Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION

Abstract

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.

Keywords: Corynebacteriales; DAP amidation; antibiotics; bacterial metabolism; cell wall; enzyme; gene knockout; glutaminase; lysozyme; peptidoglycan.

FIGURE 1.

Multiple sequence alignment of LtsA homologues from different bacterial species (ClustalW). Protein sequences are as follows: AsnB1 from L. plantarum WCFS1 (GI:28377797); LtsA from C. glutamicum ATCC 13032 (GI:62391036); LtsA from R. erythropolis PR4 (GI:226307090); and AsnB from M. tuberculosis H37RV (GI:15609338). Identical and similar amino acid residues are highlighted in blue and green, respectively. * indicates positions that have a single and fully conserved residue; : indicates conservation between groups of strongly similar properties, and . indicates conservation between groups of weakly similar properties.

FIGURE 2.

Growth curves of the wild-type (○) and ΔltsA (●) C. glutamicum strains in BHI medium at 30 °C.

FIGURE 3.

Morphologies of C. glutamicum strains. A and B, optical micrographs of exponentially growing C. glutamicum ATCC 13032 (A) and ΔltsA mutant (B) cells. Arrows indicate examples of cells exhibiting abnormal shapes. C–F, electron micrographs of frozen hydrated C. glutamicum ATCC 13032 cells (C and E) and of ΔltsA mutant cells exhibiting an irregular shape (D and F). The inset in E and F is an enlargement of the cell envelope. Arrows in C and D show the electron-dense granules.

FIGURE 4.

HPLC analysis of peptidoglycan fragments (muropeptides) generated by digestion of peptidoglycan from wild-type (WT) and ΔltsA C. glutamicum strains with muramidases (lysozyme and mutanolysin). Muropeptides were reduced by sodium borohydride and separated by HPLC on a 3-μm ODS-Hypersil column (4.6 × 250 mm), using a gradient of methanol (from 0 to 25% in 120 min) in 50 mm sodium phosphate buffer, pH 4.5, at a flow rate of 0.5 ml/min. mAU, absorbance unit × 10³ at 207 nm. Their identity is indicated in Table 1.

FIGURE 5.

MS-MS analysis of the main monomer muropeptides from wild-type and ΔltsA C. glutamicum strains. A, fragmentation of a reduced disaccharide tetrapeptide containing an amidated DAP residue. This muropeptide was isolated from wild-type C. glutamicum strain ATCC 13032 (peak C in Fig. 4). Panel a, fragmentation of the ion at m/z 940.6; panel b, inferred structure. B, fragmentation of a reduced disaccharide tetrapeptide containing a nonamidated DAP residue. This muropeptide was isolated from ΔltsA C. glutamicum strain (peak 2 in Fig. 4). Panel a, fragmentation of the ion at m/z 941.6; panel b, inferred structure.

FIGURE 6.

Bacteriolytic effect of expression of LtsA enzyme from C. glutamicum in E. coli cells. C43(DE3)(pLysS) cells carrying either the plasmid vector pET2160 (○), the pMLD288 plasmid expressing wild-type LtsA_Cg (●), or the pMLD290 plasmid expressing His-tagged LtsA_Cg (■) were grown at 37 °C in 2YT medium. When the OD₆₀₀ reached 0.2, expression of the LtsA protein was induced by addition of 1 mm IPTG. An arrest of growth followed by cell lysis was observed about 90–120 min later.

FIGURE 7.

HPLC analysis of muropeptides released by digestion of peptidoglycan from wild-type and LtsA_Cg-expressing E. coli cells with muramidases (lysozyme and mutanolysin). See the legend of Fig. 4 for details on HPLC conditions. mAU, absorbance unit × 10³ at 207 nm. The identity of the muropeptides is indicated in Table 3.

FIGURE 8.

Analysis of the pools of amidated and nonamidated forms of the peptidoglycan UDP-MurNAc pentapeptide nucleotide precursor in C. glutamicum (A) and E. coli (B) strains. Nucleotide precursors were extracted from exponentially growing cells as described under “Experimental Procedures.” Aliquots were analyzed by HPLC on a column of μ-Bondapak C₁₈ (7.8 × 300 mm). Elution at 3 ml/min was with 50 mm ammonium formate for 15 min at pH 3.35, followed by a gradient of pH, from 3.35 to 4.75, applied between 15 and 50 min.

FIGURE 9.

In vitro LtsA glutamine amidotransferase activity assays. A and B, MraY-catalyzed reaction of exchange between [¹⁴C]UMP and the UMP moiety of UDP-MurNAc pentapeptide was assayed in membrane fractions prepared from control (A) or LtsA_Cg-expressing (B) E. coli cells. Reaction mixtures containing as substrates C₅₅-P (provided by membranes), ¹⁴C-radiolabeled UMP, UDP-MurNAc pentapeptide, ATP, and glutamine were incubated with membrane extracts for 30 min at 37 °C. Amidated UDP-MurNAc pentapeptide was formed only when LtsA enzyme was present. C and D, radiolabeled UDP-MurNAc pentapeptide was incubated with the membrane extract from LtsA_Cg-expressing E. coli cells, ATP and glutamine, in the absence (C) or presence (D) of tunicamycin (an MraY inhibitor). Amidated UDP-MurNAc pentapeptide was formed only when MraY was functional, demonstrating that LtsA accepts lipid I but not UDP-MurNAc pentapeptide as a substrate. In all cases, the radiolabeled substrate and products were separated by HPLC as described under “Experimental Procedures.”

FIGURE 10.

LtsA-catalyzed amidotransferase reaction. LtsA catalyzes the transfer of an NH₂ group between l-glutamine (donor) and lipid I or II (acceptor). The NH₂ group is transferred to the carboxyl function linked to the d-carbon of the meso-DAP residue, thereby resulting in the formation of an amidated meso-DAP residue in the peptide stem. Ammonium sulfate can also act as the donor. Und, undecaprenyl.

FIGURE 11.

Lysozyme sensitivity of peptidoglycan purified from wild-type and ΔltsA mutant C. glutamicum strains. Purified peptidoglycan (∼300 μg) from wild-type (circles) and mutant (squares) strains was incubated at 37 °C in 2 ml of 25 mm potassium phosphate buffer, pH 7.8. Lysozyme (75 μg/ml, final concentration) was added at t = 0 and peptidoglycan digestion was followed by measuring the decrease of absorbance at 660 nm. Mutanolysin (50 units) was subsequently added at t = 150 min.