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PubMed2002

Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio.

Analysis of the 16S rDNAs obtained from cultures of single colonies of either type collection strains or environmental strains of the genus Vibrio revealed the presence of polymorphism in every one of the strains examined. Polymorphism was detected by visualization of heteroduplexes produced after 16S rDNA PCR amplification, a procedure that allows for the screening of a large number of isolates. Amplified 16S rDNAs obtained from both Vibrio parahaemolyticus and an environmental strain were cloned. Their nucleotide sequences revealed differences of up to 2% among 16S rDNAs from the same strain. Polymorphic sites were concentrated in a recognized variable stem-loop of bacterial 16S rRNA that contained in some cases up to 83% of the total mismatches observed. Most of the substitutions present in the stem-loop region showed compensating base covariation. The accumulation of so many compensating changes in the stem-loop region implies that the divergence of the different versions of this stem-loop is relatively ancient. This divergence could be the result of either a selection process or a lateral transfer of independently evolved genes.

DNA, Bacterial↗
PubMed1980

Formylmethionyl-tRNA alters RNA polymerase specificity.

Escherichia coli fMet-tRNAfMet alters the pattern of promoter selection of E. coli RNA polymerase (RNA nucleotidyltransferase, nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6), affecting RNA synthesis from the rRNA, suIII+tRNA, and lac promoters in different ways. The in vitro synthesis of the stable RNA species is selectively decreased, whereas that of lac RNA from both the wild-type and mutant UV5 promoters is selectively increased at high ionic strength. The functional effect of fMet-tRNAfMet resembles that of the nucleotide guanosine 3'-diphosphate 5'-diphosphate (ppGpp). This nucleotide competes with the binding of fMet-tRnafMet to RNA polymerase.

Binding, Competitive↗
PubMed1984

Identification of a promoter for transcription of the heavy strand of human mtDNA: in vitro transcription and deletion mutagenesis.

Plasmids containing the origin region of human mtDNA are specifically transcribed by the partially purified homologous mtRNA polymerase in vitro. Transcription of both the light and heavy strands initiates at the same sites previously identified as in vivo transcription start sites. The sequences responsible for initiation of heavy strand transcription are investigated in detail, since this transcript includes the rRNA cistrons and the majority of tRNA genes and potential mRNAs. Deletion mutagenesis is employed to delimit the sequences responsible for heavy strand transcription to a region of less than 35 bp surrounding the transcription start site. This region contains a repetitive sequence, AAACCCC, and shows homology to other regions of the human mtDNA that have been implicated as transcription initiation sites or that show unusual homology to other mammalian mtDNA genomes.

Base Sequence↗
PubMed2000

Function of the ski4p (Csl4p) and Ski7p proteins in 3'-to-5' degradation of mRNA.

One of two general pathways of mRNA decay in the yeast Saccharomyces cerevisiae occurs by deadenylation followed by 3'-to-5' degradation of the mRNA body. Previous results have shown that this degradation requires components of the exosome and the Ski2p, Ski3p, and Ski8p proteins, which were originally identified due to their superkiller phenotype. In this work, we demonstrate that deletion of the SKI7 gene, which encodes a putative GTPase, also causes a defect in 3'-to-5' degradation of mRNA. Deletion of SKI7, like deletion of SKI2, SKI3, or SKI8, does not affect various RNA-processing reactions of the exosome. In addition, we show that a mutation in the SKI4 gene also causes a defect in 3'-to-5' mRNA degradation. We show that the SKI4 gene is identical to the CSL4 gene, which encodes a core component of the exosome. Interestingly, the ski4-1 allele contains a point mutation resulting in a mutation in the putative RNA binding domain of the Csl4p protein. This point mutation strongly affects mRNA degradation without affecting exosome function in rRNA or snRNA processing, 5' externally transcribed spacer (ETS) degradation, or viability. In contrast, the csl4-1 allele of the same gene affects rRNA processing but not 3'-to-5' mRNA degradation. We identify csl4-1 as resulting from a partial-loss-of-function mutation in the promoter of the CSL4 gene. These data indicate that the distinct functions of the exosome can be separated genetically and suggest that the RNA binding domain of Csl4p may have a specific function in mRNA degradation.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Identification and analysis of the rnc gene for RNase III in Rhodobacter capsulatus.

The large subunit ribosomal RNA of the purple bacterium Rhodobacter capsulatus shows fragmentation into pieces of 14 and 16S, both fragments forming the functional equivalent of intact 23S rRNA. An RNA-processing step removes an extra stem-loop structure from the 23S rRNA [Kordes, E., Jock, S., Fritsch, J., Bosch, F. and Klug, G. (1994) J. Bacteriol., 176, 1121-1127]. Taking advantage of the fragmentation deficient mutant strain Fm65, we used genetic complementation to find the mutated gene responsible for this aberration. It was identified as the Rhodobacter homologue to mc from Escherichia coli encoding endoribonuclease III (RNase III). The predicted protein has 226 amino acids with a molecular weight of 25.5 kDa. It shares high homology with other known RNase III enzymes over the full length. In particular it shows the double-stranded RNA-binding domain (dsRBD) motif essential for binding of dsRNA substrates. The Fm65 mutant has a frame shift mutation resulting in complete loss of the dsRBD rendering the enzyme inactive. The cloned Rhodobacter enzyme can substitute RNase III activity in an RNase III deficient E. coli strain. Contrary to E. coli, the Rhodobacter mc is in one operon together with the lep gene encoding the leader peptidase.

Amino Acid Sequence↗
PubMed1997

Molecular analysis of toxigenic Vibrio cholerae O139 Bengal strains isolated in Bangladesh between 1993 and 1996: evidence for emergence of a new clone of the Bengal vibrios.

Vibrio cholerae O139 Bengal emerged in 1992 and rapidly spread in an epidemic form, in which it replaced existing strains of V. cholerae O1 in Bangladesh during 1992 and 1993. The subsequent emergence of a new clone of V. cholerae O1 of the El Tor biotype that transiently displaced the O139 vibrios during 1994 to 1995 and the recent reemergence of V. cholerae O139 and its coexistence with the El Tor vibrios demonstrated temporal changes in the epidemiology of cholera in Bangladesh. We studied clonal diversity among V. cholerae O139 strains isolated from cholera patients and environmental surface water since their first appearance until their transient disappearance in 1994 as well as the O139 strains that reemerged during 1995 to 1996 and were isolated in the capital Dhaka and four rural districts of Bangladesh to investigate the origin of the reemerged strains. Analysis of restriction fragment length polymorphisms in genes for conserved rRNA and cholera toxin (CT) (ctxA) or in DNA sequences flanking these genes revealed four different ribotypes and four different ctx genotypes among the 93 strains of V. cholerae O139 studied. Ribotypes I and II and ctx genotypes A through C were shared by strains isolated from the epidemic outbreak during 1992 and 1993 in Bangladesh and India, ribotype III was represented by a single CT-negative O139 strain from Argentina, and 16 of 27 (59.2%) of the reemerged strains isolated during 1995 and 1996 belonged to a new ribotype of O139 vibrios designated ribotype IV. All 16 strains belonging to ribotype IV also belonged to a new ctx genotype (genotype 4). These results provide evidence for the emergence of a new clone of toxigenic V. cholerae O139 in Bangladesh. Further analysis of the rfb gene cluster by PCR revealed the absence of a large region of the O1-specific rfb operon and the presence of an O139-specific genomic region in all O139 strains. The PCR amplicon corresponding to the rfaD gene of a CT-negative O139 strain from Argentina was smaller in length than those of the toxigenic O139 strains but was identical to those of seven non-O1 and non-O139 strains. All O139 strains except the CT-negative strain carried structural and regulatory genes for CT and toxin-coregulated pili (ctxA, tcpA, tcpI, and toxR). These results suggest that the O139 Bengal strains possibly emerged from an El Tor strain but that the CT-negative non-Bengal O139 strain might have emerged from a non-O1, non-O139 strain. Thus, strains belonging to the O139 serogroup may have emerged from similar serotype-specific genetic changes in more than one progenitor strain of V. cholerae.

Bacterial Outer Membrane Proteins↗
PubMed2000

Structural variation in the 16S-23S rRNA intergenic spacers of Vibrio parahaemolyticus.

The 16S-23S rRNA intergenic spacers (IGS) of Vibrio parahaemolyticus were PCR-amplified and cloned with pT7Blue T vector. A total of six clones were isolated dependent on size difference. The clones were different with respect to both the number and the composition of the tRNA genes included, and were designated IGS-0, IGS-E, IGS-IA, IGS-AE, IGS-EKV and IGS-EKAV. IGS-EKAV included the cluster of tRNA(Glu)-tRNA(Lys)-tRNA(Ala)-tRNA(Val); IGS-EKV, tRNA(Glu)-tRNA(Lys)-tRNA(Val); IGS-AE, tRNA(Ala)-tRNA(Glu); IGS-IA, tRNA(Ile)-tRNA(Ala); and IGS-E, tRNA(Glu). IGS-0 had no tRNA gene. Some similarities were found in the nucleotide sequence of the non-coding regions flanked by the tRNA genes. The structure difference found in the spacers is meaningful for elucidating the evolutionary line of each ribosomal RNA operon and the profile is applicable as a molecular marker of the bacterium.

Cloning, Molecular↗
PubMed1989

A novel sequence element derived from bacteriophage T7 mRNA acts as an enhancer of translation of the lacZ gene in Escherichia coli.

We recently reported that a ribosome binding site (RBS) derived from gene 10 of bacteriophage T7 (g10-L) causes a pronounced stimulation of expression when placed upstream of a variety of genes, and that this effect is probably due to a stimulation of translation efficiency in Escherichia coli (Olins, P. O., Devine, C. S., Rangwala, S. H., Kavka, K. S. (1988) Gene (Amst.) 73, 227-235). Here we present a model for the mechanism of action of the g10-L: the RBS contains a 9-base sequence which has the potential for forming a novel base-paired interaction with bases 458-466 of the 16 S rRNA of E. coli. Although such sequence homologies are rare in E. coli RBS regions, a number of similar sequences were found in the RBS regions of other bacteriophage structural genes. When an isolated homology sequence was placed upstream of a synthetic RBS, there was a 110-fold increase in the translation efficiency of the lacZ gene. Surprisingly, the homology sequence also stimulated translation when placed downstream of the initiator codon, indicating that this sequence is acting as a translational "enhancer."

Base Sequence↗
PubMed1993

Mutations at U2555, a tRNA-protected base in 23S rRNA, affect translational fidelity.

A plasmid carrying a mutation in the highly conserved base U2555 in Escherichia coli 23S rRNA was isolated by selecting for suppression of the -1 frameshift mutation trpE91. U2555 is normally protected in chemical footprinting experiments by the aminoacyl residue of A-site-bound tRNA. Substitution of U2555 by adenine or guanine (but not by cytosine) increased readthrough of all three stop codons and +1 and -1 frameshifting. These effects on translational fidelity demonstrate the importance of U2555 for selection of the correct tRNA at the ribosomal A site.

Amino Acid Sequence↗
PubMed1987

[Detection and identification of Mycoplasma infections by DNA hybridization].

Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA. The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas. This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues. A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed. This set consists of specific DNA probes and universal DNA probe. Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes. Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations. These two classes of DNA probes may be considered as complementing each other. These 32P labeled probes do not hybridize with eukaryotic DNA. The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.

Animals↗
PubMed1992

Dramatic changes in Fis levels upon nutrient upshift in Escherichia coli.

Fis is a small basic DNA-binding protein from Escherichia coli that was identified because of its role in site-specific DNA recombination reactions. Recent evidence indicates that Fis also participates in essential cell processes such as rRNA and tRNA transcription and chromosomal DNA replication. In this report, we show that Fis levels vary dramatically during the course of cell growth and in response to changing environmental conditions. When stationary-phase cells are subcultured into a rich medium, Fis levels increase from less than 100 to over 50,000 copies per cell prior to the first cell division. As cells enter exponential growth, nascent synthesis is largely shut off, and intracellular Fis levels decrease as a function of cell division. Fis synthesis also transiently increases when exponentially growing cells are shifted to a richer medium. The magnitude of the peak of Fis synthesis appears to reflect the extent of the nutritional upshift. fis mRNA levels closely resemble the protein expression pattern, suggesting that regulation occurs largely at the transcriptional level. Two RNA polymerase-binding sites and at least six high-affinity Fis-binding sites are present in the fis promoter region. We show that expression of the fis operon is negatively regulated by Fis in vivo and that purified Fis can prevent stable complex formation by RNA polymerase at the fis promoter in vitro. However, autoregulation only partially accounts for the expression pattern of Fis. We suggest that the fluctuations in Fis levels may serve as an early signal of a nutritional upshift and may be important in the physiological roles Fis plays in the cell.

Amino Acid Sequence↗
PubMed2001

Cooperative effects by the initiation codon and its flanking regions on translation initiation.

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Base Sequence↗
PubMed1997

Bacillus subtilis RNase III gene: cloning, function of the gene in Escherichia coli, and construction of Bacillus subtilis strains with altered rnc loci.

The rnc gene of Bacillus subtilis, which has 36% amino acid identity with the gene that encodes Escherichia coli RNase III endonuclease, was cloned in E. coli and shown by functional assays to encode B. subtilis RNase III (Bs-RNase III). The cloned B. subtilis rnc gene could complement an E. coli rnc strain that is deficient in rRNA processing, suggesting that Bs-RNase III is involved in rRNA processing in B. subtilis. Attempts to construct a B. subtilis rnc null mutant were unsuccessful, but a strain was constructed in which only a carboxy-terminal truncated version of Bs-RNase III was expressed. The truncated Bs-RNase III showed virtually no activity in vitro but was active in vivo. Analysis of expression of a copy of the rnc gene integrated at the amy locus and transcribed from a p(spac) promoter suggested that expression of the B. subtilis rnc is under regulatory control.

Bacillus subtilis↗
PubMed2003

[Identification of Streptococcus species and Haemophilus influenzae by direct sequencing of PCR products from 16S-23S rRNA itergenic sacer regions].

To facilitate species level identification of bacteria without the requirement of presumptive identification, the paper describes a rapid identification method of bacteria by amplification and direct sequencing 16S-23S rDNA intergenic spacer regions (ISR) of the pathogens which cause the upper respiratory tract infective disease by Streptococcus and Haemophilus. Three pairs of primer targeting conserved sequences flanking the 3' end of 16S and the 5' end of 23S rRNA were used to amplify 16S~23S rRNA ISR of 7 streptococcus strains and 8 Haemophilus strains. The PCR products were separated by 1% agarose gel electrophoresis and the polymorphisms fragments were purified with the Wizard PCR Min-Prep Kit (Promega) and Protocol-SK131(Sangon). The nucleotide sequences of ISR inserts were determined by using the XEQTM DTCS Kitjj Terminator Cycle Sequencing and a CEQTM 2000XL DNA Analysis system (Backman Coulter) automatic DAN sequencer. Then those sequences were compared with known sequences on the GenBank. The alignment of nucleotide sequence, evolutionary distances and phylogenetic trees were analyzed by software DANMAN version 4.0. The PCR products were showed polymorphism patterns with agarose gel. One band was contained in streptococcus genus. The significant variation was found among the spacer sequences of different species in Streptococcus with the lengths of the spacer varying from 269 to 446 bp. All the ISR of the streptococcal species had a tRNA Ala gene in the spacer and the sequence identities varied from 78 to 88% within genera. It was found that some spacer sequence blocks were highly conserved between operons of a genome, whereas the presence of others was variable, three regions showed significant spatial variation. Most of the differences between the sequences came from several bases insertions/deletions and substitutions. There are two major bands in the Haemophilus biotypes (515 and 884 bp), the small ISR amplicon contained one tDNA coding for tRNA(Glu). In contrast to the large one contained two tRNA genes coding for tRAN(Ala) and tRNA(Ile). Two regions of repeating motifs with only A or T were present in higher copy numbers between tRAN(Ala) and tRNA(Ile). The phylogenetic trees varied from 97.5 to 98.8%. The PCR and direct sequencing of 16S-23S rRAN ISR were successful in the pathogen species identification.

English Abstract↗
PubMed2001

Bactericidal antisense effects of peptide-PNA conjugates.

Antisense peptide nucleic acids (PNAs) can specifically inhibit Escherichia coli gene expression and growth and hold promise as anti-infective agents and as tools for microbial functional genomics. Here we demonstrate that chemical modification improves the potency of standard PNAs. We show that 9- to 12-mer PNAs, especially when attached to the cell wall/membrane-active peptide KFFKFFKFFK, provide improvements in antisense potency in E. coli amounting to two orders of magnitude while retaining target specificity. Peptide-PNA conjugates targeted to ribosomal RNA (rRNA) and to messenger RNA (mRNA) encoding the essential fatty acid biosynthesis protein Acp prevented cell growth. The anti-acpP PNA at 2 microM concentration cured HeLa cell cultures noninvasively infected with E. coli K12 without any apparent toxicity to the human cells. These results indicate that peptides can be used to carry antisense PNA agents into bacteria. Such peptide-PNA conjugates open exciting possibilities for anti-infective drug development and provide new tools for microbial genetics.

Amino Acid Sequence↗
PubMed2004

Characterization of large-insert DNA libraries from soil for environmental genomic studies of Archaea.

Complex genomic libraries are increasingly being used to retrieve complete genes, operons or large genomic fragments directly from environmental samples, without the need to cultivate the respective microorganisms. We report on the construction of three large-insert fosmid libraries in total covering 3 Gbp of community DNA from two different soil samples, a sandy ecosystem and a mixed forest soil. In a fosmid end sequencing approach including 5376 sequence tags of approximately 700 bp length, we show that mostly bacterial and, to a much lesser extent, archaeal and eukaryotic genome fragments (approximately 1% each) have been captured in our libraries. The diversity of putative protein-encoding genes, as reflected by their distribution into different COG clusters, was comparable to that encoded in complete genomes of cultivated microorganisms. A huge variety of genomic fragments has been captured in our libraries, as seen by comparison with sequences in the public databases and by the large variation in G+C contents. We dissect differences between the libraries, which relate to the different ecosystems analysed and to biases introduced by different DNA preparations. Furthermore, a range of taxonomic marker genes (other than 16S rRNA) has been identified that allows the assignment of genome fragments to specific lineages. The complete sequences of two genome fragments identified as being affiliated with Archaea, based on a gene encoding a CDC48 homologue and a thermosome subunit, respectively, are presented and discussed. We thereby extend the genomic information of uncultivated crenarchaeota from soil and offer hints to specific metabolic traits present in this group.

Adenosine Triphosphatases↗
PubMed1985

Topoisomerase I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis.

Topoisomerase I is in situ associated with DNaseI hypersensitive sites located in the promotor and terminator regions of the extrachromosomal rDNA in Tetrahymena thermophila at sites with sequences fitting the motif (sequence in text) Reconstitution experiments with purified topoisomerase I and cloned fragments of rDNA demonstrate that the enzyme exhibits the same binding and cleavage properties on naked DNA. These observations are striking as topoisomerase I previously has been found to exhibit low sequence specificity. The specific binding of the enzyme has an absolute requirement for divalent cations with a preference for Ca2+. The strong binding to the hexadecamer has been characterized by competition experiments, and it has been used to determine the molecular weight of the enzyme.

Animals↗
PubMed1989

The structural basis of the high in vivo strength of the rRNA P2 promoter of Escherichia coli.

Ribosomal RNA promoters of Escherichia coli are probably the strongest promoters in vivo and they can be used on plasmid vectors to express protein-coding sequences at a high rate. In fact, the P2 promoter of the rrnB gene is stronger (in vivo) than the tac promoter, which has a perfect consensus sequence. Conversion of the rrnB P2 promoter sequence to consensus significantly increases in vivo promoter strength. The removal of four nucleotides downstream of the -10 region also increases the strength of this promoter. On the other hand, shifting of the A + T-rich region upstream of this promoter by an 11-bp insertion drastically decreases in vivo activity. It is concluded that the two functionally important hexanucleotide sequences, -35 and -10, are necessary but not sufficient factors for the optimalization of in vivo promoter strength.

Base Sequence↗