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. 2024 Feb 29;20(2):e1011161.
doi: 10.1371/journal.pgen.1011161. eCollection 2024 Feb.

Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli

Moneca Kaul  1   2 , Suraj Kumar Meher  1   2 , Krishna Chaitanya Nallamotu  1   2 , Manjula Reddy  1   2
Affiliations

Glycan strand cleavage by a lytic transglycosylase, MltD contributes to the expansion of peptidoglycan in Escherichia coli

Moneca Kaul et al. PLoS Genet. .

Abstract

Peptidoglycan (PG) is a protective sac-like exoskeleton present in most bacterial cell walls. It is a large, covalently crosslinked mesh-like polymer made up of many glycan strands cross-bridged to each other by short peptide chains. Because PG forms a continuous mesh around the bacterial cytoplasmic membrane, opening the mesh is critical to generate space for the incorporation of new material during its expansion. In Escherichia coli, the 'space-making activity' is known to be achieved by cleavage of crosslinks between the glycan strands by a set of redundant PG endopeptidases whose absence leads to rapid lysis and cell death. Here, we demonstrate a hitherto unknown role of glycan strand cleavage in cell wall expansion in E. coli. We find that overexpression of a membrane-bound lytic transglycosylase, MltD that cuts the glycan polymers of the PG sacculus rescues the cell lysis caused by the absence of essential crosslink-specific endopeptidases, MepS, MepM and MepH. We find that cellular MltD levels are stringently controlled by two independent regulatory pathways; at the step of post-translational stability by a periplasmic adaptor-protease complex, NlpI-Prc, and post-transcriptionally by RpoS, a stationary-phase specific sigma factor. Further detailed genetic and biochemical analysis implicated a role for MltD in cleaving the nascent uncrosslinked glycan strands generated during the expansion of PG. Overall, our results show that the combined activity of PG endopeptidases and lytic transglycosylases is necessary for successful expansion of the cell wall during growth of a bacterium.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Absence of NlpI-Prc rescues the growth defects of mepS mepM mutant through MltD.
(A) mepS mepM double mutant and its derivatives lacking either nlpI or prc were tested for viability on Minimal A medium (MinA) or LB plates. Cells were grown overnight in MinA, serially diluted and 4 μl of each dilution was spotted on indicated plates and grown overnight. prc* contains a 5 amino acid deletion in the signal sequence of prc gene whereas nlpI* contains a frameshift mutation at codon 21 (B) ΔmepS ΔmepM ΔnlpI/ Para::mepS and its derivatives carrying deletion of each of the LTs were tested for viability on LB with and without arabinose (0.2%) (C,D) Cultures of WT (MG1655) and its mutant derivatives were grown overnight in MinA. Next day, they were washed and diluted 1:250 into fresh LB and growth was monitored at regular intervals. Cells were collected after 4 h of growth (marked) and visualized by DIC microscopy. Arrows indicate cell lysis. Scale bars represent 5 μm.
Fig 2
Fig 2. NlpI-Prc proteolytic system regulates MltD levels.
(A) Indicated strains carrying mltD-Flag at their native chromosomal locus were grown in LB and cells were collected between OD600 of 0.8–1.0. Normalized cell fractions were subjected to SDS-PAGE followed by western blotting as described in Materials and Methods. MepS is used as a positive control. FtsZ is used as loading control to normalize the target protein. (B) Western blots showing the growth-phase specific expression of MltD-Flag in the indicated strains. Cells were grown in LB and fractions were collected at different OD600 values. Cell lysates were processed and analyzed as described above. Bar diagrams indicate the relative fold change of respective protein levels from three replicates; *, P <0.05; ***, P <0.001; ns (not significant); n = 3.
Fig 3
Fig 3. MltD is a substrate of NlpI-Prc proteolytic system.
(A) Determination of half-life of MltD-Flag in the indicated strains was done as follows: cells were grown in LB till OD600 of 0.6 and 300 μg/ml of spectinomycin was added to block translation. Fractions were collected at indicated time points and were analyzed by western blotting as described in Materials and Methods. Error bars represent standard deviation. **, P <0.005; ***, P <0.001. FtsZ was used as a loading control. (B) In vitro degradation assay with purified MltD, Prc and NlpI proteins. The proteins were mixed in all combinations and incubated at 37°C followed by SDS-PAGE and Coomassie brilliant blue staining. Each reaction contained: MltD- 10 μg, NlpI- 1 μg and Prc- 0.4 μg. Molecular mass of the proteins is indicated in kDa.
Fig 4
Fig 4
Regulation of MltD by stationary-phase specific sigma factor, RpoS (A) Indicated strains were grown in LB and fractions were collected at different OD600 values and MltD-Flag levels were analyzed by western blotting. (B) WT and ΔrpoS mutant strain carrying either chromosomal MltD-Flag or a plasmid-borne MltD-Flag (Ptrc::mltD-Flag) were grown in LB till OD600 of 3 and normalized cell lysates were subjected to SDS-PAGE followed by western blotting. (C) Indicated strains were grown in LB and cell fractions were collected at OD600 of 1.0 and 3.0. Normalized cell fractions were used for western blot analysis. Bar diagrams indicate the relative fold change of respective protein levels from three replicates; *, P <0.05; **, P <0.005; ***, P <0.001; ****, P <0.0001; ns (not significant); n = 3.
Fig 5
Fig 5. MltD overexpression restores growth to endopeptidase-deficient mutants.
(A,B) Strains carrying pTrc99a or its mltD derivatives (WT, E125A, E125K) were grown overnight in LB with 0.2% arabinose and viability was checked on LB (A) or Minimal A (B) plates. IPTG was used at 250 μM (C) Strains were grown overnight with 0.2% arabinose and diluted 1:2500 into fresh LB containing appropriate inducers (0.2% arabinose or 250 μM IPTG) at 37°C and growth was monitored by OD600. (D, E) Cells were collected from cultures growing in LB (with arabinose or IPTG) after 3 h and subjected to DIC microscopy as described in Materials and Methods. Scale bars represent 5 μm. For panel II, cells from 20 ml culture were collected for microscopy.
Fig 6
Fig 6. Effect of mltD deletion on endopeptidase-deficient mutants.
(A) WT and its mutant derivatives were grown overnight in LB and viability assays were done on LB and NA plates at 30°C. (B) Overnight grown cultures of the above strains were washed and diluted 1:100 into fresh NB at 30°C and growth was monitored by OD600. Cells were collected after 5 h of growth and subjected to DIC microscopy as described in Materials and Methods. Arrows indicate lysed cells. (C) Indicated strains were grown in LB with 0.2% arabinose and viability was checked on indicated plates. (D) the cells were grown as described above in Minimal medium with 1:500 dilution for 10 h followed by DIC microscopy. Scale bars represent 5 μm. (E) Bar diagram depicting the incorporation of 3H-mDAP into the PG sacculi of WT and its mutant derivatives. Error bars represent standard deviation; *, P <0.05; ****, P <0.0001.
Fig 7
Fig 7. Absence of MrcA-LpoA restores growth to endopeptidase-deficient mutants overexpressing MltD.
(A) Indicated strains were grown overnight in LB with 0.2% arabinose and viability was checked on plates. IPTG was used at 100 and 200 μM for top and bottom panel respectively. (B, C) Overnight grown cultures of the above strains were diluted 1:2500 into fresh LB containing appropriate inducers (0.2% arabinose or 200 μM IPTG) at 37°C and growth was monitored by OD600. Cells were collected after 3 h of growth and subjected to DIC microscopy as described in Materials and Methods. Scale bars represent 5 μm.
Fig 8
Fig 8. Enzymatic activity of MltD.
HPLC chromatograms showing the activity of various muramidases (mutanolysin, Slt or MltD) on intact PG sacculi. Purified sacculi were treated with either mutanolysin, Slt or MltD (panel A, B, C) for 16 h at 37°C. (D) PG sacculi were treated with MepM for 16 h at 37°C followed by treatment with MltD. All proteins were used at 5 μM. *Tetra-anh (anhydro derivative of disaccharide tetrapeptide), Tetra-Tetra-anh (dimer of Tetra-anh).
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Grants and funding

This work is supported by funds from Council of Scientific and Industrial Research (MLP0141) and Department of Biotechnology (BT/PR33064/BRB/10/1819/2019), Government of India to MR. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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