The ubiquitous Cation Diffusion Facilitator proteins (CDF) play a key role in maintaining the cellular homeostasis of essential metal ions. Previous n

Pantothenate is an indispensable vitamin precursor of the synthesis of coenzyme A (CoA), a key metabolite required in over 100 metabolic reactions. β‐Alanine (β‐ala) is an indispensable component of pantothenate. Due to the metabolic relevance of this pathway, we assumed that orthologous genes for ß‐alanine synthesis would be present in the genomes of bacteria, archaea, and eukaryotes. However, comparative genomic studies revealed that orthologous gene replacement and loss of synteny occur at high frequency in panD genes. We have previously reported the atypical plasmid‐encoded location of the pantothenate pathway genes panC and panB (two copies) in R. etli CFN42. This study also revealed the unexpected absence of a panD gene encoding the aspartate decarboxylase enzyme (ADC), required for the synthesis of β‐ala. The aim of this study was to identify the source of β‐alanine in Rhizobium etli CFN42. In this study, we present a bioinformatic analysis and an experimental validation demonstrating that the source of β‐ala in this R. etli comes from β‐alanine synthase, the last enzyme of the uracil degradation pathway.

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 ofR. etliCFN42 predicts the presence ofpanCandpanBgenes clustered together on the 642 kb plasmid p42f and a second copy ofpanBon 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 theRhizobialeswith multipartite genomes, and assess the possibility that thepanCBgenes may have reached plasmids by horizontal gene transfer.Results: Analysis of mutants confirmed that thepanCandpanBgenes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location ofpanCBgenes among members of theRhizobialesshowed that onlyR. etliandR. leguminosarumstrains carrypanCBgenes in plasmids. ThepanCBphylogeny attested a common origin for chromosomal and plasmid-bornepanCBsequences, suggesting that theR. etliandR. leguminosarum panCBgenes are orthologs rather than xenologs. ThepanCBgenes 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 corepanCBgenes located in plasmids ofR. etliandR. leguminosarumare indispensable for the synthesis of pantothenate. The unusual presence ofpanCBgenes in plasmids ofRhizobialesmay 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.

Phylogenetic and functional analysis of a novel Mn²⁺/Fe²⁺-CDF transporter.

Copper (Cu) is an essential micronutrient for all aerobic forms of life. Its oxidation states (Cu ⁺ /Cu ²⁺ ) make this metal an important cofactor of enzymes catalyzing redox reactions in essential biological processes. In gram‐negative bacteria, Cu uptake is an unexplored component of a finely regulated trafficking network, mediated by protein–protein interactions that deliver Cu to target proteins and efflux surplus metal to avoid toxicity. Rhizobium etli CFN 42 is a facultative symbiotic diazotroph that must ensure its appropriate Cu supply for living either free in the soil or as an intracellular symbiont of leguminous plants. In crop fields, rhizobia have to contend with copper‐based fungicides. A detailed deletion analysis of the pRet42e (505 kb) plasmid from an R. etli mutant with enhanced CuCl ₂ tolerance led us to the identification of the ropAe gene, predicted to encode an outer membrane protein ( OMP ) with a β–barrel channel structure that may be involved in Cu transport. In support of this hypothesis, the functional characterization of ropAe revealed that: (I) gene disruption increased copper tolerance of the mutant, and its complementation with the wild‐type gene restored its wild‐type copper sensitivity; ( II ) the ropAe gene maintains a low basal transcription level in copper overload, but is upregulated when copper is scarce; ( III ) disruption of ropAe in an actP ( copA ) mutant background, defective in copper efflux, partially reduced its copper sensitivity phenotype. Finally, BLASTP comparisons and a maximum likelihood phylogenetic analysis highlight the diversification of four RopA paralogs in members of the Rhizobiaceae family. Orthologs of RopAe are highly conserved in the Rhizobiales order, poorly conserved in other alpha proteobacteria and phylogenetically unrelated to characterized porins involved in Cu or Mn uptake.

The ubiquitous cytoplasmic membrane copper transporting P _1B‐1 and P _1B‐3 ‐type ATPases pump out Cu ⁺ and Cu ²⁺ , respectively, to prevent cytoplasmic accumulation and avoid toxicity. The presence of five copies of Cu‐ATPases in the symbiotic nitrogen‐fixing bacteria Sinorhizobium meliloti is remarkable; it is the largest number of Cu ⁺ ‐transporters in a bacterial genome reported to date. Since the prevalence of multiple Cu‐ATPases in members of the Rhizobiales order is unknown, we performed an in silico analysis to understand the occurrence, diversity and evolution of Cu ⁺ ‐ATPases in members of the Rhizobiales order. Multiple copies of Cu‐ATPase coding genes (2–8) were detected in 45 of the 53 analyzed genomes. The diversity inferred from a maximum‐likelihood (ML) phylogenetic analysis classified Cu‐ATPases into four monophyletic groups. Each group contained additional subtypes, based on the presence of conserved motifs. This novel phylogeny redefines the current classification, where they are divided into two subtypes (P _1B‐1 and P _1B‐3 ). Horizontal gene transfer (HGT) as well as the evolutionary dynamic of plasmid‐borne genes may have played an important role in the functional diversification of Cu‐ATPases. Homologous cytoplasmic and periplasmic Cu ⁺ ‐chaperones, CopZ, and CusF, that integrate a CopZ‐CopA‐CusF tripartite efflux system in gamma‐proteobacteria and archeae, were found in 19 of the 53 surveyed genomes of the Rhizobiales . This result strongly suggests a high divergence of CopZ and CusF homologs, or the existence of unexplored proteins involved in cellular copper transport.