N(5)-(carboxyethyl)ornithine synthase (CeOS), an enzyme discovered in Streptococcus lactis, catalyzes the NADPH-dependent condensation between pyruvic acid and the terminal amino group of ornithine to yield N(5)-(carboxyethyl) ornithine (CeO). Polyclonal antibodies against CEOS were used to examine (by Western blot analysis) the dissemination and size of the enzyme in various tactic acid bacteria. Of the three genera and six species surveyed, CeOS was detected only in about 50% of the S. lactis strains examined. CeOS is constitutive; in several strains (e.g., K1) it exists primarily as a tetramer of 38-kDa subunits, but in other strains (e.g., 133) the subunit mass is 35-kDa. Tracer studies indicate that CeO is biosynthetically derived from exogenous arginine which, upon uptake into the cell, is rapidly converted to ornithine. Once synthesized from ornithine and pyruvate, CeO is relatively stable, which explains why it accumulates to relatively high levels (10 mM) intracellularly. As a result of experiments to determine whether CeOS was encoded on one of the plasmids in strain K1, a spontaneous derivative (K1-42) was isolated which lacked CeOS as well as the ability to ferment sucrose and synthesize the polypeptide antibiotic nisin. Hybridizations with a nisin gene probe indicated that these linked traits are encoded on the chromosome. Because the ability to ferment sucrose (and synthesize nisin) is conjugally transferable, we have proposed that these traits are located on a conjugative transposon. To further localize and clone the CeOS gene, a 39-mer based on a peptide within the aminoterminus of CeOS was synthesized. Comparison of Southern blots of the parent (K1) and the mutant (K1-42) revealed the absence of a 7-kbp EcoRI-XhoI fragment that hybridized to the labeled 39-mer. Using this probe for detection, recombinant phages were obtained which contained this latter fragment inserted into the vector Lambda ZAPII, and several of them reacted with CeOS specific antibody.
