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With the genomic era, huge amounts of information are generated. Therefore, biologists are increasingly demanding structured, exhaustive and comparative databases. The PyloriGene database (http://genolist.pasteur.fr/PyloriGene) was developed to answer to such challenges by integrating and connecting the information generated by the sequencing of two distinct strains of Helicobacter pylori, a human gastric pathogen. This pointed to the need for a general annotation consensus of both strains as their physical and functional annotation differed significantly in some cases. A revised functional classification was created in order to accommodate the existing data on both sequenced genomes and the possibility of classifying coding sequences (CDS) into several functional categories were used to harmonize CDS classification. Revision of the annotation of the two complete genomes in light of new data allowed us to reduce the percentage of hypothetical proteins from around 40% to 31%. This also resulted in a reassignment of functions or new functional assignments for a total of 108 CDS, representing around 7% of the total CDS. Interestingly, only around 5% of the total of CDS with an assigned function were based on work done on H. pylori. Finally, comparison of the two genomes revealed a significant amount of CDS size variation among corresponding (orthologues) CDS. Although a majority of these size variations were due to natural polymorphism, interestingly, as much as 113 CDS pairs differed in size due to different start codon assignment, a recurrent problem of genomes physical annotations. [ABSTRACT FROM AUTHOR]

Background: Sequence analysis of the Helicobacter pylori (Hp) genome showed that this bacterium doesn't encode for a glutaminyl-tRNA synthetase (GlnRS) but presents the particularity of possessing two closely related glutamyl-tRNA synthetases (HP#0476, named GluRS1 and HP#0643, named GluRS2; 38% amino acid identity). Method: To determine whether or not each gltX gene product is essential for the growth and survival of H. pylori, the two copies were disrupted by a non-polar kanamycin cassette. The resulting plasmids were used to transform the N6 Hp strain. To determine the substrate specificities of the two GluRSs, gltX1 and gltX2 were cloned to produce His-Tagged proteins in Escherichia coli (Ec). The two enzymes were overexpressed and purified by affinity with nickel resin. The different tRNAs (tRNAglul, tRNAglu2 and tRNAgln) were also cloned from Hp strain 26695 chromosomal DNA, overproduced in Ec and purified. Results: No colonies could be isolated after transformation of the N6 strain with the plasmids carrying the gltX1 and gltX2 mutated copies, demonstrating that both of the GluRSs are essential for Hp survival. Charging assays performed with (i) each purified GluRSs, (ii) Ec tRNA preparations, each containing tRNAglul, tRNAglu2 or tRNAgln of Hp, and (iii) 3H radiolabelled glutamate, enzymatically delineated differences between the two enzymes. GluRS1 preferentially aminoacylates tRNAglu1 and tRNAglu2 with glutamate and presents a weak activity with tRNAgln. In contrast, tRNAgln appears to be the best substrate of GluRS2 which is also capable of charging tRNAglu1 to a lesser extent. Discussion Phylogenetic studies suggest that GluRS gave rise to GlnRS after an early duplication of the gltX gene. Our work presents the first experimental evidence to suggest that the two GluRSs of Hp may be intermediates in the scale of this enzymatic evolution. [ABSTRACT FROM AUTHOR]