Extracellular zinc induces phosphoethanolamine addition to Pseudomonas aeruginosa lipid A via the ColRS two-component system

Abstract

Gram-negative bacteria survive harmful environmental stressors by modifying their outer membrane. Much of this protection is afforded upon remodeling of the lipid A region of the major surface molecule lipopolysaccharide (LPS). For example, the addition of cationic substituents, such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) and phosphoehthanolamine (pEtN) at the lipid A phosphate groups, is often induced in response to specific environmental flux stabilizing the outer membrane. The work herein represents the first report of pEtN addition to Pseudomonas aeruginosa lipid A. We have identified the key pEtN transferase which we named EptAPa and characterized its strict activity on only one position of lipid A, contrasting from previously studied EptA enzymes. We further show that transcription of eptAP a is regulated by zinc via the ColRS two-component system instead of the PmrAB system responsible for eptA regulation in E. coli and Salmonella enterica. Further, although L-Ara4N is readily added to the same position of lipid A as pEtN under certain environmental conditions, ColR specifically induces pEtN addition to lipid A in lieu of L-Ara4N when Zn2+ is present. The unique, specific regulation of eptAP a transcription and enzymatic activity described in this work demonstrates the tight yet inducible control over LPS modification in P. aeruginosa.

Fig 1

Lipid A structure of P. aeruginosa with and without inducible modifications. A) Canonical, hexa-acylated, bis-phosphorylated lipid A structure is shown in black. Phosphorylation of the lipid A phosphate groups by LpxT, which occurs in standard growth media, is indicated in brown. B) Inducible modifications to P. aeruginosa lipid A are indicated in color, including addition of a palmitate chain by PagP (green), removal of the 3-hydroxydecanoate acyl chain by PagL (pink), hydroxylation of the C12 secondary acyl chain(s) by LpxO (orange), addition of L-Ara4N at the lipid A phosphate groups by ArnT (blue), and pEtN addition by EptA (red).

Fig 2

Heterologous expression of a P. aeruginosa eptA ortholog results in pEtN addition to the lipid A. A) Cells were grown in MOPS minimal medium. Major ³²P-labeled lipid A species are indicated with a cartoon corresponding to the lipid A structure; colors of modification groups are the same as those used in Fig. 1. Expression of PA14_39020 (eptA_Pa) in P. aeruginosa results in modified lipid A species. B) MALDI-TOF MS analysis of PA14 + empty vector grown in MOPS minimal medium reveals no pEtN addition to the molecule, while C) analysis of PA14 + peptA_Pa shows pEtN modification of the lipid A. The fractions most representative of pEtN modification are shown.

Fig 3

EptA_Pa adds pEtN exclusively to the lipid A 4' phosphate group. A) Cells were grown in LB broth. Major ³²P-labeled lipid A species are indicated with a cartoon corresponding to the lipid A structure; colors of modification groups are the same as those used in Fig. 1. Heterologous expression of eptA_Pa in BN2 results in a pEtN-modified species, while expression of either the lpxE_Fn or lpxF_Fn phosphatase results in an increased of monophosphorylated species. Co-expression of lpxE_Fn and eptA_Pa results in pEtN addition to the 1-dephosphorylated lipid A molecule, while no pEtN addition of 4' –dephosphorylated species is detected. B) MALDI-TOF analysis of lipid A isolated from BN2 coexpressing lpxE_Fn and eptA_Pa corroborates the presence of a monophosphorylated, pEtN-modified species. C) MALDI-TOF analysis of lipid A isolated from BN2 coexpressing lpxF_Fn and eptA_Pa reveals that when the 4' phosphate group is removed, pEtN addition does not occur.

Fig 4

Zn²⁺ induces transcription of eptA_Pa. A) Cells were grown in LB broth. Major ³²P-labeled lipid A species are indicated with a cartoon corresponding to the lipid A structure; colors of modification groups are the same as those used in Fig. 1. Both heterologous expression of eptA_Pa as well as addition of 2mM ZnSO₄ to the media results in pEtN addition to lipid A. This modification is not detectable in the eptA_Pa mutant, but restored upon complementation with peptA_nprom. B) Relative gene expression of eptA_Pa and arnT in response to Zn²⁺. Transcription of eptA_Pa is induced by 2mM ZnSO₄ approximately 21-fold. Zn²⁺ downregulates arnT transcription by >4-fold. Ratios were standardized relative to expression of the housekeeping control gene, clpX. C), D) and E). MALDI-TOF MS analysis of lipid A prepared from cells grown in LB broth. C) Analysis of PA14 + 2mM ZnSO₄ reveals pEtN addition to lipid A, while D) ΔeptA_Pa + 2mM ZnSO₄ shows no pEtN modification, but instead L-Ara4N addition. E) Complementation of ΔeptA_Pa with peptA_nprom restores pEtN addition to the lipid A in response to Zn²⁺. The fractions most representative of pEtN modification are shown.

Fig 5

The two-component system response regulator ColR activates eptA_Pa transcription. A) Putative eptA_Pa promoter ColR binding sites are in bold and boxed; nucleotides that deviate from the conserved recognition sequence in P. putida ((T/C)(T/C)NA(C/G)NN(T/C)TTTTT(C/G)AC) are indicated in red. The number of base pairs between ColR sites or upstream of the start codon is indicated. B) Semi-quantitative RT-PCR of cDNA prepared from cells grown in MOPS minimal medium. While eptA_Pa is not transcribed in PA14, expression of colR in trans results in eptA_Pa transcription. C) Lipid A was isolated from ³²P-labeled cells grown in MOPS minimal medium and separated by TLC. Only expression of the colR response regulator, and not pmrA or phoP, results in pEtN modification of lipid A. D) MALDI-TOF MS analysis of PA14 + pcolR grown in MOPS minimal medium reveals pEtN-modified lipid A. The fraction most representative of pEtN modification is shown.

Fig 6

Deletion of colR results in loss of Zn²⁺-induced pEtN modification of P. aeruginosa lipid A. A) Lipid A was isolated from ³²P-labeled cells grown in LB broth and separated by TLC. While pEtN modification of lipid A is detectable for PA14 + 1mM ZnSO₄, no such modification occurs in PA14ΔcolR in response to Zn²⁺. Modification is restored in the complemented mutant. B) Relative gene expression of eptA_Pa and arnT in response to Zn²⁺ in the ΔcolR mutant or complemented mutant. Transcription of eptA_Pa in the presence of 1mM ZnSO₄ is induced >4-fold in a ColR-dependent manner. An approximately 10-fold decrease in arnT transcription in the presence of 1mM ZnSO₄ is also dependent on ColR. Ratios were standardized relative to expression of the housekeeping control gene, clpX. C) and D). MALDI-TOF MS analysis of lipid A prepared from cells grown in LB broth. C) No pEtN modification is detected in the PA14ΔcolR mutant grown in LB + 1mM ZnSO₄. D) Complementation of PA14ΔcolR with pcolR_nprom restores the Zn²⁺-dependent pEtN addition to the lipid A. The fractions most representative of pEtN modification are shown.

Fig 7

Proposed model of pEtN addition to P. aeruginosa lipid A. Upon sensing excess Zn²⁺, the ColS sensor kinase (green) autophosphorylates and transfers a phosphate group to the response regulator ColR (green). ColR then acts as a transcription factor, inducing transcription of eptA_Pa (red) while inhibiting that of arnT (blue). EptA_Pa protein is synthesized and transfers pEtN to the 4′-phosphate group of lipid A in the inner membrane. Lipid A is then transported to the bacterial cell surface. Following transport to the outer membrane, the 3-hydroxydecanoate acyl chain is removed by PagL (indicated in the model). In some instances, PagP can modify the lipid A (not shown). Cellular components are labelled as follows: OM, outer membrane; P, periplasm; IM, inner membrane; C, cytoplasm).