YbeA is the m3Psi methyltransferase RlmH that targets nucleotide 1915 in 23S rRNA

Pseudouridines in the stable RNAs of Bacteria are seldom subjected to further modification. There are 11 pseudouridine (Ψ) sites in Escherichia coli rRNA, and further modification is found only at Ψ1915 in 23S rRNA, where the N-3 position of the base becomes methylated. Here, we report the identity of the E. coli methyltransferase that specifically catalyzes methyl group addition to form m³Ψ1915. Analyses of E. coli rRNAs using MALDI mass spectrometry showed that inactivation of the ybeA gene leads to loss of methylation at nucleotide Ψ1915. Methylation is restored by complementing the knockout strain with a plasmid-encoded copy of ybeA. Homologs of the ybeA gene, and thus presumably the ensuing methylation at nucleotide m³Ψ1915, are present in most bacterial lineages but are essentially absent in the Archaea and Eukaryota. Loss of ybeA function in E. coli causes a slight slowing of the growth rate. Phylogenetically, ybeA and its homologs are grouped with other putative S-adenosylmethionine-dependent, SPOUT methyltransferase genes in the Cluster of Orthologous Genes COG1576; ybeA is the first member to be functionally characterized. The YbeA methyltransferase is active as a homodimer and docks comfortably into the ribosomal A site without encroaching into the P site. YbeA makes extensive interface contacts with both the 30S and 50S subunits to align its active site cofactor adjacent to nucleotide Ψ1915. Methylation by YbeA (redesignated RlmH for rRNA large subunit methyltransferase H) possibly functions as a stamp of approval signifying that the 50S subunit has engaged in translational initiation.     

Changes in the Content of Modified Nucleotides in Wheat rRNA during Greening

The modified nucleotide content of the ribosomal RNAs in wheat is greatly influenced by light. The rRNAs of etiolated seedlings contain far fewer modified derivatives. The modified nucleotide composition characteristic of green plants develops gradually as a result of irradiation. In the course of the experiments changes in the state of modification of 5.8S and 18S rRNAs were examined during the greening of etiolated wheat seedlings. Three types of minor nucleotides, O^2′-methyladenosine, O^2′-methylguanosine and pseudouridine were found in the 5.8S rRNA of green wheat leaves, none of which was detected in etiolated wheat. The minor nucleotides appeared in the 5.8S rRNA only after 48 h irradiation. The sequences of 5.8S rDNA, TTS1, ITS2 and 18S rDNA were also determined and the presence of the hyper-modified nucleotide 1-methyl-3-(α-amino-α-carboxypropyl)-pseudouridine was detected in green wheat 18S rRNA. This minor component was not demonstrable in etiolated wheat 18S rRNA, but appeared after irradiation for 48 h. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bacterial phylogeny based on 16S and 23S rRNA sequence analysis

Abstract: Molecular phylogeny increasingly supports the understanding of organismal relationships and provides the basis for the classification of microorganisms according to their natural affiliations. Comparative sequence analysis of ribosomal RNAs or the corresponding genes currently is the most widely used approach for the reconstruction of microbial phylogeny. The highly and less conserved primary and higher order structure elements of rRNAs document the history of microbial evolution and are informative for definite phylogenetic levels. An optimal alignment of the primary structures and a careful data selection are prerequisites for reliable phylogenetic conclusions. rRNA based phylogenetic trees can be reconstructed and the significance of their topologies evaluated by applying distance, maximum parsimony and maximum likelihood methods of phylogeny inference in comparison, and by fortuitous or directed resampling of the data set. Phylogenetic trees based on almost equivalent data sets of bacterial 23S and 16S rRNAs are in good agreement and their overall topologies are supported by alternative phylogenetic markers such as elongation factors and ATPase subunits. Besides their phylogenetic information content, the differently conserved primary structure regions of rRNAs provide target sites for specific hybridization probes which have been proven to be powerful tools for the identification of microbes on the basis of their phylogenetic relationships.     

Identification of pseudouridine methyltransferase in Escherichia coli

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem–loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m³Ψ) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Ψ1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m³Ψ1915 is the only methylated pseudouridine in bacteria described to date.     

乳及乳制品中阪崎肠杆菌PCR-DHPLC检测新技术的建立

为了应用PCR结合变性高效液相色谱(DHPLC)技术建立食品中阪崎肠杆菌的快速检测方法,根据阪崎肠杆菌16S-23S rRNA特异基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测.以阪崎肠杆菌等59株参考菌株做特异性试验;阪崎肠杆菌菌株稀释成不同梯度,做灵敏度试验,结果表明该方法具有很好的特异性,方法灵敏度较高,检测低限可达到为25 CFU/mL;该方法可以快速、准确检测阪崎肠杆菌,是食品中致病菌快速检测的新技术.     

我国主要生态区域绿豆慢生根瘤菌的遗传多样性和系统发育研究

利用16SrRNAPCR-RFLP、16SrRNA序列分析以及16S-23SrRNAIGS(IntergeneticSpacer)PCR-RFLP技术对分离自中国主要生态区域的44株慢生型绿豆根瘤菌和5株参比菌株进行了遗传多样性和系统发育研究。16SrRNAPCR-RFLP分析表明:在76%的相似水平上,所有供试菌株可分为三大类群:群I由LYG1等13株慢生根瘤菌组成,该群在系统发育上与B.japonicum和B.liaoningense的参比菌株存在一定的差异;群Ⅱ由XJ1等21株供试菌株、B.japonicum和B.liaoningense的代表菌株组成;群Ⅲ由10株来自广东和广西的菌株和B.elkanii的代表菌株组成。16S-23SrRNAIGSPCR-RFLP分析将供试菌株分为A、B两大群。群A由34株供试菌株、B.japonicum和B.liaoningense的代表菌株组成。在85%的相似性水平上,可再分为AⅠ、AⅡ和AⅢ3个亚群。群B由10株分离自广西和广东的菌株和B.elkanii的代表菌株组成。在85%的相似性水平上,可再分为BI和BⅡ两亚群,表现出一定的多样性。与16SrRNAPCR-RFLP相比,16S-23SrRNAIGSPCR-RFLP具有更高的解析度,供试菌株表现出更加丰富的遗传多样性。分离自中国新疆、广东和广西等地的菌株在分群上具有较为明显的地域特征。     

乳及乳制品中肺炎克雷伯氏菌PCR-DHPLC检测新技术的建立

为了应用PCR结合变性高效液相色谱(DHPLC)技术建立乳品中肺炎克雷伯氏菌的快速检测方法,根据肺炎克雷伯氏菌16S-23S rRNA特异基因序列的特点设计特异性引物,PCR扩增的产物经DHPLC技术进行快速检测。以肺炎克雷伯氏菌等57株参考菌株做特异性试验;将肺炎克雷伯氏菌菌株稀释成不同梯度,做灵敏度试验。试验结果表明该方法具有很好的特异性,灵敏度较高,检测低限可达到100 CFU/mL,可以快速、准确检测肺炎克雷伯氏菌,是乳及乳制品中致病菌快速检测的新技术。     

Identification of shiga toxin-producing bacteria by a new immuno-capture toxin gene PCR

Mechanisms and occurrence of macrolide resistance in the periodontal pathogen Treponema denticola have received little attention. In this study, erythromycin resistance due to mutations in the genes encoding T. denticola 23S rRNA was investigated. The T. denticola genome was shown to contain two copies of 23S rDNA. 23S rRNA genes of nine erythromycin-resistant isolates derived from T. denticola were amplified and sequences were analyzed. All the erythromycin-resistant strains had at least one A-->G transition mutation at the 23S rRNA gene sequence cognate to position A2058 in Escherichia coli 23S rDNA. This suggests that antibiotic pressure is sufficient to select for point mutations that confer resistance in this organism.     

Major differences between the rrnA operons of two strains of Agrobacterium vitis

The sequence of the rrnA operon and its flanking regions was determined for the Agrobacterium vitis type strain NCPPB3554. Compared to the earlier obtained rrnA sequence of A. vitis strain S4, several important differences were noted: the sequences diverged at the 5′-flanking region, within the 16S–23S intergenic region, and within the 23S rRNA sequence. The B8 stem-loop structure at the 5′-end of the 23S rRNA of strain NCPPB3554 was 142 nt shorter than that of strain S4. These findings have important consequences for the use of ribosomal RNA gene sequences in phylogenetic comparisons. Received: 16 February 1996 / Accepted: 26 April 1996     

cpcHID操纵子序列用于钝顶节旋藻品系分类与鉴定的研究

克隆并测定7株钝顶节旋藻品系的cpcHID操纵子序列,以及16SrRNA和16S-23SrRNA转录单元内间隔区(ITS)序列,进一步通过生物信息学和分子系统学等研究发现:(1)7株品系的cpcHID序列,以及16SrRNA和ITS序列具有很高的相似性。(2)基于7株品系cpcHID序列的GC含量绝对偏差平均值、碱基变异率和遗传距离系数普遍比基于16SrRNA和ITS序列的大。(3)基于cpcHID序列的分类结果与基于16SrRNA和ITS序列的十分相近。因此,cpcHID可作为节旋藻等蓝细菌分类与鉴定的一种新的分子标记,特别是以其丰富的信息量而在品系水平的分类鉴定中占有优势。     

Detection and Phylogenetic Relationships of Highly Diverse Uncultured Acidobacterial Communities in Altamira Cave Using 23S rRNA Sequence Analyses

Acidobacteria have been established as a novel and widespread bacterial phylum consistently detected during 16S rDNA-based molecular surveys. Recently, representatives of the phylum Acidobacteria were found to make up a large percentage of cloned sequences recovered from paleolithic paintings. The present study addresses the intraphylum diversity of Acidobacteria in Altamira Cave. In contrast to previous studies, we have focused on 23S rRNA-based phylogenetic analyses, exploiting the high information content of the 23S RNA gene. Comparison with existing studies was facilitated by the preparation of corresponding 16S rRNA clone libraries. Fourty-one distinct operational taxonomic units (OTU) could be identified. Of the eight described acidobacterial subgroups, five were represented in the cave; only subgroups 1, 2, and 8 were not found. Subgroup 4 was the group harbouring the highest number of OTUs. Various clusters that could not be assigned to any of the established subgroups, but were clearly grouped within the phylum and appeared to represent new taxa at elevated phylogenetic levels, were detected. Results from this study give a first insight into the enormous acidobacterial diversity existing in a single hypogean environment containing unique paleolithic paintings, the Altamira Cave. Novel taxa have been found within the phylum, suggesting a higher diversity of Acidobacteria than previously suspected even at a local scale.