Introduction
Bacteria continually encounter toxic reactive oxygen species (ROS), such as superoxide anion (O2- .), hydrogen peroxide (H2O2), 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 H2O2 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 prx1 gene, and the prx1ahpF transcription is positively regulated by V. vulnificus OxyR1, a homolog of E. coli OxyR only in cells exposed to high levels of exogenous H2O2, to result in the prx1 and prx1ahpF transcripts [16]. However, molecular analysis of the prx1ahpF transcription has not yet been experimentally verified. Here, we provided molecular genetic evidence that the V. vulnificus prx1ahpF 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 prx1 transcript and a low level of prx1ahpF transcript. Finally, the physiological role of the differential expression of prx1 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℃.
Table 1.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 of ahpF Mutant
λTo inactivate ahpF in vitro, a unique BamHI 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 BamHI 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 BamHI site of the PCR products, and ligated with SacI-SpeI-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].
Table 2.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 A600 of 0.5 were isolated using an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) [19]. When necessary, the strains were exposed to 250 µM H2O2 (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 prx1 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 prx1 was end-labeled with [γ-32P]-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 prx1 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 of prx1ahpF Operon
The pair of primers PRX1-PM01 and -PM02 (Table 2) was designed and used to amplify the 2,470bp DNA fragment encompassing the prx1ahpF operon, including its promoter region Pprx. 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 Pprx) within the Pprx on pSS1105 was mutated to -16G, -8C, and +1T, respectively (Fig. 3A) 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 prx1 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 prx1 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 A600 of 0.5 after being exposed to 250 µM H2O2 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 prx1 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 -prx1ahpF Intergenic Region-luxCDABE Transcriptional Fusions and Measurement of Cellular Luminescence
The 342 bp prx1 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 prx1ahpF intergenic region (for pSS1324), amplified by PCR with the pair of primers PRX1AHPF-03 and -04 (Table 2), or with 55 bp of deleted-prx1ahpF 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 prx1 coding region was amplified by PCR using the pair of primers PRX1-C01 and -C02 (Table 2) and fused with the same prx1 upstream region (for pSS1323). Each PCR product was ligated with SacI-SpeI-digested pBBR-lux carrying promoterless luxCDABE [18] to create pSS reporters (pSS1323, pSS1324, or pSS1326) (Table 2; Fig. 5B), 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
The V. vulnificus prx1ahpF Genes Are Transcribed into Two Transcripts
The V. vulnificus ahpF gene is located downstream of the prx1 gene, and the two coding regions of prx1 and ahpF are transcribed in the same direction with the 136 bp intergenic region (Fig. 1A) [2]. To analyze the transcription pattern of the V. vulnificus prx1ahpF, northern blot analyses were performed using the total cellular RNAs isolated from the wild type, the prx1 mutant, and the ahpF mutant. Since the expression of prx1 is known to be induced in cells exposed to high levels of exogenous H2O2 [3], the V. vulnificus cells were exposed to 250 µM H2O2 for 30 min before the RNA isolation. As shown in Fig. 1B, the PRX1P probe was hybridized to the 0.6 kb RNA corresponding to the prx1 transcript when total RNA was isolated from the wild-type cells exposed to 250 µM H2O2, reconfirming that the expression of prx1 is induced in response to exogenous H2O2. In addition, approximately 2.3 kb RNA was detected by PRX1P when the RNA was isolated from the H2O2 -exposed wild type but not ahpF mutant (Fig. 1B). On the basis of the DNA sequence of prx1ahpF, it was anticipated that a polycistronic prx1ahpF transcript would be approximately 2.3 kb long (Fig. 1A). Northern blot analysis was performed using the AHPFP probe, and a single RNA corresponding to the prx1ahpF transcript was detected in the RNA isolated from the H2O2 -exposed wild type but not ahpF mutant (Fig. 1C), indicating that the prx1ahpF genes were also transcribed as a single operon. Therefore, it appeared that the V. vulnificus prx1ahpF genes transcribed into two transcripts, a high level of prx1 transcript and a low level of prx1ahpF transcript.
Fig. 1.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 prx1ahpF Genes Are Transcribed from a Single Promoter
To clarify whether the prx1 and prx1ahpF transcripts are transcribed from each own promoter with different activity or from one promoter, the transcription start site of prx1ahpF 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 H2O2 (Fig. 2A). The 5’ end of the prx1 or prx1ahpF transcript was located 47 bp upstream of the translational initiation codon of prx1 and subsequently designated +1 (Fig. 2B). The putative promoter constituting this transcription start site was named Pprx. The sequences for the -10 and -35 regions of Pprx were assigned on the basis of similarity to consensus sequences of the E. coli σ70 promoter (Fig. 2B). In addition, the sequences extending from -73 to -37, relative to the transcription start site of Pprx (shaded boxes in Fig. 2B), 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 prx1ahpF 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 prx1 and prx1ahpF transcripts are generated from the single promoter, Pprx.
Fig. 2.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 Pprx governs the transcription of prx1ahpF in vivo, three nucleotides of Pprx, -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 prx1ahpF operon with the wild-type and the point-mutated Pprx were cloned into pRK415 to create pSS1105, pSS1110, pSS1111, and pSS1112 (Fig. 3A). The activities of each Pprx on the pSS1105, pSS1110, pSS1111, and pSS1112 were compared by northern blot analyses using the RNAs isolated from each plasmid-containing prx1 mutant. Since PRX1P did not detect any transcripts in the RNA isolated from the prx1 m utant (Fig. 1B, lane 3), the detected bands in Fig. 3B 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 Pprx [4]. As expected, the mutations of -16T to -16G (on pSS1110) and of -8T to -8C (on pSS1111) resulted in the complete abolishment of Pprx activity, as determined based on the intensities of the prx1 and prx1ahpF transcripts detected by PRX1P (Fig. 3B). In addition, the mutation of +1G to +1T (on pSS1112) resulted in the increased expression of prx1ahpF (Fig. 3B), which is possibly attributed to the increased unwinding efficiency of the DNA duplex around the transcription start site of Pprx by the mutation. Therefore, these results confirmed that the transcription of prx1ahpF results in the prx1 and prx1ahpF transcripts from the single promoter, which was determined by the primer extension analysis (Fig. 2A).
Fig. 3.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 the prx1ahpF Intergenic Region Acts as a Transcriptional Attenuator to Create prx1 and prx1ahpF Transcripts
There are still two possible ways for the transcription of prx1ahpF into the two transcripts. One is by degradation of some of the prx1ahpF transcript through an unknown mechanism, whereas another is by transcriptional attenuation at the intergenic region of prx1ahpF, resulting in the high level of prx1 transcript and the low level of prx1ahpF transcript. In the northern blot analysis, however, the prx1 transcript was still detected by PRX1P in the RNA isolated from the ahpF mutant that cannot produce the prx1ahpF transcript (Fig. 1B, 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 prx1ahpF intergenic region, using the 3’ RACE assay. Total RNA isolated from the wild type exposed to 250 µM H2O2 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. 4A) 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 prx1 transcript with the 3’ end, +689T, located 83 bp downstream of the translational stop codon of prx1 as indicated in Fig. 4B. These results suggested that the transcription of prx1ahpF can be terminated at the intergenic region to result in the prx1 transcript.
Fig. 4.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/).
Table 3.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 prx1ahpF 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. 4C). The stem-loop structure was followed by a stretch of cytosine residues (CCCC) and the 3’ end of the prx1 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 prx1ahpF intergenic region has no poly(U) tract after the stem-loop structure (Fig. 4C).
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 prx1ahpF intergenic region attenuates the transcription of prx1ahpF by not having the poly(U) tract, leading to the production of the high level of prx1 transcript and the low level of prx1ahpF transcript. In order to experimentally verify this hypothesis, the Pprx 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. 5A). Culture luminescene was used to quantify the capacity of each intergenic region to terminate transcription (Fig. 5B). The RLU of pSS1324 carrying the intact intergenic region was about 6-fold lower than that of pSS1323 carrying the DNA fragment from the prx1 coding region used as a control, indicating that the prx1ahpF 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 × 104, not fully abolished, implying that the stem-loop structure did not completely terminate the transcription to produce a low level of prx1ahpF transcript. Therefore, it was proved that the stem-loop structure functions as the attenuator of prx1ahpF transcription, which leads to the co-production of a high level of prx1 transcript and a low level of prx1ahpF transcript.
Fig. 5.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 prx1 and prx1ahpF 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 H2O2 and tert-butyl hydroperoxide [9].
In summary, our data presented here extended our understanding of the transcription of V. vulnificus prx1ahpF by demonstrating that the prx1ahpF genes are transcribed from a single promoter to produce both prx1 and prx1ahpF 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.
References
- Abe H, Aiba H. 1996. Differential contributions of two elements of rho-independent terminator to transcription termination and mRNA stabilization. Biochimie 78: 1035-1042. https://doi.org/10.1016/S0300-9084(97)86727-2
- Baek WK, Lee HS, Oh MH, Koh MJ, Kim K, Choi SH. 2009. Identification of the Vibrio vulnificus ahpC1 gene and its influence on survival under oxidative stress and virulence. J. Microbiol. 47: 624-632. https://doi.org/10.1007/s12275-009-0130-x
- Bang YJ, Oh MH, Choi SH. 2012. Distinct characteristics of two 2-Cys peroxiredoxins of Vibrio vulnificus suggesting differential roles in detoxifying oxidative stress. J. Biol. Chem. 287: 42516-42524. https://doi.org/10.1074/jbc.M112.421214
- Browning DF, Busby SJ. 2004. The regulation of bacterial transcription initiation. Nat. Rev. Microbiol. 2: 57-65. https://doi.org/10.1038/nrmicro787
- Christman MF, Morgan RW, Jacobson FS, Ames BN. 1985. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell 41: 753-762. https://doi.org/10.1016/S0092-8674(85)80056-8
- Dubbs JM, Mongkolsuk S. 2007. Peroxiredoxins in bacterial antioxidant defense. Subcell. Biochem. 44: 143-193. https://doi.org/10.1007/978-1-4020-6051-9_7
- Dubbs JM, Mongkolsuk S. 2012. Peroxide-sensing transcriptional regulators in bacteria. J. Bacteriol. 194: 5495-5503. https://doi.org/10.1128/JB.00304-12
- Fang FC. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2: 820-832. https://doi.org/10.1038/nrmicro1004
- Fukumori F, Kishii M. 2001. Molecular cloning and transcriptional analysis of the alkyl hydroperoxide reductase genes from Pseudomonas putida KT2442. J. Gen. Appl. Microbiol. 47: 269-277. https://doi.org/10.2323/jgam.47.269
- Hall A, Karplus PA, Poole LB. 2009. Typical 2-Cys peroxiredoxins - structures, mechanisms and functions. FEBS J. 276: 2469-2477. https://doi.org/10.1111/j.1742-4658.2009.06985.x
- Imlay JA. 2008. Cellular defenses against superoxide and hydrogen peroxide. Annu. Rev. Biochem. 77: 755-776. https://doi.org/10.1146/annurev.biochem.77.061606.161055
- Jacobson FS, Morgan RW, Christman MF, Ames BN. 1989. An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage. J. Biol. Chem. 264: 1488-1496.
- Jeong HS, Kim SM, Lim MS, Kim KS, Choi SH. 2010. Direct interaction between quorum-sensing regulator SmcR and RNA polymerase is mediated by integration host factor to activate vvpE encoding elastase in Vibrio vulnificus. J. Biol. Chem. 285: 9357-9366. https://doi.org/10.1074/jbc.M109.089987
- Keen NT, Tamaki S, Kobayashi D, Trollinger D. 1988. Improved broad host-range plasmids for DNA cloning in gram-negative bacteria. Gene 70: 191-197. https://doi.org/10.1016/0378-1119(88)90117-5
- Kim KS, Lee Y. 2004. Regulation of 6S RNA biogenesis by switching utilization of both sigma factors and endoribonucleases. Nucleic Acids Res. 32: 6057-6068. https://doi.org/10.1093/nar/gkh939
- Kim S, Bang YJ, Kim D, Lim JG, Oh MH, Choi SH. 2014. Distinct characteristics of OxyR2, a new OxyR-type regulator, ensuring expression of peroxiredoxin 2 detoxifying low levels of hydrogen peroxide in Vibrio vulnificus. Mol. Microbiol. 93: 992-1009. https://doi.org/10.1111/mmi.12712
- Komissarova N, Becker J, Solter S, Kireeva M, Kashlev M. 2002. Shortening of RNA: DNA hybrid in the elongation complex of RNA polymerase is a prerequisite for transcription termination. Mol. Cell 10: 1151-1162. https://doi.org/10.1016/S1097-2765(02)00738-4
- Lenz DH, Mok KC, Lilley BN, Kulkarni RV, Wingreen NS, Bassler BL. 2004. The small RNA chaperone Hfq and multiple small RNAs control quorum sensing in Vibrio harveyi and Vibrio cholera. Cell 118: 69-82. https://doi.org/10.1016/j.cell.2004.06.009
- Lim JG, Bang YJ, Choi SH. 2014. Characterization of the Vibrio vulnificus 1-Cys peroxiredoxin Prx3 and regulation of its expression by the Fe-S cluster regulator IscR in response to oxidative stress and iron starvation. J. Biol. Chem. 289: 36263-36274. https://doi.org/10.1074/jbc.M114.611020
- Markham NR, Zuker M. 2008. UNAFold: software for nucleic acid folding and hybridization, pp. 3-31. In Keith JM (ed). Bioinformatics: Structure, Functions and Applications, Vol. 453. Humana Press, Totowa, NJ.
- Miller RA, Britigan BE. 1997. Role of oxidants in microbial pathophysiology. Clin. Microbiol. Rev. 10: 1-18.
- Miller VL, Mekalanos JJ. 1988. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacteriol. 170: 2575-2583. https://doi.org/10.1128/jb.170.6.2575-2583.1988
- Milton DL, O’Toole R, Horstedt P, Wolf-Watz H. 1996. Flagellin A is essential for the virulence of Vibrio anguillarum. J. Bacteriol. 178: 1310-1319. https://doi.org/10.1128/jb.178.5.1310-1319.1996
- Mitra A, Angamuthu K, Jayashree HV, Nagaraja V. 2009. Occurrence, divergence and evolution of intrinsic terminators across Eubacteria. Genomics 94: 110-116. https://doi.org/10.1016/j.ygeno.2009.04.004
- Nathan C, Shiloh MU. 2000. Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc. Natl. Acad. Sci. USA 97: 8841-8848. https://doi.org/10.1073/pnas.97.16.8841
- Oka A, Sugisaki H, Takanami M. 1981. Nucleotide sequence of the kanamycin resistance transposon Tn903. J. Mol. Biol. 147: 217-226. https://doi.org/10.1016/0022-2836(81)90438-1
- Poole LB, Hall A, Nelson KJ. 2011. Overview of peroxiredoxins in oxidant defense and redox regulation. Curr. Protoc. Toxicol. 49: 7.9.1-7.9.15.
- Sambrook J, Russell D. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
- Storz G, Imlay JA. 1999. Oxidative stress. Curr. Opin. Microbiol. 2: 188-194. https://doi.org/10.1016/S1369-5274(99)80033-2
- Storz G, Jacobson F, Tartaglia L, Morgan R, Silveira L, Ames B. 1989. An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp. J. Bacteriol. 171: 2049-2055. https://doi.org/10.1128/jb.171.4.2049-2055.1989
- Storz G, Zheng M. 2000. Oxidative stress, pp. 47-59. In Storz G, Hengge-Aronis R (eds.). Bacterial Stress Responses. ASM Press, Washington, DC.
- Toledano MB, Kullik I, Trinh F, Baird PT, Schneider TD, Storz G. 1994. Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell 78: 897-909. https://doi.org/10.1016/S0092-8674(94)90702-1
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