Discovery of New Secondary Metabolites from Marine Bacteria Hahella Based on an Omics Strategy

Abstract

Hahella is one characteristic genus under the Hahellaceae family and shows a good potential for synthesizing new natural products. In this study, we examined the distribution of the secondary metabolite biosynthetic gene cluster (SMBGC) under Hahella with anti-SMASH. The results derived from five genomes released 70 SMBGCs. On average, each strain contains 12 gene clusters, and the most abundant ones (45.7%) are from the family of non-ribosomal peptide synthetase (NRPS) and non-ribosomal peptide synthetase hybrid with polyketide synthase (NRPS/PKS), indicating a great potential to find bioactive compounds. The comparison of SMBGC between H. chejuensis and other species showed that H. chejuensis contained two times more gene clusters than H. ganghwensis. One strain, designed as NBU794, was isolated from the mangrove soil of Dongzhai Port in Haikou (China) by iChip. The 16S rRNA gene of NBU794 exhibited 99% identity to H. chejuensis KCTC 2396 and clustered with the H. chejuensis clade on the phylogenetic trees. Genome mining on strain NBU794 released 17 SMBGCs and two groups of bioactive compounds, which are chejuenolide A-C and nine prodiginines derivatives. The prodiginines derivatives include the well-known lead compound prodigiosin and two new compounds, 2-methyl-3-pentyl-4-O-methyl-prodiginine and 2-methyl-3-octyl-prodiginine, which were identified through fragmentation analysis based on LC-MS/MS. The anti-microbial activity assay showed prodigiosin and 2-methyl-3-heptyl-prodiginine exhibited the best performance in inhibiting Escherichia coli, Salmonella paratyphi B, MASA Staphylococcus aureus, Bacillus subtilis, and Candida albicans. Moreover, the yield of prodigiosin in H. chejuensis NBU794 was also evaluated, which could reach 1.40 g/L under the non-optimized condition and increase to 5.83 g/L in the modified ISP4 medium with macroporous adsorption beads added, indicating that NBU794 is a promising source of prodigiosin.

Keywords: Hahella; LC-MS/MS; chejuenlide; genome mining; prodiginine.

Figure 1

NRPS gene clusters and NRPS/PKS hybrid gene clusters are identified in Hahella ganghwensis DSM 17,046, Hahella chejuensis KCTC 2396, Hahella chejuensis KA22, Hahella chejuensis HN01, and Hahella sp.CCB-MM4. (A) A total of 15 NRPS gene clusters are identified and classified into eight groups based on the gene composition. (B) A total of 17 NRPS/PKS hybrid gene clusters are identified and classified into six groups based on the gene composition. The functional domains are indicated in bold letters: A, adenylation domain; AT, acyl-transferase; CAL, co-enzyme A ligase domain; C, condensation domain; cMT, carbon methyltransferase; DH, dehydratase; E, epimerization; ECH, enoyl-CoA hydratase/isomerase; ER, enoylreductase domain; GNAT, GCN5-related N-acetyltransferases domain; KR, ketoreductase; KS, ketosynthase; T, thiolation domain (peptidyl carrier protein); TAD, Trans-AT docking domain; TD, Terminal reductase domain; TE, thioesterase. The modular genes are marked with red color, while the other functional enzymes are indicated by squares with different colors.

Figure 2

Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of strain NBU794 and other strains in the genus Hahella. Bar, 0.005 represents nucleotide substitution rate (Knuc) units. Strains H. chejuensis NBU794 from this study are highlighted in bold. The figures of sample collection, strain morphology on the agar plate, and transmission electron microscopy results are listed on the left top corner.

Figure 3

LC-MS/MS analysis of prodiginine derivatives in H. chejuensis NBU794. (A) The nine UV peaks corresponding prodiginine derivatives were marked with arabic numbers, and the UV−Vis spectra of nine eluent peaks are listed on the right. (B) The MS/MS spectrums are listed on the left and compound structures with fragment information are listed on the right. Peak #1: 2-methyl-3-propyl-prodiginine; Peak #4: 2-methyl-3-hexyl-prodiginine; Peak #5: 2-methyl-3-octyl-prodiginine; Peak #6: Undecylprodiginine; Peak #7: prodigiosin; Peak #8: 2-methyl-3-butyl-prodiginine; Peak #9: 2-methyl-3-heptyl-prodiginine.

Figure 4

Structure elucidation of 2-methyl-3-pentyl-4-O-methyl-prodiginine, 2-methyl-3-octyl-prodiginine with LC-MS/MS. The product ion scans are listed on the left, the MS/MS spectrums are listed in the middle, and compound structures with fragment information are listed on the right.

Figure 5

Antimicrobial assay of prodigiosin (1), 2-methyl-3-heptyl-prodiginine (2), 2-methyl-3-propyl-prodiginine (3), 2-methyl-3-butyl-prodiginine (4). (−): to use DMSO as a negative control. (+): to use nystatin as a positive control in the antifungal test against Candida albicans; to use polymyxin as a positive control in the antibacterial test against Salmonella paratyphi B, Pseudomonas aeruginosa, Methicillin-resistant Staphylococcus aureus, Bacillus subtilis, and Escherichia coli.