie in operon with genes yibN, grxC, secB and gpsA (coding respectively for: a rhodanese-related sulfurtransferase, a glutaredoxin: glutathione-dependent redoxin, an element of the protein secretion machinery, and most probably a glycerol-phosphate dehydrogenase, possibly used in phospholipids biosynthesis). Gene cysE is the start point of cysteine biosynthesis, probably derived from an ancestral serine metabolism (Wächtershäuser, 1988; Danchin, 1989).

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 3’OH positionofAPS, which is meatbolized into 3’phosphoadenosine 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.

ATP4-  +  SO42- <–> APS2- + P2O74-

GTP4-  +  H2O <–> GDP3- +HPO42- + H+

ATP4- +  APS2- <–> ADP3- + PAPS4- + H+

and a further reaction

P2O74-  +  H2O <–> 2 HPO42- 

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).

In the sulfur assimilation pathway, PAPS is reduced into SO32-, yielding adenosine 3’-5’ diphosphate (PAP) as a by-product. It will therefore be interesting to investigate the fate of PAP after sulfate reduction. This is all the more important because this same molecule is produced in another reaction starting from coenzymeA, the transfer of the 4-phosphopantetheine group on the acyl carrier protein (ACP) of the complex synthesizing fatty acids (see Sekowska et al, 2000 and below). Finally, synthesis of secondary metabolites such as peptide antibiotics, surfactin of polyketides also require the transfer of 4-phosphopantetheine from coenzymeA, producing PAP as a by-product (Reuter, 1999). This implies the existence of a 3’-5’ diphosphoadenosine phosphatase, yielding 5'-AMP and inorganic phosphate. Protein CysQ (Swiss-Prot: P22255) may be the missing phosphatase, although we did not find explicit experimental data proving this contention. York, 1995, CysQ is similar to a plant adenosine diphosphate phosphatase Gil-Mascarell, 1999 involved in the reduction of sulfite to H2S (Swiss-Prot: Q42546). This enzyme (HAL2=MET22 Glaser, 1993) is probably also present in yeast, where its inactivation lead to methionine auxotrophy. The plant enzyme (encoded by the HAL2-like gene RHL Peng, 1995) is sensitive to the presence of sodium ions and its activity is, for some unknown reason, associated with the resistance to osmotic stress. Because of its role, one may wonder whether PAP phosphatase is not present in the cell in the neighborhood of adenylate kinase, which scavenges AMP. One should finally ask whether PAP does not play a regulatory role in a cell process controlling the entry of oxidized sulfur into the cell, eventually coupling this metabolism to that of lipids. Let us note in this respect that this molecule is very similar to cyclic AMP, a well-known regulator of gene expression (Ullmann & Danchin, 1983).

The enzyme involved in the reduction of sulfate, phosphoadenosine phosphosulfate reductase (EC (thioredoxin-dependent PAPS reductase) (PADOPS reductase) is coded by gene cysH.

5'-phosphoadenosine-3'-phosphosulfate + thioredoxin reduced


phosphoadenosine phosphate + thioredoxin oxidized + sulfite

The sulfite ion is reduced by NADPH-sulfite reductase (EC, 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, 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).

Remarkably, in M. tuberculosis (and B. subtilis as well) it has been found that APS can be used as the ultimate step in sulfite synthesis. However, it is not cleaer that this is the situation in vivo. This does not seem likely as PAPS would be only used for sulfatation that produce sulfolipids, putative virulence factors, in M. tuberculosis. The cysDNC operon codes for the multifunctional enzyme complex that exhibits the three linked catalytic activities that constitute the sulfate activation pathway. In B. subtilis, the cysH gene codes for a PAPS reductase, that has a significant APS reductase activity (Berndt et al., 2004), as in M. tuberculosis (Williams et al., 2002).

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 cysteine The reaction catalyzed by CysE (serine transacetylase, EC condenses an acetyl group from acetyl CoA on the hydroxyl group of serine, forming O-acetylserine. Sulfur, reduced as H2S, reacts with O-acetylserine in E. coli to give cysteine. Genes cysK and cysM code for O-acetylserine (thiol)-lyase-A and -B, or O-acetylserine sulfhydrylase A and B (EC Lévy, 1988; Kredich, 1996). Serine transacetylase and O-acetylserine sulfhydrylase A form an enzyme complex, cysteine synthase. In contrast, O-acetylserine sulfhydrylase B does not belong to an identified enzyme complex. Both O-acetylserine sulfhydrylases use sulfide as a nucleophile, but O-acetylserine sulfhydrylase B possesses also a characteristic feature, the ability to use thiosulfate in the place of H2S, leading to the production of S-sulfocysteine. The conversion of S-sulfocysteine into cysteine has not been demonstrated in E. coli, and this casts doubt on the physiological importance of this activity. However, as we shall see later, there exist numerous sulfonatases that may act on this molecule, yielding cysteine and a sulfate ion, which would thus enter the normal pathway.

It is useful to understand the synthesis of O-acetylserine and its regulation, because the intermediary metabolites are very often regulators of gene expression, as we shall see below for the role of N-acetylserine in E. coli. N-acetylserine (that derives from O-acetylserine) is probably an inducer of the cys regulon. This molecule is formed by spontaneous cyclisation of O-acetylserine. The conversion of O-acetylserine into N-acetylserine is produced at the rate of 1% per minute at pH 7.6, and almost ten times faster at pH 8.6. The reverse reaction does not happen, which means that O-acetylserine but not N-acetylserine can serve as a sulfur acceptor. In contrast, N-acetylserine is approximatively 15-fold more efficient than O-acetylserine in its inducer action (Kredich, 1992).

Synthesis of methionine Methionine synthesis is linked to cysteine synthesis through metabolic pathways that differ according to the organisms. There often exist several different pathways in the same organism. In E. coli, the biosynthetic pathway is the following (figure 2) Greene, 1996).

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, 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, 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 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 B12.

Methionine synthesis genes, metA (homoserine O-succinyltransferase, EC, metB (cystathionine g-synthase EC, metC (cystathionine b-lyase, EC, metE (5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase, EC and metF (5,10 methylenetetrahydrofolate reductase, EC are more or less spread out in the chromosome. The operon metB comprizes another gene, metL, that codes for a bifunctional enzyme AKII-HDHII (aspartokinase II/homoserine hydrogenase II, EC and EC, belonging to the part of the aspartate derived pathway which branches out to threonine, lysine and methionine synthesis Saint-Girons, 1988).

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).

Transport of sulfur-containing molecules and sulfur scavenging

Amino-acid and peptide permeases

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 role.

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".

Sulfate and thiosulfate are transported in E. coli by proteins coded by operon cysPUWAM and by an isolated gene, spb. These transport systems are composed of a single permease (coded by genes cysUWA, the products of which are associated with the membrane) and of two periplasmic binding proteins specific for these two anions: TSBP (thiosulfate-binding protein), coded by gene cysP, and SPB (sulfate-binding protein), coded by gene spb Sirko, 1995). Another protein, CysZ, is also necessary for transporting sulfate, but its exact function is not known. The two periplasmic proteins, TSBP and SBP, share their specificity for the same substrates. This accounts for the fact that the simple mutant is always capable of transporting either anion (less efficiently, which slows down its growth as compared to the wild type). The double mutant only is incapable of transporting both anions.

Many molecules comprising sulfur in an oxidized state (sulfinates: -C-SO2; sulfonates: -C-SO3; sulfates -O-SO3; thiosulfates -S-SO3) are available as sulfur sources Cook, 1999). There must exist several families of permeases specific for these different molecules. In E. coli only one has been identified and it transports taurine van der Ploeg, 1996). One expects that other ABC (ATP Binding Cassette) permeases transport these molecules. It is therefore probable that among the permeases of unknown function one will discover some that concentrate the sulfur-containing molecules of this family. As a working hypothesis it would be interesting to see whether there would be a permease in the operon comprizing gene ycdM (coding for a protein similar to a protein annotated in Swiss-Prot as "dibenzothiophene sulfurization enzyme A").

Once in the cell, under the action of appropriate enzymes, the molecules in question liberate their sulfur, in the form of sulfate or sulfite. These anions then enter the biosynthetic pathway that we have just described.

Organosulfonates are molecules that contain the SO3- group. It is possible to class them according to the nature of the bond they form with the sulfur atom. One thus finds O-sulfonates (often named sulfates, with an oxygen bond), N-sulfonates and C-sulfonates. O-sulfonates and N-sulfonates are degraded by hydrolases of the general classes EC 3.1.6.- or EC 3.10.1.- (action on the S-N bond), respectively. C-sulfonates, which are more stable, are not submitted to hydrolysis Cook, 1999). There exist three mechanisms for degradation of these compounds: (i) activation of carbon at the —C—SO3- bond and liberation of sulfite, a reaction catalyzed by thiamine, (ii) stabilisation of the —C—SO3- bond by addition of an atom of oxygen to the carbon of this bond (directly by oxygenation), which liberates sulfite, and (iii) an unidentified mechanism of reduction. Taurine seems to be degraded, in the majority of cases, by the first mechanism Cook, 1999). In contrast, in E. coli, taurine Eichhorn, 1997) as well as most linear and aromatic sulfonates (alkyl-sulfonates and aryl-sulfonates) in most organisms, are degraded by the action of oxygenases (mono-oxygenases and dioxygenases). The general scheme for the reactions is as follows (it is sometimes repeated several times on the successive products of a same reaction):

NADH + O2 + substrate + H+ –> NAD+ + product + HSO3-

In animals, cysteine oxidized as cysteic acid is decarboxylated into taurine and excreted. Taurine is transported by an ABC-permease (coded by genes tauABC in E. coli van der Ploeg, 1996) and ssuBAC in B. subtilis van der Ploeg, 1998). It is degraded by an a-ketoglutarate-dependent dioxygenase (tauD; Eichhorn, 1997) into aminoacetaldehyde and sulfite. The fate of aminoacetaldehyde is not known, but there must exist a hydrogenase to dispose of it. Sulfite subsequently enters the pathway of mineral sulfur assimilation.

In addition to this well characterized operon, there exist several operons in E. coli with features that are reminiscent of the systems for sulfur oxide scavenging. This is the case of likely monooxygenases (coded by the gene ycbN in an operon with ycbO and ycbM), or of dioxygenases (YeaW and YeaX). Protein YcbN is a protein (monooxygenase) identifed in the study of the proteome as induced in sulfur starvation, which substantiates this hypothesis (Quadroni, 1996 and Swiss-Prot P80645). However, its substrate is not known. In the case of dioxygenases one may wonder whether a dioxygenase identified as having 3-phenylpropionate as a substrate (YfhUVWXY) Burlingame, 1983; Burlingame, 1986) may also act on sulfur-containing molecules. There exist other such enzymes as YeaW and YeaX, for which no substrate has been described. It seems interesting to perform a detailed analysis of what might be the natural substrates of reactions of this type in a biotope similar to that of E. coli. This would allow to explore much more efficiently the nature of many genes of unknown function in its genome. On the other hand, cysteine may have been oxidized to a lower state, as sulfinate, and it seems that some gene products related to the nifS gene may act as sulfinases Mihara, 1997).

Finally, it would be important to investigate the fate of O- or N-sulfonated carbohydrates, such as those present in many polysaccharides such as heparine, chondroïtine sulfate, and in plants, carragenanes or agar-agar. We have verified that E. coli cannot grow on agar-agar in the absence of sulfur source, but this does not tell us whether it can grow in presence of the corresponding monomers. Aryl-sulfonatases, most often studied for their action on xenobiotics, may well have a spectrum of action much wider than their action on aromatic substrates.