CHARACTERIZATION OF PRODIGININE COMPOUNDS PRODUCED BY A VIBRIO SPECIES ISOLATED FROM SALT FLAT SEDIMENT ALONG THE FLORIDA GULF COAST STEPHANIE MORGAN1, MATTHEW J. THOMAS2, KATHERINE M. WALSTROM1, ERIC C. WARRICK2, BRITTANY J GASPER3* 1DIVISION OF NATURAL SCIENCES, NEW COLLEGE OF FLORIDA, SARASOTA, FLORIDA 2DEPARTMENT OF NATURAL SCIENCES, STATE COLLEGE OF FLORIDA, BRADENTON, FLORIDA 3DEPARTMENT OF BIOLOGY, FLORIDA SOUTHERN COLLEGE, LAKELAND, FLORIDA MANUSCRIPT RECEIVED 30 SEPTEMBER 2016; ACCEPTED 29 DECEMBER 2016 Copyright 2017, Fine Focus. All rights reserved. 34 • FINE FOCUS, VOL. 3 (1) Prodiginines are secondary metabolites produced by several known species of bacteria. These metabolites are known for their bright pigmentation and their potential medicinal uses. Biosynthesis of prodiginine compounds, including the well-studied prodigiosin, has been well characterized in Serratia marcescens and other bacterial species, including several marine bacteria. In an effort to isolate and identify natural products from marine organisms, an environmental sample was taken from a salt flat along the Florida Gulf Coast and cultured for bacterial growth. A bacterial species that produces a vibrant pink pigment was isolated and identified as a member of the Vibrio genus and was named MI-2. Whole genome sequencing identified a 13-gene operon with homology to the S. marcescens prodigiosin biosynthetic operon. The pigment produced by MI-2 was hypothesized to be composed of prodigiosin or related prodiginine compounds and was purified by flash column chromatography and identified by mass spectrometry. ABSTRACT CORRESPONDING AUTHOR Brittany J. Gasper* bgasper@flsouthern.edu KEYWORDS • Prodigiosin • Vibrio • antimicrobial • biosynthesis • prodiginine The prodiginine family of bacterial alkaloids includes many vibrantly pigmented compounds, most of which are red, produced as secondary metabolites by a variety of bacterial species. The most notable of these is Serratia marcescens from which the best studied prodiginine, prodigiosin, was first isolated in pure form and structurally charcterized (14, 19). Interest in the prodiginines comes not only from their strong red pigment but also from their potential medicinal uses. In addition to the antimalarial activity of prodigiosin itself (4), prodigiosin and other prodiginine derivatives have been shown to have immunosuppressive functions INTRODUCTION with novel mechanisms of action (7, 21, 30, 38, 42) and apoptotic effects in human cancer cells (11, 27, 33, 42). Since the discovery of prodigiosin in S. marcescens, prodiginine compounds have been discovered in other bacterial organisms, including Streptomyces coelicor A3(2), Alteromonas rubra, Hahella chejuensis, and Vibrio gazogenes (9, 19). Biosynthesis of prodigiosin has been best studied in S. marcescens, S. coelicor, and H. chejuensis. In S. marcescens ATCC 274, the prodigiosin biosynthesis (pig) cluster consists of 14 genes abbreviated as pigA through pigN arranged as an operon that are transcribed as a single 14-gene polycistronic mRNA (17). H. chejuensis KCTC 2396 also contains 14 prodigiosin biosynthetic genes, hapA through hapN, that are similar in their gene layout to that of S. marcescens (22). In S. coelicor A3(2), the red cluster is responsible for prodiginine biosynthesis (5). The genetic arrangement of the red cluster is significantly different from S. marcescens and H. chejuensis, however the presence of 12 homologous genes between it and the pig operon suggests the two biosynthetic pathways are similar (17). Several of the known prodiginine producers, including H. chejuensis, V. gazogenes, and A. rubra are marine microorganisms. The isolation of natural and potential medicinal products from diverse marine microorganisms has been described recently, with several new products originating from microorganisms isolated from marine environments (2, 3, 45). We report here the isolation of a prodiginine-producing Vibrio species known as Marine Isolate-2 (MI-2) from a marine salt flat environment along the central Gulf Coast of Florida. Originally isolated as a marine antibiotic-producing bacterium, MI-2 was unique in its ability to produce a distinctly pink prodiginine product under appropriate media conditions. Whole genome sequencing of MI-2 identified a 13-gene prodiginine biosynthetic gene cluster. The analysis of this prodiginine biosynthetic pathway and structural identification of the purified prodiginine compounds are discussed. PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 35 ISOLATION AND GENUS IDENTIFICATION OF MI-2 MI-2 was isolated from a salt flat sediment sample aseptically obtained from Leffis Key in Bradenton, FL. The sediment sample was diluted in 0.5 M NaCl to 10-5 grams soil/ml and grown on 0.5 M NaCl potato dextrose agar (PDA) modified with the following contents: tryptone (2 g/l), NaCl (0.5 M), glucose (5 mM), and 10X Neidhardt MOPS salts (0.1X final concentration). The Neidhardt MOPS salt components were prepared as described (32). To inhibit fungal growth, 100 μg/l cycloheximide was added to the initial isolation plates. After initial isolation of MI-2, the organism was maintained on 0.5 M NaCl modified PDA plates and 0.5 M NaCl LB agar plates and stored at 25°C. MATERIALS AND METHODS 36 • FINE FOCUS, VOL. 3 (1) Identification of the genus of MI-2 was completed by whole colony PCR amplification of the 16S rDNA. The 16S primers 63F (5’- CAG GCC TAA CAC ATG CAA GTC – 3’) and 1387R (5’ - GGG CGG WGT GTA CAA GGC – 3’) obtained from Integrated DNA Technologies (IDT) were used to amplify the gene using the MyTaqTM mix obtained from Bioline (Taunton, MA). The whole colony PCR reaction conditions were: 1 cycle of 95°C for 10 minutes; 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute; and 1 cycle of 72°C for 5 minutes. Successful PCR amplification was confirmed by agarose gel electrophoresis on a 1% agarose gel in 1X TAE buffer and purified using the Isolate II PCR and Gel Kit from Bioline. Purified PCR products were sequenced at the DNA Analysis Facility on Science Hill at Yale University. MEDIA AND GROWTH EFFECT ON PIGMENT PRODUCTION The effect of media composition and NaCl concentration on the pigment production of MI-2 was determined qualitatively by quadrant streaking MI-2 onto modified PDA plates prepared as described previously with supplementation of 2%, 4%, or 6% NaCl and LB plates supplemented with 2%, 4%, or 6% NaCl. The plates were incubated at 30°C for two days before being photographed. The effect was determined quantitatively by inoculating a colony into modified Potato Dextrose Broth (PDB) and LB liquid cultures at 2%, 4%, and 6% NaCl and incubating at 30°C for 48 hours. Absorbance of the sample was taken by wavelength scan from 400 to 700 nm at 5 nm intervals. The effect of time on pigment production of MI-2 was determined qualitatively by quadrant streaking MI-2 onto modified PDA or LB plates with supplementation of 2% or 4%. The plates were incubated at 30°C for nine days, and photographs were taken after two and nine days. The effect was determined quantitatively by inoculating a colony into PDB and LB liquid cultures at 2% and 4% NaCl and incubating at 30°C for 48 hours. Absorbance of the sample was taken by wavelength scan from 400 to 700 nm at 5 nm intervals. GENOMIC SEQUENCING AND IDENTIFICATION OF A PUTATIVE PRODIGIOSIN BIOSYNTHESIS GENE CLUSTER Genomic DNA from MI-2 was extracted from an overnight culture grown in 4% NaCl LB liquid media using the phenol chloroform method (44). The extracted DNA was checked for integrity by agarose gel electrophoresis and quantified using a NanoDrop 2000 (Thermo Scientific). Whole genome sequencing of the extracted DNA was conducted using HiSeq 2500 technology at Purdue University. The resulting genomic sequence was analyzed using the sequence viewer and annotator tool Artemis (35). The genes with homology to the pig gene cluster of S. marcescens were identified manually using the National Center for Biological Information (NCBI) protein basic local alignment tool (blastp) (15). PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 37 COMPARISON OF PUTATIVE PRODIGIOSIN BIOSYNTHESIS GENE CLUSTER FROM MI-2 AND OTHER ORGANISMS Comparison of prodigiosin biosynthesis genes from MI-2 and other known prodiginine compound producing organisms Serratia marcescens ATCC 274, Hahella chejuensis KCTC 2396, and Streptomyces coelicor A3(2) was done by aligning amino acid sequences using Clustal Omega for each gene within the cluster (37). Gene sequences were acquired using the NCBI GenBank database and the accession numbers as follows: S. marcescens – AJB33002, H. chejuensis – DQ266254, and S. coelicor – AL645882. PURIFICATION OF PRODIGIOSIN An overnight culture of MI-2 in 4% NaCl LB liquid media was centrifuged at 6000 Xg for 10 minutes; the supernatant was discarded and the pellet was resuspended in approximately 2 ml of a methanol and 2 N HCl mixture (24:1). The addition of acid was necessary to break down a suspected prodigiosin-associated protein that may sequester the pigment molecule (14, 24). The resuspended pellets were combined in 35 ml scintillation vials wrapped in aluminum foil and left for 12-18 hours on an orbital rotator. Extracts were then centrifuged at 6000 Xg for 10 minutes to remove any cellular residue. The supernatant was collected, and the solvent was evaporated using a rotary evaporator. Hydrophilic impurities were removed by a series of chloroform-water liquid- liquid extractions that were repeated on the organic layer until the water layer no longer appeared cloudy (1). The chloroform was evaporated via rotary evaporation, and pigment was redissolved in acetonitrile. Flash column chromatography was performed using silica gel as the stationary phase and acetonitrile as the mobile phase. Fractions from flash chromatography that corresponded to a pigmented smear at 0.82 to 0.63 retardation factor (Rf) regions on silica thin layer chromatography (TLC) plates with acetonitrile as the mobile phase were combined and concentrated by rotary evaporation. UV-VIS PH ASSAY The pH of methanol solvents was adjusted to 1.8, 5.0, 7.0, 8.0, 11.1 and 12.0 using solutions of 1 M HCl and 1 M NaOH. One hundred μl of purified prodigiosin pigment extract was suspended in each methanol solvent, and absorbance spectra were measured from 350 nm to 700 nm at 0.25 nm intervals. A baseline correction was performed for all samples. MASS SPECTROMETRY Electrospray Ionization (ESI) tandem mass spectrometry (MS/MS or MS2) was performed using Fourier transform mass spectrometry (FTMS) mode on an LTQ- Orbitrap with helium used as the collision gas. Relative collision energy (rCE) ranged from 30-35 in the ion trap component of the instrument. High-resolution mass spectra were obtained with full width half maximum resolving power of 100,000 at 400 m/z in profile mode. 38 • FINE FOCUS, VOL. 3 (1) Figure 1. Isolation and Colony Appearance of MI-2 Panel A shows the original isolation of MI-2 from Leffis Key in Bradenton, FL on modified PDA media. The red/pink colony of MI-2 selected for pure culture isolation is shown in the black square. Panel B shows the pure culture and pink pigment production of MI-2 on modified PDA media. B)A) ISOLATION AND GENUS IDENTIFICATION OF MI-2 The marine bacterium MI-2 was originally cultured from salt flat sediment obtained at Leffis Key in Bradenton, FL on modified PDA media supplemented with 0.5 M NaCl for marine organisms and tryptone as described in the methods to support growth of fastidious organisms. Figure 1 shows the original marine sediment isolation plate in panel A and the pure culture growth of MI-2 in panel B. The purpose of this isolation was to identify antibiotic-producing microorganisms from diverse marine environments. MI-2 was identified as an antibiotic-producer (data not shown), but its ability to produce a bright pink pigment when grown on the modified PDA prompted further characterization of this microorganism. Sequencing of the 16S rRNA gene identified the organism as being a member of the Vibrio genus with Vibrio ruber being the closest related species (data not shown). RESULTS PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 39 Media 2% NaCl 4% NaCl 6% NaCl Max Abs. λ (nm) Avg. Abs. ( ± SE) Max Abs. λ (nm) Avg. Abs. ( ± SE) Max Abs. λ (nm) Avg. Abs. ( ± SE) PDB 545 1.70 ± 0.14 545 1.87 ± 0.08 540 1.73 ± 0.08 LB 500 0.72 ± 0.02 500 1.12 ± 0.03 500 1.24 ± 0.05 Figure 2. Effect of Media Composition and NaCl on MI-2 Pigment Production MI-2 was quadrant streaked onto modified PDA and LB plates at the indicated NaCl concentrations and incubated at 30°C for 48 hours. Panels A-C show growth on PDA at 2%, 4%, and 6% NaCl concentrations. Panels D-F show growth on LB at 2%, 4%, and 6% NaCl concentrations. The shade of the pigment is different when grown on PDA compared to LB. This is confirmed by the corresponding table that shows the maximum absorbance wavelength (Max Abs. λ) and the average absorbance value (Avg. Abs.) ± standard error (SE) for three replicates grown in PDB and LB at 30°C for 48 hours. B)A) D) E) C) F) 2%NaCl PDA LB 4% 6% 40 • FINE FOCUS, VOL. 3 (1) 2% NaCl PDA 4% NaCl PDA 2% NaCl LB 4% NaCl LB NaCl Concentration PDB LB Two Days Five Days Two Days Five Days Max Abs. λ (nm) Avg. Abs. ( ± SE) Max Abs. λ (nm) Avg. Abs. ( ± SE) Max Abs. λ (nm) Avg. Abs. ( ± SE) Max Abs. λ (nm) Avg. Abs. ( ± SE) 2% 545 1.70 ± 0.14 545 2.02 ± 0.30 500 0.72 ± 0.02 450 0.781 ± 0.10 4% 545 1.87 ± 0.08 540 2.30 ± 0.01 500 1.12 ± 0.03 450 1.39 ± 0.20 Figure 3. Effect of Time on MI-2 Pigment Production MI-2 was quadrant streaked onto LB and modified PDA with 2% and 4% NaCl and incubated at 30°C for nine days. Photographs were taken after two days of growth (2D) and after nine days of growth (9D). The corresponding table shows the Max Abs. λ and Avg. Abs. ± SE for three replicates grown in PDB and LB at 30°C for two days and five days The Max Abs. λ changed dramatically from two days (500 nm) to five days (450 nm) when grown in LB with 2% and 4% NaCl but not in modified PDB at the same salt concentrations. The pigment intensity appeared to be notably decreased after nine days of growth, but this decrease was not reflected quantitatively in the absorbance values. 2D 2D 2D 2D 9D 9D 9D 9D PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 41 MEDIA AND GROWTH EFFECTS ON PIGMENT PRODUCTION The effects of media (LB vs. PDA) and NaCl concentration (2%, 4%, or 6%) on pigment production of MI-2 are shown in Figure 2. While the pigment produced by MI-2 appeared bright pink on the 2% NaCl PDA plate, it was more red on the 2% NaCl LB plate. This difference was quantitatively confirmed by measuring the absorbance after growth in liquid samples (tabular data in Figure 2). The maximum absorbance wavelength (Max Abs. λ) was 540-545 for PDB and 500 for LB. There was a slight change in the Max Abs. λ for PDB at 4% (545 nm) to 6% (540 nm) which may explain the slight change seen in the pigment color from panels B to C. Pigment production on both media types visually appeared to show a decrease in intensity as the concentration of NaCl in the media increased as seen by comparing panels A to C and D to F, however this decrease was not replicated quantitatively as there is no decreased absorbance seen with higher NaCl concentrations. The effect of time on pigment production of MI-2 is shown in Figure 3. After nine days of growth, pigment appearance of MI-2 appeared notably altered from where it was after two days of growth. Extensive growth on PDA appeared to result in decreased pigment content, and altered pigment color from red-pink to red-orange was seen after growth on LB. The change in pigment appearance was more dramatic on both media types at 4% NaCl than at 2% NaCl. Quantitative analysis of this (tabular data in Figure 3) confirmed a change in the max. abs. λ from 500 nm to 450 nm when grown in LB while the max. abs. λ of PDB remained relatively unchanged by prolonged growth. Average absorbance values do not show the decreased pigment production that is visible in the figure as values for all samples actually increased from two days to five days. IDENTIFICATION OF PUTATIVE PRODIGIOSIN BIOSYNTHESIS GENE CLUSTER In order to better characterize and identify MI-2, its genome was sequenced. After whole genome sequencing and annotation, the putative prodigiosine biosynthesis gene cluster was identified based on homology to the well characterized 14 gene pig operon found in S. marcescens (17). The MI-2 prodiginine biosynthesis gene cluster is approximately 20.3 kb in length and consists of 13 genes (Figure 4). The overall structure of the MI-2 cluster is highly similar to prodiginine biosynthesis clusters in both the arrangement of protein homologs as well as in the relative sizes of proteins and intergenic spacing. Six of the open reading frames (ORF) overlap with one another, but a significant gap of 177 base pairs was found between the putative pigC and pigD. This is comparable with the structure of the pig cluster in S. marcescens as well as the hap cluster in H. chejuensis (17, 22). The major difference between the structure of the putative pig cluster in MI-2 and S. marcescens is the absence of pigA in MI-2. A nucleotide alignment of bases preceding pigB with the coding nucleotide sequence for pigA in S. marcescens ATC 2744 showed short regions of homology (data not shown). This suggests that a pigA homolog may have been present at one time but has since been lost. 42 • FINE FOCUS, VOL. 3 (1) Figure 4. Schematic Representation of Putative Prodigiosin Biosynthesis Gene Cluster in MI-2 The cluster is approximately 20.3 kb in length and contains 13 genes. The arrows show the directionality of ORFs. Letters for ORFs correspond to the pigB-N homologs. Different arrow patterns indicate the putative role of the enzyme in the bifurcated prodiginine biosynthesis pathway in which PigC condenses the compound 4-methoxy-2,2-bipyrrole-5-carboxyaldehyde resulting from synthesis of the MBC pathway with the compound 2-methyl-3-n-amyl-pyrrole resulting from the synthesis of the MAP pathway). The corresponding table shows the size of each ORF, the nucleotide start position for each ORF and the nucleotide end position for each ORF. pig Homolog ORF Size (bp) Start* End* B 2,070 1 2,070 C 2,288 2,440 4,728 D 2,628 4,905 7,533 E 2.556 7,530 10,086 F 1,010 10,170 11,186 G 204 11,197 11,461 H 1,944 11,464 13,408 I 1,467 13,410 14,877 J 2,471 14,821 17,292 K 320 17,302 17,622 L 814 17,563 18,377 M 1,220 18,210 19,430 N 1,094 19,119 20,213 *Nucleotide start and end positions are given relative to the start position of ORF B. Table rows highlighted in gray indicate an ORF that overlaps with the ORF preceding it. 1kb MBC pathway MAP pathway MBC-MAP Condensing Enzyme B C D E F G H I J K L M N PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 43 MI-2 S. marcescens H. cheuensis S. coelicor Homolog % AAI Homolog % AAI Homolog % AAI A* PigA 55 HapA 54 RedW 43 B PigB 57 HapB 38 RedS 35 C PigC 69 HapC 54 RedH 40 D PigD 73 HapD 51 - - E PigE 80 HapE 61 - - F PigF 73 HapF 58 RedI 19 G PigG 64 HapG 45 RedO 21 H PigH 72 HapH 58 RedN 59 I PigI 58 HapI 43 RedM 40 J PigJ 61 HapJ 33 RedX K PigK 68 HapK 47 RedY 45 L PigL 17 HapL 19 RedU 19 M PigM 48 HapM 33 RedV 26 N PigN 17 HapN 14 RedF 15 Table 1. AAI of Prodiginine Biosynthetic Gene Cluster Homologs The table shows the AAI of MI-2 putative prodigiosin biosynthetic gene cluster homologs compared to known prodiginine biosynthetic proteins from S. marcescens ATCC 274, H. chejuensis KCTC 2396, and S. coelicor A3(2). AAI with MI-2 homologs were calculated using Clustal Omega. PigD and PigE homologs in S. coelicor have not been found. *PigA homolog is not found within the MI-2 cluster. PROTEIN SEQUENCE COMPARISON OF THE PUTATIVE PRODIGIOSIN BIOSYNTHESIS CLUSTER The amino acid identities (AAI) of the proteins coded for by the putative prodigiosin biosynthesis genes were compared to their homologs of S. marcescens, H. chejuensis, and S. coelicor (Table 1). In addition to the structural homology of the gene cluster, there was also consistent homology between genes in the MI-2 cluster and corresponding genes in the prodiginine biosynthesis operon of the other species. The AAI between MI-2 and Pig homologs typically ranged from 50% to 80%, a comparable range compared with other studies that have shown an AAI of only 23% or more between Vibrio and Serratia species (16, 20, 39). Exceptions to this can be seen in PigL and PigN homologs, which had very low AAI; however, neither of these two proteins are required for prodigiosin production (43). 44 • FINE FOCUS, VOL. 3 (1) Figure 5. UV-Vis Spectra of MI-2 Pigment Extract at Various pHs The graph shows the absorbance from wavelengths ranging from 380 nm to 620 nm of MI-2 pigment extract in aqueous methanol solvents at varying pHs. Two maxima can be observed at 470 nm and 535 nm. Absorbance at 535 nm is greater at acidic pH while absorbance at 470 nm is greater at basic pH. An isosbestic point can be observed at 490 nm. A bs or ba nc e Wavelength (nm) 0.24 0.2 0.16 0.12 0.08 0.04 380 400 420 440 460 480 500 520 540 560 580 600 620 pH 2 pH 5 pH7 pH8 pH11 As mentioned previously, there is no homolog for PigA found in the MI-2 putative progidiosin biosynthesis gene cluster. However, a different protein found elsewhere in the MI-2 genome did show homology to PigA. It is the sequence of this putative protein that is used for PigA comparison from MI-2 in Table 1. PURIFICATION AND UV-VIS SPECTRA OF MI-2 PURIFIED PIGMENT EXTRACT AT DIFFERENT PH Extraction and purification of the MI-2 pigment was completed by flash column chromatography. The UV-Vis spectra of the purified MI-2 pigment extract resuspended in different pH solutions is shown in Figure 5. The spectra showed two main peaks that depended on the pH of the solvent. This suggests that the compound exists in either a protonated or non- protonated form (34). The peak absorbance for what was presumed to be the protonated form occurred at a wavelength of approximately 535 nm and the non-protonated peak absorbance occurred at approximately 470 nm. These results were consistent with the UV-Vis spectra for prodigiosin (18). The spectra for the MI-2 pigment extract revealed an isosbestic point at a wavelength of approximately 490 nm. This is the wavelength in which the absorbance remains constant as a conformational change occurs due to the changes in pH of the aqueous methanol. Additionally, the data suggested that the pH at PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 45 Figure 6. Electrospray Ionization Mass Spectra of MI-2 Pigment Extract The full mass spectra of the purified MI-2 pigment extract is shown. Peaks represent ions of a particular mass (x-axis) with a relative abundance (y-axis). The peak at 324 is the most prominent while peaks at 338, 352 and 371 appear as minor components. Re la tiv e ab un da nc e m/z 324.2074 338.3422 352.2387 371.1017 100 50 0 which the absorbance between the two peaks would be equal was just above pH 8, which is consistent with the known pKa value of 8.25 for prodigiosin in acidified ethanol (18). MASS SPECTRA OF MI-2 PURIFIED PIGMENT EXTRACT The full mass spectrum of purified pigment extract from MI-2 is shown in Figure 6. This spectrum revealed a mixture of four different compounds in different abundances. A compound with a mass of 324 g/mol was the main component of the mixture, and compounds with masses of 338 g/mol, 352 g/ mol and 371 g/mol were present in the extract as minor components. The tandem mass spectra of each of these four peaks are shown in Figure 7. Tandem mass spectra of compounds with a mass of 324, 338 and 352 exhibited similar fragmentation patterns that were consistent with prodiginine compounds. Specifically, all three contained a peak at 252 m/z, which can be explained by the loss of the alkyl substituent group to yield the 2-methoxy prodiginine core. Additionally, all three showed an ion at a mass consistent with the loss of the methyl group from the methoxy. Compounds 324, 338 and 352 can be represented by compounds with varying lengths of alkyl chains, as there is a mass difference of 14 that can be represented by the addition of a CH2. The compound with a mass of 271 appeared to be unrelated as it did not contain the prodiginine core 252 peak; however, further analysis would be required to fully elucidate the structure of this compound. The spectrum from this peak is not shown in Figure 7. 46 • FINE FOCUS, VOL. 3 (1) Figure 7. Tandem Mass Spectra of Peaks from the Full Mass Spectra The tandem mass spectra of the relevant prodiginine peaks from the full mass spectra in Figure 6 are shown. Peaks represent ions produced as a result of gas-induced dissociation (GID) of the parent molecule. Panel A shows the tandem mass spectra of a 324 molecular mass molecule with the proposed structure prodigiosin. The most stable ion produced appears at m/z 309 and is the ion produced by cleavage of the methyl on the methoxy group. The peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule and 292 is the loss of the oxygen. These fragmentation patterns are shown with the dashed arrows. Panel B shows the tandem mass spectra of a 338 molecular mass molecule with the proposed structure 2-methyl-3- hexyl prodiginine. The most stable ion produced appears at m/z 323 and is the ion produced by cleavage of the methyl on the methoxy group. The peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule, and the peak at 310 is likely loss of an ethene. Other peaks represented are not readily explainable given the proposed structure. It is possible this sample that is in low abundance is contaminated. Panel C shows the tandem mass spectra of a 352 molecular mass molecule with the proposed structure prodigiosin. The most stable ion produced appears at m/z 337 and is the ion produced by cleavage of the methyl on the methoxy group. The peak at 252 represents an ion created as a result of the alkyl chain being cleaved from the molecule, and 320 is produced after the loss of the oxygen. 0 100 50% 252.1134 292.1812 309.1841 H N HN N O 309 292 252 m/z A) 0 100 50 H N HN N O 337 320 252 % m/z 252.1133 320.2124 337.2151 352.2385 C) 0 100 50% 323 252H N HN N O 203.9438 237.9829 252.1132 303.3048 310.1916 323.1994 m/z B) DISCUSSION The biosynthesis of prodigiosin is an interesting metabolic phenomenon as it is a secondary metabolite with no known direct benefit to cellular growth (43). Pure non-pigmented strains of S. marcescens show no significant difference in viability from pigmented strains (41). However, in the natural environment, prodigiosin and its derivatives may serve a purpose in bacterial defense as many have antibacterial properties (12, 25). MI-2 is a prodigiosin-producing marine bacterium of the Vibrio genus. Its species designation appears to be closest to Vibrio ruber, a known prodigiosin producer (8), though its biochemical test profile differs from V. ruber in multiple ways, suggesting MI-2 is a strain of V. ruber unique from the wild type (manuscript in preparation). Although prodiginine compounds and the organisms that produce them have been studied for a long time, there is limited research on prodigiosin biosynthesis clusters in different genera. To date, prodigiosin biosynthesis clusters have only been examined in S. marcescens ATCC 274, Serratia spp. 39006, H. chejuensis KCTC 2396, and some Streptomyces species (5, 17, 22, 36, 43). The addition of another prodiginine biosynthesis cluster in Vibrio species will add more information on the potential catalytic mechanism for each enzyme by revealing key residues in conserved regions of each gene. The appearance of the prodigiosin pigment production of MI-2 is affected by the media on which it grows and the length of incubation. The pigments produced by growth in LB and modified PDB showed different max absorbances, verifying the different colored apearance seen on the plates in Figure 2. Pigment production on both LB and modified PDA media types appeared to show a decrease in intensity as the concentration of NaCl in the media increased, suggesting an inhibitory effect of excess NaCl on pigment production. This phenomenon could not be validated quantitatively in liquid broth (tabular data of Figure 2), however it is difficult to draw a comparison in intensities from the absorbance values as differences in the concentration of cells in each sample will affect the level of pigment production and therefore the absorbance values reached. These could be further complicated by the regulation of prodigiosin production by quorum sensing (43). Additionally, increasing the length of incubation from two days to nine days resulted in altered coloration, particularly in LB media, and the appearance of decreased pigment intensity in plates, though this occurrence could also not be reproduced quantitatively in liquid (Figure 3). The qualitative data suggest the pigment produced by MI-2 may not be stable for extended periods of time in the conditions it was grown (at room temperature and exposed to oxygen) as has been shown for other prodigiosin analogs (40). However further testing with a purified sample of the pigment would need to be performed to confirm this. Growth after nine days on PDA with 2% NaCl showed more red coloration on the right hand side of growth in the third quadrant compared to the rest of the plate. This difference is believed to be caused by the pattern of pigmentation fading in the colonies and not an effect of the media itself. PRODIGININE PRODUCED BY A MARINE VIBRIO SPECIES • 47 48 • FINE FOCUS, VOL. 3 (1) When comparing the structure of the putative prodigiosin biosynthesis cluster in MI-2 to S. marcescens, the most notable difference is the absence of pigA in MI-2. PigA functions as a flavoprotein desaturase whose catalytic role in the prodigiosin biosynthetic pathway occurs after PigI but before PigJ. An alternative reaction converting the substrate of PigA to the correct product without the use of PigA has been experimentally confirmed (13), perhaps negating the need for PigA in the prodigiosin biosynthetic pathway of MI-2. Additionally, as mentioned in the results, a different protein elsewhere in the genome did show homology to PigA and may be able to function in its place in prodigiosin biosynthesis. As shown in Table 1, the AAI between proteins encoded by the putative prodigiosin biosynthetic gene cluster and known prodiginine biosynthetic proteins were typically between 50 to 80%. However, AAI comparison is likely not the best method for comparing protein function. Conservation of specific domains may be a better determinant as to whether a group of proteins will serve the same function. Although H. chejuensis and S. marcescens both produced prodigiosin, the amino acid identity between Pig and Hap homologs generally ranged from only 30% to 55% (23). Despite the seemingly lower amino acid identities between these homologs, these enzymes served the same function in prodiginine biosynthesis (23, 43). A better comparison of protein function can be done by comparing specific amino acid residues that are essential to the function of the protein. According to the NCBI protein basic local alignment tool, the MI-2 homolog of PigC contained a domain belonging to the pyruvate phosphate dikinase (PPDK) superfamily. PigC performs the final reaction of the prodigiosin biosynthesis pathway, which combines the two precursor molecules 4-methoxy-2,2- bipyrrole-5-carboxyaldehyde (MBC) and 2-methyl-3-n-amyl-pyrrole (MAP) into the final prodigiosin structure (29, 43). The mechanism for PigC is similar to that of PPDK enzymes in that they phosphorylate a carbonyl by facilitating the transfer of a phosphoryl group from ATP using a histidine residue in a phosphoryl transfer domain (PTD) (6). S. marcescens strains with a pigC knockout completely lost the ability to produce the prodigiosin pigment (43). Selective mutagenesis experiments on PigC in Serratia spp. 39006 showed that replacement of His840 with an alanine residue completely eliminated activity. Similarly, residues predicted to be important for ATP binding, Glu281 and Arg295, showed significant reduction in activity when replaced with alanine residues (6). Closer analysis of the amino acid sequence in the MI-2 homolog also revealed residues His841, Glu282 and Arg296 that are analogous to key residues in PigC (data not shown). Given this similarity, it is likely that PigC and the MI-2 protein homolog function identically to one another. Investigations to experimentally confirm the function of the putative prodigiosin biosynthetic proteins in MI-2 are underway. The prodiginine compounds isolated and identified in this study have been previously recognized and documented in several other species (1, 22, 26). Given the homology between pig, hap, and MI-2 prodiginine gene clusters it is plausible that MI-2 would produce similar prodiginine derivatives to those found in Serratia species and H. chejuensis. The mass spectrum of the purified MI-2 pigments contains multiple peaks from prodiginine compounds with different alkyl chain lengths. Prodiginine compounds varying in the length of the alkyl chain substituent group on a pyrrole of the structure are well documented and have been produced as minor byproducts in other species (1, 14, 26). The difference in MI-2 appearance on the modified PDA and the LB plates may be due to the amount of REFERENCES 1. Alihosseini F., Ju K-S., Lango J., Hammock B. D., and Sun G. 2008. Antibacterial colorants: characterization of prodiginines and their applications on textile materials. Biotechnol. Prog. 24:742–47. 2. Ameri A. 2014. Marine microbial natural products. Jundishapur J. Nat. Pharm. Prod. 9:e24716. 3. Blunt J. W., Copp B. R., Keyzers R. A., Munro M. H. G., and Prinsep M. R. 2015. Marine natural products. Nat. Prod. Rep. 32:116–211. 4. Castro A. J. 1967. Antimalarial activity of prodigiosin. Nature. 213:903–4. 5. Cerdeño A. M., Bibb M. J., and Challis G. L. 2001. 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