Agrobacterium

Mol Plant Microbe Interact, 1993 May-Jun, 6(3), 393 - 402
Functional and mutated agrocinopine synthase genes on octopine T-DNAs; Paulus F et al.; Agrocinopine synthase genes have so far been detected only on the Agrobacterium tumefaciens nopaline Ti plasmid pTiC58 and on the A . rhizogenes Ri plasmid pRiA4 . The TA region of the octopine/cucumopine (o/c) A . vitis Ti plasmid pTiTm4 strongly resembles the TL DNA of biotype I octopine Ti plasmids . In addition, it carries an intact and functional agrocinopine synthase gene close to its left border . TA/TL sequence comparison shows that the biotype I TL region still carries a 5'-deleted acs gene, strongly indicating that this gene was originally part of the TL region and belongs to the "common DNA" region of octopine and nopaline Ti plasmids . Tm4 and C58C1 (pTiTm4) remove agrocinopine A from the medium indicating that pTiTm4 also carries agrocinopine uptake genes . In spite of this, Tm4 and related strains are resistant against agrocin 84 . Two o/c Ti plasmids that are closely related to pTiTm4, pTiHm1, and pTiAB3, have a mutated acs gene; whereas Hm1 can still take up agrocinopine A, AB3 cannot . These results yield new insights in the evolution of octopine, nopaline, and o/c strains.

Mol Plant Microbe Interact, 1993 May-Jun, 6(3), 358 - 67
Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene; Fang G et al.; Three versions of the zucchini yellow mosaic virus (ZYMV) coat protein gene were engineered for expression in plants: the full-length coat protein sequence, the conserved core portion of the gene, and an antisense version . These constructs were introduced into muskmelon (Cucumis melo) and tobacco plants (Nicotiana tabacum) via Agrobacterium tumefaciens-mediated transformation; gene expression was verified by Northern and Western analysis . Transgenic R0 and R1 muskmelon plants expressing the full-length coat protein gene exhibited apparent immunity to ZYMV infection: There was a lack of symptom development during a 3-mo observation period and no measurable virus accumulation as determined by ELISA . Melon plants expressing the core or antisense constructs showed a several-day delay of systemic symptom development and reduction in virus titer . Furthermore, transgenic R1 tobacco plants expressing the full-length coat protein, core, or antisense constructs of ZYMV, a nonpathogen of tobacco, showed a short delay in symptom development and reduced virus titer when inoculated with the heterologous potyviruses, potato virus Y, and tobacco etch virus . The transgenic tobacco plants were not protected against the non-potyvirus, tobacco mosaic virus.

Mol Plant Microbe Interact, 1993 May-Jun, 6(3), 323 - 30
Coat protein-mediated resistance in transgenic tobacco expressing the tobacco mosaic virus coat protein from tissue-specific promoters; Reimann-Philipp U et al.; Coat protein-mediated resistance (CP-MR) was studied in transgenic Nicotiana tabacum 'Xanthi nn' and 'Xanthi NN' that express chimeric tobacco mosaic virus (TMV) coat protein (CP) gene constructs using two different tissue-specific promoters . The Phaseolus vulgaris pal2 promoter leads to gene expression in the upper leaf epidermis and the xylem, while the rolC promoter from Agrobacterium rhizogenes leads to gene expression in pholem and leaf hair tip cells . Tissue-specific gene expression was verified using the gusA(uidA) reporter gene, while accumulation of TMV CP was verified by Western blot analysis . Transgenic Xanthi nn plants harboring the pal2-CP gene construct were partially resistant to TMV infection . On Xanthi NN plants that expressed the pal2-CP gene construct, fewer necrotic lesions were formed after TMV inoculation compared to nontransformed control plants . The level of resistance, however, was substantially less than in plant lines that expressed TMV CP from the cauliflower mosaic virus 35S promoter . By contrast, expression of the rolC-CP construct did not confer resistance in either Xanthi nn or Xanthi NN . The results provide further evidence that CP-MR to systemic TMV infection in tobacco is probably due to inhibition of infection rather than to effects on long-distance spread through the phloem.

J Bacteriol, 1993 May, 175(10), 3083 - 8
Heat shock transcription of the groESL operon of Agrobacterium tumefaciens may involve a hairpin-loop structure; Segal G et al.; The groESL operon of Agrobacterium tumefaciens was cloned and sequenced and found to be highly homologous to previously analyzed groE operons in nucleotides of the coding region and in amino acid sequence . Transcription of this operon in A . tumefaciens was considerably stimulated by heat shock . Primer extension analysis revealed that the groE transcripts from cells under heat shock were initiated from the same promoter (a sigma-70-like promoter) as transcripts from untreated cells, and no sequence homology with the Escherichia coli heat shock promoters was observed . The DNA sequence downstream of the transcription start site contains an inverted repeat that has a strong similarity to other groESL operons of both gram-positive and gram-negative bacteria (such as cyanobacteria and chlamydiae) . This conserved region is thought to form a hairpin-loop structure and may play a role in gene regulation during heat shock.

Lett Appl Microbiol, 1993 May, 16(5), 265 - 8
Rapid purification of Ti plasmids from Agrobacterium by ethidium bromide treatment and phenol extraction; Zhang L et al.; An efficient method is described for the purification of Ti plasmid DNA from Agrobacterium . The procedure is based on the relative binding capacity of ethidium bromide to supercoiled plasmid DNA and linear DNA and on the high solubility of ethidium bromide in phenol . Following treatment with ethidium bromide, more than 87% of linear chromosomal DNA and most of the RNA was present in the phenol phase, while 91% of Ti plasmid DNA was recovered from the aqueous phase . The Ti plasmid DNA was sufficiently pure for restriction endonuclease analysis and cloning . The procedure is simple, fast and provides eight times higher yield than the standard isopycnic ultracentrifugation method.

Biotechnol Prog, 1993 May-Jun, 9(3), 317 - 22
Growth of plant root cultures in liquid- and gas-dispersed reactor environments; McKelvey SA et al.; The growth of Agrobacterium transformed "hairy root" cultures of Hyoscyamus muticus was examined in various liquid- and gas-dispersed bioreactor configurations . Reactor runs were replicated to provide statistical comparisons of nutrient availability on culture performance . Accumulated tissue mass in submerged air-sparged reactors was 31% of gyratory shake-flask controls . Experiments demonstrate that poor performance of sparged reactors is not due to bubble shear damage, carbon dioxide stripping, settling, or flotation of roots . Impaired oxygen transfer due to channeling and stagnation of the liquid phase are the apparent causes of poor growth . Roots grown on a medium-perfused inclined plane grew at 48% of gyratory controls . This demonstrates the ability of cultures to partially compensate for poor liquid distribution through vascular transport of nutrients . A reactor configuration in which the medium is sprayed over the roots and permitted to drain down through the root tissue was able to provide growth rates which are statistically indistinguishable (95% T-test) from gyratory shake-flask controls . In this type of spray/trickle-bed configuration, it is shown that distribution of the roots becomes a key factor in controlling the rate of growth . Implications of these results regarding design and scale-up of bioreactors to produce fine chemicals from root cultures are discussed.

Proc Natl Acad Sci U S A, 1993 Apr 15, 90(8), 3549 - 53
VirA, the plant-signal receptor, is responsible for the Ti plasmid-specific transfer of DNA to maize by Agrobacterium; Raineri DM et al.; Agrobacteria exhibit marked Ti (tumor-inducing)/Ri (root-inducing) plasmid specificity in their interaction with the Gramineae . In this study, we have used the technique of "agroinfection," in which Agrobacterium-mediated delivery of viral genomes into plants is detected by the development of viral disease symptoms, to identify the region of the Ti plasmid which is responsible for the major differences seen in the ability of nopaline- vs . octopine-type Ti plasmids to transfer maize streak virus (MSV) DNA to maize . Introduction of fragments of the C58 (nopaline-type) Ti plasmid into strains containing an octopine-type Ti plasmid showed that a fragment containing the nopaline-type virA locus was able to complement these normally non-agroinfectious strains to high levels of MSV DNA transfer . Octopine-type virA mutant strains that express vir genes at high levels in the absence of the plant inducing compound acetosyringone also efficiently transferred MSV DNA . These findings imply a functional difference between the virA gene products encoded by octopine- and nopaline-type Ti plasmids which has a profound effect on their ability to mediate DNA transfer to maize.

Plant Mol Biol, 1993 Apr, 22(1), 113 - 27
Modulation of cellular polyamines in tobacco by transfer and expression of mouse ornithine decarboxylase cDNA; DeScenzo RA et al.; In an attempt to modulate the metabolism of polyamines in plants, Agrobacterium tumefaciens strains were produced which contained either a full-length or a 3'-truncated mouse ornithine decarboxylase (ODC) cDNA under the control of the cauliflower mosaic virus 35S promoter . Plants of Nicotiana tabacum cv . Xanthi were used for transformation with these two strains of Agrobacterium . Transformations were confirmed by Southern hybridization and amplification by polymerase chain reaction . Two plants containing the full-length cDNA (ODC-12 and ODC-30) and two containing the truncated cDNA (12701-2 and 12701-31) were selected for further experiments . Northern blot analysis indicated that transcription was occurring and western blot analysis detected a polypeptide of ca . 50 kDa that was unique to the plants transformed with truncated ODC cDNA . In order to distinguish between the native and the mouse ODC in the transformed tissues, enzyme activity was assayed at pH optima for the two enzymes, i.e . pH 8.2 and 6.8, respectively . Substantially higher levels of ODC activity were seen at pH 6.8 (optimum for mouse ODC) in the transformants as compared to the controls . This ODC activity was inhibited by alpha-difluoromethylornithine and anti-mouse ODC antisera in a manner consistent with that reported for the mouse ODC . Analysis of cellular polyamines showed significantly elevated levels (4-12-fold) of putrescine in callus derived from transformed plant tissues as compared to the controls . The modulation of polyamine biosynthesis in plants by these techniques should allow us to further analyze the role of these ubiquitous compounds in plant growth and development.

Int J Syst Bacteriol, 1993 Apr, 43(2), 305 - 13
Phylogenetic analysis of rhizobia and agrobacteria based on 16S rRNA gene sequences; Willems A et al.; The phylogenetic relationships of members of the genera Rhizobium, Agrobacterium, Bradyrhizobium, and Azorhizobium were studied by direct sequencing of their amplified 16S rRNA genes . Comparative analysis of the sequence data confirmed that the genera Bradyrhizobium and Azorhizobium belong to distinct phylogenetic lineages . The genera Rhizobium and Agrobacterium were found to be phylogenetically heterogeneous, and several subgroupings in which Rhizobium and Agrobacterium species were intermixed were evident . The present findings show that the genus and species definitions of these organisms are in need of revision . Different possibilities for this are discussed in the light of sequencing data.

Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 2970 - 4
Molecular characterization of an operon required for pertussis toxin secretion; Weiss AA et al.; Mutants of Bordetella pertussis which are defective in secretion of pertussis toxin were isolated and characterized . The region of the B . pertussis chromosome identified by mutagenesis as playing a role in transport of pertussis toxin was sequenced . Analysis of this region revealed eight open reading frames, seven of which predict a protein exhibiting homology with one of the VirB proteins of Agrobacterium tumefaciens, which are involved in the transport of the T-DNA molecule across bacterial and plant membranes . Thus a set of accessory proteins are most likely involved in the secretion of pertussis toxin, and these proteins appear to be members of a family of proteins involved in the secretion of macromolecules from bacteria.

Nature, 1993 Apr 1, 362(6419), 448 - 50
Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction; Piper KR et al.; Conjugal transfer of Ti plasmids from Agrobacterium donors to bacterial recipients is controlled by two types of diffusible signal molecules . Induction is mediated by novel compounds, called opines, that are secreted by crown gall tumours . These neoplasias result from infection of susceptible plants by virulent agrobacteria . The second diffusible signal, called conjugation factor, is synthesized by the donor bacteria themselves . Production of this factor is induced by the opine . Here we show that conjugation is regulated directly by a transcriptional activator, TraR, which requires conjugation factor as a coinducer to activate tra gene expression . TraR is a homologue of LuxR, the lux gene activator from Vibrio fischeri which also requires an endogenously synthesized diffusible coinducer . The two regulatory systems are related; the two activator proteins show amino-acid sequence similarities and the lux system cofactor, autoinducer, will substitute for conjugation factor in the TraR-dependent activation of Ti plasmid tra genes.

Nature, 1993 Apr 1, 362(6419), 446 - 8
Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones; Zhang L et al.; Conjugal opines secreted by crown gall tumours induce strains of Agrobacterium tumefaciens that are donors of Ti plasmids to produce a diffusible conjugation factor . This enhances the conjugal transfer efficiency of the Ti plasmid in other strains of A . tumefaciens . This factor behaves as a secondary messenger, transmitting the environmental information to tra genes . Here we report the use of spectrometry to show that this factor is identical to synthetic N-(beta-oxo-octan-1-oyl)-L-homoserine lactone and confirm that the synthetic compound is biologically active . N-(Hexan-1-oyl)-L-homoserine lactone has also been detected . A closely related molecule, N-(beta-oxo-hexan-1-oyl)-L-homoserine lactone, autoinduces bioluminescence in the distantly related bacterium, Vibrio fischeri . N-Acyl-homoserine lactones thus seem to be conserved molecules in which the length and nature of the lipophilic acyl chain determines the biological function to be regulated . Mutants that do not produce the factor fail to conjugate unless supplied with it in the induction medium (our unpublished data) . These data indicate that the conjugation factor is an autoinducer and a key signal molecule in the conjugation system of A . tumefaciens . It is, to our knowledge, the first example of a second messenger molecule in a bacterial conjugation system.

Plant Mol Biol, 1993 Apr, 22(1), 129 - 42
A wound-induced promoter driving npt-II expression limited to dedifferentiated cells at wound sites is sufficient to allow selection of transgenic shoots; Firek S et al.; There is much data to indicate that only a small number of cells in plant explants are competent for stable transformation by Agrobacterium . Circumstantial evidence suggests that certain cells reentering cell division at wound sites are competent for transformation by Agrobacterium . We have discovered a member of the intracellular PR gene family from asparagus (AoPR1) which is strongly expressed upon wounding and during the reactivation of the cell cycle in cultured asparagus cells, but which shows very little expression in intact plant tissues . The promoter from the AoPR1 gene was fused to an intron-containing GUS reporter gene and shown to be more strongly expressed than the commonly used CaMV 35S constitutive promoter in target cells for plant transformation . A transcriptional fusion of the AoPR1 promoter with an NPT-II gene was found to be a very efficient marker for the selection of transgenic tobacco callus . Expression of the AoPR1-NPT-II gene allowed efficient shoot formation on transgenic callus and efficient adventitious root formation on transgenic shoots . These latter observations provided firm evidence that transformation selection marker gene expression is most crucial at the early stages of the transformation process, during the establishment of transformed micro-calli.

Mol Gen Genet, 1993 Apr, 238(1-2), 209 - 17
Transposition of the maize autonomous element Activator in transgenic Nicotiana plumbaginifolia plants; Marion-Poll A et al.; The maize autonomous transposable element Ac was introduced into haploid Nicotiana plumbaginifolia via Agrobacterium tumefaciens transformation of leaf disks . All the regenerated transformants (R0) were diploid and either homozygous or heterozygous for the hygromycin resistance gene used to select primary transformants . The Ac excision frequency was determined using the phenotypic assay of restoration of neomycin phosphotransferase activity and expression of kanamycin resistance among progeny seedlings . Some of the R0 plants segregated kanamycin-resistant seedlings in selfed progeny at a high frequency (34 to 100%) and contained one or more transposed Ac elements . In the primary transformants Ac transposition probably occurred during plant regeneration or early development . Other R0 transformants segregated kanamycin-resistant plants at a low frequency (< or = 4%) . Two transformants of this latter class, containing a unique unexcised Ac element, were chosen for further study in the expectation that their kanamycin resistant progeny would result from independent germinal transposition events . Southern blot analysis of 32 kanamycin-resistant plants (R1 or R2), selected after respectively one or two selfings of these primary transformants, showed that 27 had a transposed Ac at a new location and 5 did not have any Ac element . Transposed Ac copy number varied from one to six and almost all transposition events were independent . Southern analysis of the R2 and R3 progeny of these kanamycin-resistant plants showed that Ac continued to transpose during four generations, and its activity increased with its copy number . The frequency of Ac transposition, from different loci, remained low (< or = 7%) from R0 to R3 generations when only one Ac copy was present.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetics, 1993 Apr, 133(4), 1009 - 21
TnpA trans-activates methylated maize Suppressor-mutator transposable elements in transgenic tobacco; Schlappi M et al.; The maize Suppressor-mutator (Spm) transposable element is subject to epigenetic inactivation in transgenic tobacco, as it is in maize . Spm inactivation in tobacco is correlated with increased methylation of sequences near the element's transcription start site . To determine whether element-encoded gene products can promote the reactivation of an inactive element, we investigated the effects of introducing individual CaMV 35S promoter-driven cDNAs for tnpA, tnpB, tnpC and tnpD, the element's four known protein-coding sequences . Introduction of the tnpA cDNA promoted the reactivation of the inactive resident Spm element, as judged by the appearance of regenerants with very early excision events and transposed elements . By contrast, the tnpB, tnpC and tnpD cDNAs had no affect on the activity of the resident Spm element . Similar results were obtained when the element-encoded cDNAs were introduced either by Agrobacterium-mediated retransformation or by a genetic cross . Reactivation of an inactive Spm by the tnpA cDNA is accompanied by reduced methylation of several methylation-sensitive restriction sites near the element's transcription start site . Maintenance of the reactivated Spm element in an active state requires the continued presence of the tnpA cDNA . Elimination of the tnpA cDNA locus by genetic segregation generally results in decreased element activity, as judged by a low frequency of excision events, and is accompanied by increased methylation of the element's 5'-end . Exceptions resembling the phenomenon of "presetting" are also observed in which progeny plants that did not receive the tnpA cDNA locus after meiotic segregation maintain high excision activity and exhibit low methylation levels.

Plant J, 1993 Apr, 3(4), 599 - 606
Expression of antisense nodulin-35 RNA in Vigna aconitifolia transgenic root nodules retards peroxisome development and affects nitrogen availability to the plant; Lee NG et al.; A nodulin-35 (N-35) cDNA encoding nodule-specific uricase (EC 1.7.3.3.) was isolated from a Vigna aconitifolia (mothbean) root nodule cDNA library . Sequence analysis of Vigna uricase (VN-35) cDNA revealed 90% homology to that of soybean . The VN-35 cDNA was inserted in the antisense orientation downstream of the caMV-35S promoter, and transgenic hairy roots were formed on Vigna plants using Agrobacterium rhizogenes . Infection with Bradyrhizobium (cowpea) gave rise to root nodules on transgenic hairy roots supported by the wild-type shoot . Expression of antisense VN-35 RNA was detected in transgenic nodules on individual roots using polymerase chain reaction (PCR) . The nodules expressing antisense VN-35 RNA were smaller in size and showed lower uricase activity than nodules formed on the hairy roots transformed with a binary vector containing beta-glucuronidase (GUS) gene (used as control), and the plants exhibited nitrogen deficiency symptoms . Ultrastructural analysis and immunogold labeling with antibody against soybean N-35 revealed that the growth of peroxisomes was retarded in transgenic nodules expressing antisense VN-35 RNA . These data suggest that a reduction in ureide biosynthesis limits the availability of symbiotically reduced nitrogen to the plant . The nodules of tropical legumes appear to be specialized in nitrogen assimilation and are developmentally controlled to produce and transport ureides.

Plant Mol Biol, 1993 Mar, 21(6), 973 - 84
cis-acting regulatory regions of the soybean seed storage 11S globulin gene and their interactions with seed embryo factors; Itoh Y et al.; A 2.2 kb fragment containing the 5'-flanking region of the soybean glycinin A2B1a gene and its successive deletions with a shorter 5'-flanking sequence were fused, in frame, to the beta-glucuronidase (GUS) reporter gene . The resultant fusions were introduced into tobacco plants via Agrobacterium tumefaciens . Assays of the GUS activity in seeds of transgenic tobacco showed that the upstream region, -657 to -327 (relative to the transcription initiation site {+1}), of the glycinin gene is required for optimal expression of the transformed gene . Interactions between embryo nuclear factors and DNA fragments covering the downstream region of -326, in which are included the TATA box and legumin boxes, were not apparent . The embryo factors capable of binding specifically to three subregions, -653 to -527, -526 to -422, and -427 to -321, of the upstream regulatory region were detected . Such factors appeared to be organ-specific and could be found solely in developing seeds at the early middle stage of embryogenesis (around 24 days after flowering) . Evidence obtained by characterizing the nature of the binding proteins and by gel mobility shift assays established that the same factor does interact with a consensus motif 5'-ATA/TATTTCN-/CTA-3' which occurs four times in the cis-acting regulatory region between -657 and -327 . Moreover, this conserved motif could also be found in the 5' regulatory region of another glycinin A1aB1b gene . Thus it is likely that the observed interaction between the nuclear factor and the conserved motifs would lead to activation of transcription from the glycinin genes in maturing soybean seeds.

Plant Mol Biol, 1993 Mar, 21(6), 1011 - 21
Levels and location of expression of the Agrobacterium tumefaciens pTiA6 ipt gene promoter in transgenic tobacco; Strabala TJ et al.; The location of gene expression of the Agrobacterium tumefaciens ipt gene promoter in transgenic tobacco plants was examined using the beta-glucuronidase (GUS) reporter gene . Expression of GUS was detected in every organ and most cell types examined . The highest levels of GUS activity were found in roots . To further examine the transcriptional basis of this broad expression pattern, deletions in the 5' non-coding region of the gene were translationally fused to two promoterless reporter genes, encoding the enzymes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) . Reporter enzyme assays revealed the existence of an upstream segment required for maximal promoter function, the 5' end of which is between -442 and -408 of the Pipt ATG codon . This upstream segment is required for maximal levels of GUS expression in roots, but not in other organs, and a tobacco suspension-cultured cell line . The implications of broad ipt expression on the process of crown gall tumorigenesis are discussed.

Mol Gen Genet, 1993 Mar, 237(3), 385 - 94
Restoration of fertility by antisense RNA in genetically engineered male sterile tobacco plants; Schmulling T et al.; Transgenic tobacco plants (Nicotiana tabacum L.) expressing the rolC gene of Agrobacterium rhizogenes under the transcriptional control of the 35S RNA promoter are male sterile . When these plants are genetically crossed with others containing the rolC gene linked in antisense orientation to the 35S RNA promoter, hybrid progeny display restoration of male fertility . Moreover, hybrid progeny are revertant for other features of the rolC phenotype, such as restoration of plant height, leaf pigment content and female fertility . The level of restoration of the characteristics of untransformed tobacco appeared to be independent of the steady-state level of antisense RNA . Addition of six transcriptional enhancer sequences upstream of the 35S transcriptional start region in the antisense construct led to a higher steady-state level of antisense RNA than that produced using a promoter linked to a single enhancer sequence . However no significant difference was observed in the level of attenuation of the rolC phenotype in the progeny of crosses with either one or six transcriptional enhancers linked to the antisense rolC gene . Antisense constructs comprising only 189 bp of the rolC 5' coding region appeared less efficient in attenuating the rolC phenotype than those including the whole rolC coding region as well as its 3' untranslated region . Furthermore, results from experiments on light-controlled rolC gene expression indicate that microsporogenesis is sensitive to rolC gene action during the early stages of flower development.

Plasmid, 1993 Mar, 29(2), 154 - 9
The Ti plasmid from the wide host range Agrobacterium vitis strain Tm4: map and homology with other Ti plasmids; Otten L et al.; The octopine/cucumopine (o/c) strains of Agrobacterium vitis are divided into limited (LHR) and wide host range (WHR) strains . We present here the first map of a Ti plasmid from a WHR o/c strain, Tm4 . pTiTm4 (252 kb) has homology with the octopine Ti plasmid pTiAch5, the nopaline plasmid pTiC58, and the Ri plasmid pRiHRI . Approximately 155 kb of pTiTm4 is homologous with the LHR o/c Ti plasmids pTiAB3 and pTiAg57, the remaining part is pTiTm4-specific . Our data suggest that the evolution of the o/c Ti plasmids was accompanied by large-scale changes in plasmid structure . The origin of replication of pTiTm4 was localized by a functional assay.

Mol Microbiol, 1993 Mar, 7(5), 719 - 24
The virA promoter is a host-range determinant in Agrobacterium tumefaciens; Turk SC et al.; The limited host range (LHR) Agrobacterium tumefaciens strain Ag162 is an isolate with a narrow host range . Introduction of the wide host range (WHR) virA gene is essential for extending the host range to Kalanchoe daigremontiana . In this report we show that the region upstream of the ATG start codon is responsible for the LHR phenomenon and that this is probably due to the non-inducibility of the LHRvirA promoter . By comparing the characteristics of the LHR and WHR VirA receptor proteins, it was found that the LHR VirA protein is able to activate the WHR VirG protein in the presence of acetosyringone and that this acetosyringone-dependent vir-induction is enhanced by the presence of D-glucose, as in the case of WHR VirA proteins . These results indicate that the domains, acting as receptors for sugars and phenolic signals, must be conserved between the LHR and WHR VirA receptor proteins.

Mol Microbiol, 1993 Mar, 7(5), 637 - 45
Strategies in pathogenesis: mechanistic specificity in the detection of generic signals; Duban ME et al.; The virulence genes of the plant pathogen Agrobacterium tumefaciens are induced by more than 40 low-molecular-weight phenolic compounds . The prevailing opinion is that (i) wound-derived phenols produced on breach of the integrity of the cell wall act as the initiating signal in a series of events which results in host cell transformation, and (ii) a classical membrane receptor, putatively VirA, is responsible for the recognition of all such phenolic inducers . Here, we argue that the discovery of the subset of inducers that are relatives of the dehydrodiconiferyl alcohol glucoside (DCG) growth factors redirects our attention to work on the plant wound as a site of cell division, and suggests that we further explore the implications of early work on the relationship between transformation efficiency and the status of the cell cycle of the host . In addition, we argue that the significant structural diversity allowed in the para position of the phenol ring of inducers suggests that a receptor-ligand interaction based solely on structural recognition is insufficient, but that recognition followed by a specific proton transfer event may be sufficient to explain vir induction activity . Hence, the specificity of the response of A . tumefaciens may be a consequence of the features required for a chemical reaction to occur on the receptor surface . Finally, we review affinity labelling studies which exploit this phenol detection mechanism and which provide evidence that the phenol receptor may be other than VirA, the sensory kinase of the two component regulatory system implicated in Agrobacterium virulence.(ABSTRACT TRUNCATED AT 250 WORDS)

Clin Infect Dis, 1993 Mar, 16(3), 388 - 91
Agrobacterium radiobacter: a recently recognized opportunistic pathogen; Edmond MB et al.; Over the past decade, an increasing number of infections due to Agrobacterium radiobacter have been reported . Observation of three cases of bacteremia due to this organism prompted a review of the English-language literature . Nineteen cases of significant disease have previously been reported . In more than one-half of the cases, bacteremia was the primary manifestation, often associated with the presence of an intravascular catheter . Other clinical syndromes (peritonitis, urinary tract infection, and endocarditis) have been described . Infection is strongly related to the presence of plastic foreign material, and effective treatment often requires removal of the device . Because antimicrobial sensitivity is variable, treatment must be based on sensitivity data for the individual isolate.

J Bacteriol, 1993 Mar, 175(6), 1723 - 34
The Agrobacterium tumefaciens virB4 gene product is an essential virulence protein requiring an intact nucleoside triphosphate-binding domain; Berger BR et al.; Products of the approximately 9.5-kb virB operon are proposed to direct the export of T-DNA/protein complexes across the Agrobacterium tumefaciens envelope en route to plant cells . The presence of conserved nucleoside triphosphate (NTP)-binding domains in VirB4 and VirB11 suggests that one or both proteins couple energy, via NTP hydrolysis, to T-complex transport . To assess the importance of VirB4 for virulence, a nonpolar virB4 null mutation was introduced into the pTiA6NC plasmid of strain A348 . The 2.37-kb virB4 coding sequence was deleted precisely by oligonucleotide-directed mutagenesis in vitro . The resulting delta virB4 mutation was exchanged for the wild-type allele by two sequential recombination events with the counterselectable Bacillus subtilis sacB gene . Two derivatives, A348 delta B4.4 and A348 delta B4.5, sustained a nonpolar deletion of the wild-type virB4 allele, as judged by Southern blot hybridization and immunoblot analyses with antibodies specific for VirB4, VirB5, VirB10, and VirB11 . Transcription of wild-type virB4 from the lac promoter restored virulence to the nonpolar null mutants on a variety of dicotyledonous species, establishing virB4 as an essential virulence gene . A substitution of glutamine for Lys-439 and a deletion of Gly-438, Lys-439, and Thr-440 within the glycine-rich NTP-binding domain (Gly-Pro-Iso-Gly-Arg-Gly-Lys-Thr) abolished complementation of A348 delta B4.4 or A348 delta B4.5, demonstrating that an intact NTP-binding domain is critical for VirB4 function . Merodiploids expressing both the mutant and wild-type virB4 alleles exhibited lower virulence than A348, suggesting that VirB4, a cytoplasmic membrane protein, may contribute as a homo- or heteromultimer to A . tumefaciens virulence.

Carbohydr Res, 1993 Feb 24, 240, 153 - 9
Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source; Kai A et al.; 13C-Labeled curdlans were biosynthesized by Agrobacterium sp . (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy . The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose . In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled . From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis . The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

J Mol Biol, 1993 Feb 20, 229(4), 1165 - 74
A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif; Koonin EV; A superfamily of ATPases is described with its members sharing three distinct conserved amino acid sequence motifs . The superfamily includes numerous proteins involved in active partitioning of bacterial plasmids and chromosomes, nitrogenase iron proteins (nifH gene products), the anion pump ATPase ArsA, and VirC1 proteins encoded by Agrobacterium Ti plasmids and apparently involved in formation of single-stranded plasmid DNA . A database search identified partial sequences of genes encoding putative human and archaebacterial chromosome partitioning ATPases, suggesting that these proteins fulfil a universal function in cell division . The proteins belonging to this superfamily show the transition from the classical fingerprint of the A type purine NTP-binding motif, GXXGXGK{ST}, to a significantly modified signature, KGGXXK{ST}, with the apparent preservation of the loop conformation typical of this motif . It is speculated that the ancestral form of the A motif might have comprised a loop rich in Gly residues, GXGGXGK{ST}, resembling that in NifH proteins and ArsA . Some of the Gly residues might have been differentially substituted in various evolutionary lineages of NTPases . The functional diversity of the proteins of this ATPase superfamily is comparable with the range of functions described previously for the superfamily of "UvrA-related" ATPases.

FEMS Microbiol Lett, 1993 Feb 15, 107(1), 115 - 20
Phylogenetic analysis of the family Rhizobiaceae and related bacteria by sequencing of 16S rRNA gene using PCR and DNA sequencer; Yanagi M et al.; The 16S rRNA gene sequences of 19 strains covering 97% of the molecules were determined for the members of the family Rhizobiaceae and related bacteria by PCR and DNA sequencer . The three biovars of Agrobacterium were located separately, whereas Agrobacterium rubi clustered with A . tumefaciens . Phylogenetic locations for the species of the genera Rhizobium, Sinorhizobium, Agrobacterium, Phylobacterium, Mycoplana (M . dimorpha), Ochrobactrum, Brucella and Rochalimaea (a rickettsia) were intermingled with each other with the similarity values higher than 92% . The family Rhizobiaceae should be redefined including the above-mentioned genera despite the ability for plant association and nitrogen fixation . Bradyrhizobium japonicum and Mycoplana bullata were far remote from the other species and should be excluded from this family.

Gene, 1993 Feb 14, 124(1), 75 - 81
Expression of a foreign gene in Chlamydomonas reinhardtii; Hall LM et al.; Genomic transformation of Chlamydomonas reinhardtii exposed to glass-bead abrasion was accomplished with a chimeric neomycin phosphotransferaseII (NPTII)-encoding gene (nos::npt) flanked by the nopaline synthase promoter and polyadenylation sequences obtained from the Ti plasmid of Agrobacterium tumefaciens . These sequences were in a plasmid (pGA482) which also contained gene nit1 encoding nitrate reductase of C . reinhardtii . Transformants were selected by their ability to grow on medium containing nitrate, and 52% of these was also resistant to kanamycin . Evidence for nos::npt expression includes: (1) hybridization with probes specific for npt, (2) demonstration of NPTII activity after electrophoresis of extracts, and (3) chromatographic identification of the reaction product of NPTII, kanamycin phosphate . The highly biased codon usage in Chlamydomonas does not preclude expression.

Mol Microbiol, 1993 Feb, 7(4), 555 - 62
Characterization of a virG mutation that confers constitutive virulence gene expression in Agrobacterium; Jin S et al.; Transformation of plants by Agrobacterium tumefaciens is mediated by a set of virulence (vir) genes that are specifically induced by plant signal molecules through the VirA/VirG two-component regulatory system . The plant signal is transmitted from VirA to VirG by a cascade of phosphorylation reactions followed by the sequence-specific DNA binding of the VirG protein to the vir gene promoters which then activates their transcription . In this report, we describe a VirG mutant which is able to activate vir gene expression independently of the VirA molecule and the two plant signal molecules, acetosyringone and monosaccharides . A strain of Agrobacterium containing this virG gene but lacking a functional virA gene was able to induce tumours on all three plants that were tested . A single amino acid change of asparagine (N) to aspartate (D) at position 54, adjacent to the site of VirG phosphorylation, aspartate 52, resulted in this constitutive phenotype . In vitro phosphorylation experiments showed that the mutant protein cannot be phosphorylated by VirA, suggesting that the negative charge resulting from the N to D switch mimics the phosphorylated conformation of the VirG molecule . The same amino acid change in the virG gene of the supervirulent strain A281 also resulted in a constitutive phenotype . However, the vir genes were not induced to high levels when compared with the levels of the constitutive virG of strain A348.

Plant Mol Biol, 1993 Feb, 21(4), 705 - 8
Transgenic tobacco plants regenerated from leaf disks can be periclinal chimeras; Schmulling T et al.; Amongst rolC transgenic tobacco plants regenerated from leaf disks 6.5% are periclinal chimeras, i.e . plants with genetically different cell populations in different cell layers . The expression of the rolC gene of Agrobacterium rhizogenes causes a reduction in pigment content in leaves . The chimeric composition of the regenerated plants becomes thus apparent as light green leaf tissue in the transgenic region, tissue flanked by dark green wild-type sectors . Southern and northern blot analysis confirmed the chimeric nature of such plants . Investigation of selfed progeny of chimeric plants on selective media indicates that layer invasion in reproductive tissues can occur in tobacco early during the formation of the flower buds . The results show (1) that tobacco plants regenerated from leaf disks and grown on selective media have not necessarily the same clonal origin and (2) that they can give rise to non-transgenic offspring . The chimeric plants provide insight on the effect of rolC gene expression on microsporogenesis.

Plant Mol Biol, 1993 Feb, 21(4), 655 - 63
Expression of a cysteine proteinase inhibitor (oryzacystatin-I) in transgenic tobacco plants; Masoud SA et al.; Expression of cysteine proteinase inhibitors (cystatins) in tobacco or other plants has the potential for improving resistance against pathogens and insects that possess cysteine proteinases . A chimeric gene containing a cDNA clone of rice cystatin (oryzacystatin-I; OC-I), the cauliflower mosaic virus 35S promoter, and the nopaline synthase 3' region was introduced into tobacco plants by Agrobacterium tumefaciens . The presence of the chimeric gene in transgenic plants was detected by a polymerase chain reaction-amplified assay, and transcriptional activity was shown by RNA blot analysis . Heated extracts from transgenic tobacco plants, as well as from progeny which were obtained by selfing a primary transformant, contained protein bands that corresponded in molecular mass to OC-I and reacted with antibodies raised against rOC, a recombinant OC-I protein produced by Escherichia coli . Similar bands were absent in extracts from untransformed control plants . OC-I levels reached 0.5% and 0.6% of the total soluble proteins in leaves and roots, respectively, of some progeny . On a fresh weight basis, the OC-I content was higher in leaves (50 micrograms/g) than in roots (30 micrograms/g) . OC-I was partially purified from protein extracts of rice seeds and from transgenic tobacco leaves by affinity to anti-rOC antibodies . OC-I from both sources was active against papain.

Virology, 1993 Feb, 192(2), 415 - 21
A mutation of cauliflower mosaic virus gene I interferes with virus movement but not virus replication; Thomas CL et al.; A 300-bp deletion was made in gene I of a multimeric copy of cauliflower mosaic virus (CaMV) . The deleted and wild-type constructs were mobilized into Agrobacterium tumefaciens and used for the inoculation of plants and leaf discs of the host plant, turnip, by "agroinfection." Infection induced by the wild-type construct gave characteristic viral symptoms on the inoculated and systemically infected leaves of whole plants whereas no infection was detected using the deletion mutant . The possibility that the deletion in gene I affected a cis-related function (e.g., encapsidation) was tested by complementation of mutant gene I with wild-type CaMV . Double infection experiments showed that replication and spread of both viruses occurred without detectable recombination of the mutant . In contrast to the situation in plants, agroinfection (Grimsley et al., 1986, Proc . Natl . Acad . Sci . USA 83, 3282-3286) of leaf discs, detected as the accumulation of encapsidated forms of virion DNA, was recorded for both the wild-type and the deleted constructs . The reduced level of virus accumulation from the mutant was consistent with virus replication occurring only in cells involved in the primary interaction with Agrobacterium, and accordingly progeny viral DNA was only detected in cells around the edge of the tissue disc . These data provide definitive evidence that CaMV gene I encodes a movement protein and show that this function can act in trans in a virus infection.

J Bacteriol, 1993 Feb, 175(3), 887 - 91
Construction and characterization of Tn5virB, a transposon that generates nonpolar mutations, and its use to define virB8 as an essential virulence gene in Agrobacterium tumefaciens; Dale EM et al.; Transfer of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant cells involves movement of a single-stranded DNA-protein intermediate across several membrane and cell wall barriers . The 11 VirB proteins encoded by the Ti plasmid are hypothesized to form at least part of a membrane-localized T-DNA transport apparatus . Although available genetic and biochemical analyses support this hypothesis, detailed study of this transport apparatus is hindered by the fact that most available mutations in the virB operon are in the form of transposon insertions that have polar effects . In this study we constructed a transposon, Tn5virB, that can be used to generate nonpolar insertions in operons of A . tumefaciens and used it to demonstrate that virB8 is an essential virulence gene.

J Bacteriol, 1993 Feb, 175(3), 723 - 31
Genetic analysis of the virD operon of Agrobacterium tumefaciens: a search for functions involved in transport of T-DNA into the plant cell nucleus and in T-DNA integration; Koukolikova-Nicola Z et al.; The transferred DNA (T-DNA) is transported from Agrobacterium tumefaciens to the nucleus and is stably integrated into the genome of many plant species . It has been proposed that the VirD2 protein, tightly attached to the T-DNA, pilots the T-DNA into the plant cell nucleus and that it is involved in integration . Using agroinfection and beta-glucuronidase expression as two different very sensitive transient assays for T-DNA transfer, together with assays for stable integration, we have shown that the C-terminal half of the VirD2 protein and the VirD3 protein are not involved in T-DNA integration . However, the bipartite nuclear localization signal, which is located within the C terminus of the VirD2 protein and which has previously been shown to be able to target a foreign protein into the plant cell nucleus, was shown to be required for efficient T-DNA transfer . virD4 mutants were shown by agroinfection to be completely inactive in T-DNA transfer.

Plant Mol Biol, 1993 Feb, 21(3), 429 - 35
Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco; Palmgren G et al.; We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a beta-glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation . In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression . Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week . Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression . Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low . These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.

Arch Virol, 1993, 130(3-4), 251 - 68
Immunodetection of the plum pox virus helper component in infected plants and expression of its gene in transgenic plants; Ravelonandro M et al.; Tobacco plants (Nicotiana tabacum cv . Xanthi) have been transformed via Agrobacterium tumefaciens vectors, with cDNAs corresponding to the plum pox virus (PPV) cistron 2 encoding helper component (HC-Pro) and with the first two and half cistrons of the PPV genome . Presence of the HC-Pro in PPV-infected plants and transgenic plants transformed with the gene coding for this protein was investigated using specific polyclonal antibodies produced against the PPV HC-Pro . The results suggest that two proteases are involved in the processing of the PPV N-terminal polyprotein to yield a protein of 48 k (HC-Pro) . HC-Pro autolytically cleaves at its carboxyl-terminus and a proteolytic activity, probably associated with the protein (P1) encoded by the cistron 1, is required for the cleavage in planta between the proteins derived from cistrons 1 and 2.

Transgenic Res, 1993 Jan, 2(1), 33 - 47
Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants; Lindsey K et al.; As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterless uidA (gusA) gene, encoding the beta-glucuronidase (GUS) reporter, was introduced into populations of tobacco, Arabidopsis and potato by Agrobacterium-mediated gene transfer . The objective was to generate random functional fusions following integration of the gusA gene downstream of native gene promoters . We describe here a detailed analysis of levels and patterns of gusA activation in diverse organs and cell types in those populations . gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line . The frequency of gusA activation was differentially biased in different organs in the three species . Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T2 and T3 progeny . Developmentally-regulated and environmentally-inducible expression of gusA is described for one transgenic line . Phenotypic variants were detected in the transgenic population . These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed.

Clin Infect Dis, 1993 Jan, 16(1), 112 - 7
Agrobacterium infections in humans: experience at one hospital and review; Hulse M et al.; Agrobacteria are noted primarily for their phytopathogenicity and when isolated from human clinical specimens are often considered contaminants or organisms of low pathogenicity . We report six cases at one hospital over a 6 1/2-year period in which infection was accompanied by a compatible clinical syndrome and review 19 cases reported in the literature . Fourteen of the 25 combined cases involved central venous catheter-associated infections . Six cases involved peritonitis, five of which occurred in patients undergoing continuous ambulatory peritoneal dialysis . Additional infections included two non-catheter-associated bacteremias, one prosthetic valve endocarditis, and two urinary tract infections . Most infections were community acquired, and restriction enzyme analysis of Agrobacterium isolates from eight patients at one hospital revealed unique patterns in each case without evidence for clonal dissemination of these strains . Agrobacterium isolates may be resistant to multiple antibiotics, and optimal therapy has not yet been determined . Agrobacteria should be recognized as opportunistic pathogens in the immunocompromised host, particularly in those with indwelling plastic catheters.

Arch Microbiol, 1993, 159(2), 124 - 30
Syntrophic interactions during degradation of 4-aminobenzenesulfonic acid by a two species bacterial culture; Feigel BJ et al.; During synthrophic growth of Hydrogenophaga palleronii (strain S1) and Agrobacterium radiobacter (strain S2) with 4-aminobenzene sulfonate (4ABS) only strain S1 desaminates 4ABS by regioselective 3,4-dioxygenation . The major part of the metabolite catechol-4-sulfonate (4CS) is excreted and further metabolized by strain S2 . Although both organisms harbour activities of protocatechuate pathways assimilation of the structural analog 4CS requires first of all enzyme activities with broader substrate specificity: protocatechuate 3,4-dioxygenase and carboxymuconate cycloisomerase activities were identified which in addition to the natural substrates also convert 4CS and 3-sulfomuconate respectively . 4-Carboxymethyl-4-sulfobut-2-en-4-olide (4SL) was identified as a metabolite . Its further metabolism requires a desulfonating enzyme which eliminates sulfite from (4SL) and generates maleylacetate . Convergence with the 3-oxoadipate pathway is catalyzed by a maleyl acetate reductase, which was identified in cell-free extracts of both organisms S1 and S2 . Characteristically, only strain S1 can oxidize sulfite and thus contributes to the interdependence of the two bacteria during growth with 4ABS.

Int J Syst Bacteriol, 1993 Jan, 43(1), 1 - 7
Classification of Rhizomonas suberifaciens, an unnamed Rhizomonas species, and Sphingomonas spp . in rRNA superfamily IV; van Bruggen AH et al.; Thermal melting profiles of hybrids between 3H-labeled rRNA of Rhizomonas suberifaciens, the causal agent of corky root of lettuce, and chromosomal DNAs from 27 species of gram-negative bacteria indicated that the genus Rhizomonas belongs to superfamily IV of De Ley . On the basis of the melting temperatures of DNA hybrids with rRNAs from the type strains of R . suberifaciens, Sphingomonas paucimobilis, and Sphingomonas capsulata, Rhizomonas strains constitute a separate branch in superfamily IV, which is closely related to but separate from branches containing Zymomonas mobilis, Sphingomonas spp., and S . capsulata . Sphingomonas yanoikuyae and Rhizomonas sp . strain WI4 are located toward the base of the Rhizomonas rRNA branch . DNA-DNA hybridization indicated that S . yanoikuyae is equidistant from Rhizomonas sp . strain WI4 and S . paucimobilis . Sequences of 270 bp of 16S ribosomal DNAs from eight strains of Rhizomonas spp., eight strains of Sphingomonas spp., and Agrobacterium tumefaciens indicated that S . yanoikuyae and Rhizomonas sp . strains WI4 and CA16 are genetically more closely related to R . suberifaciens than to Sphingomonas spp . Thus, S . yanoikuyae may need to be transferred to the genus Rhizomonas on the basis of the results of further study.

Arch Microbiol, 1993, 159(1), 30 - 8
Biosynthesis of cyclic beta-(1-3),beta-(1-6) glucan in Bradyrhizobium spp; de Iannino NI et al.; Inner membranes of Bradyrhizobium japonicum strain USDA 110 produced in vitro soluble and insoluble beta-(1-3),beta-(1-6) glucans . The reaction proceeded through a 90 kDa inner membrane intermediate protein; used UDP-glucose as sugar donor and required Mg2+ . Gel chromatography of soluble glucans resolved a cyclic beta-(1-3) glucan with a degree of polymerization of eleven from a family of beta-(1-3),beta-(1-6) glucans with variable degree of polymerization higher than eleven . Bradyrhizobium strains BR4406 and BR8404 isolated from tree legume nodules in Southeast Brazil produce beta-(1-3), beta-(1-6) glucans very similar to that of B . japonicum . A 100 kDa protein was identified in these strains as intermediates in the synthesis of these glucans . Inner membranes of B . japonicum USDA110, B . japonicum I17, and Bradyrhizobium strains BR4406 and BR8404 incubated with UDP-glucose were unable to synthesize beta-(1-2) glucan and lacked the 235 kDa intermediate protein known to be involved in the synthesis of beta-(1-2) glucan in Agrobacterium tumefaciens, Rhizobium meliloti and Rhizobium loti.

J Gen Virol, 1993 Jan, 74 ( Pt 1), 15 - 22
An analysis of the complete sequence of a sugarcane bacilliform virus genome infectious to banana and rice; Bouhida M et al.; The genome of sugarcane bacilliform virus (ScBV), a badnavirus, consists of a circular dsDNA . The complete sequence of a cloned infective ScBV genome is reported here . The genome is 7568 bp in size and possesses a number of features suggesting that ScBV is a pararetrovirus . A tRNA(Met)-binding site that may serve as a primer for minus-strand synthesis is present . The plus-strand of the ScBV genome contains three open reading frames (ORFs) which are capable of encoding proteins with calculated M(r) values of 22K, 13K and 215K . The 215K protein has regions with similarity to the RNA-binding domains, aspartic proteases and replicases of retro-elements . In addition, the 215K protein also has a region with restricted similarity to the intercellular transport proteins of plant viruses . Comparisons with the other sequenced badnaviruses, Commelina yellow mottle (CoYMV) and rice tungro bacilliform (RTBV) viruses, indicate that the arrangement of the ORFs in these viruses is conserved . Located next to the putative RNA-binding domain is a cysteine-rich region that is unique to the badnaviruses . When the molecular relationships of a portion of the reverse transcriptases of plant pararetroviruses were determined, two badnaviruses, CoYMV and ScBV, form one distinct cluster, whereas three caulimoviruses, cauliflower mosaic virus, carnation etched ring virus and figwort mosaic virus, form a second cluster . The badnavirus RTBV and the caulimovirus soybean chlorotic mottle virus occupy intermediate positions between the clusters . When introduced by Agrobacterium-mediated inoculation, a construct containing 1.1 copies of the cloned ScBV genome is infectious to both rice and banana.

Appl Environ Microbiol, 1993 Jan, 59(1), 150 - 5
Characterization of RFRS9, a second member of the Rhizobium fredii repetitive sequence family from the nitrogen-fixing symbiont R . fredii USDA257; Krishnan HB et al.; The genome of the nitrogen-fixing symbiont, Rhizobium fredii USDA257, contains nine copies of repetitive sequences known as the R . fredii repetitive sequence (RFRS) family . We previously sequenced RFRS3, which is linked to symbiosis plasmid-borne nodulation genes of this organism and has substantial homology to the T-DNA of Agrobacterium rhizogenes and lesser homology to reiterated sequences of Bradyrhizobium japonicum . Here we characterize a second family member, RFRS9 . The EcoRI fragment containing RFRS9 is 1,248 bp in length and contains a single 666-bp open reading frame that is flanked by perfect 8-bp inverted repeats . Nucleic and amino acid sequences corresponding to the C terminus of the putative RFRS9 protein are nearly identical to those of RFRS3, and they retain homology to DNA from A . rhizogenes . The central portion of the RFRS9 protein also appears to be related to the S locus-specific glycoprotein family of pollen stigma incompatibility glycoproteins from Brassica oleracea, which are involved in signal perception . Sequences that define the RFRS family are restricted to the open reading frame of RFRS9 and associated upstream sequences . These regions also contain a second group of repetitive sequences, which is present in four copies within the genome of USDA257 . Both families of repetitive sequences are ubiquitous in R . fredii, and they are preferentially localized on symbiosis plasmids . Southern hybridization confirms that sequences homologous to RFRS9 are present in broad-host-range Rhizobium sp . strain NGR234, in A . rhizogenes, and in two biotype 3 strains of Agrobacterium tumefaciens.

J Bacteriol, 1993 Jan, 175(2), 401 - 10
Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955; Hong SB et al.; We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR) . Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines . Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus . The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements . Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid . Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source . The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen . This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.

Biochimie, 1993, 75(8), 635 - 8
How does the T-DNA of Agrobacterium tumefaciens find its way into the plant cell nucleus?
Koukolikova-Nicola Z, Hohn B.
Agrobacterium tumefaciens causes the crown gall disease in plants by transferring a piece of DNA, the T-DNA, into the genome of the plant cell . The virulence protein VirD2, tightly linked to the T-DNA, is thought to direct it to the plant cell nucleus and to assist it in integration . The VirD2 protein contains two nuclear localization signals (NLS) which are functional both in yeast and in plant cells . One signal is located in the N-terminal part of the protein and resembles a single-cluster type NLS . The second signal is near the C-terminus and is a bipartite type NLS . The involvement of the C-terminal NLS in the entry of the T-DNA into the plant cell nucleus was directly tested in vivo.

Biochimie, 1993, 75(7), 623 - 9
Efficient pathogen-derived resistance induced by integrated potato virus Y coat protein gene in tobacco; Kollar A et al.; The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines . Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations . The transcription and expression of the integrated CP gene was detected by Northern and Western blots . Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H . The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection . Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation . Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection . This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.

Annu Rev Microbiol, 1993, 47, 167 - 97
Transport of nucleic acids through membrane channels: snaking through small holes; Citovsky V et al.; Transport of nucleic acids through cell membranes is an essential biological process that occurs in all living organisms . This review focuses on two plant systems in which nucleic acid molecules are transported through membrane channels: transport of Agrobacterium T-DNA through nuclear pores and movement of plant viruses through intracellular connections . To provide a broader perspective, nuclear uptake of animal viruses and nuclear import/export of small nuclear (sn) RNA and messenger (m) RNA are described . By comparing the examined cases of nucleic acid transport, the review proposes a set of general rules for this type of transport through membrane channels.

Antisense Res Dev, 1993 Summer, 3(2), 181 - 90
Simultaneous inhibition of two tomato fruit cell wall hydrolases, pectinmethylesterase and polygalacturonase, with antisense gene constructs; Pear JR et al.; The cloning and sequencing of two cDNAs representing pectinmethylesterase (PME) RNAs from tomato fruit is reported . The clones were used to construct chimeric antisense PME genes designed for high-level constitutive expression in plants . A full-length antisense PME gene construct, in conjunction with a chimeric antisense polygalacturonase gene, was introduced into tomato plants via Agrobacterium-mediated transformation . Simultaneous and significant reduction in the mRNA and protein levels of these normally highly abundant cell wall hydrolases of the pectin degradation pathway were observed in ripe fruit of transformants . Thus, antisense gene constructs in plants can be used to block multiple steps in metabolic pathways simultaneously.

Plant Mol Biol, 1993 Jan, 21(2), 341 - 54
Induction of a tomato anionic peroxidase gene (tap1) by wounding in transgenic tobacco and activation of tap1/GUS and tap2/GUS chimeric gene fusions in transgenic tobacco by wounding and pathogen attack; Mohan R et al.; The anionic peroxidase genes of tomato, tap1 and tap2, are induced by wounding in tomato fruits and by elicitor treatment in cell suspension cultures . These homologous genes code for anionic peroxidases that are postulated to cause polymerization of the phenolic residues into wall polymers in wound-healing and pathogen-infected tissues . An expression construct containing the entire TAP1 gene with its 5' and 3' flanking sequences was introduced into tobacco by Agrobacterium tumefaciens-mediated gene transfer . Also, constructs containing the 5' upstream regions of tap1 and tap2 including sequences coding for their respective putative leader peptides fused translationally to the beta-glucuronidase (GUS) reporter gene were made and introduced into tobacco . Northern blot analysis of transcripts from wound-healing leaf tissues of transformants containing tap1 showed that the introduced gene was being transcribed in the heterologous host . The induction of tap1 transcripts in the wound-healing transgenic tobacco tissues was observed by 48 h and increased over time period of 84 h . Wounding also led to expression of GUS in tap1/GUS and tap2/GUS transformants and GUS activity was localized to the wound site . Activation of the tap1 and tap2 promoters in wound-healing transgenic tobacco tissues showed a GUS expression profile that correlated with the postulated role for anionic peroxidases in phenolic polymerization in suberizing tissues . Inoculation of tap1/GUS and tap2/GUS transformant leaves with fungal conidia from Fusarium solani f . sp . pisi caused expression of GUS in locally inoculated regions, and GUS expression increased over a period of four days.

Plant Mol Biol, 1993 Jan, 21(1), 17 - 26
Transgene copy number can be positively or negatively associated with transgene expression; Hobbs SL et al.; Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens . High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and beta-glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression . H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F1 plants with GUS activity that equalled the parents and individual F2 plants with 50%, 100%, 150% and 200% of parental values . However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome . Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F1 and F2 generations with both types of insert had low GUS activity while F2 segregants that only had the H-type inserts had high GUS activity again . Suppression of the H-type gene was associated with increased methylation of the insert . Particle acceleration was used to introduce further copies of uidA into tissues of the transformants . Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.

Plant Mol Biol, 1993 Jan, 21(1), 109 - 19
Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea; Suzuki H et al.; SbPRP1 is a member of the soybean (Glycine max L . Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue . To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS) . This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation . Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis . A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation . Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene . Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter . Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition . These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11837 - 41
A nuclear localization signal and the C-terminal omega sequence in the Agrobacterium tumefaciens VirD2 endonuclease are important for tumor formation; Shurvinton CE et al.; The T-DNA portion of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid 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 generate linear single-stranded VirD2-bound DNA molecules that include the entire T-DNA (T-strands) . VirD2 protein contains a nuclear localization signal (NLS) near the C terminus and may direct bound T-strands to plant nuclei . We constructed mutations in virD2 and showed that the NLS was important for tumorigenesis, although T-strand production occurred normally in its absence . A tobacco etch virus NLS, substituted for the VirD2 NLS, restored tumor-inducing activity . Amino acids (the omega sequence) at the C terminus of VirD2, outside the NLS and the endonuclease domain, contributed significantly to tumorigenesis, suggesting that VirD2 may serve a third important function in T-DNA transfer.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 11799 - 803
Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition; Yun DJ et al.; The tropane alkaloid scopolamine is a medicinally important anticholinergic drug present in several solanaceous plants . Hyoscyamine 6 beta-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine . We introduced the hydroxylase gene from Hyoscyamus niger under the control of the cauliflower mosaic virus 35S promoter into hyoscyamine-rich Atropa belladonna by the use of an Agrobacterium-mediated transformation system . A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase . In the primary transformant and its selfed progeny that inherited the transgene, the alkaloid contents of the leaf and stem were almost exclusively scopolamine . Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.

Mol Gen Genet, 1992 Dec, 236(1), 1 - 7
Design of a novel system for the construction of vectors for Agrobacterium-mediated plant transformation; Mozo T et al.; The loxP-Cre site-specific recombination system of phage P1 was used to develop a novel strategy to construct cointegrate vectors for Agrobacterium-mediated plant transformation . A pTi disarmed helper plasmid (pAL1166) was constructed by replacing the oncogenic T-DNA by a loxP sequence and a spectinomycin resistance marker in the octopine-type pTiB6 plasmid . The cre gene was cloned into an unstable incP plasmid . A third plasmid, which did not replicate in Agrobacterium and contained another loxP sequence together with a kanamycin resistance marker, was used to test the system . Electroporation of this third plasmid into an Agrobacterium strain harbouring both pAL1166 and the Cre-encoding plasmid resulted in kanamycin-resistant cells containing a cointegrate between pAL1166 and the incoming plasmid . Cointegration occurred by Cre-mediated recombination at the loxP sites, and the cointegrate was stabilized in the Agrobacterium cells by the loss of the Cre-encoding plasmid shortly after the recombination event had taken place.

Plant Mol Biol, 1992 Dec, 20(6), 1203 - 7
A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome; Gleave AP; A versatile gene expression cartridge and binary vector system was constructed for use in Agrobacterium-mediated plant transformation . The expression cartridge of the primary cloning vector, pART7, comprises of cauliflower mosaic virus Cabb B-JI isolate 35S promoter, a multiple cloning site and the transcriptional termination region of the octopine synthase gene . The entire cartridge can be removed from pART7 as a Not I fragment and introduced directly into the binary vector, pART27, recombinants being selected by blue/white screening for beta-galactosidase . pART27 carries the RK2 minimal replicon for maintenance in Agrobacterium, the ColE1 origin of replication for high-copy maintenance in Escherichia coli and the Tn7 spectinomycin/streptomycin resistance gene as a bacterial selectable marker . The organisational structure of the T-DNA of pART27 has been constructed taking into account the right to left border, 5' to 3' model of T-DNA transfer . The T-DNA carries the chimaeric kanamycin resistance gene (nopaline synthase promoter-neomycin phosphotransferase-nopaline synthase terminator) distal to the right border relative to the lacZ' region . Utilisation of these vectors in Agrobacterium-mediated transformation of tobacco demonstrated efficient T-DNA transfer to the plant genome.

Plant Mol Biol, 1992 Dec, 20(6), 1149 - 57
Differential expression of a chimeric CaMV-tomato proteinase Inhibitor I gene in leaves of transformed nightshade, tobacco and alfalfa plants; Narvaez-Vasquez J et al.; The open reading frame and terminator region of a wound-inducible tomato Inhibitor I gene, regulated by the CaMV 35S promoter, was stably integrated into the genomes of nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and alfalfa (Medicago sativa), using an Agrobacterium-mediated transformation system . The expression of the foreign Inhibitor I gene in leaves of each species was studied at the mRNA and protein levels . The levels of Inhibitor I protein present in leaves of each species correlated with the levels of mRNA . The average levels of both mRNA and Inhibitor I protein were highest in leaves of transgenic nightshade plants (over 125 micrograms of Inhibitor I per g tissue), less in tobacco plants (about 75 micrograms/g tissue), and lowest in leaves of transgenic alfalfa plants (below 20 micrograms/g tissue) . Inhibitor I protein was observed in all tissues throughout transgenic plant species, but inhibitor concentration per gram of tissue was 2-3 times higher in young developing leaf tissues and floral organs . The differences in the expression of the CaMV-tomato Inhibitor I gene among the different plant genera suggests that either the rate of transcription of the foreign gene or the rate of degradation of the nascent Inhibitor I mRNA varies among genera . Using electron microscopy techniques, the newly synthesized pre-pro-Inhibitor I protein was shown to be correctly processed and stored as a mature Inhibitor I protein within the central vacuoles of leaves of transgenic nightshade and alfalfa . The results of these experiments suggest that maximal expression of foreign proteinase inhibitor genes, and perhaps other foreign defense genes, may require gene constructs that are fashioned with promoters and terminators that allow maximum expression in the selected plant species.

Plant Mol Biol, 1992 Dec, 20(6), 1071 - 87
Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens; Liu CN et al.; In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues . Four days after A . tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery . Additional copies of octopine- and agropine-type virG genes in A . tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice . This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated . For both rice and celery, we observed a greater enhancement of transformation using A . tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene . Multiple copies of virG genes contained in A . tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice . In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A . tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene . Our results show that multiple copies of virG in A . tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A . tumefaciens and by the infected plant species.

Plant Mol Biol, 1992 Dec, 20(6), 1037 - 48
Factors influencing Agrobacterium-mediated transient expression of gusA in rice; Li XQ et al.; Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria . Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A . tumefaciens . Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A . tumefaciens . A . tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity . Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A . tumefaciens . The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/l 2,4-D during cocultivation of the explants with A . tumefaciens slightly enhanced GUS expression efficiency . All 21 rice cultivars tested expressed GUS after co-cultivation with A . tumefaciens . The GUS expression frequency was highest amongst the indica cultivars . The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties . Leaf explants were more susceptible to A . tumefaciens-facilitated GUS expression than were roots or seed remnants . The vir genes of an agropine-type Ti-plasmid of A . tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective . We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector . Reasons for differences in effectiveness of these binary vectors are discussed . Using the conditions described here, A . tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

Plant Mol Biol, 1992 Dec, 20(5), 921 - 38
Translation controls the expression level of a chimaeric reporter gene; Hensgens LA et al.; Transcriptional and translational fusions between the reading frame of the beta-D-glucuronidase gene (gusA) and the 2' as well as the 1' promoter of mannopine synthase (mas), a TR locus of Agrobacterium tumefaciens, were made . The expression of these constructs was studied in the transgenic F1 offspring of independent tobacco transformants at the protein level by assaying for GUS activity and western blot analysis of the GUS protein and at the steady-state mRNA level . In leaves, stems and roots no correlation was found between steady-state levels of GUS mRNA and enzyme activity . In older tissues significantly higher GUS activities were found . This is explained by the stable character of the GUS protein together with an accumulation of protein upon ageing . Three to ten times higher GUS activities were found for in vitro grown plants than for greenhouse-grown plants of the same offspring, despite similar levels of GUS mRNA . Roots from in vitro grown plants display three to ten times higher GUS activities than stems and leaves . In transgenic plants grown in vitro, containing a translational fusion with two AUGs in phase, the initiation of translation in leaf material occurred at both AUGs . Initiation of translation at the first AUG, however, was ten times more frequent . In contrast, initiation in roots from in vitro grown plants occurred exclusively at the second AUG.

J Bacteriol, 1992 Dec, 174(24), 7941 - 7
Mechanism of action of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens: competition between cyclization and elongation reactions; Williamson G et al.; We have examined some aspects of the mechanism of cyclic beta-1,2-glucan synthetase from Agrobacterium tumefaciens (235-kDa protein, gene product of the chvB region) . The enzyme produces cyclic beta-1,2-glucans containing 17 to 23 glucose residues from UDP-glucose . In the presence of added cyclic beta-1,2-glucans (> 0.5 mg/ml) (containing 17 to 23 glucose residues), the enzyme instead synthesizes larger cyclic beta-1,2-glucans containing 24 to 30 glucose residues . This is achieved by de novo synthesis and not by disproportion reactions with the added product . This is interpreted as inhibition of the specific cyclization reaction for the synthesis of cyclic beta-1,2-glucans containing 17 to 23 glucose residues but with no concomitant effect on the elongation (polymerization) reaction . Temperature and detergents both affect the distribution of sizes of cyclic beta-1,2-glucans, but glucans containing 24 to 30 glucose residues are not produced . We suggest that the size distribution of cyclic beta-1,2-glucan products depends on competing elongation and cyclization reactions.

Genes Dev, 1992 Dec, 6(12A), 2364 - 72
Serine-to-alanine substitutions at the amino-terminal region of phytochrome A result in an increase in biological activity; Stockhaus J et al.; We have used a tobacco transgenic plant system to assay the structure/function relationship of phytochrome A (phyA), a plant photoreceptor . The amino terminus of phyA from different plant species is very rich in serine residues . To investigate whether these serine residues are required for phytochrome function, the first 10 serine codons encoding amino acid residues 2-4, 10-14, 19, and 20 in the amino-terminal domain of the rice phyA gene (phyA) were changed to alanine codons . The mutant (S/A phyA), as well as the wild-type phyA cDNA, was placed under the control of the 35S promoter, and the chimeric genes were transferred into the tobacco genome by Agrobacterium-mediated transformation . Transgenic tobacco plants expressing either wild-type or S/A phyA showed similar phenotypic alterations, including dwarfism and dark-green leaves . However, hypocotyl elongation experiments revealed that transgenic seedlings expressing S/A phyA showed a higher amplitude of the red light response with respect to the inhibition of hypocotyl elongation . The observed difference is not correlated with expression levels of the transgene . The chromophore is attached to the mutant phyA apoprotein (PHY A), and the mutant photoreceptor is photoreversible, giving a difference spectrum indistinguishable from that of the rice phyA . Our results indicate that the S/A mutant has a higher biological activity as compared with the wild-type rice phyA.

Clin Infect Dis, 1992 Dec, 15(6), 938 - 40
Agrobacterium tumefaciens peritonitis mimicking tuberculosis; Ramirez FC et al.; Agrobacterium species have been previously implicated in the development of clinical disease . We report what we believe to be the first case of ascites caused by Agrobacterium tumefaciens in a cirrhotic patient . Since the correct diagnosis was made only after laparoscopy-guided collection of specimens from two different tissues, we suggest that Agrobacterium may be an underdiagnosed pathogen in clinical situations in which tuberculosis is considered to be the cause of high-protein ascites.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11184 - 8
Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco; Cahoon EB et al.; Little is known about the metabolic origin of petroselinic acid (18:1 delta 6cis), the principal fatty acid of the seed oil of most Umbelliferae, Araliaceae, and Garryaceae species . To examine the possibility that petroselinic acid is the product of an acyl-acyl carrier protein (ACP) desaturase, Western blots of coriander and other Umbelliferae seed extracts were probed with antibodies against the delta 9-stearoyl-ACP desaturase of avocado . In these extracts, proteins of 39 and 36 kDa were detected . Of these, only the 36-kDa peptide was specific to tissues which synthesize petroselinic acid . A cDNA encoding the 36-kDa peptide was isolated from a coriander endosperm cDNA library, placed under control of the cauliflower mosaic virus 35S promoter, and introduced into tobacco by Agrobacterium tumefaciens-mediated transformation . Expression of this cDNA in transgenic tobacco callus was accompanied by the accumulation of petroselinic acid and delta 4-hexadecenoic acid, both of which were absent from control callus . These results demonstrate the involvement of a 36-kDa putative acyl-ACP desaturase in the biosynthetic pathway of petroselinic acid and the ability to produce fatty acids of unusual structure in transgenic plants by the expression of the gene for this desaturase.

Biosci Biotechnol Biochem, 1992 Dec, 56(12), 1924 - 8
Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis; Kang HW et al.; A transposon 5 (Tn5) insertion was introduced into the genome of A . tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5 . Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated . The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis . An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized . The mutant had the same growth rate as that of the parent strain in L-broth . The mutant and the parent strain had similar attachment ability to carrot root cells . Tn5 was inserted into one site of the chromosome . The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced . An open reading frame (ORF) consisting of 395 base pairs was identified . The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain . A soluble protein was predicted from the ORF . The Tn5 was inserted near the 3'-terminal of the ORF . Homology search of this ORF found no significant homology to known genes and proteins . Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A . tumefaciens.

Acta Virol, 1992 Dec, 36(6), 567 - 75
Expression of Helenium virus S coat protein in Escherichia coli, in vitro in rabbit reticulocyte lysate and transgenic tobacco; Foster GD et al.; The coat protein open reading frame (ORF) sequence of Helenium virus S (HelVS) was cloned and expressed in E . coli, rabbit reticulocyte and transgenic tobacco . In E . coli the size of the protein was identical to that obtained for the coat protein from purified virus particles and less than that predicted for the fusion protein . This may be due to ribosome binding at a potential ribosome binding site present on the viral sequence, approximately 45 nucleotides upstream from the initiating methionine of the coat protein ORF . This region of HelVS, equivalent to the 1.5 kb subgenomic RNA, also produced high levels of protein when transcribed and translated in vitro . When introduced into Nicotiana tabacum by leaf disk transformation via Agrobacterium tumefaciens, high levels of stable coat protein were detected which were identical in molecular weight to that of HelVS coat protein and constituted approximately 0.1-0.5% of the total extracted protein.

Antibiot Khimioter, 1992 Dec, 37(12), 29 - 31
{Immunomodulating action of peptidoglycan of microbial origin}; Nikitin AV et al.; The action of a high molecular weight peptidoglycan produced by Agrobacter radiobacter sp . on the functional activity parameters in leukocytes and macrophages i . e . chemotaxis and adhesion was studied . It was shown that the peptidoglycan had a stimulating action on the chemotaxis of cells of the peritoneal exudate . A marked stimulating action of the drug on the primary immune response to the tissue antigen of sheep erythrocytes was observed . The peptidoglycan stimulated the antibody titers and delayed hypersensitivity when administered in various periods after an antigenic stimulus . Multifactorial experiments on the protective action of the peptidoglycan in experimental infections were carried out . Second-order polynomial statistic models characterizing the animal survival rate were constructed and the dose-time parameters of the drug use were optimized.

Plant Mol Biol, 1992 Dec, 20(5), 897 - 910
Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses; Genschik P et al.; Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris . Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated . The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing . A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed . cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized . The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5-mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids . The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation . The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.

Science, 1992 Nov 20, 258(5086), 1350 - 3
Activation of a plant gene by T-DNA tagging: auxin-independent growth in vitro; Hayashi H et al.; A transferred DNA (T-DNA) tagging vector with the potential to produce dominant mutations was used with cocultured Agrobacterium tumefaciens and protoplasts to tag genes involved in the action of the plant growth substance auxin . Transgenic calli were selected for their ability to grow in the absence of auxin in the culture media . From one experiment, 12 calli that displayed this phenotype were recovered, of which 11 were able to regenerate into plants . In one plant studied in detail, protoplast division in the absence of auxin genetically cosegregated with a single T-DNA insert . A messenger RNA encoded by a 6.4-kilobase sequence of plant genomic DNA rescued from the mutant is overexpressed relative to untransformed plants . The genomic DNA, as well as a cognate complementary DNA, once transfected into protoplasts promote growth and cell division in vitro in the absence of exogenously added auxin.

Gene, 1992 Nov 2, 121(1), 133 - 6
Molecular analysis of the recA gene of Agrobacterium tumefaciens C58; Wardhan H et al.; The complete nucleotide sequence of the Agrobacterium tumefaciens recA gene was determined . A comparison of the translated open reading frame of the gene with other known recA sequences revealed significant sequence conservation . However, unlike its Escherichia coli equivalent, A . tumefaciens recA lacks the upstream 'SOS box', suggesting a different mechanism of regulation for this gene.

Mol Gen Genet, 1992 Nov, 235(2-3), 292 - 303
Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4; Canaday J et al.; The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization . The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found . The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids . The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue . We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene . Both sequences show homology to the octopine synthase gene . In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.

Plasmid, 1992 Nov, 28(3), 201 - 12
Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions; Farrand SK et al.; The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84 . Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens . Efficient biocontrol is dependent upon production of agrocin 84 by strain K84 . Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated . The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map . A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6 . Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region . This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84 . The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region . This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia . One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid . Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts . One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts . This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A . radiobacter strain K84 . This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.

Plant Mol Biol, 1992 Nov, 20(3), 555 - 8
Expression of desiccation-related proteins from the resurrection plant Craterostigma plantagineum in transgenic tobacco; Iturriaga G et al.; Three cDNAs encoding desiccation-induced proteins from the resurrection plant Craterostigma plantagineum were each ligated to a triplicated CaMV 35S promoter and a nopaline synthase 3'-flanking region in an Agrobacterium vector and introduced into tobacco . Transgenic plants expressed the encoded Craterostigma proteins at high levels . This did not lead to changes in the phenotype, in the growth habit or in basic photosynthetic parameters . In tobacco, one protein was targeted to the chloroplast stroma which is its normal location in Craterostigma . These desiccation-related proteins are not sufficient per se to increase drought tolerance as measured by ion-leakage tests.

J Bacteriol, 1992 Nov, 174(21), 7040 - 3
Altered-function mutations of the transcriptional regulatory gene virG of Agrobacterium tumefaciens; Han DC et al.; Three point mutations were isolated in the Agrobacterium tumefaciens virG gene by screening for vir gene expression in the absence of added phenolic inducing compounds . All three mutations were localized in the predicted amino-terminal phosphoryl receiver domain of the protein . One mutant (N54D) bypasses the requirement for VirA and phenolic inducers both for transcriptional activation of all tested vir promoters and for plant tumorigenesis . This mutant also activates vir gene expression efficiently at neutral pH, indicating that the step in induction that is normally stimulated by acid pH occurs before or during VirG phosphorylation . The other two mutants (M13T and H15R) require VirA for activity but are sensitized to low levels of inducing stimuli.

J Bacteriol, 1992 Nov, 174(21), 7033 - 9
Functional roles assigned to the periplasmic, linker, and receiver domains of the Agrobacterium tumefaciens VirA protein; Chang CH et al.; VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites . VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver . We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo . The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity . The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation . The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.

Mol Gen Genet, 1992 Nov, 235(2-3), 259 - 68
Interest in and limits to the utilization of reporter genes for the analysis of transcriptional regulation of nitrate reductase; Vaucheret H et al.; Reporter gene techniques and mutant analysis were used to identify the molecular basis of the regulation of the expression of nitrate reductase (NR) by nitrate and nitrate-, or ammonium-derived metabolites (N-metabolites), in the true diploid species Nicotiana plumbaginifolia and in the amphidiploid species Nicotiana tabacum . The N . plumbaginifolia mutant E23 results from the insertion of a Tnt1-like retrotransposon (Tnp2) in the first exon of the single-copy nia gene, which encodes nitrate reductase . One of the resulting transcripts ends in the 5' LTR (long terminal repeat) sequence of this retrotransposon, and another one in the 3' LTR . Nitrate and N-metabolites modulate the expression of these truncated transcripts, indicating that intron splicing and termination processes are not essential to these regulatory events . A GUS reporter sequence was transcriptionally linked to the promoter of the nia-1 gene of N . tabacum . This fusion was functional in transient expression assays done with protoplasts derived from mesophyll cells of N . tabacum . However none of the regulatory mechanisms known to affect steady-state levels of the nia-1 transcript were operative under these experimental conditions . Transgenic plants carrying either this fusion or translational fusions of GUS linked to the promoter of either the nia-1 or nia-2 gene of N . tabacum were obtained by Agrobacterium-mediated transfer . A low proportion of the transgenic plants (22 out of 105 independent transformants) expressed GUS activity although at a low level . Only 4 plants exhibited a detectable level of GUS mRNA . The concentration of this mRNA increased significantly in an NR-deficient background, indicating regulation by N-metabolites . Only 2 plants, however, showed regulation (induction) by nitrate . Attempts to use aux2 or nptII reporter sequences linked to either the nia-1 or nia-2 promoter as marker genes for the selection of regulatory mutants of the nitrate assimilation pathway were unsuccessful because of our inability to isolate transgenic plants in which these reporter genes were properly regulated by nitrate . The implications of these results are discussed.

Plant Mol Biol, 1992 Nov, 20(4), 631 - 9
Evidence for sense RNA-mediated protection to PVYN in tobacco plants transformed with the viral coat protein cistron; van der Vlugt RA et al.; The coat protein (CP) cistron of the tobacco veinal necrosis strain of potato virus Y (PVYN), supplemented with translational start signals, was cloned into an Agrobacterium tumefaciens Ti transformation vector . Transformation of tobacco leaf discs resulted in 99 transgenic lines which were subsequently analysed for the presence and expression, at both the transcriptional and translational level, of the CP-gene . Although CP-specific RNA transcripts were produced in all plants no CP could be detected by several sensitive immunological techniques . Upon mechanical inoculation of progeny lines of self-pollinated original transformants (S1) with PVYN, protection levels of 20 and 95%, respectively, could be observed in two out of ten lines tested . This level of protection increased to 100% in the S2 progeny obtained from self-pollination of virus-protected S1 plants . Transformation of tobacco leaf discs with a PVYN CP construct from which the ATG start codon had been removed by site-directed mutagenesis resulted in 57 transgenic lines that all produced CP-specific transcripts . Mechanical inoculation with PVYN of S1 progeny plants of several of these lines resulted in resistance to a similar level and extent as in the S1 progeny of plants transformed with the intact CP cistron . The results obtained strongly suggest that the resistance observed in the transgenic plants is principally based on the presence of PVYN CP RNA sequences rather than on the accumulation of viral coat protein.

Gene, 1992 Oct 21, 120(2), 167 - 73
Construction of a chimeric viral gene expressing plum pox virus coat protein; Ravelonandro M et al.; The capsid-encoding gene of plum pox virus (PPV) was fused with the leader sequence of the coat protein mRNA (cp) of tobacco mosaic virus by a novel mutagenesis technique which involves reverse transcription of minus-strand RNA {synthesized by in vitro transcription of a double-stranded (ds) cDNA clone}, using an ad hoc synthetic oligodeoxynucleotide as primer . The resulting cDNA was rendered ds and cloned into the plasmid, pBluescribe M13+ . Transcription of this chimeric construction produced RNA molecules of 1250 nucleotides in length, which were used as messengers in the in vitro protein-synthesizing systems . The major product of this transcript consists of a 36-kDa polypeptide and was identified as the PPV coat protein (CP) by molecular weight estimation and by immunoprecipitation with a polyclonal antiserum to PPV . Transfer of this cDNA via Agrobacterium tumefaciens into plants was successfully performed . Transgenic Nicotiana plants producing the PPV CP were subsequently obtained.

Biochemistry, 1992 Oct 20, 31(41), 9970 - 8
Inactivation of a beta-glucosidase through the accumulation of a stable 2-deoxy-2-fluoro-alpha-D-glucopyranosyl-enzyme intermediate: a detailed investigation; Street IP et al.; The inactivation of glycosidases by 2-deoxy-2-fluoroglycosides has been shown previously to occur via the accumulation of a covalent 2-deoxy-2-fluoro-alpha-D-glucopyranosyl enzyme intermediate {Withers, S . G., & Street, I . P . (1988) J . Am . Chem . Soc . 110, 8551} . Further characterization of this process with Agrobacterium beta-glucosidase is described, and the range of glycosides engaging in this behavior is examined . Inactivation is shown to be accompanied by the release of a stoichiometric "burst" of aglycon, thereby providing a new class of active site titration agents for glycosidases . The rate of inactivation is shown to be very strongly dependent on the leaving group ability of the aglycon, the slowest inactivator studied (p-nitrophenyl2-deoxy-2-fluoro-beta-D-glucopyranoside) yielding only partial inactivation due to turnover of the intermediate becoming competitive with its formation . Such turnover of the intermediate is shown to be greatly accelerated by transglycosylation to a suitable glycoside bound in the aglycon site, resulting in the release of a disaccharide product which was isolated and characterized . The pH dependences of both the formation and the hydrolysis of the 2-deoxy-2-fluoroglycosyl-enzyme closely resemble those of each step for normal catalysis, indicating that the same catalytic groups are involved in both processes . A model system for the partial "steady-state" inactivation observed previously {Withers, S . G., Rupitz, K., & Street, I . P . (1988) J . Biol . Chem . 263, 7929} with certain other glycosidases was established by incubating the enzyme with an inactivator known to undergo relatively rapid transglycosylation in the presence of various concentrations of a suitable reactivator.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1992 Oct 20, 31(41), 9979 - 85
Mechanistic consequences of mutation of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase; Withers SG et al.; The replacement of the active site nucleophile Glu 358 in Agrobacterium beta-glucosidase by Asn and Gln by site-directed mutagenesis results in essentially complete inactivation of the enzyme, while replacement by Asp generates a mutant with a rate constant for the first step, formation of the glycosylenzyme, some 2500 times lower than that of the native enzyme . This low activity is shown to be a true property of the mutant and not due to contaminating wild-type enzyme by active site titration studies and also through studies of its thermal denaturation and of the pH dependence of the reaction catalyzed . Binding of ground-state inhibitors is affected relatively little by the mutation, while binding of transition-state analogues is greatly impaired, consistent with a principal role for Glu 358 being in transition-state stabilization, not substrate binding . Determination of kinetic parameters for a series of aryl glucosides revealed that the glycosylation step is rate determining for all these substrates in contrast to the native enzyme, where a switch from rate-limiting glycosylation to rate-limiting deglycosylation was observed as substrate reactivity was increased . These results coupled with secondary deuterium kinetic isotope effects of kH/kD = 1.17 and 1.12 measured for the 2,4-dinitrophenyl and p-nitrophenyl glucosides point to a principal role of the nucleophile in stabilizing the cationic transition states and in formation of the covalent intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1992 Oct 5, 267(28), 20471 - 80
Sequence similarities between the RP4 Tra2 and the Ti VirB region strongly support the conjugation model for T-DNA transfer; Lessl M et al.; Transfer genes of the IncP plasmid RP4 are grouped in two separate regions, designated Tra1 and Tra2 . Tra2 gene products are proposed to be mainly responsible for the formation of mating pairs in conjugating cells . To provide information relevant to understanding the function of Tra2 gene products, the nucleotide sequence of the entire RP4 Tra2 region is presented here . Twelve open reading frames were identified in the Tra2 core region, being essential for intraspecific Escherichia coli matings . Predicted sizes of 11 of the 12 Tra2 polypeptides could be verified by expression in E . coli . Based on hydropathy plot analysis, most of the Tra2 open reading frames encode proteins that may interact with membranes . Interestingly, six of the predicted Tra2 gene products exhibited significant sequence similarities to gene products encoded by the VirB operon of the Agrobacterium Ti plasmid . VirB proteins are thought to function in the formation of a transmembrane structure that mediates the passage of T-DNA molecules from bacteria into plant cells . Because of this analogy and the hydropathy of Tra2 gene products, we assume that the DNA transfer machineries acting in bacterial conjugation and T-DNA transfer are structurally and functionally similar . Therefore, the data presented here, support the hypothesis that Ti vir and IncP tra genes evolved from a common ancestor . This suggestion is favored by previous findings of sequence similarities between the IncP and Ti DNA transfer system.

Plant Mol Biol, 1992 Oct, 20(1), 113 - 22
A T-DNA transfer stimulator sequence in the vicinity of the right border of pRi8196; Hansen G et al.; An 8 bp sequence repeated 6 times is present to the right of the mannopine type pRi8196 T-DNA right-border sequence . Experiments were designed to test whether these repeats have a role in T-DNA transfer . Several constructs in which different lengths of pRi8196 right-border region were linked to the cucumopine synthesis gene on an Agrobacterium-Escherichia coli shuttle vector were made . The recombinant plasmids were tested for their efficiency to act as a source of T-DNA in a binary system in which a wild-type Ri plasmid provided virulence and root-inducing functions . The T-DNA transfer efficiency of the constructs was assessed by computing the relative frequency of roots containing cucumopine . Depending on the Ri plasmid used as source of virulence functions, a high level of T-DNA transfer was observed only if 6 (pRi8196) or 5 (pRiA4) repeats were present . These results were confirmed by looking for single-stranded T-DNA molecules (T-strands) in bacteria induced for virulence . The repetition of the 8 bp unit was named 'T-DNA transfer stimulator sequence' (TSS).

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9136 - 40
Agroinfection as an alternative to insects for infecting plants with beet western yellows luteovirus; Leiser RM et al.; Beet western yellows luteovirus, like other luteoviruses, cannot be transmitted to host plants by mechanical inoculation but requires an aphid vector, a feature that has heretofore presented a serious obstacle to the study of such viruses . In this paper we describe use of agroinfection to infect hosts with beet western yellows virus without recourse to aphids . Agroinfection is a procedure for introducing a plant virus into a host via Agrobacterium tumefaciens harboring a Ti plasmid, which can efficiently transfer a portion of the plasmid (T-DNA) to plant cells near a wound . The viral genome must be inserted into the T-DNA in such a way that it can escape and begin autonomous replication, a requirement that has, so far, limited agroinfection to pathogens with a circular genome . We have cloned cDNA corresponding to the complete beet western yellows virus RNA genome between the cauliflower mosaic virus 35S promoter and the nopaline synthase transcription termination signal . In one construct, a self-cleaving (ribozyme) sequence was included so as to produce a transcript in planta with a 3' extremity almost identical to natural viral RNA . When inoculated mechanically to host plants, the naked plasmid DNA was not infectious but, when introduced into T-DNA and agroinfected to plants, both the construct with and without the ribozyme produced an infection . This approach should be applicable to virtually any plant virus with a linear plus-strand RNA genome.

J Bacteriol, 1992 Oct, 174(20), 6666 - 73
Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity; Waters VL et al.; Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons . Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2 . Mutagenesis strategies were chosen to minimize transcriptional polar effects . Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity . Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity . Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell . We conclude that in E . coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.

J Bacteriol, 1992 Oct, 174(19), 6238 - 46
The oriT region of the Agrobacterium tumefaciens Ti plasmid pTiC58 shares DNA sequence identity with the transfer origins of RSF1010 and RK2/RP4 and with T-region borders; Cook DM et al.; Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is induced by certain opines secreted from crown galls . On transmissible plasmids, DNA transfer initiates within a cis-acting site, the origin of conjugal transfer, or oriT . We have localized an oriT on the A . tumefaciens plasmid pTiC58 to a region containing the conjugal transfer loci traI and traII and acc, which is the locus encoding catabolism of the two conjugal opines, agrocinopines A and B . The smallest functional oriT clone, a 65-bp BamHI-ApaI fragment in the recombinant plasmid pDCBA60-11, mapped within the traII locus . The nucleotide sequence for a 665-bp KpnI-EcoRI fragment with oriT activity was determined . DNA sequence alignments showed identities between the pTiC58 oriT and the transfer origins of RSF1010, pTF1, and RK2/RP4 and with the pTiC58 T-region borders . The RSF1010-like sequence on pTiC58 is located in the smallest active oriT clone of pTiC58, while the sequence showing identities with the oriT regions of RK2/RP4 and with T-region borders maps outside this region . Despite their sequence similarities, pTiC58 oriT clones were not mobilized by RP4; nor could vectors containing the RK2/RP4 oriT region or the oriT-mob region from RSF1010 be mobilized by pTiC58 . In contrast, other Ti plasmids and a conjugally active Agrobacterium opine catabolic plasmid, pAtK84b, efficiently mobilized pTiC58 oriT clones . In addition, the RSF1010 derivative, pDSK519, was mobilized at moderate frequencies by an Agrobacterium strain harboring only the cryptic plasmid pAtC58 and at very low frequencies by an Agrobacterium host that does not contain any detectable plasmids.

Dev Biol, 1992 Oct, 153(2), 386 - 95
Altered morphology in transgenic tobacco plants that overproduce cytokinins in specific tissues and organs; Li Y et al.; An auxin-inducible bidirectional promoter from the soybean SAUR gene locus was fused to a reporter gene in one direction and a cytokinin biosynthetic gene in the opposite direction and the expression of these fused genes was examined in transgenic tobacco . The Escherichia coli uidA gene, which encodes the enzyme beta-glucuronidase (GUS), was used as the reporter gene and the Agrobacterium tumefaciens ipt gene, which encodes the enzyme isopentenyl transferase, was used as the cytokinin biosynthetic gene . These constructs allowed the overproduction of cytokinins in tobacco in a tissue- and organ-specific manner . Localized overproduction of cytokinins was monitored using the GUS reporter gene and measured by an ELISA assay . The tissue- and organ-specific overproduction of cytokinins produced a number of morphological and physiological changes, including stunting, loss of apical dominance, reduction in root initiation and growth, either acceleration or prolonged delayed senescence in leaves depending on the growth conditions, adventitious shoot formation from unwounded leaf veins and petioles, altered nutrient distribution, and abnormal tissue development in stems . While some of these morphological changes result directly from the localized overproduction of cytokinins, other changes probably result from the mobilization of plant nutrients to tissues rich in cytokinins.

Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9292 - 6
mu-crystallin is a mammalian homologue of Agrobacterium ornithine cyclodeaminase and is expressed in human retina; Kim RY et al.; mu-Crystallin is the major component of the eye lens in several Australian marsupials . The complete sequence of kangaroo mu-crystallin has now been obtained by cDNA cloning . The predicted amino acid sequence shows similarity with ornithine cyclodeaminases encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens . Until now, neither ornithine cyclodeaminase nor any structurally related enzymes have been observed in eukaryotes . RNA analysis of kangaroo tissues shows that mu-crystallin is expressed at high abundance in lens, but outside the lens mu-crystallin is preferentially expressed in neural tissues, retina, and brain . An almost full-length cDNA for mu-crystallin was cloned from human retina . In human tissues, mu-crystallin mRNA is present in neural tissue, muscle, and kidney . This pattern of expression and relationship to an enzyme involved in unusual amino acid metabolism suggests the interesting possibility that mammalian mu-crystallins could be enzymes participating in processes such as osmoregulation or the metabolism of excitatory amino acids.

Genetics, 1992 Oct, 132(2), 553 - 66
Somatic and germinal recombination of a direct repeat in Arabidopsis; Assaad FF et al.; Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis . The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene . This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed . Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings . Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast . Southern analysis showed that recombinants could be formed by either crossing over or gene conversion . A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events . Some evolutionary implications are discussed.

Plant Mol Biol, 1992 Oct, 20(1), 103 - 12
Promoter methylation and progressive transgene inactivation in Arabidopsis; Kilby NJ et al.; Agrobacterium-transformed Arabidopsis plants were generated and the stability of their T-DNA-encoded resistance to kanamycin was examined . Of seven families, each homozygous for a single insertion event, two showed progressive inactivation of resistance over four generations of inbreeding . Loss of resistance was associated with methylation of an Sst II site in the nos promoter of the kanamycin resistance gene . Treatment of plant roots from inactive lines with the demethylating agent 5-azacytidine restored the ability of such lines to form callus on kanamycin-containing media . These observations are consistent with the view that methylation is a factor in the progressive inactivation of transgenes in Arabidopsis.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8759 - 63
A defective replicase gene induces resistance to cucumber mosaic virus in transgenic tobacco plants; Anderson JM et al.; Nicotiana tabacum cv . Turkish Samsun NN plants were transformed with a modified and truncated replicase gene encoded by RNA-2 of cucumber mosaic virus strain Fny . The replicase gene had been modified by deleting a 94-base-pair region spanning nucleotides 1857-1950; the deletion also caused a shift in the open reading frame, resulting in a truncated translation product approximately 75% as large as the full-length protein . Upon transformation via Agrobacterium tumefaciens, transgenic plants were obtained that were resistant to virus disease when challenged with either cucumber mosaic virus virions or RNA at concentrations up to 500 micrograms/ml or 50 micrograms/ml, respectively, the highest concentrations tested . This resistance was absolute, as neither symptoms nor virus could be detected in uninoculated leaves, even after prolonged incubation (120 days after inoculation) . These data suggest, therefore, that such a "replicase-mediated" resistance strategy may be applicable to other plant and animal viruses.

Proc Natl Acad Sci U S A, 1992 Sep 15, 89(18), 8666 - 70
Mechanism of activation of Agrobacterium virulence genes: identification of phenol-binding proteins; Lee K et al.; Agrobacterium tumefaciens initiates the expression of pathogenic genes (vir genes) in response to host-derived phenolic signals through a two-component regulatory system consisting of VirA and VirG . alpha-Bromoacetosyringone (ASBr) was developed as an inhibitor of this induction process and found to be a specific and irreversible inhibitor of vir gene induction in this pathogen . Formal replacement of one of the methoxy groups of ASBr with iodine gave an equally effective inhibitor that could carry an 125I label . We report here that the resulting radiolabeled inhibitor does not react with the sensory component of this system, VirA, either in vivo or in vitro . Rather, two small proteins, p10 and p21, bind labeled inhibitor in vivo in a time period that is consistent with the exposure time required for the inhibition of vir gene expression . Labeling to these proteins was protected by preexposure to ASBr but not by alpha-bromo-3,5-dimethoxyacetophenone, a compound of comparable chemical reactivity but previously shown not to inhibit vir gene expression . Our findings suggest that proteins that are not tumor-inducing plasmid-encoded mediate vir gene activation in a step prior to the VirA/VirG two-component regulatory system.

Biochim Biophys Acta, 1992 Sep 15, 1117(2), 167 - 73
Purification and some properties of L-fucose dehydrogenase from Agrobacterium radiobacter and its application to the assay of bound-fucose in glycoconjugates; Tsuji Y et al.; L-Fucose dehydrogenase was found in the cell extract of Agrobacterium radiobacter and purified to homogeneity about 480-fold with 16% recovery . The molecular weight of the enzyme was approx . 64,000 . The enzyme was active in the neutral pH range, unlike other L-fucose or D-arabinose dehydrogenases which are active only in the alkaline pH range . Using this enzyme and alpha-L-fucosidase F-I of Bacillus circulans (Tsuji, Y., Yamamoto, K., Tochikura, T., Seno, T., Ohkubo, Y . and Yamaguchi, H . (1990) J . Biochem . 107, 324-330) simultaneously, we developed a new coupled enzymatic method in a single buffer system for determining bound-fucose in biological materials . The fucose released by alpha-L-fucosidase F-I was oxidized with L-fucose dehydrogenase in the presence of NAD+, and the NADH formed was measured by absorbance of ultraviolet or utilized to generate color in a reaction involving CuSO4 and neocuproine . Using these methods, bound-fucose in various oligosaccharides and proteins such as lacto-N-fucopentaoses and porcine gastric mucin were quantitated within 15 min.

J Bacteriol, 1992 Sep, 174(18), 5895 - 909
Origin, structure, and regulation of argK, encoding the phaseolotoxin-resistant ornithine carbamoyltransferase in Pseudomonas syringae pv . phaseolicola, and functional expression of argK in transgenic tobacco; Hatziloukas E et al.; Pseudomonas syringae pv . phaseolicola produces the tripeptide N delta(N'-sulfo-diaminophosphinyl)-ornithylalanyl-homoarginin e (phaseolotoxin), which functions as a chlorosis-inducing toxin in the bean halo blight disease by inhibiting ornithine carbamoyltransferase (OCT) . The bacterium possesses duplicate OCT genes, one of which, argK, encodes a toxin-resistant enzyme (ROCT) and imparts resistance to phaseolotoxin . We sequenced the argK gene from strain NPS3121, defined its promoter region, analyzed its regulation, and characterized its transcripts . The gene probably originated from another organism, since it is very distantly related to the argF gene encoding the housekeeping toxin-sensitive OCT and has low G+C content compared with the bacterial genome as a whole and with other protein-coding genes from P . syringae pv . phaseolicola . Optimized alignments of 13 OCT sequences allowed us to define key residues that may be responsible for toxin resistance and to identify a distinct prokaryotic amino acid signature, in ROCT, which argues for a prokaryotic origin of argK . An in-frame fusion of the argK coding region with the chloroplast transit peptide segment of the pea rbcS gene was introduced in Nicotiana tabacum by Agrobacterium-mediated transformation . The presence of an ROCT activity in transgenic plants was demonstrated by in vitro and in vivo assays . Some plants were toxin resistant, suggesting that pathogen-derived resistance to the toxin should be feasible in the pathogen's host.

J Bacteriol, 1992 Sep, 174(17), 5676 - 85
Growth inhibition and loss of virulence in cultures of Agrobacterium tumefaciens treated with acetosyringone; Fortin C et al.; Acetosyringone, a phenolic inducer of the virulence (vir) genes of Agrobacterium tumefaciens, inhibited the growth of the nopaline-type strains T37 and C58 incubated under acidic conditions . In the course of a 6-day incubation with acetosyringone, avirulent clones were produced in different proportions by strains T37 and C58 and also by a spontaneous variant of strain C58, denominated C58F . The proportion of avirulent clones in acetosyringone-treated cultures often exceeded 50% for strains T37 and C58F and was of the order of 1% for strain C58 . Control cultures not exposed to acetosyringone did not yield avirulent clones . Two other vir inducers, sinapinic acid and syringaldehyde, also inhibited growth and promoted accumulation of avirulent clones in cultures of strains C58F and T37 . On the other hand, various acetosyringone analogs reported not to induce the vir genes did not act as growth inhibitors . All of the T37 and most of the C58F avirulent clones examined still carried a Ti plasmid . In all instances examined, avirulent clones still carrying a Ti plasmid were mutated in this plasmid . Mutants of strain C58F lacked the capacity to induce a virB::lacZ fusion in the presence of acetosyringone.

Plant Mol Biol, 1992 Sep, 19(6), 985 - 1000
AT-rich promoter elements of soybean heat shock gene Gmhsp17.5E bind two distinct sets of nuclear proteins in vitro; Czarnecka E et al.; A 33 bp double-stranded oligonucleotide homologous to two AT-rich sequences located upstream (-907 to -889 and -843 to -826) to the start of transcription of heat shock gene Gmhsp17.5E of soybean stimulated transcription when placed 5' to a truncated (-140) maize Adh1 promoter . The chimeric promoter was assayed in vivo utilizing anaerobically stressed sunflower tumors transformed by a pTi-based vector of Agrobacterium tumefaciens . Nuclear proteins extracted from soybean plumules were shown to bind double-stranded oligonucleotides homologous to AT-rich sequences in the 5' flanking regions of soybean beta-conglycinin, lectin, leghemoglobin and heat shock genes . These proteins were also shown to bind AT-rich probes homologous to homeobox protein binding sites from the Antennapedia and engrailed/fushi tarazu genes of Drosophila . Binding activity specific for AT-rich sequences showed a wide distribution among various plant organs and species . Preliminary characterization indicated that two sets of nuclear proteins from soybean bind AT-rich DNA sequences: a diverse high-molecular-weight (ca . 46-69 kDa) group, and a low-molecular-weight (23 and 32 kDa) group of proteins . The latter meets the operational criteria for high-mobility group proteins (HMGs).

Plant Mol Biol, 1992 Sep, 19(6), 951 - 8
Constitutive expression of the beta-phaseolin gene in different tissues of transgenic alfalfa does not ensure phaseolin accumulation in non-seed tissue; Bagga S et al.; Phaseolin is a glycoprotein that constitutes the major storage protein in bean seeds . The phaseolin gene promoters function in a seed-specific manner . In an attempt to understand if events following transcription of the gene also contribute to the seed-specific accumulation of the phaseolin protein, we studied the effect of substituting the constitutive CaMV-35S promoter for the beta-phaseolin gene promoter on expression of the phaseolin gene in different plant organs . A chimeric gene consisting of the 35S promoter, the coding sequence of the beta-phaseolin gene (all five introns and six exons) and the 3'-flanking region of the beta-phaseolin gene, was introduced into alfalfa via Agrobacterium tumefaciens . While all organs examined shared high levels of phaseolin transcripts, the only organ that showed significant accumulation of the phaseolin protein were the mature seeds . Co-migration of the major immunoreactive polypeptides from the non-seed organs with the authentic beta-phaseolin polypeptides on SDS-PAGE indicates that the protein in non-seed organs undergoes correct post-translational processing and modification, but are more unstable in a non-seed environment.

Plant Mol Biol, 1992 Sep, 19(6), 1019 - 30
Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells; Mozo T et al.; Different factors involved in the early steps of the T-DNA transfer process were studied by using a beta-glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments . The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants . The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes . It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants . The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid . Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs . In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time . A model explaining these results is presented.

Appl Environ Microbiol, 1992 Sep, 58(9), 2717 - 22
Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction; Picard C et al.; In order to develop a rapid and specific detection test for bacteria in soil, we improved a method based on the polymerase chain reaction (PCR) . Each step of the protocol, including direct lysis of cells, DNA purification, and PCR amplification, was optimized . To increase the efficiency of lysis, a step particularly critical for some microorganisms which resist classical techniques, we used small soil samples (100 mg) and various lytic treatments, including sonication, microwave heating, and thermal shocks . Purification of nucleic acids was achieved by passage through up to three Elutip d columns . Finally, PCR amplifications were optimized via biphasic protocols using booster conditions, lower denaturation temperatures, and addition of formamide . Two microorganisms were used as models: Agrobacterium tumefaciens, which is naturally absent from the soil used and was inoculated to calibrate the validity of the protocol, and Frankia spp., an actinomycete indigenous to the soil used . Specific primers were characterized either in the plasmid-borne vir genes for A . tumefaciens or in the variable regions of the 16S ribosomal gene for Frankia spp . Specific detection of the inoculated A . tumefaciens strain was routinely obtained when inocula ranged from 10(7) to 10(3) cells . Moreover, the strong correlation we observed between the size of the inocula and the results of the PCR reactions permitted assessment of the validity of the protocol in enumerating the number of microbial cells present in a soil sample . This allowed us to estimate the indigenous population of Frankia spp . at 0.2 x 10(5) genomes (i.e., amplifiable target sequences) per g of soil.

Anticancer Res, 1992 Sep-Oct, 12(5), 1667 - 70
Inhibition of tumor induction in tobacco by Agrobacterium tumefaciens and nodulation induced by Rhizobium meliloti in the presence of phenothiazines and structurally related compounds; Szabo M et al.; Plasmids of Agrobacterium tumefaciens and Rhizobium meliloti carrying Kanamycin resistance genes were eliminated from 1.4 to 0.2% of the growing bacterial cultures by promethazine and imipramine . As a result of plasmid elimination, the A . tumefaciens plasmidless isolate was not able to induce crown gall tumor on tobacco plants . The plasmidless R . meliloti strain failed to induce nodule formation on alfalfa plants . The efficiency of nodulation was decreased when the bacteria were grown in the presence of the drugs . The antiplasmid effects of the drugs were not prevented by opines, (nopaline and octopine) in Escherichia coli F'lac cells.

Plasmid, 1992 Sep, 28(2), 146 - 56
Physical map of the vitopine Ti plasmid pTiS4; Gerard JC et al.; Within the Agrobacterium vitis group the vitopine strains represent a special subclass . Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes . The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped . Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHRI identified several regions of homology . The origin of replication was localized to within 2.5 kb.

Mol Microbiol, 1992 Sep, 6(17), 2453 - 60
Isolation and characterization of the aadA aminoglycoside-resistance gene from Salmonella choleraesuis; Leung KY et al.; The streptomycin- and spectinomycin-resistance gene of Salmonella choleraesuis was cloned and its nucleotide sequence determined . The gene is 789 bases long, encoding a protein of a predicted size of 29,353 Da . The gene product inactivated streptomycin and spectinomycin by an adenylation modification . It is homologous (c . 40% total identity) to streptomycin adenylyltransferase, a 3'(9)-O-nucleotidyltransferase (AAD(3')(9)), which is encoded by the aadA gene in Escherichia coli, Agrobacterium tumefaciens, Klebsiella pneumonia, and Serratia marcescens . The AadA protein of S . choleraesuis differs significantly from the other AadA proteins, indicating that it may have diverged from the other members of this family earlier in evolution . Southern hybridization analysis revealed that homologous aadA sequences were also present in other streptomycin-resistant Salmonella species.

J Bacteriol, 1992 Sep, 174(18), 5999 - 6003
Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells; Wagner VT et al.; Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors . Attachment of the bacteria to plant host cells is required for tumor formation . Human vitronectin and antivitronectin antibodies both inhibited the binding of A . tumefaciens to carrot cells . Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin . The binding of biotype 1 A . tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength . Detergent extraction of carrot cells removed the receptor to which the bacteria bind . The extract was found to contain a vitronectin-like protein . These results suggest that A . tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.

Mol Gen Mikrobiol Virusol, 1992 Sep-Oct, (9-10), 8 - 10
{Plasmid P85 from Azospirillum brasilense SP245: study of the circle of possible hosts and incompatibility with plasmids from Azospirillum brasilense SP7}; Katsy EI; The possibility of the stable inheritance of the plasmid p85 mobilized derivatives from Azospirillum brasilense Sp245 in the cells of the bacterial genera Rizobiaceae (Agrobacterium tumfaciens) and Pseudomonadaceae (Pseudomonas putida) has been shown . The plasmid p85 participates in coding for the physiologically active products (the plant hormones) . It is not inherited by the Escherichia coli strains . For the first time the incompatibility of azospirillium plasmids has been demonstrated on the example of the plasmid p85 from Azospirillum brasilense Sp245 and the plasmid p115 from Azospirillum brasilense Sp7.

Gene, 1992 Aug 15, 117(2), 161 - 7
Vectors for plant transformation and cosmid libraries; Ma H et al.; A series of vectors has been constructed for the purpose of introducing cloned DNAs into plant genomes, using Agrobacterium tumefaciens-mediated transformation methods . One of these vectors, pCIT20, is a plasmid that contains a multiple cloning site (MCS), and a marker (Hph) that confers hygromycin resistance to plant cells . The others are all cosmid vectors which allow insertion of up to 46 kb of plant genomic DNA, and which also contain all of the necessary sequences for A . tumefaciens-mediated plant transformation . The cosmid vectors either contain a Hph marker (pCIT30), or a kanamycin-resistance marker (pCIT101-104) . Three of the cosmid vectors (pCIT30, pCIT101, and pCIT103) carry bacteriophage T7 and SP6 promoters flanking the cloning Bg/II site, for synthesis of end-specific RNAs . The end-specific RNAs may be used as probes when labeled with radioactive or biotinylated nucleotides, for example, in a chromosome-walking experiment . The other two cosmid vectors (pCIT102 and pCIT104) carry restriction sites flanking the insertion site (XhoI) for convenient release of the insert by restriction digests . These sites, in combination with sites internal to the insert, allow the generation of end fragments for subcloning or labeling probes . These vectors should be valuable for isolation and analysis of plant genes, using transformation, library screening, and chromosome-walking approaches.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7442 - 6
The T-DNA-linked VirD2 protein contains two distinct functional nuclear localization signals; Tinland B et al.; Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece of DNA, called T-DNA, into the nucleus of the plant cell . The virulence protein VirD2 of A . tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome . Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells . One signal is located in the N-terminal part of the protein and resembles a single-cluster-type nuclear localization signal . The second signal is near the C terminus and is a bipartite-type nuclear localization signal . The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.

J Bacteriol, 1992 Aug, 174(15), 5161 - 4
The Agrobacterium tumefaciens virD3 gene is not essential for tumorigenicity on plants; Vogel AM et al.; Genetic studies indicate that three of the four polypeptides encoded within the virD operon of the Agrobacterium tumefaciens Ti plasmid are essential for virulence . In order to determine whether the fourth polypeptide, VirD3, has any role in virulence, complementation analysis was used . An A . tumefaciens strain, A348 delta D, which lacked the entire virD operon in the Ti plasmid pTiA6, was constructed . Plasmids containing defined regions of the virD operon were introduced into this strain, and virulence was tested by the strains' abilities to form tumors on Kalanchoe leaves, tomato stems, and potato tubers . As expected, deletion of the virD operon led to an avirulent phenotype . The virulence of this strain could be restored by providing virD1, virD2, and virD4 in trans . No requirement for virD3 in tumor formation was observed in these assays.

Biol Chem Hoppe Seyler, 1992 Aug, 373(8), 699 - 705
Microbial metabolism of quinoline and related compounds . XV . Quinoline-4-carboxylic acid oxidoreductase from Agrobacterium spec.1B: a molybdenum-containing enzyme; Bauer G et al.; The quinoline-4-carboxylic acid oxidoreductase from Agrobacterium spec.1B was purified 84-fold to apparent homogeneity with 15% recovery, using ammonium sulphate precipitation, heat precipitation, hydrophobic interaction, anion exchange- and gel chromatography . The molecular mass of the native enzyme was estimated to be 320 kDa by gel filtration . SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to 85, 35 and 21 kDa . Per molecule the enzyme contains 8 atoms of iron, 8 atoms of acid-labile sulphur, 2 atoms of molybdenum, 2 molecules of FAD and as molybdenum cofactor, molybdopterin cytosine dinucleotide . Besides quinoline-4-carboxylic acid the enzyme also catalysed the conversion of quinoline, 4-chloroquinoline and 4-methylquinoline to the corresponding 2-oxo-1,2-dihydroderivatives . Cyanide, methanol, 4-chloromercuribenzoate and acriflavin were effective inhibitors.

Plant Mol Biol, 1992 Aug, 19(5), 837 - 46
Functional analysis of the promoter region of a nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L; Shen WJ et al.; The 5'-flanking region of gln-gamma, the nodule-enhanced glutamine synthetase gene from Phaseolus vulgaris L., has been analysed for cis-regulatory elements using a series of 5' deletions and hybrid gln-gamma:: CaMV 35S promoters . The promoters were fused to the uidA reporter gene and their activities tested in two heterologous expression systems . In the first system, the chimaeric genes were transferred to Lotus corniculatus L . using Agrobacterium rhizogenes and their expression was studied in nodulated hairy roots . In the second system, the constructs were electroporated into tobacco mesophyll protoplasts . The results of the 5' deletion analysis showed that the sequence between -597 and -21 (relative to the ATG codon) was sufficient for nodule-specific expression of the chimaeric gene in nodulated hairy roots, and revealed the existence of at least two positive regulatory elements . Sequences located between -2000 and -597 were able to stimulate expression in nodules but not protoplasts, while the region from -597 to -354 enhanced expression in both nodules and protoplasts . Results obtained with the hybrid gln-gamma::35S promoters showed that two overlapping restriction fragments (-516/-343 and -474/-293) were able to stimulate expression from a heterologous promoter in an orientation-dependent manner . Previous work has demonstrated the presence of conserved A/T-rich binding sites for nuclear proteins in the region between -516 and -446, and their possible role in regulating gln-gamma expression is discussed.

Plant Mol Biol, 1992 Aug, 19(5), 825 - 36
Expression in transgenic tobacco of the bacterial neomycin phosphotransferase gene modified by intron insertions of various sizes; Paszkowski J et al.; A plant selectable marker gene consisting of cauliflower mosaic virus expression signals and the protein-coding sequence of bacterial neomycin phosphotransferase was modified by insertion of an intron sequence from a storage protein gene, phaseolin . Correct and efficient splicing of the resulting mosaic RNA was observed in transgenic tobacco plants . The insertion of various linkers or gradual increase of intron size by addition in both orientations of internal intron sequences from another plant gene (parsley, 4-coumarate ligase) had little or no effect on the precision of slicing . The gene activity measured by selectability assay in the protoplast transformation showed that only introns enlarged to 1161 bases and longer caused decreased selectability . The suitability of such mosaic marker genes for studies of RNA splicing, DNA recombination and early events after infection of plants with Agrobacterium is discussed.

Carbohydr Res, 1992 Jul 2, 231, 137 - 46
Cyclic (1----2)-beta-D-glucans (cyclosophorans) produced by Agrobacterium and Rhizobium species; Hisamatsu M; Neutral and acidic cyclic (1----2)-beta-D-glucans (cyclosophorans), obtained from culture filtrates and cells of Agrobacterium and Rhizobiun, are synthesised on the cell surface and then secreted . Eight cyclosophorans with dp 17-24 were isolated; all of the strains of Agrobacterium showed almost the same distribution pattern, whereas there were three other distribution patterns for the strains of Rhizobium.

Mol Microbiol, 1992 Jul, 6(13), 1755 - 68
Molecular analysis of the lac operon encoding the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter; Williams SG et al.; The genes coding for the binding-protein-dependent lactose transport system and beta-galactosidase in Agrobacterium radiobacter strain AR50 were cloned and partially sequenced . A novel lac operon was identified which contains genes coding for a lactose-binding protein (lacE), two integral membrane proteins (lacF and lacG), an ATP-binding protein (lacK) and beta-galactosidase (lacZ) . The operon is transcribed in the order lacEFGZK . The operon is controlled by an upstream regulatory region containing putative -35 and -10 promoter sites, an operator site, a CRP-binding site probably mediating catabolite repression by glucose and galactose, and a regulatory gene (lacl) encoding a repressor protein which mediates induction by lactose and other galactosides in wild-type A . radiobacter (but not in strain AR50, thus allowing constitutive expression of the lac operon) . The derived amino acid sequences of the gene products indicate marked similarities with other binding-protein-dependent transport systems in bacteria.

Mol Plant Microbe Interact, 1992 Jul-Aug, 5(4), 318 - 21
Expression of a viral avirulence gene in transgenic plants is sufficient to induce the hypersensitive defense reaction; Pfitzner UM et al.; Tobacco plants containing the N' resistance gene exhibit a hypersensitive defense reaction when infected with tomato mosaic virus (ToMV); infection results in necrotic lesions at the primary infection sites . In an attempt to investigate the molecular mechanism(s) underlying this plant-pathogen interaction, the ToMV coat protein gene was joined by a transcriptional fusion to the strong constitutive 35S RNA promoter from cauliflower mosaic virus . This chimeric gene was introduced via Agrobacterium-mediated transformation into isogenic tobacco cultivars differing only with respect to the N' gene . Strong necrotic reactions were observed on most emerging calli of the N' genotype, but never on calli lacking the N' resistance gene . These data indicate that the coat protein of ToMV is, on its own, sufficient to induce a hypersensitive reaction in tobacco . Thus, recognition of a single viral gene product may be the only prerequisite for the induction of a specific defense reaction in higher plants.

Mol Microbiol, 1992 Jul, 6(14), 1981 - 94
Transcription and autoregulation of the stabilizing functions of broad-host-range plasmid RK2 in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa; Davis TL et al.; The broad-host-range plasmid RK2 has been shown to encode several proteins important for its maintenance within bacterial populations of a number of Gram-negative bacteria . Their genes are organized into two operons: parCBA and parD . These operons have been proposed to be transcribed from two divergent promoters, p-parCBA and p-parD, located within a sequence of approximately 150 bases . In this report we identify and characterize the sequences required for regulated transcription from these promoters in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa . Both of these promoters are repressed by their own gene products in the same manner in all three bacteria tested, with ParA functioning as the primary repressor of p-parCBA and ParD functioning as the repressor of p-parD . The binding regions of these proteins were determined through deletion analyses, DNA mobility shift assays, and an examination of the effect of mutations in this region . Based on these observations, the ParA protein appears to bind to either two inverted repeat or two direct repeat sequences, one downstream from the transcriptional initiation site and the other upstream of the p-parCBA -35 box . The ParD protein appears to bind to one inverted repeat sequence, located between the -35 and -10 boxes of p-parD.

Plant J, 1992 Jul, 2(4), 571 - 81
Expression of E . coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning; Sonnewald U; Transgenic plants were constructed expressing a novel cytosolic inorganic pyrophosphatase in order to reduce the cytosolic pyrophosphate content . To this end the Escherichia coli gene ppa encoding inorganic pyrophosphatase was cloned between the 35S CaMV promoter and the poly(A) site of the octopine synthase gene and transferred into tobacco and potato plants by Agrobacterium-mediated gene transfer . Regenerated plants were tested for the expression of the ppa gene by Northern blots and activity gels . Plants expressing active inorganic pyrophosphatase showed a dramatic change in photoassimilate partitioning . In both transgenic tobacco and potato plants the ratio between soluble sugars and starch was increased by about 3-4-fold in source leaves as compared with the wild-type . However, whereas source leaves of transgenic tobacco plants accumulated much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold) and starch (up to 8-fold) this was not observed in potato plants where the change in assimilate partitioning in source leaves was due to an increase of about 2-fold in sucrose and a reduction in starch content . Expression of the cytosolic inorganic pyrophosphatase in tobacco results in stunted growth of vegetatively growing plants due to a reduced internode distance . Upon flowering the transgenic plants increase their growth rate, reaching almost the same height as control plants at the end of the growth period . Old source leaves accumulate up to 100-fold more soluble sugars than control leaves . This increase in soluble sugars is accompanied by a reduction in chlorophyll content (up to 85%) . Transgenic potato plants showed a less dramatic change in their growth behaviour . Plants were slightly reduced in size, with stems more highly branched . Tuber number increased 2-3-fold, but tuber weight was lower resulting in no net increase in fresh weight.

J Gen Microbiol, 1992 Jul, 138 ( Pt 7), 1527 - 33
Inhibition of lipopolysaccharide synthesis in Agrobacterium tumefaciens and Aeromonas salmonicida; Goldman RC et al.; Lipopolysaccharide (LPS) synthesis was inhibited, new lipid A metabolites accumulated, and growth ceased, when the plant pathogen Agrobacterium tumefaciens and the fish pathogen Aeromonas salmonicida were treated with an antibacterial agent which specifically inhibits CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthase) . The new lipid A metabolites were purified by chromatography on DEAE-cellulose and chemically analysed . Metabolites isolated from both bacterial species contained glucosamine and phosphate in a 1:1 molar ratio, and 3-OH-C14:0 was the major fatty acid present (1 mol and 1.4 mol per mol glucosamine for A . tumefaciens and A . salmonicida, respectively) . Inhibition of LPS synthesis by CMP-KDO synthase inhibitor had no effect on the initial kinetics of A . tumefaciens attachment to cultured carrot cells, but did inhibit cell aggregation normally induced by bacterial cellulose synthesis . Bacteria treated with inhibitor remained viable and able to synthesize protein at 15% the rate of control cells, indicating that the lack of cellulose-induced aggregation was not due to the inability of bacteria to make protein, but rather the inability to respond normally to the bacterial-plant cell interaction.

Science, 1992 Jun 26, 256(5065), 1802 - 5
Nuclear localization of Agrobacterium VirE2 protein in plant cells; Citovsky V et al.; The Agrobacterium single-stranded DNA (ssDNA) intermediate T-strand is likely transferred to the plant cell nucleus as a complex with a single VirD2 molecule at its 5' end and multiple VirE2 molecules along its length . VirD2 contains a nuclear localization signal (NLS); however, because the T-strand is principally coated with VirE2 molecules, VirE2 also might assist in nuclear uptake . Indeed, VirE2 fused to a reporter protein localizes to plant cell nuclei, a process mediated by two amino acid sequences with homology to the bipartite NLS of Xenopus nucleoplasmin . Moreover, tumorigenicity of an avirulent virE2 mutant is restored when inoculated on transgenic plants expressing VirE2, supporting in planta function of VirE2.

FEMS Microbiol Lett, 1992 Jun 15, 72(3), 261 - 8
Transconjugants of Agrobacterium radiobacter harbouring sym genes of Rhizobium galegae can form an effective symbiosis with Medicago sativa; Novikova N et al.; It is known that the Rhizobium galegae genomes contain megaplasmids . The suicide vector pSUP2111 with nifH gene of R . meliloti was introduced into the strains CIAM 0703 and CIAM 0711 of R . galegae inducing effective nodules on Galega orientalis plants . The formation of self-transmissible megaplasmids was observed . The megaplasmid transfer into non-nodulating R . meliloti mutants resulted in partial complementation of the nodulation defect in recipient strains though only one transconjugant showed the nitrogen-fixing activity in symbiosis with alfalfa and another one in symbiosis with G . orientalis plants . Among the Agrobacterium strains harbouring R . galegae megaplasmids there were four classes of transconjugants: (1) Nod+ Fix- in symbiosis with goat's rue plants (three strains); (2) Nod+ Fix- on Medicago sativa (two strains); (3) Nod+ Fix+ on M . sativa (five strains); (4) Nod- with both plant hosts (11 strains).

Protein Eng, 1992 Jun, 5(4), 361 - 5
Cellulose-binding domains: potential for purification of complex proteins; Greenwood JM et al.; The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively . The gene fragment encoding the CBD can be fused to the gene of a protein of interest . Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner . Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp . are dimeric proteins . The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners . Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g . CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA . CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.

Plant Mol Biol, 1992 Jun, 19(3), 421 - 32
Molecular basis for novel root phenotypes induced by Agrobacterium rhizogenes A4 on cucumber; Amselem J et al.; We have used the wild-type Agrobacterium rhizogenes strain A4 to induce roots on cucumber stem explants . Cultures of transformed roots obtained that were capable of hormone-autonomous growth could be grouped in three phenotypic classes . Of particular interest were extremely thick roots of a type not previously described . Characterization of the transferred DNA and of the expression of the corresponding genes allowed us to determine that the genes rolABC of the TL region of the Ri plasmid are sufficient to induce thin roots similar to those observed in other species, while the aux genes of the TR region are sufficient to induce thick roots . Among clones bearing the aux genes, there was a correlation between level of expression of aux2 and root phenotype.

Appl Environ Microbiol, 1992 Jun, 58(6), 1878 - 85
Hyperreiterated DNA regions are conserved among Bradyrhizobium japonicum serocluster 123 strains; Rodriguez-Quinones F et al.; We have identified and cloned two DNA regions which are highly reiterated in Bradyrhizobium japonicum serocluster 123 strains . While one of the reiterated DNA regions, pFR2503, is closely linked to the B . japonicum common and genotype-specific nodulation genes in strain USDA 424, the other, pMAP9, is located next to a Tn5 insertion site in a host-range extension mutant of B . japonicum USDA 438 . The DNA cloned in pFR2503 and pMAP9 are reiterated 18 to 21 times, respectively, in the genomes of B . japonicum serocluster 123 strains . Gene probes from the reiterated regions share sequence homology, failed to hybridize (or hybridized poorly) to genomic DNA from other B . japonicum and Bradyrhizobium spp . strains, and did not hybridize to DNA from Rhizobium meliloti, Rhizobium fredii, Rhizobium leguminosarum biovars trifolii, phaseoli, and viceae, or Agrobacterium tumefacians . The restriction fragment length polymorphism hybridization profiles obtained by using these gene probes are useful for discriminating among serologically related B . japonicum serocluster 123 strains.

J Bacteriol, 1992 Jun, 174(12), 4169 - 74
Constitutive mutations of Agrobacterium tumefaciens transcriptional activator virG; Pazour GJ et al.; The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmids are positively regulated by virG in conjunction with virA and plant-derived inducing molecules . A procedure that utilizes both genetic selection and a genetic screen was developed to isolate mutations in virG that led to elevated levels of vir gene expression in the absence of virA and plant phenolic inducers . Mutants were isolated at a frequency of 1 in 10(7) to 10(8) . Substitution mutations at two positions in the virG coding region were found to result in the desired phenotype . One mutant had an asparagine-to-aspartic acid substitution at residue 54, and the other contained an isoleucine-to-leucine substitution at residue 106 . In both cases, the mutant phenotype required the presence of the active-site aspartic acid residue at position 52 . Further analysis showed that no other substitution at residue 54 resulted in a constitutive phenotype . In contrast, several substitutions at residue 106 led to a constitutive phenotype . The possible roles of the residues at positions 54 and 106 in VirG function are discussed.

J Bacteriol, 1992 Jun, 174(12), 4110 - 9
Analysis of mutations in trfA, the replication initiation gene of the broad-host-range plasmid RK2; Lin J et al.; Plasmids with mutations in trfA, the gene encoding the replication initiation protein of the broad-host-range plasmid RK2, were isolated and characterized . Mutants identified from a nitrosoguanidine bank were defective in supporting the replication of a wild-type RK2 origin in Escherichia coli . Most of the mutations were clustered in a region of trfA corresponding to the carboxy-terminal quarter of the TrfA protein . 5' and 3' deletion mutants of trfA were also constructed . A C-terminal deletion of three amino acids of the Tr A protein was completely nonfunctional for RK2 replication . However, a deletion of 25 amino acids from the start of the 33-kDa TrfA protein was still competent for replication . Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudomonas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii . Selected mutant TrfA proteins were partially purified and characterized in vitro . Velocity sedimentation analysis of these partially purified TrfA proteins indicated that the wild-type protein and all mutant TrfA proteins examined exist as dimers in solution . Results from in vitro replication assays corroborated the experimental findings in vivo . Gel retardation results clearly indicated that the point mutant TrfA-33:151S, which was completely defective in replication of an RK2 origin in all of the bacterial hosts tested in vivo, and a carboxy-terminal deletion mutant, TrfA-33:C delta 305, were not able to bind iterons in vitro . In addition to the partially defective or could not be distinguished from the wild-type protein in binding to the origin region . The mutant proteins with apparently normal DNA-binding activity in vitro either were inactive in all four gram-negative bacteria tested or exhibited differences in functionality depending on the host organism . These mutant TrfA proteins may be altered in the ability to interact with the replication proteins of the specific host bacterium.

Appl Environ Microbiol, 1992 Jun, 58(6), 2099 - 101
Identification of Brucella spp . by using the polymerase chain reaction; Herman L et al.; The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp . The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens.

Microb Releases, 1992 Jun, 1(1), 29 - 34
Identification of closely related Agrobacterium vitis isolates by chromosomal DNA probes; Otten L et al.; Crown gall formation on grapevine by Agrobacterium vitis is an important plant disease in many regions of the world . On grapevine, octopine/cucumopine (o/c) strains are widely distributed . Here we describe two chromosomal sequences of o/c strains of 15 and 20 kb, which reveal a high degree of polymorphism in different o/c isolates . Part of the polymorphism is due to the presence or absence of a 2.2-kb-long repeated sequence, a homolog of which is also found on the octopine Ti plasmid of Ach5, immediately to the left of the left TL-region border . The occurrence and distribution of this repeat in different o/c isolates make it possible to reconstruct the evolution of these strains . The chromosomal DNA sequences outside the repeats also differ in various isolates . The two probes described here can be used to identify strains from a collection, classify new isolates, or trace a given isolate under experimental release conditions.

Plant Mol Biol, 1992 Jun, 19(3), 485 - 90
Transformation of cauliflower (Brassica oleracea L . var . botrytis)--an experimental survey; Eimert K et al.; The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation) . Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer . Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer . Expression as well as incorporation of the NPTII sequence could be demonstrated.

Cell, 1992 May 15, 69(4), 659 - 67
The A . tumefaciens transcriptional activator OccR causes a bend at a target promoter, which is partially relaxed by a plant tumor metabolite; Wang L et al.; Octopine is released from crown gall tumors as a nutrient source and a signal molecule for the plant pathogen Agrobacterium tumefaciens . Some or all octopine-inducible genes are regulated by a protein called OccR . Primer extension analysis showed that OccR protein represses the occR gene and both represses and activates the occQ operon, which is divergently transcribed from occR . These promoters initiate transcription 46 bp apart . This regulatory system was reconstituted in vitro using purified OccR protein and Escherichia coli RNA polymerase . OccR binds with high affinity to a single site overlapping these promoters . Octopine shortens the DNAase I footprint of OccR and increases the gel mobility of OccR-DNA complexes by relaxing an OccR-incited DNA bend.

Gene, 1992 May 15, 114(2), 229 - 33
Sequence analysis of an insertion element, IS1131, isolated from the nopaline-type Ti plasmid of Agrobacterium tumefaciens; Wabiko H; Transferred DNA (T-DNA) from several nopaline-type Ti plasmids of Agrobacterium tumefaciens was previously shown to be variable in size due to the insertion of extra DNA segments . We found an insertion sequence (named IS1131) in the T-DNA of the strain PO22 and determined the nucleotide (nt) sequence . IS1131 is 2773 bp long, contains four open reading frames, and is flanked by 12-bp perfect inverted repeats (IR) . An 8-bp direct repeat was found immediately outside the IR, and represents a target site of integration . Although the IS1131 nt sequence showed a limited degree of similarity to those of the previously reported IS elements of A . tumefaciens and to the central region of T-DNA, the terminal IR of IS1131 were highly homologous (up to 83%) to those of IS66 and IS866, suggesting that these three IS elements are related to each other . A number of IS1131-related copies were found in several pathogenic, as well as nonpathogenic, nopaline-type strains from different plant sources, and were distributed on both chromosomes and plasmids.

Nature, 1992 May 14, 357(6374), 173 - 6
Self-splicing introns in tRNA genes of widely divergent bacteria; Reinhold-Hurek B et al.; The organization of eukaryotic genes into exons separated by introns has been considered as a primordial arrangement but because it does not exist in eubacterial genomes it may be that introns are relatively recent acquisitions . A self-splicing group I intron has been found in cyanobacteria at the same position of the same gene (that encoding leucyl transfer RNA, UAA anticodon) as a similar group I intron of chloroplasts, which indicates that this intron predates the invasion of eukaryotic cells by cyanobacterial endosymbionts . But it is not clear from this isolated example whether introns are more generally present in different genes or in more diverse branches of the eubacteria . Many mitochondria have intron-rich genomes and were probably derived from the alpha subgroup of the purple bacteria (or Proteobacteria), so ancient introns might also have been retained in these bacteria . We describe here the discovery of two small (237 and 205 nucleotides) self-splicing group I introns in members of two proteobacterial subgroups, Agrobacterium tumefaciens (alpha) and Azoarcus sp . (beta) . The introns are inserted in genes for tRNA(Arg) and tRNA(Ile), respectively, after the third anticodon nucleotide . Their occurrence in different genes of phylogenetically diverse bacteria indicates that group I introns have a widespread distribution among eubacteria.

Appl Environ Microbiol, 1992 May, 58(5), 1592 - 600
Heavy metals alter the electrokinetic properties of bacteria, yeasts, and clay minerals; Collins YE et al.; The electrokinetic patterns of four bacterial species (Bacillus subtilis, Bacillus megaterium, Pseudomonas aeruginosa, and Agrobacterium radiobacter), two yeasts (Saccharomyces cerevisiae and Candida albicans), and two clay minerals (montmorillonite and kaolinite) in the presence of the chloride salts of the heavy metals, Cd, Cr, Cu, Hg, Ni, Pb, and Zn, and of Na and Mg were determined by microelectrophoresis . The cells and kaolinite were net negatively charged at pH values above their isoelectric points (pI) in the presence of Na, Mg, Hg, and Pb at an ionic strength (mu) of 3 x 10(-4); montmorillonite has no pI and was net negatively charged at all pH values in the presence of these metals . However, the charge of some bacteria, S . cerevisiae, and kaolinite changed to a net positive charge (charge reversal) in the presence of Cd, Cr, Cu, Ni, and Zn at pH values above 5.0 (the pH at which charge reversal occurred differed with the metal) and then, at higher pH values, again became negative . The charge of the bacteria and S . cerevisiae also reversed in solutions of Cu and Ni with a mu of greater than 3 x 10(-4), whereas there was no reversal in solutions with a mu of less than 3 x 10(-4) . The clays became net positively charged when the mu of Cu was greater than 3 x 10(-4) and that of Ni was greater than 1.5 x 10(-4) . The charge of the cells and clays also reversed in solutions containing both Mg and Ni or both Cu and Ni (except montmorillonite) but not in solutions containing both Mg and Cu (except kaolinite) (mu = 3 x 10(-4)) . The pIs of the cells in the presence of the heavy metals were at either higher or lower pH values than in the presence of Na and Mg . Exposure of the cells to the various metals at pH values from 2 to 9 for the short times (ca . 10 min) required to measure the electrophoretic mobility did not affect their viability . The specific adsorption on the cells and clays of the hydrolyzed species of some of the heavy metals that formed at higher pH values was probably responsible for the charge reversal . These results suggest that the toxicity of some heavy metals to microorganisms varies with pH because the hydrolyzed speciation forms of these metals, which occur at higher pH values, bind on the cell surface and alter the net charge of the cell.(ABSTRACT TRUNCATED AT 400 WORDS)

J Gen Virol, 1992 May, 73 ( Pt 5), 1041 - 7
The nucleotide sequence of an infectious insect-transmissible clone of the geminivirus Panicum streak virus; Briddon RW et al.; The infectious genome of a Kenyan isolate of Panicum streak virus (PSV) has been cloned and sequenced . Infection of host plants was done using an Agrobacterium binary vector containing a partial repeat of the genome . Progeny virus from resultant infections proved to be transmissible by the leafhopper Cicadulina mbila (Naude) . Comparisons of the amino acid sequences of PSV DNA-encoded proteins with those of previously characterized geminiviruses infecting monocotyledonous plants, including maize streak virus, revealed high levels of identity . The evolutionary relationship between PSV and other geminiviruses infecting monocotyledons is discussed.

Plasmid, 1992 May, 27(3), 191 - 9
Construction of a cassette enabling regulated gene expression in the presence of aromatic hydrocarbons; Keil S et al.; A high-level expression cassette has been constructed from a TOL plasmid derived from Pseudomonas putida carrying all cis- and trans-acting regulatory elements necessary for transcriptional gene activation in the presence of aromatic hydrocarbons such as toluene . Foreign DNA can be inserted at unique KpnI, SacI, and EcoRI sites 7, 13, and 15 nucleotides downstream of a ribosome binding site . The cassette, flanked by BamHI and EcoRI restriction sites, was inserted into a broad-host-range vector and its efficacy demonstrated in various purple bacteria by monitoring the expression of a reporter gene spectrophotometrically and by SDS-PAGE . High-level induction (80- to 600-fold) was detected in Enterobacteriaceae and in Pseudomonas but was absent or low in Agrobacterium tumefaciens and Rhizobium leguminosarum.

Mol Plant Microbe Interact, 1992 May-Jun, 5(3), 228 - 34
Broad host range and promoter selection vectors for bacteria that interact with plants; Van den Eede G et al.; A plasmid vector, pGV910, and a derived cosmid, pRG930, have been constructed . Both contain the ColE1 and pVS1 origins of replication and are stably maintained in Escherichia coli, Agrobacterium tumefaciens, and Azorhizobium caulinodans ORS571 . They are compatible with commonly used IncP cloning vectors, although pVS1 was classified as an IncP plasmid, unable to replicate in E . coli (Y . Itoh, J.M . Watson, D . Haas, and T . Leisinger, Plasmid 11:206-220, 1984) . Promoter selection vectors were derived from both of these plasmids by using a promoterless beta-glucuronidase and/or beta-galactosidase gene . These vectors facilitate the study of gene expression in bacteria under particular environmental conditions . This is illustrated by the expression of the gusA gene under the control of a nod promoter in A . caulinodans nodulating stem-located infection sites on Sesbania rostrata.

Mol Gen Genet, 1992 May, 233(1-2), 53 - 64
Petunia plants escape from negative selection against a transgene by silencing the foreign DNA via methylation; Renckens S et al.; Transgenic Petunia hybrida clones harbouring the T-DNA gene 2 of Agrobacterium tumefaciens were used to test a strategy for the trapping of plant transposable elements . In the Petunia line used, floral variegation is due to the presence of the non-autonomous transposable element dTph1 at the An1 locus . The gene 2 product converts the auxin precursor indole-3-acetamide and its analogue 1-naphthalene acetamide into the active auxins indole-3-acetic acid and 1-naphthalene acetic acid . Plant cells that express gene 2 can use a low concentration of the precursors as auxins and become sensitive to the toxicity of high concentrations of these compounds . By selecting protoplast-derived microcalli or seedlings able to grow on medium with high precursor concentrations, variant plants were obtained in which gene 2 was no longer expressed . Southern analysis, using gene 2-specific probes, revealed that in one variant the T-DNA was deleted . For 30 other variants no alteration in gene 2 structure was observed, indicating that transposable element insertion was not responsible for the inactivation of gene 2 . Analysis with restriction enzymes allowing discrimination between methylated or non-methylated DNA sequences showed that the inactivated gene 2 sequences were methylated . Addition of the in vivo methylation inhibitor 5-azacytidine to the medium led to reactivation of gene 2 expression in some of the variants . These observations demonstrated that reversible DNA methylation was the main cause of silencing of gene 2 in this system.

Mutat Res, 1992 May, 273(3), 271 - 80
Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E . coli DNA repair gene ada; Angelis K et al.; The protein coding region of the E . coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid . Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity . The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants . This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.

Mikrobiol Zh, 1992 May-Jun, 54(3), 40 - 3
{A toxicological evaluation of an Agrobacterium radiobacter-based biological fertilizer}; Omel'ianets TG et al.; Pathogenic properties (for warm-blooded organisms) of the industrial strain Agrobacterium radiobacter (strain 204) and toxicity of biofertilizer on its base--rhizoagrin--have been studied . It is established that the studied microorganisms are avirulent, nontoxic, nontoxicogenic and may be recommended for making biopreparations . The preparation rhizoagrin is not toxic for warm-blooded animals and may be used as an alternative of chemical mineral fertilizers.

J Bacteriol, 1992 May, 174(9), 2891 - 7
The chloramphenicol acetyltransferase gene of Tn2424: a new breed of cat; Parent R et al.; We have sequenced the gene coding for the chloramphenicol acetyltransferase of Tn2424 of plasmid NR79 . This gene codes for a protein of 23,500 Da, and the derived protein sequence is similar to those of the chromosomal chloramphenicol acetyltransferases of Agrobacterium tumefaciens and Pseudomonas aeruginosa and of unidentified open reading frames, which may encode chloramphenicol acetyltransferases, adjacent to the ermG macrolide-lincosamide-streptogramin resistance gene of Bacillus sphaericus and the vgb virginiamycin resistance gene of Staphylococcus aureus . Weaker similarity to the LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) proteins of Escherichia coli and the NodL protein of Rhizobium leguminosarum is also observed . There is no significant similarity to any other chloramphenicol acetyltransferase genes, such as that of Tn9 . The Tn2424 cat gene is part of a 4.5-kb region which also contains the aacA1a aminoglycoside-6'-N-acetyltransferase gene; Tn2424 is similar to Tn21 except for the presence of this region . Sequences flanking the cat gene are typical of those flanking other genes inserted into pVS1-derived "integrons" by a site-specific recombinational mechanism.

Plant Mol Biol, 1992 Apr, 18(6), 1113 - 20
Auxin rapidly down-regulates transcription of the tryptophan decarboxylase gene from Catharanthus roseus; Goddijn OJ et al.; The enzyme tryptophan decarboxylase (TDC) (EC 4.1.1.28) converts tryptophan into tryptamine, and thereby channels primary metabolites into indole alkaloid biosynthesis . The production of these secondary metabolites in suspension cells of Catharanthus roseus depends on medium composition . Of the possible variables, we investigated the effect of hormones on the expression of the tdc gene in cell cultures . Omission of NAA from the growth medium resulted in accumulation of tdc mRNA . The addition of 1-naphthaleneacetic acid (NAA), indoleacetic acid (IAA) or 2,4-dichlorophenoxyacetic acid (2,4-D) rapidly reduced the enhanced tdc transcript level . Cytokinin was unable to suppress the enhanced transcript level . Hairy roots transformed by Agrobacterium rhizogenes also showed a reduction of the tdc mRNA level after NAA addition . Run-off transcription experiments showed that the down-regulation takes place at the transcriptional level within 15 minutes and independent of de novo protein synthesis . Thus one of the mechanisms which control the activity of terpenoid indole alkaloid biosynthesis in C . roseus cell cultures is the negative regulation by auxin of the gene involved in the first committed step.

J Bacteriol, 1992 Apr, 174(8), 2720 - 3
The ntrA gene of Agrobacterium tumefaciens: identification, cloning, and phenotype of a site-directed mutant; Wu ZL et al.; A 3.6-kb EcoRI fragment containing the ntrA gene of Agrobacterium tumefaciens was cloned by using the homologous ntrA gene of Rhizobium meliloti as a probe . Construction of an ntrA mutant of A . tumefaciens by site-directed insertional mutagenesis demonstrated the requirement of the ntrA gene for nitrate utilization and C4-dicarboxylate transport but not for vir gene expression or tumorigenesis.

J Bacteriol, 1992 Apr, 174(8), 2631 - 9
Spontaneous mutation conferring the ability to catabolize mannopine in Agrobacterium tumefaciens; LaPointe G et al.; Two nopaline-type strains of Agrobacterium tumefaciens, C58 and T37, as well as strain A136, which is a Ti plasmid-cured derivative of strain C58, gave rise to spontaneous mutants that were able to grow on mannopine . The observation of mutagenesis with strain A136 demonstrated that the ability to acquire this new catabolic potential was independent of the presence of a Ti plasmid . The mutants were isolated after 4 weeks of incubation on minimal medium containing mannopine as the sole carbon source . They also utilized mannopinic acid, but not agropine or agropinic acid . In addition, the spontaneous mutant LM136, but not its parent strain A136, degraded many mannityl opine analogs . {14C}mannopine disappeared in the presence of LM136 cells which had been pregrown on opine or nonopine substrates . These results suggested that the catabolic system of this mutant was not subject to a stringent regulation . A clone conferring the ability to utilize mannopine on a recipient pseudomonad was selected from a genomic library from both the mutant LM136 and its parent strain . Only the LM136 clone was expressed in the parent Agrobacterium strain A136 . Southern analysis showed that the genes for mannopine catabolism in the spontaneous mutants differed from the corresponding Ti plasmid-encoded genes of octopine-type or agropine-type Agrobacterium strains . Cells of LM136 utilized {14C}mannopine without generating detectable amounts of intracellular agropine . In contrast, a major fraction of the radioactivity recovered from cells of the octopine-type strain Ach5, after incubation on {14C}mannopine, was in the form of agropine.

J Bacteriol, 1992 Apr, 174(7), 2288 - 97
Agrobacterium tumefaciens transfers extremely long T-DNAs by a unidirectional mechanism; Miranda A et al.; During crown gall tumorigenesis, part of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid, the T-DNA, integrates into plant DNA . Direct repeats define the left and right ends of the T-DNA, but tumorigenesis requires only the right-hand repeat . Virulence (vir) genes act in trans to mobilize the T-DNA into plant cells . Transfer of T-DNA begins when the VirD endonuclease cleaves within the right-hand border repeat . Although the T-DNA right-border repeat promotes T-DNA transmission best in its normal orientation, an inverted right border exhibits reduced but significant activity . Two models may account for this diminished tumorigenesis . The right border may function bidirectionally, with strong activity only in its wild-type orientation, or it may promote T-DNA transfer in a unidirectional manner such that, with an inverted right border, transfer proceeds around the entire Ti plasmid before reaching the T-DNA . To determine whether a substantial portion of the Ti plasmid is transferred to plant cells, as predicted by the unidirectional-transfer hypothesis, we examined T-DNAs in tumors induced by strains containing a Ti plasmid with a right border inverted with respect to the T-DNA oncogenes . These tumors contained extremely long T-DNAs corresponding to most or all of the Ti plasmid . To test whether the right border can function bidirectionally, we inserted T-DNAs with either a properly oriented or an inverted right border into a specific site in the A . tumefaciens chromosome . A border situated to transfer the oncogenes first directed T-DNA transfer even from the bacterial chromosome, whereas a border in the opposite (inverted) orientation did not transfer the oncogenes to plant cells . Our results indicate that the right-border repeat functions in a unidirectional manner.

J Bacteriol, 1992 Apr, 174(7), 2215 - 21
Diauxic growth of Agrobacterium tumefaciens 15955 on succinate and mannopine; Nautiyal CS et al.; Diauxic growth was observed upon incubation of Agrobacterium tumefaciens 15955 on a mixture of succinate and mannopine as the carbon source . Diauxic growth was also observed when either fumarate or L-malate was mixed with mannopine . No diauxie was detectable when A . tumefaciens 15955 was grown on a mixture of mannopine and glucose, fructose, sucrose, or L-arabinose . Preferential utilization of succinate was observed in the initial growth phase of diauxie, whereas the final growth phase occurred at the expense of mannopine . Cells harvested during the initial growth phase exhibited a capacity for uptake of {14C}succinate but not of {14C} mannopine . A capacity for {14C}mannopine uptake was expressed during the final growth phase . Extracts from cells grown on a mixture of succinate and mannopine exhibited a low level of mannopine cyclase activity in the initial phase of diauxie . This activity increased substantially in the final phase of growth . Added succinate had no effect on the rate of {14C}mannopine uptake or mannopine cyclase activities of cells previously grown on mannopine . Diauxie was also observed during growth of strain 15955 on a mixture of succinate and octopine.

Virology, 1992 Apr, 187(2), 525 - 33
Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination; Gal S et al.; Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene . The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences . In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants . The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.

Mol Microbiol, 1992 Apr, 6(7), 907 - 20
Conjugative transfer functions of broad-host-range plasmid RK2 are coregulated with vegetative replication; Motallebi-Veshareh M et al.; The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb) . Promoter probe studies indicated that kilB is transcribed clockwise from a region containing closely spaced divergent promoters, one of which is the trfA promoter . The repression of both promoters by korB suggested that kilB may also play a role in stable maintenance of RK2 . We have sequenced the region containing kilB and analysed it by deletion and insertion mutagenesis . Loss of the KilB+ phenotype does not result in decreased stability of mini RK2 plasmids . However insertion in ORFI (kilBI) of the region analysed results in a Tra- phenotype in plasmids which are otherwise competent for transfer, demonstrating that this locus is essential for transfer and is probably the first gene of the Tra2 region . From the kilBI DNA sequence KilBI is predicted to be 34995 Da, in line with M(r) = 36,000 observed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis, and contains a type I ATP-binding motif . The purified product was used to raise antibody which allowed the level of KilBI produced from RK2 to be estimated at approximately 2000 molecules per bacterium . Protein sequence comparisons showed the highest homology score with VirB11, which is essential for the transfer of the Agrobacterium tumefaciens Ti plasmid DNA from bacteria to plant cells . The sequence similarity of both KilBI and VirB11 to a family of protein export functions suggested that KilBI may be involved in assembly of the surface-associated Tra functions . The data presented in this paper provide the first demonstration of coregulation of genes required for vegetative replication and conjugative transfer on a bacterial plasmid.

Carbohydr Res, 1992 Mar 16, 226(1), 131 - 54
Evidence that the acidic polysaccharide secreted by Agrobacterium radiobacter (ATCC 53271) has a seventeen glycosyl-residue repeating unit; O'Neill MA et al.; The extracellular anionic polysaccharide produced by the bacterium Agrobacterium radiobacter (ATCC 53271) contains D-galactose, D-glucose, and pyruvic acid in the molar ratio 2:15:2 . Analysis of the methylated polysaccharide indicated the presence of terminal, non-reducing glucosyl, 3-, 4-, 6-, 2,4-, and 4,6-linked glucosyl residues, 3-linked 4,6-O-{(S)-1-carboxyethylidene}glucosyl residues, and 3-linked galactosyl residues . Partial acid hydrolysis of the methylated polysaccharide, followed by reduction with NaB2H4 and then O-ethylation, gave a mixture of alkylated oligoglycosyl alditols that were separated by reversed-phase h.p.l.c . and analyzed by 1H-n.m.r . spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis . Smith degradation of the polysaccharide gave three diglycosyl alditols that were separated by semi-preparative, high-pH anion-exchange chromatography, and were analyzed by 1H-n.m.r . spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis . The polymer obtained by NaBH4 reduction of the periodate-oxidized polysaccharide was methylated, and the noncyclic acetals were hydrolyzed with aq . 90% formic acid to generate a mixture of partially O-methylated mono- and di-glycosyl alditols . The partially O-methylated oligoglycosyl alditols were O-ethylated . The resulting alkylated oligoglycosyl alditols were separated by reverse-phase h.p.l.c . and then characterized by 1H-n.m.r . spectroscopy, g.l.c.-m.s., and glycosyl-linkage composition analysis . The results from the studies described here provide strong evidence that the acidic polysaccharide secreted by A . radiobacter (ATCC 53271) has a heptadecasaccharide repeating unit.

Plasmid, 1992 Mar, 27(2), 119 - 29
A staphylococcal multidrug resistance gene product is a member of a new protein family; Grinius L et al.; The complete nucleotide sequence (321 bp) of smr (staphylococcal multidrug resistance), a gene coding for efflux-mediated multidrug resistance of Staphylococcus aureus, was determined by using two different plasmids as DNA templates . The smr gene product (identical to products of ebr and qacC/D genes) was shown to be homologous to a new family of small membrane proteins found in Escherichia coli, Pseudomonas aeruginosa, Agrobacterium tumefaciens, and Proteus vulgaris . The smr gene was subcloned and expressed in S . aureus and E . coli and its ability to confer the multidrug resistant phenotype was demonstrated for two different lipophilic cation classes: phosphonium derivatives and quarternary amines . Expression of smr gene leads to the efflux of tetraphenylphosphonium and to a net decrease in the uptake of lipophilic cations . The deduced polypeptide sequence (107 amino acid residues, 11,665 kDa) has 46% hydrophobic residues (Phe, Ile, Leu, and Val) and 20% hydroxylic residues (Ser and Thr) . Four transmembrane segments are predicted for smr gene product . Of the charged amino acid residues, only Glu 13 is located in a transmembrane segment . This Glu 13 is conserved in all members of the family of small membrane proteins . We propose a mechanism whereby exchange of protons at the Glu 13 is a key in the efflux of the lipophilic cation . This mechanism includes the idea that protons are transported to the Glu 13 via an appropriate chain of hydroxylic residues in the transmembrane segments of Smr.

Plant Mol Biol, 1992 Mar, 18(5), 921 - 30
Expression of a chimaeric heat-shock-inducible Agrobacterium 6b oncogene in Nicotiana rustica; Tinland B et al.; The T-6b gene of Agrobacterium tumefaciens strain Tm4 induces tumours on Nicotiana rustica by an as yet unknown mechanism . These tumours cannot be regenerated into normal plants . To study the effect of the T-6b gene product on normal plant cells, the T-6b gene was placed under control of the Drosophila melanogaster hsp70 heat-shock promoter and introduced into N . rustica . Progeny of an hsp70-T-6b transformant developed into normal plants . The inducibility of the hsp70-T-6b construct was shown by northern analysis and by heat-shock-dependent growth alterations on the level of whole seedlings . Upon wounding at normal temperature conditions hsp70-T-6b plants formed small tumours on leaves and stems . Grafts between transformed plants and normal plants led to a wound callus which remained limited to transformed tissues, indicating that the T-6b gene product does not diffuse . Protoplasts of hsp70-T-6b plants divided in the same way as control protoplasts under standard culture conditions . However, when protoplast cultures were started in the absence of hormones, normal cells rapidly lost their sensitivity towards hormones, whereas hsp70-T-6b cells remained sensitive for a significantly longer period . Thus, the T-6b gene product alters hormone sensitivity during the initial phases of protoplast culture.

Microbiol Rev, 1992 Mar, 56(1), 12 - 31
Two-way chemical signaling in Agrobacterium-plant interactions; Winans SC; The discovery in 1977 that Agrobacterium species can transfer a discrete segment of oncogenic DNA (T-DNA) to the genome of host plant cells has stimulated an intense interest in the molecular biology underlying these plant-microbe associations . This attention in turn has resulted in a series of insights about the biology of these organisms that continue to accumulate at an ever-increasing rate . This excitement was due in part to the notion that this unprecedented interkingdom DNA transfer could be exploited to create transgenic plants containing foreign genes of scientific or commercial importance . In the course of these discoveries, Agrobacterium became one of the best available models for studying the molecular interactions between bacteria and higher organisms . One extensively studied aspect of this association concerns the exchange of chemical signals between Agrobacterium spp . and host plants . Agrobacterium spp . can recognize no fewer than five classes of low-molecular-weight compounds released from plants, and other classes probably await discovery . The most widely studied of these are phenolic compounds, which stimulate the transcription of the genes needed for infection . Other compounds include specific monosaccharides and acidic environments which potentiate vir gene induction, acidic polysaccharides which induce one or more chromosomal genes, and a family of compounds called opines which are released from tumorous plant cells to the bacteria as nutrient sources . Agrobacterium spp . in return release a variety of chemical compounds to plants . The best understood is the transferred DNA itself, which contains genes that in various ways upset the balance of phytohormones, ultimately causing neoplastic cell proliferation . In addition to transferring DNA, some Agrobacterium strains directly secrete phytohormones . Finally, at least some strains release a pectinase, which degrades a component of plant cell walls.

J Bacteriol, 1992 Mar, 174(5), 1478 - 86
Cloning and sequencing of an Agrobacterium tumefaciens beta-glucosidase gene involved in modifying a vir-inducing plant signal molecule; Castle LA et al.; Induction of Agrobacterium tumefaciens virulence genes by plant phenolic compounds is essential for successful T-DNA transfer to a host plant . In Douglas fir needles, the major virulence region inducer is the glycoside coniferin (J . W . Morris and R . O . Morris, Proc . Natl . Acad . Sci . USA 87:3612-3618, 1990) . Agrobacterium strains with high beta-glucosidase activity respond to coniferin and infect Douglas fir seedlings, whereas most strains with low beta-glucosidase activity fail to respond to coniferin and are avirulent on this host . We have cloned two beta-glucosidase genes from A . tumefaciens B3/73 and sequenced one of them, cbg1 . It appears to be part of a polycistronic unit and shows a high bias for GC-rich codons . When expressed in Escherichia coli, Cbg1 beta-glucosidase hydrolyzes coniferin but not cellobiose . The 88-kDa predicted product of cbg1 is highly similar to one other bacterial beta-glucosidase and several fungal beta-glucosidases . There is little homology between Cbg1 and other bacterial beta-glucosidases, including an Agrobacterium cellobiase.

J Bacteriol, 1992 Feb, 174(4), 1189 - 96
The Agrobacterium tumefaciens vir gene transcriptional activator virG is transcriptionally induced by acid pH and other stress stimuli; Mantis NJ et al.; A set of Agrobacterium tumefaciens operons required for pathogenesis is coordinately induced during plant infection by the VirA and VirG proteins . The intracellular concentration of VirG increases in response to acidic media, and this response was proposed to be regulated at the level of transcription at a promoter (P2) that resembles the Escherichia coli heat shock promoters . To test this hypothesis, we first constructed a virG-lacZ transcriptional fusion . A strain containing this fusion had higher levels of beta-galactosidase activity in acidic media than in media at neutral pH . Second, primer extension analysis of virG indicated that acidic media stimulated the transcription of this promoter . To determine whether P2 is a member of a heat shock-like regulon in A . tumefaciens, five agents that induce E . coli heat shock genes were tested for their abilities to induce a P2-lacZ fusion in A . tumefaciens . P2 was most strongly induced by low pH, was moderately stimulated by CdCl2 or mitomycin C, and was slightly induced by P2 as measured by beta-galactosidase activity and primer extension analysis . Induction by these treatments did not require any Ti plasmid-encoded function or the chromosomally encoded RecA protein . We also pulse-labeled cellular proteins after a shift to low pH and detected several proteins whose synthesis was induced by these conditions . We conclude that P2 is primarily induced by acid pH and secondarily by certain other stimuli, each of which is stressful to cell growth . This stress induction is at least partly independent of the heat shock and SOS responses.

J Bacteriol, 1992 Feb, 174(4), 1124 - 34
A self-transmissible, narrow-host-range endogenous plasmid of Rhodobacter sphaeroides 2.4.1: physical structure, incompatibility determinants, origin of replication, and transfer functions; Suwanto A et al.; Rhodobacter sphaeroides 2.4.1 naturally harbors five cryptic endogenous plasmids (C . S . Fornari, M . Watkins, and S . Kaplan, Plasmid 11:39-47, 1984) . The smallest plasmid (pRS241e), with a molecular size of 42 kb, was observed to be a self-transmissible plasmid which can transfer only to certain strains of R . sphaeroides . Transfer frequencies can be as high as 10(-2) to 10(-3) per donor under optimal mating conditions in liquid media in the absence of oxygen . pRS241e, designated the S factor, was also shown to possess a narrow host range, failing either to replicate or to be maintained in Escherichia coli, Agrobacterium tumefaciens, and Rhizobium meliloti . It was further revealed that one of the remaining four endogenous plasmids, pRS241d, was also transmissible at a frequency similar to that of the S . factor . As a cointegrate with pSUP203, S was maintained in E . coli, providing sufficient DNA from which a physical map of S could be constructed . Progressive subcloning of S-factor DNA, in conjunction with assays of plasmid transfer, led to the localization and identification of oriV (IncA), IncB, and the putative oriT locus . The DNA sequence of the 427 bp containing oriTs revealed topological similarity to other described oriT sequences, consisting of an A-T-rich DNA region, several direct and inverted repeats, and putative integration host factor (IHF)-binding sites, and was shown to be functional in promoting plasmid transfer.

J Bacteriol, 1992 Feb, 174(4), 1086 - 98
Mutation of the miaA gene of Agrobacterium tumefaciens results in reduced vir gene expression; Gray J et al.; vir regulon expression in Agrobacterium tumefaciens involves both chromosome- and Ti-plasmid-encoded gene products . We have isolated and characterized a new chromosomal gene that when mutated results in a 2- to 10-fold reduction in the induced expression of vir genes by acetosyringone . This reduced expression occurs in AB minimal medium (pH 5.5) containing either sucrose or glucose and containing phosphate at high or low concentrations . The locus was cloned and used to complement A . tumefaciens strains harboring Tn5 insertions in the gene . Sequence analysis of this locus revealed an open reading frame with strong homology to the miaA locus of Escherichia coli and the mod5 locus of Saccharomyces cerevisiae . These genes encode tRNA: isopentenyltransferase enzymes responsible for the specific modification of the A-37 residue in UNN codon tRNA species . The function of the homologous gene in A . tumefaciens was proven by genetic complementation of E . coli miaA mutant strains . tRNA undermodification in A . tumefaciens miaA mutant strains may reduce vir gene expression by causing a reduced translation efficiency . A slight reduction in the virulence of these mutant Agrobacterium strains on red potato plants, but not on tobacco, tomato, kalanchoe, or sunflower plants, was observed.

J Bacteriol, 1992 Feb, 174(3), 991 - 7
Characterization of the Agrobacterium tumefaciens heat shock response: evidence for a sigma 32-like sigma factor; Mantis NJ et al.; We have characterized the heat shock response of Agrobacterium tumefaciens and compared it with the well-characterized Escherichia coli heat shock response . Four major heat shock proteins with apparent molecular masses of 98, 75, 65, and 20 kDa were identified by pulse-labelling cultures after temperature upshift . The three largest proteins comigrated with proteins that were antigenically related to the E . coli heat shock proteins sigma 70, DnaK, and GroEL, respectively . The heat shock proteins were also strongly induced by ethanol and cadmium chloride and were mildly induced by mitomycin C . To determine whether the A . tumefaciens heat shock regulatory system was similar to that of E . coli, we introduced the E . coli dnaK gene into A . tumefaciens . The E . coli DnK protein was expressed in A . tumefaciens, and its synthesis was induced after heat shock . Primer extension analysis of the E . coli dnaK gene in A . tumefaciens indicated that transcription initiated from one or possibly both of the E . coli heat shock promoters . We conclude that A . tumefaciens has a heat shock response similar to that of E . coli, in that (i) similar proteins are induced by heat shock, (ii) synthesis of these proteins is induced in response to similar stimuli, and (iii) A . tumefaciens can recognize an E . coli heat shock promoter, suggesting that A . tumefaciens has a sigma factor similar to sigma 32.

J Bacteriol, 1992 Feb, 174(3), 841 - 9
Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens; Zanker H et al.; The occ and noc regions of octopine and nopaline Ti plasmids in Agrobacterium tumefaciens are responsible for the catabolic utilization of octopine and nopaline, respectively . Opine-inducible promoters, genes for regulatory proteins and for catabolic enzymes, had been identified in previous work . However, both regions contained additional DNA stretches which were under the control of opine-inducible promoters, but the functions were unknown . We investigated these stretches by DNA sequence and functional analyses . The sequences showed that both of the catabolic regions contain a set of four genes which are transcribed in the same direction . The occ and noc region genes are related, but the arrangement of the genes is different . The deduced polypeptides are related to those of binding protein-dependent transport systems of basic amino acids in other bacteria . The comparison suggested that three of the polypeptides are located in the membrane and that one is a periplasmic protein . We constructed cassettes which contained either the putative transport genes only or the complete occ or noc region; all constructs, however, included the elements necessary for opine-induced expression of the genes (the regulatory gene and the inducible promoters) . Uptake studies with 3H-labelled octopine showed that the putative transport genes in the occ region code for octopine uptake proteins . The corresponding studies with 3H-labelled nopaline and the noc region cassettes indicated that the uptake of nopaline requires the putative transport genes and additional functions from the left part of the noc region.

J Nat Prod, 1992 Feb, 55(2), 149 - 62
Transgenic medicinal plants: Agrobacterium-mediated foreign gene transfer and production of secondary metabolites; Saito K et al.; Agrobacterium-Ti/Ri plasmids are natural gene vectors, by which a number of attempts have been made in genetic engineering of secondary metabolism in pharmaceutically important plants in the last few years . Opines are biosynthesized by transformed crown galls and hairy roots integrated with T-DNAs of Ti/Ri plasmids . These opines are classified into five families according to their structures and biogenesis . The production of opines is a natural example of genetic engineering of the biosynthetic machinery of plant cells for the benefit of the bacterial pathogen . One recent advance in transgenic technology of potential value to pharmacognosy is an application of transgenic organ cultures such as hairy roots and shooty teratomas to over-production and biotransformation of secondary metabolites . The hairy roots induced by Ri plasmid of Agrobacterium rhizogenes have been proved to be an efficient means of producing secondary metabolites that are normally biosynthesized in roots of differentiated plants . So far the specific metabolites produced by hairy root cultures and/or plants regenerated from hairy roots of 63 species have been analyzed and reported . As an alternative means of producing metabolites normally produced in leaves of plants, the shooty teratomas incited by the tumor-forming Ti plasmid or a shooty mutant of Agrobacterium tumefaciens have been used for the de novo biosynthesis and biotransformation of some specific secondary products . A second and more direct way to manipulate secondary pathways is performed by transferring and expressing specifically modified genes into medicinal plant cells with Agrobacterium vector systems . The genes encoding neomycin phosphotransferase and beta-glucuronidase have been used as model genes under the transcriptional control of appropriate promoters . Recently some specific genes that can eventually modify the fluxes of secondary metabolism have been integrated and expressed in medicinal plant cells . Future prospects are also discussed.

Genome, 1992 Feb, 35(1), 58 - 63
Evaluation of transgenic canola plants under field conditions; Arnoldo M et al.; Eleven independent transgenic canola (Brassica napus ssp . oleifera L . cv . Westar and Regent) lines were evaluated in the field . The plants carried a neomycin phosphotransferase (NPTII) gene for kanamycin resistance that was introduced via Agrobacterium-mediated transformation . NPTII enzyme assays, Southern blot by hybridizations and progeny analysis, confirmed the stable, heritable integration and expression of the introduced NPTII gene . A number of agronomic characteristics evaluated under field conditions, including maturity yield, and oil and protein content, were all statistically comparable between the transformed and nontransforemd platns . These results indicate that canola can be genetically engineered successfully, and that the Agrobacterium-based transformation system employed does not induced any adverse effects on the intrinsic agronomic and qualitative traits critical to the agricultural industry.

Plant Mol Biol, 1992 Feb, 18(4), 629 - 37
Spatial and temporal expression patterns directed by the Agrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot; Mattsson J et al.; We have analysed the patterns of expression of a gene encoding beta-glucuronidase (GUS) fused to the promoter of the Agrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot, Daucus carota L . Gene expression was monitored by a histochemical assay of beta-glucuronidase activity . The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development . The patterns of expression however, differed between embryos in different stages of development . In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo . In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons . During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices . Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development . This difference in organ specificity of expression is likely due to a large deletion of the promoter.

Plant Mol Biol, 1992 Feb, 18(3), 447 - 51
The xylanase introns from Cryptococcus albidus are accurately spliced in transgenic tobacco plants; Laliberte JF et al.; The xylanase gene from Cryptococcus albidus contains seven introns . Genomic and cDNA clones under the control of the CaMV 35S promoter were transferred into tobacco plants using Agrobacterium-mediated cell transformation . The genes were transcribed and the mRNAs were amplified by the polymerase chain reaction using primers on each side of the intron region . About 90% of the amplification products from plants transformed with the genomic clone corresponded to the size of the pre-mRNA (1.2 kb) and 10% represented the spliced product (0.85 kb) . The 0.85 kb fragment was cloned and sequenced and the result indicated that the introns from the xylanase gene were accurately spliced by the plant cells.

Appl Microbiol Biotechnol, 1992 Feb, 36(5), 604 - 10
Microbial modification of sugars as building blocks for chemicals; Stoppok E et al.; Investigations on the microbial modification of sucrose to the corresponding 3-keto-derivative were carried out with resting cells of Agrobacterium tumefaciens NCPPB 396 . This highly specific oxidation to yield the 3-keto-derivative has been analysed kinetically with varying substrate and cell mass concentrations . The formation of the corresponding 3-keto-derivative depended strongly on the reaction time and the aeration rate and was maximal at aeration rates up to 11.5 volume air/cultivation volume per minute with resting cells . The product formation increased with increasing substrate concentrations . However, the product yield was maximal at substrate concentrations below 20 g/l . Data pertaining to the production of active cell mass as well as for maximal 3-keto-derivative formation are presented in this paper . Also included are some applications for these derivatives.

Proc Natl Acad Sci U S A, 1992 Jan 15, 89(2), 643 - 7
Opine catabolism and conjugal transfer of the nopaline Ti plasmid pTiC58 are coordinately regulated by a single repressor; Beck von Bodman S et al.; The Ti plasmids of Agrobacterium tumefaciens are conjugal elements whose transfer is strongly repressed . Transfer is induced by the conjugal opines, a group of unique carbon compounds synthesized in crown gall tumors . The opines also induce Ti plasmid-encoded genes required by the bacteria for opine catabolism . We have cloned and sequenced a gene from the Ti plasmid pTiC58, whose product mediates the opine-dependent regulation of conjugal transfer and catabolism of the conjugal opines, agrocinopines A and B . The gene, accR, is closely linked to the agrocinopine catabolic locus . A spontaneous mutant Ti plasmid, pTiC58Trac, which constitutively expresses conjugal transfer and opine catabolism, was complemented in trans by a clone of wild-type accR . Comparative sequence analysis identified a 5-base-pair deletion close to the 5' end of the mutant accR allele from pTiC58Trac . Analysis of lacZ fusions in conjugal transfer and opine catabolic structural genes demonstrated that the accR-encoded function is a transcriptional repressor . accR can encode a 28-kDa protein . This protein is related to a class of repressor proteins that includes LacR, GutR, DeoR, FucR, and GlpR that regulate sugar catabolic systems in several bacterial genera.

FEMS Microbiol Lett, 1992 Jan 15, 69(3), 253 - 7
New functional assignment of the carotenogenic genes crtB and crtE with constructs of these genes from Erwinia species; Sandmann G et al.; The role of carotenoid genes crtB and crtE has been functionally assigned . These genes were cloned from Erwinia into Escherichia coli or Agrobacterium tumefaciens . Their functions were elucidated by assaying early isoprenoid enzymes involved in phytoene formation . In vitro reactions from extracts of E . coli carrying the crtE gene or a complete carotenogenic gene cluster in which crtB was deleted showed an elevated conversion of farnesyl pyrophosphate (FPP) into geranylgeranyl pyrophosphate (GGPP) . These results strongly indicate that the crtE gene encodes GGPP synthase . Introduction of the crtB gene into A . tumefaciens led to the conversion of GGPP into phytoene . This activity was absent in similar transformants with the crtE gene . Thus, the crtB gene probably encodes phytoene synthase, which was further supported by demonstration that phytoene accumulated in E . coli harboring both the crtB and crtE genes.

Cell, 1992 Jan 10, 68(1), 109 - 18
The VirD2 protein of A . tumefaciens contains a C-terminal bipartite nuclear localization signal: implications for nuclear uptake of DNA in plant cells; Howard EA et al.; Here we show that the VirD2 protein of A . tumefaciens functions as a nuclear localizing protein in plant cells . The nuclear localization signal of VirD2 consists of two regions containing 4-5 basic amino acids (KRPR and RKRER), located within the C-terminal 34 amino acids . These regions conform to the KR/KXR/K motif required for numerous nuclear localized nonplant eukaryotic proteins . Each region independently directs a beta-glucuronidase reporter protein to the nucleus; however, both regions are necessary for maximum efficiency . VirD2 has been shown to be tightly bound to the 5' end of the single-stranded DNA transfer intermediate, T-strand, transferred from Agrobacterium to the plant cell genome . The present results imply that T-strand transport to the plant nucleus is mediated by the tightly attached VirD2 protein via an import pathway common to higher eukaryotes.

J Gen Virol, 1992 Jan, 73 ( Pt 1), 157 - 63
Expression of cowpea mosaic virus coat protein precursor in transgenic tobacco plants; Nida DL et al.; Tobacco, Nicotiana tabacum L., supports cowpea mosaic virus (CPMV) replication and cell-to-cell movement, and thus may serve as a model system to study coat protein-mediated protection against CPMV . A chimeric gene consisting of the cauliflower mosaic virus 35S promoter, CPMV 60K coat proteins-precursor (CP-P) coding region, and the nopaline synthase polyadenylation signal was transferred to tobacco cv . Burley 21 via the Agrobacterium tumefaciens binary vector system . Gene integration and expression in the transgenic tobacco plants were confirmed by Southern and RNA dot blot analyses . Accumulation of CPMV 60K CP-P in transgenic plants, up to 2 micrograms/g of wet weight tissue, was detected by ELISA and Western blots . The results of Western blots and immunosorbent electron microscopy further indicated that CPMV CP-P neither undergoes autoproteolysis to generate the mature viral coat proteins nor assembles into virus-like capsids, suggesting that processing of the CP-P may be required for virus assembly . Because CPMV neither induces symptoms in tobacco nor moves systemically, evaluation of the reactions of the transgenic plants to virus inoculation was based on virus accumulation in the inoculated leaves . Results from such infectivity experiments did not differentiate between CP-P expressers and vector-transformed plants . The transgenic tobacco plants expressing CP-P should provide valuable material for investigating comovirus polyprotein processing and capsid assembly in vivo.

Mol Plant Microbe Interact, 1992 Jan-Feb, 5(1), 34 - 40
Resistance to tomato spotted wilt virus infection in transgenic tobacco expressing the viral nucleocapsid gene; MacKenzie DJ et al.; A recombinant plasmid containing the entire tomato spotted with virus (TSWV) nucleocapsid gene, with the exception of nucleotide encoding three N-terminal amino acids, was isolated by screening a complementary DNA library, prepared against random primed viral RNA, using a specific monoclonal antibody . The insert contained in plasmid pTSW1 was repaired and amplified by polymerase chain reaction, and the complete nucleocapsid protein gene was introduced into Nicotiana tabacum 'Samsun' by leaf disk transformation using Agrobacterium tumefaciens . Transgenic plants expressing the viral nucleocapsid protein were resistant to subsequent infection following mechanical inoculation with TSWV as indicated by a lack of systemic symptoms and little or no systemic accumulation of virus as determined by double antibody sandwich enzyme-liked immunosorbent assay . These results further extend the applicability of coat protein-mediated resistance, as previously demonstrated for a number of simple plant viruses composed of a positive-sense RNA genome encapsidated with a single species of coat protein, to a membrane-encapsidated, multi-component, negative-sense RNA virus.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 239 - 48
Biolistic transformation of prokaryotes: factors that affect biolistic transformation of very small cells; Smith FD et al.; Five bacterial species were transformed using particle gun-technology . No pretreatment of cells was necessary . Physical conditions (helium pressure, target cell distance and gap distance) and biological conditions (cell growth phase, osmoticum concentration, and cell density) were optimized for biolistic transformation of Escherichia coli and these conditions were then used to successfully transform Agrobacterium tumefaciens, Erwinia amylovora, Erwinia stewartii and Pseudomonas syringae pv . syringae . Transformation rates for E . coli were 10(4) per plate per 0.8 micrograms DNA . Although transformation rates for the other species were low (less than 10(2) per plate per 0.8 micrograms DNA), successful transformation without optimization for each species tested suggests wide utility of biolistic transformation of prokaryotes . E . coli has proven to be a useful model system to determine the effects of relative humidity, particle size and particle coating on efficiency of biolistic transformation.

Mol Microbiol, 1992 Jan, 6(2), 231 - 8
Secretion of the Rhizobium leguminosarum nodulation protein NodO by haemolysin-type systems; Scheu AK et al.; The Rhizobium leguminosarum biovar viciae nodulation protein NodO is partially homologous to haemolysin of Escherichia coli and, like haemolysin, is secreted into the growth medium . The NodO protein can be secreted by a strain of E . coli carrying the cloned nodO gene plus the haemolysin secretion genes hlyBD, in a process that also requires the outer membrane protein encoded by tolC . The related protease secretion genes, prtDEF, from Erwinia chrysanthemi also enable E . coli to secrete NodO . The Rhizobium genes encoding the proteins required for NodO secretion are unlinked to nodO and are unlike other nod genes, since they do not require flavonoids or NodO for their expression . Although proteins similar to NodO were not found in rhizobia other than R . leguminosarum bv . viciae, several rhizobia and an Agrobacterium strain containing the cloned nodO gene were found to have the ability to secrete NodO . These observations indicate that a wide range of the Rhizobiaceae have a protein secretion mechanism analogous to that which secretes haemolysin and related toxins and proteases in the ENterobacteriaceae.

Plant Cell, 1992 Jan, 4(1), 17 - 27
Subdomains of the octopine synthase upstream activating element direct cell-specific expression in transgenic tobacco plants; Kononowicz H et al.; Previous work has shown that the octopine synthase (ocs) gene encoded by the Agrobacterium tumefaciens Ti-plasmid contains an upstream activating sequence necessary for its expression in plant cells . This sequence is composed of an essential 16-bp palindrome and flanking sequences that modulate the level of expression of the ocs promoter in transgenic tobacco calli . In this study, we have used RNA gel blot analysis of RNA extracted from transgenic tobacco plants to show that the octopine synthase gene is not constitutively expressed in all plant tissues and organs . This tissue-specific pattern of expression is determined, to a large extent, by the 16-bp palindrome . Histochemical analysis, using an ocs-lacZ fusion gene, has indicated that the 16-bp palindrome directs the expression of the ocs promoter in specific cell types in the leaves, stems, and roots of transgenic tobacco plants . This expression is especially strong in the vascular tissue of the leaves, leaf mesophyll cells, leaf and stem guard cells, and the meristematic regions of the shoots and roots . Sequences surrounding the palindrome in the upstream activating sequence restrict the expression of the ocs promoter to fewer cell types, resulting in a reduced level of expression of beta-galactosidase activity in the central vascular tissue of leaves, certain types of leaf trichomes, and the leaf primordia.

Folia Microbiol (Praha), 1992, 37(3), 227 - 30
Construction and use of Agrobacterium tumefaciens binary vectors with A . tumefaciens C58 T-DNA genes; Vlasak J et al.; Five plant morphoregulatory genes were isolated from the Agrobacterium tumefaciens Ti plasmid and binary plasmid vectors for plant transformation with these genes were constructed . All vectors have a similar structure with T-DNA borders, RK2 origin of replication and chimeric kanamycin resistance gene for the selection of transformed plant tissues . Over twenty vectors with single and combined morphoregulatory genes were constructed and their effects after tobacco tissue transformation studied.

J Gen Microbiol, 1992 Jan, 138 ( Pt 1), 199 - 203
Diversity of cleavage patterns of Salmonella 23S rRNA; Hsu D et al.; The recent discovery of the phenomenon that some prokaryotes fragment their 23S rRNA during post-transcriptional processing of precursor rRNA has been shown to be particularly prevalent among strains and species of Salmonella . Some strains of Salmonella cleaved 23S rRNA at multiple sites producing several fragments . The cleavage patterns of 23S rRNA differed among Salmonella strains and sometimes among the rRNA operons in the same strain . Fragmentation of 23S rRNA was not observed in strains of the closely related species Escherichia coli . Fragmentation of 23S rRNA occurred in Brucella and Agrobacterium but the cleavage pattern was not as diverse as that demonstrated in Salmonella . Introduction of cleavage sites into precursor 23S rRNA of Salmonella is probably a recent evolutionary event.

Int J Syst Bacteriol, 1992 Jan, 42(1), 93 - 6
Phylogeny of fast-growing soybean-nodulating rhizobia support synonymy of Sinorhizobium and Rhizobium and assignment to Rhizobium fredii; Jarvis BD et al.; We determined the sequences for a 260-base segment amplified by the polymerase chain reaction (corresponding to positions 44 to 337 in the Escherichia coli 16S rRNA sequence) from seven strains of fast-growing soybean-nodulating rhizobia (including the type strains of Rhizobium fredii chemovar fredii, Rhizobium fredii chemovar siensis, Sinorhizobium fredii, and Sinorhizobium xinjiangensis) and broad-host-range Rhizobium sp . strain NGR 234 . These sequences were compared with the corresponding previously published sequences of Rhizobium leguminosarum, Rhizobium meliloti, Agrobacterium tumefaciens, Azorhizobium caulinodans, and Bradyrhizobium japonicum . All of the sequences of the fast-growing soybean rhizobia, including strain NGR 234, were identical to the sequence of R . meliloti and similar to the sequence of R . leguminosarum . These results are discussed in relation to previous findings; we concluded that the fast-growing soybean-nodulating rhizobia belong in the genus Rhizobium and should be called Rhizobium fredii.

Int J Syst Bacteriol, 1992 Jan, 42(1), 133 - 43
Marine star-shaped-aggregate-forming bacteria: Agrobacterium atlanticum sp . nov.; Agrobacterium meteori sp . nov.; Agrobacterium ferrugineum sp . nov., nom . rev.; Agrobacterium gelatinovorum sp . nov., nom . rev.; and Agrobacterium stellulatum sp . nov., nom . rev; Ruger HJ et al.; Two new species of aerobic, gram-negative, peritrichously flagellated or nonmotile marine bacteria usually forming star-shaped aggregates were isolated from northeastern Atlantic Ocean bottom sediments . These organisms resembled eight star-shaped-aggregate-forming bacterial species from the Baltic Sea originally ascribed to the genus Agrobacterium but not included on the Approved Lists of Bacterial Names because of their questionable relationships to true agrobacteria . These two sets of star-shaped-aggregate-forming bacteria were compared by means of phenotypic data, DNA base compositions, DNA-DNA relatedness, and one-dimensional electrophoretic analysis of low-molecular-weight RNAs (5S rRNA and tRNA) . According to the results of genotyping, the northeastern Atlantic Ocean isolates and three of the Baltic Sea species formed a group of closely related bacteria that could not be excluded from the genus Agrobacterium with certainty . Until more genotypic data are available, these five marine species are regarded as a distinct subdivision of the genus Agrobacterium consisting of Agrobacterium atlanticum sp . nov . (type strain, 1480T = DSM 5823T), A . meteori sp . nov . (type strain, 1513T = DSM 5824T), A . ferrugineum sp . nov . nom . rev . emend . (type strain, ATCC 25652T), A . gelatinovorum sp . nov . nom . rev . emend . (type strain, ATCC 25655T), and A . stellulatum sp . nov . nom . rev . emend . (type strain, ATCC 15215T) . "A . aggregatum" proved to be a later subjective synonym of A . stellulatum, which had priority . The remaining four Baltic Sea species, "A . agile," "A . kieliense," "A . luteum," and "A . sanguineum," could not be placed in the new subdivision of Agrobacterium.

Plant J, 1992 Jan, 2(1), 35 - 42
Excision of a transposable element from a viral vector introduced into maize plants by agroinfection; Shen WH et al.; The geminivirus maize streak virus (MSV) was used as a vector to introduce the maize transposable element Dissociation (Ds) and to study its excision in maize plants . MSV carrying Ds1 in its genome was introduced into maize plants by agroinfection . Excision of the Ds1 element from the MSV genome was detected only when functions from the transposable element Activator (Ac) were supplied in trans, either endogenously by the recipient maize plant or by co-transformation with Agrobacterium carrying a genomic Ac clone . The excision of Ds1 could easily be visualized by the appearance of viral symptoms induced by the revertant virus . The junction sequences left on the MSV genome after excision revealed 'footprints' typical of transposition as described for maize . From these results, we conclude that transposition functions in our system and that the use of the MSV replicon provides a rapid and simple tool for the investigation of the excision of transposable elements in maize plants.

Mol Gen Genet, 1992 Jan, 231(2), 186 - 93
Gene targeting in Arabidopsis thaliana; Halfter U et al.; Gene targeting of a chromosomally integrated transgene in Arabidopsis thaliana is reported . A chimeric gene consisting of the promoter of the 35S RNA of CaMV, the polyadenylation signal of the octopine synthase gene and the coding region of the bacterial hygromycin phosphotransferase gene (hpt), which was rendered non-functional by deletion of 19 bp, was introduced into the genome of A . thaliana using Agrobacterium-mediated gene transfer . A total of 3.46 x 10(8) protoplasts isolated from 17 independent transgenic Arabidopsis lines harbouring the defective chimeric hpt gene were transformed via direct gene transfer using various DNA forms containing only the intact coding region of the hpt gene . Out of 150 hygromycin-resistant colonies appearing in the course of these experiments, four were the result of targeted recombination of the incoming DNA with the defective chromosomal locus as revealed by PCR and Southern blot analysis . Comparison with the number of transformants obtained when an hpt gene controlled by a promoter and terminator from the nopaline synthase gene was employed results in a maximal ratio of homologous to non-homologous transformation in A . thaliana of 1 x 10(-4).

Plant Mol Biol, 1992 Jan, 18(2), 301 - 13
Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens; Bidney D et al.; Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation . Tobacco cv . Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII . Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves . Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol . In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment . Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.

J Bacteriol, 1992 Jan, 174(1), 303 - 8
Mutational analysis of Agrobacterium tumefaciens virD2: tyrosine 29 is essential for endonuclease activity; Vogel AM et al.; Agrobacterium tumefaciens VirD2 polypeptide, in the presence of VirD1, catalyzes a site- and strand-specific nicking reaction at the T-DNA border sequences . VirD2 is found tightly attached to the 5' end of the nicked DNA . The protein-DNA complex is presumably formed via a tyrosine residue of VirD2 (F . Durrenberger, A . Crameri, B . Hohn, and Z . Koukolikova-Nicola, Proc . Natl . Acad . Sci . USA 86:9154-9158, 1989) . A mutational approach was used to study whether a tyrosine residue(s) of VirD2 is required for its activity . By site-specific mutagenesis, a tyrosine (Y) residue at position 29, 68, 99, 119, 121, 160, or 195 of the octopine Ti plasmid pTiA6 VirD2 was altered to phenylalanine (F) . The Y-29-F or Y-121-F mutation completely abolished nicking activity of VirD2 in vivo in Escherichia coli . Two other substitutions, Y-68-F and Y-160-F, drastically reduced VirD2 activity . A substitution at position 99, 119, or 195 had no effect on VirD2 activity . Additional mutagenesis experiments showed that at position 29, no other amino acid could substitute for tyrosine without destroying VirD2 activity . At position 121, only a tryptophan (W) residue could be substituted . This, however, yielded a mutant protein with significantly reduced VirD2 activity . The nicked DNA from strains bearing a Y-68-F, Y-99-F, Y-119-F, Y-160-F, Y-195-F, or Y-121-W mutation in VirD2 was always found to contain a tightly linked protein.

Plant J, 1992 Jan, 2(1), 117 - 28
The early nodulin gene SrEnod2 from Sesbania rostrata is inducible by cytokinin; Dehio C et al.; The structure and expression of the early nodulin gene Enod2 from the stem-nodulated tropical legume Sesbania rostrata (SrEnod2) was examined . Genomic clones carrying the single SrEnod2locus were isolated and the DNA sequence of the gene was determined . The SrEnod2 gene was found to lack introns and to encode a protein consisting primarily of a 55-fold repeat of short proline-rich oligopeptides . A putative signal sequence, which may be responsible for targeting of the Enod2 protein to the cell wall, was found to precede this repeat . The temporal expression of the SrEnod2 gene was found to be different in S . rostrata stem versus root nodules induced by Azorhizobium caulinodans ORS571 . SrEnod2 gene expression was shown to be induced specifically and rapidly by physiologically significant concentrations of exogenously supplied cytokinins . The SrEnod2 gene was also found to be highly expressed in S . rostrata crown gall tumors induced by wild-type Agrobacterium tumefaciens strains, but not in tumors induced by an A . tumefaciens strain carrying a mutation in the cytokinin biosynthesis gene 4 . Implications of these observations with regard to cytokinin-induced plant gene expression and a possible role for cytokinin as a symbiotic signal are discussed.

APMIS Suppl, 1992, 30, 24 - 31
Antiplasmid activity: loss of bacterial resistance to antibiotics; Molnar J et al.; The antiplasmid activity of tricyclic compounds, e.g . phenothiazines, dibenzoazepines, dibenzocykloheptene derivatives and some stereoisomers, was shown on E . coli in vitro . Some ring-substituted phenothiazine and cannabis derivatives had only an antibacterial effect . Promethazine, a selected phenothiazine, cured antibiotic resistance and lactose fermentation of E.coli, tumour inducing ability of Agrobacterium tumefaciens and nodule formation of Rhizobium meliloti . Plasmids of different E.coli strains were eliminated with varying frequency . The antiplasmid activity of the compounds can be due to the increased membrane permeability . Inhibition of DNA gyrase and complex formation with the supercoiled form of plasmid DNA can lead to the cessation of plasmid replication in the bacterial cells . In addition, in vivo plasmid curing was demonstrated at a low frequency.

Gene, 1991 Dec 30, 109(2), 239 - 42
Transcriptional interference in transgenic plants; Ingelbrecht I et al.; When a promoterless marker gene is transformed into the plant genome using the Agrobacterium vector system, on average 30% of the T-DNA inserts produce gene fusions . This suggests that the T-DNA is preferentially integrated into transcribed regions . Here, we proposed that this transcriptional activity is responsible for some of the variation in expression frequently observed among independent transformants . Using hybrid gene constructions, we show that transcriptional readthrough into a downstream gene with opposite orientation substantially reduces expression of this gene both in transient expression and in transgenic plants . Furthermore, a poly(A) signal/terminator can block readthrough and restore the expression of the gene . Finally, enzymatic analysis of calli suggests that less variation in neomycin phosphotransferase II synthesis is observed when the gene is separated from plant DNA by promoter and terminator elements.

Nucleic Acids Res, 1991 Dec 25, 19(24), 6763 - 9
Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus; Kheyr-Pour A et al.; The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced . The single circular DNA molecule comprises 2770 nucleotides . Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome . A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants . Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation . The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule . Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.

Curr Opin Genet Dev, 1991 Dec, 1(4), 530 - 3
Genetic exchange between kingdoms; Sprague GF Jr; Bacterial conjugation with two evolutionarily divergent yeasts has been observed in the laboratory . Whether such trans-kingdom conjugation events, other than the well known Agrobacterium-plant cell interaction, actually occur in nature is not known . However, a few putative events have recently been uncovered by gene (or protein) sequence analysis, suggesting that horizontal gene transfer between phylogenetic kingdoms may be a real phenomenon.

J Bacteriol, 1991 Dec, 173(23), 7736 - 40
Induction of the alkA gene of Escherichia coli in gram-negative bacteria; Fernandez de Henestrosa AR et al.; A broad-host-range plasmid containing a fusion of the alkA and lacZ genes of Escherichia coli was introduced into various aerobic and facultative gram-negative bacteria--33 species belonging to 19 genera--to study the induction of expression of the alkA gene by alkylating agents . The bacteria included species of the families Enterobacteriaceae, Pseudomonadaceae, Rhizobiaceae, Vibrionaceae, Neisseriaceae, Rhodospirillaceae, and Azotobacteraceae . Results obtained show that all bacteria tested, except Aeromonas hydrophila, Agrobacterium tumefaciens, Hafnia alvei, Rhizobium meliloti, Salmonella enteritidis, Xanthomonas campestris, and those of the genus Rhodobacter, are able to induce the alkA gene of E . coli in the presence of N-methyl-N'-nitro-N-nitrosoguanidine . All these data indicate that the adaptive response to alkylating agents is present in bacterial species of several families and that the Ada box sequence must be widely conserved.

J Bacteriol, 1991 Dec, 173(23), 7723 - 4
A semiquantitative bioassay for relative virulence of Agrobacterium tumefaciens strains on Bryophyllum daigremontiana; Minnemeyer SL et al.; An assay to determine the relative virulence of wild-type and mutant strains of Agrobacterium tumefaciens on leaves of Bryophyllum daigremontiana has been developed . This assay is reproducible, is easy to learn, is not time consuming, and requires little space . The relative virulence of cellulose-minus mutants of A . tumefaciens was investigated with this assay . Some of these mutants were unaltered in virulence, while others showed a marked reduction in virulence.

Glycobiology, 1991 Dec, 1(6), 643 - 9
Characterization of an Agrobacterium tumefaciens lectin; Depierreux C et al.; An Agrobacterium tumefaciens suspension induces a strong agglutination of aldehyde-fixed pig erythrocytes at pH 5.0 . The agglutination is inhibited by some polysaccharides, such as fucoidin, and also when the pH is raised to 7.0 . Lectins (sugar-binding proteins) associated with the bacterial cell wall of A . tumefaciens strain 84.5 were directly evidenced by spectrofluorimetry using fluoresceinylated neoglycoproteins . The specific binding of the fluorescein-labelled neoglycoprotein bearing alpha-L-fucoside residues was also optimal at pH 5.0 . A lectin was purified by affinity chromatography on agarose substituted with alpha-L-fucopyranoside . Furthermore, the haemagglutination activity of this lectin was inhibited by polysaccharides isolated from poplar leaves.

FEMS Microbiol Lett, 1991 Dec 1, 68(3), 337 - 43
Identification of 3-deoxy-lyxo-2-heptulosaric acid in the core region of lipopolysaccharides from Rhizobiaceae; Russa R et al.; A 3-deoxy-2-heptulosaric acid (DHA), very probably with the lyxo-configuration, was identified in the R-core region of lipopolysaccharides from nodulating strains of Rhizobium leguminosarum, Rhizobium meliloti and from all three biovars of the phytopathogenic Agrobacterium tumefaciens . Its structure could be deduced from the fragmentation pattern of the corresponding alditol acetates obtained after reduction of the 2-keto and the 1.7-carboxy groups by sodium borohydride or sodium borodeuteride . DHA in lipopolysaccharide was not destroyed by periodate and is therefore not in a terminal position . Two DHA-containing oligosaccharides, namely glucosyl (1----4)-3-deoxy-2-heptulosaric acid and rhamnosyl-rhamnosyl-(1----5)-3-deoxy-2-heptulosaric acid could be tentatively identified by mass spectrometric methods amongst the products of mild acidic hydrolysis of lipopolysaccharides of Rhizobium leguminosarum strain 24 . The two types of non-nodulating mutants of Rhizobium leguminosarum included in this study did not contain 3-deoxy-2-heptulosaric acid.

EMBO J, 1991 Dec, 10(13), 3983 - 91
T-DNA gene 5 of Agrobacterium modulates auxin response by autoregulated synthesis of a growth hormone antagonist in plants; Korber H et al.; Oncogenes carried by the transferred DNA (T-DNA) of Agrobacterium Ti plasmids encode the synthesis of plant growth factors, auxin and cytokinin, and induce tumour development in plants . Other T-DNA genes regulate the tumorous growth in ways that are not yet understood . To determine the function of T-DNA gene 5, its coding region was expressed in Escherichia coli . Synthesis of the gene 5 encoded protein (26 kDa) correlated with a 28-fold increase in conversion of tryptophan to indole-3-lactate (ILA), an auxin analogue . Expression of chimeric gene 5 constructs in transgenic tobacco resulted in overproduction of ILA that enhanced shoot formation in undifferentiated tissues and increased the tolerance of germinating seedlings to the inhibitory effect of externally supplied auxin . Promoter analysis of gene 5 in plants revealed that its expression was inducible by auxin and confined to the vascular phloem cells . cis-regulatory elements required for auxin regulation and phloem specific expression of gene 5 were mapped to a 90 bp promoter region that carried DNA sequence motifs common to several auxin induced plant promoters, as well as a binding site for a nuclear factor, Ax-1 . ILA was found to inhibit the auxin induction of the gene 5 promoter and to compete with indole-3-acetic acid (IAA) for in vitro binding to purified cellular auxin binding proteins . It is suggested therefore that ILA autoregulates its own synthesis and thereby modulates a number of auxin responses in plants.

Mol Gen Genet, 1991 Dec, 230(3), 475 - 85
Genetic transformation of Arabidopsis thaliana zygotic embryos and identification of critical parameters influencing transformation efficiency; Sangwan RS et al.; An efficient procedure for Agrobacterium-mediated transformation of zygotic embryos derived from three different Arabidopsis thaliana ecotypes has been developed . This procedure yielded an average transformation rate of 76% for ecotype C24, and 15-20% for ecotypes Landsberg-erecta and Columbia . A critical step for optimal transformation was the preculture of embryos on a phytohormone-containing medium . Light and electron microscopical studies showed that, during preculture, procambium cells of embryos became highly susceptible to Agrobacterium infection . Transformed cells developed calli and regenerated shoots within 4-5 weeks of culture . A total of 1500 fertile transgenic plants were regenerated . In regenerated plants the presence of inserted DNA was verified by genomic Southern blot analysis, assays of enzymatic activities of reporter genes (neomycin phosphotransferase II and beta-glucuronidase) as well as by genetic segregation tests . R1 progenies of 45 randomly chosen transformed lines and 150 independent regenerants did not show any somaclonal variations as ascertained by both morphological and cytological criteria . Short duration (7-8 weeks), high efficiency, reproducibility and low frequency of somaclonal variation makes the zygotic embryo transformation particularly well-suited for T-DNA tagging mutagenesis.

J Bacteriol, 1991 Dec, 173(24), 7887 - 95
Characterization of cytochromes c550 and c555 from Bradyrhizobium japonicum: cloning, mutagenesis, and sequencing of the c555 gene (cycC); Tully RE et al.; The major soluble c-type cytochromes in cultured cells of Bradyrhizobium japonicum USDA 110 comprised a CO-reactive c555 (Mr, approximately 15,500) and a non-CO-reactive c550 (Mr, approximately 12,500) . Levels of cytochrome per gram of soluble protein in aerobic, anaerobic, and symbiotic cells were 32, 21, and 30 nmol, respectively, for c555 and 31, 44, and 65 nmol, respectively, for c550 . The midpoint redox potentials (Em,7) of the purified cytochromes were +236 mV for c555 and +277 mV for c550 . The CO reactivity of c555 was pH dependent, with maximal reactivity at pH 10 or greater . Rabbit antiserum was produced against purified c555 and used to screen a B . japonicum USDA 110 genomic DNA expression library in lambda gt11 for a downstream portion of the c555 gene (cycC) . This sequence was then used to probe a cosmid library for the entire c555 locus . The nucleotide sequence shows an open reading frame of 149 amino acids, with an apparent signal sequence at the N terminus and a heme-binding site near the C terminus . The deduced amino acid sequence is similar to those of the cytochromes c556 of Rhodopseudomonas palustris and Agrobacterium tumefaciens . The cycC gene was mutagenized by insertion of a kanamycin resistance cassette and homologously recombined into the B . japonicum genome . The resulting mutant made no c555 but made normal amounts of c550 . The levels of membrane cytochromes were unaffected . The mutant and wild type exhibited identical phenotypes when used to nodulate plants of soybean (Glycine max L . Merr.), with no significant differences in nodule number, nodule mass, or total amount of N2 fixed.

FEBS Lett, 1991 Nov 18, 293(1-2), 175 - 8
Repression of the CaMV 35S promoter by the octopine synthase enhancer element; Gatz C et al.; A 16 base-pair palindrome upstream of the Agrobacterium tumefaciens octopine synthase (ocs) gene functions as a positive regulatory element in plant cells (Ellis et al . (1987) EMBO J . 6, 3203-3208; Fromm et al . (1989) Plant Cell 1, 977-984) . We have converted it into a negative element by locating two copies flanking the TATA-box of the constitutively expressed CaMV 35S promoter . The reduced promoter activity is very likely due to sterical hindrance of the ocs binding protein (OCSTF) x ocs complex with the transcription initiation complex . We propose that this type of constructs can be used for the identification of recognition sites for DNA-binding proteins which are labile in vitro as well as for determining the DNA-binding activity of a trans-acting factor in vivo.

Gene, 1991 Nov 15, 107(2), 181 - 8
Expression of the gene encoding the coat protein of cucumber mosaic virus (CMV) strain WL appears to provide protection to tobacco plants against infection by several different CMV strains; Namba S et al.; The gene (cp) encoding the coat protein (CP) of cucumber mosaic virus (CMV) strain WL (CMV-WL, which belongs to CMV subgroup II) was custom polymerase chain reaction (CPCR)-engineered for expression as described by Slightom {Gene 100 (1991) 251-255} . CPCR amplification was used to add 5'- and 3'-flanking NcoI sites to the CMV-WL cp gene, and cp was cloned into the expression vector, pUC18cpexp . This CMV-WL cp expression cassette was transferred into the genome of tobacco (Nicotiana tabacum cv . Havana 423) via the Agrobacterium T-DNA transfer mechanism . R0 plants that express the CMV-WL cp gene were subcloned, propagated, and challenge-inoculated with CMV-WL . Several R0 plant lines showed excellent protection against CMV-WL infection; however, plants found to accumulate the highest CP levels did not show the highest degree of protection . Thus in our case, CP levels appear not to be a useful predictor of the degree of protection . Plants from the best protected CMV-WL cp gene-expressing R0 tobacco lines were also inoculated with CMV strains belonging to the other major CMV subgroup (subgroup I), CMV-C and CMV-Chi, and compared in a parallel experiment with a transgenic tobacco plant line that expresses the CMV-C cp gene . Plants expressing the CMV-WL cp gene appeared to show a broader spectrum of protection against infection by the various CMV strains than plants expressing the CMV-C cp gene.

EMBO J, 1991 Nov, 10(11), 3125 - 8
The protein encoded by the rolB plant oncogene hydrolyses indole glucosides; Estruch JJ et al.; The rolB gene of Agrobacterium rhizogenes, whose expression stimulates the formation of roots by transformed plant tissues and other growth alterations in transgenic plants, codes for a beta-glucosidase able to hydrolyse indole-beta-glucosides . Indeed, we show that extracts of bacteria and/or plant tissue expressing the rolB protein hydrolyse indoxyl-beta-glucoside (plant indican) . Because of the structural similarity between indoxyl-beta-glucoside and indole-3-acetyl-beta-glucoside (IAA-beta-glucoside), we propose that the physiological and developmental alterations in transgenic plants expressing the rolB gene could be the result of an increased intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides . Thus two of the oncogenes carried by the T-DNA of the plant pathogen Agrobacterium rhizogenes (rolB and rolC) perturb plant growth and development by coding for beta-glucosidases with distinct specificities . Whereas the rolC beta-glucosidase releases cytokinins from their glucoside conjugates, the rolB encoded protein hydrolyses indole-beta-glucosides . The combined action of these two genes therefore is expected to modulate the intracellular concentration of two of the main growth factors active in plants.

Indian J Exp Biol, 1991 Nov, 29(11), 991 - 1001
Interaction of T-DNA border sequences and Ti-plasmid vir functions of Agrobacterium results in differential single-stranded linear T-DNA molecule production and plant transformation; Das S et al.; The mechanism of Agrobacterium mediated genetic transformation of plants is dependent upon certain genetic function of the chromosome of the bacterium as well as on Ti-plasmid borne vir loci and the border sequences of T-DNA . The organisationally variable forms of the naturally occurring border sequences amongst Ti-plasmid types are differentially responsive to gene products of vir loci concerned with T-strand production . Additionally, the production of stable transformants is dependent upon vir gene products effective after T strands are produced . The interaction of border sequences from different strains of Agrobacterium with vir proteins encoded by various helper plasmids revealed that functional differences do exist amongst vir gene products contained in the type of helper plasmids used.

Mol Plant Microbe Interact, 1991 Nov-Dec, 4(6), 535 - 44
Expression of Rhizobium galegae common nod clones in various backgrounds; Rasanen LA et al.; The cosmid clone pRg30, carrying common nodulation genes of Rhizobium galegae HAMBI 1174, and pRg33, a subclone of pRg30 that contains a 5.7-kb ClaI insert carrying nodDABC were conjugated into various Rhizobium nod- mutant strains and into a Ti plasmid-cured Agrobacterium tumefaciens . Complementation and expression of the nodABC genes of R . galegae were studied by following microscopically the infection process and the nodulation on different test plants . The nodABC genes of R . galegae complemented the nod- strains of other Rhizobium species . The presence of extra copies of common nod genes in the homologous R . galegae nodABC- strain induced an increased nodulation on Galega orientalis . However, the inserts of R . galegae in pRg30 and pRg33 do not carry sufficient genetic information for normal nodulation of test plants in an Agrobacterium background, because the Agrobacterium transconjugants induced root hair deformation on Galega plants, but no infection threads were detected and nodulelike structures developed only at low frequency . The Agrobacterium carrying the nodDABC of R . galegae did not cause the root hairs of Medigo sativa to deform.

Biol Chem Hoppe Seyler, 1991 Nov, 372(11), 1015 - 20
Microbial metabolism of quinoline and related compounds . XI . Degradation of quinoline-4-carboxylic acid by Microbacterium sp . H2, Agrobacterium sp . 1B and Pimelobacter simplex 4B and 5B; Schmidt M et al.; From soil enrichment cultures four strains, using quinoline-4-carboxylic acid as sole source of energy and carbon, have been isolated . According to their physiological properties these bacteria have been identified as Microbacterium sp . designated H2, as Agrobacterium sp . designated 1b and Pimelobacter simplex designated 4B and 5B . Metabolites of the degradation pathway of quinoline-4-carboxylic acid have been isolated and identified . With Pimelobacter simplex 4B and 5B 2-oxo-1,2-dihydroquinoline-4-carboxylic acid and 8-hydroxycoumarin-4-carboxylic acid were isolated . The Agrobacterium strain accumulated 2-oxo-1,2-dihydroquinoline-4-carboxylic acid and 2-oxo-1,2,3,4-tetrahydroquinoline-4-carboxylic acid in the media during growth; with Microbacterium sp . H2 we only found 8-hydroxycoumarin-4-carboxylic acid . With mutants of Microbacterium sp . H2 which were induced with N-methyl-N'-nitro-N-nitrosoguanidine we found 2-oxo-1,2-dihydroquinoline-4-carboxylic acid, 8-hydroxy-coumarin-4-carboxylic acid and 2,3-dihydroxyphenyl-succinic acid.

Mol Gen Genet, 1991 Nov, 230(1-2), 302 - 9
Characterization of the supervirulent virG gene of the Agrobacterium tumefaciens plasmid pTiBo542; Chen CY et al.; The virG gene of the Agrobacterium tumefaciens Ti plasmid pTiBo542 has previously been reported to elicit stronger vir gene expression than its counterpart in the pTiA6 plasmid, a property we call the "superactivator" phenotype . The DNA sequence of the pTiBo542 virG gene was determined and compared to that of the pTiA6 gene . The DNA sequences of these genes differ at 16 positions: two differences are in the promoter regions, 12 are in the coding regions, and two are in the 3' untranslated regions . The 3' end of the pTiA6 virG gene also contains a probable insertion sequence that is not found downstream of the pTiBo542 gene . The base pair differences, in the two coding regions result in only two amino acid differences, both in the amino-terminal halves of the proteins . Five hybrid virG genes were constructed and used to activate the expression of a virB::lacZ gene fusion . Differences in the coding regions of these genes accounted for most of the superactivator phenotype, while differences at the promoter and 3' untranslated regions also contributed . These findings suggest that the properties of these VirG proteins and their quantities are important for vir gene induction, and also suggest a long-term selective pressure for mutations contributing to differences between these two genes.

Mol Gen Genet, 1991 Nov, 230(1-2), 24 - 7
Mapping of the ros virulence regulatory gene of A . tumefaciens; Cooley MB et al.; Virulence functions associated with the oncogenicity of Agrobacterium tumefaciens are encoded by vir genes contained in six major operons located on the Ti plasmid . The virC and virD operons encode functions responsible for host range and T-intermediate processing . These two operons are regulated positively by the product of virG and negatively by the product of the chromosomal gene ros, which encodes a 15.5 kDa repressor . To determine the location of the ros gene we have constructed A . tumefaciens HFR strains, using transposon Tn5mob to mobilize the ros locus, and used them to map the location of ros relative to auxotrophic loci . Tight linkage was found between ros, his-34 and his-19 . A linkage map is presented showing the location of ros relative to other known chromosomal genes associated with virulence functions.

Biotechnology (N Y), 1991 Nov, 9(11), 1103 - 5
Evidence for T-DNA mediated gene targeting to tobacco chloroplasts; Venkateswarlu K et al.; The integration of foreign DNA into plant cells by Agrobacterium mediated transformation is random and normally directed at the nucleus . Here we present evidence that such transformation can be used to introduce foreign genes into higher plant chloroplasts by site-specific homologous recombination . A binary vector was made in vitro that included chloroplast ribosomal DNA (rDNA) within T-DNA borders, and transgenic tobacco plants obtained using this construct were analyzed for the targeted recombination by hybridization, polymerase chain reaction (PCR) and DNA sequencing.

Mol Gen Genet, 1991 Oct, 229(3), 357 - 64
Structural characterization of protein secretion genes of the bacterial phytopathogen Xanthomonas campestris pathovar campestris: relatedness to secretion systems of other gram-negative bacteria; Dums F et al.; The nucleotide sequence was determined of a 5.3 kb region of the Xanthomonas campestris pathovar campestris genome carrying a gene cluster encoding protein secretion and pathogenicity functions . A putative promoter sequence and five open reading frames (ORF) which may be part of an operon were revealed . The five predicted primary translation products comprise 531, 390, 147, 169 and 138 amino acids with Mr values of 58,854, 42,299, 15,548, 18,214 and 15,108 respectively . A sixth, partial ORF is also present . Between ORF1 and ORF2 is a sequence of unknown function showing 7 bp duplications . The deduced amino acid sequence of ORF1 is related to the Klebsiella pneumoniae PulE protein, to the Bacillus subtilis ComG ORF1 and to the Agrobacterium tumefaciens VirB ORF11 products . In addition, the deduced amino acid sequence of ORF2 showed homology to the PulF and to the ComG ORF2 products . The proteins encoded by ORF3, 4 and 5 showed amino acid homology to PulG, H and I products respectively . The proteins encoded by ORF2, 3, 4 and 5 showed significant hydrophobic domains which may represent membrane-spanning regions . By contrast the protein encoded by ORF1 was largely hydrophilic and had two putative nucleoside triphosphate binding sites.

J Gen Virol, 1991 Oct, 72 ( Pt 10), 2325 - 31
The nucleotide sequence and genome structure of the geminivirus miscanthus streak virus; Chatani M et al.; A tandem dimer of miscanthus streak virus (MiSV) DNA was inserted into the T-DNA of the binary plasmid vector pBIN19 and agroinoculated into several monocotyledonous plants (monocots) using Agrobacterium tumefaciens or A . rhizogenes . Disease symptoms and geminate particles were produced in maize and Panicum milaceum plants, and MiSV-specific double-stranded and single-stranded DNAs were found in these plants . The nucleotide sequence of the infectious MiSV clone, consisting of 2672 nucleotides, was determined . Four open reading frames (ORFs) for proteins of Mr greater than 10K were identified, two (V0 and V2) in the virus (+) sense and two (C1 and C2) in the complementary (-) sense, although C2 did not have an ATG start codon . Unlike other geminiviruses infecting monocots, complementary-sense ORFs did not overlap . Potential splicing donor and acceptor sites were identified in the sequence of the border region between the C terminus of ORF C1 and the N terminus of ORF C2 . Amino acid sequences predicted from three (V2, C1 and C2) of these ORFs showed significant homology with the corresponding ORFs of other geminiviruses infecting monocots . A fifth ORF (V1), which showed some homology with ORF V1 of other monocot-infecting geminiviruses despite having a coding capacity for a product of Mr 8.8K, was found just upstream of ORF V2 as observed in those geminiviruses . ORF V0 showed no significant homology with ORFs present in any other geminiviruses . A mutation of V0 indicated that the C-terminal 30% of this ORF was not necessary for infection in maize, but that sequences around the mutated LspI site might have some regulatory role.

EMBO J, 1991 Oct, 10(10), 2889 - 95
The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates; Estruch JJ et al.; The rolC gene of Agrobacterium rhizogenes, which drastically affects growth and development of transgenic plants, codes for a cytokinin-beta-glucosidase . Indeed, rolC protein expressed in Escherichia coli as a fusion protein hydrolyses cytokinin glucosides, thus liberating free cytokinins . Furthermore, beta-glucosidase activity present in E . coli extracts expressing the rolC protein was inhibited by affinity-purified antibodies specific for the rolC protein . Finally, rolC proteins expressed in transgenic plants were shown to be responsible for cytokinin-beta-glucosidase activity . Morphological and phytohormonal analysis, performed on transgenic plants that are somatic mosaics for the expression of the rolC gene, extend and confirm our interpretation that the developmental, physiological and morphological alterations caused by rolC expression in transgenic plants are primarily due to a modification of the cytokinin balance . These observations shed new light on the control of growth and differentiation in plants by growth factors.

Plant Mol Biol, 1991 Oct, 17(4), 887 - 94
Mismatch-specific DNA breakdown in nuclear extract from tobacco (Nicotiana tabacum) callus; Cerovic G et al.; Mismatch-specific enzymatic activity was sought for in nuclei from normal and transformed plant cells originating from tobacco (Nicotiana tabacum) callus and crown gall tumor induced by Agrobacterium tumefaciens . The specific enzymatic activity was assayed with substrates derived from synthetic oligonucleotides (19-mer sequences corresponding to the human K-ras gene) . Single-base changes in the middle of the sequence were the basis for creating heteroduplexes with all eight mismatches . Homo- and heteroduplexes were ligated in a size ladder and used as substrates . We detected mismatch-specific DNA breakdown and determined basic requirements for the reactions . Kinetic analysis indicates the following reactivity order of preference: C:A=C:C=C:T greater than G:T approximately A:A approximately G:A approximately G:G approximately T:T much greater than G:C . It can be said now that specific mismatch recognition and repair activities have been detected in all kingdoms of living species.

Plant Mol Biol, 1991 Oct, 17(4), 825 - 36
Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress; Yusibov VM et al.; The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors . Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance . Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants . Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others . Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants . Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins . Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants . Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.

Plant Mol Biol, 1991 Oct, 17(4), 711 - 25
Regulation of Agrobacterium tumefaciens T-cyt gene expression in leaves of transgenic potato (Solanum tuberosum L . cv . Désirée) is strongly influenced by plant culture conditions; Dymock D et al.; The promoter region of the Agrobacterium tumefaciens T-cyt gene was linked in a translational fusion to the coding DNA of the reporter gene uidA (for beta-glucuronidase or GUS protein; EC 3.2.1.31) and to nos 3' flanking DNA . The chimaeric gene was introduced by Agrobacterium transformation into potato (Solanum tuberosum L . cv . Desiree) . In nine transgenic lines, the average GUS levels were highest in extracts from stems and roots of in vitro grown plants (ca . 11,000 GUS activity units per pmol MU per mg protein per min) but lower in leaves of the in vitro grown plants (ca . 7000 units) . GUS activity was intermediate in stems and roots of plants grown in soil as well as in in vitro crown galls (ca . 3000 units) . Activity was low in tubers, irrespective of whether these developed in vitro or in soil (both ca . 100 units), and lowest of all in leaves of soil-grown plants (ca . 10-15 units) . However, in shoot cultures reestablished from soil-grown plants, GUS activity in the leaves increased to that determined in the original shoot cultures . Hence, plant culture conditions strongly influenced the expression of the T-cyt-uidA-nos gene . In particular, it was silenced in leaves of soil-grown plants . The results are compared with previous analyses of the promoter region of the wild-type T-cyt gene and with the growth properties of a large number of crown gall cell lines and crown-gall-derived plants, including over forty S . tuberosum cv . Desiree cell lines isolated in the present study that were transformed with the wild-type T-cyt gene and six promoter-mutated derivatives . A number of implications are discussed for crown gall formation and for control of expression of plant genes which contain Activator or G-box type 5' expression control sequences.

Plant Mol Biol, 1991 Oct, 17(4), 701 - 9
Expression of a Brassica napus extensin gene in the vascular system of transgenic tobacco and rape plants; Shirsat AH et al.; The organs and tissues where the Brassica napus extA extensin gene is expressed have been identified . The extA gene with 3.75 kb of 5' flanking sequence was transferred to tobacco via disarmed Agrobacterium tumefaciens vectors and transgenic plants regenerated . The gene was found to be inactive in transgenic tobacco leaf, but was active as measured by RNA transcript assays in both stem and root tissues . To determine the cell-specific expression pattern of the extA gene, a promoter-reporter gene fusion construct was made consisting of 1.0 kb of 5' extA sequence fused to the coding region of the glucuronidase (GUS) gene . This fusion construct was introduced into B . napus via Agrobacterium rhizogenes, and expression of GUS in transgenic rape hairy roots was examined . GUS activity was only seen in the vascular tissues of the rape root, and was found to be specifically localised in the phloem.

Plant Mol Biol, 1991 Oct, 17(4), 691 - 9
Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants; Visser RG et al.; Granule-bound starch synthase is the key enzyme in amylose synthesis . The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5' upstream sequence of the granule-bound starch synthase gene from potato and the beta-glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system . The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low . However, leaves from in vitro grown plantlets exhibited an elevated GUS expression . The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants . The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves . The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene . Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities . Stolons and tubers show high expression.

Indian J Biochem Biophys, 1991 Oct-Dec, 28(5-6), 449 - 55
Transformation and regeneration of mung bean (Vigna radiata); Pal M et al.; The procedure relied on a protocol in which shoot organogenesis was induced on cotyledons of mung bean genotypes selected for susceptibility to agrobacterium seems to work reproducibly if not efficiently . Approximately 4-5% of the shoots produced on the kanamycin selected cotyledons are transgenic based on assays on kanamycin resistance and GUS activity . This demonstrated that transformation and regeneration in mung bean are possible . However, raising the transformed plants in field condition is yet to be perfected.

Chem Pharm Bull (Tokyo), 1991 Oct, 39(10), 2613 - 6
Synergistic action of phenolic signal compounds and carbohydrates in the induction of virulence gene expression of Agrobacterium tumefaciens; Song YN et al.; Virulence (vir) gene expression of Agrobacterium tumefaciens is activated by plant phenolic compounds such as alpha-hydroxyacetosyringone (HOAS), acetosyringone (AS), methyl syringate, coniferyl alcohol and sinapyl alcohol . Inositol was found to be a potentiating factor of vir-inducing activity, which enhanced the vir-inducing activity of AS and HOAS in a synergistic manner, in particular at a low concentrations of AS and HOAS . Of the other sugars tested D-glucose, L-rhamnose, D-xylose and D-galacturonic acid, the main components of plant cell wall polysaccharides, remarkedly potentiated the vir-inducing activity of AS, indicating the cooperative action of the signal compounds and sugars in Agrobacterium infection to plants.

Mol Microbiol, 1991 Oct, 5(10), 2345 - 50
An Agrobacterium two-component regulatory system for the detection of chemicals released from plant wounds; Winans SC; Crown gall tumorigenesis by Agrobacterium tumefaciens requires the co-ordinate transcriptional induction of a set of pathogenesis genes . At least three classes of environmental stimuli act synergistically to induce these genes: (i) monocyclic aromatic hydrocarbons such as acetosyringone, coniferyl alcohol, and vanillin, (ii) neutral or acidic monosaccharides such as glucose and glucuronic acid, and (iii) acidic pH . Three proteins are required to sense and respond to these stimuli: (i) VirA, a transmembrane sensory protein and histidine protein kinase, (ii) VirG, a transcriptional activator which is phosphorylated by phosphoryl VirA, and (iii) ChvE, a periplasmic sugar-binding protein . VirA and VirG are members of the so-called two-component family of regulatory proteins . This regulatory system continues to offer new discoveries in the areas of signal transduction, host-microbe interactions, and host range.

Plant Mol Biol, 1991 Oct, 17(4), 865 - 74
'Phytoantibodies': a general vector for the expression of immunoglobulin domains in transgenic plants; Benvenuto E et al.; Sequences encoding the immunoglobulin heavy-chain variable (VH) domains were engineered in a new general purpose vector to transform plants via Agrobacterium . The expression of an isolated VH domain (IVD) after introduction into the plant genome has been monitored by northern, western and immunohistochemical analysis . Immunoblotting showed that the polypeptide was stably expressed and accounted for up to 1% of the soluble protein fraction . It is therefore proposed that single immunoglobulin domains of suitable specificity expressed in plants may constitute an effective system to inhibit the activity of molecules involved in plant pathology or plant development.

J Bacteriol, 1991 Oct, 173(20), 6547 - 52
Polygalacturonase is a virulence factor in Agrobacterium tumefaciens biovar 3; Rodriguez-Palenzuela P et al.; Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grapes . All biovar 3 strains, regardless of their tumorigenicity, produce in culture a single polygalacturonase with a pI around 4.5 . A . tumefaciens biovar 3 strain CG49 was mutagenized with Tn5 by using pSUP2021 as a suicide vector . A mutant strain, CG50, lacking polygalacturonase activity was isolated . The mutation was due to a single Tn5 insertion in an 8.5-kb EcoRI fragment that also contained the polygalacturonase structural gene . The polygalacturonase-encoding pehA gene was cloned in Escherichia coli by using the plasmid pBluescript as a vector . Activity-stained isoelectric focusing gel analysis demonstrated that E . coli cells harboring the pehA+ recombinant plasmid pCPP2067 produced a polygalacturonase in culture with the same pI as the enzyme produced by CG49 . The pehA gene was localized within a 2.5-kb HindIII-SalI fragment . This fragment was used as a probe in Southern hybridization analysis and showed that no closely related genes are present in A . tumefaciens biovars 1 or 2, Rhizobium leguminosarum, or Bradyrhizobium japonicum . The polygalacturonase mutant was unable to induce root decay in grapes (Vitis vinifera cv . Chardonnay) and was substantially less tumorigenic than the wild type in grape stems when low levels of inoculum were used, although both strains were equally tumorigenic in potato disc assays . The results indicate that polygalacturonase is a virulence factor in both the root decay and crown gall incited in grapes by A . tumefaciens biovar 3.

J Bacteriol, 1991 Oct, 173(20), 6398 - 405
Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6; Valdivia RH et al.; Neoplastic crown gall tumors incited by Agrobacterium tumefaciens release novel amino acid or sugar derivatives known as opines, whose synthesis is directed by genes transferred to plant cells . Agrobacterium cells can transport and catabolize these compounds as sources of carbon and nitrogen . This article describes a region of the pTiA6 plasmid which is required for catabolism of the opine octopine and whose transcription is induced by octopine . This region of the plasmid contains four open reading frames, occQ, occM, occP, and occJ, which show homology to the family of so-called shock-sensitive permeases . TnphoA mutagenesis demonstrated that the OccJ and OccM proteins lie fully or partly in the periplasmic space . The OccJ protein was identified by electrophoresis and found to be fully localized in the periplasmic space . When these proteins were expressed in Escherichia coli, radiolabeled octopine became cell-associated.

Biotechnology (N Y), 1991 Oct, 9(10), 963 - 7
A DNA transformation-competent Arabidopsis genomic library in Agrobacterium; Lazo GR et al.; We have constructed a nuclear genomic library from the cruciferous plant Arabidopsis thaliana ecotype Columbia in a cosmid vector, pLZO3, and a host organism, Agrobacterium tumefaciens AGL1, which can directly DNA-transform the parent organism, Arabidopsis . The broad host range cosmid pLZO3 carries a gentamicin acetyltransferase gene as bacterial selective marker and tandem, chimeric neomycin and streptomycin phosphotransferase genes as plant selective markers . Agrobacterium AGL1 carries the hypervirulent, attenuated tumor-inducing plasmid pTiBo542 from which T-region DNA sequences have been precisely deleted, allowing optimal DNA transformation of many dicotyledonous plants . Agrobacterium AGL1 also carries an insertion mutation in its recA general recombination gene, which stabilizes the recombinant plasmids . The Arabidopsis genomic library consists of some 21,600 clones gridded onto 96-well microtiter dishes and, if random, carries at least three genomic equivalents . When probed for the presence of several Arabidopsis low copy-number genes, the genomic library seems representative . As with the unicellular organisms Escherichia coli and Saccharomyces cerevisiae, this DNA transformation competent genomic library should expedite gene isolation, by gene rescue, in multicellular organisms like Arabidopsis.

Proc Natl Acad Sci U S A, 1991 Sep 15, 88(18), 8029 - 33
Replicational release of geminivirus genomes from tandemly repeated copies: evidence for rolling-circle replication of a plant viral DNA; Stenger DC et al.; Agrobacterium-mediated inoculation of Nicotiana benthamiana plants with Ti plasmids containing tandem genome repeats derived from different strains of the gemini-virus beet curly top virus (BCTV) resulted in the production of unit-length recombinant progeny genomes in systemically infected plants . When two putative plus-strand origins of replication were present in constructs used as inocula, a replicational escape mechanism was favored that resulted in progeny genomes of a single predominant genotype . The genotype was dependent upon the arrangement of repeated parental genomes in the inocula . Sequencing across the junction between parental BCTV strains in the recombinant progeny allowed mapping of the plus-strand origin of replication to a 20-base-pair sequence within the conserved hairpin found in all geminivirus genomes . In contrast, when inocula contained tandemly repeated BCTV genome sequences but only a single conserved hairpin, a number of different progeny genotypes were simultaneously replicated in infected plants, a result expected if unit-length viral genomes were generated by random intramolecular recombination events . These results and other considerations indicate that geminivirus DNA replication occurs by a rolling-circle mechanism.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7854 - 8
Mechanism of phenolic activation of Agrobacterium virulence genes: development of a specific inhibitor of bacterial sensor/response systems; Hess KM et al.; The aglycone of the dihydrodiconiferyl alcohol glycosides, a series of phenolic growth factors able to substitute for some of the hormone requirements of tobacco cell division, are also potent inducers of virulence gene expression in Agrobacterium tumefaciens . However, these factors do not conform to the previously established structural requirements necessary for vir expression . Systematic evaluation of the structural requirements of these inducers has led to a model detailing the role of the phenolics in induction . With this model, a specific inhibitor of vir induction has been developed . This inhibitor does not affect the induction of other genes on the Ti plasmid but irreversibly blocks vir expression . The inhibitor has been used to show that the inducing phenolics must be constantly present to maintain expression of the vir regulon.

Proc Natl Acad Sci U S A, 1991 Sep 1, 88(17), 7763 - 7
Agrobacterium rhizogenes pRi8196 T-DNA: mapping and DNA sequence of functions involved in mannopine synthesis and hairy root differentiation; Hansen G et al.; This paper presents the map and DNA sequence analysis of pRi8196 transferred DNA (T-DNA) genes encoding root-inducing and mannopine synthesis functions . A canonical 24-base-pair border repeat as well as two "pseudoborders" are present at the functional right T-DNA border . To the left of this border are homologs of the mas1' and mas2' genes of TR pRiA4 . Next to these are five open reading frames (ORFs) homologous to ORFs 10-14 of TL of pRiA4 . ORFs 10-12 (rolA, rolB, and rolC) are less related to their pRiA4 homologs than are the other large ORFs analyzed here . In contrast to T-DNA genes of pRiA4, pRi8196 T-DNA ORFs 11 and 12 (rolB and rolC) are sufficient to induce hairy roots on carrot disks.

J Bacteriol, 1991 Sep, 173(18), 5861 - 8
Broad-host-range properties of plasmid RK2: importance of overlapping genes encoding the plasmid replication initiation protein TrfA; Fang FC et al.; The trfA gene, encoding the essential replication initiation protein of the broad-host-range plasmid RK2, possesses an in-frame overlapping arrangement . This results in the production of TrfA proteins of 33 and 44 kDa, respectively . Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone . TrfA-44/98L is identical to wild-type TrfA-44 with the exception of a single conservative amino acid alteration from methionine to leucine at codon 98; this alteration removes the translational start codon for the TrfA-33 protein . Copy number and stability were virtually identical for plasmids containing both TrfA-44 and TrfA-33 proteins or TrfA-44/98L alone in Pseudomonas aeruginosa and Agrobacterium tumefaciens, two unrelated bacteria in which TrfA-33 is poorly functional . This, along with recent in vitro studies comparing TrfA-44, TrfA-33, and TrfA-44/98L, suggests that the functional activity of TrfA-44 is not significantly affected by the 98L mutation . Analysis of minimal RK2 derivatives in certain gram-negative bacterial hosts suggests a role of the overlapping arrangement of trfA in facilitating the broad host range of RK2 . RK2 derivatives encoding TrfA-44/98L alone demonstrated decreased copy number and stability in Escherichia coli and Azotobacter vinelandii when compared with derivatives specifying both TrfA-44 and TrfA-33 . A strategy employing the trfA-44/98L mutant gene and in vivo homologous recombination was used to eliminate the internal translational start codon of trfA in the intact RK2 plasmid . The mutant intact RK2 plasmid produced only TrfA-44/98L . A small reduction in copy number and beta-lactamase expression resulted in E . coli, suggesting that overlapping trfA genes also enhance the efficiency of replication of the intact RK2 plasmid.

J Bacteriol, 1991 Sep, 173(18), 5784 - 92
Plant signal molecules activate the syrB gene, which is required for syringomycin production by Pseudomonas syringae pv . syringae; Mo YY et al.; The syrB gene is required for syringomycin production by Pseudomonas syringae pv . syringae and full virulence during plant pathogenesis . Strain B3AR132 containing a syrB::lacZ fusion was used to detect transcriptional activation of the syrB gene in syringomycin minimal medium by plant metabolites with signal activity . Among 34 plant phenolic compounds tested, arbutin, phenyl-beta-D-glucopyranoside, and salicin were shown to be strong inducers of syrB, giving rise to approximately 1,200 U of beta-galactosidase activity at 100 microM; esculin and helicin were moderate inducers, with about 250 to 400 U of beta-galactosidase activity at 100 microM . Acetosyringone and flavonoids that serve as signal molecules in Agrobacterium and Rhizobium species, respectively, did not induce the syrB::lacZ fusion . All syrB inducers were phenolic glucosides and none of the aglucone derivatives were active, suggesting that the beta-glycosidic linkage was necessary for signal activity . Phenyl-beta-D-galactopyranoside containing galactose substituted for glucose in the beta-glycosidic linkage also lacked inducer activity . Phenolic signal activity was enhanced two- to fivefold by specific sugars common to plant tissues, including D-fructose, D-mannose, and sucrose . The effect of sugars on syrB induction was most noticeable at low concentrations of phenolic glucoside (i.e., 1 to 10 microM), indicating that sugars such as D-fructose increase the sensitivity of P . syringae pv . syringae to the phenolic plant signal . Besides induction of syrB, syringomycin biosynthesis by parental strain B3A-R was induced to yield over 250 U of toxin by the additions of arbutin and D-fructose to syringomycin minimal medium . These data indicate that syringomycin production by most strains of P . syringae pv . syringae is modulated by the perception of two classes of plant signal molecules and transduced to the transcriptional apparatus of syringomycin (syr) genes such as syrB.

J Bacteriol, 1991 Sep, 173(17), 5558 - 63
Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat; Shurvinton CE et al.; T-DNA transfer by Agrobacterium tumefaciens depends on the right border repeat of the T-DNA and is greatly stimulated by overdrive, an adjacent sequence . We report that the function of overdrive does not depend on helical position with respect to the border repeat . A synthetic 24-bp overdrive and a 12-bp region containing a fully conserved 8-bp core overdrive sequence stimulated virulence equally, but full function required additional bases to the left of the 24-bp sequence.

Plant Mol Biol, 1991 Sep, 17(3), 567 - 79
Auxin-induced expression of the soybean GH3 promoter in transgenic tobacco plants; Hagen G et al.; The gene encoding the auxin-responsive GH3 mRNA (G . Hagen, A . Kleinschmidt, TJ . Guilfoyle, Planta 162: 147-153 (1984} from soybean was cloned, and its sequence and transcription initiation site were determined . The promoter of the GH3 gene has been fused to the open reading frame of the Escherichia coli uidA gene which encodes beta-glucuronidase (GUS) . This fusion gene was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation, and the expression of the gene was examined by fluorometric assay and histochemical staining of young R1 tobacco seedlings and mature plants . In transgenic tobacco plants that have not been exposed to exogenous auxin, expression of the fusion gene is largely restricted to roots of young green plants and developing floral organs, including ovules, developing seeds, and pollen, of mature plants . Application of exogenous auxin to tobacco seedlings or plant organs results in a greater than 50-fold increase in expression of GUS . Auxin-induced GUS expression is greatest in vascular tissue, but not restricted to this tissue . The auxin-deduced GUS expression was characterized for kinetics, auxin specificity and dose response.

Plant Mol Biol, 1991 Sep, 17(3), 547 - 50
Cytosolic localization in transgenic plants of the rolC peptide from Agrobacterium rhizogenes; Estruch JJ et al.; The rolC gene of Agrobacterium rhizogenes codes for a peptide with an apparent molecular weight of approximately 20 kDa . Immunolocalization of the rolC peptide, in leaves of transgenic plants which are genetic mosaics for the expression of the rolC gene, is restricted to the phenotypically altered sectors . Subcellular fractionation of homogenates from 35S-rolC transgenic leaves shows the cytosolic localization of the rolC peptide.

Plant Mol Biol, 1991 Sep, 17(3), 475 - 86
Expression of a bacterial lysine decarboxylase gene and transport of the protein into chloroplasts of transgenic tobacco; Herminghaus S et al.; A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants . Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts . The ldcgene from Hafnia alvei was therefore (a) placed under the control of the 1' promoter of the bidirectional Tr promoter from Agrobacterium tumefaciens Ti-plasmid, and (b) cloned behind the rbcS promoter from potato fused to the coding region of the rbcS transit peptide . Both ldc constructs, introduced into Nicotiana tabacum with the aid of A . tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization . However, LDC activity was only detectable in plants expressing mRNA under the control of the rbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain . In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3-1% of dry mass.

Am J Kidney Dis, 1991 Sep, 18(3), 402 - 5
Agrobacterium radiobacter peritonitis in two patients maintained on chronic peritoneal dialysis; Rodby RA et al.; We report two patients with end-stage renal disease maintained on chronic peritoneal dialysis who developed peritonitis in which the infecting organism was Agrobacterium radiobacter, normally a rare pathogen in humans . Both patients initially responded to antibiotics, but later relapsed and required catheter removal . Neither had been exposed to soil or plant material . A radiobacter is yet another of a growing list of unusual organisms that infect the peritoneal cavity of peritoneal dialysis patients.

J Bacteriol, 1991 Sep, 173(17), 5449 - 56
The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity; Close SM et al.; The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J . Loper and C . Kado, J . Bacteriol . 139:591-596, 1979) . The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis . Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization . Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa . Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa . Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes . An E . coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1 . However, several sequences with similarity to the consensus -10 sequence of the A . tumefaciens vir gene promoters were found upstream of ORF1 . Potential translational start signals are upstream of ORF1 and ORF2 . These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases . However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues . Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.

Int J Dev Biol, 1991 Sep, 35(3), 265 - 8
The influence of plant growth regulators on callus induction in pumpkin (Cucurbita pepo L.) hairy roots; Katavic V et al.; Following in vitro infection with Agrobacterium rhizogenes wild strain (mannopine, 8196) and two A . tumefaciens transconjugant strains (C58C1 pArA4abc and C58C1 pArA4b) transformed (hairy) roots were induced in pumpkin (C . pepo L.) cotyledons . The presence of pRi T-DNA in pumpkin long-term hairy root cultures was determined by Southern hybridization . The influence of plant growth regulators on callus induction in root explants from hairy root lines, which differed mutually in morphology and growth rate, was tested by the addition of growth regulators to basal nutrient medium; while 2.4-D inhibited root proliferation in all hairy root lines tested, callus induction depended both on plant growth regulators and the root line.

Mol Plant Microbe Interact, 1991 Sep-Oct, 4(5), 521 - 9
Repetitive sequences with homology to Bradyrhizobium japonicum DNA and the T-DNA of Agrobacterium rhizogenes are closely linked to nodABC of Rhizobium fredii USDA257; Krishnan HB et al.; We have detected strong homology between a 9.2-kb EcoRI restriction fragment from Rhizobium fredii USDA257 that contains nodABC and eight additional EcoRI fragments in DNA digests from this organism . A series of repetitive sequences responsible for this hybridization lies within a 0.95-kb HindIII/SalI subfragment about 1-kb upstream of nodA . This subfragment also hybridizes to multiple restriction fragments from nine other strains of R . fredii, but only one is common to all strains . The 0.95-kb subfragment does not hybridize to genomic DNA from 17 other strains of fast-growing rhizobia, but there is weak homology to two fragments from Rhizobium sp . strain NGR234 . We sequenced 2,432 base pairs (bp) of the region encompassing the repetitive sequences . It contains 65 separate 8- to 11-bp inverted and direct repeats, as well as two large open reading frames (ORFs) that overlap on opposite strands . ORF1 reads in the same direction as nodABC, contains 1,071 bp, and encodes a 40.6-kD protein . It has 74% sequence homology to an ORF within the T-DNA of Agrobacterium rhizogenes and similar homology to a series of repetitive sequences from Bradyrhizobium japonicum . ORF2 (981 bp) reads in the opposite direction, encodes a 34.7-kD protein, and has partial identity with a second ORF from A . rhizogenes . We could detect no poly(A)+ nodule transcripts with homology to ORF1 and ORF2 . The eight sets of repetitive sequences found in other EcoRI fragments of the genome were cloned from USDA257 on separate cosmids . Some of these cosmids appear to overlap, and two have fragments with homology to nifKDH.

Plant Mol Biol, 1991 Sep, 17(3), 441 - 52
An alternative approach for gene transfer in trees using wild-type Agrobacterium strains; Brasileiro AC et al.; Micropropagated shoots of three forest tree species, poplar (Populus tremula x P . alba), wild cherry (Prunus avium L.) and walnut (Juglans nigra x J . regia), were inoculated each with six different wild-type Agrobacterium strains . Poplar and wild cherry developed tumors that grew hormone-independently, whereas on walnut, gall formation was weak . On poplar and wild cherry, tumors induced by nopaline strains developed spontaneously shoots that had a normal phenotype and did not carry oncogenic T-DNA . From these observations, we have established a co-inoculation method to transform plants, using poplar as an experimental model . The method is based on inoculation of stem internodes with an Agrobacterium suspension containing both an oncogenic strain that induces shoot differentiation and a disarmed strain that provides the suitable genes in a binary vector . We used the vector pBI121 carrying neo (kanamycin resistance) and uidA (beta-glucuronidase) genes to facilitate early selection and screening . Poplar plants derived from kanamycin-resistant shoots that did not carry oncogenic T-DNA, were shown to contain and to express neo and uidA genes . These results suggest that wild-type Agrobacterium strains that induce shoot formation directly from tumors can be used as a general tool for gene transfer, avoiding difficult regeneration procedures.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 6941 - 5
Mutants of Agrobacterium tumefaciens with elevated vir gene expression; Pazour GJ et al.; Expression of Agrobacterium tumefaciens virulence (vir) genes requires virA, virG, and a plant-derived inducing compound such as acetosyringone . To identify the critical functional domains of virA and virG, a mutational approach was used . Agrobacterium A136 harboring plasmid pGP159, which contains virA, virG, and a reporter virB:lacZ gene fusion, was mutagenized with UV light or nitrosoguanidine . Survivors that formed blue colonies on a plate containing 5-bromo-4-chloro-3-indolyl beta-D-galactoside were isolated and analyzed . Quantification of beta-galactosidase activity in liquid assays identified nine mutant strains . By plasmid reconstruction and other procedures, all mutations mapped to the virA locus . These mutations caused an 11- to 560-fold increase in the vegetative level of virB:lacZ reporter gene expression . DNA sequence analysis showed that the mutations are located in four regions of VirA: transmembrane domain one, the active site, a glycine-rich region with homology to ATP-binding sites, and a region at the C terminus that has homology to the N terminus of VirG.

Proc Natl Acad Sci U S A, 1991 Aug 15, 88(16), 7041 - 5
Integration and expression of a rabbit liver cytochrome P-450 gene in transgenic Nicotiana tabacum; Saito K et al.; Cytochrome P-450 is involved in the oxidative metabolism of a broad range of substrates . We have made a chimeric construct, pSN002, containing the cDNA for rabbit liver cytochrome P-450 (IIC14) under the control of the TR2' promoter for mannopine synthase in the Agrobacterium Ti plasmid . Nicotiana tabacum was transformed with Agrobacterium tumefaciens harboring a cointegrated plasmid pSN002::pGV2260 . The presence of mRNA and of the translated protein from the chimeric cytochrome P-450 gene in transgenic plants was confirmed by RNA blot hybridization and by Western blot and immunohistochemical analyses, respectively . The transformants in which the foreign cytochrome P-450 protein is expressed show marked phenotypic changes, notably a tendency rapidly to senesce . We detected 2-propenylpyrrolidine, a degradative metabolite of nicotine alkaloids, in transgenic tobacco showing this pronounced phenotypic change . Such metabolism is likely to be due to the effect of senescence and not directly to the presence of the cytochrome P-450.

Mol Gen Genet, 1991 Aug, 228(1-2), 24 - 32
Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane; Okamoto S et al.; The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls . Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium . We have examined the localization and orientation of this protein in the cell membrane . When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions . Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells . Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions . By contrast, the VirD4'/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction . Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein . These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region . In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).

J Bacteriol, 1991 Aug, 173(16), 5173 - 80
Electrophoretic separation of the three Rhizobium meliloti replicons; Sobral BW et al.; The megaplasmids and the chromosome from the bacterium Rhizobium meliloti 1021 were separated in preparative quantities by using transverse alternating-field gel electrophoresis . The genetic content of each electrophoretically separated band was determined by Southern hybridization with replicon-specific probes and by comparison with Agrobacterium tumefaciens transconjugants harboring either pSym-a or pSym-b megaplasmids . Pulsed-field gel electrophoresis analyses of PacI (5'-TTAATTAA-3') and SwaI (5'-ATTTAAAT-3') digests of the whole genome and of the separated replicons were used to calculate genome sizes in two R . meliloti strains . In these strains, PacI digestion yielded only four fragments for the entire genome . The sizes of the PacI fragments from R . meliloti 1021 in megabase pairs (Mb) were 3.32 +/- 0.30, 1.42 +/- 0.13, 1.21 +/- 0.10, and 0.55 +/- 0.08, for a total genome size of 6.50 +/- 0.61 Mb . Southern hybridization with replicon-specific probes assigned one PacI fragment to the chromosome of R . meliloti 1021, one to pRme1021a, and two to pRme1021b . PacI digestion of A . tumefaciens pTi-cured, pSym transconjugants confirmed these assignments . In agreement with PacI data, the addition of the six SwaI fragments from R . meliloti 1021 gave a genome size of 6.54 +/- 0.43 Mb . pRme1021a was calculated to be 1.42 +/- 0.13 Mb, 1.34 +/- 0.09 Mb, and 1.38 +/- 0.12 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021a, respectively . pRme1021b was calculated to be 1.76 +/- 0.18 Mb, 1.65 +/- 0.10 Mb, and 1.74 +/- 0.13 Mb on the basis of PacI digestion, SwaI digestion, and the migration of uncut pRme1021B, respectively . The R . meliloti 1021 chromosome was calculated to be 3.32 +/- 0.30 Mb, 3.55 +/- 0.24 Mb, and 3.26 +/- 0.46 Mb on the basis of PacI data, SwaI data, and the migration of uncut chromosome, respectively.

J Bacteriol, 1991 Aug, 173(16), 5110 - 20
Genetic and molecular analyses of picA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome; Rong LJ et al.; picA is an Agrobacterium tumefaciens chromosomal locus, identified by Mu d11681 mutagenesis, that is inducible by certain acidic polysaccharides found in carrot root extract . Cloning and genetic analysis of a picA::lacZ fusion defined a region of the picA promoter that is responsible for the induction of this locus . Furthermore, we identified a possible negative regulator of picA expression upstream of the picA locus . This sequence, denoted pgl, has extensive homology to polygalacturonase genes from several organisms and inhibited the induction of the picA promoter when present in multiple copies in A . tumefaciens . DNA sequence analysis indicated at least two long open reading frames (ORFs) in the picA region . S1 nuclease mapping was used to identify the transcription initiation site of picA . Mutation of ORF1, but not ORF2, of the picA locus was responsible for an increased aggregation of A . tumefaciens, forming "ropes" in the presence of pea root cap cells . In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild-type control, a further indication that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant cell extracts.

Indian J Exp Biol, 1991 Aug, 29(8), 758 - 61
Agrobacterium mediated genetic transformation of chickpea, Cicer arietinum L; Srinivasan et al.; In leaf and stem explants of chickpea, wild type strains of Agrobacteria were able to induce tumors . These tumors were capable of phytohormone independent growth . A supervirulent strain A281 was found to be most effective . Thus, using an agrobacterium R1601, which carries genes conferring supervirulent phenotype along with a plant selectable marker gene (npt II), transformed calli of chickpea were selected in the presence of 100 micrograms/ml level of kanamycin . Molecular analyses of genomic DNA from transformed calli confirmed the integration of the marker gene into chickpea genome.

Biotechnology (N Y), 1991 Aug, 9(8), 752 - 8
Protection against detrimental effects of potyvirus infection in transgenic tobacco plants expressing the papaya ringspot virus coat protein gene; Ling K et al.; We obtained transgenic tobacco plants expressing the papaya ringspot virus (PRV) coat protein (CP) gene by transformation via Agrobacterium tumefaciens . Expression was effectively monitored by enzyme-linked immunosorbent assays (ELISA) of crude tissue extracts . Subcloned plants derived from eight original Ro transformants were inoculated with potyviruses: tobacco etch (TEV), potato virus Y (PVY), and pepper mottle (PeMV) . Plants that accumulated detectable levels of the PRV CP showed significant delay in symptom development and the symptoms were attenuated . Similar results were obtained with inoculated R1 plants . We conclude that the expression of the PRV CP-gene imparts protection against infection by a broad spectrum of potyviruses.

Gene, 1991 Jul 31, 104(1), 95 - 8
Agrobacterium rhizogenes lacZ-rolC gene expression in Escherichia coli: detection of the product in transgenic plants using RolC-specific antibodies; Oono Y et al.; The rolC sequence of the Agrobacterium rhizogenes Ri plasmid was fused in-frame to the 3' end of the lacZ gene in plasmid pEX3 . The fusion protein RolC-beta-galactosidase was accumulated as insoluble inclusion bodies in Escherichia coli . Antibodies were raised in rabbits against the fusion protein . After affinity purification, RolC-specific antibodies were found to react with a 22-kDa polypeptide prepared from roots of transgenic tobacco plants possessing a rolC gene . The result of differential centrifugation suggested that RolC is present in the soluble fraction of transformed cells.

Protein Seq Data Anal, 1991 Jul, 4(1), 61 - 2
Sequence homology between a beta-galactosidase and some beta-glucosidases; Henrissat B; A significant sequence homology was found between a thermostable beta-galactosidase from Sulfolobus solfataricus and two beta-glucosidases, respectively, from Caldocellum saccharolyticum and from Agrobacterium sp . These glycosidases appear to form a new protein family, since no homology could be detected with established beta-galactosidase or beta-glucosidase families.

Plant Mol Biol, 1991 Jul, 17(1), 49 - 60
Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters; Peach C et al.; Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters . Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants . Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold . The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure . There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49 . Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes . Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants . The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.

Appl Environ Microbiol, 1991 Jul, 57(7), 2047 - 51
Survival of Agrobacterium radiobacter K84 on various carriers for crown gall control; Pesenti-Barili B et al.; Screening was performed on nine carriers to find an improved formulation for Agrobacterium radiobacter K84 cells . The survival data showed that it is possible to preserve A . radiobacter cells on dry solid supports for a long time provided that the storage temperature is 4 degrees C and that the inoculation volume for 4 x 10(9) CFU g-1 is not less than 0.15 ml g of carrier-1 . On the other hand, a substantial carrier water content was necessary for room temperature storage . Many materials proved to be suitable as microbial carriers; in some cases, vermiculite allowed long storage times comparable to those reported for peat or carboxymethyl cellulose, which are already employed in some commercial A . radiobacter K84 products . Furthermore, vermiculite assured full and immediate biological activity in the prevention of crown gall, showing that it is suitable for a new formulation of strain K84 . A hypothesis to explain the different survival abilities in wet and dry conditions is presented.

Plant Mol Biol, 1991 Jul, 17(1), 151 - 3
A quick method to estimate the T-DNA copy number in transgenic plants at an early stage after transformation, using inverse PCR; Does MP et al.; Agrobacterium-mediated transformation of plants is known to result in transgenic plants with a variable number of integrated T-DNA copies . Our aim was to obtain transgenic tobacco plants containing one integrated T-DNA copy per genome . Therefore, a quick method was developed to estimate the T-DNA copy number of young transgenic plantlets within 10 weeks after transformation . Inverse polymerase chain reaction (IPCR) was used to amplify junction fragments, i.e . plant genomic DNA sequences flanking the known T-DNA sequences.

J Bacteriol, 1991 Jul, 173(14), 4271 - 6
A novel membrane-bound glucosyltransferase from Bradyrhizobium japonicum; Cohen JL et al.; Bacteria within the family Rhizobiaceae are distinguished by their ability to infect higher plants . The cell envelope carbohydrates of these bacteria are believed to be involved in the plant infection process . One class of cell envelope carbohydrate, the cyclic beta-1,2-glucans, is synthesized by species within two genera of this family, Agrobacterium and Rhizobium . In contrast, species of the genus Bradyrhizobium, a third genus within this family, appear to lack the capacity for cyclic beta-1,2-glucan biosynthesis . Instead, these bacteria synthesize cyclic glucans containing beta-1,6 and beta-1,3 glycosidic linkages (K.J . Miller, R.S . Gore, R . Johnson, A.J . Benesi, and V.N . Reinhold, J . Bacteriol . 172:136-142, 1990) . We now report the initial characterization of a novel membrane-bound glucosyltransferase activity from Bradyrhizobium japonicum USDA 110 . Analysis of the product of this glucosyltransferase activity revealed the following: the presence of beta-1,3 and beta-1,6 glycosidic linkages, an average molecular weight of 2,100, and no detectable reducing terminal residues . The glucosyltransferase activity was found to have an apparent Km of 50 microM for for UDP-glucose, and activity was stimulated optimally by Mn2+ ions . On the basis of the structural properties of the in vitro glucan product, it is possible that this membrane-bound glucosyltransferase activity may be responsible for the biosynthesis of cyclic beta-1,6-beta-1,3-glucans by this organism.

Mol Plant Microbe Interact, 1991 Jul-Aug, 4(4), 400 - 6
Mutants of the Agrobacterium tumefaciens virA gene exhibiting acetosyringone-independent expression of the vir regulon; Ankenbauer RG et al.; Hydroxylamine-induced mutations in the virA gene of Agrobacterium tumefaciens that do not require the plant phenolic-inducing compound acetosyringone for vir regulon induction were isolated . The isolation was based on the activation of both virB::lacZ and virE::cat fusions by mutant virA loci in the absence of acetosyringone . Three of these virA(Ais) (acetosyringone-independent signaling) mutants were characterized . All three mutants expressed a virB::lacZ fusion at high levels in the absence of acetosyringone . One virA (Ais) mutant, virA112, exhibited vir gene expression in the absence of inducing monosaccharides and acidic growth conditions, both of which are normally required for vir gene induction . The phenotype of the virA112 mutant resulted from a glycine to glutamic acid change near His-474, the site of VirA autophosphorylation.

Mol Plant Microbe Interact, 1991 Jul-Aug, 4(4), 379 - 85
Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein; Habeeb LF et al.; Agrobacterium tumefaciens incites crown gall tumors on plant hosts by conjugally transferring a discrete fragment of oncogenic DNA . In addition to oncogenes, the transferred DNA contains genes that direct the synthesis and exudation of opines, which are used as nutrients by the bacteria . The bacterium contains one or more operons of Ti plasmid-encoded genes that are required for the internalization and utilization of opines, and transcription of these catabolic genes is induced by cognate opines . Here we localize the gene required for regulated expression of the octopine degradative operon of the pTiA6 plasmid to a 2-kb fragment of Ti plasmid DNA . The protein encoded by this DNA positively regulates the transcription of the catabolic operon in the presence of octopine . In addition, it negatively regulates its own gene in the presence or absence of octopine . The sequence of this gene was determined and analysis of the inferred protein sequence indicates that the gene encodes a member of the LysR family of prokaryotic transcriptional regulatory proteins.

Mol Plant Microbe Interact, 1991 Jul-Aug, 4(4), 370 - 8
Positive regulators of opine-inducible promoters in the nopaline and octopine catabolism regions of Ti plasmids; von Lintig J et al.; The noc and occ regions of nopaline and octopine Ti plasmids in Agrobacterium tumefaciens contain genes for the catabolism of nopaline and octopine, respectively . We investigated the transcriptional organization and regulation of both regions . The noc region of pTiC58 contains two nopaline-inducible promoters, and one octopine-inducible promoter was identified in the occ region of pTiAch5 . All three promoters are positively regulated in trans by constitutively expressed genes localized at the right end of the regions . The DNA sequence analysis of these parts revealed genes coding for related proteins (35.6% identity) . The two polypeptides share significant similarity with a family of other positive gene regulators, and both contain a protein motif ("LysR" signature) that is characteristic for the DNA binding domain in these polypeptides . These proteins are the only Ti plasmid functions necessary for the activation of the opine-induced promoters . We propose the names nocR and occR for the regulator genes in the noc and in the occ regions.

Mol Gen Genet, 1991 Jul, 227(3), 385 - 90
Cross-talk between the virulence and phosphate regulons of Agrobacterium tumefaciens caused by an unusual interaction of the transcriptional activator with a regulatory DNA element; Aoyama T et al.; Transcription of a virulence gene on the hairy-root-inducing plasmid A4, which is induced by plant factors in Agrobacterium tumefaciens, was also activated by phosphate limitation in both A . tumefaciens and Escherichia coli . The starting site of RNA synthesized under the two inducing conditions was the same, and an identical promoter was responsible for both inducible expressions . The response of the virulence gene to phosphate limitation did not require the positive regulator VirG for the virulence regulon, but depended entirely on the presence of PhoB protein, the positive regulator for the phosphate regulon . The DNA signal upstream of the virulence gene, which is targeted by the VirG protein, was recognized by the E . coli PhoB protein in vitro . These results indicate that cross-talk between the two regulons occurred during the recognition of a DNA signal by the regulatory protein.

Plant Mol Biol, 1991 Jul, 17(1), 1 - 8
Direct regeneration of transformed shoots in Brassica napus from hypocotyl infections with Agrobacterium rhizogenes; Damgaard O et al.; Genetically transformed root clones of rapeseed (Brassica napus) were obtained after in vitro infection of excised hypocotyl segments with a wild type strain of Agrobacterium rhizogenes and two strains of A . rhizogenes harbouring kanamycin resistance . The ability of hairy root formation was affected by light and was highly dependent on the location of the infection site at the hypocotyl . Inoculation of decapitated hypocotyls with an intact root system gave rise to direct shoot formation from the site of inoculation . Histological sections showed that several meristems were initiated at the inoculation site . Root and shoot clones were isolated and subcultured axenically in hormone-free liquid MS medium . Identification of transformed root and shoot clones was based on opine assays . Further selection was carried out in kanamycin-enriched medium . All opine-positive root clones showed NPT II (neomycin phosphotransferase) activity . Nearly half of the shoot clones expressed a strong NPT II activity while the rest gave a weak or no NPT II response.

Proc Natl Acad Sci U S A, 1991 Jun 15, 88(12), 5472 - 6
Polypeptide composition of bacterial cyclic diguanylic acid-dependent cellulose synthase and the occurrence of immunologically crossreacting proteins in higher plants; Mayer R et al.; To comprehend the catalytic and regulatory mechanism of the cyclic diguanylic acid (c-di-GMP)-dependent cellulose synthase of Acetobacter xylinum and its relatedness to similar enzymes in other organisms, the structure of this enzyme was analyzed at the polypeptide level . The enzyme, purified 350-fold by enzyme-product entrapment, contains three major peptides (90, 67, and 54 kDa), which, based on direct photoaffinity and immunochemical labeling and amino acid sequence analysis, are constituents of the native cellulose synthase . Labeling of purified synthase with either {32P}c-di-GMP or {alpha-32P}UDP-glucose indicates that activator- and substrate-specific binding sites are most closely associated with the 67- and 54-kDa peptides, respectively, whereas marginal photolabeling is detected in the 90-kDa peptide . However, antibodies raised against a protein derived from the cellulose synthase structural gene (bcsB) specifically label all three peptides . Further, the N-terminal amino acid sequences determined for the 90- and 67-kDa peptides share a high degree of homology with the amino acid sequence deduced from the gene . We suggest that the structurally related 67- and 54-kDa peptides are fragments proteolytically derived from the 90-kDa peptide encoded by bcsB . The anti-cellulose synthase antibodies crossreact with a similar set of peptides derived from other cellulose-producing microorganisms and plants such as Agrobacterium tumefaciens, Rhizobium leguminosarum, mung bean, peas, barley, and cotton . The occurrence of such cellulose synthase-like structures in plant species suggests that a common enzymatic mechanism for cellulose biogenesis is employed throughout nature.

Nucleic Acids Res, 1991 Jun 11, 19(11), 3001 - 9
In vivo analysis of plant pre-mRNA splicing using an autonomously replicating vector; McCullough AJ et al.; In this paper, we demonstrate that an autonomously replicating plant expression vector can be used for analysis of pre-mRNA splicing determinants in intact dicot cells . This vector system relies on the Agrobacterium-mediated transfection of leaf discs with the A component of the geminivirus tomato golden mosaic virus (TGMV) . Insertion of intron sequences between viral promoter and terminator sequences results in the production of high levels of pre-mRNA transcripts that are effectively and accurately spliced in vivo . Introns from the soybean B-conglycinin gene are spliced at greater than 95% efficiency indicating that the high expression levels of precursor RNA do not exceed the intron splicing capacity of these cells . Introns from the pea and wheat rbcS genes are spliced at 85% and 73% efficiency, respectively, indicating that tobacco leaf disc nuclei are capable of effectively and accurately processing particular dicot and monocot introns . Inclusion of a dicot intron in an engineered construct results in a five-fold enhancement of the level of mRNA stably expressed in dicot nuclei.

J Gen Virol, 1991 Jun, 72 ( Pt 6), 1215 - 21
Rice tungro bacilliform virus DNA independently infects rice after Agrobacterium-mediated transfer; Dasgupta I et al.; In nature, rice tungro disease is caused by an RNA and a DNA virus complex, but we have obtained an independently infectious clone of rice tungro bacilliform virus (RTBV) DNA . Infectivity could be demonstrated only when a more than unit-length copy was cloned in the Agrobacterium binary vector Bin 19 and agroinoculated into rice plants . Rice plants thus agroinfected with cloned RTBV DNA showed typical symptoms of tungro disease, presence of viral DNA and bacilliform particles, and could be used as a source of virus to infect healthy plants by the green leafhopper (Nephotettix virescens) . The importance of this infectious clone in understanding the molecular biology of RTBV and the rice tungro disease is discussed.

Appl Environ Microbiol, 1991 Jun, 57(6), 1847 - 9
Production of beta-carotene in Zymomonas mobilis and Agrobacterium tumefaciens by introduction of the biosynthesis genes from Erwinia uredovora; Misawa N et al.; The Erwinia uredovora crtB, crtE, crtI, and crtY genes required for beta-carotene biosynthesis were introduced by conjugal transfer into an ethanol-producing bacterium, Zymomonas mobilis, and a phytopathogenic bacterium, Agrobacterium tumefaciens, in which no carotenoid is synthesized . The transconjugants of Z . mobilis and A . tumefaciens carrying these genes appeared as yellow colonies and produced 220 and 350 micrograms of beta-carotene per g of dry weight, respectively, in the stationary phase in liquid culture.

Plant Mol Biol, 1991 Jun, 16(6), 1051 - 9
Environmental conditions differentially affect vir gene induction in different Agrobacterium strains . Role of the VirA sensor protein; Turk SC et al.; The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles . The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains . Data are given which indicate that these differences are due to different properties of the virA genes of these wild types . An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28 degrees C or below.

Plant Cell, 1991 Jun, 3(6), 573 - 82
Phytochrome control of the tms2 gene in transgenic Arabidopsis: a strategy for selecting mutants in the signal transduction pathway; Karlin-Neumann GA et al.; Introduction of the tms2 gene from Agrobacterium tumefaciens into Arabidopsis thaliana yields transgenic seedlings with a new selectable phenotype: the seedlings are strongly growth inhibited on micromolar concentrations of auxin amide substrates that do not significantly affect wild-type seedlings . The tms2 gene encodes an amidohydrolase that catalyzes the conversion of biologically inactive auxin amides into active auxins, which are toxic to plants at elevated concentrations . In the absence of exogenous substrate, tms2+ transgenic seedlings grow normally and are fertile . When grown on auxin amides, both etiolated and green tms2+ seedlings exhibit a variety of dose-dependent auxin toxicity effects . tms2 mRNA and the encoded amidohydrolase activity are both detectable in transgenic but not in wild-type seedlings, demonstrating that a cognate activity is lacking in wild-type Arabidopsis . Furthermore, when the introduced tms2 gene is fused to the Arabidopsis cab140 promoter, the tms2 RNA and its encoded amidohydrolase activity and, thus, the conditional lethal phenotype can be modulated by phytochrome action . The tms2 gene can, therefore, serve as a regulatable selectable marker in Arabidopsis that should be useful in isolation of trans-regulatory mutants that have lost the imposed regulation of tms2 gene activity.

J Bacteriol, 1991 Jun, 173(11), 3356 - 65
Studies of the Bradyrhizobium japonicum nodD1 promoter: a repeated structure for the nod box; Wang SP et al.; Induction of nod genes in Rhizobium and Bradyrhizobium species is dependent on the presence of plant-produced flavonoids, the NodD protein, and the cis-acting nod box promoter sequence . Although the nodD (nodD1) gene in Rhizobium species is constitutively expressed, nodD1 expression in Bradyrhizobium japonicum is inducible by isoflavones in a manner similar to that of the nodYABC operon . A consensus nod box sequence is found 5' of the nodYABC operon, whereas a presumptive, nod box-like sequence is found 5' of the nodD1 gene . As an initial step toward examining the nodD1 promoter, the transcriptional start sites of the nodD1 and nodYABC operons were determined and found to be 44 and 28 bp, respectively, downstream of their respective nod box sequences . A series of deletions of the nodD1 promoter were constructed and fused to the lacZ gene . Analysis of the activity of these deletions clearly showed that the divergent nod box sequence was essential for nodD1 induction by isoflavones or soybean seed extract . The induction of nodD1 expression requires NodD1, as tested in B . japonicum and in a heterologous system, Agrobacterium tumefaciens . On the basis of these data, we analyzed the published nod box sequences and propose a new consensus sequence composed of paired 9-bp repeats . Analysis of the nodD1 nod box and synthetic constructs of the nocYABC nod box indicate that at least two 9-bp repeats are required for NodD1-mediated induction . Furthermore, insertions between the paired repeats of the nodYABC nod box suggest that orientation of the repeats on opposite faces of the DNA helix is essential for maximum nod gene expression.

Biochem Biophys Res Commun, 1991 May 31, 177(1), 532 - 7
Cyclophellitol: a naturally occurring mechanism-based inactivator of beta-glucosidases; Withers SG et al.; The natural product cyclophellitol, isolated from the culture filtrate of a mushroom, Phellinus sp . is found to be a highly specific and effective irreversible inactivator of beta-glucosidases . It inactivates the beta-glucosidases from both almond emulsin and Agrobacter sp . according to pseudo-first order kinetics with inactivation constants of Ki = 0.34 mM, ki = 2.38 min-1, and Ki = 0.055 mM, ki = 1.26 min-1 respectively . No reactivation of the inactivated enzyme is seen upon dialysis, thus providing evidence for the irreversibility of the inactivation . The high specificity of this inactivator is evidenced by the fact that even at very high (12 mM) concentrations of cyclophellitol, no inactivation of yeast alpha-glucosidase was observed, and only extremely slow (t1/2 greater than 5 hours) inactivation of E . coli beta-galactosidase could be detected.

Gene, 1991 May 15, 101(1), 33 - 44
Characterisation of a Pseudomonas aeruginosa twitching motility gene and evidence for a specialised protein export system widespread in eubacteria; Whitchurch CB et al.; Type-4 fimbriae (pili) are associated with a phenomenon known as twitching motility, which appears to be involved with bacterial translocation across solid surfaces . Pseudomonas aeruginosa mutants which produce fimbriae, but which have lost the twitching motility function, display altered colony morphology and resistance to fimbrial-specific bacteriophage . We have used phenotypic complementation of such mutants to isolate a region of DNA involved in twitching motility . This region was physically mapped to a SpeI fragment around 20 min on the P . aeruginosa PAO chromosome, remote from the major fimbrial locus (around 75 min) where the structural subunit-encoding gene (fimA/pilA) and ancillary genes required for fimbrial assembly (pilB, C and D) are found . A gene, pilT, within the twitching motility region is predicted to encode a 344-amino acid protein which has strong homology to a variety of other bacterial proteins . These include the P . aeruginosa PilB protein, the ComG ORF-1 protein from the Bacillus subtilis comG operon (necessary for competence), the PulE protein from the Klebsiella oxytoca (formerly K . pneumoniae) pulC-O operon (involved in pullulanase export), and the VirB-11 protein from the virB operon (involved in virulence) which is located on the Agrobacterium tumefaciens Ti plasmid . We have also identified other sets of homologies between P . aeruginosa fimbrial assembly (Pil) proteins and B . subtilis Com and K . oxytoca Pul proteins, which suggest that these are all related members of a specialised protein export pathway which is widespread in the eubacteria.

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