Live Cell Discovery of Microbial Vitamin Transport and Enzyme

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Live Cell Discovery of Microbial Vitamin Transport and Enzyme-Cofactor Interactions Lindsey N. Anderson,† Phillip K. Koech,† Andrew E. Plymale,† Elizabeth V. Landorf,‡ Allan Konopka,† Frank R. Collart,‡ Mary S. Lipton,† Margaret F. Romine,† and Aaron T. Wright*,† †

Biological Sciences Division, Pacific Northwest National Laboratory, Richland, Washington 99352 United States Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439 United States

‡

S Supporting Information *

ABSTRACT: The rapid completion of microbial genomes is inducing a conundrum in functional gene discovery. Novel methods are needed to shorten the gap between characterizing a microbial genome and experimentally validating bioinformatically predicted functions. Of particular importance are transport mechanisms, which shuttle nutrients such as B vitamins and metabolites across cell membranes and are required for the survival of microbes ranging from members of environmental microbial communities to pathogens. Methods to accurately assign function and specificity for a wide range of experimentally unidentified and/or predicted membrane-embedded transport proteins, along with characterization of intracellular enzyme-cofactor associations, are needed to enable a significantly improved understanding of microbial biochemistry and physiology, microbial interactions, and microbial responses to perturbations. Chemical probes derived from B vitamins B1, B2, and B7 have allowed us to experimentally address the aforementioned needs by identifying B vitamin transporters and intracellular enzyme-cofactor associations through live cell labeling of the filamentous anoxygenic photoheterotroph, Chlorof lexus aurantiacus J-10-f l, known to employ mechanisms for both B vitamin biosynthesis and environmental salvage. Our probes provide a unique opportunity to directly link cellular activity and protein function back to ecosystem and/or host dynamics by identifying B vitamin transport and cofactor-dependent interactions required for survival.

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syntrophic metabolism, mutualism, and vitamin exchange between microbes and their hosts or other surrounding microbes. Microbial auxotrophy and opportunism for B vitamins is facilitated by transport domains that act as the cellular interface with the environment, and the conduit for the selective uptake of vital cofactors and precursors involved in both B vitamin salvage and de novo biosynthetic pathways.6,7 Indicative of the importance of transporters, 3−16% of prokaryote genomes are predicted to encode transporters;6,8 however, the complex mechanical and functional diversity of B vitamin transport binding domain systems make it challenging to accurately predict and characterize these nutrient-dependent mechanisms by gene sequence analysis.9 Matching a substrate to a predicted transporter is a significant challenge, but the biological importance of transport demands new methods to assign function and selectivity to predicted transporters in the rapidly growing number of sequenced microbial genomes.6,10,11 Comparative genomics methods, coupled with classical microbiological assays,6,12−15 have begun to reveal the broad diversity and distribution of vitamin transporters and identify novel regulators and enzymes involved in their intracellular disposition.1,16−18

he acquisition of nutrients by extracellular salvage or transport, in addition to the intracellular utilization and disposition of those nutrients as protein cofactors, is required for growth, resilience, and resistance to perturbation of microbes and microbial communities. A nutrient group essential for viability of nearly all microbes is B-type vitamins, which are water-soluble precursors to cofactors and are required by numerous enzymes involved in primary and secondary metabolism, protein repair, and protein recycling.1,2 The need to adapt to various biochemical or nutritional limitations in the environment has resulted in microbial auxotrophy of certain B vitamins; that is, microbes that are unable to synthesize the vitamin de novo require salvage systems (transporters and the enzymes that modify or use the vitamins) to acquire and utilize B vitamins available in the surrounding environment. Auxotrophy is widespread, promoting interdependence between organisms or host, and has the potential to influence the balance of microbial community membership under nutrient limited conditions.2−4 Other “opportunistic” microbes maintain an ability to switch between de novo synthesis and environmental salvage. Because salvage is energetically less demanding than biosynthesis, it is thought that this capability provides organisms a selective advantage in nutrient-rich environments.5 Understanding the mechanisms for vitamin salvage and intracellular disposition will enhance our ability to accurately predict © XXXX American Chemical Society

Received: November 6, 2015 Accepted: December 15, 2015

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dynamics by characterizing selective mechanisms for nutrient acquisition and cofactor-enzyme interactions.

However, genome analysis alone leaves an incomplete picture, failing to identify noncanonical proteins of interest. Furthermore, experimental validation of most genetic predictions is lacking, and new experimental approaches are needed to enable B vitamin transporter discovery and the functional characterization of the intracellular pathways they impact. Here, we provide experimental evidence demonstrating that an affinity-based protein profiling (ABPP) approach can be engineered to characterize live cell nutrient transport and intracellular protein interactions. We have synthesized a suite of affinity-based probes (ABPs) derived from B vitamins: B1 (thiamine), B2 (riboflavin), and B7 (biotin) for live cell labeling and subsequent identification of B vitamin transporters and intracellular binding proteins in live Chlorof lexus aurantiacus J-10-f l by fluorescence and quantitative LC-MS based proteomic measurements (Figure 1). C. aurantiacus is

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RESULTS AND DISCUSSION B Vitamin Probe Design and Synthesis. Our objective was to develop B vitamin probes that mimic the natural substrate for uptake by live cells via expressed vitamin-specific transporters. Consequently, the desired probe design must fulfill several qualifications: (i) The synthesis should be relatively straightforward, such that any modifications to the native B vitamin molecule are performed at accessible functional moieties and yield a chemically stable probe. (ii) The addition of bulky groups to the B vitamin probe should be avoided, as they are more likely to affect transport specificity and subsequent protein interactions during live cell labeling.26,27 And, (iii) the probe should have selectivity and affinity for protein targets akin to the native B vitamins.28 To fulfill these requirements, our probes were based on the B1, B2, or B7 vitamin scaffolds and include an aliphatic diazirine photo-cross-linker and a small alkyne handle (Scheme 1). The alkyne serves as a latent reactive tag for modification with reporter azides after live cell labeling via the copper-catalyzed click chemistry (CC) reaction (Figure 2).29,30 The small size of the alkyne group reduces possible inhibition of protein binding and transport and retains the flexibility of subsequent functionalization. The aliphatic diazirine is necessary to establish a covalent bond between a probe and target transporters and proteins, which is required for detection and purification of the labeled proteins for subsequent identification by mass spectrometry-based proteomic analysis.31 Upon UV irradiation, diazirines form highly reactive but short-lived carbenes, which insert into amino acid residues resulting in irreversible labeling.32 The alkyne and diazirine moieties were installed on each probe through the use of a common “linker,” which contains a reactive amine used in amide couplings to make the probe, the alkyne, and diazirine moieties (Scheme 1). Naturally occurring vitamin B1 (thiamine) is synthesized by joining 4-amino-5-hydroxy-2-methylpyrimidine (HMP) and 5-(2-hydroxyethyl)-methylthiazole (THZ). Subsequently, thiamine is phosphorylated, yielding the active coenzyme, thiamine pyrophosphate (TPP), which is an essential cofactor in microbial central metabolism.33 Alternatively, the precursors HMP and THZ can be salvaged from the environment for biosynthesis of thiamine with subsequent reactions yielding TPP. Though attempted, synthesis of a thiamine probe was intractable due to an inability to couple the HMP and THZ moieties. We were, however, successful in synthesizing a probe from the THZ moiety, using a carboxylic acid derivative coupled to our common linker to create B1-ABP. Vitamin B2 (riboflavin) is activated by sequential phosphorylation reactions to yield coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are both key components of metabolism.34 B2-ABP was synthesized by selectively protecting the primary alcohol of ribitol, followed by acetylation of the three secondary alcohols of ribitol. An acid moiety was installed on a flavin nitrogen to permit subsequent amide coupling to the common linker, followed by protecting group removal with p-toluene sulfonic acid (Scheme 1). Though it would have been more synthetically tractable to conjugate the linker directly to the ribitol portion of vitamin B2, intracellularly FMN and FAD are synthesized via conjugations to the ribitol moiety, and we did not want to disrupt that biochemistry. We also anticipated that B2-ABP would provide

Figure 1. Chemically derived B vitamin ABPs and corresponding native vitamin structures. (A) B1-ABP (thiazole) and thiamine. (B) B2-ABP and riboflavin. (C) B7-ABP and biotin.

one of the most metabolically versatile members of the phylum Chlorof lexi.19 C. aurantiacus is an abundant member of myriad microbial mat communities from diverse geochemical locations.19−22 To obtain necessary resources, C. aurantiacus is predicted to interact both opportunistically and auxotrophically with other members within natural microbial mat communities.19,20,23,24 C. aurantiacus is a known auxotroph for vitamins B1 and B7, and opportunistic for B2.25 Given C. aurantiacus’ associations in diverse environmental communities, a breadth of prior genome and functional annotation research, and its dual use of auxotrophy and de novo synthesis of B vitamins, this model organism provides a unique platform for validating our ABPP approach. Finally, we believe live cell probe labeling experiments are critical to making measurements representative of the native physiology and metabolic integrity of the microbe. As probe research is extended to uncultivated human and environmental microbial communities, we will need to maintain physiologically relevant conditions in order to elucidate the principles governing transport mechanisms and the intracellular fate of B vitamins and other nutrients. Herein, we demonstrate that our ABPP measurements provide a unique and powerful platform for directly linking activity and function back to environmental B

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ACS Chemical Biology Scheme 1. B Vitamin ABP Synthesis

Figure 2. B vitamin probe-labeled protein identification methods including (i) SDS-PAGE gel analysis using a clickable fluorophore and (ii) biotin enrichment methods for subsequent tryptic digestion of probe-labeled protein targets and quantitative LC-MS peptide analysis. Both identification methods involve live cell probe incubations followed by UV irradiation prior to downstream analyses.

serves a key role in CO2 fixation via the hydroxypropionate pathway.35,36 Biotin, in its native form, contains an aliphatic chain terminating in a carboxylic acid, which we synthetically modified to yield B7-ABP by amide coupling to the common linker (Scheme 1). Though seemingly redundant, the click moiety was retained to allow coupling to biotin-azide for

an additional functional advantage in that riboflavin remains the core structure of both cofactors FMN and FAD and, therefore, has the potential to expand our list of selective intracellular targets by incorporating specific enzyme-cofactor identifications. Vitamin B7 (biotin), required by several carboxylase enzymes, is a requirement in primary and secondary metabolism, and C

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concern that the comparatively equivalent molecular weights of the linker moiety and THZ portions of B1-ABP may result in impeded protein binding. The B1-ABP competition experiments reveal that the probe is binding at the thiazole portion of the thiamine-binding site. We also observed complete inhibition of B2 and B7 probe labeling by native riboflavin and biotin, respectively, confirming that the probes target the native binding sites of B vitamins (Figure 4B,C). Verification of probe selectivity was also performed directly in C. aurantiacus. The concentration of B vitamins needed by microbes is often nanomolar to low micromolar but can be even lower; therefore, transporter expression is down-regulated when cultured in environments replete with B vitamins. To ensure expression and functional activity of the transporters and intracellular demand for B vitamins, we cultivated C. aurantiacus in three independent defined mediums: B1, B2, and B7-limited cultures. Each B vitamin-limited culture was probe labeled with its respective probe (e.g., B1-limited culture labeled with B1-ABP), resulting in disparate gel profiles of probe labeling (Supporting Information Figure S1). Additionally, we spiked increasing concentrations of known B vitamin binding proteins into culture lysates, resulting in protein concentration-dependent labeling (Supporting Information Figure S1). Last, we validated that the structural integrity of protein targets must be preserved in order to maintain selective probe labeling events by comparing probe labeling of pure proteins and vitaminlimited lysates using a heat shock control for each (Supporting Information Figure S2). Live Cell Labeling and Proteome Analysis of C. aurantiacus Vitamin Uptake and Intracellular Interactions. To test the ability of probes to selectively capture predicted protein targets, live cell labeling studies were conducted on C. aurantiacus, an auxotroph for thiamine and biotin, and an opportunist for riboflavin.25 C. aurantiacus encodes transporters for thiamine (ThiXYZ), thiazole (ThiW), hydroxymethylpyrimidine (CytX; Figure 5A), riboflavin (RibXYZ; Figure 5B), and biotin (BioY). C. aurantiacus was cultivated in vitaminlimited media to promote B vitamin transporter expression and stimulate a need for the vitamins intracellularly for enzymecofactor associations.38 Additionally, salvage and transport occurs on a rapid scale, and via a time course gradient from 0 to 60 min, we identified optimal levels of intracellular uptake of B vitamin probes with only 10 min of incubation (Supporting Information Figure S3). Following probe labeling and UV irradiation, biotin-azide was added to probe-labeled proteins via CC for streptavidin-mediated enrichment of probe targets and subsequent label-free quantitative characterization by LC-MS based proteomics. Analysis of the B1-ABP proteomic results for labeled transporters reveals selective targeting of the substrate-binding component (ThiY, Caur_0425) of the ABC-type thiamine transporter (ThiXYZ), as well as labeling of the thiazole (ThiW, Caur_0793) transporter (Figure 5A, Supporting Information Dataset S1; includes probe-labeled values determined by the accurate mass and time (AMT) tag approach and number of unique peptide counts observed in comparison to global data). These results confirm previous functional genome predictions that characterized these proteins as the assigned thiamine and THZ transporters.25 As anticipated, B1-ABP did not label CytX, the transporter responsible for salvage of the HMP moiety of thiamine. These data affirm that the ThiY and ThiW transporters selectively identify the thiazole (THZ) portion of the probe and permit active transport.

selective enrichment of nonbiotinylated probe targets (e.g., transporters), as opposed to only evaluating endogenously biotinylated proteins. In Vitro Assays of B Vitamin Probe Specificity. To assess whether modifications made to the native B vitamin structures to yield probes impedes natural protein binding, we evaluated probe binding to recombinantly expressed and purified B1, B2, and B7 binding proteins. For B1 and B2, we used preparations of periplasmic thiamine or riboflavin transporter substrate-binding components, ThiY (Rcas_0667, R. castenholzii) and RibY (Tter_0402, T. terrenum), respectively, which previously were shown to bind the respective B vitamins.25 For B7-binding studies, streptavidin from Streptomyces avidinii was used, which has a strong affinity for biotin.37 Probes were added at increasing concentrations to a fixed concentration of protein, followed by incubation and UV irradiation; all probes demonstrated concentration-dependent labeling (Figure 3).

Figure 3. Concentration dependent labeling of (A) ThiY (Rcas_0667, 5 μM), a B1 binding protein, with B1-ABP. (B) RibY (Tter_0402, 5 μM), a B2 binding protein, with B2-ABP. (C) Streptavidin (5 μM), a B7 binding protein, with B7-ABP.

Given that the probes irreversibly bind proteins through C−H insertion via a carbene generated upon exposure to UV irradiation, we wanted to eliminate the possibility that proteins are nonspecifically probe labeled. We performed competitive inhibition experiments, whereby a probe and the respective native B vitamin at increasing concentrations were added simultaneously to purified binding proteins. The THZ-based B1-ABP labeling of ThiY was competed in the presence of increasing concentrations of thiamine, and was strongly inhibited by native thiazole (Figure 4A). We had some initial

Figure 4. Competitive inhibition of probe labeling by increasing native vitamin concentrations. (A) ThiY (Rcas_0667, 5 μM) labeled with B1-ABP (20 μM). (B) RibY (Tter_0402, 5 μM) labeled with B2-ABP (5 μM). (C) Streptavidin (5 μM) labeled with B7-ABP (5 μM). D

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Figure 5. B vitamin transport and intracellular metabolic pathways identified by probe-based (star) and global (triangle) proteomics in C. aurantiacus. (A) Thiamine. (B) Riboflavin.

several enzymes recognized to bind TPP including pyruvate dehydrogenase that catalyzes the first step in the TCA cycle, acetolactate synthase involved in branched chain amino acid synthesis, transketolase from the Calvin-Benson cycle and pentose phosphate pathway, and α-ketoglutarate dehydrogenase from the TCA cycle (Supporting Information Dataset S1). Importantly, we not only identify two “hypothetical” proteins, but also several other proteins containing domains with minimal annotation and/or anonymous functions, thereby newly assigning a B1-binding function. Overall, our B1-ABP data confirm the transport mechanism for THZ and demonstrate high probe target selectivity. To characterize an opportunistic salvage pathway,25 we used the vitamin B2 probe in live cell labeling studies of C. aurantiacus. Based on evolutionary distribution, the genomic prediction is that this organism salvages riboflavin using the ABC-type transporter, RibXYZ (Figure 5B), when nutrient limited.25 The addition of B2-ABP to live cells resulted in the selective probe labeling of ABC-type riboflavin uptake system substrate-binding component RibY (Caur_0817), which is the riboflavin-binding component of the RibXYZ transporter (Figure 5B, Supporting Information Dataset S3). As expected, intracellularly, B2-ABP did not label riboflavin biosynthetic genes, suggesting the cells salvaged B2-ABP via active transport within our vitamin limited culture conditions. Within the cell, riboflavin is metabolized to two key coenzymes that display broad cofactor-protein interactions: flavin mononucleotide (FMN) via phosphorylation of the B2 ribose moiety and flavin adenine dinucleotide (FAD) via a phosphoester bond between the ribose of riboflavin and ADP (Figure 5B). Flavoproteins are the dominant intracellular targets of B2-ABP (Supporting Information Dataset S3), suggesting that inside the cell, the probe is recognized akin to native riboflavin and may even undergo biosynthetic conversion to FAD and FMN analogs. Several other identified target proteins are subunits of multicomplex components that require FMN or FAD, suggesting that tight cofactor-specific binding complexes are labeled by the

To demonstrate that B1-ABP binds transporters and intracellular proteins in a selective manner, and not simply based upon global protein abundances, we compared peptide identifications for B1-ABP labeled proteins versus those same proteins (if identified) in global proteomic analyses of the B1-limited cultured microbe (Supporting Information Dataset S2). The data reveal, first, that the probe does not simply bind the most abundant proteins; the four most highly labeled probe targets are ThiW, hydroxyethylthiazole kinase (ThiM, Caur_0375), thiaminase II (TenA, Caur_0797), and ThiY (Figure 5A; Supporting Information Dataset S1). Second, for probe targets such as ThiW, ThiY, and TenA, we observe an increased identification of unique peptide sequences in probe data over the global proteome data (Supporting Information Dataset S1), demonstrating high probe selectivity. Looking at intracellular enzyme-cofactor interactions, we focused on the pathway that leads to the key B1-derived cellular coenzyme, thiamine pyrophosphate (TPP) (Figure 5A). We identify binding to thiaminase II (TenA, Caur_0797), which is responsible for the catabolism of thiamine to THZ and HMP, and ThiM that selectively phosphorylates thiazole (Figure 5A). Interestingly, thiamine-phosphate pyrophosphorylase (ThiE, Caur_0374), which couples phosphorylated derivatives of HMP and THZ, is not probe labeled even though it was measured with high abundance by global proteomics, indicative of high ThiE specificity for its target phosphorylated substrates. In addition, phosphomethylpyrimidine kinase (ThiD, Caur_0373), involved in two consecutive phosphorylation steps of HMP, was also found with high abundance in the global data but was not probe labeled (Supporting Information Datasets S1 and S2), again confirming strict probe specificity for THZ or thiamine pathways. Vitamin B1, primarily as TPP, is predicted to play key cofactor roles for a large number of enzymes involved in central metabolism.2,4,39 At present, the general understanding of vitamin-protein interactions is still vague, being largely dependent upon genome predictions. Using B1-ABP, we identify E

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ACS Chemical Biology Table 1. Specificity of B Vitamin ABP Targets in Biological Processesa

average protein abundance (unique peptides identified) locus

gene

pathway

B1-ABP

Caur_1880 Caur_1881 Caur_1882 Caur_0416 Caur_3677 Caur_0645 Caur_2087 Caur_3676 Caur_1983 Caur_1984 Caur_1986

SdhB SdhA SdhC BchC BchJ HemY BchP BchE NuoI NuoD NuoB

3-hydroxypropionate CO2 fixation

22.0 (10)

B2-ABP 20.3 (18) 20.0 (4)

bacteriochlorophyll biosynthesis

20.0 (17) 21.2 (9) 21.23 (19) 18.07(6) 21.2 (22)

NADH dehydrogenase (quinone) activity

19.9 (11) 17.0 (5) 18.1 (4)

B1 global

B2 global

23.4 (8) 24.4 (24)

24.4 (6) 25.6 (26)

23.3 (14) 24.3 (6) 21.4 (14)

24.5 (13) 21.8 (3) 24.3 (25)

19.9 (2) 21.8 (6)

23.1 (11) 21.4 (5)

22.5 (4)

22.3 (4)

a

Protein abundances were determined by the AMT tag approach, and are displayed in Log2 values. Shown in parentheses is the number of unique peptides for a given protein identified during a probe or global proteome analysis. Values were determined from three replicates per analysis type.

Table 2. Shared Targets of B Vitamin ABPsa average protein abundance (unique peptides identified) locus

gene

protein description

Caur_0375 Caur_0425 Caur_0793 Caur_0966 Caur_3343 Caur_1974 Caur_2840 Caur_3726

ThiM ThiY ThiW

hydroxyethylthiazole kinase ABC-type thiamine uptake system substrate-binding component ECF-type thiazole uptake system fused substrate-binding serine protein kinase/PrkA AAA cyclase family protein dihydrolipoyllysine-residue succinyltransferase dihydrolipoamide dehydrogenase 2-oxoglutarate dehydrogenase E2 (dihydrolipoamide succinyltransferase)

LpdA SucB

B1-ABP 24.6 23.6 24.6 20.6

(30) (29) (31) (54)

18.9 (9) 22.9 (25) 19.9 (13)

B2-ABP

B7-ABP

18.1 (12) 20.9 (27) 22.2 (20) 20.4 (9) 19.7 (5) 19.7 (17) 22.1 (19)

20.0 (24) 21.0 (11)

a

Protein abundances were determined by the AMT tag approach and are displayed in Log2 values. Shown in parentheses is the number of unique peptides for a given protein identified during probe analysis. Values were determined from three replicates per analysis type.

dehydrogenase that requires FAD as a cofactor (Table 2). Central to the reaction is a lipoyl domain, which forms the N-terminal part of the multidomain E2 chain and progressively moves along the active sites of all three enzymes.41 In this example, shared binding of the three protein targets with the two probes is not surprising given the tight interactions in the complex. By probe labeling, we identified shared labeling between the B1- and B2-ABPs of thiamine transport components ThiY, ThiW, and the thiazole kinase, ThiM (Table 2). Initially, we were somewhat surprised by this, but it has been reported that ThiY and RibY are closely related homologues of one another; in Chloroflexi, it has been postulated that the RibY regulatory elements evolved from the thiamine uptake transport system family and therefore may retain similar binding sites.42 Other reports in the literature have also shown exchangeable binding to transport complexes by B vitamins and/or metabolites.1,42−44 Though shared, selectivity of probe labeling becomes quite apparent when evaluating the MS-determined abundances of protein targets (Table 2). For instance, evaluating the extent of probe labeling of ThiM, ThiW, and ThiY by both probes reveals a 90-fold, 6.5-fold, and 5.3-fold difference, respectively, between B1-ABP and B2-ABP. B1-ABP always dominated the targeting of these proteins, which are annotated as thiazole or thiamine binding. Of interest is that the difference between probe labeling for the two transporter elements, ThiW and ThiY, is much less than the thiazole kinase. To investigate the shared binding events further, we performed cross-target probe labeling to our known binding proteins, ThiY (B1-binding

probe. Multicomplex components were labeled for various pathways such as bacterial chlorophyll biosynthesis, electron transfer, redox chemistry, and fatty-acid synthesis, consistent with prior reports.40 There were also several cofactordependent proteins that were not detected by global proteomics or by any other probe but were uniquely identified by B2-ABP (Supporting Information Dataset S3). Examples include succinate dehydrogenase and NADH-quinone oxidoreductase, both of which are known to play roles in redox regulation. In summary, the riboflavin probe efficiently labeled the B2 transporter and resolved the broad distribution of intracellular protein−cofactor interactions. An interesting display of B1- and B2-ABP selectivity was revealed in three separate processes (Table 1): the 3-hydroxypropionate pathway of CO2 fixation, bacteriochlorophyll biosynthesis, and NADH:quinine oxidoreductase. Several proteins involved in these processes were highly expressed in the C. aurantiacus cultures, as determined by the global proteome results.35 Despite high global abundances, specific proteins were only probe labeled, representing the appropriate vitamin cofactor required for a protein’s function (Table 1). There were also proteins undetectable by global proteomics, which were enriched to relatively high levels by probe labeling within these pathways, e.g., SdhC and BchP. Notable were cases of shared binding targets with our B1 and B2 probes: proteins Caur_1974, Caur_3726, and Caur_2840 are the three components that make up the pyruvate dehydrogenase complex, namely, a pyruvate dehydrogenase that uses TPP as its prosthetic group, a dihydrolipoyl transacetylase, and a dihydrolipoyl F

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Figure 6. Probe selectivity for B1 and B2 binding proteins. (A) B1 binding protein, ThiY (Rcas_0667, 5 μM); (B) B2 binding protein, RibY (Tter_0402, 5 μM). Both proteins were labeled with B1-ABP (20 μM) or B2-ABP (20 μM). (C and D) Competitive inhibition gradient with native B vitamins of ABP labeled B1 binding protein, ThiY (Rcas_0667, 5 μM). (C) B1-ABP (20 μM) competed with riboflavin. (D) B2-ABP (20 μM) competed with thiamine.

unknown or unrecognizable variants of these proteins, and our probe represents a way to enrich and annotate these proteins.47 There are also two cases of shared binding (Table 2). First, there is labeling of a serine protein kinase (Caur_0966) by B1- and B7-ABPs; this is an unanticipated event for which we did not find literature precedent. An analysis of the this same protein in the global proteomics (Supporting Information Dataset S2) reveals it as moderately abundant, but there are nearly 300 other proteins that are more abundant. This would seem to preclude this being a random false positive and may define a unique cofactor feature of this enzyme. The second shared binding protein is between the B2- and B7-ABPs, a generically defined cyclase family protein (Caur_3343) with no other defined annotation. Interestingly, there are reports that show biotin enhancing cyclase activities through binding,48 and riboflavin inhibiting cyclases through binding;49 these same events may be the cause of the shared binding we observe. In summary, the B7-ABP showed high selectivity for its targets during live cell labeling. Herein, we have demonstrated that carefully designed probes can be used to characterize active transport, substrate specificity, and protein-dependent interactions of B vitamins in living microbial cells. Nutrient transport dictates microbial interactions and successful growth, whether in an environmentally relevant photosynthetic microbial mat or the human microbiome. As the evolutionary uptake and efflux of B vitamins between microbes and their extracellular environment is predicted to greatly influence the biogeochemical relationships, spatiotemporal functions, and regulatory networks established within thriving ecosystems,50 identifying transport and intracellular disposition of B vitamins with novel probe approaches will provide new and rapid insights. Using newly developed probes, we have identified and experimentally characterized B1-, B2-, and B7-specific transporters and intracellular protein− protein interactions in C. aurantiacus, which uses both biosynthetic and salvage mechanisms to obtain these vitamins. Given the ever-expanding collection of microbial genomes, probes such as those described herein will become ever more important to characterizing the architecture of microbial networks, spatiotemporal nutrient distribution, uptake patterns, and adaptive responses microbes employ to stably function in their native environments.

protein, Rcas_0667) and RibY (B 2 -binding protein, Tter_0402). For both proteins, B2-ABP binds considerably stronger (Figure 6A,B); we pressed forward further evaluating ThiY probe binding in the presence of increasing concentrations of thiamine or riboflavin. Remarkably, the addition of increasing riboflavin increases binding of B1-ABP to the protein, suggesting riboflavin potentially facilitates binding through allosteric regulation (Figure 6C); however, the effect tempers quickly as exogenous addition of more than 5 μM riboflavin results in little additional binding. Interestingly, concomitant addition of thiamine and B2-ABP inhibits probe binding to the protein (Figure 6D). Together these results suggest that thiamine transport shares similar selective binding components with riboflavin, and riboflavin binding may even augment transport activity. But, the interaction with thiamine seems to be most critical, as witnessed by the inhibitory effect of added thiamine to riboflavin probe binding. Together, these results correlate with findings that have suggested nutrient transporters may contain domains permissive toward alternate, yet related, substrates.6,42,44−46 Finally, we evaluated biotin uptake and disposition with B7-ABP. C. aurantiacus is a predicted biotin auxotroph that salvages via the biotin transporter, BioY.6 Using B7-ABP, the solitary BioY transporter was identified (Supporting Information Data Sets S4 (live cell) and S5 (in vitro)). BioY is a small, hydrophobic integral inner membrane protein containing minimal unique peptides, which limits the likelihood of identification by MS. We did not observe BioY during live cell labeling, which may be due to low expression (as microbial biotin demands are very low), or more likely due to the inherent challenges of labeling and enriching this membrane protein to detectable levels. To circumvent this issue, we lysed the biotin-limited cultures to and enriched the membrane fraction by centrifugation. Labeling and LC-MS analyses of the membrane fraction resulted in the identification of BioY; no other transporters were labeled, consistent with high probe selectivity. Within the cell, numerous biotin-dependent enzymes and carrier proteins were identified and were labeled at high levels as determined by AMT tag quantification of probelabeled protein targets. For instance, acetyl-coA carboxylase, the biotin carboxylase subunits AccB and AccC, and a uniquely biotin-dependent biotin/lipoyl attachment domain-containing protein were labeled (Supporting Information Dataset S4). B7-ABP targets are representative of biotin-dependent biological functions such as CO2 fixation, fatty acid synthesis, and amino acid metabolism.2,25 The identifications by B7-ABP of biotin carboxyl carrier proteins and biotin-dependent CoAcarboxylases is quite significant, as many microbes have

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METHODS

Synthesis of B Vitamin ABPs. See the Supporting Information. Bacterial Culture. C. aurantiacus J-10-f l cells were maintained on a modified BG-11 basal medium,51 in which sodium nitrate (17.6 mM) was replaced by NaCl (17.6 mM), unless indicated otherwise, and NH4Cl (10.0 mM) was added as the inorganic nitrogen source. G

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ACS Chemical Biology

and in vivo probe-labeled samples and the global proteomic analyses. These same high confidence peptides were used for spectral counting, in which observations of one or more peptides for a given protein are tabulated. Probe specific significance metrics are discussed further in the Supporting Information. For B7-ABP, in vitro lysate analyses (Supporting Information Dataset S5) were also performed to detail with the complexities of identifying the small, hydrophobic, membrane transporter, BioY. We also show proteins unique to each vitamin limited culture condition as observed by global proteomics, which reveals key differences in the cultures that support the probe-based observations herein (Supporting Information Dataset S6). Complete details of LC-MS sample preparation and analysis can be found in the Supporting Information.

Optical densities were recorded to identify the early mid log phase for probe labeling studies (Supporting Information Figure S4). For full details, see the Supporting Information. Cloning and Protein Purification. Coding sequences for Rcas_0667 (R. castenholzii DSM 13941, ThiY) periplasmic binding protein and Tter_0402 (T. terrenum ATCC BAA-798, RibY (NCBI Project ID: 29523)) periplasmic binding protein were PCR-amplified from genomic DNA and fused to an N-terminal hexahistidine tag in an E. coli cytoplasmic expression vector (Supporting Information). Both target proteins were sequenced, and expression cultures were purified by affinity chromatography and buffer exchanged using a desalting column or dialysis and frozen in liquid nitrogen for storage until further analysis. Fluorescent Gel Imaging of Pure Proteins. Pure proteins were obtained for the B1, B2, and B7 probe validation studies. Streptavidin (G-Biosciences) was used for B 7 -ABP studies, Rcas_0667 (R. castenholzii) for B1-ABP studies, and Tter_0402 (T. terrenum) for B2-ABP studies. Pure protein stocks were normalized (5 μM) and labeled with corresponding B vitamin probes. All pure proteins (50 μL, 5 μM) were treated with individual ABPs at 37 °C for 1 h and UV irradiated at 365 nm immediately following probe labeling on ice for 7 min (10 cm). To prepare samples for fluorescent gel analysis, we first performed click chemistry mediated attachment of an azidotetramethylrhodamine fluorophore (2.65 μM) to probe-labeled proteins followed by the addition of (tris(2-carboxyethyl)phosphine) (22 μM), TBTA (Tris[(1-benzyl-1H-1,2,3-traizol-4-yl)methyl])amine) in a 4:1 solution t-butanol/DMSO (44.8 μM), and copper sulfate (45 μM), and proteins were separated using 10% Tris-Glycine SDS-PAGE gels. For determination of concentration-dependent probe labeling, pure proteins (5 μM) were labeled with increasing concentrations of corresponding probes. For competitive inhibition studies, each pure protein was treated with increasing concentrations of the corresponding native vitamin with concomitant addition of the appropriate probe. To test competitive selectivity for B1 and B2 probes in a B1-binding protein (ThiY, Rcas_0667), samples were labeled with a probe and inhibited by an individual competing native vitamin. See the Supporting Information for full details. To test selective competition of vitamin-limited lysates (25 μg) for vitamin ABPs, corresponding pure proteins were spiked into cell lysates at increasing amounts (1 μM, 3 μM, 4 μM, and 5 μM) and labeled for 30 min at 52 °C. For structural integrity studies of ABP binding, heat shock controls were denatured at 95 °C for 10 min prior to labeling. In Vivo and in Vitro Probe Labeling and LC-MS Sample Preparation of C. aurantiacus. C. aurantiacus cells were grown and maintained on BG-11 basal medium, with limited corresponding vitamin, until they reached an optical density of ∼0.5−2.0 (650 nm; Supporting Information Figure S1). The B vitamin probes were individually added to the culture media of C. aurantiacus, incubated for 10 min at 52 °C, and UV-irradiated at 365 nm for 7 min on ice. Whole cells were immediately washed with PBS, fractionated, and lysed, and biotin-azide was appended via click chemistry for subsequent enrichment of probe-labeled proteins on streptavidin agarose resin. Enriched proteins were digested with trypsin for LC-MS analysis. Proteomic Analysis of Probe Labeling. We employed spectral counting and tag-free quantitative accurate mass and time (AMT) tag proteomics as described previously,52−54 with the following modifications. Tryptic peptides from enriched proteins were separated by LC on in-house manufactured reverse phase resin columns and analyzed on a Thermo Fisher Orbitrap MS. Data were acquired for 100 min, beginning 65 min after sample injection into the LC. Spectra were collected from 400−2000 m/z at a resolution of 100k, followed by data-dependent ion trap generation of MS/MS spectra of the six most abundant ions using a collision energy of 35%. A dynamic exclusion time of 30 s was used to discriminate against previously analyzed ions. Generated MS/MS spectra were searched using the MSGF+ algorithm against the publicly available C. aurantiacus translated genome sequence.55 Identified peptides of at least six amino acids in length having a MS-GF score ≤1 × 10−10, which corresponds to an estimated FDR < 1% at the peptide level, were used to generate an AMT tag database. This database includes LC-MS measurements from in vitro

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.5b00918. Figures S1−S4, datasets S1−S6, methods, and 1H and 13 C NMR spectra (PDF) Datasets 1−6 (XLSX) Accession Codes

Proteomics data are available at PeptideAtlas, as accession number PASS00558.

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AUTHOR INFORMATION

Corresponding Author

*Address: 902 Battelle Blvd, MSIN J4-02 Richland, WA 99352. Phone: (509) 372-5920 (office). E-mail: Aaron.Wright@pnnl. gov. Funding

This research was supported by the Genomic Science Program of the U.S. DOE-OBER and is a contribution of the PNNL Foundational Scientific Focus Area. MS-based proteomic measurements used capabilities developed partially under the GSP Panomics project; MS-based measurements and microscopy were performed in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by OBER at PNNL. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank D. Bryant at Penn State University for providing C. aurantiacus J-10-f l. We also thank A. Osterman at the Sanford-Burnham Medical Research Institute for his thoughtful discussions of the experimental design and data analysis of the manuscript.

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REFERENCES

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