Lactococcus lactis is used as a starter culture in most modern types of cheese. Its primary role is to acidify milk, which in addition to the action of the proteolytic enzyme rennet, causes the milk substrate to clot and form curd. Acidification is also important for the preservation of cheese, which if left as milk, would have a much shorter shelf life. L. lactis also contributes to cheese flavour and maturation. During the cheese making process L. lactis encounters a variety of stresses including high and low temperature, osmotic, acid, oxidative, and cell envelope stresses. The result of these stressors is damage to DNA, proteins, lipid membranes, peptidoglycan and other cellular components which consequently lead to reduced growth and acidification rates and when severe enough, cell death.
Investigation of L. lactis stress resistance mechanisms has heavily relied on temperature sensitive plasmid vectors as tools for genetic manipulation. Plasmid replication is inhibited at 37.5°C which is near the upper growth limit of L. lactis MG1363. In this study, during construction of several gene deletion mutants in L. lactis MG1363 using the temperature sensitive plasmid pG+host9, additional stable spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt hypersensitive phenotypes. Accumulation of these spontaneous mutations occurred as a consequence of the high temperature incubation required for plasmid integration into the L. lactis chromosome.
Whole genome sequencing of a strain displaying both heat resistance and salt hypersensitivity, followed by gene specific PCR and sequencing of four other mutants with these phenotypes revealed independent mutations in llmg_1816 in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family which has been shown in other bacteria to have c-di-AMP specific phosphodiesterase activity and to affect cell wall homeostasis. An independent llmg_1816 disruption mutant (Δ1816), created using a suicide plasmid, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid removal. L. lactis Δ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High temperature incubation of a wild-type industrial L. lactis strain was also found to result in spontaneous mutation of llmg_1816 and heat resistant and salt hypersensitive phenotypes, suggesting that this is not a strain specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by the llmg_1816 altered strain was inhibited by lower salt concentrations as compared to the parent strain. Salt hypersensitive L. lactis variant strains show potential for production of improved dairy fermentations, can be selected for relatively easily and are not considered to be genetically modified organisms.
Incubation of L. lactis at a temperature approaching its upper limit (37.5°C) to produce mutants with a stable heat resistant phenotype was further explored. A 3-log decline in colony count and increased colony size heterogeneity were observed after incubation of L. lactis MG1363 at 37.5°C for 48 hours as compared to incubation at 30°C over the same time period. Stable spontaneous heat resistant (but not salt sensitive) mutants were isolated from cultures grown at 37.5°C and were characterised. Whole genome sequencing of five heat resistant L. lactis strains revealed mutation of 11 loci, including 9 single nucleotide polymorphisms (SNPs), a large 32-kb deletion and t712 prophage duplication. Additional PCR analysis showed that out of a total of 22 heat resistant mutants, the large 32-kb deletion was present in 8 and tandem duplication of the t712 prophage was found in 12. Individual SNPs were found at low frequency. The fact that large scale chromosomal re-arrangements occur in independent heat resistant Lactococcus mutants suggests that this is a mechanism of adaptation of this bacteria to heat stress.
This research has brought to light the development of spontaneous heat resistance mutations during high temperature incubation which is required for the effective use of temperature sensitive plasmids. Given that temperature sensitive plasmids such as pG+host9 are particularly useful for the construction of target gene and random gene mutants and that additional spontaneous mutations compromise their validity, a rational approach was employed to modify existing methods in order to avoid development of heat resistance. Key changes to the mutant construction method include selection of non-heat resistant isolates following plasmid integration and elimination of a step requiring high temperature incubation. Mutation of llmg_0342 and llmg_1593 in L. lactis MG1363, involved in methionine and cysteine transport respectively, were used as examples and problems encountered during the procedure were documented. Provided that appropriate controls are used at key steps throughout the mutation process, temperature sensitive plasmids such as pG+host9 remain a valid and useful tool for mutant construction.