Function of heterologous and truncated RNase P proteins in Bacillus subtilis

Bacterial RNase P is composed of an RNA subunit and a single protein (encoded by the rnpB and rnpA genes respectively). The Bacillus subtilis rnpA knockdown strain d7 was used to screen for functional conservation among bacterial RNase P proteins from a representative spectrum of bacterial subphyla. We demonstrate conserved function of bacterial RNase P (RnpA) proteins despite low sequence conservation. Even rnpA genes from psychrophilic and thermophilic bacteria rescued growth of B. subtilis d7 bacteria; likewise, terminal extensions and insertions between beta strands 2 and 3, in the so-called metal binding loop, were compatible with RnpA function in B. subtilis. A deletion analysis of B. subtilis RnpA defined the structural elements essential for bacterial RNase P function in vivo. We further extended our complementation analysis in B. subtilis strain d7 to the four individual RNase P protein subunits from three different Archaea, as well as to human Rpp21 and Rpp29 as representatives of eukaryal RNase P. None of these non-bacterial RNase P proteins showed any evidence of being able to replace the B. subtilis RNase P protein in vivo, supporting the notion that archaeal/eukaryal RNase P proteins are evolutionary unrelated to the bacterial RnpA protein.     

Thermostable RNase P RNAs lacking P18 identified in the Aquificales

The RNase P RNA (rnpB) and protein (rnpA) genes were identified in the two Aquificales Sulfurihydrogenibium azorense and Persephonella marina. In contrast, neither of the two genes has been found in the sequenced genome of their close relative, Aquifex aeolicus. As in most bacteria, the rnpA genes of S. azorense and P. marina are preceded by the rpmH gene coding for ribosomal protein L34. This genetic region, including several genes up- and downstream of rpmH, is uniquely conserved among all three Aquificales strains, except that rnpA is missing in A. aeolicus. The RNase P RNAs (P RNAs) of S. azorense and P. marina are active catalysts that can be activated by heterologous bacterial P proteins at low salt. Although the two P RNAs lack helix P18 and thus one of the three major interdomain tertiary contacts, they are more thermostable than Escherichia coli P RNA and require higher temperatures for proper folding. Related to their thermostability, both RNAs include a subset of structural idiosyncrasies in their S domains, which were recently demonstrated to determine the folding properties of the thermostable S domain of Thermus thermophilus P RNA. Unlike 16S rRNA phylogeny that has placed the Aquificales as the deepest lineage of the bacterial phylogenetic tree, RNase P RNA-based phylogeny groups S. azorense and P. marina with the green sulfur, cyanobacterial, and delta/epsilon proteobacterial branches.     

Differential effects of mutations in the protein and RNA moieties of RNase P on the efficiency of suppression by various tRNA suppressors

We have studied the efficiency of suppression by tRNA suppressors in vivo in strains of Escherichia coli that harbor a mutation in the rnpA gene, the gene for the protein component (C5) of RNase P, and in strains that carry several different alleles of the rnpB gene, the gene for the RNA component (M1) of RNase P. Depending on the genetic background, different efficiencies of suppression by the various tRNA suppressors were observed. Thus, mutations in rnpA have separable and distinct effects from mutations in rnpB on the processing of tRNA precursors by RNase P. In addition, the efficiency of suppression by several derivatives of E. coli tRNA(Tyr) Su3 changed as the genetic background was altered.     

By any other name: heterologous replacement of the Escherichia coli RNase P protein subunit has in vivo fitness consequences

Bacterial RNase P is an essential ribonucleoprotein composed of a catalytic RNA component (encoded by the rnpB gene) and an associated protein moiety (encoded by rnpA). We construct a system that allows for the deletion of the essential endogenous rnpA copy and for its simultaneous replacement by a heterologous version of the gene. Using growth rate as a proxy, we explore the effects on fitness of heterologous replacement by increasingly divergent versions of the RNase P protein. All of the heterologs tested complement the loss of the endogenous rnpA gene, suggesting that all existing bacterial versions of the rnpA sequence retain the elements required for functional interaction with the RNase P RNA. All replacements, however, exact a cost on organismal fitness, and particularly on the rate of growth acceleration, defined as the time required to reach maximal growth rate. Our data suggest that the similarity of the heterolog to the endogenous version--whether defined at the sequence, structure or codon usage level--not predict the fitness costs of the replacement. The common assumption that sequence similarity predicts functional similarity requires experimental confirmation and may prove to be an oversimplification.     

Possible evolutionary origin of primitive protein-encoding mRNAs as a virusoid-like ribo-organism.

E. coli rnpA and rpmH genes encoding the protein portion of ribonuclease (RNase) P and L34 ribosomal protein were found to be homologous to the entire sequence of M1 RNA and virusoids. The resulting alignment strongly suggests that most primitive mRNAs must have emerged from virusoid-like ribo-organism.     

Conserved gene arrangement in the origin region of the Streptomyces coelicolor chromosome.

A 23-kb fragment of the Streptomyces coelicolor chromosome spanning the dnaA region has been isolated as a cosmid clone. Nucleotide sequence analysis of a 5-kb portion shows that the genes for the RNase P protein (rnpA), ribosomal protein L34 (rpmH), the replication initiator protein (dnaA), and the beta subunit of DNA polymerase III (dnaN) are present in the highly conserved gene arrangement found in all eubacterial genomes studied so far. The dnaA-dnaN intergenic region is approximately 1 kb and contains a cluster of at least 12 DnaA boxes with a consensus sequence of TTGTCCACA matching the consensus DnaA box in the phylogenetically related Micrococcus luteus. Two DnaA boxes precede the dnaA sequence. We propose that the chromosomal origin (oriC) of S. coelicolor lies between dnaA and dnaN. In related work, J. Zakrzewska-Czerwinska and H. Schrempf (J. Bacteriol. 174:2688-2693, 1992) have identified the homologous sequence from the closely-related Streptomyces lividans as capable of self-replication.     

Complementation of an RNase P RNA (rnpB) gene deletion in Escherichia coli by homologous genes from distantly related eubacteria.

We report the construction of a strain of Escherichia coli in which the only functional gene for the RNA moiety of RNase P (rnpB) resides on a plasmid that is temperature sensitive for replication. The chromosomal RNase P RNA gene was replaced with a chloramphenicol acetyltransferase gene. The conditionally lethal phenotype of this strain was suppressed by plasmids that carry RNase P RNA genes from some distantly related eubacteria, including Alcaligenes eutrophus, Bacillus subtilis, and Chromatium vinosum. Thus, the rnpB genes from these organisms are capable of functioning as the sole source of RNase P RNA in E. coli. The rnpB genes of some other organisms (Agrobacterium tumefaciens, Pseudomonas fluorescens, Bacillus brevis, Bacillus megaterium, and Bacillus stearothermophilus) could not replace the E. coli gene. The significance of these findings as they relate to RNase P RNA structure and function and the utility of the described strain for genetic studies are discussed.     

Ribonuclease P RNA gene sequencing as a tool for molecular dereplication of myxobacterial strain collections

AIMS: Efficient strain dereplication is of great value during the generation of bacterial strain collections for industrial screening. We evaluated the utilization of the RNase P RNA gene (rnpB) sequence as a tool for molecular dereplication of myxobacteria. METHODS AND RESULTS: 16S rDNA (approx. 1 x 5 kbp) and rnpB (approx. 0 x 3 kbp) sequences were obtained and aligned. From 50 strains, we obtained 20 different sequences for the 16S rDNA and 24 for rnpB. Intersequence similarity was lower for rnpB than for 16S rDNA. CONCLUSIONS: rnpB allows the rapid discrimination of similar strains, with a higher resolution power as compared with 16S rRNA gene sequencing. It not only gives better discrimination, but is also faster and cheaper than 16S rDNA sequencing. SIGNIFICANCE AND IMPACT OF THE STUDY: Myxobacteria isolation and cultivation require time and experience. The application of rnpB sequencing to early myxobacterial strain dereplication may help in the generation of diverse strain libraries of these bacteria.     

The RNase P RNA from cyanobacteria: short tandemly repeated repetitive (STRR) sequences are present within the RNase P RNA gene in heterocyst-forming cyanobacteria.

The RNase P RNA gene (rnpB) from 10 cyanobacteria has been characterized. These new RNAs, together with the previously available ones, provide a comprehensive data set of RNase P RNA from diverse cyanobacterial lineages. All heterocystous cyanobacteria, but none of the non-heterocystous strains analyzed, contain short tandemly repeated repetitive (STRR) sequences that increase the length of helix P12. Site-directed mutagenesis experiments indicate that the STRR sequences are not required for catalytic activity in vitro. STRR sequences seem to have recently and independently invaded the RNase P RNA genes in heterocyst-forming cyanobacteria because closely related strains contain unrelated STRR sequences. Most cyanobacteria RNase P RNAs lack the sequence GGU in the loop connecting helices P15 and P16 that has been established to interact with the 3'-end CCA in precursor tRNA substrates in other bacteria. This character is shared with plastid RNase P RNA. Helix P6 is longer than usual in most cyanobacteria as well as in plastid RNase P RNA.     

Genes within genes within bacteria

Recently, an unusual gene structure has been described in species of the genus Thermus, in which the rpmH (ribosomal protein L34) coding sequence was found to be entirely overlapped by the unusually large rnpA (RNase P protein subunit) sequence. Gene overlap is common in viruses, but has not been seen to this extent in any bacterium.     

Analysis of conserved non-rRNA genes of Tropheryma whipplei

The causative agent of Whipple's disease, Tropheryma whipplei, is a slow-growing bacterium that remains poorly-understood. Genetic characterization of this organism has relied heavily upon rRNA sequence analysis. Pending completion of a complete genome sequencing effort, we have characterized several conserved non-rRNA genes from T. whipplei directly from infected tissue using broad-range PCR and a genome-walking strategy. Our goals were to evaluate its phylogenetic relationships, and to find ways to expand the strain typing scheme, based on rDNA sequence comparisons. The genes coding for the ATP synthase beta subunit (atpD), elongation factor Tu (tuf), heat shock protein GroEL (groEL), beta subunit of DNA-dependent RNA polymerase (rpoB), and RNase P RNA (rnpB) were analyzed, as well as the regions upstream and downstream of the rRNA operon. Phylogenetic analyses with all non-rRNA marker molecules consistently placed T. whipplei within the class, Actinobacteria. The arrangement of genes in the atpD and rpoB chromosomal regions was also consistent with other actinomycete genomes. Tandem sequence repeats were found upstream and downstream of the rRNA operon, and downstream of the groEL gene. These chromosomal sites and the 16S-23S rRNA intergenic spacer regions were examined in the specimens of 11 patients, and a unique combination of tandem repeat numbers and spacer polymorphisms was found in each patient. These data provide the basis for a more discriminatory typing method for T. whipplei.     

A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P.

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.     

Mitochondrial RNase P RNAs in ascomycete fungi: lineage-specific variations in RNA secondary structure

The RNA subunit of mitochondrial RNase P (mtP-RNA) is encoded by a mitochondrial gene (rnpB) in several ascomycete fungi and in the protists Reclinomonas americana and Nephroselmis olivacea. By searching for universally conserved structural elements, we have identified previously unknown rnpB genes in the mitochondrial DNAs (mtDNAs) of two fission yeasts, Schizosaccharomyces pombe and Schizosaccharomyces octosporus; in the budding yeast Pichia canadensis; and in the archiascomycete Taphrina deformans. The expression of mtP-RNAs of the predicted size was experimentally confirmed in the two fission yeasts, and their precise 5' and 3' ends were determined by sequencing of cDNAs generated from circularized mtP-RNAs. Comparative RNA secondary structure modeling shows that in contrast to mtP-RNAs of the two protists R. americana and N. olivacea, those of ascomycete fungi all have highly reduced secondary structures. In certain budding yeasts, such as Saccharomycopsis fibuligera, we find only the two most conserved pairings, P1 and P4. A P18 pairing is conserved in Saccharomyces cerevisiae and its close relatives, whereas nearly half of the minimum bacterial consensus structure is retained in the RNAs of fission yeasts, Aspergillus nidulans and Taphrina deformans. The evolutionary implications of the reduction of mtP-RNA structures in ascomycetes will be discussed.     

Conditional expression of RNase P in the cyanobacterium Synechocystis sp. PCC6803 allows detection of precursor RNAs. Insight in the in vivo maturation pathway of transfer and other stable RNAs

We have constructed a strain (CT1) that expresses RNase P conditionally with the aim to analyze the in vivo tRNA processing pathway and the biological role that RNase P plays in Synechocystis 6803. In this strain, the rnpB gene, coding for the RNA subunit of RNase P, has been placed under the control of the petJ gene promoter (P(petJ)), which is repressed by copper, cell growth, and accumulation of RNase P RNA is inhibited in CT1 after the addition of copper, indicating that the regulation by copper is maintained in the chimerical P(petJ)-rnpB gene and that RNase P is essential for growth in Synechocystis. We have analyzed several RNAs by Northern blot and primer extension in CT1. Upon addition of copper to the culture medium, precursors of the mature tRNAs are detected. Furthermore, our results indicate that there is a preferred order in the action of RNase P when it processes a dimeric tRNA precursor. The precursors detected are 3'-processed, indicating that 3' processing can occur before 5' processing by RNase P. The size of the precursors suggests that the terminal CCA sequence is already present before RNase P processing. We have also analyzed other potential RNase P substrates, such as the precursors of tmRNA and 4.5 S RNA. In both cases, accumulation of larger than mature size RNAs is observed after transferring the cells to a copper-containing medium.