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,1
John Cullum,2
Daslav Hranueli,3
Iain S. Hunter,4
Nata
a Peri-Concha,5
Jasenka Pigac,6
Arinthip Thamchaipenet,7
Duica Vujaklija,6 and
Paul F. Long5*
Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia,1
Department of Genetics, University of Kaiserslautern, Kaiserslautern, Germany,2
Faculty of Food Technology and Biotechnology, University of Zagreb, Zagreb, Croatia,3
Royal College, University of Strathclyde, Glasgow, Scotland,4
School of Pharmacy, University of London, London, England,5
Department of Molecular Biology, Ruðer Bokovi Institute, Zagreb, Croatia,6
Faculty of Science, Kasetsart University, Bangkok, Thailand7
SUMMARY INTRODUCTION BACKGROUND CLONING AND MOLECULAR ARCHITECTURE OF POLYKETIDE GENE CLUSTERS Cloning of Tetracycline Resistance Genes Cloning of Genes Involved in OTC Biosynthesis Gene Disruptions in the Rimocidin Biosynthetic Gene Cluster S. RIMOSUS GENOME Chromosome Plasmids Prophages Genetic Instability tRNA, rRNA, AND GENE EXPRESSION IN S. RIMOSUS SYSTEMS FOR PROMOTING GENE EXCHANGE AND CHROMOSOMAL LINKAGE MAPPING Systems Promoting Gene Exchange Chromosomal Linkage Mapping S. rimosus as a Host-Vector System Introduction of DNA into S. rimosus. Vectors for S. rimosus. MUTATION AND SELECTION Random Mutagenesis Localized Mutagenesis Mutations affecting regulatory antibiotic genes. Other mutations affecting antibiotic yield. Screening and Selection FUTURE PROSPECTS ACKNOWLEDGMENTS REFERENCES
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| INTRODUCTION |
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Bêhal and Hunter (23) estimated in 1995 that the global production of tetracyclines was in excess of 5,000,000 kg, and there is no evidence that this has diminished in recent years. Indeed, the bulk usage of tetracyclines is likely to have increased over the last 10 years, mainly due to their use in aquaculture. Although resistance to existing tetracyclines has meant that they can no longer be used effectively for many clinical infections, there are still some important high-risk infections for which tetracyclines are the drugs of choice. Among them are infection with the malaria parasite, Plasmodium falciparum (164); Lyme disease, for which the semisynthetic drug doxycycline is the agent of choice for early diagnosis when the symptoms do not involve the central nervous system (82); and rickettsial tick-borne diseases, where doxycycline is again the agent of choice (216). Tetracycline is used in the eradication of infection by Helicobacter pylori, the causative agent of most duodenal ulcers. Formulated with metronidazole and bismuth, it eradicates the infection in around 90% of cases, and new formulations of this triple therapy are being developed (79). Emerging infections, such as infections with community-acquired methicillin-resistant Staphylococcus aureus, can be treated effectively with tetracyclines (135). Should anthrax ever reemerge, or more likely be used as an infective agent in bioterrorism, doxycycline (a tetracycline derivative) or ciprofloxacin will be the drug of choice (152). Tetracyclines have an increasing role in the treatment of noninfective disease. Their roles as inhibitors of angiogenesis (130) and inhibitors of metalloproteinases have been studied intensely (e.g., see reference 131). These effects are not due to antibiotic action, since tetracycline derivatives which are structurally related but do not possess any anti-infective activity also act in this way (120). Tetracyclines (or derivatives such as anhydrotetracycline) are now used as inducers in controllable eukaryotic expression systems. These systems, adapted from the mechanism of regulation of bacterial tetracycline resistance from transposon Tn10, use a modified bacterial tet repressor that is fused to the activating domain of virion protein 16 of herpes simplex virus to generate a tetracycline-controlled transactivator that can be expressed constitutively in HeLa cells (77). These systems have been adapted for a wide range of applications, not least of which are strategies for gene therapy (78). Oxytetracycline (OTC) finds particularly heavy use in aquaculture, where 500-kg doses may be used in one campaign (124). It continues to be approved within the European Union and elsewhere for fish health care (12), despite some evidence that tetracycline-resistant infections are becoming more prevalent.
Perhaps the most significant development in the tetracycline field in recent years has been the introduction of glycyl-glycyl-9-substituted derivatives (214)—the so-called glycylcyclines—that are now marketed under the trademark Tigecycline. This family of new compounds is active against a number of clinical infections that had become resistant to conventional tetracycline therapy. Among these are multiply resistant Staphylococcus spp. (including vancomycin-insensitive strains), Streptococcus pneumoniae, Enterococcus spp. (including vancomycin-resistant strains), and some extended-spectrum-ß-lactamase-producing isolates of the Enterobacteriaceae (68). Significantly, Tigecycline was the only anti-infective agent to receive FDA approval in the first half of 2005 (235).
The primary tetracyclines, namely, TC, OTC, chlortetracycline (CTC), and 6-demethylchlortetracycline (Fig. 1A), are produced during submerged fermentations of various Streptomyces species isolated from soils during the course of natural product screening programs by industrial as well as academic research groups. The majority of commercially significant tetracyclines are produced by strains of Streptomyces aureofaciens and Streptomyces rimosus, although other species have also been employed (160). Because of the great economic value of tetracycline antibiotics, the results of strain development programs have not been disseminated widely in the open scientific literature. However, procedures developed for the genetic manipulation of Streptomyces species producing tetracyclines have been published. The number of publications concerning S. aureofaciens genetics is not significant. For this species, scientists have been more interested in elucidating the biochemical pathway leading to TC biosynthesis (see references 21 and 103 and references therein). On the other hand, >100 publications have appeared describing the genetic manipulation of OTC-producing S. rimosus strains, affording this species one of the best-developed genetic systems of any streptomycete, second only to Streptomyces coelicolor. Unlike the genetics of S. coelicolor, which has been reviewed extensively (97), there is a paucity of curated information for S. rimosus. Accordingly, the present review concentrates on S. rimosus genetics, paying particular attention to the application of modern molecular approaches that will be invaluable for the development of new tetracyclines by combinatorial biosynthesis and novel recombination approaches (109, 122). It should also be noted, however, that S. rimosus has been known for some time to be a producer of other biologically active secondary metabolites, such as the polyene antifungal agent rimocidin (51) (Fig. 1B).
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er Bokovi Institute. The majority of the important contributions to this field have been published in the English language literature, but a considerable number have also appeared elsewhere in Russian or Croatian. Among the latter, only those deemed most relevant have been translated for inclusion in this perspective. To complicate an objective review of the literature even further, these groups have used four strains of S. rimosus, but with several different designations for each. During the late 1950s and until the early 1970s, the majority of published work performed in Russia was done with S. rimosus mutants derived from a strain previously called Actinomyces rimosus LS-T118 (155). More recently, S. rimosus strain 183 has been used to study genetic instability (see reference 210 and references therein). Since the early 1960s, the groups working in Croatia have been generating mutants isolated from two independent S. rimosus strains. These strains are ATCC 10970 (NRRL 2234) from the American Type Culture Collection, whose name has been abbreviated to R7 in the majority of publications, and S. rimosus R6, which is a soil isolate from the Faculty of Food Technology and Biotechnology, University of Zagreb, used for the development of mutants for the commercial production of OTC by PLIVA (Fig. 2). The linkage maps of S. rimosus R6 and R7 are almost identical (10). During the 1970s, the group from Pfizer Central Research, in collaboration with scientists from the John Innes Centre in the United Kingdom (71) and, more recently, scientists from the Institute of Genetics, University of Glasgow, published work using mutants derived from the prototrophic strain M4018 (37), employed commercially for the production of OTC by Pfizer. In the scientific literature, these bacteria have come to be known as the Russian (LS-T118), Zagreb (R6), and Pfizer (M4018) strains (94, 117). Other strains have also been used sporadically as donors or recipients of DNA (e.g., BS-21, 907, 183, RCC, and PG3) (41, 155, 188, 198, 212), but no more data on their genetics have been published to date.
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| BACKGROUND |
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During the early 1970s, circular chromosomal linkage maps of three S. rimosus strains were constructed. After a preliminary report at the First International Symposium on the Genetics of Industrial Microorganisms in 1970 (7), these maps were independently published by Friend and Hopwood (71) and by Ala
evi and collaborators (10). Data from these maps were combined to also include the positions of markers at new loci (9). Meanwhile, optimal conditions for mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; referred to as nitrosoguanidine [NTG] in some of the cited references) and UV irradiation were also developed (8, 39, 52, 53). At about the same time, the first estimate of the size of the S. rimosus genome was published (24). In the early 1970s, studies following three avenues of research were initiated. These were the biochemical characterization of non-OTC-producing mutants and mapping of the OTC genes (180, 200), characterization and persistence of S. rimosus phages (104, 107, 193, 225), and genetic and physical characterization of circular and linear S. rimosus plasmids (31, 41, 72, 80, 201, 212).
During the last 25 years of research into the genetics of S. rimosus, procedures for in vivo and in vitro genetic manipulations have been developed (201). These include protoplast preparation and regeneration and protoplast-mediated genetic exchange via fusion (62, 105, 108, 163, 222, 223, 228, 229). Restriction-deficient mutants (115) and broad-host-range plasmids (64, 75, 126, 135) and phage vectors (208), including a bifunctional cosmid for use with S. rimosus, have also become available (40). These genetic tools and procedures have been applied to study the genetic instability of S. rimosus strains (49, 80) and for the molecular cloning and characterization of the OTC resistance gene(s) (37, 61, 167, 198) as well as the cloning of genes involved in OTC biosynthesis (29, 36, 37, 150, 177). These genes have also been used for the formulation of design rules for constructing hybrid aromatic polyketide synthases with a view to producing structural analogues, by combinatorial biosynthesis, with potentially novel biological activities which are difficult to produce by traditional synthetic routes (125, 148, 175, 177).
| CLONING AND MOLECULAR ARCHITECTURE OF POLYKETIDE GENE CLUSTERS |
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Transcriptional analysis of otrA revealed two promoters, otrAp1 and otcCp1. During exponential growth, the otrA gene is transcribed as a single cistron from otrAp1. At the beginning of the stationary phase of growth and during OTC biosynthesis, the otrAp1 promoter is silent, and otrA is transcribed as the 3' gene of a polycistronic mRNA originating from the otcCp1 promoter (60, 61, 149). This arrangement makes good teleological sense, as the otrA gene is always expressed. During exponential growth, the cells would otherwise be susceptible to the action of neighboring cells that are making OTC, whereas during the OTC production phase the continued expression of otrA is ensured, in concert with that of the other production genes. MacGregor-Pryde (137) identified a gene upstream of otrB, otrR, whose product is a putative repressor. Thus, the overall topology of the otrB-otrR region mirrors that found in Tn10, where expression of the tetracycline efflux pump is controlled by a divergent repressor.
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Disruptions of otcD1 (encoding an aromatase/cyclase) resulted in four novel carboxamido-derived polyketides with shorter chain lengths (177). This provided strong evidence that the carboxamido group was present from the start, rather than the nascent polyketide backbone being amidated later in the biosynthetic scheme. The caboxamido-derived decaketide isolated by Zhang and collaborators (239) by heterologous expression of otcY1-4 reinforces the view that the caboxamido group is present from the outset.
When the nascent polyketide chain is completed, it is subsequently reduced and folded into a tetracyclic structure by the action of the gene products of otcY2-1 (a ketoreductase) and otcD1 (cyclase/aromatase). The folded polyketides then act as substrates for tailoring enzymes (Fig. 4). These include methylation at C-6 to form the first enzyme-free intermediate, 6-methylpretetramid (6-MPT) (145). There are three methylation steps in OTC biosynthesis, including C-6 methylation and N-dimethylation of the amino group at C-4. Two gene products, OtcZ and OtcY2-5, are methyltransferase homologues. By studying otc mutants, Rhodes and collaborators (200) initially proposed that otcZ catalyzes C-6 methylation, whereas the otcD region could be involved in the dimethylation step to form 4-aminodedimethylaminoanhydrotetracycline (4-amino-ATC). However, bioinformatic analyses of the DNA sequence for the otc cluster deduced that the second methyltransferase was encoded by otcY2-5.
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Conversion of 6-MPT to 4-hydroxy-6-methylpretetramid (4-hydroxy-6-MPT) occurs through the activity of a specific hydroxylase. In the original mutagenesis studies performed by Rhodes and collaborators at Pfizer, the otcX locus was assigned to this biochemical step (200). OtcX1 has striking similarity to the S. coelicolor ActVA-ORF2 hydroxylase from the actinorhodin cluster (38, 150). It is likely that OtcX1 is an ancillary protein in the hydroxylation reaction rather than the catalyst per se. Several other genes for hydroxylase-like proteins have been identified in the otc gene cluster. These include otcC (6-hydroxylase), otcD2, and otcY1-5. It has yet to be established which gene products hydroxylate the substrate at the C-4 position at this point of the biosynthetic scheme and which add a hydroxyl group later at the C-5 position.
Reduction of the hydroxyl group at the C-4 position of 4-hydroxy-6-MPT is controlled by a gene designated otcD3, whose product shows homology to ketoreductases. Amination at C-4 results in the formation of 4-amino-anhydrotetracycline. This step is likely catalyzed by the gene product of otcX2, which has high similarity with aminotransferases. Conversion of 4-amino-ATC to ATC occurs by the introduction of two methyl groups at C-4, most likely by the product of otcZ, as discussed above.
ATC oxygenase catalyzes the hydroxylation of ATC at C-6 in the presence of oxygen and NADPH and results in the formation of 5a,11a-dehydrotetracycline (5-DHTC). The 52-kDa subunit of this enzyme has been purified, and the N-terminal amino acid sequence has been obtained (35). These data were used to design an oligonucleotide probe to isolate the gene from an S. rimosus cosmid library (29). The oligonucleotide hybridized to the DNA region between otcZ and otcX, locating otcC. The deduced DNA sequence indicates that OtcC belongs to the family of bacterial flavin-type hydroxylases. The otcC promoter is located in the region between otcC and otcX-ORF1. The otcC transcript appears to be polycistronic and includes the otrA gene. Recently, a recombinant strain of S. rimosus that was disrupted in the genomic copy of otcC was found to synthesize a novel C17 polyketide. This result indicated that the absence of the otcC gene product significantly affects the ability of the OTC minimal PKS to synthesize a polyketide product of the correct chain length, indicating that OtcC is an essential partner in the quaternary structure of the synthase complex (175).
A hydroxyl group is then added at C-5 of 5-DHTC to form 5a,11a-dehydrooxytetracycline (DHOTC). Whether the product of the otcY1-5 or otcD2 gene is involved is discussed above. The last step in OTC biosynthesis involves a dehydrogenase. The enzyme that converts 5-DHTC to TC in S. aureofaciens has been purified (165), and OtcY2-4 was assigned to this function on the basis of its amino-terminal sequence (217). After OTC has been formed, it is exported from the cell by OtrB. Located between the otcY1 and otrB genes, otrR encodes a transcriptional regulator that does not resemble typical tetracycline repressors (190) but shows some similarity to so-called multidrug repressors from Escherichia coli. The juxtaposition and divergent transcription of otrR and otrB indicate that OtrR may be the repressor for otrB, but this notion remains to be tested.
The molecular genetics of rimocidin biosynthesis has been investigated independently with both S. rimosus R7 (A. Thamchaipenet, unpublished data) and S. diastaticus var. 108 (203), with some sequence data already having been deposited (GenBank accession no. AY423269, AY701054, AY442225, and DQ174320). Disruption of rimocidin synthase (RMS) resulted in nonproducing mutants in both S. rimosus R7 (Thamchaipenet, unpublished data) and S. diastaticus var. 108 (203), validating that the RMS genes are involved in rimocidin biosynthesis.
Deduced polypeptide analysis of the rimocidin gene cluster indicated that it is synthesized by a modular type I PKS system (59), unlike OTC, which is synthesized by a type II PKS. The domains of the rimocidin synthase of S. rimosus strain R7 (Thamchaipenet, unpublished data) (Fig. 5) are organized into 14 modules in a linear manner, similar to that of other type I PKSs, and they share common features with other polyene antibiotic gene clusters, as previously described (18). The chemical structure of rimocidin indicates that the starter unit is butyryl-CoA, which is likely formed by the condensation of two acetyl-CoA units catalyzed by crotonyl-CoA reductase. If acetyl-CoA is incorporated, then the homologous compound CE108 is formed (203).
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| S. RIMOSUS GENOME |
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Most recent work on genome size and structure has used S. rimosus R6 (the Zagreb strain). Pulsed-field gel electrophoresis (PFGE) was used to construct a restriction map of the chromosome of strain R6-501 (Fig. 6) for the rarely cutting enzymes AseI and DraI (172). The map is linear, like those of other Streptomyces species. The total chromosome size was estimated to be 8 Mb, similar to that of S. coelicolor A3(2). This would correspond to a true size of about 8.6 Mb (81) because the Saccharomyces cerevisiae chromosome markers used underestimate the sizes of high-G+C-content fragments. The ends of the chromosome are inverted repeats of about 550 kb. The chromosome end (GenBank accession number AY043328) resembles that of other Streptomyces species (113), with several inverted repeats in a region of about 200 bp. The dnaA gene is located in the AseI C2 fragment, which means that the replication origin is at least 0.5 Mb from the center of the chromosome and is not as centrally located as that in S. coelicolor A3(2). However, the dnaA gene of S. avermitilis is also displaced about 0.8 Mb from the center of the chromosome. Only a small number of other genes (recA, rRNA operons, and att-pSAM2) have been localized in the chromosome of S. rimosus. It is interesting that their positions differ significantly from those of their homologues in S. coelicolor A3(2). However, too few data are available to conclude that S. rimosus possesses a chromosomal organization radically different from that of the two sequenced species, S. coelicolor A3(2) and S. avermitilis, which show a high degree of conservation of gene order (118).
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The first physical evidence of a naturally occurring plasmid in S. rimosus (Actinomyces rimosus 907) was published by Stepnov and collaborators (212). A circular DNA molecule of 55 kb was detected in crude DNA preparations. Total DNA was analyzed electrophoretically in an agarose gel, followed by electron microscopic analysis of DNA isolated from the gel. Although attempts to isolate covalently closed circular DNA from S. rimosus RCC by CsCl-ethidium bromide density gradient centrifugation were unsuccessful, Chardon-Loriaux and collaborators (41) succeeded in detecting and purifying extrachromosomal DNA by agarose gel electrophoresis and electroelution. Electron microscopy did not show any circular molecules. This was in agreement with the results of restriction mapping showing a linear plasmid DNA molecule of about 43 kb. The plasmid, named pSRM, exhibited the lethal zygosis phenomenon.
Most of the work on S. rimosus plasmids has used S. rimosus R6. R6-501, the strain used to construct the chromosomal restriction map, did not carry any detectable circular plasmids but had a 387-kb linear plasmid, pPZG101 (81). The pPZG101 plasmid has a unique central region of about 30 kb flanked by inverted repeats of about 180 kb (173). Strain R6-65, which is an ancestor of R6-501, carries a smaller linear plasmid of about 310 kb, pPZG102, which does not have extensive inverted repeats. One end of pPZG102 seems identical to the inverted repeat of pPZG101. This suggests that pPZG101 was derived from pPZG102 by a recombination event between copies of the plasmid in inverted orientation to generate the long inverted repeats. Curing of pPZG101 has been observed. When 17 auxotrophic mutant strains were analyzed, 5 no longer carried free plasmid and 2 showed no hybridization to pPZG101. The other three strains had integrated parts of the plasmid into the chromosome, indicating that there are frequent interactions between the plasmid and the chromosome. During the study of genetic instability in S. rimosus R6 (see below), a mutant was isolated that overproduced oxytetracycline. This mutant (MV17) proved to carry a 1-Mb linear plasmid, pPZG103, derived from pPZG101 and carrying the otc cluster (173). The chromosome had a parental structure, so there was an increased copy number of the otc cluster due to the extra copy on the plasmid. pPZG103 arose from a crossover between pPZG101 and the chromosome, such that about 200 kb at one end is derived from pPZG101 and the other 800 kb is the chromosomal terminal sequence. A second mutant derived in the study (MV25) proved to have replaced a chromosome end with one end of plasmid pPZG101; the details of this mutant are discussed below.
Classical genetic analysis by the Pfizer group with strain M4018 (derived from S. rimosus R7) defined plasmid SRP2 and found a derivative, SRP2', which carries the otc cluster. PFGE analysis showed that S. rimosus R7 carries a 310-kb plasmid with a restriction pattern very similar to that of pPZG102 from S. rimosus R6-65. The SRP2'-carrying strain had replaced the original linear plasmid of about 310 kb with a much larger one, of about 950 kb, suggesting that an event similar to that observed for S. rimosus R6 had occurred (81).
C31 of S. lividans (134), the phage has a broad host range and can be grown on many species of Streptomyces. If the phage has a narrow host range, it will probably be necessary to isolate a cured derivative of the host strain, which usually involves a good portion of serendipity. Hranueli and collaborators (104, 106, 107) detected and isolated free phage particles from a liquid culture of S. rimosus R7. The phage, designated RP2, appeared to be a typical temperate DNA phage producing turbid plaques in lawns of sensitive S. rimosus R6 cells. The actinophage RP2 has a very narrow host range restricted to S. rimosus strains. Later work (193) showed that S. rimosus R6 is usually lysogenic for RP2 but that the strain used in these experiments had been spontaneously cured of its prophage. Its tadpole-shaped morphology and double-stranded DNA content place RP2 in group B1 (Fig. 7A) of the bacteriophage classification system (1). Although the latent period of actinophage-actinomycete systems is usually long, RP2—with its 6-h latent period—is the slowest- multiplying actinophage described so far (see Table 1 in reference 134). The lysogenic nature of RP2 was established on the basis of the following criteria: (i) a low spontaneous lysis frequency of 2 x 10–6 per cell, (ii) resistance to curing with actinophage-specific antiserum, (iii) a low spontaneous curing frequency of <0.05%, and (iv) immunity to superinfection with the homologous phage. Treatment with UV light and other agents did not lead to induction of the prophage. Clear plaque mutants of RP2, which failed to lysogenize sensitive cultures, arose at a frequency of about 2 x 10–5 per phage particle (104, 106, 107). However, no virulent mutants that could infect lysogenic strains were observed, probably because multiple mutations would be necessary. After mutagenic treatment of a lysogenic strain with MNNG, two classes of mutants were isolated that no longer produced actinophage. Mutants belonging to the first class were phage sensitive and probably cured of the prophage, whereas those of the second class retained immunity to superinfection and presumably carried defective RP2 prophage. Lysogeny with RP2 did not affect OTC production.
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The RP2 and RP3 phages contain linear double-stranded DNA molecules of 64.7 kb and 62.4 kb, respectively, which have been mapped by restriction enzyme digestion (193). They have a G+C content of about 70%, which is indistinguishable from that of the chromosome. The DNA molecules of both phages have cohesive ends, suggesting site-specific staggered cutting of concatameric DNA resulting from rolling circle replication (42). The two phages show hardly any cross-hybridization, implying that they are not closely related. Both phages integrate into the chromosome by using specific attachment sites that have been located in the restriction maps of the phages. The positions of the integrated prophages were also localized on the chromosomal restriction map (172). DNA from RP3 was used to generate ladders of concatemers suitable for use as high-G+C-content markers for PFGE (81).
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Class III mutants show increased oxytetracycline resistance and production and form a phenotypically homogeneous class. They were analyzed by PFGE, and most showed identical restriction patterns. A 200-kb DNA fragment including the otc cluster was amplified in three or four tandem copies, and the distal chromosomal sequences had been lost. This structure resembles those seen in other species, such as S. lividans 66 (192), with reiteration of a DNA fragment and loss of the distal sequences.
However, in most other cases, the size of the reiterated sequence is much smaller (e.g., 5.7 kb in S. lividans) and the degree of amplication is much higher (e.g., >100 copies). The increased copy number of the otc cluster accounts for the increased resistance and production. The chromosomal sequences distal to the amplified sequence have been lost, and the structure of the chromosome end in class III mutants is not clear. It is possible that dynamic amplification compensates for the loss of sequences upon chromosomal replication. In one case (mutant MV25), chromosomal amplification was accompanied by recombination with the linear plasmid pPZG101 such that a plasmid end had replaced the chromosome end distal to the reiterated otc region. In MV25, the number of reiterated copies of the otc region differed between members of the population, and there were also members of the population that had lost all copies of the OTC region, resulting in a sensitive nonproducing phenotype. As mentioned above, there was another class III mutant (MV17), which carried an extra copy of the otc cluster on the linear plasmid. The use of class III strains to achieve increased production might seem attractive, but they are subject to an increased frequency of genetic instability to produce class I variants, which eliminates the advantages of increased production.
Many streptomycetes that are currently used for industrial-scale antibiotic production have probably acquired amplified DNA sequences in their genomes as a result of repeated cycles of strain selection. In many instances, fortuitously amplified sequences will be present, but in some cases the DNA amplification may play a direct role in hyperproduction of a particular product. The role of amplification in hyperproduction of an alpha-amylase inhibitor by a strain of Streptomyces tendae has been well established, and it may also account for oleandomycin hyperproduction by a strain of Streptomyces antibioticus (see reference 238 and references therein). However, genetic instability can also be a serious problem in industrial production processes. It is well known that industrial strains are susceptible to degeneration (i.e., an irreversible loss of favored characteristics), which is undoubtedly—at least in part—a phenotypic consequence of genetic instability. For example, among others, S. aureofaciens (produces CTC) and S. rimosus (produces OTC) are commercially important species that are subject to genetic instability (95, 168). It is not surprising, therefore, that the molecular mechanisms responsible for such genetic instabilities have attracted considerable attention.
In their studies, Danilenko and collaborators (50, 66, 188) have shown that a kanamycin resistance (Kmr) gene of S. rimosus ATCC 10970 is located on a 15.6-kb amplified unit of DNA (AUD). In S. rimosus ATCC 10970 variants (e.g., P3), this AUD could undergo up to 300-fold amplification after serial subculturing on media containing increasing concentrations of the antibiotic, resulting in increased activity of the encoded aminoglycoside phosphotransferase enzyme. The Kmr gene was cloned as a 9.5-kb PstI fragment from a 15.6-kb AUD into S. lividans, using the plasmid vector SLP1.2. The recombinant plasmid also underwent amplification in response to the same selection pressure in S. lividans. However, it was not completely clear whether the plasmids studied integrated into the chromosome of S. lividans, because the SLP1.2 vector on which they were based apparently had lost the sequences that normally promote integration in this organism (169). The Kmr fragment from S. rimosus P3 might conceivably promote chromosomal integration of the plasmids, or alternatively, they may simply exist as very large plasmid multimers that could have escaped detection because of their sensitivity to shearing during DNA preparation procedures. Regarding the mechanism, this approach could prove useful for constructing stable highly expressing clones in which foreign DNA fragments could be cloned and coamplified together with plasmids derived in such a way (49, 211). The activity of the Kmr enzyme is controlled by at least two different serine/threonine protein kinases (66), but it is unknown whether changes in the expression of protein kinases play a role in genetic instability.
| tRNA, rRNA, AND GENE EXPRESSION IN S. RIMOSUS |
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A study of rRNA operons in S. rimosus was initiated by Plohl and Gamulin (186). Six rRNA operons were defined by Southern blot hybridization, with the rRNA genes in the operons separated by short intergenic regions and organized in the order 16S-23S-5S. Again, the tRNA genes were not found in association with any rRNA operons. The sequence of the 3' end of the rRNA operon (rrnF) with the whole 5S rRNA gene was characterized (186). The gene is 120 bp long and differs at two positions from the S. ambofaciens 5S rRNA gene (176). The 3' end (
450 bp) of the 23S rRNA gene and a spacer between the 23S and 5S rRNA genes are located upstream of the 5S rRNA gene on the same cloned fragment. This DNA fragment also shows high homology with the S. ambofaciens 23S rRNA gene. Remarkable sequence homology was also found with the S. ambofaciens rrnD operon in the 3'-noncoding regions, including the first termination signal. The S. rimosus rrnF operon contains a second putative terminator which is absent from the S. ambofaciens rrnD operon. The G+C content was 60%, which is much lower than the average, and the 23S-5S rRNA gene spacer was only 48.6% G+C. The whole S. rimosus operon was then sequenced and analyzed by Puji and collaborators (189). The predicted order of the genes is 16S-23S-5S, and the genes are 1,529, 3,121, and 120 nucleotides long, respectively. Homology between rRNA genes from other streptomycetes is in the range of 95% for all three genes and at least 70% for known intergenic regions of Streptomyces rRNA operons.
Evidence for the distribution of the rRNA operons and one tRNA gene was obtained following physical mapping of these genes. In S. rimosus, the rrn operons are located in the central region of the chromosome, with no operon located within 2,755 kb and 3,095 kb of the respective ends of the arms (172). Conversely, in S. coelicolor A3(2), ribosomal operons rrnC and rrnE are located about 1.4 Mb and 2.1 Mb from the chromosome ends (196). Physical mapping showed that all rRNA genes could be located on four AseI and DraI fragments (172). In S. rimosus, the tRNAPro attachment site for pSAM2 (143) is located 1.8 Mb from the chromosome end, while in S. coelicolor A3(2) it is near the center of the chromosome (172). In general, comparison of these and other gene markers with the map of S. coelicolor A3(2) has suggested clear differences in genome organization between the two species (172), a point worthy of further investigation, possibly through whole-genome sequencing efforts with S. rimosus in the future.
Regulatory sequences for transcription in S. rimosus have been poorly investigated. The first extensive analysis of the DNA fragments associated with apparent Streptomyces transcriptional start sites (213) showed a wide range of sequence diversity. Only about 20% appeared to belong to a group similar to that recognized by eubacterial RNA polymerases containing sigma 70-like subunits. These promoters, designated SEPs (Streptomyces-E. coli-type promoters), contain –35 and –10 regions with a spacing of 16 to 18 bp between the two sequences (121) and are typically associated with housekeeping genes (44). In S. rimosus, there are well-studied promoters for the otcC and otcX genes associated with OTC biosynthesis (149). Both promoters contain sequences that are similar to the consensus –10 and –35 regions of the major eubacterial and SEP-like promoters.
The expression of seven previously cloned tRNA genes from S. rimosus (74, 185) was studied by deletion experiments and Northern blot hybridization. It was demonstrated that the clusters encoding tRNAGln and tRNAGlu were transcriptionally active in homologous systems and an E. coli heterologous system. All genes in the cluster were cotranscribed as one transcriptional unit from the same promoter, located 140 to 65 bp upstream of the first gene. The sequence TTGGAC-17 bp-TAATGT, resembling an SEP, was also located in this region. Two tRNAfMet genes from S. rimosus, however, were found to be transcriptionally active only in a homologous system, indicating that they have significantly different promoters which do not function in E. coli (65).
One of the six S. rimosus ribosomal operons, rrnF, has been completely sequenced and analyzed in detail (189). Only one putative promoter for this operon (P4) was identified by sequence similarity in a DNA region upstream from the start of the 16S rRNA gene. The same –10 box (TAGAGT) was found in numerous promoters of rRNA operons in different Streptomyces spp. (127, 176, 224, 237), while the –35 regions varied considerably. A 21-bp sequence defined as an rRNA processing site was found downstream from the P4 promoter of S. rimosus rrnF. This sequence is also found in front of all Streptomyces rRNA operons studied to date (176, 231). Two more DNA fragments corresponding to upstream regions of two S. rimosus 16S rRNA operons were also cloned and analyzed (189). In the first sequence (type I), four putative promoters (P1 to P4) were identified. The second DNA fragment (type II) seemed to be a deletion derivative of the type I fragment and lacked the DNA region carrying the P2 and P3 promoters. Aside from the internal deletion, the upstream regions of the fragments are highly homologous to the upstream region of the previously described rrnF operon, starting from the P4 promoter. The putative –10 boxes of the P1 to P4 promoters of S. rimosus rRNA operons were compared with all experimentally identified or proposed promoters of Streptomyces rRNA operons. The results indicated that these sequences are conserved among all the species examined to date. In the case of the P2 and P4 promoters, sequence identity extended for several nucleotides on both sides of the –10 box. Based on the results of hybridization experiments (189), a general scheme was proposed for the transcription of all six rRNA operons in S. rimosus, with three rRNA operons under the control of four promoters (P1 to P4), two operons expressed from two promoters (P1 and P4), and only the rrnF operon expressed from one promoter, P4, which is the most proximal promoter common to all six S. rimosus rRNA operons.
Two transcription start sites were identified during study of the transcription of the S. rimosus recA gene by primer extension analysis (2). The longer of the two transcripts is initiated from a distal SEP-like promoter (TTGACA-18 bp-TCTTAT) that contains a Cheo box-like sequence (GAAC-N4-ATTC). The major transcript is initiated at position –36 and increases significantly in response to DNA damage. Ahel and collaborators (2) proposed a –35 box (TTGTCA) and –10 box (TAGCGT) that are only 11 bp apart. They noticed that regulatory sequences of this putative promoter and a part of the spacer region are almost identical to those of the DNA damage-inducible promoter of the Mycobacterium tuberculosis recA gene (162), which has been well characterized (76). This sequence is perfectly conserved in all four recA genes analyzed from Streptomyces to date (4, 153, 166, 236). A recent analysis of S. rimosus showed a significant increase in transcription from the proximal promoter of the recA gene in the presence of a RecA protein that lacks 21 amino acids from the C terminus. This up-regulation, albeit less dramatic, was also observed in S. lividans, suggesting the presence of a similar regulation mechanism for the expression of recA in other streptomycetes (3). Inspection of the S. coelicolor genome sequence identified the presence of this type of promoter in the upstream regions of many potentially UV-inducible genes and some other genes/open reading frames. A consensus sequence for this proposed Streptomyces promoter is TTGTCAGTGGC-N6-TAGGGT.
| SYSTEMS FOR PROMOTING GENE EXCHANGE AND CHROMOSOMAL LINKAGE MAPPING |
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The crossing techniques used to establish conjugation in S. rimosus were adapted from those developed for S. coelicolor A3(2) and were described in detail by Hopwood (92) and Sermonti (205). These methods use spore suspensions, but the Zagreb strain (S. rimosus R6) does not sporulate well. This led to the use of modified methods based on mycelia, including a mixed-culture method (6, 10, 13, 16, 71, 180, 200) and matings on cellophane membranes (6, 10).
Based on protoplast fusion techniques developed for S. coelicolor (100), chromosomal linkage mapping for S. rimosus has also been possible. The analysis of linkage after protoplast fusion is essentially identical to that of linkage after conjugation, but treatment of S. rimosus protoplasts with 40% (wt/vol) polyethylene glycol 1550 for 30 min increases the frequency of recombination 2 to 3 orders of magnitude compared to that for conjugation, and therefore recombination is not so dependent on the plasmid compositions of the two parent strains (108).
evi and collaborators (9) introduced a computer-assisted approach to the processing of data. The mapping method gives information about gene order in the chromosome but does not give genetic distances. These analysis methods were used to construct a genetic map of Pfizer strain M4018 for 24 genetic markers (71). When crosses were carried out, there was frequent recovery of apparent recombinants that segregated parental genotypes; these were deduced to be heterokaryons. This phenomenon was not observed with S. coelicolor A3(2).
A genetic linkage map was also constructed for S. rimosus ATCC 10970 (strain R7) (6, 10). For this strain, many of the recombinants were heteroclones. Heteroclones, which are also observed in S. coelicolor A3(2), are relatively stable partial diploids that eventually segregate haploid progeny. The frequency of inclusion of any locus in the diploid region depends on its location relative to the selected markers (102), which can be used for construction of a map. Analysis of the frequencies of different classes of haploid segregants from heteroclones allows the estimation of linkage distances between genes. Similar to the case for eukaryotic genetic maps based on meiosis, the unit of distance is the centimorgan (cM), i.e., the percentage of progeny that have undergone a crossover in the region between two gen
