Mapping nif genes of Rhodobacter capsulatus, and characterization of a mutant with a pleiotropic defect in nitrogen metabolism

Abstract

Mutations affecting the ability of the photosynthetic bacterium Rhodobacter capsulatus to fix nitrogen (Nif- ) have previously been mapped into six linkage groups (I-VI) by transduction with gene transfer agent (GTA). Because this vector carries only 4600 bp of DNA, linkage can only be demonstrated for short chromosomal distances. In order to demonstrate linkage on a larger scale, the self- transmissible plasmid, pBLM2, which has chromosome mobilizing ability in R. capsulatus, was used to map these six linkage groups relative to a number of auxotrophic markers. In this way, nif genes were found to lie in three regions of the chromosome. GTA linkage groups IV and VI were found to reside in the same region, based on their common linkages to adenosine and tryptophan markers. A second region was found to contain GTA groups I and V, both of which were linked by conjugation to isoleucine/valine and leucine markers. In a third region, group III was found to be located near the structural gene for glutamine synthetase (glnA). A seventh GTA linkage group, containing mutations which cause pleiotropic defects in nitrogen metabolism, was also found to lie in the same region with group III and glnA>> Mutants in this group have functional nitrogenase and normal regulation of nif gene expression. However, they are unable to grow on N2 or on most amino acids. The only nitrogen sources found to allow photosynthetic growth of these mutants were ammonium and asparagine. One of these mutants was studied in more detail and found to be able to grow aerobically on most nitrogen sources. Studies of gene expression, using a lac gene fusion, indicated that the gene altered in this mutant may be under the control of an ntrC- like gene, and that it also may regulate its own synthesis. Information from the lac fusion also suggests that there may be two genes in group VII, both of which are required for growth on low levels of ammonia.