Characterization and engineering of branched short-chain dicarboxylate metabolism in Pseudomonas reveals resistance to fungal 2-hydroxyparaconate.

In recent years branched short-chain dicarboxylates (BSCD) such as itaconic acid gained increasing interest in both medicine and biotechnology. Their use as building blocks for plastics urges for developing microbial upcycling strategies to provide sustainable end-of-life solutions. Furthermore, many BSCD exhibit anti-bacterial properties or exert immunomodulatory effects in macrophages, indicating a medical relevance for this group of molecules. For both of these applications, a detailed understanding of the microbial metabolism of these compounds is essential. In this study, the metabolic pathway of BSCD degradation from Pseudomonas aeruginosa PAO1 was studied in detail by heterologously transferring it to Pseudomonas putida. Heterologous expression of the PA0878-0886 itaconate metabolism gene cluster enabled P. putida KT2440 to metabolize itaconate, (S)- and (R)-methylsuccinate, (S)-citramalate, and mesaconate. The functions of the so far uncharacterized genes PA0879 and PA0881 were revealed and proven to extend the substrate range of the core degradation pathway. Furthermore, the uncharacterized gene PA0880 was discovered to encode a 2-hydroxyparaconate (2-HP) lactonase that catalyzes the cleavage of the itaconate derivative 2-HP to itatartarate. Interestingly, 2-HP was found to inhibit growth of the engineered P. putida on itaconate. All in all, this study extends the substrate range of P. putida to include BSCD for bio-upcycling of high-performance polymers, and also identifies 2-HP as promising candidate for anti-microbial applications.

Multiple cis-acting sequences mediate upregulation of the MDR1 efflux pump in a fluconazole-resistant clinical Candida albicans isolate.

Overexpression of the MDR1 gene, which encodes a multidrug efflux pump of the major facilitator superfamily, is a frequent cause of resistance to the antimycotic agent fluconazole and other metabolic inhibitors in clinical Candida albicans strains. Constitutive MDR1 overexpression in such strains is caused by mutations in as yet unknown trans-regulatory factors. In order to identify the cis-acting sequences in the MDR1 regulatory region that mediate constitutive MDR1 upregulation, we performed a promoter deletion analysis in the genetic background of an MDR1-overexpressing clinical C. albicans isolate. We found that several different regions in the MDR1 promoter can mediate MDR1 overexpression in this isolate. In contrast, deletion of one of these regions abolished benomyl-induced MDR1 expression in a C. albicans laboratory strain. These results suggest that multiple transcription factors control expression of the MDR1 efflux pump in C. albicans and that the mutation(s) that causes constitutive MDR1 overexpression and drug resistance in clinical C. albicans isolates affects the activities of several of these transcription factors.

Overexpression of the MDR1 gene is sufficient to confer increased resistance to toxic compounds in Candida albicans.

Overexpression of MDR1, which encodes a membrane transport protein of the major facilitator superfamily, is one mechanism by which the human fungal pathogen Candida albicans can develop increased resistance to the antifungal drug fluconazole and other toxic compounds. In clinical C. albicans isolates, constitutive MDR1 overexpression is accompanied by the upregulation of other genes, but it is not known if these additional alterations are required for Mdr1p function and drug resistance. To investigate whether MDR1 overexpression is sufficient to confer a drug-resistant phenotype in C. albicans, we expressed the MDR1 gene from the strong ADH1 promoter in C. albicans laboratory strains that did not express the endogenous MDR1 gene as well as in a fluconazole-resistant clinical C. albicans isolate in which the endogenous MDR1 alleles had been deleted and in a matched fluconazole-susceptible isolate from the same patient. Forced MDR1 overexpression resulted in increased resistance to the putative Mdr1p substrates cerulenin and brefeldin A, and this resistance did not depend on the additional alterations which occurred during drug resistance development in the clinical isolates. In contrast, artificial expression of the MDR1 gene from the ADH1 promoter did not enhance or only slightly enhanced fluconazole resistance, presumably because Mdr1p expression levels in the transformants were considerably lower than those observed in the fluconazole-resistant clinical isolate. These results demonstrate that MDR1 overexpression in C. albicans is sufficient to confer resistance to some toxic compounds that are substrates of this efflux pump but that the degree of resistance depends on the Mdr1p expression level.