Microbial reduction and precipitation of vanadium by Shewanella oneidensis.

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V(V)). The bacterium reduces V(V) (vanadate ion) to V(IV) (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V(IV)-containing solid.

Overproduction, purification and novel redox properties of the dihaem cytochrome c, NapB, from Haemophilus influenzae.

The napB gene of the pathogenic bacterium Haemophilus influenzae encodes a dihaem cytochrome c, the small subunit of a heterodimeric periplasmic nitrate reductase similar to those found in other bacteria. In order to obtain sufficient protein for biophysical studies, we aimed to overproduce the recombinant dihaem protein in Escherichia coli. Initial expression experiments indicated that the NapB signal peptide was not cleaved by the leader peptidase of the host organism. Apocytochrome was formed under aerobic, semi-aerobic and anaerobic growth conditions in either Luria--Bertani or minimal salts medium. The highest amounts of apo-NapB were produced in the latter medium, and the bulk was inserted into the cytoplasmic membrane. The two haem groups were covalently attached to the pre-apocytochrome only under anaerobic growth conditions, and with 2.5 mM nitrite or at least 10 mM nitrate supplemented to the minimal salts growth medium. In order to obtain holocytochrome, the gene sequence encoding mature NapB was cloned in-frame with the E. coli ompA (outer membrane protein A) signal sequence. Under anaerobic conditions, NapB was secreted into the periplasmic space, with the OmpA signal peptide being correctly processed and with both haem c groups attached covalently. Unless expressed in the DegP-protease-deficient strain HM125, some of the recombinant NapB polypeptides were N-terminally truncated as a result of proteolytic activity. Under aerobic growth conditions, co-expression with the E. coli ccm (cytochrome c maturation) genes resulted in a higher yield of holocytochrome c. The pure recombinant NapB protein showed absorption maxima at 419, 522 and 550 nm in the reduced form. The midpoint reduction potentials of the two haem groups were determined to be -25 mV and -175 mV. These results support our hypothesis that the Nap system fulfils a nitrate-scavenging role in H. influenzae.

Primary structure characterization of a Rhodocyclus tenuis diheme cytochrome c reveals the existence of two different classes of low-potential diheme cytochromes c in purple phototropic bacteria.

The complete amino acid sequence of a 26-kDa low redox potential cytochrome c-551 from Rhodocyclus tenuis was determined by a combination of Edman degradation and mass spectrometry. There are 240 residues including two heme binding sites at positions 41, 44, 128, and 132. There is no evidence for gene doubling. The only known homolog of Rc. tenuis cytochrome c-551 is the diheme cytochrome c-552 from Pseudomonas stutzeri which contains 268 residues and heme binding sites at nearly identical positions. There is 44% overall identity between the Rc. tenuis and Ps. stutzeri cytochromes with 10 internal insertions and deletions. The Ps. stutzeri cytochrome is part of a denitrification gene cluster, whereas Rc. tenuis is incapable of denitrification, suggesting different functional roles for the cytochromes. Histidines at positions 45 and 133 are the fifth heme ligands and conserved histidines at positions 29, 209, and 218 and conserved methionines at positions 114 and 139 are potential sixth heme ligands. There is no obvious homology to the low-potential diheme cytochromes characterized from other purple bacterial species such as Rhodobacter sphaeroides. There are therefore at least two classes of low-potential diheme cytochromes c found in phototrophic bacteria. There is no more than 11% helical secondary structure in Rc. tenuis cytochrome c-551 suggesting that there is no relationship to class I or class II c-type cytochromes.

A membrane-bound flavocytochrome c-sulfide dehydrogenase from the purple phototrophic sulfur bacterium Ectothiorhodospira vacuolata.

The amino acid sequence of Ectothiorhodospira vacuolata cytochrome c-552, isolated from membranes with n-butanol, shows that it is a protein of 77 amino acid residues with a molecular mass of 9,041 Da. It is closely related to the cytochrome subunit of Chlorobium limicola f. sp. thiosulfatophilum flavocytochrome c-sulfide dehydrogenase (FCSD), having 49% identity. These data allowed isolation of a 5.5-kb subgenomic clone which contains the cytochrome gene and an adjacent flavoprotein gene as in other species which have an FCSD. The cytochrome subunit has a signal peptide with a normal cleavage site, but the flavoprotein subunit has a signal sequence which suggests that the mature protein has an N-terminal cysteine, characteristic of a diacyl glycerol-modified lipoprotein. The membrane localization of FCSD was confirmed by Western blotting with antibodies raised against Chromatium vinosum FCSD. When aligned according to the three-dimensional structure of Chromatium FCSD, all but one of the side chains near the flavin are conserved. These include the Cys 42 flavin adenine dinucleotide binding site; the Cys 161-Cys 337 disulfide; Glu 167, which modulates the reactivity with sulfite; and aromatic residues which may function as charge transfer acceptors from the flavin-sulfite adduct (C. vinosum numbering). The genetic context of FCSD is different from that in other species in that flanking genes are not conserved. The transcript is only large enough to encode the two FCSD subunits. Furthermore, Northern hybridization showed that the production of E. vacuolata FCSD mRNA is regulated by sulfide. All cultures that contained sulfide in the medium had elevated levels of FCSD RNA compared with cells grown on organics (acetate, malate, or succinate) or thiosulfate alone, consistent with the role of FCSD in sulfide oxidation.