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PubMed2005

Crystal structure of a bacterial ribonuclease P RNA.

The x-ray crystal structure of a 417-nt ribonuclease P RNA from Bacillus stearothermophilus was solved to 3.3-A resolution. This RNA enzyme is constructed from a number of coaxially stacked helical domains joined together by local and long-range interactions. These helical domains are arranged to form a remarkably flat surface, which is implicated by a wealth of biochemical data in the binding and cleavage of the precursors of transfer RNA substrate. Previous photoaffinity crosslinking data are used to position the substrate on the crystal structure and to identify the chemically active site of the ribozyme. This site is located in a highly conserved core structure formed by intricately interlaced long-range interactions between interhelical sequences.

Bacterial Proteins↗
PubMed2002

A comparison of enzymatic and molecular approaches to characterize the cellulolytic microbial ecosystems of the rumen and the cecum.

We used RNA probes and enzyme activities to compare the cellulolytic microbial ecosystems of the rumen and the cecum. Four rumen- and cecum-cannulated wethers were fed a diet of barley plus hay (60:40). Digesta samples were collected 1 h before feeding and 3, 6, and 9 h after feeding for measurements on microbial populations, and 1 h before feeding and 3 and 6 h after feeding for digestion measurements, pH, and VFA. Polysaccharidase and glycosidase specific activities of solid-adherent microorganisms were measured respectively by the amount of reducing sugars released from xylan or avicel or p-nitrophenol from the p-nitrophenol derivatives of xylose and glucose. The distribution and amounts of the three main cellulolytic bacterial species (Fibrobacter succinogenes, Ruminococcus albus, and Ruminococcus flavefaciens) were determined by dot-blot hybridization using specific 16SrRNA-targeting probes. Enzyme activities were higher in the rumen than in the cecum and before feeding than at 3 h after feeding. The sum of the three cellulolytic bacterial species represented, on average, 4.5% of the total bacterial RNA in the two compartments and did not vary with sampling time. The cellulolytic bacterial community structure was different in the two compartments, with F. succinogenes as the main species in the rumen and R. flavefaciens in the cecum. The lower cellulolytic activity in the cecum than in the rumen could not be ascribed to any difference in the structure of the cellulolytic bacterial community between these two compartments, and other hypotheses related to digestion are proposed.

Animal Feed↗
PubMed2005

Tracking RNA polymerase, one step at a time.

Although the structural analysis of transcription has attained atomic resolution, techniques to manipulate single molecules involved in transcription have lagged behind. A recent paper in Nature by Abbondanzieri et al. (2005) closes this gap. This study finds that individual molecules of bacterial RNA polymerase move in single base-pair steps and concludes that RNA polymerase acts as a Brownian ratchet that is driven forward by the binding of incoming nucleoside triphosphates.

Base Pairing↗
PubMed1992

Effects of amino acid substitutions in the -10 binding region of sigma E from Bacillus subtilis.

The sigma subunit of bacterial RNA polymerase is required for specific binding to promoters. One region in most sigma factors makes sequence-specific contacts at the -10 region of its cognate promoters. To test the role of the amino acids in this -10 binding region, we examined the effects of 49 single-amino-acid substitutions in sigma E from Bacillus subtilis. We assayed the effect of each amino acid substitution on spore formation because sigma E is essential for endospore formation in B. subtilis. Our results showed that substitutions at several positions, including the highly conserved aromatic amino acid at position 102, had little or no detectable effect. Substitutions at another position, position 117, produced dominant negative mutations; we suggest that these mutations allow RNA polymerase containing the mutant sigma factor to bind specifically to promoters but prevent transcription initiation. Of the recessive defective alleles, those that produced substitutions at positions 113, 115, and 120 produced the most defective sigma factors. These results suggest that the residues at or near these positions in wild-type sigma E play important roles in sigma E function.

Alleles↗
PubMed2006

Molecular basis for temperature sensing by an RNA thermometer.

Regulatory RNA elements, like riboswitches, respond to intracellular signals by three-dimensional (3D) conformational changes. RNA thermometers employ a similar strategy to sense temperature changes in the cell and regulate the translational machinery. We present here the first 3D NMR structure of the functional domain of a highly conserved bacterial RNA thermometer containing the ribosome binding site that remains occluded at normal temperatures (30 degrees C). We identified a region adjacent to the Shine-Dalgarno sequence that has a network of weak hydrogen bonds within the RNA helix. With the onset of heat shock at 42 degrees C, destabilisation of the RNA structure initiates at this region and favours the release of the ribosome binding site and of the start codon. Deletion of a highly conserved G residue leads to the formation of a stable regular RNA helix that loses thermosensing ability. Our results indicate that RNA thermometers are able to sense temperature changes without the aid of accessory factors.

Base Sequence↗
PubMed1971

Bacterial mutants in which the gene N function of bacteriophage lambda is blocked have an altered RNA polymerase.

Bacterial mutants have been isolated, called groN, that block phage development by interference with the action of the product of the phage N gene. lambdatrp phages, which depend on the N product for the synthesis of tryptophan enzymes, do not make these enzymes in groN bacteria. Two type of phage mutants have been isolated that can overcome the groN block. One type makes an altered N product, the other contains an N-bypass mutation. The groN mutation is closely linked to the rifamycin-resistance locus in Escherichia coli. Purified RNA polymerase from the groN mutant is less activated by salt and more sensitive to rifamycin than is the polymerase from gro(+). This suggests that the groN mutation produces a structural change in the bacterial RNA polymerase such that it can no longer interact properly with the phage N product.

Bacterial Proteins↗
PubMed2005

Detection of viral, bacterial, and parasitological RNA or DNA of nine intestinal pathogens in fecal samples archived as part of the english infectious intestinal disease study: assessment of the stability of target nucleic acid.

Fecal samples were collected from cases and controls as part of the Infectious Intestinal Disease (IID) study in England and were stored as frozen suspensions for 8 to 12 years. The purpose of this study was to apply PCR-based procedures to assess the stability of pathogen-specific nucleic acid sequences present in this archive. Samples from which Cryptosporidium, Giardia, Salmonella, Campylobacter, enteroaggregative Escherichia coli (EAggEC), enterotoxigenic Clostridium perfringens, rotaviruses, noroviruses, or sapoviruses had been previously detected during the IID study using conventional methods were selected from the archive. A generic nucleic acid extraction method to recover RNA or DNA was used. Complementary DNA was generated from RNA by reverse transcription with random priming. Block-based and real-time PCR assays were used to amplify and detect gene fragments from each of these pathogens. The percentage reconfirmation of target was as follows: Giardia duodenalis 68%, Cryptosporidium 96%, Campylobacter 98%, Salmonella 98%, enterotoxigenic C perfringens 34%, EAggEC 93.3%, rotavirus 95%, norovirus 73%, and sapovirus 85%. This study has shown that nucleic acid can be extracted and specific sequences amplified and detected from archived fecal samples. The IID archive therefore represents a valuable resource for further studies, especially the investigation of the samples from which no pathogens had previously been detected.

Cryopreservation↗
PubMed1994

Inhibition of in vivo transcription by actinomycin D treatment of ethylenediaminetetraacetic acid-permeabilized Escherichia coli: effects on bacteriophage proliferation.

Rifampicin, which is able to block DNA-dependent RNA synthesis, has been widely used to selectively inhibit host protein synthesis in RNA bacteriophage-infected Escherichia coli without affecting the viral specific protein synthesis. However, in many cases it is necessary to increase rifampicin levels to 200 micrograms/ml in order to obtain an almost complete suppression of bacterial protein synthesis, and these high antibiotic concentrations cause at the same time a strong inhibition of phage proliferation resulting in a 50- to 100-fold reduction of phage yields. We have partially avoided this difficulty by using actinomycin D after permeabilization of bacteria by a brief incubation with EDTA. To optimize the method the effects of changing EDTA and actinomycin concentrations as well as the duration of the permeabilization period have been studied. With this procedure it has been possible to shut off bacterial RNA and protein synthesis with phage yields about 10 times higher than those observed in the presence of high levels of rifampicin. The usefulness of the described method is particularly evident when working with rifampicin-resistant strains of E. coli.

Coliphages↗
PubMed2004

Molecular mechanism of transcription inhibition by peptide antibiotic Microcin J25.

21 amino acid peptide Microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). MccJ25-resistance mutations cluster in the RNAP secondary channel through which incoming NTP substrates are thought to reach the catalytic center and the 3' end of the nascent RNA is likely to thread in backtracked transcription complexes. The secondary channel also accepts transcript cleavage factors GreA and GreB. Here, we demonstrate that MccJ25 inhibits GreA/GreB-dependent transcript cleavage, impedes formation of backtracked complexes, and can be crosslinked to the 3'-end of the nascent RNA in elongation complexes. These results place the MccJ25 binding site within the secondary channel. Moreover, single-molecule assays reveal that MccJ25 binding to a transcribing RNAP temporarily stops transcript elongation but has no effect on the elongation velocity between pauses. Kinetic analysis of single-molecule data allows us to put forward a model of transcription inhibition by MccJ25 that envisions the complete occlusion of the secondary channel by bound inhibitor.

Anti-Bacterial Agents↗
PubMed1975

Conversion of Bacillus subtilis RNA polymerase activity in vitro by a protein induced by phage SP01.

A protein fraction from B. subtilis infected with phage SP01 (fraction LGG) stimulates the activity of RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase) core from uninfected bacteria. Fraction LGG contains a protein (P-28, molecular weight 28,000) that is labeled after phage infection and binds tightly to RNA polymerase core at a relatively high ionic strength. B. subtilis RNA polymerase core with bound P-28 has the transcription specificity of the previously purified, phage-modified B-P RNA polymerase; the latter contains two subunits, v-28 and v-13 (molecular weights 28,000 and 13,000, respectively) that are synthesized after phage infection. Both enzymes transcribe SP01 DNA preferentially and direct the asymmetric synthesis of viral middle RNA. P-28, like v-28, binds more tightly to B. subtilis RNA polymerase core than the B. subtilis initiation factor, sigma, at higher ionic strength. We propose that P-28 and v-28 are the same protein. P-28 and, by implication, v-28 suffice to endow the bacterial RNA polymerase core with a novel transcription specificity.

Ammonium Sulfate↗
PubMed2006

Differential expression of key pneumococcal virulence genes in vivo.

Few studies have examined in vivo virulence gene expression in Streptococcus pneumoniae. In this study, expression of key pneumococcal virulence genes cbpA, pspA, ply, psaA, cps2A, piaA, nanA and spxB in the nasopharynx, lungs and bloodstream of mice was investigated, following intranasal challenge with the serotype 2 strain D39. Bacterial RNA was extracted, linearly amplified and assayed by real-time RT-PCR. At 72 h, cbpA mRNA was present at higher levels in the nasopharynx and lungs than in the blood. At this time-point, the mRNAs for PspA and PiaA were most abundant in the nasopharynx, whereas no significant difference in gene expression between niches was observed for ply, psaA and cps2A. Both nanA and spxB mRNAs were present in higher amounts in the nasopharynx than in the lungs or blood. These findings illustrate the dynamic nature of pneumococcal virulence gene expression in vivo.

Animals↗
PubMed1976

Changes in transfer ribonucleic acids of Bacillus subtilis during different growth phases.

The transfer ribonucleic acids (tRNAs) of B. subtilis at different growth phases are examined for changes in the composition and the methylation of minor constituents. The composition of the tRNAs indicates about equal amounts of adenosine and uridine, and of guanosine and cytidine. About 3-4 residues are present as modified bases in the average tRNA molecule. The net composition of tRNAs appears to remain unaltered during different growth phases. In vitro methylation of tRNAs indicates lack of methyl groups in both exponentially growing cells and spores. In vivo methylation studies show tRNA methylation occurs during the stationary phase in the absence of net tRNA synthesis. Thus, both in vitro and in vivo methylation indicate that the tRNAs in exponentially growing cells do not contain their full complement of modified bases. More complete modification is noted in tRNAs from stationary cells or spores. Hence, tRNA mofifications in general are preserved with fidelity even in the dormant spore but the possibility is left open that specific modifications of selected isoacceptors of tRNAs may occur.

Bacillus subtilis↗
PubMed1999

Analysis of the CoIE1 stability determinant Rcd.

Multimer formation is an important cause of instability for many multicopy plasmids. Plasmid CoIE1 is maintained stably because multimers are converted to monomers by Xer-mediated site-specific recombination at the cer site. However, multimer resolution is not the whole story; inactivation of a promoter (Pcer) within cer causes plasmid instability even though recombination is unaffected. The promoter directs the synthesis of a short transcript (Rcd) which is proposed to delay the division of multimer-containing cells. Mapping of the 5' terminus of Rcd confirms that transcription initiates from Pcer. The 3' terminus shows considerable heterogeneity, consistent with a primary transcript of 95 nt being degraded via intermediates of 79 and 70 nt. Secondary structure predictions for Rcd are presented. Of four mutations which abolish Rcd-mediated growth inhibition, one reduces the activity of Pcer while the other three map to the rcd coding sequence and reduce the steady-state level of the transcript. RNA folding analysis suggests that these three mutant transcripts adopt a common secondary structure in which the major stem-loop differs from that of wild-type Rcd. A survey of 24 cer-like multimer resolution sites revealed six which contain Pcer-like sequences. The putative transcripts from these sites have similar predicted secondary structures to Rcd and contain a highly conserved 15 base sequence. To test the hypothesis that Rcd acts as an anti-sense RNA, interacting with its target gene(s) through the 15 nt sequence, we used DNA hybridization and sequence analysis to find matches to this sequence in the Escherichia coli chromosome. Our failure to find plausible anti-sense targets has led to the suggestion that Rcd may interact directly with a protein target.

Bacteriocin Plasmids↗
PubMed1977

Biochemical properties of Clostridium bifermentans spores.

As previously found for spores of Bacillus species, dormant spores of Clostridium bifermentans contained essentially no adenosine triphosphate, a high level of adenosine monophosphate, a high level of 3-phosphoglyceric acid, and much transfer ribonucleic acid lacking a 3'-terminal adenosine monophosphate residue. As in spores of Bacillus species, germination of C. bifermentans spores was accompanied by utilization of the 3-phosphoglyceric acid, a large increase in the adenosine triphosphate level, and the disappearance of defective transfer ribonucleic acid. In contrast to spores of Bacillus species, dormant spores of C. bifermentans contained little free amino acid.

Adenine Nucleotides↗
PubMed1986

Transcriptional control of the Bacillus subtilis spoIID gene.

We cloned the wild-type allele of the spoIID locus of Bacillus subtilis. This DNA region was shown to be transcribed beginning within an hour after the onset of sporulation. The amount of spoIID mRNA present in cells at 1 h after the end of growth was more than 50-fold greater than it was growing cells; the pool of this mRNA decreased steadily after 1.5 h after the end of growth. spoIID mRNA was present in stationary-phase cells of sporulation mutants with lesions in the spo0J and spoIIB genes but was absent in cells carrying spo0B, spo0H, spoIIA, spoIIE, spoIIG, or spoIIIA mutations. In vitro runoff transcription with the E sigma 55, E sigma 37, E sigma 32, and E sigma 29 forms of RNA polymerase indicated that only the E sigma 29 form was able to transcribe the spoIID gene. This result is consistent with results of studies with the Spo- mutants, because only mutants that produced E sigma 29 were able to produce spoIID mRNA in vivo. In the course of this work, two additional transcription units were discovered in the DNA region neighboring the spoIID gene. One of these was expressed during vegetative growth; the other was expressed early during sporulation and corresponded to an in vitro transcript produced by the E sigma 29 forms of RNA polymerase.

Bacillus subtilis↗
PubMed2003

Protein synthesis in Escherichia coli with mischarged tRNA.

Two types of aspartyl-tRNA synthetase exist: the discriminating enzyme (D-AspRS) forms only Asp-tRNA(Asp), while the nondiscriminating one (ND-AspRS) also synthesizes Asp-tRNA(Asn), a required intermediate in protein synthesis in many organisms (but not in Escherichia coli). On the basis of the E. coli trpA34 missense mutant transformed with heterologous ND-aspS genes, we developed a system with which to measure the in vivo formation of Asp-tRNA(Asn) and its acceptance by elongation factor EF-Tu. While large amounts of Asp-tRNA(Asn) are detrimental to E. coli, smaller amounts support protein synthesis and allow the formation of up to 38% of the wild-type level of missense-suppressed tryptophan synthetase.

Aspartate-tRNA Ligase↗
PubMed2002

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain.

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.

Adjuvants, Immunologic↗