Effect of the expression of Escherichia coli fhuA gene in Rhizobium sp. IC3123 and ST1 in planta: Its role in increased nodule occupancy and function in pigeon pea R. Geetha, Anjana J. Desai, G. Archana * Department of Microbiology and Biotechnology Centre, The Maharaja Sayajirao University of Baroda, Vadodara 390002, Gujarat, India 1. Introduction Although iron is the fourth most abundant element on the Earth’s surface, its bioavailability in aerobic environments and at neutral pH is severely limited due to the presence of virtually insoluble ferric iron–hydroxide complexes under these conditions. Iron limitation is thus a major deterrent for optimum growth of most microorganisms as this metal is indispensable for vital functions like electron transport and DNA synthesis (Ratledge and Dover, 2001). Nitrogen-fixing rhizobia, living as endosymbionts in root nodules of legume host plants, have a high demand for iron because a number of ‘symbiotic’ proteins contain iron or heme (Guerinot, 1993). Diverse types of siderophores are produced by the different rhizobial genera, e.g. Rhizobium leguminosarum bv. viciae, the symbiont of peas, lentils, vetches and some beans, synthesizes a cyclic trihydroxamate type siderophore called vicibactin which is taken up by cells via outer and inner membrane machinery encoded by fhuABCD operon (Dilworth et al., 1998). Rhizobia to be used as biofertilizer should be able to colonize efficiently in the soil. Iron availability in aerobic environments and at neutral pH, is 10 18 M(Guerinot and Yi, 1994) which may be one of the factors affecting rhizospheric colonization (Guerinot, 1991), due to the presence of iron rich proteins involved in the symbiotic nitrogen fixation. As endosymbionts, rhizobia must have efficient mechanisms to acquire iron from host plant due to their high iron requirement to produce cytochromes three times more in comparison to their free living form (Sangwan and O’Brian, 1992) and synthesize nitrogenase (containing at least 30 iron atoms), which constitutes 10–12% of the total bacterial protein (Verma and Long, 1983). Mechanisms employed by rhizobia and other microorganisms to acquire iron include, production of siderophores for solubilizing iron (Dilworth et al., 1998; Jadhav and Desai, 1992; Berraho et al., 1997; Guerinot et al., 1990) and cross-utilization of siderophores produced by accompanying soil and rhizosphere microorganism (Plessner et al., 1993; Loper and Henkels, 1999; Jurkevitch et al., 1992). Applied Soil Ecology 43 (2009) 185–190 ARTICLE INFO Article history: Received 1 April 2008 Received in revised form 20 June 2009 Accepted 22 July 2009 Keywords: E. coli fhuA expression in rhizobia Ferrichrome uptake Pigeon pea rhizobia Siderophores Nodule occupancy ABSTRACT The inability to utilize a fungal siderophore as source of iron nutrition by most of the rhizobial cultures isolated from pigeon pea, could be considered a negative fitness factor since hydroxamate siderophores are found in significant amounts in natural soils. Thus these cultures were engineered to use ferrichrome a prototype of hydroxamate type siderophore. Pigeon pea Rhizobium spp. IC3123 and ST1 harboring Escherichia coli fhuA gene, responsible for uptake of Fe 3+ -ferrichrome, were obtained by transformation with pGR1, a broad host range plasmid carrying the fhuA gene under the control of the lac promoter of E. coli. Expression of fhuA in transformed rhizobial strains IC3123::pGR1 and ST1::pGR1 was confirmed by the ability of the plasmid-bearing strains to utilize iron bound to ferrichrome. Inoculation of pigeon pea plants with fhuA expressing rhizobial strains in pot experiments showed a significant increase in plant growth as well as nodule density as compared to those inoculated with the parent as well as the empty vector-bearing strain. Inoculation of pigeon pea seedlings with IC3123::pGR1 and ST1::pGR1 led to marked increase in shoot fresh weight, nodule number per plant, chlorophyll content of leaves and effective nodule symbiosis when compared with plants inoculated with the parent strains IC3123 and ST1. The positive effect of IC3123::pGR1 and ST1::pGR1 treatment on plant growth was more significantly observed when ferrichrome producing Ustilago maydis, known to secrete ferrichrome, was co-inoculated along with the transformed rhizobia. The presence of fhuA gene in rhizobial strains also led to an increased survival and root colonization. ß 2009 Elsevier B.V. All rights reserved. * Corresponding author. Tel.: +91 265 2794396; fax: +91 265 2792508. E-mail addresses: geetha2578@yahoo.co.in (R. Geetha), archana.gayatri@yahoo.co.in (G. Archana). Contents lists available at ScienceDirect Applied Soil Ecology journal homepage: www.elsevier.com/locate/apsoil 0929-1393/$ – see front matter ß 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2009.07.005