Cobalamin cbiP mutant shows decreased tolerance to low temperature and copper stress in Listeria monocytogenes

Abstract

Background: Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans. This pathogen activates multiple regulatory mechanisms in response to stress, and cobalamin biosynthesis might have a potential role in bacterial protection. Low temperature is a strategy used in the food industry to control bacteria proliferation; however, L. monocytogenes can grow in cold temperatures and overcome different stress conditions. In this study we selected L. monocytogenes List2-2, a strain with high tolerance to the combination of low temperature + copper, to understand whether the cobalamin biosynthesis pathway is part of the tolerance mechanism to this stress condition. For this, we characterized the transcription level of three cobalamin biosynthesis-related genes (cbiP, cbiB, and cysG) and the eutV gene, a transcriptional regulator encoding gene involved in ethanolamine metabolism, in L. monocytogenes strain List2-2 growing simultaneously under two environmental stressors: low temperature (8 °C) + copper (0.5 mM of CuSO₄ × 5H₂O). In addition, the gene cbiP, which encodes an essential cobyric acid synthase required in the cobalamin pathway, was deleted by homologous recombination to evaluate the impact of this gene in L. monocytogenes tolerance to a low temperature (8 °C) + different copper concentrations.

Results: By analyzing the KEGG pathway database, twenty-two genes were involved in the cobalamin biosynthesis pathway in L. monocytogenes List2-2. The expression of genes cbiP, cbiB, and cysG, and eutV increased 6 h after the exposure to low temperature + copper. The cobalamin cbiP mutant strain List2-2ΔcbiP showed less tolerance to low temperature + copper (3 mM) than the wild-type L. monocytogenes List2-2. The addition of cyanocobalamin (5 nM) to the medium reverted the phenotype observed in List2-2ΔcbiP.

Conclusion: These results indicate that cobalamin biosynthesis is necessary for L. monocytogenes growth under stress and that the cbiP gene may play a role in the survival and growth of L. monocytogenes List2-2 at low temperature + copper.

Keywords: Cobalamin; Copper; Listeria monocytogenes; Low temperature.

Fig. 1

Temporal expression pattern of cobalamin biosynthesis-related genes in response to copper (0.5 mM) of L. monocytogenes List2-2 growing at 8 °C. A cysG, B cbiP, C cbiB, D eutV. Data were expressed as fold-change between copper treated at different times and untreated (time zero without copper). Different letters indicate significant differences comparing different times (1, 6 and 24 h). P-value: < 0.05

Fig. 2

Effect of different copper concentrations on the growth of L. monocytogenes strain List2-2 and List2-2ΔcbiP cultured in TSBYe agar at 8 °C without (A) or with (B) cyanocobalamin 5 nM cyanocobalamin. The blue rectangle shows the differences in bacterial growth between List2-2 wild-type and List2-2ΔcbiP in agar media without cobalamin. The red rectangle shows the bacterial growth between List2-2 wild-type and List2-2ΔcbiP in agar media with cobalamin