-
Proceedings of the National Academy of... Jun 2000We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted...
We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells.
Topics: Chromosomes, Bacterial; DNA Nucleotidyltransferases; Escherichia coli; Integrases; Lac Operon; Mutation; Operon; Plasmids; Polymerase Chain Reaction; Recombinases; Recombination, Genetic
PubMed: 10829079
DOI: 10.1073/pnas.120163297 -
FEMS Microbiology Reviews Sep 2010This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operon's... (Review)
Review
This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiological states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.
Topics: Allosteric Site; Amino Acid Sequence; AraC Transcription Factor; Arabinose; DNA, Bacterial; Escherichia coli; Escherichia coli Proteins; Fluorescence; Gene Expression Regulation, Bacterial; Models, Molecular; Molecular Sequence Data; Operon; Protein Binding; Protein Multimerization; Protein Structure, Tertiary; Sequence Alignment; Trans-Activators
PubMed: 20491933
DOI: 10.1111/j.1574-6976.2010.00226.x -
Cellular and Molecular Life Sciences :... Dec 2009Operons (clusters of co-regulated genes with related functions) are common features of bacterial genomes. More recently, functional gene clustering has been reported in... (Review)
Review
Operons (clusters of co-regulated genes with related functions) are common features of bacterial genomes. More recently, functional gene clustering has been reported in eukaryotes, from yeasts to filamentous fungi, plants, and animals. Gene clusters can consist of paralogous genes that have most likely arisen by gene duplication. However, there are now many examples of eukaryotic gene clusters that contain functionally related but non-homologous genes and that represent functional gene organizations with operon-like features (physical clustering and co-regulation). These include gene clusters for use of different carbon and nitrogen sources in yeasts, for production of antibiotics, toxins, and virulence determinants in filamentous fungi, for production of defense compounds in plants, and for innate and adaptive immunity in animals (the major histocompatibility locus). The aim of this article is to review features of functional gene clusters in prokaryotes and eukaryotes and the significance of clustering for effective function.
Topics: Animals; Eukaryota; Fungi; Genome; Models, Genetic; Multigene Family; Operon; Plants
PubMed: 19662496
DOI: 10.1007/s00018-009-0114-3 -
WormBook : the Online Review of C.... Apr 2015Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where... (Review)
Review
Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.
Topics: Animals; Caenorhabditis elegans; Genes, Helminth; Multigene Family; Operon
PubMed: 25936768
DOI: 10.1895/wormbook.1.175.1 -
Cold Spring Harbor Perspectives in... Jan 2016The cytoplasm of prokaryotes contains many molecular machines interacting directly with the chromosome. These vital interactions depend on the chromosome structure, as a... (Review)
Review
The cytoplasm of prokaryotes contains many molecular machines interacting directly with the chromosome. These vital interactions depend on the chromosome structure, as a molecule, and on the genome organization, as a unit of genetic information. Strong selection for the organization of the genetic elements implicated in these interactions drives replicon ploidy, gene distribution, operon conservation, and the formation of replication-associated traits. The genomes of prokaryotes are also very plastic with high rates of horizontal gene transfer and gene loss. The evolutionary conflicts between plasticity and organization lead to the formation of regions with high genetic diversity whose impact on chromosome structure is poorly understood. Prokaryotic genomes are remarkable documents of natural history because they carry the imprint of all of these selective and mutational forces. Their study allows a better understanding of molecular mechanisms, their impact on microbial evolution, and how they can be tinkered in synthetic biology.
Topics: Biological Evolution; Chromosome Segregation; Chromosomes; DNA Replication; Escherichia coli; Gene Transfer, Horizontal; Genome; Genome Size; Models, Genetic; Operon; Ploidies; Prokaryotic Cells; Recombination, Genetic
PubMed: 26729648
DOI: 10.1101/cshperspect.a018168 -
PloS One 2022Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas...
Advanced periodontitis has been shown to have strong association with the residence of the bacterial consortia known as the red complex comprised by Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola. T. forsythia shares a distant genetic linkage to Bacteroidetes thetaiotaomicron and may therefore produce analogous polysaccharide utilization loci (PUL) which enable complex carbohydrate degradation, import, and use, although this capacity has yet to be demonstrated. Chondroitin sulfate A is a linear, sulfated carbohydrate linked to periodontal disease as the principal species of glycosaminoglycan appended on the surface of cortical bone of teeth and in supporting dental ligaments. Through genomic comparisons with B. thetaiotaomicron, a new PUL-like operon (Bfo2285-Bfo2295, and Bfo3043) was identified in T. forsythia and the crystal structure of two proteins from this PUL-like operon, Bfo2290 and Bfo2294, were reported using X-ray crystallography. Enzyme kinetics for Bfo2290 were reported using a pH-dependent assay and suggested a Km of 0.75 mg/ml ± 0.60 mg/ml, Kcat of 3.74 min-1 ± 0.88 min-1, and Vmax of 7.48 μM/min ± 1.76 μM/min with partially degraded chondroitin sulfate A. Fluorophore-assisted carbohydrate electrophoresis was used to show the processive degradation of chondroitin sulfate A by the proteins encoded in T. forsythia PUL-like operon, and revealed Bfo2291 and Bfo2290 to be an endolytic chondroitin sulfate A lyase and exolytic ΔDi-4S chondroitin sulfate A sulfatase, respectively.
Topics: Chondroitin Sulfates; Lyases; Operon; Sulfatases; Tannerella forsythia
PubMed: 36112631
DOI: 10.1371/journal.pone.0272904 -
MSystems Dec 2022Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize...
Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their host's CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often colocalize to form operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can (i) predict Acas and their associated operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multistep filtering, 36 high-confident new Aca families were identified, which is three times that of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (, , and ) and their phages, while most known Aca families were from and . A complex association network between Acrs and Acas was revealed by analyzing their operonic colocalizations. It appears very common in evolution that the same genes can recombine with different genes and to form diverse operon combinations. At least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of operons, Aca can be viewed as anti-anti-CRISPRs, with great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved helix-turn-helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex operonic colocalization network between different families of Acrs and Acas.
Topics: Humans; CRISPR-Cas Systems; Bacteria; Bacteriophages; Operon; Prophages
PubMed: 36413017
DOI: 10.1128/msystems.00817-22 -
Molekuliarnaia Biologiia 2018The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past... (Review)
Review
The origin of bioluminescence in living organisms was first mentioned by Charles Darwin (1859) and remains obscure despite significant success achieved over the past decades. Here we discuss the mechanisms of bacterial bioluminescence. We have the main results from structural and functional analysis of the genes of lux operons, enzymes (luciferase), and mechanisms of bioluminescence in several species of marine bacteria, which belong to three genera, Vibrio, Aliivibrio, and Photobacterium (A. fischeri, V. harveyi, P. leiognathi, and P. phosphoreum), and in terrestrial bacteria of the genus Photorhabdus (Ph. luminescens). The structure and mechanisms for the regulation of the expression of the lux operons are discussed. The fundamental characteristics of luciferase and luciferase-catalyzed reactions (stages of FMNH2 and tetradecanal oxidation, dimensional structure, as well as folding and refolding of the macromolecule) are described. We also discuss the main concepts of the origin of bacterial bioluminescence and its role in the ecology of modern marine fauna, including its involvement in the processes of detoxification of the reactive oxygen species and DNA repair, as well as the bait hypothesis.
Topics: Aliivibrio; Bacterial Proteins; DNA, Bacterial; Genes, Bacterial; Luciferases; Luminescence; Operon; Photobacterium; Vibrio
PubMed: 30633237
DOI: 10.1134/S0026898418060186 -
WormBook : the Online Review of C.... Jun 2005About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the... (Review)
Review
About 70% of C. elegans mRNAs are trans-spliced to one of two 22 nucleotide spliced leaders. SL1 is used to trim off the 5' ends of pre-mRNAs and replace them with the SL1 sequence. This processing event is very closely related to cis-splicing, or intron removal. The SL1 sequence is donated by a 100 nt small nuclear ribonucleoprotein particle (snRNP). This snRNP is structurally and functionally related to the U snRNAs (U1, U2, U4, U5 and U6) that play key roles in intron removal and trans-splicing, except that it is consumed in the process of splicing. More than half of C. elegans pre-mRNAs are subject to SL1 trans-splicing. About 30% are not trans-spliced at all. The remaining genes are trans-spliced by SL2. These genes are all downstream genes in closely spaced gene clusters similar to bacterial operons. They are transcribed from a promoter at the 5' end of the cluster of between 2 and 8 genes. This transcription makes a polycistronic pre-mRNA that is co-transcriptionally processed by cleavage and polyadenylation at the 3' end of each gene, and this event is closely coupled to the SL2 trans-splicing event that occurs only approximately 100 nt further downstream. Recent studies on the mechanism of SL2 trans-splicing have revealed that one of the 3' end formation proteins, CstF, interacts with the only protein known to be specific to the SL2 snRNP. The operons contain primarily genes whose products are needed for mitochondrial function and the basic machinery of gene expression: transcription, splicing and translation. Many operons contain genes whose products are known to function together. This presumably provides co-regulation of these proteins by producing a single RNA that encodes both.
Topics: Animals; Caenorhabditis elegans; Evolution, Molecular; Operon; RNA, Helminth; Ribonucleoproteins, Small Nuclear; Trans-Splicing
PubMed: 18050426
DOI: 10.1895/wormbook.1.5.1 -
FEMS Microbiology Reviews Apr 1997Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have... (Review)
Review
Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistance to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.
Topics: Biological Evolution; DNA Transposable Elements; Drug Resistance, Microbial; Environmental Microbiology; Gene Transfer, Horizontal; Gram-Negative Bacteria; Gram-Positive Bacteria; Mercury; Operon; Oxidation-Reduction; Species Specificity
PubMed: 9167257
DOI: 10.1111/j.1574-6976.1997.tb00300.x