A P-type ATPase importer that discriminates between essential and toxic transition metals

Transition metals, although being essential cofactors in many physiological processes, are toxic at elevated concentrations. Among the membrane-embedded transport proteins that maintain appropriate intracellular levels of transition metals are ATP-driven pumps belonging to the P-type ATPase superfamily. These metal transporters may be differentiated according to their substrate specificities, where the majority of pumps can extrude either silver and copper or zinc, cadmium, and lead. In the present report, we have established the substrate specificities of nine previously uncharacterized prokaryotic transition-metal P-type ATPases. We find that all of the newly identified exporters indeed fall into one of the two above-mentioned categories. In addition to these exporters, one importer, Pseudomonas aeruginosa Q9I147, was also identified. This protein, designated HmtA (heavy metal transporter A), exhibited a different substrate recognition profile from the exporters. In vivo metal susceptibility assays, intracellular metal measurements, and transport experiments all suggest that HmtA mediates the uptake of copper and zinc but not of silver, mercury, or cadmium. The substrate selectivity of this importer ensures the high-affinity uptake of essential metals, while avoiding intracellular contamination by their toxic counterparts

Genomic Analyses of Transport Proteins in Ralstonia metallidurans

Ralstonia (Wautersia, Cupriavidus) metallidurans (Rme) is better able to withstand high concentrations of heavy metals than any other well-studied organism. This fact renders it a potential agent of bioremediation as well as an ideal model organism for understanding metal resistance phenotypes. We have analysed the genome of Rme for genes encoding homologues of established and putative transport proteins; 13% of all genes in Rme encode such homologues. Nearly one-third of the transporters identified (32%) appear to function in inorganic ion transport with three-quarters of these acting on cations. Transporters specific for amino acids outnumber sugar transporters nearly 3 : 1, and this fact plus the large number of uptake systems for organic acids indicates the heterotrophic preferences of these bacteria. Putative drug efflux pumps comprise 10% of the encoded transporters, but numerous efflux pumps for heavy metals, metabolites and macromolecules were also identified. The results presented should facilitate genetic manipulation and mechanistic studies of transport in this remarkable bacterium

Comparison of Oropharyngeal Microbiota from Children with Asthma and Cystic Fibrosis

A genuine microbiota resides in the lungs which emanates from the colonization by the oropharyngeal microbiota. Changes in the oropharyngeal microbiota might be the source of dysbiosis observed in the lower airways in patients suffering from asthma or cystic fibrosis (CF). To examine this hypothesis, we compared the throat microbiota from healthy children (n=62) and that from children with asthma (n=27) and CF (n=57) aged 6 to 12 years using 16S rRNA amplicon sequencing. Our results show high levels of similarities between healthy controls and children with asthma and CF revealing the existence of a core microbiome represented by Prevotella, Streptococcus, Neisseria, Veillonella, and Haemophilus. However, in CF, the global diversity, the bacterial load, and abundances of 53 OTUs were significantly reduced, whereas abundances of 6 OTUs representing opportunistic pathogens such as Pseudomonas, Staphylococcus, and Streptococcus were increased compared to those in healthy controls controls and asthmatics. Our data reveal a core microbiome in the throat of healthy children that persists in asthma and CF indicating shared host regulation favoring growth of commensals. Furthermore, we provide evidence for dysbiosis with a decrease in diversity and biomass associated with the presence of known pathogens consistent with impaired host defense in children with CF

ZnuA and zinc homeostasis in pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous environmental bacterium and a clinically significant opportunistic human pathogen. Central to the ability of P. aeruginosa to colonise both environmental and host niches is the acquisition of zinc. Here we show that P. aeruginosa PAO1 acquires zinc via an ATP-binding cassette (ABC) permease in which ZnuA is the high affinity, zinc-specific binding protein. Zinc uptake in Gram-negative organisms predominantly occurs via an ABC permease, and consistent with this expectation a P. aeruginosa ΔznuA mutant strain showed an ~60% reduction in cellular zinc accumulation, while other metal ions were essentially unaffected. Despite the major reduction in zinc accumulation, minimal phenotypic differences were observed between the wild-type and ΔznuA mutant strains. However, the effect of zinc limitation on the transcriptome of P. aeruginosa PAO1 revealed significant changes in gene expression that enable adaptation to low-zinc conditions. Genes significantly up-regulated included non-zinc-requiring paralogs of zinc-dependent proteins and a number of novel import pathways associated with zinc acquisition. Collectively, this study provides new insight into the acquisition of zinc by P. aeruginosa PAO1, revealing a hitherto unrecognized complexity in zinc homeostasis that enables the bacterium to survive under zinc limitation.Victoria G. Pederick, Bart A. Eijkelkamp, Stephanie L. Begg, Miranda P. Ween, Lauren J. McAllister, James C. Paton, Christopher A. McDevit

Interplay of the Czc System and Two P-Type ATPases in Conferring Metal Resistance to Ralstonia metallidurans

Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA

Characterization of the 101-Kilobase-Pair Megaplasmid pKB1, Isolated from the Rubber-Degrading Bacterium Gordonia westfalica Kb1

The complete sequence of the circular 101,016-bp megaplasmid pKB1 from the cis-1,4-polyisoprene-degrading bacterium Gordonia westfalica Kb1, which represents the first described extrachromosomal DNA of a member of this genus, was determined. Plasmid pKB1 harbors 105 open reading frames. The predicted products of 46 of these are significantly related to proteins of known function. Plasmid pKB1 is organized into three functional regions that are flanked by insertion sequence (IS) elements: (i) a replication and putative partitioning region, (ii) a putative metabolic region, and (iii) a large putative conjugative transfer region, which is interrupted by an additional IS element. Southern hybridization experiments revealed the presence of another copy of this conjugational transfer region on the bacterial chromosome. The origin of replication (oriV) of pKB1 was identified and used for construction of Escherichia coli-Gordonia shuttle vectors, which was also suitable for several other Gordonia species and related genera. The metabolic region included the heavy-metal resistance gene cadA, encoding a P-type ATPase. Expression of cadA in E. coli mediated resistance to cadmium, but not to zinc, and decreased the cellular content of cadmium in this host. When G. westfalica strain Kb1 was cured of plasmid pKB1, the resulting derivative strains exhibited slightly decreased cadmium resistance. Furthermore, they had lost the ability to use isoprene rubber as a sole source of carbon and energy, suggesting that genes essential for rubber degradation are encoded by pKB1