Control Of Tac Promoter Research Articles

Reserpine Is the New Addition into the Repertoire of AcrB Efflux Pump Inhibitors

Acriflavine resistance protein B (AcrB) serves as prototype for multidrug resistance (MDR) efflux transporters of resistance nodulation division (RND) superfamily. AcrB has been proven as potential drug target with many synthetic and natural inhibitors have been identified such as those belonging to pyranopyridine, naphthamide and pimozide classes. The plant derived alkaloid inhibitors represented by reserpine has been found to inhibit both ATP binding cassette and major facilitator efflux transporters. In this study we report the reserpine induced inhibition of RND transporter AcrB. The preliminary docking analysis hints that reserpine shares its binding site with ciprofloxacin, a known substrate of AcrB and could possibly act as competitive inhibitor. For in vitro validation, AcrB from Salmonella typhi was cloned under the control of tac promoter and resulting vector was introduced into E. coli C41(DE3). Under autoinduced conditions, cells overexpressing AcrB transporter were subjected to combined dose of ciprofloxacin and reserpine. The combined exposure resulted in enhanced ciprofloxacin-induced growth inhibition of cells expressing AcrB transporter as compared to control cells transformed with vector of backbone sequence. Time kill analysis further confirmed these findings. To the best of our knowledge, this is first study to show that exposure to reserpine induces inhibition of AcrB. The assay developed in this study allows simple and reproducible detection of substrate/inhibitor effects upon AcrB and related efflux transporters.

Improvement of desulfurizing activity of haloalkaliphilic Thialkalivibrio versutus SOB306 with the expression of Vitreoscilla hemoglobin gene.

To construct efficient transformation and expression system and further improve desulfurizing activity of cells through expression of Vitreoscilla hemoglobin (VHb) in haloalkaliphilic Thialkalivibrio versutus SOB306. We transferred plasmids pKT230 and pBBR-smr into T. versutus SOB306 via a conjugation method. We identified four promoters from among several predicted promoters by scoring for streptomycin resistance, and finally selected tac and p3 based on the efficiency of expression of red fluorescent protein (RFP). Expression of RFP when regulated by tac was more than three times that of p3 in SOB306. Further, we expressed VHb under the control of tac promoter in SOB306. Expression of VHb was verified using CO-difference spectra. The results showed that VHb expression can boost sulfur metabolism, as evidenced by an increase of about 11.7±1.8% in the average rate of thiosulfate removal in the presence of VHb. A conjugation transfer and an expression system for Thialkalivibrio, has been developed for the first time and used for expression of VHb to improve desulfurizing activity.

Enhanced 1-Butanol Production in Engineered Klebsiella pneumoniae by NADH Regeneration

1-Butanol, as a next-generation biofuel, is an important target product of biorefinery research. With the introduction of the CoA-dependent pathway or Ehrlich pathway, many engineered strains have been developed to produce 1-butanol, although cofactor imbalance occurred in engineered strains by the introduction of the 1-butanol synthesis pathway. Several studies have been performed to regenerate NADH by overexpressing NAD+-dependent enzymes. However, the significant role of cofactor regeneration in 1-butanol production and the transcription level of the target genes have seldom been studied. The 1-butanol producer, recombinant Klebsiella pneumoniae (KLA), was constructed by overexpressing the genes kivd, leuABCD, and adhE1 under the control of tac promoter in this study, and several NADH regeneration strategies were adopted to solve the problem of NADH imbalance, including the introduction of NAD+-dependent enzymes (formate dehydrogenase, pyridine nucleotide transhydrogenase, and glucose dehydrogenase) or elimination of the NADH competition pathway (1,3-propanediol synthesis). The resultant NADH/NAD+ ratio, 1-butanol production, and transcription levels have been significantly affected. In comparison to the wild-type strain, the NADH/NAD+ ratio in the reengineered strains was increased by 78–135%, and the transcript levels of target genes have been obviously interfered. Moreover, the resultant 1-butanol titer was increased by 83–114% in comparison to KLA.

Production of DNA polymerase from Thermus aquaticus in recombinant Escherichia coli

Among dozens of DNA polymerases cloned from thermophilic bacteria, Taq DNA polymerase from Thermus aquaticus has been most frequently used in polymerase chain reaction (PCR) that is being applied to gene cloning, DNA sequencing, gene expression analysis, and detection of infectious and genetic diseases. Since native Taq DNA polymerase is expressed at low level in T. aquaticus, recombinant Escherichia coli system was used to produce Taq DNA polymerase in a large amount. Taq DNA polymerase was expressed as a soluble form under the control of tac promoter in E. coli, and purified by heat treatment and ion exchange chromatographies. The purified Taq DNA polymerase was nearly homogeneous and exhibited a similar DNA amplification activity with a commercial Taq DNA polymerase.

Type 2 IDI performs better than type 1 for improving lycopene production in metabolically engineered E. coli strains

In this study a comparison was made between type 1 and type 2 isopentenyl diphosphate isomerases (IDI) in improving lycopene production in Escherichia coli. The corresponding genes of Bacillus licheniformis and the host (i(Bl) and i(Ec), respectively) were expressed in lycopene producing E. coli strains by pTlyci(Bl) and pTlyci(Ec) plasmids, under the control of tac promoter. The results showed that the overexpression of i(Ec) improved the lycopene production from 33 ± 1 in E. coli Tlyc to 68 ± 3 mg/gDCW in E. coli Tlyci(Ec). In contrast, the expression of i(Bl) increased the lycopene production more efficiently up to 80 ± 9 mg/gDCW in E. coli Tlyci(Bl). The introduction of a heterologous mevalonate pathway to elevate the IPP abundance resulted in a lycopene production up to 132 ± 5 mg/gDCW with i(Ec) in E. coli Tlyci(Ec)-mev and 181 ± 9 mg/gDCW with i(Bl) in E. coli Tlyci(Bl)-mev, that is, 4 and 5.6 times respectively. When fructose, mannose, arabinose, and acetate were each used as an auxiliary substrate with glycerol, lycopene production was inhibited by different extents. Among auxiliary substrates tested, only citrate was an improving one for lycopene production in all strains with a maximum of 198 ± 3 mg/gDCW in E. coli Tlyci(Bl)-mev. It may be concluded that the type 2 IDI performs better than the type 1 in metabolic engineering attempts for isoprenoid production in E. coli. In addition, the metabolic engineering of citrate pathway seems a promising approach to have more isoprenoid accumulation in E. coli.

Expression of Clostridium acetobutylicum butanol synthetic genes in Escherichia coli

A recombinant butanol pathway composed of Clostridium acetobutylicum ATCC 824 genes, thiL, hbd, crt, bcd-etfB-etfA, and adhe1 (or adhe) coding for acetyl-CoA acetyltransferase (THL), beta-hydroxybutyryl-CoA dehydrogenase (HBD), 3-hydroxybutyryl-CoA dehydratase (CRT), butyryl-CoA dehydrogenase (BCD), butyraldehyde dehydrogenase (BYDH), and butanol dehydrogenase (BDH), under the tac promoter control was constructed and was introduced into Escherichia coli. The functional expression of these six enzymes was proved by demonstrating the corresponding enzyme activities using spectrophotometric, high performance liquid chromatography and gas chromatography analyses. The BCD activity, which was not detected in E. coli previously, was shown in the present study by performing the procedure from cell extract preparation to activity measurement under anaerobic condition. Moreover, the etfA and etfB co-expression was found to be essential for the BCD activity. In the case of BYDH activity, the adhe gene product was shown to have higher specificity towards butyryl-CoA compared to the adhe1 product. Butanol production from glucose was achieved by the highly concentrated cells of the butanologenic E. coli strains, BUT1 with adhe1 and BUT2 with adhe, under anaerobic condition, and the BUT1 and BUT2 strains were shown to produce 4 and 16-mM butanol with 6- and 1-mM butyrate as a byproduct, respectively. This study reports the novel butanol production by an aerobically pregrown microorganism possessing the genes of a strict anaerobe, Clostridium acetobutylicum.

The Role of Human HtrA1 in Arthritic Disease

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms.

A two-stage refinement approach for the enhancement of excretory production of an exoglucanase from Escherichia coli

Hyper-expression of a secretory exoglucanase, Exg, encoded by the cex gene of Cellulomonas fimi was previously shown to saturate the SecYEG pathway and result in dramatic cell death of recombinant Escherichia coli (Z.B. Fu, K.L. Ng, T.L. Lam, W.K.R. Wong, Cell death caused by hyper-expression of a secretory exoglucanase in Esherichia coli, Protein Expr. Purif. 42 (2005) 67–77). We propose here that the cell lysate ratio (Pre/Mat RQ) of the unprocessed precursor Exg protein (Pre-Exg) and its processed mature product (Mat-Exg) reflects the capacity of E. coli to secrete Exg. A Pre/Mat RQ of 20/80, designated the “Critical Value,” was an important threshold measurement. A rise in the Pre/Mat RQ triggered a mass killing effect. The use of various secretion signal peptides did not improve the viability of cells expressing high levels of Pre-Exg under strong tac promoter control. However, use of the weaker vegG promoter in conjunction with a change in start codon of the spa leader sequence from ATG to TTG in a pM1vegGcexL plasmid construct resulted in a high level (0.9 U ml −1) of excreted Exg in shake-flask cultures. This was 50% higher than the best result obtained from plasmid construct lacUV5par8cex, using the lacUV5 promoter and the ompA leader sequence. Variations in the excreted Exg activities were attributable to differences in the Pre/Mat RQ values of the induced cultures harboring pM1vegGcexL and lacUV5par8cex. These values were 18/82 and 10/90, respectively. Employing fed-batch cultivation in two-liter fermentors, an induced JM101(pM1vegGcexL) culture yielded 4.5 U ml −1 of excreted Exg, which was over six fold greater that previously reported. Our results illustrate the successful application of the Pre/Mat RQ ratio as a guide to the attainment of a maximum level of secreted/excreted Exg.

Cloning and expression of Klebsiella phage K11 lysozyme gene

Previously, the lysozyme gene of the Klebsiella phage K11 was partially sequenced in our lab. Using the sequence information the lysozyme gene of the Klebsiella phage K11 was amplified and cloned using the polymerase chain reaction of the pfu DNA polymerase. The nucleotide sequence of phage K11 lysozyme gene was determined. The open reading frame corresponds to a polypeptide with 151 amino acids and molecular weight of 16,932 Da. The deduced amino acid sequence of this polypeptide shows 74–75% homologies to the T7 and T3 phage lysozymes. Although the gene was efficiently expressed under the control of tac promoter in Escherichia coli XL1-blue cells at 37 °C, most of the K11 lysozyme produced was insoluble. When the temperature of cell growth was lowered, however, solubility of the K11 lysozyme was increased gradually. The insoluble protein expressed at 37 °C was solubilized in 5 M guanidine–HCl and refolded in the presence of oxido-shuffling agent (GSH/GSSG). Through the refolding process the recombinant lysozyme was solubilized and purified. The purified K11 lysozyme showed transcription inhibition of K11 RNA polymerase as well as amidase activity. These results showed that the lysozyme of bacteriophage K11 is a bifunctional protein that cuts a bond in the bacterial cell wall and selectively inhibits K11 phage RNA polymerase. Also, transcription inhibition ability of K11 lysozyme with T7 or SP6 phage RNA polymerase was measured. T7 RNA polymerase was less inhibited than K11 RNA polymerase by K11 lysozyme. But SP6 RNA polymerase was not nearly inhibited by K11 lysozyme.

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His) 6-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni 2+–nitrilotriacetic acid agarose. The purity, as determined by SDS–PAGE analysis, was greater than 80%. The yield was 0.7–1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca 2+ was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.

Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus.

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.

Cloning, analysis and expression of the gene for β-glycosidase of Thermus caldophilus GK24 and properties of the enzyme

The gene (bglT) encoding Thermus caldophilus GK24 β-glycosidase (Tca β-glycosidase) was cloned and sequenced. The gene contains an open reading frame encoding 431 amino acids with a Mr of 48 658 Da. The bglT gene was expressed under the control of tac promoter on a high-copy plasmid in E. coli. The recombinant Tca β-glycosidase was purified 41.5-fold with a 59% yield and a specific activity of 83 U mg−1 protein.

Functional mapping against Escherichia coli for the broad-spectrum antimicrobial peptide, thanatin, based on an in vivo monitoring assay system.

Previously, we established for the first time an in vivo monitoring assay system conjugated with random mutagenesis in order to study the structure-function relationship of the antimicrobial peptide, apidaecin [Taguchi et al. (1996) Appl. Environ. Microbiol. 62, 4652-4655]. In the present study, this methodology was used to carry out the functional mapping of a second target, thanatin, a 21-residue peptide that exhibits the broadest antimicrobial spectrum so far observed among insect defense peptides [Fehlbaum et al. (1996) Proc. Natl. Acad. Sci. USA 93, 1221-1225]. First, a synthetic gene encoding thanatin was expressed in a fused form with Streptomyces protease inhibitor protein, SSI, under the control of tac promoter in Escherichia coli JM109. Expression of the thanatin-fused protein was found to depend on the concentration of the transcriptional inducer, isopropyl-beta-D-thio-galactopyranoside (IPTG), and to parallel the degree of growth inhibition of the transformant cells. When a PCR random mutation was introduced into the structural gene for thanatin, diminished growth inhibition of the IPTG-induced transformed cells was mostly observed in variants as measured by colony size (plate assay) or optical density (liquid assay) in comparison with the wild-type peptide, possibly depending on the decreased antimicrobial activity of each variant. Next, wild-type thanatin and three variants screened by the in vivo assay, two singly mutated proteins (C11Y and M21R) and one doubly mutated protein (K17R/R20G), were stably overproduced with a fusion partner protein resulting in the efficient formation of inclusion bodies in E. coli BL21(DE3). The products were isolated in large amounts (yield 30%) from the fused protein by successive chemical and enzymatic digestions at the protein fusion linker site. Anti-E. coli JM109 activities, judged by minimum inhibitory concentration, of the purified peptides were in good agreement with those estimated semi-quantitatively by the in vivo assay. Based on the NMR solution structure and molecular dynamics, the structure-function relationship of thanatin is discussed by comparing the functional mapping data obtained here with the previous biochemical data. The functional mapping newly suggests the importance of a hydrogen bonding network formed within the C-terminal loop joining the beta-strands arranged antiparallel to one another that are supposed to be crutial for exhibiting anti-E. coli activity.

Expression of Trigonopsis variabilis D-amino acid oxidase gene in Escherichia coli and characterization of its inactive mutants.

The D-amino acid oxidase cDNA gene (daao) of Trigonopsis variabilis was prepared by reverse transcriptase-polymerase chain reaction (PCR) and cloned into Escherichia coli expression vector, pTrc99A, under the control of tac promoter. Expression of daao gene significantly affected the growth and morphology of E. coli. The highest D-amino acid oxidase (DAAO) activity was 705 U (mg of protein)(-)(1), which was about 12-fold higher than that of D-alanine-induced T. variabilis. The DAAO protein exhibited activity on native-PAGE and had a M(r)value of 39.3 kDa. We also constructed an expression plasmid, pKm-DAAO, in which kanamycin instead of ampicillin was used as the selective marker. High-performance liquid chromatography (HPLC) analysis demonstrated that cephalosporin C could be converted to 7-glutarylcephalosporanic acid by cell-free extract of E. coli harboring pKm-DAAO. Four inactive DAAO mutants were obtained by error-prone PCR. Sequence analysis of these four DAAO mutants indicated the occurrence of mutations at Val-167, Pro-291, Pro-309, and Ala-343 residues. The His(6)-tagged DAAOs were expressed in E. coli and purified by nickel ion affinity chromatography. The results showed that all DAAO mutants lost their enzymatic activities and characteristic adsorption spectra for flavoenzyme. Based on the crystal structure of a homologous protein, pig DAAO, it is suggested that these four residues may play essential structural roles in DAAO conformation, thereby influencing DAAO's catalytic activity.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from γ-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a γ-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 291–299, 2000.

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation fromγ-aminobutyrate and glutamate

To provide 4-hydroxybutyryl-CoA for poly(3-hydroxybutyrate-co-4-hydroxybutyrate) formation from glutamate in Escherichia coli, an acetyl-CoA:4-hydroxybutyrate CoA transferase from Clostridium kluyveri, a 4-hydroxybutyrate dehydrogenase from Ralstonia eutropha, a gamma-aminobutyrate:2-ketoglutarate transaminase from Escherichia coli, and glutamate decarboxylases from Arabidopsis thaliana or E. coli were cloned and functionality tested by expression of single genes in E. coli to verify enzymatic activity, and uniquely assembled as operons under the control of the lac promoter. These operons were independently transformed into E. coli CT101 harboring the runaway replication vector pJM9238 for polyhydroxyalkanoate (PHA) production. Plasmid pJM9238 contains the PHA biosynthetic operon of R. eutropha under tac promoter control. Polyhydroxyalkanoate formation was monitored by nuclear magnetic resonance (NMR) spectroscopic analysis of the chloroform extracted and ethanol precipitated polyesters. Functionality of the biosynthetic pathway for copolymer production was demonstrated through feeding experiments using various carbon sources that supplied different precursors within the 4HB-CoA biosynthetic pathway.

Soluble expression of cloned phage K11 RNA polymerase gene in Escherichia coli at a low temperature.

The gene 1 of the Klebsiella phage K11 encoding the phage RNA polymerase was amplified using the polymerase chain reaction of the Pfu DNA polymerase, cloned and expressed under the control of tac promoter in Escherichia coli. Although the gene was efficiently expressed in E. coli BL21 cells at 37 degrees C, most of the K11 RNA polymerase produced was insoluble, in contrast to soluble expression of the cloned T7 RNA polymerase gene. Coexpression of the bacterial chaperone GroES and GroEL genes together did not help solubilize the K11 RNA polymerase. When the temperature of cell growth was lowered, however, solubility of the K11 RNA polymerase was increased substantially. It was found much more soluble when expressed at 25 degrees C than at 30 and 37 degrees C. Thus, the cloned K11 RNA polymerase gene was expressed in E. coli mostly to the soluble form at 25 degrees C. The protein was purified to homogeneity by chromatography using DEAE-Sephacel and Affigel-blue columns and was found to be active in vitro with the K11 genome or a K11 promoter. The purified K11 RNA polymerase showed highly stringent specificity for the K11 promoter. Low-level cross-reactivity was shown with the SP6 and T7 consensus promoters, while no activity shown with the T3 consensus promoter at all.

High level expression of soluble angiogenin in Escherichia coli.

Human angiogenin was genetically engineered and contained the E. coli Omp A signal sequence for secreting soluble angiogenin to the periplasm under tac promoter control. The angiogenin sequence was encoded in a single gene and expressed as a 14.4 kilodalton soluble protein in E. coli. It was purified by CM-Sepharose ion-exchange chromatography and by a heparin-Sepharose affinity chromatography procedure. The biological activity of angiogenin was established by its ability to inhibit mRNA-dependent rabbit reticulocyte cell-free translation.

Immunogenicity of a Salmonella typhi CVD 908 candidate vaccine strain expressing the major surface protein gp63 of Leishmania mexicana mexicana

Attenuated Salmonella typhi are attractive for use as live vector vaccines to express protozoal antigens and deliver them to the human immune system. The gene encoding the mature form of Leishmania mexicana mexicana gp63 under control of tac promoter was integrated into the ΔaroC locus of the chromosome of attenuated ΔaroC, ΔaroD S. typhi strain CVD 908. After oral immunization of BALB c mice with two 1 × 10 9 colony forming unit doses given 21 days apart, CVD 908Ω( ΔaroC:: P tac -gp63) elicited a broad T cell-mediated immune response against L. m. mexicana gp63 as demonstrated by: (1) lymphoproliferative response to fixed whole L. m. mexicana promastigotes; (2) activation of IL-2 (but not IL-4)-producing lymphocytes; (3) appearance of cytotoxic T cells against mouse mastocytoma cells expressing gp63. This T-cell mediated immune response was associated with significant protection in F1(BALB/cXC57Bl/6) mice challenged in their footpads with a wild type strain of L. m. mexicana.

A spectinomycin resistance determinant from the spectinomycin producer Streptomyces flavopersicus.

The spectinomycin (sp) resistance determinant from Streptomyces flavopersicus was cloned into Streptomyces lividans using the plasmid vector pIJ699. A plasmid, pDGL15, with a 3.65 kb insert from S. flavopersicus conferring resistance to Sp was isolated. DNA sequence analysis of the 3651 1 bp DNA insert revealed four open reading frames (ORFs). The amino acid sequence deduced from one ORF (SpcN) showed a high degree of similarity to an aminoglycoside phosphotransferase (StrN) and from a second one (SpcR) to a regulatory protein (StrR) of the streptomycin biosynthesis gene cluster from S. griseus. The two other ORFs were incomplete and the deduced amino acid sequences showed similarities to an amidinotransferase encoded in the streptomycin biosynthesis gene cluster of S. griseus and to the transposase of IS112, respectively. Expression of the spcN gene in E. coli under the control of tac promoter conferred Sp resistance to the cells. An enzymic assay confirmed that the gene product of spcN is an ATP-dependent aminoglycoside phosphotransferase which phosphorylates Sp and actinamine, the aminocyclitol moiety of Sp.