Four independent menaquinone (vitamin K
2
)-deficient mutants of
Escherichia coli
, blocked in the conversion of
o
-succinylbenzoate (OSB) to 1,4-dihydroxy-2-naphthoate (DHNA), were found to represent two distinct classes. Enzymatic complementation was observed when a cell-free extract of one mutant was mixed with extracts of any of the remaining three mutants. The missing enzymes in the two classes were identified by in vitro complementation with preparations of OSB-coenzyme A (CoA) synthetase or DHNA synthase isolated from
Mycobacterium phlei
. Mutants lacking DHNA synthase (and therefore complementing with
M. phlei
DHNA synthase) were designated
menB
, and the mutant lacking OSB-CoA synthetase (and therefore complementing with
M. phlei
OSB-CoA synthetase) was designated
menE
. The
menB
mutants produced only the spirodilactone form of OSB when extracts were incubated with [2,3-
14
C
2
]OSB, ATP, and CoA; the OSB was unchanged on incubation with an extract from the
menE
mutant under these conditions. Experiments with strains lysogenized by a λ
men
transducing phage (λG68) and transduction studies with phage P1 indicated that the
menB
and
menE
genes form part of a cluster of four genes, controlling the early steps in menaquinone biosynthesis, located at 48.5 min in the
E. coli
linkage map. Evidence was obtained for the clockwise gene order
gyrA
....-B-D, where the asterisk denotes the uncertain position of
menE
relative to
menC
and
menB
. The transducing phage (λG68) contained functional
menB, menC
, and
menE
genes, but only part of the
menD
gene, and it was designated λ
menC B(D)
.