rnpB Sequence Research Articles

Multi-locus sequence analysis of ‘Candidatus Mycoplasma haematomacacae’ in free-ranging macaques from Thailand suggestive of a closer relationship to hemotropic mycoplasmas in capuchins and potential origin from bats

Although ‘Candidatus Mycoplasma haematomacacae’ (formerly known as ‘Candidatus Mycoplasma haemomacaque’) has been reported on extensively in macaques from Thailand, the USA, Japan, and Brazil, its genetic characterization has primarily been restricted to the 16S rRNA sequences with no exploration on multi-locus sequence analysis. The primary goal of this study was to characterize ‘Ca. M. haematomacacae’ among Thai macaques based on multiple genetic markers. Between April 2018 and November 2021, blood samples were taken from 580 free-ranging macaques (560 Macaca fascicularis and 20 Macaca nemestrina) in 15 locations encompassing 10 provinces throughout Thailand. Using the conventional PCR assay targeting the 16S ribosomal RNA (16S rRNA) gene, 338 out of 580 macaques (58.27 %) tested hemoplasma-positive. Of these, 40 positive samples were further subjected to DNA sequencing, and all were identified as ‘Ca. M. haematomacacae’. Subsequently, the partial nucleotide sequences of 23S ribosomal RNA (23S rRNA) and RNase P RNA (rnpB) genes of this particular hemoplasma species were amplified through nested PCR assay. The analysis of multi-locus genetic markers revealed that the 23S rRNA and rnpB sequences exhibited higher levels of genetic diversity than the 16S rRNA sequences. Furthermore, the 16S rRNA analyses demonstrated that ‘Ca. M. haematomacacae’ infecting Old World monkeys (Macaca spp.) was most closely related to hemotropic Mycoplasma spp. in black-capped capuchins (Sapajus apella) and Marcgrave's capuchins (Sapajus flavius) from Brazil, as well as establishing a common ancestor clade with hemotropic Mycoplasma spp. from the Neotropical bats in Belize and Peru and an Old World bat in Spain. The 23S rRNA analyses likewise evidenced that ‘Ca. M. haematomacacae’ formed a sister clade with hemotropic Mycoplasma spp. in Neotropical bats from Belize and Panama. Thus, the present findings, based on multi-locus sequence analysis, suggest a potential origin of ‘Ca. M. haematomacacae’ from Neotropical and Old World bats. To the best of the authors’ knowledge, this study provided the largest dataset so far of multi-locus genetic sequences of ‘Ca. M. haematomacacae’ isolated from Thai macaques and enhanced the accuracy of phylogenetic analyses, providing insights into their origins among hemotropic Mycoplasma spp. discovered worldwide.

Comparison of species identification of endocarditis associated viridans streptococci using rnpB genotyping and 2 MALDI-TOF systems

Streptococcus spp. are important causes of infective endocarditis but challenging in species identification. This study compared identification based on sequence determination of the rnpB gene with 2 systems of matrix-assisted laser desorption ionization–time of flight mass spectrometry, MALDI Biotyper (Bruker) and VITEK MS IVD (bioMérieux). Blood culture isolates of viridans streptococci from 63 patients with infective endocarditis were tested. The 3 methods showed full agreement for all 36 isolates identified in the Anginosus, Bovis, and Mutans groups or identified as Streptococcus cristatus, Streptococcus gordonii, or Streptococcus sanguinis. None of the methods could reliably identify the 23 isolates to the species level when designated as Streptococcus mitis, Streptococcus oralis, or Streptococcus tigurinus. In 7 isolates classified to the Mitis group, the rnpB sequences deviated strikingly from all reference sequences, and additional analysis of sodA and groEL genes indicated the occurrence of yet unidentified Streptococcus spp.

Construction of an Efficient In Vitro System for Analysis of Transcription from Sigma 54-Dependent pspA Promoter

IbsC is a small toxin protein in Escherichia coli, whose expression is repressed by a cis-acting small noncoding RNA, SibC (renamed from RygC or QUAD1c). Overexpression of IbsC or the absence of SibC transcription induces the expression of both psp operon (pspABCDE) and pspG gene encoding phage shock proteins, whose expression were known to be induced by multiple environment stresses or agents such as filamentous phage infection, osmotic shock, continued incubation in stationary phase, heat shock, and ethanol treatment. Although exogenous overexpression of IbsC leads to cell death, physiological roles of IbsC remain unknown especially because the strain without ibsC gene in the chromosome shows no growth defects. Considering that only Psp proteins are induced by expression of IbsC, not by other similar small toxin proteins such as LdrD, ShoB, and TisB, the role of IbsC might specifically be related to phage shock proteins. The psp operon is transcribed from pspA promoter by RNA polymerase holoenzyme containing the alternative stress-responsive sigma factor 54 (σ). Transcription from pspA promoter requires PspF as an activator protein that binds to the upstream region of the core promoter elements. Furthermore, PspA inhibits the σ-dependent pspA transcription by interacting with PspF and exerting its negative effects on transcription activation by PspF. Since the IbsC expression causes the induction of pspA transcription, it is attempting to see how the signal of IbsC gets transduced into the transcription activation. The mechanism involved in linking IbsC to pspA transcription is essential for understanding not only the physiological functions of IbsC, but also the regulation mechanism of transcription from σdependent promoters in response to environmental stresses. To understand the molecular mechanism of the pspA induction by IbsC, it is necessary to know which factors are involved in the activation of pspA transcription under conditions of IbsC expression. Although an in vitro transcription system for pspA promoter analysis was previously set up, it is difficult to identify in vitro transcripts because their transcription termination sites are unclear. To overcome the drawback of the previous in vitro transcription system, in this study, we constructed plasmid pPR56 containing a transcription fusion of pspA promoter and rnpB terminator by replacing the rnpB promoter-containing DNA fragment of pLMd23-wt with the pspA promoter-containing fragment. The pspA promoter-containing fragment spanning from −320 through +56 of pspA, which also includes two UAS sequences (UAS I and UAS II) as PspF-binding sites and the IHF binding site, was subcloned into the BamHI/ EcoRI linearlized pLMd23-wt not having the rnpB promotercontaining DNA fragment, to generate the fusion plasmid pPR56 (Fig. 1). The rnpB terminator region in the fusion construct contained the rnpB sequence from +331 to +1286, which includes the three rnpB terminators T1, T2, and T3 leading to transcription termination at +413, +526, and +638, respectively. Therefore, this pPR56 construct was designed to generate pspA-rnpB fusion transcripts of 146 nt terminating at T1 (pspAT1), 259 nt at T2 (pspAT2), and 371 nt at T3 (pspAT3) if transcription starts at the transcription initiation site of pspA. Using supercoiled plasmid pPR56 DNA as a template, in vitro transcription was carried out by adding Eσ and PspF. The pspAT1 transcript of 145 nt was produced as a major band although minor pspAT2 and pspAT3 products were also observed (Fig. 2). The increased abundance of three transcripts was observed with the incremental amount of PspF protein, while RNA I of 108 nt (a transcript transcribed from its own σ specific promoter of the plasmid DNA) was not produced. In contrast, the same transcription reaction performed with Eσ did not produce the pspAT transcripts. Instead, this reaction generated RNA I. All our data confirmed that the pspA transcripts were produced from the σ-specific pspA promoter of pPR56. To

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

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. 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. 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. 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.

Toward a quantitative DNA-based definition of pneumococcal pneumonia: a comparison of Streptococcus pneumoniae target genes, with special reference to the Spn9802 fragment

The current shift from phenotypically toward genotypically based microbial diagnosis is not unproblematic. A novel quantitative real-time polymerase chain reaction (PCR) assay based on the Spn9802 DNA fragment was therefore developed for detection of Streptococcus pneumoniae. Out of 44 bacterial species, only S. pneumoniae and Streptococcus pseudopneumoniae were positive in Spn9802 PCR. In an evaluation on nasopharyngeal aspirates from 166 patients with community-acquired pneumonia, the assay was positive in 49 of 50 culture-positive cases. Of 19 culture-negative but Spn9802 PCR-positive cases, 12 were confirmed as S. pneumoniae by rnpB sequence analysis. With an expanded reference standard, including culture and rnpB sequencing, Spn9802 had a sensitivity of 94% and a specificity of 98%. A cutoff for clinically significant positivity was 10 4 DNA copies/mL, giving 71% sensitivity and 100% specificity. In conclusion, Spn9802 real-time PCR is highly sensitive and specific. The quantification it provides enables differentiation between pneumococcal pathogenicity and commensalism.

Phylogenetic relationships and species differentiation of 39 Legionella species by sequence determination of the RNase P RNA gene rnpB

The rnpB gene is ubiquitous in Bacteria, Archaea and Eucarya and encodes the RNA component of RNase P, an endoribonuclease P that consists of one RNA and one protein subunit (C5). In this study, partial rnpB genes were sequenced from 39 type strains and 16 additional strains of the genus Legionella. Models of the putative secondary structures of the RNase P RNA in the genus Legionella are proposed and possible interactions between RNase P RNA and C5 are discussed. The phylogenetic relationships within the genus Legionella were examined and rnpB sequences indicated six main clades that together comprised 27 of the 39 species examined. The phylogenetic relationships were further inferred by analysing combined datasets of sequences from the rnpB, mip, 16S rRNA and rpoB genes. It is concluded that rnpB is suitable for use in phylogenetic studies of closely related species and that it exhibits the potential to discriminate between Legionella species.

Conserved and variable domains within divergent rnase P RNA gene sequences of Prochlorococcus strains.

RNase P RNA gene (rnpB) sequences were PCR-amplified from different members of the Prochlorococcus group. Aligned nucleotide sequences revealed a variance of up to 27% for rnpB. Comparative secondary structure analysis showed that domains P12, P18 and P19 of these novel ribozyme sequences in particular are highly divergent. Thus, these regions in RNase P RNA might serve as potential targets for deoxyoligonucleotide primers for the identification of specific genotypes of Prochlorococcus and for probing field populations. Phylogenetic trees constructed from RNase P RNA sequences were similar to, but not fully congruent with, those derived previously using sequences of the 16S rRNA gene. However, the application of rnpB sequences allowed a better resolution within clades of very closely related genotypes. As is known from 16S rRNA-based phylogenetic trees, sequences from individual strains clustered according to their physiology and the conditions at the original site of isolation, rather than their geographical origin. All sequences obtained from high-light-adapted strains formed a single coherent clade, as did the four sequences from low-light-adapted strains that were previously isolated from the North Atlantic and the subtropical North Pacific. This suggests a remarkable genetic stability of Prochlorococcus genotypes that thrive under identical ecological conditions.

Characterization of the rnpB gene and RNase P RNA in the order Chlamydiales.

The sequence of the RNase P RNA gene (rnpB) was determined for 60 strains representing all nine species in the family Chlamydiaceae and for the related Chlamydiales species, Parachlamydia acanthamoebae and Simkania negevensis. These sequences were used to infer evolutionary relationships among the Chlamydiaceae. The analysis separated Chlamydophila and Chlamydia into two lineages, with Chlamydophila forming three distinct clusters: the Chlamydophila pneumoniae strains; the Chlamydophila pecorum strains; and a third cluster comprising the species Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila caviae and Chlamydophila felis. The Chlamydia line of descent contained two clusters, with the Chlamydia suis strains distinctly separated from strains of Chlamydia trachomatis and Chlamydia muridarum. This analysis indicated that the rnpB sequence and structure are distinctive markers for species in the Chlamydiaceae. It was also demonstrated that the RNase P RNA derived from Chlamydia trachomatis is able to cleave a tRNA precursor in the absence of protein. These findings are discussed in relation to the structure of Chlamydia RNase P RNA.

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis

The genes encoding the RNA ( rnpB) and protein ( rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.