Cloning and identification of an Agrobacterium radiobacter 5D-1 chromosome fragment involved in control of nitrogen metabolism, biosynthesis of indolylacetic acid and replication of ColE1 plasmids

Abstract

Pleiotropic chromosomal mutations were earlier identified in saprophytic associative bacterium Agrobacterium radiobacter 5D-1. The mutations changed nitrogen metabolism, disturbed synthesis of indolylacetic acid (IAA), and conferred the ability to sustain replication of ColE1 plasmid derivatives, which are not normally maintained in bacteria other than Escherichia. The mutations were designated Nr (Nitrogen metabolism) and assigned to a single cluster on an A. radiobacter genetic map. A 420-bp fragment AGH23.1.1 was cloned from an agrobacterial genomic library. Introduced in the Nr mutants as a part of a pUC18-based recombinant plasmid, the AGH23.1.1 fragment complemented the Nr mutations with respect to nitrogen metabolism and IAA biosynthesis, but transformants still sustained replication of ColE1 plasmids. Transformation with the linear AGH23.1.1 fragment was due to substitution of a mutant allele of the nr gene with its wild-type counterpart as a result of recombination and completely restored the wild type in the Nr mutants, including the inability to maintain ColE1 plasmids. The AGH23.1.1 fragment and its flanking regions were sequenced. The established sequence was shown to contain two open reading frames (ORFs) coding for proteins with unknown functions. Thus, the cloned fragment contained a gene(s) that controls nitrogen metabolism and IAA synthesis and prevent replication of ColE1 plasmids in A. radiobacter cells. Possible variants of the genetic control of these processes are considered.