Helge K. Abicht

Response of Gram-positive bacteria to copper stress

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.

Killing of bacteria by copper surfaces involves dissolved copper

ABSTRACT 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.

Genome Sequence of Desulfosporosinus sp. OT, an Acidophilic Sulfate-Reducing Bacterium from Copper Mining Waste in Norilsk, Northern Siberia

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.

Non-enzymic copper reduction by menaquinone enhances copper toxicity in Lactococcus lactis IL1403.

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-reductase-deficient 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 ΔmenE 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 ΔmenE 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 ΔmenE 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.

Responses of Lactic Acid Bacteria to Heavy Metal Stress

Lactic acid bacteria (LAB) are of great importance in the manufacture of food and dairy products, but also for an increasing number of biotechnological applications. When applied to industrial processes, these bacteria face various stress conditions, such as low pH, high temperature, osmotic shock, and metal stress. Of the last category, exposure to copper has received wide attention and detailed mechanistic insight is available. We thus have a comprehensive understanding of copper extrusion by adenosine triphosphatases, gene regulation by copper, and intracellular copper chaperoning. 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 in general. Also, recent biochemical work has shed new light on the mechanism of copper toxicity. The response of LAB to metals other than copper has received only little attention and will be discussed for other organisms to the extent that it could be relevant for LAB.

Genome Sequence of Desulfovibrio sp. A2, a Highly Copper Resistant, Sulfate-Reducing Bacterium Isolated from Effluents of a Zinc Smelter at the Urals

Desulfovibrio sp. A2 is an anaerobic gram-negative sulfate-reducing bacterium with remarkable tolerance to copper. It was isolated from wastewater effluents of a zinc smelter at the Urals. Here, we report the 4.2-Mb draft genome sequence of Desulfovibrio sp. A2 and identify potential copper resistance mechanisms.

A copper-induced quinone degradation pathway provides protection against combined copper/quinone stress in Lactococcus lactis IL1403

Quinones are ubiquitous in the environment. They occur naturally but are also in widespread use in human and industrial activities. Quinones alone are relatively benign to bacteria, but in combination with copper, they become toxic by a mechanism that leads to intracellular thiol depletion. Here, it was shown that the yahCD‐yaiAB operon of Lactococcus lactis IL1403 provides resistance to combined copper/quinone stress. The operon is under the control of CopR, which also regulates expression of the copRZA copper resistance operon as well as other L. lactis genes. Expression of the yahCD‐yaiAB operon is induced by copper but not by quinones. Two of the proteins encoded by the operon appear to play key roles in alleviating quinone/copper stress: YaiB is a flavoprotein that converts p‐benzoquinones to less toxic hydroquinones, using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as reductant; YaiA is a hydroquinone dioxygenase that converts hydroquinone putatively to 4‐hydroxymuconic semialdehyde in an oxygen‐consuming reaction. Hydroquinone and methylhydroquinone are both substrates of YaiA. Deletion of yaiB causes increased sensitivity of L. lactis to quinones and complete growth arrest under combined quinone and copper stress. Copper induction of the yahCD‐yaiAB operon offers protection to copper/quinone toxicity and could provide a growth advantage to L. lactis in some environments.

Copper resistance and its regulation in the sulfate-reducing bacterium Desulfosporosinus sp. OT.

Desulfosporosinus sp. OT is a Gram-positive, acidophilic sulfate-reducing firmicute isolated from copper tailings sediment in the Norilsk mining-smelting area in Siberia and represents the first Desulfosporosinus species whose genome has been sequenced. Desulfosporosinus sp. OT is exceptionally copper resistant, which made it of interest to study the resistance mechanism. It possesses a copUAZ operon which is shown here to be involved in copper resistance. The copU gene encodes a CsoR-type homotetrameric repressor. By electrophoretic mobility shift assay, it was shown that CopU binds to the operator/promoter region of the copUAZ operon in the absence of copper and is released from the DNA by Cu+ or Ag+, implying that CopU regulates the operon in a copper/silver-dependent manner. DOT_CopA is a P1B-type ATPase related to other characterized, bacterial copper ATPases. When expressed in a copper-sensitive Escherichia coli ΔcopA mutant, it restores copper resistance to WT levels. His-tagged DOT_CopA was expressed from a plasmid in E. coli and purified by Ni-NTA affinity chromatography. The purified enzyme was most active in the presence of Cu(I) and bacterial phospholipids. These findings indicate that the copUAZ operon confers copper resistance to Desulfosporosinus sp. OT, but do not per se explain the basis of the high copper resistance of this strain.