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Selective Toxicity Alteration of a Highly Toxic Antibiotic by an Enzyme Catalyzing Antibiotic Modification

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Selective Toxicity Alteration of a Highly Toxic Antibiotic by an Enzyme Catalyzing Antibiotic Modification Actinomycetologica (2008) 22:50–55 Copyright Ó 2008 The Society for Actinomycetes Japan VOL. 22, NO. 2 Award Lecture Selective toxicity alteration of a highly toxic antibiotic by an enzyme catalyzing antibiotic modification Yoshimitsu Hamanoà Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui 910-1195, Japan. (Received Sep. 27, 2008 / Accepted Sep. 29, 2008 / Published Dec. 25, 2008) INTRODUCTION moiety of -lysine(s) has been shown to play a crucial role in antibiotic activity. On the other hand, Inamori et al. Streptothricins (STs) (Fig. 1) are broad-spectrum (Inamori et al., 1988) and Taniyama et al. (Taniyama et al., antibiotics that were first isolated from Streptomyces 1971) have independently reported that ST-F-acid (Fig. 1, lavendulae in 1943 (Waksman, 1943). All STs consist of termed as racenomycin-A-acid in their studies)—chemi- a carbamoylated D-gulosamine to which the -lysine cally prepared from ST-F—did not exhibit antibiotic homopolymer (1 to 7 residues) and the amide form of the activity against bacteria, fungi, and plants; however, the unusual amino acid ‘‘streptolidine lactam’’ are attached. biological activity of ST-D-acid was not tested. This result STs inhibit protein biosynthesis in prokaryotic cells; in confirmed that streptolidine lactam is essential for antibiotic addition, they strongly inhibit the growth of eukaryotes activity. We therefore hypothesized that microorganisms such as yeasts (Goldstein & McCusker, 1999; Hentges showing resistance to STs through alternative resistance et al., 2005; Shen et al., 2005), fungi (Idnurm et al., 2004), mechanisms might produce an enzyme that hydrolyzes protozoa (Joshi et al., 1995), insects (Takemoto et al., streptolidine lactam, thereby inactivating STs. Actinomy- 1980) and plants (Chamberlain et al., 1994). Therefore, STs cetes are known to produce many natural products with are used as effective selective agents for recombinant DNA structural diversity occurring due to the unique substrate work in some of these organisms. However, STs are not specificities of the enzymes. Therefore, we focused on currently used therapeutically due to their nephrotoxicity Streptomyces, the representative strains belonging to acti- (Hoffmann et al., 1986a and 1986b; Hartl et al.). nomycetes, to efficiently identify our target enzyme. To date, many ST-resistance genes have been identified Here, we describe the cloning of a gene whose product in transposons such as Tn1825 and Tn1826, which have confers ST resistance through the modification of strepto- been isolated from bacteria that are resistant to ST lidine lactam, as expected (Hamano et al., 2006). Addi- (Partridge & Hall, 2005); such transposons have also been tionally, we used the recombinant enzyme of this gene isolated from human pathogens such as Shiga toxin- product to investigate its functions and properties. We also producing Escherichia coli (Singh et al., 2005) and the discuss an interesting observation regarding the selective Shigella strain (Peirano et al., 2005). Bacterial resistance to toxicity of an ST compound that was converted by the gene antibiotics that inhibit protein biosynthesis (e.g., amino- product. glycosides) can occur as a result of decreased antibiotic uptake and accumulation, modification of 16S RNA or Cloning and sequencing analysis of the ST-resistance ribosomal proteins, or enzymatic modification of the gene antibiotics (Vakulenko & Mobashery, 2003). However, in To obtain our target gene that confers ST resistance via a the case of bacterial resistance to STs, only one resistance novel mechanism, we focused on ST-nonproducing Strep- mechanism has yet been identified: the resistance is due to tomyces strains since we had anticipated that the isolation a modification of the ST molecule by monoacetylation at of our target gene could be hindered by genes encoding the -amino group of -lysine(s). In fact, in ST producers NAT in ST producers. Based on the present studies of such as S. lavendulae (Horinouchi et al., 1987), S. rochei MICs for STs in the Streptomyces strains that have not (Ferna´ndez-Moreno et al., 1997) and S. noursei (Krugel been accepted as ST producers, Streptomyces albulus et al., 1988; Grammel et al., 2002), the ST-resistance genes NBRC14147 was found to be more resistant to STs than encoding N-acetyltransferase (NAT) have been identified the ST producer S. lavendulae NBRC12789 (Table 1). and their role in self-resistance against their own STs has PCR using primers designed for genes that encode NATs been investigated. Based on this resistance mechanism for STs and the genomic DNA of the NBRC14147 strain as and the fact that streptothricin D (ST-D, Fig. 1) is a more a template did not show any amplified fragments, whereas a effective antibiotic than streptothricin F (ST-F, Fig. 1), the specific amplified fragment was detected when the genomic ÃCorresponding author: Phone: +81-776-61-6000. Fax: +81-776-61-6015. E-mail address: [email protected] 50 ACTINOMYCETOLOGICA VOL. 22, NO. 2 carbamoylated streptolidine streptolidine D-gulosamine lactam O O O OH H O OH H N N NH hydrolysis of OH H2N O O N H2N O O N streptolidine lactam N N NH2 H H OH SttH OH OH OH HN H HN H N N ST-F (n=1) H ST-F-acid (n=1) H ST-D (n=3) O NH2 n ST-D-acid (n=3) O NH2 n β-lysine Fig. 1. Chemical structure of streptothricins (STs). DNA of S. lavendulae NBRC12789 was used (data not shown). Therefore, since the NBRC14147 strain was Table 1. ST resistance profiles of the Streptomyces strains suggested to have no gene encoding NAT, this strain was MIC (mg/mL) ST selected as a source of the novel ST-resistance gene. To Streptomyces strains of STs production obtain the novel ST-resistance gene, the NBRC14147 S. lavendulae NBRC12789 400à yes strain genomic library was constructed with the pWHM3 S. albulus NBRC14147 >400à no plasmid carrying the thiostrepton-resistance gene. This S. lividans TK23 6.25à no library was introduced into Streptomyces lividans TK23, + pWHM3 6.25y — which is sensitive to STs and thiostrepton, and trans- + pWHM3-st11 >400y — formants resistant to both thiostrepton (20 mg/ml) and + pWHM3-orf2-3 >400y — STs (>400 mg/ml, a mixture of ST-F and ST-D with + pWHM3-orf1 6.25y — ST-F:ST-D ratio of approximately 5:1) were isolated. From ÃATCC medium No. 5 plates containing STs (0–400 mg/mL) these transformants, we selected one which harbored the were used. yATCC medium No. 5 plates containing STs pWHM3 plasmid carrying a 2.9-kb fragment (pWHM3- (0–400 mg/mL) and thiostrepton (20 mg/mL) were used. The st11, Fig. 2 and Table 1) for further experiments because MICs were determined after incubation for 3 days at 30 C. Southern blotting using this fragment as a probe revealed that all plasmids isolated from these transformants carried 0.5-kb ORF1 sttH (ORF2) ORF3 Eco RI Kpn I Bam HI/Sau 3AI Sau 3AI/Bam HI pWHM3-st11 pWHM3 1 2913-bp pWHM3-orf2-3 pWHM3 pWHM3-orf1 pWHM3 amino acid molecular ORFs homologous proteins (identity, accession no. in the UniProt database) residues weight ORF1 401 42328 esterase from; - Streptomyces avermitilis MA4680 (46%, Q82NJ9) - Streptomyces chrysomallus (44%, O87861) - Streptomyces coelicolor A3(2) (34%, Q9RKC5) β-lactamase from; - Mycobacterium paratuberculosis (44%, Q73XM6 ) - Pseudomonas fluorescens PfO-1 (34%, Q3KGY6) - Anaeromyxobacter dehalogenans 2CP-C (33%, Q4NPE1) ORF2 (sttH ) 234 23567 isochorismatase-like - Shewanella amazonensis SB2B (31%, Q3QE02) hydrolase from; - Burkholderia cenocepacia AU 1054 (30%, Q44WW2) - Pseudomonas putida F1 (28%, Q2XEN3) ORF3 -- lipase from; - Trichodesmium erythraeum IMS101 (29%, Q3H7D3) (partial) - Rhodopirellula baltica (28%, Q7UQZ0 ) Fig. 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. 51 ACTINOMYCETOLOGICA VOL. 22, NO. 2 the 2.9-kb fragment (data not shown). (mV) ST-F The sequencing analysis of the 2.9-kb DNA fragment 100 (A) and frame analysis with the codon usage for Streptomyces strains revealed two ORFs (ORF1 and 2) and one partial 50 ORF (ORF3) (Fig. 2). In order to predict the functions of 0 the individual ORFs, we searched the relevant databases (UniProt) with their translated products using BLAST; the ST-F (B) results are summarized in Fig. 2. In brief, the three ORFs 100 showed similarity to esterase and -lactamase (ORF1), 50 isochorismatase-like hydrolase (ORF2) and lipase (ORF3). Thus, this fragment contained no gene homologous to the 0 nat gene. Since the amino acid sequence of ORF1 was 100 (C) similar to those of the -lactamases, this ORF was thought product to be our target. To address this, we constructed plasmids pWHM3-orf1 and pWHM3-orf2-3, which carried ORF1 50 and ORF2-ORF3, respectively (Fig. 2), and introduced 0 them into S. lividans TK23. However, unexpectedly, MIC 0 5 10 15 20 25 (min) studies confirmed that the transformant harboring pWHM3- orf2-3 showed ST resistance (Table 1). Considering the Fig. 3. HPLC analysis of the product formed by rSttH. ST-F was fact that the pWHM3-orf2-3 plasmid carried only a partial incubated with (C) or without (B) rSttH, and the reaction mixtures form of ORF3, ORF2 was found to confer STs resistance and ST-F standard (A) were then analyzed by reverse-phase and was designated as sttH, a novel ST-resistance gene. HPLC. Functional analysis of SttH We constructed a recombinant SttH (rSttH) as N- does not possess the nat gene homolog that is normally terminal 6  His-tagged fusion proteins. A highly purified clustered with the biosynthetic genes for ST in ST- rSttH obtained by Ni-affinity chromatography was incu- producing Streptomyces strains, and (ii) ST-related com- bated with ST-F.
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