Exceptional production of both prodigiosin and cycloprodigiosin as major metabolic constituents by a novel marine bacterium, Zooshikella rubidus S1-1

Abstract

A Gram-negative, red-pigment-producing marine bacterial strain, designated S1-1, was isolated from the tidal flat sediment of the Yellow Sea, Korea. On the basis of phenotypic, phylogenetic, and genetic data, strain S1-1 (KCTC 11448BP) represented a new species of the genus Zooshikella. Thus, we propose the name Zooshikella rubidus sp. nov. Liquid chromatography and mass spectrometry of the red pigments produced by strain S1-1 revealed that the major metabolic compounds were prodigiosin and cycloprodigiosin. In addition, this organism produced six minor prodigiosin analogues, including two new structures that were previously unknown. To our knowledge, this is the first description of a microorganism that simultaneously produces prodigiosin and cycloprodigiosin as two major metabolites. Both prodigiosin and cycloprodigiosin showed antimicrobial activity against several microbial species. These bacteria were approximately 1.5-fold more sensitive to cycloprodigiosin than to prodigiosin. The metabolites also showed anticancer activity against human melanoma cells, which showed significantly more sensitivity to prodigiosin than to cycloprodigiosin. The secondary metabolite profiles of strain S1-1 and two reference bacterial strains were compared by liquid chromatography-mass spectrometry. Multivariate statistical analyses based on secondary metabolite profiles by liquid chromatography-mass spectrometry indicated that the metabolite profile of strain S1-1 could clearly be distinguished from those of two phylogenetically related, prodigiosin-producing bacterial strains.

Fig. 1.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) results of the extracts from Zooshikella rubidus S1-1 were compared to those from Zooshikella ganghwensis KCTC 12044^T. (A) LC chromatogram of extracts from Zooshikella rubidus S1-1 (top) and Zooshikella ganghwensis KCTC 12044^T (bottom). The major compounds were separated by HPLC at the indicated retention times of 7.08 min (m/z 322, cycloprodigiosin) and 7.86 min (m/z 324, prodigiosin). (B) Mass spectra (top) and MS/MS fragmentation patterns (bottom) of cycloprodigiosin (left) and prodigiosin (right). (C) Molecular structures of cycloprodigiosin (left) and prodigiosin (right) identified by the MS/MS fragmentation study.

Fig. 2.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and identified structures of prodigiosin and six prodigiosin analogues in the extract of Zooshikella rubidus S1-1. The extracted mass chromatogram (first column), mass spectrum (second column), and MS/MS spectrum (third column) of prodigiosin (C) and six prodigiosin analogues (A, B, D, E, F, and G) were separated by HPLC at the indicated retention times. The predicted molecular structures are shown in the last column.

Fig. 3.

Effects of prodigiosin and cycloprodigiosin on microorganisms and cancer cell lines. Comparison of the antimicrobial activities of prodigiosin and cycloprodigiosin purified from Zooshikella rubidus S1-1 (A). DMSO was used as a vehicle solvent and negative control. Human melanoma cancer cells were treated with prodigiosin (B) and cycloprodigiosin (C) at various concentrations or with vehicle solvent (0.1% DMSO). All assays were performed in triplicate, and error bars represent standard deviations from the means.

Fig. 4.

The distinctiveness of Zooshikella rubidus S1-1. (A) Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the positions of Zooshikella rubidus S1-1, Zooshikella ganghwensis KCTC 12044^T, and other related taxa. Bootstrap values (expressed as percentages of 1,000 replications) that were greater than 50% are shown at branch points. Bar, 0.01 substitutions per nucleotide position. (B) Principal component analysis (PCA) score plot of LC-MS profiles of three prodigiosin-producing strains. The PCA shows differences in metabolite profiles among Hahella chejuensis KCTC 2396^T (▵), Zooshikella rubidus S1-1 (□), and Zooshikella ganghwensis KCTC 12044^T (○) in marine broth 2216 (n = 9). (C) Dendrogram based on hierarchical cluster analysis of metabolite profiles. (D) Thumbnail image of metabolite array.