Molecular cloning, overexpression and mapping of the slt gene encoding the soluble lytic transglycosylase of Escherichia coli

Summary The gene of the major autolysin of Escherichia coli, the soluble lytic transglycosylase (Slt), was isolated from an expression gene library. The cloned slt gene was used to determine its chromosomal map position adjacent to trpR at 99.7 min on the E. coli linkage map.     

Murein-metabolizing enzymes from Escherichia coli: existence of a second lytic transglycosylase.

In addition to the soluble lytic transglycosylase, a murein-metabolizing enzyme with a molecular mass of 70 kDa (Slt70), Escherichia coli possesses a second lytic transglycosylase, which has been described as a membrane-bound lytic transglycosylase (Mlt; 35 kDa; EC 3.2.1.-). The mlt gene, which supposedly encodes Mlt, was cloned, and the complete nucleotide sequence was determined. The open reading frame, identified on a 1.7-kb SalI-PstI fragment, codes for a protein of 323 amino acids (M(r) = 37,410). Two transmembrane helices and one membrane-associated helix were predicted in the N-terminal half of the protein. Lysine and arginine residues represent up to 15% of the amino acids, resulting in a calculated isoelectric point of 10.0. The deduced primary structure did not show significant sequence similarity to Slt70 from E. coli. High-level expression of the presumed mlt gene was not paralleled by an increase in murein hydrolase activity. To clarify the identity of the second transglycosylase, we purified an enzyme with the specificity of a transglycosylase from an E. coli slt deletion strain. The completely soluble transglycosylase, with a molecular mass of approximately 35 kDa, was designated Slt35. Its determined 26 N-terminal amino acids showed similarity to a segment in the middle of the Slt70 primary structure. Polyclonal anti-Mlt antibodies, which had been used for the isolation of the mlt gene, were found to cross-react with Mlt as well as with Slt35, suggesting that the previously described Mlt preparation was contaminated with Slt35. We conclude that the second transglycosylase of E. coli is not a membrane-bound protein but rather is a soluble protein.     

Crystallization of the soluble lytic transglycosylase from Escherichia coli K12

Lytic transglycosylases degrade the murein polymer of the bacterial cell wall to 1,6-anhydromuropeptides. These enzymes are of significant medical interest, not only because they are ideal targets for the development of new classes of antibiotics, but also because the low molecular weight products of their catalytic action can cause diverse biological activities in humans, which can be either beneficial or toxic. A soluble lytic transglycosylase was purified from an overproducing Escherichia coli strain and X-ray quality crystals were obtained at room temperature from hanging drops by vapor diffusion against 20 to 25% (NH4)2SO4, in 100 mM-sodium acetate buffer, pH 5.0. The crystals diffract in the X-ray beam to 2.8 A resolution. Their space group is P2(1)2(1)2(1) with cell dimensions a = 81 A, b = 88 A and c = 135 A. Assuming one monomer (Mr 70,362) per asymmetric unit, the solvent content of these crystals is 63%.     

Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein.     

Purification and properties of a membrane-bound lytic transglycosylase from Escherichia coli.

A membrane-bound lytic transglycosylase (Mlt) has been solubilized in the presence of 2% Triton X-100 containing 0.5 M NaCl from membranes of an Escherichia coli mutant that carries a deletion in the slt gene coding for a 70-kDa soluble lytic transglycosylase (Slt70). The enzyme was purified by a four-step procedure including anion-exchange (HiLoad SP-Sepharose and MonoS), heparin-Sepharose, and poly(U)-Sepharose 4B column chromatography. The purified protein that migrated during denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band corresponding to an apparent molecular mass of about 38 kDa is referred to as Mlt38. Optimal activity was found in buffers with a pH between 4.0 and 4.5. The enzyme is stimulated by a factor of 2.5 in the presence of Mg2+ at a concentration of 10 mM and loses its activity rapidly at temperatures above 30 degrees C. Besides insoluble murein sacculi, the enzyme was able to degrade glycan strands isolated from murein by amidase treatment. The enzymatic reaction occurred with a maximal velocity of about 2.2 mg/liter/min with murein sacculi as a substrate. The amino acid sequences of four proteolytic peptides showed no identity with known sequences in the data bank. With Mlt38, the number of proteins in E. coli showing lytic transglycosylase activity rises to three.     

Control of the activity of the soluble lytic transglycosylase by the stringent response in Escherichia coli

The soluble lytic transglycosylase (Slt) of Escherichia coli is known to be a powerful murein hydrolase in vitro. It is shown here to act as an autolysin in vivo as well. Rapid autolysis of Slt overproducing cells was induced by protein biosynthesis inhibitors, which also block the fomration of guanosine-5'-diphosphate-3'-diphosphate (ppGpp). When amino acid starvation was used to inhibit protein synthesis, autolysis was suppressed in relA+ but not in relA- cells. These findings indicate that the stringent control modulates the enzymatic activity of the soluble lytic transglycosylase in vivo.     

Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease.

The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orf17-orf26-ruvC-orf23 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orf17, orf26, and ruvC (orf19) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.     

Cloning and identification of the Escherichia coli murB DNA sequence, which encodes UDP-N-acetylenolpyruvoylglucosamine reductase.

The murB gene, which complemented the UDP-N-acetylenolpyruvoylglucosamine reductase (EC 1.1.1.158) mutation in Escherichia coli ST5, was cloned from an E. coli chromosomal library. murB was subcloned on a 2.8-kb PvuII fragment into pUC19 and sequenced. A 1,029-bp open reading frame encoded a 342-amino-acid polypeptide of 37,859 Da. A DNA sequence homology search revealed that murB had almost 100% homology with a previously reported unidentified open reading frame, ORFII, at 89.9 min. Physical and genetic mapping results were consistent with this map position, and minicell analyses of murB subclones showed a plasmid-encoded protein of approximately 37,000 Da, which closely matched the calculated size of the murB protein.     

Revised nucleotide sequence of the gltP gene, which encodes the proton-glutamate-aspartate transport protein of Escherichia coli K-12.

The gene encoding the proton-glutamate carrier (GltP) of Escherichia coli K-12 was sequenced, and the primary structure of the protein was analyzed. The nucleotide sequence was found to differ in several aspects from the previously published sequence (B. Wallace, Y. Yang, J. Hong, and D. Lum, J. Bacteriol. 172:3214-3220, 1990). The corrected open reading frame encodes a protein of 437 (instead of 395) amino acids. Hydropathy analysis predicts 12 membrane-spanning alpha-helical regions. The complementary strand does contain an open reading frame possibly encoding a highly hydrophilic polypeptide of 272 amino acids.     

Crystal structure of MltA from Escherichia coli reveals a unique lytic transglycosylase fold

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the beta-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydromuropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0A resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi beta-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a beta-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H.insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.     

High-resolution crystal structure of MltE, an outer membrane-anchored endolytic peptidoglycan lytic transglycosylase from Escherichia coli

The crystal structure of the first endolytic peptidoglycan lytic transglycosylase MltE from Escherichia coli is reported here. The degradative activity of this enzyme initiates the process of cell wall recycling, which is an integral event in the existence of bacteria. The structure sheds light on how MltE recognizes its substrate, the cell wall peptidoglycan. It also explains the ability of this endolytic enzyme to cleave in the middle of the peptidoglycan chains. Furthermore, the structure reveals how the enzyme is sequestered on the inner leaflet of the outer membrane.     

Sequence and overexpression of the menD gene from Escherichia coli.

The menD gene of Escherichia coli codes for the first enzyme of menaquinone biosynthesis, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) synthase. DNA sequence analysis of menD shows an open reading frame encoding a 52-kilodalton protein. Possible promoter and ribosome binding sites are present. Insertion of the menD gene into a tac promoter expression vector leads to nearly a 100-fold increase in the level of SHCHC synthase activity upon induction with isopropyl-beta-D-thiogalactoside (IPTG). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins shows a 61-kilodalton protein produced upon induction of the menD-containing expression vector. This is the first reported sequence analysis of a men gene and the first significant amplification of any of the menaquinone biosynthetic enzymes.     

Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase.

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUV5 promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.     

Cloning, nucleotide sequence, and overexpression of the L-rhamnose isomerase gene from Pseudomonas stutzeri in Escherichia coli

The gene encoding L-rhamnose isomerase (L-RhI) from Pseudomonas stutzeri was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the L-RhI gene revealed an open reading frame of 1,290 bp coding for a protein of 430 amino acid residues with a predicted molecular mass of 46,946 Da. A comparison of the deduced amino acid sequence with sequences in relevant databases indicated that no significant homology has previously been identified. An amino acid sequence alignment, however, suggested that the residues involved in the active site of L-RhI from E. coli are conserved in that from P. stutzeri. The L-RhI gene was then overexpressed in E. coli cells under the control of the T5 promoter. The recombinant clone, E. coli JM109, produced significant levels of L-RhI activity, with a specific activity of 140 U/mg and a volumetric yield of 20,000 U of soluble enzyme per liter of medium. This reflected a 20-fold increase in the volumetric yield compared to the value for the intrinsic yield. The recombinant L-RhI protein was purified to apparent homogeneity on the basis of three-step chromatography. The purified recombinant enzyme showed a single band with an estimated molecular weight of 42,000 in a sodium dodecyl sulfate-polyacrylamide gel. The overall enzymatic properties of the purified recombinant L-RhI protein were the same as those of the authentic one, as the optimal activity was measured at 60 degrees C within a broad pH range from 5.0 to 11.0, with an optimum at pH 9.0.