Molecular analysis of the amy gene locus of Thermoanaerobacterium thermosulfurigenes EM1 encoding starch-degrading enzymes and a binding protein-dependent maltose transport system

Abstract

A gene of Thermoanaerobacterium thermosulfurigenes EM1 encoding a protein with similarity to the maltose-binding protein of Escherichia coli was cloned and sequenced. It was located in the amy gene region of the chromosome downstream of the pullulanase-encoding amyB gene and upstream of amyDC, encoding membrane components of an ABC transport system, and the alpha-amylase gene amyA. The gene was designated amyE. Analysis of mRNA by Northern (RNA) blotting revealed that expression of the amy gene region is repressed during growth on glucose. Maximum levels of mRNA were detected with maltose as a substrate. An operon which was transcribed in the order amyBEDC was identified. However, an additional transcription start point was found in front of amyE. The amyA gene represented a monocistronic operon. Putative -35 and -10 promoter sites were deduced from the three transcription start sites of the amy gene region, and possible regulatory regions mediating induction by maltose and catabolite repression by glucose were identified by sequence analysis and comparison. The biochemical characterization of maltose uptake in T. thermosulfurigenes EM1 revealed two transport systems with Km values of 7 microM (high affinity) and 400 microM (low affinity). We conclude that the high-affinity system, which is specific for maltose and maltotriose, is a binding-protein-dependent transporter encoded by amyEDC. The gene for the putative ATP-binding protein has not yet been identified, and in contrast to similar systems in other bacteria, it is not located in the immediate vicinity of the chromosome.