JOURNAL OF BACTERIOLOGY, Jan. 2011, p. 460-472 0021-9193/11/$12.00 doi:10.1128/JB.01010-10 Copyright 2011, American Society for Microbiology. All Rights Reserved.
argC Orthologs from Rhizobiales Show Diverse Profiles of Transcriptional Efficiency and Functionality in Sinorhizobium melilotiᰔ† Rafael Díaz, Carmen Vargas-Lagunas, Miguel Angel Villalobos,‡ Humberto Peralta, Yolanda Mora, Sergio Encarnacio'n, Lourdes Girard, and Jaime Mora* Programa de Geno'mica Funcional de Procariotes, Centro de Ciencias Geno'micas, Universidad Nacional Auto'noma de Me'xico, Av. Universidad, Cuernavaca, Morelos C.P. 62210, Mexico Received 24 August 2010/Accepted 31 October 2010 Several factors can influence ortholog replacement between closely related species. We evaluated the tran-
scriptional 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.
Synteny, gene neighboring, or conservation of chromosomal ment. We hypothesized that the species signature represents gene order has been proposed to be a result of the interde- the proportion of a particular sequence that responds to ad- pendence between a gene product and its genomic context.
aptation (32); it is a useful evolutive measure because we found Conservation of a syntenic block could be favored by selective a high direct correlation with the nonsynonymous substitution pressure because the arrangement allows the sequential or rate (Peralta et al., unpublished). Although orthologous genes coordinated expression of genes that correctly integrate impor- of Rhizobiales encode proteins with high degrees of identity tant metabolic functions (41, 64, 69). By a comparative analysis among several species, the proteins differ in many respects, of four Rhizobiales species (namely, Sinorhizobium meliloti, including isoelectric point. Small differences in protein se- Agrobacterium tumefaciens, Mesorhizobium loti, and Brucella quence may be the result of evolutionary adaptation in re- melitensis), we found that syntenic genes exhibit striking dif- sponse to the particular intracellular environment of a given ferences compared to nonsyntenic genes, including increased species. In this scenario, specific amino acid changes would be operon and network organization, high sequence conservation, selected for optimal performance in a particular genetic back- diminished evolutionary rates, essential functional role, and ground. Knight et al. (40) carried out a virtual analysis of specific phylogenetic associations (32; H. Peralta, G. Guerrero, proteomes of nearly 100 organisms and showed that there was A. Aguilar, and J. Mora, unpublished data).
a correlation between theoretical proteomes and ecological Orthologs encode similar functions sharing a common an- niches; conversely, there was no correlation between phylog- cestor and exhibiting various degrees of conservation, due inlarge part to functional adaptation and cellular role in different eny and differences observed in the theoretical proteomes. Our species (45). We have proposed a new parameter termed "spe- previous work has shown that mutation of a single gene, aniA cies signature" to extract the amount of amino acid residues (a carbon flux regulator), produced a proteomic alteration of specific for a given species, based on multiple sequence align- approximately 800 proteins (16, 22), indicating that the ab-sence or modification of a single gene can result in complexchanges in global gene expression. In this context, syntenic * Corresponding author. Mailing address: Programa de Geno'mica orthologs are ideal to evaluate the functional importance of Funcional de Procariotes, Centro de Ciencias Geno'micas-UNAM, species signature in related organisms.
Apdo. postal 565-A, Cuernavaca, Morelos C.P. 62220, Me'xico. Phone: The Rhizobiales order is a versatile group of bacteria that 52 777 3114661. Fax: 52 777 3175094. E-mail:
† Supplemental material for this article may be found at http://jb present very interesting features, such as a huge amount of genes, abundance of genes acquired by horizontal transfer ‡ Present address: Centro de Investigacio'n en Biotecnología events, a symbiotic or pathogenic association with higher or- Aplicada-IPN, Ex Hacienda San Juan Molino, Tlaxcala C.P. 90700, ganisms such as plants or animals, and the ability for nodula- ᰔ Published ahead of print on 12 November 2010.
tion and nitrogen fixation in some species (9, 13, 29, 31, 50, 54, EXPRESSION OF argC ORTHOLOGS FROM RHIZOBIALES TABLE 1. Bacterial strains and plasmids used in this study Sinorhizobium meliloti argC mutant S. meliloti 1021 derivative, argC::⍀Sp supE44lacU169(␾80lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 mB ) recA13 ara-14 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 ⌬placZ pBBR1MCS5 derivative, carrying the promoterless gus gene; Gmr Plasmid containing the ⍀-Sp interposon; Spr pBBR1MCS3 derivative with S. meliloti speB-argC genes pBBR1MCS3 derivative with A. tumefaciens speB-argC genes pBBR1MCS3 derivative with R. etli speB-argC genes pBBR1MCS3 derivative with M. loti speB-argC genes pBBMCS53 derivative plac::gus transcriptional fusion pBBMCS53 derivative pargCSm::gus transcriptional fusion pBBMCS53 derivative pspeBSm::gus transcriptional fusion pBBMCS53 derivative plac-argCSm::gus transcriptional fusion pBBMCS53 derivative plac-argCAt::gus transcriptional fusion pBBMCS53 derivative plac-argCRe::gus transcriptional fusion pBBMCS53 derivative plac-argCMl::gus transcriptional fusion pBBMCS53 derivative pargC-argCSm::gus transcriptional fusion pBBMCS53 derivative pargC-argCAt::gus transcriptional fusion pBBMCS53 derivative pargC-argCRe::gus transcriptional fusion pBBMCS53 derivative pargC-argCMl::gus transcriptional fusion pBBMCS53 derivative pspeB-argCSm::gus transcriptional fusion pBBMCS53 derivative pspeB-argCAt::gus transcriptional fusion pBBMCS53 derivative pspeB-argCRe::gus transcriptional fusion pBBMCS53 derivative pspeB-argCMl::gus transcriptional fusion pBBMCS53 derivative carrying the S. meliloti argC gene under its promoter pBBMCS53 derivative carrying the A. tumefaciens argC gene under the control of the S. meliloti argC promoter.
pBBMCS53 derivative carrying the R. etli argC gene under the control of the pBBMCS53 derivative carrying the M. loti argC gene under the control of the pBBMCS53 derivative pargCAt::gus transcriptional fusion pBBMCS53 derivative pargCRe::gus transcriptional fusion pBBMCS53 derivative pargCMl::gus transcriptional fusion a Tcr, tetracycline resistance; Apr, ampicillin resistance; Smr, streptomycin; Spr, spectinomycin resistance; Kmr, kanamycin resistance; Gmr, gentamicin resistance.
65). In addition to a circular chromosome, some rhizobia carry given species, both intracellular (interactions with other enzymes secondary chromosomes or plasmids of high molecular size.
and metabolites) and extracellular. The effect of heterologous Arginine is an essential amino acid in bacteria, and its synthesis complementation of an S. meliloti argC mutant with the corre- requires a great deal of energy and reducing power (14, 49, 63).
sponding ortholog from other Rhizobiales species (A. tumefaciens, Arginine is the most common nitrogen storage compound and R. etli, and M. loti) was compared in terms of growth, excretion of precursor of polyamine synthesis (48, 61). argC genes are com- organic compounds, transcriptional and enzymatic activity, and monly organized in operons, exemplified by the Escherichia molecular parameters such as mRNA and translation efficiency.
coli argCBH operon (56). In R. etli, the argC mutation affects An analysis of the putative speB-argC operon was also carried out growth capacity using ammonium as the sole nitrogen source to define a novel functional argC promoter.
and results in failure to nodulate the common bean root (23).
We were interested in exploring whether the species signature MATERIALS AND METHODS
(particular amino acid sequence variations) in orthologous syn- Bacterial strains and growth conditions. Bacterial strains and plasmids used in
tenic genes reflects adaptations to the particular conditions of a the present study are listed in Table 1. The S. meliloti argC mutant was obtained


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