Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis.

Constitutive biosynthesis of lipid A via the Raetz pathway is essential for the viability and fitness of Gram-negative bacteria, includingChlamydia trachomatis Although nearly all of the enzymes in the lipid A biosynthetic pathway are highly conserved across Gram-negative bacteria, the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN (UDP-DAGn) to form lipid X is carried out by two unrelated enzymes: LpxH in beta- and gammaproteobacteria and LpxI in alphaproteobacteria. The intracellular pathogenC. trachomatislacks an ortholog for either of these two enzymes, and yet, it synthesizes lipid A and exhibits conservation of genes encoding other lipid A enzymes. Employing a complementation screen against aC. trachomatisgenomic library using a conditional-lethallpxHmutantEscherichia colistrain, we have identified an open reading frame (Ct461, renamedlpxG) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function inE. coliand catalyzes the conversion of UDP-DAGn to lipid Xin vitro LpxG shows little sequence similarity to either LpxH or LpxI, highlighting LpxG as the founding member of a third class of UDP-DAGn hydrolases. Overexpression of LpxG results in toxic accumulation of lipid X and profoundly reduces the infectivity ofC. trachomatis, validating LpxG as the long-sought-after UDP-DAGn pyrophosphatase in this prominent human pathogen. The complementation approach presented here overcomes the lack of suitable genetic tools forC. trachomatisand should be broadly applicable for the functional characterization of other essentialC. trachomatisgenes.IMPORTANCEChlamydia trachomatisis a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of manyChlamydiagenes remain uncharacterized, including the essential gene encoding the UDP-DAGn pyrophosphatase activity for the biosynthesis of lipid A, the membrane anchor of lipooligosaccharide and the predominant lipid species of the outer leaflet of the bacterial outer membrane. We designed a complementation screen against theC. trachomatisgenomic library using a conditional-lethal mutant ofE. coliand identified the missing essential gene in the lipid A biosynthetic pathway, which we designatedlpxG We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activityin vitro Overexpression of LpxG inC. trachomatisleads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating thein vivofunction of LpxG and highlighting the importance of regulated lipid A biosynthesis inC. trachomatis.

Mount Carmel in the Commune: Promoting the Holy Land in Central Italy in the 13th and 14th Centuries

The Carmelite friars were the last of the major mendicant orders to be established in Italy. Originally an eremitical order, they arrived from the Holy Land in the 1240s, decades after other mendicant orders, such as the Franciscans and Dominicans, had constructed churches and cultivated patrons in the burgeoning urban centers of central Italy. In a religious market already saturated with friars, the Carmelites distinguished themselves by promoting their Holy Land provenance, eremitical values, and by developing an institutional history claiming to be descendants of the Old Testament prophet Elijah. By the end of the 13th century the order had constructed thriving churches and convents and leveraged itself into a prominent position in the religious community. My dissertation analyzes these early Carmelite churches and convents, as well as the friars’ interactions with patrons, civic governments, and the urban space they occupied. Through three primary case studies – the churches and convents of Pisa, Siena and Florence – I examine the Carmelites’ approach to art, architecture, and urban space as the order transformed its mission from one of solitary prayer to one of active ministry.

My central questions are these: To what degree did the Carmelites’ Holy Land provenance inform the art and architecture they created for their central Italian churches? And to what degree was their visual culture instead a reflection of the mendicant norms of the time?

I have sought to analyze the Carmelites at the institutional level, to determine how the order viewed itself and how it wanted its legacy to develop. I then seek to determine how and if the institutional model was utilized in the artistic and architectural production of the individual convents. The understanding of Carmelite art as a promotional tool for the identity of the order is not a new one, however my work is the first to consider deeply the order’s architectural aspirations. I also consider the order’s relationships with its de facto founding saint, the prophet Elijah, and its patron, the Virgin Mary, in a more comprehensive manner that situates the resultant visual culture into the contemporary theological and historical contexts.