16S rRNA Molecules Research Articles

Sulfate-reducing bacteria methylate mercury at variable rates in pure culture and in marine sediments.

Differences in methylmercury (CH(3)Hg) production normalized to the sulfate reduction rate (SRR) in various species of sulfate-reducing bacteria (SRB) were quantified in pure cultures and in marine sediment slurries in order to determine if SRB strains which differ phylogenetically methylate mercury (Hg) at similar rates. Cultures representing five genera of the SRB (Desulfovibrio desulfuricans, Desulfobulbus propionicus, Desulfococcus multivorans, Desulfobacter sp. strain BG-8, and Desulfobacterium sp. strain BG-33) were grown in a strictly anoxic, minimal medium that received a dose of inorganic Hg 120 h after inoculation. The mercury methylation rates (MMR) normalized per cell were up to 3 orders of magnitude higher in pure cultures of members of SRB groups capable of acetate utilization (e.g., the family Desulfobacteriaceae) than in pure cultures of members of groups that are not able to use acetate (e.g., the family Desulfovibrionaceae). Little or no Hg methylation was observed in cultures of Desulfobacterium or Desulfovibrio strains in the absence of sulfate, indicating that Hg methylation was coupled to respiration in these strains. Mercury methylation, sulfate reduction, and the identities of sulfate-reducing bacteria in marine sediment slurries were also studied. Sulfate-reducing consortia were identified by using group-specific oligonucleotide probes that targeted the 16S rRNA molecule. Acetate-amended slurries, which were dominated by members of the Desulfobacterium and Desulfobacter groups, exhibited a pronounced ability to methylate Hg when the MMR were normalized to the SRR, while lactate-amended and control slurries had normalized MMR that were not statistically different. Collectively, the results of pure-culture and amended-sediment experiments suggest that members of the family Desulfobacteriaceae have a greater potential to methylate Hg than members of the family Desulfovibrionaceae have when the MMR are normalized to the SRR. Hg methylation potential may be related to genetic composition and/or carbon metabolism in the SRB. Furthermore, we found that in marine sediments that are rich in organic matter and dissolved sulfide rapid CH(3)Hg accumulation is coupled to rapid sulfate reduction. The observations described above have broad implications for understanding the control of CH(3)Hg formation and for developing remediation strategies for Hg-contaminated sediments.

Multiplex PCR for the detection of Lactobacillus pontis and two related species in a sourdough fermentation.

A specific multiplex PCR assay based on the amplification of parts of the 16S rRNA molecule was designed. Primers derived from variable regions of the 16S rRNA provided a means of easily differentiating the species Lactobacillus pontis and Lactobacillus panis. They could be clearly discriminated from the phylogenetically related species Lactobacillus vaginalis, Lactobacillus oris, and Lactobacillus reuteri and from other lactobacilli commonly known to be present in sourdough. Other strains isolated together with L. pontis from an industrial sourdough fermentation could be clearly separated from these species by comparative sequence analysis and construction of a specific PCR primer. For a fast identification a DNA isolation protocol based on the ultrasonic lysis of cells from single colonies was developed. To demonstrate the potential of such techniques for tracking these organisms in a laboratory-scale fermentation, we combined the specific PCR assay with direct DNA extraction from the organisms in the sourdough without previous cultivation.

Problems associated with the direct viable count procedure applied to gram-positive bacteria

Despite the numerous advantages of fluorescent in situ hybridization (FISH) for identifying a single bacterial cell with 16S rRNA probes, problems are encountered with starving bacteria in natural samples. The original direct viable count procedure (DVC) includes a revivification step in the presence of an antibiotic inhibiting cell division. Cells elongate and accumulate ribosomes. This results in a natural amplification of 16S rRNA molecules (target of FISH). However, it is limited to gram-negative bacteria which are sensitive to nalidixic acid. The objective of this study was to develop a procedure for estimating the number of metabolically active gram-positive Staphylococcus aureus and Enterococcus faecalis cells by the use of a method which combines the number of substrate-responsive cells and their identification by FISH. It was observed that no single published DVC method could apply to taxonomically different gram-positive bacteria. Since cells were not counted, the revivification step in presence of nalidixic acid will be referred to as revivification without cell division. For each species, different low-nutrient media and complex media, different fluoroquinolones and beta-lactam antibiotics, concentrations of antibiotics, combinations of antibiotics, temperature and time were evaluated using bacteria in different physiological states and in natural samples. Enumeration of bacteria by plate counts and direct FISH were compared. The improved procedure should yield information about the physiological state, the taxonomic identity, and the enumeration of viable gram-positive bacteria. The application of DVC to an entire ecosystem is presently still a challenge.

Updated phylogenetic description of the Mycoplasma hominis cluster (Weisburg et al. 1989) based on 16S rDNA sequences.

The fastidious nature of the mollicutes (mycoplasmas), their lack of a classic bacterial cell wall, and their very small genome, make phylogenetic placements of new species in this enlarging group of prokaryotes an important and valuable aid in their classification. In this report we have determined the phylogeny of the Mycoplasma hominis cluster of the hominis group. The 16S rDNA sequences from several previously described Mycoplasma species were determined and ten species were found to belong to the M. hominis cluster. With almost complete sequences available, the phylogenetic analysis revealed that the M. hominis cluster currently comprises 19 species, forming a distinct clade as judged from branch lengths, bootstrap percentage values, nucleotide signature analysis, and structural elements in the 16S rRNA molecule. The 16S rRNA gene sequences of species in the M. hominis cluster were found to be > or = 94% similar and the range within which similarities can be used in the classification of new species is discussed. Members of the M. hominis cluster all share a major biochemical property of M. hominis, in that they hydrolyse arginine and are incapable of fermenting glucose. This consistency in phenotypic pattern has not been found in any of the other phylogenetic clusters of the hominis group. Two species, the non-cultivable agent of Grey Lung disease in rodents (tentatively named 'Candidatus Mycoplasma ravipulmonis') and the avian species Mycoplasma gypis strain B1/T1T, were regarded as close relatives to the M. hominis cluster, but are clearly separated from the species of this cluster. Both species formed early branches of the M. hominis cluster and should be regarded as individual lines containing one species.

In situ reverse transcription for the specific detection of bacteria and protozoa.

In situ hybridization experiments with oligonucleotide probes directed against the 16S and 18S rRNA molecules have been used successfully to identify specific organisms in mixed microbial populations. However, there are limitations in applying these techniques to environmental samples. In the present study we have examined the possibility of using in situ reverse transcription as an alternative to hybridization methods for the rapid detection of Escherichia coli and the waterborne parasite Cryptosporidium parvum. Following fixation and permeabilization of the cells, extension reactions were performed with species-specific primers, AMV reverse transcriptase and either cy3-AP3-dUTP or fluorescein-11-dUTP at 45 degrees C for 45 min. The cells or oocysts were then filtered onto Costar metallic membrane filters and images captured with a CCD camera. The results have shown that this technique can successfully detect E. coli cells and C. parvum oocysts in under 2 h.

Characterization of transient RNA-RNA interactions important for the facilitated structure formation of bacterial ribosomal 16S RNA.

The co-transcribed leader sequences of bacterial rRNA are known to affect the structure and function of the small ribosomal subunits. Base changes in the leader nut -like sequence elements have been shown to cause misfolded but correctly processed 16S rRNA structures at low growth temperature. Transient interactions of leader sequences with the nascent 16S rRNA are considered to guide rRNA folding and to facilitate correct structure formation. In order to understand this chaperone-like activity of the leader RNA we have analyzed the thermodynamic stabilities of wild-type and mutant leader transcripts. We show here that base changes cause subtle differences in the melting profiles of the corresponding leader transcripts. Furthermore, we show that direct interaction between leader transcripts and the 16S rRNA is limited to the 5'-domain of the 16S rRNA for both wild-type and mutant leaders. Binding studies of mutant and wild-type leader transcripts to 16S rRNA revealed small changes in the affinities and the thermal stabilities as a consequence of the base changes. Different complex stabilities as a function of the Mg(2+) ion concentration indicated that mutant and wild-type leader transcripts interact differently with the 16S rRNA, consistent with a less stable and tightly folded structure of the mutant leader. Employing time-resolved oligonucleotide hybridization assays we could show different folding kinetics for 16S rRNA molecules when linked to wild-type leader, mutant leader or in the absence of leader RNA. The studies help to understand how bacterial rRNA leader transcripts may affect the folding of the small subunit rRNA.

Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water

A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V1region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water. The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia , Klebsiella , Enterobacter and Citrobacter . In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system. The results obtained enabled the specific detection of E. coli in under 3 h. Hybridizations were also performed on cells which were treated with chlorine (1·5 mg l−1) for up to 30 min. Escherichia coli cells were still detected after storage for 14 days at room temperature. No cells were detected by conventional plate count or the «Colilert» assay, a method currently used for the routine detection of E. coli and coliforms in the water industry. Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration. Only cells that had not been chlorinated were detected. These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells. However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ . Hybridization experiments were also performed successfully on seeded tap water samples. The number of fluorescent cells detected correlated well with those obtained by plate count analysis. This represents the first reported use of PNA oligonucleotides forin situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches.

Biochemical analysis of a 5S rRNA-associated sub-particle from trypsinized eukaryotic ribosomes.

On limited trypsinization, eukaryotic ribosomes released sub-particles that comprised a 5S rRNA molecule and two peptides (a 32 kDa and a 14 kDa). By tryptic finger-printing and amino-terminal sequence analysis, these two peptides were determined to be derived from large subunit ribosomal protein L5 (rpL5). The 32 kDa peptide represents the rpL5 protein minus the amino terminal eight residues and the carboxyl terminal ends (approximately 21 residues), whereas the 14 kDa peptide comprised near the amino-terminal region. The time course of ribosome trypsinization revealed that the two peptides were released kinetically. The indicated that the amino and carboxyl terminal ends of rpL5 were the first to be hydrolyzed, suggesting that the two ends of the rpL5 protein were exposed on the surface of ribosomes. Exposure of the carboxyl-terminal end was confirmed by use of an anti-L5c antibody raised against the carboxyl terminal region of rpL5. The kinetic data also revealed that the nearby amino terminal region of rpL5 (represented by the 14 kDa peptide) was the last part of rpL5 to be hydrolyzed, which was considered to be the 5S rRNA binding site.

RRNA probe‐based cell fishing of bacteria

We have developed a new, cultivation-independent, fast and flexible method for the rRNA-targeted probe-based enrichment of bacteria. The target cells were labelled by in situ hybridization with biotinylated polyribonucleotide probes. These probes were generated by in vitro transcription of amplified rDNA of a variable region in domain III of the 23S rRNA molecules. The probes were about 300 nucleotides in length and were labelled by incorporation of biotin-UTP during the transcription. Probes were hybridized with bacterial cells and incubated with paramagnetic streptavidin-coated particles. The labelled target cells can be separated in a column filled with steel wool inserted into the field of a permanent magnet. Unlabelled, non-target cells pass through the column, whereas labelled cells are retained. They were eluted from the column after removal of the magnetic field. Up to now, the method has been tested with mixtures of different pure cultures. For the first time, transcript probes have been used for the labelling of the target cells and for their specific separation. The enrichment of the target cells can be monitored by a streptavidin-fluorescein staining of the biotinylated target cells and/or by a subsequent in situ hybridization with fluorescently labelled oligonucleotide probes. Enrichment rates of up to 90-fold, depending on the original abundance of the cells of interest, could be determined. To demonstrate that the sorted cells were amenable to molecular analysis, we amplified and sequenced a part of the tuf gene of enriched Acinetobacter calcoaceticus cells.

Fragmentation heterogeneity of 23S ribosomal RNA in Haemophilus species.

The fragmentation of 23S rRNA of 23 Haemophilus influenzae strains and eight strains belonging to other Haemophilus species was investigated. Instead of intact molecules, the 23S rRNA molecules were found to be cleaved into two to five smaller conserved fragments in most strains examined, especially in H. influenzae type b (5/6) and nontypeable strains (5/5). One or two conserved potential cleavage sites were identified by PCR analysis of the strains showing a fragmented 23S rRNA pattern. The relevant nucleotide sequences were determined and compared to H. influenzae Rd, which contains intact 23S rRNA molecules. An identical 112bp long intervening sequence (IVS) at position 542 and a conserved 121-123bp IVS sequence at position 1171 were found in two H. influenzae type b strains and one nontypeable strain. Among the strains with fragmented 23S rRNA, nearly half showed a heterogeneous cleavage pattern due to the dispersion of IVSs among different 23S rRNA operons. The localization of the conserved H. influenzae IVSs coincided well with the extensively studied IVSs among other bacteria, but differed in nucleotide sequence from any other reported IVSs. Therefore, the IVSs of Haemophilus 23S rRNA may originate from a common source that is independent of other bacteria.

A possible new mtrRNA mutation site for aminoglycoside-induced deafness syndrome.

The aminoglycoside group of antibiotics include some of the most powerful agents in the arsenal available for use against gram-negative bacterial infections and include such compounds as gentamycin, tobramycin, amikacin, netilmicin, kanamycin, streptomycin and neomycin. Their primary intracellular site of action has been shown to be the 30S ribosomal subunit, consisting of 21 proteins and a single 16S molecule of RNA, the 16SrRNA molecule. Bacterial resistance to aminoglycosides has been associated with mutations in both the protein subunits and the 16SrRNA. Gentamycin has been advocated as an antibiotic for aggressive treatment of lung infections in Cystic Fibrosis (CF) patients. This same antibiotic is widely applied in a range of other situations. The primary intracellular site of the antimicrobial action of aminoglycosides is the bacterial 30S ribosomal subunit, consisting of 21 proteins and a single 16S RNA molecule. One unfortunate but relatively rare side-effect of these antibiotics is their ototoxicity; deafness is induced in approximately 1 in 10,000 individuals treated with these antibiotics. Another reported side-effect of the aminoglycosides, nephrotoxicity, may be induced by a similar mechanism to that suggested for ototoxicity. There is now considerable evidence that the aminoglycoside-induced deafness is of mitochondrial origin. The human mitochondrial 12S subunit of ribosomal RNA (rRNA) transcribed from mitochondrial DNA (mtDNA) shares more homology with bacterial 16S rRNA than with the human cytosolic 18S rRNA. As reported by Hutchin et al. (1993) and Prezant et al. (1993), the mtDNA of a group susceptible individuals was shown to contain a mutation, corresponding to nucleotide position 1555 of the 12S rRNA sequence. The mutation was shown to exhibit homoplasmic expression—all copies of the mitochondrial gene in the tissues examined contained the mutation. It was proposed that the slight change in the 12SRNA sequence resulting from this mutation was sufficient to alter the nucleic acid conformation to a more “bacterial-like” structure. Since then there have been a number of reports of other mutated 12S rRNA sites which confer susceptibility to aminoglycoside-induced deafness (Table 1) along with one report of a 16S rRNA mutation associated with this syndrome. It is believed that these mutated structures fail to bind appropriately to the (nuclear-encoded) ribosomal protein components of the mitochondrial translation process. It has been proposed that this leads to the formation of stable mismatched aminoacyl-tRNAs in the 70S ribosome, thereby causing misreading of the (mitochondrially-transcribed) mRNA during protein synthesis.

The environment of 5S rRNA in the ribosome: cross-links to 23S rRNA from sites within helices II and III of the 5S molecule.

Three contiguous fragments of Escherichia coli 5S rRNA were prepared by T7 transcription from synthetic DNA templates. The central fragment, comprising residues 33-71 of the molecule, was transcribed in the presence of 4-thiouridine triphosphate together with [32P]UTP. The three transcripts were ligated together, yielding a 5S rRNA analogue carrying 4-thiouridine residues at positions 40, 48, 55 and 65 in helices II and III. After ligation, the 4-thiouridine residues were derivatised with p -azidophenacyl bromide. The modified 5S rRNA was reconstituted into 50S subunits and these subunits were used to prepare 70S ribosomes in the presence or absence of tRNA and mRNA. The azidophenyl groups were then photoactivated by mild irradiation at 300 nm and the products of cross-linking analysed by our standard procedures. Multiple cross-links from 5S rRNA to two distinct regions of the 23S rRNA were observed. The first region was located in helix 38 in Domain II of the 23S molecule, with cross-links at sites between nucleotides 885 and 922. The second region covered helices 81-85 in Domain V, with sites between nucleotides 2272 and 2345. Taken together with previous data, these results serve to define the arrangement of the 5S rRNA molecule relative to the 23S rRNA within the 50S subunit.

To Knot or Not to Knot? Examination of 16S Ribosomal RNA Models

The presence of topological knots in large RNA structures is highly unlikely given that 1) no RNA structures determined thus far contain topological knots, 2) secondary structure maps for most RNA molecules are knot free, 3) there are no known RNA topoisomerases, and 4) it is difficult to imagine how knots could be formed specifically and uniquely during transcription. Since native RNA structures probably lack topological knots, models of these RNA molecules should be free of knots as well. Therefore, we have examined four existing models for the 30S ribosomal subunit to determine if any of the three domains of the 16S rRNA molecule is knotted. We found that all but one model had at least one knotted domain. We conclude that models of large RNA molecules should be examined for knotting before publication.

Non-random fragmentation of ribosomal RNA in Helicobacter pylori during conversion to the coccoid form.

The integrity of DNA and ribosomal RNAs in exponentially growing (bacillary) and ageing stationary phase (coccoid) cultures of Helicobacter pylori type strain CCUG 17874 was investigated. Extensive non-random fragmentation of rRNAs was observed during the conversion to the coccoid form. Beside a small proportion of full-length 16S and 23S rRNA that was always present, the majority of both 16S and 23S rRNA molecules showed distinct highly specific fragmentation patterns. The 16S rRNA fragmentation was characterised in detail by means of Northern blot and primer extension analysis. One cleavage site was located within the highly conserved U5 region (position about 920). The results could not be attributed to the presence of intervening sequences in the 16S and 23S rRNA genes.

Non-random fragmentation of ribosomal RNA in Helicobacter pylori during conversion to the coccoid form

The integrity of DNA and ribosomal RNAs in exponentially growing (bacillary) and ageing stationary phase (coccoid) cultures of Helicobacter pylori type strain CCUG 17874 was investigated. Extensive non-random fragmentation of rRNAs was observed during the conversion to the coccoid form. Beside a small proportion of full-length 16S and 23S rRNA that was always present, the majority of both 16S and 23S rRNA molecules showed distinct highly specific fragmentation patterns. The 16S rRNA fragmentation was characterised in detail by means of Northern blot and primer extension analysis. One cleavage site was located within the highly conserved U5 region (position about 920). The results could not be attributed to the presence of intervening sequences in the 16S and 23S rRNA genes.

Limitations of Metazoan 18S rRNA Sequence Data: Implications for Reconstructing a Phylogeny of the Animal Kingdom and Inferring the Reality of the Cambrian Explosion

We document the phylogenetic behavior of the 18S rRNA molecule in 67 taxa from 28 metazoan phyla and assess the effects of among-site rate variation on reconstructing phylogenies of the animal kingdom. This empirical assessment was undertaken to clarify further the limits of resolution of the 18S rRNA gene as a phylogenetic marker and to address the question of whether 18S rRNA phylogenies can be used as a source of evidence to infer the reality of a Cambrian explosion. A notable degree of among-site rate variation exists between different regions of the 18S rRNA molecule, as well as within all classes of secondary structure. There is a significant negative correlation between inferred number of nucleotide substitutions and phylogenetic information, as well as with the degree of substitutional saturation within the molecule. Base compositional differences both within and between taxa exist and, in certain lineages, may be associated with long branches and phylogenetic position. Importantly, excluding sites with different degrees of nucleotide substitution significantly influences the topology and degree of resolution of maximum-parsimony phylogenies as well as neighbor-joining phylogenies (corrected and uncorrected for among-site rate variation) reconstructed at the metazoan scale. Together, these data indicate that the 18S rRNA molecule is an unsuitable candidate for reconstructing the evolutionary history of all metazoan phyla, and that the polytomies, i.e., unresolved nodes within 18S rRNA phylogenies, cannot be used as a single or reliable source of evidence to support the hypothesis of a Cambrian explosion.

Spatial Homogeneity of Abundant Bacterial 16S rRNA Molecules in Grassland Soils

The variability of prominent bacterial 16S rRNA molecules from environmental soil samples was investigated. Ribosomes and genomic DNA were extracted from 160 soil samples derived from three different test fields in the Drentse A grasslands (The Netherlands). After amplification of bacterial 16S rRNA molecules by reverse transcription and PCR, the products were separated by temperature-gradient gel electrophoresis. Characteristic and complex band patterns were obtained, indicating high bacterial diversity. The fingerprints from soil samples from plots, taken in regular patterns, were almost identical. Reproducible differences between the three test fields of different history were obtained. A parallel approach with PCR-amplified genomic 16S rDNA led to similar results. The presence and activity of prominent bacteria in test fields of several hundred m2 were constant. Only one gram of soil was needed to represent the prominent bacteria in large homogeneous grassland areas. The spatial distribution of bacterial ribosomes in soil at this site was homogeneous, suggesting the presence and activity of the dominant soil bacteria was the same.

Secondary structure of some elements of 18S rRNA suggests that strongylid and a part of rhabditid nematodes are monophyletic

Analysis of the secondary structure of 18S rRNA molecules in nematodes revealed some new traits in the secondary structure peculiar to their hairpin 17. Some of them are characteristic of all the nematodes, whereas others are characteristic exclusively of the order Rhabditida. The loss of a nucleotide pair in the highly conservative region of hairpin 17 distinguishes 18S rRNA of the Strongylida and some species of the Rhabditida from other nematodes and, moreover, from all other organisms. Hence, it is possible to regard the Strongylida and a part of the Rhabditida including Caenorhabditis elegans as a new monophyletic taxon.

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes.

Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the alpha-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Development and evaluation of a PCR-based assay for detection of Haemobartonella felis in cats and differentiation of H. felis from related bacteria by restriction fragment length polymorphism analysis.

The 16S rRNA gene of Haemobartonella felis was amplified by using universal eubacterial primers and was subsequently cloned and sequenced. Based on this sequence data, we designed a set of H. felis-specific primers. These primers selectively amplified a 1,316-bp DNA fragment of the 16S rRNA gene of H. felis from each of four experimentally infected cats at peak parasitemia. No PCR product was amplified from purified DNA of Eperythrozoon suis, Mycoplasma genitalium, and Bartonella bacilliformis. Blood from the experimental cats prior to infection was negative for PCR products and was greatly diminished or absent 1 month after doxycycline treatment. The overall sequence identity of this fragment varied by less than 1.0% among experimentally infected cats. By taking into consideration the secondary structure of the 16S rRNA molecule, we were able to further verify the alignment of nucleotides and quality of our sequence data. In this PCR assay, the minimum detectable number of H. felis organisms was determined to be between 50 and 704. The potential usefulness of restriction enzymes DdeI and MnlI for distinguishing H. felis from closely related bacteria was examined. This is the first report of the utility of PCR-facilitated diagnosis and discrimination of H. felis infection in cats.