Lipopolysaccharide is one of the major constituents of the Gram-negative bacterial outer membrane and is a potent stimulator of the host innate immune response. The biosynthesis of the lipid A moiety of lipopolysaccharide is a complex process in which multiple gene products are involved. Two late lipid A acyl transferases, LpxL and LpxM, were first identified in Escherichia coli and shown to be responsible for the addition of secondary acyl chains to the 2 and 3 positions of lipid A, respectively. Here, we describe the identification of two lpxL homologues in the genome of Bordetella pertussis. We show that one of them, LpxL2, is responsible for the addition of the secondary myristate group that is normally present at the 2 position of B. pertussis lipid A, whereas the other one, LpxL1, mediates the addition of a previously unrecognized secondary 2-hydroxy laurate at the 2 position. Increased expression of lpxL1 results in the appearance of a hexa-acylated lipopolysaccharide form with strongly increased endotoxic activity. In addition, we show that an lpxL1-deficient mutant of B. pertussis displays a defect in the infection of human macrophages.Pertussis or whooping cough is a severe acute respiratory illness that is characterized by paroxysmal coughing and a distinctive "whooping" sound when air is subsequently inhaled. The disease is highly contagious and most severe in neonates and children younger than one year. Pertussis is caused by the Gram-negative bacterium Bordetella pertussis. While the genus Bordetella currently encompasses nine species, apart from B. pertussis only three other members, Bordetella bronchiseptica, Bordetella parapertussis, and Bordetella holmesii, have been associated with respiratory infections in humans and other mammals (1). The Gram-negative bacterial cell envelope is composed of two membranes, the inner and the outer membrane, which are separated by the periplasm. The inner membrane is a symmetrical bilayer composed of phospholipids, whereas the outer membrane is asymmetric and consists of phospholipids in the inner leaflet and lipopolysaccharide (LPS) 2 in the outer leaflet. LPS, which is also known as endotoxin, consists of three distinct structural domains: lipid A, the core, and the O-antigen (2). The first domain, lipid A, functions as a hydrophobic membrane anchor and forms the bioactive component of the molecule (3). The structure of lipid A is conserved among different bacterial groups, indicating its importance for the correct functioning of the outer membrane. Generally, lipid A consists of a ␤-1Ј,6-linked D-glucosamine (GlcN) disaccharide carrying ester-and amide-linked 3-hydroxyl fatty acids at the C-2, C-3, C-2Ј, and C-3Ј positions, and phosphate groups at positions C-1 and C-4Ј. The endotoxic activity of LPS is based on the recognition of lipid A by the TLR4/MD-2 complex of the host, which leads to the activation of NF-B and, consequently, to an increased production and secretion of proinflammatory cytokines, such as IL-6, tumor necrosis factor-␣, and IL-1␤ (4). C...

SUMMARYA strategy for gene cloning in the cyanobacterium Anacystis nidulans R2 was developed which made use of a gene library constructed in a shuttle cosmid vector. The method involved phenotypic complementation of mutants with pooled cosmid DNA. The development of the procedure and its application to the cloning of a third gene involved in nitrate reduction are described.