Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers.

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity.

Pharmacological validation of Trypanosoma brucei phosphodiesterases B1 and B2 as druggable targets for African sleeping sickness.

Neglected tropical disease drug discovery requires application of pragmatic and efficient methods for development of new therapeutic agents. In this report, we describe our target repurposing efforts for the essential phosphodiesterase (PDE) enzymes TbrPDEB1 and TbrPDEB2 of Trypanosoma brucei , the causative agent for human African trypanosomiasis (HAT). We describe protein expression and purification, assay development, and benchmark screening of a collection of 20 established human PDE inhibitors.

An alternative pathway for reduced folate biosynthesis in bacteria and halophilic archaea.

Whereas tetrahydrofolate is an essential cofactor in all bacteria, the gene that encodes the enzyme dihydrofolate reductase (DHFR) could not be identified in many of the bacteria whose genomes have been entirely sequenced. In this communication we show that the halophilic archaea Halobacterium salinarum and Haloarcula marismortui contain genes coding for proteins with an N-terminal domain homologous to dihydrofolate synthase (FolC) and a C-terminal domain homologous to dihydropteroate synthase (FolP). These genes are able to complement a Haloferax volcanii mutant that lacks DHFR.

Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli.

A strain of Escherichia coli missing three members of the thioredoxin superfamily, thioredoxins 1 and 2 and glutaredoxin 1, is unable to grow, a phenotype presumed to be due to the inability of cells to reduce the essential enzyme ribonucleotide reductase. Two classes of mutations can restore growth to such a strain. First, we have isolated a collection of mutations in the gene for the protein glutaredoxin 3 that suppress the growth defect.

Functions of thiol-disulfide oxidoreductases in E. coli: redox myths, realities, and practicalities.

A large family of enzymes contributes to the thiol-disulfide redox environment of the cells of most organisms. These proteins belong to pathways that carry out a variety of reactions, including the promotion of disulfide bond formation in extracytoplasmic proteins, the isomerization of proteins with incorrect disulfide bonds, and the reduction of disulfide bonds in the active sites of cytoplasmic proteins. Although the redox activities of these proteins measured in vitro often is consistent with the role (oxidant or reductant) these proteins perform in vivo, this is not always the case.

Development of a gene knockout system for the halophilic archaeon Haloferax volcanii by use of the pyrE gene.

So far, the extremely halophilic archaeon Haloferax volcanii has the best genetic tools among the archaea. However, the lack of an efficient gene knockout system for this organism has hampered further genetic studies. In this paper we describe the development of pyrE-based positive selection and counterselection systems to generate an efficient gene knockout system.

Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni.

A search of the complete genome sequence of the halophilic archaeon Halobacterium salinarum failed to identify a gene homologous to the thymidylate synthase (thyA) gene present in the closely related Haloferax volcanii. To understand the source of thymidine synthesis in Hbt. salinarum, a genomic library of Hbt.