SOIL MICROBIOLOGY 1-Aminocyclopropane-1-Carboxylate (ACC) Deaminase Genes in Rhizobia from Southern Saskatchewan Jin Duan & Kirsten M. Müller & Trevor C. Charles & Susanne Vesely & Bernard R. Glick Received: 17 January 2008 / Accepted: 17 May 2008 / Published online: 12 June 2008 # Springer Science + Business Media, LLC 2008 Abstract A collection of 233 rhizobia strains from 30 different sites across Saskatchewan, Canada was assayed for 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, with 27 of the strains displaying activity. When all 27 strains were characterized based on 16S rRNA gene sequences, it was noted that 26 strains are close to Rhizobium leguminosarum and one strain is close to Rhizobium gallicum. Polymerase chain reaction (PCR) was used to rapidly isolate ACC deaminase structural genes from the above-mentioned 27 strains; 17 of them have 99% identities with the previously characterized ACC deaminase structural gene (acdS) from R. leguminosarum bv. viciae 128C53K, whereas the other ten strains are 84% identical (864~866/1,020 bp) compared to the acdS from strain 128C53K. Southern hybridization showed that each strain has only one ACC deaminase gene. Using inverse PCR, the region upstream of the ACC deaminase structural genes was characterized for all 27 strains, and 17 of these strains were shown to encode a leucine-responsive regulatory protein. The results are discussed in the context of a previously proposed model for the regulation of bacterial ACC deaminase in R. leguminosarum 128C53K. Introduction The microbial enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase (EC: 4.1.99.4) cleaves ACC, the imme- diate precursor of ethylene in plants. This enzyme was first isolated from Pseudomonas sp. strain ACP [22] and since then, it has been detected in a wide range of microbes including the fungus Penicillium citrinum [23], the yeast Hansenula saturnus [45], and a large number of bacteria including Rhizobium leguminosarum bv. viciae, Rhizobium hedysari, and Mesorhizobium loti [3, 4, 7, 10–12, 24, 26–28, 30, 35, 38, 58, 60, 62]. A model describing the role of ACC deaminase in plant-growth-promoting bacteria suggests that Rhizobacteria, attached to the surface of the plants seeds or roots, can take up some of the ACC that is exuded from the seeds or roots and cleave it thereby decreasing the level of ethylene in the plant and its inhibitory effect on root elongation in particular and plant growth in general [14, 15]. It has been known for some time that ethylene inhibits nodulation by rhizobia in various legumes [21]. For example, ethylene has been shown to inhibit nodule development in Medicago sativa [51], Pisum sativum [16, 18, 19, 33], and Trifolium repens [16]. A Medicago truncatula hypernodulat- ing mutant, sickle, has been demonstrated to be ethylene insensitive [49] and showed altered auxin transport regula- tion during nodulation [53]. However, the mechanisms by which ethylene controls nodulation is not yet fully known. Based on the model described for free-living bacteria, it was suggested that strains of rhizobia that have ACC deaminase activity may have the ability to lower ethylene levels in their host-specific legumes and overcome some of the negative effects of ethylene on nodulation [36, 37]. In 2003, the first report documenting the presence of ACC deaminase in Rhizobium spp. showed that five of 13 strains of rhizobia that were tested were observed to have active ACC deaminase [35]. The ACC deaminase genes from R. leguminosarum bv. viciae 128C53K and 99A1 have 64% identities to the gene from the well-known plant-growth- promoting bacterium, Pseudomonas putida UW4 [57]. In addition, all of these rhizobial strains had relatively low J. Duan (*) : K. M. Müller : T. C. Charles : S. Vesely : B. R. Glick Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON N2L 3G1, Canada e-mail: jduan@sciborg.uwaterloo.ca Microb Ecol (2009) 57:423–436 DOI 10.1007/s00248-008-9407-6