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.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.

The correlation between the chemical stability and the microbiological activity of polymyxin B in phosphate buffer pH 6.0 and in proprietary eye drops was evaluated. High Performance Liquid Chromatography (HPLC) was used to quantify the amount of the main components in samples stored at 43,50,55 and 60°C for a period of 500 h. The data indicated that there are significant differences in chemical stability among the different proprietary eye drops. The accurate decomposition rate constants and shelf-lives (~o) at 4°C of two of the six formulated eye drops and the standard polymyxin B solution stored in glass containers at pH 6.0 were established. It was concluded that microbiological assay by agar diffusion was unsuitable for determining the activity of control polymyxin B in phosphate buffer and polymyxin B in eye drops. Killing time determinations for polymyxin B against cell suspensions of P. aeruginosa NCTC 6750 were consequently used. Thioglycollate broth containingp- aminobenzoic acid (PABA) 0.16 %w/v and magnesium sulphate 1 %w/v was used as an inactivating recovery medium. The effect of preservatives and of second antibacterials contained in the eye drops were tested individually and combined with polymyxin B. Thiomersal 0.001 %w/v, trimethoprim 0.02 %w/v and thiomersal 0.001 %w/v plus trimethoprim 0.02 %w/v did not have an effect on the activity of polymyxin B 2000 U/ml. Neomycin was an exception and at the concentrations in the range 0.0192 to 0.16 %w/v exhibited an antagonistic effect. Chemical interaction between polymyxin B and neomycin could not be detected and it was considered that the inhibitory effect of neomycin may be the result of competition between polymyxin B and neomycin for the same binding sites on the cell surface. Gentamicin is active against P. aeruginosa NCTC 6750 and at concentrations of 0.075 and 0.036 %w/v it exhibited an additive effect with polymyxin B 2000 U/ml against the test organism. The results obtained with the samples stored at 55°C for a period of 500 h demonstrated the critical effect of pH. At a pH of 6.0 microbiological activity and chemical stability appeared optimal. The chemical stability data of five eye drop samples correlated with microbiological activity data. Exceptions were polymyxin B in one eye drop sample and control polymyxin B. These extensively decomposed samples showed good antibacterial activity which appeared to result from the activity of decomposition products. Chemical stability data for standard polymyxin B solution at pH 6.0 also correlated to microbiological activity data over the temperature range of 92 - 115°C. The polymyxin B retained detectable microbiological activity when the amount of PB1 was greater than 20%. It is suggested that the decomposition products which occurred at these higher temperatures did not possess antibacterial activity.

Pseudomonas saccharophila could fix N$ sb2$ under micro-aerobic conditions, heterotrophically and chemolithotrophically. Uptake hydrogenase activity under heterotrophic conditions had no effect on the O$ sb2$ sensitivity of nitrogenase. H$ sb2$ induced whereas sucrose and O$ sb2$ repressed hydrogenase synthesis. Sucrose and O$ sb2$ did not inhibit hydrogenase activity. Hydrogenase and urease were located in the membrane and soluble fractions, respectively. Nickel stimulated growth, hydrogenase expression, and nitrogenase activity under N-limited chemolithotrophic conditions. Hydrogenase synthesis specifically required nickel and its repression by O$ sb2$ was alleviated by increasing the nickel concentration. Incorporated $ sp{63}$Ni$ sp{2+}$ was about 3 times higher in the soluble than in the membrane fraction. The short-term uptake of nickel was energy-independent and had an apparent $K sb{m}$ of 31.7 uM and $V sb{max}$ of 3.8 nmol Ni$ sp{2+}$ (mg protein)$ sp{-1}$min$ sp{-1}$. / A counting method for heterotrophic and chemolithotrophic N$ sb2$-fixing H$ sb2$-oxidizing bacteria was developed. The white bean rhizosphere soil showed relatively high numbers of these bacteria.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

We studied the host specificity, interactions with plant immune systems, and virulence factors of the phytopathogenic Type III secretion system-carrying bacterium Pseudomonas syringae. In studying host specificity, we ran growth and pod assays using seventeen pathovars of P. syringae on kidney bean hosts. We tracked bacterial growth numbers over six days and compared pathovar growth patterns. To study immune interactions with host plants, we performed effector-triggered immunity induction and suppression assays with individual effectors in Arabidopsis thaliana to determine whether effector evolutionary age was related to resultant plant immune responses. No correlations were observed. To generate candidate virulence effectors, we sequenced mRNA from seven P. syringae pathovars grown in inducing media and pulled out hits to virulence-related genes.

Pseudomonas aeruginosa is a versatile Gram-negative pathogen that can infect a diversity of immunocompromised patients. Interest in alternatives to traditional antibiotics has inspired our investigation of R- and F-type pyocins as novel therapeutics. These phage tail-like bacteriocins are produced by P. aeruginosa to kill competing strains via pore formation in target cells. We aimed to characterize the diversity of pyocins and bacteriophages generated by diverse P. aeruginosa strains so as to identify pyocins of therapeutic value. Strategies to delineate pyocin and phage activities included physical methods, the modulation of pyocin regulation, and antibody-based detection of tail-like pyocins. We have identified the dominance of R- and F-type pyocins in impacting P. aeruginosa populations and revealed a small number of strains producing particularly potent pyocins. In addition, the co-regulation of phages and pyocins, the dependence of pyocins on pili for activity, and the striking diversity of pyocin susceptibility have all been recognized.

I have investigated the evolutionary response of Pseudomonas aeruginosa to salt (NaCl) stress, and the physiological mechanisms responsible for this adaptation. Populations of P. aeruginosa founded from the same ancestral genotype were selected at three different concentrations of NaCl, low, moderate and high for about 660 generations with four independent replicates for each concentration. Adaptation was measured as the fitness of the evolved populations relative to the ancestor assessed in direct, head-to-head competition experiments conducted in the same environment in which they were selected (direct response) as well as in all alternative environments (correlated response). Results suggest that selection in each salt environment led to adaptation to that environment and a modest degree of specialization that evolved because correlated responses to selection were smaller than direct responses. In order to identify the physiological mechanisms contributing to the populations' adaptation in high NaCl concentration, I chose a sample of evolved lines that showed the strongest evidence for specialization to salt and competed them against the common ancestor in KCl and sucrose. Results suggested that increased Na+ /H+ antiporter activity is probably the primary mechanism behind adaptation to high NaCl concentration, however alternative mechanisms cannot be excluded. Tolerance curves, which measure the performance of a genotype across a gradient of salt concentrations, suggested no change in the high salt group’s ability to tolerate extreme concentrations of NaCl. We conclude that high salt evolved population showed improvements to its ionic/osmotic stress resistance strategies mainly to Na+ efflux strategies but with no changes to salt niche.

ABSTRACT Sclerotinia sclerotiorum is a ubiquitous ascomycetous fungal pathogen that causes disease in over 400 crop species, specifically in soybean and canola plants, where stem rot is the most common disease symptom. Pseudomonas chlororaphis PA23 was previously isolated from the rhizosphere of soybean and has demonstrated excellent antifungal activity against S. sclerotiorum in vitro, greenhouse and field experiments. To elucidate the molecular mechanisms involved in PA23 biocontrol, random mutagenesis experiments were initiated. Several mutants were isolated that could be divided into three general classes. Biocontrol activity of various Pseudomonas spp. is highly regulated by a GacS/GacA two-component global regulatory system. Class I PA23 mutants harboured Tn5 insertions in the gacS-coding region, resulting in pleiotropic defects including deficiency in secondary metabolite production and biocontrol activity. Complementation with the wild type gacS allele in trans restored wild type phenotypes. These findings suggest that the ability of P. chlororaphis PA23 to suppress S. sclerotiorum causing stem rot in canola is dependent on a functional GacS/GacA global regulatory system. This is the first study assessing disease symptoms on canola (Brassica napus L.) plants inoculated with a gacS minus strain of P. chlororaphis. Phenazine compounds are considered to be a key secondary metabolite contributing to the antagonistic and antifungal activity of P. chlororaphis. In P. chlororaphis PA23, mutations in phenazine biosynthetic genes exhibited equal or more antifungal activity in vitro, compared to the wild type. To assess the effect of the deficiency in phenazine production, a Class II mutant , harbouring a Tn5 insertion in phzE was tested for a number of biocontrol traits including secondary metabolite production, motility, and suppression of Sclerotinia pathogenic traits. Since no other traits were markedly affected beyond phenazine production, it was concluded that phenazine is not the major product contributing to S. sclerotiorum biocontrol. A single Class III mutant was isolated harbouring a Tn5 insertion in a gene encoding a transcriptional regulator of the LysR family. This mutant exhibited no antifungal activity on plate assays and was unable to protect against S. sclerotiorum in green house assays. A number of secondary metabolites were no longer produced by this mutant, suggesting that this LysR-type transcriptional regulator is either directly or indirectly involved in controlling several genes in P. chlororaphis PA23.