FEMS Microbiology Letters 17 (1983) 63-67 63 Published by Elsevier Biomedical Press Recombinant plasmids containing menaquinone biosynthetic genes of Escherichia coli Duncan J. Shaw, E. Clare Robinson, R. Meganathan *, Ronald Bentley * and John R. Guest Department of Microbiologr', University of Sheffield, Sheffield SIO 2TN, U.K., and * Department of Biological Sciences, Universi(v of Pittsburgh, Pittsburgh, PA 15260, U.S.A. Received 14 September 1982 Accepted 20 September 1982 1. INTRODUCTION Menaquinone (ML; Vitamin K2) is an essential electron carrier in the anaerobic ATP-generating fumarate reductase system of Escherichia coli [1-3] and it is also involved in the anaerobic biosynthe- sis of pyrimidines, porphyrins and succinyl-CoA [4-6]. Previous studies have identified five mena- quinone biosynthetic genes (menA, B, C, D, E) whose roles in the pathway are as follows [7-12]: menC, D menE chorismate ~ o-succinylbenzoate ---, (osB) menB o-succinylbenzoyl-coenzyme A --* (OSB-CoA) menA 1,4-dihydroxy-2-naphthoate (DHN) demethylmenaquinone ~ menaquinone (DMK) (MK) The menA gene is located at 88 min in the E. coli linkage map and the menB, C, D and E genes are clustered at 48.5 min with the clockwise order Correspondence to: Prof. John R. Guest, Department of Mi- crobiology, Sheffield University, Western Bank, Sheffield, S10 2TN, U.K. menC-E*-B-D [ 12,13]; the asterisk denotes that the position of menE is not known relative to menC and menB. To facilitate studies on the enzymology of menaquinone biosynthesis and on their organisa- tion and expression, the men genes have been cloned. The isolation and properties of a recombi- nant lambda transducing phage containing the menB, C and E genes, but only part of the menD gene, have been reported previously [12,14]. This paper describes the isolation of two ColEl-men + hybrid plasmids, one of which contains the entire menD gene. Also described is the alignment of the genetic and restriction site maps of the men region and the amplification of menaquinone biosyn- thetic enzyme activities in plasmid-carrying strains. 2. MATERIALS AND METHODS 2.1. Bacteria, phage and plasmids Strains of E. coli K-12 used for genetic testing, isolation of recombinant plasmids, and phage propagation are shown in Table 1. A mixture rep- resenting all the colonies of the Clarke-Carbon ColE1-E. coli gene bank [15] was kindly provided by Dr. S.T. Cole. The XmenCE*B(D) transducing phage (XG68) carrying the menB, C and E but only part of D genes has been described previously [12,14]. 0378-1097/83/0000-0000/$03.00 © 1983 Federation of European Microbiological Societies Downloaded from https://academic.oup.com/femsle/article-abstract/17/1-3/63/549241 by guest on 20 May 2020