CHARACTERIZATION OF METHANOBACTERIUM-THERMOAUTOTROPHICUM MARBURG MUTANTS DEFECTIVE IN REGULATION OF L-TRYPTOPHAN BIOSYNTHESIS

Three nitrosoguanidine-induced mutants of the archaeon Methanobacteriu m thermoautotrophicum Marburg resistant to 5-methyltryptophan were iso lated and characterized. They were found to take up L-tryptophan, as w ild-type cells, via an energy-dependent, low-affinity transport system specific for L-tryptophan, with a K-m of 300 mu M and a V-max of 7 nm ol/mg (dry weight)/min. Resistance to 5-methyltryptophan was not due t o feedback-resistant anthranilate synthase but to constitutive express ion of the trp genes, as measured by the specific activities of anthra nilate synthase and tryptophan synthase, the enzymes encoded by trpEG and trpB, respectively, of the trpEGCFBAD gene cluster. Estimation of trpE mRNA obtained from mutant cells grown in minimal medium with or w ithout L-tryptophan suggested that constitutive expression resulted fr om deficient transcriptional regulation. The enhanced expression of th e trp genes in the mutants was found to result in intracellular L-tryp tophan pools that were two- to fourfold higher than in the wild type. Sequencing of the region upstream of trpE revealed in two mutants poin t mutations mapping on the 5'-side of the archaeal box A, whereas in t he third mutant this region did not differ from that of the wild type. These results suggest that (i) in M. thermoautotrophicum the 5-methyl tryptophan-resistant phenotype arises from lesions in components of a regulatory system controlling transcription of the trp genes and (ii) cis-acting sequence elements in front of the trpE promoter may form pa rt of this system.