Transcriptomic and metabolomic analyses to study the key role by which Ralstonia insidiosa induces Listeria monocytogenes to form suspended aggregates

Abstract

Ralstonia insidiosa can survive in a wide range of aqueous environments, including food processing areas, and is harmful to humans. It can induce Listeria monocytogenes to form suspended aggregates, resulting from the co-aggregation of two bacteria, which allows for more persistent survival and increases the risk of L. monocytogenes contamination. In our study, different groups of aggregates were analyzed and compared using Illumina RNA sequencing technology. These included R. insidiosa under normal and barren nutrient conditions and in the presence or absence of L. monocytogenes as a way to screen for differentially expressed genes (DEGs) in the process of aggregate formation. In addition, sterile supernatants of R. insidiosa were analyzed under different nutrient conditions using metabolomics to investigate the effect of nutrient-poor conditions on metabolite production by R. insidiosa. We also undertook a combined analysis of transcriptome and metabolome data to further investigate the induction effect of R. insidiosa on L. monocytogenes in a barren environment. The results of the functional annotation analysis on the surface of DEGs and qPCR showed that under nutrient-poor conditions, the acdx, puuE, and acs genes of R. insidiosa were significantly upregulated in biosynthetic processes such as carbon metabolism, metabolic pathways, and biosynthesis of secondary metabolites, with Log₂FC reaching 4.39, 3.96, and 3.95 respectively. In contrast, the Log₂FC of cydA, cyoB, and rpsJ in oxidative phosphorylation and ribosomal pathways reached 3.74, 3.87, and 4.25, respectively. Thirty-one key components were identified while screening for differential metabolites, which mainly included amino acids and their metabolites, enriched to the pathways of biosynthesis of amino acids, phenylalanine metabolism, and methionine metabolism. Of these, aminomalonic acid and Proximicin B were the special components of R. insidiosa that were metabolized under nutrient-poor conditions.

Keywords: Listeria monocytogenes; Ralstonia insidiosa; metabolome; sterile supernatants; suspended aggregates; transcriptome.

Figure 1

Formation of different suspended aggregates and changes of aggregation index. (A) Aggregates formed by bacteria cultured in tryptic soy broth (TSB); (B) Aggregates formed by bacteria cultured in 10% TSB; (C) The aggregation index of different groups; (D) Change in aggregation index every 6 h. 1–5 in (C, D) are R. insidiosa in TSB, R. insidiosa in 10% TSB, R. insidiosa with L. monocytogenes in TSB, R. insidiosa with L. monocytogenes in 10% TSB, and L. monocytogenes in 10% supernatant of R. insidiosa, respectively. *P < 0.05, **P < 0.01; NS, means not significant.

Figure 2

TEM and SEM images of suspended aggregates. (a, b) TEM of the aggregates formed by R. insidiosa and L. monocytogenes in 10% TSB with different scales; (c, d) TEM of the aggregates formed by L. monocytogenes with RIS in 10% TSB with different scales; (e) SEM of the planktonic bacteria R. insidiosa in 10% TSB; (f) SEM of the aggregates formed by R. insidiosa in 10% TSB; (g) SEM of the aggregates formed by R. insidiosa and L. monocytogenes in 10% TSB; (h) L. monocytogenes with RIS in 10% TSB.

Figure 3

Changes in DEGs expression. (A) Upregulation and downregulation of DEGs; (B) Venn diagram showing the co-regulation of DEGs in all comparison groups; (C) Volcano map of differentially expressed genes (significantly different genes with red notes indicate upregulation and blue dots indicate downregulation; the abscissa represents the fold change in gene expression in different samples, and the ordinate represents the statistical significance of the differences in gene expression); (D) Heatmaps of DEGs compared between different groups. Sample information: X was extracted from aggregates of R. insidiosa in TSB, Y was extracted from aggregates of R. insidiosa in 10% TSB, and Z was extracted from aggregates of R. insidiosa co-cultured with L. monocytogenes in 10% TSB.

Figure 4

Gene Ontology (GO) distribution map of differentially expressed genes (DEGs) in three main categories. The ordinate is the GO term, and the abscissa is the log₁₀(p-value) of DEGs in the term. X vs. Y is TSB vs. 10% TSB; Y vs. Z is RI vs. RI with LM.

Figure 5

Scatter plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment for DEGs. The number of DEGs in each pathway is closely related to the size of dots, and the color of dots reflects different q values. The rich factor was positively correlated with the enrichment degree. The smaller the q-value, the more significant the enrichment. X vs. Y is TSB vs. 10% TSB; Y vs. Z is RI vs. RI with LM.

Figure 6

Quality control of metabolomics data. (A) Heatmap showing the results of the clustering analysis of DAMs (horizontal: sample information, vertical: metabolite information, scale: the value of metabolite relative content after standardized processing; the redder the color is, the higher the content); (B) PCA score of DAMs (horizontal and vertical axes refer to the first and second principal components, respectively, and percentages; the values contributed by the principal components to the sample differences; the same color means the same component). A is TSB without inoculation; B is RIS of TSB; C is RIS of 20% TSB; D is RIS of 10% TSB.

Figure 7

Upregulation and downregulation of DAMs in different treatment groups. (A, B) Volcano plots of the regulated metabolites; (C, D) Heatmaps of the regulated metabolites; (E, F) Variations of the top 10 DAMs. The first row is the B vs. C group, and the second row is the B vs. D group. B vs. C is RIS of TSB vs. RIS of 20% TSB; B vs. D group is RIS of TSB vs. RIS of 10% TSB.

Figure 8

Different metabolite KEGG enrichment maps. The horizontal axis is the Rich factor corresponding to different pathways, the vertical axis is the name of the pathway, the shade of the color is proportional to the degree of enrichment, and the area of the dots reacts to the number of differential metabolites. B vs. C is RIS of TSB vs. RIS of 20% TSB; B vs. D group is RIS of TSB vs. RIS of 10% TSB.

Figure 9

Correlation analysis of transcriptomic and metabolomic. (A–C) The nine-quadrant diagram: horizontal coordinates represent the log₂FC of genes, and vertical coordinates represent the log₂FC of metabolites; (D–F) The bar charts of KEGG enrichment analysis. The horizontal coordinate represents the rich factor of differential metabolites and DEGs enriched to the pathway, and the vertical coordinate represents the name of the KEGG pathway.

Figure 10

Gene regulation of the formation of the suspended aggregates. The three connected squares represent the relevant gene changes in different groups. Red indicates upregulation, blue indicates downregulation, and green indicates both up and downregulation. Boxes with sharp corners indicate genes, and boxes with obtuse corners indicate metabolites.