The Glucuronic Acid Utilization Gene Cluster from Bacillus stearothermophilus T-6

Abstract

ABSTRACT A λ-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting β-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a β-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of α- d -glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon ( orf1 , - 2 , - 3 , and - 4 ) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2- O -α-(4- O -methyl-α- d -glucuronosyl)-xylotriose] (MeGlcUAXyl 3 ). The following two genes code for an intracellular α-glucuronidase ( aguA ) and a β-xylosidase ( xynB ). Five more genes ( kdgK , kdgA , uxaC , uxuA , and uxuB ) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR , the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl 3 , the most likely molecular inducer.