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

Abstract

Background and aimsTo 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.MethodsSignature-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.ResultsOf 5508 signature-tagged mutants, microarrays implicated 50 as having decreased rhizosphere fitness. Growth tests identified six mutants with rhizosphere-specific phenotypes. The mutation in one of the genes (araE) was in an arabinose catabolism operon and blocked growth on arabinose. The mutation in another gene (pcaM), encoding a predicted solute binding protein for protocatechuate and hydroxybenzoate uptake, decreased growth on protocatechuate. Both mutants were decreased for nodule infection competitiveness with mixed inoculants, but nodulated peas normally when inoculated alone. Other mutants with similar phenotypes had mutations predicted to affect secondary metabolism.ConclusionsCatabolism of arabinose and protocatechuate in the pea rhizosphere is important for competitiveness of R.l. viciae. Other genes predicted to be involved in secondary metabolism are also important.

Specificity traits consistent with legume‐rhizobia coevolution displayed by Ensifer meliloti rhizosphere colonization

“…That other genes found to be relevant to the colonization of pea roots by R. leguminosarum bv. viciae – such as araE (arabinose catabolism) and pcaM (protocatechuate transport; [Garcia‐Fraile et al ., ]) – did not prove to be as relevant for the colonization of alfalfa roots by E. meliloti is indeed of interest. In view of these data, future studies should be performed to investigate if the compositional profiles of root exudates from different legumes correlate with the catabolic capabilities of the associated symbiont rhizobia.…”

“…Using a phenomic approach, we could identify the collection of genetic markers involved in the rhizobial colonization of rhizospheres. By means of a similar strategy, several mutants of R. leguminosarum bv viciae with decreased rhizosphere fitness had been recently reported (Garcia-Fraile et al, 2015). The experimental protocol that was used in the present work combined the power of high-throughput nucleotide sequencing to quantify (i.e., count) the presence of specific mutants in the rhizosphere, with the possibility of simultaneously investigating the colonization phenotype of ca.…”

“…Based conceptually on a different experimental approach, the screening on an omic scale of mutants created by signature-tagged mutagenesis (STM) can also serve to identify clones that express specific phenotypes (Lehoux et al, 1999;Pobigaylo et al, 2006;van Opijnen and Camilli, 2013). The use of such tools adapted to the analysis of rhizosphere colonization was accordingly able to identify six R. leguminosarum biovar viceae mutants with rhizosphere-specific phenotypes and to demonstrate the relevance of arabinose and protocatechuate metabolism to the colonization of pea rhizospheres by those rhizobia (Garcia-Fraile et al, 2015). In the example of E. meliloti, the use of STM has thus far concentrated on the screening for genes relevant to symbiosis and competitiveness mediated by the use of signature amplification and microarray technologies (Pobigaylo et al, 2008).…”

What makes rhizobia rhizosphere colonizers?

“…The mutant library employed here, had been previously used to test for genes required for competitive nodulation (Pobigaylo et al, 2008). Although the size of the library was similar to that used by Garcia-Fraile et al (2015), the authors found here many more genes potentially implicated in rhizosphere colonization, possibly due to the use of NGS instead of microarray hybridization for calculating the abundance of specific mutants in the output samples. By testing samples taken at 3 and 7 days after inoculation, the authors identified a total of 175 different insertion mutants putatively affected in alfalfa rhizosphere colonization.…”

“…A signature tag mutagenesis (STM) approach was used by Garcia‐Fraile et al . () to identify Rhizobium leguminosarum mutants affected in pea rhizosphere colonization. 5508 mutants were screened in colonization experiments by hybridization of microarrays and 50 mutants impaired in colonization were initially isolated.…”