beta-Ketothiolase genes in Azotobacter vinelandii

Azotobacter vinelandii is proposed to contain a single beta -ketothiolase a ctivity participating in the formation of acetoacetyl-CoA, a precursor for poly-beta -hydroxybutyrate (PHB) synthesis, and in beta -oxidation (Manchak , J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotob acter vinelandii strain UWD. Microbiology 140, 953-963). We designed a dege nerate oligonucleotide from a highly conserved region among bacterial beta -ketothiolases and used it to identify bktA, a gene with a deduced protein product with a high similarity to beta -ketothiolases. Immediately downstre am of bktA, we identified a gene called hbdH, which encodes a protein exhib iting similarity to beta -hydroxyacyl-CoA and beta -hydroxybutyryl-CoA dehy drogenases. Two regions with homology to bktA were also observed. One of th ese was cloned and allowed the identification of the phbA gene, encoding a second beta -ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbd H and phbA mutations, respectively, as well as strain EG1 carrying both bkt A and phbA mutations, were constructed. The hbdH mutation had no effect on beta -hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilati on. The bktA mutation had no effect on beta -ketothiolase activity, PHB syn thesis or fatty acid assimilation, whereas the phbA mutation significantly reduced beta -ketothiolase activity and PHB accumulation, showing that this is the beta -ketothiolase involved in PHB biosynthesis. Strain EG1 was fou nd to grow under beta -oxidation conditions and to possess beta -ketothiola se activity. Taken together, these results demonstrate the presence of thre e genes coding for beta -ketothiolases in A. vinelandii. (C) 2000 Elsevier Science B.V. All rights reserved.