The Gram-positive bacteria Enterococcus hirae, Lactococcus lactis, and Bacillus subtilis have received wide attention in the study of copper homeostasis. Consequently, copper extrusion by ATPases, gene regulation by copper, and intracellular copper chaperoning are understood in some detail. This has provided profound insight into basic principles of how organisms handle copper. It also emerged that many bacterial species may not require copper for life, making copper homeostatic systems pure defense mechanisms. Structural work on copper homeostatic proteins has given insight into copper coordination and bonding and has started to give molecular insight into copper handling in biological systems. Finally, recent biochemical work has shed new light on the mechanism of copper toxicity, which may not primarily be mediated by reactive oxygen radicals.

Bacteria are rapidly killed on copper surfaces. However, the mechanism of this process remains unclear. Using Enterococcus hirae, the effect of inactivation of copper homeostatic genes and of medium compositions on survival and copper dissolution was tested. The results support a role for dissolved copper ions in killing.

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Background: ␣-Proteobacteria, extant relatives of mitochondria, are model organisms for studying assembly of bacterial and mitochondrial metalloenzymes. Results: Periplasmic thioredoxin TlpA is a specific reductant for copper chaperone ScoI and cytochrome oxidase subunit II (CoxB). Conclusion: Cysteines in the copper-binding sites of ScoI and CoxB must be reduced prior to metallation. Significance: Structures of TlpA-ScoI and TlpA-CoxB intermediates reveal mechanistic details of the reduction process.

Lactococcus lactis possesses a pronounced extracellular Cu 2+ -reduction activity which leads to the accumulation of Cu + in the medium. The kinetics of this reaction were not saturable by increasing copper concentrations, suggesting a non-enzymic reaction. A copper-reductasedeficient mutant, isolated by random transposon mutagenesis, had an insertion in the menE gene, which encodes O-succinylbenzoic acid CoA ligase. This is a key enzyme in menaquinone biosynthesis. The DmenE mutant was deficient in short-chain menaquinones, and exogenously added menaquinone complemented the copper-reductase-deficient phenotype. Haem-induced respiration of wild-type L. lactis efficiently suppressed copper reduction, presumably by competition by the bd-type quinol oxidase for menaquinone. As expected, the DmenE mutant was respiration-deficient, but could be made respiration-proficient by supplementation with menaquinone. Growth of wild-type cells was more copper-sensitive than that of the DmenE mutant, due to the production of Cu + ions by the wild-type. This growth inhibition of the wild-type was strongly attenuated if Cu + was scavenged with the Cu(I) chelator bicinchoninic acid. These findings support a model whereby copper is non-enzymically reduced at the membrane by menaquinones. Respiration effectively competes for reduced quinones, which suppresses copper reduction. These findings highlight novel links between copper reduction, respiration and Cu + toxicity in L. lactis.

The mechanisms underlying the biogenesis of the structurally unique, binuclear Cu1.5+•Cu1.5+ redox center (CuA) on subunit II (CoxB) of cytochrome oxidases have been a long-standing mystery. Here, we reconstituted the CoxB•CuA center in vitro from apo-CoxB and the holo-forms of the copper transfer chaperones ScoI and PcuC. A previously unknown, highly stable ScoI•Cu2+•CoxB complex was shown to be rapidly formed as the first intermediate in the pathway. Moreover, our structural data revealed that PcuC has two copper-binding sites, one each for Cu1+ and Cu2+, and that only PcuC•Cu1+•Cu2+ can release CoxB•Cu2+ from the ScoI•Cu2+•CoxB complex. The CoxB•CuA center was then formed quantitatively by transfer of Cu1+ from a second equivalent of PcuC•Cu1+•Cu2+ to CoxB•Cu2+. This metalation pathway is consistent with all available in vivo data and identifies the sources of the Cu ions required for CuA center formation and the order of their delivery to CoxB.

Lactococcus lactis cannot synthesize haem, but when supplied with haem, expresses a cytochrome bd oxidase. Apart from the cydAB structural genes for this oxidase, L. lactis features two additional genes, hemH and hemW (hemN), with conjectured functions in haem metabolism. While it appears clear that hemH encodes a ferrochelatase, no function is known for hemW. HemW-like proteins occur in bacteria, plants and animals, and are usually annotated as CPDHs (coproporphyrinogen III dehydrogenases). However, such a function has never been demonstrated for a HemW-like protein. We here studied HemW of L. lactis and showed that it is devoid of CPDH activity in vivo and in vitro. Recombinantly produced, purified HemW contained an Fe-S (iron-sulfur) cluster and was dimeric; upon loss of the iron, the protein became monomeric. Both forms of the protein covalently bound haem b in vitro, with a stoichiometry of one haem per monomer and a KD of 8 μM. In vivo, HemW occurred as a haem-free cytosolic form, as well as a haem-containing membrane-associated form. Addition of L. lactis membranes to haem-containing HemW triggered the release of haem from HemW in vitro. On the basis of these findings, we propose a role of HemW in haem trafficking. HemW-like proteins form a distinct phylogenetic clade that has not previously been recognized.

We have sequenced the genome of Desulfosporosinus sp. OT, a Gram-positive, acidophilic sulfate-reducing Firmicute isolated from copper tailing sediment in the Norilsk mining-smelting area in Northern Siberia, Russia. This represents the first sequenced genome of a Desulfosporosinus species. The genome has a size of 5.7 Mb and encodes 6,222 putative proteins.Members of the genus Desulfosporosinus are sulfate-reducing bacteria, often found in microbial communities associated with mining environments and involved in the bioremediation of metal-contaminated water and sediments (1,(5)(6)(7)(8)12). In these environments, enriched with SO 4 2Ϫ , sulfate reduction contributes to precipitation of metal sulfides and thereby the immobilization of toxic metals. Recently, Desulfosporosinus bacteria were identified as key players in microbial sulfate reduction in a low-sulfate peatland (13). Here we announce the first draft genome of a member of the genus Desulfosporosinus. Desulfosporosinus sp. OT was isolated from acidic sediment (sample T9) of a copper-mining waste site in Norilsk, Russia (10).Genomic DNA was isolated by alkaline lysis (4). Sequencing was carried out by pyrosequencing, using an FLX genome sequencer (Roche). A total of 357,734 reads with an average read length of 400 bp resulted in 142,885,740 sequenced bases. Sequence assembly was performed with GS De Novo Assembler version 2.3 software with default settings, yielding 304 contigs with an average size of 22,674 nucleotides and an average of 42% GC content. The number of the bases in all contigs totaled 5,705,508, corresponding to 25-fold sequencing coverage. Identification of open reading frames and annotation were performed by the annotation service for microbial genomes of the Institute for Genome Sciences (IGS), School of Medicine, University of Maryland. The annotation yielded 6,222 open reading frames for proteins, 74 tRNA genes, and 3 rRNA genes. The 16S RNA sequence of Desulfosporosinus sp. OT is 99.4% and 98.1% identical with those of Desulfosporosinus sp. 5apy (GenBank accession no. AF159120) and Desulfosporosinus lacus (GenBank accession no. AJ582757) (14), respectively, which are the most closely related microbial isolates.Desulfosporosinus sp. OT withstands copper concentrations of up to 236 mM, which is severalfold higher than the concentrations so far reported for other sulfate-reducing bacteria such as Desulfovibrio sp. A2 (genome announcement submitted for publication) or Desulfovibrio sp. R2, also isolated from metal-contaminated habitats (9, 11). Desulfosporosinus sp. OT harbors two CopA-like CPx-type ATPases (see reference 16 for a review), DOT_2451 and DOT_2536, and a polyphosphate kinase-phosphatase couple, DOT_3559 and DOT_4690, as present in Acidithiobacillus ferrooxidans. These systems appear to play a key role in copper tolerance (2). As would be expected for a Gram-positive organism, no CueO-like multicopper oxidase system or cus-like copper efflux system, both of which serve in the control of periplasmic copper in Escherichia coli...