Short branched-chain C6 carboxylic acids result in increased growth, novel ‘unnatural’ fatty acids and increased membrane fluidity in a Listeria monocytogenes branched-chain fatty acid-deficient mutant

Abstract

Listeria monocytogenes is a psychrotolerant food borne pathogen, responsible for the high fatality disease listeriosis, and expensive food product recalls. Branched-chain fatty acids (BCFAs) of the membrane play a critical role in providing appropriate membrane fluidity and optimum membrane biophysics. The fatty acid composition of a BCFA-deficient mutant is characterized by high amounts of straight-chain fatty acids and even-numbered iso fatty acids, in contrast to the parent strain where odd-numbered anteiso fatty acids predominate. The presence of 2-methylbutyrate (C5) stimulated growth of the mutant at 37°C and restored growth at 10°C along with the content of odd-numbered anteiso fatty acids. The C6 branched-chain carboxylic acids 2-ethylbutyrate and 2-methylpentanoate also stimulated growth to a similar extent as 2-methylbutyrate. However, 3-methylpentanoate was ineffective in rescuing growth. 2-ethylbutyrate and 2-methylpentanoate led to novel major fatty acids in the lipid profile of the membrane that were identified as 12-ethyltetradecanoic acid and 12-methylpentadecanoic acid respectively. Membrane anisotropy studies indicated that growth of strain MOR401 in the presence of these precursors increased its membrane fluidity to levels of the wild type. Cells supplemented with 2-methylpentanoate or 2-ethylbutyrate at 10°C shortened the chain length of novel fatty acids, thus showing homeoviscous adaptation. These experiments use the mutant as a tool to modulate the membrane fatty acid compositions through synthetic precursor supplementation, and show how existing enzymes in L. monocytogenes adapt to exhibit non-native activity yielding unique ‘unnatural’ fatty acid molecules, which nevertheless possess the correct biophysical properties for proper membrane function in the BCFA-deficient mutant.

Broad substrate specificity of phosphotransbutyrylase from Listeria monocytogenes: A potential participant in an alternative pathway for provision of acyl CoA precursors for fatty acid biosynthesis

“…Extensive studies on medium supplementation by Kaneda [12] in B. subtilis and by Sen et al [13] in L. monocytogenes show that addition of a specific BCCA results in an increase in membrane fatty acids biosynthesized from that particular BCCA. The carboxylic acids that are capable of causing an alteration of membrane fatty acid composition are not restricted to the normal products of BCAA metabolism but also include unnatural C6 BCCAs that have a similar structure such as 2-ethylbutyrate, 2-methylpentanoate, and straight-chain carboxylic acids (SCCAs) such as butyrate and propionate [12,13].…”

“…The carboxylic acids that are capable of causing an alteration of membrane fatty acid composition are not restricted to the normal products of BCAA metabolism but also include unnatural C6 BCCAs that have a similar structure such as 2-ethylbutyrate, 2-methylpentanoate, and straight-chain carboxylic acids (SCCAs) such as butyrate and propionate [12,13]. Incorporation of novel fatty acids into the membrane as a result of elongation of unnatural C6 BCCAs is indicative of the entry of the supplements into the FAS II pathway as their respective acyl CoA derivatives, since the only known source of membrane BCFAs is via biosynthesis [6].…”

“…Incorporation of novel fatty acids into the membrane as a result of elongation of unnatural C6 BCCAs is indicative of the entry of the supplements into the FAS II pathway as their respective acyl CoA derivatives, since the only known source of membrane BCFAs is via biosynthesis [6]. Formation of acyl CoA derivatives despite the absence of functional Bkd confirms the presence of an alternate pathway which catalyzes the conversion of supplemented carboxylic acids into their respective acyl CoA end products [10,13]. Willecke and Pardee [11] first suggested the involvement of an alternate system in the conversion of BCCAs into precursors of fatty acids in a bkd mutant of B. subtilis .…”

“…Willecke and Pardee [11] first suggested the involvement of an alternate system in the conversion of BCCAs into precursors of fatty acids in a bkd mutant of B. subtilis . Integration of the products of such a wide range of substrates into the membrane implies that the pathway involved in their activation must be remarkably flexible in its substrate specificity [10,13]. This pathway has not been characterized in L. monocytogenes thus far despite ample evidence of its existence.…”

“…The results indicate that Ptb demonstrates broad substrate specificity, which is consistent with our hypothesis of its role in the incorporation of exogenous carboxylic acids into the membrane fatty acids. SCCAs such as propionate and butyrate, which are used as food preservatives, additionally may contribute to the control of L. monocytogenes by resulting in the biosynthesis of straight-chain fatty acids that rigidify the membrane [10,13]. The substantial activity with these substrates suggests that Ptb may play a role in this process.…”