argC Orthologs from Rhizobiales show diverse profiles of transcriptional efficiency and functionality in Sinorhizobium meliloti

Abstract

Several factors can influence ortholog replacement between closely related species. We evaluated the transcriptional expression and metabolic performance of ortholog substitution complementing a Sinorhizobium meliloti argC mutant with argC from Rhizobiales (Agrobacterium tumefaciens, Rhizobium etli, and Mesorhizobium loti). The argC gene is necessary for the synthesis of arginine, an amino acid that is central to protein and cellular metabolism. Strains were obtained carrying plasmids with argC orthologs expressed under the speB and argC (S. meliloti) and lac (Escherichia coli) promoters. Complementation analysis was assessed by growth, transcriptional activity, enzymatic activity, mRNA levels, specific detection of ArgC proteomic protein, and translational efficiency. The argC orthologs performed differently in each complementation, reflecting the diverse factors influencing gene expression and the ability of the ortholog product to function in a foreign metabolic background. Optimal complementation was directly related to sequence similarity with S. meliloti, and was inversely related to species signature, with M. loti argC showing the poorest performance, followed by R. etli and A. tumefaciens. Different copy numbers of genes and amounts of mRNA and protein were produced, even with genes transcribed from the same promoter, indicating that coding sequences play a role in the transcription and translation processes. These results provide relevant information for further genomic analyses and suggest that orthologous gene substitutions between closely related species are not completely functionally equivalent.

FIG. 1.

Species signature of ArgC from Rhizobiales. A multiple alignment was carried out using CLUSTAL W. Species signatures were determined with a perl custom script. Yellow blocks indicate identical residues in at least two species. Colored residues for each organism denote species signature as follows: orange, R. etli; red, A. tumefaciens; blue, S. meliloti; gray, B. melitensis; and green, M. loti. Residues not in blocks are gaps in some organism or nonhomologous sequences.

FIG. 2.

Phylogenetic analysis of ArgC from Rhizobiales. The alignment was done using the MUSCLE program. Phylogeny was generated by the PhyML method. The tree was drawn with TreeView. Bootstrap analysis was performed with 100 replicates. Supporting values are shown in the main branches. The E. coli sequence was taken as the outgroup. The bar represents substitutions per residue.

FIG. 3.

Transcriptional analysis of argC promoter activity. Expression of the indicated argC promoter-gus (β-glucuronidase) gene fusions is depicted as follows: first group, in the genetic background of the indicated species; second group, in S. meliloti; and third group, in M. loti. Bars: black, S. meliloti; gray, A. tumefaciens; empty, R. etli; patterned, M. loti.

FIG. 4.

Growth curves of S. meliloti argC-complemented strains. The left panels depict growth in MM supplemented with 10 mM succinic acid and 10 mM ammonium chloride. The right panels depict growth in MM supplemented with 10 mM mannose, 10 mM potassium nitrate. (A and B) argC under the control of the S. meliloti argC promoter; (C and D) argC under the control of the E. coli lac promoter; (E and F) argC under the control of the S. meliloti speB promoter. Each panel represents the argC mutant strain complemented with argC gene from S. meliloti (⧫), A. tumefaciens (▪), R. etli (□), and M. loti (•); the S. meliloti wild-type strain (○); and the argC mutant strain (▴).