Demonstration of novel enoyl-acyl carrier protein reductases in Vibrio cholerae

One of the growing interests in bacterial fatty acid biosynthesis is due to the fact that an increasing number of both synthetic and natural antibacterial compounds specifically target fatty acid biosynthesis enzymes. However, the investigations this approach has engendered have uncovered diversity in this relatively conserved pathway. The enoyl-acyl carrier protein (ACP) reductase catalyses the last step in the elongation cycle of fatty acids. It is not only a key enzyme in the pathway, but also the most diverse enzyme across bacterial species and the target of many antimicrobial agents. Despite this diversity, advanced bioinformatics tools failed to detect any of the known enoyl-ACP reductase (ENR) isoforms in Vibrio cholerae. This dissertation describes the discovery of two novel ENR isozymes by functional complementation of an Escherichia coli fabI(Ts) mutant strain with a V. cholerae genomic library. We first uncovered and named FabV, a new class of triclosan-resistant enoyl-ACP reductases. In vivo and in vitro analysis of the purified protein showed that FabV is an NADH-dependent ENR, and belongs to the same superfamily as FabI and FabL, two of the three previously characterized ENRs. Unlike the Escherichia coli ENR (FabI), FabV is intrinsically resistant to triclosan, the common household antibacterial agent and expression of FabV renders its host resistant to triclosan. Interestingly, we found that FabV is present in a number of important Gram-negative pathogens. Building on our work, the FabV ENR activity was subsequently confirmed by other groups in two important pathogens: Burkholderia mallei and Pseudomonas aeruginosa (Lu and Tonge 2010; Zhu, Lin et al. 2010). However, fabV was not the only ENR expressed in Vibrio cholerae. Screening of another cosmid library with higher genome coverage identified yet another gene able to restore de novo fatty acid biosynthesis in the ENR mutant strain. The product of this gene called FabS is a novel NADPH-dependent triclosan-sensitive ENR. Unlike FabV, FabS does not belong to any of the known superfamilies of proteins associated with known ENRs (short chain dehydrogenase reductase, SDR or nitropropane dioxygenase, NPD). Interestingly, the analysis of the conserved domains present in the FabS protein sequence links FabS to NADH-quinone reductases. Comparing the presence of conserved domains present in all known ENRs (Marchler-Bauer, Lu et al. 2011) suggests that the ENR activity may result from convergent evolution involving protein domain shuffling. We conclude with preliminary data suggesting the existence of other active ENRs in Vibrio cholerae, uncovering an even greater diversity. The potential physiological relevance of such diversity in this essential enzyme is also discussed.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Microbiology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2011

Author dc:creator

• Massengo-Tiasse, Romelle Prisca

Contributors dc:contributor

• Cronan, John E.
• Salyers, Abigail A.
• Gardner, Jeffrey F.
• Miller, Charles G.

• Enoyl-acyl carrier protein reductase
• vibrio cholerae
• fatty acid biosynthesis
• triclosan

Statement dc:rights

• Copyright 2011 Romelle Prisca Massengo-Tiasse

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/24519

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/24519