Poster Abstracts, Beta-lactam Antibiotics

Genetic Analysis of the Putative Thioesterase Region Of ACV Synthetase

Bahram Arezi and Geoffrey Turner. Department of Molecular Biology and Biotechnology, University of Sheffield, S10 2UH

The acvA gene encodes -(L-( -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) which catalyses the first step in -lactam biosynthesis pathways. The non-ribosomal synthesis of the tripeptide ACV follows the thiotemplate mechanism in which the release of the ACV from the enzyme, presumably bound as a thioester, requires the activity of a thioesterase. Inspection of the amino acid sequence revealed the motif GX-S-X-G in the C-terminal region which is found in a number of thioesterase enzymes. To determine the significance of this putative thioesterase in the C-terminal region of ACVS, deletions were made from the 3' end using the available full length cloned P. chrysogenum acvA gene. The first deletion removed the thioesterase motif and all sequences 3' to it. The second deletion removed the 3' end of the acvA but it retained the motif. Both of these deletions removed most of the ACVS activity, as determined by penicillin bioassay in a strain of A.nidulans in which the acvA was precisely deleted.

Attempts were made to complement the deleted strains with the expressed C-terminal end of ACVS. Expression under the control of the alca promoter was detected by SDS-PAGE and western blotting.

This strain was crossed with the deleted strains and analysis of progeny by PCR made it possible to obtain a strain in which the separated domains were co-expressed. Penicillin bioassay showed no changes in penicillin production compared to the deleted strains. This suggests that the expression of the C-terminal end and deleted ACVS in trans cannot restore penicillin production.

Further purification of the truncated enzyme may reveal whether the ACV is trapped on the enzyme and if so, whether the valine is in the L- or D form in order to establish when epimerization occurs.

Cloning and Analysis of an Intron Containing Peptide Synthetase Gene from the Entomopathogenic Fungus Metarhizum anisopliae

A.M. Bailey, M.J. Kershaw, S.E. Reynolds, A.K. Charnley and J.M. Clarkson. School of Biology and Biochemistry, University of Bath, Bath BA2 7AY UK

The entomopathogenic fungus Metarhizium anisopliae produces a family of related cyclic hexapeptide toxins called destruxins. The precise role of these toxins is unclear, with reported effects including immunosuppression, causing paralysis and impairing osmotic control of the host. The significance of these effects in the disease process is not clear and in order to define the role of the toxin more fully we aim to disrupt the gene(s) responsible for destruxin synthesis.

Many peptide synthetase genes have been extensively characterised and several conserved regions identified within each of their domains. Oligonucleotides designed against two of these conserved areas have been used to screen a genomic EMBLIII library and positive clones identified. A region of more than 12kb has been completely sequenced and this spans three of the six predicted domains. These domains are typical of peptide synthetase genes, containing all of the expected core sequences. In contrast to the other peptide synthetase genes characterised to date, the open reading frame of this gene is split by at least two introns. These show typical fungal intron consensus sequences and their presence has been confirmed bv RT-PCR, also confirming that this gene is transcribed. Progress towards the complete sequencing and disruption of this gene will be presented.

Comparison of the Penicillin Gene Clusters and RFLP Patterns of Penicillin Gene Specific PCR Products of P. nalgiovense and P. chrysogenum

P.Farber and R. Geisen Federal Research Centre for Nutrition, Institute for Hygiene and Toxicology, Engesserstraf3e 20, D-76131 Karlsruhe, Germany

Penicillium nalgiovense a terverticillate species of the genus Penicillium is a frequently used starter culture for mold ripened foods. This fungal species can preferably be isolated from fermented meat products f.e. salami. From the physiological as well as the genetical point of view P. nalgiovense seems to be related very closely to P. chrysogenum a well known producer of the a-lactam antibiotic penicillin. It was possible to prove the complete set of genes -(L-( -aminoadipyl)-L-cysteinyl-D-valine synthetase (acvA), isopenicillase N synthase (ipna) and acylCoA:6-aminopenicillanic acid acyltransferase (aat) necessary for biosynthesis of penicillin in P. nalgiovense as well by gene specific polymerase chain reactions using oliginucleotide primers derived from sequences of P. chrysogenum, as in Southern hybridization experiments using DIG labeled DNA probes. For further judging the degree of relation between these two species cluster analysis of penicillin biosynthetic genes in P. nalgiovense have been made and results of these experiments have been compared to the penicillin gene cluster in P. chrysogenum. These investigations showed that penicillin genes in P. nalgiovense are clustered in nearly the same cluster as in P. chrysogenum. RFLP analysis of PCR fragments of about 1 kb showed that PCR products from P. nalgiovense and P. chrysogenum yielded exactly the same restriction fragments when they have been digested by enzyme MboI, but a slightly different pattern of restriction fragments could be found when enzyme HaeIII have been used. The results of these investigations further confirm the assumption that P. nalgiovense and P. chrvsogenum are two very closely related fungal species.

The Penicillin Gene Cluster Is Amplified in Tandem Repeats Linked by Conserved Sequences

Francisco Fierro*, JL. Barredo#, B Diez#, S. Gutierrez*, F.J Fernandez* K. Kosalkova and J. F. Martin *. *Area de Microbiologia e Instituto de Biotecnologia (INBIOTEC), Universidad de Leon, Facultad de Biologia, 24071 Leon, Spain.
#Antibioticos S.A., Avenida de Antibioticos 56, Leon, Spain.

The penicillin biosynthetic genes (pcbAB, pcbc and penDE) of Penicillium chysogenum AS-P-78 were located in a 106.5 kb DNA region that is amplified in tandem repeats (five or six copies) linked by conserved TTTACA sequences. The wild type strains P. chrysogenuni NRRL 1951 and P. notatum ATCC 9478 contain a single copy of the 106.5 kb region. This region was bordered bv the same TTTACA hexanucleotide found between the repeats in the strain AS-P-78. A penicillin overproducer strain, P. chrysogenum El, contains a large number of copies in tandem of a 57.9 kb DNA fragment that mostly overlaps the right half of the amplified region in the strain AS-P-78. The tandem repeats in strain El are linked by the sequence TGTAAA, which is the reverse complementary of that appearing in strains AS-P-78 and NRRL 1951. The occurrence of one or the other pattern of amplification seems to depend on the orientation of a 3.4 kb fragment (shift fragment: SF) located at the right border of the amplified region. The penicillin non-producer mutant npe10 showed a deletion of a 57.9 kb fragment that corresponds exactly to the amplified region in strain E I. The conserved hexanucleotide sequence was reconstituted at the deletion site in strain npe10. The amplification has occurred within a single chromosome (chromosome 1), no other copies were located in any of the remaining chromosomes in the strains studied. The tandem reiteration and deletion appear to arise by mutation-induced site-specific recombination at the conserved hexanucleotide sequences.

Isolation and Characterization of Mutants Affecting the Expression of the ACVS Gene in Aspergillus nidulans.

Joel Harper and Geoff Turner. Department of Molecular Biology and Biotechnology, University Of Sheffield, Sheffield SIO 2UH, England

The expression of penicillin biosynthetic genes in Aspergillus nidulans has been examined by the use of the reporter genes lacZ and uidA which have been fused in frame to the promoters of the acvA and ipnA genes1 The acvA gene encodes for the enzyme -(L-( -aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) - and is the first of the three key enzymes responsible for penicillin production in Aspergillus nidulans.

In an attempt to find regulatory genes affecting acvA expression, mutants of the strain carrying the acvA-lacZ fusion were produced by UV irradiation, and those that showed either an increased expression of lacZ or those showing a loss of expression were selected for further analysis. Mutants showing loss of -galactosidase activity were probed to check for the loss of the fusion gene, and those retaining the sequence were analyzed by sexual crosses. All mutations appeared to be linked to the fusion gene, and the promoter was sequenced to look for mutations.

The mutants that displayed an increase in -galactosidase activity were crossed to establish the location of the mutation. Those showing a mutation not linked to the fusion gene were used to form diploids in an attempt to map the linkage group of the mutation, whilst those that showed linkage were sequenced to detect the position of the mutation.

1 Brakhage et al, 1992

Functional Consequences of Structural Variation of the Thioesterase Domain of ACV Synthetase

W Kallow1, H. von Dohren1, J Kennedy2, G. Turner2, 1 Institute of Biochemistry and Molekular Biology, TU Berlin, Franklinstr. 29, 1058 7 Berlin, FRG, 2 Department of Molecular Biology & Biotechnology, University Sheffield, P.O. Box 594, Western Bank, Sheffield SI 0 2 UH, UK

ACV synthetase forms the tripeptide -(L-( -aminoadipyl)-L-cysteinyl-D-valine (LLD-ACV) from the respective amino acids and ATP. It is considered the rate-limiting enzyme in penicillin and cephalosporin fermentation, and serves as a model system for peptide biosynthetic systems. We are studying the involvement of the thioesterase-like site in the terminating tripeptide cleavage and the mechanism of epimerization of the valine residue by site directed mutagenesis.

We have constructed a strain of A.nidulans in which the acvA gene encoding ACV synthetase has been deleted. If a wild-type P. chrysogenum acvA gene is reintroduced by transformation, penicillin production is restored to wild type levels. Mutations can be introduced in vitro anywhere in the acvA gene and studied for expression and functional properties. We have altered one of the functionally important sefine residues of the thioesterase to alanine. This strain shows a strongly reduced production of antibiotic. The altered multienzyme has been isolated and characterized. Evidence is presented for the intermediate -(L-( -aminoadipyl)-L-cysteinyl-D-valine (LLL-ACV) and impaired peptide release. The data support the timing of epimerization at the tripeptide stage.

Construction of Hybrid Peptide Synthetases

Jonathan Kennedy and Geoffrey Turner. Department of Molecular Biology and Biotechnology, , University of Sheffield, Sheffield SI 0 2 UH, U K.

Peptide synthetases catalyse the non-ribosomal synthesis of small peptides. The peptides produced by such systems include amino acids and hydoxy acids unavailable to the ribosomal system such as the D isomers of, and methylated amino acids. They also often have important biological activities, acting as antibiotics, immunosuppressants, plant toxins, or insect toxins. The primary structure of peptide synthetases reveals the presence of repeated similar domains, with one domain for each amino or hydroxy acid added to the chain.

ACV synthetase, which catalyses the production of the tripeptide ACV, the first step in penicillin biosynthesis, was used as a test system for the construction of hybrid peptide synthetases. Two different strategies have been employed in order to produce hybrid systems. The first of these involved the expression of ACV synthetase as two separate polypeptides. In order to mimic the systems found in gramicidin and tyrocidin biosynthesis in which two or three enzymes interact to produce the peptide product. The activity was monitored in a strain of A. nidulans in which the acvA gene had been deleted, with penicillin production indicating the presence of ACV synthetase activity. The second method involved the production of a fusion gene in which domains of the acvA gene from different organisms were linked. This was again analysed in the AacvA strain.

It was found that the expression of ACV synthetase as two separate polypeptides failed to produce any penicillin while the hybrid ACV svnthetase was found to be active. Work is now progressing towards the construction of hybrid peptide synthetase genes, mixing domains from entirely different veptide svnthetases, for the biosynthesis of novel peptide products.

Identification of Upstream Activating Regions Important in Cis-acting Regulation of Expression of pcbAB Gene in Penicillium chrysogenum

Katarina Kosalkova, Ana T. Marcos, Santiago Gutierrez, Javier Velasco, Francisco Fierro, Javier Casqueiro, Oscar Banuelos and Juan F. Martin. Area of Microbiology, Faculty of Biology, University of Leon and Institute of Biotechnology, INBIOTEC, 24071 Leon Spain.

Expression of the Penicillium chrysogenum gene pcbAB, encoding the -aminoadipyl-cysteinyl-valine synthetase (ACVS) that catalyzes the first step of penicillin biosynthesis was analysed since the control of expression of this gene is particulary important. Expression of the pcbAB gene is believed to be limiting for penicillin biosynthesis. Accumulating evidence suggests that the 1. 1 6 -kb intergenic region between the pcbAB and pcbC genes regulates their expression. Functional elements in the pcbAB upstream region have been defined by assaying -galactosidase activity in extracts from recombinant strains carrying deletion derivates of the pcbAB promoter fused to the Escherichia coli lacZ as a reporter gene. Transformants with a single copy of the lacZ gene at the pyrG locus were selected. Strains were grown in penicillin production medium under carbon catabolite repressing or derrepressing condition. Analysis of the -galactosidase activity of several single copy transformants revealed the presence of two upstream regions that are important for expression of the pcbAB gene and both regions appear to be involved in glucose catabolite regulation of expression of the pcbAB gene. Both fragments were shifted in a gel retardation assays by DNA-binding proteins.

Regulation of the Penicillin Biosynthesis Gene aat (PenDE) of Asperillus nidulans Encoding Acyl Coenzyme A:6-aminopenicillanic Acid Acyltransferase

Olivier Litzka and Axel A. Brakhage. Lehrstuhl fur Mikrobiologie, Universitat Munchen, Maria-Ward-Str. la, D80638 Munchen, F.R.G.

Penicillin is produced as a classical secondary metabolite by some filamentous fungi. Because penicillin is still an important antibiotic for the therapy of infectious diseases, knowledge about the genetics of its biosynthesis will have an impact on production and modification of this -lactam antibiotic.

The last reaction of the 3-step biosynthetic pathway of penicillin is catalysed by acyl coenzyme A:6-aminopenicillanic acid acyltransferase (AAT). The enzyme is encoded by the aat (penDE) gene which is located together with the two other penicillin biosynthesis genes acva (pcbAB) and ipna (penDE) in a single gene cluster. To analyse the expression of the aat gene, its promoter region was fused in frame with the E. coli lacZ reporter gene. In a fermentation run, the expression of aat-lacZ gene fusions was maximal during the first 24 h when the growth of mycelia was maximal as well. The use of glucose instead of lactose as the carbon source led to reduced AAT specific activity. The aat-lacZ expression, however, was not affected by the carbon source, indicating that the glucose effect is posttranscriptionally mediated. For the identification of cis-acting regulatory sequences in the promoter of the aat gene and of corresponding DNA binding proteins, band shift experiments with partially purified protein extracts of A. nidulans were carried out. By analysing different DNA fragments spanning the whole or parts of the aat promoter, a recognition site for a DNA binding protein was localised within a 20 bp DNA fragment. The introduction of mutations in this DNA fragment abolished the binding of the protein in vitro. An aat-lacZ gene fusion carrying the same mutations in the aat promoter region showed a different expression pattern compared with that of a wild-type gene fusion, providing evidence for the significance of the identified protein binding site in vivo. Taken together, a comprehensive picture of the molecular regulation of this secondary metabolism gene is emerging.

Transcription of the pcbAB, pcbC and penDE Genes of Penicillium chrysogenum is Strongly Repressed by Glucose and the Repression is Not Reversed by Alkaline pHs

Ana T. Marcos Santiago Gutierrez, Katarina Kosalkova, Francisco J. Fernandez, Javier Velasco, Clara Arenos and Juan F. Martin. Area of Microblology, Faculty of Biology, University of Leon and Institute of Biotechnology, INBIOTEC, 24071 Leon, Spain

Glucose repressed transcription of the penicillin biosynthesis genes pcbAB, pcbc and penDE genes in Penicillium chrysogenum when added at inoculation time but it had little repressive effect when added at 12 h to batch cultures and no effect when added at 24 or 36 h. The rate of penicillin biosynthesis and expression of the three genes pcbAB, pcbc and penDE was reduced at pH6.0 as compared to pH 7.0. A slight increase of the expression of pcbC and penDE (and to a smaller extent of pcbAB) was observed in glucose grown cultures at pH values 7.1, 7.8 and 8.5 but alkaline pHs did not override the strong repression exerted by glucose. Transcription of the actin gene used as control was not significantly affected by glucose or alkaline pH values. Glucose repression and the lack of reversal of repression by alkaline pHs was also observed when the promoters of the pcbAB, pcbc and penDE genes were coupled to the LacZ reporter gene and the fusions integrated as single copy transformants (by directed targeting) at the pyrG locus. Glucose repression of the three genes encoding enzymes of penicillin biosynthesis appears, therefore, to be exerted by a regulatory mechanism independent from pH regulation.

Structure-function Analysis of ACV Synthetase from Penicillium chrysogenum Substrate Specificity of the Third Domain by Fragment Expression

M.T. Tavanlar1,3, J. Kennedy2, G. Turner2, H. von Dohren 3, A.K Raymundol

l National Institutes of Biotechnology and Applied Microbiology, University of the Philippines Los Banos, College, Laguna 4031, Phillippines. 2 Department of Molecular Biology and Biotechnology, University of Sheffield, P.O.Box 594, Western

Bank, Sheffield SIO 2UH, U.K. 3 Institute of Biochemistry and Molecular Biology, Technical University, Franklinstr. 29, D10587 Berlin, FRG

Biosynthesis of the penicillin precursor -(L-( -aminoadipyl)-L-cysteinyl-D-valine (ACV) is catalyzed from the constituent L-amino acids and ATP by a multifunctional peptide synthetase (ACV synthetase, ACVS). Its structure basically consists of three modules, each containing key motifs involved in ATP-binding, acyladenylate formation and aminoacylation. The modules have been tentatively associated with activation of the respective amino acids in order of their sequence in the peptide, and this association has been proved by limited proteolysis for the middle domain activating cysteine. Investigation on the substrate specificities of the other modules has been approached by fragment expression. We here present data on the third module thought to activate L-valine. The adenylate region has been subcloned and is expressed in Aspergillus nidulans. Unexpectedly also -aminoadipic acid forms an adenylate.

Use of Reporter Genes to Identify Recessive Trans-acting Mutations Specifically Involved in the Regulation of Aspergillus nidulans Penicillin Biosynthesis Genes

Jan Van den Brulle and Axel A. Brakhage. Lehrstuhl fur Mikrobiologie, Universitat Munchen, Maria-Ward-Str. I a, D80638 Munchen, F.R.G.

Starting from three amino acid precursors, penicillin biosynthesis is catalysed by three enzymes which are encoded by the following three genes: acvA (pcbAB), ipnA (pcbC) and aat (penDE). To identify trans-acting mutations which are specifically involved in the regulation of these secondary metabolism genes, a molecular approach was employed by using an Aspergillus nidulans strain (AXTII9) carrying acvA-uidA and ipnA-lacZ gene fusions integrated in double copies at the chromosomal argB gene. On minimal agar plates supplemented with X-Gal, colonies of such a strain stained blue, indicative of ipnA-lacZ expression. After mutagenesis with UV-light, colonies were isolated on agar plates with lactose as carbon source, which produced only a faint blue colour or no colour at all. Such mutants (named Prg for penicillin regulation) most likely were defective in trans-acting genes. Control experiments revealed that the mutants studied still carried the correct number of gene fusions. In a fermentation run, mutants Prg-I and Prg-6 exhibited only 20 to 50% of the ipnA-lacZ expression of the wild-type strain and produced only 20 to 30% of the penicillin produced by the wild-type strain. Western blot analysis showed that these mutants contained reduced amounts of ipnA gene product, i.e., isopenicillin N synthase. Both mutant Prg- I and mutant Prg-6 also differed in acvA-uidA expression levels from the wild-type. Segregation analysis indicated that for both mutants the Prg phenotype resulted from mutation of a single gene. Two different complementation groups, which were designated prgA1 and prgB1, were identified.

The identification and characterisation of cosmid clones complementing the prgA1 mutation will be presented.

Brakhage, A. A. and J. Van den Brulle (1995). J. Bacteriol., 177: 27812788

The Aspergillus nidulans lysF Gene Encodes Homoaconitase an Enzyme Involved in the Fungal Specific L-lysine Biosynthesis

Gerhard Weidner and Axel A. Brakhage. Lehrstuhl fur Mikrobiologie, Universitat Munchen, Maria-Ward-Str. la, D80638 Munchen, F.R.G.

In Aspergillus nidulans, the biosynthesis of L-lysine proceeds via the L-2aminoadipate pathway which is specific for higher fungi. L-2-Aminoadipic acid is a key intermediate of the L-lysine biosynthetic pathway and simultaneously, besides L-cysteine and L-valine, one of the precursor amino acids of the penicillin biosynthesis. Hence, L-2-aminoadipic acid is a branch point of primary and secondary metabolism. The analysis of regulation of genes / enzymes leading to formation of L-2-aminoadipic acid thus represents an excellent tool to investigate how the regulation of primary and secondary metabolism interacts, in order to coordinate the supply of L-2aminoadipic acid for both L-lysine and penicillin biosynthesis. In Saccharomyces cerevisiae, the expression of nearly all genes encoding enzymes of the L-lysine biosynthetic pathway is repressed by L-lysine. Interestingly, in A. nidulans the expression of the penicillin biosynthesis genes acvA (pcbAB) and ipnA (pcbC) is regulated by L-lysine, too.

Because, so far, nothing is known about the molecular basis of the L-lysine biosynthesis in filamentous fungi, we cloned the lysF gene by complementation of the corresponding lysine auxotrophic mutant of A. nidulans. Biochemical analysis revealed that lysF affects a step before formation of L-2-aminoadipic acid of the L-lysine biosynthetic pathway. Genomic DNA used for complementation derived from a chromosome specific cosmid library of A. nidulans. Subcloning from a 19 kbp HindlII fragment and DNA sequence analysis revealed an open reading fraine encoded by at least 1.8 kbp. Within the deduced amino acid sequence of lysF, a motif characteristic of an iron-sulfur cluster is present. Computer analysis did not reveal major homologies of the deduced amino acid sequence of lysF to any other known gene. Further biochemical analysis indicated that lysF encodes homoaconitase. We thus present the first cloning of a gene encoding homoaconitase activity. The addition of L-lysine to the fermentation medium led to drastically reduced homoaconitase specific activity implying that the level of homoaconitase is regulated by L-lysine. The physiological role of the lysF gene product for both L-lysine and penicillin biosynthesis will be discussed.

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