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PubMed1998

RNA polymerase mutants that destabilize RNA polymerase-promoter complexes alter NTP-sensing by rrn P1 promoters.

Mutations in Escherichia coli rpoB or rpoC, selected for the ability to confer prototrophy on relA spoT strains, were found to affect transcription from rrn P1 promoters. Two mutant strains (beta RH454 and beta' delta 215-220) reduced transcription of rrn P1 core promoter-lacZ fusions but not of control promoter-lacZ fusions. Purified mutant RNAPs formed complexes with rrn P1 promoters that were much less stable than those formed by wild-type RNAP and required high concentrations of the initiating NTP for efficient rrn P1 transcription. The instability of the rrn P1 core promoter complexes with the mutant RNAPs and their altered regulatory properties support a recently proposed model for the control of rRNA transcription by changing concentrations of the initiating NTPs. We further suggest that destabilization of promoter complexes by the mutant RNAPs mimics effects of ppGpp, decreasing or increasing transcription depending on the kinetic properties of the specific promoter.

Adenosine Triphosphate↗
PubMed1984

Nucleotide sequence of the rpoA-rplQ DNA of Escherichia coli: a second regulatory binding site for protein S4?

The "alpha-operon" of E.coli is a unit of regulation comprising the following known genes, mostly encoding ribosomal proteins (in order of transcription, and with their products named in brackets): rpsM (S13), rpsK (S11), rpsD (S4), rpoA (alpha-subunit of RNA polymerase), rplQ (L17). There is evidence that S4 tightly regulates all of these genes, except rpoA, by repressing translation of the polycistronic mRNA. Binding of S4 to the S13 start-site is thought to regulate the first three genes. We have extended the 'rpsD-rpoA' sequences previously determined by others, to include all of rpoA and rplQ. The rpoA-rplQ intercistronic region shows strong primary, and potential secondary structural homologies with the S4-binding sites on 16S rRNA and S13 mRNA. We suggest that S4 represses L17 translation directly.

Bacterial Proteins↗
PubMed1982

Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena.

In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region. A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid. Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized. When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing. The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons. We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions. We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds. The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.

Animals↗
PubMed1983

Unbalanced ribosomal protein synthesis in a strain of Escherichia coli containing a cloned, truncated 16-S ribosomal RNA gene.

An Escherichia coli K12 strain, carrying the promotor and proximal portion of the 16-S rRNA gene from rrnB cloned in the high-copy-number plasmid psF2124, has been examined for abnormalities in ribosome biogenesis. Both ribosomal RNA accumulation and ribosome content are depressed in this strain as compared to the control strain carrying the plasmid vector alone. The rate of total protein synthesis, however, appears to be normal. In contrast, the rate of ribosomal protein synthesis, relative to total protein synthesis, is elevated. The rates of synthesis of individual ribosomal proteins were determined and found to vary greatly, ranging from severe under-synthesis (displayed especially by proteins L7/L12) to massive over-synthesis (displayed particularly in the case of protein S7). Analysis of the rates of synthesis of other proteins coded for by the S12 operon revealed that protein S12 was moderately over-produced, but elongation factors EF-G and EF-Tu appear to be synthesized at the same rate as EF-Ts, all three being moderately under-synthesized relative to total soluble proteins.

Bacterial Proteins↗
PubMed1984

Transcription of the mouse ribosomal spacer region.

This paper describes experiments designed to test the hypothesis that DNA sequences upstream from the mouse rRNA promoter are transcribed in vivo or in vitro. Plasmid pB28 contains a SalI restriction fragment that extends from -169 to -1,894 base pairs, with respect to the origin of transcription of pre-rRNA. Labeled RNA synthesized in intact cells does not hybridize to this region. Neither S1 nuclease mapping nor RNA dot blot hybridization revealed the presence of sequences complementary to this region. Transcriptional studies carried out in vitro indicated that this region is not transcribed under conditions that are optimal for utilization of the authentic rRNA promoter. Moreover, this region does not appear to form stable transcription complexes with RNA polymerase I transcription components. These data indicate that the mouse rDNA repeating unit differs from those of Xenopus spp. and Drosophila melanogaster in that reduplicated RNA polymerase I promoters are not found in the mouse rDNA spacer region.

Animals↗
PubMed1990

Cloning and analysis of the Bacillus subtilis rpsD gene, encoding ribosomal protein S4.

The rpsD gene, encoding ribosomal protein S4, was isolated from Bacillus subtilis by hybridization with oligonucleotide probes derived from the S4 amino-terminal protein sequence. Sequence analysis of the cloned DNA indicated that rpsD is likely to be monocistronic, in contrast to Escherichia coli rpsD, which is located in the alpha operon and is the translational regulator for alpha operon ribosomal protein gene expression in E. coli. The cloned gene was shown to map at position 263 degrees on the B. subtilis chromosome, at the position to which mutations conferring alterations in the electrophoretic mobility of protein S4 were localized. A promoter was identified upstream of the rpsD coding sequence; initiation of transcription at this promoter would result in a transcript containing a leader region 180 bases in length. Immediately downstream of the rpsD coding region were two sequences resembling transcriptional terminators. An open reading frame homologous to tyrosyl-tRNA synthetase (tyrS) genes was identified downstream of rpsD but in the opposite orientation. The leader region of rpsD mRNA is predicted to have extensive secondary structure, resembling a region of B. subtilis 16S rRNA where S4 is likely to bind; similar mRNA features have been found to be important in ribosomal gene regulation in E. coli. These results provide the first steps toward analysis of the regulation of rpsD gene expression in B. subtilis.

Amino Acid Sequence↗
PubMed2005

Isolation and transcriptional analysis of novel tetrachloroethene reductive dehalogenase gene from Desulfitobacterium sp. strain KBC1.

Strain KBC1, an anaerobic bacterium, that dechlorinates tetrachloroethene (PCE) to trichloroethene was isolated. This strain also dechlorinated high concentrations of PCE at a temperature range of 10 to 40 degrees C and showed high oxygen tolerance. Based on the 16S rRNA gene sequence analysis, this microorganism was identified as a species of the genus Desulfitobacterium. Several species of this genus have been reported to be potent ortho-chlorophenol and PCE dechlorinators; however, the gene coding PCE-specific dehalogenase had not been cloned thus far. In this report, we identified a novel PCE reductive dehalogenase (PrdA) gene from the Desulfitobacterium sp. strain KBC1. These prd genes, including putative membrane anchor protein, were classified as novel type of PCE reductive dehalogenase (approximately 40% homology with the general PCE dehalogenase). It was revealed that the two open reading frames had been transcribed as identical mRNA and were induced strictly in the presence of PCE. This transcriptional regulation appeared to be controlled by the transcriptional activator located downstream of prdAB operon. According to the substrate utility of the strain KBC1 and phylogenetic analysis of PrdA, this microorganism may be expected to play the role of a primary dechlorinator of PCE in the environment.

Amino Acid Motifs↗
PubMed2004

Can whole genome analysis refine the taxonomy of the genus Rhodococcus?

The current systematics of the genus Rhodococcus is unclear, partly because many members were originally included before the application of a polyphasic taxonomic approach, central to which is the acquisition of 16S rRNA sequence data. This has resulted in the reclassification and description of many new species. Hence, the literature is replete with new species names that have not been brought together in an organized and easily interpreted form. This taxonomic confusion has been compounded by assigning many xenobiotic degrading isolates with phylogenetic positions but without formal taxonomic descriptions. In order to provide a framework for a taxonomic approach based on multiple genetic loci, a survey was undertaken of the known genome characteristics of members of the genus Rhodococcus including: (i) genetics of cell envelope biosynthesis; (ii) virulence genes; (iii) gene clusters involved in metabolic degradation and industrially relevant pathways; (iv) genetic analysis tools; (v) rapid identification of bacteria including rhodococci with specific gene RFLPs; (vi) genomic organization of rrn operons. Genes encoding virulence factors have been characterized for Rhodococcus equi and Rhodococcus fascians. Based on peptide signature comparisons deduced from gene sequences for cytochrome P-450, mono- and dioxygenases, alkane degradation, nitrile metabolism, proteasomes and desulfurization, phylogenetic relationships can be deduced for Rhodococcus erythropolis, Rhodococcus globerulus, Rhodococcus ruber and a number of undesignated Rhodococcus spp. that may distinguish the genus Rhodococcus into two further genera. The linear genome topologies that exist in some Rhodococcus species may alter a previously proposed model for the analysis of genomic fingerprinting techniques used in bacterial systematics.

Actinobacteria↗
PubMedDate not supplied

Antigenic variation and strain heterogeneity in Borrelia spp.

Antigenic variation and strain heterogeneity have been demonstrated for the pathogenic Borrelia species, i.e. B. burgdorferi and the relapsing fever borreliae. In relapsing fever, new borrelia serotypes emerge at a high rate spontaneously, a mechanism that is caused by DNA rearrangements on linear plasmid translocating genes coding for variable major proteins from previous silent to expression sites (i.e. from inner sites to telomeric sites of the plasmid). As a result of this variation, the borreliae escape the immune response of the host, thus leading to the relapse phenomenon. In B. burgdorferi, which is the causative agent of the multisystem disorder Lyme borreliosis, there is also a growing body of findings that antigenic variation is involved in pathogenesis of the disease. Phenotypic variation of strains in vitro concerns the size and the amount of surface-associated proteins (OspA, OspB and pC). There are indications that OspA and OspB truncations are due to deletions within the ospAB operon caused by recombination events, and that OspA/OspB-less mutants lack the 49-kb plasmid that bears the ospAB operon. With the increasing number of isolates obtained from various geographic and biological sources, it became apparent that B. burgdorferi is immunologically and genetically more heterogeneous, as previously believed. The major outer surface proteins OspA and OspB (which have been efficient antigens in vaccine studies) are heterogeneous at a genetic level. The same degree of genetic non-identity was observed for the pC protein. Other proteins like flagellin and the highly specific immunodominant p100 range protein show a lower degree of non-identity. Recombinant OspA, pC, p100 range protein and flagellin have been hyperexpressed in E. coli and these proteins are immunologically reactive. This allows further research for development of vaccines and diagnostic tools. B. burgdorferi isolates have been investigated with genotyping (DNA hybridization, PCR and 16S rRNA analysis) as well as serotyping by various authors. Comparison of the different methods has shown good agreement when the same strains have been investigated. No correlation could be found between different phenotypic and genotypic groups with respect to the ability to cause arthritis in SCID mice. A serotyping system based on immunological differences in OspA detected by a panel of monoclonal antibodies has been proposed. Serotyping a large number of B. burgdorferi isolates has shown a striking predominance of the OspA serotype 2 among European isolates from human skin, in contrast to isolates from ticks or CSF.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigenic Variation↗
PubMed1991

The Ams (altered mRNA stability) protein and ribonuclease E are encoded by the same structural gene of Escherichia coli.

The in vitro and in vivo analysis of the ribonuclease E-deficient (rne-) and the altered mRNA stability protein-deficient (ams-) strains of Escherichia coli has demonstrated that they carry mutations in the same structural gene. Strains encoding either thermolabile RNase E (rne-3071) or Ams protein (ams-1) are defective in both rRNA processing and mRNA turnover. Immediately after a shift to the nonpermissive temperature, the chemical decay rate of bulk mRNA is slowed 2- to 3-fold, and within 70 min, precursors to 5S rRNA begin to accumulate. In addition, all of the phenotypes associated with either the rne-3071 or the ams-1 alleles were complemented by a recombinant plasmid carrying ams+. When taken together with previous genetic studies, these results suggest that the role of ribonuclease E in mRNA turnover involves endonucleolytic cleavages at the proposed ACAG(A/U)AUUUG consensus sequence.

Bacterial Proteins↗
PubMed1994

Structural elements of rps0 mRNA involved in the modulation of translational initiation and regulation of E. coli ribosomal protein S15.

Previous experiments showed that S15 inhibits its own translation by binding to its mRNA in a region overlapping the ribosome loading site. This binding was postulated to stabilize a pseudoknot structure that exists in equilibrium with two stem-loops and to trap the ribosome on its mRNA loading site in a transitory state. In this study, we investigated the effect of mutations in the translational operator on: the binding of protein S15, the formation of the 30S/mRNA/tRNA(fMet) ternary initiation complex, the ability of S15 to inhibit the formation of this ternary complex. The results were compared to in vivo expression and repression rates. The results show that (1) the pseudoknot is required for S15 recognition and translational control; (2) mRNA and 16S rRNA efficiently compete for S15 binding and 16S rRNA suppresses the ability of S15 to inhibit the formation of the active ternary complex; (3) the ribosome binds more efficiently to the pseudoknot than to the stem-loop; (4) sequences located between nucleotides 12 to 47 of the S15 coding phase enhances the efficiency of ribosome binding in vitro; this is correlated with enhanced in vivo expression and regulation rates.

Base Sequence↗
PubMed1999

Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods.

Cystic fibrosis (CF) predisposes patients to bacterial colonization and infection of the lower airways. Several species belonging to the genus Burkholderia are potential CF-related pathogens, but microbiological identification may be complicated. This situation is not in the least due to the poorly defined taxonomic status of these bacteria, and further validation of the available diagnostic assays is required. A total of 114 geographically diverse bacterial isolates, previously identified in reference laboratories as Burkholderia cepacia (n = 51), B. gladioli (n = 14), Ralstonia pickettii (n = 6), B. multivorans (n = 2), Stenotrophomonas maltophilia (n = 3), and Pseudomonas aeruginosa (n = 11), were collected from environmental, clinical, and reference sources. In addition, 27 clinical isolates putatively identified as Burkholderia spp. were recovered from the sputum of Dutch CF patients. All isolates were used to evaluate the accuracy of two selective growth media, four systems for biochemical identification (API 20NE, Vitek GNI, Vitek NFC, and MicroScan), and three different PCR-based assays. The PCR assays amplify different parts of the ribosomal DNA operon, either alone or in combination with cleavage by various restriction enzymes (PCR-restriction fragment length polymorphism [RFLP] analysis). The best system for the biochemical identification of B. cepacia appeared to be the API 20NE test. None of the biochemical assays successfully grouped the B. gladioli strains. The PCR-RFLP method appeared to be the optimal method for accurate nucleic acid-mediated identification of the different Burkholderia spp. With this method, B. gladioli was also reliably classified in a separate group. For the laboratory diagnosis of B. cepacia, we recommend parallel cultures on blood agar medium and selective agar plates. Further identification of colonies with a Burkholderia phenotype should be performed with the API 20NE test. For final confirmation of species identities, PCR amplification of the small-subunit rRNA gene followed by RFLP analysis with various enzymes is recommended.

Bacterial Typing Techniques↗
PubMed2002

PNPase activity determines the efficiency of mRNA 3'-end processing, the degradation of tRNA and the extent of polyadenylation in chloroplasts.

The exoribonuclease polynucleotide phosphorylase (PNPase) has been implicated in mRNA processing and degradation in bacteria as well as in chloroplasts of higher plants. Here, we report the first comprehensive in vivo study of chloroplast PNPase function. Modulation of PNPase activity in Arabidopsis chloroplasts by a reverse genetic approach revealed that, although this enzyme is essential for efficient 3'-end processing of mRNAs, it is insufficient to mediate transcript degradation. Surprisingly, we identified PNPase as also being indispensable for 3'-end maturation of 23S rRNA transcripts. Analysis of tRNA amounts in transgenic Arabidopsis plants suggests a direct correlation of PNPase activity and tRNA levels, indicating an additional function of this exoribo nuclease in tRNA decay. Moreover, the extent of polyadenylated mRNAs in chloroplasts is negatively correlated with PNPase activity. Together, our results attribute novel functions to PNPase in the metabolism of all major classes of plastid RNAs and suggest an unexpectedly complex role for PNPase in RNA processing and decay.

Arabidopsis↗
PubMedDate not supplied

Requirement for an upstream element for optimal transcription of a bacterial tRNA gene.

Bacterial promoters are the sites at the 5' end of each gene that bind RNA polymerase and direct the initiation of transcription. The functional elements of Escherichia coli promoters are two highly conserved sequences, each about six nucleotides long, usually centred at sites -10 and -35, +1 being the initiating nucleotide. We have been interested in the structure of promoters of genes that are subject to stringent control, that is whose expression is reduced in conditions of amino acid shortage, such as rRNA and tRNA genes. We have therefore mapped the sequences involved in promoting in vivo transcription of a bacterial tRNATyr (tyrT) gene by fusing the tyrT promoter region to a galactokinase (galK) gene, and using in vivo expression of galactokinase activity to measure promoter strength. We show here that efficient expression from the tyrT promoter requires specific sequences upstream of the canonical promoter elements, and we suggest that these sequences constitute an extended promoter structure.

Base Sequence↗
PubMed2003

Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium species and their direct application for species identification.

We analyzed the tuf gene, encoding elongation factor Tu, from 33 strains representing 17 Lactobacillus species and 8 Bifidobacterium species. The tuf sequences were aligned and used to infer phylogenesis among species of lactobacilli and bifidobacteria. We demonstrated that the synonymous substitution affecting this gene renders elongation factor Tu a reliable molecular clock for investigating evolutionary distances of lactobacilli and bifidobacteria. In fact, the phylogeny generated by these tuf sequences is consistent with that derived from 16S rRNA analysis. The investigation of a multiple alignment of tuf sequences revealed regions conserved among strains belonging to the same species but distinct from those of other species. PCR primers complementary to these regions allowed species-specific identification of closely related species, such as Lactobacillus casei group members. These tuf gene-based assays developed in this study provide an alternative to present methods for the identification for lactic acid bacterial species. Since a variable number of tuf genes have been described for bacteria, the presence of multiple genes was examined. Southern analysis revealed one tuf gene in the genomes of lactobacilli and bifidobacteria, but the tuf gene was arranged differently in the genomes of these two taxa. Our results revealed that the tuf gene in bifidobacteria is flanked by the same gene constellation as the str operon, as originally reported for Escherichia coli. In contrast, bioinformatic and transcriptional analyses of the DNA region flanking the tuf gene in four Lactobacillus species indicated the same four-gene unit and suggested a novel tuf operon specific for the genus Lactobacillus.

Adult↗
PubMed1982

Nucleotide sequences of transcription and translation initiation regions in Bacillus phage phi 29 early genes.

phi 29 DNA directs the synthesis of three major proteins of Mr = 22,400, 13,900, and 10,500 in a cell-free transcription-translation system derived from Bacillus subtilis. We have determined the locations of the coding regions for these early proteins on the phi 29 genome, and our results are in agreement with genetic evidence that the 22.4-kilodalton protein is the product of cistron 17 (p17) and the 13.9-kilodalton protein is the product of cistron 6 (p6). The 13.9-kilodalton and 10.5-kilodalton proteins are encoded on a polycistronic mRNA previously designated G3b RNA. We have determined the nucleotide sequence of a HindIII restriction fragment of phi 29 DNA, the H fragment, that encodes the 13.9-kilodalton protein and contains two early promoters, G3a and G3b. The nucleotide sequence of the ribosome binding site contains a polypurine region capable of forming a very stable complex with nine bases on the 3' end of B. subtilis 16 S rRNA. This strong Shine-Dalgarno complementarity supports the hypothesis that an extensive mRNA . rRNA interaction is a requirement for efficient translation by B. subtilis ribosomes (McLaughlin, J. R., Murray, C. L., and Rabinowitz, J. C. (1981) J. Biol. Chem. 256, 11283-11291). The nucleotide sequences of both promoters are very similar to the "consensus" sequence for Escherichia coli promoters. While E. coli RNA polymerase initiates transcription from both of these promoters in vitro, only the G3b promoter is utilized by B. subtilis RNA polymerase under the same conditions.

Bacillus subtilis↗
PubMed2006

A single monooxygenase, ese, is involved in the metabolism of the organochlorides endosulfan and endosulfate in an Arthrobacter sp.

In this paper we describe isolation of a bacterium capable of degrading both isomers of the organochloride insecticide endosulfan and its toxic metabolite, endosulfate. The bacterium was isolated from a soil microbial population that was enriched with continuous pressure to use endosulfate as the sole source of sulfur. Analysis of the 16S rRNA sequence of the bacterium indicated that it was an Arthrobacter species. The organochloride-degrading activity was not observed in the presence of sodium sulfite as an alternative sulfur source, suggesting that the activity was part of the sulfur starvation response of the strain. A gene, ese, encoding an enzyme capable of degrading both isomers of endosulfan and endosulfate was isolated from this bacterium. The enzyme belongs to the two-component flavin-dependent monooxygenase family whose members require reduced flavin for activity. Nuclear magnetic resonance analyses identified the metabolite of endosulfan as endosulfan monoalcohol and the metabolite of endosulfate as endosulfan hemisulfate. The ese gene was located in a cluster of 10 open reading frames encoding proteins with low levels of sulfur-containing amino acids. These open reading frames were organized into two apparent divergently orientated operons and a gene encoding a putative LysR-type transcriptional regulator. The operon not containing ese did contain a homologue whose product exhibited 62% amino acid identity to the ese-encoded protein.

Arthrobacter↗
PubMed1992

Quantitative analysis of transcription and RNA levels of 15 barley chloroplast genes. Transcription rates and mRNA levels vary over 300-fold; predicted mRNA stabilities vary 30-fold.

Higher plant plastid genomes encode rRNAs, tRNAs, and protein subunits of the RNA polymerase, ribosomes, and the photosynthetic apparatus which vary over 1000-fold in abundance. Quantitative analysis of transcription and RNA levels was carried out on 15 plastid genes which are located in 14 different transcription units covering 50% of the barley plastid genome. Transcription of 16S rRNA, trnfM-trnG, and trnK was high relative to most other plastid genes. Transcription of trnfM-trnG was 5 times greater than trnK indicating that differences in tRNA levels in plastids could be due, in part, to differences in transcription. Among the protein coding genes, mRNA levels varied over 900-fold and transcription over 300-fold. The gene showing the lowest transcription rate and mRNA level, rpoB, is located in a gene cluster which encodes subunits of the plastid RNA polymerase (rpoB-rpoC1-rpoC2). RpoA, which encodes the alpha subunit of the RNA polymerase, was located in a gene cluster encoding ribosomal proteins (rpl23, rps19, rpl16) and infA. RNA from this gene cluster is 30-fold more abundant than rpoB mRNA, suggesting that expression of rpoA is regulated at the level of translation or protein stability. Polycistronic operons encoding subunits of the photosynthetic apparatus (psbB-psbH-petB-petD; psbK-psbI-psbD-psbC; atpB-atpE; psaA-psaB) had higher transcription rates and correspondingly higher mRNA levels than genes which encode ribosomal proteins or RNA polymerase subunits. RbcL and psbA, which are located in separate transcription units, exhibited the highest transcription rates and mRNA levels. Correspondence between transcription rate, mRNA level, and protein abundance indicates that transcription is a primary determinant of barley plastid gene expression. In addition, a 30-fold variation in predicted mRNA stability was observed which further increases the dynamic range of plastid mRNA abundance.

Base Sequence↗