New Recombination Methods for Sinorhizobium meliloti Genetics

Abstract

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative ␣-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.Sinorhizobium meliloti is a gram-negative bacterium that is best known for forming nitrogen-fixing symbiotic relationships with legumes, such as alfalfa (25). When not in symbiosis, these rhizobia are part of the normal, free-living soil microflora. S. meliloti cells detect root exudates in the soil and migrate toward, attach to, and invade the root hairs of alfalfa. This process ultimately leads to the formation of specialized root organs called nodules. During the development of nodules, S. meliloti cells differentiate into endosymbiotic forms called bacteroids, which can reduce N 2 to NH 4 ϩ through an energetically expensive process requiring eight low-potential electrons and at least 16 ATP molecules per molecule of N 2 reduced. The bacteroids provide this fixed nitrogen (NH 4 ϩ ) to the plant, and the plant delivers organic acids and perhaps other carbon and energy sources to the bacteroids (6, 10).The DNA sequence of the S. meliloti strain 1021 genome, which consists of a chromosome (3.65 Mb) and two megaplasmids, pSymA (1.35 Mb) and pSymB (1.68 Mb), has been determined (9). This 6.7-Mb genome was predicted to contain at least 6,207 protein-coding genes along with various insertion sequence elements and phage sequences (9). Having the sequence available is a major step toward describing the biology of the bacteria, but new methods for analyzing the genome are needed in order to obtain a more functional description of the roles played by each of these genetic elements. Manipulating a genome requires extensive use of oligonucleotides, over 12,000 for the S. meliloti genome. The expense of these and the attendant cost of cloning and other standard manipulations are a considerable barrier to working with the entire sequence. However, methods b...

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