β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes

Myronovskyi, Maksym; Welle, Elisabeth; Fedorenko, Victor; Luzhetskyy, Andriy

doi:10.1128/aem.00434-11

Cited by 176 publications

(168 citation statements)

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“…To confirm the likely direct regulatory targets of PspR and σ PspX , a series of promoter fusions were made to a version of gusA encoding β-glucuronidase (GUS) that had been codon-optimized for expression in streptomycetes (34). The constitutive promoter of ermE (P ermE* ) (35) was cloned upstream of gusA in pGUS (34), yielding pIJ10740, which was integrated into the ΦC31 chromosomal attachment site (attB) of S. coelicolor M1152 to provide a positive control strain.…”

Section: Resultsmentioning

confidence: 99%

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

Sherwood

Bibb

2013

Proc. Natl. Acad. Sci. U.S.A.

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Planosporicin is a ribosomally synthesized, posttranslationally modified peptide lantibiotic produced by the actinomycete Planomonospora alba. It contains one methyl-lanthionine and four lanthionine bridges and inhibits cell wall biosynthesis in other Gram-positive bacteria probably by binding to lipid II, the immediate precursor for cell wall biosynthesis. Planosporicin production, which is encoded by a cluster of 15 genes, is confined to stationary phase in liquid culture and to the onset of morphological differentiation when P. alba is grown on agar. This growth phase-dependent gene expression is controlled transcriptionally by three pathway-specific regulatory proteins: an extracytoplasmic function σ factor (PspX), its cognate anti-σ factor (PspW), and a transcriptional activator (PspR) with a C-terminal helix-turn-helix DNA-binding domain. Using mutational analysis, S1 nuclease mapping, quantitative RT-PCR, and transcriptional fusions, we have determined the direct regulatory dependencies within the planosporicin gene cluster and present a model in which subinhibitory concentrations of the lantibiotic function in a feed-forward mechanism to elicit high levels of planosporicin production. We show that in addition to acting as an antibiotic, planosporicin can function as an extracellular signaling molecule to elicit precocious production of the lantibiotic, presumably ensuring synchronous and concerted lantibiotic biosynthesis in the wider population and, thus, the production of ecologically effective concentrations of the antibiotic.

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Section: Resultsmentioning

confidence: 99%

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

Sherwood

Bibb

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…We previously applied a system based on the gene gusA, coding for a ␤-glucuronidase (5). Colonies of clones which still carry the suicide plasmid would occur in blue when overlaid with 5-bromo-4-chloro-1H-indol-3-yl ␤-D-glucopyranosiduronic acid (X-Gluc), while clones which lost it would keep their natural yellow color (5,27). The main problem with this approach was the low homologous recombination rate, since about 100 colonies had to be screened to identify a single one that lost the suicide plasmid, as was demonstrated in this study with the deletion of the yagRST cluster.…”

Section: Discussionmentioning

confidence: 99%

“…Alternatively, it is possible to make the homogenotes differ visibly from heterogenotes. This was achieved with enzymes such as a ␤-glucuronidase (27), the green fluorescent protein (28), or luciferase (29).…”

mentioning

confidence: 99%

Development of an Improved System for the Generation of Knockout Mutants of Amycolatopsis sp. Strain ATCC 39116

Meyer

Pupkes

Steinbüchel

2017

Appl Environ Microbiol

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The Gram-positive actinomycete Amycolatopsis sp. strain ATCC 39116 is used for the industrial production of natural vanillin. Previously, the only gene deletion performed in this strain targeted the gene vdh, coding for a vanillin dehydrogenase. The generation of this mutant suffered from a high number of illegitimate recombinations and a low rate of homologous recombination. To alleviate this, we constructed an optimized deletion system based on a modified suicide vector. Thereby, we were able to increase the rate of homologous integration from less than 1% of the analyzed clones to 20% or 50%, depending on the targeted gene. We were furthermore able to reduce the screening effort needed to identify homogenotes through the use of the rpsL gene from Saccharopolyspora erythraea, which confers streptomycin sensitivity on clones still carrying the suicide vector. The new suicide vector is p6SUI5ERPSL, and its applicability was demonstrated by the deletion of three Amycolatopsis gene clusters. The deletion of the first of the gene clusters, coding for an aldehyde oxidase (yagRST), led to no altered phenotype compared to the parent strain; deletion of the second, coding for a vanillic acid decarboxylase (vdcBCD), led to a phenotype that was strongly impaired in its growth with vanillic acid as the sole carbon source and also unable to form guaiacol; and deletion of the third, coding for a vanillate demethylase (vanAB), led to only a negligible impact in comparison. Therefore, we showed that decarboxylation of vanillic acid is the main degradation pathway in Amycolatopsis sp. ATCC 39116 while the demethylation plays only a minor role and does not compensate the deletion of vdcBCD.IMPORTANCE Amycolatopsis sp. ATCC 39116 is an important microorganism used for the production of natural vanillin from ferulic acid. In contrast to this importance, it has previously been shown that this strain is hard to manipulate on a genetic level. We therefore generated an optimized system to facilitate the deletion of genes in this strain. This allowed us to greatly reduce the time and work requirements for generating deletions. This could allow the improvement of vanillin production in the future and also the elucidation of metabolic pathways. To test our deletion system, we deleted three gene clusters in Amycolatopsis sp. ATCC 39116. One showed no involvement in the metabolism of vanillin, while the second proved to be the main pathway of vanillic acid degradation and completely stopped the formation of the off-flavor guaiacol. The third appeared to have only a negligible impact on the degradation of vanillic acid.

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“…All pSCAK plasmids have a pSETGUS (25) vector backbone, and DNA fragments were cloned using either BamHI or XbaI or both sites. The hrdBp promoter was amplified from the genome in the form of an ϳ0.3-kb EcoRI-XbaI DNA fragment to replace the ermE*p sequence in the pCK plasmids mentioned above.…”

Section: Methodsmentioning

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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β-Glucuronidase as a Sensitive and Versatile Reporter in Actinomycetes

Cited by 176 publications

References 50 publications

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

The antibiotic planosporicin coordinates its own production in the actinomycete Planomonospora alba

Development of an Improved System for the Generation of Knockout Mutants of Amycolatopsis sp. Strain ATCC 39116

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

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