Growth fitness, heme uptake and genomic variants in mutants of oxygen-tolerant Lacticaseibacillus casei and Lactiplantibacillus plantarum strains.

Adaptive Laboratory Evolution (ALE) is a powerful tool to improve the fitness of industrially relevant microorganisms, because it circumvents some of the problems related to the use of genetically modified strains. In this study, we used an ALE strategy involving serial batch cultivations in aerobic and respiratory conditions to generate spontaneous mutants from the respiration-competent strain Lacticaseibacillus casei N87. Genotypic changes in selected mutants were investigated using whole genome sequencing (WGS). The O2-tolerant Lactiplantibacillus plantarum C17 and its mutant C17-m58 (obtained from a previous ALE study) were included in heme uptake experiments and in WGS and variant calling analyses. Several Lcb. casei N87 mutants cultivated under aerobic and respiratory conditions showed improved biomass production, O2 uptake and oxidative stress tolerance compared to the parental strain. Mutants of Lcb. casei and Lpb. plantarum differed from the parental strains in the ability to use heme and menaquinone. High heme concentrations (> 10 mg/L), however, were toxic for all strains. Single nucleotide modifications (SNPs) were detected in some genes encoding for proteins and transcriptional regulators involved in carbon metabolism, oxidative stress, redox balance, and cell wall properties, but their role in the evolved phenotypes needs further investigations. We conclude that prolonged adaptation to aerobic and respiratory life-style may be used as natural strategy to generate strains with improved O2-consuming ability and oxidative stress tolerance, two important features to develop robust cultures and to reduce oxidative processes in foods.

Aerobic and respirative growth of heterofermentative lactic acid bacteria: A screening study.

Heterofermentative lactic acid bacteria (76 strains) belonging to Lactobacillus, Leuconostoc and Weissella species which are important in fermentation, spoilage or as probiotics were screened in a factorial experiment for their ability to grow, produce catalase and consume oxygen in aerobiosis or in anaerobiosis, with or without supplementation with hemin and/or menaquinone in a medium containing glucose as a carbohydrate source. Aerobiosis improved growth with a few exceptions. The effect of supplementation with heme and/or menaquinone was strain specific and clear evidence of heme-boosted respiration was found in some cases. Heme-catalase was produced by strains of L. brevis, W. minor and Leuc. mesenteroides; some strains of the latter species produced non-heme catalase. Shaken flasks experiments showed that aerobic growth resulted in increased maximum growth rate and in a limited increase in biomass. Heme supplementation during aerobic growth resulted in a further increase in growth rate and final biomass only for a few strains; this was often related to catalase, which was also responsible for increased tolerance of H2O2. In both experiments we found evidence of heme toxicity, especially in anaerobiosis and in absence of menaquinone. Dose response curves for aerobic growth in the presence of combinations of hemin and menaquinone were non-monotonic, with growth stimulation at low doses of heme (<2.5 mg/l) and toxicity at higher doses. Menaquinone at 0.25-8 mg/l increased growth stimulation and partially reduced toxicity.

Investigation of Factors Affecting Aerobic and Respiratory Growth in the Oxygen-Tolerant Strain Lactobacillus casei N87.

Aerobic and respiratory cultivations provide benefits for some lactic acid bacteria (LAB). Growth, metabolites, enzymatic activities (lactate dehydrogenase; pyruvate and NADH oxidases, NADH peroxidase; catalase), antioxidant capability and stress tolerance of Lactobacillus casei N87 were evaluated in anaerobic, aerobic and respiratory (aerobiosis with heme and menaquinone supplementation) batch cultivations with different dissolved oxygen (DO) concentrations. The expression of pox (pyruvate oxidase) and cydABCD operon (cytochrome bd oxidase complex) was quantified by quantitative Real Time polymerase chain reaction. Respiration increased biomass production compared to anaerobiosis and unsupplemented aerobiosis, and altered the central metabolism rerouting pyruvate away from lactate accumulation. All enzymatic activities, except lactate dehydrogenase, were higher in respiratory cultures, while unsupplemented aerobiosis with 60% of DO promoted H2O2 and free radical accumulation. Respiration improved the survival to oxidative and freeze-drying stresses, while significant numbers of dead, damaged and viable but not cultivable cells were found in unsupplemented aerobic cultures (60% DO). Analysis of gene expression suggested that the activation of aerobic and respiratory pathways occurred during the exponential growth phase, and that O2 and hemin induced, respectively, the transcription of pox and cydABCD genes. Respiratory cultivation might be a natural strategy to improve functional and technological properties of L. casei.

Draft Genome Sequence of the Respiration-Competent Strain Lactobacillus casei N87.

Lactobacillus casei is used as a starter, adjunct, and/or probiotic culture in the production of fermented and functional foods. Here, we report the draft genome sequence of the respiration-competent strain L. casei N87, isolated from infant feces. This genome information may be useful for the study of respiratory metabolism in lactic acid bacteria.

Assessment of aerobic and respiratory growth in the Lactobacillus casei group.

One hundred eighty four strains belonging to the species Lactobacillus casei, L. paracasei and L. rhamnosus were screened for their ability to grow under aerobic conditions, in media containing heme and menaquinone and/or compounds generating reactive oxygen species (ROS), in order to identify respiratory and oxygen-tolerant phenotypes. Most strains were able to cope with aerobic conditions and for many strains aerobic growth and heme or heme/menaquinone supplementation increased biomass production compared to anaerobic cultivation. Only four L. casei strains showed a catalase-like activity under anaerobic, aerobic and respiratory conditions and were able to survive in presence of H2O2 (1 mM). Almost all L. casei and L. paracasei strains tolerated menadione (0.2 mM) and most tolerated pyrogallol (50 mM), while L. rhamnosus was usually resistant only to the latter compound. This is the first study in which an extensive screening of oxygen and oxidative stress tolerance of members of the L. casei group has been carried out. Results allowed the selection of strains showing the typical traits of aerobic and respiratory metabolism (increased pH and biomass under aerobic or respiratory conditions) and unique oxidative stress response properties. Aerobic growth and respiration may confer technological and physiological advantages in the L. casei group and oxygen-tolerant phenotypes could be exploited in several food industry applications.