THE SULFUR-OXIDIZING PROKARYOTESBiological oxidation of hydrogen sulfide to sulfate is one of the major reactions of the global sulfur cycle. Reduced inorganic sulfur compounds (referred to below as sulfur) are exclusively oxidized by prokaryotes, and sulfate is the major oxidation product. Sulfur oxidation in members of the Eukarya is mediated by lithoautotrophic bacterial endosymbionts (44).The sulfur-oxidizing prokaryotes are phylogenetically diverse ( Fig. 1). In the domain Archaea aerobic sulfur oxidation is restricted to members of the order Sulfolobales (21, 58), and in the domain Bacteria sulfur is oxidized by aerobic lithotrophs or by anaerobic phototrophs. The nonphototrophic obligate anaerobe Wolinella succinogenes oxidizes hydrogen sulfide to polysulfide during fumarate respiration (41). The ecology, physiology, and biochemistry of sulfur-oxidizing bacteria have been reviewed previously. The neutrophilic chemolithotrophic bacteria have been reviewed by Kelly et al. (31,32,35) and Takakuwa (63), the acidophilic sulfur-oxidizing bacteria have been reviewed by Harrison (23) and Pronk et al. (46), and the molecular genetics of Acidithiobacillus ferrooxidans has been reviewed by Rawlings and Kusano (49). The sulfur metabolism of phototrophic bacteria has been reviewed by Brune (9,10) and Trüper and Fischer (65). The physiology and genetics of both phototrophic and lithotrophic sulfur-oxidizing prokaryotes have been discussed recently (18).Prokaryotes oxidize hydrogen sulfide, sulfur, sulfite, thiosulfate, and various polythionates under alkaline (57), neutral, or acidic conditions (23). Aerobic sulfur-oxidizing prokaryotes belong to genera like

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APO-1 is a 48-kDa cell-membrane protein identical to the Fas antigen now designated CD95. It is a member of the NGF/TNF receptor superfamily. Anti-APO-1 monoclonal antibody induces apoptosis in a variety of cell types expressing this antigen. We immunohistochemically investigated APO-1 expression in normal colon mucosa, 20 adenomas, 258 colon carcinomas and 10 liver metastases and carried out in vitro studies using a panel of colon-carcinoma cell lines. Immunohistochemically, APO-1 was regularly expressed at the basolateral membrane of normal colon epithelia. In a minor fraction of colon adenomas and in 39.1% of colon carcinomas APO-1 expression was diminished and in 48.1% of carcinomas, predominantly of the non-mucinous type, APO-1 expression was completely abrogated. The normal level of APO-1 in carcinomas was correlated with the mucinous type. Reduced/lost APO-1 expression was more frequent in rectal carcinomas. Complete loss of APO-1 was more frequent in tumors that had already metastasized. APO-1 expression in liver metastases essentially corresponded to that of the primary tumors. Comparative analysis with data from previous studies revealed that the mode of APO-1 expression is correlated with that of HLA-A,B,C./beta 2m, HLA-DR, HLA-D-associated invariant chain and of the secretory component. Surface expression of APO-1 was heterogeneous in colon-carcinoma cell lines; SW480 expressed considerable amounts of APO-1 on all cells, while HT-29 constitutively did less so and only in a minority of cells. Surface density of APO-1 and the fraction of positive cells in HT-29 was enhanced by interferon-gamma (IFN-gamma) and, additively, by tumor necrosis factor-alpha (TNF-alpha), whereas in SW480 APO-1 expression was not modulated by these cytokines. We conclude that neoplastic transformation of colon epithelium often leads to a loss of the physiologic, high level of surface APO-1 by giving rise either to a stable lack of APO-1 or to an IFN-gamma/TNF-alpha-sensitive phenotype of inducible APO-1 expression.

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The novel genes soxFGH were identified, completing the sox gene cluster of Paracoccus pantotrophus coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction. soxF coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome c reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in soxF and complementation analysis, it was evident that the soxFGH gene products were not required for lithotrophic growth with thiosulfate.

The periplasmic sulfite dehydrogenase of Paracoccus pantotrophus GB17 was purified to homogeneity by a four-step procedure from cells grown lithoautotrophically with thiosulfate. The molecular mass of native sulfite dehydrogenase was 190 kDa as determined by native gradient PAGE. SDS-PAGE showed sulfite dehydrogenase to comprise two subunits with molecular masses of 47 kDa and 50 kDa, suggesting an alpha2beta2 structure. The N-terminal amino acid sequence and immunochemical analysis using SoxC-specific antibodies identified the 47-kDa protein as the soxC gene product. SoxD-specific antibodies identified the 50-kDa protein as SoxD. Based on the molecular masses deduced from the nucleotide sequence for mature SoxC (43,442 Da) and SoxD (37,637 Da) sulfite dehydrogenase contained 1.30 mol molybdenum/mol alpha2beta2 sulfite dehydrogenase. The iron content was 3.17 mol/mol alpha2beta2 sulfite dehydrogenase, and 3.53 mol heme/mol alpha2beta2 sulfite dehydrogenase was determined by pyridine hemochrome analysis. These data are consistent with the two heme-binding domains (CxxCH), characteristic for c-type cytochromes, deduced from the soxD nucleotide sequence. Electrospray ionization revealed two masses for SoxC of 43,503 and 43,897 Da. The difference in molecular mass was attributed to the molybdenum cofactor of SoxC. For SoxD a mass of 38,815 Da was determined; this accounted for the polypeptide and two covalently bound hemes. Reconstitution of the catalytic activity of sulfite dehydrogenase required additional fractions; these eluted from Q Sepharose at 0.05, 0.25, and 0.30 M NaCl. The K(m) of sulfite dehydrogenase for sulfite was 7.0 microM and for cytochrome c 19 microM. Sulfite dehydrogenase activity was inhibited by sulfate and phosphate. The structural and catalytic properties make sulfite dehydrogenase from P. denitrificans GB17 distinct from sulfite oxidases of other prokaryotic or eukaryotic sources.

Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide-, sulfur-, thiosulfate-and sulfitedependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein-bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10 977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N-Ethylmaleimide inhibited thiosulfate-oxidation and the kinetics suggested a turn-over-dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation. ß

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