Polyphasic Taxonomy: A Tool to Identify and Characterize Members of Complex Microbial Communities

Johnson, Crystal

Polyphasic Taxonomy: A Tool to Identify and Characterize Members of Complex Microbial Communities

dc.contributor.advisor	Lawson, Paul
dc.contributor.author	Johnson, Crystal
dc.contributor.committeeMember	Tanner, Ralph
dc.contributor.committeeMember	Broughton, Richard
dc.contributor.committeeMember	Wawrik, Boris
dc.contributor.committeeMember	Stevenson, Bradley
dc.date.accessioned	2014-12-11T20:24:23Z
dc.date.available	2014-12-11T20:24:23Z
dc.date.issued	2014-12
dc.date.manuscript	2014-12
dc.description.abstract	Systematics is a fundamental discipline that underpins the science of microbiology, providing a framework that allows for the identification, classification, taxonomy, and nomenclature of single cell organisms. Since the second half of the twentieth century, systematics has made revolutionary insights into microbial relationships, revealing the vast diversity on planet Earth. Culture-independent methods based on the small subunit of the ribosomal RNA (16S rRNA) gene have since been developing at an ever-accelerating rate. With what are now considered common technologies, Sanger sequencing, Pyrosequencing, and Next Generation sequencing have revealed that the majority of microbial life remains yet to be cultured in the laboratory. Consequently, taxonomists have realized the enormity of cataloguing this diversity and have developed a polyphasic methodology for the description and classification of new taxa. Although molecular methods are freely available to researchers for identification purposes, novel organisms cannot be described in the scientific literature purely on sequence information alone. Instead, the application of a polyphasic taxonomic approach emphasizes the use of classical methods married with modern genetic fingerprinting. Traditional techniques include morphological and biochemical descriptions, as well as chemotaxonomic features that encompass cell wall, polar lipid, fatty acid, and respiratory menoquinones. These important diagnostic biomarkers aid in the general assignment of isolates to the correct taxa. Specifically, chemotaxonomic characteristics are especially useful in reflecting phylogenies at the genus/family level. Modern techniques use the 16S rRNA gene amplified by the polymerase chain reaction (PCR) as a useful tool for comparing evolutionary distances. Acting as a molecular time chronometer, variable and conserved regions can then be mathematically assessed by comparing multiple sequence alignments and viewed as phylogenetic trees. But these taxonomic classifications do not necessarily define the expected physiological traits since closely related organisms isolated from different locations can have very distinct physiologies and metabolic processes. Therefore, laboratory investigations with these isolates and comparisons to reference strains of closely related organisms remain essential. When investigating any complex ecosystem, cultivation methods include the use of enrichment media that cast a nutritionally wide net through the use of various substrate and energy source amendments. Diverse conditions can be established by altering incubation temperatures, pH values, headspace compositions (N2, H2, and CO2 gas) as well as internal vessel pressure during incubation. Some information on the types of organisms present within an environment may be derived from 16S gene sequence molecular inventories, and therefore microbial groups of interest may be specially targeted with particular media amendments and selective growth conditions. Isolates can then be obtained by subculturing single colonies from plates and roll tubes, as well as through the use of dilutions to isolate dominant organisms. While rRNA gene-based phylogenies have revolutionized microbial taxonomy, they continue to be reinforced by chemotaxonomic and biochemical considerations. A combination of bacterial isolation and cultivation, descriptive classical techniques, and modern molecular sequencing methods has thus resulted in the comprehensive classification of new taxa. The laboratory studies presented in this dissertation used these evolved taxonomic tools to validate the discovery of a number of novel, or previously uncultured, isolates from complex ecosystems. Chapters 1 and 2 describe the isolation of a novel bacterial species with family reclassification and the isolation of a novel genus, respectively. Pure cultures, obtained from swine manure storage pits, were found to represent previously uncultivated taxa. Physiological, biochemical, and genetic features were investigated in order to determine phylogeny and demonstrate uniqueness. One organism, strain SF-S1T, was related to members of the genus Peptoniphilus which was accommodated within a new family, Peptoniphilaceae fam. nov. A second organism, strain Con12T, was classified as a novel genus and species, Savagea faecisuis gen. nov., sp. nov. Additionally, a new species from an Alaskan oil production pipeline was isolated in pure culture and characterized physiologically in order to investigate its role in the environment. This work stemmed from a collaborative effort within the OU Biocorrosion Center that focused on the molecular -dependent and -independent detection of microbial populations and their activities associated with biocorrosion. As such, the goal of the cultivation component presented here was to explore the microbiology of pipeline effluent for previously unidentified cultivars. Phylogenetic analysis based on 16S rRNA gene sequences from this isolate indicated that strain PE-10T belonged within the genus Proteiniphilum, most closely to Proteiniphilum acetatigenes (94% similarity). Chapter 3 provides evidence that PE-10 T represents only the second cultivated species of this genus. Pyrosequencing and qPCR data suggested that Proteiniphilum-like organisms comprised 11% of the population of this particular sample. Furthermore, incubation experiments with carbon steel suggested that PE-10T might be involved in biofilm production. The isolation of Proteiniphilum alaskensis strain PE-10T represented the cultivation of an abundant molecular phylotype, therefore providing a tangible microbe with which to conduct corrosion experiments. Towards this effort, Chapter 4 provides an investigation of parameters useful in the assessment of Microbially Influenced Corrosion (MIC). The objectives were two-fold: first, to evaluate various ways to measure MIC, and second, to demonstrate these methods by measuring the differential corrosion activity between two model organisms. Accomplishment of these efforts helped structure the tools necessary for evaluating the nature of novel taxa found to be associated with pipeline corrosion. In this study, biofilm characteristics, metal dissolution, and the damage to steel surfaces were analyzed in order to demonstrate the biocorrosive potential of bacteria. Desulfovibrio indonesiensis and Desulfovibrio alaskensis, chosen because they are sulfate reducing bacteria (SRB) associated with oil production systems, were shown to interact differently with carbon steel as measured with various Scanning Electron Microscopy (SEM) techniques, iron analysis, and surface profilometry. The study presented here used a series of measurements to directly characterize the corrosive capabilities of microbial assemblages. Ultimately, these parameters will be essential for determining the risk of resident bacteria as well as for assessing the efficacy of current corrosion detection and mitigation strategies. In the appendix, descriptions of two additional novel species are included. Here, previously uncultivated bacterial isolates were obtained from the water of a saltern in Santa Pola, Alicante, Spain and from the subgingival plaque of a canine Labrador Retriever. These species have been validated and appear in the formats of The International Journal for Systematic Evolutionary Microbiology (IJSEM) and Anaerobe, respectively. The breadth of taxonomic discoveries has never been more realized, and remains an encouragement for scientists to continue to identify and characterize members of complex microbial communities.	en_US
dc.identifier.uri	http://hdl.handle.net/11244/13869
dc.language	en_US	en_US
dc.subject	Taxonomy	en_US
dc.subject	Anaerobic Microbiology	en_US
dc.subject	Systematics	en_US
dc.subject	Biocorrosion	en_US
dc.thesis.degree	Ph.D.	en_US
dc.title	Polyphasic Taxonomy: A Tool to Identify and Characterize Members of Complex Microbial Communities	en_US
ou.group	College of Arts and Sciences::Department of Microbiology and Plant Biology	en_US
shareok.orcid	0000-0003-1044-5241	en_US