Characterization Of The Azotobacter Vinelandii Algc Gene

FEMS Microbiology Letters 238 (2004) 199–206 www.fems-microbiology.org

Characterization of the Azotobacter vinelandii algC gene involved in alginate and lipopolysaccharide production
´˜ Gerardo Gaona a,1, Cinthia Nunez a, Joanna B. Goldberg b, Alicia S. Linford b, a ´ ´ ´ Rebeca Najera , Miguel Castaneda c, Josefina Guzman a, Guadalupe Espın a, ˜ a,* ´ ´ Gloria Soberon-Chavez
a

´ ´ ´ ´Departamento de Microbiologıa Molecular, Instituto de Biotecnologıa, Universidad Nacional Autonoma de Mexico, Cuernavaca, Morelos, Mexico b Department of Microbiology, University of Virginia, Health Science Center, Charlottesville, VA 22908, USA c ´ Centro de Investigaciones en Ciencias Microbiologicas, Instituto de Ciencias, BUAP, Puebla, Mexico Received 10 July 2004; accepted 16 July 2004 Firstpublished online 30 July 2004

Abstract Azotobacter vinelandii is a soil c-proteobacteria that fixes nitrogen and forms desiccation-resistant cysts. The exopolysaccharide alginate is an integral part of the layers surrounding the cysts. Here, we reported the cloning of A. vinelandii algC, encoding the enzyme catalyzing the second step of alginate pathway. We showed that AlgC is involved not only inalginate production, but also in lipopolysaccharide (LPS) synthesis and that it seems to have both phosphomannomutase and phosphoglucomutase activities. The transcriptional analysis of the A. vinelandii algC gene showed that it contained two start sites, one of which was dependent on the alternative sigma factor AlgU/AlgT. This finding explains why alginate biosynthesis is dependent on AlgUactivity, since all other alginate biosynthetic genes have been characterized previously and algC is the only alginate structural gene that is directly transcribed by this sigma factor. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
Keywords: Alginate; Azotobacter vinelandii; Extracytoplasmic function sigma factor

1. Introduction Azotobactervinelandii is a soil bacterium belonging to the c-proteobacteria. It fixes nitrogen under aero-

´ Corresponding author at: Departamento de Biologıa Molecular y ´ ´ Biotecnologıa, Instituto de Investigaciones Biomedicas, UNAM, Apdo ´ Postal 70-228, Ciudad de Mexico, 04510, Mexico. Tel.: +52 55 5622 3870; fax: +52 55 5622 3855. ´ ´ E-mail address: gloria@biomedicas.unam.mx (G. Soberon-Chavez). 1 ´Present address: Departamento de Biotecnologıa, Facultad de ´ ´ Ciencias Quımicas, Universidad Autonoma de Coahuila, Saltillo, Coahuila.

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bic conditions due to an increased respiration rate under conditions of nitrogen starvation [1]. Under unfavorable environmental conditions A. vinelandii forms desiccation-resistant cysts [2], which are differentiated cells surrounded by two capsule-likelayers containing a high proportion of the exopolysaccharide alginate [3]. Alginate is essential for the encystment process, since non-mucoid strains fail to encyst [4–6]. Recently the entire sequence of the 4.5Mb chromosome of strain UW was determined (www. azotobacter.org/wat). The molecular genetics of A. vinelandii alginate has been described in some detail [4,7–14], including its biosyntheticpathway [15]. It has been reported that the

0378-1097/$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsle.2004.07.044

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G. Gaona et al. / FEMS Microbiology Letters 238 (2004) 199–206

genes encoding for the enzymes involved in alginate synthesis, with the exception of algC which encodes for the secondenzyme in this biosynthetic route, are present in a gene cluster [4,5,7,8], that is arranged as three operons [5,7,8]. One of these transcriptional units includes the algD gene, coding for GDP-mannose dehydrogenase, alone [4]. The alternative sigma factor rE (also known as AlgU or AlgT) [9], which belongs to the family of sigma factors that regulate the expression of functions related to the...