Thiosulfate, polythionates and elemental sulfur assimilation and ...

FEMS MicrobiologyReviews75 (1990) 351-382 Published by Ehevier

351

FEMSRE 00154

Tbiosulfate, polythionates and elemental sulfur assimilation and reduction in the bacterial world A. Le F a o u 1, B.S. Rajagopal 2,., U Daniels 2 a n d G. F a u q u e 3 . . . a Laboratoire de Bactdriologie de la Facult~ de Mcl~ecine, Strasbourg. France, z V_tniversityof lowo, Department of Micrvbiotogy, Iowa City, IA, U.S.A., and J A R B S et Service de Radioagronomie, CEN Cadarache, Saint Paul-lez-Durance, France

Received6 October 1989 Accepted14 December 1989 Key words: Thiosulfate reductase; Polythionate reductase; Thiosulfate sulfur transferase; Sulfur compounds assimilation; Eubacteria; Archaebaeteria

1. S U M M A R Y

Among sulfur compounds, thiosulfate and polythionates are present at least transiently in many environments. These compounds have a similar chemical structure and their metabolism appears closely related. They are commonly used as energy sources for photoautotrophic or chemolithotrophic microorganisms, but their assimilation has been seldom studied and their importance in bacterial physiology is not well understood. lmost all bacterial strains are able to cleave these compotmds since they possess thiosnlfate sulfur transferase, ~ o s u l f a t e reductase or S-sulfocysteive synthase activities. However, the role of these enzymes in the assimilation of thiosulfate or polythionates has not always been clearly established.

Correspondence to: A.E. Le Faou, Laboratoirede Bact~iologie,

Facolt6de M&lecine,3 rue Koeberi~,67000Strasbourg. France. * Present address: Department of Microbiologyand Dentistry, 18-246 Moos Tower, University of Minnesota, Minneapolis, MN 55455, U.S.A. * * Present addzees:Centre d'Oc&mologiede Marseille,CNRS UA 41, Fazult6 des Sciencesde Luminy, Case 901, 13288 MatseilleCedex9, France.

Elemental sulfur is, on the contrary, very common in the environment. It is an energy source for sulfur-reducing eubacteria and archaebacteria and many sulfur-oxidizing archaebacteria. A phenomenon still not well understood is the "excessive assimilatory sulfur metabofism" as observed in methanogens which perform a sulfur reduction which exceeds their anabolic needs without any apparent benefit. In heterotrophs, assimilation of elemental sulfur is seldom described and it is uncertain whether this process actually has a physiological significance. Thus, reduction of thiosnlfate and elemental sulfur is a common but incompletely understood feature among bacteria. These activities could give bacteria a selective advantage, but further investigations are needed to clarify this possibility. Presence of thiosulfate, polythionates and sulfur reductase activities does not imply obligatorily that these activities play a role in thiosulfate, polythionates or sulfur assimilation as these compounds could be merely intermediates in bacterial metabolism. The possibility also exists that the assimilation of these sulfur compounds is just a side effect of an enzymatic activity with a completely different function.

0168-6445/90/$03.50 O 1990 Federation of European MicrobiologicalSocieties

352 As long as these questions remain unanswered, our understanding of sulfur and thiosulfate metabolism will remain incomplete.

2. INTRODUCTION Reduction of sulfur compounds is a vital process for many bacteria and an essential step in the global sulfur cycle. This reduction is due mainly to sulfate- and sulfur-reducing bacteria which perform oxidative phosphorylation in anaerobiosis in the presence of sulfate, sulfite or elemental sulfur as electron acceptors. These microorganisms produce large amounts of sulfide, the oxidation of which permits energy generation by chemolithotrophic and photosynthetic bacteria. Thiosulfate is also present in the environment. It is an electron donor for either respiration by chemolitho:rophs (e.g. Thiobacillus) or photosynthesis by phototrophs (photosynthetic bacteria). Some eubacterial sulfate reducers gain their energy by a disproportionation of this compound to sulfate and sulfide [1]. Sulfate assimilation provides bacteria with the sulfide necessary for cysteine synthesis and other biosynthetic needs. Among eubacteria, almost all species are able to reduce sulfate and the biochemistry, genetics and physiology of this process are well understood [2]. A few heterotrophs, (e.g. Legionella [3], FranciseUa [4] and some species of Neisseria [5,6]) are unable to assimilate sulfate and are thus considered 'cysteine-requiring'. The photosynthetic Chlorobium is also unable to assimilate sulfate but does not use cysteine as sole source of sulfur and necessitates sulfide, thiosulfate or elemental sulfur for growth [7]. However, it has been shown in few bacteria that thiosulfate can serve as an alternative mineral sulfur source [8-13]. Elemental sulfur, which is common in the environment, is an energy source for photo- and chemolithotrophs, but its assimilation by heterotrophic bacteria is very seldom described [14-16]. About a decade ago, a 8teat accumulation of physiological and biochemical evidence led to the major revision of the concepts of bacterial relationships [17]: the procaryotic kingdom was shown to be most appropriately divided into the eubac-

teria and archaebacteria, based on major cell wall and membrane structural differences, different molecular properties, and different biochemical processes, including in some cases novel coenzymes. The majority of organisms discussed above, and those known to most microbiologists are eubacteria: Desulfovibrio and related species, photosynthetic bacteria, Thiobacillus and all het.erotrophic medically important organisms. However, the archaebacteria are a much less studied group, encompassing the aerobic extreme halophiles, the anaerobic methanogenic bacteria and a group of obligate and facultative thermophilic anaerobes, termed the 'sulfur-dependent archaebacteria', which can carry out respiratory dissimilatory sulfur metabolism. The methanogenic bactc~a and sulfur-dependent archaebacteria are major actors in the microbial metabolism of sulfur compounds, as discussed below. The microbial metabolism of sulfur compounds comprises a major portion of the global sulfur cycle. The biochemistry and chemistry of this cycle is complex and still undefined in some areas. Part of the complexity arises from the multiple oxidation states of sulfur, and the fact that some of the transformations involve chemical as well as biological reactions in the environment; also, some of the more important microbes and enzymes involved have not been well studied. Figure 1 describes the global sulfur cycle. Microbes can participate in this cycle in two clearly different ways: assimilatory, where the sulfur compound is taken up for use in b'msynthesis of cellular material, and is not transformed as an energy-ganerating mechanism; and dissimilatory, -where the sulfur compound is transformed as part of a respiratory energy generating mechanism. There are also a few examples that we will discuss where much more sulfur compound is metabolized than is needed for biosynthetic purposes, but the organism is growing on another energy source, and has not been demonstrated to use the energy available from the sulfur transformation; we will term this 'excessive assimilatory sulfur metabolism'. As shown in Fig. 1, the global sulfur cycle involves the reduction of both sulfate and elemental sulfur by bacteria; in many cases, especially the sulfate reducers and the sulfur-dependent

353 OXIC

3. CHEMISTRY OF THIOSULFATE~ POLYTHIONATES, A N D E L E M E N T A L S U L F U R

.....//

~--

\

\_.\ .... \ \

\ ...................\,, ...... R-SH o~.d.,~,,-"" H=S

\.==_.J_.J

Fig. 1. The biological sulfur cycle. * Thinsulfate and poly-

thionatcs are not involved in all the represented metabolic pathways, how,~er, as they are sulfur sources fur both assimilative a~sd dlssimilative processes, they were placed concurrently with sulfate in the sulfur cycle.

archaebacteria, eners~y is obtained by oxidative phosphorylation in ane :robiosis in the presence of sulfate, sulfite or elemental sulfur. These bacteria produce great amounts of sulfide, the oxidation of which permits energy generation by aerobic chemolithotrophic bacteria (e.g. Thiobacillus) and photosynthetic bacteria. As well, the chemical oxidation in air of sulfide to So is appreciable; in the interface between aerobic and anaerobic zones, this reaction can be significant. Both thiosulfate and polythionates can be produced in such environment. Elemental sulfur occurs as large deposits or in warm springs; it can be an energy source for many bacteria by either oxidative or reductive processes. In this review, we will cover the bacterial assimilation of thiosulfate, polythionates and elemental sulfur, but will concentrate on the biochemistry and physiology of the reduction and dismutation of these compounds.

The major available forms of sulfur in nature are generally sulfate or sulfide in soil or water, and sulfur dioxi,~e in the atmosphere [18,19]; thiosulfate, polythionate~ and elemental sulfur apparently play a smaller, however significant, role. Thiosulfate is an asynunetri¢ molecule which possesses a sulfane (outer) sulfur and a sulfonyl (inner) sulfur atom [20,21]. Polythionates derive from polysulfanes but they are also similar to thiosulfate, since they possess one sulfcnyl sulfur at each end of the molecule (Fig. 2). Many polythionates are known and the length of the chain of sulfane sulfur atoms varies from one to 10 and more (dithionate, $202- , is not a polythionate and has completely different chemical properties). However, two polythionates of interest are easily synthesized and well characterized: trithionate ($3O 2 - ) and tetrathionate ($402-); both are commonly used for physiological and biochemical studies. Thiosulfate is the more stable at neutral and basic pH, while polythionates are stable in a crystallized f~)rm at + 4°C and in acidic aqueous solutions, and are readily decomposed at basic pH. Thiosulfate and polythionates are produced TI-~SUL¢ATE

I°1 O-S--S

O

m~n. ,w~ N m POLYlI-IIONATES

I

o

ol~-

o-s--cs~--s-o I

o I~n.llOl~'rE

I O0

o

I

12.

Fig. 2. Chemical structuresof thios~fate, p o l ~ t e s ditbinzmt¢.

and

354 by chemical and biochemical przcesses and are present at least transiently in the environment [22,23]. In humans, they are produced by metabofism and belong to the sulfane pool [24] although their concentrations in body fluids are always low [251. The stable form of elemental sulfur is the 8 atom ring which constitutes orthorhombic crystals [20,21]. Many other chemical forms of sulfur are known but they are not stable and give more or less rapidly the orthorhombic form. Stendel et al. [26,27] have proposed the following formula for the elemental sulfur which is present as hydrophilic globules formed in cultures of Thiobacillus

(T.) ferrooxidans: xS8 • yH2SnO6 • zH20-Ss the sulfur globules consist of long-chained polythionate ions attached to a hydrophobic core with mainly Ss-rings. The elemental sulfur, which is very sparingly soluble in water, can be rapidly reactivated according to the following equation: HSsSO~" -- S8 + HSO~and then can easily penetrate biological membranes. Sulfur melts at 119°C. It has a strong propensity to concatenation, forming rings or polysulfide chains. Above 80°C a spontaneous reduction of elemental sulfur is observed [28]. Heating also changes the structure of sulfur which presents a monoclinic form before melting. Colloidal sulfur, commonly used as a sulfur source in microbial systems, is the 'soluble form' of elemental sulfur and is stable at low salt concentrations (less than 0.1 M). Hydrophilic colloids of sulfur are usually prepared by adding hydrochloric acid to a solution of thiosulfate. These colloids consist of chains of atoms associated with thiosulfuric acid [29]. Sulfur flower is obtained by condensation of sulfur vapors. It is stable and consists of orthorhombic sulfur associated in variable proportions to free chains of sulfur atoms [21]. Colloidal sulfur, sulfur flower or orthorhombic sulfur are used for growing bacteria and for biochemical studies. It has to be noted that these physical forms of sulfur are not obligatorily identical to the ones encountered by bacteria in the environment and that media

preparation (e.g. autoclaving) could alter the sulfur source added.

4. BIOCHEMISTRY OF THIOSULFATE AND POLYTHIONATES REDUCTION

4.1. Thiosulfate sulfur transferase (rhodanese) The enzyme rhodanese [thiosuifate sulfur transferase (TST); EC 2.8.1.11 has been extensively studied in eucaryotes. Its catalytic properties and its structure are well known [30,31]; it is a holoprotein with a molecular mass of 32.9 kDa. The presence of two zinc per molecule of enzyme has been reported [32] but elucidation of its three-dimensional structure has shown that the enzyme does not contain any metal atom [31]. The rhodanese molecule comprises two domains with similar tertiary structures although their primary structurcs show little homology. Only the C=terminal feld has catalytic properties and harbors the active sites, whereas the N-terminal domain appears to be necessary for the enzyme to function. TST accepts a wide range of sulfane radical donors and acceptors between which it ensures a transfer of this radical [30]: any sulfane-containing compound can be a sulfur donor substrate. The TST performs a non-reducing thiosulfate cleavage according to the following mechanism:

s~o~- + E~-S--+ En~-S.S- + SO~A sulfane acceptor A (e.g. cyanide) restores the

native enzyme: Enz-S-S- + A - ~ Enz-S- + A-SIt has been shown that these reactions correspond to a double displacement mechanism [30,33]. When

sulfane acceptors" s u c h as r e d u c e d lipoate

or reduced glutathione are used, a persulfide is formed. Oxidation of the acceptor permits the production of sulfide (this reaction could be a non-enzymatic process): R-S-S - + R-S ----,R-S-S-R + S2In this case the net reaction of thiosulfate cleavage $202- + 2 R - S - ~ SO32- + R-S-S-R + S2-

355 is equivalent to a thiosulfate reductase activity CI'SR). Since its first description in 7". thiooxidans by M c C h e s n e y in 1958 [34], the TST has been detected in numerous mieroorgnnisms: chemofithotrophic and photosynthetic bacteria and heterotrophs with aerobic or anaerobic metabolism [35-49]; its presence in methanogenic bacteria has been demonstrated (Berlier, Y., personal communication). However, in some genera the activity is so low that its presence is questionable (e.g.

Salmonella, sulfate-reducing bacteria): this could b e due to spontaneous non-enzymatic interferences [50]. The presence of rhodanese activity in almost all bacteria with a n aerobic oxidative metabolism (e.g. Pseudomonas, Nebseria, Acinetobacter...) has also to b e noted [42-44,46,47~ TST activity is generally measured b y the formarion of thiocyanate in presence of thiosulfate and cyanide according to S~Srbo's method [51], sometimes modified for better sensitivity [35,47].

Table 1 Properties of ba~eriM and eucaryotic thiomdfate sulfur transferases oqpmim~

~

x.s.,o~ -

[refereace! Procaryotes

(kDa)

(raM)

Optimum pH

Inhibitors

Actlvaton

Sulfite NEM; IA Sulfite NEM; IAM N.R.

tiME; GSH

N.FU N.R.

N.IL N.R.

9-10

Sulfite

N.IL

7.5

Sulfite

fl-ME

Sulfite NEM; IA

N.R.

Sulfite

fl-ME; GSH

NEM; IA

C~teine

> 10

Sulfite IAM Cyste~e

None

>8

Sulfite IA; tAM Cystetne

N.R.

8.5

Cysu~e GSH

N.R.

9.6

N.R.

N.R.

Thiobadllw ~irnficans [351

38

N.I~

8-9

T. ferrooxidam [38]

N.R.

0.58

7.5-9

N.R.

0.6

8.7

34 39

5 0.25

C/u'oma6umsp. [37] CMon~um vi~'iolorme[49] ~

a s jm/mtrb [41] Desu/foto~dum nigrificans [39] C~dmn-d~nm~ bacteria [45]

45

N.R. 15-17

3.3 120 3.7

N.R. N.R.

11 •

N.R. N.IL

Acitwtobacter ca/coaceticm [44]

35

26.3

8-8.5

Neisseria /~orr/weae [7]

N.R.

0.4

Bovineliver [30]

32.9

7

Trametes sangu/nea [.¢~]

N.R.

8.3

37

3.1

Cercopithet~t7 aeth/ops [57]

N.I~ - m reported, NEbI= N*cthyl-malc~mid~IA = ~ t ¢ , , mer~tocflumol.

IAM = ~ d e ,

GSH = mduaxl $ 1 u l a ~

fl*MF..-fl-

356 TST is a soluble enzyme which has been reported to be periplasmic in Pseudomonas (Ps.) aeruginosa [52] or cytoplasmic in Bacillus subtilis [36]. A membrane-bound activity, which coexists with a soluble one, has been described in Thiocapsa (Tcp.) roseopersicina [53]. Rhodanese is a constitutive enzyme and its specific activity is enhanced only 2-3 times by varying culture conditions: e.g. in T. denitrificans a higher medium pH, increase in thiosulfate concentration [54], or addition of cyanide [35]; in Tcp. roseopersicina during anaerobic autotrophic growth [53]; in Neisseria ( N.) gonorrhoeae, grown with thiosulfate or sulfur flower (in place of cysteine) [6]. Rhodanese has been purified to homogeneity [35,44,49] but very little is known about its physico-chemical properties. This enzyme is present in low amount in bacterial extracts. Purification steps lead to a considerable loss of activity [6,44,49]. As a consequence, catalytic properties are often studied in partially purified extracts. Despite some differences in kinetic properties, this enzyme exhibits some common characteristics: the optimum pH is basic and sulfite is a very potent non-competitive inhibitor (Table 1). The discrepancies between these results could be related, at least partially, to the conditions used for the kinetic studies. In N. gonorrhoeae a competitive inhibition by phosph¢te (which is commonly used as buffer for studying TST activity) or sulfate ions has been observed [6]: this could be related to the structural analogy of these ions with thioosulfate. Eucaryotic TST [55-57] resemble the bacterial enzymes (Table 1). The molecular weight of the bacterial TST is most commonly about 35 kDa [35,39,44,49]. Multiple forms of rhodanese have been described iii cucaryotes [57--60], They differ by their electrophoretic mobilities or chromatographic elution profiles even though their molecu-, lar weight remains the same. This phenomenon has been related: (i) to the presence of a dissociative form of the molecule (which gives a 19-kDa protein) and a non-dissociative form corresponding to the native enzyme [59], (fi) to a deamidation of the native enzyme [58] and (iii), to the existence of conformational isomers [60]. Ptoteolvtic cleavage of rhodanes¢ induced the same phenomenon [61]. In bacteria many peaks of TST activity were

eluted from a DEAE cellulose column [6,37]. It is not known if this is due to either enzyme structure or the purification process (presence of protease in crude extracts or chemical modification of the molecule). In T. denitrificans the TST (38 kDa) is said to consist of 4 subunits (10 kDa) which can be further dissociated in smaller fragments (7 and 2 kDa, respectively) [35] but no other bacterial enzymes have such a characteristic. In Chlorobium ( Chb.) vibrioforme f. thiosulfatophilum two different enzymes exist, one with acidic and the other one with basic pH [49]. Ploegman et al. [62] have hypothesized that the two domains of eucaryotic rhodanese originate from a common ancestor via gene duplication. It would be interesting to investigate the physico-chemical properties and sequence of bacterial rhodanese to know if low molecular weight enzymes could correspond to a monomeric form. Mercaptopyruvate sulfur transferase is a sulfur transferase [24] which has been purified from Escherichia (E.) cell [63]. It is a zinc-containing protein (MW 23 kDa) consisting of two subunits' (MW 12 kDa). Mercaptopyruvate is the only sulfane donor used by the enzyme but the sulfane accepter has no strict specificity: it could be sulfite (with formation of thiosulfate) or cyanide (with formation of thiocyanate) [30].

4.2. Thiosulfate reductase: Thiosulfate reductase (TSR) performs the reaction szo~- + 2 e - - , SO~- + S~The molecular mechanism of reductive cleavage of uhiosulfate is not yet known in bacteria. However, the eucaryotic enzymes reduce thiosulfate by a one-step process unlike rhodanese. The TSR present in animal tissues is very labile [24] but the yeast enzyme is stable and has been purified [64,65]. It is a soluble holoprotein (MW 24 kDa) which uses reduced glutathione or cystcine as electron donor. Cyanide or sulfite are not sulfane accepters. Tlfiosulfate reductase activity is found in numerous microorganisms [54,66-77]. Two different classes of enzymes have been described. In

357

Proteus ( P.) mirabilis [69,70], Desulfovibrio ( D.) gigas [71,72] and D. vulgaris Miyazaki F [77], a high molecular weight protein (85-220 kDa) linked, at least pa~!ially, to the cytoplasmic membrane is present. These proteins also possess a tetrathionate reductase activity. The P. mirabilis enzyme, which synthesis is repressed by oxygen, also has trithionate reductase activity. The synthesis of these proteins is repressed by oxygen and induced by anaerobiosis or in presence of tetrathionate or thiosulfate. A second class of TSR has also been detected in anaerobic (Desulfotomaculure (Din.) nigrificans [67], D. oulgaris Hildenbor-

Table 2 Comparison of bacterial tad eucaryotic thiosulfate reductase activities Organisms [reference]

Procaryotes Desulfotomaculum nigriflcans 1671 DesulfooibriotJulgaris Hildenborough 168] D. uulgaris Miyaza~ F [77] D. gigas [Tll [721 R.hodopseudomonas palustris [411 Proteusmirabilis [69] Escherichiacoli [1191 Pseudomoaas strain 16B [811 Neisseria gonorrhoeae 17] Eucaryotes Chlorellafusca [761

Yeast [64,651

MW (kDa)

Km $20~(mM)

N.R.

1.3

Specific activity in crude extracts a

50

16.3

N.R.

N.IL

85-89

N.R.

280

0.5 0.05

200 280

220 186 90

N.R.

15

133

0.26

530

N.R.

N.R.

1

N.R.

N.R.

20

N.R.

2

24 26.5 28 55 24

0.164 0.156 N.R. N.R. 3.7-6.2

2

N.R.

220

• The specific activities are expressed in nmol of B2S produoed, min-Lm8 protein-I. N.R. m not reported

ough [68]) and aerobic (N. gonorrhoeae [73], and N. meningitidis [74]) bacteria. Their specific activity in crude extracts is low and varies little according to growth conditions [6]. The K m for th/osulfate is about 10 times higher than for the membrane-related enzymes, which could indicate that these soluble enzymes have an assinfiIatory role. They are poorly characterized, which makes comparison difficult between these two classes of proteins (Table 2). It is not known if thiosulfate reductase activities in the presence of G S H are related to an enzyme resembling the yeast enzyme. Four distinct proteins with "rSR activity have been isolated from the green alga Cidorella fusca strain 211-8b [76]. They have different molecular weights (24-55 kDa) and pH optima (8.5-9.5) [76]. Thiosulfate and polythionates reduct~se activities are generally determined manometrically with reduced methyl (or benzyl) viologen and hydrogenase as electron donor [78]. H o ~ v e r , T S R activity in the presence of cysteme, dithionite, reduced glutathione, or reduced fipoate has been also reported [6,74].

4.3. Trithionate reductase Trithionate reductase (TRTR) is a more elusive enzyme. A single protein able to specifically redace trithionate has never been described. The reduction of this compound by D. g/gas was observed in vitro by thiosulfate reductase and bisulfite reductase in the presence of G S H ; however, this could be a non-enzymatic process [72]. A related phenomenon has also been described in D. oulgaris Hildenborough [79]. N. gonorrhoeae possesses a soluble T R T R with a very low specific activity [47].

4.4. Tetrathionate reductase Tetrathionate reductase ( T r R ) activity has been described in numerous genera of microorganisms. It is a dissimilative enzyme, membrane-associated, which permits an anaerobic oxidative metabolism in bacteria [80]. A marine heterotroph possesses a constitutive soluble T I l L analogous to the Thiobacillus enzyme, which also oxidizes thiosulfate [81]. In photosynthetic bacteria, a thiosulfate:

358 acceptor oxidoreductase which can oxidize thiosulfate into tetrathionate is present; however, it is not known if this enzyme performs the reduction of tetrathionate [82]. Tetrathionate assimilation has not been investigated in heterotrophs. A soluble T T R activity has been demonstrated in N. gonorrhoeae [47] but has not been further characterized. 4.5. S-suifocysteine synthase S.sulfocysteine synthase (SSCS) forms Ssulfoeysteine in presence of thiosulfate and Oacetylserine [83,84]: $2032- + O-acetylserine --, S-suifocysteine + acetate This enzyme is also known as O-acetylserine sulfhydrylase B or cysteine synthase B since it synthesizes cysteine in the presence of sulfur and O-acetylserine; cysteine synthase A or O-acetylsefine sulfhydrylase A, forms a complex with sefine acetyl transferase and does not use thiosulfate as substrate [2]. SSCS is present along with cysteine synthase A [83,85]. However, this enzyme is lacking in many genera [6,83]. The specific activity of SSCS is not related to the sulfur source present in the medium for Ps. aeruginosa [83] but in Salmonelia (S.) typ~imurium, it is repressed by cysteine, as well as the sulfate assimilation pathway, under the control of the gene Cys B [86]. The physiological role of SSCS is not clearly understood. It can replace cysteine synthase A for cysteine biosynthesis when, after mutation, this activity is lacking [86]. The importance of SSCS in thiosulfate assimilation is not well investigated. SSCS is a soluble enzyme with a molecular weight of about 50 kDa [87,88]. It binds 2 tool of pyridoxal phosphate per tool of enzyme and it can be dissociated into two subunits [88].

5. BIOCHEMISTRY O F E L E M E N T A L SULFUR REDUCTION Sulfur reductase has been purified and characterized from a few eubacterial facultative

Table 3 Bacterial sulfur reductases Organisms [references] Spirillum 5175 [111] Desulfovibrio sp [89,90,93,97) Desulfuromonas acetoxidans [~,1o71

Composition

Localization

N.R.

Membraae

Tetrahemecytochrome c3 Periplasm (mainly) c-type cytochrome

Cytoplasm Membrane

Wollinella succinogenes [99-101] Iron-sulfur protein Membrane Chromatium vinosum [116] Flavocyrochrome c552(c) Membrane Chlorobium thiosulfatophilum [117.118] Flavocytochrome c553(c) Soluble Spirillum 5175 [111]

Desulfovibrio sp [89,90,93,97] Desulfuromonas acetoxidans [96,1o71 Wollinella suceinogenes [99-101] Chromatilb*n vinosum [116]

-

-

2700 69 b 26

+

N.R.

N.R.

N.R.

Chlorobium thiosuifatophilum [117,118] N.R.

N.R.

• S~cific activity expret~d in nanomol H2 consumed'rain -!" mg-1 protein. b For D. bacu/atus DSM 1743. ¢ Possessesalso sulfide: cytochrome c reductase activity [88a116]. N.R. - not reported sulfur-reducing microorganisms (Table 3). The tetraheme cytochrome c 3 is the sulfur reductase of some strains of sulfate reducers of the genus Desulfovibrio, such as D. baculatus D S M 1743 and Norway 4 [89], D. g/gas [89], D. salexigens British Guiana, and D. africanus Benghazi [G. Fauque and J. Le Gall, unpublished data], from which the sulfur reductase activity is purified together with

359 the tetraheme cytochrome c3. The sulfur reductase activities of the tetraheme cytochrome c3 from D. baculatus and D. gigas correlate well with the ability of these organisms to grow on elemental sulfur (see Section 6.1.3.). Membranes isolated from D. gigas and D. baculatus Norway 4 contain hydrogenase and c-type cytochromes and catalyze the reduction of sulfur to sulfide coupled with the oxidation of dihydrogen [90]: H 2 + S --~ H2S(AG~ ~ - 2 8 kJ/moi H2) A specific interaction exists between the membranes of these two strains and the sulfur micelles. An oxidative phosphorylation linked to the dissimilatory reduction of elemental sulfur has been described with a particulate fraction of D. gi'gas [90]. A P/2 e- ratio of 0.1 was obtained, a value similar to the one found with the H2/SO 2- and H2/NH2OH systems [9!,92]. An uncout~'-~ngof phosphorylation in the H2/S system was observed with pentachlorophenol and after addition of methyl viologen or D. gigas tetraheme cytochrome c3 [90]. A mechanism of sulfur reduction by an exposed, low-potential heine of D. baculatus Norway 4 tetraheme cytoehrome c3 has been proposed; this reaction may involve insoluble $8 molecules as intermediates [93]. The triheme cytochrome c~ from Desulfuromonas ( Drm.) acetoxidans, which is lacking a re.'3' !ow-poten:ia! heine [94,951, does rLot reduce colloidal sulfur [96]. In the same way, the high-potential monoheme cytochrome css3 from D. vulgaris Hildenborough [97] and cytoehromes cs53os0) from 1). baculatus Norway 4 and DSM 1743 [98] are not able to reduce elemental sulfur. A sulfur reductase has also been isolated from Wolinella (W.) succinogenes grown with formate and elemental sulfur [99], according to the reaction: Formate + S --* Co2 + H2S(AGo = - 2 3 kJ/mol formate) The sulfur reductase has been solubilized from W. succ/nogenes membranes with Triton X-100 and partially purified (20-fold, by means of chromatofocusmg). The active enzyme has a molecular mass of 200 kDa and consists of a dimer with one type of subunit with a Mr of 85 kDa. The enzyme

contains equal amounts of free iron and sulfide (120/~mol/g protein), but no b- or c-type cytochrome [99]. The electron transport chain catalyzing the reduction of sulfur by formate consists merely of formate dehydrogenase and sulfur reductase. The sulfur reductase could be fully incorporated into sonic liposomes together with formate dehydrogenase and these liposomes could catalyze the electron transfer from formate to elemental sulfur [99-101]. In the obligate dissinfilatory sulfur-reducing bacterium Drm. acetoxidans, different results have been reported on the localization of the sulfur reductase. The cytoplasmic frac~:on of Drm. acetoxidans contains large amounts of low-spin cytochromes, mainly of the c-type, such as the cytochrome c7 [102,103], and iron-sulfur proteins such as rubredoxin and ferredoxin [103,104]. Two c-type cytochrome fractions "css3" and ' R ' have been isolated from the cytoplasm of Drm. acetoxidans strain 5071 (DSM 1675) grown on acetate as carbon and energy sources and mutate as electron acceptor [96]. These cytoehrome fractions were able to reduce colloidal sulfur to hydrogen sulfide in presence of the trtheme cytochrome c~ from Drm. acetoxidans and the periplasn~c (NiFe) hydrogenase from D. gigas [96]. The particulate fraction of Drr~ aceto~idans contains menaquinone (MK-8) [105,106] and minor amounts of cytoehrome b [103]. The membrane fraction of Drm. acetoxidans grown on acetate and sulfur catalyzes the ATP-dependent oxidation of succinate with elemental sulfur or NAD as electron acceptors [107]. A H2S dehydrogenase and a sulfur reductase are present in the membrane [107]. A pathway of electrons from the intermediates of the citric-acid cycle to the terminal electron acceptor SO has been proposed for D r ~ acetoxidans [108,109]. An electron carrier, probably a cytochrome c [110], could connect sulfur reductase and NADH dehydrogcoase. The activation and the reduction of elemental sulfur have been studied in fi,:e strains of eubecteria: Spirillum 5175, D. baculatus, D.,vn. ocetoxidans, DrnL acetexigens, and Drm~ suceinoxidans [111]. Spirillum 5175 grown with elemental sulfur has the most active sulfur oxidoreductase with a specific activity of 270 nmol of hydrogen con-

360 sumed per win per mg protein. This activity is also present with Spirillum 5175 grown on fumarate and nitrate, showing that the sulfur reductase is a constitutive enzyme in this species [111]. The sulfur reductase has also been reported to be constitutive in D. baculatus [89], Drm. acetoxidans (R. Bache and N. Pfennig, personal communication) and in different genera of methanogenic bacteria (see Section 6.2.1.). In contrast the sulfur reductase is induced by the growth conditions in W. succinogenes [99]. With Spirillum 5175, the sulfur oxidoreductase activity is localized in the membrane fraction and the pH optimum for the reduction of elemental sulfur is at pH 8.9 [111]. There is a lag phase in the reaction which is pH-dependent, with a change in the turbidity of the solution due to the conversion of the substrate into a more active form accompanied by a change in the size of the colloidal particles. Similar observations have been reported during the reduction of colloidal sulfur by Desulfovibrio species [112] and Drm. acetoxidans [96]. Different preparations of elemental sulfur have been utilized and contradictory results have been reported concerning their activities. According to ZOphel et al. [113] the highest activity and the most reproducible results for Spirillum 5175 were obtained with the colloidal sulfur prepared after Janek [113] and with tetrasulfide and polysulfides. The sulfur suspension made according to Odtn [see 114] was not very active and with the hydrophilic sulfur prepared according to Roy and Trudinger [115], the activities were generally lower and the results less reproducible. In contrast, the sulfur from Roy and Trudinger's method has been reported to be more active than Janek's sulfur with the tetraheme cytochromes c3 from D. baculatus Norway 4 and DSM 1743 [96,97]. An enzymatic reduction of elemental sulfur occurs during the assimilation of So by Propionibacterium (Pb.) shermanii and the sulfur reductase is localized in the membrane fraction facilitating the passing of sulfur into the cell [14]. Fukumori and Yamanaka [116] have found that the flavocytochrome css2 from Chromatium (Ch.) vinosum has elemental sulfur reductase activity besides sulfide: cytochrome c reductase activity. Another fiavocytochrome c, the cytochrome c5s3 from Chb. thio-

suifatophilum (now called Chb. iimicola forma sp. thiosulfatophilum) also catalyzes the reduction of elemental sulfur to sulfide with reduced benzyl viologen as electron donor [117,118]. Very little is known about sulfur reduction in heterotrophs. A mutant of E. coil B [15], N. gonorrhoeae and Ps. aeruginosa assimilate elemental sulfur [16]. In N. gonorrhoeae, a sulfur reductase activity has been found in the particulate fraction [6].

6. PHYSIOLOGICAL IMPORTANCE OF THE REDUCTION OF THIOSULFATE, POLYTHIONATES AND ELEMENTAL SULFUR Due to the major differences between the eubacteria and archaebacteria, we will divide our discussion on this topic into two parts dealing with each kingdom in turn.

6.1 Eubacteria 6.1.1. Thiosulfate assimilation Clarke [119] and Olitski [120] were the first to demonstrate that resting cells of ' n o n H2S-producing' bacteria form sulfide when thiosulfate is present in the incubation medium. However, such a production is always weak when compared to that observed with bacteria which accumulate sulfide in a thiosulfate-containing medium during growth. Later, utilization of 35S-thiosulfate demonstrated assimilation of the two sulfur atoms into proteins [8,12,121]. If a sulfite reductase is lacking, as in N. gonorrhoeae, only the sulfane sulfur of thiosulfate is incorporated, and when bacteria are growing in the presence of thiosulfate, sulfite accumulates in the medium [12]. All bacteria so far studied, that possess at least one of the activities described in Section 4, are able to perform cleavage of thiosulfate and to use it as sole sulfur source. Thus, thiosulfate assimilation could be a widespread characteristic among bacteria. A specific thiosulfate reductase has been purified from sulfate-reducing bacteria of the genus Desulfovibrio [68,71,72]. However, two different pathways are proposed for the dissimilatory re-

361 duction of bisulfite in sulfate reducers: either a direct 6-electron reduction of SO2- to S 2- or the trithionate pathway involving trithionate and thiosulfate as intermediates [122]. Several arguments are in favor of a direct bisulfite reduction for Desulfovibrio species (even if a trithionate reductase system has been characterized in D. vulgaris Hildenborough [79]) and of an active trithionate pathway ip. Desulfo¢omaculum s0. Although thiosuifate reductase is not connected to oxidative phosphorylation, sulfate-reducing bacteria are able to grow with thiosulfate as the only electron acceptor [123]; in this case energy could originate from the reduction of suifite produced by tbe reduction of thiosulfate. From the perspective of microbial ecology, sulfide production during growth with thiosulfate could give an organism a selective advantage by inhibiting microorganisms sensitive to this compound. Also the formation of sulfide allows the establishment of a symbiotic system with sulfur-oxidizing bacteria in sulfureta [123]. Thiosulfate oxidation by chemo- and photolithotrophs is commonly considered to begin with a splitting of thiosulfate into sulfite and sulfur prior to its oxidation [82,124-126]. A non-reducing reaction is energetically intecesting and, in T. denitrificans, rhodanese has been implicated in this process [35]. However, a periplasmic 'thiosulfate-oxidizing multienzyme system' has been described in T. versutus in which rhodanese does not play any role [127,128]. It is not known, in this case, whether the cleavage of the sulfur-sulfur bound occurs prior to or after oxidation of the molecule [129]. According to Kelly [124] direct tetrathionate oxidation into sulfate is a reaction which is not likely to occur in chemolithotrophs; thus TTR in these bacteria would be the first step in tetrathionate oxidation. In photosynthetic bacteria the picture is somewhat identical. Oxidation of thiosulfate into sulfate occurs without any evidence of tetrat~fionate as an intermediate: this latter compound accumulates under very particular conditions (e.g. when grown in high thiosulfate concentration) and few species are able to oxidize tetrathionate [82]. Thus oxidation of thiosulfate by photosynthetic bacteria occurs likely via a splitting of this compound, but the enzyme(s) involved

in this process (thiosulfate reductase, rhoda~-lese or another enzyme) is (are) not yet known [82]. Among bacteria from the environment, assimilation of sulfur compounds has not been thoroughly investigated. In sulfate-reducing bacteria, two different sulfite reductases ('assim/lative' and 'dissimilative') have been described whose significance is not well understood [122]. The complex serine acetyltransferase-cysteine synthase exists in D. vulgaris Hildenborough [130]. In Rhodopseudomonas ( R.) sulfldophila the sulfate assimilation pathway, identical to the one present in S. typhimurium, is repressed by reduced sulfur compounds (sulfite or sulfide) [131]. In heterotrophs which assimilate thiosulfate, evidence exists that some TSR act~viti~ may be related to the presence of a rhodanese activity: (i) the observation with crude extracts of E. coli that H2S production, in the presence of thiosulfate is enhanced by pyruvate, TPP and Mg 2+ suggests that a rhodanese is probably responsible for the thiosulfate reductase activity [66]; (ii) the thiosulfate reductase activity of Tcp. roseopersicina [53], N. gonorr:~oeae [6] and N. meningitidis [74] observed in the presence of dithionite, reduced lipoate, GSH or dithioerythritol could be related to the presence of rhodanese; (iii) rhodanese and TSR present some common properties in N. gonorrhoeae [6]; and (iv) rhodanese specific activity is higher in thiosulfate-grown cells as compared to cysteine-grown bacteria [6]. In spite of these results, it is difficult to ascribe a definitive role for rhodanese in thiosulfate assimilation since the presence of a thiosulfate reductase with a low specific activity cannot be ruled cut. For example, TSR activity was not associated with purified rhodanese of Acinetobacter calcoaceticus [44], and in Din. nigrificans TST and TSR activities can be separated [39]. However, the activities so far described are not sufficiently characterized to clearly demarcate the existence of a specific enzyme responsible for thiosulfate cleavage. In the environment the concentrations of thiosulfate are low, but if a chemical (or biochemical) reaction produces this compound continuously, it could be a source for either dissimilatory or assimilatory metabofism of bacteria. Permeation systems, which can be specific or not for thiosulfate,

362 could permit growth on low substrate concentration [132,133]. Human pathofens have numerous sulfur sources available in body fluids [134]. For example N. meningitidis can grow on low thiosulfate concentrations (0.125 raM) [6,13]. On the con~ary, N. gonorrhoeae assimilates thiosulfate only if its concentration is equal or greater than 2 mM [6]. When growing with thiosulfate, ultrastructural changes are seen when compared to cysteine-grown cells [6]; they could be related to sulfite accumulation in the culture medium [135]. Thus, N. gonorrhoeae in a patient cannot rely on thiosulfate for growth since its concentration is too low; because of this, thiosulfate assimilation by the gonococcus probably does not have any physiological significance, but c o u l d indicate merely that this bacterium has retained the ability of cysteine synthesis. This could be of importance in vivo where N. gonorrhoeae could use reduced sulfur compounds (e.g. sulfides) present in its environment. The role of rhodanese in cyanide detoxific~tion, which is considered essential for eucaryotes [134], does not appear to have great importance for bacteria. Cyanide enhances the specific activity of rhodanese about 3-fold in T. denitrificans [35]; however, rhodanese has not been found in some cyanide-sensitive bacteria [43]. In eucaryotes, rhodanese, besides other sulfur transferases, ensures the sulfane sulfur transfer between components of the sulfur pool since it has a wide substrate specificity [24]. Moreover, rhodanese is said to provide some proteins with the sulfur contained in F e / S clusters, although such a role is still disputed [136,137]. Such an activity has never been demonstrated in bacteria and the role of rhodanese in thiosulfate assimilation is not clearly established. Thus, it has to be admitted that the exact function of rhodanese in bacteria remains obscure. In S. typhimurium, after its formation by Ssulfocysteine synthase, S-sulfocysteine is reduced to ¢.ysteine plus sulfite by either reduced ghtathlone or a system involving thioredoxin and a thioredoxin reductase [138]. In the latter case, the mechanism is close to the one involved in sulfate assimilation in E. coli [139]: thioredoxin, a carrier protein [140], is the sulfonyl acceptor after PAPS reduction. Thi~ thioredoxin-bound sulfonyl radical

is eventually reduced by a 'thiosulfonate reductase'. This enzyme, which is improperly named (it should be $-alkyl thiosulfate reductase [llSD, could correspond to the sulfite reductase. However, the bound intermediates in sulfate assimilat i o n were not found in S. typhimurium [141]. S-sulfocysteinc synthase, which permits direct thiosulfate assimilation and can replace cysteinc synthase, could give a selective advantage to the bacteria. However~ its exact physiological significance remains unknown. A novel type of energy metabofism, involving the fermentation of inorganic sulfur compounds, has been discovered in a new species of Desulfovibrio (1). sulfodismutans) which is able to grow under strict anaerobic conditions by carrying out a disproportionation (or dismutation) of thiosulfate and sulfite into sulfate and sulfide [1,142]. Actually, calling this mode of growth 'fermentation' may not be appropriate, since electron transport phosphorylation probably is still responsible for energy generation; this definition or semantic problem is found in several groups of bacteria.

6.1.2. Polythionate assimilation The assimilation of polythionates has been seldom described. N. gonorrhoeae is able to assimilate trithionate but the growth rate is about half that of thiosulfate-grown cells [6]. It is possible that soluble TRTR and TTR activities present in N. gonorrhoeae are also due to rhodanese since polythionates are substrates for the latter enzyme [241.

6.1.3. Eleme,~tal sulfur reduction The reduction of elemental sulfur in anaerobic environments has largely been ignored since colloidal sulfur may be reduced by a wide variety of microorganisms by a mechanism assumed not to be of physiological importance [143-147]. However, a process of respiratory utilization of elemental sulfur is known, in addition to this incidental sulfur reduction. The dissimilatory sulfur-reducing bacteria are physiological assemblages of mostly obligatory anaerobic microorganisms which conserve mergy by reduction of elemental sulfur to hydrogen sulfide linked to the oxidation of organic compounds [148-153]. The dissimilatory eubacte-

363 rial sulfur reducers comprise both facultative and true (or strict) respiratory organisms. The facultadve dissimilatory sulfur-reducing bacteria are able to use elemental sulfur as the terminal electron acceptor in the absence ef other po~ible electron acceptors (e.g. sulfite, sulfate or nitrate). Two groups of facultative mesophilic sulfur reducers are actually known: some sulfatereducing bacteria and some spirilloid microorganisms [152,153]. Biebl and Pfennig [154] first showed that some strains of sulfate reducers are also able to grow with elemental sulfur (sulfur flower) as electron acceptor. Four strains (D. baculatus strains DSM 1743 and Norway 4, sulfate-reducing bacteria strains 4474 and 5174) are straight rods and desulfoviridin-negative[153]. Three strains are vibrio-shaped and contain desulfoviridin as dissimilatory bisuifite reductase (D. gigas DSM 496 [154], D. multispirans NCIB 12078 [155] and D. fructovoran~ DSM 3604 [156]). The spore-forming, Gram-negative, motile, rod-shaped Dm. sapomandens strain Pato (DSM 3223) is also able to reduce elemental sulfur to sulfide [!57]. A Gram-negative, non-motile, rod-shaped, asporulated sulfate reducer D. carbinolicus DSM 3852 (formerly strain EDK 82), capable of growth with methanol as the sole source of energy can also utilize elemental sulfur [158,159]. Recently, Nanninga and Gottschal have reported that two other sulfate reducers, namely D. desulfuricans subsp. desulfuricans strain DK 81 and D. sapovorans strain DKbu 14, use elemental sulfur as electron aeceptor [159]. Slow growth in the presence of elemental sulfur has been observed with D. vulgaris Woolwich (NCIB 8457) [160]. Four unidentified saccharolytic dissimilatory sulfate-reducing strains isolated from an anaerobic digestor were als~ reported to reduce elemental sulfm"when lactate was used as carbon and energy source [161]. These four strains were motile, Gram-negative, nonsporulating rods, mesophilic and differed markedly from known sulfate-reducing bacteria, especially with respect to carbon source utilization and sulfur sources which could be reduced [161]. The type strains of D. desulfurieans, 1). vulgaris, Desu,~fonema pigra eald Dnt nigrificans did not grow with sulfur as a respiratory electron acceptor although some sulfide was formed [154]. Elemental sulfur

has been reported to inhibit growth of some sulfate-reducing bacteria in the presence of sulfate (e.g.D. sapooorans [153], Din. acetoxidans [162], Desuifonema species [163], and Desulfobacter post-

gatei [164}). Three types of organisms belong to the spirilloid sulfur-reducing bacteria: gpirillum 5175 [165], W. succinogenes [166] and a free-living Campylobacter species (DSM 806) [167]. The cells are small, curved with a single, polar flagellum [168] and are able to oxidize formate and hydrogen with sulfur, nitrate, fumarate and malate as terminal electron acceptors. Most of the spirilloid sulfur reducers are able to use dimethylsulfoxide as the electron acceptor and to reduce it to dimethylsulfide [153,169]. Some thermophilic anaerobic microorganisms have also been reported to be able to facultatively reduce elemental sulfur to sulfide. The strain NS-E is a strictly heterotrophic non-motile bacterium of marine origin with an optimum temperature for growth of 77°C [170]. The addition of elemental sulfur to the yeast extract-glucose medium of strain '.;S-E did not increase the growth rate but resulted in a 4-fold increase of cell densities; this strain has been recently named Thermotoga ( Tt.) neapolitana DSM 4359 [171]. The type specL~ of the genus Thermotoga, Tt. maritima DSM 3109, is also able to reduce elemental sulfur without increase in the growth yield [172]. The formation of H2S in the genus Thermotogo has been postulated to be a kind of detoxification reaction preventing inhibition of these bacteria by hydrogen [172]. Five anaerobic thermophilic saccharolytic bacteria [Clostridium ( C.) thermosaccharolyticum DSM 571, C thermohydrosulfuricum DSM 567, Thermoanaerobium ( Ta.) brockii 33075, Ta. lactoethylicum ZE-1 and ,~..ermobacteroides acetoethylicus ATCC 33265] were also shown to be capable of reducing elemental sulfur [173]. Several groups of microorganisms are supposed to profit from the reduction of elemental sulfur although they do not multiply by this reaction under anaerobic conditions [1~3,174]. Some colorless sulfur bacte~a of the genus Beggiatoa may reduce stored sulfur globules under anaerubiosis resulting in a distinct, increase of the cell dry mass as in the case of Begg~atoa (B.) cf. leptomitiformis

364 [175]. In B. alba, sulfur stored by filaments was reduced to sulfide under short-terra anoxic conditions [176]; this sulfur reduction was linked to the endogenous oxidation of stored carbon, possibly p9ly-B-hydroxybutyric acid [176a] and to hydrogen oxidation [176]. The cyanobacterium Oscillatoria limnetica is able, in the dark, under anaerobic conditions to reduce sulfur to sulfide with intracellular polyglucose as electron donor [177]. A very slow H,S production from intracellular and extracellular elemental sulfur has also been obser':ed in the course of the fermentative, anaerobic dark metabolism of phototrophic green and purple sulfur bacteria [174,178-181]. The true or obligate dissimilatory sulfur-reducing microorganisms are strict anaerobic bacteria for which the reduction of elemental sulfur is the essential metabolism for growth. These strict sulfur reducers belong to ~,he genus Desuifuromonas, which is a rather homogeneous g~oup of rodshaped to slightly curved motile organisms [151153]. The genus Desulfuromonas comprises three species: Drm. acetoxidans, Drm. acetexigens and Drm. succinoxidans; all the species oxidize acetate to CO: linked with the reduction of So to H2S. DesulJuromonas species are not able to reduce sulfite, sulfate, thiosulfate or other oxidized sulfur or nitrogen compounds [151,153]. Most Desulfuromonas species are capable of growth with fumarate or malate as terminal electron acceptor instead of sulfur [153]. Drm. acetoxidans strain PM1 has recently been found to be able to convert betaine into trimethylamine and carbon dioxide in the presence of elemental sulfur as electron act ptot [182]. Strictly assimilative processes in the presence of elemental sulfur have seldom been described. However, it is possible that such an ability is actually underestimated since it is often not investigated in the characterization of microorganisms. R. sulfoviridis is not able to use sulfate as sole sulfur source but it can grow with colloidal sulfur [10] and photoheterotrophicaily-grown cultures of R. adriatica are able to assimilate the latter compound [183]. Assimilation of elemental sulfur has also been described in the propionic acid bacterium Pb. shermanii [14,184]. A mutant of E. coli B can assimilate elemental sulfur but

not the parental strain [15]; such a peculiarity has been related to an outer membrane alteration. N. gonorrhoeae and Ps. aeruginosa grow satisfactorily with sulfur flower; however, important alterations of their ultrastructures have been observed [6,16]. The physiology of sulfur assimilation is poorly understood and it is not known how sulfur granules can reach the cytoplasmic membrane where their reduction would occur or if a cytochrome is involved in sulfur assimilation. Since sulfur is very insoluble, extracellular enzymes might be involved ;.n its utilization.

6.2. Archaebacteria The archaebaeteria consist of three main physiological groups: the methanogens, the sulfur-dependent extreme thermophiles and the extreme halophiles; only the genus Thermoplasma does not fall into any of these groups but occupies an intermediate position [185-191]. The present understanding of reduetive sulfur metabohsm in this group of microorganisms is reviewed in this section.

6.2.1. Methanogenic bacteria Methanogenie bacteria make up one of tile larger divisions of the archaebaeteria [192-196]. Virtually all methanogens can carry out the overall reaction: 4 H2 + CO2 -'* CH4 + 2 H20 They use this reaction to generate energy. The present understanding of sulfur nutrition and metabolism in these bacteria is limi'ed [194]. All methanogens can use sulfide as a sulfur source. Sulfate is generally not a sulfur source for methanogens [194,197-204]. Metbanococcus ( Mc.)

thermolithotrophicus, Mc. jannaschii, Methanob.:cierium (Mb.) thermoautotrophicum strains Ma':burg and z3H, Methanobrevibacter (Mbr.) ruminantium and Methanosarcina (Ms.) barkeri strain 227 grow well with elemental sulfur, sulfite or thiosulfate as the sole sulfur source [202,203]. Some species are also able to use cysteine [197,198,203208] or methionine [201,207]. Ms. barkeri strain Fusaro (DSM 804) was able to use thio.~ulfate, but not sulfate or sulfite, as the sole sulfur source

365 [204]. The ability to reduce sulfite, thiosulfate and elemental sulfur, b u t not sulfate, appears to be widespread a m o n g methanogens [202-204,209211]; only Me. thermolithotrophicus and Mbr. ruminantium have been demonstrated to use sulfate. Thiosulfate and sulfite reductase activities have been detected in the crude extracts of Ms. barkeri strain MS (DSM 800) [212], Ms. barkeri strain 227, Mb. thermoautotrophicum strain Marburg and Me. thermolithotrophicus (B.S. Rajagopal a n d L. Daniels, unpubfished results; Table 4). Ms. barkeri strain Fusaro has only thiosnlfate but not sulfate or suifite reductase activity [204]. A sulfite reductase, the P590, has been isolated and characterized from Ms. barkeri D S M 800 [212-214]. It is a low molecular weight (23 kDa), low-spin "assimilatory-type' sulfite reductase containing one siroheme and one 4 Fe-4 S cluster per polypeptide chain, which has also been found in Drm. acetoxidans [214]. The physiolosical sisnlficance

of this enzyme is still not understood. Peck a n d Lissolo i215] have suggested that this new group of low-spin sulfite reductases may functioa in a dissiwilatory mode possibly in the anaerobic disproportionation of thiosulfate a a d sulfite to sulfate and sulfide. Sulfur reductase acfi-~ty has not been well studied in methanogvns. In culture, most species can produce high levels of sulfide ( > 5 raM) from elemental sulfur, using H 2 as the source of electrons. Although the reaction: H 2 + S O~ H2S(z~G~ = - 2 7 . 9 k J / m o l ) could in theory provzde a source of cnersy, this has not been examined fully in methanogens. Tiffs is an example of "excessive assimilatory sulfur metabofism" alluded to in the introduction. T h e sulfur reductase activity is present in crude extracts of Ms. barkeri strain 227, Mb. thermoautotrophicum strain Marburg a n d Mc. thermolifhotrophicus (Table 4) and this activity appears to be

Table 4 Reduction by hydrogen of inorganic sulfur compounds by crude extracts of methanogens Substrates

Specifi,: activity (nmol H2 oxidized-min - I. ms protein - t ) Methanosarcinabarkeri DSM 800 a 227 b

None Na2SO3 Na2S203 Na 2S306 Na2SO4 + ATP

0 18.0 5.0 8.0 0

S°

N.D.

Substrates

None Na2SO3 Na2S203 Na2S306 Na2SO 4 + ATP s°

Fusaro c (DSM 804)

Methanococc~ thermolithotrophicusb

0 7.5 3.0 N.D. 0

0 0 5.21 N.D. 0

0 12.0 6.0 N.D. 3.0

6.0

N.D.

8.5

Specific activity (nmol H 2 oxidized-ndn - I. mg protein - ]) Methanobacteriumthermo¢"totrophicum Marburg b AH c

Methanobacterium strain HU e

0 10.5 4.0 N.D.

0 N.D. 2.28 N.D.

0 N.D. 24.25 N.D.

0 8.0

N.D. N.D.

N.D. N.D.

° Manometric assay [212]. b Sulfide determination (Rajagopal, B.S. and DaniOs, L., ui+ablished results). © Sulfide determination [204]. N.D. = not determined

366 constitutive (B.S. Rajagopal and L. Daniels, unpublished results). Interestingly, Methanospiriilum (Msp.) hungatei also showed sulfur reduetase activity in extracts, although it was not able to use elemental sulfur as the sole sulfur source for growth [202]. The characterization of enzymes capable of reducing su~f'ar in methanogens a n d sulfur-dependent extreme thermophiles is a n interesting topic for future studies. Thus, the recent data suggest that methanogens have a variety of enzymes capable of reducing various sulfur compounds like elemental sulfur, sulfite and thiosulfate. Sulfate cannot be used as a significant source of sulfur by most methanogens [197-204,208,216-218]. The marine environment is high in sulfate and it is reasonable t o expect that some marine isolates of methanogens are able to reduce sulfate and use it as sulfur source. However, only one out of six marine isolates so far examined (i.e. Mc. thermolithotrophicus) use,.; sulfate as the sole source of sulfur [202,203]. This suggests that it is not a c o m m o n attribute ~vithin methanogens in this environment. However, since in some anaerobic environments sulfate is reduced to sulfide by sulfa:e reducers, this will be available to methanogens in nature. As well, in an environment at the interface between aerobic and anaerobic zones, e.g. at the waterline on offshore platforms and piers, sulfide produced will be oxidized at some times by air, chemically, to form S °, which in an appropriate micrcenviro~ament can serve as a sulfur source for methanogens. 6.2.2. Sulfur-dependent extreme thermophilic bacteria The sulfur-dependent archaebacteria include sulfur-oxidizing and -reducing Suifolobales and sulfur-reducing Thermoproteales and Thermococcales [185-191,219]. Table 5 presents information about growth conditions and substrates for a variety of genera a~ld species of sulfur-dep rodent archaebacteria. Table 6 describes the energy.yielding reactions used by these archaebacteria. 6.2.2.1. Suifolobales. The genus Sulfolobus comprises three reco_~aized species, namely Sulfolobus (Sb.) acidocaldarius, Sb. solfataricus and Sb. brierleyi, which are characterized by a n irregular coccoid shape of the cells, the low p H and high

Table 5 Requirements and modes of growth of sulfur-dependent archaebacteria Organisms [reference] Sulfolobales Acidianus infernus [227,228] Ac. brierleyi [227,228] Desulfurolobus ambivalens [231,232] Sulfolobus sp. [187,222,223,256] Thermoproteales Desulfurococcus sp. [238] Dc. amylolyticus [239] Dc. raucosus [187,237] Dc. mobilis [187,237] Pyrobaculum islandicum [246] Py. organotrophum [246] p~rodictiumbrockii [187,243-245] Pd. occultum [187,243-245] Thermodisces spp. [187,243-245| Td. maritimus [187,243-245l Thermofilumpenaens [187,240] Thermoproteuatenax [187,235] Tpr. neutrophilus [187,235,245] Staphylothermus marinus [248] Thermococcales Pyrococcusfuriosus [242] Pc. woesei [191] Thermococcusceler [241] Other Caldococcuslitoralis [251]

Temperature (°C) pH Optimal Range Optimal Range

85

65- 95 2.0

1.0-5.0

75

?

1.0-7

80

70- 87 2.5

1.0-3.5

70-80

55- 87 2 -3.5

1.0-5.9

85-90

55- 94 7.0-7.5

4.5-7.0

90-92

?

?

85

75- 93 6.0

4.5-7.0

85

75- 93 6.0

4.5-7.0

100

7-102 6.0

5.0-7.0

100

?-102 6.0

5.0-7.0

105

85-110 5.5

5.0-7.0

105

85-310 5.5

5.0-7.0

85

75- 98 5.5

5.0-7.0

88

75- 98 5.5

5.0-7.0

88

80- 95 5.5

4.0-6.5

88

80- 95 5.0

2.5-6.0

85

80- 95 6.8

5.0-7.5

92

65- 98 6.5

4.5-8.5

100

?-103 7.0

5.0-9.0

100

7-105 6.2

6.0-6.5

92

7- 93 5.8

4.0-7.0

88

55-100 6.4

5.9-7.0

3.0

6.4

367 Table 5 (continued) Organisms [references]

Table 5 (continued) Growth Heterotrophic (by SO respiralion)

Sulfolobales Acidianu~ infernus [227,228] Ac. brierleyi [227,228] Desu/furo/obus ambivalens 1231,232] Sulfolobus sp. [187,222, 223,256] +

Chemolitho- In Stimutrophic absence latory (so/H2 of So factors autotrophy)

+

=

+=

+ =

-

YE

+b

YE

-

YE

[251]

+

YE

+

+

-

+

-

+

YE

+

-

+

YE

+

+

-

-

+

-

-

-

-

+

-

YE

(obfigate) Pd occultum [187,243-245] Thermod/scus Spp. [187,243-245] TeL maritimus [187,243-245] Thermofllum pendens [187,237]

-

+ (obfigate)

-

YE

+

-

+

YE

+

YE

+

+

+

-

sucrose, starch

+

+

YE

+ (obligate)

YE

Thermoprmeus teno~¢

[187,235] Tpr. neutrophilus [187,235,245] Staphylothermus mar/nus

[248]

Therm,ococcales Pyrococcus furious 12421 Pc. woesei [191] Therraococc~ ~e/er 1241]

+

_

Growth Heretotrophic (by S° respiration)

Chemolitho- In Sfimutrophic absence latory (S°/H2 of S° factors autotrophy)

+

-

+

+

-

+

+

-

+

Other Cal~c~c~z litoralis

+ (some)

Therraoproteales Desulfurococcus sp. [238] Dc. amylolyticus [239] De. "nucosus [187,237] De. mobilis [187,237] Pyrobacu/um islandicum [246] Py.orsanotrophum [2,~1 Pyrodictium brockii [187,243-245]

Organ~ms [references I

+

+

YE

a Facultative aerobe. b Aerobic (02 respiration). ? = no data. YE = yeast extract.

t e m p e r a t u r e o p t i m a for g r o w t h , t h e l/pid c o m p o s i tion a n d t h e c o m m o n m o d e o f c h e m o l i t h o t r o p l f i c energy conservation by oxidation of elemental s u l f u r [187,219-226]. Alternatively, t h e s e rn/croo r g a n i s m s are able to g r o w u s i n g o r g a n i c m a t e r i a l s in t h e a b s e n c e o f s u l f u r a s t h e e l e c t r o n d o n o r [222,226]. Segerer et al. [227] h a v e isolated a g r o u p o f t h e r m o p h i l i c solfatarlc a x c h a e b a e t e H a t h a t axe able to g r o w either strictly a n a e r o b i c a l l y b y r e d u c tion, o r fully aerobically b y o x i d a t i o n o f m o l e c u l a r sulfur, d e p e n d i n g o n o x y g e n s u p p l y . T h e abifiO to g r o w in t h e s e two w a y s is s h a r e d b y Sb. brierleyL Recently, Segerer et al. [228] a s s i g n e d t h e s e n e w isolates t o g e t h e r w R h Sb. brierleyi to a n e w g e n u s Acidianus and described Acidianus ( Ac.) infernus and Ac. brierleyi. Earlier this g e n u s w a s referred to as Acidothermuz [229,230]. Ac. infernus is a n o b l i g a t e s u l f u r - d e p e n d a n t chemoiithotroph g:owing by either oxidation or r e d u c t i o n o f e l e m e n t a l s u l f u r [227,228]. U n d e r a n a e r o b i c c o n d i t i o n s , g r o w t h d e p e n d s strictly o n h y d r o g e n a n d sulfur; h y d r o g e n c a n n o t b e r e p l a c e d b y a n y o r g a n i c s u b s t r a t e (glucose, lactose, acetate,

368 Table 6 Energy-yielding reactions of sulfur-metabolizing archaebacteria Mode of nutrition

Metabolism

Energy-yielding reaction Energy available per 2 • (AG~ in kJ)

Organisms [references]

Chemolithotrophic

S°/H2 autotrophy

H 2 + S° ---*H2S (-27.9)

Pyrodictium occultum and PcL brockii [244] Thermoproteus tenax ~ [235,245] Tpr. neutrophilus[245] Acidianus infernus b and Ac. brierleyi b [227,228] Desulfurolobus ambivalens b

[231,232] Pyrobaculum islandicum [246] Thermodiscus maritimus a

[187,243-2451 Sulfur oxidation

2 S ° + 3 02 + 2 H20 ~ 2 H2SO4 (-182.4)

Sulfolobus acidocaldarius a, and Sb. solfataricus a

1221-224,2261 Sulfolobus isolates TH2 m

and Kra 23 [233]

Heterotrophic

Pyrite oxidation

4 FeS2 + 15 02 +2 H20 2 Fe2(SO4) 3 +2H2SO4 (9.)

Sulfur respiration

'organic' [H] + SO--, H2S + CO2 e.g. pyruvate + 3 H 2 0 + 5 So 5 H2S+3 CO 2 (-14.3)

Unknown anaerobic respiration

Yeast extract ---,CO 2 + ? (9.)

Ferraentation

Yeast extract --* CO 2 + 9.(9)

Ae. brierleyi a,b [227,228] Ac. infernus b [227,228] Ds. ambivalens b [231,232] Sulfolobus isolates TH2 a,

Kxa 23 and VE 2 [233] Tpr.tenax a [235] Desulfurococcur mobilis and Dc. mucosus [237] Desulfurococcus sp. [238[ Dc. amylolyticus [239] Thermofilum pendens [240] Thermococcur celer [241] Pyrococcus furiosus ¢ [2421 Pc. woesei a [191] Py. islandicum a and Py. organotrophum [246] Therrr.odiscus sp. and Td maritimus a

[187,230,243-2451 Caldococcus litoralis [251] Dc. mobilis and De. mucosus

[2371 Dc. amylolyticus [239] Tc. celer [241] Thermodiscus species and T¢£ mc,ritimus a [187,230,244] Pc, furiosus ©[242] Pc. woesei d [191] Stophylothermus marinus ©

[24S] Glucose ~ H 2 + C O 2 (9.)

Cd. litoralis [251] Dc. saccharovorans [187,229]

369 Table 6 (continued) Mode of nutrition

Metabolism

Respiration

Energy-yieldh~g reaction Energy available per 2 e(AG~ in IcY) Oxygen respiration 'Organic' [ H ] + O 2 --*2 H 2 0 + C O 2 e.g.Glucose+ 6 0 2 ~ 6 H 2 0 + 6 C O 2 (-470.3)

Molybdate respiration (?) Dissimilatory sulfate reduction

Carbohydrate [H] + SO~- ~ S2e.g. 2 Lactic+ SO4z2 Acetic + COz + S2- + 2 H20 ( - 29.8)

Orgamsms [references[

Sb. acidocaldarius a and Sb. solfataricus a [222-224,2261 Sulfolobus isolate TH 2 [233] Ac. brierleyi a [228] Sb. acidocaldari~ [221] Ac. brierleyi and Ac. infernr¢ [228] Archaeoglobuxfulgidus [252.254[

a Facultatively autotrophic. b Facultatively aerobic. c Growth with SOonly in presence of excessive amounts of hydrogen:hydrogen strongly inhibits growth and is removed due to formation of H2S. d G:~wth with yeast extract or polysaccharides in the absence of sulfur, provided hydrogen is present. e Obfigatofily dependent on elemental sulfur.

peptone, etc.) and elemental sulfur cannot b e replaced by thiosulfate, sulfite or sulfate. Ac. brierleyi is also able to grow by reduction of sulfur with hydrogen, b u t Sb. acidocaldaritts and Sb. solfataricus are unable to grow by this metabolic pathway. U n d e r aerobic conditions, Ac. infernus and Ac. brierleyi grow by oxidation of sulfur forming sulfuric acid [227,228]. In contrast to Sb. acidocaldarius, Sb. solfataricus and Ac. brierleyi, Ac. infernus is not able to oxidize organic comp o u n d s without sulfur and is therefore a strict chemofithotroph. Zfllig et al. [231] have also isolated a strain from a solfataric water hole in Iceland (which a p p e a r e d along with Thermoproteus in a n anaerobic enrichment v~ture) that was also capable of chemolithotrophic growth by either sulfur oxidation or reduction. This isolate, provisionally called Sb. ambivalens, has been n a m e d Desulfurolobus (Ds.) ambivalens [232]. Ds. ambivalens is a facultative anaerobe capable of utilizing C O 2 as the sole carbon source a n d either sulfur and oxygen (yielding sulfuric acid) or sulfur a n d hydrogen (yielding H2S ) as energy sources. It contains several plasmids, one of which CuSL 10)

is amplified during anaerobic growth. A subclone of Ds. ambivalens, without the pSL 10 plasmid, was still able to grow by oxidation or reduction of sulfur ruling out a role for this plasmid in chemolithoautotrophic growth [231,232]. The relationship of Ds. ambivalens to Ac. infernus and Ac. brierleyi is yet to be determined quantitatively. Their O + C% are similar (32.7 tool% for Ds. ambivalens, a n d 31 tool% for Ac. infernus and Ac. brierleyi) b u t different from the values characteristic of Sulfolobus species (about 38 tool%) [226-228,232]. However, their maximum growth temperatures are different (73°C, 87°C and up to 95°C for Ac. brierlyi, Ds. ambivalens and Ac. infernus, respectively) a n d Ds. ambivalens and Ac. infernus are obfigately chemolithotrophic and sulfur-dependent (under b o t h aerobic a n d anaerobic conditions); in contrast Ac. brierleyi is capable of organotrophic growth without sulfur in the presence of oxygen a n d yeast extract, peptone, tryptone, beef extract or casamino acids [228,233]. The absence of a significant D N A - D N A cross-hybridization, the large disparity in thermotolerance and the differences in the mobilities of homologous R N A polymerase components rule out a

370

close relationship of Ac. brierleyi and Ds. ambivalens [232]. The latter is probably more closely related to Ac. infernus.

6.2.2.2. Thermoproteales and Thermococcales. All characterized Thermoproteales and Thermococcales grow by sulfur respiration of organic matter or by a pure chemolithotrophic metabolism using hydrogen and elemental sulfur as energy source and CO 2 as carbon source [187,229,230,233,234]. Thermoproteales comprises three genera: Thermoproteus [235,236], Desulfurococcus [237-239], and Thermofilum [240]. Thermococcales are represented by: Thermococcus [241] and Pyrococcus species [191,242]. Pyrodictium and Thermodiscus species [243-245] are considered to be members of Thermoproteales because of their oxidative metabolism in the presence of sulfur [187]. However, their taxonomic position remains to be more precisely determined; according to the study of 16 S and 5 S rRNA catalogues (still preliminary), these species appear more closely related to Sulfolobus than to Thermoproteus [187,188,229,230,234]. The most recently described Pyrobaculum species resembles Thermoproteus by its sulfur oxidative metabolism and its morphology [246]. However, the phylogenetic distance between these two species is evident since they lack significant DNA homology [246]. Pyrodictium and Pyrobaculum have higher optimal growth temperatures (100105°C) than Thermoproteus spp. (88°C). Thus, Pyrodictium and Pyrobaculum together with Thermodiscus probably represent an intermediate group between Sulfolobales and Thermoproteales. The exact taxonomic position can be determined only after complete phylogenetic analysis between these organisms and members of Sulfolobales and Ther-

moproteales. Thermoproteus (Tpr.) tenax and Tpr. neutrophilus are able to grow chemolithotrophically with hydrogen plus elemental sulfur as energy source and CO2 as the sole carbon source [235,245]. Under these conditions, a trace of yeast extract (0.02~) stimulates growth, but is not essential. Most Thermoproteus species grow heterotrophicall), by sulfur respiration of organic compounds forming CO2 and H2S, and Tpr. tenax is able to use many organic compounds (e.g. glucose, methanol, fumarate) and even carbon monoxide

as sources of energy and carbon [235]. In the absence of sulfur, cysteine or malate can serve as the terminal electron acceptor [187,235]. Some isolates do not form significant amounts of H2S during heterotrophic growth, even in the presence of sulfur, while autotrophic growth is strictly dependent on H2S formation [230]. ChemoHthotrophlc growth has not been o',=~served with Desulfurococcus, Thermofilum, Thermococcus and Pyrococcus species, all of which live by sulfur respiration of substrates like yeast extract, peptides (tryptone) or proteins (casein) but not of casamino acids [191,237-242]. Desulfurococcus [237] and Thermococcus [241] can grow in the absence of sulfur, though with considerably lower rate and yield. Little CO 2 is produced under these conditions, implying that energy is conserved by some unknown mode of fermentation. Thermofilum has unusual growth factor requirements and it can be grown as a pure culture by the addition of cell debris or polar lipid fractions of Tpr. tenax, but not from Thermoplasma (Tp.) acidophilum [240]. Pyrococcus (Pc.) furiosus is capable of beterotrophic growth on peptone, tryptone, yeast extract, meat extract, extracts of eubacterial and archaebacterial cells, casein, maltose and starch as carbon and energy sources [242]. The presence of hydrogen as a fermentation product strongly inhibits growth. This phenomenon can be prevented by addition of sulfur since hydrogen is removed due to formation of H2S. However, Pc. furiosus does not appear to obtain energy from sulfur respiration since growth rates are shnilar both in the presence and absence of sulfur when hydrogen is removed by flushing with nitrogen [242]. In contrast Pc. woesei grows by sulfur respiration, preferably with tryptone or yeast extract as carbon sources and grows poorly on polysaccharides, unless hydrogen and sulfur are present [191]. In the absence of sulfur, it can grow with yeast extract, but not tryptone, provided hydrogen is present. A marine archaebacterial isolate, strain NS-C, grew better in the presence of sulfur, with exponential H2S production [247]. Staphylothermus (St.) marinus is an obligate heterotroph, growing optimally on a mixture of yeast extract (0.1~) and peptone (0.5%) in sea water; nonetheless it shows

an o~ligate dependence on elemental sulfur, although only relatively low amounts of H2S are formed during growth [248]. The type of metabolism of St. marinus is still unknown: CO2, acetate and isovalerate have been detected as products suggesting a fermentative metabolism. Pyrodictium species [Pyrodictium ( Pd.) occulture and Pd brockii] grow by a strict sulfur-hydrogen autotrophy similar to Tpr. tenax and Tpr. neutrophilus [243,245]. Yeast extract and peptone stimulate the growth and production of sulfide, but these substrates are unable to serve as energy sources [244,249,250]. Pyrodictium species have an optimal growth temperature around 105°C [244] and sulfide production (chemical) in uninoculated controls is noticeable at these temperatures, but substantially below the levels observed in inoculated media [249,250]. Thermodiscus can grow heterotrophically with yeast extract and sulfur by sulfur respiration [187,229,230,234] but all strains also show good growth on yeast extract without sulfur and hydrogen [187,230]. Sometimes, Thermodiscus species do not form significant amounts of H2S even at optimal growth. In some isolates, growth is stimulated by hydrogen. A new marine, halophilic and extremely thermophilic, coccoid sulfur-reducing arehaebacterium, Caldococcus litoralis strain Z1301, has been recently described [251], It differs from all other extremely thermophilic organoheterotrophic sulfur-reducing cocci previously characterized (e.g. Desuifurococcus and Thermococcus species) and can be considered as a representative of an independent taxon [251]. Solfite, thiosulfate and sulfate do not ~rve as electron aeccptors for the chemolithotro~hic or organotrophic growth of Thermoproteales and Thermococcales [187]. However, some strains of the recently isolated Pyroboculum species are capable of using sulfite or thiosulfate as electron accep'.ors during organotrophic growth on yeast extract, peptone and extracts of eubacterial or archaebacterial cells [246]. These species also grow heterotrophically by respiration of elemental sulfur, L(-)cystine or oxidized glutathione. Pyrobaculum species also grow chemolithotrophically with sulfur and hydrogen as energy source and CO 2 as the carbon source. Sulfur could not be

replaced by any other mineral or organic sulfur compounds during chemolithotrophic growth of Pyrobaculum species [246]. The members of Thermeproteales, espe~ally Therraoproteus species, are able to emulsify the sulfur used as electron a~-ptor [235,237,240,241]. In growing cultures of Therraofilum (Tf.) pendens, the usually evenly distributed sulfur changes its structure drastically, forming either needles with rounded edges or even semisolid droplets at temperatures where normally the orthorhombic forms of sulfur are stable [240]. During chemolithotrophic growth of Thermoproteus, about 1.5 tool of H,S are produced per gram of cell mass (dry weight) formed [235]. The corresponding figure for growth by sulfur respiration is 0.4 tool for Therraoproteus [235] and 0.044 tool of H2S per gram of cell mass for Desulfuwcoccus [237]. At extreme temperatures (96°C), the growth yield of Thermoproteus species (heterotrophic growth) relative to H2S formation decreases steeply during stationary phase, suggesting uncoupled H2S production. High concentration of H2S (more than 0.2 arm.) inhibits growth of Thermoproteus and Desuifurococcus [235,237]. Hydrophobic sulfur compounds such as (CH2)2S3 and (CH2)2S5 have been isolated from the residual sulfur of chemolithotrophi¢ Thermoproteus cultures [187]. Soon after the start of such cultures, the previously fight yellow sulfur turns grayish grecn and becomes evenly distributed in an almost colloidal manner and the smooth surface (previous) of the sulfur grains turns rough [187]. Desuifurococcus [237] and Thermococcus [241] produce a characteristically fetid compound, possibly a mercaptan, when grown by sulfur respiration. Archaeoglobus ( Ag.) fulgidus, the recently isolated sulfate-reducing archaebacterium [252-254] represents a novel phenotype and does not fit into any of the three basic phenotypes (methanogens, snlfur-dependent extreme thermophiles or extreme halol:b.fles). This organism is able to grow by dissimilatory reduction of sulfate, sulfite or thiosulfate but not elemental sulfur, and makes very small quantities of methane, although it lacks some of the cofactors associated with methanogenesis [252,254]. 16 S rRNA sequencing indicates that the lineage represented by Ag. fu/g/dus arises be-

372 tween Methanococcus (deepest of the methanogens branchings) and Thermococcus (the deepest of all branchings on the methanogen side of the tree) on the phylogenetic tree for archaebacteria [253]. Based on its intermediate metabolism between methanogenesis and sulfur reduction of extreme thermophiles, and phylogenetic considerations, Ag. fulgidus may represent a transition stage in the evolution of (mesophilic) methanogenesis from sulfur based thermophific metabolism [2522541.

6.2.3. Extreme halophiles and Thermoplasma Nothing much is know~ about sulfur metabofism in halobacteria [255] and Thermoplasma species [187,256,257]. Brock has observed that although the thermoacidophile Thermoplasma did not grow on elemental sulfur, it reduced sulfur to H2S in presence of yeast extract [256]. Anaerobic growth of Tp. acidophilum and Tp. volcanium is strongly enhanced by elemental sulfur, which is reduced to H2S [257].

7. CONCLUSIONS Thiosulfate, polythionates or elemental sulfur are essential for the growth of numerous bacteria and they can be used instead of sulfate by many others. In some environments, thiosulfate or elemental sulfur could be the only sulfur sources that microorganisms can utilize. The ability for metabolizing these compounds is then a selective advantage. In all bacteria so far studied, thiosulfate cleavage is performed by one of the enzymes described in Section 4.: thiosulfate reductase a n d / o r polythionate reductase, thiosulfate sulfur transferase and S-sulfocysteine synthase. The broad substrate specificity of these enzymes is noteworthy. It could be related to an analogy of the chemical structures of thiosulfate and polythionates. However, for the moment, an enzyme s~r~fi~.~h'y responsible for the assimilation of thiosulfate and/or polythionates has not been described. Thus the possibility exists that assimilation of these compounds is just an artifact observed in vitro due to enzyme activities which are

not present in the bacterial environment. However, given the widespread ability among genera for the reduction or the cleavage of these molecules their metabolism could be a fundamental process, using them either as intermediate compounds or as substitute sulfur sources. According to this hypothesis, the importance of thiosulfate and polythionate metabolism could be underestimated. The assimilation of elemental sulfur is a somewhat di.fferent problem since this element is very common in the environment. Although few data are available, sulfur reduction could also be a widespread characteristic in procaryotes. Methanogens are similar to the anaerobic sulfur-reducing archaebacteria by virtue of their ability to reduce molecular sulfur at high rate. The archaebacteria are thought to have diverged from other microorganisms early in the evolution of the biosphere and the reduction of sulfate (and sulfite) is also a very ancient process [258]. It could then be possible that Ag. fulgidus, a true archaebacterinm able to reduce sulfate, is an ancestral sulfate re~ ducer. It is possible that sulfur reduction is a primitive means of energy conservation and may have been the forerunner of energetically more efficient methanogenesis [259]. Alternatively, the ability of methanogens to reduce sulfur may be the result of high levels of enzymes that function in an unrelated path, and by chance are able to reduce sulfur. Comparison of both metabolic types at the molecular level may shed further light on the evolution of both groups of organisms. However, the physiological significance of elemental sulfur assimilation remains unknown and it is not elucidated if a sulfur reductase is involved specifically in this process.

APPENDIX

Abbreviations TST, thiosulfate sulfur transferase; TSR, thiosulfate reductase; TRTR, trithionate reductase; TTR, tetrathionate reductase; SSCS, S-sulfocysteine synthase; TPP, thiamine pyrophosphate; PAPS, phosphoadenosine phosphosulfate; GSH,

3"/3 r e d u c e d glutathione; IA, i o d o a c e t a t e ; I A M , iodoacetamide; /3-ME, fl-mercaptoethanol; N E M , N-ethyl-maleimide; YE, yeast extract; D S M , Deutsche Sammlung yon Mikroorganismen, Braunschweig, F.R.G.; N C I B , N a t i o n a l Collection o f Industrial Bacteria, A b e r d e e n , U.K.; A T C C , A m e r i c a n T y p e Culture Collection, Rockvillc, M D . , U.S.A.

Genus names Acidianus, Ac.; Acinetobacter, A.; Archaeoglobus, Ag.; Beggiatoa, B.; Caldococcus, Cd.; Chlorobium, Chb.; Ckromatium, Ch.; Clostridium, C.; Desulfotomaculum, Dm.; Desulfovibrio, D.; Desulfurococcus, Dc.; Desulfurolobus, Ds.; Desulfuromonus, Drm.; Escherichia, E.; Methanobacterium, Mb.; Methanobrevibacter, Mbr.; Methanococcus, Mc.; Methanosarcina, Ms.; Methanospirillum, Msp.; Neisseria, N.; Propionibacterium, Pb.; Proteus, P.; Pseudomonas, Ps.; Pyrobaculum, Py.; Pyrococcus, Pc.; Pyrodictium, Pd.; Rhodopseudomonas, R.; Salmonella, S.; StaphyIothermus, St.; Sulfolobus, Sb., Thermoanaerobium, Ta.; Thermococcus, Tc.; Thermodiscus, Td.; Thermofilum, Tf.; Thermoplusma, Tp.; Thermoproteus, Tpr.; Thermotoga, Tt.; Thiobacillus, T.; Thiocapsa, Tcp.; Wolinella, W. G i v e n the s t r o n g morphological similarities bet w e e n D. baculatus ( D S M 1743) and D. desulfuricans N o r w a y 4 ( D S M 1741), we have followed Postgate's suggestion [123] that the latter strain should b e reclassified as D. baculatus.

ACKNOWLEDGEMENTS W e t h a n k Pr. J. Le Gall for having p r o p o s e d the topic o f this review ~nd for his s u p p o r t a n d e n c o u r a g e m e n t t h r o u g h o u t its completion. P a r t o f this w o r k was m a d e possible t h a n k s to a special collaborative a g r e e m e n t b e t w e e n the University o f Georgia: (U.S.A.) a n d l'Associafion p o u r la Rec h e r c h e e n Bio~nergie Solai.-e (~"rance) (B.S.R. a n d G.F.). L.D. a n d B.S.R. are appreciative o f s u p p o r t f r o m Office o f N a v a l R e s e a r c h G r a n t No. 0001488-K-0195.

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