Common biosynthetic feature of fortimicin-group antibiotics

The fortimicin (FTM) group antibiotics are produced by actinomycetes belonging to various genera. FTM's and sannamycins are the products of Micromonospora olivasterospora and Streptomyces sannanensis, respectively. The possible presence of common features of the biosynthesis of these structurally related antibiotics in the two producers was examined. Enzymatic studies were done by the bioconversion experiments using FTM precursors and the washed cells of S. sannanensis. Ion pair/reverse-phase HPLC was employed as the analytical tool. S. sannanensis could convert almost all FTM precursors to the expected structures in the down stream of the pathway except one step. Thus the sets of the biosynthetic enzymes of the two bacteria share the common substrate specificity and react alternatively.     

Cloning of midecamycin(MLS)-resistance genes from Streptomyces mycarofaciens, Streptomyces lividans and Streptomyces coelicolor A3(2)

DNA containing genes for midecamycin(Mdm)-resistance was cloned from Streptomyces mycarofaciens ATCC 21454 (mdmA gene), Streptomyces lividans 66 (lrm gene) and Streptomyces coelicolor A3(2). The phenotype imparted to S. lividans and Streptomyces griseofuscus transformants by the cloned DNA segments indicates that they encode an MLS-type of resistance activity. The mdmA and lrm genes could be distinguished by the phenotype they conferred in S. lividans and S. griseofuscus, whereas the S. lividans lrm and S. coelicolor MLS genes appears to be identical on the basis of their restriction maps and behavior in S. lividans and S. griseofuscus. The DNA sequence of a 1.4-kb BamH I DNA fragment containing the mdmA gene indicates the presence of one complete orf whose deduced product exhibits a high similarity to the deduced product of the Streptomyces thermotolerans carB gene and several other bacterial MLS-resistance genes.     

Lobophorins E and F, new spirotetronate antibiotics from a South China Sea-derived Streptomyces sp. SCSIO 01127

The strain SCSIO 01127, isolated from the South China Sea sediment, was identified as a member of Streptomyces by the 16S rDNA sequence analysis. Two new spirotetronate antibiotics lobophorins E (1) and F (2), along with two known analogs lobophorins A (3) and B (4), were isolated from Streptomyces sp. SCSIO 01127. Their structures were elucidated on the basis of detailed IR, NMR and MS spectroscopic analyses. The new compound lobophorin F (2) showed antibacterial activities against Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 with MIC values of 8?μg?ml(-1) for both the strains, better than that of lobophorin B (4). Lobophorin F (2) also displayed better cytotoxic activities than lobophorin B (4), with IC(50) of 6.82, 2.93 and 3.16?μM against SF-268, MCF-7 and NCI-H460, respectively.     

Cloning and characterization of the sisomicin-resistance gene from Micromonospora inyoensis

We cloned DNA fragments of sisomicin-producing Micromonospora inyoensis into Streptomyces plasmid vectors and identified Streptomyces lividans TK24 transformants expressing the M. inyoensis sisomicin-resistance (sisA) gene. The sisA gene was compared to the previously reported Micromonospora purpurea Kan-Gen (kanamycin-gentamicin)-resistance gene. While the restriction endonuclease digestion patterns of the two determinants appear to be divergent, the genes are nonetheless closely related, based on similar patterns and levels of aminoglycoside-resistance and their ability to cross-hybridize under stringent conditions. We have transformed recombinant plasmid pMD5-2, which carries the sisA gene, into our M. purpurea gentamicin-production strain and determined that gentamicin biosynthesis was not improved.     

Antifungal anthracycline antibiotics, spartanamicins A and B from Micromonospora spp.

Spartanamicins A and B, two antifungal antibiotics, were produced by a culture of Micromonospora spp. strain No. MSU-43097 (ATCC 53803), isolated from a potted soil containing asparagus (Asparagus officinalis L.) plants. The antibiotics were isolated from the mycelial cake using organic solvents. The structures of spartanamicins A and B were determined by spectral and chemical means. Spartanamicin B is more active as an antifungal compound than it's analogue, A. The minimum inhibitory concentration for spartanamicin B on Candida albicans and Aspergillus, Cladosporium, Cryptococcus, Rhodotorula and Staphylococcus spp. ranged from 0.2 to 1 microgram/ml. It was not active against Staphylococcus aureus, Escherichia coli and Citrobacter spp. but some strains of S. aureus were sensitive.     

The leptomycin gene cluster and its heterologous expression in Streptomyces lividans

Leptomycin exerts its antifungal and anti-tumoral activity via inhibiting nucleo-cytoplasmic translocations in eukaryotic cells. To learn more about the biosynthesis of leptomycin and in an effort to generate leptomycin analogues through genetic engineering, 90 kb segment of DNA containing the putative leptomycin (lep) biosynthesis cluster from Streptomyces sp. ATCC 39366 was cloned and sequenced. The lep cluster consist of 12 polyketide synthase (PKS) modules distributed in four genes (lepA, B, C and D) and a P450 encoding gene. The lep gene cluster was confirmed by its successful expression in Streptomyces lividans, where it directed the production of the two natural congeners-leptomycins A and B. The production of leptomycin B showed that the host has the capability to synthesize ethylmalonyl-CoA.     

Cloning from Streptomyces cellulosae of the gene encoding beta-lactamase, a blue-dextran binding protein.

A beta-lactamase gene was cloned from Streptomyces cellulosae as a 2.3-kb DNA fragment using Streptomyces lividans 1326 and PIJ385 as a host-vector system. During the course of cloning, a part of the chromosomal DNA fragment cloned together with a part of the vector plasmid were deleted, indicating instability of this contiguous DNA region. The enzyme from the clone showed similar properties with respect to binding of blue dextran and isoelectric point to the enzyme from S. cellulosae. The cloned gene hybridized not only to DNA of S. cellulosae, the source of DNA, but also to DNAs of several Streptomyces species, irrespective of their formation of beta-lactamase. These results suggest that this gene may have homology to genes other than the one for beta-lactamase.     

Antimicrobial Screening of Actinobacteria using a Modified Cross-Streak Method

Out of the 30 actinobacterial cultures screened for antimicrobial activity, 28 cultures were found to produce active products against various pathogenic microorganisms such as Gram-negative, Gram-positive bacteria and yeast, using a modified cross streak method. The modified method helped in easy quantification of results and also in ruling out probable mutual antibiosis. The actinobacterial strains that showed the ability to produce antimicrobial compounds belonged to Streptomyces (53%), Micromonospora (13%) and Actinomadura (10%) genera. Streptomyces sp. strain MMA-5 showed the highest multispecific antibiosis efficiency score value. Broad antibiotic spectrum activity was exhibited by Streptomyces sp. strain MMA-2 and Micromonospora sp. strain MMA-8. The multidrug resistant human pathogenic yeast strain Candida albicans was inhibited by 18 actinobacterial strains.     

Cloning of antibiotic-resistance genes in Streptomyces

Antibiotic-resistance genes were shotgun cloned from antibiotic-producing Streptomyces sp. using pock-forming plasmids (pSF689 and pSF765), as cloning vectors. Streptomyces chartreusis SF1623 and S. lividans 66 were used as host strains. The ribostamycin (RSM) resistance gene was cloned from S. ribosidificus SF733 DNA (on a 2.3 Md PstI fragment) into both S. chartreusis SF1623 and S. lividans 66, using pSF689 as vector. Kanamycin (KM), novobiocin (NB), destomycin (DM) and racemomycin (RM) resistance genes were cloned from S. kanamyceticus M1164, S. spheroides M1469, S. rimofaciens M1470 and S. lavendulae A249 genomic DNA into S. lividans 66, using pSF765 as vector. Furthermore two types of KM resistance determinants derived from S. kanamyceticus M1164 were cloned using S. lividans 66, the pSF689 vector. The RSM resistance gene showed no homology to plasmid pSF733 of S. ribosidificus SF733, but hybridized to PstI or BclI digested total DNA of S. ribosidificus SF733.     

小单孢菌FIM98-160产生的抗分枝杆菌抗生素labilomycin

在筛选抗结核杆菌药物的过程中 ,从福州水库底土样品中分离到小单孢菌FIM98 1 60 ,对该菌株作分类鉴定 ,抗菌物质的分离纯化 ,所得化合物含有三个组分 ,其中A组分经理化性质、光谱特征和生物学活性研究 ,证明它与链霉菌产生的labilomycin同质。它是首次从小单孢菌代谢产物中分离得到。     

海洋放线菌NRPS基因的筛选

目的 建立快速、高效的NRPS基因筛选体系,从海洋微生物中筛选具有潜在合成多肽类次级代谢产物的活性菌株,为定向获得新的含氨基酸抗生素奠定基础.方法 采用选择性培养分离获得海洋放线菌,通过快速抗菌活性初筛,设计特异性引物利用PCR技术对初筛活性菌株进行NRPS基因筛选,进一步对阳性菌株进行16S rDNA排重.结果 利用建立的NRPS基因筛选体系对初筛具有抗菌活性的295株放线菌进行筛选,得到12株阳性菌株,分别属于小单孢菌属、链霉菌属和疣孢菌属.结论 利用PCR技术建立了快速、高效的NRPS基因筛选体系,为多肽类活性化合物的获得提供了基因学的依据.     

Strain- and species-specific distribution of the streptomycin gene cluster and kan-related sequences in Streptomyces griseus

The streptomycin (SM) gene cluster was investigated for its distribution in streptomycetes by Southern hybridization using nick-translated DNA probes, which were isolated from the SM-6-phosphotransferase (SPH) and amidinotransferase (ADT) regions of the SM gene cluster of Streptomyces griseus SS-1198. Bgl II-digested genomic DNAs from SM-producing strains of S. griseus yielded the same size fragment (7.0 kb) which hybridized to both the SPH and ADT probes as expected from the restriction endonuclease cleavage map of the SM gene cluster. By contrast, no genomic DNA fragments from heterologous Streptomyces strains hybridized to the probes. Thus, only SM-producing strains of S. griseus possess the highly homologous SM gene cluster. Similarly, distribution of DNA sequences homologous to the kanamycin (KM)-resistance determinant (kan) from a KM-resistant regenerant of S. griseus SS-1198 protoplasts was also examined. Using the kan gene fragment as the probe it was revealed that the kan-related sequences are present in all the strains of S. griseus tested, irrespective of the type of antibiotics they produce. However, no hybridization to the kan gene probe (KAN) was observed with DNA digests derived from other Streptomyces species.     

Biological conversion of erythronolide B, an intermediate of erythromycin biogenesis, into new "hybrid" macrolide antibiotics

Transformation of erythronolide B to new antibiotics was attempted by feeding this compound during the fermentation of Streptomyces antibioticus ATCC31771, a blocked mutant of an oleandomycin producing strain. As a result, four new active compounds were obtained with hybrid structures between erythromycin and oleandomycin. They were identified as 3-O-oleandrosyl-5-O-desosaminyl-15-hydroxyerythronolide B (I), 3-O-oleandrosyl-5-O-desosaminylerythronolide B (II), 3-O-oleandrosyl-5-O-desosaminyl-(8S)-8-hydroxyerythronolide B (III) and 3-O-oleandrosyl-5-O-desosaminyl-(8R)-8,19-epoxyerythronolide B (IV). They were found to be less active, but more stable to acid, than erythromycin A. From their relative biogenetical relationship together with the structure elucidated some hypotheses about late stages of oleandomycin biosynthesis are inferred too.     

广西茅尾海红树林根围淤泥放线菌多样性及抗菌活性

目的 对广西茅尾海红树林保护区植物根部淤泥中的放线菌进行分离鉴别,研究放线菌的多样性和抗菌活性。方法 选用10种分离培养基,经稀释涂布法分离放线菌;通过PCR扩增,测序后获得菌株16S rRNA基因序列;邻接法构建系统发育树并进行同源性分析,探测放线菌的多样性;针对发酵液经乙酸乙酯萃取后的有机相、水相及菌丝丙酮浸泡液共3类样品,采用纸片扩散法开展抗菌活性研究。结果 从8份红树林植物根围淤泥样品中分离纯化得到244株放线菌,分布于5个目13个科29个属,优势菌属为链霉菌属和小单孢菌属;16S rRNA基因序列相似性低于98.65%的放线菌共有4株,可能为潜在新物种;抗菌活性筛选结果表明,83株放线菌的抗菌活性阳性率达71.1%。结论 广西茅尾海红树林保护区植物根围淤泥中抗菌活性放线菌的多样性丰富。     

O-demethylpaulomycins A and B, U-77,802 and U-77,803, paulomenols A and B, new metabolites produced by Streptomyces paulus

O-Demethylpaulomycin A (C33H44N2O17S), O-demethylpaulomycin B (C32H42N2O17S), paulomenol A (C29H43NO16), paulomenol B (C28H41NO16), and the hydrogen sulfide adducts of paulomycin A (U-77,802, C34H48N2O17S2), and paulomycin B (U-77,803, C33H46N2O17S2) have been isolated from fermentations of Streptomyces paulus strain 273. The structure of these compounds was determined by 1H and 13C NMR and fast atom bombardment mass spectrum spectroscopic techniques and degradative studies. The antibacterial properties of these new metabolites, which are related to paulomycins A and B (J. Antibiotics 35: 285-294, 1982), are briefly discussed.     

Increased production of aminoglycosides associated with amplified antibiotic resistance genes

The 6'-N-acetyltransferase derived from Streptomyces kanamyceticus strain M1164 was cloned on to the high copy plasmid vector pIJ702 and introduced into S. kanamyceticus (ATCC 12853, a kanamycin producer) and S. fradiae (ATCC 10745, a neomycin producer). In both cases transformants containing the recombinant plasmid showed increased resistance to a number of aminoglycoside antibiotics and substantially increased production of kanamycin and neomycin. This demonstrates that specific amplification of gene products associated with antibiotic biosynthesis provides a means for improving antibiotic production.     

Discovery, isolation and structure of novel cephamycins of Streptomyces chartreusis

By the use of HPLC technique after treatment with beta-lactamases, two novel cephamycins, SF-1623 and SF-1623B, were discovered and isolated from the fermentation broth of Streptomyces chartreusis SF-1623. The structures of SF-1623 and SF-1623B were determined to contain 3-sulfothiomethyl and 3-hydroxymethyl groups respectively, by chemical and enzymatic transformation reactions. Studies on the fermentation condition and process for the large scale preparation of antibiotic SF-1623 are also described.     

NMR analysis of quinocycline antibiotics: structure determination of kosinostatin,an antitumor substance from Micromonospora sp. TP-A0468

A quinocycline antibiotic, kosinostatin, was isolated from the culture broth of Micromonospora sp. TP-A0468 along with isoquinocycline B. Structure of kosinostatin was determined to be the stereoisomer of isoquinocycline B regarding to the stereochemistry at the C-2' spiro carbon by NMR analysis. Kosinostatin isomerizes to isoquinocycline B through the inversion of the stereocenter at C-2'. Comparison of physico-chemical properties indicated that kosinostatin is presumably identical with quinocycline B isolated by CELMER et al. from Streptomyces aureofaciens.     

新来源棘白霉素B脱酰基酶产生菌的筛选和转化条件优化

目的筛选放线菌来源的棘白霉素B脱酰基酶产生菌,并对其转化条件进行优化。方法根据菌株转化产物对白念珠菌的抑制活性进行初筛,再以HPLC、LC-MS检测确定目的菌株;对目的菌株转化棘白霉素B的培养基、转化温度和时间、缓冲液、甲醇含量等条件进行优化以提高转化率。结果筛选得到4株可产生脱酰基酶的阳性菌株,其中,N07W-.61为小单孢菌属菌株,其余3株为链霉菌属菌株;通过转化条件优化,菌株N07W-61对棘白霉素B的转化率从2.60%提高至61.24%。结论小单孢菌属菌株产生棘白霉素B脱酰基酶为首次报道;该属菌株N07W-61在优化转化条件下对棘白霉素B的转化率有了大幅提高。     

Molecular cloning of a D-cycloserine resistance gene from D-cycloserine-producing Streptomyces garyphalus

A 3.5-kb DNA fragment that confers resistance to D-cycloserine (DCS) was cloned from the chromosomal DNA of a DCS-producing Streptomyces garyphalus into Streptomyces lividans by a shot-gun cloning technique. Nucleotide sequence analysis revealed the existence of four open reading frames (ORFs B, C, D, and E), together with two incomplete ORFs, A and F. By introduction of the cloned fragment into Escherichia coli, the host obtained resistance to DCS. We showed that ORF B, which consists of 903 bp, is a DCS resistance gene. The hydropathy plot analysis of a protein deduced from ORF B revealed that the protein carries membrane-integral domains spanning the membrane 10 times, which suggests that the DCS-resistance determinant may be a factor associated with DCS transport.