FEMS Microbiology Letters 77 (1991) 209-212 © 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50 ADONIS 0378109791000855

FEMSLE 04264

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pH Regulation of penicillin production in Aspergillus nidulans

Ajit J. Shah 1, Joan Tilburn 2, Maxwell W. Adlard 1 and Herbert N. Arst, Jr. 2

School of Biological and Health Sciences, Polytechnic of Central London, London, and " Department of Bacteriology, Royal Postgraduate Medical School, London, U.K.

Received 30 August 1990 Accepted 31 August 1990

Key words: Aspergillus nidulans; Penicillin; pH regulation; Mutation

1. SUMMARY

As shown by both bioassay and high-perfor- mance liquid chromatographic (HPLC) analysis, penicillin G production by Aspergillus nidulans is subject to regulation by the pH of the growth medium. Penicillin titres were highest at alkaline pH and in strains carrying mutations in the regu- latory gene pacC which mimics the effects of growth at alkaline pH. They were lowest at acid pH and in strains carrying mutations in the palA, palB, palC, palE or palF genes which mimic the effects of growth at acid pH.

2. INTRODUCTION

All microorganisms must contend with varia- tions of pH in their environments. Most, probably all, have a pH homeostatic mechanism restricting the range of pH variation encountered by in- tracellular molecules. Molecules located at or be- yond the cell's permeability barrier are not, how- ever, similarly protected from extremes of pH.

Correspondence to." H.N. Arst, Department of Bacteriology, Royal Postgraduate Medical School, Ducane Road, London W12 ONN, U.K.

Therefore it is advantageous for an organism to possess a regulatory system for sensing environ- mental pH and ensuring appropriate syntheses of molecules destined for location at or beyond the cell membrane, enabling, for example, secretion of acid phosphatases in acidic environments and al- kaline phosphatases in alkaline environments.

In the ascomycete fungus Aspergillus nidulans mutations specifically affecting pH regulation of gene expression have been identified [1]. There are six genes where mutations impair pH regulation of the syntheses of extracellular enzymes and per- meases. Putative loss-of-function mutations in pacC mimic the effects of growth at alkaline pH whereas those in palA, palB, palC, palE and palF are phenotypically indistinguishable from each other and mimic the effects of growth at acid pH [1]. A model consistent with available data proposes that the pacC gene encodes a protein which regulates syntheses of certain activities (e.g. acid phosphatase, acid phosphodiesterase, y- aminobutyrate permease) in a positive fashion and others (e.g. secreted alkaline phosphatase) in a negative fashion [1]. The products of the palA, B, C, E and F genes have been proposed to mediate synthesis of an effector molecule able to inactivate the pacC product, preventing both its positive and negative actions [1]. The effector must be present (and effective) at alkaline pH.

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Here we show that the pH regulatory system of A. nidulans also affects titres of penicillin G pro- duced by this organism, levels being highest at alkaline pH. Thus the regulatory domain of the pH system extends to relatively small secreted molecules, a point of practical as well as scientific importance for the production of penicillin and possibly other compounds synthesised by micro- organisms.

3. MATERIALS AND METHODS

3.1. Genetic techniques, growth testing and strains Genetic techniques were modified after Ponte-

corvo et al. [2], McCully and Forbes [3] and Clut- terbuck [4]. Growth testing of and markers carried by Aspergillus nidulans strains have been described previously [1,5,6]. For gene symbol definitions Clutterbuck [6] should be consulted, Full geno- types of strains used for penicillin titre determina- tions given in Tables 1 and 2 were pabaA1 (wild type), pabaA1 palG21 pacCll pantoB100, yA2 pacC14 choA1, biA1 wA3 palA1 and biA1 pabaA1 chaA1 palB7. A number of additional strains of each relevant genotype was also tested by bioassay. Co-segregation of penicillin bioassay ,phenotype and other phenotypic characteristics was followed through crosses in the cases of pacC5, pacC11 pacC14, palA1, palC4 and palF15. The solid and liquid complete media de- scribed by Cove [7] were used. Phenylacetic acid wgs added to all complete media at a final con- centration of 3 g/1.

3.2. Penicillin fermentations One-ml suspensions containing approximately

107 conidiospores were inoculated into 25 ml liquid complete medium containing a final concentration of 500 mM buffer (citrate at pH 5.1 and 6.5; Tris-HC1 at pH 8.1) in 100 ml Erlenmeyer flasks and incubated at 26°C in an LH Fermentation (Stoke Poges, U.K.) incubator-shaker model MKX at 200 rpm. Cultures were harvested after 72 h. Bioassays were carried out on fermentation media using essentially the method of Smith et al. [8] with Staphylococcus aureus NCTC 6751 as indica- tor organism incubated for 24 h at 36 ° C. Both

antibiotic activity in bioassays and peaks corre- sponding to penicillin G in HPLC chromatograms disappeared after treatment with/~-lactamase [9].

3.3. HPLC analysis Penicillin G levels in fermentation broths were

determined by HPLC using pre-column derivatisa- tion with 1-hydroxybenzotriazole-mercury (II) chloride as described previously [9] with the fol- lowing modifications: (1) The Concentration of the mercury (II) chloride solution was 10 mM. (2) A Perkin-Elmer LC 850 diode array detector fitted with an 18/~1 flow cell was used. (3) The flow rate was 1.0 m l / m i n throughout the HPLC run. (4) The elution programme was linear for the first 25.0 min and isocratic for the following 10.0 min.

4. RESULTS

Bioassay data in Table 1 illustrate the effects of growth culture pH and mutations affecting pH regulation on titres of a /~-lactamase-sensitive an- tibiotic, principally penicillin G (vide infra), pro- duced by A. nidulans. Penicillin titres increased markedly as the culture pH was raised from 5.1 to 8.1 despite the greater lability of penicillin G at pH 8 as compared to pH 6 [10]. In agreement with this effect of increasing pH, pacC- mutations (which mimic growth at alkaline pH) strongly elevated penicillin production at pH 6.5 whereas a

Table 1

/~-lactam antibiotic titres as determined by bioassay of culture medium after growth of wild type and pH regulatory mutants of A. nidutans

Relevant genotype

fl-lactam titre after growth at pH

5.1 6.5 8.1

Wild-type 0.9 2.5 16.1 pacCll n.d. 17.6 16.5 pacC14 3.1 11.4 22.9 palA1 n.d. n.d. - palB7 n.d. n.d. -

The results, averaged from two experiments agreeing within 5%, are expressed in gg penicillin G/ml/g dry weight, n.d., not detectable; -, not done because pal- strains grow very poorly and accumulate suppressor mutations at alkaline pH [11.

palA- and a palB-mutation (which mimic growth at acid pH)e l imina ted it. Elevation of penicillin titres is likely to be a general Property of pacC- mutations because it also resulted from a third such allele pacC5 (results not shown),

It should be stressed that these differences in penicillin production represent genuine effects on penicillin levels: growth rates were comparable between mutants and wild type and, for each strain, over the assayed pH range. Moreover, they were seen whether mycelia used for bioassay originated from shaken culture (as in Table 1) or from colonies grown on agar discs (used in genetic analysis where bioassays were performed on large numbers of strains).

Null mutations in all five genes putatively in- volved in effector biosynthesis virtually abolished penicillin production: in addition to palA1 and palB7 strains shown in Table 1, palC4, palEll and palF15 strains all failed to produce penicillin as judged by bioassay (results not shown). Indeed, we have found that the penicillin non-producing mutations npeCO07 and npeDO045 [11-14] are palA- and palF- alleles, respectively: they are phenotypically indistinguishable from palA-, B-, C-, E- and F - mutations using the criteria de- scribed by Dorn [15,16] and Caddick et al. [1]. They fail to complement palA1 and palF15, re- spectively, in diploids and are tightly linked ( < 0.1 centiMorgan) to the mutations they fail to com- plement. In agreement with the allocation of palF15 by Dorn [15] and subsequent meiotic stud- ies [6,15,17], npeDO045 was allocated, using para- sexual analysis [3], to linkage group VII. This disagrees with the allocation to linkage group II reported by Makins et al. [14]. The npeCO07 mu- tation was allocated to linkage groups II and III, a translocation (whose relationship to the npeCO07 mutation has not been established) preventing re- assortment of these two linkage groups. This allo- cation agrees with that by Dorn [15] and meiotic recombination studies [6,15,18] for palA1 in link- age group III. However, it disagrees with the allo- cation of npeCO07 to linkage group IV by Makins et al. [14].

Penicillin G determinations using HPLC are presented in Table 2. Some differences of detail would be expected between HPLC and bioassay

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Table 2

Penicillin G titres as determined by HPLC analysis of culture medium after growth of wild type and pH regulatory mutants of A. nidulans

Relevant genotype

Penicillin G titre after growth at pH

5.1 6.5 8.1

Wild-type 2.26_+0.11 3.32_+0.25 9.91_+1.15 pacC11 1.72-+0.04 9.52-+0.10 17.3 _+1.0 pacC14 8.96_+0.34 16.2 _+2.5 23.5 _+0.0 palB7 0.04 + 0.05 0.20 _+ 0.25 -

The results _+1 S.D. are averages of two independent de- terminations expressed in ~g penicillin G / m l / g dry weight. - , not done because pal strains grow very poorly and accu- mulate suppressor mutat ions at alkaline pH [1].

data as the bioassay measures other (]3-1actamase- sensitive) antibiotics (including the intermediate isopenicillin N) in addition to penicillin G. Never- theless, the broad trends are similar. High pH and pacC- mutations favoured penicillin production whereas low pH and pal- mutations reduced it.

5. DISCUSSION

Formally, penicillin G production by A. nidu- lans follows the same pattern of pH regulation as biosynthesis of alkaline phosphatase with highest levels achieved at alkaline pH and in the presence of mutations mimicking it. Thus although the site(s) of pH regulation are not yet known, the pacC + product apparently acts in a negative fash- ion in influencing penicillin titres.

Why penicillin production should be subject to pH regulation is not immediately obvious. How- ever, as a secreted compound, its biological action occurs outside fungal cells. Bacterial competition with fungi is more intense at alkaline than at acid pH [19] and fl-lactam antibiotics are more toxic, at least to many Salmonella spp., at alkaline than at acid pH [20]. Although characterisation of the pH regulatory system centred initially on regu- lation of permeases and extracellular enzymes, it is clear that the system can, in principle, also par- ticipate in regulation of the syntheses of secreted metabolites, penicillin being the first example.

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A C K N O W L E D G E M E N T S

This work was supported by grants to the laboratories of M.W.A. and H.N.A. from the Bio- technology Directorate of the Science and En- gineering Research Counci l through its "Ant ib io - tics Ini t iat ive" in association with ICI, Smi thKl ine Beecham, Glaxo and Celltech to whom we are very grateful. We thank Jeevan Mungroo for tech- nical assistance and Michael Dona ld son for assis- tance with fermenta t ions in l iquid media.

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