Transcriptome Analysis of a Spray Drying-Resistant Subpopulation Reveals a Zinc-Dependent Mechanism for Robustness in L. lactis SK11

The viability of starter cultures is essential for an adequate contribution to the fermentation process and end-product. Therefore, robustness during processing and storage is an important characteristic of starter culture strains. For instance, during spray drying cells are exposed to heat and oxidative stress, generally resulting in loss of viability. In this study, we exposed the industrially relevant but stress-sensitive Lactococcus lactis strain SK11 to two cycles of heat stress, with intermediate recovery and cultivation at moderate temperatures. After these two cycles of heat exposure, the abundance of robust derivatives was increased as compared with the original culture, which enabled isolation of heat-resistant subpopulations displaying up to 1,000-fold enhanced heat stress survival. Moreover, this heat-resistant subpopulation demonstrated an increased survival during spray drying. Derivatives from two independent lineages displayed different transcriptome changes as compared with the wild type strain, indicating that the increased robustness within these lineages was established by different adaptive strategies. Nevertheless, an overlap in differential gene expression in all five derivatives tested in both lineages included three genes in an operon involved in zinc transport. The link between zinc homeostasis and heat stress survival in L. lactis was experimentally established by culturing of the wild type strain SK11 in medium with various levels of zinc ions, which resulted in alterations in heat stress survival phenotypes. This study demonstrates that robust derivatives of a relatively sensitive L. lactis strain can be isolated by repeated exposure to heat stress. Moreover, this work demonstrates that transcriptome analysis of these robust derivatives can provide clues for improvement of the robustness of the original strain. This could boost the industrial application of strains with specific desirable traits but inadequate robustness characteristics

Strain-Dependent Transcriptome Signatures for Robustness in Lactococcus lactis.

Recently, we demonstrated that fermentation conditions have a strong impact on subsequent survival of Lactococcus lactis strain MG1363 during heat and oxidative stress, two important parameters during spray drying. Moreover, employment of a transcriptome-phenotype matching approach revealed groups of genes associated with robustness towards heat and/or oxidative stress. To investigate if other strains have similar or distinct transcriptome signatures for robustness, we applied an identical transcriptome-robustness phenotype matching approach on the L. lactis strains IL1403, KF147 and SK11, which have previously been demonstrated to display highly diverse robustness phenotypes. These strains were subjected to an identical fermentation regime as was performed earlier for strain MG1363 and consisted of twelve conditions, varying in the level of salt and/or oxygen, as well as fermentation temperature and pH. In the exponential phase of growth, cells were harvested for transcriptome analysis and assessment of heat and oxidative stress survival phenotypes. The variation in fermentation conditions resulted in differences in heat and oxidative stress survival of up to five 10-log units. Effects of the fermentation conditions on stress survival of the L. lactis strains were typically strain-dependent, although the fermentation conditions had mainly similar effects on the growth characteristics of the different strains. By association of the transcriptomes and robustness phenotypes highly strain-specific transcriptome signatures for robustness towards heat and oxidative stress were identified, indicating that multiple mechanisms exist to increase robustness and, as a consequence, robustness of each strain requires individual optimization. However, a relatively small overlap in the transcriptome responses of the strains was also identified and this generic transcriptome signature included genes previously associated with stress (ctsR and lplL) and novel genes, including nanE and genes encoding transport proteins. The transcript levels of these genes can function as indicators of robustness and could aid in selection of fermentation parameters, potentially resulting in more optimal robustness during spray drying

Draft genome sequences of 24 Lactococcus lactis strains

Contains fulltext : 169962.pdf (publisher's version ) (Open Access)The lactic acid bacterium Lactococcus lactis is widely used for the production of fermented dairy products. Here, we present the draft genome sequences of 24 L. lactis strains isolated from different environments and geographic locations

Generic correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).

<p>Generic correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).</p

Effect of pH on final OD.

<p>Boxplots of final optical density (OD_final) of strains MG1363, IL1403, KF147 and SK11 in fermentations with an initial pH of 6.0 or 6.5.</p

Individual correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).

<p>Individual correlating gene expressions with robustness towards heat stress (A) or oxidative stress (B).</p

Heat stress survival of KF147 and SK11.

<p>Boxplots of robustness phenotypes towards 10 minutes of heat stress at relatively low and high oxygen levels for strain KF147 (A) and at various fermentation temperatures for strain SK11 (B). Robustness is expressed as the difference of log CFU/ml after stress (N_t) and before stress (N₀).</p

Effect of temperature on growth rate.

<p>Boxplots of maximum growth rate (μ_max in h^-1) of strains MG1363, IL1403, KF147 and SK11 in fermentations at 27, 30 and 35°C.</p

Fermentation conditions, growth characteristics and stress survival.

<p>Fermentation conditions, growth characteristics and stress survival.</p