Identification and phylogenetic sorting of bacterial lineages with universally conserved genes and proteins.
Molecular characterizations of bacteria often employ ribosomal DNA (rDNA) to establish the identity and relationships among organisms, but the use of rRNA sequences can be problematic as the result of alignment ambiguities caused by indels, the lack of informative characters, and varying functional constraints over the molecule. Although protein-coding regions have been used as an alternative to rRNA, there is neither consensus among the genes examined nor ways to rapidly obtain sequence information for such genes from uncharacterized bacterial species. To standardize the set of protein-coding loci assayed in bacterial genomes, we examined over 100 widely distributed genes to identify sets of universal primers for use in the PCR amplification of protein coding regions that are common to virtually all bacteria. From this set, we developed primer sets that each target of 10 genes spanning an array of genomic locations and functional categories. Although many of the primers contain sequence degeneracies that aid in targeting genes across diverse taxa, most are adequate for direct sequencing of amplification products, thereby eliminating intermediate cloning before sequence determination. We foresee the analysis of these protein-coding regions as being complementary to ribosomal DNA for answering questions pertaining to bacterial identification, classification, phylogenetics and evolution.
Santos
Scott R
SR
Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, AZ 85721, USA.
Ochman
Howard
H
D001419:Bacteria / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000235:genetics*; D015373:Bacterial Typing Techniques; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005798:Genes, Bacterial; D020653:Peptide Elongation Factor G / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011938:Rec A Recombinases / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C482638:LepA protein, Legionella pneumophila; D020653:Peptide Elongation Factor G; D012269:Ribosomal Proteins; C029466:ribosomal protein L2; C071446:RecG protein, E coli; D011938:Rec A Recombinases; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D027081:DNA Gyrase