Volume 2, Number 3—July 1996
Legionella-Like Amebal Pathogens––Phylogenetic Status and Possible Role in Respiratory Disease
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1Genomic DNA was isolated by standard methods and extracted with phenol/chloroform. A 1400-bp 16S rDNA segment was amplified by using primers specific for Eubacteria and based on a consensus of Legionella 16S rRNA sequences. Each strain was sequenced at least 3 times to ascertain accuracy of obtained results. 16S rRNA sequences for all Legionella species, those of some previously sequenced LLAPs and Coxiella burnetii were obtained from GenBank/EMBL and aligned with sequences generated in this study, using the GCG sequence analysis package (version 8.0). Comparisons were performed at NCBI using the BLAST network service (32). Ambiguous and hypervariable regions were removed before phylogenetic analysis, which was carried out on 53 strains for 1303 nucleotides by the neighbor-joining method from the Phylogeny Inference Package (Phylip) (33), version 3.5. Multiple datasets (X100) were analyzed, and different distance models were compared to ensure reliability. Some of the 16S rRNA sequences included in the data analysis are of unpublished and undescribed stains. Accession numbers for sequences included in this study are as follows: LLAP-1 U64034, LLAP-2 U44909, LLAP-3 X60080, LLAP-4 X97357, LLAP-6 X97357, LLAP-7 U44910, LLAP-8 U64035, LLAP- 9 U44911, LLAP-10 X97363, LLAP-11 X97362, LLAP-12 X97366. L. adelaidensis Z49716, L.anisa X733394, L. birminghamensis Z49717, L. brunensis X73403, L. cherrii X73404, L. cincinnatiensis X73407, Coxiella burnetii M21291, L. donaldsonii Z49724, L.dumoffii X73405, L.erythra Z32638, L. fairfieldensis Z49722, L. feelii X73395, L. geestiana Z49723, L. gormanii Z32639, L. gratiana Z49725, L. hackeliae M36028, L. israelensis X73408, L. jamestowniensis X73409, L. jordanis X73396, L. lansingensis Z49727, L. londiniensis Z49728, L. longbeachae M36029, L. maceachernii X60081, L. micdadei M36032, L. moravica Z49729, L. nautarum Z49730, L. oakridgensis X73397, L. parisiensis Z49731, L. pneumophila M59157, L. quateirensis Z49732, L. worsliensis Z49739, L. quinlivanii Z49733, L. rubrilucens X73398, L. santicrucis Z49735, Sarcobium lyticum X66835, L. shakespearei Z49736, L. spiritensis M36030, L. steigerwaltii X73400, L. sainthelensi Z49734, L. tusconensis Z32644, L. wadsworthii X73401.
2The Remel Poly-ID kit used in this study is designed to identify 22 species and 31 serogroups of Legionella (34,35).