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Lactobacillus johnsonii

ABSTRACT: Genome-wide transcriptome analysis of the oxygen response of L. johnsonii reveals that NADH oxidase is a secondary H2O2 source.

PROVIDER: PRJNA230384 | ENA |

REPOSITORIES: ENA

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Project description:Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus species is a well-known issue in the food industry. In this study, the transcriptional response to oxygen of Lactobacillus johnsonii, one of the H2O2-producing strains used in the food industry, was analyzed. It was found that aerobic growth conditions led to a more than two-fold downregulation of 45 genes as compared to anaerobic growth, whereas 6 genes were more than twofold upregulated. Among the upregulated genes were two genes that displayed significant homology to NADH-dependent oxidoreductase (NOX). The postulated transcriptional regulation of the nox promoter by oxygen was studied using a GUS-reporter construct, confirming a 2.1-fold upregulated GUS-expression upon aerobic growth. Exposure to sublethal levels of hydrogen peroxide did not result in significant regulation of the nox promoter. In a previous study of hydrogen peroxide production by L. johnsonii, a NADH flavin reductase (NFR) was identified to be involved in hydrogen peroxide production. An NFR-deficient derivative was strongly impaired in H2O2 production, but regained a partial H2O2 producing capacity upon prolonged oxygen exposure. The nox-promoter appeared to be 3.6-fold upregulated under aerobic conditions in the NFR-deficient background, which may imply a role of this gene in the regained H2O2 production. Indeed, deletion of the nox-gene in the NFR-deletion background, resulted in a strain that no longer produced H2O2, also during prolonged exposure to oxygen. The double-mutant (nfr, nox) displayed strongly impaired aerobic growth and oxygenation induced rapid growth stagnation that is not caused by H2O2. We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but can also involve an oxygen-inducible NADH oxidase under aerobic conditions. Moreover, our results imply that H2O2 production plays a prominent role in oxygen tolerance of L. johnsonii. loop design of the samples including two shortcuts

Project description:Work with pathogens like Vibrio cholerae has shown major differences between gene expression in bacteria grown in vitro and in vivo. To explore this subject for commensals, we investigated the transcription of the Lactobacillus johnsonii NCC533 genome during in vitro using the microarray technology. In broth growth, 537, 626 and 277 of the 1756 tested genes were expressed during exponential, “adaptation” (early stationary phase) and stationary phase, respectively. One hundred-one, 150 and 33 genes, respectively, were specifically transcribed in these three phases. Forty-four per cent of the NCC533 genome were not detectably transcribed under any of the investigated conditions. Non-transcribed genes were clustered on the genome and enriched in the variable genome part. For gene expression profiling experiments, cells were grown in a Sixfors fermentor system, composed of four individual 500 ml vessels (Infors, Bottmingen, Switzerland). Growth curves were performed at least in triplicate using the four separate fermentation vessels (fermentor 1 to 4), which were inoculated at 0.4% (v/v) with four individual overnight cultures in order to reach a starting optical density (OD600nm) of 0.05. Samples were taken at regular intervals from the four vessels to measure the OD600 nm and determine the CFU/ml until 36 h of fermentation. Aliquots of 15 ml (for early (T1-T2) and mid-exponential phases (T3-T4)) and 10 ml (for adaptation (T5), mid-stationary (T6) and late stationary phases (T7)) were centrifuged for 5 min at 10,000 x g at 4°C. Cell pellets were snap frozen in liquid nitrogen and stored at –80°C until further use.L. johnsonii NCC533 was grown in MRS broth supplemented with 2 % glucose as carbon source and 0.05% cysteine as redox buffer. For mRNA isolation L. johnsonii NCC533 cells were harvested from the broth culture several time points after inoculation (Fig. 1). In a total of 27 hybridization experiments. Genes were scored as expressed when their signal was detected in all hybridization experiments (with two technical replicates per slide) with mRNA preparations obtained from three independent growth experiments. Keywords: Growth phases of Lactobacillus johnsonii NCC533 in MRS

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Lactobacillus johnsonii

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