Transcription regulation of virulence gene expression in Francisella tularensis

Francisella tularensis is a small, aerobic, Gram-negative bacterium and a facultative intracellular pathogen of animals. It is the etiologic agent of the disease tularemia, which can be fatal in humans. The first identified regulator of virulence gene expression in F. tularensis was named the macrophage growth locus A (MglA) protein because it is required for replication within macrophages. MglA positively influences expression of multiple virulence genes, but the mechansim of this MglA-dependent regulation is unknown. MglA is an ortholog of the Escherichia coli stringent starvation protein A (SspA), which associates with E. coli RNA polymerase (RNAP) and is thought to regulate the expression of particular genes under conditions of stress. F. tularensis also contains an MglA-like protein annotated as SspA. Co-purification studies indicated that MglA and SspA are RNAP-associated proteins in F. tularensis and that the association of MglA with RNAP depends on the presence of SspA. Bacterial two-hybrid assays showed that MglA and SspA directly interact with one another, and genome-wide transcription analyses revealed that the MglA and SspA regulons are nearly identical. Together, these data support the idea that in F. tularensis , MglA and SspA interact with one another to form a complex that associates with RNAP to regulate the expression of virulence genes. F. tularensis also contains two additional critical regulators of virulence gene expression: ppGpp (a small molecule that signals nutrient stress) and PigR (a putative DNA-binding protein). Genome-wide transcription analyses indicated that MglA, SspA, PigR, and ppGpp regulate the same set of genes. Bacterial two-hybrid assays showed that MglA, SspA, and PigR form a three-protein complex, and in vivo crosslinking experiments showed that ppGpp facilitates the interaction between PigR and the MglA-SspA complex. These findings provide a framework for understanding the mechanistic basis for virulence gene control in F. tularensis and uncover a novel mechanism by which ppGpp influences gene expression--by modulating the interaction between a transcription activator and the RNAP-associated MglA-SspA complex.