Molecular analyses ofpicA, a plant-inducible locus on the Agrobacterium tumefaciens chromosome

Lijun Rong, Purdue University

Abstract

The synthesis of about a dozen polypeptides of A. tumefaciens A136 was increased in the presence of crude carrot root extract in comparison to bacteria grown in AB minimal medium, as measured by one-dimensional SDS-polyacrylamide gel electrophoresis. One plant-inducible locus, picA, (for plant-inducible, chromosomal), was identified by transposon Mu dI1681 mutagenesis of A. tumefaciens A136. This locus was inducible 10- to 50-fold by crude carrot root extract. Mutation of picA had no detectable effect upon bacterial growth. However, A. tumefaciens cells harboring a mutated picA locus aggregated into long "ropes" when incubated with pea root tip cells. Such aggregation was not displayed by the parental strain. In addition, a potato tuber disk virulence assay indicated that a preinduced picA mutant was more virulent than was the wild type control. These results suggested that the picA locus regulates the surface properties of the bacterium in the presence of plant cells or plant extracts. 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. S1 nuclease mapping was used to identify the transcription initiation site of picA. 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. A preliminary characterization of the inducing compound in the carrot root extract suggests that the active substance is an acidic polysaccharide. In addition, commercial polygalacturonic acid can induce picA. When QAE-Sephadex-purified inducing component from carrot root extract was compared to that from commercial polygalacturonic acid, we found that both were eluted from the column at the same salt concentration, and that in both cases, $\beta$-galactosidase activity of the fractions correlated well with the uronic acid concentration of the fractions. These results suggested that the active factor in carrot root extract is related to polygalacturonic acid. However, the inducing compound in carrot root extract was at least 100-fold more potent for inducing picA than was commercial polygalacturonic acid, suggesting that the inducer in carrot root extract is different from polygalacturonic acid. It was calculated that the inducing compound from carrot root extract can act as an inducer at a concentration of as low as 1 $\mu$M, suggesting that it is a signal molecule, rather than a metabolite.

Degree

Ph.D.

Advisors

Gelvin, Purdue University.

Subject Area

Molecular biology

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