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

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Journal of Bacteriology, June 1999, p. 3695-3704, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Smadar Shulami,¹ Orit Gat,¹^, Abraham L. Sonenshein,² and Yuval Shoham¹^,^*

Department of Food Engineering and Biotechnology, Technion-Israel Institute of Technology, Haifa 32000, Israel,¹ and Department of Molecular Biology and Microbiology, Tufts University, Boston, Massachusetts 02111²

Received 4 February 1999/Accepted 13 April 1999

A $lambda$ -EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independentclones exhibiting $beta$ -xylosidase activity were isolated. The clonesoverlap each other and together represent a 23.5-kb chromosomalsegment. The segment contains a cluster of xylan utilization genes,which are organized in at least three transcriptional units. Theseinclude the gene for the extracellular xylanase, xylanase T-6;part of an operon coding for an intracellular xylanase and a $beta$ -xylosidase;and a putative 15.5-kb-long transcriptional unit, consisting of12 genes involved in the utilization of $alpha$ -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 characteristiccomponents of the binding-protein-dependent transport systems.The most likely natural substrate for this transport system isaldotetraouronic acid [2-O- $alpha$ -(4-O-methyl- $alpha$ -D-glucuronosyl)-xylotriose](MeGlcUAXyl₃). The following two genes code for an intracellular $alpha$ -glucuronidase (aguA) and a $beta$ -xylosidase (xynB). Five more genes(kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologousto 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 determinedby primer extension analysis and is located 349 bp from the initialATG codon. The potential operator site is a perfect 12-bp invertedrepeat located downstream from the promoter between nucleotides+170 and +181. Gel retardation assays indicated that UxuR bindsspecifically to this sequence and that this binding is efficientlyprevented in vitro by MeGlcUAXyl₃, the most likely molecularinducer.

^* Corresponding author. Mailing address: Department of Food Engineering & Biotechnology, Technion, Haifa 32000, Israel. Phone:972-4-8293072. Fax: 972-4-8320742. E-mail: yshoham{at}tx.technion.ac.il.

Present address: Department of Biochemistry, Israel Institute for Biological Research, Ness-Ziona 70400,Israel.

Journal of Bacteriology, June 1999, p. 3695-3704, Vol. 181, No. 12
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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