Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Streptococcus pneumoniae requires manganese for colonization of the human host, but the underlying molecular basis for this requirement has not been elucidated. Recently, it was shown that zinc could compromise manganese uptake and that zinc levels increased during infection by S. pneumoniae in all the niches that it colonized. Here we show, by quantitative means, that extracellular zinc acts in a dose dependent manner to competitively inhibit manganese uptake by S. pneumoniae, with an EC50 of 30.2 µM for zinc in cation-defined media. By exploiting the ability to directly manipulate S. pneumoniae accumulation of manganese, we analyzed the connection between manganese and superoxide dismutase (SodA), a primary source of protection for S. pneumoniae against oxidative stress. We show that manganese starvation led to a decrease in sodA transcription indicating that expression of sodA was regulated through an unknown manganese responsive pathway. Intriguingly, examination of recombinant SodA revealed that the enzyme was potentially a cambialistic superoxide dismutase with an iron/manganese cofactor. SodA was also shown to provide the majority of protection against oxidative stress as a S. pneumoniae ΔsodA mutant strain was found to be hypersensitive to oxidative stress, despite having wild-type manganese levels, indicating that the metal ion alone was not sufficiently protective. Collectively, these results provide a quantitative assessment of the competitive effect of zinc upon manganese uptake and provide a molecular basis for how extracellular zinc exerts a ‘toxic’ effect on bacterial pathogens, such as S. pneumoniae.

Cadmium is a transition metal ion that is highly toxic in biological systems. Although relatively rare in the Earth's crust, anthropogenic release of cadmium since industrialization has increased biogeochemical cycling and the abundance of the ion in the biosphere. Despite this, the molecular basis of its toxicity remains unclear. Here we combine metal-accumulation assays, high-resolution structural data and biochemical analyses to show that cadmium toxicity, in Streptococcus pneumoniae, occurs via perturbation of first row transition metal ion homeostasis. We show that cadmium uptake reduces the millimolar cellular accumulation of manganese and zinc, and thereby increases sensitivity to oxidative stress. Despite this, high cellular concentrations of cadmium (B17 mM) are tolerated, with negligible impact on growth or sensitivity to oxidative stress, when manganese and glutathione are abundant. Collectively, this work provides insight into the molecular basis of cadmium toxicity in prokaryotes, and the connection between cadmium accumulation and oxidative stress.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S . pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S . pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S . pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S . pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

WalKR (YycFG) is the only essential two-component regulator in the human pathogen Staphylococcus aureus . WalKR regulates peptidoglycan synthesis, but this function alone does not explain its essentiality. Here, to further understand WalKR function, we investigate a suppressor mutant that arose when WalKR activity was impaired; a histidine to tyrosine substitution (H271Y) in the cytoplasmic Per-Arnt-Sim (PAS CYT ) domain of the histidine kinase WalK. Introducing the WalK H271Y mutation into wild-type S. aureus activates the WalKR regulon. Structural analyses of the WalK PAS CYT domain reveal a metal-binding site, in which a zinc ion (Zn 2+ ) is tetrahedrally-coordinated by four amino acids including H271. The WalK H271Y mutation abrogates metal binding, increasing WalK kinase activity and WalR phosphorylation. Thus, Zn 2+ -binding negatively regulates WalKR. Promoter-reporter experiments using S. aureus confirm Zn 2+ sensing by this system. Identification of a metal ligand recognized by the WalKR system broadens our understanding of this critical S. aureus regulon.

The new agreement specifically addresses what authors can do with different versions of their manuscript -e.g. use in theses and collections, teaching and training, conference presentations, sharing with colleagues, and posting on websites and repositories. The terms under which these uses can occur are clearly identified to prevent misunderstandings that could jeopardize final publication of a manuscript (Section II, Permitted Uses by Authors). Easy Reference User Guide Posting Accepted and Published Works on Websites and Repositories:A digital file of the Accepted Work and/or the Published Work may be made publicly available on websites or repositories (e.g. the Author's personal website, preprint servers, university networks or primary employer's institutional websites, third party institutional or subject-based repositories, and conference websites that feature presentations by the Author(s) based on the Accepted and/or the Published Work) under the following conditions:• It is mandated by the Author(s)' funding agency, primary employer, or, in the case of Author(s) employed in academia, university administration.• If the mandated public availability of the Accepted Manuscript is sooner than 12 months after online publication of the Published Work, a waiver from the relevant institutional policy should be sought. If a waiver cannot be obtained, the Author(s) may sponsor the immediate availability of the final Published Work through participation in the ACS AuthorChoice program-for information about this program see http://pubs.acs.org/page/policy/authorchoice/index.html.• If the mandated public availability of the Accepted Manuscript is not sooner than 12 months after online publication of the Published Work, the Accepted Manuscript may be posted to the mandated website or repository. The following notice should be included at the time of posting, or the posting amended as appropriate: "This document is the Accepted Manuscript version of a Published Work that appeared in final form in [JournalTitle], copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see [insert ACS Articles on Request authordirected link to Published Work, see http://pubs.acs.org/page/policy/articlesonrequest/index.html]." • The posting must be for non-commercial purposes and not violate the ACS' "Ethical Guidelines to Publication of Chemical Research" (see http://pubs.acs.org/ethics).• Regardless of any mandated public availability date of a digital file of the final Published Work, Author(s) may make this file available only via the ACS AuthorChoice Program. For more information, see http://pubs.acs.org/page/policy/authorchoice/index.html. sensor provides a significant advantage in that it allows the use of nL sampling when compared to ICP-MS (mL) and . This work paves the way to a generic approach for developing surface-based ion sensors using a range of sensor molecules, which can be attached to a surface without the need for its chemical modifica...

Zinc is an essential nutrient in biological systems due to its structural or catalytic requirement in proteins involved in diverse cellular processes. To meet this cellular demand, microbes must acquire sufficient zinc from their environment. However, many environments have low zinc availability. One of the mechanisms used by bacteria to acquire zinc is through the production of small molecules known as zincophores. Similar to bacterial siderophores used for iron uptake, zincophores are synthesized by the bacterium and exported and then reimported as zincophore-zinc complexes. Thus far, only four zincophores have been described, including two from the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa, in which they play a critical role in zinc acquisition during infection, and one in a soil bacterium. To determine what other microbes may produce zincophores, we used bioinformatic analyses to identify new zincophore biosynthetic gene clusters (BGCs) and predict the diversity of molecules synthesized. Genome neighborhood network analysis identified approximately 250 unique zincophore-producing species from actinobacteria, firmicutes, proteobacteria, and fusobacteria. This indicates that zincophores are produced by diverse bacteria that inhabit a broad range of ecological niches. Many of the BGCs likely produce characterized zincophores, based on similarity to the characterized systems. However, this analysis also identified numerous BGCs that, based on the colocalization of additional modifying enzymes and sequence divergence of the biosynthetic enzymes, are likely to produce unique zincophores. Collectively, these findings provide a comprehensive understanding of the zincophore biosynthetic landscape that will be invaluable for future research on these important small molecules. IMPORTANCE Bacteria must acquire essential nutrients, including zinc, from their environment. For bacterial pathogens, this necessitates overcoming the host metal-withholding response known as nutritional immunity. A novel type of zinc uptake mechanism that involves the bacterial production of a small zinc-scavenging molecule was recently described in the human pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis, as well as the soil-associated bacterium Paenibacillus mucilaginosus. This suggests that zincophores may be important for zinc acquisition in diverse environments. In this study, we sought to identify other zincophore-producing bacteria using bioinformatics. We identified almost 250 unique zincophore-producing species, including human and animal pathogens, as well as isolates from soil, rhizosphere, plant, and marine habitats. Crucially, we observed diversity at the amino acid and gene organization levels, suggesting that many of these species are producing unique zincophores. Together, our findings highlight the importance of zincophores for a broad array of bacteria living in diverse environments.

The host restricts the availability of zinc to prevent infection. To overcome this defense, Staphylococcus aureus and Pseudomonas aeruginosa rely on zincophore-dependent zinc importers. Synthesis of the zincophore staphylopine by S. aureus and its import are both necessary for the bacterium to cause infection. In this study, we sought to elucidate how loss of zincophore efflux impacts bacterial resistance to host-imposed zinc starvation. In culture and during infection, mutants lacking CntE, the staphylopine efflux pump, were more sensitive to zinc starvation imposed by the metal-binding immune effector calprotectin than those lacking the ability to import staphylopine. However, disruption of staphylopine synthesis reversed the enhanced sensitivity phenotype of the ΔcntE mutant to calprotectin, indicating that intracellular toxicity of staphylopine is more detrimental than the impaired ability to acquire zinc. Unexpectedly, intracellular accumulation of staphylopine does not increase the expression of metal importers or alter cellular metal concentrations, suggesting that, contrary to prevailing models, the toxicity associated with staphylopine is not strictly due to intracellular chelation of metals. As P. aeruginosa and other pathogens produce zincophores with similar chemistry, our observations on the crucial importance of zincophore efflux are likely to be broadly relevant. IMPORTANCE Staphylococcus aureus and many other bacterial pathogens rely on metal-binding small molecules to obtain the essential metal zinc during infection. In this study, we reveal that export of these small molecules is critical for overcoming host-imposed metal starvation during infection and prevents toxicity due to accumulation of the metal-binding molecule within the cell. Surprisingly, we found that intracellular toxicity of the molecule is not due to chelation of cellular metals.