<i>Streptomyces</i> temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms)

Baltz, Richard H.

doi:10.1007/s10295-011-1069-6

Cited by 73 publications

(55 citation statements)

References 109 publications

(157 reference statements)

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“…The hygromycin resistance cassette from pMD19-hyg was inserted into the SmaI site of pRT801 (39,40) to obtain plasmid pDZL803. The rifZ gene with its intact promoter region was amplified with primers 0655C-F/0655C-R using Phanta super-fidelity DNA polymerase (Vazyme) and inserted into the EcoRV site of pDZL803, producing plasmid pDZL8031.…”

Section: Characterization Of the Transposon Insertional Sites In Mutamentioning

confidence: 99%

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Liu

Shao

et al. 2017

Appl Environ Microbiol

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Rifamycin and its derivatives are particularly effective against the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium leprae. Although the biosynthetic pathway of rifamycin has been extensively studied in Amycolatopsis mediterranei, little is known about the regulation in rifamycin biosynthesis. Here, an in vivo transposon system was employed to identify genes involved in the regulation of rifamycin production in A. mediterranei U32. In total, nine rifamycin-deficient mutants were isolated, among which three mutants had the transposon inserted in AMED_0655 (rifZ, encoding a LuxR family regulator). The rifZ gene was further knocked out via homologous recombination, and the transcription of genes in the rifamycin biosynthetic gene cluster (rif cluster) was remarkably reduced in the rifZ null mutant. Based on the cotranscription assay results, genes within the rif cluster were grouped into 10 operons, sharing six promoter regions. By use of electrophoretic mobility shift assay and DNase I footprinting assay, RifZ was proved to specially bind to all six promoter regions, which was consistent with the fact that RifZ regulated the transcription of the whole rif cluster. The binding consensus sequence was further characterized through alignment using the RifZ-protected DNA sequences. By use of bionformatic analysis, another five promoters containing the RifZ box (CTACC-N8-GGATG) were identified, among which the binding of RifZ to the promoter regions of both rifK and orf18 (AMED_0645) was further verified. As RifZ directly regulates the transcription of all operons within the rif cluster, we propose that RifZ is a pathwayspecific regulator for the rif cluster.IMPORTANCE To this day, rifamycin and its derivatives are still the first-line antituberculosis drugs. The biosynthesis of rifamycin has been extensively studied, and most biosynthetic processes have been characterized. However, little is known about the regulation of the transcription of the rifamycin biosynthetic gene cluster (rif cluster), and no regulator has been characterized. Through the employment of transposon screening, we here characterized a LuxR family regulator, RifZ, as a direct transcriptional activator for the rif cluster. As RifZ directly regulates the transcription of the entire rif cluster, it is considered a pathway-specific regulator for rifamycin biosynthesis. Therefore, as the first regulator characterized for direct regulation of rif cluster transcription, RifZ may provide a new clue for further engineering of highyield industrial strains.

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Section: Characterization Of the Transposon Insertional Sites In Mutamentioning

confidence: 99%

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Liu

Shao

et al. 2017

Appl Environ Microbiol

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“…Indeed, 70% of clinically useful antibiotics come from Streptomyces spp. Genetic tools derived from Streptomyces phages have been invaluable in investigations aimed at understanding the fundamental biology of these bacteria and in manipulating antibiotic pathways (3,4). Phages were used initially as cloning vectors, but since the early 1990s, integrating plasmid vectors based on phage-encoded site-specific recombination systems have been widely used, enabling the stable insertion of gene constructs into a defined site (attB) in the chromosome (5,6).…”

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confidence: 99%

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp

et al. 2013

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eThe genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, Hau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage Hau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species. Bacteriophages are the most abundant and diverse genetic entities on planet Earth. Despite this, it has been proposed that all double-stranded DNA (dsDNA) phages share the same gene pool and that there are clear examples where common ancestry is evident between orthologous gene pairs from very different phages (for example, the capsid-encoding genes from coliphage HK97 and Streptomyces phage C31) (1, 2). Understanding the mechanisms through which phages generate such vast diversity is still a challenge, but the drivers of selection and adaptation to growth in different bacterial hosts are clearly strong influences. We are interested in the adaptations of phages that infect the Actinobacteria, a class of high-GC, Gram-positive bacteria that have very diverse morphology, physiology, and growth properties.Actinobacteria, particularly those in the genus Streptomyces, have a remarkable capacity to synthesize a diverse array of secondary metabolites of all the major classes. Indeed, 70% of clinically useful antibiotics come from Streptomyces spp. Genetic tools derived from Streptomyces phages have been invaluable in investigations aimed at understanding the fundamental biology of these bacteria and in manipulating antibiotic pathways (3, 4). Phages were used initially as cloning vectors, but since the early 1990s, integrating plasmid vectors based on phage-encoded site-specific recombination systems have been widely used, enabling the stable insertion of gene constructs into a defined site (attB) in the chromosome (5, 6). Moreover, as streptomycetes are mycelial and undergo a developmental cycle, phages that infect this group of bacteria are likely to have undergone host-specific evolu...

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“…The S. griseus draft genome was searched for two genetic elements for chromosomal incorporation of exogenous DNA. The first element was a phage ⌽C31 attB site that allows the integration of pSET vector-carried DNA into a host chromosome (38). A 51-bp sequence in a hypothetical protein gene (39), highly similar to the attB sequences found in other Streptomyces genomes, was identified (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

Kulkarni

Zeng

Zhou

et al. 2016

Appl Environ Microbiol

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c Albomycin (ABM), also known as grisein, is a sulfur-containing metabolite produced by Streptomyces griseus ATCC 700974. Genes predicted to be involved in the biosynthesis of ABM and ABM-like molecules are found in the genomes of other actinomycetes. ABM has potent antibacterial activity, and as a result, many attempts have been made to develop ABM into a drug since the last century. Although the productivity of S. griseus can be increased with random mutagenesis methods, understanding of Streptomyces sulfur amino acid (SAA) metabolism, which supplies a precursor for ABM biosynthesis, could lead to improved and stable production. We previously characterized the gene cluster (abm) in the genome-sequenced S. griseus strain and proposed that the sulfur atom of ABM is derived from either cysteine (Cys) or homocysteine (Hcy). The gene product, AbmD, appears to be an important link between primary and secondary sulfur metabolic pathways. Here, we show that propargylglycine or iron supplementation in growth media increased ABM production by significantly changing the relative concentrations of intracellular Cys and Hcy. An SAA metabolic network of S. griseus was constructed. Pathways toward increasing Hcy were shown to positively impact ABM production. The abmD gene and five genes that increased the Hcy/Cys ratio were assembled downstream of hrdBp promoter sequences and integrated into the chromosome for overexpression. The ABM titer of one engineered strain, SCAK3, in a chemically defined medium was consistently improved to levels ϳ400% of the wild type. Finally, we analyzed the production and growth of SCAK3 in shake flasks for further process development. The Streptomyces genus was established at the beginning of the golden age of antibiotic discovery (1). Streptomyces griseus, a representative organism of the genus (2), was shown during this time to produce streptomycin (3), which has been clinically used to treat bacterial infections and other human diseases. It is well known that Streptomyces organisms in general have a large biosynthetic potential, being capable of producing many bioactive secondary metabolites-the S. griseus strains producing albomycin (ABM) are no exception. ABM was named by former Soviet Union scientists and underwent clinical investigations well before the information was released to the English-speaking scientific world (4). The biological activity and chemical constitution of ABM were later reported to be nearly identical to those of grisein, which was isolated from a distinct subtype of S. griseus by U.S. scientists in the 1940s (5-7). Despite its remarkable properties, continued studies of ABM were not pursued, partly because of the poor yields (8) and unpublicized research on the microorganism (9, 10). At present, the wealth of Streptomyces genomic information that is publically available provides the possibility of using a systems biology approach to uncover new aspects of Streptomyces metabolism and potentially overcome the production bottleneck.ABM and ABM-like secondary metabolites...

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Streptomyces temperate bacteriophage integration systems for stable genetic engineering of actinomycetes (and other organisms)

Cited by 73 publications

References 109 publications

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

RifZ (AMED_0655) Is a Pathway-Specific Regulator for Rifamycin Biosynthesis in Amycolatopsis mediterranei

Evolutionary Relationships among Actinophages and a Putative Adaptation for Growth in Streptomyces spp

A Branch Point of Streptomyces Sulfur Amino Acid Metabolism Controls the Production of Albomycin

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