Disruption of the copper efflux pump (CopA) of Serratia marcescens ATCC 274 pleiotropically affects copper sensitivity and production of the tripyrrole secondary metabolite, prodigiosin

The prodigiosin biosynthetic gene cluster (pig cluster) of Serratia marcescens ATCC 274 (Sma 274) is flanked by cueR/copA homologues. Inactivation of the copA homologue resulted in an increased sensitivity to copper, confirming that CopA is involved in copper homeostasis in Sma 274. The effect of copper on the biosynthesis of prodigiosin in Sma 274 and the copA mutant strain was investigated. Increased levels of copper were found to reduce prodigiosin production in the wild type Sma 274, but increase production in the copA mutant strain. The physiological implications for CopA mediated prodigiosin production are discussed. We also demonstrate that the gene products of pigB–pigE of Sma 274 are sufficient for the biosynthesis of 2-methyl-3-n-amyl-pyrrole and condensation with 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde to form prodigiosin, as we have shown for Serratia sp. ATCC 39006.     

Genome-Wide Screening and Identification of Factors Affecting the Biosynthesis of Prodigiosin by Hahella chejuensis,Using Escherichia coli as a Surrogate Host

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.Harmful algal blooms (HABs), commonly called red tide, are a phenomenon in which toxin-producing marine algae rapidly proliferate in the offshore area. The HAB-causing phytoplanktons are reported to interact with other organisms such as bacteria and fungi. Among them, the marine bacteria are known to play important roles in decreasing or developing HABs (3, 5, 14). For instance, Hahella chejuensis, isolated from the coastal area of Marado in South Korea (15), is capable of killing Cochlodinium polykrikoides (12). C. polykrikoides is a major microalga that causes HABs, especially in the Northeast Pacific coastal area (8). The bacterial determinant that kills C. polykrikoides was further characterized as a red pigment referred to as prodigiosin (12). Prodigiosin belongs to a family of tripyrrole antibiotic molecules called prodiginines, which have potential as anticancer agents or immunosuppressants (24). The prodigiosin congener isolated from H. chejuensis also exerts an immunosuppressive effect (11).Through completed genome sequencing of H. chejuensis and its functional analysis, the genomic region involved in biosynthesis of prodigiosin was elucidated (12). This complete set of prodigiosin-biosynthetic genes was named the hap gene cluster. The red pigment prodigiosin was further characterized structurally, and the biosynthetic pathway was proposed by Kim and colleagues (13, 14). Genes of the hap cluster share homology with those in the pig cluster and the red cluster which are involved in prodiginine-biosynthetic intermediates of Serratia marcescens and Streptomyces coelicolor, respectively (7, 23, 25). Enzymes encoded by the genes in the pig and red clusters have been characterized (24). However, gene expression of the hap cluster can be tightly controlled, based on the observation that heterologous expression of the hap cluster alone failed to produce the pigment in Escherichia coli. The recombinant E. coli was able to produce the pigment only when the culture filtrate of H. chejuensis was added to the growth media (12). This result indicates that another regulatory cue is needed for prodigiosin biosynthesis, which prompted us to search for regulatory factors that modulate prodigiosin biosynthesis in H. chejuensis.In this study, regulatory factors for biosynthesis of prodigiosin in H. chejuensis were identified by functional screening. To search for such factors, a plasmid library derived from the genomic DNA of H. chejuensis was constructed and transformed into E. coli strains carrying the hap cluster. In the cases of Serratia marcescens and Streptomyces coelicolor, molecular inputs, such as cell-produced quorum-sensing signal molecules or two-component systems (TCSs) for signal transduction, have been verified as key regulatory signals for prodigiosin biosynthesis so far (4, 9, 10, 20-22). Similarly, some clones of interest uncovered in this study include molecular factors such as those that belong to the TCS. Also, we elucidated that an apparently noncoding region in the hap cluster functions as a key factor of prodigiosin biosynthesis.     

Evolution of the RpoS Regulon: Origin of RpoS and the Conservation of RpoS-Dependent Regulation in Bacteria

The RpoS sigma factor in proteobacteria regulates genes in stationary phase and in response to stress. Although of conserved function, the RpoS regulon may have different gene composition across species due to high genomic diversity and to known environmental conditions that select for RpoS mutants. In this study, the distribution of RpoS homologs in prokaryotes and the differential dependence of regulon members on RpoS for expression in two γ-proteobacteria (Escherichia coli and Pseudomonas aeruginosa) were examined. Using a maximum-likelihood phylogeny and reciprocal best hits analysis, we show that the RpoS sigma factor is conserved within γ-, β-, and δ-proteobacteria. Annotated RpoS of Borrelia and the enteric RpoS are postulated to have separate evolutionary origins. To determine the conservation of RpoS-dependent gene expression across species, reciprocal best hits analysis was used to identify orthologs of the E. coli RpoS regulon in the RpoS regulon of P. aeruginosa. Of the 186 RpoS-dependent genes of E. coli, 50 proteins have an ortholog within the P. aeruginosa genome. Twelve genes of the 50 orthologs are RpoS-dependent in both species, and at least four genes are regulated by RpoS in other γ-proteobacteria. Despite RpoS conservation in γ-, β-, and δ-proteobacteria, RpoS regulon composition is subject to modification between species. Environmental selection for RpoS mutants likely contributes to the evolutionary divergence and specialization of the RpoS regulon within different bacterial genomes.     

The PhoBR two-component system regulates antibiotic biosynthesis in Serratia in response to phosphate

Background  

Secondary metabolism in Serratia sp. ATCC 39006 (Serratia 39006) is controlled via a complex network of regulators, including a LuxIR-type (SmaIR) quorum sensing (QS) system. Here we investigate the molecular mechanism by which phosphate limitation controls biosynthesis of two antibiotic secondary metabolites, prodigiosin and carbapenem, in Serratia 39006.     

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator,DR1558

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H₂O₂). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H₂O₂, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.     

Comparative Genome Analyses of Serratia marcescens FS14 Reveals Its High Antagonistic Potential

S. marcescens FS14 was isolated from an Atractylodes macrocephala Koidz plant that was infected by Fusarium oxysporum and showed symptoms of root rot. With the completion of the genome sequence of FS14, the first comprehensive comparative-genomic analysis of the Serratia genus was performed. Pan-genome and COG analyses showed that the majority of the conserved core genes are involved in basic cellular functions, while genomic factors such as prophages contribute considerably to genome diversity. Additionally, a Type I restriction-modification system, a Type III secretion system and tellurium resistance genes are found in only some Serratia species. Comparative analysis further identified that S. marcescens FS14 possesses multiple mechanisms for antagonism against other microorganisms, including the production of prodigiosin, bacteriocins, and multi-antibiotic resistant determinants as well as chitinases. The presence of two evolutionarily distinct Type VI secretion systems (T6SSs) in FS14 may provide further competitive advantages for FS14 against other microbes. To our knowledge, this is the first report of comparative analysis on T6SSs in the genus, which identifies four types of T6SSs in Serratia spp.. Competition bioassays of FS14 against the vital plant pathogenic bacterium Ralstonia solanacearum and fungi Fusarium oxysporum and Sclerotinia sclerotiorum were performed to support our genomic analyses, in which FS14 demonstrated high antagonistic activities against both bacterial and fungal phytopathogens.     

Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly ¹⁴C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [carboxy-¹⁴C]-, and [Me-¹⁴C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.