Transposon sequencing analysis of Bradyrhizobium diazoefficiens 110spc4

Claudine Baraquet,Caroline S Harwood,Weijun Dai,Kieran Pechter,Jose Mendiola

doi:10.1038/s41598-021-92534-z

Abstract

Bradyrhizobium diazoefficiens USDA110 is one of the most effective nitrogen-fixing symbionts of soybeans. Here we carried out a large-scale transposon insertion sequencing (Tn-seq) analysis of strain Bd110spc4, which is derived from USDA110, with the goal of increasing available resources for identifying genes crucial for the survival of this plant symbiont under diverse conditions. We prepared two transposon (Tn) insertion libraries of Bd110spc4 with 155,042 unique Tn insertions when the libraries were combined, which is an average of one insertion every 58.7 bp of the reference USDA110 genome. Application of bioinformatic filtering steps to remove genes too small to be expected to have Tn insertions, resulted in a list of genes that were classified as putatively essential. Comparison of this gene set with genes putatively essential for the growth of the closely related alpha-proteobacterium, Rhodopseudomonas palustris, revealed a small set of five genes that may be collectively essential for closely related members of the family Bradyrhizobiaceae. This group includes bacteria with diverse lifestyles ranging from plant symbionts to animal-associated species to free-living species.

Highlights

Bradyrhizobium diazoefficiens, formerly named Bradyrhizobium japonicum[1], is an alpha-proteobacterium that infects soybeans (Glycine max) to form nitrogen-fixing root nodules that provide useable nitrogen to the host plant
We found immediate utility for the list of Bd110spc[4] putative essential genes when we used multiple BLAST comparisons to identify a small set of five genes that we hypothesize to be collectively or conditionally essential for a subset of related species in the family Bradyrhizobiaceae with diverse lifestyles but not for other alpha-proteobacteria
Individual conjugative mating mixtures were plated on yeast extract-arabinosegluconate (AG) agar supplemented with kanamycin (Km) to select for Tn insertions and chloramphenicol to

Summary

Introduction

Bradyrhizobium diazoefficiens, formerly named Bradyrhizobium japonicum[1], is an alpha-proteobacterium that infects soybeans (Glycine max) to form nitrogen-fixing root nodules that provide useable nitrogen to the host plant. To provide a community resource that we or others can use to identify genes important for fitness, we generated a transposon insertion library of Bd110spc[4], mapped the sites of Tn insertions, and applied bioinformatic analysis to estimate a set of putative essential genes required for aerobic growth on yeast extract- arabinose-gluconate (AG) medium. Bd110spc[4] is a spontaneous spectinomycin resistant mutant of B. diazoefficiens USDA11013 This baseline data set can be used in conjunction with the Bd110spc[4] transposon insertion library that we generated to identify genes that are needed for Bd110spc[4] growth or survival in natural conditions or in a specific condition in the laboratory. We found immediate utility for the list of Bd110spc[4] putative essential genes when we used multiple BLAST comparisons to identify a small set of five genes that we hypothesize to be collectively or conditionally essential for a subset of related species in the family Bradyrhizobiaceae with diverse lifestyles but not for other alpha-proteobacteria

Methods

Results

Conclusion