Rifamycin Biosynthesis Research Articles

Deciphering the Timing of Naphthalenic Ring Formation in the Biosynthesis of 8-Deoxyrifamycins.

Although the biosynthesis of rifamycin has been studied for three decades, the biosynthetic formation of the naphthalenic ring remains unclear. In this study, by deletion of all post-PKS modification genes, we identified macrolactam precursors released from rif PKS. Isolated prorifamycins (M3 and M4) have a benzenic chromophore and exist in two sets of macrocyclic atropisomers. The transformation from prorifamycins to benzenoid (5) and naphthalenoid (6) was suggested to be a non-enzymatic process, which is an off-PKS assembly.

Rifamycin W Analogues from Amycolatopsis mediterranei S699 Δrif-orf5 Strain.

Rifamycin W, the most predominant intermediate in the biosynthesis of rifamycin, needs to undergo polyketide backbone rearrangement to produce rifamycin B via an oxidative cleavage of the C-12/C-29 double bond. However, the mechanism of this putative oxidative cleavage has not been characterized yet. Rif-Orf5 (a putative cytochrome P450 monooxygenase) was proposed to be involved in the cleavage of this olefinic moiety of rifamycin W. In this study, the mutant strain Amycolatopsis mediterranei S699 Δrif-orf5 was constructed by in-frame deleting the rif-orf5 gene to afford thirteen rifamycin W congeners (1–13) including seven new ones (1–7). Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Presumably, compounds 1–4 were derivatized from rifamycin W via C-5/C-11 retro-Claisen cleavage, and compounds 1–3, 9 and 10 featured a hemiacetal. Compounds 5–7 and 11 showed oxygenations at various sites of the ansa chain. In addition, compounds 1–3 exhibited antibacterial activity against Staphylococcus aureus with minimal inhibitory concentration (MIC) values of 5, 40 and 0.5 µg/mL, respectively. Compounds 1 and 3 showed modest antiproliferative activity against HeLa and Caco-2 cells with half maximal inhibitory concentration (IC50) values of about 50 µM.

Differential mass spectrometry-based proteome analyses unveil major regulatory hubs in rifamycin B production in Amycolatopsis mediterranei

Rifamycin B is produced by Amycolatopsis mediterranei S699 as a secondary metabolite. Its semi-synthetic derivatives have been used for curing tuberculosis caused by Mycobacterium tuberculosis. But the emergence of rifampicin-resistant strains required analogs of rifamycin B to be developed by rifamycin biosynthetic gene cluster manipulation. In 2014 genetic engineering of the rifamycin polyketide synthase gene cluster in S699 led to a mutant, A. mediterranei DCO#34, that produced 24-desmethylrifamycin B. Unfortunately, the productivity was strongly reduced to 20 mgL−1 as compared to 50 mgL−1 of rifamycin B. To understand the mechanisms leading to reduced productivity and rifamycin biosynthesis by A. mediterranei S699 during the early and late growth phase we performed a proteome study for wild type strain S699, mutant DCO#34, and the non-producer strain SCO2-2. Proteins identification and relative label-free quantification were performed by nLC-MS/MS. Data are available via ProteomeXchange with identifier PXD016416. Also, in-silico protein-protein interaction approach was used to determine the relationship between different structural and regulatory proteins involved in rifamycin biosynthesis. Our studies revealed RifA, RifK, RifL, Rif-Orf19 as the major regulatory hubs. Relative abundance expression values revealed that genes encoding RifC-RifI and the transporter RifP, down-regulated in DCO#34 and genes encoding RifR, RifZ, other regulatory proteins up-regulated. SignificanceThe study is designed mainly to understand the underlying mechanisms of rifamycin biosynthesis in Amycolatopsis mediterranei. This resulted in the identification of regulatory hubs which play a crucial role in regulating secondary metabolism. It elucidates the complex mechanism of secondary metabolite biosynthesis and their conversion and extracellular transportation in temporal correlation with the different growth phases. The study also elucidated the mechanisms leading to reduced production of analog, 24-desmethylrifamycin B by the genetically modified strain DCO#34, derivatives of which have been found effective against rifampicin-resistant strains of Mycobacterium tuberculosis. These results can be useful while carrying out genetic manipulations to improve the strains of Amycolatopsis to produce better analogs/drugs and promote the eradication of TB. Thus, this study is contributing significantly to the growing knowledge in the field of the crucial drug, rifamycin B biosynthesis by an economically important bacterium Amycolatopsis mediterranei.

8-Deoxy-Rifamycin Derivatives from Amycolatopsis mediterranei S699 ΔrifT Strain.

Proansamycin X, a hypothetical earliest macrocyclic precursor in the biosynthesis of rifamycin, had never been isolated and identified. According to bioinformatics analysis, it was proposed that RifT (a putative NADH-dependent dehydrogenase) may be a candidate target responsible for the dehydrogenation of proansamycin X. In this study, the mutant strain Amycolatopsis mediterranei S699 ΔrifT was constructed by deleting the rifT gene. From this strain, eleven 8-deoxy-rifamycin derivatives (1–11) and seven known analogues (12–18) were isolated. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and high-resolution ESI mass spectra. Compound 1 is a novel amide N-glycoside of seco-rifamycin. Compounds 2 and 3 feature conserved 11,12-seco-rifamycin W skeleton. The diverse post-modifications in the polyketide chain led to the production of 4–11. Compounds 2, 3, 5, 6, 13 and 15 exhibited antibacterial activity against Staphylococcus aureus (MIC (minimal inhibitory concentration) values of 10, 20, 20, 20, 40 and 20 μg/mL, respectively). Compounds 14, 15, 16, 17 and 18 showed potent antiproliferative activity against KG1 cells with IC50 (half maximal inhibitory concentration) values of 14.91, 44.78, 2.16, 18.67 and 8.07 μM, respectively.

Prediction of Transcription Factors and Their Involvement in Regulating Rifamycin Production in Amycolatopsis mediterranei S699.

Amycolatopsis mediterranei S699 produces rifamycin B and successors of this strain are in use for the industrial production of rifamycin B. Semisynthetic derivatives of rifamycin B are used against Mycobacterium tuberculosis that causes tuberculosis. Although the rifamycin biosynthetic gene cluster was characterized two decades ago, the regulation of rifamycin B biosynthesis in Amycolatopsis mediterranei S699 is poorly understood. In this study, we analysed the genome and proteome of Amycolatopsis mediterranei S699 and identified 1102 transcription factors which comprise about 10% of the total genome. Using interactomics approaches we delineated 30 unique transcription factors directly involved in secondary metabolism that regulate rifamycin B biosynthesis. We also predict the role of RifN as hub in controlling the regulation of other genes involved in rifamycin biosynthesis. RifN is important for maintaining the integrity of the rifamycin-network. Thus, these transcription factor can be exploited to improve rifamycin B production in Amycolatopsis mediterranei S699.

GlnR Dominates Rifamycin Biosynthesis by Activating the rif Cluster Genes Transcription Both Directly and Indirectly in Amycolatopsis mediterranei.

Because of the remarkable efficacy in treating mycobacterial infections, rifamycin and its derivatives are still first-line antimycobacterial drugs. It has been intensely studied to increase rifamycin yield from Amycolatopsis mediterranei, and nitrate is found to provide a stable and remarkable stimulating effect on the rifamycin production, a phenomenon known as “nitrate-stimulating effect (NSE)”. Although the NSE has been widely used for the industrial production of rifamycin, its detailed molecular mechanism remains ill-defined. And our previous study has established that the global nitrogen regulator GlnR may participate in the NSE, but the underlying mechanism is still enigmatic. Here, we demonstrate that GlnR directly controls rifamycin biosynthesis in A. mediterranei and thus plays an essential role in the NSE. Firstly, GlnR specifically binds to the upstream region of rifZ, which leads us to uncover that rifZ has its own promoter. As RifZ is a pathway-specific activator for the whole rif cluster, GlnR indirectly upregulates the whole rif cluster transcription by directly activating the rifZ expression. Secondly, GlnR specifically binds to the upstream region of rifK, which is also characterized to have its own promoter. It is well-known that RifK is a 3-amino-5-hydroxybenzoic acid (AHBA, the starter unit of rifamycin) synthase, thus GlnR can promote the supply of the rifamycin precursor by directly activating the rifK transcription. Notably, GlnR and RifZ independently activate the rifK transcription through binding to different sites in rifK promoter region, which suggests that the cells have a sophisticated regulatory mechanism to control the AHBA biosynthesis. Collectively, this study reveals that GlnR activates the rif cluster transcription in both direct (for rifZ and rifK) and indirect (for the whole rif cluster) manners, which well interprets the phenomenon that the NSE doesn’t occur in the glnR null mutant. Furthermore, this study deepens our understanding about the molecular mechanism of the NSE.

34a-Hydroxylation in Rifamycin Biosynthesis Catalyzed by Cytochrome P450 Encoded by rif-orf13

34a-Hydroxylation in Rifamycin Biosynthesis Catalyzed by Cytochrome P450 Encoded by <i>rif-orf13</i>

GlnR positive transcriptional regulation of the phosphate-specific transport system pstSCAB in Amycolatopsis mediterranei U32.

Amycolatopsis mediterranei U32 is an important industrial strain for the production of rifamycin SV. Rifampicin, a derivative of rifamycin SV, is commonly used to treat mycobacterial infections. Although phosphate has long been known to affect rifamycin biosynthesis, phosphate transport, metabolism, and regulation are poorly understood in A. mediterranei. In this study, the functional phosphate transport system pstSCAB was isolated by RNA sequencing and inactivated by insertion mutation in A. mediterranei U32. The mycelium morphology changed from a filamentous shape in the wild-type and pstS1+ strains to irregular swollen shape at the end of filamentous in the ΔpstS1 strain. RT-PCR assay revealed that pstSCAB genes are co-transcribed as a polycistronic messenger. The pstSCAB transcription was significantly activated by nitrate supplementation and positively regulated by GlnR which is a global regulator of nitrogen metabolism in actinomycetes. At the same time, the yield of rifamycin SV decreased after mutation (ΔpstS1) compared with wild-type U32, which indicated a strong connection among phosphate metabolism, nitrogen metabolism, and rifamycin production in actinomycetes.

Deciphering the late steps of rifamycin biosynthesis

Rifamycin-derived drugs, including rifampin, rifabutin, rifapentine, and rifaximin, have long been used as first-line therapies for the treatment of tuberculosis and other deadly infections. However, the late steps leading to the biosynthesis of the industrially important rifamycin SV and B remain largely unknown. Here, we characterize a network of reactions underlying the biosynthesis of rifamycin SV, S, L, O, and B. The two-subunit transketolase Rif15 and the cytochrome P450 enzyme Rif16 are found to mediate, respectively, a unique C–O bond formation in rifamycin L and an atypical P450 ester-to-ether transformation from rifamycin L to B. Both reactions showcase interesting chemistries for these two widespread and well-studied enzyme families.

A feedback regulatory model for RifQ-mediated repression of rifamycin export in Amycolatopsis mediterranei

BackgroundDue to the important role of rifamycin in curing tuberculosis infection, the study on rifamycin has never been stopped. Although RifZ, which locates within the rifamycin biosynthetic cluster, has recently been characterized as a pathway-specific regulator for rifamycin biosynthesis, little is known about the regulation of rifamycin export.ResultsIn this work, we proved that the expression of the rifamycin efflux pump (RifP) was regulated by RifQ, a TetR-family transcriptional regulator. Deletion of rifQ had little impact on bacterial growth, but resulted in improved rifamycin production, which was consistent with the reverse transcription PCR results that RifQ negatively regulated rifP’s transcription. With electrophoretic mobility shift assay and DNase I Footprinting assay, RifQ was found to directly bind to the promoter region of rifP, and a typical inverted repeat was identified within the RifQ-protected sequences. The transcription initiation site of rifP was further characterized and found to be upstream of the RifQ binding sites, well explaining the RifQ-mediated repression of rifP’s transcription in vivo. Moreover, rifamycin B (the end product of rifamycin biosynthesis) remarkably decreased the DNA binding affinity of RifQ, which led to derepression of rifamycin export, reducing the intracellular concentration of rifamycin B as well as its toxicity against the host.ConclusionsHere, we proved that the export of rifamycin B was repressed by RifQ in Amycolatopsis mediterranei, and the RifQ-mediated repression could be specifically relieved by rifamycin B, the end product of rifamycin biosynthesis, based on which a feedback model was proposed for regulation of rifamycin export. With the findings here, one could improve the antibiotic yield by simply inactivating the negative regulator of the antibiotic transporter.

Amexanthomycins A-J, pentangular polyphenols produced by Amycolatopsis mediterranei S699∆rifA.

Ten new pentangular polyphenols, namely amexanthomycins A-J (1-10) were isolated from the strain Amycolatopsis mediterranei S699∆rifA constructed by deleting the polyketide synthase genes responsible for the biosynthesis of rifamycins. Their structures were elucidated on the basis of 1D and 2D NMR spectroscopic data and high-resolution ESIMS. Amexanthomycins A-C (1-3) showed inhibitory activity against human DNA topoisomerases.

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei.

Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathway-specific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of high-yield industrial strains.

Evaluation of the Synthetic Potential of an AHBA Knockout Mutant of the Rifamycin Producer Amycolatopsis mediterranei.

Supplementing an AHBA(-) mutant strain of Amycolatopsis mediterranei, the rifamycin producer, with a series of benzoic acid derivatives yielded new tetraketides containing different phenyl groups. These mutasynthetic studies revealed unique reductive properties of A. mediterranei towards nitro- and azidoarenes, leading to the corresponding anilines. In selected cases, the yields of mutaproducts (fermentation products isolated after feeding bacteria with chemically prepared analogs of natural building blocks) obtained are in a range (up to 118 mg L(-1)) that renders them useful as chiral building blocks for further synthetic endeavors. The configuration of the stereogenic centers at C6 and C7 was determined to be 6R,7S for one representative tetraketide. Importantly, processing beyond the tetraketide stage is not always blocked when the formation of the bicyclic naphthalene precursor cannot occur. This was proven by formation of a bromo undecaketide, an observation that has implications regarding the evolutionary development of rifamycin biosynthesis.

Genome sequencing and annotation of Amycolatopsis azurea DSM 43854(T).

We report the 9.2 Mb genome of the azureomycin A and B antibiotic producing strain Amycolatopsis azurea isolated from a Japanese soil sample. The draft genome of strain DSM 43854T consists of 9,223,451 bp with a G + C content of 69.0% and the genome contains 3 rRNA genes (5S–23S–16S) and 58 aminoacyl-tRNA synthetase genes. The homology searches revealed that the PKS gene clusters are supposed to be responsible for the biosynthesis of naptomycin, macbecin, rifamycin, mitomycin, maduropeptin enediyne, neocarzinostatin enediyne, C-1027 enediyne, calicheamicin enediyne, landomycin, simocyclinone, medermycin, granaticin, polyketomycin, teicoplanin, balhimycin, vancomycin, staurosporine, rubradirin and complestatin.

Identification and functional analysis of a nitrate assimilation operon nasACKBDEF from Amycolatopsis mediterranei U32

Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the "nitrate stimulated" rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism

Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10,236,715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9,228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.

A complex role of Amycolatopsis mediterranei GlnR in nitrogen metabolism and related antibiotics production

Amycolatopsis, genus of a rare actinomycete, produces many clinically important antibiotics, such as rifamycin and vancomycin. Although GlnR of Amycolatopsis mediterranei is a direct activator of the glnA gene expression, the production of GlnR does not linearly correlate with the expression of glnA under different nitrogen conditions. Moreover, A. mediterranei GlnR apparently inhibits rifamycin biosynthesis in the absence of nitrate but is indispensable for the nitrate-stimulating effect for its production, which leads to the hyper-production of rifamycin. When glnR of A. mediterranei was introduced into its phylogenetically related organism, Streptomyces coelicolor, we found that GlnR widely participated in the host strain's secondary metabolism, resemblance to the phenotypes of a unique S. coelicolor glnR mutant, FS2. In contrast, absence or increment in copy number of the native S. coelicolor glnR did not result in a detectable pleiotrophic effect. We thus suggest that GlnR is a global regulator with a dual functional impact upon nitrogen metabolism and related antibiotics production.

Identification of tailoring genes involved in the modification of the polyketide backbone of rifamycin B by Amycolatopsis mediterranei S699

Rifamycin B biosynthesis by Amycolatopsis mediterranei S699 involves a number of unusual modification reactions in the formation of the unique polyketide backbone and decoration of the molecule. A number of genes believed to be involved in the tailoring of rifamycin B were investigated and the results confirmed that the formation of the naphthalene ring moiety of rifamycin takes place during the polyketide chain extension and is catalysed by Rif-Orf19, a 3-(3-hydroxyphenyl)propionate hydroxylase-like protein. The cytochrome P450-dependent monooxygenase encoded by rif-orf5 is required for the conversion of the Delta12, 29 olefinic bond in the polyketide backbone of rifamycin W into the ketal moiety of rifamycin B. Furthermore, Rif-Orf3 may be involved in the regulation of rifamycin B production, as its knock-out mutant produced about 40 % more rifamycin B than the wild-type. The work also revealed that many of the genes located in the cluster are not involved in rifamycin biosynthesis, but might be evolutionary remnants carried over from an ancestral lineage.

Engineered biosynthesis of an ansamycin polyketide precursor in Escherichia coli.

Ansamycins such as rifamycin, ansamitocin, and geldanamycin are an important class of polyketide natural products. Their biosynthetic pathways are especially complex because they involve the formation of 3-amino-5-hydroxybenzoic acid (AHBA) followed by backbone assembly by a hybrid nonribosomal peptide synthetase/polyketide synthase. We have reconstituted the ability to synthesize 2,6-dimethyl-3,5,7-trihydroxy-7-(3'-amino-5'-hydroxyphenyl)-2,4-heptadienoic acid (P8/1-OG), an intermediate in rifamycin biosynthesis, in an extensively manipulated strain of Escherichia coli. The parent strain, BAP1, contains the sfp phosphopantetheinyl transferase gene from Bacillus subtilis, which posttranslationally modifies polyketide synthase and nonribosomal peptide synthetase modules. AHBA biosynthesis in this host required introduction of seven genes from Amycolatopsis mediterranei, which produces rifamycin, and Actinosynnema pretiosum, which produces ansamitocin. Because the four-module RifA protein (530 kDa) from the rifamycin synthetase could not be efficiently produced in an intact form in E. coli, it was genetically split into two bimodular proteins separated by matched linker pairs to facilitate efficient inter-polypeptide transfer of a biosynthetic intermediate. A derivative of BAP1 was engineered that harbors the AHBA biosynthetic operon, the bicistronic RifA construct and the pccB and accA1 genes from Streptomyces coelicolor, which enable methylmalonyl-CoA biosynthesis. Fermentation of this strain of E. coli yielded P8/1-OG, an N-acetyl P8/1-OG analog, and AHBA. In addition to providing a fundamentally new route to shikimate and ansamycin-type compounds, this result enables further genetic manipulation of AHBA-derived polyketide natural products with unprecedented power.

Stability of plasmid pA387 derivatives in Amycolatopsis mediterranei producing rifamycin.

Genetic studies on the biosynthesis of rifamycins in producer strains such as Amylcolaptopsis mediterranei U-32 are severely hampered by the availability of efficient transformation procedures and stable plasmid vectors. Using an efficient electroporation procedure we have studied the replication and stability of a pA387 derivative, pDXM32. This plasmid confers enhanced plasmid stability and copy number compared to pA387 derivatives commonly used as cloning vectors in A. mediterranei. Deletion derivatives in the region previously identified as being a minimal replication origin were also examined with respect to their ability to transform A. mediterranei and at least one locus was essential for replication. A 5.4 kbp DNA fragment was sequenced and annotated encoding the replication and plasmid stability functions. A parA homologue was identified which is likely to confer plasmid stability.