Submitted to: Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/7/2004
Publication Date: 2/1/2005
Citation: Zhu, K., Bayles, D.O., Xiong, A., Jayaswal, R.K., Wilkinson, B.J. 2005. Precursor and temperature modulation of fatty acid composition and growth of listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain alpha-keto acid dehydrogenase. Microbiology. 151:615-623. Interpretive Summary: Listeria monocytogenes is the causative agent of listeriosis, a food-borne illness that has a high rate of fatality compared to other food-borne infections. L. monocytogenes can grow at refrigeration temperatures making it difficult to prevent the growth of this pathogen. To understand how L. monocytogenes can grow at low temperature, mutant strains were created that could not grow at refrigeration temperature. Two of these mutants were found to be deficient in their ability to make the membrane modifications necessary for growth at low temperature. The nature of this defect was confirmed by DNA sequencing, by genetic complementation, and by providing the cells with specific precursors needed for making the membrane modifications essential for low temperature growth. The two described mutations occur in two genes that code for different peptide subunits of a protein responsible for generating the fatty acid precursors needed for making the modified membrane lipids essential for low temperature growth. This work has increased our understanding of how L. monocytogenes regulates the membrane modification process, which allows the organism to grow over a wide range of temperatures. The studies using fatty acid precursors indicate that adding certain fatty acid precursors might provide a method for inhibiting the low temperature growth of L. monocytogenes.
Technical Abstract: Branched-chain fatty acids (BCFAs) typically comprise more than 90% of the fatty acids of Listeria monocytogenes. We 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 alpha-keto acid dehydrogenase cluster (bkd) in these two mutants. The cold-sensitivity and BCFA deficiency of cld-1, where Tn917 was inserted in bkdB, were complemented in trans by cloned bkdB. The growth and corresponding BCFA content of the mutants at 37°C was stimulated by fatty acid precursors downstream of 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, alpha-ketomethylvalerate, alpha-ketoisovalerate and alpha-ketoisocaproate, had much poorer activity in stimulating the growth and BCFA content of the mutants. At 26°C only 2-methylbutyrate and isovalerate stimulated the growth, and at 10°C only 2-methylbutyrate stimulated the growth of the mutants. Pyruvate depressed the BCFA content of cld-2 from 33% to about 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 cld-2 was provided with the BCFA precursors, it was able to increase its anteiso-C15:0 fatty acid content at 10°C compared to 37°C, which is the characteristic response of L. monocytogenes to low temperature. This implies that the thermal regulation of fatty acid composition occurs downstream from the Bkd complex.