Characterization and interactions of lactic acid bacteria and bacteriophages isolated from a mixed-strain dairy starter culture

Tanya Ann Lodics, Purdue University

Abstract

Mixed-strain dairy starter cultures are complex ecosystems consisting of undefined mixtures of lactococcal and leuconostoc strains. They also often contain lytic bacteriophages that coexist with the bacterial strains in a phage-carrier state. We isolated and characterized lactic acid bacteria and carried bacteriophages from such a commercial starter. Interactions between the phages and various bacterial isolates were also investigated. We classified bacterial strains into genera and species by classical microbiological methods. Plasmid DNA analysis aided grouping of related isolates. The dominant flora consisted of 24 groups of Lactococcus lactis ssp. cremoris, 1 group of Lactococcus lactis ssp. lactis, 4 groups of Lactococcus lactis ssp. lactis biovar. diacetylactis, and 1 group of Leuconostoc cremoris strains. Strains within groups varied in their fermentation properties and bacteriophage resistance. We characterized one temperate and eight lytic bacteriophages by morphology, host ranges, growth characteristics, genomic physical structure, and protein profiles. Lytic phages belonged to two different groups based on morphotypes and DNA-DNA homology. Six L. lactis ssp. cremoris-specific phages were related to type phage P008. Two others specific to L. lactis ssp. lactis biovar. diacetylactis formed a unique type. The temperate phage shared slight DNA homology with representatives from both lytic phage morphotypes. All phage types had some DNA homology with type phage P270. To gain insight into the evolution of indigenous populations within such a complex mixture, we investigated how the starter culture populations changed with repeated transfer in milk. After sixty transfers, the bacterial population was less heterogenous, but the dominant surviving flora was phage-resistant. Representatives of two different phage morphotypes were carried throughout subculturing, but no newly-evolved phages were found. Phage resistance mechanisms of several lactococcal isolates were investigated. Mechanisms found include restriction-modification systems, reduced phage adsorption, and abortive infection. Because many genes are plasmid-encoded in the lactococci, we tested strains for conjugal transfer of phage resistance plasmids, and subjected them to plasmid-curing conditions. Most strains were either nonconjugative, or if conjugative, did not transfer phage resistance. Many had resilient plasmids that were difficult to cure. Nevertheless, we identified and mapped a plasmid encoding abortive infection.

Degree

Ph.D.

Advisors

Steenson, Purdue University.

Subject Area

Food science|Microbiology

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