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Do most bacteria have plasmids?

Do most bacteria have plasmids?


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Do most bacteria have plasmids? I googled that and I found this

Yes, Plasmids naturally exist in all bacterial cells. Here

But I know some of them don't!

Is this sentence scientifically correct?

Most bacteria have plasmids. If you study a random bacterium, you will probably find plasmid(s) in it.


I think there might be some confusion between individual bacterial cells and "Bacteria" in general. The link you shared is either oversimplifying to the point of losing touch with the truth, or they simply used inappropriate word choice. It's unfortunate that they didn't seem to include a primary source to backup that claim.

For a more general statement like, "Most bacteria have plasmids," I would interpret that as meaning plasmids have been found in most bacterial lineages, not necessarily that most individual bacterial cells have plasmids. But I could see how that might confuse some readers and/or educational content creators.

I will share this for reference. The distribution of plasmids is typically heterogeneous within populations, and variable between populations. For any given plasmid, it's common for only a fraction of the population to carry it. Sometimes nearly 100% of a population carries has the same plasmid, but in other cases it can be 10% or less. I would wager that it depends on how mobile that plasmid is and on the selective pressures specific to the environment in which they are living.

I have personally observed this heterogeneity even in laboratory strains under antibiotic selection. I once determined that less than 20% of my E. coli cells in a culture were actually carrying the plasmid under selection (I did this quick experiment because I was not able to recover detectable amounts of the protein that I was trying to express in my strain).

The reference I linked might be a useful resource for the OP. In case that link doesn't work, here's the reference.

Holger Heuer, Zaid Abdo, Kornelia Smalla, Patchy distribution of flexible genetic elements in bacterial populations mediates robustness to environmental uncertainty, FEMS Microbiology Ecology, Volume 65, Issue 3, September 2008, Pages 361-371, https://doi.org/10.1111/j.1574-6941.2008.00539.x


When people work with E. coli in labs, they often use a strain initially without plasmids - which is very easily infected if someone else nearby is working with a strain that does have a plasmid.


4: Cells: structure and function (mostly Bacterial)


A. structure: phospholipid bilayer and proteins based on fluid mosaic model. Hydrophobic core
made of HC tails of fatty acid residues of phospholipids (review). Hydrophilic heads associate
with water molecules fig.


B. functions: semi- or selectively permeable membrane, controls movement of substances into
and out of cell. Site for electron transport chain and photosystems in some
bacteria and of flagellar base in flagellated bacteria and more
-location of membrane transport proteins essential for moving large polar or
charged substances across membrane


C. More on transport later&hellip


D. Damage to cytoplasmic membrane may kill bacteria


E. Cytoplasmic membranes are relatively weak and are vulnerable to osmotic lysis
Vulnerability of cytoplasmic membranes: osmosis and osmotic lysis
-osmosis: diffusion of water from area of high water concentration to area of low water
concentration through selectively permeable membrane (e.g. cytoplasmic membrane)
-effects of isotonic, hypertonic and hypotonic solutions on cells (aka isoosmotic, hyperosmotic, hypoosmotic):
-Osmotic lysis:
-most bacteria live in hypotonic environments

-concentration of solutes inside cell is higher than concentration of solutes
outside cell, consequently concentration of water outside cell is greater than
concentration of water inside cell, consequently&hellip
-net flow of water will be from outside cell into cell across cytoplasmic
membrane consequently

> water pressure inside cell continues to increase
until cytoplasmic membrane bursts, cell undergoes &ldquolysis&rdquo. This process is called
&ldquoosmotic lysis&rdquo and will kill bacterium.


F. How do bacteria prevent osmotic lysis? Most common solution is formation of &ldquocell wall&rdquo (see
next section)

V. Cell walls of Domain Bacteria


A. components of bacterial cell wall: peptidoglycan (&ldquopg&rdquo)


1. only members of Domain Bacteria synthesize peptidoglycan

2. function: prevention of osmotic lysis shape of bacterium

3. Peptidoglycan structure: alternating covalently linked
-N-acetylglucosamine (NAG or G) and
-N-acetylmuramic acid (NAM or M only found in Domain Bacteria!) with
-tetrapeptide &ldquotails&rdquo

4. peptide cross-links essential for strength of pg crosslinks formed by bacterial enzymes, &ldquotranspeptidases&rdquo (aka bacterial PBP Penicillin Binding Proteins)

a. beta-lactam antibiotics (ex penicillin, ampicillin, amoxicillin) irreversibly bind the bacterial transpeptidases so they cannot form peptide crosslinks in pg, thus weakening pg and leading to osmotic lysis
of growing bacteria. Beta-lactams are not effective at killing bacteria in &ldquostationary stage&rsquo ie bacteria which are not actively growing, nor are they active against bacteria lacking cell walls ex Mycoplasma.

b. Penicillin was discovered by Sir Alexander Fleming

c. Vancomycin also interferes with crosslinking of pg, leading to osmotic lysis of bacteria (different mechanism than beta-lactam antibiotics however)


5. Lysozyme (also discovered by Alexander Fleming) is an enzyme found in tears, saliva, sweat. Lysozyme cleaves the covalent glycosidic bonds between NAM and NAG, weakening cell wall, leading to osmotic lysis of some bacteria.


B. Different types of Bacterial cell walls


1. Gram- positive cell walls: thick layer of peptidoglycan teichoic and lipoteichoic acids some have additional carbohydrates some have cell wall proteins (ex M protein of Streptococcus pyogenes)

2. Gram-negative cell walls: thin layer of peptidoglycan connected by lipoproteins to outer membrane. Space between cell membrane and outer membrane is called the &ldquoperiplasmic space&rdquo

a. outer membrane components:
i. lipopolysaccharide aka &ldquoLPS&rdquo, &ldquoendotoxin&rdquo when released from dying gram- negative bacteria, triggers massive cytokine release leading to vasodilation and increased capillary permeability and &ldquoDIC&rdquo disseminated intravascular coagulation (blood clots form). Hypotension, decreased tissue perfusion, multiple organ system failure, shock (endotoxic shock) and death may result as a consequence (&ldquoendotoxemia). LPS bind to host cell leukocyte Tolllike receptors to trigger cytokine flood (more later).LPS is also used in &ldquoserotyping&rdquo gram-negative bacteria ( &ldquoO&rdquo somatic antigens more later). LPS found only in gram-negative bacteria.
ii. phospholipids
iii. porins: protein channels through which hydrophilic substances can
cross outer membrane


b. outer membrane functions: prevents diffusion of secreted enzymes protects
bacterium against toxic substances (ex some antibiotics such as penicillin,
lysozyme, bile)


3. Acid fast cell wall: e.g.,Mycobacterium tuberculosis, leprae: peptidoglycan covered by
mycolic acid-lipid bilayer. Creates hydrophobic barrier against antibiotics , chemicals, stains,
drying. Nutrients slow to pass barrier therefore these bacteria are very slow growing (difficult to
culture and perform antibiotic sensitivity testing). Patients infected with Mycobacterium are often
on long term antimicrobial therapy (months, years!) Protein porins in mycolic acid-lipid layer
permit passage of some hydrophilic substances. Described as a &lsquowaxy or lipid-rich&rdquo cell wall.
Requires special staining procedure (acid-fast stain see p109 in textbook).


4. Some Bacteria lack cell walls, e.g., Mycoplasm, Chlamydia and &ldquoL-forms&rdquo. If a patient suffers
from infection with such bacteria, treatment with beta-lactam antibiotics would have no effect as
these bacteria lack the target of the antibiotics. (recall Mycoplasma &ldquosteal&rdquo cholesterol from their
animal hosts to incorporate into their cell membranes to strengthen membranes in absence of cell
wall)


5. Domain Archaea: Archaea do not synthesize true peptidoglycan-
External Structures of Prokaryotic cells


VI. Glycocalyx= &ldquosugar cup&rdquo, a &ldquosticky&rdquo covering found on some prokaryotes


Two types of glycocalyces: capsules (tightly attached) and slime layers (loosely attached).
Capsule/Slime layers: outermost layer, covers cell wall, produced by many but not all bacteria.
Structure: Usually polysaccharides, some exceptions (Bacillus anthracis produces
capsule of poly-D-glutamic acid)
-weakly antigenic, the &ldquoK&rdquo antigens of Enterobacteriaceae


Function:
&bull prevents desiccation/drying out
&bull adherence to surfaces (oral streptococci make sticky capsule/slime
layer which permits adherence to tooth surfaces),
&bull antiphagocytic : inhibits phagocytosis by leukocytes , essential for
pathogenicity of many bacteria


VII. Flagella (plural singular=flagellum) Read as homework

  • function: motility.
  • structure= flagellin protein subunits make up 20nm diameter filament, attached to hook and basal
  • body . Basal body consists of protein rings and shaft embedded in cell wall/cell membrane (more
  • in lab). Flagellar proteins act as antigens (trigger antibody production). &ldquoH&rdquo or &ldquoHauch&rdquo antigens
  • of Enterobacteriaceae are the flagellar antigens used in serotyping (eg. E . coli O157:H7).
  • Flagellar arrangements: monotrichous( single polar flagellum), amphitrichous (both ends),
  • lophotrichous (tuft), peritrichous (flagella all over) - Flagella rotates similar to boat propeller
  • Chemoreceptors located in cell membrane permit bacteria to detect concentration gradients of
  • chemicals in environment. Chemotaxis is movement in response to chemical gradients. Positive
  • chemotaxis: movement in direction of increasing concentration gradient ex nutrient molecules.
  • Negative chemotaxis: movement down concentration gradient ex toxin molecules
  • Axial filaments or endoflagella : spirochetes are spiral shaped bacteria. Examples are Treponema
  • pallidum (causes syphilis) and Borrelia burgdorferi (causes Lyme Disease). These bacteria have
  • bundles of endoflagella attached at both ends of their cells covered by an outer sheath forming an
  • axial filament . Rotation of the endoflagella causes axial filament to rotate around spirochete,
  • permitting the bacteria to &ldquocorkscrew&rdquo through their environment, often thick mucous blankets,
  • perhaps even through tissues.

VIII. Pili and fimbriae

  • function: attachment.
  • structure= pilin protein subunits form hollow tubes projecting from surface of cell.
  • adhesins: Specific proteins in pili called adhesins permit attachment to surfaces in environment, including host cells. Adhesins often bind to specific receptors on host cell surfaces
  • fimbriae: usually numerous, relatively short, used to attach to surfaces in environment, including
  • other cells ex Neisseria gonorrhoeae uses fimbriae to attach to cells of host mucous membranes.
  • Reports have suggested Neisseria can change the types of adhesins expressed on its fimbriae so it
  • can first attach to mucosal cells of genital tract, then to cells of oral region, then cells of eye. What would happen if a mutation inhibited production of fimbriae by Neisseria?
  • Role of fimbriae in Biofilms
  • sex pilus (gram -negative bacteria aka conjugation pilus, &ldquoF&rdquo pilus): attaches one
  • bacteria to another, facilitates exchange of genetic information/DNA. example.
  • Involved in transfer of antibiotic resistance genes between bacteria.
  • Inside the bacterial cell


IX. Cytoplasm and internal structures: 90% water. Contains chromosome, plasmids, ribosomes, enzymes, nutrients, waste products, inclusion bodies


X. Chromosome

most bacteria have single, circular double stranded DNA chromosome ( a few have linear chromosomes). DNA carries genetic information. DNA base sequence determines amino acid sequence of proteins. (more later -antibiotics fluoroquinolones such as ciprofloxacin used to treat anthrax victims are bacterial DNA gyrase inhibitors these antibiotics prevent &ldquorelaxation&rdquo of supercoiled bacterial DNA required for DNA replication and transcription (other DNA gyrase inhibitors include nalidixic acid and novobiocin)


XI. Plasmids

extrachromosomal, circular, self-replicating DNA. Frequently carry &ldquoextra&rdquo genetic
information example antibiotic resistance genes (&ldquoR&rdquo or resistance plasmids). May be passed
from one bacterium to another resulting in spread of antibiotic resistance. Conjugative plasmids
carry genes for synthesis of sex pili and proteins involved in transfer of bacterial DNA from
donor to recipient (more later in genetics)


XII. Ribosomes

70S ribosomes (compared to larger 80s cytoplasmic ribosomes of eukaryotes)

  • -structure: 2 subunits, 50S and 30S made of ribosomal RNA/rRNA and ribosomal proteins
  • -site of protein synthesis
  • -S-Svedberg unit, used to express sedimentation rates using ultracentrifuges.
  • -70S bacterial ribosomes are the target of many antibiotics examples tetracycline,
  • chloramphenicol, macrolides (erythromycin, azithromycin) aminoglycosides ex gentamicin,
  • kanamycin. These antibiotics inhibit protein synthesis by bacteria

XIII. Endospores

resistant, dormant /resting structures, protect bacterium&rsquos DNA, under harsh
conditions. Layers of protein, peptidoglycan, high calcium ion contenet and dipicolinic acid, low water
contenet. .Bacillus and Clostridium are endospore formers. Endospores can germinate to produce new
metabolically active, replicating vegetative cells. If inhaled, endospores of Bacillus anthracis will
germinate in lungs causing pneumonia and may spread throughout body (usually lethal). Unfavorable conditions trigger vegetative cells to sporulate and produce endospores. Contain very little water and dipicolinic acid (heat resistance)
See: anthrax and clostridial diseases


XIV. Inclusions (not on exam 1)


XV. Cytoskeleton read (not on exam 1)


Addition: Homework transport of substances across membranes and eukaryotic cells-read sections in
textbook
Transport of substances across cell membranes is presented following discussion of eukaryotic cells in the
lecture PowerPoint, however a few notes regarding specialized transport in bacteria follow:
Reference: Protein Secretion Systems in Gram-negative bacteria Source p63-65 Prescott, Harley and
Klein&rsquos Microbiology Wiley et al ed 2008 McGraw Hill Publ
Type III protein secretion systems of some gram-negative bacterial pathogens: these systems
permit injection of virulence factors (ex toxins) into host target cells. Structurally complex,
similar to hypodermic syringe/needle mutation in genes for Type III secretion may have permitted
evolution of bacterial flagella.
Type IV secretion systems: used to transport proteins AND to transfer DNA during conjugation
components form syringe-like structure similar to Type III system

Bio 440 Eukaryotic Cells: Ch 4 Tortora


I. Eukaryotic microorganisms: organisms with membrane bound nucleus.
Domain Eukarya
Kingdoms: ( out-of-date" Protista" "algae", "protozoa"), Fungi, Plantae, Animalia


II. Evolution of endomembrane system
Primitive prokaryotic cell: in-folding of cell membrane--> nuclear membrane, endoplasmic reticulum, Golgi body, vesicles, lysosomes. Compartmentalization of functions.


III. Nucleus

A. Nuclear membrane with pores
B. Chromosomes and reproduction: multiple, linear chromosomes of double-stranded DNA split into
coding exons and non-coding introns. DNA associated with histone proteins.
Eukaryotes which reproduce sexually normally are &ldquodiploid&rdquo, ie cells contain 2 copies of each
chromosome, (one copy of each chromosome donated by each parent). Therefore there are usually 2 copies
of each gene, and the genes may not be identical. Haploid gametes are formed during sexual reproduction,
containing one copy of each chromosome. 2 haploid gametes fuse to form a diploid zygote (fusion of
gamete nuclei form zygote nucleus). Some eukaryotic microbes can reproduce asexually and have haploid
cells. Some organisms can reproduce sexually or asexually (ex fungi) and therefore may have either
diploid or haploid cells. HOMEWORK read mitosis and meiosis in textbook

IV. Endoplasmic reticulum

A. Continuous w/ nuclear membrane
B. 2 types

1. RER=Rough Endoplasmic Reticulum: &ldquostudded&rdquo with ribosomes -synthesis of proteins destined for export, incorporation into membranes or delivery to other organelles
2. SER= Smooth endoplasmic reticulum -lipid synthesis, detoxification


V. Golgi Body

receive proteins/lipids from ER via transport vesicle processing and shipping to final
destination (secretion, membranes, other organelles)


VI. Lysosomes and Peroxisomes

Lysosomes (animal cells): vesicles filled with hydrolytic enzymes. May fuse with phagosomes to hydrolyze nutrient molecules or destroy invading microorganisms (phagocytosis). Peroxisomes :vesicles containing peroxidase/catalase and other enzymes. Plants: oxidize fats. Animals: oxidize amino acids.


VII. Ribosomes

80S cytoplasmic ribosomes sites of protein synthesis. Free in cytoplasm or fixed to RER. In mitochondria and chloroplasts, 70S-like ribosomes.


VIII. Mitochondria

all aerobic respiring eukaryotes (exception: Giardia lacks mitochondria)
A. &ldquoPowerhouse&rdquo of cell, site of ATP generation via aerobic respiration
C6H12O6 + 6O2--> 6CO2 + 6 H2O + Energy (heat + ATP)
B. Evolved from primitive prokaryotic cell? see Theory of Endosymbiosis/Endosymbiotic Theory


IX. Chloroplast: photosynthetic plants and algae

A. Sites of oxygenic photosynthesis: 6CO2 + 6H2O--light energy--> C6H12O6 + 6O2
B. Evolved from primitive cyanobacteria? Theory of Endosymbiosis/Endosymbiotic Theory

X. Theory of Endosymbiosis/Endosymbiotic

primitive nucleated cell phagocytizes (&ldquoeats&rdquo)
primitive aerobic respiring bacterium. Bacterium becomes an endosymbiont, living within host cell,
generates ATP for host, host provides protection for bacterium. Bacterium eventually evolves into
mitochondrion. Similar story for chloroplast evolution. Primitive nucleated cell with mitochondria
phagocytize primitive photosynthetic cyanobacterium. Bacterium becomes endosymbiont, evolves into
chloroplast.


Evidence to support Theory of Endosymbiosis:
1. mitochondria (mito.) and chloroplast (chloro) are self replicating-divide independently of host
cell
2. mito. and chloro. have own self-replicating, circular chromosomes similar to prokaryotic
chromosomes
3. mito/chlor. size similar to bacteria
4. membrane arrangement of mito./chlor. fit theory of bacterium engulfed in phagosome
5. mito./chlor. have ribosomes similar to prokaryotic 70S ribosomes and are inhibited by
antibiotics targeting 70S ribosomes
6. mito/chloro. ribosomal RNA sequences similar to Bacteria rRNA sequences.


XI. Cytoskeleton

A. Microtubules of tubulin subunits: mitotic spindles, flagella and cilia (protein dynein associated w/fl & cilia).
B. Microfilaments of actin subunits: cytoplasmic streaming pseudopodia formation f amoeba and slime molds
C. Intermediate fibers: variety ex keratin: rigidity


XII. Appendages


A. Flagella: motility. Structurally very different from bacterial flagella. Microtubules (9 doublets + 2
central) covered with cell membrane, flex/beat (do not rotate/turn like bacterial flagella). ex Protozoa
B. Cilia: very similar to flagella except shorter, more numerous. Protozoa ciliates
Ciliated epithelium of respiratory tract important part of mucociliary escalator destroyed by viruses,
smoking, predisposes to bacterial respiratory infections. Ciliated epithelium of oviduct important for
moving egg to uterus. Pathogens such as Chlamydia and Neiserria gonorrhoeae cause destruction of cilia,
results in ectopic pregnancy, sterility.


XIII. Cell wall: provides shape, resists turgor pressure/prevents osmotic lysis

A. Animals lack cell wall some protozoa have protein layer called pellicle
B. Fungi and algae have cell wall
1. fungi cell wall: may contain chitin, polymer of N-acetylglucosamine (NAG) nitrogencontaining
polysaccharide
2. some algal cell walls and some fungal walls contain cellulose


XIV. Cell membrane phospholipid bilayer with proteins based on fluid mosaic model. Proteins may move
laterally. Consistency of membrane is like thin layer of oil. Primary function is to control movement of
substances into and out of cell


A. Homework: Movement of substances across membranes text

1. nonpolar substances may cross: diffusion, passive process
-Oxygen, carbon dioxide, ethanol and medium-length fatty acids may diffuse across
membrane. (A small amount of water may also diffuse through phospholipid bilayer )
2. water may cross rapidly via water-transmitting pores (aquaporins&rdquo)
-osmosis, passive. Know lysis, plasmolysis and when each happens
- &ldquoosmosis&rdquo and &ldquotonicity&rdquo (know)
3. Hydrophilic substances ie charged or polar substances, may not cross membrane unless assisted
by transport proteins ex protein pores/carrier proteins/&rdquopermeases&rdquo
4. Note: whenever substances are moved against their concentration gradient( that is from an area
of low concentration to an area of high concentration), energy must be expended.

passive transport: substance moved from area / Transport requiring energy/active transport
of high concentration to low. No NRG req&rsquod. / (energy=ATP, proton/chemical gradient)
1. Simple diffusion / 4. Active transport: Substance moved from low to
2. Osmosis / high concentration. Specific protein carriers. sugars,
amino acids, vitamins
3. Facilitated diffusion: /
- protein mediates diffusion. / 5. group translocation: (primarily in bacteria) --
Specific channels/pores or / -substance to be transported is modified
carrier proteins. / as it crosses membrane. ex glucose

B. Specialized transport: Animal cells/ protozoa lacking cell walls: endocytosis (pinocytosis and
phagocytosis) and exocytosis require cytoskeleton rearrangements and energy expenditure
endocytosis :taking in material in membrane in-folding ->vesicles


-: engulf cell/solid= phagocytosis engulf liquid =pinocytosis
ex phagocytic cells of immune system: monocytes-macrophages and
neutrophils: recognize &ldquoforeign&rdquo bacterial invaders, attach, phagocytize
and destroy via phagosomes and lysosomes
-also receptor mediated endocytosis
-exocytosis: expel/secrete substances from cell via vesicles


Plasmids in Bacteria: Properties, Types and Replication

Plasmids are defined as extra-chromosomal genetic elements, occurring chiefly in bacteria and rarely in eukaryotic organisms. In bacteria, plasmids are circular double-stranded DNA molecules which contain genes controlling a wide variety of functions. In yeast (Saccharomyces cerevisiae) an RNA plasmid has been found.

Plasmids are self-replicating elements, yet they are largely dependent on the host cell for their reproduction, because they use the host cell replication machinery. The first plasmid to be discovered was the sex-factor or F plasmid (F stands for fertility) of E. coli K12. This plasmid confers the ability to an E. coli cell (F + ) to conjugate with another lacking this plasmid (P cell). The F-plasmid can exist in two alternative states, viz. it can either remain free in the cell or it can be integrated into the E. coli chromosome. Plasmids with such property are known as episomes.

Generally, the bacterial plasmids are 1 to 5% of the chromosomal DNA in size. Plasmids vary widely in size. The smaller plasmids have molecular weights ranging between 4 to 5 x 10 6 Daltons, while the larger ones have molecular weights of 25 to 95 x 10 6 Daltons.

Plasmids not only vary in size, but also in copy number which denotes the number of copies of a specific plasmid in a cell. Copy number is discontinuously variable i.e. some plasmids generally the smaller ones — have a high copy number, while the larger plasmids have characteristically a low copy number. Those having a high copy number are known as relaxed plasmids and those having c low copy number are called stringent plasmids.

Whatever may be the copy number, plasmids are generally distributed equally in the daughter cell during cell division. Rarely, a plasmid-free cell may arise spontaneously at a frequency of about 1 in 10 4 -cells. Plasmid-free cells may also be produced artificially by the use of mutagens.

The process is commonly called curing of plasmids. Usually, the low-copy number large plasmids have one or two copies per cell and are easier to be cured. The smaller plasmids, in contrast, may have 10 to 100 copies per cell.

So far as the biological functions of plasmids are concerned, they are not indispensable constituents of the bacteria. This is proved by the fact that the bacteria cured of plasmids can grow normally without any difficulty. However, the genes carried on the plasmid DNA confer special properties to the host bacteria and such properties may become advantageous under special environmental conditions.

For example, bacteria carrying the R-plasmids (resistance plasmids) can survive when the environment contains inhibitory concentrations of one or more antibiotics. Obviously, the R-plasmid-less bacteria are destroyed under such conditions.

Another example is provided by the plasmids of some species of Pseudomonas which carry genes for production of enzymes catalyzing degradation of complex hydrocarbons. Bacteria carrying such plasmids are capable of using such unusual substrates for growth and, obviously, enjoy special advantage over others lacking them.

The F-plasmid gives the power to carry out a type of sexual reproduction to bacteria making it possible to exchange genetic materials leading to genetic recombination. Again, some bacteria, like E. coli, Pseudomonas, Lactobacillus etc. produce special type of proteins, called bacteriocins which are coded by plasmid genes. These proteins are able to kill other closely related bacteria and, thereby, they can eliminate competition for food and space.

Thus it is seen that even though plasmids are not absolutely essential for the life of bacteria under normal conditions of growth, their presence may become valuable and advantageous for the host under special conditions, or may even prove critical for survival as in case of the R-plasmids. The R-plasmids with the help of the resistance genes produce proteins which can inactivate or destroy specific antibiotics.

Besides the advantageous properties attributable to the plasmids, these extra-chromosomal genetic elements have played an important role in the development of recombinant DNA technology. In this technology, the plasmids are used as vectors for transferring a gene of interest from one organism to another organism. Such transfer of a gene is possible, not only from one bacterium to another, but also from an eukaryotic organism to a bacterium, or vice versa.

A segment of DNA containing the specific gene is isolated from a suitable donor and inserted by recombinant DNA technology into a plasmid. The recombinant plasmid is next introduced into a suitable host cell where the gene is expressed producing the gene product.

In this way, several human genes producing therapeutically important proteins have been introduced into bacteria. Also, some bacterial genes have been transferred to eukaryotic hosts, like plants, and some viral genes have been transferred to yeasts. In most of such gene transfers, plasmids play a key role as vectors or carriers.

Types of Plasmids:

(i) F-Plasmid:

The F-plasmid, also known as the fertility factor or sex-factor, determines the sex of E. coli bacteria. The cells containing this plasmid are designated as F + and those without it as F – . F + bacteria are considered as male, because they can act as donor of not only the plasmid, but also chromosomal genes to the F – cells which act as recipient and are, therefore, considered as female.

The process of transfer takes place by conjugation of the F + cell with the F – cell. The F-plasmid is a conjugative plasmid.

we know that a characteristic feature of the F-plasmid is that it can either remain as an independent entity replicating separately along with the chromosomal DNA, or it can be inserted into the chromosome as its integral part. When an F-plasmid is integrated into the E. coli chromosome, the bacterial cell changes from P to an Hfr-strain (high frequency of recombination).

There are many sites on the E. coli chromosome where the F-plasmid can be integrated. Depending on the site, each integration gives rise to a different Hfr-strain. In F + x F – conjugation, the plasmid alone is transmitted, but in Hfr x F – conjugation, chromosomal genes are transmitted and rarely also the F-plasmid.

The F-plasmid is a large self-transmissible plasmid having a double-stranded circular DNA molecule. Its molecular weight is 63 x 10 6 Daltons and it contains about genes controlling the transfer of the plasmid from the donor to the recipient. A mutation in any of the essential genes results in the loss of transmissibility of the plasmid.

Just as an F-plasmid can be integrated into the chromosome of the host cell, so it can though on rare occasions, be separated or excised from the E. coli chromosome in the free state to form the circular plasmid. It has been observed that the excision process is sometimes imperfect in the sense that some parts of the E. coli chromosome adjoining the linearly inserted F-plasmid are included in the excised F-DNA and at the same time, parts of the plasmid DNA are retained in the E. coli chromosome.

The F-plasmids containing parts of the chromosomal DNA are designated as F’-plasmids. When an F-plasmid loses some of its essential genes during the excision process, the plasmid is rendered incapable of independent existence and is, ultimately, eliminated during cell division.

When an F-plasmid is transmitted by conjugation to an F-recipient, it can transfer the chromosomal genes carried by it. Thereby, the recipient becomes diploid in respect of these transferred genes (because it now contains one copy of its own and another copy of the same gene transmitted by the F -plasmid). Thus, exchange of chromosomal genes may occur through F’-plasmids. This has been described as sex-duction.

(ii) R-Plasmids:

R-plasmids conferring resistance to various drugs individually or multiple resistance to several antibacterial agents were first discovered in Japan in the 1950s in the gastroenteritis-causing Shigella dysenteriae. Since then these plasmids have been found in E. coli and other enteric bacteria. Such plasmids have proved a great threat to the medical science.

The large R-plasmids having molecular weights ranging between 30 x 10 6 Daltons are self- transmissible by conjugation with other bacteria. They are, therefore, conjugative plasmids, like the F-plasmid.

Smaller R-plasmids having molecular weights of about 5 to 6 x 10 6 Daltons are non- transmissible. Most of the self-transmissible large plasmids like R100 of Shigella conferring multiple drug resistance are co-integrates of two DNA segments joined to each other by covalent linkage to form a single double-stranded circular molecule.

One DNA segment is called the resistance transfer factor (RTF), while the other segment contains the drug-resistance genes. The RTF is mainly involved in the transfer function of the R-plasmid and contains a number of genes (the transfer genes) and some others controlling replication of the plasmid in the host cell.

The resistance genes located in the other segment elaborate enzymes for destruction of the antibacterial drugs, like penicillins, streptomycin, chloramphenicol, tetracyclines, kanamycin, sulfonamides etc. (Fig. 9.89).

In some drug-resistant bacteria, such as Salmonella typhimurium strain 29, the resistance genes are located not in the same plasmid, but in separate plasmids of different size. This is sometimes known as plasmid aggregation.

The transposable elements that complex transposons may carry genes for drug resistance. Such elements can be integrated into plasmids giving rise to a drug-resistance plasmid. Thus, R plasmids may be made up of a collection of transposons, each of which may carry one or more genes for antibiotic resistance. For example, Tn 5 carrying a gene for kanamycin resistance may be inserted into the plasmid R100 of Shigella making the plasmid able to resist the antibiotic.

Besides drug resistance, plasmids may also make bacterial hosts resistant to the toxic effects of heavy metals. Plasmid-coded resistance to nickel, cobalt, mercury, arsenic and cadmium has been reported in different species belonging to the genera Pseudomonas, Escherichia, Salmonella and Staphylococcus.

(iii) Col-Plasmids:

The Col-plasmids are present in different strains of E. coli and they contain genes controlling synthesis of a class of proteins called colicines. Colicines are able to inhibit the growth of related bacteria which lack a Col-plasmid (Cor).

Several different types of Col-plasmids have been discovered, each of which produces colicines having a different mode of inhibition of susceptible bacteria. For example, Col B induces a damage of the cytoplasmic membrane of the target bacteria and Col E2 and Col E3 cause degradation of nucleic acids.

Like R-plasmids, Col-plasmids may be self-transmissible or non-self transmissible. Large Col plasmids, like Col I and Col V-K94 having molecular weights of 60 x 10 6 Daltons or above are self- transmissible. They have a small copy number, usually 1 to 3 copies per cell. Small Col-plasmids, like Col-El, have molecular weight weighs of about 4 to 5 x 10 6 Daltons.

They have a high copy number, usually 10 to 30 copies per cell. They are self-non-transmissible, but may be mobilized with the help of F-plasmid. This means that when an F + -cell contains also a Col El plasmid and conjugates with an F- cell, the Col El plasmid can be transferred to the recipient through the mating bridge constructed by the F-plasmid. Obviously, an F-ColEl + cell is unable to mobilize the Col-plasmid to another cell, because it is unable to build a mating bridge.

In contrast, the large Col-plasmids are self-transmissible, because they have the genes for building the conjugation apparatus themselves and do not depend on the F-plasmid for transfer to other cells. Like F and large R-plasmids, the large Col-plasmids are also conjugative plasmids.

Colicins belong to a general class of proteins, called bacteriocins. Many bacteria have been found to elaborate bacteriocins which are able to kill other related or even unrelated bacteria. Such proteins are coded by genes present in bacteriocinogenic plasmids.

Bacteriocins produced by different bacteria are sometimes given different names, like pyocine produced by Pseudomonas aeruginosa, megasine elaborated by Bacillus megaterium, nisin by lactobacilli, etc. In general, bacteriocins exert their antibacterial action by binding to the cell wall of the target cells and by inhibiting one of the vital metabolic processes, like replication of nucleic acids, transcription, protein synthesis or energy metabolism.

Bacteriocins produced by enteric bacteria help to maintain a healthy ecological balance in the human colon. Other bacteriocins produced by bacteria under natural environmental conditions probably function by eliminating competitors. Nisin produced by lactic acid bacteria has been commercially used for preservation of food and dairy products.

(iv) Degradative Plasmids:

Degradation or dissimilation of organic compounds in course of mineralization is often controlled by plasmid-borne genes in many microorganisms. Such plasmids with genes coding for enzymes that catabolize complex organic molecules are known as degradative or dissimilation plasmids. For example, in species of Pseudomonas, both chromosomal and plasmid genes produce enzymes for break­down of complex compounds.

Some of the plasmid genes code for enzymes which degrade such unusual compounds like camphor, toluene, naphthalene, salicylate and complex hydrocarbons of crude petroleum. With the help of these enzymes, the bacteria can utilize these compounds as source of carbon and energy.

As a result, bacteria possessing such degradative plasmids stand a much better chance of survival under conditions where only such unusual compounds are available. Normal bacteria without such plasmid-coded enzymes would perish under similar conditions.

The capability of organisms carrying degradative plasmids to metabolize unusual diverse complex compounds suggests the possibility of employing them as means of bioremediation of the polluted environment. The development of genetic engineering techniques has encouraged scientists to develop genetically improved strains of bacteria containing plasmids capable of degradation of an array of complex compounds, such as those occurring in crude petroleum.

A synthetic strain of Pseudomonas has been developed by Anandamohan Chakraborty of the university of Illinois, USA offering prospects of practical use in removing oil-spills in the oceans, caused by leakage of crude petroleum from tankers. Oil-spills prove a great danger to marine life, both plants and animals.

(v) Ti-Plasmid of Agrobacterium:

Ti-plasmid is a tumour-inducing large extra-chromosomal double stranded circular DNA which is present in Agrobacterium tumefaciens, a plant-pathogenic bacterium causing the crown-gall disease in many dicotyledonous species. Crown-gall is a tumour produced at the collar region of plants by agrobacteria which possess the Ti-plasmid. Bacteria lacking the plasmid are non-virulent.

Ti-plasmid is about 200 kilo base-pair long circular DNA. Only a small part of this large molecule, a 30 kilo base-pair long fragment is responsible for tumour formation. This fragment is called the T-DNA (T stands for transformation). When Agrobacterium infects a susceptible host plant, the Ti-plasmid is released in the host cell and a copy of the T-DNA is integrated into the genome of the host plant.

The integrated T-DNA then stimulates cellular atrophy producing eventually a tumour, called a crown gall. The T-DNA insertion in plant host genome is the first instance of an inter-kingdom genetic exchange by natural means.

A notable feature of T-DNA is that once it is incorporated into the host genome, the presence of the pathogenic organism is no longer necessary for induction of tumour. Thus, a close parallelism with cancer induction in animal cell is observed. The T-DNA segment of the Ti-plasmid contains genes controlling synthesis of phytohormones, like indole acetic acid and cytokinins, as well as several other compounds, called opines. Opines, such as octopine and nopaline are used as growth substrates by agrobacteria.

The rest of the Ti-plasmid contains several genes controlling virulence (vir genes). These genes control T-DNA transfer to the host. Other genes of the plasmid control functions relating to bacterial conjugation, DNA replication and catabolism of the opines synthesised by gene products of the T-DNA segment.

The T-DNA acts as a mobile unit like a transposon, but it does not have a gene, like transposase to mediate its own mobilization . Its mobilization is effected by genes located in the Ti-plasmid, but outside T-DNA. The 30 kilo base long T-DNA is flanked on either side by 25 base pair imperfect direct repeats forming T-DNA borders.

The vir genes of Ti-plasmid are involved in the generation of a transferable copy of T-DNA and its transfer to plant cell through the cell membrane and the nuclear membrane, as well as through the bacterial and plant cell walls. T-DNA is transferred as a single-stranded copy.

The copy is separated from T-DNA segment, capped at the 5′-end by a protein coded by a vir gene (vir D2) and covered by a large number of protein molecules coded by another vir gene (vir E2). This T-complex is transported to the plant cell through a membrane pore produced by another vir gene. The T-complex (ss-DNA + proteins) is about 3.6 μm long and less than 2 nm thick.

A gross structure of the Ti-plasmid and generation of the T-complex have been diagrammatically represented in Fig. 9.90:

The ability of Agrobacterium tumefaciens to transfer its Ti-plasmid to many dicotyledonous plants (but not monocotyledonous ones) opened up the possibility of introducing foreign genes into the hosts using the Ti-plasmid as a vehicle (vector).

This has been practically employed to insert a gene of interest into the T-DNA segment by recombinant DNA technology. The tumour-inducing genes and other unnecessary genes of T-DNA are removed and replaced by the gene chosen for insertion. Several foreign genes have been introduced into a variety of hosts to produce transgenic plants.

Among the notable achievements are productions of transgenic plants resistant to the herbicide glyophosate and to feeding insects. Glyophosate resistance gene was isolated from Salmonella and the insect-resistance gene from Bacillus thuringiensis which synthesise an insecticidal protein. Another interesting achievement though not of practical significance was production of bioluminescent tomato plants by introducing the gene controlling bioluminescence in fire-fly.

(vi) Eukaryotic Plasmids:

Plasmids occur rarely in eukaryotic cells. Some plasmids have been found in yeast (Saccharomyces cerevisiae) and in several plants. The only RNA plasmid discovered till now has been found in yeast. It is a double-stranded RNA having a molecular weight of 15 x 10 6 Daltons. It contains 10 genes including one coding for a bacteriocin-like protein. The protein can kill other yeast cells lacking the plasmid. This yeast plasmid has been designated as killer particle.

Yeast also contains small DNA plasmids with high copy number. They are located in the nucleus and like the chromosomal DNA are associated with basic proteins — histones. Some yeast DNA plasmids have been genetically engineered in such a way that they are capable of multiplication in both E. coli and yeast.

One such engineered yeast plasmid is Yep which can function as a shuttle vector. This plasmid has been used in transfer of useful genes from other organisms into yeast cells via E. coli for production of valuable therapeutically important proteins. A successful application of the Yep plasmid is the transfer of the gene coding the coat glycoprotein of hepatitis B virus to yeast.

The transgenic yeast can express the gene successfully with production of the viral glycoprotein. The glycoprotein has been used for preparation of hepatitis B vaccine for human application. Shuttle vectors are specially useful in transferring eukaryotic genes, because such genes are often not successfully expressed in bacterial hosts.

Besides yeast, DNA plasmids have been discovered in several plants, like maize and sorghum, as also in several fungi. These plasmids are made of usually linear double stranded DNA molecules where as all bacterial plasmids are circular.

Replication of Plasmids:

In the non-dividing plasmids, the double-stranded DNA exists as a right-handed super-helical coil having 400-600 base pairs per turn of the coil. During replication, the plasmids can multiply autonomously, although replication requires the host cell enzymes. That is why plasmids can multiply only within host cells.

Each plasmid has its own origin of replication. Some plasmids also have genes which code for proteins necessary for their own multiplication. This is proved by the fact that a temperature-sensitive mutant of F-plasmid (F + ts) is unable to replicate at 42°C, although it can function normally at 37°C.

Different aspects of plasmid replication are briefly discussed below:

(i) Non-Transmissible Plasmids:

Replication of plasmid DNA starts at the site of origin and may proceed either bi-directionally as in case of bacterial chromosome, or may proceed unidirectionally depending on the nature of plasmid. In bidirectional replication, replication terminates when the two replication forks meet each other. In unidirectional replication, termination occurs when the replication fork reaches the site of origin. In both cases, the circularity of the plasmid DNA is maintained throughout the process.

(ii) Self-Transmissible Plasmids:

In case of conjugative plasmids, like F-plasmid or R-plasmid, replication occurs by the rolling- circle model. The supercoiled DNA undergoes a nick in one of the strand resulting in relaxation of the super coiled state to form an open circle. The enzyme catalyzing the nick remains attached to the 5′-P end of the relaxed molecules. Such a single-stranded nick becomes necessary for transfer of a copy of the plasmid during conjugation to the mating partner.

By rolling circle replication, the donor cell retains its double-stranded plasmid, while a single-stranded copy is transferred through the mating bridge to the recipient cell, where a complimentary strand is synthesized and ligated to form a double-stranded copy of the plasmid. Replication of an F-plasmid is diagrammatically shown in Fig. 9.91.

(iii) Control of Copy Number:

The large plasmids are characterized by low copy number (one to few) and small plasmids by high copy number (10 to 100). The copy number is controlled by an inhibitor coded by the plasmid DNA itself. The inhibitor concentration in the bacterial cell determines the rate of initiation of plasmid replication.

When a cell containing two large plasmids divides to produce two daughter cells, each having one plasmid, the inhibitor concentration in these cells is the same as that of the mother cell. Now, the daughter cells grow in size to attain maturity resulting in lowering of the inhibitor concentration in the cytoplasm.

As a consequence, DNA synthesis is initiated in the plasmid leading to its replication producing two copies. As each plasmid copy possesses an inhibitor gene, the production of inhibitor doubles and the inhibitor concentration becomes high enough to stop plasmid DNA synthesis and further replication. Thus, the copy number is restricted to two per cell.

A similar mechanism of control of copy member is believed to operate in case of high copy number plasmids also. However, in this case, the inhibitor concentration must reach a higher threshold level to stop initiation of plasmid DNA synthesis in comparison to that of low copy number plasmids.

(iv) Plasmid Amplification:

Another important point of plasmid replication is that chromosomal DNA synthesis and plasmid DNA synthesis are independent of each other, though, in both, DNA synthesis is followed by replication. Thus it is possible to stop chromosomal DNA synthesis and replication without affecting
plasmid DNA synthesis and replication.

Such situation can be practically created by adding chloramphenicol to a bacterial culture. This antibiotic specifically inhibits prokaryotic protein synthesis. When it is added to a growing bacterial culture, chromosomal DNA synthesis is inhibited, but plasmid DNA synthesis and replication continue at the cost of the available replication proteins which are not used for chromosomal DNA synthesis.

The net result is that each bacterial cell contains large number of plasmid copies. This is known as plasmid amplification. When a specific gene which has been transferred (cloned) to a plasmid requires to be isolated, plasmid amplication becomes a useful tool, because of high plasmid DNA concentration in the total cellular DNA.

(v) Transfer of Non-Self Transmissible Plasmids:

There are some plasmids which do not possess genes for self-transmission, but can be transferred to other cells with the help of a self-transmissible plasmid when both plasmids occur in the same cell. They are known as mobilizable plasmids.

Such plasmids possess genes for proteins needed for nicking its own DNA at the site of origin of replication, but lack in genes needed for building the conjugation tube. When they coexist with a self-transmissible plasmid, like F or R, the latter can build the mating bridge through which a copy of the mobilizable plasmid produced by rolling-circle replication is transferred to a recipient cell (Fig. 9.92).

A different type of mobilization occurs when a donor cell having a self-transmissible plasmid conjugates with a recipient having a mobilizable plasmid. In this type of conjugation, both the donor and the recipient acquire a copy of both types of plasmids by a process of retro transfer. First, the self- transmissible plasmid replicates by rolling-circle model and a single-stranded copy is transferred through the mating bridge to the recipient, where it forms a complimentary strand leading to the formation of a copy of the self-transmissible plasmid in the usual way.

The mobilizable plasmid in the recipient cell then replicates and a single-stranded copy is transferred to the other cell which now acts as the recipient of the mobilizable plasmid. Finally, the two cells separate and each has a copy of the self- transmissible plasmid and a copy of the mobilizable plasmid (Fig. 9.93).

Incompatibility of Plasmids:

Generally, two closely related plasmids cannot coexist in a bacterial cell. In the population of progeny cells derived from a cell containing two such plasmids, the proportion of cells having only one of the two plasmids increases with every cell division. This is known as plasmid incompatibility.

On the other hand, two different unrelated plasmids, e.g. F plasmid and ColEl can exist together without any difficulty, because these plasmids belong to two different incompatibility groups. Whereas, two F-plasmids cannot coexist in the same cell.

One mechanism by which a plasmid already resident in a cell prevents the entry of a second similar plasmid into the same cell is by surface exclusion. For example, an F-plasmid of E. coli does not allow entry of another F-plasmid by inhibiting it from leaving the cell where it is already located. The effect is mediated at the surface of the cell whereby the F-DNA cannot come out of the cell.

A different mechanism operates when a cell already has two closely related plasmids, say X and Y. We know that the copy number of plasmids is controlled by specific inhibitors coded by the plasmid itself.

As X and Y are two closely related plasmids, it would be expected that their inhibitors would also be closely similar and that replication of both the plasmids would be regulated by the inhibitor produced either by X or Y.

During replication, X and Y may be selected at random, so that, during first replication of the plasmid, a cell initially containing one copy of each plasmid may produce two copies of either X or Y, so that the cell has now two copies of either X or Y and one copy of the un-replicated plasmid i.e. 2X + Y or X + 2Y. In the second round of plasmid replication, each cell will contain 4 plasmids, but depending on which plasmid is replicated, the combination may be X + 3Y, 2x + 2Y or 3X + Y.

Now the cell divides to produce two daughter cells, each with 2 plasmids and the plasmid combinations of the daughter cells may be X + X, X + Y or Y + Y. Thus the probability of progeny cells having either two X plasmids or two Y plasmids is equal to those having two different plasmids i.e. X + Y. In other words, the probability of elimination of one plasmid is 50%. Such probability increases with more cell generations.

The events leading to plasmid elimination are shown in Fig. 9.94:

Plasmid Library:

A plasmid library is a gene library which contains a collection of bacterial cultures, each of which contains a plasmid, but plasmid of one culture differs from that of another in having a separate DNA fragment of a genome of an organism. The total genome isolated from an organism is fragmented and the fragments are inserted (cloned) separately into individual plasmids.

These recombinant plasmids are then introduced into suitable host bacteria. Thus each bacterial culture contains a plasmid with a fragment. The total collection of cultures would be expected to contain the entire genome of an organism and would constitute a gene library of the particular organism. This is schematically represented in Fig. 9.95.


How many plasmids can a competent bacteria take - (Aug/22/2008 )

i would appreciate if any body can tell me how many plasmids a competent bacteria can take.
i am actually transforming 2 plasmids which differ only in a tag and i cant separate them before transformation bcz i myself add the tag. so i just want to know if competent bacteria take only one plasmid or they can take several, if the first one is true i can simply separat them with colony picking. otherwise gel purification and dna sequencing must come to help me work it out

If you asking . if I have 2 different plasmids in my mix, will both be taken up in the same bug? The answer is YES.

what is your tag . if it is a genetic tag, you could use PCR to identify which clone contains only the tagged-plasmid.

I was been taught that it is very low chance for one competent cell to take more than 2 different plasmids.

I also believe that this is a rare problem. Even if it were common, you can achieve your desired result by transforming with serial dilutions of your ligation, and picking colonies from the more dilute transformation plates.

to my knowledge transformation doesnot depends on kind of plasmid. any kind of plasmid can be inserted into bacteria. its a physical process.

There's a difference between putting all of the plasmids into a cell at the same time and putting them in one at a time. For inserting five, you would do them one at a time with selection at each stage. I thought the question concerned the insertion of more than one plasmid from solution in the same transformation round.

The plasmids must be of different incompatibility groups to stably coexist in the same cell.

many thanks to your replies,
both plasmids are pcDNA 3.1(+), the insert is also the same but there is a flag tag only in one of them.
i am transforming them at the same time and i cant separate them before transformation.
i cant get what u mean by "The plasmids must be of different incompatibility groups to stably coexist in the same cell"

Say we call one plasmid "A" and the other "B". At the time of transformation, you may have a single cell with both "A" and "B" resident. However, it is unlikely that this situation is what you need rather, I'm guessing you probably want this cell to divide, and for each of the daughter cells to also contain "A" and "B", and for these daughter cells to divide and each of their siblings to contain "A" and "B", etc., etc., and for each of the plasmids to express the genes they carry.

A problem with this scenario arises if plasmids "A" and "B" belong to the same incompatibility (Inc) group. Plasmids do not exist singly in a cell, but have a genetically determined copy number they strive to maintain. Plasmids belonging to the same incompatibility group interfere with the stable segregation of each other into progeny cells so that, over time, one or the other plasmid is disproportionately represented in any given cell many times one or the other plasmid is completely displaced and thus lost in the population.

For more information, see the "Incompatibility Groups" section here, see the sections on "Incompatibility testing" and "Incompatibility groups" in the Google Books preview of Plasmid Biology by Gregory Phillips, Barbara E. Funnell here, or Google "plasmid incompatibility groups".


Contents

Resistance plasmids by definition carry one or more antibiotic resistance genes. They are frequently accompanied by the genes encoding virulence determinants, specific enzymes or resistance to toxic heavy metals. Multiple resistance genes are commonly arranged in the resistance cassettes. The antibiotic resistance genes found on the plasmids confer resistance to most of the antibiotic classes used nowadays, for example, beta-lactams, fluoroquinolones and aminoglycosides. [1] [3]

It is very common for the resistance genes or entire resistance cassettes to be re-arranged on the same plasmid or be moved to a different plasmid or chromosome by means of recombination systems. Examples of such systems include integrons and transposons. [3]

Most of the resistance plasmids are conjugative, meaning that they encode all the needed components for the transfer of the plasmid to other bacterium. Other smaller plasmids (usually < 10 kb in size) can be mobilized by a conjugative plasmid (usually > 30 kb) in order to be transferred. [3]

Members of family Enterobacteriaceae, for example, Escherichia coli or Klebsiella pneumoniae pose the biggest threat regarding plasmid-mediated resistance in hospital- and community-acquired infections. [1]

Beta-lactam resistance Edit

Both narrow spectrum beta-lactamases (e.g. penicillinases) and extended spectrum beta-lactamases (ESBL) are common for resistance plasmids in Enterobacteriaceae. Often multiple beta-lactamase genes are found on the same plasmid hydrolyzing a wide spectrum of beta-lactam antibiotics. [1]

Extended spectrum beta-lactamases (ESBL) Edit

ESBL enzymes can hydrolyze all beta-lactam antibiotics, including cephalosporins, except for the carpabepenems. The first clinically observed ESBL enzymes were mutated versions of the narrow spectrum beta-lactamases, like TEM and SHV. Other ESBL enzymes originate outside of family Enterobacteriaceae, but have been spreading as well. [1]

In addition, since the plasmids that carry ESBL genes also commonly encode resistance determinants for many other antibiotics, ESBL strains are often resistant to many non-beta-lactam antibiotics as well, [4] leaving very few options for the treatment.

Carbapenemases Edit

Carbapenemases represent type of ESBL which are able to hydrolyze carbapenem antibiotics that are considered as the last-resort treatment for ESBL-producing bacteria. KPC, NDM-1, VIM and OXA-48 carbapenemases have been increasingly reported worldwide as causes of hospital-acquired infections. [1]

Quinolone resistance Edit

Quinolone resistance genes are frequently located on the same plasmid as the ESBL genes. Examples of resistance mechanisms include different Qnr proteins, aminoglycose acetyltransferase aac(6')-Ib-cr that is able to hydrolyze ciprofloxacin and norfloxacin, as well as efflux transporters OqxAB and QepA. [1]

Aminoglycoside resistance Edit

Aminoglycoside resistance genes are also commonly found together with ESBL genes. Resistance to aminoglycosides is conferred via numerous aminoglycoside-modifying enzymes and 16S rRNA methyltransferases. [1]


DNA study shows bacteria mixing and matching genes to survive

The smallest creatures can have some of the biggest impacts on the planet. You can get a sense of how healthy a given environment is by taking a census of the types of microorganisms that call it home. Now researchers at Berkeley Lab have developed a new way to get a larger snapshot of what's going on, by looking specifically at the genes bacteria pass around to help each other adapt to a changing world.

While most species pass genes down "vertically" from parent to child, bacteria have the ability to share them "horizontally," swapping DNA packages called plasmids to essentially teach each other new tricks. This sneaky skill-swapping is how bacteria are quickly becoming resistant to antibiotics.

Since they're shared so widely, studying these plasmids can give scientists a window into the environment as a whole, by looking at what kind of pressures the organisms there are facing. If, for example, a pond is particularly salty, you might find a high level of plasmids that help bacteria survive saltiness.

"When you want to learn about a microbial community, focusing specifically on their plasmids allows you to get a sense of the suite of capabilities that a community wants to keep mobile perhaps because they are needed periodically," says Aindrila Mukhopadhyay, lead researcher on the study. "Studying plasmids is like looking inside someone's backpack to see what they're keeping handy to use themselves and potentially share with another person. Say you look inside and find an umbrella. It may not be raining at the time, but the umbrella suggests that it rains from time to time."

This overall network of plasmids is known as the plasmidome. While this isn't the first time scientists have studied it, the team developed and refined a new method to help separate it from the wider pool of chromosomal DNA in a sample. The new method lets scientists detect different-sized plasmids even in environments with relatively few bacteria.

Lead researcher Aindrila Mukhopadhyay, left, and lead author of the paper, Ankita Kothari, right

The team tested the technique in samples of groundwater from several wells at the Oak Ridge Field Research Center, an environment that's laden with heavy metals. Hundreds of different plasmids were found, and similar ones showed up in different samples regardless of the variety of bacteria species present.

The most common plasmids found were those that gave bacteria resistance to mercury, but interestingly, the team didn't detect any mercury in the water. The fact that these genes had spread around so widely indicated that the water must have been contaminated by mercury in the past, and the microbial community is still hanging onto the resistance in case it happens again.

Knowing these kinds of things can give scientists a snapshot of the past and present of a site and the organisms that live there, as well as a glimpse at how well they might be able to adapt to certain changes in future. On top of that, the study might help uncover new plasmids that could be put to work cleaning up the environment, for example, or treating sewage.

"These mobile genetic packages present a way to manipulate these organisms naturally," says Ankita Kothari, lead author of the study. "So, if you want to go examine an ecosystem at a molecular level and you need genetic tools to do that, the answer could be in the plasmidome already."

The research was published in the journal mBio.

The smallest creatures can have some of the biggest impacts on the planet. You can get a sense of how healthy a given environment is by taking a census of the types of microorganisms that call it home. Now researchers at Berkeley Lab have developed a new way to get a larger snapshot of what's going on, by looking specifically at the genes bacteria pass around to help each other adapt to a changing world.

While most species pass genes down "vertically" from parent to child, bacteria have the ability to share them "horizontally," swapping DNA packages called plasmids to essentially teach each other new tricks. This sneaky skill-swapping is how bacteria are quickly becoming resistant to antibiotics.

Since they're shared so widely, studying these plasmids can give scientists a window into the environment as a whole, by looking at what kind of pressures the organisms there are facing. If, for example, a pond is particularly salty, you might find a high level of plasmids that help bacteria survive saltiness.

"When you want to learn about a microbial community, focusing specifically on their plasmids allows you to get a sense of the suite of capabilities that a community wants to keep mobile perhaps because they are needed periodically," says Aindrila Mukhopadhyay, lead researcher on the study. "Studying plasmids is like looking inside someone's backpack to see what they're keeping handy to use themselves and potentially share with another person. Say you look inside and find an umbrella. It may not be raining at the time, but the umbrella suggests that it rains from time to time."

This overall network of plasmids is known as the plasmidome. While this isn't the first time scientists have studied it, the team developed and refined a new method to help separate it from the wider pool of chromosomal DNA in a sample. The new method lets scientists detect different-sized plasmids even in environments with relatively few bacteria.

Lead researcher Aindrila Mukhopadhyay, left, and lead author of the paper, Ankita Kothari, right

The team tested the technique in samples of groundwater from several wells at the Oak Ridge Field Research Center, an environment that's laden with heavy metals. Hundreds of different plasmids were found, and similar ones showed up in different samples regardless of the variety of bacteria species present.

The most common plasmids found were those that gave bacteria resistance to mercury, but interestingly, the team didn't detect any mercury in the water. The fact that these genes had spread around so widely indicated that the water must have been contaminated by mercury in the past, and the microbial community is still hanging onto the resistance in case it happens again.

Knowing these kinds of things can give scientists a snapshot of the past and present of a site and the organisms that live there, as well as a glimpse at how well they might be able to adapt to certain changes in future. On top of that, the study might help uncover new plasmids that could be put to work cleaning up the environment, for example, or treating sewage.

"These mobile genetic packages present a way to manipulate these organisms naturally," says Ankita Kothari, lead author of the study. "So, if you want to go examine an ecosystem at a molecular level and you need genetic tools to do that, the answer could be in the plasmidome already."


Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community

Conjugal plasmids can provide microbes with full complements of new genes and constitute potent vehicles for horizontal gene transfer. Conjugal plasmid transfer is deemed responsible for the rapid spread of antibiotic resistance among microbes. While broad host range plasmids are known to transfer to diverse hosts in pure culture, the extent of their ability to transfer in the complex bacterial communities present in most habitats has not been comprehensively studied. Here, we isolated and characterized transconjugants with a degree of sensitivity not previously realized to investigate the transfer range of IncP- and IncPromA-type broad host range plasmids from three proteobacterial donors to a soil bacterial community. We identified transfer to many different recipients belonging to 11 different bacterial phyla. The prevalence of transconjugants belonging to diverse Gram-positive Firmicutes and Actinobacteria suggests that inter-Gram plasmid transfer of IncP-1 and IncPromA-type plasmids is a frequent phenomenon. While the plasmid receiving fractions of the community were both plasmid- and donor- dependent, we identified a core super-permissive fraction that could take up different plasmids from diverse donor strains. This fraction, comprising 80% of the identified transconjugants, thus has the potential to dominate IncP- and IncPromA-type plasmid transfer in soil. Our results demonstrate that these broad host range plasmids have a hitherto unrecognized potential to transfer readily to very diverse bacteria and can, therefore, directly connect large proportions of the soil bacterial gene pool. This finding reinforces the evolutionary and medical significances of these plasmids.

Figures

Typical transconjugal microcolonies for plasmid…

Typical transconjugal microcolonies for plasmid pKJK5:: gfp introduced through E. coli MG1655:: lacI…

FACS sorting of transconjugal cells…

FACS sorting of transconjugal cells from a mating mixture initiated with soil bacteria…

Principal coordinate analysis (PCoA) of…

Principal coordinate analysis (PCoA) of individual transconjugal pools, as well as of the…

Phylogenetic tree showing all identified…

Phylogenetic tree showing all identified transconjugant OTUs for three different plasmids (pKJK5, RP4…

Phylogenetic tree showing all identified…

Phylogenetic tree showing all identified transconjugant OTUs for the same plasmid (pKJK5) introduced…

Venn diagram of transconjugal pools…

Venn diagram of transconjugal pools for plasmid pKJK5 transferred from three different donor…


Bacterial Transformation

Transformation is the genetic alteration of a cell by the update of DNA from the environment. This process can occur naturally in some types of bacteria, but is typically rare. In a lab, we can subject bacteria to conditions that will cause them to take up DNA from the environment (to become “transformed”). There are several ways to transform bacteria in a lab setting, but one of the most common involves changing the concentration of ions in the bacteria’s surroundings and then heating the cells in a specific way. Bacteria that are able to easily take up DNA from the environment are called “competent”. Making cells competent renders their cell membrane more permeable to DNA. After the new DNA has entered the bacteria, it is used by the cell to make RNA and then protein. The new proteins produced from this DNA are what cause the change in the traits of the cells.

Plasmids

In addition to their DNA genome (which is circular), bacteria can also contain additional smaller circles of DNA called plasmids. A plasmid is a small, circular piece of double-stranded DNA that can be copied by bacterial cells. Plasmids occur naturally in bacteria and they are widely used by scientists as a method of for introducing foreign DNA into these cells because the sequence of DNA within the plasmid can be modified in the lab. Once a plasmid has entered a cell, it is copied by the cell’s DNA replication machinery. When the bacterial cell divides, each new daughter cell receives copies of the plasmid. One original transformed bacteria will divide to form a visible colony made up of one million or more transformed bacteria, which each contain a copy of the plasmid (Figure 3).

Figure 2: Diagram of a bacteria that contains bacterial genomic DNA and three plasmids. Note that the plasmids are not to scale and would typically be much smaller than the bacterial genome. When the bacteria divides, the daughter cells receive a copy of the genomic DNA and of any plasmids present in the cell. Credit: Spaully CC SA 2.5 Plasmid Replication

Figure 3: E. coli growing on a nutrient plate. Each spot is one isolated colony (one distinct circular spot). Each colony is made up of 10’s or 100’s of thousands of cells that grew from a single original bacterial cell. Picture modified from: Madprime “K12 E coli colonies on plate Public Domain.

Selecting for transformed bacteria

In order to transform bacteria using plasmid DNA, biotechnologists must overcome two problems. First, cells that contain plasmid DNA have a disadvantage since cellular resources (such as energy) are being used to replicate the plasmid and to synthesize the proteins that are encoded for by the plasmid’s DNA. If a mixed population of cells with plasmids and cells without plasmids is grown together in the presence of plenty of nutrients, then the cells without the plasmids grow faster because they are not wasting energy on a plasmid that they do not need (Figure 4). Therefore, there is always tremendous pressure on cells to get rid of their plasmids. If they are able to get rid of the plasmid, they will grow faster on a nutrient plate (or in the environment). However, getting rid of the plasmid is exactly what we do not want them to do. To overcome the pressure to get rid of the plasmid, we must provide an advantage to the cells that have and keep the plasmid.

Figure 4: When bacteria with and without a plasmid are grown on an LB nutrient plate (no antibiotic present), the bacteria without the plasmid will grow more quickly because it is not using energy to replicate the plasmid and to make proteins encoded by the plasmid. Photo Credit: Lisa Bartee, 2020, CCBY.

Second, we need to be able to determine which bacteria received the plasmid. In a typical transformation, billions of bacteria are treated to make them competent and then exposed to plasmid DNA. Typically, fewer than 1 in 1000 bacteria will acquire the plasmid (Figure 5). We need a way to get rid of the untransformed bacteria (greater than 99% of the total bacteria present) so that we are left with only the bacteria that were transformed with the plasmid. If we do not get rid of the untransformed bacteria, we will not be able to see the transformed bacteria since they are such a small percentage of the total number.

Figure 5: Only a few bacteria in a transformation (typically less than 1%) will actually take up the plasmid and become transformed. We need a way to get rid of the bacteria that have not taken up the plasmid so that we are left with only transformed bacteria. Photo Credit: Lisa Bartee, 2020, CCBY.

Antibiotic resistance genes provide a means of finding the bacteria that acquired the plasmid DNA in the midst of all those bacteria that did not. Antibiotics are chemicals that inhibit the growth of or kill bacteria. If the plasmid contains a gene for resistance to an antibiotic, then after transformation, bacteria grown on a nutrient plate containing the antibiotic will not be inhibited or killed by it. This means that bacteria that took up the plasmid during transformation can be distinguished from bacteria that did not by growing the bacteria on a nutrient plate containing the antibiotic (Figure 6). Only the bacteria that were transformed with the plasmid will survive the killing effect of the antibiotic and grow to form visible colonies on the plate. Remember that a colony is formed from more than one million genetically identical bacterial cells. This means that the only colonies growing on a nutrient + antibiotic plate after a transformation are the bacteria that acquired and kept the plasmid. Using an antibiotic in the nutrient plate and an antibiotic resistance gene in the plasmid accomplishes our two goals of giving an advantage to cells that have a plasmid so the plasmid is retained and of having a marker so we know our cells contain new DNA. Resistance to an antibiotic is known as a selectable marker because we can select for cells that contain it.

Figure 6: When bacteria with and without a plasmid are grown on an LB nutrient plate containing antibiotic, the bacteria without the plasmid will all be killed by the antibiotic. Only bacteria containing the antibiotic will be able to grow. Photo Credits: Lisa Bartee, 2020, CCBY. Gabriel Van Helsing, CCSA 3.0, Skull and Crossbones.

This is what it using an antibiotic to select transformed cells that contain a plasmid would look like:

For Our Lab:

The plasmid that we will be using is called pGLO (available from Bio-Rad). This plasmid contains several important pieces:

  • Ori – an origin of replication, which allows the plasmid to be copied when the bacteria divide.
  • GFP (green fluorescent protein) gene – the GFP protein gives a green glow in the presence of UV light.
  • bla gene – the enzyme beta-lactamase is produced from this gene. This enzyme breaks down some antibiotics such as ampicillin when they are present in the environment before they can kill the bacteria.
  • araC gene – the AraC protein produced by this gene turns on the GFP gene when arabinose is present in the environment.

Bacteria that are transformed with this plasmid will have two new traits: they will fluoresce green under UV light and they will be resistant to the antibiotic ampicillin.

The basic steps in the process of bacterial transformation are:

  • Mix actively growing bacteria with the plasmid DNA you want to insert in a tube containing CaCl2 (calcium chloride) solution.
  • “Heat shock” the bacteria by rapidly heating and then cooling them. This process causes the plasmid to enter the bacteria.
  • Transfer the bacteria to an LB nutrient plate (containing nutrients) so that they can recover and express their newly acquired genes.

After the bacterial transformation procedure has been carried out, cells that contain the plasmid are selected for by growing the bacteria on LB nutrient plates that contain ampicillin. The ampicillin kills any cell that did not get transformed with the plasmid. This means that the only bacteria which can grow to form visible colonies on a plate containing LB nutrients and ampicillin are transformed cells. These cells will produce GFP at very low levels and will appear whitish when viewed under UV light.

Arabinose is a type of sugar that can be added to the plates when they are poured. Although arabinose is a sugar, it is not being used as a nutrient source in this experiment. When transformed bacteria are grown on plates containing LB nutrients + ampicillin + arabinose, the arabinose interacts with the araC protein (which is produced from the araC gene). The interaction of arabinose + araC protein stimulates transcription of the GFP gene. This results in a brilliant green glow when the bacteria are viewed under a UV light source.

Samples in our experiment

In our lab, we will compare transformed (+pGLO) and non-transformed (-pGLO) bacteria grown on several different types of plate. Here are the key points to remember:


Protein expression from bacteria which has two plasmids? - Incompatibility of plasmids (Jul/27/2007 )

Hi!
I am planning to express two plasmids in bacteria. I will have two different pET vectors with F1 origin. They have diffrent antibiotic resistance. Has anybody had `bad`or `good`experience in expressing two pET vectors. I have read that it might be problematic to express two plasmids in bhttp://www.protocol-online.org/forums/style_images/1/folder_post_icons/icon2.gif
http://www.protocol-online.org/forums/styl. con2.gifacteria due to the incompatibility of two plasmids. But I could not find any info about the criteria to be considered as incompatible.
I would be grateful for your replies.
Thank you

I dont really get it. Do you mean by transforming 2 plasmids inside the bacteria? Hmm.. this is the first time I heard about it. In my own opinion, I don't think it will be a very good idea to express 2 plasmids at one time. Expressing 1 plasmid itself is tough.

5 plasmids? Hmm what kind of bacteria can do so? I am just curious. Not an expert in it. =)

What you want to do is possible, as each plasmid type has its own antibiotic selection marker. However, the system is unstable as two plasmids from the same incompatability group interefere with the stable inheritance of each other. Thus the ratio of plasmid A to plasmid B will vary from cell to cell. some cells will have more plasmid A and others less. Which in turn results in the ratio of proteins expressed by each plasmid to differ between cells.

There are about 30 incompatability groups. So theoratically I could have

30 plasmids living side by side, as long as each plasmid comes from a different incompatability group.

Still, 5 is a lot. The most I have read about is 4. The e coli cells weren't too happy. Too many antibiotics.

You could maintain 2 plasmids inside 1 E.coli, but you need:
- Different replicator factors (diffrent ori)
- Different selection markers.

Since you said that you have 2 diffrent pET vector with F1 origin, I take it that the ori region for both vectors is the same? If so, the two plasmids are incompatible, can't maintain for long in the same bacteria. Choose different ori.


Eva Top

My research is currently focused on the evolution and ecology of plasmids that transfer to and replicate in a broad range of bacteria. Plasmids are mobile genetic elements found in most bacteria. Because they readily transfer between different types of bacteria under natural conditions, they play an important role in rapid bacterial adaptation to changing environments. A good example is the current epidemic of multiple antibiotic resistance in human pathogens, which is largely due to the spread of multi-drug resistance plasmids. Although plasmid-mediated gene transfer is now recognized as a key mechanism in the alarming rise of antibiotic resistance, little is known about their host range, their ability to invade bacterial populations in the absence of selection, and their genetic diversity. We are addressing these questions using various Proteobacteria and plasmids as model systems.

Selected Publications

Selection of publications in peer-reviewed journals (from over 100 total)

  • Loftie-Eaton, W., K. Bashford, H. Quinn, K. Dong, J. Millstein, S. Hunter, M. Thomason, H. Merrikh, H., J.M. Ponciano, and E.M. Top. 2017. Compensatory mutations improve general permissiveness to antibiotic resistance plasmids. Nature Ecol. Evol. 1: 1354&ndash1363.
  • Stalder, T., L. M. Rogers, C. Renfrow, H. Yano, Z. Smith, and E.M. Top. 2017. Emerging patterns of plasmid-host coevolution that stabilize antibiotic resistance. Scientific Reports 7: 4853.
  • Thomas, C.M., N. R. Thomson, A. M. Cerdeño-Tárraga, C. J. Brown, E.M. Top, and L. S. Frost. 2017. Annotation of Plasmid Genes. Plasmid 91:61-67.
  • Ridenhour, B., G. Metzger, M. France, K. Gliniewicz, J. Millstein, L. Forney, E.M. Top. 2017.Persistence of antibiotic resistance plasmids in bacterial biofilms. Evol. Appl. 10:640-647.
  • Stalder, T., and E.M. Top. 2016. Plasmid transfer in biofilms: A perspective on limitations and opportunities. NPJ Biofilms and Microbiomes. 16022: 1-5.
  • Yano H., K. Wegrzyn, W. Loftie-Eaton, J. Johnson, G.E. Deckert, L.M. Rogers, I. Konieczny, and E.M. Top. 2016. Evolved plasmid-host interactions reduce plasmid interference cost. Mol. Microbiol. 101: 743-756.
  • Loftie-Eaton, W. H. Yano, S. Burleigh, R.S. Simmons, J.M. Hughes, L.M. Rogers, S.S. Hunter, M.L. Settles, L.J. Forney, J.M. Ponciano, and E.M. Top. 2016. Evolutionary paths that expand plasmid host-range: implications for spread of antibiotic resistance. Mol. Biol. Evol. 33: 885&ndash897.
  • Li, X., Y. Wang, C. Brown, F. Yao, Y. Jiang, E.M. Top, and H. Li. 2016. Diversification of broad host range plasmids correlates with the presence of antibiotic resistance genes. FEMS Microbiol. Ecol. 92: fiv151.
  • Li, X., E. M. Top, Y. Wang, C. J. Brown, Y. Jiang, and H. Li. 2015. The broad-host-range plasmid pSFA231 isolated from petroleum-contaminated sediment represents a new member of the PromA plasmid family. Frontiers in Microbiology 5: 777 (1-12).
  • Loftie-Eaton, W., H. Suzuki, K. Bashford, H. Heuer, P. Stragier, P. De Vos, M.L. Settles, and E. M. Top 2015. Draft genome sequence of Pseudomonas sp. nov. H2. Genome Announcement 3: e00241-15.
  • Loftie-Eaton, W., A. Tucker, A. Norton, and E. M. Top. 2014. Flow cytometry and Real-Time qPCR as tools for assessing plasmid persistence. Appl Environ Microbiol. 80: 5439-5446 (PMCID: PMC4136099).
  • Hunter, S., H. Yano, W. Loftie-Eaton, J. Hughes, L. De Gelder, P. Stragier, P. De Vos, M. Settles, and E. M. Top. 2014. Draft genome sequence of Pseudomonas moraviensis R28. Genome Announcement 2: e00035-14. (PMCID: PMC3931354).
  • Brown, C. J., D. Sen, H. Yano, M. L. Bauer, L. M. Rogers, G. A. Van der Auwera, and E. M. Top. 2013. Diverse broad-host-range plasmids from freshwater carry few accessory genes. Appl. Environ. Microbiol. 79: 7684-7695. (PMCID: PMC3837812)
  • Yano, H., L.M. Rogers, M.G. Knox, H. Heuer, K. Smalla, C.J. Brown, and E. M. Top. 2013. Host range diversification within the IncP-1 plasmid group. Microbiology 159: 2303-2315. (PMCID: PMC3836486)
  • Oliveira, C.S., A. Moura, I. Henriques, C.J. Brown, L.M. Rogers, E. M. Top, and A. Correia. 2013. Comparative genomics of IncP-1&epsilon plasmids from water environments reveals diverse and unique accessory genetic elements. Plasmid 70: 412-149.
  • Król, J. E., A. J. Wojtowicz, L. M. Rogers, H. Heuer, K. Smalla, S. M. Krone, and E. M. Top 2013. Invasion of E. coli biofilms by multidrug resistance plasmids. Plasmid 70: 110&ndash119. (PMCID: PMC3687034)
  • Sen, D., C.J. Brown, E. M. Top and J. Sullivan. 2013. Inferring the evolutionary history of IncP-1 plasmids despite incongruence among backbone gene trees. Mol. Biol. Evol. 30: 154-166. (PMCID: PMC3525142)
  • Yano H, Genka H, Ohtsubo Y, Nagata Y, Top E.M., Tsuda M. 2013. Cointegrate-resolution of toluene-catabolic transposon Tn4651: Determination of crossover site and the segment required for full resolution activity. Plasmid 69: 24&ndash35.
  • Hughes, J.M., B.K. Lohman, G.E. Deckert, E.P. Nichols, M. Settles, Z. Abdo, and E. M. Top. 2012. The role of clonal interference in the evolutionary dynamics of plasmid-host adaptation. mBio 3(4): e00077-12.
  • Stolze, Y., F. Eikmeyer, D. Wibberg, G. Brandis, C. Karsten, I. Krahn, S. Schneiker-Bekel, P. Viehöver, A. Barsch, M. Keck, E. Top, K. Niehaus, A.Pühler, and A. Schlüter. 2012. IncP-1beta plasmids of Comamonas sp. and Delftia sp. strains isolated from a wastewater treatment plant mediate resistance to and decolorization of the triphenylmethane dye crystal violet.Microbiology.158: 2060-2072.
  • Van Meervenne, E., E. Van Coillie, F. Devlieghere, L. Herman, L.S.P. De Gelder, E. M. Top, and N. Boon. Strain specific transfer of antibiotic resistance from an environmental plasmid to foodborne pathogens. J. Biomed. Biotechnol. 2012: ID 834598.
  • Yano, H., G. E. Deckert, L. M. Rogers, and E. M. Top. 2012. The role of long and short replication initiation protein in the fate of IncP-1 plasmids. J. Bacteriol. 194: 1533&ndash1543.
  • Eikmeyer, F.G., R. Szczepanowski, D.Wibberg, A. Hadiati, S. Schneiker-Bekel, L. M. Rogers, C.J. Brown, E. M. Top, A. Pühler, A. Schlüter. 2012. The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution. Plasmid 68: 13-24
  • Heuer, H., C.T.T. Binh, S. Jechalke, C. Kopmann, U. Zimmerling, E. Krögerrecklenfort, T. Ledger, B. Gonzalez, E. M. Top, K. Smalla. 2012. IncP-1&epsilon plasmids are important vectors of antibiotic resistance genes in agricultural systems: diversification driven by class 1 integron gene cassettes. Frontiers in Microbiology 3: Article 2. http://www.ncbi.nlm.nih.gov/pubmed/22279444
  • Król, J.E., J.T. Penrod, H. McCaslin, L.M. Rogers, H. Yano, W. Dejonghe, C.J. Brown, R.E. Parales, S. Wuertz, E. M. Top. 2012. Genomic and functional analysis of the IncP-1&beta plasmids pNB8c and pWDL7::rfp explains their role in 3-chloroaniline catabolism. Appl. Environ. Microbiol. 78: 828-838. http://www.ncbi.nlm.nih.gov/pubmed/22101050
  • Zhong, X., J. Droesch, R. Fox, E. M. Top, and S M. Krone. 2012. On the meaning and estimation of plasmid transfer rates for surface-associated and well-mixed bacterial populations. J. Theor. Biol. 294: 144&ndash152. http://www.ncbi.nlm.nih.gov/pubmed/22085738

Research Projects

Dr. Top's lab is a member of Biology, Institute for Bioinformatics and Evolutionary Studies (IBEST) and the Bioinformatics & Computational Biology (BCB) Graduate Programs.



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