Where, within the nucleolus, are the rRNA genes located?
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Animals↗
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Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli.
Assessment of 16S-23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5' terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S-23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.
Bacillus↗
Short peptides conferring resistance to macrolide antibiotics.
Translation of specific short peptides can render the ribosome resistant to macrolide antibiotics such as erythromycin. Peptides act in cis upon the ribosome on which they have been translated. Amino acid sequence and size are critical for peptide activity. Pentapeptides with different consensus sequences confer resistance to structurally different macrolide antibiotics, suggesting direct interaction between the peptide and the drug on the ribosome. Translation of resistance peptides may result in expulsion of the macrolide antibiotics from the ribosome. The consensus sequence of peptides conferring erythromycin resistance is similar to the sequence of the leader peptide involved in translational attenuation of erythromycin resistance genes, indicating that a similar type of interaction between the nascent peptide and antibiotics can occur in both cases.
Anti-Bacterial Agents↗
Characterization of atypical Aeromonas salmonicida by different methods.
Fifty two isolates of atypical Aeromonas salmonicida, recovered from a wide range of hosts and geographical locations, were heterogeneous in terms of molecular and phenotypic characteristics, and represented taxa which could not be accommodated by the current classification of four subspecies. Generally, there was incongruence between the molecular (PCR, RAPD and ribotyping) and phenotypic methods in terms of cluster membership. By PCR, 6 groups were described of which Group 1 encompassed 12 isolates including the type strain of A. salmonicida subsp. smithia. Group 2 accommodated 23 isolates including the reference cultures of subspecies achromogenes and masoucida. The named culture of Haemophilus piscium was recovered in Group 6. By ribotyping and RAPD, the reference cultures were recovered in separate groups. All methods pointed to the uniqueness of subspecies smithia. Most isolates contained 2-6 plasmids, of 2.3 to 150 kb in length. Nevertheless, all isolates possessed certain key characteristics, including Gram-negativity, and the absence of motility.
Aeromonas↗
A qualitative evaluation of the published oligonucleotides specific for the 16S rRNA gene sequences of the ammonia-oxidizing bacteria.
Over the past few years, there has been an increasing interest in making oligonucleotides specific for ammonia-oxidizing bacteria (AOB), in order to detect and monitor these slow growing bacteria in environmental samples, in enrichment cultures and in wastewater treatment plants. Based on 16S rDNA sequences, a broad selection of oligonucleotides have been designed, either encompassing all known AOB in the beta-subgroup of the Proteobacteria (beta AOB), or subclasses within beta AOB. Thirty different oligonucleotides have so far been published, with varying specificity. The first AOB-specific oligonucleotides published were obtained as a result of an alignment of only eleven 16S rDNA sequences from AOB. Including the present study, there are now forty nearly full length 16S rDNA sequences available from these bacteria, in addition to a number of partial sequences, so that an improved evaluation of the published oligonucleotides can be done. Two new 16S rRNA gene sequences from Nitrosospira are presented here, in a phylogenetic analysis containing every 16S rRNA gene sequences (> 1 kb) available from AOB. On the basis of an alignment of all these sequences, combined with searches in the nucleotide sequence databases, an evaluation of the thirty published oligonucleotides is presented. The analysis expose the strength and weakness of each oligonucleotide and discuss the use of oligonucleotides specific for 16S rRNA genes in future studies of AOB. The present work also identifies one new, broad range primer, specific for the AOB in the beta-subgroup of the Proteobacteria.
Ammonia↗
Identification of Leptospira inadai by continuous monitoring of fluorescence during rapid cycle PCR.
Seven new Leptospira isolates from rats, a buffalo, and contaminated media showed either reactive serology against more than 1 serogroup or no reactive serology against a reference panel of 22 serovars in the microscopic agglutination test (MAT). Because of these inconclusive results, the 16S rDNA sequences of these isolates were determined and found to resemble that of the type strain of Leptospira inadai (L. inadai), serovar lyme strain 10, which is considered to be nonpathogenic for humans. Comparative analyses of other Leptospira 16S rDNA sequences from databases revealed a L. inadai-specific signature sequence, against which an amplification primer was designed. This primer when used in conjunction with an universal primer enabled the trial of a rapid PCR protocol in which fluorescence emissions due to binding of SYBR Green I dye to PCR products were continuously monitored during rapid thermal cycling. A melting curve acquired immediately after PCR was used to distinguish the intended product. The thermal cycling and continuous monitoring of fluorescence emission were accomplished by the LightCycler; the whole procedure of 30 PCR cycles and melting curve acquisition required only 20 minutes. The primer achieved the required specificity, as the intended PCR product resulted only from 6 confirmed L. inadai reference strains and 7 field isolates that had been verified as L. inadai by the 16S rDNA sequencing, but not from 16 reference strains of Leptospira belonging to 7 other genospecies. Furthermore, these experiments showed that the PCR protocol was robust because target DNA of different conditions, which were extracted by either 1 of the 4 methods used, could be detected.
Animals↗
Eyeing bacterial genomes.
The density of information in a bacterial genome allows its history, organization and encoded functions to be distilled into a single graphical representation. These features have made it possible to discern the forces acting in and on bacterial genomes at levels not attainable in eukaryotes.
Bacteria↗
Analysis of the Fis-dependent and Fis-independent transcription activation mechanisms of the Escherichia coli ribosomal RNA P1 promoter.
The role of the curved DNA sequence upstream to the Escherichia coli ribosomal RNA P1 promoter in transcription activation was studied. This sequence region had been shown to activate transcription from P1 in vivo and in vitro and to harbor binding sites for the trans-activating protein Fis. We have constructed a series of linker scanning mutants spanning the region -104 to -47, relative to the transcription start site. DNA fragments carrying the mutations show altered gel electrophoretic mobilities, consistent with reduced DNA bending angles compared to the wild-type sequence. Using gel retardation assays, qualitative as well as quantitative differences in the binding of the trans-activating protein Fis to the mutant DNA fragments could be observed. The effects of the mutations on rrnB P1 promoter activation were studied in vivo in fis+ and fis- backgrounds. A reduction in the promoter strength for some of the linker mutants correlates with altered Fis binding to two of the known Fis binding sites. Shifting the Fis binding region by half a helical turn, relative to the promoter core sequence, abolishes Fis-mediated activation almost totally, whereas activation is partly restored by a shift of a complete helical turn. For one mutant, which does not show alterations in Fis binding, a decrease in the promoter strength was observed in a fis- strain. From the results, we conclude that two upstream activating mechanisms, one Fis-dependent and one Fis-independent, influence the rrnB P1 promoter strength. Sequence determinants for the Fis-independent mechanism are closer to the promoter core region than the Fis binding sites. In addition, the study demonstrates that both the helical geometry and the absolute distance of the UAS region relative to the promoter are crucial for transcription activation.
Base Sequence↗
Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance.
Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region. These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence. Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP element-containing promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase. We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperature-sensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes. Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction.
Base Sequence↗
The mediator for stringent control, ppGpp, binds to the beta-subunit of Escherichia coli RNA polymerase.
BACKGROUND: Inhibition of transcription of rRNA in Escherichia coli upon amino acid starvation is thought to be due to the binding of ppGpp to RNA polymerase. However, the nature of this interaction still remains obscure. RESULTS: Here, the azido-derivative of ppGpp was synthesized from azido-GDP and [gamma-32P]ATP by way of the phosphate transfer reaction of the RelA enzyme. The product was subsequently characterized by one and two-dimensional chromatography. The resulting compound [32P]azido-ppGpp, where the azido group is attached to the base moiety, was purified to homogeneity and was photo-crosslinked to Escherichia coli RNA polymerase. SDS-PAGE analysis of the azido-ppGpp-bound enzyme, tryptic digestion and Western blot analysis suggested that azido-ppGpp binds to the beta-subunit of RNA polymerase. CONCLUSION: It was observed that both the N-terminal and C-terminal domains of the beta-subunit were labelled with azido-ppGpp in the native enzyme. However, under denaturing conditions only the C-terminal part from amino acid residue 802 to residue 1211/1216/1223 was predominantly crosslinked to azido-ppGpp. The excess of unlabelled ppGpp competes with azido-ppGpp for binding to the enzyme. azido-ppGpp inhibits single-round transcription at the stringent promoter like rrnBP1. In addition, ribosomal protein genes were also found to be inhibited by N3ppGpp. On the other hand, transcription at the lac UV5 promoter remained unaffected upon the addition of azido-ppGpp.
Binding Sites↗
Identification and analysis of PCB dechlorinating anaerobic enrichments by amplification: accuracy of community structure based on restriction analysis and partial sequencing of 16S rRNA genes.
The composition of polychlorinated biphenyl (PCB) dechlorinating mixed communities was analysed by restriction fragment length polymorphism of PCR amplified rDNAs (ARDRA) and partial sequencing of 16S rRNA genes amplified from PCB degrading enrichments. Restriction analysis confirms that the 16S rRNA genes amplified from PCB dechlorinating communities vary depending on the PCB congener dechlorinated. Comparison of 16S rRNA sequences to published ribosomal databases indicates that the two most abundant Operational Taxonomic Units (OTUs) appear to be species of the genus Clostridium. The amount that the amplification procedure contributed to this result was determined by varying the amplification procedure and by creating an artificial template mixture. Varying the amount of template by sixfold in the amplification did not affect the distribution of OTUs but the number of OTUs observed decreased with decreasing template concentration. Comparison of products amplified from mixtures of 16S rDNA clones indicates that the more abundant Clostridium OTU did not amplify more efficiently than those of less abundant OTUs. Hybridization to a probe designed to detect the most abundant OTUs indicates that two other OTUs are closely related to this Clostridium species.
Bacteria, Anaerobic↗
Effects of the Escherichia coli DNA-binding protein H-NS on rRNA synthesis in vivo.
The Escherichia coli DNA-binding protein H-NS is known to interact specifically with the upstream region of ribosomal RNA transcription units, where it causes transcriptional repression in vitro. Here, we present results demonstrating the effect of H-NS on rRNA transcription in vivo. rRNA synthesis rates were compared in cells that differ in the expression of functional H-NS or FIS molecules. We could show that in the absence of H-NS derepression of rRNA synthesis occurs at low growth rates. During the cell cycle H-NS is responsible for the rapid shut-off of rRNA synthesis at the end of the exponential phase. As it is known for FIS-dependent activation, the inhibitory function of H-NS is specific for P1, the first of the tandem rRNA promoters. The effect of H-NS on rRNA synthesis was further assessed under stress conditions. While under osmotic upshift the reduction in rRNA synthesis is clearly H-NS-dependent, no such influence could be detected at cold shock. Determination of the cellular ppGpp concentrations revealed that H-NS does not mediate its function via alterations in the synthesis of the global effector ppGpp.
Bacterial Proteins↗
The quaternary structure of RNase G from Escherichia coli.
RNase G is the endoribonuclease responsible for forming the mature 5' end of 16S rRNA. This enzyme shares 35% identity with and 50% similarity to the N-terminal 470 amino acids encompassing the catalytic domain of RNase E, the major endonuclease in Escherichia coli. In this study, we developed non-denaturing purifications for overexpressed RNase G. Using mass spectrometry and N-terminal sequencing, we unambiguously identified the N-terminal sequence of the protein and found that translation is initiated at the second of two potential start sites. Using velocity sedimentation and oxidative cross-linking, we determined that RNase G exists largely as a dimer in equilibrium with monomers and higher multimers. Moreover, dimerization is required for activity. Four of the six cysteine residues of RNase G were mutated to serine. No single cysteine to serine mutation resulted in a complete loss of cross-linking, dimerization or activity. However, multiple mutations in a highly conserved cluster of cysteines, including C405 and C408, resulted in a partial loss of activity and a shift in the distribution of RNase G multimers towards monomers. We propose that many of the cysteines in RNase G lie on its surface and define, in part, the subunit-subunit interface.
Cysteine↗
Nucleotide sequence of the nifH gene coding for nitrogen reductase in the acetic acid bacterium Acetobacter diazotrophicus.
The nifH gene sequence of the nitrogen-fixing bacterium Acetobacter diazotrophicus was determined with the use of the polymerase chain reaction and universal degenerate oligonucleotide primers. The gene shows highest pair-wise similarity to the nifH gene of Azospirillum brasilense. The phylogenetic relationships of the nifH gene sequences were compared with those inferred from 16S rRNA gene sequences. Knowledge of the sequence of the nifH gene contributes to the growing database of nifH gene sequences, and will allow the detection of Acet. diazotrophicus from environmental samples with nifH gene-based primers.
Acetobacter↗
Transmissibility of Pseudomonas cepacia infection in clinic patients and lung-transplant recipients with cystic fibrosis.
BACKGROUND: In patients with cystic fibrosis, infection with Pseudomonas cepacia is associated with poor outcomes. However, the extent of person-to-person transmission and the source of P. cepacia infection after lung transplantation are not well defined. Using DNA-based typing systems, we sought to determine the genetic relatedness of P. cepacia infection at one cystic fibrosis center. METHODS: We analyzed 65 P. cepacia isolates gathered over a period of eight years at a single cystic fibrosis center from 17 clinic patients and from 5 patients who underwent double-lung transplantation. The isolates were analyzed by ribotyping and chromosomal fingerprinting based on pulsed-field gel electrophoresis. RESULTS: Analyses of serial isolates revealed that each clinic patient and transplant recipient harbored a different P. cepacia clone that was persistent. In the transplant recipients, the preoperative and postoperative isolates were identical. In the two patients with disseminated infection after lung transplantation, isolates from multiple sites were identical and indicated clonal expansion of the previous respiratory P. cepacia strain. Pulsed-field gel electrophoresis proved both more discriminative and more practical than ribotyping as a means of defining the genetic relatedness of the P. cepacia isolates. CONCLUSIONS: Our serial analyses in patients with cystic fibrosis at one center found distinct strains of P. cepacia persistently infecting each patient and no evidence of person-to-person transmission of this organism. P. cepacia infection after lung transplantation was due to the persistence of the strain present before transplantation.
Bacterial Typing Techniques↗
Rearrangements in the genome of the bacterium Salmonella typhi.
We have determined the genomic map of the bacterium Salmonella typhi Ty2, the causal organism of typhoid fever, by using pulsed-field gel electrophoresis. Digestion of the Ty2 genome with endonucleases Xba I, Bln I, and Ceu I yielded 33, 26, and 7 fragments, respectively, that were placed in order on a circular chromosome of 4780 kb. Transposon Tn10 was inserted in specific genes of Salmonella typhimurium and transduced into S. typhi, and thus, the positions of 37 S. typhi genes were located through the Xba I and Bln I sites of the Tn10. Gene order on chromosomes of Escherichia coli K-12 and S. typhimurium LT2 is remarkably conserved; however, the gene order in S. typhi Ty2 is different, suggesting it has undergone major genomic rearrangements during its evolution. These rearrangements include inversions and transpositions in the 7 DNA fragments between the seven rrn operons for rRNA (postulated to be due to homologous recombination in these rrn genes), another inversion that covers the replication terminus region (resembling inversions found in other enteric bacteria), and at least three insertions, one as large as 118 kb. Partial digestion of genomic DNA with the intron-encoded endonuclease I-Ceu I, which cuts only in rrn genes, shows chromosomal rearrangements, apparently due to homologous recombination in the rrn genes, that were detected in all wild-type strains of S. typhi tested. These rearrangements may have been selected to compensate for the insertions that otherwise would have altered the locations of genes with respect to the origin and terminus of replication. These observations are relevant to our view of the evolution of the bacterial genome and may be significant in the virulence of S. typhi.
Chromosomes, Bacterial↗
Stringent control and growth-rate-dependent control have nonidentical promoter sequence requirements.
Escherichia coli uses at least two regulatory systems, stringent control and growth-rate-dependent control, to adjust rRNA output to amino acid availability and the steady-state growth rate, respectively. We examined transcription from rrnB P1 promoters containing or lacking the cis-acting UP element and FIS protein binding sites after amino acid starvation. The "core promoter" responds to amino acid starvation like the full-length wild-type promoter; thus, neither the UP element nor FIS plays a role in stringent control. To clarify the relationship between growth-rate-dependent regulation and stringent control, we measured transcription from growth-rate-independent promoters during amino acid starvation. Four rrnB P1 mutants defective for growth-rate control and two other growth-rate-independent promoters (rrnB P2 and pS10) still displayed stringent regulation. Thus, the two systems have different promoter determinants, consistent with the idea that they function by different mechanisms. Two mutations disrupted stringent control of rrnB P1: (i) a multiple base change in the "discriminator" region between the -10 hexamer and the transcription start site and (ii) a double substitution making the promoter resemble the E sigma 70 consensus promoter. These results have important implications for the mechanisms of both stringent control and growth-rate-dependent control of rRNA transcription.
Base Sequence↗
Bipartite modular structure of intrinsic, RNA hairpin-independent termination signal for phage RNA polymerases.
The phage SP6 RNA and T7 RNA polymerases, which are closely related to each other, intrinsically stop at two signals in the Escherichia coli rrnB terminator t1 through different mechanisms. The downstream signal functioned without an RNA secondary structure formation, in which the signal was still active when separated from the upstream, hairpin-forming signal, and IMP incorporation enhanced its efficiency. The sequence from -15 to -1 was essential for the downstream, hairpin-independent termination (at -1). The results of SP6 transcription with heteroduplex templates and ribonucleotide analogs suggested that the downstream signal consists of two functionally different modules. The effects of iodo-CMP or IMP incorporation into RNA on termination efficiency were not sensitive to incorporation at -9 and upstream, but they were reactive to incorporation at -6 and -2, as reflected by strong iodo-rC:dG and weak rI:dC base pairing. Thus, the downstream module (from -8 approximately -6 to -1) appears to facilitate the release of RNA. Mismatches in the templates at -6 to +1 allowed for efficient termination, unlike those upstream of the sequence. The upstream module (from -15 to -9 approximately -7) functions as a duplex. Pausing of the SP6 elongation complex at the termination site was detected when RNA release was suppressed by the incorporation of 5-bromo-UMP, and it was dependent on the upstream module. Results of single-round SP6 transcriptions using 3'-deoxynucleotides and immobilized templates indicated that RNA was not released from the elongation complexes halted at the termination site on the template variants carrying mutations in the upstream or downstream module, whereas such complexes on the wild type template were dissociated. Thus, halting or simple pausing was not sufficient for termination even when the downstream module was intact. The upstream module appears to mediate such conformation change necessary for termination.
Base Pairing↗