Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli.
On the basis of the published N-terminal amino acid sequence of the soluble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open reading frame (ORF) was cloned from the 60.8 min region of the E. coli chromosome. The nucleotide sequence of the ORF, containing a putative lipoprotein-processing site, was shown by [3H]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A larger protein, presumably the prolipoprotein form, accumulated in the presence of globomycin. Over-expression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase in murein hydrolase activity in the membrane fraction and resulted in rapid cell lysis. After membrane fractionation by sucrose-density-gradient centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernatant could be inhibited by the addition of the serine-protease inhibitor phenylmethylsulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the murein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB gene showed no obvious phenotype.
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D005798:Genes, Bacterial; D016695:Glycosyltransferases / Q000235:genetics*; D008074:Lipoproteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009238:N-Acetylmuramoyl-L-alanine Amidase / Q000235:genetics*; D010641:Phenotype; D016133:Polymerase Chain Reaction
D017931:DNA Primers; D004269:DNA, Bacterial; D008074:Lipoproteins; D016695:Glycosyltransferases; C021815:murein transglycosylase; D009238:N-Acetylmuramoyl-L-alanine Amidase