RESUMO
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.
Assuntos
Listeria monocytogenes , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Cobre , Humanos , Listeria monocytogenes/genética , Temperatura , Vitamina B 12/genética , Vitamina B 12/metabolismoRESUMO
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 List2-2Δ cbiP 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 List2-2Δ cbiP . 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.
Assuntos
Humanos , Listeria monocytogenes/genética , Temperatura , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vitamina B 12/genética , Vitamina B 12/metabolismo , Temperatura Baixa , CobreRESUMO
BACKGROUND: Despite the fact that most vitamins are present in a variety of foods, malnutrition, unbalanced diets or insufficient intake of foods are still the cause of vitamin deficiencies in humans in some countries. Vitamin B12 (Cobalamin) is a complex compound that is only naturally produced by bacteria and archea. It has been reported that certain strains belonging to lactic acid bacteria group are capable of synthesized water-soluble vitamins such as those included in the B-group, as vitamin B12. In this context, the goal of the present paper was to evaluate and characterize the production of vitamin B12 in Lactobacillus coryniformis CRL 1001, a heterofermentative strain isolated from silage. RESULTS: Cell extract of L. coryniformis CRL 1001, isolated from silage, is able to correct the coenzyme B12 requirement of Salmonella enterica serovar Typhimurium AR 2680 in minimal medium. The chemical characterization of the corrinoid-like molecule isolated from CRL 1001 cell extract using HPLC and mass spectrometry is reported. The majority of the corrinoid produced by this strain has adenine like Coα-ligand instead 5,6-dimethylbenzimidazole. Genomic studies revealed the presence of the complete machinery of the anaerobic biosynthesis pathway of coenzyme B12. The detected genes encode all proteins for the corrin ring biosynthesis and for the binding of upper (ß) and lower (α) ligands in one continuous stretch of the chromosome. CONCLUSIONS: The results here described show for the first time that L. coryniformis subsp. coryniformis CRL 1001 is able to produce pseudocobalamin containing adenine instead of 5,6-dimethlbenzimidazole in the Coα-ligand. Genomic analysis allowed the identification and characterization of the complete de novo biosynthetic pathway of the corrinoid produced by the CRL 1001 strain.
Assuntos
Lactobacillus/genética , Lactobacillus/metabolismo , Família Multigênica , Vitamina B 12/biossíntese , Vitamina B 12/genética , Adenina/metabolismo , Anaerobiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Benzimidazóis/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Cobamidas/genética , Cobamidas/metabolismo , Genes Bacterianos , Lactobacillus/isolamento & purificação , Espectrometria de Massas/métodos , Fases de Leitura Aberta , Salmonella enterica/genética , Salmonella enterica/metabolismo , Salmonella typhimurium/genética , Análise de Sequência de Proteína , Silagem/microbiologia , Vitamina B 12/química , Vitamina B 12/isolamento & purificaçãoRESUMO
This review examines the evidence suggesting that the anaerobic biosynthesis of cobalamin (vitamin B12) evolved during early stages of cell evolution and was quickly recruited in the pathway leading to deoxyribonucleotides, the building blocks of DNA genomes. Biochemical evolution preceding the synthesis of the heme group and related molecules is discussed within the framework of geological evolution in which the appearance and accumulation of an oxygen-rich atmosphere stands as one of the major events in the evolution of the planet and the biosphere.
Assuntos
Corrinoides/metabolismo , Heme/metabolismo , Animais , Corrinoides/genética , DNA/genética , DNA/metabolismo , Evolução Molecular , Evolução Planetária , Heme/genética , Humanos , Vitamina B 12/genética , Vitamina B 12/metabolismoRESUMO
BACKGROUND: Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class, which is at the base of the fungal phylogenetic tree. In this sense, some ancestral characteristics of fungi and animals or fungi and plants could have been retained in this aquatic fungus and lost in members of late-diverging fungal species. To identify in B. emersonii sequences associated with these ancestral characteristics two approaches were followed: (1) a large-scale comparative analysis between putative unigene sequences (uniseqs) from B. emersonii and three databases constructed ad hoc with fungal proteins, animal proteins and plant unigenes deposited in Genbank, and (2) a pairwise comparison between B. emersonii full-length cDNA sequences and their putative orthologues in the ascomycete Neurospora crassa and the basidiomycete Ustilago maydis. RESULTS: Comparative analyses of B. emersonii uniseqs with fungi, animal and plant databases through the two approaches mentioned above produced 166 B. emersonii sequences, which were identified as putatively absent from other fungi or not previously described. Through these approaches we found: (1) possible orthologues of genes previously identified as specific to animals and/or plants, and (2) genes conserved in fungi, but with a large difference in divergence rate in B. emersonii. Among these sequences, we observed cDNAs encoding enzymes from coenzyme B12-dependent propionyl-CoA pathway, a metabolic route not previously described in fungi, and validated their expression in Northern blots. CONCLUSION: Using two different approaches involving comparative sequence analyses, we could identify sequences from the early-diverging fungus B. emersonii previously considered specific to animals or plants, and highly divergent sequences from the same fungus relative to other fungi.