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Identification of novel tylosin analogues generated by a wblA disruption mutant of Streptomyces ansochromogenes.

Lu C, Liao G, Zhang J, Tan H - Microb. Cell Fact. (2015)

Bottom Line: One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool.Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA.Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Lucheng522@aliyun.com.

ABSTRACT

Background: Streptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces.

Results: The wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae.

Conclusions: Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.

No MeSH data available.


Related in: MedlinePlus

Effects of wblA disruption on nikkomycin production. a The bioassay of nikkomycin against Alternaria longipes. b The bioassay of nikkomycin against Candida albicans. c HPLC analysis of nikkomycin. d Transcription analysis of genes related to nikkomycin biosynthesis by qRT-PCR; the transcript of hrdB was used as an internal control. (I): ΔwblA, (II): S. ansochromogenes 7100, (III): complemented strain by integrating a copy of wblA into the chromosome of ΔwblA, (IV): the control strain by integrating pSET152 vector into the chromosome of ΔwblA. Arrows indicate the peak of nikkomycin on HPLC produced by S. ansochromogenes 7100
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Fig2: Effects of wblA disruption on nikkomycin production. a The bioassay of nikkomycin against Alternaria longipes. b The bioassay of nikkomycin against Candida albicans. c HPLC analysis of nikkomycin. d Transcription analysis of genes related to nikkomycin biosynthesis by qRT-PCR; the transcript of hrdB was used as an internal control. (I): ΔwblA, (II): S. ansochromogenes 7100, (III): complemented strain by integrating a copy of wblA into the chromosome of ΔwblA, (IV): the control strain by integrating pSET152 vector into the chromosome of ΔwblA. Arrows indicate the peak of nikkomycin on HPLC produced by S. ansochromogenes 7100

Mentions: In order to identify whether the metabolite profile could be affected by wblA disruption in S. ansochromogenes 7100, ΔwblA was constructed via homologous recombination. As expected, ΔwblA failed to form grey spores and spore chains on minimal medium (MM) agar in comparison with wild-type (WT) strain (Fig. 1a–c). On the other hand, nikkomycin, the only secondary metabolite identified so far from this strain, was examined. Cultures from the same time-course experiments were subjected to bioassays against Alternaria longipes and Candida albicans for nikkomycin activity test (Fig. 2a, b). In contrast to WT strain, no inhibition zone was observed against above two indicator strains with the fermentation filtrate of ΔwblA. High-performance liquid chromatography (HPLC) analysis demonstrated that the production of nikkomycin was completely abolished in ΔwblA (Fig. 2c). To further verify the effect of wblA disruption on nikkomycin production, the transcription profile of genes involved in nikkomycin biosynthesis was analyzed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The biosynthetic gene cluster of nikkomycin includes one pathway-specific regulatory gene (sanG) and 21 structural genes consisting of three transcriptional units (sanO-V, sanN-I and sanF-X) [16]. The first gene of each transcriptional unit was chosen to examine the transcription of corresponding genes. The results showed that transcriptions of sanG and other three genes (sanN, sanO and sanF) situated in each transcriptional unit were all not detected in ΔwblA, whereas the transcription of hrdB as internal control, encoding the principal sigma-like factor, was not affected by the disruption of wblA (Fig. 2d). Complementary experiment was performed by integrating a copy of wblA and pSET152 vector into the chromosome of ΔwblA, respectively. As expected, nikkomycin production in ΔwblA was restored as that in WT strain (Fig. 2a–c). These results demonstrated that wblA is essential for nikkomycin biosynthesis in S. ansochromogenes 7100. Disruption of this gene affected not only the spore formation but also the nikkomycin biosynthesis, implying that wblA possesses multiple functions.Fig. 1


Identification of novel tylosin analogues generated by a wblA disruption mutant of Streptomyces ansochromogenes.

Lu C, Liao G, Zhang J, Tan H - Microb. Cell Fact. (2015)

Effects of wblA disruption on nikkomycin production. a The bioassay of nikkomycin against Alternaria longipes. b The bioassay of nikkomycin against Candida albicans. c HPLC analysis of nikkomycin. d Transcription analysis of genes related to nikkomycin biosynthesis by qRT-PCR; the transcript of hrdB was used as an internal control. (I): ΔwblA, (II): S. ansochromogenes 7100, (III): complemented strain by integrating a copy of wblA into the chromosome of ΔwblA, (IV): the control strain by integrating pSET152 vector into the chromosome of ΔwblA. Arrows indicate the peak of nikkomycin on HPLC produced by S. ansochromogenes 7100
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4630966&req=5

Fig2: Effects of wblA disruption on nikkomycin production. a The bioassay of nikkomycin against Alternaria longipes. b The bioassay of nikkomycin against Candida albicans. c HPLC analysis of nikkomycin. d Transcription analysis of genes related to nikkomycin biosynthesis by qRT-PCR; the transcript of hrdB was used as an internal control. (I): ΔwblA, (II): S. ansochromogenes 7100, (III): complemented strain by integrating a copy of wblA into the chromosome of ΔwblA, (IV): the control strain by integrating pSET152 vector into the chromosome of ΔwblA. Arrows indicate the peak of nikkomycin on HPLC produced by S. ansochromogenes 7100
Mentions: In order to identify whether the metabolite profile could be affected by wblA disruption in S. ansochromogenes 7100, ΔwblA was constructed via homologous recombination. As expected, ΔwblA failed to form grey spores and spore chains on minimal medium (MM) agar in comparison with wild-type (WT) strain (Fig. 1a–c). On the other hand, nikkomycin, the only secondary metabolite identified so far from this strain, was examined. Cultures from the same time-course experiments were subjected to bioassays against Alternaria longipes and Candida albicans for nikkomycin activity test (Fig. 2a, b). In contrast to WT strain, no inhibition zone was observed against above two indicator strains with the fermentation filtrate of ΔwblA. High-performance liquid chromatography (HPLC) analysis demonstrated that the production of nikkomycin was completely abolished in ΔwblA (Fig. 2c). To further verify the effect of wblA disruption on nikkomycin production, the transcription profile of genes involved in nikkomycin biosynthesis was analyzed by quantitative Real Time Polymerase Chain Reaction (qRT-PCR). The biosynthetic gene cluster of nikkomycin includes one pathway-specific regulatory gene (sanG) and 21 structural genes consisting of three transcriptional units (sanO-V, sanN-I and sanF-X) [16]. The first gene of each transcriptional unit was chosen to examine the transcription of corresponding genes. The results showed that transcriptions of sanG and other three genes (sanN, sanO and sanF) situated in each transcriptional unit were all not detected in ΔwblA, whereas the transcription of hrdB as internal control, encoding the principal sigma-like factor, was not affected by the disruption of wblA (Fig. 2d). Complementary experiment was performed by integrating a copy of wblA and pSET152 vector into the chromosome of ΔwblA, respectively. As expected, nikkomycin production in ΔwblA was restored as that in WT strain (Fig. 2a–c). These results demonstrated that wblA is essential for nikkomycin biosynthesis in S. ansochromogenes 7100. Disruption of this gene affected not only the spore formation but also the nikkomycin biosynthesis, implying that wblA possesses multiple functions.Fig. 1

Bottom Line: One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool.Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA.Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China. Lucheng522@aliyun.com.

ABSTRACT

Background: Streptomyces, as the main source of antibiotics, has been intensively exploited for discovering new drug candidates to combat the evolving pathogens. Disruption of wblA, an actinobacteria-specific gene controlling major developmental transition, can cause the alteration of phenotype and morphology in many species of Streptomyces. One wblA homologue was found in Streptomyces ansochromogenes 7100 by using the Basic Local Alignment Search Tool. It is interesting to identify whether novel secondary metabolites could be produced by the wblA disruption mutant as evidenced in other Streptomyces.

Results: The wblA disruption mutant of S. ansochromogenes 7100 (ΔwblA) was constructed by homologous recombination. ΔwblA failed to produce spores and nikkomycin, the major product of S. ansochromogenes 7100 (wild-type strain) during fermentation. Antibacterial activity against Staphylococcus aureus and Bacillus cereus was observed with fermentation broth of ΔwblA but not with that of the wild-type strain. To identify the antibacterial compounds, the two compounds (compound 1 and compound 2) produced by ΔwblA were characterized as 16-membered macrolides by mass spectrometry and nuclear magnetic resonance spectroscopy. The chemical structure of these compounds shows similarity with tylosin, and the bioassays indicated that the two compounds inhibited the growth of a number of gram-positive bacteria. It is intriguing that they displayed much higher activity than tylosin against Streptococcus pneumoniae.

Conclusions: Two novel tylosin analogues (compound 1 and 2) were generated by ΔwblA. Bioassays showed that compound 1 and 2 displayed much higher activity than tylosin against Streptococcus pneumoniae, implying that these two compounds might be used to widen the application of tylosin.

No MeSH data available.


Related in: MedlinePlus