Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens

Scheeren-Groot, E. P.; Rodenburg, Kees W.; Dulk‐Ras, Amke den; Turk, Stefan C. H. J.; Hooykaas, Paul J. J.

doi:10.1128/jb.176.21.6418-6426.1994

Cited by 33 publications

(31 citation statements)

References 52 publications

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“…Identity of or similarity between mutants recovered from the same tumor suggested that these mutants were of clonal origin and had benefited from a selective advantage over wild-type cells in the plant (15,17,19). The positions of the point mutations, each mutation occurring in a different clone, are indicated by the arrows.…”

Section: Resultsmentioning

confidence: 99%

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Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

et al. 1995

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Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant.The virulence (vir) and transferred (T) regions of Agrobacterium tumefaciens Ti plasmids contain genes directly involved in plant transformation. Expression of the vir genes by the bacterium is induced through the action of the virA-virG twocomponent regulatory system in response to plant phenolic compounds such as acetosyringone. The T-DNA, produced from the T region at the direction of the vir genes, becomes integrated into plant chromosomal DNA, where it determines the synthesis of plant growth hormones responsible for crown gall tumor development. The T-DNA also confers on tumor cells the capacity to produce unusual metabolites called opines. One of these opines, nopaline, is synthesized through the reductive condensation of arginine and ␣-ketoglutaric acid. Other Ti plasmid-encoded genes, which are not transferred to plant cells, enable the bacterial pathogen to utilize opines for growth and tumor colonization (for reviews, see references 5, 6, and 20). In spite of the enhanced colonization potential associated with expression of the opine catabolic genes, pathogenic Agrobacterium strains are difficult to recover from certain types of tumors in which nonpathogenic, opine-utilizing agrobacteria often prevail (4, 14). To account for the frequent occurrence of nonpathogenic agrobacteria, it is proposed here that some plant infections with A. tumefaciens result in the modification of the bacterial genome. Conceivably, nonpathogenic mutants produced by A. tumefaciens in association with the host plant could be altered either in the vir or T region of the Ti plasmid or in the chromosome. Chromosomal genes that are required for efficient plant transformation include chvE, which encodes a protein that potentiates the effect of plant phenolic compounds on vir gene induction (11).Our recent observatio...

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Section: Resultsmentioning

confidence: 99%

“…1B). A previous study (19) on chemical mutagenesis of virG showed that point mutations inactivating protein function were distributed randomly over the coding sequence.…”

Section: Discussionmentioning

confidence: 99%

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

et al. 1995

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“…According to the structure predicted by SWISS-MODEL (Guex & Peitsch, 1997;Schwede et al, 2003), a replacement of Asn877 by Ser increases its contact with the site of phosphorylation, Asp875, through hydrogen bonding. Different substitutions at the corresponding positions in VirG (N54D) (Han et al, 1992;Jin et al, 1993;Pazour et al, 1992;Scheeren-Groot et al, 1994), CheB (E58K) (Stewart, 1993) and CheY (N59K, N49R) (Silversmith et al, 2001) also have an activating effect, thereby outlining the importance of this non-conserved residue in the activation of response regulator domains.…”

Section: Discussionmentioning

confidence: 99%

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

et al. 2005

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Mutations in rcsC that result in constitutive colanic acid capsule synthesis were obtained in Salmonella enterica serovar Typhimurium. Most rcsC alleles were dominant; however, recessive rcsC alleles were also found, in agreement with the postulated double role (positive and negative) of RcsC on the activation of the RcsB/C phosphorelay system. Salmonella rcsC mutants with constitutive activation of the Rcs system are severely attenuated for virulence in BALB/c mice and their degree of attenuation correlates with the level of Rcs activation. Partial relief of attenuation by a gmm mutation indicates that capsule overproduction is one of the factors leading to avirulence in constitutively activated rcsC mutants. INTRODUCTIONThe Rcs phosphorelay system was initially characterized in Escherichia coli as a two-component positive regulator of colanic acid capsule synthesis encoded by rcsC and rcsB (Brill et al., 1988;Gottesman et al., 1985;Stout & Gottesman, 1990). The sensor protein RcsC, a hybrid histidine kinase, and the transcriptional activator RcsB are the main components of the system, which also includes a second transcriptional activator, RcsA (Stout et al., 1991) and an intermediate phosphotransmitter, YojN (Takeda et al., 2001). The Rcs system is also a negative regulator of the flagellar master operon flhDC (Francez-Charlot et al., 2003), whose products are necessary for the expression of genes involved in flagellum synthesis, motility and chemotaxis.The first environmental signal shown to strongly and rapidly induce colanic acid synthesis was osmotic upshift (Sledjeski & Gottesman, 1996). This effect was mediated by the Rcs system. More recently it has been shown that the Rcs phosphorelay can be activated when wild-type cells are grown at 20 uC in the presence of glucose and a high concentration of Zn 2+ (Hagiwara et al., 2003). Furthermore, several genetic conditions that result in alterations in envelope composition or integrity are known to activate the Rcs system. For example, a mutation in the mdoH gene involved in the biosynthesis of membrane-derived oligosaccharides (Ebel et al., 1997) Here, we describe the isolation of Salmonella rcsC mutants with constitutive activation of the Rcs system, and the characterization of amino acid substitutions that lead to different degrees of activation of the system. Virulence trials with constitutively activated rcsC mutants provide a direct demonstration of the link between activation of the Rcs system and avirulence in mice. We also show that virulence attenuation associated with activation of the RcsB/C system is partially due to colanic acid overproduction. METHODSBacterial strains, bacteriophages and strain construction.S. enterica serovar Typhimurium strains used in this study are described in Table 1. Unless otherwise indicated, the strains derive from the mouse-virulent strain 14028. Transductional crosses using phage P22 HT 105/1 int201 (Schmieger, 1972) were used for strain construction operations. The transduction protocol was described by Maloy (199...

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“…A constitutive VirG mutant, VirG(N54D) (191,192,246,374,439), binds to vir box DNA approximately 10-fold more tightly than does wild-type VirG (192). Since the mutation introduces a new negative charge adjacent to the site of phosphorylation, it is thought that this mutation mimics the phosphorylated form of the protein.…”

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confidence: 99%

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

Brencic

Winans

2005

Microbiol Mol Biol Rev

339

208

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Diverse interactions between hosts and microbes are initiated by the detection of host-released chemical signals. Detection of these signals leads to altered patterns of gene expression that culminate in specific and adaptive changes in bacterial physiology that are required for these associations. This concept was first demonstrated for the members of the family Rhizobiaceae and was later found to apply to many other plant-associated bacteria as well as to microbes that colonize human and animal hosts. The family Rhizobiaceae includes various genera of rhizobia as well as species of Agrobacterium. Rhizobia are symbionts of legumes, which fix nitrogen within root nodules, while Agrobacterium tumefaciens is a pathogen that causes crown gall tumors on a wide variety of plants. The plant-released signals that are recognized by these bacteria are low-molecular-weight, diffusible molecules and are detected by the bacteria through specific receptor proteins. Similar phenomena are observed with other plant pathogens, including Pseudomonas syringae, Ralstonia solanacearum, and Erwinia spp., although here the signals and signal receptors are not as well defined. In some cases, nutritional conditions such as iron limitation or the lack of nitrogen sources seem to provide a significant cue. While much has been learned about the process of host detection over the past 20 years, our knowledge is far from being complete. The complex nature of the plant-microbe interactions makes it extremely challenging to gain a comprehensive picture of host detection in natural environments, and thus many signals and signal recognition systems remain to be described

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Mutational analysis of the transcriptional activator VirG of Agrobacterium tumefaciens

Cited by 33 publications

References 52 publications

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

Detection of and Response to Signals Involved in Host-Microbe Interactions by Plant-Associated Bacteria

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