John Battista - Academia.edu (original) (raw)
Papers by John Battista
Humana Press eBooks, Nov 14, 2003
Genetics, Feb 1, 2004
We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radio... more We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV-and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the -subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced Ͼ250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N Ј-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.
Proceedings of the National Academy of Sciences of the United States of America, Aug 12, 2002
Understanding biological systems and the roles of their constituents is facilitated by the abilit... more Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.
The FASEB Journal, Dec 18, 1997
IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation ... more IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation in an uncharacterized locus designated irrE. Five overlapping cosmids capable of restoring ionizing radiation resistance to IRS24 were isolated from a genomic library. The ends of each cloned insert were sequenced, and these sequences were used to localize irrE to a 970-bp region on chromosome I of D. radiodurans R1. The irrE gene corresponds to coding sequence DR0167 in the R1 genome. The irrE gene encodes a 35,000-Da protein that has no similarity to any previously characterized peptide. The irrE locus of R1 was also inactivated by transposon mutagenesis, and this strain was sensitive to ionizing radiation, UV light, and mitomycin C. Preliminary findings indicate that IrrE is a novel regulatory protein that stimulates transcription of the recA gene following exposure to ionizing radiation.
We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumu... more We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.
<div><p>(A) This set of reactions uses the labeled duplex DNA illustrated. The oligos... more <div><p>(A) This set of reactions uses the labeled duplex DNA illustrated. The oligos annealed to form this DNA are 51 and 37 nt in length and pair so as to leave a 14-nt 3′ extension. The shorter DNA is 5′ end–labeled. The first lane contains unreacted DNA, showing both the annealed duplex and the unannealed single-stranded DNA. The second lane shows the DNA after treatment with 3 units of exonuclease I for 7 min in a 15-μl reaction mixture. Note that the duplex DNA in the upper band has been shortened by removal of the single-stranded extension. In lanes 3 and 4, the DdrA protein (4 μM) has been incubated with the DNA, without and with the 3 units of exonuclease I, respectively. The DNA is bound by DdrA and shifted to the top of the gel. The reactions shown in lanes 5 and 6 are identical to those in lanes 3 and 4, but with SDS and proteinase K added to disrupt the DdrA–DNA complexes and reveal that the DNA has been minimally affected by exonuclease I. The final lane shows another reaction of the DNA with 3 units of exonuclease I, in the presence of 4 μM bovine serum albumin. Exonuclease I degrades single-stranded DNA in the 3′ to 5′ direction.</p> <p>(B) The protein bound to the duplex DNA is DdrA. The reaction of lane 3 in (A) was scaled up and the protein–DNA complex excised from the gel as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020304#s4" target="_blank">Materials and Methods</a>. The protein in this complex was subjected to electrophoresis on an SDS-polyacrylamide gel, shown here (lane 3). The control lanes contained prestained protein standards (lane 1) and purified DdrA protein (lane 2). The gel-extracted protein comigrated with DdrA.</p></div
We have previously generated four replicate populations of ionizing radiation (IR)- resistantEsch... more We have previously generated four replicate populations of ionizing radiation (IR)- resistantEscherichia colithough directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR isolated from population IR-3-20 can play a role in IR resistance. An FNR variant is unique to IR-3-20 and suggests a role for altered global metabolism through the FNR regulon as a means for experimentally-evolved IR resistance.
PLOS ONE, 2019
We have previously generated four replicate populations of ionizing radiation (IR)-resistant Esch... more We have previously generated four replicate populations of ionizing radiation (IR)-resistant Escherichia coli though directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR, unique to and isolated from population IR-3-20, plays a role in IR resistance. The F186I allele of FNR exhibits a diminished ability to activate transcription from FNR-activatable promoters, and furthermore reduces levels of intracellular ROS. The FNR F186I variant is apparently capable of enhancing resistance to IR under chronic irradiation conditions, but does not increase cell survival when exposed to acute irradiation. Our results underline the importance of dose rate on cell survival of IR exposure.
Bergey's Manual of Systematics of Archaea and Bacteria, 2015
Journal of Bacteriology, 1994
Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of eit... more 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.
94598, 5 DNASTAR, Inc., Madison, WI 53705 USA 6Roche NimbleGen Inc, 500 S. 18 Rosa Rd., Madison, ... more 94598, 5 DNASTAR, Inc., Madison, WI 53705 USA 6Roche NimbleGen Inc, 500 S. 18 Rosa Rd., Madison, WI 53711, 7Laboratory of Genetics, University of Wisconsin, 425G 19 Henry Mall, Madison, WI 53706 20 21 22 Running Title: Directed evolution of ionizing radiation resistance 23 24 *Corresponding authors 25 26 For purposes of submission: 27 Michael M. Cox 28 Department of Biochemistry 29 University of Wisconsin-Madison 30 433 Babcock Drive 31 Madison, WI 53575 USA 32 phone:608-262-1181 33 fax:608-265-2603 34 email:cox@biochem.wisc.edu 35 36 Current addresses: 37 Reece J. Goiffon: Campus Box 8226, Bio & Biomed Science Grad Affairs, Washington 38 University School of Medicine, St. Louis, MO 63110. rjgoif@gmail.com 39 Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Bacteriol. doi:10.1128/JB.00502-09 JB Accepts, published online ahead of print on 5 June 2009
The primary objectives of this proposal was to define the subset of proteins required for the ion... more The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy. Summary of the Most Significant Results 1. Identifying the proteins required for ionizing radiation resistance in D. radiodurans R1: At the outset of these studies, we knew very little about why D. radiodurans' was so much more IR resistance than other species, but it was clear that proteins needed for cell survival were synthesized in cultures exposed to IR. Irradiated cultures cannot recover in the presence of chloramphenicol, this antibiotic preventing restitution of IR-induced single-strand and doublestrand DNA breaks. During the first three years of the funding period, we made identifying the proteins induced in response to ionizing radiation a priority in our efforts to better define D. radiodurans' mechanisms of IR resistance. We described the genomic expression profile of D. radiodurans R1 cultures as they recover from a sub-lethal dose of IR, comparing that profile with R1 cultures recovering from desiccation and with irradiated cultures of LSU2030, an IR sensitive strain of R1 that lacks the transcriptional activator IrrE. We compared unirradiated R1 cultures in exponential phase growth with age matched R1 cultures during the first hour after exposure to a non-lethal 3 kGy dose of γ radiation. This time course was deemed appropriate because it was determined that most DNA repairs had been completed. To determine how global transcription in D. radiodurans R1 changes in response to IR, competitive hybridizations were repeated for 6 independent experimental trials. We identified those genes whose mean Cy5/Cy3 ratios increased 3-fold or higher in the irradiated population relative to the unirradiated population. Seventy-two genes (2.2% of the genome) respond with increased expression within the first hour after exposure. A detectable protein product, an accurate mass tag, has been reported for 65% of these loci, verifying that these open reading frames encode a protein product. The genes induced in response to IR were grouped into nine categories based on their similarity to known proteins with 54 of the 72 loci falling into one of three categories. The largest group (44%) encodes proteins of unknown function. These loci are among the most highly induced; 20 being induced greater than 5-fold in the irradiated population. Six genes (recA,
Microbial Evolution under Extreme Conditions, 2015
Molecular Microbiology, 2005
Humana Press eBooks, Nov 14, 2003
Genetics, Feb 1, 2004
We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radio... more We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV-and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the -subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced Ͼ250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N Ј-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.
Proceedings of the National Academy of Sciences of the United States of America, Aug 12, 2002
Understanding biological systems and the roles of their constituents is facilitated by the abilit... more Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organism's dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.
The FASEB Journal, Dec 18, 1997
IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation ... more IRS24 is a DNA damage-sensitive strain of Deinococcus radiodurans strain 302 carrying a mutation in an uncharacterized locus designated irrE. Five overlapping cosmids capable of restoring ionizing radiation resistance to IRS24 were isolated from a genomic library. The ends of each cloned insert were sequenced, and these sequences were used to localize irrE to a 970-bp region on chromosome I of D. radiodurans R1. The irrE gene corresponds to coding sequence DR0167 in the R1 genome. The irrE gene encodes a 35,000-Da protein that has no similarity to any previously characterized peptide. The irrE locus of R1 was also inactivated by transposon mutagenesis, and this strain was sensitive to ionizing radiation, UV light, and mitomycin C. Preliminary findings indicate that IrrE is a novel regulatory protein that stimulates transcription of the recA gene following exposure to ionizing radiation.
We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumu... more We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.
<div><p>(A) This set of reactions uses the labeled duplex DNA illustrated. The oligos... more <div><p>(A) This set of reactions uses the labeled duplex DNA illustrated. The oligos annealed to form this DNA are 51 and 37 nt in length and pair so as to leave a 14-nt 3′ extension. The shorter DNA is 5′ end–labeled. The first lane contains unreacted DNA, showing both the annealed duplex and the unannealed single-stranded DNA. The second lane shows the DNA after treatment with 3 units of exonuclease I for 7 min in a 15-μl reaction mixture. Note that the duplex DNA in the upper band has been shortened by removal of the single-stranded extension. In lanes 3 and 4, the DdrA protein (4 μM) has been incubated with the DNA, without and with the 3 units of exonuclease I, respectively. The DNA is bound by DdrA and shifted to the top of the gel. The reactions shown in lanes 5 and 6 are identical to those in lanes 3 and 4, but with SDS and proteinase K added to disrupt the DdrA–DNA complexes and reveal that the DNA has been minimally affected by exonuclease I. The final lane shows another reaction of the DNA with 3 units of exonuclease I, in the presence of 4 μM bovine serum albumin. Exonuclease I degrades single-stranded DNA in the 3′ to 5′ direction.</p> <p>(B) The protein bound to the duplex DNA is DdrA. The reaction of lane 3 in (A) was scaled up and the protein–DNA complex excised from the gel as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020304#s4" target="_blank">Materials and Methods</a>. The protein in this complex was subjected to electrophoresis on an SDS-polyacrylamide gel, shown here (lane 3). The control lanes contained prestained protein standards (lane 1) and purified DdrA protein (lane 2). The gel-extracted protein comigrated with DdrA.</p></div
We have previously generated four replicate populations of ionizing radiation (IR)- resistantEsch... more We have previously generated four replicate populations of ionizing radiation (IR)- resistantEscherichia colithough directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR isolated from population IR-3-20 can play a role in IR resistance. An FNR variant is unique to IR-3-20 and suggests a role for altered global metabolism through the FNR regulon as a means for experimentally-evolved IR resistance.
PLOS ONE, 2019
We have previously generated four replicate populations of ionizing radiation (IR)-resistant Esch... more We have previously generated four replicate populations of ionizing radiation (IR)-resistant Escherichia coli though directed evolution. Sequencing of isolates from these populations revealed that mutations affecting DNA repair (through DNA double-strand break repair and replication restart), ROS amelioration, and cell wall metabolism were prominent. Three mutations involved in DNA repair explained the IR resistance phenotype in one population, and similar DNA repair mutations were prominent in two others. The remaining population, IR-3-20, had no mutations in the key DNA repair proteins, suggesting that it had taken a different evolutionary path to IR resistance. Here, we present evidence that a variant of the anaerobic metabolism transcription factor FNR, unique to and isolated from population IR-3-20, plays a role in IR resistance. The F186I allele of FNR exhibits a diminished ability to activate transcription from FNR-activatable promoters, and furthermore reduces levels of intracellular ROS. The FNR F186I variant is apparently capable of enhancing resistance to IR under chronic irradiation conditions, but does not increase cell survival when exposed to acute irradiation. Our results underline the importance of dose rate on cell survival of IR exposure.
Bergey's Manual of Systematics of Archaea and Bacteria, 2015
Journal of Bacteriology, 1994
Two new loci, irrB and irrI, have been identified in Deinococcus radiodurans. Inactivation of eit... more 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.
94598, 5 DNASTAR, Inc., Madison, WI 53705 USA 6Roche NimbleGen Inc, 500 S. 18 Rosa Rd., Madison, ... more 94598, 5 DNASTAR, Inc., Madison, WI 53705 USA 6Roche NimbleGen Inc, 500 S. 18 Rosa Rd., Madison, WI 53711, 7Laboratory of Genetics, University of Wisconsin, 425G 19 Henry Mall, Madison, WI 53706 20 21 22 Running Title: Directed evolution of ionizing radiation resistance 23 24 *Corresponding authors 25 26 For purposes of submission: 27 Michael M. Cox 28 Department of Biochemistry 29 University of Wisconsin-Madison 30 433 Babcock Drive 31 Madison, WI 53575 USA 32 phone:608-262-1181 33 fax:608-265-2603 34 email:cox@biochem.wisc.edu 35 36 Current addresses: 37 Reece J. Goiffon: Campus Box 8226, Bio & Biomed Science Grad Affairs, Washington 38 University School of Medicine, St. Louis, MO 63110. rjgoif@gmail.com 39 Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. J. Bacteriol. doi:10.1128/JB.00502-09 JB Accepts, published online ahead of print on 5 June 2009
The primary objectives of this proposal was to define the subset of proteins required for the ion... more The primary objectives of this proposal was to define the subset of proteins required for the ionizing radiation (IR) resistance of Deinococcus radiodurans R1, characterize the activities of those proteins, and apply what was learned to problems of interest to the Department of Energy. Summary of the Most Significant Results 1. Identifying the proteins required for ionizing radiation resistance in D. radiodurans R1: At the outset of these studies, we knew very little about why D. radiodurans' was so much more IR resistance than other species, but it was clear that proteins needed for cell survival were synthesized in cultures exposed to IR. Irradiated cultures cannot recover in the presence of chloramphenicol, this antibiotic preventing restitution of IR-induced single-strand and doublestrand DNA breaks. During the first three years of the funding period, we made identifying the proteins induced in response to ionizing radiation a priority in our efforts to better define D. radiodurans' mechanisms of IR resistance. We described the genomic expression profile of D. radiodurans R1 cultures as they recover from a sub-lethal dose of IR, comparing that profile with R1 cultures recovering from desiccation and with irradiated cultures of LSU2030, an IR sensitive strain of R1 that lacks the transcriptional activator IrrE. We compared unirradiated R1 cultures in exponential phase growth with age matched R1 cultures during the first hour after exposure to a non-lethal 3 kGy dose of γ radiation. This time course was deemed appropriate because it was determined that most DNA repairs had been completed. To determine how global transcription in D. radiodurans R1 changes in response to IR, competitive hybridizations were repeated for 6 independent experimental trials. We identified those genes whose mean Cy5/Cy3 ratios increased 3-fold or higher in the irradiated population relative to the unirradiated population. Seventy-two genes (2.2% of the genome) respond with increased expression within the first hour after exposure. A detectable protein product, an accurate mass tag, has been reported for 65% of these loci, verifying that these open reading frames encode a protein product. The genes induced in response to IR were grouped into nine categories based on their similarity to known proteins with 54 of the 72 loci falling into one of three categories. The largest group (44%) encodes proteins of unknown function. These loci are among the most highly induced; 20 being induced greater than 5-fold in the irradiated population. Six genes (recA,
Microbial Evolution under Extreme Conditions, 2015
Molecular Microbiology, 2005