Pyruvate Formate-Lyase Enables Efficient Growth of Escherichia coli on Acetate and Formate.

Pyruvate formate-lyase (PFL) is a ubiquitous enzyme that supports increased ATP yield during sugar fermentation. While the PFL reaction is known to be reversible in vitro, the ability of PFL to support microbial growth by condensing acetyl-CoA and formate in vivo has never been directly tested. Here, we employ Escherichia coli mutant strains that cannot assimilate acetate via the glyoxylate shunt and use carbon labeling experiments to unequivocally demonstrate PFL-dependent co-assimilation of acetate and formate.

Measurement and manipulation of cell size parameters in fission yeast.

Cells usually grow to a certain size before they divide. The fission yeast Schizosaccharomyces pombe is an established model to dissect the molecular control of cell size homeostasis and cell cycle. In this chapter, we describe two simple methods to: (1) precisely compute geometrical parameters (cell length, diameter, surface, and volume) of single growing and dividing fission yeast cells with image analysis scripts and (2) manipulate cell diameter with microfabricated chambers and assess for cell size at division.

Silencing of antibiotic resistance in E. coli with engineered phage bearing small regulatory RNAs.

In response to emergent antibiotic resistance, new strategies are needed to enhance the effectiveness of existing antibiotics. Here, we describe a phagemid-delivered, RNA-mediated system capable of directly knocking down antibiotic resistance phenotypes. Small regulatory RNAs (sRNAs) were designed to specifically inhibit translation of chloramphenicol acetyltransferase and kanamycin phosphotransferase.

In situ characterization of mycobacterial growth inhibition by lytic enzymes expressed in vectorized E. coli.

The emergence of extremely drug resistant Mycobacterium tuberculosis necessitates new strategies to combat the pathogen. Engineered bacteria may serve as vectors to deliver proteins to human cells, including mycobacteria-infected macrophages. In this work, we target Mycobacterium smegmatis, a nonpathogenic tuberculosis model, with E.