Cell cycle progression in Caulobacter crescentus depends on the master regulator, CtrA. During the transition from swarmer to stalk cell (G1 to S phase), CtrA is degraded by the AAA+ protease ClpXP and levels rise again in the predivisional stage. The focus of this work is to explore how cyclic, regulated degradation is controlled. CtrA is known to bind to the origin of replication, thereby suppressing replication, so we first asked if DNA binding had an effect on CtrA stability. CtrA is readily degraded by ClpXP on its own, but when bound to DNA containing the proper binding sites, degradation is inhibited. Stabilization is dependent on DNA binding, as CtrA mutants deficient in DNA binding show the same degradation regardless of addition of DNA, as does CtrA in the presence of a mutant origin sequence lacking CtrA binding sites. Looking closely at CtrA degradation in the presence of auxiliary factors suggests that higher order complex formation may be a mechanism of protecting critical cell cycle regulators from premature proteolysis. In vivo study of over-expression of CtrA mutants revealed that accumulation of non-degradable CtrA, CtrA-DD, perturbs the cell cycle, leading to filamentation and a G2 arrest. Over-expression of the DNA-binding domain alone showed filamentation but no G2 arrest, suggesting that CtrA-DD is detrimental for reasons including, but likely not limited to, its ability to bind DNA. Exogenously expressing other domains of CtrA may further elucidate the mechanism of its regulated degradation in vivo.

Introduction: A Neisseria meningitidis carrier study has been conducted among students at Örebro university in Sweden in 2018 and 2019. Pharyngeal samples were collected from 3489 students. PCR for the genes ctrA and crgA was run on all samples. The positive samples were then cultured on agar plates to find the N. meningitidis. In 349 of the PCR positive samples, no N. meningitidis could be isolated, which raised the question if other bacteria could have these genes. The most likely bacteria to have these genes were assumed to be other species within the genus Neisseria. Aim: To identify whether other neisserial species have the ctrA and crgA genes. Methods: The 349 samples were cultured on agar plates for two days. The species were then identified by MALDI-TOF MS. The isolated Neisserial species and some other species as controls were saved. PCR for ctrA and crgA genes were then run on these bacteria to determine whether they possessed these genes. Results: Five N. meningitidis that had been missed by the first round of culture were identified. Seventy-five other colonies of neisserial species were isolated. N. subflava (n=40) were the most common. Nine (12 %) were crgA positive but none were ctrA positive. At least one crgA positive colony was found in four of the five different non-meningococcal neisserial species isolated in this study. Conclusion: The crgA gene seems quite common among non-meningococcal neisserial species while ctrA seems to be specific for N. meningitidis

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