Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N

The nucleotide sequence of the pepN gene from lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated Mr of 95,368, which agrees with the apparent Mr of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

Indigenous and Environmental Modulation of Frequencies of Mutation in Lactobacillus plantarum▿ †

Reliability of microbial (starter) strains in terms of quality, functional properties, growth performance, and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing, such as acid, osmotic, temperature, and oxidative stresses. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution for survival under stress conditions. However, this may lead to accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effects of specific fermentation or processing conditions on the mutation frequency is an important step toward preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (N-methyl-N′-nitro-N-nitrosoguanidine) and by controlled expression of endogenous candidate mutator genes (e.g., a truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, under low-pH conditions, at high salt concentrations, or under starvation conditions did not have a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide resulted in a 100-fold increase in the mutation frequency compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42°C prior to treatment with sublethal levels of hydrogen peroxide, there was a 10-fold increase in survival after peroxide treatment, and there was a concomitant 50-fold decrease in the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be obtained by pretreatment of cultures with other, nonmutagenic stress conditions

Use of propidium monoazide for selective profiling of viable microbial cells during Gouda cheese ripening

DNA based microbial community profiling of food samples is confounded by the presence of DNA derived from membrane compromised (dead or injured) cells. Selective amplification of DNA from viable (intact) fraction of the community by propidium monoazide (PMA) treatment could circumvent this problem. Gouda cheese manufacturing is a proper model to evaluate the use of PMA for selective detection of intact cells since large fraction of membrane compromised cells emerges as a background in the cheese matrix during ripening. In this study, the effect of PMA on cheese community profiles was evaluated throughout manufacturing and ripening using quantitative PCR (qPCR). PMA effectively inhibited the amplification of DNA derived from membrane compromised cells and enhanced the analysis of the intact fraction residing in the cheese samples. Furthermore, a two-step protocol, which involves whole genome amplification (WGA) to enrich the DNA not modified with PMA and subsequent sequencing, was developed for the selective metagenome sequencing of viable fraction in the Gouda cheese microbial community. The metagenome profile of PMA treated cheese sample reflected the viable community profile at that time point in the cheese manufacturing

Low doses of diarrhoeagenic E. coli induce enhanced monocyte and mDC responses and prevent development of symptoms after homologous rechallenge

The experimental challenge with attenuated enterotoxigenic E. coli strain E1392/75-2A prevents diarrhea upon a secondary challenge with the same bacteria. A dose-response pilot study was performed to investigate which immunological factors are associated with this protection. Healthy subjects were inoculated with increasing E. coli doses of 1E6-1E10 CFU, and three weeks later, all participants were rechallenged with the highest dose (1E10 CFU). Gastrointestinal discomfort symptoms were recorded, and stool and blood samples were analyzed. After the primary challenge, stool frequency, diarrhea symptom scores, and E. coli-specific serum IgG (IgG-CFA/II) titer increased in a dose-dependent manner. Fecal calprotectin and serum IgG-CFA/II response after primary challenge were delayed in the lower dose groups. Even though stool frequency after the secondary challenge was inversely related to the primary inoculation dose, all E. coli doses protected against clinical symptoms upon rechallenge. Ex vivo stimulation of PBMCs with E. coli just before the second challenge resulted in increased numbers of IL-6+/TNF-α+ monocytes and mDCs than before the primary challenge, without dose-dependency. These data demonstrate that primary E. coli infection with as few as 1E6 CFU protects against a high-dose secondary challenge with a homologous attenuated strain. Increased serum IgG-CFA/II levels and E. coli-induced mDC and monocyte responses after primary challenge suggest that protection against secondary E. coli challenges is associated with adaptive as well as innate immune responses

Total fecal wet weight (A), % of fecal wet weight (%WW) (B), delta % fecal wet weight (Δ%WW) in interval I (C), delta % fecal wet weight (Δ%WW) in interval II (D), stool frequency (E), and stool consistency (BSS) (F), before and after dia.

E. coli challenges. The changes in total fecal wet weight, % fecal wet weight, and average Bristol Stool Score (BSS) were not dose-dependent after primary or secondary challenges. The stool frequency was positively related to the primary dia. E. coli dose (pE. coli challenge dose in interval I and decreased in interval II; checking the linear trend using the one-way ANOVA post-test, showed significance for interval II (p<0.05) but not interval I. The dotted lines represent challenges on day 14 and day 35 of the study, and each symbol represents the mean and SEM of the group. Data (A, B, E, and F) were analyzed using repeated-measures Generalized Estimating Equations (GEE) model and one-way ANOVA post-test was applied to check the linear trend the data in (C and D).</p

Variation in the percentage of double-positive monocytes within different primary infection group doses.

After ex vivo stimulation of PBMCs with either medium (RPMI-1640), 200 ng/mL of LPS, 500 ng/mL of Flagellin, or 1E7 CFU/well of E. coli (strain E1392/75-2A), the percentage of double-positive monocytes increased in all dose groups. This increase was significant after stimulation with flagellin and E. coli in group dose 1E6 and 1E7, flagellin in group dose 1E8, and following LPS, flagellin, ETEC stimulation in group 1E10. Data are shown in Whisker-plots with a median, 25% and 75% quartile. * p (TIF)</p

Baseline characteristics of study participants.

Baseline characteristics of study participants.</p

GSRS domain diarrhea (A), GSRS domain abdominal pain (B), and GSRS total daily score (C) before and after dia.

E. coli challenges. The increase in GSRS domain diarrhea score was significantly dose-dependent (P<0.05) after the first challenge but not after the second one. Following the primary challenge, both GSRS domain abdominal pain and GSRS total daily score also increased, without dose-dependency, where an increase was not noted after the second challenge. The dotted lines represent challenge days 14 and 35 of the study, and each symbol represents the mean and one-sided SEM of the group. Data were analyzed using repeated-measures Generalized Estimating Equations (GEE) model.</p