Research Article: Strand specific RNA-sequencing and membrane lipid profiling reveals growth phase-dependent cold stress response mechanisms in Listeria monocytogenes

Date Published: June 29, 2017

Publisher: Public Library of Science

Author(s): Patricia Hingston, Jessica Chen, Kevin Allen, Lisbeth Truelstrup Hansen, Siyun Wang, Shihui Yang.


The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and grow during refrigerated storage. Increased knowledge of the cold-stress response of this pathogen will enhance the ability to control it in the food-supply-chain. This study utilized strand-specific RNA sequencing and whole cell fatty acid (FA) profiling to characterize the bacterium’s cold stress response. RNA and FAs were extracted from a cold-tolerant strain at five time points between early lag phase and late stationary-phase, both at 4°C and 20°C. Overall, more genes (1.3×) were suppressed than induced at 4°C. Late stationary-phase cells exhibited the greatest number (n = 1,431) and magnitude (>1,000-fold) of differentially expressed genes (>2-fold, p<0.05) in response to cold. A core set of 22 genes was upregulated at all growth phases, including nine genes required for branched-chain fatty acid (BCFA) synthesis, the osmolyte transporter genes opuCBCD, and the internalin A and D genes. Genes suppressed at 4°C were largely associated with cobalamin (B12) biosynthesis or the production/export of cell wall components. Antisense transcription accounted for up to 1.6% of total mapped reads with higher levels (2.5×) observed at 4°C than 20°C. The greatest number of upregulated antisense transcripts at 4°C occurred in early lag phase, however, at both temperatures, antisense expression levels were highest in late stationary-phase cells. Cold-induced FA membrane changes included a 15% increase in the proportion of BCFAs and a 15% transient increase in unsaturated FAs between lag and exponential phase. These increases probably reduced the membrane phase transition temperature until optimal levels of BCFAs could be produced. Collectively, this research provides new information regarding cold-induced membrane composition changes in L. monocytogenes, the growth-phase dependency of its cold-stress regulon, and the active roles of antisense transcripts in regulating its cold stress response.

Partial Text

The human pathogen Listeria monocytogenes continues to pose a challenge in the food industry, where it is known to contaminate ready-to-eat foods and to grow during refrigerated storage. Ingestion of L. monocytogenes by susceptible individuals can cause potentially fatal food-borne infections with mortality rates as high as 40% reported [1]. The ubiquitous nature of this pathogen makes it difficult to eliminate from our food systems however, post-processing levels of L. monocytogenes contamination are often low [2–4] and unlikely to cause disease [5, 6]. Furthermore, the risks associated with L. monocytogenes contamination can be addressed by heating foods to an appropriate temperature [7]. Therefore, the ability of L. monocytogenes to grow to unsafe levels in ready-to-eat foods during the shelf-life of products, pose the greatest concern to consumer health.

Here we present results from the first time-course study to investigate the transcriptional response and associated cold-induced membrane FA changes of L. monocytogenes, from early growth phases through late stationary-phase. To the best of our knowledge this is also the first study to look at asRNA expression in L. monocytogenes during cold stress and across multiple growth phases. Our results revealed that the L. monocytogenes transcriptomic response to cold stress is most active during late stationary-phase survival. Similarly, we show that the L. monocytogenes cold-stress regulon differs greatly across growth phases, highlighting the importance of carefully selecting appropriate time points when designing and conducting transcriptome studies.