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At least 919 records · Page 51Linked to original sources
PubMed1999

Transcriptional induction by aromatic amino acids in Saccharomyces cerevisiae.

Aromatic aminotransferase II, product of the ARO9 gene, catalyzes the first step of tryptophan, phenylalanine, and tyrosine catabolism in Saccharomyces cerevisiae. ARO9 expression is under the dual control of specific induction and nitrogen source regulation. We have here identified UASaro, a 36-bp upstream element necessary and sufficient to promote transcriptional induction of reporter gene expression in response to tryptophan, phenylalanine, or tyrosine. We then isolated mutants in which UASaro-mediated ARO9 transcription is partially or totally impaired. Mutations abolishing ARO9 induction affect a gene called ARO80 (YDR421w), coding for a Zn2Cys6 family transcription factor. A sequence highly similar to UASaro was found upstream from the YDR380w gene encoding a homolog of bacterial indolepyruvate decarboxylase. In yeast, this enzyme is postulated to catalyze the second step of tryptophan catabolism to tryptophol. We show that ARO9 and YDR380w (named ARO10) have similar patterns of transcriptional regulation and are both under the positive control of Aro80p. Nitrogen regulation of ARO9 expression seems not directly to involve the general factor Ure2p, Gln3p, Nil1p, Uga43p, or Gzf3p. ARO9 expression appears, rather, to be mainly regulated by inducer exclusion. Finally, we show that Gap1p, the general amino acid permease, and Wap1p (Ycl025p), a newly discovered inducible amino acid permease with broad specificity, are the main aromatic amino acid transporters for catabolic purposes.

Amino Acid Sequence↗
PubMed1991

Interaction of a regulatory protein with a DNA target containing two overlapping binding sites.

The LexA repressor from Escherichia coli regulates the transcription of about 20 different genes upon binding to single or multiple operators. In this work we study the interaction of LexA with the control region of the caa gene (coding for the bacterial toxin colicin A) that contains two operators (O1 and O2) which overlap by at least 2 base pairs relevant for sequence specific DNA recognition. This arrangement raises the question of how the LexA molecules which bind to the central overlapping part of the two operators avoid steric clashes and further, of whether the interaction of LexA with the two operators is cooperative or not. To address these questions we have constructed two mutant operators (O1+O2- and O1-O2+) for which the two most strongly conserved base pairs in each of the external operator half-sites have been mutated. Using methylation interference with the complex formation of LexA with the wild-type and these two mutant operators we could show: 1) that the two mutant operators behave symmetrically in that the methylation of one crucial guanine base in both operator half-sites interferes strongly with complex formation, 2) but that in the wild-type operator (containing four functional operator half-sites) only the two external half-operators give rise to interference if this crucial guanine base is methylated, whereas methylation of the two equivalent guanine bases within the two central (overlapping) operator half-sites does not lead to interference with the formation of a complex where both operators are occupied simultaneously. These data suggest that the centrally bound LexA molecules adopt a somewhat different binding mode than those bound to the external half-operators in order to avoid steric clashes and/or to optimize protein-protein contacts which are likely to be at the origin of the binding cooperativity that we could demonstrate by quantitative DNase I footprinting and gel retardation experiments. While the methylation interference experiments revealed a non-equivalence for the binding of externally and centrally bound LexA molecules, both methylation protection and hydroxyl radical footprinting were unable to reveal this difference, suggesting that the difference between the two binding modes should be fairly subtle.

Bacterial Proteins↗
PubMed2026

Landscape of retron diversity across the SPIRE microbial metagenome resource reveals candidate novel type XI-like lineages.

Retrons are bacterial genetic elements encoding a specialized reverse transcriptase (RT) that synthesizes multicopy single-stranded DNA and are increasingly recognized as components of bacterial anti-phage defense systems. However, their diversity and ecological distribution across large-scale genomic resources remain poorly characterized. Here, we surveyed retron RTs across the SPIRE representative metagenome collection, a non-redundant, species-level data set spanning diverse microbial habitats. Using a curated panel of type-specific hidden Markov models, we identified retrons representing all canonical types together with additional divergent lineages. Retron distribution showed strong taxonomic and ecological structuring, with some groups restricted to specific bacterial phyla, whereas others were broadly distributed across environmental categories. Systematic novelty assessment identified two candidate type XI-like lineages, TXI_C2like and TXI_noncan_h, characterized by protease-independent architectures and distinct accessory modules associated with WYL- and DnaB_C-containing proteins, respectively. De novo covariance-based analyses further identified candidate msr/msd-like non-coding RNA structures in both lineages, supporting conservation of the canonical RT-ncRNA organizational framework despite extensive sequence divergence. Together, these findings expand the known diversity of retron systems and identify type XI-like retrons as a dynamic and previously underexplored evolutionary group.IMPORTANCERetrons are bacterial genetic elements that are increasingly exploited as programmable tools for genome editing, molecular recording, and biosensing in addition to their natural role in anti-phage defense. Despite this growing biotechnological interest, the true diversity of retrons across the bacterial world has remained largely unmapped. By mining a resource of over 100,000 processed microbial metagenomes, we uncovered thousands of retron sequences spanning known types as well as previously unrecognized lineages and found that their distribution is strongly shaped by both bacterial taxonomy and ecological niche. Among these, we identified two candidate new lineages related to type XI retrons that lack the protease domain typical of this group but instead carry distinct accessory proteins, expanding the known architectural diversity of these systems. These findings broaden the catalog of retron diversity available for functional characterization and biotechnological engineering and provide a framework for prioritizing candidate lineages for future experimental validation.

effectors↗
PubMed1999

Evidence for genetic drift in endosymbionts (Buchnera): analyses of protein-coding genes.

Buchnera, the bacterial endosymbionts of aphids, undergo severe population bottlenecks during maternal transmission through their hosts. Previous studies suggest an increased effect of drift within these strictly asexual, small populations, resulting in an increased fixation of slightly deleterious mutations. This study further explores sequence evolution in Buchnera using three approaches. First, patterns of codon usage were compared across several homologous Escherichia coli and Buchnera loci, in order to test the prediction that selection for the use of optimal codons is less effective in small populations. A chi 2-based measure of codon bias was developed to adjust for the overall A + T richness of silent positions in the endosymbionts. In contrast to E. coli homologues, adaptive codon bias across Buchnera loci is markedly low, and patterns of codon usage lack a strong relationship with gene expression level. These data suggest that codon usage in Buchnera has been shaped largely by mutational pressure and drift rather than by selection for translational efficiency. One exception to the overall lack of bias is groEL, which is known to be constitutively overexpressed in Buchnera and other endosymbionts. Second, relative-rate tests show elevated rates of sequence evolution of numerous protein-coding loci across Buchnera, compared to E. coli. Finally, consistently higher ratios of nonsynonymous to synonymous substitutions in Buchnera loci relative to the enteric bacteria strongly suggest the accumulation of nonsynonymous substitutions in endosymbiont lineages. Combined, these results suggest a decreased effectiveness of purifying selection in purging endosymbiont populations of slightly deleterious mutations, particularly those affecting codon usage and amino acid identity.

Animals↗
PubMed1980

Arrangement of coding and non-coding sequences in the DNA molecules coding for rRNAs in Oxytricha sp. DNA of ciliated protozoa. VII.

All of the genes in the macronucleus of Oxytricha sp. occur on physically separate "gene-sized" DNA molecules. We have inserted the DNA molecule that codes for rRNA into a bacterial plasmid in order to study its structure and function. Using restriction nuclease mapping and hybridization of 125I-rRNAs to gel separated DNA fragments we have determined that the intact rDNA is 8,140+/-50 base pairs (bp) in length. Reading from one end, the molecule consists of approximately 1,540 bp of non-coding DNA, approximately 2,100+/-50 bp that code for 19S rRNA, approximately 3,700+/-50 bp that code for 25 rTNA, and approximately 620+/-50 bp of non-coding DNDA. The 5.8S rRNA coding sequence (approximately 150 bp) occurs at one end of the 25S RNA coding region but which end is not known yet. All three rRNAs are encoded in the same strand of the DNA molecule, and transcription is in the order: 19S leads to 25S.

Animals↗
PubMed2002

Targeting mammary epithelial cells using a bacterial artificial chromosome.

We describe the generation of transgenic mouse lines expressing Cre recombinase in epithelial cells of the lactating mammary gland. As an expression vector, we used a P1-derived bacterial artificial chromosome (PAC) which harbors the gene for the secretory milk protein, whey acidic protein (Wap). Using homologous recombination in E. coli, the PAC was modified to carry the improved coding sequence of Cre recombinase (iCre). Transgenic lines carrying the WAPiCre PAC express Cre recombinase efficiently in the majority of mammary epithelial cells upon lactation. Of only four transgenic lines produced, three express Cre recombinase to a high efficiency. LoxP-flanked DNA sequences are recombined in virtually all epithelial cells of WAPiCre transgenic mice at lactation day 3.

Animals↗
PubMed1998

Phylogeny of the main bacterial 16S rRNA sequences in Drentse A grassland soils (The Netherlands).

The main bacteria in peaty, acid grassland soils in the Netherlands were investigated by ribosome isolation, temperature gradient gel electrophoresis, hybridization, cloning, and sequencing. Instead of using only 16S rDNA to determine the sequences present, we focused on rRNA to classify and quantify the most active bacteria. After direct ribosome isolation from soil, a partial amplicon of bacterial 16S rRNA was generated by reverse transcription-PCR. The sequence-specific separation by temperature gradient gel electrophoresis yielded soil-specific fingerprints, which were compared to signals from a clone library of genes coding for 16S rRNA. Cloned 16S rDNA sequences matching with intense bands in the fingerprint were sequenced. The relationships of the sequences to those of cultured organisms of known phylogeny were determined. Most of the amplicons originated from organisms closely related to Bacillus species. Such sequences were also detected by direct dot blot hybridization on soil rRNA: a probe specific for Firmicutes with low G+C content counted for about 50% of all bacterial rRNA. The bacterial activity in Drentse A grassland soil could be estimated by direct dot blot hybridization and sequencing of clones; it was found that about 65% of all the bacterial ribosomes originated from Firmicutes. The most active bacteria apparently were Bacillus species, from which about half of the sequences derived. Other sequences similar to those of gram-positive bacteria were only remotely related to known Firmicutes with a high G+C content. Other sequences were related to Proteobacteria, mainly the alpha subclass.

Bacteria↗
PubMed2002

Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis.

Current views of bacterial chromosome segregation vary in respect of the likely presence or absence of an active segregation mechanism involving a mitotic-like apparatus. Furthermore, little is known about cis-acting elements for chromosome segregation in bacteria. In this report, we show that two separate DNA regions, a 3' coding region of dnaA and the AT-rich sequence between dnaA and dnaN (the initial opening site of duplex DNA during replication), are necessary for efficient segregation of the chromosome in Bacillus subtilis. When a plasmid replicon was integrated into argG, far from oriC, on the chromosome and then the oriC function was disrupted, the oriC-deleted mutant formed anucleate cells at 5% possibly because of defects in chromosome segregation. However, when the two DNA sequences were added near oriN, frequency of anucleate cells decreased to 1%. In these cells, the origin (argG) regions were localized near cell poles, whereas they were randomly distributed in cells without the two DNA sequences. These results suggest that the two DNA sequences in and downstream of the dnaA gene participate in correct positioning of the replication origin region within the cell and that this function is associated with accurate chromosome segregation in B. subtilis.

Bacillus subtilis↗
PubMed2003

Identification and characterization of a unique adenosine kinase from Mycobacterium tuberculosis.

Adenosine kinase (AK) is a purine salvage enzyme that catalyzes the phosphorylation of adenosine to AMP. In Mycobacterium tuberculosis, AK can also catalyze the phosphorylation of the adenosine analog 2-methyladenosine (methyl-Ado), the first step in the metabolism of this compound to an active form. Purification of AK from M. tuberculosis yielded a 35-kDa protein that existed as a dimer in its native form. Adenosine (Ado) was preferred as a substrate at least 30-fold (Km = 0.8 +/- 0.08 microM) over other natural nucleosides, and substrate inhibition was observed when Ado concentrations exceeded 5 micro M. M. tuberculosis and human AKs exhibited different affinities for methyl-Ado, with Km values of 79 and 960 microM, respectively, indicating that differences exist between the substrate binding sites of these enzymes. ATP was a good phosphate donor (Km = 1100 +/- 140 microM); however, the activity levels observed with dGTP and GTP were 4.7 and 2.5 times the levels observed with ATP, respectively. M. tuberculosis AK activity was dependent on Mg2+, and activity was stimulated by potassium, as reflected by a decrease in the Km and an increase in Vmax for both Ado and methyl-Ado. The N-terminal amino acid sequence of the purified enzyme revealed complete identity with Rv2202c, a protein currently classified as a hypothetical sugar kinase. When an AK-deficient strain of M. tuberculosis (SRICK1) was transformed with this gene, it exhibited a 5,000-fold increase in AK activity compared to extracts from the original mutants. These results verified that the protein that we identified as AK was coded for by Rv2202c. AK is not commonly found in bacteria, and to the best of our knowledge, M. tuberculosis AK is the first bacterial AK to be characterized. The enzyme shows greater sequence homology with ribokinase and fructokinase than it does with other AKs. The multiple differences that exist between M. tuberculosis and human AKs may provide the molecular basis for the development of nucleoside analog compounds with selective activity against M. tuberculosis.

Adenosine↗
PubMed2004

cDNA cloning, gene structure, and expression of Broad-Complex (BR-C) genes in the silkworm, Bombyx mori.

To clarify the molecular mechanisms of metamorphosis, we analyzed the Broad-Complex (BR-C) gene in the silkworm, Bombyx mori. We cloned cDNAs for the full coding regions of the Z1, Z2, and Z4 isoforms of BR-C. The Z3 zinc finger sequence was found in the 3'UTR of the Z2 isoform. The predicted amino acid sequence showed high homology with Drosophila and Manduca BR-C proteins. Five bacterial artificial chromosome (BAC) clones were screened from a Bombyx BAC library. Restriction enzyme cleavage maps of 170 kb regions were constructed, and a total of 25.8 kb were sequenced. The BAC analysis showed that the 5'UTR of the BR-C gene consists of the first two exons, while the coding region contains a core region domain with five exons and four zinc finger exons in the order Z1, Z4, Z2, and Z3. Expression analysis revealed 9.5, 6.5, and 5.5 kb BR-C transcripts. These increased during the spinning ecdysone peak on day 6 of the fifth instar when pupal commitment occurs in the Bombyx epidermis. In addition, a small amount of BR-C mRNA was detected in the epidermis before this peak. BR-C mRNA was also expressed in the fat body from day 1 in the fourth instar to day 7 in the fifth instar.

Amino Acid Sequence↗
PubMed1992

The product of unr, the highly conserved gene upstream of N-ras, contains multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif.

We show that the open reading frame transcribed from the unr gene (immediately upstream of N-ras) in mammals consists of multiple repeats similar to the cold-shock domain (CSD), a putative DNA-binding motif found in prokaryotic cold-shock proteins, and eukaryotic DNA-binding proteins. Alignment of the CSD sequences of unr with those from other proteins reveals a core of similarity for which a consistent secondary structure prediction can be derived. This prediction suggests that the CSD consists primarily of beta-sheet, in contrast to most known eukaryotic DNA-binding proteins. Sequence analysis of the 3' end of the guinea pig unr gene shows that the core of one CSD repeat is encoded in a single exon, consistent with the modular assembly of the gene from ancestral CSD-coding units.

Amino Acid Sequence↗
PubMed1999

Genome degradation is an ongoing process in Rickettsia.

To study reductive evolutionary processes in bacterial genomes, we examine sequences in the Rickettsia genomes which are unconstrained by selection and evolve as pseudogenes, one of which is the metK gene, which codes for AdoMet synthetase. Here, we sequenced the metK gene and three surrounding genes in eight different species of the genus Rickettsia. The metK gene was found to contain a high incidence of deletions in six lineages, while the three genes in its surroundings were functionally conserved in all eight lineages. A more drastic example of gene degradation was identified in the metK downstream region, which contained an open reading frame in Rickettsia felis. Remnants of this open reading frame could be reconstructed in five additional species by eliminating sites of frameshift mutations and termination codons. A detailed examination of the two reconstructed genes revealed that deletions strongly predominate over insertions and that there is a strong transition bias for point mutations which is coupled to an excess of GC-to-AT substitutions. Since the molecular evolution of these inactive genes should reflect the rates and patterns of neutral mutations, our results strongly suggest that there is a high spontaneous rate of deletions as well as a strong mutation bias toward AT pairs in the Rickettsia genomes. This may explain the low genomic G + C content (29%), the small genome size (1.1 Mb), and the high noncoding content (24%), as well as the presence of several pseudogenes in the Rickettsia prowazekii genome.

Base Sequence↗
PubMed2000

Characterization of the ccpA gene of Enterococcus faecalis: identification of starvation-inducible proteins regulated by ccpA.

Inactivation of ccpA in Enterococcus faecalis leads to reduction of the growth rate, derepression of the galKETR operon in the presence of a mixture of glucose and galactose, and reduction of transcription of ldh in the presence of glucose. Moreover, the E. faecalis ccpA gene fully complements a Bacillus subtilis ccpA mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and the ccpA mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for L-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from Deinococcus radiodurans.

Bacillus subtilis↗
PubMed1997

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus are subjective synonyms.

The tannin-degrading species Streptococcus gallolyticus and Streptococcus caprinus have been shown to be subjective synonyms on the basis of their levels of 16S rRNA sequence similarity (98.3%) and DNA-DNA homology (> 70%) and the phenotypes of their type strains. S. gallolyticus has nomenclatural priority according to Rule 24b(2) of the International Code of Nomenclature of Bacteria.

Animals↗
PubMed1999

A polyketide synthase gene required for biosynthesis of fumonisin mycotoxins in Gibberella fujikuroi mating population A.

Fumonisins are toxins associated with several mycotoxicoses and are produced by the maize pathogen Gibberella fujikuroi mating population A (MP-A). Biochemical analyses indicate that fumonisins are a product of either polyketide or fatty acid biosynthesis. To isolate a putative polyketide synthase (PKS) gene involved in fumonisin biosynthesis, we employed PCR with degenerate PKS primers and a cDNA template prepared from a fumonisin-producing culture of G. fujikuroi. Sequence analysis of the single PCR product and its flanking DNA revealed a gene (FUM5) with a 7.8-kb coding region. The predicted FUM5 translation product was highly similar to bacterial and fungal Type I PKSs. Transformation of a cosmid clone carrying FUM5 into G. fujikuroi enhanced production in three strains and restored wild-type production in a fumonisin nonproducing mutant. Disruption of FUM5 reduced fumonisin production by over 99% in G. fujikuroi MP-A. Together, these results indicate that FUM5 is a PKS gene required for fumonisin biosynthesis.

3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗