Pseudobulbiferamides: Plasmid-Encoded Ureidopeptide Natural Products with Biosynthetic Gene Clusters Shared Among Marine Bacteria of Different Genera

Ureidopeptidic natural products possess a wide variety of favorable pharmacological properties. In addition, they have been shown to mediate core physiological functions in producer bacteria. Here, we report that similar ureidopeptidic natural products with conserved biosynthetic gene clusters are produced by different bacterial genera that coinhabit marine invertebrate microbiomes. We demonstrate that a Microbulbifer strain isolated from a marine sponge can produce two different classes of ureidopeptide natural products encoded by two different biosynthetic gene clusters that are positioned on the bacterial chromosome and on a plasmid. The plasmid encoded ureidopeptide natural products, which we term the pseudobulbiferamides (5–8), resemble the ureidopeptide natural products produced by Pseudovibrio, a different marine bacterial genus that is likewise present in marine sponge commensal microbiomes. Using imaging mass spectrometry, we find that the two classes of Microbulbifer-derived ureidopeptides occupy different physical spaces relative to the bacterial colony, perhaps implying different roles for these two compound classes in Microbulbifer physiology and environmental interactions.


Figure S10 .
Figure S10.Marfey's analysis to determine the absolute configuration of the Phe residue in 5. Extracted ion chromatograms (EICs) demonstrating the retention time of the 2-4-dinitrophenyl-5-L-alanine amidederivitized (DAA-derivatized) Phe residue resulting from the acid hydrolysis of 5 (top), retention time of DAA-derivatized standard of L-Phe (middle), and the retention time of the similarly derivatized standard of D-Phe (bottom).Of note, although only one Phe is present in 5, both a major peak of L-Phe and a minor one of D-Phe were detected by retention time matching.We deduced that racemization of L-Phe took place during acidic hydrolysis, which is akin to homophymamide A that has been verified by chemical synthesis, due to their exocyclic amino acid residue structures attached to the ureido bond. 1 Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S11 .
Figure S11.Marfey's analysis to determine the absolute configuration of the Arg residue in 5. Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Arg residue resulting from the acid hydrolysis of 5 (top), retention time of DAA-derivatized standard of L-Arg (middle), and the retention time of the similarly derivatized standard of D-Arg (bottom).By retention time matching, the Arg residue in 5 was determined to be L-Arg.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S12 .
Figure S12.Marfey's analysis to determine the absolute configuration of the Pro residue in 5. Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Pro residue resulting from the acid hydrolysis of 5 (top), retention time of DAA-derivatized standard of L-Pro (middle), and the retention time of the similarly derivatized standard of D-Pro (bottom).By retention time matching, the Pro residue in 5 was determined to be L-Pro.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S13 .
Figure S13.Marfey's analysis to determine the absolute configuration of the Gln residue in 5.During acid hydrolysis, Gln is converted to Glu.Hence, Glu standards are used here.Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Glu residue resulting from the acid hydrolysis of 5 (top), retention time of DAA-derivatized standard of L-Glu (middle), and the retention time of the similarly derivatized standard of D-Glu (bottom).By retention time matching, the Gln residue in 5 was determined to be L-Gln.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S32 .
Figure S32.Marfey's analysis to determine the absolute configuration of the Phe residue in 7. Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Phe residue resulting from the acid hydrolysis of 7 (top), retention time of DAA-derivatized standard of L-Phe (middle), and the retention time of the similarly derivatized standard of D-Phe (bottom).Akin to 5, the racemization of L-Phe in 7 took place. 1 Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S33 .
Figure S33.Marfey's analysis to determine the absolute configuration of the Ala residue in 7. Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Ala residue resulting from the acid hydrolysis of 7 (top), retention time of DAA-derivatized standard of L-Ala (middle), and the retention time of the similarly derivatized standard of D-Ala (bottom).By retention time matching, the Ala residue in 7 was determined to be L-Ala.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S34 .
Figure S34.Marfey's analysis to determine the absolute configuration of the Arg residue in 7. From top to bottom-EICs demonstrating retention time of DAA-derivitized Arg residue obtained by acid hydrolysis of 7, DAA-derivitized standard for L-Arg spiked with the derivatized acid hydrolysate of 7, and DAAderivitized standard for D-Arg spiked with the derivatized acid hydrolysate of 7. By retention time matching, the Arg residue in 7 was determined to be L-Arg.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S35 .
Figure S35.Marfey's analysis to determine the absolute configuration of the Pro residue in 7. Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Pro residue resulting from the acid hydrolysis of 7 (top), retention time of DAA-derivatized standard of L-Pro (middle), and the retention time of the similarly derivatized standard of D-Pro (bottom).By retention time matching, the Pro residue in 7 was determined to be L-Pro.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Figure S36 .
Figure S36.Marfey's analysis to determine the absolute configuration of the Gln residue in 7.During acid hydrolysis, Gln is converted to Glu; hence, Glu standards are used here.Extracted ion chromatograms (EICs) demonstrating the retention time of the DAA-derivatized Glu residue resulting from the acid hydrolysis of 7 (top), retention time of DAA-derivatized standard of L-Glu (middle), and the retention time of the similarly derivatized standard of D-Glu (bottom).By retention time matching, the Gln residue in 7was determined to be L-Gln.Separation was achieved using the Agilent Poroshell EC-C18 (100×4.6 mm, 2.7 µm) column.Mass spectrometry data were acquired in the negative ionization mode.

Table S2 .
Identification numbers for MS/MS spectra deposition for pseudobulbiferamides in the GNPS