Identification of a d-Arabinose-5-Phosphate Isomerase in the Gram-Positive Clostridium tetani

Abstract

d-Arabinose-5-phosphate (A5P) isomerases (APIs) catalyze the interconversion of d-ribulose-5-phosphate and d-arabinose-5-phosphate. Various Gram-negative bacteria, such as the uropathogenic Escherichia coli strain CFT073, contain multiple API paralogs (KdsD, GutQ, KpsF, and c3406) that have been assigned various cellular functions. The d-arabinose-5-phosphate formed by these enzymes seems to play important roles in the biosynthesis of lipopolysaccharide (LPS) and group 2 K-antigen capsules, as well as in the regulation of the cellular d-glucitol uptake and uropathogenic infectivity/virulence. The genome of a Gram-positive pathogenic bacterium, Clostridium tetani, contains a gene encoding a putative API, C. tetani API (CtAPI), even though C. tetani lacks both LPS and capsid biosynthetic genes. To better understand the physiological role of d-arabinose-5-phosphate in this Gram-positive organism, recombinant CtAPI was purified and characterized. CtAPI displays biochemical characteristics similar to those of APIs from Gram-negative organisms and complements the API deficiency of an E. coli API knockout strain. Thus, CtAPI represents the first d-arabinose-5-phosphate isomerase to be identified and characterized from a Gram-positive bacterium.IMPORTANCE The genome of Clostridium tetani, a pathogenic Gram-positive bacterium and the causative agent of tetanus, contains a gene (the CtAPI gene) that shares high sequence similarity with those of genes encoding d-arabinose-5-phosphate isomerases. APIs play an important role within Gram-negative bacteria in d-arabinose-5-phosphate production for lipopolysaccharide biosynthesis, capsule formation, and regulation of cellular d-glucitol uptake. The significance of our research is in identifying and characterizing CtAPI, the first Gram-positive API. Our findings show that CtAPI is specific to the interconversion of arabinose-5-phosphate and ribulose-5-phosphate while having no activity with the other sugars and sugar phosphates tested. We have speculated a regulatory role for this API in C. tetani, an organism that does not produce lipopolysaccharide.

Keywords: Clostridium tetani; aldose-ketose isomerase; arabinose-5-phosphate; isomerase; kinetics; ribulose-5-phosphate.

FIG 1

Multiple-sequence alignment of the sequences of CtAPI and the APIs of E. coli. Sequences were aligned using Clustal W (27). An asterisk denotes residues absolutely conserved, a colon denotes conservative replacements, and a period denotes semiconservative replacements.

FIG 2

Genomic contexts of gutQ in E. coli K-12 MG1655 (A) and the gene encoding CtAPI in C. tetani (B). Genes potentially involved in sugar metabolism and transport are shaded in dark gray.

FIG 3

Complementation of the A5P deficiency of E. coli strain TCM15 on an LB agar plate. (Left side of plate) Agar containing LB medium supplemented with 15 μM A5P, 10 μM G6P, and 0.1 mg/ml ampicillin. (Right side of plate) Agar containing LB medium supplemented with 0.1 mg/ml ampicillin. On both sides, the wedges were streaked with E. coli TCM15 harboring pT7-7-c3406 (positive control, wedge 1), E. coli TCM15 harboring pT7-7 (vector control, wedge 2), and E. coli TCM15 harboring pT7-7-CtAPI (CtAPI, wedge 3).