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At least 667 records · Page 37Linked to original sources
PubMed1997

A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rRNA gene mutations in clarithromycin-resistant Helicobacter pylori.

We have developed a rapid PCR-oligonucleotide ligation assay that can discriminate single base substitutions that are associated with clarithromycin resistance in Helicobacter pylori. Susceptible isolates were wild type at positions 2143 and 2144 (cognate to 2058 and 2059 in Escherichia coli), while 93% of the resistant isolates contained A-to-G mutations at either position and 7% of the isolates contained A-to-C mutations at position 2143. In addition, the MIC for 86% of the resistant isolates with an A2143 mutation was > or = 64 micrograms per ml, and that for 89% of the resistant isolates with an A2144 mutation was < or = 32 micrograms per ml.

Anti-Bacterial Agents↗
PubMed2002

Interaction of avilamycin with ribosomes and resistance caused by mutations in 23S rRNA.

The antibiotic growth promoter avilamycin inhibits protein synthesis by binding to bacterial ribosomes. Here the binding site is further characterized on Escherichia coli ribosomes. The drug interacts with domain V of 23S rRNA, giving a chemical footprint at nucleotides A2482 and A2534. Selection of avilamycin-resistant Halobacterium halobium cells revealed mutations in helix 89 of 23S rRNA. Furthermore, mutations in helices 89 and 91, which have previously been shown to confer resistance to evernimicin, give cross-resistance to avilamycin. These data place the binding site of avilamycin on 23S rRNA close to the elbow of A-site tRNA. It is inferred that avilamycin interacts with the ribosomes at the ribosomal A-site interfering with initiation factor IF2 and tRNA binding in a manner similar to evernimicin.

Anti-Bacterial Agents↗
PubMed2002

Fitness cost of chromosomal drug resistance-conferring mutations.

To study the cost of chromosomal drug resistance mutations to bacteria, we investigated the fitness cost of mutations that confer resistance to different classes of antibiotics affecting bacterial protein synthesis (aminocyclitols, 2-deoxystreptamines, macrolides). We used a model system based on an in vitro competition assay with defined Mycobacterium smegmatis laboratory mutants; selected mutations were introduced by genetic techniques to address the possibility that compensatory mutations ameliorate the resistance cost. We found that the chromosomal drug resistance mutations studied often had only a small fitness cost; compensatory mutations were not involved in low-cost or no-cost resistance mutations. When drug resistance mutations found in clinical isolates were considered, selection of those mutations that have little or no fitness cost in the in vitro competition assay seems to occur. These results argue against expectations that link decreased levels of antibiotic consumption with the decline in the level of resistance.

Anti-Bacterial Agents↗
PubMed1998

Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales.

A fosmid library with inserts containing approximately 40 kb of marine bacterial DNA (J. L. Stein, T. L. Marsh, K. Y. Wu, H. Shizuya, and E. F. DeLong, J. Bacteriol. 178:591-599, 1996) yielded four clones with 16S rRNA genes from the order Planctomycetales. Three of the clones belong to the Pirellula group and one clone belongs to the Planctomyces group, based on phylogenetic and signature nucleotide analyses of full-length 16S rRNA genes. Sequence analysis of the ends of the genes revealed a consistent mismatch in a widely used bacterium-specific 16S rRNA PCR amplification priming site (27F), which has also been reported in some thermophiles and spirochetes.

Bacteria↗
PubMed1999

Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238 E. coli isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.

Animals↗
PubMed2005

Low intraspecific diversity in a polynucleobacter subcluster population numerically dominating bacterioplankton of a freshwater pond.

Cultivation-dependent and -independent methods were combined to investigate the microdiversity of a Polynucleobacter subcluster population (Betaproteobacteria) numerically dominating the bacterioplankton of a small, humic freshwater pond. Complete coverage of the population by cultivation allowed the analysis of microdiversity beyond the phylogenetic resolution of ribosomal markers. Fluorescent in situ hybridization with two probes specific for the narrow subcluster C (PnecC bacteria) of the Polynucleobacter cluster revealed that this population contributed up to 60% to the total number of bacterioplankton cells. Microdiversity was investigated for a date at which the highest relative numbers of PnecC were observed. A clone library of fragments of the ribosomal operon (16S rRNA genes, complete 16S-23S internal transcribed spacer 1 [ITS1], partial 23S rRNA genes) amplified with universal bacterial primers was constructed. The library was stepwise screened for fragments from PnecC bacteria and for different ITS genotypes of PnecC bacteria. The isolated PnecC strains were characterized by sequencing of the 16S rRNA genes and the ITS1. Both the clone library and the established culture collection contained only the same three ITS genotypes, and one of them contributed 46% to the entire number of clones. Genomic fingerprinting of the isolates with several methods always resulted in the detection of only one fingerprint per ITS genotype. We conclude that a Polynucleobacter population with an extremely low intraspecific diversity and an uneven structure numerically dominated the bacterioplankton community in the investigated habitat. This low intraspecific diversity is in strong contrast to the high intraspecific diversities found in marine bacterial populations.

Animals↗
PubMed1993

DNA binding and bending are necessary but not sufficient for Fis-dependent activation of rrnB P1.

The Escherichia coli Fis protein binds to three sites in the upstream activation region of the rrnB P1 promoter and enhances transcription 5- to 10-fold in vivo. In this report, we investigate the mechanism of Fis-dependent activation of transcription. We show that stimulation of rrnB P1 transcription by Fis can occur on linear DNA templates and does not require DNA upstream of the promoter-proximal Fis site I. Mutants of Fis defective for Hin-mediated recombination have been isolated previously and have defined an N-terminal domain required for DNA inversion by Hin in addition to the C-terminal domain which is required for DNA binding. Several of these mutants were found to be defective in stimulation of rrnB P1 transcription in vivo and in vitro. Activation-defective mutants fall into three classes: those that fail to bind to the upstream activation region, those that bind but fail to bend the DNA normally, and those that bind and bend but still fail to activate transcription. We conclude that it is unlikely that Fis functions by simply bringing upstream sequences or bound factors into the proximity of RNA polymerase to activate transcription. Rather, the data are most easily interpreted in terms of transcription activation by direct interactions between Fis and RNA polymerase, requiring precise positioning of the two proteins facilitated by bending of the DNA binding site.

Carrier Proteins↗
PubMed1996

A positive control mutant of the transcription activator protein FIS.

The FIS protein is a transcription activator of rRNA and other genes in Escherichia coli. We have identified mutants of the FIS protein resulting in reduced rrnB P1 transcription activation that nevertheless retain the ability to bind DNA in vivo. The mutations map to amino acid 74, the N-terminal amino acid of the protein's helix-turn-helix DNA binding motif, and to amino acids 71 and 72 in the adjoining surface-exposed loop. In vitro analyses of one of the activation-defective mutants (with a G-to-S mutation at position 72) indicates that it binds to and bends rrnB P1 FIS site I DNA the same as wild-type FIS. These data suggest that amino acids in this region of FIS are required for transcription activation by contacting RNA polymerase directly, independent of any other role(s) this region may play in DNA binding or protein-induced bending.

Binding Sites↗
PubMed1996

Upstream interactions at the lambda pRM promoter are sequence nonspecific and activate the promoter to a lesser extent than an introduced UP element of an rRNA promoter.

The rightward regulatory region of bacteriophage lambda contains two promoters, pRM and pR, which direct the synthesis of nonoverlapping divergent transcripts from start sites 82 bp apart. Each of the two promoters has an upstream (A+T)-rich region (ATR) within the sequence from -40 to -60 where in the rrnB P1 promoter a stretch of 20 (A+T) bp greatly stimulates promoter function. Here we present an investigation of the possible functional significance of pRM's ATR. We determined the effects on RNA polymerase-pRM promoter interaction both of (G+C) substitutions in the ATR and of amino acid substitutions in the alpha subunit, known to affect the upstream interaction. We find small (two- to threefold) effects of selected mutations in the alpha subunit on open complex formation at pRM. However, the (presumably upstream) interactions underlying these effects are sequence nonspecific, as they are not affected by (G+C) substitutions in the ATR. Substitution of the 20-bp UP element of the rrnB P1 promoter between positions -40 and -60 at pRM stimulates open complex formation to a considerably greater extent (5- to 10-fold). Results from kinetic studies indicate that on this construct the UP element mainly accelerates a step subsequent to the binding of RNA polymerase, although it may also facilitate the binding event itself. Less extensive studies likewise provide evidence for a two- to threefold activation of pR by upstream interactions. The possible involvement of the alpha subunit in the previously characterized (e.g., B. C. Mita, Y. Tang, and P. L. deHaseth, J. Biol. Chem. 270:30428-30433, 1995) interference of pR-bound RNA polymerase with open complex formation at pRM is discussed.

Amino Acid Sequence↗
PubMed1997

Promoters and transcripts associated with the aroP gene of Escherichia coli.

Analysis of in vitro transcriptional events initiating within the region immediately upstream of the aroP coding region has revealed the presence of three promoters, P1, P2, and P3. Both P1 and P2 give rise to mRNA encoding the AroP protein, whereas P3 initiates transcription in the opposite direction. Both P1 and P3 contain UP elements which contribute to promoter strength. Regulation of expression from these three promoters has been examined in vitro by using supercoiled DNA templates and in vivo by using lacZ transcriptional fusions and specific promoter mutants. Expression from P2 is partially repressed by TyrR alone both in vitro and in vivo. Addition of the aromatic amino acid tyrosine, phenylalanine, or tryptophan further increases this repression. P1 is not repressed by TyrR alone but is repressed in vivo in the presence of phenylalanine, tyrosine, or tryptophan. This also occurs in vitro but requires Ca2+ ions in the reaction mixture for its demonstration. Under these conditions, transcription from P3 is enhanced by TyrR protein with phenylalanine, tyrosine, or tryptophan. However, we were unable to demonstrate P3 expression in vivo. Under repressing conditions, there is no production of truncated RNA molecules (from P1), which would be expected if repression involved a roadblock mechanism.

Amino Acid Transport Systems↗
PubMed1998

Activation of Escherichia coli rRNA transcription by FIS during a growth cycle.

rRNA transcription in Escherichia coli is activated by the FIS protein, which binds upstream of rrnp1 promoters and interacts directly with RNA polymerase. Analysis of the contribution of FIS to rrn transcription under changing physiological conditions is complicated by several factors: the wide variation in cellular FIS concentrations with growth conditions, the contributions of several other regulatory systems to rRNA synthesis, and the pleiotropy of fis mutations. In this report, we show by in vivo footprinting and Western blot analysis that occupancy of the rrnBp1 FIS sites correlates with cellular levels of FIS. We find, using two methods of measurement (pulse induction of a FIS-activated hybrid promoter and primer extension from an unstable transcript made from rrnBp1), that the extent of transcription activation by FIS parallels the degree of FIS site occupancy and therefore cellular FIS levels. FIS activates transcription throughout exponential growth at low culture density, but rrnp1 transcription increases independently of FIS immediately following upshift, before FIS accumulates. These results support the model that FIS is one of a set of overlapping signals that together contribute to transcription from rrnp1 promoters during steady-state growth.

Base Sequence↗
PubMed1999

Transcriptional organization of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea.

The transcriptional organization of the erythromycin biosynthetic gene (ery) cluster of Saccharopolyspora erythraea has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a S. erythraea transcriptional terminator within the eryAI, eryAIII, eryBIII, eryBIV, eryBV, eryBVI, eryCIV, and eryCVI genes and additionally by an eryAI -10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream ery biosynthetic genes. Our results demonstrate that the ery gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from eryAI to eryG. Two overlapping polycistronic transcripts extending from eryBIV to eryBVII were identified. In addition, seven ery cluster promoter transcription start sites, one each beginning at eryAI, eryBI, eryBIII, eryBVI, and eryK and two beginning at eryBIV, were determined.

Base Sequence↗
PubMed2000

Distribution of intervening sequences in the genes for 23S rRNA and rRNA fragmentation among strains of the Salmonella reference collection B (SARB) and SARC sets.

Intervening sequences (IVSs) occur sporadically in several bacterial genera in the genes for 23S rRNA at relatively conserved locations. They are cleaved after transcription and lead to the presence of fragmented rRNA, which is incorporated into the ribosomes without religation but is nevertheless functional. The fragmentation of rRNA and the number of IVSs in all 72 strains of the Salmonella Reference Collection B set and 16 strains of the Salmonella Reference Collection C set, which have been established on the basis of multilocus enzyme electrophoresis (MLEE), were analyzed in the present study. Fragmentation of 23S rRNA was restricted to conserved cleavage sites located at bp 550 (helix 25) and bp 1170 (helix 45), locations where IVSs have been reported. Random cleavage at sites where IVSs could not be detected was not seen. Uncleaved IVSs were not detected in any case; thus, the IVSs invariably led to rRNA fragmentation, indicating a strong selection for maintenance of RNase III cleavage sites. The distribution of the number of IVSs carried by the different strains in the seven rrl genes is diverse, and the pattern of IVS possession could not be related to the MLEE pattern among the various Salmonella strains tested; this indicates that the IVSs are frequently exchanged between strains by lateral transfer. All eight subspecies of the genus Salmonella, including subspecies V represented by Salmonella bongori, have IVSs in both helix 25 and helix 45; this indicates that IVSs entered the genus after its divergence from Escherichia coli (more than 100 million years ago) but before separation of the genus Salmonella into many forms or that they were in the ancestor but have been lost from Escherichia.

Base Pairing↗
PubMed2001

Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli.

The high activity of the rrnB P1 promoter in Escherichia coli results from a cis-acting DNA sequence, the UP element, and a trans-acting transcription factor, FIS. In this study, we examine the effects of FIS and the UP element at the other six rrn P1 promoters. We find that UP elements are present at all of the rrn P1 promoters, but they make different relative contributions to promoter activity. Similarly, FIS binds upstream of, and activates, all seven rrn P1 promoters but to different extents. The total number of FIS binding sites, as well as their positions relative to the transcription start site, differ at each rrn P1 promoter. Surprisingly, the FIS sites upstream of site I play a much larger role in transcription from most rrn P1 promoters compared to rrnB P1. Our studies indicate that the overall activities of the seven rrn P1 promoters are similar, and the same contributors are responsible for these high activities, but these inputs make different relative contributions and may act through slightly different mechanisms at each promoter. These studies have implications for the control of gene expression of unlinked multigene families.

Base Sequence↗
PubMed2001

Regulation of rRNA transcription correlates with nucleoside triphosphate sensing.

We have previously shown that the activity of the Escherichia coli rRNA promoter rrnB P1 in vitro depends on the concentration of the initiating nucleotide, ATP, and can respond to changes in ATP pools in vivo. We have proposed that this nucleoside triphosphate (NTP) sensing might contribute to regulation of rRNA transcription. To test this model, we have measured the ATP requirements for transcription from 11 different rrnB P1 core promoter mutants in vitro and compared them with the regulatory responses of the same promoters in vivo. The seven rrnB P1 variants that required much lower ATP concentrations than the wild-type promoter for efficient transcription in vitro were defective for response to growth rate changes in vivo (growth rate-dependent regulation). In contrast, the four variants requiring high ATP concentrations in vitro (like the wild-type promoter) were regulated with the growth rate in vivo. We also observed a correlation between NTP sensing in vitro and the response of the promoters in vivo to deletion of the fis gene (an example of homeostatic control), although this relationship was not as tight as for growth rate-dependent regulation. We conclude that the kinetic features responsible for the high ATP concentration dependence of the rrnB P1 promoter in vitro are responsible, at least in part, for the promoter's regulation in vivo, consistent with the model in which rrnB P1 promoter activity can be regulated by changes in NTP pools in vivo (or by hypothetical factors that work at the same kinetic steps that make the promoter sensitive to NTPs).

Adenosine Triphosphate↗
PubMed2002

Mutations in the GTPase center of Escherichia coli 23S rRNA indicate release factor 2-interactive sites.

Mutations in the GTPase center of Escherichia coli 23S rRNA were characterized in vivo as UGA-specific nonsense suppressors. Some site-directed mutations did not exhibit suppressor activity and were interspersed among suppressor mutations. Our results demonstrate the involvement of the two adjacent loops of this conserved rRNA structure in UGA-dependent translation termination and, taken with previous in vitro analyses and with consideration of the crystal structure of the GTPase center RNA, indicate that nucleotides 1067, 1093, 1094, and 1095 are sites of interaction with release factor 2.

Binding Sites↗
PubMed2003

Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616.

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.

Amino Acids↗
PubMed2003

Atypical 16S rRNA gene copies in Ochrobactrum intermedium strains reveal a large genomic rearrangement by recombination between rrn copies.

Ochrobactrum intermedium is an opportunistic human pathogen belonging to the alpha 2 subgroup of proteobacteria. The 16S rDNA sequences of nine O. intermedium isolates from a collection of clinical and environmental isolates exhibited a 46-bp insertion at position 187, which was present in only one sequence among the 82 complete or partial 16S rDNA sequences of Ochrobactrum spp. available in data banks. Reverse transcription-PCR experiments showed that the 46-bp insertion remained in the 16S rRNA. The inserted sequence folded into a stem-loop structure, which took place in and prolonged helix H184 of the 16S rRNA molecule. Helix H184 has been described as conserved in length among eubacteria, suggesting the idiosyncratic character of the 46-bp insertion. Pulsed-field gel electrophoresis experiments showed that seven of the clinical isolates carrying the 46-bp insertion belonged to the same clone. Insertion and rrn copy numbers were determined by hybridization and I-CeuI digestion. In the set of clonal isolates, the loss of two insertion copies revealed the deletion of a large genomic fragment of 150 kb, which included one rrn copy; deletion occurred during the in vivo evolution of the clone. Determination of the rrn skeleton suggested that the large genomic rearrangement occurred during events involving homologous recombination between rrn copies. The loss of insertion copies suggested a phenomenon of concerted evolution among heterogeneous rrn copies.

Alphaproteobacteria↗