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 ofListeria monocytogenes. The authors have previously described two Tn917-induced, cold-sensitive, BCFA-deficient (<40 %)L. monocytogenesmutants (cld-1andcld-2) with lowered membrane fluidity. Sequence analyses revealed that Tn917was inserted into different genes of the branched-chainα-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency ofcld-1, in which Tn917was inserted intobkdB, were complementedin transby clonedbkdB. The growth and corresponding BCFA content of the mutants at 37 °C 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,α-ketomethylvalerate,α-ketoisovalerate andα-ketoisocaproate, exhibited much poorer activity. At 26 °C, 2-methylbutyrate and isovalerate stimulated the growth of the mutants, and at 10 °C, only 2-methylbutyrate stimulated growth. Pyruvate depressed the BCFA content ofcld-2from 33 % to 27 %, which may be close to the minimum BCFA requirement forL. monocytogenes. The transcription ofbkdwas enhanced by Bkd substrates, but not by low temperature. When provided with the BCFA precursors,cld-2was able to increase its anteiso-C15 : 0fatty acid content at 10 °C compared to 37 °C, which is the characteristic response ofL. monocytogenesto low temperature. This implies that Bkd is not the major cold-regulation point of BCFA synthesis.