Relationship of the Genetic Determination of Impaired Penicillin Production in Naturally Occurring Strains to that in Induced Mutants of Aspergillus nidulans

Cole, Duncan; Holt, G.; Macdonald, K. D.

doi:10.1099/00221287-96-2-423

Cited by 21 publications

(11 citation statements)

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“…Southern blot analysis was also carried out on genomic DNA digests from six independently isolated, naturally occurring npeA strains (Birmingham Strain nos 49,108,112,123,132 and 136) all belonging to heterokaryoncompatibility group F (Cole et al, 1976). The banding patterns observed were identical to those seen for the naturally occurring npeAO0491l mutation, indicating a similar deletion of this 20 kb genomic region in all seven of the wild isolate npeA mutants analysed (unpublished data).…”

Section: Resultsmentioning

confidence: 73%

“…Of these, the majority of penicillin non-producing mutations fall into the npeA category. In addition, there exist naturally occurring penicillin non-producers, which have been shown to correspond to mutational defects mapping at the npeA locus (Cole et al, 1976). Biochemical work has implicated this locus in the formation of the tripeptide b(L-aoaminoadipyl)-L-cysteinyl-D-valine (ACV).…”

Section: Introductionmentioning

confidence: 99%

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The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis

1990

Trends in Genetics

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Clones of Aspergillus nidulans genomic DNA spanning 20 kb have been isolated and shown by a combination of classical and molecular genetic means to represent the npeA locus, previously found to be one of four loci (npeA, npeB, npeC and npeD) involved in the synthesis of penicillin. As well as containing the gene encoding the second enzyme for penicillin biosynthesis, namely isopenicillin N synthetase (IPNS) (designated ipnA), our results show that these clones (pSTA200, pSTA201 and pSTA207) contain two more genes to form a cluster of three contiguous penicillin biosynthetic genes. Our evidence suggests that these genes encode 5(L-aaminoadipyl)-L-cysteinyl-D-vaiine synthetase (ACVS) and acyl transferase (ACYT) (designated acvA and acyA respectively), the first and third enzymes required for penicillin biosynthesis, with the gene order being acvA -ipnA -acyA. Transcripts have been identified for the three genes and their approximate sizes determinedacvA 9.5 kb, ipnA 1.4 kb and acyA 1.6 kb. All three mRNA species are observed in cells grown in fermentation medium but not in cells grown in minimal medium, suggesting that the control of penicillin biosynthesis is, in part, at the level of mRNA accumulation. Finally our results show that acvA and ipnA genes are divergently transcribed, whilst acyA is transcribed in the same orientation as ipnA.

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Section: Resultsmentioning

confidence: 73%

Section: Introductionmentioning

confidence: 99%

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis

1990

Trends in Genetics

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“…npeE1 defines a positive-acting regulatory gene of ipnA transcription essential for penicillin production. Mutations impairing penicillin biosynthesis have been already described for A. nidulans (14) and belong to four different complementation groups, denoted npeA, npeB, npeC, and npeD (11), none of which is located in chromosome IV (24), to which the npeE1 mutation has been assigned by haploidization analysis. Therefore, npeE1 identifies a new npe gene.…”

Section: Discussionmentioning

confidence: 99%

“…involving a number of auxotrophies. An example of the difficulty in genetically analyzing regulatory mutations in this pathway is provided by the penicillin-nonproducer mutations npeC007 and npeD0045 (11), which were incorrectly assigned to chromosomes IV and II, respectively, by haploidization and meiotic linkage analysis (24). However, using more sensitive scoring techniques available for pal mutations, Shah et al (28) showed them to be palA mutant (palA is located in chromosome III) and palF mutant (palF is in chromosome VII) alleles, respectively.…”

Section: Discussionmentioning

confidence: 99%

A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway

1995

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Secondary metabolism, usually superfluous under laboratory conditions, is intrinsically elusive to genetic analysis of its regulation. We describe here a method of analyzing regulatory mutations affecting expression of secondary metabolic genes, with an Aspergillus nidulans penicillin structural gene (ipnA [encoding isopenicillin N-synthase]) as a model. The method is based on a targeted double integration of a lacZ fusion reporter gene in a chromosome different from that containing the penicillin gene cluster. The trans-acting regulatory mutations simultaneously affect lacZ expression and penicillin biosynthesis. One of these mutations (npeE1) has been analyzed in detail. This mutation is recessive, prevents penicillin production and ipnA::lacZ expression, and results in very low levels of the ipnA message at certain times of growth. This indicates that npeE positively controls ipnA transcription. We also show that this tandem reporter fusion allows genetic analysis of npeE1 by using the sexual and parasexual cycles and that lacZ expression is an easily scorable phenotype. Haploidization analysis established that npeE is located in chromosome IV, but npeE1 does not show meiotic linkage to a number of known chromosome IV markers. This method might be of general applicability to genetic analysis of regulation of other fungal secondary metabolic pathways.Secondary metabolism in microbes is often elusive to genetic analysis, because most, if not all, pathways classified in this category are dispensable under laboratory conditions. The penicillin biosynthetic pathway (22) is a prototype of such pathways in filamentous ascomycetes and has been extensively used as a model for at least four reasons. (i) It is a rather simple pathway, and only three enzymes are required to convert primary metabolites (three amino acids) into penicillin; (ii) the corresponding structural genes, which are clustered, have been cloned and characterized from several species; (iii) the end product can be sensitively detected with a bioassay; and (iv) it is of obvious biotechnological interest. As a consequence, a wealth of information about this pathway has been accumulated. In contrast, regulation of penicillin biosynthesis is largely unelucidated, possibly because the absence of a sexual cycle in Penicillium chrysogenum has hindered formal genetic analysis of the putative regulatory mechanisms. This problem has been circumvented by using Aspergillus nidulans (26), a closely related plectomycete amenable to formal genetic studies (9) and for which sophisticated molecular biology techniques are available (31).By using molecular techniques to analyze transcription of the A. nidulans ipnA gene (encoding isopenicillin N-synthase, a key enzyme catalyzing the central step in the pathway), we have described two modes of transcriptional regulation of a penicillin structural gene. Carbon regulation in response to the availability of a preferred carbon source modulates ipnA expression through the action of a yet undefined negative-acting regulatory ge...

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“…This locus, designated npeA, has also been found to be affected in wild-type strains of Asp. nidulans incapable of synthesizing detectable amounts of penicillin (Cole et al, 1976).…”

Section: Discussionmentioning

confidence: 99%

Genetic and Biochemical Studies of Mutants of Penicillium chrysogenum Impaired in Penicillin Production

Normansell¹,

Normansell²,

Holt³

1979

Journal of General Microbiology

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Seventy-eight mutants of Penicillium chrysogenum strain NRRL 195 1, that were impaired in penicillin production, were isolated following treatment with various mutagens. Twelve that yielded about 10% of their parental penicillin titre were studied in detail. Analyses of heterozygous diploids formed between them revealed the existence of at least five complementation groups with respect to penicillin production -V, W, X, Y and Z. Most mutants belonged to group Y. A biochemical investigation of the intracellular peptides in strains representing the five groups demonstrated the absence of the tripeptide a-aminoadipoylcysteinyl-valine from mutants of groups X, Y and Z. Extracts of mutants of groups W, Y and Z were able to catalyse a penicillin acyl-exchange reaction, a mutant of group V showed only a trace of activity and a mutant from group X completely lacked this ability.

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Relationship of the Genetic Determination of Impaired Penicillin Production in Naturally Occurring Strains to that in Induced Mutants of Aspergillus nidulans

Cited by 21 publications

References 4 publications

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis

The Aspergillus nidulans npeA locus consists of three contiguous genes required for penicillin biosynthesis

A lacZ reporter fusion method for the genetic analysis of regulatory mutations in pathways of fungal secondary metabolism and its application to the Aspergillus nidulans penicillin pathway

Genetic and Biochemical Studies of Mutants of Penicillium chrysogenum Impaired in Penicillin Production

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