Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain α-keto acid dehydrogenase

Abstract

Branched-chain fatty acids (BCFAs) typically constitute more than 90 % of the fatty acids of Listeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %) L. monocytogenes mutants (cld-1 and cld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917 was inserted into different genes of the branched-chain a-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, in which Tn917 was inserted into bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37 6C were stimulated by fatty acid precursors bypassing Bkd, 2-methylbutyrate (precursor for odd-numbered anteiso-fatty acids), isobutyrate (precursor for even-numbered iso-fatty acids) and isovalerate (precursor for odd-numbered iso-fatty acids). In contrast, the corresponding Bkd substrates, a-ketomethylvalerate, a-ketoisovalerate and a-ketoisocaproate, exhibited much poorer activity. At 26 6C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 6C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content of cld-2 from 33 % to 27 %, which may be close to the minimum BCFA requirement for L. monocytogenes. The transcription of bkd was enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors, cld-2 was able to increase its anteiso-C 15 : 0 fatty acid content at 10 6C compared to 37 6C, which is the characteristic response of L. monocytogenes to low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis. INTRODUCTIONListeria monocytogenes causes the potentially life-threatening infection known as listeriosis, which has a fatality rate of 20-25 % (Mead et al., 1999). Listeriosis outbreaks have been linked to the consumption of food contaminated with this organism. Nowadays, there is an increased consumption of ready-to-eat, chilled and frozen foods, with decreased use of chemical microbial control agents. The ability of L. monocytogenes to grow at refrigeration temperatures is a critical aspect of its role as a food-borne pathogen, since refrigeration temperature inhibits the growth of most other food-borne pathogens (Bryan, 2004). However, the underlying psychrotolerance mechanism is not completely understood, and a better understanding may result in a novel strategy for controlling the growth of L. monocytogenes at low temperatures.Low temperature has profound effects on all aspects of microbial cell structure and function, involving the structural integrity of macromolecules, macromolecular assemblies, protein synthesis and nutrient uptake (Panoff et al., 1998;Weber & Marahiel, 2002 Two Tn917 transposon-induced mutants (cld-1 and cld-2) have lost the ability to grow at 4 u C on solid media, but are not defective in the induction of cold-shock proteins (Bayles et al., 1996). These mutants are deficient in the production of odd-numbered BCFAs, and exhibit atypical am...

Role of Branched-Chain Fatty Acids in pH Stress Tolerance in Listeria monocytogenes

“…pH stress did not induce gross changes in the total amounts of unsaturated FAs. This is interesting because pH stress induces considerable changes in total unsaturated FA content in other bacteria, and L. monocytogenes does make such changes in response to other environmental stresses (4,13,25,26,29,32,49). The absence of significant differences between the responses of L. monocytogenes 10403S and the isogenic sigB null mutant suggests that sigB does not have a major role in pH-induced fatty acid modulation in L. monocytogenes.…”

“…Thus, these two enzymes, BcaT and Bkd, are critical for BCFA biosynthesis in L. monocytogenes and represent a possible mechanism for stress regulation and modification of FA profiles in this pathogen (8,28,36,37). The differences between the growth rates of BCFA-deficient mutants and the BCFA-competent parent strain under pH stress and the resolution of such differences by the provision of exogenous 2MBA, bypassing the branched-chain ␣-keto acid dehydrogenase step in the biosynthesis of BCFA (49,50), demonstrated the close correlation between membrane BCFA content and the ability of L. monocytogenes to grow under such adverse environmental conditions.…”

“…In L. monocytogenes (49,50) (and also in Bacillus subtilis [12]), such enzymes and their products have vital roles in membrane adaptation to highand low-temperature stress. BCFA synthesis involves the transamination of branched-chain amino acids such as isoleucine, valine, and leucine by a branched-chain amino acid transaminase (BcaT) (12,23) and subsequent oxidative decarboxylation by the branched-chain ␣-keto acid dehydrogenase (Bkd) (8,28,36,37).…”

“…Washed cells from mid-exponential-phase cultures of L. monocytogenes 10403S, an isogenic sigB null mutant (3), and isogenic non-BCFA-producing cld-1 and cld-2 mutants (1,49) were inoculated into preheated (30°C) 100-ml volumes of the buffered MBHIB and grown (30°C/200 rpm) to an optical density at 600 nm of 0.5 to 0.6. Growth rates of cultures (doubling times per hour of cultures in exponential growth) were calculated (2).…”

“…Growth rates of cultures (doubling times per hour of cultures in exponential growth) were calculated (2). Mid-exponential-phase cells were recovered by centrifugation at 8,000 ϫ g for 10 min at 4°C and washed three times with distilled water.The FAs in washed-cell pellets were saponified, methylated, and extracted as described previously (1,49,50). Methyl ester mixtures were separated using an Agilent 5890 dual-tower gas chromatograph with split/splitless injector, flame ionization detector, 25-m by 0.2-mm Ultra 2 capillary column (HewlettPackard), and automatic sampler/integrator and analyzed using an FA identification program (MIDI; Sherlock 4.5 Microbial Identification System).…”