No description available.

No description available.

No description available.

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.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Biological control is an intriguing alternative to the use of chemical pesticides as it represents a safer, more environmentally friendly approach to managing plant pathogens. Pseudomonas chlororaphis strain PA23 was isolated from soybean root tips and it was found to be an excellent antagonist of sclerotinia stem rot. Our studies have shown that pyrrolnitrin (PRN) is the key metabolite required for S. sclerotiorum inhibition, while phenazine (PHZ) is important for biofilm establishment. For this reason, research efforts were directed towards elucidating the mechanisms governing PA23-mediated antibiotic production. To determine how these compounds were regulated, QS-deficient strains and an rpoS mutant were generated. The QS-deficient strains no longer inhibited the fungal pathogen S. sclerotiorum in vitro and exhibited reduced PRN, PHZ and protease production. Analysis of transcriptional fusions revealed that RpoS has a positive and negative effect on phzI and phzR, respectively. In a reciprocal manner, RpoS is positively regulated by QS. Characterization of a phzRrpoS double mutant showed reduced antifungal activity as well as PRN and PHZ production, similar to the QS-deficient strains. Furthermore, phzR but not rpoS was able to complement the phzRrpoS double mutant for the aforementioned traits, indicating that the Phz QS system is a central regulator of PA23-mediated antagonism. GacS/GacA, PsrA, RpoS and the PhzI/PhzR QS are members of a complex regulatory hierarchy that influence secondary metabolite production in PA23. An additional system, termed Rsm, was identified, adding yet another layer of complexity to the regulatory network. The Rsm system in PA23 appears to be comprised of a single small non-coding regulatory RNA termed RsmZ, and two RNA binding proteins RsmA and RsmE. We discovered that the expression of rsmZ, rsmA and rsmE all require GacA. In addition, both PsrA and QS were shown to positively regulate rsmZ transcription. For rsmE, GacA may indirectly regulate expression through PsrA, RpoS and QS, as all three regulators control rsmE transcription. Furthermore, we believe that the positive effects of PsrA and QS on rsmE transcription are likely mediated through RpoS as only RpoS show direct activation of rsmE in an E. coli background.

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such inter-species interactions. Biotic elicitation mimics the inter-species interactions in nature by introducing cell extracts, parts of cell wall, or dead cells into the culture of another species thus resulting in complex metabolic responses in the elicited microorganisms. In this thesis we report the exploitation of the interspecies interaction in order to enhance the antibiotic production by the model organism Streptomyces coelicolor. It produces four known antibiotics: actinorhodin, undecylprodigioisn, methylenomycin and the calcium dependent antibiotic. We investigated the production of actinorhodin and undecylprodigiosin only because of the lack of quantitative analytical methods for the other two. The pure cultures of S. coelicolor in a defined medium produce higher concentrations of actinorhodin compared with undecylprodigiosin. However, undecylprodigiosin is more important due to its antitumor activities. We introduced live and dead cells of E. coli, Bacillus subtilis and Staphylococcus aureus, separately, to the S. coelicolor culture. Investigations were performed on Petri dishes, shake flasks and 2 L bioreactors. The suitable amount of each elicitor bacterium was first determined based on its ability to grow in the S. coelicolor medium so that it did not overtake the growth of S. coelicolor. Growth of S. coelicolor and glucose consumption of the elicited cultures were studied and compared with those in the pure culture. Our results revealed an alteration in the antibiotic production pattern by S. coelicolor, such that undecylprodigiosin production was significantly enhanced and actinorhodin decreased. The maximum enhancement occurred in the culture elicited with the live cells of E. coli with an increase of 3.5-fold, whereas the minimum was with elicitation using S. aureus cells (2.1-fold increase). Also, a considerable suppression in the production of actinorhodin was observed upon elicitation with live cells of E. coli or S. aureus. Furthermore, another positive outcome of the elicitation was the earlier onset of undecylprodigiosin production by 24-35h compared to the pure culture of S. coelicolor. Moreover, this study showed that the dead cells of B. subtilis and S. aureus had the same elicitation effects as the live cells, contrary to the heat-killed cells of E. coli that had no such effect. Some optimisation experiments on the amount and the timing of the elicitation were performed and the optimal conditions were chosen that would increase undecylprodigiosin production. Elicitation in the bioreactor resulted in as much as six-fold increase in the production of undecylprodigiosin compared with the pure culture and approximately double that obtained in the shake flasks. The antimicrobial activities of the extracted actinorhodin and undecylprodigiosin on the elicitor bacteria were tested in agar diffusion tests. Undecylprodigiosin always inhibited the growth of the elicitor bacteria whereas actinorhodin was less effective. In addition, our results indicated that the interaction between S. coelicolor and E. coli was mediated via a molecule present in the E. coli culture, while no such evidence was found in the case of interaction with B. subtilis or S. aureus. The results showed that the elicitation with the cells of B. subtilis and S. aureus was not due to peptidoglycan or N-acetyl glucoseamine which is the constituents of the cell wall that may have been released by lyses during the culture process. Such inter-species interactions may form the basis of new strategies in the search for novel antibiotics and other bioactive compounds. They can also be used to increase the productivity of existing processes for antibiotics as it was found in this work.

The Streptomyces are invaluable as a natural source of antibiotics and other bioactive compounds used in medicine and agriculture. S. coelicolor is the model streptomycete, and is studied for its complex secondary metabolism and multicellular life cycle. The subject of this work is bldB, a gene essential for development and antibiotic production in S. coelicolor, and one of its many homologues, located in the abaA antibiotic regulatory locus. The aim was to study the transcriptional regulation of bldB using a luminescent reporter, and investigate the role of each of the genes in the abaA cluster in regulation of antibiotic production, in order to understand the function and mechanism of action of bldB and its homologues. Individual deletion of each of the four genes in the abaA cluster resulted in varying effects on production of the antibiotic CDA. The bldB homologue, SCO0703, was shown to be a positive regulator of CDA, as the null mutant was severely defective in CDA production. It was found that bldB is expressed in most other bld developmental mutants, with the exception of bldD. There was no direct interaction observed between BldD and the bldB promoter, and possible mechanisms of indirect regulation are proposed. / Thesis / Master of Science (MSc)