Browsing by Author "Clark, Stewart James"

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Denaturing gradient gel electrophoresis (DGGE) can rapidly display the bacterial diversity contained in 16S rDNA clone libraries
(2006-04) Burr, Mark D.; Clark, Stewart James; Camper, Anne K.
Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.
Keys to unlocking the biofilm phenotype of virulent environmental isolate of Salmonella
(Montana State University - Bozeman, College of Letters & Science, 2008) Clark, Stewart James; Chairperson, Graduate Committee: Anne Camper
The aim of this research was to elucidate the phenotypic adaptation of an environmental isolate of Salmonella enterica grown in a single species biofilm using transcriptomic analysis. This environmental isolate was obtained from an outbreak in Gideon, MO, and was classified as Salmonella enterica serotype Missouri. Gene expression profiles obtained from this environmental isolate were compared with profiles of the ATCC type strain Salmonella enterica serotype Typhimurium LT2 grown under the same conditions. It was shown that there were distinct transcriptional differences in both of the strains between the biofilm and planktonic phenotypes. Both strains exhibited the strong up-regulation of several gene pathways that were unique to the biofilm phenotype. These included genes responsible for the cobalamin-dependent anaerobic utilization of 1,2-propanediol (cob-cbi-pdu), type III secretion system apparatus and effector proteins located on Salmonella Pathogenicity Island 2 (SPI-2) and the well characterized csg operon largely responsible for biofilm formation in Salmonella. A significant proportion of the genes present on the virulence plasmid PSLT were shown to be exclusively up-regulated in the biofilm phenotype of Salmonella Typhimurium LT2, illustrating the tendency of this pathogen to exhibit a promiscuous lifestyle whilst in the non-host environment. It was further demonstrated that the environmental isolate exhibited a more tenacious biofilm-forming tendency and overall greater survivability than the type strain in a low nutrient, non-host environment. It appeared from the transcriptional profile of Salmonella Typhimurium LT2 during planktonic growth that the organism struggled to adapt and survive under low nutrient conditions as evidenced by the increased expression of ribosomal subunit operons rps and rpl and several stress-related genes including dnaK and htp. The conclusion may be drawn that Salmonella Missouri has developed several key systems differentiating the biofilm and planktonic phenotypes and affording it a competitive advantage. While some of these traits have previously been studied exclusively in the context of host pathogenicity, this research indicates that perhaps these so-called virulence strategies may afford the pathogen enhanced survival in non-host environments as well. Therefore, these findings suggest that the use of excessively sub-cultured laboratory strains may be inappropriate surrogates for studying the behavior of real-world pathogens.