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dc.contributor.authorBuchan, Alison
dc.description.abstractThe degradation of lignin-related aromatic compounds is an important ecological process in the highly productive salt marshes of the southeastern U. S., yet little is known of the mediating organisms or their catabolic pathways. An initial study of six isolates suggested that members of the roseobacter lineage, a dominant marine clade within the ?- Proteobacteria, could degrade aromatic compounds via the ?-ketoadipate pathway, a catabolic route that has been well characterized in soil microbes. Four of the roseobacter group isolates had inducible protocatechuate 3,4-dioxygenase activity, a key enzyme in the pathway, in cell-free extracts when grown on p-hydroxybenzoate. The pcaHG genes encoding this ring-cleavage enzyme were cloned and sequenced from two isolates and in both cases the genes could be expressed in E. coli to yield dioxygenase activity. Evidence of genes encoding for protocatechuate 3,4-dioxygenase was found in all six roseobacter isolates by detection of pcaH by Southern hybridization or PCR amplification. These results suggested this ecologically important marine lineage compose a significant fraction of the aromatic compound degrading community in coastal systems. To test this hypothesis we investigated the diversity of pcaH amplified from bacterial communities associated with decaying Spartina alterniflora, the salt marsh grass dominating these coastal systems, as well as from enrichment cultures with aromatic substrates. Sequence analysis of 149 pcaH clones revealed 85 unique sequences. Fifty-eight percent of the clones matched sequences amplified from a collection of 36 bacterial isolates obtained from seawater or from senescent Spartina. Fifty-two percent of the pcaH clones could be assigned to the roseobacter group. Another 6% matched genes retrieved from non-roseobacter isolates cultured from decaying Spartina and 42% could not be assigned to a cultured bacterium based on sequence identity. These findings revealed a high diversity of genes encoding a single step in aromatic compound degradation in this coastal marsh and that many of the genes were indeed harbored by members of the roseobacter lineage. In a final study, we explored the genetic diversity of the ?-ketoadipate pathway among eight members of the roseobacter lineage (pairwise sequence identities of the 16S rDNA gene ranged from 92 to 99%). Genomic fragments containing gene clusters of this pathway were isolated and characterized by targeting pcaH. Sequence analysis revealed five unique gene arrangements. Identical gene clusters were found mostly between isolates demonstrating species-level identity (i.e. >99% similarity of 16S rDNA). In one isolate, six functionally related genes were identified: pcaQ, pobA, pcaD, -C, -H, and -G. The remaining seven isolates lacked at least one of these six genes within their respective gene clusters, however, gene order was consistent with this isolate s orientation. These results portray the dynamic nature of a set of genes that potentially play a central catabolic role in coastal marine environments.
dc.subjectaromatic compound degradationmarine bacteriaprotocatechuate dioxygenasemicrobial ecology
dc.titleEcology and genetics of aromatic compound degradtion in the ecologically important roseobacter lineage of marine bacteria
dc.description.departmentSchool of Marine Programs
dc.description.majorMarine Sciences
dc.description.advisorMary Ann Moran
dc.description.committeeMary Ann Moran
dc.description.committeeEllen Neidle
dc.description.committeeJames T. Hollibaugh
dc.description.committeeBrian Binder
dc.description.committeeWilliam Whitman

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