ABSTRACT
Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.
Subject(s)
Agrobacterium tumefaciens/genetics , Genome, Bacterial , Sequence Analysis, DNA , Agrobacterium tumefaciens/classification , Agrobacterium tumefaciens/pathogenicity , Agrobacterium tumefaciens/physiology , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carrier Proteins/chemistry , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle , Chromosomes, Bacterial/genetics , DNA Replication , Genes, Bacterial , Molecular Sequence Data , Phylogeny , Plant Tumors/microbiology , Plants/microbiology , Plasmids , Replicon , Rhizobiaceae/genetics , Signal Transduction , Sinorhizobium meliloti/genetics , Synteny , Telomere , Virulence/geneticsABSTRACT
We determined the nucleotide (nt) sequence of a mutation that confers proline overproduction and enhanced tolerance of osmotic stress on bacteria. The mutation, designated as proB74, is an allele of the Escherichia coli proB gene which results in a loss of allosteric regulation of the protein product, gamma-glutamyl kinase. Our sequencing indicated that the proB74 mutation is a substitution of an A for a G at nt position 319 of the coding strand of the gene, resulting in a change of an aspartate to an asparagine at amino acid (aa) residue 107 of the predicted protein product. Rushlow et al. [Gene 39 (1984) 109-112] determined that another proB mutation (designated as DHPR), that resulted in a loss of allosteric inhibition by proline of the E. coli gamma-glutamyl kinase, was due to a substitution of an alanine for a glutamate at aa residue 143. Therefore, even though both the DHPR and the proB74 mutations caused a loss of allosteric inhibition of gamma-glutamyl kinase, they are due to different amino acid substitutions.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Genes, Regulator , Mutation , Phosphotransferases (Carboxyl Group Acceptor) , Proline/biosynthesis , Alleles , Allosteric Regulation , Base Sequence , Crosses, Genetic , Escherichia coli/enzymology , Genes , Osmolar Concentration , Phosphotransferases/genetics , Phosphotransferases/metabolismABSTRACT
We constructed plasmids carrying the Escherichia coli proB gene that encodes gamma-glutamyl kinase, under the control of the yeast GAL1 promoter. This construction was carried out with both the wild-type proB+ gene and a mutant allele, proB74, that specifies an enzyme resistant to feedback inhibition by proline. Yeast pro1 mutants harboring these plasmids are proline prototrophs. We conclude that the pro1 mutation results in a deficiency in the gamma-glutamyl kinase activity in Saccharomyces cerevisiae. Expression of the proB74 allele in yeast resulted in enhanced resistance to the proline analogue L-azetidine-2-carboxylate and in a 2.4-fold elevation of the intracellular free proline levels. This result suggests that gamma-glutamyl kinase is the rate limiting step in proline biosynthesis in yeast.
Subject(s)
Escherichia coli/genetics , Genes, Bacterial , Genes , Mutation , Phosphotransferases (Carboxyl Group Acceptor) , Phosphotransferases/genetics , Proline/metabolism , Saccharomyces cerevisiae/genetics , Alleles , Cloning, Molecular , DNA Restriction Enzymes , Escherichia coli/enzymology , Genotype , PlasmidsABSTRACT
A combined genetic and physical map of the Agrobacterium tumefaciens A348 (derivative of C58) genome was constructed to address the discrepancy between initial single-chromosome genetic maps and more recent physical mapping data supporting the presence of two nonhomologous chromosomes. The combined map confirms the two-chromosome genomic structure and the correspondence of the initial genetic maps to the circular chromosome. The linear chromosome is almost devoid of auxotrophic markers, which probably explains why it was missed by genetic mapping studies.