Emergence of Cu(++)-tolerant mutants defective in gellan synthesis in Cu(++)-stressed cultures of Sphingomonas paucimobilis.
Cells defective in gellan synthesis appeared during cultivation of the gellan gum-producing strain Sphingomonas paucimobilis R40 with inhibitory concentrations of copper, supplied as CuCl2. The percentage of less mucoid colonial variants dramatically increased with the increase in Cu++ supplementation, reaching 85% of total viable cells at the maximal concentration for growth. Results reported in this work indicate that emergence of colonial variants defective in gellan synthesis results from Cu(++)-induced mutation and the growth advantage of these mutants in Cu(++)-stressed cultures. In fact, DNA homologous recombination strongly increased with the increase in copper supplementation as indicated by the regeneration of kanamycin-resistant cells of R40 harbouring plasmid pBX404-7, which carries two non-overlapping truncated genes derived from a gene conferring kanamycin resistance. The four major groups of colonial mutants that emerged from Cu(++)-stressed cultures of R40 exhibited reduced growth rate and biomass yield in the absence of Cu++ stress and produced decreased levels of exopolysac-charide (EPS) which yielded solutions of lower or negligible viscosity. The level of increased Cu++ tolerance of these mutants, assessed by the inhibitory effect of Cu++ on growth, correlated with the degree of loss of the ability to secrete high-molecular-mass EPS. Consistent with the growth advantage of gellan-defective mutants in Cu(++)-stressed cultures, the non-producing strain RP10, spontaneously obtained during extended cultivation of R40, also exhibited a higher tolerance to Cu++. In addition, its non-mucoid phenotype was stably maintained during Cu(++)-stressed cultivation despite the stimulation of homologous recombination by Cu++.
Laboratório de Engenharia Bioquímica, Instituto Superior Técnico, Lisboa.
J A
JA
Richau
D
D
Choquenet
A M
AM
Fialho
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I
Sá-Correia
D003300:Copper / Q000494:pharmacology*; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D010957:Plasmids; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D011549:Pseudomonas / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D011995:Recombination, Genetic
D011135:Polysaccharides, Bacterial; C048288:gellan gum; D003300:Copper