UspA2H-Negative Variant of Moraxella catarrhalis Strain O46E Has a Deletion in a Homopolymeric Nucleotide Repeat Common to uspA2H Genes

• Published in: Infection and Immunity 2007-04, Vol.75 (4), p.2035-2045
• Main Authors: Wang, Wei, Pearson, Melanie M, Attia, Ahmed S, Blick, Robert J, Hansen, Eric J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Artificial Gene Fusion; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Base Sequence; beta-Galactosidase - analysis; beta-Galactosidase - genetics; Biological and medical sciences; Blotting, Northern; Codon, Initiator; Fundamental and applied biological sciences. Psychology; Gene Expression; Genes, Reporter; Microbiology; Molecular Pathogenesis; Molecular Sequence Data; Moraxella (Branhamella) catarrhalis - genetics; Moraxella catarrhalis; Mutagenesis, Site-Directed; Open Reading Frames; Poly A - genetics; Repetitive Sequences, Nucleic Acid - genetics; RNA, Bacterial - analysis; RNA, Bacterial - genetics; RNA, Bacterial - metabolism; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Deletion; Transcription Initiation Site; Transcription, Genetic
• ISSN: 0019-9567; 1098-5522
• DOI: 10.1128/IAI.00609-06

Moraxella catarrhalis strains can express either a UspA2 protein or a UspA2H protein. The latter protein is encoded by a gene that possesses a homopolymeric nucleotide tract containing eight adenine (A) residues [i.e., a poly(A) tract] which is located near the 5' end. A spontaneous UspA2H-negative variant of M. catarrhalis strain O46E, designated O46E.U2V, was found to have a uspA2H poly(A) tract that contained seven A residues. Northern blot analysis of total RNA from the O46E parent strain revealed a readily detectable uspA2H mRNA transcript, whereas little or no uspA2H transcript was detectable in total RNA from the UspA2H-negative variant O46E.U2V. The 5' end of the uspA2H genes from both the O46E parent strain and the O46E.U2V variant were ligated to a promoterless lacZ gene to prepare translational fusions for use as reporter constructs. The level of β-galactosidase activity expressed by the fusion construct containing eight A residues in its poly(A) tract was 200-fold greater than that obtained with the construct that had seven A residues. Site-directed mutagenesis of the 5' end of the uspA2H gene confirmed that translation was initiated at a GTG codon located 21 nucleotides (nt) upstream of the poly(A) tract. Primer extension analysis determined that the transcriptional start site of the uspA2H gene was located 291 nt upstream from the GTG translational start codon. This poly(A) tract was also found to be present in the uspA2H genes of other M. catarrhalis strains.