Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans

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MODEL

Research in Microbiology xx (2017) 1e12 www.elsevier.com/locate/resmic

Original Article

Identification and characterization of the genes encoding carbon monoxide Q1 Q8 dehydrogenase in Terrabacter carboxydivorans Jae Ho Lee a,1, Sae Woong Park a,2, Young Min Kim a, Jeong-Il Oh b,*

Q7

b

a Department of Systems Biology, Yonsei University, Seoul 03722, Republic of Korea Department of Microbiology, Pusan National University, Busan 46241, Republic of Korea

Received 8 September 2016; accepted 20 January 2017 Available online ▪ ▪ ▪

Abstract Terrabacter carboxydivorans is able to grow aerobically at low concentrations of carbon monoxide (CO) as a sole source of carbon and energy. The genes for carbon monoxide dehydrogenase (CO-DH) were cloned from T. carboxydivorans and analyzed. The operon encoding T. carboxydivorans CO-DH was composed of three structural genes with the transcriptional order of cutB, cutC and cutA, as well as an additional accessory gene (orf4). Phylogenetic analysis of CutA revealed that T. carboxydivorans CO-DH was classified into a group distinct from previously characterized CO-DHs. Expression of antisense RNA for the cutB or cutA gene in T. carboxydivorans led to a decrease in CO-DH activity, confirming that cutBCA genes are the functional genes encoding CO-DH. The CO-DH operon was expressed even in the absence of CO and further inducible by CO. In addition, CO-DH synthesis was increased in the stationary phase compared to the exponential phase during heterotrophic growth on glucose and glycerol. Point mutations of a partially inverted repeat sequence (TCGGA-N6-GCCCA) in the upstream region of the cutB gene almost abolished expression of the CO-DH operon, indicating that the inverted-repeat sequence might be a cis-acting regulatory site for the positive regulation of the CO-DH operon. © 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Keywords: Carbon monoxide dehydrogenase; Carboxydobacteria; Terrabacter carboxydivorans

Q2

1. Introduction Carboxydobacteria include both carboxydotrophs and carboxydovores. Carboxydotrophic bacteria are a group of aerobic bacteria capable of growing chemolithotrophically with carbon monoxide (CO) as a sole source of carbon and energy using CO dehydrogenase (CO-DH) [1,2]. Carboxydovores are the bacteria that cannot grow with CO as the sole carbon and energy source but exploit CO mixotrophically as a supplementary

* Corresponding author. E-mail addresses: [email protected] (J.H. Lee), [email protected] (S.W. Park), [email protected] (Y.M. Kim), [email protected] (J.-I. Oh). 1 Present address: R&BD Center, Korea Yakult Co., Ltd., Yongin, Gyeonggi, Republic of Korea. 2 Present address: Department of Microbiology and Immunology, Weill Cornell Medical College, New York, NY 10065, USA.

energy source [3]. Studies on CO oxidation by bacteria were initiated by Kistner [4] and the isolation and identification of carboxydobacteria began in the early 1970s [5]. Many carboxydobacteria have been identified from Gram-negative proteobacteria and Gram-positive actinobacteria, including mycobacteria [6e16]. Although studies for carboxydobacteria have been performed mainly on carboxydotrophs grown at high concentrations of CO (>10%), CO oxidizers enriched with low concentrations of CO (40e400 p.p.m.) have been also reported [3,11,17]. Terrabacter carboxydivorans used in this study was also isolated from soil after enrichment with 400 p.p.m. of CO. This strain could utilize CO and grow at low concentrations of CO (400 p.p.m.), but did not grow at high concentrations of CO (30% COe70% air) [17]. The key enzyme for CO oxidation in aerobic carboxydobacteria is CO-DH, which catalyzes the oxidation of CO to CO2 using H2O as an oxidant [1,2]. Two electrons from CO

http://dx.doi.org/10.1016/j.resmic.2017.01.004 0923-2508/© 2017 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved. Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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oxidation are transferred to the CO-insensitive respiratory electron transport chain to generate proton motive force, and the produced CO2 is assimilated by the CalvineBensoneBassham cycle [1,2]. The CO-DH enzymes consist of three non-identical subunits with a quaternary structure of a2b2g2. The large subunit (a, catalytic subunit) is a molybdoprotein containing molybdopterin cytosine dinucleotide (MCD). The medium (b) and small (g) subunits are a flavoprotein with a molecule of flavin adenine dinucleotide (FAD) and an ironesulfur protein with two [2Fee2S] centers, respectively [18e20]. On the basis of phylogenetic analysis of large subunits of CO-DHs and the conserved active site motifs, grouping of CODHs into two clades was suggested by King [13]. While the CO-DHs belonging to the OMP (Oligotropha, Mycobacterium and Pseudomonas) group have the active site motif, AYRCSFR, those of the BMS (Bradyrhizobium, Mesorhizobium and Sinorhizobium) group contain AYRGAGR. The CO-DHs belonging to OMP and BMS groups are also referred to as form I and form II CO-DHs, respectively [3]. Recently, CO-DHs were classified into three groups (group I [mycobacterial CO-DHs], group II [Gram-negative bacterial CODHs] and group III [Gram-positive bacterial CO-DHs other than mycobacteria]) by Kim and Park [21], based on phylogenetic analysis on large subunits and immunological relatedness of CO-DHs. Especially, the mycobacterial CO-DHs have unique properties distinguished from other groups of CO-

DHs, in that the mycobacterial CO-DHs have no immunological relatedness with those of Gram-negative bacteria (not tested for Gram-positive bacterial CO-DHs) [22] and possess nitric oxide dehydrogenase activity [23]. In this study, we cloned the genes encoding CO-DH in T. carboxydivorans, and demonstrated that T. carboxydivorans contains the active form II CO-DH. Furthermore, we report the cis-acting regulatory sequences and the regulation of the CODH genes in T. carboxydivorans. 2. Materials and methods 2.1. Bacterial strains, plasmids and cultivation conditions The bacterial strains and plasmids used in this study are listed in Table 1. T. carboxydivorans was grown at 30
C in standard mineral base (SMB) medium [18] supplemented with 400 p.p.m. CO and 0.005% (w/v) yeast extract (SMB-CO), with 0.2% (w/v) glucose (SMB-glucose) or 0.2% (v/v) glycerol (SMB-glycerol), or in nutrient broth (NB). Mycobacterium sp. strain JC1 DSM 3803 and Oligotropha carboxidovorans were grown at 37
C in SMB medium with a gas mixture of 30% (v/v) COe70% (v/v) air (SMB-CO) or in SMB-glucose [8,24,25]. Hydrogenophaga pseudoflava was cultivated at 37
C in SMB medium with 0.2% (w/v) succinate (SMB-succinate) [9]. Escherichia coli strains were grown at

Table 1 Bacterial strains and plasmids used in this study. Strain or plasmid Strains Terrabacter carboxydivorans Mycobacterium sp. strain JC1 Oligotropha carboxidovorans Hydrogenophaga pseudoflava E. coli DH5a BL21 (DE3) Plasmids pBluescript II KS(þ) pCutBIRpm1 pCutBIRpm2 pCutBup192 pCutBup67 pET-11a pHisCutA pMV261LacZ pPL pRH1351 pTA pTAcutA pTAHisCutA pTE pTEaRcutA pTEaRcutB

Q6

Description

Reference or source

Wild Wild Wild Wild

[17] [8,25] [24] [9]

type type type type

supE44 lac16 (f80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 F ompT hsdS B(rB, mB) gal dcm (DE3)

[46] Promega

Cloning vector for E. coli; Ampr pTE::236-bp DNA fragment containing the cutB and orfZ intergenic region including point mutations at inverted repeat 1 pTE::236-bp DNA fragment containing the cutB and orfZ intergenic region including point mutations at inverted repeat 2 pTE::236-bp DNA fragment containing the cutB and orfZ intergenic region pTE::111-bp DNA fragment containing the cutB and orfZ intergenic region Expression vector for E. coli; His-tag; Ampr pET-11a::2432-bp DNA fragment containing the intact cutA 7570-bp plasmid which replicated in T. carboxydivorans; hsp60 promoter; lacZ; Kanr Promoterless vector derived from pTE; Genr E. coliemycobacterial shuttle vector; Genr PCR product cloning vector; Ampr pTA::1314-bp DNA fragment containing the partial cutA from T. carboxydivorans pTA::2439-bp DNA fragment containing the intact cutA 7515-bp plasmid derived from pMV261LacZ; hsp60 promoter; lacZ; Genr Antisense RNA experiment pTE::225-bp DNA fragment containing the 74-bp of the cutB and cutA intergenic region and 151-bp of the cutA Antisense RNA experiment, pTE::212-bp DNA fragment containing the 93-bp of the cutB and orfZ intergenic region and 119-bp of the cutB

Stratagene This study This study This study This study Stratagene This study [33] This study [32] RBC Bioscience This study This study This study This study This study

Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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37
C in Luria-Bertani medium (LB). E. coli DH5a was used as a host for all plasmid constructions. E. coli BL21 (DE3) was used for overexpression of CutA. Growth was measured spectrophotometrically by determining the turbidity of cultures at 600 nm (OD600). Ampicillin (50 mg/ml) or gentamicin (250 mg/ml for T. carboxydivorans and 30 mg/ml for E. coli) were added to culture medium, when required.

2.4. Enzyme assays

2.2. DNA manipulation and electroporation techniques

2.4.2. b-Galactosidase b-Galactosidase activity was spectrophotometrically assayed by determining the initial conversion rate of the substrate analog o-nitrophenyl-D-galactopyranoside (o-NPG) to o-nitrophenol at 420 nm and 30
C for 1 min as described previously [29].

Genomic DNA of T. carboxydivorans was isolated from cells grown in NB by the CTAB method [26]. Plasmids were isolated from E. coli by the alkaline lysis method [27]. T. carboxydivorans was transformed by electroporation in a 0.2 cm cuvette using the Gene Pulser™ apparatus (BIO-RAD) at 2.5 kV/cm, 25 mF and 1000 U. Competent T. carboxydivorans cells for electroporation were prepared as follows. T. carboxydivorans grown to the exponential phase (OD600 of 0.5e0.6) in 300 ml of SMB-glucose medium was harvested by centrifugation at 1600  g for 10 min at 4
C. Harvested cells were washed three times with ice-cold 10% (v/v) glycerol. Finally, T. carboxydivorans cells were resuspended in 3 ml of ice-cold 10% (v/v) glycerol and stored in aliquots at 80
C. 2.3. Cloning of CO-DH genes of T. carboxydivorans The degenerative PCR primers, BMS-F (50 -GGCGGCTT[C/ T]GG[C/G]TC[C/G]AAGAT-30 ) and O/B-R (50 -[C/T]TCGA[C/ T]GATCATCGG[A/G]TTGA-30 ), were used to amplify a part of the CO-DH large subunit (CutA) gene of T. carboxydivorans [13]. PCR was carried out using the genomic DNA of T. carboxydivorans as a template. The PCR product was cloned into pTA (RBC Bioscience), yielding the plasmid pTAcutA. Southern blot hybridization was performed using the PCR product labeled with [a-32P]dCTP as a probe; 4 mg of total DNA from T. carboxydivorans digested by NotI, XmaI, BglII, BamHI/PstI, or BglII/PstI for 10 h was subjected to agarose gel electrophoresis and then transferred to Hybond-Nþ membranes (Amersham) by using a semi-dry transfer apparatus (BIO-RAD). Hybridization and washing were carried out at 65
C following the manufacturer's instructions. DNA fragments around the gel regions where hybridization signals had been detected in Southern blot analysis were eluted from the agarose gel and purified with Megaquickspin™ kit (Intron Biotechnology). The purified fragments were ligated into pBluescript II KS(þ) vector to construct plasmid clone libraries. E. coli DH5a was transformed with these plasmid libraries and transformants were selected on LB plates for ampicillin resistance. All colonies grown on LB plates with ampicillin were screened by colony lift hybridization. Colony lift hybridization was performed using the same probe for Southern blot analysis. The clone libraries on LB plates were incubated for 1 h at 4
C and lifted by Hybond-Nþ membranes. The membranes holding colonies were soaked twice in denaturing solution (0.5 M NaOH and 1 M NaCl) for 5 min and neutralizing solutions (1 M TriseHCl, pH 7.4, 1 M NaCl) for 5 min. The membranes were fixed under UV illumination. Hybridization and washing were performed as described above.

2.4.1. CO-DH CO-DH activity was assayed spectrophotometrically by measuring the CO-dependent reduction of 2-(4-indophenyl)-3(4-nitrophenyl)-2H-tetrazolium chloride (INT; 3 496 ¼ 17.981 mM1 cm1) by the method described previously [28].

2.5. Activity staining of CO-DH Cell-free extracts of T. carboxydivorans were subjected to non-denaturing PAGE and staining of the gel by CO-DH activity was performed with 0.05% (w/v) phenazine methosulfate and 0.25% (w/v) nitroblue tetrazolium in 50 mM TriseHCl (pH 7.5) flushed with CO as described previously [1]. 2.6. Generation of antisera against T. carboxydivorans CutA A 2439-bp fragment containing the cutA gene was amplified using the primers, 50 -CATATGACGACGCAGATGT TCGGCGC-30 (the NdeI site is underlined) and 50 -GGATTC TGCGGTCGGCTCCTTCGA-30 (the BamHI site is underlined). The PCR product was cloned into pTA, yielding pTAHisCutA. The sequence of the cloned insert was verified by DNA sequencing. After digestion of pTAHisCutA with NdeI and BamHI, 2432-bp DNA fragments were purified and inserted into pET-11a restricted with the same enzymes to yield pHisCutA. pHisCutA was introduced into E. coli BL21 (DE3) and CutA expression was induced with 0.2 mM IPTG for 21 h at 20
C. The overexpressed His6-tagged CutA was purified by affinity chromatography using a Talon column (Clontech) according to the manufacturer's instructions. Purified CutA was sent to the company (Young-In Frontier) to raise its polyclonal antibody from a rabbit. 2.7. Phylogenetic analysis Phylogenetic analysis was performed by using the MEGA program (version 3.1) [30] after multiple alignment of the sequences of the CutA homologs using the CLUSTAL_X program [31]. The tree was generated by neighbor joining (Poisson correction model). The bootstrap values were calculated from 1000 replicates. 2.8. Expression of antisense RNAs for the CO-DH genes in T. carboxydivorans To express antisense RNAs for the cutA and cutB genes, an expression plasmid usable in T. carboxydivorans was developed

Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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as follows. A DNA fragment containing a gentamicin-resistant gene and its promoter was amplified from pRH1351 [32] using the primers, 50 -GCTAGCGATGATCGTGCCGTGATC-30 (the NheI site is underlined) and 50 -ACTAGTCCGATCTCGGCTTGAACG-30 (the SpeI site is underlined). The PCR product was cloned into pMV261LacZ [33] digested with NheI and SpeI to replace the kanamycin-resistant gene on pMV261LacZ by the gentamicin-resistant gene. The resulting vector pTE contains the constitutively active hsp60 promoter of Mycobacterium bovis BCG and the lacZ gene downstream of the promoter. pTEaRcutA and pTEaRcutB are the plasmids that were used for expression of antisense RNAs for cutA and cutB of T. carboxydivorans, respectively. For the construction of pTEaRcutA, a 225-bp DNA fragment containing the 50 portion of cutA and its Shine-Dalgarno sequence was amplified by PCR using the primers, 50 -CTGCAGATCGTCAAGAGCGTGCTG-30 (the PstI site is underlined) and 50 -GGATCCTGTCGACGATGCGG GCGT-30 (the BamHI site is underlined). The PCR product was cloned into pTE with cutA oriented in the opposite direction to the hsp60 promoter, yielding pTEaRcutA. The pTEaRcutB plasmid expressing the antisense RNA for cutB was constructed in the same way as pTEaRcutA, except for a 212-bp DNA fragment amplified by PCR using the primers 50 CTGCAGGGTCGTTCACGGCATAAG-30 (the PstI site is underlined) and 50 -GGATCCATCGGGATGAGGCTCTGA-30 (the BamHI site is underlined). 2.9. Construction of cutB::lacZ transcriptional fusion plasmids pCutBup192 is a lacZ transcriptional fusion plasmid that contains a 236-bp insert consisting of the 50 portion of cutB and the 192-bp upstream region of cutB. A DNA fragment containing the putative 35 and 10 regions and two partially inverted repeats (IR1 and IR2) was amplified by PCR using the primers, 50 -TCTAGATCTACCAGGACCAGCCGGGCG-30 (the XbaI site is underlined) and 50 -GGATCCTCGACGGT0 GAGCGGTGCGTG-3 (the BamHI site is underlined), restricted with XbaI and BamHI and then cloned into pTE to generate pCutBup192. For the construction of pCutBup67, a 111-bp DNA fragment containing the putative 10 region and IR2 was amplified by PCR using the primers, 50 -TCTAGAAGCCCACGTGCAGCGATGCA-30 (the XbaI site is underlined) and 50 -GGATCCTCGACGGTGAGCGGTGC GTG-30 (the BamHI site is underlined) and cloned into pTE in the same way as pCutBup192 to yield pCutBup67. pCutBIRpm1 and pCutBIRpm2 are the same constructs as pCutBup192 except for mutations in IR1 and IR2, respectively. To construct pCutBIRpm1, a 167-bp PCR product containing base substitutions within IR1 was obtained by PCR using the primers, 50 TCCGATCCCATATCGGGGG-30 (the substituted sequences are underlined) and 50 -GGATCCTCGACGGTGAGCGGTG CGTG-30 (the BamHI site is underlined). The 167-bp PCR product was used as a primer for the second PCR with the primer 50 -TCTAGATCTACCAGGACCAGCCGGGCG-30 (the XbaI

site is underlined). The final PCR product digested with XbaI and BamHI was cloned into pTE to generate pCutBIRpm1. The construction of pCutBIRpm2 was performed in the same way as pCutBIRpm1 using the primer 50 -AAGCCCACACTTAGCG ATG-30 (the substituted sequences are underlined) in place of 50 TCCGATCCCATATCGGGGG-30 . For construction of pPL, a promoterless derivative of the pTE vector, pTE was subjected to restriction with XbaI and BamHI and subsequent treatment with the Klenow fragment of E. coli DNA polymerase I. The reaction products were selfligated to yield pPL. 2.10. RNA isolation and primer extension analysis RNA isolation and primer extension analysis were performed as described previously [25]. The primer PE-1 (50 CGACCTCGGTGAGGACGGCGACGGC-30 ), complementary to the nucleotide sequence at positions 49 to 73 of cutB, was used for both sequencing and primer extension. 3. Results 3.1. Cloning and sequence analysis of CO-DH genes from T. carboxydivorans To gain insight into CO oxidation in T. carboxydivorans, CO-DH genes were cloned from T. carboxydivorans and analyzed. PCR was carried out with two sets of primers, BMSF (50 -GGCGGCTT[C/T]GG[C/G]TC[C/G]AAGAT-30 ) and O/ B-R (50 -[C/T]TCGA[T/C]GATCATCGG[A/G]TTGA-30 ) or OMP-F (50 -GGCGGCTT[C/T]GG[C/G]AA[C/G]AAGGT-30 ) and O/B-R [13]. A 1314-bp DNA fragment was successfully amplified with primers BMS-F (specific to the BMS group of CO-DHs) and O/B-R, but not with OMP-F (specific to the OMP group of CO-DHs) and O/B-R (Fig. 1a), indicating that T. carboxydivorans contains only form II CO-DH that belongs to the BMS group. BLAST analysis on the nucleotide sequence of the PCR product showed that it had the highest degree of identity (77%) to blasa_0145 encoding the large subunit of CO-DH in Blastococcus saxobsidens, implying that the amplified PCR product encodes the putative large subunit (CutA) of T. carboxydivorans CO-DH. To clone the entire CODH gene cluster, genomic DNA of T. carboxydivorans was digested with restriction enzymes in several combinations and subjected to Southern blot analysis. Southern blot analysis showed that the probe hybridized with 3.7-kb, 8.0-kb, >10-kb, 10-kb and 2.2-kb DNA fragments resulting from restriction by NotI only, BamHI/PstI, BglII/PstI, BglII only and XmaI only, respectively (Fig. 1b). Partial genomic libraries were constructed using the NotI- and BamHI/PstI-digested fragments and screened by colony lift hybridization. One and five positive clones from the NotI- and BamHI/PstI-partial genomic libraries, respectively, were identified by colony lift hybridization (data not shown) and all the DNA inserts were sequenced. Four open reading frames (cutB, cutC, cutA and

Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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Fig. 1. Cloning of CO-DH genes from T. carboxydivorans. (a) PCR for the detection of the large subunit gene (cutA) of CO-DH with the BMS-F and O/B-R primers (lane 1) or the OMP-F and O/B-R primers (lane 2). (b) Southern blot analysis using a partial cutA gene as a probe. The genomic DNA of T. carboxydivorans was digested with several combinations of restriction enzymes (N, NotI; BP, BamHI and PstI; GP, BglII and PstI; G, BglII; X, XmaI). M: size marker. (c) The genetic organization of the CO-DH structural genes and an accessory gene in T. carboxydivorans. Open reading frames are displayed as arrows indicating their transcriptional direction. Genes encoding three subunits of CO-DH are shown as solid black arrows. Short vertical lines below the arrows indicate the positions of restriction enzyme sites for BamHI, BglII, NotI, PstI, or XmaI. Thick black lines indicate DNA fragments used to determine the nucleotide sequences. The probe binding site in Southern hybridization is marked by the gray line at the bottom.

orf4) related to CO oxidation were identified from clones BP-4 and N-1 (Fig. 1c). DNA sequencing revealed that the CO-DH structural genes are arranged in the order of cutB (medium subunit)-cutC (small subunit)-cutA (large subunit), and that an additional open reading frame (orf4) showing homology with coxG of O. carboxidovorans occurs with overlapping of the cutA gene (Fig. 1c). BLAST analysis revealed that the genes upstream and downstream of the cutBCA-orf4 gene cluster are not related to CO metabolism. The open reading frame downstream of orf4 showed homology with nitrilase/cyanide hydratase, and the gene upstream of cutB had homology with uracilexanthine permease. The cutB, cutC, cutA and orf4 genes were composed of 894, 552, 2433 and 660 nucleotides, respectively. Molecular weights of the proteins deduced from the nucleotide sequences of cutB, cutC, cutA and orf4 were calculated to be 30,618, 19,616, 87,776 and 21,691 Da, respectively. The amino acid sequences of CutA, CutB and CutC of T. carboxydivorans CO-DH were 34, 34 and 50% identical to those of large, medium and small subunits of Mycobacterium sp. strain JC1 CO-DH, and 31, 35 and 47% identical to those of H. pseudoflava CO-DH, respectively. T. carboxydivorans CutA contained the conserved MCD-binding amino acids and segments (Q217, G245GGFGVKI, Q491GQGHETV, A532SRAA, C673GTMIN, A748GE). CutB and CutC had the putative FAD-binding motif (A32GGQ and T111TVG) and two putative [2Fee2S] binding motifs (C48-X4C-X2-C-X11-C and C116-X2-C-X31-C-X-C), respectively [19,20,34]. The GenBank accession number for the sequence reported in this paper is KC012617.

3.2. Evidence for the cutBCA genes as the functional structural genes encoding CO-DH To examine whether the cloned CO-DH genes encode functional CO-DH in T. carboxydivorans, the plasmid constructs expressing antisense RNAs for cutB (pTEaRcutB) and cutA (pTEaRcutA) were introduced into T. carboxydivorans.

Fig. 2. Inhibition of CO-DH synthesis by expression of antisense RNAs for cutA and cutB. Repression of CO-DH expression was examined by performing CO-DH enzyme assay and activity staining with cell-free extracts from T. carboxydivorans grown to the stationary phase (OD600 ¼ 1.2) in SMBglucose. Wild type, T. carboxydivorans containing no plasmid; vector, T. carboxydivorans with the empty vector pTE as a control; AR cutB, T. carboxydivorans with pTEaRcutB; AR cutA, T. carboxydivorans with pTEaRcutA. The same amounts of proteins for each strain were used for PAGE. All values provided are averages of results from three independent determinations. Error bars indicate standard deviations.

Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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T. carboxydivorans strain containing empty vector pTE exhibited approximately the same CO-DH activity as the same strain without the vector (Fig. 2). T. carboxydivorans strains expressing antisense RNAs of cutB and cutA showed a decrease in CO-DH activity, by 32% and 31%, respectively,

compared to the control strain with the empty vector, which was also confirmed by activity staining of CO-DH (Fig. 2). This result indicates that the cloned CO-DH genes are the functional genes encoding the active CO-DH in T. carboxydivorans.

Fig. 3. Phylogenetic tree of deduced amino acid sequences of the catalytic (large) subunits of CO-DHs. Amino acid sequence alignments were performed using CLUSTAL X. The tree was generated by neighbor joining (Poisson correction model) using the MEGA 3.1 program. Gaps in the alignment were completely deleted. GenBank accession numbers for the sequences are given in parentheses. Bootstrap values were calculated from 1000 replicates. Bradyrhizobium japonicum: B. japonicum USDA 110; Burkholderia xenovorans: B. xenovorans LB400.

Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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3.3. Comparison of T. carboxydivorans CO-DH with other carboxydobacterial CO-DHs Phylogenetic analysis of large subunits of aerobic CO-DHs was performed to examine the evolution of T. carboxydivorans CO-DH. In this phylogenetic analysis, CO-DHs were grouped into 5 clades (Fig. 3). Group I represents mycobacterial CODHs and their structural genes are arranged in the transcriptional order of cutB-cutC-cutA (mycobacterial group). This group also contains one of three CO-DHs found in Rhodococcus jostii. Group II consists of CO-DHs in Gram-negative bacteria and their structural genes are organized in the order of cutB-cutC-cutA (Gram-negative BCA group). Some CO-DHs from Gram-positive bacteria constitute group III. The structural genes of the CO-DHs in this group are arranged in the transcriptional order of cutC-cutA-cutB (Gram-positive CAB group). Group IV is made up of CO-DHs from Gram-negative rhizobia, bacteria belonging to the marine Roseobacter clade and Rhodospirillum centenum. The structural genes for the CO-DHs are arranged in the order of cutC-cutA-cutB (Gramnegative CAB group). T. carboxydivorans CO-DH forms group V together with CO-DHs from several Gram-positive bacteria (R. jostii, Rhodococcus equi, Saccharomonospora marina and Saccharomonospora xinjiangenesis). The structural genes of the CO-DHs in group V are arranged in the order of cutB-cutC-cutA (Gram-positive BCA group). Although CO-DH activity and carboxydotrophic growth were observed for T. carboxydivorans [17], the CO-DH protein was not detected in T. carboxydivorans cell-free extracts by western blotting analysis using antibodies against CO-DHs of Mycobacterium sp. strain JC1 (group I) and H. pseudoflava (group II) (Fig. 4). Furthermore, polyclonal antibodies against T. carboxydivorans CutA cross-reacted with neither CO-DHs of Mycobacterium sp. strain JC1 nor H. pseudoflava (Fig. 4). This result implies that form II CO-DH in T. carboxydivorans is not immunologically related to the mycobacterial (group I)

Fig. 4. Western blot analysis to determine immunological relatedness between CO-DHs. Cell-free extracts (with the same amounts of proteins) prepared from T. carboxydivorans cells grown in SMB-glucose (lane 1), H. pseudoflava grown in SMB-succinate (lane 2) and Mycobacterium sp. strain JC1 grown in SMB-glucose (lane 3) were subjected to non-denaturing PAGE on 7.5% acrylamide gels, followed by Coomassie brilliant blue (CBB) staining and Western blotting analysis. Western blot analysis was performed using antisera against T. carboxydivorans CutA (Ab_Tc), H. pseudoflava CO-DH (Ab_Hp) and Mycobacterium sp. strain JC1 CO-DH (Ab_JC1).

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and Gram-negative bacterial (group II) form I CO-DHs. Since antibodies against group III and group IV CO-DHs were not available, we did not assess the immunological relation of T. carboxydivorans CO-DH to form II CO-DHs of group III and IV. 3.4. Expression analysis of CO-DH in T. carboxydivorans To analyze expression of CO-DH in T. carboxydivorans grown on different carbon and energy sources, CO-DH activity staining and enzyme assay were performed using cell-free extracts prepared from T. carboxydivorans grown to the exponential phase (OD600 of 0.5e0.6) in SMB-CO, SMBglucose and SMB-glycerol. Both analyses demonstrated that CO-DH activity was highest in T. carboxydivorans grown in SMB-CO, and that CO-DH was synthesized in T. carboxydivorans grown heterotrophically even in the absence of CO (Fig. 5). In the case of CO-DH in Mycobacterium sp. strain JC1, the cellular level of CO-DH and expression of the CODH genes were known to be significantly increased in the strain grown to the stationary phase compared to those in the strain grown to the exponential phase [35]. To examine whether this was also the case for T. carboxydivorans, CO-DH enzyme assay, activity staining and western blotting were performed using cell-free extracts from T. carboxydivorans

Fig. 5. Cellular levels and activities of CO-DH in T. carboxydivorans grown on different carbon sources. CO-DH activity staining (a) and enzyme assay (b) were performed with cell-free extracts prepared from cells of T. carboxydivorans grown to the exponential phase in SMB-CO, SMB-glucose and SMB-glycerol. For CO-DH activity staining, cell-free extracts (with the same amounts of proteins) were subjected to non-denaturing PAGE on 7.5% acrylamide gels. The gels were then cut into two strips for CBB staining (lane 1) and activity staining of CO-DH (lane 2).

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grown heterotrophically with glucose and glycerol (Fig. 6). When T. carboxydivorans was grown in SMB-glucose and SMB-glycerol, CO-DH activity was increased in the stationary phase relative to that in the exponential phase, by 1.8- and 2.9fold, respectively, which was confirmed by activity staining and western blotting analyses. The result indicates that CODH in T. carboxydivorans is expressed even in the absence of CO, and that its synthesis is induced in the stationary growth phase. 3.5. Transcription analysis of the CO-DH genes It was previously reported that the CO-DH structural genes in mycobacterial and Gram-negative BCA groups constitute an operon [20,25]. The cutB, cutC, cutA and orf4 genes in T. carboxydivorans overlap each other by 8, 4 and 4 bp, respectively, implying the possibility that the four genes are co-transcribed into a single polycistronic mRNA. RT-PCR was performed to examine the co-transcription of cutB, cutC, cutA and orf4. RT-PCR revealed that cutBCA and orf4 were transcribed together to form the CO-DH operon (data not shown). The transcriptional start point (TSP) of the CO-DH operon was determined by primer extension analysis performed with total RNA isolated from T. carboxydivorans grown to the stationary phase in SMB-glucose. As shown in Fig. 7, primer extension analysis revealed that transcription of the CO-DH operon begins at nucleotide ‘C’ located 30 bp upstream of cutB. The DNA sequence upstream of cutB was analyzed to identify cis-acting elements implicated in expression of the CO-DH operon. The promoter region of cutB around 35 and 10 bp upstream of TSP did not show sequences similar to the

Fig. 6. Induction of CO-DH synthesis in T. carboxydivorans in the stationary growth phase and in the presence of CO. CO-DH enzyme assay activity staining and western blot analysis were performed with cell-free extracts from T. carboxydivorans grown to the exponential (Exp) and stationary phase (Station) in SMB-glucose or SMB-glycerol.

Fig. 7. Promoter region of the CO-DH operon in T. carboxydivorans. (a) Mapping of transcription start point of the CO-DH operon by primer extension analysis. The total RNA isolated from T. carboxydivorans grown in SMBglucose was subjected to primer extension analysis. The boxed base with an asterisk indicates the transcriptional start point of the CO-DH operon. PE: primer extension. (b) The nucleotide sequence of the upstream region of cutB. The position of the identified transcriptional start site is marked by þ1. The putative 35 and 10 regions are boxed. The translational start codon of cutB is indicated by the arrow with the gene name. The partially inverted repeat sequences (IR1 and IR2), which are similar to the CRP-binding motif, are indicated by arrows above their sequences in bold face. The putative ShineDalgarno sequence was marked in bold face with the asterisks.

consensus sequences of the well-known E. coli 35 and 10 regions (TTGACA-N17-TATAAT) for the housekeeping sigma factor. Since the sequence (TTGCCA) around the position at 43 is very similar to the consensus sequence of the 35 region, we designated this sequence as a putative 35 region. Two partially inverted repeat sequences (IR1 and IR2), which are similar to the binding site ([C/T]GTGA-N6-TCAC[G/A]) Q3 of the cAMP receptor protein (Crp), were identified 95e110 bp and 46e61 bp upstream of cutB, respectively. To examine the role of the cis-acting elements upstream of the CO-DH operon, promoter assay using several cutB::lacZ translational fusions was carried out (Fig. 8). Basal levels of bgalactosidase activity were detected in the T. carboxydivorans strain with the empty vector, pPL. The T. carboxydivorans strain harboring pCutBup192 showed that b-galactosidase activity in cells grown in glycerol plus CO was 1.4-fold higher than that of cells grown in glycerol alone (Fig. 8a), indicating that the CO-DH operon is inducible by CO and that the 192-bp DNA fragment fused to lacZ contains the regulatory elements involved in expression and regulation of the CO-DH operon. When the putative 35 region and its upstream region were removed from pCutBup192 (pCutBup67), b-galactosidase activity was reduced to the basal level (Fig. 8b). Point mutations within the IR1 sequence led to a significant decrease in the promoter activity of the CO-DH operon (pCutBIRpm1),

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Fig. 8. Transcription analysis of the CO-DH genes. (a) Induction of CO-DH gene expression by CO was examined. T. carboxydivorans strains containing the cutB::lacZ transcriptional fusion plasmid (pCutBup192) were grown to the exponential phase (OD600 of 0.5e0.6) in SMB-glycerol and SMB-glycerol with CO. (b) Effect of mutations in the cutB promoter region on cutB expression was determined. b-Galactosidase activities were measured with T. carboxydivorans strains harboring pPL, pCutBup192, pCutBup67, pCutBIRpm1 and pCutBIRpm2. The strains were grown to the exponential phase (OD600 of 0.5e0.6) in SMB-glucose. The partially inverted repeats (IR1 and IR2) are indicated by lines above the sequences in bold face. The mutated sequences within IR1 and IR2 are marked by asterisks. The gray boxes indicate the putative 35 or 10 regions. pPL is a promoterless vector for a negative control. All values provided are averages of results from three independent determinations. Error bars indicate the standard deviations. Asterisks (***) indicate a statistically significant change (p  0.001).

while mutations within IR2 did not affect expression of the CO-DH operon (pCutBIRpm2), indicating that IR1 may act as an activator-binding site for the expression of the CO-DH operon (Fig. 8b). 4. Discussion 4.1. Classification of CO-DHs Previously, aerobic CO-DHs were classified into either two groups (OMP and BMS) by King [13] or three groups (mycobacterial CO-DHs, Gram-negative bacterial CO-DHs and Gram-positive bacterial CO-DHs other than mycobacteria) by Kim and Park [21]. Grouping by King is based mainly on the conserved active site motifs involved in the copper coordination in the large subunits of CO-DHs, while grouping by Kim and Park is based on phylogenetic analysis of the large subunits of CO-DHs, as well as the immunological relatedness of CO-DHs. Since many putative CO-DH genes have been identified from prokaryotes including T. carboxydivorans as a result of genome sequencing, the demand for a new grouping of CO-DHs to more systematically classify them has increased. We herein suggested that CO-DHs could be classified into 5 groups based on comprehensive phylogenetic analysis, including newly identified CO-DHs that had not been used in previous grouping analyses (Fig. 3). The CO-DHs of group I are the CO-DHs found in mycobacteria, which possess NO dehydrogenase activity in addition to CO-DH activity [23]. The CO-DHs of groups II and IV are Gramnegative bacterial CO-DHs in proteobacteria. The CO-DH of

Bradyrhizobium japonicum USDA 110 (group IV) was shown to be immunologically related to that of O. carboxidovorans (group II) [10], which raises the possibility that CO-DHs in groups II and IV might be immunologically related to each other. The CO-DHs of groups III and V are Gram-positive bacterial CO-DHs in actinobacteria. It is noteworthy that the CO-DHs in the same group share common features such as the same gene order of the CO-DH structural genes, conservation of the same active site motifs in the large subunits and Gramstaining properties of the bacteria containing them (Table 2), indicating that our new classification of CO-DHs is more pertinent for reflecting the functional and evolutionary aspects of CO-DHs than the previous classification. As mentioned above, King's grouping of CO-DHs is based on the different amino acid sequences of the active site motifs. Table 2 Comparison of CO-DHs in different groups. Group

Gene arrangement

Active sitea

Grouping by King

Grouping by Kim and Park

I II III IV V

B-C-Ab B-C-A C-A-B C-A-B B-C-A

AYACSFR AYRCSFRc AYRGAGR AYRGAGR PYRGAGR

OMP OMP BMS BMS BMS

Mycobacterial CO-DH Gram () CO-DH Gram (þ) CO-DH Gram () CO-DH Gram (þ) CO-DH

a The prediction of the active site was based on the site in CoxL of O. carboxidovorans [34]. b B, C, and A mean the structural genes for medium, small, and large subunits of CO-DH, respectively. c AYRCSFR is located at positions 385e391 in CoxL of O. carboxidovorans.

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CO-DHs of groups I and II have the active site motif of “AY(A/R)CSFR” and therefore belong to the OMP group (Table 2). This active site motif is uniquely conserved in CODHs among molybdenum hydroxylase family enzymes [3]. The CO-DHs in the OMP group are also known as form I CODHs. In contrast, those of groups III and IV contain the “AYRGAGR” motif that is characteristic of form II CO-DHs in the BMS group (Table 2). The “AYRGAGR” motif also occurs in many molybdenum hydroxylases other than CO-DH [3]. The CO-DH of the OMP group was demonstrated to be the key enzyme for CO utilization in mycobacterial and Gramnegative carboxydotrophic bacteria. All studied carboxydobacteria harboring form I CO-DHs were demonstrated to be capable of CO oxidation, even though many of them cannot grow lithoautotrophically with CO as the sole carbon and energy source [3]. The failure of lithoautotrophic growth can be explained by the absence of CO-fixation pathways or COinsensitive terminal oxidases. In contrast, functional studies on form II CO-DHs of the BMS group have not been done beyond the identification of CO-DH genes from genomic sequences, except for the CO-DH of B. japonicum USDA 110 [10]. Since bacteria other than B. japonicum, which contain only form II CO-DH genes, were shown to be unable to oxidize CO, the notion that form II CO-DHs possess catalytic activity for CO oxidation [3,10,16] remains subject to debate. In the case of form I CO-DHs containing the “AY(A/R)CSFR” motif in the active site, the central cysteine in the motif coordinates the copper ion of the catalytically essential bimetallic [CuSMoO2] center [36,37]. The copper ion of the bimetallic center was suggested to be required for initial CO binding, and therefore to be essential for CO oxidation by CODH [36]. Absence of the corresponding cysteine in “(A/P) YRGAGR”-containing form II CO-DHs might account for the inability of most bacteria with only form II CO-DH genes to oxidize CO. It is noteworthy that T. carboxydivorans, with only form II CO-DH, is capable of lithoautotrophic growth with CO, the phenotype of which was previously observed only in B. japonicum USDA 110 [10,38]. It remains elusive as to how B. japonicum USDA 110 and T. carboxydivorans can utilize CO. Further study regarding the occurrence and nature of the bimetallic center in purified CO-DH of T. carboxydivorans is required to answer this question. Some carboxydobacteria such as Ruegeria pomeroyi, R. jostii and Stappia sp. contain both form I and form II CO-DHs (Fig. 3; [3]), which might be the result of horizontal gene transfer [28]. Burkholderia xenovorans LB400 has two paralogous form I CO-DHs like Mycobacterium sp. strain JC1 [25], which might result from gene duplication during evolution. However, the physiological role of multiple CO-DHs in these bacteria remains unclarified. The carboxydobacteria with form II CO-DHs of the BMS group are known to normally have a small number or the absence of CO-DH accessory genes near the CO-DH structural genes, and the structural genes of CO-DH are arranged in the order of cutC-cutA-cutB (or coxS-coxL-coxM ), while the structural genes of form I CO-DHs occur in the order of cutBcutC-cutA (or coxM-coxS-coxL) [3]. T. carboxydivorans CO-

DH is assigned to the BMS group, because its gene was amplified with the BMS and O/B-R primers and the large subunit has the active site motif similar to that found in CODHs of the BMS group (Table 2). The CO-DH in T. carboxydivorans has no immunological relatedness to form I CO-DHs of groups I and II that belong to the OMP group (Fig. 4). Furthermore, only one CO-DH accessory gene, orf4 (coxG homolog), was found downstream of the CO-DH structural genes in T. carboxydivorans. However, the transcriptional order of the CO-DH structural genes of T. carboxydivorans is arranged in the transcriptional order of cutB-cutC-cutA, as in the case of the CO-DHs in the OMP group. Taken together, these findings, as well as results from phylogenetic analysis, led us to classify T. carboxydivorans CO-DH together with CO-DHs of R. jostii, R. equi, S. marina and S. xinjiangenesis into a new group separated from the other groups of CO-DHs (Fig. 3). 4.2. Regulation of CO-DH genes in T. carboxydivorans There are two regulatory systems that have been reported to regulate the genes (cut or cox) encoding aerobic CO-DHs: CutR and CRP (cAMP receptor protein) in Mycobacterium sp. strain JC1 and RcoM in B. xenovorans. The CutR protein is a LysR-type transcriptional regulator and serves as a transcriptional activator for expression of two cutBCA operons in Mycobacterium sp. strain JC1. The cutR gene is conserved in mycobacteria and has not been identified in any carboxydotrophic bacteria other than mycobacteria. It remains undefined as to whether CutR directly senses CO. The conserved CRP binding sites (TGTGA-N6-TCACA) were reported to occur upstream of the cutBCA operons in fast-growing mycobacteria such as Mycobacterium sp. strain JC1 and Mycobacterium smegmatis. Mutations of the CRP-binding site abolished the induction of cutBCA expression in Mycobacterium sp. strain JC1 grown to the stationary phase [35]. The cutBCA operon was not expressed in M. smegmatis, when the crp gene (msmeg_6189) was inactivated by deletion (unpublished result). Both results strongly suggest that CRP positively regulates expression of cutBCA in mycobacteria and that induction of cutBCA expression in the stationary phase is likely mediated by CRP. RcoM is a hemoprotein that acts as a transcriptional activator. When CO binds to the heme moiety within its N-terminal PAS fold, RcoM is activated to serve as a transcriptional activator for the coxMSL operon in B. xenovorans [39]. With regard to anaerobic CO-DHs, two regulatory systems (CooA in Rhodospirillum rubrum and CorQR in Thermococcus onnurineus) have been identified [40,41]. The homodimeric CooA regulator is a heme-containing CO sensor and distantly related to the CRP/FNR transcriptional factors. As in the case of RcoM, binding of CO to CooA brings about conformational changes which activate its transcriptional activity for coo genes. The corQ and corR genes were identified immediately upstream of the CO-DH genes in T. onnurineus. The CorQ and CorR proteins were shown to be necessary for CO-dependent induction of CO-DH gene expression in this bacterium [41]. However, the mechanism by which CorQ and CorR perceive CO remains to be elucidated.

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The addition of CO to glycerol-grown cultures of T. carboxydivorans led to a modest increase in expression of the CO-DH genes (Fig. 8), indicating the presence of (a) COresponsive regulatory system(s). Interestingly, the induction of CO-DH synthesis was also observed when heterotrophic cultures of T. carboxydivorans reached the stationary phase in which the supplied organic carbon sources such as glucose and glycerol might be limiting (Fig. 6), implying that expression of the CO-DH genes is likely subjected to catabolite repression. This expression pattern of CO-DH genes was also observed in Mycobacterium sp. strain JC1 and H. pseudoflava [25,35,42,43]. From these results, we assume that the strong induction of CO-DH synthesis in T. carboxydivorans grown lithoautotrophically with CO appears to result from the combinatory effect of both CO induction and the abolishment of catabolite repression in the absence of organic substrates. Two partially inverted repeat sequences, IR1 (TcGGa-N6gCCcA) and IR2 (cGTGC-N6-GCACa), were identified 65e80 bp and 16e31 bp upstream of the transcription start point for the CO-DH genes, respectively (Fig. 7). Since the sequences of IR1 and IR2 are similar to the binding motifs of CRP of E. coli (TGTGA-N6-TCACA) and Mycobacterium tuberculosis ([C/T]GTGA-N6-TCAC[G/A]) [44] and synthesis of CO-DH in T. carboxydivorans was increased in the stationary phase of growth (Fig. 6), there is a possibility that the CO-DH genes in T. carboxydivorans might be subjected to catabolite repression through positive regulation by CRP as in mycobacteria [35]. Promoter assay with mutated IR1 and IR2 sequences revealed that IR1 serves as a cis-acting regulatory sequence involved in positive regulation of the CO-DH genes (Fig. 8). Transcriptional activators normally bind to positions between 80 and 30 relative to the transcriptional start points to recruit RNA polymerase or to facilitate open complex formation [45]. In this regard, the position of IR1 is pertinent to the cis-acting element where a transcriptional activator binds. Since the genome sequence of T. carboxydivorans is not available at present, we do not know whether the homologs of the known CO-responsive regulators, such as CRP, CutR, RcoM and CooA, are present in T. carboxydivorans. Study on the transcriptional regulator acting on IR1 is under way. Conflict of interest The authors declare no conflicts of interest. References [1] Kim YM, Hegeman GD. Oxidation of carbon monoxide by bacteria. Int Rev Cytol 1983;81:1e32. [2] Meyer O, Schlegel HG. Biology of aerobic carbon monoxide-oxidizing bacteria. Annu Rev Microbiol 1983;37:277e310. [3] King GM, Weber CF. Distribution, diversity and ecology of aerobic COoxidizing bacteria. Nat Rev Microbiol 2007;5:107e18. [4] Kistner A. On a bacterium oxidizing carbon monoxide. K Nederl Akad Wetenschap Proc Ser C 1953;56:443e50. [5] Sanzhiev EU, Zavarzin GA. Bacterium oxidizing carbon monoxide. Dokl Akad Nauk SSSR 1971;196:956e8.

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Please cite this article in press as: Lee JH, et al., Identification and characterization of the genes encoding carbon monoxide dehydrogenase in Terrabacter carboxydivorans, Research in Microbiology (2017), http://dx.doi.org/10.1016/j.resmic.2017.01.004

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