Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode

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Stable isotope-assisted metabolic profiling reveals growth mode dependent differential metabolism and multiple catabolic pathways of L-phenylalanine in Rubrivivax benzoatilyticus JA2 Lakshmi Prasuna Mekala, Mujahid Mohammed, Sasikala Chintalapati, and Venakata Ramana Chintalapati J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.7b00500 • Publication Date (Web): 18 Oct 2017 Downloaded from http://pubs.acs.org on October 19, 2017

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Journal of Proteome Research

Stable isotope-assisted metabolic profiling reveals growth mode dependent differential

metabolism

and

multiple

catabolic

pathways

of

L-

phenylalanine in Rubrivivax benzoatilyticus JA2 Lakshmi Prasuna Mekala1#, Mujahid Mohammed1#, Sasikala Chintalapati2, Venkata Ramana Chintalapati1*

1

Department of Plant Sciences, School of Life Sciences, University of Hyderabad, P.O.

Central University, Hyderabad 500 046, India. 2

Bacterial Discovery Laboratory, Centre for Environment, Institute of Science & Technology,

Jawaharlal Nehru Technological University, Hyderabad, Kukatpally, Hyderabad 500 085, India. #

These authors contributed equally to this work

*

Corresponding author:

Prof. Ch.V. Ramana, Department of Plant Sciences, School of Life Sciences, University of Hyderabad, Hyderabad-500 046, Telangana, India. E-mail: [email protected] Tel phone : +91 040 23134502 Fax: +91 040 23010120 & 23010145,

ACS Paragon Plus Environment

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ABSTRACT Anoxygenic phototrophic bacteria are metabolically versatile, survive under different growth modes using diverse organic compounds, yet their metabolic diversity is largely unexplored. In the present study, we employed stable isotope-assisted metabolic profiling to unravel the L-phenylalanine catabolism in Rubrivivax benzoatilyticus JA2 under varying growth modes. Strain JA2 grows under anaerobic and aerobic conditions by utilizing L-phenylalanine as a nitrogen source.

Further, ring labeled

13

C6-phenylalanine feeding followed by LC-MS

exometabolite profiling revealed 60 labeled metabolic features(M+6/M+12/M+18) derived solely from L-phenylalanine, of which 11 were identified, 7 putatively identified and 42 unidentified under anaerobic, aerobic conditions.

However, labeled metabolites were

significantly higher in aerobic compared to anaerobic conditions. Furthermore, detected metabolites, enzyme activities indicated multiple L-phenylalanine catabolic routes mainly, Ehrlich, homogentisate-dependent melanin, benzenoid and unidentified pathways operating under anaerobic and aerobic conditions in strain JA2. Interestingly, the study indicated Lphenylalanine-dependent and independent benzenoid biosynthesis in strain JA2 and differential flux of L-phenylalanine to Ehrlich and benzenoid pathways under anaerobic/aerobic conditions. Additionally, unidentified labeled metabolites strongly suggest the presence of unknown phenylalanine catabolic routes in strain JA2. Overall, the study uncovered the L-phenylalanine catabolic diversity in strain JA2 and demonstrated the potential of stable isotope-assisted metabolomics in unraveling the hidden metabolic repertoire.

KEYWORDS: Stable isotope-assisted metabolic profiling, Phenylalanine, differential metabolism, anoxygenic photosynthetic bacteria, benzenoids, Ehrlich pathway, melanin.


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INTRODUCTION Bacteria are one of the most diverse groups of microorganisms survive under a wide range of environmental conditions by using the extended range of substrates. The remarkable metabolic flexibility and sophisticated metabolic regulations enable bacteria to exploit the nutrient resources for their growth and adapt to the changing environment1-4. Though the bacterial metabolism has a profound effect on human health and ecology5, 6, our understanding of bacterial metabolism is limited and mostly restricted to few model organisms studied under a defined set of conditions7, 8. Homology-based annotations drive the current metabolic networks of newly sequenced bacteria, as new gene sequences have little to no homology with database entries and thereby limiting the identification of new biochemical reactions or pathways8-10. Thus, in the metabolic charts, central metabolic pathways are well predicted while accessory or conditionally induced pathways are underrepresented7, 8. Untargeted metabolomics is an emerging tool to discover the novel metabolic pathways and new enzyme functions, wherein the identification of a different class of known and unknown metabolites is possible at a time8, 9. However, label-free untargeted metabolomics undermines the biosynthetic origin/substrate fate and metabolic flux of known and unknown pathways8. Using untargeted metabolomics in conjunction with stable isotope substrate feeding allows 1. identification of tracer derived unknown/known metabolites, 2. metabolic flux, 3. discovery of hidden metabolic networks under a given set of conditions1115

. Untargeted stable isotope-assisted metabolic profiling (SIMP) have been fundamental in

understanding the bacterial central metabolic networks in model organisms16-18. However, employing SIMP to poorly characterized organisms with unique physiological traits or exposing them to different growth conditions has high potential to discover conditionally induced new metabolic pathways and hidden metabolic versatility7, 12. One such metabolically versatile group is anoxygenic phototrophic bacteria (APB), capable of surviving under diverse conditions19, 20. Although they display growth on various organic substrates under different growth modes, their metabolic flexibility is largely unexplored. Rubrivivax benzoatilyticus JA2, a metabolically versatile anoxygenic phototrophic bacterium grows under different growth conditions by utilizing a broad range of organic compounds21. Strain JA2 has the remarkable aromatic compound metabolism; it transforms aniline to acetanilide22, L-tryptophan to indoles19,

23

and L-phenylalanine to

phenolic compounds24, 25. SIMP studies on tryptophan catabolism in strain JA2 revealed multiple tryptophan catabolic pathways19 and a novel enzyme, tryptophan ammonia lyase26.


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Stress-induced overproduction of indole derivatives was reported in strain JA2 by yet unknown metabolic regulation mechanism27. The previous study on L-phenylalanine catabolism revealed that strain JA2 could not utilize phenylalanine as the sole source of carbon instead uses as a nitrogen source under anaerobic condition24. Strain JA2 transforms phenylalanine to phenyllactic acid via phenylpyruvate24 and also produces a novel phenolic compound, Rubrivivaxin, through yet unknown pathway under anaerobic condition25. Overall, these findings suggest the potential of strain JA2 in metabolizing the aromatic compounds under anaerobic condition. Though strain JA2 grows under anaerobic and aerobic conditions, studies on its aromatic metabolism are restricted only to anaerobic conditions. Interestingly, the genome of strain JA2 indicated the presence of genes involved in aerobic aromatic metabolic routes. To this end we hypothesize that strain JA2 may have adopted different catabolic routes to metabolize growth substrates under anaerobic and aerobic conditions and this aspect is not studied so far. To test this hypothesis, in the present study, we employed untargeted SIMP to analyze the exometabolome of strain JA2 grown in the presence of labeled l-phenylalanine under anaerobic and aerobic conditions. Untargeted exometabolite profiling facilitated the detection of the

13

C labelled metabolites and thus

captured the biochemical events associated with phenylalanine catabolism in strain JA2. The current study revealed the surprising versatility in L-phenylalanine catabolism and presence of unexpected new pathways. Our study also demonstrated catabolic multitasking (catabolism of a compound via multiple pathways) and differential metabolism of L-phenylalanine under anaerobic and aerobic conditions in strain JA2.

METHODS Organism and growth conditions Rubrivivax benzoatilyticus JA2 (ATCC BAA-35) was regularly grown photoheterotrophically under anaerobic conditions at 30±2°C, light 2400 Lux on mineral media supplemented with malate (22 mM) as a carbon source and ammonium chloride (7 mM) as a nitrogen source at pH 6.8 in culture bottles (250 ml)24. In the present study, all the experiments were performed using photoheterotrophically grown log phase cultures (OD 0.2 at 660 nm) as inoculum. Ammonium chloride was replaced with L-phenylalanine (1mM) as a nitrogen source whenever required. Anaerobic growth was achieved in filled 250 ml culture bottles without any air gap and tightly sealed, incubated at 30˚C. Aerobic growth was achieved in 500 ml flasks containing 200 ml of medium and flasks were incubated at 30˚C in a rotary incubator shaker (New Brunswick, Innova) operating at 180 rpm for 48h. For, stable isotope profiling


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studies, media was amended with 1 mM of unlabeled L-phenylalanine (12C6) or 13C6 labeled L-phenylalanine (Ring-13C6, 99% Cambridge Isotope laboratories Inc. USA). Stable isotope feeding experiments were carried out in biological replicates, and the growth was measured turbidometrically at O.D 660nm. For metabolite and pigment extraction cells were harvested after 48h (stationary phase) of growth. Estimation of phenylalanine and total phenols Phenylalanine was estimated directly from the supernatants through HPLC according to Prasuna et al. (2012). Total phenolic content of the culture supernatants was estimated using Folin-Ciocalteau (FC) reagent as per standard protocol28 with slight modifications and expressed as µg mg dry weight of the cells. Briefly, to 0.5 ml of supernatants, 0.5 ml FC reagent and 2 ml of Na2CO3 (20%) were added, incubated for 5 minutes without warming the samples. The absorbance was measured at 650 nm against a reagent blank and gallic acid as a standard. Extraction of metabolites Cultures grown in the presence or absence of L-phenylalanine (control; mineral media with ammonium chloride) under anaerobic and aerobic conditions were harvested after 48h (stationary phase) by centrifugation (10000 g, 4○C, 10 min) and the supernatants were acidified (pH 2) with 5N HCl. Supernatants were extracted thrice with ethyl acetate (100 ml), and the extracts were pooled. The combined extracts evaporated under vacuum using a rotary flash evaporator (Heidolph, Germany) at 35○C. Finally, the dried extracts were dissolved in 1 ml of LC-MS grade methanol, filtered through 0.22 µm membrane filters (Supor, PallScientifics), stored at -20○C until further analysis. Melanin pigment purification, Electron Spin Resonance (ESR) spectroscopic and SEM analysis The pigment was purified from supernatants obtained from aerobic cultures grown in the presence of L-phenylalanine. After 48 h of growth, cells were harvested by centrifugation (10000 g for 10 min, 4°C), the supernatant was acidified (pH 2) using 5N HCl and stored at 4°C for 5 days. The brown precipitate formed was separated by centrifugation (12000 g, 4°C for 10 min) and the pigment was purified by successive washing steps (twice) with 5 ml of 100% organic solvents (hexane, chloroform, ethyl acetate, ethanol) followed by Milli-Q water (thrice with 5 ml). Finally, the total purified pigment was freeze-dried for further analysis and solubility of dried pigment was tested in 1 ml of NaOH (1N). ESR spectrum of purified brown pigment was recorded on JEOL X-band ESR spectrometer.


The dried

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pigment (30 mg) was loaded onto ESR tube, and the spectrum was recorded using standard method and parameters29. SEM analysis was performed by mounting the dried sample on glass pieces (0.5x0.5 cm) and fixed on stubs followed by gold sputtering. Specimens were examined using SEM (Philips-XL30 series) at 10 different random locations under different magnifications. HPLC analysis HPLC analysis was done according to Mujahid et al.30 In brief, HPLC analysis was carried out on Prominence system (Shimadzu, Japan) consisted Photodiode Array detector and Phenomenex C-18 column (Luna, 5µm, 250 x 4.6 mm). Linear gradient programme was employed to separate the metabolites. 1% (v/v) acetic acid in water (solvent A) and acetonitrile (100%, solvent B) (LC-MS grade Merck) was used as mobile phase with 1.5 ml/min flow rate. The absorption spectra were recorded with a photodiode array detector, and the metabolites were quantified using HPLC based on the peak areas of the known concentrations of authentic standards (Sigma Aldrich). LC-MS-ESI analysis Ethyl acetate extracts obtained from cultures grown in the presence of unlabeled(12C) or labeled (13C) L-phenylalanine were subjected to LC-MS analysis. First, unlabeled and labeled samples were individually run on LC-MS followed by the pooled sample (1:1 ratio) of unlabeled and labeled fractions for unequivocal detection of isotopically enriched metabolites. Mass spectral analysis was performed on 6520 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, USA) coupled to HPLC (Agilent 1200 series) equipped with Photodiode Array detector; an autosampler was used to inject the 2 µl sample for chromatographic separation at 25°C. Metabolites were separated on reverse phase column (Phenomenex), C-18 (Luna, 5 µm, 150 x 4.6 mm) with a constant flow rate of 0.8 ml.min-1 using 0.1% acetic acid in water (v/v) (eluent A) and acetonitrile (eluent B). Metabolites were eluted using a gradient programme: Initially, the mobile phase composition was 1% eluant B followed by a linear gradient of 55% within 48 min and then step gradient to 100% within 55 min, and held for 5 min. Finally, the column was washed and re-equilibrated (1% eluant B) for 5 min. Metabolites were detected, and the absorption spectra were recorded using a Photodiode Array detector (220-600 nm). The separated compounds were infused into the electron spray ionization (ESI) ion source operated under full spectrum scan mode from 501000 m/z. Data acquisitions were collected under both positive and negative ionization modes in

separate

runs.

Two

reference

masses:

121.0509 m/z (C5H4N4);

922.0098 m/z (C18H18O6N3P3F24) were measured throughout the sample run for mass


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correction and obtained the accurate mass. Mass spectrometer conditions: nitrogen as drying gas at a constant flow of 10 L.min-1, nebulizing at 45 psi respectively, drying gas temperature (300°C), ion source capillary voltage (4 kV) and fragmentor voltage (144 V). acquisition

were

acquired

using

MassHunter

workstation

(version.06.0,

Data Agilent

Technologies, USA). Data analysis and metabolite identification All the raw data files were analyzed using Agilent MassHunter Qualitative software (version.06.0, Agilent Technologies, USA). Data (labeled-unlabeled mix) was manually inspected for

12

C6 monoisotopic ions(M) and respective

13

C6 isotopic pattern (M+6/M+12)

13

expected for one or two C6-aromatic rings incorporated metabolites. Molecular ion masses of isotopically enriched metabolic features (M+6/M+12) were predicted from measured m/z values of the putative unlabeled features (M). Metabolic feature pairs (unlabeled and labeled) were considered with defined criteria; (i) similar retention times with co-elution, (ii) similar chromatographic peak shape, (iii) identical absorption spectra, (iv) theoretical mass shift ∆m/z 6.0201/12.0402. Peak area(ion intensity) of

12

13

C and

C metabolic features were

obtained from Extracted Ion Chromatograms (EICs). MAVEN31 software package was also employed to confirm the

12

C and

13

C metabolic feature pairs. Molecular formula generator

(MFG) algorithm of MassHunter was used to generate the tentative molecular formulae for the metabolic features (12C). MFG uses monoisotopic mass accuracy, isotopic abundance ratios and spacing between isotopic peaks to assign the molecular formulae and score. Higher the MFG score (highest 100) of a candidate formula, it is likely to be correct and indicates accurate mass analysis. Criteria to access the metabolite identity- confirmed: m/z, absorbance, predicted formula, database match, standards; putative: m/z, absorbance, predicted formula, database match; unidentified:

m/z, absorbance, predicted formula.

METLIN(https://metlin.scripps.edu), MassBank(www.massbank.jp), HMDB(www.hmdb.ca), KEGG(www.genome.jp/kegg/pathway)

database

entries

were

used

for

metabolite

identification. Enzymatic assays Enzymatic assays were performed using the cell lysate obtained from anaerobic or aerobic cultures grown in the presence of L-phenylalanine. The cell lysate was prepared according to Prasuna et al24 and obtained cell lysate was used as an enzyme source. Aromatic amino acid aminotransferase activity was done according to Prasuna et al. 201224 using L-tyrosine (2 mM) as substrate. Phenylpyruvate decarboxylase reaction mixture contained phenylpyruvate (2.5 mM), MgCl2 (0.05 mM) and 0.1 mM thiamine diphosphate in 3 ml volume.


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Phenylacetaldehyde dehydrogenase assay mixture consisted of 2 mM phenylacetaldehyde, 0.5 mM NAD. Hydroxyphenylpyruvate dioxygenase assay mixture consisted of 4hydroxyphenylpyruvate (1 mM), ferrous sulfate (1 mM), and ascorbic acid (5 mM). Chorismate pyruvate-lyase assay mixture consisted of 1 mM chorismate. All the enzymatic assays were carried out in 3 ml reaction volume using 50 mM Tris buffer pH 7.5, and the reactions were initiated by the addition of appropriate amount of enzyme source to the reaction mixture and incubated for 30 min at 37°C. Reactions were stopped with 300 µl of 5N HCl, and the blank reaction consisted of pre-denatured enzyme source. The reaction mixture was centrifuged at 10,000 rpm for 10 min, and the supernatant of the reaction mixture was used to detect the products. The supernatants obtained from assays were extracted with ethyl acetate(3 ml thrice), and the pooled extracts were evaporated to dryness under vacuum using flash evaporator. Finally, extracts were dissolved in 100 µl of methanol (HPLC-grade)24. Products, 4-hydroxyphenylpyruvic acid, phenylacetic acid, homogentisic acid and 4– hydroxybenzoic acid formation was detected using HPLC and co-eluted with authentic standards while pyruvate decarboxylase product, phenylacetaldehyde was detected using the GC-MS method described by Mujahid et al22.

RESULTS Growth, phenylalanine utilization, and phenols production Growth and phenylalanine utilization by strain JA2 were studied in anaerobic and aerobic conditions. Strain JA2 utilized L-phenylalanine as a sole source of nitrogen under both the conditions with the simultaneous growth (Figure 1). Strain JA2 utilized 0.96 mM of Lphenylalanine under anaerobic (Figure 1A) and completely (1 mM) under aerobic (Figure 1B) conditions. Anaerobic cultures reached highest optical density compared to aerobic (Figure 1). Strain JA2 produced phenolic compounds with the concomitant utilization of Lphenylalanine under both the conditions. Phenols production was significantly higher in Lphenylalanine amended conditions compared to control (Figure 1). L-phenylalanine amended aerobic cultures (168 µg. mg dry wt-1) showed higher phenolic content compared to anaerobic cultures (72 µg. mg dry wt-1) (Figure 1C, 1D). Untargeted stable isotope-assisted metabolite profiling in strain JA2 Initially, ethyl acetate extracts of anaerobic and aerobic cultures were subjected to HPLC analysis to check the possible variations in exometabolite profiles. HPLC analysis revealed a significant difference in metabolic profiles (at 260 nm) of aerobic and anaerobic cultures ( Figure S1). Metabolic profiling showed more metabolites in phenylalanine-amended aerobic


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and anaerobic cultures compared to respective controls (Figure S1). The variations observed in metabolic profiles of phenylalanine-amended cultures compared to control most likely due to metabolites produced as a result of phenylalanine catabolism. To unequivocally demonstrate the phenylalanine catabolism, we employed an untargeted SIMP study wherein anaerobic and aerobic cultures were grown in the presence of

12

C6 or 13C6 L-phenylalanine.

The total ion chromatograms (TICs) of specific conditions in positive and negative ionization modes showing marked variations were represented (Figure 2A). From the TICs, phenylalanine derived metabolic features were identified based on the mass difference of 6.0201 or 12.0402 units compared to 12C-isotopologues describing the incorporation of one or two aromatic rings. Typical labeling pattern of unlabeled and labeled metabolites was described, wherein 4-hydroxybenzoic acid (4-HBA) showed only unlabeled EIC 137[M+0] while PLA showed both unlabeled 165[M+0] and labeled 171[M+6] EICs, showing 6 units mass difference (Figure S2). More labeled metabolic features were detected in negative compared to positive ionization mode in anaerobic as well as aerobic conditions, while few were ionized in both ionization modes (Figure 2B). A typical Rt vs. m/z profile of labeled metabolic features from L-phenylalanineamended anaerobic and aerobic cultures was represented (Figure. 3A). The profiles showed clear differences in mass distribution and relative polarity of the metabolites, wherein highly non-polar metabolites observed only in aerobic conditions (Figure 3A). Amongst the labeled metabolic features, 39 were detected only in aerobic, 15 in anaerobic and 7 were found in both conditions (Figure 3B). Further, under anaerobic condition, 18 metabolic features showed an increase in mass by +6 units and 2 by +12 mass units compared to unlabeled. Similarly, 28 metabolic features showed an increase in mass by +6 units, while 15 showed +12 units and one showed +18 mass units increase in aerobic conditions (Figure 3C). Increase in mass by +18 indicates that metabolic feature incorporated with three aromatic rings from phenylalanine. Further, absorption spectra were compared to determine the variations in labeled metabolic features of both conditions.

Metabolic features showed

absorption in UV and visible regions and displayed broad spectral differences indicating that they are chemically distinct. Based on the absorption maxima, the metabolic features were categorised into 3 groups, 240-280 nm, 280-320 nm, >350 nm. Metabolic features showing an absorption >350 nm were observed only in aerobic conditions (Figure 3D). Based on the criteria of an increase in mass by 6 or 12 units, a total of 60 labeled metabolic features were detected from both conditions, identified, putatively annotated and unidentified metabolites were listed in Table 1.


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Isotopic enrichment of benzenoids Few metabolites displayed the differential labeling under anaerobic and aerobic conditions, and their isotopic enrichment was determined. Labeled and unlabeled ion intensities of metabolites were acquired from respective EICs to measure the isotopic enrichment. Surprisingly, benzenoids such as 4-HBA, 4-hydroxybenzaldehyde (4-HBAld) and benzoic acid (BA) displayed both unlabeled (M+0) and labeled (M+6) ion signals (Figure S3) from 13C6 L-phenylalanine fed anaerobic as well as aerobic conditions. Labeled isotopic signal (M+6) for 4-HBA was not detected in anaerobic cultures amended with

13

C6 L-

phenylalanine, while the significant labeling signal was observed in aerobic cultures (63%) (Figure 4A, B). The isotopic abundance of 4-HBAld and BA were significantly high under aerobic (97% and 98%) compared to anaerobic conditions (28% and 29%) (Figure 4A, B). Quantification of the aryl metabolites Further, the aryl metabolites produced by strain JA2 in the presence of Lphenylalanine under anaerobic and aerobic conditions were quantified by HPLC analysis. Phenyl derivatives such as phenyllactic acid (PLA), phenylpyruvate (PPY), phenylacetic acid (PAA) and 4-hydroxyphenylpyruvic acid (4-HPPY) were detected only in cultures grown in the presence of L-phenylalanine while they were absent in control cultures. PLA levels were significantly higher in anaerobic conditions compared to aerobic (Figure 4C) while PPY and 4-HPPY were detected only in anaerobic but not in aerobic cultures (Figure 4C). Further, PAA levels were higher in aerobic compared to anaerobic conditions (Figure 4C). Interestingly, 4-HBA, 4-HBAld, BA were detected in L-phenylalanine-amended and unamended (control) anaerobic as well as aerobic cultures (Figure 4D). Under anaerobic conditions, 4-HBA levels were not altered significantly (

Stable Isotope-Assisted Metabolic Profiling Reveals Growth Mode

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