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Molecular Analysis of Promoter and Intergenic Region Attenuator of the Vibrio vulnificus prx1ahpF Operon
Molecular Analysis of Promoter and Intergenic Region Attenuator of the Vibrio vulnificus prx1ahpF Operon
Journal of Microbiology and Biotechnology. 2015. Sep, 25(8): 1380-1389
Copyright © 2015, The Korean Society For Microbiology And Biotechnology
  • Received : March 11, 2015
  • Accepted : March 29, 2015
  • Published : September 28, 2015
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About the Authors
Hyun Sung Lee
Korea Food Research Institute, Seongnam 463-746, Republic of Korea
Jong Gyu Lim
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 151-921, Republic of Korea
Kook Han
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
Younghoon Lee
Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 305-701, Republic of Korea
Sang Ho Choi
National Research Laboratory of Molecular Microbiology and Toxicology, Department of Agricultural Biotechnology, and Center for Food Safety and Toxicology, Seoul National University, Seoul 151-921, Republic of Korea
choish@snu.ac.kr

Abstract
Prx1, an AhpF-dependent 2-Cys peroxiredoxin (Prx), was previously identified in Vibrio vulnificus , a facultative aerobic pathogen. In the present study, transcription of the V. vulnificus prx 1 ahpF genes, which are adjacently located on the chromosome, was evaluated by analyzing the promoter and intergenic region of the two genes. Northern blot analyses revealed that transcription of prx 1 ahpF results in two transcripts, the prx 1 and prx 1 ahpF transcripts. Primer extension analysis and a point mutational analysis of the promoter region showed that the two transcripts are generated from a single promoter. In addition, the 3’ end of the prx 1 transcript at the prx 1 ahpF intergenic region was determined by a 3’RACE assay. These results suggested that the prx 1 ahpF genes are transcribed as an operon, and the prx 1 transcript was produced by transcriptional termination in the intergenic region. RNA secondary structure prediction of the prx 1 ahpF intergenic region singled out a stem-loop structure without poly(U) tract, and a deletion analysis of the intergenic region showed that the atypical stem-loop structure acts as the transcriptional attenuator to result in the prx 1 and prx 1 ahpF transcripts. The combined results demonstrate that the differential expression of prx 1 and ahpF is accomplished by the cis -acting transcriptional attenuator located between the two genes and thereby leads to the production of a high level of Prx1 and a low level of AhpF.
Keywords
Introduction
Bacteria continually encounter toxic reactive oxygen species (ROS), such as superoxide anion (O 2 - . ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (·OH), which are generated by incomplete reduction of oxygen during respiration and aerobic metabolism [29 , 31] . Oxidative stress caused by increased levels of ROS can lead to the damage of cellular components, including metal centers, protein, DNA, and membrane lipid [29 , 31] . In particular, pathogenic bacteria have to cope with oxidative stress imposed by immune systems to survive host environments and in turn to ensure developing illness [8 , 21 , 25] . Therefore, pathogens have evolved sophisticated mechanisms to overcome oxidative stress, and the mechanisms are closely linked to their virulence [8 , 25] .
The mechanisms of bacterial defense against oxidative stress include highly specific and effective antioxidant enzymes such as superoxide dismutase, catalase, and peroxiredoxin (Prx) [6 , 11] . Among these, Prxs are a ubiquitous family of cysteine-based peroxidases that catalyze the reduction of peroxides such as H 2 O 2 and organic hydroperoxide [6 , 27] . Typical 2-Cys Prxs, the largest group of Prxs, have two conserved catalytic cysteines, peroxidatic and resolving cysteines. Peroxidatic cysteine reacts with peroxides and forms a cysteine sulfenic acid intermediate, which is followed by the formation of an intermolecular disulfide bond with resolving cysteine from another subunit. Disulfide-bonded 2-Cys Prxs are subsequently reduced and reactivated by thiol-containing reductants such as thioredoxin (Trx) and alkyl hydroperoxidase subunit F (AhpF) [10] .
AhpC (alkyl hydroperoxidase subunit C), originally identified from Escherichia coli and Salmonella Typhimurium, is one of the best characterized 2-Cys Prxs and utilizes AhpF as a reductant to compose an NADH-dependent peroxidase system [5 , 12 , 30] . AhpF is a flavoprotein with NADH:disulfide oxidoreductase activity and restores the disulfide bond in AhpC to the reduced form by transferring electrons from NADH to AhpC [6] . In most cases, the ahpF gene is located adjacent downstream of the ahpC gene on the chromosome and is co-transcribed with ahpC as an operon [6] . The expression of ahpCF is activated by OxyR, which is a central regulator of the oxidative stress response when exposed to exogenous oxidants in a number of bacteria [7] . However, studies about the exact molecular mechanisms of ahpCF transcription are still limited [6] .
In the facultative aerobic pathogen Vibrio vulnificus , a 2-Cys Prx, which is highly homologous to other bacterial Prxs such as E. coli AhpC (78% identity in amino acid sequences) was previously identified and designated as Prx1 (formerly V. vulnificus AhpC) [2] . Prx1, forming an NADH-dependent peroxide reductase system with AhpF, is effective at decomposing large amounts of peroxides rapidly and contributes to not only the growth and survival of the pathogen under exogenous oxidative stress but also virulence in mice [2 , 3]. The V. vulnificus ahpF gene is located downstream of the prx 1 gene, and the prx 1 ahpF transcription is positively regulated by V. vulnificus OxyR1, a homolog of E. coli OxyR only in cells exposed to high levels of exogenous H 2 O 2 , to result in the prx 1 and prx 1 ahpF transcripts [16] . However, molecular analysis of the prx 1 ahpF transcription has not yet been experimentally verified. Here, we provided molecular genetic evidence that the V. vulnificus prx 1 ahpF genes are transcribed as an operon from a single promoter, and a stem-loop structure located between the two genes attenuates the transcription to result in a high level of prx 1 transcript and a low level of prx 1 ahpF transcript. Finally, the physiological role of the differential expression of prx 1 and ahpF is discussed.
Materials and Methods
- Strains, Plasmids, and Culture Conditions
The strains and plasmids used in this study are listed in Table 1 . Unless noted otherwise, the V. vulnificus strains were grown aerobically in Luria-Bertani (LB) medium supplemented with 2.0% (w/v) NaCl (LBS) at 30℃.
Plasmids and bacterial strains used in this study.
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a Kmr , kanamycin-resistant; Cmr , chloramphenicol-resistant; Apr , ampicillin-resistant; Tcr , tetracycline-resistant. b Shown are the nucleotide positions within Pprx, where +1 is the transcription start site of Pprx.
- Generation ofahpFMutant
λTo inactivate ahpF in vitro , a unique Bam HI site was introduced into the open reading frame (ORF) of ahpF using the PCR-mediated linker-scanning mutation method as described previously [16] . Briefly, pairs of primers AHPF0801 and AHPF0802 for amplification of the 5’ amplicon or AHPF0803 and AHPF0804 for amplification of the 3’ amplicon were designed and used ( Table 2 ). The ahpF with Bam HI site was amplified by PCR using a mixture of both amplicons as the template and AHPF0801 and AHPF0804 as primers. The ahpF :: nptI was constructed by insertion of a 1.2 kb nptI DNA conferring resistance to kanamycin [26] into the Bam HI site of the PCR products, and ligated with Sac I- Spe I-digested pDM4 [23] to form pWK0805 ( Table 1 ). The E. coli SM10 λ pir, tra strain [22] containing pWK0805 was used as a conjugal donor to V. vulnificus MO6-24/O to generate the ahpF mutant BK081 ( Table 1 ). The conjugation and isolation of the transconjugants were conducted as previously described [16] .
Oligonucleotides used in this study.
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a The oligonucleotides were designed using the V. vulnificus MO6-24/O genomic sequence (GenBank Accession No. CP002469 and CP002470; http://www.ncbi.nlm.nih.gov). b Regions of oligonucleotides not complementary to the corresponding genes are underlined. c Shown are the nucleotide positions within the Pprx, where +1 is the transcription start site of Pprx.
- RNA Purification and Transcript Analysis
Total cellular RNAs from the V. vulnificus strains grown to an A 600 of 0.5 were isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) [19] . When necessary, the strains were exposed to 250 µM H 2 O 2 (Sigma, St. Louis, MO, USA) for 30min and then harvested. For northern blot analysis, reactions were performed according to standard procedures [28] with 15 µg of RNA. The DNA probes, PRX1P and AHPFP, were prepared respectively by labeling DNA fragments containing the prx 1 and ahpF coding regions with [α- 32 P]-dCTP, and used for hybridization as previously described [16] . For primer extension analysis, the 24-base oligonucleotide primer PRX1-PE02 ( Table 2 ) complementary to the coding region of prx 1 was end-labeled with [γ- 32 P]-ATP and added to the RNA. The primer was then extended with SuperScript II RNase H reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The cDNA products were purified and resolved on a sequencing gel alongside sequencing ladders generated from pSS1050 with the same primer. The plasmid pSS1050 was constructed by cloning the 410bp prx 1 upstream region extending from −271 to +139, amplified by PCR using a pair of primers, PRX1-PE01 and -PE02 ( Table 2 ), into pGEM-T Easy (Promega, Madison, WI, USA). The northern blots and primer extension gels were visualized using a phosphorimage analyzer (BAS1500; Fuji Photo Film Co. Ltd., Tokyo, Japan).
- Mutational Analysis of the Promoter Region ofprx1ahpFOperon
The pair of primers PRX1-PM01 and -PM02 ( Table 2 ) was designed and used to amplify the 2,470bp DNA fragment encompassing the prx 1 ahpF operon, including its promoter region P prx . The PCR product was cloned into the broad-host-range vector pRK415 [14] to create pSS1105 ( Table 1 ). Each of the three nucleotide bases -16T, -8T, and +1G (where +1 is the transcription start site of P prx ) within the P prx on pSS1105 was mutated to -16G, -8C, and +1T, respectively ( Fig. 3 A) using a QuikChange site-directed mutagenesis kit (Agilent Technologies, Loveland, CO, USA) [19] . The complementary mutagenic primers listed in Table 2 were used in conjunction with the plasmid pSS1105 (as a template DNA) to create pSS1110 (for the -16T → G mutation), pSS1111 (for the -8T → C mutation), and pSS1112 (for the +1G → T mutation) ( Table 2 ). The mutations were confirmed by DNA sequencing. The pSS1105, pSS1110, pSS1111, or pSS1112 was transferred into the prx 1 mutant OH0701 ( Table 1 ) by conjugation, and the total RNAs from each strain were subjected to northern blot analyses as described above.
- Rapid Amplification of cDNA 3’ ends (3’ RACE) Assay
The 3’ RACE assay to determine the 3’ end of the prx 1 transcript was performed as described previously [15] , with minor modification. Briefly, total cellular RNA (3 µg) from the wild-type V. vulnificus grown to an A 600 of 0.5 after being exposed to 250 µM H 2 O 2 for 30min was dephosphorylated with 0.01 U of calf intestine alkaline phosphatase (New England Biolabs, Ipswich, MA, USA) and then ligated with 500 pmol of 3’ adaptor RNA (5’-GCU GAU GGC GAU GAA UGA ACA CUG CUU UGA UGA AA-3’) (Bioneer, Seoul, South Korea) with 50 U of T4 RNA ligase (New England Biolabs) in a 50 µl reaction. The adaptor-ligated RNA was reverse-transcribed and amplified with adaptor-specific primer RACE-ASP and prx 1 gene-specific primer RACE-GSP ( Table 2 ) using a One-step reverse-transcription PCR (RT-PCR) kit (Qiagen) according to the manufacturer’s instructions. The PCR product was separated on 2% agarose gel, purified, and analyzed by DNA sequencing after cloning into the pGEM-T Easy vector.
- Construction of Pprx-prx1ahpFIntergenic Region-luxCDABETranscriptional Fusions and Measurement of Cellular Luminescence
The 342 bp prx 1 upstream region extending from −162 to +177, amplified by PCR using the pair of primers PRX1AHPF-01 and -02 ( Table 2 ), was fused with 136 bp of the intact prx 1 ahpF intergenic region (for pSS1324), amplified by PCR with the pair of primers PRX1AHPF-03 and -04 ( Table 2 ), or with 55 bp of deleted- prx 1 ahpF intergenic region (for pSS1326), amplified with the pair of primers PRX1AHPF-03 and -05 ( Table 2 ), using the PCR-mediated linkerscanning method as described above. As a control, a 136 bp DNA fragment within the prx 1 coding region was amplified by PCR using the pair of primers PRX1-C01 and -C02 ( Table 2 ) and fused with the same prx 1 upstream region (for pSS1323). Each PCR product was ligated with Sac I- Spe I-digested pBBR- lux carrying promoterless luxCDABE [18] to create pSS reporters (pSS1323, pSS1324, or pSS1326) ( Table 2 ; Fig. 5 B), which were then transferred into V. vulnificus MO6-24/O by conjugation. The cellular luminescence of the cultures was measured with a luminometer (Lumat model 9507, Berthold, Germany) and expressed in arbitrary relative luminescence unit (RLU), as described previously [13] .
- Data Analyses
Averages and standard errors of the mean (SEM) were calculated from at least three independent experiments. All data were analyzed by Student's t tests with the SAS program (SAS software; SAS Institute Inc.). Significance of differences between experimental groups was accepted at a P value of <0.005.
Results and Discussion
- TheV. vulnificus prx1ahpFGenes Are Transcribed into Two Transcripts
The V. vulnificus ahpF gene is located downstream of the prx 1 gene, and the two coding regions of prx 1 and ahpF are transcribed in the same direction with the 136 bp intergenic region ( Fig. 1 A) [2] . To analyze the transcription pattern of the V. vulnificus prx 1 ahpF , northern blot analyses were performed using the total cellular RNAs isolated from the wild type, the prx 1 mutant, and the ahpF mutant. Since the expression of prx 1 is known to be induced in cells exposed to high levels of exogenous H 2 O 2 [3] , the V. vulnificus cells were exposed to 250 µM H 2 O 2 for 30 min before the RNA isolation. As shown in Fig. 1 B, the PRX1P probe was hybridized to the 0.6 kb RNA corresponding to the prx 1 transcript when total RNA was isolated from the wild-type cells exposed to 250 µM H 2 O 2 , reconfirming that the expression of prx 1 is induced in response to exogenous H 2 O 2 . In addition, approximately 2.3 kb RNA was detected by PRX1P when the RNA was isolated from the H 2 O 2 -exposed wild type but not ahpF mutant ( Fig. 1 B). On the basis of the DNA sequence of prx 1 ahpF , it was anticipated that a polycistronic prx 1 ahpF transcript would be approximately 2.3 kb long ( Fig. 1 A). Northern blot analysis was performed using the AHPFP probe, and a single RNA corresponding to the prx 1 ahpF transcript was detected in the RNA isolated from the H 2 O 2 -exposed wild type but not ahpF mutant ( Fig. 1 C), indicating that the prx 1 ahpF genes were also transcribed as a single operon. Therefore, it appeared that the V. vulnificus prx 1 ahpF genes transcribed into two transcripts, a high level of prx 1 transcript and a low level of prx 1 ahpF transcript.
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Genetic organization and transcript analysis of V. vulnificus prx1ahpF. (A) The shaded arrows represent the transcriptional directions and the coding regions of the V. vulnificus prx1 (VVMO6_03966) and ahpF (VVMO6_03967). Locus tag numbers based on the database of the V. vulnificus MO6-24/O genome sequence (GenBank Accession No. CP002469 and CP002470) are shown above each coding region. The DNA probes, PRX1P and AHPFP, used for northern blot analyses are depicted below each coding region by shaded bars. (B and C) Total RNAs were isolated from the strains grown to an A600 of 0.5 after being exposed to 250 µM H2O2 for 30 min as indicated. RNAs were resolved and hybridized to a 32P-labeled DNA probe corresponding to the internal coding regions of prx1 (PRX1P, B) or ahpF (AHPFP, C). The RNA size markers (Invitrogen) and prx1 and prx1ahpF transcripts are shown in kilobases. prx1, prx1 mutant; ahpF, ahpF mutant.
- V. vulnificus prx1ahpFGenes Are Transcribed from a Single Promoter
To clarify whether the prx 1 and prx 1 ahpF transcripts are transcribed from each own promoter with different activity or from one promoter, the transcription start site of prx 1 ahpF genes was determined by primer extension analysis. A single transcript was produced from primer extension of RNA isolated from the wild type exposed to 250 µM H 2 O 2 ( Fig. 2 A). The 5’ end of the prx 1 or prx 1 ahpF transcript was located 47 bp upstream of the translational initiation codon of prx 1 and subsequently designated +1 ( Fig. 2 B). The putative promoter constituting this transcription start site was named P prx . The sequences for the -10 and -35 regions of P prx were assigned on the basis of similarity to consensus sequences of the E. coli σ 70 promoter ( Fig. 2 B). In addition, the sequences extending from -73 to -37, relative to the transcription start site of P prx (shaded boxes in Fig. 2 B), showed 87.5% similarity to a consensus sequence of the E. coli OxyR-binding site [32] , indicating that, consistent with our previous report [16] , the V. vulnificus OxyR1 may activate the expression of prx 1 ahpF by direct binding to the promoter region. Using different sets of primers, no other transcription start sites were identified by primer extension analyses (data not shown). These results suggested that the prx 1 and prx 1 ahpF transcripts are generated from the single promoter, P prx .
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Transcription start sites and sequence analysis of the prx1ahpF promoter region. (A) Transcription start site of Pprx, indicated by the asterisk, was determined by primer extension of the RNA derived from the wildtype V. vulnificus grown to an A600 of 0.5 after being exposed to 250 µM H2O2 for 30 min. Lanes C, T, A, and G represent the nucleotide sequencing ladders of pSS1050. (B) The transcription start site of Pprx (+1) is indicated by a bent arrow, and the positions of the putative -10 and -35 regions are underlined. The putative sequences for binding of OxyR1 are presented as shaded boxes. The consensus sequences of theE. coli OxyR-binding site are indicated above the V. vulnificus DNA sequence. The ATG translation initiation codons and the putative ribosome-binding site (SD) are also shown in boldface.
To confirm whether the P prx governs the transcription of prx1ahpF in vivo , three nucleotides of P prx , -16T, -8T, and +1G, which are thought to be important in the transcription, were selected and mutated to -16G, -8C, and +1T, respectively. The DNA fragments encompassing the prx 1 ahpF operon with the wild-type and the point-mutated P prx were cloned into pRK415 to create pSS1105, pSS1110, pSS1111, and pSS1112 ( Fig. 3 A). The activities of each P prx on the pSS1105, pSS1110, pSS1111, and pSS1112 were compared by northern blot analyses using the RNAs isolated from each plasmid-containing prx 1 mutant. Since PRX1P did not detect any transcripts in the RNA isolated from the prx 1 m utant ( Fig. 1 B, lane 3), the detected bands in Fig. 3 B represent the transcripts that were transcribed from each plasmid. The -16T, a first base of the extended -10 region (normally, TGn) that is recognized by domain 3 of the RNA polymerase σ subunit and the -8T, a fifth base of the -10 region, may specify the initial binding of RNA polymerase to the P prx [4] . As expected, the mutations of -16T to -16G (on pSS1110) and of -8T to -8C (on pSS1111) resulted in the complete abolishment of P prx activity, as determined based on the intensities of the prx 1 and prx 1 ahpF transcripts detected by PRX1P ( Fig. 3 B). In addition, the mutation of +1G to +1T (on pSS1112) resulted in the increased expression of prx 1 ahpF ( Fig. 3 B), which is possibly attributed to the increased unwinding efficiency of the DNA duplex around the transcription start site of P prx by the mutation. Therefore, these results confirmed that the transcription of prx 1 ahpF results in the prx 1 and prx 1 ahpF transcripts from the single promoter, which was determined by the primer extension analysis ( Fig. 2 A).
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Effect of the Pprx mutation on the prx1ahpF transcription. (A) A set of DNA fragments encompassing the wild type or mutated Pprx and the prx1ahpF operon was subcloned into pRK415 to create pSS1105, pSS1110, pSS1111, or pSS1112 for northern blot analyses as shown in shaded box. The transcription start site of Pprx (+1) and the putative -10 and extended -10 (TGn) regions are underlined. (B) Total RNAs were isolated from the exponential phase culture (A600 = 0.5) of the prx1 mutant containing each plasmid and then resolved and hybridized to PRX1P. The results are presented as described in Fig. 1B.
- Stem-Loop Structure in theprx1ahpFIntergenic Region Acts as a Transcriptional Attenuator to Createprx1 andprx1ahpFTranscripts
There are still two possible ways for the transcription of prx 1 ahpF into the two transcripts. One is by degradation of some of the prx 1 ahpF transcript through an unknown mechanism, whereas another is by transcriptional attenuation at the intergenic region of prx 1 ahpF , resulting in the high level of prx 1 transcript and the low level of prx 1 ahpF transcript. In the northern blot analysis, however, the prx 1 transcript was still detected by PRX1P in the RNA isolated from the ahpF mutant that cannot produce the prx 1 ahpF transcript ( Fig. 1 B, lane 4): therefore, the former hypothesis is not the case. To verify the latter hypothesis, we first examined whether there is any transcriptional termination site within the prx 1 ahpF intergenic region, using the 3’ RACE assay. Total RNA isolated from the wild type exposed to 250 µM H 2 O 2 was ligated to a 3’ adaptor, followed by RT-PCR amplification, and the resulting PCR products were then analyzed on an agarose gel. As a result, only one band was detected at comparable levels ( Fig. 4 A) and was then eluted from the gel and subjected to the DNA sequencing analysis ( Table 3 ). The sequencing data suggested that the band was derived from the prx 1 transcript with the 3’ end, +689T, located 83 bp downstream of the translational stop codon of prx 1 as indicated in Fig. 4 B. These results suggested that the transcription of prx 1 ahpF can be terminated at the intergenic region to result in the prx 1 transcript.
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Determination of the 3’end of the prx1 transcript and sequence analysis of the prx1ahpF intergenic region. (A) The 3’ RACE product of the RNA derived from the wild-type V. vulnificus grown to an A600 of 0.5 after being exposed to 250 µM H2O2 for 30 min was separated on a 2% agarose gel and indicated by an arrow. The DNA size markers (100 bp DNA ladder, New England Biolabs) are shown in kilobases. (B) The coding regions of prx1ahpF genes and the chromosomal DNA are indicated by arrow boxes and a thick line, respectively. DNA sequences of the 136 bp prx1ahpF intergenic region, indicated by a black box, are shown. The 3’ end position of the prx1 transcript (T) that is determined by 3’ RACE assay (Table 3), the translational stop codon of prx1 (TAA), and the translational initiation codon of ahpF (ATG) are indicated in boldface. Black arrows indicate a palindrome in the prx1ahpF intergenic region. (C) The RNA secondary structure of the RNA deduced from the 56 bp DNA sequence, indicated by a shaded box in panel B, was predicted by the UNAfold software [20] (http://www.bioinfo.rpi.edu/applications/mfold/).
Sequencing analysis of the 3’ RACE products.
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a The RACE products shown in Fig. 4A were cloned into pGEM-T Easy vector and analyzed by DNA sequencing. b Shown are the nucleotide positions within the Pprx, where +1 is the transcription start site of Pprx. c The numbers in parentheses indicate the frequency of occurrence.
The possible mechanism of the transcriptional termination at the intergenic region of prx 1 ahpF was further investigated by predicting the secondary structure of the RNA deduced from the 137 bp intergenic region DNA. As a result, a stemloop structure comprising 49 bp RNA with an 18 base pairstem formed by a palindrome region and a 13-bases loop was singled out ( Fig. 4 C). The stem-loop structure was followed by a stretch of cytosine residues (CCCC) and the 3’ end of the prx 1 transcript (U), which is likely to serve as a rho-independent intrinsic terminator of transcription. The canonical intrinsic terminator of E. coli is known to include a poly(U) tract following a palindrome on the transcript [24] , and the poly(U) tract is known to contribute to the termination of transcription by reducing the stability the RNA-DNA hybrid [17] . Therefore, it is highly intriguing that the putative terminator in the prx 1 ahpF intergenic region has no poly(U) tract after the stem-loop structure ( Fig. 4 C).
It has been reported that disruption of the poly(U) tract of the intrinsic terminator reduces its transcriptional termination activity significantly but not completely [1] . Therefore, it is reasonable to hypothesize that the putative atypical terminator in the prx 1 ahpF intergenic region attenuates the transcription of prx 1 ahpF by not having the poly(U) tract, leading to the production of the high level of prx 1 transcript and the low level of prx 1 ahpF transcript. In order to experimentally verify this hypothesis, the P prx ligated with 136 bp of the intact intergenic region or 55 bp of the deleted intergenic region, in which the downstream half of the palindrome was deleted, was fused with the luxCDABE reporter genes ( Fig. 5 A). Culture luminescene was used to quantify the capacity of each intergenic region to terminate transcription ( Fig. 5 B). The RLU of pSS1324 carrying the intact intergenic region was about 6-fold lower than that of pSS1323 carrying the DNA fragment from the prx 1 coding region used as a control, indicating that the prx 1 ahpF intergenic region is indeed important for the transcriptional termination. For pSS1326 carrying the deleted intergenic region, which could not form the stemloop structure, the RLU was about 8-fold higher compared with pSS1323. This result suggested that the stem-loop structure is indispensable for the transcriptional termination. It is noteworthy that, although pSS1324 contained the intact stem-loop structure, its RLU was a considerable quantity of 1.0 × 10 4 , not fully abolished, implying that the stem-loop structure did not completely terminate the transcription to produce a low level of prx 1 ahpF transcript. Therefore, it was proved that the stem-loop structure functions as the attenuator of prx 1 ahpF transcription, which leads to the co-production of a high level of prx 1 transcript and a low level of prx 1 ahpF transcript.
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Deletion analysis of the prx1ahpF intergenic region. (A) Pprx-prx1ahpF intergenic region-luxCDABE transcriptional fusion pSS reporters were created as described in Materials and Methods. The promoter region (Pprx) and intergenic region of prx1ahpF are indicated by dark gray boxes and black boxes, respectively. Lollipops on the black boxes represent the stem-loop structure between the two genes, predicted in Fig. 4C. The 136 bp DNA fragment within the coding region of prx1 used as a positive control (for pSS1323) is presented as a dotted box. (B) Cellular luminescence was determined from the log phase culture (A600 = 0.5) of V. vulnificus containing each pSS reporter as indicated. Error bars represent the SEM. **, p < 0.005 relative to the V. vulnificus containing pSS1324.
The differential expression of prx 1 and prx 1 ahpF transcripts by the cis -acting intrinsic terminator, which leads to the production of a high level of Prx1 and a low level of AhpF, could have considerable benefits. First, the intrinsic terminator, an ancient regulatory mechanism that does not require any additional factors for its activity, is largely economical and efficient [24] . Second, the ratio of the amount of cellular Prx1 and AhpF proteins that resulted from the controlled expression by the terminator can be maintained stably, regardless of environmental conditions that bacteria can encounter. This is noticeable, because a high production of AhpF, the NADH-consuming protein, more than needed could cause disruption of the intracellular NADH homeostasis. Therefore, the quantitative control of AhpF may be important to avoid unnecessary loss of NADH and inappropriate flow of electrons in the cell. Consistent with this, it has been reported that E. coli cells overexpressing Pseudomonas putida ahpF were more sensitive to oxidants such as H 2 O 2 and tert -butyl hydroperoxide [9] .
In summary, our data presented here extended our understanding of the transcription of V. vulnificus prx 1 ahpF by demonstrating that the prx 1 ahpF genes are transcribed from a single promoter to produce both prx 1 and prx 1 ahpF transcripts by intergenic stem-loop structure-mediated transcriptional attenuation. The resulting precisely controlled expression of Prx1 and AhpF may be crucial for maintaining the NADH homeostasis of V. vulnificus and thereby further contribute to the ROS-scavenging activity of the pathogen under oxidative stress.
Acknowledgements
This work was supported by a grant from the Public Welfare and Safety Research Program (2012M3A2A1051679) and a grant from the Mid-career Research Program (2010-0029167) through the National Research Foundation funded by the Ministry of Science, ICT, and Future Planning, and a grant (14162MFDS972) from the Ministry of Food and Drug Safety, Republic of Korea.
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