Chitin Degradation Machinery and Secondary Metabolite Profiles in the Marine Bacterium Pseudoalteromonas rubra S4059

Abstract

Genome mining of pigmented Pseudoalteromonas has revealed a large potential for the production of bioactive compounds and hydrolytic enzymes. The purpose of the present study was to explore this bioactivity potential in a potent antibiotic and enzyme producer, Pseudoalteromonas rubra strain S4059. Proteomic analyses (data are available via ProteomeXchange with identifier PXD023249) indicated that a highly efficient chitin degradation machinery was present in the red-pigmented P. rubra S4059 when grown on chitin. Four GH18 chitinases and two GH20 hexosaminidases were significantly upregulated under these conditions. GH19 chitinases, which are not common in bacteria, are consistently found in pigmented Pseudoalteromonas, and in S4059, GH19 was only detected when the bacterium was grown on chitin. To explore the possible role of GH19 in pigmented Pseudoalteromonas, we developed a protocol for genetic manipulation of S4059 and deleted the GH19 chitinase, and compared phenotypes of the mutant and wild type. However, none of the chitin degrading ability, secondary metabolite profile, or biofilm-forming capacity was affected by GH19 deletion. In conclusion, we developed a genetic manipulation protocol that can be used to unravel the bioactive potential of pigmented pseudoalteromonads. An efficient chitinolytic enzyme cocktail was identified in S4059, suggesting that this strain could be a candidate with industrial potential.

Keywords: Pseudoalteromonas; chitin; chitin degradation machinery; chitinase; secondary metabolites.

Figure 1

Comparison of identified proteins in the culture supernatant (A) and cells (B) of Pseudoalteromonas rubra S4059 when grown on chitin compared to on mannose. The dotted lines indicated that a threshold value for the cut off was a combination of p-value ≤ 0.05 and Log₂ fold-change ≥ 1.5. Red dots represent upregulated proteins, and blue dots represent downregulated proteins. Chitinolytic enzymes with significant fold changes are highlighted by enlarged dots with protein names in (A,B). Heat map comparison of identified chitinolytic enzymes in the supernatant and cells of Pseudoalteromonas rubra S4059 is color-coded by increasing abundance (C). C–WT–cell: cells of S4059 grown on chitin; M–WT–cell: cells of S4059 grown on mannose; C–WT–sup: culture supernatant of S4059 grown on chitin; M–WT–sup: culture supernatant of S4059 grown on mannose. White denotes proteins not detected under this condition. ‘Overlap’ indicates these proteins were detected in both chitin and mannose-grown samples, and an asterisk (*) highlights proteins significantly upregulated when grown on chitin compared to on mannose.

Figure 2

The in-frame deletion of the GH19 chitinase gene in Pseudoalteromonas rubra S4059 was verified by PCR with primers P1 and P2 that target the left and right homology arm of the GH19 chitinase gene (A). The PCR products were analyzed by electrophoresis (B). M: DNA ladder; NC: negative control with water as the PCR template; WT: PCR products with gDNA of wild type strain S4059 as a template; ΔGH19: PCR products with gDNA of ΔGH19 as a template. Growth kinetics of Pseudoalteromonas rubra S4059 wild type (red lines) and ΔGH19 (black lines) when grown in a marine minimal medium containing (C) colloidal chitin or (D) crystalline chitin (shrimp chitin) without casamino acids at 25 °C for 24 h, shaking at 200 rpm. Data were analyzed on three biological replicates, and the error bars represent the standard deviation.

Figure 3

Growth and attachment of Pseudoalteromonas rubra S4059 wild type (red) and mutant ∆GH19 (black) on Vannamei shrimp shells. Long dash lines: samples from liquid surrounding shrimp shells before sonication. Solid lines: samples from liquid surrounding shrimp shells after sonication. Short dash lines: samples from 3% Sigma sea salt without shrimp shells as a control. Data were analyzed on three biological replicates, and the error bars represent standard deviation.