Agrobacterium

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, which is consistent with the view that Agrobacterium pili are required for transfer of DNA to plant cells in a process similar to that of conjugation.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 8889 - 94
Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex; Anderson LB et al.; Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants . These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate . To study the function of the VirB proteins in DNA transfer, we developed an expression system in A . tumefaciens . Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins . The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol . Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond . Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.

Mol Biotechnol, 1996 Aug, 6(1), 17 - 30
Transgene inheritance in plants genetically engineered by microprojectile bombardment; Pawlowski WP et al.; Microprojectile bombardment to deliver DNA into plant cells represents a major breakthrough in the development of plant transformation technologies and accordingly has resulted in transformation of numerous species considered recalcitrant to Agrobacterium- or protoplast-mediated transformation methods . This article attempts to review the current understanding of the molecular and genetic behavior of transgenes introduced by microprojectile bombardment . The characteristic features of the transgene integration pattern resulting from DNA delivery via microprojectile bombardment include integration of the full length transgene as well as rearranged copies of the introduced DNA . Copy number of both the transgene and rearranged fragments is often highly variable . Most frequently the multiple transgene copies and rearranged fragments are inherited as a single locus . However, a variable proportion of transgenic events produced by microprojectile bombardment exhibit Mendelian ratios for monogenic and digenic segregation vs events exhibiting segregation distortion . The potential mechanisms underlying these observations are discussed.

Protein Expr Purif, 1996 Aug, 8(1), 109 - 18
Preproricin expressed in Nicotiana tabacum cells in vitro is fully processed and biologically active; Tagge EP et al.; Ricin, the highly toxic glycoprotein expressed in the endosperm of castor seeds, is composed of a galactose-binding lectin B chain (RTB) disulfide linked to a RNA N-glycosidase A chain (RTA) . Chemically modified ricin has been conjugated to monoclonal antibodies and used for targeted therapy of cancer and autoimmune diseases . Replacement of chemically coupled molecules with a genetically engineered targeted ricin would improve homogeneity and yield and permit structural changes in the fusion toxin to be introduced readily by oligonucleotide-directed mutagenesis . Previous methods of expression of ricin fusion proteins have been limited to expression of RTA or RTB moieties alone or expression of incompletely processed toxin in Xenopus laevis oocytes . In the present study, we introduced the cDNA encoding preproricin into cultured tobacco cells via Agrobacterium tumefaciens-mediated gene transfer . Yields of ricin in soluble cell extracts were 1 microg/g in cells or, approximately, 0.1% of the total soluble protein . The ricin was partially purified by P2 monoclonal antibody anti-RTB affinity chromatography . The RTA and RTB immunoreactive material migrated on SDS-PAGE at 65 kDa under nonreducing conditions and at 32-35 kDa under reducing conditions . The tobacco ricin bound to immobilized asialofetuin as avidly as castor bean ricin, suggesting intact sugar binding . Tobacco ricin inhibited rabbit reticulocyte lysate protein translation similar to castor bean ricin (IC50 of 3 x 10(-12) M for tobacco ricin and 1 x 10(-11) M for castor bean ricin) . The human cutaneous T cell lymphoma cell line HUT102 showed similar sensitivity to tobacco ricin when compared to castor bean ricin (IC50 = 9 x 10(-13) and 2 x 10(-12) M, respectively) . The efficiency of gene transfer, reasonable levels of expression, and full post-translational processing indicate that this expression system is suitable for production of ricin fusion toxins for therapeutic applications.

Arch Microbiol, 1996 Aug, 166(2), 92 - 100
Purification and characterization of a novel type of protocatechuate 3,4-dioxygenase with the ability to oxidize 4-sulfocatechol; Hammer A et al.; 4-Aminobenzenesulfonate is degraded via 4-sulfocatechol by a mixed bacterial culture that consists of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2 . From the 4-sulfocatechol-degrading organism A . radiobacter strain S2, a dioxygenase that converted 4-sulfocatechol to 3-sulfomuconate was purified to homogeneity . The purified enzyme also converted protocatechuate with a similar catalytic activity to 3-carboxy-cis, cis-muconate . Furthermore, the purified enzyme oxidized 3, 4-dihydroxyphenylacetate, 3,4-dihydroxycinnamate, catechol, and 3- and 4-methylcatechol . The enzyme had a mol . wt . of about 97,400 as determined by gel filtration and consisted of two different types of subunits with mol . wt . of about 23,000 and 28,500 . The NH2-terminal amino acid sequences of the two subunits were determined . An isofunctional dioxygenase was partially purified from H . palleronii strain S1 . A . radiobacter strain S2 also induced, after growth with 4-sulfocatechol, an rising dbl quote, "ordinary" protocatechuate 3,4-dioxygenase that did not oxidize 4-sulfocatechol . This enzyme was also purified to homogeneity, and its catalytic and structural characteristics were compared to the "4-sulfocatechol-dioxygenase" from the same strain.

J Bacteriol, 1996 Aug, 178(15), 4717 - 20
Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene; Chang CH et al.; Transcription of the virG gene initiates from two tandem promoters, designated P1 and P2, that are located 50 nucleotides apart . Transcription of the P2 promoter is induced by extracellular acidity . cis-acting sites required for P2 activity were identified by constructing and assaying a series of 5' and 3' resections and site-directed nucleotide substitutions . Nucleotides between positions -9 and -37 were sufficient for regulated promoter activity . Within this region, nucleotide substitutions at the predicted -10 and -35 regions strongly reduced P2 expression . In addition, alterations in the region between nucleotides -24 and -32 also eliminated or strongly reduced promoter activity . These data suggest that this promoter may be regulated by a positive transcription factor that binds to nucleotide residues in this interval.

J Bacteriol, 1996 Aug, 178(15), 4710 - 6
Pleiotropic phenotypes caused by genetic ablation of the receiver module of the Agrobacterium tumefaciens VirA protein; Chang CH et al.; The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites . VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module . We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation . In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module . We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module . This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module . These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously . The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.

J Bacteriol, 1996 Aug, 178(15), 4661 - 9
Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in Xanthomonas campestris pv . vesicatoria avrBs2; Swords KM et al.; Molecular characterization of the avrBs2 locus from Xanthomonas campestris pv . vesicatoria has revealed that expression of this gene triggers disease resistance in Bs2 pepper (Capsicum annuum) plants and contributes to virulence of the pathogen . Deletion analysis and site-directed mutagenesis established the avrBs2 gene as a 2,190-bp open reading frame encoding a putative 80.1-kDa protein . Two classes of Xanthomonas pathogens evading Bs2 host resistance and displaying reduced fitness were found to be specifically mutated in avrBs2 . Members of one class contained a 5-bp insertion, while the second class was distinguished by a divergent 3' region of avrBs2; both mutant classes were complemented in trans by a plasmid-borne copy of avrBs2 . A divergent avrBs2 homolog was cloned from the Brassica pathogen X . campestris pv . campestris . The predicted AvrBs2 proteins from the two Xanthomonas pathovars were strongly conserved and had predicted sequence similarity with both Agrobacterium tumefaciens agrocinopine synthase and Escherichia coli UgpQ, two enzymes involved in the synthesis or hydrolysis of phosphodiester linkages . On the basis of homology with agrocinopine synthase and UgpQ and the dual phenotype of avirulence and virulence, several models for the function of AvrBs2 are proposed.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 8145 - 50
Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport; Krysan PJ et al.; The transferred DNA (T-DNA) of Agrobacterium tumefaciens serves as an insertional mutagen once integrated into a host plant's genome . As a means of facilitating reverse genetic analysis in Arabidopsis thaliana, we have developed a method that allows one to search for plants carrying F-DNA insertions within any sequenced Arabidopsis gene . Using PCR, we screened a collection of 9100 independent T-DNA-transformed Arabidopsis lines and found 17 T-DNA insertions within the 63 genes analyzed . The genes surveyed include members of various gene families involved in signal transduction and ion transport . As an example, data are shown for a T-DNA insertion that was found within CPK-9, a member of the gene family encoding calmodulin-domain protein kinases.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7512 - 7
Intermolecular disulfide bonds stabilize VirB7 homodimers and VirB7/VirB9 heterodimers during biogenesis of the Agrobacterium tumefaciens T-complex transport apparatus; Spudich GM et al.; The Agrobacterium tumefaciens VirB7 lipoprotein contributes to the stabilization of VirB proteins during biogenesis of the putative T-complex transport apparatus . Here, we report that stabilization of VirB7 itself is correlated with its ability to form disulfide cross-linked homodimers via a reactive Cys-24 residue . Three types of beta-mercaptoethanol-dissociable complexes were visualized with VirB7 and/or a VirB7::PhoA41 fusion protein: (i) a 9-kDa complex corresponding in size to a VirB7 homodimer, (ii) a 54-kDa complex corresponding in size to a VirB7/VirB7::PhoA41 mixed dimer, and (iii) a 102-kDa complex corresponding to a VirB7::PhoA41 homodimer . A VirB7C24S mutant protein was immunologically undetectable, whereas the corresponding VirB7C24S::PhoA41 derivative accumulated to detectable levels but failed to form dissociable homodimers or mixed dimers with wild-type VirB7 . We further report that VirB7-dependent stabilization of VirB9 is correlated with the ability of these two proteins to dimerize via formation of a disulfide bridge between reactive Cys-24 and Cys-262 residues, respectively . Two types of dissociable complexes were visualized: (i) a 36-kDa complex corresponding in size to a VirB7/VirB9 heterodimer and (ii) an 84-kDa complex corresponding in size to a VirB7/VirB9::PhoA293 heterodimer . A VirB9C262S mutant protein was immunologically undetectable, whereas the corresponding VirB9C262S::PhoA293 derivative accumulated to detectable levels but failed to form dissociable heterodimers with wild-type VirB7 . Taken together, these results support a model in which the formation of disulfide cross-linked VirB7 dimers represent critical early steps in the biogenesis of the T-complex transport apparatus.

Proc Natl Acad Sci U S A, 1996 Jul 9, 93(14), 7321 - 6
A family of lysozyme-like virulence factors in bacterial pathogens of plants and animals; Mushegian AR et al.; We describe a conserved family of bacterial gene products that includes the VirB1 virulence factor encoded by tumor-inducing plasmids of Agrobacterium spp., proteins involved in conjugative DNA transfer of broad-host-range bacterial plasmids, and gene products that may be involved in invasion by Shigella spp . and Salmonella enterica . Sequence analysis and structural modeling show that the proteins in this group are related to chicken egg white lysozyme and are likely to adopt a lysozyme-like structural fold . Based on their similarity to lysozyme, we predict that these proteins have glycosidase activity . Iterative data base searches with three conserved sequence motifs from this protein family detect a more distant relationship to bacterial and bacteriophage lytic transglycosylases, and goose egg white lysozyme . Two acidic residues in the VirB1 protein of Agrobacterium tumefaciens form a putative catalytic dyad, Each of these residues was changed into the corresponding amide by site-directed mutagenesis . Strains of A . tumefaciens that express mutated VirB1 proteins have a significantly reduced virulence . We hypothesize that many bacterial proteins involved in export of macromolecules belong to a widespread class of hydrolases and cleave beta-1,4-glycosidic bonds as part of their function.

Indian J Exp Biol, 1996 Jul, 34(7), 716 - 8
Direct somatic embryogenesis and transformation in Cicer arietinum L; Ramana RV et al.; Somatic embryos were induced directly from immature cotyledons of the genotype of chickpea ICC 4918 (annigiri) on B5 medium supplemented with 2,4,5-T or 2,4-D in combination with BA or KN . Successful transformation was achieved via somatic embryogenesis using Agrobacterium tumefaciens strain LBA4404, carrying a binary plasmid vector system containing neomycin phosphotransferase (NPT II) gene as the selectable marker and beta-glucuronidase (GUS) as a reporter gene . Histochemical staining for GUS expression was observed as primary evidence for transformation.

Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 617 - 29
Characterization of the biosynthesis of beta(1-2) cyclic glucan in R . Fredii . Beta(1-2) glucan has no apparent role in nodule invasion of Mc Call and Peking soybean cultivars; Inon de Iannino N et al.; Three wild type strains of Rhizobium fredii, USDA 191, USDA 257 and HH 303, do not synthesize in vivo or in vitro beta(1-3), beta(1-6) cyclic glucans, all strains form in vitro and in vivo cyclic beta(1-2) glucans . Approximately 80% of the recovered R . fredii cellular cyclic beta(1-2) glucans were anionic and the substituent was identified as phosphoglycerol . Inner membranes prepared from these R . fredii strains have a beta(1-2) glucan-intermediate-protein with apparent molecular mass undistinguishable from Agrobacterium tumefaciens beta(1-2) glucan intermediate protein . Studies of the degree of polymerization of the oligosaccharides recovered from the protein-intermediate after short pulse incubations with UDP-14C-glucose suggested that the rate limiting step in the biosynthesis of cyclic glucan is cyclization . Kinetic studies revealed that the K(m) for UDP-glucose was 0.33 mM . No difference was detected between the K(m) for initiation/elongation and cyclization reactions . Nodulation studies of a ndvB R . fredii mutant with Mc Call and Peking soybean cultivars, revealed that beta(1-2) glucans do not seem to be required for normal nodule invasion of these soybean cultivars.

Plant Mol Biol, 1996 Jul, 31(4), 887 - 95
Expression of two heterologous promoters, Agrobacterium rhizogenes rolC and cauliflower mosaic virus 35S, in the stem of transgenic hybrid aspen plants during the annual cycle of growth and dormancy; Nilsson O et al.; We monitored, for the first time, the activity of two model heterologous promoters, the Agrobacterium rhizogenes rolC and the cauliflower mosaic virus (CaMV) 35S, throughout the annual cycle of growth and dormancy in a perennial species, hybrid aspen . Each promoter was fused to the uidA beta-glucuronidase (GUS) reporter gene and the constructs were introduced into the hybrid aspen genome by Agrobacterium-mediated transformation . Both wild-type and transgenic plants were cultivated under different regimes of photoperiod and temperature to induce passage through one growth-dormancy-reactivation cycle, and at intervals GUS staining was assessed in stem sections . In rolC::uidA transformants, GUS activity in rapidly growing current-year shoots was not only tissue-specific, being localized to the phloem, but also cell-specific at the shoot base, where it was present only in the companion cells . However, during the onset of dormancy induced by short photoperiod, GUS activity shifted laterally from the phloem to include the cortex and pith . After subsequent exposure to chilling temperatures to induce the transition between the dormancy stages of rest and quiescence, GUS activity almost disappeared from all stem tissues, but regained its original phloem specificity and intensity after the shoots were reactivated by exposing them to long photoperiod and high temperatures . In contrast, GUS activity in the stem of 35S::uidA transformants was strong in all tissues except for the vascular cambium and xylem, and did not vary in intensity during the growth-dormancy-reactivation cycle . The lateral shift and increased intensity of GUS activity in the stem of rolC::uidA transformants during dormancy induction was shown to be associated with the accumulation of starch, and to be mimicked by incubating stem sections in sucrose, as well as glucose and fructose, but not sorbitol, prior to the GUS assay . Our results demonstrate that the activities of the rolC and 35S promoters varied in very different, unpredictable ways during the annual cycle of growth and dormancy in a perennial species, and indicate that the spatial and temporal variation in rolC promoter activity that we observed in the stem of transgenic hybrid aspen plants is attributable to cellular and seasonal changes in sucrose content.

Can J Microbiol, 1996 Jul, 42(7), 621 - 7
Changes in the surface charge of bacteria caused by heavy metals do not affect survival; Collins YE et al.; Bacillus subtilis and Agrobacterium radiobacter remained viable when exposed to Ni (1 x 10(-4)M; ionic strength (mu) = 3 x 10(-4)) at pH values known to cause a change of the net negative charge of the cells to a net positive charge (charge reversal) . The gross morphology, as determined by scanning electron microscopy, of these and other bacteria and of Saccharomyces cerevisiae was not altered in the presence of Ni, Cu, and Zn (1 x 10(-4) M; mu = 3 x 10(-4)), which caused a charge reversal at pH values between 6.0 and 9.0 . Similar results were obtained in the presence of Na and Mg, which did not cause charge reversal at the same mu and pH values . These results confirmed that cells remain viable when their surface charge is changed in the presence of some heavy metals at high pH values.

J Bacteriol, 1996 Jul, 178(14), 4248 - 57
The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes; Alt-Morbe J et al.; We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer . Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58 . One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore . Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid . An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids . We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources . A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems . The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids . We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.

J Bacteriol, 1996 Jul, 178(14), 4233 - 47
The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F; Farrand SK et al.; The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems . One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another . The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58 . By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region . Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units . We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra . The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site . One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG . Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested . Proteins of the predicted sizes were expressible from traA, traC, traD, and traG . The products of several Ti plasmid tra genes are related to those of other conjugation systems . The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F . The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system . Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function . traB, while not essential, is required for maximum frequency of transfer . Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.

Plant J, 1996 Jul, 10(1), 165 - 74
Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers; Komari T et al.; Novel 'super-binary' vectors that carried two separate T-DNAs were constructed . One T-DNA contained a drug-resistance, selection-marker gene and the other contained a gene for beta-glucuronidase (GUS) . A large number of tobacco (Nicotiana tabacum L.) and rice (Oryza sativa L.) transformants were produced by Agrobacterium tumefaciens LBA4404 that carried the vectors . Frequency of co-transformation with the two T-DNAs was greater than 47% GUS-positive, drug-sensitive progeny were obtained from more than half of the co-transformants . Molecular analyses by Southern hybridization and polymerase chain reactions confirmed integration and segregation of the T-DNAs . Thus, the non-selectable T-DNA that was genetically separable from the selection marker was integrated into more than a quarter of the initial, drug-resistant transformants . Since various DNA fragments may be inserted into the non-selectable T-DNA by a simple procedure, these vectors will likely be very useful for the production of marker-free transformants of diverse plant species . Delivery of two T-DNAs to plants from mixtures of A . tumefaciens was also tested, but frequency of co-transformation was relatively low.

Microbiology, 1996 Jul, 142 ( Pt 7), 1705 - 13
Diversity of repC plasmid-replication sequences in Rhizobium leguminosarum; Turner SL et al.; Homologues of the plasmid replicator gene repC were detected and characterized in a sample of Rhizobium leguminosarum strains . Conserved PCR primers were designed from published sequences of repC; they amplified a fragment of about 750 bp from 39 out of 41 strains tested, and also from several Sinorhizobium strains, including S . meliloti . Restriction endonuclease digestion showed that the PCR product from individual strains, though uniform in size, was often heterogeneous in sequence . PCR products from 24 field isolates of R . leguminosarum from France, Germany and the UK were cloned and partially sequenced from both ends . Phylogenies constructed from the 5' and 3' ends (200 bp each) were largely congruent and demonstrated four clearly defined groups plus several unique strains . Published Agrobacterium repC sequences fall within the phylogeny of R . leguminosarum sequences, though not within any of the four groups . Specific pairs of PCR primers were designed for each of the four groups; 29 out of 41 R . leguminosarum strains gave a PCR product of the expected size with more than one group-specific primer pair . We hypothesize that the sequence groups correspond to incompatibility groups of Rhizobium plasmids.

Mol Plant Microbe Interact, 1996 Jul, 9(5), 401 - 8
Characterization and distribution of tartrate utilization genes in the grapevine pathogen Agrobacterium vitis; Salomone JY et al.; Agrobacterium vitis is a common pathogen of grapevine . Most strains utilize tartrate, an abundant compound in grapevine . Strain AB3 carries two tartrate utilization (or TAR) regions: TAR-I (on the large pTrAB3 plasmid) and TAR-II (on the AB3 Ti plasmid) . TAR-I and TAR-II were structurally and functionally analyzed and are similar to the TAR-III region from the tartrate utilization plasmid pTrAB4 of the nopaline-type A . vitis strain AB4 (Crouzet and Otten, J . Bacteriol . 1995, 177:6518-6526) . The minimal tartrate utilization region of TAR-I contains four genes (ttuA-ttuD) . The ttuC gene is homologous to the tartrate dehydrogenase gene from Pseudomonas putida . Outside the minimal region a second ttuC-like gene is found (ttuC') which is transcribed and complements a ttuC mutant . Most grapevine isolates carry one or two of the three characterized TAR regions and show a considerable degree of polymorphism around these regions.

Arch Microbiol, 1996 Jul, 166(1), 68 - 70
Major differences between the rrnA operons of two strains of Agrobacterium vitis; Otten L et al.; The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554 . Compared to the earlier obtained rrnA sequence of A . vitis strain S4, several important differences were noted: the sequences diverged at the 5'-flanking region, within the 16S-23S intergenic region, and within the 23S rRNA sequence . The B8 stem-loop structure at the 5'-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4 . These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons.

Science, 1996 Jun 14, 272(5268), 1655 - 8
Enzymatic synthesis of a quorum-sensing autoinducer through use of defined substrates; More MI et al.; Many bacteria, including several pathogens of plants and humans, use a pheromone called an autoinducer to regulate gene expression in a cell density-dependent manner . Agrobacterium autoinducer {AAI, N-(3-oxo-octanoyl)-L-homoserine lactone} of A . tumefaciens is synthesized by the Tral protein, which is encoded by the tumor-inducing plasmid . Purified hexahistidinyl-Tral (H6-Tral) used S-adenosylmethionine to make the homoserine lactone moiety of AAI, but did not use related compounds . H6-Tral used 3-oxo-octanoyl-acyl carrier protein to make the 3-oxo-octanoyl moiety of AAI, but did not use 3-oxo-octanoyl-coenzyme A . These results demonstrate the enzymatic synthesis of an autoinducer through the use of purified substrates.

Biochim Biophys Acta, 1996 Jun 4, 1290(2), 177 - 83
Purification and properties of L(-)-carnitine dehydrogenase from Agrobacterium sp; Hanschmann H et al.; L(-)-Carnitine:NAD+ oxidoreductase, EC 1.1.1.108, from Agrobacterium sp . catalyzes the oxidation of L(-)-carnitine to 3-dehydrocarnitine as initial step of L(-)-carnitine degradation . The enzyme was purified 76-fold by four chromatographic steps . A high substrate specificity for L(-)-carnitine and NAD+ was observed . The molecular mass of the native enzyme is 114 kDa and it consists of two identical subunits as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis . The isoelectric point was found to be 5.2-5.4 . The optimum temperature is 45 degrees C and the optimum pH for the oxidation and the reduction reaction are 9.5 and 5.5-6.5, respectively . Kinetic parameters and amino-terminal sequence were determined . The oxidation reaction is inhibited by D(+)-carnitine, trimethylamine, several metal ions and cetyltrimethylammoniumbromide (CTAB).

Mol Microbiol, 1996 Jun, 20(6), 1199 - 210
Localization of OccR-activated and TraR-activated promoters that express two ABC-type permeases and the traR gene of Ti plasmid pTiR10; Fuqua C et al.; Conjugation of Agrobacterium tumefaciens wide-host-range octopine-type Ti plasmids is regulated by the LuxR-type transcriptional activator TraR in conjunction with an acylated homoserine lactone designated AAI . Expression of traR in octopine-type Ti plasmids is stimulated by OccR in response to octopine, an opine released from crown gall tumours, and is also positively autoregulated by TraR and AAI . Genetic and physical mapping of these promoters indicates that the OccR-activated promoter lies 14.5 kb upstream of traR, while the TraR-activated promoter lies 6 kb upstream . The upstream portion of the 14.5 kb operon contains seven previously characterized genes that direct the uptake and catabolism of octopine . The TraR-activated promoter lies just downstream from the octopine catabolic genes, and transcribes six genes in addition to traR, including five genes (ophABCDE) that show strong homology to oligo-peptide permeases of Salmonella typhimurium and Bacillus subtilis . Several TraR-regulated promoters overlap with 18 bp inverted repeats called tra boxes . In contrast, the traR autoregulatory promoter is not associated with a consensus tra box.

Plant Mol Biol, 1996 Jun, 31(3), 677 - 81
Deviating T-DNA transfer from Agrobacterium tumefaciens to plants; van der Graaff E et al.; We analyzed 29 T-DNA inserts in transgenic Arabidopsis thaliana plants for the junction of the right border sequences and the flanking plant DNA . DNA sequencing showed that in most lines the right border sequences transferred had been preserved during integration, corroborating literature data . Surprisingly, in four independent transgenic lines a complete right border repeat was present followed by binary vector sequences . Cloning of two of these T-DNA inserts by plasmid rescue showed that in these lines the transferred DNA consisted of the complete binary vector sequences in addition to the T-region . On the basis of the structure of the transferred DNA we propose that in these lines T-DNA transfer started at the left-border repeat, continued through the vector part, passed the right border repeat, and ended only after reaching again this left-border repeat.

Biodegradation, 1996 Jun, 7(3), 223 - 9
Degradation of 4-aminobenzenesulfonate by a two-species bacterial coculture . Physiological interactions between Hydrogenophaga palleronii S1 and Agrobacterium radiobacter S2; Dangmann E et al.; The mutualistic interactions in a 4-aminobenzenesulfonate (sulfanilate) degrading mixed bacterial culture were studied . This coculture consisted of Hydrogenophaga palleronii strain S1 and Agrobacterium radiobacter strain S2 . In this coculture only strain S1 desaminated sulfanilate to catechol-4-sulfonate, which did not accumulate in the medium but served as growth substrate for strain S2 . During growth in batch culture with sulfanilate as sole source of carbon, energy, nitrogen and sulfur, the relative cell numbers (colony forming units) of both strains were almost constant . None of the strains reached a cell number which was more than threefold higher than the cell number of the second strain . A mineral medium with sulfanilate was inoculated with different relative cell numbers of both strains (relative number of colony forming units S1:S2 2200:1 to 1:500) . In all cases, growth was found and the proportion of both strains moved towards an about equal value of about 3:1 (strain S1:strain S2) . In contrast to the coculture, strain S1 did not grow in a mineral medium in axenic culture with 4-aminobenzenesulfonate or any other simple organic compound tested . A sterile culture supernatant from strain S2 enabled strain S1 to grow with 4-aminobenzenesulfonate . The same growth promoting effect was found after the addition of a combination of 4-aminobenzoate, biotin and vitamin B12 . Strain S1 grew with 4-aminobenzenesulfonate plus the three vitamins with about the same growth rate as the mixed culture in a mineral medium . When (resting) cells of strain S1 were incubated in a pure mineral medium with sulfanilate, up to 30% of the oxidized sulfanilate accumulated as catechol-4-sulfonate in the culture medium . In contrast, only minor amounts of catechol-4-sulfonate accumulated when strain S1 was grown with 4ABS in the presence of the vitamins.

J Bacteriol, 1996 Jun, 178(12), 3671 - 5
Conservation of PcaQ, a transcriptional activator of pca genes for catabolism of phenolic compounds, in Agrobacterium tumefaciens and Rhizobium species; Parke D; In Agrobacterium tumefaciens A348, control of five genes for catabolism of the phenolic compound protocatechuate to beta-ketoadipate is exerted by the gene pcaQ . The product of pcaQ is a transcriptional activator which is distinct from regulators of the beta-ketoadipate pathway characterized in other bacterial groups . An investigation of whether pcaQ is present and conserved in related Rhizobium species employed Southern hybridization and an agrobacterial pcaD::LacZ promoter probe plasmid . These studies revealed that homologs of the activator are widespread among members of the family Rhizobiaceae, being present in Rhizobium leguminosarum, Rhizobium fredii, Rhizobium meliloti, Rhizobium etli, and Rhizobium tropici.

J Bacteriol, 1996 Jun, 178(12), 3634 - 40
Heat shock activation of the groESL operon of Agrobacterium tumefaciens and the regulatory roles of the inverted repeat; Segal G et al.; Deletions were constructed in the conserved inverted repeat (IR) found in the groESL operon of Agrobacterium tumefaciens and in many other groE and dnaK operons and genes in eubacteria . These deletions affected the level of expression of the operon and the magnitude of its heat shock activation . The IR seems to operate at the DNA level, probably as an operator site that binds a repressor under non-heat shock conditions . The IR was also found to function at the mRNA level, since under non-heat shock conditions transcripts containing deletions of one side of the IR had longer half-lives than did transcripts containing the wild-type IR . Under heat shock conditions, the half-life of the mRNA was unaffected by this deletion because of heat shock-dependent cleavage . However, the groESL operon was found to be heat shock activated even after most of the IR was deleted . This observation, together with the fact that the groESL operon of A . tumefaciens was heat shock activated in Escherichia coli and vice versa, suggests that a heat shock promoter regulates the heat shock activation of this operon . The primary role of the IR appears to be in reducing the MRNA levels from this promoter under non-heat shock conditions.

J Bacteriol, 1996 Jun, 178(11), 3285 - 92
A Ti plasmid-encoded enzyme required for degradation of mannopine is functionally homologous to the T-region-encoded enzyme required for synthesis of this opine in crown gall tumors; Kim KS et al.; The mocC gene encoded by the octopine/mannityl opine-type Ti plasmid pTi15955 is related at the nucleotide sequence level to mas1' encoded by the T region of this plasmid . While Mas1 is required for the synthesis of mannopine (MOP) by crown gall tumor cells, MocC is essential for the utilization of MOP by Agrobacterium spp . A cosmid clone of pTi15955, pYDH208, encodes mocC and confers the utilization of MOP on strain NT1 and on strain UIA5, a derivative of NT1 lacking the 450-kb cryptic plasmid pAtC58 . NT1 or UIA5 harboring pYDH208 with an insertion mutation in mocC failed to utilize MOP as the sole carbon source . Plasmid pSa-C, which encodes only mocC, complemented this mutation in both strains . This plasmid also was sufficient to confer utilization of MOP on NT1 but not on UIA5 . Computer analysis showed that MocC is related at the amino acid sequence level to members of the short-chain alcohol dehydrogenase family of oxidoreductases . Lysates prepared from Escherichia coli cells expressing mocC contained an enzymatic activity that oxidizes MOP to deoxyfructosyl glutamine (santhopine {SOP}) in the presence of NAD+ . The reaction catalyzed by the MOP oxidoreductase is reversible; in the presence of NADH, the enzyme reduced SOP to MOP . The apparent Km values of the enzyme for MOP and SOP were 6.3 and 1.2 mM, respectively . Among analogs of MOP tested, only N-1-(1-deoxy-D-lyxityl)-L-glutamine and N-1-(1-deoxy-D-mannityl)-L-asparagine served as substrates for MOP oxidoreductase . These results indicate that mocC encodes an oxidoreductase that, as an oxidase, is essential for the catabolism of MOP . The reductase activity of this enzyme is precisely the reaction ascribed to its T-region-encoded homolog, Mas1, which is responsible for biosynthesis of mannopine in crown gall tumors.

J Bacteriol, 1996 Jun, 178(11), 3275 - 84
Ti plasmid-encoded genes responsible for catabolism of the crown gall opine mannopine by Agrobacterium tumefaciens are homologs of the T-region genes responsible for synthesis of this opine by the plant tumor; Kim KS et al.; Agrobacterium tumefaciens NT1 harboring pSaB4, which contains the 14-kb BamHI fragment 4 from the octopine/mannityl opine-type Ti plasmid pTi15955, grew well with agropine (AGR) but slowly with mannopine (MOP) as the sole carbon source . When a second plasmid encoding a dedicated transport system for MOP was introduced, these cells grew well with both AGR and MOP . Transposon insertion mutagenesis and subcloning identified a 5.7-kb region of BamHI fragment 4 that encodes functions required for the degradation of MOP . DNA sequence analysis revealed seven putative genes in this region: mocD (moc for mannityl opine catabolism) and mocE, oriented from right to left, and mocRCBAS, oriented from left to right . Significant identities exist at the nucleotide and derived amino acid sequence levels between these moc genes and the mas genes that are responsible for opine biosynthesis in crown gall tumors . MocD is a homolog of Mas2, the anabolic conjugase encoded by mas2' . MocE and MocC are related to the amino half and the carboxyl half, respectively, of Mas1 (MOP reductase), the second enzyme for MOP biosynthesis . These results indicate that the moc and mas genes evolved from a common origin . MocR and MocS are related to each other and to a putative repressor for the AGR degradation system encoded by the rhizogenic plasmid pRiA4 . MocB and MocA are homologs of 6-phosphogluconate dehydratase and glucose-6-phosphate dehydrogenase, respectively . Mutations in mocD and mocE, but not mocC, are suppressed by functions encoded by the chromosome or the 450-kb megaplasmid present in many Agrobacterium isolates . We propose that moc genes derived from genes located elsewhere in the bacterial genome and that the tumor-expressed mas genes evolved from the bacterial moc genes.

J Bacteriol, 1996 Jun, 178(11), 3168 - 76
The Agrobacterium tumefaciens VirB7 lipoprotein is required for stabilization of VirB proteins during assembly of the T-complex transport apparatus; Fernandez D et al.; The Agrobacterium tumefaciens virB7 gene product is a lipoprotein whose function is required for the transmission of oncogenic T-DNA to susceptible plant cells . Three lines of study provided evidence that VirB7 interacts with and stabilizes other VirB proteins during the assembly of the putative T-complex transport apparatus . First, a precise deletion of virB7 from the pTiA6NC plasmid of wild-type strain A348 was correlated with significant reductions in the steady-state levels of several VirB proteins, including VirB4, VirB9, VirB10, and VirB11; trans expression of virB7 in the delta virB7 mutant partially restored the levels of these proteins, and trans coexpression of virB7 and virB8 fully restored the levels of these proteins to wild-type levels . Second, modulation of VirB7 levels resulted in corresponding changes in the levels of other VirB proteins in the following cell types: (i) a delta virB7 mutant expressing virB7 and virB8 from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible Plac and other virB genes from acetosyringone (AS)-inducible PvirB; (ii) a delta virB operon mutant expressing virB7 and virB8 from Plac and virB9, virB10, and virB11 from PvirB; and (iii) a delta virB operon mutant expressing virB7 from IPTG-inducible Pklac and virB9 from an AS-inducible PvirB . Third, the synthesis of a VirB7::PhoA fusion protein in strain A348 was correlated with a significant reduction in the steady-state levels of VirB4, VirB5, and VirB7 through VirB11; these cells also exhibited a severely attenuated virulence phenotype, indicating that synthesis of the fusion protein perturbs the assembly of VirB proteins into a stabilized protein complex required for T-complex transport . Extracts of AS-induced cells electrophoresed under nonreducing conditions possessed undetectable levels of the 32-kDa VirB9 and 4.5-kDa VirB7 monomers and instead possessed a 36-kDa complex that cross-reacted with both VirB7 and VirB9 antisera and accumulated as a function of virB7 expression . Our results are consistent with a model in which VirB7 stabilizes VirB9 by formation of a covalent intermolecular cross-link; in turn, the VirB7-VirB9 heterodimer promotes the assembly of a functional T-complex transport machinery.

J Bacteriol, 1996 Jun, 178(11), 3156 - 67
The Agrobacterium tumefaciens virB7 gene product, a proposed component of the T-complex transport apparatus, is a membrane-associated lipoprotein exposed at the periplasmic surface; Fernandez D et al.; The Agrobacterium tumefaciens virB7 gene product contains a typical signal sequence ending with a consensus signal peptidase II cleavage site characteristic of bacterial lipoproteins . VirB7 was shown to be processed as a lipoprotein by (i) in vivo labeling of native VirB7 and a VirB7::PhoA fusion with {3H}palmitic acid and (ii) inhibition of VirB7 processing by globomycin, a known inhibitor of signal peptidase II . A VirB7 derivative sustaining a Ser substitution for the invariant Cys-15 residue within the signal peptidase II cleavage site could not be visualized immunologically and failed to complement a delta virB7 mutation, establishing the importance of this putative lipid attachment site for VirB7 maturation and function . VirB7 partitioned predominantly with outer membrane fractions from wild-type A348 cells as well as a delta virB operon derivative transformed with a virB7 expression plasmid . Expression of virB7 fused to phoA, the alkaline phosphatase gene of Escherichia coli, gave rise to high alkaline phosphatase activities in E . coli and A . tumefaciens cells, providing genetic evidence for the export of VirB7 in these hosts . VirB7 was shown to be intrinsically resistant to proteinase K; by contrast, a VirB7::PhoA derivative was degraded by proteinase K treatment of A . tumefaciens spheroplasts and remained intact upon treatment of whole cells . Together, the results of these studies favor a model in which VirB7 is topologically configured as a monotopic protein with its amino terminus anchored predominantly to the outer membrane and with its hydrophilic carboxyl domain located in the periplasmic space . Parallel studies of VirB5, VirB8, VirB9, and VirB10 established that each of these membrane-associated proteins also contains a large periplasmic domain whereas VirB11 resides predominantly or exclusively within the interior of the cell.

Ann N Y Acad Sci, 1996 May 25, 792, 62 - 71
Bioproduction of human enzymes in transgenic tobacco; Cramer CL et al.; Transgenic plants have significant potential in the bioproduction of complex human therapeutic proteins due to ease of genetic manipulation, lack of potential contamination with human pathogens, conservation of eukaryotic cell machinery mediating protein modification, and low cost of biomass production . Tobacco has been used as our initial transgenic system because Agrobacterium-mediated transformation is highly efficient, prolific seed production greatly facilitates biomass scale-up, and development of new "health-positive" uses for tobacco has significant regional support . We have targeted bioproduction of complex recombinant human proteins with commercial potential as human pharmaceuticals . Human protein C (hPC), a highly processed serum protease of the coagulation/anticoagulation cascade, was produced at low levels in transgenic tobacco leaves . Analogous to its processing in mammalian systems, tobacco-synthesized hPC appears to undergo multiple proteolytic cleavages, disulfide bond formation, and N-linked glycosylation . Although tobacco-derived hPC has not yet been tested for all posttranslational modifications or for enzymatic (anticlotting) activity, these results are promising and suggest considerable conservation of protein processing machinery between plants and animals . CropTech researchers have also produced the human lysosomal enzyme glucocerebrosidase (hGC) in transgenic tobacco . This glycoprotein has significant commercial potential as replacement therapy in patients with Gaucher's disease . Regular intravenous administration of modified glucocerebrosidase, derived from human placentae or CHO cells, has proven highly effective in reducing disease manifestations in patients with Gaucher's disease . However, the enzyme is expensive (dubbed the "world's most expensive drug" by the media), making it a dramatic model for evaluating the potential of plants to provide a safe, low-cost source of bioactive human enzymes . Transgenic tobacco plants were generated that contained the human glucocerebrosidase cDNA under the control of an inducible plant promoter . hGC expression was demonstrated in plant extracts by enzyme activity assay and immunologic cross-reactivity with anti-hGC antibodies . Tobacco-synthesized hGC comigrates with human placental-derived hGC during electrophoretic separations, is glycosylated, and, most significantly, is enzymatically active . Although expression levels vary depending on transformant and induction protocol, hGC production of > 1 mg/g fresh weight of leaf tissue has been attained in crude extracts . Our studies provide strong support for the utilization of tobacco for high-level production of active hGC for purification and eventual therapeutic use at potentially much reduced costs . Furthermore, this technology should be directly adaptable to the production of a variety of other complex human proteins of biologic and pharmaceutical interest.

Virology, 1996 May 15, 219(2), 387 - 94
Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin; Briddon RW et al.; The genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced . TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies) . Infectivity of the cloned viral genome was demonstrated by Agrobacterium-mediated inoculation of several host species . Progeny virus was transmissible by the treehopper vector of TPCTV, Micrutalis malleifera (Fowler) . The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae . The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whitefly Bemisia tabaci Genn . The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus.

Proc Natl Acad Sci U S A, 1996 May 14, 93(10), 5055 - 60
Two different but related mechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination; Puchta H et al.; Genomic double-strand breaks (DSBs) are key intermediates in recombination reactions of living organisms . We studied the repair of genomic DSBs by homologous sequences in plants . Tobacco plants containing a site for the highly specific restriction enzyme I-Sce I were cotransformed with Agrobacterium strains carrying sequences homologous to the transgene locus and, separately, containing the gene coding for the enzyme . We show that the induction of a DSB can increase the frequency of homologous recombination at a specific locus by up to two orders of magnitude . Analysis of the recombination products demonstrates that a DSB can be repaired via homologous recombination by at least two different but related pathways . In the major pathway, homologies on both sides of the DSB are used, analogous to the conservative DSB repair model originally proposed for meiotic recombination in yeast . Homologous recombination of the minor pathway is restricted to one side of the DSB as described by the nonconservative one-sided invasion model . The sequence of the recombination partners was absolutely conserved in two cases, whereas in a third case, a deletion of 14 bp had occurred, probably due to DNA polymerase slippage during the copy process . The induction of DSB breaks to enhance homologous recombination can be applied for a variety of approaches of plant genome manipulation.

J Appl Bacteriol, 1996 May, 80(5), 517 - 28
Characterization of Rhizobium 'hedysari' by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting; Selenska-Pobell S et al.; The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers . For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari' . As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used . By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh . 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position . The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible . In parallel, analysis of the same strains was performed by PCR amplification of their DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR) . By both AP and rep-PCR an identification of every particular strain was achieved . In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons . On the basis of the results presented here it can be concluded that AP and rep-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.

Plant Cell, 1996 May, 8(5), 873 - 86
Early transcription of Agrobacterium T-DNA genes in tobacco and maize; Narasimhulu SB et al.; We developed a sensitive procedure to investigate the kinetics of transcription of an Agrobacterium tumefaciens transferred (T)-DNA-encoded beta-glucuronidase gusA (uidA) gene soon after infection of plant suspension culture cells . The procedure uses a reverse transcriptase-polymerase chain reaction and enables detection of gusA transcripts within 18 to 24 hr after cocultivation of the bacteria with either tobacco or maize cells . Detection of gusA transcripts depended absolutely on the intact virulence (vir) genes virB, virD1/virD2, and virD4 within the bacterium . Mutations in virC and virE resulted in delayed and highly attenuated expression of the gusA gene . A nonpolar transposon insertion into the C-terminal coding region of virD2 resulted in only slightly decreased production of gusA mRNA, although this insertion resulted in the loss of the nuclear localization sequence and the important omega region from VirD2 protein and rendered the bacterium avirulent . However, expression of gusA transcripts in tobacco infected by this virD2 mutant was more transient than in cells infected by a wild-type strain . Infection of tobacco cells with an Agrobacterium strain harboring a mutant virD2 allele from which the omega region had been deleted resulted in similar transient expression of gusA mRNA . These data indicate that the C-terminal nuclear localization signal of the VirD2 protein is not essential for nuclear uptake of T-DNA and further suggest that the omega domain of VirD2 may be required for efficient integration of T-DNA into the plant genome . The finding that the initial kinetics of gusA gene expression in maize cells are similar to those shown in infected tobacco cells but that the presence of gusA mRNA in maize is highly transient suggests that the block to maize transformation involves T-DNA integration and not T-DNA entry into the cell or nuclear targeting.

Mol Plant Microbe Interact, 1996 May, 9(4), 310 - 3
Translocation and exudation of tumor metabolites in crown galled plants; Savka MA et al.; Crown gall tumors are induced on susceptible plants by pathogenic strains of Agrobacterium . These neoplastic plants cells produce metabolites, called opines, which provide a source of nutrients to colonizing agrobacteria . Opine production previously has been shown to influence microbial communities in the immediate vicinity of the tumor . We have obtained evidence for opine translocation to and exudation from distal uninfected regions of the plant host . Grafted plants made from an opine-producing transgenic scion with a wild-type stock, or with a wild-type scion and an opine-producing stock accumulate opines in the wild-type portions of the plant . Moreover, opines were detected in root, stem, and leaf tissues of nontransgenic plants on which stem crown galls had been induced by pathogenic strains of Agrobacterium . These plants exuded opines from their roots as a component of their root exudates . Translocation of opines from the tumor to other parts of the plant, and their exudation from roots, indicates that these biologically active compounds are available to opine-catabolizing microbes that have not induced the tumors but are present in the rhizosphere or on portions of the plant distant from the site of the gall.

Mol Gen Genet, 1996 Apr 24, 251(1), 99 - 107
Sequence and characterisation of a ribosomal RNA operon from Agrobacterium vitis; Otten L et al.; One of the four ribosomal RNA operons (rrnA) from the Agrobacterium vitis vitopine strain S4 was sequenced, rrnA is most closely related to the rrn operons of Bradyrhizobium japonicum and Rhodobacter sphaeroides and carries an fMet-tRNA gene downstream of its 5S gene, as in the case of R . sphaeroides . The 16S rRNA sequence of S4 differs from the A . vitis K309 type strain sequence by only one nucleotide, in spite of the fact that S4 and K309 have very different Ti plasmids . The predicted secondary structure of the S4 23S rRNA shows several features that are specific for the alpha proteobacteria, and an unusual branched structure in the universal B8 stem . The 3' ends of the three other rrn copies of S4 were also cloned and sequenced . Sequence comparison delimits the 3' ends of the four repeats and defines two groups: rrnA/rrnB and rrnC/rrnD.

J Biol Chem, 1996 Apr 19, 271(16), 9326 - 31
Topological mapping of the cysteine residues of N-carbamyl-D-amino-acid amidohydrolase and their role in enzymatic activity; Grifantini R et al.; The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production Of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli . The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide . To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser . Only the mutant carrying the Cys172 substitution was catalytically inactive, and the other mutants maintained the same specific activity as the wild type enzyme . The crucial role of Cys172 in enzymatic activity was also confirmed by chemical derivatization of the protein with iodoacetate . Furthermore, chemical derivatizations using both acrylamide and Ellman's reagent revealed that (i) none of the five cysteines is engaged in disulfide bridges, (ii) Cys172 is easily accessible to the solvent, (iii) Cys193 and Cys250 appear to be buried in the protein core, and (iv) Cys243 and Cys279 seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions . These data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction.

Gene, 1996 Apr 17, 170(1), 57 - 62
Genetic organization of a DNA-processing region required for mobilization of a non-self-transmissible plasmid, pEC3, isolated from Erwinia carotovora subsp . carotovora; Nomura N et al.; A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp . carotovora, can be mobilized by an IncP-type plasmid . The hybrid plasmid vector, pETC3, constructed from pEC3 by fusion to markers conferring TcR and CmR, was transferred by conjugation from Escherichia coli (Ec) to various genera of Enterobacteriaceae and to other genera of Gram(-) bacteria which included Xanthomonas, Agrobacterium and Rhizobium . Deletion analysis and successive subcloning of pEC3 revealed that a cis-acting locus, oriT and a trans-acting locus, mob, were involved in mobilization of pEC3 . Five open reading frames (ORFs) were found in the mob region, of which four were identified as mobA, B, C and D . The mobA gene overlapped with mobC, B, D and ORF1 that were transcribed polycistronically from upstream from mobC . The nature of the four products of mob genes, MobA, B, C and D, was verified by use of the T7 promoter system in Ec.

J Bacteriol, 1996 Apr, 178(8), 2427 - 30
Purification and characterization of catabolic mannopine cyclase encoded by the Agrobacterium tumefaciens Ti plasmid pTi15955; Hong SB et al.; Catabolic mannopine (MOP) cyclase encoded by certain Agrobacterium Ti and Ri plasmids lactonizes MOP to agropine (AGR) . The enzyme, purified to homogeneity from a recombinant clone, has a molecular mass of 45 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography . The enzyme catalyzed the lactonization of MOP to AGR without the need for any cofactors . The enzyme also converted AGR to MOP with the lactonizing activity being predominant over the reverse reaction . MOP cyclase is specific for imine conjugates of D-hexose and L-glutamine and was not inhibited by sugars or amino acids . The enzyme lactonized deoxyfructosyl glutamine, a natural intermediate of MOP synthesis and catabolism, to a product indistinguishable from chrysopine, a newly discovered crown gall opine . The enzyme also lactonized N-l-(1,2-dideoxy-D-mannityl)-L-glutamine, indicating that a hydroxyl group at carbon atom 2 of the sugar moiety is not required for the enzymatic reaction.

J Bacteriol, 1996 Apr, 178(7), 1872 - 80
Identification of Agrobacterium tumefaciens genes that direct the complete catabolism of octopine; Cho K et al.; Agrobacterium tumefaciens R10 was mutagenized by using the promoter probe transposon Tn5-gusA7, and a library of approximately 5,000 transcriptional fusions was screened for octopine-inducible patterns of gene expression . Twenty-one mutants carrying strongly inducible gusA fusions, 20 of which showed defects in the catabolism of octopine or its metabolites, were obtained . One group of mutants could not use octopine as a carbon source, while a second group of mutants could not utilize arginine or ornithine and a third group could not utilize octopine, arginine, ornithine, or proline as a carbon source . Utilization of these compounds as nitrogen sources showed similar but not identical patterns . Fifteen fusions were subcloned together with adjacent DNA . Sequence analysis and further genetic analysis indicated that insertions of the first group are localized in the occ region of the Ti plasmid . Insertions of the second group were localized to a gene encoding ornithine cyclodeaminase . This gene is very similar to, but distinct from, a homolog located on the Ti plasmid . This gene is located immediately downstream from a gene encoding an arginase . Genetic experiments indicated that this arginase gene is essential for octopine and arginine catabolism . Insertions of the third group was localized to a gene whose product is required for degradation of proline . We therefore have identified all steps required for the catabolism of octopine to glutamate.

Proc Natl Acad Sci U S A, 1996 Mar 19, 93(6), 2392 - 7
Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells; Zupan JR et al.; Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2 . VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed . The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA . In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2 . Microinjected ssDNA alone remained cytoplasmic . Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 444 - 7
Cloning and nucleotide sequence of the AgeI methylase gene from Agrobacterium gelatinovorum IAM 12617, a marine bacterium; Suzuki T et al.; The AgeI restriction-modification system from a marine bacterium, Agrobacterium gelatinovorum IAM 12617, recognizes the nucleotide sequence ACCGGT . The gene coding for the AgeI methylase (M.AgeI) was cloned into Escherichia coli DH5 alpha MCR, and the nucleotides of the gene were sequenced . The M.AgeI gene coded for a protein of 429 amino acid residues (molecular mass, 47,358 daltons) . The deduced amino acid sequence of M.AgeI was compared with those of other methylases and showed that there are high degrees of similarity in some cytosine-5 methylases.

Plant Cell, 1996 Mar, 8(3), 363 - 73
Transport of DNA into the nuclei of xenopus oocytes by a modified VirE2 protein of Agrobacterium; Guralnick B et al.; We used Agrobacterium T-DNA nuclear transport to examine the specificity of nuclear targeting between plants and animals and the nuclear import of DNA by a specialized transport protein . Two karyophilic Agrobacterium virulence (Vir) proteins, VirD2 and VirE2, which presumably associate with the transported T-DNA and function in many plant species, were microinjected into Drosophila embryos and Xenopus oocytes . In both animal systems, VirD2 localized to the cell nuclei and VirE2 remained exclusively cytoplasmic, suggesting that VirE2 nuclear localization signals may be plant specific . Repositioning one amino acid residue within VirE2 nuclear localization signals enabled them to function in animal cells . The modified VirE2 protein bound DNA and actively transported it into the nuclei of Xenopus oocytes . These observations suggest a functional difference in nuclear import between animals and plants and show that DNA can be transported into the cell nucleus via a protein-specific pathway.

Cell Mol Biol (Noisy-le-grand), 1996 Mar, 42(2), 151 - 8
Sensitivity of ribosomes from Agrobacterium tumefaciens to the ribosome-inactivating protein crotin 2 depending on the translocational state; Alegre C et al.; The GTP analog guanylylmethylene diphosphonate (GppCH2p) strongly inhibited polyuridylic acid-directed polypeptide synthesis in a cell-free translation system prepared from Agrobacterium tumefaciens . Fusidic acid increased even further the inhibitory action . The pre-translocational ribosomal complexes formed with the GppCH2p and the elongation factor G protected the ribosome against the depurinating action of crotin 2 assayed as the acid-dependent release of the RNA fragment whose terminal sequence is 5'-GAGGACCGGGAUGGAC-3' . The results allowed to conclude that the interaction of both crotin 2 and the elongation factor G with the A . tumefaciens ribosomes in the pre-translocational state must take place at overlapping, either sterically or allosterically, ribosomal sites which are equally accessible to the RIP.

J Bacteriol, 1996 Mar, 178(6), 1498 - 504
Temperature affects the T-DNA transfer machinery of Agrobacterium tumefaciens; Fullner KJ et al.; Early studies on Agrobacterium tumefaciens showed that development of tumors on plants following infection by A . tumefaciens was optimal at temperatures around 22 degrees C and did not occur at temperatures above 29 degrees C . To assess whether this inability to induce tumors is due to a defect in the T-DNA transfer machinery, mobilization of an incompatibility group Q (IncQ) plasmid by the T-DNA transfer machinery of A . tumefaciens was tested at various temperatures . Optimal transfer occurred when matings were performed at 19 degrees C, and transfer was not seen when matings were incubated above 28 degrees C . Transfer of the IncQ plasmid was dependent upon induction of the virB and virD operons by acetosyringone but was not dependent upon induction of the tra genes by octopine . However, alterations in the level of vir gene induction could not account for the decrease in transfer with increasing temperature . A . tumefaciens did successfully mobilize IncQ plasmids at higher temperatures when alternative transfer machineries were provided . Thus, the defect in transfer at high temperature is apparently in the T-DNA transfer machinery itself . As these data correlate with earlier tumorigenesis studies, we propose that tumor suppression at higher temperatures results from a T-DNA transfer machinery which does not function properly.

Eur J Biochem, 1996 Mar 1, 236(2), 620 - 5
A 13C-NMR study of the mechanism of bacterial metabolism of monomethyl sulfate; Higgins TP et al.; Two different mechanisms have been proposed previously for initiating the biodegradation of monomethyl sulfate (MeSO4) in bacteria . For a Hyphomicrobium species, a sulfatase enzyme has been proposed to hydrolyse MeSO4 to methanol and inorganic sulfate . For an Agrobacterium sp., an alternative proposal involves monooxygenation of MeSO4 (hydroxylation) to produce methanediol monosulfate, which decomposes spontaneously to formaldehyde and inorganic sulfate . In the present study, 13C-NMR was used to monitor metabolic intermediates of {13C}MeSO4 in real time in each species in order to resolve the issue of mechanism of biodegradation . Agrobacterium sp . M3C grew on MeSO4 but not on methanol . MeSO4-grown cells catabolised {13C}MeSO4 but not {13C}methanol, and {13C}methanol did not accumulate from MeSO4 in the presence of a known inhibitor of methanol dehydrogenase (cyclopropanol) . Hyphomicrobium MS223 grew on MeSO4 and, in contrast with the Agrobacterium sp., also on methanol . The normally rapid metabolism of {13C}methanol by methanol-grown cells was arrested by cyclopropanol, but metabolism of {13C}MeSO4 by MeSO4-grown cells was unaffected . Moreover there was no accumulation of {13C}methanol from {13C}MeSO4 under conditions in which methanol dehydrogenase was shown to be inactive . The results provided strong evidence against the intermediacy of methanol in the biodegradation of MeSO4 in either species, and thereby render untenable mechanisms involving sulfatase-mediated hydrolysis of MeSO4 . The data are consistent with the hydroxylation of MeSO4 via a monooxygenation mechanism and subsequent spontaneous hydrolysis of the methanediol monosulfate intermediate.

J Nutr, 1996 Mar, 126(3), 728 - 40
The expressed protein in glyphosate-tolerant soybean, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp . strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice; Harrison LA et al.; The safety of 5-enolpyruvylshikimate-3-phosphate synthase enzyme derived from Agrobacterium sp . strain CP4 (CP4 EPSPS) was assessed . CP4 EPSPS is the only protein introduced by genetic manipulation that is expressed in glyphosate-tolerant soybeans, which are being developed to provide new weed-control options for farmers . Expression of this protein in plants imparts high levels of glyphosate tolerance . The safety of CP4 EPSPS was ascertained by evaluating both physical and functional characteristics . CP4 EPSPS degrades readily in simulated gastric and intestinal fluids, suggesting that this protein will be degraded in the mammalian digestive tract upon ingestion as a component of food or feed, There were no deleterious effects due to the acute administration of CP4 EPSPS to mice by gavage at a high dosage of 572 mg/kg body wt, which exceeds 1000-fold tha anticipated consumption level of food products potentially containing CP4 EPSPS protein . CP4 EPSPS does not pose any important allergen concerns because this protein does not possess characteristics typical of allergenic proteins . These data, in combination with seed compositional analysis and animal feeding studies, support the conclusion that glyphosate-tolerant soybean are as safe and nutritious as traditional soybeans currently being marketed.

J Nutr, 1996 Mar, 126(3), 717 - 27
The feeding value of soybeans fed to rats, chickens, catfish and dairy cattle is not altered by genetic incorporation of glyphosate tolerance; Hammond BG et al.; Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate . These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived . Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially . In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet . The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish) . Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines . Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows . In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line . These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients . They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp . strain CP4.

Proc Natl Acad Sci U S A, 1996 Feb 20, 93(4), 1613 - 8
Agrobacterium tumefaciens-mediated transformation of yeast; Piers KL et al.; Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis . A . tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants . We show that the host range of A . tumefaciens can be extended to include Saccharomyces cerevisiae . Additionally, we demonstrate that while T-DNA transfer into S . cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved . The Ti plasmid-encoded vir genes of A . tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S . cerevisiae, as is vir gene induction . However, mutations in the chromosomal virulence genes of A . tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S . cerevisiae . We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.

Mol Gen Genet, 1996 Feb 5, 250(2), 169 - 79
Identification of a partition and replication region in the Alcaligenes eutrophus megaplasmid pMOL28; Taghavi S et al.; A 4.64 kb region of the 180 kb heavy metal resistance plasmid pMOL28 of Alcaligenes eutrophus CH34, previously shown to be able to replicate autonomously, was sequenced and analyzed . Three genes involved in plasmid maintenance were identified: parA28 and parB28 are involved in plasmid partitioning and stability, while repA28 encodes a protein required for replication . In addition to the par AB28 genes, a third locus, parS28, required in cis active partitioning was identified . The parABS28 locus of pMOL28 shows strong similarity in organization to the sop, par and rep regions, respectively, of the Escherichia coli F-factor, the E.coli P1 and P7 prophages and the Agrobacterium pTiB6S3 and pRiA4b plasmids . The ParAB28 proteins of pMOL28 also show similarity to the proteins encoded by two conserved open reading frames present in the replication regions of the Pseudomonas putida and Bacillus subtilis chromosomes . The functionality of the pMOL28 par region was examined by performing stability and incompatibility tests between pMOL28 and pMOL846 or pMOL850 which contain the 4.64 EcoRI replicon fragment of pMOL28, cloned in opposite orientations into pSUP202, which is itself unable to replicate in A . eutrophus . The RepA2 8 replication protein showed similarity to the RepL protein of P1, which is required for lytic replication of this E . coli phage . The replication origin of pMOL28, oriV28, seems to be located within the repA28 coding region, and pMOL28 replication may depend on transcriptional activation of oriV28.

Gene, 1996 Feb 2, 168(1), 49 - 53
The phzI gene of Pseudomonas aureofaciens 30-84 is responsible for the production of a diffusible signal required for phenazine antibiotic production; Wood DW et al.; The production of phenazine (Ph) antibiotics in Pseudomonas aureofaciens (Pau) 30-84 is positively regulated by PhzR, a protein belonging to the LuxR family of transcriptional activators . We have now identified phzI, a second gene required for PH production . The product of phzI is a member of the LuxI family of N-acyl-homoserine lactone (N-acyl-HSL) synthases . Inactivation of phzI results in the loss of Ph production in Pau 30-84 . The presence of phzI in Escherichia coli is sufficient for the production of a diffusible signal which activates phzB expression in Pau 30-84 and traA expression in a N-acyl-HSL-dependent reporter strain of Agrobacterium tumefaciens . In addition, synthetic N-(3-oxohexanoyl)-L-HSL induces phzB expression in Pau 30-84 . These results suggest that Pau 30-84 produces a N-acyl-HSL signal that regulates Ph production, and that phzI plays a central role in this signaling pathway.

Biosci Biotechnol Biochem, 1996 Feb, 60(2), 322 - 4
Cloning and expression in Escherichia coli of sucrose phosphorylase gene from Leuconostoc mesenteroides No . 165; Kawasaki H et al.; The sucrose phosphorylase gene of an isolate, Leuconostoc mesenteroides No . 165, was amplified by PCR, cloned on pUC118, and expressed in E . coli . The nucleotide sequence of the gene showed 96.3% similarity to that of L . mesenteroides ATCC12291 and 67% to that of Streptococcus mutans, but low similarity to the Agrobacterium vitis gene . The cloned gene, which fusing with lacZalpha, was expressed inducibly with IPTG in E . coli to produce an active enzyme in large quantities that accounted for about 50% of the total cell protein.

Trends Microbiol, 1996 Feb, 4(2), 64 - 8
Adaptation of a conjugal transfer system for the export of pathogenic macromolecules; Winans SC et al.; Conjugal transfer of bacterial plasmids requires a pore through which DNA can traverse the envelopes of the donor and recipient cells . Recent studies indicate that these pores, which are composed of approximately ten proteins, are evolutionarily related to the transport systems required for the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to plant cells and for toxin secretion from Bordetella pertussis.

J Ind Microbiol, 1996 Feb, 16(2), 129 - 33
Production of an extracellular polysaccharide by Agrobacterium sp DS3 NRRL B-14297 isolated from soil; Hou CT et al.; A bacterium isolated from soil and identified as Agrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions . Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.5:2.4:1, together with 3.7% (w/w) pyruvic acid . Methylation analyses showed the presence of (1-->3)-, (1-->4)- and (1-->6)-linked glucose, (1-->3)- and (1-->4, 1-->6)-linked galactose and a small portion of (1-->3)-linked mannose residues . Succinic acid was not present . The molecular weight of the polysaccharide was estimated by light scattering to be 2 x 10(6) Da . The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55 degrees C . Maximum yield of the polysaccharide, 20 g L-1, was reached in 48 h at 30 degrees C incubation.

Genetika, 1996 Feb, 32(2), 204 - 10
{Construction and analysis of transgenic plants of Nicotiana tabacum L . expressing a bacterial gene for beta-1,3-glucanase . II . Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes}; Darbinian NS et al.; The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum . The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively . These constructions were used for transformation of agrobacteria followed by transfer into plants . In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants . The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.

J Bacteriol, 1996 Feb, 178(4), 961 - 70
Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens; Doty SL et al.; The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS) . In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites . To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated . Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain . Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2 . Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose . This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE . Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA . These mutants were defective in the vir gene response to AS . These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain . Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.

J Bacteriol, 1996 Feb, 178(4), 1207 - 12
VirE1 protein mediates export of the single-stranded DNA-binding protein VirE2 from Agrobacterium tumefaciens into plant cells; Sundberg C et al.; Agrobacterium tumefaciens transfers single-stranded DNAs (T strands) into plant cells . VirE1 and VirE2, which is a single-stranded DNA binding protein, are important for tumorigenesis . We show that T strands and VirE2 can enter plant cells independently and that export of VirE2, but not of T strands, depends on VirE1.

Proc Natl Acad Sci U S A, 1996 Jan 9, 93(1), 126 - 30
Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens; Rossi L et al.; Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus . The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell . This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome . Here, we analyze different putative roles of the VirE2 protein in the plant cell . In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated . We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus . Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2 . In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent . We conclude that the VirE2 protein of A . tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Diagn Microbiol Infect Dis, 1996 Jan, 24(1), 43 - 5
Bacteremia due to Agrobacterium tumefaciens (radiobacter) . Report of infection in a pregnant women and her stillborn fetus; Southern PM Jr; Agrobacterium tumefaciens (radiobacter) is usually a plant pathogen, but is isolated occasionally from human clinical specimens, frequently along with other bacteria . Agrobacterium tumefaciens (radiobacter) has been isolated from blood, central intravenous catheters, peritoneal fluid, urine, and cellulitis aspirates, often in immunocompromised individuals . This report details the isolation of A . tumefaciens (radiobacter) from the blood of a pregnant woman, as well as from the blood of her stillborn, premature fetus . It is, to our knowledge, the first report of such an occurrence.

DNA Seq, 1996, 6(6), 337 - 46
Cloning, DNA sequence, and regulation of expression of a gene encoding beta-galactosidase from Lactococcus lactis; Griffin HG et al.; The beta-galactosidase from Escherichia coli is one of the most important enzymes in molecular biology . Here we report the cloning and sequencing of a gene encoding beta-galactosidase from Lactococcus lactis and compare the predicted amino acid sequence to that from other organisms . The beta-galactosidase from L . lactis was found to be a protein of 996 residues with 68.7% similarity to the E . coli enzyme and 65.8% similarity to the enzyme from Klebsiella pneumoniae . The lactococcal beta-galactosidase has lower similarity (approx 55%) to the enzymes from other lactic acid bacteria and no significant similarity to the beta-galactosidase enzymes from Agrobacterium radiobacter, Bacillus stearothermophilus, or Clostridium thermosulfurogenes . Expression of the lacZ gene from L . lactis was found to be higher when cells were grown in medium containing lactose than when grown in glucose, and expression was higher when cells were grown at 30 degrees C than at 35 degrees C.

Chin J Biotechnol, 1996, 12(2), 67 - 72
Insect-resistant tobacco plants expressing insect-specific neurotoxin AaIT; Yao B et al.; The recombinant plant expression vector pNGY-2 with designed and synthesized AaIT gene had been constructed . The AaIT gene was fused behind the sequence of TMV and inserted into expression vector under the control of two linked 35s promoters . The recombinant plasmid pNGY-2 was transferred into tobacco NC89 by agrobacterium-mediated transfer system . The GUS activity analysis and Southern blotting of regenerated plants indicated that AaIT gene had been integrated into tobacco genome . Insect bioassays showed that some transgenic plants had notable insect-resistant activity.

Annu Rev Microbiol, 1996, 50, 727 - 51
Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators; Fuqua C et al.; The importance of accurate demographic information is reflected in the United States Constitution, Article 1, which provides for a decennial census of this country's human population . Bacteria also conduct a census of their population and do so more frequently, more efficiently, and as far we know, with little if any of the political contentiousness caused by human demographers . Many examples have been found of particular bacterial genes, operons, or regulons that are expressed preferentially at high cell densities . Many of these are regulated by proteins related to the LuxR and LuxI proteins of Vibrio fischeri, and by a diffusible pheromone called an autoinducer . LuxR and LuxI and their cognate autoinducer (3-oxohexanoyl homoserine lactone, designated VAI-1) provide an important model to describe the functions of this family of proteins . LuxR is a VAI-1 receptor and a VAI-1-dependent transcriptional activator, and LuxI directs the synthesis of VAI-1 . VAI-1 diffuses across the bacterial envelope, and intracellular concentrations of it are therefore strongly increased by nearby VAI-1-producing bacteria . Similar systems regulate pathogenesis factors in Pseudomonas aeruginosa and Erwinia spp., as well as T1 plasmid conjugal transfer in Agrobacterium tumefaciens, and many other genes in numerous genera of gram-negative bacteria . Genetic analyses of these systems have revealed a high degree of functional conservation, while also uncovering features that are unique to each.

Arch Virol, 1996, 141(9), 1637 - 50
Infectivity and complete nucleotide sequence of the genome of a genetically distinct strain of maize streak virus from Reunion Island; Peterschmitt M et al.; A complete infectious genome of an isolate of maize streak subgroup 1 geminivirus from Reunion Island (MSV-R) was cloned and sequenced . Using an Agrobacterium tumefaciens Ti plasmid delivery system, the cloned 2.7 kb circular DNA was shown to be infectious in maize . The agroinfected virus could be transmitted by Cicadulina mbila, the most common vector species of MSV in Reunion . Analysis of open reading frames (ORFs) revealed seven potential coding regions including the 4 ORFs conserved in all geminiviruses infecting monocotyledonous plants, the 2 on the viral "+" strand (MP, CP), and the 2 on the complementary "-" strand (RepA, RepB) . The nucleotide sequence of MSV-R was compared to previously determined sequence of three African clones from Nigeria (MSV-N), Kenya (MSV-K), and South Africa (MSV-S) . More similarity was found between the African clones (97.0-97.3%) than between these and MSV-R (94.4-95.3%) . Nucleotide substitutions were frequent in the large intergenic region, particularly in and around the most likely TATA box for the complementary sense genes, and in the 5' end of ORF V1 . The comparison of the predicted peptide sequences of the proteins encoded by ORFs MP, RepA and RepB confirmed the higher similarity between the African clones (97.8-99.3%) than between these and MSV-R (95.1-97.1%) . However the amino acid sequences of the protein encoded by ORF CP (capsid protein) were very conserved among all the 4 clones, suggesting a high selection pressure on this ORF.

Chin J Biotechnol, 1996, 12(1), 39 - 45
Agrobacterium tumefaciens mediated transformation of Orychophragmus violaceus cotyledon and regeneration of transgenic plants; Zhou J et al.; Excised cotyledons of Orychophragmus violaceus were used as explants for tissue culture . They were cultured on the MS medium supplemented with BA (3 mg/L) and NAA (0.2 mg/L) . When the regenerated buds were 2 cm long, they were excised and transferred onto 1/2 MS medium with IBA (0.03 mg/L), then the whole plants were regenerated . The frequency of plant regeneration was 100% . Subsequently, the genetic transformation of O . violaceus was studied . After 2-3 days of cocultivation with Agrobacterium tumefaciens strain A208se (pTiT37, pROA93), the cotyledons were transferred onto the selection medium containing 25 mg/L Km and 250 mg/L Ap . After shoots emerged, they were excised and transferred onto the rooting medium containing 25 mg/L Km and 100 mg/L Cef . The roots were formed within 4-5 weeks . The whole plants were transplanted into pots and grew well . The frequency of plant regeneration was about 51% . The regenerated plants showed high enzymatic activities of beta-glucuronidase and neomycine phosphotransferase II . Southern blot analysis confirmed that NPTII gene had been stably integrated into the chromosomal genome of O . violaceus . The transformation frequency was 5.6% . The first transgenic plant of O . violaceus is being reported.

Plant Cell Physiol, 1996 Jan, 37(1), 103 - 6
Trans-kingdom conjugation between Agrobacterium tumefaciens and Saccharomyces cerevisiae, a bacterium and a yeast; Sawasaki Y et al.; For conjugation between prokaryotic Agrobacterium tumefaciens and eukaryotic Saccharomyces cerevisiae, we constructed two novel conjugative plasmids . A . tumefaciens transmitted the plasmids to S . cerevisiae with the aid of tra genes on a helper plasmid . The transmitted plasmids retained their original structure and function in transconjugant yeasts . The presence of Ti plasmid barely affected the trans-kingdom conjugation.

Yi Chuan Xue Bao, 1996, 23(1), 77 - 83
{Transgenic Brassica napus resistant to turnip mosaic virus}; Lu A et al.; A system for obtaining regenerated plantlets of "double low" Brassica napus by using cotyledonary petioles as material was established . The turnip mosaic virus coat protein (TuMV-CP) gene inserted in the binary vector pBTu in the Agrobacterium tumefaciens LBA 4404 was integrated into Brassica napus through co-culture of cotyledonary petioles with LBA4404, and the material in co-culture was selected under the stress of Kanamycin . Regenerated plantlets were obtained, and the specific TuMV-CP gene was proved to be integrated into the genomic DNA of the regenerated plants by the specific PCR amplification, dot hybridization and Southern blot . All these transgenic plants were proved to be resistant to virulent TuMV by virus challenge in different degree.

Plant Mol Biol, 1996 Jan, 30(1), 77 - 96
Sequence-specific interactions of wound-inducible nuclear factors with mannopine synthase 2' promoter wound-responsive elements; Ni M et al.; A 318 bp mannopine synthase 2' (mas2') promoter element from the T-DNA of Agrobacterium tumefacians can direct wound-inducible and root-preferential expression of a linked uidA gene in transgenic tobacco plants . Wound inducibility is further enhanced by sucrose in the medium . Promoter deletion analysis indicated that the sucrose enhancement is conferred by a region extending from -318 to -213 . DNase I footprinting indicated that an A/T-rich DNA sequence in this region is protected by tobacco nuclear factors . Regions extending from -103 to +66 and from -213 to -138 directed wound-inducibile expression of a linked uidA gene when placed downstream of a CaMV 35S enhancer or upstream of a truncated (-209) CaMV 35S promoter, respectively . DNase I footprinting analyses indicated that proteins from wounded tobacco leaves specifically bound to three contiguous motifs downstream of the mas2' TATA box . In addition to a common retarded band formed by the upstream wound-responsive element complexed with proteins from either wounded or unwounded tobacco leaves, two unique retarded bands were observed when this element was incubated with protein from wounded leaves . Methylation interference analysis additionally identified an unique motif composed of promoter elements and nuclear factors derived specifically from wounded tobacco leaves . We propose a model to describe the involvement of nuclear factors with mas2' promoter elements in wound-inducible gene expression.

Plant Mol Biol, 1996 Jan, 30(1), 125 - 34
Tissue-specific expression of the rolA gene mediates morphological changes in transgenic tobacco; Guivarc'h A et al.; The spatial and temporal activity of the entire and individual promoter domains of the rolA gene of Agrobacterium rhizogenes was investigated and correlated with the distinctive features of the phenotypes of transgenic tobacco plants . The GUS assay was performed in the presence of an oxidative catalyst during the development of transgenic plants expressing chimeric genes containing the beta-glucuronidase coding sequence under the control of the different promoter domains . In situ hybridization was also used on transgenic plants harbouring rolA under the control of the entire or deleted promoter . This paper demonstrates for the first time that the entire rolA promoter, composed of domains, A, B and C, is silent in seeds, then activated at the onset of germination in the cotyledons and in the elongation zone of the radicle and is finally expressed throughout the vegetative and floral phases . Domains B + C, which were sufficient to induce wrinkled leaves and short internodes, were active in all the stem tissues, but only in the companion cells of the phloem strands of the leaves . Domain C, which specified a dwarf phenotype with normal leaves, was weakly expressed in the stem vascular bundles and in the leaf internal phloem . These results indicate that the vascular bundles are the primary targets for the generation of the short internode phenotype . Furthermore, the local expression of rolA in the stem vascular bundles induced a size reduction of the surrounding parenchyma cells, suggesting the existence of some diffusible factor(s) associated with the expression of the rolA gene.

FEMS Microbiol Lett, 1996 Jan 1, 135(1), 85 - 92
Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens; Chen CY et al.; The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens . osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell . When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter . Osa protein is produced, enabling us to determine its subcellular location . Immunoblot analyses located Osa protein at the inner membrane of both A . tumefaciens and Escherichia coli . Because Osa inhibits oncogenicity of A . tumefaciens, and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa . Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes . These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.

Transgenic Res, 1996 Jan, 5(1), 57 - 65
Expression of the isopentenyl transferase gene is regulated by auxin in transgenic tobacco tissues; Zhang XD et al.; The isopentenyl transferase gene (ipt) from Agrobacterium tumefaciens was isolated and introduced, via a disarmed binary vector, into tobacco using the Agrobacterium tumefaciens-mediated gene transfer system . The expression of the ipt gene was monitored by RNA hybridization, western blotting and cytokinin analysis . The addition of auxin to the media rapidly reduced the level of cytokinins in the transgenic tissues and this was associated with a reduction in IPT mRNA and protein levels . It is concluded that the hormone auxin can regulate expression of a gene involved in biosynthesis of the second hormone cytokinin . Although exogenous benzyladenine did not directly affect ipt gene expression, it did antagonize the effect of auxin on levels of cytokinins and IPT mRNA and protein.

J Bacteriol, 1996 Jan, 178(2), 435 - 40
Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes; Fuqua C et al.; Ti plasmids of Agrobacterium tumefaciens, in addition to transferring oncogenic DNA to the nuclei of infected plant cells, can conjugally transfer between agrobacteria . Conjugation of wide-host-range octopine-type Ti plasmids requires a tumor-released arginine derivative called octopine . Octopine stimulates expression of the traR gene, whose product directly activates other tra genes in the presence of an acylated homoserine lactone called Agrobacterium autoinducer (AAI) . We have localized the transcription starts of three tra promoters and find conserved elements (tra boxes) at virtually identical positions upstream of each promoter . Disruption of these tra boxes abolished induction of each promoter . Deletion analysis of the traI promoter indicates that tra boxes are the only upstream elements required for transcriptional activation . Since Ti plasmid donor cells both produce and respond to AAI, we tested whether expression of tra promoters was enhanced by high concentrations of bacteria . Both tra gene expression and conjugation itself were strongly stimulated either by high donor densities or by exogenous AAI.

J Bacteriol, 1996 Jan, 178(2), 366 - 71
Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors; Schripsema J et al.; Small bacteriocin was isolated from the culture broth of the gram-negative bacterium Rhizobium leguminosarum, which forms symbiotic nitrogen-fixing root nodules on a number of leguminous plants . The structure of the molecule was elucidated by spectroscopic methods and identified as N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone . The absolute configuration of both asymmetric carbon atoms in the molecule was determined by the use of the chiral solvating agents S-(+)- and R-(-)-2,2,2-trifluoro-1-(9-anthryl)-ethanol . small bacteriocin is structurally related to the quorum sensing co-transcription factors for genes from other bacteria such as Vibrio fischeri, Pseudomonas aeruginosa, Erwinia carotovora, and Agrobacterium tumefaciens which are involved in animal-microbe or plant-microbe interactions . The mechanism of regulation of such interactions by this kind of co-transcription factors is still unknown in R . leguminosarum.

J Bacteriol, 1996 Jan, 178(1), 266 - 72
Characterization of PcaQ, a LysR-type transcriptional activator required for catabolism of phenolic compounds, from Agrobacterium tumefaciens; Parke D; Previous work demonstrated that catabolism of the phenolic compounds p-hydroxybenzoate and protocatechuate via the beta-ketoadipate pathway in Agrobacterium tumefaciens is mediated by a regulatory gene, pcaQ, that acts in trans to elicit expression of many of the enzymes encoded by the pca genes . There was evidence that five pca structural genes are organized in a polycistronic operon transcribed in the order pcaDCHGB . The pcaQ gene is upstream of this operon . The activator encoded by pcaQ was novel in having the metabolite beta-carboxy-cis,cis-muconate as a coinducer . This communication reports the nucleotide sequence of pcaQ and identifies its deduced polypeptide product as a member of the LysR family of regulatory molecules . PcaQ has a calculated molecular weight of 33,546, which is consistent with the size of LysR relatives . Like many other LysR members, PcaQ serves as an activator at the level of transcription, it has a conserved amino-terminal domain, and its gene is transcribed divergently from the operon that it regulates and is subject to negative autoregulation . Studies of coinducer specificity identified an unstable pathway metabolite, gamma-carboxymuconolactone, as a second coinducer . Analysis of expression from a pcaD::lacZ promoter probe plasmid revealed that PcaQ and the coinducer exert their effect on a 133-nucleotide region upstream of pcaD . The nucleotide sequence of this region in a mutant strain constitutive for enzymes encoded by the pcaDCHGB operon identified nucleotides likely to be involved in the pcaDCHGB promoter and substantiated the inclusion of five pca structural genes in the operon.

Proc Natl Acad Sci U S A, 1995 Dec 19, 92(26), 12245 - 9
Genetic evidence for direct sensing of phenolic compounds by the VirA protein of Agrobacterium tumefaciens; 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 . However, it is not clear how the phenolic compounds are sensed by the VirA/VirG system . We tested the vir-inducing abilities of 15 different phenolic compounds using four wild-type strains of A . tumefaciens--KU12, C58, A6, and Bo542 . We analyzed the relationship between structures of the phenolic compounds and levels of vir gene expression in these strains . In strain KU12, vir genes were not induced by phenolic compounds containing 4'-hydroxy, 3'-methoxy, and 5'-methoxy groups, such as acetosyringone, which strongly induced vir genes of the other three strains . On the other hand, vir genes of strain KU12 were induced by phenolic compounds containing only a 4'-hydroxy group, such as 4-hydroxyacetophenone, which did not induce vir genes of the other three strains . The vir genes of strains KU12, A6, and Bo542 were all induced by phenolic compounds containing 4'-hydroxy and 3'-methoxy groups, such as acetovanillone . By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic-sensing determinant is associated with Ti plasmid . Subcloning of Ti plasmid indicates that the virA locus determines which phenolic compounds can function as vir gene inducers . These results suggest that the VirA protein directly senses the phenolic compounds for vir gene activation.

Proc Natl Acad Sci U S A, 1995 Dec 5, 92(25), 11786 - 90
Arabidopsis mutants deficient in T-DNA integration; Sonti RV et al.; Arabidopsis thaliana mutants originally isolated as hypersensitive to irradiation were screened for the ability to be transformed by Agrobacterium transferred DNA (T-DNA) . One of four UV-hypersensitive mutants and one of two gamma-hypersensitive mutants tested showed a significant reduction in the frequency of stable transformants compared with radioresistant controls . In a transient assay for T-DNA transfer independent of genomic integration, both mutant lines took up and expressed T-DNA as efficiently as parental lines . These lines are therefore deficient specifically in stable T-DNA integration and thus provide direct evidence for the role of a plant function in that process . As radiation hypersensitivity suggests a deficiency in repair of DNA damage, that plant function may be one that is also involved in DNA repair, possibly, from other evidence, in repair of double-strand DNA breaks.

Int J Biol Macromol, 1995 Dec, 17(6), 357 - 63
NMR studies of succinoglycan repeating-unit octasaccharides from Rhizobium meliloti and Agrobacterium radiobacter; Chouly C et al.; Complete 1H and 13C-nuclear magnetic resonance assignments have been obtained for the octasaccharide repeating units of the bacterial polysaccharide succinoglycan from Rhizobium meliloti Rm1021 and Agrobacterium radiobacter NCIB 11883 . The assignments were used to determine the locations of the O-succinyl and O-acetyl substituents . The O-acetyl substituent in Rm1021 was attached to the 3rd residue from the reducing end, and the O-succinyl group was attached to the 7th residue in both octasaccharides . The structure of the Rm1021 octasaccharide is as shown below: {formula: see text} A small amount of succinate was also attached to C6 of the 6th residue in both octasaccharides.

Plant Mol Biol, 1995 Dec, 29(6), 1299 - 304
5'-regulatory region of Agrobacterium tumefaciens T-DNA gene 6b directs organ-specific, wound-inducible and auxin-inducible expression in transgenic tobacco; Bagyan IL et al.; The regulatory activity of a 826 bp DNA fragment located upstream of the pTiBo542 TL-DNA gene 6b coding region was analyzed in transgenic tobacco, using beta-glucuronidase (gus) as a reporter gene . The region was shown to drive organ-specific, wound- and auxin-inducible expression of the reporter, the effect being dependent on the type and concentration of auxin.

Biosci Biotechnol Biochem, 1995 Dec, 59(12), 2336 - 8
Blasticidin S deaminase gene (BSD): a new selection marker gene for transformation of Arabidopsis thaliana and Nicotiana tabacum; Tamura K et al.; Arabidopsis thaliana and Nicotiana tabacum were transformed to blasticidin S (BS) resistance with BSD (the BS deaminase gene from Aspergillus terreus) using the Agrobacterium-mediated transformation method . Expression of BSD allowed direct selection of transformants by the fungicide, and both kinds of transgenic plants showed high level of resistance phenotype at 100 ppm of BS sprayed on the leaves . Using Botrytis cinerea, the causal fungus of gray mold disease, it was exemplified that application of BS could control the disease in transgenic tobacco with negligible phytotoxicity.

Cytometry, 1995 Dec 1, 21(4), 363 - 73
Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album; Veuskens J et al.; The preparation of stable chromosome suspensions and flow cytometric sorting of both the Y sex chromosome of the white campion, Melandrium album, and the deleted Y chromosome of an asexual mutant, 5K63, is described . The principle has been to maintain transformed roots in vitro, synchronise and block mitosis, reduce cells to protoplasts, and lyse these to release chromosomes . Such in vitro material, unlike many cell suspensions, showed a stable karyotype . Factors critical to producing high-quality chromosome suspensions from protoplasts include osmolality of isolation solutions and choice of spindle toxin and of lysis buffer . Agrobacterium rhizogenes transformed young growing root cultures were synchronised at G1/S with 50 microM aphidicolin for 24 h and released to a mitotic block with 30 microM oryzalin for 11 h . Protoplast preparations from such tissue routinely had metaphase indices reaching 15% . Suspensions of intact metaphase chromosomes, with few chromatids, were obtained by lysing swollen mitotic protoplasts in a citric acid/disodium phosphate buffer . Except for the presence of clumps of autosomal chromosomes near the X and Y chromosome zones, monoparametric histograms of fluorescence intensities of suspensions stained with 4',6-diamino-2-phenylindole showed profiles similar to theoretical flow karyotypes . Two types of Y chromosomes, one full-length and one partially deleted (from the asexual mutant), could be sorted at 90% purity (21-fold enrichment of Y) . These results are discussed in the context of sex determination and differentiation in higher plants.

Appl Microbiol Biotechnol, 1995 Dec, 44(3-4), 466 - 72
Heat-inducible expression system for a foreign gene in cultured tobacco cells using the HSP18.2 promoter of Arabidopsis thaliana; Yoshida K et al.; A system for the controlled expression of a foreign gene in cultured tobacco cells (Nicotiana tabacum, BY2) by temperature shift was constructed . A 925-base-pair (bp) DNA fragment containing the 5'-flanking region of a low-molecular-mass heat-shock protein gene (HSP18.2) of Arabidopsis thaliana was inserted upstream of the beta-glucuronidase reporter gene (GUS) . The resulting HSP18.2-GUS construct was introduced into BY2 cells by electroporation or Agrobacterium-mediated transformation . Transient expression of the HSP18.2 promoter in protoplasts was very low regardless of the heat shock . Although expression of the HSP18.2-GUS chimeric gene in the stable transformants of BY2 was hardly detected in culture at 25 degrees C, the expression increased rapidly on the transcriptional level when the incubation temperature was shifted to 35-37 degrees C . The optimal temperature for heat-shock induction was 37 degrees C . After a 2-h incubation at 37 degrees C, GUS activity was about 1000-fold greater than that before heat shock . The amount of GUS mRNA was maximum 2 h after heat shock, and then decreased gradually.

Lett Appl Microbiol, 1995 Dec, 21(6), 402 - 5
Variability of the 5'-end of the large subunit rDNA and presence of a new short class of rRNA in Rhizobiaceae; Evguenieva-Hackenberg E et al.; A highly variable region of DNA was found between positions 115 and 388 (Escherichia coli numeration) of the large subunit (ls) rRNA genes of 55 rhizobial and agrobacterial strains . In each case this heterogeneity was accompanied by the presence of a new rRNA species approximately 130 bp long . This novel rRNA species corresponded to the 5'-end of the ls rRNA genes . An additional rRNA processing site was located in the central region of the remaining ls rRNA of many of the Rhizobium leguminosarum and Rh . etli strains, and in all of the agrobacteria studied, excepting the type strain of Agrobacterium vitis NCPPB 3554 and Agrobacterium sp . strain ChAg4.

Plant Physiol, 1995 Dec, 109(4), 1353 - 61
Structural requirements of oleosin domains for subcellular targeting to the oil body; van Rooijen GJ et al.; We have investigated the protein domains responsible for the correct subcellular targeting of plant seed oleosins . We have attempted to study this targeting in vivo using "tagged" oleosins in transgenic plants . Different constructs were prepared lacking gene sequences encoding one of three structural domains of natural oleosins . Each was fused in frame to the Escherichia coli uid A gene encoding beta-glucuronidase (GUS) . These constructs were introduced into Brassica napus using Agrobacterium-mediated transformation . GUS activity was measured in washed oil bodies and in the soluble protein fraction of the transgenic seeds . It was found that complete Arabidopsis oleosin-GUS fusions undergo correct subcellular targeting in transgenic Brassica seeds . Removal of the C-terminal domain of the Arabidopsis oleosin comprising the last 48 amino acids had no effect on overall subcellular targeting . In contrast, loss of the first 47 amino acids (N terminus) or amino acids 48 to 113 (which make up a lipophilic core) resulted in impaired targeting of the fusion protein to the oil bodies and greatly reduced accumulation of the fusion protein . Northern blotting revealed that this reduction is not due to differences in mRNA accumulation . Results from these measurements indicated that both the N-terminal and central oleosin domain are important for targeting to the oil body and show that there is a direct correlation between the inability to target to the oil body and protein stability.

J Bacteriol, 1995 Dec, 177(23), 6993 - 8
Fragmentations of the large-subunit rRNA in the family Rhizobiaceae; Selenska-Pobell S et al.; A 130-nucleotide-long rRNA species corresponding to the 5' end of the 23S rRNA gene was found in 96 strains belonging to different Rhizobium, Bradyrhizobium, and Agrobacterium species . Additional fragmentation in the central region of the large-subunit rRNA occurred in all agrobacteria, except Agrobacterium vitis, and in most Rhizobium leguminosarum and Rhizobium etli strains but did not occur in any of the other rhizobia and bradyrhizobia studied.

J Bacteriol, 1995 Dec, 177(23), 6952 - 7
Cloning and characterization of the sigA gene encoding the major sigma subunit of Rhizobium meliloti; Rushing BG et al.; Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti . The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R . meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to that of SigA of the closely related bacterium Agrobacterium tumefaciens . Through Southern analysis, we located the gene on the R . meliloti main chromosome rather than on one of the megaplasmids . The sigA locus does not appear to be part of a macromolecular synthesis operon (MMS), as in many other bacterial species, but rather lies downstream of a partial open reading frame showing similarity to the threonine dehydrogenase gene (tdh) of E . coli.

Mol Gen Genet, 1995 Nov 27, 249(3), 265 - 73
Horizontal gene transfer: regulated expression of a tobacco homologue of the Agrobacterium rhizogenes rolC gene; Meyer AD et al.; A tobacco homologue (trolC) of the rolC gene of the Agrobacterium rhizogenes Ri-plasmid was cloned and sequenced from Nicotiana tabacum L . cv . Havana 425 . The coding region of trolC is similar in sequence (69-87% for DNA and 54-89% for the deduced amino acid sequence) to rolC genes of the agropine, mannopine, and mikimopine strains of Ri-plasmids and the N . glauca rolC homologue . Southern analyses showed that trolC is encoded by a small gene family derived from the tomentosiformis ancestor of tobacco . This suggests that trolC resulted from an ancient transfer of DNA between A . rhizogenes and a progenitor of modern tobacco . Transcripts of trolC were detected in three morphologically distinct cultivars of tobacco . trolC mRNA accumulated in young leaves and shoot tips, but not in lower leaves and roots of mature plants . Accumulation of trolC mRNA in cultured leaf tissues was strongly down-regulated by auxin and induced by cytokinin . These results are of particular interest because they suggest that a gene of bacterial origin introduced during evolution can have a function in a modern plant.

Biochem Biophys Res Commun, 1995 Nov 22, 216(3), 1095 - 100
Properties of D-hydantoinase from Agrobacterium tumefaciens and its use for the preparation of N-carbamyl D-amino acids; Durham DR et al.; Agrobacterium tumefaciens strain 47C expresses an inducible D-hydantoinase that catalyzes the formation of optically pure N-carbamyl D-amino acids from racemic hydantoin precursors . The D-Hydantoinase was shown to be active and stable at elevated temperatures and pH values, thus affording favorable bioreaction conditions that result in the racemization of DL-hydantoins to the utilizable D-isomer . The enzyme demonstrated optimal reaction kinetics at pH 10 and 70 degrees C, was not activated by metal ions, and exhibited a distinctive substrate specificity . A . tumefacins hydantoinase was most active on 5,6-dihydrouracil and DL-5-methylhydantoin with only slight activity on DL-benzylhydantoin . Extracts or whole cells of A . tumefaciens were used as biocatalyst to mediate the stereospecific conversion of DL-phenylhydantoin or DL-5-methylhydantoin to the respective N-carbamyl D-amino acids . In addition, immobilized cell systems were shown to be useful for biocatalyst reuse.

Nucleic Acids Res, 1995 Nov 11, 23(21), 4383 - 90
Isolation and sequence analysis of rpoH genes encoding sigma 32 homologs from gram negative bacteria: conserved mRNA and protein segments for heat shock regulation; Nakahigashi K et al.; The rpoH genes encoding homologs of Escherichia coli sigma 32 (heat shock sigma factor) were isolated and sequenced from five gram negative proteobacteria (gamma or alpha subgroup): Enterobacter cloacae (gamma), Serratia marcescens (gamma), Proteus mirabilis (gamma), Agrobacterium tumefaciens (alpha) and Zymomonas mobilis (alpha) . Comparison of these and three known genes from E.coli (gamma), Citrobacter freundii (gamma) and Pseudomonas aeruginosa (gamma) revealed marked similarities that should reflect conserved function and regulation of sigma 32 in the heat shock response . Both the sequence complementary to part of 16S rRNA (the 'downstream box') and a predicted mRNA secondary structure similar to those involved in translational control of sigma 32 in E.coli were found for the rpoH genes from the gamma, but not the alpha, subgroup, despite considerable divergence in nucleotide sequence . Moreover, a stretch of nine amino acid residues Q(R/K)(K/R)LFFNLR, designated the 'RpoH box', was absolutely conserved among all sigma 32 homologs, but absent in other sigma factors; this sequence overlapped with the segment of polypeptide thought to be involved in DnaK/DnaJ chaperone-mediated negative control of synthesis and stability of sigma 32 . In addition, a putative sigma E (sigma 24)-specific promoter was found in front of all rpoH genes from the gamma, but not alpha, subgroup . These results suggest that the regulatory mechanisms, as well as the function, of the heat shock response known in E.coli are very well conserved among the gamma subgroup and partially conserved among the alpha proteobacteria.

J Mol Biol, 1995 Nov 10, 253(5), 691 - 702
The sixty nucleotide OccR operator contains a subsite essential and sufficient for OccR binding and a second subsite required for ligand-responsive DNA bending; Wang L et al.; OccR is a transcriptional regulatory protein of Agrobacterium tumefaciens that activates the occQ operon in response to octopine, an arginine derivative released from plant tumors . OccR binds to its operator with similar affinity and the same stoichiometry in the presence or absence of octopine, but octopine shortens the protein's DNase I footprint and partially relaxes an OccR-incited DNA bend . In this study, resections and other alterations of the operator were used to demonstrate that 19 nucleotides near the end of the operator furthest from the occQ promoter were essential for high affinity OccR binding . This sequence, denoted the high affinity subsite, was sufficient for binding, provided that the deleted operator sequences were replaced with vector-derived DNA . The same number of OccR monomers bound to resected operators as to the wild-type operator, and OccR was able to protect vector-derived sequences adjacent to the high affinity subsite . Sequences at the promoter proximal end of the operator were required for wild-type patterns of ligand-responsive DNA bending . A sequence alteration at the end furthest from the high affinity subsite caused a partially locked low angle DNA bend, while two more centrally localized mutations caused fully or partially locked high angle bends . This suggests that the promoter proximal half of the operator may contain at least two sites required for wild-type ligand-responsive DNA bending . These mutations also provided evidence that the partial relaxation of this bend by octopine may be essential for occQ activation.

Biochemistry, 1995 Nov 7, 34(44), 14547 - 53
Substrate-induced inactivation of a crippled beta-glucosidase mutant: identification of the labeled amino acid and mutagenic analysis of its role; Gebler JC et al.; The beta-glucosidase from Agrobacterium sp . catalyzes the hydrolysis of beta-glucosides via a covalent alpha-D-glucopyranosyl-enzyme intermediate involving Glu358 . Hydrolysis of 2,4-dinitrophenyl beta-D-glucopyranoside by the low activity Glu358Asp mutant of Agrobacterium beta-glucosidase is accompanied by time-dependent inactivation of the enzyme . Through kinetic studies, labeling, and sequence analysis, inactivation is shown to be a consequence of the occasional (1 time in 1100) attack of Tyr298 on the anomeric center of the substrate, in place of the catalytic nucleophile, with formation of a stable alpha-D-glucopyranosyl tyrosine residue . Tyr298 is conserved throughout family 1 of glycoside hydrolases, an indication of a possible role in catalysis . Results of a kinetic analysis of the Tyr298Phe mutant are consistent with a function of Tyr298 in both orienting the nearby nucleophile Glu358 and stabilizing its deprotonated state in the free enzyme.

FEMS Microbiol Lett, 1995 Nov 1, 133(1-2), 53 - 8
The replicator region of the Rhizobium leguminosarum cryptic plasmid pRL8JI; Turner SL et al.; The replicator region of the cryptic plasmid pRL8JI from Rhizobium leguminosarum strain 3841 was cloned and sequenced . The recombinant plasmid (pYK3) was selected by function from a partial EcoRI library of total DNA cloned in pSUP202 and shows incompatibility with plasmid pRL8JI when conjugated into R . leguminosarum strains 3841 and its derivative 1062 . The cloned insert (approximately 10.5 kb) comprises five EcoRI fragments none of which confers replicative stability when cloned individually . A single 5.0-kb BamHI fragment, that spans all five EcoRI fragments and confers replicative stability on pSUP202 in R . leguminosarum, has been sequenced . This replicator region shows organisational and sequence similarity to the replicator regions of the Agrobacterium plasmids pTiB6S3 and pRiA4b . It has three open reading frames (repA, repB, repC) and a conserved intergenic sequence.

Mol Gen Genet, 1995 Nov 1, 249(1), 102 - 10
Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending; Kreusch D et al.; The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium . The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs . We have investigated heterologous interactions between the regulators and the promoters . Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopaline, NocR, and the occ promoter led to limited promoter activation . We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo . The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect . Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine . The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations . Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters . No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated . These findings suggest that bend relaxation may be an integral part of promoter activation.

Plant Mol Biol, 1995 Nov, 29(4), 759 - 72
The promoter of the Vicia faba L . VfENOD-GRP3 gene encoding a glycine-rich early nodulin mediates a predominant gene expression in the interzone II-III region of transgenic Vicia hirsuta root nodules; Kuster H et al.; We recently reported on the broad bean gene VfENOD-GRP3 encoding a glycine-rich early nodulin . This gene was predominantly expressed in the interzone II-III region of Vicia faba root nodules . The VfENOD-GRP3 promoter contained several sequence motifs potentially involved in the regulation of gene expression . To investigate the molecular basis for the specific VfENOD-GRP3 expression, defined VfENOD-GRP3 promoter fragments were fused to an intron-containing gusAint gene . Agrobacterium rhizogenes ARqual strains carrying these fusions integrated into the TL DNA were used to generate hairy roots on Vicia hirsuta, which subsequently were nodulated . Histochemical analysis of transgenic nodules indicated that a strong gusAint expression in the interzone II-III region was mediated by the -1252/+10 VfENOD-GRP3 promoter region . This reporter gene expression in V . hirsuta was comparable to the location of VfENOD-GRP3 transcripts in V . faba nodules . An analysis of defined promoter fragments revealed that a strong gusAint expression in the interzone II-III region was also mediated by the -737/+10 promoter, whereas the -239/+10 promoter only mediated a weak gusAint expression in the interzone II-III region . Since the -239/+10 promoter fragment did not resemble published nodulin gene promoters, we propose that it contains new sequence motifs involved in mediating gene expression in the interzone II-III region of Vicia nodules.

J Bacteriol, 1995 Nov, 177(22), 6575 - 84
Structure and functional analysis of a marine bacterial carotenoid biosynthesis gene cluster and astaxanthin biosynthetic pathway proposed at the gene level; Misawa N et al.; A carotenoid biosynthesis gene cluster for the production of astaxanthin was isolated from the marine bacterium Agrobacterium aurantiacum . This cluster contained five carotenogenic genes with the same orientation, which were designated crtW, crtZ, crtY, crtI, and crtB . The stop codons of individual crt genes except for crtB overlapped the start codons of the following crt genes . Escherichia coli transformants carrying the Erwinia uredovora carotenoid biosynthesis genes provide suitable substrates for carotenoid biosynthesis . The functions of the five crt genes of A . aurantiacum were determined through chromatographic and spectroscopic analyses of the pigments accumulated in some E . coli transformants carrying various combinations of the E . uredovora and A . aurantiacum carotenogenic genes . As a result, the astaxanthin biosynthetic pathway is proposed for the first time at the level of the biosynthesis genes . The crtW and crtZ gene products, which mediated the oxygenation reactions from beta-carotene to astaxanthin, were found to have low substrate specificity . This allowed the production of many presumed intermediates of astaxanthin, i.e., adonixanthin, phoenicoxanthin (adonirubin), canthaxanthin, 3'-hydroxyechinenone, and 3-hydroxyechinenone.

J Bacteriol, 1995 Nov, 177(22), 6518 - 26
Sequence and mutational analysis of a tartrate utilization operon from Agrobacterium vitis; Crouzet P et al.; The grapevine is the natural host of the tumorigenic bacterium Agrobacterium vitis . Most of the A . vitis isolates can use tartrate, an unusually abundant compound in grapevine . The nopaline strain, AB4, contains a 170-kb conjugative plasmid (pTrAB4) encoding tartrate utilization . A 5.65-kb pTrAB4 region which enables non-tartrate-utilizing Agrobacterium tumefaciens to grow on tartrate was sequenced and mutagenized with the transcriptional fusion transposon Tn5-uidA1 . This DNA fragment contains four intact open reading frames (ORFs) (ttuABCD) required for tartrate-dependent growth . The mutant phenotypes of each ORF, their homologies to published sequences, and their induction patterns allowed us to propose a model for tartrate utilization in A . vitis . ttuA encodes a LysR-like transcriptional activator and is transcribed in the absence of tartrate . ttuB codes for a protein with homology to transporter proteins and is required for entry of tartrate into bacteria . ttuC codes for a tartrate dehydrogenase, while ttuD lacks homology to known sequences; the growth properties of ttuD mutants suggest that TtuD catalyzes the second step in tartrate degradation . A fifth incomplete ORF (ttuE) encodes a pyruvate kinase which is induced by tartrate and required for optimal growth . Although the ttuABCD fragment allows growth of A . tumefaciens on tartrate, it does not provide full tartrate utilization in the original A . vitis background.

Transgenic Res, 1995 Nov, 4(6), 388 - 96
Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter; Elmayan T et al.; In order to study the expression in plants of the rolD promoter of Agrobacterium rhizogenes, we have constructed chimaeric genes placing the coding region of the gusA (uidA) marker gene under control of two rolD promoter fragments of different length . Similar results were obtained with both genes . Expression studies were carried out in transformed R1 progeny plants . In mature transformed tobacco plants, the rolD-gus genes were expressed strongly in roots, and to much lower levels in stems and leaves . This pattern of expression was transmitted to progeny, though the ratio of the level of expression in roots relative to that in leaves was much lower in young seedlings . The degree of root specificity in rolD-gus transformants was less than that of a gene constructed with domain A of the CaMV 35S promoter, domA-gus, but the level of root expression was much higher than with the latter gene . However, the level of expression of the rolD-gus genes was less than that of a gus gene with a 35S promoter with doubled domain B, 35S2-gus . The rolD-gus genes had a distinctive pattern of expression in roots, compared to that of the two other genes, with the strongest GUS activity observed in the root elongation zone and in vascular tissue, and much less in the root apex.

Nucleic Acids Res, 1995 Oct 25, 23(20), 4087 - 91
Mutational analysis of a conserved motif of Agrobacterium tumefaciens VirD2; Vogel AM et al.; The VirD2 polypeptide from Agrobacterium tumefaciens, in the presence of VirD1, introduces a site- and strand-specific nick at the T-DNA borders . A similar reaction at the origin of transfer (oriT) of plasmids is essential for plasmid transfer by bacterial conjugation . A comparison of protein sequences of VirD2 and its functional homologs in bacterial conjugation and in rolling circle replication revealed that they share a conserved 14 residue segment, HxDxxx(P/u)HuHuuux {residues 126-139 of VirD2; Ilyina, T.V . and Koonin, E.V . (1992) Nucleic Acids Res . 20, 3279-3285} . A mutational approach was used to test the role of these residues in the endonuclease activity of VirD2 . The results demonstrated that the two invariant histidine residues (H133 and H135) are essential for activity . Mutations at three sites, histidine 126, aspartic acid 128 and aspartic acid 130, that are conserved in a subfamily of the plasmid mobilization proteins, led to the loss of VirD2 activity . Aspartic acid at position 130, could be substituted with glutamic acid and to a much lesser extent, with tyrosine . In contrast, another conserved residue, asparagine 139, tolerated many different amino acid substitutions . The non-conserved residues, arginine 129, proline 132 and leucine 134, were also found to be important for function . Isolation of null mutations that map throughout this conserved domain confirm the hypothesis that this region is essential for function.

Mol Gen Genet, 1995 Oct 25, 248(6), 649 - 56
Transgene inactivation in Petunia hybrida is influenced by the properties of the foreign gene; Elomaa P et al.; Petunia mutant RL01 was transformed with maize A1 and gerbera gdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity . The same Agrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments . Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants . However, the A1 and gdfr transformants showed clearly different phenotypes . The flowers of the primary A1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of the gdfr transformants showed intense and highly stable coloration . The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment . As previously reported by others, the instability in pigmentation in the A1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself . In the gdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome . Only rarely was partial methylation of the 35S promoter detected, while the gdfr cDNA always remained in an unmethylated state . We conclude that the properties of the transgene itself strongly influence the inactivation process . The dicotyledonous gdfr cDNA with a lower GC content and fewer possible methylation sites is more 'compatible' the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.

J Mol Biol, 1995 Oct 13, 253(1), 32 - 8
High angle and ligand-induced low angle DNA bends incited by OccR lie in the same plane with OccR bound to the interior angle; Wang L et al.; The OccR protein of Agrobacterium tumefaciens activates transcription of the occQ operon in response to octopine . Octopine shortens the DNase I footprint of OccR and relaxes an OccR-incited DNA bend . In this study, we used hydroxyl radical footprinting to show that OccR contacts only one face of the operator . This contact spans 50 nucleotide pairs in the absence of octopine, and 39 nucleotide pairs in the presence of octopine . Octopine enhanced protection of the central region of the footprint . OccR blocked intercalation by methidiumpropyl EDTA along most of the operator, but did not block intercalation at the bend center . OccR protected DNA against both reagents far more completely at the promoter-distal half of the operator than at the promoter-proximal half . Experiments to alter the phasing between the operator bend and an adjacent sequence-directed bend indicated that the high angle DNA bend in the absence of octopine, and the low angle bend in the presence of octopine, lie in the same plane . Operator DNA was bent toward the helical face that OccR protected from hydroxyl radicals, indicating that OccR occupies the interior angle of this bend.

Antonie Van Leeuwenhoek, 1995 Oct, 68(3), 237 - 43
Bacteriolytic activities of the free-living soil amoebae, Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis; Weekers PH et al.; Bacteriolytic activities of axenically grown free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and Hartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined . A spectrophotometric assay revealed that the specific bacteriolytic activities of both Acanthamoeba species were higher as those of the three Hartmannella strains . Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus and Pseudomonas fluorescens were more easily lysed than the other bacteria tested . Agrobacterium tumefaciens, Klebsiella aerogenes and Serratia marcescens were hardly affected at all by the amoebal bacteriolytic activities . Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis . Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3-10 was performed to separate the bacteriolytic isoenzymes of amoebae . Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay . The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae . Distinct differences between typical pI points of bacteriolytic activities in Acanthamoeba and Hartmannella were shown . Bacteriolytic activities of Hartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0-9.3 while for Acanthamoeba the range was pI 4.5-8.9.

J Bacteriol, 1995 Oct, 177(20), 5952 - 8
The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter; Segal G et al.; The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons . Transcription of this operon in A . tumefaciens was stimulated by heat shock as well as by exposure to ethanol and hydrogen peroxide . There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing only the dnaK gene . Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells . The upstream regulatory region of the dnaKJ operon of A . tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons . Sequence analysis of the promoter region of several groESL and dnaK operons from alpha-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor . This promoter may constitute the heat shock promoter of these alpha-purple proteobacteria.

Microbiology, 1995 Oct, 141 ( Pt 10), 2601 - 10
Agrobacterium radiobacter and related organisms take up fructose via a binding-protein-dependent active-transport system; Williams SG et al.; Washed cells of Agrobacterium radiobacter prepared from a fructose-limited continuous culture (D 0.045 h-1) transported D(-){U-14C}fructose in a linear manner for up to 4 min at a rate several-fold higher than the rate of fructose utilization by the growing culture . D(-){U-14C}Fructose transport exhibited a high affinity for fructose (KT < 1 microM) and was inhibited to varying extents by osmotic shock, by the uncoupling agent carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose > D-glucose/D-galactose/D-xylose; no inhibition by D-arabinose) . Prolonged growth of A . radiobacter in fructose-limited continuous culture led to the selection of a novel strain (AR100) which overproduced a fructose-binding protein (FBP) and showed an increased rate of fructose transport . FBP was purified from osmotic-shock fluid using anion-exchange fast protein liquid chromatography (FPLC) . The monomeric protein (M(r) 34,200 by SDS-PAGE and 37,700 by gel-filtration FPLC) bound D-{U-14C}-fructose stoichiometrically (1.17 nmol nmol FBP-1) and with high affinity (KD 0.49 microM) as shown by equilibrium dialysis . Binding of D-{U-14C}fructose by FBP was variably inhibited by unlabelled sugars (D-fructose/D-mannose > D-ribose > D-sorbose; no inhibition by D-glucose, D-galactose or D-arabinose) . The N-terminal amino acid sequence of FBP (ADTSVCLI-) was similar to that of several sugar-binding proteins from other species of bacteria . Fructose transport and FBP were variably induced in batch cultures of A . radiobacter by growth on different carbon sources (D-fructose > D-ribose/D-mannose > D-glucose; no induction by succinate) . An immunologically similar protein to FBP was produced by Agrobacterium tumefaciens and various species of Rhizobium following growth on fructose . It is concluded that fructose is transported into A . radiobacter and related organisms via a periplasmic fructose/mannose-binding-protein-dependent active-transport system, in contrast to the phosphotransferase system used by many other species of bacteria.

Microbiology, 1995 Oct, 141 ( Pt 10), 2425 - 32
Study of the organization of the genomes of Escherichia coli, Brucella melitensis and Agrobacterium tumefaciens by insertion of a unique restriction site; Jumas-Bilak E et al.; Tn5Map, a Tn5 derivative containing the 18 bp I-SceI site, was delivered from a RP4-mobilizable, RK6-derived suicide vector to Escherichia coli HB101, Brucella melitensis and Agrobacterium tumefaciens C58, which all lack natural I-SceI sites in their genomes . Digestion of the DNA from Tn5Map-containing strains and analysis by pulsed-field gel electrophoresis (PFGE) revealed that these derivatives contained a single transposon insertion . These digests also gave direct and independent proof for the single circular chromosome of E . coli, and for the presence of two circular chromosomes in B . melitensis and of a circular and a linear chromosome in A . tumefaciens C58 (which also contains two large circular plasmids) . This rapid and versatile technique is potentially applicable to the study of the genomic organization in all Gram-negative bacteria which support Tn5 transposition . Moreover, linearization of circular replicons could be the first step for a rapid method of physical mapping.

Plant Mol Biol, 1995 Oct, 29(2), 279 - 92
A novel wound-inducible extensin gene is expressed early in newly isolated protoplasts of Nicotiana sylvestris; Parmentier Y et al.; A cDNA clone (6PExt 1.2) encoding a novel extensin was isolated from a cDNA library made from 6 h old mesophyll protoplasts of Nicotiana sylvestris . The screening was performed with a heterologous probe from carrot . The encoded polypeptide showed features characteristic of hydroxyproline-rich glycoproteins such as Ser-(Pro)4 repeats and a high content in Tyr and Lys residues . The presence of four Tyr-X-Tyr-Lys motifs suggests the possibility for intramolecular isodityrosine cross-links whereas three Val-Tyr-Lys motifs may participate in intermolecular cross-links . The analysis of genomic DNA gel blots using both the N . sylvestris and the carrot clones as probes showed that the 6PExt 1.2 gene belongs to a complex multigene family encoding extensin and extensin-related polypeptides in N . sylvestris as well as in related Nicotianeae including a laboratory hybrid . This was confirmed by the analysis of RNA gel blots: a set of mRNAs ranging in size from 0.3 kb to 3.5 kb was found by the carrot extensin probe . The 6PExt 1.2 probe found a 1.2 kb mRNA in protoplasts and in wounded tissues as well as a 0.9 kb mRNA which seemed to be stem-specific . The gene encoding 6PExt 1.2 was induced by wounding in protoplasts, in leaf strips and after Agrobacterium tumefaciens infection of stems.

Plant Mol Biol, 1995 Oct, 29(1), 99 - 108
Site-directed mutagenesis of the enhancer region of the 780 gene promoter of T-DNA; O'Grady K et al.; Potential regulatory sequences within the enhancer-like region of the 780 gene promoter (Agrobacterium tumefaciens T-DNA) were identified by site-directed mutagenesis . Transcriptional activity of the mutated promoter was analyzed by S1 nuclease mapping of RNA from crown gall tumors of sunflower incited using a T-DNA-based vector . Variability in expression levels were minimized by the use of an internal reference gene and the pooling of at least 200 tumors per construct tested . This approach identified numerous sequences that influence transcriptional activity in either a positive or negative manner . Eight regions of positive influence and three of negative were identified from analysis of those mutations that exhibited low variability in expression (P < 0.005) and affected activity by at least 20%.

Plant Mol Biol, 1995 Oct, 29(1), 125 - 33
Rice scutellum induces Agrobacterium tumefaciens vir genes and T-strand generation; Vijayachandra K et al.; For successful transformation of a plant by Agrobacterium tumefaciens it is essential that the explant used in cocultivation has the ability to induce Agrobacterium tumour-inducing (Ti) plasmid virulence (vir) genes . Here we report a significant variation in different tissues of Indica rice (Oryza sativa L . cv . Co43) in their ability to induce Agrobacterium tumefaciens vir genes and T-strand generation, using explants preincubated in liquid Murashige and Skoog (MS) medium . An analysis of rice leaf segments revealed that they neither induced vir genes nor inhibited vir gene induction . Of different parts of rice plants of different ages analysed only scutellum from four-day old rice seedlings induced vir genes and generation of T-strands . We observed that the physical presence of preincubated scutella is required for vir gene induction . Conditioned medium from which preincubated scutella were removed did not induce the vir genes . Scutellum-derived calli, cultured for 25 days on medium containing 2,4-D, also induced virE to an appreciable level . These results suggest that scutellum and scutellum-derived calli may be the most susceptible tissues of rice for Agrobacterium-mediated transformation.

J Bacteriol, 1995 Oct, 177(19), 5485 - 94
Identification of Rhizobium-specific intergenic mosaic elements within an essential two-component regulatory system of Rhizobium species; Osteras M et al.; Analysis of the DNA regions upstream of the phosphoenolpyruvate carboxykinase gene (pckA) in Rhizobium meliloti and Rhizobium sp . strain NGR234 identified an open reading frame which was highly homologous to the Agrobacterium tumefaciens chromosomal virulence gene product ChvI . A second gene product, 500 bp downstream of the chvI-like gene in R . meliloti, was homologous to the A . tumefaciens ChvG protein . The homology between the R . meliloti and A . tumefaciens genes was confirmed, because the R . meliloti chvI and chvG genes complemented A . tumefaciens chvI and chvG mutants for growth on complex media . We were unable to construct chvI or chvG insertion mutants of R . meliloti, whereas mutants carrying insertions outside of these genes were readily obtained . A 108-bp repeat element characterized by two large palindromes was identified in the chvI and chvG intergenic regions of both Rhizobium species . This element was duplicated in Rhizobium sp . strain NGR234 . Another structurally similar element with a size of 109 bp was present in R . meliloti but not in Rhizobium sp . strain NGR234 . These elements were named rhizobium-specific intergenic mosaic elements (RIMEs), because their distribution seems to be limited to members of the family Rhizobiaceae . A homology search in GenBank detected six more copies of the first element (RIME1), all in Rhizobium species, and three extra copies of the second element (RIME2), only in R . meliloti . Southern blot analysis with a probe specific to RIME1 showed the presence of several copies of the element in the genome of R . meliloti, Rhizobium sp . strain NGR234, Rhizobium leguminosarum, and Agrobacterium rhizogenes, but none was present in A . tumefaciens and Bradyrhizobium japonicum.

J Bacteriol, 1995 Oct, 177(19), 5419 - 26
Cloning, sequencing, and phenotypic analysis of laf1, encoding the flagellin of the lateral flagella of Azospirillum brasilense Sp7; Moens S et al.; Azospirillum brasilense can display a single polar flagellum and several lateral flagella . The A . brasilense Sp7 gene laf1, encoding the flagellin of the lateral flagella, was isolated and sequenced . The derived protein sequence is extensively similar to those of the flagellins of Rhizobium meliloti, Agrobacterium tumefaciens, Bartonella bacilliformis, and Caulobacter crescentus . An amino acid alignment shows that the flagellins of these bacteria are clustered and are clearly different from other known flagellins . A laf1 mutant, FAJ0201, was constructed by replacing an internal part of the laf1 gene by a kanamycin resistance-encoding gene cassette . The mutant is devoid of lateral flagella but still forms the polar flagellum . This phenotype is further characterized by the abolishment of the capacities to swarm on a semisolid surface and to spread from a stab inoculation in a semisolid medium . FAJ0201 shows a normal wheat root colonization pattern in the initial stage of plant root interaction.

Int J Syst Bacteriol, 1995 Oct, 45(4), 706 - 11
Bradyrhizobium liaoningense sp . nov., isolated from the root nodules of soybeans; Xu LM et al.; Seventeen strains of extra-slowly growing (ESG) soybean rhizobia isolated from root nodules of Glycine soja and Glycine max growing in five provinces (Liaoning, Heilongjiang, Shanxi, Hubei, and Anhui) in the People's Republic of China were compared with 48 reference strains belonging to the genera Bradyrhizobium, Rhizobium, and Agrobacterium by performing a numerical analysis of 191 phenotypic features . Our results showed that all of the ESG strains examined clustered closely in the genus Bradyrhizobium but were separated from Bradyrhizobium japonicum at the species level and that they could be differentiated from Rhizobium and Agrobacterium species at the genus level . On the basis of the results of our numerical taxonomy analysis, a genomic DNA G & C content analysis, DNA-DNA hybridization experiments, a partial 16S rRNA sequence analysis, a serological analysis, an N and C content analysis, and an N/C ratio analysis of members of the three groups of soybean rhizobia, we propose the name Bradyrhizobium liaoningense sp . nov . for the ESG strains; the type strain of this species is strain 2281.

J Biol Chem, 1995 Sep 15, 270(37), 21928 - 33
Construction of chimeric beta-glucosidases with improved enzymatic properties; Singh A et al.; The amino acid sequences of beta-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show about 40% similarity . The pH/temperature optima and stabilities and substrate specificities of the two enzymes are quite different . C . gilvus beta-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35 degrees C, whereas the corresponding values for A . tumefaciens are 7.2-7.4 and 60 degrees C, respectively . The substrate specificity of A . tumefaciens enzyme toward different aryl glycosides is broader than C . gilvus enzyme . To analyze these properties further, three chimeric beta-glucosidases were constructed by substituting segments from the C-terminal homologous region of C . gilvus beta-glucosidase gene with that of A . tumefaciens . The chimeric enzymes were characterized with respect to pH/temperature activity and stability and substrate specificity . Chimeric enzymes exhibited chromatographic behavior similar to that of C . gilvus enzyme . However, enzymatic properties of chimeras were admixtures of those of the two parents . The chimeric enzymes were optimally active at 45-50 degrees C and pH 6.6-7.0 . Km values of chimeric enzymes for the various saccharides were admixtures of both parental enzymes . These results suggest that the two domains of C . gilvus and A . tumefaciens enzymes probably can fold independently . The homologous C-terminal region in beta-glucosidase appears to play an important role in determining enzyme characteristics . Changes in the properties on substitution of segments in this region might be related to the enzyme specificity, and beta-glucosidases with improved properties can be prepared by manipulating this region.

Transgenic Res, 1995 Sep, 4(5), 315 - 23
Overexpression of a tryptophan decarboxylase cDNA in Catharanthus roseus crown gall calluses results in increased tryptamine levels but not in increased terpenoid indole alkaloid production; Goddijn OJ et al.; The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) catalyses a key step in the biosynthesis of terpenoid indole alkaloids in C . roseus by converting tryptophan into tryptamine . Hardly any tdc mRNA could be detected in hormone-independent callus and cell suspension cultures transformed by the oncogenic T-DNA of Agrobacterium tumefaciens . Supply of tryptamine may therefore represent a limiting factor in the biosynthesis of alkaloids by such cultures . To investigate this possibility, chimaeric gene constructs, in which a tdc cDNA is linked in the sense or antisense orientation to the cauliflower mosaic virus 35S promoter and terminator, were introduced in C . roseus cells by infecting seedlings with an oncogenic A . tumefaciens strain . In the resulting crown gall tumour calluses harbouring the tdc sense construct, an increased TDC protein level, TDC activity and tryptamine content but no significant increase in terpenoid indole alkaloid production were observed compared to empty-vector-transformed tumour calluses . In tumour calluses containing the tdc antisense construct, decreased levels of TDC activity were measured . Factors which might be responsible for the lack in increased terpenoid indole alkaloid production in the tdc cDNA overexpressing crown gall calluses are discussed.

J Anim Sci, 1995 Sep, 73(9), 2752 - 9
A biotechnological approach to improving the nutritive value of alfalfa; Tabe LM et al.; The postruminal supply of the sulfur-containing amino acids, methionine and cysteine, has been reported to be a major limitation to wool growth in sheep . We aim to improve the protein quality of forage for ruminants by introducing into alfalfa chimeric genes encoding a ruminally stable, sulfur amino acid-rich protein from sunflower seeds . Four gene constructs were transferred to Australian commercial cultivars of alfalfa using Agrobacterium tumefaciens-mediated transformation and selection with phosphinothricin (PPT) . Modification of the sunflower seed albumin protein-coding region by addition of the coding information for an endoplasmic reticulum (ER) retention signal was found to greatly increase the level to which the sulfur amino acid-rich protein accumulated in the leaves of transgenic alfalfa plants . The Cauliflower Mosaic Virus (CaMV) 35S promoter and two light-regulated plant gene promoter regions were compared for their ability to direct high-level expression of the introduced genes in alfalfa leaves . The highest expression of sunflower seed albumin was found in transformants bearing a gene incorporating the promoter from the Arabidopsis thaliana ats1A gene, which encodes the ribulose bisphosphate carboxylase small subunit . The highest level of sunflower seed albumin found in transgenic alfalfa leaves was estimated to constitute .1% of soluble leaf protein . This level of accumulation of the foreign protein would be predicted to supply an extra 40 mg of sulfur amino acids daily to sheep fed the modified forage . Published studies in which wool growth rates were significantly increased employed supplementation of approximately 1 to 2 g of sulfur amino acids daily.

Plant Cell Physiol, 1995 Sep, 36(6), 1003 - 12
The regulatory functions of the rolB and rolC genes of Agrobacterium rhizogenes are conserved in the homologous genes (Ngrol) of Nicotiana glauca in tobacco genetic tumors; Nagata N et al.; To compare patterns of expression between the Ngrol genes of N . glauca and the Rirol genes of Agrobacterium rhizogenes, we performed fluorometric and histochemical analysis of transgenic genetic tumors on the hybrid of Nicotiana glauca x N . langsdorffii (F1) that harbored a beta-glucuronidase (GUS) reporter gene fused to the promoter of NgrolB, NgrolC, RirolB or RirolC . The promoters of NgrolB and NgrolC had 2- to 3-fold lower activity than those of RirolB and RirolC . However, the changes in patterns of GUS activity caused by deletion of NgrolB and NgrolC promoters were similar to those of RirolB and RirolC promoters . This result suggests that the cis-acting sequences that regulate the level of expression of RirolB and RirolC are conserved in the NgrolB and NgrolC promoters . Furthermore, an auxin dependent (NAA-dependent) increase in GUS activity was observed in the case of NgroB-GUS and RirolB-GUS . Histochemical analysis showed GUS activity encoded by both NgrolB-GUS and RirolB-GUS in normal-type F1 transgenic plants was located in meristematic zones, while that encoded by NgrolC-GUS and RirolC-GUS was detected mainly in vascular systems of various organs . Thus, the patterns of expression of the Ngrol genes were the same as those of the Rirol genes in terms of promotion by auxin and tissue-specificity, indicating that regulatory mechanisms for both sets of genes have been conserved during the evolution of the genus Nicotiana after transfer from a progenitor of Agrobacterium to that of Nicotiana.

J Bacteriol, 1995 Sep, 177(17), 4890 - 9
Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the IncQ plasmid RSF1010; Binns AN et al.; The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid . The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2 . The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system . Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11 . Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules . By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A . tumefaciens were also monitored . The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants . Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation . Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.

J Bacteriol, 1995 Sep, 177(17), 4881 - 9
Interactions of VirB9, -10, and -11 with the membrane fraction of Agrobacterium tumefaciens: solubility studies provide evidence for tight associations; Finberg KE et al.; The eleven predicted gene products of the Agrobacterium tumefaciens virB operon are believed to form a transmembrane pore complex through which T-DNA export occurs . The VirB10 protein is required for virulence and is a component of an aggregate associated with the membrane fraction of A . tumefaciens . Removal of the putative membrane-spanning domain (amino acids 22 through 55) disrupts the membrane topology of VirB10 (J . E . Ward, E . M . Dale, E . W . Nester, and A . N . Binns, J . Bacteriol . 172:5200-5210, 1990) . Deletion of the sequences encoding amino acids 22 to 55 abolishes the ability of plasmid-borne virB10 to complement a null mutation in the virB10 gene, suggesting that the proper topology of VirB10 in the membrane may indeed play a crucial role in T-DNA transfer to the plant cell . Western blot (immunoblot) analysis indicated that the observed loss of virulence could not be attributed to a decrease in the steady-state levels of the mutant VirB10 protein . Although the deletion of the single transmembrane domain would be expected to perturb membrane association, VirB10 delta 22-55 was found exclusively in the membrane fraction . Urea extraction studies suggested that this membrane localization might be the result of a peripheral membrane association; however, the mutant protein was found in both inner and outer membrane fractions separated by sucrose density gradient centrifugation . Both wild-type VirB10 and wild-type VirB9 were only partially removed from the membranes by extraction with 1% Triton X-100, while VirB5 and VirB8 were Triton X-100 soluble . VirB11 was stripped from the membranes by 6 M urea but not by a more mild salt extraction . The fractionation patterns of VirB9, VirB10, and VirB11 were not dependent on each other or on VirB8 or VirD4 . The observed tight association of VirB9, VirB10, and VirB11 with the membrane fraction support the notion that these proteins may exist as components of multiprotein pore complexes, perhaps spanning both the inner and outer membranes of Agrobacterium cells.

Mol Plant Microbe Interact, 1995 Sep-Oct, 8(5), 792 - 4
Synthetic antimicrobial peptide design; Powell WA et al.; To guide the design of potential plant pathogen-resistance genes, synthetic variants of naturally occurring antimicrobial gene products were evaluated . Five 20-amino acid (ESF1, ESF4, ESF5, ESF6, ESF13), one 18-amino acid (ESF12), and one 17-amino acid (ESF17) amphipathic peptide sequences were designed, synthesized, and tested with in vitro bioassays . Positive charges on the hydrophilic side of the peptide were shown to be essential for antifungal activity, yet the number of positive charges could be varied with little or no change in activity . The size could be reduced to 18 amino acids, but at 17 amino acids a significant reduction in activity was observed . ESF1, 5, 6, and 12 peptides were inhibitory to the germination of conidia from Cryphonectria parasitica, Fusarium oxysporum f . sp . lycopersici, and Septoria musiva but did not inhibit the germination of pollen from Castanea mollissima and Salix lucida . ESF12 also had no effect on the germination of Malus sylvestris and Lycopersicon esculentum pollen, but inhibited the growth of the bacteria Agrobacterium tumefaciens, Erwinia amylovora, and Pseudomonas syringae . The minimal inhibitory concentrations of the active ESF peptides were similar to those of the naturally occurring control peptides, magainin II and cecropin B . The significant differential in sensitivity between the microbes and plant cells indicated that the active ESF peptides are potentially useful models for designing plant pathogen-resistance genes.

Mol Plant Microbe Interact, 1995 Sep-Oct, 8(5), 788 - 91
Regulation of Agrobacterium tumefaciens virulence gene expression: isolation of a mutation that restores virGD52E function; Gubba S et al.; Expression of Agrobacterium tumefaciens virulence (vir) genes is controlled by virA, virG, and a plant inducer . Isolation of two constitutive mutants of the transcriptional activator virG, virGN54D, and virGI106L, that support vir gene expression in a virA independent manner has previously been reported . Characterization of virGN54D by several groups showed considerable variation in its ability to activate vir gene transcription . In this study we demonstrate that these differences can be accounted for by plasmid copy number . We report the isolation of a third constitutive mutation, virGI77V, that partially restores transcription activation function of a nonfunctional virG mutant, virGD52E . The second regulator, VirA, in its extreme C-terminus, contains a domain that is homologous to the N-terminal domain of VirG . Deletion of this domain of VirA leads to a fully constitutive phenotype.

Mol Plant Microbe Interact, 1995 Sep-Oct, 8(5), 747 - 54
Pleiotropic effects of regulatory ros mutants of Agrobacterium radiobacter and their interaction with Fe and glucose; Brightwell G et al.; Four exo mutants of Agrobacterium radiobacter, defective in the synthesis of acidic exopolysaccharide were complemented by a gene from that species, which is similar to the transcriptional regulator, ros, of A . tumefaciens . It was confirmed that this A . radiobacter gene, which we term rosAR, like ros, repressed its own transcription as well as that of virC and virD, two loci involved in tumorigenesis . The sequence of RosAR suggested that it might bind to a transition metal and its repressor abilities were shown to require Fe in the medium; repression was also enhanced with increasing levels of glucose . Certain rosAR mutants, in which its 3' end was removed were dominant; i.e., when plasmids containing such mutant forms of the gene were introduced into wild-type A . radiobacter, the transconjugants were nonmucoid . Such effects were also seen in a wide range of bacteria, including Escherichia coli and Xanthomonas . Several mutants that were complementd by rosAR also accumulated protoporphyrin, suggesting a defect in haem synthesis.

Eur J Biochem, 1995 Sep 1, 232(2), 536 - 44
Quinoline 2-oxidoreductase and 2-oxo-1,2-dihydroquinoline 5,6-dioxygenase from Comamonas testosteroni 63 . The first two enzymes in quinoline and 3-methylquinoline degradation; Schach S et al.; The enzymes catalysing the first two steps of quinoline and 3-methylquinoline degradation by Comamonas testosteroni 63 were investigated . Quinoline 2-oxidoreductase, which catalyses the hydroxylation of (3-methyl-)quinoline to (3-methyl-)2-oxo-1,2-dihydroquinoline, was purified to apparent homogeneity . The native enzyme, with a molecular mass of 360 kDa, is composed of three non-identical subunits (87, 32, and 22 kDa), occurring in a ratio of 1.16:1:0.83 . Containing FAD, molybdenum, iron, and acid-labile sulfur in the stoichiometric ratio of 2:2:8:8, the enzyme belongs to the molybdo-iron/sulfur flavoproteins . Molybdopterin cytosine dinucleotide is the organic part of the pterin molybdenum cofactor . Comparison of N-terminal amino acid sequences revealed similarities to a number of procaryotic molybdenum-containing hydroxylases . Especially the N-termini of the beta-subunits of the quinoline 2-oxidoreductases from Comamonas testosteroni 63, Pseudomonas putida 86, and Rhodococcus spec . B1, and of quinoline-4-carboxylic acid 2-oxidoreductase from Agrobacterium spec . 1B showed striking similarities . Further degradation of (3-methyl-)2-oxo-1,2-dihydroquinoline proceeds via dioxygenation at the benzene ring, i.e . at 5,6-position {Schach, S., Schwarz, G., Fetzner, S . & Lingens, F . (1993) Biol . Chem . Hoppe-Seyler 374, 175-181} . 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase was partially purified; NADH and oxygen are required for the reaction, and the enzymic activity is enhanced 1.5-fold by addition of Fe2+ ions . Unexpectedly, this aromatic ring dioxygenase did not separate into distinct protein components, but is apparently a single-component enzyme . The molecular mass was estimated to be about 260 kDa . 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is very thermolabile . However, dithioerythritol and low concentrations of substrate had a moderately stabilizing effect . 2-Oxo-1,2-dihydroquinoline 5,6-dioxygenase is inhibited by sulfhydryl-blocking agents, by metal-chelating agents, and by the flavin analogues quinacrine and acriflavin.

Plant Mol Biol, 1995 Sep, 28(6), 1149 - 54
Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA; Ramanathan V et al.; We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6 . Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner . Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences . These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.

Plant Mol Biol, 1995 Sep, 28(6), 1103 - 10
A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and nod factor induced expression; Vijn I et al.; ENOD12 is one of the first nodulin genes expressed upon inoculation with Rhizobium and also purified Nod factors are able to induce ENOD12 expression . The ENOD12 gene family in pea (Pisum sativum) has two members . A cDNA clone representing PsENOD12A {26} and a PsENOD12B genomic clone {7} have been previously described . The isolation and characterization of a PsENOD12A genomic clone is presented in this paper . By using a Vicia hirsuta-Agrobacterium rhizogenes transformation system it is shown that both genes have a similar expression pattern in transgenic V . hirsuta root nodules . Promoter analyses of both PsENOD12 promoters showed that the 200 bp immediately upstream of the transcription start are sufficient to direct nodule-specific and Nod factor-induced gene expression.

Plant Physiol, 1995 Sep, 109(1), 41 - 52
Stress activation of a bean hydroxyproline-rich glycoprotein promoter is superimposed on a pattern of tissue-specific developmental expression; Wycoff KL et al.; The HRGP4.1 gene, which encodes a cell wall hydroxyproline-rich glycoprotein, was isolated from a genomic library of bean (Phaseolus vulgaris L.) . Two transcripts, one induced by wounding and one by elicitation, were transcribed from the same initiation site . The gene encodes a polypeptide of 580 amino acids with the amino terminal half consisting of repeats of the sequence serine-(proline)4-lysine-histidine-serine-(proline)4-(tyrosine)3-histidi ne and the carboxyl-terminal half composed of repeats of the sequence serine-(proline)4-valine-tyrosine-lysine-tyrosine-lysine . A 964-bp upstream promoter fragment was translationally fused to the beta-glucuronidase reporter gene (Escherichia coli uidA) and transferred into tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation . Analysis of beta-glucuronidase activity showed that wounding caused local activation of the HRGP4.1 promoter in the phloem . Infection by tobacco mosaic virus was a less effective inducer than wounding . Stress induction was superimposed on tissue-specific developmental expression in stem nodes and root tips, suggesting that HRGP4.1 may have specific structural roles in development as well as protective functions in defense . Deletion analysis showed that control of tissue specificity and wound inducibility lies in a region between -94 and -251 relative to the transcription start site and that activation by infection lies outside that region.

Appl Microbiol Biotechnol, 1995 Aug-Sep, 43(4), 706 - 12
The effect of pH and oxygen concentration on the formation of 3-ketodisaccharides by Agrobacterium tumefaciens; Stoppok E et al.; The further optimization of 3-ketodisaccharide formation with sucrose, leucrose and iso maltulose was studied with special regard to pH and oxygen concentration in the reaction mixture with resting cells of Agrobacterium tumefaciens . It was found that the optimal pH values for the highest reaction rate and highest yield were different as the pH affected the stability of the 3-keto derivatives formed . A pH shift to 5.0 clearly reduced the enzymatic degradation of the 3-keto derivatives thus stabilizing them . The influence of constant oxygen concentrations on 3-ketosucrose formation was tested showing results not explicable with normal Michaelis-Menten kinetics . For each substrate a maximum of reaction rate and yield were obtained at very low oxygen concentrations.

Appl Environ Microbiol, 1995 Aug, 61(8), 2879 - 84
Universal PCR primers for detection of phytopathogenic Agrobacterium strains; Haas JH et al.; Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains . The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested . PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes . The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction . One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA . The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.

EMBO J, 1995 Jul 17, 14(14), 3585 - 95
The Agrobacterium tumefaciens virulence D2 protein is responsible for precise integration of T-DNA into the plant genome; Tinland B et al.; The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T-DNA during its transfer to the plant cell nucleus . We analyze here its participation in the integration of T-DNA by using a virD2 mutant . This mutation reduces the efficiency of T-DNA transfer, but the efficiency of integration of T-DNA per se is unaffected . Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration . These results indicate that the wild-type VirD2 protein participates in ligation of the 5'-end of the T-strand to plant DNA and that this ligation step is not rate limiting for T-DNA integration.

EMBO J, 1995 Jul 3, 14(13), 3206 - 14
Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae; Bundock P et al.; Agrobacterium tumefaciens transfers part of its tumour-inducing (Ti) plasmid, the transferred or T-DNA, to plants during tumourigenesis . This represents the only example of naturally occurring trans-kingdom transfer of genetic material . Here we report that A.tumefaciens can transfer its T-DNA not only to plant cells, but also to another eukaryote, namely the yeast Saccharomyces cerevisiae . The Ti plasmid virulence (vir) genes that mediate T-DNA transfer to plants were found to be essential for transfer to yeast as well . Transgenic S.cerevisiae strains were analysed for their T-DNA content . Results showed that T-DNA circles were formed in yeast with precise fusions between the left and right borders . Such T-DNA circles were stably maintained by the yeast if the replicator from the yeast 2 mu plasmid was present in the T-DNA . Integration of T-DNA in the S.cerevisiae genome was found to occur via homologous recombination . This contrasts with integration in the plant genome, where T-DNA integrates preferentially via illegitimate recombination . Our results thus suggest that the process of T-DNA integration is predominantly determined by host factors.

Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 576 - 83
Isolation of ropB, a gene encoding a 22-kDa Rhizobium leguminosarum outer membrane protein; Roest HP et al.; As judged from immunochemical detection, the levels of outer membrane antigen groups II and III of Rhizobium leguminosarum bv . viciae strain 248 decrease during bacteroid differentiation (R . A . de Maagd, R . de Rijk, I . H . M . Mulders, and B . J . J . Lugtenberg, J . Bacteriol . 171:1136-1142, 1989) . Using a newly developed colony blot assay, a cosmid clone expressing the Mab8 epitope of the outer membrane antigen group II of R . l . bv . viciae strain 248 was selected in Rhizobium meliloti LPR2120 . From this cosmid the gene encoding this epitope was cloned and characterized . An open reading frame of 636 nucleotides was found and predicted to encode a protein with a calculated molecular mass of 22.5 kDa . After subtraction of the predicted 23 amino acid signal peptide, a M(r) of 20.3 kDa was calculated for the mature protein . This gene, designated ropB, was not active in Escherichia coli under the control of its own promoter . The C-terminal amino acid of the protein is a phenylalanine residue which is required for efficient translocation of outer membrane proteins . Membrane spanning amphipathic beta-sheets are predicted to represent a major part of the secondary structure of the protein . A model of the topology of the protein is presented . We determined the start of transcription in order to analyze the promoter region . No homology was found with other known promoter sequences . The ropB gene appeared to be well-conserved in R . leguminosarum and Agrobacterium tumefaciens strains . An attempt was made to mimic the immunochemical decrease of RopB ex planta . Neither the various growth conditions tested nor the addition of nodule or plant extracts resulted in a reduction of the Mab8 epitope to bacteroid levels.

Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 538 - 48
T-DNA genes responsible for inducing a necrotic response on grape vines; Deng W et al.; Agrobacterium tumefaciens supervirulent strain A281 induces a progressive necrotic response, rather than tumor formation, when inoculated on stems of several grape cultivars . The Ti plasmid, and specifically its T-DNA, is required for the process . In the present study, 40 T-DNA insertion mutants of A281 were generated via transposon mutagenesis and tested for their necrosis-inducing ability on grape stems in vitro . Ten mutants were attenuated in inducing necrogenesis . Restriction mapping and DNA sequencing revealed that at least two genes, tms1 and 6b, whose gene products are involved in the synthesis and activity modulation of auxin, are responsible for inducing necrogenesis . Double mutants of tms1 and 6b were totally non-necrogenic . The orientation of grapevine stem explants showed strong effects on the occurrence and progress of necrogenesis . Inoculation of Agrobacterium on physiological basal ends resulted in the greatest degree of necrogenesis . In addition, gene 5 of T-DNA, which modulates auxin responses in plants by the autoregulated synthesis of an auxin antagonist, was found to be separated from other TL-DNA genes by a novel insertion sequence, IS1312 . Since a T-DNA borderlike sequence occurs in IS1312, gene 5 might not always be transferred into plants . Based on the accumulated data, we propose that the necrogenesis induced by Agrobacterium results from the sensitivity of grapevine cells to elevated levels of auxin or a precursor of auxin.

Mol Plant Microbe Interact, 1995 Jul-Aug, 8(4), 532 - 7
Hairy root nodulation of Casuarina glauca: a system for the study of symbiotic gene expression in an actinorhizal tree; Diouf D et al.; The purpose of this study was to establish a fast system for producing transgenic actinorhizal root nodules of Casuarina glauca . Agrobacterium rhizogenes strain A4RS carrying the p35S-gusA-int gene construct was used to induce hairy roots on hypocotyls of 3-week-old C . glauca seedlings . Three weeks after wounding, the original root system was excised, and composite plants consisting of transgenic roots on untransformed shoots were transferred to test tubes to be inoculated with Frankia . The actinorhizal nodules formed on transformed roots had the nitrogenase activity and morphology of untransformed nodules . beta-Glucuronidase (GUS) activity was examined in transgenic roots and nodules by fluorometric and histochemical assays . The results indicate that transgenic nodules generated with this root transformation system could facilitate the molecular study of symbiotic nitrogen fixation in actinorhizal trees.

Res Microbiol, 1995 Jul-Aug, 146(6), 485 - 92
Utilization of cellobiose and other beta-D-glucosides in Agrobacterium tumefaciens; Marasco R et al.; Agrobacterium tumefaciens strain C58 was able to utilize carbon from cellobiose and some other beta-D-glucosides as efficiently as from glucose . beta-D-glucoside utilization was partially inducible and the induction was subject to catabolite repression by glucose, independently of the presence of cyclic AMP in the medium . It was also independent of Ti plasmid-encoded functions . beta-D-glucosides were hydrolysed by a single, cytoplasmic and constitutively expressed beta-glucosidase, which was active on non-phosphorylated substrates and insensitive to glucose inhibition.

Plant Physiol, 1995 Jul, 108(3), 929 - 37
Activation of two osmotin-like protein genes by abiotic stimuli and fungal pathogen in transgenic potato plants; Zhu B et al.; Osmotin-like proteins are encoded by at least six members of a multigene family in Solanum commersonii . A genomic clone (lambda pGEM2a-7) that contains two osmotin-like protein genes (OSML13 and OSML81) arranged in the same transcriptional orientation has been isolated . Restriction mapping and sequence analysis indicated that the two intronless genes correspond to the previously characterized pA13 and pA81 cDNAs . To study the transcriptional activation of OSML13 and OSML81 promoters, the 5' flanking DNA sequence (-1078 to +35 of OSML13 and -1054 to +41 of OSML81) was fused to the beta-glucoronidase (GUS) coding region, and the chimeric gene fusions were introduced into wild potato (S . commersonii) plants via Agrobacterium-mediated transformation . Analysis of the chimeric gene expression in transgenic potato plants showed that both 5' flanking DNA sequences are sufficient to impart GUS inducibility by abscisic acid, NaCl, salicylic acid, wounding, and fungal infection . Low temperature activated both chimeric genes only slightly . Infection with Phytophthora infestans resulted in strong GUS expression from both chimeric genes primarily in the sites of pathogen invasion, suggesting a limited diffusion of fungal infection-mediated signals . The expression patterns of both osmotin-like protein genes implicate their dual functions in osmotic stress and plant pathogen defense.

J Bacteriol, 1995 Jul, 177(13), 3808 - 17
Supraoperonic clustering of pca genes for catabolism of the phenolic compound protocatechuate in Agrobacterium tumefaciens; Parke D; The protocatechuate branch of the beta-ketoadipate pathway comprises the last six enzymatic steps in the catabolism of diverse phenolic compounds to citric acid cycle intermediates . In this paper, the regulation and tight supraoperonic clustering of the protocatechuate (pca) genes from Agrobacterium tumefaciens A348 are elucidated . A previous study found that the pcaD gene is controlled by an adjacent regulatory gene, pcaQ, which encodes an activator . The activator responded to beta-carboxy-cis,cis-muconate and was shown to control the synthesis of at least three genes (pcaD and pcaHG) . In this work, eight genes required for the catabolism of protocatechuate were localized within a 13.5-kb SalI region of DNA . Isolation and characterization of transposon Tn5 mutant strains facilitated the localization of pca genes . Five structural genes were found to respond to the tricarboxylic acid and to be contiguous in an operon transcribed in the order pcaDCHGB . These genes encode enzymes beta-ketoadipate enol-lactone hydrolase, gamma-carboxymuconolactone decarboxylase, protocatechuate 3,4-dioxygenase (pcaHG), and beta-carboxy-cis,cis-muconate lactonizing enzyme, respectively . Approximately 4 kb from the pcaD gene are the pcaIJ genes, which encode beta-ketoadipate succinyl-coenzyme A transferase for the next-to-last step of the pathway . The pcaIJ genes are transcribed divergently from the pcaDCHGB operon and are expressed in response to beta-ketoadipate . The pattern of induction of pca genes by beta-carboxy-cis,cis-muconate and beta-ketoadipate in A . tumefaciens is similar to that observed in Rhizobium leguminosarum bv . trifolii and is distinct from induction patterns for the genes from other microbial groups.

J Bacteriol, 1995 Jul, 177(13), 3752 - 7
Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors; Belanger C et al.; Little is known about the effect of the host on the genetic stability of bacterial plant pathogens . Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect . Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A . tumefaciens . Here we report that five different A . tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants . Two of these five strains were also found to produce mutants on pear and/or blackberry plants . Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG . Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells . This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant.

Mol Microbiol, 1995 Jul, 17(2), 259 - 69
An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants; Pan SQ et al.; Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS) . virJ lies between virA and virB, and is transcribed in the same direction . The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence . virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis . Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains . While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS . VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction . The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A . tumefaciens to plant cells . acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear . We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.

Mol Gen Genet, 1995 Jun 10, 247(5), 555 - 64
A two-element Enhancer-Inhibitor transposon system in Arabidopsis thaliana; Aarts MG et al.; The Enhancer-Inhibitor (En-I), also known as Suppressor-mutator (Spm-dSpm), transposable element system of maize was modified and introduced into Arabidopsis by Agrobacterium tumefaciens transformation . A stable En/Spm transposase source under control of the CaMV 35S promoter mediated frequent transposition of I/dSpm elements . Transposition occurred continuously throughout plant development over at least seven consecutive plant generations after transformation . New insertions were found at both linked and unlinked positions relative to a transposon donor site . The independent transposition frequency was defined as a transposition parameter, which quantified the rate of unique insertion events and ranged from 7.8% to 29.2% in different populations . An increase as well as a decrease in I/dSpm element copy number was seen at the individual plant level, but not at the population level after several plant generations . The continuous, frequent transposition observed for this transposon system makes it an attractive tool for use in gene tagging in Arabidopsis.

Jpn J Genet, 1995 Jun, 70(3), 409 - 22
A conditional negative selection for Arabidopsis expressing a bacterial cytosine deaminase gene; Kobayashi T et al.; The enzyme activity for cytosine deaminase, which converts cytosine to uracil in bacterial, is usually undetected in higher plants and animals . The enzyme also catalyzes conversion of non-toxic 5-fluorocytosine (5-FC) to 5- fluorouracil (5-FU), a toxic compound for plant growth . The gene encoding cytosine deaminase (codA) from Escherichia coli was fused to cauliflower mosaic virus (CaMV) 35S promoter (P35S), and cloned into a binary vector pLABR101 . The resulting plasmid pLABR102 contained two marker genes for plants: a positive marker gene, bialaphos resistance (bar) gene driven by the promoter from nopaline synthase gene (Pnos) and a negative one, P35S-codA . The binary vector pLABR102 was transformed into Arabidopsis thaliana via Agrobacterium-mediated transformation . In transgenic progenies (T3) of the second (T2) generation heterozygous for a single T-DNA insertion, a 3:1 segregation ratio was observed on both bialaphos (resistance to sensitive) and 5-FC (sensitive to unaffected) . From T2 plants homozygous for the T-DNA insert, on the other hand, no segregation was detected: all the T3 seedlings were resistant to bialaphos and sensitive to 5-FC . PCR and Northern analyses showed that the 5-FC sensitivity in transgenic descendants was caused by the integration and expression of the chimeric codA gene in the Arabidopsis genome . The results indicated that cytosine deaminase from E . coli is functional and useful for negative selection in Arabidopsis, and that sensitivity to 5-FC as well as the positive bialaphos resistance are dominant traits in Arabidopsis.

J Biol Chem, 1995 May 26, 270(21), 12339 - 42
Divergent transcription and a remote operator play a role in control of expression of a nopaline catabolism promoter in Agrobacterium tumefaciens; Marincs F et al.; The nocP-nocR divergent gene arrangement of the nopaline catabolism (noc) operon of the Agrobacterium tumefaciens Ti plasmid pTiT37 was examined with respect to the expression of the nocP promoter . Under repressive conditions, i.e . in the absence of nopaline, four distinct levels of PnocP expression were observed . The lowest level of expression, i.e . full repression, was detected in the presence of the NocR repressor, together with the remote noc operator and productive transcription from the divergent nocR promoter . The next level was observed in the absence of either the NocR protein or of the operator or of both . The third level was detected when abortive transcription from the nocR promoter occurred, irrespective of the presence or absence of the NocR protein . The highest level of PnocP expression was observed in the absence of both productive transcription from PnocR and the operator sequence, whether or not the NocR protein was present . Under inductive conditions, i.e . in the presence of nopaline, expression of PnocP was activated if both the NocR protein and the operator were present . Absence of either NocR or the operator resulted in lack of inducibility of the nocP promoter . Transcription from the divergent nocR promoter had no influence on the activation of PnocP . It was also found that the absence of the operator affected plasmid supercoiling in vivo . The results suggest that DNA topology has a role in the regulation of the nocP promoter.

Gene, 1995 May 19, 157(1-2), 283 - 7
Effect of the M-EcoRII methyltransferase-encoding gene on the phenotype of Nicotiana tabacum transgenic cells; Buryanov YI et al.; The EcoRII DNA methyltransferase (M-EcoRII; MTase) modifies a cytosine in the DNA sequence CCWGG which contains a CNG methylation motif characteristic of plant DNA . The gene (ecoRIIM) encoding this MTase has been cloned into the T-DNA of the wild-type Agrobacterium Ti-plasmid pTiC58 downstream from the plant expression nopaline synthase-encoding gene promoter . Nicotiana tabacum cells have been transformed with Agrobacterium tumefaciens harbouring this recombinant Ti-plasmid . The primary transformed tabacco tissue line has given rise to novel stable lines which are morphologically distinctive . Southern hybridization analysis of all transformed tissue lines has shown the presence, in each of them, of ecoRIIM . The tissue studied differed in morphology in callus culture, dependence on phytohormones and the ability to synthesize nopaline.

Arch Microbiol, 1995 May, 163(5), 345 - 51
Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213; Russa R et al.; Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium deoxycholate-PAGE pattern and composition . LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region . LPS-P contained large amounts of 6-deoxy-L-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio approximately 30:1), both of which were completely absent in LPS-W . Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-Me-6dTal) probably localized at the non-reducing end of the O-chain . This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by 13C NMR analysis . The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid . Lipid A was of the lipid ADAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-D-glucose-containing lipid A backbone . Lipid ADAG is widespread among species of the alpha-2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.

Plant Physiol, 1995 May, 108(1), 203 - 10
The choice of reducing substrate is altered by replacement of an alanine by a proline in the FAD domain of a bispecific NAD(P)H-nitrate reductase from birch; Schondorf T et al.; Differences in the amino acid sequence between the bispecific NAD(P)H-nitrate reductase of birch (Betula pendula Roth) and the monospecific NADH-nitrate reductases of a variety of other higher plants have been found at the dinucleotide-binding site in the FAD domain . To pinpoint amino acid residues that determine the choice of reducing substrate, we introduced mutations into the cDNA coding for birch nitrate reductase . These mutations were aimed at replacing certain amino acids of the NAD(P)H-binding site by conserved amino acids located at identical positions in NADH-monospecific enzymes . The mutated cDNAs were integrated into the genome of tobacco by Agrobacterium-mediated transformation . Transgenic tobacco (Nicotiana tabacum) plants were grown on a medium containing ammonium as the sole nitrogen source to keep endogenous tobacco nitrate reductase activity low . Whereas some of the mutated enzymes showed a slight preference for NADPH, as does the nonmutated birch enzyme, the activity of some others greatly depended on the availability of NADH and was low with NADPH alone . Comparison of the mutations reveals that replacement of a single amino acid in the birch sequence (alanine871 by proline) is critical for the use of reducing substrate.

Plant J, 1995 May, 7(5), 761 - 70
Functional expression of Saccharomyces cerevisiae CYP51A1 encoding lanosterol-14-demethylase in tobacco results in bypass of endogenous sterol biosynthetic pathway and resistance to an obtusifoliol-14-demethylase herbicide inhibitor; Grausem B et al.; Nicotiana tabacum protoplasts have been transformed by Agrobacterium tumefaciens containing a T-DNA in which the gene CYP51A1 encoding lanosterol-14-demethylase (LAN14DM) from Saccharomyces cerevisiae is under the control of a cauliflower mosaic virus (CaMV) 35S promoter . Two transformants strongly expressed the LAN14DM as shown by Northern and Western experiments . These transgenic calli were killed by LAB 170250F (LAB) (a phytotoxic fungicide inhibiting both plant obtusifoliol-14-demethylase (OBT14DM) and LAN14DM) but were resistant to gamma-ketotriazole (gamma-kt), a herbicide which has been shown to inhibit OBT14DM but not LAN14DM at a concentration that was lethal to control calli . However, these transgenic calli were killed by mixtures of gamma-kt plus fungicide inhibitors of LAN14DM such as ketoconazole, itraconazole or flusilazole which alone were not effective . Further analysis of the transgenic calli grown in the presence of gamma-kt showed that their delta 5-sterol content was close to that of untreated control calli obtained from protoplasts transformed with control plasmid; this is in agreement with evidence that the LAN14DM expressed from the transgene could bypass the blocked OBT14DM by using the plant substate obtusifoliol . In contrast, control calli when treated with gamma-kt, displayed a sterol content strongly enriched in 14 alpha-methyl sterols and depressed in physiological delta 5-sterols . When the transgenic calli were cultured in mixtures of gamma-kt and LAN14DM inhibitors sterol compositions enriched in 14 alpha-methyl sterols were obtained, reflecting a strong inhibition of both 'endogenous' OBT14DM and 'exogenous' LAN14DM . Taken together these results show that in tobacco calli transformed with CYP51A1, resistance to a triazole herbicide arises from expression of a functional LAN14DM enzyme; its activity in transgenic tissues creates a bypass of the sterol biosynthetic pathway at the 14-demethylase level when this latter is blocked by an OBT14DM herbicide inhibitor.

FEMS Microbiol Lett, 1995 May 1, 128(2), 151 - 5
Marine algae that display anti-tumorigenic activity against Agrobacterium tumefaciens; el-Masry MH et al.; Thirty-five extracts representing different seasonal growths of 17 marine algal species collected from the Alexandria coast were tested for anti-tumorigenic activity against Agrobacterium tumefaciens galls on potato discs . Eleven extracts (nine species) displayed > 20% inhibition of tumor initiation, with three of these (Codium tomentosum, winter; Jania rubens, summer; Padina pavonia, winter) displaying relatively high activity . Bacterial viability tests showed that the inhibitory effects were directly due to anti-tumorigenesis rather than an indirect result of anti-bacterial activity.

J Bacteriol, 1995 May, 177(9), 2554 - 9
Sequence and distribution of IS1312: evidence for horizontal DNA transfer from Rhizobium meliloti to Agrobacterium tumefaciens; Deng W et al.; Two novel insertion sequences, IS1312 and IS1313, were found in pTiBo542, the Ti plasmid of Agrobacterium tumefaciens strains Bo542 and A281 . Nucleotide sequencing and Southern hybridization revealed that IS1312 and IS1313 are homologous to Rhizobium meliloti ISRm1 and ISRm2, respectively . IS1312, ISRm1, and another Agrobacterium insertion sequence, IS426, belong to the same IS3 family of insertion sequences; however, IS1312 is more closely related to the Rhizobium ISRm1 than it is to the Agrobacterium IS426 . The distribution patterns of these insertion elements and their sequence similarities suggest that IS1312 and IS1313 were horizontally transferred from R . meliloti to A . tumefaciens.

Mol Microbiol, 1995 May, 16(4), 615 - 24
The bacterial 'enigma': cracking the code of cell-cell communication; Salmond GP et al.; In recent years it has become clear that the production of N-acyl homoserine lactones (N-AHLs) is widespread in Gram-negative bacteria . These molecules act as diffusible chemical communication signals (bacterial pheromones) which regulate diverse physiological processes including bioluminescence, antibiotic production, plasmid conjugal transfer and synthesis of exoenzyme virulence factors in plant and animal pathogens . The paradigm for N-AHL production is in the bioluminescence (lux) phenotype of Photobacterium fischeri (formerly classified as Vibrio fischeri) where the signalling molecule N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) is synthesized by the action of the LuxI protein . OHHL is thought to bind to the LuxR protein, allowing it to act as a positive transcriptional activator in an autoinduction process that physiologically couples cell density (and growth phase) to the expression of the bioluminescence genes . Based on the growing information on LuxI and LuxR homologues in other N-AHL-producing bacterial species such as Erwinia carotovora, Pseudomonas aeruginosa, Yersinia enterocolitica, Agrobacterium tumefaciens and Rhizobium leguminosarum, it seems that analogues of the P . fischeri lux autoinducer sensing system are widely distributed in bacteria . The general physiological function of these simple chemical signalling systems appears to be the modulation of discrete and diverse metabolic processes in concert with cell density . In an evolutionary sense, the elaboration and action of these bacterial pheromones can be viewed as an example of multicellularity in prokaryotic populations.

Biochem Biophys Res Commun, 1995 Apr 26, 209(3), 867 - 76
Canthaxanthin biosynthesis by the conversion of methylene to keto groups in a hydrocarbon beta-carotene by a single gene; Misawa N et al.; Compounds that include (a) keto group(s) in a molecule are ubiquitous natural components . A novel gene involved in ketocompound biosynthesis, designated crtW, was isolated from the marine bacteria Agrobacterium aurantiacum and Alcaligenes PC-1 that produce ketocarotenoids such as astaxanthin . When this gene was introduced into Escherichia coli that accumulated beta-carotene due to the Erwinia carotenogenic genes, the E . coli transformants synthesized canthaxanthin, one of ketocarotenoids, which was identified after purification by its visible, FD-MS and 1H-NMR spectral analysis . It has been demonstrated for the first time that one gene encodes an enzyme "ketolase" that catalyzes the conversion of methylene groups of a hydrocarbon beta-carotene to keto groups for synthesizing canthaxanthin via echinenone.

Mol Gen Genet, 1995 Apr 20, 247(2), 206 - 15
A homolog of the Rhizobium meliloti nitrogen fixation gene fixN is involved in the production of a microaerobically induced oxidase activity in the phytopathogenic bacterium Agrobacterium tumefaciens; Schluter A et al.; Hybridization analysis using the Rhizobium meliloti nitrogen fixation gene fixN as a probe revealed the presence of a homologous DNA region in the phytopathogenic bacterium Agrobacterium tumefaciens . Hybridization signals were also detected with total DNAs of Rhizobium leguminosarum bv . phaseoli, Rhodobacter capsulatus and Escherichia coli, but not those of Xanthomonas campestris pv . campestris and Pseudomonas putida . The hybridizing fragment from A . tumefaciens was cloned and sequenced . The predicted gene product of one of the two open reading frames identified on the sequenced fragment shows homology to FixN of different Rhizobiaceae as well as a low but significant similarity to subunit I of heme copper oxidases from various bacteria . The presence of five strictly conserved histidine residues previously implicated in forming ligands to heme and CuB in oxidases and the predicted membrane topology provide evidence that the A . tumefaciens fixN-like gene product is a component of the heme copper oxidase superfamily . The incomplete open reading frame starting only 8 nucleotides downstream of the fixN-like gene exhibits homology to Rhizobium fixO . Using an uidA (GUS) gene fusion it could be shown that the A . tumefaciens fixN-like gene is preferentially expressed under microaerobic conditions . Expression of the uidA fusion is abolished in R . meliloti fixJ and fixK mutants, indicating that an Fnr-like protein is involved in transcriptional regulation of the fixN-like gene in A . tumefaciens . The presence of an upstream DNA sequence motif identical to the Fnr-consensus binding site (anaerobox) further supports this hypothesis . A . tumefaciens mutated in the fixN-like gene shows decreased TMPD-specific oxidase activity under microaerobic conditions, indicating that the fixN-like gene or operon codes for proteins involved in respiration under reduced oxygen availability.

Anal Biochem, 1995 Apr 10, 226(2), 235 - 40
A general method for detecting and sizing large plasmids; Barton BM et al.; We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE) . Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol . Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels . S1 nuclease converted supercoiled plasmids into full-length linear molecules . Large plasmids migrated as discrete bands that were readily observed after ethidium staining . Their sizes were reliably estimated by comparison with linear DNA markers . Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic . S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus) . The procedure gave size values consistent with previous estimates for characterized megaplasmids . Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus . S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites . It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.

Plant Mol Biol, 1995 Apr, 28(1), 123 - 36
Expression of Agrobacterium rhizogenes auxin biosynthesis genes in transgenic tobacco plants; Gaudin V et al.; Plant oncogenes aux1 and aux2 carried by the TR-DNA of Agrobacterium rhizogenes strain A4 encode two enzymes involved in the auxin biosynthesis pathway in transformed plant cells . The short divergent promoter region between the two aux-coding sequences contains the main regulatory elements . This region was fused to the uidA reporter gene and introduced into Nicotiana tabacum in order to investigate the regulation and the tissue specificity of these genes . Neither wound nor hormone induction could be detected on transgenic leaf discs . However, phytohormone concentration and auxin/cytokinin balance controlled the expression of the chimaeric genes in transgenic protoplasts . The expression was localised in apical meristems, root tip meristems, lateral root primordia, in cells derived from transgenic protoplasts and in transgenic calli . Histological analysis showed that the expression was located in cells reactivated by in vitro culture . Experiments using cell-cycle inhibitors such as hydroxyurea or aphidicolin on transgenic protoplast cultures highly decreased the beta-glucuronidase activity of the chimaeric genes . These results as well as the histological approach suggest a correlation between expression of the aux1 and aux2 genes and cell division.

Microbiology, 1995 Apr, 141 ( Pt 4), 853 - 61
IS292: a novel insertion element from Agrobacterium; Ponsonnet C et al.; A new insertion sequence, IS292, located in the 6b gene of a nopaline-type Agrobacterium strain (X88-292) isolated from poplar was identified and sequenced . IS292 is 2494 bp long, has 21 bp inverted terminal repeats with two mismatches, and generates 10 bp direct repeats upon integration . No sequence similarity was found between IS292 and other insertion elements associated with Agrobacterium, but it shows strong similarity with ISR/1 from Rhizobium leguminosarum bv . viciae . The occurrence of IS292-like sequences in various Agrobacterium isolates, especially different Agrobacterium strains isolated from the same biotope, was demonstrated by DNA hybridization.

Plant Physiol, 1995 Apr, 107(4), 1041 - 7
Transfer of T-DNA from Agrobacterium to the plant cell; Zupan JR et al.; Agrobacterium tumefaciens is the causative agent of crown gall, a disease of dicotyledonous plants characterized by a tumorous phenotype . Earlier in this century, scientific interest in A . tumefaciens was based on the possibility that the study of plant tumors might reveal mechanisms that were also operating in animal neoplasia . In the recent past, the tumorous growth was shown to result from the expression of genes coded for by a DNA segment of bacterial origin that was transferred and became stably integrated into the plant genome . This initial molecular characterization of the infection process suggested that Agrobacterium might be used to deliver genetic material into plants . The potential to genetically engineer plants generated renewed interest in the study of A . tumefaciens . In this review, we concentrate on the most recent advances in the study of Agrobacterium-mediated gene transfer, its relationship to conjugation, DNA processing and transport, and nuclear targeting . In the following discussion, references for earlier work can be found in more comprehensive reviews (Hooykaas and Schilperoort, 1992; Zambryski, 1992; Hooykaas and Beijersbergen, 1994).

Int J Syst Bacteriol, 1995 Apr, 45(2), 334 - 41
Taxonomic study of bacteria isolated from plants: proposal of Sphingomonas rosa sp . nov., Sphingomonas pruni sp . nov., Sphingomonas asaccharolytica sp . nov., and Sphingomonas mali sp . nov; Takeuchi M et al.; The taxonomic positions of 10 strains of 3-ketolactose-forming bacteria which were isolated from the roots of plants (Rosa sp., Psychotria nairobiensis, Ardisia crispa, Prunus persica, and apple trees) were investigated . The DNA base compositions of these strains ranged from 64.0 to 65.7 mol%, the isoprenoid quinone of each strain was ubiquinone 10, 3-hydroxy fatty acids were lacking in the cellular fatty acids of these organisms, and all of the strains contained a sphingolipid with the long-chain base dihydrosphingosin . These are characteristics of the genus Sphingomonas . On the basis of morphological, physiological, and chemotaxonomic characteristics, together with DNA-DNA hybridization and 16S ribosomal DNA sequence comparison data, we propose the following four new species of the genus Sphingomonas: Sphingomonas rosa (type strain, IFO 15208) for the strains isolated from rose plants and formerly named {Agrobacterium rhizogenes}; Sphingomonas pruni (type strain, IFO 15498) for the strains isolated from Prunus persica; and Sphingomonas asaccharolytica (type strain, IFO 15499) and Sphingomonas mali (type strain, IFO 15500) for the strains isolated from apple trees . Two strains which were isolated from Psychotria nairobiensis and formerly named {Chromobacterium lividum} were identified as Sphingomonas yanoikuyae strains.

J Bacteriol, 1995 Mar, 177(5), 1367 - 73
Activity of the Agrobacterium Ti plasmid conjugal transfer regulator TraR is inhibited by the product of the traM gene; Fuqua C et al.; The Agrobacterium Ti plasmid tra regulon was previously found to be positively regulated by the TraR protein in the presence of a diffusible N-acyl homoserine lactone designated Agrobacterium autoinducer (AAI) . TraR and AAI are similar to LuxR from Vibrio fischeri and the Vibrio autoinducer (VAI), which regulate target bioluminescence (lux) genes in a cell density-dependent manner . We now show that tra genes are also regulated by a second protein, designated TraM, which acts to antagonize TraR-dependent activation . The traM gene is closely linked to traR, and the two genes are transcribed convergently . The predicted TraM proteins of two different Ti plasmids are 77% identical but are not significantly similar to other protein sequences in the database, and thus TraM may represent a novel regulatory protein . Null mutations in traM cause strongly increased conjugation, tra gene transcription, and AAI production . A functional copy of traM introduced into traM mutants decreased conjugation, tra gene transcription, and AAI synthesis . TraM inhibits transcription of traA, traI, and traM . Although traM was first identified by its octopine-inducible promoter, we now show that induction by octopine requires traR, strongly suggesting that TraR is the direct traM activator.

Plant Mol Biol, 1995 Mar, 27(6), 1163 - 72
Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells; Matsumoto S et al.; Erythropoietin (Epo), a glycoprotein that regulates the formation of erythrocytes in mammals, was produced in cultured tobacco BY2 cells (Nicotiana tabacum L . cv . Bright Yellow 2) by introducing human Epo cDNA via Agrobacterium tumefaciens-mediated gene transfer . Epo was correctly processed and subsequently penetrated the plasma membrane of tobacco cells . However, it remained attached to the cell wall and was not released into the culture medium . Although Epo produced by tobacco cells was glycosylated with N-linked oligosaccharides, these carbohydrates were smaller than those of the recombinant Epo produced in mammalian cells . Epo produced in tobacco exhibited in vitro biological activities by inducing the differentiation and proliferation of erythroid cells . However, it had no in vivo biological activities . A lectin-binding assay indicated the lack of sialic acid residues in the N-linked oligosaccharides of Epo, suggesting that Epo was removed from the circulation before it reached erythropoietic tissues.

Plant Mol Biol, 1995 Mar, 27(6), 1071 - 83
Transgene-mediated auxin overproduction in Arabidopsis: hypocotyl elongation phenotype and interactions with the hy6-1 hypocotyl elongation and axr1 auxin-resistant mutants; Romano CP et al.; Transgenic Arabidopsis thaliana plants constitutively expressing Agrobacterium tumefaciens tryptophan monooxygenase (iaaM) were obtained and characterized . Arabidopsis plants expressing iaaM have up to 4-fold higher levels of free indole-3-acetic acid (IAA) and display increased hypocotyl elongation in the light . This result clearly demonstrates that excess endogenous auxin can promote cell elongation in a whole plant . Interactions of the auxin-overproducing transgenic plants with the phytochrome-deficient hy6-1 and auxin-resistant axr1-3 mutations were also studied . The effects of auxin overproduction on hypocotyl elongation were not additive to the effects of phytochrome deficiency in the hy6-1 mutant, indicating that excess auxin does not counteract factors that limit hypocotyl elongation in hy6-1 seedlings . Auxin-overproducing seedlings are also qualitatively indistinguishable from wild-type controls in their response to red, far-red, and blue light treatments, demonstrating that the effect of excess auxin on hypocotyl elongation is independent of red and blue light-mediated effects . All phenotypic effects of iaaM-mediated auxin overproduction (i.e . increased hypocotyl elongation in the light, severe rosette leaf epinasty, and increased apical dominance) are suppressed by the auxin-resistant axr1-3 mutation . The axr1-3 mutation apparently blocks auxin signal transduction since it does not reduce auxin levels when combined with the auxin-overproducing transgene.

Appl Microbiol Biotechnol, 1995 Mar, 42(6), 895 - 900
Use of a polymerase-chain-reaction-amplified DNA probe from Pseudomonas putida to detect D-hydantoinase-producing microorganisms by direct colony hybridization; LaPointe G et al.; Pseudomonas putida strain DSM 84 produces N-carbamyl-D-amino acids from the corresponding D-5-monosubstituted hydantoins . The sequence of the D-hydantoinase gene from this strain (GenBank accession number L24157) was used to develop a DNA probe of 122 base pairs (bp) that could detect D-hydantoinase genes in other bacterial genera by DNA and by colony hybridization . Under conditions tolerating 32% mismatch, the probe was specific for all strains that expressed D-hydantoinase activity . These include Pseudomonadaceae of all rRNA groups, and bacteria belonging to the genera Agrobacterium, Serratia, Corynebacterium, and Arthrobacter . Environmental sampling was simulated by screening a mixture of unknown microorganisms from commercial inocula for the biodegradation of industrial, municipal and domestic wastes . The 122-bp probe was specific for microorganisms that subsequently demonstrated D-hydantoinase activity . Bacterial species from four different genera were detected, which were Pseudomonas, Klebsiella, Enterobacter, and Enterococcus.

Mol Plant Microbe Interact, 1995 Mar-Apr, 8(2), 322 - 6
Isolation and characterization of a locus from Azospirillum brasilense Sp7 that complements the tumorigenic defect of Agrobacterium tumefaciens chvB mutant; Raina S et al.; The chromosomal virulence gene chvB of Agrobacterium tumefaciens is required for pathogenesis . A DNA fragment from the chvB locus can hybridize to DNA from Azospirillum brasilense Sp7 . This DNA fragment could restore the tumorigenic activity of the chvB mutant strain A . tumefaciens A1011 towards leaf disks of Nicotiana tabacum . An NH2-terminal open reading frame, 480 codons long, was most likely responsible for the restoration of the tumorigenic activity . The A . brasilense sequence showed good homology with the NH2-terminal region of the ndvB gene of Rhizobium meliloti.

Mol Plant Microbe Interact, 1995 Mar-Apr, 8(2), 311 - 21
Novel Ti plasmids in Agrobacterium strains isolated from fig tree and chrysanthemum tumors and their opinelike molecules; Vaudequin-Dransart V et al.; Galls naturally induced on Fig and chrysanthemum plants by strains of Agrobacterium contained, in addition to other well-characterized opines such as nopaline, three tumor-specific opinelike molecules . These molecules were identified as deoxy-fructosyl-glutamine (dfg), deoxy-fructosyl-5-oxo-proline (dfop), and chrysopine (Chilton et al., unpublished) . Strains isolated from Fig tree and chrysanthemum tumors harbored different and unrelated Ti plasmids as judged by hybridization with various vir and T-DNA probes . They also exhibited different opine-catabolic properties . The strains isolated from chrysanthemum plants (Chry strains) and Fig trees degraded chrysopine, but only the Chry strains used dfg and dfop . Remarkably, other strains of Agrobacterium catabolized these two molecules: dfg was degraded by most pathogenic and nonpathogenic Agrobacterium strains, and dfop by all Agrobacterium strains degrading the opine agropinic acid . These results have strong ecological and evolutionary inferences which fit previous speculation on the origin of opine-related functions.

Mol Plant Microbe Interact, 1995 Mar-Apr, 8(2), 286 - 91
Immunity to potato mop-top virus in Nicotiana benthamiana plants expressing the coat protein gene is effective against fungal inoculation of the virus; Reavy B et al.; Nicotiana benthamiana stem tissue was transformed with Agrobacterium tumefaciens harboring a binary vector containing the potato mop-top virus (PMTV) coat protein (CP) gene . PMTV CP was expressed in large amounts in some of the primary transformants . The five transgenic lines which produced the most CP were selected for resistance testing . Flowers on transformed plants were allowed to self-fertilize . Transgenic seedlings selected from the T1 seed were mechanically inoculated with two strains of PMTV . Virus multiplication, assayed by infectivity, was detected in only one transgenic plant of 98 inoculated . T1 plants were also highly resistant to graft inoculation; PMTV multiplied in only one plant of 45 inoculated . Transgenic T1 seedlings were challenged in a bait test in which they were grown in soil containing viruliferous spores of the vector fungus Spongospora subterranea . In these tests only two plants out of 99 became infected . Of the five transgenic lines tested, plants of three lines were immune to infection following manual, graft, or fungal inoculation.

Mol Plant Microbe Interact, 1995 Mar-Apr, 8(2), 267 - 77
Molecular analysis of the Rhizobium meliloti mucR gene regulating the biosynthesis of the exopolysaccharides succinoglycan and galactoglucan; Keller M et al.; The Rhizobium meliloti Tn5 mutant Rm3131, producing galactoglucan (EPS II) instead of succinoglycan (EPS I), was complemented by a 3.6-kb EcoRI-fragment of the Rhizobium meliloti genome . Sequencing of this fragment revealed six open reading frames (ORFs) . The ORF found to be affected in the mutant Rm3131 codes for a putative protein of 15.7 kDa and forms a monocistronic transcriptional unit . Further genetic analysis revealed that the gene mutated in Rm3131 is identical to the previously described R . meliloti mucR gene (H . Zhan, S.B . Levery, C . C . Lee, and J.A . Leigh, 1989, Proc . Natl . Acad . Sci . USA 86:3055-3059) . By hybridization it was shown that a mucR homologous gene is present in several rhizobacteria . The deduced amino acid sequence of MucR showed nearly 80% identity to the Agrobacterium tumefaciens Ros protein, a negative regulator of vir genes and necessary for succinoglycan production . MucR contains like Ros a putative zinc finger sequence of the C2H2 type . Transcriptional fusions of genes for EPS I and EPS II synthesis, the so-called exo and exp genes, with the marker gene lacZ were used to delineate the role of mucR for exo and exp gene expression . It was found that exp genes are negatively regulated by MucR on the transcriptional level, whereas a posttranscriptional regulation by MucR is assumed for exo genes . Furthermore, mucR is negatively regulating its own transcription.

Can J Microbiol, 1995 Mar, 41(3), 284 - 93
A full factorial analysis of nine factors influencing in vitro antagonistic screens for potential biocontrol agents; Dickie GA et al.; The effect of nine factors on the outcome of classic in vitro screens testing the antagonistic action of endophytic bacterial isolates from grape vines against virulent Agrobacterium vitis has been examined . These factors were (i) the strain of A . vitis, (ii) the strain of endophyte, (iii) the growth medium of the pathogen, (iv) the growth medium of the endophyte, (v) the temperature of growth of the pathogen, (vi) the temperature of growth of the endophyte, (vii) the pH of growth of the pathogen, (viii) the pH of growth of the endophyte, and (ix) the medium of the assay plate . Analyses of variance of the full factorial design incorporating main effects and two- and three-way interactions accounted for 66% of the variance . All nine factors had a significant effect on the diameter of inhibition zones (p < 0.001) . An examination of the three-way interactions revealed that generalizations were difficult to draw; each target agrobacterium had a specific response to a given antagonistic isolate . It was possible to determine that the growth history of bacterial strains, before they were administered to an assay plate to test for antagonism (especially the composition of the growth medium and the temperature of growth), had a profound effect on the outcome of the test . Generally the more chemically defined media produced less inhibition whereas the lower growth temperature of 15 degrees C produced more inhibition . These findings could be relevant to in situ inhibitory activity . The method used to conduct the inhibitory screen (order of strain application and the medium of the assay plate) had a profound influence on the results . These influences add to the caution necessary in the use of in vitro antagonistic screens for finding successful biocontrol agents.

Plant Cell, 1995 Mar, 7(3), 259 - 70
Impaired photoassimilate partitioning caused by phloem-specific removal of pyrophosphate can be complemented by a phloem-specific cytosolic yeast-derived invertase in transgenic plants; Lerchl J et al.; Constitutive expression of the Escherichia coli ppa gene encoding inorganic pyrophosphatase resulted in sugar accumulation in source leaves and stunted growth of transgenic tobacco plants . The reason for this phenotype was hypothesized to be reduced sucrose utilization and loading into the phloem . To study the role of PPi in phloem cells, a chimeric gene was constructed using the phloem-specific rolC promoter of Agrobacterium rhizogenes to drive the expression of the ppa gene . Removal of cytosolic PPi in those cells resulted in photoassimilate accumulation in source leaves, chlorophyll loss, and reduced plant growth . From these data, it was postulated that sucrose hydrolysis via sucrose synthase is essential for assimilate partitioning . To bypass the PPi-dependent sucrose synthase step, transgenic plants were produced that express various levels of the yeast suc2 gene, which encodes cytosolic invertase, in their phloem cells . To combine the phloem-specific expression of the ppa gene and the suc2 gene, crosses between invertase- and pyrophosphatase-containing transgenic plants were performed . Analysis of their offspring revealed that invertase can complement the phenotypic effects caused by the removal of PPi in phloem cells.

Enferm Infecc Microbiol Clin, 1995 Mar, 13(3), 157 - 9
{Agrobacterium radiobacter bacteremia . Study of 2 cases and review of the literature}; Fernandez-Guerrero ML et al.; BACKGROUND: Agrobacteria are small gramnegative bacilli which produce tumors in different vegetal species and occasionally are human pathogens . METHODS: A retrospective review was carried out and the contribution of 2 cases of bacteremia by Agrobacterium radiobacter reported . RESULTS: Both patients developed uncomplicated bacteremia easily controlled by antimicrobian treatment without requiring removal of the intravascular device in one . CONCLUSIONS: Agrobacterium is a rare cause of bacteremic infection in immunosuppressed patients with intravenous catheters . Although the isolations are sensitive to different antibiotics some strains present resistance to cefalosporines, aminoglycosides and cloramphenicol . The mortality of these infections is slight.

Transgenic Res, 1995 Mar, 4(2), 132 - 41
Expression of giant silkmoth cecropin B genes in tobacco; Florack D et al.; Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora cecropia . To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion . All constructs were cloned in a plant expression vector and introduced in tobacco via Agrobacterium tumefaciens . A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels . Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide . In none of the transgenic plants could the cecropin B peptide be detected . This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts . This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide . In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation . Nevertheless, transgenic tobacco plants were evaluated for resistance to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P . syringae pv . tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in vitro . No resistance was found . These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.

Mol Gen Genet, 1995 Feb 20, 246(4), 509 - 13
Phenotypic instability of transgenic tobacco plants and their progenies expressing Arabidopsis thaliana small GTP-binding protein genes; Aspuria ET et al.; Chimeric genes consisting of the cauliflower mosaic virus 35S promoter, a cDNA encoding a small GTP-binding protein from Arabidopsis thaliana (ara-2 or ara-4) and the terminator of the nopaline synthase gene were cloned into a binary vector . Tobacco leaf tissues were transformed with this plasmid via Agrobacterium-mediated transformation . Transgenic plants possessing either ara-2 or ara-4 occasionally showed morphological abnormalities in leaves and other organs . However, such alterations were not always associated with co-transferred characters, such as kanamycin tolerance, and they arose in no more than 10% of the transgenic plants . Such phenomena were also observed in the progenies of the primary transgenic plants . Despite such unusual inheritance of the phenotypic abnormalities, GTP-binding activity of the inserted ara gene products was detected in all plants tested.

Proc Natl Acad Sci U S A, 1995 Feb 14, 92(4), 1167 - 71
Targeting the maize T-urf13 product into tobacco mitochondria confers methomyl sensitivity to mitochondrial respiration; Chaumont F et al.; The URF13 protein, which is encoded by the maize mitochondrial T-urf13 gene, is thought to be responsible for pathotoxin and methomyl sensitivity and male sterility . We have investigated whether T-urf13 confers toxin sensitivity and male sterility when expressed in another plant species . The coding sequence of T-urf13 was fused to a mitochondrial targeting presequence, placed under the control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation . Plants expressing high levels of URF13 were methomyl sensitive . Subcellular analysis indicated that URF13 is mainly associated with the mitochondria . Adding methomyl to isolated mitochondria stimulated NADH-linked respiration and uncoupled oxidative phosphorylation, indicating that URF13 was imported into the mitochondria, and conferred toxin sensitivity . Most control plants, which expressed the T-urf13c construct lacking the mitochondrial presequence, were methomyl sensitive and contained URF13 in a membrane fraction . Subcellular fractionation by sucrose gradient centrifugation showed that URF13 sedimented at several positions, suggesting the protein is associated with various organelles, including mitochondria . No methomyl effect was observed in isolated mitochondria, however, indicating that URF13 was not imported and did not confer toxin sensitivity to the mitochondria . Thus, URF13 confers toxin sensitivity to transgenic tobacco with or without import into the mitochondria . There was no correlation between the expression of URF13 and male sterility, suggesting either that URF13 does not cause male sterility in transgenic tobacco or that URF13 is not expressed in sufficient amounts in the appropriate anther cells.

Mol Gen Genet, 1995 Feb 6, 246(3), 309 - 15
Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli; Das A et al.; Two spontaneous deletions of a derivative of the broad-host-range plasmid RK2 were isolated from Agrobacterium tumefaciens . The two deletions have lost 56 and 505 bp, respectively, near the origin of replication (oriV) . Of the eight 17-bp repeats present in the RK2 oriV, the smaller deletion has lost the first two while the larger one has lost the first three . The deletions led to a significant increase (3- to 7-fold) in plasmid copy number in A . tumefaciens, indicating their importance in copy number control . While the smaller deletion could replicate in Escherichia coli, the larger one could not . The role of the oriV sequences in the replication of pRK2 in A . tumefaciens and in E . coli is discussed.

Plant Mol Biol, 1995 Feb, 27(3), 457 - 66
Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity; Macknight RC et al.; The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors . Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure . The transgenic roots were then nodulated . The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots . The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained . The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT . When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level . Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression . Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region . The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2 . This suggests that the lupin factor identified here is a NAT2 homologue . No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.

J Bacteriol, 1995 Feb, 177(4), 892 - 7
The octopine-type Ti plasmid pTiA6 of Agrobacterium tumefaciens contains a gene homologous to the chromosomal virulence gene acvB; Kalogeraki VS et al.; Although the majority of genes required for the transfer of T-DNA from Agrobacterium tumefaciens to plant nuclei are located on the Ti plasmid, some chromosomal genes, including the recently described acvB gene, are also required . We show that AcvB shows 50% identity with the product of an open reading frame, designated virJ, that is found between the virA and virB genes in the octopine-type Ti plasmid pTiA6 . This reading frame is not found in the nopaline-type Ti plasmid pTiC58 . acvB is required for tumorigenesis by a strain carrying a nopaline-type Ti plasmid, and virJ complements this nontumorigenic phenotype, indicating that the products of these genes have similar functions . A virJ-phoA fusion expressed enzymatically active alkaline phosphatase, indicating that VirJ is at least partially exported . virJ is induced in a VirA/VirG-dependent fashion by the vir gene inducer acetosyringone . Primer extension analysis and subcloning of the virJ-phoA fusion indicate that the acetosyringone-inducible promoter lies directly upstream of the virJ structural gene . Although the roles of the two homologous genes in tumorigenesis remain to be elucidated, strains lacking acvB and virJ (i) are proficient for induction of the vir regulon, (ii) are able to transfer their Ti plasmids by conjugation, and (iii) are resistant to plant wound extracts . Finally, mutations in these genes cannot be complemented extracellularly.

J Bacteriol, 1995 Feb, 177(4), 1076 - 81
Mechanism of cellulose synthesis in Agrobacterium tumefaciens; Matthysse AG et al.; Extracts of Agrobacterium tumefaciens incorporated UDP-{14C}glucose into cellulose . When the extracts were fractionated into membrane and soluble components, neither fraction was able to synthesize cellulose . A combination of the membrane and soluble fractions restored the activity found in the original extracts . Extracts of cellulose-minus mutants showed no significant incorporation of UDP-glucose into cellulose . When mixtures of the extracts were made, the mutants were found to fall into two groups: extracts of mutants from the first group could be combined with extracts of the second group to obtain cellulose synthesis . No synthesis was observed when extracts of mutants from the same group were mixed . The groups of mutants corresponded to the two operons identified in sequencing the cel genes (A . G . Matthysse, S . White, and R . Lightfoot . J . Bacteriol . 177:1069-1075, 1995) . Extracts of mutants were fractionated into membrane and soluble components, and the fractions were mixed and assayed for the ability to synthesize cellulose . When the membrane fraction from mutants in the celDE operon was combined with the soluble fraction from mutants in the celABC operon, incorporation of UDP-glucose into cellulose was observed . In order to determine whether lipid-linked intermediates were involved in cellulose synthesis, permeablized cells were examined for the incorporation of UDP-{14C}glucose into material extractable with organic solvents . No radioactivity was found in the chloroform-methanol extract of mutants in the celDE operon, but radioactive material was recovered in the chloroform-methanol extract of mutants in the celABC operon . The saccharide component of these compounds was released after mild acid hydrolysis and was found to be mainly glucose for the celA insertion mutant and a mixture of cellobiose, cellotriose, and cellotetrose for the celB and celC insertion mutants . The radioactive compound extracted with chloroform-methanol form the celC insertion mutant was incorporated into cellulose by membrane preparations from celE mutants, which suggests that this compound is a lipid-linked intermediate in cellulose synthesis.

J Bacteriol, 1995 Feb, 177(4), 1069 - 75
Genes required for cellulose synthesis in Agrobacterium tumefaciens; Matthysse AG et al.; A region of the chromosome of Agrobacterium tumefaciens 11 kb long containing two operons required for cellulose synthesis and a part of a gene homologous to the fixR gene of Bradyrhizobium japonicum has been sequenced . One of the cellulose synthesis operons contained a gene (celA) homologous to the cellulose synthase (bscA) gene of Acetobacter xylinum . The same operon also contained a gene (celC) homologous to endoglucanase genes from A . xylinum, Cellulomonas uda, and Erwinia chrysanthemi . The middle gene of this operon (celB) and both the genes of the other operon required for cellulose synthesis (celDE) showed no significant homology to genes contained in the databases . Transposon insertions showed that at least the last gene of each of these operons (celC and celE) was required for cellulose synthesis in A . tumefaciens.

Biochem J, 1995 Feb 1, 305 ( Pt 3), 715 - 9
Construction and characterization of a chimeric beta-glucosidase; Singh A et al.; The amino acid sequences of beta-glucosidases from Cellvibrio gilvus and Agrobacterium tumefaciens show significant similarity in most of the parts . However, the pH/temperature optima and stabilities of the two enzymes are quite different . C . gilvus beta-glucosidase exhibits an optimum pH of 6.2-6.4 and temperature of 35 degrees C, whereas the corresponding values for A . tumefaciens are 7.2-7.4 and 60 degrees C respectively . To analyse these properties further, a chimeric beta-glucosidase was constructed by replacing a segment from the C-terminal region of C . gilvus beta-glucosidase gene with that of A . tumefaciens . The partially purified chimeric enzyme was characterized with respect to pH/temperature activity and stability and substrate affinity . Our results suggest that C-terminal segment(s) might be important in beta-glucosidase specificity, and shuffling of even a small segment of gene in this region might significantly alter or improve the enzymic properties such as thermal stability.

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