Proteomic analysis of Lactobacillus casei GCRL163 cell-free extracts reveals a SecB homolog and other biomarkers of prolonged heat stress

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.

Genes Contributing to the Unique Biology and Intrinsic Antibiotic Resistance of Enterococcus faecalis

“…BLAST analysis shows that EF0742 is conserved throughout enterococci and related species (vagococci, streptococci, and lactococci) emerging from the same phylogenetic branch. A divergent homolog was studied in Lactobacillus casei and was found to share structural resemblance to SecB and to be important for surviving prolonged heat stress ( 75 ).…”

“…Included within this group is the adjacent gene, msrA (EF1681), which encodes methionine sulfoxide reductase A. MsrA and MsrB (EF3164) have been shown to be important for repairing oxidative damage and to contribute to H 2 O 2 challenge survival ( 76 ). In the competitive growth environment of Tn-seq, insertions in either msrA or msrB are highly compromising (although it is still possible to isolate and study mutants [ 75 ]), highlighting the oxidative stress challenge enterococci experience even when growing in nutrient medium. That study further showed that EF1683 to EF1680 occur in an operon expressed in that gene order, which, along with the specificity of the effect, supports the finding that EF1680 itself is a determinant of polymyxin and daptomycin resistance (as opposed to a polar effect on EF1681).…”

Lactobacillus , Bifidobacterium and Lactococcus response to environmental stress: Mechanisms and application of cross‐protection to improve resistance against freeze‐drying

“…Notably, during the heat stress adaptation, LAB are known to downregulate UFA/FA to enhance the membrane rigidity by adjusting fluidity. Besides, the proteins within the phosphotransferase Accepted Article system (PTS) system and the ATP-binding cassette (ABC) transporter system are upregulated at elevated temperatures (Adu et al 2018). During heat stress, L. casei GCRL163 induces a complicated network of metabolic processes to generate adequate energy as per the physiological needs.…”

Ribosome Profiling and RNA Sequencing Reveal Genome-Wide Cellular Translation and Transcription Regulation Under Osmotic Stress in Lactobacillus rhamnosus ATCC 53103

“…However, these DEGs involved in fatty acid biosynthesis and metabolism pathways were downregulated at both levels. This reflected that L. rhamnosus needed to save cellular metabolic energy and increase environmental adaptation by limiting fatty acid biosynthesis ( Adu et al, 2018 ). Purine metabolism was activated in the bacteria in response to environmental stress ( Goncheva et al, 2019 ).…”