Proteomic and Transcriptomic Analyses to Decipher the Chitinolytic Response of Jeongeupia spp

Abstract

In nature, chitin, the most abundant marine biopolymer, does not accumulate due to the action of chitinolytic organisms, whose saccharification systems provide instructional blueprints for effective chitin conversion. Therefore, discovery and deconstruction of chitinolytic machineries and associated enzyme systems are essential for the advancement of biotechnological chitin valorization. Through combined investigation of the chitin-induced secretome with differential proteomic and transcriptomic analyses, a holistic system biology approach has been applied to unravel the chitin response mechanisms in the Gram-negative Jeongeupia wiesaeckerbachi. Hereby, the majority of the genome-encoded chitinolytic machinery, consisting of various glycoside hydrolases and a lytic polysaccharide monooxygenase, could be detected extracellularly. Intracellular proteomics revealed a distinct translation pattern with significant upregulation of glucosamine transport, metabolism, and chemotaxis-associated proteins. While the differential transcriptomic results suggested the overall recruitment of more genes during chitin metabolism compared to that of glucose, the detected protein-mRNA correlation was low. As one of the first studies of its kind, the involvement of over 350 unique enzymes and 570 unique genes in the catabolic chitin response of a Gram-negative bacterium could be identified through a three-way systems biology approach. Based on the cumulative data, a holistic model for the chitinolytic machinery of Jeongeupia spp. is proposed.

Keywords: chitin; chitinase; chitinolytic; glycosidic hydrolase family 18; lytic polysaccharide monooxygenase; omics; proteomics; transcriptomics.

Figure 1

Predicted signal peptides of Jeongeupia wiesaeckerbachi in the chitin-induced extracellular proteome. Enzymes predicted to be translocated with the classical secretory pathways Sec, TAT, and Pilin or non-classical pathways (“Other”) by SignalP 6.0 are illustrated on the left side. The sum of all classically secreted enzymes (SignalP), non-classically secreted enzymes predicted by SecretomeP 2.0 with scores of >0.5, and actual non-classically secreted enzymes through comparison of the two algorithms are on the right. Non-secreted enzymes are assembled by the total minimal chitin induced proteome count (386) subtracted by the sum of all classical (148) and non-classical (44) translocated enzymes.

Figure 2

Heatmap (A) and volcano plot (B) of the differential intracellular proteomic analysis of Jeongeupia wiesaeckerbachi with chitin and glucose minimal media. (A) The heatmap was generated with PEAKS studios with the following parameters: Fold Change >2, Significance >20, Significance method ANOVA. Upregulated proteins are depicted in red and downregulated proteins in green. (B) Volcano plot of the same dataset. Dots in red represent proteins with a fold change >2 and a significance of >20% under glucose media; blue dots represent proteins, which are upregulated under chitin media conditions applying the same statistic thresholds. The top 5 most significant proteins are labelled with their respective gene ID. Refer to Table 2 for detailed information on gene functions.

Figure 3

Functional classification of intra- and extracellular proteins of Jeongeupia wiesaeckerbachi under chitin conditions according to GO-terms. Both datasets were gathered in quadruplicates. Intracellular proteomics data were evaluated differentially to glucose-supplied cells, with significance values >20% and >2 log2 fold changes; refer to the heatmap (Figure 2). Extracellular proteins depicted were shared amongst all samples. Category counts are shown in each individual pie chart. Totals under the pie charts refer to the annotated fractions of the total protein entries submitted to BlastKOALA, with 203 intracellularly and 386 extracellularly, corresponding to approximately 70% annotated proteins each.

Figure 4

Differential transcriptomics results of Jeongeupia wiesaeckerbachi supplied with glucose or chitin. (A) Heatmap of the top 1000 most variable transcripts detected in duplicates, (B) Volcano plot of the differential transcriptomic dataset. The respective top 5 most significantly transcribed genes in glucose (red dots) or chitin (blue dots) media are labelled with their gene accession number.

Figure 5

Schematic illustration of the proposed chitinolytic machinery of Jeongeupia wiesaeckerbachi. Extracellular proteins, depicted outside the bacteria cell, were detected in all triplicates cultivated in minimal media with colloidal chitin and a single sample cultivated with unbleached, decalcified crab shells. Intracellular proteins, illustrated inside the bacteria cell, were differentially upregulated with chitin minimal media compared to on glucose cultivated cells. Lightning bolts indicate signal transduction. Larger symbols indicate a higher significance or fold-change compared to enzymes of the same classification. Figure created with BioRender and Inkscape.