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

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

The design and application of a 50 K SNP chip for a threatened Aotearoa New Zealand passerine, the hihi

Next-generation sequencing has transformed the fields of ecological and evolutionary genetics by allowing for cost-effective identification of genome-wide variation. Single nucleotide polymorphism (SNP) arrays, or “SNP chips”, enable very large numbers of individuals to be consistently genotyped at a selected set of these identified markers, and also offer the advantage of being able to analyse samples of variable DNA quality. We used reduced representation restriction-aided digest sequencing (RAD-seq) of 31 birds of the threatened hihi (Notiomystis cincta; stitchbird) and low-coverage whole genome sequencing (WGS) of 10 of these birds to develop an Affymetrix 50 K SNP chip. We overcame the limitations of having no hihi reference genome and a low quantity of sequence data by separate and pooled de novo assembly of each of the 10 WGS birds. Reads from all individuals were mapped back to these de novo assemblies to identify SNPs. A subset of RAD-seq and WGS SNPs were selected for inclusion on the chip, prioritising SNPs with the highest quality scores whose flanking sequence uniquely aligned to the zebra finch (Taeniopygia guttata) genome. Of the 58,466 SNPs manufactured on the chip, 72% passed filtering metrics and were polymorphic. By genotyping 1,536 hihi on the array, we found that SNPs detected in multiple assemblies were more likely to successfully genotype, representing a cost-effective approach to identify SNPs for genotyping. Here, we demonstrate the utility of the SNP chip by describing the high rates of linkage disequilibrium in the hihi genome, reflecting the history of population bottlenecks in the species

Elucidation of novel physiological and genetic elements associated with the cold adaptability and survival of Listeria monocytogenes in the food processing continuum

Novel physiological and genetic factors associated with the survival of Listeria monocytogenes in the food-processing continuum were investigated, with an emphasis on its cold-growth ability. Food-related L. monocytogenes strains (n=166) were sequenced and subsequently evaluated on their ability to tolerate cold (4°C), salt (6% NaCl, 25°C), acid (pH 5, 25°C), and desiccation (33% RH, 20°C) stress. Stress tolerances were associated with serotype, clonal complex, full-length inlA profiles, and plasmid harbourage. Notably, strains possessing full length inlA (as opposed to a truncated version) exhibited significantly (p<0.001) enhanced cold tolerance and plasmid-positive strains demonstrated enhanced (p=0.013) acid tolerance. Relative gene expression indicated that several plasmid-encoded genes (e.g., NADH peroxidase, clpL, proW) are induced in L. monocytogenes during growth in 6% NaCl and at pH 5. Additionally, a whole-genome sequence phylogeny revealed closely related stress sensitive and tolerant strains, highlighting that minor genetic differences impact strain phenotypes. Strand-specific RNA sequencing showed that L. monocytogenes suppresses 1.3× more genes than it induces at 4°C relative to 20°C. The largest number (n=1,431) and greatest magnitude (>1,000-fold) of differentially expressed (e.g., >2-fold, p<0.05) genes occurred in late stationary-phase cells. A core set of 22 genes were upregulated at all five growth phases investigated and included nine genes required for branched-chain fatty acid (BCFA) synthesis. Correspondingly, BCFA levels increased by 15% during cold stress exposure. Transcription of antisense RNA (asRNA) was 2.5× higher in cells grown at 4°C relative to 20°C, with the most asRNA transcripts upregulated in lag phase cells. Spontaneous L. monocytogenes variants displaying enhanced cold tolerance (ECT) were isolated from a cold-sensitive strain culture following 84 days of storage at 4°C. While the parent strain had an impaired ability to produce BCFAs, the ECT variants were able to overcome this limitation which is hypothesized to be a result of mutations identified in acetyl-coA carboxylase. Collectively this work has improved our understanding of the response of L. monocytogenes to to cold stress and genotypes associated with stress-tolerance phenotypes. This information may be useful for developing biomarkers to quickly predict the risks associated with food isolates, or aid in developing new and/or improved intervention strategies.Land and Food Systems, Faculty ofGraduat

Comparative Analysis of Listeria monocytogenes Plasmids and Expression Levels of Plasmid-Encoded Genes during Growth under Salt and Acid Stress Conditions

Listeria monocytogenes strains are known to harbour plasmids that confer resistance to sanitizers, heavy metals, and antibiotics; however, very little research has been conducted into how plasmids may influence L. monocytogenes’ ability to tolerate food-related stresses. To investigate this, a library (n = 93) of L. monocytogenes plasmid sequences were compared. Plasmid sequences were divided into two groups (G1 and G2) based on a repA phylogeny. Twenty-six unique plasmid types were observed, with 13 belonging to each of the two repA-based groups. G1 plasmids were significantly (p 2-fold induction of plasmid-contained genes encoding an NADH peroxidase, cadmium ATPase, multicopper oxidase, and a ClpL chaperone protein during growth under salt (6% NaCl) and acid conditions (pH 5) and ProW, an osmolyte transporter, under salt stress conditions. No differences in salt and acid tolerance were observed between plasmid-cured and wildtype strains. This work highlights the abundance of specific plasmid types among food-related L. monocytogenes strains, the unique characteristics of G1 and G2 plasmids, and the possible contributions of plasmids to L. monocytogenes tolerance to food-related stresses.Land and Food Systems, Faculty ofNon UBCReviewedFacult

Genotypes associated with Listeria monocytogenes isolates displaying impaired or enhanced tolerances to cold, salt, acid, or desiccation stress

The human pathogen Listeria monocytogenes is a large concern in the food industry where its continuous detection in food products has caused a string of recalls in North America and Europe. Most recognized for its ability to grow in foods during refrigerated storage, L. monocytogenes can also tolerate several other food-related stresses with some strains possessing higher levels of tolerances than others. The objective of this study was to use a combination of phenotypic analyses and whole genome sequencing to elucidate potential relationships between L. monocytogenes genotypes and food-related stress tolerance phenotypes. To accomplish this, 166 L. monocytogenes isolates were sequenced and evaluated for their ability to grow in cold (4°C), salt (6% NaCl, 25°C), and acid (pH 5, 25°C) stress conditions as well as survive desiccation (33% RH, 20°C). The results revealed that the stress tolerance of L. monocytogenes is associated with serotype, clonal complex (CC), full length inlA profiles, and the presence of a plasmid which was identified in 55% of isolates. Isolates with full length inlA exhibited significantly (p < 0.001) enhanced cold tolerance relative to those harboring a premature stop codon (PMSC) in this gene. Similarly, isolates possessing a plasmid demonstrated significantly (p = 0.013) enhanced acid tolerance. We also identified nine new L. monocytogenes sequence types, a new inlA PMSC, and several connections between CCs and the presence/absence or variations of specific genetic elements. A whole genome single-nucleotide-variants phylogeny revealed sporadic distribution of tolerant isolates and closely related sensitive and tolerant isolates, highlighting that minor genetic differences can influence the stress tolerance of L. monocytogenes. Specifically, a number of cold and desiccation sensitive isolates contained PMSCs in σB regulator genes (rsbS, rsbU, rsbV). Collectively, the results suggest that knowing the sequence type of an isolate in addition to screening for the presence of full-length inlA and a plasmid, could help food processors and food agency investigators determine why certain isolates might be persisting in a food processing environment. Additionally, increased sequencing of L. monocytogenes isolates in combination with stress tolerance profiling, will enhance the ability to identify genetic elements associated with higher risk strains

Primers used for quantitative PCR validation of RNA-seq data.

<p>Primers used for quantitative PCR validation of RNA-seq data.</p

Transcription regulators significantly (p<0.05^*) overrepresented among genes differentially expressed at 4°C vs. 20°C.

<p>Transcription regulators significantly (p<0.05^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0180123#t007fn001" target="_blank">*</a>}) overrepresented among genes differentially expressed at 4°C vs. 20°C.</p