Characterization of Methanobacterium thermoautotrophicum Marburg mutants defective in regulation of L-tryptophan biosynthesis

Abstract

Three nitrosoguanidine-induced mutants of the archaeon Methanobacterium thermoautotrophicum Marburg resistant to 5-methyltryptophan were isolated and characterized. They were found to take up L-tryptophan, as wild-type cells, via an energy-dependent, low-affinity transport system specific for L-tryptophan, with a Km of 300 microM and a Vmax of 7 nmol/mg (dry weight)/min. Resistance to 5-methyltryptophan was not due to feedback-resistant anthranilate synthase but to constitutive expression of the trp genes, as measured by the specific activities of anthranilate 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 without L-tryptophan suggested that constitutive expression resulted from deficient transcriptional regulation. The enhanced expression of the trp genes in the mutants was found to result in intracellular L-tryptophan pools that were two- to fourfold higher than in the wild type. Sequencing of the region upstream of trpE revealed in two mutants point mutations mapping on the 5'-side of the archaeal box A, whereas in the third mutant this region did not differ from that of the wild type. These results suggest that (i) in M. thermoautotrophicum the 5-methyltryptophan-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 part of this system.