Tandem repeats of the tetramer 5'-CAAT-3' present in lic2A are required for phase variation but not lipopolysaccharide biosynthesis in Haemophilus influenzae.
A novel lipopolysaccharide (LPS) biosynthesis gene, lic2B, which is required for the biosynthesis of a phase-variable LPS structure expressed by Haemophilus influenzae RM7004 is described. The product of this gene is homologous to Lic2A and the recently described LPS biosynthetic enzymes, LgtB from Neisseria gonorrhoea and LgtE from Neisseria meningitidis, and LpsA from Pasteurella haemolytica. Of this family of enzymes only Lic2A contains the repetitive tetrapeptide motif (SINQ)(n) encoded by multiple tandem repeats of 5'-CAAT-3'. This observation suggested that (SINQ)(n) might not be a prerequisite for the catalytic activity of this protein. To address this hypothesis, we deleted the 5'-CAAT-3' repeats from lic2A so that the protein encoded by the modified gene was analogous to Lic2B. This mutation had no apparent effect on the overall apparent molecular weight of LPS as judged by Tricine-SDS-PAGE and did not affect ability to react with monoclonal antibody 4C4. It was therefore concluded that (SINQ)(n) is not a prerequisite for the enzymatic function of Lic2A and that the 5'-CAAT-3' repeats in lic2A function solely as a mechanism for generating phase variation. This observation suggested that wide variation in the number of 5'-CAAT-3' repeats might be tolerated in lic2A, and this was confirmed by surveying the number of 5'-CAAT-3' repeats in a range of different H. influenzae strains. The predicted secondary structure of (SINQ)(n) indicates that it forms a highly flexible random coiled structure, which is unlikely to impede formation of the domains that may be required for catalytic activity. This characteristic is also a feature of repetitive tetrapeptides encoded by other tetrameric repeats located within coding sequences present on the chromosome of H. influenzae Rd.
High
N J
NJ
Department of Paediatrics, University of Oxford, Headington, Oxford, UK.
Jennings
M P
MP
Moxon
E R
ER
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D001483:Base Sequence; D005798:Genes, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D017433:Protein Structure, Secondary; D012091:Repetitive Sequences, Nucleic Acid; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C102237:lic2A protein, Haemophilus influenzae; C104912:lic2B protein, Haemophilus influenzae