Emergence of Cu ++-tolerant mutants defective in gellan synthesis in Cu ++-stressed cultures of Sphingomonas paucimobilis

Abstract

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++.