In Escherichia coli, the integral outer membrane (OM) enzyme PagP covalently modifies lipid A by incorporating a phospholipid-derived palmitate chain to fortify the OM permeability barrier. We perturbed the bacterial OM in order to activate PagP and examined if it exerts transcriptional regulation through either of its extracellular or periplasmic active sites. Data from RNA-seq revealed the differential expression of 50 genes upon comparing the E. coli imp4213 (lptD4213) strain NR760∆pagPλInChpagP (shortened to NR760λp in this work), in which PagP is constitutively activated, and the mutant NR760λpY87F carrying the periplasmic residue mutation Y87F. 40 genes were upregulated, and encoded proteins related to anaerobic processes, whereas 10 genes were downregulated, and encoded proteins related to aerobic processes. RNA-seq was followed by a study of differential gene expression using the NanoString nCounter system. Results confirmed a 2.7-fold upregulation of dmsA when we compared the strains NR760λp to NR760λpY87F. We also found a 2.5-fold repression of dmsA transcription when we compared the lptD+ parental strain NR754λp to NR754λpS77A carrying the mutation S77A in the extracellular active site of PagP. We then investigated dmsA transcription using a lacZ reporter gene in plasmid pRS551-lacZ. High basal β-galactosidase activity became attenuated in pagP null mutants. OM perturbation using pentamidine showed that dmsA transcription was repressed. Complementation of the chromosomal ∆pagP deletion with a single copy pBADGr plasmid, expressing PagP under the control of an arabinose-inducible promoter, restored dmsA β-galactosidase activity. We observed partial complementation with the downstream cspE gene, which identified a polar effect of the ∆pagP allele. Through deletion of rpoS, rcsB, cpxA, cpxR, pmrA, pmrB, and fadD in E. coli MC4100, we showed that β-galactosidase activity of pdmsA-lacZ was affected by all of these regulators. Our results indicate that these regulators are involved in PagP-mediated regulation of dmsA transcription under aerobic conditions. / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/24064 |
| Date | January 2018 |
| Creators | Maldonado Alvarez, Liset |
| Contributors | Bishop, Russell, Chemistry and Chemical Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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