Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

Alduina, Rosa; Giardina, Anna; Gallo, Giuseppe; Renzone, Giovanni; Ferraro, Clelia; Contino, Antonio; Scaloni, Andrea; Donadio, Stefano; Puglia, Anna Maria

doi:10.1007/s00253-005-1929-y

Cited by 23 publications

(20 citation statements)

References 22 publications

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“…Streptomyces species offer many potential advantages as hosts for the heterologous expression of secondary metabolite genes and we previously have successfully demonstrated heterologous expression of actinomycete genes in S. lividans [12]. …”

Section: Discussionmentioning

confidence: 99%

“…Plasmid DNA, extracted from EC-40 by alkaline lysis method [25] and digested with Bam HI, was ligated to Bam HI-digested and dephosphorylated ESAC vector and the ligation mixture was used to transform Escherichia coli ElectroMAX DH10B cells, as described in Alduina et al ., 2005 [12]. Streptomyces protoplast formation, transformation and regeneration were carried out according to the methods of Kieser et al .…”

Section: Methodsmentioning

confidence: 99%

“…Previous results [10-12] have shown that E. coli - Streptomyces artificial chromosomes (ESACs), carrying large inserts of actinomycete DNA, can be introduced into a genetically accessible strain such as Streptomyces lividans , where they are stably maintained as an integrated form in its chromosome.…”

Section: Introductionmentioning

confidence: 99%

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Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

et al. 2010

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BackgroundA bacterial artificial chromosomal library of Planobispora rosea, a genetically intractable actinomycete strain, was constructed using Escherichia coli-Streptomyces artificial chromosome (ESAC) and screened for the presence of genes known to be involved in the biosynthesis of antibiotics.ResultsOne clone with a 40 kb insert showed antimicrobial activity against Gram positive bacteria. Insert sequence analysis and subcloning experiments revealed that the bioactivity was due to a 3.5 kb DNA fragment containing two open reading frames. These orfs encode two proteins with high similarity to a putative membrane protein of Streptomyces coelicolor and to the nogalamycin resistance protein SnorO of Streptomyces nogalater, respectively. The role of these two Orfs is unknown in Planobispora. Disruption and complementation experiments revealed that both proteins are necessary for the antibacterial activity and chemical analysis demonstrated that the antibiotic activity was due to thiostrepton, antibiotic used as recombinant clone selection marker.ConclusionTwo Planobispora rosea orfs are responsible for increasing intracellular amounts and storage of thiostrepton in Streptomyces lividans.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

et al. 2010

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“…ATCC 39727 genome, a BamHI‐restricted genomic library in ESAC E. coli / Streptomyces sp. shuttle vector was used (Alduina et al ., 2005). Screening was carried out by using a 424 bp pnp ‐specific probe obtained by PCR amplification of Nonomuraea DNA with heterologous primers.…”

Section: Methodsmentioning

confidence: 99%

Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

Siculella¹,

Damiano²,

Summa³

et al. 2010

Molecular Microbiology

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SummaryWith the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5Ј-diphosphate 3Ј-diphosphate (ppGpp) and guanosine 5Ј-triphosphate 3Ј-diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)-encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild-type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic clusterspecific dpgA mRNA were stabilized during the idiophase in the wild-type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse-labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.

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“…When the same methodology was applied to Nonomuraea sp. ATCC 39727, it was not successful, since its DNA undergoes degradation during PFGE [29, 37]. The PFGE step was therefore omitted, and a high-quality, high molecular weight genomic library of 2,051 recombinant clones with inserts larger than 30 up to 155 kb with an average insert size of 57 kb was constructed in pPAC-S2.…”

Section: Actinomycete Genomic Libraries Constructed In Artificial mentioning

confidence: 99%

Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes

Alduina

Gallo

2012

Journal of Biomedicine and Biotechnology

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Actinomycetes are an important source of biologically active compounds, like antibiotics, antitumor agents, and immunosuppressors. Genome sequencing is revealing that this class of microorganisms has larger genomes relative to other bacteria and uses a considerable fraction of its coding capacity (5–10%) for the production of mostly cryptic secondary metabolites. To access actinomycetes biosynthetic capabilities or to improve the pharmacokinetic properties and production yields of these chemically complex compounds, genetic manipulation of the producer strains can be performed. Heterologous expression in amenable hosts can be useful to exploit and to explore the genetic potential of actinomycetes and not cultivable but interesting bacteria. Artificial chromosomes that can be stably integrated into the Streptomyces genome were constructed and demonstrated to be effective for transferring entire biosynthetic gene clusters from intractable actinomycetes into more suitable hosts. In this paper, the construction of several shuttle Escherichia coli-Streptomyces artificial chromosomes is discussed together with old and new strategies applied to improve heterologous production of secondary metabolites.

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Expression in Streptomyces lividans of Nonomuraea genes cloned in an artificial chromosome

Cited by 23 publications

References 22 publications

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

Two heterologously expressed Planobispora rosea proteins cooperatively induce Streptomyces lividans thiostrepton uptake and storage from the extracellular medium

Guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a ‘rare’ actinomycete

Artificial Chromosomes to Explore and to Exploit Biosynthetic Capabilities of Actinomycetes

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