Construction of a Knockout Mutant of the Streptothricin-Resistance Gene in Streptomyces Albulus by Electroporation

Actinomycetologica (2006) 20:35–41 VOL. 20, NO. 2

Construction of a Knockout Mutant of the Streptothricin-Resistance Gene in Streptomyces albulus by Electroporation

Yoshimitsu Hamano*, Chitose Maruyama and Hisashi Kimoto

Department of Bioscience, Fukui Prefectural University, Fukui, Japan

(Received Sep. 25, 2006 / Accepted Oct. 4, 2006)

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the b-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. In a previous study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus by shotgun cloning with a heterologous host. In vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. We suggested that the true role of SttH may not be its involvement in resistance against STs; instead, it may catalyze the hydrolysis of naturally occurring cyclic amide compounds in the metabolism of S. albulus. In this study, we therefore constructed an sttH gene-knockout mutant to better understand the true biological role of SttH in S. albulus and to clarify whether or not S. albulus possesses different ST-resistance gene(s). The minimum inhibitory concentration MIC of STs in the sttH gene-knockout mutant was determined to be 6.25 mg/ml, whereas the MIC of STs in the S. albulus wild strain was determined to be >400 mg/ml. This result clearly demonstrated that sttH is the sole gene responsible for ST resistance in the S. albulus strain. Moreover, we developed a transformation system for S. albulus by electroporation in this study.

INTRODUCTION this homolog is normally clustered with the ST biosynthetic genes in ST-producing Streptomyces strains, and (ii) ST- Streptothricins (STs) produced by Streptomyces strains related compounds were undetectable in the fermentation are broad-spectrum antibiotics in prokaryotes and eukary- broth. This raises the possibility that the true role of SttH may otes1–8). All STs consist of a carbamoylated D-gulosamine to not be its involvement in self-resistance against STs pro- which the b-lysine homopolymer (1 to 7 residues) and the duced by the organism; instead, it may catalyze the hydroly- amide form of the unusual amino acid streptolidine (strepto- sis of naturally occurring cyclic amide compounds in the lidine lactam) are attached (Fig. 1). Although many ST- metabolism of S. albulus. We also demonstrated that the resistance genes have been identified in bacteria, including deduced primary structure of SttH resembles those of ether clinically isolated pathogens9–11) and ST-producing Strep- hydrolases of the isochorismatase superfamily. Isochoris- tomyces strains12–15), only one resistance mechanism has matase (EC 3.3.2.1) is an enzyme (EntB) occurring in been identified. This mechanism involves the modification Escherichia coli and is produced during the biosynthesis of of the ST molecule by monoacetylation of the moiety of the enterobactin, an iron-chelating product derived from choris- b-lysine(s). Recently, we successfully isolated a novel ST- mic acid and involved in the transport of iron from the resistance gene (sttH) from Streptomyces albulus, which is bacterial environment into the cell cytoplasm17–19). The phzD a known ST nonproducer16). The in vitro analysis of SttH gene encoding an isochorismatase-related enzyme, which demonstrated that this enzyme catalyzes the hydrolysis of the shares 46% identity with the EntB isochorismatase, is also amide bond of streptolidine lactam, thereby conferring ST known to participate in phenazine biosynthesis in Pseudo- resistance (Fig. 1). Interestingly, the selective toxicity of ST- monas strains20). Parsons et al. recently reported that the D possessing the 3×b-lysine moiety was altered from broad- structure of PhzD is remarkably similar to other structures spectrum to bacterial-specific by the hydrolysis of streptoli- from a subfamily of a/b-hydrolase fold enzymes, whose dine lactam, although ST-F (1×b-lysine) was detoxified by members are known to hydrolyze amides, phosphates, phos- SttH in both prokaryotes and eukaryotes (yeasts)16). phonates, epoxides, and C-X bonds20). However, to the best Actinomycetes that produce antibiotics are known to of our knowledge, no amide hydrolysis reactions have been possess self-resistance genes against biosynthesized anti- observed in EntB or PhzD. On the other hand, the recent biotics. However, we believe S. albulus NBRC14147 to be genome sequencing projects in bacteria have shown that the an ST nonproducer based on the following observations: (i) genes encoding proteins belonging to the isochorismatase- this strain does not possess the nat gene homolog, which like hydrolase superfamily exist in almost all bacteria, encodes N-acetyltransferase, for the STs’s self-resistance; including enterobactin- or phenazine-producing bacteria. In

*Corresponding author. Phone: +81-776-61-6000, Fax: +81-776-61-6015, E-mail: [email protected] 35 ACTINOMYCETOLOGICA VOL. 20, NO. 2

Fig. 1. Chemical structure of streptothricins (STs). fact, Streptomyces avermitilis MA-4680 possesses two puta- pGEM11Z (Promega, Tokyo, Japan) were used for the sub- tive isochorismatase-like hydrolase genes, SAV1838 and cloning experiments and for sequencing analysis. The cos- SAV1388, and the deduced amino acid sequence of SttH mid pOJ44623) was used for the preparation of a genomic exhibited weak similarity with those of SAV1838 (22% library, and was used as template DNA in PCR to amplify identity) and SAV1388 (16%). Streptomyces coelicolor the apramycin-resistance gene (apr). The plasmid pMODTM- A3(2) also possess the gene SCO2330 (25% identity with 2 (Epicentre, Madison, WI, USA) was used for construction SttH). In addition, the E. coli w3110 strain possesses the iso- of a transposon carrying the apr gene. E. coli ATCC BAA- chorismatase-like hydrolase gene, yecD (19% identity with 525 (E. coli ET12567)24) was used as a host to isolate a non- SttH). To date, however, only SttH has been known to be methylated plasmid. The plasmid pTSR193, which has the involved in hydrolysis of STs. Although bacteria commonly thiostrepton-resistance gene (tsr) and does not have a repli- thus possess isochorismatase-like hydrolase(s), except for con for Streptomyces strains, was used for a gene knockout EntB or PhzD, the functions of these proteins remain unclear. experiment as a suicide vector. The plasmid pLAE003 was Therefore, SttH is the first enzyme whose function was deter- employed as a control plasmid to optimize electroporation mined among these isochorismatase-like hydrolases that conditions. Recombinant DNA procedures for the E. coli and commonly exist in bacteria. These findings also suggest that Streptomyces strains were performed using standard tech- some of the isochorismatase-like hydrolases of unknown niques25,26). Southern blotting was performed using the function from other bacteria could also hydrolyze cyclic enhanced chemiluminescence (ECL) direct nucleic acid amides. labeling and detection system (Amersham Bioscience, In this study, to provide insights into the biological role of Piscataway, NJ, USA). these isochorismatase-like hydrolases that commonly occur in bacteria and to facilitate the understanding of the true Cloning of the sttH gene flanking region from S. albulus biological role of SttH in S. albulus, we constructed an sttH NBRC14147 gene-knockout mutant. We also describe the development of To clone the sttH gene flanking region from S. albulus a transformation system using electroporation to facilitate NBRC14147, the genomic library of S. albulus CR1 was the construction of a gene knockout mutant in S. albulus. constructed as follows: The genomic DNA of S. albulus NBRC14147 was partially digested with Sau3AI. Sau3AI MATERIALS AND METHODS fragments larger than 25-kb were ligated into the BamHI site of the cosmid pOJ446, and the ligation mixture was subse- Bacterial Strains, Plasmids, and General Techniques for quently used for in vitro packaging with Gigapack III Gold DNA Manipulation Packaging Extracts (Stratagene, La Jolla, CA, USA). A cos- S. albulus NBRC14147 was used as the DNA source for mid clone pSttHCOS1 was screened by colony hybridization cloning the sttH gene with the flanking region. The cryptic using the sttH gene as a probe (accession no. AB248874). plasmid pNO33-curing S. albulus strain CR121), which is Several restriction enzyme-digested fragments, which derived from S. albulus NBRC14147, was used in the sttH hybridized to the probe, were subcloned into pUC19. After gene inactivation experiment. The media and growth con- the construction of a series of plasmids, sequencing was ditions for S. albulus NBRC14147 have been described carried out. Finally, the DNA sequences of the NotI-BamHI previously22). Escherichia coli XL1-Blue MRF' (Toyobo, 4.2-kb fragment were analyzed (Fig. 2, A). Osaka, Japan) and the plasmids pUC118, pUC19, and

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Fig. 2. Inactivation of the sttH gene. The sttH gene inactivation was performed as represented in the schematic diagram (A) and confirmed by Southern blot analysis with the genomic DNA of the sttH gene-knockout mutant (B). NotI-BamHI digests of the genomic DNA from S. albulus CR1 (lane 1) and the sttH gene-knockout mutant S. albulus CRM002 (lane 2) were subjected to Southern blot analysis using the sttH gene as the probe. The arrows indicate the hybridized fragment sizes. The hybridized fragments are schematically represented.

Electroporation Construction of a transposon carrying the apr gene S. albulus strain CR1 spores (500 ml; 1×1010 spores/ml) To amplify the apr gene, the following set of primers was were inoculated into 50 ml of SLB medium consisting of designed and used for PCR: 5'-GAAAGTCTACACGAAT- sucrose (10.3%), Bacto tryptone (1%; Difco), Bacto yeast TCTTTGGCAAAATC-3' (forward) and 5'-CGCGGT- extract (0.5%; Difco), and NaCl (0.5%) (pH 7.0) and were GAGTTCAAGCTTTTTCATATCTCA-3' (reverse). Re- cultivated for 5 h at 30ºC. The germinated spores were col- striction enzyme sites (shown in italics) were introduced into lected by centrifugation, washed twice with chilled water, these primers. The PCR with pOJ446 as a template DNA was and suspended with 10% glycerol (1 ml; 5×109 spores/ml). carried out under standard conditions. An amplified DNA Then, 50 ml of the suspension was mixed with 1–5 ml of DNA fragment was digested with EcoRI and HindIII, and was lig- at 0ºC, after which the mixture was pipetted into a chilled ated into pUC118. After sequence confirmation of the result- electroporation cuvette (inter-electrode distance =0.1 cm; ing plasmid, a 1.2-kbp EcoRI-HindIII fragment was inserted Bio-Rad Laboratories, Hercules, CA, USA) and subjected to into the same site of pMOD TM-2. The resulting plasmid was a single voltage shock using a Gene Pulser II (Bio-Rad). In digested with PvuII to obtain the 1.3-kb PvuII fragment, and the electrotransformation using plasmid pLAE003 carrying this fragment was used as the transposon carrying the apr the neomycin resistance gene, 250 ml of SLB medium was gene in the following experiments. added immediately after the pulse. The sample was incubated at 30ºC for 3 h and then plated on the PLP medium27) Construction of sttH gene-knockout mutant containing neomycin (400 mg/ml). In the case of transfor- The NotI-BamHI 4.2-kb fragment carrying the sttH gene mation with a pTSR193-derived plasmid carrying the tsr was inserted into the same sites of pGEM11Z. The resulting gene, the sample was incubated at 30ºC for 12 h and then plasmid was digested with SphI and BamHI, and this fragment plated on the PLP medium containing thiostrepton (5 mg/ml). containing the sttH gene was subcloned into the same sites

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Table 1. Bacterial strains and plasmids used in this study and their relevant characteristics and sources Reference Strain or plasmid Relevant characteristicsa or source S. albulus strains CR1 cryptic plasmid (pNO33) free 21) CRM002 CR1 sttH::apr This study E. coli strains XL1-blue MRF' subcloning host, ∆(mcrA)183 Stratagene ∆(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F' proAB lacIqZ∆M15 Tn10 (Tetr)] ET12567 DNA methylase genes-deficient mutant, F- 24) dam-13::Tn9 dcm-6 hsdM hsdR zjj-202::Tn10 recF143 galK2 galT22 ara-14 lacY1 xyl-5 leuB6 thi-1 tonA31 rpsL136 hisG4 tsx-78 mtl-1 glnV44) Plasmids pUC118 Ampr pUC19 Ampr pGEM11Z Ampr pNEB193 Ampr New England Biolabs pMODTM-2 Ampr, transposon construction vector Epicentre pOJ446 Apmr, cosmid used for the construction of the genomic library of S. albulus CR1 23) pSttHCOS1 Apmr, derivative of pOJ446 containing ~35-kb DNA fragment flanking the sttH gene This study pTSR193 Tspr, derivative of pNEB193 plasmid harboring tsr gene This study pLAE003 Neor (aphII), oriT of RK2, E. coli-S. albulus shuttle vector harboring pNO33 replicon 21) a Tetr, tetracycline-resistant; Ampr, ampicillin-resistant; Apmr, apramycin-resistant; Tspr, thiostrepton-resistant; Neor, neomycin-resistant

spores. Diluted spores were plated on ATCC medium 5 Table 2. ST resistance profiles of the Streptomyces strains plates, and the colonies showing apramycin resistance and Streptomyces strains MIC (mg/mL) of STs* thiostrepton sensitivity were further selected. Insertional S. albulus CR1 >400 mutations in the sttH gene were confirmed by Southern blot S. albulus CRM002 6.25 analysis; the schematic diagram is presented in Fig. 2. S. lividans TK23 6.25 *ATCC medium 5 plates containing STs (0–400 mg/mL) were used. RESULTS AND DISCUSSION The MICs were determined after incubation for 3 days at 30ºC. Cloning of the sttH gene with the flanking region from S. albulus NBRC14147 Investigation into the gene organization of the flanking of pTSR193, which has the thiostrepton-resistance gene region sometimes provides important information about the (tsr). To inactivate the sttH gene on the resulting plasmid, function of a target gene. To gain insight into the biological pTSR193-sttH, the transposon carrying the apr gene was role of isochorismatase-like hydrolases that commonly occur randomly inserted into pTSR193-sttH by in vitro transposon in bacteria and to better understand the true biological role insertion reaction with EZ-Tn5 Transposase (Epicentre). By of SttH in S. albulus, we cloned the flanking region of the restriction enzyme digestions and sequencing analysis, the sttH gene. In a previous study, we cloned the Sau3AI 2.9-kb pTSR193-sttH::apr plasmid, in which the transposon had fragment carrying the sttH gene16). Therefore, the Sau3AI been inserted into the sttH gene, was selected. This plasmid 2.9-kb fragment was used as a probe to screen an S. albulus was subsequently introduced into the E. coli ET12567 strain, CR1 cosmid library. From 12 positive clones, one cosmid which is a deficient mutant of DNA methylase genes, and clone (pSttHCOS1) containing a 4.2-kb NotI-BamHI hy- was prepared for use in further experiments. bridizing fragment was selected (Fig. 2, A). Sequencing The plasmid pTSR193-sttH::apr was introduced into S. analysis of this fragment was carried out, and frame analy- albulus CR1 by electroporation (25 mF, 100 Ω, 15 kV/cm). sis with the codon usage for Streptomyces strains28) revealed A transformant exhibiting resistance against thiostrepton and two additional ORFs (ORF-4 and -5) (Fig. 2, A). A database apramycin was cultivated in SLB medium only with apramycin search with BLAST29) showed that the deduced amino acid (50 mg/ml). After cultivation three times, an aliquot of the sequence of ORF-5 (partial form) has a significant similarity culture was plated on ATCC medium 5 plates to obtain the to the sequences of iron-sulphur binding subunits [2Fe-2S]

38 ACTINOMYCETOLOGICA VOL. 20, NO. 2 of xanthine dehydrogenases (XDH) or oxidoreductases from Germination of S. albulus CR1 occurred after cultivation Streptomyces coelicolor A3(2) (Gene ID no. SCO0689, 71% for 4 h with SLB medium. Therefore, to obtain spores from identity), S. coelicolor A3(2) (SCO1134, 67%), Strepto- different germination stages, spores after cultivation for 4, 5, myces avermitilis MA-4680 (SAV1540, 68%), and S. aver- 6, and 7 h were collected (Fig. 3) and used in the transfor- mitilis MA-4680 (SAV7382, 62%). The deduced amino acid mation experiment. The spore suspensions (50 ml) were sequences of ORF-4 exhibited 44%, 41%, and 37% identity mixed with the plasmid pLAE003 (0.02 mg), which is iso- to hypothetical proteins with unknown functions from S. lated from S. albulus CR1 and used as a standard plasmid to coelicolor A3(2) (Gene ID no. SCO7065), S. avermitilis optimize transformation conditions by electroporation, and MA-4680 (SAV7192), and S. coelicolor A3(2) (SCO7287), were each subjected to a single voltage shock with fixed elec- respectively. tric parameters (25 mF, 100 Ω, 17.5 kV/cm). Immediately Our previous in vitro analysis of SttH demonstrated that after the pulse, 250 ml of SLB medium was added. The sam- SttH catalyzes the hydrolysis of the amide bond of streptoli- ple was incubated at 30ºC for 3 h and then plated on PLP dine lactam in STs (Fig. 1). However, we speculate that the medium containing neomycin (400 mg/ml). Fortunately, true substrate of SttH may not be STs; instead, it may cat- after incubation at 30ºC for 3 days, transformants were suc- alyze the hydrolysis of naturally occurring cyclic amide cessfully obtained from spores at all germination stages. In compounds in the metabolism of S. albulus. In the present particular, spores after 5 h cultivation gave the highest trans- study, ORF-4 was found to show a significant similarity to formation efficiency (1.7×104 transformants/mg DNA). The the sequences of iron-sulphur binding subunits [2Fe-2S] of efficiency was 4- to10-fold higher than those obtained from XDH, which is known to be involved in purine metabolism the spores of the other germination stages. Therefore, the and to be essential enzyme for growth in bacteria. XDH is a spores cultivated for 5 h were used for further experiments. complex metallo-flavoprotein that catalyzes the hydroxyla- Because electric field strength is the most crucial parameter tion of hypoxanthine and xanthine, the last two steps in the in the electrotransfer of DNA into bacteria, we next exam- formation of urate, using a water molecule as the ultimate ined the effect of field strength on transformation efficiency. source of oxygen incorporated into the product30). Because By testing field strength from 5 kV/cm to 25 kV/cm, we these purines are also cyclic amide compounds, SttH could found the electrotransfer to be effectual at 10 to 20 kV/cm, be involved in purine metabolisms, particularly regarding the with a peak transformant yield at 15 kV/cm. The effect of the degradation of purine compounds. Furthermore, considering time constant on the transformation efficiency was next stud- that isochorismatase-like hydrolases such as SttH commonly ied by varying the resistance (100–400 Ω). The efficiency exist in bacteria, we were interested to learn whether or not decreased in accordance with the increase in the time con- the genes encoding isochorismatase-like hydrolases are es- stant. The transformant yield was highest when the resistance sential for growth. To address this question, we attempted to was 100 Ω, giving a 2.4 msec time constant. Finally, we inactivate the sttH gene in S. albulus. obtained a constant transformation efficiency of 2×104 to 5×104/mg DNA. Transformation of S. albulus CR1 by electroporation For the construction of the knockout mutant in S. albulus Construction of the sttH gene-knockout mutant CR1, an efficient genetic system for this strain was required. The plasmid pTSR193-sttH::apr, in which the transposon Until now, we have developed transformation systems for was inserted into the sttH gene, was constructed and intro- DNA delivery into the S. albulus strain based on both poly- duced into S. albulus CR1 by electroporation (25 mF, 100 Ω, ethylene glycol (PEG)-mediated protoplast transformation 15 kV/cm). The transformant exhibiting thiostrepton and and intergeneric conjugation from Escherichia coli. How- apramycin was selected, cultivated in SLB medium only ever, in the present study we developed a transformation sys- with apramycin, and plated on ATCC medium 5 plates. tem by electroporation to facilitate the construction of a gene About 1% of the transformants were thiostrepton-sensitive knockout mutant in S. albulus. In recent years, transforma- and apramycin-resistant, and these were isolated. The tion by electroporation has been rendered possible for several genomic DNA of these transformants was thus obtained, and gram-negative and gram-positive bacteria. Electroporation was prepared and digested with NotI and BamHI, then involves the application of a brief, high-voltage pulse to a hybridized by Southern blotting using the sttH gene as the suspension of cells and DNA, resulting in transient mem- probe. As shown in Fig. 2, the sttH gene-knockout mutant, brane pores and subsequent DNA uptake. Therefore, this which is designated S. albulus CRM002 in this study, was method is less tedious and time-consuming than PEG-medi- successfully constructed. In the previous study16), we cloned ated protoplast transformation and intergeneric conjugation. only the sttH gene by shotgun cloning with Streptomyces livi- To date, in Streptomyces strains, there are some reports of dans TK23, which is sensitive to STs; namely, no additional transformations by electroporation31–33). On the basis of these gene conferring ST resistance was identified in the shotgun procedures, we initially attempted to introduce a plasmid cloning experiment. Therefore, to clarify whether or not the DNA into the mycelium of S. albulus CR1. However, no S. albulus CR1 possesses a different ST-resistance gene, we transformant was obtained despite extensive efforts. We investigated the ST susceptibility in S. albulus CRM002. The therefore tested germinated spores for transformation. minimum inhibitory concentration (MIC) of STs in S.

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Fig. 3. Phase-contrast microscopy of the germinated spores. The S. albulus strain CR1 spores of 500 ml (1×1010 spores/ml) was inoculated into 50 ml of SLB medium and cultivated for 4 h (photo A), 5 h (B), 6 h (C), and 7 h (D) at 30ºC. albulus CRM002 was determined to be 6.25 mg/ml, where- resistance cassettes for gene disruption in Saccharomyces as the MIC of STs in S. albulus CR1 was determined to be cerevisiae. Yeast 15: 1541–1553, 1999 >400 mg/ml. This result clearly demonstrated that the sttH 3) Hentges, P.; B. Van Driessche, L. Tafforeau, J. Vandenhaute gene is the sole gene responsible for ST resistance in the S. & A. M. Carr: Three novel antibiotic marker cassettes for gene Schizosaccharomyces albulus strain. In addition, no morphological changes were disruption and marker switching in pombe. Yeast 22: 1013–1019, 2005 observed in this mutant. 4) Shen, J.; W. Guo & J. R. Kohler: CaNAT1, a heterologous The fact that we were able to construct the sttH gene- dominant selectable marker for transformation of Candida knockout mutant is of great interest, because this result albicans and other pathogenic Candida species. Infect. implies that the sttH gene is not essential for growth. In this Immun. 73: 1239–1242, 2005 study, the true biological role of the sttH gene is still unclear. 5) Idnurm, A.; J. L. Reedy, J. C. Nussbaum & J. Heitman: Crypto- However, by using this mutant, we could find a naturally coccus neoformans virulence gene discovery through inser- occurring cyclic amide compound that would be a true tional mutagenesis. Eukaryot. Cell 3: 420–429, 2004 substrate for SttH. Investigation into the metabolites in S. 6) Joshi, P. B.; J. R. Webb, J. E. Davies & W. R. McMaster: The albulus CRM002 is now under way. gene encoding streptothricin acetyltransferase (sat) as a selectable marker for Leishmania expression vectors. Gene 156: REFERENCES 145–149, 1995 7) Takemoto, T.; Y. Inamori, Y. Kato, M. Kubo, K. Morimoto, K. Morisaka, M. Sakai, Y. Sawada & H. Taniyama: Physio- 1) Waksman, S. A.: Production and activity of streptothricin. J. logical activity of streptothricin antibiotics. Chem. Pharm. Bacteriol. 46: 299–310, 1943 Bull. (Tokyo) 28: 2884–2891, 1980 2) Goldstein, A. L. & J. H. McCusker: Three new dominant drug

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