Glycerol-3-phosphate transport in Haemophilus influenzae: cloning, sequencing, and transcription analysis of the glpT gene.

Abstract

The presence of a functional glpT gene in Haemophilus influenzae could be questioned, since there is only what appears to be a truncated glpT (HI0686, 143 nt in the 5'-end) available in the H. influenzae Rd genome database (Fleischmann et al. , 1995). For cloning of the glpT gene from H. influenzae type b strain Eagan, an isogenic glpT, rec-1 double mutant and a selective medium for detection of the glpT mutant strains were constructed. The recombinant plasmid carrying glpT was able to complement the isogenic glpT mutant to wild-type levels of G3P uptake and permitted growth on a selective medium with G3P as a major carbon source. The nucleotide sequences of the glpT gene were determined both directly from PCR products and from the cloned DNA insert of strain Eagan. An identical 1440 bp open reading frame with 480 deduced amino acids, highly homologous to other bacterial G3P permeases, was identified. A Northern blot analysis showed that the glpT genes in both Eagan and Rd strains were transcribed on a RNA of approximately 1.4 kb in size. Thus, it is likely that HI0686 sequence originates from a mutated glpT clone in Escherichia coli.