E. coli (electron micrograph)
|( Migula 1895) Castellani & Chalmers 1919|
Escherichia coli (abbreviated E. coli ) - also Kolibakterium called - is a gram-negative , acidifying and peritrich begeißeltes bacterium that normally in the human and animal intestine occurs. For this reason, among other things, this non-enteric bacterium is also considered a faecal indicator . E. coli and other facultative anaerobic organisms make up about 1 ‰ of the intestinal flora .
Within the enterobacteria family ( ancient Greek ἕντερον , Latin enteron "intestine") E. coli belongs to the important genus Escherichia and is its type species . It was named after the German pediatrician Theodor Escherich , who first described it. Coli is the Latin genitive of colon (to English colon ), a part of the large intestine .
E. coli is known as a vitamin producer, especially vitamin K , in the human intestinal flora . Most of these species are not disease-causing, but there are also many different pathogenic strains. It is one of the most common causes of human infectious diseases. The base sequence of the genome of some strains has been fully elucidated. As a model organism, it is one of the best-studied prokaryotes and plays an important role as a host organism in molecular biology. The Nobel Prize in Physiology or Medicine was awarded to numerous researchers who have studied the biology of E. coli .
E. coli is in the form of straight, cylindrical rods with rounded ends. The diameter is 1.1–1.5 µm and the length 2.0–6.0 µm. They come in pairs or individually. In the Gram stain they behave negatively ( Gram- negative). It does not form bacterial spores . The cells consist predominantly (70-85%) of water, with 96% of the dry matter consisting of polymers , among which the proteins dominate. There are 4288 different proteins annotated . In the cytoplasm as well as in the cell envelope (consisting of cell membrane , periplasm , outer membrane ) they fulfill structural , enzymatic and regulatory functions. The genome spans about 4600 kilobase pairs and occurs as a covalently self-contained bacterial chromosome .
Many tribes have fimbriae ( pili ). A cell of strain K-12 typically contains about 100-500 type 1 fimbriae 0.2-2.0 µm in length and about 7 nm in diameter. There are more than 30 different types of fimbriae, which are divided into two are classified according to their adhesive properties on red blood cells : MS (mannose-sensitive), which cannot clump red blood cells in the presence of mannose ( haemagglutination ) and MR (mannose-resistant), which do not mind the presence of sugar. Type 1 fimbriae, which belong to the MS fimbriae, occur in both symbiotic and pathogenic strains and are therefore not used for differentiation. MR fimbriae are serologically diverse and often act as virulence factors. Their attachment is both species and organ specific. In addition, E. coli also forms a sex pilus (also F-pilus , F for fertility ) with which cell-cell contacts for the exchange of genetic information ( conjugation ) are possible. In addition, the F-Pilus also serves as a receptor for some bacteriophages, after which the virus DNA is introduced ( transduction ).
E. coli cells can actively move (they are motile ) through peritrichal flagella or - less often - they are incapable of active movement. Motile E. coli move with their proteinaceous flagellum , repeatedly changing direction: A bacterium moves in one direction by bundling the flagella and working together. The locomotion is temporarily interrupted by staggering, as the flagella bundle dissolves and the individual flagella turn in different directions. After that, the flagella bundle reforms and accelerates the bacterium in a new direction. The stability of the bundle is enhanced by chemoreceptors . If the bacteria are offered a nutrient, the stability of the flagella bundle is further strengthened and the bacteria accumulate.
E. coli is chemotactic : if individuals swim in a concentration gradient of an attractant in the direction of increasing concentration, they change direction less frequently. If they swim down a concentration gradient, their movement pattern cannot be distinguished from that in an isotropic solution, and they change direction more frequently. In addition to positive chemotaxis , E. coli can also actively remove itself from pollutants (negative chemotaxis), with low concentrations of pollutants not being attractants and high concentrations of nutrients not being repulsive. There are mutants who do not recognize certain pollutants and non-chemotactic mutants who cannot recognize attractants either. The process requires L - methionine .
The signal transduction for accurate chemotactic responses has been in evolution for optimal performance with minimal protein expression developed. Due to the high selection pressure, chemotaxis in E. coli is very sensitive, has a quick response and is perfectly adapted. In addition, the arrangement within the bacterial chemosensory system seems to be highly conserved.
E. coli has transport proteins in the cell membrane for the exchange of substances . Outer membrane proteins OmpF and OmpC dominate among the porins , although they are not substrate-specific , but prefer cationic and neutral ions and do not accept hydrophobic compounds. The number of copies depends on the osmolarity of the environment and is used to adapt to the habitat. Under the conditions in the large intestine ( hyperosmolarity , higher temperature), OmpC channels predominate. If the bacterium leaves its host and finds itself in a less preferred habitat, e.g. B. a body of water (lower osmolarity and temperature) so the OmpF synthesis is promoted. For substrates that are not transported at all or inadequately by the unspecific porins, there are substrate-specific porins. In the case of phosphate deficiency , E. coli expresses the protein PhoE. Together with maltodextrins , this results in maltoporins, which also function as receptors for the lambda phage and are therefore also called LamB. Strains that can utilize sucrose take it up via the channel protein ScrY. Long-chain fatty acids are transported into the cell with FadL.
E. coli is heterotrophic , facultative anaerobic and has the ability to gain energy both through the respiratory chain and through " mixed acid fermentation ". The fermentation balance in E. coli looks like this:
Glucose is fermented by E. coli with the formation of acid, which can be detected with methyl red as a pH indicator . In addition to acid, E. coli also forms gas from glucose . The indole test for tryptophanase is positive. The Voges-Proskauer reaction for the detection of acetoin formation is negative. No discoloration is visible on Simmons Citrate Agar , as E. coli cannot use citrate as the sole source of energy. In addition, it can not utilize malonate . Acetate and tartrate can be metabolized (test with methyl red according to Jordan). Nitrate can be reduced to nitrite . No hydrogen sulfide is formed on Triple Sugar Iron Agar . E. coli cannot hydrolyze urea or gelatin , but some strains can hydrolyze esculin . Lysine is decarboxylated by many strains , ornithine only by a few. In potassium cyanide growth assay is growing E. coli not. It has no phenylalanine deaminase , no lipase and no DNase in the narrower sense. The oxidase test with Kovacs reagent is always negative. Furthermore, L - arabinose , lactose , maltose , D - mannitol , D - mannose , mucic acid , D - sorbitol , trehalose and D - xylose can be fermentatively used by most of the strains .
Serotyping is a useful way of classifying E. coli based on the numerous differences in the antigen structure on the bacterial surface.
There are four groups of serotypes :
- flagellar H antigens for the flagella derived from "growing with breath bacteria", since they generate by their active transport on an agar plate a matt crimp pattern that looks like a tinged glass plate. They are protein antigens.
- somatic O antigens, derived from “without a touch” for the lipopolysaccharides that are on the surface of the cell wall. Their specificity is determined by carbohydrate side chains. About 190 different O antigens are currently known.
Are rarely used for diagnostics:
- K antigens for the capsule consisting of polysaccharides are constructed
- fimbrial F antigens for the fimbriae
E. coli occurs as a universal and commensal companion in the lower intestinal tract of warm-blooded animals (including humans). There are around 10 8 -10 9 colony-forming units per g in the stool . It can survive in other habitats as well . In newborns, it plays an important role as the first colonizer. Although it is only available in small numbers, it is used to settle obligate anaerobes , which have physiological importance in digestion . Despite its small percentage in the intestine, E. coli occupies a dominant position in the intestine, which it colonizes in humans within 40 hours of birth via food, water or other individuals. The ability to adhere to the mucus allows E. coli to reside in the intestine for a long time. Although a great deal is known about the organism, relatively little is known about its ecology in the intestine.
Sporadic outbreaks of enterotoxic strains (ETEC) transmitted through drinking water are known. In addition, ETEC is transmitted through consumption of soft cheese and raw vegetables . Enteropathogenic strains (EPEC) outbreaks are often associated with contaminated drinking water and some meat products . Enterohaemorrhagic E. coli (EHEC) infections often come from food or occur through water. Frequently infected foods are undercooked ground beef , raw milk , cold sandwiches , water, unpasteurized apple juice , sprouts and raw vegetables. There were also epidemics in connection with hamburgers , roast beef , cabbage rolls and raw sausage ( tea sausage ).
The gastric acid-resistant strain Escherichia coli O157: H7 (EHEC), which is harmless to cattle, can be detected in 1–2% of cattle faeces, which can also contaminate meat during slaughter and cause severe food poisoning in humans. The reason for this is that often starchy grain is fed, which is incompletely broken down in the rumen and fermented to acid, so that acidophilic bacteria accumulate there. Feeding hay or grass reduces the number of human pathogenic strains.
Since a complete EHEC remediation of the livestock is not possible, prophylaxis must start with slaughter hygiene. Beef products should be cooked through for at least 10 minutes at a minimum of 70 ° C. Due to the high level of environmental resistance of the pathogen, food manufacturers should carry out exposure tests and HACCP analyzes. Risk groups (children under 6 years of age and immunocompromised people) should not consume raw products.
According to the EU Bathing Water Ordinance of 2008, the following limit values apply to E.coli:
- "Excellent quality" up to 500/100 ml
- "Good quality" up to 1000/100 ml
In the case of drinking water , on the other hand, a restrictive limit of 0/100 ml applies.
The strain Escherichia coli Alfred Nissle 1917 (trade name Mutaflor) is one of the most frequently examined probiotics . During the Balkan War he was isolated from the chair of a soldier who, unlike his comrades, did not suffer from diarrhea. The tribe has now been sequenced and has six different systems for picking up iron, knocking out competitors. It has adhesins for effective colonization and blocks the attachment and penetration of pathogenic bacteria to the epithelial cells of the intestine. It also has an anti-inflammatory effect on T cells - proliferation . It also stimulates the production of human β-defensin 2 , which, as a broad-spectrum antibiotic, kills both gram-positive and -negative bacteria, fungi and viruses.
Most E. coli strains are non-pathogenic and therefore harmless. However, some serotypes play an important role in diseases inside and outside the gut . E. coli is an opportunistic pathogen in hosts with immunodeficiency , which means that it can only become effective when the host is weakened. Uropathogenic E. coli (UPEC) are responsible for uncomplicated urinary tract infections . E. coli (NMEC) causing neonatal meningitis can cross the blood-brain barrier and cause meningitis in newborns. NMEC and UPEC cause sepsis in the bloodstream .
It is assumed that E. coli is associated with chronic inflammatory bowel diseases such as Crohn's disease and ulcerative colitis , since, in addition to genetic predisposition and environmental factors, a dysregulated immune response of the mucous membrane against commensal bacteria could also be involved in the development of the disease . The patient's mucosa is abnormally colonized with adherent invasive E. coli (AIEC) which attach to and invade the epithelial cells.
The intestinal pathogenic E. coli are divided into five different patho groups. Worldwide, they cause 160 million diarrheal diseases and 1 million deaths every year. In most cases, children under 5 are affected in developing countries.
Enteropathogenic E. coli
Enteropathogenic E. coli (EPEC for short) cause severe diarrhea in young children, which is rare in industrialized societies, but is often responsible for child deaths in underdeveloped countries. With the help of the EPEC Adhesion Factor (EAF), the EPEC attach to the epithelial cells of the small intestine and then inject toxins into the enterocytes using a type III secretion system . There are also so-called atypical EPECs. They show the serotypes common at STEC as well as virulence and pathogenicity factors such as the eae gene. However , they have probably lost the Stx prophage and the associated stx genes that are characteristic of STEC .
Enterotoxic E. coli
Enterotoxic E. coli (ETEC for short) are more common pathogens of travelers' diarrhea ("Montezuma's revenge"). The reason for this disease is a heat-labile enterotoxin of the A / B type (LT I and LT II) and a heat-stable enterotoxin (ST). This 73 kDa protein has two domains , one of which was a G to ganglioside of the target cell binding ( B indende domain). The other domain is the A ctive component similar to the cholera toxin (approximately 80% gene homology) the adenylate cyclase activated. The approximately 15-20 amino acid long ST activates guanylate cyclase. The activation of adenylate cyclase and guanylate cyclase ends in secretory diarrhea in which a lot of water and electrolytes are lost. The bacterium receives the genetic information from a lysogenic phage through transduction .
Enteroinvasive E. coli
Enteroinvasive E. coli (EIEC for short) penetrate the epithelial cells of the colon and multiply there. Actin tails form within the cell, which, like Listeria and Shigella, penetrate into neighboring epithelial cells. Inflammation and ulceration develop, with blood, mucus and white blood cells ( granulocytes ) being secreted . In addition, EIEC can release enterotoxins that lead to electrolyte and water loss. The clinical picture resembles a bacterial dysentery with fever and bloody-slimy diarrhea, whereby weakened symptoms are often accompanied by watery diarrhea.
Enterohemorrhagic E. coli
Enterohaemorrhagic E. coli (EHEC for short) are Shiga toxin- producing E. coli (STEC) with additional pathogenicity factors . Shigatoxin has an enterotoxic and cytotoxic effect and shows similarities to the toxin produced by Shigella . VTEC ( verotoxin producing E. coli ) are named analogously . Intestinal diseases caused by EHEC are primarily known as enterohaemorrhagic colitis . EHEC infections are one of the most common causes of food poisoning . The pathogen is highly infectious: 10-100 individuals are sufficient for one disease. The low infectious dose favors transmission from person to person. However, infection can also occur through contact with animals ( zoonosis ) or through swallowing bath water. Typical clinical pictures are thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). HUS is particularly feared because of the possibility of death from terminal kidney damage. All age groups are affected, but especially children under 6 years of age. The kidney failure runs in 10-30% of cases with the patient's death within one year after the onset of the disease.
Enteroaggregative E. coli
Enteroaggregative E. coli (EAggEC or EAEC abbreviated) have the ability to auto-aggregate. They attach themselves to the small intestinal epithelium with specific fimbriae . Characteristic is the increased production of mucus by the mucosal cells , which delays excretion. Secretory-type diarrhea due to enterotoxins (EAST) occurs. EAEC causes both acute and chronic recurrent diarrheal diseases that can drag on for weeks. In addition to watery, slimy diarrhea, fever and vomiting or bloody stools can also occur. In immunocompromised (. Eg HIV -patients) is the most common pathogen EAEC a bacterial enteritis .
E. coli is responsible for a wide variety of infectious diseases in animals. Specific veterinary clinical pictures are:
- Coliruhr the suckling and weaning pig
- Colisepticemia of the calves and lambs
- Colisepticemia in poultry
- Colimastitis in cows
Coliform bacteria are used as an indicator of the sanitary water quality and hygiene in food processing. Faecal coliforms are the standard indicator of contamination , especially in shellfish . E. coli indicates faecal contamination as well as unsanitary processing. Classically, the biochemical methods for the detection of E. coli , total coliforms or fecal coliforms are based on the utilization of lactose . Counting is possible using the MPN method . Common is a brilliant green - bile lactose broth in which gas formation is observed. However, there are also special culture media such as eosin methylene blue , VRB agar ( crystal violet - neutral red gall agar), MacConkey agar and endo agar that indicate lactose utilization. The IMViC test can be performed to differentiate from other enterobacteria .
Pathogenic E. coli are also first enriched. The ETEC heat-labile enterotoxin (LT) can be detected by a Y-1 adrenal cell assay, latex agglutination assay and ELISA . The heat-stable toxin (ST) of the ETEC can also be detected using ELISA or the young mouse assay. The genes for LT and ST are known and can be detected using PCR or using a gene probe . In connection with plating on agar culture media, ETEC-positive colonies can be counted. EIEC are non-motile and anaerogenic in that they do not decarboxylate lysine and do not ferment lactose. The invasive phenotype of EIEC, which is encoded by a high molecular weight plasmid, can be detected using HeLa cells or Hep-2 tissue cell cultures. Alternatively, PCR and probe methods can be used for the invasion genes. The EPEC Intimin protein is encoded by an eae gene that can be tested for with PCR. Furthermore, the EPEC adherent factor (EAF) is encoded using a plasmid. The protein can be detected with Hep-2 cells. EHEC can be detected via the Shiga toxins (Stx). In particular, Stx1 and Stx2 are associated with human diseases, with numerous variants of Stx2 existing. The production of Stx1 and Stx2 can be done with cytotoxicity tests on Vero cells or Hela tissue cultures as well as with ELISA and latex aggulation tests. There are also PCR assays for Stx1, Stx2 or other characteristic markers. EHEC are also characterized by no or slow fermentation of sorbitol. Another biochemical differentiation is the LST-MUG assay, which is based on the enzymatic activity of β-glucuronidase (GUD). GUD converts the substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) into 4-methylumbelliferone , which shows a blue fluorescence at 365 nm (UV light) . GUD is produced by> 95% of E. coli strains (including those that do not produce gas), but not by EHEC of the serotype O157: H7, which is why it can be used here for differentiation.
Therapy for facultative pathogenic strains should always be targeted according to an antibiogram . E. coli species (especially ESBL strains in contrast to the wild type, which is highly sensitive to cephalosporins ) have antibiotic resistance through the formation of numerous β-lactamases that are able to cleave β-lactam antibiotics (ESBL strains, which are resistant to all β-lactama antibiotics except carbapenem are also often multi-resistant - also to quinolones ).
The drugs of choice are aminopenicillins , ureidopenicillins , cephalosporins (ideally from the 2nd generation), carbapenems , quinolone antibiotics and cotrimoxazole . Aminoglycosides are combined in exceptional situations.
If the urinary tract is affected, cotrimoxazole (if sensitive) and cefuroxime are particularly suitable, or alternatively levofloxacin , ciprofloxacin and fosfomycin ; in bacteremia and sepsis cefotaxime and ceftriaxone , and alternatively levofloxacin and ciprofloxacin. Ceftriaxone, cefotaxime and alternatively meropenem are suitable for meningitis caused by E. coli . Infections with ESBL-positive strains are treated with ertapenem , imipenem , meropenem, levofloxacin or (if sensitive) with ciprofloxacin.
In obligatory pathogenic E. coli strains, the gastroenteritidia are self-limiting. However, the severe fluid loss needs to be treated, especially in infants and young children. Oral rehydration solutions are ideal for water and salt loss . Antibiotics (e.g. ciprofloxacin and co-trimoxazole) administered twice within 24 hours can relieve the duration and severity of the disease.
A rapid reduction in the number of germs can only be achieved with very early use. The therapy of enterohaemorrhagic E. coli with antibiotics can lead to complications due to the increased release of verotoxin . Particularly when fluoroquinolones , co-trimoxazole, aminoglycosides and fosfomycin are administered, the unfavorable effects predominate. This does not apply equally to carbapenems . The use of newer macrolides and possibly clindamycin as well as rifampicin and rifaximin for a given indication (e.g. rehabilitation of meningococcal carriers) does not seem contraindicated. However, the use of these substances in the sense of weakening EHEC virulence by reducing verotoxin production remains controversial.
In Germany, direct or indirect evidence of Escherichia coli (enterohaemorrhagic strains (EHEC) and other enteric pathogenic strains) must be reported by name in accordance with the Infection Protection Act , provided the evidence indicates an acute infection.
E. coli was discovered in 1885 by Theodor Escherich , who then called it "Bacterium coli commune". In 1919 it was renamed in his honor. In 1892 it was proposed by Shardinger to use E. coli as an indicator organism for fecal contamination. In practice, however, it was difficult to differentiate E. coli from other enterobacteria using purely biochemical detection methods , which is why the non-taxonomic group of bacteria coliforms was defined. In 1997 the DNA sequence was determined . This took 15 years.
|Cladogram with 26 completely sequenced E. coli strains, calculated by determining the orthology of all genes. Extracted with the help of Dendroscope from the phylogenetic tree of the OMA Orthologs Project (accessed March 11, 2012).|
The nuclear genome, which is found in all strains, makes up only 6% of the gene families. Over 90% of the genes are variable. The diversity within the species and the overlapping gene content with related species suggest a transition instead of a sharp species delimitation within the Enterobacteriaceae . In particular between the genus Shigella and enteroinvasive E. coli there is a close evolutionary relationship both in the chromosomal DNA and in the virulence plasmid.
A distinction is made between four main groups (A, B1, B2, and D) on the basis of phylogenetic analyzes, with the virulent extraintestinal strains mainly belonging to groups B2 and D. Within the genus Escherichia , E. fergusonii is the closest related species.
The extraintestinal pathogenic (ExPEC) serotype O7: K1 (strain IAI39) causes haemorrhagic septicemia in domestic pigs . It has no ETEC (enterotoxic) or EDEC (edema disease-causing E. coli ) virulence factors. Instead, he has P-fimbriae and aerobactin strain SMS-3-5 / SECEC was isolated in an industrial area near the coast, contaminated with heavy metals . It is resistant to numerous antibiotics in high concentrations.
Serotype O127: H6, strain E2348 / 69 was the first enteropathogenic E. coli (EPEC) to be sequenced and best studied .
In comparison with other pathogenic strains, CFT073 of the serotype O6: H1 shows the absence of a type III secretion system and no phage- or plasmid-coded toxins. Instead, it has fimbrial adhesins , car transporters, iron sequestering systems, and recombinases . In conclusion, one can say that extraintestinal pathogenic E. coli developed independently of one another. The different pathotypes have a high syntany , which has arisen through vertical gene transfer and forms a common backbone, which is interrupted by numerous islands due to horizontal gene transfer .
The serotype O45: K1 (strain S88 / ExPEC) was isolated in 1999 from the cerebrospinal fluid of a late onset of meningitis in France. He belongs to the phylogenetic group B2. The serotype O1: K1 causes diseases in birds and is called "avian pathogen Escherichia coli " (APEC). It is closely related to three human uropathogens (UPEC). UTI89 is a uropathogenic E. coli strain isolated from a patient with acute bladder infection. In contrast, strain ED1a is non-pathogenic and was isolated from the stool of a healthy man. The uropathogenic strain 536 (O6: K15: H31) was originally derived from a patient with acute pyelonephritis . It is a model organism for extra-intestinal E. coli . The genome contains five well-characterized islands of pathogenicity and a newly discovered sixth that is the key virulence factor.
The dreaded food poisoner E. coli O157: H7 has been fully sequenced to understand its pathogenicity. The explanation is a massive lateral gene transfer . Over 1000 new genes are found in this strain-specific cluster. It contains possible virulence factors, alternative metabolic abilities and numerous prophages that can be used for food monitoring. The virulence plasmid pO157 has 100 open reading frames . An unusually large gene has a putative active site , which with the family of large clostridial toxin (LCT) and proteins as ToxA and B from Clostridium difficile is related. The strain TW14359 was isolated from spinach in 2006 after an outbreak of E. coli O157: H7 in the USA. New gene segments were found which explain the increased ability to trigger the haemolytic uremic syndrome or, alternatively, the adaptation to plants. The strain also contains genes for intact anaerobic nitrite reductases .
The serotype O139: H28 (strain E24377A / ETEC) is an enterotoxic isolate. It has the colonization factor antigen (CFA) pili to attach to. 4% of the genome consists of insertion sequences . Presumably the strain can use propanediol as the only carbon source. Strain 55989 / EAEC is an enteroaggregative strain that was isolated in 2002 in the Central African Republic from the stool of an HIV-positive adult suffering from severe watery diarrhea.
Serotype O8 strain IAI1 is a human commensal strain isolated from the faeces of a healthy French man in the 1980s. Bacteria of the strain SE11 of the serotype O152: H28 have also been isolated from the faeces of a healthy human. Compared to the laboratory strain K-12 MG1655, the strain has additional genes for car transporters and fimbriae to attach to the intestinal cells. He also has more genes that are important for carbohydrate metabolism. Everything indicates that this strain has adapted to the human intestine. Bacteria of the serotype O103: H2 (strain 12009) were isolated in 2001 in Japan from a patient with sporadic bloody diarrhea. Apparently, EHEC strains with the same pathotypes of different origins were created independently of one another by lambda phages, insertion elements and virulence plasmids.
Crooks (ATCC 8739 / DSM 1576) is a fecal strain that is used to test the effectiveness of antimicrobial agents. It has an insertion element within ompC and can therefore only express ompF as an outer membrane porin. Strain B serves as a research model for phage sensitivity, restriction modification systems and bacterial evolution. It lacks proteases . In addition, it produces little acetate when it is offered a lot of glucose. Because of a simple cell surface, the selective permeability is improved. Therefore, it is often used for recombinant protein expression on a laboratory scale and on an industrial scale. The K12 strain of MC4100 is very widely used. Genome sequencing showed that during its adaptation to the laboratory environment, it developed additional differences that were not artificially created. The K12 strain was isolated from a patient with diphtheria in 1922 and added to the Stanford strain collection in 1925. Since it is prototrophic and easy to grow in a defined medium with short generation times, it quickly became the most widely studied organism and, since the 1950s, has been used to understand numerous fundamental biochemical and molecular processes. In addition, it has the ability to recombine, which is rare among e-coli wild types . The strain DH10B is a derivative of K12, which is widely used due to its ease of transformation, especially of large plasmids (useful in genome sequencing ). The sequence was put together from “contamination sequences” from the genome sequencing of bovine animals.
Serotype O9: H4 (strain HS) was isolated by a laboratory scientist from the Walter Reed Military Hospital . In human experiments it was shown that the HS strain colonizes the gastrointestinal tract, but does not cause any symptoms. The strain UMN026 is an extraintestinal pathogenic strain (ExPEC) that was isolated in the USA in 1999 from a patient with acute cystitis . It belongs to the phylogenetic group D and has the serotype O17: K52: H18. He is a representative of a group of resistant pathogens.
The serotype O104: H4 clone HUSEC041 (sequence type 678) contains virulence factors that are typical for both STEC and EaggEC. In connection with the HUS epidemic in 2011 in northern Germany, this hybrid is held responsible for the particularly severe form.
Richard Lenski has been conducting a long-term experiment on the evolution of E. coli since 1988 . Every day the cultures are inoculated into a fresh medium and frozen. In this way, new, better adapted tribes can again compete against their ancestors and check whether they have adapted better to their Erlenmeyer flask environment. The changes in the genome are determined in parallel, the innovation rate continuously decreasing the better the cultures had adapted. Parallel experiments were also made with Myxococcus xanthus , which hunted and eats E. coli , and varied the temperature and medium. Under constant conditions without predators and only one sugar ( glucose ), citrate was also added to the nutrient medium , although E. coli cannot utilize it. In 2003, a citrate-utilizing mutant suddenly dominated , which can be proven to be derived from the original culture.
Using genetically modified E. coli , it is possible to produce biosensors for heavy metals such as arsenic . The natural mechanisms are coupled with reporter genes such as β-galactosidase , bacterial luciferases (lux) or the green fluorescent protein (GFP). In this way it is possible to inexpensively detect arsenites and arsenates below one microgram .
The combination of circular DNA present separately in the bacterial cell, the so-called plasmids , with the discovery of the restriction enzyme Eco RI , which specifically cuts double-stranded DNA and leaves identical protruding ends, marked the birth of genetic engineering . In order to obtain recombinant DNA (artificially produced DNA), in a typical cloning first the DNA fragment of choice and then the plasmid DNA of a so-called cloning vector are cut with restriction enzymes. The ends of the DNA fragment and the plasmid are joined by a ligation and then transformed into E. coli . In this way, for example, alien genes can be introduced into E. coli so that it can synthesize the foreign proteins encoded on the DNA. The restriction enzyme Eco RV is also used from E. coli .
Modern artificially produced plasmids ( vectors or “gene ferries”) such as pUC19 contain additional antibiotic resistance genes to kill bacterial cells without cloned plasmids on selective culture media. Another possibility for selecting transformed colonies is blue-white screening . The lacZ gene encodes the protein β-galactosidase , which is synthesized in the presence of IPTG and cleaves the artificial glycoside X-Gal into a blue dye. Non-transformed cells therefore appear blue on a culture medium with X-Gal. If a restriction enzyme cuts within the lacZ gene and a foreign gene is successfully introduced, the sequence for expression of β-galactosidase is destroyed to such an extent that α complementation is no longer possible. So successfully transformed cells appear white. The lacZ thus serves as a reporter gene . The test only works with the deletion mutant lacZΔM15 , whose defective β-galactosidase is dependent on α complementation.
Forcing E. coli to overexpress heterologous proteins can lead to problems with protein folding . There is an accumulation of misfolded and thus biologically inactive proteins ( inclusion bodies ). One strategy to increase the yield of soluble protein is cultivation at low temperatures. For this purpose, the folding helper proteins ( chaperonins ) Cpn10 and Cpn60 from the psychrophilic bacterium Oleispira antarctica , which work at 4–12 ° C, are co-expressed in mesophilic E. coli . They are sold by Agilent under the brand name ArcticExpress . In addition, strains are offered that have additional tRNAs in order to counteract the limiting codon bias in the translation of DNA from foreign organisms into recombinant proteins .
E. coli XL1-Red is a strain that is used in molecular biology and genetic engineering for undirected mutagenesis . Due to defects in the DNA repair mechanism , the strain shows a 5000-fold mutation rate compared to the wild type, but also a significantly lower growth rate (doubling every 90 minutes at 37 ° C). The defects in the repair mechanism of DNA replication are due to three mutations in genomic DNA. The MutS gene contains mutations in the DNA mismatch repair proteins . Due to the mutation, there is no defective base repair after DNA replication . MutD causes a defect in the 3'-5 ' exonuclease activity of DNA polymerase III . MutT is responsible for the inability to hydrolyze the base analog oxo-dGTP . Neither mutagens nor carcinogens are required to trigger mutations . The generation of gene mutations is a classic means in molecular biology research to characterize general or partial functions of the corresponding gene.
The first commercial biotechnological application was the production of the human hormone somatostatin by Genentech using genetically modified E. coli . The large-scale production of insulin and growth hormones followed shortly thereafter. The insulin preparation produced in this way is used in the treatment of diabetes mellitus . This is E. coli particularly suitable because it is part of the intestinal flora of humans and virtually no allergies caused. Genetically modified E. coli bacteria are also used in the industrial production of amino acids , interferon and other fine chemicals , enzymes and pharmaceuticals . Nine of 31 therapeutic proteins that received drug approval between 2003 and 2006 were produced in E. coli .
In order to be able to handle E. coli more easily in biotechnological applications, a team led by Frederick Blattner ( University of Wisconsin ) bred a strain whose genome has been reduced by around 15 percent compared to naturally occurring variants, and which is still viable and reproductive. For this purpose, two different E. coli strains were compared and those genes were removed that do not have a homolog in the other strain and thus seem to be dispensable.
The production of biofuels from proteins is possible with genetically modified E. coli , which contain three exogenous transamination and deamination cycles . The proteins are initially produced by Saccharomyces cerevisiae , Bacillus subtilis , micro algae (for simultaneous CO 2 fixation) or E. coli itself. The proteins could also come from waste from fermentation, food processing, and bioethanol production . The hydrolysates are then converted into C4 and C5 alcohols with 56% of the theoretical yield. The resulting nitrogen can be converted into fertilizer.
Changes in the metabolism of E. coli can produce higher alcohols such as isobutanol , 1-butanol , 2-methyl-1-butanol , 3-methyl-1-butanol and 2-phenylethanol from the carbon source glucose . For this, the highly active biosynthetic pathway for amino acids is used and the 2- keto acid intermediates are used for alcohol synthesis. Compared to the classic biofuel ethanol , the higher energy density and the low hygroscopicity should be emphasized. In addition, branched-chain alcohols have higher octane numbers .
Cooperation in case of nutrient deficiency
Bacteria can help each other when there is a lack of nutrients. In the case of genetically modified Escherichia coli and Acinetobacter baylyi, it was observed that E. coli connected with Acinetobacter baylyi through up to 14 micrometers long nanochannels in order to exchange cytoplasmic components. The two bacteria were changed in such a way that they could no longer produce the amino acids necessary for themselves, but produced the amino acids necessary for the other species. E. coli then formed nanochannels to connect with Acinetobacter baylyi and survive. It is still unclear whether bacteria can specifically control which cell they attach to and whether this connection is of a parasitic nature.
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