Lex2B, a phase-variable glycosyltransferase, adds either a glucose or a galactose to Haemophilus influenzae lipopolysaccharide.
ABSTRACT Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second beta-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.
Article: Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis.[show abstract] [hide abstract]
ABSTRACT: We utilized the recently described tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (TSDS-PAGE) system to study the lipooligosaccharides (LOS) and lipopolysaccharides (LPS) of gram negative bacteria. TSDS-PAGE resulted in a high degree of resolution of LOS and LPS in the 'mini-gel' format. TSDS-PAGE resulted in the LOS and LPS migrating as a function of their Mr during electrophoresis and allowed estimation of Mr from a protein standard. Several species of LOS were analyzed. The newly described procedure allowed a more rapid and accurate analysis of LOS and the core region of LPS.Journal of Immunological Methods 02/1990; 126(1):109-17. · 2.20 Impact Factor
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ABSTRACT: Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.Journal of Molecular Biology 07/1983; 166(4):557-80. · 4.00 Impact Factor
Article: Haemophilus influenzae type b lipooligosaccharide: stability of expression and association with virulence.[show abstract] [hide abstract]
ABSTRACT: Spontaneous antigenic and phenotypic variations in the lipooligosaccharide (LOS) of two strains of Haemophilus influenzae type b (Hib) were previously shown to be associated with changes in virulence (A. Kimura and E.J. Hansen, Infect. Immun. 51:69-79, 1986). The goal of the present study was to define further the stability of LOS expression by this pathogen and the role of Hib LOS in virulence. Variation in LOS antigenic reactivity, as detected with LOS-specific monoclonal antibodies, was observed in 3 of 30 Hib strains after single-colony passage. When large numbers of individual colonies from seven other Hib strains were screened, however, spontaneous LOS antigenic variation was detected in all of the strains. Antigenic variation was not consistently associated with an altered LOS phenotype, as determined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis and silver staining of LOS preparations. Changes in the LOS antigenic phenotype were correlated with altered virulence potential in two strains. In these strains, acquisition of reactivity with certain LOS-directed monoclonal antibodies was associated with the synthesis of a higher-molecular-weight LOS, enhanced virulence, and increased resistance to serum killing involving the classical complement pathway.Infection and Immunity 09/1987; 55(9):1979-86. · 4.16 Impact Factor