Directed construction and analysis of a Sinorhizobium meliloti pSymA deletion mutant library.

Resources from the Sinorhizobium meliloti Rm1021 open reading frame (ORF) plasmid libraries were used in a medium-throughput method to construct a set of 50 overlapping deletion mutants covering all of the Rm1021 pSymA megaplasmid except the replicon region. Each resulting pSymA derivative carried a defined deletion of approximately 25 ORFs. Various phenotypes, including cytochrome c respiration activity, the ability of the mutants to grow on various carbon and nitrogen sources, and the symbiotic effectiveness of the mutants with alfalfa, were analyzed. This approach allowed us to systematically evaluate the potential impact of regions of Rm1021 pSymA for their free-living and symbiotic phenotypes.

Construction and expression of sugar kinase transcriptional gene fusions by using the Sinorhizobium meliloti ORFeome.

The Sinorhizobium meliloti ORFeome project cloned 6,314 open reading frames (ORFs) into a modified Gateway entry vector system from which the ORFs could be transferred to destination vectors in vivo via bacterial conjugation. In this work, a reporter gene destination vector, pMK2030, was constructed and used to generate ORF-specific transcriptional fusions to beta-glucuronidase (gusA) and green fluorescent protein (gfp) reporter genes. A total of 6,290 ORFs were successfully transferred from the entry vector library into pMK2030. To demonstrate the utility of this system, reporter plasmids corresponding to 30 annotated sugar kinase genes were integrated into the S. meliloti SM1021 and/or SM8530 genome. Expression of these genes was measured using a high-throughput beta-glucuronidase assay to track expression on nine different carbon sources. Six ORFs integrated into SM1021 and SM8530 had different basal levels of expression in the two strains. The annotated activities of three other sugar kinases were also confirmed.

Development of a functional genomics platform for Sinorhizobium meliloti: construction of an ORFeome.

The nitrogen-fixing, symbiotic bacterium Sinorhizobium meliloti reduces molecular dinitrogen to ammonia in a specific symbiotic context, supporting the nitrogen requirements of various forage legumes, including alfalfa. Determining the DNA sequence of the S. meliloti genome was an important step in plant-microbe interaction research, adding to the considerable information already available about this bacterium by suggesting possible functions for many of the >6,200 annotated open reading frames (ORFs). However, the predictive power of bioinformatic analysis is limited, and putting the role of these genes into a biological context will require more definitive functional approaches. We present here a strategy for genetic analysis of S. meliloti on a genomic scale and report the successful implementation of the first step of this strategy by constructing a set of plasmids representing 100% of the 6,317 annotated ORFs cloned into a mobilizable plasmid by using efficient PCR and recombination protocols. By using integrase recombination to insert these ORFs into other plasmids in vitro or in vivo (B. L. House et al., Appl. Environ. Microbiol. 70:2806-2815, 2004), this ORFeome can be used to generate various specialized genetic materials for functional analysis of S. meliloti, such as operon fusions, mutants, and protein expression plasmids. The strategy can be generalized to many other genome projects, and the S. meliloti clones should be useful for investigators wanting an accessible source of cloned genes encoding specific enzymes.

New recombination methods for Sinorhizobium meliloti genetics.

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 alpha-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.