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Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008. / Committee Chair: Jordan, I. King; Committee Member: Borodovsky, Mark; Committee Member: Bunimovich, Leonid; Committee Member: Choi, Jung; Committee Member: McDonald, John.

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.

Thesis (Ph.D.) -- University of Montana, 2008. / Title from author supplied metadata. Description based on contents viewed on June 26, 2009. Includes bibliographical references.

Embryonic chromatin diminution, the selective excision of large amounts of heterochromatic DNA from presomatic cell lineages, provides an example of an unusually large augmentation of the germline genome and raises questions regarding the source of the increased amount of DNA and its relevance to the biology of the organism. DNA levels in adult germ cell nuclei of the copepod Cyclops kolensis were determined by DNA-Feulgen cytophotometry and compared with those of somatic nuclei of adults and both pre- and postdiminuted embryos from the same mothers. Almost 75 pg DNA/nucleus is excised by diminution, resulting in the return of each generation to the approximately 1 pg DNA/nucleus level found for adult soma. To account for the increase in DNA levels of germ cells observed here, we propose alternative hypotheses to the original model of chromatin diminution: (1) repetitive endocycles or (2) proliferation of genetic elements. Specific tests for these hypotheses using next-generation sequencing and quantitative cytophotometry, as well as the functional significance of germ cell DNA augmentation to the copepod, are discussed.

The gene encoding resistance to β-lactam antibiotics in the staphylococci is found on the chromosome in a genomic island designated Staphylococcal Chromosome Cassette mec or SCCmec. In addition to the resistance gene, mecA, SCCmec also contains site specific recombinase genes, ccrAB, that are capable of catalyzing the chromosomal excision and integration of SCCmec. The increasing prevalence of methicillin-resistant Staphylococcus aureus infections may be due, in part, to the transfer of SCCmec into successful methicillin-sensitive S. aureus lineages. In this work we attempt to better characterize the factors associated with SCCmec transfer, beginning with CcrAB-mediated SCCmec excision in a collection of MRSA containing type IV SCCmec. CcrAB-mediated excision of type IV SCCmec was not demonstrated for all strains tested, as a subset of S. aureus strains with type IV SCCmed did not excise their element. These strains are all highly related and represent a lineage of successful community associated pathogens. In addition, the inability to excise SCCmec in these strains is associated with the presence of a presumptive mobile element containing the gene for staphylococcal enterotoxin H (seh) immediately outside of SCCmec on the chromosome. Staphylococcus aureus strain USA300 contains SCCmec type IV and a genomic island containing an arginine deiminase pathway, known as ACME, inserted adjacent to one another in the SCCmec chromosomal attachment site. Each element was site-specifically excised from the chromosome by CcrAB, resulting in two independent, extra-chromosomal, circularized elements. Therefore the presence of ACME did not disrupt SCCmec excision.Next, we describe three MRSA strains that become resistant to vancomyein during passage on increasing concentrations of the drug. In each case, mecA was lost during passage. Strain 5836VR lost mecA by the site-specific chromosomal excision of SCCmec while the other two strains (3130VR and VP32) deleted portions of their SCCmec elements in a manner that appears to involve IS431. Conversion to vancomycin resistance caused a decrease in growth rate that was partially compensated for by deletion of mecA. In mixed culture competition experiements, vancomycin resistant strains that lacked mecA readily out-competed their mecA-containing counterparts, suggesting that the loss of mecA during conversion to vancomycin resistance was advantageous to the organism.Finally, we examined the genetic structure surrounding the SCCmec attachment site in a diverse collection of methicillin-sensitive S. aureus isolates. This region of the chromosome varies greatly from strain to strain and appaers to be prone to recomination. Open reading frames found in this region were homologous to enterotoxins, restriction-modification enzymes, and transposases. Several open reading frames that have not been previously reported in staphylococci were also present in this region. 28 out of the 42 isolates examined did not contain the attachment site sequence found in S. aureus isolates known to be capable of CcrAB-mediated SCCmec integration or excision. This may indicate that these strains do not contain a functional attachment site and therefore may not have the potential to acquire SCCmec by CcrAB-mediated recombination.