In previous work (D. R. Harris et al., J Bacteriol 191:5240–5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans. After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans. Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident. IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000 Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus. Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.

Manganese is an essential trace nutrient for organisms, because of its role in cofactoring enzymes and providing protection against reactive oxygen species (ROS). Many bacteria require manganese to form pathogenic or symbiotic interactions with eukaryotic host cells. However, excess manganese is toxic, requiring cells to have manganese export mechanisms. Bacteria are currently known to possess two widely-distributed classes of manganese export proteins, MntP and MntE, but other types of transporters likely exist. Moreover, the structure and function of MntP is not well understood. Here, we characterized the role of three structurally related proteins known or predicted to be involved in manganese transport in bacteria from the MntP, UPF0016 and TerC families. These studies used computational analysis to analyze phylogeny and structure, physiological assays to test sensitivity to high levels of manganese and ROS, and ICP-MS to measure metal levels. We found that MntP alters cellular resistance to ROS. Moreover, we used extensive computational analyses and phenotypic assays to identify amino acids required for MntP activity. These negativelycharged residues likely serve to directly bind manganese and transport it from the cytoplasm through the membrane. We further characterized two other potential manganese transporters associated with a Mn-sensing riboswitch, and found that the UPF0016 family of proteins has manganese export activity. We provide the first phenotypic and biochemical evidence for the role of Alx, a member of the TerC family, in manganese homeostasis. It does not appear to export manganese, rather it intriguingly facilitates an increase in intracellular manganese concentration. These findings expand knowledge about the identity and mechanisms of manganese homeostasis proteins across bacteria and show that proximity to a Mnresponsive riboswitch can be used to identify new components of the manganese homeostasis machinery. INTRODUCTIONTransition metals are essential for life as they play important roles as enzyme cofactors and structural components of proteins and RNAs. Reflecting this, one third of the proteomes of organisms from bacteria to humans consist of metalloproteins (1,2). In bacteria, metal availability is intimately involved in pathogenesis. Bacteria unable to maintain proper metal homeostasis are less virulent, and mammalian hosts actively seek to withhold essential metals from invading bacteria (3,4). Yet in excess, metals are toxic to cells. This toxicity typically results from metaldependent oxidative damage (e.g., the Fenton reaction) and/or the displacement of cognate metals from their binding sites by the metal that is in excess (3,(5)(6)(7)(8). Thus, cells have a battery of metal importers, exporters, sequestration factors, and regulators to carefully control the intracellular level of each metal (1,2,9,10).

The GLUT (SLC2) family of membrane-associated transporters are described as glucose transporters. However, this family is divided into three classes, and although the regulated transporter activity of Class I proteins is becoming better understood, Class III protein functions continue to be obscure. We have cataloged the relative expression and splicing of SLC2 mRNA isomers in tumors and normal tissues, with a focus on breast tumors and cell lines. mRNA for the Class III protein GLUT8 is the predominant SLC2 species expressed alongside GLUT1 in many tissues but GLUT8 exists mostly as an untranslated splice form in tumors. We confirm that GLUT8 is not presented at the cell surface and does not transport glucose directly. However, we reveal a lysosome-dependent reaction that cleaves the GLUT8 protein and releases the carboxy-terminal peptide to a separate vesicle population. Given the localization of GLUT8 at a major metabolic hub (the late endosomal/lysosomal interface), and its regulated cleavage reaction, we evaluated TXNIP-mediated hexosamine homeostasis, and speculate that GLUT8 may function as a sensory component of this reaction.

Precursor mRNA (pre-mRNA) splicing is an essential process for gene expression in eukaryotes catalyzed by the spliceosome in two transesterification steps. The spliceosome is a large, highly dynamic complex composed of 5 small nuclear RNAs and dozens of proteins, some of which are needed throughout the splicing reaction while others only act during specific stages. The human protein FAM192A was recently proposed to be a splicing factor that functions during the second transesterification step, exon ligation, based on analysis of cryo-electron microscopy (cryo-EM) density. It was also proposed that Fyv6 might be the functional S. cerevisiae homolog of FAM192A; however, no biochemical or genetic data has been reported to support this hypothesis. Herein, we show that Fyv6 is a splicing factor and acts during exon ligation. Deletion of FYV6 results in genetic interactions with the essential splicing factors Prp8, Prp16, and Prp22; decreases splicing in vivo of reporter gene harboring intron substitutions that limit the rate of exon ligation; and changes 3' splice site (SS) selection. Together, these data suggest that Fyv6 is a component of the spliceosome and the potential functional and structural homolog of human FAM192A.