As part of a large-scale regulatory investigation, cysteine biosynthesis was examined in a soil bacterium, Acinetobacter baylyi ADP1. This pathway involves the reductive assimilation of sulfate to meet the essential biological requirement for sulfur. As reported here, there are significant differences in genetic organization, enzymatic steps, and transcriptional regulation in cysteine biosynthesis by A. baylyi when compared to well-characterized enteric bacteria such as Escherichia coli. To identify the role of a LysR-type regulator, CysB (encoded by ACIAD2597), gene expression was assessed with transcriptional reporters, and mutations in the chromosomal cysB gene were generated and characterized. Electrophoretic mobility gel shift assays were used to assess protein-DNA interactions. In addition, growth in defined media was used to characterize the ability of the wild type and mutant strains to utilize a wide range of diverse sulfur sources. These studies have broader implications for biotechnology, metabolic engineering, and novel approaches to drug development.