Engineering mammalian cells for solid-state sensor applications.

A fundamental advance in the development and application of cell- and tissue-based biosensors would be the ability to achieve air-dry stabilization of mammalian (especially human) cells with subsequent recovery following rehydration. The would allow for the preparation of sensors with extended shelf lives, only requiring the addition of water for activation. By understanding and subsequently employing the tactics used by desiccation-tolerant extremophiles, it may be possible to design stabilized mammalian cell-based biosensors. The approaches required to realize this goal are discussed and illustrated with several examples.

Inactivation of two homologues of proteins presumed to be involved in the desiccation tolerance of plants sensitizes Deinococcus radiodurans R1 to desiccation.

Mutational inactivation of the genes designated DR1172 and DRB0118 in Deinococcus radiodurans R1 greatly sensitizes this species to desiccation, but not to ionizing radiation. These genes encode proteins that share features with the desiccation-induced LEA76 proteins of many plants and the PCC13-62 protein of Craterostigma plantagineum, suggesting that D. radiodurans may serve as a useful model for the study of desiccation tolerance in higher organisms.

Inhibition of metabolism and growth of Mycobacterium leprae by gamma irradiation.

Mycobacterium leprae is uncultivable on artificial medium, but viability can be maintained without multiplication for a limited time in vitro. In this study, we evaluated gamma-irradiation (gamma-irr) as a means to kill this slowly growing organism. Freshly harvested, viable, athymic, nu/nu mouse-derived M. leprae were exposed to varying doses of gamma-irr from a 60Co source. Two indicators of bacterial viability were determined: metabolism, measured by oxidation of 14C-palmitic acid to 14CO2 in the BACTEC 460 system, and multiplication, measured by titration in the mouse foot pad. gamma-Irr of both M. leprae and M. lufu, a cultivable control mycobacterium, resulted in a dose-dependent inhibition of viability. gamma-Irr of up to 10(3) rad had little effect on the metabolic activity of either organism. For M. leprae, 10(4)-10(5) rad caused an intermediate inhibitory effect; whereas 10(6) rad yielded almost total inhibition. In the mouse foot pad assay, up to 10(4) rad had little effect on M. leprae growth; however, 10(5) rad resulted in at least a 2-log reduction in the number of bacilli recovered and no M. leprae growth was measurable after exposure to 10(6) rad. With M. lufu, 10(5) rad inhibited metabolic activity by 99% and caused > or = 2-log reduction in the number of colony forming units (CFU). No CFU of M. lufu were recovered after exposure to 10(6) rad. Scanning electron microscopy revealed the presence of some aberrant protrusions on the cell surface of lethally irradiated M. leprae; whereas boiling and autoclaving caused obvious morphological denaturation. These data suggest that gamma-irr is an effective way to kill M. leprae without causing extensive damage to the cell architecture. Killing M. leprae by gamma-irr may be preferable when comparing cellular responses to live versus dead bacilli in vitro and in vivo.

Radiation resistance: the fragments that remain.

The complete genome sequence of the bacterium, Deinococcus radiodurans R1 has been released. This achievement will greatly aid efforts to study this organism, but analysis of the sequence reveals little that helps explain the extreme ionizing radiation resistance of this species.

Protection of the DNA during the exposure of Escherichia coli cells to a toxic metabolite: the role of the KefB and KefC potassium channels.

The effect of the toxic metabolite methylglyoxal on the DNA of Escherichia coli cells has been investigated. Exposure of E. coli cells to methylglyoxal reduces the transformability of plasmid DNA and results in the degradation of genomic DNA. The activity of the KefB and KefC potassium channels protects E. coli cells against methylglyoxal and limits the amount of DNA damage. In mutants lacking KefB and KefC, methylglyoxal-induced DNA damage was reduced by incubation with a weak acid that lowers the pHi to the same extent as through KefB and KefC activation. This provides evidence that acidification of the cytoplasm protects E. coli DNA against methylglyoxal. By the analysis of cells lacking UvrA, we demonstrate that this repair protein is required for the degradation of the DNA upon methylglyoxal exposure. However, protection by KefB and KefC occurred independently of UvrA. Although we present evidence that exposure of E. coli cells to methylglyoxal results in DNA degradation, our results suggest this event is not essential for methylglyoxal-induced death. The implications of these findings will be discussed.

Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus.

In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA-DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant.

Why is Deinococcus radiodurans so resistant to ionizing radiation?

When exponential-phase cultures of Deinococcus radiodurans are exposed to a 5000-Gray dose of gamma radiation, individual cells suffer massive DNA damage. Despite this insult to their genetic integrity, these cells survive without loss of viability or evidence of mutation, repairing the damage by as-yet-poorly-understood mechanisms.

Against all odds: the survival strategies of Deinococcus radiodurans.

Bacteria of the genus Deinococcus exhibit an extraordinary ability to withstand the lethal and mutagenic effects of DNA damaging agents-particularly the effects of ionizing radiation. These bacteria are the most DNA damage-tolerant organisms ever identified. Relatively little is known about the biochemical basis for this phenomenon; however, available evidence indicates that efficient repair of DNA damage is, in large part, responsible for the deinococci's radioresistance. Obviously, an explanation of the deinococci's DNA damage tolerance cannot be developed solely on the basis of the DNA repair strategies of more radiosensitive organisms. The deinococci's capacity to survive DNA damage suggests that (a) they employ repair mechanisms that are fundamentally different from other prokaryotes, or that (b) they have the ability to potentiate the effectiveness of the conventional complement of DNA repair proteins. An argument is made for the latter alternative.

Radioresistance of Deinococcus radiodurans: functions necessary to survive ionizing radiation are also necessary to survive prolonged desiccation.

Forty-one ionizing radiation-sensitive strains of Deinococcus radiodurans were evaluated for their ability to survive 6 weeks of desiccation. All exhibited a substantial loss of viability upon rehydration compared with wild-type D. radiodurans. Examination of chromosomal DNA from desiccated cultures revealed a time-dependent increase in DNA damage, as measured by an increase in DNA double-strand breaks. The evidence presented suggests that D. radiodurans' ionizing radiation resistance is incidental, a consequence of this organism's adaptation to a common physiological stress, dehydration.

Genetic characterization of forty ionizing radiation-sensitive strains of Deinococcus radiodurans: linkage information from transformation.

Natural transformation was used to help define a collection of ionizing radiation-sensitive strains of Deinococcus radiodurans. Three putative rec mutations were identified, as were three pol alleles. Forty of the ionizing radiation-sensitive strains were placed into 16 linkage groups, and evidence obtained indicates that each linkage group consists of a cluster of mutations not more than 1,000 bp apart. In addition, a new class of D. radiodurans mutant was described that, although radioresistant, appears to recover from ionizing radiation-induced DNA damage slowly relative to other strains of D. radiodurans.

Peroxynitrite causes DNA nicks in plasmid pBR322.

Peroxynitrite causes single-strand breaks in pBR322 supercoiled DNA as evidenced by agarose gel electrophoresis analysis. The effect of three free radical scavengers, namely mannitol, benzoate and dimethylsulfoxide, were studied. Mannitol failed to protect DNA from damage by peroxynitrite while benzoate and dimethylsulfoxide amplified the damage. These results suggest the damage caused by peroxynitrite alone is not mediated by free radicals since typical free radical scavengers fail to prevent the damage.

Novel ionizing radiation-sensitive mutants of Deinococcus radiodurans.

Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of either locus results in a partial loss of resistance to ionizing radiation. The magnitude of this loss is locus specific and differentially affected by inactivation of the uvrA gene product. An irrB uvrA double mutant is more sensitive to ionizing radiation than is an irrB mutant. In contrast, the irrI uvrA double mutant and the irrI mutant are equally sensitive to ionizing radiation. The irrB and irrI mutations also reduce D. radiodurans resistance to UV radiation, this effect being most pronounced in uvrA+ backgrounds. Subclones of each gene have been isolated, and the loci have been mapped relative to each other. The irrB and irrI genes are separated by approximately 20 kb of intervening sequence that encodes the uvrA and pol genes.

Dominant negative umuD mutations decreasing RecA-mediated cleavage suggest roles for intact UmuD in modulation of SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. The UmuD protein shares homology with a family of proteins that includes LexA and several bacteriophage repressors. UmuD is posttranslationally activated for its role in mutagenesis by a RecA-mediated proteolytic cleavage that yields UmuD'. A set of missense mutants of umuD was isolated and shown to encode mutant UmuD proteins that are deficient in RecA-mediated cleavage in vivo. Most of these mutations are dominant to umuD+ with respect to UV mutagenesis yet do not interfere with SOS induction. Although both UmuD and UmuD' form homodimers, we provide evidence that they preferentially form heterodimers. The relationship of UmuD to LexA, lambda repressor, and other members of the family of proteins is discussed and possible roles of intact UmuD in modulating SOS mutagenesis are discussed.

Genetic analyses of cellular functions required for UV mutagenesis in Escherichia coli.

In Escherichia coli, most UV and chemical mutagenesis is not a passive process and requires the participation of the umuD and umuC gene products. However, the molecular mechanism of UV mutagenesis is not yet understood and the roles of the UmuD and UmuC proteins have not been elucidated. The umuDC operon is induced by UV irradiation and regulated as part of the SOS response. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. The RecA protein appears to have a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD at the time of SOS induction. In addition, we have obtained evidence which indicates that the GroEL and GroES proteins also play a role in UV mutagenesis. Similarities of the amino acid sequence of UmuD to the sequence of gene 45 protein of bacteriophage T4 and of the sequence of UmuC to those of the gene 44 and gene 62 proteins suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.

Amino acid similarities to other proteins offer insights into roles of UmuD and UmuC in mutagenesis.

The products of the umuD and umuC genes are required for most uv and chemical mutagenesis in Escherichia coli. The genes are organized in an operon that is repressed by LexA and regulated as part of the SOS response. The umuD protein shares homology with the carboxyl-terminal domain of LexA. Genetic evidence now indicates that RecA-mediated cleavage activates UmuD for its role in mutagenesis. The COOH-terminal fragment of UmuD is both necessary and sufficient for this role. Similarities of UmuD to gene 45 protein of bacteriophage T4 and of UmuC to gene 44 protein and gene 62 protein suggest possible roles for UmuD and UmuC in mutagenesis that are supported by preliminary evidence.

RecA-mediated cleavage activates UmuD for mutagenesis: mechanistic relationship between transcriptional derepression and posttranslational activation.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli. It has been shown that the UmuD protein shares homology with LexA, the repressor of the SOS genes. In this paper we describe a series of genetic experiments that indicate that the purpose of RecA-mediated cleavage of UmuD at its bond between Cys-24 and Gly-25 is to activate UmuD for its role in mutagenesis and that the COOH-terminal fragment of UmuD is necessary and sufficient for the role of UmuD in UV mutagenesis. Other genetic experiments are presented that (i) support the hypothesis that the primary role of Ser-60 in UmuD function is to act as a nucleophile in the RecA-mediated cleavage reaction and (ii) raise the possibility that RecA has a third role in UV mutagenesis besides mediating the cleavage of LexA and UmuD.

Prostaglandin H synthase-dependent epoxidation of aflatoxin B1.

This report demonstrates that aflatoxin B1 (AFB1) is cooxidized by prostaglandin H (PGH) synthase to form 2,3-dihydro-2,3-epoxy-aflatoxin B1 oxide. Using ram seminal vesicle microsomes as a source of PGH synthase, our results demonstrate that AFB1 is converted to a mutagen during arachidonate turnover that we identify as the epoxide by isolating adducts formed to DNA. The efficiency of AFB1 epoxidation by PGH synthase is assessed and compared with mixed-function oxidase-dependent metabolic activation of this compound.