EXPRESSION OF THE PUTA GENE ENCODING PROLINE DEHYDROGENASE FROM RHODOBACTER-CAPSULATUS IS INDEPENDENT OF NTRC REGULATION BUT REQUIRES AN LRP-LIKE ACTIVATOR PROTEIN

Four Rhodobacter capsulatus mutants unable to grow with proline as the sole nitrogen source were isolated by random Tn5 mutagenesis. The Tn5 insertions were mapped within two adjacent chromosomal EcoRI fragments, DNA sequence analysis of this region revealed three open reading frames designated selD, putR, and putA, The putA gene codes for a protein of 1,127 amino acid residues which is homologous to PutA of Salmonella typhimurium and Escherichia coli, The central part of R. capsulatus PutA showed homology to proline dehydrogenase of Saccharomyces cerevisiae (Put1) and Drosophila melanogaster (SlgA). The C-terminal part of PutA exhibited homology to Put2 (pyrroline-5-carboxylate dehydrogenase) of S. cerevisiae and to aldehyde dehydrogenases from different organisms, Therefore, it seems likely that in R, capsulatus, as in enteric bacteria, both enzymatic steps for proline degradation are catalyzed by a single polypeptide (PutA), The deduced amino acid sequence of PutR (154 amino acid residues) shelved homology to the small regulatory proteins Lrp, BkdR, and AsnC, The putR gene, which is divergently transcribed from putA, is essential for proline utilization and codes for an activator of putA expression. The expression of putA was induced by proline and was not affected by ammonia or other amino acids. In addition, putA expression was autoregulated by PutA itself, Mutations in glnB, nifR1 (ntrC), and nifR4 (ntrA encoding sigma(54)) had no influence on put gene expression, The open reading frame located downstream of R, capsulatus putR exhibited strong homology to the E. coli selD gene, which is involved in selenium metabolism, R. capsulatus selD mutants exhibited a Put(+) phenotype, demonstrating that selD is required neither for viability nor for proline utilization.