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Regulation of internal promoters in a zinc-responsive operon is influenced by transcription from upstream promoters.

ABSTRACT: In the cyanobacterium Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120), a zinc-responsive operon (all4725-all4721) has been described, which contains 4 distinct promoters. The two most upstream ones bind Zur with high affinity, whereas the other two do not or do so with a very low affinity. In this paper, a detailed characterization of the four promoters is presented, showing that all four were induced by metal depletion, and they were constitutively derepressed in a zur mutant, despite the two downstream promoters not being direct targets for this regulator. Crucially, induction by metal depletion of the two downstream promoters was abrogated when transcription initiated at the upstream promoters was interrupted by a polar insertion midway in the operon. In contrast, insertion of a nitrogen-responsive promoter at a roughly similar position provoked the two downstream promoters to adopt a regulatory pattern mimicking that of the inserted promoter. Thus, regulation of the two downstream promoters is apparently influenced by transcription from promoters upstream. Evidence is presented indicating that the activity of the two downstream promoters is kept basal in Anabaena by repression. A regulatory model compatible with these results is proposed, where promoters controlled by repression in bacterial operons may be subjected to a hierarchical regulation depending on their position in the operon. According to this model, internal promoters may respond to stimuli governing the activity of promoters upstream by an indirect regulation and to specific stimuli by a direct regulation.

SUBMITTER: Napolitano M

PROVIDER: S-EPMC3591996 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.

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Regulation of internal promoters in a zinc-responsive operon is influenced by transcription from upstream promoters.

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