The Limits of Acidification, Growth, and Stationary Phase Survival of Lactococcus cremoris

An important question in microbiology is if bacteria can optimize different traits simultaneously, or if this is prevented by a trade-off between these traits. For the dairy fermentation industry, it is of interest whether traits like acidification rate, flavor formation, and stationary phase survival trade-off, to see if all of these traits can be improved for increased functionality in fermentations. In this thesis we studies these traits for Lactococcus cremoris. The turbidity of milk prohibits the use of optical density measurements to characterize traits like: the inoculation density, the time to reach the maximal growth rate (lag-time), the specific acidification rate, and the maximal growth rate. To solve this issue, we cleared milk through centrifugation, resulting in a clear milk-like liquid, termed milk serum. We found that the behavior of lactic acid bacteria in milk serum was predictive of that in milk for 39 Lactococcus lactis strains (R2=0.81) and to a lesser extent for 42 Lactiplantibacillus plantarum strains (R2 = 0.49). Transcriptome and multi-sugar growth studies suggest that Lactococcus cremoris represses genes involved in the catabolization of lower growth rate supporting (lower quality) sugars in a hierarchical order. Vice versa, L. cremoris appears to often express genes related to the consumption of higher growth rate supporting (higher quality) sugars. We found that L. cremoris nearly always prepares for higher quality sugars, while most lower quality sugars were not prepared for. However, cells were never prepared for the high-quality sugar trehalose even during growth on lower quality sugars. In contrast, cells were always prepared for growth on fructose and lactose, even during growth on the highest quality sugar, glucose. These findings show that the catabolization hierarchy does not strictly follow what one would predict based on literature and it is also not strictly linked to the sugar quality. While starved cells have previously been found to synthesize stress-related proteins, acid-stationary cells did not. We found that only after six days protein synthesis capacity aids cell survival during starvation. We also found that subpopulations of stationary cells can synthesize protein without a nitrogen source, and that this subpopulation size decreases with increasing stationary phase length. Finally, we showed that acid-induced stationary cells remain energized and can maintain a pH gradient over their membrane, whereas starved cells cannot. We assumed throughout this thesis that the bacterial growth rate was limited by constraints on the cytosolic protein pool. We found that overexpressing dummy proteins to more than 5% of the total proteome did not decrease the growth rate of L. cremoris growing on glucose or galactose in chemically defined medium, which contains 20 amino acids. Similarly, we found no growth rate reduction upon protein overexpression in the rich medium M17 with glucose as carbon source. However, in contrast to CDMpc, in M17 nisin itself reduced the growth rate, potentially altering the proteome constraint with respect to M17 without nisin. We conclude that constraints on the cytosolic protein pool do not limit L. cremoris NZ9000 growth in CDMpc, which is in contrast with protein allocation studies in E. coli but consistent with earlier findings in L. lactis. Overall, this thesis shows that L. cremoris does not appear to behave optimally in regard to proteome allocation theory and growth rate maximization in chemically defined medium. Although in milk such a conclusion is more difficult to draw, the result of this thesis give hope that the growth rate and associated acidification rate can be increased further, without impacting traits like flavor formation and stationary phase survival, as long as these are also selected for. This points towards unused potential for industrial strain improvement.