Inhibition of Lipopolysaccharide Biosynthesis and Cell Growth following Inactivation of the kdtA Gene in Escherichia coli

Belunis, Charles; Clementz, Tony; Carty, Sherry M.; Raetz, Christian R. H.

doi:10.1074/jbc.270.46.27646

Cited by 115 publications

(144 citation statements)

References 29 publications

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“…The essential role of Kdo was first established by the characterization of Salmonella mutants conditionally blocked in the biosynthesis of this sugar acid (Rick and Osborn, 1977). Recently, Belunis et al (1995) confirmed this data with a temperature-sensitive knockout system for the Kdo transferase of Escherichia coli. Based on these findings, the enzymes involved in the biosynthesis and activation of Kdo evoked increasing interest as potential targets for new types of antibiotics with broad reactivity against all gram-negative bacteria (Goldman et al, 1987;Hammond et al, 1987).…”

supporting

confidence: 65%

Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

Brabetz¹,

Brade²

1997

European Journal of Biochemistry

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The kdsA gene encoding 3-deoxy-~-manno-2-octulosonate-8-phosphate (Kdo-8-P) synthase of Chlamydia psittaci 6BC was cloned by complementing the temperature-sensitive kdsA mutant Salmonella enterica serovar Typhimurium AG701i50. The sequence analysis of a recombinant DNA fragment revealed an open reading frame of 807 nucleotides which codes for a polypeptide of 269 amino acids with a high degree of similarity to known KdsA proteins. In addition, alignments of Kdo-8-P synthases with bacterial and fungal 3-deoxy-~-arabino-2-heptulosonate-7-phosphate (Dha-7-P) synthases suggested that both classes of enzymes are structurally related and may belong to a family of 2-keto-3-deoxy-aldonic acid synthases. The chlamydial protein was overexpressed and functionally characterized in vitro to synthesize Kdo-8-P from D-arabinose 5-phosphate and phosphoennlpyruvate. A chlamydial DNA region upstream of the gene exhibiting similarities to the consensus sequence of a ' " promoters of Escherichia coli was responsible for the heterologous expression of kdsA.Keywords: 3-deoxy-~-manno-2-octulosonate-8-phosphate synthase; lipopolysaccharide biosynthesis; Chlamydia.Lipopolysaccharides (LPS) are amphiphilic molecules anchored in the outer leaflet of the outer membrane of gram-negative bacteria (Rietschel et al., 1994). In general, the architecture of LPS consists of a phosphorylated and acylated p(l+6)-linked glucosainine disaccharide, termed lipid A, to which a variable carbohydrate moiety is covalently linked. For genetic and biosynthetic reasons the sugar moiety may be further divided into a lipid-A proximal core and an outer 0-antigen region (Schnaitman and Klena, 1993). As surface structures, LPS play an important role in the interaction of gram-negative bacteria with higher organisms and are responsible for many immunological and pathophysiological effects which are observed during infectious diseases. On the other hand, many cellular characteristics of gram-negative bacteria like motility or the resistance to antibiotics may be correlated with biophysical and biochemical properities of LPS (Schnaitman and Klena, 1993;Nikaido, 1994). In particular, it is known from the structural analysis of various species, as well as mutants with defects in the biosynthesis of the inner core, that 3-deoxy-D-manno-2-octu~osonic acid (Kdo) linked to lipid A is a basic constituent of all LPS and Abbreviations. Dhd-7-P, 3-deoxy-~-arabino-2-heptulosonate-7-phosphate; Kdo, 3-deoxy-~-manno-2-octulosonate ; Kdo-k-P, 3-deoxy-D-manno-2-octulosonate-8-phosphate; LPS, lipopolysaccharide.Enzymes. 3-Deoxy-~-arabino-2-heptulosonate-7-phosphate synthase (EC 4.1.2.15); 3-deoxy-~-munno-2-octulosonate-8-phosphate synthase (EC 4.1.2.1 6 ) ; CTP: 3-deoxy-D-matmo-2-octu~osonate cytidylyltransferase, CMP-Kdo synthetase (EC 2.7.7.38); 3-deoxy-~-mannu-2-octulosonate transferase (EC

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confidence: 65%

Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

Brabetz¹,

Brade²

1997

European Journal of Biochemistry

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“…Furthermore, the E. coli Kdo transferase (KdtA) (34,35) requires the presence of an unsubstituted 4Ј-phosphate moiety in its acceptor substrates, 2 consistent with the idea that incorporation of the L-Ara4N moiety at the 4Ј-phosphate position occurs later than Kdo addition during normal lipopolysaccharide biosynthesis. Conversely, given the structures of lipid A precursors in Kdodeficient mutants of S. typhimurium in which the pEtN substituent occurs solely at the 4Ј-position (6,19), it appears that pEtN addition to the 1-phosphate must also be Kdo-dependent, whereas pEtN transfer to the 4Ј-phosphate is not.…”

Section: Discussionmentioning

confidence: 76%

Lipid A Modifications in Polymyxin-resistant Salmonella typhimurium

Zhou

Ribeiro

Lin

et al. 2001

Journal of Biological Chemistry

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128

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Salmonella typhimurium and related organisms synthesize lipid A by the same pathway as Escherichia coli K-12 (1, 2), but they usually modify the final product with additional covalent appendages (Fig. 1A), such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) 1 (3-7), phosphoethanolamine (pEtN) (4 -6), (S)-2-hydroxymyristate (8, 9), and palmitate (5, 6, 10 -12). Different combinations of these substituents account for the remarkable heterogeneity of lipid A molecules found in S. typhimurium.The biosynthesis of lipid A modifications is under the control of the PhoP/PhoQ and the PmrA/PmrB two-component signaling systems (13-15). Addition of the L-Ara4N unit is required for resistance to polymyxin (16 -18). Incorporation of the palmitoyl chain confers resistance to certain cationic anti-microbial peptides (11). Modification of lipid A with L-Ara4N, pEtN, and/or palmitate may also occur in E. coli K-12, but only under special circumstances, as in polymyxin-resistant (pmrA constitutive) mutants (17) or in wild type cells exposed to ammonium metavanadate (7,19).With the exception of PagP, the outer membrane enzyme that incorporates palmitate (12, 20), the enzymes responsible for the covalent modifications of lipid A have not been identified. The L-Ara4N residue is attached primarily to the 4Ј-phosphate group of lipid A in wild type S. typhimurium or in metavanadate-treated E. coli, whereas pEtN is usually attached to the 1-phosphate (19). However, in S. typhimurium mutants defective in Kdo biosynthesis, lipid A precursors accumulate in which L-Ara4N is linked exclusively to the 1-phosphate, and pEtN is attached mainly to the 4Ј-phosphate (5,6,19). The enzymatic pathways that account for these interesting and complex structural anomalies are unknown.An important clue to the origin of the L-Ara4N moiety has emerged from the discovery of the pmrE and pmrF genes, which are required for the maintenance of polymyxin resistance and the biosynthesis of L-Ara4N-modified lipid A (18). The pmrE (ugd) gene encodes UDP-glucose dehydrogenase (18), suggesting that L-Ara4N is derived from UDP-glucuronic acid. The pmrF gene encodes a homologue of yeast dolichyl phosphate-mannose synthase and is part of an operon (18) that includes additional open reading frames hypothesized to encode other putative enzymes required for L-Ara4N biosynthesis and attachment to lipid A (7, 21). The operon is regulated directly by PmrA (18), which in turn may be activated by PhoP/PhoQ, low pH, or ferric ions (14,22,23). So far, no in vitro assays have been developed to validate the functions of the proteins encoded by the pmrF operon.Because of its heterogeneity (Fig. 1A), S. typhimurium lipid * This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H.R.), AI-30479 (to S. I. M.), and GM54882 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.** To w...

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“…Galactosyltransferase reactions were identical but also included 1.0 mM UDP-galactose in addition to the above components. Distal Kdo-transferase assays contained all the galactosyltransferase reaction components plus 2 mM Kdo, 5 mM CTP, 10 mM MgCl 2 , and 1.8 milliunits of partially purified CMP-Kdo synthase per 10 l. CMP-Kdo is generated in situ because of its short half-life (minutes) (30).…”

Section: Methodsmentioning

confidence: 99%

“…The labeled lipid IV A was converted to Kdo 2 -[4Ј-32 P]lipid IV A using purified E. coli Kdo-transferase (30,31). The products were purified by preparative thin layer chromatography and stored at Ϫ20°C as an aqueous dispersion (30,31). Prior to each use, these substrates were subjected to ultrasonic irradiation in a water bath for 60 s.…”

Section: Methodsmentioning

confidence: 99%

“…Efficient incorporation of these sugars is dependent upon both the inclusion of R. leguminosarum membranes and the appropriate sugar nucleotides. In the case of CMP-Kdo, a generating system must be used because of the short half-life of this compound (30,31,51,52). lpcA, lpcB, and lpcC must be glycosyltransferase structural genes (Fig.…”

Section: Sequential Addition Of Mannose Galactose and Kdo To The Acmentioning

confidence: 99%

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Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

Kadrmas¹,

Allaway²,

Studholme³

et al. 1998

Journal of Biological Chemistry

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The lipopolysaccharide (LPS) core of the Gram-negative bacterium Rhizobium leguminosarum is more amenable to enzymatic study than that of Escherichia coli because much of it is synthesized from readily available sugar nucleotides. The inner portion of the R. leguminosarum core contains mannose, galactose, and three We have also discovered the new gene (lpcC) that encodes the mannosyltransferase. The gene is separated by several kilobase pairs from the lpcAB cluster. All three glycosyltransferases are carried on cosmid pIJ1848, which contains at least 20 kilobase pairs of R. leguminosarum DNA. Transfer of pIJ1848 into R. meliloti 1021 results in heterologous expression of all three enzymes, which are not normally present in strain 1021. Expression of the lpc genes individually behind the T7 promoter results in the production of each R. leguminosarum glycosyltransferase in E. coli membranes in a catalytically active form, demonstrating that lpcA, lpcB, and lpcC are structural genes.

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Inhibition of Lipopolysaccharide Biosynthesis and Cell Growth following Inactivation of the kdtA Gene in Escherichia coli

Cited by 115 publications

References 29 publications

Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

Molecular Cloning, Sequence Analysis and Functional Characterization of the Gene Kdsa, Encoding 3‐Deoxy‐D‐Manno‐2‐Octulosonate‐8‐Phosphate Synthase of Chlamydia Psittaci 6BC

Lipid A Modifications in Polymyxin-resistant Salmonella typhimurium

Cloning and Overexpression of Glycosyltransferases That Generate the Lipopolysaccharide Core of Rhizobium leguminosarum

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