SSB binds to the RecG and PriA helicases in vivo in the absence of DNA.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 m ammonium sulfate must be present, bringing into question the validity of these results as these are nonphysiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using the purification of tagged complexes, our results show that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the 'storage form' of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB, which, by virtue of its high affinity for single-stranded DNA, allows these helicases to outcompete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

Fluorescent single-stranded DNA-binding proteins enable in vitro and in vivo studies.

Fluorescent single-stranded DNA-binding proteins (SSB) that have a defined number of fluorophores per tetramer are invaluable tools to understand biochemical mechanism and biological function. Here, we describe the purification of fluorescent SSB chimeras with a unique number of fluorescent subunits incorporated per tetramer. We describe the use of these tetramers to enable clear visualization of SSB in vivo. Purified chimeras also facilitate single molecule studies (Liu et al., Protein Sci 20:1005-1020, 2011).

Profile analysis and modeling of reduced tillage effects on soil nitrous oxide flux.

The impact of no-till (NT) and other reduced tillage (RT) practices on soil to atmosphere fluxes of nitrous oxide (N(2)O) are difficult to predict, and there is limited information regarding strategies for minimizing fluxes from RT systems. We measured vertical distributions of key microbial, chemical, and physical properties in soils from a long-term tillage experiment and used these data as inputs to a process-based model that accounts for N(2)O production, consumption, and gaseous diffusion. The results demonstrate how differences among tillage systems in the stratification of microbial enzyme activity, chemical reactivity, and other properties can control N(2)O fluxes. Under nitrification-dominated conditions, simulated N(2)O emissions in the presence of nitrite (NO(2)(-)) were 2 to 10 times higher in NT soil compared to soil under conventional tillage (CT). Under denitrification-dominated conditions in the presence of nitrate (NO(3)(-)), higher bulk density and water content under NT promoted higher denitrification rates than CT. These effects were partially offset by higher soluble organic carbon and/or temperature and lower N(2)O reduction rates under CT. The NT/CT ratio of N(2)O fluxes increased as NO(2)(-) or NO(3)(-) was placed closer to the surface. The highest NT/CT ratios of N(2)O flux (>30:1) were predicted for near-surface NO(3)(-) placement, while NT/CT ratios < 1 were predicted for NO(3)(-) placement below 15 cm. These results suggest that N(2)O fluxes from RT systems can be minimized by subsurface fertilizer placement and by using a chemical form of fertilizer that does not promote substantial NO(2)(-) accumulation.