Membrane-bound lytic endotransglycosylase in Escherichia coli - PubMed (original) (raw)

Membrane-bound lytic endotransglycosylase in Escherichia coli

A R Kraft et al. J Bacteriol. 1998 Jul.

Abstract

The gene for a novel endotype membrane-bound lytic transglycosylase, emtA, was mapped at 26.7 min of the E. coli chromosome. EmtA is a lipoprotein with an apparent molecular mass of 22kDa. Overexpression of the emtA gene did not result in bacteriolysis in vivo, but the enzyme was shown to hydrolyze glycan strands isolated from murein by amidase treatment. The formation of tetra- and hexasaccharides, but no disaccharides, reflects the endospecificity of the enzyme. The products are characterized by the presence of 1,6-anhydromuramic acid, indicating a lytic transglycosylase reaction mechanism. EmtA may function as a formatting enzyme that trims the nascent murein strands produced by the murein synthesis machinery into proper sizes, or it may be involved in the formation of tightly controlled minor holes in the murein sacculus to facilitate the export of bulky compounds across the murein barrier.

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Figures

FIG. 1

FIG. 1

Murein glycan chain-degrading activities in membrane extracts of an mltA mutant. E. coli LT12 harboring pAK5 (b) or pJFK118EH as a control (a and c) were induced by the addition of 1 mM IPTG, and membrane extracts were prepared as described in Materials and Methods. Extracts corresponding to 1 μl (a and b) or 100 μl (c) of culture were incubated for 30 min at 30°C with radiolabeled murein glycan chains with a length of seven disaccharide units terminating with a 1,6-anhydromuramic acid residue. The breakdown products, all terminating with 1,6-anhydromuramic acid, were analyzed by reversed-phase HPLC. The numbers 2 to 7 indicate the degree of polymerization in the disaccharide units. Thus, for example, the “3” indicates a 1,6-anhydrohexasaccharide consisting of (GlcNAc-MurNAc)2–GlcNAc-(1,6-anhydro)MurNAc.

FIG. 2

FIG. 2

Nucleotide sequence of the emtA gene and derived amino acid sequence. The consensus sequence of the lipoprotein processing site is shown in bold italics, with an asterisk indicating the modified cysteine. Regions of high similarity to other known lytic transglycosylases are underlined (12, 35).

FIG. 3

FIG. 3

Expression of the cloned lytic transglycosylase gene. E. coli LT12 harboring pJFK118EH (lanes b and d) or pAK5 (lanes c and e) was induced for 4 h by the addition of 1 mM IPTG. Cell homogenates (lanes b and c) or membrane extracts (lanes d and e) were separated by SDS–12% PAGE and stained with Coomassie brilliant blue. Lane a shows molecular mass markers. The position of the cloned lytic transglycosylase is indicated by an arrow.

FIG. 4

FIG. 4

[3H]palmitate labeling of overexpressed EmtA. E. coli LT12 harboring pJFK118EH (lanes a and b) or pAK5 (lanes c to e) were labeled by growth in the presence of [3H]palmitate (5 mCi/ml). Cells were induced for 30 min by the addition of 1 mM IPTG (lanes b, d, and e). One culture (lane e) received 100 μg of globomycin per ml to inhibit the processing of lipoproteins. Separation of the proteins by SDS–15% PAGE and fluorography was performed as described in Materials and Methods. Molecular masses of prestained marker proteins and the position of EmtA are indicated on the right.

FIG. 5

FIG. 5

Kinetics of glycan chain degradation by the lytic endotransglycosylase. E. coli LT12(pAK5) was induced by the addition of 1 mM IPTG, and membrane extracts were prepared as described in Materials and Methods. Extracts corresponding to 1 μl (b and c) or 100 μl (d and e) of culture were incubated with radiolabeled murein glycan chains with a length of seven disaccharide units terminating with 1,6-anhydromuramic acid (a) for 5 min (b and d) or 30 min (c and e) at 30°C. The breakdown products were analyzed by reversed-phase HPLC. Numbers 1 to 7, (GlcNAc-MurNAc)0–6–GlcNAc-(1,6-anhydro)MurNAc (as described in Fig. 1).

FIG. 6

FIG. 6

Zymogram analysis of overexpressed EmtA. E. coli LT12 harboring pJFK118EH (lane a) or pAK5 (lane b) was induced by the addition of 1 mM IPTG. Cell homogenates were separated by SDS–12% PAGE in gels containing 0.2% lyophilized cells of M. lysodeikticus. Renaturation of the separated proteins and staining of the gel were performed as described in Materials and Methods. The stained gel was scanned and inverted to obtain better contrast. Molecular masses of prestained marker proteins are indicated on the right.

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