Plant J, 1997 Apr, 11(4), 717 - 28 Gene targeting and instability of Agrobacterium T-DNA loci in the plant genome; Risseeuw E et al.; To develop a model system for studies of homologous recombination in plants, transgenic Nicotiana tabacum and Nicotiana plumbaginifolia lines were generated harbouring a single target T-DNA containing the negative selective codA gene encoding cytosine deaminase (CD) and the beta-glucuronidase (GUS) gene . Subsequently, the target lines were transformed with a replacement-type T-DNA vector in which the CD gene and the GUS promoter had been replaced with a kanamycin-resistance gene . For both Nicotiana species kanamycin-resistant lines were selected which had lost the CD gene and the GUS activity . One tobacco line was the result of a precise gene targeting event . However, most other lines were selected due to a chromosomal deletion of the target locus . The deletion frequency of the target locus varied between target lines, and could be present in up to 20% of the calli which were grown from leaf protoplasts . T-DNA transfer was not required for induction of the deletions, indicating that the target loci were unstable . A few lines were obtained in which the target locus had been deleted partially . Sequence analysis of the junctions revealed deletion of DNA sequences between microhomologies . We conclude that T-DNAs, which are stable during plant development as well as in transmission to the offspring, may become unstable during propagation in callus tissue . The relationships between callus culture, genetic instability and the process of T-DNA integration and deletion in the plant genome are discussed. Microbiology, 1997 Apr, 143 ( Pt 4), 1115 - 24 Periplasmic cyclic 1,2-beta-glucan in Brucella spp . is not osmoregulated; Briones G et al.; Biosynthesis of periplasmic cyclic 1,2-beta-glucans in Brucella ovis strain REO198 and B . abortus strain 519 was found to be carried out by membrane-bound enzymes that use UDP-glucose (UDP-Glc) as donor substrate . Contrary to what happens in species of the genera Agrobacterium and Rhizobium, the accumulation of the reaction products in Brucella appeared not to be osmotically regulated . Incubation of permeabilized cells with UDP-{14C}Glc led to the formation of soluble neutral cyclic 1,2-beta-glucans and {14C}glucose-labelled glucoproteins . PAGE of pulse-chase experiments carried out with permeabilized cells showed that the molecular mass of the labelled protein was indistinguishable from Agrobacterium tumefaciens A348 and Rhizobium fredii USDA191 glucoproteins known to be intermediates in the synthesis of cyclic glucans . Brucella total membrane preparations were less efficient than permeabilized cells in the formation of cyclic glucan; this was attributed to defective cyclization . Accumulation of protein intermediates having oligosaccharides of high molecular mass that were not released from the protein was observed after chase with 2 mM UDP-Glc . This defect was not observed when permeabilized cells were used as enzyme preparation, thus suggesting that in Brucella a factor(s) that was lost or inactivated upon the preparation of membranes was required for the effective regulation between elongation and cyclization reactions. Proc Natl Acad Sci U S A, 1997 Apr 1, 94(7), 3459 - 64 Hrp pilus: an hrp-dependent bacterial surface appendage produced by Pseudomonas syringae pv . tomato DC3000; Roine E et al.; Hypersensitive response and pathogenicity (hrp) genes control the ability of major groups of plant pathogenic bacteria to elicit the hypersensitive response (HR) in resistant plants and to cause disease in susceptible plants . A number of Hrp proteins share significant similarities with components of the type III secretion apparatus and flagellar assembly apparatus in animal pathogenic bacteria . Here we report that Pseudomonas syringae pv . tomato strain DC3000 (race 0) produces a filamentous surface appendage (Hrp pilus) of 6-8 nm in diameter in a solid minimal medium that induces hrp genes . Formation of the Hrp pilus is dependent on at least two hrp genes, hrpS and hrpH (recently renamed hrcC), which are involved in gene regulation and protein secretion, respectively . Our finding of the Hrp pilus, together with recent reports of Salmonella typhimurium surface appendages that are involved in bacterial invasion into the animal cell and of the Agrobacterium tumefaciens virB-dependent pilus that is involved in the transfer of T-DNA into plant cells, suggests that surface appendage formation is a common feature of animal and plant pathogenic bacteria in the infection of eukaryotic cells . Furthermore, we have identified HrpA as a major structural protein of the Hrp pilus . Finally, we show that a nonpolar hrpA mutant of P . syringae pv . tomato DC3000 is unable to form the Hrp pilus or to cause either an HR or disease in plants. J Bacteriol, 1997 Apr, 179(7), 2452 - 8 The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon; Kemner JM et al.; The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment . The region downstream of the A . tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis . Three open reading frames transcribed in the same direction as chvE were revealed . The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter . The third open reading frame encodes a protein of unknown function . The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake . We have named these genes gguA, -B, and -C, for glucose galactose uptake . Mutations in gguA, gguB, or gguC do not affect virulence of A . tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. J Bacteriol, 1997 Apr, 179(7), 2373 - 81 Generation of buds, swellings, and branches instead of filaments after blocking the cell cycle of Rhizobium meliloti; Latch JN et al.; Inhibition of cell division in rod-shaped bacteria such as Escherichia coli and Bacillus subtilis results in elongation into long filaments many times the length of dividing cells . As a first step in characterizing the Rhizobium meliloti cell division machinery, we tested whether R . meliloti cells could also form long filaments after cell division was blocked . Unexpectedly, DNA-damaging agents, such as mitomycin C and nalidixic acid, caused only limited elongation . Instead, mitomycin C in particular induced a significant proportion of the cells to branch at the poles . Moreover, methods used to inhibit septation, such as FtsZ overproduction and cephalexin treatment, induced growing cells to swell, bud, or branch while increasing in mass, whereas filamentation was not observed . Overproduction of E . coli FtsZ in R . meliloti resulted in the same branched morphology, as did overproduction of R . meliloti FtsZ in Agrobacterium tumefaciens . These results suggest that in these normally rod-shaped species and perhaps others, branching and swelling are default pathways for increasing mass when cell division is blocked. J Bacteriol, 1997 Apr, 179(7), 2305 - 13 Variable efficiency of a Ti plasmid-encoded VirA protein in different agrobacterial hosts; Belanger C et al.; The transconjugant CB100, harboring the Ti plasmid from the Agrobacterium tumefaciens biovar 2 strain D10B/87 in the chromosomal background of the biovar 1 strain C58, was defective in vir gene induction . This defect was corrected in the presence of virA from pTiA6 . Based on this complementation result and an analysis of the induction requirements of the transconjugant CB100 and its parent strains, it was hypothesized that the defective vir gene induction in CB100 was related to a dysfunctional interaction between the pTi-encoded D10B/87 VirA and the chromosome-encoded C58 ChvE . To verify this hypothesis, D10B/87 and C58 virA were compared, and conclusions from this first set of analyses were then corroborated by comparing D10B/87 and C58 chvE . Whereas only a few nucleotide differences were identified in the promoters and 5' ends of the coding regions of D10B/87 and C58 virA, analysis of hybrid virA genes showed that these differences collectively accounted for the poor vir gene induction of strain CB100 . In contrast with the sequence similarity of the VirA proteins, extensive divergence was seen between the chromosome-encoded D10B/87 and C58 ChvE . Although D10B/87 chvE introduced in trans had little effect on vir gene induction of CB100, it enhanced the induction response of a strain CB100 derivative in which the chromosomal C58 chvE had been inactivated by marker exchange . These results suggest that chromosomal backgrounds provided by different strains of A . tumefaciens are not equivalent for VirA function . Following conjugative transfer of certain Ti plasmids to a new agrobacterial host, evolution of the newly introduced virA, or coevolution of chvE and virA, may lead to optimization of ChvE-VirA interaction and vir gene induction levels. Gene, 1997 Mar 25, 188(1), 69 - 75 Suicide plasmids containing promoterless reporter genes can simultaneously disrupt and create fusions to target genes of diverse bacteria; Kalogeraki VS et al.; We describe several plasmids that are designed to create fusions between chromosomal or plasmid-encoded genes and the lacZ, phoA or gfp reporter genes . These plasmids all contain the vegetative origin of R6K, but lack the R6K pir gene, and therefore fail to replicate in strains lacking pir . Fragments of target genes are introduced into these plasmids, and fusions are created in a single step as a consequence of (Campbell-type) integration of the entire plasmid by homologous recombination . Cloned fragments containing either an intact 5'-end of the target gene including its promoter or an intact 3'-end of the gene preserve a functional copy of that gene, while fragments lacking both 5'- and 3'-ends of the target gene cause a gene disruption . In addition to facilitating measurements of gene expression, some plasmids create translational fusions to beta-galactosidase or alkaline phosphatase and are therefore useful in studying the membrane topology of a target protein . We demonstrate the utility of these plasmids by constructing and testing two operon fusions and two protein fusions between the virG gene of Agrobacterium tumefaciens and lacZ. J Biol Chem, 1997 Mar 7, 272(10), 6128 - 35 In vitro characterization of astaxanthin biosynthetic enzymes; Fraser PD et al.; Escherichia coli strains expressing the marine bacteria (Agrobacterium aurantiacum and Alcaligenes sp . strain PC-1) astaxanthin biosynthetic genes (crtZ and W), Haematococcus pluvialis bkt, and Erwinia uredovora crtZ genes were used for in vitro characterization of the respective enzymes . Specific enzyme assays indicated that all of the enzymes are bifunctional, in that the CrtZ enzymes formed zeaxanthin from beta-carotene via beta-cryptoxanthin, as well as astaxanthin from canthaxanthin via phoenicoxanthin (adonirubin) . The BKT/CrtW enzymes synthesized canthaxanthin via echinenone from beta-carotene and 4-ketozeaxanthin (adonixanthin) with trace amounts of astaxanthin from zeaxanthin . Comparison of maximum catalytic activities as well as selectivity experiments carried out in the presence of both utilizable substrates indicated that the CrtZ enzymes from marine bacteria converted canthaxanthin to astaxanthin preferentially, whereas the Erwinia CrtZ possessed a favorability to the formation of zeaxanthin from beta-carotene . The CrtW/BKT enzymes were not so defined in their substrate preference, responding readily to fluctuations in substrate levels . Other properties obtained indicated that the enzymes were strictly oxygen-requiring; and a cofactor mixture of 2-oxoglutarate, ascorbic acid, and Fe2+ was beneficial to activity . Based on enzymological data, a predicted pathway for astaxanthin biosynthesis is described, and it is proposed that CrtZ-like enzymes be termed carotenoid 3, (3')-beta-ionone ring hydroxylase and CrtW/BKT carotenoid 4, (4')-beta-ionone ring oxygenase. Tsitol Genet, 1997 Mar-Apr, 31(2), 17 - 22 {The genetic transformation of Lycopersicon peruvianum var . dentatum by using Agrobacterium tumefaciens carrying a plasmid with the human beta-interferon gene}; Rudas VA et al.; Wild tomato plants of Lycopersicon peruvianum var . dentatum were transformed with pG11 plasmid having human beta-interferon (hIFN-beta) and NPTII genes . Transformation was demonstrated by polymerase chain reaction (PCR) and ELISA assay . Expression of hIFN-beta gene was identified by Western blot analysis . Transgenic plants produced seeds and F1 generation inherited integrated traits. Plant Mol Biol, 1997 Mar, 33(4), 729 - 35 Expression patterns of vascular-specific promoters RolC and Sh in transgenic potatoes and their use in engineering PLRV-resistant plants; Graham MW et al.; The expression patterns of GUS fusion constructs driven by the Agrobacterium rhizogenes RolC and the maize Sh (Shrunken; sucrose synthase-1) promoters were examined in transgenic potatoes (cv . Atlantic) . RolC drove high-level gene expression in phloem tissue, bundle sheath cells and vascular parenchyma, but not in xylem or non-vascular tissues . Sh expression was exclusively confined to phloem tissue . Potato leafroll luteovirus (PLRV) replicates only in phloem tissues, and we show that when RolC is used to drive expression of the PLRV coat protein gene, virus-resistant lines can be obtained . In contrast, no significant resistance was observed when the Sh promoter was used. Plant Mol Biol, 1997 Mar, 33(4), 667 - 78 A novel glycine-rich/hydrophobic 16 kDa polypeptide gene from tobacco: similarity to proline-rich protein genes and its wound-inducible and developmentally regulated expression; Yasuda E et al.; We have isolated a cDNA clone, NT16, encoding a novel glycine-rich/hydrophobic protein from tobacco crown gall tumor tissues, which was induced by the T-DNA genes of Agrobacterium tumefaciens . The accumulation of NT16 transcripts was high in unorganized callus as well as in shoot-forming calli . In normal tobacco plants, the transcript levels were high in roots, and low in stems, whereas virtually no transcript accumulation was found in flowers or leaves . In leaves, however, NT16 transcript accumulation was induced by mechanical wounding . These results show that NT16 expression is developmentally regulated and induced by wound-stress conditions . Sequence analysis suggests that NT16 encodes a putative 16 kDa polypeptide that is apparently composed of 3 structural domains: two hydrophobic regions separated by a glycine-rich region . The NT16 polypeptide displays similarity to a number of proteins in its hydrophobic domains, but is unique in its glycine-rich domain which, in the corresponding domains of the homologous proteins, are mostly proline-rich . Since both glycine-rich and proline-rich proteins are generally reported to be mostly cell wall proteins, the NT16 gene may be involved in shoot and root formation and in wound-healing process by modifying cell wall composition. Plant Mol Biol, 1997 Mar, 33(5), 835 - 46 Developmental regulation of two tomato lipoxygenase promoters in transgenic tobacco and tomato; Beaudoin N et al.; Two lipoxygenase (LOX) genes (tomloxA and tomloxB) are expressed in ripening tomato fruit, and tomloxA is also expressed in germinating seedlings . The 5'-upstream regions of these genes were isolated to study the regulatory elements involved in coordinating tomlox gene expression . Sequence analysis of the promoters did not reveal any previously characterized regulatory elements except for TATA and CAAT boxes . However, the sequence motif GATAcAnnAAtnTGATG was found in both promoters . Chimeric gene fusions of each tomlox promoter with the beta-glucuronidase reporter gene (gus) were introduced into tobacco and tomato plants via Agrobacterium-mediated transformation . GUS activity in tomloxA-gus plants during seed germination peaked at day 5 and was enhanced by methyl jasmonate (MeJa) treatment . No GUS activity was detected in tomloxB-gus seedlings . Neither wounding nor abscisic acid (ABA) treatment of transgenic seedlings modified the activity of either promoter . During fruit development, GUS expression in tomloxA-gus tobacco fruit increased 5 days after anthesis (DAA) and peaked at 20 DAA . In tomloxB-gus tobacco fruit, GUS activity increased at 10 DAA and peaked at 20 DAA . In transgenic tomato fruit, tomloxA-gus expression was localized to the outer pericarp during fruit ripening, while tomloxB-gus expression was localized in the outer pericarp and columella . These data demonstrate that the promoter regions used in these experiments contain cis-acting regulatory elements required for proper regulation of tomlox expression during development and for MeJa-responsiveness. Plant Cell, 1997 Mar, 9(3), 317 - 33 Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration; Nam J et al.; We show that among ecotypes of Arabidopsis, there is considerable variation in their susceptibility to crown gall disease . Differences in susceptibility are heritable and, in one ecotype, segregate as a single major contributing locus . In several ecotypes, recalcitrance to tumorigenesis results from decreased binding of Agrobacterium to inoculated root explants . The recalcitrance of another ecotype occurs at a late step in T-DNA transfer . Transient expression of a T-DNA-encoded beta-glucuronidase gusA gene is efficient, but the ecotype is deficient in crown gall tumorigenesis, transformation to kanamycin resistance, and stable GUS expression . This ecotype is also more sensitive to gamma radiation than is a susceptible ecotype . DNA gel blot analysis showed that after infection by Agrobacterium, less T-DNA was integrated into the genome of the recalcitrant ecotype than was integrated into the genome of a highly susceptible ecotype. Transgenic Res, 1997 Mar, 6(2), 133 - 41 Effects of seed-specific expression of a cytokinin biosynthetic gene on canola and tobacco phenotypes; Roeckel P et al.; The Agrobacterium tumefaciens isopentenyl transferase gene (ipt), a cytokinin biosynthetic gene, was placed under the control of 1.9 kb of promoter sequence from the 2S albumin AT2S1 gene isolated from an Arabidopsis thaliana library . The construct was introduced into canola (Brassica napus) and tobacco (Nicotiana tabacum) . ipt transcripts were followed during embryo development of transgenic plants by northern hybridizations . The phenotype of transformed plants from the T1 generation was analysed and we observed an increased branching of inflorescences in tobacco and canola plants expressing the ipt gene . Comparing with controls, the average number of capsules and siliques in AT2S1-ipt plants was 82.6 and 24.8% higher, respectively . This result was correlated with an increase in cytokinin levels in transgenic plants, as revealed by RIA . Indeed, cytokinin contents of T1 AT2S1-ipt B . napus seeds were found 2.2-fold higher than cytokinin contents of control seeds, and T1 AT2S1-ipt N . tabacum capsules contained 2.6-fold more cytokinins than control capsules . In tobacco, the average seed weight per capsule was lower in AT2S1-ipt plants while the seed number per silique and the average seed weight were not modified in canola carrying this construct . The average seed yield per plant was not significantly increased in AT2S1-ipt tobacco or canola plants. Mol Plant Microbe Interact, 1997 Mar, 10(2), 221 - 7 Discrete regions of the sensor protein virA determine the strain-specific ability of Agrobacterium to agroinfect maize; Heath JD et al.; The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection . In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection . The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection . The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348,to agroinfection competence . The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6 . In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background . The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined. Mol Plant Microbe Interact, 1997 Mar, 10(2), 180 - 6 rosR, a determinant of nodulation competitiveness in Rhizobium etli; Bittinger MA et al.; We previously described a Tn5 mutant of Rhizobium etli strain CE3, designated CE3003, that is decreased in nodulation competitiveness, reduced in competitive growth in the rhizosphere, and has a hydrophobic cell surface (R . S . Araujo, E . A . Robleto, and J . Handelsman, Appl . Environ . Microbiol., 60:1430-1436, 1994) . To determine the molecular basis for the mutant phenotypes, we identified a 1.2-kb fragment of DNA derived from the parent that restored the wild-type phenotypes to the mutant . DNA sequence analysis indicated that this 1.2-kb fragment contained a single open reading frame that we designated rosR . The Tn5 insertion in CE3003 was within rosR . We constructed a derivative of CE3 that contained a deletion in rosR, and this mutant was phenotypically indistinguishable from CE3003 in cell surface and competitive characteristics . Based on the nucleotide sequence, the deduced RosR amino acid sequence is 80% identical to that of the Ros protein from Agrobacterium tumefaciens and the MucR protein from Rhizobium meliloti . Both Ros and MucR are transcriptional repressors that contain a putative zinc-finger DNA-binding domain . This study defines a gene, rosR, that is homologous to a family of transcriptional regulators and contributes to nodulation competitiveness of R . etli . Moreover, we established that a single gene affects nodulation competitiveness, competitive growth in the rhizosphere, and cell surface hydrophobicity. J Bacteriol, 1997 Mar, 179(5), 1573 - 9 Characterization of the promoter of the Rhizobium etli recA gene; Tapias A et al.; The promoter of the Rhizobium etli recA gene has been identified by primer extension and by making deletions affecting several regions located upstream of its coding region . A gel mobility shift assay carried out with crude extracts of cells of R . etli has been used to show that a DNA-protein complex is formed in the R . etli recA promoter region in vitro . Analysis of the minimal region of the recA promoter giving rise to this DNA-protein complex revealed the presence of an imperfect palindrome corresponding to the sequence TTGN11CAA . Site-directed mutation of both halves of this palindrome indicated that both motifs, TTG and CAA, are necessary for both normal DNA-protein complex formation in vitro and full DNA damage-mediated inducibility of the recA gene in vivo . However, the TTG motif seems to be more dispensable than the CAA one . The presence of this same palindrome upstream of the recA genes of Rhizobium meliloti and Agrobacterium tumefaciens, whose expression is also regulated in R . etli cells, suggests that this TTGN11CAA sequence may be the SOS box of at least these three members of the Rhizobiaceae. Mol Cells, 1997 Feb 28, 7(1), 84 - 9 Characterization of a hexamer motif and b element of the nopaline synthase (nos) promoter; Kim Y et al.; Nopaline synthase (nos) is of bacterial origin and expressed in plant tissues to generate an unusual compound, nopaline, which is secreted into the environment where Agrobacterium tumefaciens uses it as a nutrient source . The nos promoter contains three distinctive regions which are important for the promoter activity . Among these, the upstream region between -131 and -112 is essential for the nos promoter . The upstream region consists of two hexamer motifs which are separated by an eight nucleotide sequence . In this study, we have investigated the role of the hexamer motif that is located between -117 and -112 . Increasing the distance between the hexamer and the downstream regulatory region significantly reduced the promoter activity, indicating that a proper distance between the regulatory elements is needed for an optimum level of activity . Insertion of a single copy of the hexamer enhanced the promoter in both orientations . Point mutations in the inserted hexamer sequence reduced the enhancing effect . These results confirmed that the hexamer motif is important for the nos promoter activity . The b sequence that is located immediately downstream of the hexamer motif did not function by itself, but together with the hexamer it appeared to enhance the promoter activity . These indicate that the sequence surrounding of the upstream regulatory element is also involved in controlling the promoter function. Mol Cells, 1997 Feb 28, 7(1), 21 - 7 Promoter sequences of two homologous pectin esterase genes from Chinese cabbage (Brassica campestris L . ssp . pekinensis) and pollen-specific expression of the GUS gene driven by a promoter in tobacco plants; Kim HU et al.; The promoter regions of two genomic clones, GBAN215-6 and GBAN215-12 from Chinese cabbage (Brassica campestris L . ssp . pekinensis), were sequenced . The nucleotide sequences of their promoter regions were compared with that of the Bp19 pollen-specific gene of Brassca napus . High nucleotide sequence homologies were observed among these three genes in the region between 210 bp upstream and the putative transcription start site . A sequence motif TGTGGTG, which is similar to that of the PB core motif (TGTGGTT) of two tomato pollen-specific genes, LAT52 and LAT56, was present in these two cloned genes . To determine regulatory sequences responsible for the anther-specific expression of the gene BAN215-6, two recombinant plasmids, pBPE3 (-274- + 109) and pBPE4 (-816- + 109) containing different lengths of the promoter fused with the GUS gene, were constructed and introduced into tobacco plants by Agrobacterium-mediated transformation . The result showed that the 383 bp (-274- + 109) of the BAN215-6 promoter region was sufficient for the anther-specific expression of the GUS gene . The GUS expression in a tobacco plants transformed with these constructs was first detected in uninucleate microspores and persisted at in vitro germinated pollen tubes . The expression level was increased during anther development, reaching the highest level in mature pollens. Mol Gen Genet, 1997 Feb 20, 253(5), 609 - 14 In vivo expression of a full-length cDNA copy of potato leafroll virus (PLRV) in protoplasts and transgenic plants; Prufer D et al.; A full-length cDNA copy (PLRVfl) of potato leafroll virus (PLRV) was constructed and examined in vivo for its biological activities by transient expression experiments with plasmid DNA or in vitro transcribed RNA . In addition, PLRVfl cDNA was stably introduced into the genome of potato plants by Agrobacterium-mediated leaf disc transformation . Both transient and stable expression of PLRVfl resulted in the synthesis of genomic and subgenomic PLRV RNAs . Transgenic plants accumulated the 17-kDa movement protein and displayed the typical symptoms of PLRV infection . This is the first example of the constitutive expression of a phloem-limited virus in planta. Enzyme Microb Technol, 1997 Feb 15, 20(3), 207 - 13 The role of liquid mixing and gas-phase dispersion in a submerged, sparged root reactor; Tescione LD et al.; An Agrobacterium-transformed root culture of Solanum tuberosum was grown in a 15-1 bubble column . The specific respiration rate decreased by a factor of ten as the tissue grew over a 25-day culture period . On days 5, 8, 13, and 21, respiration was shown to be independent of aeration rate over a range of 0.05-0.4 vvm (volume of air per volume of liquid min-1) . Gas dispersion measured from argon tracer residence time distributions increased fourfold due to increased stagnation and channeling of gas through the bed of growing roots; however, introduction of an antifoam surfactant on day 20 greatly reduced dispersion with no accompanying change in respiration . Taken together, the gas dispersion and respiration studies suggest that the gas-liquid interface is not the dominant resistance to oxygen mass transfer . Liquid mixing time measured with a dye tracer increased from 1.45 +/- 0.45 min with no root tissue to 40.2 +/- 1.6 min with 180 g FW l-1 of roots in the column . In addition, the oxygen uptake rate of growing tips (5.2 +/- 0.2 mm) of individual root segments of S . tuberosum measured in a stirred microcell (600 microliters) increased with the oxygen tension of the medium . Based on these results, the role of liquid mixing, gas-phase dispersion, and diffusion in the tissue in the scaleup of root culture is discussed. Genet Res, 1997 Feb, 69(1), 11 - 5 Mutational analysis of the rolA gene of Agrobacterium rhizogenes in tobacco: function of the rolA pre-mRNA intron and rolA proteins defective in their biological activity; Spena A et al.; The rolA gene of Agrobacterium rhizogenes contains in its untranslated leader region a spliceosomal intron, which is spliced in Arabidopsis and in Nicotiana tabacum . Expression under the control of the 35S promoter from cauliflower mosaic virus of a rolA gene derivative defective in splicing still causes alterations of growth in transgenic tobacco plants . Splicing of rolA mRNA is required for efficient expression of the rolA phenotype in vivo . Moreover, splicing is required for efficient in vitro translation of the rolA mRNA . In contrast, expression of a 35S-rolA gene derivative with the ATG initiation codon replaced by ATA does not cause any phenotypical alteration . Mutations leading to amino acid substitutions at positions 37 and 40 of the rolA coding region were isolated as null mutants in Arabidopsis plants transgenic for the rolA gene . However, when expressed in tobacco under the control of the 35S promoter, they cause a rolA phenotype reduced in the expressivity of its traits . The molecular characterization of rolA mutants might be useful for understanding the biochemical function of the rolA protein. Mol Microbiol, 1997 Feb, 23(3), 579 - 90 Characterization of flagella genes of Agrobacterium tumefaciens, and the effect of a bald strain on virulence; Chesnokova O et al.; Agrobacterium tumefaciens produces flagella that are arranged circumthecally near one end of the bacilliform cell . The flagella are required for motility to facilitate reaching the root surface, and possibly aid in orientating the bacterial cells at various sites for infection . We have identified three flagella genes designated flaA, flaB, and flaC . Mutations in flaA, flaB and flaC result in abberant swimming behaviour . Electron microscopic examination of these mutants revealed the defective flagella . A non-motile, bald mutant strain was generated by deleting all three fla genes . Nucleotide sequencing of flaA, flaB, and flaC showed that they have a potential coding capacity for polypeptides of 307, 321, and 314 amino acid residues, respectively . The predicted amino acid sequences of the A . tumefaciens FlaA and FlaB proteins are similar (66% average identity) to the FlaA and FlaB proteins encoded by flaA and flaB genes, respectively, in Rhizobium meliloti . There was no counterpart FlaC protein reported in R . meliloti, but the A . tumefaciens FlaC is similar in amino acid sequence to the R . meliloti FlaA (59.8% identity) and FlaB (66.7% identity) . Distinct from FlaA and FlaB of R . meliloti is the absence of histidine and cysteine residues and their shorter length (by 88 amino acid residues fewer than FlaA and FlaB of R . meliloti) . The transcriptional start sites of each fla gene determined by primer extension revealed consensus-sequence boxes representing potential binding sites for sigma 28 RNA polymerase (RNAP) upstream of the transcriptional start of each fla gene . Besides the potential sigma 28-binding site upstream of flaC, also present are additional putative conserved sequences, GC at -11 and GG at -21 from the transcriptional start, that resemble potential binding motifs for sigma 54 . Because the sigma 54 promoter is associated with genes regulated by physiological changes in various bacteria, the flaC gene might be similarly regulated in response to A . tumefaciens responding to host plant stimuli . Virulence studies showed that the bald strain was consistently reduced in virulence below that of the parental wild-type strain by at least 38% . The difference is statistically significant and suggests that the flagella may play a role in facilitating virulence. J Bacteriol, 1997 Feb, 179(4), 1291 - 7 Ti plasmid conjugation is independent of vir: reconstitution of the tra functions from pTiC58 as a binary system; Cook DM et al.; Two regions of the nopaline-type Ti plasmid pTiC58 are important for conjugal transfer of this element to recipient bacteria . These two regions were cloned into two independent replicons to produce a binary transfer system . For one region, oriT/tra, we constructed two derivatives, pFRtra and pDCtra-5 . Each contains the oriT site and the two flanking, divergently transcribed tra operons that encode the DNA processing functions associated with the relaxosome . These two plasmids also carry traR, which encodes the transcriptional activator necessary for expression of transfer genes . The two plasmids differ by the amounts of traB sequence or sequence downstream of traG present in the construct . The second replicon, pPLE2, carries the traI/trb region . The traI gene confers production of the Agrobacterium tumefaciens N-acyl homoserine lactone autoinducer, while the remaining genes in the trb operon encode components of the mating bridge . Donors harboring the two plasmids mobilized the transfer of the plasmid carrying the oriT/tra region to an A . tumefaciens recipient at frequencies similar to that at which the intact Ti plasmid transferred . Plasmid pFRtra, which encodes most of traB, was mobilized at a frequency almost 10-fold higher than was pDCtra-5, which lacks most of the gene . A . tumefaciens donors also mobilized pFRtra to Escherichia coli and Pseudomonas fluorescens recipients at frequencies similar to those observed with A . tumefaciens recipients . Rhizobium meliloti harboring the binary system also transferred the oriT/tra component to these recipients . However, E . coli or P . fluorescens donors harboring the binary system did not transfer pFRtra to any of the recipients . Furthermore, while the A . tumefaciens and R . meliloti donors produced high levels of the autoinducer, the P . fluorescens and E . coli donors produced only trace amounts of this signal molecule . These results indicate that the tra system of pTiC58 is fully contained within the characterized tra and trb regions of the Ti plasmid, that conjugation does not require functions encoded by the vir system for maximal activity, and that while the Ti plasmid tra system recognizes diverse gram-negative bacteria as recipients, of the hosts tested, it functions only in members of the family Rhizobiaceae. J Bacteriol, 1997 Feb, 179(4), 1211 - 8 The lipoprotein VirB7 interacts with VirB9 in the membranes of Agrobacterium tumefaciens; Baron C et al.; VirB9 and VirB7 are essential components of the putative VirB membrane channel required for transfer of the T-complex from Agrobacterium tumefaciens into plants . In this report, we present a biochemical analysis of their interaction and cellular localization . A comparison of relative electrophoretic mobilities under nonreducing and reducing conditions suggested that they form thiol-sensitive complexes with other proteins . Two-dimensional gel electrophoresis identified one complex as a heterodimer of VirB9 and VirB7 covalently linked by a disulfide bond, as well as VirB7 homodimers and monomers . Immunoprecipitation with VirB9-specific antiserum isolated the heterodimeric VirB9-VirB7 complex . Incubation with reducing agent split the complex into its constituent VirB9 and VirB7, which further confirmed linkage via cysteine residues . The interaction between VirB9 and VirB7 also was observed in the yeast two-hybrid system . Membrane attachment of VirB9-VirB7 may be conferred by lipoprotein modification, since labeling with {3H}palmitic acid in A . tumefaciens verified that VirB7 is a lipoprotein associated with VirB9 . VirB9 and VirB7 showed equal distribution between inner and outer membranes, in accord with their proposed association with the transmembrane VirB complex. J Bacteriol, 1997 Feb, 179(4), 1203 - 10 VirB1, a component of the T-complex transfer machinery of Agrobacterium tumefaciens, is processed to a C-terminal secreted product, VirB1; Baron C et al.; During genetic transformation of plant cells by Agrobacterium tumefaciens, 11 VirB proteins and VirD4 are proposed to form a transmembrane bridge to transfer a DNA-protein complex (T-complex) into the plant cytoplasm . In this study, the localization of the first product of the virB operon, VirB1, was studied in detail . While full-length VirB1 localized mostly to the inner membrane, an immunoreactive VirB1 product was found as soluble processed form, designated VirB1* . Equal amounts of VirB1* could be detected in concentrated culture supernatants versus associated with the cell . VirB1* was purified from the supernatant of vir-induced cells by ammonium sulfate precipitation and Q-Sepharose chromatography . Sequence analysis of the N terminus of VirB1* localized the processing site after amino acid 172 of VirB1 . Cell-associated VirB1* was partly removed by vortexing, suggesting a loose association with the cell or active secretion . However, cross-linking and coimmunoprecipitation showed a close association of cell-bound VirB1* with the VirB9-VirB7 heterodimer, a membrane-associated component of the T-complex transfer machinery . Homologies of the N-terminal part of VirB1 to bacterial transglycosylases suggest that it may assist T-complex transfer by local lysis of the bacterial cell wall, whereas the exposed localization of the C-terminal processing product VirB1* predicts direct interaction with the plant . Thus, VirB1 may be a bifunctional protein where both parts have different functions in T-complex transfer from Agrobacterium to plant cells. J Bacteriol, 1997 Feb, 179(4), 1165 - 73 Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2; Dombek P et al.; The transferred DNA (T-DNA) portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid enters infected plant cells and integrates into plant nuclear DNA . Direct repeats define the T-DNA ends; transfer begins when the VirD2 endonuclease produces a site-specific nick in the right-hand border repeat and attaches to the 5' end of the nicked strand . Subsequent events liberate the lower strand of the T-DNA from the Ti plasmid, producing single-stranded DNA molecules (T strands) that are covalently linked to VirD2 at their 5' ends . A . tumefaciens appears to transfer T-DNA into plant cells as a T-strand-VirD2 complex . The bacterium also transports VirE2, a cooperative single-stranded DNA-binding protein, into plant cells during infection . Both VirD2 and VirE2 contain nuclear localization signals that may direct these proteins, and bound T strands, into plant nuclei . Here we report the locations of functional regions of VirE2 identified by eight insertions of XhoI linker oligonucleotides, and one deletion mutation, throughout virE2 . We examined the effects of these mutations on virulence, single-stranded DNA (ssDNA) binding, and accumulation of VirE2 in A . tumefaciens . Two of the mutations in the C-terminal half of VirE2 eliminated ssDNA binding, whereas two insertions in the N-terminal half altered cooperativity . Four of the mutations, distributed throughout virE2, decreased the stability of VirE2 in A . tumefaciens . In addition, we isolated a mutation in the central region of VirE2 that decreased tumorigenicity but did not affect ssDNA binding or VirE2 accumulation . This mutation may affect export of VirE2 into plant cells or nuclear localization of VirE2, or it may affect an uncharacterized activity of VirE2. J Bacteriol, 1997 Feb, 179(3), 583 - 91 Characterization of membrane and protein interaction determinants of the Agrobacterium tumefaciens VirB11 ATPase; Rashkova S et al.; The VirB11 ATPase is a putative component of the transport machinery responsible for directing the export of nucleoprotein particles (T complexes) across the Agrobacterium tumefaciens envelope to susceptible plant cells . Fractionation and membrane treatment studies showed that approximately 30% of VirB11 partitioned as soluble protein, whereas the remaining protein was only partially solubilized with urea from cytoplasmic membranes of wild-type strain A348 as well as a Ti-plasmidless strain expressing virB11 from an IncP replicon . Mutations in virB11 affecting protein function were mapped near the amino terminus (Q6L, P13L, and E25G), just upstream of a region encoding a Walker A nucleotide-binding site (F154H;L155M), and within the Walker A motif (P170L, K175Q, and delta GKT174-176) . The K175Q and delta GKT174-176 mutant proteins partitioned almost exclusively with the cytoplasmic membrane, suggesting that an activity associated with nucleotide binding could modulate the affinity of VirB11 for the cytoplasmic membrane . The virB11F154H;L155M allele was transdominant over wild-type virB11 in a merodiploid assay, providing strong evidence that at least one form of VirB11 functions as a homo- or heteromultimer . An allele with a deletion of the first half of the gene, virB11 delta1-156, was transdominant in a merodiploid assay, indicating that the C-terminal half of VirB11 contains a protein interaction domain . Products of both virB11 delta1-156 and virB11 delta158-343, which synthesizes the N-terminal half of VirB11, associated tightly with the A . tumefaciens membrane, suggesting that both halves of VirB11 contain membrane interaction determinants. Biochem J, 1997 Jan 15, 321 ( Pt 2), 319 - 24 Purification of a protein from Agrobacterium tumefaciens strain A348 that binds phenolic compounds; Dye F et al.; In order to induce tumours on dicotyledonous plants, the bacterium Agrobacterium tumefaciens needs to be able to sense signal molecules, i.e . phenolic compounds . In order to identify putative chemoreceptors or environmental sensors involved in vir gene induction, we undertook the purification of a phenol-binding protein by affinity chromatography on a syringamide Ultrogel A4 column equilibrated at pH 5.6 . A mild extraction of bacterial proteins with a Tris/HCl buffer at pH 9.0 led to the purification of a 39 kDa protein (Pbp39) with a pl of 4.3 after specific elution of the affinity matrix with sodium syringate . When the affinity chromatography was performed at neutral pH, barely any protein was isolated, indicating the importance of an acidic pH for optimal affinity . A microplate binding experiment revealed that both syringlyl biotinylated-BSA and sinapyl-biotinylated-BSA bound at pH 5.6 to the plate coated with Pbp39. Biochim Biophys Acta, 1997 Jan 4, 1337(1), 133 - 42 Purification and characterization of D(+)-carnitine dehydrogenase from Agrobacterium sp.--a new enzyme of carnitine metabolism; Hanschmann H et al.; D(+)-Carnitine dehydrogenase from Agrobacterium sp . catalyzes the oxidation of D(+)-carnitine to 3-dehydrocarnitine as initial step of D(+)-carnitine degradation . The NAD(+)-specific, cytosolic enzyme was purified 126-fold to apparent electrophoretic homogeneity by 4 chromatographic steps . The molecular mass of the native enzyme was estimated to be 88 kDa by size-exclusion chromatography . It seems to be composed of 3 identical subunits with a relative molecular mass of 28 kDa as found by sodium dodecyl sulfate polyacrylamide gel electrophoresis and laser-induced mass spectrometry . The isoelectric point was found to be 4.7-5.0 . The optimum temperature is 37 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.0-9.5 and 5.5-6.5, respectively . The purified enzyme was further characterized with respect to substrate specificity, kinetic parameters and amino terminal sequence . Analogues of D(+)-carnitine (L(-)-carnitine, crotonobetaine, gamma-butyrobetaine, carnitine amide, glycine betaine, choline) are competitive inhibitors of D(+)-carnitine oxidation . The equilibrium constant of the reaction of D(+)-carnitine dehydrogenase was determined to be 2.2 x 10(-12) . The purified D(+)-carnitine dehydrogenase has similar kinetic properties to the L(-)-carnitine dehydrogenase from the same microorganism as well as to L(-)-carnitine dehydrogenases of other bacteria. Chin J Biotechnol, 1997, 13(1), 31 - 6 The role of heterologous nifAc product in the regulation of nif expression in Agrobacterium tumefaciens; Zhang J et al.; The plasmids pCK5, pCK3, pSZ36, and pSZ23-CA, which carried constitutive nifAc gene of Azotobacter chroococcum and Klebsiella pneumoniae were transferred into A . tumefaciens C58/pGV3850 with triparental mating . The growth rate of these transconjugants was similar to the wild type . Nitrogenase synthesis was demonstrated by Western blotting, in the presence of 10 mmol/L NH4+, and the nitrogenase activity was restored to 73%, 24%, 11%, and 62%, respectively . The results showed that the regulative gene of nitrogen fixation in A . chroococcum and K . pneumoniae played a regulative role for the expression of A . tumefaciens nitrogen fixation gene . Among them, the role of A . chroococcum nifAc gene was the strongest, the fusion plasmid pSZ23-CA which carried nifA-ntrC gene of K . pneumoniae was stronger, and the nifAc gene of K . pneumoniae was weak. Plasmid, 1997, 38(1), 53 - 9 Complete nucleotide sequence of the replicator region of Paracoccus (Thiobacillus) versutus pTAV1 plasmid and its correlation to several plasmids of Agrobacterium and Rhizobium species; Bartosik D et al.; The complete nucleotide sequence of the replicator region of pTAV1, a cryptic, low copy number plasmid of Paracoccus versutus, was determined . The minimal replicon sequence (3149 bp) included in pTAV203/18 contains two open reading frames with coding capabilities for putative polypeptides of 23.8 (RepX) and 46 kDa (RepC') . The two genes have the same transcriptional polarity and both seem to be essential for replication of pTAV203 . The predicted amino acid sequence of RepC' shows significant homology with the major replication-associated proteins of several Agrobacterium and Rhizobium plasmids . A probable origin of replication (oriV) was proposed to be localized at the 3' terminal end of the repC' gene. Plasmid, 1997, 37(3), 181 - 8 The hydrophobic TraM protein of pKM101 is required for conjugal transfer and sensitivity to donor-specific bacteriophage; Cellini C et al.; pKM101 is a self-transmissible plasmid of the IncN incompatibility group . Analysis of the DNA sequences of the genes required for conjugal transfer suggested the existence of a previously uncharacterized open reading frame, designated traM, that might be required for conjugation . Merodiploid strains containing transposon insertion mutations either in traM or in neighboring tra genes were used to demonstrate that traM constitutes a new complementation group essential for conjugation and donor phage sensitivity . The hydrophobicity profile of TraM suggests that it contains a signal sequence . The remainder of TraM is also composed predominantly of hydrophobic amino acids but contains one possible surface exposed loop . TraM-alkaline phosphatase and TraM-beta-galactosidase fusion proteins supported the hypothesis that TraM has a small cytoplasmic loop . We were unable to detect heterologous complementation between any tra mutation and its homolog from the virB operon of Agrobacterium tumefaciens. Biochimie, 1997, 79(1), 3 - 6 Alkylsyringamides, new inducers of Agrobacterium tumefaciens virulence genes; Dye F et al.; The virulence genes of Agrobacterium tumefaciens are specifically activated by plant phenolic compounds and allow this organism to genetically transform plant cells . New types of phenolic compounds, three phenol amides derived from syringic acid, were synthesized . Introduction of an amide group in syringic acid strongly enhances its vir gene inducing activity. Genet Eng (N Y), 1997, 19, 201 - 14 Nucleic acid transport in plant-pathogen interactions; Lartey R et al.; Inter- and intracellular transport of nucleic acids during plant-pathogen interaction is described on the examples of cell-to-cell movement of plant viruses and nuclear import of Agrobacterium T-DNA . In both cases, the transport process is mediated by specialized proteins produced by the pathogen . Plant virus movement occurs through the intercellular connections, plasmodesmata . In this process, the viral genomic nucleic acid is bound by virus-encoded movement protein . The nucleoprotein complex is then targeted to plasmodesmata, potentially via interaction with the host cell cytoskeleton . Prior to translocation, the plasmodesmal channel is dilated by the movement of protein . Nuclear import of Agrobacterium T-DNA is also mediated by bacterial proteins associated with the transported nucleic acid molecule . Specifically, the VirD2 and VirE2 proteins complex with the transferred DNA, providing it with the nuclear localization signals (NLSs) . The VirD2 NLS is an evolutionarily conserved signal, active both in plant and animal cells . In contrast, the VirE2 NLS is plant-specific . Both VirD2 and VirE2 NLSs most likely interact with the plant cell nuclear import machinery to initiate the transport process. Planta, 1997, 201(4), 424 - 33 Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L . plants triggers changes in ammonium assimilation and plant development; Vincent R et al.; A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L . plants . Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots . To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of TL- and TR-DNA sequences were chosen for further analyses . These plants had a 50-80% increase in total leaf GS activity . Plants were grown under different nitrogen regimes (4 or 12 mM NH4+) and aspects of carbon and nitrogen metabolism were examined . In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH4+ in comparison to the wild type grown under the same conditions . Labelling experiments using 15NH4+ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids . This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS . It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation . Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants . As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium. Transgenic Res, 1997 Jan, 6(1), 41 - 50 Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L . cultivars and in vitro evaluation of salt tolerance; Bordas M et al.; An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized . The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-beta-glucuronidase (gus)- . The entire construct was introduced into commercial cultivars of melon . Transformants were selected for their ability to grow on media containing kanamycin . Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization . Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period . The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv . 'Pharo' . Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants . A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer . In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 gl-1 NaCl . Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants. Biosci Biotechnol Biochem, 1997 Jan, 61(1), 152 - 7 A novel NADP(+)-dependent serine dehydrogenase from Agrobacterium tumefaciens; Chowdhury EK et al.; NADP(+)-dependent serine dehydrogenase {EC 1.1.1.-}, which catalyzes the oxidation of the hydroxyl group of serine to form 2-aminomalonate semialdehyde, was purified to homogeneity from a crude extract of Agrobacterium tumefaciens ICR 1600 . The enzyme had a molecular mass of about 100 kDa and consisted of four identical subunits . In addition to L-serine, D-serine, L-glycerate, D-glycerate, and 2-methyl-DL-serine were substrates . However, O-methyl-DL-serine and L-threonine were inert . The enzyme showed maximal activity at about pH 9 for the oxidation of L-serine . The enzyme required NADP+ as a coenzyme, NAD+ was inert . The enzyme was not inhibited by EDTA, o-phenanthroline, or alpha,alpha'-dipyridyl, but was inhibited by HgCl2, p-chloromercuribenzoate, L-cysteine, D-cysteine, malonate, 2-methylmalonate, and tartronate . The Michaelis constants for L-serine, D-serine, and NADP+ were 42, 44, and 0.029 mM, respectively. Plant J, 1997 Jan, 11(1), 15 - 29 T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs; De Neve M et al.; Nicotiana protoplasts and Arabidopsis leaf discs or roots were co-cultivated with two Agrobacterium strains each carrying a different T-DNA . Co-transformed plants were selected and the integration of the different T-DNAs was analysed at the genetic and genomic level . Genetic analysis showed that the T-DNAs derived from different bacteria were frequently integrated at the same locus, independent of the plant species or transformation method used . Southern analysis revealed that 12 out of 27 Arabidopsis transformants contained the co-transferred T-DNAs linked to each other in all possible configurations but with a preference for those with at least one right border involved in linkage . Overall, our data support the hypothesis that ligation of separate T-DNAs is a dominant mechanism in formation of the frequently observed repeats of identical T-DNAs . We propose a scheme which could explain the formation of T-DNA repeats and the preferential involvement of right borders in T-DNA linkages. J Gen Virol, 1997 Jan, 78 ( Pt 1), 229 - 36 Two mRNAs are transcribed from banana bunchy top virus DNA-1; Beetham PR et al.; We have mapped the mRNA transcripts of banana bunchy top virus (BBTV) DNA-1 . Northern hybridization and 3' RACE analysis identified two poly-adenylated RNAs associated with BBTV DNA-1 . Previously, one major ORF in the virion sense of DNA-1 had been identified, which encoded a putative replication protein (Rep) . An mRNA was identified in BBTV infected bananas that was clearly transcribed from this Rep ORF . Further, a second transcript was identified which mapped to an ORF completely within the Rep ORF . This encoded a putative 5 kDa protein of unknown function . Both these transcripts were also identified in a tobacco plant that had been transformed with Agrobacterium tumefaciens harbouring a binary construct containing the Rep ORF from BBTV DNA-1 . This Rep ORF was inserted 3' of a cauliflower mosaic virus 35S promoter and 5' of a vegetable storage protein terminator . The transcripts mapped from these tobacco plants were identical at the 3' end to the transcripts from BBTV infected banana plants . The site of polyadenylation for the Rep ORF was at base 963 immediately 3' of the translational stop codon confirming that the polyadenylation signals for this transcript were all within the ORF . However, the internal ORF had a large untranslated region of 272 bases with its site of polyadenylation at nucleotide 803 and a polyadenylation signal 3' of the translational stop codon . A possible upstream termination signal (A/TTGTAA) was identified and was conserved within BBTV DNA-1 sequences from different international isolates. Mol Plant Microbe Interact, 1997 Jan, 10(1), 69 - 78 Characterization of a salicylic acid-insensitive mutant (sai1) of Arabidopsis thaliana, identified in a selective screen utilizing the SA-inducible expression of the tms2 gene; Shah J et al.; Salicylic acid (SA) plays an important signaling role in the resistance of many plants to pathogen invasion . Increases in endogenous SA levels have been associated with the hypersensitive response as well as systemic acquired resistance (SAR) . SA also induces the expression of a subset of the pathogenesis-related (PR) genes . However, relatively little is known about the events occurring subsequent to SA accumulation during a resistance response . In order to identify mutations in components of the SA signal transduction pathway, we have developed a genetic screen in Arabidopsis thaliana that utilizes the Agrobacterium tumefaciens tms2 gene as a counter-selectable marker . SA-inducible expression of the tms2 gene from the tobacco PR-1a promoter confers sensitivity to alpha-naphthalene acetamide (alpha-NAM), resulting in inhibition of root growth in germinating transgenic Arabidopsis seedlings . Mutants in which root growth is insensitive to alpha-NAM have been selected from this PR-1a:tms2 transgenic line with the expectation that a subset will lack a regulatory component downstream of SA . The sail mutant so identified expressed neither the PR-1a:tms2 transgene nor the endogenous Arabidopsis PR-1, PR-2, and PR-5 genes in response to SA . These genes also were not induced in sai1 by 2,6-dichloroisonicotinic acid (INA) or benzothiadiazole (BTH), two chemical inducers of SAR . As expected of a mutation acting downstream of SA, sai1 plants accumulate SA and its glucoside in response to infection with an avirulent pathogen and are more susceptible to this avirulent pathogen than the wild-type parent . sai1 is allelic to npr1, a previously identified SA-noninducible mutation . The recessive nature of the noninducible sai1 mutation suggests that the wild-type SAI1 gene acts as a positive regulator in the SA signal transduction pathway. J Bacteriol, 1997 Jan, 179(2), 453 - 62 The VirB4 ATPase of Agrobacterium tumefaciens is a cytoplasmic membrane protein exposed at the periplasmic surface; Dang TA et al.; The VirB4 ATPase of Agrobacterium tumefaciens, a putative component of the T-complex transport apparatus, associates with the cytoplasmic membrane independently of other products of the Ti plasmid . VirB4 was resistant to extraction from membranes of wild-type strain A348 or a Ti-plasmidless strain expressing virB4 from an IncP replicon . To evaluate the membrane topology of VirB4, a nested deletion method was used to generate a high frequency of random fusions between virB4 and 'phoA, which encodes a periplasmically active alkaline phosphatase (AP) deleted of its signal sequence . VirB4::PhoA hybrid proteins exhibiting AP activity in Escherichia coli and A . tumefaciens had junction sites that mapped to two regions, between residues 58 and 84 (region 1) and between residues 450 and 514 (region 2) . Conversely, VirB4::beta-galactosidase hybrid proteins with junction sites mapping to regions 1 and 2 exhibited low beta-galactosidase activities and hybrid proteins with junction sites elsewhere exhibited high beta-galactosidase activities . Enzymatically active VirB5::PhoA hybrid proteins had junction sites that were distributed throughout the length of the protein . Proteinase K treatment of A . tumefaciens spheroplasts resulted in the disappearance of the 87-kDa VirB4 protein and the concomitant appearance of two immunoreactive species of approximately 35 and approximately 45 kDa . Taken together, our data support a model in which VirB4 is topologically configured as an integral cytoplasmic membrane protein with two periplasmic domains. J Bacteriol, 1997 Jan, 179(2), 439 - 44 pigB determines a diffusible factor needed for extracellular polysaccharide slime and xanthomonadin production in Xanthomonas campestris pv . campestris; Poplawsky AR et al.; Seven xanthomonadin transcriptional units (pigA through pigG) were identified by transposon saturation mutagenesis within an 18.6-kbp portion of the previously identified 25.4-kbp pig region from Xanthomonas campestris pv . campestris (strain B-24) . Since marker exchange mutant strains with insertions in one 3.7-kbp portion of pig could not be obtained, mutations in this region may be lethal to the bacterium . Complementation analyses with different insertion mutations further defined and confirmed the seven transcriptional units . Insertional inactivation of one of the transcriptional units, pigB, resulted in greatly reduced levels of both xanthomonadins and extracellular polysaccharide slime, and a pigB-encoding plasmid restored both traits to these strains . pigB mutant strains could also be restored extracellularly by growth adjacent to strains with insertion mutations in any of the other six xanthomonadin transcriptional units, the parent strain (B-24), or strains of five different species of Xanthomonas . Strain B-24 produced a nontransforming diffusible factor (DF), which could be restored to pigB mutants by the pigB-encoding plasmid . Several lines of evidence indicate that DF is a novel bacterial pheromone, different from the known signal molecules of Vibrio, Agrobacterium, Erwinia, Pseudomonas, and Burkholderia spp. J Bacteriol, 1997 Jan, 179(1), 78 - 89 Interactions between VirB9 and VirB10 membrane proteins involved in movement of DNA from Agrobacterium tumefaciens into plant cells; Beaupre CE et al.; The 11 VirB proteins from Agrobacterium tumefaciens are predicted to form a membrane-bound complex that mediates the movement of DNA from the bacterium into plant cells . The studies reported here on the possible VirB protein interactions in such a complex demonstrate that VirB9 and VirB10 can each form high-molecular-weight complexes after treatment with a chemical cross-linker . Analysis of nonpolar virB mutants showed that the formation of the VirB10 complexes does not occur in a virB9 mutant and that VirB9 and VirB10 are not components of the same cross-linked complex . VirB9, when stabilized by the concurrent expression of VirB7, was shown to be sufficient to permit VirB10 to cross-link into its usual high-molecular-weight forms in the absence of other Vir proteins . Randomly introduced single point mutations in virB9 resulted in Agrobacterium strains with severely attenuated virulence . Although some of the mutants contained wild-type levels of VirB9 and displayed an unaltered VirB9 cross-linking pattern, VirB10 cross-linking was drastically reduced . We conclude that specific amino acid residues in VirB9 are necessary for interaction with VirB10 resulting in the capacity of VirB10 to participate in high-molecular-weight complexes that can be visualized by chemical cross-linking. J Bacteriol, 1997 Jan, 179(1), 1 - 8 Transcriptional regulation and locations of Agrobacterium tumefaciens genes required for complete catabolism of octopine; Cho K et al.; By screening for octopine-inducible gene expression, we previously identified all the genes required for utilization of octopine as a source of carbon, nitrogen, and energy . They are (i) octopine oxidase, which converts octopine to arginine and pyruvate and is encoded by the ooxAB operon, (ii) arginase, which converts arginine to ornithine and urea and is encoded by arcA, (iii) ornithine cyclodeaminase, which converts ornithine to proline and ammonia and is encoded by the homologous arcB and ocd genes, and (iv) proline dehydrogenase, which converts proline to glutamate and is encoded by putA . Here we describe the regulation and localization of each of these genes . The ooxA-ooxB-ocd operon was previously shown to reside on the Ti plasmid and to be directly inducible by octopine . The arcAB operon is directly inducible by arginine, while it is induced by octopine only in strains that can convert octopine to arginine . Ornithine may also be a direct inducer of arcAB . putA is directly inducible by proline, while induction by octopine and by arginine (and probably by ornithine) requires their conversion to proline . Genetic studies indicate that arcAB and putA are localized on a conjugal genetic element . This element can be transferred to other Agrobacterium tumefaciens strains by a mechanism that does not require recA-dependent homologous recombination . Transfer of this genetic element from A . tumefaciens R10 requires at least one tra gene found on its Ti plasmid, indicating that this element is not self-transmissible but is mobilizable by the Ti plasmid . The DNA containing the arcAB and putA genes comigrates with a 243-kb linear molecular weight standard on field inversion electrophoretic gels. Appl Environ Microbiol, 1997 Jan, 63(1), 338 - 46 Characterization of the Agrobacterium vitis pehA gene and comparison of the encoded polygalacturonase with the homologous enzymes from Erwinia carotovora and Ralstonia solanacearum; Herlache TC et al.; DNA sequencing of the Agrobacterium vitis pehA gene revealed a predicted protein with an M(r) of 58,000 and significant similarity to the polygalacturonases of two other plant pathogens, Erwinia carotovora and Ralstonia (= Pseudomonas or Burkholderia) solanacearum . Sequencing of the N terminus of the PehA protein demonstrated cleavage of a 34-amino-acid signal peptide from pre-PehA . Mature PehA accumulated primarily in the periplasm of A . vitis and pehA+ Escherichia coli cells during exponential growth . A . vitis PehA released dimers, trimers, and monomers from polygalacturonic acid and caused less electrolyte leakage from potato tuber tissue than did the E . carotovora and R . solanacearum polygalacturonases. Cell, 1996 Dec 27, 87(7), 1307 - 16 Recognition of the bacterial avirulence protein AvrBs3 occurs inside the host plant cell; Van den Ackerveken G et al.; The molecular mechanism by which bacterial avirulence genes mediate recognition by resistant host plants has been enigmatic for more than a decade . In this paper we provide evidence that the Xanthomonas campestris pv . vesicatoria avirulence protein AvrBs3 is recognized inside the plant cell . Transient expression of avrBs3 in pepper leaves, using Agrobacterium tumefaciens for gene delivery, results in hypersensitive cell death, specifically on plants carrying the resistance gene Bs3 . In addition, for its intracellular recognition, AvrBs3 requires nuclear localization signals that are present in the C-terminal region of the protein . We propose that AvrBs3 is translocated into plant cells via the Xanthomonas Hrp type III secretion system and that nuclear factors are involved in AvrBs3 perception. Proc Natl Acad Sci U S A, 1996 Dec 24, 93(26), 15272 - 5 Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination; Bundock P et al.; Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination . Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA . We showed previously that T-DNA can also be transferred from A . tumefaciens to Sac-charomyces cerevisiae and integrates into the yeast genome via homologous recombination . We show here that when the T-DNA lacks homology with the S . cerevisiae genome, it integrates at random positions via illegitimate recombination . From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences . The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized . It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation . Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved. Dev Biol, 1996 Dec 15, 180(2), 693 - 700 The plant oncogene rolD stimulates flowering in transgenic tobacco plants; Mauro ML et al.; The Agrobacterium rhizogenes T-DNA oncogene rolD under the control of its own 5' regulatory region was transferred to day-neutral tobacco plants . The main trait induced by rolD in transgenic plants is a striking precocity in flower setting and a strong enhancement of the flowering potential . In rolD plants, early flowering is followed by the very rapid growth of numerous lateral inflorescences . The analysis of several morphological and histological parameters suggests that some characteristic morphological abnormalities observed in rolD plants can be accounted for by their early reproductive phase transition and points to the involvement in the transition of a greater portion of the plant body than is the case for untransformed tobacco . The in vitro morphogenic potential of tissues from rolD plants was also tested . Superficial thin cell layer explants from rolD plants show an earlier and much enhanced flower organogenesis, compared to controls, both on flowering and on hormone-free medium. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14978 - 83 "Agrolistic" transformation of plant cells: integration of T-strands generated in planta; Hansen G et al.; We describe a novel plant transformation technique, termed "agrolistic," that combines the advantages of the Agrobacterium transformation system with the high efficiency of biolistic DNA delivery . Agrolistic transformation allows integration of the gene of interest without undesired vector sequence . The virulence genes virD1 and virD2 from Agrobacterium tumefaciens that are required in bacteria for excision of T-strands from the tumor-inducing plasmid were placed under the control of the CaMV35S promoter and codelivered with a target plasmid containing border sequences flanking the gene of interest . Transient expression assays in tobacco and in maize cells indicated that vir gene products caused strand-specific nicking in planta at the right border sequence, similar to VirD1/VirD2-catalyzed T-strand excision observed in Agrobacterium . Agrolistically transformed tobacco calli were obtained after codelivery of virD1 and virD2 genes together with a selectable marker flanked by border sequences . Some inserts exhibited right junctions with plant DNA that corresponded precisely to the sequence expected for T-DNA (portion of the tumor-inducing plasmid that is transferred to plant cells) insertion events . We designate these as "agrolistic" inserts, as distinguished from "biolistic" inserts . Both types of inserts were found in some transformed lines . The frequency of agrolistic inserts was 20% that of biolistic inserts. Proc Natl Acad Sci U S A, 1996 Dec 10, 93(25), 14648 - 53 cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors; Censini S et al.; cagA, a gene that codes for an immunodominant antigen, is present only in Helicobacter pylori strains that are associated with severe forms of gastroduodenal disease (type I strains) . We found that the genetic locus that contains cagA (cag) is part of a 40-kb DNA insertion that likely was acquired horizontally and integrated into the chromosomal glutamate racemase gene . This pathogenicity island is flanked by direct repeats of 31 bp . In some strains, cag is split into a right segment (cagI) and a left segment (cagII) by a novel insertion sequence (IS605) . In a minority of H . pylori strains, cagI and cagII are separated by an intervening chromosomal sequence . Nucleotide sequencing of the 23,508 base pairs that form the cagI region and the extreme 3' end of the cagII region reveals the presence of 19 ORFs that code for proteins predicted to be mostly membrane associated with one gene (cagE), which is similar to the toxin-secretion gene of Bordetella pertussis, ptlC, and the transport systems required for plasmid transfer, including the virB4 gene of Agrobacterium tumefaciens . Transposon inactivation of several of the cagI genes abolishes induction of IL-8 expression in gastric epithelial cell lines . Thus, we believe the cag region may encode a novel H . pylori secretion system for the export of virulence determinants. Mol Biotechnol, 1996 Dec, 6(3), 335 - 45 Manipulating photosynthesis; Knight JS et al.; The levels of individual photosynthetic proteins can be independently decreased by the Agrobacterium-mediated transformation of plants with antisense RNA constructs . Protocols for the introduction of such constructs into Agrobacterium, the Agrobacterium-mediated transformation of tobacco leaf disks, and the screening and analysis of the transgenic plants produced are described. Appl Microbiol Biotechnol, 1996 Dec, 46(5-6), 660 - 6 Biodehalogenation of low concentrations of 1,3-dichloropropanol by mono- and mixed cultures of bacteria; Fauzi AM et al.; The degradation of low concentrations of 1,3-dichloro-2-propanol (1,3-DCP) and related halohydrins by whole cells and cell-free extracts of soil bacteria has been investigated . Three bacteria (strains A1, A2, A4), isolated from the same soil sample, were distinguished on the basis of cell morphology, growth kinetics and haloalcohol dehalogenase profiles . Strain A1, probably an Agrobacterium sp., dehalogenated 1,3-DCP with the highest specific activity (0.33 U mg protein-1) and also had the highest affinity for 1,3-DCP (Km, 0.1 mM) . Non-growing cells of this bacterium dehalogenated low concentrations of 1,3-DCP with a first-order rate constant (kl) of 1.13 h-1 . The presence of a non-dehalogenating bacterium, strain G1 (tentatively identified as Pseudomonas mesophilius), did not enhance the dehalogenation rate of low 1,3-DCP concentrations . However, the mixed-species consortium of strains A1 and G1 had greater stability than the mono-species culture at DCP concentrations above 1.0 gl-1. Plant Mol Biol, 1996 Dec, 32(6), 1197 - 203 Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression; Cluster PD et al.; Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants . Presented here are the full details of T-DNA structural organization in that population . Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups . Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both . Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats . Large fragments of binary-vector sequences were linked to the T-DNA in seven plants. Plant Mol Biol, 1996 Dec, 32(6), 1135 - 48 T-DNA integration into genomic DNA of rice following Agrobacterium inoculation of isolated shoot apices; Park SH et al.; This paper establishes that the isolated shoot meristem of monocotyledons can be infected and transformed using Agrobacterium . Since this explant from nearly any cereal cultivar can rapidly regenerate into a plant, using this explant effectively eliminates the genotype regeneration restrictions to cereal crop transformation allowing direct transformation of elite germplasm . Shoot apices of Oryza sativa L . Tropical Japonica, cv . Maybelle were explants used for cocultivation, and gene transfer was accomplished using Agrobacterium containing plasmids for the bar gene expression driven by the CaMV 35S promoter or by the rice actin 1 promoter . Experiments to determine the survival rates of isolated shoot apices on media containing the herbicide, glufosinate-ammonium (PPT), established that no shoot apices survived on 0.5 or 1.0 mg/l PPT . After shoot apices were cocultivated with Agrobacterium, 2.8% (overall 20 out of 721 shoot apices) survived on 0.5 mg/l PPT . Results demonstrated that the use of the actin 1 promoter-based expression vector and an extra-wounding treatment of the meristematic cells appeared to be most effective in promoting transformation . Integration, expression and transmission of the transferred foreign genes in primary, R1 and R2 generation plants were confirmed by molecular analyses and herbicide application tests . A germination test of R2 progeny from one of the transgenic plants (R1) established a phenotype segregation ratio showing a non-Mendelian inheritance pattern . Inactivation of the transferred foreign gene in R2 progeny appeared to result from transgene methylation. Plant Mol Biol, 1996 Dec, 32(6), 1029 - 35 Analysis of kafirin promoter activity in transgenic tobacco seeds; DeRose RT et al.; Sequences corresponding to 855 bp of 5' promoter region and the transit peptide from lambdaGK.1,a genomic clone encoding a 22 kDa alpha-kafirin seed protein from sorghum, were translationally fused to a cloned beta-glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation . No GUS expression was detectable at any stage of growth in stems or leaves of these plants . However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10-15 days after flowering . Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument . These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region. Curr Biol, 1996 Dec 1, 6(12), 1567 - 9 Plant transformation: a pilus in Agrobacterium T-DNA transfer; Baron C et al.; Agrobacterium tumefaciens transfers a protein-DNA complex to plant cells in a process similar to bacterial conjugation; the mechanism of transfer is beginning to be unravelled by biochemical, genetic and electron microscopic studies. Plant Mol Biol, 1996 Dec, 32(5), 785 - 96 Expression analysis of an Arabidopsis C2H2 zinc finger protein gene; Tague BW et al.; C2H2 zinc finger protein genes encode nucleic acid-binding proteins involved in the regulation of gene activity . AtZFP1 (Arabidopsis thaliana zinc finger protein 1) is one member of a small family of C2H2 zinc finger-encoding sequences previously characterized from Arabidopsis . The genomic sequence corresponding to the AtZFP1 cDNA has been determined . Molecular analysis demonstrates that AtZFP1 is a unique, intronless gene which encodes a 1100 nucleotides mRNA highly expressed in roots and stems . A construct in which 2.5 kb of AtZFP1 upstream sequences is linked to the beta-glucuronidase gene was introduced into Arabidopsis by Agrobacterium-mediated transformation of roots . Histochemical analysis of transgenic Arabidopsis carrying the AtZFP1 promoter: beta-glucuronidase fusion shows good correlation with RNA blot hybridization analysis . This transgenic line will be a useful tool for analyzing the regulation of AtZFP1 to further our understanding of its function. Mol Microbiol, 1996 Dec, 22(5), 1025 - 33 The putA gene of Agrobacterium tumefaciens is transcriptionally activated in response to proline by an Lrp-like protein and is not autoregulated; Cho K et al.; The Agrobacterium tumefaciens putA gene, which encodes proline dehydrogenase, is transcriptionally induced by exogenous proline . In contrast to the putA genes of enteric bacteria, the A . tumefaciens putA gene is not regulated by the PutA protein, as the putA promoter remained strongly proline inducible in strains lacking PutA . A putA null mutation increased the expression of the putA promoter under a variety of conditions . However, this mutation is predicted to increase the cytoplasmic concentration of proline, and this alone probably accounts for its effects on putA expression . The putA promoter was also strongly induced by valine, and the putA genotype did not affect expression by this gratuitous inducer . An open reading frame (ORF) encoding an Lrp-like protein was found transcribed divergently from putA . Disruption of this ORF, designated putR, abolished induction of the putA promoter by proline or valine . In addition to activating putA, PutR also repressed its own transcription, and this autorepression was only slightly affected by exogenous proline . The transcription start sites for the putA and putR genes are separated by 64 nucleotides, suggesting that PutR could regulate both promoters by binding to a single operator. J Bacteriol, 1996 Dec, 178(24), 7138 - 43 Cloning of the rpoD analog from Rhizobium etli: sigA of R . etli is growth phase regulated; Luka S et al.; Rhizobium bacteria fix atmospheric nitrogen during symbiosis with legume plants only after bacterial division is arrested . The role of the major vegetative sigma factor, SigA, utilized by Rhizobium bacteria during symbiosis is unknown . By using PCR technology, a portion of the sigA gene corresponding to domain II was directly amplified from Rhizobium etli total DNA by using two primers designed in accordance with the published sequence of sigA from Agrobacterium tumefaciens . The amplified fragment was cloned and used as a hybridization probe for cloning of the R . etli sigA gene . Sequencing data revealed an open reading frame of 2,055 bp showing extensive similarity to various vegetative sigma factors . The 5' end of the sigA transcript was determined and revealed a long, seemingly untranslated region of 170 nucleotides . Quantitative analysis of the sigA transcript by RNase protection and by primer extension assays indicated its down-regulation during entry into the stationary phase . On the basis of the structures of various vegetative sigma factors and considering previous information on heterologous expression, we speculate on the function of domain I of vegetative sigma factors. J Bacteriol, 1996 Dec, 178(24), 7080 - 9 Repressor for the sn-glycerol 3-phosphate regulon of Escherichia coli K-12: primary structure and identification of the DNA-binding domain; Zeng G et al.; The nucleotide sequence of the glpEGR operon of Escherichia coli was determined . The translational reading frame at the beginning, middle, and end of each gene was verified . The glpE gene encodes an acidic, cytoplasmic protein of 108 amino acids with a molecular weight of 12,082 . The glpG gene encodes a basic, cytoplasmic membrane-associated protein of 276 amino acids with a molecular weight of 31,278 . The functions of GlpE and GlpG are unknown . The glpR gene encodes the repressor for the glycerol 3-phosphate regulon, a protein predicted to contain 252 amino acids with a calculated molecular weight of 28,048 . The amino acid sequence of the glp repressor was similar to several repressors of carbohydrate catabolic systems, including those of the glucitol (GutR), fucose (FucR), and deoxyribonucleoside (DeoR) systems of E . coli, as well as those of the lactose (LacR) and inositol (IolR) systems of gram-positive bacteria and agrocinopine (AccR) system of Agrobacterium tumefaciens . These repressors constitute a family of related proteins, all of which contain approximately 250 amino acids, possess a helix-turn-helix DNA-binding motif near the amino terminus, and bind a sugar phosphate molecule as the inducing signal . The DNA recognition helix of the glp repressor and the nucleotide sequence of the glp operator were very similar to those of the deo system . The presumptive recognition helix of the glp repressor was changed by site-directed mutagenesis to match that of the deo repressor or, in a separate construct, to abolish DNA binding . Neither altered form of the glp repressor recognized the glp or deo operator, either in vivo or in vitro . However, both altered forms of the glp repressor were negatively dominant to the wild-type glp repressor, indicating that the inability to bind DNA with high affinity was due to alteration of the DNA-binding domain, not to an inability to oligomerize or instability of the altered repressors . For the first time, analysis of repressors with altered DNA-binding domains has verified the assignment of the helix-turn-helix motif of the transcriptional regulators in the deoR family. J Clin Microbiol, 1996 Dec, 34(12), 3212 - 3 Case of bacterial endophthalmitis caused by an Agrobacterium radiobacter-like organism; Miller JM et al.; A case of postsurgical endophthalmitis caused by Agrobacterium radiobacter in a 70-year-old male is reported . A . radiobacter organisms are normally environmental bacteria but may occasionally be opportunistic pathogens . Infection in this case occurred after the patient was discharged following routine cataract surgery . The infection cleared after empiric therapy intraocular administration of vancomycin and gentamicin. FEMS Microbiol Lett, 1996 Nov 15, 145(1), 55 - 62 Identification, sequencing and mutagenesis of the gene for a D-carbamoylase from Agrobacterium radiobacter; Buson A et al.; A clone positive for D-carbamoylase activity (2.7 kb HindIII-BamHI DNA fragment) was obtained by screening a genomic library of Agrobacterium radiobacter in Escherichia coli . This DNA fragment contains an open reading frame of 912 bp which is predicted to encode a peptide of 304 amino acids with a calculated molecular mass of 34247 Da . The D-carbamoylase gene, named cauA, was placed under the control of T7 RNA-dependent promoter and expressed in E . coli BL21(DE3) . After induction with isopropyl-thio-beta-D-galactopyranoside, the synthesis of D-carbamoylase in E . coli reached about 40% of the total protein . The expressed protein was shown to possess a molecular mass, on SDS-PAGE, of 36 kDa and showed an enhanced stability with respect to that of the wild-type enzyme derived from A . radiobacter . Site-directed mutagenesis experiments allowed us to establish that a Pro14-->Leu14 exchange leads to an inactive enzyme species, while a Cys279-->Ser279 exchange did not impair the functional properties of the enzyme. Gene, 1996 Nov 7, 179(1), 83 - 8 The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein; Lee YW et al.; The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins . Although it is clear that the monosaccharides require binding to a periplasmic binding protein before they can interact with the sensor VirA protein, it is not certain whether the phenolic compounds also interact with a binding protein or directly interact with the sensor protein . To shed light on this question, we tested the vir-inducing abilities of several different phenolic compounds using two wild-type strains of A . tumefaciens, KU12 and A6 . We found that several compounds such as 4-hydroxyacetophone and p-coumaric acid induced the vir of KU12, but not A6 . On the other hand, acetosyringone and several other phenolic compounds induced the vir of A6, but not KU12 . By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic sensing determinant is associated with the Ti plasmid . Subcloning of the Ti plasmid indicated that the virA locus determines which phenolic compounds can function as vir inducers . These results suggest that VirA directly senses the phenolic compounds for vir activation. Mol Microbiol, 1996 Nov, 22(4), 655 - 66 A putative helical domain in the MalK subunit of the ATP-binding-cassette transport system for maltose of Salmonella typhimurium (MalFGK2) is crucial for interaction with MalF and MalG . A study using the LacK protein of Agrobacterium radiobacter as a tool; Wilken S et al.; The ATP-binding-cassette (ABC) protein LacK of Agrobacterium radiobacter displays high sequence similarity to the MalK subunit of the Salmonella typhimurium maltose-transport system (MalFGK2) . We have used LacK as a tool to identify sites of interaction of MalK with the membrane-integral components MalF and MalG . Small amounts of LacK, resulting from the expression of the plasmid-borne lacK gene, proved to be sufficient for partial restoration of growth of a malK strain of S . typhimurium on maltose . LacK failed to substitute for MalK in regulating the expression of maltose-inducible genes but the hybrid complex MalFGLacK2 was sensitive to inducer exclusion . The lacK gene also complemented a ugpC mutant of Escherichia coli to growth on sn-glycerol-3-phosphate as the phosphate source . Partially purified LacK exhibited a spontaneous ATPase activity comparable to that of MalK . A MalK"-'LacK chimeric protein was isolated (by in vivo recombination) in which the N-terminal 140 amino acids of MalK are fused to residues 141-363 of LacK . The protein substituted for MalK in maltose transport considerably better than LacK . Furthermore, random mutagenesis of the plasmid-borne lacK gene yielded three clones that were superior to wild-type lacK in complementing a malK mutation . Single mutations (V114M or L123F) substantially improved the growth of a malK strain on maltose, whereas a double mutation (L123F, S295N) resulted in growth and transport rates that were indistinguishable from those obtained with MalK . In contrast, the introduction of the single change S295N into LacK had no effect but combination with the V114M mutation led to a further twofold increase in transport activity . These results indicate that a putative helical domain in MalK, encompassing residues 89-140, is crucial for a functional, high-affinity interaction with MalF and MalG. Plant Physiol, 1996 Nov, 112(3), 939 - 51 Exogenous phytohormone-independent growth and regeneration of tobacco plants transgenic for the 6b gene of Agrobacterium tumefaciens AKE10; Wabiko H et al.; The 6b gene of Agrobacterium tumefaciens AKE10 (AK-6b) induces crown gall tumors on certain plants but so far there have been no reports of the gene being able to induce tumors on culture medium . We cloned T-DNA segments containing the 6b gene but lacking the auxin and cytokinin biosynthesis genes from A . tumefaciens AKE10 . Tobacco (Nicotiana tabacum) leaf discs infected with A . tumefaciens LBA4404 carrying the clones produced shooty calli on hormone-free Murashige-Skoog medium . The relevant T-DNA segment was integrated into plant DNA as determined by Southern hybridization . Some of these immature shoots spontaneously developed into mature shoots, of which several leaves displayed morphological abnormalities . When leaf discs of these mature plants were placed onto the same medium numerous shoots developed from the wounding sites, indicating that the transgenic plants possessed a high regenerative potential . Northern blot and reverse transcriptase-polymerase chain reaction analyses showed a large accumulation of the AK-6b transcripts in the shooty calli, but only a limited degree in mature plants, demonstrating that AK-6b expression is regulated in plants and essential for the early stages of regeneration . Cytokinin levels in the shooty calli were comparable to those in normal shoots, suggesting that shoot regeneration is not mediated by the modulation of cytokinin content. J Bacteriol, 1996 Nov, 178(21), 6192 - 9 A chimeric disposition of the elongation factor genes in Rickettsia prowazekii; Syvanen AC et al.; An exceptional disposition of the elongation factor genes is observed in Rickettsia prowazekii, in which there is only one tuf gene, which is distant from the lone fus gene . In contrast, the closely related bacterium Agrobacterium tumefaciens has the normal bacterial arrangement of two tuf genes, of which one is tightly linked to the fus gene . Analysis of the flanking sequences of the single tuf gene in R . prowazekii shows that it is preceded by two of the four tRNA genes located in the 5' region of the Escherichia coli tufB gene and that it is followed by rpsJ as well as associated ribosomal protein genes, which in E . coli are located downstream of the tufA gene . The fus gene is located within the str operon and is followed by one tRNA gene as well as by the genes secE and nusG, which are located in the 3' region of tufB in E . coli . This atypical disposition of genes suggests that intrachromosomal recombination between duplicated tuf genes has contributed to the evolution of the unique genomic architecture of R . prowazekii. Mol Plant Microbe Interact, 1996 Nov, 9(8), 704 - 12 HrpG, a key hrp regulatory protein of Xanthomonas campestris pv . vesicatoria is homologous to two-component response regulators; Wengelnik K et al.; Xanthomonas campestris pv . vesicatoria is the causal agent of bacterial spot disease of pepper and tomato plants . Expression of its basic pathogenicity genes, the hrp genes, is induced in planta and in XVM2 medium and is dependent on the hrp regulatory gene hrpXv for five out of six loci in the 23-kb hrp cluster . Here we describe the isolation of a novel hrp gene, hrpG, that was identified after chemical mutagenesis and that is located next to the hrpXv gene . In a hrpG mutant induction of expression of the seven loci hrpA to hrpF, and hrpXv is abolished, suggesting that hrpG functions at the top of the hrp gene regulatory cascade . hrpG is the only gene in the locus and encodes a putative protein of 263 amino acids with a molecular mass of 28.9 kDa . The HrpG amino acid sequence shows similarity to response regulator proteins of the OmpR subclass of two-component systems, being mostly related to the ChvI proteins of Agrobacterium tumefaciens and Rhizobium spp., and TctD of Salmonella typhimurium . Expression of hrpG is low in complex medium, is increased in XVM2 by a factor of four, and is independent of other hrp loci . A model on hrp gene regulation in Xanthomonas campestris pv . vesicatoria is discussed. FEMS Microbiol Lett, 1996 Oct 15, 144(1), 1 - 11 Bartonella bacilliformis: dangerous pathogen slowly emerging from deep background; Ihler GM; Bartonella bacilliformis was perhaps the most lethal bacterial human pathogen in the pre-antibiotic era, but infections were and are limited to a specific geographical area, largely in Peru, corresponding to the range of its sand fly vector . B . bacilliformis targets both red cells and endothelial cells . Recent phylogenetic realignments have revealed a close genetic relationship to other bacteria which cause human diseases, including bacterial angiomatosis, to the former Grahamella species which infect red cells in other mammals, and to plant pathogens and symbionts including Agrobacterium tumefaciens and Rhizobium meliloti . Features of B . bacilliformis that contribute to its pathogenesis are slowly coming into view, and are here reviewed. Virology, 1996 Oct 1, 224(1), 130 - 8 Resistance to tomato yellow leaf curl geminivirus in Nicotiana benthamiana plants transformed with a truncated viral C1 gene; Noris E et al.; The C1 gene of tomato yellow leaf curl geminivirus (TYLCV) encodes a multifunctional protein (Rep) involved in replication . A truncated form of this gene, capable of expressing the N-terminal 210 amino acids (aa) of the Rep protein, was cloned under the control of the CaMV 35S promoter and introduced into Nicotiana benthamiana using Agrobacterium tumefaciens . The same sequence was also cloned in antisense orientation . When self-pollinated progeny of 19 primary transformants were tested for resistance to TYLCV by agroinoculation, some plants proved to be resistant, particularly in the sense lines . Two such lines were further studied . The presence of the transgene was verified and its expression was followed at intervals . All plants that were resistant to TYLCV at 4 weeks postinoculation (wpi) contained detectable amounts of transgenic mRNA and protein at the time of infection . Resistance was overcome in a few plants at 9 wpi, and in most at 15 wpi . Infection of leaf discs derived from transgenic plants showed that expression of the transgene correlated with a substantial reduction of viral DNA replication . Cotransfections of tobacco protoplasts demonstrated that inhibition of viral DNA replication requires expression of the truncated Rep protein and suggested that the small ORF C4, also present in our construct, plays no role in the resistance observed . The results obtained using both transient and stable gene expression systems show that the expression of the N-terminal 210 aa of the TYLCV Rep protein efficiently interferes with virus infection. J Bacteriol, 1996 Oct, 178(20), 6043 - 8 Cyclic beta-(1,2)-glucan synthesis in Rhizobiaceae: roles of the 319-kilodalton protein intermediate; Castro OA et al.; Cyclic beta-(1,2)-glucans are synthesized by members of the Rhizobiaceae family through protein-linked oligosaccharides as intermediates . The protein moiety is a large inner membrane molecule of about 319 kDa . In Agrobacterium tumefaciens and in Rhizobium meliloti the protein is termed ChvB and NdvB, respectively . Inner membranes of R . meliloti 102F34 and A . tumefaciens A348 were first incubated with UDP-{14C}Glc and then solubilized with Triton X-100 and analyzed by polyacrylamide gel electrophoresis under native conditions . A radioactive band corresponding to the 319-kDa protein was detected in both bacteria . Triton-solubilized inner membranes of A . tumefaciens were submitted to native electrophoresis and then assayed for oligosaccharide-protein intermediate formation in situ by incubating the gel with UDP-{14C}Glc . A {14C}glucose-labeled protein with an electrophoretic mobility identical to that corresponding to the 319-kDa {14C}glucan protein intermediate was detected . In addition, protein-linked radioactivity was partially chased when the gel was incubated with unlabeled UDP-Glc . A heterogeneous family of cyclic beta-(1,2)-glucans was formed upon incubation of the gel portion containing the 319-kDa protein intermediate with UDP-{14C}Glc . A protein with an electrophoretic behavior similar to the 319-kDa protein intermediate was "in gel" labeled by using Triton-solubilized inner membranes of an A . tumefaciens exoC mutant, which contains a protein intermediate without nascent glucan . These results indicate that initiation (protein glucosylation), elongation, and cyclization were catalyzed in situ . Therefore, the three enzymatic activities detected in situ reside in a unique protein component (i.e., cyclic beta-(1,2)-glucan synthase) . It is suggested that the protein component is the 319-kDa protein intermediate, which might catalyze the overall cyclic beta-(1,2)-glucan synthesis. J Bacteriol, 1996 Oct, 178(19), 5706 - 11 VirB2 is a processed pilin-like protein encoded by the Agrobacterium tumefaciens Ti plasmid; Jones AL et al.; The mechanism of DNA transmission between distinct organisms has remained a subject of long-standing interest . Agrobacterium tumefaciens mediates the transfer of plant oncogenes in the form of a 25-kb T-DNA sector of a resident Ti plasmid . A growing body of evidence leading to the elucidation of the mechanism involved in T-DNA transfer comes from studies on the vir genes contained in six major operons that are required for the T-DNA transfer process . Recent comparative amino acid sequence studies of the products of these vir genes have revealed interesting similarities between Tra proteins of Escherichia coli F factor, which are involved in the biosynthesis and assembly of a conjugative pilus, and VirB proteins encoded by genes of the virB operon of A . tumefaciens pTiC58 . We have previously identified VirB2 as a pilin-like protein with processing features similar to those of TraA of the F plasmid and have shown that VirB2 is required for the biosynthesis of pilin on a flagella-free Agrobacterium strain . In the present work, VirB2 is found to be processed and localized primarily to the cytoplasmic membrane in E . coli . Cleavage of VirB2 was predicted previously to occur between alanine and glutamine in the sequence -Pro-Ala-Ala-Ala-Glu-Ser- . This peptidase cleavage sequence was mutated by an amino acid substitution for one of the alanine residues (D for A at position 45 {A45D}), by deletion of the three adjacent alanines, and by a frameshift mutation 22 bp upstream of the predicted Ala-Glu cleavage site . With the exception of the frameshift mutation, the alanine mutations do not prevent VirB2 processing in E . coli, while in A . tumefaciens they result in VirB2 instability, since no holo- or processed protein is detectable . All of the above mutations abolish virulence . The frameshift mutation abolishes processing in both organisms . These results indicate that VirB2 is processed into a 7.2-kDa structural protein . The cleavage site in E . coli appears to differ from that predicted in A . tumefaciens . Yet, the cleavage sites are relatively close to each other since the final cleavage products are similar in size and are produced irrespective of the length of the amino-terminal portion of the holoprotein . As we observed previously, the similarity between the processing of VirB2 in A . tumefaciens and the processing of the propilin TraA of the F plasmid now extends to E . coli. Mol Cell Biol, 1996 Oct, 16(10), 5924 - 32 Integration of an insertion-type transferred DNA vector from Agrobacterium tumefaciens into the Saccharomyces cerevisiae genome by gap repair; Risseeuw E et al.; Recently, it was shown that Agrobacterium tumefaciens can transfer transferred DNA (T-DNA) to Saccharomyces cerevisiae and that this T-DNA, when used as a replacement vector, is integrated via homologous recombination into the yeast genome . To test whether T-DNA can be a suitable substrate for integration via the gap repair mechanism as well, a model system developed for detection of homologous recombination events in plants was transferred to S . cerevisiae . Analysis of the yeast transformants revealed that an insertion type T-DNA vector can indeed be integrated via gap repair . Interestingly, the transformation frequency and the type of recombination events turned out to depend strongly on the orientation of the insert between the borders in such an insertion type T-DNA vector. Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10280 - 4 Plant virus DNA replication processes in Agrobacterium: insight into the origins of geminiviruses? Rigden JE, Dry IB, Krake LR, Rezaian MA. Agrobacterium tumefaciens, a bacterial plant pathogen, when transformed with plasmid constructs containing greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indistinguishable from those produced in infected plants . The accumulation of the viral DNA species depends on the presence of two origins of replication in the DNA constructs and is drastically reduced by introducing mutations into the viral replication-associated protein (Rep or C1) ORF, indicating that an active viral replication process is occurring in the bacterial cell . The accumulation of these viral DNA species is not affected by mutations or deletions in the other viral open reading frames . The observation that geminivirus DNA replication functions are supported by the bacterial cellular machinery provides evidence for the theory that these circular single-stranded DNA viruses have evolved from prokaryotic episomal replicons. Nucleic Acids Res, 1996 Sep 15, 24(18), 3649 - 50 A PCR-based DNA fingerprinting technique: AFLP for molecular typing of bacteria; Lin JJ et al.; Amplified restriction fragment polymorphism (AFLP) is a PCR-based DNA fingerprinting technique . In AFLP analysis, bacterial genomic DNA is digested with restriction enzymes, ligated to adapters, and a subset of DNA fragments are amplified using primers containing 16 adapter defined sequences with one additional arbitrary nucleotide . Polymorphisms of different Escherichia coli strains or Agrobacterium tumefaciens strains were demonstrated as distinct, unique bands in a denaturing sequencing gel using AFLP . The polymorphisms detected between BL21 and BL21F'IQ and between DH5 alpha and DH5 alpha F'IQ strains indicated that AFLP is able to resolve differences in F' episomal DNA (approximately 100 kb). Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9975 - 9 Stable transfer of intact high molecular weight DNA into plant chromosomes; Hamilton CM et al.; In conjunction with an enhanced system for Agrobacterium-mediated plant transformation, a new binary bacterial artificial chromosome (BIBAC) vector has been developed that is capable of transferring at least 150 kb of foreign DNA into a plant nuclear genome . The transferred DNA appears to be intact in the majority of transformed tobacco plants analyzed and is faithfully inherited in the progeny . The ability to introduce high molecular weight DNA into plant chromosomes should accelerate gene identification and genetic engineering of plants and may lead to new approaches in studies of genome organization. Microbiology, 1996 Sep, 142 ( Pt 9), 2621 - 9 Expression of the exoY gene, required for exopolysaccharide synthesis in Agrobacterium, is activated by the regulatory ros gene; Tiburtius A et al.; Some mutants of Agrobacterium radiobacter, defective in exopolysaccharide synthesis, were phenotypically complemented by two different regions of cloned chromosomal DNA . One of these had been shown to contain a gene termed ros, a novel class of transcriptional regulator . The other contains a gene termed exoY which encodes a glycosyltransferase that is involved in one of the early steps in exopolysaccharide synthesis . Mutations in ros reduced the expression of exoY and a model to account for the complementation of certain exo alleles by both ros and exoY is presented . TnphoA insertions into exoY which expressed alkaline phosphatase activity were isolated and mapped, confirming the membrane location of the exoY gene product . Some of these mutations were dominant, causing merodiploids to be non-mucoid . exoY is linked to two genes, one encoding an omega-aminotransferase and the other encoding an aldehyde dehydrogenase. J Bacteriol, 1996 Sep, 178(17), 5302 - 8 Requirement for genes with homology to ABC transport systems for attachment and virulence of Agrobacterium tumefaciens; Matthysse AG et al.; Transposon mutants of Agrobacterium tumefaciens which were avirulent and unable to attach to plant cells were isolated and described previously . A clone from a library of Agrobacterium tumefaciens DNA which was able to complement these chromosomal att mutants was identified . Tn3HoHo1 insertions in this clone were made and used to replace the wild-type genes in the bacterial chromosome by marker exchange . The resulting mutants were avirulent and showed either no or very much reduced attachment to carrot suspension culture cells . We sequenced a 10-kb region of this clone and found a putative operon containing nine open reading frames (ORFs) (attA1A2BCDEFGH) . The second and third ORFs (attA2 and attB) showed homology to genes encoding the membrane-spanning proteins (potB and potH; potC and potI) of periplasmic binding protein-dependent (ABC) transport systems from gram-negative bacteria . The homology was strongest to proteins involved in the transport of spermidine and putrescine . The first and fifth ORFs (attA1 and attE) showed homology to the genes encoding ATP-binding proteins of these systems including potA, potG, and cysT from Escherichia coli; occP from A . tumefaciens; cysA from Synechococcus spp.; and ORF-C from an operon involved in the attachment of Campylobacte jejuni . The ability of mutants in these att genes to bind to host cells was restored by addition of conditioned medium during incubation of the bacteria with host cells. Curr Microbiol, 1996 Sep, 33(3), 156 - 62 Octopine- and Nopaline-Inducible Proteins in Agrobacterium tumefaciens Are Also Induced by Arginine Palanichelvam K, Veluthambi K. Octopine induced the synthesis of 83, 76, 62, 58, 44, 42, 31, and 22 kDa proteins in Agrobacterium tumefaciens strains harboring the tumor-inducing (Ti) plasmids pTiA6 and pTiAch5 . Nopaline induced the synthesis of 83, 76, 62, 58, 56, 44, 42, 31, and 22 kDa proteins in A . tumefaciens strains harboring the Ti plasmids pTiC58 and pTiT37 . The molecular masses of proteins induced by octopine and nopaline were very similar . In accordance with the 'opine concept', octopine and nopaline were found to induce protein synthesis only in strains harboring the respective Ti plasmids . Arginine, a common catabolic product of octopine and nopaline, induced the synthesis of most of the proteins induced by the two opines . Our results show that only the initial step(s) of octopine and nopaline catabolism are induced by specific opines in the respective strains . The subsequent steps are likely to be regulated by arginine in both strains. Science, 1996 Aug 23, 273(5278), 1107 - 9 Pilus assembly by Agrobacterium T-DNA transfer genes; Fullner KJ et al.; Agrobacterium tumefaciens can genetically transform eukaryotic cells . In many bacteria, pili are required for interbacterial DNA transfer . The formation of pili by Agrobacterium required induction of tumor-inducing (Ti) plasmid-encoded virulence genes and growth at low temperature . A genetic analysis demonstrated that virA, virG, virB1 through virB11, and virD4 are the only Ti plasmid genes necessary for pilus assembly . The loss and gain of pili in various mutants correlated with the loss and gain of transferred DNA (T-DNA) transfer functions,