Sequence determinants of promoter activity

Little is known about the effect of the host on the genetic stability of bacterial plant pathogens. Crown gall, a plant disease caused by Agrobacterium tumefaciens, may represent a useful model to study this effect. Indeed, our previous observations on the natural occurrence and origin of nonpathogenic agrobacteria suggest that the host plant might induce loss of pathogenicity in populations of A. tumefaciens. Here we report that five different A. tumefaciens strains initially isolated from apple tumors produced up to 99% nonpathogenic mutants following their reintroduction into axenic apple plants. Two of these five strains were also found to produce mutants on pear and/or blackberry plants. Generally, the mutants of the apple isolate D10B/87 were altered in the tumor-inducing plasmid, harboring either deletions in this plasmid or point mutations in the regulatory virulence gene virG. Most of the mutants originating from the same tumor appeared to be of clonal origin, implying that the host plants influenced agrobacterial populations by favoring growth of nonpathogenic mutants over that of wild-type cells. This hypothesis was confirmed by coinoculation of apple rootstocks with strain D10B/87 and a nonpathogenic mutant.The virulence (vir) and transferred (T) regions of Agrobacterium tumefaciens Ti plasmids contain genes directly involved in plant transformation. Expression of the vir genes by the bacterium is induced through the action of the virA-virG twocomponent regulatory system in response to plant phenolic compounds such as acetosyringone. The T-DNA, produced from the T region at the direction of the vir genes, becomes integrated into plant chromosomal DNA, where it determines the synthesis of plant growth hormones responsible for crown gall tumor development. The T-DNA also confers on tumor cells the capacity to produce unusual metabolites called opines. One of these opines, nopaline, is synthesized through the reductive condensation of arginine and ␣-ketoglutaric acid. Other Ti plasmid-encoded genes, which are not transferred to plant cells, enable the bacterial pathogen to utilize opines for growth and tumor colonization (for reviews, see references 5, 6, and 20). In spite of the enhanced colonization potential associated with expression of the opine catabolic genes, pathogenic Agrobacterium strains are difficult to recover from certain types of tumors in which nonpathogenic, opine-utilizing agrobacteria often prevail (4, 14). To account for the frequent occurrence of nonpathogenic agrobacteria, it is proposed here that some plant infections with A. tumefaciens result in the modification of the bacterial genome. Conceivably, nonpathogenic mutants produced by A. tumefaciens in association with the host plant could be altered either in the vir or T region of the Ti plasmid or in the chromosome. Chromosomal genes that are required for efficient plant transformation include chvE, which encodes a protein that potentiates the effect of plant phenolic compounds on vir gene induction (11).Our recent observatio...

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“…1C). Other examples of promoter-down point mutations located immediately upstream of the Ϫ10 consensus region are known (21).…”

Section: Resultsmentioning

confidence: 99%

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

et al. 1995

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“…Within these regions, blocks of nucleotides which are important for interaction with the polymerase or ribosome, such as the promoter -35 and -10 regions and the Shine-Dalgarno (S-D) (2,6) sequence of ribosome binding sites have been identified by a combination of approaches. These include protection from nuclease digestion (7), analysis of spontaneous mutants (8,9,10), biochemical methods (7,11,12,13) and deletion mapping (1,5,14).…”

Section: Introductionmentioning

confidence: 99%

“…It appears that the most important features which determine the strength of E.coli promoters are homology with the -35 (TTGACA) and -10 (TATAATG) 'consensus' sequences, and an ideal spacing of 17 nucleotides betwe-en these areas (10,14,15,16).…”

Section: Introductionmentioning

confidence: 99%

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Warburton¹,

Boseley²,

Porter³

1983

Nucl Acids Res

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A strategy for local mutagenesis of DNA has been developed. The lac promoter in phage M13mp9 was replaced with the E.coli trp promoter. A restriction fragment bearing only the trp promoter region was mutagenized with nitrous acid, religated to the unmutagenized vector and transfected into E.coli. Several clones which give darker blue plaques on indicator media, suggesting increased a-galactosidase synthesis, were selected for DNA sequencing. One clone has a G--A transition on the 3' side of the 'Pribnow box' which results in a constitutive promoter. Two clones have different point mutations (C--T and T--C) between the Shine-Dalgarno sequence and initiation codon which raise expression of a-galactosidase two-fold.A secondary structure model suggests that the latter two mutations could exert their effect by destabilizing base-pairing of the lac Z coding region with the ribosome binding site (RBS), thereby allowing easier access to ribosomes. Support for the model comes from the finding that neither of the RBS mutations increase expression of a different downstream gene which forms no obvious secondary structure with the RBS region, whether or not the mutations are present.These results strengthen the hypothesis that secondary structure masking is a major determinant of RBS strength.

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“…Plasmids pMS200 {Pant +) and pMS206 (Pant ~RE167)have the 818-bp Sau3A1 fragment containing Pant and Ps,~ inserted into the BamHI site of pMS99 which is flanked by EcoRI sites (Youderian et al 1982). The 832-bp EcoRI fragment was purified from a 5% acrylamide gel following EcoRI digestion of pMS200.…”

Section: Dna Templates and Enzymesmentioning

confidence: 99%

Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.

Liao¹,

Wu²,

Chiang³

et al. 1987

Genes Dev.

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The characterization in vitro of a newly discovered promoter {P,~} in the bacteriophage P22 immI region is described. P.,, is located within the ant gene and is directed toward the major imml promoter, P.~t. The entire intercistronic region between the P22 arc and ant genes (69 bp) is transcribed. The initiation and termination of sar (small antisense regulatory) RNA transcription are unusual. Frequent abortive initiation occurs in the presence of all four NTPs; RNA products 3-13 nucleotides in length are produced in about 15-to 25-fold larger numbers than full-length transcripts. Termination of sar RNA synthesis occurs after transcription of the first and second Ts of a TTTA sequence following a region of hyphenated dyad symmetry. The effects of convergent transcription between P~t and P.~ were investigated on linear and supercoiled templates. Active transcription from P~.t interferes with full-length transcription from Psi,; several factors that interfere with P~t initiation (e.g., P~.t down-mutation, Mnt repressor protein, Arc repressor protein} result in indirect activation of sat RNA synthesis. The sar RNA pairs rapidly with ant mRNA to form a stable stoichiometric complex. The location and properties of P.~, suggest an important regulatory function for sar RNA as a negative effector of ant expression. The results of Wu et al. (this issue} support this suggestion.

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Sequence determinants of promoter activity

Cited by 144 publications

References 40 publications

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

Genetic analysis of nonpathogenic Agrobacterium tumefaciens mutants arising in crown gall tumors

Increased expression of a cloned gene by local mutagenesis of Its promoter and ribosome binding site

Control of gene expression in bacteriophage P22 by a small antisense RNA. I. Characterization in vitro of the Psar promoter and the sar RNA transcript.

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