Nucleic Acids Res, 1984 Jun 11, 12(11), 4665 - 77 Nucleotide sequence of the tmr locus of Agrobacterium tumefaciens pTi T37 T-DNA; Goldberg SB et al.; The nucleotide sequence of the tmr locus from the nopaline-type pTi T37 plasmid of Agrobacterium tumefaciens was determined . Examination of this sequence allowed us to identify an open reading frame of 720 nucleotides capable of encoding a protein with a derived molecular weight of 27025 d . Comparison of the pTi T37 tmr sequence with the published sequence of the pTi Ach5 tmr locus shows over 88% homology in the 240 bases 5' to the translational initiation codon and over 91% homology in the coding sequences . The 3' nontranslated regions show less than 50% homology as expected for the 3' regions of divergent related genes . The possible significance of areas of conserved sequences, particularly in the 5' regulatory regions, is discussed. J Bacteriol, 1984 Jun, 158(3), 1133 - 43 Rhizobium meliloti nodulation genes allow Agrobacterium tumefaciens and Escherichia coli to form pseudonodules on alfalfa; Hirsch AM et al.; Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation . The A . tumefaciens and E . coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants . A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid . These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules . Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules . There was no evidence of bacterial release from the infection threads . In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells . No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed . In contrast to the A . tumefaciens-induced pseudonodules , the E . coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules . Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa. Can J Microbiol, 1984 May, 30(5), 676 - 81 Flagella-specific bacteriophages of Agrobacterium tumefaciens: demonstration of virulence of nonmotile mutants; Bradley DE et al.; Bacteriophages GS2 and GS6 for Agrobacterium tumefaciens were shown by electron microscopy to adsorb to flagella . This specificity was confirmed by the finding that phage-resistant mutants were nonmotile . Such mutants retained tumor-inducing virulence and ability to attach to plant cells, indicating that motility was not required for these properties . Both phages had contractile tails and appeared similar in the electron microscope. J Bacteriol, 1984 May, 158(2), 650 - 8 Mannityl opine analogs allow isolation of catabolic pathway regulatory mutants; Chilton WS et al.; Five virulent Agrobacterium spp . strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds . Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs . In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP . Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs . The analogs allowed selection of mutants of all strains . Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806 . Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested . The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted . Only ketose analogs were refused . Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways. J Bacteriol, 1984 May, 158(2), 754 - 6 trans-Acting virulence functions of the octopine Ti plasmid from Agrobacterium tumefaciens; Hille J et al.; All Ti plasmid-encoded virulence functions that were studied act in trans . An octopine Ti plasmid-specific vir operon, called vir-O, located on an EcoRI restriction fragment has been characterized . Sequences with promoter activity in Escherichia coli were identified for a second vir operon, called vir-C, which was located close to the position of vir-O. EMBO J, 1984 May, 3(5), 1029 - 37 A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors; Koncz C et al.; A simple method for inserting foreign genes into the T-region of Agrobacterium Ti-plasmids is described . A modified cosmid (pHC 79) was introduced into a predetermined site of the T-region of pTi C58 . An Agrobacterium strain harboring this modified Ti-plasmid was used as an acceptor strain into which genes, cloned in pBR322, can be introduced by mobilization from Escherichia coli . pBR322-derived plasmids cannot replicate in Agrobacterium, but can be maintained by integration into the T-region of the modified Ti-plasmids by homologous recombination . This method was used to introduce the genes for ovalbumin and alpha-actin from chicken into tobacco tumors . Southern blotting and re-isolation of the inserted genes by reverse cloning showed that the animal DNA was transferred and integrated into the plant genome without rearrangements . The alpha-actin gene is not transcribed in plant tumors, whereas transcription of the ovalbumin gene was observed, however the initiation point of transcription was different from the one used in the chicken oviduct . The RNA transcribed from the chicken ovalbumin gene is polyadenylated and ranges in size between 2 and 7 kb. Ann Microbiol (Paris), 1984 May-Jun, 135A(3), 427 - 42 {Taxonomic position of Agrobacterium strains of hospital origin}; Popoff MY et al.; A collection of 31 strains received as Agrobacterium tumefaciens and A . radiobacter was subjected to detailed phenotypic and genomic studies . These strains were recovered from plants, soil, water and clinical specimens . Type strains of A . tumefaciens, A . radiobacter, A . rhizogenes and A . rubi were also included . The strains were tested for their ability to use 169 organic compounds as sources of carbon and energy . In addition, 11 conventional characters were studied for each strain . Relatedness among the strains was assessed by determining the extent of reassociation in heterologous DNA preparations . S1 nuclease and diethylaminoethyl-cellulose filters were used to separate reassociated from non-reassociated nucleotide sequences, and to determine the thermal stability of related nucleotide sequences . The resultant data revealed the following points: regardless of their phytopathogenic effects, 3-ketolactose-producing A . tumefaciens and A . radiobacter strains group into one species; this species contains 9 taxa which can be differentiated from each other by phenotypic and genomic characters; clinical isolates did not induce tumours on plants and clustered in three taxa of this species; the clinical and ecological significance of these organisms is not known; the present classification of the genus Agrobacterium is based on phytopathogenicity and does not reflect the phylogenetic relationships amongst these bacteria; as proposed previously by several workers, the genus Agrobacterium should be divided into 3 species on the basis of phenotypic and genomic characteristics . Different aspects of the classification and nomenclature of Agrobacterium are discussed. Plasmid, 1984 May, 11(3), 206 - 20 Genetic and molecular characterization of the Pseudomonas plasmid pVS1; Itoh Y et al.; A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed . Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P . fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1 . The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21 . A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P . aeruginosa PAO but selective pressure was needed for plasmid maintenance . The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent . Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P . aeruginosa, P . putida, P . fluorescens, P . acidovorans, P . cepacia, P . mendocina, P . stutzeri, P . syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum. Plasmid, 1984 May, 11(3), 195 - 205 A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants; Hooykaas PJ et al.; Transposon-insertion mutants with vir- Ti plasmids were characterized and then used in complementation experiments . One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF . The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species . Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction . Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid . The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir- nopaline Ti plasmid . Also the transfer of an Ri plasmid to a large number of different vir- octopine or nopaline Ti plasmid mutants rendered these strains virulent . These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids . In complementation experiments between vir- octopine Ti plasmid mutations and vir- nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids . The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci . One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific) . For the other locus the name virC was retained . Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica . VirO- mutants produced rooty tumors on Kalanchoe tubiflora. J Bacteriol, 1984 Apr, 158(1), 383 - 5 Transfer of the octopine T-DNA segment to plant cells mediated by different types of Agrobacterium tumor- or root-inducing plasmids: generality of virulence systems; Hoekema A et al.; Genetic complementation studies demonstrated that the transfer to plant cells of the octopine T-DNA, entirely present as the only part of the tumor-inducing (Ti) plasmid on the plasmid pAL1050, was effected by the virulence systems from related plasmids, viz . the nopaline Ti plasmid pTiC58, the limited host range plasmid pTiAg57, and the root-inducing (Ri) plasmid pRi1855 . Rhizobium symbiosis plasmids were not capable of effecting the introduction of pAL1050 into plant cells. Microbiol Sci, 1984 Apr, 1(1), 1 - 4 Agrocins and the biological control of crown gall; Kerr A et al.; Agrocin 84 is a plasmid-encoded, fraudulent adenine nucleotide antibiotic responsible for the preventative biological control of the plant cancer, crown gall . It has bacteriocin-like selectivity which is dependent on a Ti-plasmid-encoded permease in pathogenic agrobacteria . Other nucleotide agrocins have been described and partially characterized; more may be confidently predicted. Arch Microbiol, 1984 Apr, 137(4), 338 - 43 Changes in properties of phytopathogenic bacteria effected by plasmid pRD1; Kozyrovskaya NA et al.; Transfer of plasmid pRD1 from Escherichia coli K 12J62 -1 to phytopathogenic bacteria, Agrobacterium tumefaciens, Xanthomonas beticola and Erwinia carotovora subsp . carotovora caused changes in conjugant properties not determined by the plasmid and the emergence of the properties not present in the parent strains . Clones have been obtained with intermediate properties between donor and recipient, including those with altered or lost virulence . In transconjugants of A . tumefaciens virulence increased . In transconjugants of X . beticola and E . carotovora subsp . carotovora highly virulent as well as avirulent forms have been observed . The loss of virulence in X . beticola correlated with the Nif+ phenotype . Plasmid pRD1 also affected the biochemical properties of the new hosts. EMBO J, 1984 Apr, 3(4), 835 - 46 The complete nucleotide sequence of the TL-DNA of the Agrobacterium tumefaciens plasmid pTiAch5; Gielen J et al.; We have determined the complete primary structure (13 637 bp) of the TL-region of Agrobacterium tumefaciens octopine plasmid pTiAch5 . This sequence comprises two small direct repeats which flank the TL-region at each extremity and are involved in the transfer and/or integration of this DNA segment in plants . TL-DNA specifies eight open-reading frames corresponding to experimentally identified transcripts in crown gall tumor tissue . The eight coding regions are not interrupted by intervening sequences and are separated from each other by AT-rich regions . Potential transcriptional control signals upstream of the 5' and 3' ends of all the transcribed regions resemble typical eukaryotic signals: (i) transcriptional initiation signals ('TATA' or Goldberg- Hogness box) are present upstream to the presumed translational start codons; (ii) ' CCAAT ' sequences are present upstream of the proposed 'TATA' box; (iii) polyadenylation signals are present in the 3'-untranslated regions . Furthermore, no Shine-Dalgarno sequences are present upstream of the presumed translational start codons. Nucleic Acids Res, 1984 Mar 12, 12(5), 2317 - 25 DNA sequence analysis of crown gall tumor T-DNA encoding the 0.7 kb transcript; McPherson JC; Crown gall tumor formation involves integration into the plant genome of DNA sequences (the T-region) of tumor-inducing (Ti) plasmids present in Agrobacterium tumefaciens . The T-DNA of the tumor expresses several gene products . Little is known about the function or regulation of expression of the 0.7kb transcript, which represents a relatively abundant T-DNA transcript in octopine-type tumors . In this report, a detailed structural analysis of the gene encoding the 0.7 kb transcript has been obtained by DNA sequence analysis of T-DNA isolated from A6S/2 tumor line . An indication of the structural characteristics of the protein product is obtained from the predicted amino acid sequence . The sequences flanking the open reading frame show characteristics with other eucaryotic genes . The corresponding DNA sequence of the inducing Ti plasmid (pTiA6) is identical with that of the DNA sequence from the tumor . Comparison of this gene sequence with the corresponding region of another Ti plasmid (pTiAch5) shows several differences in the 5' flanking sequence, but the nucleotide sequence of the coding region and 3' flanking region are identical. Plasmid, 1984 Mar, 11(2), 130 - 40 T-DNA fragments of hairy root plasmid pRi8196 are distantly related to octopine and nopaline Ti plasmid T-DNA; Lahners K et al.; Agrobacterium Ti (tumor-inducing) and Ri (root-inducing) plasmids transform dicot plant cells by insertion of a specific plasmid sector called T-DNA (transferred DNA) into host plant nuclear DNA . The mannopine -type Ri plasmid pRi8196 contains four BamHI fragments that encompass core T-DNA . We report Southern hybridization studies that show that these four fragments have no strong homology to octopine-, nopaline-, or agropine -type Ti plasmids . We detected and mapped very weak homology regions, most of which are assignable to opine synthase or opine catabolic functions on the Ti plasmid . We found no homology between Ri T-DNA and the region of Ti T-DNA that encodes tumor morphology functions. J Bacteriol, 1984 Mar, 157(3), 739 - 45 Characterization of the replication and stability regions of Agrobacterium tumefaciens plasmid pTAR; Gallie DR et al.; A 5.4-kilobase region containing the origin of replication and stability maintenance of the 44-kilobase Agrobacterium tumefaciens plasmid pTAR has been mapped and characterized . Within this region is a 1.3-kilobase segment that is capable of directing autonomous replication . The remaining segment contains the stability locus for maintenance of pTAR during nonselective growth . Approximately 35% of pTAR shares sequence homology with pAg119, a 44-kilobase cryptic plasmid in grapevine strain 1D1119 . However, no homology was detected between pTAR DNA and several Ti plasmids or several other small cryptic plasmids in many A . tumefaciens strains . A recombinant plasmid containing the origin of replication and stability maintenance region of pTAR was compatible with pTiC58, pTi15955, and pTi119 and incompatible with pAg119 . A new compatibility group, Inc Ag-1, is discussed. Proc Natl Acad Sci U S A, 1984 Feb, 81(4), 1035 - 9 Generation of a Tn5 promoter probe and its use in the study of gene expression in Caulobacter crescentus; Bellofatto V et al.; A promoter probe, Tn5-VB32, was constructed and placed in a P group R plasmid containing bacteriophage Mu sequences, allowing transfer of the transposon to bacteria such as Caulobacter, Rhizobium, and Agrobacterium without retention of the plasmid . The probe carries an altered Tn5 transposon that allows detection of chromosomal promoter regions by virtue of acquired kanamycin resistance . A fragment of DNA containing the neomycin phosphotransferase II (NPT II) gene from Tn5, lacking its promoter region but retaining its translation initiation signal, was inserted into a Tn5 derivative that lacked the entire NPT II gene and a large portion of the IS50L sequence while retaining its ability to transpose . This Tn5 derivative also contained the intact tetracycline resistance-encoding region of the transposon Tn10 . Transposition of the Tn5-VB32 promoter probe into the Caulobacter crescentus chromosome generated auxotrophic and motility mutants and Southern blot analysis of DNA from these mutants showed Tn5-VB32 sequences in random-sized chromosomal restriction fragments . Transcriptional regulation by exogenous cysteine of NPT II gene expression was demonstrated in a cysteine auxotroph generated by Tn5-VB32 insertional inactivation . NPT II synthesis, measured by agar plate assays of kanamycin resistance and by immunoprecipitation of the NPT II protein, was repressed in the presence of cysteine and derepressed in its absence . Several fla- mutants were also isolated by Tn5-VB32 mutagenesis and shown to confer kanamycin resistance . Insertions within temporally regulated genes, such as those involved in flagellar biosynthesis and chemotaxis functions, can now be used directly to monitor transcriptional regulation from Caulobacter promoter sequences. J Bacteriol, 1984 Feb, 157(2), 357 - 62 Succinamopine: a new crown gall opine; Chilton WS et al.; Agrobacterium tumefaciens strains can incite plant tumors consisting of transformed cells that synthesize novel metabolites called opines . The pattern of opine synthesis is dictated by plasmid-borne genes in the pathogen; additional plasmid genes confer on the pathogen the ability to catabolize the same pattern of opines synthesized . One group of A . tumefaciens strains, AT181, EU6, and T10/73, contains closely related tumor-inducing (Ti) plasmids that encode the ability to degrade the opine nopaline; but tumors incited by these strains do not synthesize nopaline . We demonstrated by Southern blot hybridization that AT181(pTi) has no DNA homologous to the nopaline synthase gene of pTi T37, a nopaline Ti plasmid that appears to be most closely related to this group based on fingerprint analysis . Tumors incited by these seemingly anomalous strains contain a new opine that we designate succinamopine . Its structure is analogous to that of nopaline, with asparagine replacing arginine . Evidence for the structure of succinamopine, as well as those of two related metabolites, succinamopine lactam and succinopine lactam, will be published elsewhere . Ability to catabolize succinamopine, succinamopine lactam, and succinopine lactam is encoded by pTi AT181, pTi EU6, and pTi T10/73, but not by any of 15 other Ti and root-inducing plasmids tested . Three avirulent strains tested did not catabolize succinamopine, succinamopine lactam, or succinopine lactam . We propose that pTi AT181, pTi EU6, and pTi T10/73 be designated the succinamopine Ti plasmids. Eur J Biochem, 1984 Jan 16, 138(2), 387 - 91 The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid; Schroder G et al.; Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems . In transformed plant cells it participates in auxin-controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000 . We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis . Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole-3-acetamide into a substance which was unambiguously identified as indole-3-acetic acid . The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene . Extracts from tobacco crown gall cells, but not from non-transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole-3-acetic acid . The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole-3-acetic acid. J Biochem (Tokyo), 1984 Jan, 95(1), 13 - 8 D-glucosaminate dehydratase: spectrometric properties of the enzyme-bound pyridoxal 5'-phosphate; Iwamoto R et al.; The holoenzyme of D-glucosaminate dehydratase {EC 4.2.1.26} from Agrobacterium radiobacter showed absorption peaks at 280 and 415 nm with a shoulder in the region of 320 to 330 nm . The treatment of the enzyme with hydroxylamine followed by dialysis led to disappearance of both the absorption peak at 415 nm and the shoulder, giving the apoenzyme . The fluorescence excitation maximum of the holoenzyme was at 320 nm with a shoulder at 420 nm (emission at 510 nm), and the emission maxima were at 420 nm with a shoulder at 370 nm (excitation at 320 nm) and at 510 nm (excitation at 420 nm) . The holoenzyme showed a negative circular dichroic band at 418 nm and a positive shoulder at around 320 nm . Reduction of the holoenzyme with sodium borohydride caused a loss of the absorption peak at 415 nm with a concomitant increase of 325 nm absorbance and an irreversible loss of the activity . The occurrence of epsilon-N-pyridoxyllysine in the acid hydrolysate of the reduced enzyme showed that D-glucosaminate dehydratase contains a catalytically essential lysine residue whose epsilon-amino group binds the 4-formyl group of pyridoxal 5'-phosphate to form a Schiff base . The plots of absorption of the apoenzyme against the amount of pyridoxal 5'-phosphate added showed that four and two molar equivalents of the cofactor bind to the apoenzyme and subunit, respectively . The biphasic nature of the spectrometric titration curve of the apoenzyme with pyridoxal 5'-phosphate and the two Km values obtained for the cofactor suggest the occurrence of two distinct types of binding sites for pyridoxal 5'-phosphate in the enzyme. J Bacteriol, 1984 Jan, 157(1), 269 - 76 Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids; Huffman GA et al.; A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants . The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones . Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b . As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids . About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens . Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b . The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b . A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr . A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37. J Bacteriol, 1984 Jan, 157(1), 134 - 42 Transfer of Rhizobium meliloti pSym genes into Agrobacterium tumefaciens: host-specific nodulation by atypical infection; Truchet G et al.; The pSym megaplasmid of Rhizobium meliloti 2011 mobilized by plasmid RP4, or plasmid pGMI42, an RP4-prime derivative which carries a 290-kilobase pSym fragment including nitrogenase and nod genes, was introduced into Agrobacterium tumefaciens . The resulting transconjugants induced root deformations specifically on the homologous hosts Medicago sativa and Melilotus alba and not on the heterologous hosts Trifolium pratense and Trifolium repens . The root deformations were shown to be genuine nodules by physiological and cytological studies . Thus, host specificity nodulation genes are located on the pSym megaplasmid . Host nodulation specificity did not seem to require recognition at the root hair level since no infection threads could be detected in the root hairs . Cytological observations indicated that bacteria penetrated only the superficial layers of the host root tissue by an atypical infection process . The submeristematic zone and the central tissue of the nodules were bacteria free . Thus, nodule organogenesis was probably triggered from a distance by the bacteria . Agrobacterium transconjugants carrying pSym induced the formation of more numerous and larger nodules than those carrying the RP4-prime plasmid pGMI42, suggesting that some genes influencing nodule organogenesis are located in a pSym region(s) outside that which has been cloned into pGMI42. Zentralbl Mikrobiol, 1984, 139(5), 375 - 82 {Degradation and utilization of 2,4-dioxohexahydro-1,3,5-triazine (DHT) by soil microorganisms}; el-Dahtory TA et al.; The biodegradation and utilization of the antiphytoviral substance 2,4-dioxohexahydro-1,3,5-triazine (DHT) by soil microorganisms was investigated . Mixed cultures of microorganisms deriving from different soils diminish in nutrient broth the content of DHT with increasing duration of culture . Microorganisms from an Egyptian garden soil fully degrade 10(-3) mol/1 DHT in a culture without additional aeration within 28 days . Also in deficient media the mixed microorganisms reduce the amount of DHT, reaching in nitrogen free nutrient solution even a degradation rate up to 12 mg DHT per liter and day . Pure cultures of Rhizobium leguminosarum, Proteus vulgaris, Saccharomyces cerevisiae and especially Agrobacterium radiobacter diminish the content of DHT in nitrogen free media, too . No such effect was detectable in cultures of four other species of soil bacteria . The DHT degradation by the microorganisms is connected with significant cell multiplication, e.g . A . radiobacter in shaking cultures with DHT as sole source of nitrogen shows a typical growth cycle with a lag-phase of 24 hours . The short persistence time of DHT in soils is concluded to be mainly due to biodegradation by microorganisms. J Mol Appl Genet, 1984, 2(4), 354 - 62 Nucleotide sequence and transcript mapping of the tmr gene of the pTiA6NC octopine Ti-plasmid: a bacterial gene involved in plant tumorigenesis; Lichtenstein C et al.; The nucleotide sequence of a tumor morphology gene, tmr, from the Agrobacterium tumefaciens Ti-plasmid, pTiA6NC, and its flanking 5' region was determined by M13 "dideoxy" procedures . The DNA sequence reveals an open reading frame capable of encoding a 240 amino acid protein . We have identified the polyadenylated transcript initiation and termination sits by S1 nuclease mapping . The extent of the sequence required for transcription 5' to the start of transcription has been delimited by two transposon insertions . The first of these maps at -- 121 with respect to transcription initiation and results in the wild-type phenotype, the second insertion maps at about -85 and results in a tmr phenotype. Plasmid, 1984 Jan, 11(1), 17 - 27 Physical map of the Agrobacterium rhizogenes strain 8196 virulence plasmid; Koplow J et al.; Virulence of Agrobacterium rhizogenes, agent of hairy root disease, is conferred by large plasmids called Ri (root-inducing) plasmids . We have determined the BamHI fragment map of pRi8196, MW 143 Mda, principally by analysis of recombinant plasmids containing overlapping BamHI partial-digest fragments . Clones containing solitary BamHI inserts of remaining unmapped fragments were used to probe a series of Southern-blotted, pRi8196-derived EcoRI, PstI, HindIII, SalI, or SmaI digests . Continguous hybridized bands represented complements of EcoRI, PstI, HindIII, SalI, or SmaI fragments which bridged the unmapped BamHI fragments . We present, in addition, a detailed map of the core T-DNA region with respect to the restriction endonucleases SalI, EcoRI, HpaI, and HindIII. Mol Gen Genet, 1984, 193(3), 535 - 7 Studies on Tn951 (lac+) expression in Agrobacterium; Borland PA et al.; None of the Agrobacterium tumefaciens and A . rubi strains tested produces detectable amounts of beta-galactosidase although they are capable of utilizing lactose as sole source of carbon . This opportunity was taken to investigate the expression of lac transposon Tn951 (Cornelis et al . 1978) in Agrobacterium with the ultimate goal of using this system to investigate alien gene expression . When the transposon was introduced with the help of a broad-host range plasmid, RP1, the transconjugants produced significant quantities of beta-galactosidase which was inducible by isopropyl-beta-D-thiogalactopyranoside . Tn951 was capable of restoring the Lac+ phenotype to an A . tumefaciens mutant not capable of using lactose . Cellobiose, a known inducer of aldohexopyranoside: cytochrome c oxidoreductase (which regulates the characteristic 3-ketolactose production in Agrobacterium; van Beeumen and De Ley (1968), had no effect on beta-galactosidase activity. Mol Gen Genet, 1984, 193(1), 1 - 7 Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region; Lundquist RC et al.; Mutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors . Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain . Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion . The analysis identified six complementation groups . Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus . The mutation in one complementation group showed variation in host range. J Mol Appl Genet, 1984, 2(6), 549 - 62 A cauliflower mosaic virus promoter directs expression of kanamycin resistance in morphogenic transformed plant cells; Koziel MG et al.; The promoter region of the CaMV inclusion body protein gene was modified for use in chimeric gene fusions . The modified promoter was used to construct a selectable marker for plant transformation based on the Tn 5 kanamycin resistance gene . This chimeric selectable marker was introduced into plant cells using oncogenic and deoncogenized strains of Agrobacterium tumefaciens . Both types of transformation produced kanamycin-resistant cell lines . The resistant cell lines derived from the deoncogenized strains were used to regenerate shoots . A second type of selection based on the ability of octopine synthase to detoxify aminoethyl cysteine was also used to select transformants in both oncogenic and nononcogenic transformation. J Mol Appl Genet, 1984, 2(5), 465 - 70 The T-DNA of Agrobacterium rhizogenes is transmitted through meiosis to the progeny of hairy root plants; Costantino P et al.; Plants regenerated from tobacco hairy root callus cultures were fertilized by self-pollination, and healthy, morphologically normal R1 offspring were obtained . Three of these R1 seedlings were analyzed for the presence of T-DNA and synthesis of the T-DNA-specific compound agropine . All three R1 plants analyzed contained the same full-length T-DNA as the parental regenerant, while only two showed agropine synthesis. Nucleic Acids Res, 1983 Sep 24, 11(18), 6211 - 23 Nucleotide sequence of the Agrobacterium tumefaciens octopine Ti plasmid-encoded tmr gene; Heidekamp F et al.; The nucleotide sequence of the tmr gene, encoded by the octopine Ti plasmid from Agrobacterium tumefaciens (pTiAch5), was determined . The T-DNA, which encompasses this gene, is involved in tumor formation and maintenance, and probably mediates the cytokinin-independent growth of transformed plant cells . The nucleotide sequence of the tmr gene displays a continuous open reading frame specifying a polypeptide chain of 240 amino acids . The 5'- terminus of the polyadenylated tmr mRNA isolated from octopine tobacco tumor cell lines was determined by nuclease S1 mapping . The nucleotide sequence 5'-TATAAAA-3', which sequence is identical to the canonical "TATA" box, was found 29 nucleotides upstream from the major initiation site for RNA synthesis . Two potential polyadenylation signals 5'-AATAAA-3' were found at 207 and 275 nucleotides downstream from the TAG stopcodon of the tmr gene . A comparison was made of nucleotide stretches, involved in transcription control of T-DNA genes. Eur J Biochem, 1983 Sep 15, 135(2), 321 - 30 The amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185; Tempst P et al.; The evidence for the amino acid sequence of cytochrome c-556 from Agrobacterium tumefaciens strain Apple 185 is reported . The sequence was determined by manual Edman degradation of tryptic and chymotryptic peptides using the DABITC/PITC double-coupling method; some peptides were further cleaved by partial acid hydrolysis and with Staphylococcus aureus protease . The sequence overlaps 13-15, 83-85 and 106-108 as well as the region 113-118 involving the haem-binding sequence Cys-Xaa-Xaa-Cys-His were deduced by homology with cytochrome c-556 from Agrobacterium tumefaciens strain B2a . The identity of histidine at position 6 has been inferred from fast-atom bombardment experiments on the N-terminal tryptic peptide, and Asp-63 was deduced from the electrophoretic mobility of the peptides in which it occurs . The cytochrome from A . tumefaciens Apple 185 contains 125 amino acids of which 71 are identical in the protein from strain B2a . Together with cytochrome c-556 from the photosynthetic prokaryote Rhodopseudomonas palustris strain 2.1.37, the presently studied protein is the third known example of a monohaem class II cytochrome of the low-spin type having the single haem group covalently linked near the C terminus of the polypeptide chain . The only methionine residue in the Apple protein, methionine-13, is the most likely candidate to be the sixth haem ligand and therefore to be responsible for the low-spin character of the haem iron. J Cell Biol, 1983 Sep, 97(3), 787 - 94 Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid; Wong CH et al.; We examined expression of the megaplasmid pRme41b of Rhizobium meliloti in two different Rhizobium sp . Strains and in Agrobacterium tumefaciens . Transfer of pRme41b into these bacteria was facilitated by insertion of a recombinant plasmid coding for mobilization functions of RP4 into the nif region (Kondorosi, A., E . Kondorosi, C.E . Pankhurst, W . J . Broughton, and Z . Banfalvi, 1982, Mol . Gen . Genet., 188:433-439) . In all cases, transconjugants formed nodule-like structures on the roots of Medicago sativa . These structures were largely composed of meristematic cells but they were not invaded by bacteria . Bacteria were found only within infection threads in root hairs, and within intercellular spaces of the outermost cells of the structures . The donor strain of R . meliloti containing pAK11 or pAK12 in pRme41b initially produced nodules on M . sativa that did not fix nitrogen (Fix-) . In these nodules, bacteria were released from infection threads into the host cells but they did not multiply appreciably . Any bacteroids formed degenerated prematurely . In some cases, however, reversion to a Fix+ phenotype occurred after 4 to 6 wk . Bacteria released into newly infected cells in these nodules showed normal development into bacteriods. Gene, 1983 Sep, 23(3), 315 - 30 A general method for the transfer of cloned genes to plant cells; Shaw CH et al.; This paper describes a method for the transfer to plant cells of any cloned gene, regardless of its termini or internal restriction enzyme cleavage sites . A broad host-range intermediate vector, pGV1117, was constructed containing HindIII-23, a right-end T-region fragment of the nopaline plasmid pTiC58 . Using in vivo protection by EcoRI methylase and EcoRI linker ligation, a fragment of rabbit chromosomal DNA, carrying the beta-globin gene, was inserted into plasmid pGV1117 . Following transmission to Agrobacterium tumefaciens, insertion of the gene into the T-region of pTiC58 occurred via in vivo recombination . Infection of axenic tobacco seedlings resulted in the transfer to the plant genome of an intact beta-globin gene, as part of the T-DNA . Although the gene was stably maintained during tissue culture, beta-globin-specific transcripts were not detected in the transformed plant cells. Plasmid, 1983 Sep, 10(2), 119 - 29 Restriction endonuclease mapping of the root-inducing plasmid of Agrobacterium rhizogenes 1855; Pomponi M et al.; The root-inducing plasmid of the agropine type Agrobacterium rhizogenes 1855 was mapped by means of the restriction endonuclease EcoRI . The circular arrangement of the more than 60 fragments generated by this enzyme was established by electrophoretic analysis of pBR322 clones harboring overlapping segments of pRi1855 derived by partial digestion with EcoRI . A large region of the plasmid comprising the T-DNA was mapped with two additional enzymes, BamHI and HindIII, by means of Southern blot hybridizations between the fragments generated by the three enzymes. Gene, 1983 Sep, 24(1), 53 - 9 Construction of a broad-host-range kanamycin-resistant plasmid vector; Roychoudhury R et al.; A new 10.2-kb plasmid, pIRL2, was constructed by using a 2140-bp DNA fragment carrying the Kmr gene and BamHI cohesive ends . These BamHI cohesive ends were used to trap the replicating DNA fragment from a partial Sau3A digest of the plasmid R300B . The plasmid contains unique EcoRI, SstI, HindIII, SmaI, SalI, and XhoI sites . These sites can be used as cloning sites without the loss of Kmr . A unique BglII site can be used as a cloning site by insertional inactivation of the Kmr structural gene, coding for neomycin phosphotransferase type II . The new plasmid carries the Sur and Smr genes of R300B . The direction of transcription from the neo promoter is clockwise, the same as that from the sul promoter . The plasmid retains the broad-host-range function of R300B, and thus it may be used for gene cloning in Rhizobium and Agrobacterium for genetic engineering of plant cells. J Gen Microbiol, 1983 Aug, 129 (Pt 8), 2535 - 43 Transfer of an indigenous plasmid of Rhizobium loti to other rhizobia and Agrobacterium tumefaciens; Pankhurst CE et al.; Rhizobium loti strains NZP2037 and NZP2213 were each found to contain a single large plasmid: pRlo2037a (240 MDal) and pRlo2213a (120 MDal), respectively . Plasmid DNA present in crude cell lysates of each strain and purified pRlo2037a DNA did not hybridize with pID1, a recombinant plasmid containing part of the nitrogen fixation (nif) region of R . meliloti, indicating that nif genes were not present on these plasmids . The transposon Tn5 was inserted into pRlo2037a and this plasmid was then transferred into R . leguminosarum, R . meliloti and Agrobacterium tumefaciens . All transconjugants failed to nodulate Lotus pedunculatus, suggesting that the ability to nodulate this legume was also not carried on pRlo2037a . Transfer of pRlo2037a to R . loti strain NZP2213 did not alter the Nod+ Fix- phenotype of this strain for L . pedunculatus . Determinants for flavolan resistance, believed to be necessary for effective nodulation of L . pedunculatus, were not carried on pRlo2037a . These data suggest that nodulation, nitrogen fixation and flavolan resistance genes are not present on the large plasmid in R . loti strain NZP2037. Proc Natl Acad Sci U S A, 1983 Aug, 80(15), 4803 - 7 Expression of bacterial genes in plant cells; Fraley RT et al.; Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques . The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II . The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A . tumefaciens cells with protoplast-derived cells . Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues . Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics . Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions . The significance of these results for plant genetic engineering is discussed. Plasmid, 1983 Jul, 10(1), 21 - 30 A new technique for genetic engineering of Agrobacterium Ti plasmid; Comai L et al.; A new technique is described that allows easy introduction of foreign genetic elements into specific regions of Agrobacterium tumefaciens DNA . It uses plasmids that (1) can be introduced, but not maintained in A . tumefaciens, (2) have a region homologous to the genome of the recipient, and (3) have an appropriate marker . Selection for the marker will yield transconjugants in which the introduced plasmid has recombined with the host genome . Applications of the technique are described. J Bacteriol, 1983 Jul, 155(1), 196 - 202 Ti plasmid and chromosomal ornithine catabolism genes of Agrobacterium tumefaciens C58; Schardl CL et al.; The pTiC58 plasmid noc genes of Agrobacterium tumefaciens C58 code for nopaline oxidase (nocC), nopaline permease (nocP), the inducible periplasmic protein n1 (nocB), and a function(s) required for ornithine catabolism (nocA) . In addition, strains C58 and Ach-5 of A . tumefaciens have chromosomal ornithine catabolism genes . The chromosomal orc gene codes for ornithine dehydrogenase . Strain C58 is normally orc, but orc+ mutants can be selected . We have characterized both chromosomal orc and pTiC58 nocA plasmid genes . Complementation of most chromosomal orc mutants by pTiC58 restored growth on both nopaline and L-ornithine but did not restore ornithine dehydrogenase activity . We conclude that ornithine is an intermediate of nopaline degradation and that the Ti plasmid and chromosome both code for ornithine-degradative enzymes . A model for nopaline catabolism is presented. J Bacteriol, 1983 May, 154(2), 906 - 15 Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection; Matthysse AG; During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils . We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose . Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface . The wild-type bacteria became surrounded by fibrils within 2 h after attachment . No fibrils were seen with the cellulose-minus mutants . Prolonged incubation of wild-type A . tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells . No such aggregates were formed after the incubation of carrot cells with cellulose-minus A . tumefaciens . The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells . Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent . However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water . Thus, a major role of the cellulose fibrils synthesized by A . tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors. J Bacteriol, 1983 May, 154(2), 693 - 701 Site-directed mutagenesis in Escherichia coli of a stable R772::Ti cointegrate plasmid from Agrobacterium tumefaciens; Hille J et al.; The host range of an octopine Ti plasmid is limited to Rhizobiaceae . This has been extended also to Escherichia coli in the form of a stable cointegrate with the wide-host-range plasmid R772 . Its structure was studied by constructing a physical map of R772 and of the R772::pTiB6 cointegrate . An insertion sequence present in R772, called IS70, turned out to be involved in cointegrate formation . We found one intact copy of IS70 and a small segment of IS70, respectively, at the junctions of R772 and Ti DNA . The absence of a complete second copy of IS70 is a likely explanation for the stability of the cointegrate plasmid . A procedure for site-directed mutagenesis of this cointegrate plasmid in E . coli is described . The effect of mutations in the Ti plasmid part can be studied subsequently by transferring the cointegrate into Agrobacterium tumefaciens . The advantage of this procedure for Ti plasmids over other methods used at present is discussed. Antimicrob Agents Chemother, 1983 Apr, 23(4), 598 - 602 Sulfur metabolism in the biosynthesis of monobactams; O'Sullivan J et al.; We studied the biosynthesis of monobactams with respect to sulfur metabolism in Chromobacterium violaceum, Acetobacter sp., and Agrobacterium radiobacter . All three organisms used inorganic sulfur for monobactam production . When sulfur-containing amino acids were assayed as a source of sulfur for monobactam production, C . violaceum used cystine but not cysteine or methionine, Acetobacter sp . used all three compounds, and A . radiobacter used none . 35S from cysteine, methionine, and sodium sulfate was incorporated into monobactam by Acetobacter sp . Cell-free extracts of all three organisms were shown to possess cysteine desulfhydrase activity . In Acetobacter sp., this activity was constitutive, required pyridoxal phosphate, and had a pH optimum of 9.5 . Extensive loss of 3H from L-{3-3H}cysteine was seen upon desulfhydration; no evidence of serine formation was found . Active sulfate was formed in cell-free extracts of A . radiobacter, and, since inorganic sulfur was used by all three organisms, it is likely that the sulfamate group of monobactams is produced via active sulfate. Cell, 1983 Apr, 32(4), 1057 - 67 Genetic analysis of T-DNA transcripts in nopaline crown galls; Joos H et al.; Plant crown gall tumor cells result from the insertion and expression of a defined DNA sequence, called T-DNA, which is derived from the Ti plasmid, harbored by Agrobacterium tumefaciens strains . To study the function of the genes of the T-DNA of the nopaline Ti plasmid, pTiC58, a collection of mutants was isolated so that T-DNA genes are inactivated either separately or in various combinations . It was found that no single T-DNA gene or T-region border is absolutely essential for stable tumor formation . We have identified the gene responsible for synthesis in transformed cells of the phosphorylated sugar, agrocinopine, and at least three additional genes controlling the morphology of plant tumors . Two of these latter genes work together to inhibit shoot formation and ensure efficient tumorous growth . Inactivation of these genes can be suppressed by the addition of auxins . The third gene inhibits root formation and appears to play a role in the cytokinin-independent growth of transformed cells . Mutants missing all three genes do not induce tumors, nor shoot or root formation, although the mutant T-DNA sequence is transferred to plant cells. Cell, 1983 Apr, 32(4), 1033 - 43 Regeneration of intact tobacco plants containing full length copies of genetically engineered T-DNA, and transmission of T-DNA to R1 progeny; Barton KA et al.; Cloned DNA sequences encoding yeast alcohol dehydrogenase and a bacterial neomycin phosphotransferase have been inserted into the T-DNA of Agrobacterium tumefaciens plasmid pTiT37 at the "rooty" locus . Transformation of tobacco stem segments with the engineered bacterial strains produced attenuated crown gall tumors that were capable of regeneration into intact, normal tobacco plants . The yeast gene and entire transferred DNA (T-DNA) were present in the regenerated plants in multiple copies, and nopaline was found in all tissues . The plants were fertile, and seedlings resulting from self-pollination also contained intact and multiple copies of the engineered T-DNA . Expression of nopaline in the germinated seedlings derived from one regenerated plant was variable and did not correlate with the levels of T-DNA present in the seedlings . Preliminary evidence indicates that nopaline in progeny of other similarly engineered plants is more uniform . The disarming of pTiT37 by insertions at the "rooty" locus thus appears to produce a useful gene vector for higher plants. Ann Microbiol (Paris), 1983 Mar-Apr, 134A(2), 141 - 7 {Demonstration of a metabolic property of Rhibozium meliloti usable for its classification}; Courtois B et al.; Fast-growing Rhizobium and Agrobacterium strans were studied for their behaviour towards two carbohydrates (glucose and fructose) according to the following criteria: acidification and soluble exopolysaccharide production by resting cells . R . meliloti and Agrobacterium spp . showed a similar behaviour: in glucose-containing medium, acidification occurred and few exopolysaccharides were produced, whereas in medium supplemented with fructose, a neutral pH was maintained and large amounts of exopolysaccharides were synthesized . Other fast-growing Rhizobium spp . did not acidify these media and produced variable amounts of exopolysacchrides. Proc Natl Acad Sci U S A, 1983 Mar, 80(6), 1660 - 4 Multiple mutations in the T region of the Agrobacterium tumefaciens tumor-inducing plasmid; Ream LW et al.; Three genetic loci affecting tumor morphology lie within pTiA6NC T-DNA: tms, tmr, and tml . Using deletions and multiple transposon insertions, we constructed tumor-inducing (Ti) plasmids representing every possible double and triple mutant combination . tms tmr and tms tmr tml mutants did not incite tumors on most plants and produced a very weak response on a few other hosts but tms tml and tmr tml mutants were virulent . Thus, either tms+ or tmr+ alone can promote significant tumor growth but tml+ by itself is not sufficient . On hosts where tms mutants induce tumors accompanied by shoot proliferation, addition of a tml mutation reduces or eliminates shoot proliferation, suggesting that tml+ promotes shoot development . The small calli incited by tms tmr and tms tmr tml mutants contain agropine, an indication that these plant cells incorporate T-DNA in the absence of substantial tumor growth. J Bacteriol, 1983 Mar, 153(3), 1535 - 42 Comparison of Ti plasmids from three different biotypes of Agrobacterium tumefaciens isolated from grapevines; Knauf VC et al.; Twenty-six plasmids from grapevine isolates of Agrobacterium tumefaciens were analyzed by SmaI fingerprinting and by hybridization of nick-translated DNA to DNA of another plasmid . These experiments established that octopine Ti plasmids are not highly conserved, although octopine Ti plasmids from biotype 1 A . tumefaciens strains appeared to be very similar . Octopine Ti plasmids from biotype 3 strains are more variable in terms of host range and SmaI fingerprints, but share extensive DNA homology . Fingerprints of nopaline Ti plasmids from strains of a given biotype resemble each other but not fingerprints of Ti plasmids from strains of the other two biotypes . The wide host range octopine Ti plasmid from the biotype 3 strain Ag86 shares more DNA homology with narrow host range Ti plasmids, nopaline Ti plasmids, and octopine catabolism plasmids than with the wide host range octopine Ti plasmid from biotype 1 strain 20/1 . pTiAg86 does share homology with the portion of pTi20/1 integrated and expressed in plant tumor cells . Since all wide host range Ti plasmids studied contain these sequences, we suggest that natural selection for a wide host range resulted in the presence of the common sequences in distantly related plasmids . The lack of homology between this "common DNA" and limited host range Ti plasmids shows that the DNA sequences per se are not required for tumorigenesis. J Bacteriol, 1983 Mar, 153(3), 1451 - 60 Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens; Pischl DL et al.; We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome . First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45 . We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5 . By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations . We also constructed isolates of A . tumefaciens containing an insertion of Tn5 in the chromosome . We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients . Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested . Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies . Donors were shown to transfer chromosomal markers in a polarized fashion . Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent . The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A . tumefaciens chromosomal functions. J Bacteriol, 1983 Feb, 153(2), 878 - 83 Mutational analysis of the virulence region of an Agrobacterium tumefaciens Ti plasmid; Klee HJ et al.; Forty-nine Tn3 and Tn5 transposition insertion mutations were introduced into the virulence region of the pTiA6NC plasmid of Agrobacterium tumefaciens . Five Tn5 transposition mutations from an earlier study (D . Garfinkel and E . Nester, J . Bacteriol . 144:732-743, 1980) were also mapped more accurately . These mutations defined five separate loci within the virulence region . Two Tn3 insertions into one of these loci, virA, result in a strain which is only weakly virulent; however, a Tn5 insertion into this locus eliminates virulence . One Tn5 insertion into another locus, virC, results in a strain which is weakly virulent . Two additional Tn5 insertions into this locus eliminate virulence . Insertions into the remaining three loci eliminate virulence entirely. Nucleic Acids Res, 1983 Jan 25, 11(2), 369 - 85 Structure and transcription of the nopaline synthase gene region of T-DNA; Bevan M et al.; We present the DNA sequence and plant-tumor transcription pattern of some 2400 base pairs from the right border region of pTi T37 DNA from the virulent Agrobacterium tumefaciens strain T37 . This region includes the entire transcription unit encompassing the nopaline synthase gene, together with parts of other transcription units . The strategy used to determine the sequence also produced two opposing series of defined, asymmetric deletions across the target DNA region, some of which may serve future purposes in the exploitation of this sequence, which is known to be expressed in a wide variety of host plant tissues. Nucleic Acids Res, 1983 Jan 11, 11(1), 159 - 74 Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors; Gelvin SB et al.; Methylation of the T-DNA in Agrobacterium tumefaciens and in four octopine-type (A6S/2, E9, 15955/1, 15955/01) and one nopaline-type (HT37#15) crown gall tumors was investigated using the isoschizomeric restriction endonucleases Msp I and Hpa II . T-DNA in the octopine-type Ti-plasmid pTiB6(806) was not methylated at the sequence 5'CCGG3' in Agrobacterium . With two possible exceptions, neither was the T-DNA of the nopaline-type Ti-plasmid pTiT37 methylated in the bacterium . In all tumor lines investigated, at least one copy of the T-DNA was not methylated . DNA methylation was not detected in the lines A6S/2, 15955/1, HT37#15, and the TL region of E9 . DNA methylation of some copies of TR in the E9 tumor line, and possibly in the 15955/01 line, was detected . The methylation of some copies of TR in the E9 line may indicate that not all copies of TR are transcribed in this tumor. J Mol Appl Genet, 1983, 2(2), 201 - 9 Structure of T-DNA in roots transformed by Agrobacterium rhizogenes; Byrne MC et al.; DNA isolated from hairy roots incited on Daucus carota by Agrobacterium strains harboring the Ri plasmid was probed with Ri plasmid fragments to identify plasmid sequences transferred and integrated into the plant genome . One hairy root line was incited by an A . rhizogenes natural isolate strain harboring two large plasmids in addition to the Ri plasmid . DNA from hairy roots incited by this strain contained a total of five nonidentical copies of T-DNA which together comprised a T-DNA complement representing a 34-42 kb segment of the Ri plasmid . A second hairy root line was incited by a Ti plasmidless A . tumefaciens strain into which the Ri plasmid had been conjugated . Hairy root DNA incited by this strain contained four identical T-DNA copies 17-18 kb in length and one truncated copy . T-DNA borders occurred at preferential sites of the Ri plasmid . T-DNA structure in these hairy root lines is similar to T-DNA structure in tumors incited by Ti plasmids of A . tumefaciens. Nucleic Acids Symp Ser, 1983, (12), 111 - 4 Relationship between adenylate cytokinin production and Ti plasmid of Agrobacterium tumefaciens; Sonoki S et al.; To know whether the tumor-inducing plasmid of Agrobacterium tumefaciens carries genetic information of the biosynthesis of cytokinins, the levels of 6-(3-methyl-2-butenyl-amino)purine (iPAde) and its 4-hydroxy derivative trans-zeatin (trans-Z) and its p-beta-D-ribofuranoside (trans-ZR) produced in media by wild-type virulent strain, plasmid-cured avirulent strain and the deletion mutant were compared . The highest levels of iPAde and trans-Z were found in the culture filtrate of late-log phase growth of plasmid-containing virulent strain, then the levels of iPAde and trans-Z were reduced rapidly at stationary phase . The plasmid-cured avirulent strain and deletion mutant had low levels of iPAde and trans-Z throughout the growth . Results obtained here showed Ti plasmid plays an important role in cytokinin biosynthesis. Mol Gen Genet, 1983, 191(1), 10 - 6 A functional map of the nopaline catabolism genes on the Ti plasmid of Agrobacterium tumefaciens C58; Schardl CL et al.; The nopaline catabolism (noc) genes are located in a 14.4 kb region on the pTiC58 plasmid of A . tumefaciens C58 . These genes permit the bacterium to grow on nopaline N2-(1,3-dicarboxylpropyl) arginine, a substrate produced in plant tumors initiated by strain C58 . The functions of the noc genes include the use of nopaline and L-ornithine as sole carbon and nitrogen sources . Using Tn5 insertional mutants, we have identified and mapped the positions of the genes that are responsible for nopaline catabolism (NopC), ornithine catabolism (OrnC) and nopaline uptake (NopU) . A polar relationship was found between these phenotypes, which extended leftward over the noc region to the T-region . The NopC mutants were also deficient in nopaline oxidase, an enzyme that liberates free arginine from nopaline . The noc region also encodes the synthesis of a periplasmic protein, n1 that was induced by nopaline . Tn5 insertional mutations and molecular cloning were used to map the n1 production locus . The recombinant plasmids, pSa4480 and pSa4481, containing the 8.9 kb right-hand end of the noc region, conferred n1 production when introduced into a pTi-free strain of A . tumefaciens . Production of n1 by the strains carrying these plasmids required nopaline induction . We have identified in toto three noc loci: nocB, nocC, and nocA, which confer n1 production, nopaline oxidase production and ornithine catabolism respectively . A model is proposed whereby the noc genes of pTiC58 are contained on a leftward reading operon in the order nocB, nocC, and nocA. J Mol Appl Genet, 1983, 2(2), 211 - 24 Nopaline Ti-plasmid, pTiT37, T-DNA insertions into a flax genome; Hepburn AG et al.; The FT37/1 plant tumor line induced on flax epicotyls by T37, a nopaline strain of Agrobacterium tumefaciens, contains multiple copies of the pTiT37, T-DNA . There are three to four distinct full-length insertions and one tandem insertion . Allowing for the different copy numbers of the inserts, this amounts to seven to eight T-DNA copies per genome unit . The genome unit in this case is the haploid DNA value (7 X 10(8) bp) which predicts a T-DNA copy number of 14-16 per diploid cell . Three novel types of abnormal insertion are also present . FIL (one copy per basic genome) comprises 3.04-4.47 kb of T-DNA derived from the left end, with a normal left border and an abnormal right border . FIR (four copies per basic genome) comprises 5.88-6.47 kb of T-DNA derived from the right end, with a normal right border and an abnormal left border . The third abnormality is represented by fragment "X," a HindIII fragment of 4.90 kb which contains homology with several noncontiguous regions of the T-DNA and which may derive from a tandem insertion . Of the two possible left-border sites (primary and secondary) in which fusion with plant DNA sequences has been observed, only the primary is used. Eur J Biochem, 1983 Jan 1, 129(3), 603 - 14 The complete amino-acid sequence of the low-spin class II cytochrome c-556 from Agrobacterium tumefaciens strain B2a; Tempst P et al.; The amino acid sequence of the soluble monohaem cytochrome c-556 from Agrobacterium tumefaciens, strain B2a, has been determined . The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin . Sequencing of the various peptides was achieved by a combination of manual dansyl-Edman degradation and automatic liquid-phase sequence analysis . The main characteristic of this cytochrome is that the haem-binding sequence Cys-Xaa-Yaa-Cys-His occurs in the C-terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C-terminal lysine-122 . As such, the protein belongs to cytochrome c sequence class II (sensu Ambler) . The cytochrome c-556 is the first example known of a class II cytochrome of the low-spin type isolated from an obligate aerobic organism. J Mol Appl Genet, 1983, 2(3), 315 - 29 The role of cytosine methylation in the control of nopaline synthase gene expression in a plant tumor; Hepburn AG et al.; The FT37/1 tumor line induced by Agrobacterium tumefaciens on flax epicotyls contains 22-24 copies of the T-DNA encoded nopaline synthase gene per cell . All the gene copies are methylated to some extent but the methylation is not uniform, nor does it reflect the methylation level of the flanking plant DNA . This extensive methylation correlates with an extremely low level of expression of the nopaline synthase gene . Treatment of the tumor line with the in vivo demethylating drug 5-azacytidine at a concentration of 3 X 10(-5) M, resulted in the demethylation of, on average, one copy of the nopaline synthase gene per cell . This demethylation was paralleled by an increase in the transcription of the gene and indicates that cytosine methylation is capable of suppressing the expression of plant genes in vivo. Cell, 1982 Dec, 31(3 Pt 2), 605 - 12 Variation in hormone autonomy and regenerative potential of cells transformed by strain A66 of Agrobacterium tumefaciens; Binns AN et al.; Mutant Agrobacterium tumefaciens strain A66 is shown to differ from its wild-type progenitor (strain A6) by a spontaneous 2.7 kb DNA insert into the T-DNA region of its Ti plasmid . Tobacco stems transformed by A66 exhibit an attenuated response characterized by slow growth and shoot proliferation . Clonal analysis demonstrates that this response is due to an alteration in the growth and regenerative potential of transformed cells, rather than to variation in the frequency of fully autonomous cells within the primary tumor . Cloned A66 transformed tobacco cells exhibit an auxin requirement for growth that can be overcome by shoot proliferation . Other host species, however, may complement the A66 mutation yielding fully auxin-independent tumors when transformed by this bacterium. J Bacteriol, 1982 Dec, 152(3), 1265 - 75 Agrobacterium tumefaciens mutants affected in attachment to plant cells; Douglas CJ et al.; An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A . tumefaciens codes for a specific ability of this bacterium to attach to plant cells . This ability is associated with tumorigenesis by A . tumefaciens, the ability of avirulent A . tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages . A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells . The attachment of A . tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells . Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806 . Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability . In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages . The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages . Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process . Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated. J Nat Prod, 1982 Nov-Dec, 45(6), 679 - 86 Modification and evaluation of the potato disc assay and antitumor screening of euphorbiaceae seeds; Ferrigni NR et al.; Galsky et al . (10, 11) have reported that the inhibition and growth of crown gall tumors, initiated on potato discs by Agrobacterium tumefaciens, apparently has good agreement with 3PS in vivo antileukemic activity in mice . We have now modified and evaluated this assay for its potential use as a prescreen and fractionation monitor of plant extracts for 3PS activity . The modified assay was performed on a series of natural compounds and plant extracts (known to have 3PS activity) and on extracts of seeds of 41 Euphorbiaceae species . The results suggest that the potato disc assay is a safe, simple, rapid, in-house, low cost, prescreen for 3PS antitumor activity; it detects some false positives, but few false negatives; it is statistically much more predictive (p = 0.002) of 3PS activity than either the 9KB (p = 0.140) or the 9PS (p = 0.114) cytotoxicity assays . Its extended use could easily obviate the expense and extensive need for animals in the initial stages of antitumor screening and plant fractionation. Agents Actions, 1982 Oct, 12(4), 543 - 51 Antimicrobial, insect sterilizing and ovicidal activity of some oxo-vanadium(IV) and oxo-vanadium(V) complexes; Datta S et al.; Twenty-three newly synthesized mixed-ligand complexes of oxo-vanadium(IV) and oxo-vanadium(V) were studied for antimicrobial activity . Eight of these complexes were found to have microbicidal properties . The complexes {NH4}{VO(gl)2}H2O (gl-H2 = glycolic acid) and {VO(ACOAP)(acac-H)}H20 (ACOAP-H2=Schiff base of acetylacetone and orthoaminophenol, acac-H=acetyl-acetone) show broad bactericidal spectra, while the complexes {VO(ACSAM)2}OH (ACSAM-H = Schiff base of acetylacetone and sulphanilamide) and {VO(CSSAM-H)2}H2O (CSSAM-H =Schiff base of 3-carboxy salicylaldehyde and sulphanilamide) possess pronounced antidermatophytic properties . The latter is inhibitory to plant pathogenic fungi as well . Plant tumour producing Agrobacterium tumefaciens is effectively inhibited in vitro by the complex {VO(ACTSC-Na)(acac)}H2O (ACTSC-H2 = condensation product of acetylacetone and thiosemicarbazide) . Minimum inhibitory concentrations of all the active complexes are within the values of 0.125-2.00 mg/ml . Out of the 7 active complexes tested for 50% inhibition of conidial germination of Helminthosporium oryzae, a rice plant pathogen, only 1 complex, viz . {VO(acac) (ACACAACD)} (ACACAACD(H)NH4 equal Schiff base of acetylacetone and ammonium 2-amino-1-cyclopentene-1-dithiocarboxylate) shows a positive result . The effective concentration is 0.55 mg/ml . Three vanadium complexes were tested for insect sterilant and ovicidal properties on the red cotton bug, Dysdercus koenigi . The complex {VO(HASA-Na) (acac)}H2O (HASA-H2 = Schiff base of orthohydroxyacetophenone and anthr anilic acid) was found to be a suitable male sterilant. Cell, 1982 Sep, 30(2), 589 - 97 T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum; Ooms G et al.; Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells . Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous . Detailed analysis of T-DNA organization showed that TL- or "core" T-DNA was always present at one or two copies per diploid genome . However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated . TR-DNA was not detected . The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived. J Antibiot (Tokyo), 1982 Jul, 35(7), 814 - 21 Distribution of beta-lactam and beta-lactone producing bacteria in nature; Wells JS et al.; Over one million bacteria were isolated from a large variety of soil, plant and water samples collected from different environments and examined in an extremely sensitive and highly specific screen for beta-lactam production . A group of seven related monocyclic beta-lactams (monobactams) were isolated from strains representing four genera-Agrobacterium, Chromobacterium, Gluconobacter and Pseudomonas . Monobactam-producing strains of Agrobacterium and Pseudomonas were isolated only rarely . Producing strains of Chromobacterium were isolated from a relatively limited number of habitats while the Gluconobacter strains appeared to be widespread in nature . In addition, three closely related beta-lactone-containing molecules were isolated from strains representing three genera-Arthrobacter, Bacillus and Pseudomonas . The Bacillus and Pseudomonas strains were isolated infrequently but from a variety of samples . The producing strain of Arthrobacter was isolated only once. Cell, 1982 Jul, 29(3), 1005 - 14 DNA from the A6S/2 crown gall tumor contains scrambled Ti-plasmid sequences near its junctions with plant DNA; Simpson RB et al.; The A6S/2 tumor incited on tobacco by Agrobacterium tumefaciens harboring the octopine-type A6 Ti plasmid contains one insert of Ti-plasmid sequences (the T DNA) . This 13 kb insert is derived from a colinear sequence in the Ti plasmid (the T region) and becomes attached to plant DNA in the nucleus of the host cell . We have determined the DNA sequence encompassing the left end of the T region of the A6 Ti plasmid and the corresponding portion of the A6S/2 T DNA . The two sequences are identical for at least 806 bp . To the left of the divergence point, the tumor contains five partially overlapping sequences that are direct or inverted repeats of sequences to the right of the divergence point . The Ti plasmid contains only the right member of each of these repeats . We have also performed heteroduplex studies that indicate that this T DNA has a 520 bp inverted repeat of an internal sequence at the right end near its junction with plant DNA . The repeated sequences near the ends of the T DNA resemble the repeats of adenovirus type 12 sequences found near its junction with host DNA . We discuss data suggesting that the 23 bp to the immediate right of the divergence point of the A6 left junction form a site important in some step in the transfer of T-region DNA from the bacteria to the plant. J Bacteriol, 1982 Jul, 151(1), 343 - 50 Characteristics of Ti plasmids from broad-host-range and ecologically specific biotype 2 and 3 strains of Agrobacterium tumefaciens; Perry KL et al.; Agrobacterium tumefaciens strains isolated from crown gall tumors on grapevines in California were consistently of the biotype 3 group . All 11 of these strains were limited in their host range and harbored Ti plasmids with molecular masses between 119 and 142 megadaltons (Mdal) as well as a larger cryptic plasmid of greater than 200 Mdal; occasionally a smaller cryptic plasmid of 65 Mdal was also present . Ti plasmids o these strains have DNA sequences in common with Ti plasmids of octopine and nopaline strains belonging to the biotype 1 group and exhibited sequence homologies with the conserved region of the T-DNA . Ten of the 11 strains utilized octopine as a sole source of carbon and nitrogen and 3 strains catabolized both octopine and nopaline, whereas 1 strain catabolized only nopaline . All of these strains were resistant to the bacteriocin agrocin-84, except one grapevine strain that belonged to the biotype 1 group and was agrocin sensitive; it is also differed in its plasmid and virulence characteristics . Isolations from Rubus ursinus ollalieberry galls yielded exclusively biotype 2 strains . These strans were insensitive to agrocin-84, utilized nopaline as a sole carbon and nitrogen source, and were highly virulent on all host plants tested . They contained Ti plasmids ranging between 100 and 130 Mdal and occasionally a cryptic plasmid of 69 Mdal . Their Ti plasmids have DNA sequences in common with Ti plasmids of biotype 1 strains and with the conserved region of the T-DNA. Arch Microbiol, 1982 May, 131(3), 271 - 7 R68.45 mediated chromosomal gene transfer in Agrobacterium tumefaciens; Bryan J et al.; A large plasmid enables its host Agrobacterium tumefaciens to cause tumorous condition in a wide variety of dicotyledonous plants{see Ooms et al . Gene 14:33--50 (1981) )) . The location and role of chromosomal genes in this phenomenon are not known . As the first stage in studying this aspect, a project was initiated to investigate the chromosomal genetics of the bacterium . R68.45, a P group plasmid, was chosen as a transmission agent . After a preliminary assessment it was decided to use C58 as a standard strain to carry out the mapping . The plasmid itself, as judged by the presence of antibiotic markers, appears to be stable in A . tumefaciens; its ability to promote chromosomal mobilisation, however, remains only in 60--80% transconjugants . Good Agrobacterium donors are capable of transferring chromosomal genes at a frequency varying between 10(-5) to 10(-6) per recipient . The recombinants are stable even under non-selective conditions . A linear linkage map consisting of 16 markers was built using coinheritance frequencies obtained from 21 four-point crosses. J Bacteriol, 1982 Apr, 150(1), 327 - 31 Complementation analysis of Agrobacterium tumefaciens Ti plasmid mutations affecting oncogenicity; Klee HJ et al.; A wide host range cosmid vector has been constructed by insertion of the lambda cos site into the plasmid pRK2501 . This cosmid, which is maintained in Agrobacterium tumefaciens and is compatible with the Ti plasmid, has been used to make a clone bank of the A . tumefaciens pTiA6 plasmid . Several pTiA6 cosmids have been used to complement Tn5-induced Ti plasmid mutations . Five avirulent mutations which map outside of the region of the plasmid maintained in plant tumours (T-DNA) could be complemented in a trans orientation . Two mutations which are located on a single HpaI restriction fragment outside of the T-DNA, as well as three mutations which map within the T-DNA region, could not be complemented in a trans orientation in a REC- host. J Bacteriol, 1982 Apr, 150(1), 395 - 7 Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti; Hooykaas PJ et al.; Eight different derivatives of R plasmid RP1 with thermosensitive mutations affecting maintenance in Escherichia coli and Pseudomonas aeruginosa were introduce into Rhizobium meliloti . None of the plasmids showed a thermosensitive character in R . meliloti . On the other hand, a certain deletion mutation in RP1 was found to cause plasmid instability in rhizobia and agrobacteria, but not in E . coli. Antimicrob Agents Chemother, 1982 Apr, 21(4), 558 - 64 Biosynthesis of monobactam compounds: origin of the carbon atoms in the beta-lactam ring; O'Sullivan J et al.; The biosynthesis of monobactams by strains of Chromobacterium violaceum, Acetobacter sp., and Agrobacterium radiobacter was studied . Monobactams were produced during logarithmic growth by C . violaceum and Acetobacter sp . and during late log growth on glycerol and in stationary phase by A . radiobacter . The addition of various amino acids failed to significantly stimulate monobactam production in any of the producing organisms . Several 14C-amino acids and pyruvate were incorporated in vivo into monobactams . Serine, glycine, and cysteine were better incorporated than alanine or aspartate, whereas an excess of nonradioactive serine depressed the incorporation of labelled cysteine, glycine, and pyruvate . A comparison of {1-14C} glycine and {2-14C} glycine incorporation data suggests that glycine was first converted to serine . With a mixture of {U-14C{serine and {3-3H}serine, C . violaceum synthesized a monobactam with a complete retention of tritium, whereas with a {U-14C} cystine and {3-3H} cystine mixture, there was an extensive loss of C-3 tritium . Acetobacter sp . and A . radiobacter also utilized the double-labeled serine without the loss of tritium in their respective monobactams . It appears, therefore that in the three organisms, the carbon atoms of the beta-lactam ring of the monobactam are derived directly from serine without the loss of the C-3 hydrogen atoms, probably by an SN2 ring closure mechanism . With {methyl-14C} methionine, most of the radioactivity in the monobactam from Acetobacter sp . was in the methyl moiety of the beta-lactam ring methoxyl group. Nucleic Acids Res, 1982 Mar 11, 10(5), 1679 - 89 T-DNA of pTi-15955 from Agrobacterium tumefaciens is transcribed into a minimum of seven polyadenylated RNAs in a sunflower crown gall tumor; Murai N et al.; Northern blot hybridization analysis of polysomal polyadenylated RNA isolated from sunflower crown gall tumor PSCG-15955 demonstrated that a minimum of seven RNAs were transcribed from T-DNA of pTi-15955 from Agrobacterium tumefaciens . The sizes of the T-DNA transcripts were 1.8, 1.6, 1.5, 1.1, 1.0 kilo bases (kb) and two transcripts of 0.8 kb long . The relative abundance of these polyadenylated RNAs varied greatly, the 1.0 kb RNA being the most abundant and the 1.6 kb RNA being the least abundant . Assignment of map locations of the seven polyadenylated RNAs indicated that the conserved region of T-DNA which may play a central role in tumorigenesis contained four RNAs of 1.8, 1.1, 0.8(a) and a portion of 0.8(b) kb long. Kosm Biol Aviakosm Med, 1982 Mar-Apr, 16(2), 45 - 8 {Physiological and biochemical characteristics of the cells of a carrot gall tumor developing in weightlessness}; Tairbekov MG et al.; The paper presents the results of experiments with the carrot tissues infected with Agrobacterium tumefaciens flown onboard the biosatellite Cosmos-1129 in cooperation with the US scientists . Postflight, the respiratory activity of tumour cells was determined and K+ and Na+ permeability of cell membranes was measured . The resulting data give evidence that in weightlessness the development of the carrot gall tumour is accompanied by changes in the above physiological and biochemical parameters . The changes are, however, within the physiological limits, leading to no pathologies of the whole cell. J Bacteriol, 1982 Mar, 149(3), 1129 - 34 Relationship between Nif plasmids of fast-growing Rhizobium species and Ti plasmids of Agrobacterium tumefaciens; Prakash RK et al.; By use of the Southern blot hybridization technique the extent of DNA homology was determined between the Nif plasmid of a number of fast-growing Rhizobium species and Ti plasmids of the octopine (pTiAch5) and nopaline (pTiC58) type . DNA sequences common to these plasmids were located on functional maps of the Ti plasmids . No homology between Nif plasmids and the T region of Ti plasmids was detected. Eur J Biochem, 1982 Mar, 123(1), 73 - 80 Metal coordination centres of class II cytochromes c; Moore GR et al.; The class II cytochromes Rhodospirillum molischianum cytochrome c', Rhodopseudomonas palustris cytochrome C556 and Agrobacterium tumefaciens (B2a) cytochrome c556 have been investigated with a variety of spectroscopic techniques . The cytochrome c' was found to be high-spin and the two cytochromes c556 were found to be mainly low-spin and sx-coordinate with the fifth and sixth ligands being histidine and methionine . The implications of the different types of iron coordination are discussed. J Bacteriol, 1982 Feb, 149(2), 771 - 4 Ferrisiderophore reductase activity in Agrobacterium tumefaciens; Lodge JS et al.; Reduction of the iron in ferriagrobactin by the cytoplasmic fraction of Agrobacterium tumefaciens strictly required NaDH as the reductant . Addition of flavin mononucleotide and anaerobic conditions were necessary for the reaction; when added with flavin mononucleotide, magnesium was stimulatory . This ferrisiderophore reductase activity may be a part of the iron assimilation process in A . tumefaciens. Infect Immun, 1982 Feb, 35(2), 528 - 32 Pulmonary toxicity of endotoxins: comparison of lipopolysaccharides from various bacterial species; Helander I et al.; Lipopolysaccharides from three gram-negative bacteria isolated from bale cotton and piggery air were analyzed for their chemical composition, and their pulmonary toxicity for guinea pigs, lethal toxicity for mice, and pyrogenicity for rabbits were measured . Lipopolysaccharides from Enterobacter agglomerans and Citrobacter freundii had closely related chemical compositions; both were pyrogenic for rabbits and caused a dose-dependent influx of polymorphonuclear leukocytes into the airways of guinea pigs . The lethal toxicities of these lipopolysaccharides in mice were comparable to that of Salmonella typhimurium lipopolysaccharide, which was used as a reference . Lipopolysaccharide from Agrobacterium sp . was chemically different from those of E . agglomerans and C . freundii, did not induce any influx of polymorphonuclear leukocytes, and was only weakly toxic or pyrogenic . The low biological activity of the agrobacterial lipopolysaccharide may be due to its different chemical composition. J Mol Appl Genet, 1982, 1(6), 561 - 73 Nopaline synthase: transcript mapping and DNA sequence; Depicker A et al.; The DNA sequence of the nopaline synthase gene (nos) from Agrobacterium tumefaciens Ti plasmid pTiT37 and adjacent regions up to the right border of the T-DNA was determined . The 5' and 3' termini of the polyadenylated nos mRNA, isolated from a T37 tobacco teratoma tumor line, were localized by S1 mapping . The final mRNA is unspliced, encoded by a region of about 1450 bp, and specifies an open reading frame of 413 amino acids . Potential transcriptional signals in the 5' flanking DNA, such as CATAAA ("TATA box") and GGTCACTAT ("CAT box"), bear close resemblance to other eukaryotic promoters . Two putative polyadenylation signals, AATAAA and AATAAT, are found about 135 and 50 bp from the 3' end, respectively . This study may provide information for the development of expression vectors for genes in plant cells; moreover, the structural gene can be used as an easy screenable marker. J Mol Appl Genet, 1982, 1(6), 539 - 46 Multiple transcripts of T-DNA detected in nopaline crown gall tumors; Bevan MW et al.; Crown gall plant tumors contain neoplastic cells transformed by incorporation of a foreign DNA element, T-DNA, derived from a large tumor-inducing plasmid in the inciting Agrobacterium strain . T-DNA is covalently joined to the nuclear DNA of the tumor cell, and RNA transcripts from T-DNA are present in polyadenylated form on polysomes . This paper presents a detailed analysis of those parts of T-DNA transcribed in a nopaline-type tobacco teratoma, BT37, whose T-DNA has been mapped and cloned . Northern blots of polyA+ RNA were probed with 21 different nick-translated T-DNA fragments, and at least 13 well-defined transcripts were visualized. J Mol Appl Genet, 1982, 1(6), 499 - 511 Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene; De Greve H et al.; We have determined the complete nucleotide sequence of the gene for the crown gall enzyme, octopine synthase . The sequence was derived from cloned fragments of the Agrobacterium tumefaciens Ti plasmid Ach5 . It displayed a continuous open reading frame encoding a polypeptide chain of 358 amino acids . The nucleotide positions corresponding to the 5' end and poly(A) addition site of the mature octopine synthase mRNA from a tobacco tumor cell line were determined by S1 nuclease mapping . Two sequences closely resembling transcriptional control regions found in eukaryotic genes transcribed by RNA polymerase II were identified in the flanking genomic DNA: a sequence 5'-TATTTAAA-3' was located 32 base pairs upstream from the initiation site of transcription, and a hexanucleotide 5'-AATAAT-3' occurred 17 base pairs in front of the poly(A) addition site . No Shine-Dalgarno sequence was present in the untranslated 5' leader sequence . The observations indicate that this DNA sequence, although naturally carried by a bacterial plasmid, is programmed as a functional plant gene. Microbios, 1982, 34(136), 113 - 32 Passage of bacterial DNA into host cells during in vitro transformation of Nicotiana tabacum by Agrobacterium tumefaciens; Sigee DC et al.; Tumorigenic and non-tumorigenic strains of Agrobacterium tumefaciens were labelled with tritiated thymidine and added separately to suspension cultures of X-D line habituated tobacco cells . The subsequent association of bacteria with plant host cells and the passage of bacteria and bacterial DNA into these cells was examined by conventional light and electron microscopy and light microscope autoradiography . The results suggest that both strains of bacterium enter damaged host cells (via wall lesions), where they remain restricted to cell vacuoles . By 48 h, the radioactive label has spread from the vacuoles of infiltrated cells to the non-vacuolar cytoplasm of the whole callus tissue . Tumorigenic bacteria differ from the non-tumorigenic strain in promoting a significant increase in host cell nuclear volume, which is paralleled by clear and specific nuclear labelling . It is proposed that tumorigenic DNA, but not non-tumorigenic DNA, is specifically incorporated into, and activates, the host cell nucleus. J Mol Appl Genet, 1982, 1(4), 361 - 70 Tumor induction by Agrobacterium tumefaciens: analysis of the boundaries of T-DNA; Zambryski P et al.; Molecular cloning has been used to isolate the ends of that portion of the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens which has been designated T-DNA and which has been transferred to the genome of tobacco crown gall tumor cells . Analysis of the DNA sequences of the plant border clones compared with the corresponding sequences of the Ti plasmid suggests that the mechanism of transferred DNA integration and subsequent stabilization is precise at the right border and imprecise on the left . The T-DNA junction occurs within a variation of a single base pair (bp) on the right but varies over at least 70 bp on the left . In addition, there are several sequences which are repeated near the ends of the T-DNA region in the Ti plasmid . Seemingly, there is no specificity with regard to the site of integration in the plant genome. J Biochem (Tokyo), 1982 Jan, 91(1), 283 - 9 D-glucosaminate dehydratase from Agrobacterium radiobacter . Physicochemical and enzymological properties; Iwamoto R et al.; The bacterial distribution of D-glucosaminate dehydratase {EC 4.2.1.26} was investigated and Agrobacterium radiobacter (IAM 1526) was found to have the highest enzyme activity . The enzyme was formed inducibly in a glycerol-urea medium by D-glucosamine, D-galactosamine, and D-glucosamine, but not by D-mannosamine . The enzyme purified from the cells grown in the glucosamine-glycerol-urea medium was shown to be homogeneous by ultracentrifugation . The molecular weight was determined to be about 66,000 by the sedimentation equilibrium method, and 72,800 by the gel permeation chromatography low-angle light scattering method . The pH optimum is 8.3-9.0 . The enzyme catalyzed the dehydration of D-glucosaminate (relative activity: 100, Km: 2.8 mM), D-galactosaminate (31.5, 5.0 mM), D-mannosaminate (17.5, 29 mM), D-threonine (5.1, 4.8 mM), D-serine (3.2, 0.026 mM), and L-serine (1.1, ND), but not L-threonine . The reverse reaction does not occur . The enzyme is inhibited by typical inhibitors of pyridoxal 5'-phosphate enzymes, such as L'penicillamine, and also by carbonyl reagents, thiol reagents, divalent metals, and several D-amino acids and D-amino sugars. Infect Immun, 1982 Jan, 35(1), 359 - 62 Ultrastructure of Gram-negative cotton bacteria with different pulmonary toxicities; Lounatmaa K et al.; The shedding of outer membrane material was observed by electron microscopy in typical gram-negative cotton bacteria, except Agrobacterium sp . This finding is in accordance with the relative pulmonary toxicities of these bacteria . The presence of capsules did not seem to be correlated with the acute pulmonary toxicity of the bacteria. Mol Gen Genet, 1982, 188(3), 425 - 32 Indoleacetic acid complementation and its relation to host range specifying genes on the Ti plasmid of Agrobacterium tumefaciens; Kao JC et al.; Host range variations were noted when 23 wild-type strains of Agrobacterium tumefaciens were tested on 27 different plant species . Because we have shown previously that host range specificity is conferred by the pTi plasmid, these variations in host specificity implicated genetic differences among pTi plasmids within the A . tumefaciens population that was tested . Host specificity was independent of the type of opine utilized and biotype of the strain used . These data suggested that separate genetic determinants operate for host specificity . This hypothesis was confirmed by Tn5 mutagenesis of the pTi plasmid, which generated mutants affected in host specificity . The regions of host specifying genes were located by displacement analysis of mutant pTi-plasmid-DNA restriction fragments . There are at least two sites on the pTiC58 plasmid: one within the T-region and the other about 75-77 kb to the right of this region . Mutations within the T-region were chemically complemented by indoleacetic acid, which restored the host range of the mutants . Such complementations were not observed with mutants outside the T-region. Mol Gen Genet, 1982, 188(3), 418 - 24 Units of genetic expression in the virulence region of a plant tumor-inducing plasmid of Agrobacterium tumefaciens; Iyer VN et al.; The effect of a large number of Tn3 insertions in the vir region of the Ti plasmid pTiA6NC on the virulence of Agrobacterium was determined . The Vir- insertions were mapped in three of the five loci that have been defined previously . Merodiploid Rec- strains carrying one insertion mutation on the Ti plasmid and another insertion mutation (or the homologous wild-type region) on a compatible plasmid were constructed and used in complementation tests for virulence in test plants . This analysis has revealed that there are ten units of gene expression, presumably transcription units in the vir region . Mutation in one of these units is confirmed to be dominant while those in all others are recessive . Co-infection of test plants with pairs of insertion mutants did not restore virulence. Annu Rev Genet, 1982, 16, 357 - 84 T-DNA of the Agrobacterium Ti and Ri plasmids; Bevan MW et al.; The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation . It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes . The opine synthases are enzymes encoded in T-DNA that function in the plant cell . Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector . What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A . tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes. Mol Gen Genet, 1982, 186(1), 10 - 5 Genetic map of the crown gall suppressive IncW plasmid pSa; Tait RC et al.; A genetic map of the W incompatibility group plasmid pSa has been prepared through the construction of deletion derivatives of pSa and the cloning of various fragments of pSa in pBR322 . Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to chloramphenicol, sulfonamides, spectinomycin, streptomycin, kanamycin, gentamycin, and tobramycin . Information sufficient for the replication of the plasmid in both Escherichia coli and Agrobacterium tumefaciens is contained within a 4 kilobase pair region . Two regions have been identified as involved in the transfer of the plasmid; one of these regions is also involved in the inhibition of oncogenesis by pSa when it is present in an oncogenic strain of A . tumefaciens . Certain of the deletion derivatives of pSa are potential vectors for the cloning and analysis of A . tumefaciens Ti plasmid DNA. Acta Microbiol Pol, 1982, 31(2), 145 - 51 Studies on superoxide dismutase activities in virulent and avirulent strains of Agrobacterium tumefaciens and also in normal and crown gall tumor cells of Bryophyllum calycinum; Banerjee D et al.; Superoxide dismutase activity in virulent strains of Agrobacterium tumefaciens was found to be higher than that in avirulent strains . Polyacrylamide gel electrophoresis revealed two isoenzymes in both these strains . These isoenzymes are suggested to be iron and manganese containing superoxide dismutases . Crown gall tumor cells of the plant Bryophyllum calycinum were found to have higher superoxide dismutase activity than the normal plant cells . Polyacrylamide gel electrophoresis revealed two isoenzymes in both normal and crown gall tumor cells . Advantages of the higher superoxide dismutase activities in respect of the survival of virulent strains of A . tumefaciens and crown gall tumor growth have been discussed. Nucleic Acids Res, 1981 Dec 21, 9(24), 6763 - 72 A coupled transcription-translation system from Agrobacterium tumefaciens and its application to study Ti plasmid expression in vitro; Kartasova T et al.; A coupled transcription-translation system was isolated from A . tumefaciens . Expression of plasmids pBR322 and pKT212 from E.coli, cloned fragments of Ti plasmid (plasmids pSS155 and pSS156) and Ti plasmid derivatives pAL2802, pAL2811, pAL2821 and pAL2832 was analysed in an A . tumefaciens cell-free system and compared with their expression in an E.coli cell-free system . New proteins of 41K and 44K appeared in A tumefaciens extracts, as the result of Ti plasmid expression . These proteins were not found in E.coli extracts. Gene, 1981 Nov, 15(2-3), 279 - 83 Genetic transformation of Rhizobium meliloti by plasmid DNA; Selvaraj G et al.; A procedure for the genetic transformation of Rhizobium meliloti by plasmid DNA is described . It is an adaptation of a procedure used originally for Escherichia coli and later for Agrobacterium tumefaciens . Depending on the R . meliloti isolate used as the recipient, the efficiency of transformation was in the range of 7 x 10(1) to 3.7 x 10(3) per microgram of plasmid pRK248 DNA and 10(-6) to 10(-9) per viable cell . A number of different naturally occurring strains could be transformed . Plasmids related to pRK248 and varying in size from 9.6 to 56 kb could be used, suggesting that the procedure will be useful for molecular cloning of genes in R . meliloti. Biochemistry, 1981 Oct 13, 20(21), 6097 - 102 Agrobacterium tumefaciens RNA polymerase: a new purification procedure and a study of the stable binding sites on homologous deoxyribonucleic acid; Cardarelli M et al.; RNA polymerase (RNA nucleotidyltransferase, EC 2.7.7.6) of Agrobacterium tumefaciens has been purified according to a fast and efficient procedure . The method involves only two chromatographic steps and yields a highly active enzyme . The RNA polymerase was studied with respect to the ability to bind its homologous genome . A . tumefaciens deoxyribonucleic acid (DNA) binds the enzyme even when fragmented at undergenic size (300 base pairs) . The general binding is unspecific and very labile at low concentrations of heparin (0.66 micrograms/mL) . The number and distribution of the stable binding sites, class A sites {Hinkle, D., & Chamberlin, M . J . (1972) J . Mol . Biol . 70, 157-185}, have been calculated from the heparin-induced dissociation kinetics of binary complexes formed between the enzyme and DNA fragments of various sizes . A total of 3.5 x 10(3) class A sites (forming binary complexes with a half-life of 16.6 min) are present on A . tumefaciens genome, a large number of which show a distribution of 800-1000 base pairs . The rest have a more widely spaced distribution . The interactions between Escherichia coli RNA polymerase and the A . tumefaciens template have also been examined, and it has been observed that E . coli holoenzyme forms stable complexes with a shorter half-life and recognizes a lower number of class A sites on A . tumefaciens genome. Biochemistry, 1981 Oct 13, 20(21), 6012 - 7 Distribution of cytokinin-active nucleosides in isoaccepting transfer ribonucleic acids from Agrobacterium tumefaciens; Morris RO et al.; The cytokinin-active isoprenoid nucleosides of Agrobacterium tumefaciens transfer ribonucleic acid were identified by high-pressure liquid chromatography, permethylation, and mass spectroscopy . Besides the expected 6-{(3-methylbut-2-enyl)amino}-9-(beta-D-ribofuranosyl)purine (i6A) and its 2-methylthio derivative (ms2i6A), substantial amounts of cis- and trans-ribosylzeatin (io6A) and cis-2-(methylthio)ribosylzeatin (c-ms2io6A) were present . These hydroxylated side chain derivatives are normally characteristic of plant tRNA . Fractionation of the total bacterial tRNA on BD-cellulose and RPC-5 allowed isolation of purified iso-accepting species whose cytokinin nucleoside contents were then determined . Distribution of the isoprenoid nucleosides among the U-group tRNA species was not uniform . cis-Ribosylzeatin was found almost exclusively in one tRNASer while ms2io6A was found predominantly in tRNAPhe, tRNASer, and tRNATyr . Not all cytokinin-active species were found in every member of the U-group tRNAs . The only species present in tRNATrp was i6A; it contained no zeatin derivatives . The hydroxylation and methylthiolation processes appear to be highly specific and dependent upon tRNA structure or sequence. Cell, 1981 Jun, 24(3), 719 - 27 Retention of tumor markers in F1 progeny plants from in vitro induced octopine and nopaline tumor tissues; Wullems GJ et al.; Tumorous tobacco shoots have been derived from callus tissues produced by Agrobacterium tumefaciens--induced transformation of tobacco protoplasts and by fusion of normal protoplasts with those from crown gall tumors . The continued presence of T-DNA sequences in shoots is directly demonstrated by Southern blotting and is also revealed by the presence of the tumor markers octopine and nopaline . When grafted onto normal tobacco plants, both octopine- and nopaline-type shoots (including those from somatic hybrids) produced flowers and set seed . Germination of these seeds gave F1 progeny that showed retention of morphological markers of their parental shoots, and one seedling retained the ability to synthesize nopaline . The data demonstrate that T-DNA markers can be retained during meiosis and are expressed in F1 plants. Gene, 1981 Jun-Jul, 14(1-2), 33 - 50 Grown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions; Ooms G et al.; Ti plasmid mutants derived from Agrobacterium tumefaciens strain Ach5 that induce tumors of abnormal morphology have been analyzed . On tobacco, A . tumefaciens mutant strain LBA4060 induces tumors that specifically give rise to shoots . Shoots continue to grow from in vitro cultured bacteria-free tumor tissue derived from such tumors . The mutant character is shown to be correlated with the insertion of an A . tumefaciens IS element, IS60, into the left arm of the T-region of the octopine Ti plasmid . Evidence is presented showing that IS60 is transferred into the plant cell DNA as part of the T-DNA . A second Ti plasmid insertion mutant A . tumefaciens strain LBA4210, with a Tn904 transposon in the center of the T-region, induces tumors that specifically exhibit a root development on tobacco plants . T-DNA has been detected in sterile amorphous crown-gall tissue derived from these tumors . The transposon Tn904 insertion was shown to result a changed "core" T-DNA . Abnormal tumor morphologies induced by these mutant strains have been observed also on Kalanchoe stems . On tomato plants the mutants induce small unorganized tumors while on Nicotiana rustica unorganized tumors, nearly equal in size to those caused by the wild-type strain have been induced . LBA4060 was shown to be avirulent on Kalanchoe leaves and LBA4210 was weakly virulent . Infection of Kalanchoe leaves or tomato plants with a mixture of separately grown cultures of both mutants resulted in the formation of more or less normal tumors . The exposure of a tomato plant to naphthalene acetic acid (NAA), a synthetic auxin, during development of tumors induced by LBA4060 stimulated tumor formation . Tumor growth induced by LBA4210 was found to be stimulated by kinetin. J Bacteriol, 1981 May, 146(2), 484 - 93 Relationship between the limited and wide host range octopine-type Ti plasmids of Agrobacterium tumefaciens; Thomashow MF et al.; The relationship between the limited host range octopine Ti plasmids and the wide host range octopine Ti plasmids pTiB6806 and pTiA6 was studied . The limited host range Ti plasmids shared extensive deoxyribonucleic acid homology; pTiAg63 and pTiAg162 were essentially completely homologous with pTiAg158 while pTiAg57 shared approximately 64% homology with pTiAg158 . In contrast, the limited host range octopine Ti plasmids only shared 6 to 15% homology with the wide host range octopine Ti plasmid pTiB6806 . Thus, limited and wide host range octopine Ti plasmids comprise distinct families of plasmids . The deoxyribonucleic acid homology shared between the limited host range Ti plasmids and pTiB6806, however, was distributed over some 50% of pTiB6806, suggesting that both families of plasmids evolved from a common progenitor plasmid . The limited host range Ti plasmids showed relatively strong homology with pTiB6806 HpaI fragment 7, a region which codes for octopine utilization by the bacterium, but showed only weak homology with pTiB6806 HpaI fragment 12, a region required for virulence . In addition, homology between the limited host range octopine Ti plasmids and the "common deoxyribonucleic acid," sequences shown to have a central role in plant cell transformation, was barely detectable when stringent hybridization conditions were used . We therefore conclude that a highly conserved version of the common deoxyribonucleic acid is not required for crown gall tumorigenesis on all plant species. Eur J Biochem, 1981 Apr, 115(3), 539 - 43 Substituents at N6 and C-5' control selective uptake and toxicity of the adenine-nucleotide bacteriocin, agrocin 84, in Agrobacteria; Murphy PJ et al.; The inhibition of a sensitive strain of Agrobacterium radiobacter by the nucleotide bacteriocin agrocin 84 has been studied . A structure-function study of the agrocin 84 molecule was undertaken . Two agrocin 84 nucleotide fragments lacking either the N6 or 5'-phosphoramidate substit