We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D 37 values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

Natural resistance-associated macrophage protein (Nramp) family transporters catalyze uptake of essential divalent transition metals like iron and manganese. To discriminate against abundant competitors, the Nramp metal-binding site should favor softer transition metals, which interact either covalently or ionically with coordinating molecules, over hard calcium and magnesium, which interact mainly ionically. The metal-binding site contains an unusual, but conserved, methionine, and its sulfur coordinates transition metal substrates, suggesting a vital role in their transport. Using a bacterial Nramp model system, we show that, surprisingly, this conserved methionine is dispensable for transport of the physiological manganese substrate and similar divalents iron and cobalt, with several small amino acid replacements still enabling robust uptake. Moreover, the methionine sulfur's presence makes the toxic metal cadmium a preferred substrate. However, a methionine-to-alanine substitution enables transport of calcium and magnesium. Thus, the putative evolutionary pressure to maintain the Nramp metal-binding methionine likely exists because it-more effectively than any other amino acid-increases selectivity for low-abundance transition metal transport in the presence of high-abundance divalents like calcium and magnesium.transition metals | MntH | divalent metal transporter DMT1 | hard-soft acid-base theory | ion selectivity filters A ll organisms require transition metal ions as cofactors in proteins that perform a variety of essential cellular tasks. Through evolution, organisms have developed mechanisms to acquire, transport, and safely store essential metals such as manganese, iron, cobalt, and zinc. The natural resistance-associated macrophage protein (Nramp) family of metal transporters represents a common transition metal acquisition strategy conserved across all kingdoms of life (1). The first discovered mammalian Nramp (Nramp1) is expressed in phagosomal membranes and likely extracts essential metals to help kill engulfed pathogens (2, 3). Mammals use Nramp2, an essential gene also called DMT1, to absorb dietary iron into the enterocytes that line the small intestine (4) and to extract iron from transferrin-containing endosomes in all tissues. Bacteria express their own Nramp homologs, which they typically use to scavenge manganese and other first row divalent transition metals (5, 6). Last, most plants have several Nramp homologs that take up iron and manganese, the essential cofactor in photosystem II, from the soil or vacuolar stores (7,8).Nramps are generally thought to function as metal-proton symporters (1) and are able to bind and/or transport a wide range of divalent transition metal substrates, including the biologically useful metals Mn 2+ , Fe 2+ , Co 2+ , Ni 2+ , Cu 2+ , and Zn 2+ , as well as the toxic heavy metals Cd 2+ , Pb 2+ , and Hg 2+ (4, 9-13). Nramps do discriminate against the divalent alkaline earth metal ions Mg 2+ and Ca 2+ (9, 14), which are typically several orders of magnitude mor...

Summary The widely-conserved natural resistance associated macrophage protein (Nramp) family of divalent metal transporters enables manganese import in bacteria and dietary iron uptake in mammals. We determined the crystal structure of the Deinococcus radiodurans Nramp homolog (DraNramp) in an inward-facing apo state, including the complete transmembrane (TM) segment 1a—absent from a previous Nramp structure. Mapping our cysteine accessibility scanning results onto this structure, we identified the metal permeation pathway in the alternate outward-open conformation. We investigated the functional impact of two natural anemia-causing glycine-to-arginine mutations, which impaired transition metal transport in both human Nramp2 and DraNramp. The TM4 G153R mutation perturbs the closing of the outward metal permeation pathway and alters the selectivity of the conserved metal-binding site. In contrast, the TM1a G45R mutation prevents conformational change by sterically blocking the essential movement of that helix, thus locking the transporter in an inward-facing state.

Natural resistance-associated macrophage protein (Nramp) transporters enable uptake of essential transition metal micronutrients in numerous biological contexts. These proteins are believed to function as secondary transporters that harness the electrochemical energy of proton gradients by “coupling” proton and metal transport. Here we use the Deinococcus radiodurans (Dra) Nramp homologue, for which we have determined crystal structures in multiple conformations, to investigate mechanistic details of metal and proton transport. We untangle the proton-metal coupling behavior of DraNramp into two distinct phenomena: ΔpH stimulation of metal transport rates and metal stimulation of proton transport. Surprisingly, metal type influences substrate stoichiometry, leading to manganese-proton cotransport but cadmium uniport, while proton uniport also occurs. Additionally, a physiological negative membrane potential is required for high-affinity metal uptake. To begin to understand how Nramp’s structure imparts these properties, we target a conserved salt-bridge network that forms a proton-transport pathway from the metal-binding site to the cytosol. Mutations to this network diminish voltage and ΔpH dependence of metal transport rates, alter substrate selectivity, perturb or eliminate metal-stimulated proton transport, and erode the directional bias favoring outward-to-inward metal transport under physiological-like conditions. Thus, this unique salt-bridge network may help Nramp-family transporters maximize metal uptake and reduce deleterious back-transport of acquired metals. We provide a new mechanistic model for Nramp proton-metal cotransport and propose that functional advantages may arise from deviations from the traditional model of symport.

Many bacteria take up DNA from their environment as part of the process of natural transformation. DNA uptake allows microorganisms to gain genetic diversity and can lead to the spread of antibiotic resistance or virulence genes within a microbial population. Development of genetic competence (Com) in Bacillus subtilis is a highly regulated process that culminates in expression of several late competence genes and formation of the DNA uptake apparatus. The late competence operon comF encodes a small protein of unknown function, ComFB. To gain insight into the function of ComFB, we determined its 3D structure via X-ray crystallography. ComFB is a dimer and each subunit consists of four α-helices connected by short loops and one extended β-strand-like stretch. Each subunit contains one zinc-binding site formed by four cysteines, which are unusually spaced in the primary sequence. Using structure- and bioinformatics-guided substitutions we analyzed the inter-subunit interface of the ComFB dimer. Based on these analyses, we conclude that ComFB is an obligate dimer. We also characterized ComFB in vivo and found that this protein is produced in competent cells and is localized to the cytosol. Consistent with previous reports, we showed that deletion of ComFB does not affect DNA uptake function. Combining our results, we conclude that ComFB is unlikely to be a part of the DNA uptake machinery under tested conditions and instead may have a regulatory function.