Amplifiable Unit Of DNA Research Articles

Amplification of the entire kanamycin biosynthetic gene cluster during empirical strain improvement of Streptomyces kanamyceticus

Streptomyces kanamyceticus 12-6 is a derivative of the wild-type strain developed for industrial kanamycin (Km) production. Southern analysis and DNA sequencing revealed amplification of a large genomic segment including the entire Km biosynthetic gene cluster in the chromosome of strain 12-6. At 145 kb, the amplifiable unit of DNA (AUD) is the largest AUD reported in Streptomyces. Striking repetitive DNA sequences belonging to the clustered regularly interspaced short palindromic repeats family were found in the AUD and may play a role in its amplification. Strain 12-6 contains a mixture of different chromosomes with varying numbers of AUDs, sometimes exceeding 36 copies and producing an amplified region >5.7 Mb. The level of Km production depended on the copy number of the Km biosynthetic gene cluster, suggesting that DNA amplification occurred during strain improvement as a consequence of selection for increased Km resistance. Amplification of DNA segments including entire antibiotic biosynthetic gene clusters might be a common mechanism leading to increased antibiotic production in industrial strains.

Spontaneous chromosome circularization and amplification of a new amplifiable unit of DNA belonging to the terminal inverted repeats in Streptomyces ambofaciens ATCC 23877.

In Streptomyces, the linear chromosomal DNA is highly unstable and undergoes large rearrangements usually at the extremities. These rearrangements consist of the deletion of several hundred kilobases, often associated with the amplification of an adjacent sequence, AUD ( amplifiable unit of DNA). In Streptomyces ambofaciens, two amplifiable regions (AUD6 and AUD90), located approximately 600 kb and 1,200 kb from the right chromosomal end respectively, have been characterized. Here, the isolation and molecular characterization of a new S. ambofaciens mutant strain exhibiting a green-pigmented phenotype is described; the wild-type produces a gray pigment. In this mutant, both chromosome ends were deleted, which probably led to circularization of the chromosome. These deletions were associated with amplification of a sequence belonging to the chromosomal terminal inverted repeats (TIRs), which might constitute the new fragment generated by the chromosomal circularization.

Genetic instability associated with insertion of IS6100 into one end of the Streptomyces lividans chromosome.

Analysis of 548 recombinant strains of Streptomyces lividans carrying chromosomal insertions of IS6100 revealed that six mutants contained DNA amplifications. The amplifications differed in size but included IS6100 sequences. Hybridization with representative cosmid clones containing sequences from the unstable regions of the chromosome indicated that, in each mutant, DNA rearrangements affected just one of the chromosome ends. The amplifications were derived either from a region immediately proximal to the terminal inverted repeat (TIR) or further distal, from a previously characterized type I amplifiable unit of DNA. There was no evidence for extensive deletions accompanying the amplifications and chromosome linearity was maintained with, at least in five mutants, clear evidence for no loss of either TIR. The nature of the rearrangements provides evidence that insertions affecting the integrity of a chromosome end can contribute to genetic instability in Streptomyces.

Stimulation of genetic instability and associated large genomic rearrangements in Streptomyces ambofaciens by three fluoroquinolones

In Streptomyces ambofaciens NSA2002, pigmented wild-type colonies spontaneously give rise to pigment-negative (Pig-) mutants at a frequency of about 0.5%. This genetic instability is related to large deletions which can be associated with amplifications of DNA sequences. The influence of three fluoroquinolones (ciprofloxacin, enoxacin, and norfloxacin) on this property was investigated. At a survival rate higher than 60%, most colonies showed a patchwork phenotype consisting of phenotypically heterogeneous colonies harboring numerous mutant sectors. Moreover, the frequency of Pig- mutants rose to more than 90% at survival rates equal to or higher than 10%. Induced Pig- mutants showed the same phenotypical features as did spontaneous mutants. Most of them also harbored deletions, associated in some cases with DNA amplifications, in two loci of the large unstable region, AUD6 and AUD90 (derived from amplifiable unit of DNA). The size of deletions in induced mutants could rise to 1.5 Mb. These results show that ciprofloxacin, enoxacin, and norfloxacin greatly stimulate genetic instability and the occurrence of DNA rearrangements in S. ambofaciens. Moreover, these three fluoroquinolones had the same rank order for both toxic (i.e., antibacterial) and genotoxic activities. If the antibacterial effect of fluoroquinolones in S. ambofaciens is due to their interference with DNA gyrase, as shown for some other organisms, the genotoxic effect observed could be due to their interaction with this type II topoisomerase. This suggests that DNA gyrase is involved in the process of genetic instability in S. ambofaciens.

Primary structure analysis of a duplicated region in the amplifiable AUD6 locus of Streptomyces ambofaciens DSM40697.

In Streptomyces ambofaciens, an amplifiable unit of DNA (AUD6) contains two homologous sequences, one located on the right extremity of the AUD (S1R), the other being internal (IHS). This paper presents the molecular analysis of this duplication. The nucleotide sequences are almost identical (95%) and each contains an ORF of about 330 codons, the two ORFs being nearly identical. The two hypothetical proteins, deduced from these sequences, show about 30% identity with different bacterial repressors. They also show a particularly strong similarity (90% identity between the full-length sequences) with hypothetical proteins of Streptomyces lividans 66 encoded by sequences also present on an amplifiable DNA region (AUD1).

Analysis of a genetically unstable region inStreptomyces lividans 66-TK64

Genetic and molecular analyses of an unstable region encompassing the gene loci cml arg and a 5.7 kb amplifiable unit of DNA were done. Spontaneous mutants from Cm1R →CmlS and the revertants from CmlS →CmlR were analysed for mutations at arg locus and amplification of amplifiable unit of DNA. Twenty-one revertants were analysed. Two of these had large-scale amplification and one of these was also Arg-. Nine of the revertants which were Arg+ had low-level or intermediate-level amplification of the 5.7 kb DNA sequence but no deletions of the flanking sequences were detected. Five of the CmIR’ revertants, which were also Arg+, had lost one of the two copies from the doublet of amplifiable unit of DNA. The remaining five revertants did not show any other change. The amplifiable unit of DNA, therefore, not only undergoes amplification but can also suffer specific deletion of one copy. Thus, this region as a whole is characterized by instability and the events appear to take place at more than one locus concomitantly with a high frequency.

A chromosomal hotspot for multiple rearrangements associated with genetic instability of Streptomyces ambofaciens DSM 40697

Genetic instability in Strepfomyces ambofaciens DSM 40697 involves genomic rearrangements such as amplifications and deletions of particular DNA sequences. Most amplifications were located in two amplifiable regions, one of which, called AUD6 (amplifiable unit of DNA no. 6) was revealed to be a rearrangement hotspot. Indeed, 30% of the mutant strains studied had amplifications, deletions or both at the AUD6 locus. This locus contains several reiterations which are specific to this AUD. Moreover, one of the endpoints of the AUD6 shows homology with an internal sequence. Deletions occurred exclusively at one side of the amplified DNA sequence (ADS) and removed part of the proximal copy of this ADS, leading to the conclusion that multiple rearrangements can occur at this AUD locus.

Amplification on the Amycolatopsis (Nocardia) mediterranei plasmid pMEA100: Sequence similarities to actinomycete att sites

An amplification of a 2.0-kb fragment was found on the plasmid pMEA100 isolated from a subculture of the wild-type strain LBG A3136 of Amycolatopsis (Nocardia) mediterranei. Plasmid preparations contained a mixture of molecules with copy numbers of the amplified unit in the range of 2 to 10. The amplification on pMEA100 was stable; propagation of cells for many generations did not change the pattern of the amplified DNA. Fragments of the plasmids containing the amplifiable unit of DNA (AUD) and the amplified DNA sequence (ADS) were subcloned and characterized. Sequencing of the AUD terminal regions and the junction between ADS units showed that the amplifiable unit of DNA was flanked by 12-bp direct repeats. The DNA segments adjacent to the 12-bp sequence common to the left and right AUD terminal regions also showed significant similarities. In addition, the left AUD terminal region flanking the 12-bp repeat exhibited considerable sequence similarity to actinomycete plasmid attachment sites, particularly to the pMEA100 att site.

DNA amplification in Streptomyces achromogenes subsp. rubradiris is accompanied by a deletion, and the amplified sequences are conditionally stable and can be eliminated by two pathways.

Southern blot analysis of BglII-digested DNA isolated from wild-type Streptomyces achromogenes, which harbors the 8.8-kilobase amplifiable unit of DNA, AUD-Sar 1, and of similarly digested DNA from 12 strains carrying an array of 200 to 300 tandem copies of a specific AUD-Sar 1-derived 8.0-kilobase DNA sequence, ADS-Sar 1, revealed the absence of the 12.4-kilobase BglII AUD-Sar 1-chromosome right junction band in the latter strains, whereas the corresponding 26.0-kilobase left junction band remained unaltered. Further Southern analyses indicated in all of the seven amplified strains tested the occurrence of a deletion of at least 10 kilobases of the DNA adjacent to the right side of the AUD. The deletion has one endpoint in the vicinity of the ADS array. Corroborating and expanding upon previously reported results, we found that the amplified DNA of strain C010 was stably maintained for at least 20 transfers when the transfers involved mycelia propagated in spectinomycin-free liquid medium. In contrast, when strain C010 was subjected separately to one cycle of protoplast formation and regeneration or to three cycles of spore germination, aerial mycelium formation, and sporulation on spectinomycin-free media, only approximately 20% of the protoplast regenerants and spores retained the reiterated DNA sequences and the ability subsequently to form colonies on media containing high levels of spectinomycin. Approximately 80% of these units completely deleted the reiterated DNA and left adjacent sequences and exhibited sensitivity to 25 micrograms of spectinomycin per ml. One among 24 protoplast-derived deletants apparently retained the left portion of the AUD-ADS left direct repeat plus left adjacent sequences.

Characterization of two families of spontaneously amplifiable units of DNA in Streptomyces ambofaciens.

Four highly amplified DNA sequences (ADS) ranging from 5.8 to 24.8 kb were found in spontaneous mutant strains of Streptomyces ambofaciens DSM 40697. Restriction patterns of total DNA were hybridized with purified ADS6 (24.8 kb) as a probe to detect the amplifiable regions in the wild-type (WT) genome. The results suggested that the amplifiable unit of DNA (AUD) was present as a single copy in the WT genome. Moreover, similarities suggested by the restriction maps of three of the ADS were confirmed by hybridization experiments. The fourth ADS did not hybridize with the three others. Therefore, two families of DNA sequences are potentially amplifiable in the S. ambofaciens genome.

Genetic instability and DNA amplification in Streptomyces lividans 66.

Streptomyces lividans 66 exhibits genetic instability, involving sequential loss of resistance to chloramphenicol (Cams) and subsequent mutation of argG. Associated with this instability is the amplification of a 5.7-kilobase (kb) amplified DNA sequence (ADS). We have characterized a second, independent pathway of genetic instability, involving sequential loss of resistance to tetracycline (Tets) followed by mutation in nitrogen assimilation (Ntr). We detected DNA amplification in many of these mutant strains, as well as other reiterations coresident with the 5.7-kb ADS in Cams Arg mutants. However, in contrast to the 5.7-kb ADS, none of the novel elements were observed to amplify at high frequency. The mutation of argG is due to a deletion, one endpoint of which is defined by the 5.7-kb ADS. This amplification derives from a structure, the tandemly duplicated amplifiable unit of DNA (AUD), present in the wild-type genome. We found that progenitor strains containing just a single-copy AUD failed to reproducibly generate amplification of this element in Cams argG mutants, and DNA deletion endpoints proximal to the element were found to be unspecific. These results suggest that a duplicated AUD structure is required for high-frequency amplification and that this reiteration can subsequently buffer the extent of deletion formation in the relevant chromosomal region.

Spectinomycin resistance and associated DNA amplification in Streptomyces achromogenes subsp. rubradiris.

Streptomyces achromogenes subsp. rubradiris plated at low density on 1,000 micrograms of spectinomycin per ml initially produces slow-growing, bald colonies from which arise, in a spatially and temporally random fashion, foci of rapidly growing aerial mycelium-forming cells whose DNA contains an approximately 200- to 300-fold amplification of an 8-kilobase (kb) sequence. This sequence was cloned in Escherichia coli on pBR322 and physically characterized. It was separately cloned also in Streptomyces lividans as a BglII fragment and shown to impart high-level resistance to spectinomycin in an orientation-independent manner when present in either the high-copy-number vector pIJ702 or the unit-copy-number vector pIJ943. A spectinomycin resistance determinant was shown to reside on a 1.7-kb SphI-BglII subfragment. Analysis of Southern blots of restriction enzyme digests of wild-type S. achromogenes DNA probed with the labeled 8-kb DNA sequence resulted in the identification and subsequent cloning in S. lividans of a 10.4-kb BamHI fragment which probably includes the complete 8.8-kb amplifiable unit of DNA. This unit is present in wild-type S. achromogenes and in the initially slow-growing, bald colonies arising on 1,000 micrograms of spectinomycin per ml as a single copy. It carries two 0.8-kb direct repeats at its termini as well as the spectinomycin resistance determinant close to one of these termini. About 5% of protoplast regenerants from wild-type S. achromogenes and 77% of protoplast regenerants from the rapidly growing strains lost both the ability to grow on spectinomycin at 10 micrograms/ml and the sequences that hybridize with the 8-kb probe DNA. The 1.7-kb Bg/II-SphI resistance fragment, when introduced via the vector pIJ702 into an S. achromogenes strain sensitive to 10 microgram of spectinomycin per ml, permitted its vigorous growth on 1,000 micrograms of the antibiotic per ml.

High frequency conjugal transfer of tylosin genes and amplifiable DNA in Streptomyces fradiae.

Genes encoding enzymes for tylosin biosynthesis, genes involved in the expression of resistance to tylosin (Tyl), hygromycin B (Hm), chloramphenicol (Cm), and mitomycin C (MC), and a single copy of an amplifiable unit of DNA (AUD) were jointly transferred at very high frequencies by conjugation from several different Streptomyces fradiae strains to S. fradiae JS85, a mutant defective in many or possibly all tylosin biosynthetic reactions and containing a multiple tandem reiteration of the AUD. No recombination was observed between nar, rif and spc genes in conjugal matings, but recombination was observed between these genes after protoplast fusion. Tylosin biosynthetic genes were transferred at a much lower frequency to S. fradiae JS87, another mutant defective in many or all tylosin biosynthetic reactions, but deleted for the AUD and other DNA sequences. These findings suggest that tylosin structural genes, several genes encoding antibiotic resistance determinants, and amplifiable DNA are present on a self-transmissible element that does not mobilize chromosomal genes, and that JS85 and JS87 contain deletions, and JS85 an amplification, of overlapping portions of this element.