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PubMed2002

Analyses of microbial activity in biomass-recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products.

The relationship between mixed microbial community structure and physiology when grown under substrate-limited conditions was investigated using continuous-flow bioreactors with 100% biomass recycle. Community structure was analyzed by denaturing gradient gel electrophoresis (DGGE) of the PCR and RT-PCR amplified V3 region of 16S rDNA and 16S rRNA templates, respectively. Comparisons were made of communities exposed to different types of transient conditions (e.g., long- and short-term starvation, increasing nutrients). With progressively more stringent substrate limitation over time, the specific content of community RNA declined by more than 10-fold and closely followed the decline in specific growth rate. In contrast, the DNA content was variable (up to 3-fold differences) and did not follow the same trend. Cluster analysis of the presence or absence of individual bands indicated that the fingerprints generated by the two templates were different, and community response was first observed in the rRNA fraction. However, both the rDNA and rRNA fingerprints provided a picture of temporal population dynamics. Dice similarity coefficients gave a quantitative measure of the differences and changes between the communities. In comparison, standard cultivation techniques yielded only a quarter of the phylotypes detected by DGGE, but included the most dominant population based on rRNA. Nucleotide-sequence analyses of the almost complete 16S rRNA genes of these isolates place them in the same group of organisms that is typically cultivated from environmental samples: alpha, beta, and gamma Proteobacteria and the high GC and the low GC Gram-positive divisions.

Bacteria↗

PubMed1991

A functional pseudoknot in 16S ribosomal RNA.

Several lines of evidence indicate that the universally conserved 530 loop of 16S ribosomal RNA plays a crucial role in translation, related to the binding of tRNA to the ribosomal A site. Based upon limited phylogenetic sequence variation, Woese and Gutell (1989) have proposed that residues 524-526 in the 530 hairpin loop are base paired with residues 505-507 in an adjoining bulge loop, suggesting that this region of 16S rRNA folds into a pseudoknot structure. Here, we demonstrate that Watson-Crick interactions between these nucleotides are essential for ribosomal function. Moreover, we find that certain mild perturbations of the structure, for example, creation of G-U wobble pairs, generate resistance to streptomycin, an antibiotic known to interfere with the decoding process. Chemical probing of mutant ribosomes from streptomycin-resistant cells shows that the mutant ribosomes have a reduced affinity for streptomycin, even though streptomycin is thought to interact with a site on the 30S subunit that is distinct from the 530 region. Data from earlier in vitro assembly studies suggest that the pseudoknot structure is stabilized by ribosomal protein S12, mutations in which have long been known to confer streptomycin resistance and dependence.

Base Sequence↗

PubMed1979

Alteration in thermal stability of ribosomes from Drosophila melanogaster with age.

Thermal analysis of high salt (0.5 M) washed ribosomal monomers from young and old male Drosophila melanogaster revealed an 8 degrees C downshift in the mean temperature of denaturation (Tm). Moreover, there was observed a marked loss in the ability of ribosomes extracted from older flies to reassociate upon cooling. These observations suggest that age-dependent alterations in the structural integrity of the rRNA-r protein complex could, at least in part, be responsible for the diminished capacity for protein synthesis in this species with advancing age.

Aging↗

PubMed1991

Characterization of a 54-nucleotide gap region in the 28S rRNA gene of Schistosoma mansoni.

We have analyzed 572 bp in the 28S rDNA of the human blood fluke Schistosoma mansoni which correspond to expansion segment 5 of domain IV as defined by Clark et al. for the Xenopus laevis 28S rRNA. S1 nuclease mapping and primer extension analysis comparing this region with the mature 28S rRNA indicate that there are 54 nucleotides present in the 28S rDNA which are absent from the mature rRNA. This defines a gap that creates two 28S rRNA subunits (28S alpha and 28S beta). Comparison of the S. mansoni sequence with rDNAs of other organisms which contain gaps in their 28S rRNA shows that the overall features are conserved except that the S. mansoni gap is less A + T-rich. The conserved features include: (1) the location of the gap within the 28S rRNA; (2) the predicted secondary structure of the gap, containing a stem-loop with a UAAU sequence within the loop; and (3) a conserved CGAAAGGG on the 3' side of the gap.

Animals↗

PubMed1992

Evidence for a close genealogical relationship between Afipia (the causal organism of cat scratch disease), Bradyrhizobium japonicum and Blastobacter denitrificans.

The primary structures of the small subunit rRNA of Bradyrhizobium japonicum and Blastobacter denitrificans were determined by direct sequencing of enzymatically amplified DNA. Comparative sequence analysis revealed that Bradyrhizobium japonicum and Blastobacter denitrificans are members of the alpha-2 subgroup of the Proteobacteria and show a very close phylogenetic relationship with the genus Afipia.

Animals↗

PubMed2004

Microbial communities in oil-contaminated seawater.

Although diverse bacteria capable of degrading petroleum hydrocarbons have been isolated and characterized, the vast majority of hydrocarbon-degrading bacteria, including anaerobes, could remain undiscovered, as a large fraction of bacteria inhabiting marine environments are uncultivable. Using culture-independent rRNA approaches, changes in the structure of microbial communities have been analyzed in marine environments contaminated by a real oil spill and in micro- or mesocosms that mimic such environments. Alcanivorax and Cycloclasticus of the gamma-Proteobacteria were identified as two key organisms with major roles in the degradation of petroleum hydrocarbons. Alcanivorax is responsible for alkane biodegradation, whereas Cycloclasticus degrades various aromatic hydrocarbons. This information will be useful to develop in situ bioremediation strategies for the clean-up of marine oil spills.

Bacteria↗

PubMed2002

Limitation of ribosomal protein L11 availability in vivo affects translation termination.

Historically referred to as "the GTPase center", the L11 binding region (L11BR) of Escherichia coli 23 S rRNA is a highly conserved structure that has been implicated in several essential functions during protein synthesis. Here, in vivo expression of an RNA fragment containing that structure was found to affect translation termination in a codon-specific manner. The cause of these effects appeared to be titration of ribosomal protein L11, since normal phenotypes could be restored by simultaneous overproduction of wild-type L11 but not mutant L11. Subsequently, altered termination phenotypes were produced when the availability of L11 was limited by overexpression of RNA antisense to L11 mRNA and, finally, by inactivation of the chromosomal L11 gene, and they too were reversible by simultaneous expression of cloned L11. Our results indicate that in the intact cell the L11BR is an integral functional unit important for translation termination and that the presence of L11 in ribosomes is required for UAG-dependent termination and is somewhat inhibitory of UGA-dependent termination.

Base Sequence↗

PubMed1979

Properties of Escherichia coli 16S ribosomal ribonucleic acid treated with 4,5',8-trimethylpsoralen and light.

16S rRNA reacted with the furocoumarin 4,5',8-trimethylpsoralen (trioxsalen) and 360-nm light showed a number of chemical and physical differences from untreated RNA. After extensive irradiation, five molecules of trioxsalen were bound per molecule of RNA. The trioxsalen-treated RNA had an altered ultraviolet absorption spectrum and a distinctive fluorescence emission spectrum. The modified RNA was significantly more resistant to T1 ribonuclease digestion than was control RNA. Treated RNA, when mixed with purified ribosomal proteins, was not functional in the in vitro reconstitution of 30S subunits and yielded more slowly sedimenting particles which were inactive in protein synthesis assays. By contrast, 16S rRNA within the 30S subunit structure did not exhibit these changes when reacted with the same dose of trioxsalen and light, suggesting that the ribosomal proteins were effective in protecting the RNA from interaction with the drug.

Cell Fractionation↗

PubMed2005

Genomic and proteomic comparisons between bacterial and archaeal genomes and related comparisons with the yeast and fly genomes.

Bacterial, archaeal, yeast, and fly genomes are compared with respect to predicted highly expressed (PHX) genes and several genomic properties. There is a striking difference in the status of PHX ribosomal protein (RP) genes where the archaeal genome generally encodes more RP genes and fewer PHX RPs compared with bacterial genomes. The increase in RPs in archaea and eukaryotes compared with that in bacteria may reflect a more complex set of interactions in archaea and eukaryotes in regulating translation, e.g., differences in structure requiring scaffolding of longer rRNA molecules, expanded interactions with the chaperone machinery, and, in eukaryotic interactions with endoplasmic reticulum components. The yeast genome is similar to fast-growing bacteria in PHX genes but also features several cytoskeletal genes, including actin and tropomyosin, and several signal transduction regulatory proteins from the 14.3.3 family. The most PHX genes of Drosophila encode cytoskeletal and exoskeletal proteins. We found that the preference of a microorganism for an anaerobic metabolism correlates with the number of PHX enzymes of the glycolysis pathway that well exceeds the number of PHX enzymes acting in the tricarboxylic acid cycle. Conversely, if the number of PHX enzymes of the tricarboxylic acid cycle well exceeds the PHX enzymes of glycolysis, an aerobic metabolism is preferred. Where the numbers are approximately commensurate, a facultative growth behavior prevails.

Animals↗

PubMed2002

The ribosome filter hypothesis.

A variety of posttranscriptional mechanisms affects the processing, subcellular localization, and translation of messenger RNAs (mRNAs). Translational control appears to occur primarily at the initiation rather than the elongation stage. It has been suggested that translation is mediated largely by means of a cap-binding/scanning mechanism. On the basis of recent findings, we propose here that differential binding of particular mRNAs to eukaryotic 40S ribosomal subunits before translation may also selectively affect rates of polypeptide chain production. In this view, ribosomal subunits themselves are considered to be regulatory elements or filters that mediate interactions between particular mRNAs and components of the translation machinery. Differences in these interactions affect how efficiently individual mRNAs compete for ribosomal subunits. These competitive interactions would depend in part on the complementarity between sequences in mRNA and rRNA, as well as on structural differences among ribosomes in different cell types. By these means, translation may either be enhanced through increased recruitment of ribosomes or inhibited through strong interactions that sequester mRNAs. We propose that ribosomal filters may be important in cell differentiation and describe experimental tests for the filter hypothesis.

Models, Biological↗

PubMed1988

Evidence that glutamic acid 167 is an active-site residue of Shiga-like toxin I.

Escherichia coli Shiga-like toxin I, a close relative of Shiga toxin and a distant relative of the ricin family of plant toxins, inhibits eukaryotic protein synthesis by catalyzing the depurination of adenosine 4324 in 28S rRNA. By comparing the crystallographic structure of ricin with amino acids conserved between the Shiga and ricin toxin families, we identified seven potential active-site residues of Shiga-like toxin I. The structural gene encoding Shiga-like toxin I A chain (Slt-IA), the enzymatically active subunit, was engineered for high expression in E. coli. Oligonucleotide-directed mutagenesis of the gene for Slt-IA was used to change glutamic acid 167 to aspartic acid. As measured by an in vitro assay for inhibition of protein synthesis, the specific activity of mutant Slt-IA was decreased by a factor of 1000 compared to wild-type Slt-IA. Immunoblots showed that mutant and wild-type Slt-IA were synthesized as full-length proteins and were processed correctly by signal peptidase. Both proteins were equally susceptible to trypsin digestion, suggesting that the amino acid substitution did not produce a major alteration in Slt-IA conformation. We conclude that glutamic acid 167 is critical for activity of the Shiga-like toxin I A chain and may be located at the active site.

Bacterial Toxins↗

PubMed2000

Primary structure and function analysis of the Abrus precatorius agglutinin A chain by site-directed mutagenesis. Pro(199) Of amphiphilic alpha-helix H impairs protein synthesis inhibitory activity.

Abrus agglutinin (AAG), a low-toxicity protein from the plant Abrus precatorius, is less lethal than abrina (ABRa) in mice (LD(50) = 5 mg/kg versus 20 microg/kg of body weight). Nucleotide sequence analysis of a cDNA clone encoding full-length AAG showed an open reading frame with 1641 base pairs, corresponding to a 547-amino acid residue preproprotein containing a signal peptide and a linker region (two amino acid residues) between the AAG-A and AAG-B subunits. AAG had high homology to ABRa (77.8%). The 13 amino acid residues involved in catalytic function, which are highly conserved among abrins and ricins, were also conserved within AAG-A. The protein synthesis inhibitory activity of AAG-A (IC(50) = 3.5 nM) was weaker than that of ABRa-A (0.05 nM). Molecular modeling followed by site-directed mutagenesis showed that Pro(199) of AAG-A, located in amphiphilic helix H and corresponding to Asn(200) of ABRa-A, can induce bending of helix H. This bending would presumably affect the binding of AAG-A to its target sequence, GpApGpAp, in the tetraloop structure of the 28 S rRNA subunit and could be one of the major factors contributing to the relatively weak protein synthesis inhibitory activity and toxicity of AAG.

Abrin↗

PubMed2026

Concurrent ecological and evolutionary processes contribute to mutualism breakdown between legumes and rhizobia.

Though they jointly shape community responses to environmental perturbations, ecology and evolution are often examined separately, even in microorganisms where both occur over short timescales. Here we examine ecological and evolutionary responses to 33 years of nitrogen fertilization using the legume-rhizobium mutualism. Pairing a manipulative inoculation study with full-length 16S rRNA gene amplicon sequencing and structural equation modeling allows us to synthesize across biological scales: whole bacterial community, genus Rhizobium, Rhizobium ASVs, and symbiosis plasmids. Clover's preferred partner decreases in N-addition soils, limiting host growth, while a diverse and largely uncharacterized Rhizobium community increases. This ecological change is compounded by a concurrent evolutionary degradation of symbiont partner quality via changing frequencies of symbiotic plasmids. Ecological (rarer symbionts) and evolutionary (inferior symbionts) processes each accounted for roughly half of this loss of host benefit, revealing that ecology and evolution jointly shape mutualism breakdown over the short timescales typical of microbial systems.

ecology↗

PubMed2006

An improved statistical method for detecting heterotachy in nucleotide sequences.

The principle of heterotachy states that the substitution rate of sites in a gene can change through time. In this article, we propose a powerful statistical test to detect sites that evolve according to the process of heterotachy. We apply this test to an alignment of 1289 eukaryotic rRNA molecules to 1) determine how widespread the phenomenon of heterotachy is in ribosomal RNA, 2) to test whether these heterotachous sites are nonrandomly distributed, that is, linked to secondary structure features of ribosomal RNA, and 3) to determine the impact of heterotachous sites on the bootstrap support of monophyletic groupings. Our study revealed that with 21 monophyletic taxa, approximately two-thirds of the sites in the considered set of sequences is heterotachous. Although the detected heterotachous sites do not appear bound to specific structural features of the small subunit rRNA, their presence is shown to have a large beneficial influence on the bootstrap support of monophyletic groups. Using extensive testing, we show that this may not be due to heterotachy itself but merely due to the increased substitution rate at the detected heterotachous sites.

Chi-Square Distribution↗

PubMed1994

Different forms of U15 snoRNA are encoded in the introns of the ribosomal protein S1 gene of Xenopus laevis.

Recent cloning and sequencing of one of the two Xenopus gene copies (S1b) coding for the ribosomal protein S1 has revealed that its introns III, V and VI carry a region of about 150 nt that shares an identity of 60%. We show here the presence in Xenopus oocytes and cultured cells of a 143-147 nt long RNA species encoded by these three repeated sequences on the same strand as the S1 mRNA and by at least one repeat present in the S1 a copy of the r-protein gene. We identify these RNAs as forms of the small nucleolar RNA U15 (U15 snoRNA) because of their sequence homology with an already described human U15 RNA encoded in the first intron of the human r-protein S3 gene, which is homologous to Xenopus S1. Comparison of the various Xenopus and human U15 RNA forms shows a very high conservation in some regions, but considerable divergence in others. In particular the most conserved sequences include two box C and two box D motifs, typical of most snoRNAs interacting with the nucleolar protein fibrillarin. Adjacent to the two D boxes there are two sequences, 9 and 10 nt in length, which are perfectly complementary to an evolutionary conserved sequence of the 28S rRNA. Modeling the possible secondary structure of Xenopus and human U15 RNAs reveals that, in spite of the noticeable sequence diversity, a high structural conservation in some cases may be maintained by compensatory mutations. We show also that the different Xenopus U15 RNA forms are expressed at comparable levels, localized in the nucleoli and produced by processing of the intronic sequences, as recently described for other snoRNAs.

Animals↗

PubMed1995

Role for the highly conserved region of domain IV of 23S-like rRNA in subunit-subunit interactions at the peptidyl transferase centre.

The function of the highly conserved and accessible region of domain IV of 23S rRNA (positions 1900-1981 in Escherichia coli 23S rRNA) was investigated by subjecting it to a random mutagenesis procedure that produced single-site mutations efficiently. Nine single-site mutants were selected that were recessive lethal. High levels of mutated 23S rRNA were expressed in E. coli and extracted ribosomes were investigated for their content of mutated rRNA. The peptidyl transferase activity of the ribosomes was also estimated using a newly developed method involving selective inhibition of chromosome-encoded ribosomes by clindamycin. Two of the mutants, U1940A and U1955G, yielded 50S subunits that were defective in subunit-subunit association but active in peptidyl transferase activity and five, U1926C, U1946C, U1979C, U1982A and G1984A, produced 50S subunits that were defective in both subunit-subunit interactions and peptidyl transferase activity. We infer that the large conserved rRNA region generates a complex structure that plays an essential role in maintaining and modulating subunit-subunit interactions and argue that its involvement in the peptidyl transferase centre is secondary, possibly involving the correct alignment of protein L2.

Base Sequence↗

PubMed1979

Analysis of RNA secondary structure by photochemical reversal of psoralen crosslinks.

Aminomethyltrioxsalen (AMT), a psoralen, is known to cause interstrand crosslinks in double stranded nucleic acids. We have demonstrated the photochemical reversal of this reaction, and have used this result to develop a method for identification of specific sequences which are adjacent because of RNA secondary structure formation. E. coli 5S rRNA is used as a model system. We isolated and characterized a product that is derived from the stem region of 5S RNA.

Base Composition↗

PubMed1981

Multiple ribosomal RNA cleavage pathways in mammalian cells.

The sequence content of mouse L cell pre-rRNA was examined by RNA gel transfer and blot hybridization. Nuclear RNAs were separated by agarose gel electrophoresis, transferred to diazo-paper, and hybridized to twelve different restriction fragments that are complementary to various sections of 45S pre-rRNA. An abundant new 34S pre-rRNA and less abundant new 37S, 26S and 17S pre-rRNAs were detected. The presence of these new pre-rRNAs suggests the existence of at least two new pre-rRNA cleavage pathways. 34S and 26S pre-rRNAs were also detected in HeLa cells suggesting that these new cleavage pathways are characteristic of mammalian cells. Further, an abundant new 12S precursor to 5.8S rRNA was also detected and is common to all the proposed cleavage pathways. The previously identified 45S, 41S, 32S and 20S pre-rRNAs were readily detected and their general structure confirmed. The 20S pre-rRNA is characteristic of the known pathway used by HeLa and other cells, and its presence suggests that growing mouse L cells use this pre-rRNA cleavage pathway. The 36S pre-rRNA characteristic of the previously described mouse L cell cleavage pathway was not detected. In all these cleavage pathways pre-rRNA cleavage sites are apparently identical and occur at or near the termini of the mature 18S, 5.8S and 28S rRNA sequences. The pathways differ only in the temporal order of cleavage at these sites. The position of the 5.8S rRNA sequence was located within the internal transcribed spacer. The known and conserved sequence of 5.8S rRNA from several organisms predicts a characteristic pattern of restriction enzyme sites for 5.8S rDNA. Internal transcribed spacer rDNA was mapped with restriction enzymes, and the characteristic pattern was found near the midpoint of the internal transcribed spacer. This places the 5.8S rRNA sequence at or near the 5' terminus of 32S pre-rRNA.

Animals↗