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. 2016 Feb;99(4):700-18.
doi: 10.1111/mmi.13258. Epub 2015 Nov 19.

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria

Rachel Yunck  1 , Hongbaek Cho  1 , Thomas G Bernhardt  1
Affiliations

Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria

Rachel Yunck et al. Mol Microbiol. 2016 Feb.

Abstract

Bacterial cells are fortified against osmotic lysis by a cell wall made of peptidoglycan (PG). Synthases called penicillin-binding proteins (PBPs), the targets of penicillin and related antibiotics, polymerize the glycan strands of PG and crosslink them into the cell wall meshwork via attached peptides. The average length of glycan chains inserted into the matrix by the PBPs is thought to play an important role in bacterial morphogenesis, but polymerization termination factors controlling this process have yet to be discovered. Here, we report the identification of Escherichia coli MltG (YceG) as a potential terminase for glycan polymerization that is broadly conserved in bacteria. A clone containing mltG was initially isolated in a screen for multicopy plasmids generating a lethal phenotype in cells defective for the PG synthase PBP1b. Biochemical studies revealed that MltG is an inner membrane enzyme with endolytic transglycosylase activity capable of cleaving at internal positions within a glycan polymer. Radiolabeling experiments further demonstrated MltG-dependent nascent PG processing in vivo, and bacterial two-hybrid analysis identified an MltG-PBP1b interaction. Mutants lacking MltG were also shown to have longer glycans in their PG relative to wild-type cells. Our combined results are thus consistent with a model in which MltG associates with PG synthetic complexes to cleave nascent polymers and terminate their elongation.

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Figures

Figure 1
Figure 1. Lytic transglycosylase reaction
Diagram showing the lytic transglycosylase reaction. Strand cleavage results in the formation of an anhMurNAc sugar at what would normally be the reducing end of the polysaccharide. Shown below the chemical structures are cartoon representations of the substrate and products.
Figure 2
Figure 2. MltG overproduction is lethal in cells defective for PBP1b
A–D. Cells of TU122/pDY1 were transformed with the multicopy E. coli library and plated on LB agar supplemented with IPTG and Xgal. A typical screening plate is shown in panel A, and close-up images highlighting the different colony phenotypes are shown in B–D. See text for details. E. Diagram of the genomic locus resulting in a multicopy lethal phenotype in ΔponB cells. F. Cells of MG1655 or its ΔponB derivative containing plasmid pRY53 [Para::mltG] were grown overnight in M9 minimal maltose medium supplemented with chloramphenicol. Cells in the resulting cultures were harvested, washed twice in an equal volume of M9 salts, and resuspended in M9 salts at an OD600 of 1.0. The resulting cell suspensions were subjected to serial dilution, and 5μL of each dilution were spotted onto M9 agar plates containing 0.2% glucose or arabinose as indicated. Plates were incubated at 30°C for 2 days prior to imaging. G. Overnight cultures of the strains in F as well as the same strains carrying an empty vector control were diluted 1:100 in M9 maltose medium with chloramphenicol and grown to mid-log with shaking at 37°C. The cells were harvested, washed as above, and resuspended to an OD600 of 0.02 in M9 arabinose medium. Growth at 37°C was then monitored by following culture optical density (OD600). H–I. ΔponB cells carrying either an empty vector pCM6 [Para::empty] or plasmid pRY53 [Para::mltG] were grown on LB glucose plates, and then scraped and resuspended in M9 salts for imaging on agarose pads using DIC optics. Bars equal 4 microns.
Figure 3
Figure 3. MltG is structurally similar to SleB
A. Shown is a space-filling model of the MltG structure (PDB 2r1f) colored to indicate amino acid conservation using ConSurf (Ashkenazy et al., 2010). Note the high conservation of residues in the cleft-forming region. B. Ribbon diagrams showing the structural alignment of the proposed catalytic region of MltG (boxed in A) (burgundy) with the catalytic domain of the SleB lytic transglycosylase (grey) (Li et al., 2012; Jing et al., 2012). The catalytic glutamate is shown in stick form and highlighted with an asterisk. This structural homology was also reported previously by Jing and co-workers (Jing et al., 2012).
Figure 4
Figure 4. MltG degrades cell wall in vitro
Dye-labeled PG sacculi were incubated with the indicated proteins (4 μM) or buffer alone for the indicated times. Reactions were terminated, undigested PG was pelleted by centrifugation, and the absorbance of the supernatant at 595 nm was measured. Shown are the results from reactions performed in triplicate, with the error bars representing the 95% confidence interval of the measurements.
Figure 5
Figure 5. MltG is a lytic transglycosylase with endoglycosidase activity
A–B. Extractedion chromatograms resulting from the digestion of purified, unlabeled PG with MltG (A) or Slt (B). Traces are for ions with an m/z of 922.38 corresponding to LT degradation products. C. Shows the identity of the peaks in A–B along with relevant mass data, including the experimental mass to charge ratio, charge (z), peak identity, and cartoon structure of product molecules.
Figure 6
Figure 6. MltG is widely conserved in the bacterial domain
Bacterial phylogenetic tree constructed using iTOL (Letunic and Bork, 2011) and a diversity set of 150 strains. The presence of MltG or other LT family members in a given species is indicated by the colored regions at the outer edge of the tree. A color-coded legend is given in the lower right portion of the panel.
Figure 7
Figure 7. MltG is an inner membrane protein
A–D. Cytological assay for determining the localization of MltG in the cell envelope. Cells expressing mCherry fusions to MltG (A), Slt (B), the wildtype signal-sequence of Pal for outer membrane targeting [ssPal(OM)] (C), or a modified version of ssPal retained in the inner membrane [ssPal(IM)] (D) were grown to an OD600 of 0.3–0.4, osmotically shocked by resuspension in plasmolysis buffer (30% sucrose, 50 mM HEPES (pH 7.4), 40 mM sodium azide), and imaged by mCherry (panel 1) or phase contrast (panel 2) optics. Slt-mCherry serves as a periplasmic control. ssPal(OM)-mCherry and ssPal(IM)-mCherry serve as controls for outer and inner membrane localized proteins, respectively. Arrows highlight signals that track with the inner membrane in plasmolysis bays. Bar equals 4 microns. E. Cells expressing the indicated variants of MltG were harvested 1 hr post induction with 100μM IPTG. The whole cells were left either untreated or treated with 10mM MTSES. After washing out excess MTSES, treated and untreated cells were lysed, their proteins were denatured, and the lysates were exposed to 5mM PEG-malemide (mPEG) (2kDa). Following TCA precipitation, equivalent amounts of total protein were separated by SDS-PAGE and blotted to membranes. MltG variants were detected with affinity purified anti-MltG antibodies. A cartoon diagram of the MltG membrane topology suggested by the SCAM analysis is shown below the blot image with the location of the Cys residue in each variant indicated. Asterisk marks the bands of MltG shifted to a higher molecular weight following reaction with mPEG.
Figure 8
Figure 8. Alteration of muropeptide composition and glycan strand length in the PG of MltG defective cells
A. The abundance of each muropeptide species in WT and ΔmltG PG preparations was determined. Shown are the ratios of selected groupings of muropeptide species in ΔmltG versus WT PG. Pentapeptide, Tetrapeptide, and Tripeptide indicate all muropeptide species (crosslinked and uncrosslinked) containing the indicated type of peptide. Similarly, Anhydo indicates all anhMurNAc containing species, and Tri-Lys-Arg indicates species that were linked to the outer membrane lipoprotein Lpp. Tabulated relative abundances for the complete set of known muropeptide species for each strain are presented in Table S1. B. Detailed comparison of pentapeptide containing muropeptides between the indicated strains showing the differential change between crosslinked and uncrosslinked species. C. PG preparations from the indicated strains were treated with the amidase AmiD to remove the peptide stems. The resulting peptide-free glycan strands were then separated according to length and quantified using a previously described HPLC protocol (Harz et al., 1990). Quantified peak areas for representative samples are shown. Chromatograms are presented in Figure S4.
Figure 9
Figure 9. Bacterial two-hybrid interaction of MltG with PBP1b
BTH101 cells containing plasmids producing the indicated T25 and T18 fusions were grown to saturation in LB with 50μg/ml amp, 25 μg/ml kan, and 500 μM IPTG and 5 μl of each culture was spotted on LB agar containing 40 μg/ml Amp, 25 μg/ml kan, 500μM IPTG, and 40 μg/ml X-gal.
Figure 10
Figure 10. Nascent PG degradation by Slt and MltG following cefsulodin treatment
Cells of TU278 [ΔlysA ΔampD] or its indicated derivatives were pulse labeled with [3H]-mDAP with or without prior treatment with cefsulodin (100 μg/ml). Soluble metabolites were then extracted with hot water, separated by HPLC, and turnover products were quantified. Radiolabel incorporation into PG was determined by digesting the pellets resulting from the hot water extraction with lysozyme and quantifying the amount of label released into the supernatant by scintillation counting. Results are the average of three independent experiments with the error bars representing the standard deviation. See text and Material and Methods for experimental details.
Figure 11
Figure 11. Model for MltG functioning as a PG terminase
Shown is a model detailing the potential role of MltG as a terminase for PG polymerization. The PG matrix is drawn as in Figure 1 with GlcNAc in orange, MurNAc in red, and anhMurNAc in blue. Small circles attached to MurNAc and anhMurNAc represent the peptide side chains. On the left, an aPBP (green) is polymerizing a nascent glycan strand using its GT domain (circle closest to membrane). Although not shown, the polymers are extended by adding new dissacharides from the precursor lipid II to the growing chain at the membrane-proximal (MurNAc) end. We propose that MltG is recruited to the active polymerase via direct or indirect interactions. There, it cleaves the growing strand endolytically to terminate its elongation. It is not clear where along the strand MltG might cut. The site shown is merely to present the concept of a termination reaction. How much of the polymer remains associated with the polymerase post MltG cleavage and whether this portion of the chain can continue being elongated following cleavage also requires further investigation.
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