Correction: characterization of the virB operon from Agrobacterium tumefaciens Ti plasmid.
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Amino Acid Sequence↗
Biomedical subjects
J E Ward
Publications and source records attributed to J E Ward.
Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.
The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity.
Bacterial Proteins↗
Identification of a virB10 protein aggregate in the inner membrane of Agrobacterium tumefaciens.
Products of the virB operon are proposed components of a membrane-associated T-DNA transport apparatus in Agrobacterium tumefaciens. Here we identified the virB10 gene product and raised specific antiserum to the protein. While the virB10 reading frame contains two potential ATG translation start sites located 32 codons apart, we found that only the downstream ATG was required for efficient VirB10 synthesis. Cellular localization studies and analysis of translational fusions with the Escherichia coli alkaline phosphatase gene (phoA) indicated that VirB10 was anchored in the inner membrane and contained a periplasmic domain. This work also demonstrated the utility of alkaline phosphatase as a reporter for secreted proteins in A. tumefaciens. Several high-molecular-weight forms of VirB10 were observed after treatment of A. tumefaciens whole cells or inner membranes with protein cross-linking agents, suggesting that VirB10 exists as a native oligomer or forms an aggregate with other membrane proteins. These results provide the first biochemical evidence that a VirB protein complex is membrane associated in A. tumefaciens.
Amino Acid Sequence↗
Removable partial denture design and fabrication survey of the prosthodontic specialist.
In an effort to better understand and define the present standard of practice for removable partial denture design and fabrication, a questionnaire was prepared and distributed to prosthodontic specialists and graduate students or residents attending the American College of Prosthodontists annual meeting in 1987. The survey was designed to determine the philosophies and techniques used by prosthodontic specialists in treatment involving the removable partial denture. There were 195 questionnaires completed and used in determining the results. The results indicate areas of general agreement. Comparison with other data shows areas of controversy, but prosthodontists tend to follow techniques and philosophies similar to what is taught in most U.S. dental schools and what is recommended by the Academy of Denture Prosthetics.
Adult↗
A gene required for transfer of T-DNA to plants encodes an ATPase with autophosphorylating activity.
The virB operon of the Agrobacterium tume-faciens pTiA6NC plasmid likely plays a role in directing T-DNA transfer events at the bacterial membrane, as determined previously by mutagenesis and cellular fractionation studies and by DNA sequence analysis of the approximately 12-kilobase-pair operon. The DNA sequence analysis also revealed consensus mononucleotide binding domains in the deduced virB5 and virB11 gene products, suggesting that one or both of these proteins couple energy, by means of nucleotide triphosphate (NTP) hydrolysis, to T-DNA transport. In this report, the product of virB11, an essential virulence gene, was overproduced in Escherichia coli and purified by using immunoaffinity chromatography. The immunoaffinity purified protein, as well as NaDodSO4/polyacrylamide gel-eluted protein, bound and hydrolyzed ATP in the absence of DNA effectors. VirB11 protein also demonstrated in vitro autophosphorylation activity. VirB11 protein was localized primarily to the cytoplasmic membrane by immunoblot analysis of membrane fractions. The deduced VirB11 protein exhibits sequence similarity to comG ORF1, a protein required for uptake of DNA by competent Bacillus subtilis cells. These findings suggest that phosphorylation may serve to activate a component(s) of the A. tumefaciens T-DNA transport apparatus and may also represent a general activation mechanism of other bacterial DNA transport systems.
Adenosine Triphosphatases↗
A protein required for transcriptional regulation of Agrobacterium virulence genes spans the cytoplasmic membrane.
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.
Bacterial Proteins↗
Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid.
The virulence genes of the Agrobacterium tumefaciens Ti plasmid are grouped into six transcription units and direct the transfer of T-DNA into plant cells. We report here the nucleotide sequence of the largest vir operon, virB, from the Ti plasmid pTiA6NC. This operon contains 11 open reading frames, 7 of which show evidence of translational coupling. trpE::virB gene fusions were used to confirm the reading frames of genes virB2, 4, 5, 6, 7, 8, 10, and 11. In addition, the native gene products of virB6 and virB9 were identified using maxicell and in vitro transcription-translation techniques, and the VirB9 protein was found to be proteolytically processed. The codon usage of the predicted virB genes is very similar to the other pTiA6 vir genes and is much less biased than Escherichia coli. Since many of the virB gene products have secretion signals common to exported bacterial proteins, it is likely that they will be membrane-associated. We propose that the VirB proteins are involved in the formation of a transmembrane structure which mediates the passage of the transferred T-DNA molecule through the bacterial and plant cell membranes.
Base Sequence↗
The Agrobacterium tumefaciens virE2 gene product is a single-stranded-DNA-binding protein that associates with T-DNA.
Agrobacterium tumefaciens transfers T-DNA into the plant genome by a process mediated by Ti plasmid-encoded vir genes. Cleavage at T-DNA border sequences by the VirD endonuclease generates linear, single-stranded T-DNA molecules. In the work described in this report, we used electrophoretic mobility shift assays to show that the purified virE2 gene product binds to single-stranded DNA. VirE2 protein associates with T-DNA as shown by immunoprecipitation studies with VirE2-specific antiserum. The VirE2 protein was detected primarily in the cytoplasm, but also in the inner and outer membrane and periplasmic fractions. Virulence of a virE2 mutant was restored by mixed infection with strains carrying an intact vir region, but not with virA, virB, virD, virE, or virG mutants or chvA, chvB, or exoC mutants. We propose that the VirE2 protein is involved in the processing of T-DNA and in T-strand protection during transfer to the plant cell.
Bacterial Proteins↗
Characterization of the virA locus of Agrobacterium tumefaciens: a transcriptional regulator and host range determinant.
The virulence (vir) region of Agrobacterium tumefaciens mediates the transfer of a defined segment of plasmid DNA (the T-DNA) into the plant genome. The vir genes are specifically induced by molecules produced by wounded plant cells, and virA is required for this induction. We have determined the nucleotide sequence of virA loci from limited (pTiAg162) and wide (pTiA6) host range tumor-inducing (Ti) plasmids, each of which encodes a single protein of 92,000 daltons. Using antibody directed against the virA gene product, we have localized the VirA protein to the bacterial inner membrane. VirA is homologous to at least four bacterial proteins which play a role in the transcriptional regulation of diverse families of genes. Based on its role in vir gene induction, homology to transcriptional regulators and membrane localization, we propose that VirA acts as an environmental sensor of plant-derived inducer molecules and transmits this information to the level of vir gene expression. The pTiAg162 virA locus was shown to be ineffective at directing vir gene induction, suggesting that this may in part contribute to the narrow host range conferred by this plasmid.
Bacterial Proteins↗
Analysis of cell division gene ftsZ (sulB) from gram-negative and gram-positive bacteria.
The ftsZ (sulB) gene of Escherichia coli codes for a 40,000-dalton protein that carries out a key step in the cell division pathway. The presence of an ftsZ gene protein in other bacterial species was examined by a combination of Southern blot and Western blot analyses. Southern blot analysis of genomic restriction digests revealed that many bacteria, including species from six members of the family Enterobacteriaceae and from Pseudomonas aeruginosa and Agrobacterium tumefaciens, contained sequences which hybridized with an E. coli ftsZ probe. Genomic DNA from more distantly related bacteria, including Bacillus subtilis, Branhamella catarrhalis, Micrococcus luteus, and Staphylococcus aureus, did not hybridize under minimally stringent conditions. Western blot analysis, with anti-E. coli FtsZ antiserum, revealed that all bacterial species examined contained a major immunoreactive band. Several of the Enterobacteriaceae were transformed with a multicopy plasmid encoding the E. coli ftsZ gene. These transformed strains, Shigella sonnei, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes, were shown to overproduce the FtsZ protein and to produce minicells. Analysis of [35S]methionine-labeled minicells revealed that the plasmid-encoded gene products were the major labeled species. This demonstrated that the E. coli ftsZ gene could function in other bacterial species to induce minicells and that these minicells could be used to analyze plasmid-endoced gene products.
Bacterial Proteins↗
Structure and expression of the cell division genes ftsQ, ftsA and ftsZ.
The essential cell division genes ftsQ, ftsA and ftsZ map in a cluster of cell envelope genes located at two minutes on the Escherichia coli genetic map and appear to constitute an atypical operon. These closely clustered genes are all transcribed in the same direction and yet each of these genes was independently cloned on a multicopy plasmid and found to be expressed. Tn5 insertion mutagenesis of this region confirmed that these genes were independently expressed, indicating that each of these genes has its own promoter. However, maximum expression of the distal ftsZ gene required the promoter for the proximal ftsQ gene. The proximal ftsZ promoter was located within the ftsA structural gene and the proximal ftsA promoter was located within the ftsQ structural gene. No transcription terminators were evident from the DNA sequence analysis of this region. The sequence analysis also revealed that the termination codon for ftsQ overlapped the initiation codon of the ftsA gene and that the ftsA and ftsZ genes were separated by 60 base-pairs. The ftsQ gene product has a calculated Mr of 31,432 and the ftsA gene product has a calculated Mr of 45,327. The structure and expression of these genes are discussed.
Autoradiography↗
Overproduction of FtsZ induces minicell formation in E. coli.
The ftsZ gene in E. coli K-12 is an essential cell division gene. We report that a two to sevenfold increase in the level of the FtsZ protein resulted in induction of the minicell phenotype. An increase in the level of FtsZ beyond this range resulted in an inhibition of all cell division. Unlike the classical minicell mutant, the formation of minicells induced by increased levels of FtsZ did not occur at the expense of normal divisions, indicating that increasing FtsZ resulted in additional division events per cell cycle. In addition, increased FtsZ caused cell division to be initiated earlier in the cell cycle. These results are consistent with the level or activity of FtsZ controlling the frequency of cell division in E. coli.
Bacterial Proteins↗
A lacZ-ftsZ gene fusion is an analog of the cell division inhibitor sulA.
An in-frame lacZ-ftsZ gene fusion under lac control was fortuitously constructed by subcloning an EcoRI fragment that contains approximately 90% of the ftsZ gene. The identity of the gene fusion was confirmed by isolating an amber mutation in the hybrid gene and then using it to reconstruct the ftsZ gene, which now contained an amber mutation. The hybrid protein (ZZ), which does not possess ftsZ activity, contains seven amino acids of lacZ at its amino terminal end, followed by 35,000 daltons of the carboxyl end of the ftsZ protein. Induction of the hybrid protein resulted in a rapid cessation of cell division which could be reversed by removing the lac inducer. This inhibition of division could be prevented by an increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ, which is known to code for an altered but functional ftsZ protein. An increased gene dosage of ftsZ or the presence of the sulB allele of ftsZ is known to overcome sulA-mediated inhibition of division during the SOS response. Thus, our results suggest that ZZ is an analog of sulA and may aid in determining how sulA inhibits cell division.
Bacterial Proteins↗
A preextraction profile record.
The preextraction profile record is a means of easily, quickly, and inexpensively recording clinical data prior to extraction of the natural teeth. The record contains a great deal of preextraction information and can be easily stored in the patient's chart. However, the preextraction profile record does not obviate the need for other preextraction records such as radiographs, photographs, and diagnostic casts. The information recorded in preextraction records allows the dentist to have access to and compensate for the progressive changes which occur when the natural teeth are extracted.
Cephalometry↗
Fabrication of a crown and fixed partial denture remount cast.
A technique has been described for fabricating an accurate working cast in which the finished crowns are easily removable.
Acrylic Resins↗
Simplified technique for production of a distal-extension removable partial denture remounting cast.
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Dental Casting Technique↗
Effect of time lapse between mixing and loading on the flow of tissue conditioning materials .
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Analysis of Variance↗
Pros and cons of hard and resilient denture base materials.
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Aluminum↗