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Journal of Clinical Microbiology, June 2005, p. 2881-2885, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2881-2885.2005

Multiple Copies of the 16S rRNA Gene in Nocardia nova Isolates and Implications for Sequence-Based Identification Procedures

Patricia S. Conville^* and Frank G. Witebsky

Microbiology Service, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda, Maryland

Received 4 August 2004/ Returned for modification 5 December 2004/ Accepted 14 January 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Molecular investigation of two Nocardia patient isolates showed unusual restriction fragment length polymorphism patterns with restriction endonuclease assays (REA) using an amplified portion of the 16S rRNA gene. Patterns typical of Nocardia nova were obtained with REA of an amplified portion of the 65-kDa heat shock protein gene. Subsequent sequence analysis of the 16S rRNA gene regions of these isolates showed the presence of ambiguous bases within an expected restriction endonuclease recognition site which were not able to be resolved on repeat testing. Cloning of amplified regions of the 16S rRNA genes and subsequent sequencing of the resulting clones from the two patient isolates showed three different 16S rRNA gene sequences which corresponded to sequences found in N. nova, a molecular variant of N. nova, and a previously undescribed sequence. Hybridization studies using a DNA probe corresponding to an 89-bp conserved region of the 16S rRNA gene confirmed the presence of at least two copies of the 16S rRNA gene in the N. nova type strain, in a patient isolate identical to the molecular variant of N. nova, and in the two other patient isolates. All isolates were found to belong to the species N. nova as determined by DNA-DNA hybridization. Because minimal variation has been found in the 16S rRNA gene sequences of different species of Nocardia, those laboratories employing molecular methods for identification of these species must be aware of the potential identification complications that may be caused by the presence of differing 16S rRNA genes in the same isolate.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nocardia species have been implicated in a wide variety of infectious diseases in both immunocompetent and immunocompromised patients. In recent years the number of recognized clinically relevant Nocardia species has dramatically increased, and the resulting difficulties in determining an accurate identification for many of these isolates by conventional methodologies have been well documented (4, 8, 14). The use of molecular techniques, including restriction endonuclease assays (REA) using amplified portions of both the heat shock protein gene and the 16S rRNA gene, has greatly improved the reliability of the identifications obtained for these species (8, 16). To date, however, the method that provides the most definitive identification of most Nocardia isolates is sequence analysis of the 16S rRNA gene (14). In many cases, analysis of even a segment of the gene gives sufficient information to allow an accurate identification (6). However, in our experience with 16S rRNA gene sequencing of Nocardia isolates, we have occasionally observed the occurrence of ambiguous bases which are not able to be resolved, even with repeated sequencing. Occasionally, these ambiguous bases occur within a recognition sequence of a restriction endonuclease, resulting in restriction fragment length polymorphism (RFLP) patterns that show more bands than expected. We have recently observed this problem with several isolates suspected to be related to Nocardia nova; RFLP patterns obtained from DpnII digests of an amplified region of the 16S rRNA genes of these isolates corresponded both to those obtained from N. nova and to an RFLP variant of N. nova. Sequence analysis confirmed the presence of at least one ambiguous base occurring within the recognition sequence of the endonuclease. We determined that these isolates contained at least two copies of the 16S rRNA gene and that the sequence of the gene at the restriction site was different for the two copies. We here report the detailed molecular analysis of three patient isolates and of the N. nova type strain.

(The possible existence of such isolates was previously reported [Abstr. 104th Gen. Meet. Am. Soc. Microbiol., abstr. U-075, 2004].)

Top Abstract Introduction Materials and Methods Results Discussion References

 
Organisms. The American Type Culture Collection type strain of Nocardia nova (ATCC 33726^T) was used as the organism to which all other Nocardia nova-like organisms were compared. Three patient isolates were examined. Isolate A was obtained from a patient being treated at the Clinical Center of the National Institutes of Health, and isolates B and C were referred to the University of Texas Health Center at Tyler for identification and/or susceptibility testing. All isolates were grown on Sabouraud dextrose agar, Emmons modification (Hardy Diagnostics, Santa Monica, Calif.); organisms were modified acid-fast stain positive, and colonies showed aerial hyphae. Molecular studies on all isolates were performed on subcultures derived from a single colony.

Restriction endonuclease assay and direct 16S rRNA gene sequencing. DNA from all isolates was extracted as previously described (8). All organisms were initially identified using PCR of a 999-bp region of the 16S rRNA gene and a 440-bp region of the 65-kDa heat shock protein (HSP) gene and subsequent REA as previously described (8, 16). Briefly, the amplified region of the 16S rRNA gene was digested with the endonucleases HinP1I, DpnII, BstEII, and SphI (New England Biolabs, Beverly, Mass.); the amplified region of the HSP gene was digested with the endonucleases MspI and HinfI (New England Biolabs). Digests were electrophoresed for 2 h on a 2% MetaPhor agarose gel (Cambrex Bio Science Rockland, Inc., Rockland, Maine). Gels were analyzed using the Bio-Rad Molecular Analyst System (Bio-Rad Laboratories, Hercules, Calif.); resulting RFLP patterns were compared to patterns obtained with the type strains of various Nocardia species known to cause human disease. The sequences of a 1,463-bp region of the 16S rRNA genes of the type strain and patient isolates were determined using procedures previously described (7, 8).

Cloning. PCR was performed using the DNA prepared for use for molecular identification; the DNA of isolates B and C was extracted as previously described (8). Amplification of a 532-bp region of the 16S rRNA gene (corresponding to bases 2 through 533 of the sequence of Nocardia asteroides ATCC 19247^T; GenBank accession number X84850) was performed using the primers 5'-CGA-ACG-CTG-GCG-GCG-TGC-TTA-AC-3' and 5'-ACC-GCC-TAC-AAG-CTC-TTT-ACG-CC-3' (Research Genetics, Huntsville, AL), each at a concentration of 0.25 µM. PCR was performed using puReTaq Ready-To-Go PCR Beads (Amersham Biosciences, Buckinghamshire, United Kingdom) with 2.5 µl extracted DNA. The DNA was denatured for 5 min at 94°C and then subjected to 40 cycles of amplification (94°C for 60 s, 68°C for 45 s, and 72°C for 60 s) followed by a 10-min extension at 72°C. Cloning was performed using the TA cloning kit (Invitrogen Corporation, Carlsbad, Calif.). Briefly, amplified DNA was ligated into pCR2.1 (Invitrogen Corporation) and transformed into One Shot INVF' chemically competent Escherichia coli (Invitrogen Corporation). Transformants were plated on Luria-Bertani agar with ampicillin, X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside), and IPTG (isopropyl-ß-D-thiogalactopyranoside) (KD Medical, Columbia, MD), and colonies showing inserts were subcultured to Lennox L broth (Quality Biologicals, Gaithersburg, MD) and incubated at 35°C overnight. Plasmids were recovered using the QIAprep spin miniprep kit (QIAGEN Inc., Valencia, Calif.), and inserts were tested for appropriate size by digestion with the restriction endonuclease EcoRI (New England Biolabs). Nineteen plasmid clones from both isolates B and C were sequenced directly using "reaction 1" sequencing primers with tails containing M13 forward binding sites. One clone of each sequence type was further sequenced with the "reaction 1" sequencing primers containing the M13 forward and reverse binding sites as previously described (8).

Restriction digests of genomic DNA and hybridization studies. Genomic DNA from all isolates was extracted as previously described (7, 9, 10). DNA was quantitated and digested for 2 h using the restriction endonuclease SphI (New England Biolabs) according to the recommendations of the manufacturer. This endonuclease was chosen because analysis of 16S rRNA gene sequences of all four isolates showed no SphI recognition sites within the region studied. Digests were electrophoresed overnight on a 0.5% SeaKem GTG gel (Cambrex Bio Science Rockland, Inc.) containing 2 µg ethidium bromide (Amresco, Solon, Ohio) at 20 V. Digests were visualized using the Kodak Image Station 440CF (Eastman Kodak Company, Rochester, NY). DNA fragments were transferred and fixed to a positively charged nylon membrane (Roche Diagnostics, Mannheim, Germany) (15). A DNA probe was designed which was complementary to an 89-bp conserved region of the nocardial 16S rRNA gene corresponding to bases 1098 through 1186 of the sequence of N. asteroides ATCC 19247^T (GenBank accession number X84850). The probe sequence was 5'-GAG-ACT-GCC-GGG-GTC-AAC-TCG-GAG-GAA-GGT-GGG-GAC-GAC-GTC-AAG-TCA-TCA-TGC-CCC-TTA-TGT-CCA-GGG-CTT-CAC-ACA-TGC-TAC-AAT-GG-3' (Midland Certified Reagent Co., Crawford, Tex.). The probe was labeled at the 3' end with digoxigenin-labeled dideoxyuridine triphosphate by using the DIG Oligonucleotide Tailing Kit, 2nd Generation (Roche Diagnostics) according to the instructions of the manufacturer. Hybridization using 10 pmol of the labeled probe in 7.5 ml DIG Easy Hyb (Roche Diagnostics) with 0.1 mg/ml poly(A) (Roche Diagnostics) was performed overnight in a rotating hybridization chamber at 55°C. Stringency washes were performed as recommended (Roche Diagnostics). Chemiluminescent detection was performed using the DIG Luminescent Detection Kit (Roche Diagnostics), according to the manufacturer's instructions. Luminescent products were visualized and digitized using the Kodak Image Station 440CF.

DNA-DNA hybridization. Genomic DNA from all isolates was extracted as previously described (7, 9, 10). DNA obtained from the type strain of N. nova was labeled with [³²P]dCTP by using a nick translation kit (Invitrogen Corporation). The hybridization method for determination of DNA relatedness by absorbance to hydroxyapatite has been described previously (3). All reactions were performed in duplicate at 70°C. The relative binding ratio ([percentage of heterologous DNA bound to hydroxyapatite/percentage of homologous DNA bound to hydroxyapatite] x 100) was calculated by the method of Brenner et al. (2). The percent divergence (calculated to the nearest 0.5%) was determined by assuming that each degree of heteroduplex instability, compared to the melting temperature of the homologous duplex, was caused by 1% unpaired bases (2).

Sequence analysis. Sequences were assembled using SeqMan II software (DNASTAR, Inc., Madison, Wis.) and aligned using Megalign software (DNASTAR, Inc.) using the Clustal V algorithm. The presence of SphI recognition sites within the 16S rRNA gene sequence was evaluated using MapDraw software (DNASTAR).

Top Abstract Introduction Materials and Methods Results Discussion References

 
REA. HinP1I digests of the amplified region of the 16S rRNA gene showed identical RFLP patterns for the type strain of N. nova and the three patient isolates (bands of approximately 420, 350, and 225 bp) (data not shown). DpnII digests of the amplified region of the 16S rRNA gene showed three different RFLP patterns (Fig. 1), one for the N. nova type strain, one for the N. nova molecular variant, and a third for isolates B and C. The N. nova type strain and isolate A (designated "N. nova variant") each showed three bands of 640, 200, and 95 bp and 700, 200, and 95 bp, respectively. Isolates B and C showed identical patterns of four bands (700, 640, 200, and 95 bp) that appeared to be a combination of the patterns observed with N. nova and the N. nova variant. MspI and HinfI digests of the amplified region of the HSP gene showed identical patterns for all isolates, corresponding to the pattern expected for N. nova (16) (data not shown).

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FIG. 1. DpnII digests of a 999-bp amplified region of the 16S rRNA gene. Lane 1, base pair marker; lane 2, N. nova ATCC 33726T; lane 3, isolate A (N. nova variant); lane 4, isolate B; lane 5, isolate C; lane 6, base pair marker.

16S rRNA gene sequencing. BLAST searches of the sequences obtained for isolates A, B, and C all showed closest similarity to N. nova. Alignment of 1,369-bp sequences (sequences were trimmed to the size of the shortest sequence) of isolate A and the N. nova type strain showed two base discrepancies (>99.9% similarity) (Table 1); one of these base discrepancies occurred within a recognition sequence of the endonuclease DpnII. The 1,369-bp sequences of isolates B and C each showed two base discrepancies from the sequence of the type strain of N. nova (>99.9% similarity); each of these base discrepancies were ambiguous bases which were not able to be resolved (Fig. 2). All of the discrepancies for all isolates occurred within the regions corresponding to bases 102 to 154 of N. asteroides ATCC 19247^T (GenBank accession number X84850). No SphI recognition sites were detected within the 16S rRNA gene sequences of any of the isolates.

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TABLE 1. Base discrepancies among the N. nova type strain and patient isolates B and C

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FIG. 2. Sequence trace data for isolates B and C, showing direct sequencing with ambiguous bases and sequence data for individual clones. The region corresponds to the gene region between bases 152 and 156 of the sequence of N. asteroides ATCC 19247T (GenBank accession no. X84850).

Clones. Analysis of the sequences of the 19 clones of isolate B showed two different sequence patterns (Table 1). The sequences of 10 of the clones were identical to the sequence obtained for the type strain of N. nova; the sequences of 9 of the clones were identical to the sequence obtained for isolate A, the N. nova variant. For isolate C, two different base sequence patterns were also found with the 19 clones sequenced (Table 1). The sequences of 14 of the clones were identical to the sequence obtained for the type strain of N. nova, and the sequences of the five remaining clones showed two base discrepancies from the N. nova type strain. These two discrepancies were in adjacent bases, both corresponding to the recognition site of DpnII (Table 1). Base discrepancies noted for the cloned plasmids of isolates B and C correspond to the ambiguous bases noted in the direct sequences of isolates B and C (Table 1; Fig. 2).

Hybridization assay with digested genomic DNA. The labeled probe hybridized with two regions of the SphI-digested genomic DNAs of all isolates (Fig. 3).

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FIG. 3. DNA hybridization of SphI digests of genomic DNA with a probe complementary to an 89-bp region of the 16S rRNA gene. Lane 1, N. nova ATCC 33726T; lane 2, isolate A (N. nova variant); lane 3, isolate B; lane 4, isolate C; lane 5, positive control.

DNA-DNA hybridization. DNA hybridization results for isolates A, B, and C with the N. nova type strain showed that all three patient isolates belonged to the species N. nova. Compared to the type strain of N. nova, isolates A, B, and C showed relative binding ratios (percent divergences) of 95 (2.0), 100 (2.0), and 86 (1.0), respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The presence of multiple copies of the 16S rRNA gene has been documented for numerous bacterial species; recent reports have shown that within some species, the multiple copies show distinct sequence differences (1, 5, 11-13, 17). We are unaware of any previous documentation of the presence of more than one copy of the 16S rRNA gene in members of the genus Nocardia. The three patient isolates included in this study were proven to be members of the species N. nova by DNA-DNA hybridization. These three isolates and the N. nova type strain all contained at least two copies of the 16S rRNA gene. For the N. nova type strain and isolate A (an isolate determined to be the N. nova variant), the within-organism 16S rRNA gene copies are identical to each other as determined by the distinct RFLP patterns obtained by REA and by the lack of ambiguous bases in the gene sequences. The N. nova variant has previously been considered to be related to the type strain of N. nova based on the similarities of the 16S rRNA sequences (8); the N. nova variant, however, showed a different RFLP pattern from the type strain of N. nova when REA was performed using a DpnII digest of the amplified region of the 16S rRNA gene. The sequences of the 16S rRNA genes of the N. nova type strain and isolate A differed by two bases; one of these base differences occurred within the recognition sequence of DpnII, resulting in the different RFLP patterns obtained for the two isolates.

The patient isolates B and C were also shown to possess at least two copies of the 16S rRNA gene. Cloning experiments showed that the two genes in each of the isolates were different from each other. For each isolate, the sequence of one of the genes was identical to that of the N. nova type strain. For isolate B, the sequence of the second gene was identical to that of isolate A, the N. nova variant. For isolate C, the sequence of the second gene was unlike either the sequence of the N. nova type strain or that of the N. nova variant.

We are confident that the sequence differences that we note here represent real base differences between the two 16S rRNA gene copies and not mixed cultures or polymerase transcription errors. All molecular work was initiated from subcultures of single colonies. The 16S rRNA gene sequences obtained for the multiple gene copies were reproduced by analyzing multiple clones from each isolate, and multiple sequences from each clone were studied.

We were unable to unequivocally determine if there are more than two copies of the 16S rRNA gene present in these isolates. DNA hybridization with a probe complementary to a conserved region of the 16S rRNA gene showed two distinct regions of hybridization for each isolate. The SphI endonuclease does not have a restriction site within any of the 16S rRNA genes we sequenced, so the double bands shown in Fig. 2 indicate that each of the isolates we studied contains at least two separate 16S rRNA genes. Although it seem improbable, it is remotely possible that the hybridized area of the blot may contain more than one band of nearly identical size that was not clearly resolved. Preliminary work with other patient isolates not included in this study suggests that some Nocardia isolates may possess more than two copies of the 16S rRNA gene (data not shown). Further study using digestion with additional restriction endonucleases may allow more definitive determination of the total number of 16S rRNA copies with the N. nova isolates studied here.

Cilia et al. reported that with the Enterobacteriaceae, which are known to have multiple 16S rRNA copies, the sequences of the multiple within-organism copies tend to be different within the most variable regions of the gene (5), and they noted that these regions represent the locations of most mutations. This is also the case with the isolates presented here. The DpnII recognition site is located within the "variable region" of the 16S rRNA gene of Nocardia (8); it is this region which tends to vary from species to species and which is particularly useful in the identification of various Nocardia species by sequence analysis.

The presence of multiple copies of the 16S rRNA gene has some specific implications for the widespread use of molecular methods for the identification of Nocardia species. If both gene copies of a particular isolate are of the same sequence, identification of the isolate should be straightforward by any method, as long as that isolate belongs to a previously described species. The presence of multiple copies with base differences within a restriction endonuclease recognition site may result in uninterpretable RFLP patterns when REA testing of the 16S rRNA gene is performed; frequently these isolates show RFLP patterns with extra bands resulting from amplification and digestion of both gene regions. In such cases, when more than one gene is present, the sum of the sizes of all bands may exceed the size of the original amplified product. It is important to be aware of the fact that RFLP patterns will not appear unusual for isolates that have more than one copy of the 16S rRNA gene unless those genes have base changes within a restriction endonuclease recognition site. If sequencing is performed to identify isolates with multiple gene copies, ambiguous bases will be seen in the tracings where there are base discrepancies (Fig. 2). Care should be taken to analyze these sequences carefully and to attempt resolution of any ambiguous bases by repeat testing, if necessary. This is especially important when attempting to identify isolates such as Nocardia which have been shown to have particularly small 16S rRNA gene sequence differences among species (7). Cilia et al. noted that when analyzing phylogenies of closely related species, attention should be paid to the method used for obtaining sequence data, as sequences derived from clones may be more definitive than sequence data derived from direct sequencing (5).

The presence of multiple 16S rRNA gene copies within a particular organism could be of particular importance if, as we suspect is possible, the sequence of one or more of the copies is unlike the sequence of any known organism. Under these circumstances, only DNA-DNA hybridization would be able to provide definitive identification of the isolate.

Because of the difficulties encountered in conventional identification of Nocardia species and the recent reliance on molecular methods for accurate species determination, the awareness of the presence of multiple copies of the 16S rRNA gene in at least some isolates of Nocardia species should enhance the reliability of identifications obtained using these procedures. Laboratorians must be diligent when analyzing and using 16S rRNA sequences for the identification of Nocardia species and should carefully analyze any ambiguities or discrepancies detected.

 
We thank Richard Wallace, Barbara Brown-Elliott, and Rebecca Wilson of the University of Texas Health Care Center at Tyler for supplying the isolates resembling N. nova that were used in this study. We thank Stephen Fischer and Li Li of the Microbiology Service, Department of Laboratory Medicine, Clinical Center, NIH, for technical advice concerning probe hybridization procedures. We thank Patrick Murray, Microbiology Service, Department of Laboratory Medicine, Clinical Center, NIH, for critically reviewing the manuscript.

 
* Corresponding author. Mailing address: 10 Center Drive, MSC 1508, National Institutes of Health, Bethesda, MD 20892-1508. Phone: (301) 496-4433. Fax: (301) 402-1886. E-mail: pconville{at}nih.gov.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, June 2005, p. 2881-2885, Vol. 43, No. 6
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.6.2881-2885.2005

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