INTRODUCTION

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Proteases are physiologically necessary for living organisms in which these enzymes display a variety of physiological functions, including pathogenesis and virulence. Alteromonas sp. strain O-7 is a gram-negative, flagellated, motile, and aerobic rod-shaped bacterium of marine origin (31). The strain secretes two serine proteinases (AprI and AprII) into the culture medium. We have already cloned and sequenced the genes (aprI and aprII) and characterized the relationship between structure and function of these enzymes (30, 32). These proteases were produced as large precursors consisting of four domains: the signal sequence, the N-terminal proregion, the mature AprI and AprII, and the conserved C-terminal extension. The C-terminal proregions were characterized by two repeated sequences which showed high sequence similarities with those of the C-terminal proregions from other known bacteria, such as Vibrio vulnificus (20), Vibrio cholerae (13), and Xanthomonas campestris (18). The homologous C-terminal proregion of V. vulnificus metalloprotease was essential for efficient attachment to insoluble protein substrates and erythrocyte membranes (20). Recently, many members of a subtilisin-like superfamily (subtilases) have been cloned and sequenced (24). Siezen and Leunissen classified the subtilases into families A (subtilisin family), B (thermitase family), C (proteinase K family), D (lantibiotic peptidase family), E (Kexin family), and F (pyrolysin family) based on amino acid sequence similarity (28). The mature AprI and AprII belong to families B and C, respectively.

Recently, we found that the ferric uptake regulator (Fur) box-like sequence was located upstream of the aprII gene. In most bacteria, iron-dependent regulation of genes depends to a large extent on the Fur repressor protein (9, 17). The Fur protein of Escherichia coli is a cytoplasmic 17-kDa polypeptide which binds iron as corepressor and consequently binds to the consensus sequence 5'-GATAATGATAATCATTATC-3', the so-called Fur box, repressing gene transcription under iron-rich conditions (2, 8, 22, 26). The Fur protein consists of two different domains, the N-terminal DNA binding domain and the C-terminal dimerization or metal binding domain (29). In E. coli more than 36 genes are transcriptionally regulated by the Fur protein (3). Therefore, the Fur protein plays an essential role in the iron acquisition system (9, 35). However, the regulation of the microbial serine protease-encoding gene by the Fur protein has not been reported. Here we describe how the aprII gene from Alteromonas sp. strain O-7 is regulated by Fur. The results of Western and Northern blot analyses demonstrated that aprII is a member of the iron regulon and plays an important role in the iron acquisition system of the strain. Furthermore, the nucleotide sequence of fur from the strain was determined, and the deduced amino acid sequence of Fur was compared with those of other microbial Fur proteins.

	MATERIALS AND METHODS

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Bacterial strains, plasmids, growth conditions, and DNA manipulations. Alteromonas sp. strain O-7 was cultured at 27°C in Bacto Marine Broth 2216 (Difco). E. coli JM109 was grown at 37°C on Luria-Bertani (LB) medium for the selection of transformants. E. coli H1780 (fur fiu::placMu13) (12) was kindly provided by S. Yamamoto (Okayama University, Okayama, Japan). Iron-depleted conditions were achieved by supplementing LB or Bacto Marine Broth 2216 medium with the iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) at a final concentration of 10 µg per ml, and iron-rich conditions were achieved by supplementing the above medium with FeSO₄ at a final concentration of 60 µM. Plasmids pUC18 and pUC19 were used as the cloning vectors. General DNA manipulations were carried out according to the method of Sambrook et al. (25).

Nucleotide sequence determination. Nucleotide sequencing was carried out by the dideoxy chain termination method with the Thermo Sequenase fluorescence-labeled primer cycle sequencing kit (Amersham Pharmacia Biotech) on a DNA sequencer (Hitachi SQ3000). The sequence data were analyzed using the GENETYX-WIN program (Software Development Co., Ltd.).

Western blotting and immunodetection. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done by the method of Laemmli (14). A prestained protein marker (low range, nacalai tesque) was used as a standard. Alteromonas sp. strain O-7 was cultured in iron-depleted or iron-rich Bacto Marine Broth 2216 medium until the optical density at 600 nm reached 1.5. The extracellular fraction was collected by centrifugation (24,650 × g for 5 min at 4°C), and 0.1 volume of 20% trichloroacetic acid was added to the supernatant. After centrifugation (24,650 × g for 5 min at 4°C), the pellet was directly dissolved with SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE and transferred to Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad Laboratories) with a semidry blotting apparatus (AE-6670; ATTO, Tokyo, Japan). The membrane was incubated for 1 h at room temperature with anti-AprII polyclonal mouse antiserum diluted to 1:25,000 in phosphate-buffered saline containing 0.1% Triton X-100. Bound antibody was detected by incubation for 1 h at room temperature with peroxidase-conjugated goat anti-mouse immunoglobulin G diluted to 1:2,000 in the same buffer. Horseradish peroxidase activity was detected by using 3-amino-9-ethylcarbazole as a substrate. The amount of production of AprII was measured by Image Gauge (version 3.0; Fuji Film, Tokyo, Japan).

Northern blot analysis. Total RNA was extracted from 1.5 ml of cell suspensions of Alteromonas sp. strain O-7 by using the SV total RNA isolation system (Promega) according to the manufacturer's instructions. The total RNA (5 µg) was separated electrophoretically in a 1.2% formaldehyde-containing agarose gel. RNA was transferred to a positively charged nylon membrane (Hybond-N+ membrane; Amersham Pharmacia Biotech) by VacuGene XL (Amersham Pharmacia Biotech). The HindIII-XbaI fragment (0.8 kb) from pAP661 carrying aprII was used as a probe (30). The fragment was labeled with alkaline phosphatase according to the manufacturer's instruction (AlkPhos Direct; Amersham Pharmacia Biotech). Alkaline phosphatase activity was visualized fluorescently by using CDP-Star chemiluminescent reagent (Amersham Pharmacia Biotech) and exposure to film (Hyperfilm-MP; Amersham Pharmacia Biotech). Perfect RNA markers (0.2 to 10 kb; Novagen) were used as a standard. The amount of transcript of aprII was measured by Image Gauge (version 3.0; Fuji Film).

Primer extension. About 5.0 µg of RNA was used to map the 5' end of the aprII transcript. Reverse transcription was initiated from the fluorescein isothiocyanate (FITC)-labeled primer, 5'-GATCATCGATTGCTTTGTTTGAC-3', complementary to the 5' end of the aprII coding region. The reaction was carried out at 50°C for 60 min using avian myeloblastosis virus reverse transcriptase (Promega). The primer extension and the sequencing reaction products were analyzed on a 6.0% denaturing polyacrylamide gel by DNA sequencer (Hitachi SQ3000). The sequence reaction was performed with the same primer.

Cloning and nucleotide sequencing of fur from Alteromonas sp. strain O-7. Alteromonas sp. strain O-7 total DNA, prepared as described previously, was used as a template DNA (28). The bidirectional degenerated oligonucleotide primers [furF, 5'-AA(A/G)AA(C/G/T)GC(A/C/T)GG(C/T)TT(A/G/T)AA(A/G)GT(A/T)AC-3'; furR, 5'-C(A/C/T)A(A/G)(A/G)TG(A/G)TC(A/G)TG(A/G)TGC(A/G)TG(A/C/G)TG-3'] were synthesized based on the highly conserved amino acid sequence in gram-negative Fur proteins. PCR amplification was performed for 20 cycles consisting of 94°C for 30 s, 50°C for 2 s, and 72°C for 25 s. The 192-bp product of the central region of Fur was cloned by using a pMOSBlue blunt-ended cloning kit (Amersham Pharmacia Biotech), sequenced, and used as a probe. Genomic DNA was digested with various restriction enzymes and electrophoresed on a 0.6% agarose gel. The probe hybridized strongly with 4.0-kb EcoRI fragment. The fragments in the range of 3.6 to 4.2 kb were excised from the gel and purified with QIAquick gel extraction kit (Qiagen). These were ligated into the dephosphorylated EcoRI site of pUC19 for the construction of plasmid DNA library. To obtain the additional downstream sequence, PCR was performed using the plasmid DNA library. Primers (furC, 5'-GATAATCAACACATCAGCGCAG-3'; M4, M13 universal primer) were designed based on the extract sequence of the 192-bp fragment and pUC19, respectively. Again, PCR was performed to clone the 5' upstream region of fur gene in the same manner as described above. furN (5'-ATACGAAGATCAGCTGGAATTG-3') and RV (M13 universal primer) were used as primers.

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession numbers AB040411 and AB040412.

	RESULTS

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Production of AprII under iron-depleted or -rich conditions. A map of the locus containing aprII gene is shown in Fig. 1. Sequence analysis of the upstream region of aprII demonstrated that the Fur box-like sequence was located 287 bp upstream of the ATG translation initiation codon of aprII. In most bacteria, iron-acquisition systems are negatively regulated by Fur. Under iron-rich conditions, the Fur-Fe²⁺ complex binds to the target DNA sequence (Fur box) in the promoter region of iron-regulated genes (9, 35). Therefore, we investigated whether the production of AprII is regulated by the environmental iron concentrations. This strain was cultured in iron-depleted or -rich medium, and then the level of AprII in the culture supernatants was analyzed by Western blotting (Fig. 2). The production of AprII was significantly repressed by 50% under iron-rich conditions. Furthermore, to determine whether the expression of aprII is regulated by iron at a transcriptional level, we performed Northern analysis of total RNA from Alteromonas sp. strain O-7 grown under iron-depleted or -rich conditions (Fig. 3). The RNA transcript of aprII was repressed by 60% under iron-rich conditions in comparison with that under iron-depleted conditions. These results indicate that AprII biosynthesis is regulated by iron through the control of transcription.

View larger version (9K): [in this window] [in a new window]   FIG. 1.   (A) Restriction map of pAP661; (B) domain structure of AprII. (A) Arrows indicate the ORF and the direction of transcription. aprII, serine protease-encoding gene; aprR, putative transcriptional regulator-encoding gene. (B) The arrows indicate the repeat amino acid sequence. Abbreviations: SP, signal peptide; AprII-N, N-terminal proregion; AprII-M, mature AprII; AprII-C, C-terminal proregion.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Western blot analysis of AprII. (A) Coomassie blue staining. Alteromonas sp. strain O-7 was cultured in iron-depleted () or iron-rich (+) medium. Each of the culture supernatants (900 µl) was added to 100 µl of 20% trichloroacetic acid and centrifuged. The pellets were dissolved with 10 µl of SDS-PAGE sample buffer and subjected to SDS-12% PAGE. Lane M, prestained molecular weight marker. (B) Western blot analysis of AprII. Samples were subjected to SDS-PAGE followed by immunodetection with 1:25,000 dilutions of AprII antibodies.

View larger version (70K): [in this window] [in a new window]   FIG. 3.   Northern blot analysis of the aprII transcript. Alteromonas sp. strain O-7 was cultured in iron-depleted () or iron-rich (+) medium. Lane M, RNA marker. (A) Formaldehyde-agarose gel electrophoresis of the total RNA from the strain; (B) Northern blot analysis.

Primer extension analysis. The Fur boxes of iron-regulated genes generally exist in close vicinity to their promoter regions (9, 35). However, the Fur box-like sequence of Alteromonas sp. strain O-7 was located far upstream of the ATG translation initiation codon of aprII. Thus, primer extension analysis was carried out to determine the transcriptional start site and to locate the promoter of aprII. Primer extension analysis determined the transcriptional start point of aprII to be the A that was 172 bp upstream from the initiation codon (Fig. 4). Although no 35 or 10 consensus sequence typical of prokaryotic promoters was present in front of the start point, TTTATT (positions 33 to 28 with respect to the transcriptional start point +1) and TATCCT (positions 11 to 6) with a 17-bp space similar to the consensus sequence seem to be the promoter of aprII. These results indicate that the Fur box-like sequence was not located immediately upstream of the putative 35 and 10 sequences and was 115 bp distal to it.

View larger version (40K): [in this window] [in a new window]   FIG. 4.   Determination of the transcription start site of aprII. (A) Primer extension and nucleotide sequencing were performed with the same FITC-labeled primer. The nucleotide sequence around the transcription start site is shown in lanes A, C, G, and T. The transcriptional start site is shown by an arrow (lane P). (B) Nucleotide sequence of the 5' upstream regions of aprII. The deduced amino acid sequence of AprII is shown below the nucleotide sequence. The Fur box-like sequence is boxed. The transcriptional start site is indicated with +1. The position of the FITC-labeled primer is shown below the nucleotide sequence by an arrow. The putative 35 and 10 regions are marked with solid and broken lines.

Expression of aprII in a fur mutant of E. coli. In order to confirm that the expression of aprII is under the regulation of Fur, we performed Northern blot analysis of the total RNA prepared from E. coli JM109(pAP661) and E. coli H1780(pAP661) grown under iron-depleted or -rich conditions. Plasmid pAP661 is a recombinant DNA containing aprII (Fig. 1). E. coli H1780 is a fur null mutant strain, and thus, under iron-rich conditions, there is no repression of the Fur-regulated gene (12). The total RNAs were prepared from these transformants grown under different conditions and were analyzed by Northern hybridization using aprII as a probe (Fig. 5). Under iron-depleted conditions, high-level transcription of aprII was detected in both E. coli JM109(pAP661) and E. coli H1780(pAP661). On the other hand, under iron-rich conditions, the transcription level of aprII was remarkably repressed in E. coli JM109(pAP661); however, in E. coli H1780(pAP661) the transcription of aprII was independent of iron. These results indicate that the expression of aprII is also regulated by Fur in a heterologous genetic background.

View larger version (69K): [in this window] [in a new window]   FIG. 5.   Transcriptional regulation of aprII in E. coli. E. coli JM109(pAP661) and E. coli H1780(pAP661) were cultured in iron-depleted () or iron-rich (+) LB medium. Lane M, RNA marker. (A) Formaldehyde-agarose gel electrophoresis of the total RNA; (B) Northern blot analysis of the aprII transcript.

Cloning and nucleotide sequence of fur from Alteromonas sp. strain O-7. In both Alteromonas sp. strain O-7 and E. coli JM109, the expression of the aprII gene was dependent on iron concentration in the medium. Thus, it was presumed that aprII expression is directly regulated by Fur of Alteromonas homologous to that of E. coli. To isolate fur from the genomic library of Alteromonas sp. strain O-7, degenerated primers (furF and furR, shown in Fig. 6) were synthesized based on the highly conserved amino acid sequence in gram-negative Fur proteins. The amplified DNA was about 200 bp, and its nucleotide sequence was determined. Although its deduced amino acid sequence revealed significant similarity with microbial Fur (11, 16, 26, 34), the PCR product was a truncated gene with the deletion of the 5' upstream and 3' downstream regions. To obtain the full length of the fur gene from Alteromonas sp. strain O-7, PCR was performed two times using the plasmid DNA library. The nucleotide sequence of the fur gene of the strain is shown in Fig. 6. The open reading frame (ORF) has an ATG start codon at position 497, which is preceded by a possible ribosome-binding site (GAGA) at a distance of 8 nucleotides. It could encode a protein of 148 amino acids with a calculated molecular mass of 16,682 Da. The molecular mass of the translated protein was close to that of E. coli Fur (26). A perfect inverted repeat sequence was located downstream of the fur terminal codon (TAA), which is likely to take part in rho-independent transcriptional termination of the fur. Comparison of the deduced amino acid sequence of the ORF with the BLAST database revealed that the gene encoded a protein homologous to Fur proteins from other gram-negative bacteria, such as E. coli (70% identity) (26), V. vulnificus (71% identity) (16), and Vibrio parahaemolyticus (71% identity) (34). His-90 was conserved in Alteromonas Fur, which is the putative Fe²⁺ binding amino acid residue of microbial Fur. A partial ORF which was located upstream of the fur ORF was identified as a flavodoxin gene (fldA) by the computer analysis (BLAST search program) (1). Flavodoxin together with ferredoxin- or flavodoxin-NADP⁺ reductase functions as a reduction system for many metalloproteins (1, 35). In E. coli, fldA was located 287 bp upstream from the fur start codon, whereas fldA of Alteromonas sp. strain O-7 was located 103 bp from the fur start codon of Alteromonas sp. strain O-7.

View larger version (31K): [in this window] [in a new window]   FIG. 6.   Nucleotide sequence of fur and the 3' region of fldA. The deduced amino acid sequences of Fur and FldA are shown below the nucleotide sequence. The inverted repeat sequence is indicated by convergent dashed arrows. The PCR primers furF, furR, furC, and furN are shown below the nucleotide sequence by arrows. The putative Shine-Dalgarno sequence is marked with a broken line.

	DISCUSSION

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The limiting factor in iron availability is the extremely low solubility of Fe³⁺ at neutral pH. This is true in marine and freshwater environments. Regardless of its deficiencies, iron is essential to marine organisms because it is a component of metalloproteins involved in many life processes, such as photosynthesis, respiration, and nitrogen fixation. Thus, marine organisms have evolved an elaborate mechanism to acquire iron, which is precisely regulated and exquisitely selective.

The results presented in this report clearly indicate that AprII from Alteromonas sp. strain O-7 is an iron-regulated serine protease and plays an important role to acquire iron in the marine environment. The Fur protein is a global regulator, which functions as a negative regulator in iron acquisition systems (5, 9, 33). Under iron-rich conditions, the Fur protein binds a ferrous ion and acquires a conformation able to bind target DNA sequences, termed the Fur box, of the iron-regulated genes (2, 8, 22, 26). As a consequence, the high concentration of iron leads to a shutoff of the expression of many genes involved in iron uptake (9). We have found that the Fur box-like sequence was located upstream of the aprII gene. The Fur-box like sequence shared a high degree of similarity with the E. coli Fur box consensus sequence (GATAATGATAATCATTATC) (2, 8, 22, 26). Thus, we examined whether the expression of aprII is regulated by iron concentrations. As expected, Western blot analysis revealed that the production of AprII was significantly reduced under iron-rich conditions. Furthermore, Northern blot analysis demonstrated that the expression of aprII was regulated at the transcriptional level. The Fur boxes are generally found in the promoter region of iron-regulated genes (26). Escolar et al. have recently shown that the basis of the mechanism of repression used by Fur in the aerobactin promoter is direct competition between RNA polymerase and Fur-Fe²⁺ for the same target sites around the 35 hexamer of the major aerobactin promoter (7). To determine the putative 35 and 10 sequences of aprII, primer extension analysis was performed. Unlike those of the Fur boxes so far reported, the Fur box-like sequence from the present strain was located 115 bp upstream from the transcriptional start point, indicating that Fur could not independently prevent RNA polymerase from commencing synthesis of mRNA. This suggests that there exists another regulatory protein in the regulation of aprII expression, which binds in the region between the Fur box-like sequence and promoter and changes the DNA structure to prevent the binding of RNA polymerase. Sequence analysis of the region downstream of the aprII gene revealed an ORF encoding a polypeptide of 279 amino acids with a calculated molecular mass of 31,232 Da (Fig. 1). The BLAST search program revealed that the protein, designated AprR, belongs to the AraC/XylS family of transcriptional regulators (10). Among these regulator proteins, regulation of the araBAD (P_araBAD) and araC (P_araC) promoters by the AraC protein has been extensively characterized (4, 19). In the absence of arabinose, one monomer of the AraC dimer occupies the araI₁ site, while the other occupies a half-site approximately 200 bp away, known as araO₂. The dimer bound to target sequence in this way generates a DNA loop, which prevents transcription from P_araBAD and P_araC. In analogy with the AraC protein, AprR might generate a DNA loop together with Fur under iron-rich conditions and block the access of RNA polymerase to the promoter of aprII. The precise role of AprR in the regulation of AprII biosynthesis in Alteromonas sp. strain O-7 remains to be determined.

We cloned a gene encoding Alteromonas Fur that is structurally similar to that of E. coli. In E. coli fur, the Fur box was located in the promoter region, and transcription of the gene is autoregulated by the protein itself (6). However, the Fur box could not be identified in the upstream region of Alteromonas fur. This may indicate that Alteromonas Fur recognizes a sequence different from that recognized by E. coli Fur or that the expression of Alteromonas fur may be regulated by another gene product involved in the iron acquisition systems of the strain. To examine functionality of the cloned Alteromonas Fur, the gene was inserted in the expression vector pGEX-6P-1 (Amersham Pharmacia Biotech) and transformed in E. coli H1780, which carries a lacZ gene under the control of Fur in a background without fur (12). High expression of -galactosidase causes colonies to appear red on MacConkey agar plates, whereas reduced expression results in a pale colony color (12). The fur transformant showed a red and a pale color under iron-depleted and iron-rich conditions, respectively, indicating that Alteromonas Fur can bind to the E. coli Fur box (data not shown).

In order to acquire iron from the extracellular milieu, many bacteria have the ability to secrete siderophores, which are low molecular mass and bind ferric ions with very high affinity (15, 21, 23). Alteromonas sp. strain O-7 was grown on a chrome azurol S plate for detecting siderophore excretion (27). Orange halos around the colonies were formed, demonstrating that the strain produced siderophores (data not shown). Several lines of evidence allow us to speculate that AprII biosynthesis is activated under iron-depleted conditions and iron is released from metalloproteins by proteolysis of AprII. Subsequently, siderophores chelate the released iron, and the cell recovers the iron-siderophore complexes through specific outer membrane receptors.