Characterization of Theacc (Agrocinopine Catabolism) Locus on the Nopaline-Type Ti Plasmid From Agrobacterium Tumefaciens Strain C58

Kim, Heenam

Permalink

https://hdl.handle.net/2142/86732 Copy

Description

Title

Characterization of Theacc (Agrocinopine Catabolism) Locus on the Nopaline-Type Ti Plasmid From Agrobacterium Tumefaciens Strain C58

Author(s)

Kim, Heenam

Issue Date

1998

Doctoral Committee Chair(s)

Farrand, Stephen K.

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

Biology, Molecular

Language

eng

Abstract

The acc locus from the classic nopaline-type Ti plasmid pTiC58 confers utilization of agrocinopines A+B and susceptibility to an antibiotic called agrocin 84. DNA sequence analyses revealed that acc is composed of eight genes, acc R, and accABCDEFG. accR codes for the repressor which regulates this locus. The predicted proteins from accABCDE are related to each component of ABC-type transport systems, while the predicted products of accF and accG are related to catabolic enzymes. Genetic analyses of insertion mutations showed that accABCDE are required for uptake of both agrocin 84 and agrocinopines, whereas accF and accG are required for utilization of the opines. The accF mutant was not susceptible to agrocin 84 although it took up the antibiotic. This suggests that agrocin 84 must be activated by functions coded for by accF after it is transported into the bacterium. Analysis of lacZ fusions showed that acc is induced in response to agrocinopines and to phosphate starvation. Analyses of subclones of acc revealed that promoters are present in the regions upstream of accR, accA, and accF. Two such elements located upstream of accR are responsible for regulation of expression of the entire acc operon by agrocinopines and by phosphate starvation. $P\sb{accR1}$, consists of typical $-$10 and $-$35 sequences and is required for repression of expression by AccR, whereas the other, $P\sb{accR2}$, which is composed of a typical $-$10 sequence and a pho box-like element, most likely is responsible for induction of acc in response to low phosphate levels. Two related candidate operators, an inverted repeat (IR) sequence that overlaps the $-$10-like element of the $P\sb{accR1}$ promoter, and an imperfect IR about 110 bp upstream from the proximal IR, are located in the untranslated region 5$\prime$ to accR. Strains harboring clones containing only the proximal IR, showed constitutive levels of expression of the acc::lacZ reporters under phosphate-rich conditions. Purified His-6-C-tagged AccR bound DNA fragments containing the two IRs specifically in the absence of agrocinopines, but did not bind the fragments when the opines are present. These results suggest that AccR represses expression of acc by binding to both IR elements located in the promoter region of accR. Agrobacterium strains were attracted to several opines tested. The determinants for chemotaxis to these opines were localized to opine catabolic regions of the Ti plasmids. Analysis of insertion mutations and subclones of acc showed that accA, which codes for the periplasmic agrocinopine binding protein, is required for chemotaxis to agrocinopines, but transport and catabolism of these opines are not required for the chemotactic activity. I propose that occJ, nocT, and motA that code for periplasmic binding proteins for octopine, nopaline, and mannopine, respectively, also are required for chemotaxis to their cognate opines.

Graduation Semester

1998

Type of Resource

text

Permalink

http://hdl.handle.net/2142/86732

Owning Collections