The atypical peptidoglycan of Francisella tularensis

Peptidoglycan (PG), the main component of the bacterial cell wall, plays a fundamental role in maintaining cell shape, integrity, and resistance to osmotic pressure. Preliminary observations suggested that unlike typical bacteria, Francisella tularensis infrequently produces detectable PG while growing in rich liquid media. However, increasing salt concentration induces the production of PG in all bacteria. In this study, we confirmed and extended these observations by incubating bacteria with fluorescent D-alanine (which integrates into the PG peptide stem during synthesis). Fluorescence microscopy revealed that very few F. tularensis bacteria produce detectable PG in low salt media, while seemingly all individual bacteria produce this layer in high salt media. A transposon mutagenesis screen yielded sixteen F. tularensis mutant isolates capable of growing immediately on high salt media. Interestingly, isolate Tn4 did not appear to synthesize any detectable levels of PG regardless of the level of salt in the media. Sequencing and subsequent microscopy revealed that this transposon abrogated expression of FTL_1305 (mviN). To identify candidate genes responsible for peptidoglycan modulation, RNA-seq was conducted on F. tularensis bacteria cultured in low or high salt conditions. This analysis revealed several ORFs of interest. Future LC/MS studies will examine the makeup of muropeptides to investigate whether the composition of the tetrapeptide of PG crosslinks is modulated.