INTRODUCTION

The ability to acquire iron from the very limited free iron sources in the tissues and fluids of mammalian hosts is an important component of bacterial virulence (1, 2). Highly virulent strains of the fish pathogen Vibrio anguillarum possess a very efficient plasmid-mediated high affinity iron uptake system, which can compete for the complexed iron with the host high affinity iron-binding proteins such as transferrin, lactoferrin, and ferritin (2, 3). There are two components of this plasmid-mediated iron uptake system. One is the siderophore anguibactin, the actual iron scavenger; the other is an energy-coupled iron transport system that processes the iron-anguibactin complex (2, 4, 5). The iron transport region of pJM1 encodes the fatA, fatB, fatC, and fatD genes. FatA is the receptor for ferric anguibactin complexes (6); FatB is a membrane-located lipoprotein that shares domain homology with periplasmic binding proteins (7); and FatC and FatD are cytoplasmic integral membrane proteins (8).

Despite the fact that iron is an essential nutrient, it is also potentially toxic, since it promotes the formation of reactive hydroxy radicals. It is not surprising then that the levels of cellular entry and storage are tightly controlled by the iron concentration of the cell (9). In Escherichia coli and other bacteria, global iron-responsive transcription occurs via the Fur repressor protein (2, 10, 11). In the presence of Fe²⁺, Fur acts as a repressor via sequence-specific protein-DNA interactions at the promoter regions of Fur-regulated genes (12, 13, 14). In V. anguillarum, regulation is governed by both negative and positive factors (15). Positive regulation is mediated by the pJM1 plasmid-encoded 110-kDa protein AngR and the TAF products, which act synergistically (16, 17). Negative regulation is mediated by the chromosome-encoded Fur protein and by the plasmid-encoded antisense RNA (18, 19, 20). RNA is a 650-nucleotide RNA, which is encoded in the fatB coding region in the complementary strand and is preferentially expressed under iron-rich conditions. We recently constructed a plasmid in which RNA is under the control of an external promoter (19). By using this construct we were able to show that the presence of RNA led to a dramatic reduction of FatA and FatB expression, probably by interaction between the polycistronic fatDCBA mRNA and RNA, which may result in a modification of the secondary structure of this polycistronic mRNA (19, 20). This change appears to enhance processing upstream of the fatA coding region, resulting in a concomitant inhibition of FatA synthesis and a degradation of the fatB region in this mRNA (19, 20). Since that work was carried out by using fusions of RNA with external promoters, very little was known about its physiological role in the iron repression of the pJM1 system and in the mechanism by which iron or other products regulate its synthesis.

In this work we demonstrate that RNA indeed plays an important biological role on the negative regulation of the iron transport genes fatA and fatB under physiological conditions and show that the Fur protein is essential for RNA transcription initiation, while iron plays a role in increasing the RNA stability.

EXPERIMENTAL PROCEDURES

The bacterial strains and plasmids used in this study are listed in Table I.

Table I. Bacterial strains and plasmids Strain or plasmid Relevant characteristic(s) Source E. coli HB101 F thr1, leuB6, dam4, thi1, hsdS1, lacY1, tonA21, , supE44 Ref. 40 JM109 recA1, endA1, gyrA96, thi1, hsdR17, (rk, mk+), supE44, relA1, (lac-proAB), [F traD36, proAB, lacIqlacZM15] Ref. 41 V. anguillarum 775 Natural isolate, prototype, (pJM1) Ref. 42 H775-3 Plasmidless derivative of 775 Ref. 5 775MET11 fur mutant isolated in the presence of 10 mM MnCl2 Ref. 18 pBluescript SK+ Cloning vector Stratagene pBR325 Cloning vector Ref. 43 pKK232-8 Promoterless cat vector Ref. 44 pQC6.1 189-bp HindIII-Sau3AI fragment containing the transcriptional start points b and c for RNA cloned in pKK232-8 This work pQC6.2 390-bp Sau3AI fragment containing the transcriptional start point a for RNA cloned in pKK232-8 This work pQC6.4 579-bp HindIII-Sau3AI fragment of the RNA promoter region cloned in pKK232-8 This work pQC6.5 2.7-kb PvuII-StuI fragment of the RNA promoter region cloned in pKK232-8 This work pJHC-LW217 866-bp HindIII fragment of the RNA promoter region cloned in pKK232-8 Ref. 20 pJHC-A122 Truncated RNA which lacks 100 nucleotides from the 5 end, cloned in pBR325 Ref. 19 pMET13.1 92-bp ClaI-SalI fragment of the fatA gene cloned in pBluescript SK+ Ref. 19 pJHC-S400 135-bp SalI-HindIII fragment of the fatB region cloned in pBluescript SK+ Ref. 27 pQC10 270-bp HindIII-EcoRI fragment of the cat gene cloned in pBluescript SK+ This work pQC3.5 420-bp SalI-ClaI fragment of the aroC gene cloned in pBluescript SK+ This work

E. coli strains were grown at 37 °C in Luria broth or on Luria broth solidified with 1.5% (w/v) agar (Difco). V. anguillarum strains were grown at 26 °C in Trypticase soy broth supplemented with 1% (w/v) sodium chloride (TSBS), Trypticase soy agar supplemented with 1% (w/v) sodium chloride (TSAS), or M9 minimal medium (3) supplemented with 0.2% casamino acids (Difco). Iron-limiting conditions were established by adding the iron chelator ethylenediamine-di-(o-hydroxyphenylacetic) acid (EDDA)¹ (Sigma) to a final concentration of 0.5-1 µM in M9 minimal medium. Iron-rich conditions were obtained by adding 100 µg/ml ferric ammonium citrate to M9 minimal medium. Antibiotics were added to the culture medium at the following concentrations: ampicillin, 500 µg/ml; chloramphenicol (Cm), 30 µg/ml.

Plasmid DNA was prepared by the method of Birnboim and Doly (21). Restriction endonuclease digestion of DNA was performed under the conditions recommended by the supplier (Life Technologies, Inc.). Transformation was carried out as described by Cohen et al. (22). Conjugation was carried out as described by Tolmasky et al. (23).

Overnight cultures of V. anguillarum in M9 minimal medium with the appropriate antibiotics were diluted 50-fold into 50 ml of fresh M9 minimal medium plus either 0.5-1 µM EDDA or 100 µg/ml ferric ammonium citrate and grown to an optical density of 0.4-0.6 at 600 nm. Cells were chilled on ice and pelleted by centrifugation. Total RNA was extracted using the hot phenol method as described by von Gabain et al. (24). RNase protection assays were carried out as described previously (20). The fatA-specific riboprobe was made from plasmid pMET13.1, linearized with ClaI, and transcribed with T3 RNA polymerase. The fatB-specific riboprobe was prepared from plasmid pJHC-S400, linearized with SalI, and transcribed with T3 RNA polymerase. The RNA-specific riboprobe was prepared from plasmid pJHC-S400, linearized with HindIII, and transcribed with T7 RNA polymerase. The cat-specific riboprobe was prepared from pQC10, linearized with PvuII, and transcribed with T3 RNA polymerase. The aroC-specific riboprobe was used as an internal control and prepared from plasmid pQC3.5, linearized with RsaI, and transcribed with T3 RNA polymerase.

Overnight cultures of V. anguillarum in M9 minimal medium with the appropriate antibiotics were diluted 50-fold into two flasks each containing 400 ml of fresh M9 minimal medium, one with the addition of 1 µM EDDA, the other with the addition of 100 µg/ml ferric ammonium citrate, and cultures were grown to an optical density of 0.4-0.6 at 600 nm. Rifampicin was then added at a final concentration of 200 µg/ml, and 40-ml aliquots were removed from the culture at various times (0, 0.25, 0.5, 0.75, 1, 2, 4, 8, and 12 min), and immediately placed in dry ice with ethanol. Total RNAs were prepared by the hot phenol method as described by von Gabain et al. (24). The level of RNA was measured by RNase protection experiments using an RNA-specific riboprobe.

Site-directed Mutagenesis of the RNA Promoter

A 189-bp HindIII-Sau3AI DNA fragment containing the promoter region of RNA was cloned in pBluescript SK⁺ to generate pQC7, which was used as the template to perform site-directed mutagenesis. The specific mutations were generated by using the Muta-Gene Phagemid in vitro mutagenesis kit (Bio-Rad Laboratories, Richmond, CA) and synthetic mutagenic oligonucleotides. Mutations were confirmed by DNA sequencing using the dideoxy chain-termination method (25) and the Sequencing Kit (U.S. Biochemical Corp.) with the T3 or T7 sequencing primers. The mutated HindIII-Sau3A1 fragment was recloned back to pKK232-8 to generate mutated RNA promoter-cat fusion plasmids and transferred by conjugation to V. anguillarum H775-3 as described previously (23). The transconjugant strains were tested for the minimal inhibitory concentration for chloramphenicol resistance and the level of chloramphenicol acetyltransferase (CAT) was determined by the ELISA method.

10 µg of purified plasmid pQC6.1 DNA was mutagenized with hydroxylamine as described by Rose and Fink (26). Mutated DNA was transformed into E. coli HB101 and then conjugated to a Fur-deficient V. anguillarum 775MET11 and selected for the chloramphenicol resistance (Cm^r) phenotype on TSAS plates containing 5 µg/ml of Cm and 5 mM of MnCl₂. DNAs were then purified from Cm^r colonies, and the 189-bp HindIII-Sau3AI insert was cloned in pBluescript SK⁺ to perform sequencing analysis. The mutated HindIII-Sau3AI DNA fragment was recloned into the wild type vector pKK232-8 to exclude the possibility of mutated vector sequences. The promoter activity was assessed by the cat mRNA level as detected by using a RNase protection assay with a cat-specific riboprobe.

V. anguillarum strains were cultured in TSBS or M9 minimal medium under either iron-rich or iron-limiting conditions for 16 h. 1.5 ml of the culture was microcentrifuged and used to prepare cell lysates and perform the ELISA assay using the CAT ELISA kit (Boehringer Mannheim) following the supplier's instructions.

RESULTS

Identification and Mutational Studies of RNA Promoters

We have previously mapped by primer extension three possible transcription start points for RNA: Ra, Rb, and Rc (27). In order to more precisely define the promoter region, we subcloned a 390-bp Sau3AI DNA fragment containing the transcriptional start point Ra and its promoter region, pa. We also subcloned a 189-bp HindIII-Sau3AI fragment containing the transcriptional start points Rb plus Rc and their promoter regions pb and pc, since their respective 10 and 35 regions overlap. These fragments were cloned into plasmid vector pKK232-8, which has a promoterless chloramphenicol acetyltransferase gene (cat). The cloning orientation of the fragments containing the putative RNA promoter sequences was confirmed by sequencing analysis. Plasmids were conjugated into V. anguillarum H775-3, and promoter strength was assessed by either the CAT levels or by the chloramphenicol resistance phenotype. We found that the major promoter activity is associated with the fragment carrying the promoters pb and pc (designated pb/c) in plasmid pQC6.1. This plasmid confers to V. anguillarum resistance to 10 µg/ml of Cm on TSAS plates, while the subclone carrying the pa promoter region, pQC6.2, only shows weak promoter activity (cells harboring pQC6.2 are sensitive to 5 µg/ml of Cm on TSAS plates) (Fig. 1). To discriminate which promoter (pb or pc) is active in pQC6.1, we recloned the 189-bp HindIII-SalI fragment into pBluescript SK⁺ to generate, by site-directed mutagenesis, a series of mutations on the 10 and/or 35 regions of pb/c. Base pair substitutions were made on nucleotides considered to be among the most highly conserved bases in the E. coli 10 and 35 sequences. The mutated HindIII-SalI fragment was cloned back into pKK232-8 and conjugated into V. anguillarum H775-3 to determine promoter activity by measuring CAT levels using the ELISA method. Mutations in the 10 regions of pb (AATAGGCT to gAgAGGCg) and pc (TAAACT to TcAACg), respectively, resulted in a reduction in promoter activity of ~8-fold for the pb mutant and of only 1.4-fold for the pc mutant (Fig. 2, see mutants b1 and c). Combining the mutations of both 10 regions of pb and pc resulted in a 73-fold reduction of the promoter activity (Fig. 2, see mutants b/c and b1/c). These results suggest that both promoters pb and pc must act synergistically. It is of interest that mutations in the 35 regions of pc did not appreciably change the promoter activities (Fig. 2, see mutant c2).

Physical and genetic map of the pJM1 iron transport region and construction of RNA-

Mutational analysis of the RNA promoter.

Iron Regulation of the RNA Promoter

We have previously shown that RNA is preferentially found under iron-rich conditions (19, 20, 27). This enhancing effect of iron on the steady state levels of RNA could be due to regulatory mechanisms occurring at either the transcription initiation or posttranscriptional level. To identify whether the RNA promoter is regulated by iron, we first measured the highly active RNA pb/c promoter activity under iron-rich and iron-limiting conditions. We used the cat fusion plasmid pQC6.1 in V. anguillarum 775 and determined CAT by the ELISA method. Table II shows that the promoter pb/c is fully active and that there is no significant difference of promoter activity under iron-rich and iron-limiting conditions. However, it is still possible that there is a cis element required for iron regulation of the RNA promoter that is missing in the pQC6.1, since it contains only 64 nucleotides upstream of the start point Rb. Therefore, we generated other RNA-cat fusion constructs containing 454-1800 bp upstream of the start point Rb (Fig. 1) and conjugated them into the V. anguillarum 775 strain. It is clear from Table II that the promoter activities from all of these fusion constructs are not iron-regulated. The same results were obtained by measuring cat mRNA level of the V. anguillarum 775 harboring these RNA-cat fusion constructs grown under either iron-rich or iron-limiting conditions (Fig. 7B and data not shown). These results demonstrate that the promoter activity of RNA is not regulated by iron.

Table II. Regulation of CAT activity from RNA-cat fusion constructs in V. anguillarum 775  V. anguillarum strains harboring various RNA-cat fusion constructs were cultured for 16 h in M9 minimal medium with either addition of 100 µg/ml of ferric ammonium citrate (iron-rich) or 0.5 µM EDDA (iron-limiting). The CAT activity was measured using the ELISA method as described under ``Experimental Procedures.'' CAT activity (A410) (Fe/EDDA ratios)a pQC6.1 0.87  ± 0.20 pQC6.4 0.76  ± 0.12 pQC6.5 0.76  ± 0.15 a  Values shown were obtained from CAT activity under iron-rich conditions normalized to that under iron-limiting conditions.

Influence of the Iron Status of the Cell on the Stability of RNA

In the previous section we presented results demonstrating that the RNA promoter is not activated by increasing iron concentrations. However, there is a dramatic increase in the steady state amount of RNA when cells are grown under iron-rich conditions. It is possible, therefore, that the iron concentration of the cell plays a role in maintaining the steady state levels of RNA posttranscriptionally rather than at the transcription initiation level. To investigate this possibility, we studied the half-life of RNA in the presence of rifampicin under iron-rich and iron-limiting conditions. RNAs were prepared from rifampicin-treated cultures of V. anguillarum 775, harboring the pJM1 plasmid, grown under either iron-rich or iron-limiting conditions. RNA levels were determined by RNase protection assay using a riboprobe for specific recognition of RNA (Fig. 3A). The results, shown in Fig. 3A, demonstrate that the half-life of RNA is about 1 min under iron-rich conditions. Conversely, under iron limitation, most of the RNA is already degraded by the 0 time point. RNA was also detected at 30 s under iron-limiting conditions for an unexplainable reason. These results indicate that high iron concentrations, either directly or indirectly, must play an important role in protecting RNA from degradation. In order to further confirm that the regulation of RNA by iron is mostly at the posttranscriptional level, we investigated the stability of RNA synthesized from pJHC-A122, a construct that encodes an active RNA that lacks the first 100 nucleotides from the 5 end as well as its indigenous promoter. In this construct, RNA is synthesized under the control of a foreign promoter, the tetracycline resistance gene promoter, and thus we are sure that there is no regulation at the initiation level. RNAs were prepared from a strain of V. anguillarum 775 harboring pJHC-A122, and RNA levels were determined by a RNase protection assay using the same RNA-specific riboprobe as that used in Fig. 3A. As shown in Fig. 3B, the half-life of RNA is less than 2 min under iron-limiting conditions, whereas under iron-rich conditions, the level of RNA is only slightly decreased up to 12 min. This result confirms that the regulation of RNA by iron is mostly at the posttranscriptional level by enhancing the stability of RNA. It is of interest that, although conserving the increased stability under iron-rich conditions, the RNA synthesized under the control of the tetracycline promoter is more stable as a whole than that synthesized under the control of the indigenous promoter, under both iron-rich and iron-limiting conditions. It is possible that the 5 fusion of vector sequences may result in a hybrid RNA with a more stable secondary structure, albeit not so different as to change the ancestral requirement for increasing iron concentrations for overall stability.

Decay of RNA under iron-rich and iron-limiting conditions.

Regulation by Fur of Antisense RNA Synthesis and Iron Transport Gene Transcription

We have identified a chromosome-encoded Fur protein in V. anguillarum 775 that plays a role in the negative regulation of the synthesis of iron transport proteins and catechol production (18, 19). Since the expression of the iron transport genes fatA and fatB is also negatively regulated by RNA, while RNA expression is regulated by iron, it was of interest to determine whether Fur intervenes in the regulation of RNA synthesis under iron-rich conditions. Therefore, we measured the synthesis of RNA as well as the synthesis of fatA and fatB-specific mRNA as a function of the iron concentration in a fur mutant strain of V. anguillarum 775MET11 that is deficient in the synthesis of Fur protein. Total RNA was isolated from V. anguillarum 775 and 775MET11 cultured in M9 minimal medium under either iron-rich or iron-limiting conditions. The RNA levels were determined by RNase protection assays with probes complementary to the sense RNA in the fatA and fatB regions and to RNA (Fig. 4). We found that RNA could be detected in the wild type strain 775 preferentially under iron-rich conditions, while RNA could not be detected either in iron-rich or iron-limiting conditions in the Fur-deficient mutant 775MET11, even after very long exposures of the x-ray film, in which the signal for RNA in the wild type strain had been greatly intensified (Fig. 4 and data not shown). These results strongly suggest that the Fur product is essential for RNA synthesis independently of the iron concentration of the cell. Conversely, the fatA and fatB specific mRNA was constitutively expressed. This result demonstrates that Fur regulates the expression of fatA and fatB genes at the transcriptional level. However, under iron-limiting conditions, the mRNA levels of these iron transport genes were considerably higher in the Fur- and RNA-deficient mutant as compared with that in the wild type strain (Fig. 4, compare lane 4 with lane 2 and lane 9 with lane 7). These differences in levels are inversely related to the levels of RNA found in the mutant and the wild type strain. The same results have been obtained from experiments in which the aroC probe was used as an internal control, since aroC transcription is independent of the iron status of the cell (Ref. 28 and data not shown). The fact that the expression of the iron transport genes fatA and fatB is greatly enhanced in the absence of RNA suggests that RNA may play an important role on the negative regulation of these iron transport genes under physiological conditions.

and RNA expression by

In order to further determine the contribution of Fur and RNA on the negative regulation of the iron transport genes, we used a construct, pJHC-A122, that synthesizes the RNA independently of the iron status of the medium. By introducing this construct into the Fur-deficient V. anguillarum strain 775MET11, we examined the expression of iron transport genes by RNase protection, using a riboprobe specific for the fatB region (Fig. 5). Total RNAs were isolated from V. anguillarum 775 and 775MET11 with or without harboring pJHC-A122, cultured in M9 minimal medium under either iron-rich or iron-limiting conditions. We found that RNA is constitutively expressed at a high level in both the Fur-deficient mutant and in the wild type strain harboring pJHC-A122 (Fig. 5A), while fatB-specific transcripts were greatly degraded in those strains harboring the pJHC-A122 plasmid independently of whether they had a deficiency in Fur (Fig. 5B). These results confirmed not only the important role of RNA in the negative regulation of the iron transport genes but also that Fur is essential for RNA synthesis, possibly required in an interaction with the promoter region of RNA, which in turn activates transcription.

Fur in strains harboring constitutive high levels of RNA.

Fur Is Essential for RNA Transcription Initiation

In the previous section we showed that Fur is essential for the expression of RNA. In order to determine whether Fur regulates RNA synthesis at the transcription initiation level, we conjugated pQC6.1, in which the RNA promoter pb/c is fused to the cat gene, into the Fur-deficient V. anguillarum strain 775MET11. Promoter activity was measured by CAT ELISA and RNase protection of cat mRNA. Fig. 6A shows that the RNA promoter (pb/c) activity was 7-12-fold less as compared with that in the wild type strain harboring pQC6.1. The similar result was obtained from RNase protection studies of cat mRNA (Fig. 7B). These results demonstrated that Fur either directly or indirectly is essential for initiation of transcription of RNA. Inspection of Figs. 6A and 7B also shows that this Fur-mediated regulation is independent of iron. It is possible that an interaction between Fur and the DNA upstream of the RNA coding sequences is necessary for this transcription initiation activity. Therefore, sequences upstream of RNA were searched for homology to the E. coli Fur box. Fig. 6B shows the comparison of the putative Fur box with the consensus sequence for the E. coli Fur box. Only 10 out of 19 nucleotides were identical to the E. coli Fur consensus sequence. This result is not surprising, since the Fur requirement for RNA transcription initiation is independent of the iron concentration of the cell and the Fur box is recognized by the complex Fur-Fe²⁺. Therefore, it is likely that a binding site for the holoprotein may have a different sequence or, alternatively, that this pleiotropic protein may be regulating another gene whose product is the actual regulator of RNA synthesis.

Regulation of RNA promoter activity by

To further investigate the mechanism of Fur action on the control of the RNA transcription initiation, we first investigated whether the RNA promoter is active in the E. coli Fur-deficient strain BN4020. The RNA promoter-cat fusion plasmid pQC6.1 was transformed into the isogenic strains of E. coli Fur-proficient BN402 and Fur-deficient BN4020, and we examined whether the transformants were Cm^r. It was found that pQC6.1 endowed both BN402 and BN4020 with a resistance to 30 µg/ml Cm in Luria agar plates, indicating that the RNA promoter pb/c is active in E. coli independently of Fur. This result also suggests that if activation of RNA transcription in V. anguillarum requires Fur, which acts to facilitate the interaction of RNA polymerase with the RNA promoter, then this requirement is not necessary with the E. coli transcription machinery. Alternatively, if Fur activates RNA transcription via repression of an intermediate repressor in V. anguillarum, then this repressor does not exist in E. coli.

Next, we intended to isolate promoter mutations that did not require Fur, which could be interpreted as indicating a positive regulatory function for Fur, as is the case for the catabolite gene activator protein (CAP). Plasmid pQC6.1 DNA was mutagenized with hydroxylamine. Mutated pQC6.1 DNA was first transformed into E. coli HB101 and then conjugated into the V. anguillarum Fur-deficient 775MET11. The activated RNA promoter pb/c mutants in 775MET11 were selected on TSAS plates containing 5 µg/ml of Cm and 5 mM of MnCl₂. 50 Cm^r colonies were isolated. 10 of them were selected to clone the 189-bp promoter insert in pBluescript SK⁺ to perform sequencing analysis. A single mutation, from TAGGCT to TAGaCT, in the 10 region of promoter pb was found in all 10 clones (Fig. 7A). To ensure that the mutation phenotype resided only within the promoter insert, the 189-bp insert was recloned into pKK232-8 that had not been exposed to the mutagen, creating plasmid pQC-HA6.1. This plasmid was then transferred to V. anguillarum 775MET11 by conjugation. RNAs were prepared from V. anguillarum strains 775 harboring pQC6.1, 775MET11 harboring pQC6.1, and 775MET11 harboring pQC-HA6.1, cultured in M9 minimal medium under either iron-rich or iron-limiting conditions. The cat mRNA level was determined by a RNase protection assay, using a riboprobe for specific detection of the cat transcript. It was found that the mutated promoter pb/c of pQC-HA6.1 in 775MET11 had much higher activity than the wild type promoter pb/c of pQC6.1 in 775 and 775MET11 (Fig. 7B). This result indicates that this mutation in the promoter region greatly affected RNA polymerase binding to the promoter region, thus enhancing RNA polymerase function significantly. It also suggests that, if Fur is directly required for activation of transcription of RNA, then this requirement is not necessary when the single nucleotide from TAGGCT to TAGaCT, in the 10 region of promoter pb, was mutated. This nucleotide change could lead to a conformational change of DNA, thus promoting the interaction of RNA polymerase with the RNA promoter region independently of the presence of Fur. A similar kind of mutations (class III mutations) in the 10 region of the lac promoter that facilitated lac expression in the absence of activator catabolite gene activator protein (CAP)-cAMP complex have also been isolated in E. coli (29). Alternatively, the mutation might have affected a putative repressor binding site, thus greatly increasing promoter activity.

DISCUSSION

Antisense RNA has been demonstrated to play a number of regulatory roles including the control of transcription, translation, transposition, plasmid copy number and plasmid killing as well as a role in the control of plasmid replication and in the lysis/lysogeny cycle (30, 31). However, very little is known about the mechanisms of regulation of the expression of antisense RNA. RNA is the first antisense RNA that was reported to play a role in the repression of the expression of iron transport genes (27). The expression of RNA has been shown to be regulated by iron (19, 20, 27), and three possible transcription start points have been mapped at Ra, Rb, and Rc (27). By subcloning a fragment containing promoter regions corresponding to each transcription start point upstream of the cat gene, we were able to demonstrate in this work that the major promoter activity is associated with the region that we designated pb/c. Mutagenesis analysis of each 10 region of pb and pc allowed us to identify that both pb and pc are active. RNA is mainly detected under iron-rich conditions; therefore, we attempted to understand the nature of this differential behavior by studying the promoter activity of various RNA-cat gene fusion plasmids under iron-rich and iron-limiting conditions. Our results demonstrated that iron does not regulate RNA transcription initiation. It became clear, however, by studying the half-life of RNA, that iron regulates RNA synthesis at the posttranscriptional level by affecting RNA stability regardless of whether RNA synthesis is under the control of the indigenous or of an external promoter. However, the mechanism of the stabilization by iron of the RNA half-life is not clear. It is known that in eukaryotic mRNAs, iron-regulatory elements (IREs) can modulate the expression of the transferrin receptor and ferritin genes posttranscriptionally dependent on the location of IRE within the context of the transcript. Binding of the iron-regulatory protein to IRE at the 5 end of a ferritin mRNA inhibits translation initiation by blocking ribosome binding, while binding of the iron-regulatory protein to IRE at the 3 end of a transferrin receptor mRNA stabilizes this mRNA by blocking nuclease access to mRNAs (32). The specific secondary structure of a hairpin with a terminal hexaloop of the sequence CAGUG(U/C) is an important feature of IREs (32). We have found by sequencing analysis that in RNA there are several stem-loop structures at the 3 end as well as in other regions of the RNA transcript, and the stem-loop structures at the 3 end are also present on a truncated RNA molecule that lacks the first 100 nucleotides at the 5 end but that still has inhibitory functions.² This truncated RNA molecule, synthesized under the control of an external promoter, is also stabilized by iron. Therefore, these secondary structures may play an important role in RNA stability. One of the possible mechanisms of iron stabilization of RNA is that there is a protein factor that is only able to bind RNA in the presence of iron. This binding prevents RNA from ribonuclease attack either by a secondary structure change of RNA or by a steric hindrance effect of this protein factor.

Previous work, using a construct in which RNA synthesis was under the control of a strong external promoter, the tetracycline resistance gene promoter, showed that a high level of RNA leads to a reduced expression of the fatA and fatB genes (19, 20). However, direct evidence of the physiological role of RNA under the control of its own promoter was lacking. Therefore, we initiated experiments to understand the contribution of RNA in the control of the expression of the iron transport genes under physiological conditions. We first found that RNA was not synthesized in a Fur-deficient strain of V. anguillarum. By using the RNA promoter pb/c-cat gene fusion studies we were able to demonstrate that the Fur requirement was at the RNA transcription initiation level. This Fur regulation was independent of the iron concentration of the cell. It was of interest that the lack of RNA in the Fur-deficient mutant led to a dramatic enhancement in the levels of the fatA and fatB transcripts (Fig. 4), strongly suggesting that RNA may play an important role in the negative regulation of the expression of the iron transport genes fatA and fatB under physiological conditions.

The question is, of course, how Fur regulates the initiation of transcription of RNA. Fur is a pleiotropic protein that regulates many iron-regulated and some non-iron-regulated genes (11, 33, 34). In most cases, Fur acts as a transcriptional repressor with Fe²⁺ as a cofactor. Recently, a number of studies indicated that Fur could also act as an activator (35, 36). For example, sodB gene expression is positively regulated by Fur, possibly via a direct DNA-protein interaction (35). Since no Fur box has been found in the sodB gene, it is possible that a different mechanism might be involved in the positive regulation of the sodB gene by Fur (35). Our results in this work showed that Fur is essential for RNA synthesis at the transcription initiation level, yet only a very poor Fur box has been found in the RNA promoter region. Several possibilities come to mind with respect to the mechanisms of Fur action on RNA synthesis. First, Fur could serve as an activator via a direct interaction with the RNA promoter; second, Fur could be a repressor of another unidentified factor, which would be an actual repressor for the promoter region of RNA; the presence of Fur would lead to repression of this additional repressor gene, resulting in expression of RNA. Several other scenarios, in which Fur could play a role as an activator of other genes that may be involved in RNA expression, are also possible. In the effort to distinguish these possibilities, we have isolated, by hydroxylamine mutagenesis of the RNA promoter, 50 RNA promoter mutants that are active in the V. anguillarum Fur-deficient strain. All of the mutants mapped at a single nucleotide within the 10 region of the promoter pb, from TAGGCT to TAGaCT, in 10 mutants that had been sequenced. This mutation greatly enhanced the promoter activity in the Fur-deficient strain compared with the wild type promoter activity in the wild type strain (Fig. 7B). It is possible that the nucleotide G to A change leads to a favorable structure for the RNA polymerase function. It is known that CAP is essential for transcription initiation of the lac promoter. In the presence of cAMP, CAP binds to a site located at the 35 region of the lac promoter. The binding of the CAP-cAMP complex stimulates the productive interaction of RNA polymerase with the promoter possibly involved in a protein-protein interaction with RNA polymerase or a DNA distortion that facilitates transcription initiation (37, 38, 39). However, certain types of mutations, class III mutations, have been isolated that facilitate lac expression in the absence of the CAP-cAMP complex (29). These mutations were mapped within the 10 region of the lac promoter and thought to affect open-complex formation of RNA polymerase, which is the rate-determining step for the wild type lac promoter and which is also influenced by the CAP-cAMP complex. Therefore, the fact that we were able to isolate RNA promoter mutants similar to class III mutations of the lac promoter suggests that Fur could have a function similar to that of CAP for the lac system, in the activation of RNA expression. We also found that the activation of RNA transcription initiation is independent of Fur in E. coli, possibly because of the existence of a different transcription machinery, i.e. the RNA polymerase in E. coli may not require Fur to activate transcription initiation of RNA. The absence of a ``Fur box'' at the RNA promoter region and the fact that the regulation of RNA by Fur is independent of iron indicate that a unique mechanism must be involved in this regulation in which Fur interaction with DNA may occur by binding with sequences different from the ``Fur box.'' However, these experiments did not exclude the possibility that Fur could also function as a repressor of another gene that encodes a repressor of RNA synthesis or as an activator of another activator for RNA synthesis. Further experiments to understand the mechanism of Fur in the control of the synthesis of RNA are currently under way.