Genetic and Physiological Responses of Bifidobacterium animalis subsp. lactis to Hydrogen Peroxide Stress

Abstract

bConsumer interest in probiotic bifidobacteria is increasing, but industry efforts to secure high cell viability in foods is undermined by these anaerobes' sensitivity to oxidative stress. To address this limitation, we investigated genetic and physiological responses of two fully sequenced Bifidobacterium animalis subsp. lactis strains, BL-04 and DSM 10140, to hydrogen peroxide (H 2 O 2 ) stress. Although the genome sequences for these strains are highly clonal, prior work showed that they differ in both intrinsic and inducible H 2 O 2 resistance. Transcriptome analysis of early-stationary-phase cells exposed to a sublethal H 2 O 2 concentration detected significant (P < 0.05) changes in expression of 138 genes in strain BL-04 after 5 min and 27 genes after 20 min. Surprisingly, no significant changes in gene expression were detected in DSM 10140 at either time. Genomic data suggested that differences in H 2 O 2 stress resistance might be due to a mutation in a BL-04 gene encoding long-chain fatty acid coenzyme A (CoA) ligase. To explore this possibility, membrane fatty acids were isolated and analyzed by gas chromatography-mass spectrometry (GC-MS). Results confirmed that the strains had significantly different lipid profiles: the BL-04 membrane contained higher percentages of C 14:0 and C 16:0 and lower percentages of C 18:1n9 . Alteration of the DSM 10140 membrane lipid composition using modified growth medium to more closely mimic that of BL-04 yielded cells that showed increased intrinsic resistance to lethal H 2 O 2 challenge but did not display an inducible H 2 O 2 stress response. The results show that deliberate stress induction or membrane lipid modification can be employed to significantly improve H 2 O 2 resistance in B. animalis subsp. lactis strains. Bifidobacteria are Gram-positive rods of irregular shape with a GϩC content of 55 to 67% and are part of the normal gastrointestinal flora in human infants and adults (1, 2). Bifidobacteria have been associated with several health-related benefits, including a decrease in severity of the side effects associated with use of antibiotics, reduced incidence of infection in patients receiving irradiation therapy, decrease in the duration of diarrhea due to various etiologies, reduced frequency of allergic reactions, and alleviation of constipation (3-8). Although no conclusive data regarding a minimal effective dose of probiotics in humans are available, results from clinical trials suggest a direct dose-effect correlation with probiotic efficacy (9, 10). This means that bifidobacteria likely need to be consumed at very high levels (Ͼ10 7 CFU) in bioactive foods to effect a probiotic outcome. At present, yogurt and fermented milks are the most common foods for delivery of probiotic bifidobacteria, but their incorporation into other foods is increasing. A major obstacle to production and storage of bioactive foods containing bifidobacteria is the susceptibility of these cells to oxidative stress. Bifidobacteria are anaerobic and therefore lack common enzyme...

Effects of Linoleic Acid on Gut-Derived Bifidobacterium breve DSM 20213: A Transcriptomic Approach

“…Since LA exposure influenced genes and regulators involved in lipid metabolism, one could speculate that the cell membrane might be a target of LA action. Previous studies demonstrate that several kinds of stresses, including osmotic or acid stress, can prompt modifications in the bacterial cell membrane composition-particularly fatty acid chain length, saturation, and cyclopropanation-to decrease membrane permeability to oxidative free radicals and increase membrane resistance to lipid peroxidation [51]. We observed that LA induced downregulation of genes, such as the TadE gene, encoding type IVb tight adherence pili, which is in accordance with another transcriptional analysis showing reduced expression of these genes in response to stress conditions [52].…”

Transcriptome analysis of Bifidobacterium longum strains that show a differential response to hydrogen peroxide stress

“…RNA isolation was performed as previously described (Oberg et al, 2013). Cells were grown in batch culture under pH control to early stationary phase and then samples (5 mL) were harvested by centrifugation at 7500 × g for 10 min.…”

“…cDNA synthesis and labeling was performed as described previously (Oberg et al, 2013). cDNA was synthesized and labeled as recommended by the Affymetrix (Santa Clara, CA) protocol for prokaryotic target preparation in the GeneChip Expression Analysis Technical Manual (media.affymetrix.com).…”

“…DNA microarray hybridization and statistical analysis was performed as described previously (Oberg et al, 2013). Sample hybridization was performed at the Center for Integrated Biosystems at Utah State University against a custom Affymetrix bifidobacterial DNA microarray designed to include 3113 shared and unique chromosomal genes predicted to occur in B. longum NCC2705 and D2957.…”

Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria

“…Cultivation of lactic acid bacteria and Bifidobacteria has been the subject of numerous studies considering their importance as probiotics [17,21,24,26]. Besides this application, some specific strains of Bifidobacteria are also becoming popular as functional foods [19].…”