Questionable 16S ribosomal RNA gene annotations are frequent in completed microbial genomes

According to recent reports, many ribosomal RNA gene annotations are still questionable, and the use of inappropriate tools for annotation has been blamed. However, we believe that the abundant 16S rRNA partial sequence in the databases, mainly created by culture-independent PCR methods, is another main cause of the ambiguous annotations of 16S rRNA. To examine the current status of 16S rRNA gene annotations in complete microbial genomes, we used as a criterion the conserved anti-SD sequence, located at the 3′ end of the 16S rRNA gene, which is commonly overlooked by culture-independent PCR methods. In our large survey, 859 16S rRNA gene sequences from 252 different species of the microbial complete genomes were inspected. 67 species (234 genes) were detected with ambiguous annotations. The common anti-SD sequence and other conserved 16S rRNA sequence features could be detected in the downstream-intergenic regions for almost every questionable sequence, indicating that many of the 16S rRNA genes were annotated incorrectly. Furthermore, we found that more than 91.5% of the 93,716 sequences of the available 16S rRNA in the main databases are partial sequences. We also performed BLAST analysis for every questionable rRNA sequence, and most of the best hits in the analysis were rRNA partial sequences. This result indicates that partial sequences are prevalent in the databases, and that these sequences have significantly affected the accuracy of microbial genomic annotation. We suggest that the annotation of 16S rRNA genes in newly complete microbial genomes must be done in more detail, and that revision of questionable rRNA annotations should commence as soon as possible.     

16S rRNA序列分析法在医学微生物鉴定中的应用

１６Ｓ ｒＲＮＡ序列分析作为微生物系统分类的主要依据已得到了广泛认同,随着微生物核糖体数据库的日益完善,该技术成为细菌分类和鉴定的一个有力工具。本文概述了 １６５ ｒＲＮＡ序列分析法的技术步骤以及该技术在医学微生物研究中的应用,总结了目前文献报导的各种致病微生物种属特异性 １６５ ｒＲＮＡ引物和探针序列,同时分析了该技术在应用中存在的一些问题。     

小鲵科线粒体16S rRNA基因序列分析及其系统发育

To study the phylogeny of Hynobiidae, we amplified DNA fragments of 470 bp 16S ribosomal RNA (16S rRNA) gene on mitochondrial DNA from Ranodon sibiricus and Ranodon tsinpaensis. PCR products were cloned into PMD18 T vector after purification. These sequences were determined and deposited in the GenBank (accession numbers: AY373459 for Ranodon sibiricus, AY372534 for Ranodon tsinpaensis). By comparing the nucleotide differences of 16S ribosomal RNA sequences among Liua shihi, Pseudohynobius flavomaculatus and Batrachuperus genus from GenBank database, we analyzed the divergences and base substitution among these sequences with the MEGA software. The molecular results support that B. tibetanus, B. pinchonii and B. karlschmidti are classified into three valid species. Liua shihi has closer phylogenetic relationships to Ranodon tsinpaensis than to other species. More our results reveal that Pseudohynobius flavomaculatus is not a synonym of Ranodon tsinpaensis. [Acta Zoologica Sinica 50 (3) : 464 - 469,2004].     

Role of N-terminal helix in interaction of ribosomal protein S15 with 16S rRNA

The position and conformation of the N-terminal helix of free ribosomal protein S15 was earlier found to be modified under various conditions. This variability was supposed to provide the recognition by the protein of its specific site on 16S rRNA. To test this hypothesis, we substituted some amino acid residues in this helix and assessed effects of these substitutions on the affinity of the protein for 16S rRNA. The crystal structure of the complex of one of these mutants (Thr3Cys S15) with the 16S rRNA fragment was determined, and a computer model of the complex containing another mutant (Gln8Met S15) was designed. The available and new information was analyzed in detail, and the N-terminal helix was concluded to play no significant role in the specific binding of the S15 protein to its target on 16S rRNA.     

A novel method for determining microflora composition using dynamic phylogenetic analysis of 16S ribosomal RNA deep sequencing data

Deep sequencing of the 16S rRNA gene provides a comprehensive view of bacterial communities in a particular environment and has expanded our ability to study the impact of the microflora on human health and disease. Current analysis methods rely on comparisons of the sequences generated with an expanding but limited set of annotated 16S rRNA sequences or phylogenic clustering of sequences based on arbitrary similarity cutoffs. We describe a novel approach to characterize bacterial composition using deep sequencing of 16S rRNA gene. Our method defines operational taxonomic units based on phylogenetic tree reconstruction and dynamic clustering of sequences using solely sequencing data. These OTUs can be used to identify differences in bacteria abundance between environments. This approach can perform better than previous phylogenetic methods and will significantly improve our understanding of the microfloral role on human diseases by providing a comprehensive analysis of the microbial composition from various bacterial communities.     

Involvement of lysine and arginine residues in the binding of yeast ribosomal protein YL3 to 5S RNA

The contribution of lysine and arginine residues to the formation of yeast ribonucleoprotein complex 5S RNA. protein YL3 has been investigated by determining the effects on complex formation of modification with chemical reagents specific for either lysine or arginine. Treatment of protein YL3 with acetic anhydride, malefic anhydride or phenylglyoxal is accompanied by loss of its capacity to bind to 5S RNA. This effect is accomplished by modification with phenylglyoxal of only 3 arginine residues per YL3 molecule. In contrast, a large number of protein YL3 amino groups [16] must be modified by acetic anhydride to prevent complex formation.     

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.     

基于18S rRNA和线粒体16S rRNA基因序列的柳蚕进化分析

为探讨柳蚕Actias selene Hübner与鳞翅目昆虫的系统发育关系,本研究利用PCR扩增获得了柳蚕核糖体18S rRNA和线粒体16S rRNA基因的部分序列,长度分别为391bp和428bp。并采用邻近距离法(NJ)、最大简约法(MP)、类平均聚类法(UPGMA)构建系统进化树。结果表明,柳蚕线粒体16SrRNA基因序列与大蚕蛾科昆虫的16SrRNA基因序列均表现出偏好于碱基AT的倾向。柳蚕与所研究的其它蚕类的遗传距离介于0.016至0.140之间,其中与温带柞蚕Antheraea roylii的遗传距离最小,与野桑蚕Bombyx mandarina的遗传距离最大。而基于鳞翅目昆虫18S rRNA基因部分序列的进化分析显示,柳蚕与柞蚕Antheraea pernyi之间的遗传距离最小(0.010),与蓖麻蚕Samia ricini的遗传距离最大(0.017)。     

Identification of probiotic lactobacilli used for animal feeds on the basis of 16S ribosomal RNA gene sequence

The use of probiotics such as Lactobacillus in animal feeds has gained popularity in recent years. In this study the 16S rRNA gene sequence of L. acidophilus in two commercial agents which have been used in animal feeds, LAB‐MOS and Ghenisson 22, was determined. Phylogenetic tree analysis revealed that the two agents, strain MNFLM01 in LAB‐MOS and strain GAL‐2 in Ghenisson 22, belonged to L. rhamnosus (a member of the L. casei group) and L. johnsonii (a member of the L. acidophilus group), respectively. Biochemical tests assigned the two as L. rhamnosus and ambiguously as L. acidophilus. The data suggest that 16S rRNA gene sequence analysis provides more accurate identification of Lactobacillus species than biochemical tests and would allow quality assurance of relevant commercial products. The 16S rRNA gene sequences of strains MNFLM01 and GAL‐2 determined in this study have been submitted to the DDBJ/EMBL/GenBank accession numbers under accession numbers AB288235 and AB295648, respectively.     

Rapid and specific detection of pathogenicleptospira species by amplification of ribosomal sequences

We have developed an assay for the detection of pathogenicLeptospira that is based on the polymerase chain reaction. With the combination of agarose gel electrophoresis and blotting, pathogenicLeptospira can be discriminated specifically from nonpathogenicLeptospira and other bacterial species. This method, based on the amplification of 16S ribosomal RNA sequences, is able to detect 10 leptospiral cells/mL in cattle urine samples and 100 leptospiral cells/mL in pig urine samples. Using this assay leptospires were detected in urine samples from cattle that were experimentally infected withLeptospira interrogate serovarhardjo type hardjobovis.     

Post-maturation of the plastid ribosomal RNA in the plant kingdom

Summary The in vivo fragmentation of the plastid rRNA from plants situated at different places in the evolutionary scale, with the exception ofAlgae, was analysed by electrophoresis using fully denaturing conditions. This fragmentation corresponds to an in vivo post-maturation. It exists only in some bacteria and is not random. Five main groups of fragments with the following real molecular weights (Mr) are found in 23 S:ca 0.9 × 10⁶; 0.7 × 10⁶; 0.45 × 10⁶; 0.35 × 10⁶ and 0.15 × 10⁶. The existence of a large fragment (Mr = 0.9 × 10⁶) corresponds to a primitive type of fragmentation found in some archaic plants. Dicotyledons and several other groups have the same pattern of 23 S fragmentation, often comprising all the fragments mentioned above, whilstGraminaceae (Monocotyledons) constitute a special group with a very predominant 0.35 × 10⁶ dalton fragment and the absence of the 0.45 × 10⁶ dalton fragment. The plastid 16 S rRNA in all plants studied here has aMr of 0.54 × 10⁶ which is smaller than the 16 S ofEscbericbia coli taken as reference (0.56 × 10⁶ dalton).     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Comparing aminoglycoside binding sites in bacterial ribosomal RNA and aminoglycoside modifying enzymes

Aminoglycoside antibiotics are used against severe bacterial infections. They bind to the bacterial ribosomal RNA and interfere with the translation process. However, bacteria produce aminoglycoside modifying enzymes (AME) to resist aminoglycoside actions. AMEs form a variable group and yet they specifically recognize and efficiently bind aminoglycosides, which are also diverse in terms of total net charge and the number of pseudo‐sugar rings. Here, we present the results of 25 molecular dynamics simulations of three AME representatives and aminoglycoside ribosomal RNA binding site, unliganded and complexed with an aminoglycoside, kanamycin A. A comparison of the aminoglycoside binding sites in these different receptors revealed that the enzymes efficiently mimic the nucleic acid environment of the ribosomal RNA binding cleft. Although internal dynamics of AMEs and their interaction patterns with aminoglycosides differ, the energetical analysis showed that the most favorable sites are virtually the same in the enzymes and RNA. The most copied interactions were of electrostatic nature, but stacking was also replicated in one AME:kanamycin complex. In addition, we found that some water‐mediated interactions were very stable in the simulations of the complexes. We show that our simulations reproduce well findings from NMR or X‐ray structural studies, as well as results from directed mutagenesis. The outcomes of our analyses provide new insight into aminoglycoside resistance mechanism that is related to the enzymatic modification of these drugs. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A novel mutation 3090 G>A of the mitochondrial 16S ribosomal RNA associated with myopathy

We describe a young woman who presented with a progressive myopathy since the age of 9. Spectrophotometric analysis of the respiratory chain in muscle tissue revealed combined and profound complex I, III, II+III, and IV deficiency ranging from 60% to 95% associated with morphological and histochemical abnormalities of the muscle. An exhaustive screening of mitochondrial transfer and ribosomal RNAs showed a novel G>A substitution at nucleotide position 3090 which was detected only in urine sediment and muscle of the patient and was not found in her mother's blood cells and urine sample. We suggest that this novel de novo mutation in the 16S ribosomal RNA, a nucleotide which is highly conserved in different species, would impair mitochondrial protein synthesis and would cause a severe myopathy.     

Evolution according to large ribosomal subunit RNA

Evolutionary trees were constructed, by distance methods, from an alignment of 225 complete large subunit (LSU) rRNA sequences, representing Eucarya, Archaea, Bacteria, plastids, and mitochondria. A comparison was made with trees based on sets of small subunit (SSU) rRNA sequences. Trees constructed on the set of 172 species and organelles for which the sequences of both molecules are known had a very similar topology, at least with respect to the divergence order of large taxa such as the eukaryotic kingdoms and the bacterial divisions. However, since there are more than ten times as many SSU as LSU rRNA sequences, it is possible to select many SSU rRNA sequence sets of equivalent size but different species composition. The topologies of these trees showed considerable differences according to the particular species set selected.The effect of the dataset and of different distance correction methods on tree topology was tested for both LSU and SSU rRNA by repetitive random sampling of a single species from each large taxon. The impact of the species set on the topology of the resulting consensus trees is much lower using LSU than using SSU rRNA. This might imply that LSU rRNA is a better molecule for studying wide-range relationships. The mitochondria behave clearly as a monophyletic group, clustering with the Proteobacteria. Gram-positive bacteria appear as two distinct groups, which are found clustered together in very few cases. Archaea behave as if monophyletic in most cases, but with a low confidence.Abbreviations LSU rRNA large subunit ribosomal RNA - SSU rRNA small subunit ribosomal RNA - JC Jukes and Cantor - JN Jin and Nei Correspondence to: R. De Wachter     

The adenosine wedge: A new structural motif in ribosomal RNA

Here, we present a new recurrent RNA arrangement, the so-called adenosine wedge (A-wedge), which is found in three places of the ribosomal RNA in both ribosomal subunits. The arrangement has a hierarchical structure, consisting of elements previously described as recurrent motifs, namely, the along-groove packing motif, the A-minor and the hook-turn. Within the A-wedge, these elements are involved in different types of cause–effect relationships, providing together for the particular tertiary structure of the motif.