Arabinose and protocatechuate catabolism genes are important for growth of Rhizobium leguminosarum biovar viciae in the pea rhizosphere

Paula Garcia-Fraile 0 1 3 Jonathan C. Seaman 0 1 3 Ramakrishnan Karunakaran 0 1 3 Anne Edwards 0 1 3 Philip S. Poole 0 1 3 J. Allan Downie 0 1 3 Responsible Editor: Katharina Pawlowski. 0 Present Address: P. Garcia-Fraile Institute of Microbiology, Academy of Sciences of Czech Republic , Videnska 1083, 142 20 Prague , Czech Republic 1 J. C. Seaman School of Biological Sciences, University of Reading , Reading RG6 6AJ , UK 2 ) Department of Molecular Microbiology, John Innes Centre , Norwich Research Park, Norwich NR4 7UH , UK 3 Present Address: P. S. Poole Department of Plant Sciences, University of Oxford , South Parks Road, Oxford OX1 3RB , UK Background and aims To form nitrogen-fixing nodules on pea roots, Rhizobium leguminosarum biovar viciae must be competitive in the rhizosphere. Our aim was to identify genes important for rhizosphere fitness. Methods Signature-tagged mutants were screened using microarrays to identify mutants reduced for growth in pea rhizospheres. Candidate mutants were assessed relative to controls for growth in minimal medium, growth in pea rhizospheres and for infection of peas in mixed inoculants. Mutated genes were identified by DNA sequencing and confirmed by transduction. - Rhizobia are soil bacteria studied primarily because of their ability to infect the roots of leguminous plants, producing nitrogen-fixing nodules. Prior to infecting legume roots, rhizobia grow in the rhizosphere (the region of soil in close proximity to roots) using nutrients secreted from the plant roots. Rhizobia in the rhizosphere are motile and can attach to roots and root hairs where they can grow to form a biofilm. These attached rhizobia are well positioned to detect flavonoids, isoflavonoids and related compounds that induce the bacterial genes required for legume nodulation (Downie 2010). Each nodule produced on legume roots is usually the result of a clonal infection event and typically a legume such as pea grown in the field produces around 150 nodules (Bourion et al. 2007). This corresponds to about 150 successful infections, but since there can be around 104106 pea-nodulating bacteria (Rhizobium leguminosarum biovar viciae) per g of soil (and at least a Kg of soil will be occupied by a mature pea root system), it is clear that the vast majority of R.l. viciae bacteria in the soil will not infect peas in any given growing season. Therefore rhizobia must be able to survive and grow well in the rhizosphere, without necessarily infecting legumes. Although a great deal is known about rhizobial genes required for nodule infection and nitrogen fixation (Downie 2010), less is known about genes required for growth and survival in the rhizosphere, because their identification is relatively difficult. Different approaches have been taken to identify genes required for successful growth in the rhizosphere. For example, promoter trapping approaches (referred to as in vivo expression technology, IVET) have been used to identify rhizosphereexpressed promoters in Pseudomonas fluorescens (Varivarn et al. 2013) and in R.l. viciae (Barr et al. 2008) and a recombination-based variation of IVET has been used to analyse gene expression in S. meliloti during rhizosphere and symbiotic growth of S. meliloti (Gao and Teplitski 2008). Microarray analyses of bacterial RNA has given insights into genes expressed following growth of Pseudomonas aeruginosa in a root exudate (Mark et al. 2005) and of R.l. viciae grown in root exudate and in different rhizospheres (Ramachandran et al. 2011). These studies led to the identification of genes induced in these environments and were followed up by the construction of targeted mutations, several of which decreased rhizosphere fitness. An alternative reverse genetics approach has been to use comparative genomics, in an attempt to identify genes prevalent in bacteria that grow in the rhizosphere (Redondo-Nieto et al. 2013; Silby et al. 2009). These reverse genetics approaches can identify many genes likely to be involved in rhizosphere growth or survival, but they require targeted mutagenesis to determine whether the genes are required for the bacteria to be competitive in the rhizosphere. Direct approaches to identification of mutations that decrease the growth of bacteria in specific environments include signature-tagged mutagenesis (Pobigaylo et al. 2008) and direct sequencing of transposon insertions in large populations of transposon-mutagenized bacteria (Barquist et al. 2013). In both these approaches poorly growing representatives can be identified based on the depletion of bacteria containing specific transposon insertions within a population of bacteria in the tested environment. The depleted bacteria can be detected based on decreased levels of specific transposon insertions identified from parallel DNA sequencing of populations or by using microarrays to identify depletion of tagged transposon insertions in defined groups of mutants. To complement our previous work on IVET selection of rhizosphere-expressed genes (Barr et al. 2008) and microarray analysis and mutagenesis of genes induced in the rhizosphere of peas (Ramachandran et al. 2011), here we have used signature tagged mutagenesis to identify genes which, when mutated, decrease the fitness of R.l. viciae in the pea rhizosphere. Materials and methods Bacterial strains and growth conditions R.l. viciae and Escherichia coli strains used in this work are listed in Table 1. E. coli strains were grown at 37 C in L medium (Sambrook et al. 1989) and R.l. viciae strains were grown at 28 C either in TY medium (Beringer 1974) or in AMS minimal medium containing 10 mM NH4Cl and carbon sources as indicated (Poole et al. 1994). Antibiotics were used at the following concentrations (g ml1): spectinomycin (Spc: 100); streptomycin (Str: 500) and neomycin (Nm: 160) unless specified otherwise. Growth was monitored at 28 C using an InfiniteF200 microtitre plate shaker/reader (Tecan, Reading, RG7 5AH, UK), measuring absorbance at 600 nm every 40 min. Transductions were done using phage RL38 as described previously (BuchananWollaston 1979). All plant tests were done with Pisum sativum cv. Avola peas in a growth chamber at 22 C with a 16 h-light 8 h-dark light cycle. Bacterial growth in pea rhizospheres was done with pea seedlings grown in 50 ml screw-capped Falcon centrifuge tubes (Fisher Scientific UK Ltd Loughborough LE11 5RG). Washed vermiculite was added to the 30 ml mark on each tube, 10 ml of nitrogen-free FP medium Table 1 Bacterial strains used in this study Strain number (mutant ID) Donor strain for conjugation of STM plasmids Supercompetent E.coli cells (Fahraeus 1957) was added and the tubes were capped and autoclaved. A single sterile germinating pea seed was added to each tube and after 1 week in a growth chamber, the tubes were inoculated with about 104 bacteria. One week later, bacteria were recovered from the rhizosphere by cutting off the plant shoots, adding 18 ml of ph (...truncated)