Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

No abstract

Termination of RNA Polymerase II (Pol II) activity serves a vital cellular role by separating ubiquitous transcription units and influencing RNA fate and function. In the yeast Saccharomyces cerevisiae, Pol II termination is carried out by cleavage and polyadenylation factor (CPF-CF) and Nrd1-Nab3-Sen1 (NNS) complexes, which operate primarily at mRNA and non-coding RNA genes, respectively. Premature Pol II termination (attenuation) contributes to gene regulation, but there is limited knowledge of its prevalence and biological significance. In particular, it is unclear how much crosstalk occurs between CPF-CF and NNS complexes and how Pol II attenuation is modulated during stress adaptation. In this study, we have identified an attenuator in the DEF1 DNA repair gene, which includes a portion of the 5′-untranslated region (UTR) and upstream open reading frame (ORF). Using a plasmid-based reporter gene system, we conducted a genetic screen of 14 termination mutants and their ability to confer Pol II read-through defects. The DEF1 attenuator behaved as a hybrid terminator, relying heavily on CPF-CF and Sen1 but without Nrd1 and Nab3 involvement. Our genetic selection identified 22 cis-acting point mutations that clustered into four regions, including a polyadenylation site efficiency element that genetically interacts with its cognate binding-protein Hrp1. Outside of the reporter gene context, a DEF1 attenuator mutant increased mRNA and protein expression, exacerbating the toxicity of a constitutively active Def1 protein. Overall, our data support a biologically significant role for transcription attenuation in regulating DEF1 expression, which can be modulated during the DNA damage response.