Glycoside Hydrolase Genes Are Required for Virulence of Agrobacterium tumefaciens on Bryophyllum daigremontiana and Tomato.

Agrobacterium tumefaciens is a rhizosphere bacterium that can infect wound sites on plants. The bacterium transfers a segment of DNA (T-DNA) from the Ti plasmid to the plant host cell via a type IV secretion system where the DNA becomes integrated into the host cell chromosomes. The expression of T-DNA in the plant results in tumor formation. Although the binding of the bacteria to plant surfaces has been studied previously, there is little work on possible interactions of the bacteria with the plant cell wall. Seven of the 48 genes encoding putative glycoside hydrolases (Atu2295, Atu2371, Atu3104, Atu3129, Atu4560, Atu4561, and Atu4665) in the genome of A. tumefaciens C58 were found to play a role in virulence on tomato and Bryophyllum daigremontiana Two of these genes (pglA and pglB; Atu3129 and Atu4560) encode enzymes capable of digesting polygalacturonic acid and, thus, may play a role in the digestion of pectin. One gene (arfA; Atu3104) encodes an arabinosylfuranosidase, which could remove arabinose from the ends of polysaccharide chains. Two genes (bglA and bglB; Atu2295 and Atu4561) encode proteins with ß-glycosidase activity and could digest a variety of plant cell wall oligosaccharides and polysaccharides. One gene (xynA; Atu2371) encodes a putative xylanase, which may play a role in the digestion of xylan. Another gene (melA; Atu4665) encodes a protein with α-galactosidase activity and may be involved in the breakdown of arabinogalactans. Limited digestion of the plant cell wall by A. tumefaciens may be involved in tumor formation on tomato and B. daigremontianaIMPORTANCEA. tumefaciens is used in the construction of genetically engineered plants, as it is able to transfer DNA to plant hosts. Knowledge of the mechanisms of DNA transfer and the genes required will aid in the understanding of this process. Manipulation of glycoside hydrolases may increase transformation and widen the host range of the bacterium. A. tumefaciens also causes disease (crown gall tumors) on a variety of plants, including stone fruit trees, grapes, and grafted ornamentals such as roses. It is possible that compounds that inhibit glycoside hydrolases could be used to control crown gall disease caused by A. tumefaciens.

Exopolysaccharides of Agrobacterium tumefaciens.

Agrobacterium exopolysaccharides play a major role in the life of the cell. Exopolysaccharides are required for bacterial growth as a biofilm and they protect the bacteria against environmental stresses. Five of the exopolysaccharides made by A. tumefaciens have been characterized extensively with respect to their structure, synthesis, regulation, and role in the life of the bacteria. These are cyclic-ß-(1, 2)-glucan, cellulose, curdlan, succinoglycan, and the unipolar polysaccharide (UPP). This chapter describes the structure, synthesis, regulation, and function of these five exopolysaccharides.

Adherence of Bacteria to Plant Surfaces Measured in the Laboratory.

This manuscript describes a method to measure bacterial binding to axenic plant surfaces in the light microscope and through the use of viable cell counts. Plant materials used include roots, sprouts, leaves, and cut fruits. The methods described are inexpensive, easy, and suitable for small sample sizes. Binding is measured in the laboratory and a variety of incubation media and conditions can be used. The effect of inhibitors can be determined. Situations that promote and inhibit binding can also be assessed. In some cases it is possible to distinguish whether various conditions alter binding primarily due to their effects on the plant or on the bacteria.

A comparison of the retention of pathogenic Escherichia coli†O157 by sprouts, leaves and fruits.

The retention (binding to or association with the plant) of Escherichia coli by cut leaves and fruits after vigorous water washing was compared with that by sprouts. Retention by fruits and leaves was similar but differed from retention by sprouts in rate, effect of wounding and requirement for poly-ß,1-6-N-acetyl-D-glucosamine. Escherichia coli was retained by cut ends of lettuce leaves within 5 min while more than 1 h was required for retention by the intact epidermis of leaves and fruits, and more than 1 day for sprouts. Retention after 5 min at the cut leaf edge was specific for E. coli and was not shown by the plant-associated bacteria Agrobacterium tumefaciens and Sinorhizobium meliloti. Escherichia coli was retained by lettuce, spinach, alfalfa, bean, tomato, Arabidopsis thaliana, cucumber, and pepper leaves and fruits faster than by sprouts. Wounding of leaves and fruits but not sprouts increased bacterial retention. Mutations in the exopolysaccharide synthesis genes yhjN and wcaD reduced the numbers of bacteria retained. PgaC mutants were retained by cut leaves and fruits but not by sprouts. There was no significant difference in the retention of an O157 and a K12 strain by fruits or leaves. However, retention by sprouts of O157 strains was significantly greater than K12 strains. These findings suggest that there are differences in the mechanisms of E coli retention among sprouts, and leaves and fruits.

Attachment of Agrobacterium to plant surfaces.

Agrobacterium tumefaciens binds to the surfaces of inanimate objects, plants, and fungi. These bacteria are excellent colonizers of root surfaces. In addition, they also bind to soil particles and to the surface of artificial or man-made substances, such as polyesters and plastics. The mechanisms of attachment to these different surfaces have not been completely elucidated. At least two types of binding have been described unipolarpolysaccharide-dependent polar attachment and unipolar polysaccharide-independent attachment (both polar and lateral). The genes encoding the enzymes for the production of the former are located on the circular chromosome, while the genes involved in the latter have not been identified. The expression of both of these types of attachment is regulated in response to environmental signals. However, the signals to which they respond differ so that the two types of attachment are not necessarily expressed coordinately.

Sulfide oxidation, nitrate respiration, carbon acquisition, and electron transport pathways suggested by the draft genome of a single orange Guaymas Basin Beggiatoa (Cand. Maribeggiatoa) sp. filament.

A near-complete draft genome has been obtained for a single vacuolated orange Beggiatoa (Cand. Maribeggiatoa) filament from a Guaymas Basin seafloor microbial mat, the third relatively complete sequence for the Beggiatoaceae. Possible pathways for sulfide oxidation; nitrate respiration; inorganic carbon fixation by both Type II RuBisCO and the reductive tricarboxylic acid cycle; acetate and possibly formate uptake; and energy-generating electron transport via both oxidative phosphorylation and the Rnf complex are discussed here. A role in nitrite reduction is suggested for an abundant orange cytochrome produced by the Guaymas strain; this has a possible homolog in Beggiatoa (Cand. Isobeggiatoa) sp. PS, isolated from marine harbor sediment, but not Beggiatoa alba B18LD, isolated from a freshwater rice field ditch. Inferred phylogenies for the Calvin-Benson-Bassham (CBB) cycle and the reductive (rTCA) and oxidative (TCA) tricarboxylic acid cycles suggest that genes encoding succinate dehydrogenase and enzymes for carboxylation and/or decarboxylation steps (including RuBisCO) may have been introduced to (or exported from) one or more of the three genomes by horizontal transfer, sometimes by different routes. Sequences from the two marine strains are generally more similar to each other than to sequences from the freshwater strain, except in the case of RuBisCO: only the Guaymas strain encodes a Type II enzyme, which (where studied) discriminates less against oxygen than do Type I RuBisCOs. Genes subject to horizontal transfer may represent key steps for adaptation to factors such as oxygen and carbon dioxide concentration, organic carbon availability, and environmental variability.

Why orange Guaymas Basin Beggiatoa spp. are orange: single-filament-genome-enabled identification of an abundant octaheme cytochrome with hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities.

Orange, white, and yellow vacuolated Beggiatoaceae filaments are visually dominant members of microbial mats found near sea floor hydrothermal vents and cold seeps, with orange filaments typically concentrated toward the mat centers. No marine vacuolate Beggiatoaceae are yet in pure culture, but evidence to date suggests they are nitrate-reducing, sulfide-oxidizing bacteria. The nearly complete genome sequence of a single orange Beggiatoa ("Candidatus Maribeggiatoa") filament from a microbial mat sample collected in 2008 at a hydrothermal site in Guaymas Basin (Gulf of California, Mexico) was recently obtained. From this sequence, the gene encoding an abundant soluble orange-pigmented protein in Guaymas Basin mat samples (collected in 2009) was identified by microcapillary reverse-phase high-performance liquid chromatography (HPLC) nano-electrospray tandem mass spectrometry (µLC-MS-MS) of a pigmented band excised from a denaturing polyacrylamide gel. The predicted protein sequence is related to a large group of octaheme cytochromes whose few characterized representatives are hydroxylamine or hydrazine oxidases. The protein was partially purified and shown by in vitro assays to have hydroxylamine oxidase, hydrazine oxidase, and nitrite reductase activities. From what is known of Beggiatoaceae physiology, nitrite reduction is the most likely in vivo role of the octaheme protein, but future experiments are required to confirm this tentative conclusion. Thus, while present-day genomic and proteomic techniques have allowed precise identification of an abundant mat protein, and its potential activities could be assayed, proof of its physiological role remains elusive in the absence of a pure culture that can be genetically manipulated.

attG and attC mutations of Agrobacterium tumefaciens are dominant negative mutations that block attachment and virulence.

The cryptic plasmid (pAT) of Agrobacterium tumefaciens was not required for virulence or attachment to plant surfaces. However, mutations in the attC and attG genes located on pAT caused the bacteria to become avirulent and non-attaching on tomato, carrot, and Bryophyllum daigremontiana. This was the case whether the mutation was in the copy of the genes located on pAT or whether it was carried in a second copy of the attA-G operon located on a plasmid in cells that contained a wild-type copy of pAT. Thus attC and attG mutations are dominant negative mutations. The mechanism by which these mutations block attachment and virulence is unknown.

Polysaccharides cellulose, poly-beta-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.

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-beta-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.

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.

Differential binding of Escherichia coli O157:H7 to alfalfa, human epithelial cells, and plastic is mediated by a variety of surface structures.

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.

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.

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.

A functional cellulose synthase from ascidian epidermis.

Among animals, urochordates (e.g., ascidians) are unique in their ability to biosynthesize cellulose. In ascidians cellulose is synthesized in the epidermis and incorporated into a protective coat know as the tunic. A putative cellulose synthase-like gene was first identified in the genome sequences of the ascidian Ciona intestinalis. We describe here a cellulose synthase gene from the ascidian Ciona savignyi that is expressed in the epidermis. The predicted C. savignyi cellulose synthase amino acid sequence showed conserved features found in all cellulose synthases, including plants, but was most similar to cellulose synthases from bacteria, fungi, and Dictyostelium discoidium. However, unlike other known cellulose synthases, the predicted C. savignyi polypeptide has a degenerate cellulase-like region near the carboxyl-terminal end. An expression construct carrying the C. savignyi cDNA was found to restore cellulose biosynthesis to a cellulose synthase (CelA) minus mutant of Agrobacterium tumefaciens, showing that the predicted protein has cellulose synthase activity. The lack of cellulose biosynthesis in all other groups of metazoans and the similarity of the C. savignyi cellulose synthase to enzymes from cellulose-producing organisms support the hypothesis that the urochordates acquired the cellulose biosynthetic pathway by horizontal transfer.

Agrobacterium-mediated root transformation is inhibited by mutation of an Arabidopsis cellulose synthase-like gene.

Agrobacterium-mediated plant genetic transformation involves a complex interaction between the bacterium and the host plant. Relatively little is known about the role plant genes and proteins play in this process. We previously identified an Arabidopsis mutant, rat4, that is resistant to Agrobacterium transformation. We show here that rat4 contains a T-DNA insertion into the 3'-untranslated region of the cellulose synthase-like gene CSLA9. CSLA9 transcripts are greatly reduced in the rat4 mutant. Genetic complementation of rat4 with wild-type genomic copies of the CSLA9 gene restores both transformation competence and the wild-type level of CSLA9 transcripts. The CSLA9 promoter shows a distinct pattern of expression in Arabidopsis plants. In particular, the promoter is active in the elongation zone of roots, the root tissue that we previously showed is most susceptible to Agrobacterium-mediated transformation. Disruption of the CSLA9 gene in the rat4 mutant results in reduced numbers and rate of growth of lateral roots and reduced ability of the roots to bind A. tumefaciens cells under certain conditions. No major differences in the linkage structure of the non-cellulosic polysaccharides could be traced to the defective CSLA9 gene.

Identification of Arabidopsis rat mutants.

Limited knowledge currently exists regarding the roles of plant genes and proteins in the Agrobacterium tumefaciens-mediated transformation process. To understand the host contribution to transformation, we carried out root-based transformation assays to identify Arabidopsis mutants that are resistant to Agrobacterium transformation (rat mutants). To date, we have identified 126 rat mutants by screening libraries of T-DNA insertion mutants and by using various "reverse genetic" approaches. These mutants disrupt expression of genes of numerous categories, including chromatin structural and remodeling genes, and genes encoding proteins implicated in nuclear targeting, cell wall structure and metabolism, cytoskeleton structure and function, and signal transduction. Here, we present an update on the identification and characterization of these rat mutants.

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.

Attachment to roots and virulence of a chvB mutant of Agrobacterium tumefaciens are temperature sensitive.

Agrobacterium tumefaciens chvB mutants are unable to produce beta-1,2 glucan. They are nonattaching and avirulent and show reduced motility at room temperature. At lower temperatures (16 degrees C), chvB mutants became virulent on Bryophyllum daigremontiana and Lycopersicon esculentum and were able to attach to L. esculentum, Arabidopsis thaliana, Daucus carota, and Tagetes erecta roots. The mutant bacteria also recovered wild-type motility at lower temperatures. Two other nonattaching mutants of A. tumefaciens, AttR and AtrA, were unaffected by the lowered temperature, remaining nonattaching and avirulent.