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PubMed2005

Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis.

The transcriptional start sites of 27 promoters in Helicobacter pylori strain 4187E have been successfully identified using a non-radioactive primer extension protocol. The technique involves reverse transcribing mRNA with a sequence-specific FAM-labelled primer. The length of the FAM-labelled cDNA primer extension product can be analysed on a standard DNA sequencer using GeneScan software. This information can be used in conjunction with DNA sequencing data to identify the transcriptional start site of a promoter. Total bacterial RNA produced more specific primer extension products with stronger FAM signals than a population enriched for mRNA. Using this technology, it is not necessary to complete the DNA sequencing reactions in parallel with the primer extension experiments. The FAM-labelled primer extension products do not require a PCR amplification step prior to analysis on a sequencing gel, and no phenol/chloroform purifications are required at any stage of the procedure. Fluorescent-based primer extension methods have obvious advantages over the conventional radioactive protocols, and this report extends the currently used methodologies in this field.

Base Sequence↗
PubMed2005

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome.

The conserved 900 tetraloop that caps helix 27 of 16S ribosomal RNA (rRNA) interacts with helix 24 of 16S rRNA and also with helix 67 of 23S rRNA, forming the intersubunit bridge B2c, proximal to the decoding center. In previous studies, we investigated how the interaction between the 900 tetraloop and helix 24 participates in subunit association and translational fidelity. In the present study, we investigated whether the 900 tetraloop is involved in other undetected interactions with different regions of the Escherichia coli 16S rRNA. Using a genetic complementation approach, we selected mutations in 16S rRNA that compensate for a 900 tetraloop mutation, A900G, which severely impairs subunit association and translational fidelity. Mutations were randomly introduced in 16S rRNA, using either a mutagenic XL1-Red E. coli strain or an error-prone PCR strategy. Gain-offunction mutations were selected in vivo with a specialized ribosome system. Two mutations, the deletion of U12 and the U12C substitution, were thus independently selected in helix 1 of 16S rRNA. This helix is located in the vicinity of helix 27, but does not directly contact the 900 tetraloop in the crystal structures of the ribosome. Both mutations correct the subunit association and translational fidelity defects caused by the A900G mutation, revealing an unanticipated functional interaction between these two regions of 16S rRNA.

Base Sequence↗
PubMed2004

Simultaneous analysis of host and pathogen interactions during an in vivo infection reveals local induction of host acute phase response proteins, a novel bacterial stress response, and evidence of a host-imposed metal ion limited environment.

A fundamental goal in the study of infections is to understand the dynamic interplay between host and pathogen; however, direct in vivo interrogation of this disease process via transcriptional profiling has been lacking. Here we describe the development and application of novel bacterial RNA amplification technology to simultaneously identify key elements of both host and pathogen responses in a murine infection model. On the bacterial side, we found induction of an unusual pattern of stress response genes, a response to host-induced metal ion limitation, and a failure to achieve stationary phase in vivo. On the mammalian side, we observed the surprising induction of several genes encoding acute phase response proteins including hepcidin, haptoglobin, complement C3 and metallothionein 1 at the site of infection, as well as other mediators of innate immunity. Thus, our results reveal host-pathogen cross-talk not predicted by previous in vitro analyses and provide the framework to eavesdrop on a broad array of host-pathogen interactions in vivo. As described here, the comprehensive examination of host-pathogen interactions during an infection is critical to the discovery of novel approaches for intervention not predicted by current models.

Acute-Phase Proteins↗
PubMed1995

RNA degradation in Escherichia coli regulated by 3' adenylation and 5' phosphorylation.

Although polyadenylation has commonly been regarded as a special feature of eukaryotic messenger RNA, there are many reports of polyA tails on bacterial RNA (for example, refs 3-8). In Escherichia coli, adenylation mediated by the pcnB gene greatly accelerates decay of RNA I, an antisense repressor of replication of ColE1 type plasmids that resembles highly structured transfer RNA but shows the rapid turnover characteristic of mRNA. Here we report that both 3' adenylation and 5' phosphorylation affect the rate of digestion of RNA I by the 3' exonuclease, polynucleotide phosphorylase; conversely, mutation of the polynucleotide phosphorylase-encoding pnp gene affects ribonuclease acting at the 5' end. Together these findings indicate that enzymes attacking RNA I at its separate termini can interact functionally. Additionally, our discovery that adenylation-mediated degradation by polynucleotide phosphorylase imparts an mRNA-like half-like to RNA I suggests a possible mechanism to account for the rapid decay of mRNA in E. coli.

Adenine↗
PubMed2004

Benefits and pitfalls of using microarrays to monitor bacterial gene expression during infection.

The understanding of bacterial pathogenesis is dependent on techniques that elucidate the underlying genetic and biochemical mechanisms. To study the mechanism of bacterial survival and proliferation within host cells we need accurate tools that tell us what is occurring within the infecting organism. It has now become possible to determine the transcriptional status of in vivo-derived bacteria at the level of the whole genome. Such expression profiles serve as a monitor of the host cell environment as well as an indicator of the bacterial adaptation to its intracellular niche. Here, we review the methods used to produce microarray data for defining the bacterial intracellular transcriptome, and examine the pitfalls in extracting bacterial RNA from the infected host compartment.

Animals↗
PubMed1970

Continued expression of the ribonucleic acid control gene during inhibition of Escherichia coli ribonucleic acid and protein synthesis.

The effect of the ribonucleic acid (RNA) control (RC) gene on the biosynthesis of viral RNA has been examined in an RC(str) and an RC(rel) host infected with R17 RNA bacteriophage under conditions in which host RNA and protein synthesis were inhibited by the addition of rifampicin. Methionine and isoleucine starvation depressed viral RNA biosynthesis in an RC(str) host but not in an RC(rel) host. However, histidine starvation had little effect on viral RNA and protein synthesis in both RC(str) and RC(rel) cells, although it had a marked effect on host protein and RNA synthesis in an RC(str) host. Chloramphenicol relieved the effect of amino acid starvation on viral RNA synthesis in an RC(str) host. It is concluded that stringent control of viral RNA biosynthesis does not require the continued biosynthesis of the RC gene product (RNA or protein) and that a preformed RC gene product can regulate the biosynthesis of the exogenous RNA. It is suggested that the amino acid dependence of viral RNA biosynthesis is due to its obligatory coupling with the translation of the viral coat protein which lacks histidine. It may be inferred that the amino acid requirement of bacterial RNA is due to its coupling with the translation of a host-specific protein (other than the RC gene product) which requires a full complement of amino acids. Since chloramphenicol is known to permit ribosome movement in the absence of protein synthesis, it is suggested that ribosome movement along the nascent RNA chain is a sufficient condition for the continuation of RNA synthesis.

Alanine↗
PubMed2005

Exoribonuclease R interacts with endoribonuclease E and an RNA helicase in the psychrotrophic bacterium Pseudomonas syringae Lz4W.

Endoribonuclease E, a key enzyme involved in RNA decay and processing in bacteria, organizes a protein complex called degradosome. In Escherichia coli, Rhodobacter capsulatus, and Streptomyces coelicolor, RNase E interacts with the phosphate-dependent exoribonuclease polynucleotide phosphorylase, DEAD-box helicase(s), and additional factors in an RNA-degrading complex. To characterize the degradosome of the psychrotrophic bacterium Pseudomonas syringae Lz4W, RNase E was enriched by cation exchange chromatography and fractionation in a glycerol density gradient. Most surprisingly, the hydrolytic exoribonuclease RNase R was found to co-purify with RNase E. Co-immunoprecipitation and Ni(2+)-affinity pull-down experiments confirmed the specific interaction between RNase R and RNase E. Additionally, the DEAD-box helicase RhlE was identified as part of this protein complex. Fractions comprising the three proteins showed RNase E and RNase R activity and efficiently degraded a synthetic stem-loop containing RNA in the presence of ATP. The unexpected association of RNase R with RNase E and RhlE in an RNA-degrading complex indicates that the cold-adapted P. syringae has a degradosome of novel structure. The identification of RNase R instead of polynucleotide phosphorylase in this complex underlines the importance of the interaction between endo- and exoribonucleases for the bacterial RNA metabolism. The physical association of RNase E with an exoribonuclease and an RNA helicase apparently is a common theme in the composition of bacterial RNA-degrading complexes.

Adenosine Triphosphate↗
PubMed1975

Effect of the rifamycin dimers on the activities of nucleic acid polymerases from various sources. Relation between lipophily and toxicity.

The chemical dimers of rifamycin SV resembled the corresponding monomeric analogs with respect to the inhibitory properties versus the nucleic acid polymerases. At low doses, such compounds blocked the initiation step of the DNA transcription catalyzed by the bacterial RNA polymerase, as observed for the parental antibiotic and its derivative rifampicin which are largely used in therapy. At concentrations one to two orders of magnitude higher, the chemically modified rifamycins inhibited also other nucleotidyltransferases. The widespread toxicity of the dimeric and monomeric semisynthetic rifamycins versus these enzymes was not causally related with an enhancement of their lipophily. The observed effects might be due to a loss of selectivity in the inhibition mechanism which was originally specific for the RNA polymerase from E. coli at the beginning of its catalysis. The rifamycin derivatives might then react with the catalytic portion of other nucleotidyltransferases interfering adversely with the enzyme activity in a number of ways and/or at different levels.

Animals↗
PubMed1999

Codon usage and lateral gene transfer in Bacillus subtilis.

Bacillus subtilis possesses three classes of genes, differing by their codon preference. One class corresponds to prophages or prophage-like elements, indicative of the existence of systematic lateral gene transfer in this organism. The nature of the selection pressure that operates on codon bias is beginning to be understood.

Bacillus subtilis↗
PubMed1975

On the mode of action of lomofungin, an inhibitor of RNA synthesis in yeast.

Lomofungin is a potent inhibitor of RNA synthesis in yeast. Studies on the mode of action of the inhibitor were carried out using yeast RNA polymerases A and B and bacterial RNA polymerase. In vitro inhibition of RNA synthesis is independent of the nature and concentration of the template used and of the nucleoside triphosphate concentration. The extent of inhibition is strongly dependent upon the nature and concentration of divalent cations used to simulate transcription. The three RNA polymerases were inhibited to the same extent in the presence of Mn2+ ions whereas little inhibition was observed with Mg2+ ions. Spectrophotometric studies reveal the formation of different complexes between lomofungin and divalent cations (Mn2+, Mg2+, or Zn2+) with the respective stoichiometries of 0.5, 1, and 2 divalent cations per molecule of lomofungin. The complexes formed depend upon the nature of the divalent cation involved. No direct interaction between lomofungin and DNA could be observed in the presence of divalent cations but evidence is presented that lomofungin interacts with yeast RNA polymerase A. Inhibition of RNA synthesis occurs at the level of both chain initiation and elongation.

Chelating Agents↗
PubMed1969

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin.

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.

Amino Acids↗
PubMed1992

Mutant sigma factor blocks transition between promoter binding and initiation of transcription.

The sigma subunit of bacterial RNA polymerase is required for specific binding of the enzyme to promoters. This specificity is probably directed by two regions of most sigma factors that make sequence-specific contacts at two regions of promoters, the -10 and -35 regions. We found that a single amino acid substitution in the -10 recognition region of sigma E from Bacillus subtilis trapped RNA polymerase in a stable complex with promoter DNA in which it was unable to initiate transcription. Our results are consistent with the view that promoter utilization by RNA polymerase proceeds through several intermediate steps and suggest that the -10 recognition region of sigma factors may participate in a step that follows initial promoter binding.

Amino Acid Sequence↗
PubMed2004

Invasion of porcine brain microvascular endothelial cells by Streptococcus suis serotype 2.

Streptococcus suis is an important swine pathogen that mainly causes meningitis and occasionally causes other infections, such as endocarditis, arthritis, and pneumonia. The pathogenesis of S. suis infection has not been completely defined. However, in order to cause meningitis, S. suis has to cross the blood-brain barrier (BBB) made up of brain microvascular endothelial cells. The objective of this work was to study the interactions of S. suis serotype 2 with porcine brain microvascular endothelial cells (PBMEC). The ability of North American and European S. suis serotype 2 strains to adhere to PBMEC and, most importantly, to invade PBMEC was demonstrated by using an antibiotic protection assay and was confirmed by electron microscopy. The polysaccharide capsule of S. suis seemed to partially interfere with the adhesion and invasion abilities of the bacterium. Our results showed that intracellular viable S. suis could be found in PBMEC up to 7 h after antibiotic treatment. Inhibition studies demonstrated that invasion of PBMEC by S. suis required actin microfilaments but not microtubular cytoskeletal elements or active bacterial RNA or protein synthesis. At high bacterial doses, suilysin-positive strains were toxic for PBMEC. The role of suilysin in cytotoxicity was confirmed by using purified suilysin, electron microscopy, and the lack of toxicity of a suilysin-negative mutant. In swine, the invasion of endothelial cells of the BBB could play an important role in the pathogenesis of the meningitis caused by S. suis.

Actin Cytoskeleton↗
PubMed1999

Structure-activity relationships of novel 2-substituted quinazoline antibacterial agents.

High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.

Animals↗
PubMed1969

Control of gene function in bacteriophage T4. I. Ribonucleic acid and deoxyribonucleic acid metabolism in T4rII-infected lambda-lysogenic hosts.

Deoxyribonucleic acid (DNA) synthesis in T4rII-infected, lambda-lysogenic strains of Escherichia coli proceeds with one-half the rate of T4 wild-infected bacteria and stops 16 min after infection at 37 C. The rates of ribonucleic acid (RNA) synthesis, however, are the same with T4rII and T4 wild. The turnover of pulse-labeled RNA is slow in K strains (half-lives 10 to 20 min) as compared with B strains (half-lives 2.5 to 6 min). Lambda-lysogeny increases the apparent messenger (m) RNA half-lives in pulse-chase experiments. The shutoff of host RNA synthesis in T4rII infected K(lambda) is incomplete. Moreover, the preferential transcription of T4 DNA ceases 13 min after infection, and transcription of host and prophage lambda DNA is resumed. The T4 RNA synthesized in rII-infected K(lambda) contains no late T4 mRNA. The early portion of the T4 genome, however, is transcribed completely. The T4-induced early modification of bacterial RNA polymerase does occur. Resumption of host DNA transcription at 13 min after infection is not associated with a reversal of the above polymerase modification. It is concluded that in lambdalysogenic bacteria T4rII infections are abortive because RNA polymerase is prevented from transcribing late T4 genes.

Centrifugation, Density Gradient↗
PubMed1997

Regions of the Escherichia coli primary sigma factor sigma70 that are involved in interaction with RNA polymerase core enzyme.

BACKGROUND: The sigma factors of bacterial RNA polymerase are required for recognition of promoters in transcription initiation. Most sigma factors share several regions with significant homology in their amino acid sequences (regions 1-4). Some primary sigma factors carry a large nonconserved segment between regions 1 and 2. The binding of an sigma factor to the core enzyme alters the structure and properties of the sigma factor, but little is known about the binding mechanism and subsequent reactions. In this report, we employed the protein footprinting method to investigate the alteration of the structure and function of Escherichia coli sigma70 by binding to core enzyme and promoter DNA. RESULTS: A segment between regions 1.1 and 1.2, and that in region 3.2, were preferentially cleaved by hydroxyl radicals. Upon binding to the core enzyme, segments in regions 1.1, 2, 3 and 4 were substantially protected, while cleavage at a small segment in region 4.2 was weakly enhanced. In a binary complex of holoenzyme and promoter DNA, additional segments in regions 2.4 and 4.2 were protected, while the protection at region 1.1 disappeared. The nonconserved acidic region of sigma70 in the holoenzyme became hypersensitive upon binding to DNA. CONCLUSIONS: These results suggest that not only the conserved region 2, but also regions 1.1, 3 and 4 of the sigma factor are involved in binding to the core enzyme. The nonconserved acidic region is likely to be more exposed by further binding of sigma factor to promoter DNA.

Binding Sites↗