Chemical-based methods have been developed for transformation of DNA into log phase cells of the budding yeast Saccharomyces cerevisiae with high efficiency. Transformation of early stationary phase cells, e.g., cells grown in overnight liquid cultures or as colonies on plates, is less efficient than log phase cells but is simpler and more adaptable to high throughput projects. In this study we have tested different approaches for transformation of early stationary phase cell cultures and identified a method utilizing polyethylene glycol (PEG), lithium acetate and dimethyl sulfoxide (DMSO) as most efficient. Plasmid DNA transformations using this method could be improved modestly by allowing cells to recover from the chemical treatment in rich broth before plating to selective media. Strong increases in transformation efficiencies were observed when cells were treated briefly with dithiothreitol (DTT). Tests using several different yeast strain backgrounds indicated that DTT treatment could enhance transformation efficiencies by up to 40-fold. Evaluation of multiple parameters affecting the efficiency of the method led to development of an optimized protocol achieving >50,000 transformants per µg DNA in most backgrounds tested.

Bacterial antibiotic persistence is a transient physiological state wherein cells become dormant and thereby evade being killed by antibiotics. Once the antibiotic is removed, bacterial persisters are able to resuscitate and repopulate.

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Edited by Michael IbbaThe Helicobacter pylori Asp-tRNA Asn /Glu-tRNA Gln amidotransferase (Gat-CAB) utilizes an uncommonly hydrophilic,~40 A ammonia tunnel for ammonia/ammonium transport between isolated active sites. Hydrophilicity of this tunnel requires a distinct ammonia transport mechanism, which hypothetically occurs through a series of deprotonation and protonation steps. To explore the initiation of this relay mechanism, the highly conserved tunnel residue D185 (in the GatA subunit) was enzymatically and computationally investigated by comparing D185A, D185N, and D185E mutant enzymes to wild-type GatCAB. Our results indicate that D185 acts as an acid/base residue, participating directly in catalysis. To our knowledge, this is the first example of acid/base chemistry in a glutamine-dependent amidotransferase ammonia tunnel.

Many plasmids used for gene cloning and heterologous protein expression in E. coli cells are low copy number or single copy number plasmids. Extraction of these types of plasmids from small bacterial cell cultures produces low DNA yields. In this study we have quantitated yields of low copy and single copy number plasmid DNAs after growth of cells in four widely used broths (SB, SOC, TB and 2xYT) and compared results to those obtained with LB, the most common E. coli cell growth medium. TB (terrific broth) consistently generated the greatest amount of plasmid DNA, in agreement with its ability to produce higher cell titers. The superiority of TB was primarily due to its high levels of yeast extract (24 g/L) and was independent of glycerol, a unique component of this broth. Interestingly, simply preparing LB with similarly high levels of yeast extract (LB24 broth) resulted in plasmid yields that were equivalent to those of TB. In contrast, increasing ampicillin concentration to enhance plasmid retention did not improve plasmid DNA recovery. These experiments demonstrate that yields of low and single copy number plasmid DNAs from minipreps can be strongly enhanced using simple and inexpensive media.