Functional characterization of a Penicillium chrysogenum mutanase gene induced upon co-cultivation with Bacillus subtilis

Abstract

Background: Microbial gene expression is strongly influenced by environmental growth conditions. Comparison of gene expression under different conditions is frequently used for functional analysis and to unravel regulatory networks, however, gene expression responses to co-cultivation with other microorganisms, a common occurrence in nature, is rarely studied under laboratory conditions. To explore cellular responses of the antibiotic-producing fungus Penicillium chrysogenum to prokaryotes, the present study investigates its transcriptional responses during co-cultivation with Bacillus subtilis.

Results: Steady-state glucose-limited chemostats of P. chrysogenum grown under penillicin-non-producing conditions were inoculated with B. subtilis. Physiological and transcriptional responses of P. chrysogenum in the resulting mixed culture were monitored over 72 h. Under these conditions, B. subtilis outcompeted P. chrysogenum, as reflected by a three-fold increase of the B. subtilis population size and a two-fold reduction of the P. chrysogenum biomass concentration. Genes involved in the penicillin pathway and in synthesis of the penicillin precursors and side-chain were unresponsive to the presence of B. subtilis. Moreover, Penicillium polyketide synthase and nonribosomal peptide synthase genes were either not expressed or down-regulated. Among the highly responsive genes, two putative α-1,3 endoglucanase (mutanase) genes viz Pc12g07500 and Pc12g13330 were upregulated by more than 15-fold and 8-fold, respectively. Measurement of enzyme activity in the supernatant of mixed culture confirmed that the co-cultivation with B. subtilis induced mutanase production. Mutanase activity was neither observed in pure cultures of P. chrysogenum or B. subtilis, nor during exposure of P. chrysogenum to B. subtilis culture supernatants or heat-inactivated B. subtilis cells. However, mutanase production was observed in cultures of P. chrysogenum exposed to filter-sterilized supernatants of mixed cultures of P. chrysogenum and B. subtilis. Heterologous expression of Pc12g07500 and Pc12g13330 genes in Saccharomyces cerevisiae confirmed that Pc12g07500 encoded an active α-1,3 endoglucanase.

Conclusion: Time-course transcriptional profiling of P. chrysogenum revealed differentially expressed genes during co-cultivation with B. subtilis. Penicillin production was not induced under these conditions. However, induction of a newly characterized P. chrysogenum gene encoding α-1,3 endoglucanase may enhance the efficacy of fungal antibiotics by degrading bacterial exopolysaccharides.

Figure 1

Dynamics of P. chrysogenum and B. subtilis during co-cultivation. At t = 0 h, a steady-state, glucose-limited chemostat culture of P. chrysogenum, grown in the absence of phenylacetate and at a dilution rate of 0.03 h^-1, was inoculated with B. subtilis cultures. The data represents the t = 0 h normalized biomass concentration of P. chrysogenum (○) and B. subtilis (●). The dashed line represents wash-out at zero specific growth rate. Data points represent average and mean deviation of duplicate experiments.

Figure 2

Change of B. subtilis morphology in chemostat cultures. A: Mixed culture of B. subtilis and P. chrysogenum at 0 h; B. subtilis cells were pregrown in shake-flask culture and addition to a P. chrysogenum glucose-limited chemostat culture. B: Mixed culture of B. subtilis and P. chrysogenum at 72 h. C: pure culture of B. subtilis after 72 h of glucose-limited cultivation. Pictures taken under oil immersion and 1000X magnification using a Zeiss Axio imager D1 and an Axio Camera.

Figure 3

K-mean clustered gene expression profiles of groups of specific interest (Clusters 1 (A), 3 (B) and 4 (C) over the course of co-cultivation of P. chrysogenum with B. subtilis and the results of hypergeometric distribution analysis for enrichment of functional categories. The thick line represents the average of the mean normalized expression data of the genes comprising the cluster. The y-axis represents normalized expression values. Functional categories are mentioned together with their P-value, k representing the number of genes of the corresponding category within the set of differentially expressed genes and n representing the number of genes in the respective functional category in the whole genome.

Figure 4

Expression of P. chrysogenum secondary metabolism genes during co-cultivation with B. subtilis. Heat map of transcript levels of genes encoding NRPS (and -like), PKS (and -like) and hybrid NRPS-PKS in strain Wisconsin 54–1255, grown in aerobic glucose-limited chemostat cultures, 0 h, 5 h, 24 h, 48 h and 72 h after addition of B. subtilis. The expression data were derived from hybridization intensity of Affymetrix DNA microarrays (DSM PENa520255F) (n = 3). The transcript data were Z-normalized for each experiment. Genes that were not expressed in the mixed culture are marked with *and the seven genes whose transcript profile was deemed significantly different from the null situation are marked with #. The numbers between brackets indicate the numbers of NRPS, NRP-like, PKS, PKS-like and hybrids catalogued in the P. chrysogenum genome.

Figure 5

Mutanase production by P. chrysogenum during co-cultivation with B. subtilis. A- fungal biomass (○) and mutanase activity (●) after 0 h, 5 h, 24 h, 48 h and 72 h of co-cultivation with B. subtilis in aerobic, glucose-limited chemostat cultures. B- Expression of the six putative mutanase genes found in the genome of P. chrysogenum during co-cultivation. (●) Pc18g06380, (○) Pc12g13330, (×) Pc13g0113, (∆) Pc15g0400 (▲) Pc12g07500, (□) Pc13g12810. Data are presented as mean and standard deviation of three independent replicates.

Figure 6

Mutanase production by P. chrysogenum after addition of cell-free supernatants from mixed cultures of P. chrysogenum and B. subtilis. Mutanase activity was measured after addition of supernatant samples of mixed cultures of P. chrysogenum and B. subtilis grown in shake flasks (○), of a 24-h mixed chemostat culture (●) to a 24-h batch culture of P. chrysogenum and in culture grown in presence of 0.1% of mutan (α 1,3 glucan) (▲), of 0.1% of pullulan (α-1,4-α-1,6-glucan), of 0.1% of dextran (α1,6 glucan) (×) and 0.1% of β1,3 glucan (∆) The presented data are averages and standard deviations of triplicate experiments. We It is worth mentioning that the residual mutanase activity (0.020 U.ml^-1) observed at the start of the P. chrysogenum shake flask cultures spiked with supernatant of mixed culture originated from the inoculum. Data are presented as average ± mean deviation of three independent experiments.

Figure 7

Comparison of putative mutanase (α-1,3 glucanase) encoding genes in P. chrysogenum. Top panel- Phylogenetic tree based on Clustal-X alignment of predicted amino acid sequences of P. chrysogenum Pc12g07500 (accession number: CAP80377.1), Pc12g13330 (CAP80960), Pc13g12810 (CAP92350), Pc13g01130 (CAP91182), Pc15g01400 (CAP83026), Pc18g06380 (CAP94862), Trichoderma harzianum MutA (ADZ45396.1) and Talaromyces purpurogenus MutA (AF214481_1). The tree was constructed using TREECON for Windows. Bottom panel- Amino acid sequence alignment of the previously mentioned proteins. Amino acids highlighted in green represent positions that are conserved in at least 50% of the aligned sequences.