A cosmid (pND320) bearing 42.5 kb of Escherichia coli chromosomal DNA, including the genes between xylE and ssb near minute 92 on the linkage map, was isolated by selection for complementation of a dnaB mutation. Known nucleotide (nt) sequences were used to align restriction maps in this region to the physical map of the chromosome (coordinates 4319.5 to 4362 kb), and to locate precisely and define the orientations of 19 genes. Predicted physical linkage of sequenced genes across unsequenced gaps of defined length was confirmed by the nt sequence analysis of fragments subcloned from pND320. Mutant complementation by plasmids showed that ubiA is located between malM and plsB. A previously sequenced long open reading frame that encodes the C-terminal portion of the E. coli ubiA product (4-hydroxybenzoate polyprenyltransferase, HPTase) shows a high degree of sequence identity with the corresponding segment of yeast HPTase (the COQ2 gene product). Comparison of homologous regions from E. coli and Salmonella typhimurium was used to locate precisely the gene alr that encodes alanine racemase (ARase) between dnaB and tyrB. Subcloning of alr downstream from tandem bacteriophage lambda promoters produced a plasmid that directed high-level overproduction of a soluble approx. 40-kDa protein with ARase activity.