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During entry into stationary phase, Escherichia coli expresses many genes which impart cellular resistance to numerous environmental stresses such as oxidative or acid stress. Many ofthese genes are regulated by the alternative sigma factor, RpoS To identify additional genes regulated by RpoS, a phenotype independent genetic screen was previously employed (L. Wei Masters thesis). The identities ofthe ten most highly RpoSdependent fusions were determined by DNA sequencing and subsequent sequence analysis using the BLAST algorithm Three fusions map to genes previously known to be RpoSdependent while the remaining seven represent new members ofthe regulon The expression of many ofthe RpoS-dependent fusions remained growth phase dependent even in the rpoS background This suggests that other growth phase regulatory factors in addition to RpoS may coordinate stationary phase gene expression Upon sequencing the remaining rsd fusions, three mutants mapped to narY which is part ofthe narZYWV operon encoding the secondary nitrate reductase Z (NRZ) This operon was selected for further investigation since NRZ has been previously reported to be constitutively expressed Expression studies using promoter lacZ fusions and nitrate reductase assays reveal that NRZ is induced ten-fold at the onset ofstationary phase and twenty-fold in the presence of nitrate Like other rsd fusions, growth phase dependent expression was observed in an rpoS background indicating that other regulatory factors may be involvedn the regulation of NRZ Northern analyses using probes specific to NRZ confirmed that transcription of NRZ is indeed dependent on RpoS. These results suggest that RpoS mediated regulation of NRZ may be an important physiological adaptation to reduced oxygen levels during transition to stationary phase / Thesis / Master of Science (MSc)

BiophotonicE. coli O157:H7(BEC) was evaluated for growth and stability to evaluate the effectiveness of BEC as a tool to validate pathogen reduction interventions. Escherichia coliO157:H7 containing Xen14 and pAK1 lux plasmids were monitored for growth and stability over 10 d at 2.2˚C and 37˚C. Concentration of BECat 2.2˚C remained constant (P> 0.05) but photonic emissions (RLU/s) changed (P<0.05), resulting in a positive correlation between RLU/s and CFU/mL (P< 0.01; r=0.5718). BEC grown at 37˚C yielded no change after a decrease on day 2 (P>0.05). In phase I, purge was monitored for photonic emission after trim was inoculated with BEC. BECwas found at 4 h in Combo C and D. Phase II purge collection used inoculated trim indicative of trim used currently in the beef industry stored in simulated combos. Neither photonic emission nor CFU’s were enumerated in phase II.

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In this study, we report that expression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon is induced in stationary phase cultures and that this increase is largely dependent on RpoS, the alternative stress sigma factor. Using combinatorial 𝘢𝘳𝘨𝘙 and 𝘳𝘱𝘰𝘚 mutants, we evaluated the relative contributions of these two regulators to the expression of 𝘢𝘳𝘨𝘏 using operon 𝘭𝘢𝘤𝘡 fusions. While ArgR was found to be the main factor responsible for de-repression of the 𝘢𝘳𝘨𝘊𝘉𝘏 operon, RpoS was required for full expression of this biosynthetic operon at concentrations below 10 μg arginine ml⁻¹, a level at which growth of an arginine auxotroph was arginine limited. At high arginine concentrations (>10 μg ml⁻¹) 𝘢𝘳𝘨𝘊𝘉𝘏 expression was strongly repressed as expected by ArgR. 𝘢𝘳𝘨𝘊𝘉𝘏 expression was 30 fold higher in Δ𝘢𝘳𝘨𝘙 mutants relative to a wild type fully repressed strain and this expression was independent of RpoS. These results indicate that RpoS plays an important role in the regulation of arginine biosynthesis, particularly when the operon is partially de-repressed as would be the case in starvation conditions. / Thesis / Master of Science (MS)

The Ad5 E1B antigens of MW 58000 and 19000 are known to be involved in oncogenic transformation of mammalian cells. To obtain sufficient quantities of these proteins for biochemical studies on their mechanism of action, I have attempted to express the Ad5 E1B genes in Escherichia coli. Using the strategy developed by Guarente et al. (1980), I have constructed plasmids which have the trancriptional and translational controls of the E. coli lac operon linked 5' of the 19k and 58k coding sequences. One plasmid was shown to synthesize high levels of a stable, immunoreactive 19k analogue consisting of 19k with 29 adenovirus-coded ribosome-binding sequence is functional in directing translation of this protein. Synthesis of 58k was not demonstrated, perhaps the result of [protein instability in E. coli. However, immunoreactive proteins which may correspond to the amino terminal region of 58k were demonstrated. / Thesis / Master of Science (MS)

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