Ann G. Matthysse

Differential Binding of Escherichia coli O157:H7 to Alfalfa, Human Epithelial Cells, and Plastic Is Mediated by a Variety of Surface Structures

ABSTRACT Escherichia coli O157:H7 carried on plant surfaces, including alfalfa sprouts, has been implicated in food poisoning and outbreaks of disease in the United States. Adhesion to cell surfaces is a key component for bacterial establishment and colonization on many types of surfaces. Several E. coli O157:H7 surface proteins are thought to be important for adhesion and/or biofilm formation. Therefore, we examined whether mutations in several genes encoding potential adhesins and regulators of adherence have an effect on bacterial binding to plants and also examined the role of these genes during adhesion to Caco-2 cells and during biofilm formation on plastic in vitro. The genes tested included those encoding adhesins (cah, aidA1, and ompA) and mediators of hyperadherence (tdcA, yidE, waaI, and cadA) and those associated with fimbria formation (csgA, csgD, and lpfD2). The introduction of some of these genes (cah, aidA1, and csg loci) into an E. coli K-12 strain markedly increased its ability to bind to alfalfa sprouts and seed coats. The addition of more than one of these genes did not show an additive effect. In contrast, deletion of one or more of these genes in a strain of E. coli O157:H7 did not affect its ability to bind to alfalfa. Only the absence of the ompA gene had a significant effect on binding, and the plant-bacterium interaction was markedly reduced in a tdcA ompA double mutant. In contrast, the E. coli O157:H7 ompA and tdcA ompA mutant strains were only slightly affected in adhesion to Caco-2 cells and during biofilm formation. These findings suggest that some adhesins alone are sufficient to promote binding to alfalfa and that they may exist in E. coli O157:H7 as redundant systems, allowing it to compensate for the loss of one or more of these systems. Binding to the three types of surfaces appeared to be mediated by overlapping but distinct sets of genes. The only gene which appeared to be irreplaceable for binding to plant surfaces was ompA.

Characterization of the Binding of Diarrheagenic Strains of E. coli to Plant Surfaces and the Role of Curli in the Interaction of the Bacteria with Alfalfa Sprouts

Diarrheagenic Escherichia coli were able to bind to plant surfaces, including alfalfa sprouts and open seed coats, and tomato and Arabidopsis thaliana seedlings incubated in water. The characteristics of the binding differed with the bacterial strain examined. Laboratory K12 strains of E. coli failed to show significant binding to any of the plant surfaces examined, suggesting that some of the genes present and expressed in pathogenic strains and absent or unexpressed in K12 strains may be required for binding to plants. When a plasmid carrying the mlrA gene (a positive regulator of curli biosynthesis) or a plasmid carrying the operons that encode the synthesis of curli (csgA-G) was introduced into K12 strains, the bacteria acquired the ability to bind to sprouts. CsgA mutants of an avian pathogenic E. coli and an O157:H7 strain showed no reduction in their ability to bind to sprouts. Thus, the production of curli appears to be sufficient to allow K12 strains to bind, but curli are not necessary for the binding of pathogenic strains, suggesting that pathogenic strains may have more than one mechanism for binding to plant surfaces.

The Effect of Cellulose Overproduction on Binding and Biofilm Formation on Roots by Agrobacterium tumefaciens

Agrobacterium tumefaciens growing in liquid attaches to the surface of tomato and Arabidopsis thaliana roots, forming a biofilm. The bacteria also colonize roots grown in sterile quartz sand. Attachment, root colonization, and biofilm formation all were markedly reduced in celA and chvB mutants, deficient in production of cellulose and cyclic beta-(1,2)-D-glucans, respectively. We have identified two genes (celG and cell) in which mutations result in the overproduction of cellulose as judged by chemical fractionation and methylation analysis. Wild-type and chvB mutant strains carrying a cDNA clone of a cellulose synthase gene from the marine urochordate Ciona savignyi also overproduced cellulose. The overproduction in a wild-type strain resulted in increased biofilm formation on roots, as evaluated by light microscopy, and levels of root colonization intermediate between those of cellulose-minus mutants and the wild type. Overproduction of cellulose by a nonattaching chvB mutant restored biofilm formation and bacterial attachment in microscopic and viable cell count assays and partially restored root colonization. Although attachment to plant surfaces was restored, overproduction of cellulose did not restore virulence in the chvB mutant strain, suggesting that simple bacterial binding to plant surfaces is not sufficient for pathogenesis.

Polysaccharides Cellulose, Poly-β-1,6-N-Acetyl-d-Glucosamine, and Colanic Acid Are Required for Optimal Binding of Escherichia coli O157:H7 Strains to Alfalfa Sprouts and K-12 Strains to Plastic but Not for Binding to Epithelial Cells

ABSTRACT When Escherichia coli O157:H7 bacteria are added to alfalfa sprouts growing in water, the bacteria bind tightly to the sprouts. In contrast, laboratory K-12 strains of E. coli do not bind to sprouts under similar conditions. The roles of E. coli O157:H7 lipopolysaccharide (LPS), capsular polysaccharide, and exopolysaccharides in binding to sprouts were examined. An LPS mutant had no effect on the binding of the pathogenic strain. Cellulose synthase mutants showed a significant reduction in binding; colanic acid mutants were more severely reduced, and binding by poly-β-1,6-N-acetylglucosamine (PGA) mutants was barely detectable. The addition of a plasmid carrying a cellulose synthase gene to K-12 strains allowed them to bind to sprouts. A plasmid carrying the Bps biosynthesis genes had only a marginal effect on the binding of K-12 bacteria. However, the introduction of the same plasmid allowed Sinorhizobium meliloti and a nonbinding mutant of Agrobacterium tumefaciens to bind to tomato root segments. These results suggest that although multiple redundant protein adhesins are involved in the binding of E. coli O157:H7 to sprouts, the polysaccharides required for binding are not redundant and each polysaccharide may play a distinct role. PGA, colanic acid, and cellulose were also required for biofilm formation by a K-12 strain on plastic, but not for the binding of E. coli O157:H7 to mammalian cells.

A region of the Agrobacterium tumefaciens chromosome containing genes required for virulence and attachment to host cells.

A 29 kb region of the circular chromosome of Agrobacterium tumefaciens containing genes required for bacterial attachment to host cells and virulence has been sequenced. Transposon mutants in many of the genes have been obtained. The mutants can be divided into two groups: those which can be complemented by conditioned medium and those whose phenotype is unaffected by conditioned medium. The first group includes mutants in genes with homology to ABC transporters, one possible transcriptional regulator, and some closely linked genes immediately downstream. The second group includes mutants in two possible transcriptional regulators, one ATPase, and a number of biosynthetic genes including a transacetylase required for the formation of an acetylated capsular polysaccharide. There are also several genes with no homology to genes of identified function. The presence of such a large number of genes required for attachment to host cells suggests that the ability of A. tumefaciens to bind to plant cells may play an important role in the life of these bacteria.

The FNR‐type transcriptional regulator SinR controls maturation of Agrobacterium tumefaciens biofilms

Agrobacterium tumefaciens is a plant pathogen that persists as surface‐associated populations on plants or soil particles. A genetic screen for A. tumefaciens mutants deficient for surface interactions identified a mutant that forms thin, sparsely populated biofilms, but is proficient for initial attachment. The mutant is disrupted in a gene designated sinR, encoding a member of the DNR subfamily of FNR‐type transcription regulators. SinR is required for normal maturation of A. tumefaciens biofilms on both inert surfaces and plant tissues, and elevated sinR expression results in accelerated biofilm formation. Expression of sinR is increased close to 30‐fold in cultures grown in oxygen‐limited environments and is also induced within biofilms grown under oxic conditions. A consensus FNR box, the presumptive binding site for FNR‐type proteins, is located upstream of the sinR promoter. FnrN, a second A. tumefaciens FNR‐like regulator, is required for induction of sinR in oxygen‐limited cultures, whereas SinR negatively influences its own expression. FnrN influences biofilm formation, but its effects are less dramatic than those of SinR. We propose a model in which a signal cascade, responsive to oxygen limitation and initiated by FnrN, activates sinR expression in response to decreased oxygen levels, and influences the formation of A. tumefaciens biofilms.

Binding of Agrobacterium tumefaciens to carrot protoplasts

Abstract Agrobacterium tumefaciens attached both to intact carrot cells in vitro and to carrot protoplasts. After attachment to the protoplast membrane the bacteria elaborated cellulose fibrils, which anchored them to the surface of the plant cell. These fibrils entrapped other bacteria forming large bacterial clusters on the plant cell surface. The kinetics and bacterial strain specificity of attachment to protoplasts were similar to those observed with intact cells. Thus the plant cell membrane may contain a specific receptor for virulent Agrobacterium cells. It is likely that the transfer of tumor-inducing plasmid DNA from the bacterium to the plant cell would be facilitated by the tight binding of large numbers of bacteria to the plant cell membrane.

The Effect of the Agrobacterium tumefaciens attR Mutation on Attachment and Root Colonization Differs between Legumes and Other Dicots

ABSTRACT Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus,A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.

Plant cell range for attachment of Agrobacterium tumefaciens to tissue culture cells

Abstract Agrobacterium tumefaciens attached to tissue culture cells of the dicotyledons carrot, tobacco, Bryophyllum daigremontianum, Vinca rosea and soybean. With the exception of soybean all of these plants responded to infection with A. tumefaciens in vivo by the formation of tumors. Fewer bacteria attached to tissue culture cells of the monocotyledon oat and corn, than to dicotyledon cells. Inoculation of these monocotyledons with A. tumefaciens did not induce tumors. Carrot embryos in tissue culture also bound fewer A. tumefaciens than did callus cells and the bacteria did not induce tumors on germinating carrot embryonic axes. Thus, in general, the range of attachment of A. tumefaciens to tissue culture cells in vitro parallels the host range for tumor formation by A. tumefaciens in vivo . This suggests that the availability of a reasonable number of accessible bacterial binding sites on the plant cell surface may be one important factor in determining the host range of the bacterium.

Proteome analysis of plant-induced proteins of Agrobacterium tumefaciens

Abstract A proteome study of Agrobacterium tumefaciens exposed to plant roots demonstrated the existence of a plant-dependent stimulon. This stimulon was induced by exposure to cut roots and consists of at least 30 soluble proteins (pI 4-7), including several proteins whose involvement in agrobacteria-host interactions has not been previously reported. Exposure of the bacteria to tomato roots also resulted in modification of the proteins: Ribosomal Protein L19, GroEL, AttM, and ChvE, indicating the significance of protein modifications in the interactions of agrobacteria with plants.