The Role of SufS Is Restricted to FeâS Cluster Biosynthesis in

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The role of SufS is restricted to FeS cluster biosynthesis in Escherichia coli Martin Bühning, Angelo Valleriani, and Silke Leimkühler Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00040 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 27, 2017

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The role of SufS is restricted to Fe-S cluster biosynthesis in Escherichia coli Martin Bühning§, Angelo Valleriani¶ and Silke Leimkühler§* §

Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany,

¶

Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam 14476, Germany

ACS Paragon Plus Environment

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ABSTRACT

In Escherichia coli two different systems have been identified that are important for the coordinate formation of Fe-S clusters, namely the ISC and SUF systems. The ISC system is the housekeeping Fe-S machinery, which provides Fe-S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for numerous sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS lead to an overall decrease of Fe-S cluster enzymes and molybdoenzyme activity in addition to a decrease in Fe-S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe-S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe-S containing proteins, including the MoaA protein, which is involved in the conversion of 5'GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share high similarities, we show that the function of their respective L-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot replace the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4thiouridine or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe-S cluster assembly in the cell.


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INTRODUCTION Sulfur-containing molecules, like iron sulfur (Fe-S) clusters, the molybdenum cofactor (Moco) or thionucleosides in tRNA are important for the viability of living cells. The incorporation of sulfur into these biomolecules requires coordinated synthesis routes and regulated sulfur relay systems.1 In Escherichia coli two major sulfur-mobilization systems from L-cysteine have been identified, namely the iron sulfur cluster (ISC) system and the sulfur mobilization (SUF) system.2 The central component of these systems are the two L-cysteine desulfurases IscS or SufS that mobilize sulfur from L-cysteine to form L-alanine and a protein-bound persulfide intermediate.1 The persulfide, in turn, is transferred to different sulfur-acceptor proteins that deliver the sulfur specifically to the biosynthetic pathways among which are the abovementioned biomolecules. For Fe-S cluster biosynthesis, E. coli IscS was shown to act as housekeeping L-cysteine desulfurase. IscS interacts with its scaffold protein IscU, where iron and sulfur are combined to form either [2Fe-2S] or [4Fe-4S] clusters.3 The reduction equivalent required for sulfur delivery from IscS for Fe-S formation on IscU is most likely provided by the [2Fe-2S] cluster containing protein ferredoxin (Fdx).4 Additionally, IscS also binds to CyaY and IscX, two proteins that were shown to be involved in Fe-S cluster formation, however, their direct involvement in this process remains controversial.5 In contrast, the SUF pathway encoded by the sufABCDSE operon was identified as alternative Fe-S cluster assembly route. In this system, SufS recruits the sulfur acceptor protein SufE, which acts as an enhancer to stimulate SufS activity.6 After sulfur transfer from SufS to SufE, SufB most likely accepts the sulfur from SufE. In concert with SufC and SufD Fe-S clusters are finally build on SufB which in turn can be delivered to acceptor proteins. 7,8


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Further, IscS has been identified as initial sulfur donor for tRNA thiolation. The formation of thionucleosides in tRNA occurs via two different routes.9 Firstly, IscS can transfer the sulfur in form of a persulfide directly to the sulfurtransferases TusA or ThiI. For 5-methylaminomethyl-2thiouridine formation at position 34 (mnm5s2U34) of tRNALys, Glu, Gln, the persulfide of TusA is delivered via the TusBCD complex and TusE to MnmA.10 The binding site of TusA on IscS has been revealed by the crystal structure of the IscS-TusA complex.11 ThiI, in contrast, is involved in sulfur insertion to form 4-thiouridine at position 8 (s4U8) of tRNA.12 Secondly, the synthesis of the thiomodifications 2-thiocytidine at position 32 (s2C32) and 2-methylthio-N6 isopentenyladenosine at position 37 (ms2i6A37) of certain tRNAs are Fe-S cluster dependent. In E. coli, these tRNA modifications involve TtcA for the synthesis of s2C32 and MiaB for ms2i6A37 modification. TtcA contains one [4Fe-4S] cluster, while MiaB contains two [4Fe-4S] clusters and is a member of the radical S-adenosyl-methionine (SAM) superfamily of proteins. 13,14

Both MiaB and TtcA were suggested to require Fe-S clusters for their activity as

thiomodification enzymes.13,14 In addition to tRNA thiolation of s2C32 and ms2i6A37, Fe-S clusters are also essential for Moco biosynthesis. In the first step of Moco biosynthesis, MoaA and MoaC convert 5'-GTP to cyclic pyranopterin monophosphate (cPMP). Similar to MiaB, MoaA is a radical SAM enzyme containing two [4Fe-4S] clusters that are essential for MoaA activity.15 In the second step, two sulfur atoms are inserted into cPMP by the enzyme molyptopterin (MPT) synthase and MPT is produced.16 In the third step, molybdenum is ligated to MPT to form Moco. Finally, in the fourth step of bacterial Moco biosynthesis, Moco is further modified by the addition of CTP or GTP forming the MPT cytosine dinucleotide (MCD) cofactor or the bis-MPT guanine dinucleotide (bis-MGD) cofactor, respectively. Bis-MGD thereby is the predominant form of Moco in E. coli


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and is present in enzymes like nitrate reductase (NR) or trimethylamine-N-oxide reductase (TMAOR).17 Previous results identified the small sulfur transferring protein TusA to be involved in the second step of Moco biosynthesis. Here, the MPT synthase consisting of two MoaD and MoaE subunits performs the insertion of two sulfur atoms into cPMP to form MPT. The sulfur originates from IscS and is most likely transferred via TusA directly to form the C-terminal thiocarboxylate group on MoaD. The sulfur of the MoaD-thiocarboxylate is then inserted into MoaE-bound cPMP. It has been shown that a deletion of tusA resulted in an overall reduced sulfuration level of MPT synthase, cPMP accumulation and largely reduced molybdoenzyme activities under aerobic conditions. In contrast, in cells grown anaerobically the role of TusA for sulfur transfer to MoaD was suggested to be partially replaced by other enzymes, since molybdoenzyme activities were retained to a level of 50%. Based on genomic and transcriptional analysis it has been suggested that SufS in concert with alternative sulfurtransferases is able to replace TusA in its sulfur transfer role under these conditions.18,19 This assumption was based on the fact that higher levels of sufS mRNA were identified in a ∆tusA deletion strain. Here, we analyzed the role of the SUF system for its role in Moco biosynthesis and tRNA thiolation under aerobic and anaerobic conditions. We chose a system to express the sufABCDSE operon in selected E. coli deletion strains for its ability to provide the sulfur for thiomodifications in tRNA and to synthesize the dithiolene group of Moco. As expected, the SUF system was able to replace IscS in its role for Fe-S cluster biosynthesis. However, the results demonstrate that components of the SUF system were unable to transfer the sulfur to either ThiI or TusA for both thionucleoside formation or sulfur insertion into cPMP, restricting its role to Fe-S cluster biosynthesis.


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EXPERIMENTAL PROCEDURES Bacterial Strains, Plasmids, Media, and Growth Conditions. Strains and plasmids used in this study are listed in Table 1. All isogenic BW25113 mutant strains (Keio collection) were obtained from the National BioResource Project (National Institute of Genomics, Japan).20 When required, the T7 promoter was introduced into E. coli strains BW25113 (wild type), ∆tusA, ∆thiI, ∆iscU, ∆cyaY, ∆iscS and ∆fdx by using the λDE3 lysogenization kit (Novagen). E. coli cultures were grown in LB medium under aerobic or anaerobic conditions at 37°C. When required, 150 µg/mL ampicillin, 25 g/mL kanamycin or 50 g/mL chloramphenicol were added to the medium during growth. 15 mM potassium nitrate or 15 mM trimethylamine-N-oxide (TMAO) were added as indicated. Protein expression was induced by the addition of 10 µM IPTG.

Protein concentration quantification. Protein concentrations were determined using the Bradford Reagent Coomassie Plus™ Protein Assay Reagent (Thermo) with bovine serum albumin (BSA) as a standard following manufacture’s instructions.

Quantification of NR and TMAOR activities. The activity of NR or TMAOR was measured in crude extracts obtained from E. coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS and ∆fdx after aerobic or anaerobic growth for 8 h in the presence of 15 mM potassium nitrate or 15 mM TMAO (cells were harvested at stationary growth phase at OD600 = 2.0 - 3.3 for aerobic cultures or OD600 = 0.8 - 1.8 for anaerobic cultures). Cells were harvested by centrifugation and resuspended in 50 mM Tris-HCl (pH 7.5) (for NR activities) or 100 mM phosphate buffer (pH 6.5) (for TMAOR activities). Cell lysates were obtained by sonification, transferred into an anaerobic chamber and incubated at 4 °C for at least 3 h. 50 µL of each cell lysate was analyzed


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for NR or TMAOR activity in a volume of 4 ml containing 0.3 mM benzylviologen, 10 mM KNO3 or 5 mM TMAO in 20 mM Tris-HCl (pH 6.8) or 100 mM phosphate buffer (pH 6.5), respectively. The assay was initiated by injecting sodium dithionite to the anaerobic reaction mixture until OD600 of of 0.8-0.9 for reduced benzyl viologen was reached. After the addition of crude extract, the oxidation of benzylviologen was recoded at 600 nm for 30 sec. The activity was calculated using the equation U = 0.5 x (∆Abs600/min)/ε600(benzyl viologen)/V, using the extinction coefficient for benzyl viologen of 7.4 mmol-1 x cm-1. One Unit is defined as the oxidation of 1 µmol reduced benzyl viologen per minute. The activity was normalized to the OD600 of the cells before harvesting.16

Detection of Moco and cPMP in cell extracts. To determine the total Moco content (as MPT and Moco) 50 ml cultures of E. coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS or ∆fdx were grown for 8 h (cells were harvested at stationary growth phase at OD600 = 2.0 - 3.3 for aerobic cultures or OD600 = 0.8 - 1.8 for anaerobic cultures) under aerobic or anaerobic conditions in the presence of either 15 mM potassium nitrate or 15 mM TMAO, harvested by centrifugation, and resuspended in 100 mM Tris-HCl, pH 7.2. Cells were lysed by sonification and the cell debris was removed by centrifugation. Samples were oxidized in the presence of acidic iodine at 95 °C for 30 min and excess iodine was removed by the addition of 55 µL of 1% (w/v) ascorbic acid. The pH of the samples was adjusted with 1 M Tris-HCl to pH 8.3. After addition of 40 mM MgCl2 and 1 unit of fast alkaline phosphatase, FormA was obtained. For purification of FormA, the samples were loaded onto a 500 µl QAE ion exchange resin (Sigma) equilibrated in water. The column was washed with 10 column volumes of water and with 1300 µL 10 mM acetic acid. FormA was eluted with 6 x 500 µL 10 mM acetic acid. The fractions


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were separated on a C18 reversed phase HPLC column (4.6 x 250 mm ODS Hypersil, particle size 5 µm) equilibrated with 5 mM ammonium acetate, 15% (v/v) methanol at a flow rate of 1 ml/min. Elution of FormA was monitored by an Agilent 1100 series fluorescence detector with an excitation at 383 nm and emission at 450 nm. The total FormA content was normalized to the total protein concentration determined by Bradford. Total cPMP can be converted into its oxidized fluorescent derivate CompoundZ with acidic iodine. CompoundZ was isolated from crude extracts as described above for FormA with slight modifications. Prior to elution from the QAE column, the column was washed with 10 column volumes water and 10 mM acetic acid before CompoundZ was eluted with 5 x 1000 µL 100 mM HCl. The fractions were separated on a C18 reversed phase HPLC column (4.6 x 250 mm ODS Hypersil, particle size 5 µm) equilibrated with 10 mM potassium dihydrogen phosphate (pH 3.0), 1% (v/v) methanol at a flow rate of 1 ml/min. CompoundZ was monitored by its fluorescence with an excitation at 383 nm and emission at 450 nm. The total CompoundZ content was normalized to the total protein concentration determined by Bradford.

Quantification of thionucleosides in tRNA. Total tRNA was extracted using the TriFast (peqLab) reagent form cell pellets of E. coli strains BW25113 (wild type), ∆tusA, ∆thiI, ∆iscU, ∆cyaY, ∆iscS and ∆fdx. Each strain was either cultivated in a volume of 50 mL aerobically or in a volume of 150 mL anaerobically at 37 °C for 8 h (cells were harvested at stationary growth phase at OD600 = 2.0 - 3.3 for aerobic cultures or OD600 = 0.8 - 1.8 for anaerobic cultures). Cells were harvested by centrifugation and suspended in 25 mL TriReagent. Total RNA was precipitated form the aqueous phase with isopropanol after addition of chloroform. The obtained pellet was washed twice with 75% ethanol and solved in 2x RNA loading dye (95% formamid,


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0.025% SDS, 0.025% xylene cyanole, 0.025% bromophenol, 0.5 mM EDTA (pH 8.0)). RNA species were separated by 10% Urea-PAGE at 200 V and the bands corresponding to tRNAs were eluted from the gels over night in 50 mM sodium acetate, 150 mM sodium chloride (pH 7.0). tRNAs were precipitated with isopropanol, washed twice with 75% ethanol, resolved in 50 mM sodium acetate, 50 mM zinc acetate (pH 5.3) and the concentration of tRNA was determined spectrophotometrically. An amount of 200 µg tRNA were digested over night at 37 °C in the same buffer with 4 U P1 nuclease (Sigma) and 4 U fast alkaline phosphate (Sigma) after adjusting the pH to 8.5. The obtained nucleosides were separated on a C18 reversed phase LiChrospher column (4.6x250 nm; particle size 5 µm) at 1 ml/min using 10 mM ammonium phosphate (pH 5.3)/methanol after the protocol reported previously with slight modifications.21 The elution of the nucleosides was followed at 254 nm, 274 nm and 330 nm, the amount quantified by peak integration and normalized to the pseudouridine peak at 254 nm.

Expression and Purification of Proteins. IscS, SufS, SufE, TusA and ThiI were expressed in BL21(DE3) cells and purified following previously described procedures.19,22,23

Quantification of L-cysteine desulfurase activity. The activity of IscS and SufS in the presence of TusA, ThiI or/and SufE was quantified either as methylene blue or by L-alanine quantification by derivatization with NDA following published procedures.24,25 Here, 1 µM IscS or SufS were incubated with 2 µM TusA, ThiI or/and SufE for 10 min at 30 °C in the presence of 0.5 mM DTT. The reaction was stopped and each product quantified by using a standard calibration curve.


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Detection of protein-protein interactions. To test the complex formation between IscS or SufS with either TusA, ThiI and/or SufE, 30 µM of IscS or SufS was incubated with 60 µM TusA, ThiI and/or SufE for 25 min at 37 °C. The protein mixture was injected onto a Superdex 200 column connected to an Aekta purifier system, which has been equilibrated in 50 mM Tris-HCl, 100 mM NaCl, 10 mM β-mercaptoethanol (pH 8.0). Proteins were separated at a flow rate of 1 ml/min and the elution profile was recorded at 280 nm. The proteins in the elution fractions were separated by 15% SDS-PAGE.

Immunodetection of IscS, SufS and GroEL. E.coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS and ∆fdx were cultivated under aerobic or anaerobic conditions in the presence of 15 mM potassium nitrate or 15 mM TMAO. Cells were harvested after 8 h (cells were harvested at stationary growth phase at OD600 = 2.0 - 3.3 for aerobic cultures or OD600 = 0.8 - 1.8 for anaerobic cultures), lysed by sonification in 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP40 (v/v)(pH 8.0) and cell debris removed by centrifugation. Protein concentration was quantified by Bradford. 50 µg of the cell extracts were separated by 12% SDS-PAGE and transferred to nitrocellulose or PVDF membranes (Amersham). The membrane was blocked with 5% BSA in TBST for 1 h at room temperature, rinsed with TBST and incubated with chicken anti-SufS serum (1:2000), rabbit anti-IscS (1:5000) serum or rabbit anti-GroEL antibodies (Abcam)(1:10000) over night at 4 °C. The blot was washed with TBST and incubated with horseradish peroxidase (HRP)- conjugated goat anti-chicken (Abcam)(1:5000) or goat anti-rabbit secondary antibodies (Thermo Scientific)(1:5000). Target proteins were visualized by enhanced chemiluminescence.


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Quantification of β-galactosidase activities. For the construction of an iscR-lacZ fusion, the respective promoter region 200 bp upstream of iscR including the ATG starts codon but excluding the ryhB binding side was PCR amplified and cloned into the EcoRI/BamHI sites of pGE593. The strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS and ∆fdx were transformed either with the transcriptional fusion or with the pGE593 vector control or a plasmid containing the suf operon. Cells were grown under aerobic or anaerobic conditions in the presence of 15 mM potassium nitrate or 15 mM TMAO at 37 °C until mid-exponential phase (34 h). Cells were permeabilized using Chloroform/SDS in 100 mM Sorensen Buffer (pH 7.0) supplemented with KCl, MgSO4 and β-mercaptoethanol. The reaction was started by the addition of 400 µg ortho-Nitrophenyl-β-galactoside (ONPG) and was stopped by addition of 1 M Na2CO3. The amount of formed ortho-Nitrophenol was measured at 420 nm, corrected for light scattering at 550 nm and normalized to the volume of cells, their optical density at 600 nm and the reaction time (Miller units). For each strain a respective blank reaction containing cells transformed with the vector control was subtracted.

Detection of aconitase and malate dehydrogenase activities. The enzymatic activity of aconitase and malate dehydrogenase was measured in crude extracts. E. coli strains BW25113 (wild type), ∆iscU, ∆cyaY, ∆iscS and ∆fdx were cultivated under aerobic conditions for 6 h (early stationary growth phase at OD600 = 1.6 - 3.0) and lysed by sonification in 50 mM Tris-HCl, 150 mM NaCl, 0.5% NP-40 (v/v) (pH 8.0). Aconitase activity was determined in a coupled enzymatic assay monitoring NADPH production at 340 nm from the oxidation of produced isocitrate by isocitrate dehydrogenase. 50 µL cell lysate were incubated for 5 min in 450 µL 50 mM Tris-HCl, 50 mM NaCl, 5 mM MgCl2, 0.5 mM NADP and 0.05 U isocitrate dehydrogenase


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(pH 8.0). The reaction was started by addition of 500 µL 2.5 mM cis-aconitate in the same buffer. 1 Unit is defined as 1 µmol NADPH formed in 1 min. Aconitase activity was normalized to total protein concentration used in the assay. Malate dehydrogenase activity was detected by activity staining. 25 µg total protein was separated by a 7.5% native PAGE at 4°C. Native polyacrylamide gels were incubated at 37°C in 50 mM Tris-HCl, 5 mg/mL malate, 0.6 mg/mL NAD, 0.5 mg/mL nitro tetrazolium blue, 0.04 mg/mL phenazine methosulfate (pH 8.0) for 5 min.

RESULTS Quantification of SufS protein levels in different E. coli deletion strains. Since previous results suggested that SufS might have an additional role in Moco biosynthesis by replacing IscS under certain conditions, we wanted to investigate the role of SufS in providing the sulfur for both nucleosides in tRNA and Moco further. Microarray analyses showed a twofold increase of the suf operon mRNA in tusA deficient strain under anaerobic conditions. We therefore wanted to test the endogenous expression of SufS in the ∆tusA mutant strain. As controls we analyzed the SufS levels in strains with deletions in iscU, cyaY, iscS or fdx since the proteins encoded by these genes influence Fe-S cluster assembly. Further, the influence of oxygen was analyzed in cells grown on nitrate or TMAO, for the induction of the expression of NR and TMAOR. Overall endogenous SufS levels were low under all conditions tested including the ∆tusA strain, however, elevated levels of SufS were observed in ∆iscU and ∆fdx strains (Supporting Information Figure S1). Additionally, we overexpressed the sufABCDSE operon, the expression of which is controlled by an IPTG inducible promoter. We successfully confirmed the expected elevated levels of SufS in the tested E. coli strains. As a protein loading control, the


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amount of GroEL was detected with a specific antibody, showing the same protein loading in all protein extracts tested (Supporting Information Figure S1).

NR and TMAOR activities depend on the proteins IscS, IscU and Fdx of the Fe-S cluster assembly. To test whether elevated levels of SufS can restore molybdoenzyme activity in a ∆tusA strain, we analyzed the activities of NR and TMAOR after introduction of the suf operon to different E. coli deletion strains. To overcome possible regulatory effects, an approach was chosen in which the whole sufABCDSE operon was introduced to these strains, the expression of which was induced by the addition of 10 µM IPTG. The activities of both enzymes were compared, since E. coli NR enzymes require Fe-S clusters for their activities, while E. coli TMAOR enzymes contain only the bis-MGD cofactor. We analyzed the activities of both enzymes under aerobic and anaerobic conditions, since the SUF and ISC operons are differently regulated by the presence of oxygen. The cells were grown in the presence and absence of oxygen and the expression of NR or TMAOR were induced by the addition of either nitrate or TMAO during growth. As expected, enzyme activities were lower under aerobic conditions, with 10% of the overall activity for NR and 5% of the activitiy for TMAOR in comparison to the anaerobic cultures. Further, the results in Figure 1 show that TMAOR and NR activities were largely reduced in strains carrying a deletion in either iscS or iscU. While the role of IscS was not restored by the complementation of the suf operon in this deletion strain, expression of the suf operon in the ∆iscU strain restored NR and TMAOR activity up to 80% of wild type activity. In contrast, deletion of the fdx gene had no influence on NR activity under aerobic conditions, while under anaerobic conditions NR activity was 90% reduced in this strain in comparison to the respective wild type control. Further, a


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functional Fdx protein is required for an active TMAOR under both conditions tested (Figure 1B). Here, the suf operon was able to restore the effect of the fdx deletion for both NR and TMAOR activity. A deletion of cyaY had no impact on NR activity, while TMAOR activity was reduced in a cyaY deletion strain about 30%. In addition, TMAOR activity was significantly increased in a cyaY deletion strain when the suf operon was present (Figure 1B). This effect was much more pronounced under aerobic conditions than under anaerobic conditions. The deletion of tusA resulted in a major decrease of NR and TMAOR activities (Figure 1), which confirms previously published results.19 While under aerobic conditions both molybdoenzymes were mainly inactive, under anaerobic conditions an activity of 45 - 75% was retained in comparison to the wild type strain. Here, expression of the suf operon in the ∆tusA deficient strain did not result in an increase of enzyme activities. The results therefore suggest that both TusA and IscS are essential for the activity of NR and TMAOR in a route independent from the assembly of FeS clusters, since the suf operon was unable to rescue the negative effect caused by a deletion of both genes individually.

Accumulation of cPMP correlates with decreased Moco levels in ∆tusA and ∆iscS strains. Fe-S clusters are also required for the activity of MoaA in the first step of Moco biosynthesis. MoaA harbors two [4Fe-4S] clusters and catalyzes the conversion of 5’GTP to cPMP. To further dissect the role of TusA, IscS, IscU, CyaY and Fdx on the biosynthesis of Moco, we analyzed the effect of the expression of the suf operon on the amount of cPMP and Moco in the respective deletion strains. The amounts of cPMP and Moco produced in the respective strains were quantified after aerobic or anaerobic growth in the presence of either nitrate or TMAO. For the quantification of produced Moco or accumulated cPMP, both molecules were converted into


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their oxidized fluorescent derivatives FormA and CompoundZ, respectively, purified from crude extracts and separated via HPLC. The results show an accumulation of cPMP in ∆tusA cells grown on nitrate (Figure 2A) or TMAO (Figure 2B), as reported previously.19 cPMP was also accumulated in a ∆iscS strain, however, only after the expression of the suf operon. All other strains grown with nitrate or TMAO did not accumulate cPMP, independent on the presence of the suf operon or oxygen during growth. In addition, the amount of formed Moco was quantified. The results shown in Figure 3 correlate well with the activity of NR and TMAOR and the cPMP levels reported above. However, by this method the cellular Moco content of all molybdoenzymes is quantified. The results in Figure 3 show that generally the Moco content is largely reduced in ∆tusA and ∆iscS strains after growth on either nitrate or TMAO. The effect of a deletion of tusA or iscS was not rescued by expression of the suf operon (Figures 3A and B). In cells lacking iscU, the amount of Moco was more than 50% decreased in comparison to the wild type strain under all conditions tested. In contrast, deletion of cyaY had only a minor effect on anaerobically produced Moco in the presence of TMAO. Interestingly, Fdx played only a role for Moco production when the cells were grown anaerobically, but independent from nitrate or TMAO respiration. Here, the ∆fdx strain contained only 40-50% Moco in relation to the corresponding wild type. The introduction of the suf operon in a ∆fdx, ∆iscU and ∆cyaY strain was able to rescue this defect in Moco formation. From these results it can be concluded that IscS and TusA are exclusively involved in the conversion of cPMP to MPT, a role that cannot be replaced by the components of the SUF system. However, the suf operon is able to provide Fe-S clusters for MoaA activity which is obvious by the accumulation of cPMP in selected mutant strains.


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Fe-S cluster independent thiomodifications depend exclusively on the IscS protein. Besides Moco biosynthesis, TusA and IscS are also involved in the formation of mnm5s2U34 modified uridines at position 34 of tRNAs charged with Lys, Gln and Glu.10 In addition, we quantified the levels of s4U8, a modification that depends on IscS and ThiI.12 Total tRNAs were isolated from the respective E. coli deletion strains, cleaved, de-phosphorylated into single nucleosides by P1 nuclease and phosphatase treatment. The different nucleosides were separated by HPLC and quantified by their absorbance. The results in Figure 4A and B show that the amounts of mnm5s2U34 or s4U8 tRNA modifications remained unaffected in deletion strains of iscU, cyaY or fdx. In contrast, in a ∆iscS mutant strain mnm5s2U34 and s4U8 modifications were not detectable. Similarly, the modifications mnm5s2U34 or s4U8 were not detected in ∆tusA or ∆thiI strains, respectively. Further, the levels of modified tRNAs proved to be independent of the expression of the suf operon in the iscS, tusA or thiI deficient strain. These findings underline the essential role of IscS for all tRNA modifications. These thiomodifications further depend on functional TusA or ThiI proteins, which specific roles in sulfur transfer and/or roles in tRNA activation can not be replaced by the SUF system.

TusA and ThiI specifically interact with IscS. The absence of mnm5s2U34 and s4U8 in the ∆iscS strain overexpressing the suf operon showed that SufSE of the SUF machinery is not able to replace IscS in its sulfur transfer role. While the interaction side of TusA and ThiI on IscS was mapped, we were interested to analyze whether TusA and ThiI are unable to interact with SufS or the SufSE complex.11


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Since TusA was reported previously to enhance the activity of IscS, the influence of TusA and ThiI on the activities of IscS, SufS and SufSE were quantified. Two methods were chosen to analyze the L-cysteine desulfurase activities, which either quantify released sulfide as methylene blue or directly detect the amount of produced L-alanine in the presence of DTT. The results in Figure 5 show that IscS alone had an activity of 0.26-0.28 U/mg. After addition of TusA or ThiI an increase of activity of 2 and 7 fold, respectively, was obtained which is consistent with previous reports.10 SufS alone showed a low L-cysteine desulfurase activity of 0.06-0.08 U/mg, which was increased 12 fold by the addition of SufE. In contrast to IscS, the presence of neither TusA nor ThiI resulted in an increase of the activity of SufS or the SufSE complex, respectively. Further, we analyzed whether TusA or ThiI are able to form a complex with either SufS alone or with the SufSE complex. Proteins were incubated and analyzed for complex formation by size exclusion chromatography. The results showed that SufS readily formed a complex with SufE (Supporting Information Figure S2). In contrast, an interaction of SufS or SufSE with TusA or ThiI was not detected. In summary, IscS is the exclusive binding partner for both TusA and ThiI.

The SUF machinery can provide Fe-S clusters for proteins involved in tRNA thiomodifications. Recent publications reported that the tRNA-thiolase TtcA and the tRNA-methylthiolase MiaB are involved in the tRNA thiomodifications of s2C32 and ms2i6A37 in a Fe-S cluster-dependent manner, respectively.13,26 To analyze whether SUF can replace ISC in providing Fe-S clusters for proteins involved in tRNA thiomodifications, the activities of the two Fe-S cluster containing enzymes TtcA and MiaB was analyzed in the respective deletion strains.


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As shown in Figure 6A and B, the relative amounts of s2C32 and ms2i6A37 were more than 60% reduced in ∆iscU and ∆iscS strains in comparison to the wild type strain, both under aerobic and anaerobic conditions. This effect was restored by the expression of the suf operon in these strains, which resulted in wild type levels of s2C32 and ms2i6A37. Interestingly, the ∆fdx strain showed a reduction of s2C32 and ms2i6A37 levels of 80% only under anaerobic conditions, an effect that was also restored by the introduction of the suf operon. Levels of Fe-S cluster dependent thiomodifications were not changed in ∆cyaY strain. Measurements for the accumulation of the ms2i6A37 precursor i6A37 thereby correlated well with the reduced amounts of ms2i6A37 in Figure 6B (Supporting Information Figure S3). Thus, the SUF machinery seems to be able to replace the ISC system in providing Fe-S clusters for both TtcA and MiaB.

Aconitase activity was increased in Fe-S cluster deficient strains after introduction of the suf operon. To further analyze the effect of the SUF system on Fe-S cluster-containing enzymes, we measured the overall activity of the two [4Fe-4S] cluster containing aconitases AcnA and AcnB in iscU, cyaY, iscS and fdx deletion strains in the presence or absence of the suf operon. In this assay, however, mainly AcnB is measured under our growth conditions, while AcnA shows only minor activities (data not shown). Figure 7A shows that expression of the suf operon did not influence aconitase activity in the wild type strain. As expected, aconitase activity in the ∆iscU, ∆iscS and ∆cyaY strains was lower as compared to the wild type strain. A residual activity of 22% and 8% of wild type activity was obtained in the ∆iscU and ∆iscS mutant strains, respectively, whereas a deletion of cyaY resulted in a 50% reduced activity. The absence of Fdx had no impact on aconitase activity. By


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overexpression of the suf operon, a two- to four-fold increase in aconitase activity was obtained in the ∆iscU, ∆cyaY and ∆iscS strains. The results therefore confirm the previous results obtained for MoaA shown above, showing that IscU and IscS can bee replaced by the suf operon in providing [4Fe-4S] clusters for aconitase activity. We further tested the activity of the housekeeping enzyme malate dehydrogenase in an in-gel activity stain. Figure 7B shows that the malate dehydrogenase activity was comparable in all strains analyzed, so that the overall cell metabolism remained mainly unaffected in the deletion strains.

Expression of the suf operon decreases the expression of an iscR-lacZ fusion. In addition, we also tested the maturation of the IscR, the transcriptional regulator of the iscRSUA-AhscABfdx-iscX operon. IscR itself in its [2Fe-2S] cluster-bound state negatively regulates the transcription of the isc operon. When Fe-S clusters are limiting and no [2Fe-2S] clusters are bound to IscR, the expression of the isc operon is induced. For that purpose an iscRpromoter lacZ-fusion was constructed and β-galactosidase activity was measured. The results in Figure 8 show that in ΔiscU and ΔiscS strains the β-galactosidase activity was 45 fold increased both under aerobic and anaerobic conditions, while deletion of fdx only resulted in an increased expression under anaerobic conditions. Further, expression of iscR-lacZ was independent on the presence of TusA and only slightly induced in the ∆cyaY strain. In contrast, we observed a reduction of iscR-lacZ expression by the introduction of the suf operon in the ∆iscS and ∆fdx strains, while the β-galactosidase activity was only slightly decreased in the ∆iscU strain. This shows that similar to the aconitase activities, the Fe-S cluster for IscR can be provided by the SUF system and effectively inserted into apo-IscR.


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Since the expression levels of the isc operon and the suf operon are tightly co-regulated in the cell, we also analyzed the effect of the protein levels of IscS in the deletion strains before and after the introduction of the suf operon by immunodetection (Supporting Information Figure S4). The detected IscS concentrations correlated well with the above described β-galactosidase activities for iscR-lacZ fusion. The reduction of IscS protein concentrations was shown to be more pronounced when the cells were grown with nitrate in comparison to cells grown with TMAO. Interestingly, all strains grown anaerobically showed that the overall amount of IscS was reduced when the suf operon was expressed (Supporting Information Figure S4).

DISCUSSION SufS is unable to provide the sulfur for Moco or thionucleosides in tRNA. In this study we analyzed the E. coli SUF and ISC systems in their roles to transfer sulfur for FeS cluster, Moco and thionucleosides in tRNA. Especially the role of the L-cysteine desulfurases IscS and SufS of both systems have been investigated in detail in the past. While IscS serves as the universal sulfur donor for FeS clusters, nucleosides in tRNA, biotin, thiamin, lipoic acid and Moco, the role of SufS, in contrast, has been mainly assigned to a role in FeS cluster biosynthesis (Figure 9). E. coli cells with only one of the systems were shown to be viable due to their functional redundancy.27 In contrast, simultaneous inactivation of the ISC and SUF system is lethal to the cell. That's why we were choosing an approach to complement single mutant strains. By introducing of the whole suf operon and testing for functional complementation of the respective role of single genes we were able to dissect their roles in FeS cluster assembly, Moco biosynthesis and thiolation of tRNA. 28-31


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Especially the function of the L-cysteine desulfurases IscS and SufS has been investigated in detail in the past. While IscS serves as the universal sulfur donor for Fe-S clusters, nucleosides in tRNA, biotin, thiamin, lipoic acid and Moco, the role of SufS, in contrast, has been mainly assigned to a role in Fe-S cluster biosynthesis (Figure 9).8,23 In our studies, we investigated the role of SufS as a sulfur donor for pathways beyond Fe-S cluster biosynthesis. This especially was of interest since under specific cellular conditions "backup systems" were proposed to exist in the cell that ensure basic levels of the important sulfur-containing cofactors and thionucleosides, that might enable cellular viability under specific conditions. This has become clear by the example of Moco biosynthesis, for which a remaining 50% activity of NR and TMAOR under anaerobic conditions was identified in a ∆tusA mutant strain. Here, microarray analyses revealed that the mRNA of the components of the SUF system was increased under those conditions19. However, the results in this study clearly show that only IscS is capable of fulfilling these multiple roles in cellular sulfur trafficking, since SufS was not able to replace IscS in its role for Moco biosynthesis and thionucleoside formation for certain tRNAs (Figure 9). The results showed that overexpression of the suf operon in an iscS or tusA deficient strain did not result in the production of active Moco. The accumulation of cPMP in this strain, however, demonstrated that the suf system was able to produce the Fe-S clusters required for the activity of MoaA, which produced cPMP from 5'GTP under the conditions tested. Further, the protein levels of SufS were not increased in these mutant strains. The reason why in a ∆tusA mutant under anaerobic conditions more than 50% NR and TMAOR activities were obtained in comparison to the wild type strain still remain elusive. This implies that still other sulfur transfer systems in the cell like rhodanese-like proteins might exist that can substitute for the role of


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TusA in Moco biosynthesis, as proposed previously.18,19 Since Moco is an important cofactor for numerous enzymes in E. coli catalyzing important cellular functions, an alternative sulfurtransfer route independent from TusA might be advantageous under certain conditions, which seems to be restricted to Moco biosynthesis, since thiomodifications of tRNA were not detected in the tusA deletion strain. Similar to Moco biosynthesis, 4-thiouridine formation was not detected in the absence of iscS, underlining the specificity of the IscS-ThiI interaction. We were able to confirm with the purified proteins that a productive complex of SufS with neither TusA nor ThiI was formed, independent on the presence of SufE. Thus, SufE is so far the only specific interaction partner of SufS in E. coli.23

The SUF system can replace the role of IscS for Fe-S clusters containing proteins. In our study we confirmed previous studies showing that the SUF machinery can replace the ISC system in Fe-S cluster biosynthesis.31 Analyses of the activities of Fe-S-cluster containing NR and Fe-S cluster-free TMAOR in different mutants strains in genes of the isc operon, like ∆iscU, ∆fdx or ∆cyaY resulted in largely reduced overall activities of both enzymes under aerobic and anaerobic conditions. However, by overexpression of the suf operon in the iscU deficient strain, both molybdoenzyme activities were restored. For ms2i6A37 and s2C32 thiomodifications of tRNA, the proteins MiaB and TtcA require [4Fe4S] clusters for their activities.26 Analysis of the different thionucleosides showed that IscS and IscU are essential for MiaB and TtcA activity. The suf operon expressed in the iscU or iscS deficient strains was able to overcome the gene defects of the ISC system and ms2i6A37 in s2C32 were detected in the respective strains.


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Recently, CyaY has been discussed as novel component of the ISC system.5 In these studies, deletion of cyaY under iron-rich conditions resulted in decreased activities of Fe-S cluster proteins, like Nuo, IscR and Sdh.5 In our studies, the absence of CyaY resulted in a decreased TMAOR activity, whereas NR activity was retained to wild type levels in this strain. Strikingly, in our studies the effect of CyaY on TMAOR activity was compensated by overexpression of the suf operon. The decrease in activity thereby correlated with a decrease in total MPT/Moco levels in this strain. Thus, CyaY might influence Fe-S cluster formation for Moco biosynthesis only under specific conditions (like TMAO respiration). However for tRNA thiolation, a requirement for CyaY could not be demonstrated, since wild type levels of s2C32 and ms2i6A37 were detected in a cyaY deficient strain. Further, a recent report identified Fdx as non-essential component of the ISC system under anaerobic conditions in the absence of the SUF operon.31 Fdx thereby harbors a [2Fe-2S] cluster and has been proposed to act as the donor of reductive equivalents during Fe-S cluster assembly.4 However, analysis of the ∆fdx strain under anaerobic conditions showed a decrease of Fe-S cluster-dependent thiomodifications and Moco levels. Since overexpression of the suf operon in this strain rescued the effect of the fdx deletion it can be concluded that Fdx is essential at least for the formation of the [4Fe-4S] cluster for TtcA, MiaB and MoaA. Under aerobic conditions the ∆fdx strain showed wild type levels of MPT, s2C32 and ms2i6A37 revealing that the role of Fdx is not required under these conditions or can be fully replaced by other proteins.31

The co-regulation of the SUF and ISC systems Barras and coworkers recently reported on the insertion of Fe-S clusters into the two transcriptional regulators NsrR and IscR. Their study showed that in an iscUA deficient strain,


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IscR lost its ability to repress iscSUA transcription. However, by introduction of the suf operon active IscR was restored and repressed isc transcription.32 In our study, we show that overexpression of the suf operon resulted in a decrease of IscS expression (as observed by immunodetection). It is therefore concluded that elevated concentrations of the components of the SUF machinery result in higher levels of Fe-S cluster insertion into apo-IscR and, in turn, a repression of isc transcription. Previous reports identified that [2Fe-2S] and [4Fe-4S] clusters are stably bound to SufB.33 Since in our studies IscS levels decreased after overexpression of the suf operon, it is likely that SufB is capable of binding a stable [2Fe-2S] cluster that is directly or indirectly via A-type carrier proteins, like SufA, transferred to IscR. Our immunoblot analysis confirmed the coregulation of suf induction and isc repression, which is based on the regulation by IscR and the presence of oxygen.

The role of SufS is restricted to Fe-S cluster formation in E. coli. The two major Fe-S cluster assembly pathways for cellular Fe-S cluster containing proteins are the ISC and the SUF system. The phylogenetic distribution of these two systems, however, is very complex. While in Cyanobacteria the SUF pathway is the major Fe-S assembly system compared to the ISC pathway, in E. coli the relative importance of SUF and ISC system is reversed. Furthermore, organisms exist in which the SUF system is the only Fe-S cluster assembly system, like in some archaea.34,35 In eukaryotes, the occurrence of the two systems is restricted to specific organelles. Here, homologues of the ISC system are present in mitochondria, while the SUF system is restricted to chloroplasts in photosynthetic organisms.36,37 In organisms that contain exclusively the SUF system, disruption of any of the genes was shown to be lethal to the cells.38 In contrast to organisms that are only dependent on the SUF system for


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Fe-S cluster formation, other bacteria use two or more systems in parallel. It has been shown for E. coli that the SUF system is mainly active under stress conditions, whereas its housekeeping Fe-S cluster machinery encoded in the isc operon, is responsible for the maturation of most Fe-S cluster containing proteins under normal growth conditions.34 However, the L-cysteine desulfurase IscS of the E. coli ISC system has additionally evolved to fulfill numerous cellular roles as major sulfur mobilizing enzyme not only for Fe-S clusters, but also for sulfur-containing cofactors in including Moco and thiamin in addition to thio-modifications of nucleosides in tRNA (Figure 9). To accomplish this role, IscS interacts with numerous sulfur accepting proteins. So far the interactions with TusA, CyaY, ThiI and Fdx were mapped on IscS.11,39,40 Xray crystallography of the IscS-IscU and IscS-TusA complexes revealed that although the binding sites for IscU and TusA were both located in the area surrounding the active site cysteine (Cys328), their binding sites were not overlapping.11 The larger flexibility of IscS to deliver the sulfur to numerous different proteins thereby seems to be realized by the active-site loop that in IscS is long and flexible. In contrast, other cysteine desulfurases like SufS have a relatively short loop thereby restricting its interaction partners to a single protein (SufE). It has further been proposed that by providing multiple binding sites on IscS to several sulfur-accepting proteins, E. coli can not only regulate the activity of IscS, but also the accessibility of its interaction partners.41 It has been suggested that to ensure cell viability under conditions in which the supply of sulfur is limited, sulfur delivery to IscU is preferred to the other interaction partners. Restricting SufS as a backup system for Fe-S cluster formation thereby underlines the importance to fine-tune sulfur distribution to react to different growth conditions in the cell. How is the sulfur transfer realized in other organisms, in which the SUF system is the main machinery for Fe-S cluster assembly? In organisms like Bacillus subtilis the inability of SufS to


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interact with multiple sulfur acceptor proteins seems to be compensated by the presence of numerous L-cysteine desulfurases that are specific for the synthesis of one sulfur-containing molecule.42 In B. subtilis, three different L-cysteine desulfurases in addition to SufS are present, namely YrvO, NifZ, and NifS. YrvO thereby is specific for 2-thiouridine and Moco formation while NifZ is specific for 4-thiouridine formation.43-45 NifS, in contrast, is essential for Fe-S clusters for the synthesis of NAD+.46 Further, in B. subtilis the genes for the L-cysteine desulfurases and the respective interaction partners are organized in operon structures on the genome, to ensure a tight co-regulation of synthesis of the proteins only under the conditions when the proteins are required for the synthesis of the specific molecule.42 Also here, it seems to be not advantageous to the cell, that the L-cysteine desulfurases can replace each other but are rather specific for the synthesis of a particular sulfur-containing cofactor. In summary, our results contribute to the understanding of the complex interplay between the different Fe-S cluster machineries and sulfur relay systems for the synthesis of sulfur containing biomolecules in E.coli.


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AUTHOR INFORMATION Corresponding Author

*To whom correspondence should be addressed: Silke Leimkühler, Institute of Biochemistry and Biology, Department of Molecular Enzymology, University of Potsdam, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany, Telephone: +49-331-977-5603; Fax: +49-331 977-5128; Email: [email protected]

Funding

The research leading to these results has received funding from the International Max Planck Research School on Multiscale Biosystems to M.B, A.V. and S.L. This work was supported by the Deutsche Forschungsgemeinschaft grant LE1171/15-1 and LE1171/11-1 to SL.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

We thank Frederic Barras (CNRS Marseille, France) for providing plasmid pET-Ehis, Petra Hänzelmann (Würzburg) for providing plasmid pPH151 and Prof. F. Wayne Outten (University of South Carolina) for providing the SufS anti-serum. Angelika Lehmann (University of Potsdam) and Natalie Lupilov (Ruhr University Bochum) are thanked for their technical assistance.


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Supporting Information available Figure S1: Quantification of SufS levels by immunodetection in different E. coli deletion strains Figure S2: Analysis of interactions between TusA, ThiI, SufS and SufE by size exclusion chromatography. Figure S3: Quantification of i6A37 in different E. coli strains Figure S4: Quantification of IscS levels by immunodetection in different E. coli deletion strains.

ABBREVIATIONS ferredoxin (Fdx) molybdenum cofactor (Moco) cyclic pyranopterin monophosphate (cPMP) molyptopterin (MPT) cytosine dinucleotide (MCD) bis- MPT guanine dinucleotide (bis-MGD) nitrate reductase (NR) trimethyl-N-oxide reductase (TMAOR)


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(44) Black, K. A., and Santos, Dos, P. C. (2015) Abbreviated Pathway for Biosynthesis of 2Thiouridine in Bacillus subtilis. J. Bacteriol. 197, 1952–1962. (45) Martin, H. L., Black, K. A., and Santos, Dos, P. C. (2015) Functional investigation of Bacillus subtilis YrkF’s involvement in sulfur transfer reactions. Peptidomics 2, 45–51. (46) Sun, D., and Setlow, P. (1993) Cloning, nucleotide sequence, and regulation of the Bacillus subtilis nadB gene and a nifS-like gene, both of which are essential for NAD biosynthesis. J. Bacteriol. 175, 1423–1432. (47) Hänzelmann, P., Hernández, H. L., Menzel, C., Garcia-Serres, R., Huynh, B. H., Johnson, M. K., Mendel, R. R., and Schindelin, H. (2004) Characterization of MOCS1A, an oxygensensitive iron-sulfur protein involved in human molybdenum cofactor biosynthesis. J. Biol. Chem. 279, 34721–34732.


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TABLES Table 1. List of used strains and plasmids. Plasmid/Strain

Genotype / relevant characterization

Reference

Plasmid pMB23

Gene region -200 bp to -1 upstream iscR transcriptional start cloned into EcoRI/BamHI

This study

sites of pGE593, AmpR pPH151

sufABCDSE operon cloned into NcoI/EcoRI 47

sites of pACYCDuet1, Cm pSL223

R

thiI gene cloned into NdeI/BamHI sites of This study pET15b, AmpR

pSL209

iscS gene cloned into NcoI/BamHI sites of 22

pET15b, AmpR pJD34

tusA gene cloned into NdeI/BamHI sites of 19

pET11b, AmpR pSL213

sufS gene cloned into NdeI/BamHI sites of 22

pET15b, Amp pET-Ehis

R

sufE gene cloned into NdeI/XhoI sites of 6

pET22b, Amp

R

Strains BW25113

laclq rrnBT14 lacZWJ16 hsdR514 20

araBADAH33 rhaBADLD78 JW3435 (∆tusA)

BW25113 tusA::km 

20


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JW2513 (∆iscU)

BW25113 iscU::km 

20

JW3779 (∆cyaY)

BW25113 cyaY::km 

20

JW2514 (∆iscS)

BW25113 iscS::km 

20

JW2509 (∆fdx)

BW25113 fdx::km 

20

JW0413 (∆thiI)

BW25113 thiI::km 

20


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FIGURE LEGENDS Figure 1. Analysis of the activities of NR or TMAOR in different E. coli strains The activities of NR (A) or TMAOR (B) were determined in E. coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS or ∆fdx containing the suf operon (+) or the corresponding vector control (-). Cells were grown in the presence of 10 µM IPTG, 15 mM potassium nitrate or 15 mM TMAO under aerobic (dark grey) or anaerobic (light grey) conditions. NR and TMAOR activity was measured in crude extracts following the oxidation of dithionite-reduced benzylviologen at 600 nm. The activities were normalized to the OD 600 nm of the cells after 8h growth.

Figure 2. Quantification of cPMP in different E. coli strains The E. coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS or ∆fdx were either transformed with the suf operon (+) or with the corresponding vector control (-). The strains were grown aerobically (dark grey) or anaerobically (light grey) in the presence of 15 mM nitrate (A) or 15 mM TMAO (B) containing 10 µM IPTG. Total cPMP was oxidized over night into its fluorescent derivate CompoundZ with acidic iodine. CompoundZ was isolated and its fluorescence was monitored by excitation at 383 nm and emission at 450 nm on a HPLC. The corresponding peak area was related to the total soluble protein concentration used in the assay.

Figure 3. Quantification of Moco in different E. coli strains The E. coli strains BW25113 (wild type), ∆tusA, ∆iscU, ∆cyaY, ∆iscS or ∆fdx were either transformed with the suf operon (+) or with the corresponding vector control (-). The strains were grown aerobically (dark grey) or anaerobically (light grey) in the presence of 15 mM nitrate (A) or 15 mM TMAO (B) containing 10 µM IPTG. Total Moco in the obtained crude extracts was


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oxidized over night with acidic iodine into its fluorescence derivative FormA. FormA was isolated and its fluorescence monitored by excitation at 383 nm and emission at 450 nm on a HPLC. The corresponding peak area was related to the total soluble protein concentration used in the assay.

Figure 4. Quantification of Fe-S cluster independent thiomodifications E. coli strains The E. coli strains BW25113 (wild type), ∆tusA, ∆thiI, ∆iscU, ∆cyaY, ∆iscS or ∆fdx were either transformed with the suf operon (+) or with the corresponding vector control (-). The strains were grown aerobically (dark grey) or anaerobically (light grey) in the presence of 10 µM IPTG. Total RNA was isolated by phenol/chloroform extraction and total tRNAs were separated from other RNA species by 10% Urea-PAGE. tRNAs were digested into nucleosides and separated by HPLC. The elution of mnm5s2U34 was followed at 274 nm (A) or s4U8 at 330 nm (B). The corresponding peaks were integrated and normalized to the internal pseudouridine standard.

Figure 5. Influence of TusA, ThiI and SufE on IscS and SufS activity The activity of 1 µM IscS or SufS was quantified in the presence of 2 µM TusA, ThiI and/or SufE. The proteins were incubated for 10 min at 30 °C, the reaction and the amount of produced sulfide quantified via methylenblue at 670 nm (dark grey) or after derivatisation of the released L-alanine with NDA by excitation at 419 nm and emission at 493 nm (light grey). 1 U is defined as 1 µmol sulfide or L-alanine produced in 1 min. The symbols + and - are used to indicate the presence and absence of a protein in the assay, respectively.

Figure 6. Quantification of Fe-S cluster dependent thionucleosides in different E. coli strains


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The E. coli strains BW25113 (wild type), ∆tusA, ∆thiI, ∆iscU, ∆cyaY, ∆iscS or ∆fdx were either transformed with the suf operon (+) or with the corresponding vector control (-). The strains were grown aerobically (dark grey) or anaerobically (light grey) in the presence of 10 µM IPTG. Total RNA was isolated by phenol/chloroform extraction and total tRNAs were separated from other RNA species by 10% Urea-PAGE. tRNAs were digested into nucleosides and separated by HPLC. The elution of s2C32 (A) or ms2i6A37 (B) was followed at 254 nm. The corresponding peaks were integrated and normalized to the internal pseudouridine standard.

Figure 7. Quantification of aconitase and malate dehydrogenase activity in different E. coli strains The E. coli strains BW25113 (wild type), ∆iscU, ∆cyaY, ∆iscS or ∆fdx were either transformed with the suf operon (+) or with the corresponding vector control (-). The strains were grown aerobically (dark grey) or anaerobically (light grey) containing 10 µM IPTG until midexponential phase. (A) Aconitase activity was indirectly measured in crude extract in a coupled enzymatic reaction with isocitrate dehydrogenase and the formation of NADPH was followed at 340 nm. The activity was normalized to total protein amount in the reaction. (B) 25 µg total protein were natively separated and malate dehydrogenase was stained using formation of formazan at 37 °C.

Figure 8. Analysis of the regulation of the iscR promoter in different E. coli strains The E. coli strains ∆tusA, ∆iscU, ∆cyaY, ∆iscS and ∆fdx as well as the corresponding wild type strain BW25113 were either transformed with the suf operon (+) or with the corresponding vector control (-). A second plasmid contains the promoter region of the iscR promoter fused to


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the lacZ reporter gene. The expression of the iscR promoter was measured in strains grown aerobically (dark grey) or anaerobically (light grey) in the presence of 15 mM nitrate (A) and 15 mM TMAO (B) until mid-log phase. β-galactosidase activity was measured using ONPG as substrate. The formation of o-nitrophenol was quantified at 420 nm and corrected for light scattering at 550 nm. β-galactosidase activity was standardized to miller units by normalizing to required reaction time, OD600 of used cell culture and amount of cells. Background reactions were subtracted by the corresponding control strains transformed with the plasmid-encoded lacZ gene only.

Figure 9. Model for the sulfur transfer pathways in E. coli Fe-S clusters are mainly assembled via two different pathways, namely the ISC and SUF. Proteins from each system are encoded in the receptive operons and their synthesis is regulated with respect to Fe-S cluster demand via the transcription factor IscR. Assembled Fe-S clusters can be inserted into different proteins, like aconitase or proteins that are involved in tRNA thiolation. Further, Fe-S clusters are also required for MoaA activity that catalyzes the conversion of GTP into cPMP in the first step of Moco biosynthesis. cPMP is further converted to MPT by sulfur transfer from MoaD of the MPT synthase complex. IscS/TusA thereby are the sulfur donors for MoaD. Molybdenum is incorporated into MPT and the formed Mo-MPT can be further modified. IscS is also essential for 4-thiouridine and 2-thiouridine synthesis by sulfur transfer to the proteins ThiI and TusA.


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Graphic for the Table of Contents

The role of SufS is restricted to Fe-S cluster biosynthesis in Escherichia coli Martin Bühning§, Angelo Valleriani¶ and Silke Leimkühler§* §

Institute of Biochemistry and Biology, University of Potsdam, D-14476 Potsdam, Germany,

¶

Department of Theory and Bio-Systems, Max Planck Institute of Colloids and Interfaces, Potsdam 14476, Germany

For Table of Contents Use Only


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Figure 1 175x171mm (300 x 300 DPI)


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Figure 3 175x170mm (300 x 300 DPI)


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Figure 5 105x121mm (300 x 300 DPI)


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Figure 7 133x119mm (300 x 300 DPI)


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Figure 9 145x93mm (300 x 300 DPI)


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