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

Genome-wide prediction and validation of sigma70 promoters in Lactobacillus plantarum WCFS1

Contains fulltext : 108029.pdf (publisher's version ) (Open Access)BACKGROUND: In prokaryotes, sigma factors are essential for directing the transcription machinery towards promoters. Various sigma factors have been described that recognize, and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor sigma(70) is commonly involved in transcription of the cell's housekeeping genes, which is mediated by the conserved sigma(70) promoter sequence motifs. In this study the sigma(70)-promoter sequences in Lactobacillus plantarum WCFS1 were predicted using a genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with transcription start sites inferred from the 5'-ends of transcripts detected by high-resolution tiling array transcriptome datasets. RESULTS: To identify sigma(70)-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focussing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved sigma(70)-promoter consensus. Position weight matrices-based models of the recovered sigma(70)-promoter sequence motif were employed to identify 3874 motifs with significant similarity (p-value<10(-4)) to the model-motif in the L. plantarum genome. Genome-wide transcript information deduced from whole genome tiling-array transcriptome datasets, was used to infer transcription start sites (TSSs) from the 5'-end of transcripts. By this procedure, 1167 putative TSSs were identified that were used to corroborate the transcriptionally active fraction of these predicted promoters. In total, 568 predicted promoters were found in proximity (</= 40 nucleotides) of the putative TSSs, showing a highly significant co-occurrence of predicted promoter and TSS (p-value<10(-263)). CONCLUSIONS: High-resolution tiling arrays provide a suitable source to infer TSSs at a genome-wide level, and allow experimental verification of in silico predicted promoter sequence motifs

Flow chart of promoter validation.

<p>Genome-wide motif search using MEME and MAST provided 3874 and 14962 predicted σ⁷⁰-promoters at p-value cut-offs of 10⁻⁴ and (10⁻⁴≤p-value<10⁻³), respectively. For validation of the predicted promoters, transcriptionally active regions (TARs) were detected using tiling array data of <i>L. plantarum</i> WCFS1 and for each TAR the transcription start site (TSS) was determined. After filtering the TSSs for low-expressed TARs, 621 TSSs were obtained for validation of predicted σ⁷⁰-promoters. At a low p-value cut-off (p-value<10⁻⁴), 568 σ⁷⁰-promoters were found that co-localized within a distance of 40 nt of TSSs. An additional 120 σ⁷⁰-promoters were found to co-localized within a distance of 40 nt of TSSs after raising the p-value cut-off (10⁻⁴≤p-value<10⁻³). For 94 TSSs, no co-localized σ⁷⁰-promoter (within a distance of 40 nt) was found.</p

Distribution of distances between promoters and TSSs.

<p>Distribution of distances (in nt) for 568 cases where a predicted σ⁷⁰-promoter (genome-wide search, p-values<10⁻⁴) and a TSS were found in proximity (≤60 nt) of each other. TSSs are divided into 5 groups (A, B, C, D, E) defined by the differences between the means of signal intensities upstream and downstream of the TSS. The 5 groups are the inter-percentile ranges (E: 0–20%, D: 21–40%, C:41–60%, B: 61–80%, A: 81–100%) of the ranked mean differences (see Methods). At distances above 40 nt the observed number of co-localized TSSs and promoters (≤10 counts) is equal to the expected number co-localized TSSs and promoters (see Methods).</p

Highly conserved motifs.

<p>Highly conserved motifs (E-value<10⁻⁸⁰) detected by MEME search in the upstream regions of protein-encoding genes (for a full list see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045097#pone.0045097.s004" target="_blank">Figure S1</a> in Supplementary results). Motifs are shown as Seqlogos <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045097#pone.0045097-Crooks1" target="_blank">[45]</a>.</p