Genetics of Streptomyces rimosus, the Oxytetracycline Producer

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.     

Identification of some streptomycetes producing oxytetracycline

A study was made of Streptomyces rimosus and mutant strains to compare the phenotype of high and low oxytetracycline producers. Strains were identified using a probabilistic identification matrix for the genus Streptomyces. Mutant strains separated into two groups: high-titre strains and blocked mutants. The former identified with the S. rimosus cluster whereas the latter were not identified. Two further oxytetracycline producers identified with the Streptomyces lydicus cluster.     

Molecular cloning and functional characterization of an ATP-binding cassette transporter OtrC from Streptomyces rimosus

ABSTRACT: BACKGROUND: The otrC gene of Streptomyces rimosus was previously annotated as an oxytetracycline (OTC) resistance protein. However, the amino acid sequence analysis of OtrC shows that it is a putative ATP-binding cassette (ABC) transporter with multidrug resistance function. To our knowledge, none of the ABC transporters in S. rimosus have yet been characterized. In this study, we aimed to characterize the multidrug exporter function of OtrC and evaluate its relevancy to OTC production. RESULTS: In order to investigate OtrC's function, otrC is cloned and expressed in E. coli The exporter function of OtrC was identified by ATPase activity determination and ethidium bromide efflux assays. Also, the susceptibilities of OtrC-overexpressing cells to several structurally unrelated drugs were compared with those of OtrC-non-expressing cells by minimal inhibitory concentration (MIC) assays, indicating that OtrC functions as a drug exporter with a broad range of drug specificities. The OTC production was enhanced by 1.6-fold in M4018 (P = 0.000877) and 1.4-fold in SR16 (P = 0.00973) duplication mutants, while it decreased to 80% in disruption mutants (P = 0.0182 and 0.0124 in M4018 and SR16, respectively). CONCLUSIONS: The results suggest that OtrC is an ABC transporter with multidrug resistance function, and plays an important role in self-protection by drug efflux mechanisms. This is the first report of such a protein in S. rimosus, and otrC could be a valuable target for genetic manipulation to improve the production of industrial antibiotics.     

The genome of <Emphasis Type="Italic">Streptomyces rimosus</Emphasis> subsp. <Emphasis Type="Italic">rimosus</Emphasis> shows a novel structure compared to other <Emphasis Type="Italic">Streptomyces</Emphasis> using DNA/DNA microarray analysis

DNA/DNA genome microarray analysis together with genome sequencing suggests that the genome of members of the genus Streptomyces would seem to have a common structure including a linear genomic structure, a core of common syntenous Actinomycete genes, the presence of species specific terminal regions and two intermediate group of syntenous genes that seem to be genus specific. We analyzed Streptomyces species using DNA/DNA microarray comparative genome analysis. Only Streptomyces rimosus failed to give a congruent genome pattern for the genes found in Streptomyces coelicolor. We expanded the analysis to include a number of strains related to the type strain of S. rimosus and obtained a similar divergence from the main body of Streptomyces species. These strains showed very close identity to the original strain with no gene deletion or duplication detected. The 16S rRNA sequences of these S. rimosus strains were confirmed as very similar to the S. rimosus sequences available from the Ribosomal Database Project. When the SSU ribosomal RNA phylogeny of S. rimosus is analyzed, the species is positioned at the edge of the Streptomyces clade. We conclude that S. rimosus represents a distinct evolutionary lineage making the species a worthy possibility for genome sequencing.     

Selection of the optimal conditions for the procurement and regeneration of protoplasts of the industrial strain of Streptomyces rimosus, the producer of oxytetracycline, and the effect of the protoplasting process on the antibiotic activity]

Optimal conditions for protoplasting of the Streptomyces rimosus industrial strain No. 1 producing oxytetracycline were developed. Observation of the early stages of the protoplast regeneration in microchambers showed that there were two regeneration types: normal and anomalous. The latter was likely defined by the glycine effect on cell wall synthesis. It was accompanied by the stage in which the protoplasts had the form of multiplying protoplast-like cells. The protoplasting of the S. rimosus culture producing oxytetracycline resulted in an increase in the variability of an antibiotic producing property and the frequency of low active variants.     

适合龟裂链霉菌~(13)C代谢通量分析合成培养基的优化及应用

为开发一种适合龟裂链霉菌13C代谢通量分析的合成培养基,以龟裂链霉菌模式菌株M4018为研究对象,比较其在各种有机氮源和无机氮源的生长和土霉素合成特性。首次筛选到以硝酸钾为主要氮源的合成培养基,通过响应面分析法进一步优化,将土霉素合成能力由75.2 mg/L提高到145.6 mg/L。并应用到100%的1-13C葡萄糖标记实验,首次从同位素标记代谢流分析上证实了龟裂链霉菌中不存在2-酮-3-脱氧-6-磷酸葡糖酸裂解途径(Entner-Doudoroff pathway,ED),为龟裂链霉菌13C代谢通量分析提供了重要基础。     

Molecular cloning of chlortetracycline resistance gene from chlortetracycline producer Streptomyces aureofaciens

The chlortetracycline (CT) resistance gene ctr was cloned from S. aureofaciens 633, a strain producing the antibiotic. The 6.6-kb DNA Bam HI fragment containing the resistance gene was cloned with the plasmid vector pIJ699. Comparison of the restriction maps of the cloned gene and the oxytetracycline (OT) resistance gene otrA from S. rimosus revealed their similarity which enabled identification of the cloned resistance gene as otrA. Investigation of the resistance determinants in S. aureofaciens 633 made it possible to identify a mtr gene(s). It was demonstrated that introduction of a ctrA gene into S. lividance provided a simultaneous increase in the resistance of the recipient strain to CT and a number of macrolide antibiotics. The CT resistance determinants in S. lividans TK64 showed properties of exogenous induction by CT and the macrolide antibiotics similar to the properties of the mtr gene(s) of S. aureofaciens. Possible adaptation properties of mtr genes are discussed.     

龟裂链霉菌zwf2基因阻断提高土霉素生物合成

葡萄糖-6-磷酸脱氢酶(G6PDH)是链霉菌磷酸戊糖途径中第一个酶("看家"酶),也是形成NADPH的关键酶,由zwf1和zwf2基因编码.以温敏型质粒pKC1139为基础构建了用于阻断龟裂链霉菌zwf2的重组质粒pKC1139-zwf2',通过大肠杆菌GM2929去甲基化pKC1139-zwf2'后电转至原始龟裂链霉菌M4018感受态细胞,筛选得到转化子.转化子进一步通过PCR鉴定和点杂交印迹分析鉴定,证明是zwf2基因阻断的阳性突变子命名为M4018-△zwf2.以原始菌株为对照,突变子摇瓶发酵结果表明:突变子的葡萄糖-6-磷酸脱氢酶酶活是原始菌的50%左右,但土霉素生物合成水平则提高了27%;在细胞生长方面,二者均在第4d进入生长稳定期而开始大量合成土霉素,发酵结束时细胞菌体浓度基本相同,但突变子的单位菌丝体土霉素生物合成能力则提高了31%.因此,zwf2的阻断有利于土霉素的生物合成,而对细胞生长没有明显影响.     

工程改造龟裂链霉菌提高土霉素产量

土霉素是由龟裂链霉菌合成的一类广谱性抗生素,前期研究工作证明其生物合成受其自身途径特异性调控蛋白OtcR的直接调节,OtcR能够激活和促进土霉素合成基因簇的转录表达。在龟裂链霉菌M4018宿主内利用强启动子单独过表达OtcR蛋白,使土霉素的产量提高到原来产量的4倍;为了进一步提高土霉素产量,在M4108宿主内表达乙酰辅酶A羧化酶基因,提高其胞内土霉素合成的前体物丙二酸单酰辅酶A的含量。对出发菌株M4018进行工程改造,同时过表达途径特异性调控蛋白OtcR和乙酰辅酶A羧化酶,发酵检测改造后的重组工程菌株土霉素的产量由1.37g/L提高到9.09g/L,该研究策略对工程改造龟裂链霉菌提高土霉素的产量具有重要的指导意义。     

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c . 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.     

Actinomyces rimosus resistance to oxytetracycline

High frequency of spontaneous and UV-and acridine dye-induced variants susceptible to oxytetracycline (OTC) and deprived of the capacity for synthesizing this antibiotic was observed in strain LST-118 of Actinomyces rimosus. The cells of strain LST-118 of Act. rimosus contained extrachromosomal DNA not found in its OTC susceptible variant BS87, which provides evidence in favour of participation of the extrachromosomal genetic elements in control of OTC resistance of the cells of Act. rimosus, LST-118. The OTC resistance in strain LST-118 is of inducable character. The resistance level is increasing from the beginning of the antibiotic synthesis and initially the subinhibitory concentrations of OTC in the medium were the inductors triggering cellular mechanisms ensuring resistance of the cell to the increasing concentrations of OTC in the medium. The capacity for absorption of OTC in Act rimosus is 2--3 times lower than that in E. coli. The experiments with labeled tetracycline showed that the cells of the actinomycete absorbed OTC when it was present in the medium. The absorption of the main amount of the antibiotic was registered during the first 5 minutes. The difference in absorption of OTC by the cells of the antibiotic resistant and sensitive strains was insignificant.     

Mutagenic action of N-nitrosodimethylurea on Actinomyces rimosus and Penicillium chrysogenum]

High mutagenic activity of N-nitrozodimethylurea (NDMU), an agent of the group of the nitrozo compounds not studied in detail was shown with respect to prototrophic and auxotrophic strains of Actinomyces rimosus, an organism producing oxytetracycline and Penicillium chrysogenum, an organism producing penicillin. The rate of direct and back mutations in the auxotrophic strain of Act. rimosus under the effect of NDMU was many times higher than that of spontaneous mutations. NDMU was used at one of the selection stages at which a more active variant of Act. rimosus was obtained. This is evident of a possible use of the mutagen for induction of variation with respect to the quantitative feature of oxytetracycline production. A great number of morphologically changed forms and biochemical mutants of Pen. chrysogenum formed under the effect of this substance. NDMU induced a mutant of Pen. chrysogenum capable of selective synthesis of 6-aminopenicillinic acid without addition of the precursor.     

Determinant of resistance to kanamycin in Streptomycin rimosus: amplification in the chromosome and reversible genetic instability

The study on the kanamycin resistance determinant (Kanr) in an oxytetracycline--producing strain of S. rimosus showed that it was capable of amplifying in the chromosome during selection for increasing the antibiotic resistance level. The amplification of the DNA fragment with a molecular weight of 10.3 MDa containing Kanr amounted to 300 copies per genome, which resulted in a more than 1000-fold increase in kanamycin resistance level. Cloning of the Kanr determinant on plasmid SLP1.2 in S. lividans strain 66 was performed. In Streptomyces lividans strain 66 the Kanr determinant preserved the capacity for amplification in the hybrid plasmid pSU10 integrated into the chromosome. The Kanr determinant in the strains of S. rimosus and S. lividans was characterized by transfers Kanr in equilibrium Kans with a frequency of 1 X 10(-3). It was shown that the mutation in S. lividans strain 66 resulting in phenotype Kans was not connected with the structural Kanr gene on plasmid pSU10 but was localized on the chromosome. Phenotype Kans was promoted by a decrease in the number of the copies of the regulatory genetic element designated RES1. The reverse to phenotype Kanr might be due to one of the following events: amplification to the initial level of RES1 and amplification up to 200 copies per the genome of the hybrid plasmid pSU10 containing the Kanr determinant. Amplification of the Kanr determinant with preserved initial level of RES1 element resulted in a more than 1000 times increase in the resistance level.     

Genetic interactions in Streptomyces rimosus mediated by conjugation and by protoplast fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.     

Effect of the aeration conditions on the biosynthesis of oxytetracycline and the formation of organic acids by a culture of Actinomyces rimosus]

The effect of the aeration conditions on oxytetracycline biosynthesis and production of organic acids by Act. rimosus was studied. Intensive biosynthesis of oxytetracycline in shaken flasks with concentrated complex media was observed at the rate of oxygen dissolution in the liquid ranging from 14 to 25 mg/1/min. Lower rates of the oxygen dissolution up to 7 mg/1/min resulted in decreased rates of the culture growth and the medium component consumption, decreased antibiotic levels, production of significant amounts of pyruvic and acetic acids.     

Effect of liquid culture requirements on antifungal antibiotic production by Streptomyces rimosus MY02

Streptomyces rimosus MY02 was isolated from a soil sample which was collected from the northeast of China. The effect of medium components (i.e. carbon and nitrogen sources) and other culture requirements (i.e. initial pH and temperature) on production of antifungal antibiotics by S. rimosus MY02 was investigated in our work. The best conditions for the strain MY02 in 250-ml Erlenmeyer flask, for example, initial pH, temperature, medium capacity, agitation rate, seed age, inoculum size and growth period, were 6.0, 28 degrees C, 50 ml, 180 rpm, 4 days, 10% (v/v) and 120 h, respectively. Components and dosage of the medium, which effect antibiotic production, were determined by uniform design combined with regression analysis; meanwhile, a regression model was established. The components and dosage of the best medium were starch, 53.313 g; defatted peanut powder, 9.376 g; (NH(4))(2)SO(4), 6.244 g; and NaCl, 5.836 g; in 1l of distilled water. Residual values obtained between the observed values by experiments and predicted values by the model are very low, and this result showed that the experimental results were well in consistence with the calculation results via the model. The antifungal antibiotic production by S. rimosus MY02 was improved by optimization of the components and culture requirements. The diameter of inhibition zone of the culture supernatant from S. rimosus MY02 against Fusarium oxysporium f sp. cucumarinum was 33.19 mm.