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What pathway would E. coli use to make pyruvate from acetate? I have found several papers that refer to a possible mechanism that could move acetate to pyruvate via a three-step process:
Step 1: Acetate Kinase (EC 18.104.22.168)
Step 2: Phosphate acetyltransferase (EC 22.214.171.124)
Step 3: Ferredoxin oxidoreductase (EC 126.96.36.199)
Is this the pathway that would be used in an E. coli system?
Several pathways/metabolic reactions are required for bacteria to convert acetate to pyruvate - or more pertinently to the gluconeogenic pathway - but the key is the glyoxylate cycle or shunt. The sequence of reactions is:
- 2 acetate → 2 acetyl CoA (acetyl CoA synthetase)
- 1 acetyl CoA + oxaloacetate → citrate → isocitrate (entry to Krebs TCA cycle)
- isocitrate → glyoxylate + succinate (glyoxylate shunt)
- succinate → oxaloacetate (Krebs TCA cycle)
- 1 acetyl CoA + glyoxylate → malate (glyoxylate shunt)
- malate → oxaloacetate (Krebs TCA cycle)
Thus, the two molecules of acetate replenish the original molecule of oxaloacetate used for one of them to enter the tricarboxylic acid (TCA) cycle, with - in addition - a second molecule of oxaloacetate. This is available for:
- oxaloacetate → phosphoenolpyruvate (PEP carboxykinase of gluconeogenesis)
The phosphoenolpyruvate (PEP) could, in principle, be converted to pyruvate:
- phosphoenolpyruvate → pyruvate (pyruvate kinase of glycolysis)
Although this sequence of reactions would normally be used to generate glucose etc via gluconeogenesis.
The link betweeen the TCA cycle and glucose metabolism
In addition to oxidizing acetyl CoA, the Krebs tricarboxylic acid cycle (TCA cycle) can act as source of certain metabolic intermediates for other pathways. As shown below, oxaloacetate (OAA) can be converted to phosophoenol pyruvate (PEP), in a reaction that is part of the gluconeogenic conversion of pyruvate to PEP.
Although certain bacteria can convert acetate into acetyl CoA in a reaction catalysed by acetyl CoA synthetase the TCA cycle per se does not perform a net conversion acetyl CoA to OAA, because a molecule of OAA is needed for acetylCoA to enter the TCA cycle and must be regenerated. The TCA cycle, in effect, oxidizes acetyl CoA to carbon dioxide. The TCA cycle, per se, can only convert to OAA and PEP those intermediates that enter the cycle directly after the acetyl CoA stage.
The glyoxylate shunt
The foregoing explains why most animals cannot convert fat to glucose. (Acetyl CoA cannot be converted to pyruvate by pyruvate dehydrogenase is this reaction is effectively irreversible.) However plant and certain bacteria (including Escherichia coli) can overcome this limitation by means of the glyoxylate shunt. A text available online that explains this is Berg et al., in a section entitled 'The Glyoxylate Cycle Enables Plants and Bacteria to Grow on Acetate'. To help clarify this I have prepared my own diagram, below.
This involves the conversion:
oxaloacetate + 2 acetyl CoA → 2 oxaloacetate
i.e. the net conversion of
2 acetyl CoA → oxaloacetate
The glyoxylate shunt reactions are imposed on the TCA cycle with a pink overlay. One molecule of acetyl CoA condenses with OAA to enter the TCA cycle in the normal manner. However the isocitrate formed from citrate, instead of being decarboxylated, is converted to glyoxylate and succinate. The succinate - a dicarboxylic acid - regenerates the original molecule of OAA used to allow acetyl CoA to enter the TCA cycle.
What of the glycoxylate produced in the isocitrate lyase reaction? It reacts with a second molecule of acetyl CoA to produce the dicarboxylic acid malate in a reaction catalysed by malate synthase, the second unique enzyme of the shunt. This TCA cycle intermediate is converted to OAA. This second OAA is a net gain from the metabolism and can be converted to PEP, as indicated in the first diagram.
The poster asks about production of pyruvate from acetate. This is technically possible through the pyruvate kinase reaction. However I do not quite see the point of this. I would normally expect PEP to be channeled allong the gluconeogenic pathway towards glucose.
Coda: Pyruvate:ferredoxin oxidoreductase
The poster suggests a series of reactions to convert acetate to pyruvate, the final one being that catalysed by pyruvate:ferredoxin oxidoreductase. Although the pyruvate:ferredoxin oxidoreductase (EC 188.8.131.52) reaction is reversible, it generally functions to oxidize pyruvate to acetyl CoA. It is used as an alternative to pyruvate dehydrogenase by bacteria under anaerobic conditions (as it does not require NAD+), and this is regarded as its role in E. coli, and to an even greater extent in obligate anaerobes such as Clostridium.
The reverse reaction suggested by the poster, involving the synthesis of pyruvate from acetyl CoA, is restricted to autotrophs
Escherichia coli is a heterotrophic organism, meaning that it obtains its food from a different source. This source is most often its host organism. And from their host, they obtain Carbon via biosynthesis of organic molecules that were ingested by their host. Carbon is very important to E. coli because the bacterial cell is composed almost ENTIRELY of carbon molecules bound to other important elements ( CHNOPS , anyone?)
E. coli's main source of carbon comes from glucose molecules ingested by its host organism. This is then broken down into useable carbon by means of central metabolism , which consists of three steps:
1. Embden-Meyerhof-Parnas (EMP) Pathway : converts glucose to pyruvate
2. Tricarboxylic Acid (TCA) cycle : oxidizes Acetyl CoA to CO2
3. Pentose Phosphate Cycle (PPP) : oxidizes glucose to CO 2
Copyright-free image retrieved from BioMed Central (View Permission Here )
E. coli can also utilize the carbon from gluconate , and this is metabolized through the use of enzymes from the Entner-Doudoroff pathway (E. coli therefore doesn't directly use this pathway). The Entner-Doudoroff is similar to the EMP pathway in that gluconate is converted into pyruvate, but what is different is that the pyruvate is converted to CO2 and acetaldehyde . Then the acetaldehyde is further changed to ethanol .
E. coli can also convert the pyruvate that it generates through the EMP pathway into ATP through fermentation (which is easy to understand since E. coli is a facultatively anaerobic organism). High ATP production is essential for ensuring that the organism will have sufficient energy to allow metabolism to occur.
* CHNOPS stands for C arbon, H ydrogen, N itrogen, O xygen, P hosphorus, and S ulfur
Photo from a Public Doman, retrieved from Wikimedia Commons
Growing E. coli is easy and fast
Scientists first chose to work with E. coli because it was easy and fast to grow in the laboratory.
There are several features of E. coli that make it easy to culture:
- It likes it warm – but not too warm. Because E. coli is a gut bacterium, it grows best at body temperature (37.4ºC). This is an easy temperature for scientists to work with in the laboratory.
- It isn’t fussy about nutrition.E. coli can obtain energy from a wide variety of sources. In its natural environment (the gut), it consumes digested foodstuffs. In a laboratory context, E. coli can be fed easily and cheaply – think chicken soup for bacteria
- It can grow with or without oxygen. In the gut, E. coli grows anaerobically (in the absence of oxygen). However, unlike some anaerobic bacteria E. coli also grows well in aerobic environments, such as a culture flask in a laboratory.
- It grows fast. Under ideal conditions, individual E. coli cells can double every 20 minutes. At that rate, it would be possible to produce a million E. coli cells from one parent cell within about 7 hours. Fast growth means that experiments involving E. coli can be done quickly, conveniently and cheaply.
Experimental determination of the ATP cost under anaerobic conditions
For experimental calculations one needs to know how to convert from the amount of carbon source consumed to the amount of ATP produced. It is easiest to calculate YATP under anaerobic conditions because ATP is only generated outside the mitochondria (substrate phosphorylation). The amount of ATP produced per substrate molecule is well established (Stouthamer 1979). For example, the anaerobic breakdown of glucose into pyruvate (glycolysis) yields 2 ATP. E. coli can produce another ATP by further conversion of the carbon source into acetate, for a total of 3 ATP/glucose. However, it is important to remember that not all glucose is used to create ATP glucose is both the energy and the carbon source in the experimental data presented below. By knowing the carbon content of the cell biomass, one can infer the net amount of carbons that become part of the cell’s dry mass, and therefore the remaining part is the amount of glucose which goes into the creation of ATP for energy. The basic input into these calculations is the amount of dry cell weight produced per mole of substrate consumed, also known as Yglu for a glucose substrate. The amount of substrate consumed can be measured experimentally, which enables the transformation of Yglu to YATP (Stouthamer and Bettenhausen 1973).
YATP values were found to be dependent on the growth rate. At higher growth rates, less ATP was needed to form cells. With growth rates ranging from 0.570 to 0.087 per hour, the YATP values ranged from 7.4 to 3.0 grams of cells per mole of ATP, respectively, on glucose and minimal media in continuous culture (Hempfling and Mainzer 1975). The slower the growth rate, the more ATP it takes to make one gram of cells. Unless the composition of cells is changing at different growth rates, this increased ATP requirement/cell most likely reflects the longer duplication time, which requires much higher maintenance costs per cell duplication. These numbers need to be corrected for the extra maintenance costs implicit in longer doubling times. It was recognized that the slope of a plot of 1/YATP vs. 1/μ, where μ is the growth rate, is equivalent to a value of the growth-rate-independent energy requirement, or maintenance energy (Pirt 1965). This is used to calculate a value of YATPmax, the maximum yield of grams of cells per mol of ATP which aims not to include the maintenance energy costs.
When YATP is corrected for maintenance costs, the value for duplicating a cell’s contents (now referred to as YATPmax after the correction) is 10.3 grams of cells per mole of ATP (Hempfling and Mainzer 1975). This is equivalent to 16.4 billion ATP/cell (BNID 101983). Under similar conditions (glucose-limited anaerobic growth in a chemostat), a different set of authors determined a YATPmax value of 8.5 (19.8 billion ATP/cell) (Stouthamer and Bettenhausen 1977). Therefore, under glucose-limited anaerobic growth, it requires
16-20 billion ATP molecules to generate a new E. coli.
Overexpression of the genes and overproduction of their coded proteins involved in glycerol uptake and metabolism were detected. These proteins are responsible for the transport and incorporation of glycerol as DHAP, one of the metabolites of the glycolytic pathway. Overexpression of several glycolytic and gluconeogenic genes in the upper part of the glycolytic pathway, especially fbaA, fbaB, fbp and pgi, are responsible for the production of G6P from DHAP. Low F1,6P/F6P levels could be the signal for the induction of some of these regulons when strain JM101 is growing on glycerol. This phenomena is reinforced by the differential expression of some genes regulated by Cra which respond to low F1,6P concentrations.
The detected overexpression of the mal/lam and mgl/gal regulons and the overproduction of their coded proteins and some genes regulated by RpoS, indicate that JM101 apparently induced a “carbon stress and carbon scavenging response” when growing on glycerol as the sole carbon source, indicating as reported that this carbohydrate is a poor carbon source. This proposition is in agreement with the involvement of RpoS, the master regulator of stress response in glycerol fermentation, since its inactivation reduced 10% the μ and delayed by two hours the growth of JM101ΔrpoS.
The detected overexpression of poxB, acs, pta, actP, acnB and the glyoxylate shunt genes (aceBA and glcB), some of them transcribed by RpoS, indicates that JM101 is apparently producing and simultaneously consuming acetate when growing on glycerol as the sole carbon source. In agreement with this proposal, was the result that no acetate was detected when growing on glycerol, and acetate can be coutilized with glycerol as carbon sources. It has been proposed that when E. coli is growing slowly on glucose apparently reduces the carbon flux through the Pdh system, which yields AcCoA directly from PYR, and diverts part of the carbon flux via PoxB that synthesizes acetate from PYR, with a concomitant reduction of quinones at the membrane. Acetate, is then utilized by Acs and transformed into AcCoA, apparently creating a “carbon acetate recycling” mechanism which is also apparently present in PB11 (a derivative of JM101 lacking PTS) that grows slowly on glucose. Therefore, it appears that in addition to the glycolytic metabolism that is functioning in JM101 when growing on glycerol, carbon scavenging responses are also observed in this strain when it grows on this poor carbon source. Consistent with this proposed metabolic response, in JM101 cultures grown on glycerol as mentioned, no acetate was detected, because this strain probably recycles acetate through the PoxB-Acs-glyoxylate shunt enzymes and is capable of coutilizing glycerol and acetate. In accordance, JM101 derivatives with inactive poxB or pckA genes accumulated acetate and their specific growth rates were affected. The induction of this mechanism apparently permitted a more efficient carbon utilization and acetate recycling in these growing conditions. In agreement, the downregulation of icd and lpdA coding for IcdA and LpdA (part of the SucABCD complex), supports the proposition that the carbon flux is reduced through the lower section of the TCA cycle, thus enabling carbon gluconeogenic recycling through the glyoxylate shunt, since aceBA and glcB were overexpressed. As a result, if this hypothesis is correct, less carbon should be lost as CO2 in the lower section of the TCA pathway.
In agreement with a reduced TCA cycle during the growth of JM101 on glycerol, it appears that relatively low production of C4 carbon metabolites occurred, given that the μ of this strain was enhanced in cultures grown on glycerol when succinate, malate or aminoacids derived from 2-oxoglutarate were added to the fermentation. This supports the hypothesis that when E. coli grows slowly, part of the carbon is recycled, preserved through the glyoxylate shunt and not lost as CO2 in the TCA cycle.
Indole production in JM101 grown on glycerol indicates an important carbon flux through the aromatic amino acids pathway. Indole is a signaling molecule that activates Crl for modulating the expression of certain RpoS-Crl regulons however, a signaling role of this metabolite is not completely clear at the moment. It has been proposed that the expression of rpoS is not only negatively controlled by cAMP-CRP high levels but also inversely correlated with growth rate. Since glycerol is a relatively poor carbon source, intracellular high levels of cAMP-CRP are expected in JM101. Indole synthesis could stimulate RpoS activity under these non favorable growth conditions. Additional studies should be conducted to gain a better understanding into the role of this signal. Nevertheless, the detected overproduction of Cdd, DeoD, and Upp suggests that the carbon flux through the pentose-phosphate pathway is reduced when glycerol is used as the sole carbon source as compared to the flux when glucose is utilized. It appears that when glycerol is used as the only carbon source, a carbon stress mechanism occurs. In this condition, RpoS regulons could be also indirectly activated by indole, allowing a more adequate response to growth on carbon limited conditions. Importantly, and in agreement with an increased carbon flux through the aromatic pathway growing on glycerol when JM101 is transformed with plasmid pJLBaroG fbr tktA that redirects and enhances carbon flux into the aromatic pathway, this strain showed a yield increase of aromatic compounds almost 9-fold as compared to the production of these metabolites when glucose is used as the sole carbon source.
The transcription levels of most of the measured genes correlated with the detected values of the proteins produced in the analyzed growth conditions, using glycerol as the only carbon source. Also, the specific activities of various measured proteins correlated with these values.
In this contribution we described new features of E. coli physiology during the growth on glycerol, as detected through a proteomic-transcriptional study and kinetic-stoichiometric evaluation of strain JM101 and some isogenic mutants in certain key PEP-PYR genes (poxB, ppc, pckA, pykA and pykF) and in rpoS. It appears that when glycerol is used as the sole carbon source in addition to the glycolytic metabolism, a carbon stress response occurs that includes carbon scavenging and acetate gluconeogenic carbon recycling responses mediated mainly by RpoS. In addition, this regulator could also be activated by Crl through indole, allowing a more adequate response to growth on glycerol, a carbon limited condition. The simultaneous utilization of various metabolic redundant alternative mechanisms when growing on glycerol indicates metabolic plasticity of E. coli. Understanding these capacities advances the knowledge on the physiological responses E. coli is capable of, and enhances our capacities for developing more advanced metabolic engineering strategies using this bacterium for the production of specific metabolites.
ACADEMIC EDITOR: Raul Rivas, Editor in Chief
PEER REVIEW: Four peer reviewers contributed to the peer review report. Reviewers’ reports totaled 870 words, excluding any confidential comments to the academic editor.
FUNDING: Authors disclose no external funding sources.
COMPETING INTERESTS: Authors disclose no potential conflicts of interest.
Paper subject to independent expert single-blind peer review. All editorial decisions made by independent academic editor. Upon submission manuscript was subject to anti-plagiarism scanning. Prior to publication all authors have given signed confirmation of agreement to article publication and compliance with all applicable ethical and legal requirements, including the accuracy of author and contributor information, disclosure of competing interests and funding sources, compliance with ethical requirements relating to human and animal study participants, and compliance with any copyright requirements of third parties. This journal is a member of the Committee on Publication Ethics (COPE).
Conceived and designed the experiments: VK and VKRV. Analyzed the data: VK and VKRV. Wrote the first draft of the manuscript: VK and VKRV. Contributed to the writing of the manuscript: VK and VKRV. Agree with manuscript results and conclusions: VK and VKRV. Jointly developed the structure and arguments for the paper: VK and VKRV. Made critical revisions and approved final version: VK and VKRV. Both authors reviewed and approved of the final manuscript.
6.10.4 Biological suitability test for water
Principle: cultures of Enterobacter aerogenes are made in liquid media one medium is made with the water to be tested, the other with high-quality reference distilled water (e.g. high-quality water for pharmaceutical/injection use, codex guaranteed).
A difference greater than 15 to 20% between the numbers of colonies after 24 hours in the test culture and in the control gives evidence of toxicity (See ISO 9998-1991, Annex B).
This test should be carried out on commissioning of the apparatus and after any subsequent changes. Testing may also be required during the investigation of any quality control problems.
In the latest work, Milo and his team used a mix of genetic engineering and lab evolution to create a strain of E. coli that can get all its carbon from CO2. First, they gave the bacterium genes that encode a pair of enzymes that allow photosynthetic organisms to convert CO2 into organic carbon. Plants and cyanobacteria power this conversion with light, but that wasn’t feasible for E. coli. Instead, Milo’s team inserted a gene that lets the bacterium glean energy from an organic molecule called formate.
Even with these additions, the bacterium refused to swap its sugar meals for CO2. To further tweak the strain, the researchers cultured successive generations of the modified E. coli for a year, giving them only minute quantities of sugar, and CO2 at concentrations about 250 times those in Earth’s atmosphere. They hoped that the bacteria would evolve mutations to adapt to this new diet. After about 200 days, the first cells capable of using CO2 as their only carbon source emerged. And after 300 days, these bacteria grew faster in the lab conditions than did those that could not consume CO2.
The CO2-eating, or autotrophic, E. coli strains can still grow on sugar — and would use that source of fuel over CO2, given the choice, says Milo. Compared with normal E. coli, which can double in number every 20 minutes, the autotrophic E. coli are laggards, dividing every 18 hours when grown in an atmosphere that is 10% CO2. They are not able to subsist without sugar on atmospheric levels of CO2 — currently 0.041%.
Milo and his team hope to make their bacteria grow faster and live on lower levels of CO2. They are also trying to understand how the E. coli evolved to eat CO2: changes in just 11 genes seemed to allow the switch, and they are now working on determining how.
The work is a “milestone” and shows the power of melding engineering and evolution to improve natural processes, says Cheryl Kerfeld, a bioengineer at Michigan State University in East Lansing and the Lawrence Berkeley National Laboratory in California.
Already, E. coli is used to make synthetic versions of useful chemicals such as insulin and human growth hormone. Milo says that his team’s work could expand the products the bacteria can make, to include renewable fuels, food and other substances. But he doesn’t see this happening soon.
“This is a proof-of-concept paper,” agrees Erb. “It will take a couple years until we see this organism applied.”
Escherichia coli bacteria
Four different strains of Escherichia coli on Endo agar with biochemical slope (see here). Glucose fermentation with gas production, urea and H2S negative, lactose positive (with exception of strain D - "late lactose fermenter" on Endo agar it looks like lactose negative). All four strains are mannitol positive (best seen in fig. D), cellobiose negative (strains A, B). Approximately 50% of E.coli strains are sucrose negative (fig.A, D). Colonies of some strains have typical greenish metallic sheen (fig.E) but many of them grow without it (fig.F).
Escherichia coli (commonly abbreviated E. coli) is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans. The harmless strains are part of the normal flora of the gut, and can benefit their hosts by producing vitamin K2, and by preventing the establishment of pathogenic bacteria within the intestine. E. coli and related bacteria constitute about 0.1% of gut flora, and fecal-oral transmission is the major route through which pathogenic strains of the bacterium cause disease.
Certain strains of E. coli, such as O157:H7, O104:H4, O121, O26, O103, O111, O145, and O104:H21, produce potentially lethal toxins. Food poisoning caused by E. coli can result from eating unwashed vegetables or undercooked meat. O157:H7 is also notorious for causing serious and even life-threatening complications such as hemolytic-uremic syndrome. The O104:H4 strain is equally virulent. It is the strain behind the deadly June 2011 E. coli outbreak in Europe.
- Enterotoxigenic E. coli (ETEC)
- Enteropathogenic E. coli (EPEC)
- Enteroinvasive E. coli (EIEC)
- Enterohemorrhagic E. coli (EHEC)
- Enteroaggregative E. coli (EAEC)
Escherichia coli and Urinary Tract Infections (UTIs)
Escherichia coli and urinary tract infections are often discussed together as E. coli (uropathogenic E. coli, UPEC) is often indicated as the major cause of UTIs. Basically, the urinary tract comprises the parts of the body responsible for the removal of body waste and excess water, and the maintenance of electrolyte balance in the body. The urinary tract includes the kidneys, the bladder, the urethra and the ureters.
Causes of UTIs
Naturally, the urinary system is immune to infections therefore, certain microorganisms must invade it before it can be infected. In ascending infections, fecal bacteria colonize the urethra and spread up the urinary tract to the bladder as well as to the kidneys (causing pyelonephritis), or the prostate in males. Because women have a shorter urethra than men, they are more likely to suffer from an ascending UTI. Escherichia coli are the main causative bacteria of UTIs they are responsible for 4 out of 5 cases of the infections. Uropathogenic E. coli use P fimbriae (pyelonephritis-associated pili) to bind urinary tract endothelial cells and colonize the bladder. Uropathogenic E. coli often produce alpha- and beta-hemolysins, which cause lysis of urinary tract cells.
Apart from E. coli, other bacteria like Pseudomonas aeruginosa, Staphylococcus saprophyticus, Neisseria gonorrhoeae, Klebsiella pneumoniae, Enterococcus faecalis, Proteus spp. are also capable of causing urinary tract infections. The most common yeasts causing complicated and uncomplicated urinary tract infections are Candida albicans and other Candida species (e.g., C.glabrata). Rarely they may be due to viral or parasitic infections.
Symptoms of UTIs
In some cases, urinary tract infections and other Escherichia coli infections are often unnoticed as they show no symptoms. However, their common symptoms include:
- Burning sensations during urination
- Feverish conditions
- Constant, strong urge to urinate
- In male – pain in the rectal region
- In female – pain in the pelvis
- Frequency, intense passing out of small amounts of urine
- Urine with appearances of blood and/or foul odor
- Pain around the hips, abdomen, or lower back region
There are rarely any major complications associated with urinary tract infections. However, if the infections are left untreated for long, chronic infections may develop with conditions such as kidney stone, abscesses, fistulas and, in some rare cases, cancer of the bladder, kidney damages or death.
Treatment of Urinary Tract Infections
The infections are generally treated with the aid of antibiotics which are substances capable of destroying bacteria and other related organisms in the body. Antibiotics can either be given orally (e.g., nitrofurantoin) or intravenously based on the severity of the infections. They are given orally if the infections are still at their mild state, while they would be given via the vein (intravenous mode) in severe cases.
Preventions of Urinary Tract Infections
Escherichia coli basic characteristics
- GRAM-NEGATIVE RODS
- CATALASE: POSITIVE
- OXIDASE: NEGATIVE
- FACULTATIVELY ANAEROBIC
Escherichia coli biochemical identification
- POS. positive ( > 90% of strains are positive)
- D most positive (51 - 89%)
- d most negative (11 - 50%)
- NEG. negative (0 - 10%)
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