A resident of Florida returned from a short visit to southern Africa to find a male Amblyomma hebraeum tick attached to the skin behind her knee. Amblyomma hebraeum is a major vector of 2 pathogens that cause important diseases in southern Africa, heartwater of ruminants and African tick-bite fever of humans. The tick was tested by polymerase chain reaction assay for evidence of infection with Cowdria ruminantium and Rickettsia africae (the causative agents of heart-water and African tick-bite fever, respectively) and was found to be negative for both agents. This is the second record of the exotic tick, A. hebraeum, being introduced into the United States on a human host.

DNA replication is a fundamental biological process that is tightly regulated in all living cells. In bacteria, the master regulator DnaA controls when and where replication begins by building a step-wise complex that loads the replicative helicase onto chromosomal DNA. In many bacteria, DnaA requires the adaptor proteins DnaD and DnaB to aid DnaA during helicase loading. How DnaA, its adaptors, and the helicase form a complex at the origin is largely unknown. In this study, we addressed this long-standing question by disassembling the initiation proteins into their individual domains and testing all possible pair-wise combinations in a bacterial two-hybrid assay. Here we report a full description of the cryptic interaction sites used by the helicase loading machinery from Bacillus subtilis. In addition, we investigated how complex formation of the helicase loading machinery is regulated by the checkpoint protein SirA, which is a potent replication inhibitor in sporulating cells. We found that SirA and the DnaD adaptor bind overlapping sites on DnaA, and therefore SirA acts as a competitive inhibitor to block initiation. The interaction between DnaA and DnaD was also mapped to the same DnaA surface in the human pathogen Staphylococcus aureus, demonstrating the broad conservation of this interface. Therefore, our approach has unveiled key protein interactions essential for initiation and is widely applicable for mapping interactions in other signaling pathways that are governed by cryptic binding surfaces.Author SummaryIn order to proliferate, bacteria must first build a step-wise protein complex on their chromosomes that determines when and where DNA replication begins. This protein complex is assembled through dynamic interactions that have been difficult to study and remain largely uncharacterized. Here we show that by deconstructing the proteins into their constituent domains, the interactions used to build the initiation complex can be readily detected and mapped to single amino acid resolution. Using this approach, we demonstrate that DNA replication is controlled through conformational changes that dictate the availability of interaction surfaces. In addition, negative regulators can also block DNA replication by influencing complex formation so that cells survive inhospitable conditions. Initiation proteins from the model organism B. subtilis and the human pathogen S. aureus were both used to underscore the general applicability of the results to different bacterial systems. Furthermore, our general strategy for mapping dynamic protein interactions is suitable for many different signaling pathways that are controlled through cryptic interaction surfaces.

16The DNA damage response is a signaling pathway found throughout biology. In many bacteria 17 the DNA damage checkpoint is enforced by inducing expression of a small, membrane bound 18 inhibitor that delays cell division providing time to repair damaged chromosomes. How cells 19 sense successful DNA repair and promote checkpoint recovery is unknown. By using a high-20 throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that 21 promote DNA damage checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads 22 to accumulation of the checkpoint protein YneA. DNA damage sensitivity and increased cell 23 elongation in protease mutants depends on yneA. Further, expression of YneA in protease 24 mutants was sufficient to inhibit cell proliferation. Finally, we show that one of the two 25 proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for 26 DNA damage checkpoint recovery in bacteria that use membrane bound cell division inhibitors. 27