Drug efflux represents an important protection mechanism against antibiotics
and environmental toxic compounds in bacteria. Efflux genes constitute from 6%
to 18% of all transporters in bacterial genomes, yet their regulation, natural
substrates, and physiological functions are poorly understood. Among the 20
chromosomally encoded efflux genes in Escherichia coli K-12, only the
AcrAB-TolC efflux system is constitutively expressed under the ordinary
laboratory growth of E. coli. To explore conditions and circumstances that trigger
the expression of additional efflux genes as well as their physiological functions, I
examined the expression of all 20 efflux genes under a physiologically relevant
circumstance for E. coli, which is anaerobic condition in this study. I found that
expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold
when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed
that the anaerobically induced expression of mdtEF is subject to the regulation of
the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance
assay showed that anaerobically grown E. coli cells display an increased efflux
activity and enhanced drug tolerance in an MdtEF dependent manner, confirming
the functional up-regulation of the efflux pump MdtEF in the anaerobic
physiology of E. coli.
Since the up-regulation of mdtEF by anaerobic growth occurs in the absence
of antibiotics and drugs, I speculate that MdtEF has physiological functions under
the anaerobic growth of E. coli. To explore this, I first compared the viability of
ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell
survival during prolonged anaerobic growth of E. coli. Interestingly, this defect
became more pronounced when cells grow in the presence of 10 mM nitrate, but
no defect was observed in ΔmdtEF strain when cells grow in the presence of 40
mM fumarate under the same anaerobic conditions, suggesting that MdtEF has
physiological roles relevant to the anaerobic respiration of nitrate. I further found
that E. coli cells harboring the deletion of mdtEF are susceptible to indole
nitrosative derivatives, a class of toxic by-products formed and accumulated
within E. coli when the bacterium respires nitrate under anaerobic conditions, and
deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores
the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate.
Taken together, I conclude that the multidrug efflux pump MdtEF expels the
nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since
the production and accumulation of nitrosyl indole derivatives is ascribed to the
reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the
up-regulated multidrug efflux pump MdtEF functions to protect E. coli from
nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/182276 |
| Date | January 2012 |
| Creators | Zhang, Yiliang, 张毅良 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B4775266X |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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