Michael
L. Kahn
Institute
of Biological Chemistry
Washington State University,
Pullman, WA , 99164
A New Platform for Genomic Manipulation of Sinorhizobium meliloti
The availability of genome sequences of many bacteria has created a need for
strategies that allow large-scale genetic manipulation of these bacteria. Various
goals of such manipulation include generating nucleic acid arrays for monitoring
transcription and for using hybridization approaches to examine the genomes
of related bacteria, expressing proteins predicted from the genomic sequence,
mutating predicted genes, and constructing reporter fusions to allow gene expression
to be monitored. Sinorhizobium meliloti is a Gram negative soil bacterium
that can form nitrogen-fixing symbiotic relationships with important forage
crops such as alfalfa and the model legume, Medicago truncatula. The
DNA sequence of S. meliloti 1021 is predicted to contain over 6000
open reading frames on the chromosome and two “megaplasmids”. We
have constructed several new plasmids based on the GATEWAY integrase-mediated
recombination cloning system developed by Invitrogen that have been designed
to make this system more compatible for use in E. coli and other bacteria, including
S. meliloti. These new plasmids have been used in manipulating a cluster of
about 60 genes located on one of the “megaplasmids”, pSymA, that
are involved in denitrification and the response to microaerobic conditions.
From these preliminary results, we conclude that these new plasmids will be
useful in large- and small-scale analysis of the genomic sequences of S.
meliloti.
Approaching the Genetic Basis of Medicago’s Contribution to Symbiotic Effectiveness
When plant cultivars and bacterial strains are tested in different
combinations, no one cultivar or strain is typically found to be superior and
there is little understanding of which symbiont traits contribute to the success
of a particular interaction. In addition, breeding more effective plants has
been hampered by the complexity of using nitrogen fixation as a quantitative
trait. In previous work, we developed an isotope-dilution mass spectrometry
method for measuring nitrogen isotopes in chlorophyll (pheophytin), and used
this assay to estimate the contribution of symbiotic nitrogen fixation to plant
nitrogen content.
To investigate plant determinants of effectiveness, we screened 16 isolates
of Sinorhizobium meliloti for differential performance on two lines
of Medicago truncatula, Jemalong-6 and DZA315.16. Following infection
of plants grown on 15N-urea, 15N content of pheophytin was measured. When Jemalong-6
was infected with a citrate synthase point mutant, S. meliloti MK506,
only 36% of pheophytin nitrogen was from fixation, much lower than the 63% value
obtained for DZA315.16. In contrast, Jemalong-6 obtained 63% of its nitrogen
from fixation when infected with S. meliloti strain USDA1600 compared to 44%
for DZA315.16.
The assay measures a parameter that should correlate closely with the contribution
that nitrogen fixation makes to plant nutrition. With it, we hope to be able
to measure enough samples in a set of recombinant inbred progeny lines from
a cross between these parental lines to establish the linkage relationships
responsible for the differences in symbiotic performance.
Carnitine metabolism
In E. coli, the use of carnitine as a terminal electron acceptor depends on a functional caiTABCDE operon. It had been suggested that the adjacent but divergent fixABCX operon is also required for carnitine metabolism, perhaps to provide electrons for carnitine reduction. We have constructed E. coli fixA and fixB mutants and find that they are unable to reduce carnitine to g-butyrobetaine under anaerobic conditions.
