Genetic Indicators for Calcium Signaling Studies in Toxoplasma gondii.

Fluctuations of the cytosolic calcium ion (Ca2+) concentration regulate a variety of cellular functions in all eukaryotes. Cells express a sophisticated set of mechanisms to balance the cytosolic Ca2+ levels and the signals that elevate Ca2+ in the cytosol are compensated by mechanisms that reduce it. Alterations in Ca2+-dependent homeostatic mechanisms are the cause of many prominent diseases in humans, such as heart failure or neuronal death.The genetic tractability of Toxoplasma gondii and the availability of genetic tools enabled the use of Genetically Encoded Calcium Indicators (GECIs) expressed in the cytoplasm, which started a new era in the studies of Toxoplasma calcium signaling. It was finally possible to see Ca2+ oscillations prior to exit of the parasite from host cells. Years after Endo et al showed that ionophores triggered egress, the assumption that oscillations occur prior to egress from host cells has been validated by experiments using GECIs. GECIs allowed the visualization of specific Ca2+ signals in live intracellular parasites and to distinguish these signals from host cell calcium fluctuations. In this chapter we present an overview describing "tried and true" methods of our lab who pioneered the first use of GECI's in Toxoplasma, including GECI choice, methodology for transfection and selection of ideal clones, their characterization, and the use of GECI-expressing parasites for fluorometric and microscopic analysis.

Novel heterologous bacterial system reveals enhanced susceptibility to DNA damage mediated by yqgF, a nearly ubiquitous and often essential gene.

Despite its presence in most bacteria, yqgF remains one of only 13 essential genes of unknown function in Escherichia coli. Predictions of YqgF function often derive from sequence similarity to RuvC, the canonical Holliday junction resolvase. To clarify its role, we deleted yqgF from a bacterium where it is not essential, Acinetobacter baylyi ADP1. Loss of yqgF impaired growth and increased the frequency of transformation and allelic replacement (TAR). When E. coli yqgF was inserted in place of its A. baylyi chromosomal orthologue, wild-type growth and TAR were restored. Functional similarities of yqgF in both gamma-proteobacteria were further supported by defective 16S rRNA processing by the A. baylyi mutant, an effect previously shown in E. coli for a temperature-sensitive yqgF allele. However, our data question the validity of deducing YqgF function strictly by comparison to RuvC. A. baylyi studies indicated that YqgF and RuvC can function in opposition to one another. Relative to the wild type, the ΔyqgF mutant had increased TAR frequency and increased resistance to nalidixic acid, a DNA-damaging agent. In contrast, deletion of ruvC decreased TAR frequency and lowered resistance to nalidixic acid. YqgF, but not RuvC, appears to increase bacterial susceptibility to DNA damage, including UV radiation. Nevertheless, the effects of yqgF on growth and TAR frequency were found to depend on amino acids analogous to catalytically required residues of RuvC. This new heterologous system should facilitate future yqgF investigation by exploiting the viability of A. baylyi yqgF mutants. In addition, bioinformatic analysis showed that a non-essential gene immediately upstream of yqgF in A. baylyi and E. coli (yqgE) is similarly positioned in most gamma- and beta-proteobacteria. A small overlap in the coding sequences of these adjacent genes is typical. This conserved genetic arrangement raises the possibility of a functional partnership between yqgE and yqgF.