Physiology and Genetics of Sulfur-oxidizing Bacteria

Physiology and Genetics of Sulfur-oxidizing Bacteria Cornelius G. Friedrich

Lehrstuhl fur Technische Mikrobiologie, Fachbereich Chemietechnik, Universitat Dortmund, 0-44221 Dortmund, Germany

ABSTRACT Reduced inorganic sulfur compounds are oxidized by members of the domains Archaea and Bacteria. These compounds are used as electron donors for anaerobic phototrophic and aerobic chemotrophic growth, and are mostly oxidized to sulfate. Different enzymes mediate the conversion of various reduced sulfur compounds. Their physiological function in sulfur oxidation is considered (i) mostly from the biochemical characterization of the enzymatic reaction, (ii) rarely from the regulation of their formation, and (iii) only in a few cases from the mutational gene inactivation and characterization of the resulting mutant phenotype. In this review the sulfur-metabolizingreactions of selected phototrophic and of chemotrophic prokaryotes are discussed. These comprise an archaeon, a cyanobacterium, green sulfur bacteria, and selected phototrophic and chemotrophic proteobacteria. The genetic systems are summarized which are presently available for these organisms, and which can be used to study the molecular basis of their dissimilatory sulfur metabolism. Tbo groups of thiobacteria can be distinguished: those able to grow with tetrathionate and other reduced sulfur compounds, and those unable to do so. This distinction can be made irrespective of their phototrophic or chemotrophic metabolism, neutrophilic or acidophilic nature, and may indicate a mechanism different from that of thiosulfate oxidation. However, the core enzyme for tetrathionate oxidation has not been identified so far. Several phototrophic bacteria utilize hydrogen sulfide, which is considered to be oxidized by flavocytochrome c owing to its in vitro activity. However, the function of flavocytochrome c in vivo may be ADVANCES IN MICROBIALPHYSIOLOGY VOL 39 ISBN 0-12-027739-5

Copyright 0 1998 Academic Prws Limited All rights of reproduction in MYform reserved

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different, because it is missing in other hydrogen sulfide-oxidizing bacteria, but is present in most thiosulfate-oxidizingbacteria. A possible function of flavocytochrome c is discussed based on biophysical studies, and the identification of a flavocytochrome in the operon encoding enzymes involved in thiosulfate oxidation of Paracoccus denitrificans. Adenosine-5’-phosphosulfatereductase thought to function in the ‘reverse’ direction in different phototrophic and chemotrophic sulfur-oxidizing bacteria was analysed in Chromatiurnvinosum. Inactivation of the corresponding gene does not affect the sulfite-oxidizing ability of the mutant. This result questions the concept of its ‘reverse’function, generally accepted for over three decades. 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Aerobic sulfur oxidation in Sulfolobales . . . . . . . . . . . . . . . . . . . . . . . 2.1. Sulfur oxygenase-reductaseof Acidianus ambivalens . . . . . . . . . . . . . 2.2. Gene transfer systems in Archaea . . . . . . . . . . . . . . . . . . . . . . . 3. Sulfur oxidation in cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Hydrogen su1fide:quinone reductase of Oscillatoris limnetica . . . . . . . . . 3.2. Gene transfer systems in cyanobacteria . . . . . . . . . . . . . . . . . . . . 4. Sulfur oxidation in green sulfur bacteria . . . . . . . . . . . . . . . . . . . . . . . 4.1. Sulfur-oxidizingenzymes of Chlorobium . . . . . . . . . . . . . . . . . . . . 4.2. Gene transfer systems in green sulfur bacteria . . . . . . . . . . . . . . . . 5. Sulfur oxidation in Proteobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Phototrophic Proteobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Sulfur oxidation in nonphototrophic bacteria . . . . . . . . . . . . . . . . . 6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

236 238 239 243 244 244 245 248 248 249 251 252 259 274 276 276

1. INTRODUCTION Inorganic reduced sulfur compounds can be used by various prokaryotes as electron donors for phototrophic or lithotrophic growth. Dissimilatory sulfur oxidation occurs under anaerobic and aerobic growth conditions, mostly with sulfate as the major oxidation product. The capacity for sulfur oxidation is found in the domains of the Arrhaea and Bacteria, while in the Eukarya dissimilatory sulfur oxidation is mediated by lithotrophic bacterial endosymbionts. Within the domain Archuea, dissimilatory sulfur metabolism is present in the Crenarchueota and Euryarchueota. Sulfate or sulfur reduction is common in anaerobic metabolism among different genera of thermophilic or extreme thermophilic Archaea with molecular hydrogen, carbohydrates or peptides as electron donors. However, aerobic lithoautotrophic sulfur oxidation is restricted within the

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SULFUR-OXIDIZING BACTERIA

CO, HCOOH, H3COH

Figure 1 Interrelationships of lithoautotrophic, organoautotrophic and phototrophic metabolisms with respect to their electron donating growth substrates.

Crenarchueotu to members of the extreme thermophilic obligate acidophilic Sulfolobales. Within the domain Bacteria, reduced inorganic sulfur compounds such as hydrogen sulfide, polysulfide, elemental sulfur, sulfite, thiosulfate or polythionates serve as electron donors for anaerobic phototrophic or aerobic lithotrophic growth. However, in most phototrophic or lithotrophic microorganisms,additional electron donors can be used. Different bacteria may obtain energy from light and use molecular hydrogen, carbon monoxide or other reduced one-carbon compounds, ferrous iron, manganese or other metals as electron donors for anaerobic phototrophic or aerobic lithotrophic growth. Different types of energy metabolism may be found in one strain (Fig. 1); for example, the purple non-sulfur bacterium Rhodobacter capsulatus grows anaerobically in the light or aerobically in the dark with either molecular hydrogen or hydrogen sulfide, and the obligate lithotrophic acidophile Thiobacillus fermoxidans oxidizes aerobically ferrous iron, sulfur, hydrogen or formate. The various lithotrophic or phototrophic metabolisms are not specific properties of specialized prokaqotes but may function in one strain as an alternative mode of life in Bacteria and - with respect to aerobic hydrogen metabolism - also in some Archaea (Huber et al., 1992a). Sulfur oxidation is a major energy source not only for free-living thiobacteria but also for special thiobacteria living in symbiotic, host-specific relationships with invertebrates in hydrothermal vents (reviewed by Nelson and Fisher, 1995; Nelson and Hagen, 1995).The evolutionary relationshipsof the microorganismshave been reviewed by Woese (1987), and with emphasis on sulfur-oxidizingbacteria by Lane et al. (1992). Wherever practical, selected sulfur-oxidizing microorganisms are treated here according to their phylogeneticposition. The physiology and taxonomy of the Archaea have been reviewed previously (Blochl et al., 1995; Schonheit and Schafer, 1995; Stetter et al., 1990). The ecology, physiology and genetics of the cyanobacteria have been reviewed by Cohen and Gurevitz (1992). For detailed

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information on the physiology of phototrophic bacteria and their sulfurmetabolizing enzymes the reader is refened to reviews by Brune (1989), Triiper and Fischer (1982) and Pfennig and Triiper (1992). Assays for several sulfurmetabolizing enzymes have been elaborated as summarized by Dahl and Triiper (1994). The enzymes characterized so far from the colourless thiobacteria have been reviewed by Kelly (1982, 1988, 1990) and Takakuwa (1992), and from the acidophilic thiobacteria by Harrison (1984) and Pronk et al. (1990b). Biochemical studies have merged in hypothetical pathways of sulfur oxidation in various bacteria. However, the significance of some sulfur-metabolizingenzymes and their specific linkage to energy metabolism is still a matter of debate. To elucidate the function and regulation of formation of the enzymes described so far, genetic approaches are indispensable. Gene transfer systems or molecular genetics have been reviewed for the cyanobacteria (Haselkom, 1991;Cohen and Gurevitz, 1992),for Rhodospirillaceae (Donohue and Kaplan, 1991), and for Thiobacillusferrooxidans (Rawlings and Kusano, 1994).The application of genetic and molecular biological techniques has substantially enhanced our understanding of aerobic and anaerobic metabolism of hydrogen oxidation (Friedrich and Friedrich, 1990), methylotrophy (Harms and van Spanning, 1991),other types of energy metabolism,and nitrogen fixation. With respect to sulfur oxidation, however, genetics and molecular analyses are in their infancy due to problems in plating efficiencies, sufficient gene transfer rates via conjugation and lack of alternative energy sources for obligate lithotrophs. The following discussion relates to selected prokaryotes that have been characterized with respect to their physiology or biochemistry of sulfur oxidation. The unique anaerobic energy metabolism via disproportionation of inorganic sulfur compounds as described for Desulfovibrio sulfodismutans (Bak and Cypionka, 1987) is not treated here. This chapter focuses on systems where, besides biochemical characterization, genetics and molecular biology have advanced the understanding of these systems, and points to thiobacteria where genetic systems are available that may have been developed to investigate other characteristics of these strains.

2. AEROBIC SULFUR OXIDATION IN SULFOLOBALES

The unique combination of dissimilatory aerobic sulfur oxidation and anaerobic sulfate respiration in one strain is exclusively found in the order Sulfolobaleswithin the Crenarchaeota (Segerer er al., 1985; Zillig et al., 1985). Desulfurolobus ambivalens is a strictly acidophilic extreme thermophilic and obligately chemolithoautotrophic archaeon able to obtain energy from the aerobic oxidation of sulfur and hydrogen sulfide to sulfuric acid. Desulfurolobus mbivalens also grows lithoautotrophically under anaerobic conditions, reducing sulfate to hydrogen

SULFUR-OXIDIZING BACTERIA

239

sulfide with molecular hydrogen as the electron donor (Zillig et al., 1986).and this anaerobic autotrophy is correlated with the amplification of plasmid pDLlO (Zillig et al., 1985). Desulfurolobus ambivalens resembles Acidianus infernus in its principal physiological properties with respect to sulfate dissimilation and sulfur oxidation. The common physiology of the two strains is in accordance with their close phylogenetic relationship based on 16s rDNA homology, and led to the reclassification of D. ambivalens as Acidianus ambivalens (Fuchs et al., 1996). 2.1. Sulfur Oxygenase-reductase of Acidianus ambivalens

Two enzymes involved in sulfur oxidation have been described from the genus Acidianus (Table 1). Sulfolobus brierleyi was reclassified as Acidianus brierleyi (Segerer et al., 1986). This strain exhibits the same physiological characteristics as A. ambivalens and contains a sulfur oxygenase which has been purified from extracts of aerobically grown cells. In enzyme assays with elemental sulfur as the substrate, formation of sulfite and traces of thiosulfate were reported. Convincing evidence was obtained that sulfur oxygenase requires molecular oxygen as demonstrated by 1 8 0 2 incorporation into sulfur dioxide. The enzyme has a M,of 560000 and is composed of one type of subunit of Mr 35 OOO (Emmel et al., 1986). From A. ambivalens, a sulfur oxygenase-reductase (SOR) was described by Kletzin (1989). The SOR was exclusively present in the soluble fraction of aerobically grown cells and was purified to homogeneity. In the presence of oxygen (but not under a hydrogen atmosphere) SOR produces sulfite plus thiosulfate and hydrogen sulfide in a ratio of about 1.2:1 to 1.3:1 . The proposed reaction of SOR is given in equation 1.

+ 40H- + HSO; + S20:- + 2HS- + H+ [S] + HSO; + HS20;

5[S]+ 0

(1) (2) The thiosulfate formed may originate from a non-enzymatic conversion of sulfur and sulfite as given in equation 2, leading to an overall stoichiometryof 1 :1 between oxidized and reduced products (Kletzin, 1994). The molecular mass of SOR as determined by gel filtration is 550 kDa, consisting of identical subunits of 40 kDa. The enzyme does not require a cofactor for activity and does not contain a chromophore (Kletzin, 1989). Sulfur oxygenase-reductasefrom A . ambivalens and sulfur oxygenase from A . brierleyi are obviously the same enzymes, while from the latter hydrogen sulfide formation was not recognized. The sor gene region was cloned with the help of an oligonucleotide probe derived from the amino-terminal amino-acid sequence. Of four open reading frames (Fig. 2) detected by DNA sequencing, the sor gene was identified on the basis of matching amino-acid sequences. The sor gene has the coding capacity for a 35 3 17 Da protein, which is in agreement with the size determined by SDS-PAGE. The SOR sequence is the first known from an archaeal sulfur-oxidizing enzyme. It 2

Table 1 Physiological characteristics and inorganic sulfur-metabolizing enzymes of selected sulfur-oxidizing archaea and bacteria

Photo- or lithotrophic growth or oxidation

Strain Archaea Sulfolobales: AcidiMus ambivalens AcidiMus brierleyi Bacteria Cyanobacteria. Oscillarorialimnetica Green sulfur bacteria: Chlombiumthwsulfatophilum

C.limicola f. sp. thiosulfiatophilum

Chlorobiwntepidum Proteobacteria Photatrophic bacteria: Chromatiurn vinosum

Thiocapsa roseopersicina Ectothiorfrodospirashapovnikovii Rhudoinacter capsulatus

Growth on

Cl Metabolism S2- So SO:- TS 'IT HZ compound pH

Enzymes metabolizing inorganic T ( T ) sulfur compounds

Reference

OL OL

+ + + + + - + + +

2.5 1.8

94 70

Sulfur oxygendreductase Sulfur oxygenase

OP

+

7.5

35

Hydrogen sulfide:quinone reductase

[31

OP

+ + -

6.8 25-35 Flavocytochrome c Thiosu1fate:cytochrome c oxidoreductase

[41 [51

OP

7.0

30

OP

+ + - + + + + - +

7.2

48

FP

+ + + + - +

FP

+ + + + + + +

7.0 30-35 Adenosine 5'-phosphosulfate (APS) reductase Flavocytochrome c Sirohaem sulfite reductase Su1fite:acceptor oxidoreductase Thiosu1fate:cytochrome c oxidoreductase 7.3 20-35 Su1fite:acceptor oxidoreductase

FP FP

+

-

-

-

-

-

-

+ +

-

-

+ + +

-

+

Hydrogen su1fide:quinone reductase

8.0 %35 7.0 30-35 Hydrogen su1fide:quinone reductase

Photo-or lithotrophic growth or oxidation

Strain Rhodobactersulfabphilus

Growth on Cl Metabolism S2- So SO:- TS ‘IT H2 compound pH

OP

+ - - + - + + +

FP

+ + + - - + + + + + +

Rhodopseudomows palustris Rhodospirillwn rubrum Non-phototrophic bacteria: Aquaspirillwn autotrophicwn Aquifex pymphilus Bosea thiooxidarts Hydrogenobacteracidophilus Hydrogenobacter thennophilus Paracoccus denitrijcans

FL

OL

FL

OL OL

FL

Paracoccus versutus

FL

Pseudomonaspseudoflava GA3 Thiobacillusacidophilus

FL FL

Thiobacilluscaldus KU Thiobacillusdenitrijicans

OL OL

Thiobacillusneapolitanur Thiobacillusnovellus

OL

Thiobacillusferrooxidans

OL

FL

+ + - + + + + + +* + + + - + + - + + + + + + + + + -

+ +

30

8.0

+

6.8 7.4 3.5 7.0 8.0

30 85 30 65 70 30

-

+

8.0

30

+ + + + + + + + +

+ + + + + + + + + + + + + + + +

+

+ -

2.5

8.0

30 30

-

2.5 7.0

45 30

-

7.0

7.0

30 30

+

2.5

30

+

Reference

Hydrogen sulfide:quinone oxidoreductase 7.0 30-37 Thiosu1fate:cytochrome c oxidoreductase 6.8 30

7.0

-

+

Enzymes metabolizing inorganic

T(T) sulfur compounds

’Ihiosulfateoxidase. sulfite oxidase Thiosulfate-oxidizingenzyme system; sulfite dehydrogenase Thiosulfate-oxidizingenzyme system; sulfite dehydrogenase Tetra-, trithionate hydrolase; thiosulfate dehydrogenase APS reductase Sulfite oxidase Sirohaem sulfite reductase

Su1fite:cytochrome c oxidoreductase SulfurFe(III) oxidoreductase, sulfide:Fe(III) oxidoreductase Su1fite:FflII) oxidoreductase

Table 1 continued.

Photo- or lithotrophic growth or oxidation Strain

Metabolism S2- So S&-

H2

Enzymes metabolizing inorganic

T(T) sulfurcompounds

FL

+ + + + + + + + -

-

6.0 7.0

30-35

FL

+ + + + + + + + -

-

5.4 2.5

51 30

7.0

30

30

Thiobacilluspemmetabolis Thiobacillustepidarius

OL

Thiobacilh themsulfatus Thiobacillusthiooxidans

OL

Marine pseudomod strain 16B

H

Xanthobacterautotrophicus GZ29 Gram-positive bacteria: Bacillus schlegclii

TS TT

Grow* on Cl compound pH

FL FL

+ +

+

44

+ + +

+

8.0

+

+

7.0 59-72

+

Trithionate hydrolase, tetrathionate synthase, sulfite dehydrogenase

Reference [391 1471

Su1fite:cytochrome c oxidoreductase Sulfur-oxidizing enzyme system Thiosulfate-oxidizingenzyme, tetrathionate reductase thiosulfate reductase

FL, facultativelithotroph; FP, facultative phototroph; H, heterotroph; OL, obligate lithotroph; OP.obligate phototroph; TS, thiosulfate; 'IT, tetrathionate; +, -, utilized or not for lithotrophic andor phototrophic growth. *Aerobic hydrogen lithotrophy exclusively in the presence of sulfur or thiosulfate. References: [l] Kletzin, 1989; [2] Emmel et al., 1986; [3] Arieli et al., 1994; [4] Kusai and Yamanaka,1973a; [5] Kusai and Yamanaka, 1973b; [6] Shahak etal., 1992; 171 Wahlund et al., 1991; [8] Dahl, 1996; (91 Dolataet al., 1993; [lo] Fukumori and Yamanaka, 1979; [ l l ] Schedel et al., 1979; [I21Triiper [I61 Schiitz eta1.,1997; [17] Imhoff, 1989b; and Wscher, 1982; 1131 Schmitt et al., 1981; [14] Petushkova and Ivanovsky, 1976; [IS] Imhoff, 1 9 8 9 ~ [18] Brune and Triiper, 1986; [19] Appelt etal.. 1979; [ a ] Taper and Imhoff, 1989; [21) Friedrich and Mitrenga, 1981; [22] Aragno and Schlegel, 1978; [23] Huber elal., 1992b; [24] Das and Mishra, 1996; [25] Das et al., 19%; [26] Shima and Suzuki, 1993; [27] Alfredsson et al., 1986; [28] Bonjour and Aragno, 1986; [29] Chandraand Friedrich, 1986; [30] Wodara et al., 1994; [31] Lu and Kelly, 1983b; [32] Lu and Kelly, 1984a; 1331 Auling et al., 1978; [34] Medenberg et aL. 1992b; [35]Medenberg ct al., 1993; [36] Hallberg and Lindstrlirn, 1994; [37] Hallberg et aL, 19% [38] Bowen et al., 1966; [39] Kelly and Harrison, 1989; [40] Aminuddin and Nicholas, 1974; [41] Schedel and Ttiiper, 1979; [42] Charles and Suzuki, 1966b; [43] Toghrol and Southerland, 1983; [44] Sugio et al., 1989; 1451 Sugio el al., f992a; [46] Sugio et al., t992b; [47] Lu and Kelly, 1988b; [48] Shooner et al., 1996; [49] Nakamuraelal., 1995; [SO] K d a m a , 1969; I511 Whited and Tuttle, 1983; [52] Wiegel CI d.,1978; [53] Aragno, 1991; [SS] Hudson et al., 1988.

243

SULFUR-OXIDIZING BACTERIA

I

1 kb

i

Figure 2 The sor gene region of Acidianus ambivalenscoding for sulfur oxygenasdreductase with three open reading frames of unknown function (accession number X56616).

does not exhibit homology to known proteins (Kletzin. 1992).The sorgene and the three other open reading frames are transcribed separately. The sor gene transcript of approximately loo0 nucleotides in length is amplified about 40-fold in cells cultivated aerobically. This result is consistent with the observation that anaerobically grown cells are devoid of SOR activity. Transcription of the three other open reading frames does not respond to oxygen (Kletzin, 1992). The biological significance of SOR remains questionable. Its role in energy metabolism is not clear since no link to the electron transport chain is yet reported (Kletzin, 1992). Three findings would present evidence for SOR function: (i) isolation of a component that brings about electron transfer to the respiratory chain; (ii) characterization of a sulfur-oxidizingactivity different from SOR; and (iii) the inactivation of the sor gene and the characterization of the mutant phenotype. The final product of sulfur oxidation by A. ambivalens is sulfate. The SOR may supply the cells with hydrogen sulfide, the actual substrate for oxidation to sulfate, and additional reductant may be obtained from sulfite oxidation.

2.2. Gene Transfer Systems in Archaea The function of a gene can often be deduced from its inactivation. Directed mutagenesis, however, requires DNA transfer and homologous recombination. In the Archaea a few genetic systems are in the process of being developed. Plating techniques have been established for strains of the genus Sulfolobus, providing a potential tool for the physiological and genetic manipulation of these extreme acidophiles (Grogan, 1989). From the thermoacidophile Sulfolobus islandicus, multicopy plasmids were isolated that may be useful as cloning vectors in Sulfolobales. The smallest 5.3 kb plasmid. pRNl, was completely sequenced (Keeling et al., 1996) and protocols for electrotransformationhave been elaborated and used with pRNl or viruses. Other viruses and plasmids of Sulfolobales strains have been recently isolated and characterized (Schleper et al., 1992; Zillig et al., 1994). The ‘virus-like’particle SSV1. produced by Sulfolobus shibatae, has been sequenced (Palm etal., 1991). Transfectionof SSVl to S. solfataricus was obtained after electroporation and this particle integrates in a site-specific manner (Schleper et al., 1992). Horizontal plasmid transfer by mating within the genus Sulfolobus was reported

244

CORNELIUS G. FRIEDRICH

for a heterotrophic Sulfolobus isolate from a Japanese hot spring to the recipient Sulfolobus solfataricus and to other Sulfolobus strains. The isolate Sulfolobus NOB8H2 harbours 2 0 4 0 copies of a 45 kb plasmid, pNOB8, which confers marked growth retardation to its host. Transconjugants were shown to serve as donors for further transmission of pNOB8 (Schleper et al., 1995). Characterization of these genetic elements including properties of transfer and recombination is necessary to develop specific tools for gene transfer and genetic analysis of extreme thermophilic and acidophilic sulfur-oxidizing microorganisms. The ultimate in genetics of an organism would be the complete genomic nucleotide sequence as published for Methanobacteriumjannaschii (Bult et al., 1996). A chromosomal map of Sulfolobus acidocaldarius has been constructed. The map indicated a circular chromosome of 2.7 Mb on which rRNA genes have been located (Kondo et al., 1993). Recently, the partial nucleotide sequence of 156 kb of the Sulfolobus solfataricus €2genome has been obtained and within this sequence several genes like the virus-like particle SSVl have been located by Sensen et al. (1996).

3. SULFUR OXIDATION IN CYANOBACTERIA

Cyanobacteria perform oxygenic photosynthesis and oxidize water via photosystem (PS) I and PS I1 to molecular oxygen and reductant for carbon dioxide fixation and metabolic processes. However, the filamentous cyanobacterium Oscillatoria limnetica can also perform anoxygenic photosynthesis using only PS I and hydrogen sulfide as the electron donor to form elemental sulfur as the product (Cohen et al., 1975a,b). Subsequently, hydrogen sulfide oxidation was detected in many unicellular and filamentous cyanobacteria (Garlick et al.. 1977). Depending on the physiological conditions, the resulting reductant is transferred (i) to carbon dioxide to yield cell material, or (ii) to protons to yield molecular hydrogen in the absence of carbon dioxide (Belkin and Padan, 1978) or (iii) to dinitrogen to yield ammonia when combined nitrogen is absent (Belkin et al., 1987). The ecological significance of phototrophic hydrogen sulfide oxidation is seen in the flexibility of cyanobacteria to adjust to changing environmental conditions of aerobiosis and anoxygenic phototrophy in hydrogen sulfide-rich environments at low redox values (Garlick et al., 1977). 3.1. Hydrogen Su1fide:Quinone Reductase of Oscillatoria limnetica Oscillatoria limnetica, a filamentous non-heterocyst-forming cyanobacterium, specifically induces anoxygenic photosynthesis in a process that requires anaerobiosis, the presence of light, hydrogen sulfide, and protein synthesis. Under these

SU LFU R-OX1DIZING BACTERIA

245

conditions 0. limnetica forms su1fide:plastoquinone oxidoreductase (SQR; EC 1.85-), which catalyses the hydrogen sulfide-dependent reduction of plastoquinone- 1and most probably catalyses the initial step in sulfide-dependentanoxygenic photosynthesis. The enzyme in its active form is bound to thylakoid membranes and is composed of a single polypeptide of 57 kDa. The amino-terminal (Nterminal) amino-acid sequence contains the highly conserved fingerprint of the NADFAD-binding domain (Arieli et al., 1994). The enzyme exhibits UV and visible light absorption and fluorescence spectra characteristic of flavoproteins and is inhibited by potassium cyanide. Therefore, SQR was suggested to be a flavoprotein which binds sulfide and quinone and transfers electrons via FAD (Arieli et al., 1994). Knowledge of the N-terminal amino-acid sequence of SQR facilitates the cloning of the SQR structural gene via reverse genetics. As described below, SQR is not unique to 0. limnetica;its presence has also been demonstrated in the green sulfur bacterium Chlorobiurn lirnicola f. sp. thiosulfatophilum and in the phototrophic proteobacteria Rhodobacter capsulatus and Paracoccus denitrijicans. 3.2. Gene Transfer Systems in Cyanobacteria

Gene transfer systems like conjugation and transformation have been elaborated for different strains of cyanobacteria as reviewed by Haselkorn (1991) and Cohen and Gurevitz (1992), and several protocols for chemical mutagenesis and gene transfer methods have been summarized by Golden (1988). A physical and genetic map of the circular 6.4 Mb chromosome of Anabaena PCC 7120 has been obtained based on pulsed-field gel electrophoresis and about 30 genes or gene clusters were localized (Bancroft et al., 1989). A combined physical and genetic map of the chromosome of the unicellular cyanobacterium Synechocystis PCC 6803 was also constructed. The estimated genome size is 3.82 Mb (Churin etal., 1995). Moreover, from this strain the nucleotide sequence of the entire genome was reported and potential protein coding regions assigned (Kaneko et al., 1996). Genetic studies have advanced the understanding of the structure and function of the chlorophyll protein complexes PS I and PS 11, of the nitrogen fixation systems, of metal transport, and of other systems. Protocols for transformation and electrotransformation have been primarily developed for strains of unicellular Synechococcus (Table 2; Golden and Sherman, 1984). Conditions for conjugation for the filamentous heterocyst-forming Anabaena as a recipient are well defined (Wolk et al., 1984). Also, shuttle vectors were constructed with multiple cloning sites that can be efficiently transformed in Synechococcus PCC 7942 (Laudenbach et al., 1990). Mutagenesis of cyanobacteria is possible but is hampered by a highly effective repair system and the polyploidy of the cyanobacteria. Therefore, extensive segregation over as many as seven generations is necessary to select for mutants.

Table 2 Gene transfer systems reported from selected sulfur-oxidizing archaea, phototrophic and colourless bacteria

Strain Archaea Sulfurolobus solfataricus Bacteria cyanobacteria: Synechococcus PCc7942 Green sulfur bacteria: Chlombiumlimicola DSM 245 Chlombiumtepidurn Chlombiumvibrioforme Phototrophic bacteria: Chromatium vinosum Rhodobacter capsulatus Rhodobactersphaemides RhodopseudomoMsviridis Rhodospirillum rubnun Non-phototrophic bacteria: Bosea thiooxidons Paracoccus denitrijicansPD1207 Paracoccus denitrificansPD1236 Paracoccus denitrijicansGB 17

Transfer system

Transfer of vector or plasmid

Conjugation Electrotransformation Electrotransfection

pNOB8 pRN1. pRN2 SSVl

Transfer efficiency (transconjugant per donor or recipient) or (transformant per Pg DNA) lo-' 106

Transformation

References Schleper et al. (1995) Zillig et al. (1994) Schleper el al. (1992) Haselkorn (1991)

Transformation Conjugation Electrotransformation

14 kb plasmid (Tie+) hcQ pVS2, pKKL3.14

Conjugation Conjugation

1 IncP. Q IncP. Tnl, Tn3, Tn5, Tn903 pRK2013, pSUP.202 Imp, Q. W 1.5 x 104-104 pBR325derivatives,pSUP5011 pSUP.202, RK2derivatives IMP, Q, W

Pattaragulwanit and Dahl(l995) Donohue and Kaplan (1991) Kaufmann et al. (1984) Miller and Kaplan (1978) Davis et al. (1988) Lang and Oestehelt (1989) Donohue and Kaplan (1991)

pBR325::TnS-mb(=pSUPSO11)2.2 x lo4 RSF1010, RSF101O:TnS < 4.3 x lo4

Das and Mishra (19%) DeVries et al.(1989) DeVries et al.(1989) Chandra and Friedrich (1986)

Conjugation Conjugation Conjugation Conjugation Conjugation Conjugation Conjugation

RSF1010. RSF101O:TnS pBR325::TnS-mb

2 x 10-4 10-*-10-~

1.7 xlO-', 5.7 x 1o-~-1o - ~

Mtndez-Alvarez et al.(1994) Wahlund and Madigan (1995) Kjaerulff et al. (1994)

Table 2 continued.

Strain

Transfer system

pRK2013::Tnl721 pSUP50 11 IncP, Q.W Pseudomomspseudof2ava GA3 pBR325: :TnS-mb Thiobacillus acidophilus IncP hcP, Q. W Thiobacillus neapolitanus Conjugation IncP hcp, Q.W Electrotransformation IncP, pRK415Km-derivative Thwbacillusnovellus Conjugation IncP, pVKlOO RP4.8::TnSOI Thiobacillusferrooxidans Conjugation IncP, Q. pSUP1011, RP1::TnS Electrotransformation pUC18::mer Thiobacillus thiooxidans Conjugation IncP Xanthobacter autotrophicus GZ29 Generalized transduction Chromosome Conjugation Chromosome Paracoccus versutus

Conjugation Conjugation Electrotransformation Conjugation Conjugation

Transfer of vector or plasmid

Transfer efficiency (transconjugant per donor or recipient) or (transformant per Pg DNA) 1.5 x 1 0 - ~ 1.2 x 10-8 3.0 x lo3 10-8 10-5-3 x 10" 8.5 x 10-~

6.8 x lo3 1o-~- 10" 6.3 x 10" 10-5-10-7 2 x Id 10-~-10-~ 10-4 10~-10-~

References Davidson et al. (1985) Chandra and Friedrich (1986) Wlodarczyk et al. (1994) Chandra and Friedrich (1986) Quentmeier and Friedrich (1994) Davidson and Summers (1983) Kulpa et al. (1983) Davidson and Summers (1983) English et al. (1995) Davidson et al. (1985) Davidson el al. (1985) Peng et al. (1994) Kusano et al. (1992) Jin et al. (1992) Wilke and Schlegel(l979) Wilke (1980)

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Transposon mutagenesis has been applied in addition to chemical mutagenesis: transposon Tn5 coding for kanamycin resistance has been introduced to Anubuena by conjugation (Elhai and Wolk, 1990). Numerous methods are available to study a variety of cyanobacterial genes via their insertional inactivation, deletion or modification, or to study the expression of foreign genes in cyanobacteria (Cohen and Gurevitz, 1992). So far these methods have not been adopted for genetic analysis of SQR of 0. limneticu or for the genetic analysis of anaerobic phototrophic hydrogen sulfide oxidation.

4. SULFUR OXIDATION IN GREEN SULFUR BACTERIA

The green sulfur bacteria are strictly anaerobic and obligately phototrophic; they perform anoxygenic photosynthesis, and are incapable of respiratory metabolism in the dark. They are able to use hydrogen sulfide and elemental sulfur as photosynthetic electron donors which are oxidized to sulfate. Also, molecular hydrogen can serve as the electron donor. Thiosulfate is oxidized by two strains that originated from one strain, the forma species Chlorobium limicolu forma Species thiosulfatophilum. This culture had to be divided and associated with C. limicolu and C. vibrioforme, and the two strains are now designated C. limicolu f. sp. thiosulfatophilum and C. vibrioforme f. sp. thiosulfatophilum (Pfennig and Triiper, 1974; Triiper and Fischer, 1982). Both strains are distinguished from C. limicola and C. vibrioforme by their ability to utilize thiosulfate (see Table 1). Unlike other phototrophic or lithoautotrophicbacteria which fix carbon dioxide via the reductive pentose-phosphate cycle, Chlorobium limicolu assimilates carbon dioxide via the reductive citric acid cycle (Fuchs et ul., 1980). For detailed information on the components of the phototrophic electron transport chain, and the generation of a transmembranous gradient, the reader is referred to reviews by Blankenship (1985) and Brune (1989). 4.1. Sulfur-oxidizing Enzymes of Chlorobium

Three enzyme activities have been described in the genus Chlorobium: thiosulfate:cytochrome c reductase, soluble flavocytochrome c and a membrane-bound su1fide:quinone reductase (SQR). Also, three unusual c-type cytochromes have been found in C. thiosulfutophilum, a culture not differentiated at that time to the two forma species: cytochrome c-551 (45 m a ) , flavocytochrome c (50 kDa) and cytochrome c-555 (10 kDa) (Meyer et ul., 1968). The thiosu1fate:cytochrome c reductase of C. thiosulfatophilum possibly comprises an enzyme system consisting of a colourless 80 kDa protein which reduces cytochrome c-55 1. The reaction rate and the extent of cytochrome c-55 1 reduction

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are enhanced significantly by catalytic amounts of cytochrome c-555. This cytochrome, however, is considered not to be reduced by the 80 kDa protein and possibly acts as an effector or eliminates some reaction product. The thiosulfateoxidizing enzyme does not exhibit sulfite dehydrogenase activity, and sulfite inhibits this reaction severely. The product of the reaction has so far not been identified. However, polythionates like di-, tri- or tetrathionate are not detected (Kusai and Yamanaka, 1973b). Cytochrome c-551 may be essential for thiosulfate oxidation since its substitution with c-type cytochromes from Rhodospirillum rubrum or Thiobacillus novellus resulted in low oxidation rates. Moreover, cytochrome c-551 is exclusively found in cells able to utilize hosulfate, not in cells unable to oxidize thiosulfate (Steinmetzand Fischer, 1981,1982a). Tetrathionate has not been described to be an intermediate of sulfur metabolism in the Chlorobiaceae. However, it can be used by resting cells for carbon dioxide fixation at hi h light intensities (Khanna and Nicholas, 1982). Using thiosulfate labelled with ' S at the sulfane sulfur ( [ 3 s S - S 0 3 ] 2 - ) and at the sulfone sulfur ([S-35S03]23, evidence was obtained for a reductive cleavage of thiosulfate yielding sulfide and sulfite, which become oxidized to sulfate (Khanna and Nicholas, 1982). Flavocytochrome c-553 from C. thiosulfatophilum catalyses the transfer of two electrons from hydrogen sulfide to cytochrome c at micromolar concentrations with polysulfide or elemental sulfur as oxidation products. The enzyme was, therefore, characterizedas su1fide:cytochromec reductase (Kusai and Yamanaka, 1973a).The heterodimeric enzyme (Mrof 58 OOO) was purified from the soluble fraction of cell free extracts. It is composed of a flavoprotein subunit (Mr47 000)and a haemoprotein (Mr 11OOO) (Yamanakaet al., 1979).The enzyme SQR is present in Chlorobium limicola f. sp. thiosulfatophilum. Membranes catalyse the electron transfer from hydrogen sulfide to externally added quinones in the dark. The enzyme SQR is most active with ubiquinone 2 and plastoquinone 1 and 2, and shows little activity with ubiquinone 1 and menadione (Shahak et al., 1992). Flavocytochromec and SQR are both sulfide-oxidizingactivities and are present in a single strain of Chlorobium. The activity crucial for phototrophic energy metabolism has not been identified so far. It is interesting to note that flavocytochrome c is apparently absent from several green sulfur bacteria - Pelodictyon luteolum (Steinmetz and Fischer, 1982b) and C. vibrioforme - which oxidize hydrogen sulfide to sulfate but are unable to utilize thiosulfate (Steinmetz et al., 1983; Table 3). As outlined below, flavocytochrome c may play a role in non-phototrophic thiobacteria able to oxidize thiosulfate.

Q

4.2. Gene Transfer Systems in Green Sulfur Bacteria

The occurrence of conjugation and transformationhas been reported in green sulfur bacteria. Natural transformation has been reviewed by Ormerod (1988). Transformation as well as molecular biological approaches have been employed to identify,

Table 3 Occurrence of flavocytochrome c and su1fide:quinone reductase in sulfur-oxidizing bacteria The data for products of phototrophic electron donors were taken from Triiper (1981).

Strain Oscillatorialimnetica Chlorobiwn lhicola f. sp. thiosuifatophilwn Chlorobim vibriofom Chlorobiwn vibrioforme f. sp. thiosulfatophilm Pelodctyon l~leolwn Chmmatim vinosum Chmmatim watmingii Ectothiorhhpira halophila Paracoccus denitrifcans GB 17 Rhohbacter capsulatus Rhohbacter sphaemides Rhohbacter sulfidophilus Rhodospirillum d r u m Thiocapsu pfennigii Thiocapsu mopersicinn

Substrate

Product

Flavocytochrome Sulfide: quinone c (subunit oxidoreductase ma) OrW

References

SGSO sdsG-

Key: +. present;-, not present; + (n.r.), present, molecular mass not reported. *Deduced moIecular mass of gene sequence 47 kDa (Schutz et al., 1997). References: [l] E. Padan, personal communication; [2] Arieli et al., 1994; [3] Yamanaka et al., 1979; [4] Shahak et al.. 1992; [5] Steinmetz er al., 1983; [6] Steinmetz and Fischer, 1982a, (71 Steinme& and Fischer, 1982h [S] Wermter and Fischer, 1983; [9] Meyer, 1985; [lo] A. Quentmeier and C. Friedrich, unpublished data; [ll] G. Hauska, personal communication; [ 121 Meyer and Cusanovich, 1985; [13] Schiitz et al., 1997; 1141 Neutzling, 1985. cited in Brune, 1989; [ 151 Brune and Triiper, 1986; [ 161 Meyer et al., 1973; [ 171 Rscher and Triiper, 1979.

SULFUR-OXIDIZING BACTERIA

251

clone and sequence several genes of an operon coding for the photosynthetic reaction centre (Buttner et al., 1992). Also, genes coding for proteins of the antenna system have been identified from C. vibrioforme and C. tepidum (Chung et al., 1994). Natural transformation frequencies are low in green sulfur bacteria. However, an efficient and reproducible protocol for transformation of plasmid DNA by electroporation has been described for C. vibrioforme. With the aid of this method the essential role of cytochrome c-551, a component of the photosynthetic reaction centre complex, in phototrophic growth was analysed. A 9 kb plasmid carrying the pscC gene coding for cytochrome c-551 was transformed to and integrated by homologous recombination into the C. vibrioforme chromosome (Kjaerulff et al., 1994). An interesting observation concerning thiosulfate utilization by C. limicola was reported by MCndez-Alvarez et al., (1994). The DNA of a 14 kb plasmid was isolated from C. limicola f. sp. thiosulfatophilumand used to transform C. limicola unable to grow on thiosulfate. The resulting transformants acquired the ability to grow with thiosulfate, and this plasmid was reisolated from the transformants. This plasmid codes probably either for the ability to use thiosulfate or for some regulator for activation or increased expression of silent genes required for thiosulfate utilization. It also constitutes a potential cloning vector (Mkndez-Alvarez et al., 1994). Chlorobium tepidum is a thermophilic green bacterium isolated from New Zealand hot springs and able to use hydrogen sulfide and thiosulfate, and to fix dinitrogen (Wahlund et al., 1991; Wahlund and Madigan, 1993). For this strain an efficient plating technique was established by supplying hydrogen sulfide from the gas phase and using a special strain with high plating efficiency. Conjugal transfer of broad host range plasmids of the incompatibility group Q from Escherichia coli to C. tepidum was reported at frequencies of lo-' to lo4 exconjugants per donor cell. With favourable growth characteristics and transfer frequencies, this strain appears to be amenable to genetic studies (Wahlund and Madigan, 1995).

5. SULFUR OXIDATION IN PROTEOBACTERIA

The class of Proteobacteria has been suggested as a new, higher taxon to circumscribe the alpha, beta, gamma and delta groups that are included among the phylogenetic relatives of the photosynthetic bacteria and as a suitable collective name for reference to that group. The group names were proposed to remain at the level of a subclass (Stackebrandt et al., 1988). In this overview selected phototrophic and non-phototrophic proteobacteria are discussed separately.The strains were selected with respect to the available data on the enzymology and genetics of sulfur metabolism. In this class, anoxygenic phototrophy is present in phylogenetically

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different bacteria (Stackebrandt et al., 1996). Also, lithotrophic sulfur metabolism is found in bacteria of different subclasses (Lane et al., 1992; Fig. 3).

5.1. Phototrophic Proteobacteria The phototrophic proteobacteria include the families of the Chromatiaceae, Ectothiorhodospiraceae and Rhodospirillaceae. The Chromatiaceae are within the gamma subdivision of the purple bacteria. They generally use hydrogen sulfide or elemental sulfur as phototrophicelectron donors which are oxidized to sulfate. With hydrogen sulfide as substrate, sulfur is first accumulated in globules inside the cell and then further oxidized when hydrogen sulfide is depleted. Some species can also use sulfite and thiosulfate, while tetrathionate is not metabolized. l b o groups of the Chromatiaceae can be distinguished on the basis of their chemotrophic growth (Kampf and Pfennig, 1980). The physiology of the phototrophic sulfur-oxidizing purple bacteria, their excreted intermediates of sulfur oxidation, their substrates, and the enzymes involved in sulfur metabolism have been discussed in a number of reviews (Triiper and Fischer, 1982; Brune, 1989; Dahl and Triiper, 1994). Table 1 lists selected strains, some of their physiological properties, and enzymes of the respective strains converting inorganic sulfur compounds. The Ectothiorhodospiraceaeare phylogeneticallyclosely related to the Chromatiaceae, are members of the gamma subdivision,and photooxidizehydrogen sulfide to sulfate. However, elemental sulfur is transiently accumulated in globules outside the cell, and subsequently oxidized to sulfate upon depletion of hydrogen sulfide. Some strains also oxidize sulfite or thiosulfate to sulfate, and some are facultatively chemotrophic. Ectothiorhodospirahalochloris and E. abdelmalekii were reported not to be true photoautotrophs,growing mixotrophically in the light with hydrogen sulfide and an organic carbon source (Then and Triiper, 1983,1984). Rhodospirillaceae are within the alpha and beta subdivisions of the Proteobacteria. Except for a few strains, most members of the Rhodospirillaceae are able to grow with hydrogen sulfide as the electron donor, albeit at concentrations of less than 1 m.Hydrogen sulfide is photooxidized to elemental sulfur, sulfate, thiosulfate or tetrathionate, depending on the strain and growth condition (Imhoff and Triiper, 1989). Elemental sulfur is not accumulated in globules inside or outside the cell. Some strains, however, form sulfur as an intermediate, and a few utilize thiosulfate, e.g. Rhodobacter adriaticus, R. suljidophilus (reclassified as Rhodovulum sulfidophilum) (Imhoff, 1989b; Hirashi and Umeda, 1994), Rhodobacter veldkampii, Rhodopseudomnas palustris and Rhoahpseudomonas sulfoviridis. Most species can also grow with molecular hydrogen and many strains fix dinitrogen. In Rhodobacter capsulatus and Rhodospirillum rubrum aerobic chemotrophic growth in the dark has been demonstrated, hence these strains are facultative phototrophs (Siefert and Pfennig, 1979; Madigan and Gest, 1979).

A

B

Figure 3 The 16.9 rRNA based relationships among members of the alpha (A) and beta and gamma (B) subclass of the Proteobacteria.The horizontal components of the branch lengths are proportional to evolutionarydistances. Adapted from Lane et al. (1992) with permission.

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CORNELIUS G . FRIEDRICH

5.1.1. Chromatium vinosum Chromatiumvinosum is facultatively photoautotrophic, able to grow anaerobically in the light with hydrogen sulfide, sulfite, thiosulfate and with molecular hydrogen, but not with tetrathionate (see Table 1). This organism is also capable of aerobic chemotrophic growth in the dark with thiosulfate and carbon dioxide, provided the oxygen partial pressure is low (Kampf and Pfennig, 1980, 1986). The pathway of sulfur oxidation was proposed to proceed in three steps: (i) oxidation of hydrogen sulfide or thiosulfate to elemental sulfur, which is deposited intracellularly; (ii) oxidation of hydrogen sulfide or elemental sulfur to sulfite; and (iii) oxidation of sulfite to sulfate, the final product (Dahl and Triiper, 1994). Thiosulfate may be metabolized by the three-step pathway or may be converted to tetrathionate. Several enzymes have been described in C. vinosum grown phototrophically with hydrogen sulfide or thiosulfate. Flavocytochrome c has been shown to catalyse the hydrogen sulfide-dependent reduction of cytochrome c with elemental sulfur or polysulfide as product (Fukumori and Yamanaka, 1979).Flavocytochrome c also strongly binds sulfite and thiosulfate as well as other nucleophiles. It is composed of a flavoprotein subunit of 46 kDa and a haemoprotein subunit of 21 kDa (Yamanaka etal., 1979; Cusanovich etal., 1991; Meyer et al., 1991). Dolata et al. (1993) have cloned and sequenced the corresponding gene region. Sequence analysis suggests a periplasmic location of the enzyme. As summarized in Table 3, flavocytochrome c has not been detected in other thiobacteria using hydrogen sulfide for phototrophic or chemolithotrophic growth, as was reported of Ectothiorhodospira halophila (Meyer, 1985), Thiocapsa roseopersicina (Fischer and Triiper, 1979), Chromatiumwarmingii (Wermter and Fischer, 1983) and Thiocapsa pfennigii (Meyer et al., 1973). The high affinity for (and the reduction of) flavocytochrome c by hydrogen sulfide was considered as a reaction functioning in vivo, because flavin accepts two electrons simultaneously and thus avoids sulfide radical intermediates which would otherwise occur from one electron transfer to monohaem cytochromes (Brune, 1989). If the physiological role of flavocytochrome c is different from the catalytic activity in vitro, the question arises as to which is the core protein involved in hydrogen sulfide oxidation. Sirohaem sulfite reductase (SR) catalyses the reduction of sulfite in vitro to hydrogen sulfide, the major reaction product, while thiosulfate and trithionate are also formed. This enzyme was suggested to function in the reverse direction in vivo, oxidizing hydrogen sulfide to sulfite. The proposed 'reverse' function is based on the regulation of SR formation since its activity is exclusively present in C. vinosum cells grown autotrophically on hydrogen sulfide, and it is not found in extracts from cells grown photoheterotrophically with malate and sulfate. Therefore, an assimilatory role of SR is unlikely (Schedel et al., 1979). The SR was purified to near homogeneity and was characterized as a sirohaem-containing enzyme of M, 280 000 with a- and P-subunits of M, 37 000 and 43 000, suggesting a subunit stoichiometry of a4P4.

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Oxidation of sulfite to sulfate is catalysed by su1fite:acceptor oxidoreductase. This enzyme activity has been detected in crude extracts of C. vinosum (Triiper and Fischer, 1982). Data on the purified enzyme are not available yet, and measurement of su1fite:acceptor oxidoreductase from crude extracts is strongly influenced by non-enzymatic artefacts (Dahl, 1996). A second sulfite-oxidizingactivity has been studied from C. vinosum: the membrane-bound adenylylsulfate (adenosine 5‘phosphosulfate,APS) reductase (EC 1.8.99.2).The enzyme APS reductase catalyses the oxidation of sulfite with adenosine 5’-monophosphate (AMP) and ferricyanide as electron acceptor (Schwenn and Biere, 1979). The enzyme ADP sulfurylase catalyses the conversion of APS with inorganic phosphate to yield adenosine 5’-diphosphate (ADP) and sulfate. The enzyme ATP sulfurylase converts APS in an analogous reaction with pyrophosphateto yield adenosine 5’-triphosphate (ATP) and sulfate. Enzymes that may be involved in sulfite oxidation of phototrophic sulfur bacteria have been tabulated by Dahl and Triiper (1994). The significance of the ‘reverse’ function of APS reductase in sulfite oxidation and photolithoautotrophic growth has not been convincingly solved biochemically, and APS reductase activity is absent in several thiobacteria. As outlined in section 5.1.2, genetic and molecular biological studies cast doubt on such a function. Thiosulfate is completely oxidized by C. vinosum to tetrathionate when the cells are exposed to mildly acidic conditions (pH 6). In cell-free extracts, tetrathionate is formed by a thiosulfate oxidoreductase with ferricyanide as electron acceptor (equation 3) (Smith, 1966).

~[s-sot ~)I[O~S-S-S-SO~]~~+ 2e-

(3)

2e- + 9 2 + 2Ht + 20H- t)H20 + 20H(4) Thiosu1fate:ferricyanide oxidoreductase has an optimum activity around pH 5.0 and a K,,,for thiosulfate of 1.5 m ~ The . characteristics of the enzyme in vitm correlate with the conversion of thiosulfate to tetrathionate in situ under acidic conditions (Smith, 1966). The physiological significance of this reaction may be in the escape of the organism from acidic conditions, since the oxidation of thiosulfate to tetrathionate yields hydroxyl anions (equation 4) instead of protons from sulfuric acid. Thus, tetrathionate is formed although it is not used by C. vinosum for phototrophic growth. Moreover, the low affinity of thiosulfate oxidoreductase for thiosulfate results in the preference of thiosulfate conversionby other enzymes with high affinity for thiosulfate. Thiosu1fate:acceptor oxidoreductase activity differing from that described above was reported by Schmitt et al. (1981). The colourless enzyme of 35 kDa transfers electrons from thiosulfate to c-type cytochromes and was purified from the soluble fraction. The enzyme revealed a K,,,for thiosulfate of 2 pM, and was most active in cytochrome c reduction when flavocytochrome c-552 was added. Flavocytochromec-552 was isolated from chromatophores,with a native molecular mass of 71.5 kDa, composed of two non-identical subunits of 45 kDa and 20 kDa. These data differ from other reports (Bartsch, 1978;Dolata etal., 1993)with respect

2 56

CORNELIUS G. FRIEDRICH

to the cellular location of flavocytochrome c. The isolation of flavocytochrome c from the membrane fraction under the conditions applied may be indicative of a complex of different proteins. This view could explain the appearance of different protein bands upon mild treatment of the enzyme with sodium dodecylsulfate (SDS) (Schmitt ef al., 1981). 5.1.2. Gene Transfer in Chromatium vinosum A physical map of the chromosome of C. vinosum has now been obtained. The sum of sizes from the restriction fragments revealed a genome size of 3.64 Mb. Three rRNA genes have been located so far (Gaju et al., 1995). No gene transfer system for C. vinosum was available until recently, but a protocol for conjugal gene transfer has now been reported for C. vinosum strain D by Pattaragulwanitand Dahl(l995). Broad host range IncP plasmids and IncQ vectors were transferred from E. coli to C. vinosum Mobilization was achieved with the aid of the transfer functions of plasmid RP4 either present extrachromosomally or present in the chromosome of E. coli S17-1. Conjugation efficiencies of up to 1 were achieved. Back transfer to E. coli and transfer to Rhodospirillum rubrum were also achieved (Pattaragulwanit and Dahl, 1995). The aprBA locus coding for APS reductase was identified in a DNA library of C. vinosum using an oligonucleotideprobe derived from conserved sequences of aprB of APS reductase from other sources. The aprBA genes have been shown to be present as a single copy in this strain. The aprBA locus was cloned 2 cartridge. The and the apsB gene inactivated by insertion of a kanamycin f mutation was introduced into the C. vinosum chromosome by double homologous recombination. Enzymological studies demonstrated the absence of APS reductase activity from the mutants. Further phenotypic characterization showed no significant effect of APS deficiency on the sulfite-oxidizing ability of the mutant cells under photolithoautotrophic growth conditions. Thus, convincing evidence was presented that APS reductase was not essential for this type of metabolism (Dahl, 1996). In light of this important result, the possible role of su1fite:acceptor oxidoreductase was examined. Chromatiurn vinosum grows with sulfite as the phototrophic electron donor. This rare physiological trait among sulfur-oxidizing bacteria enables discrimination of the significance of sulfite oxidation to sulfate from other reactions. Tungstate specifically inhibits molybdoenzymes (Rajagopalan, 1980) and inhibits growth with thiosulfate of various colourless thiobacteria (Friedrich ef al., 1986). Also, tungstate inhibits growth of C. vinosum with sulfite. Su1fite:acceptoroxidoreductasefrom Thiobacillus novellus and from other sources contains the molybdenum cofactor (Rajagopalan, 1980; Toghrol and Southerland, 1983). and this result was taken as physiological evidence that su1fite:acceptor oxidoreductase plays a crucial role in phototrophic sulfur metabolism (Dahl, 1996). From C. vinosum, the gene locus for flavocytochrome c has been identified by reverse genetics using oligonucleotides as probes derived from the amino-acid sequence of an internal peptide of its cytochrome subunit. The cloned region

SULFUR-OXIDIZING BACTERIA

I

1 kb

257

I

Figure 4 The gene region of the flavocytochromec of Chmmatium vinosum strain D. The size of the open reading frame 1 (ORF1) is inferred by analogy to a 201 amino-acid tetrahaem cytochrome of Pseudomonas stutzeri; O R R is homologous to human ankyrin, fccA codes for the haemoprotein, and fccB for the flavoprotein subunit. The size of fccB. indicated by the dashed arrow,is based on the size of the purified protein. Adapted from Dolata et al. (1993) with permission.

contains four open reading frames which encode the carboxyl-terminal amino-acid residues of a tetrahaem cytochrome (ORFl), and a human ankyrin homologue ( O W ) . Downstream of O W the fccA gene is located coding for the 21 kDa dihaem cytochrome subunit. Adjacent to fccA an incomplete gene fccB is located coding for 27% of the flavoprotein sequence (Fig. 4). Messenger RNA analysis suggested that ORFl and ORF2 are not cotranscribed with fccAB, and it was concluded that the two flavocytochromec genes form a separate operon. From the deduced amino-acid sequences of fccAB it was evident that both proteins contain signal peptides, which apparently direct the proteins to the periplasmic space (Dolata et al., 1993). Within the 30-residue signal peptide of the flavoprotein subunit, a conserved motif, RRDFIK, was detected, which is essential for export of the protein to the periplasmic space and identical to the motif of the napA gene product of Alcaligenes eutmphus, the molybdenum cofactor-carrying subunit of the periplasmic nitrate reductase (Siddiqui et al., 1993) and similarly typical for proteins with complex redox centres (Voordouw, 1992;Berks, 1996).The periplasmic location of flavocytochrome c has an important functional implication since the potential product, elemental sulfur, is accumulated in C. vinosum in the cytoplasm. The true physiological role of this flavoproteinmay therefore be related to its ability to bind sulfite, thiosulfate and mercaptans (Dolata et al., 1993). The possible function of flavocytochrome c in thiosulfate utilization of Paracoccus denitrificans is discussed below.

5.1.3. Sulfir Oxidation in Rhodospirillaceae Members of the Rhodospirillaceae are either obligately or facultatively phototrophic and can use a wide variety of organic substrates for anaerobic photoheterotrophic growth or hydrogen for photolithotrophic growth (Klemme, 1968). They

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CORNELIUS G. FRIEDRICH

are also capable of anaerobic respiration (reviewed by Ferguson et al., 1987), of anaerobic dark growth with carbon monoxide (Uffen, 1976) and of anaerobic fermentation (Schultz and Weaver, 1982; reviewed by Willison, 1993). Under microaerophilic conditions several species grow chemotrophically with molecular hydrogen, or by organoautotrophic growth with formate or methanol (Siefert and Pfennig, 1979). Moreover, the ability to fix dinitrogen in the absence of combined nitrogen appears to be a general trait of Rhodospirillaceae, with few exceptions (Masters and Madigan, 1983). Photolithotrophic growth on hydrogen sulfide was reported in 1972 for Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum and Rhodopseudomonaspalustris when this electron donor was supplied at low concentrations in a chemostat (Hansen and van Gemerden, 1972). Generally, hydrogen sulfide is toxic to cells especially when it is undissociated at low pH, and different degrees of tolerance are observed. Most strains require concentrations below 0.5 m~ sodium sulfide. Rhodobacter capsulatus tolerates up to 2 mM sodium sulfide in the medium. In Rhodobacter sphaeroides and Rhodopseudomonas palustris, the sulfide-oxidizing flavocytochrome c has not been detected (Meyer and Cusanovich, 1985). It may well be that the inability of these strains to utilize thiosulfate correlates with the absence of flavocytochrome c. With the exception of Thiocapsa roseopersicina this is seemingly a general correlation (see Table 3). Also, for the few strains studied, su1fide:quinone reductase which - like flavocytochrome c - yields elemental sulfur as reaction product in vitro, has been detected irrespective of the ability to oxidize either hydrogen sulfide or thiosulfate to sulfate (Table 3). If the apparent correlation is meaningful, then flavocytochrome c plays an essential role in thiosulfate metabolism and should be present in phototrophic and non-phototrophic bacteria able to utilize thiosulfate. If exceptions are meaningful, then thiosulfate could be metabolized by different components. From R. palustris, thiosu1fate:cytochrome c oxidoreductase has been partially purified from the soluble fraction. The 93 kDa enzyme requires a separately purified and characterized cytochrome c as electron acceptor for thiosulfate-oxidizing activity (Appelt et al., 1979). With respect to the requirement of the additional cytochrome c-555, this system resembles the one described in Chlorobium thiosulfatophilum (Kusai and Yamanaka, 1973b). Su1fide:quinone reductase has been examined in Rhodobacter capsulatus DSM155 and purified to homogeneity from chromatophores. It is composed of a single polypeptide with an apparent molecular mass of about 55 kDa exhibiting fluorescence spectra characteristic of a flavoprotein, and is similar to the SQR from Oscillatoria limnetica. The structural gene was identified with an oligonucleotide as probe deduced from amino-terminal and internal amino-acid sequences. The molecular mass of the polypeptide as deduced from the nucleotide sequence is 47 kDa. The SQR of R. capsulatus is 48% identical to the enzyme of 0. limnetica and contains the typical FAD/NAD(P) binding site, and exhibits further similarities to other flavoproteins (Schiitz et al., 1997).

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5.1.4. Gene Transfer Systems in Rhodospirillaceae Genetic systems are available for strains of the Rhodospirillaceae that can use hydrogen sulfide for photolithotrophic growth. Rhodobacter capsulatus and R. sphaeroides have been studied extensively owing to their favourable growth characteristics and their physiological versatility (reviewed by Donohue and Kaplan, 1991; Willison, 1993). Most information is available for these strains, while som: genetic studies deal with Rhodospirillum rubrum and Rhodopseudomonas viridis, of which the latter grows only photoheterotrophically (Triiper and Imhoff, 1989). A phage-like gene transfer agent, GTA, is released from Rhodobacter capsulatus in the stationary growth phase. The GTA mediates generalized transduction in R. capsulatus and was used for mapping mutations in photosynthetic (Marrs, 1974) and nitrogen fixation loci (Wall et al., 1984). The GTA was also used to introduce interposon-mutatedgenes to R. capsulatus (Young et al., 1989) in addition to other vectors constructed for DNA mobilization (Marrs, 1981). An efficient conjugational system has been developedby Simon et al. (1983) with E. coli S 17-1 as donor, which harbours the transfer function of plasmid RP4 on the chromosome, and ColEl plasmids which do not replicate in R. capsulatus. R. sphaeroides and Rhodospirillum rubrum. This system has been successfully applied to DNAtransfer from E. coli S 17-1 into these and many other Gram-negative strains (Kaufmann et al., 1984; Davis et al., 1988; Klipp et al., 1988) and also to Rhodopseudomonas viridis (Lang and Oesterhelt, 1989). Plasmids of the incompatibility group P, Q and W can also be used for gene transfer within this group of organisms. Finally, a map of the Rhodobacter capsulatus chromosome was established, and the positions of genetic markers were generally determined by conjugation with the R plasmid derivative pTH10. Map positions of genes are located coding for (for example) photosystem I, nitrogen fixation, hydrogen metabolism, carbon dioxide fixation, ammonia-dependent growth and house-keeping genes, reflecting the interests in the different systems (Willison et al., 1985). In conclusion, a wide range of genetic tools may be applied to study genetic aspects of sulfide oxidation and its regulation and the role of SQR for hydrogen sulfide-dependentphototrophic growth. Genetic systems have not been applied so far for strains that also use thiosulfate for photolithotrophic growth, such as Rhodobacter adriaticus, Rhodovulum (Rhodobacter) suljidophilum, Rhodobacter veldkampii and Rhodopseudomonas sulfoviridis.

5.2. Sulfur Oxidation in Non-phototrophic Bacteria The non-phototrophic or colourless thiobacteria use inorganic reduced sulfur compounds as electron donors for facultative or obligate lithoautotrophic growth. Without exception, carbon dioxide is fixed in these strains via the ribulose 1,5-bisphosphate cycle. These thiobacteria are either neutrophilic or acidophilic, mesophilic or moderately thermophile. They are mostly Gram-negative. A few

260

CORNELIUS G. FRIEDRICH

Gram-positive, endospore-forming sulfur-oxidizing bacilli have been described (Aragno, 1991). The physiology of the colourless neutrophilicthiobacteria and their biochemistry of sulfur oxidation has been reviewed by Kelly (1982, 1988, 1990) and Takakuwa (1992). The acidophilicthiobacteriahave been reviewed by Harrison (1984) and Pronk et al. (199Ob), and the molecular genetics of Thiobacillus ferrooxidans has been reviewed by Rawlings and Kusano (1994). The special characteristics of hydrogen sulfide or thiosulfate oxidation from so far unculturable endosymbionts of the invertebrate Rijlia pachyptila, the mussel Bathymodiolus thermophilus,and Calyptogena mugnijica and others living in the deep-sea vents and seeps, and their impact on carbon dioxide fixation, have been reviewed (Nelson and Fisher, 1995; Nelson and Hagen, 1995). The genus name Thiobacillus initially referred to the lithoautotrophic sulfuroxidizing property of a strain (Beijerinck, 1904, cited by Kelly, 1990). It was later used as a taxonomic marker irrespectiveof the systematicaffiliation of new isolates - and still is. Sulfur oxidation is certainly a major mode of energy conversion for obligately lithotrophic thiobacteria such as Thiobacillus thiooxidans, T ferrooxidans and T neapolitanus. Facultatively lithotrophic thiobacteria may use sulfur oxidation as an alternative mode of energy transformation depending on the environment. Thus, this chemolithotrophic ability is not unique to the species classified therein (Friedrich and Mitrenga, 1981). As emphasized, different lithotrophic abilities may reside in a single strain. Thus, different species assigned to the genus Thiobacillusare distributed over the phylogenetic tree of the Proteobacteria, and are within the alpha, beta and gamma subclasses (see Fig. 3). This distribution applies to other lithotrophic characters which are also harboured by systematically diverse bacteria, such as aerobic hydrogen-oxidation (Aragno and Schlegel, 1981). carbon monoxide oxidation (Meyer and Schlegel, 1983; Gadkari et al., 1990), iron oxidation (Blake et al., 1993), and to the phototrophic character (Stackebrandt et al., 1996). N o groups of thiobacteria can be distinguished on the basis of their ability to grow on tetrathionate. This distinction is independent of the neutrophilic, acidophilic, obligate or facultative lithotrophic or phototrophic character, while acidophilic thiobacteria appear to be generally capable of tetrathionate utilization (see Table 1). The sulfur-oxidizing ability is not restricted to strains which had been enriched on reduced sulfur compounds but is also present in several hydrogenoxidizing bacteria. These bacteria are thought to live in microaerophilic environments that contain hydrogen evolved from anaerobic processes (Schlegel, 1974). Reduced sulfur compounds produced by sulfate-reducing bacteria are also abundant in these environments. Different strains of hydrogen-oxidizing bacteria are capable of oxidizing thiosulfate such as Aquaspirillumautotmphicum,Paracoccus denitrijicans, Pseudomonas palleroni, Pseudomonas pseudojlava GA3, Xanthobacter autotrophicus(Friedrich and Mitrenga, 1981). Aquifex pyrophilus (Huber et al., 1992b), Bacillus schlegelii (Aragno. 1991), Hydrogenobacter acidophilus (Shima and Suzuki. 1993),Hydrogenobacterthennophilus(Alfredssonet al., 1986;

SULFUR-OXIDIZING BACTERIA

261

Bonjour and Aragno, 1986), Thiobacillus caldus (Hallberg and Lindstrom, 1994), Thiobacillusferrooxidans (Drobner et al., 1990) and Thiobacillus plumbophilus (Drobner et al., 1992) (see Table 1). Most studies were performed with neutrophilic colourless thiobacteria and thiosulfate as the substrate, which is stable under aerobic conditions, although acid-labile.Tetrathionateis stable at pH 2 and therefore a favourable substrate under acidic conditions. Several enzymes have been characterized catalysing reactions that involve inorganic sulfur compounds. Nevertheless, the physiological significance of these reactions remained a matter of debate. The following studies may shed some light on the physiological role of sulfur-metabolizing enzymes: (i) the regulation of enzyme expression under selective growth conditions, provided the organism has an alternative energy-conserving metabolism; (ii) isolation and characterization of mutants deficient in sulfur-oxidizing ability; and (iii) the identification of the mutated gene and its molecular analysis. The mere existence of an enzyme activity in vitro does not enable its physiological role to be deduced. Rhodanese represents one example where the description of the enzymic reaction does not allow conclusions to be drawn about the physiological role of the enzyme. Rhodanese is a sulfur transferase and catalyses the cyanide-dependent cleavage of thiosulfate to yield sulfite and thiocyanate (equation 5 ) . Sz0;- + CN- t)SCN- + SO:(5) Although widely distributed in bacteria, plants and animals, the physiological role of the sulfur transferase has not been established. Rhodanese is effective in the synthesis in vitro of heterometallic clusters of Clostridiumpasteurianum or spinach ferredoxins, as well as in reactivation of nitrogenase lacking the full complement of iron and sulfide (Pagani et al., 1984; Bonomi et al., 1985). Its role in thiosulfate metabolism has been questioned. This enzyme is not required for thiosulfate oxidation in vitro in a reconstituted enzyme system of Thiobacillus versutus (Lu and Kelly, 1983c), reclassified as Paracoccus versutus (Katayama et al., 1995). Rhodanese activity is not regulated during anaerobic growth phases of Thiobacillus denitrificans growing on thiosulfate, while thiosulfate reductase is, and no products (sulfide, sulfite or polythionates) other than elemental sulfur are formed (Schedel and Triiper, 1980). The appearance of sulfur, however, was taken as evidence that rhodanese is involved in thiosulfate oxidation of T. denitrificans (Schedel and Triiper, 1980). In Paracoccus denitrificans GB17 rhodanese formation is not regulated. Hydrogen sulfide and thiosulfate-oxidizing enzymes are specifically induced by thiosulfate, while rhodanese is not (Chandra and Friedrich, 1986). 5.2.1. Sulfur Oxidation in Neutrophilic Thiobacteria 5.2.1.1. Thiobacillus novellus Thiobacillus novellus was the first thiobacterium described to be facultatively lithoautotrophic(Starkey, 1935).The bacterium grows heterotrophically with a variety of sugars and organic acids, including

262

CORNELIUS G. FRIEDRICH

oxalate and formate. Formamide and methanol are also used for organoautotrophic growth, and sulfide, thiosulfate, and tetrathionate as lithotrophic substrates (Taylor and Hoare, 1969; Chandra and Shetna, 1977). Thiosulfate-grown cells of T. novellus oxidize thiosulfate and tetrathionate to sulfate, while in a cell-free extract only thiosulfate is metabolized (Charles and Suzuki, 1966a). This enzyme system is associated with the cytoplasmic membrane. This system oxidizes 1 mol of thiosulfate with 2 mol of oxygen to 2 mol of sulfate (equation 6).

-S-SO;

+ 202 + H20 t)2SOf- + 2H+

(6)

) be separated from sulfite: The thiosulfate-oxidizing activity (Km 0.12 m ~ can cytochrome c oxidoreductase by gel filtration. The latter is required for thiosulfate oxidation (Oh and Suzuki, 1977a). The components of the thiosulfate-oxidizing activity in isolated membrane vesicles comprise a membrane-bound rhodanese, considered as the initial step of thiosulfate cleavage. Although membranes did not exhibit a sulfite-oxidizing activity, some activity appeared after desoxycholate treatment, and this activity was significantly enhanced by addition of cytochrome c. Sulfur oxygenase was released from the membranes after mild trypsin treatment (Oh and Suzuki, 1977b). Sulfur oxygenase forming sulfite as proposed by the authors would not allow energy conservation and would decrease the cellular yield by half. The molar growth yield of T novellus during lithoautotrophic growth on thiosulfate, expressed as gram dry cell weight per mole of thiosulfate (YmTS) is 5.7 g mol-' (Leefeldt and Matin, 1980).This value correlates with that of Paracoccus versutus (Y,Ts 5.5 g mol-'; Kuenen, 1979) and Paracoccus denitrificans ( Y m ~ s 4.5 g mol-'; Friedrich and Mitrenga, 1981). The proposed mechar,ism of the involvement of molecular oxygen in sulfur oxidation conflicts with that proposed in F! versutus (see Fig. 5; Kelly, 1988) and in t?denitrificans (see later, Fig. 7). Both strains differ from T. novellus in their inability to utilize polythionates (see Table 1). Attempts to reconstitute the resolved thiosulfate oxidizing activity of T. novellus in vitro were not successful (Oh and Suzuki, 1977b). Sulfite is oxidized by T. novellus,and purified su1fite:cytochromec oxidoreductase specifically reacts with sulfite as electron donor and with femcyanide or cytochrome c as electron acceptor (Charles and Suzuki, 1966b). Su1fite:cytochromec is a monomeric 40 kDa protein and thus smaller than the enzymes from other sources. The enzyme contains the molybdenum cofactor and a haem moiety (Yamanaka et al., 1981; Toghrol and Southerland, 1983). 5.2.1.2. Thiobacillus tepidarius Thiobacillus tepidarius is an obligately lithoautotrophic, moderate thermophile, isolated from the hot springs at Bath, UK. This strain utilizes hydrogen sulfide, thiosulfate, trithionate, tetrathionate and a wide range of other polythionates for growth, and oxidizes sulfite. It has a cellular yield with thiosulfate of Y,,Ts 6.9 g mol-I. In general, the cellular yields of T. tepidariuswith reduced sulfur compounds are about 40% higher than observed for other thiobacteria with the exception of Thiobacillus denitrifcans (Justin and Kelly, 1978; Wood and Kelly, 1986). Therefore, the mechanism of sulfur oxidation and

SULFUR-OXIDIZING BACTERIA

263

energy coupling was investigated. In the first step, thiosulfate is completely oxidized by thiosulfate oxidoreductase (TSO) to tetrathionate (equation 3) which is subsequently oxidized to sulfate (Lu and Kelly, 1988a). The preferred electron acceptor in the in vitro assay of TSO is fenicyanide. The soluble 138 kDa enzyme consists of 45 kDa subunits and is presumably located in the periplasm. Trithionate is hydrolysed to thiosulfate and to sulfate by trithionate hydrolase (equation 7), and thiosulfate is then further oxidized to tetrathionate. -O$-S-SO;

+ H 2 0 t)-S-SO; + SO:- + 2H+

(7)

It was not possible to demonstrate tetrathionate oxidizing activity in cell-free extracts. Evidence was obtained from inhibition studies with whole cells that tetrathionate oxidation is linked to the cytoplasmic membrane. In fact, cytochrome c-dependent sulfite dehydrogenase activity is present in membrane preparations (Lu and Kelly, 1988b). 5.2.1.3. Thiobacillus thioparus Thiobacillus thioparus is obligately Iithoautotrophic and exhibits similar physiological characteristics to T tepidarius; it grows with tetrathionate but is not a moderate thermophile. The thiosulfateoxidizing system described for this obligate lithoautotroph is also similar to that described in T. tepidarius. The similarity of TSO encompasses the molecular mass (1 15 kDa), affinity for thiosulfate as substrate, and preference for ferricyanide as electron acceptor (Lyric and Suzuki, 1970). Also, APS reductase was purified. The enzyme (170 kDa) contains per mol of enzyme, 1 mol FAD, 8-10 mol iron, and 4-5 mol acid-labile sulfur (Adachi and Suzuki, 1977). The crucial activity for lithoautotrophic growth appears to be that of tetrathionate oxidation to sulfate. This core activity, however, has so far not been resolved biochemically from this or any other strain, and the genetic resolution of the enzyme system is hampered by the lack of an alternative lithotrophy other than sulfur oxidation. 5.2.1.4. Marine pseudomonad strain 16B The marine heterotrophic pseudomonad strain 16B is able to use thiosulfate as an additional - but not as the sole - source of energy, and thiosulfate oxidation is coupled to ATP synthesis (Tuttle, 1980). In this strain, the regulation of formation of these enzymes was investigated as well as their biochemistry. Addition of thiosulfate to heterotrophic growth conditions increases both the growth rate and the yield of 16B in dilute organic media owing to a carbon-sparingeffect; i.e. energy derived from thiosulfate oxidation permits carbon to be conserved for anabolic metabolism rather than respired (Tuttle e f al., 1974). Several sulfur-metabolizing enzyme activities have been described in this strain grown under aerobic and anaerobic conditions. Sulfur metabolism in the heterotroph 16B is unique in that it represents a composite of activities found in obligate aerobes and anaerobes. The thiosulfate-oxidizing enzyme (TSO) forms tetrathionate according to equation 3. It is a soluble protein of 132 kDa and constitutively formed under aerobic or anaerobic growth conditions (Tuttle, 1980). As shown from the purified enzyme, TSO mediates tetrathionate reduction under anaerobic conditions provided a suitable reductant is present

264

CORNELIUS G. FRIEDRICH

(Tuttle et al., 1983; Whited and Tuttle, 1983). From anaerobically grown cells, a second tetrathionate-reducing activity (TTR) was detected, present in the membrane and the cytoplasmic fraction, and differed from TSO in catalysing the reverse reaction. Tetrathionate reductase is induced by tetrathionate and by thiosulfate, although the true inducer is not clear since both substrates are interconvertible and the reaction is repressed under aerobic conditions irrespective of the presence of an inducer. From anaerobic thiosulfate- or tetrathionate-induced cells thiosulfate reductase (TSR) is also found (Whited and Tuttle, 1983). In this strain the sulfur-oxidizing system and energy coupling should be amenable to genetic analysis because the basic requirements are met. However, such an approach has not been reported. 5.2.1.5. Thiobacillus denitrificans Thiobacillus denitrijicans is an obligately lithoautotrophic, facultative, anaerobic thiobacterium able to grow with tetrathionate and other reduced sulfur compounds (see Table 1). Under anaerobic conditions, this strain grows on thiosulfate as reductant and nitrate as the electron acceptor. The strain does not grow on hydrogen sulfide, elemental sulfur, sulfite or polythionates, but oxidizes transiently deposited sulfur (Schedel and Triiper, 1980). Sirohaem sulfite reductase (SR)was believed to function in the ‘reverse’direction according to equation 8 owing to the fundamental differences of the enzyme characteristics compared with assimilatory enzymes. Sz-

+ 3H20 t)SO:- + 6H’ + 2e-

(8)

The SR of Z denitrificans has been purified to homogeneity. It has a tetrameric a& structure of 160 kDa and subunits of 38 kDa and 43 kDa. In an anaerobic assay with a hydrogen atmosphere, and with viologens reduced by Desuljovibrio hydrogenase, the enzyme reduces sulfite to sulfide, thiosulfate and trithionate (Schedel and Triiper, 1979). The concept of the reverse function has been considered since APS reductase appears to be present in Z denitrificans in rather large amounts (4-5 % of total protein). The purified APS reductase was characterized as a flavoprotein containing non-haem iron (Bowen et al., 1966). Thiosulfate reductase activity abruptly declines upon depletion of thiosulfate in the medium. This enzyme was discussed as a second activity of rhodanese (Schedel and Triiper, 1980). However, the kinetics of decrease suggest a first-order inactivation after thiosulfate exhaustion, while rhodanese activity remains constant. From experiments using 35S-SO:- it is clear that 35S-(sulfane)-sulfur is assimilated, suggesting its conversion to 35S-hydrogen sulfide. Formation of sulfide from elemental sulfur or thiosulfate, however, was not proposed. An AMP-independent sulfite oxidase was described by Aminuddin and Nicholas (1974). This membranebound enzyme uses nitrate, oxygen or femcyanide as an electron acceptor and does not require AMP. The purified enzyme, however, functions only with ferricyanide. This activity deserves revived attention in light of the results obtained from APS reductase inactivation in Chromatiurn vinosum. Thus, the concept of the ‘reverse’ function of sirohaem sulfite reductase in Z denitrificans awaits a new approach.

SULFUR-OXIDIZING BACTERIA

265

5.2.1.6. Paracoccus versutus The genus Paracoccus contains three species, of which I! versufus (formerly Thiobacillus versutus; Katayama et al., 1995) and I! denitrificans (van Verseveld and Stouthamer, 1992) are facultative lithoautotrophs, able to grow on a variety of organic carbon sources and lithoautotrophically on thiosulfate but not polythionates. Growth of I! versutus on thiosulfate has been studied extensively (Wood and Kelly, 1977, 1981; Kuenen, 1979); it requires molybdate (Friedrich et al., 1986) and manganese (11) ions (Cammack et al., 1989). The thiosulfate-oxidizingenzyme system of C versutus was the first to be analysed with respect to the proteins involved, the reconstitution of the catalytic activity with the different components involved, and electron transfer. Evidence was obtained that the components of the thiosulfate-oxidizing enzyme system are located in the periplasm (Lu, 1986). The enzyme system is composed of five components: enzyme A, enzyme B, cytochrome c-551, cytochrome c-552.5 and sulfite dehydrogenase (Kelly, 1988, 1990;Fig. 5 ) . Enzyme A is a colourless 16 kDa protein which binds thiosulfate at the sulfone moiety in a 1:1 molar ratio with an apparent Kd of 70 pM.Binding of thiosulfate is competitively inhibited by sulfite (Ki25 pM;Lu ef al., 1985). Enzyme B is a colourless 64 kDa protein and has a dimeric composition of 32 kDa subunits (Lu and Kelly, 1983a,b; Fig. 5). Convincing evidence was presented for a binuclear manganese cluster in enzyme B (Cammack et al., 1989). The enzymatic reaction of enzyme B has so far not been established. Cytochrome c-552.5 is a homodimer of 56 kDa with subunits of 29 kDa, and cytochrome c-55 1 is a hexamer of 260 kDa composed of 43 kDa subunits (Lu and Kelly, 1984b). The enzyme complex mediates thiosulfate oxidation and the transfer of 8 mol of electrons per mol of thiosulfate to the final acceptor cytochrome c (Lu and Kelly, 1983a; Cammack et al., 1989). Su1fite:cytochrome c oxidoreductase, designated sulfite dehydrogenase, is a 44 kDa protein which is intimately associated with cytochrome c-551. Separation of the two proteins greatly decreases sulfite dehydrogenase activity, which is not restored by remixing them. Addition of sulfite dehydrogenase to the reconstituted thiosulfate-oxidizingenzyme system increases the thiosulfate oxidation rate by 2&25%. Since no sulfite and no other possible intermediates of thiosulfate oxidation are observed, the function and the true substrateof sulfite dehydrogenase in vivo are still in question (Lu and Kelly, 1984a). From the data presented it is clear that sulfur oxidation occurs without release of intermediates. The role of enzyme B and the mechanism of thiosulfate cleavage remain to be solved. 5.2.1.7. Paracoccus denitrificans GB17 Paracoccus denitrificans grows autotrophically with molecular hydrogen, thiosulfate and methanol. The components of the respiratory chain and the genes involved in one-carbon metabolism and in denitrificationhave been intensively investigated as reviewed by Stouthamer (1992). Paracoccus denirrijhns strain GB 17,originally isolated by Robertson and Kuenen (1983) and designated Thiosphaerapantotropha GB 17, was described as differing from the genus Paracoccus and from C versutus (Kuenen and Robertson, 1989). Re-examination of I: pantotropha revealed that the 16s rRNA sequence is

266

CORNELIUS G. FRIEDRICH

-s-so,-

U

cytochromc c - Sulphilc: oxidorcductw W, 4-%OW cytochrome Cytochromc c,,, oxidrr - (M, 260.000: of six polypeptides of

0 0

ENZYME A (M, 16.000) Binds thionrlphate [A-SO~-S-l ina I : I m o b ratio

U

I

ENZYME

0 0

[I

n

JHlO

B

(MI 63,000; subunits M, 32,000; contains Mn in a 1:1 molar ratio I

2so:IOH'

Outer membrane

=regate

M, 43,000; 4-S hrem and 6-7 Fe per mole;

Two mid-point rcdox

A

+240mV cytochromc

potential centres,

- En,, - I I S M d /

(001)

I

8C-

CIS1

I

Cytochrome cJJU (M, 56,000; subunitrhf, 29,000; up to 3 h a m and 3 Fe per mole; At least two mid-point mdox potential ccntrcs.EE,, - 2 l S m d + 2 2 0 m V ~~

Periplum

Cytoplasmic membrane

Figure 5 The periplasmic thiosulfate-oxidizing multi-enzyme system of Paracoccus versutus. From Kelly (1988) with permission.

identical to that off? denitrificans type strain, and the DNA homology is 85%. In agreement with the report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics (Wayne e f al., 1987), Thiosphuera pantotropha GB17 was reclassified as Paracoccus denitrificans GB 17 (Ludwig et al., 1993). According to the 16s rRNA sequence and DNA-DNA homology, Paracoccus versutus (formerly Thiobacillus versutus, Thiobacillus A2) is the closest relative of R denitrijkans (Katayama et al., 1995). From the similarity of both strains it is likely that their enzyme system for thiosulfate oxidation is similar or even identical. Attempts to use f? versutus in gene transfer experiments were not very promising (Davidson et al., 1985; Chandra and Friedrich, 1986; DeVries et al., 1989). Therefore, R denirrifcans GB17 was chosen as a genetic model (see Table 3). Using the system of Simon et al. (1983), transposon Tn5-mob mutagenesis yielded three classes of well-defined mutants in thiosulfate and hydrogen sulfide oxidation (Sox-): (i) mutants with specific lesions in sulfur metabolism; (ii) mutants that are pleiotropic and unable to oxidize thiosulfate and molecular hydrogen, or to reduce nitrite anaerobically, suggesting a lesion in a common cytochrome; and (iii) pleiotropic mutants with deficiencies in growth with formate, nitrate and

SULFUR-OXIDIZING BACTERIA

267

xanthine, owing to the lack of a functional molybdenum cofactor (Chandra and Friedrich, 1986; Friedrich et al., 1986). With the aid of a class (i) mutant, a 13 kb DNA region was cloned from the wild-type. This piece of DNA carries part of the genes required for the Sox character (Mittenhuber ef al., 1991). Partial sequence analysis revealed three open reading frames (OW),designated s o d , B and C. The protocol for purification of the thiosulfate-oxidizing enzyme system (Lu and Kelly, 1983a,b) was modified to stabilize the components of the corresponding system of I? denitrificans GB17 and to enhance separation of the proteins. A 29 kDa subunit of cytochrome c, required for thiosulfate oxidation, is encoded by s o d as evident from amino-acid sequences of internal peptides. Moreover, a native 30 kDa protein separates into two peptides of about 12 kDa and 16 kDa by SDS-PAGE, of which the 16 kDa protein appears to be equivalent to enzyme A of I? versutus as judged from the size of the protein and from N-terminal amino-acid sequence comparison. However, a heterodimer has now been clearly demonstrated in I? denitrificuns GB17 (A. Quentmeier et al., unpublished data). The size of the protein deduced from soxB is approximately 60.5 kDa, suggesting the presence of a leader peptide containing 16 amino acids, and a processed size of 59 kDa. The SoxB protein is closely related to enzyme B of I? versutus as deduced from comparison with amino-acid sequences of peptides (Wodara et al., 1994). The putative signal peptide supports the periplasmic location of the enzyme complex in I? versutus (Lu, 1986). Immunochemical studies confirmed the size of 59 kDa for the mature SoxB of I? denitrificans GB17 and I? versutus (C. Friedrich et ul., unpublished data). The class (i) mutants are unable to oxidize thiosulfate and hydrogen sulfide (Chandra and Friedrich. 1986). SoxB was proposed to function as sulfide dehydrogenase in I? denitrificans GB 17, and biochemical evidence suggested the hydrogen sulfide-dependent activity was linked to a 32 kDa protein (Schneider and Friedrich, 1994). In light of recent immunochemical data confirming SoxB to be a 59 kDa protein (C. Friedrich et al., unpublished data) and the detection of SQR activity from this strain (M. Schiitz and G. Hauska, personal communication), the assignment of sulfide dehydrogenase activity to SoxB appears to be questionable. As noted below, the gene coding for flavocytochrome c has been detected, also performing sulfide-dependent cytochrome c reduction. The soxC gene encodes for a 47 kDa protein with a putative signal peptide of 40 amino acids, and 43.7 kDa for the mature protein. The deduced amino-acid sequence was identical to rat liver sulfite oxidase, suggesting that SoxC functions as su1fite:cytochrome c oxidoreductase, referred to as sulfite dehydrogenase (STD). The significance of STD in thiosulfate oxidation is evident from a mutant GBsoxCA with an in-frame deletion in soxC at a putative molybdenum cofactor binding domain (Barber and Neame, 1990), which prevented growth of the mutant on thiosulfate and STD activity in vitro, and led to a marked decrease in thiosulfate oxidation rate (Wodara et al., 1997). Keeping in mind the similarity of the systems oft? versutus and I? denitr@cuns GB17, this result demonstrates the significance of STD for thiosulfate oxidation. Since induced cells of strain GBsoxCd still

268

CORNELIUS G. FRIEDRICH

I kb

$;

I

sod

SOXB

L V

soxc

h

)

V

SOXD

h

h

V

V

] soxE

>I

soxF

Figure 6 The sox gene region of Paracoccus denitrij?cans GB17: SOA, partial open reading frame coding for a 29 kDa dihaem cytochromec;sox& 60 kDa SoxB protein; SOXC, 47 kDa sulfite dehydrogenase; SOXD,40 kDa cytochrome c; s o d , 26 kDa cytochrome c; SOXF,partial open reading frame of a flavocytochrome.

oxidize thiosulfate at a low rate, the role of STD may be in the detoxification of sulfite during growth with thiosulfate. Further sequence analysis revealed three designated soxD, E and F SOXDand soxE additional ORFs downstream of SOXC, code for periplasmic c-type cytochromesof 40 kDa and 26 kDa respectively,which contain putative signal peptides and conserved haem-binding sites. At present only a partial sequence is available for soxF, which is predicted to encode a protein with extensive similarity to the flavoprotein of C. vinosum (Fig. 6 ) (C. Wodara ef al., unpublished data; Dolata et al., 1993). As outlined above, flavocytochrome c is associated with a haemoprotein at least in the cases so far investigated. Therefore, the presence of an equivalent protein is predicted in I! denitrificans GB17. Recently, using antibodies against an immunogenic epitope of the deduced amino-acid sequence of SOXF,immunochemical data demonstrate that the soxF gene product is coregulated with expression of soxB and soxC (C. Friedrich, unpublished data). From the sequence analysis and biochemical data it is evident that the sulfur-oxidizing system of I! denitrificans GB17 is more complex than that described for f? versutus. The requirement for sulfite dehydrogenase has definitely been demonstrated by the phenotype of a mutant, impaired in the soxC gene. Flavocytochromes have so far only been investigated in phototrophic bacteria which (with the exceptions listed in Table 3) are able to oxidize thiosulfate. Preliminary sequence data suggest that a flavocytochromegene is present in the I! denitrificansGB 17 operon coding for thiosulfate and hydrogen sulfide oxidation. From the data elaborated so far in I! denitrificans GB 17 we share the reservations of Dolata et al. (1993) that this protein functions in vivo as sulfide dehydrogenase. Flavocytochrome c of C. vinosum strongly binds sulfite and thiosulfate (Cusanovich et al., 1991; Meyer et al., 1991). The close link of the putative flavocytochrome c with thiosulfate oxidation on the genetic level suggests its specific function in thiosulfate metabolism, and this may be an initial step of thiosulfate oxidation. Our present view of the thiosulfate-oxidizing system of I! denitrificans GB 17 is summarized in Fig. 7. The data of Lu and Kelly (1983a,b) and our own data suggest a covalent link of thiosulfate to a protein of the system. Two alternatives exist for such a covalent

SULFUR-OXIDIZING BACTERIA

269

Figure 7 Model of the periplasmic thiosulfate-oxidizing enzyme system of Paracoccus

denitnflcunsGB 17. Proteins were designated according to the gene designation given in Fig.

6. A, 29 kDa dihaem cytochrome c; B, 60 kDa SoxB protein; C, 47 kDa sulfite dehydrogenase;D.40kDa cytochrome c; E,26 kDa cytochrome c; F, putative flavoprotein.

bond: (i) oxidation of the sulfane sulfur of thiosulfate with a sulfide moiety of the protein (equation 9), hydrolysis of the sulfone (equation 10) and successive oxidation of the persulfide to sulfonate (equation 11) with subsequent hydrolysis to yield sulfate analogous to equation 10; (ii) addition of thiosulfate with concomitant reductive cleavage of a disulfide bond. The observations of Meyer et al. (1991) suggest interaction of a disulfide bond of the Chromatiurn vinusurn flavocytochrome c with sulfite and with thiosulfate. By analogy, a disulfide bond may be reduced according to equation 12. E-S- + %SO; t)E-S-S-SO; + 2e- (E= enzyme) (9)

E-S-S-SO; + H2O C) E-S-S- + H2S04 E-S-S- + 3H20 t)E-S-SO; + 6e- + 6H' E-S-S-E + -S-SO; C) E-S- + E-S-S-SO;

5.2.2.

(10) (11) (12)

Gene Transfer Systems in Neutrophilic Thiubacteria

The genetics of bacterial sulfur oxidation has only a brief history. Prerequisites given for other strains are mostly absent from neutrophilic and from acidophilic thiobacteria. Not all strains form colonies on solid media, and if they do, growth is sparse, since inorganic sulfur compounds allow only a low energy yield. Moreover, oxidation of reduced sulfur yields mostly sulfuric acid, acidifying the medium. Mutants unable to oxidize inorganic sulfur compounds cannot be identified on the

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basis of loss of acid production owing to the rapid diffusion of acid and buffer. Therefore, chemical mutagenesis and conventional methodologies for mutant selection are not practical. The application of transposon mutagenesis is a straightforward approach, which requires reliable conjugation or transformation systems. These systems have been applied to few thiobacteria differing in their physiology and biochemistry of sulfur oxidation. Broad host range plasmids of the incompatibility groups (Inc) P and Q and their derivatives - and more seldomly IncW plasmids - mostly carrying antibiotic resistance markers are transferred by conjugation from E. coli to facultative and obligate lithotrophic thiobacteria at efficiencies of lo-* to lo4 transconjugants per donor or recipient (Table 3). This has been demonstrated for different strains of Paracoccus denitripcans (Chandra and Friedrich, 1986; DeVries et al., 1989) and for t? versutus (Davidson et al., 1985). Davidson and Summers (1983) have surveyed the transfer of different broad host range plasmids from E. coli to neutrophilic members of the genus Thiobacillus and Thiobacillus acidophilus, and have examined the stability of these plasmids and the expression of their markers. Plasmids R388, RP4 and different RP4 derivatives are transmissible by conjugation to Thiobacillus novellus at frequencies of per recipient. The cosmid cloning vector pVKl00 can be mobilized with the aid of the ‘helper’ plasmid pRK2013 in triparental crosses, and is stably maintained in T. novellus while pRK2013 is not. Plasmids were not transmissible from E. coli to Thiobacillus intermedius, Thiobacillus perometabolis, and to the obligate lithoautotroph Thiobacillus neapolitanus, although they can be mated from T. novellus into these strains. With the exceptions of ampicillin and chloramphenicol resistance, a variety of useful resistance loci including tetracycline, gentamicin, trimethoprim, mercury and tellurite are expressed in T. novellus (Davidson and Summers, 1983). Using E. coli ROE531 or Pseudomonas aeruginosa PA02 as donor, Kulpa et al. (1983) demonstrated transmission of RP1 from both strains to I: neapolitanus at reasonable frequencies of 4.8-8.5 x lo-’, while back-transfer from T. neapolitanus is significantly less efficient. Plasmids are transferred to T. versutus only at low frequencies (Davidson and Summers, 1983; Chandra and Friedrich, 1986), while transformation by electroporation is an efficient method for transfer of plasmids of 5 kb to 100 kb (Wlodarczyk et al., 1994). Electrotransformation was also achieved for T. neupolitanus and T intermedius, using broad host range plasmid pRK415 and cloning vectors designed for use in E. coli and the thiobacteria. The plasmids are maintained in the strains under selective conditions and were used to generate mutants in carboxysome formation (English et al., 1995). The nitrogen-fixing hydrogen bacterium Xanthobacter autotrophicus GZ29 oxidizes thiosulfate at a high rate but grows slowly with thiosulfate (Friedrich and Mitrenga, 1981; C.G. Friedrich, unpublished data). This strain harbours a small phage capable of generalized transduction at high efficiency but with limited capacity for DNA to be cotransduced (Wilke and Schlegel, 1979). A low-frequency conjugational system was also described for this strain (Wilke, 1980).

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Transpositional mutagenesis has been successfully applied to a wide range of diverse Gram-negative bacteria and has proved a useful tool for a few thiobacteria. Suicide vectors could not be successfully applied for transpositional mutagenesis of I: novellus but this was achieved by means of the incompatibility of IncP plasmids. Insertion of Tn501 coding for mercury resistance yielded different types of mutants, of which one showed a reduced ability to oxidize elemental sulfur, thiosulfate and tetrathionate, and to fix carbon dioxide. Transposon TnSOl cannot be used for mutagenesis of F! versutus, since this strain exhibits a natural mercury resistance, and is resistant to arsenate and gentamicin. A suicide vector system Tnl721 coding for tetracycline resistance can be introduced into I: versutus. Among the mutants isolated, none was impaired in sulfur-oxidizing ability (Davidson et al., 1985). A heterotrophic bacterium, auxotrophic for glutamate and able to oxidize thiosulfate, has been isolated, and is designated Bosea thiooxidans. Based on 16s rRNA analysis, B. thiooxidans strain BI-42 is a distinct taxon and belongs to the alpha subclass of the Proteobacteria (Das et al., 1996). To analyse the thiosulfateoxidizing system in B. thiooxidans, the conjugation system developed by Simon et al. (1983) was used with E. coli S17-1 as donor for transfer of the suicide plasmid pSUP5011, carrying transposon Tn5-mob, as previously successfully used for F! denitrijicans GB 17. Using strain BI-50,Das and Mishra (1996) isolated three types of mutants impaired in thiosulfate metabolism. These mutations cause deficiency either in thiosulfate oxidase or cytochrome content. One leaky mutant deposits elemental sulfur on the surface of the colonies. This thiosulfate-oxidizing system is potentially interesting but needs description of the biochemistry of the enzymes involved. DeVries et al. (1989) described a straightforward method to increase transfer efficiencies of plasmids and transposons into €? denitrificans DSM413 to isolate mutants in methanol utilization. Using E. coli as donor, about 10” transconjugants per recipient cell were observed with IncPl and IncQ broad host range plasmids, and attempts to isolate transposon-induced mutations using suicide vectors were unsuccessful. Mutants defective in restriction and modification were isolated after chemical mutagenesis of F! denitrificans DSM413 and these exhibit transfer efficiencies increased by over four orders of magnitude (DeVries et al., 1989). 5.2.3. Sulfur Oxidation in Acidophilic Thiobacteria The acidophilic thiobacteria grow best at pH 2-3. The acidophilic, obligately lithoautotrophic thiobacteria, Thiobacillusferrooxidans and Thiobacillus thiooxiduns, and the facultatively lithoautotrophic Thiobacillus acidophilus, have been described in detail with respect to their physiology, industrial and ecological significance in mineral leaching (Harrison, 1984) and the biochemistry of sulfur oxidation (Pronk et al., 1990b). These strains use sulfide, elemental sulfur, thiosulfate and tetrathionate for lithoautotrophic growth. While T. thiooxidans is

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restricted to these substrates, I: fermoxidans is highly versatile and in addition grows on rather insoluble heavy metal sulfide ores such as pyrite, chalcopyrite, sphalerite and others, and oxidizes sulfide to sulfate (Torma, 1988). Thiobacillus fermoxidans oxidizes ferrous to ferric iron aerobically (Ingledew, 1982),and under anaerobic conditions oxidizes sulfur and reduces ferric to ferrous iron (Pronk et al., 1991b, 1992; Suzuki et al., 1990). Also, molybdenum (V)ions can be oxidized (Sugio et al., 1992a). Thiobacillus fermoxidans also grows with molecular hydrogen as electron donor (Drobner et al., 1990). Thiobacillus acidophilus is unable to oxidize ferrous iron, and grows well with pentoses, hexoses, and glutamate (Guay and Silver, 1975). Formate is undissociated at low pH (p& 3.8), uncouples energy conservation at low pH, and causes bacteriostasis. Organoautotrophic growth of I:fermoxiduns and T. acidophilus with formate was discovered when formate was supplied at concentrations of less than 100 PM in a chemostat (Pronk et al., 1991a). The physiological versatility of I: fermoxidans is brought about by specific enzymes for sulfur oxidation (Sugio et al., 1992a,b), for hydrogen oxidation (Fischer et al., 1996) and for formate oxidation. Some of the enzymes catalysing these reactions have been characterized. These relate to sulfur metabolism dependent on iron, molybdenum, or copper (Sugio et al., 1990, 1992a). The existence of a hydrogen su1fide:ferric ion oxidoreductase was reported from washed cell experiSu1fur:femc ion oxidoreductasecatalyses the oxidation ments (Sugio et al., 1992~). of elemental sulfur to sulfite with ferric ion as electron acceptor (Sugio etal., 1989). Su1fur:ferric ion oxidoreductase was purified to homogeneity, catalysing anaerobically the oxidation of 1 mol elemental sulfur to sulfite with concomitant reduction of 4 mol ferric ion to yield ferrous ion (Sugio et al., 1987). The existence of a new type of sulfite oxidase which transfers electrons to femc ion was proposed by Sugio et al. (1987). Oxidation of sulfite may occur in vivo by su1fite:ferric ion oxidoreductase (SFOR) which differs from other sulfite oxidases described so far. It has a molecular mass of 650 kDa and is composed of two non-identical subunits of 61 kDa and 59 kDa. The catalytic activity of this enzyme suffers from end-product inhibition by ferrous ion (Sugio et al., 1992b). The enzymology of thiosulfate and tetrathionate metabolism in T.fermoxidans has not been analysed so far. Thiobacillus thiooxiduns grows well with elemental sulfur which is readily oxidized to sulfite by washed cell suspensions when sulfite oxidation is inhibited by 2-n-heptyl-4-hydroxyquinolineN-oxide (HQNO; Suzuki et al., 1992). The strain requires molybdate for lithoautotrophic growth with elemental sulfur (Takakuwa et al., 1977). Also, thiosulfate conversion has been investigated from whole cells and cell-free extracts and partially purified preparations (Chan and Suzuki, 1994). Oxidation of inorganic sulfur compounds by thiobacilli has been reviewed by Suzuki et al. (1992, 1993), who summarized the evidence for their conclusion that sulfur and sulfite oxidation are the key reactions in the oxidation of sulfide, thiosulfate and tetrathionate. However, the crucial activity has not been identified so far.

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Thiobacillus acidophilus, characterized in detail by Guay and Silver (1975). Mason and Kelly (1988) and Pro& et al. (199Oa). oxidizes hydrogen sulfide, thiosulfate and trithionate at a high rate at pH 2. Tetrathionateand elemental sulfur are oxidized at intermediate rates, while sulfite is oxidized at a low rate even at its optimal pH of 6. Tetrathionate is metabolized to stoichiometric amounts of thiosulfate, sulfate and elemental sulfur under anaerobic conditions. During oxidation of hydrogen sulfide, elemental sulfur appears as an intermediate. Oxidation of thiosulfate or trithionate yields tetrathionate which is then oxidized to sulfate (Meulenberget al., 1992a). Thiobacillusacidophilus hydrolyses trithionate to thiosulfate and sulfate (equation 7). Thiosulfate is oxidized to tetrathionate (equation 3). Purified trithionate hydrolase (99 kDa) is composed of subunits of 34 kDa, has a pH optimum of about 4, and does not hydrolyse tetrathionate (Meulenberg et al., 1992b). Apparently homogenous thiosulfate dehydrogenase (thiosu1fate:acceptor oxidoreductase) has a native molecular mass of 102 kDa, contains 5.3 mol of haem per mol of enzyme, is composed of subunits of 24 kDa and 20 kDa, and is thought to be located in the periplasm. Both subunits contain c-553-type haem. The enzyme is strongly inhibited by sulfite, and significantly inhibited by trithionate (Meulenberg et al., 1993). Thiosulfate dehydrogenase yields two electrons which can be coupled to energy conservation. The catalysis differs from that of trithionate hydrolase. However, the catalysis by both enzymes merges in tetrathionate formation which is converted to sulfur under anaerobiosis. Hydrogen sulfide, elemental sulfur and tetrathionate metabolism may yield a common sulfur intermediate which is then further oxidized to sulfate (Meulenberg et al., 1992a). The formation of elemental sulfur under anaerobic conditions may be regarded as an artificial reaction when an appropriate electron acceptor is missing. Formation of sulfur at low aeration rates is also observed from Thiobacillus caMus growing on tetrathionate (C. Friedrich, J. Fischer and B. Heller, unpublished data). In conclusion, the enzymes catalysing the conversion of tetrathionate to sulfur and sulfate are yet to be identified in I: acidophilus and in other tetrathionate-utilizingstrains. 5.2.4. Gene Transfer Systems in Acidophilic Thiobacteria Generally, conjugation occurs under optimum physiological conditions for both donor and recipient: for acidophilic bacteria, these conditions are acidic. If DNAis to be transferred from the neutrophile E. coli these conditions are detrimental to the physiological requirements of the donor. Identification of the transconjugants requires post-mating prerequisites such as a high plating efficiency of the acidophilic recipients. Early attempts to transfer plasmids directly from E. coli into acidophilic strains have failed. Broad host range plasmids of incompatibility group IncP and IncQ can, however, be transferred into Thiobacillus novellus, and from this strain these plasmids are mated into I: acidophilus (Davidson and Summers,

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1983). Using a different medium, direct transfer of RP4 and pVKlOl from E. coli K12J53 into T. ucidophifus results in transfer efficiencies of 1.1 x and 2.9 x lo4 transconjugants per recipient, respectively. The IncQ plasmid pSUP106 is transferred to T. ucidophifus in triparental matings with pRK2013 as ‘helper’ plasmid, and the resistance markers are expressed (Quentmeier and Friedrich, 1994). Using T. thiooxidans as recipient, RP4 and other IncP plasmids are transferred from E. coli C600 into this strain at efficiencies of 1.3 x lo-’ to 1.4 x lo-’ and are maintained in this strain. The tetracycline resistance marker is expressed in T. thiooxiduns, while ampicillin resistance is not. Back-transfer from T. thiooxiduns to E. coli is more efficient (1.0 x Jin et al., 1992). The molecular genetics of T. ferrooxiduns have been reviewed comprehensively by Rawlings and Kusano (1994) and this section is restricted to gene transfer systems into this strain. Numerous reports deal with the plating efficiency of T. ferrooxiduns and previous attempts have failed to establish a natural gene transfer system. Therefore, electrotransformationwas applied using plasmid pKT240 with an IncQ-type replicon and the determinants for mercury resistance. Electrotransformation into a mercury-sensitive strain resulted only in low efficiencies of up to 200 colonies per pg of DNA (Kusano et ul., 1992). Recently, a solid medium of pH 4.64.8 was described for this strain containing both ferrous iron plus thiosulfate, and either kanamycin or streptomycinto select for recombinants(Peng el al., 1994). Using the filter mating technique, IncP and IncQ plasmids are transferred from E. coli to T.ferrooxidunsat efficiencies of up to 2 x 10-~transconjugants per recipient and are stably maintained in this strain. Back-transfer to E. coli is two orders of magnitude more efficient. With this conjugational system, using the suicide vector pSUPlOll (Simon et uf., 1983) carrying Tn5 coding for kanamycin resistance, transposon Tn5 was introduced to T. ferrooxiduns with apparent transposition frequencies of up to 2.7 x lo4 (Peng et ul., 1994). Thiobucillus ferrooxiduns requires highly acid conditions for optimal growth; it exhibits resistance to extreme concentration of toxic heavy metals, and owing to its mineral leaching capacity is of industrial importance. Previously, genes of T. ferrooxiduns have been studied in E. coli,and this efficient DNA transfer system has opened I:ferrooxidans to genetic manipulation and analysis of the various characteristics that make this strain so special.

6. CONCLUSIONS

In the past, oxidation of inorganic reduced sulfur compounds in phylogenetically different bacteria was taken as evidence for different biochemical pathways (Schlegel, 1975). In a historical review, Kelly (1982) summarized the different and complex pathways proposed earlier. Meanwhile, new proteins have been characterized involved in sulfur metabolism in different phototrophic or chemotrophic

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bacteria. Elemental sulfur is converted by sulfur oxygenase/reductaseto sulfite and to hydrogen sulfide in Acidianus ambivalens; this enzyme is so far unique. Its significance for energy conservation remains either to be established or will lead to identification of an activity oxidizing hydrogen sulfide to sulfate. Hydrogen sulfide is oxidized by su1fide:quinone reductase and by flavocytochrome c, in phototrophic and presumably chemotrophic bacteria, and both activities may be present in one strain. Analysis of the gene region involved in thiosulfate oxidation in Paracoccus denitrificans GB17 revealed a gene coding for a flavoprotein as deduced from the degree of identity to the flavocytochrome c of C . vinosurn. The function of flavocytochrome c as sulfide dehydrogenase has been questioned on the basis of biophysical data. Its genetic link in I? denitrifcans GB17 strongly suggests a function in thiosulfate oxidation, and inactivation of the flavocytochrome will resolve this question. The detection of APS reductase, of ADP and ATP sulfurylase in phototrophic and in chemotrophic thiobacteria, and of sirohaem sulfite reductase, led to the concept of the ‘reverse’ function of these enzymes, which was accepted for three decades. Chromatiurn vinosurn exhibits all the physiological characteristics required for genetic work, and this strain was selected to establish a genetic system. A single report by Dahl (1996) has presented convincing evidence that APS reductase does not function in sulfur oxidation in C. vinosum. In this report, Dahl questioned the concept of the ‘reverse’function of this enzyme. The function of sirohaem sulfite reductase also needs to be investigated at a genetic level. It seems likely that the diversity of mechanisms of sulfur oxidation proposed earlier may be less than was apparent. ‘The reactions about which least are known are those affecting thiosulphate cleavage, and those converting sulphur to sulphite’ (Kelly, 1972). Kelly (1982) confirmed this view. It is still true today for phototrophic and for chemotrophic thiobacteria oxidizing reduced sulfur compounds. However, new perspectives have emerged. Gene transfer systems have been established for different thiobacteria and existing systems can be improved. Chlorobium limicola f. sp. thiosulfatophilurncan be readily transformed and grows with hydrogen sulfide, thiosulfate and tetrathionate. The sulfur-oxidizing system would be worth analysing, as part of the information for growth with thiosulfate resides on a plasmid (MCndez-Alvarez et al., 1994). Genetic systems for members of the Rhodospirillaceae able to oxidize thiosulfate to sulfate need to be established. Good candidates are Rhodobacfer sulfdophilum or Rhodopseudomonas palustris, since these are metabolically highly versatile. Of the colourless thiobacteria, the facultative lithoautotrophs are suited for analysis of their sulfur-oxidizing system. Of these, Thiobacillus novellus grows with tetrathionate, and a genetic system for transfer of plasmids is already well characterized. Several bacteria convert thiosulfate to tetrathionate, and the respective enzymes have been characterized from several sources (Lyric and Suzuki, 1970; Schmitt et al., 1981; Lu and Kelly, 1988b; Meulenberg etal., 1993). A likely intermediate of tetrathionate oxidation may be elemental sulfur. This intermediate, however, is observed upon limited supply of an electron acceptor. The

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actual proteins involved in the oxidation of tetrathionate have not been characterized and are likely to differ from a thiosulfate-oxidizing enzyme system. The enzyme system of Paracoccus versutus analysed by Lu and Kelly (1983a,b,c) is one of the best characterized. Paracoccus versutus is closely related to I! denitrificans; however, its thiosulfate-oxidizingenzyme system appears to be more complex as evident from genetic analysis. The F! denitrificans system may either differ in several details, or the enzyme preparations from F! versutus may have contained traces of other proteins. A previous survey of sulfur-oxidizing systems in various bacteria used relevant sox-DNA of F! denitrificans GB17 as probes (Mittenhuber et al., 1991). This method, however, was insufficient to discriminate between different systems. However, this method clearly demonstrated the close relationship of the systems with respect to DNA homology to F! versutus and to Rhodobacter capsulatus which are members of the alpha-3 subclass of the Proteobacteria. Since R. cupsulatus oxidizes only hydrogen sulfide to elemental sulfur, the hybridization may lead to the enzyme responsible for this step. In conclusion, an immunochemical analysis will help to further elucidate the relationship of the thiosulfate and of the tetrathionate-oxidizing enzyme systems in the different bacteria.

I thank T. Beffa, H. Cypionka, C. Dahl, G. Hauska, H. W. Jannasch, D. P. Kelly, J. G. Kuenen, D. C. Nelson, I. Suzuki, T. Sugio, S. Takakuwa and H. G. Triiper for reprints; M. Schutz, G. Hauska and E. Padan for information prior to publication; D. P. Kelly and T. E. Meyer for permission to reproduce figures; W. Hammer and H.-K. Tang for help in literature acquisition, and F. Bardischewsky for preparation of the figures. I am especially indebted to C. Wodara and A. Quentmeier from whose unpublished results I have quoted essential parts, and I thank B. Friedrich for critically reading the manuscript.

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