Kegg Pathway: Aminoglycosides - Streptomyces

Mini Rev Med Chem. 2009 Aug; 9(9): 1040-51
Ostash B, Korynevska A, Stoika R, Fedorenko V

This review covers existing literature (from 1990 to 2008) on landomycins (LS), a family of glycosylated angucyclines, with an emphasis on the bioactivity scope of landomycin (La)-like structures accessible via biocombinatorial manipulations. Some LS display strong antitumor activity and have inspired several chemical studies focused mainly on their unusual deoxysugar chains. A decade of genetic studies on La-producing bacteria has provided many novel molecules with altered structure and activity. A complex nonlinear correlation between the length of the carbohydrate tail of LS and their antitumor activity has also been revealed. It implies that simpler LS than the largest member of the family, LaA, are still potential drug leads. Combinatorial biosynthesis appears to be a powerful tool to search the chemical space around the La scaffold.

Angew Chem Int Ed Engl. 2009; 48(35): 6507-10
Donohoe TJ, Flores A, Bataille CJ, Churruca F

J Biosci Bioeng. 2009 Aug; 108(2): 92-8
Malla S, Niraula NP, Liou K, Sohng JK

To enhance doxorubicin (DXR) production, the structural sugar biosynthesis genes desIII and desIV from Streptomyces venezuelae ATCC 15439 and the glycosyltransferase pair dnrS/dnrQ from Streptomyces peucetius ATCC 27952 were cloned into the expression vector pIBR25, which contains a strong ermE promoter. The recombinant plasmids pDnrS25 and pDnrQS25 were constructed for overexpression of dnrS and the dnrS/dnrQ pair, whereas pDesSD25 and pDesQS25 were constructed to express desIII/desIV and dnrS/dnrQ-desIII/desIV, respectively. All of these recombinant plasmids were introduced into S. peucetius ATCC 27952. The recombinant strains produced more DXR than the S. peucetius parental strain: a 1.2-fold increase with pDnrS25, a 2.8-fold increase with pDnrQS25, a 2.6-fold increase with pDesSD25, and a 5.6-fold increase with pDesQS25. This study showed that DXR production was significantly enhanced by overexpression of potential biosynthetic sugar genes and glycosyltransferase.

Electrophoresis. 2009 Jul; 30(14): 2454-9
Liang J, Niu Q, Xu X, Luo Y, Zhou X, Deng Z, Wang Z

Nucleic acids remaining within bacterial protein samples from Streptomyces lividans and Escherichia coli were found to interfere significantly with blue native polyacrylamide gel electrophoresis (BN-PAGE), a technique used frequently for analyzing bacterial protein complexes in proteomics studies. We have used ultracentrifugation and/or precipitation of cell lysates with streptomycin sulfate to eliminate nucleic acids from total and/or membrane protein samples. Nucleic acid-binding proteins were first enriched by precipitation with streptomycin sulfate, and contaminating nucleic acids were then eliminated by precipitation by adding polyethyleneimine. The performance of BN-PAGE was found to be dramatically improved by these sample preparation steps.

J Ind Microbiol Biotechnol. 2009 Oct; 36(10): 1257-65
Singh B, Oh TJ, Sohng JK

Thorough investigation of Streptomyces peucetius ATCC 27952 genome revealed a sesquiterpene synthase, named spterp13, which encodes a putative protein of 732 amino acids with significant similarity to S. avermitilis MA-4680 (SAV2163, GeoA) and S. coelicolor A3(2) (SCO6073). The proteins encoded by SAV2163 and SCO6073 produce geosmin in the respective strains. However, the spterp13 gene seemed to be silent in S. peucetius. Deletion of the doxorubicin gene cluster from S. peucetius resulted in increased cell growth rate along with detectable production of geosmin. When we over expressed the spterp13 gene in S. peucetius DM07 under the control of an ermE* promoter, 2.4 +/- 0.4-fold enhanced production of geosmin was observed.

Protein Expr Purif. 2009 Oct; 67(2): 132-8
Ajith VK, Prasad R

An antitumour chemotherapeutic, daunorubicin (DNR), produced by Streptomyces peucetius exhibits cytotoxic activity through topoisomerase-mediated interaction with DNA, thereby inhibiting DNA replication and repair and RNA and protein synthesis. It is synthesized by the type II polyketide pathway. Understanding molecular mechanisms that drive expression of antibiotic biosynthetic genes in response to diverse signals and chemical inducers is of considerable interest. Intergenic DNA between regulatory genes dnrN and dnrO of DNR biosynthesis pathway in S. peucetius has a promoter for transcription of dnrN in one strand and three promoters in the opposite strand for dnrO. Studies have shown that DnrO binds to a specific sequence in this region to activate transcription of dnrN. In the present study, using biotinylated intergenic DNA in combination with streptavidin magnetic beads, we have purified a protein that binds to this target sequence. The protein has been characterized by nano LC ESI MS/MS mass spectrometry. Sequence similarity searches for effective identification of protein by genome databases comparisons led to identification of a sequence-specific DNA binding protein that exhibits dihydrolipoamide dehydrogenase (DLDH) activity suggesting that this protein may be involved in regulation of DNR biosynthesis.

Eur J Mass Spectrom (Chichester, Eng). 2009; 15(2): 73-81
Kelso C, Rojas JD, Furlan RL, Padilla G, Beck JL

Cultures of cosmomycin D-producing Streptomyces olindensis ICB20 that were propagated for many generations underwent mutations that resulted in production of a range of related anthracyclines by the bacteria. The anthracyclines that retained the two trisaccharide chains of the parent compound were separated by HPLC. Exact mass determination of these compounds revealed that they differed from cosmomycin D (CosD) in that they contained one to three fewer oxygen atoms (loss of hydroxyl groups). Some of the anthracyclines that were separated by HPLC had the same mass. The location from which the hydroxyl groups had been lost relative to CosD (on the aglycone and/or on the sugar residues) was probed by collisionally-activated dissociation using an electrospray ionisation linear quadrupole ion trap mass spectrometer. The presence of anthracyclines with the same mass, but different structure, was confirmed using an electrospray ionisation travelling wave ion mobility mass spectrometer.

Antimicrob Agents Chemother. 2009 Jul; 53(7): 3173-7
Rezzonico F, Stockwell VO, Duffy B

Streptomycin is used in plant agriculture for bacterial disease control, particularly against fire blight in pome fruit orchards. Concerns that this may increase environmental antibiotic resistance have led to bans or restrictions on use. Experience with antibiotic use in animal feeds raises the possible influence of formulation-delivered resistance genes. We demonstrate that agricultural streptomycin formulations do not carry producer organism resistance genes. By using an optimized extraction procedure, Streptomyces 16S rRNA genes and the streptomycin resistance gene strA were not detected in agricultural streptomycin formulations. This diminishes the likelihood for one potential factor in resistance development due to streptomycin use.

Microbiology. 2009 Jul; 155(Pt 7): 2197-210
Hara H, Ohnishi Y, Horinouchi S

A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) is a microbial hormone that triggers morphological differentiation and secondary metabolism in Streptomyces griseus. The effects of A-factor on global gene expression were determined by DNA microarray analysis of transcriptomes obtained with the A-factor-deficient mutant DeltaafsA. A-factor was added at a concentration of 25 ng ml(-1) to mutant DeltaafsA at the middle of the exponential growth phase, and RNA samples were prepared from the cells grown after A-factor addition for a further 5, 15 and 30 min, and 1, 2, 4, 8 and 12 h. The effects of A-factor on transcription of all protein-coding genes of S. griseus were evaluated by comparison of the transcriptomes with those obtained from cells grown in the absence of A-factor. Analysis of variance among the transcriptomes revealed that 477 genes, which were dispersed throughout the chromosome, were differentially expressed during the 12 h after addition of A-factor, when evaluated by specific criteria. Quality threshold clustering analysis with regard to putative polycistronic transcriptional units and levels of upregulation predicted that 152 genes belonging to 74 transcriptional units were probable A-factor-inducible genes. Competitive electrophoretic mobility shift assays using DNA fragments including putative promoter regions of these 74 transcriptional units suggested that AdpA bound 37 regions to activate 72 genes in total. Many of these A-factor-inducible genes encoded proteins of unknown function, suggesting that the A-factor regulatory cascade of S. griseus affects gene expression at a specific time point more profoundly than expected.

Methods Enzymol. 2009; 458: 143-57
Hsiao NH, Gottelt M, Takano E

Antibiotic production is regulated by numerous signals, including the so-called bacterial hormones found in antibiotic producing organisms such as Streptomyces. These signals, the gamma-butyrolactones, are produced in very small quantities, which has hindered their structural elucidation and made it difficult to assess whether they are being produced. In this chapter, we describe a rapid small-scale extraction method from either solid or liquid cultures in scales of one plate or 50 ml of medium. Also described is a bioassay to detect the gamma-butyrolactones by determining either the production of pigmented antibiotic of Streptomyces coelicolor or kanamycin resistant growth on addition of the gamma-butyrolactones. We also describe some insights into the identification of the gamma-butyrolactone receptor and its targets and also the gel retardation conditions with three differently labeled probes.

Methods Enzymol. 2009; 459: 459-91
Wehmeier UF, Piepersberg W

The classical Aminoglycosides are, with very few exceptions, typically actinobacterial secondary metabolites with antimicrobial activities all mediated by inhibiting translation on the 30S subunit of the bacterial ribosome. Some chemically related natural products inhibit glucosidases by mimicking oligo-alpha-1,4-glucosides. The biochemistry of the aminoglycoside biosynthetic pathways is still a developing field since none of the pathways has been analyzed to completeness as yet. In this chapter we treat the enzymology of aminoglycoside biosyntheses as far as it becomes apparent from recent investigations based on the availability of DNA sequence data of biosynthetic gene clusters for all major structural classes of these bacterial metabolites. We give a more general overview of the field, including descriptions of some key enzymes in various aminoglycoside pathways, whereas in Chapter 20 provides a detailed account of the better-studied enzymology thus far known for the neomycin and butirosin pathways.

Appl Microbiol Biotechnol. 2009 Jul; 83(6): 1067-76
Erb A, Krauth C, Luzhetskyy A, Bechthold A

A lanGT4 mutant of the landomycin A producer Streptomyces cyanogenus S136 was constructed, leading to the production of landomycin D with two deoxy sugars in the side chain and proving that LanGT4 is responsible for attaching the third deoxy sugar of the hexasaccharide side chain. Heterologous expression of lndGT4 of the landomycin E producer Streptomyces globisporus 1912 in the lanGT4 mutant restored landomycin A production, indicating that LndGT4, like LanGT4, also has the ability to work iteratively. A S. cyanogenus S136 mutant with a mutation in lanGT1, encoding a D: -olivosyltransferase, was shown to produce landomycin I with one deoxy sugar and, surprisingly, a new landomycin derivative (landomycin L) containing a D: -olivose followed by an L: -rhodinose. Heterologous expression of lndGT1 of S. globisporus 1912 in the lanGT1 mutant did not restore landomycin A production but led to the formation of a second new landomycin derivative (landomycin K) containing an unusual pentasaccharide chain (D: -olivose-D: -olivose-L: -rhodinose-D: -olivose-L: -rhodinose). The formation of landomycin L and landomycin K is most probably attributed to the high substrate flexibility of the rhodinosyltransferase LanGT4.

J Antibiot (Tokyo). 2009 May; 62(5): 233-7
Ji Z, Wang M, Wei S, Zhang J, Wu W

Five streptothricin acids (compounds 1-5) were isolated by ion-exchange resin chromatography and preparative RP-HPLC from the fermentation broth of Streptomyces qinlingensis. Their structures were elucidated mainly by analyses of the IR, HR-EIS-MS and NMR spectral data. They were deduced as two known compounds, streptothricin F acid (1) and streptothricin D acid (2), and three new compounds, 12-carbamoylstreptothricin E acid (3), 12-carbamoylstreptothricin D acid (4) and N-acetyl-streptothricin D acid (5), respectively. The antibacterial activities of 1-5 against Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Bacillus cereus and Pseudomonas aeruginosa were assayed by micro-broth dilution. Comparison of the MICs with streptothricin F and D showed that the antimicrobial activities of 1-5 were decreased significantly but varied with the structures.

Chembiochem. 2009 Mar 23; 10(5): 813-9
He QL, Jia XY, Tang MC, Tian ZH, Tang GL, Liu W

Acta Crystallogr Sect F Struct Biol Cryst Commun. 2009 Mar 1; 65(Pt 3): 256-9
Koskiniemi H, Grocholski T, Schneider G, Niemi J

12-deoxy-nogalonic acid oxygenase (SnoaB) catalyzes the oxygenation of 12-deoxy-nogalonic acid at position 12 to yield nogalonic acid, which is one of the steps in the biosynthesis of the polyketide nogalamycin in Streptomyces nogalater. SnoaB belongs to a family of small cofactor-free oxygenases which carry out oxygenation reactions without the aid of any prosthetic group, cofactor or metal ion. Recombinant SnoaB was crystallized in space group P2(1)2(1)2, with unit-cell parameters a = 58.8, b = 114.1, c = 49.5 A, and these crystals diffracted to 2.4 A resolution. Recombinant SnoaB does not contain any methionine residues and three double mutants were designed and produced for the preparation of selenomethionine-substituted samples. The selenomethionine-substituted mutant F40M/L89M crystallized in the same space group as the native enzyme.

Antibiot Khimioter. 2008; 53(7-8): 3-7
Terekhova LP, Galatenko OA, Trenin AS, Tolstykh IV, Zenkova VA, Zhukhlistova NE, Ol'khovatova OL, Malkina ND, Boĭkova IuV, Ustinova EV, Katrukha GS

Taxonomic properties of strain INA-1132 producing antibiotic INA-1132 are described. The antibiotic showed activity against grampositive bacteria and fungi. The strain was classified as belonging to the genus Streptomyces and by its taxomic characteristics is most close to S. baarnensis. The experiments with the bacterial culture Halobacterium salinarum (previously H. halobium) revealed hypolipidemic activity of the antibiotic, i. e. its ability to inhibit biosynthesis of sterols. Conditions for the production of the antibiotic, methods of its isolation and purification, as well as the results of the chemical structure elucidation are described. By its physicochemical properties the antibiotic is identical to chlorothricin. The structure of antibiotic INA-1132 was ascertained by X-ray analysis. Conformation of the molecule of chlorothricin (antibiotic 1132) was determined for the first time.

Rapid Commun Mass Spectrom. 2009 Mar; 23(6): 907-14
Kawano S

Impurities in streptomycin (STR) and dihydrostreptomycin (DHS) were investigated by hydrophilic interaction chromatography/electrospray ionization quadrupole ion trap/time-of-flight mass spectrometry (HILIC/ESI-QIT/TOFMS). Samples were separated on a fused-core silica column (100 mmx2.1 mm i.d., particle size: 2.7 microm) with isocratic elution using 200 mM ammonium formate buffer (pH 4.5) and acetonitrile as mobile phase. Constant neutral loss survey in accurate mass measurement was carried out by QIT/TOFMS. Formulae, chemical structures of impurities in an STR sample were suggested with supporting results on the probable pathways of STR biosynthesis by Streptomyces griseus.

Biotechnol Lett. 2009 Jun; 31(6): 869-75
Jnawali HN, Subba B, Liou K, Sohng JK

A putative aminotransferase gene, kanB, lies in the biosynthetic gene cluster of Streptomyces kanamyceticus ATCC 12853 and has 66% identity with neo6 in neomycin biosynthesis. Streptomyces fradiae Deltaneo6::tsr was generated by disrupting neo6 in the neomycin producer Streptomyces fradiae. Neomycin production was completely abolished in the disruptant mutant but was restored through self-complementation of neo6. S. fradiae HN4 was generated through complementation with kanB in Streptomyces fradiae neo6::tsr. Based on metabolite analysis by ESI/MS and LC/MS, neomycin production was restored in Streptomyces fradiae HN4. Thus, like neo6, kanB also functions as a 2-deoxy-scyllo-inosose aminotransferase that has dual functions in the formation of 2-deoxy-scyllo-inosose (DOS).

Appl Microbiol Biotechnol. 2009 May; 83(2): 305-13
Zhao GR, Luo T, Zhou YJ, Jiang X, Qiao B, Yu FM, Yuan YJ

Streptolydigin, a secondary metabolite produced by Streptomyces lydicus, is a potent inhibitor of bacterial RNA polymerases. It has been suggested that streptolydigin biosynthesis is associated with polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS). Thus, there is great interest in understanding the role of fatty acid biosynthesis in the biosynthesis of streptolydigin. In this paper, we cloned a type II fatty acid synthase (FAS II) gene cluster of fabDHCF from the genome of S. lydicus and constructed the SlyfabCF-disrupted mutant. Sequence analysis showed that SlyfabDHCF is 3.7 kb in length and encodes four separated proteins with conserved motifs and active residues, as shown in the FAS II of other bacteria. The SlyfabCF disruption inhibited streptolydigin biosynthesis and retarded mycelial growth, which were likely caused by the inhibition of fatty acid synthesis. Streptolydigin was not detected in the culture of the mutant strain by liquid chromatography-mass spectrometry. Meanwhile, the streptolol moiety of streptolydigin accumulated in cultures. As encoded by fabCF, acyl carrier protein (ACP) and beta-ketoacyl-ACP synthase II are required for streptolydigin biosynthesis and likely involved in the step between PKS and NRPS. Our results provide the first genetic and metabolic evidence that SlyfabCF is shared by fatty acid synthesis and antibiotic streptolydigin synthesis.

Mol Cells. 2009 Jan; 27(1): 83-8
Nepal KK, Oh TJ, Subba B, Yoo JC, Sohng JK

Amino acid homology analysis predicted that rbmD, a putative glycosyltransferase from Streptomyces ribosidificus ATCC 21294, has the highest homology with neoD in neomycin biosynthesis. S. fradiae BS1, in which the production of neomycin was abolished, was generated by disruption of the neoD gene in the neomycin producer S. fradiae. The restoration of neomycin by self complementation suggested that there was no polar effect in the mutant. In addition, S. fradiae BS6 was created with complementation by rbmD in S. fradiae BS1, and secondary metabolite analysis by ESI/MS, LC/MS and MS/MS showed the restoration of neomycin production in S. fradiae BS6. These gene inactivation and complementation studies suggested that, like neoD, rbmD functions as a 2-N-acetlyglucosaminyltransferase and demonstrated the potential for the generation of novel aminoglycoside antibiotics using glycosyltransferases in vivo.