Construction of a new cloning vector utilizing a cryptic plasmid and the highly expressed melanin-synthesizing gene operon from Streptomyces castaneoglobisporus

Streptomyces castaneoglobisporus HUT6202 overproduces a diffusible melanin pigment and harbors a cryptic 7.4-kb plasmid, pHY6202. We constructed a Streptomyces cloning vector, pSY10CMM, consisting of the HUT6202 rep gene, the thiostrepton resistance gene and an operon encoding the synthesis of melanin pigment, abbreviated mel, from S. castaneoglobisporus. The vector, which has SphI and BamHI sites as cloning sites with insertional inactivation of the mel, is a more convenient cloning vector than commonly used pIJ702, because of its broad host range for antibiotic-producing Streptomyces strains and its much greater production of diffusible melanin pigment.     

Expression ofStreptomyces melC andchoA genes by a clonedStreptococcus thermophilus promoter STP2201

Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected inEscherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3. Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone ST_P2201 as a strong promoter inE. coli. Subcloning of a ST_P2201-melC DNA fragment into the pMEU-seriesS. thermophilus-E. coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel⁺ phenotype toE. coli. The pEU5aML2201a was further shown to afford a high level of tyrosinase production (2 units mg^–1 protein) inE. coli, and to produce an apparently inactivemelC gene product that reacts with anti-tyrosinase antiserum inS. thermophilus. SubstitutingmelC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to anE. coli transformant at a level of (1.06±0.15)×10^–7 units mg^–1 protein. Introduction of this plasmid intoS. thermophilus by electrotransformation yielded ChoA transformant that produced the enzyme at about 25% of the level found inE. coli.     

Cloning of a tyrosinase gene in Streptococcus thermophilus

Summary The streptococcal cloning vector pIL253 (4.96-kbp, Em^r) was used to introduce the Streptomyces antibioticus tyrosinase (mel) gene (1.56-kbp) into S. thermophilus, an important microbe in dairy fermentations. Electrotransformants of S. thermophilus ST128 contained 6.51-kbp recombinant plasmids which probed positively in Southern hybridizations with the biotin-labeled mel fragment. Western blots of cell extracts resolved by SDS-PAGE showed the presence of a ca. 31-kDa band thus confirming the synthesis of tyrosinase protein by genetic transformants.     

An efficient method for the overexpression and purification of active tyrosinase from Streptomyces castaneoglobisporus

The melanin-synthesizing gene operon cloned from Streptomyces castaneoglobisporus HUT6202 consists of two genes, designated tyrC and orf378, which encode apotyrosinase (TYRC) and its activator protein (ORF378), respectively. We have suggested that ORF378 may facilitate the incorporation of Cu(II) into apotyrosinase to express tyrosinase activity. To overproduce ORF378 and TYRC in Escherichia coli BL21(DE3)-pLysS, tyrC, and orf378 were independently but not polycistronically placed under the control of a T7 promoter in a vector, pET-21a(+). His(6)-tagged TYRC and His(6)-tagged ORF378 were simultaneously overproduced in an E. coli strain harboring a plasmid, designated pET-mel2, and the two proteins were co-purified with a Ni(II)-bound affinity column. Gel filtration analysis revealed that the two proteins form a heterodimer complex. The complexed protein was retrieved at a high efficiency (11 mg/L). To obtain an active TYRC, which is a Cu(II)-bound form of tyrosinase, we constructed pET-mel3 that carries orf378 without His(6)-tag and His(6)-tagged tyrC. After the cell-free extract from E. coli harboring pET-mel3 was subjected to Cu(II)-bound affinity column chromatography, His(6)-tagged TYRC, eluted from the column, exhibited the tyrosinase activity. The k(cat) and K(m) values for l-3,4-dihydroxyphenylalanine (l-DOPA) of His(6)-tagged TYRC, which catalyzes the oxidation of l-DOPA to dopaquinone, were 880+/-80s(-1) and 8.1+/-0.9 mM, respectively.     

The nucleotide sequence of the tyrosinase gene from Streptomyces antibioticus and characterization of the gene product

The sequence of a 1.56-kb DNA fragment containing the tyrosinase gene (mel) from Streptomyces antibioticus was determined and the Mr (30612) and amino acid (aa) sequence of the protein were deduced from the nucleotide (nt) sequence. Intracellular and extracellular tyrosinase from S. antibioticus, transformed with pIJ702 (containing mel), were purified to homogeneity; the Mr (29 500), as determined by Sephadex G-75 chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was consistent with the value derived from the nt sequence. Edman degradation established that the N-terminal sequence of both the intracellular and extracellular forms of tyrosinase are identical and correspond to the aa sequence derived from the structural gene. In addition, this sequence exhibits striking homology to the N-terminal region of the intracellular and extracellular enzyme purified from Streptomyces glaucescens (Crameri et al., 1982). An additional open reading frame (ORF438) upstream of the mel gene, was also identified that appears to code for a protein (Mr = 14 754) with a putative signal sequence.     

Cloning and expression of the tyrosinase gene from Streptomyces antibioticus in Streptomyces lividans

In two separate studies a BclI-generated DNA fragment coding for the enzyme tyrosinase, responsible for melanin synthesis, was cloned from Streptomyces antibioticus DNA into two SLP1.2-based plasmid vectors (pIJ37 and pIJ41) to generate the hybrid plasmids, designated pIJ700 and pIJ701, using S. lividans 66 as the host. The fragment (1.55 kb) was subcloned into the multicopy plasmid pIJ350 (which carries thiostrepton resistance and has two non-essential BclI sites) to generate four new plasmids (pIJ702-pIJ705) with the tyrosinase insert located in either orientation at each site. All six plasmids conferred melanin production (the Mel+ phenotype) on their host. As in the S. antibioticus parent, strains of S. lividans carrying the gene specifying tyrosinase synthesis possessed an enzyme activity which was inducible. Most of the tyrosinase activity was secreted during growth of S. antibioticus; in contrast, the majority remained intracellular in the S. lividans clones. The specific activity of the induced tyrosinase activity (intracellular) was higher (up to 36-fold) when the gene was present on the multicopy vector in comparison with its location on the low copy plasmids, pIJ700 or pIJ701, or in S. antibioticus. Restriction mapping of the tyrosinase fragment in pIJ702 revealed endonuclease cleavage sites for several enzymes, including single sites for BglII, SphI and SstI that are absent from the parent vector (pIJ350). Insertion of DNA fragments at any one of these sites abolished the Mel+ phenotype. The results indicate that pIJ702 is a useful cloning vector with insertional inactivation of the Mel+ character as the basis of clone recognition.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

Metagenomes of complex microbial consortia derived from different soils as sources for novel genes conferring formation of carbonyls from short-chain polyols on Escherichia coli

Metagenomic DNA libraries from three different soil samples (meadow, sugar beet field, cropland) were constructed. The three unamplified libraries comprised approximately 1267000 independent clones and harbored approximately 4.05 Gbp of environmental DNA. Approximately 300000 recombinant Escherichia coli strains of each library per test substrate were screened for the production of carbonyls from short-chain (C2 to C4) polyols such as 1,2-ethanediol, 2,3-butanediol, and a mixture of glycerol and 1,2-propanediol on indicator agar. Twenty-four positive E. COLI clones were obtained during the initial screen. Fifteen of them contained recombinant plasmids, designated pAK201-215, which conferred a stable carbonyl-forming phenotype on E. coli Sequencing revealed that the inserts of pAK201-215 encoded 26 complete and 14 incomplete predicted protein-encoding genes. Most of these genes were similar to genes with unknown functions from other microorganisms or unrelated to any other known gene. The further analysis was focused on the 7 plasmids (pAK204, pAK206, pAK208, and pAK210-213) recovered from the positive clones, which exhibited an NAD(H)-dependent alcohol oxidoreductase activity with polyols or the correlating carbonyls as substrates in crude extracts. Three genes (ORF6, ORF24, and ORF25) conferring this activity were identified during subcloning of the inserts of pAK204, pAK211, and pAK212. The sequences of the three deduced gene products revealed no significant similarities to known alcohol oxidoreductases, but contained putative glycine-rich regions, which are characteristic for binding of nicotinamide cofactors.     

Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport

A 2.7 kb fragment of Helicobacter pylori UA802 chromosomal DNA was cloned and sequenced. Three open reading frames (designated ORF1, oRF2 and ORF3, respectively) were predicted from the DNA sequence, of which ORF1 and ORF2 appeared to be located within the same operon. The deduced 611-amino-acid sequence of ORF1, a P-type ATPase (designated hpCopA), had striking homology (29-38%) with several bacterial P-type ATPases and contained the potential functional domains conserved in P-type ATPases from various sources ranging from bacterial to human. A protein of 66 amino acids (designated hpCopP) encoded by ORF2 shared extensive sequence similarity with MerP, a periplasmic mercuric ion-transporting protein, and contains the heavy metal-binding motif. Disruption of ORF1 with a chloramphenicol-resistance cassette (CAT) rendered the H. pylori mutants more susceptible to cupric ion than their parental strains, whereas there is no significant alternation of susceptibility to Ni²⁺, Cd²⁺ and Hg²⁺ between the mutants and the parental strains. The results obtained indicate that ORF1 and ORF2 comprise a cation-transporting system which is associated with copper export out of the H. pylori cells.     

The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus.

The melC operon of Streptomyces antibioticus is unstable, undergoing frequent spontaneous deletions. All the delta melC mutants analyzed also lost 2-kb V1 DNA, which contained two open reading frames (ORFs) homologous to ORF4 (a putative oxidoreductase gene) and ORF5 (a putative AraC-type regulatory gene) of Tn4811. The two ORFs may constitute an accessory unit of a different transposon.     

Characterisation of the cry54Ab1 operon of Bacillus thuringiensis subsp. sichuansis strain MC28

A novel cry gene operon harbouring two open reading frames (ORFs) was discovered in a large plasmid of Bacillus thuringiensis (Bt) subsp. sichuansis MC28 (pMC189). The first ORF encoded 743 amino acids. It had a deduced molecular mass of 84.5 kDa and 73.2% identity with Cry54Aa1 protein as indicated by amino acid homology. This cry gene was designated as cry54Ab1 by the Bt Toxin Nomenclature Committee. The second ORF encoded 503 amino acids and had a deduced molecular mass of 58.2 kDa. It is here called as orf2. The whole cry54Ab1 operon, cry54Ab1 and orf2 were digested with BamHI/SalI and inserted into a shuttle vector, pSTK. The recombinant plasmids were transferred into an acrystalliferous mutant Bt HD73^?. Parasporal inclusions were observed only under expression of the whole cry54Ab1 operon. The bioassay experiments showed that expressed products of the cry54Ab1 and the whole cry54Ab1 operon were all toxic to Culex quinquefasciatus (Diptera).     

Sequencing and analysis of aMycoplasma gallisepticum A5969 chromosome region containing the S10 andrrn23-5 operons

A 20,115-nt region of theMycoplasma gallisepticum A5959 genome was sequenced (GenBank accession no. AF036708). The region contains therrn23-5 and S10 operons, the lactate dehydrogenase gene, and two open reading frames (ORF293 and ORF129/ORF171) coding for proteins of unknown function. Therrn23-5 operon includes genes for 23S and 5S rRNAs. The S10 operon includes genes for 20 ribosomal proteins, Sec Y transport protein, adenylate kinase, and methionine aminopeptidase, and lacks theinfA-rpl36-rps13-rpoA-rpl17 genes found in the S10 operon ofM. genitalium, M. pneumoniae, andBacillus subtilis. The product ofM. gallisepticum ldh is equally similar to the corresponding proteins of mycoplasmata andB. subtilis but contains only a part of the motif characteristic of the active center of lactate dehydrogenases. The chromosome region adjacent to the sequenced one containsuvrA,nrdE,nrdF, andptsI.     

A Non-Restricting and Non-Methylating <Emphasis Type="Italic">Escherichia coli</Emphasis> Strain for DNA Cloning and High-Throughput Conjugation to <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

Escherichia coli strains are used in secondary metabolism research for DNA cloning and transferring plasmids by intergeneric conjugation. Non-restricting strains are desirable for DNA cloning and non-methylating strains are beneficial for transferring DNA to methyl-restricting hosts, like Streptomyces coelicolor. We have constructed a non-methylating E. coli strain, JTU007, by deleting the DNA methylation genes dcm and dam from the widely used non-restricting cloning host DH10B. JTU007 was tested as donor for the conjugative transfer of a plasmid containing the 39 kb actinorhodin biosynthesis gene cluster to S. lividans and S. coelicolor. The Dcm⁻ Dam⁻ strain JTU007 transferred DNA into S. coelicolor A(3)2 derivatives at high frequency. To demonstrate the usefulness of E. coli JTU007 for gene cloning, we constructed a comprehensive S. toxytricini genomic cosmid library, and transferred it using high-throughput conjugation to the methyl-restricting S. coelicolor. One of the cosmid clones produced a brown pigment, and the clone was revealed to carry a tyrosinase operon. JTU007 is more useful than ET12567 because it does not restrict methylated DNA in primary cloning, and gives higher transformation and cosmid infection frequencies.     

Interspecific transfer and expression of melanin gene(s) on cosmids in Streptomyces strains

Summary Cosmid pR4C1 has been constructed from a multicopy plasmid pIJ365 and the cohesive ends site of an actinophage R4 (Morino et al. 1985). The cosmid can be transferred to various Streptomyces strains by R4 phage-mediated transduction. Melanin-synthesizing gene(s) originated from Streptomyces antibioticus was inserted into the cosmid and succesfully transferred by tranduction to 7 different strains out of 10 strains examined. The features of melanin-gene(s) expresion were examined in those cells. Tyrosinase activities from the melanin-gene(s) were detected in culture media and/or cell extracts although the extents of mel gene expression and tyrosinase excretion vary strain to strain. The cosmid transfer system described in this paper will be promising to survey suitable hosts for the maximum expression of cloned genes among Streptomyces strains.