The psychrotolerance mechanisms of Listeria monocytogenes were investigated by modulation of cell membrane fatty acid composition, studying the activity of branched-chain α-keto acid decarboxylase, and analyzing the data from genome-wide transcriptional profiling of the organism in response to cold stress. Supplementation of medium with branched-chain amino acids and short branched-chain carboxylic acids induced the biosynthesis of specific groups of fatty acids: isoleucine or 2-methylbutyrate increased fatty acids anteiso-C15:0 and anteiso-C17:0, leucine or isovalerate increased fatty acids iso-C15:0 and iso-C17:0 , and valine or isobutyrate increased fatty acids iso-C16:0 and iso-C14:0. Three branched C6-fatty acid precursors (2-ethylbutyric acid, 2-methylpentanoic acid, and 3-methylpentanoic acid) were found to be incorporated into long-chain fatty acids, each of them caused a new set of two fatty acids to be produced. Short and medium straight chain carboxylic acids significantly inhibited growth, but had minimal effects on fatty acid composition. Butyrate significantly increased the amount of the fatty acids n-C14:0 and n-C16:0, particularly at 37°C, but to a lesser extent at 10°C. This suggests that L. monocytogenes has the capacity to sustain the amount of fatty acid anteiso-C 15:0 to at least the minimum level required for growth at low temperature.
The BkdE1α and BkdE1β polypeptides of branched-chain alpha keto acid dehydrogenase from L. monocytogenes 10403S revealed the consensus amino acid sequences of a thiamine binding motif and a proton channel on the α and β subunits, respectively. The maximum activity of BkdE1 at 30°C was observed with α-keto methylvalerate (KMV) and the least with pyruvate. This implies that the best substrate for BkdE1 is a C5 substrate having the branched-chain at the α carbon atom (KMV), which is a precursor of odd-numbered anteiso fatty acids. The activity was reduced as the carbon chain reduced to C4 (KIV) or having the branching at the β carbon atom (KIC). The activity was low with an unbranched substrate (α-ketoglutarate), or when the chain length was reduced to C3 (pyruvate), which was a very low activity substrate. Purified mixed BkdE1α and BkdE1β showed no activity at low temperature. However, BkdE1 activity at low temperature was detected using a cell free extract of E. coli carrying the overexpression vector. These data suggests that some factors in the cell free extract may facilitate the enzyme function at low temperature.
Data from genome-wide transcriptional profiling was used to study gene expression of L. monocytogenes subjected to cold shock and cold acclimation. Genes encoding AA3-600 quinol oxidase, ferrichrome hydroxamate ABC transporter, pyrimidine biosynthesis, an operon for maintaining pH homeostasis, DNA gyrase, DNA repair, and chaperones were upregulated in response to cold shock. Genes related to fibronectin-binding protein and PrfA were downregulated in cold acclimated cultures. This suggests that L. monocytogenes is less virulent when grown at low temperature. The branched-chain α-keto acid dehydrogenase operon and a fatty acid biosynthesis operon were upregulated in response to cold shock, but were downregulated in cold acclimated cultures. In contrast, a mannose-specific phosphotransferase system (PTS) operon and the Kdp-ATPase system (kdpEDCBA) showed underexpression in response to cold shock, but overexpression in cold acclimation. A number of genes encoding ribosomal proteins and flagellar biosynthesis were upregulated for both cold stress conditions.
L. monocytogenes has been listed as a priority food-borne pathogen by the National Institute of Allergy and Infectious disease (NIAID). It is important to understand the psychrotolerance mechanism and to find a way to control its growth at low temperature. Our data suggest the idea that medium straight chain carboxylic acid can inhibit growth effectively. Leucine and isovalerate can inhibit growth and change the fatty acid composition of L. monocytogenes at low temperature.
|Advisor:||Wilkinson, Brian J.|
|School:||Illinois State University|
|School Location:||United States -- Illinois|
|Source:||DAI-B 69/11, Dissertation Abstracts International|
|Subjects:||Molecular biology, Microbiology|
|Keywords:||Fatty acids, Listeria monocytogenes, Methylvalerate, Psychrotolerance|
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