The Tol-Pal system is required for peptidoglycan-cleaving enzymes to complete bacterial cell division

Abstract

Tol-Pal is a multiprotein system present in the envelope of Gram-negative bacteria. Inactivation of this widely conserved machinery compromises the outer membrane (OM) layer of these organisms, resulting in hypersensitivity to many antibiotics. Mutants in the tol-pal locus fail to complete division and form cell chains. This phenotype along with the localization of Tol-Pal components to the cytokinetic ring in Escherichia coli has led to the proposal that the primary function of the system is to promote OM constriction during division. Accordingly, a poorly constricted OM is believed to link the cell chains formed upon Tol-Pal inactivation. However, we show here that cell chains of E. coli tol-pal mutants are connected by an incompletely processed peptidoglycan (PG) layer. Genetic suppressors of this defect were isolated and found to overproduce OM lipoproteins capable of cleaving the glycan strands of PG. Among the factors promoting cell separation in mutant cells was a protein of previously unknown function (YddW), which we have identified as a divisome-localized glycosyl hydrolase that cleaves peptide-free PG glycans. Overall, our results indicate that the cell chaining defect of Tol-Pal mutants cannot simply be interpreted as a defect in OM constriction. Rather, the complex also appears to be required for the activity of several OM-localized enzymes with cell wall remodeling activity. Thus, the Tol-Pal system may play a more general role in coordinating OM invagination with PG remodeling at the division site than previously appreciated.

Keywords: cell wall; colicin; divisome; peptidoglycan.

Fig. 1.

Disruption of Tol-Pal leads to a septal PG splitting defect. Images of HADA labeled PG sacculi isolated from TB28 (WT) (A), AA40 (ΔtolQRA) (B), and MT91 (Δtol-pal) (C) cells grown at 42 °C in the presence of 500 μM HADA for 3 h. Larger fields of view are presented in SI Appendix, Fig. S1. (Scale bar: 5 μm.)

Fig. 2.

Growth and division defects of a Δtol-pal mutant are suppressed by mutations in prc and nlpI. (A) Serial dilutions of TB28 (WT), MT91 (Δtol-pal), and a representative MT91 suppressor mutant prc(Y366C) were spotted on LB0N agar and grown at the indicated temperature. (B) Phase-contrast images of MT91 [Δtol-pal] and a representative MT91 suppressor mutant grown in LB0N at 42 °C for 3 h. (Scale bars: 5 μm.) (C) Schematic showing the location of suppressor mutations.

Fig. 3.

Suppression of growth and division defects of a Δtol-pal mutant by overexpression of MltB or DigH. (A) Serial dilutions of TB28 (WT) or MT91 (Δtol-pal) harboring either the empty vector (ev) pCM6 or derivatives pAAY245 (mltB) or pAAY246 (digH [yddW]) harboring the indicated gene with its native upstream region for expression were spotted on LB0N agar and grown at the indicated temperature. (B) Phase-contrast images of MT91 cells with the same vectors following growth at 42 °C for 3 h. (Scale bars: 5 μm.)

Fig. 4.

Evidence that DigH is a glycosyl hydrolase that cleaves denuded peptidoglycan. (A) Schematic of the domain organization of DigH highlighting its single GHL10 domain and predicted lipoprotein signal sequence. The putative lipobox is indicated in bold red letters, and the putative signal peptidase cut site is marked with an arrow. (B) RBB-labeled sacculi were incubated for ∼24 h with 200 μg/mL lysozyme, 8 μM DigH, or 0.5 μM AmiD along with 0, 2, 4, 6, or 8 μM WT DigH or 8 μM DigH(D236N). Upon quenching of the reaction with ethanol, the undigested PG was pelleted and released material was quantified by measuring dye absorbance at 600 nm. Data for three technical replicates, with the error bars representing the SE are shown. (C) An overnight culture of AAY119(attλAAY264) (ΔdigH [P_lac::digH-mCherry]) was diluted 1:2,000 in M9 medium supplemented with 0.2% maltose and 250 μM isopropyl beta-D-1-thiogalacactopyranoside (IPTG). Cells were grown for 5 h at 37 °C and then imaged on agarose pads by phase contrast and fluorescence microscopy. Midcell localization of DigH-mCherry was observed in 27.3 ± 4.8% cells (7 images analyzed, >200 cells each). (Scale bar: 3 μm.)

Fig. 5.

SPOR domain localization in Δtol-pal cells. Overnight cultures of TB28 (WT) and MT51 (Δtol-pal) harboring the integrated expression construct attHKNP78 (P_lac::dsbA^ss-mCherry-FtsN^SPOR) were subcultured 1:2,000 in LB0N supplemented with 100 μM IPTG and grown for 2 h at 30 °C and then for 3 h at 42 °C. Cells were placed on agarose pads and imaged with phase contrast and epifluorescence optics. (Scale bar: 5 μm.) Arrows point to membrane blebs emanating from septa and mCherry-containing vesicles.