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Rockett, K.A.1
Awburn, M.M.2
Cowden, W.B.3
Clarke, I.A.4
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9
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0024006934
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A S. typhimurium 14028s Mud J transposon library containing ∼50,000 independent insertions was constructed by use of S. typhimurium TT10388, as reported [K. Hughes and J. M. Roth, Genetics 119, 9 (1988)]. The library was subcultured in M9 medium. GSNO was added to logarithmically dividing cells at a final concentration of 2 mM (which is not inhibitory for rapidly dividing cells) One hour later, cycloserine was added to a final concentration of 100 mg/liter. Cyclosenne is specifically bactericidal for dividing cells, as described [F. Bolivar, R. L. Rodriguez, M. C. Betlach, H. W. Boyer, Gene 2, 75 (1977)] Therefore, this protocol provides a positive selection for mutants with enhanced GSNO susceptibility. After 1 hour of exposure to cycloserine, the cells were washed, allowed to enter stationary phase, diluted into fresh medium, and enriched for a second time
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Genetics
, vol.119
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Hughes, K.1
Roth, J.M.2
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10
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0017584021
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A S. typhimurium 14028s Mud J transposon library containing ∼50,000 independent insertions was constructed by use of S. typhimurium TT10388, as reported [K. Hughes and J. M. Roth, Genetics 119, 9 (1988)]. The library was subcultured in M9 medium. GSNO was added to logarithmically dividing cells at a final concentration of 2 mM (which is not inhibitory for rapidly dividing cells) One hour later, cycloserine was added to a final concentration of 100 mg/liter. Cyclosenne is specifically bactericidal for dividing cells, as described [F. Bolivar, R. L. Rodriguez, M. C. Betlach, H. W. Boyer, Gene 2, 75 (1977)] Therefore, this protocol provides a positive selection for mutants with enhanced GSNO susceptibility. After 1 hour of exposure to cycloserine, the cells were washed, allowed to enter stationary phase, diluted into fresh medium, and enriched for a second time
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(1977)
Gene
, vol.2
, pp. 75
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Bolivar, F.1
Rodriguez, R.L.2
Betlach, M.C.3
Boyer, H.W.4
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11
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4243128306
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note
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After enrichment, the library was plated as individual colonies that were screened for GSNO susceptibility. Seven clones with highly increased susceptibility to GSNO were identified. The attL-junction fragments were cloned and sequenced as described (2) All seven clones were shown to have identical insertions between nucleotides 325 and 326 of the S. typhimurium metL gene. The sequenced region was shown to be 80% identical at the nucleotide level and >90% identical at the amino acid level to the corresponding region of E coli metL (22). We used bacteriophage P22 to transduce the metL.:Mud J insertion into a new wild-type background, 100% cotransduction of GSNO hypersusceptibility was confirmed.
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12
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4243105186
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note
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Oligonucleotide primers 5′-AAGCTTGGAGGGTA-AAAATGAGTGTGAGTGTGATTGCGCAGG-3′ and 5′-GGATCCCCGGGCATTATTAAATTTCTGAAATT-ACAA-3′ were used to amplity the E. coli metL gene (22) from DH5α genomic DNA in a polymerase chain reaction (PCR). The first primer incorporates a ribosomal binding sequence to allow metL expression, the native motL gene is ordinarily expressed as part of the metBL operon. The 2 4-kb PCR product was cloned into pBLUESCRIPT (Stratagene) and introduced into metL mutant S. typhimurium.
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15
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0021718304
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I. Saint-Girons, N Duchange, G N. Cohen, M M Zakin, J. Biol Chem. 259, 14282 (1984).
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Saint-Girons, I.1
Duchange, N.2
Cohen, G.N.3
Zakin, M.M.4
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0020374667
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Mutant S typhimurium strains SA2824 (LT2 thrA13), DS163 (LT2 metJ713), and DS1373 (LT2 metK36) were obtained from K. Sanderson at the Salmonella Genetic Stock Centre (Calgary, Alberta, Canada). Salmonella typhimurium LT2 was used as the wild-type control for comparative GSNO susceptibility studies with these strains. M9 medium was supplemented with 500 μM threonine for studies with S typhimurium SA2824; threonine supplementation has no effect on the GSNO inhibitory zone diameter obtained for wild-type S. typhimurium. All GSNO susceptibility studies were done a minimum of three times. Mutations in met J and metK both derepress methionine biosynthetic genes [J. T. Mulligan, W. Margolin, J. H. Krueger, G. C Walker, J. Bacteriol. 151, 609 (1982)].
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Mulligan, J.T.1
Margolin, W.2
Krueger, J.H.3
Walker, G.C.4
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18
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0025809651
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12-dependent transmethylase. The metH..lacZ plasmid pGS201 carries a metH transcriptional fusion with lacZYA, as described [K. A. Byerly, M L Urbanowski, G V. Stauffer, ibid. 173, 3547 (1991)]
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J. Bacteriol.
, vol.173
, pp. 3547
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Byerly, K.A.1
Urbanowski, M.L.2
Stauffer, G.V.3
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T G. Evans, L. Thai, D. L. Granger, J B. Hibbs Jr , J. Immunol. 151, 907 (1993); S Formica, T. I A. Roach, J M Blackwell, Immunology 82, 42 (1994), C. Nathan, Cell 82, 873 (1995)
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, vol.151
, pp. 907
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Evans, T.G.1
Thai, L.2
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Hibbs Jr., J.B.4
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20
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T G. Evans, L. Thai, D. L. Granger, J B. Hibbs Jr , J. Immunol. 151, 907 (1993); S Formica, T. I A. Roach, J M Blackwell, Immunology 82, 42 (1994), C. Nathan, Cell 82, 873 (1995)
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, vol.82
, pp. 42
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Formica, S.1
Roach, T.I.A.2
Blackwell, J.M.3
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21
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T G. Evans, L. Thai, D. L. Granger, J B. Hibbs Jr , J. Immunol. 151, 907 (1993); S Formica, T. I A. Roach, J M Blackwell, Immunology 82, 42 (1994), C. Nathan, Cell 82, 873 (1995)
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, pp. 873
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4243116501
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note
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3 metE mutant S. typhimurium killed two of four mice and made all of the infected mice ill.
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23
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K. S McCully, Am. J. Pathol. 56, 111 (1969); G. H J. Boers, A. G. H. Smals, F. J M. Tnjberis, N. Engl. J. Med 313, 709 (1985), R. Clarke et al., ibid. 324, 1149 (1991); J. Selhub et al , ibid. 332, 286 (1995).
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K. S McCully, Am. J. Pathol. 56, 111 (1969); G. H J. Boers, A. G. H. Smals, F. J M. Tnjberis, N. Engl. J. Med 313, 709 (1985), R. Clarke et al., ibid. 324, 1149 (1991); J. Selhub et al , ibid. 332, 286 (1995).
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Boers, G.H.J.1
Smals, A.G.H.2
Tnjberis, F.J.M.3
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K. S McCully, Am. J. Pathol. 56, 111 (1969); G. H J. Boers, A. G. H. Smals, F. J M. Tnjberis, N. Engl. J. Med 313, 709 (1985), R. Clarke et al., ibid. 324, 1149 (1991); J. Selhub et al , ibid. 332, 286 (1995).
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K. S McCully, Am. J. Pathol. 56, 111 (1969); G. H J. Boers, A. G. H. Smals, F. J M. Tnjberis, N. Engl. J. Med 313, 709 (1985), R. Clarke et al., ibid. 324, 1149 (1991); J. Selhub et al , ibid. 332, 286 (1995).
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0003785155
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Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Each β-galactosidase assay was performed three times
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J. H. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1972) Each β-galactosidase assay was performed three times
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Miller, J.H.1
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33
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0017807890
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pFF200 contains the neomycin gene promoter of transposon Tn5 transcriptionally fused to the E. coli lacZ gene on a pACYC 184 replicon [A. C. Chang and S N. Cohen, J. Bacteriol. 134, 1141 (1978)].
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Aminoguanidine is a moderately selective inhibitor of the cytokine-inducible form of NO synthase (NOS II) that can be orally administered [J. Chan, K Tanaka, D. Carroll, J. Rynn, B. R. Bloom, Infect Immun. 63, 736 (1995)]
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Chan, J.1
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Bloom, B.R.5
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35
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note
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We are grateful to D Granger, E. Groisman, and J. Stamler for helpful suggestions and to K. Sanderson and A. Hessel for gifts of bactenal strains. Supported in part by grants from the U.S. Department of Agriculture (9401954), the National Institutes of Health (A132463), and the Thorkildsen Research Fellowship Program.
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