Siddhartha Dan, MIAZ, M.Tech (I.K Gujral Punjab Technical University, Jalandhar, Punjab) Microbiology Industrial Importances of E. coli (Morphology, Growth Requirements, and Genetics) Microbial Biotechnology

3. Morphology,
Growth Requirements,
Genetics
E.coli

5. Industrial microbiology is a branch of biotechnology and
microbiology, which mainly deals with the study of various
microorganisms and its applications in industrial processes.
In large-scale industrial processes, microbes are widely used to synthesize a number of
products valuable to human beings. There are numerous industrial products that are
derived from microbes such as:
• Food additives.
• Alcoholic and non-alcoholic beverages.
• Biofuels, metabolites, and biofertilizers.
• Few Chemicals, Enzymes and other Bioactive Molecules.
• Vaccines and other Antibiotics to kill or retard the growth of disease-causing microbes.
These microbes play a crucial role in the fermentation process to obtain a number of
products, obtained by fermentation process through industrial processes are fermented
beverages, malted cereals, broths, fruit juices, antibiotics, Vitamins, Enzymes etc.

6. Today Lecture
 Habitat of E. coli
 Morphology of E. coli
 Antigenic Structure of E. coli
 Cultural Characteristics of E. coli
 Growth Factors Requirements
 Cell Cycle
 DNA Replication
 Genetics Material
 Industrial Importances
 Treatment of E. coli infections
 Prevention and Control of E. coli infections

7. 1.E. coli is the normal flora of the human body.
2.Most of them are harmless and opportunistic.
3.E. coli constitute about 0.1% to 1% of GI tract bacteria.
4.It is the largest group of bacteria living in the intestine.
5.E. coli was discovered by Theodor Escherich in 1885 after isolating it from the feces of newborns.
6.The niche of E. coli depends upon the availability of the nutrients within the intestine of host organisms.
7.Primary habitat of E. coli is in the gastrointestinal tract of humans and many other warm-blooded animals.
8.It is found in the mucus or the epithelium on the wall of the intestine and colon of the large intestine
9.E. coli helps with the absorption of vitamin K and other vitamins in the colon.
10.E. coli is also found in human feces and ground meats due to slaughterhouse processing
11.When E. coli is excreted from the intestinal tract, the bacteria are able to survive only for a few hours.
12.E. coli is found outside the body in faecally contaminated environments such as water or mud or sediments.
13.If E. coli comes in contact with raw vegetables, it has the potential to attach itself to the leaves of the vegetable.
14.E. coli can also be found in environments at a higher temperature, such as on the edge of hot springs.
Habitat of E. coli

8. Morphology of E. coli
1. E. coli is gram-negative (-ve) rod-shaped bacteria.
2. It is 1-3 x 0.4-0.7 µm in size and 0.6 to 0.7 µm in volume.
3. It is arranged singly or in pairs.
4. It is motile due to peritrichous flagella.
5. Some strains are non-motile.
6. Some strains may be fimbriated.
7. Fimbriae are of type 1 (hemagglutinating & mannose-sensitive)
and are present in both motile and non-motile strains.
1. Some strains of E. coli isolated from extra-intestinal infections
have a polysaccharide capsule.
1. They are non-sporing and facultative anaerobes
2. They have a thin cell wall with only 1 or 2 layers of peptidoglycan.
3. Growth occurs over a wide range of temperatures from 15-45°C.

9. Antigenic Structure of E. coli
Heat Stable Lipopolysaccharide (LPS) is the major cell wall
antigen of E. coli & E. coli possesses 4 antigens; H, O, K and F.
H or Flagellar Antigen
 Heat and alcohol labile protein
 Present on the flagella
 Genus specific
 Present as monophasic
 75 ‘H’ antigens have been recognized
O or Somatic Antigen
 Heat stable, resistant to boiling up to 2 hrs. 30 min.
 Occur on the surface of the outer membrane
 An integral part of the cell wall
 173 ‘O’ antigens have been recognized
K or Capsular Antigen
 Heat labile
 Acidic polysaccharide antigen present in the envelope
 Boiling removes the K antigen
 Inhibit phagocytosis
 103 ‘K’ antigens have been recognized
F or Fimbrial Antigen
 Heat labile proteins
 Present in the fimbriae
 K88, K99 antigens

10. Cultural Characteristics of E. coli
 E. coli is a facultative anaerobe.
 Its optimum growth temperature is 37°C and ranges from 10°C to 40°C.
E. coli on Nutrient Agar (NA)
1. They appear large, circular, low convex, grayish, white, moist, smooth, and opaque.
2. They are of 2 forms: Smooth (S) form and Rough (R) form.
3. Smooth forms are emulsifiable in saline.
4. Due to repeated subculture, there is smooth to rough variation (S-R variation).
E. coli on Blood Agar (BA)
1. Colonies are big, circular, gray and moist.
2. Beta (β) hemolytic colonies are formed.
E. coli on MacConkey Agar (MAC)
1. Colonies are circular, moist, smooth and of entire margin.
2. Colonies appear flat and pink.
3. They are lactose fermenting colonies.

11. E. coli on Mueller Hinton Agar (MHA)
1. Colonies are pale straw colored.
E. coli on Eosin Methylene Blue (EMB) Agar
1. Green Metallic sheen colonies are formed.
E. coli on m-ENDO Agar
1. Colonies are green metallic sheen.
2. Metabolise lactose with the production of aldehyde and acid.
E. coli on Violet Red Bile Agar (VRBA)
1. Red colonies (pink to red) are formed.
2. Bluish fluorescence around are seen around colonies under UV.
E. coli on Cystine Lactose Electrolyte-Deficient (CLED) Agar
1. They give lactose positive yellow colonies.
E. coli on Liquid Media
1. They show homogenous turbid growth within 12-18 hours.
2. R form agglutinate spontaneously, forming sediment on the bottom of the test tubes.
3. After prolonged incubation (>72 hrs), pellicles are formed on the surface of liquid media.
4. Heavy deposits are formed which disperses on shaking.

12. Growth Factors
1. C, O, N, H, P, and S are chemical elements required to build nearly all cellular components.
2. In addition to these major elements, elements like Fe, selenium, Ca, Na, and several others
are required to build specific structures and perform specific processes.
1. E. coli, along with all other living organisms, requires environmental sources of all of these elements in order to survive.
2. Most of these elements come from food sources, like carbohydrates, proteins, and fats.
3. Growth factors can include amino acids, nucleotides, fatty acids, or vitamins.
4. The naturally occurring (wild-type) strain of E. coli doesn't require any growth factors. If given the appropriate elements and
an energy source, E. coli can synthesize all 20 amino acids, all vitamins, all nucleotides, and all fatty acids that it uses during
growth and metabolism.
5. E. coli's ability to make its own growth factors, there is an advantage to supplying them.
6. E. coli population can double in size about every 20 min. In the colon, where E. coli has to compete for growth factors with
other intestinal bacteria and the host, it can take around 12 hours for the population to double.
Temperature:
1. E. coli evolved in human colon, it isn't a stretch to find that the optimum temperature for its growth is around 37 Deg. Cel.
2. Bacteria that thrive at these temperatures are called mesophiles. Mesophile is able to grow and divide between 10-45 Deg. Cel.
3. An E. coli cell will not die if it is removed from a 37-degree environment, but its growth rate will slow down.
Oxygen Concentration
1. The colon is, for the most part, an anaerobic environment. The absence of oxygen is obviously not a problem for E. coli,
considering this is where it evolved to live.
2. These areas are exposed to atmospheric oxygen concentrations. But E. coli can handle these wild swings in oxygen
concentration no problem.

13. Water availability
 Natural or substrate‐induced (salt or sugar) low water activity (or potential) controls what microbes have the potential to grow given that
all other factors are in acceptable tolerance ranges.
 Rehydration can cause an anoxic environment around the cells, therefore, E. coli and other bacteria need to adjust their membranes and
gene regulation to adapt to the desiccation and rehydration cycles.
 Growth of E. coli in the soil environment was negatively influenced by soil desiccation; while E. coli survival rates were not different
between dried and wet soils.
 Upon rehydration, E. coli that survived in desiccated soil showed growth, indicating that water availability is critical for E. coli to grow.
Nutrient availability
 The availability of nutrients such as carbon, nitrogen and phosphorus is also an important factor influencing E. coli survival and growth in
the environment.
 In addition, E. coli showed a catabolic flexibility under glucose‐limited conditions, resulting in the efficient uptake of diverse carbon
sources
pH
 Environmental pH can also influence the survival and growth of E. coli in soil, and the level of pH resistance varies by strains.
 Escherichia coli serotype O157:H7 strains showed superior survival at low pH, as compared to non‐O157 E. coli strains.
 Similar to acidophiles, some E. coli O157:H7 strains can survive better at low pH than at relatively high pH.
Solar radiation
 Solar radiation is the most effective abiotic factor causing death of FIB in environmental waters. The inactivation process of FIB by
sunlight involves three major mechanisms utilizing photobiological, photooxidative and photochemical pathways. Solar radiation,
especially those in the lower wavelengths (i.e. ultraviolet (UV) light) can directly cause DNA damage and oxidation of cellular contents,
but these mechanisms are effective only at depths to which sunlight reaches (e.g. upper surface water).
 Since water is an effective filter of light, this mostly occurs in the upper water column (<22 cm depth) or on the soil surface. In the
photochemical inactivation process, oxygen‐free radicals (O•) and hydrogen peroxides (H2O2) are produced when oxygen (O2) and organic
matter are exposed to sunlight. These destructive chemicals can be delivered to the deeper areas (90 cm depth) of the water environment.

14. Presence of other micro‐organisms
 Escherichia coli interacts with other micro‐organisms in all natural habitats. E. coli can be predated by protozoa and lysed by phages. These
two biological mechanisms have been reported to be responsible for up to 70% of the FIB removal over 120 h in river water.
 E. coli also needs to compete with indigenous micro‐organisms for limited nutrient sources, and defend themselves from antagonism in the
environment. E. coli populations grew much better in sterile vs nonsterile soils, indicating that microbiota has a crucial effect
on E. coli survival.
Ability to form biofilms
 Biofilms formed by E. coli on surfaces in aquatic environments, such as sediments, is a well‐known factor contributing to the persistence
of E. coli in natural environments.
 Biofilms protect the bacteria from hostile environmental conditions such as UV rad., desiccation, protozoan predators, and Chemicals.
 They also may provide bacteria with a source of nutrients. Bacterial cells detached from mature biofilms can be transported to other locations
by increased flow rates in aquatic environments and result in the building of new biofilms, indicating that biofilm‐borne E. coli can be
transported to alternate sites and observed without evidence of faecal contamination in the environment.
Differential survival/growth ability among Escherichia coli strains
 The ability of E. coli strain to acquire nutrients, compete with other micro‐organisms, survive and grow in the environment is likely to vary by
strains and genotype. Thus, differential survival/growth ability among E. coli strains could cause a shift in E. coli populations in wastewater or
faecal‐impacted sediments and in manure‐amended soil

16. E. coli, like most bacteria, has a single origin of replication on its
chromosome. The origin is about 245245245 base pairs long and has
mostly A/T base pairs (which are held together by fewer hydrogen bonds
than G/C base pairs), making the DNA strands easier to separate.
Specialized proteins recognize the origin, bind to this site, and open up the
DNA. As the DNA opens, two Y-shaped structures called replication
forks are formed, together making up what's called a replication bubble.
The replication forks will move in opposite directions as replication
proceeds.
1. Helicase opens up the DNA at the replication fork.
2. Single-strand binding proteins coat the DNA around the replication fork
to prevent rewinding of the DNA.
3. Topoisomerase works at the region ahead of the replication fork to prevent
supercoiling.
4. Primase synthesizes RNA primers complementary to the DNA strand.
5. DNA polymerase III extends the primers, adding on to the 3' end, to make
the bulk of the new DNA.
6. RNA primers are removed and replaced with DNA by DNA polymerase I.
7. The gaps between DNA fragments are sealed by DNA ligase.

17. Cell cycle
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. 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.
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. However, asynchrony can be
caused by mutations to for instance DnaA or DnaA initiator-
associating protein DiaA.

19. 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.
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. 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. 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.

20. Gene nomenclature
1. 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.
2. 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.
3. 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). Hence, recD = b2819 = JW2787. Note,
however, that most databases have their own numbering system, e.g. the EcoGene database uses EG10826 for recD. Finally,
ECK numbers are specifically used for alleles in the MG1655 strain of E. coli K-12. Complete lists of genes and their
synonyms can be obtained from databases such as EcoGene or Uniprot.
Proteome
1. 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. 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.
2. 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.
3. The genome also contains insertion sequence (IS) elements, phage remnants, and many other patches of unusual composition
indicating genome plasticity through horizontal transfer.
Interactome
1. 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.

21. Treatment of E. coli infections
1. The sulfonamides, ampicillin, cephalosporins, fluoroquinolones, and aminoglycosides have
marked antibacterial effects against the enterics, but variation in susceptibility is great, and
laboratory tests for antibiotic susceptibility are essential.
2. E. coli meningitis requires antibiotics, such as third-generation cephalosporins (eg, ceftriaxone).
3. E. coli pneumonia requires respiratory support, adequate oxygenation, and antibiotics, such as
third-generation cephalosporins or fluoroquinolones.
4. In most cases of diarrheal disease, antibiotics are not prescribed. The best way to treat E
coli infection is to drink plenty of fluids to avoid dehydration and to get as much rest as possible.
However, patients should avoid dairy products because those products may induce temporary
lactose intolerance, and therefore make diarrhea worse.
Prevention and Control of E. coli infections
1. It is widely recommended that caution be observed in regard to food and drink in areas where
environmental sanitation is poor and that early and brief treatment (eg, with ciprofloxacin or
trimethoprim-sulfamethoxazole) be substituted for prophylaxis.
2. Their control depends on handwashing, rigorous asepsis, sterilization of equipment, disinfection,
restraint in intravenous therapy, and strict precautions in keeping the urinary tract sterile (ie,
closed drainage).

22. https://scholar.google.com/citations?user=UrHZPW8AAAAJ&hl=en