Genetic organization of encapsulated and non-encapsulated Haemophilus influenzae strains. Analysis of the glpTQ and 23S rRNA operons

Sammanfattning: Encapsulated Haemophilus influenzae type b (Hib) and non-encapsulated H. influenzae (NTHi) are important pathogens causing invasive and mucosal infections in children. A 42 kDa surface-exposed lipoprotein, protein D, has been found to be a potential virulence factor of NTHi. The gene encoding protein D (hpd) is homologous to the glpQ gene encoding glycerophosphodiester phosphodiesterase in Escherichia coli. The E. coli glpQ and the upstream glpT gene, encoding glycerol-3-phosphate (G3P) permease, consist of one operon and are involved in the catabolic metabolism of G3P and glycerolphosphodiesters. The hpd (glpQ) gene from three Hib and three NTHi strains was cloned and sequenced, and the respective DNA sequences manifested a high degree of homology. The glpT gene of Hib was cloned into an isogenic glpT mutant selected from a spontaneous fosfomycin-resistant isolate. The DNA sequence of glpT was highly conserved between Hib and NTHi, and our results provided an intact glpT sequence missing from the H. influenzae genome sequence. However, a striking difference between Hib and NTHi in the glpTQ operons is that a 1.4 kbp fragment containing two putative ORFs was found in the glpTQ intergenic region of Hib whereas it is lacking in NTHi. The 1.4 kbp glpTQ intergenic region might be acquired by lateral transfer from an organism with a lower G + C content. Transcriptional organization and regulation for glpTQ operons were analyzed by Northern blotting, primer extension and RT-PCR. In Hib Eagan, glpT and glpQ transcribed separately, and the expression of glpT was reduced by glucose in comparison to G3P. In NTHi 772, glpT and glpQ co-transcribed and they appeared to be enhanced by glucose. Multiple potential promoter sites for glpT were found in both strains and an unique site was only found in 772. Although no apparent phenotype change was found from an isogenic 1.4 kbp glpTQ intergenic region mutant strain, the presence of the 1.4 kbp region apparently separated the glpT and glpQ transcription in Hib. Fragmentation of 23S rRNA in H. influenzae was caused by the existence of two intervening sequences (IVSs) in the 23S rRNA coding regions. Except for H. influenzae biotype IV strains, all of Hib and NTHi strains tested were found carrying fragmented 23S rRNA, and half of them manifested a heterogeneous cleavage pattern. Fragmentation of 23S rRNA is a common phenomenon in H. influenzae but is rarely found in other Haemophilus species.