Description of global changes in metabolome
The nutritional composition of standard laboratory growth media such as LB is inadequate in modeling bacterial physiology during infection, as nutrient availability can vary markedly between infection sites and standard growth media. To overcome this challenge, the previously developed SCFM2 was used to model the physical and chemical environment of human sputum from patients with cystic fibrosis. In this study, we employed an untargeted metabolomic approach to compare the metabolomes of three different strains of B. cenocepacia namely, C5424, K56-2, and J2315 when cultured in SCFM2 and LB in triplicate (Fig. 1a). These strains are clonal and are associated with the highly transmissible, epidemic ET12 lineage of B. cenocepacia19. In addition, comparative metabolomic analysis was also carried out in the presence and absence of trimethoprim, which is an antibiotic used clinically for treatment of Burkholderia infections40. This antibiotic is known to upregulate biosynthetic pathways involved in production of secondary metabolites in B. thailandensis and in select strains of B. cenocepacia in LB, but its effect has not been investigated in SCFM241-43. In effort to capture a diverse range of compound classes, extractions were performed on bacterial cultures using both liquid-liquid (with ethyl acetate, EtOAc) and solid-phase extraction (SPE) methods. Extracts were then analyzed using high resolution tandem mass spectrometry coupled with ultra-high-performance liquid chromatography (UHPLC-HRMS/MS). Metabolite features representing analytes detected at a unique m/z and retention time were extracted, aligned, and quantified using the open-source MZmine2 software and feature-based molecular networking was performed (Figure 1b)44,45.
We first compared the metabolome of each strain irrespective of media type, extraction method, and exposure to antibiotic (Fig. 2a). The largest number of unique metabolite features (20.5%) were detected exclusively in the extracts of the strain K56-2, whereas 8.5% unique features were detected in the extracts of J2315 strain and 1.1% in the extracts of the strain C5424. While 30.2% of features were shared by all three strains, the largest number of features were shared between the strains J2315 and C54524 (31.7%). Among other factors, the similar metabotype of these two strains may be reflective of their ability to produce a brown pigment known as pyomelanin, which is not produced by the strain K56-246,47. Next, we compared the metabolomes acquired using the SPE and EtOAc extraction methods separately at 48h post-inoculum. This analysis revealed that many metabolite features are exclusively detected in extracts generated using SPE as compared to extracts generated using with EtOAc (Fig. 2b). A total of 32.5% of the metabolite features were detected only with the SPE method, while 9.5% of the total features were unique to EtOAc extraction method (Fig. 2b). Thus, the extraction method employed results in biased metabolomics comparisons when only one type of extraction strategy is employed. Lastly, we generated UpSet plots to visualize the number of features unique to each strain under different growth conditions as well as exposure to sub-lethal dose of antibiotic trimethoprim after subtraction of media background (Fig. 2c)48. This analysis revealed that media specific differences were the largest driver of metabolomic diversity within this study, with 878 features shared between all LB samples (●) and 563 features shared between all SCFM2 samples (●). In comparison, 513 features were detected in all samples. The UpSet plot also demonstrates that trimethoprim induces a unique metabolomic response in the K56-2 strain, with 362 metabolomic features exclusively detected in SCFM2 samples (●), 59 exclusive to LB samples (●), and 44 detected in both media types (●). Interestingly, 456 metabolomic features were uniquely detected in all LB samples except for K56-2 with trimethoprim (●) and 37 in all SCFM2 samples except for K56-2 with trimethoprim (●). While our prior studies have shown that K56-2 exhibits a unique metabolomic response to trimethoprim, our analysis demonstrates that this response is even more apparent in an environment representative of CF sputum43. Another pattern emerging from the UpSet plot is that many features are uniquely detected as shared between the pigmented J2315 and C5424 strains, with 203 features uniquely detected in LB (●), 90 in SCFM2 (●), and 93 across both media types (●). An additional 59 features were uniquely detected in C5424 and J2315 strains cultured in LB and SCFM2 in the presence of trimethoprim (●), hinting at a distinct response to trimethoprim that is associated with pyomelanin production. Such responses are hallmark of the personalized phenotypes of Burkholderia strains observed during infection49-52. Future investigations into phenotypic differences as well as differences in gene expression via transcriptomics will provide insights into the biochemical underpinnings driving these observations. Specific metabolites underlying these personalized chemotypes are discussed below.
Differences in siderophore production between LB and SCFM2 cultures
Siderophores are compounds secreted by bacteria to acquire iron from the surrounding environment, and were among the metabolites which were differentially produced by B. cenocepacia strains in LB and SCFM2 in this study. In healthy mammalian hosts, the pool of free iron is limited due to poor solubility of iron in its ferric state under physiological conditions, and because the majority of iron is either located in intracellular compartments or bound by host proteins such as hemoglobin, transferrin, lactoferrin, and ferritin53,54. In contrast, levels of both free and ferritin-bound iron are higher in CF sputum compared to sputum from healthy hosts55,56. Since this cofactor is essential for many important biological processes, iron-acquisition is required for survival in the host environment and can influence microbe-microbe interactions54,57. Siderophores which are known to be produced by Burkholderia include pyochelin, ornibactins, salicylic acid, and cepabactin43,54,58. Among these, B. cenocepacia have been shown to produce ornibactin and pyochelin54. In our study, various structural analogs of pyochelin were detected exclusively in SCFM2 in the presence of trimethoprim (Fig. 3). Pyochelin itself was not detected in this study, despite prior reports of low-level production by B. cenocepacia. These prior reports did not use mass spectrometry or NMR to report pyochelin production, but relied on fluorescence-based thin layer chromatography59. Using MSn analysis, we report production of a methylated derivative of pyochelin rather than pyochelin by B. cenocepacia strains used in this study as described below.
The feature with m/z 339.083 had a database annotation as a pyochelin methyl ester in GNPS (Supplementary Fig. S1a)60. However, pyochelin methyl ester has only been reported as a synthetic product generated to facilitate NMR characterization, and there is no available biosynthetic evidence that would support methylation on the carboxylic acid to produce an ester61. This feature was detected exclusively in cultures grown in SCFM2 supplemented with trimethoprim in both the K56-2 and C5424 strains, albeit low levels were observed in C5424 (Fig. 3a). Comparisons with the GNPS library MS2 spectra of pyochelin revealed that the fragment peaks with m/z 120.045, 180.048, and 190.032 are shared between the two molecules while others were shifted by 14.015 Da (-CH2), supporting the annotation as a methylated analog of pyochelin (Supplementary Fig. S1b). A structure search of pyochelin in SciFinder revealed thiazostatin as a likely candidate. Thiazostatin A/B (1) are previously reported stereoisomeric natural products that are related to pyochelin by an additional C4”-methylation of the thiazolidine ring (Fig. 3a)62,63. This thiazolidine C4”-methylation has also been observed in structurally homologous metabolites including isopyochelin, watasemycin, and yersiniabactin64-66. By MS2 analysis alone, the location of the methylation cannot be unambiguously determined, although the mass shifts of the fragment ion with m/z 146.027 in the MS2 spectrum of pyochelin to m/z 160.043 in our unknown metabolite’s MS2 spectrum suggests it is found on the terminal thiazolidine ring. To confirm the position of methyl group, we conducted MS3 analysis on the fragment ion with m/z 186.058. This analysis revealed that fragmentation of 186.058 yields a MS3 ion with m/z 158.063, which indicates methylation on the thiazolidine ring rather than at the carboxylic acid (Supplementary Fig. S2). Thus, this feature is reported as thiazostatin A/B. Thiazostatin A/B have been found to display antioxidant activity, and further screening is necessary to determine if this compound possess additional bioactivities62. Using MASST through the GNPS platform, we searched the MS2 spectrum of this compound against all public spectral datasets (Supplementary Fig. S3)67. This MASST search found dataset matches in extracts of Pseudomonas spp. grown in vitro, as well as in datasets analyzing the metabolomes of humans with various inflammatory diseases (including CF, diabetes, irritable bowel syndrome, rheumatoid arthritis, and HIV). Detection of this molecule in humans with inflammatory disease raises the possibility that this molecule may be important in influencing host microbiome structure, although further studies will be needed to explore whether this observation is truly associated with any biological significance.
Another metabolite with m/z 307.021 was detected exclusively in SCFM2 media extracts in both the B. cenocepacia strains K56-2 and J2315. In our molecular network, this feature matched with 2′-(2-hydroxyphenyl)-4′-thiazolyl-2,4-thiazolinyl-4-carboxylic acid (HPTzTn-COOH, 2) in the GNPS spectral library, which was further verified by manually comparing experimental MS2 spectra with a previously published MS2 spectra (Fig. 3a, Supplementary Fig. S4a)68. Like pyochelin, HPTzTn-COOH is a siderophore that is dependent on salicylic acid and cysteine as biosynthetic precursors and is capable of chelating Fe3+ in addition to other metal ions including Al3+, Ni2+, and Ca2+ 68,69. HPTzTn-COOH was detected exclusively in SCFM2 media, and primarily in the K56-2 strain, although also at low levels in J2315 (Fig 3a). This metabolite exhibited increased production in the presence of trimethoprim for the K56-2 strain and was not detected in the absence of trimethoprim for the J2315 strain.
Next, a feature with m/z 222.022 was detected exclusively in K56-2 cultures grown in SCFM2 media in the presence of trimethoprim (Fig. 3b, Supplementary Fig. S4b). This feature was annotated as aeruginoic acid (3), which is a shunt product in the pyochelin biosynthesis pathway observed in Pseudomonas and Burkholderia spp.70,71. Aeruginoic acid is the oxidized form of aeruginaldehyde (also known as the integrated quorum sensing signal, or IQS), which has been proposed as a “fourth QS molecule” in P. aeruginosa, although this claim is disputed72,73. The reactive aldehyde moiety of aeruginaldehyde also leads it to become incorporated into more complex natural products (such as malleonitrone or mindapyrrole B)74,75. Interestingly, pyochelin has been shown to spontaneously undergo cleavage and subsequent transformation into aeruginaldehyde when incubated in buffer solution at 30°C 74. Another shunt product of pyochelin biosynthesis with m/z 210.058 was detected under the same conditions as aeruginoic acid, and annotated as aerugine (4) (Fig. 3b, Supplementary Fig. S4c). Aerugine has been previously isolated from Pseudomonas fluorescens and exhibits selective antifungal activity76. Recent work by Kaplan et. al has indicated that aeruginaldehyde, aerugine, and aeruginoic acid all have iron-binding activity with aeruginoic acid binding to Fe3+ with a 1:1 ratio, aeruginaldehyde binding with a 2:1 ratio, and aerugine binding with a 3:1 ratio71. Unlike pyochelin, aeruginoic acid has a specific affinity for iron compared to other biologically relevant metals71. The largest abundance of thiazostatin is observed in the strain K56-2, which is likely why these intermediates are also detected in the strain K56-2.
In contrast to metabolites from the pyochelin pathway described above, the ornibactin class of siderophores was detected in the culture extracts of LB medium and not detected in culture extracts of SCFM2 (Supplementary Fig. S5). The SCFM2 media is prepared by adding 3.60 µM iron in the form of Fe3SO4, which was observed to be the average iron concentration present in expectorated sputum collected from CF patients23. Recently, it has been reported that commercial sources of mucin can be contaminating sources of iron that lead to altered siderophore production in P. aeruginosa cultured in SCFM222. To determine whether the concentration of iron might account for the differential production of siderophores observed between SCFM2 and LB media, inductively coupled plasma mass spectrometry (ICP-MS) was performed on media aliquots. ICP-MS analysis revealed that SCFM2 contained a mean iron concentration of 5.25 µM (standard deviation of 0.52 µM) while LB contained a similar mean concentration of 4.73 µM (standard deviation of 0.62 µM) (Supplementary Fig. S6). Therefore, factors other than iron availability likely play a role in the expression of the pyochelin and ornibactin biosynthetic gene clusters. QS systems have been previously implicated in regulating production of both pyochelin and ornibactin, with the CepR transcriptional regulator repressing production of ornibactin and the CepR2 regulator activating production of pyochelin in B. cenocepacia H11177. In our experiment, production of siderophores was observed to be dependent on strain and nutritional environment. The production of metabolites from the pyochelin pathway was further induced by the antibiotic trimethoprim, while ornibactins were not. Thus, a specific chemical cue in SCFM2 in presence of trimethoprim might play a role in induction of pyochelin production under these conditions and warrants detailed investigation in the future with knock strains that lack QS circuitry. This observation is noteworthy as pyochelin production by B. cepacia was previously suggested to be correlated with morbidity and mortality in patients with CF78. Mechanistic investigations into selective induction of siderophore biosynthesis pathways are critical to understanding their relevance to infections, and as such require further inquiry.
N-acyl-homoserine lactones production in LB compared to SCFM2
QS mediates bacterial response to changing environmental conditions through cell-density dependent global changes in gene expression79,80. Burkholderia can utilize QS to coordinate various metabolic processes and modulate cellular phenotypes such as swarming, aggregation, spatial structuring, and biofilm formation81,82. QS also plays an important role in infection by regulating genes involved in virulence, and so establishing how the external environment influences production of QS signals is important to understanding their role in pathogenesis83-85. The CepIR and CciIR QS systems have been described in B. cenocepacia strains which primarily produce and sense N-octanoyl-homoserine lactone (C8-AHL, 5) and N-hexanoyl-homoserine lactone (C6-AHL) respectively. In addition, an orphan CepR2, which is inactivated by C8-AHL, is also present in these strains 77,86-88. The gene for the orphan CepR2 lacks adjacent gene required for synthesis for N-acyl-homoserine lactone. In a previous untargeted metabolomic experiment, we observed production of a wide diversity of AHLs by Burkholderia spp. grown in LB media, as well as their corresponding acyl-homoserine products formed by hydrolysis of the lactone ring (referred to hereafter as “hydrolyzed AHLs”)43. In this study, we queried whether these signals are differentially detected in SCFM2 compared to LB media. The C8-AHL (m/z 228.160, 5) was detected in both LB and SCFM2 media, and its hydrolyzed form (6) was detected in LB media alone (Fig. 4). Additionally, we detected hydrolyzed C13-AHL (7), hydrolyzed C13-AHL:1db (8), the sodium adduct of hydrolyzed C13-AHL (9), and the sodium adduct of hydrolyzed 3-OH C13-AHL (10) exclusively in SCFM2 when trimethoprim was present (Fig. 4). Thus, the production of these C13-AHLs were induced by trimethoprim only in SCFM2. Detection of C8-AHL, hydrolyzed C8-AHL and hydrolyzed C13-AHL was confirmed using commercial AHL standards. These AHL standards were treated with sodium hydroxide to promote hydrolysis of the lactone ring to verify the detection of hydrolyzed AHLs (Supplementary Fig. S7). Naturally produced AHLs with an odd number of carbons in the acyl sidechain are relatively rare, and their functions are not well-characterized89. In a previous untargeted metabolomics study of 10 different Burkholderia strains grown in LB media, we detected hydrolyzed 3-oxo-C13-AHL:1db exclusively in extracts of B. thailandensis E26443. To our knowledge, the present study represents the first description of C13 AHLs being produced by B. cenocepacia. Further studies will be needed to explore the function and biochemical basis for production of C13-AHLs by B. cenocepacia in SCFM2 media, and identify the mechanism by which trimethoprim upregulates the production of this AHL.
Fragin and pyrazine secondary metabolites
Fragin (11) is a diazeniumdiolate metallophore with antifungal activity that is produced by Burkholderia and Pseudomonas spp.90. In a previous untargeted metabolomics study, we reported significantly increased production of fragin and its structural analogs in B. cenocepacia K56-2 cultures grown in LB media when supplemented with the antibiotic trimethoprim43. Fragin production was only observed in the K56-2 strain, despite the fact that the ham gene cluster responsible for fragin biosynthesis is identical in the closely related J2315 and C5424 strains. This result highlighted that even genetically similar Burkholderia strains can exhibit markedly different responses to external stimuli, such as trimethoprim exposure. In the current study, fragin was detected in both media conditions, but the majority of other nodes in the cluster (11/18) were detected exclusively in either LB or SCFM2 media (Fig. 5a). These analogs differ in the length of the acyl group added by the HamF enzyme, likely a result of differential availability of compounds containing variable acyl chain lengths which act as substrates for HamF. The MS2 spectra of these analogs showed a characteristic fragment corresponding to the loss of NO group (29.998 Da), that was also observed in the MS1 spectrum as an in-source fragment43. Fragin analogs with differential production in the two media types include nodes with m/z 302.243 (and the corresponding in-source fragment with m/z 272.246), 316.223 (in-source fragment with m/z 286.225), and 318.239 (in-source fragment with m/z 288.240) (Fig. 5a).
We discovered another cluster in our molecular network containing several molecules which, like fragin, are exclusively detected in K56-2 samples and show increased production in the presence of trimethoprim (Fig. 5b). These metabolites were reported by our group in a previous study as unknown metabolites43. Here we employed the recently developed CANOPUS tool to classify these unknown compounds into ClassyFire chemical classes, leading to their annotation as pyrazines91,92. This information led us to conduct a literature search of pyrazines produced by Burkholderia, enabling annotation of these metabolites as pyrazine N-oxides (PNOs), which was verified using standards provided by Li and colleagues (Supplementary Figs. S8, S9)93. The expression of pvfB and pvfC genes in animal pathogen Pseudomonas entomophila L48 and the plant pathogen Pseudomonas syringae pv. syringae UMAF0158 were previously shown to lead to production of a family of PNOs, namely PNO B (12, m/z 181.135), PNO A (13, m/z 197.126), and dPNO (14, m/z 199.145)93. The pvfB and pvfC genes are homologous to the Burkholderia genes hamC and hamD present in the biosynthetic gene cluster of fragin. Disruption of pvfA-D cluster in animal pathogen Pseudomonas entomophila L48 and the plant pathogen Pseudomonas syringae pv. syringae UMAF0158 was shown to significantly reduce the virulence of these strains93. Subsequent studies found that the pvf gene cluster is involved in synthesis of a signaling molecule which regulates production of small molecule virulence factors such as monalysin in P. entomophilia and mangotoxin in P. syringae94-96. Both dPNO and PNO B appeared in the feature-based molecular network, while PNO A did not due to low abundance. However, a node corresponding to PNO A was observed when data was analyzed with a classical molecular network (Fig. 5b). Two additional nodes in this network were putatively annotated as 2-isopropyl-3- methoxypyrazine (m/z 153.102, 15) and 2,5-diisopropylpyrazine (m/z 165.138, 16) based upon available literature of bacterially produced pyrazines (Fig. 5b, Supplementary Fig. S9)97. The MS2 spectra of these related molecules did not have fragment ions in common, and so molecular networking methods alone failed to highlight the structural relatedness of these three compounds. Nevertheless, CANOPUS enabled us to independently predict that each of these compounds were pyrazines, ultimately leading to their annotation.
These observations reveal that the ham gene cluster that is responsible for fragin biosynthesis also leads to production of PNOs in B. cenocepacia. PNO production was also induced by the addition of trimethoprim in both the SCFM2 and LB culture media. Thus, unlike the siderophores described above which showed media-dependent induction by trimethoprim, fragin and PNOs are similarly induced by antibiotic trimethoprim in both LB and SCFM2. The ability of trimethoprim to induce production of fragin and pyrazines including PNOs in both SCFM2 and LB media highlights that antibiotics can serve as signaling molecules capable of significantly modulating expression of the genes encoding virulence factors across multiple nutritional environments.
Differential detection of lipids in LB and SCFM2
The overall lipid composition of bacterial cells has been reported to be influenced by several local environmental factors, including pH, nutrient availability, oxygen levels, temperature, and buildup of metabolic waste products98. Several clusters with hits to lipids in the GNPS spectral database were detected at higher levels in bacterial extracts grown in SCFM2, including hopanoids, phytomonic acid, monosaturated monoacylglycerols (MGs), and phosphatidylethanolamines (PEs), described below (Fig. 6).
The annotation of hopanoid cluster was performed by first searching for candidate features with m/z calculated for known bacterial hopanoids. Identified candidate features were then verified by comparing experimental MS2 spectra to spectra previously published in literature99,100. Once annotations were supported through spectral matching, these features were used to further propagate annotations to connected nodes within the molecular network based on mass differences. Hopanoids are pentacyclic triterpenoids frequently found in bacterial membranes101,102. These polycyclic lipids are structurally analogous to eukaryotic sterols and are thought to have similar functions in regulating fluidity, permeability, and stabilization of bacterial membranes. Hopanoid biosynthesis has been previously reported in B. cenocepacia, where they are involved in resistance to low pH and antibiotics while also being important for swimming and swarming motility101,102. A metabolite feature with m/z 708.540 is annotated as bacteriohopanetetrol (BHT) cyclitol ether (17) and was detected only in extracts of SCFM2 (Fig. 6a, Supplementary Fig. S10a)99,102. Production of BHT cyclitol ether has been previously reported in B. cenocepacia, and our predicted annotation was supported by comparing experimental MS2 spectra with previously published spectra available in literature99,102. In addition, we also annotated the node with m/z 706.526 as unsaturated BHT cyclitol ether (18) (Fig. 6a). Both Δ6 and Δ11 monosaturated BHT analogs have been previously characterized in bacteria100. Although our mass spectrometry analysis alone is insufficient to pinpoint the location of this unsaturation, we have putatively annotated this feature as bacteriohop-6-enetetrol cyclitol ether (18) since unsaturation at this location has been reported in B. cepacia strains102,103. This feature was detected in all three B. cenocepacia strains when grown in SCFM2 media with the highest intensity observed in the K56-2 strain, but when cultured in LB it was only detected in J2315 cultures at low levels. Annotation of another feature with m/z 724.535 is consistent with a gain of a hydroxyl group from BHT cyclitol ether (17). This feature was exclusively detected in K56-2 strains grown in SCFM2 media and the associated MS2 spectra indicates this feature is likely bacteriohopanepentol (BHP) cyclitol ether (19) (Supplementary Fig. S10b). BHP derivatives have been fully characterized only in Acetobacter spp., Azotobacter vinelandii, and Nostoc spp., although our observation is supported by prior evidence suggesting BHP derivatives are produced by B. cepacia strains as well103-106. The feature with m/z 722.520 was detected in all three B. cenocepacia strains (largely in K56-2 samples) grown in SCFM2 media, which is presumably bacteriohop-6-enepentol cyclitol ether (20) although the location of unsaturation cannot be unambiguously determined as mentioned above. Next, a feature with m/z 750.551 was detected exclusively in SCFM2 containing cultures with a mass difference of 42.011 (C2H2O) from BHT cyclitol ether representative of acetylation. The final feature in the hopanoid cluster with m/z of 748.543 is annotated as unsaturated analog of the acetylated BHT cyclitol ether (m/z 750.551), with the unsaturation most likely occurring at the C6 position.
Another metabolite in the lipid family is annotated as phytomonic acid (21) based on the spectral match to the MS2 spectrum in the GNPS database. This annotation was confirmed using a commercial analytical standard (Fig. 6b, Supplementary Fig. S11a). Phytomonic acid was detected in both LB and SCFM2 media, although consistently higher levels were detected in SCFM2 media for all three strains (Supplementary Fig. S11b). Production of cyclopropane acids have been previously reported in B. multivorans107. Cyclopropane fatty acids such as phytomonic acid are suggested to regulate membrane fluidity and stability and have been shown to increase extracellular survival in acidic or under conditions of high osmolarity108,109. These lipids also are major components of the membranes of intracellular pathogens such as Brucella abortus and Mycobacterium tuberculosis108,109. It is interesting to note that similar to B. abortus and M. tuberculosis, Burkholderia spp. are also capable of intracellularly infecting macrophage cells. The specific role of cyclopropane fatty acids in B. cenocepacia virulence requires further investigation.
Multiple monounsaturated monoacylglycerol (MG) lipids including nonadecenoyl-glycerol (19:1 MG, 22), octadecenoyl-glycerol (18:1 MG, 23), heptadecenoyl-glycerol (17:1 MG, 24), hexadecanoyl-glycerol (16:1 MG, 25), and the sodium adduct of heptadecenoyl-glycerol (17:1 MG, 26) were detected only during growth in SCFM2. Annotations for this class of lipids was performed with a commercial standard of octadecenoyl(d7)-glycerol (18:1-d(7) MG) (Fig. 6c, Supplementary Fig. S12). For both 19:1 MG (22) and 17:1 MG (24), two unique metabolic features were detected with identical masses and MS2 patterns but slightly different retention times, possibly corresponding to distinct cis/trans isomers. Burkholderia spp. are known to produce the lipase LipA, which yields monoacylglycerols as a product during degradation of di- and tri-acylglycerides110,111. In B. cenocepacia, production of the LipA lipase is induced as part of the CepIR quorum sensing system 86,112. Due to the ability of monoacylglycerols to destabilize bacterial cell membranes, several have been reported to demonstrate antimicrobial activities which vary based on chain length and degree of unsaturation113,114.
Finally, several phosphatidylethanolamines (PEs) were detected in this study, all consistently detected under similar conditions. In Gram-negative bacteria, the inner leaflet of the outer membrane is made up of phospholipids, of which PEs are the major component115. These PEs varied by the length of their acyl-chains, and were annotated as 2-OH-PE(16:1) (27), 2-OH-PE(16:0) (28), 2-OH-PE(17:1) (29), 2-OH-PE(18:1) (30), 2-OH-PE(18:0) (31), and 2-OH-PE(19:1) (32) based on GNPS library matching and manual annotations based on characteristic MS2 fragmentation patterns described in literature (Fig. 6d)116. Production of 2-OH-PEs has been observed in B. cepacia, and is reported to increase during stress observed under growth in high temperature117.
Differential metabolism of the antibiotic trimethoprim between LB and SCFM2
Several compounds that were differentially detected between growth in SCFM2 and LB were found to be related to trimethoprim as evidenced by MS2 spectral similarity. As previously described, MS2LDA was used to discover metabolites that contained trimethoprim as substructure. Thus, being higher in molecular weight than trimethoprim, these metabolites represent biochemical transformations of trimethoprim itself by B. cenocepacia bacteria43. In this study, trimethoprim substructure was annotated using MS2LDA as Mass2Motif 543. We compared the presence of these metabolites in the extracts of bacteria cultured in LB and SCFM2 (Fig. 7). Metabolism of trimethoprim was carried out by only the pigmented strains J2315 and C5424 and not by the non-pigmented K56-2 strain, as reported previously43. We generated a volcano plot of molecules containing the trimethoprim motif to visualize compounds which were differentially detected across the two media conditions (Fig. 7a). This analysis revealed that majority of the trimethoprim metabolites showed significantly higher production in SCFM2 as compared to LB. Structural characterization of these metabolites will provide insight into the pathways used by pigmented Bcc strains to metabolize xenobiotic compounds like the antibiotic trimethoprim and will facilitate future studies exploring how biotransformation will impact antibacterial activity.