Psychrotrophic strain of Janthinobacterium lividum from a cold Alaskan soil produces prodigiosin

Abstract

We have explored the microbial community in a nonpermafrost, cold Alaskan soil using both culture-based and culture-independent approaches. In the present study, we cultured >1000 bacterial isolates from this soil and characterized the collection of isolates phylogenetically and functionally. A screen for antibiosis identified an atypical, red-pigmented strain of Janthinobacterium lividum (strain BR01) that produced prodigiosin when grown at cool temperatures as well as strains (e.g., strain BP01) that are more typical of J. lividium, which produce a purple pigment, violacein. Both purple- and red-pigmented strains exhibited high levels of resistance to beta-lactam antibiotics. The prodigiosin pathway cloned from J. lividium BR01 was expressed in the heterologous host, Escherichia coli, and the responsible gene cluster differs from that of a well-studied prodigiosin producer, Serratia sp. J. lividum BR01 is the first example of a prodigiosin-producer among the beta-Proteobacteria. The results show that characterization of cultured organisms from previously unexplored environments can expand the current portrait of the microbial world.

FIG. 1.

The relative abundance of each phylum in the Alaskan soil culture collection isolated on 1/10th-tryptic soy agar (n = 273 16S rRNA gene sequences) and in a culture-independent, PCR-derived clone library (n = 1033 16S rRNA gene sequences) (Schloss and Handelsman, 2006). White bars represent sequences identified by culture-dependent methods, and black bars represent sequences identified by culture-independent methods. Candidate phyla OP10, WCHB1, BD group, and ACE have no sequenced representatives.

FIG. 2.

Maps of open reading frames (ORFs) from pBRbeta (33,133 bp) and pBPbeta (38,875 bp), aligned across homologous regions. The ORFs with dotted diagonal lines in each map encode putative β-lactamase repressor proteins (A), carboxypeptidases (B), and metallo-β-lactamases (LRA-20 and LRA-21). The asterisk below the map of pBPbeta designates the ORF that is missing in pBRbeta. ORFs filled with black, gray, and black dots have homology to hypothetical ORFs, regulatory proteins, and transport proteins, respectively. Those ORFs lacking a color have homology to a methyltransferase (1), hemerythrin-like protein (2), oxidoreductase (3), branched-chain amino acid aminotransferase (4), ribonuclease I (5), AMP-dependent synthetase and ligase (6), electron chain protein (7), adventurous gliding motility protein (8, 11), TPR repeat protein (9), alcohol dehydrogenase (10), ferrichrome-iron receptor (12), and isochorismate hydrolase (13).

FIG. 3.

Structures of compounds isolated in this study. Prodigiosin (A) and heptylprodigiosin (B) were purified from a culture of Janthinobacterium lividum BR01, and violacein (C) was purified from a culture of J. lividum BP01.

FIG. 4.

Maps of ORFs from pBRred (32,611 bp) (A) and pBPpur (32,163 bp) (B). The ORFs with diagonal lines correspond to homologues of the pig pathway, which encodes prodigiosin biosynthetic genes, and the ORFs with grid lines correspond to homologues of the vio pathway, which encodes violacein biosynthetic genes. ORFs filled with black, gray, and black dots have homology to hypothetical ORFs, regulatory proteins, and transport proteins, respectively. Those ORFs lacking fill have homology to a D-aspartate O-methyltransferase (1), rhodanase (2), glyoxalase (3), osmotically inducible protein OsmC (4), L-sorbosone dehydrogenase (5), NAD(+) kinase (6), RecN (7), class C β-lactamase (8), α-amylase (9), transposase (10, 11), DNA polymerase III ɛ subunit (12), ribonuclease H (13), and methyltransferase (14).