Regulation of fatty acid synthesis in enterococcus faecalis

Fatty acid biosynthesis (FAB) is an essential cellular pathway for providing precursors for synthesis of membrane phospholipids and other acylated molecules. Acyl carrier proteins (ACPs) play a key role in these pathways. The Gram-positive pathogen Enterococcus faecalis encodes two ACPs named AcpA and AcpB. The acpA gene is within the fab gene cluster which encodes most of the enzymes of fatty acid biosynthesis while the acpB gene is located near the plsX gene that encodes the phosphate acyltransferase. In vitro reactions showed that both AcpA and AcpB were functional acyl carrier proteins. Further study through ACP malonylation and acyl transferring reactions proved that AcpA is responsible for de novo fatty acid biosynthesis while AcpB plays a key role in incorporation of exogenous fatty acids. The constructed ∆acpA strain showed growth deficiency whereas the ∆acpB strain grew similarly to wild-type strain. Further purification of ACPs from E. faecalis wild-type strain (FA2-2) verified that both AcpA and AcpB were functionally expressed in vivo and the level of AcpA protein decreased tremendously in the presence of oleic acid. This effect was lost in ∆fabT strain lacking the MarR transcription repressor FabT. AcpA could play a role in incorporation of exogenous fatty acids when the acpB gene is deleted. The phospholipid acyl chains of Enterococcus faecalis can be derived either by de novo synthesis or by incorporation of exogenous fatty acids through the fatty acid kinase complex (Fak)-phosphate acyltransferase (PlsX) pathway. Exogenous fatty acids suppress fatty acid synthesis through the transcriptional repressor FabT, the loss of which eliminated regulation of de novo fatty acid biosynthesis and resulted in decreased incorporation of exogenous unsaturated fatty acids. Purified FabT bound to the promoters of several fatty acid synthesis genes that contain a specific palindromic sequence and binding was enhanced by acylated derivatives of acyl carrier protein B (acyl-AcpB). The loss of the PlsX pathway blocked FabT-dependent transcriptional repression in the presence of oleic acid. Transcriptional repression was partially retained in the E. faecalis ∆acpB strain which showed decreased fatty acid biosynthesis in the presence of exogenous unsaturated fatty acids. The fact that FabT-dependent activity remained in the ∆acpB strain indicates that acylated derivatives of AcpA were weak enhancers of FabT binding although AcpA is believed to primarily function in de novo fatty acid synthesis. Acyl-acyl carrier protein (ACP) phosphate acyltransferase PlsX plays an important role in phospholipid synthesis and exogenous fatty acid incorporation in Enterococcus faecalis. Loss of plsX seriously blocked bacterial growth and led to auxotrophy of phospholipid fatty acyl chains, which could not be relieved by further introduction of a ∆fabT mutation. The ∆plsX strain accumulated suppressor mutants. One of these encoded a truncated β-ketoacyl-acyl carrier protein synthase II (FabO) which restored normal growth and recovered de novo phospholipid acyl chain synthesis by increasing saturated acyl-ACP synthesis. This restoration was also achieved through overexpression of an E. faecalis enoyl-ACP reductase either FabK or FabI which results in high levels of saturated fatty acid synthesis. A thioesterase encoded by tesE gene plays a key role in cleavage of acyl-ACP to provide free fatty acids for fatty acid kinase (FakAB)-catalyzed conversion to acyl-phosphates essential in initiation of phospholipid synthesis. High level production of saturated acyl-ACPs is required to offset the marked preference of the TesE thioesterase for unsaturated acyl-ACPs, indicating the preference of saturated fatty acyl chains for the sn1-position of E. faecalis phospholipids.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biochemistry

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2023

Author dc:creator

• Zou, Qi

Contributors dc:contributor

• Cronan, John E
• Imlay, James A
• Sligar, Stephen G
• Nair, Satish K

• Fatty Acid Synthesis
• Acyl Carrier Protein
• Phospholipid Synthesis
• Enterococcus Faecalis
• Transcription Repression
• Acyltransferase

Statement dc:rights

• Copyright 2023 Qi Zou

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/120226

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F120226