Excision of Anabaena PCC 7120 nifD element in Escherichia coli: Growth kinetics and RecA regulated xisA expression and DNA rearrangement R. Karunakaran a,1 , Omita Mehta a,2 , Prashant Kunjadia a,3 , Shrikumar Apte b , G. Nareshkumar a, * a Molecular Microbial Biochemistry Laboratory, Department of Biochemistry, Faculty of Science, M.S. University of Baroda, Vadodara 390 002, India b Molecular Biology Division, Bhabha Atomic Research Center, Trombay, Mumbai 400 085, India Received 6 December 2006; received in revised form 29 May 2007; accepted 6 July 2007 Abstract Anabaena PCC 7120 nifHDK operon is interrupted by an 11 kb DNA element which is excised during the development of heterocysts by Excisase A, encoded by the xisA gene residing on the element. The excision is a site-specific recombination event that occurs at the 11 base pair direct repeats flanking the element. Earlier work showed the excision of the 11 kb element in Escherichia coli at a frequency 0.3%. We report here the excision of this element at 1.1% and 1.98% in E. coli DH5a, and 1.9% and 10.9% in E. coli JM 101 when grown on Luria broth and minimal media, respectively. Excision of nifD element in isogenic recA  (RK1) and recA + (RK2) E. coli JM101 P1 transductants, showed similar results to that of E. coli JM101 and DH5a, respectively. A plasmid pMX32, carrying a xisA defective 11 kb element, showed no excision in E. coli RK2 strain. In contrast to Anabaena PCC 7120, excision of nifD element did not increase in E. coli DH5a grown in iron-deficient conditions. A PxisA::lacZ transcriptional fusion, used to detect the expression of elusive xisA gene, showed maximal b-galactosidase activity in the stationary phase. The results suggest that the excision event in E. coli may involve addi- tional factors, such as RecA and that the physiological status can influence the excision of nifD element. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Rearrangement; NifD; Anabaena PCC7120 1. Introduction Anabaena sp. strain PCC 7120 is a heterocystous cyano- bacterium that forms heterocysts in response to a step down in combined nitrogen source and fixes nitrogen in heterocysts (Golden and Yoon, 2003; Meeks and Elhai, 2002). Nitrogen fixation (nif) genes in Anabaena PCC 7120 are widely dispersed in the genome (Kaneko et al., 2001). Genome of the heterocysts undergoes three indepen- dent DNA rearrangements in the later stages of heterocyst differentiation in response to nitrogen starvation (Carrasco and Golden, 1985; Golden et al., 1985). The nifD, fdxN, and hupL genes are interrupted by DNA elements, which are present in vegetative cell chromosome from nt 1,700,623 to 1,711,900 (11,278 bp), 1,716,797–1,776,224 (59,428 bp) and 785,538–794,956 (9419 bp) (Kaneko et al., 2001). These elements are flanked by direct repeat of nucleotides of 11 bp, 5 bp, and 16 bp, respectively. The recombinase genes located within the aforesaid the ele- ments encode XisA, XisF and XisC proteins, respectively. 0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.07.031 * Corresponding author. Tel.: +91 265 2795594; mobile: +91 9227104967. E-mail address: gnaresh_k@yahoo.co.in (G. Nareshkumar). 1 Present address: School of Animal and Microbial Sciences, University of Reading, Whiteknights, P.O. Box 228, Reading RG6 6AJ, UK. 2 Present address: Department of Pathology, University of Chicago, 5841 S Maryland Avenue, Chicago, IL 60637, USA. 3 Present address: N.V. Patel College of Pure and Applied Sciences (NVPAS). Charutar Vidya Mandal, Vallabh Vidyanagar 388120, Anand, India. Bioresource Technology xxx (2007) xxx–xxx ARTICLE IN PRESS Please cite this article in press as: Karunakaran, R. et al., Excision of Anabaena PCC 7120 nifD element in Escherichia ..., Bioresour. Technol. (2007), doi:10.1016/j.biortech.2007.07.031