The chvH Locus of Agrobacterium Encodes a Homologue of an Elongation Factor Involved in Protein Synthesis

Abstract

The virulence of Agrobacterium tumefaciens depends on both chromosome-and Ti plasmid-encoded gene products. In this study, we characterize a chromosomal locus, chvH, previously identified by TnphoA mutagenesis and shown to be required for tumor formation. Through DNA sequencing and comparison of the sequence with identified sequences in the database, we show that this locus encodes a protein similar in sequence to elongation factor P, a protein thought to be involved in peptide bond synthesis in Escherichia coli. The analysis of vir-lacZ and vir-phoA translational fusions as well as Western immunoblotting revealed that the expression of Vir proteins such as VirE2 was significantly reduced in the chvH mutant compared with the wild-type strain. The E. coli efp gene complemented detergent sensitivity, virulence, and expression of VirE2 in the chvH mutant, suggesting that chvH and efp are functionally homologous. As expected, ChvH exerts its activity at the posttranscriptional level. Southern analysis suggests that the gene encoding this elongation factor is present as a single copy in A. tumefaciens. We constructed a chvH deletion mutant in which a 445-bp fragment within its coding sequence was deleted and replaced with an omega fragment. On complex medium, this mutant grew more slowly than the wild-type strain, indicating that elongation factor P is important but not essential for the growth of Agrobacterium.Agrobacterium tumefaciens causes crown gall disease in a wide range of dicotyledonous plants. The disease, characterized by neoplastic transformation at the site of infection, results from the transfer and expression of oncogenes from the bacterium to susceptible plant cells (for a review, see reference 27). This transfer process is governed primarily by the products of the vir genes located on the Ti plasmid. These genes are tightly regulated and are expressed to a significant level only in the presence of plant signal molecules synthesized by wounded plant cells. The vir genes are transcriptionally regulated by the Ti plasmid-encoded VirA/VirG two-component regulatory system (26, 52). The VirA protein senses the plant signal molecules and then transduces the signal by phosphate transfer to the response regulator, the VirG protein. The activated VirG protein is a positive transcriptional activator of itself and all other Ti plasmid-encoded vir operons.Numerous chromosomal virulence genes (chv) have also been shown to play important roles in the ability of Agrobacterium to transform plants (for a review, see reference 39). In general, the functions of chromosomal virulence genes have not been well elucidated, and mutations in these genes are pleiotropic. Consequently, their precise roles in tumor formation have been difficult to assess. An analysis of a limited number of chv mutants suggests that whereas vir genes on the Ti plasmid are dedicated solely to specific steps in the interaction of Agrobacterium with host plants, the chromosomal virulence genes play important roles in the general physiology of Agroba...

Loss of Elongation Factor P Disrupts Bacterial Outer Membrane Integrity

“…These findings were corroborated by a recent study which reported changes of equally small magnitudes in the soluble proteome of an E. coli efp mutant (60). This finding is also consistent with data obtained for the efp (chvH) mutant of Agrobacterium tumefaciens, where few differences were observed in the protein profiles of the mutant and wild-type strains using one-dimensional SDS-PAGE, except for a single, unidentified 32-kDa protein that was expressed at much higher levels in the mutant (47).…”

“…It is possible that a difference in strain background or growth condition, such as high temperature or low-osmolarity medium, led to the earlier conclusion that this factor is essential. In addition to Salmonella, EF-P has been successfully knocked out in E. coli, Agrobacterium, and Acinetobacter (5,17,47,59). In the case of the Acinetobacter efp mutant, suppressor mutations conveying enhanced growth were obtained after a few generations (17).…”

“…However, a systematic high-throughput attempt to disrupt every nonessential gene in E. coli found that at least one strain of E. coli could tolerate a disruption of efp and remain viable (5,59). Furthermore, while a transposon insertion in the Agrobacterium homolog of efp, chvH, did not affect bacterial viability, the chvH mutant displayed reduced virulence and expression of the critical virulence factor VirE2, as well as increased sensitivity to detergents (47). Disruption of the efp gene in Acinetobacter also does not affect bacterial viability, although fitness is adversely affected (17).…”

Molecular evidence of genetic modification of Sinorhizobium meliloti: enhanced PCB bioremediation

“…The plasmid pE43 carried the Pseudomonas aeruginosa strain 142 ohb genes cloned into the broad-host-range vector pSP329, carrying a multiple cloning site and lacZ a-complementation fragment cloned into the HaeII site of the plasmid pTJS75, a derivative of R-factor RK2 [5].…”

Elongation Factor P and Modifying Enzyme PoxA Are Necessary for Virulence of Shigella flexneri

“…Properly modified EF-P is necessary for the virulence of several bacterial species, including Salmonella enterica and Agrobacterium tumefaciens (29,30). Here we investigate the role of EF-P and its modification in the virulence of S. flexneri.…”