In this work we describe the isolation and use of panC and panB mutants to analyze the involvement of these plasmid-encoded genes in pantothenate biosynthesis. A survey of the localization of panCB genes among members of the Rhizobiales with multipartite genomes allowed us to infer a panCB phylogeny and
to establish the probable chromosomal origin of these plasmid-borne genes. We also report that the panCB genes could not totally restore the growth in minimal medium (MM) of a strain cured of TEW-7197 plasmid p42f, suggesting that other functions essential for growth in MM are encoded in this plasmid. Results Functional characterization of plasmid p42f encoded panCB genes The predicted function of the product see more of panC (RHE_PF00001) annotated as PBAL, is the catalysis of the last step of pantothenate synthesis. This PBAL (298 amino acids) showed 43% identity and 62% similarity over 279 amino acids with the functionally characterized PBAL of E. coli K12 (284 amino acids). A search for conserved domains (CD-search) at NCBI-CDD revealed the presence of a typical pantoate-binding site. The panB gene (RHE_PF00002) is located immediately downstream of panC. The four nucleotide overlap between the panC TGA codon and panB ATG codon suggest that these genes might be transcribed as an operon. The panB gene encodes
a putative MOHMT, the first enzyme of the pantothenate pathway. A BlastP comparison between the functionally characterized MOHMT of E. coli K12 (264 amino acids) and the putative MOHMT encoded on plasmid p42f of R. etli CFN42 (273 amino acids) showed
37% identity and 56% similarity over a length of 240 amino acids. A CD-search indicated that in the putative MOHMT of R. etli CFN42 the magnesium binding and active site domains are conserved. Additionally, Paralog Search (KEGG SSDB) and pathway tools predicted a second probable MOHMT, encoded on plasmid p42e (locus tag RHE_PE00443). Both proteins are similar in length (273 and 270 aa for the products encoded by panB and RHE_PE00443, respectively). However, a BlastP comparison of these mTOR inhibitor sequences showed only 36% identity and 56% similarity over a tract of 140 amino acids. A CD-search revealed that only 5 of 12 of the invariable residues present in the active site domain are conserved in RHE_PE00443. The metal binding domain could not be detected by the CD-search. To determine whether the panC and panB genes located on plasmid p42f are required for pantothenate synthesis, OICR-9429 nmr mutations in these genes were generated by site-directed vector integration mutagenesis via a single cross-over recombination (see details in Material and Methods and Table 1). Mutants ReTV1 (panC -) and ReTV2 (panB – ) were unable to grow in minimal medium (MM) lacking calcium pantothenate (Figure 1a). Supplementation of MM with 1 μM calcium pantothenate allowed the panC and panB mutants to recover their wild-type growth rate (Figure 1b).