Báo cáo khoa học: A novel ATP-binding cassette transporter is responsible for resistance to viologen herbicides in the cyanobacterium Synechocystis sp. PCC 6803

A novel ATP-binding cassette transporter is responsible for resistance to viologen herbicides in the cyanobacterium Synechocystis sp PCC 6803 Jana Prosecka1,2, Artem V Orlov3, Yuri S Fantin3, Vladislav V Zinchenko3, Michael M Babykin4 and Martin Tichy1,2 Department of Autotrophic Microorganisms, Institute of Microbiology, Trebon, Czech Republic Institute of Physical Biology, University of South Bohemia, Nove Hrady, Czech Republic Department of Genetics, Moscow State University, Russia International Biotechnological Centre, Moscow State University, Russia Keywords ABC-type transporter; cyanobacteria; DUF990 family proteins; oxidative stress; viologen herbicide resistance Correspondence M Tichy, Laboratory of Photosynthesis, Institute of Microbiology, Opatovicky mlyn, Trebon 379 81, Czech Republic Fax: +400 384 340415 Tel: +400 384 340433 E-mail: tichym@alga.cz (Received 23 January 2009, revised 18 May 2009, accepted 22 May 2009) doi:10.1111/j.1742-4658.2009.07109.x The charged quaternary ammonium compounds – methyl, ethyl and benzyl viologens – generate reactive oxygen species in photosynthetic cells Three independent methyl viologen-resistant spontaneous mutants of Synechocystis sp PCC 6803 were identified, in which the conserved R115 residue of the Slr1174 protein was replaced with G115, L115 and C115 The Slr1174 protein of the DUF990 family is related to the permease subunit of an ABC-2-type transporter and its R115 mutation was found to be solely responsible for the observed methyl viologen resistance Bioinformatic analysis showed that in various bacterial genomes, two genes encoding another permease subunit and the ATPase component of an ATP-binding cassette transporter form putative operons with slr1174 orthologs, suggesting that the protein products of these genes may form functional transporters The corresponding genes in Synechocystis sp PCC 6803, i.e slr0610 for the permease and slr1901 for the ATPase, did not form such an operon However, insertional inactivation of any slr1174, slr0610 or slr1901 genes in both the wild-type and the R115-resistant mutant resulted in increased sensitivity to methyl, ethyl and benzyl viologens; moreover, single- and double-insertion mutants did not differ in their viologen sensitivity Our data suggest that Slr1901, Slr1174 and Slr0610 form a heteromeric ATP-binding cassette-type viologen exporter, in which each component is critical for viologen extrusion Because the greatest increase in mutant sensitivity was observed in the case of ethyl viologen, the three proteins have been named EvrA (Slr1901), EvrB (Slr1174) and EvrC (Slr0610) This is the first report of a function for a DUF990 family protein Introduction Molecular oxygen is essential for most organisms However, during aerobic respiration or oxygenic photosynthesis, reactive oxygen species, including the superoxide anion radical (O2)), are formed In the photosynthetic electron transport chain, instead of NADP, O2 may accept an electron from the reduction Abbreviations ABC, ATP-binding cassette; BV, benzyl viologen; DQ, diquat; EV, ethyl viologen; MATE, multidrug and toxic compounds extrusion; MDT, multidrug transporter; MFS, major facilitator superfamily; MV, methyl viologen; NBD, nucleotide binding domain; SMR, small multidrug resistance; TMD, transmembrane domain; TMH, transmembrane helices FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4001 Viologen ABC transporter in Synechocystis J Prosecka et al side of photosystem I to form O2) [1] This so-called Mehler reaction [2] becomes a major electron-transfer route in the presence of the redox cycling agent methyl viologen (MV) This nonselective herbicide, also known as paraquat (1,1-dimethyl-4,4¢-bipyridinium dichloride), is a charged quaternary ammonium compound that generates O2) under aerobic conditions by cyclic univalent reduction and reoxidation [3] As such, MV is commonly used to mimic and magnify the oxidative stress that cells normally encounter during photosynthesis and respiration Organisms usually cope with MV by impairing MV uptake [4,5] or enhancing MV efflux from the cell [6–12], both of which lower the intracellular MV concentration Drug efflux pumps (also known as drug efflux carriers, drug transporters or exporters) are believed to play a major role in cell resistance to various toxic agents in all domains of life Of the more than 900 known families of transport systems, at least 35 include toxic ion and drug efflux carriers Moreover, in Gram-negative bacteria with two cellular membranes, auxiliary transport proteins, such as membrane fusion proteins and outer membrane channels (b-barrel porins; subclass 1.B), may be involved in the extrusion of antibiotics and other toxic compounds (Transporter Classification Database, TCDB: http:// www.tcdb.org/) [13,14] Although some efflux pumps are selective for a given substrate, many transporters are able to extrude a plethora of structurally unrelated drugs, conferring the organism with a multidrugresistance phenotype [14,15] On the basis of bioenergetic and structural criteria, multidrug transporters (MDTs) can be divided into two major classes: (a) primary ATP-binding cassette (ABC)-type MDTs that use the free energy of ATP hydrolysis to pump drugs out of the cell, and (b) secondary MDTs that utilize the transmembrane electrochemical gradient of protons or sodium ions to extrude drugs from the cell [13,16–20] The majority of MDTs identified and characterized to date are energized by the proton motive force (secondary transporters) Depending on their size, similarity in primary structure and topology, they can be divided into four main transporter families or superfamilies [21]: major facilitator superfamily (MFS) [22], small multidrug resistance (SMR) [23], resistance-nodulation-cell division [24], and multidrug and toxic compound extrusion (MATE) [25] ABC transporters generally have two similar halves, each of which contains two parts – a hydrophobic transmembrane domain (TMD) and a nucleotide-binding domain (NBD) Two TMDs form a passageway for cargo, and two NBDs located in the cytoplasm bind and hydrolyze ATP In most cases, 4002 eukaryotic ABC exporters are expressed with all four domains in a single TMD ⁄ NBD ⁄ TMD ⁄ NBD structure [26] In bacteria, one gene can encode a protein with a single combined TMD ⁄ NBD to generate the functional homodimer, or two genes may separately encode NBD and TMD Moreover, the TMD ⁄ TMD and NBD ⁄ NBD structures are described as components of fullsize ABC transporters [27] Recently, several bacterial genes have been isolated that encode transporters conferring MV resistance These include emrB of Escherichia coli, smvA of Salmonella enterica [6], pqrB of Streptomyces coelicolor [7] and pqrA of Ochrobactrum anthropi [8,9], all of which encode MFS proteins Another example is the E coli emrE (or mvrC) gene which encodes a protein belonging to the SMR transporter family; this family is restricted to prokaryotic cells, and its members are the smallest multidrug efflux pumps with only four helices and no significant extramembrane domain [10] It has been shown that the SMR exporter YddG of S enterica extrudes MV in cooperation with OmpD porin, whereas the less abundant TolC porin is involved in MV import into the cell [5] In general, more than half of the known SMR proteins can export MV [27] Full genome analysis of the cyanobacterium Synechocystis sp PCC 6803 (hereafter Synechocystis sp.) has revealed several putative multidrug resistance exporters (http://www.membranetransport.org): five TMDs of ABC transporters; and five, three and two proteins from the MFS, resistance-nodulation-cell division and MATE families, respectively It is surprising that the complement of MFS transporters in Synechocystis sp is much fewer than the 70 found in E coli Moreover, no putative MV efflux pumps were found based on homology to known MV transporters A search for spontaneous MV-resistant Synechocystis sp mutants led to the identification of the MATE family PrqA protein, which functions as an MV exporter The prqA gene is cotranscribed with prqR, which encodes a TetR-like repressor protein A point mutation in prqR affects the putative DNA-binding domain of the PrqR repressor and results in derepression of the prqRA operon, leading to increased MV resistance [11,12] Our search for new MV-resistant mutants of Synechocystis sp with mutations that not map within the prqR gene yielded three MV-resistant strains Each of these carries a different substitution of a single amino acid residue in the Slr1174 permease protein that represented the TMD of an ABC transporter A bioinformatics search revealed two potential structural partners of this TMD, i.e Slr0610 and Slr1901 (another TMD and NBD), which could form a functional ABC-type viologen exporter The existence of FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS J Prosecka et al A Log D 750 this heteromeric exporter in Synechocystis sp was confirmed by insertional mutagenesis in which inactivation of each subunit in the MV-resistant mutant led to the MV-sensitive phenotype In this study, we demonstrate that even the native exporter is involved in the efflux of MV and other quaternary ammonium herbicides, and document how a single amino acid substitution influences the substrate specificity of the transporter Because the greatest increase in mutant sensitivity was observed for ethyl viologen (EV), the three proteins were named EvrA (Slr1901), EvrB (Slr1174) and EvrC (Slr0610) Viologen ABC transporter in Synechocystis –0.2 μM –0.4 0.3 μM 0.6 μM –0.6 1.2 μM –0.8 –1 –1.2 –1.4 10 Results In a previous study, the MV-resistant phenotype of a Synechocystis sp Prq20 mutant was a result of elevated expression of a MATE family drug exporter encoded by the prqA gene, derepressed because of the L17Q substitution in the PrqR repressor of the TetR family [11,12] Also, the first MV-resistant mutant isolated in this study mapped to the prqR gene, leading to the Y52E substitution in PrqR The phenotype of this new Prq mutant was identical to that of the previously described Prq20 mutant, suggesting that both the L17Q and Y52E substitutions derepress the prqRA operon To identify new proteins providing protection against MV, two strategies were employed to avoid the characterization of other prqR mutants In the first approach, the prqR gene from the MV-resistant strain was amplified by PCR and examined for its ability to transform wild-type cells into MV-resistant ones The second approach was based on the generation of MVresistant mutants in a DprqA background Using these approaches, we isolated three independent spontaneous mutants with increased resistance to MV; two in the wild-type background and one in the DprqA background Surprisingly, in all three MV-resistant mutants, the mutation was localized in a single gene, slr1174, which has been renamed evrB Moreover, sequencing of evrB from all three mutants revealed that the three different point mutations replaced the same amino acid (R115) in the EvrB protein with three different amino acids, i.e G115, L115 and C115, respectively To transfer the C115 mutation from the DprqA background into the wild-type background, PCR-amplified evrB of the C115 strain was used to transform wild-type cells The point evrB MV-resistant mutants have been named EvrB–R115G, EvrB–R115L and EvrB–R115C 30 20 Time (h) 30 B Prq20 EvrB-R115L –0.2 WT Log D 750 Mapping of the MV-resistant mutants 20 Time (h) –0.4 ΔevrC –0.6 EvrB-R115L/ΔevrA ΔevrB –0.8 –1 –1.2 –1.4 10 Fig Growth curves of wild-type and mutant strains in the presence of MV (A) Growth of the wild-type at different MV concentrations (B) Growth of the wild-type and mutants in the presence of 0.6 lM MV All three substitutions in EvrB provided a similar level of protection to MV in three different sensitivity assays: doubling time in the presence of MV (Fig 1), MV disk-diffusion assay (Fig 2) and threshold MV concentration (Table 1) (only results for EvrB–R115C are shown) This resistance was comparable with that of the Prq20 mutant with the derepressed MATE-type exporter PrqA Wild-type cells grew poorly in the presence of 0.6 lm MV, and no growth impairment was apparent in the EvrB–R115C and Prq20 mutants at the same MV concentration (Fig 1) In the disk-diffusion assays, at least 10-fold higher concentrations of MV were needed to obtain the same level of growth inhibition (Fig 2) In EvrB–R115C, a 25-fold higher threshold concentration of MV was needed to inhibit growth on plates (Table 1) The fact that EvrB–R115C and Prq20 can tolerate a much higher MV concentration than the wild-type without inducing the cell stress response was also FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4003 Viologen ABC transporter in Synechocystis J Prosecka et al WT 0.1 50 0.5 25 20 10 EvrB protein Prq20 EvrB-R115C organic peroxide or MV [28–30] In the wild-type, we detected a 10–15-fold increase in the slr1544 transcript level after treatment with lm MV for 20 No such increase in the slr1544 transcript level was observed in the EvrB–R115C and Prq20 mutants after the same treatment, indicating that, unlike the wildtype, mutant cells were not stressed Fig The disk-diffusion assay was used to compare the sensitivity of the wild-type, EvrB–R115C and Prq20 mutants to MV Aliquots (0.1 mL) of a particular exponential culture (adjusted to D750 = 0.5) were spread on BG11–agar plates, grown for one day, treated with lL of MV (at concentrations of 0.1, 0.5, 1, 5, 10, 15, 20, 25 and 50 lM) and incubated for days demonstrated by following expression of the slr1544 gene, which is transiently induced by various stresses such as high light, low temperature, high salinity, The EvrB protein is annotated as a hypothetical protein of the DUF990 family of functionally uncharacterized proteins that are related to the permease subunit of ABC-2-type transporters [31] This indicates that EvrB may be a component of an ABC transporter involved in MV transport To determine whether the observed MV resistance was caused by increased MV export or impaired import, the evrB gene was inactivated in the wild-type background (Fig S1) The resulting DevrB strain did not exhibit any obvious alteration in phenotype with respect to its growth rate or pigment content However, it exhibited moderately increased sensitivity to MV, which was reflected in a decreased MV threshold concentration in comparison with the wild-type (Table 1) evrB inactivation in the EvrB–R115C mutant background led to the same MV sensitivity as in the DevrB, indicating that the R115 substitution in EvrB is solely responsible for the elevated MV resistance in EvrB–R115C (Fig and Table 1) This suggested that EvrB is involved in MV efflux in the wild-type and that this efflux is greatly enhanced in R115 point mutants Table Threshold concentrations of methyl viologen (MV), ethyl viologen (EV) and benzyl viologen (BV) for wild-type, EvrB–R115C point mutant and mutants with insertion inactivation of the evrA, evrB and evrC genes Threshold concentrations were determined by serial dilutions as described in Experimental procedures Threshold concentration (lM) MV EV BV 3.9 97 2.3 2.3 2.3 2.9 2.9 2.9 2.3 97 97 134 134 0.8 0.8 0.8 1.1 1.1 1.1 0.8 134 134 4.9 4.9 0.1 0.1 0.1 0.2 0.2 0.2 0.1 4.9 4.9 Strain wild-type EvrB–R115C DevrA DevrB DevrC EvrB–R115C ⁄ DevrA EvrB–R115C ⁄ DevrC DevrA ⁄ DevrB DevrB ⁄ DevrC EvrB–R115C ⁄ evrA+ EvrB–R115C ⁄ evrC+ 4004 FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS J Prosecka et al The inhibitory effect of MV on photosynthetic organisms involves accepting electrons from photosystem I, followed by reduction of oxygen to the superoxide anion radical, leading to oxidative damage to various cellular components To address the possible role of EvrB in the adaptive response of cyanobacterium to oxidative stress, we followed evrB expression after treatment with lm MV The treatment did not influence the relative transcript abundance of evrB, indicating the absence of a link between evrB expression and oxidative stress caused by MV Putative EvrABC transporter Because most ABC transporters consist of four domains – two TMDs and two NBDs – the EvrB may require protein partner(s) to form an active ABC-type MV exporter The STRING search tool was used to retrieve possible interacting genes ⁄ proteins [32] This tool predicted two potential functional partners of EvrB, Slr0610 (renamed EvrC) and Slr1901 (renamed EvrA), which had unknown functions Interestingly, EvrC represents another putative permease of the DUF990 family, whereas EvrA is a putative ATPbinding protein of an ABC transporter Advanced membrane topology algorithms based on a combination of a cascade-neural network and hidden Markov models using the evolutionary information from multiple sequence alignments predicted the presence of six or seven a-helical transmembrane helices (TMH) for EvrB (not shown) It is proposed that the protein is embedded in the cytoplasmic membrane with the N-terminus located inside the cell The six-TMH structure was also predicted for EvrC Based on this, there would be 12 TMH in the EvrB–EvrC heteromeric transporter, in agreement with the canonical ABC exporter topology Functional analysis of the genes encoding the heteromeric ABC-type exporter EvrABC If all three components, EvrA, EvrB and EvrC, are necessary for building the functional EvrABC transporter, inactivation of any of the subunits should result in a loss of drug efflux function and an increase in mutant sensitivity Moreover, no increase in mutant sensitivity should be observed after consecutive inactivation of an additional subunit To confirm the existence of the hypothetical MV exporter and to elucidate its role in Synechocystis sp., functional analysis of genes encoding putative transporter components was carried out Therefore, the evrC and evrA genes were inactivated by insertions in the wild-type background Viologen ABC transporter in Synechocystis and in mutants with increased and decreased MV resistance – EvrB–R115C and DevrB The resulting DevrC and DevrA strains did not exhibit any obvious alteration in the phenotype As mentioned above, a point mutation in the R115 evrB mutants resulted in increased cell resistance to MV Remarkably, such mutations did not influence the resistance to other diquaternary ammonium herbicides that are structurally similar to MV such as EV and benzyl viologen (BV) (Table 1) However, inactivation of evrB in the wild-type background increased the relative sensitivity to these compounds by 160- and 50-fold, respectively This is a much more pronounced increase than that observed with MV (Table 1), indicating lower affinity of the wild-type EvrB for MV Inactivation of evrC and evrA, each of which encodes two putative EvrABC exporter subunits, decreased the resistance to all three viologens to similar levels in both the wild-type and EvrB–R115C backgrounds (Table 1) As in the case of the DevrB mutant, the slightly enhanced MV sensitivity of the DevrA and DevrC strains was strikingly different from their EV or BV sensitivity, which again increased by 160- and 50-fold, respectively, relative to the wild-type strain Moreover, no significant differences in viologen sensitivity were detected among the single- and double-insertion mutants, i.e DevrA, DevrB, DevrC, EvrB–R115C ⁄ DevrA, EvrB–R115C ⁄ DevrC, DevrA ⁄ DevrB and DevrB ⁄ DevrC (Table 1) To prove that evrA and evrC inactivations are responsible for the observed sensitive phenotypes, the EvrB–R115C ⁄ DevrA and EvrB–R115C ⁄ DevrC mutants were complemented by intact evrA and evrC from the recombinant plasmids, yielding EvrB–R115C ⁄ evrA+ and EvrB– R115C ⁄ evrC+ strains The complemented strains exhibited the same viologen resistance as the EvrB– R115C mutant (Table 1) The subunit inactivation data suggest that the EvrA (NBD), EvrB (TMD) and EvrC (another TMD) proteins form a heteromeric ABC-type viologen exporter in which each component is necessary for viologen extrusion Most of the known MV exporters belong to a heterogeneous group of multidrug efflux pumps that confer resistance to a variety of structurally unrelated compounds [27] In order to determine the substrate specificity of the EvrABC exporter, the wild-type, EvrB–R115C strain and insertion mutants with inactivated exporter subunits were tested using the disk-diffusion assay for growth in the presence of an oxygen stressor (hydrogen peroxide), superoxide generators (pyrogallol, menadione and duroquinone) and quaternary ammonium compounds (acriflavine, ethidium bromide and diquat) Diquat (DQ) was used because it FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4005 Viologen ABC transporter in Synechocystis J Prosecka et al represents the 2,2¢-bipyridine derivative that is structurally related to viologens, which are 4,4¢-bipyridines No difference was observed in the sensitivity of the tested strains to this set of toxic compounds, indicating that the substrate specificity of the EvrABC exporter is probably rather narrow, limited to particular viologens Different substrate specificity was observed for the MATE-type exporter PrqA derepressed in the Prq20 mutant Here, we observed 30-fold increase in the resistance to both MV and DQ, but only twofold increase in the resistance to BV in comparison with the wild-type Discussion The novel ABC transporter EvrABC A novel ABC-type drug exporter that confers resistance to the diquaternary ammonium herbicides MV, EV and BV in Synechocystis sp was identified by the insertional inactivation of genes encoding the putative exporter components This insertion always abolished the function of the transporter Two of the genes, evrB and evrC, encode different permease subunits (TMDs) from the DUF990 family of proteins of unknown function, and the third gene, evrA, encodes the ATP-binding subunit (NBD) of an ABC transporter Functional association of the evrA, evrB and evrC genes was predicted by the STRING database based on the observation that in various bacterial genomes, the genes encoding the orthologous counterparts of the EvrA, EvrB and EvrC proteins are organized in putative operons The gene organization in such operons varied considerably among organisms (Fig 3), suggesting functional relevance of these gene clusters Also, the tree view option of the pair-wise alignment in the NCBI BLASTP database search (http://blast.ncbi.nlm nih.gov/Blast.cgi) produced almost the same tree topologies for all three proteins (not shown), implying that they coevolved as a functional complex Among cyanobacteria, the two proteins from the DUF990 family were found in all strains with known genome sequences, with the exception of Thermosynechococ- cus elongatus It is interesting to note that the genes encoding the three transporter subunits, which form operons in most organisms, are dispersed all over the Synechocystis sp genome Apparently, in Synechocystis sp., most genes of the ancestral operons are scattered throughout the chromosome, possibly because of the presence of abundant repetitive elements that can lead to genome rearrangements [33,34] By selecting spontaneous MV-resistant mutants, one can generally expect two types of mutations In the first type, the ability to transport or metabolize MV is altered, whereas in the second type, the ability to deal with reactive oxygen species produced by MV is improved Surprisingly, only mutants affecting MV transport were found in numerous studies [5–10] In Synechocystis sp., two classes of mutants with increased resistance to MV were found The first class of mutants mapped onto the prqR gene, encoding a regulator from the TetR family that controls expression of the MATE-type exporter PrqA [11,12] The second class of mutants characterized in this study mapped onto the evrB gene, encoding a subunit of an ABC transporter Also, for evrB R115 mutants, enhanced MV export is the most likely explanation for the observed MV resistance We believe that the circumstantial evidence that evr genes encode a true viologen exporter is rather convincing, although we cannot exclude some indirect effects of the EvrABC transporter on viologen resistance The observed viologen resistance was not caused by impaired viologen import because inactivation of any of the evrA, evrB or evrC genes resulted in a decrease and not an increase in viologen resistance Unlike the wild-type, evrB R115 mutants were not stressed in the presence of MV, suggesting that there was a low intracellular concentration of superoxide anion radical in the mutant cells However, resistance to the superoxide anion radical itself did not change in any null Devr mutant, excluding a direct role of the transporter in superoxide resistance Finally, the different sensitivity of the wild-type and the R115 evrB mutants for structurally and chemically similar viologens can be best Fig Gene organization in the evr cluster in various organisms The position and size of the genes encoding proteins that are most homologous to EvrA, EvrB and EvrC in different organisms are shown Note that the genes tend to cluster together despite changes in the gene order or in the gene orientation (in Thermus thermophilus) The disconnected genes are present at different locations of the genome 4006 FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS J Prosecka et al explained by direct interaction of viologens with the EvrABC transporter Role of R115 in the substrate selectivity of EvrB permease Interestingly, the R115 residue replaced by G115, L115 or C115 in EvrB, is highly conserved in the cyanobacterial cluster despite the lower overall EvrB homology (36% identity) There was only one conservative replacement for K115 found in Trichodesmium erythraeum Moreover, a similar conserved R116 residue is observed at the same location in the EvrC protein This suggests a potential functional importance of this amino acid Irrespective of the six- or seven-TMS EvrB model, the conserved R115 is located on the cytoplasmic side of the third TMS It is probable that the R115 substitutions in the EvrB of the MV-resistant mutants influence the substrate specificity of the transporter In all three reported R115 substitutions (R115G, R115L and R115C), a positively charged arginine residue was replaced, which resulted in the same mutant phenotype, although the properties of the three amino acids are quite different: glycine is tiny, whereas leucine is large and hydrophobic and cysteine is polar It seems that there is no strict requirement for a particular amino acid at position 115 and that removal of the positively charged arginine from the cytoplasmic side of the EvrABC exporter is required to increase the specificity or processivity for MV DQ, a structural analog of viologens, did not seem to be transported by the native EvrABC transporter This is different from the Prq20 MV-resistant mutant, which was also partially characterized in this study Here DQ and MV, but not BV, were efficiently removed by MATE-type exporter PrqA High selectivity and a large difference in the transport efficiency of two closely related substrates is unusual among exporters of quaternary ammonium compounds, with members of the MFS family generally conferring a broader resistance phenotype than members of the SMR family [35] All of these results are related to secondary MDTs that utilize the transmembrane electrochemical gradient to drive the export There are no data on the involvement of ABC transporters in the transport of diquaternary ammonium compounds to date DUF990 family proteins The DUF990 family is formed by two groups of proteins with the same predicted secondary structure, but with weak mutual homology Interestingly, in most organisms, there is a single protein from each group, Viologen ABC transporter in Synechocystis indicating that both groups may originate from an ancient duplication event For example, protein FN0879 from Fusobacterium nucleatum is paralogously related to FN0881 (E-value = 9e)10) in the same putative operon and is also significantly homologous to both EvrB (E-value = 1e)14) and EvrC (E-value = 4e)7) Currently, DUF990 family proteins have been identified in 135 species across the bacterial kingdom, implying their potential functional importance [31] No function has yet been assigned to transporters containing DUF990 proteins; however, based on the much higher conservancy observed among NBDs than TMDs, EvrA belongs to the ABC-2 subfamily of NBD components of bacterial ABC transport systems [36] Of the proteins from this subfamily whose functions are known, the EvrA protein is most similar to NatA and DrrA NatA is an NBD component of the NatAB system catalyzing ATP-dependent Na+ extrusion [37] and DrrA is a part of the DrrAB bacterial exporter that confers resistance to the antibiotics daunorubicin and doxorubicin [38] Regarding the function of the EvrABC transporter in Synechocystis sp and other (cyano)bacteria, it is questionable whether its native function is to export viologens, because viologens are not normally present in bacterial cells However, we believe that the information obtained from model viologen transport may help us to establish the true role of the EvrABC transporter and determine its natural substrate Altogether, our data suggest the existence of a new heteromeric ABC-type drug exporter that, to our knowledge, is the first ABC transporter shown to be involved in the export of diquaternary ammonium viologen herbicides This is also the first time that a function has been associated with the widely distributed putative permease subunit from the DUF990 family Experimental procedures Growth conditions and generation of spontaneous mutants Wild-type and mutants of Synechocystis sp were grown autotrophically in liquid BG11 medium supplemented with TES buffer (10 mm, pH 7.8) at 30 C and at a light intensity of 40 lmolỈm)2Ỉs)1 Solid media were supplemented with 1% agar Measurements of autotrophic growth rates were performed in microtitre plates (culture volume 0.25 mL) with intense shaking (900 rpm) Attenuance values at 750 nm were measured using a microplate reader (Tecan Sunrise, Vienna, Austria) The values plotted against time were used to calculate the doubling time Spontaneous MV-resistant mutants were obtained by consecutive cultivation of the FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4007 Viologen ABC transporter in Synechocystis J Prosecka et al wild-type or DprqA strain [12] in liquid media, with increasing MV concentrations from 0.5 to 10 lm After plating on BG11–agar containing 20 lm MV, single colonies were picked and grown on plates containing up to 60 lm MV All MV-resistant mutants were maintained in media supplemented with 20 lm MV Sensitivity to MV and other toxic compounds The disk-diffusion assay used to estimate the sensitivity of the wild-type and mutant strains to various toxic compounds was a modification of standard methods [39,40] An 0.1-mL aliquot of a particular exponential culture (adjusted to D750 = 0.5) was spread on a BG11–agar plate, grown for day, treated with lL of a solution containing an appropriate concentration of the test compound and incubated for days The threshold concentrations of MV, BV and EV for each strain were determined by serial dilution The exponential liquid cultures were diluted to D750 = 0.2 and further diluted 10-fold to D750 = 0.02 and D750 = 0.002 A 3-lL aliquot from each dilution was spotted on BG11–agar plates containing different concentrations of the inhibitor The lowest concentration of MV, BV or EV that inhibited cell growth at all three dilutions was defined as the threshold concentration The values reported here are representative of those obtained in three independent experiments Mapping of MV-resistant mutants Spontaneous MV-resistant mutants were mapped as described previously [41,42] Briefly, the isolated chromosomal DNA of a MV-resistant mutant was digested with a set of restriction enzymes The restriction fragments were fractionated by agarose gel electrophoresis and used to transform the wild-type cells The transformed cells were plated on BG11 plates covered with nitrocellulose filters (0.85 lm) After days, the filters were transferred to plates containing 20 lm MV The filter was subsequently transferred to plates containing 40 and 60 lm on the eighth and tenth day, respectively The sizes of the restriction fragments yielding MV-resistant transformants were compared with an ordered database of chromosomal restriction fragments and yielded putative chromosome fragments carrying the resistance mutation The genome region containing the resistance mutation was determined as the intersection of these sets of putative transforming fragments resulting from individual restriction digests using the vhelper restriction analysis program (http://orlovsergei.com/Progs/VH/VH.zip) Mutant construction The DNA fragments carrying the evrA, evrB and evrC genes were amplified from the Synechocystis sp wild-type genomic DNA by PCR using the following primers: 4008 for evrA, 5¢-TTTGTCAGGTCAGTCGGGTGATG-3¢ and 5¢-CTGGAGCCGGTTTCTCGTAGTTTG-3¢; for evrB, 5¢-GCCAACGGGAAGAAGCCAAGAC-3¢ and 5¢-TTGCCGGATATCAAAGCCCAAG-3¢; and for evrC, 5¢-TGATCCTTTACCTGTGGCCCTGAC-3¢ and 5¢-GCCGCCCTTGACTGAACTTTG-3¢ The evrA gene was cloned into pTZ18R (Pharmacia, Uppsala, Sweden) as a whole 1.7 kb PCR fragment, whereas evrB and evrC were cloned as 1.4 kb SmaI–SalI and 2.0 kb DraI–Ecl136II fragments, respectively The evrA gene was interrupted by insertion of a 1.3 kb kanamycin-resistance cassette For the evrB interruption, a portion of gene between nucleotides 210 and 584 was removed by inverse PCR (divergent primers 5¢-GAC CTTTCCCGACGCAG-3¢ and 5¢-CGCACCACAAAGA CAGC-3¢) and replaced with a 1.8 kb spectinomycin-resistance cassette The evrC gene was interrupted by insertion of a 0.9 kb gentamycin-resistance cassette The resulting plasmids were transformed into Synechocystis wild-type cells to generate single Devr mutants To generate double mutants, the plasmids carrying DevrA and DevrC constructs were transformed into the EvrB–R115C point mutant and into the DevrB insertion mutant The transformants were selected and segregated on plates containing the appropriate antibiotic (up to lgỈmL)1 gentamycin, 20 lgỈmL)1 spectinomycin or 50 lgỈmL)1 kanamycin) The genotypes of the single- and double-insertion mutants were confirmed by PCR analysis (data not shown) The relevant chromosomal regions of the Devr mutants are shown in Fig S1 Gene expression, RNA isolation, reverse transcription and quantitative PCR Wild-type and mutants growing exponentially were treated with lm MV when D750 = 0.5 and harvested at 0, 20 min, h and h A 5-mL aliquot of Synechocystis sp cells at D750 = 0.5 was cooled on ice, and the cells were harvested by centrifugation at 3000 g for at C The pellets were immediately frozen in liquid nitrogen, and the samples were stored at )75 C The total RNA was extracted using the modified hot phenol method [43] Briefly, frozen cells were thawed on ice and washed twice with the resuspension buffer, lysed in SDS lysis buffer, heated twice and extracted with hot acid phenol and chloroform Total RNA was precipitated by LiCl and ethanol Twenty nanograms of purified RNA was used for cDNA synthesis using random primers and SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) Real timequantitative PCR was performed on the Rotor-Gene 3000 system using the iQ SYBR Green Supermix (BioRad, Hercules, CA, USA) Each quantitative PCR experiment was performed in duplicate for two independent RNA isolations from the same culture 16S rRNA was used as the reference Its level was found to be proportional to that of total RNA (estimated on an RNA agarose gel) under all FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS J Prosecka et al conditions The DDCt method was used to calculate the gene expression levels Secondary structure prediction of membrane proteins The web application server TRAMPLE, which is dedicated to the detection and annotation of transmembrane protein sequences, was used to predict the overall membrane topology and the ability to form TMHs [44] TRAMPLE provides different membrane topology algorithms based on a combination of a cascade-neural network and hidden Markov models The methods were tested on the recently published structure of the S aureus Sav1866 protein, which is a probable bacterial multidrug ABC transporter, and good results were obtained [45] Acknowledgements The authors are grateful to Prof Teruo Ogawa for providing the original evrA mutant and to Eva Prachova for technical assistance This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (project nos MSM6007665808 and ME 881) and by the Czech Academy of Sciences (AV0Z50200510) It was also supported by a grant from the Russian Foundation for Basic Research 07-04-00117 and the program ‘Leading Scientific Schools’ (RI-112 ⁄ 001 ⁄ 211) References Asada K (2000) The water–water cycle as alternative photon and electron sinks Phil Trans R Soc Lond B Biol Sci 355, 1419–1431 Mehler AH (1951) Studies on reactions of illuminated chloroplasts I Mechanism of the reduction of oxygen and other Hill reagents Arch Biochem 33, 65–77 Hassan HM & Fridovich I (1978) Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli J Biol Chem 253, 8143–8148 Kao SM & Hassan HM (1985) Biochemical characterization of a paraquat-tolerant mutant of Escherichia coli J Biol Chem 260, 10478–10481 Santiviago CA, Fuentes JA, Bueno SM, Trombert AN, Hildago AA, Socias LT, Youderian P & Mora GC (2002) The Salmonella enterica sv Typhimurium smvA, yddG and ompD (porin) genes are required for the efficient efflux of methyl viologen Mol Microbiol 46, 687– 698 Hongo E, Morimyo M, Mita K, Machida I, HamaInaba H, Tsuji H, Ichimura S & Noda Y (1994) The methyl viologen-resistance-encoding gene smvA of Salmonella typhimurium Gene 148, 173–174 Viologen ABC transporter in Synechocystis Cho YH, Kim EJ, Chung HJ, Choi JH, Chater KF, Ahn BE, Shin JH & Roe JH (2003) The pqrAB operon is responsible for paraquat resistance in Streptomyces coelicolor J Bacteriol 185, 6756–6763 Jo J, Won SH, Son D & Lee BH (2004) Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene Biotechnol Lett 26, 1391–1396 Won SH, Lee BH, Lee HS & Jo J (2001) An Ochrobactrum anthropi gene conferring paraquat resistance to the heterologous host Escherichia coli Biochem Biophys Res Commun 285, 885–890 10 Lomovskaya O & Lewis K (1992) emr, an Escherichia coli locus for multidrug resistance Proc Natl Acad Sci USA 89, 8938–8942 11 Babykin MM, Sidoruk KV, Zinchenko VV, Nefedova LN, Cerff R & Shestakov SV (2003) On the involvement of the regulatory gene prqR in the development of resistance to methyl viologen in cyanobacterium Synechocystis sp PCC6803 Genetika 39, 25–32 12 Nefedova LN, Fantin Iu S, Zinchenko VV & Babykin MM (2003) The prqA and mvrA genes encoding carrier proteins control resistance to methyl viologen in the cyanobacterium Synechocystis sp PCC6803 Genetika 39, 336–340 13 Saier MH Jr (2000) A functional–phylogenetic classification system for transmembrane solute transporters Microbiol Mol Biol Rev 64, 354–411 14 Van Bambeke F, Glupczynski Y, Plesiat P, Pechere JC & Tulkens PM (2003) Antibiotic efflux pumps in prokaryotic cells: occurrence, impact on resistance and strategies for the future of antimicrobial therapy J Antimicrob Chemother 51, 1055–1065 15 Poole K (2005) Efflux-mediated antimicrobial resistance J Antimicrob Chemother 56, 20–51 16 Baranova NN & Neyfakh AA (1997) Apparent involvement of a multidrug transporter in the fluoroquinolone resistance of Streptococcus pneumoniae Antimicrob Agents Chemother 41, 1396–1398 17 De Rossi E, Branzoni M, Cantoni R, Milano A, Riccardi G & Ciferri O (1998) mmr, a Mycobacterium tuberculosis gene conferring resistance to small cationic dyes and inhibitors J Bacteriol 180, 6068–6071 18 Heir E, Sundheim G & Holck AL (1998) The Staphylococcus qacH gene product: a new member of the SMR family encoding multidrug resistance FEMS Microbiol Lett 163, 49–56 19 Littlejohn TG, Paulsen IT, Gillespie MT, Tennent JM, Midgley M, Jones IG, Purewal AS & Skurray RA (1992) Substrate specificity and energetics of antiseptic and disinfectant resistance in Staphylococcus aureus FEMS Microbiol Lett 74, 259–265 20 Paulsen IT, Brown MH & Skurray RA (1996) Protondependent multidrug efflux systems Microbiol Rev 60, 575–608 FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4009 Viologen ABC transporter in Synechocystis J Prosecka et al 21 Putman M, van Veen HW & Konings WN (2000) Molecular properties of bacterial multidrug transporters Microbiol Mol Biol Rev 64, 672–693 22 Marger MD & Saier MH Jr (1993) A major superfamily of transmembrane facilitators that catalyse uniport, symport and antiport Trends Biochem Sci 18, 13–20 23 Paulsen IT, Skurray RA, Tam R, Saier MH Jr, Turner RJ, Weiner JH, Goldberg EB & Grinius LL (1996) The SMR family: a novel family of multidrug efflux proteins involved with the efflux of lipophilic drugs Mol Microbiol 19, 1167–1175 24 Saier MH Jr, Tam R, Reizer A & Reizer J (1994) Two novel families of bacterial membrane proteins concerned with nodulation, cell division and transport Mol Microbiol 11, 841–847 25 Brown MH, Paulsen IT & Skurray RA (1999) The multidrug efflux protein NorM is a prototype of a new family of transporters Mol Microbiol 31, 394–395 26 Hollenstein K, Dawson RJ & Locher KP (2007) Structure and mechanism of ABC transporter proteins Curr Opin Struct Biol 17, 412–418 27 Borges-Walmsley MI, McKeegan KS & Walmsley AR (2003) Structure and function of efflux pumps that confer resistance to drugs Biochem J 376, 313–338 28 Hihara Y, Sonoike K, Kanehisa M & Ikeuchi M (2003) DNA microarray analysis of redox-responsive genes in the genome of the cyanobacterium Synechocystis sp strain PCC 6803 J Bacteriol 185, 1719–1725 29 Li H, Singh AK, McIntyre LM & Sherman LA (2004) Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp strain PCC 6803 J Bacteriol 186, 3331–3345 30 Murata N & Suzuki I (2006) Exploitation of genomic sequences in a systematic analysis to access how cyanobacteria sense environmental stress J Exp Bot 57, 235– 247 31 Finn RD, Tate J, Mistry J, Coggill PC, Sammut SJ, Hotz HR, Ceric G, Forslund K, Eddy SR, Sonnhammer EL et al (2008) The Pfam protein families database Nucleic Acids Res 36, D281–D288 32 von Mering C, Jensen LJ, Kuhn M, Chaffron S, Doerks T, Kruger B, Snel B & Bork P (2007) STRING 7– recent developments in the integration and prediction of protein interactions Nucleic Acids Res 35, D358–D362 33 Fujibuchi W, Ogata H, Matsuda H & Kanehisa M (2000) Automatic detection of conserved gene clusters in multiple genomes by graph comparison and P-quasi grouping Nucleic Acids Res 28, 4029–4036 34 Itoh T, Takemoto K, Mori H & Gojobori T (1999) Evolutionary instability of operon structures disclosed by sequence comparisons of complete microbial genomes Mol Biol Evol 16, 332–346 35 Anthonisen IL, Sunde M, Steinum TM, Sidhu MS & Sorum H (2002) Organization of the antiseptic resis- 4010 36 37 38 39 40 41 42 43 44 45 tance gene qacA and Tn552-related beta-lactamase genes in multidrug-resistant Staphylococcus haemolyticus strains of animal and human origins Antimicrob Agents Chemother 46, 3606–3612 Reizer J, Reizer A & Saier MH Jr (1992) A new subfamily of bacterial ABC-type transport systems catalyzing export of drugs and carbohydrates Protein Sci 1, 1326–1332 Cheng J, Guffanti AA & Krulwich TA (1997) A twogene ABC-type transport system that extrudes Na+ in Bacillus subtilis is induced by ethanol or protonophore Mol Microbiol 23, 1107–1120 Guilfoile PG & Hutchinson CR (1991) A bacterial analog of the mdr gene of mammalian tumor cells is present in Streptomyces peucetius, the producer of daunorubicin and doxorubicin Proc Natl Acad Sci USA 88, 8553–8557 Bauer AW, Kirby WM, Sherris JC & Turck M (1966) Antibiotic susceptibility testing by a standardized single disk method Am J Clin Pathol 45, 493–496 Buchmeier N, Bossie S, Chen CY, Fang FC, Guiney DG & Libby SJ (1997) SlyA, a transcriptional regulator of Salmonella typhimurium, is required for resistance to oxidative stress and is expressed in the intracellular environment of macrophages Infect Immun 65, 3725– 3730 Ermakova-Gerdes S & Vermaas W (1999) Inactivation of the open reading frame slr0399 in Synechocystis sp PCC 6803 functionally complements mutations near the Q(A) niche of photosystem II – a possible role of slr0399 as a chaperone for quinone binding J Biol Chem 274, 30540–30549 Tichy M & Vermaas W (2000) Combinatorial mutagenesis and pseudorevertant analysis to characterize regions in loop E of the CP47 protein in Synechocystis sp PCC 6803 Eur J Biochem 267, 6296–6301 Mohamed A & Jansson C (1989) Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803 Plant Mol Biol 13, 693–700 Fariselli P, Finelli M, Rossi I, Amico M, Zauli A, Martelli PL & Casadio R (2005) TRAMPLE: the transmembrane protein labelling environment Nucleic Acids Res 33, W198–W201 Dawson RJ & Locher KP (2006) Structure of a bacterial multidrug ABC transporter Nature 443, 180–185 Supporting information The following supplementary material is available Fig S1 Schematic representation of the procedure used for the insertional inactivation of the evrB (A), evrA (B) and evrC (C) genes in the Synechocystis genome FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS J Prosecka et al This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and Viologen ABC transporter in Synechocystis may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 4001–4011 ª 2009 The Authors Journal compilation ª 2009 FEBS 4011