Radiolabelling of the monate moiety in the study of pseudomonic acid biosynthesis

Abstract Monic acid A was isolated from a Pseudomonas fluorescens fermentation in which pseudomonic acid A (PA) was the principal secondary metabolite. [3-¹⁴C]3-Hydroxy-3-methyl-glutaric acid (HMG) given early in the idiophase radiolabelled PA (1.1% incorporation), confirming experimentally the putative direct involvement of HMG in the biosynthesis of PA, but contributed relatively insignificant radiolabel to the monic acid extracted from the broth at the end of the fermentation. Ethionine inhibited (80%) PA biosynthesis and correspondingly reduced incorporation of [¹⁴C]HMG. In contrast, ethionine increased incorporation of [methyl-¹⁴C]methionine into PA and enhanced specific radioactivity of the antibiotic 8-fold. Ethionine inhibition of secondary metabolite methylations did not divert pseudomonate biosynthesis to give unusual analogues, implying that methylation of a putative pentaketide precursor of the monate moiety forms a vital intermediate of the pseudomonate pathway, but caused a new [¹⁴C]HMG-derived polar metabolite of biosynthetic interest to become evident.     

Inhibition of isoleucyl-transfer ribonucleic acid synthetase in Echerichia coli by pseudomonic acid

The mode of action of the antibiotic pseudomonic acid has been studied in Escherichia coli. Pseudomonic acid strongly inhibits protein and RNA synthesis in vivo. The antibiotic had no effect on highly purified DNA-dependent RNA polymerase and showed only a weak inhibitory effect on a poly(U)-directed polyphenylalanine-forming ribosomal preparation. Chloramphenicol reversed inhibition of RNA synthesis in vivo. Pseudomonic acid had little effect on RNA synthesis in a regulatory mutant, E. coli B AS19 RC(rel), whereas protein synthesis was strongly inhibited. In pseudomonic acid-treated cells, increased concentrations of ppGpp, pppGpp and ATP were observed, but the GTP pool size decreased, suggesting that inhibition of RNA synthesis is a consequence of the stringent control mechanism imposed by pseudomonic acid-induced deprivation of an amino acid. Of the 20 common amino acids, only isoleucine reversed the inhibitory effect in vivo. The antibiotic was found to be a powerful inhibitor of isoleucyl-tRNA synthetase both in vivo and in vitro. Of seven other tRNA synthetases assayed, only a weak inhibitory effect on phenylalanyl-tRNA synthetase was observed; this presumably accounted for the weak effect on polyphenylalanine formation in a ribosomal preparation. Pseudomonic acid also significantly de-repressed threonine deaminase and transaminase B activity, but not dihydroxyacid dehydratase (isoleucine-biosynthetic enzymes) by decreasing the supply of aminoacylated tRNA(Ile). Pseudomonic acid is the second naturally occurring inhibitor of bacterial isoleucyl-tRNA synthetase to be discovered, furanomycin being the first.     

Cloning and characterization of a polyketide synthase gene from Streptomyces fradiae Tü2717, which carries the genes for biosynthesis of the angucycline antibiotic urdamycin A and a gene probably involved in its oxygenation.

A DNA fragment was cloned as cosmid purd8, which encodes a polyketide synthase involved in the production of the angucycline antibiotic urdamycin from Streptomyces fradiae Tü2717. Deletion of the polyketide synthase genes from the chromosome abolished urdamycin production. In addition, purd8 conferred urdamycin resistance on introduction into Streptomyces lividans TK24. Sequence analysis of 5.7 kb of purd8 revealed six open reading frames transcribed in the same direction. The deduced amino acid sequences of the six open reading frames strongly resemble proteins from known type II polyketide synthase gene clusters: a ketoacyl synthase, a chain length factor, an acyl carrier protein, a ketoreductase, a cyclase, and an oxygenase. Heterologous expression of the urdamycin genes encoding a ketoacyl synthase and a chain length factor in Streptomyces glaucescens tetracenomycin C-nonproducing mutants impaired in either the TcmK ketoacyl synthase or TcmL chain length factor resulted in the production of tetracenomycin C. Heterologous expression of a putative oxygenase gene from the urdamycin gene cluster in S. glaucescens GLA.O caused production of the hybrid antibiotic 6-hydroxy tetracenomycin C.     

Isoleucyl-tRNA synthetase of Methanobacterium thermoautotrophicum Marburg. Cloning of the gene, nucleotide sequence, and localization of a base change conferring resistance to pseudomonic acid

The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced. ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae. The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast. The archaebacterial enzyme fitted well into this group of enzymes. It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family. Comparison between the isoleucyl-tRNA synthetases of M. thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli. The ileS gene of the pseudomonic acid-resistant M. thermoautotrophicum mutant MBT10 was also sequenced. The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence. The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme. Both the mutant and the wild-type ileS gene were expressed in E. coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid.     

The role of monic acid A in pseudomonic acid A biosynthesis inPseudomonas fluorescens

Summary The putative role of monic acid A as a biosynthetic intermediate of the antibiotic pseudomonic acid A, providing a C₁₇ moiety requiring only esterification with a C₉ fatty acid, has been tested by administration of [¹⁴C]monic acid A early in the pseudomonic acid A idiophase of aPseudomonas fluorescens fermentation. [¹⁴C]Monic acid A was not taken up by the cells and the pseudomonic acid A subsequently accumulated was not radiolabelled. Experimental demonstration of the biosynthetic role of monic acid A and the potential use of monate analogues in biotransformations will require unexpectedly elaborate strategies to ensure the uptake of these compounds into bacterial protoplasts. The impermeability to monic acid A explains for the first time why it is not an antibiotic.     

A role for 3-hydroxy-3-methyl-glutaric acid in the biosynthesis of pseudomonic acid A

Abstract [3-¹⁴C]3-Hydroxy-3-methyl-glutaric acid (HMG) was incorporated (0.9%) into pseudomonic acid A when administered to a shaken flask fermentation of Pseudomonas fluorescens 4, 9 and 14 h after inoculation. [2-¹⁴C]-Mevalonic acid was not incorporated into pseudomonic acid. Experimental evidence supports the previous deduction that pseudomonic acid biosynthesis might directly involve HMG, an apparently unusual intermediary metabolite in prokaryotes.     

Generation of dominant selectable markers for resistance to pseudomonic acid by cloning and mutagenesis of the ileS gene from the archaeon Methanosarcina barkeri fusaro

Currently, only one selectable marker is available for genetic studies in the archaeal genus Methanosarcina. Here we report the generation of selectable markers that encode resistance to pseudomonic acid (PA(r)) in Methanosarcina species by mutagenesis of the isoleucyl-tRNA synthetase gene (ileS) from Methanosarcina barkeri Fusaro. The M. barkeri ileS gene was obtained by screening of a genomic library for hybridization to a PCR fragment. The complete 3,787-bp DNA sequence surrounding and including the ileS gene was determined. As expected, M. barkeri IleS is phylogenetically related to other archaeal IleS proteins. The ileS gene was cloned into a Methanosarcina-Escherichia coli shuttle vector and mutagenized with hydroxylamine. Nine independent PA(r) clones were isolated after transformation of Methanosarcina acetivorans C2A with the mutagenized plasmids. Seven of these clones carry multiple changes from the wild-type sequence. Most mutations that confer PA(r) were shown to alter amino acid residues near the KMSKS consensus sequence of class I aminoacyl-tRNA synthetases. One particular mutation (G594E) was present in all but one of the PA(r) clones. The MIC of pseudomonic acid for M. acetivorans transformed with a plasmid carrying this single mutation is 70 microgram/ml of medium (for the wild type, the MIC is 12 microgram/ml). The highest MICs (560 microgram/ml) were observed with two triple mutants, A440V/A482T/G594E and A440V/G593D/G594E. Plasmid shuttle vectors and insertion cassettes that encode PA(r) based on the mutant ileS alleles are described. Finally, the implications of the specific mutations we isolated with respect to binding of pseudomonic acid by IleS are discussed.     

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.     

Isolation of clustered genes that are notably homologous to the eicosapentaenoic acid biosynthesis gene cluster from the docosahexaenoic acid-producing bacterium Vibrio marinus strain MP-1

A 40-kbp DNA fragment was isolated from the cosmid library of Vibrio marinus strain MP-1. Among the 22 putative open reading frames (ORFs) in this fragment, ORFs 8, 9, 10 and 11 had high homology with ORFs 5, 6, 7 and 8 of the eicosapentaenoic acid biosynthesis gene cluster, respectively. Then, we speculate that these ORFs are responsible for docosahexaenoic acid biosynthesis in this bacterium.     

Isolation of two Pseudomonas strains producing pseudomonic acid A

Two novel Pseudomonas strains were isolated from groundwater sediment samples. The strains showed resistance against the antibiotics tetracycline, cephalothin, nisin, vancomycin, nalidixic acid, erythromycin, lincomycin, and penicillin and grew at temperatures between 15 and 37 °C and pH values from 4 to 10 with a maximum at pH 7 to 10. The 16S ribosomal RNA gene sequences and the substrate spectrum of the isolates revealed that the two strains belonged to the Pseudomonas fluorescens group. The supernatants of both strains had an antibiotic effect against Gram-positive bacteria and one Gram-negative strain. The effective substance was produced under standard cultivation conditions without special inducer molecules or special medium composition. The antibiotically active compound was identified as pseudomonic acid A by off-line high performance liquid chromatography (HPLC) and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). The measurement on ultra performance liquid chromatography (UPLC, UV–vis detection) confirmed the determination of pseudomonic acid A which was produced by both strains at 1.7–3.5 mg/l. Our findings indicate that the ability to produce the antibiotic pseudomonic acid A (Mupirocin) is more spread among the pseudomonads then anticipated from the only producer known so far.     

The stachydrine catabolism region in Sinorhizobium meliloti encodes a multi-enzyme complex similar to the xenobiotic degrading systems in other bacteria

Stachydrine (proline betaine) can be used by Sinorhizobium meliloti as a source of carbon and nitrogen. Catabolism depends on an initial N-demethylation, after which the resultant N-methyl proline enters general metabolism. Deletion and insertion mutagenesis demonstrated that the information necessary for catabolism is carried on the symbiotic plasmid (pSym) distal to nodD2 and the nod-nif cluster. Sequencing of an 8.5kb fragment spanning this region revealed four open reading frames with functional homology to known proteins, including a putative monooxygenase and a putative NADPH-FMN-reductase, which were shown by insertional and frame-shift mutagenesis to be necessary for stachydrine catabolism. Other open reading frames, encoding a putative flavoprotein and a repressor, were judged not to be required for stachydrine catabolism, since they were not included in a fragment capable of complementing a deletion of the entire stc region. Sequence and mutagenesis data suggest that stachydrine is demethylated by an iron-sulfur monooxygenase of the Rieske type with a requirement for a specific reductase. The stc catabolic cluster, therefore, resembles xenobiotic degradation in other bacteria and recalls rhizopine catabolism in S. meliloti. Stachydrine appears to have multiple roles in osmoprotection, nutrition and nodulation. Genes involved in stachydrine catabolism are also necessary for carnitine degradation; thus, they could be important in the catabolism of a variety of root exudates and mediate other relationships.     

Cloning, sequencing, and characterization of the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2

Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[alpha]naphthacenequinone aglycone substituted with D-alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB (ketosynthases alpha and beta). The pradimicin gene cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA.     

Synthesis and antibacterial properties of beta-diketone acrylate bioisosteres of pseudomonic acid A.

A series of beta-diketone acrylate bioisosteres 4 of pseudomonic acid A 1 have been synthesized and evaluated for their ability to inhibit bacterial isoleucyl-tRNA synthetase and act as antibacterial agents. A number of analogues have excellent antibacterial activity. Selected examples were shown to afford good blood levels and to be effective in a murine infection model.     

Nucleotide sequences and expression of genes from Streptomyces purpurascens that cause the production of new anthracyclines in Streptomyces galilaeus.

Six open reading frames, rdmA to rdmF, in a 6,077-bp segment of Streptomyces purpurascens DNA which caused the production of hybrid anthracyclines were identified. The minimal fragment that produced anthracyclines modified at the 10th position contained rdmB to rdmD; rdmE is the gene for aklavinone-11-hydroxylase. RdmC is similar to a putative open reading frame in the daunorubicin biosynthetic cluster of Streptomyces peucetius and is likely to participate in the removal of the side chain at the 10th position.     

Cloning and sequencing of a putative Escherichia coli [NiFe] hydrogenase-1 operon containing six open reading frames.

DNA encompassing the structural genes of an Escherichia coli [NiFe] hydrogenase has been cloned and sequenced. The genes were identified as those encoding the large and small subunits of hydrogenase isozyme 1 based on NH2-terminal sequences of purified subunits (kindly provided by K. Francis and K. T. Shanmugam). The structural genes formed part of a putative operon that contained four additional open reading frames. We have designated the operon hya and the six open reading frames hyaA through F. hyaA and hyaB encode the small and large structural subunits, respectively. The nucleotide-derived amino acid sequence of hyaC has a calculated molecular mass of 27.6 kilodaltons, contains 20% aromatic residues, and has four potential membrane-spanning regions. Open reading frames hyaD through F could encode polypeptides of 21.5, 14.9, and 31.5 kilodaltons, respectively. These putative peptides have no homology to other reported protein sequences, and their functions are unknown.     

Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus.

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6-dimethyl-3'-amino-3'-deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Deltapur4, Deltapur5 and Deltapur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d-rhamnose and alpha-p-coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.     

Inactivation of Kex2p diminishes the virulence of Candida albicans

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.     

A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic

BackgroundUnderstanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin.Conclusions/SignificancePlasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.     

Enteropathogenic Escherichia coli: identification of a gene cluster coding for bundle-forming pilus morphogenesis.

Sequence flanking the bfpA locus on the enteroadherent factor plasmid of the enteropathogenic Escherichia coli (EPEC) strain B171-8 (O111:NM) was obtained to identify genes that might be required for bundle-forming pilus (BFP) biosynthesis. Deletion experiments led to the identification of a contiguous cluster of at least 12 open reading frames, including bfpA, that could direct the synthesis of a morphologically normal BFP filament. Within the bfp gene cluster, we identified open reading frames that share homology with other type IV pilus accessory genes and with genes required for transformation competence and protein secretion. Immediately upstream of the bfp gene cluster, we identified a potential replication origin including genes that are predicted to encode proteins homologous with replicase and resolvase. Restriction fragment length polymorphism analysis of DNA from six additional EPEC serotypes showed that the organization of the bfp gene cluster and its juxtaposition with a potential plasmid origin of replication are highly conserved features of the EPEC biotype.