Agrobacterium genetically transforms its hosts by transferring a segment of DNA (T-DNA) into the host cell and integrating it into the host genome. Integration requires a close interaction between T-DNA, which is packaged into a nucleoprotein complex (T-complex) by bacterial virulence (Vir) proteins, and the host chromatin. This interaction is facilitated by the host protein VIP 1, which binds both to the major protein component of the T-complex, VirE2, and to the core histones. Recently, VIP1 has been demonstrated to mediate the interaction between plant nucleosomes and VirE2-DNA complexes (i.e., synthetic T-complex-like structures) in vitro. Here, we discuss major implications of these observations—such as the possible role of core histone modifications, proteasomal uncoating of the T-complex mediated by the bacterial F-box protein VirF, and the need for changes in chromatin structure to render it accessible to the T-DNA integration—for the process of chromatin targeting of foreign DNA and its integration into the eukaryotic genome.
histones; nucleosomes; VirE2-interacting protein 1 (VIP1); VirE2; chromatin targeting; T-complex
Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.
To genetically transform plants, Agrobacterium transfers its T-DNA into the host cell and integrates it into the plant genome, resulting in neoplastic growths. Over the past two decades, a great deal has been learned about the molecular mechanism by which Agrobacterium produces T-DNA and transports it into the host nucleus. However, T-DNA integration, which is the limiting, hence, the most critical step of the transformation process, largely remains an enigma. Increasing evidence suggests that Agrobacterium utilizes the host DNA repair machinery to facilitate T-DNA integration. Meanwhile, it is well known that chromatin modifications, including the phosphorylation of histone H2AX, play an important role in DNA repair. Thus, by implication, such epigenetic codes in chromatin may also have a considerable impact on T-DNA integration, although the direct evidence to demonstrate this hypothesis is still lacking. In this review, we summarize the recent advances in our understanding of Agrobacterium T-DNA integration and discuss the potential link between this process and the epigenetic information in the host chromatin.
Agrobacterium; T-DNA integration; DSB repair; chromatin modifications; histone codes
In plant-pathogen interactions, the host defends against the invading pathogen and the pathogen aims to suppress or subvert this defense. Whereas the defense suppression strategy is relatively well understood for many pathogens, the mechanisms by which pathogens can actively utilize the defense machinery of the host remain obscure. We report that Agrobacterium, a microorganism that elicits neoplastic growths on many plant species, induces expression of a plant defense-related F-box protein, VBF, which it incorporates into its own pathway for genetic transformation. Our data suggest that VBF may function to uncoat the bacterial transferred DNA from its associated virulence VirE2 and host VIP1 proteins via the SCFVBF pathway. Suppression of VBF elevates the intracellular content of VIP1, but renders the plant largely resistant to Agrobacterium, indicating that, in the infection pathway, VBF is functionally epistatic to VIP1. When expressed in Agrobacterium and exported into the plant cell, VBF facilitates tumor formation.
The VirA protein is one of two proteins required for transcriptional activation of Agrobacterium tumefaciens virulence genes in response to phenolic compounds released by plants during infection. We describe two experimental approaches which indicate that this protein has a transmembrane topology. First, spheroplasts of Escherichia coli or wild-type A. tumefaciens expressing the VirA protein were treated with proteinase K to digest periplasmic proteins, and the remaining proteins were immunologically stained on Western blots (immunoblots) by using anti-VirA antibody. Second, transposon TnphoA was used to generate translational fusions between virA and phoA, the latter of which is the structural gene for alkaline phosphatase. Both techniques indicated that VirA spans the cytoplasmic membrane, with approximately 275 amino acids near the amino terminus being localized in the periplasmic space and the rest of the protein being localized in the cytoplasm. We also show that overexpression of VirA in E. coli is deleterious to cell growth and that this phenomenon depends on the synthesis of either the second hydrophobic core or some nearby portion of the VirA protein.
The transfer of DNA from Agrobacterium tumefaciens into a plant cell requires the activities of several virulence (vir) genes that reside on the tumor-inducing (Ti) plasmid. The putative transferred intermediate is a single-stranded DNA (T strand), covalently attached to the VirD2 protein and coated with the single-stranded DNA-binding protein, VirE2. The movement of this intermediate out of Agrobacterium cells and into plant cells requires the expression of the virB operon, which encodes 11 proteins that localize to the membrane system. Our earlier studies showed that the IncQ broad-host-range plasmid RSF1010, which can be transferred from Agrobacterium cells to plant cells, inhibits the transfer of T-DNA from pTiA6 in a fashion that is reversed by overexpression of virB9, virB10, and virB11. Here, we examined the specificity of this inhibition by following the transfer of other T-DNA molecules. By using extracellular complementation assays, the effects of RSF1010 on movement of either VirE2 or an uncoated T strand from A. tumefaciens were also monitored. The RSF1010 derivative plasmid pJW323 drastically inhibited the capacity of strains to serve as VirE2 donors but only partially inhibited T-strand transfer from virE2 mutants. Further, we show that all the virB genes tested are required for the movement of VirE2 and the uncoated T strand as assayed by extracellular complementation. Our results are consistent with a model in which the RSF1010 plasmid, or intermediates from it, compete with the T strand and VirE2 for a common transport site.
Similar to Agrobacterium tumerfaciens, Agrobacterium rhizogenes can transfer foreign DNAs into plant cells based on the autonomous root-inducing (Ri) plasmid. A. rhizogenes can cause hairy root formation on plant tissues and form composite plants after transformation. On these composite plants, some of the regenerated roots are transgenic, carrying the wild type T-DNA and the engineered binary vector; while the shoots are still non-transgenic, serving to provide energy and growth support. These hairy root composite plants will not produce transgenic seeds, but there are a number of important features that make these composite plants very useful in plant research. First, with a broad host range,A. rhizogenes can transform many plant species, especially dicots, allowing genetic engineering in a variety of species. Second, A. rhizogenes infect tissues and explants directly; no tissue cultures prior to transformation is necessary to obtain composite plants, making them ideal for transforming recalcitrant plant species. Moreover, transgenic root tissues can be generated in a matter of weeks. For Medicago truncatula, we can obtain transgenic roots in as short as three weeks, faster than normal floral dip Arabidopsis transformation. Overall, the hairy root composite plant technology is a versatile and useful tool to study gene functions and root related-phenotypes. Here we demonstrate how hairy root composite plants can be used to study plant-rhizobium interactions and nodulation in the difficult-to-transform species M. truncatula.
An isolation technique for Chlamydia trachomatis using McCoy cells is described. In contrast to earlier techniques employing such cells, no pretreatment of the cells was used. The glutarimide antibiotic cycloheximide was added to the culture medium used for incubating the cells after infection. Cycloheximide was used at concentrations that depressed, but did not completely inhibit, the metabolism of the eucaryotic host cells. In studies on different immunotypes of C. trachomatis cultured in the yolk sac of embryonated hen eggs, the cycloheximide technique was compared with a method using pretreatment of cells with 5-iodo-2-deoxyuridine. The cycloheximide method gave greater numbers of inclusion-forming units per cover slip for all the immunotypes of trachoma-inclusion conjunctivitis agents tested, i.e., A through I. In a study of 194 cervical and urethral specimens from women, cycloheximide treatment of McCoy cells was found to be more efficient than 5-iodo-2-deoxyuridine treatment for the isolation of C. trachomatis.
Migrastatin (1), iso-migrastatin (5) and lactimidomycin (7) are all glutarimide-containing polyketides known for their unique structures and cytotoxic activities against human cancer cell lines. Migrastatin, a strong inhibitor of tumor cell migration, has been an important lead in the development of antimetastatic agents. Yet studies of the related 12-membered macrolides iso-migrastatin, lactimidomycin and related analogs have been hampered by their limited availability. We report here the production, isolation, structural characterization and biological activities of iso-migrastatin, lactimidomycin, and 23 related congeners. Our studies showed that, as a family, the glutarimide-containing 12-membered macrolides are extremely potent cell migration inhibitors with some members displaying activity on par or superior to that of migrastatin as exemplified by compounds 5, 7, and 9–12. On the basis of these findings, the structures and activity of this family of compounds as cell migration inhibitors are discussed.
Agrobacterium tumefaciens causes crown gall disease on a variety of plants. During the infection process Agrobacterium transfers a nucleoprotein complex, the VirD2 T-complex, and at least two Vir proteins, VirE2 and VirF, into the plant cell via the VirB/VirD4 type IV secretion system. Recently, we found that T-DNA could also be transferred from Agrobacterium to Saccharomyces cerevisiae. Here, we describe a novel method to also detect trans-kingdom Vir protein transfer from Agrobacterium to yeast, using the Cre/lox system. Protein fusions between Cre and VirE2 or VirF were expressed in Agrobacterium. Transfer of the Cre-Vir fusion proteins from Agrobacterium to yeast was monitored by a selectable excision event resulting from site-specific recombination mediated by Cre on a lox-flanked transgene in yeast. The VirE2 and VirF proteins were transported to yeast via the virB-encoded transfer system in the presence of coupling factor VirD4, analogous to translocation into plant cells. The yeast system therefore provides a suitable and fast model system to study basic aspects of trans-kingdom protein transport from Agrobacterium into host cells. Using this method we showed that VirE2 and VirF protein transfer was inhibited by the presence of the Osa protein. Besides, we found evidence for a novel third effector protein, VirE3, which has a similar C-terminal signature to VirE2 and VirF.
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.
A method is described for autoclaving low levels of solid infectious, radioactive waste. The method permits steam penetration to inactivate biologic waste, while any volatile radioactive compounds generated during the autoclave process are absorbed. Inactivation of radiolabeled infectious waste has been problematic because the usual sterilization techniques result in unacceptable radiation handling practices. If autoclaved under the usual conditions, there exists a high probability of volatilization or release of radioisotopes from the waste. This results in the radioactive contamination of the autoclave and the laboratory area where steam is released from the autoclave. Our results provide a practical method to inactivate and dispose of infectious radioactive waste. For our research, Bacillus pumilus spore strips and vaccinia virus were used as more heat-resistant surrogates of the human immunodeficiency virus (HIV). These surrogates were used because HIV is difficult to grow under most conditions and is less heat tolerant than the surrogates. In addition, B. pumilus has defined cell death values, whereas such values have not been established for HIV. Both B. pumilus and vaccinia virus are less hazardous to work with. The autoclave method is time efficient and can be performed by laboratory personnel with minimal handling of the waste. Furthermore, waste site handlers are able to visually inspect the solid waste containers and ascertain that inactivation procedures have been implemented.
The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes.
The importation of genetic material into cells is a common and fundamental mechanism occurring in bacterial conjugation, DNA uptake, and Agrobacterium plant infection and is, for instance, responsible for antibiotic resistance spread. Previous studies suggested that this process relied only on the activity of complex molecular machines pumping the single-stranded DNA (ssDNA) into the recipient cell. Here, we show that proteins provided by the pathogenic organism and translocated prior to the arrival of ssDNA into the recipient cell also play a fundamental role. These proteins not only bind to ssDNA to protect it but also rearrange ssDNA into a compact helix, thus generating a contractile force that pulls the DNA into the host. Interestingly, the production of mechanical energy occurs solely through the free-energy gain during the binding of VirE2 to ssDNA without the need for an external source of energy, such as nucleotide hydrolysis.
Combining single-molecule techniques and biological assays, the authors show that VirE2 proteins could mediate the active translocation ofAgrobacterium's genetic material into plant cells.
Response regulators are the ultimate modulators in two-component signal transduction pathways. The N-terminal receiver domains generally accept phosphates from cognate histidine kinases to control output. VirG for example, the response regulator of the VirA/VirG two-component system in Agrobacterium tumefaciens, mediates the expression of virulence genes in response to plant host signals. Response regulators have a highly conserved structure and share a similar conformational activation upon phosphorylation, yet the sequence and structural features that determine or perturb the cooperative activation events are ill defined. Here we use VirG and the unique features of the Agrobacterium system to extend our understanding of the response regulator activation. Two previously isolated constitutive VirG mutants, VirGN54D and VirGI77V/D52E, provide the foundation for our studies. In vivo phosphorylation patterns establish that VirGN54D is able to accumulate phosphates from small-molecule phosphate donors, such as acetyl phosphate, while the VirGI77V/D52E allele carries conformational changes mimicking the active conformation. Further structural alterations on these two alleles begin to reveal the changes necessary for response regulator activation.
Superoxide dismutase (SOD) is a critical enzyme associated with controlling oxygen toxicity arising out of oxidative stress in any living system. A hyper-thermostable SOD isolated from a polyextremophile higher plant Potentilla atrosanguinea Lodd. var. argyrophylla (Wall. ex Lehm.) was engineered by mutation of a single amino acid that enhanced the thermostability of the enzyme to twofold. The engineered enzyme was functional from sub-zero temperature to >50°C, tolerated autoclaving (heating at 121°C, at a pressure of 1.1 kg per square cm for 20 min) and was resistant to proteolysis. The present work is the first example to enhance the thermostability of a hyper-thermostable protein and has potential to application to other proteins for enhancing thermostability.
Johnson, R. C. (Fort Detrick, Frederick, Md.) and N. D. Gary. Nutrition of Leptospira pomona. I. A chemically defined substitute for rabbit serum ultrafiltrate. J. Bacteriol. 83:668–672. 1962.—Poor growth of Leptospira pomona, strain Wickard, in a medium containing exhaustively dialyzed rabbit serum was corrected by addition of rabbit serum ultrafiltrate. A dialyzed serum medium was used to investigate the nutritional qualities of various compounds when tested as substitutes for the ultrafiltrate fraction. l-Asparagine was the only amino acid that markedly stimulated growth. l-Glutamine was active only when autoclaved (converted to the ammonium salt of pyrrolidone carboxylic acid), and NH4Cl and urea satisfactorily replaced l-asparagine. Results indicated that amino acids functioned primarily as a nitrogen source in this medium.
Thiamine was the only vitamin required for growth. The lag phase was usually decreased by 1 day with NaHCO3.
A medium composed of 0.02 m phospate buffer (pH 7.4), 15% dialyzed rabbit serum, thiamine (5 μg/ml), and NH4Cl (10−3m) supported growth equivalent to that obtained with undialyzed rabbit serum medium. L. pomona was transferred six times, in this medium and seven other serotypes were transferred three times, without any decrease in amount of growth. Growth of L. pomona was initiated with 20 organisms per ml.
A transformation system which is free of in vitro plant regeneration following Agrobacterium infection is established for the forage legume, Sunnhemp (Crotalaria juncea L.) where in the entire embryo axis of the germinating seed was used as the target tissue for transformation. After standardization of transformation conditions, the cotyledonary node of the embryo axis was infected with Agrobacterium host LBA 4404 harboring the recombinant vector pCAMBIA 2301. The bivalent 1D gene of the two major foot and mouth disease virus (FMDV) serotypes ‘O’ and ‘A22’ and the neomycin phosphotransferase (nptII) gene were used as the markers for optimization of the protocol. The embryo axes were pricked randomly on the cotyledonary node and co-cultivated with Agrobacterium. The germlings were then allowed to grow under standard growth room conditions in to mature fertile plants. 60 T0 plants were established from 3 separate experiments. Three hundred seeds from the 60 T0 plants were sown to raise the T1 generation of which 180 were analyzed for integration of bivalent FMDV gene 1D “O” and “A22” and the nptII gene. Eighteen out of these 180 plants amplified both the marker genes. Two independent transgenic lines 24 and 37, showed elevated levels of expression of 12 μg and 8 μg (per gm of fresh leaf) of the bivalent ID antigen “O” and “A22” . The results showed that the transformation efficiency was 3 %. To the best of our knowledge, this is the first successful attempt of Agrobacterium tumefaciens mediated transformation of Sunnhemp. The protocol can generate whole plant transformants with relative ease and should be compatible to all genotypes of Sunnhemp.
Sunnhemp; Agrobacterium tumefaciens; FMDV -1D gene; nptII gene markers; in planta transformation
Development of transgenics in pigeon pea remains dogged by poor plant regeneration in vitro from transformed tissues and low frequency transformation protocols. This article presents a non-tissue culture-based method of generating transgenic pigeon pea (Cajanus cajan (L.) Millisp.) plants using Agrobacterium-Ti plasmid-mediated transformation system. The protocol involves raising of whole plant transformants (T0 plants) directly from Agrobacterium-infected young seedlings. The plumular and intercotyledonary meristems of the seedling axes are targeted for transformation. The transformation conditions optimized were, pricking of the apical and intercotyledonary region of the seedling axes of two-day old germinating seedlings with a sewing needle, infection with Agrobacterium (LBA4404/pKIWI105 carrying uid A and npt II genes) in Winans’ AB medium that was added with wounded tobacco leaf extract, co-cultivation in the same medium for 1h and transfer of seedlings to soilrite for further growth and hardening and subsequent transfer of seedlings to soil in pots in the greenhouse. Out of the 22–25 primary transformants that survived infection-hardening treatments from each of the three experiments, 15 plants on the average established on the soil under greenhouse conditions, showed slow growth initially, nevertheless grew as normal plants, and flowered and set seed eventually. Of the several seeds harvested from all the T0 plants, six hundred were sown to obtain progeny (T1) plants and 350 of these were randomly analysed to determine their transgenic nature. PCR was performed for both gus (uid A) and npt II genes. Forty eight of the 350 T1 plants amplified both transgenes. Southern blot analysis substantiated the integration and transmission of these genes. The protocol ensured generation of pigeon pea transgenic plants with considerable ease in a short time and is applicable across different genotypes/cultivars of the crop and offers immense potential as a supplemental or an alternative protocol for generating transgenic plants of difficult-to-regenerate pigeon pea. Further, the protocol offers the option of doing away with a selection step in the procedure and so facilitates transformation, which is free of marker genes.
Cajanus cajan; Transformation; Tissue culture-independent plant regeneration
Agrobacterium tumefaciens causes crown gall disease by transferring oncogenic, single-stranded DNA (T strand), covalently attached to the VirD2 protein, across the bacterial envelope into plant cells where its expression results in tumor formation. The single-stranded DNA binding protein VirE2 is also transferred into the plant cell, though the location at which VirE2 interacts with the T strand is still under investigation. The movement of the transferred DNA and VirE2 from A. tumefaciens to the plant cell depends on the membrane-localized VirB and VirD4 proteins. Further, the movement of the IncQ broad-host-range plasmid RSF1010 between Agrobacterium strains or from Agrobacterium to plants also requires the virB-encoded transfer system. Our earlier studies showed that the presence of the RSF1010 plasmid in wild-type strains of Agrobacterium inhibits both their virulence and their capacity to transport VirE2, as assayed by coinfection with virE mutants. Here we demonstrate that the capacity to form a conjugal intermediate of RSF1010 is necessary for this inhibition, suggesting that the transferred form of the plasmid competes with the VirD2-T strand and/or VirE2 for a common export site.
The vir gene products of Agrobacterium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
VIP1 (VirE2 interacting protein 1), initially discovered as a host protein involved in Agrobacterium-plant cell DNA transfer, is a transcription factor of the basic leucine-zipper (bZIP) domain family that regulates several defence-related genes in Arabidopsis. We have developed assays to assess VIP1 binding to its DNA target in vitro and transcriptional activation efficiency in planta. Several point mutations in the VIP1 response element VRE affected the VIP1 activity, and a strong correlation between VIP1-VRE binding and transcriptional activation levels was observed. Promoter activation by VIP1 was influenced by bacterial and plant proteins known to interact with VIP1 during Agrobacterium infection, i.e., VirE2, VirF and VIP2. VirF, an F-box protein, strongly decreased VIP1 transcriptional activation ability, but not its binding to VRE
in vitro, most likely by triggering proteasomal degradation of VIP1. Finally, activation of a VRE-containing promoter was observed in dividing cells, probably resulting from activation of endogenous VIP1.
The Gram negative plant pathogen Agrobacterium tumefaciens is uniquely capable of genetically transforming eukaryotic host cells during the infection process. DNA and protein substrates are transferred into plant cells via a type IV secretion system (T4SS), which forms large cell-envelope spanning complexes at multiple sites around the bacterial circumference. To gain a detailed understanding of T4SS positioning, the spatial distribution of fluorescently labeled T4SS components was quantitatively assessed to distinguish between random and structured localization processes. Through deconvolution microscopy followed by Fourier analysis and modeling, T4SS foci were found to localize in a non-random periodic pattern. These results indicate that T4SS complexes are dependent on an underlying scaffold or assembly process to obtain an organized distribution suitable for effective delivery of substrates into host cells.
To provide multiple conjugating sites on cyclic peptides for their increasing biomedical applications, a tailed cyclic RGD peptide, c[RGDfE(GGGKK-NH2)] was designed with c(RGDfE) linked through Glu to a tail consisting of a spacer of three Gly residues and a linker of two Lys residues. The spacer is used to increase the mobility and binding ability of the c(RGDfE) ligand, and the linker is used to proved multiple active sites for conjugating other molecules or biomaterials. We found that the sequence of Glu(Gly)-OAll leads to glutarimide formation, which disrupts the formation of cyclic RGD peptides. However, our results show that glutarimide formation is sequence dependent and can be inhibited by incorporating an amino acid like Lys(Boc) with steric hindrance from the protecting group. To prevent glutarimide formation, Ser(tBu) was used to replace the glycine in the GGG spacer adjacent to the residue of Glu, and a tailed cyclic RGD peptide, c[RGDfE(SGGKK-NH2)] was successfully obtained.
Glutarimide formation; Cyclic RGD peptide; Solid-phase peptide synthesis; Glutamic acid; Allyl protecting group
We have developed a transformation system for the yeast Candida utilis. A novel strategy was applied to construct the transformation system, since auxotrophic mutants which could be used as hosts for transformation are not available. A gene encoding the ribosomal protein L41 was cloned from C. utilis, which is sensitive to cycloheximide, and used as a marker gene conferring cycloheximide resistance after modification of its amino acid sequence. The marker gene was constructed by substitution of the proline codon at position 56 with the glutamine codon by in vitro mutagenesis, as it had been reported previously that the 56th amino acid residue of L41 is responsible for the cycloheximide sensitivity of various organisms (S. Kawai, S. Murao, M. Mochizuki, I. Shibuya, K. Yano, and M. Takagi, J. Bacteriol. 174:254-262 1992). The ribosomal DNA (i.e., DNA coding for rRNA) of C. utilis was also cloned and used as a multiple-copy target for the integration of vector DNA into the genome, which resulted in a high transformation efficiency. Transformants were obtained by electroporation with a maximum efficiency of approximately 1,400 transformants per 1 microgram of linearized DNA carrying the gene for cycloheximide resistance and part of the ribosomal DNA. No transformants were obtained with intact plasmids. Multiple copies of the linearized plasmid were integrated into the host chromosome by homologous recombination. Southern analysis of the transformants in which vector DNA was integrated at the L41 gene locus indicated that there are two copies of gene for the L41 protein per cell, suggesting that C. utilis is diploid. Transformants were obtained from a variety of C. utilis strains, indicating that this method is applicable to the transformation of other C. utilis strains, even though there is significant heterogeneity in chromosomal karyotypes among these strains.
VirB5 is a type 4 secretion system protein of Agrobacterium located on the surface of the bacterial cell. This localization pattern suggests a function for VirB5 which is beyond its known role in biogenesis and/or stabilization of the T-pilus and which may involve early interactions between Agrobacterium and the host cell. Here, we identify VirB5 as the first Agrobacterium virulence protein that can enhance infectivity extracellularly. Specifically, we show that elevating the amounts of the extracellular VirB5—by exogenous addition of the purified protein, its overexpression in the bacterium, or transgenic expression in and secretion out of the host cell—enhances the efficiency the Agrobacterium-mediated T-DNA transfer, as measured by transient expression of genes contained on the transferred T-DNA molecule. Importantly, the exogenous VirB5 enhanced transient T-DNA expression in sugar beet, a major crop recalcitrant to genetic manipulation. Increasing the pool of the extracellular VirB5 did not complement an Agrobacterium virB5 mutant, suggesting a dual function for VirB5: in the bacterium and at the bacterium-host cell interface. Consistent with this idea, VirB5 expressed in the host cell, but not secreted, had no effect on the transformation efficiency. That the increase in T-DNA expression promoted by the exogenous VirB5 was not due to its effects on bacterial growth, virulence gene induction, bacterial attachment to plant tissue, or host cell defense response suggests that VirB5 participates in the early steps of the T-DNA transfer to the plant cell.