Selection of nitrogen-fixing deficient Burkholderia vietnamiensis strains by cystic fibrosis patients: involvement of nif gene deletions and auxotrophic mutations Aymeric Menard, 1 Claire Monnez, 1 Paulina Estrada de los Santos, 1† Christine Segonds, 2 Jesus Caballero-Mellado, 3 John J. LiPuma, 4 Gerard Chabanon 2 and Benoit Cournoyer 1 * 1 Research group on «Bacterial Opportunistic Pathogens and Environment», Université de Lyon, Lyon, F-69003, France; Université Lyon 1, Villeurbanne, F-69622, France; CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, F-69622; Ecole Nationale Vétérinaire de Lyon, Marcy L’étoile, F-69280, France. 2 Observatoire cepacia, Laboratoire de Bacteriologie- Hygiene, Institut Federatif de Biologie, Hôpital Purpan, TSA 40031, 31059 Toulouse Cedex 9, France. 3 Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Ap. Postal 565-A, Cuernavaca, Morelos, Mexico. 4 Department of Pediatrics and Communicable Diseases, University of Michigan, Ann Arbor, MI 48109, USA. Summary Burkholderia vietnamiensis is the third most prevalent species of the Burkholderia cepacia complex (Bcc) found in cystic fibrosis (CF) patients. Its ability at fixing nitrogen makes it one of the main Bcc species showing strong filiations with environmental reser- voirs. In this study, 83% (29 over 35) of the B. vietnam- iensis CF isolates and 100% of the environmental ones (over 29) were found expressing the dinitroge- nase complex (encoded by the nif cluster) which is essential in N 2 fixation. Among the deficient strains, two were found growing with ammonium chloride suggesting that they were defective in N2 fixation, and four with amino acids supplements suggesting that they were harbouring auxotrophic mutations. To get insights about the genetic events that led to the emer- gence of the N2-fixing defective strains, a genetic analysis of B. vietnamiensis nitrogen-fixing property was undertaken. A 40-kb-long nif cluster and nif regulatory genes were identified within the B. viet- namiensis strain G4 genome sequence, and analysed. Transposon mutagenesis and nifH genetic marker exchanges showed the nif cluster and several other genes like gltB (encoding a subunit of the glutamate synthase) to play a key role in B. vietnamiensis ability at growing in nitrogen-free media. nif cluster DNA probings of restricted genomic DNA blots showed a full deletion of the nif cluster for one of the N 2-fixing defective strain while the other one showed a genetic organization similar to the one of the G4 strain. For 17% of B. vietnamiensis clinical strains, CF lungs appeared to have favoured the selection of mutations or deletions leading to N2-fixing deficiencies. Introduction The Burkholderia genus is part of the Betaproteobacteria and consists of more than 30 bacterial species. It includes a tight phylogenetic complex of more than 10 species which is named the Burkholderia cepacia complex (Bcc). Most of these Bcc species can infect cystic fibrosis (CF) patients. They can also infect immunocompromised indi- viduals or patients with chronic granulomatous disease (for review of Bcc infections see Mahenthiralingam et al., 2005). Burkholderia vietnamiensis is the third most preva- lent species of the Bcc found among CF patients (LiPuma, 2005). The most damaging and prevalent Bcc species in CF patients are B. cenocepacia and B. multivorans. Burkholderia vietnamiensis was also reported in bacter- aemia (Magalhães et al., 2003) and a neck abscess (Gillis et al., 1995). Other Burkholderia species like B. gladioli and B. pseudomallei were also recorded in CF patients (Bauernfeind et al., 1998; O’Carroll et al., 2003). Burkholderia pseudomallei is the aetiological agent of melioidosis (Brygoo, 1953). Several Burkholderia species including those of the Bcc are well known for their potential benefits in agriculture and the chemical industries. Burkholderia spp. can also be nitrogen fixers, converting N 2 into NH3. Only one species of the Bcc, B. vietnamiensis, is, these days, con- sidered able to fix nitrogen (Perin et al., 2006). However, the literature published prior to the definition of the Bcc Received 4 October, 2006; accepted 14 December, 2006. *For correspondence. E-mail cournoye@biomserv.univ-lyon1.fr; Tel. (+33) 4 72 43 14 95; Fax (+33) 4 72 43 12 23. † Present address: Vrij Universiteit Brussel, Pleinlaan 2, B-1050, Brussel, Belgium. Environmental Microbiology (2007) 9(5), 1176–1185 doi:10.1111/j.1462-2920.2007.01240.x © 2007 The Authors Journal compilation © 2007 Society for Applied Microbiology and Blackwell Publishing Ltd