The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.

The stringent response (SR) is a global regulatory mechanism that allows bacteria to survive starvation. The plant surface is one environment where a fluctuation in nutrient availability is experienced. Because both Pseudomonas sp. DF41 and Pseudomonas chlororaphis PA23 are able to protect canola from the fungal pathogen Sclerotinia sclerotiorum when applied as a foliar spray, we sought to investigate the impact of this response on the antifungal activities of these two biocontrol strains. The SR exerts its effects on gene transcription through production of the alarmone(p)ppGpp. Metabolism of (p)ppGpp is governed by two enzymes; RelA acts as a synthetase, while SpoT can function as either a synthetase or a hydrolase. To investigate how the SR affects the ability of strains DF41 and PA23 to inhibit the fungal pathogen,relA and relAspoT mutants were generated through allelic exchange. Strain DF41 relA and relAspoT mutants were found to exhibit increased antifungal activity due to enhanced lipopeptide (LP) antibiotic production. Addition of relA, but not spoT in trans restored the mutant phenotype to that of the parent. The influence of the SR on the regulatory mechanisms governing strain DF41 biocontrol was also investigated. It was determined that relA forms part of the Gac regulon while RpoS is under SR control. In fact, addition of rpoS in trans restored protease activity to wild-type levels, but did not attenuate antifungal activity. The SR mutants PA23relA and PA23relAspoT, also exhibited increased growth inhibition of S. sclerotiorum in vitro compared to the wild type. Both mutants showed enhanced production of the antifungal factors pyrrolnitrin, lipase and protease and were complemented by the addition of relA but not spoT. Herein, the SR was found to regulate that Gac system, QS, and RpoS. The presence of gacS or rpoS in multicopy restored the mutant phenotype to that of the wild type.In summary, these findings suggest that the SR negatively influences the biocontrol activities of strains DF41 and PA23. It is evident that the SR is merely one mechanism by which DF41 and PA23-mediated antagonism is regulated.

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Pseudomonads, particularly the fluorescent pseudomonads, are common rhizosphere bacteria accounting for a significant portion of the culturable rhizosphere bacteria. The presence and diversity of Pseudomonas spp. in the rhizosphere is important because of their ability to influence plant and soil health. Diversity is generated as the result of mutation, the rearrangement of genes within the genome and the acquisition of genes by horizontal transfer systems, e.g. plasmids, bacteriophages, transposons or integrons. The purpose of this study was to examine the phenotypic and genotypic diversity of a subset of pseudomonads (N=133) isolated from the rhizosphere and root-interior of four cultivars of field-grown canola. Pseudomonads were analyzed according to their 16S rRNA and cpn60 gene sequences and selected phenotypic properties (fatty acid methyl ester (FAME) profiles, antibiotic resistance, extracellular enzyme production and carbon substrate utilization). On the basis of 16S rRNA and cpn60 gene sequences, two major clusters were observed, the Pseudomonas fluorescens complex and the P. putida complex. Phylogenetic analysis of the partial gene sequences suggested that the phylogeny of root-associated pseudomonads had no effect on their associations with different cultivars or root zones (i.e. rhizosphere and root interior). Principal component analysis (PCA) of their phenotypic properties revealed little variation among the pseudomonads associated with different canola cultivars. Importantly, while little difference was observed in isolates from different cultivars significant phenotypic variation was observed in isolates from different root zones. Cluster analysis of their phenotypic properties exhibited little correlation with their phylogenetic relationships. In the majority of situations, the isolates grouped into a phylogenetic cluster had less than 75-80% similarity among their phenotypic traits despite a close evolutionary relationship as determined by 16S rRNA and cpn60 gene sequencing. The results indicated that the genotype of the rhizosphere pseudomonads was not accurately reflected in their phenotype. Analysis of the mobile genetic elements (MGEs) associated with a randomly selected subset of the pseudomonad isolates (N=66) revealed that 58% (N=38) contained plasmids, 50% (N=33) contained inducible prophages, 24% (N=16) contained integrons and 23% (N=15) contained transposons. Examination of the MGEs associated with a subset of rhizosphere pseudomonads revealed that MGEs were present in the isolates independent of the degree of similarity between their phenotypic and phylogenetic relationships. Therefore, mutation and genomic rearrangement appear to be the major influences in the observed incongruence between the phylogenetic and the phenotypic relationships of the bacteria examined.

Pseudomonads, particularly the fluorescent pseudomonads, are common rhizosphere bacteria accounting for a significant portion of the culturable rhizosphere bacteria. The presence and diversity of Pseudomonas spp. in the rhizosphere is important because of their ability to influence plant and soil health. Diversity is generated as the result of mutation, the rearrangement of genes within the genome and the acquisition of genes by horizontal transfer systems, e.g. plasmids, bacteriophages, transposons or integrons. The purpose of this study was to examine the phenotypic and genotypic diversity of a subset of pseudomonads (N=133) isolated from the rhizosphere and root-interior of four cultivars of field-grown canola. Pseudomonads were analyzed according to their 16S rRNA and cpn60 gene sequences and selected phenotypic properties (fatty acid methyl ester (FAME) profiles, antibiotic resistance, extracellular enzyme production and carbon substrate utilization). On the basis of 16S rRNA and cpn60 gene sequences, two major clusters were observed, the Pseudomonas fluorescens complex and the P. putida complex. Phylogenetic analysis of the partial gene sequences suggested that the phylogeny of root-associated pseudomonads had no effect on their associations with different cultivars or root zones (i.e. rhizosphere and root interior). Principal component analysis (PCA) of their phenotypic properties revealed little variation among the pseudomonads associated with different canola cultivars. Importantly, while little difference was observed in isolates from different cultivars significant phenotypic variation was observed in isolates from different root zones. Cluster analysis of their phenotypic properties exhibited little correlation with their phylogenetic relationships. In the majority of situations, the isolates grouped into a phylogenetic cluster had less than 75-80% similarity among their phenotypic traits despite a close evolutionary relationship as determined by 16S rRNA and cpn60 gene sequencing. The results indicated that the genotype of the rhizosphere pseudomonads was not accurately reflected in their phenotype. Analysis of the mobile genetic elements (MGEs) associated with a randomly selected subset of the pseudomonad isolates (N=66) revealed that 58% (N=38) contained plasmids, 50% (N=33) contained inducible prophages, 24% (N=16) contained integrons and 23% (N=15) contained transposons. Examination of the MGEs associated with a subset of rhizosphere pseudomonads revealed that MGEs were present in the isolates independent of the degree of similarity between their phenotypic and phylogenetic relationships. Therefore, mutation and genomic rearrangement appear to be the major influences in the observed incongruence between the phylogenetic and the phenotypic relationships of the bacteria examined.

Aspartate transcarbamoylase (ATCase) was purified from 16 selected bacterial species including existing Pseudomonas species and former species reassigned to new genera. An enormous diversity was seen among the 16 enzymes with each class of ATCase being represented. The smallest class, class C, with a catalytically active homotrimer, at 100 kDa, was found in Bacillus and other Gram positive bacteria. In this report, the ATCases from the Gram negatives, Shewanella putrefaciens and Stenotrophomonas maltophilia were added to class C membership. The enteric bacteria typify class B ATCases at 310 kDa, with a dodecameric structure composed of two catalytic trimers coupled to three regulatory dimers. A key feature of class B ATCases is the dissociability of the holoenzyme into regulatory and catalytic subunits which were enzymatically active. In this report, the ATCase from Pseudomonas indigofera was added to class B ATCases. The largest class, at 480 kDa, class A, contains the fluorescent Pseudomonas including most members of the 16S rRNA homology group I. Two polypeptides are produced from overlapping pyrBC' genes. The former, pyrB, encodes a 34 kDa catalytic polypeptide while pyrC' encodes a 45 kDa dihydroorotase-like polypeptide. Two non active trimers are made from six 34 kDa chains which are cemented by six 45 kDa chains to form the active dodecameric structure. Dissociation of the holoenyzme into its separate active subunits has not been possible. In this report, the ATCases from Comamonas acidovorans and C. testosteroni, were added to the class A enzymes. An even larger class of ATCase than class A at 600 kDa was discovered in Burkholderia cepacia. Stoichiometric measurements predict a dodecamer of six 39 kDa polypeptides and six 60 kDa polypeptides. Unlike other large pseudomonads ATCases, the enzyme from B. cepacia was dissociable into smaller active forms. Both the holoenzyme and its dissociated forms were regulated by nucleotide effectors. A new class of ATCase was proposed for B. cepacia type enzymes.

Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2008. / Bibliography: p. 257-274. / Literature review -- General materials and methods -- Characterisation of strain collection -- Distribution of integrons and gene cassettes in pseudomonas -- Genomic context of pseudomonas integrons -- Evolutionary analysis of pseudomonas spp. integrons 199 -- Final discussion -- Appendix -- References. / Integrons associated with mobile genetic elements have played a central role in the emergence and spread of multiple antibiotic resistance in many pathogenic bacteria. However, the discovery of integrons in the chromosomes of diverse, non-pathogenic bacteria suggests that integrons have a broader role in bacterial evolution. The Pseudomonas stutzeri species complex is a well studied model for bacterial diversity. Members of the complex are genetically closely related, but sub-taxa are not able to be defined by exclusively shared sets of phenotypic characters. Rather, on the basis of total DNA:DNA similarity, Ps. stutzeri strains have been divided into 17 different groups (termed genomovars). Two Ps. stutzeri strains have been found to contain Chromosomal Integrons (CIs). This thesis involved exploration of the hypothesis that a CI was present in the common ancestor of the Ps. stutzeri species complex and assessed the impact of integrons on diversity across all Pseudomonads. The history and significance of integrons is discussed in Chapter 1 as part of a literature review, and general materials and methods are provided in Chapter 2. Chapters 3 - 6 comprise the sections in which data generated during my PhD project are presented. A comprehensive analysis of the relationships between the strains being analysed is presented in Chapter 3. In Chapter 4, results of PCR and hybridisation screening for integrons across the strain collection are presented. In Chapter 5 the recovery of additional integrons and in depth sequence analysis of the recovered integrons are described. Finally, Chapter 6 contains statistical analyses of integron-associated genes and Chapter 7 contains a final discussion the most significant findings. Twenty-three Pseudomonas spp. strains were screened for the presence of integrons. All but three were found to contain integron-like sequences; however, most integron sequences recovered contained inactivated core integrons. viii Despite having a chromosomal locus, integrons in Pseudomonas were found to have properties indicative of frequent horizontal transfer. Evidence was also obtained which suggests that integrons have been acquired at the same locus on multiple independent occasions. This has not been observed in other families of chromosomal integrons and suggests that the loci at which integrons in Pseudomonas are found are hotspots for recombination. / Mode of access: World Wide Web. / xiii, 274 p. ill