Downloaded from www.microbiologyresearch.org by IP: 54.224.135.207 On: Mon, 09 May 2016 00:01:42 A previously uncharacterized tetratricopeptide-repeat-containing protein is involved in cell envelope function in Rhizobium leguminosarum Kara D. Neudorf, 1 Elizabeth M. Vanderlinde, 2 Dinah D. Tambalo 1 and Christopher K. Yost 1 Correspondence Christopher K. Yost chris.yost@uregina.ca Received 15 July 2014 Accepted 30 October 2014 1 Department of Biology, University of Regina, Regina, Saskatchewan S4S 0A2, Canada 2 Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5A2, Canada Rhizobium leguminosarum is a soil bacterium that is an intracellular symbiont of leguminous plants through the formation of nitrogen-fixing root nodules. Due to the changing environments that rhizobia encounter, the cell is often faced with a variety of cell altering stressors that can compromise the cell envelope integrity. A previously uncharacterized operon (RL3499–RL3502) has been linked to proper cell envelope function, and mutants display pleiotropic phenotypes including an inability to grow on peptide-rich media. In order to identify functional partners to the operon, suppressor mutants capable of growth on complex, peptide-rich media were isolated. A suppressor mutant of a non-polar mutation to RL3500 was chosen for further characterization. Transposon mutagenesis, screening for loss of the suppressor phenotype, led to the identification of a Tn5 insertion in an uncharacterized tetratricopeptide-repeat-containing protein RL0936. Furthermore, RL0936 had a 3.5-fold increase in gene expression in the suppressor strain when compared with the WT and a 1.5-fold increase in the original RL3500 mutant. Mutation of RL0936 decreased desiccation tolerance and lowered the ability to form biofilms when compared with the WT strain. This work has identified a potential interaction between RL0936 and the RL3499–RL3502 operon that is involved in cell envelope development in R. leguminosarum, and has described phenotypic activities to a previously uncharacterized conserved hypothetical gene. INTRODUCTION Rhizobium leguminosarum is a Gram-negative bacterium that can form a symbiotic relationship with legumes such as peas and lentils. R. leguminosarum infects the plant root through an infection thread, enters root cells through endocytosis and differentiates into bacteroids culminating in the formation of a root nodule structure. Within the nodule the bacteroids supply fixed atmospheric nitrogen to the plant, while the plant supplies carbon to the bacteroids (Gibson et al., 2008). Free-living rhizobia can be found in the rhizosphere, where they experience a dynamic growth environment including fluctuations in water availability, temperature and nutrient limitation due to competition with other microbes. Since bacteria often must respond to changing environments, they have evolved a complex cell envelope that adapts under stressful conditions to maintain cell envelope integrity in order to sustain its primary function of permitting the select passage of nutrients and waste products (Silhavy et al., 2010; Storz & Hengge- Aronis, 2000). The genetic network involved in cell envelope development and stress adaptation is only partially described in rhizobia, despite its function for cell survival in the soil and during symbiosis. Using genetic approaches, we have initiated a programme to identify novel genes involved in cell envelope development. Our earlier work and that of others has identified a link between Rhizobium genes involved in cell envelope development and their requirement for growth on complex peptide rich media such as tryptone-yeast extract medium (TY). Specific examples include genes involved in the synthesis of the very long chain fatty acid attached to the lipid A of the outer membrane lipopoly- saccharide (Vanderlinde et al., 2009), a gene with homo- logy to the cell-envelope-associated carboxyl-terminal protease (ctpA) (Gilbert et al., 2007), a two-component Abbreviations: NPN, 1-N-phenylnaphthylamine; TPR, tetratricopeptide repeat; vWFA, von Willebrand factor type A. One supplementary figure is available with the online Supplementary Material. Microbiology (2015), 161, 148–157 DOI 10.1099/mic.0.082420-0 148 082420 G 2015 The Authors Printed in Great Britain