E.Coli and its life cycle

SUBHRADEEP SARKARM.Sc Applied Genetics

Biology of E.coli• Escherichia coli ( commonly abbreviated E. coli) is a Gram-negative,

facultative anaerobic ,non-sporulating and rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms .

• Most E. coli strains are harmless, but some 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.

• Cells are able to survive outside the body for a limited amount of time .There is, however, a growing body of research that has examined environmentally persistent E. coli which can survive for extended periods of time outside of the host .

• The bacterium can also be grown easily and inexpensively in a laboratory setting, and has been intensively investigated for over 60 years.

Biology of E.coli• E. coli is the most widely studied prokaryotic model organism, and an

important species in the fields of biotechnology and microbiology, where it has served as the host organism for the majority of work with recombinant DNA.

• Cells are typically rod-shaped, and are about 2.0 μm long and 0.5 μm in diameter.

• It can live on a wide variety of substrates. • E. coli uses mixed-acid fermentation in anaerobic conditions, producing

lactate, succinate, ethanol, acetate and carbon dioxide. • Optimal growth of E. coli occurs at 37°C (98.6°F) but some laboratory strains

can multiply at temperatures of up to 49°C (120.2°F).• Growth can be driven by aerobic or anaerobic respiration• Strains that possess flagella are motile. • E. coli and related bacteria possess the ability to transfer DNA via bacterial

conjugation, transduction or transformation, which allows genetic material to spread horizontally through an existing population

Life cycle of E.coli• E. coli reproduces by two means: cell division, and the transfer of genetic material through a sex

pilus (conjugation).

• In most bacteria, conjugation depends on a fertility (F) factor(a plasmid) that is present in the donor cell and absent in the recipient cell. Cells that contain F are referred to as F, and cells lacking F are F.

• The F factor contains an origin of replication and a number of genes required for conjugation .

• For example, some of these genes encode sex pili , slender extensions of the cell membrane.

• A cell containing F produces the sex pili, which makes contact with a receptor on an F cell and pulls the two cells together. DNA is then transferred from the F cell to the F cell. Conjugation can take place only between a cell that possesses F and a cell that lacks F.

• Once inside the recipient cell, the single strand is replicated, producing a circular, double-stranded copy of the F plasmid .

• If the entire F factor is transferred to the recipient F -cell, that cell becomes an F+ cell.

Conjugation between an F+ and F- cell.◗

Conjugation between Hfr and F- cell• In Hfr cells are those in which the F factor is integrated into the bacterial chromosome .

• Hfr cells behave as F+ cells, forming sex pili and undergoing conjugation with F- cells.

• In conjugation between Hfr and F- cells ,the integrated F factor is nicked, and the end of the nicked strand moves into the F- cell ,just as it does in conjugation between F+ and F- cells.

• In the Hfr cells, the F factor is linked to the bacterial chromosome, so the chromosome follows it into the recipient cell.

• How much of the bacterial chromosome is transferred depends on the length of time that the two cells remain in conjugation.

• Once inside the recipient cell, the donor DNA strand is replicated ,and crossing over between it and the original chromosome of the F- cell may take place.

• When crossing over has taken place in the recipient cell, the donated chromosome is degraded, and the recombinant recipient chromosome remains to be replicated and passed to later generations by binary fission..

• In a mating of Hfr x F-, the F- cell almost never becomes F or Hfr, because the F factor is nicked in the middle during the initiation of strand transfer, placing part of F at the beginning and part at the end of the strand to be transferred.

• To become F or Hfr, the recipient cell must receive the entire F factor, requiring that the entire bacterial chromosome is transferred.

• This event happens rarely, because most conjugating cells break apart before the entire chromosome has been transferred.

Advantages of E.coli as a model organism

•Rapid reproduction and small size.•Under optimal conditions , they can reproduce every 20 mts. , in a mere 7 hrs., a single bacterial cell can give rise to more than 2 millions descendants.•Thus enormous numbers of cells can be grown quickly , so that even very rare mutations will appear in a short period of time .•Consequently , numerous mutations in E. coli everything from colony appearance to drug resistance , have been isolated and characterized.•E.coli is easy to culture in the laboratory in liquid medium or on solid medium within petriplates.•In liquid culture , E.coli cells will grow to aconcentration of a billion cells per milliliter, and trillion of bacterial cells can be easily grown on a single test tube.•When E. coli ells are diluted and spread onto the solid medium of a petri dish , individual bacteria reproduce asexually , giving rise to a concentrated clump of 10 million -100million genetically identical cells , called a colony.•This colony formation makes it easy to isolate genetically pure strains of the bacteria.

Role of E.coli in biotechnology• E. coli plays an important role in modern biological engineering and

industrial microbiology.• 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.

• Considered a very versatile host for the production of heterologous proteins, researchers can introduce genes into the microbes using plasmids, allowing for the mass production of proteins in industrial fermentation processes.

• Genetic systems have also been developed which allow the production of recombinant proteins using E. coli. One of the first useful applications of recombinant DNA technology was the manipulation of E. coli to produce human insulin.

• Modified E. coli cells have been used in vaccine development, bioremediation, and production of immobilised enzymes.

• E. coli cannot, however, be used to produce some of the larger, more complex proteins which contain multiple disulfide bonds or proteins that also require post-translational modification for activity.

E.coli Genome• The first complete DNA sequence of an E. coli genome (laboratory strain K-

12 derivative MG1655) was published in 1997. • Despite having been the subject of intensive genetic analysis for

approximately 40 years, a large number of these genes were previously unknown.

• When the genome was sequenced as part of Human genome project ,it was found to be 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.

• The coding density was found to be very high, with a mean distance between genes of only 118 base pairs.

• Greater gene density is due to a combination of factors: (1) bacterial genes have no introns, (2) neighboring genes are very close together throughout the genome; i.e., there are hardly any big regions of non-coding DNA between genes.

E.coli Genome• The genome was observed to contain a significant number of

transposable genetic elements, repeat elements, cryptic prophages, and bacteriophage remnants.

• Today, over 60 complete genomic sequences of Escherichia and Shigella species are available. (Shigella is a genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella.)

• Comparison of these sequences shows a remarkable amount of diversity; only about 20% of each genome represents sequences that are present in every one of the isolates, while approximately 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 pan-genome) exceeds 16,000.

• This very large variety of component genes has been interpreted to mean that two-thirds of the E. coli pan-genome originated in other species and arrived through the process of horizontal gene transfer.

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Pathogenic E.coli• The predominant facultative anaerobe resident in the human colon is

the Gram-negative motile bacillus Escherichia coli. E. coli colonises the infant gut within hours of birth.

• However, E. coli has a dichotomous existence; while the majority of E. coli strains exist within the mammalian intestinal tract as harmless commensals, paradoxically several evolutionary lineages have deviated from this harmless lifestyle to become pathogens.

• Current dogma suggests that such latter strains of E. coli have acquired additional genetic elements, encoding specific virulence factors, which enable the organism to cause disease when infecting an otherwise healthy individual.

• The resulting clinical syndromes include extraintestinal infections, such as urinary tract infections, septicaemia and meningitis, and intestinal infections mediating diarrhea.

Pathogenic E.coli• Those strains causing intestinal infections can be divided into six separate and

major categories or pathotypes viz. enteroaggregative E. coli (EAEC), enteroinvasive (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC) and diffuse adhering E. coli (DAEC).

• A volunteer study demonstrated that EAEC strain 042 elicited diarrhea in the majority of volunteers .

• Since these ground-breaking observations many studies have demonstrated the association of EAEC and diarrhea in both developing countries and industrialised nations.

• Thus, EAEC have been significantly associated with (i) endemic diarrhea in infants in developing and industrialised nations, particularly persistent diarrhea, (ii) persistent diarrhea in HIV-positive patients, (iii) traveller's diarrhea, (iv) food/water-borne outbreaks and (v) sporadic cases of diarrhea .

Genome of a Pathogenic E.coli( EAEC 042 )

• The genome of EAEC 042 consists of a circular chromosome of 5,241,977 bp and one plasmid pAA of 113,346 bp.

• .A total of 4,886 genes were identified in the chromosome• 556 (11%) are conserved hypothetical proteins, with no known

function and only 481 (10%) seem to be mobile elements such as integrases, transposases, or phage related.

• 78 genomic islands have been identified in EAEC 042 that are differentially distributed/represented and/or sequence diverged among the sequenced E. coli genomes, these islands are designated regions of difference (ROD) .

• The overall size of these RODs is 1.26 Mb (24% of the chromosome).

Genome of a Pathogenic E.coli( EAEC 042 )

• The RODs encode virulence determinants, metabolic proteins, proteins with no obvious functions and mobile elements such as prophage and a conjugative transposon.

• The conjugative transposon Tn2411 (within ROD66) is highly similar to Tn21 and carries a variety of genes encoding antibiotic resistance .

• Nine prophage regions, designated 042p1–042p9, were identified in the EAEC 042 genome .Four of the prophage were lambdoid in nature (042p2, 042p3, 042p4 and 042p6) and were highly similar to each other and three (042p1, 042p3 and 042p6) appeared to carry cargo genes .

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