Identification of a new locus involved in expression of Haemophilus influenzae type b lipooligosaccharide

Abstract

Previous studies have shown that changes in the expression of the Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS) epitope reactive with monoclonal antibody (MAb) 5G8 can be correlated with alterations in the virulence of some Hib strains. To identify the locus involved in expression of this particular LOS epitope, a genomic library was constructed in the plasmid shuttle vector pGJB103 from Hib strain DL42, which constitutively expressed LOS reactive with MAb 5G8. This library was used to transform a second Hib

Use of the complete genome sequence information of Haemophilus influenzae strain Rd to investigate lipopolysaccharide biosynthesis

“…The lgtC locus had an associated tetranucleotide repeat motif; there were 23 copies of GACA in strain Rd, and 22 and 32 copies in strains RM153 and RM7004, respectively (Hood et al, 1996). The repeat sequences were located at the extreme 5Ј end of the predicted translated reading frame in a similar fashion to the CAAT and GCAA repeats reported previously for other LPS-associated genes (Weiser et al, 1989;Jarosik and Hansen, 1994). An RM7004 strain mutated in orfM, the ORF adjacent to lgtC, produces an LPS gel-migration pattern similar, but not identical, to that from RM7004lgtC.…”

“…The molecule lacks the O-specific side chain characteristic of the LPS of the enteric bacteria. Several of the surface-exposed epitopes of terminal hexose sugars are subject to high-frequency phase variation (Weiser et al, 1989;Jarosik and Hansen, 1994), an adaptive mechanism which is advantageous for survival of bacteria confronted by the differing microenvironments and immune responses of the host. This heterogeneity has complicated the structural and biological analysis of H. influenzae LPS; therefore the construction of mutant strains, with defined core structures, has been a priority.…”

“…1). These included the lic genes (Weiser et al, 1989;High et al, 1993) and the lex2 gene (Jarosik and Hansen, 1994) involved with LPS phase variation, the lsg locus (Spinola et al, 1990), galE (Maskell et al, 1992), and rfaE (Lee et al, 1995), rfaD (A. Preston, personal communication) and isn (Preston et al, 1996;Zwahlen et al, 1985) genes which are involved with heptose biosynthesis. The majority of biosynthetic functions remain to be Identification of LPS-related genes from the H. influenzae strain Rd genome sequence The sequences of a wide range of LPS biosynthetic genes from Gram-negative bacteria were used as 'database probes' to screen for homologous genes, by DNA or amino acid similarity, in the H. influenzae strain Rd genome database prior to its annotation.…”

Molecular analysis of a locus for the biosynthesis and phase‐variable expression of the lacto‐N‐neotetraose terminal lipopolysaccharide structure in Neisseria meningitidis

“…Restriction endonuclease sites are indicated above map: A, AIwNI; AI, Alul; E, EcoNI; H, Hindlll; Ha IpsA gene of Pasteurella haemolytica (GenBank accession number U15958) and the recently described IgtB and IgtE genes of Neisseria gonorrhoeae (Ng;Gotschiich, 1994). The lex2B gene of Hi, which is also involved in LPS biosynthesis (Jarosik and Hansen, 1994), does not show a high level of overall homology but when aligned does show homology with some of the regions which are highly conserved between the sequences displayed in Fig. 2 (result not shown).…”

Tandem repeats of the tetramer 5′‐CAAT‐3’present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae

“…Another example of a tetrameric repeat associated with H. influenzae LPS biosynthesis is 5'-GCAA-3, which encodes the tetrapeptide Ala-Ser-Glu-Gln (ASKQ). Multiple repeats of this tetramer have been found within the 5' end of ORF1 in the /ex-2 locus, which is required for the biosynthesis of the phase-variable LPS structure recognized by mAb 5G8 (Jarosik and Hansen, 1994). The use of repetitive DNA sequences as a mechanism for generating phase variation of cell-surface structures is also a feature of Neisseria gonorrhoeae and Neisseria meningitidis (for a review see Robertson and Meyer, 1992).…”