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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 pathogen\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            lacks 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 a\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            genomic library using a conditional-lethal\n            <jats:italic>lpxH<\/jats:italic>\n            mutant\n            <jats:named-content content-type=\"genus-species\">Escherichia coli<\/jats:named-content>\n            strain, we have identified an open reading frame (Ct461, renamed\n            <jats:italic>lpxG<\/jats:italic>\n            ) encoding a previously uncharacterized enzyme that complements the UDP-DAGn hydrolase function in\n            <jats:named-content content-type=\"genus-species\">E. coli<\/jats:named-content>\n            and catalyzes the conversion of UDP-DAGn to lipid X\n            <jats:italic>in vitro<\/jats:italic>\n            . 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 of\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            , 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 for\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            and should be broadly applicable for the functional characterization of other essential\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            genes\n            <jats:italic>.<\/jats:italic>\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            <jats:named-content content-type=\"genus-species\">Chlamydia trachomatis<\/jats:named-content>\n            is a leading cause of infectious blindness and sexually transmitted disease. Due to the lack of robust genetic tools, the functions of many\n            <jats:italic>Chlamydia<\/jats:italic>\n            genes 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 the\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            genomic library using a conditional-lethal mutant of\n            <jats:named-content content-type=\"genus-species\">E. coli<\/jats:named-content>\n            and identified the missing essential gene in the lipid A biosynthetic pathway, which we designated\n            <jats:italic>lpxG<\/jats:italic>\n            . We show that LpxG is a member of the calcineurin-like phosphatases and displays robust UDP-DAGn pyrophosphatase activity\n            <jats:italic>in vitro<\/jats:italic>\n            . Overexpression of LpxG in\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            leads to the accumulation of the predicted lipid intermediate and reduces bacterial infectivity, validating the\n            <jats:italic>in vivo<\/jats:italic>\n            function of LpxG and highlighting the importance of regulated lipid A biosynthesis in\n            <jats:named-content content-type=\"genus-species\">C. trachomatis<\/jats:named-content>\n            .\n          <\/jats:p>","DOI":"10.1128\/mbio.00090-16","type":"journal-article","created":{"date-parts":[[2016,3,23]],"date-time":"2016-03-23T01:51:16Z","timestamp":1458697876000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":27,"title":["Discovery of the Elusive UDP-Diacylglucosamine Hydrolase in the Lipid A Biosynthetic Pathway in Chlamydia trachomatis"],"prefix":"10.1128","volume":"7","author":[{"given":"Hayley E.","family":"Young","sequence":"first","affiliation":[{"name":"Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jinshi","family":"Zhao","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jeffrey R.","family":"Barker","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ziqiang","family":"Guan","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Raphael H.","family":"Valdivia","sequence":"additional","affiliation":[{"name":"Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7823-3416","authenticated-orcid":false,"given":"Pei","family":"Zhou","sequence":"additional","affiliation":[{"name":"Department of Biochemistry, Duke University Medical Center, Durham, North Carolina, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_2_2_2","doi-asserted-by":"publisher","DOI":"10.1086\/652397"},{"key":"e_1_3_2_3_2","first-page":"139","volume-title":"Chlamydia: intracellular biology, pathogenesis, and immunity","author":"Schachter J","year":"1999","unstructured":"Schachter J. 1999. 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