Browsing by Author "Todd Klaenhammer, Committee Member"

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Carbohydrate Utilization Pathway Analysis in the Hyperthermophile Thermotoga maritima
(2006-03-01) Conners, Shannon Burns; Todd Klaenhammer, Committee Member; Robert Kelly, Committee Chair; Greg Gibson, Committee Member; Bruce Weir, Committee Member; Jason Osborne, Committee Member
Carbohydrate utilization and production pathways identified in Thermotoga species likely contribute to their ubiquity in hydrothermal environments. Many carbohydrate-active enzymes from Thermotoga maritima have been characterized biochemically; however, sugar uptake systems and regulatory mechanisms that control them have not been well defined. Transcriptional data from cDNA microarrays were examined using mixed effects statistical models to predict candidate sugar substrates for ABC (ATP-binding cassette) transporters in T. maritima. Genes encoding proteins previously annotated as oligopeptide/dipeptide ABC transporters responded transcriptionally to various carbohydrates. This finding was consistent with protein sequence comparisons that revealed closer relationships to archaeal sugar transporters than to bacterial peptide transporters. In many cases, glycosyl hydrolases, co-localized with these transporters, also responded to the same sugars. Putative transcriptional repressors of the LacI, XylR, and DeoR families were likely involved in regulating genomic units for beta-1,4-glucan, beta-1,3-glucan, beta-1,4-mannan, ribose, and rhamnose metabolism and transport. Carbohydrate utilization pathways in T. maritima may be related to ecological interactions within cell communities. Exopolysaccharide-based biofilms composed primarily of β-linked glucose, with small amounts of mannose and ribose, formed under certain conditions in both pure T. maritima cultures and mixed cultures of T. maritima and M. jannaschii. Further examination of transcriptional differences between biofilm-bound sessile cells and planktonic cells revealed differential expression of beta-glucan-specific degradation enzymes, even though maltose, an alpha-1,4 linked glucose disaccharide, was used as a growth substrate. Higher transcripts of genes encoding iron and sulfur compound transport, iron-sulfur cluster chaperones, and iron-sulfur cluster proteins suggest altered redox environments in biofilm cells. Further direct comparisons between cellobiose and maltose-grown cells suggested that transcription of cellobiose utilization genes is highly sensitive to the presence of cellobiose, or a cellobiose-maltose mixture. Increased transcripts of genes related to polysulfide reductases in cellobiose-grown cells and biofilm cells suggested that T. maritima cells in pure culture biofilms escaped hydrogen inhibition by preferentially reducing sulfur compounds, while cells in mixed culture biofilms form close associations with hydrogen-utilizing methanogens. In addition to probing issues related to the microbial physiology and ecology of T. maritima, this work illustrates the strategic use of DNA microarray-based transcriptional analysis for functional genomics studies.
Effects of Organic Acids and Atmosphere on the Survival of Escherichia coli O157:H7 Under Conditions Similar to Acidified Foods
(2009-12-03) Kreske, Audrey Christina; Fred Breidt, Committee Chair; Roger McFeeters, Committee Member; Todd Klaenhammer, Committee Member; Hosni Hassan, Committee Member
The ability of Escherichia coli O157:H7 to survive in acidified vegetable products is of concern because of previously documented outbreaks associated with fruit juices. A study was conducted to determine the survival of E. coli O157:H7 in organic acids at pH values typical of acidified vegetable products (pH 3.2 and 3.7), under different atmospheres, and a range of ionic strengths (0.086 to 1.14). Determination of internal pH and catalase activity in acid solutions can explain how dissolved oxygen or D- versus L-lactic acid reduces the survival of Escherichia coli O157:H7 when the organism is exposed to low pH (3.2) values typical of fermented and acidified foods. All solutions contained 20 mM gluconic acid, which was used as a non-inhibitory low pH buffer to compare the individual acid effect to that of pH alone on the survival of E. coli O157:H7. E. coli O157:H7 cells challenged in buffered solution with ca. 5 mg/L dissolved oxygen (present in tap water) over a range of ionic strengths at pH 3.2 exhibited a decrease in survival as the ionic strength was increased over 6 h. Overall, under oxygen limiting conditions in an anaerobic chamber, there is no significant difference in the loss of viability of E. coli O157:H7 cells regardless of pH, acid type, concentration, or ionic strength. For lactate concentrations up to 40 mM, there was no significant difference in internal pH values when cells were incubated in D- versus L-lactic acid solutions. Unexpectedly, cells incubated under aerobic conditions maintained a significantly higher internal pH (ca. 5.8) than cells incubated under anaerobic conditions (ca. 5.4), regardless of the isomer of lactic acid used. Overall, catalase activity was higher when cells were in the presence of L-lactic acid versus D-lactic acid however there is a decrease in survival when cells are incubated in L-lactic acid. Under the conditions tested, differences in survival of E. coli O157:H7 between isomers of lactic acid and atmospheres is not a result of internal pH or catalase activity. However, the lower internal pH maintained by cells incubated anaerobically (5 mM lactic acid) results in a smaller pH gradient and decreases the accumulation of acid anions inside the cell, which may contribute to increased survival under anaerobic conditions. Many acid and acidified foods are sold in hermetically sealed containers with oxygen limiting conditions. Our results demonstrate that E. coli O157:H7 may survive better than previously expected from studies with acid solutions containing dissolved oxygen.
Erythromycin Susceptibility and Genomic Regions Characterization of Campylobacter coli
(2007-11-01) Chan, Kamfai; Todd Klaenhammer, Committee Member; Fred Breidt, Committee Member; Sophia Kathariou, Committee Chair; Craig Altier, Committee Member
Campylobacter jejuni and Campylobacter coli are major bacterial food-borne pathogens that cause enteric diseases worldwide, resulting in significant public health and economic burden. These two closely related species colonize a wide range of farm animals including turkeys, pigs, chickens, and cattle. Consumption of meat (especially poultry) contaminated with C. jejuni or C. coli has been implicated as the major route of infection. When needed, antibiotics used for treatment are fluoroquinolones or macrolides such as erythromycin. Recent studies have shown that the percentage of C. coli that have resistance to antimicrobials, including those typically used for treatment of human campylobacteriosis, has increased, making it a top priority to investigate the mechanisms for acquisition and dissemination of antimicrobial resistance in these pathogens. Certain Campylobacter strains harbor a transcribed intervening sequence (IVS) in their 23S rRNA genes. Following transcription, the IVS is excised, leading to fragmentation of the 23S rRNA. The origin and possible functions of the IVS are unknown. Furthermore, the distribution of IVS-harboring strains within Campylobacter populations is poorly understood. In this study, strains of Campylobacter coli from turkeys, representing numerous different multilocus sequence typing (MLST)-based sequence types (STs), were characterized in terms of IVS content and erythromycin susceptibility. We identified strains that harbored IVSs in all three 23S rRNA genes, as well as strains lacked IVSs from at least one of the genes. The STs of the latter strains belonged to an unusual cluster of C. coli STs ('cluster II'), earlier found primarily in turkey strains, and characterized by presence of the C. jejuni aspA103 allele. The majority of strains harboring IVSs in all three 23S rRNA genes were resistant to erythromycin. In contrast, cluster II strains, which harbored at least one IVS-free 23rRNA gene, were susceptible to the antibiotic. Cluster II strains could be transformed to erythromycin resistance with genomic DNA from C. coli that harbored IVS and the A2075G transition in the 23S rRNA gene, associated with resistance to erythromycin in Campylobacter. Erythromycin-resistant transformants harbored both the A2075 transition and IVS. The findings suggest that absence of IVS in C. coli from turkeys is characteristic of a unique clonal group of erythromycin-susceptible strains, and that IVS can be acquired by these strains via natural transformation to erythromycin resistance. Analysis of C. coli and C. jejuni strains isolated from broilers, turkeys and swine has shown the association of the lack of IVS and erythromycin susceptibility is unique to C. coli from turkeys. The presence of the C. jejuni aspA103 allele in the chromosome of cluster II C. coli strains is a unique characteristic of this clonal group. To further characterize the genome composition in the aspA region in cluster II strains, we determined the corresponding DNA sequences from two turkey-derived cluster II C. coli strains (6979 and 7474, of ST-1150 and ST-1161, respectively). Genomic organization upstream of the aspA gene was divergent between these two cluster II strains and the reference strain C. coli RM2228, the genome sequence of which has been completed. Genes encoding a putative Crp-family transcriptional regulator (CCO0137) and a conserved hypothetical protein (CCO0138) that were present in C. coli RM2228 and C. coli 6818 were not found in the same genomic region in C. coli 6979 or C. coli 7474. Moreover, single nucleotide polymorphism (SNP) analysis revealed that genes encoding subunit II of cytochrome d ubiquinol oxidase (cydB) and a putative aspartate racemase (CJ0085c) harbored numerous C. jejuni-specific SNPs. Interestingly, genes encoding subunit I of cytochrome d ubiquinol oxidase (cydA) and uracil-DNA glycosylase (ung) harbored C. coli-specific SNPs in their 5' portions but C. jejuni-specific SNPs in their 3' portions, suggesting that these may be hybrid genes that were originated from C. jejuni and C. coli. Our data suggest the presence of recombination events in the genomic region between cydA and ung in cluster II strains of C. coli. Such genomic features may contribute to the observed prevalence of cluster II strains among C. coli from turkeys, and to the characteristic susceptibility to erythromycin exhibited by these strains.
Redox Potential Trends of Cucumber Fermentation as Influenced by Microbial Growth and Gas Purging
(2008-12-05) Olsen, Maegan Jessie; Ilenys M. PÃƒÂ©rez-DÃƒÂaz, Committee Chair; Roger McFeeters, Committee Member; Todd Klaenhammer, Committee Member
Determining the redox potential of a system can be a useful tool in evaluating the thermodynamic changes that occur over a period of time. Currently, fermentation industries use pH measurements to assess the progress of fermentation and growth of lactic acid bacteria (LAB). Redox potential (Eh) measures the transfer of electrons within a system, rather than solely the concentration of hydrogen ions, as measured by pH. Continuous monitoring of fermentation by Eh may allow for more descriptive analysis of the metabolic process and could offer a method for the earlier prediction of spoilage by yeasts. The trends in redox potential of fermenting cucumbers were observed to evaluate the possible application of this parameter in monitoring the development of fermentation. Additionally, an evaluation of the effect of gas purging on microbial growth during the fermentation was conducted using redox potential trends as a monitoring tool. Cucumbers were packed and brined using sodium chloride, calcium chloride, and acetic acid and the redox potential monitored during fermentation. Examination of both pasteurized-inoculated jars and non-pasteurized jars was conducted in this study. In addition, changes in redox potential were measured in fermentations that were purged with nitrogen, oxygen, hydrogen, or left untreated. Brine samples were analyzed for microbial counts, pH and substrates and products of fermentation. Under conditions representative of a standard cucumber fermentation, a dramatic decrease in the redox potential was observed during the first day, concomitantly with the initiation of the log phase of bacterial growth. However, in the presence of spoilage yeast, redox potential remained low over this initial period. The progress of a fermentation sparged with nitrogen, oxygen, or no treatment produced similar Eh trends. The Eh trend was more reduced under hydrogen sparging. A heightened growth rate for LAB, more efficient production of lactic acid, and lower production of ethanol occurred with nitrogen sparging. The evidence suggests that the growth of yeasts in a cucumber fermentation could be detected based upon differences in the redox potential. Distinct variations in Eh were still recorded after pH values decreased and remained constant, suggesting the valuable possible application of Eh to monitor industrial cucumber fermentations. Additionally, changes in redox potential were affected by gases introduced during cucumber fermentations and sparging with nitrogen could have substantial benefits to the industry.
Transcriptome Analysis of Saccharomyces cerevisiae under Bacterial Contamination and Lactic Acid Stress during Ethanolic Fermentation.
(2010-10-22) Ivey, Melissa; Trevor Phister, Committee Chair; Ilenys Perez Diaz, Committee Member; Todd Klaenhammer, Committee Member