Comparative Analysis of Three Bradyrhizobium diazoefficiens Genomes Show Specific Mutations Acquired during Selection for a Higher Motility Phenotype and Adaption to Laboratory Conditions

Abstract

Microbial genomes are being extensively studied using next-generation sequencing technologies in order to understand the changes that occur under different selection regimes. In this work, the number and type of mutations that have occurred in three Bradyrhizobium diazoefficiens USDA 110^T strains under laboratory conditions and during selection for a more motile phenotypic variant were analyzed. Most of the mutations found in both processes consisted of single nucleotide polymorphisms, single nucleotide deletions or insertions. In the case of adaptation to laboratory conditions, half of the changes occurred within intergenic regions, and around 80% were insertions. When the more motile phenotypic variant was evaluated, eight single nucleotide polymorphisms and an 11-bp deletion were found, although none of them was directly related to known motility or chemotaxis genes. Two mutants were constructed to evaluate the 11-bp deletion affecting the alpha subunit of 2-oxoacid:acceptor oxidoreductase (AAV28_RS30705-blr6743). The results showed that this single deletion was not responsible for the enhanced motility phenotype. IMPORTANCE The genetic and genomic changes that occur under laboratory conditions in Bradyrhizobium diazoefficiens genomes remain poorly studied. Only a few genome sequences of this important nitrogen-fixing species are available, and there are no genome-wide comparative analyses of related strains. In the present work, we sequenced and compared the genomes of strains derived from a parent strain, B. diazoefficiens USDA 110, that has undergone processes of repeated culture in the laboratory environment, or phenotypic selection toward antibiotic resistance and enhanced motility. Our results represent the first analysis in B. diazoefficiens that provides insights into the specific mutations that are acquired during these processes.

Keywords: comparative genomics; laboratory adaptation; single nucleotide polymorphism; swimming motility.

FIG 1

Maximum-likelihood phylogenetic tree generated using the SNPs of the core genome. Snippy-core was used to generate the alignment of the core genome SNPs between the sequences of the reference strains USDA 110, K USDA 110 (Kaneko et al.) (10), and DR USDA 110 (Davis-Richardson et al.) (11) and the draft genome sequences of LP USDA 110, LP 3004, and LP 3008. The shading corresponds to the conservation level. The letters in red indicate the mutation on the rpsL gene of LP 3004 and LP 3008 which provides streptomycin resistance. The numbers at the nodes indicate bootstrap support.

FIG 2

Comparison of the draft genome sequences of LP USDA 110, LP 3004, and LP 3008. (A) Venn diagram of the DNA polymorphisms found comparing the reference strain DR USDA 110 and each of the strains sequenced, using Snippy software. The overlapping areas show the number polymorphisms shared between the three sequenced strains of this study. (B) Genome comparison between LP USDA 110, LP 3004, and LP 3008 and the reference strain DR USDA 110. The innermost to outermost circles indicate the position of the single nucleotide insertions (SNIs, squares), single nucleotide polymorphisms (SNPs, circles), and single nucleotide deletions (SNDs, triangles). Multiple nucleotide deletions are represented by yellow triangles. The genome assemblies were mapped against the reference genome (black line). The locus tag (old annotation) of the genes presenting a polymorphism is shown. The colors indicate the strains as follows: blue, LP USDA 110; red, LP 3004; and green, LP 3008. The orange circles represent the SNPs on the rpsL gene.

FIG 3

Phenotypic characterization of B. diazoefficiens blr6743 mutants in different genomic backgrounds. Swimming plates in 0.3% (wt/vol) agar containing Götz medium incubated at 28°C were performed for LP 3008 and LP 3008 carrying blr6743 wt (A) and LP 3004 and LP 3004 Δ6743-6744 (B). (C) Swimming performance of LP 3008, LP3008 blr6743 wt, LP 3004, and LP 3004 Δ6743-6744. Cells of each of these strains were inoculated individually onto a plate, and the diameter of the halo was measured at the indicated times. Four technical replicates of each strain were performed. (D) SDS-PAGE of extracellular B. diazoefficiens proteins of LP 3004 (lane 1), LP 3004 Δ6743-6744 (lane 2), LP 3008 (lane 3), LP 3008 blr6743 wt (lane 4), and a positive control (+) with both flagellin proteins of B. diazoefficiens. Each experiment was repeated twice.