Information

What is the mechanism of oxygen uptake in E. coli?


How does E. coli uptake oxygen? Most of the literature I found is concerned with response to oxygen level supplied in the medium, as opposed to how much is actually transported inside. Can they shut down the oxygen uptake if required? For example, to avoid damaging effects of reactive oxygen species.

There exist bacteria which die in presence of oxygen (strict anaerobe), while some use oxygen when available but also can survive in absence of it (facultative anaerobes, like E. coli). Are there bacteria which cannot consume oxygen for metabolism, but when exposed to oxygen they don't die, rather simply discard the oxygen and carry out fermentation? In a sense, they are strict anaerobe (their mode of metabolism is anaerobic), but they can still survive in presence of oxygen in environment.


This question got me thinking about what are the metabolic enzymes that take oxygen up in E.coli. I searched the metacyc database for reactions that consume molecular oxygen and there are only 3 that take in oxygen and one that produces oxygen.

All three consumers of oxygen in E.coli are the oxidation of ubiquinone by at two sites in cytochrome-bcl or by cytochrome-bo. All of these export protons to create the gradient that drives ATP formation by F₀F₁ ATPase in the periplasm (the region between E.coli 's inner and outer membrane).

On the production side, superoxide dismutase reduces superoxide to oxygen to control oxidation damage. Apparently the innards of E.coli are somewhat oxygen tolerant.


The plasma membrane is quite permeable to oxygen and thus oxygen enters the cell simply by diffusion. Reactive oxygen species can be reduced enzymatically in aerobic organisms. Obligate anaerobes lack or don't produce sufficient quantities of these enzymes. An organism that doesn't use oxygen for metabolism but is also not relatively harmed by it can be classified as aerotolerant.

Image from: http://en.m.wikipedia.org/wiki/Anaerobic_organism

Original Caption: Aerobic and anaerobic bacteria can be identified by growing them in test tubes of thioglycollate broth: 1: Obligate aerobes need oxygen because they cannot ferment or respire anaerobically. They gather at the top of the tube where the oxygen concentration is highest. 2: Obligate anaerobes are poisoned by oxygen, so they gather at the bottom of the tube where the oxygen concentration is lowest. 3: Facultative anaerobes can grow with or without oxygen because they can metabolise energy aerobically or anaerobically. They gather mostly at the top because aerobic respiration generates more ATP than either fermentation or anaerobic respiration. 4: Microaerophiles need oxygen because they cannot ferment or respire anaerobically. However, they are poisoned by high concentrations of oxygen. They gather in the upper part of the test tube but not the very top. 5: Aerotolerant organisms do not require oxygen as they metabolise energy anaerobically. Unlike obligate anaerobes however, they are not poisoned by oxygen. They can be found evenly spread throughout the test tube.


I am adding a supplement/concluding remarks to the existing answers.

Some points to be considered:

  • Electron transport chain can work without oxygen and can use other chemical species as electron acceptors.
  • E.coli is capable of aerobic respiration, anaerobic (anoxic) respiration and fermentation (substrate level phosphorylation).

How does E. coli uptake oxygen?

Short answer as already mentioned by canadianer: Passive diffusion

Can they shut down the oxygen uptake if required?

Oxygen is the primary electron acceptor and E.coli would prefer oxygen to other electron acceptors. Only in the absence of oxygen is the FNR regulon switched on. Other bacteria can show negative aerotaxis i.e. running away from oxygen (they obviously have to be motile) [ref]. This paper also comments that positive and negative aerotaxis happens via a common mechanism- sensitivity of proton motive force to O₂. However, ROS has no role to play in it. It is most likely that at high oxygen levels E.coli will try to jack up anti-oxidant response. To conclude- they cannot regulate oxygen uptake.

Aerotolerant anaerobes: Clostridium perfringens, Clostridium intestinale.


AN EXPERIMENTAL PROCEDURE FOR QUANTIFICATION OF INDIVIDUAL HYPERBARIC FACTORS AFFECTING THE PHYSICAL PERFORMANCE OF THE RAT

RESULTS

Resting and peak exercise oxygen uptakes are summarized in Table 2 .

TABLE 2 . Resting and peak oxygen uptake during exercise in 10 environmental conditions.

RestExercise
Gas compositions1 ATA3 ATA10 ATA1 ATA3 ATA10 ATA
Air29±1.224±2.6 (a) 20±2.1 (b) 63±3.442±2.6 (c) 23±2.1 (c)
20% O2 in He25±1.423±1.324±1.160±1.756±1.733±2.7 (c)
1% O2 in N2 22±1.2 (b) 48±2.4 (b)
1% O2 in He 24±1.7 50±3.1 (b)
2% O2 in N2 24±1.9 (a) 32±4.4 (c)
2% O2 in He 20±1.8 (a) 42±1.3 (c)

Values are X ± 1 S.E. for 6 rats in each experimental condition. The body weight of the rats was 344±2.4 (60 rats) g. and ranged from 305 to 390 g. Oxygen uptake is measured in ml/min/kg (STPD).

(a), (b), and (c) indicates P&lt0.05, P&lt0.01, and P&lt0.001, when compared to their respective inert gas mixtures at 1 ATA, by grouped t-tests.

Oxygen uptake at rest in 3 and 10 ATA normoxic nitrogen environments was depressed by 24% and 17%, respectively whereas in hyperoxic nitrogen it was 17% and 31% respectively. The corresponding changes in the helium environments were much less than those in nitrogen environments and a significant depression of oxygen uptake 20% was observed in the 10 ATA normoxic heliox condition.

Peak oxygen uptake during exercise in 3 and 10 ATA hyperoxic nitrogen (compressed air) was depressed 33% and 63% respectively compared to 1 ATA air. Under normoxic nitrogen conditions this decrease was 24% and 49%, respectively at 3 and 10 ATA. The depressive effect on exercise oxygen uptake of hyperbaric helium was 17% and 30% at normoxic 3 and 10 ATA and 7% and 45% at hyperoxic 3 and 10 ATA.

Total effect of compressed air and compressed helium containing 20% oxygen on oxygen uptake is complex, because elevation of inspired PO2, PN2 or PHe, and gas density are all expected concurrent to the elevation of ambient pressure. Utilizing the data presented above, the quantification of the individual effects of hydrostatic pressure, gas density, and hyperoxia on the ability to take up oxygen was made by the following procedures:

Effect of hyperoxia was determined by comparing oxygen uptakes for varied PO2 at a given ambient pressure, inert gas, and gas density ( Fig. 1 ).

Fig. 1 . Oxygen uptake at rest and during peak exercise in normoxic and hyperoxic nitrogen environments. Vertical bars indicate ± 1. S.E. of mean (6 rats).

In the nitrogen environment, an increase in PO2 from 150 to 450 mm Hg depressed oxygen uptake by 6 units in ml/min/kg of body weight and an increased in PO2 from 150 to 1500 mm Hg depressed oxygen uptake by 9 ml/min/kg. At rest the effect of hyperoxia was small and was statistically not significant. A similar comparison can be made for the hyperoxic effect in helium environments. However, this comparison was not as clean as we would have liked it to be because in the case of helium, the gas density of the hyperoxic mixture was about 2 times that of the normoxic mixture at these pressures.

Effect of inspired gas density on oxygen uptake was analyzed by utilizing the data obtained under normoxic conditions, and plotting the oxygen uptake as a function of relative gas density on a log scale. Oxygen uptake decreased as gas density increased in each inert gas environment during exercise. However, an insignificant effect was seen at rest ( Fig. 2 ). The effect of elevated gas density was obtained by comparing the oxygen uptakes at the same ambient pressure. This so called “isobaric comparison” is necessary in order to exclude the pressure factor. During exercise at 3 ATA, the peak oxygen uptake was depressed by 2 ml/min/kg with an increase in gas density of 2.4 units, thus depression of 2/2.4 in oxygen uptake per unit increase in gas density is realized. By calculation, an increase in gas density of 2 units (from 1 ATA to 3 ATA) resulted in depression of oxygen uptake by 1.7 units. Similarly, comparisons of oxygen uptake at 10 ATA showed that an increase in gas density of 8.3 units caused a decrease in oxygen uptake of 10 units. Therefore, an increase of gas density of 9 units (from 1 ATA to 10 ATA) resulted in depression of oxygen uptake by 11 (=10(10 – 1)/8.3) units.

Fig. 2 . Oxygen uptake at rest and during peak exercise as a function of gas density. Effect of gas density was determined by comparing oxygen uptake under normoxic and isobaric conditions and the effect of hydrostatic pressure was determined by comparing oxygen uptake under normoxic and iso-density conditions. Means of 6 rats ± 1 S.E. are indicated.

Effect of ambient pressure per se on oxygen uptake in the rat is obtained by comparing the difference in oxygen uptake at a given gas density and PO2 (normoxic). For example, given a relative gas density of 1.0, the pressure difference between the nitrogen and the helium mixture was 3.8 ATA ( Fig. 2 ). For a gas density of 1.7, the pressure difference between these two gas mixtures was 8.3 ATA. The differences in oxygen uptake between these two environments represent the effect of hydrostatic pressure per se. With the pressure difference ranging from 3.8 to 8.3 ATA, the depression of oxygen uptake was found to be between 14 to 16 units at peak exersice, and between 6 to 7 units at rest ( Fig. 2 ).

Summation of the effects of elevated ambient pressure, gas density, and PO2 is sufficient to explain the total observed effect on resting oxygen uptake in various hyperbaric nitrogen environments, as well as exercise uptake at 3 ATA in air. As measured experimentally, a statistically insignificant underestimation of the total effect was seen in 10 ATA air ( Fig. 3 ). Summation of the effects of these three factors can not be made for hyperoxic heliox conditions due to difficulties in estimation of PO2 effect (see above).

Fig. 3 . Comparison of the measured total effects of air under hyperbaric conditions (T) with the estimated individual effects of hyperoxia (PO2), density (D), and hydrostatic pressure (P). Lines indicated by normoxic, isobaric and iso-density represent stepwise removal of the effects of hyperoxia, hydrostatic pressure, and gas density, respectively.


Escherichia Coli

Although most strains of E. coli bacteria are harmless and live in the intestines of healthy humans and animals, several strains can produce powerful toxins and cause severe illness in humans. This versatile pathogen is best known for being transmitted to humans through contaminated foods — such as undercooked meat and unpasteurized fruit juice — and has attracts much attention when serious outbreaks occur.

E. coli is capable of causing a wide variety of diseases — from urinary tract infections to meningitis. A considerable amount of media coverage has recently been devoted to one particular strain of E. coli, responsible for an estimated 73,000 cases of infection and 61 deaths in the United States each year. Knowing more about the biology, the evolution, and the genetic basis of this pathogen is crucial to future prevention of infection and illness.

Pathogenic E. coli is a unique, comprehensive analysis of the biology and molecular mechanisms that enable this ubiquitous organism to thrive. Leading investigators in the field discuss the molecular basis of E. coli pathogenesis followed by chapters on genomics and evolution. Detailed descriptions of distinct strains reveal the molecular pathogenesis of each and the causes of intestinal and extra-intestinal infections in humans. Pathogenic E. coli concludes with a presentation of virulance factors, common to two or more pathotypes. This unique collection presents timely and vital information on understanding the inner workings of E. coli, which will lend key insights into disease prevention research.

Although most strains of E. coli bacteria are harmless and live in the intestines of healthy humans and animals, several strains can produce powerful toxins and cause severe illness in humans. This versatile pathogen is best known for being transmitted to humans through contaminated foods — such as undercooked meat and unpasteurized fruit juice — and has attracts much attention when serious outbreaks occur.

E. coli is capable of causing a wide variety of diseases — from urinary tract infections to meningitis. A considerable amount of media coverage has recently been devoted to one particular strain of E. coli, responsible for an estimated 73,000 cases of infection and 61 deaths in the United States each year. Knowing more about the biology, the evolution, and the genetic basis of this pathogen is crucial to future prevention of infection and illness.

Pathogenic E. coli is a unique, comprehensive analysis of the biology and molecular mechanisms that enable this ubiquitous organism to thrive. Leading investigators in the field discuss the molecular basis of E. coli pathogenesis followed by chapters on genomics and evolution. Detailed descriptions of distinct strains reveal the molecular pathogenesis of each and the causes of intestinal and extra-intestinal infections in humans. Pathogenic E. coli concludes with a presentation of virulance factors, common to two or more pathotypes. This unique collection presents timely and vital information on understanding the inner workings of E. coli, which will lend key insights into disease prevention research.


Systems biology approach reveals that overflow metabolism of acetate in Escherichia coli is triggered by carbon catabolite repression of acetyl-CoA synthetase

Background: The biotechnology industry has extensively exploited Escherichia coli for producing recombinant proteins, biofuels etc. However, high growth rate aerobic E. coli cultivations are accompanied by acetate excretion i.e. overflow metabolism which is harmful as it inhibits growth, diverts valuable carbon from biomass formation and is detrimental for target product synthesis. Although overflow metabolism has been studied for decades, its regulation mechanisms still remain unclear.

Results: In the current work, growth rate dependent acetate overflow metabolism of E. coli was continuously monitored using advanced continuous cultivation methods (A-stat and D-stat). The first step in acetate overflow switch (at μ = 0.27 ± 0.02 h(-1)) is the repression of acetyl-CoA synthetase (Acs) activity triggered by carbon catabolite repression resulting in decreased assimilation of acetate produced by phosphotransacetylase (Pta), and disruption of the PTA-ACS node. This was indicated by acetate synthesis pathways PTA-ACKA and POXB component expression down-regulation before the overflow switch at μ = 0.27 ± 0.02 h(-1) with concurrent 5-fold stronger repression of acetate-consuming Acs. This in turn suggests insufficient Acs activity for consuming all the acetate produced by Pta, leading to disruption of the acetate cycling process in PTA-ACS node where constant acetyl phosphate or acetate regeneration is essential for E. coli chemotaxis, proteolysis, pathogenesis etc. regulation. In addition, two-substrate A-stat and D-stat experiments showed that acetate consumption capability of E. coli decreased drastically, just as Acs expression, before the start of overflow metabolism. The second step in overflow switch is the sharp decline in cAMP production at μ = 0.45 h(-1) leading to total Acs inhibition and fast accumulation of acetate.

Conclusion: This study is an example of how a systems biology approach allowed to propose a new regulation mechanism for overflow metabolism in E. coli shown by proteomic, transcriptomic and metabolomic levels coupled to two-phase acetate accumulation: acetate overflow metabolism in E. coli is triggered by Acs down-regulation resulting in decreased assimilation of acetic acid produced by Pta, and disruption of the PTA-ACS node.


Concluding remarks

In conclusion, mechanisms affecting the barrier properties of the OM lipid bilayer itself or the expression and/or function of the general diffusion porin channels residing in the OM have an impact on the sensitivity of Gram-negative bacteria to many different types of antibiotics. Clearly any weakening of the LPS bilayer by targeting LPS synthesizing enzymes will sensitize bacteria to hydrophobic and some hydrophilic antibiotics, leading to the possibility of combinatorial drug therapy. A better understanding of the function of general diffusion porins, and in particular of the parameters that might lead to porin closure or inactivation, will allow a reassessment of the efficiency of penetration of the antibiotics using this pathway in different conditions. It is hoped that, as we further understand at the molecular level the structure and function of these OM macromolecules and of those that regulate them, scientists will be able to refine the current drug therapies or design new types of antibiotics that target these surface exposed entities.


Contents

Type and morphology Edit

E. coli is a Gram-negative, facultative anaerobe (that makes ATP by aerobic respiration if oxygen is present, but is capable of switching to fermentation or anaerobic respiration if oxygen is absent) and nonsporulating bacterium. [17] 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 3 . [18] [19] [20]

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 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. [15]

Strains that possess flagella are motile. The flagella have a peritrichous arrangement. [21] It also attaches and effaces to the microvilli of the intestines via an adhesion molecule known as intimin. [22]

Metabolism Edit

E. coli can live on 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. [23]

In addition, E. coli's metabolism can be rewired to solely use CO2 as 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. [24]

E. coli have 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 the EDP is the more thermodynamically favorable 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. [25]

Catabolite Repression Edit

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 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 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. [26]

Culture growth Edit

Optimum growth of E. coli occurs at 37 °C (98.6 °F), but some laboratory strains can multiply at temperatures up to 49 °C (120 °F). [27] E. coli grows in 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 the reduction of substrates such as oxygen, nitrate, fumarate, dimethyl sulfoxide, and trimethylamine N-oxide. [28] 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. The ability to continue growing in the absence of oxygen is an advantage to bacteria because their survival is increased in environments where water predominates. [15]

Cell cycle Edit

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 the conclusion of DNA replication and the end of cell division. [29] 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 the DNA and overlapping cell cycles. [30]

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 is then referred to being asynchronous. [31] However, asynchrony can be caused by mutations to for instance DnaA [31] or DnaA initiator-associating protein DiaA. [32]

Genetic adaptation Edit

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, [33] is where the spread of the gene encoding for 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, [34] and E. coli remains one of the most diverse bacterial species: only 20% of the genes in a typical E. coli genome is shared among all strains. [35]

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. [36] 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 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 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. [12] [13] 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.

Serotypes Edit

A common subdivision system of E. coli, but not based on evolutionary relatedness, 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). [37] It is, however, common to cite only the serogroup, i.e. the O-antigen. At present, about 190 serogroups are known. [38] The common laboratory strain has a mutation that prevents the formation of an O-antigen and is thus not typeable.

Genome plasticity and evolution Edit

Like all lifeforms, new strains of E. coli evolve through 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. [39] 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 a bout of diarrhea that is often self-limiting in healthy adults but is frequently lethal to children in the developing world. [40] More virulent strains, such as O157:H7, cause serious illness or death in the elderly, the very young, or the immunocompromised. [40] [41]

The genera Escherichia and Salmonella diverged around 102 million years ago (credibility interval: 57–176 mya), which coincides with the divergence of their hosts: the former being found in mammals and the latter in birds and reptiles. [42] 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. [43]

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 the laboratory. [44] 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 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 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. [45]

Neotype strain Edit

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 the genus Enterobacter + "i" (sic.) + "aceae", but from "enterobacterium" + "aceae" (enterobacterium being not a genus, but an alternative trivial name to enteric bacterium). [46] [47]

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 T , [48] also known under the deposit names DSM 30083, [49] ATCC 11775, [50] and NCTC 9001, [51] which is pathogenic to chickens and has an O1:K1:H7 serotype. [52] 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. [48]

Phylogeny of E. coli strains Edit

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. the inferred evolutionary history, as shown below where the species is divided into six groups. [53] [54] Particularly the use of whole genome sequences yields highly supported phylogenies. Based on such data, five subspecies of E. coli were distinguished. [48]

The link between phylogenetic distance ("relatedness") and pathology is small, [48] 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 a single subspecies of E. coli in a phylogenomic study that included the type strain, [48] and for this reason an according reclassification is difficult. 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).

E. coli S88 (O45:K1. Extracellular pathogenic)

E. coli UMN026 (O17:K52:H18. Extracellular pathogenic)

E. coli (O19:H34. Extracellular pathogenic)

E. coli (O7:K1. Extracellular pathogenic)

E. coli GOS1 (O104:H4 EAHEC) German 2011 outbreak

E. coli ATCC8739 (O146. Crook's E.coli used in phage work in the 1950s)

E. coli K-12 W3110 (O16. λ − F − "wild type" molecular biology strain)

E. coli K-12 DH10b (O16. high electrocompetency molecular biology strain)

E. coli K-12 DH1 (O16. high chemical competency molecular biology strain)

E. coli K-12 MG1655 (O16. λ − F − "wild type" molecular biology strain)

E. coli BW2952 (O16. competent molecular biology strain)

E. coli B REL606 (O7. high competency molecular biology strain)

E. coli BL21-DE3 (O7. expression molecular biology strain with T7 polymerase for pET system)

The first complete DNA sequence of an E. coli genome (laboratory strain K-12 derivative MG1655) was published in 1997. It is a circular DNA molecule 4.6 million base pairs in length, containing 4288 annotated protein-coding genes (organized into 2584 operons), seven ribosomal RNA (rRNA) operons, and 86 transfer RNA (tRNA) genes. Despite having been the subject of intensive genetic analysis for about 40 years, many of these genes were previously unknown. The coding density was found to be very high, with a mean distance between genes of only 118 base pairs. The genome was observed to contain a significant number of transposable genetic elements, repeat elements, cryptic prophages, and bacteriophage remnants. [55]

More than three hundred complete genomic sequences of Escherichia and Shigella species are known. The genome sequence of the type strain of E. coli was added to this collection before 2014. [48] Comparison of these sequences shows a remarkable amount of diversity only about 20% of each genome represents sequences present in every one of the isolates, while around 80% of each genome can vary among isolates. [35] Each individual genome contains between 4,000 and 5,500 genes, but the total number of different genes among all of the sequenced E. coli strains (the pangenome) exceeds 16,000. This very large variety of component genes has been interpreted to mean that two-thirds of the E. coli pangenome originated in other species and arrived through the process of horizontal gene transfer. [56]

Genes in E. coli are usually named by 4-letter acronyms that derive from their function (when known) and italicized. For instance, recA is named after its role in homologous recombination plus the letter A. Functionally related genes are named recB, recC, recD etc. The proteins are named by uppercase acronyms, e.g. RecA, RecB, etc. When the genome of E. coli was sequenced, all genes were numbered (more or less) in their order on the genome and abbreviated by b numbers, such as b2819 (= recD). The "b" names were created after Fred Blattner, who led the genome sequence effort. [55] Another numbering system was introduced with the sequence of another E. coli strain, W3110, which was sequenced in Japan and hence uses numbers starting by JW. (Japanese W3110), e.g. JW2787 (= recD). [57] Hence, recD = b2819 = JW2787. Note, however, that most databases have their own numbering system, e.g. the EcoGene database [58] uses EG10826 for recD. Finally, ECK numbers are specifically used for alleles in the MG1655 strain of E. coli K-12. [58] Complete lists of genes and their synonyms can be obtained from databases such as EcoGene or Uniprot.

Proteome Edit

Several studies have investigated the proteome of E. coli. By 2006, 1,627 (38%) of the 4,237 open reading frames (ORFs) had been identified experimentally. [59] The 4,639,221–base pair sequence of Escherichia coli K-12 is presented. Of 4288 protein-coding genes annotated, 38 percent have no attributed function. Comparison with five other sequenced microbes reveals ubiquitous as well as narrowly distributed gene families many families of similar genes within E. coli are also evident. The largest family of paralogous proteins contains 80 ABC transporters. The genome as a whole is strikingly organized with respect to the local direction of replication guanines, oligonucleotides possibly related to replication and recombination, and most genes are so oriented. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition indicating genome plasticity through horizontal transfer. [55]

Interactome Edit

The interactome of E. coli has been studied by affinity purification and mass spectrometry (AP/MS) and by analyzing the binary interactions among its proteins.

Protein complexes. A 2006 study purified 4,339 proteins from cultures of strain K-12 and found interacting partners for 2,667 proteins, many of which had unknown functions at the time. [60] A 2009 study found 5,993 interactions between proteins of the same E. coli strain, though these data showed little overlap with those of the 2006 publication. [61]

Binary interactions. Rajagopala et al. (2014) have carried out systematic yeast two-hybrid screens with most E. coli proteins, and found a total of 2,234 protein-protein interactions. [62] This study also integrated genetic interactions and protein structures and mapped 458 interactions within 227 protein complexes.

E. coli belongs to a group of bacteria informally known as coliforms that are found in the gastrointestinal tract of warm-blooded animals. [63] E. coli normally colonizes an infant's gastrointestinal tract within 40 hours of birth, arriving with food or water or from the individuals handling the child. In the bowel, E. coli adheres to the mucus of the large intestine. It is the primary facultative anaerobe of the human gastrointestinal tract. [64] (Facultative anaerobes are organisms that can grow in either the presence or absence of oxygen.) As long as these bacteria do not acquire genetic elements encoding for virulence factors, they remain benign commensals. [65]

Therapeutic use Edit

Due to the low cost and speed with which it can be grown and modified in laboratory settings, E. coli is a popular expression platform for the production of recombinant proteins used in therapeutics. One advantage to using E. coli over another expression platform is that E. coli naturally does not export many proteins into the periplasm, making it easier to recover a protein of interest without cross-contamination. [66] The E. coli K-12 strains and their derivatives (DH1, DH5α, MG1655, RV308 and W3110) are the strains most widely used by the biotechnology industry. [67] Nonpathogenic E. coli strain Nissle 1917 (EcN), (Mutaflor) and E. coli O83:K24:H31 (Colinfant) [68] [69] ) are used as probiotic agents in medicine, mainly for the treatment of various gastrointestinal diseases, [70] including inflammatory bowel disease. [71] It is thought that the EcN strain might impede the growth of opportunistic pathogens, including Salmonella and other coliform enteropathogens, through the production of microcin proteins the production of siderophores. [72]

Most E. coli strains do not cause disease, naturally living in the gut, [73] but virulent strains can cause gastroenteritis, urinary tract infections, neonatal meningitis, hemorrhagic colitis, and Crohn's disease. Common signs and symptoms include severe abdominal cramps, diarrhea, hemorrhagic colitis, vomiting, and sometimes fever. In rarer cases, virulent strains are also responsible for bowel necrosis (tissue death) and perforation without progressing to hemolytic-uremic syndrome, peritonitis, mastitis, sepsis, and Gram-negative pneumonia. Very young children are more susceptible to develop severe illness, such as hemolytic uremic syndrome however, healthy individuals of all ages are at risk to the severe consequences that may arise as a result of being infected with E. coli. [64] [74] [75] [76]

Some strains of E. coli, for example O157:H7, can produce Shiga toxin (classified as a bioterrorism agent). The Shiga toxin causes inflammatory responses in target cells of the gut, leaving behind lesions which result in the bloody diarrhea that is a symptom of a Shiga toxin-producing E. coli (STEC) infection. This toxin further causes premature destruction of the red blood cells, which then clog the body's filtering system, the kidneys, in some rare cases (usually in children and the elderly) causing hemolytic-uremic syndrome (HUS), which may lead to kidney failure and even death. Signs of hemolytic uremic syndrome include decreased frequency of urination, lethargy, and paleness of cheeks and inside the lower eyelids. In 25% of HUS patients, complications of nervous system occur, which in turn causes strokes. In addition, this strain causes the buildup of fluid (since the kidneys do not work), leading to edema around the lungs, legs, and arms. This increase in fluid buildup especially around the lungs impedes the functioning of the heart, causing an increase in blood pressure. [77] [22] [78] [79] [80] [75] [76]

Uropathogenic E. coli (UPEC) is one of the main causes of urinary tract infections. [81] It is part of the normal microbiota in the gut and can be introduced in many ways. In particular for females, the direction of wiping after defecation (wiping back to front) can lead to fecal contamination of the urogenital orifices. Anal intercourse can also introduce this bacterium into the male urethra, and in switching from anal to vaginal intercourse, the male can also introduce UPEC to the female urogenital system.

Enterotoxigenic E. coli (ETEC) is the most common cause of traveler's diarrhea, with as many as 840 million cases worldwide in developing countries each year. The bacteria, typically transmitted through contaminated food or drinking water, adheres to the intestinal lining, where it secretes either of two types of enterotoxins, leading to watery diarrhea. The rate and severity of infections are higher among children under the age of five, including as many as 380,000 deaths annually. [82]

In May 2011, one E. coli strain, O104:H4, was the subject of a bacterial outbreak that began in Germany. Certain strains of E. coli are a major cause of foodborne illness. The outbreak started when several people in Germany were infected with enterohemorrhagic E. coli (EHEC) bacteria, leading to hemolytic-uremic syndrome (HUS), a medical emergency that requires urgent treatment. The outbreak did not only concern Germany, but also 15 other countries, including regions in North America. [83] On 30 June 2011, the German Bundesinstitut für Risikobewertung (BfR) (Federal Institute for Risk Assessment, a federal institute within the German Federal Ministry of Food, Agriculture and Consumer Protection) announced that seeds of fenugreek from Egypt were likely the cause of the EHEC outbreak. [84]

Some studies have demonstrated an absence of E.coli in the gut flora of subjects with the metabolic disorder Phenylketonuria. It is hypothesized that the absence of these normal bacterium impairs the production of the key vitamins B2 (riboflavin) and K2 (menaquinone) - vitamins which are implicated in many physiological roles in humans such as cellular and bone metabolism - and so contributes to the disorder. [85]

Incubation period Edit

The time between ingesting the STEC bacteria and feeling sick is called the "incubation period". The incubation period is usually 3–4 days after the exposure, but may be as short as 1 day or as long as 10 days. The symptoms often begin slowly with mild belly pain or non-bloody diarrhea that worsens over several days. HUS, if it occurs, develops an average 7 days after the first symptoms, when the diarrhea is improving. [86]

Diagnosis Edit

Diagnosis of infectious diarrhea and identification of antimicrobial resistance is performed using a stool culture with subsequent antibiotic sensitivity testing. It requires a minimum of 2 days and maximum of several weeks to culture gastrointestinal pathogens. The sensitivity (true positive) and specificity (true negative) rates for stool culture vary by pathogen, although a number of human pathogens can not be cultured. For culture-positive samples, antimicrobial resistance testing takes an additional 12-24 hours to perform.

Current point of care molecular diagnostic tests can identify E. coli and antimicrobial resistance in the identified strains much faster than culture and sensitivity testing. Microarray-based platforms can identify specific pathogenic strains of E. coli and E. coli-specific AMR genes in two hours or less with high sensitivity and specificity, but the size of the test panel (i.e., total pathogens and antimicrobial resistance genes) is limited. Newer metagenomics-based infectious disease diagnostic platforms are currently being developed to overcome the various limitations of culture and all currently available molecular diagnostic technologies.

Treatment Edit

The mainstay of treatment is the assessment of dehydration and replacement of fluid and electrolytes. Administration of antibiotics has been shown to shorten the course of illness and duration of excretion of enterotoxigenic E. coli (ETEC) in adults in endemic areas and in traveller's diarrhea, though the rate of resistance to commonly used antibiotics is increasing and they are generally not recommended. [87] The antibiotic used depends upon susceptibility patterns in the particular geographical region. Currently, the antibiotics of choice are fluoroquinolones or azithromycin, with an emerging role for rifaximin. Oral rifaximin, a semisynthetic rifamycin derivative, is an effective and well-tolerated antibacterial for the management of adults with non-invasive traveller's diarrhea. Rifaximin was significantly more effective than placebo and no less effective than ciprofloxacin in reducing the duration of diarrhea. While rifaximin is effective in patients with E. coli-predominant traveller's diarrhea, it appears ineffective in patients infected with inflammatory or invasive enteropathogens. [88]

Prevention Edit

ETEC is the type of E. coli that most vaccine development efforts are focused on. Antibodies against the LT and major CFs of ETEC provide protection against LT-producing, ETEC-expressing homologous CFs. Oral inactivated vaccines consisting of toxin antigen and whole cells, i.e. the licensed recombinant cholera B subunit (rCTB)-WC cholera vaccine Dukoral, have been developed. There are currently no licensed vaccines for ETEC, though several are in various stages of development. [89] In different trials, the rCTB-WC cholera vaccine provided high (85–100%) short-term protection. An oral ETEC vaccine candidate consisting of rCTB and formalin inactivated E. coli bacteria expressing major CFs has been shown in clinical trials to be safe, immunogenic, and effective against severe diarrhoea in American travelers but not against ETEC diarrhoea in young children in Egypt. A modified ETEC vaccine consisting of recombinant E. coli strains over-expressing the major CFs and a more LT-like hybrid toxoid called LCTBA, are undergoing clinical testing. [90] [91]

Other proven prevention methods for E. coli transmission include handwashing and improved sanitation and drinking water, as transmission occurs through fecal contamination of food and water supplies. Additionally, thoroughly cooking meat and avoiding consumption of raw, unpasteurized beverages, such as juices and milk are other proven methods for preventing E.coli. Lastly, avoid cross-contamination of utensils and work spaces when preparing food. [92]

Because of its long history of laboratory culture and ease of manipulation, E. coli plays an important role in modern biological engineering and industrial microbiology. [93] The work of Stanley Norman Cohen and Herbert Boyer in E. coli, using plasmids and restriction enzymes to create recombinant DNA, became a foundation of biotechnology. [94]

E. coli is a very versatile host for the production of heterologous proteins, [95] and various protein expression systems have been developed which allow the production of recombinant proteins in E. coli. Researchers can introduce genes into the microbes using plasmids which permit high level expression of protein, and such protein may be mass-produced in industrial fermentation processes. One of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin. [96]

Many proteins previously thought difficult or impossible to be expressed in E. coli in folded form have been successfully expressed in E. coli. For example, proteins with multiple disulphide bonds may be produced in the periplasmic space or in the cytoplasm of mutants rendered sufficiently oxidizing to allow disulphide-bonds to form, [97] while proteins requiring post-translational modification such as glycosylation for stability or function have been expressed using the N-linked glycosylation system of Campylobacter jejuni engineered into E. coli. [98] [99] [100]

Modified E. coli cells have been used in vaccine development, bioremediation, production of biofuels, [101] lighting, and production of immobilised enzymes. [95] [102]

Strain K-12 is a mutant form of E. coli that over-expresses the enzyme Alkaline Phosphatase (ALP). [103] The mutation arises due to a defect in the gene that constantly codes for the enzyme. A gene that is producing a product without any inhibition is said to have constitutive activity. This particular mutant form is used to isolate and purify the aforementioned enzyme. [103]

Strain OP50 of Escherichia coli is used for maintenance of Caenorhabditis elegans cultures.

Strain JM109 is a mutant form of E. coli that is recA and endA deficient. The strain can be utilized for blue/white screening when the cells carry the fertility factor episome [104] Lack of recA decreases the possibility of unwanted restriction of the DNA of interest and lack of endA inhibit plasmid DNA decomposition. Thus, JM109 is useful for cloning and expression systems.

Model organism Edit

E. coli is frequently used as a model organism in microbiology studies. Cultivated strains (e.g. E. coli K12) are well-adapted to the laboratory environment, and, unlike wild-type strains, have lost their ability to thrive in the intestine. Many laboratory strains lose their ability to form biofilms. [105] [106] These features protect wild-type strains from antibodies and other chemical attacks, but require a large expenditure of energy and material resources. E. coli is often used as a representative microorganism in the research of novel water treatment and sterilisation methods, including photocatalysis. By standard plate count methods, following sequential dilutions, and growth on agar gel plates, the concentration of viable organisms or CFUs (Colony Forming Units), in a known volume of treated water can be evaluated, allowing the comparative assessment of materials performance. [107]

In 1946, Joshua Lederberg and Edward Tatum first described the phenomenon known as bacterial conjugation using E. coli as a model bacterium, [108] and it remains the primary model to study conjugation. [109] E. coli was an integral part of the first experiments to understand phage genetics, [110] and early researchers, such as Seymour Benzer, used E. coli and phage T4 to understand the topography of gene structure. [111] Prior to Benzer's research, it was not known whether the gene was a linear structure, or if it had a branching pattern. [112]

E. coli was one of the first organisms to have its genome sequenced the complete genome of E. coli K12 was published by Science in 1997 [55]

From 2002 to 2010, a team at the Hungarian Academy of Science created a strain of Escherichia coli called MDS42, which is now sold by Scarab Genomics of Madison, WI under the name of "Clean Genome. E.coli", [113] where 15% of the genome of the parental strain (E. coli K-12 MG1655) were removed to aid in molecular biology efficiency, removing IS elements, pseudogenes and phages, resulting in better maintenance of plasmid-encoded toxic genes, which are often inactivated by transposons. [114] [115] [116] Biochemistry and replication machinery were not altered.

By evaluating the possible combination of nanotechnologies with landscape ecology, complex habitat landscapes can be generated with details at the nanoscale. [117] On such synthetic ecosystems, evolutionary experiments with E. coli have been performed to study the spatial biophysics of adaptation in an island biogeography on-chip.

Studies are also being performed attempting to program E. coli to solve complicated mathematics problems, such as the Hamiltonian path problem. [118]

In other studies, non-pathogenic E. coli has been used as a model microorganism towards understanding the effects of simulated microgravity (on Earth) on the same. [119] [120]

In 1885, the German-Austrian pediatrician Theodor Escherich discovered this organism in the feces of healthy individuals. He called it Bacterium coli commune because it is found in the colon. Early classifications of prokaryotes placed these in a handful of genera based on their shape and motility (at that time Ernst Haeckel's classification of bacteria in the kingdom Monera was in place). [91] [121] [122]

Bacterium coli was the type species of the now invalid genus Bacterium when it was revealed that the former type species ("Bacterium triloculare") was missing. [123] Following a revision of Bacterium, it was reclassified as Bacillus coli by Migula in 1895 [124] and later reclassified in the newly created genus Escherichia, named after its original discoverer. [125]

In 1996, the world's worst to date outbreak of E. coli food poisoning occurred in Wishaw, Scotland, killing 21 people. [126] This death toll was exceeded in 2011, when the 2011 Germany E. coli O104:H4 outbreak, linked to organic fenugreek sprouts, killed 53 people.


Flux balance analysis is based on constraints

The first step in FBA is to mathematically represent metabolic reactions (Box 1). The core feature of this representation is a tabulation, in the form of a numerical matrix, of the stoichiometric coefficients of each reaction ( Fig. 1a,b ). These stoichiometries impose constraints on the flow of metabolites through the network. Constraints such as these lie at the heart of FBA, differentiating the approach from theory-based models based on biophysical equations that require many difficult-to-measure kinetic parameters 8, 9 .

Box 1: Mathematical representation of metabolism

Metabolic reactions are represented as a stoichiometric matrix (S), of size m*n. Every row of this matrix represents one unique compound (for a system with m compounds) and every column represents one reaction (n reactions). The entries in each column are the stoichiometric coefficients of the metabolites participating in a reaction. There is a negative coefficient for every metabolite consumed, and a positive coefficient for every metabolite that is produced. A stoichiometric coefficient of zero is used for every metabolite that does not participate in a particular reaction. S is a sparse matrix since most biochemical reactions involve only a few different metabolites. The flux through all of the reactions in a network is represented by the vector v, which has a length of n. The concentrations of all metabolites are represented by the vector x, with length m. The system of mass balance equations at steady state (dx/dt = 0) is given in Fig. 1c 23 :

Any v that satisfies this equation is said to be in the null space of S. In any realistic large-scale metabolic model, there are more reactions than there are compounds (n > m). In other words, there are more unknown variables than equations, so there is no unique solution to this system of equations.

Although constraints define a range of solutions, it is still possible to identify and analyze single points within the solution space. For example, we may be interested in identifying which point corresponds to the maximum growth rate or to maximum ATP production of an organism, given its particular set of constraints. FBA is one method for identifying such optimal points within a constrained space ( Fig. 2 ).

The conceptual basis of constraint-based modeling and FBA. With no constraints, the flux distribution of a biological network may lie at any point in a solution space. When mass balance constraints imposed by the stoichiometric matrix S (1) and capacity constraints imposed by the lower and upper bounds (ai and bi) (2) are applied to a network, it defines an allowable solution space. The network may acquire any flux distribution within this space, but points outside this space are denied by the constraints. Through optimization of an objective function, FBA can identify a single optimal flux distribution that lies on the edge of the allowable solution space.

FBA seeks to maximize or minimize an objective function Z = c T v, which can be any linear combination of fluxes, where c is a vector of weights, indicating how much each reaction (such as the biomass reaction when simulating maximum growth) contributes to the objective function. In practice, when only one reaction is desired for maximization or minimization, c is a vector of zeros with a one at the position of the reaction of interest ( Fig. 1d ).

Optimization of such a system is accomplished by linear programming ( Fig. 1e ). FBA can thus be defined as the use of linear programming to solve the equation Sv = 0 given a set of upper and lower bounds on v and a linear combination of fluxes as an objective function. The output of FBA is a particular flux distribution, v, which maximizes or minimizes the objective function.

Formulation of an FBA problem.(a) First, a metabolic network reconstruction is built, consisting of a list of stoichiometrically balanced biochemical reactions. (b) Next, this reconstruction is converted into a mathematical model by forming a matrix (labeled S) in which each row represents a metabolite and each column represents a reaction. (c) At steady state, the flux through each reaction is given by the equation Sv = 0. Since there are more reactions than metabolites in large models, there is more than one possible solution to this equation. (d) An objective function is defined as Z = c T v, where c is a vector of weights (indicating how much each reaction contributes to the objective function). In practice, when only one reaction is desired for maximization or minimization, c is a vector of zeros with a one at the position of the reaction of interest. When simulating growth, the objective function will have a 1 at the position of the biomass reaction. (e) Finally, linear programming can be used to identify a particular flux distribution that maximizes or minimizes this objective function while observing the constraints imposed by the mass balance equations and reaction bounds.

Constraints are represented in two ways, as equations that balance reaction inputs and outputs and as inequalities that impose bounds on the system. The matrix of stoichiometries imposes flux (that is, mass) balance constraints on the system, ensuring that the total amount of any compound being produced must be equal to the total amount being consumed at steady state ( Fig. 1c ). Every reaction can also be given upper and lower bounds, which define the maximum and minimum allowable fluxes of the reactions. These balances and bounds define the space of allowable flux distributions of a system—that is, the rates at which every metabolite is consumed or produced by each reaction. Other constraints can also be added 10 .


Generating controlled reducing environments in aerobic recombinant Escherichia coli fermentations: Effects on cell growth, oxygen uptake, heat shock protein expression, and in vivo CAT activity

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742Search for more papers by this author

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742

Medical Biotechnology Center, University of Maryland Biotechnology Institute and Department of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, Maryland 21228

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742

Center for Agricultural Biotechnology, University of Maryland Biotechnology Institute and Department of Chemical Engineering, University of Maryland, College Park, Maryland 20742Search for more papers by this author

Abstract

The independent control of culture redox potential (CRP) by the regulated addition of a reducing agent, dithiothreitol (DTT) was demonstrated in aerated recombinant Escherichia coli fermentations. Moderate levels of DTT addition resulted in minimal changes to specific oxygen uptake, growth rate, and dissolved oxygen. Excessive levels of DTT addition were toxic to the cells resulting in cessation of growth. Chloramphenicol acetyltransferase (CAT) activity (nmoles/μg total protein min.) decreased in batch fermentation experiments with respect to increasing levels of DTT addition. To further investigate the mechanisms affecting CAT activity, experiments were performed to assay heat shock protein expression and specific CAT activity (nmoles/μg CAT min.). Expression of such molecular chaperones as GroEL and DnaK were found to increase after addition of DTT. Additionally, sigma factor 32 (σ 32 ) and several proteases were seen to increase dramatically during addition of DTT. Specific CAT activity (nmoles/μg CAT min.) varied greatly as DTT was added, however, a minimum in activity was found at the highest level of DTT addition in E. coli strains RR1 [pBR329] and JM105 [pROEX-CAT]. In conjunction, cellular stress was found to reach a maximum at the same levels of DTT. Although DTT addition has the potential for directly affecting intracellular protein folding, the effects felt from the increased stress within the cell are likely the dominant effector. That the effects of DTT were measured within the cytoplasm of the cell suggests that the periplasmic redox potential was also altered. The changes in specific CAT activity, molecular chaperones, and other heat shock proteins, in the presence of minimal growth rate and oxygen uptake alterations, suggest that the ex vivo control of redox potential provides a new process for affecting the yield and conformation of heterologous proteins in aerated E. coli fermentations. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59: 248–259, 1998.


5 Major Metabolic Pathways in Organisms| Microbiology

The following points highlight the five major pathways in organisms. The pathways are: 1. Glycolysis 2. Pentose Phosphate Pathway 3. Entner-Doudoroff Pathway 4. Tricarboxylic Acid Cycle 5. Glyoxylate Cycle.

Metabolic Pathway # 1. Glycolysis:

Glycolysis (glyco-sugar of sweet, lysis-breakdown) is the initial phase of metabolism during which the organic molecule glucose and other sugar are partially oxidized to smaller molecules e.g. pyruvate usually with the generation of some ATP and reduced coenzymes. Microorganisms employ several metabolic pathways to catabolize glucose and other sugars.

There are three important routes of glucose conversion to pyruvate such as glycolysis or Embden-Myerhof pathway (BMP) pathway, pentose phosphate pathway, and Entner-Doudroff pathway. Glycolysis is the most important type of mechanism by which organisms obtain energy from organic compounds in absence of molecular oxygen. As it occurs in the absence of oxygen, therefore, it is also called anaerobic fermentation.

Since living organisms arose in the environment lacking oxygen, anaerobic fermentation was the only method to obtain energy. However, glycolysis or anaerobic fermentation is present in both aerobic and anaerobic organisms.

Most higher organisms have retained the glycolytic pathway of degradation i.e. glucose to pyruvic acid as a preparatory pathway for complete aerobic catabolism of glucose. Glycolysis also serves as an emergency mechanism in anaerobic organisms to produce energy in the absence of oxygen.

(i) EMP Pathways:

In case of aerobic catabolic carbohydrate metabolism (aerobic respira­tion), some bacteria such as E. coli, Azotobacter spp., Bacillus eutrophus, etc. exhibit EMP pathway whereas, ED pathway (phosphorylated) is followed by the species of Alcaligenes, Rhizobium, Xanthomonas, etc. The non-phosphorylated ED pathway occurs in archaea (Pyrococcus spp., Thermoplasma spp, etc.). It is interesting to note that no archaeobacteria uses EMP pathway.

EMP pathway in bacteria initiates by using the phosphoenol pyruvate phosphotransferase system (PEP: PTS) that converts glucose to glucose 6-phosphate during nutrient transport across the cell membrane.

The glucose 6-phosphate is then isomerized to fructose 6-phosphate which is further converted to fructose 1, 6-bi-phosphate. This conversion requires ATP as a source of energy and an enzyme called phosphofructokinase.

It is essentially the reversal of glycolysis, which fulfills a similar anaplerotic role. It is particularly important during growth on pyruvate related C3 compounds and C2 units. The several class of flow of carbon from pyruvate maintains a supply of hexoses. These are required for cell wall and its component synthesis.

The complete pathway of glycolysis from glucose to pyruvate (Fig. 12.4) were elucidated by Gustav Embden (who gave the manner of cleavage of fructose 1, 6-diphosphate and pattern of subsequent steps) and Otto Meyerhof (who confirmed Embden’s work and studied the energetics of glycolysis), in late 1953s. Therefore the sequence reaction from glucose to pyruvate is also called Embden-Meyerhof pathway or glycolysis (EMP).

The overall balance sheet of glycolysis is given below:

Glucose + 2ADP + 2Pi + 2NAD + → Pyruvate + 2ATP + 2NADH + 2H +

In anaerobic organisms pyruvate is further converted to lactate or other organic compounds like alcohol, etc., after using NADH and H + formed during glycolysis:

Pyruvate + NADH + H+ ↔ Lactate + NAD +

In aerobes the pyruvate is converted to acetyl CoA as a preparatory step for entrance into tricarboxylic acid cycle, for complete oxidation of glucose.

Pyruvate + NAD + + CoA → Acetyl CoA + NADH + H + + CO2

Glycolysis is carried out by the help of ten enzymes for ten reactions of the glycolytic pathway. These enzymes are present in the soluble portion of the cytoplasm. All the mtermediates of the glycolytic pathway are phosphorylated compounds. The most important use of phosphate groups is in the production of ATP from ADP and phosphate.

The complete reactions of glycolytic pathway can be divided into two stages. In the first stage, ATP is utilized and glucose is converted into two molecules of three carbon compounds, glyceraldehyde 3-phosphate and dihydroxy acetone phosphate. The glyceraldehyde 3-phosphate is converted into pyruvic acid resulting in a net synthesis of two molecules of ATP.

The complete reaction with respective enzyme is shown in Fig. 12.4:

Apart from glucose, other types of sugar (monosaccharides, disaccharides, polysaccharides) can also enter the glycolytic pathway.

(а) Polysaccharides e.g. Glycogen:

Glucose-6-phosphate Glucose 6 – phosphate can enter as an intermediate of glycolysis.

(b) Disaccharides e.g. Sucrose:

The three key regulatory enzymes, hexokinase, phosphofructokinase and pyruvate kinase act irreversibly and rest of the steps are reversible.

(c) Homo-saccharides: e.g. Fructose:

Fructose can enter the glycolysis by changing to glyceraldehyde 3-phosphate.

Dihydroxyacetone phosphate can enter the glycolysis after enzymatically converting to dihydroxyacetone phosphate.

(ii) Alternate EMP Pathway-Methyl Glyoxal Pathway:

The methyl glyoxal pathway is an alternate of the EMP pathway. It Operates in the presence of low concentration of phosphate to the bacteria, E. coli, Clostridium spp., Pseudomonas spp. etc. In this pathway, dihydroxyacetone so formed converted to methyl glyoxal which later on gives rise to pyruvate.

Hence, there is complete absence of the phosphorylation step in which glyceraldehyde 3-phosphate forms 1, 3-bis-phospho- glycerate. The methyl glyoxal pathway consumes O2 and ATP and no ATP is generated in this pathway (Fig. 12.5).

Metabolic Pathway # 2. Pentose Phosphate Pathway (PPP):

Pentose phosphate pathway is an alternative of glucose degradation. This pathway, also called hexose monophosphate shunt (HMP) or phosphogluconate pathway is not the major pathway, but is a multipurpose pathway. Its main function is to generate reducing power in the extra-mitochondrial cytoplasm in the form of NADH. Its second function is to convert hexoses into pentoses, required in synthesis of nucleic acids.

Its third function is complete oxidative degradation of pentose. The reactions of phosphogluconate pathway take place in the soluble portion of extra-mitochondrial cytoplasm of cells.

The bacteria which show PPP are Bacillus subtilis, E. coli. Streptococcus faecalis and Leuconostoc mesenteroides. Apart from microorganisms the prominent tissues which show PPP are liver, mammary gland and adrenal cortex. The complete PPP is given in Fig 12.6.

There are three enzymes involved in PPP i.e. transketolase, transaldolase and ribulosephosphate 3-epimerase. Ribulose phosphate 3-epimerase catalyzes the conversion of ribulose 5-phosphate into the epimer xylulose 5-phosphate. Transketolase transfers the glycoaldehyde group (CH, OH—CO—) from xylulose 5-phosphate to ribose 5-phosphate to yield sedoheptulose 7-phosphate and glyceraldehyde-3-phosphate.

Transketolase also catalyzes the transfer of glycoaldehyde group from a number of 2-keto sugar phosphate to carbon atom one of a number of different aldose phosphate. Transaldolase acts on the products of transketolase and transfer dihydroxyacetone group to form fructose 6-phosphate and erythrose 4-phosphate (Fig. 12.6).

Fig. 12.6 : The Pentose phosphate pathway.

Pentose phosphate pathway thus functions according to the needs of the cell. If the requirement of reducing power is more then it proceeds towards the formation of NADPH but if pentoses are required it functions in the direction of formation of pentose. But if the cell requires instant energy the PPP stops and glycolysis and TCA proceed.

(a) To anabolic reactions that require electron donors

(b) To Calvin-Benson Cycle (dark reactions of photosynthesis)

(c) To synthesis of nucleotides and nucleic acids

(d) To step e of glycolysis

(e) To glucose 6-phosphate which can enter the pentose phosphate pathway or glycolysis

(f) To synthesis of several amino acids.

Metabolic Pathway # 3. Entner-Doudoroff Pathway:

Apart from glycolysis, Entner-Doudoroff pathway is another pathway for oxidation of glucose to pyruvic acid. This pathway is found in some Gram-negative bacteria like Rhizobium, Agrobacterium and Pseudomonas and is absent in Gram-positive bacteria. In this pathway each molecule of glucose, forms two molecules of NADPH and one molecule of ATP. The complete pathway is shown in the Fig. 12.7.

In this pathway glucose 6-phosphate is oxidized to 6-phosphogluconate, then converted to 2- keto-3-deoxy-6-phosphoglucose (kDPG) cleaved using enzyme to give rise glyceraldehyde’s 3- phosphate and pyruvate directly without generation of ATP. The catabolism of glucose results in production of only one ATP molecule whereas in EMP pathway, two ATP molecules are produced. This seems that EMP pathway more efficient than that of ED pathway.

Further, difference between ED pathway and PP pathway is the generation of reduced NADPH from NADP in the former. It is interesting to note that coenzyme NADP+ and NADPH are used in anabolic reactions. Thus, the ED pathway provides an important mechanism for producing NADPH and the 3-carbon building blocks used in biosynthetic reactions etc.

Partially non-phosphorylated ED pathway is involved in some bacteria such as Clostridium spp. Achromobacter spp., Alcaligens spp. and Archaea (Halobacterium spp.) In this case, intermediate product formed prior to kDPG is non-phosphorylated, and phosphogluconate is dehydrated to give rise kDPG, which gives to pyruvate.

In later steps, the reactions of ED pathway are followed. This pathway is also found in other bacteria such as Pseudomonas aeruginosa, Azotobacter, and Enterococcus faecalis, and an anaerobic bacterium Zymomonas mobilis.

Metabolic Pathway # 4. Tricarboxylic Acid Cycle:

The tricarboxylic acid cycle was first given by H.A. Krebs in 1937. H.A. Krebs then gave the name citric acid cycle. Because of citric acid is the first product of Krebs cycle, is also known as TCA cycle due to presence of three carboxylic groups in a molecule of citric acid.

The cycle is of universal occurrence in all the aerobic organisms and leads to complete oxidation of glucose to CO2 and H2O while glycolysis leads to incomplete oxidation of glucose to pyruvate.

Tri-carboxyhc acid cycle completely oxidises it to release large amount of energy in the form of NADH + H + mainly and GTP. NADH + H + enter the respiratory chain where each NADH + H + produces three ATP molecules. GTP is converted to ATP by substrate level oxidation. Another form of energy is in the form of substrate of FADH 2 , which also enters the respiratory chain to form two molecules of ATP.

All the reactions of tricarboxylic acid cycle take place in the inner compartment of mitochondrion. Some of these enzymes occur in the matrix of inner compartment, while rest of them occur on the inner mitochondrial membrane. For the start of the cycle, the pyruvate formed in the glycolysis is first converted to acetyl Co-A by preparatory reaction.

Pyruvate + NAD + + CoA → acetyl CoA + NADH + H+ + CO2

The reaction is irreversible and is not itself a part of the tricarboxylic acid cycle. It is carried out with the help of the enzyme pyruvate dehydrogenase. Acetyl CoA then enters the cycle after combining with oxaloacetate to form citrate, after which a cycle of reactions occurs (Fig. 12.8) leading to the formation of six CO2, eight NADH + H + , one FADH2 and one molecule of glucose.

There are few key steps in the tricarboxylic acid cycle which control the cycle as per need of the cell. The first of these controls is the preparatory reaction. The activity of pyruvate dehydrogenase is reduced in the presence of excess ATP and again increases in the absence of ATP.

There are two more steps which can control the cycle. These are the isocitrate dehydrogenase reaction (which requires ADP as positive regulation), and succinate dehydrogenase reaction (promoted by succinate, phosphate and ATP). However, the key control of the cycle is the reaction carried out by citrate synthase. This is the primary control of the cycle.

Metabolic Pathway # 5. Glyoxylate Cycle:

It is anaplerotic reaction in which oxaloacetate is taken from TCA cycle to meet out the demand of carbon requirement for amino acid biosynthesis. Hence, these intermediates have to be replenished via an alternate route, called anaplerotic pathway i.e. glyoxylate pathway. This cycle operates for glucoiieogenesis. Glyoxylate cycle was given first by Krebs and H.R. Kornberg.

This cycle is a modified form of tricarboxylic acid cycle found in plants and those microorganisms which utilize fatty acids as the source of energy in the form of acetyl Co A.

In this cycle the CO2 evolving steps of tricarboxylic acid cycle were by-passed and instead a second molecule of acetyl CoA is utilized (which condenses with glyoxylate to form malate). Succinate is a by product, used for biosynthesis, particularly in gluconeogenesis.

The overall reaction of glyoxylate cycle is given below:

2 Acetyl Co-A + NAD + + 2H2O → Succinate + 2CoA + NADH + H +

The two key enzymes, isocitrate lyase and malate synthase of glyoxylate cycle are localised in cytoplasmic organelles called glyoxysomes. Glyoxylate cycle goes on simultaneously with the tricarboxylic acid cycle, while tricarboxylic acid provides energy glyoxylate cycle provides succinate for the formation of new carbohydrate from fats as shown in Fig. 12.9.


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