Studies of bacterial homeostasis Sinorhizobium meliloti and Escherichia coli

Abstract

The symbiosis between Sinorhizobium meliloti and its plant host Medicago sativa, offers a tractable model to explore the bacterial requirements for endocytic survival in a eukaryotic host. It has been shown that during development of this symbiosis, M. sativa releases an oxidative burst that S. meliloti must be able to overcome in order for symbiotic development to continue. Employing a novel two-part screen, I identified Sinorhizobium meliloti mutants that were both sensitive to oxidative stress and symbiotically defective on the host plant Medicago sativa. The mutants affect a wide variety of cellular processes and represent both novel and previously identified genes important in symbiosis. One mutant I identified was disrupted in sitA, which encodes the periplasmic binding protein of the putative iron/manganese ABC transporter SitABCD. Disruption of sitA causes elevated sensitivity to the reactive oxygen species hydrogen peroxide and superoxide. Disruption of sitA leads to elevated catalase activity and a severe decrease in superoxide dismutase B (SodB) activity and protein level. The decrease in SodB level strongly correlates with the superoxide sensitivity of the sitA mutant. I demonstrate that all free-living phenotypes of the sitA mutant can be rescued by the addition of exogenous manganese but not iron, a result that strongly implies SitABCD plays an important role in manganese uptake in S. meliloti. A second mutant I identified in my screen was disrupted in a previously unexplored orf, SMc0lll3. SMc0lll3 produces anl8 kD protein that is a member of a highly conserved family, universal among bacteria. In addition to being required for S. meliloti symbiosis with alfalfa, SMc01113 is also required to protect the bacterium from a wide range of environmental stresses.

(cont.) Our findings support a role for this novel protein in RNA and/or phospholipid metabolism. The striking pleiotropy of the SMc01113 mutant lead me to further investigate the molecular function of SMc01113. I show that the SMc01113 protein is part of a large Cluster of Orthologous Group (COG), COG0319 and that homologs of this protein are functionally equivalent. Using the model system of Escherichia coli, I demonstrate that the E. coli homolog, YbeY, is required for ribosome maturation. Loss of YbeY activity affects maturation of both 16S and 23S rRNA and causes a severe loss of polysomes. 70S ribosomes formed in a [Delta]ybeY mutant show reduced translational activity and fidelity. I further demonstrate the human homolog, C21 orf57, may play a similar role in human mitochondria. While investigating the [Delta]ybeY mutant, I found that, in contrast to the wide range of stresses it was sensitive to, the [Delta]ybeY mutant was very resistant to the DNA replication inhibitor hydroxyurea. Using a systems-level analysis of the genomic transcriptional response to hydroxyurea, I show that hydroxyurea triggers pathways involved in both cell survival and cell death, and suggest a model where, for any given bacterium in a population, hydroxyurea can induce a molecular switch from a survival mode to a programmed cell death mode. I use this model to explore possible mechanisms for the increased resistance of the [Delta]ybeY mutant to hydroxyurea.