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. The hanging-drop vapour-diffusion method was used to produce crystals that were suitable for X-ray diffraction. Data were collected to 2.0 A resolution. The crystal belonged to space group C2, with unit-cell parameters a = 208.1, b = 57.7, c = 59.9 A, beta = 97.5 degrees .

Streptomyces venezuelae 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.A.1) through sequence homology. The protein is encoded by jadL, situated within the jadomycin biosynthetic gene cluster. JadL has, therefore, been assigned a putative role in host defense by exporting its probable substrates, the jadomycins, a family of secondary metabolites produced by Streptomyces venezuelae ISP5230. Herein, we evaluate this assumption through the construction and analysis of a jadL disrupted mutant, S. venezuelae VS678 (ΔjadL::aac(3)IV). Quantitative determination of jadomycin production with the jadL disrupted mutant did not show a significant decrease in production in comparison to the wildtype strain, as determined by HPLC and by tandem mass spectrometry. These results suggest that efflux of jadomycin occurs upon disruption of jadL, or that JadL is not involved in jadomycin efflux. Potentially, other transporters within S. venezuelae ISP5230 may adopt this role upon inactivation of JadL to export jadomycins.

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. Here we report the X-ray crystal structure of CmlS determined at 2.2 A resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, the C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4alpha)-OOH intermediate. Strikingly, the 8alpha carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.

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. The altered media will facilitate the isolation of related jadomycin B congeners.

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. These angucycline intermediates were also converted to jadomycin A by transformant of the heterologous host Streptomyces lividans expressing the jadFGH oxygenases in vivo and by its cell-free extracts in vitro; thus the three gene products JadFGH are implicated in catalysis of the post-polyketide synthase biosynthetic reactions converting UWM6 to jadomycin aglycone. Genetic and biochemical analyses indicate that JadH possesses dehydrase activity, not previously associated with polyketide-modifying oxygenase. Since the formation of aromatic polyketides often requires multiple dehydration steps, bifunctionality of oxygenases modifying aromatic polyketides may be a general phenomenon.

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. Insertional inactivation of jadW(1) reduced the growth rate of S. venezuelae cultures in aerated liquid media containing complex nitrogen sources and altered growth morphology in minimal medium. It also affected sporulation on agar media. Cultures of jadW(1)-disrupted mutants grown under conditions supporting biosynthesis of JdB or chloramphenicol by the wild-type strain failed to produce either of the antibiotics. Complementing the disrupted strain by transformation with pJV435, containing a cloned copy of the gene, improved sporulation and restored antibiotic biosynthesis in transformants to titres close to those of the wild-type similarly transformed with pJV435 as a control. The results are consistent with a role for jadW(1) in regulating morphological and metabolic differentiation. Further sequence analysis of jadR(2), which functions with jadR(1) in stress-induced activation of JdB biosynthesis, indicated that this gene encodes a gamma-butyrolactone receptor homologue. The growth-rate-sensitive phenotype of the jadW(1)-disrupted mutant, and the proximity of jadW(1) to jadR(2) indicate that this region of the jad gene cluster contains a regulatory mechanism incorporating gamma-butyrolactone signalling and sensitivity to environmental stress.

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. When used to probe Southern blots of restriction-enzyme-digested DNA, the [alpha-(32)P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes.

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. Analysis by HPLC and NMR of intermediates accumulated by cultures of the insertionally inactivated Streptomyces venezuelae mutants indicated that jadO, P, Q, S, T, U and V mediate formation of the dideoxysugar moiety of jadomycin B and its attachment to the aglycone. Based on these results and sequence similarities to genes described in other species producing deoxysugar derivatives, a biosynthetic pathway is proposed in which the jadQ product (glucose-1-phosphate nucleotidyltransferase) activates glucose to its nucleotide diphosphate (NDP) derivative, and the jadT product (a 4,6-dehydratase) converts this to NDP-4-keto-6-deoxy-D-glucose. An NDP-hexose 2,3-dehydratase and an oxidoreductase, encoded by jadO and jadP, respectively, catalyse ensuing reactions that produce an NDP-2,6-dideoxy-D-threo-4-hexulose. The product of jadU (NDP-4-keto-2,6-dideoxy-5-epimerase) converts this intermediate to its L-erythro form and the jadV product (NDP-4-keto-2,6-dideoxyhexose 4-ketoreductase) reduces the keto group of the NDP-4-hexulose to give an activated form of the L-digitoxose moiety in jadomycin B. Finally, a glycosyltransferase encoded by jadS transfers the activated sugar to jadomycin aglycone. The function of jadX is unclear; the gene is not essential for jadomycin B biosynthesis, but its presence ensures complete conversion of the aglycone to the glycoside. The deduced amino acid sequence of a 612 bp ORF (jadR*) downstream of the dideoxysugar biosynthesis genes resembles many TetR-family transcriptional regulator sequences.

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. venezuelae gave mutant strains VS1111 and VS1112, each producing a similar series of Cm analogues in which unhalogenated acyl groups replaced the dichloroacetyl substituent of Cm. 1H-NMR established that the principal metabolite in the disrupted strains was the alpha-N-propionyl analogue. The sequence of CmlK implicated the protein in adenylation, and involvement in halogenation was inferred from biosynthesis of analogues by the cmlK-disrupted mutant. A role in generating the dichloroacetyl substituent was supported by partial restoration of Cm biosynthesis when a cloned copy of cmlK was introduced in trans into VS1111. Complementation of the mutant also indicated that inactivation of cmlK rather than a polar effect of the disruption on cmlS expression had interfered with dichloroacetyl biosynthesis. The deduced CmlS sequence resembled sequences of FADH2-dependent halogenases. Conjugal transfer of cmlK or cmlS into S. venezuelae cml-2, a chlorination-deficient strain with a mutation mapped genetically to the Cm biosynthesis gene cluster, did not complement the cml-2 lesion, suggesting that one or more genes in addition to cmlK and cmlS is needed to assemble the dichloroacetyl substituent. Insertional inactivation of ORF13 did not affect Cm production, and the products of ORF14 and ORF15 matched Streptomyces coelicolor A3(2) proteins lacking plausible functions in Cm biosynthesis. Thus cmlS appears to mark the downstream end of the gene cluster.

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. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd(-)-complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (Mr 35484), was identified as the gene for Asd by comparing its sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the S. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the S. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in S. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.

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. Upstream of ORF2 is a small ORF encoding 18 amino acids that include three adjacent Trp residues; in addition the ORF contains inverted repeats with sequence and positional similarity to the products of attenuator (trpL) regions that regulate tryptophan biosynthesis in other bacteria. In cultures of a trpC mutant of S. venezuelae, increasing the concentration of exogenous tryptophan decreased the formation of anthranilate synthase; similar evidence of endproduct repression was obtained in a trpCER mutant of E. coli transformed with a vector containing the cloned DNA fragment from S. venezuelae. The anthranilate synthase activity in S. venezuelae cell extracts was inhibited by tryptophan, although only at high concentrations of the amino acid. A two-base deletion introduced into the cloned S. venezuelae DNA fragment prevented complementation of a trpE mutation in E. coli. However, S. venezuelae transformants in which the two-base deletion had been introduced by replacement of homologous chromosomal DNA did not exhibit a Trp- phenotype. The result implies that S. venezuelae has one or more additional genes for anthranilate synthase. In alignments with anthranilate synthase genes from other organisms, ORF2 from S. venezuelae most closely resembled genes for phenazine biosynthesis in Pseudomonas. The results bear on the function of the gene in S. venezuelae.

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. The cloned S. venezuelae DNA fragment was subcloned and trimmed to the minimum size conferring pab complementation. The resulting 2.8 kb BamHl-Sacl fragment was sequenced. Codon preference analysis showed one complete ORF encoding a polypeptide of 670 amino acids. Comparison of the deduced amino acid sequence with database proteins indicated that the N- and C-terminal regions resembled PabA and PabB, respectively, of numerous bacteria. The gene product showed overall sequence similarity to the product of a fused pabAB gene associated with secondary metabolism in Streptomyces griseus. Insertion of an apramycin resistance gene into pabAB cloned in a segregationally unstable vector and replacement of the S. venezuelae chromosomal pabAB with the disrupted copy lowered sulfanilamide resistance from 25 to 5 micrograms mL-1 and blocked Cm production.

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). The jadM ORF lay immediately downstream of, and partially overlapped, jadL. It contained 786 nucleotides encoding an amino acid sequence like those of enzymes in the phosphopantetheinyl transferase family. The jadN ORF contained 1794 nucleotides and encoded an amino acid sequence resembling acyl-CoA decarboxylases, thus suggesting a role in polyketide condensation reactions. The jadX ORF was not identified, but the partial jadO showed marked similarities in its deduced amino acid sequence to NDP-hexose-2,3-dehydratases, indicating a role in forming the sugar component of jadomycin B. Expression of jadM in Escherichia coli and examination of the product by SDS-PAGE established that the ORF encoded a 29.1 kDa protein, corresponding in size to the 262 amino acid polypeptide deduced from the jadM sequence. Evidence from a Northern hybridization indicated that jadM expression is correlated with jadomycin B synthesis. Cultures of S. venezuelae ISP5230 disrupted in jadM produced only 2-5% of the wild-type titre of jadomycin B, but grew well and produced chloramphenicol normally. The authors conclude that jadM encodes a holo-ACP synthase needed primarily for jadomycin B biosynthesis.

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. Shunt pathways are proposed to account for the formation of the metabolites from p-aminophenylalanine.

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. The deduced polypeptide encoded 483 amino acids with a calculated M(r) of 54,200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.

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. Since a plausible biosynthetic pathway for jadomycin B includes an angular polyketide intermediate that undergoes oxidative ring fission before condensation with an amino acid, we subcloned one of the presumptive oxygenase genes (ORF6) in a segregationally unstable shuttle vector (pHJL400) and disrupted it by inserting the gene for apramycin resistance. Transformation of S. venezuelae with the disruption vector and selection for apramycin resistance gave mutants blocked in jadomycin biosynthesis. Southern hybridization confirmed that gene replacement had occurred. Cultures of the mutants accumulated a metabolite identified by comparison with an authentic sample as rabelomycin, a non-nitrogenous polyketide-derived antibiotic originally isolated from Streptomyces olivaceus.

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.