Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Nov 15;24(22):16355.
doi: 10.3390/ijms242216355.

NlpI-Prc Proteolytic Complex Mediates Peptidoglycan Synthesis and Degradation via Regulation of Hydrolases and Synthases in Escherichia coli

Xinwei Liu  1 , Tanneke den Blaauwen  1
Affiliations

NlpI-Prc Proteolytic Complex Mediates Peptidoglycan Synthesis and Degradation via Regulation of Hydrolases and Synthases in Escherichia coli

Xinwei Liu et al. Int J Mol Sci. .

Abstract

Balancing peptidoglycan (PG) synthesis and degradation with precision is essential for bacterial growth, yet our comprehension of this intricate process remains limited. The NlpI-Prc proteolytic complex plays a crucial but poorly understood role in the regulation of multiple enzymes involved in PG metabolism. In this paper, through fluorescent D-amino acid 7-hydroxycoumarincarbonylamino-D-alanine (HADA) labeling and immunolabeling assays, we have demonstrated that the NlpI-Prc complex regulates the activity of PG transpeptidases and subcellular localization of PBP3 under certain growth conditions. PBP7 (a PG hydrolase) and MltD (a lytic transglycosylase) were confirmed to be negatively regulated by the NlpI-Prc complex by an in vivo degradation assay. The endopeptidases, MepS, MepM, and MepH, have consistently been demonstrated as redundantly essential "space makers" for nascent PG insertion. However, we observed that the absence of NlpI-Prc complex can alleviate the lethality of the mepS mepM mepH mutant. A function of PG lytic transglycosylases MltA and MltD as "space makers" was proposed through multiple gene deletions. These findings unveil novel roles for NlpI-Prc in the regulation of both PG synthesis and degradation, shedding light on the previously undiscovered function of lytic transglycosylases as "space makers" in PG expansion.

Keywords: E. coli; endopeptidase; lytic glycosidase; peptidoglycan; periplasmic protease; proteolytic control.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Abundance changes in PBPs in ΔnlpI, Δprc, and ΔnlpI-prc mutant strains: (a) Phase-contrast images of WT and mutant strains. The strains were initially cultured in LB medium for 3 h from overnight cultures. After washing two times with LB medium without NaCl, strains were incubated in LB medium with or without NaCl at temperatures of 37 °C and 42 °C for an additional 2 h. The cultures were fixed with 2.8% formaldehyde and 0.04% glutaraldehyde (FA/GA) and imaged using phase-contrast microscopy. The scale bar equals 5 µm and (b) Bocillin-FL binding to penicillin-binding proteins (PBPs). Strains cultured as indicated were washed twice with PBS and incubated with PBS containing Bocillin-FL for 10 min at room temperature (RT). The PBPs of different strains were visualized by a 10% SDS-PAGE. The abundance of PBPs in WT cultured in different conditions serve as control for mutans cultured in the same condition.
Figure 2
Figure 2
Midcell localization of PBP3 is undermined and the activity of transpeptidases is weaker in ΔnlpI cultured in LB medium without NaCl at 42 °C compared with wild-type (BW25113) cells cultured in the same condition: (a) Cells of both WT and ΔnlpI strains, cultured in LB medium without NaCl for 1 h, were fixed using FA/GA and subjected to immunolabeling using antibodies specific to PBP3. Phase-contrast images, corresponding fluorescence images, and demographs showing the fluorescence localization of PBP3, with cells sorted according to their cell length. The scale bar equals 5 µm. The numbers of cells analysed were 2822 and 1450 for WT and ΔnlpI, respectively; (b) The cell length and fluorescence per µm3 of WT and ΔnlpI strains, immunolabeled with PBP3 antibody; (c) Phase-contrast and fluorescence images of cells cultured in LB medium with or without NaCl containing 250 μM HADA at 37 and 42 °C. The scale bar equals 5 µm. Demographs of the concentration of HADA in the strains sorted according to cell length. The white line indicates the length of the cells. The number of cells analysed were 2235 and 504 for WT, 2636 and 993 for ΔnlpI grown in LB medium at 37 °C and LB medium without NaCl at 42 °C, respectively; and (d) The cell length and fluorescence per µm3 of HADA in WT and ΔnlpI cultured in LB medium with or without NaCl at 37 and 42 °C. The fluorescence concentration of WT cells cultured in different conditions serve as control for the ΔnlpI strain cultures in the same condition. Statistical significance, determined through unpaired t-test, was indicated as **** p ≤ 0.0001.
Figure 3
Figure 3
The activity of transpeptidases is weaker in Δprc cultured in LB medium without NaCl at 42 °C compared with wild-type (BW25113) cells cultured in the same condition: (a) HADA incorporation in WT and Δprc cultured in LB at 37 °C and LB without NaCl at 42 °C. Values in violin bar graphs represent mean fluorescence concentration quantified from more than 300 cells. The fluorescence concentration of WT cultured in different conditions serve as control for Δprc cultured in the same condition. Statistical significance determined using an unpaired t test, was indicated as **** p ≤ 0.0001; (b) Demographs of the concentration of HADA in the strains sorted according to cell length. The white line indicates the length of the cells The number of cells analysed were 315 and 364 for WT, 814 and 530 for ΔnlpI, for the cells grown in LB medium at 37 °C and LB medium without NaCl at 42 °C, respectively; (c) Phase-contrast and fluorescence images of cells cultured in medium containing 250 μM HADA. The scale bar equals 5 µm; and (d) Growth curve of strains in LB medium with or without NaCl at 42 °C. The growth curve of WT cultured in different conditions serve as positive control for mutants cultured in the same condition. The growth curves were performed in triplicate for each mutant. The solid lines and their corresponding shaded areas represent the mean ± S.D.
Figure 4
Figure 4
PBP7 can be degraded by Prc in an NlpI-independent manner. WT, ΔnlpI, Δprc, and ΔnlpI prc mepS were cultured in LB medium at 37 °C, at an OD600 of ≈0.6, 400 μg/mL spec was added to block new proteins synthesis and samples were collected at indicated time points: (a) PBP7 and FtsN were detected using immunoblot analysis with specific antibodies against PBP7 and FtsN, respectively. FtsN was used as a loading control. The experiment was performed three times, and representative images are presented; (b) The normalized amount of PBP7 at indicated time points in different strains. The grey value of each strain at 0 min was set as 1 (control), and the change in grey value at each time point was plotted for each strain. Significance determined using an unpaired t test, was indicated as, ** p ≤ 0.01, *** p ≤ 0.001; and (c) The normalized amount of PBP7 in different strains in the absence of spec. The grey value of WT strain was set as 1 (control), and the relative grey value was plotted for each strain. Significance determined using an unpaired t test, was indicated as, * p ≤ 0.05.
Figure 5
Figure 5
The absence NlpI-Prc alleviates the lethality of the mepS mepM mepH mutant strain. (a) Spot dilution assay on LB agar for WT, ΔnlpI prc mepS mepM and ΔnlpI prc mepS mepM mepH. Viability of WT cells in LB agar serves as a control for the mutants and (b) Phase-contrast images for the different strains cultured in LB medium at 37 °C. The scale bar equals 5 µm.
Figure 6
Figure 6
The absence of NlpI-Prc in ΔmepS suppresses its sensitivity to EDTA and the absence of MepH, MltA, or MltD in the ΔmepS nlpI prc restored the strains to high sensitivity to EDTA. (a,b) Strains cultured in LB medium at 37 °C for overnight were serially 10-fold diluted from 10−1 to 10−6 before spotting them onto LB plate and LB plate containing 1 mM EDTA. The viability of WT cells cultured in LB with 1 mM EDTA agar serves as a positive control for mutants cultured in same condition. The viability of strains cultured in LB agar serves as a cell loading control.
Figure 7
Figure 7
MltD is a substrate of the NlpI-Prc complex and complements the EDTA sensitivity of ΔmepS: (a) WT and ΔnlpI prc mepS harbouring a plasmid expressing HA-MltD were cultured in LB medium at 37 °C, when the OD600 reached 0.3, 400 μg/mL spec was added to block new proteins synthesis, and samples were collected at indicated time points. HA-MltD and FtsN were detected using immunoblot analysis with specific antibodies against the HA tag and FtsN, respectively. FtsN was used as a loading control and (b) The normalized amount of HA-MltD at indicated time points in different strains. Left panel: the grey value of each strain at 0 min was set as 1 (control) and the change in grey value at each time point was plotted for each strain. Right panel: the grey value of WT strain was set as 1 (as a control), and the relative grey value was plotted for ΔnlpI prc mepS. (c) The WT and mepS mutant cells harbouring the empty vector (EV) or vector expressing wild-type MltD were serially 10-fold diluted from 10−1 to 10−6 before spotting them onto LB plate and LB plate containing 1 mM EDTA. The viability of WT (EV) serves as a positive control. The viability of ΔmepS (EV) serves as a negative control.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Vollmer W., Blanot D., de Pedro M.A. Peptidoglycan structure and architecture. FEMS Microbiol. Rev. 2008;32:149–167. doi: 10.1111/j.1574-6976.2007.00094.x. - DOI - PubMed
    1. Alcorlo M., Martínez-Caballero S., Molina R., Hermoso J.A. Carbohydrate recognition and lysis by bacterial peptidoglycan hydrolases. Curr. Opin. Struct. Biol. 2017;44:87–100. doi: 10.1016/j.sbi.2017.01.001. - DOI - PubMed
    1. Höltje J.V. Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli. Microbiol. Mol. Biol. Rev. 1998;62:181–203. doi: 10.1128/MMBR.62.1.181-203.1998. - DOI - PMC - PubMed
    1. Scheffers D.J., Pinho M.G. Bacterial cell wall synthesis: New insights from localization studies. Microbiol. Mol. Biol. Rev. 2005;69:585–607. doi: 10.1128/MMBR.69.4.585-607.2005. - DOI - PMC - PubMed
    1. Vollmer W., Bertsche U. Murein (peptidoglycan) structure, architecture and biosynthesis in Escherichia coli. Biochim. Biophys. Acta. 2008;1778:1714–1734. doi: 10.1016/j.bbamem.2007.06.007. - DOI - PubMed

MeSH terms

LinkOut - more resources

-