Abstract
Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163.
Highlights
Common global responses include protection of macromolecules involved in metabolic activities or cellular structural integrity; modulation of metabolic activity and energy production to ameliorate the impact of stress, typically around the fate of pyruvate; altered uptake of alternative carbon sources to facilitate increased energy production; and increased proteolysis and catabolism of amino acids
While growth in bioreactors at 40 ̊C was marginally impaired, 45 ̊C caused significant decline in growth rate and rapid entry into stationary phase. The proteomes for these two temperatures were markedly different to cells cultured at lower temperatures and to each other, while sharing many common trends with the global responses documented for lactic acid bacteria (LAB), in greater abundance of ATP-binding cassette (ABC)- and PTS-transporters plus rerouting carbohydrate metabolism
The gene cluster including the hrcA heat inducible regulator, grpE, dnaK and dnaJ are considered to form an operon which is conserved across Lactobacillus species [8], our proteomic data indicated separate regulation of these genes as growth temperature increased, an observation supported by detecting promoters in intergenic regions, which has not been reported for heat shock responses in LAB
Summary
Lactobacillus casei and related species occur in fermented milk and cheese products as nonstarter, adventitious microbiota where they improve ripening and flavour development [1]. To confer probiotic and other functional traits, LAB must survive exposure to heat and other stressors during manufacturing processes, as well as harsh conditions encountered in the gastrointestinal tract following consumption. Under these conditions, several physiological and molecular responses are induced to enhance growth, adaptation and survival so alleviating impacts of stress [6]. Proteomics of Lactobacillus casei GCRL163 unveils biomarkers of prolonged heat stress of most proteins in fatty acid synthesis and non-differential modulation of most proteins involved in glycolysis. Changes in fatty acid composition were observed to be growth phaseand temperature-dependent as previously reported in other bacterial species [19, 20], with reduced vaccenic acid synthesis in cells from 45 ̊C culture and detection of cyclopropane derivatives of C18:0 fatty acids and previously unreported cyclopentenyl moieties
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