CLONING AND EXPRESSION OF A MUREIN HYDROLASE LIPOPROTEIN FROM ESCHERICHIA-COLI

On the basis of the published N-terminal amino acid sequence of the so luble lytic transglycosylase 35 (Slt35) of Escherichia coli, an open r eading 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 [H-3]-palmitate labelling to encode a lipoprotein with an apparent molecular mass of 36 kDa. A lar ger protein, presumably the prolipoprotein form, accumulated in the pr esence of globomycin. Overexpression of the gene, designated mltB (for membrane-bound lytic transglycosylase B), caused a 55-fold increase i n murein hydrolase activity in the membrane fraction and resulted in r apid cell lysis. After membrane fractionation by sucrose-density-gradi ent centrifugation, most of the induced enzyme activity was present in the outer and intermediate membrane fractions. Murein hydrolase activ ity in the soluble fraction of a homogenate of cells induced for MltB increased with time. This release of enzyme activity into the supernat ant could be inhibited by the addition of the serine-protease inhibito r phenylmethylsulphonyl fluoride. It is concluded that the previously isolated Slt35 protein is a proteolytic degradation product of the mur ein hydrolase lipoprotein MltB. Surprisingly, a deletion in the mltB g ene showed no obvious phenotype.