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53-455: The lac repressor (LacI) operates by a helix-turn-helix motif in its DNA-binding domain , binding base-specifically to the major groove of the operator region of the lac operon , with base contacts also made by residues of symmetry-related alpha helices, the "hinge" helices, which bind deeply in the minor groove. This bound repressor can reduce transcription of the Lac proteins by occluding

106-420: A bout of diarrhea that is often self-limiting in healthy adults but is frequently lethal to children in the developing world. More virulent strains, such as O157:H7 , cause serious illness or death in the elderly, the very young, or the immunocompromised . The genera Escherichia and Salmonella diverged around 102 million years ago (credibility interval: 57–176 mya), an event unrelated to

159-525: A combination of free diffusion in 3D and 1D-sliding on the DNA. During sliding the repressor is in contact with the DNA helix, sliding around and tracking its major groove, which speeds up the search process by extending the target length when the TF slides in onto the operator from the side. In vivo single-molecule experiments with E. coli cells have now tested and verified the facilitated diffusion model, and shown that

212-563: A common 20–25 amino acid sequence that facilitates DNA recognition. The helix-turn-helix motif is a DNA-binding motif. The recognition and binding to DNA by helix-turn-helix proteins is done by the two α helices, one occupying the N-terminal end of the motif, the other at the C-terminus . In most cases, such as in the Cro repressor, the second helix contributes most to DNA recognition, and hence it

265-520: A laboratory setting, and has been intensively investigated for over 60 years. E. coli is a chemoheterotroph whose chemically defined medium must include a source of carbon and energy . E. coli is the most widely studied prokaryotic model organism , and an important species in the fields of biotechnology and microbiology , where it has served as the host organism for the majority of work with recombinant DNA . Under favourable conditions, it takes as little as 20 minutes to reproduce. E. coli

318-453: A short strand of amino acids , that bind to the major groove of DNA. The HTH motif occurs in many proteins that regulate gene expression . It should not be confused with the helix–loop–helix motif. The discovery of the helix-turn-helix motif was based on similarities between several genes encoding transcription regulatory proteins from bacteriophage lambda and Escherichia coli : Cro, CAP , and λ repressor , which were found to share

371-425: A single subspecies of E. coli in a phylogenomic study that included the type strain. All commonly used research strains of E. coli belong to group A and are derived mainly from Clifton's K-12 strain (λ F ; O16) and to a lesser degree from d'Herelle 's " Bacillus coli " strain (B strain; O7). There have been multiple proposals to revise the taxonomy to match phylogeny. However, all these proposals need to face

424-516: A single tetramer induces DNA looping. Each monomer has 360 amino acids, so it has 1440 amino acids in total, and 154,520 Dalton of atomic mass. LacI finds its target operator DNA surprisingly fast. In vitro the search is 10-100 times faster than the theoretical upper limit for two particles searching for each other via diffusion in three dimensions (3D). To explain the fast search, it was hypothesized that LacI and other transcription factors (TFs) find their binding sites by facilitated diffusion,

477-414: A variety of defined laboratory media, such as lysogeny broth , or any medium that contains glucose , ammonium phosphate monobasic , sodium chloride , magnesium sulfate , potassium phosphate dibasic , and water . Growth can be driven by aerobic or anaerobic respiration , using a large variety of redox pairs , including the oxidation of pyruvic acid , formic acid , hydrogen , and amino acids , and

530-519: A wide variety of substrates and uses mixed acid fermentation in anaerobic conditions, producing lactate , succinate , ethanol , acetate , and carbon dioxide . Since many pathways in mixed-acid fermentation produce hydrogen gas, these pathways require the levels of hydrogen to be low, as is the case when E. coli lives together with hydrogen-consuming organisms, such as methanogens or sulphate-reducing bacteria . In addition, E. coli ' s metabolism can be rewired to solely use CO 2 as

583-421: Is a subgroup within the species that has unique characteristics that distinguish it from other strains . These differences are often detectable only at the molecular level; however, they may result in changes to the physiology or lifecycle of the bacterium. For example, a strain may gain pathogenic capacity , the ability to use a unique carbon source , the ability to take upon a particular ecological niche , or

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636-407: Is a general process, affecting prokaryotes and eukaryotes alike. E. coli and related bacteria possess the ability to transfer DNA via bacterial conjugation or transduction , which allows genetic material to spread horizontally through an existing population. The process of transduction, which uses the bacterial virus called a bacteriophage , is where the spread of the gene encoding for

689-405: Is a gram-negative, facultative anaerobe , nonsporulating coliform bacterium . Cells are typically rod-shaped, and are about 2.0 μm long and 0.25–1.0  μm in diameter, with a cell volume of 0.6–0.7 μm . E. coli stains gram-negative because its cell wall is composed of a thin peptidoglycan layer and an outer membrane. During the staining process, E. coli picks up the color of

742-514: Is by serotype, which is based on major surface antigens (O antigen: part of lipopolysaccharide layer; H: flagellin ; K antigen : capsule), e.g. O157:H7 ). It is, however, common to cite only the serogroup , i.e. the O-antigen . At present, about 190 serogroups are known. The common laboratory strain has a mutation that prevents the formation of an O-antigen and is thus not typeable. Like all lifeforms, new strains of E. coli evolve through

795-429: Is formed by a 3-helical bundle and a 3- or 4-strand beta-sheet (wing). The topology of helices and strands in the wHTH motifs may vary. In the transcription factor ETS wHTH folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold arranged in the order α1-β1-β2-α2-α3-β3-β4 where the third helix is the DNA recognition helix . Other derivatives of the helix-turn-helix motif include

848-540: Is found in the transcriptional activator Myb . The tetra-helical helix-turn-helix motif has an additional C-terminal helix compared to the tri-helical motifs. These include the LuxR-type DNA-binding HTH domain found in bacterial transcription factors and the helix-turn-helix motif found in the TetR repressors. Multihelical versions with additional helices also occur. The winged helix-turn-helix (wHTH) motif

901-448: Is often called the "recognition helix". It binds to the major groove of DNA through a series of hydrogen bonds and various Van der Waals interactions with exposed bases . The other α helix stabilizes the interaction between protein and DNA, but does not play a particularly strong role in its recognition. The recognition helix and its preceding helix always have the same relative orientation. Several attempts have been made to classify

954-412: Is pathogenic to chickens and has an O1:K1:H7 serotype . However, in most studies, either O157:H7 , K-12 MG1655, or K-12 W3110 were used as a representative E. coli . The genome of the type strain has only lately been sequenced. Many strains belonging to this species have been isolated and characterised. In addition to serotype ( vide supra ), they can be classified according to their phylogeny , i.e.

1007-459: Is small, e.g. the O157:H7 serotype strains, which form a clade ("an exclusive group")—group E below—are all enterohaemorragic strains (EHEC), but not all EHEC strains are closely related. In fact, four different species of Shigella are nested among E. coli strains ( vide supra ), while E. albertii and E. fergusonii are outside this group. Indeed, all Shigella species were placed within

1060-421: Is targeted to one of several operator DNA sequences (known as O 1 , O 2 and O 3 ). The O 1 operator sequence slightly overlaps with the promoter, which increases the affinity of RNA polymerase for the promoter sequence such that it cannot enter elongation and remains in abortive initiation . Additionally, because each tetramer contains two DNA-binding subunits, binding of multiple operator sequences by

1113-638: Is then referred to being asynchronous. However, asynchrony can be caused by mutations to for instance DnaA or DnaA initiator-associating protein DiaA . Although E. coli reproduces by binary fission the two supposedly identical cells produced by cell division are functionally asymmetric with the old pole cell acting as an aging parent that repeatedly produces rejuvenated offspring. When exposed to an elevated stress level, damage accumulation in an old E. coli lineage may surpass its immortality threshold so that it arrests division and becomes mortal. Cellular aging

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1166-499: Is this probability to recognize the operator that changes with DNA sequence, while the time the TF remains in the bound conformation on the operator changes less with sequence. The TF often leaves the sequence it is intended to regulate, but at a strong target site, it almost always make a very short journey before finding the way back again. On the macroscopic scale, this looks like a stable interaction. This binding mechanism explains how DNA binding proteins manage to quickly search through

1219-483: The DNA and overlapping cell cycles. The number of replication forks in fast growing E. coli typically follows 2n (n = 1, 2 or 3). This only happens if replication is initiated simultaneously from all origins of replications , and is referred to as synchronous replication . However, not all cells in a culture replicate synchronously. In this case cells do not have multiples of two replication forks . Replication initiation

1272-459: The RNA polymerase binding site or by prompting DNA looping. When lactose is present, allolactose binds to the lac repressor, causing an allosteric change in its shape. In its changed state, the lac repressor is unable to bind tightly to its cognate operator. Thus, the gene is mostly off in the absence of inducer and mostly on in the presence of inducer, although the degree of gene expression depends on

1325-654: The Shiga toxin from the Shigella bacteria to E. coli helped produce E. coli O157:H7 , the Shiga toxin-producing strain of E. coli. E. coli encompasses an enormous population of bacteria that exhibit a very high degree of both genetic and phenotypic diversity. Genome sequencing of many isolates of E. coli and related bacteria shows that a taxonomic reclassification would be desirable. However, this has not been done, largely due to its medical importance, and E. coli remains one of

1378-503: The DNA-binding domain found in MarR , a regulator of multiple antibiotic resistance , which forms a winged helix-turn-helix with an additional C-terminal alpha helix. Escherichia coli Escherichia coli ( / ˌ ɛ ʃ ə ˈ r ɪ k i ə ˈ k oʊ l aɪ / ESH -ə- RIK -ee-ə KOH -lye ) is a gram-negative , facultative anaerobic , rod-shaped , coliform bacterium of

1431-556: The EDP is the more thermodynamically favourable of the three pathways, E. coli do not use the EDP for glucose metabolism , relying mainly on the EMPP and the OPPP. The EDP mainly remains inactive except for during growth with gluconate . When growing in the presence of a mixture of sugars, bacteria will often consume the sugars sequentially through a process known as catabolite repression. By repressing

1484-515: The TF scans on average 45 bp during each sliding event, before the TF detaches spontaneously and resumes exploring the genome in 3D. These experiments also suggest that LacI slides over the O 1 operator several times before binding, meaning that different DNA sequences can have different probabilities to be recognized at each encounter with the TF. This implies a trade-off between fast search on nonspecific sequences and binding to specific sequences. In vivo and in vitro experiments have shown that it

1537-484: The ability to resist antimicrobial agents . Different strains of E. coli are often host-specific, making it possible to determine the source of fecal contamination in environmental samples. For example, knowing which E. coli strains are present in a water sample allows researchers to make assumptions about whether the contamination originated from a human, another mammal , or a bird . A common subdivision system of E. coli , but not based on evolutionary relatedness,

1590-412: The absence of oxygen is an advantage to bacteria because their survival is increased in environments where water predominates. The bacterial cell cycle is divided into three stages. The B period occurs between the completion of cell division and the beginning of DNA replication . The C period encompasses the time it takes to replicate the chromosomal DNA. The D period refers to the stage between

1643-425: The bacterium cause disease. Cells are able to survive outside the body for a limited amount of time, which makes them potential indicator organisms to test environmental samples for fecal contamination . A growing body of research, though, has examined environmentally persistent E. coli which can survive for many days and grow outside a host. The bacterium can be grown and cultured easily and inexpensively in

Lac repressor - Misplaced Pages Continue

1696-631: The bacterium on glucose and lactose , where E. coli will consume glucose before lactose . Catabolite repression has also been observed in E. coli in the presence of other non-glucose sugars, such as arabinose and xylose , sorbitol , rhamnose , and ribose . In E. coli , glucose catabolite repression is regulated by the phosphotransferase system , a multi-protein phosphorylation cascade that couples glucose uptake and metabolism . Optimum growth of E. coli occurs at 37 °C (99 °F), but some laboratory strains can multiply at temperatures up to 49 °C (120 °F). E. coli grows in

1749-431: The conclusion of DNA replication and the end of cell division. The doubling rate of E. coli is higher when more nutrients are available. However, the length of the C and D periods do not change, even when the doubling time becomes less than the sum of the C and D periods. At the fastest growth rates, replication begins before the previous round of replication has completed, resulting in multiple replication forks along

1802-409: The counterstain safranin and stains pink. The outer membrane surrounding the cell wall provides a barrier to certain antibiotics such that E. coli is not damaged by penicillin . The flagella which allow the bacteria to swim have a peritrichous arrangement . It also attaches and effaces to the microvilli of the intestines via an adhesion molecule known as intimin . E. coli can live on

1855-411: The expression of the genes involved in metabolizing the less preferred sugars, cells will usually first consume the sugar yielding the highest growth rate, followed by the sugar yielding the next highest growth rate, and so on. In doing so the cells ensure that their limited metabolic resources are being used to maximize the rate of growth. The well-used example of this with E. coli involves the growth of

1908-433: The genome in E. coli cells less accessible for the repressor. The existence of hopping, where the protein slips out of the major groove of DNA to land in another nearby groove along the DNA chain, has been proven more directly in vitro , where the lac repressor has been observed to bypass operators, flip orientation, and rotate with a longer pitch than the 10.5 bp period of DNA while moving along it. The lac repressor

1961-494: The genome of the cell without getting stuck too long at sequences that resemble the true target. An all-atom molecular dynamics simulation suggests that the transcription factor encounters a barrier of 1 k B T during sliding and 12 k B T for dissociation, implying that the repressor will slide over 8 bp on average before dissociating. The in vivo search model for the lac repressor includes intersegment transfer and hopping as well as crowding by other proteins which make

2014-442: The genus Enterobacter + "i" (sic.) + " aceae ", but from "enterobacterium" + "aceae" (enterobacterium being not a genus, but an alternative trivial name to enteric bacterium). The original strain described by Escherich is believed to be lost, consequently a new type strain (neotype) was chosen as a representative: the neotype strain is U5/41 , also known under the deposit names DSM 30083 , ATCC 11775 , and NCTC 9001, which

2067-406: The genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms. Most E. coli strains are harmless, but some serotypes such as EPEC and ETEC are pathogenic, can cause serious food poisoning in their hosts and are occasionally responsible for food contamination incidents that prompt product recalls. Most strains are part of the normal microbiota of

2120-433: The gut and are harmless or even beneficial to humans (although these strains tend to be less studied than the pathogenic ones ). For example, some strains of E. coli benefit their hosts by producing vitamin K 2 or by preventing the colonization of the intestine by pathogenic bacteria . These mutually beneficial relationships between E. coli and humans are a type of mutualistic biological relationship — where both

2173-403: The helix-turn-helix motifs based on their structure and the spatial arrangement of their helices. Some of the main types are described below. The di-helical helix-turn-helix motif is the simplest helix-turn-helix motif. A fragment of Engrailed homeodomain encompassing only the two helices and the turn was found to be an ultrafast independently folding protein domain. An example of this motif

Lac repressor - Misplaced Pages Continue

2226-434: The humans and the E. coli are benefitting each other. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for three days, but its numbers decline slowly afterwards. E. coli and other facultative anaerobes constitute about 0.1% of gut microbiota , and fecal–oral transmission is the major route through which pathogenic strains of

2279-404: The inferred evolutionary history, as shown below where the species is divided into six groups as of 2014. Particularly the use of whole genome sequences yields highly supported phylogenies. The phylogroup structure remains robust to newer methods and sequences, which sometimes adds newer groups, giving 8 or 14 as of 2023. The link between phylogenetic distance ("relatedness") and pathology

2332-465: The laboratory. For instance, E. coli typically do not have the ability to grow aerobically with citrate as a carbon source , which is used as a diagnostic criterion with which to differentiate E. coli from other, closely, related bacteria such as Salmonella . In this experiment, one population of E. coli unexpectedly evolved the ability to aerobically metabolize citrate , a major evolutionary shift with some hallmarks of microbial speciation . In

2385-547: The microbial world, a relationship of predation can be established similar to that observed in the animal world. Considered, it has been seen that E. coli is the prey of multiple generalist predators, such as Myxococcus xanthus . In this predator-prey relationship, a parallel evolution of both species is observed through genomic and phenotypic modifications, in the case of E. coli the modifications are modified in two aspects involved in their virulence such as mucoid production (excessive production of exoplasmic acid alginate ) and

2438-578: The most diverse bacterial species: only 20% of the genes in a typical E. coli genome is shared among all strains. In fact, from the more constructive point of view, the members of genus Shigella ( S. dysenteriae , S. flexneri , S. boydii , and S. sonnei ) should be classified as E. coli strains, a phenomenon termed taxa in disguise . Similarly, other strains of E. coli (e.g. the K-12 strain commonly used in recombinant DNA work) are sufficiently different that they would merit reclassification. A strain

2491-558: The much earlier (see Synapsid ) divergence of their hosts: the former being found in mammals and the latter in birds and reptiles. This was followed by a split of an Escherichia ancestor into five species ( E. albertii , E. coli , E. fergusonii , E. hermannii , and E. vulneris ). The last E. coli ancestor split between 20 and 30 million years ago. The long-term evolution experiments using E. coli , begun by Richard Lenski in 1988, have allowed direct observation of genome evolution over more than 65,000 generations in

2544-498: The natural biological processes of mutation , gene duplication , and horizontal gene transfer ; in particular, 18% of the genome of the laboratory strain MG1655 was horizontally acquired since the divergence from Salmonella . E. coli K-12 and E. coli B strains are the most frequently used varieties for laboratory purposes. Some strains develop traits that can be harmful to a host animal. These virulent strains typically cause

2597-560: The number of repressors in the cell and on the repressor's DNA-binding affinity. Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a commonly used allolactose mimic which can be used to induce transcription of genes being regulated by lac repressor. Structurally, the lac repressor protein is a homotetramer . More precisely, the tetramer contains two DNA-binding subunits composed of two monomers each (a dimer of dimers). Each monomer consists of four distinct regions: DNA binding occurs via an N-terminal helix-turn-helix structural motif and

2650-428: The reduction of substrates such as oxygen , nitrate , fumarate , dimethyl sulfoxide , and trimethylamine N-oxide . E. coli is classified as a facultative anaerobe . It uses oxygen when it is present and available. It can, however, continue to grow in the absence of oxygen using fermentation or anaerobic respiration . Respiration type is managed in part by the arc system . The ability to continue growing in

2703-751: The source of carbon for biomass production. In other words, this obligate heterotroph's metabolism can be altered to display autotrophic capabilities by heterologously expressing carbon fixation genes as well as formate dehydrogenase and conducting laboratory evolution experiments. This may be done by using formate to reduce electron carriers and supply the ATP required in anabolic pathways inside of these synthetic autotrophs. E. coli has three native glycolytic pathways: EMPP , EDP , and OPPP . The EMPP employs ten enzymatic steps to yield two pyruvates , two ATP , and two NADH per glucose molecule while OPPP serves as an oxidation route for NADPH synthesis. Although

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2756-519: The suppression of the OmpT gene, producing in future generations a better adaptation of one of the species that is counteracted by the evolution of the other, following a co-evolutionary model demonstrated by the Red Queen hypothesis . E. coli is the type species of the genus ( Escherichia ) and in turn Escherichia is the type genus of the family Enterobacteriaceae , where the family name does not stem from

2809-453: Was first isolated by Walter Gilbert and Benno Müller-Hill in 1966. They showed that in vitro the protein bound to DNA containing the lac operon, and it released the DNA when IPTG (an analog of allolactose) was added. Helix-turn-helix Helix-turn-helix is a DNA-binding domain (DBD). The helix-turn-helix ( HTH ) is a major structural motif capable of binding DNA . Each monomer incorporates two α helices , joined by

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