The Agrobacterium tumefaciens vir gene transcriptional activator virG is transcriptionally induced by acid pH and other stress stimuli

Abstract

A set of Agrobacterium tumefaciens operons required for pathogenesis is coordinately induced during plant infection by the VirA and VirG proteins. The intracellular concentration of VirG increases in response to acidic media, and this response was proposed to be regulated at the level of transcription at a promoter (P2) that resembles the Escherichia coli heat shock promoters. To test this hypothesis, we first constructed a virG-lacZ transcriptional fusion. A strain containing this fusion had higher levels of I-galactosidase activity in acidic media than in media at neutral pH. Second, primer extension analysis of virG indicated that acidic media stimulated the transcription of this promoter. To determine whether P2 is a member of a heat shock-like regulon in A. tumefaciens, five agents that induce E. coli heat shock genes were tested for their abilities to induce a P2-lacZ fusion in A. tumefaciens. P2 was most strongly induced by low pH, was moderately stimulated by CdCI2 or mitomycin C, and was slightly induced by alkaline pH or ethanol. Heat shock or growth at high temperature did not cause detectable induction of P2 as measured by I-galactosidase activity and primer extension analysis. Induction by these treatments did not require any Ti plasmid-encoded function or the chromosomally encoded RecA protein. We also pulse-labeled cellular proteins after a shift to low pH and detected several proteins whose synthesis was induced by these conditions. We conclude that P2 is primarily induced by acid pH and secondarily by certain other stimuli, each of which is stressful to cell growth. This stress induction is at least partly independent of the heat shock and SOS responses.Establishment of crown gall tumors on plant hosts by Agrobacterium tumefaciens requires the recognition of a plant wound susceptible to infection, transfer of a discrete segment of DNA (T-DNA) to the plant cell nucleus, and integration of T-DNA into the plant genome (for reviews, see references 2, 12, 23, 43, and 66). The genes required for T-DNA transfer (vir genes) are located on a large plasmid, called the Ti plasmid. The vir genes are transcriptionally induced in response to a family of phenolic compounds produced by wounded plant cells (50, 51). Induction also requires acidic growth medium (52) and is potentiated by certain monosaccharides (3,47).Two members of the vir regulon, virA and virG, are required for vir gene induction (44,53,63) and encode proteins that are members of the family of bacterial twocomponent regulatory systems (28, 37, 42, 62). For some vir promoters, VirA and VirG are the only known regulatory proteins (34, 64), while the virC and virD promoters are also controlled by a repressor encoded by the chromosomal ros gene (5, 7). VirA is a transmembrane protein kinase that can phosphorylate itself and VirG (19,21,22,36). VirG specifically binds cis-acting regulatory sequences involved in transcriptional activation of the virulence genes (22,40,41). Recent evidence indicates that vir expression is rate limited by the pool siz...

Agrobacterium-Mediated Plant Transformation: Biology and Applications

“…In addition, the affinity of the ChvE protein for sugar acids is increased at low pH, suggesting a link between the sugar and acidity response in A. tumefaciens (Hu et al, 2012). A. tumefaciens utilizes the ChvG/ChvI two-component system to activate the transcription of virG and induces expression of several other bacterial genes (Mantis and Winans, 1992;Chang and Winans, 1996), including genes involved in chemotaxis and motility, several chromosome-encoded virulence genes, succinoglycan biosynthesis genes, and the gene cluster encoding the type VI secretion system (T6SS) (Yuan et al, 2007a;Wu et al, 2008;Wu et al, 2012). Recent studies further identified ExoR as a periplasmic negative regulator acting upstream of the ChvG sensor kinase to repress acid-inducible gene expression (Wu et al, 2012;Heckel et al, 2014) and uncovered a role for the T6SS in interbacterial competition activity during the plant colonization process (Ma et al, 2014).…”

Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene

“…Induction of virG by extracellular acidity occurs at a promoter designated P2 and does not require any Ti plasmidencoded protein (23). The virG P1 promoter, in contrast, is activated by phospho-VirG (16,37,43,45) and is also induced by phosphate starvation, probably via a homolog of the E. coli PhoB protein (4,43).…”

“…Because the induction of virG by acidity and by phosphate starvation does not require the VirG protein, these stimuli have been viewed as necessary to establish a pool of VirG sufficiently large to induce the vir regulon. This pool of VirG, upon phosphorylation, can then more strongly express virG in a positively autoregulated fashion (23).…”

Reconstitution of Acetosyringone-Mediated Agrobacterium tumefaciens Virulence Gene Expression in the Heterologous Host Escherichia coli

“…The expression of virulence genes in A. tumefaciens is known to be affected by changes in temperature (23) and pH (32), with 28°C and pH 5.5 being optimal. The thermosensitive nature of virulence expression in A. tumefaciens is due to a reversible inactivation of VirA protein (23).…”

“…Once phosphorylated, VirG activates transcription from promoters containing a specific 12-bp sequence called the vir box, which is present in the promoters of all vir genes (25,40). This expression is augmented by the presence of certain monosaccharides (5,43) and an acidic pH (32), which is characteristic of plant wound sites. A periplasmic sugar-binding protein, ChvE, which is highly homologous to glucose-binding protein of Escherichia coli, interacts with the periplasmic portion of the VirA molecule in the presence of certain monosaccharides, including glucose and arabinose (2,15).…”