Abstract
The activities of fumarase- and manganese-cofactored superoxide dismutase (SOD), encoded by the fumC and sodA genes in Pseudomonas aeruginosa, are elevated in mucoid, alginate-producing bacteria and in response to iron deprivation (D. J. Hassett, M. L. Howell, P. A. Sokol, M. L. Vasil, and G. E. Dean, J. Bacteriol. 179:1442-1451, 1997). In this study, a 393-bp open reading frame, fagA (Fur-associated gene), was identified immediately upstream of fumC, in an operon with orfX and sodA. Two iron boxes or Fur (ferric uptake regulatory protein) binding sites were discovered just upstream of fagA. Purified P. aeruginosa Fur caused a gel mobility shift of a PCR product containing these iron box regions. DNA footprinting analysis revealed a 37-bp region that included the Fur binding sites and was protected by Fur. Primer extension analysis and RNase protection assays revealed that the operon is composed of at least three major iron-regulated transcripts. Four mucoid fur mutants produced 1.7- to 2.6-fold-greater fumarase activity and 1.7- to 2.3-greater amounts of alginate than wild-type organisms. A strain devoid of the alternative sigma factor AlgT(U) produced elevated levels of one major transcript and fumarase C and manganase-cofactored SOD activity, suggesting that AlgT(U) may either play a role in regulating this transcript or function in some facet of iron metabolism. These data suggest that the P. aeruginosa fagA, fumC, orfX, and sodA genes reside together on a small operon that is regulated by Fur and is transcribed in response to iron limitation in mucoid, alginate-producing bacteria.
Full Text
The Full Text of this article is available as a PDF (2.0 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Ater J. L., Herbst J. J., Landaw S. A., O'Brien R. T. Relative anemia and iron deficiency in cystic fibrosis. Pediatrics. 1983 May;71(5):810–814. [PubMed] [Google Scholar]
- Bayer A. S., Eftekhar F., Tu J., Nast C. C., Speert D. P. Oxygen-dependent up-regulation of mucoid exopolysaccharide (alginate) production in Pseudomonas aeruginosa. Infect Immun. 1990 May;58(5):1344–1349. doi: 10.1128/iai.58.5.1344-1349.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baynham P. J., Wozniak D. J. Identification and characterization of AlgZ, an AlgT-dependent DNA-binding protein required for Pseudomonas aeruginosa algD transcription. Mol Microbiol. 1996 Oct;22(1):97–108. doi: 10.1111/j.1365-2958.1996.tb02659.x. [DOI] [PubMed] [Google Scholar]
- Berry A., DeVault J. D., Chakrabarty A. M. High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains. J Bacteriol. 1989 May;171(5):2312–2317. doi: 10.1128/jb.171.5.2312-2317.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
- Calderwood S. B., Mekalanos J. J. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J Bacteriol. 1987 Oct;169(10):4759–4764. doi: 10.1128/jb.169.10.4759-4764.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chitnis C. E., Ohman D. E. Cloning of Pseudomonas aeruginosa algG, which controls alginate structure. J Bacteriol. 1990 Jun;172(6):2894–2900. doi: 10.1128/jb.172.6.2894-2900.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chu L., May T. B., Chakrabarty A. M., Misra T. K. Nucleotide sequence and expression of the algE gene involved in alginate biosynthesis by Pseudomonas aeruginosa. Gene. 1991 Oct 30;107(1):1–10. doi: 10.1016/0378-1119(91)90290-r. [DOI] [PubMed] [Google Scholar]
- Clare D. A., Duong M. N., Darr D., Archibald F., Fridovich I. Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal Biochem. 1984 Aug 1;140(2):532–537. doi: 10.1016/0003-2697(84)90204-5. [DOI] [PubMed] [Google Scholar]
- Compan I., Touati D. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J Bacteriol. 1993 Mar;175(6):1687–1696. doi: 10.1128/jb.175.6.1687-1696.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeVault J. D., Kimbara K., Chakrabarty A. M. Pulmonary dehydration and infection in cystic fibrosis: evidence that ethanol activates alginate gene expression and induction of mucoidy in Pseudomonas aeruginosa. Mol Microbiol. 1990 May;4(5):737–745. doi: 10.1111/j.1365-2958.1990.tb00644.x. [DOI] [PubMed] [Google Scholar]
- DeVries C. A., Ohman D. E. Mucoid-to-nonmucoid conversion in alginate-producing Pseudomonas aeruginosa often results from spontaneous mutations in algT, encoding a putative alternate sigma factor, and shows evidence for autoregulation. J Bacteriol. 1994 Nov;176(21):6677–6687. doi: 10.1128/jb.176.21.6677-6687.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deretic V., Dikshit R., Konyecsni W. M., Chakrabarty A. M., Misra T. K. The algR gene, which regulates mucoidy in Pseudomonas aeruginosa, belongs to a class of environmentally responsive genes. J Bacteriol. 1989 Mar;171(3):1278–1283. doi: 10.1128/jb.171.3.1278-1283.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deretic V., Gill J. F., Chakrabarty A. M. Gene algD coding for GDPmannose dehydrogenase is transcriptionally activated in mucoid Pseudomonas aeruginosa. J Bacteriol. 1987 Jan;169(1):351–358. doi: 10.1128/jb.169.1.351-358.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Deretic V., Schurr M. J., Boucher J. C., Martin D. W. Conversion of Pseudomonas aeruginosa to mucoidy in cystic fibrosis: environmental stress and regulation of bacterial virulence by alternative sigma factors. J Bacteriol. 1994 May;176(10):2773–2780. doi: 10.1128/jb.176.10.2773-2780.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flynn J. L., Ohman D. E. Cloning of genes from mucoid Pseudomonas aeruginosa which control spontaneous conversion to the alginate production phenotype. J Bacteriol. 1988 Apr;170(4):1452–1460. doi: 10.1128/jb.170.4.1452-1460.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Flynn J. L., Ohman D. E. Use of a gene replacement cosmid vector for cloning alginate conversion genes from mucoid and nonmucoid Pseudomonas aeruginosa strains: algS controls expression of algT. J Bacteriol. 1988 Jul;170(7):3228–3236. doi: 10.1128/jb.170.7.3228-3236.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franklin M. J., Ohman D. E. Identification of algF in the alginate biosynthetic gene cluster of Pseudomonas aeruginosa which is required for alginate acetylation. J Bacteriol. 1993 Aug;175(16):5057–5065. doi: 10.1128/jb.175.16.5057-5065.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Franklin M. J., Ohman D. E. Identification of algI and algJ in the Pseudomonas aeruginosa alginate biosynthetic gene cluster which are required for alginate O acetylation. J Bacteriol. 1996 Apr;178(8):2186–2195. doi: 10.1128/jb.178.8.2186-2195.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldberg J. B., Gorman W. L., Flynn J. L., Ohman D. E. A mutation in algN permits trans activation of alginate production by algT in Pseudomonas species. J Bacteriol. 1993 Mar;175(5):1303–1308. doi: 10.1128/jb.175.5.1303-1308.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goldberg J. B., Ohman D. E. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Govan J. R., Deretic V. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev. 1996 Sep;60(3):539–574. doi: 10.1128/mr.60.3.539-574.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haas B., Murphy E., Castignetti D. Siderophore synthesis by mucoid Pseudomonas aeruginosa strains isolated from cystic fibrosis patients. Can J Microbiol. 1991 Aug;37(8):654–657. doi: 10.1139/m91-111. [DOI] [PubMed] [Google Scholar]
- Hantke K. Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. Mol Gen Genet. 1987 Nov;210(1):135–139. doi: 10.1007/BF00337769. [DOI] [PubMed] [Google Scholar]
- Hassett D. J., Sokol P. A., Howell M. L., Ma J. F., Schweizer H. T., Ochsner U., Vasil M. L. Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J Bacteriol. 1996 Jul;178(14):3996–4003. doi: 10.1128/jb.178.14.3996-4003.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hassett D. J., Woodruff W. A., Wozniak D. J., Vasil M. L., Cohen M. S., Ohman D. E. Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese superoxide dismutase activity in alginate-producing bacteria. J Bacteriol. 1993 Dec;175(23):7658–7665. doi: 10.1128/jb.175.23.7658-7665.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hiratsu K., Amemura M., Nashimoto H., Shinagawa H., Makino K. The rpoE gene of Escherichia coli, which encodes sigma E, is essential for bacterial growth at high temperature. J Bacteriol. 1995 May;177(10):2918–2922. doi: 10.1128/jb.177.10.2918-2922.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Holloway B. W., Krishnapillai V., Morgan A. F. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. doi: 10.1128/mr.43.1.73-102.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kato J., Chu L., Kitano K., DeVault J. D., Kimbara K., Chakrabarty A. M., Misra T. K. Nucleotide sequence of a regulatory region controlling alginate synthesis in Pseudomonas aeruginosa: characterization of the algR2 gene. Gene. 1989 Dec 7;84(1):31–38. doi: 10.1016/0378-1119(89)90136-4. [DOI] [PubMed] [Google Scholar]
- Knutson C. A., Jeanes A. A new modification of the carbazole analysis: application to heteropolysaccharides. Anal Biochem. 1968 Sep;24(3):470–481. doi: 10.1016/0003-2697(68)90154-1. [DOI] [PubMed] [Google Scholar]
- Krieg D. P., Bass J. A., Mattingly S. J. Aeration selects for mucoid phenotype of Pseudomonas aeruginosa. J Clin Microbiol. 1986 Dec;24(6):986–990. doi: 10.1128/jcm.24.6.986-990.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin D. W., Holloway B. W., Deretic V. Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J Bacteriol. 1993 Feb;175(4):1153–1164. doi: 10.1128/jb.175.4.1153-1164.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Martin D. W., Schurr M. J., Mudd M. H., Govan J. R., Holloway B. W., Deretic V. Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8377–8381. doi: 10.1073/pnas.90.18.8377. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Meyer J. M., Hohnadel D., Khan A., Cornelis P. Pyoverdin-facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdin receptor. Mol Microbiol. 1990 Aug;4(8):1401–1405. doi: 10.1111/j.1365-2958.1990.tb00719.x. [DOI] [PubMed] [Google Scholar]
- Mian F. A., Jarman T. R., Righelato R. C. Biosynthesis of exopolysaccharide by Pseudomonas aeruginosa. J Bacteriol. 1978 May;134(2):418–422. doi: 10.1128/jb.134.2.418-422.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mohr C. D., Martin D. W., Konyecsni W. M., Govan J. R., Lory S., Deretic V. Role of the far-upstream sites of the algD promoter and the algR and rpoN genes in environmental modulation of mucoidy in Pseudomonas aeruginosa. J Bacteriol. 1990 Nov;172(11):6576–6580. doi: 10.1128/jb.172.11.6576-6580.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ochsner U. A., Vasil A. I., Vasil M. L. Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol. 1995 Dec;177(24):7194–7201. doi: 10.1128/jb.177.24.7194-7201.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ochsner U. A., Vasil M. L. Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc Natl Acad Sci U S A. 1996 Apr 30;93(9):4409–4414. doi: 10.1073/pnas.93.9.4409. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prince R. W., Cox C. D., Vasil M. L. Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol. 1993 May;175(9):2589–2598. doi: 10.1128/jb.175.9.2589-2598.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Prince R. W., Storey D. G., Vasil A. I., Vasil M. L. Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01. Mol Microbiol. 1991 Nov;5(11):2823–2831. doi: 10.1111/j.1365-2958.1991.tb01991.x. [DOI] [PubMed] [Google Scholar]
- Schurr M. J., Yu H., Boucher J. C., Hibler N. S., Deretic V. Multiple promoters and induction by heat shock of the gene encoding the alternative sigma factor AlgU (sigma E) which controls mucoidy in cystic fibrosis isolates of Pseudomonas aeruginosa. J Bacteriol. 1995 Oct;177(19):5670–5679. doi: 10.1128/jb.177.19.5670-5679.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schurr M. J., Yu H., Martinez-Salazar J. M., Boucher J. C., Deretic V. Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J Bacteriol. 1996 Aug;178(16):4997–5004. doi: 10.1128/jb.178.16.4997-5004.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schweizer H. D. Small broad-host-range gentamycin resistance gene cassettes for site-specific insertion and deletion mutagenesis. Biotechniques. 1993 Nov;15(5):831–834. [PubMed] [Google Scholar]
- Schweizer H. P. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195–1204. doi: 10.1111/j.1365-2958.1992.tb01558.x. [DOI] [PubMed] [Google Scholar]
- Schwyn B., Neilands J. B. Universal chemical assay for the detection and determination of siderophores. Anal Biochem. 1987 Jan;160(1):47–56. doi: 10.1016/0003-2697(87)90612-9. [DOI] [PubMed] [Google Scholar]
- Silver S., Johnseine P., Whitney E., Clark D. Manganese-resistant mutants of Escherichia coli: physiological and genetic studies. J Bacteriol. 1972 Apr;110(1):186–195. doi: 10.1128/jb.110.1.186-195.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tardat B., Touati D. Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA. Mol Microbiol. 1993 Jul;9(1):53–63. doi: 10.1111/j.1365-2958.1993.tb01668.x. [DOI] [PubMed] [Google Scholar]
- Terry J. M., Piña S. E., Mattingly S. J. Environmental conditions which influence mucoid conversion Pseudomonas aeruginosa PAO1. Infect Immun. 1991 Feb;59(2):471–477. doi: 10.1128/iai.59.2.471-477.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Terry J. M., Piña S. E., Mattingly S. J. Role of energy metabolism in conversion of nonmucoid Pseudomonas aeruginosa to the mucoid phenotype. Infect Immun. 1992 Apr;60(4):1329–1335. doi: 10.1128/iai.60.4.1329-1335.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wozniak D. J., Ohman D. E. Involvement of the alginate algT gene and integration host factor in the regulation of the Pseudomonas aeruginosa algB gene. J Bacteriol. 1993 Jul;175(13):4145–4153. doi: 10.1128/jb.175.13.4145-4153.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wozniak D. J., Ohman D. E. Transcriptional analysis of the Pseudomonas aeruginosa genes algR, algB, and algD reveals a hierarchy of alginate gene expression which is modulated by algT. J Bacteriol. 1994 Oct;176(19):6007–6014. doi: 10.1128/jb.176.19.6007-6014.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Xie Z. D., Hershberger C. D., Shankar S., Ye R. W., Chakrabarty A. M. Sigma factor-anti-sigma factor interaction in alginate synthesis: inhibition of AlgT by MucA. J Bacteriol. 1996 Aug;178(16):4990–4996. doi: 10.1128/jb.178.16.4990-4996.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yu H., Boucher J. C., Hibler N. S., Deretic V. Virulence properties of Pseudomonas aeruginosa lacking the extreme-stress sigma factor AlgU (sigmaE). Infect Immun. 1996 Jul;64(7):2774–2781. doi: 10.1128/iai.64.7.2774-2781.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zielinski N. A., Chakrabarty A. M., Berry A. Characterization and regulation of the Pseudomonas aeruginosa algC gene encoding phosphomannomutase. J Biol Chem. 1991 May 25;266(15):9754–9763. [PubMed] [Google Scholar]
- de Lorenzo V., Giovannini F., Herrero M., Neilands J. B. Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30. J Mol Biol. 1988 Oct 20;203(4):875–884. doi: 10.1016/0022-2836(88)90113-1. [DOI] [PubMed] [Google Scholar]