The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6

Abstract

A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-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 beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 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 characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic 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 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 MeGlcUAXyl3, the most likely molecular inducer.

FIG. 1

A proposed degradation pathway of MeGlcUAXyl₃ in B. stearothermophilus T-6. (A) Xylan is composed of β-1,4-linked xylopyranose units which can be substituted with l-arabinofuranosyl, methyl-d-glucuronic acid, and acetyl side chains. The key enzyme in the degradation of xylan is an extracellular endo-1,4-β-xylanase (xynA). This enzyme releases short xylose units (xylobiose, xylotriose, and xylotetraose) which can be substituted with various side chains such as l-arabinose, d-glucuronic acid, or its 4-O-methyl ether, as in 2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose (MeGlcUAXyl₃). (B) MeGlcUAXyl₃ is demethylated and enters the cell via a specific transporting system. This system resembles the binding-protein-dependent transport systems in which a solute-binding lipoprotein interacts with integral membrane protein components that are involved in translocating the substrate across the membrane (22). Inside the cell, GlcUAXyl₃ is cleaved by α-glucuronidase to yield xylotriose and d-glucuronic acid. The xylo-oligomers are hydrolyzed to xylose by intracellular xylanase and β-xylosidase. Xylose is converted into xylulose-5-phosphate, which can enter the pentose cycle. d-Glucuronic acid is converted into KDG by a three-step pathway catalyzed by uronate isomerase (uxaC), d-mannonate oxidoreductase (uxuB), and d-mannonate hydrolase (uxuA). KDG is then phosphorylated by KDG kinase (kdgK) to give KDGP, which is finally cleaved by KDGP aldolase (kdgA) to yield glyceraldehyde 3-phosphate (GA3P) and pyruvate. The latter two compounds can enter the Embden-Meyerhof-Parnas pathway.

FIG. 2

Genetic map of the 23.5-kb segment containing xylan and glucuronic acid utilization genes. (A) Fragments cloned directly from phage DNA into pKS or pSL301 are shown as open rectangles. (B) The positions of the 19 ORFs. The sequence of the N terminus of the xynB2 gene product is incomplete. The letter P indicates the proposed promoter regions, and Ω indicates rho-independent terminator-like transcription terminators. (C) The number of amino acids and the calculated molecular weight (in thousands) of the putative protein encoded by each gene are given below it. (D) Detection of xynB, uxaC, and xynA expression at the RNA level by slot blot RNA. Total RNA was isolated from mid-exponential-phase cultures of B. stearothermophilus T-6 grown in BSM supplemented with 0.5% xylose and 0.5% glucose (a) or with 0.5% glucose as the sole carbon source (b), applied to Schleicher & Schuell BA85 nitrocellulose, and annealed separately to three different ³⁵S-labeled DNAs for xynB, uxaC, and xynA. The hybridization solution contained about 5,000 cpm of each probe per ml.

FIG. 3

(A) Mapping the 5′ termini of the GlcUA cluster by primer extension analysis. Total RNA was isolated from mid-exponential-phase cultures of B. stearothermophilus T-6 grown in BSM supplemented with 0.5% xylose and 0.5% glucose (lane 1) or with 0.5% glucose as the sole carbon source (lane 2). Dideoxynucleotide sequence reactions were carried out with the same primer used for the reverse transcriptase reactions. The position of the transcriptional start point is indicated with an asterisk on the inferred nontemplate strand sequence. (B) Sequence data for the regulatory region. The transcriptional start point (+1) is indicated by a vertical arrowhead. The −35 and −10 regions, the proposed ribosome binding site (RBS), the initiating methionine codon, and the potential CREs are in boldface. The CRE sequence is TGT/AAANC|GNTNA/TCA, where underlined letters represent the most critical bases, N is any base, and the vertical line denotes an axis of symmetry (28, 68). The GlcUA operator is indicated by horizontal arrowheads above the inverted repeat. The sequence of the primer used for primer extension experiments is underlined.

FIG. 4

(A) Gel retardation of a ³²P-labeled GlcUA operator fragment by crude extracts from E. coli cells producing UxuR. All lanes contained about 4 fmol (0.08 ng) of radioactively labeled DNA fragment containing the synthetic GlcUA operator. Lane 1 contained no extract. Lane 2 contained 20 μg of crude extract from cells carrying only the vector (pET11d). Lanes 3 to 8 contained different amounts of crude extracts from E. coli producing UxuR. The shifted bands are indicated by arrows: a, higher-mobility band; b, lower-mobility band; c, free DNA (not shifted). (B) Competition experiments using unlabeled synthetic GlcUA operator. Lane 1 contained 0.62 μg of crude extract from cells carrying only the vector (pET11d). Lanes 2 to 9 contained crude extracts from E. coli producing UxuR together with different amounts of the unlabeled synthetic GlcUA operator. (C) Competition experiments using synthetic araD operator. Lanes 1 to 6 contained the ³⁵S-labeled synthetic GlcUA operator with no extract (lane 1); extract from cells carrying only the vector (pET11d) (lane 2); and extracts from E. coli producing UxuR together with 0, 500, 1,000, and 2,000 fmol of unlabeled synthetic araD operator, (lanes 3 to 6, respectively). Lanes 7 and 8 contained 4 fmol of ³⁵S-labeled synthetic araD operator without (lane 7) or with (lane 8) 20 μg of crude extracts from E. coli producing UxuR.

FIG. 5

Binding of UxuR to the GlcUA operator in the presence of various sugars. All lanes contained 0.08 ng of radioactively labeled DNA. Lane 1 contained no extract. Lane 2 contained 0.16 μg of crude extract from cells carrying only the vector (pET11d). Lanes 3 to 11 contained 0.16 μg of crude extracts from E. coli producing UxuR (pET11d-uxuR) together with a 10 mM concentration of the following sugars: aldotetraouronic acid [2-O-α-(4-O-methyl-α-d-glucuronosyl)-xylotriose (MeGlcUAXyl₃)] (lane 3), a solution of aldobiouronic acid (MeGlcUAXyl₁) (20%) and aldotriouronic acid (MeGlcUAXyl₂) (80%) (lane 4), xylose (Xyl) (lane 5), xylobiose (Xyl₂) (lane 6), xylotriose (Xyl₃) (lane 7), xylotetraose (Xyl₄) (lane 8), d-glucuronic acid (GlcUA) (lane 9), d-glucose (Glc) (lane 10), and l-arabinose (Ara) (lane 11).