Downloaded from www.microbiologyresearch.org by IP: 54.237.90.162 On: Sat, 09 Jul 2016 19:35:22 Microbiology (1997), 143, 957-969 Printed in Great Britain The Bacillus subtilis L-arabinose (ara) operon : nucleotide sequence, genetic organization and expression Isabe1 Sd-Nlogueira,l Teresa V. Nogueira,'t S6nia Soares'If: and Herminia de Lencastre1t2 Author for correspondence: Isabel Sa-Nogueira. Tel: +351 1 4426171. Fax: +351 1 4428766. e-mail : sanoguei @itqb.unl.pt lnstituto de Tecnologia Quimica e Bioldgica, Universidade Nova de Lisboa. Apartado 127,2780 Oeiras Codex, Portugal The Rockefeller Un iversity, Laboratory of Microbiology, 1230 York Avenue, New York, NY 10021 -6399, USA The Bacillus subtilis L-arabinose metabolic genes araA, araB and araD, encoding L-arabinose isomerase, L-ri bulokinase and ~-ribulose-5-phosphate 4-epimerase, respectively, have been cloned previously and the products of araB and araD were shown to be functionally homologous to their Escherichia coli counterparts by complementation experiments. Here we report that araA, araB and araD, whose inactivation leads to an Ara' phenotype, are the first three ORFs of a nine cistron transcriptional unit with a total length of 11 kb. This operon, called ara, is located at about 256" on the B. subtilis genetic map and contains six new genes named araL, araM, araN, araP, araQ and abfA. Expression of the ara operon is directed by a strong &like promoter identified within a 150 bp DNA fragment upstream from the translation start site of araA. Analysis of the sequence of the ara operon showed that the putative products of araN, araP and araQ are homologous to bacterial components of binding-protein-dependent transport systems and abfA most probably encodes an a-L-arabinofuranosidase. The functions of araL and araM are unknown. An in vitro-constructed insertion-deletion mutation in the region downstream from araD allowed us to demonstrate that araf, araM, araN, araP, araQ and abfA are not essential for L-arabinose utilization. Studies with strains bearing transcriptional fusions of the operon to the E. coli lacZ gene revealed that expression from the ara promoter is induced by L-arabinose and repressed by g I ucose. Keywords : Bacillus subtilis, L-arabinose (ara) operon, expression, catabolite repression INTRODUCTION Bacillus subtilis, an endospore-forming Gram-positive bacterium, is able to grow on L-arabinose as sole carbon source. L-Arabinose residues are found widely distri- buted among many heteropolysaccharides of different plant tissues, such as arabinans, arabinogalactans, xylans and arabinoxylans. Bacillus species in their t Present address: lnstitut de Biologie Physico-Chimique,, 13 Rue Pierre et Marie Curie, 75005 Paris, France. *Present address: Public Health Research Institute, 455 First Avenue, New York, NY 10016, USA. Abbreviations: Cm, chloramphenicol; Em, erythromycin; Km, kanamycin; Sp, spectinomycin. The accession numbers for the nucleotide sequences reported in this paper are X89408 (araA, B and D) and X89810 (araL, M, N, P, Q ,and abfA). natural reservoir, the soil, participate in the early stages of plant material decomposition and B. subtilis secretes three enzymes, an endo-arabanase and two arabino- sidases, capable of releasing arabinosyl oligomers and L- arabinose from plant cell walls (Kaji & Saheki, 1975; Weinstein & Albersheim, 1979). The pathway of L- arabinose utilization in B. subtilis has been described by Lepesant & Dedonder (1967a). After entering the cell, L- arabinose is sequentially converted to L-ribulose, L- ribulose 5-phosphate, and D-XylUlOSe 5-phosphate by the action of L-arabinose isomerase, L-ribulokinase and ~-ribulose-5-phosphate 4-epimerase, respectively. D-XY- lulose 5-phosphate is further catabolized through the pentose phosphate pathway. Mutants unable to use L- arabinose as sole carbon source, deficient in one of the three enzymes involved in L-arabinose catabolism, have been characterized, as well as constitutive mutants for 0002-1 125 0 1997 SGM 957