Altered lipopolysaccharide characteristic of the I69 phenotype in Haemophilus influenzae results from mutations in a novel gene, isn

Abstract

The I69 phenotype of Haemophilus influenzae results from a mutation leading to a lipopolysaccharide molecule consisting only of lipid A and a single phosphorylated 2-keto-3-deoxyoctulosonic acid residue. In this paper we describe the identification of a gene which, when mutated, results in the I69 phenotype. We have named the gene isn. The predicted amino acid sequence of Isn is homologous to the product of the lmbN gene involved in the biosynthesis of the sugar-containing antibiotic lincomycin by Streptomyces lincolnensis. isn is situated between two loci that are homologous to the dpp and art periplasmic permease systems in Escherichia coli. Northern (RNA) blot and primer extension analyses reveal that isn is transcribed as a monocistronic mRNA. Potential functions of Isn protein are discussed.Haemophilus influenzae is a gram-negative bacterium often found as a commensal of the human upper respiratory tract. In infants, serotype b (Hib) strains are a major cause of invasive diseases such as meningitis, cellulitis, epiglottitis, and pneumonia, whilst nontypeable strains are a common cause of otitis media and lower respiratory tract infections, particularly in the developing world (36). Surface molecules such as capsular polysaccharide and lipopolysaccharide (LPS) are important contributors to the virulence of the bacterium (26,27).H. influenzae LPS consists of a toxic lipid A molecule linked via a single phosphorylated 2-keto-3-deoxyoctulosonic acid (KDO) residue to a branched-chain oligosaccharide (26). Microheterogeneity and phase-variable expression of oligosaccharide epitopes have led to difficulty in solving the fine structure of the oligosaccharide, but recently partial structures have been deduced for several strains (29,30).Molecular genetic studies have identified a number of loci necessary for the biosynthesis of H. influenzae LPS. Among these, the lic and lsg loci have been cloned and sequenced, and these data have led to hypotheses about LPS biosynthesis, structure, and mechanisms of phase variation (2,3,10,15,21,22,35,(40)(41)(42). Of the loci identified, one of the least studied at the DNA level is that defined by the I69 mutation. During the construction of a genomic library of chromosomal DNA from an H. influenzae type b strain in a vector, it was observed that transformation of the DNA from one of the recombinant clones back into wild-type H. influenzae produced an opaque colony phenotype. This clone was called I69 and contained a 9.4-kb EcoRI restriction fragment (44). Subsequent analysis of the I69 transformants revealed that their LPS consisted of only lipid A and a single molecule of phosphorylated KDO (13). H. influenzae transformants displaying the I69 phenotype are serum sensitive and avirulent, failing to colonize infant rats after intranasal, intraperitoneal, or intravascular challenge (44).In this report, we show that the I69 phenotype results from the mutation of a previously unidentified gene situated between homologs of the Escherichia coli dpp and art loci, which encode dipeptide ...

The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

“…Plasmid DNA was prepared from E. coli strains by the alkaline-lysis method (Sambrook et al, 1989). Chromosomal DNA was prepared from H. influenzae by the method described by Preston et al (1996). Plasmid DNA was introduced into E. coli by transformation after CaCl 2 treatment (Sambrook et al, 1989) and into H. influenzae by transformation using the MIV procedure (Herriott et al, 1970).…”

Antigenic Variation among Bordetella

“…One gel was visualized by silver staining as described previously (21). Membranes were blocked with 5% nonfat dried milk in 0.1% Triton X-100 in PBS and incubated with the indicated pooled serum at a dilution of 1:3000 in blocking buffer at 4°C overnight.…”

“…Generation of Immune Sera-Groups of five C57/Bl.6 mice (Jackson Laboratory, Bar Harbor, ME) lightly sedated with isoflurane (IsoFlo, Abbott) were infected by intranasal instillation of 5 (21) and transferred to polyvinylidene difluoride membranes (Immobilon-P, Millipore, Fisher). One gel was visualized by silver staining as described previously (21).…”

Post-assembly Modification of Bordetella bronchiseptica O Polysaccharide by a Novel Periplasmic Enzyme Encoded by wbmE

“…for SDS-PAGE analysis was obtained from B. bronchiseptica using a modification of the method of Hitchcock and Brown (31) as has been described (32). SDS-PAGE of LPS was performed using Novex precast 16% Tricine gels (Invitrogen).…”