Regulation and evolution of the penicillin biosynthesis gene cluster of Aspergillus nidulans

Penicillin is one of the most important antibiotics and consequently, its biosynthesis is probably the best understood pathway in fungal secondary metabolism. However, there is little knowledge about the environmental signals and their transmission modulating the expression of the structural genes. In this work, the light-dependent regulator velvet A (VeA) and a central protein kinase C (PkcA) were found to be part of the penicillin biosynthesis regulating network in the fungus Aspergillus nidulans. Evolution of the gene cluster that contains genes of apparently both bacterial and eukaryotic origin has not been fully elucidated yet. The final step of penicillin biosynthesis is catalysed by isopenicillin N acyltransferase, which was characterised in this work, and that is encoded by the aatA gene. Because there is no bacterial homolog, its evolutionary origin remained obscure. As shown in this work, disruption of aatA still enabled penicillin production. Genome mining led to the discovery of the aatB gene which has a similar structure and expression pattern as aatA. Disruption of aatB resulted in a reduced penicillin titre. Surface plasmon resonance analysis and Northern blot analysis indicated that the promoters of both aatA and aatB are bound and regulated by the same transcription factors AnCF and AnBH1. In contrast to aatA, aatB does not encode a peroxisomal targeting signal (PTS1). Overexpression of a mutated aatBPTS1 gene in an aatA disruption strain (leading to peroxisomal localisation of AatB) increased the penicillin titre more than overexpression of the wild-type aatB. Homologs of aatA are exclusively part of the penicillin biosynthesis gene cluster, whereas aatB homologs also exist in non-producing fungi. These findings suggest that aatB is a paralog of aatA. They extend the model of evolution of the penicillin biosynthesis gene cluster by recruitment of a biosynthesis gene and its cis-regulatory sites upon gene duplication

Protein Kinase C (PkcA) of Aspergillus nidulans Is Involved in Penicillin Production

The biosynthesis of the β-lactam antibiotic penicillin in the filamentous fungus Aspergillus nidulans is catalyzed by three enzymes that are encoded by the acvA, ipnA, and aatA genes. A variety of cis-acting DNA elements and regulatory factors form a complex regulatory network controlling these β-lactam biosynthesis genes. Regulators involved include the CCAAT-binding complex AnCF and AnBH1. AnBH1 acts as a repressor of the penicillin biosynthesis gene aatA. Until now, however, little information has been available on the signal transduction cascades leading to the transcription factors. Here we show that inhibition of protein kinase C (Pkc) activity in A. nidulans led to cytoplasmic localization of an AnBH1-enhanced green fluorescent protein (EGFP) fusion protein. Computer analysis of the genome and screening of an A. nidulans gene library revealed that the fungus possesses two putative Pkc-encoding genes, which we designated pkcA and pkcB. Only PkcA showed all the characteristic features of fungal Pkc's. Production of pkcA antisense RNA in A. nidulans led to reduced growth and conidiation in Aspergillus minimal medium, while in fermentation medium it led to enhanced expression of an aatAp-lacZ gene fusion, reduced pencillin production, and predominantly cytoplasmic localization of AnBH1. These data agree with the finding that inhibition of Pkc activity prevented nuclear localization of AnBH1-EGFP. As a result, repression of aatA expression was relieved. The involvement of Pkc in penicillin biosynthesis is also interesting in light of the fact that in the yeast Saccharomyces cerevisiae, Pkc plays a major role in maintaining cell integrity

Contribution of Peroxisomes to Penicillin Biosynthesis in Aspergillus nidulans▿ †

Peroxisomal localization of the third enzyme of the penicillin biosynthesis pathway of Aspergillus nidulans, acyl-coenzyme A:IPN acyltransferase (IAT), is mediated by its atypical peroxisomal targeting signal 1 (PTS1). However, mislocalization of IAT by deletion of either its PTS1 or of genes encoding proteins involved in peroxisome formation or transport does not completely abolish penicillin biosynthesis. This is in contrast to the effects of IAT mislocalization in Penicillium chrysogenum

Phenotypic variation of salmonella in host tissues delays eradication by antimicrobial chemotherapy

Antibiotic therapy often fails to eliminate a fraction of transiently refractory bacteria, causing relapses and chronic infections. Multiple mechanisms can induce such persisters with high antimicrobial tolerance in vitro, but their in vivo relevance remains unclear. Using a fluorescent growth rate reporter, we detected extensive phenotypic variation of Salmonella in host tissues. This included slow-growing subsets as well as well-nourished fast-growing subsets driving disease progression. Monitoring of Salmonella growth and survival during chemotherapy revealed that antibiotic killing correlated with single-cell division rates. Nondividing Salmonella survived best but were rare, limiting their impact. Instead, most survivors originated from abundant moderately growing, partially tolerant Salmonella. These data demonstrate that host tissues diversify pathogen physiology, with major consequences for disease progression and control