It has been established that 16S rRNA gene-based phytogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the β-subclass of the Proteobacteria. 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 23S 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 16S and 23S rRNA genes of the AOB were shown to be arranged in the classical way: a 16S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears to 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 16S 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 16S rRNA gene-based phytogeny but has the benefit of giving a higher resolution.
BarryT.,
ColleranG.,
GlennonM.,
DunicanL. K.,
GannonF.1991; The 16S/23S ribosomal spacer region as a target for DNA probes to identify eubacteria. PCR Methods Appl 1:51–56
BercovierH.,
KafriO.,
Sela S.1986; Mycobacteria possess a surprisingly small number of ribosomal RNA genes in relation to the size of their genome. Biochem Biophys Res Commun 136:1136–1141
EdwardsU.,
RogallT.,
BlockerH.,
EmdeM.,
BottgerE. C.1989; Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853
FoxG. E.,
WisotzkeyJ. D.,
JurtshukP.Jr1992; How close is close: 16S rRNA sequence identity may not be sufficient to guarantee species identity. Int J Syst Bacteriol 42:166–170
GrahamT.,
Golsteyn-ThomasE. J.,
GannonV. J.,
ThomasJ. E.1996; Genus- and species specific detection of Listeria monocytogenes using polymerase chain reaction assays targeting the 16S/23S intergenic spacer region of the rRNA operon. Can J Microbiol 42:1155–1162
HeadI. M.,
HiornsW. D.,
EmbleyT. M.,
McCarthyA. J.,
SaundersJ. R.1993; The phylogeny of autotrophic ammonia-oxidizing bacteria as determined by analysis of 16S ribosomal RNA gene sequences. J Gen Microbiol 139:1147–1153
HeadI. M.,
HiornsW. D.,
EmbleyT. M.,
McCarthyA. J.,
SaundersJ. R.1995; In Validation of the Publication of New Names and New Combinations Previously Effectively Published Outside the IJSB List no. 54. Int J Syst Bacteriol 45:619–620
JiangQ. Q.1996; Nitrosospira from terrestrial environments, its urease activity and nitrous oxide production. Doctor Scientarium thesis 1996:21, Agricultural University of Norway
KoopsH.-P.,
MöllerU. C.1992; The lithotrophic ammoniaoxidizing bacteria. In The Prokaryotes2625–2637BalowsA.,
TrüperH. G.,
DworkinM.,
HarderW.,
SchleiferK. H.
New York: Fischer-Verlag;
LiX.,
De BoerS. H.1995; Selection of polymerase chain reaction chain reaction primers from an RNA intergenic spacer region for specific detection of Clavibacter michiganensis subsp. sepedonicus. Phytopathology 85:837–842
LillehaugD.,
NesI. F.,
BirkelandN. K.1997; A highly efficient and stable system for site-specific integration of genes and plasmids into the phage phiLC3 attachment site (attB) of the Lactococcus lactis chromosome. Gene 188:129–36
MobarryB. K.,
WagnerM.,
UrbainV.,
RittmannB. E.,
StahlD. A.1996; Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl Environ Microbiol 62:2156–2162
MoreiraD.,
AmilsR.1996; PCR-mediated detection of the chemolithotrophic bacterium Thiobacillus cuprinus using 23S rDNA and 16S/23S rDNA intergenic spacer region-targeted oligonucleotide primers. FEMS Microbiol Lett 142:289–293
NavarroE.,
SimonetP.,
NormandP.,
Bard inR.1992b; Characterization of natural populations of Nitrobacter spp. using PCR/RFLP analysis of the ribosomal intergenic spacer. Arch Microbiol 157:107–115
RijpensN. P.,
JannesG.,
Van AsbroeckM.,
RossauR.,
HermanL. M. F.1996; Direct detection of Brucella spp. in raw milk by PCR and reverse hybridization with 16S-23S rRNA spacer probes. Appl Environ Microbiol 62:1683–1688
RudnerR.,
StudamireB.,
JarvisE. D.1994; Determination of restriction fragment length polymorphism in bacteria using ribosomal RNA genes. Methods Enzymol 235:184–196
SmartC. D.,
SchneiderB.,
BlomquistC. L.,
GuerraL. J.,
HarrisonN. A.,
AhrensU.,
LorenzK. H.,
SeemullerE.,
KirkpatrickB. C.1996; Phytoplasma-specific PCR primers based on sequences of the 16S-23S rRNA spacer region. Appl Environ Microbiol 62:2988–2993
TeskeA.,
AimE.,
ReganJ. M.,
TozeS.,
RittmannB. E.,
StahlD. A.1994; Evolutionary relationships among ammonia- and nitrite-oxidizing bacteria. J Bacteriol 176:6623–6630
UtåkerJ. B.,
NesI. F.1998; A qualitative evaluation of the published oligonucleotides specific for the 16S rDNA gene sequences of the ammonia-oxidizing bacteria. Syst Appl Microbiol 21:72–88
YoonJ.-H.,
LeeS. T.,
KimS.-B.,
GoodfellowM.,
ParkY.-H.1997; Inter- and intraspecific genetic analysis of the genus Saccharomonospora with 16S to 23S ribosomal DNA (rDNA) and 23S to 5S rDNA internally transcribed spacer sequences. Int J Syst Bacteriol 47:661–669