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Originally published In Press as doi:10.1074/jbc.M602294200 on April 26, 2006

J. Biol. Chem., Vol. 281, Issue 25, 16842-16848, June 23, 2006
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A Novel Enzyme Conferring Streptothricin Resistance Alters the Toxicity of Streptothricin D from Broad-spectrum to Bacteria-specific*

Yoshimitsu Hamano{ddagger}1, Nobuyasu Matsuura§, Miwa Kitamura{ddagger}, and Hiroshi Takagi{ddagger}2

From the {ddagger}Department of Bioscience, Fukui Prefectural University, Fukui 910-1195, Japan and the §Department of Life Science, Okayama University of Science, Okayama 700-0005, Japan

Received for publication, March 10, 2006 , and in revised form, April 24, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Streptothricins (STs)3 (Fig. 1) are broad-spectrum antibiotics that were first isolated from Streptomyces lavendulae in 1943 (1). All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. STs inhibit protein biosynthesis in prokaryotic cells; in addition, they strongly inhibit the growth of eukaryotes such as yeast (2–4), fungi (5), protozoa (6), insects (7), and plants (8). Therefore, STs are used as effective selective agents for recombinant DNA work in some of these organisms. However, STs are not currently used therapeutically due to their nephrotoxicity (9–11).

To date, many ST-resistance genes have been identified in transposons such as Tn1825 and Tn1826, which have been isolated from bacteria that are resistant to ST (12); such transposons have also been isolated from human pathogens such as Shiga toxin-producing Escherichia coli (13) and the Shigella strain (14). Bacterial resistance to antibiotics that inhibit protein biosynthesis (e.g. aminoglycosides) can occur as a result of decreased antibiotic uptake and accumulation, modification of 16 S RNA or ribosomal proteins, or enzymatic modification of the antibiotics (15). However, in the case of bacterial resistance to STs, only one resistance mechanism has yet been identified: the resistance is due to a modification of the ST molecule by monoacetylation at the beta-amino group (position 16) of beta-lysine(s) (Fig. 1). In fact, in ST producers such as S. lavendulae (16), Streptomyces rochei (17), and Streptomyces noursei (18, 19), the ST-resistance genes encoding N-acetyltransferase (NAT) have been identified and their role in self-resistance against their own STs has been investigated. Based on this resistance mechanism and the fact that streptothricin D (ST-D) is a more effective antibiotic than streptothricin F (ST-F), the moiety of beta-lysine(s) has been shown to play a crucial role in antibiotic activity. On the other hand, Inamori et al. (20) and Taniyama et al. (21) have independently reported that ST-F-acid (Fig. 1, termed as racenomycin-A-acid in their studies), chemically prepared from ST-F, did not exhibit antibiotic activity against bacteria, fungi, and plants; however, the biological activity of ST-D-acid was not tested. This result confirmed that streptolidine lactam is essential for antibiotic activity. We therefore hypothesized that microorganisms showing resistance to STs through alternative resistance mechanisms might produce an enzyme that hydrolyzes streptolidine lactam, thereby inactivating STs. Actinomycetes are known to produce many natural products with structural diversity occurring due to the unique substrate specificities of the enzymes. Therefore, we focused on Streptomyces, the representative strains belonging to actinomycetes, to efficiently identify our target enzyme.

Here, we describe the cloning of a gene whose product confers ST resistance through the modification of streptolidine lactam, as expected. Additionally, we used the recombinant enzyme of this gene product to investigate its functions and properties. We also discuss an interesting observation regarding the selective toxicity of an ST compound that was converted by the gene product.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals—STs (clonNAT, a mixture of ST-F and ST-D; the ST-F: ST-D ratio was ~5:1) were obtained from Werner BioAgents (Jena, Germany). All other chemicals used were of analytical grade.


Figure 1
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  		FIGURE 1.
Chemical structure of STs.

 
Bacterial Strains, Plasmids, and General Techniques for DNA Manipulation—Streptomyces albulus NBRC14147 was used as the DNA source for cloning of the sttH gene. The media and growth conditions for S. albulus NBRC14147 have been described previously (22). The E. coli-Streptomyces shuttle vector pWHM3 (23) and Streptomyces lividans TK23 (23) were employed for cloning the sttH gene and S. lavendulae NBRC12789 was used as an ST producer. The nat gene encoding NAT for STs was obtained from the pHN15 plasmid (Werner BioAgents). The pQE30 plasmid, E. coli M15(pREP4) (Qiagen, Tokyo, Japan), and E. coli XL1-Blue MRF' (Toyobo, Osaka, Japan) were used to overexpress the recombinant protein. Recombinant DNA procedures for the E. coli and Streptomyces strains were performed using standard techniques (23, 24). Southern blotting was performed using the enhanced chemiluminescence (ECL) direct nucleic acid labeling and detection system (Amersham Biosciences). The Saccharomyces cerevisiae CKY8 strain (MAT{alpha} ura3-52 leu2-3112) and the yeast episomal plasmid pAD4 were employed for the expression of the sttH and nat genes in S. cerevisiae. This plasmid is known as the E. coli-Saccharomyces shuttle vector and contains selection markers, specifically, the ampicillin-resistance gene (for E. coli) and the LEU2 gene (for S. cerevisiae). The CKY8 strain was transformed using the Yeastmaker Yeast Transformation System 2 (Clontech). S. cerevisiae CKY8 strains possessing pAD4 derivatives were grown in a synthetic complete medium (25) lacking L-leucine (SC-Leu) or yeast extract-peptone-dextrose (YPD) medium (25). The following microorganisms were used for minimum inhibitory concentration (MIC) studies with STs and ST-acids: S. cerevisiae S288C (same genetic background as strain CKY8), Schizosaccharomyces pombe L972, E. coli W3110, Bacillus subtilis NBRC13169, Staphylococcus aureus AB, and S. aureus FIR1169 (26).

PCR Amplification of the Genes Encoding NATs in the Streptomyces Strains—Based on the highly conserved amino acid sequence of the NATs of S. lavendulae (16), S. rochei (17), and S. noursei (18, 19), we designed primers 5'-GACGC(G/C)GA(A/G)GC(G/C)ATCGA(A/G)G (G/C)(G/C)CT(G/C)GA-3' and 5'-GTTST(C/T)GTT(G/C)GT(G/C) AC(C/T)TC(G/C)AGCCA-3'. PCR amplification was performed under the following conditions: 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min.

Cloning of the sttH Gene of S. albulus NBRC14147—The genomic DNA of S. albulus NBRC14147 was partially digested with Sau3AI. The Sau3AI fragments that were larger than 2.0 kb were then ligated into the BamHI site of the pWHM3 plasmid carrying the thiostrepton-resistance gene. S. lividans TK23 was transformed using this ligated DNA, and transformants that were resistant to both STs, i.e. to ST-D and ST-F (100 µg/ml of a mixture of ST-F and ST-D with an ST-F:ST-D ratio of ~5:1) and thiostrepton (20 µg/ml) were isolated on R5 agar medium (23). Of the 13 transformants, one harboring a plasmid containing a 2.9-kb insert (pWHM3-st11) was selected for further experiments (Fig. 2). After we determined the complete nucleotide sequence of the 2.9-kb fragment, one plasmid carrying open reading frame-2 (ORF2)-ORF3 (pWHM3-orf2–3) and another carrying ORF1 (pWHM3-orf1) were constructed using pWHM3 (Fig. 2).

Estimation of the Start Codon of the sttH (ORF2) Gene—To estimate the start codon in the sttH gene, seven forward primers and one reverse primer were designed, as shown schematically in Fig. 2, and were used to amplify the sttH gene. Additional BamHI sites (5'-GGGGGATCC-3') were attached to all forward primers, and an additional HindIII site (5'-ACCAAGCTT-3') was attached to the reverse primer. PCR cycles were performed under standard conditions. After sequence confirmation, each of the seven amplified fragments was inserted into the same site of pQE30 to obtain the following plasmids: pQE30-SHF1R (carrying a PCR fragment amplified with primers SH-F1 and SH-R), pQE30-SHF2R (SH-F2 and SH-R), pQE30-SHF3R (SH-F3 and SH-R), pQE30-SHF4R (SH-F4 and SH-R), pQE30-SHF5R (SH-F5 and SH-R), pQE30-SHF6R (SH-F6 and SH-R), and pQE30-SHF7R (SH-F7 and SH-R). Each resulting plasmid was introduced into E. coli XL1-Blue MRF'. In these transformants, the MICs for STs were determined on Luria-Bertani (LB) agar plates (24) containing ampicillin (100 µg/ml), 0.1 mM isopropyl-beta-D-thiogalactopyranoside, and STs (0–100 µg/ml, a mixture of ST-F and ST-D with a ST-F:ST-D ratio of ~5:1).

Identification of ST-F- and ST-D-derived Compounds Formed by rSttH, and Kinetic Studies of rSttH—Following the protocol specified by the manufacturer (Qiagen), the recombinant SttH (rSttH) overexpressed as the N-terminal His6-tagged fusion protein was purified from the cell-free extract of E. coli M15(pREP4) harboring pQE30-SHF6R. The reaction mixture (500 µl) contained 100 mM sodium phosphate buffer (pH 6.5), 1 mg/ml ST-F or ST-D, and 100 µg/ml purified rSttH. After incubation of the reaction mixture with or without rSttH at 30 °C for 1 h, chloroform extraction was carried out to remove proteins. The water layers were then analyzed by reverse-phase high performance liquid chromatography (HPLC) using an ion-pair reagent. The analytical conditions were as follows: column, C18 reverse-phase Cosmosil 5C18-AR-II (250 x 4.6 mm) (Nacalai Tesque, Kyoto, Japan); column temperature, 30 °C; detection, 210 nm; and flow rate, 1 ml/min. We used two different mobile phases: 0.1% heptafluorobutyric acid, 18% acetonitrile (for reaction with ST-F) and 0.1% heptafluorobutyric acid, 23% acetonitrile (for reaction with ST-D). Kinetic assays were performed under conditions identical to those described above, except that the enzyme concentration (2 µg/ml) and the reaction time (5 min) were reduced to enable measurement of steady-state kinetic parameters. All assays were carried out under linear conditions. The reactions were terminated by the addition of 15 µl of 2 N HCl, and the products were then analyzed by HPLC. A Lineweaver-Burk plot was used to estimate the kinetic constants. The native molecular mass of rSttH was estimated by gel filtration using a Cosmosil 5Diol-300 column (7.8 x 600 mm; Nacalai Tesque) equilibrated with 20 mM sodium phosphate buffer (pH 7.0) containing 100 mM Na2SO4.

Purification of ST Compounds—ST-F and ST-D were purified from commercially available STs (a mixture of ST-F and ST-D) by reverse-phase HPLC under conditions identical to those described above, except for column size (250 x 10 mm), flow rate (4.72 ml/min), and acetonitrile concentration (25%). After removal of the organic solvent from the HPLC fractions, the aqueous layer was freeze-dried to yield the compounds in the form of a white powder. The enzymatically synthesized ST-F-acid and ST-D-acid were purified by the same procedure as that described for ST-F and ST-D purification.

Determination of ST-F-acid and ST-D-acid Structures—Positive ion electrospray ionization tandem mass spectra (ESI-MS/MS) images of the ST compounds were obtained in the positive mode using a Finnigan MAT TSQ 7000 (Quadrapole Tandem MS/MS Analyzer). The 1H NMR spectral data for ST-F-acid and ST-D-acid were recorded at 500 MHz using a JEOL LNM-LA500 spectrometer and were the following: (i) ST-F-acid, 1H NMR (500 MHz, D2O) {delta}: 1.60 (4H, m, H-17, 18), 2.51 (1H, dd, J = 8 and 17 Hz, H-15), 2.62 (1H, dd, J = 5 and 17 Hz, H-15), 2.86 (2H, br s, H-19), 2.94 (1H, dd, J = 10 and 13 Hz, H-4), 3.08 (1H, dd, J = 3 and 13 Hz, H-3), 3.49 (1H, m, H-16), 3.55 (2H, d, J = 6 Hz, H-12), 3.98 (2H, m, H-5), 3.99 (1H, t, J = 3, Hz, H-9), 4.05 (1H, dd, J = 3 and 10 Hz, H-8), 4.14 (1H, t, J = 6 Hz, H-11), 4.31 (1H, d, J = 5 Hz, H-2), 4.59 (1H, d, J = 4 Hz, H-10), 4.95 (1H, d, J = 9 Hz, H-7). (ii) ST-D-acid, 1H NMR (500 MHz, D2O) {delta}: 1.43 (4H, m, H-18, 24), 1.51 (4H, m, H-17, 23), 1.60 (4H, m, H-29, 30), 2.40 (1H, dd, J = 8 and 16 Hz, H-27), 2.43 (1H, dd, J = 8 and 16 Hz, H-21), 2.48 (1H, dd, J = 8 and 16 Hz, H-15), 2.50 (1H, dd, J = 5 and 17 Hz, H-27), 2.53 (1H, dd, J = 5 and 17 Hz, H-21), 2.57 (1H, dd, J = 5 and 17 Hz, H-15), 2.85 (2H, m, H-31), 2.86 (2H, br s, H-19), 2.94 (1H, dd, J = 10 and 13 Hz, H-4), 3.05 (3H, m, acetyl), 3.08 (1H, dd, J = 3 and 13 Hz, H-3), 3.45 (4H, m, H-19, 25), 3.48 (3H, m, H-16, -22, and -28), 3.54 (2H, d, J = 6 Hz, H-12), 3.98 (1H, t, J = 3 Hz, H-9), 3.99 (2H, m, H-5), 4.06 (1H, dd, J = 3 and 10 Hz, H-8), 4.14 (1H, t, J = 6 Hz, H-11), 4.31 (1H, d, J = 5 Hz, H-2), 4.58 (1H, d, J = 4 Hz, H-10), and 4.94 (1H, d, J = 9 Hz, H-7).

Investigation of the ST-D Resistance Profile in the E. coli and S. cerevisiae Strains Overexpressing SttH or NAT—To construct an E. coli strain that overexpressed NAT, the following set of primers was designed and used for PCR: 5'-GGGGGATCCACCACTCTTGACGACACGGCT-3' (forward) and 5'-ACCAAGCTTTCAGGGGCAGGGCATGCTCAT-3' (reverse). Restriction enzyme sites (underlined) and a stop codon (boldface) were introduced into these primers. An expression vector (pQE30-nat) possessing a fragment amplified with these primers was constructed using pQE30. The MICs of ST-F and ST-D in the E. coli XL1-Blue MRF' strain harboring pQE30-nat or pQE30-SHF6R were determined on LB agar plates containing ampicillin (100 µg/ml), 0.1 mM isopropyl-beta-D-thiogalactopyranoside, and STs (0–4 mM).

We also constructed a S. cerevisiae CKY8 strain overexpressing NAT or SttH by the following procedure. The following two sets of primers with an additional restriction site (underlined) and a stop codon (boldface) were designed and used for PCR: 5'-ACCAAGCTTAATATGACCACTCTTGACGACACG-3' (forward primer for the nat gene), 5'-AAACTGCAGTCAGGGGCAGGGCATGCTCAT-3' (reverse primer for the nat gene); 5'-ACCAAGCTTACCATGCCCCCCGAGACCGCCGCG-3' (forward primer for the sttH gene), 5'-AAACTGCAGTCAGCGCGCTGGAGCGGGCGG-3' (reverse primer for the sttH gene). After sequence confirmation, the amplified fragments were inserted into the same sites of pAD4 to yield pAD4-nat and pAD4-sttH in which these genes were expressed under the control of the constitutive yeast promoter of alcohol dehydrogenase. An S. cerevisiae CKY8 strain harboring pAD4-nat or pAD4-sttH was grown in SC-Leu medium containing STs (ST-F or ST-D, 0–4 mM), and the MICs for ST-F and ST-D were then determined.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing Analysis of the ST-resistance Gene—Based on the present studies of MICs for STs in the Streptomyces strains that have not been accepted as ST producers, S. albulus NBRC14147 was found to be more resistant to STs than the ST producer S. lavendulae NBRC12789 (Table 1). Furthermore, PCR using primers designed for genes that encode NATs for STs and the genomic DNA of the NBRC14147 strain as a template did not show any amplified fragments. However, a specific amplified fragment was detected when the genomic DNA of S. lavendulae NBRC12789 was used (data not shown). Therefore, because the NBRC14147 strain was not believed to contain a homologous gene encoding NAT, this strain was selected as a source of the novel ST-resistance gene. To obtain the ST-resistance gene, the NBRC14147 strain genomic library was constructed with the pWHM3 plasmid carrying the thiostrepton-resistance gene. This library was introduced into S. lividans TK23, which is sensitive to STs and thiostrepton, and transformants resistant to both thiostrepton (20 µg/ml) and STs (>400 µg/ml, a mixture of ST-F and ST-D with ST-F:ST-D ratio of ~5:1) were isolated. From these transformants, we selected one that harbored the pWHM3 plasmid carrying a 2.9-kb fragment (pWHM3-st11, Fig. 2 and Table 1) for further experiments because Southern blotting using this fragment as a probe revealed that all plasmids isolated from these transformants carried the 2.9-kb fragment (data not shown).


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  		TABLE 1
ST resistance profiles of the Streptomyces strains

 
The sequencing analysis of the 2.9-kb DNA fragment and frame analysis with the codon usage for Streptomyces strains (27) revealed two ORFs (ORF1 and -2) and one partial ORF (ORF3) (Fig. 2). To determine the functions of the individual ORFs, we searched the relevant databases (UniProt) with their translated products using BLAST (28); the results are summarized in Fig. 2. In brief, the three ORFs showed similarity to esterase and beta-lactamase (ORF1), isochorismatase-like hydrolase (ORF2), and lipase (ORF3). Thus, this fragment contained no gene homologous to the nat gene. To verify the genes that were responsible for ST resistance, we constructed plasmids pWHM3-orf1 and pWHM3-orf2–3, which carried ORF1 and ORF2-ORF3, respectively (Fig. 2), and introduced them into S. lividans TK23. MIC studies confirmed that the transformant harboring pWHM3-orf2–3 showed ST resistance (Table 1). Considering the fact that the pWHM3-orf2–3 plasmid carried only a partial form of ORF3, ORF2 was considered to confer STs resistance and was designated as sttH, a novel ST-resistance gene.

Estimation of the Start Codon of the sttH Gene—Based on the results of the sequencing analysis and frame analysis, eight possible start codons (ATG and GTG, positions 1 to 8; Fig. 2) were identified in the sttH gene. Furthermore, the absence of the combination of a promoter region and a ribosome binding site, which is generally found in Streptomyces strains, made it difficult to identify the start codon. Therefore, we constructed six types of rSttH as N-terminal His6-tagged fusion proteins, each of which had a different N-terminal region, to estimate a start codon based on their enzymatic activities indicated by MIC values. We constructed the expression plasmids for E. coli, pQE30-SHF1R (carrying the sttH gene from position 1), pQE30-SHF2R (position 2), pQE30-SHF3R (positions 3 and 4), pQE30-SHF4R (position 5), pQE30-SHF5R (positions 6 and 7), and pQE30-SHF6R (position 8); we also constructed pQE30-SHF7R even though it was not expected that the GTG codon at position 9 would be a possible start codon because the resultant peptide product was extremely short. After introducing these 7 plasmids and pQE30 (no insert) into E. coli, the MICs of STs (a mixture of ST-F and ST-D) were determined to be 12.5 µg/ml (E. coli strain possessing pQE30), 50 µg/ml (in each of pQE30-SHF1R, pQE30-SHF2R, and pQE30-SHF3R), 100 µg/ml (in each of pQE30-SHF4R and pQE30-SHF5R), >100 µg/ml (pQE30-SHF6R), and 12.5 µg/ml (pQE30-SHF7R). This suggests that the position 8 codon could function as a start codon in S. albulus NBRC14147.


Figure 2
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  		FIGURE 2.
Schematic organization of the cloned 2.9-kb fragment involved in ST resistance, and ORFs deduced by sequencing analysis. The hatched boxes represent the cloned fragments in the pWHM3 plasmid. The possible start codons (position 1 to 8) in the sttH gene are highlighted with gray boxes. The PCR primers used are shown schematically by arrows.

 


Figure 3
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  		FIGURE 3.
HPLC analysis of the product formed by rSttH. ST-F was incubated with (C) or without (B) rSttH, and the reaction mixtures and ST-F standard (A) were then analyzed by reverse-phase HPLC. The HPLC conditions are described under "Experimental Procedures."

 


Figure 4
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  		FIGURE 4.
ESI-MS/MS analysis of the rSttH product from ST-F (ST-F-acid) and ST-D (ST-D-acid). The ESI-MS and -MS/MS spectra of ST-F (A), ST-F-acid (B), ST-D (C), and ST-D-acid (D), which were dissolved in 0.2% formic acid, 50% acetonitrile, were obtained.

 
Identification and Structure Determination of the ST-F- and ST-D-derived Compounds Converted by rSttH—A highly purified rSttH obtained by nickel affinity chromatography was incubated with ST-F. An rSttH-dependent product eluted with a retention time longer than that of ST-F on reverse-phase HPLC was specifically detected in the reaction mixture without any additives such as cofactors or metal ions (Fig. 3). Similarly, an rSttH-dependent product was also detected when ST-D was used as a substrate (data not shown). To determine the structures of the rSttH-dependent products thus obtained, these compounds were purified and analyzed by ESI-MS and NMR. The molecular masses of ST-F and ST-F-derived compounds were determined to be 503 (m/z = 504 [M + H]+) and 521 (m/z = 522 [M + H]+), respectively, by ESI-MS analysis (Fig. 4, A and B). In addition, ESI-MS/MS spectra also revealed that this change in the molecular mass (18 Da) had occurred in the streptolidine lactam moiety. A correlative observation was also noted in the ST-D-derived compound (Fig. 4, C and D). This, together with the finding that the primary structure of SttH is similar to that of isochorismatase-like hydrolase, indicates that rSttH catalyzes the hydrolysis of the amide bond of streptolidine lactam. To confirm these predicted structures, we performed NMR analysis, finding that the obtained 1H NMR spectral features of the streptolidine moiety (see "Experimental Procedures") were in complete agreement with those of a chemically synthesized streptolidine (Fig. 1) reported by Jackson et al. (29). The rSttH products from ST-F and ST-D were thus determined to be ST-F-acid and ST-D-acid, respectively.

Enzymatic Properties of rSttH—The optimal pH was measured in two buffers (100 mM) at various pH values: sodium phosphate, pH 4.5–7, and Tris-HCl, pH 7–10. Maximum enzyme activity was observed at pH 6.5 and was rapidly lost with a decrease in pH (around 4). The effect of temperature on the enzyme activity was also investigated over the temperature range of 25–75 °C in 100 mM sodium phosphate buffer (pH 6.5). Although the enzyme activity was maximal at 45 °C, an activity level of ~90% was detected at 65 °C. The enzyme dialyzed against sodium phosphate buffer (pH 6.5) containing 10 mM EDTA showed no decrease in activity, demonstrating that this enzyme does not require metal ions for its activity (data not shown). The kinetic parameters are summarized and shown in Table 2. The Km values of rSttH were calculated to be 0.96 ± 0.19 mM for ST-F and 5.74 ± 0.99 mM for ST-D; this shows that the enzyme has a higher affinity for the ST compound having a shorter chain of the beta-lysine polymer. However, the Vmax value of rSttH for ST-D was slightly higher than that for ST-F. The calculated Vmax/Km value of the reaction with ST-F was 4-fold higher than that of the reaction with ST-D. The native molecular mass of rSttH was estimated to be 50 kDa by gel filtration (data not shown), suggesting that rSttH was present as a homodimer.


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  		TABLE 2
Kinetic parameters for STs

Kinetic parameters were determined with 100 mM sodium phosphate buffer (pH 6.5) at 30 °C.

 
Investigation of the ST-D-resistance Profile in E. coli and Yeast Cells Overexpressing the sttH or nat Genes and in Other Microorganisms—Although the biological activity of chemically prepared ST-F-acid has been reported to be negligible in microorganisms and plants (20, 21), the biological activity of chemically prepared ST-D-acid remains unclear. The sttH gene conferred resistance against the mixture of ST-F and ST-D (ST-F:ST-D ratio was ~5:1) in the S. lividans and E. coli strains as described above. However, it is still unclear whether SttH detoxified ST-D. Therefore, we determined the MICs of both ST-F and ST-D for E. coli and S. cerevisiae strains expressing SttH (Table 3). In this experiment, we also employed the E. coli and S. cerevisiae strains expressing NAT as controls. The E. coli (pQE-nat) and S. cerevisiae (pAD4-nat) strains expressing NAT showed resistance to both ST-F and ST-D as reported previously. Interestingly, the MIC value of ST-D was extremely low for E. coli (pQE30-SHF6R) expressing rSttH; this was in contrast to the case of S. cerevisiae (pAD4-sttH) and indicates that the ST-D-acid formed by SttH was still active as an antibacterial agent against prokaryotic cells. To confirm this, we investigated the selective toxicity of ST-acids and STs against various microorganisms, including Gram-positive and Gram-negative bacteria, clinically isolated pathogenic bacteria, and yeast. The MIC studies revealed that in contrast to the almost complete loss of the antibacterial activity of ST-F-acid against both prokaryotic and eukaryotic cells, ST-D-acid showed a high activity against bacteria, such as E. coli, B. subtilis, and S. aureus, but not against eukaryotic cells such as S. cerevisiae and S. pombe (Table 3).


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  		TABLE 3
MICs of the ST compound in prokaryotic and eukaryotic cells

Yeast and bacteria were grown for 2 days at 30 °C and for 1 day at 37 °C, respectively.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
To obtain our target gene that confers ST resistance via a novel mechanism, we focused on ST-nonproducing Streptomyces strains because we had anticipated that the isolation of our target gene could be hindered by genes encoding NAT in ST producers. Indeed, a DNA fragment conferring ST resistance was successfully obtained from S. albulus NBRC14147, which has not been accepted as an ST producer, and no homologous genes encoding NAT were detected on this fragment. Because the amino acid sequence of ORF1 was similar to those of the beta-lactamases, this ORF was thought to be our target. However, unexpectedly, the product of ORF2 (sttH) was found to catalyze the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Actinomycetes producing antibiotics are known to possess self-resistance genes against the antibiotic biosynthesized. However, we believe S. albulus NBRC14147 to be an ST nonproducer based on the following observations: (i) this strain does not possess the nat gene homolog that is normally clustered with the biosynthetic genes for ST in ST-producing Streptomyces strains, and (ii) ST-related compounds were undetectable in the fermentation broth. This raises the possibility that the true role of SttH may not be its involvement in self-resistance against STs produced by the organism; instead, it may catalyze the hydrolysis of naturally occurring cyclic amide compounds in the metabolism of S. albulus. In the present study, it was shown that the deduced primary structure of SttH resembles those of ether hydrolases that belong to the isochorismatase superfamily. Isochorismatase (EC 3.3.2.1 [EC] ) is an enzyme (EntB) occurring in E. coli and is produced during the biosynthesis of enterobactin, an iron-chelating product derived from chorismic acid and involved in the transport of iron from the bacterial environment into the cell cytoplasm (30–32). The phzD gene encoding an isochorismatase-related enzyme, which shares 46% identity with the EntB isochorismatase, is also known to participate in phenazine biosynthesis in Pseudomonas strains (33). Parsons et al. (33) have recently reported that the structure of PhzD is remarkably similar to other structures from a subfamily of {alpha}/beta-hydrolase-fold enzymes, whose members are known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds. However, to the best of our knowledge, no amide hydrolysis reactions have been observed in EntB or PhzD. On the other hand, the recent genome sequencing projects in bacteria have shown that the genes encoding proteins belonging to the isochorismatase-like hydrolase superfamily exist in almost all bacteria including enterobactin- or phenazine-producing bacteria. Although bacteria commonly possess isochorismatase-like hydrolase(s), except for EntB or PhzD, the functions of these proteins remain unclear. Their three-dimensional structures have been determined (34, 35); however, their enzymatic functions and substrates have not yet been clarified by structural data. Interestingly, an analysis using 3D-PSSM (36) demonstrated that the deduced three-dimensional structure of SttH is considerably similar to isochorismatase-like hydrolases of unknown function from Enterococcus faecalis v583 (Protein Data Bank accession number 2A67) and E. coli (PDB accession number 1J2R [PDB] ), indicating that SttH belongs to this superfamily. Thus, SttH is the first enzyme whose function was determined among these isochorismatase-like hydrolases that commonly exist in bacteria. These findings also suggest that some of the isochorismatase-like hydrolases of unknown function from other bacteria could also hydrolyze cyclic amides. We investigated the enzymatic properties of SttH by using its recombinant enzyme that was translated from the codon at position 8 (Fig. 2). Because this codon was estimated to be the start codon based on its catalytic activity indicated by MIC values, it is not certain whether this codon is the appropriate start codon. Therefore, SttH translated from a different position might have a different substrate specificity. A further in-depth enzymatic characterization of SttH and sequencing analysis of the regions flanking the sttH gene should facilitate the understanding of the true biological role of SttH in S. albulus. Such investigation might also be able to provide insights into the biological role of these isochorismatase-like hydrolases that commonly occur in bacteria.

Kinetic studies demonstrated that the Vmax/Km value for ST-D (12.0) is 4-fold lower than that for ST-F (44.0) (Table 2). In fact, the MIC values of ST-F (0.25 mM) and ST-D (<0.03 mM) in rSttH-overexpressing E. coli (Table 3) were in good agreement with the results from this kinetic study. However, surprisingly, ST-D was completely detoxified by SttH in the S. cerevisiae CKY8 strain. In contrast, the nat gene encoding NAT was found to confer ST-D resistance in both E. coli and S. cerevisiae. Based on these results, we hypothesized that ST-D-acid must remain active in prokaryotes such as E. coli, but not in eukaryotes such as yeast; the selective activity of ST-D would be altered by the hydrolysis of streptolidine lactam. To verify this hypothesis, MIC studies using ST-acids and STs were carried out in various microorganisms. As shown in Table 3, the inactivation ratios of ST-F-acid and ST-D-acid were calculated for each microorganism. The inactivation ratios of ST-D-acid for yeast (125–250) were found to be ~4–17-fold higher than those for bacteria (15–31.3). In particular, the antibacterial activities of ST-D-acid have been found to be robust against the clinically isolated pathogenic bacteria S. aureus AB (unpublished enterotoxin AB-producing strain) and S. aureus FIR1169 (toxic shock syndrome exotoxins-producing strain) (26). STs have not been clinically developed due to their toxicities in mammals. However, in the present study, we found that ST-D-acid exhibits a potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid has potential for clinical development or for use as a new lead compound for drug discovery. Moreover, although STs and the nat gene are currently being used in recombinant DNA work in prokaryotes and eukaryotes, they may be superseded by the combination of ST-D (a more active antibiotic than ST-F) and the sttH gene in eukaryotic cells, particularly in yeast.


   FOOTNOTES
 
* This work was supported in part by grants from the Chisso Corporation and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) (to H. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB248874 [GenBank] (the DNA sequence of the 2.9-kb Sau3AI fragment containing the sttH gene). Back

1 To whom correspondence may be addressed: 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Fukui 910-1195, Japan. Tel.: 81-776-61-6000; Fax: 81-776-61-6015; E-mail: hamano{at}fpu.ac.jp. 2 To whom correspondence may be addressed. E-mail: hiro{at}fpu.ac.jp.

3 The abbreviations used are: STs, streptothricins; ST-F, streptothricin F; ST-D, streptothricin D; MIC, minimum inhibitory concentration; NAT, N-acetyltransferase; rSttH, recombinant SttH; HPLC, high performance liquid chromatography; ORF, open reading frame; ESI-MS, electrospray ionization tandem mass spectra. Back


   ACKNOWLEDGMENTS
 
We are grateful to C. A. Kaiser (Massachusetts Institute of Technology, Boston, MA), S. Takeuchi (Fukui Prefectural University, Fukui, Japan), and J. Nikawa (Kyushu Institute of Technology, Fukuoka, Japan) for providing the S. cerevisiae strain, the S. aureus strains, and the plasmid pAD4, respectively. We also thank M. Sassa (University of Fukui, Fukui, Japan) for assistance with ESI-MS/MS data collection.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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