Plasmid genetics of Leuconostoc mesenteroides
Abstract
The contribution of Leuconostoc bacteria in milk fermentations is secondary, but essential for flavor and texture development. Genetic studies of Leuconostoc for strain improvement purposes have received only limited attention. The goal of this work was to generate gene transfer protocols (conjugation and transformation) and information concerning plasmid biology for recombinant DNA technology applications in this microorganism. Three broad-host-range self-transmissible plasmids were used to develop a conjugation protocol between Lactococcus and Leuconostoc species. High transfer frequencies (10$\sp{-1}$ to 10$\sp{-3}$ transconjugants per donor cell) were observed from Lactococcus lactis subsp. lactis to Leuconostoc mesenteroides subsp. cremoris. Reverse intergeneric transfer from Leu. mesenteroides subsp. cremoris to Lc. lactis subsp. lactis occurred at frequencies up to 10$\sp{-4}$ transconjugants per donor cell. Intrageneric transfer between Leu. mesenteroides subsp. cremoris strains was detected at frequencies as high as 10$\sp{-3}$ transconjugants per donor cell. Plasmid DNA was also introduced in Leu. mesenteroides by electroporation with frequencies ranging from $<$15 to 10$\sp6$ transformants per $\mu$g of DNA. The efficiency of such transfer procedures in Leu. mesenteroides should allow the implementation of cloning strategies already developed in other Gram-positive bacteria. Citrate metabolism, responsible for flavor production, was unstable and suspected to be linked to plasmid DNA in five strains of Leu. mesenteroides. Plasmid and restriction analysis revealed the loss of a 23-kb (pSD201) or a 21-kb (pSD211) plasmid in citrate-negative (Cit$\sp{-}$) mutants of Leu. mesenteroides subsp. mesenteroides. Hybridization experiments with an oligonucleotide probe based on a lactococcal citrate permease (citP) gene sequence indicated that both plasmids encoded a citrate permease. Apparent plasmid loss was not observed in Cit$\sp{-}$ isolates of Leu. mesenteroides subsp. cremoris or Leu. mesenteroides subsp. dextranicum. Neither plasmid nor genomic DNA from these subspecies hybridized with the probe suggesting that their citP genes differ from those in Lactococcus or Leu. mesenteroides subsp. mesenteroides. Subsequent genetic analyses (partial cloning and sequencing) revealed that the citP gene of pSD201 is very similar to those in other lactic acid bacteria.
Degree
Ph.D.
Advisors
Nelson, Purdue University.
Subject Area
Genetics|Food science
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