Regulation of production, maturation and lytic release of the Rhodobacter capsulatus gene transfer agent

The Rhodobacter capsulatus gene transfer agent (RcGTA) is a phage-like particle that mediates horizontal gene transfer between R. capsulatus strains, and its production is regulated by several bacterial systems, including quorum sensing and the CckA-ChpT-CtrA phosphorelay. This thesis presents evidence that RcGTA is released from cells by cell lysis, that lysis is modulated by the concentration of inorganic phosphate in the growth medium and that lysis requires an endolysin and holin gene. The expression of the lysis genes is regulated by the histidine kinase CckA, the phosphotransferase ChpT and the response regulator CtrA, and requires phosphorylation of CtrA. The endolysin and holin were characterized by expression in E. coli. High resolution electron microscopy images of affinity-purified RcGTA confirmed that RcGTA contains tail fibers and head spikes, and newly revealed the presence of a baseplate-like structure. RcGTA was found to undergo a maturation process similar to that of phages, and this maturation was regulated by the CckA-ChpT-CtrA phosphorelay. Cells lacking CckA produced tail-less particles containing DNA and polytube structures. During particle assembly spikes are attached to the head of RcGTA, and spike formation required ghsA and ghsB, which appear to be co-transcribed and regulated by CckA-ChpT-CtrA and quorum sensing. Spikes were required for efficient binding of RcGTA to the R. capsulatus capsular polysaccharide. Two new regulators of RcGTA, ClpX and DivL, were identified. ClpX was required for transduction, and capsid formation in cells lacking ClpX appeared to halt RcGTA production prior to DNA packaging. DivL appeared to be involved in regulating the CckA kinase activity; however a loss of DivL resulted in opposite phenotypes for an RcGTA overproducer and a wild type strain. Additionally, RcGTA production was found to be stimulated by temporary depletion of amino acids, but did not require the (p)ppGpp-mediated stringent response or a homologue of the general stress response sigma factor EcfG.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

A Sterol Panel for Multiple Rare Lipid Disorders: Validation and Application for Sitosterolemia, Cerebrotendinous Xanthomatosis and Smith-Lemli-Opitz Syndome

BACKGROUND. Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoration of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements are limited in many parts of the world. METHODS. A simple and rapid method to extract free sterols from human serum and quantitate their concentration using isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be “compatible” with routine workflows (eg. 96-well format) in a clinical lab and was extensively validated. Serum from 73 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n=7), Smith-Lemli-Optiz syndrome (SLOS; n=1) and cerebrotendinous xanthomatosis (CTX; n=1) were analyzed. RESULTS. All seven sitosterolemia patients were measured to have greatly elevated levels of free plant sterols (sitosterol, campesterol and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC and an unidentified compound (likely 8-dehydrocholesterol). CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful in the diagnosis of desmosterolosis and lathosterolosis. CONCLUSION. The reported method is a relatively simple and fast method capable of quantitating diagnostically important sterols and differentiating patients with several rare lipid disorders from controls