RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach

Abstract

It has been established that 165 rRNA gene-based phylogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the &subclass of the Proteobacferia. In this study, 12 isolates of AOB were ribotyped, and the sequences of the 16s-23s rDNA intergenic spacer region (ISR) were determined and used in a phylogenetic study. 16s and 235 rDNA ribotyping revealed that the AOB studied contain only one rrn operon per genome, in contrast to most bacteria, which have 5-10 copies of the rRNA genes per genome. It is likely that the presence of only one set of rRNA genes is related to the slow growth of the AOB. The 165 and 235 rRNA genes of the AOB were shown to be arranged in the classical way: a 16s rRNA gene, an ISR and a 235 rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears t o vary considerably. The size of the ISR was approximately 400 bp in the Nitrosomonas isolates and 645-694 bp in the Nitrosospira isolates, suggesting a species-specific size difference in the ISR. The ISR contained two potential tRNA genes in the 5' end in all isolates studied. The similarity values between the ISR sequences of the AOB are low (42*9-96*2°/~) compared with the 165 rDNA sequence similarity values, and therefore the ISR sequences are valuable as a complementary phylogenetic tool in combination with 16s rRNA gene sequences. The phylogenetic analysis of the AOB based on ISR sequences confirms the 165 rRNA gene-based phylogeny but has the benefit of giving a higher resolution.

16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads

“…For each lineage, the minimum 16S rRNA sequence similarity between two of its members is depicted. Sequences included in the analysis were published by Aakra et al (1999a, b), Head et al (1993), Juretschko et al (1998), Purkhold et al (2000), Sorokin et al (1998), Suwa et al (1997) and Yamagata et al (1999). Sequences of strains CNS326, G1, K1, IWT202, TK794, WH-2, Koll-21, DYS317, DYS323, IWT514, TNO632, marine bacteria C-45, NH4W, 122, URW, NO3W, C-113, TT140-098-2, TT140-98A and estuarine bacteria TA 921-i-NH4, B19-i-NH4, C-17 are unpublished but are available at GenBank.…”

“…Filled and empty circles indicate parsimony bootstrap values (100 resamplings) above 90 and 70 %, respectively. Sequences included in the analysis were published by Aakra et al (1999aAakra et al ( , b, 2001b, Head et al (1993), Teske et al (1994), Tokuyama et al (1997) and Utå ker et al (1995). Sequences of strains GS833, E12, NRS527, NpAV, RY6A, RY3C, TCH716 and PJA1 are unpublished but are available at GenBank.…”

A Universal Method for the Identification of Bacteria Based on General PCR Primers

“…DNA sequencing and sequence alignment have been widely applied and accepted as methods of bacterial detection and identification [2,8,18,21,[24][25][26]. Phylogenetic analysis of bacteria can be based on their 16S DNA sequences [2].…”

“…To solve this problem, investigators [4,17,20,25,27,[29][30][31] sought universal primers. Unfortunately, ''Universal Primers'' do not live up to their name since they do not cover all bacteria [30].…”

“…Detection and identification of non-target bacteria require a different approach; degenerate primers are used to amplify gyr B for taxonomic analysis of Pseudomonas putida [22] while PCR coupled to sequencing of the 16S rDNA is used to identify pathogenic Pseudomonas spp. [24] and others [25,26]. PCR-RFLP analysis of 16S genes is used in the analysis of Campylobacter, Helicobacter, and Arcobacter [27,28].…”