3-Ketoglucose reductase of Agrobacterium tumefaciens

Two kinds of 3-ketoglucose-reducing enzyme were partially purified from the sonic extract of Agrobacterium tumefaciens IAM 1525 grown on a sucrose-containing medium. Both enzymes have a specific requirement for reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a hydrogen donor and catalyze the reduction of 3-ketoglucose to glucose but do not reduce 3-ketoglucosides such as 3-ketosucrose, 3-ketoglucose-1-phosphate, 3-ketotrehalose, and 3-ketocellobiose. From the requirement and substrate specificity of the enzymes, the name NADPH:3-ketoglucose oxidoreductase (trivial name, 3-ketoglucose reductase) was proposed. By diethylaminoethyl-cellulose column chromatography, two reductases were separated, and the early and late eluted enzymes were designated reductase I and II, respectively. K(m) values of reductase I and II were as follows: for 3-ketoglucose both had an identical value of 2.5 x 10(-5)m, and for NADPH the values were 1.0 x 10(-5)m and 1.5 x 10(-5)m, respectively. Optimal pH values were also identical: pH 4.8 to 5.0 in 10(-2)m phosphate buffer. Intracellular localization of the enzymes is discussed.     

Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.     

Transposon-Induced Catalase-Deficient Agrobacterium tumefaciens

Agrobacterium tumefaciens MKR, a nonpathogenic strain, has three catalase isozymes and one superoxide dismutase but no detectable peroxidase activity. A large number (8400) of transconjugants were obtained with pSUP1011::Tn5 suicide vector. The transposition frequencies were found to be greater in biparental mating than in triparental mating with helper plasmid. Mutants MLA31, MLA32, MLA41, and MLA41(a), generated by transposon mutagenesis, all lacked one of the catalase isozymes. Mutants were more susceptible to cell death than the wild type upon direct exposure to 10.0 mmol L⁻¹ H₂O₂. The specific activity of the enzyme catalase was found to be higher in nitrogen-rich growth medium than carbon-rich growth medium. Received: 28 January 1997 / Accepted: 25 February 1997     

Conditions for Production of 3-Ketomaltose from Agrobacterium tumefaciens

Up to 39% yields of 3-ketomaltose were achieved in 18 to 22 hr when Agrobacterium tumefaciens NRRL B-36 was cultured at 25 to 28 C in a simple medium containing 4.0 to 8.0% maltose, 0.09% urea, 0.5% CaCO(3), 0.6% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O. For maximum production of 3-ketomaltose the culture had to be maintained approximately at pH 7.0.     

Transfection in Agrobacterium tumefaciens

Intact cells of Agrobacterium tumefaciens were examined for ability to take up biologically active LR-4 phage deoxyribonucleic acid (DNA) from the surrounding medium. DNA incorporation as measured by subsequent plaque formation (transfection) failed to occur when the bacteria were grown in defined minimal salts media, and was restricted to a 4-hr period in the early log phase of growth in enriched media. In the latter case, maximal transfection frequencies were obtained after a 25- to 30-min incubation with 22.5 mug of phage DNA/ml. Higher DNA concentrations or longer incubation times were inhibitory. Transfection was completely inhibited by deoxyribonuclease but not by ribonuclease, trypsin, or phage-specific antisera.     

Murine Toxicity of Agrobacterium tumefaciens

Eleven strains of the crown gall organism, Agrobacterium tumefaciens, tested by intraperitoneal injection into mice, were lethal within 48 hr. Five other species had some lethal strains. The lethal effect of A. tumefaciens appeared to be the result of a toxic rather than an infectious process, since histopathological anomalies were not found in mice injected with live cultures and since heat-killed cultures were lethal. The murine toxin disappeared when A. tumefaciens was grown at 36 C and reappeared when the organism was subsequently incubated below 30 C. The murine toxin itself was not inactivated by exposure to 100 C for 30 min. The toxin was associated with the cells and was not excreted into the medium. Centrifugal fractionation revealed that the toxin was associated with the smaller cells in 3-day stationary-phase cultures. These data suggested a possible relationship between toxin production and the production of the agents responsible for the initiation of plant tumors.     

The T-pilus of Agrobacterium tumefaciens

T-pilus biogenesis uses a conserved transmembrane nucleoprotein- and protein-transport apparatus for the transport of cyclic T-pilin subunits to the Agrobacterium cell surface. T-pilin subunits are processed from full-length VirB2 pro-pilin into a cyclized peptide, a rapid reaction that is Agrobacterium specific and can occur in the absence of Ti-plasmid genes.     

Broad-Host-Range Agrocin of Agrobacterium tumefaciens

Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed.     

Stress-induced proteins of Agrobacterium tumefaciens

The pattern of proteins produced by bacteria represents the physiological state of the organism as well as the environmental conditions encountered. Environmental stress induces the expression of several regulons encoding stress proteins. Extensive information about the proteins which constitute these regulons (or stimulons) and their control is available for very few bacteria, such as the Gram-positive Bacillus subtilis and the Gram-negative Escherichia coli (gamma-proteobacteria) and is minimal for all other bacteria. Agrobacterium tumefaciens is a Gram-negative plant pathogen of the alpha-proteobacteria, which constitutes the main tool for plant recombinant genetics. Our previous studies on the control of chaperone-coding operons indicated that A. tumefaciens has unique features and combines regulatory elements from both B. subtilis and E. coli. Therefore, we examined the patterns of proteins induced in A. tumefaciens by environmental changes using two-dimensional gel electrophoresis and dual-channel image analysis. Shifts to high temperature, oxidative and mild acid stresses stimulated the expression of 97 proteins. The results indicate that most of these stress-induced proteins (80/97) were specific to one stress stimulon. Only 10 proteins appear to belong to a general stress regulon.     

ACC deaminase activity in avirulent Agrobacterium tumefaciens D3

Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58.     

Stimulation of Agrobacterium tumefaciens virulence with indole-3-acetic acid

The phytopathogen Agrobacterium tumefaciens incites the production of crown-gall on a wide range of dicotyledonous plants. Gall formation is dependent upon indole-3-acetic acid (IAA) and cytokinin production by the transformed plant cells. Upon incubation of Agrobacterium tumefaciens C58 with the plant hormone indole-3-acetic acid (IAA), bacterial virulence on cucumber plants was stimulated up to tenfold. Stimulation was maximized after exposure of bacteria to 50 or 100 μg ml^-1 IAA for 3 h. This was shown to be at the early log phase of bacterial growth.
The authors suggest that the excretion of IAA by the transformed plant cells stimulates bacterial virulence mechanism(s) encoded by the Ti plasmid, the chromosome, or both.     

Occurrence of Agrobacterium tumefaciens Biovar 3 on Grapevine in Brazil

Occurrence of crown gall on grape was observed in a vineyard in Minas Gerais State, Brazil. Isolations of the pathogen yielded a bacterium which incited galls on grapevine, tomato and bryophillum. The bacterium was confirmed as Agrobacterium tumefaciens and its characterization showed it to be biovar 3. This is the first report on the occurrence of the biovar 3 of A. tumefaciens on grapevine in Brazil.     

Chloroplast transformation by Agrobacterium tumefaciens

A chimeric gene consisting of the promoter region of the nopaline synthase gene (Pnos) fused to the coding sequence of the chloramphenicol acetyltransferase gene (cat gene) of Tn9 was introduced by co-cultivation in tobacco protoplasts followed by selection with 10 μg/ml chloramphenicol. The chloramphenicol-resistant plants derived from these selected calli were unable to transmit the Cm^R phenotype through pollen. A typically maternal inheritance pattern was observed. Southern blot analysis showed that the chimeric Pnos-cat gene was present in the chloroplasts of these resistant plants. Furthermore, the chloramphenicol acetyltransferase activity was shown to be associated with the chloroplast fraction. These observations are the first proof that the Agrobacterium Ti-plasmid vectors can be used to introduce genes in chloroplasts.     

l-Sorbose metabolism in Agrobacterium tumefaciens

The pathway of l-sorbose metabolism in Agrobacterium tumefaciens strain B6 was determined to be: l-sorbose d-glucitol (sorbitol) d-fructose d-fructose-6-phosphate d-glucose-6-phosphate. The reduction of l-sorbose and the oxidation of d-glucitol were mediated by NADPH- and NAD⁺-linked oxidoreductases, respectively. The intermediates, d-glucitol and d-fructose, were isolated from in vitro reaction mixtures by column chromatography on Dowex 1-borate, and identified enzymatically. d-Fructose was identified chemically by its ¹H-NMR spectrum and the IR spectrum and the melting point of the fructosazone. d-Glucitol was characterized chemically by the melting point and the IR spectrum of its hexaacetate. A. tumefaciens ICPB TT111, a representative of another genetic race of Agrobacterium, lacked l-sorbose reductase and therefore failed to grow on l-sorbose; it grew normally on d-glucitol.     

Carbohydrate metabolism in Agrobacterium tumefaciens

The activity of pentose cycling (PC) reactions in Agrobacterium tumefaciens is much greater than that normally found in bacteria, and in this regard the organism represents a unique category. Equations specifically derived from radiorespirometric data for bacteria with high PC activity in the presence of an alternate pathway are presented. A. tumefaciens utilizes d-glucose by strictly aerobic mechanisms involving the Entner-Doudoroff (ED) and PC pathways; relative participation by the ED pathway is 55% and by the PC cycle, 44%. The 3-ketoglycose-synthesizing system in the bacterium does not affect the relative participation of these two pathways. Radiorespirometric and enzymatic analyses clearly demonstrate that the Embden-Meyerhof-Parnas pathway does not function. Studies on the oxidation of pyruvic, acetic, succinic, and glutamic acids show that terminal respiration includes both the tricarboxylic acid and glyoxylic acid cycles.     

Hexuronic acid dehydrogenase of Agrobacterium tumefaciens

Growth of Agrobacterium tumefaciens on d-glucuronic acid (GlcUA) or d-galacturonic acid (GalUA) induces formation of hexuronic acid dehydrogenase [d-aldohexuronic acid: nicotinamide adenine dinucleotide (NAD) oxidoreductase]. The dehydrogenase, which irreversibly converts GlcUA or GalUA to the corresponding hexaric acid with the concomitant reduction of NAD, but not of nicotinamide adenine dinucleotide phosphate was purified 60-fold by MnCl(2) treatment, (NH(4))(2)SO(4) fractionation, chromatography on diethylaminoethyl Sephadex and negative adsorption with Ca(3)(PO(4))(2) gel. The pH optimum is 8.0. Other uronic acids, aldohexoses, aldopentoses, and polyols, are not substrates. Reduced nicotinamide adenine dinucleotide is an inhibitor strictly competitive with NAD. Kinetic data indicate that the dehydrogenase induced by growth on GlcUA may not be identical with that induced by growth on GalUA.     

Biotypes of Agrobacterium tumefaciens in Hungary

Isolates of Agrobacterium tumefaciens from Hungary were separated into three biotypes on the basis of their physiological characters. Biotypes 1 and 2 corresponded with those of Keane et al . (1970). The most common isolates were of biotype 2. Isolates from grapevines formed a separate biotype which might be distinguished from biotype 1 by D-(–)tartrate and malonate utilization. Many isolates with biotype-intermediate characters were found. Isolates utilizing D-(–)tartrate, erythritol and malonate were included into biotype 2, although many of them were 3–ketolactose positive. Biotypes were not separated geographically and biotype 1 and 2 apparently occurred together.