The bialaphos biosynthetic genes ofStreptomyces hygroscopicus: Molecular cloning and characterization of the gene cluster

Summary We have isolated and studied the organization ofStreptomyces hygroscopicus genes responsible for the biosynthesis of the antibiotic herbicide bialaphos. Bialaphos production genes were cloned from genomic DNA using a plasmid vector (pIJ702). Three plasmids were isolated which restored productivity toS. hygroscopicus mutants blocked at different steps of the biosynthetic pathway. Subcloning experiments using other nonproducing mutants showed that four additional bialaphos production genes were also contained on these plasmids. A gene conferring resistance to bialaphos, which was independently cloned using the plasmid vector pIJ61, and an antibiotic-sensitive host (S. lividans), was also linked to the production genes. Cosmids were isolated which defined the location of these genes in a 16 kb cluster.     

Molecular cloning and identification of the laspartomycin biosynthetic gene cluster from Streptomyces viridochromogenes

The biosynthetic gene cluster for laspartomycins, a family of 11 amino acid peptide antibiotics, has been cloned and sequenced from Streptomyces viridochromogenes ATCC 29814. Annotation of a segment of 88912bp of S. viridochromogenes genomic sequence revealed the putative lpm cluster and its flanking regions which harbor 43 open reading frames. The lpm cluster, which spans approximately 60 kb, consists of 21 open reading frames. Those include four NRPS genes (lpmA/orf18, lpmB/orf25, lpmC/orf26 and lpmD/orf27), four genes (orfs 21, 22, 24 and 29) involved in the lipid tail biosynthesis and attachment, four regulatory genes (orfs 13, 19, 32 and 33) and three putative exporters or self-resistance genes (orfs 14, 20 and 30). In addition, the gene involved in the biosynthesis of the nonproteinogenic amino acid Pip was also identified in the lpm cluster while the genes necessary for the biosynthesis of the rare residue diaminopropionic acid (Dap) were found to reside elsewhere on the chromosome. Interestingly, the dabA, dabB and dabC genes predicted to code for the biosynthesis of the unusual amino acid diaminobutyric acid (Dab) are organized into the lpm cluster even though the Dab residue was not found in the laspartomycins. Disruption of the NRPS lpmC gene completely abolished laspartomycin production in the corresponding mutant strain. These findings will allow molecular engineering and combinatorial biosynthesis approaches to expand the structural diversity of the amphomycin-group peptide antibiotics including the laspartomycins and friulimicins.     

Molecular cloning and characterization of a 2-deoxystreptamine biosynthetic gene cluster in gentamicin-producing Micromonospora echinospora ATCC15835

The organization of the 2-deoxystreptamine (DOS) biosynthetic gene cluster of Micromonospora echinospora has been determined. Sequencing of a 14.04 kb-region revealed twelve open reading frames (ORFs): four putative DOS biosynthetic genes (gtmA, B, C, and D), five amino sugars biosynthetic genes (gtmE, G, H, I, and gacB), two aminoglycoside resistance genes (gtmF and J) as well as a hypothetical ORF (gacA). One of the putative DOS biosynthetic genes, gtmA, was expressed in Escherichia coli, and the purified protein was shown to convert glucose-6-phosphate (G-6-P) to 2-deoxy-scyllo-inosose (DOI), a key step in DOS biosynthesis. In addition gtmJ was expressed in Streptomyces lividans and shown to confer gentamicin resistance. Thus gtmA and gtmJ are implicated in the biosynthesis of gentamicin and in resistance to it, respectively.     

Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: Analysis of genes flanking the polyketide synthase

Analysis of the gene cluster from Streptomyces hygroscopicus that governs the biosynthesis of the polyketide immuno-suppressant rapamycin (Rp) has revealed that it contains three exceptionally large open reading frames (ORFs) encoding the modular polyketide synthase (PKS). Between two of these lies a fourth gene (rapP) encoding a pipecolate-incorporating enzyme that probably also catalyzes closure of the macrolide ring. On either side of these very large genes are ranged a total of 22 further ORFs before the limits of the cluster are reached, as judged by the identification of genes clearly encoding unrelated activities. Several of these ORFs appear to encode enzymes that would be required for Rp biosynthesis. These include two cytochrome P-450 monooxygenases (P450s), designated RapJ and RapN, an associated ferredoxin (Fd) RapO, and three potential SAM-dependent O-methyltransferases (MTases), RapI, RapM and RapQ. All of these are likely to be involved in ‘late’ modification of the macrocycle. The cluster also contains a novel gene (rapL) whose product is proposed to catalyze the formation of the Rp precursor, L-pipecolate, through the cyclodeamination of L-lysine. Adjacent genes have putative roles in Rp regulation and export. The codon usage of the PKS biosynthetic genes is markedly different from that of the flanking genes of the cluster     

Molecular cloning and characterization of the human beta-like globin gene cluster

The genes encoding human embryonic (epsilon), fetal (G gamma, A gamma) and adult (delta, beta) beta-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage lambda libraries of high molecular weight (15-20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five beta-like polypeptides, including the embryonic epsilon-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5'-epsilon-(13.3 kb)-G gamma-(3.5 kb)-A gamma-(13.9 kb)-delta-(5.4 kb)-beta-3'. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known beta-like globin genes, we have detected two other beta-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the A gamma-delta intergenic region while the other is located 6-9 kb 5' to the epsilon gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence (*) which is interspersed with the globin genes in the following manner: 5'-**epsilon-*G gamma-A gamma*-**delta-beta*-3'. Fine structure mapping of the region located 5' to the delta-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.     

Molecular cloning and characterization of an ML-236B (compactin) biosynthetic gene cluster in Penicillium citrinum

Cloning of genes encoding polyketide synthases (PKSs) has allowed us to identify a gene cluster for ML-236B biosynthesis in Penicillium citrinum. Like lovastatin, which is produced by Aspergillus terreus, ML-236B (compactin) inhibits the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Genomic sequencing and Northern analysis showed that nine predicted genes for ML-236B biosynthesis were located within a 38-kb region and were transcribed when ML-236B was produced. The predicted amino acid sequences encoded by these nine genes, designated mlcA- mlcH and mlcR, were similar to those encoded by the genes for lovastatin synthesis, and were therefore assumed to be involved either directly or indirectly in ML-236B biosynthesis. Targeted disruption experiments provided evidence that two PKS genes in the cluster, mlcA and mlcB, are required for the biosynthesis of the nonaketide and the diketide moieties, respectively, of ML-236B, suggesting that the gene cluster as a whole is responsible for ML-236B biosynthesis in P. citrinum. Bioconversion of some of the predicted intermediates by an mlcA-disrupted mutant was also investigated in order to analyze the ML-236B biosynthetic pathway. The molecular organization of the gene cluster and proposed functions for the ML-236B biosynthetic genes in P. citrinum are described.     

Molecular cloning and characterization of two sets of alpha-theta genes in the rat alpha-like globin gene cluster

The highly heterogeneous rat hemoglobin system was investigated at the gene level. Two regions of the alpha-like globin gene cluster from a Wistar rat were isolated. Four lambda Dash recombinant clones carrying rat alpha-like globin genes were localized on two distinct gene regions. A region of approximately 16kb was found to contain the 5'-IIalpha1-psi theta 1-3' loci, and another of approximately 24kb the 5'-IIalpha2-psi theta2-psiI alpha3-3' loci. Both IIalpha1 and IIalpha2 are considered to be active, coding the IIalpha-globin chain. The nt sequences of IIalpha1 and IIalpha2 are identical except for six nt in the non-coding region. The psiI alpha3 locus is a truncated pseudogene. The putative promoter region of an alpha-like globin gene is joined directly to the third exon, homologous to that of Ialpha-globin cDNA. psi theta1 and psi theta2 are also pseudogenes, as evidenced by several deletions located in the protein-coding regions of these loci. The psi theta1 and psi theta2 loci exhibit extensive homology, but the restriction maps of these genes and their flanking regions differ considerably. Genomic Southern blot analyses of the total liver DNA from six rats showed the existence of three theta-globin-related genes, including psi theta1 and psi theta2. These results indicate that the two gene regions investigated are not allelic variants, but may be generated by block duplication. This is the first report of the existence of rodent theta-globin genes.     

Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin

Streptomyces viridifaciens MG456-hF10 produces the antibiotic valanimycin, a naturally occurring azoxy compound. Valanimycin is known to be derived from valine and serine with the intermediacy of isobutylamine and isobutylhydroxylamine, but little is known about the stages in the pathway leading to the formation of the azoxy group. In previous studies, a cosmid containing S. viridifaciens DNA was isolated that conferred valanimycin production upon Strepto-myces lividans TK24. Subcloning of DNA from the valanimycin-producing cosmid has led to the identi-fication of a 22 kb segment of DNA sufficient to allow valanimycin production in S. lividans TK24. Sequencing of this DNA segment and the surrounding DNA revealed the presence of 20 genes. Gene disruption experiments defined the boundaries of the valanimycin gene cluster, which appears to contain 14 genes. The cluster includes an amino acid decar-boxylase gene (vlmD), a valanimycin resistance gene (vlmF ), at least two regulatory genes (vlmE, vlmI ), two genes encoding a flavin monooxygenase (vlmH, vlmR), a seryl tRNA synthetase gene (vlmL ) and seven genes of unknown function. Overproduction and characterization of VlmD demonstrated that it catalyses the decarboxylation of l-valine. An unusual feature of the valanimycin gene cluster is that four genes involved in branched amino acid biosynthesis are located near its 5' end.     

Molecular basis for chloronium-mediated meroterpene cyclization: cloning, sequencing, and heterologous expression of the napyradiomycin biosynthetic gene cluster

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.     

An approach for cloning biosynthetic genes of 2-deoxystreptamine-containing aminocyclitol antibiotics: isolation of a biosynthetic gene cluster of tobramycin from Streptomyces tenebrarius

Genes homologous to 2-deoxystreptamine (DOS) biosynthetic genes were isolated from aminoglycoside producers, Micromonospora and Streptomyces spp., using PCR primers based on the core sequences of 2-deoxy-scyllo-inosose (DOI) synthase and L-glutamine: scyllo-inosose aminotransferase (GIA). Identities of 40-45% were observed for DOI synthases, and 65-75% were observed for GIAs. The gene cluster of tobramycin biosynthesis was isolated from the genomic library of Streptomyces tenebrarius using DOI synthase as a probe. Sequencing of 33.9 kb revealed 24 putative open reading frames including the tobramycin biosynthetic gene cluster (13.8 kb) and a transport protein. This cluster encodes proteins homologous to 2-deoxystreptamine biosynthetic enzymes, glycosyltransferase and other aminocyclitols biosynthetic enzymes. Sequence analysis revealed the evolution of DOI synthases from 3-dehydroquinate (DHQ) synthases in actinomycetes. DOI synthases and GIA are therefore useful for cloning biosynthetic genes of DOS-containing aminocyclitol antibiotics or for screening such metabolites producers.     

Molecular cloning and characterization of ovine homeo-box-containing genes

The sheep genome contains at least eleven homeo-boxes (hox). Using two hox-specific 36-mer oligodeoxynucleotides to screen a sheep genomic library, constructed in lambda Charon28, clones of nine of the hox were identified. Six of the hox clones were analysed by nucleotide sequencing, Southern-blot hybridization and Northern-blot analysis. Two of the hox appear to be cognates of the human Hu-1 (or mouse Hox 2.1) and the mouse Hox 1-3, while another is closely related to the mouse Hox 1-4. These results suggest that there is strong sequence conservation in the hox-containing genes of different mammals, and highlight the possible occurrence of an ubiquitous set of hox-containing genes in mammals. Northern-blot analysis of four sheep hox-containing genes indicates that they are all expressed during embryogenesis and that expression is temporally regulated allowing hierarchical-regulatory interaction. Interestingly, none of the cloned hox-containing sequences contain repetitive sequences.     

Molecular cloning of menaquinone biosynthetic genes of Escherichia coli K12

Summary A tranducing phage carrying some of the genes (men) defining the early stages of menaquinone biosynthesis was isolated from a pool of recombinant lambda phages that had been constructed from R.HindIII digests of E. coli DNA and the corresponding insertion vector. The lesions of menB and menC mutants were complemented by the phage but menD mutants were transduced either at low frequencies or not at all. This indicates that the transducing phage contains functional menB and menC genes but that only part of the menD gene had been cloned. The phage (G68) was accordingly disignated menCB(D). Studies with the transducing phage enabled earlier mapping data (Guest 1979) to be reinterpreted in favour of the gene order nalA.... menC..menB..menD.... purF. Restriction analyses established the presence of a bacterial DNA fragment (11.5 kb) linked by a R.HindIII target to the right arm of the genome but fused to the left arm of the vector. Hybridization studies confirmed that the cloned DNA was derived from a larger R.HindIII fragment (21 kb). A physical map of the men region was constructed and some flanking and overlapping fragments were identified.