Abstract
Plant-associated pseudomonads are commonly exposed to copper bactericides, which are applied to reduce the disease incidence caused by these bacteria. Consequently, many of these bacteria have acquired resistance or tolerance to copper salts. We recently conducted a survey of 37 copper-resistant (Cur) Pseudomonas spp., including P. cepacia, P. fluorescens, P. syringae, and P. viridiflava, and found that a subset of the P. syringae strains showed a dramatic increase in exopolysaccharide (EPS) production on mannitol-glutamate medium containing CuSO4 at 250 micrograms/ml. A modified carbazole assay indicated that the EPS produced on copper-amended media contained high levels of uronic acids, suggesting that the EPS was primarily alginic acid. Uronic acids extracted from selected strains were further confirmed to be alginate by demonstrating their sensitivity to alginate lyase and by descending paper chromatography following acid hydrolysis. Subinhibitory levels of arsenate, cobalt, lithium, rubidium, molybdenum, and mercury did not induce EPS production, indicating that alginate biosynthesis is not induced in P. syringae cells exposed to these heavy metals. A 200-kb plasmid designated pPSR12 conferred a stably mucoid phenotype to several P. syringae recipients and also increased their resistance to cobalt and arsenate. A cosmid clone constructed from pPSR12 which conferred a stably mucoid phenotype to several P. syringae strains but not to Pseudomonas aeruginosa was obtained. Results obtained in this study indicate that some of the signals and regulatory genes for alginate production in P. syringae differ from those described for alginate production in P. aeruginosa.
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- Anwar H., Strap J. L., Costerton J. W. Kinetic interaction of biofilm cells of Staphylococcus aureus with cephalexin and tobramycin in a chemostat system. Antimicrob Agents Chemother. 1992 Apr;36(4):890–893. doi: 10.1128/aac.36.4.890. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Barta T. M., Kinscherf T. G., Willis D. K. Regulation of tabtoxin production by the lemA gene in Pseudomonas syringae. J Bacteriol. 1992 May;174(9):3021–3029. doi: 10.1128/jb.174.9.3021-3029.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender C. L., Cooksey D. A. Indigenous plasmids in Pseudomonas syringae pv. tomato: conjugative transfer and role in copper resistance. J Bacteriol. 1986 Feb;165(2):534–541. doi: 10.1128/jb.165.2.534-541.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender C. L., Cooksey D. A. Molecular cloning of copper resistance genes from Pseudomonas syringae pv. tomato. J Bacteriol. 1987 Feb;169(2):470–474. doi: 10.1128/jb.169.2.470-474.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bender C. L., Young S. A., Mitchell R. E. Conservation of Plasmid DNA Sequences in Coronatine-Producing Pathovars of Pseudomonas syringae. Appl Environ Microbiol. 1991 Apr;57(4):993–999. doi: 10.1128/aem.57.4.993-999.1991. [DOI] [PMC free article] [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]
- Boyd A., Ghosh M., May T. B., Shinabarger D., Keogh R., Chakrabarty A. M. Sequence of the algL gene of Pseudomonas aeruginosa and purification of its alginate lyase product. Gene. 1993 Sep 6;131(1):1–8. doi: 10.1016/0378-1119(93)90662-m. [DOI] [PubMed] [Google Scholar]
- Brumbley S. M., Carney B. F., Denny T. P. Phenotype conversion in Pseudomonas solanacearum due to spontaneous inactivation of PhcA, a putative LysR transcriptional regulator. J Bacteriol. 1993 Sep;175(17):5477–5487. doi: 10.1128/jb.175.17.5477-5487.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Buckmire F. L. Influence of nutrient media on the characteristics of the exopolysaccharide produced by three mucoid Pseudomonas aeruginosa strains. Microbios. 1984;41(163):49–63. [PubMed] [Google Scholar]
- Cha J. S., Cooksey D. A. Copper resistance in Pseudomonas syringae mediated by periplasmic and outer membrane proteins. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8915–8919. doi: 10.1073/pnas.88.20.8915. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chan R., Lam J. S., Lam K., Costerton J. W. Influence of culture conditions on expression of the mucoid mode of growth of Pseudomonas aeruginosa. J Clin Microbiol. 1984 Jan;19(1):8–16. doi: 10.1128/jcm.19.1.8-16.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Costerton J. W., Cheng K. J., Geesey G. G., Ladd T. I., Nickel J. C., Dasgupta M., Marrie T. J. Bacterial biofilms in nature and disease. Annu Rev Microbiol. 1987;41:435–464. doi: 10.1146/annurev.mi.41.100187.002251. [DOI] [PubMed] [Google Scholar]
- Darzins A., Chakrabarty A. M. Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol. 1984 Jul;159(1):9–18. doi: 10.1128/jb.159.1.9-18.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Davies D. G., Chakrabarty A. M., Geesey G. G. Exopolysaccharide production in biofilms: substratum activation of alginate gene expression by Pseudomonas aeruginosa. Appl Environ Microbiol. 1993 Apr;59(4):1181–1186. doi: 10.1128/aem.59.4.1181-1186.1993. [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]
- Evans L. R., Linker A. Production and characterization of the slime polysaccharide of Pseudomonas aeruginosa. J Bacteriol. 1973 Nov;116(2):915–924. doi: 10.1128/jb.116.2.915-924.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fett W. F., Osman S. F., Dunn M. F. Characterization of exopolysaccharides produced by plant-associated fluorescent pseudomonads. Appl Environ Microbiol. 1989 Mar;55(3):579–583. doi: 10.1128/aem.55.3.579-583.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fett W. F., Osman S. F., Fishman M. L., Siebles T. S. Alginate production by plant-pathogenic pseudomonads. Appl Environ Microbiol. 1986 Sep;52(3):466–473. doi: 10.1128/aem.52.3.466-473.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fett W. F., Wijey C., Lifson E. R. Occurrence of alginate gene sequences among members of the pseudomonad rRNA homology groups I-IV. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):151–157. doi: 10.1016/0378-1097(92)90017-i. [DOI] [PubMed] [Google Scholar]
- Fialho A. M., Zielinski N. A., Fett W. F., Chakrabarty A. M., Berry A. Distribution of alginate gene sequences in the Pseudomonas rRNA homology group I-Azomonas-Azotobacter lineage of superfamily B procaryotes. Appl Environ Microbiol. 1990 Feb;56(2):436–443. doi: 10.1128/aem.56.2.436-443.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gacesa P., Wusteman F. S. Plate assay for simultaneous detection of alginate lyases and determination of substrate specificity. Appl Environ Microbiol. 1990 Jul;56(7):2265–2267. doi: 10.1128/aem.56.7.2265-2267.1990. [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]
- Govan J. R., Fyfe J. A. Mucoid Pseudomonas aeruginosa and cystic fibrosis: resistance of the mucoid from to carbenicillin, flucloxacillin and tobramycin and the isolation of mucoid variants in vitro. J Antimicrob Chemother. 1978 May;4(3):233–240. doi: 10.1093/jac/4.3.233. [DOI] [PubMed] [Google Scholar]
- Hoyle B. D., Williams L. J., Costerton J. W. Production of mucoid exopolysaccharide during development of Pseudomonas aeruginosa biofilms. Infect Immun. 1993 Feb;61(2):777–780. doi: 10.1128/iai.61.2.777-780.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jones J. D., Gutterson N. An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a. Gene. 1987;61(3):299–306. doi: 10.1016/0378-1119(87)90193-4. [DOI] [PubMed] [Google Scholar]
- KING E. O., WARD M. K., RANEY D. E. Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med. 1954 Aug;44(2):301–307. [PubMed] [Google Scholar]
- Kao C. C., Barlow E., Sequeira L. Extracellular polysaccharide is required for wild-type virulence of Pseudomonas solanacearum. J Bacteriol. 1992 Feb;174(3):1068–1071. doi: 10.1128/jb.174.3.1068-1071.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lee Y. A., Hendson M., Panopoulos N. J., Schroth M. N. Molecular cloning, chromosomal mapping, and sequence analysis of copper resistance genes from Xanthomonas campestris pv. juglandis: homology with small blue copper proteins and multicopper oxidase. J Bacteriol. 1994 Jan;176(1):173–188. doi: 10.1128/jb.176.1.173-188.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Liao C. H., McCallus D. E., Fett W. F. Molecular characterization of two gene loci required for production of the key pathogenicity factor pectate lyase in Pseudomonas viridiflava. Mol Plant Microbe Interact. 1994 May-Jun;7(3):391–400. doi: 10.1094/mpmi-7-0391. [DOI] [PubMed] [Google Scholar]
- Martin D. W., Schurr M. J., Yu H., Deretic V. Analysis of promoters controlled by the putative sigma factor AlgU regulating conversion to mucoidy in Pseudomonas aeruginosa: relationship to sigma E and stress response. J Bacteriol. 1994 Nov;176(21):6688–6696. doi: 10.1128/jb.176.21.6688-6696.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- May T. B., Chakrabarty A. M. Isolation and assay of Pseudomonas aeruginosa alginate. Methods Enzymol. 1994;235:295–304. doi: 10.1016/0076-6879(94)35148-1. [DOI] [PubMed] [Google Scholar]
- May T. B., Shinabarger D., Maharaj R., Kato J., Chu L., DeVault J. D., Roychoudhury S., Zielinski N. A., Berry A., Rothmel R. K. Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients. Clin Microbiol Rev. 1991 Apr;4(2):191–206. doi: 10.1128/cmr.4.2.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mittelman M. W., Geesey G. G. Copper-binding characteristics of exopolymers from a freshwater-sediment bacterium. Appl Environ Microbiol. 1985 Apr;49(4):846–851. doi: 10.1128/aem.49.4.846-851.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Osman S. F., Fett W. F., Fishman M. L. Exopolysaccharides of the phytopathogen Pseudomonas syringae pv. glycinea. J Bacteriol. 1986 Apr;166(1):66–71. doi: 10.1128/jb.166.1.66-71.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Phillis J. W., Smith-Barbour M., O'Regan M. H., Perkins L. M. Amino acid and purine release in rat brain following temporary middle cerebral artery occlusion. Neurochem Res. 1994 Sep;19(9):1125–1130. doi: 10.1007/BF00965145. [DOI] [PubMed] [Google Scholar]
- Schiller N. L., Monday S. R., Boyd C. M., Keen N. T., Ohman D. E. Characterization of the Pseudomonas aeruginosa alginate lyase gene (algL): cloning, sequencing, and expression in Escherichia coli. J Bacteriol. 1993 Aug;175(15):4780–4789. doi: 10.1128/jb.175.15.4780-4789.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Selvaraj G., Iyer V. N. Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria. J Bacteriol. 1983 Dec;156(3):1292–1300. doi: 10.1128/jb.156.3.1292-1300.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Staskawicz B. J., Dahlbeck D., Keen N. T. Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L.) Merr. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6024–6028. doi: 10.1073/pnas.81.19.6024. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sundin G. W., Bender C. L. Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae. Appl Environ Microbiol. 1993 Apr;59(4):1018–1024. doi: 10.1128/aem.59.4.1018-1024.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sundin G. W., Demezas D. H., Bender C. L. Genetic and plasmid diversity within natural populations of Pseudomonas syringae with various exposures to copper and streptomycin bactericides. Appl Environ Microbiol. 1994 Dec;60(12):4421–4431. doi: 10.1128/aem.60.12.4421-4431.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- TREVELYAN W. E., PROCTER D. P., HARRISON J. S. Detection of sugars on paper chromatograms. Nature. 1950 Sep 9;166(4219):444–445. doi: 10.1038/166444b0. [DOI] [PubMed] [Google Scholar]
- Thomassen M. J., Demko C. A., Boxerbaum B., Stern R. C., Kuchenbrod P. J. Multiple of isolates of Pseudomonas aeruginosa with differing antimicrobial susceptibility patterns from patients with cystic fibrosis. J Infect Dis. 1979 Dec;140(6):873–880. doi: 10.1093/infdis/140.6.873. [DOI] [PubMed] [Google Scholar]
- Ullrich M., Bereswill S., Völksch B., Fritsche W., Geider K. Molecular characterization of field isolates of Pseudomonas syringae pv. glycinea differing in coronatine production. J Gen Microbiol. 1993 Aug;139(8):1927–1937. doi: 10.1099/00221287-139-8-1927. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Whitfield C. Bacterial extracellular polysaccharides. Can J Microbiol. 1988 Apr;34(4):415–420. doi: 10.1139/m88-073. [DOI] [PubMed] [Google Scholar]
