scholarly journals Reading canonical and modified nucleotides in 16S ribosomal RNA using nanopore direct RNA sequencing

2017 ◽  
Author(s):  
Andrew M. Smith ◽  
Miten Jain ◽  
Logan Mulroney ◽  
Daniel R. Garalde ◽  
Mark Akeson
Keyword(s):  
16S Rrna ◽  
Rna Sequencing ◽  
Ionic Current ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Rapid Identification ◽  
Rrna Genes ◽  
Aminoglycoside Resistance ◽  
Base Pairs ◽  
E Coli

The ribosome small subunit is expressed in all living cells. It performs numerous essential functions during translation, including formation of the initiation complex and proofreading of base-pairs between mRNA codons and tRNA anticodons. The core constituent of the small ribosomal subunit is a ∼1.5 kb RNA strand in prokaryotes (16S rRNA) and a homologous ∼1.8 kb RNA strand in eukaryotes (18S rRNA). Traditional sequencing-by-synthesis (SBS) of rRNA genes or rRNA cDNA copies has achieved wide use as a ‘molecular chronometer’ for phylogenetic studies 1, and as a tool for identifying infectious organisms in the clinic 2. However, epigenetic modifications on rRNA are erased by SBS methods. Here we describe direct MinION nanopore sequencing of individual, full-length 16S rRNA absent reverse transcription or amplification. As little as 5 picograms (∼10 attomole) of E. coli 16S rRNA was detected in 4.5 micrograms of total human RNA. Nanopore ionic current traces that deviated from canonical patterns revealed conserved 16S rRNA base modifications, and a 7-methylguanosine modification that confers aminoglycoside resistance to some pathological E. coli strains. This direct RNA sequencing technology has promise for rapid identification of microbes in the environment and in patient samples.

scholarly journals Aminoglycoside Resistance with Homogeneous and Heterogeneous Populations of Antibiotic-Resistant Ribosomes

2001 ◽  
Vol 45 (9) ◽  
pp. 2414-2419 ◽  
Author(s):  
Michael I. Recht ◽  
Joseph D. Puglisi
Keyword(s):  
16S Rrna ◽  
Small Subunit ◽  
Aminoglycoside Resistance ◽  
Antibiotic Resistant ◽  
Partial Dominance ◽  
E Coli ◽  
Bacterial Ribosome ◽  
High Level ◽  
Decoding Region ◽  
Level Resistance

ABSTRACT Aminoglycosides bind to rRNA in the small subunit of the bacterial ribosome. Mutations in the decoding region of 16S rRNA confer resistance to specific subsets of aminoglycoside antibiotics. The two major classes of 2-deoxystreptamine aminoglycosides are the 4,5- and the 4,6-disubstituted antibiotics. Antibiotics of the 4,5-disubstituted class include neomycin, paromomycin, and ribostamycin. Gentamicins and kanamycins belong to the 4,6-disubstituted class of aminoglycosides. Structural studies indicated the potential importance of position 1406 (Escherichia coli numbering) in the binding of ring III of the 4,6-disubstituted class of aminoglycosides to 16S rRNA. We have introduced a U1406-to-A mutation in a plasmid-encoded copy of E. coli 16S rRNA which has been expressed either in a mixture with wild-type ribosomes or in a strain in which all rRNA is transcribed from the plasmid-encoded rrn operon. High-level resistance to many of the 4,6-disubstituted aminoglycosides is observed only when all the rRNA contains the U1406-to-A mutation. In contrast to the partial dominance of resistance observed with other mutations in the decoding region, there is a dominance of sensitivity with the 1406A mutation. Chemical footprinting experiments indicate that resistance arises from a reduced affinity of the antibiotic for the rRNA target. These results demonstrate that although position 1406 is an important determinant in the binding and action of the 4,6-disubstituted aminoglycosides, other rRNA mutations that perturb the binding of ring I of both classes of 2-deoxystreptamine aminoglycosides confer higher levels of resistance as well as a partial dominance of resistance.

Protein-induced conformational changes in 16S rRNA during assembly of the Escherichia Coli 30S ribosomal subunit: A STEM study

1987 ◽  
Vol 45 ◽  
pp. 910-911
Author(s):  
M. Boublik ◽  
V. Mandiyan ◽  
J.F. Hainfeld ◽  
J.S. Wall
Keyword(s):  
16S Rrna ◽  
Conformational Changes ◽  
Ribosomal Proteins ◽  
Structural Changes ◽  
Ribosomal Subunit ◽  
Compact Structure ◽  
E Coli ◽  
Freeze Dried ◽  
16S Rna ◽  
30S Subunit

The aim of this study is to understand the mechanism of 16S rRNA folding into the compact structure of the small 30S subunit of E. coli ribosome. The assembly of the 30S E. coli ribosomal subunit is a sequence of specific interactions of 16S rRNA with 21 ribosomal proteins (S1-S21). Using dedicated high resolution STEM we have monitored structural changes induced in 16S rRNA by the proteins S4, S8, S15 and S20 which are involved in the initial steps of 30S subunit assembly. S4 is the first protein to bind directly and stoichiometrically to 16S rRNA. Direct binding also occurs individually between 16S RNA and S8 and S15. However, binding of S20 requires the presence of S4 and S8. The RNA-protein complexes are prepared by the standard reconstitution procedure, dialyzed against 60 mM KCl, 2 mM Mg(OAc)2, 10 mM-Hepes-KOH pH 7.5 (Buffer A), freeze-dried and observed unstained in dark field at -160°.

Functional and Structural Characterization of Acquired Pan-Aminoglycoside Resistance 16S rRNA Methyltransferase NpmB1

2021 ◽  
Author(s):  
Akito Kawai ◽  
Masahiro Suzuki ◽  
Kentaro Tsukamoto ◽  
Yusuke Minato ◽  
Yohei Doi
Keyword(s):  
Amino Acid ◽  
16S Rrna ◽  
Amino Acid Identity ◽  
Single Amino Acid ◽  
Aminoglycoside Resistance ◽  
E Coli ◽  
The United Kingdom ◽  
Amino Acid Variant ◽  
A Site

Post-translational methylation of the A site of 16S rRNA at position A1408 leads to pan-aminoglycoside resistance encompassing both 4,5- and 4,6-disubstituted 2-deoxystreptamine (DOS) aminoglycosides. To date, NpmA is the only acquired enzyme with such function. Here, we present function and structure of NpmB1 whose sequence was identified in Escherichia coli genomes registered from the United Kingdom. NpmB1 possesses 40% amino acid identity with NpmA1 and confers resistance to all clinically relevant aminoglycosides including 4,5-DOS agents. Phylogenetic analysis of NpmB1 and NpmB2, its single amino acid variant, revealed that the encoding gene was likely acquired by E. coli from a soil bacterium. The structure of NpmB1 suggests that it requires a structural change of the β6/7 linker in order to bind to 16S rRNA. These findings establish NpmB1 and NpmB2 as the second group of acquired pan-aminoglycoside resistance 16S rRNA methyltransferases.

scholarly journals Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

2006 ◽  
Vol 50 (2) ◽  
pp. 445-452 ◽  
Author(s):  
Daniel Criswell ◽  
Virginia L. Tobiason ◽  
J. Stephen Lodmell ◽  
D. Scott Samuels
Keyword(s):  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Type Strain ◽  
Wild Type Strain ◽  
Small Subunit ◽  
Wild Type ◽  
Spectinomycin Resistance ◽  
E Coli ◽  
Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

scholarly journals A new species of the Dendropsophus parviceps group from the western Amazon Basin (Amphibia: Anura: Hylidae)

Zootaxa ◽  
2012 ◽  
Vol 3249 (1) ◽  
pp. 18 ◽  
Author(s):  
ANA P. MOTTA ◽  
SANTIAGO CASTROVIEJO-FISHER ◽  
PABLO J. VENEGAS ◽  
VICTOR G.D. ORRICO ◽  
JOSÉ M. PADIAL
Keyword(s):  
New Species ◽  
Maximum Likelihood ◽  
16S Rrna ◽  
Amazon Basin ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Terra Firme ◽  
Base Pairs ◽  
White Spots ◽  
A New Species

We describe Dendropsophus frosti sp. nov. from lowland terra firme rainforests of the headwaters of the Amazon RiverBasin in Colombia and Peru. The new species is known from only two localities, the type locality near Leticia (Departa-mento Amazonas, Colombia, 04° 06' 24.2" S, 69° 56' 57.4" W; 103 m.a.s.l.), and the paratopotypic locality, Piedras in thePutumayo basin (Departamento Loreto, Peru, 02.79278° S, 72.91750° W; 90–170 m.a.s.l.). Maximum likelihood and par-simony analyses of 2436 aligned base pairs of the 12S and 16S rRNA genes recovered the new species as a member of D.parviceps group and sister to D. brevifrons. The new species is most closely related to D. parviceps, D. brevifrons, and D.koechlini, and it can be readily distinguished from these and all other members of the D. parviceps group by, among othercharacters, its plain dorsal light brown coloration, copper iris, plain immaculate pale yellow to white venter coloration, lack of flash marks on groin and axillae, and absence of white spots on lips.

scholarly journals Effect of gamma irradiation on viability and DNA of Staphylococcus epidermidis and Escherichia coli

2006 ◽  
Vol 55 (9) ◽  
pp. 1271-1275 ◽  
Author(s):  
Andrej Trampuz ◽  
Kerryl E. Piper ◽  
James M. Steckelberg ◽  
Robin Patel
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Gamma Irradiation ◽  
Staphylococcus Epidermidis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Molecular Diagnostic ◽  
Molecular Diagnostic Techniques ◽  
Bacterial Cells ◽  
E Coli

Gamma irradiation is widely used for sterilization; however, its effect on elimination of amplifiable DNA, an issue of relevance to molecular diagnostic approaches, has not been well studied. The effect of gamma irradiation on the viability of Staphylococcus epidermidis and Escherichia coli (using quantitative cultures) and on their DNA (using quantitative 16S rRNA gene PCR) was evaluated. Viability was abrogated at 2.8 and 3.6 kGy for S. epidermidis and E. coli, respectively. The radiation dose required to reduce viable bacteria by one log10 (D 10 value) was 0.31 and 0.35 kGy for S. epidermidis and E. coli, respectively. D 10 values for amplifiable DNA extracted from bacteria were 2.58 and 3.09 kGy for S. epidermidis and E. coli, respectively, whereas D 10 values for amplifiable DNA were significantly higher for DNA extracted from irradiated viable bacterial cells (22.9 and 52.6 kGy for S. epidermidis and E. coli, respectively; P<0.001). This study showed that gamma irradiation of DNA in viable bacterial cells has little effect on amplifiable DNA, was not able to eliminate amplifiable 16S rRNA genes at a dose of up to 12 kGy and cannot therefore be used for elimination of DNA contamination of PCR reaction components or laboratory equipment when this DNA is present in microbial cells. This finding has practical implications for those using molecular diagnostic techniques in microbiology.

scholarly journals Fitness Cost and Interference of Arm/Rmt Aminoglycoside Resistance with the RsmF Housekeeping Methyltransferases

2012 ◽  
Vol 56 (5) ◽  
pp. 2335-2341 ◽  
Author(s):  
Belen Gutierrez ◽  
Jose A. Escudero ◽  
Alvaro San Millan ◽  
Laura Hidalgo ◽  
Laura Carrilero ◽  
...  
Keyword(s):  
16S Rrna ◽  
Pathogenic Bacteria ◽  
Binding Pocket ◽  
Maldi Mass Spectrometry ◽  
Fitness Cost ◽  
Resistance Pattern ◽  
Aminoglycoside Resistance ◽  
Content Type ◽  
E Coli ◽  
High Level

ABSTRACTArm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants ofEscherichia coli, corresponding to the genotypesrsmF+, ΔrsmF,rsmF+rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmFbacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility inE. coli. Competition experiments between the isogenicE. colistrains showed that, contrary to expectation, acquisition ofrmtCdoes not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

scholarly journals Succession of Microbial Communities during Hot Composting as Detected by PCR–Single-Strand-Conformation Polymorphism-Based Genetic Profiles of Small-Subunit rRNA Genes

2000 ◽  
Vol 66 (3) ◽  
pp. 930-936 ◽  
Author(s):  
Sabine Peters ◽  
Stefanie Koschinsky ◽  
Frank Schwieger ◽  
Christoph C. Tebbe
Keyword(s):  
16S Rrna ◽  
18S Rrna ◽  
Single Strand Conformation Polymorphism ◽  
Small Subunit ◽  
Ssu Rdna ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Small Subunit Rrna ◽  
Genetic Profiles ◽  
Conformation Polymorphism

ABSTRACT A cultivation-independent technique for genetic profiling of PCR-amplified small-subunit rRNA genes (SSU rDNA) was chosen to characterize the diversity and succession of microbial communities during composting of an organic agricultural substrate. PCR amplifications were performed with DNA directly extracted from compost samples and with primers targeting either (i) the V4–V5 region of eubacterial 16S rRNA genes, (ii) the V3 region in the 16S rRNA genes of actinomycetes, or (iii) the V8–V9 region of fungal 18S rRNA genes. Homologous PCR products were converted to single-stranded DNA molecules by exonuclease digestion and were subsequently electrophoretically separated by their single-strand-conformation polymorphism (SSCP). Genetic profiles obtained by this technique showed a succession and increasing diversity of microbial populations with all primers. A total of 19 single products were isolated from the profiles by PCR reamplification and cloning. DNA sequencing of these molecular isolates showed similarities in the range of 92.3 to 100% to known gram-positive bacteria with a low or high G+C DNA content and to the SSU rDNA of γ-Proteobacteria. The amplified 18S rRNA gene sequences were related to the respective gene regions of Candida krusei and Candida tropicalis. Specific molecular isolates could be attributed to different composting stages. The diversity of cultivated bacteria isolated from samples taken at the end of the composting process was low. A total of 290 isolates were related to only 6 different species. Two or three of these species were also detectable in the SSCP community profiles. Our study indicates that community SSCP profiles can be highly useful for the monitoring of bacterial diversity and community successions in a biotechnologically relevant process.

scholarly journals Human ERAL1 is a mitochondrial RNA chaperone involved in the assembly of the 28S small mitochondrial ribosomal subunit

2010 ◽  
Vol 430 (3) ◽  
pp. 551-558 ◽  
Author(s):  
Sven Dennerlein ◽  
Agata Rozanska ◽  
Mateusz Wydro ◽  
Zofia M. A. Chrzanowska-Lightowlers ◽  
Robert N. Lightowlers
Keyword(s):  
16S Rrna ◽  
Cellular Localization ◽  
Mitochondrial Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Mitochondrial Rna ◽  
Rna Chaperone ◽  
Stem Loop ◽  
Loop Region

The bacterial Ras-like protein Era has been reported previously to bind 16S rRNA within the 30S ribosomal subunit and to play a crucial role in ribosome assembly. An orthologue of this essential GTPase ERAL1 (Era G-protein-like 1) exists in higher eukaryotes and although its exact molecular function and cellular localization is unknown, its absence has been linked to apoptosis. In the present study we show that human ERAL1 is a mitochondrial protein important for the formation of the 28S small mitoribosomal subunit. We also show that ERAL1 binds in vivo to the rRNA component of the small subunit [12S mt (mitochondrial)-rRNA]. Bacterial Era associates with a 3′ unstructured nonanucleotide immediately downstream of the terminal stem–loop (helix 45) of 16S rRNA. This site contains an AUCA sequence highly conserved across all domains of life, immediately upstream of the anti-Shine–Dalgarno sequence, which is conserved in bacteria. Strikingly, this entire region is absent from 12S mt-rRNA. We have mapped the ERAL1-binding site to a 33 nucleotide section delineating the 3′ terminal stem–loop region of 12S mt-rRNA. This loop contains two adenine residues that are reported to be dimethylated on mitoribosome maturation. Furthermore, and also in contrast with the bacterial orthologue, loss of ERAL1 leads to rapid decay of nascent 12S mt-rRNA, consistent with a role as a mitochondrial RNA chaperone. Finally, whereas depletion of ERAL1 leads to apoptosis, cell death occurs prior to any appreciable loss of mitochondrial protein synthesis or reduction in the stability of mitochondrial mRNA.

scholarly journals Selective and Sensitive Method for PCR Amplification of Escherichia coli 16S rRNA Genes in Soil

2000 ◽  
Vol 66 (2) ◽  
pp. 844-849 ◽  
Author(s):  
G. Sabat ◽  
P. Rose ◽  
W. J. Hickey ◽  
J. M. Harkin
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Pcr Amplification ◽  
Pcr Primers ◽  
Enteric Bacteria ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Selective Amplification ◽  
E Coli

ABSTRACT A set of PCR primers targeting 16S rRNA gene sequences was designed, and PCR parameters were optimized to develop a robust and reliable protocol for selective amplification of Escherichia coli 16S rRNA genes. The method was capable of discriminatingE. coli from other enteric bacteria, including its closest relative, Shigella. Selective amplification of E. coli occurred only when the annealing temperature in the PCR was elevated to 72°C, which is 10°C higher than the optimum for the primers. Sensitivity was retained by modifying the length of steps in the PCR, by increasing the number of cycles, and most importantly by optimizing the MgCl2 concentration. The PCR protocol developed can be completed in less then 2 h and, by using Southern hybridization, has a detection limit of ca. 10 genomic equivalents per reaction. The method was demonstrated to be effective for detectingE. coli DNA in heterogeneous DNA samples, such as those extracted from soil.

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