Control of lipopolysaccharide biosynthesis by FtsH-mediated proteolysis of LpxC is conserved in enterobacteria but not in all gram-negative bacteria

Abstract

Despite the essential function of lipopolysaccharides (LPS) in Gram-negative bacteria, it is largely unknown how the exact amount of this molecule in the outer membrane is controlled. The first committed step in LPS biosynthesis is catalyzed by the LpxC enzyme. In Escherichia coli, the cellular concentration of LpxC is adjusted by the only essential protease in this organism, the membrane-anchored metalloprotease FtsH. Turnover of E. coli LpxC requires a length- and sequence-specific C-terminal degradation signal. LpxC proteins from Salmonella, Yersinia, and Vibrio species carry similar C-terminal ends and, like the E. coli enzyme, were degraded by FtsH. Although LpxC proteins are highly conserved in Gram-negative bacteria, there are striking differences in their C termini. The Aquifex aeolicus enzyme, which is devoid of the C-terminal extension, was stable in E. coli, whereas LpxC from the alphaproteobacteria Agrobacterium tumefaciens and Rhodobacter capsulatus was degraded by the Lon protease. Proteolysis of the A. tumefaciens protein required the C-terminal end of LpxC. High stability of Pseudomonas aeruginosa LpxC in E. coli and P. aeruginosa suggested that Pseudomonas uses a proteolysis-independent strategy to control its LPS content. The differences in LpxC turnover along with previously reported differences in susceptibility against antimicrobial compounds have important implications for the potential of LpxC as a drug target.

FIG. 1.

Comparison of the C-terminal sequences of LpxC from selected Gram-negative bacteria. Sequences identical to LpxC_Ec are underlined, and residues conforming to the C-terminal degradation signal of LpxC_Ec are shown in bold. The length of the LpxC proteins is given as the number of amino acids.

FIG. 2.

Functional expression of LpxC variants from Gram-negative bacteria in E. coli. (A) Expression of LpxC from S. enterica (LpxC_Se; pBO1142) and V. cholerae (LpxC_Vc; pBO1146) impaired growth of E. coli W3110 cells as effectively as the expression of E. coli LpxC (LpxC_Ec; pBO110). Growth of cells containing the vector control (pBO197) is given as a reference. Cultures carrying the corresponding plasmids were grown in LB medium at 30°C supplemented with 0 (▪), 0.1 (⧫), or 0.5% arabinose (▴) for protein induction. (B) E. coli BL21 or W3110 cells showed increased sensitivity against bile salts in MacConkey agar when LpxC from Y. pseudotuberculosis (LpxC_Yp; pBO1173) or A. aeolicus (LpxC_Aa; pBO2381) and R. capsulatus (LpxC_Rc; pBO1718) was expressed. Serial dilutions of cultures (OD₅₈₀ of 0.5) containing the corresponding plasmids were spotted on MacConkey agar plates with 0 and 0.1 mM IPTG or 0 and 100 ng ml⁻¹ AHT. Cells harboring the plasmids pET19b or pBO1721 were used as vector controls, and pBO1702 or pBO2382 was used as a positive control. (C) Induction of active LpxC from A. tumefaciens (LpxC_At; pBO1144) and P. aeruginosa (LpxC_Pa; pBO1177) with 0 (light gray), 0.1 (gray), and 0.5% arabinose (black) resulted in accumulating KDO amounts per cell compared to the vector control (pBO197), as has been shown for LpxC_Ec (pBO110) (16). t, time.

FIG. 3.

Degradation experiments of LpxC proteins from Gram-negative bacteria in E. coli. Stability of plasmid-encoded LpxC from different bacteria was measured in E. coli W3110 or BL21 (WT) and the corresponding ΔftsH strain depending on the expression system. LpxC was encoded on pBO1142 (LpxC_Se), pBO1173 (LpxC_Yp), pBO1146 (LpxC_Vc), pBO1177 (LpxC_Pa), pBO1718 (LpxC_Rc), pBO2381 (LpxC_Aa), and pBO1144 (LpxC_At). Half-lives (T_1/2) of LpxC variants were analyzed using in vivo degradation experiments and Western blot analysis. Chloramphenicol (Cm) was used to block translation. The sample taken before induction of LpxC expression is indicated by a minus sign. Standard deviations were calculated from at least three independent experiments. The asterisk indicates that LpxC_Pa turned out to be a poor protease substrate with a half-life of 78 ± 9.9 min when degradation experiments were performed for 120 min.

FIG. 4.

LpxC proteins from A. tumefaciens and R. capsulatus are Lon substrates in E. coli. Stabilities of LpxC_At and LpxC_Rc were measured in the E. coli Δlon strain and the corresponding parental strain (RH166; WT). The half-lives were analyzed using in vivo degradation experiments and Western blot analysis. Chloramphenicol (Cm) was used to block translation. The sample taken before induction of LpxC expression is indicated by a minus sign. Standard deviations were calculated from three independent experiments.

FIG. 5.

The LpxC C terminus is conserved as a degradation signal. Truncation of 5 or 13 amino acids from the C terminus from LpxC_Se (pBO1172) or LpxC_At (pBO1187), respectively, led to stabilization of these proteins in E. coli. Half-lives of LpxC variants were analyzed using in vivo degradation experiments and Western blot analysis. Chloramphenicol (Cm) was used to block translation. The sample taken before induction of LpxC expression is indicated by a minus sign. Standard deviations were calculated from at least three independent experiments.

FIG. 6.

LpxC_Se is a protease substrate in S. enterica, and the C terminus is crucial for degradation. LpxC_Se or LpxC_SeΔC5 encoded on pBO1142 or pBO1172, respectively, was expressed in S. enterica cells. Chloramphenicol (Cm) was used to block translation. Half-lives were analyzed using in vivo degradation experiments and Western blot analysis. The sample taken before induction of LpxC expression is indicated by a minus sign. Standard deviations were calculated from at least three independent experiments.

FIG. 7.

LpxC_Pa is a poor protease substrate in P. aeruginosa, and overexpression of this enzyme is not toxic in this organism. (A) Stability of LpxC_Pa was measured in P. aeruginosa using gentamicin (Gm) to block translation. The sample taken before induction of LpxC expression is indicated by a minus sign. (B) Overexpression of LpxC_Pa in P. aeruginosa is not toxic. Cultures carrying pBO1745 were grown in LB medium at 30°C supplemented with 0 (▪), 0.5 (⧫), or 1% arabinose (▴) for LpxC_Pa protein induction. Growth of cells containing the vector control (pHERD20T) is given as a reference. Samples of P. aeruginosa cells harboring pBO1745 were taken after 445 min (indicated by the arrow), and LpxC_Pa production was verified using Western blot analysis (C).