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4-26-2017 Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface-binding Teal Furnholm University of New Hampshire, Durham

Medhat Rehan University of New Hampshire, Durham

Jessica Wishart University of New Hampshire, Durham

Louis S. Tisa University of New Hampshire, Durham, [email protected]

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Recommended Citation Furnholm, T., M. Rehan, J. Wishart, and L. S. Tisa. 2017. Pb+2 Tolerance by Frankia sp. strain EAN1pec involves a Surface-Binding. Microbiology 163:472-487. DOI: 10.1099/mic.0.000439

This Article is brought to you for free and open access by the Molecular, Cellular and Biomedical Sciences at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Molecular, Cellular and Biomedical Sciences Scholarship by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. RESEARCH ARTICLE Furnholm et al., Microbiology 2017;163:472–487 DOI 10.1099/mic.0.000439

Pb2+ tolerance by Frankia sp. strain EAN1pec involves surface- binding

Teal Furnholm,1 Medhat Rehan,1,2,3 Jessica Wishart1,4 and Louis S. Tisa1,*

Abstract Several Frankia strains have been shown to be lead-resistant. The mechanism of lead resistance was investigated for Frankia sp. strain EAN1pec. Analysis of the cultures by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDAX) and Fourier transforming infrared spectroscopy (FTIR) demonstrated that Frankia sp. strain EAN1pec undergoes surface modifications and binds high quantities of Pb+2. Both labelled and unlabelled shotgun proteomics approaches were used to determine changes in Frankia sp. strain EAN1pec protein expression in response to lead and zinc. Pb2+ specifically induced changes in exopolysaccharides, the stringent response, and the phosphate (pho) regulon. Two metal transporters (a Cu2+-ATPase and cation diffusion facilitator), as well as several hypothetical transporters, were also upregulated and may be involved in metal export. The exported Pb2+ may be precipitated at the cell surface by an upregulated polyphosphate kinase, undecaprenyl diphosphate and inorganic diphosphatase. A variety of metal chaperones for ensuring correct placement were also upregulated with both Pb+2 and Zn+2 stress. Thus, this Pb+2 resistance mechanism is similar to other characterized systems. The cumulative interplay of these many mechanisms may explain the extraordinary resilience of Frankia sp. strain EAN1pec to Pb+2. A potential transcription factor (DUF156) was identified in association with several proteins identified as upregulated with heavy metals. This site was also discovered, for the first time, in thousands of other organisms across two kingdoms.

INTRODUCTION information and have been used in genome mining [8, 11, 12], comparative genomics [6, 8], transcriptomics [13–17] The actinorhizal symbiosis involves a mutualism between the nitrogen-fixing , Frankia, and a variety of and proteomics approaches [18], and have provided a base- woody dicotyledonous plants, termed actinorhizal plants, line for this study. that results in the development of a root nodule structure Most studies on the effects of metals on Frankia physiol- [1]. The symbiosis is responsible for the ability of the acti- ogy have centred on essential metals: Fe+3 acquisition via norhizal plants to inhibit harsh environments. Actinorhizal siderophores [19], Ni+2 and hydrogenase activity [20, 21], plants are able to colonize mine tailings and lands highly Ca+2 in vesicle development and function [22]. Metals and contaminated with industrial waste with the help of Frankia Frankia–plant interactions have also focused on nodulation – [2 4]. Besides their symbiont lifestyle, Frankia is also found effects with B, Zn+2, Ca+2 or acidic soils [23–25]. A few as a member of the soil environment although less informa- studies have centred on toxic metals that have no known tion is known about this lifestyle [5]. essential functions [26–30]. Previously, we investigated the Elucidation of three Frankia genomes has revealed new metal tolerance levels of 12 Frankia strains using plate potential in respect to metabolic diversity, natural assays and found that Frankia cultures exhibit elevated lev- , and stress tolerance, which may aid the cos- els of tolerance to various heavy metals including Pb+2, +2 mopolitan nature of the actinorhizal symbiosis [6–8]. In SeO2, Cu and AsO4 [26]. A binding or sequestration recent years, several more Frankia strains have been mechanism for Pb+2-resistance was proposed in that study. sequenced [9, 10]. These databases are providing a wealth of A bioinformatics analysis of the Frankia genomes

Received 18 November 2016; Accepted 23 January 2017 Author affiliations: 1Department of Cellular, Molecular, and Biomedical Sciences, University of New Hampshire, Durham, NH, USA; 2Department of Genetics, College of Agriculture, Kafrelsheikh University, Egypt; 3Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University, Saudi Arabia; 4Department of Microbiology, Oregon State University, Corvallis, OR, USA. *Correspondence: Louis S. Tisa, [email protected] Keywords: Actinorhizal symbiosis; nitrogen fixation; metal resistance; soil microbe; Bioremediation; proteomics. Abbreviations: EDAX, energy dispersive X-ray spectroscopy; EPS, exopolysaccharide; FTIR, Fourier transforming infrared spectroscopy; MTC, maxi- mum tolerance concentration; PPX, exopolyphosphatase; SEM, scanning electron microscopy; TFBS, transcription factor binding site; TMS, trans- membrane segment. Three supplementary figures and three supplementary tables are available with the online Supplementary Material.

000439 ã 2017 The Authors

472 Furnholm et al., Microbiology 2017;163:472–487 predicted several metal tolerance mechanisms that need dehydration steps (30, 50, 70, 90 and 100 %). The dehy- experimental validation [31]. The Cu+2 resistance mecha- drated samples were layered with t-butyl for freeze- nism in Frankia sp. strain EuI1c has been shown to drying and sputter coated with gold. The samples were involve surface binding and transport proteins [32]. The viewed at Â1000 to Â20 000 magnification on a scanning goal of this study was to further investigate the mecha- electron microscope (AMRAY 3300FE). nisms of Pb+2-resistance and to identify potentially SEM-energy dispersive X-ray spectroscopy (EDAX) involved in these mechanism. For these experiments, samples were coated with METHODS and subjected to elemental composition analysis by the use of an EDAX microanalysis system (PGT/Brucker/imix-PC) Frankia strains and growth conditions coupled to the scanning electron microscope according to Frankia sp. strain EAN1pec [33] was used in this study. the method described by Kessi et al. [38]. Frankia cultures Stock cultures were grown and maintained in basal MP were exposed to 0, 3 or 5 mM Pb(NO3)2 for 15 days. Precip- growth medium with NH4Cl as the nitrogen source and itate formed in cell-free culture media with 8 mM Pb was 20 mM fructose as a carbon and energy source, as described also analysed by EDAX. Intensities of each element were previously [34]. Basal MP growth medium consisted of averaged from four fields (Â10 000 magnification) for each 50 mM morpholinopropanesulfonic acid (MOPS) and condition. 10 mM K H-KH PO buffer pH 6.8. To this was added 2 2 4 Fourier transforming infrared spectroscopy (FTIR) separately sterilized (final concentrations) 2.0 mM MgSO4, 20 µM FeCl3-100 µM nitrilotriacetic acid (NTA), 1.0 mM Cells were grown in medium containing different concen- À1 Na2MoO4 . 2H2O, 1.0 ml l of a trace salts mixture, 20 mM trations (0, 3, 5 or 8 mM) of Pb(NO3)2. Five day-old cultures fructose and 5.0 mM NH4Cl. The concentrated trace salts were harvested by centrifugation at 13 000 g for 10 min and À1 solution contained (g l ) 2.5 Fe2(SO4)3 . 7H2O, 5.0 MnCl2 washed Â3 in sterile distilled H2O. The washed cells were –  . 2H2O, 1.0 ZnSO7 . 7H2O, 0.2 Co(NO3)2 . 6H2O and 10.0 frozen at 80 C and lyophilized for 48 h in a Freezezone CaCl2 .H2O. 6Lplus freeze dryer (LABConco). FTIR analysis of the lyophilized samples were performed as described previously Lead sensitivity assay [35] on a diamond attenuated total reflectance (ATR) Nico- A 24-well growth assay was used to determine lead toler- let iS10 (Thermo Scientific). The average FTIR spectrum for ance levels of Frankia as described previously [35]. Briefly, three replicates and replicate spectrum variation were deter- cells were grown for 15 days in MP medium containing dif- mined by the use of the Omnic software package (Thermo ferent concentrations of Pb(NO3)2 or Pb-acetate and growth Scientific). was measured by total cellular protein content as described below. The effect of phosphate on lead toxicity was tested in Protein preparation for proteomics studies MP medium containing ultra-low, low and medium phos- For these studies, cultures were incubated for 5 days under phate levels (0.1, 1.0 and 10.0 mM, respectively). Cultures metal stress conditions [3 mM Pb(NO3)2 or 3 mM ZnSO4] were tested under nitrogen-sufficient (5 mM NH4Cl as a or under no metal stress (control). The cells were har- nitrogen source) and nitrogen-deficient (N2 as a nitrogen vested by centrifugation at 1000 g and washed with cold source) conditions. Growth yield was determined by sub- TE buffer (20 mM Tris, 10 mM EDTA, pH 7.5) to remove tracting the protein content of the inoculum. The effect of non-specifically bound metals. The washed cells were col- ZnCl2 on growth was also determined. lected by centrifugation at 1000 g, the pellet was resus- pended in 10 ml protein extraction solution [4 % CHAPS, Total cellular protein determination 20 mM Tris-HCl, 8M urea, 1 mM MgCl2, dithiothreitol Protein content was measured by the BCA (bicinchoninic (DTT), phenylmethylsulfonyl fluoride (PMSF), and a pro- acid) method [36]. tein inhibitor cocktail (Sigma)], and passed through a French pressure cell press at 20 000 psi and 4 C. The crude Scanning electron microscopy (SEM) cell lysate was centrifuged 10 000 g and 4 C for 15 min to For these experiments, Frankia cultures were grown for remove unbroken cells and the supernatant fluid was 2 weeks in medium supplemented with or without 0, 3 or transferred to a new 50 ml plastic conical tube. Proteins 5 mM Pb(NO3)2. Cells were harvested by centrifugation at were precipitated with 40 ml cold acetone for 1 h and cen- 10 000 g for 10 min. Harvested samples were processed via a trifuged at 10 000 g and 4 C for 15 min. After the acetone critical point drying method [37]. Briefly, after the cells was discarded, the protein pellet was dried and stored at were washed three times with phosphate-buffered saline À60 C until digestion and multidimensional protein iden- (PBS, pH 7.4), the samples were fixed by incubation in mod- tification technology (MudPIT) analysis. ified Karnovsky¢s fixative solution (2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1 M phosphate buffer, pH Isobaric tagging for relative and absolute 7.4) for 4 h. The samples were washed with PBS, followed quantitation (iTRAQ) protein analysis by a distilled water wash. The washed fixed cells were dehy- The acetone-precipitated sample pellets were re-dissolved in drated by critical point drying through a series of alcohol varying volumes (as required to dissolve the pellets) of 8 M

473 Furnholm et al., Microbiology 2017;163:472–487 urea, 0.3 M triethylammonium bicarbonate (TEAB). An 0.25 µg was analysed using LC-MS/MS on a Thermo Scien- analysis was performed on 2 µl of each sample tific LTQ Orbitrap which was also equipped with a Waters after acid hydrolysis on a Hitachi L-8900 A Amino Acid nanoAQCUITY UPLC system, and a Waters Symmetry Analyzer. Based on these results, 25 µg of each sample was C18 180 µm Â20 mm trap column and a 1.7 µm, used for enzymatic digestion and iTRAQ labelling. Prior to 75 µmÂ250 mm nanoACQUITY UPLC column (35 C) for digestion, the samples were reduced using 1 µl of 45 mM separation. Analysis was performed in triplicate dithiothreitol (#20290; Pierce Thermo Scientific), and incu- after randomization and with two blanks runs inbetween  bated at 37 C for 20 min. The reaction mixture was alky- samples. Trapping was done at 7 µl minÀ1, 99 % Buffer A lated with the addition of 2 µl of 100 mM iodoacetamide (100 % water, 0.1 % formic acid) for 3 min with peptide sep- (#I1149; Sigma-Aldrich) and incubated in the dark at room aration at 300 nl minÀ1 with Buffer A (100 % water, 0.1 % temperature for 20 min. The urea concentration was diluted formic acid) and Buffer B (100 % acetonitrile, 0.075 % for- to 2 M by adding 12 µl water. Samples were enzymatically mic acid). A linear gradient (91 min) was run with 5 % digested with the addition of 1.25 µg lysyl endopeptidase  Buffer B at initial conditions, 40 % Buffer B at 90 min, and (#125-02543; Wako) and incubated at 37 C for 5 h. This 85 % Buffer B at 91 min. treatment was followed by the addition of 1.25 µg trypsin (Promega Seq. Grade, 25 µl of 1 mg mlÀ1 in 50 mM acetic The LC-MS/MS data were processed with the use of Progen- acid) and incubation at 37 C for 16 h. esis LCMS software [Nonlinear Dynamics. (www.nonlinear. com)]. Protein identification was performed using the Mas- Each dried 8-plex iTRAQ label vial was dissolved in 50 µl of cot search algorithm. Search parameters were 15 p.p.m. pep- 100 % 2-propanol. After vortexing, this mixture was trans- tide tolerance and 0.6 Da MS/MS tolerance, up to +7 charge ferred to the appropriate vial (C7-21 113; C 7-22 114; Pb 7- state, and variable carbamidomethylated cysteine and oxi- 21 115; Pb 7-22 116; Zn 7-21 117; Zn 7-22 118) and dized methionine. Data were standardized to a single sam- incubated at room temperature for 2 h. For technical repli- ple to minimize retention time (RT) variability between cates, two of the samples were labelled with two different runs. Mass accuracy was <3 p.p.m. Features within RT tags (C-7-21 119; Pb 7-22 121). After labelling, the tagged ranges of 0–25 min were filtered out, as were features with samples were combined and acidified (2 µl of 1 M phospho- charge +6 and +1. A normalization factor was calculated ric acid). Cation exchange chromatography of the combined for each run to account for differences in sample load sample was performed on an Applied Biosystems Vision between injections. The experimental design was set up to Workstation. This system uses a 2.1Â200 mm PolySul- foethyl A column (PolyLC) with a linear 118 min gradient group multiple injections from each run. (Buffer A: 10 mM potassium phosphate, 25 % acetonitrile pH 3.0; Buffer B: 10 mM potassium phosphate, 25 % aceto- Bioinformatics analysis of the proteome data nitrile pH 3.0, plus 1 M potassium chloride). For each feature in the dataset, the normalized abundances, Twenty cation-exchange fractions were collected, Speedvac maximum fold change, and ANOVA values were calculated. dried, and resuspended in 5 µl of 70 % formic acid. The frac- These data were imported into Mascot Distiller for ion iden- tions were diluted to 15 µl with 0.1 % trifluoroacetic acid tification using the NCBI Frankia sp. strain EAN1pec geno- (TFA) prior to loading 30 % of each fraction onto an mic database (www.ncbi.nlm.nih.gov/nuccore/NC_009921), Applied Biosystems QSTAR Elite [hybrid Liquid chroma- and proteins with a P<0.05 and peptide ions scores of 25 tography–mass spectrometry (LC-MS/MS)] interfaced with were included in the final analysis. Proteins identified using a Waters nanoACQUITY UPLC system. Trapping was done MASCOT were imported into the Progenesis LCMS soft- on a Waters Symmetry C18 180 µmÂ20 mm trap column at ware, where search hits are assigned to corresponding fea- À1 15 µl min , 99 % Buffer A (100 % water, 0.1 % formic acid) tures (Intensities). The exponentially modified protein for 1 min. Peptide separation was performed on a 1.7 µm, abundance index (emPAI) scores were also used for com- 75 µm  250 mm nanoACQUITY UPLC column (35 C) at À1 parative quantitation from the resulting MASCOT file [40]. 500 nl min with Buffer A (100 % water, 0.1 % formic acid) The ion score cut-off was set to 25, and those data with and Buffer B (100 % acetonitrile, 0.1 % formic acid). The P>0.05 were filtered out. A separate analysis of normalized gradient was 99 % Buffer A at initial conditions to 2 min from the label-free sample data was also con- with a linear gradient to 50 % Buffer B at 60 min, and 85 % ducted. An in-house PERL algorithm was used to assign Buffer B at 61 min. AB SCIEX ProteinPilot version 3.0 soft- peptides to Frankia sp. strain EAN1pec proteins and deter- ware was used for protein identification and iTRAQ quanti- mine changes in expression by spectral counts. A minimum tation. All iTRAQ results were uploaded into the Yale Protein Expression database (YPED; http://yped.med.yale. spectra count of 3 and E-value cut-off of 0.5 per peptide was edu/repository/) [39]. All reported iTRAQ data was at an used during protein assignment, and proteins with two FDR of 1 %. unique peptides were selected for comparative analysis. Pro- teins only found in metal conditions or with at least 2 Label-free protein quantitation greater intensity, emPAI, iTRAQ, or spectra ratios com- A separate 1.4 µg aliquot of each sample was digested as pared to the control were considered differentially above, but using 0.07 µg of lysyl endopeptidase. A total of expressed.

474 Furnholm et al., Microbiology 2017;163:472–487

Identification of a transcription factor binding site Green PCR Master Mix. Parameters for the Agilent MP3000 were as follows: (1) 95 C 15 min, (2) 40 cycles of 95 C for An in-house PERL program was used to analyse intergenic  regions of Frankia sp. strain EAN1pec potential metal resis- 15 s and 60 C for 30 s, and (3) finally, a thermal disassocia- tion cycle of 95 C for 60 s, 55 C for 30 s, and incremental tance genes for potential transcription factor binding sites. A  self-complementary palindromic motif was discovered increases temperature to 95 C for 30 s. Reactions were per- upstream of Cu2+ P-type ATPase genes. To determine formed in triplicate and the comparative threshold-cycle whether or not this motif is specific to Frankia sp. strain method was used to quantify expression. The results were standardized with gyrB expression levels. Relative EAN1pec, the search was expanded to upstream regions DD (300 bp) of a total of 35 784 genes including: 1. CopA- or expression (fold changes) was determined by the Ct ZntA-like (COG2217) P-type ATPase transporters; 2. CopZ- method [42], with the control (no metal stress) as the like (COG2608) metal chaperones; and 3. orthologues to the calibrator. 2+ Pb upregulated CopA-associated secreted protein (Fra- Statistical analysis nean1_5746). The intergenic regions were downloaded from Statistical tests were performed using JMP software utilizing the IMG database (http://img.jgi.doe.gov/). The PERL pro- Fit Model analysis with standard least squares coupled gram used the regular expression ‘ATACCC (2,8) GGGTAT’ with restricted maximum-likelihood (REML) to estimate (with one allowed bp substitution) and identified 8126 genes random variance components. Significance threshold was set with intergenic regions containing the motif. The list was at a P-value of 0.05. A two-way ANOVA was conducted on reduced to one species per genera and sequences were sorted growth and gene expression experiments and levels of signifi- by distance of motif to start codon. cance were determined by the Tukey-Kramer HSD test. Genes potentially containing the motif were analysed using Phylogibbs software (www.phylogibbs.unibas.ch/cgi-bin/ RESULTS phylogibbs.pl) [41] and a conserved motif sequence logo Effect of phosphate levels on Pb2+ resistance by was created. A search was conducted in the Frankia sp. Frankia sp. strain EAN1pec strain EAN1pec genome to determine whether other genes are potentially co-regulated by this motif. Frankia sp. strain Previous studies with a plate assay indicate that many EAN1pec genes containing a minimum of five complemen- Frankia strains exhibited high levels of lead tolerance, while tary palindromic basepairs from the conserved motif with a the other strains tested were sensitive [26]. A 24-well plate maximum of two base substitutions, and between 13–17 bp assay (Fig. S1), showed that Frankia growth was signifi- 2+ in length were identified using the PERL program. Genes cantly stimulated by low levels of Pb for both Pb(NO3)2 were searched manually to identify those with proteins and Pb(C2H3O2)2 exposure. The nitrogen or phosphorous upregulated with Pb2+ or Zn2+, or those in potential operons status of the media had no effect on this increased growth at with upregulated proteins. low levels of Pb(NO3)2 or Pb(C2H3O2)2. The minimum inhibitory concentration (MIC) and maximum tolerance Gene expression analyses concentration (MTC) values for Pb2+ under nitrogen- For these experiments, all solutions and material were sufficient conditions were similar to those observed with DEPC-treated to prevent RNA degradation. For the RNA Frankia growth on solid medium [26]. At 0.1, 1.0 and experiments, replicate Frankia sp. strain EAN1pec cultures 10 mM phosphate levels, the MTC values were 1, 2 and 3, (n=3) were grown under Pb2+ challenge for 3 days. RNA respectively. At 0.1 mM phosphate, the MIC value was extractions were performed by the Triton X-100 method as 5 mM and it was greater than 9 mM for the other two phos- previously described [11]. To remove contaminating DNA, phate levels. the RNA was subjected to treatment with DNAse I (New Pb2+ induced morphological changes in the Frankia England Biolabs) according to manufacturer’s specifica- cell surface tions. The RNA concentration for each sample was deter- 2+ mined by a Nanodrop 1000 spectrophotometer (Thermo When observed under phase-contrast microscopy, Pb - Scientific). RNA from each replicate was pooled and con- resistant Frankia sp. strain EAN1pec formed a precipitate that was associated with its hyphae. Fig. S2 shows verted to cDNA using random hexamer primers, 500 ng Frankia sp. strain EAN1pec grown in medium with RNA and SuperScript III reverse transcriptase according to 2+ ’ 8.0 mM Pb(NO3)2 or 6.0 mM ZnSO4. The Pb precipitate the manufacturer s instructions (Invitrogen). The cDNA 2+ À1 was distinctly different from that of Zn , which was held was diluted to 10 ng µl in RNAse-free H2O and stored at 2+  in an amorphous extracellular matrix. The Pb precipitates À20 C until use. were further investigated at a higher resolution. SEM pho- Frankia gene expression analyses were performed by qRT- tomicrographs of Frankia sp. strain EAN1pec grown under PCR using specific primers (Table S1, available in the online 3.0 mM Pb2+-stress (Fig. 1) revealed that a stable Pb2+ pre- Supplementary Material) and SYBR Green PCR Master Mix cipitate was attached to the cell surface. These results are (Applied Biosystems) as described previously [12]. Briefly, similar to those observed with Bacillus [43]. SEM-EDAX each 25 µl reaction contained 100 ng template cDNA, was used to determine the elemental composition of these 300 nM of the forward and reverse primer mix, and SYBR structures (Fig. 2) and they exhibited an elevated Pb2+

475 Furnholm et al., Microbiology 2017;163:472–487

(a) (b) Of the 1430 positively regulated proteins, 428 proteins responded only to Pb2+, and 437 responded only to Zn2+. Table 1 shows the results for upregulated proteins that may significantly contribute to metal resistance in Frankia sp. strain EAN1pec. Pb2+induced three copper-related proteins including a fused CopC/D domain transporter (Fra- nean1_7241), a Cu2+ P-type ATPase (Franean1_1679) and a negatively charged secreted protein (Franean1_5746) that is found almost exclusively in association with Cu2+ P-type Fig. 1. SEM of Frankia sp. strain EAN1pec grown underPb2+-stress. ATPases (Table 1a). A Kup-type potassium transporter (Fra- Frankia sp. strain EAN1pec was grown for 15 days in basal growth nean1_4647) was also upregulated with Pb2+. Under osmotic + medium with or without 3.0 mM Pb(NO3)2 as described in Methods. (a) and acid stress conditions, Kup permeases import K and Control conditions [0 mM Pb(NO3)2]; (b) 3.0 mM Pb(NO3)2. Bars, 10 µm. may provide the high K+ levels needed for exopolyphospha- tase (PPX) activity [46]. Zn2+ stress did not exclusively induce any metal transporters, but caused upregulation of the Zn2+- 2+ content as expected. Precipitates formed in the Frankia and Ni -binding hydrogenase maturation factor (HypA, Fra- nean1_2483). In Frankia sp. strain EAN1pec, other metal- medium resembled pyromorphite [Pb5(PO4)3Cl], a stable precipitate formed under typical surface geochemical con- binding proteins including bacterioferritins (Franean1_2250, ditions [44]. Unlike the medium-formed precipitate, there Franean1_2437), cobaltochelatase (CobN, Franean1_4862), is a complete absence of detectable chloride ions in the ferric-iron-binding protein (FbpA; Franean1_4453), and iron- 2+ sulfur assembly proteins (Franean1_2086, Franean1_2088) Frankia-bound Pb precipitate (Fig. 2). Treatment with +2 EDTA also failed to remove this membrane-bound precipi- were upregulated in response to either Zn alone or both tate (data not shown). These results indicate that the metals (Table 1a). Exposure to both metals also induced the Frankia Pb2+ precipitate did not simply form from the upregulation of a cation diffusion facilitator (CDF, Fra- medium as pyromorphite, but was generated from some nean1_2205), which functions as a general divalent metal cellular process. The EDAX results also indicated that the exporter [47]. proportion of phosphate was much higher for Frankia cul- Among the Pb2+- and Zn2+-upregulated proteins, 321 (22 %) tures exposed to higher Pb2+ concentrations which suggest were hypothetical proteins and 143 of these proteins have no that different Pbx(PO4)x compounds form and bind to the significantly homologous functional domains. There were 92 Frankia cell surface depending on Pb2+concentration. hypothetical proteins containing transmembrane segments To examine surface property changes, FTIR analysis was (TMS), and based on individual inspection of predicted mem- used to characterize the general types of present brane orientation, 35 contained large extracellular regions or based on specific wave number areas [45]. FTIR spectra were potential transporters (Table S2a). Some of the hypothet- were collected for Frankia sp. strain EAN1pec cells exposed ical proteins demonstrated a limited (<30 %) homology to to Pb2+-stress and no Pb2+ control cells (Fig. S3). Exposures other known proteins, including: 1. cell wall modification of 3 to 8 mM Pb(NO ) caused several changes in the spec- (Franean1_5160, Franean1_6801); 2. surface lipid 3 2 modification enzymes (Franean1_0195, Franean1_2417, Fra- tral pattern for Frankia. Specific changes were observed in nean1_7260); and 3. some very large membrane-anchored the wavelength regions of 742, 776, 974, 1028, 1386, 1454, À1 proteins that were possibly involved in divalent cation- or 1543 and 1719 cm . polysaccharide-binding (Franean1_0453, Franean1_5995). Small proteins with high percentage cysteine (metallothio- +2 Pb -induced proteins neins) or histidine (metallohistins) are known mechanisms of Relative protein abundance was determined by 2-D HPLC metal resistance, and have previously been predicted in MS/MS in both labelled (iTRAQ) and unlabelled samples Frankia [48]. Using an in-house PERL algorithm, the amino for Frankia sp. strain EAN1pec cells exposed to either acid composition for all the Frankia sp. strain EAN1pec pro- 3 mM Pb2+ or Zn2+. For comparative purposes, Zn2+ was teins was determined (data not shown). Among the 137 hypo- included as a divalent, beneficial metal that is toxic at ele- thetical Frankia sp. strain EAN1pec proteins that score in the vated levels. The MTC and MIC values for Zn2+ were 3 and top 1 % for cysteine (>3 %) or histidine (>5 %) content, 14 8 mM, respectively (data not shown). From these experi- have increased levels of expression with metals in this study ments, 1430 statistically significant proteins (those exhibit- (Table 1b). ing at least a twofold change in expression) were identified, Heavy metals are known to affect sulfur homeostasis by oxi- and represent 19.9 % of the Frankia sp. EAN1pec predicted dizing disulfide and iron-sulfur bonds and binding to thiol proteins. Only 395 of the identified proteins were deter- amino acids and other cellular thiol molecules (e.g. glutathi- mined in the labelled (iTRAQ) samples, likely due to high one, mycothiol) [48]. Pb2+ induced alkanesulfonate mono- abundance proteins overwhelming other signals in the oxygenase (Franean1_3757) expression (Table 1c), which pooled samples (Fig. 3). indicates that the cell was undergoing sulfur starvation [49].

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Several sulfur assimilation proteins including a sulfate trans- sp. strain EAN1pec, one of the two phosphate metabolism- porter (SulP, Franean1_0735), sulfite reductase (Fra- related proteins upregulated with Zn+2 was alkaline phos- nean1_6105), and cobalamin-independent methionine phatase (Franean1_1306; the other upregulated protein was synthase (Franean1_1081) were upregulated with Pb2+ a guanosine pentaphosphate [(p)ppGpp] synthase (Fra- induction (Table 1c). Zn2+ also upregulated two proteins nean1_5139) (Table 1c). from the sulfur assimilation pathway including a sulfate Pb+2 also induced (p)ppGpp synthase (Franean1_5138) and adenylyltransferase (Franean1_4225), and a cobalamin- other proteins involved in phosphorous metabolism, includ- dependent methionine synthase (Franean1_1643). Disulfide bond (DsbA, Franean1_3881), which is ing a phosphate starvation protein (PhoH, Franean1_0834), a responsible for correct formation of disulfide bonds in polyphosphate kinase (Ppk, Franean1_1116), and a polyphos- nascent proteins, was upregulated with Pb2+ inductionPb2+ phate phosphatase (Ppx, Franean1_6144) (Table 1c). Phos- induction (Table 1c). DsbA has been linked to Zn2+ resis- phate starvation leads to (p)ppGpp increases, which positively tance and alkaline phosphatase production [50]. In Frankia regulates the gene ppk, leading to a temporary polyphosphate

(a) (b)

KeV KeV (c) (d)

KeV KeV Elemental composition of Frankia sp. EAN1pec exposed to Pb(II) 0 mM 3 mM 5 mM Precipitate C 22 754±5496 6915±5504 5756±2631 2439±2512 O 18 920±4855 6614±2273 5814±1203 2886±1480 P 4532±2724 3418±1087 7370±2177 2590±529 Pb/S 20 099±9634 5119±1165 5276±1203 3684±339 Cl 8415±4064 0±0 0±0 2634±1972

Fig. 2. SEM-EDAX of Frankia sp. strain EAN1pec grown under Pb2+-stress. Frankia sp. strain EAN1pec was grown for 15 days in basal

growth medium with 0, 3 or 5 mM Pb(NO3)2 as described in Methods. For comparative purposes, lead precipitate formed in the absence of Frankia was also included. Panels (a)À(d), show EDAX spectra for control cells and corresponding element analysis: +2 (a) 0 mM Pb(NO3)2; (b) 3 mM Pb(NO3)2; (c) 5 mM Pb(NO3)2; (d) Pb precipitate formed in the absence of Frankia. Table shows mean 2+ intensity levels of the different elements of the precipitate from Pb -stressed cells. Values represent the mean of four replicates ±SD.

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Pb(NO3)2 function (COG R) and energy metabolism (COG C) enzymes, EMPAI Intensity including a variety of , and dehy- drogenases. Those proteins that were upregulated by both metals were primarily involved in lipid metabolism (COG I), iTRAQ 111 17 Spectral count protein translation (COG J) machinery (ribosomes), and 12 protein modification (COG O) mechanisms involved in pro- 8 4 tein folding (e.g. GroES/EL chaperonins). Secondary metabo- 40 0 52 567 lism proteins (COG Q) were also upregulated with both metals, many falling into two predicted biosynthesis clusters. 11 The first were from a biosynthesis cluster (Fra- 1 139 nean1_2389, Franean1_2392, Franean1_2396), which is pre- 0 14 dicted to produce a spore pigment [8]. The second included members of an erythronolide biosynthesis cluster (Fra- 8 nean1_3887–Franean1_3896), whose predicted product was a specialized lipid [8]. ZnSO 4 Surface modification proteins EMPAI Intensity Several other important differences in surface and lipid pro- tein expression patterns for Pb2+ and Zn2+ inductions were iTRAQ 79 13 Spectral count also observed. Pb2+ exposure upregulated nearly twice as 23 many (32 vs 18) surface sugar- and lipid-related proteins 1 8 compared to Zn2+ (Table S2b). These proteins included sev- eral families of , extracellular polysac- 23 537 4 114 charide synthesis (EPS)-related, sugar phosphate permeases, and mycolic acid lipid exporters. 23 +2 3 146 Zn -induction caused an upregulation of squalene-phy- 0 15 toene synthase cluster of proteins (Franean1_3971, Fra- 13 nean1_5716+17, Franean1_5128+32) (Table 1d), which are predicted to be involved in carotenoid and hopanoid lipid production [8]. Pb2+ and Zn2+ induction resulted in upregu- Fig. 3. Distribution of Pb2+- or Zn2+-upregulated proteins identified by lation of several proteins involved in phytoene precursor analysis type. Values represent number of upregulated proteins identi- biosynthesis proteins as well (Franean1_0162, Fra- fied with two peptides from labelled (iTRAQ) or unlabelled samples. nean1_0845, Franean1_0798 and Franean1_1170). Polyke- Proteins only found in metal conditions or with at least 2Â greater tide-based secondary metabolite genes are also upregulated iTRAQ, EMPAI, intensity, or spectra ratios compared to the control with Pb2+ and Zn2+, including polyketide cyclase and several were considered upregulated. erythronolide synthetase (Table 1d). and other secondary metabolites have been shown to be significantly increased in microbial communities exposed to heavy met- accumulation [51]. High levels of polyphosphate are known als [55, 56]. to chelate metals (e.g. Zn) in the cytoplasm. When expressed 2+ 2+ Recognition of a metal-induced regulatory binding with Ppx, polyphosphate increases Cu and Cd resistance site [52, 53]. Both Pb2+- and Zn2+-induction caused an upregula- An in-house PERL algorithm was used to find potential tion of PhoU (Franean1_6147). This Zn2+-binding, self-regu- intergenic regulatory sites of metal-related genes in the lating global regulator is known to help repress phosphate Frankia sp. EAN1pec genome. A potential transcription fac- uptake and secondary metabolism under normal conditions tor binding site (TFBS) was discovered upstream of each of [54]. Therefore, PhoU upregulation indicates these processes the three operons containing Cu2+ P-type ATPases (CopA1: have been de-repressed. Franean1_1679, CopA2: Franean1_5747, CopA3: Fra- nean1_6538). This TFBS was also identified upstream of a COG distribution of upregulated proteins metal-sensing repressor (DUF156) orthologue (Fra- The distribution of the upregulated proteins into COG catego- nean1_3360) and a potential Pb2+-precipitation protein ries was determined and the results are shown in Fig. 4. Based undecaprenyl diphosphatase (UppP, Franean1_2469). on COG distribution of upregulated proteins, Pb2+ induced: When the whole Frankia sp. strain EAN1pec genome was (1) gene regulation proteins (COG T) such as histidine or ser- analysed, 32 genes were identified as potential regulated by ine/threonine kinases; (2) DNA replication and repair mecha- this TFBS, 13 of which had Pb2+- or Zn2+-upregulated pro- nisms (COG L); and (3) a variety of inorganic metabolism teins (Table S3). Orthologues of Frankia sp. strain EAN1pec proteins (COG P). Zn2+ primarily upregulated general heavy-metal-resistance genes that have this motif upstream

478 Furnholm et al., Microbiology 2017;163:472–487

Table 1. Selected Frankia sp. EAN1pec proteins positively regulated with Pb2+ or Zn2+ potentially involved in metal resistance

Locus Tag Product* Size† COG TMS‡ Pb§ Zn|| HighScore¶ iTRAQ#

(a) Metal homeostasis and resistance Lead Franean1_7241 Copper Resistance Protein CopCD 649 P Y 1 0 Y N Franean1_5746 Heavy Metal Associated Secreted Protein 314 Y 2 0 Y N Franean1_1679 Heavy Metal Translocating P-Type ATPase 810 P Y 2 0 Y N Franean1_4647 Potassium (K+) Transporter 654 P Y 1 0 Y N Zinc Franean1_2250 Bacterioferritin 157 P N 0 1 Y N Franean1_2086 FeS Assembly Protein SufD 381 O N 0 1 Y N Franean1_2483 Hydrogenase Expression Synthesis HypA 110 R N 0 1 Y N Both Franean1_2437 Bacterioferritin 157 P N 1 1 Y N Franean1_2205 Cation Diffusion Facilitator 317 P Y 1 2 Y N Franean1_4862 Cobaltochelatase CobN Subunit 1266 H N 2 2 Y N Franean1_4453 FbpA iron(III) transport system SBP 356 P Y 1 2 Y N Franean1_2088 FeS Assembly ATPase SufC 260 O N 1 1 Y Y (b) Potential metallothioneins and metallohistins Lead Franean1_2772 Hypothetical Protein (5C 4 h) 94 N 1 0 Y N Franean1_5045 Hypothetical Protein (6C 9 h) 171 N 1 0 N N Franean1_2475 Hypothetical Protein (1C 5 h) 83 N 1 0 Y N Franean1_4430 Hypothetical Protein (1C 10 h) 86 N 2 0 Y N Franean1_1391 Protein Of Unknown Function DUF83 (5C 10 h) 171 L N 1 0 Y N Zinc Franean1_4967 Hypothetical Protein (4C 5 h) 84 S N 0 2 N N Franean1_2030 Hypothetical Protein (4C 4 h) 98 N 0 1 Y N Franean1_0040 Hypothetical Protein (16C 19 h) 504 N 0 2 Y N Franean1_1687 Hypothetical Protein (0C 12 h) 169 N 0 1 Y N Franean1_0871 Hypothetical Protein 4Fe-4S Ferredoxin Domain Protein (8C 0 h) 107 C N 0 2 N N Both Franean1_4168 Hypothetical Protein (4C 31 h) 524 S N 1 2 Y N Franean1_2951 Hypothetical Protein (13C 4 h) 228 N 1 1 Y N Franean1_0080 Hypothetical Protein (3C 5 h) 122 N 1 2 Y N Franean1_2790 Hypothetical Protein (6C 3 h) 167 N 1 1 Y N (c) Phosphate and sulfur metabolism Lead Franean1_1116 Polyphosphate Kinase 738 P N 1 0 Y N Franean1_6144 Ppx GppA Phosphatase 322 F P N 1 0 Y N Franean1_0834 Phosphate Starvation Inducible PhoH Family Protein 430 T N 1 0 Y Y Franean1_5138 ppGpp Synthetase I SpoT RelA 927 K T N 2 0 Y N Franean1_3757 Alkanesulfonate Monooxygenase Sulfur Starvation Response 400 C N 1 0 Y N Franean1_0735 Sulphate Transporter 554 P Y 1 0 Y N Franean1_6105 Sulfite Reductase 586 P N 1 0 Y N Franean1_1081 Methionine Synthase Vitamin-B12 Independent 372 N 1 0 Y N Zinc Franean1_1306 Putative Secreted Alkaline Phosphatase 573 P N 0 1 Y N Franean1_5139 ppGpp Synthetase I SpoT RelA 879 K T N 0 1 Y N Franean1_1643 Methionine Synthase 1262 E N 0 2 Y N Franean1_3881 DsbA Oxidoreductase 69 N 0 2 Y N Franean1_4225 Sulfate Adenylyltransferase Large Subunit 647 P N 0 1 Y Y Both Franean1_6147 Phosphate Uptake Regulator PhoU 214 P N 1 2 Y N Franean1_0302 Inorganic Diphosphatase 180 C N 1 1 Y Y (d) and secondary metabolites Lead Franean1_0116 Aminoglycoside Phosphotransferase 379 R N 1 0 N N Franean1_3590 Aminoglycoside Phosphotransferase 370 R N 1 0 Y N Franean1_6964 Efflux Transporter RND Family MFP Subunit 500 M Y 1 0 Y N Franean1_3471 Erythronolide Synthase Predicted Product: Specialized Lipid 1071 Q N 1 0 Y N Franean1_5969 Geranylgeranyl Reductase 409 C N 1 0 Y N Franean1_1871 Glyoxalase Bleomycin Resistance Protein Dioxygenase 138 R N 1 0 Y N Franean1_3988 Major facilitator superfamily MFS_1 482 G Y 1 0 N N

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Table 1. cont.

Locus Tag Product* Size† COG TMS‡ Pb§ Zn|| HighScore¶ iTRAQ#

Franean1_6823 MFS Drug Resistance Transporter EmrB QacA Subfamily 558 G Y 1 0 Y N Franean1_1925 ABC Transporter Related Multidrug/Peptide Exporter 726 V Y 1 0 Y N Franean1_1926 ABC Transporter Related Multidrug/Peptide Exporter 588 V Y 1 0 Y N Franean1_3763 ABC Transporter Related Multidrug/Peptide Exporter 984 V Y 1 0 Y N Franean1_0376 ABC Transporter Related Bacteriocin/Lantibiotic Exporters 318 V N 1 0 Y N Franean1_0377 ABC Transporter Related Bacteriocin/Lantibiotic Exporters 1264 V Y 1 0 Y N Franean1_0056 Lantibiotic Dehydratase Domain Protein 1035 N 1 0 Y N Franean1_1332 Lantibiotic Dehydratase Domain Protein 1066 N 2 0 Y N Franean1_6754 Lanthionine Synthetase C Family Protein 358 N 1 0 Y N Zinc Franean1_4888 ABC Transporter Related Multidrug/Peptide Exporter 842 V M Y 0 1 Y N Franean1_6413 ABC Transporter Related Multidrug/Peptide Exporter 577 V Y 0 1 Y N Franean1_2248 Linocin_M18 Bacteriocin Protein 276 S N 0 1 Y N Franean1_4774 Enterobactin Exporter Ents 443 Y 0 1 N N Franean1_5184 Deoxyxylulose-5-Phosphate Synthase 650 H I N 0 2 Y N Franean1_5201 Inositol-Phosphate Phosphatase 304 G N 0 1 Y N Franean1_5202 UDP-N-Acetylglucosamine 2-Epimerase 584 M N 0 2 Y N Franean1_7227 I 455 C N 0 1 Y N Franean1_3971 Phytoene Desaturase 468 C N 0 1 Y N Franean1_5716 Squalene Phytoene Synthase Predicted Product: Hopanoids 300 I N 0 2 Y N Franean1_5717 Squalene Phytoene Synthase Predicted Product: Hopanoids 379 I N 0 2 Y Y Franean1_5128 Zeta-Phytoene Desaturase Predicted Product: Carotenoids 506 Q N 0 1 Y N Franean1_5132 Squalene Phytoene Synthase Predicted Product: Carotenoids 326 I N 0 1 Y N Both Franean1_0845 Hydroxymethylbutenyl Pyrophosphate Reductase 338 I M N 2 2 Y Y Franean1_0798 4-Diphosphocytidyl-2C-Methyl-D-Erythritol Synthase 249 I N 2 2 Y Y Franean1_1170 1-Hydroxy-2-Methyl-2-(E)-Butenyl 4-Diphosphate Synthase 384 I N 1 2 Y N Franean1_0162 Geranylgeranyl Reductase 392 C N 1 1 Y N Franean1_7229 Citrate (Si)-Synthase 364 C N 3 2 Y N Franean1_7318 Myo-Inositol-1-Phosphate Synthase 357 I N 2 2 Y N Franean1_1659 Glutamine-Scyllo-Inositol Transaminase 388 M N 2 2 Y N Franean1_3043 Siderophore-Interacting Protein 623 P N 1 1 Y N Franean1_2389 Polyketide Synthesis Cyclase Predicted Product: Spore Pigment 125 N 1 2 Y N Franean1_2392 Cupin 2 Conserved Barrel Domain Protein (1C 8 h) Predicted 140 S N 1 2 Y N Product: Spore Pigment Franean1_2396 H. M. P. Antibiotic Biosynthesis Monooxygenase (0C 8 h) 107 N 1 2 Y N Predicted Product: Spore Pigment Franean1_5372 Mycocerosate Synthase 438 Q N 1 1 Y N Franean1_3887 Erythronolide Synthase Predicted Product: Specialized Lipid 3158 Q N 1 2 Y N Franean1_3888 Beta-ketoacyl synthase Erythronolide cluster. Predicted Product: 3254 Q N 1 2 Y Y Specialized Lipid Franean1_3889 Beta-ketoacyl synthase Erythronolide cluster. Predicted Product: 3493 Q N 2 1 Y N Specialized Lipid Franean1_3896 H. M. P. 1TMS Erythronolide cluster. Predicted Product: 405 Y 1 1 Y N Specialized Lipid Franean1_2119 Undecaprenyl Diphosphate Synthase 333 I N 1 1 Y N (e) Oxidative, thiol, and general stress proteins Lead Franean1_3800 Heat Shock Protein 70 880 O N 1 0 Y N Franean1_1571 Heat Shock Protein DnaJ Domain Protein 348 O N 1 0 Y N Franean1_2109 Heat-Inducible Transcription Repressor HrcA 339 K N 1 0 Y N Franean1_6169 Rhodanese Domain Protein 277 P N 1 0 Y Y Franean1_1940 Tellurite Resistance TerB 151 P N 1 0 Y N Zinc Franean1_2110 Chaperone Protein DnaJ 380 O N 0 2 Y N Franean1_3906 Rubredoxin-Like Zinc Ribbon Domain 143 R N 0 2 Y N Franean1_4493 Rubredoxin-Like Zinc Ribbon Domain 148 R N 0 2 Y N Franean1_6385 Tellurite Resistance TerD 568 T R N 0 1 Y N

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Table 1. cont.

Locus Tag Product* Size† COG TMS‡ Pb§ Zn|| HighScore¶ iTRAQ#

Both Franean1_1864 Alkyl Hydroperoxide Reductase 152 O N 1 2 Y N Franean1_1533 Catalase Peroxidase HPI 743 P N 1 2 Y Y Franean1_1569 Chaperone Protein DnaK 663 O N 1 1 Y N Franean1_0233 Chaperone Protein DnaK 612 O N 2 2 Y Y Franean1_6002 Chaperonin Cpn10 101 O N 2 2 Y Y Franean1_6001 Chaperonin GroEL 549 O N 1 2 Y N Franean1_3353 Chaperonin GroEL 542 O N 2 2 Y N Franean1_0175 Chaperonin GroEL 540 O N 3 2 Y N Franean1_3854 Chaperonin GroEL 545 O N 3 2 Y Y Franean1_1801 Cytochrome C Oxidase Subunit 288 C Y 3 2 Y Y Franean1_1946 Heat Shock Protein 70 957 P O N 1 2 Y N Franean1_5291 Heat Shock Protein Hsp20 199 O N 1 2 Y N Franean1_3387 Heat Shock Protein Hsp90 643 O N 2 2 Y Y Franean1_7212 Heat Shock Protein Hsp90-Family 1315 N 2 1 Y N Franean1_5485 Glutathione Peroxidase 178 O N 3 2 Y Y Franean1_3666 Peroxidase 208 O N 3 2 Y Y Franean1_0149 Rhodanese Domain Protein 116 P N 1 2 Y N Franean1_0977 Superoxide Dismutase 132 N 2 1 Y Y Franean1_0900 Tellurite Resistance TerD 479 T N 1 2 Y N Franean1_1054 Tellurite Resistance TerD 191 T N 1 1 N N Franean1_7328 Thioredoxin Reductase 348 O N 2 1 Y Y Franean1_7329 Thioredoxin 108 O N 1 2 Y N Franean1_6387 Toxic Anion Resistance Family Protein TelA 409 P N 1 2 Y N Franean1_2430 Tellurite Resistance TerB 332 P N 1 2 Y N *Protein name and relevant manual annotations. Bold and highlighted (grey) data represent a potential functional group. †Amino acid length of protein. ‡Transmembrane segments (TMS) present (Y) or absent (N) in protein. §Number of samples from Pb2+-exposed cells containing indicated protein (max. 3). ||Number of samples from Zn2+-exposed cells containing indicated protein (max. 2). ¶Protein score for sample is in top 20 % of all upregulated proteins for at least one sample. #Protein was identified as upregulated in labelled (ITRAQ) experiment. were analysed for the presence of the TFBS. Thousands of experiments, it was selected as a control. The gpx tran- Cu2+-type ATPase (CopA) and Zn-type ATPase (ZntA) script exhibited 5.0-, 7.4- and 6.9- fold increase with transporters, CopZ-like metal chaperones, and potential expose to 3, 5 and 8 mM Pb2+ stress, respectively. heavy-metal-associated (PHMA) secreted and lipoproteins Under 3 mM Pb2+ stress, the mRNA levels of both the were found with this TFBS upstream. The motif was highly csoR and uppS genes showed at 2.5- and 2.1-fold increase, conserved among many different types of and even respectively, which was followed by a negative dose-depen- some eukaryotes (Fig. 5). DUF156 orthologues (Pfam02583) dent response to Pb2+. None of the tested metal exporters with this TFBS motif have been found in other bacteria, were significantly upregulated under 3 mM Pb+2 stress, but including the Ni2+/Co2+-responsive regulator NcrB in Lep- the mRNA levels of copA2 (Franean1_5747), copA3 (Fra- tospirillium ferriphilum [57], the Cd2+/Zn2+/Ni2+-responsive nean1_6538), and cdf1 (Franean1_2205) significantly regulator MreA in Pseudomonas putida [58], and the Cu+2- increased under exposure to 8 mM Pb2+. responsive regulator CsoR in Streptomyces lividans [59]. From these analyses, it was concluded that the identified DISCUSSION palindrome was a CsoR/DUF156 repressor binding site. Frankia improves the survival of actinorhizal plants in the +2 2+ presence of high concentrations of Pb and other contami- Gene expression in response to Pb -stress nants [2, 3, 60–62]. There also appears to be a general Transcript levels of several metal-upregulated proteins increase in growth on both Pb(C2H3O2)2 and Pb(NO3)2 for were detected by qRT-PCR (Fig. 6). One protein, glutathi- up to 2 mM Pb+2. Frankia strains were shown to have a pos- one peroxidase (GPx, Franean1_5485), was upregulated itive growth response to other metals including 2.5 mM with both metals and in both labelled and unlabelled Ni+2 and 10 mM Al+3 [20, 27, 28]. This increased growth

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20 significant changes in mRNA levels and protein expression 18 were observed in Frankia sp. strain EAN1pec. Fig. 7 shows the interplay of the Pb+2-upregulated proteins in Frankia 16 sp. strain EAN1pec that contribute to its high level of Pb2+ 14 resistance. Pb Zn 2+ 12 Both Pb export and precipitation mechanisms 10 Frankia sp. strain EAN1pec does not contain Zn/Cd/Pb- type ATPases, it does contain the three Cu-ATPase (CopA) 8 paralogues. Based on the qRT-PCR results, none of the CogDistribution (%) 6 copA genes were upregulated at 3 mM Pb2+ (Fig. 6), but the CopA1 protein was identified as upregulated with Pb2+ in 4 the proteome (Table 1a). At higher Pb2+ concentrations, 2 both copA2 and copA3 showed a significant increase in 0 mRNA levels (Fig. 6). Therefore, expression of heavy metal R I C J Q K T L E G O H S M P F U D V A exporters and phosphate precipitation may be more import- ant at higher Pb2+ levels, while the surface and stress-related

2+ changes may be sufficient to provide resistance at lower Fig. 4. COG categories distribution of proteins upregulated by Pb or Pb2+ levels. Zn2+. COG categories for proteins upregulated with Pb2+, Zn2+, or both metals were downloaded from the IMG database. The number of pro- CsoR/DUF156 is known to detect a variety of metals [64]. teins belonging to each functional category is shown as percentage of The CsoR/DUF156 orthologue could be involved in Pb2+- total identified proteins from each condition. Categories include: R, sensing in Frankia. This gene had elevated mRNA levels at General Function; I, Lipid Metabolism; C, Energy Production; J, Transla- 2+ tion and Ribosomes; Q, Secondary Metabolism; K, Transcription; T, Sig- 3 mM Pb exposure, but was downregulated at higher nal Transduction; L, DNA Replication and Repair; E, Amino Acid metal concentrations (Fig. 6). Furthermore, a CsoR-like Metabolism; G, Carbohydrate Metabolism; O, Protein Modification and metal regulatory site was found upstream of all three CuAT- Turnover; H, Coenzyme Metabolism; S, Unknown Function; M, Cell Wall Pase-containing operons and UppP (Table S3). The inverse and Membrane; P, Inorganic Metabolism; F, Nucleotide Metabolism; U, correlation of CsoR and CopA/CDF1 expression, and the Intracellular Protein Trafficking; D, Defence Mechanisms; V, Cell Divi- presence of potential CsoR regulatory sites near metal trans- sion and Growth; A, RNA Metabolism. port (CopA) and precipitation mechanisms (UppP) support the possibility that metal export and precipitation are related in Frankia under high levels of metal exposure. with toxic metals could be due to metal stress-induced Other lead-response orthologues of transport proteins includ- changes in sugar and phosphate metabolism observed by ing CopCD, and the sugar-phosphate major facilitator super- FTIR, EDAX, and MudPIT in Frankia sp. strain EAN1pec 2+ 2+ +2 family (MFS) transporters were upregulated with Pb . Pb (discussed below). The levels of Pb resistance are signifi- upregulated seven hypothetical proteins that are potential cant. In the soil, the EPA has set a 0.96 µM limit and many transporters based on the presence of multiple predicted +2 – Pb -contaminated sites are in the 5 250 µM range. transmembrane segments (TMS), including one (Fra- In this study, Frankia sp. strain EAN1pec was shown to nean1_5744) that has significant metal-binding potential. Sev- absorb significant amounts of Pb2+ from its surrounding eral Gram-positive and Gram-negative bacteria exploit the 2+ 2+ environment, and precipitate Pb+2 on the cell surface high affinity of Pb for phosphate to detoxify Pb as intra- (Fig. 1). Our results also show that the bound precipitate and extracellular precipitates [65]. In a similar mechanism to was stable through several washings of water, acid, EDTA, Ralstonia metallidurans [66], one mechanism for Frankia is 2+ or alcohol. This result indicates the presence of a specific that Pb export is followed by precipitation by the metal- metal-binding mechanism rather than transient sorption of induced inorganic phosphatase (Franean1_0302), or the Pb+2 to surface macromolecules. Based on the EDAX undecaprenyl phosphatase (UppP). Another potential mecha- nism is that undecaprenyl diphosphate itself could play an results, there was nearly half as much phosphate per quan- 2+ tity of Pb+2 at 3 mM Pb+2 than at 5 mM Pb+2. FTIR analy- additional role in Pb resistance. An undecaprenyl diphos- sis also showed a smaller phosphate peak at 3 mM Pb+2. phate synthase (UppS) protein was upregulated with both These observed Pb+2 precipitation and concomitant metals (Table 1d). The mRNA level of the uppS gene was also changes in surface polysaccharides, phosphates, and sulfur- upregulated 2.1-fold as well (Fig. 6). ligands also occurs with several other bacterial and fungal Metal-induced polyphosphate degradation and resultant species [43, 63]. metal-phosphate efflux has also been linked to extracellular metal precipitation [51]. In Frankia, Pb2+ upregulated the Frankia +2 transcript and proteome response to Pb stringent response regulator (p)ppGpp synthetase In the absence of genetic tools, qRT-PCR and shotgun pro- (Table 1), which in turn may have upregulated a variety teomics approaches were used to determine whether cellular responses including polyphosphate production.

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Fig. 5. Regulatory motif of heavy metal P-type ATPase transporters and their associated proteins. Intergenic regions up to 200 bp upstream of CopA- or ZntA-like (COG2217) P-type ATPase transporters, CopZ-like (COG2608) metal chaperones, and orthologues to the Pb2+-upregulated CopA-associated secreted protein (Franean1_5746) [indicated with asterisk] from various genera of eukaryotes and were searched for palindromic sequences as potential transcription factor binding sites. The identified self-complemen- tary palindromic motif was found at a variable distance from the start codon, only those found within 80 bp from the start codon are displayed. A sequence logo [lower right] of the conserved motif created from Phylogibbs software is also included.

Cytoplasmic Pb2+ could be detoxified either directly by bind- precipitates by the Pb2+-upregulated polyphosphate phospha- ing polyphosphate or by the formation Pb2+-phosphate tase (Ppx). The strong FTIR peak at ~974 cmÀ1 with Pb2+, likely a P–O bond, was also seen with extracellular metal poly- phosphates [67].

10 0 mM 3 mM Exopolysaccharides and metal adsorption 5 mM 8 8 mM There were nearly twice as many surface sugar, cell-wall and lipid-related proteins upregulated with Pb2+ than with Zn2+. 6 A mixture of surface glycoproteins and glycolipids (collec- tively exopolysaccharides) were shown to contribute signifi- Fold change Fold 4 cantly to Pb2+ resistance in other bacteria [68]. Both the production of an extracellular milieu (Fig. S2) and 2 sugar–phosphate peaks identified by FTIR (Fig. S3) impli- cate a role for exopolysaccharides (EPS) in surface Pb2+ 0 2+ copA1 copA2 copA3 cdf1 gpx uppS csoR binding. Pb binding to EPS is known to occur in other microbes [69, 70]. Potential EPS-related hypothetical sur- face proteins were also expressed with metals, and include +2 Fig. 6. Transcriptional changes of selected Frankia genes under Pb cell wall (Franean1_5160, Franean1_6801), glycoprotein challenge. Values represent fold changes in gene transcript levels (Franean1_0453), or lipid modification enzymes (Fra- compared to the control for Frankia sp. EAN1pec cells exposed to Pb+2 (0, 3, 5 and 8 mM). Gene transcript levels were normalized to the nean1_0195, Franean1_2417, Franean1_7260) (Table S2a). housekeeping gene gyrB for each Pb+2 concentration. Error bars rep- Collectively, these extracellular proteins could contribute to resent 1 sd (n=2 or 3). observed metal-sorbing extracellular matrix, either acting in a protective capacity or as a site for EPS attachment.

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Fig. 7. Proposed system for Frankia sp. strain EAN1pec Pb2+-resistance. Low phosphate conditions due to Pb-bound phosphate in the media stimulate the pho regulon which upregulates both 1) the high affinity phosphate transporter PstABC (Franean1_ 0353–55) that imports phosphate, and 2) the polyphosphate kinase (PPK, Franean1_1116) which converts the phosphate to 3) polyphosphate. Poly- phosphate binds Pb2+ and, using 4) exopolyphosphatase (PPX, Franean1_6144), is converted to either 5) ATP or to 6) metal phosphates that are exported via 7) Pho84 (Franean1_3988, Franean1_6944). PPX need high levels of K+ that is provided by 8) the high affinity potassium transporter Kup (Franean1_4647). Pb2+ can also be exported by heavy metal transporters 9) CDF (Franean1_2205) or 10) CopA (Franean1_1679, Franean1_5747, Franean1_6538). 11) The classic model for extracellular Pb2+ precipitation is that exported Pb2 + is precipitated onto the cell surface using undecaprenyl diphosphatase (UppP, Franean1_0102, Franean1_2469, Franean1_3860). 12) SpoT (Franean1_5138) produces the alarmone 13) guanosine pentaphosphate [(P)PPGPP]. 14) (P)PPGPP temporarily represses PPX activity to allow polyphosphate accumulation, and 15) turns on the stringent response. The stringent response upregulates lipid, sugar, and cell-wall modification processes (see Tables 1d and S2d). 16) UppS (Franean1_2119) produces undecaprenyl diphosphate which, in addition to being modified by UppP, can 17) carry a variety exopolysaccharides (EPS) which may adsorb Pb2+. 18) Undecaprenyl diphosphate also has high affinity to lantibiotics (Franean1_0056, Franean1_1332, Franean1_6754), which could be exported via

484 Furnholm et al., Microbiology 2017;163:472–487

upregulated 19) antimictobial peptide ABC transporters (Franean1_0376, Franean1_1925–26, Franean1_3763). Lantibiotics could bind Pb2+ and leave a phosphate exposed for further Pb2+ binding. 20) Other lipids including mycoserosate (Franean1_5372) or specialized lipids (Franean1_3887–96), could be exported by 21) RND lipid transporters (Franean1_1576, Franean1_2271, Franean1_5721) and may contribute to Pb2+-adsorbing EPS or reduce cell permeability to Pb2+. 22) Pb2+ induced many sugar- and cell-wall-related genes that may contribute to EPS as well. Metals generate significant thiol stress. Thiol homeostasis is partially regulated by PhoU (Franean1_6147). To compensate, 23) SulP increases sulfate import which is 24) reduced to sulfide by SirA (Franean_6105) for incor- poration into cellular thiols (e.g. glutathione) and proteins. 25) PhoU also regulates secondary metabolites like the aforementioned myoserosate as well as siderophores and pigments (Table 1d) that are exported via 26) major facilitator superfamily (MFS) permeases (Franean1_3988, Franean2_6823). Secondary metabolites like siderophores can then chelate and detoxify metals. All the listed mecha- nisms were identified as upregulated with Pb2+ except 1) PstABC and 11) UppP.

Metal-related secondary metabolite production References 1. Normand P, Benson DR, Berry AM, Tisa LS. Family Frankiaceae. 2+ There were specific secondary metabolite responses to Pb In: Rosenberg E, DeLong EF, Lory S, Stackebrandt E and Thomp- and to Zn2+, including changes in lipids that may be due to son F (editors). The – Actinobacteria. Berlin: Springer; the stringent response (Table 1d). In Streptomyces, phytoene 2014. pp. 339–356. synthesis is under the control of SigR, an oxidative-thiol 2. Ridgway KP, Marland LA, Harrison AF, Wright J, Young JP et al. stress regulator [71]. Mycocerosate is another large lipid Molecular diversity of Frankia in root nodules of Alnus incana grown with inoculum from polluted urban soils. FEMS Microbiol that acts as a protective mechanism by reducing cell perme- Ecol 2004;50:255–263. ability [72]. Visual inspection by light microscopy shows 3. Roy S, Khasa DP, Greer CW. 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Symbiosis 2016;70: 2+ Frankia sp. strain EAN1pec to Pb . 5–16. 11. Niemann J, Tisa LS. Nitric oxide and oxygen regulate truncated hemoglobin gene expression in Frankia strain CcI3. J Bacteriol Funding information 2008;190:7864–7867. Partial funding was provided by the New Hampshire Agricultural 12. Perrine-Walker F, Doumas P, Lucas M, Vaissayre V, Beauchemin Experiment Station. This is Scientific Contribution Number 2631. This et al. work was supported by the USDA National Institute of Food and Agri- NJ Auxin carriers localization drives auxin accumulation in culture Hatch 022821. M. R. was supported by an Egyptian Channel plant cells infected by Frankia in Casuarina glauca actinorhizal Fellowship from The Egyptian Cultural Affairs and Missions Sectors. nodules. Plant Physiol 2010;154:1372–1380. 13. Alloisio N, Queiroux C, Fournier P, Pujic P, Normand P et al. The Acknowledgements Frankia alni symbiotic transcriptome. Mol Plant Microbe Interact We thank Robert Mooney for his help with the photography, Nancy 2010;23:593–607. Chemin for her help with the electron microscopy, and Alix Konisky, 14. Bickhart DM, Benson DR. Transcriptomes of Frankia sp. strain Rebecca Wagers, Joel Richards and Glenn Krumholz for their initial CcI3 in growth transitions. BMC Microbiol 2011;11:192. contributions to this project (all from UNH – University of New Hampshire). 15. Popovici J, Comte G, Bagnarol E, Alloisio N, Fournier P et al. Dif- ferential effects of rare specific flavonoids on compatible and Conflicts of interest incompatible strains in the Myrica gale-Frankia actinorhizal symbi- The authors declare that there are no conflicts of interest. osis. Appl Environ Microbiol 2010;76:2451–2460.

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