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Journal of Bacteriology, June 2008, p. 4272-4280, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00162-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Characterization of abn2 (yxiA), Encoding a Bacillus subtilis GH43 Arabinanase, Abn2, and Its Role in Arabino-Polysaccharide Degradation{triangledown}

José Manuel Inácio1 and Isabel de Sá-Nogueira1,2*

Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa. Avenida de República-EAN, 2780-157 Oeiras, Portugal,1 Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Quinta da Torre, 2829-516 Caparica, Portugal2

Received 1 February 2008/ Accepted 5 April 2008

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-{alpha}-1,5-L-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-{alpha}-1,5-L-arabinan as the preferred substrate, the enzyme exhibited an apparent Km of 2.0 mg ml–1 and Vmax of 0.25 mmol min–1 mg–1 at pH 7.0 and 50°C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a {sigma}A-dependent and a {sigma}H-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

* Corresponding author. Mailing address: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida de República-EAN, 2780-157 Oeiras, Portugal. Phone: (351)-21-4469524. Fax: (351)-21-4411277. E-mail: sanoguei{at}itqb.unl.pt

{triangledown} Published ahead of print on 11 April 2008.

Journal of Bacteriology, June 2008, p. 4272-4280, Vol. 190, No. 12
0021-9193/08/$08.00+0     doi:10.1128/JB.00162-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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