The viable-but-nonculturable condition is induced by copper in Agrobacterium tumefaciens and Rhizobium leguminosarum.

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved Agrobacterium tumefaciens and Rhizobium leguminosarum cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.     

The Viable-but-Nonculturable Condition Is Induced by Copper in Agrobacterium tumefaciens and Rhizobium leguminosarum

Many bacteria respond to changes in environmental conditions by entering the viable-but-nonculturable state. We have determined that copper can induce nutrient-starved Agrobacterium tumefaciens and Rhizobium leguminosarum cells to become viable but nonculturable. This is the first report of a chemical inducer of this condition.     

Transfer of the symbiotic plasmid from Rhizobium leguminosarum biovar trifolii to Agrobacterium tumefaciens

This study examined the symbiotic properties of Agrobacterium transconjugants isolated by transferring a Tn5-mob-marked derivative of the 315 kb megaplasmid pRt4Sa from Rhizobium leguminosarum bv. trifolii 4S (wild-type strain) to Agrobacterium tumefaciens A136 as the recipient. The genetic characteristics of the AT4S transconjugant strains were ascertained by random amplified polymorphic DNA (RAPD) analyses and Southern hybridization using Tn5-mob and nod genes as probes. Several of these AT4S transconjugants carrying pRt4Sa were able to nodulate roots of the normal legume host, white clover. In addition, some AT4S transconjugant strains were able to induce nodules on other leguminous plants, including alfalfa and hairy vetch. A characteristic bacteroid differentiation was observed in clover and alfalfa nodules induced by the AT4S-series strains, although nitrogen-fixing activity (acetylene reduction) was not found. Furthermore, strain H1R1, obtained by retracing transfer of the pRt4Sa::Tn5-mob from strain AT4Sa to strain H1 (pRt4Sa cured derivative of 4S), induced Fix(+) nodules on clover roots. These results indicate the evidence that only nod genes can be expressed in the Agrobacterium background.     

Identification of the sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other Rhizobia and Agrobacterium tumefaciens

A plasmid of 150 Mdal from Rhizobium leguminosarum RCC1001 was found to be a Sym plasmid (pSym1) carrying genes for root nodulation and nitrogen fixation on plants of the pea vetch cross-inoculation group. The plasmid was expressed not only in different R. leguminosarum and R. trifolii hosts, but also in Agrobacterium tumefaciens and R. meliloti, although in root nodules induced by A. tumefaciens and R. meliloti hosts no nitrogen was fixed. The host range for root nodule induction appeared to be determined by pSym1 and only included plants of the pea vetch cross-inoculation group; in contrast, the host range for the induction of root hair deformations, which was found also to be determined by pSym1 was less restricted and included besides plants of the pea vetch group in addition plants of the clover group. This corroborates previous findings that host specificity for nodulation and nitrogen fixation is exerted at a stage after the induction of root hair deformations.     

Serological studies with Agrobacterium radiobacter,A. tumefaciens,and Rhizobium strains

Summary Antisera were prepared against cell material from 3 strains of A. radiobacter and 6 of A. tumefaciens. Agar diffusion and immune absorption techniques revealed 3 antigens common to each strain of these organisms. However, 5 different lipopolysaccharide antigens occurred in the 9 test strains. There was no obvious species differences in the distribution of these antigens. Mannose, and possibly glucuronic acid were immunologically active in one of the lipopolysaccharides.The agrobacterial antisera were further crosstested with antigenic material for 34 strains of Rhizobium. Fast-growing rhizobia showed extensive cross-reaction, but only one of 7 R. lupini strains tested reacted with any antiserum.     

R-plasmid-mediated chromosomal gene transfer in Agrobacterium tumefaciens.

Although several techniques are available for transferring the Ti plasmids from one strain of agrobacterium tumefaciens to another, there are no reproducible methods for analysis of chromosomal markers in this phytopathogen. The R plasmid, R68.45, is known to show chromosomal mobilizing ability in several bacterial genera including the closely related Rhizobia. R68.45 was transferred into the prototrophic A. tumefaciens strain 15955. Ten kanamycin-resistant transconjugant clones were tested for chromosomal mobilizing ability by mating with strain SA10, rifampin- and streptomycin-resistant histidine auxotroph of strain 15955. Of the 10 donor clones, 2 showed high chromosomal mobilizing ability. Between 1,000 and 2,000 His+ colony-forming units per ml were obtained, a value 10 to 20 times greater than can be accounted for by spontaneous reversion. Sequential recloning and matings resulted in the isolation of relatively stable donor cultures. Chromosome gene transfer is dependent upon the presence in the donor of R68.45. Donors lacking an R plasmid or harboring the closely related plasmid RP4 failed to yield His+ transconjugants. With strain SA11, a methionine auxotroph of strain SA10, coinheritance of histidine and methionine independence could be demonstrated. Approximately half of the transconjugants also inherited R68.45. These results indicate that A. tumefaciens 15955 is capable of undergoing host chromosomal genetic exchange.     

Ti plasmid-controlled chromosome transfer in Agrobacterium tumefaciens.

In octopine-type A. tumefaciens R10, transfer of chromosomal arginine degradation genes (arc genes) was observed under conditions in which Ti plasmid transfer took place. However, transconjugants that had acquired the arc genes but not the Ti plasmid were recovered. During this process, several other chromosomal genes, such as genes encoding phage resistances or genes complementing a galactose utilization mutation or a glycine-serine auxotrophy, were transferred from strain R10 to the recipient.     

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.     

Developmental Effects of Zeatin, Ribosyl-Zeatin, and Agrobacterium tumefaciens B(6) on Certain Mosses

Eight species of mosses studied were divided into two groups on the basis of their developmental responses to ribosyl-trans-zeatin and Agro-bacterium tumefaciens B₆. All eight produced either gametophores or callus on the protonema in response to 6-(γ,γ-dimethylallylamino) purine and trans-zeatin. Three which produced normal gametophores with A. tumefaciens yielded callus or abnormal gametophores with ribosyl-trans-zeatin. Ribosyl-trans-zeatin and A. tumefaciens were relatively ineffective on five other mosses. Characteristics of protonemal growth common to each of these two groups are described.     

Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens.

Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A. tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C. To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A. tumefaciens was tested at various temperatures. Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C. Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine. However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature. A. tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided. Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself. As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly.     

Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens.

Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281. Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively. IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426. The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R. meliloti to A. tumefaciens.     

Expression of an agrocin-encoding plasmid of Agrobacterium tumefaciens in Rhizobium meliloti

Plasmid RP4 was used to mobilize the agrocin 84-encoding plasmid, pAg396, from Agrobacterium tumefaciens strain 396 to A. tumefaciens C58 and C58CI as well as Rhizobium meliloti. It was transferred to, but not stably maintained in, R. leguminosarum. It could not be transferred to R. lupini, R. japonicum or R. trifolii. Plasmid pAg396 did not segregate in R. meliloti and produced levels of agrocin comparable to the parental strain A. tumefaciens 396. The potential of agrocin producing R. meliloti in biological control of crown gall is being investigated.     

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens.

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.     

Structure and biosynthesis of the ribosomal ribonucleic acids from the oncogenic bacterium Agrobacterium tumefaciens.

The rRNA of the oncogenic bacterium Agrobacterium tumefaciens was extracted by several methods and analysed by polyacrylamide-gel electrophoresis. The large rRNA of this bacterium is degraded in vivo during the maturation of the ribosome. The influence of Mg2+ and denaturation on degradation of 23S RNA was studied. In pulse and chase experiments, we identified two precursors of the rRNA with mol.wts. of 1.04 x 10(6) and 0.70 x 10(6). From studies of the structure of the large rRNA, we propose that it could have arisen from a gene duplication. This structure is discussed in relation to a recent hypothesis involving such gene duplication as a means of origin of 23S rRNA.     

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called 'opines', e.g. octopine (N-alpha-(D-1-carboxyethyl)-L-arginine) and nopaline (N-alpha-(1,3-dicarboxypropyl)-L-argine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a 'genetic colonization' of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.     

Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus.

Agrobacterium tumefaciens and Rhizobium meliloti carry related genetic loci which have important roles in virulence and symbiosis. Previously, it was shown that two virulence loci of A. tumefaciens, chvA and chvB, are related to two R. meliloti symbiosis loci, ndvA and ndvB, respectively (T. Dylan, L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta, Proc. Natl. Acad. Sci. USA 83:4403-4407, 1986). Here we show that these two phytobacteria possess additional related virulence/symbiosis genes. Results of genetic complementation and DNA hybridization experiments indicate that the pscA virulence locus of A. tumefaciens is structurally and functionally related to the exoC symbiosis locus of R. meliloti. Thus, A. tumefaciens and R. meliloti bear at least three related genetic loci that have crucial roles in establishing the interactions that each bacterium has with its respective host plants.     

Discrimination of Rhizobium japonicum,Rhizobium lupini,Rhizobium trifolii,Rhizobium leguminosarum and of bacteriods by uptake of 2-ketoglutaric acid,glutamic acid and phosphate

Rhizobium strains (one each of Rh.japonicum, Rh. lupini, Rh. leguminosarum) take up 2-ketoglutaric acid in general much faster and from lower concentrations in the medium than strains of Escherichia coli, Bacillus subtilis and Chromobacterium violaceum. A strain of Enterobacter aerogenes, however, is more similar to some Rhizobium strains. The same strains of Rhizobium take up also phosphate much faster and from lower concentrations than the other bacteria tested. 4 strains of Rh. lupini proved to be significantly different from 4 strains of Rh. trifolii in taking up l-glutamic acid from three to ten times lower concentration within 5 h. A similar difference was noticed between 5 strains of Rh. leguminosarum and 2 strains of Rh. japonicum for the uptake of 2-ketoglutaric acid and of l-glutamic acid. Isolated bacteriods from nodules of Glycine max var. Chippeway have a reduced uptake capacity for glutamic acid and for 2-ketoglutaric acid during the first 10–12 h, but reach the same value after 24 h as free living Rh. japonicum cells. The differences in the uptake kinetics are independent of cell concentration. The group II Rhizobium strains (Rh. japonicum and Rh. lupini, slow growing Rhizobium) are characterized by a rapid uptake of glutamic acid to a lowremaining concentration of 1–3×10^-7 M and an uptake of 2-ketoglutaric acid to a remaining concentration of 2–5×10^-7 M. The group I Rhizobium strains (Rh. trifolii and Rh. leguminosarum, fast growing Rhizobium), can be characterized by a much slower uptake of both substances with a more than ten times higher concentration of both metabolites remaining in the medium after the same time.