Identification of the lipopolysaccharide modifications controlled by the Salmonella PmrA/PmrB system mediating resistance to Fe(III) and Al(III)

Abstract

Iron is an essential metal but can be toxic in excess. While several homeostatic mechanisms prevent oxygen-dependent killing promoted by Fe(II), little is known about how cells cope with Fe(III), which kills by oxygen-independent means. Several Gram-negative bacterial species harbour a regulatory system - termed PmrA/PmrB - that is activated by and required for resistance to Fe(III). We now report the identification of the PmrA-regulated determinants mediating resistance to Fe(III) and Al(III) in Salmonella enterica serovar Typhimurium. We establish that these determinants remodel two regions of the lipopolysaccharide, decreasing the negative charge of this major constituent of the outer membrane. Remodelling entails the covalent modification of the two phosphates in the lipid A region with phosphoethanolamine and 4-aminoarabinose, which has been previously implicated in resistance to polymyxin B, as well as dephosphorylation of the Hep(II) phosphate in the core region by the PmrG protein. A mutant lacking the PmrA-regulated Fe(III) resistance genes bound more Fe(III) than the wild-type strain and was defective for survival in soil, suggesting that these PmrA-regulated lipopolysaccharide modifications aid Salmonella's survival and spread in non-host environments.

Fig. 1

Increased Fe(III) binding to the bacterial cell surface of Fe(III) hypersensitive mutants and rescue by polymyxin B nonapeptide. A. Association of ⁵⁹Fe with wild-type (14028s), ΔpmrAB (EG13937), ΔpmrC (EG16626), Δugd (EG16627), ΔpmrG (EG16628) and ΔpmrC ugd pmrG (EG16639) strains, and with the ΔpmrC ugd pmrG mutant expressing the pmrC (EG15771), ugd (EG15753), or pmrG (EG15751) genes. B. Survival of wild-type(14028s), ΔpmrAB (EG13937) and ΔpmrC ugd pmrG (EG16639) strains after incubation in the presence of FeCl₃ in the presence or absence of the non-toxic polymyxin B nonapeptide or with no compounds added.

Fig. 2

PmrG is a phosphatase that targets the Hep(II) phosphate and localizes to the periplasm. A. Schematic representation of the LPS structure indicating the phosphates that are targeted by the Fe(III) resistance determinants. The PmrC protein mediates the modification of the lipid A phosphates with phosphoehanolamine whereas the Ugd and PbgPE proteins modifies the lipid A phosphates with 4-aminoarabinose. The PmrG protein removes the phosphate from Hep(II) in the inner core region of the LPS, which is normally introduced by the RfaY protein. B. SDS-PAGE gel of the purified PmrG-His₆ protein prepared as described in Experimental procedures. C. Phosphatase activity of the purified PmrG-His protein was determined on core OS prepared from wild-type (14028s) or the phosphate-deficient Hep(II) ΔrfaY mutant (EG16916). The PhoP-His₆ protein was used as negative control. D. Subcellular localization of chromosomally encoded PmrG-FLAG protein was determined by Western blotting of cytoplasmic and periplasmic extracts from strain EG16142. Purity of the preparations was verified by measuring the β-galactosidase activity of the cytoplasmic and periplasmic fractions.

Fig. 3

Fe(III) resistance determinants are necessary for survival in soil. Fe(III) sensitive mutants are impaired for growth in soil (EG13937 and EG16639). Survival ratio is the ratio of mutants to wild-type Salmonella (14028s).