Abstract
The genome of Lactococcus lactis encodes a single long chain 3-ketoacyl-acyl carrier protein synthase. This is in contrast to its close relative, Enterococcus faecalis, and to Escherichia coli, both of which have two such enzymes. In E. faecalis and E. coli, one of the two long chain synthases (FabO and FabB, respectively) has a role in unsaturated fatty acid synthesis that cannot be satisfied by FabF, the other long chain synthase. Since L. lactis has only a single long chain 3-ketoacyl-acyl carrier protein synthase (annotated as FabF), it seemed likely that this enzyme must function both in unsaturated fatty acid synthesis and in elongation of short chain acyl carrier protein substrates to the C18 fatty acids found in the cellular phospholipids. We report that this is the case. Expression of L. lactis FabF can functionally replace both FabB and FabF in E. coli, although it does not restore thermal regulation of phospholipid fatty acid composition to E. coli fabF mutant strains. The lack of thermal regulation was predictable because wild-type L. lactis was found not to show any significant change in fatty acid composition with growth temperature. We also report that overproduction of L. lactis FabF allows growth of an L. lactis mutant strain that lacks the FabH short chain 3-ketoacyl-acyl carrier protein synthase. The strain tested was a derivative (called the ∆fabH bypass strain) of the original fabH deletion strain that had acquired the ability to grow when supplemented with octanoate. Upon introduction of a FabF overexpression plasmid into this strain, growth proceeded normally in the absence of fatty acid supplementation. Moreover, this strain had a normal rate of fatty acid synthesis and a normal fatty acid composition. Both the ∆fabH bypass strain that overproduced FabF and the wild type strain incorporated much less exogenous octanoate into long chain phospholipid fatty acids than did the ∆fabH bypass strain. Incorporation of octanoate and decanoate labeled with deuterium showed that these acids were incorporated intact as the distal methyl and methylene groups of the long chain fatty acids.
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References
Alberts AW, Bell RM, Vagelos PR (1972) Acyl carrier protein. XV. Studies of β-ketoacyl-acyl carrier protein synthetase. J Biol Chem 247:3190–3198
Allen EE, Bartlett DH (2000) FabF is required for piezoregulation of cis-vaccenic acid levels and piezophilic growth of the deep-sea bacterium Photobacterium profundum strain SS9. J Bacteriol 182:1264–1271
Arnvig Mcguire K, McGuire JN, von Wettstein-Knowles P (2000) Acyl carrier protein (ACP) inhibition and other differences between β-ketoacyl synthase (KAS) I and II. Biochem Soc Trans 28:607–610
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917
Bolotin A et al (2001) The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 11:731–753
Campbell JW, Cronan JE Jr (2001) Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu Rev Microbiol 55:305–332
Christie WW (2003) Lipid analysis: isolation, separation, identification and structural analysis of lipids, 3rd edn. The Oily Press, Bridgewater
de Mendoza D, Cronan JE (1983) Temperature regulation of membrane fluidity in bacteria. Tren Biochem Sci 8:49–52
de Mendoza D, Klages Ulrich A, Cronan JE Jr (1983) Thermal regulation of membrane fluidity in Escherichia coli. Effects of overproduction of β-ketoacyl-acyl carrier protein synthase I. J Biol Chem 258:2098–2101
Edwards P, Nelsen JS, Metz JG, Dehesh K (1997) Cloning of the fabF gene in an expression vector and in vitro characterization of recombinant fabF and fabB encoded enzymes from Escherichia coli. FEBS Lett 402:62–66
Garwin JL, Klages AL, Cronan JE Jr (1980) β-Ketoacyl-acyl carrier protein synthase II of Escherichia coli. Evidence for function in the thermal regulation of fatty acid synthesis. J Biol Chem 255:3263–3265
Guzman L-M, Belin D, Carson MJ, Beckwith J (1995) Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 177:4121–4130
Hooper SD, Berg OG (2003) Duplication is more common among laterally transferred genes than among indigenous genes. Genome Biol 4:R48
Jackowski S, Murphy CM, Cronan JE Jr, Rock CO (1989) Acetoacetyl-acyl carrier protein synthase. A target for the antibiotic thiolactomycin. J Biol Chem 264:7624–7629
Jackowski S, Zhang YM, Price AC, White SW, Rock CO (2002) A missense mutation in the fabB β-ketoacyl-acyl carrier protein synthase I) gene confers thiolactomycin resistance to Escherichia coli. Antimicrob Agents Chemother 46:1246–1252
Kutchma AJ, Hoang TT, Schweizer HP (1999) Characterization of a Pseudomonas aeruginosa fatty acid biosynthetic gene cluster: purification of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD). J Bacteriol 181:5498–5504
Lai CY, Cronan JE (2003) β-Ketoacyl-acyl carrier protein synthase III (FabH) is essential for bacterial fatty acid synthesis. J Biol Chem 278:51494–51503
Lu YJ, White SW, Rock CO (2005) Domain swapping between Enterococcus faecalis FabN and FabZ proteins localizes the structural determinants for isomerase activity. J Biol Chem 280:30342–30348
Makarova K et al (2006) Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci USA 103:15611–15616
McGuire KA, Siggaard-Andersen M, Bangera MG, Olsen JG, von Wettstein-Knowles P (2001) β-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket. Biochemistry 40:9836–9845
McIntyre DA, Harlander SK (1989) Improved electroporation efficiency of intact Lactococcus lactis subsp. lactis cells grown in defined media. App Env Microbiol 55:2621–2626
Que Y-A, Haefliger J-A, Francioli P, Moreillon P (2000) Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector. Infect Immun 68:3516–3522
Sweetman G et al (1996) Electrospray ionization mass spectrometric analysis of phospholipids of Escherichia coli. Mol Microbiol 20:233–238
Terzaghi BE, Sandine WE (1975) Improved medium for lactic streptococci and their bacteriophages. Appl Environ Microbiol 29:807–813
Tsay JT, Rock CO, Jackowski S (1992) Overproduction of β-ketoacyl-acyl carrier protein synthase I imparts thiolactomycin resistance to Escherichia coli K-12. J Bacteriol 174:508–513
Ulrich AK, de Mendoza D, Garwin JL, Cronan JE Jr (1983) Genetic and biochemical analyses of Escherichia coli mutants altered in the temperature-dependent regulation of membrane lipid composition. J Bacteriol 154:221–230
Wang H, Cronan JE (2004) Functional replacement of the FabA and FabB proteins of Escherichia coli fatty acid synthesis by Enterococcus faecalis FabZ and FabF homologues. J Biol Chem 279:34489–34495
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R.M-K. was supported in part by a National Science and Engineering Research Council of Canada Postdoctoral Fellowship. This work was supported by NIH grant AI15650.
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Communicated by Jorge Membrillo-Hernández.
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Morgan-Kiss, R.M., Cronan, J.E. The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis . Arch Microbiol 190, 427–437 (2008). https://doi.org/10.1007/s00203-008-0390-6
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DOI: https://doi.org/10.1007/s00203-008-0390-6