ppGpp is a dual-role regulator involved in balancing iron absorption and prodiginine biosynthesis in Pseudoalteromonas.

Iron is an essential element for microbial survival and secondary metabolism. However, excess iron availability and overloaded secondary metabolites can hinder microbial growth and survival. Microorganisms must tightly control iron homeostasis and secondary metabolism. Our previous studies have found that the stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis by activating iron uptake in Pseudoalteromonas sp. strain R3. It is believed that the interaction between SspA and the small nucleotide ppGpp is important for iron to exert regulation functions. However, the roles of ppGpp in iron absorption and prodiginine biosynthesis, and the underlying relationship between ppGpp and SspA in strain R3 remain unclear. In this study, we found that ppGpp accumulation in strain R3 could be induced by limiting iron. In addition, ppGpp not only positively regulated iron uptake and prodiginine biosynthesis via increasing the SspA level but also directly repressed iron uptake and prodiginine biosynthesis independent of SspA, highlighting the finding that ppGpp can stabilize both iron levels and prodiginine production. Notably, the abolishment of ppGpp significantly increased prodiginine production, thus providing a theoretical basis for manipulating prodiginine production in the future. This dynamic ppGpp-mediated interaction between iron uptake and prodiginine biosynthesis has significant implications for understanding the roles of nutrient uptake and secondary metabolism for the survival of bacteria in unfavorable environments.

ROK family regulator NagC promotes prodigiosin biosynthesis independent of N-acetylglucosamine in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 is an important model strain for the study of prodigiosin production, whose prodigiosin biosynthesis genes (pigA-O) are arranged in an operon. Several transcription factors have been shown to control the transcription of the pig operon. However, since the regulation of prodigiosin biosynthesis is complex, the regulatory mechanism for this process has not been well established. In most γ-proteobacteria, the ROK family regulator NagC acts as a global transcription factor in response to N-acetylglucosamine (GlcNAc). In Serratia sp. ATCC 39006, NagC represses the transcription of two divergent operons, nagE and nagBAC, which encode proteins involved in the transport and metabolism of GlcNAc. Moreover, NagC directly binds to a 21-nt region that partially overlaps the -10 and -35 regions of the pig promoter and promotes the transcription of prodigiosin biosynthesis genes, thereby increasing prodigiosin production. Although NagC still acts as both repressor and activator in Serratia sp. ATCC 39006, its transcriptional regulatory activity is independent of GlcNAc. NagC was first found to regulate antibiotic biosynthesis in Gram-negative bacteria, and NagC-mediated regulation is not responsive to GlcNAc, which contributes to future studies on the regulation of secondary metabolism by NagC in other bacteria. IMPORTANCE: The ROK family transcription factor NagC is an important global regulator in the γ-proteobacteria. A large number of genes involved in the transport and metabolism of sugars, as well as those associated with biofilm formation and pathogenicity, are regulated by NagC. In all of these regulations, the transcriptional regulatory activity of NagC responds to the supply of GlcNAc in the environment. Here, we found for the first time that NagC can regulate antibiotic biosynthesis, whose transcriptional regulatory activity is independent of GlcNAc. This suggests that NagC may respond to more signals and regulate more physiological processes in Gram-negative bacteria.

Functional connexion of bacterioferritin in antibiotic production and morphological differentiation in Streptomyces coelicolor.

BACKGROUND: Several two-component systems of Streptomyces coelicolor, a model organism used for studying antibiotic production in Streptomyces, affect the expression of the bfr (SCO2113) gene that encodes a bacterioferritin, a protein involved in iron storage. In this work, we have studied the effect of the deletion mutant ∆bfr in S. coelicolor. RESULTS: The ∆bfr mutant exhibits a delay in morphological differentiation and produces a lesser amount of the two pigmented antibiotics (actinorhodin and undecylprodigiosin) compared to the wild type on complex media. The effect of iron in minimal medium was tested in the wild type and ∆bfr mutant. Consequently, we also observed different levels of production of the two pigmented antibiotics between the two strains, depending on the iron concentration and the medium (solid or liquid) used. Contrary to expectations, no differences in intracellular iron concentration were detected between the wild type and ∆bfr mutant. However, a higher level of reactive oxygen species in the ∆bfr mutant and a higher tolerance to oxidative stress were observed. Proteomic analysis showed no variation in iron response proteins, but there was a lower abundance of proteins related to actinorhodin and ribosomal proteins, as well as others related to secondary metabolite production and differentiation. Additionally, a higher abundance of proteins related to various types of stress, such as respiration and hypoxia among others, was also revealed. Data are available via ProteomeXchange with identifier PXD050869. CONCLUSION: This bacterioferritin in S. coelicolor (Bfr) is a new element in the complex regulation of secondary metabolism in S. coelicolor and, additionally, iron acts as a signal to modulate the biosynthesis of active molecules. Our model proposes an interaction between Bfr and iron-containing regulatory proteins. Thus, identifying these interactions would provide new information for improving antibiotic production in Streptomyces.

An overview of the two-component system GarR/GarS role on antibiotic production in Streptomyces coelicolor.

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein-protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. KEY POINTS: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED.

Insights into the Genome of a New Strain Serratia rubidaea XU1 Isolated from Radioactive Soil and its Prodigiosin Production and Antimicrobial Properties.

The genus Serratia is a typical red bacterium involved in prodigiosin synthesis. Here, we report the genome sequence of Serratia rubidaea XU1, which was isolated from radiation-contaminated soil in Xinjiang, China. The genome of XU1 is composed of 4,972,898 base pairs with a GC content of 59.25%. The genome sequence contains 4707 genes and encodes 4573 proteins, 79 tRNAs, and 17 rRNAs. The prodigiosin biosynthesis gene cluster was identified and analyzed, showing a sequence similarity of 85.55-96.02% with Serratia rubidaea. After optimizing the biosynthesis process, XU1 was able to achieve a maximum titer of 574 units/cell of prodigiosin at a pH of 7.5 and a temperature of 25 °C for 36 h. Glycerol at 20 g/L and beef extract at 5 g/L were used as the carbon and nitrogen sources, respectively. Prodigiosin extracted from XU1 demonstrated inhibition of Escherichia coli, Staphylococcus aureus, Enterococcus faecalis and Pseudomonas aeruginosa. The availability of the sequenced genome of XU1 will be greatly beneficial and contribute to complementary studies on the biosynthetic mechanisms of prodigiosin.

PsrA is a novel regulator contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in Serratia marcescens.

Serratia marcescens is a Gram-negative bacterium of the Enterobacteriaceae family that can produce numbers of biologically active secondary metabolites. However, our understanding of the regulatory mechanisms behind secondary metabolites biosynthesis in S. marcescens remains limited. In this study, we identified an uncharacterized LysR family transcriptional regulator, encoding gene BVG90_12635, here we named psrA, that positively controlled prodigiosin synthesis in S. marcescens. This phenotype corresponded to PsrA positive control of transcriptional of the prodigiosin-associated pig operon by directly binding to a regulatory binding site (RBS) and an activating binding site (ABS) in the promoter region of the pig operon. We demonstrated that L-proline is an effector for the PsrA, which enhances the binding affinity of PsrA to its target promoters. Using transcriptomics and further experiments, we show that PsrA indirectly regulates pleiotropic phenotypes, including serrawettin W1 biosynthesis, extracellular polysaccharide production, biofilm formation, swarming motility and T6SS-mediated antibacterial activity in S. marcescens. Collectively, this study proposes that PsrA is a novel regulator that contributes to antibiotic synthesis, bacterial virulence, cell motility and extracellular polysaccharides production in S. marcescens and provides important clues for future studies exploring the function of the PsrA and PsrA-like proteins which are widely present in many other bacteria.

Improving Bioprocess Conditions for the Production of Prodigiosin Using a Marine Serratia rubidaea Strain.

The enormous potential attributed to prodigiosin regarding its applicability as a natural pigment and pharmaceutical agent justifies the development of sound bioprocesses for its production. Using a Serratia rubidaea strain isolated from a shallow-water hydrothermal vent, optimization of the growth medium composition was carried out. After medium development, the bacterium temperature, light and oxygen needs were studied, as was growth inhibition by product concentration. The implemented changes led to a 13-fold increase in prodigiosin production in a shake flask, reaching 19.7 mg/L. The conditions allowing the highest bacterial cell growth and prodigiosin production were also tested with another marine strain: S. marcescens isolated from a tide rock pool was able to produce 15.8 mg/L of prodigiosin. The bioprocess with S. rubidaea was scaled up from 0.1 L shake flasks to 2 L bioreactors using the maintenance of the oxygen mass transfer coefficient (kLa) as the scale-up criterion. The implemented parameters in the bioreactor led to an 8-fold increase in product per biomass yield and to a final concentration of 293.1 mg/L of prodigiosin in 24 h.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4'-phosphopantetheine, 4'-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker-ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein-protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

Two component system CpxR/A regulates the prodigiosin biosynthesis by negative control in Serratia marcescens FS14.

Prodigiosin is a tripyrrole red secondary metabolite synthesized by many microorganisms, including Serratia marcescens. In this study, we found that the deletion of the gene of sensor kinase CpxA dramatically decreased the prodigiosin production, while the deletion of the gene of the response regulator CpxR or both genes of CpxRA has no effect on prodigiosin production, the kinase function of CpxA is not essential for its regulation on prodigiosin production while the phosphorylation site of CpxR is required. We further demonstrated that the CpxA regulates the prodigiosin biosynthesis at the transcriptional level and the phosphatase activity of CpxA plays vital roles in the regulation of prodigiosin biosynthesis. Finally, we proposed that CpxR/A regulates the prodigiosin biosynthesis by negative control and the phosphorylation level of CpxR may determine the positive or negative control of the genes it regulated.

Stepwise genetic engineering of Pseudomonas putida enables robust heterologous production of prodigiosin and glidobactin A.

Polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS) comprise biosynthetic pathways that provide access to diverse, often bioactive natural products. Metabolic engineering can improve production metrics to support characterization and drug-development studies, but often native hosts are difficult to genetically manipulate and/or culture. For this reason, heterologous expression is a common strategy for natural product discovery and characterization. Many bacteria have been developed to express heterologous biosynthetic gene clusters (BGCs) for producing polyketides and nonribosomal peptides. In this article, we describe tools for using Pseudomonas putida, a Gram-negative soil bacterium, as a heterologous host for producing natural products. Pseudomonads are known to produce many natural products, but P. putida production titers have been inconsistent in the literature and often low compared to other hosts. In recent years, synthetic biology tools for engineering P. putida have greatly improved, but their application towards production of natural products is limited. To demonstrate the potential of P. putida as a heterologous host, we introduced BGCs encoding the synthesis of prodigiosin and glidobactin A, two bioactive natural products synthesized from a combination of PKS and NRPS enzymology. Engineered strains exhibited robust production of both compounds after a single chromosomal integration of the corresponding BGC. Next, we took advantage of a set of genome-editing tools to increase titers by modifying transcription and translation of the BGCs and increasing the availability of auxiliary proteins required for PKS and NRPS activity. Lastly, we discovered genetic modifications to P. putida that affect natural product synthesis, including a strategy for removing a carbon sink that improves product titers. These efforts resulted in production strains capable of producing 1.1 g/L prodigiosin and 470 mg/L glidobactin A.

Regulator RcsB Controls Prodigiosin Synthesis and Various Cellular Processes in Serratia marcescens JNB5-1.

Prodigiosin (PG), a red linear tripyrrole pigment normally secreted by Serratia marcescens, has received attention for its reported immunosuppressive, antimicrobial, and anticancer properties. Although several genes have been shown to be important for prodigiosin synthesis, information on the regulatory mechanisms behind this cellular process remains limited. In this work, we identified that the transcriptional regulator RcsB encoding gene BVG90_13250 (rcsB) negatively controlled prodigiosin biosynthesis in S. marcescens Disruption of rcsB conferred a remarkably increased production of prodigiosin. This phenotype corresponded to negative control of transcription of the prodigiosin-associated pig operon by RcsB, probably by binding to the promoter region of the prodigiosin synthesis positive regulator FlhDC. Moreover, using transcriptomics and further experiments, we revealed that RcsB also controlled some other important cellular processes, including swimming and swarming motilities, capsular polysaccharide production, biofilm formation, and acid resistance (AR), in S. marcescens Collectively, this work proposes that RcsB is a prodigiosin synthesis repressor in S. marcescens and provides insight into the regulatory mechanism of RcsB in cell motility, capsular polysaccharide production, and acid resistance in S. marcescensIMPORTANCE RcsB is a two-component response regulator in the Rcs phosphorelay system, and it plays versatile regulatory functions in Enterobacteriaceae However, information on the function of the RcsB protein in bacteria, especially in S. marcescens, remains limited. In this work, we illustrated experimentally that the RcsB protein was involved in diverse cellular processes in S. marcescens, including prodigiosin synthesis, cell motility, capsular polysaccharide production, biofilm formation, and acid resistance. Additionally, the regulatory mechanism of the RcsB protein in these cellular processes was investigated. In conclusion, this work indicated that RcsB could be a regulator for prodigiosin synthesis and provides insight into the function of the RcsB protein in S. marcescens.

Stringent Starvation Protein Regulates Prodiginine Biosynthesis via Affecting Siderophore Production in Pseudoalteromonas sp. Strain R3.

Prodiginines are a family of red-pigmented secondary metabolites with multiple biological activities. The biosynthesis of prodiginines is affected by various physiological and environmental factors. Thus, prodiginine biosynthesis regulation is highly complex and multifaceted. Although the regulatory mechanism for prodiginine biosynthesis has been extensively studied in Serratia and Streptomyces species, little is known about that in the marine betaproteobacterium Pseudoalteromonas In this study, we report that stringent starvation protein A (SspA), an RNA polymerase-associated regulatory protein, is required for the biosynthesis of prodiginine in Pseudoalteromonas sp. strain R3. The strain lacking sspA (ΔsspA) fails to produce prodiginine, which resulted from the downregulation of the prodiginine biosynthetic gene (pig) cluster. The effect of SspA on prodiginine biosynthesis is independent of histone-like nucleoid structuring protein (H-NS) and RpoS (σS). Further analysis demonstrates that the ΔsspA strain has a significant decrease in the transcription of the siderophore biosynthesis gene (pvd) cluster, leading to the inhibition of siderophore production and iron uptake. The ΔsspA strain regains the ability to synthesize prodiginine by cocultivation with siderophore producers or the addition of iron. Therefore, we conclude that SspA-regulated prodiginine biosynthesis is due to decreased siderophore levels and iron deficiency. We further show that the iron homeostasis master regulator Fur is also essential for pig transcription and prodiginine biosynthesis. Overall, our results suggest that SspA indirectly regulates the biosynthesis of prodiginine, which is mediated by the siderophore-dependent iron uptake pathway.IMPORTANCE The red-pigmented prodiginines are attracting increasing interest due to their broad biological activities. As with many secondary metabolites, the biosynthesis of prodiginines is regulated by both environmental and physiological factors. At present, studies on the regulation of prodiginine biosynthesis are mainly restricted to Serratia and Streptomyces species. This work focused on the regulatory mechanism of prodiginine biosynthesis in Pseudoalteromonas sp. R3. We found that stringent starvation protein A (SspA) positively regulates prodiginine biosynthesis via affecting the siderophore-dependent iron uptake pathway. The connections among SspA, iron homeostasis, and prodiginine biosynthesis were investigated. These findings uncover a novel regulatory mechanism for prodigiosin biosynthesis.

Enhanced Prodigiosin Production in Serratia marcescens JNB5-1 by Introduction of a Polynucleotide Fragment into the pigN 3' Untranslated Region and Disulfide Bonds into O-Methyl Transferase (PigF).

In Serratia marcescens JNB5-1, prodigiosin was highly produced at 30°C, but it was noticeably repressed at ≥37°C. Our initial results demonstrated that both the production and the stability of the O-methyl transferase (PigF) and oxidoreductase (PigN) involved in the prodigiosin pathway in S. marcescens JNB5-1 sharply decreased at ≥37°C. Therefore, in this study, we improved mRNA stability and protein production using de novo polynucleotide fragments (PNFs) and the introduction of disulfide bonds, respectively, and observed their effects on prodigiosin production. Our results demonstrate that adding PNFs at the 3' untranslated regions of pigF and pigN significantly improved the mRNA half-lives of these genes, leading to an increase in the transcript and expression levels. Subsequently, the introduction of disulfide bonds in pigF improved the thermal stability, pH stability, and copper ion resistance of PigF. Finally, shake flask fermentation showed that the prodigiosin titer with the engineered S. marcescens was increased by 61.38% from 5.36 to 8.65 g/liter compared to the JNB5-1 strain at 30°C and, significantly, the prodigiosin yield increased 2.05-fold from 0.38 to 0.78 g/liter at 37°C. In this study, we revealed that the introduction of PNFs and disulfide bonds greatly improved the expression and stability of pigF and pigN, hence efficiently enhancing prodigiosin production with S. marcescens at 30 and 37°C. IMPORTANCE This study highlights a promising strategy to improve mRNA/enzyme stability and to increase production using de novo PNF libraries and the introduction of disulfide bonds into the protein. PNFs could increase the half-life of target gene mRNA and effectively prevent its degradation. Moreover, PNFs could increase the relative intensity of target genes without affecting the expression of other genes; as a result, it could alleviate the cellular burden compared to other regulatory elements such as promoters. In addition, we obtained a PigF variant with improved activity and stability by the introduction of disulfide bonds into PigF. Collectively, we demonstrate here a novel approach for improving mRNA/enzyme stability using PNFs, which results in enhanced prodigiosin production in S. marcescens at 30°C.

Enhanced production of prodigiosin by Serratia marcescens UCP 1549 using agrosubstrates in solid-state fermentation.

This work aimed to investigate the production of prodigiosin by S. marcescens UCP 1549 in solid-state fermentation (SSF), as a sustainable alternative for reducing the production costs and environmental impact. Thus, different agro-industrial substrates were used in the formulation of the prodigiosin production medium, obtaining the maximum yield of pigment (119.8 g/kg dry substrate) in medium consisting of 5 g wheat bran, 5% waste soybean oil and saline solution. The pigment was confirmed as prodigiosin by the maximum absorbance peak at 535 nm, Rf 0.9 in TLC, and the functional groups by infrared spectrum (FTIR). Prodigiosin demonstrated stability at different values of temperature, pH and NaCl concentrations and antimicrobial properties, as well as not show any toxicity. These results confirm the applicability of SSF as a sustainable and promising technology and wheat bran as potential agrosubstrate to produce prodigiosin, making the bioprocess economic and competitive for industrial purposes.

High-level production of microbial prodigiosin: A review.

Prodigiosin is a natural red pigment derived primarily from secondary metabolites of microorganisms, especially Serratia marcescens. It can also be chemically synthesized. Prodigiosin has been proven to have antitumor, antibacterial, antimalaria, anti-insect, antialgae, and immunosuppressive activities, and is gaining increasing important in the global market because of its great potential application value in clinical medicine development, environmental treatment, preparation of food additives, and so on. Due to the low efficiency of prodigiosin chemical synthesis, high-level prodigiosin of production by microorganisms are necessary for prodigiosin applications. In this paper, the production of prodigiosin by microorganism in recent decades is reviewed. The methods and strategies for increasing the yield of prodigiosin are discussed from the aspects of medium composition, additives, factors affecting production conditions, strain modification, and fermentation methods.

2-Keto-D-gluconic acid and prodigiosin producing by a Serratia marcescens.

Microbial fermentation has become the main method to produce target compound. In this study, a 2-Keto-D-gluconic acid (2-KGA) producing mutant strain was obtained by mutation with rational screening methods. Meanwhile, prodigiosin was produced when the nitrogen source in the medium was changed to peptone and its fermentation conditions were evaluated to achieve high-efficient accumulation. The mutant strain SDSPY-136 was firstly identified as Serratia marcescens by morphological observation and 16S rDNA sequencing. The 2-KGA synthetic capacity of S. marcescens SDSPY-136 was evaluated by shake fermentation with 110 g/L glucose as substrates. For fermentation, 2-KGA yield, conversation rate and purity of SDSPY-136 reached 104.60 g/L, 95.10%, 99.11% in 72 h. The red pigment was extracted from the fermentation broth using acidic methanol and identified as prodigiosin by FT-IR. The optimal conditions were as follows: glycerol 20 g/L, peptone 20 g/L, MgSO415 g/L, pH 6.0, a 2% (v/v) inoculum, 30 °C and 200 rpm of shaking culture. Eventually, prodigiosin reached a yield of 9.89 g/Lafter shake culturing for 50 h under this condition. The mutant S. marcescens SDSPY-136 was shown to be promising for 2-KGA and prodigiosin production and a suitable object for prodigiosin metabolism research of S. marcescens.

The FloR master regulator controls flotation, virulence and antibiotic production in Serratia sp. ATCC 39006.

Serratia sp. ATCC 39006 produces intracellular gas vesicles to enable upward flotation in water columns. It also uses flagellar rotation to swim through liquid and swarm across semi-solid surfaces. Flotation and motility can be co-regulated with production of a ß-lactam antibiotic (carbapenem carboxylate) and a linear tripyrrole red antibiotic, prodigiosin. Production of gas vesicles, carbapenem and prodigiosin antibiotics, and motility are controlled by master transcriptional and post-transcriptional regulators, including the SmaI/SmaR-based quorum sensing system and the mRNA binding protein, RsmA. Recently, the ribose operon repressor, RbsR, was also defined as a pleiotropic regulator of flotation and virulence factor elaboration in this strain. Here, we report the discovery of a new global regulator (FloR; a DeoR family transcription factor) that modulates flotation through control of gas vesicle morphogenesis. The floR mutation is highly pleiotropic, down-regulating production of gas vesicles, carbapenem and prodigiosin antibiotics, and infection in Caenorhabditis elegans, but up-regulating flagellar motility. Detailed proteomic analysis using TMT peptide labelling and LC-MS/MS revealed that FloR is a physiological master regulator that operates through subordinate pleiotropic regulators including Rap, RpoS, RsmA, PigU, PstS and PigT.

Substrate Flexibility of the Flavin-Dependent Dihydropyrrole Oxidases PigB and HapB Involved in Antibiotic Prodigiosin Biosynthesis.

In the biosynthesis of the tripyrrolic pigment prodigiosin, PigB is a predicted flavin-dependent oxidase responsible for the formation of 2-methyl-3-amylpyrrole (MAP) from a dihydropyrrole. To prove which dihydropyrrole is the true intermediate, both possibilities, 5-methyl-4-pentyl-3,4-dihydro-2H-pyrrole (5 a, resulting from transamination of the aldehyde of 3-acetyloctanal) and 2-methyl-3-pentyl-3,4-dihydro-2H-pyrrole (6, resulting from transamination of the ketone), were synthesised. Only 5 a restored pigment production in a strain of Serratia sp. ATCC 39006 blocked earlier in MAP biosynthesis. PigB is membrane-associated and inactive when its transmembrane domain was deleted, but HapB, its homologue in Hahella chejuensis, lacks the transmembrane domain and is active in solution. Two colourimetric assays for PigB and HapB were developed, and the HapB-catalysed reaction was kinetically characterised. Ten analogues of 5 a were synthesised, varying in the C2 and C3 side chains, and tested as substrates of HapB in vitro and for restoration of pigment production in Serratia ΔpigD in vivo. All lengths of side chain tested at C3 were accepted, but only short side chains at C2 were accepted. The knowledge that 5 a is an intermediate in prodigiosin biosynthesis and the ease of synthesis of analogues of 5 a makes a range of prodigiosin analogues readily available by mutasynthesis.

Purification and Kinetic Characterization of the Essential Condensation Enzymes Involved in Prodiginine and Tambjamine Biosynthesis.

Prodiginines and tambjamines are related families of bioactive alkaloid natural products with pharmaceutical potential. Both compound families result from a convergent biosynthetic pathway ending in the condensation of a conserved bipyrrole core with a variable partner. This reaction is performed by unique condensation enzymes, and has the potential to be manipulated to produce new pyrrolic compounds. We have purified and reconstituted the in vitro activity of the condensation enzymes PigC and TamQ from Pseudoalteromonas sp., which are involved, respectively, in the prodiginine and tambjamine biosynthetic pathways. Kinetic analysis confirmed a Uni Uni Bi Uni ping-pong reaction sequence with competitive and uncompetitive substrate inhibition for PigC and TamQ respectively. The kinetic parameters of each enzyme provide insight into their differing substrate scope, and suggest that TamQ may have evolved a wide substrate tolerance that can be used for the production of novel prodiginines and tambjamines.

RedH and PigC Catalyze the Biosynthesis of Hybrubins via Phosphorylation of 4'-Methoxy-2,2'-Bipyrrole-5'-Carbaldehyde.

Hybrubins are "unnatural" alkaloids with the same 4'-methoxy-2,2'-bipyrrole-5'-methine moiety found in prodiginines and a different ring derived from tetramic acids. Here, we demonstrated that RedH, a homologue of prodigiosin synthetase PigC, was responsible for the biosynthesis of hybrubins A and B in Streptomyces lividansIn vitro reactions indicated that RedH and PigC catalyzed the intermolecular condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and (Z)-5-ethylidenetetramic acid (ETA) to produce hybrubin B. Moreover, we demonstrated that RedH and PigC activated MBC via phosphorylation of the aldehyde group to form an intermediate Pi-MBC and that the subsequent condensation between Pi-MBC and (Z)-5-ethylidenetetramic acid occurs in a nonenzymatic way.IMPORTANCE Hybrubins are an emerging class of prodiginines possessing a new C ring derived from 5'-substituted tetramic acids and the methylene bridge connecting the C ring at a different position. We have supposed that condensation between 4'-methoxy-2,2'-bipyrrole-5'-carbaldehyde (MBC) and 5-ethylidenetetramic acid (ETA) yields the hybrid natural products hybrubins, which was proposed to be catalyzed by the undecylprodigiosin synthetase RedH. However, it is doubted whether RedH is able to catalyze another type of condensation between MBC and tetramic acids. In this study, we have demonstrated that the MBC-ETA condensation proceeds through RedH/PigC-catalyzed enzymatic activation of MBC via phosphorylation and a nonenzymatic condensation of Pi-MBC with ETA. Since MBC analogues have been shown to be accepted by PigC, more hybrubin analogues might be produced by using combinations of MBC analogues and other tetramic acids in future studies.