ISP5230 Maintains Excretion of Jadomycin upon Disruption of the MFS Transporter JadL Located within the Natural Product Biosynthetic Gene Cluster.

JadL was identified as a Major Facilitator Superfamily (MFS) transporter (T.C. 2.

Chloramphenicol biosynthesis: the structure of CmlS, a flavin-dependent halogenase showing a covalent flavin-aspartate bond.

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group.

Expression, purification and preliminary diffraction studies of CmlS.

CmlS, a flavin-dependent halogenase (FDH) present in the chloramphenicol-biosynthetic pathway in Streptomyces venezuelae, directs the dichlorination of an acetyl group. The reaction mechanism of CmlS is of considerable interest as it will help to explain how the FDH family can halogenate a wide range of substrates through a common mechanism. The protein has been recombinantly expressed in Escherichia coli and purified to homogeneity.

Culture conditions improving the production of jadomycin B.

The jadomycins are a unique family of benzoxazolophenanthridine antibiotics produced by Streptomyces venezuelae ISP5230 following heat or ethanol shock or phage infection. We have modified the culture conditions by altering the carbon source, buffer, inoculum size, and timing of ethanol shock, thereby reducing growing times and improving jadomycin B production. Our optimized conditions use glucose as the carbon source, MOPS as buffer, low concentrations of phosphate, a defined inoculum concentration and an immediate ethanol shock to induce jadomycin B production; results that contrast previous studies.

Functional analyses of oxygenases in jadomycin biosynthesis and identification of JadH as a bifunctional oxygenase/dehydrase.

A novel angucycline metabolite, 2,3-dehydro-UWM6, was identified in a jadH mutant of Streptomyces venezuelae ISP5230. Both UWM6 and 2,3-dehydro-UWM6 could be converted to jadomycin A or B by a ketosynthase alpha (jadA) mutant of S. venezuelae.

Biosynthesis of the dichloroacetyl component of chloramphenicol in Streptomyces venezuelae ISP5230: genes required for halogenation.

Five ORFs were detected in a fragment from the Streptomyces venezuelae ISP5230 genomic DNA library by hybridization with a PCR product amplified from primers representing a consensus of known halogenase sequences. Sequencing and functional analyses demonstrated that ORFs 11 and 12 (but not ORFs 13-15) extended the partially characterized gene cluster for chloramphenicol (Cm) biosynthesis in the chromosome. Disruption of ORF11 (cmlK) or ORF12 (cmlS) and conjugal transfer of the insertionally inactivated genes to S.

Control of growth, secondary metabolism and sporulation in Streptomyces venezuelae ISP5230 by jadW(1), a member of the afsA family of gamma-butyrolactone regulatory genes.

Three new genes (jadW(1), jadW(2) and jadW(3)) were isolated from a region of the Streptomyces venezuelae ISP5230 chromosome at the left-hand end of the jad cluster for jadomycin B (JdB) biosynthesis. The deduced amino acid sequence of jadW(1) showed strong similarity to gene products associated in several streptomycetes with gamma-butyrolactone autoregulators controlling morphological differentiation and secondary metabolism. Examination of JadW(1) for conserved domains detected a repeat sequence characteristic of proteins in the AfsA regulatory family.

Metabolites of a blocked chloramphenicol producer.

Addition of p-aminophenylalanine (4), an advanced biosynthetic precursor of the antibiotic chloramphenicol (5), to a Streptomyces venezuelae pabAB mutant (VS629) restored chloramphenicol production and led to formation of the non-chlorinated analogue corynecin II (6) and four acetanilide derivatives: p-(acetylamino)phenylalanine (7), p-(acetylamino)benzyl alcohol (13), p-(acetylamino)benzoic acid (14), and p-(acetylamino)phenol (acetaminophen, 16). Metabolite structures were deduced from NMR and MS-MS data and established by chromatographic and spectroscopic comparisons with authentic samples. Reference compound 13 was synthesized by reducing the acid chloride of 14.

Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230.

Consensus amino acid sequences of FADH(2)-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a beta-alpha-beta fold present in FAD-binding sites of certain monooxygenases.

Biosynthesis of the dideoxysugar component of jadomycin B: genes in the jad cluster of Streptomyces venezuelae ISP5230 for L-digitoxose assembly and transfer to the angucycline aglycone.

Eight additional genes, jadX, O, P, Q, S, T, U and V, in the jad cluster of Streptomyces venezuelae ISP5230, were located immediately downstream of jadN by chromosome walking. Sequence analyses and comparisons implicated them in biosynthesis of the 2,6-dideoxysugar in jadomycin B. The genes were cloned in Escherichia coli, inactivated by inserting an apramycin resistance cassette with a promoter driving transcription of downstream genes, and transferred into Streptomyces venezuelae by intergeneric conjugation.

Isolation of 3' -O-acetylchloramphenicol: a possible intermediate in chloramphenicol biosynthesis.

3' -O-acetylchloramphenicol, commonly formed from chloramphenicol by resistant bacteria, has been isolated from the antibiotic-producing organism. Biosynthetic experiments suggest that it is a protected intermediate in chloramphenicol biosynthesis, implicating acetylation as a self-resistance mechanism in the producing organism.

Cloning and functional analysis of a phosphopantetheinyl transferase superfamily gene associated with jadomycin biosynthesis in Streptomyces venezuelae ISP5230.

Sequence analysis of a XhoI/SacI fragment of chromosomal DNA downstream of jadL in the Streptomyces venezuelae ISP5230 gene cluster for jadomycin biosynthesis detected a partial ORF similar in its deduced amino acid sequence to the hetI product involved in synthesizing a regulator of heterocyst spacing in ANABAENA: By probing a phage library of S. venezuelae DNA with the XhoI/SacI fragment, the authors identified and isolated a hybridizing clone. The nucleotide sequence of its DNA contained three complete ORFs (jadM, N and X) and one incomplete ORF (jadO).

Regulation of an anthranilate synthase gene in Streptomyces venezuelae by a trp attenuator.

The nucleotide sequence of a 2-4 kb BamHI-SalI fragment of Streptomyces venezuelae ISP5230 DNA that complements trpE and trpG mutations in Escherichia coli contains two ORFs. The larger of these (ORF2) encodes a 624 amino acid sequence similar to the overall sequence of the two subunits of anthranilate synthase. The two-thirds nearest the amino terminus resembles the aminase subunit; the remaining one-third resembles the glutamine amidotransferase subunit.

A role for pabAB, a p-aminobenzoate synthase gene of Streptomyces venezuelae ISP5230, in chloramphenicol biosynthesis.

Mutagenesis of Streptomyces venezuelae ISP5230 and selection for P-aminobenzoic acid-dependent growth in the presence of sulfanilamide yielded pab mutants (VS519 and VS620) that continued to produce chloramphenicol (Cm), although with increased medium dependence. Transforming the mutants with pDQ102 or pDQ103, which carried a pab-complementing fragment from S. venezuelae ISP5230 in alternative orientations, restored uniformly high Cm production in VS620, but did not alter the medium dependence of Cm production in VS519.

Streptomyces akiyoshiensis differs from other gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids.

A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39,000 transformants tested grew on LB medium lacking diaminopimelate.

Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis.

Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence.

Accumulation of the angucycline antibiotic rabelomycin after disruption of an oxygenase gene in the jadomycin B biosynthetic gene cluster of Streptomyces venezuelae.

DNA from a region downstream of and overlapping the polyketide synthase (PKS) gene cluster for jadomycin B biosynthesis in Streptomyces venezuelae was cloned and sequenced. Analysis of the nucleotide sequence located one complete ORF (ORF6), an incomplete one representing the 3' region of ORF4 in the PKS cluster, and a second incomplete one (ORF7). The deduced amino acid sequences for ORFs 6 and 7 resemble those of oxygenases.