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Journal of Bacteriology, April 1999, p. 2001-2007, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

acs1 of Haemophilus influenzae Type a Capsulation Locus Region II Encodes a Bifunctional Ribulose 5-Phosphate Reductase- CDP-Ribitol Pyrophosphorylase

Anja Follens,1 Maria Veiga-da-Cunha,2 Rita Merckx,1 Emile van Schaftingen,2 and Johan van Eldere1,*

Rega Institute for Medical Research, Catholic University of Leuven, B-3000 Leuven,1 and C. de Duve Institute of Cellular Pathology, Université Catholique de Louvain 7539, B-1200 Brussels,2 Belgium

Received 23 December 1998/Accepted 19 January 1999

The serotype-specific, 5.9-kb region II of the Haemophilus influenzae type a capsulation locus was sequenced and found to contain four open reading frames termed acs1 to acs4. Acs1 was 96% identical to H. influenzae type b Orf1, previously shown to have CDP-ribitol pyrophosphorylase activity (J. Van Eldere, L. Brophy, B. Loynds, P. Celis, I. Hancock, S. Carman, J. S. Kroll, and E. R. Moxon, Mol. Microbiol. 15:107-118, 1995). Low but significant homology to other pyrophosphorylases was only detected in the N-terminal part of Acs1, whereas the C-terminal part was homologous to several short-chain dehydrogenases/reductases, suggesting that Acs1 might be a bifunctional enzyme. To test this hypothesis, acs1 was cloned in an expression vector and overexpressed in Escherichia coli. Cells expressing this protein displayed both ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities, whereas these activities were not detectable in control cells. Acs1 was purified to near homogeneity and found to copurify with ribitol 5-phosphate dehydrogenase and CDP-ribitol pyrophosphorylase activities. These had superimposable elution profiles from DEAE-Sepharose and Blue-Sepharose columns. The dehydrogenase activity was specific for ribulose 5-phosphate and NADPH in one direction and for ribitol 5-phosphate and NADP+ in the other direction and was markedly stimulated by CTP. The pyrophosphorylase showed activity with CTP and ribitol 5-phosphate or arabitol 5-phosphate. We conclude that acs1 encodes a bifunctional enzyme that converts ribulose 5-phosphate into ribitol 5-phosphate and further into CDP-ribitol, which is the activated precursor form for incorporation of ribitol 5-phosphate into the H. influenzae type a capsular polysaccharide.

* Corresponding author. Mailing address: Rega Institute for Medical Research, Catholic University of Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Phone: 32 16 337372. Fax: 32 16 337320. E-mail: Johan.VanEldere{at}rega.kuleuven.ac.be.

Journal of Bacteriology, April 1999, p. 2001-2007, Vol. 181, No. 7
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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