Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H₂O₂). Each strain was exposed to a range of H₂O₂ concentrations (0-10 mM) to evaluate and compare intrinsic resistance to H₂O₂. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H₂O₂ concentration for 20 or 60 min followed by challenge at a lethal H₂O₂ concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H₂O₂ resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H₂O₂ resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H₂O₂ after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H₂O₂ treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.

T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04 T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BIphosphohydrolase and b-galactosidase activities. Total genomic DNA was sequenced from strain WDC04 T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer.

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...

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.