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Gram-positive bacteria, including Listeria monocytogenes, adjust membrane fluidity by shortening fatty acid chain length and increasing the proportional production of anteiso fatty acids at lower growth temperatures. The first condensation reaction in fatty acid biosynthesis is carried out by β-ketoacyl-acyl carrier protein synthase III (FabH), which determines the type of fatty acid produced in bacteria. Here we measured initial rates of FabH catalyzed condensation of malonyl-acyl carrier protein and alternate branched-chain precursor acyl-CoAs utilizing affinity-purified His-tagged L. monocytogenes FabH heterologously expressed in Escherichia coli. L. monocytogenes FabH exhibited preference for 2-methylbutyryl-CoA, the precursor of odd-numbered anteiso fatty acids, at 30 °C that was further increased at low temperature (10 °C) suggesting that temperature-dependent substrate selectivity of FabH underlies the increased formation of anteiso branched-chain fatty acids during low temperature adaptation. The increased FabH preferential condensation of 2-methylbutyryl-CoA could not be attributed to a significant higher availability of this fatty acid precursor as acyl-CoA pool levels were reduced similarly for all fatty acid precursors at low temperatures.
Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that causes severe invasive disease in humans and other animal species. The majority of human listeriosis cases are caused by consumption of contaminated food products (Mead et al., 1999). A major factor in this is the relatively robust growth of Listeria at refrigeration temperature (Junttila et al., 1988; Ferreira et al., 2001; Bryan, 2004). Although adaptation to low temperature growth is complex involving many aspects of the molecular biology and biochemistry of the cell, the ability to modulate membrane fatty acid composition and hence membrane fluidity is a critical component of temperature adaptation (Suutari & Laakso, 1994; Zhang & Rock, 2008). Unsaturated, shorter, and anteiso-branched fatty acids all increase membrane fluidity compared to saturated, longer, and iso-branched fatty acids, respectively (Suutari & Laakso, 1994; Zhang & Rock, 2008). Iso- and anteiso-fatty acids are branched chain fatty acids with one methyl group at the penultimate (iso-) or antepenultimate (anteiso-) position of the fatty acid chain. L. monocytogenes is a Gram-positive bacterium and high contents of iso- and anteiso-branched fatty acids are characteristic of many Gram-positive bacteria including L. monocytogenes (Suutari & Laakso, 1994). An increase in the proportion of anteiso- and a decrease in the proportion of iso-fatty acids in response to the lowering of growth temperature is a common mode of adaptation to lower growth temperatures in Gram-positive bacteria in general (Suutari & Laakso, 1994), and in L. monocytogenes (Annous et al., 1997; Nichols et al., 2002; Zhu et al., 2005; Mastronicolis et al, 2005, 2008).
Anteiso fatty acids are produced from the primer 2-methylbutyryl-CoA, which is derived from the branched-chain amino acid isoleucine, whereas odd-numbered iso-branched fatty acids are produced from isovaleryl-CoA derived from leucine, and even-numbered iso- branched fatty acids are produced from isobutyryl-CoA, derived from valine (Kaneda, 1991). The type of branched-chain fatty acid produced is determined by FabH, which carries out the first condensation reaction in the fatty acid biosynthesis pathway (Zhang & Rock, 2008). FabHs from branched-chain fatty acid-producing bacteria prefer branched-chain acyl-CoA primers over acetyl-CoA (Choi et al., 2000). The importance of FabH in substrate specificity is clearly shown by the deficiency in branched-chain fatty acid synthesis when the FabH of Streptomyces coelicolor is replaced with E. coli FabH (Li et al., 2005). When growth temperature is lowered, 2-methylbutyryl-CoA, the precursor of anteiso fatty acids, is used increasingly over the precursors of iso fatty acids by an unknown mechanism (Suutari & Laakso, 1994). This study shows that FabH exhibits an increased preference for 2-methylbutyryl-CoA at low temperatures, and thus likely contributes to an increase in anteiso fatty acids, which is a part of temperature adaptation in L. monocytogenes.
Acetyl-CoA, butyryl-CoA, isobutyryl-CoA, isovaleryl-CoA, and malonyl-CoA substrates were purchased from Sigma (St. Louis, MO, USA). 2-methylbutyryl-CoA was synthesized as described previously (Stadtman, 1957).
Based on the fabH DNA sequence of L. monocytogenes strain EGD-e, two oligonucleotide primers (upstream 5′-GGATCCATGAACGCAGGAATTTTAGC-3′ and downstream (5′-GAATTCTTACTTACCCCAACGAATG-3′; restriction sites for cloning underlined) were designed for the amplification of the fabH gene of L. monocytogenes 10403S strain. The fabH gene (GenBank FJ749129) was cloned in expression vector pRSETa and expressed as an amino-terminal His-tagged protein in E. coli BL21 (plysS). FabH protein was purified by nickel chelate affinity chromatography to yield a homogeneous enzyme of expected molecular mass (~37 kDa) based on sodium dodecyl sulfate gel electrophoresis as previously described (Heath & Rock, 1995).
FabH assays were performed using purified L. monocytogenes His-tagged-FabH and the assay conditions described previously (Heath & Rock, 1996; Choi et al., 2000). Briefly, an assay mixture contained 0.1 M sodium phosphate buffer (pH 7.0), 65 μM acyl carrier protein, 250 μM dithiothreitol, 70 μM [2-14C] malonyl-CoA (52 mCi mmol−1), 100 μM NADPH, FabD (1 μg), FabG (50 ng), 45 μM of either isovaleryl-CoA, 2-methylbutyryl-CoA, or isobutyryl-CoA when reactions were performed at 30 °C. The substrate concentrations (isovaleryl-CoA, 2-methylbutyryl-CoA, or isobutyryl-CoA) were increased to 1 mM at 10 °C. Reactions were initiated by adding L. monocytogenes FabH and incubated for 20 min; assays were stopped by adding 13.3 μL of 4x sample loading buffer and 50 μL aliquots were loaded onto a 13% polyacrylamide gel containing 0.5 M urea. Gels were dried and products were quantified via PhosphoImager. Specific activities were calculated from the slopes of the plot of product formation versus L. monocytogenes FabH concentration in the assay. The Km values were determined by varying the substrate concentration in the assay up to 200 μM with 160 ng of L. monocytogenes FabH (10 °C), or 80 μM with 20 ng of L. monocytogenes FabH (30 °C). Acetyl-CoA was examined up to 200 μM with 640 ng of L. monocytogenes FabH (30 °C).
L. monocytogenes strain 10403S was grown in 500 mL of BHI medium in a 2-L Erlenmeyer flask with shaking (180 r.p.m.) at 37 °C and 10 °C in triplicate to an OD600 nm of 0.45. The cells were harvested by centrifugation at 10 000 g at 0 °C and washed twice with cold sterile water. Cold methanol [50 % (vol/vol), stored at −20 °C] was added rapidly, centrifuged at 15 000 g and supernatants were collected. The samples were extracted further as described previously with minor modifications (Roessner et al., 2000, 2001). Briefly, the cell pellets were suspended in 1.4 mL of 70% methanol and 10 μL of 0.2 mg mL−1 malonyl-CoA was added as internal standard. The cells were sonicated for 30 min at 4 °C. The mixture was extracted for additional 15 min at 4 °C, centrifuged at 15 000 g. The supernatants were subject to dryness in vacuum and the dry residues were resuspended in water. After centrifugation at 10 000 g, the collected supernatants were used for liquid chromatography/mass spectrometry (LC/MS) analysis. LC/MS analysis was performed on an Agilent LC/ion trap MS system. An Agilent 1100 series LC system (Santa Clara, CA, USA) was used for sample separation and introduction to mass spectrometry. The sample mixture were placed in the cooled sample tray (4 °C) and an aliquot of 5 μL sample was injected into the Alltech Prevail C18 column (150 × 4.6 mm, 5-micro) (Deerfield, IL, USA). The Alltech Prevail C18 column was equilibrated with 75% solvent A (15 mM ammonia formate) and 25% solvent B (90% methanol and 10% 10 mM ammonia formate), and eluted at ambient temperature with a 300 μL min−1 flow rate. The linear gradient was as follows: 0 min, 25% B; 10–13 min, 100% B; 15–21 min, 25% B. A clean run of 100% methanol for 8 min was performed after every 4 samples followed by a blank run to ensure optimum column performance.
Mass spectra were acquired using an Agilent MSD Trap XCT Plus mass spectrometer equipped with an ESI source (Santa Clara, CA, USA). For best sensitivity, ESI signals from standards were tuned with the use of a Kd Scientific 789100A model syringe pump (Holliston, MA, USA) connected directly to the ion source via PEEK tubing. Nitrogen was used as the nebulizer gas (30 psi) and drying gas (9 L min−1). The capillary voltage was set to 4.5 kV. The heated capillary of the ESI source was kept at 350 °C during the analysis. Coenzymes A were detected in multiple reactions monitoring mode under negative ESI with the following transitions: isovaleryl- and 2-methylbutyryl-CoA, m/z 850.6 → 503.3; butyryl- and isobutyryl-CoA, m/z 836.5 → 489.0; acetyl-CoA, m/z 808.6 → 461.1; malonyl-CoA, m/z 852.5 → 808.5.
At 30 °C, L. monocytogenes FabH had a clear preference for branched-chain-CoA precursors versus acetyl-CoA (Table 1). The order of catalytic efficiency (i.e., kcat/Km) for the various substrates (in decreasing order) was: 2-methylbutyryl-CoA > isovaleryl-CoA > isobutyryl-CoA acetyl-CoA (Table 1). The L. monocytogenes FabH acyl-CoA preference was similar to that observed for B. subtilis (Choi et al., 2000) and S. aureus (Qiu et al., 2005), two related Gram-positive bacteria. In contrast, E. coli FabH clearly prefers acetyl-CoA as a substrate and has low activity with branched-chain acyl-CoAs (Choi et al., 2000). The catalytic efficiency of FabH for the same substrates was also determined at 10 °C, which revealed striking alterations in kinetic parameters for the different fatty acid substrates. For example, effective FabH condensation of isovaleryl-CoA was decreased 20-fold, whereas its activity with 2-methylbutyryl-CoA only decreased ~5-fold. Thus, L. monocytogenes FabH prefers 2-methylbutyryl-CoA at both 10 °C and 30 °C, but this selectivity for 2-methylbutyryl-CoA becomes even more pronounced at lower temperature. This result was consistent with the prevalence of anteiso fatty acids at all temperatures, and consistent with the increase in the proportion of anteiso fatty acids produced at lower temperatures (Annous et al., 1997; Nichols et al., 2002; Zhu et al., 2005; Mastronicolis et al., 2005, 2008).
We observed that FabH had a decreased apparent affinity for isobutyryl-CoA at lower temperatures (KmIB = 24.3 and 30.7 at 30 °C and 10 °C, respectively) (Table 1). Although even numbered iso-fatty acids are only a minor component of the total fatty acids of L. monocytogenes, this apparent reduction in affinity for isobutyryl-CoA may be important as it reduces the ability of isobutyryl-CoA to compete with 2-methylbutyryl-CoA for the active site on FabH, which could otherwise hinder production of odd-numbered anteiso fatty acids.
The estimated pool concentrations of the acyl-CoAs at 37 °C (Table 2) were of the same order as the Km values of FabH (Table 1). We are not aware of other determinations of the levels of branched acyl-CoAs, but the levels found at 37 °C are of the same order as those of other acyl-CoAs in other bacteria (Newton et al., 1996; Chohnan et al., 1997). The levels of all CoA derivatives were substantially reduced at 10 °C compared to values obtained at 37 °C (Table 2). The largest decline was in the levels of acetyl-CoA (~20-fold), whereas the other acyl-CoA derivatives decreased five- to seven-fold in cells grown at 10 °C. Despite these differences in the absolute levels of acyl-CoA precursors at the two temperatures, their relative abundances compared to each other remained essentially unchanged.
The FabH preference for 2-methylbutyryl-CoA at 10 °C supports the idea that FabH substrate specificity contributes to the increased proportion of anteiso fatty acids produced by bacteria during cold adaptation. We think that these basic observations on substrate specificity will also apply to the FabH enzymes of other bacteria that increase the content of anteiso fatty acids at lower growth temperatures, such as B. subtilis (Klein et al., 1999), S. aureus (Joyce et al., 1970), and other Gram positive bacteria (Suutari & Laakso, 1994).
This work was supported by award 2006–35201–17386 from the National Research Initiative Competitive Grants Program of the United States Department of Agriculture, National Institutes of Health Grants GM 34496 and Cancer Center (CORE) Support Grant CA 21765, and the American Lebanese Syrian Associated Charities.