*4.13. Chemical Analysis by UHPLC-HRMS*

The chemical analysis approach was modified from Holm et al. [55]. Chemical analysis was performed on a Bruker maXis 3G orthogonal acceleration quadrupole timeof-flight mass spectrometer (Bruker Daltonics, Billlerica, MA, USA) equipped with an electrospray ionization (ESI) source and connected to an Ultimate 3000 UHPLC system (Dionex, Sunnyvale, CA, USA). The column used was a reverse-phase Kinetex 1.7 μm C18, 100 mm × 2.1 mm (Phenomenex). The column temperature was kept at 40 ◦C throughout the analysis. A linear gradient of LC-MS grade water and acetonitrile both buffered with formic acid was used, starting at 10% (*v/v*) acetonitrile and increased to 100% in 10 min, maintaining this rate for 3 min before returning to the starting conditions in 0.1 min and staying there for 2.4 min before the following run. A flow rate of 0.40 mL/min was used. Time-of-flight mass spectrometry (TOFMS) was performed in ESI+ with a data acquisition range of 10 scans per second at *m*/*z* 75–1250. The TOFMS was calibrated using the Bruker Daltonics high precision calibration algorithm by means of the internal standard sodium formate, which was automatically infused before each run. This provided a mass accuracy of better than 1.5 ppm in MS mode. UV-visible spectra were collected at wavelengths from 200 to 700 nm. Data processing was performed using DataAnalysis 4.0 (Bruker Daltonics, Billerica, MA, USA) and Target Analysis 1.2 software (Bruker Daltonics). Tandem MS spectra were acquired on an Agilent 6545 QTOF-MS using the method described in Isbrandt et al. (2020) [56].

**Supplementary Materials:** The following are available online at https://www.mdpi.com/1660-339 7/19/2/108/s1, Table S1. The predicted biosynthetic gene clusters (BGCs) of Pseudoalteromonas rubra S4059 by antiSMASH 6.0. Table S2. The fold change of six significantly upregulated chitinolytic enzymes in cell or supernatant samples. Table S3. Significantly up-and downregulated proteins in Pseudoalteromonas rubra S4059 proteome when grown on chitin compared to on mannose. Table S4. Antibiotic sensitivity of Pseudoalteromonas rubra S4059 growth on an MB agar plate. Table S5. Bacteria and plasmids used in this study. Table S6. Primers used in this study. Table S7. The normalized protein abundance of chitinolytic enzymes in S4059. Figure S1. (A) Base peak chromatograms of Pseudoalteromonas rubra S4059 wild type (WT) and ΔGH19 mutant when cultivated on a marine minimal medium using mannose, crystalline chitin, colloidal chitin, or Nacetyl glucosamine (NAG) as the carbon source. The red pigment prodigiosin and two of its analogs (hexyl prodigiosin and heptyl prodigiosin) could be identified in the extract and confirmed based on MS/MS experiments and the acquired absorption spectra. Experiments were done in biological triplicates, and a reference chromatogram of the sterile growth medium was included to show media components also present in the experiments. (B) Tandem MS spectra recorded for (1) prodigiosin, (2) hexyl-prodigiosin, and (3) heptyl-prodigiosin. The prodigiosin MS/MS spectra matched our in-house MS/MS library, and the characteristic fragment m/z 252.11 and loss of CH3 (m/z 15.02) additionally matched those previously reported for prodigiosin and analogs [57]. (C) Recorded UV-Vis absorption spectrum of prodigiosin. All proposed prodigiosins share identical absorption spectra. The spectrum is in agreement with previously reported literature [58]. Figure S2. (A–D) Growth kinetics of Pseudoalteromonas rubra S4059 wild type (WT) and ΔGH19 mutant in mannose (A), colloidal chitin (B), crystalline chitin (crab chitin) (C), and NAG (chitin monomer) (D) containing

a marine minimal medium (MMM) with casamino acids. (E,F) WT and ΔGH19 growth in crystalline chitin (E) or colloidal chitin containing MMM without casamino acid. Square: WT; Triangle: ΔGH19. The points are bio-replicates, and error bars are standard deviation. (G,H) Chitin degradation activities of wild type and the mutant on colloidal chitin plate (G) and crystalline chitin plate (H). Figure S3. Biofilm formation on the microtiter-well plastic surface of Pseudoalteromonas rubra S4059 wild type and ΔGH19 mutant in four different sole carbon contained medium as determined by the O'Toole and Kolter crystal violet assay. (A) in mannose; (B) in NAG (chitin monomer); (C) in colloidal chitin; (D) in crab chitin. All experiments were in bio-triplicates, and error bars are standard deviation.

**Author Contributions:** X.W., S.S.P., S.-D.Z., and L.G. conceived and design the experiments; X.W. performed the experiments; T.I. and T.O.L. performed the chemical analysis; E.M.S. and M.W.N. performed the proteomic analyses. M.L.S. assembled the genome of *P. rubra* S4059 and supervised the statistical analysis. Y.B. assisted in approving that GH19 was expressed in *P. rubra* S4059 when grown on chitin. X.W. wrote the first manuscript draft; S.-D.Z. and L.G. supervised the edited and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by the Chinese Scholarship Council (CSC scholarship No. 201706170066), the Danish National Research Foundation (DNRF137) for the Center for Microbial Secondary Metabolites, the Independent Research Fund Denmark (grant DFF–7017-00003), and the European Union's Horizon 2020 research and Innovation Programme under the Marie Sklodowska-Curie grant agreement no. 713683 (COFUND fellows DTU) via the H. C. Ørsted fellowship program.

**Data Availability Statement:** Proteomic data are available via ProteomeXchange with identifier PXD023249 (http://www.proteomexchange.org/) and the genome of *Pseudoalteromonas rubra* S4059 is available at the National Center for Biotechnology Information (NCBI) under the accession number CP045429 (https://www.ncbi.nlm.nih.gov/nuccore/CP045429.1/ (accessed on 23 November 2020)) and CP045430 (https://www.ncbi.nlm.nih.gov/nuccore/CP045430.1/ (accessed on 23 November 2020)). Further inquiries can be directed to the corresponding author/s.

**Acknowledgments:** We gratefully acknowledge Youming Zhang and Zhen Li from the Shandong University for kindly providing the RecET direct cloning strains and vectors.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

