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.

Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059

“…S5C). According to previous proteomic data on P. rubra S4059 (data are available via ProteomeXchange with identifier PXD023249), four extracellular proteases with high relative abundance were found in the samples of wild-type S4059 culture supernatants, including two peptidases ( A0A5S3UZL3 and A0A5S3V0P4 ), one putative alkaline serine protease ( A0A5S3V092 ), and a putative serine protease ( A0A5S3UW99 ) ( 20 ). The predicted molecular weights of those proteases were 53 to 64 kDa without signal peptides.…”

“…Not all bacteriocins are inactivated by treatment with proteinase K and pepsin as reported for several lactic acid bacteria ( 30 ). Previous proteomic analysis on P. rubra S4059 ( 20 ) (data are available via ProteomeXchange with identifier PXD023249) indicated that the strain produced several proteases that may have the same antibacterial function as that described in P. piscicida ( 25 ). Further studies with the molecular weight cutoff and protein analyses found that the antimicrobial activities may also be due to extracellular proteases/peptidases, which were often secreted by marine bacteria and reported with potent antibacterial activity ( 31 – 33 ).…”

“…The gene knockout strategy is followed from Wang et al ( 20 ). Briefly, approximately 1-kb upstream and downstream regions flanking pigC gene were amplified and fused by overlap extension PCR, and the recombining segment was inserted into pDM4 vector to construct the suicide plasmid by direct cloning method ( 36 , 38 ).…”

“…The protocol was modified from Wang et al ( 20 ). Briefly, P. rubra S4059 and the prodigiosin-deficient mutant Δ pigC were cultured in 100 ml MMM containing 0.2% mannose at 25°C, 200 rpm and samples were taken after 1, 2, and 3, days.…”

Molecular Characterization of Four Alkaline Chitinases from Three Chitinolytic Bacteria Isolated from a Mudflat

“…GH18 chitinases have been found in various organisms from bacteria to humans, including plants, molds, and vertebrates, while GH19 chitinases are found mainly in plants and are not common in bacteria, having recently been found in only a few strains, such as Chitinophaga sp. [ 4 ], Pseudoalteromonas rubra , [ 5 ], and Streptomyces alfalfa [ 6 ].…”