Summary Poly‐γ‐glutamate (PGA), a natural polymer, is synthesized by several bacteria (all Gram‐positive), one archaea and one eukaryote. PGA has diverse biochemical properties, enabling it to play different roles, depending on the organism and its environment. Indeed, PGA allows bacteria to survive at high salt concentrations and may also be involved in virulence. The minimal gene sets required for PGA synthesis were recently defined. There are currently two nomenclatures depending on the PGA final status: cap, for ‘capsule’, when PGA is surface associated or pgs, for ‘polyglutamate synthase’, when PGA is released. The minimal gene sets contain four genes termed cap or pgs B, C, A and E. The PGA synthesis complex is membrane‐anchored and uses glutamate and ATP as substrates. Schematically, the reaction may be divided into two steps, PGA synthesis and PGA transport through the membrane. PGA synthesis depends primarily on CapB‐CapC (or PgsB‐PgsC), whereas PGA transport requires the presence, or the addition, of CapA‐CapE (or PgsAA‐PgsE). The synthesis complex is probably responsible for the stereochemical specificity of PGA composition. Finally, PGA may be anchored to the bacterial surface or released. An additional enzyme is involved in this reaction: either CapD, a γ‐glutamyl‐transpeptidase that catalyses anchorage of the PGA, or PgsS, a hydrolase that facilitates release. The anchoring of PGA to the bacterial surface is important for virulence. All cap genes are therefore potential targets for inhibitors specifically blocking PGA synthesis or anchorage.

Summary Several examples of bacterial surface‐structure anchoring have been described, but they do not include polyglutamate capsule. Bacillus anthracis capsule, which is composed only of poly‐γ‐ d‐glutamate, is one of the two major virulence factors of the bacterium. We analysed its anchoring. We report that the polyglutamate is anchored directly to the peptidoglycan and that the bond is covalent. We constructed a capD mutant strain, capD being the fourth gene of the capsule biosynthetic operon. The mutant bacilli are surrounded by polyglutamate material that is not covalently anchored. Thus, CapD is required for the covalent anchoring of polyglutamate to the peptidoglycan. Sequence similarities suggest that CapD is a γ‐glutamyltranspeptidase. Furthermore, CapD is cleaved at the γ‐glutamyltranspeptidase consensus cleavage site, and the two subunits remain associated, as necessary for γ‐glutamyltranspeptidase activity. Other Gram‐positive γ‐glutamyltranspeptidases are secreted, but CapD is located at the Bacillus surface, associated both with the membrane and the peptidoglycan. Polyglutamate is hydrolysed by CapD indicating that it is a CapD substrate. We suggest that CapD catalyses the capsule anchoring reaction. Interestingly, the CapD– strain is far less virulent than the parental strain.

Francisella tularensis is a highly infectious bacterial pathogen, responsible for the zoonotic disease tularemia. We screened a bank of transposon insertion mutants of F. tularensis subsp. holarctica LVS for colony morphology alterations and selected a mutant with a transposon insertion in wbtA, the first gene of the predicted lipopolysaccharide O-antigen gene cluster. Inactivation of wbtA led to the complete loss of O antigen, conferred serum sensitivity, impaired intracellular replication, and severely attenuated virulence in the mouse model. Notably, this mutant afforded protection against a challenge against virulent LVS.