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Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences
F A Rainey1, N L Ward-Rainey, P H Janssen
1DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany, rainey@gbf-braunschweig.de
Abstract:
Sequence analysis of the cloned 16S rRNA genes of Clostridium paradoxum DSM 7308T revealed the presence of 15 different sequences in variable region I (Escherichia coli position 73-97) of the 16S rRNA. The majority of the cloned genes contained intervening sequences (IVSs), which varied in length from 120-131 nt, and were present in the DNA obtained from single colonies of C. paradoxum. The absence of IVSs in the mature rRNA was demonstrated by Northern hybridization and sequence analysis of the 16S rRNA reverse transcriptase (RT)-PCR product. This finding was supported by the failure of oligonucleotide probes specific for certain IVSs to hybridize to the RT-PCR product obtained from C. paradoxum. Alterations in culture conditions (temperature, pH, salt) or culture age did not lead to expression of RNA containing IVSs, as indicated by the size of RT-PCR products. Hybridization of the restriction-enzyme-digested genomic DNA of C. paradoxum with probes derived from the IVSs demonstrated that the 16S rRNA genes containing different IVSs are located at different sites on the chromosome.
Insights
Clostridium paradoxum possesses 16S ribosomal RNA (rRNA) genes with intervening sequences (IVSs). These IVSs are absent in mature rRNA, indicating a unique gene expression mechanism in this bacterium.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The 16S ribosomal RNA (rRNA) gene is a crucial molecular marker for bacterial identification and phylogenetic analysis.
- Intervening sequences (IVSs) are non-coding sequences within genes that are typically spliced out during RNA processing.
- The presence and processing of IVSs in bacterial 16S rRNA genes are rare and not fully understood.
Purpose of the Study:
- To investigate the presence and nature of intervening sequences (IVSs) in the 16S rRNA genes of Clostridium paradoxum DSM 7308T.
- To determine if these IVSs are present in the mature 16S rRNA molecule.
- To explore the genomic location and potential regulation of these IVS-containing genes.
Main Methods:
- Cloning and sequencing of 16S rRNA genes from Clostridium paradoxum.
- Northern hybridization and reverse transcriptase PCR (RT-PCR) to analyze mature rRNA.
- Oligonucleotide probing to detect IVS-specific sequences in rRNA.
- Restriction enzyme digestion and Southern hybridization of genomic DNA.
Main Results:
- Fifteen distinct sequences, including variable intervening sequences (IVSs) of 120-131 nucleotides, were identified in the cloned 16S rRNA genes.
- Northern hybridization and RT-PCR confirmed the absence of IVSs in the mature 16S rRNA, indicating efficient splicing.
- Culture condition modifications did not alter the presence or absence of IVSs in the expressed rRNA.
- Genomic DNA analysis revealed that 16S rRNA genes containing different IVSs are located at distinct chromosomal positions.
Conclusions:
- Clostridium paradoxum DSM 7308T possesses 16S rRNA genes with unique, variable intervening sequences.
- These IVSs are effectively removed during RNA processing, resulting in mature rRNA devoid of these sequences.
- The differential genomic organization of IVS-containing 16S rRNA genes suggests a complex regulatory mechanism.