Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in Rhizobium etli and Rhizobium leguminosarum

Abstract

Background: A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer.

Results: Analysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.

Conclusions: This study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The unusual presence of panCB genes in plasmids of Rhizobiales may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.

Figure 1

Pantothenate auxotrophy of R. etli CFN42 panC and panB mutants. Growth of the R. etli CFN42 wild-type strain and its derivative panC (ReTV1) and panB (ReTV2) mutants in: (a) minimal medium, (b) minimal medium supplemented with 1 μM calcium pantothenate. Values represent the means of three independent experiments; error bars show standard deviations.

Figure 2

Comparison of phylogenetic trees constructed from core and panCB genes. Maximum-likelihood phylogenetic trees of 16 Rhizobiales constructed using the concatenated nucleic acid sequences of 10 housekeeping genes (a) or panC and panB concatenated genes (b). Bootstrap values are shown over each branch (based on 100 pseudo-replicates).

Figure 3

panCB genes do not fully restore the growth deficiency of CFNX186. Growth of R. etli CFN42 wild-type strain, its p42f-cured derivative CFNX186, CFNX186 complemented with the panCB genes (CFNX186-4) and CFNX186 complemented with a 20 kb EcoRI fragment of plasmid p42f containing the panC, panB, oxyR and katG genes (CFNX186-24) in: (a) minimal medium, (b) minimal medium supplemented with 1 μM pantothenate. Growth curves are the mean of at least three independent experiments; error bars represent standard deviations.