Dynamic expression of heat-shock and acid-tolerance related genes of Lactobacillus delbrueckii ssp. bulgaricus LBB.B5 in milk
Abstract
Lactobacillus delbrueckii ssp. bulgaricus is a central species in the production of fermented dairy food. However, from the point of view of starter culture production this species is extremely sensitive to freeze-drying. Therefore, it is essential to understand the physiological processes and adaptation mechanisms of the bacterial cell, especially its response to factors such as heat-, cold- and acid stress. Here we report the results of the dynamic expression monitoring of heat/cold-shock related genes (hsp60 and cspA) and genes putatively contributing to acid-tolerance (ornB, thrB and thrC) in L. delbr. ssp. bulgaricus LBB.B5 grown in milk. These genes were selected as hsp60 and cspA encode a heat-shock and a cold-shock protein, respectively, while ornB encodes an ornithine decarboxylase, related to acid tolerance in this species. Genes thrB and thrC are involved in threonine synthesis and are of particular interest as unlike the majority of amino-acids, the threonine synthesis pathway is conserved in all L. delbr. ssp. bulgaricus strains. Expression levels were monitored by RT-qPCR for 7 hours of fermentation at 42oC and then until the 24th hour under cold storage at 4oC. Samples at 3h were used as control.
Two distinct patterns in the expression dynamics of the analysed genes were observed. Genes cspA, ornB, thrB and thrC, followed a pattern with maximal expression at 5h of the fermentation with levels of 11.6, 6.8, 3.9 and 2.4 times the control, respectively. Maximal expression levels coincided with the transition of the culture from exponential to stationary phase at a pH threshold of 5.0. Gene hsp60 showed a different pattern with gradually increasing expression throughout the hole fermentation process, including after cold storage when expression level reached 6.4 times the control.
The upregulation of threonine biosynthesis and of a cold-shock protein synthesis with the onset of the stationary phase may suggest that together with the activity of ornithine decarboxylase, they go beyond their function of serving the amino-acid anabolism or managing cold stress, but rather facilitate the transition of the cells to stationary phase and/or their adaptation to acidic conditions. The gradual upregulation of hsp60 on the other hand may reflect the adaptation of the cells to growth at higher temperature (42oC) and subsequent transfer to cold storage.
Keywords
acid-tolerance, gene expression, heat-shock, Lactobacillus bulgaricus
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