Efficient PCR-based gene targeting in isolates of the nonconventional yeast Debaryomyces hansenii

Debaryomyces hansenii is a yeast with considerable biotechnological potential as an osmotolerant, stress-tolerant oleaginous microbe. However, targeted genome modification tools are limited and require a strain with auxotrophic markers. Gene targeting by homologous recombination has been reported to be inefficient, but here we describe a set of reagents and a method that allows gene targeting at high efficiency in wild-type isolates. It uses a simple polymerase chain reaction (PCR)-based amplification that extends a completely heterologous selectable marker with 50 bp flanks identical to the target site in the genome. Transformants integrate the PCR product through homologous recombination at high frequency (>75%). We illustrate the potential of this method by disrupting genes at high efficiency and by expressing a heterologous protein from a safe chromosomal harbour site. These methods should stimulate and facilitate further analysis of D. hansenii strains and open the way to engineer strains for biotechnology

Peroxisomal NAD(H) homeostasis in the yeast debaryomyces hansenii depends on two redox shuttles and the NAD+ carrier, Pmp47

Debaryomyces hansenii is considered an unconventional yeast with a strong biotechnological potential, which can produce and store high amounts of lipids. However, relatively little is known about its lipid metabolism, and genetic tools for this yeast have been limited. The aim of this study was to explore the fatty acid β-oxidation pathway in D. hansenii. To this end, we employed recently developed methods to generate multiple gene deletions and tag open reading frames with GFP in their chromosomal context in this yeast. We found that, similar as in other yeasts, the β-oxidation of fatty acids in D. hansenii was restricted to peroxisomes. We report a series of experiments in D. hansenii and the well-studied yeast Saccharomyces cerevisiae that show that the homeostasis of NAD+ in D. hansenii peroxisomes is dependent upon the peroxisomal membrane protein Pmp47 and two peroxisomal dehydrogenases, Mdh3 and Gpd1, which both export reducing equivalents produced during β-oxidation to the cytosol. Pmp47 is the first identified NAD+ carrier in yeast peroxisomes

Impact of Sequential Passaging on Protein Expression of E. coli Using Proteomics Analysis

Urinary tract infection (UTI) is one of the most prevalent bacterial infections in the world affecting the bladder and the kidney. Escherichia coli (E. coli) is the main causative agent of 80–90% of community-acquired UTIs, about 40% of nosocomial UTIs, and 25% of recurrent UTIs. The field of proteomics has emerged as a great tool to analyze expressed proteins to identify possible biomarkers associated with many pathological states and, to the same extent, those associated with bacterial pathogenesis and their ability to cause recurrent infections. Here, in a descriptive cross-sectional pilot study, we employed proteomic techniques to investigate the effects of environmental stress on protein profiles of E. coli simulated by sequential passaging of samples from patients with UTIs to screen for unique proteins that arise under stressful environment and could aid in the early detection of UTIs. Four urine samples were collected from individuals with recurrent UTI and sequentially subcultured; protein samples were extracted from bacterial pellets and analyzed using 2-dimensional gel electrophoresis (2DGE). Protein spots of interest arising from changes in the protein profile were analyzed using liquid chromatography-mass spectrometry (LC-MS/MS) and matched against known databases to identify related proteins. We identified ATPB_ECOBW, ASPA ECOLI, DPS ECOL6, and DCEB ECOLI as proteins associated with higher passaging. We concluded that passaging resulted in identifiable changes in the protein profile of E. coli, namely, proteins that are associated with survival and possible adaptation of bacteria, suggestive of factors contributing to antibiotic resistance and recurrent UTIs. Furthermore, our method could be further used to identify indicator-protein candidates that could be a part of a growing protein database to diagnose and identify causative agents in UTIs