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

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.

Biological Potential of Chitinolytic Marine Bacteria.

Chitinolytic microorganisms secrete a range of chitin modifying enzymes, which can be exploited for production of chitin derived products or as fungal or pest control agents. Here, we explored the potential of 11 marine bacteria (Pseudoalteromonadaceae, Vibrionaceae) for chitin degradation using in silico and phenotypic assays. Of 10 chitinolytic strains, three strains, Photobacterium galatheae S2753, Pseudoalteromonas piscicida S2040 and S2724, produced large clearing zones on chitin plates. All strains were antifungal, but against different fungal targets. One strain, Pseudoalteromonas piscicida S2040, had a pronounced antifungal activity against all seven fungal strains. There was no correlation between the number of chitin modifying enzymes as found by genome mining and the chitin degrading activity as measured by size of clearing zones on chitin agar. Based on in silico and in vitro analyses, we cloned and expressed two ChiA-like chitinases from the two most potent candidates to exemplify the industrial potential.

Production of the antimicrobial compound tetrabromopyrrole and the Pseudomonas quinolone system precursor, 2-heptyl-4-quinolone, by a novel marine species Pseudoalteromonas galatheae sp. nov.

Novel antimicrobials are urgently needed due to the rapid spread of antibiotic resistant bacteria. In a genome-wide analysis of Pseudoalteromonas strains, one strain (S4498) was noticed due to its potent antibiotic activity. It did not produce the yellow antimicrobial pigment bromoalterochromide, which was produced by several related type strains with which it shared less than 95% average nucleotide identity. Also, it produced a sweet-smelling volatile not observed from other strains. Mining the genome of strain S4498 using the secondary metabolite prediction tool antiSMASH led to eight biosynthetic gene clusters with no homology to known compounds, and synteny analyses revealed that the yellow pigment bromoalterochromide was likely lost during evolution. Metabolome profiling of strain S4498 using HPLC-HRMS analyses revealed marked differences to the type strains. In particular, a series of quinolones known as pseudanes were identified and verified by NMR. The characteristic odor of the strain was linked to the pseudanes. The highly halogenated compound tetrabromopyrrole was detected as the major antibacterial component by bioassay-guided fractionation. Taken together, the polyphasic analysis demonstrates that strain S4498 belongs to a novel species within the genus Pseudoalteromonas, and we propose the name Pseudoalteromonas galatheae sp. nov. (type strain S4498T = NCIMB 15250T = LMG 31599T).

Marine Chitinolytic Pseudoalteromonas Represents an Untapped Reservoir of Bioactive Potential.

Chitin is the most abundant polymer in the marine environment and a nutrient-rich surface for adhering marine bacteria. We have previously shown that chitin can induce the production of antibiotic compounds in Vibrionaceae, suggesting that the discovery of novel bioactive molecules from bacteria can be facilitated by mimicking their natural habitat. The purpose of this study was to determine the glycosyl hydrolase (GH) profiles of strains of the genus Pseudoalteromonas to enable selection of presumed growth substrates and explore possible links to secondary metabolism. Genomic analyses were conducted on 62 pigmented and 95 nonpigmented strains. Analysis of the total GH profiles and multidimensional scaling suggested that the degradation of chitin is a significant trait of pigmented strains, whereas nonpigmented strains seem to be driven toward the degradation of alga-derived carbohydrates. The genomes of all pigmented strains and 40 nonpigmented strains encoded at least one conserved chitin degradation cluster, and chitinolytic activity was phenotypically confirmed. Additionally, the genomes of all pigmented and a few nonpigmented strains encoded chitinases of the rare GH family 19. Pigmented strains devote up to 15% of their genome to secondary metabolism, while for nonpigmented species it was 3% at most. Thus, pigmented Pseudoalteromonas strains have a bioactive potential similar to that of well-known antibiotic producers of the Actinobacteria phylum. Growth on chitin did not measurably enhance the antibacterial activity of the strains; however, we demonstrated a remarkable co-occurrence of chitin degradation and the potential for secondary metabolite production in pigmented Pseudoalteromonas strains. This indicates that chitin and its colonizers of the Pseudoalteromonas genus represent a so far underexplored niche for novel enzymes and bioactive compounds.IMPORTANCE Infectious bacteria are developing and spreading resistance to conventional treatments at a rapid pace. To provide novel potent antimicrobials, we must develop new bioprospecting strategies. Here, we combined in silico and phenotypic approaches to explore the bioactive potential of the marine bacterial genus Pseudoalteromonas We found that pigmented strains in particular represent an untapped resource of secondary metabolites and that they also harbor an elaborate chitinolytic machinery. Furthermore, our analysis showed that chitin is likely a preferred substrate for pigmented species, in contrast to nonpigmented species. Potentially, chitin could facilitate the production of new secondary metabolites in pigmented Pseudoalteromonas strains.