Abstract
Antibiotic production kinetics of Streptomyces strain JS1 (an isolate selected from an industrial screen for biocontrol activity) and Streptomyces hygroscopicus (a culture collection strain with similar biocontrol activity to JS1) were examined. Growth-associated niphimycin production was observed in both strains during carbon-limited batch culture. Growth associated antibiotic production in carbon-limited medium has not been reported elsewhere and the antibiotic production physiology of biocontrol isolates has not been extensively studied in other laboratories. Increases in biomass and antibiotic production occurred simultaneously in JS1 (24h) and S. hygroscopicus (40h) carbon-limited cultures. Specific growth and antibiotic production rates peaked simultaneously (35h in S. hygroscopicus, 30h in JS1). Examination of the correlation between intracellular protein synthesis rate and niphimycin production rate was consistent with the relationship between these parameters proposed (in our laboratory and elsewhere) for the more frequently reported phenomenon of growth- dissociated antibiotic production. Evidence was obtained which resulted in a hypothesis that the unusual antibiotic production kinetics were a result of the unusually low affinity of JS1 for glucose. A novel approach (multi-compartment nonlinear modelling) to the determination of substrate affinity constants yielded a Ks value of 2.9mM which compares to 7.55muM (i.e. significantly higher affinity) value for Saccharopolyspora erythraea, a species which demonstrates the more common, growth dissociated form of production. Antibiotic production in nitrogen-limited culture was also growth- associated, but this has been reported elsewhere. Work reported here suggests that affinity for nitrogen substrate is significantly lower than that for glucose in S. erythraea (4.45mM compared to 7.55muM) a strain that also exhibits growth-associated antibiotic production under nitrogen limitation. This presumably explains the more growth-associated production kinetics observed in nitrogen-limited cultures. It is tempting to speculate a link between antibiotic production kinetics and biocontrol potential. A micro-organism capable of releasing anti-microbial product in synchrony with cell growth would presumably have more effect in reducing the rhizosphere microflora, prior to colonising the habitat, than a species producing the antibiotic as a secondary metabolite. If this hypothesis is justified, then it may explain the success of JS1 and S. hygroscopicus in the industrial screen. A mutant of JS1, unable to produce niphimycin, displayed diminished biocontrol capability, indicating that niphimycin has a role in the observed biological control effect, in this instance. An attempt to increase the biocontrol effectiveness of JS1 by enhancing niphimycin production in hydroponic and agar tomato culture systems was unsuccessful due to the production kinetics displayed by JS1. Manipulating culture conditions for increased niphimycin production inevitably resulted in increased JS1 growth which was associated with plant death. Electron microscopy suggested that this enhanced growth resulted in excessive colonisation of the root system, possibly resulting in plant death due to root oxygen starvation. The fungal pathogens Phytophthora capsici and Fusarium oxysporum, used as challenge organisms in the industrial screen, had significantly higher affinities for the substrates examined (15muM and <10muM, respectively for glucose and 22muM and <38muM, respectively, for nitrate) compared to the affinities of JS1 (2.9mM and 2.4mM, respectively, for glucose and nitrate) indicating that competition for nutrients was unlikely to account for the success of JS1 in the screen. An additional novel concept explored in this work was the use of a fractional factorial medium design procedure (the Plackett-Burman technique) for the attempted identification of nutrients that could be used to simultaneously enhance growth of biocontrol agents whilst inhibiting the growth of target pathogens. Nutritional requirements thus elucidated were compared to those of variant strains of S. hygroscopicus and other Streptomyces species.