Biomedical subjects

Eugene W Nester

Publications and source records attributed to Eugene W Nester.

10 recordsLinked to original sources

PubMed2006

A catalytic carbohydrate contributes to bacterial antibiotic resistance.

Penicillins are widespread in nature and lethal to growing bacteria. Because of the severe threat posed by these antibiotics, bacteria have evolved a wide variety of strategies for combating them. Here, we describe one unusual strategy that involves the activity of a catalytic carbohydrate. We show that the cyclic oligosaccharide, beta-cyclodextrin (betaCD), can hydrolyze, and thereby inactivate, penicillin in vivo. Moreover, we demonstrate that this catalytic activity contributes to the antibiotic resistance of a bacterium that synthesizes this oligosaccharide in the laboratory. Taken together, these data not only expand our understanding of the biochemistry of penicillin resistance, but also provide the first demonstration of natural carbohydrate-mediated catalysis in a living system.

Ampicillin↗

PubMed2006

Indoleacetic acid, a product of transferred DNA, inhibits vir gene expression and growth of Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens induces crown gall tumors by transferring a piece of its tumor-inducing plasmid into plant cells. This transferred DNA encodes the synthesis of indole acetic acid (IAA) and cytokinin, and their overproduction results in tumor formation. The transfer is initiated by a two-component regulatory system, VirA/G recognizing plant signal molecules in the plant rhizosphere and activating a regulon on the tumor-inducing plasmid, which is required for the processing and transfer of DNA and protein. Although a great deal is known about vir gene activation, nothing is known about whether or how the vir gene regulon is inactivated after plant cell transformation. Presumably, just as a mechanism exists for activating the vir gene regulon only when a plant is in the immediate environment, a mechanism should exist for inactivating the same regulon once it has fulfilled its mission to transferred DNA into plant cells. We now show that IAA inactivates vir gene expression by competing with the inducing phenolic compound acetosyringone for interaction with VirA. IAA does not inhibit the vir genes in cells containing a constitutive sensor virA locus, which does not require any signal molecules to become phosphorylated. At higher concentrations, IAA inhibits the growth of Agrobacterium and many other plant-associated bacteria but not the growth of bacteria that occupy other ecological niches. These observations provide the missing link in the cycle of vir gene activation and inactivation.

Agrobacterium tumefaciens↗

PubMed2006

The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens.

The pathogen Agrobacterium tumefaciens infects a broad range of plants, introducing the T-DNA into their genome. Contrary to all known bacterial phytopathogens, A. tumefaciens lacks the hypersensitive response-inducing hrp genes, although it introduces numerous proteins into the plant cell through a type IV secretion system. To understand the timing and extent of the plant transcriptional response to this unusual pathogen, we used an Arabidopsis 26,000-gene oligonucleotide microarray. We inoculated Arabidopsis cell cultures with an oncogenic Agrobacterium strain and analyzed four biological replicates to identify two robust sets of regulated genes, one induced and the other suppressed. In both cases, the response was distinct at 48 h after infection, but not at 24 h or earlier. The induced set includes genes encoding known defense proteins, and the repressed set is enriched with genes characteristic of cell proliferation even though a growth arrest was not visible in the inoculated cultures. The analysis of the repressed genes revealed that the conserved upstream regulatory elements Frankiebox (also known as "site II") and Telobox are associated with the suppression of gene expression. The regulated gene sets should be useful in dissecting the signaling pathways in this plant-pathogen interaction.

Agrobacterium tumefaciens↗

PubMed2005

Citrate synthase mutants of Agrobacterium are attenuated in virulence and display reduced vir gene induction.

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brucella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.

Agrobacterium tumefaciens↗

PubMed2005

Phosphoenolpyruvate carboxykinase is an acid-induced, chromosomally encoded virulence factor in Agrobacterium tumefaciens.

The pckA gene, encoding phosphoenolpyruvate carboxykinase, catalyzes the reversible decarboxylation and phosphorylation of oxaloacetate to form phosphoenolpyruvate. Located on the circular chromosome of Agrobacterium, this locus is adjacent to the loci chvG and chvI, encoding a two-component regulatory system that has been shown to be important in virulence. Using a reporter gene fusion, studies showed that the pckA gene is induced by acidic pH but not by acetosyringone. This acid induction is regulated by the chvG-chvI regulatory system, which controls acid-inducible genes. A pckA mutant had no demonstrable PckA enzyme activity and grew on AB minimal medium with glucose but did not grow on the same medium with succinate as the sole carbon source and was more inhibited in its growth than the wild-type strain by an acidic environment. A pckA mutant was highly attenuated in tumor-inducing ability on tobacco leaf disks and was severely attenuated in vir gene expression. Although vir gene induction was completely restored when a constitutive virG gene was introduced into the mutant strain, virulence was only partially restored. These results suggest that avirulence may be due to a combination of the inhibition of this mutant in the acidic plant wound environment and the poor induction of the vir genes.

Agrobacterium tumefaciens↗

PubMed2004

DARTs: A DNA-based in vitro polypeptide display technology.

Display technologies link proteins with the genes that encode them, providing a means of selecting proteins with desired properties through the process of directed evolution. Here, we describe DNA/protein attachment and recovery tools (DARTs), a novel polypeptide display technology that utilizes the Agrobacterium tumefaciens protein VirD2 to generate DNA-protein hybrid molecules. The resulting DNA-protein hybrids are small, robust, and are not expected to be subject to the synthesis and selection biases associated with viral- and cell-based display systems. We demonstrated that these DNA-protein hybrids could be used to display a variety of peptides that bind to appropriate antibodies for immunodetection and immunopurification. Further, the DNA components of the hybrid molecules can hybridize to complementary DNA molecules in solution or on a solid substrate. Because full-length VirD2 self-associated, we constructed a truncation that did not self-associate but still exhibited DNA linking activity and efficiently displayed peptides. Finally, we purified DNA-protein hybrids using their displayed peptide epitopes and amplified their DNA components by polymerase chain reaction. We suggest that the DART polypeptide display system will be valuable for performing directed evolution and generating protein arrays.

Base Sequence↗

PubMed2003

Purine synthesis and increased Agrobacterium tumefaciens transformation of yeast and plants.

The bacterium Agrobacterium tumefaciens transforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeast Saccharomyces cerevisiae as a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity to A. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity to A. tumefaciens transformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility of A. tumefaciens in genetic engineering.

Agrobacterium tumefaciens↗

PubMed2002

A global pH sensor: Agrobacterium sensor protein ChvG regulates acid-inducible genes on its two chromosomes and Ti plasmid.

A sensor protein ChvG is part of a chromosomally encoded two-component regulatory system ChvG/ChvI that is important for the virulence of Agrobacterium tumefaciens. However, it is not clear what genes ChvG regulates or what signal(s) it senses. In this communication, we demonstrate that ChvG is involved in the regulation of acid-inducible genes, including aopB and katA, residing on the circular and linear chromosomes, respectively, and the tumor-inducing (Ti)-plasmid-harbored vir genes, virB and virE. ChvG was absolutely required for the expression of aopB and very important for the expression of virB and virE. However, it was responsible only for the responsiveness of katA and, to a limited extent, the vir genes to acidic pH. ChvG appears to play a role in katA expression by repressing katA at neutral pH. ChvG had no effect on the expression of two genes that were not acid-inducible. Because ChvG regulates unlinked acid-inducible genes encoding different functions in different ways, we hypothesize that ChvG is a global sensor protein that can directly or indirectly sense extracellular acidity. We also analyzed the re-sequenced chvG and found that ChvG is more homologous to its Sinorhizobium meliloti counterpart ExoS than was previously thought. Full-length ChvG is conserved in members of the alpha-proteobacteria, whereas only the C-terminal kinase domain is conserved in other bacteria. Sensing acidity appears to enable Agrobacterium to coordinate its coping with the environment of wounded plants to cause tumors.

Agrobacterium tumefaciens↗

PubMed2002

Agrobacterium type IV secretion is a two-step process in which export substrates associate with the virulence protein VirJ in the periplasm.

Type IV secretion systems are virulence determinants in many bacteria and share extensive homology with many conjugal transfer systems. Although type IV systems and their homologues have been studied widely, the mechanism by which substrates are secreted remains unclear. In Agrobacterium, we show that type IV secretion substrates that lack signal peptides form a soluble complex in the periplasm with the virulence protein VirJ. Additionally, these proteins co-precipitate with constituents of the type IV transporter: the VirB pilus and the VirD4 protein. Our findings suggest that the substrate proteins localized to the periplasm may associate with the pilus in a manner that is mediated by VirJ, and suggest a two-step process for type IV secretion in Agrobacterium. Our analyses of protein-protein interactions in a variety of mutant backgrounds indicate that substrates are probably secreted independently of one another.

Bacterial Proteins↗

PubMed2002

A new type IV secretion system promotes conjugal transfer in Agrobacterium tumefaciens.

Two DNA transfer systems encoded by the tumor-inducing (Ti) plasmid have been previously identified in Agrobacterium tumefaciens. The virB operon is required for the transfer of transferred DNA to the plant host, and the trb system encodes functions required for the conjugal transfer of the Ti plasmid between cells of Agrobacterium. Recent availability of the genome sequence of Agrobacterium allowed us to identify a third system that is most similar to the VirB type IV secretion system of Bartonella henselae. We have designated this system avhB for Agrobacterium virulence homologue virB. The avhB loci reside on pAtC58 and encode at least 10 proteins (AvhB2 through AvhB11), 7 of which display significant similarity to the corresponding virulence-associated VirB proteins of the Ti plasmid. However, the AvhB system is not required for tumor formation; rather, it mediates the conjugal transfer of the pAtC58 cryptic plasmid between cells of Agrobacterium. This transfer occurs in the absence of the Ti plasmid-encoded VirB and Trb systems. Like the VirB system, AvhB products promote the conjugal transfer of the IncQ plasmid RSF1010, suggesting that these products comprise a mating-pair formation system. The presence of plasmid TiC58 or plasmid RSF1010 reduces the conjugal transfer efficiency of pAtC58 10- or 1,000-fold, respectively. These data suggest that complex substrate interactions exist among the three DNA transfer systems of Agrobacterium.

Agrobacterium tumefaciens↗