Analysis of rpoB polymorphism and PCR-based approaches for the identification of Leuconostoc mesenteroides at the species and subspecies level.

Leuconostoc mesenteroides includes the subsp. cremoris, subsp. dextranicum, subsp. mesenteroides and subsp. jonggajibkimchii, but the identification at the subspecies level using current phenotypic and/or genotypic methods is still difficult. In this study, a polyphasic approach based on the analysis of rpoB gene polymorphism, Multiplex-PCR and phenotypic tests was optimised and used to identify a collection of Leuc. mesenteroides strains at the species and subspecies levels. The annotation of published Leuc. mesenteroides genomes was also revised. A polymorphic region of rpoB gene was effective in separating Leuc. mesenteroides strains at the species (rpoB-species-specific-PCR) and subspecies (phylogenetic comparison) levels. Multiplex-PCR discriminated the subsp. mesenteroides from subsp. cremoris, but strains of uncertain attribution were found among subsp. dextranicum and subsp. jonggajibkimchii. Most of phenotypic features were not suitable for subspecies discrimination. Our assays may provide a rapid and reliable identification of subsp. mesenteroides and subsp. cremoris strains in fermented foods. The discrimination of subsp. dextranicum and subsp. jonggajibkimchii suffered from several limitations (e.g. low number of available strains and genomes, phenotypic profile close to subsp. mesenteroides, discrepancy between genotypic and phenotypic traits) and further investigations are needed to clarify their delineation and taxonomical position.

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.