Genome-wide screening and identification of factors affecting the biosynthesis of prodigiosin by Hahella chejuensis, using Escherichia coli as a surrogate host

Abstract

A marine bacterium, Hahella chejuensis, recently has attracted attention due to its lytic activity against a red-tide dinoflagellate. The algicidal function originates from its red pigment, prodigiosin, which also exhibits immunosuppressive or anticancer activity. Genome sequencing and functional analysis revealed a gene set contained in the hap gene cluster that is responsible for the biosynthesis of prodigiosin. To screen for the factors affecting the prodigiosin biosynthesis, we constructed a plasmid library of the H. chejuensis genomic DNA, introduced it into Escherichia coli strains harboring the hap cluster, and observed changes in production of the red pigment. Among the screened clones, hapXY genes whose products constitute a two-component signal transduction system were elucidated as positive regulators of the pigment production. In addition, an Hfq-dependent, noncoding region located at one end of the hap cluster was confirmed to play roles in regulation. Identification of factors involved in the regulation of prodigiosin biosynthesis should help in understanding how the prodigiosin-biosynthetic pathway is organized and controlled and also aid in modulating the overexpression of prodigiosin in a heterologous host, such as E. coli, or in the natural producer, H. chejuensis.

FIG. 1.

Effects of two-component signal transduction system genes on prodrigiosin biosynthesis. (A) Domains of the histidine kinase HapX and the response regulator HapY. (B) Construction of in-frame deletion mutants. p8E02-G3 contains genes involved in a two-component signal transduction system that consists of a histidine kinase and a response regulator. A mutant, p8E02-G3ΔHK, lacking the His kinase A domain of the histidine kinase was constructed. Transcriptional regulatory domain for DNA binding of the response regulator was deleted to produce another mutant, p8E02-G3ΔRR. Arrowheads, directions of the plasmid-encoded lac promoters. (C) Spectrophotometric and colorimetric analysis for prodigiosin production by HC81008E02 containing p8E02-G3, p8E02-G3ΔHK, or p8E02-G3ΔRR. Prodigiosin production assay was conducted as described in Materials and Methods. Samples were prepared in three replicates, and error bars indicate the standard errors of the means. HC81008E02 does not produce prodigiosin, based on LC-MS/MS analysis (S.-K. Kwon and J. F. Kim, unpublished observation). HATPase, histidine kinase-like ATPase domain; HisKA, histidine kinase A domain; HAMP, histidine kinases, adenylyl cyclases, methyl binding proteins, and phosphatases domain; TcR, transcriptional regulatory protein, C terminal; RRR, response regulator receiver domain.

FIG. 2.

A regulatory region in the hap cluster that affects prodigiosin biosynthesis. (A) Candidate clones containing a region in the hap cluster that alters prodigiosin biosynthesis. Six plasmid clones contain hap genes or genes located in the hap cluster. Plasmids positively or negatively modulating pigment production share the common overlapping region, but these two types of clones have opposite directions of the plasmid-encoded lac promoters (shown by arrowheads). Plasmids having the promoter in the direction of hap transcription negatively affected pigment production; those having the promoter opposite the direction of hap transcription positively affected it. Filled arrows indicate the hap genes homologous to the pig genes of Serratia marcescens and to the red genes of Streptomyces coelicolor A3(2). (B) Plasmid maps of pSKK1 and pSKK2 and pigment production of HC81006F09 R4 (pSKK1), HC81008E02 (pSKK2), and HC81002H12 (pSKK2). The overlapping region common to all of these plasmids was cloned into pUC19 and pUC18 to produce pSKK1 and pSKK2, respectively. HC81006F09 R4 transformed with pSKK1 was turned off in pigment production. HC81008E02 or HC81002H12 carrying pSKK2 became constitutively red.

FIG. 3.

Effect of a noncoding region part of the hap cluster on prodigiosin biosynthesis. (A) Plasmid map of pSKK2/EAB150; (B) pigment production of HC81008E02 (pSKK2/EAB150) and HC81002H12 (pSKK2/EAB150). Through serial deletions of pSKK2, the minimum region that affects prodigiosin biosynthesis was elucidated. pSKK2/EAB150 carries this genomic region, and its effect on prodigiosin biosynthesis was confirmed by pigment production after transformation of this plasmid into HC81008E02 and HC81002H12.

FIG. 4.

Open reading frames present in the sequence of the pSKK2/EAB150 insert. Two ORFs that start with the CTG initiation codon, preceded by appropriate sequences that resemble the ribosome-binding site, were found in this intergenic region of 124 bases. Those ORFs were 36 and 15 codons long. Frame shift mutations were introduced into these ORFs to test the possibility that either of the two ORFs encodes a small protein (asterisks represent the bases deleted to release a mutant of each ORF).

FIG. 5.

Prodigiosin production by wild type and hfq mutants of HC81008E02, HC81002H12, and HC81006F09 R4 with or without the region carried by pSKK2 or pSKK1. Prodigiosin production assay was conducted as described in Materials and Methods (asterisks represent the samples whose ethanol extracts showed no indication of pigment production). Samples were prepared in three replicates, and error bars indicate the standard errors of the means.