Mutations and Their Chemical Basis

Mutations and Their Chemical Basis

 Mutations and Mutagenesis

 Morphological mutations change the microorganism’s colonial or cellular morphology.
 Lethal mutations, when expressed, result in the death of the microorganism. Lethal mutations are recovered only if they are recessive in diploid organisms or conditional (see the following) in haploid organisms.


 Conditional mutations are those that are expressed only under certain environmental conditions. For example, a conditional lethal mutation in E. coli might not be expressed under permissive conditions such as low temperature but would be expressed under restrictive conditions such as high temperature.



 Biochemical mutations are those causing a change in the biochemistry of the cell. Since these mutations often inactivate a biosynthetic pathway, they frequently make a microorganism unable to grow on a medium lacking an adequate supply of the pathway’s end product.

 The mutant cannot grow on minimal medium and requires nutrient supplements. Such mutants are called auxotrophs, whereas microbial strains that can grow on minimal medium are prototrophs.



Mutations occur in one of two ways.

(1) Spontaneous mutations: arise occasionally in all cells and develop in the absence of any added agent.
(2) Induced mutations: on the other hand, are the result of exposure of the organism to some physical or chemical agent called a mutagen.






 Spontaneous Mutations

 Spontaneous mutations arise without exposure to external agents.
 This class of mutations may result from errors in DNA replication, or even from the action of transposons



 Generally replication errors occur when the base of a template nucleotide takes on a rare tautomeric form. Tautomerism is the relationship between two structural isomers that are in chemical equilibrium and readily change into one another.

 These tautomeric shifts change the hydrogen-bonding characteristics of the bases, allowing purine for purine or pyrimidine for pyrimidine substitutions that can eventually lead to a stable alteration of the nucleotide sequence. Such substitutions are known as transition mutations and are relatively common, although most of them are repaired by various proofreading functions



 In transversion mutations, a purine is substituted for a pyrimidine, or a pyrimidine for a purine. These mutations are rarer due to the steric problems of pairing purines with purines and pyrimidines with pyrimidines.



 Spontaneous mutations also arise from frameshifts, usually caused by the deletion of DNA segments resulting in an altered codon reading frame



 it is possible for purine nucleotides to be depurinated—that is, to lose their base. This results in the formation of an apurinic site, which will not base pair normally and may cause a transition type mutation after the next roundof replication.



 Cytosine can be deaminated to uracil, which is then removed to form an apyrimidinic site.

 Reactive forms of oxygen such as oxygen free radicals and peroxides are produced by aerobic metabolism. These may alter DNA bases and cause mutations.

o For example, guanine can be converted to 8-oxo-7,8-dihydrodeoxyguanine, which often pairs with adenine rather than cytosine during replication.


 Induced Mutations


 Mutagens can be conveniently classified according to their mechanism of action. Four common modes of mutagen action are incorporation of---
1. base analogs
2. specific mispairing,
3. intercalation bypass of replication

A. Base analogs are structurally similar to normal nitrogenous bases and can be incorporated into the growing polynucleotide chain during replication. E.g-

 A widely used base analog is 5-bromouracil (5-BU), an analog of thymine. It undergoes a tautomeric shift from the normal keto form to an enol much more frequently than does a normal base.


B. Specific mispairing is caused when a mutagen changes a base’s structure and therefore alters its base pairing characteristics.e.g------
1.-methyl-nitrosoguanidine, an alkylating agent that adds methyl groups to guanine, causing it to mispair with thymine
2.-Hydroxylamine hydroxylates the C-4 nitrogen of cytosine, causing it to base pair like thymine.

C. Intercalating agents distort DNA to induce single nucleotide pair insertions and deletions
 Intercalating agents include acridines such as proflavin and acridine orange Other e.g-
UV radiation generates cyclobutane type dimers, usually thymine dimers, between adjacent pyrimidines ionizing radiation and carcinogens such as aflatoxin B1 and other benzo (a) pyrene derivatives

 The Expression of Mutations
 A mutation from the most prevalent gene form, the wild type, to a mutant form is called a forward mutation.
 Later, a second mutation may make the mutant appear to be a wild-type organism again. Such a mutation is called a reversion mutation because the organism seems to have reverted back to its original phenotype.
 True back mutation converts the mutant nucleotide sequence back to the wild-type sequence.
 The wild-type phenotype also can be regained by a second mutation in a different gene, a suppressor mutation, which overcomes the effect of the first mutation (table 11.2). If the second mutation is within the same gene, the change may be called a second site reversion or intragenic suppression.


 Point mutations
 Most mutations affect only one base pair in a given location and therefore are called point mutations.


Types of point mutations


 One kind of point mutation that could not be detected until the advent of nucleic acid sequencing techniques is the silent mutation.

 If a mutation is an alteration of the nucleotide sequence of DNA, mutations can occur and have no visible effect because of code degeneracy.

 A second type of point mutation is the missense mutation. This mutation involves a single base substitution in the DNA that changes a codon for one amino acid into a codon for another

 For example, the codon GAG, which specifies glutamic acid, could be changed to GUG, which codes for valine. The expression of missense mutations can vary. Certainly the mutation is expressed at the level of protein structure.


 Mutations also occur in the regulatory sequences responsible for the control of gene expression and in other noncoding portions of structural genes
 Constitutive lactose operon mutants in E. coli are excellent examples. These mutations map in the operator site and produce altered operator sequences that are not recognized by the repressor protein, and therefore the operon is continuously active in transcription. If a mutation renders the promoter sequence nonfunctional, the coding region of the structural gene will be completely normal, but a mutant phenotype will result due to the absence of a product


Type of Mutation Result and Example

Forward Mutations
Single Nucleotide-Pair (Base-Pair) Substitutions

At DNA Level




Transition Purine replaced by a different purine, or pyrimidine replaced by a different
pyrimidine (e.g., AT GC).



Transversion Purine replaced by a pyrimidine, or pyrimidine replaced by a purine


At Protein Level
Silent mutation Triplet codes for same amino acid:
AGG CGG
both code for Arg
Neutral mutation Triplet codes for different but functionally equivalent amino acid:
AAA (Lys) AGA (Arg)
Missense mutation Triplet codes for a different amino acid.
Nonsense mutation Triplet codes for chain termination:
CAG (Gln) UAG (stop)


Single Nucleotide-Pair Addition or Deletion: Frameshift Mutation -------Any addition or deletion of base pairs that is not a multiple of three results in a frameshift in reading the DNA segments that code for protein




Intragenic Addition or Deletion of Several to Many Nucleotide Pairs



Reverse Mutations

True Reversion AAA (Lys) -----forward-------GAA (Glu) ------reverse--- AAA (Lys)
wild type mutant wild type







Equivalent Reversion UCC (Ser) ------------forward UGC (Cys) ------------reverseAGC (Ser)
wild type mutant wild type





Suppressor Mutations


Intragenic Suppressor Mutations
Frame shift of opposite sign at site within gene. Addition of X
to the base sequence shifts the reading frame from the CAT
codon to XCA followed by TCA codons. The subsequent
deletion of a C base shifts the reading frame back to CAT.








Extragenic Suppressor Mutations
Nonsense suppressors Gene (e.g., for tyrosine tRNA) undergoes mutational event in its anticodon
region that enables it to recognize and align with a mutant nonsense codon
(e.g., UAG) to insert an amino acid (tyrosine) and permit completion of the
translation.







Physiological suppressors A defect in one chemical pathway is circumvented by another mutation—for
example, one that opens up another chemical pathway to the same product, or
one that permits more efficient uptake of a compound produced in small
quantities because of the original mutation










 Detection and Isolation of Mutants
 Suitable detection system for the mutant phenotype under study also is needed. Since mutations are generally
rare, about one per 107 to 1011 cells,

 Many proteins are still functional after the substitution of a single amino acid, but this depends on the type and location of the
amino acid.
For instance, replacement of a nonpolar amino acid in the protein’s interior with a polar amino acid probably will drastically alter the protein’s three-dimensional structure and
therefore its function.


 A third type of point mutation causes the early termination of translation and therefore results in a shortened polypeptide. Such mutations are called nonsense mutations because they involve the conversion of a sense codon to a nonsense or stop codon.





 Frame shift mutations arise from the insertion or deletion of one or two base pairs within the coding region of the gene. Since the code consists of a precise sequence of triplet codons, the addition or deletion of fewer than three base pairs will cause the reading frame to be shifted for all codons downstream



 The replica plating technique is used to detect auxotrophic mutants. It distinguishes between mutants and the wild-type strain based on their ability to grow in the absence of a particular biosynthetic end product

 A lysine auxotroph, for instance, will grow on lysine-supplemented media but not on a medium lacking an adequate supply of lysine because it cannot synthesize this amino acid.


 Carcinogenicity Testing

 The Ames test, developed by Bruce Ames in the 1970s, has been widely used to test for carcinogens. The Ames test is a mutational reversion assay employing several special strains of Salmonella typhimurium, each of which has a different mutation in the histidine biosynthesis operon.



 SUMMARY

• A mutation is a stable, heritable change in the nucleotide sequence of the genetic material, usually DNA.
• Mutations can be divided into many categories based on their effects on the phenotype, some major types are morphological, lethal, conditional, biochemical, and resistance mutations.
• Spontaneous mutations can arise from replication errors (transitions, transversions, and frameshifts), from DNA lesions (apurinic sites, apyrimidinic sites, oxidations), and from insertions.
• Induced mutations are caused by mutagens. Mutations may result from the incorporation of base analogs, specific mispairing due to alteration of a base, the presence of intercalating agents, and a bypass of replication because of severe damage. Starvation and environmental stresses may stimulate mutator genes and lead to hypermutation.
• The mutant phenotype can be restored to wild type by either a true reverse mutation or a suppressor mutation.
• There are four important types of point mutations: silent mutations, missense mutations, nonsense mutations, and frameshift mutations .
• It is essential to have a sensitive and specific detection technique to isolate mutants; an example is replica plating for the detection of auxotrophs (a direct detection system).
• One of the most effective isolation techniques is to adjust environmental conditions so that the mutant will grow while the wild-type organism does not.
• Because many carcinogens are also mutagenic, one can test for mutagenicity with the Ames test and use the results as an indirect indication of carcinogenicity.
• Mutations and DNA damage are repaired in several ways; for example: proofreading by replication enzymes, excision repair, removal of lesions (e.g., photoreactivation), postreplication repair (mismatch repair), and recombination repair.







JSR

Microbial Nutrition

Microbial Nutrition

 macroelements or macronutrients--carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron

 micronutrients or trace elements---—manganese, zinc, cobalt, molybdenum, nickel, and copper(Micronutrients are normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and maintenance of protein structure)
 zinc (Zn2+) is present at the active site of some enzymes but is also involved in the association of regulatory and catalytic subunits in E. coli aspartate carbamoyltransferase.
 Manganese (Mn2+) aids many enzymes catalyzing the transfer of phosphate groups.
 Molybdenum (Mo2+) is required for nitrogen fixation.
 cobalt (Co2+) is a component of vitamin B12

Particular requirements

 Diatoms need silicic acid (H4SiO4) to construct their beautiful cell walls of silica [(SiO2)n].
 Although most bacteria do not require large amounts of sodium, many bacteria growing in saline lakes and oceans (see pp. 123, 461) depend on the presence of high concentrations of sodium ion (Na+).

Requirements for Carbon, Hydrogen,and Oxygen

 Carbon is needed for the skeleton or backbone of all organic molecules, and molecules serving as carbon sources normally also contribute both oxygen and hydrogen atoms

 They are the source of all three(C,H,O) elements. Because these organic nutrients are almost always reduced and have electrons that they can donate to other molecules, they also can serve as energy sources.
 Indeed, the more reduced organic molecules are, the higher their energy content (e.g., lipids have a higher energy content than carbohydrates).

 electron transfers release energy when the electrons move from reduced donors with more negative reduction potentials to oxidized electron acceptors with more positive potentials.

 One important carbon source that does not supply hydrogen or energy is carbon dioxide (CO2). This is because CO2 is oxidized and lacks hydrogen.

 only autotrophs can use CO2 as their sole or principal source of carbon

 The reduction of CO2 is a very energy-expensive process. Thus many microorganisms cannot use CO2 as their sole carbon source but must rely on the presence of more reduced, complex molecules such as glucose for a supply of carbon

 most heterotrophs use reduced organic compounds as sources of both carbon and energy.
 For example, the glycolytic pathway produces carbon skeletons for use in biosynthesis and also releases energy as ATP and NADH.

aboratory experiments indicate that there is no naturally occurring organic molecule that cannot be used by some microorganism.
• Actinomycetes will degrade amyl alcohol, paraffin, and even rubber.

• Burkholderia cepacia can use over 100 different carbon compounds.
• some bacteria are exceedingly fastidious and catabolize only a few carbon compounds. Cultures of methylotrophic bacteria metabolize methane, methanol, carbon monoxide, formic acid, and related one-carbon molecules.


• Parasitic members of the genus Leptospira use only long-chain fatty acids as their major source of carbon and energy.


Indigestible molecules sometimes are oxidized and degraded in the presence of a growthpromoting nutrient that is metabolized at the same time, a process called cometabolism .The products of this breakdown process can then be used as nutrients by other microorganisms.











Nutritional Types of Microorganisms

Carbon Sources


Autotrophs CO2 sole or principal biosynthetic carbon source

Heterotrophs Reduced, preformed, organic molecules from other organisms

Energy Sources
Phototrophs Light
Chemotrophs Oxidation of organic or inorganic compounds

Electron Sources
Lithotrophs Reduced inorganic molecules
Organotrophs Organic molecules

Major Nutritional Types of Microorganisms



Major Nutritional Types Sources of Energy, Hydrogen/Electrons, and Carbon Representative Microorganisms
Photolithotrophic autotrophy

(Photolithoautotrophy) • Light energy
• Inorganic hydrogen/electron (H/e–) donor Purple and green sulfur bacteria CO2 carbon source • Algae
• Purple and green sulfur bacteria
• Cyanobacteria
Photoorganotrophic heterotrophy (Photoorganoheterotrophy) • Light energy

• Organic H/e– donor Organic carbon source (CO2 may also be used) • Purple nonsulfur bacteria

• Green nonsulfur bacteria
Chemolithotrophic autotrophy (Chemolithoautotrophy) • Chemical energy source (inorganic)

• Inorganic H/e– donor CO2 carbon source • Sulfur-oxidizing bacteria Hydrogen bacteria

• Nitrifying bacteria Iron-oxidizing bacteria
Chemoorganotrophic heterotrophy
(Chemoorganoheterotrophy) • Chemical energy source (organic

• Organic H/e– donor Organic carbon source • Protozoa
• Fungi
• Most nonphotosynthetic bacteria (including most pathogens)


 Eucaryotic algae and cyanobacteria employ water as the electron donor and release oxygen

 Purple and green sulfur bacteria cannot oxidize water but extract electrons from inorganic donors like hydrogen, hydrogen sulfide, and elemental sulfur.

 It should be noted that essentially all pathogenic microorganisms are chemoheterotrophs.

 These photoorganotrophic heterotrophs (photoorganoheterotrophs) are common inhabitants of polluted lakes and streams. Some of these bacteria also can grow as photoautotrophs with molecular hydrogen as an electron donor.


 The chemolithotrophic autotrophs (chemolithoautotrophs), oxidizes reduced inorganic compounds such as iron, nitrogen, or sulfur molecules to derive both energy and electrons for biosynthesis.

 Chemolithotrophs contribute greatly to the chemical transformations of elements (e.g., the conversion of ammonia to nitrate or sulfur to sulfate) that continually occur in the ecosystem.


Some microorganism changes their mode of nutrition on course of environment

 many purple nonsulfur bacteria act as photoorganotrophic heterotrophs in the absence of oxygen but oxidize organic molecules and function chemotrophically at normal oxygen levels. When oxygen is low, photosynthesis and oxidative metabolism may function simultaneously.

 bacteria such as Beggiatoa that rely on inorganic energy sources and organic (or sometimes CO2) carbon sources. These microbes are sometimes called mixotrophic because they combine chemolithoautotrophic and heterotrophic metabolic processes.




Requirements for Nitrogen, Phosphorus, and Sulfur


 Many microorganisms can use the nitrogen in amino acids, and ammonia often is directly incorporated through the action of such enzymes as glutamate dehydrogenase or glutamine synthetase and glutamate synthase

 Almost all microorganisms use inorganic phosphate as their phosphorus source and incorporate it directly.

 Low phosphate levels actually limit microbial growth in many aquatic environments.

 Phosphate uptake by E. coli has been intensively studied. This bacterium can use both organic and inorganic phosphate.

 Some organophosphates such as hexose 6-phosphates can be taken up directly by transport proteins.

 Other organophosphates are often hydrolyzed in the periplasm by the enzyme alkaline phosphatase to produce inorganic phosphate, which then is transported across the plasma membrane.

 At high phosphate concentrations, transport probably is due to the Pit system. When phosphate concentrations are low, the PST, (phosphate-specific transport) system is more important.

 The PST system has higher affinity for phosphate; it is an ABC transporter and uses a periplasmic binding protein.

 Sulfur is needed for the synthesis of substances like the amino acids cysteine(reduced form) and methionine, some carbohydrates, biotin, and thiamine.

Growth Factors:

 There are three major classes of growth factors:
(1) amino acids,
(2) purines and pyrimidines
(3) vitamins.


 Other growth factors are also seen; heme (from hemoglobin or cytochromes) is required by Haemophilus influenzae, and some mycoplasmas need cholesterol

Functions of Some Common Vitamins in Microorganisms

Vitamin Functions
Biotin Carboxylation (CO2 fixation) One-carbon metabolism
Cyanocobalamin (B12) Molecular rearrangements One-carbon metabolism—carries methyl groups
Folic acid One-carbon metabolism
Lipoic acid Transfer of acyl groups
Pantothenic acid Precursor of coenzyme A—carries acyl groups
(pyruvate oxidation, fatty acid metabolism)
Pyridoxine (B6) Amino acid metabolism (e.g., transamination)
Niacin (nicotinic acid) Precursor of NAD and NADP—carry electrons
and hydrogen atoms
Riboflavin (B2) Precursor of FAD and FMN—carry electrons or hydrogen atoms
Thiamine (B1) Aldehyde group transfer (pyruvate decarboxylation, -keto acid oxidation)



 Good examples of such vitamins and the microorganisms that synthesize them are:
• Riboflavin by (Clostridium, Candida, Ashbya, Eremothecium)
• coenzyme A by(Brevibacterium)
• vitamin B12 by (Streptomyces, Propionibacterium, Pseudomonas)
• vitamin C by (Gluconobacter, Erwinia, Corynebacterium)
• B-carotene (Dunaliella)
• vitamin D (Saccharomyces).


Uptake of Nutrients by the Cell
1. facilitated diffusion,
2. active transport,
3. group translocation.

Eucaryotic microorganisms do not appear to employ group translocation but take up nutrients by the process of endocytosis .

A. Passive diffusion
 Passive diffusion, often simply called diffusion, is the process in which molecules move from a region of higher concentration to one of lower concentration because of random thermal agitation.
 The rate of passive diffusion is dependent on the size of the concentration gradient between a cell’s exterior and its interior
 A fairly large concentration gradient is required for adequate nutrient uptake by passive diffusion (i.e., the external nutrient concentration must be high)
 Very small molecules such as H2O, glycerol,O2, and CO2 often move across membranes by passive diffusion.


 The rate of diffusion across selectively permeable membranes is greatly increased by using carrier proteins, sometimes called permeases, which are embedded in the plasma membrane.

• Because a carrier aids the diffusion process, it is called facilitated diffusion. The rate of facilitated diffusion increases with the concentration gradient much more rapidly and at lower concentrations of the diffusing molecule than that of passive diffusion

The two most widespread MIP- (major intrinsic protein) channels in bacteria are aquaporins that transport water and glycerol facilitators, which aid glycerol diffusion


B. Active Transport
 Active transport is the transport of solute molecules to higher concentrations, or against a concentration gradient, with the use of metabolic energy input.


 Binding protein transport systems or ATP-binding cassette transporters (ABC transporters) are active in bacteria, archaea, and eucaryotes.

 ABC transporters employ special substrate binding proteins, which are located in the periplasmic space of gram-negative bacteria) or are attached to membrane lipids on the external face of the gram-positive plasma membrane.

 These binding proteins, which also may participate in Chemotaxis

 E. coli transports a variety of sugars (arabinose, maltose, galactose, ribose) and amino acids (glutamate, histidine, leucine) by this mechanism.

 It should be noted that eucaryotic ABC transporters are sometimes of great medical importance.

 Some tumor cells pump drugs out using these transporters.
 Cystic fibrosis results from a mutation that inactivates an ABC transporter that acts as a chloride ion channel in the lungs.

 linked transport of two substances in the same direction is called symport.(lactose &H+ in E.coli)

 linked transport in which the transported substances move in opposite directions is termed antiport.(Na ion &H+ in E.coli))

C. Group Translocation

 A process in which a molecule is transported into the cell while being chemically altered (this can be classified as a type of energy-dependent transport because metabolic energy is used).


 The best-known group translocation system is the phosphoenolpyruvate: sugar phosphotransferase system (PTS).

 It transports a variety of sugars into procaryotic cells while phosphorylating them using phosphoenolpyruvate (PEP) as the phosphate donor

PEP +sugar (outside) pyruvate + sugar +P (inside)

 The PTS is quite complex. In E. coli and Salmonella typhimurium, it consists of two enzymes and a low molecular weight heat-stable protein (HPr). HPr and enzyme I (EI) are cytoplasmic. Enzyme II (EII) is more variable in structure and often composed of three subunits or domains.

 PTSs are widely distributed in procaryotes. Except for some species of Bacillus that have both glycolysis and the phosphotransferase system, aerobic bacteria seem to lack PTSs.
 Besides their role in transport, PTS proteins can act as chemoreceptors for chemotaxis


D. Iron Uptake

 Almost all microorganisms require iron for use in cytochromes and many enzymes.

 Iron uptake is made difficult by the extreme insolubility of ferric iron (Fe3+) and its derivatives, which leaves little free iron available for transport
 Many bacteria and fungi have overcome this difficulty by secreting siderophores [Greek for iron bearers].
 Siderophores are low molecular weight molecules that are able to complex with ferric iron and supply it to the cell.
 These iron-transport molecules are normally either hydroxamates or phenolatescatecholates.
 Ferrichrome is a hydroxamate produced by many fungi; enterobactin is the catecholate formed by E. coli






Culture Media

Synthetic or Defined Media
 Such a medium in which all components are known is a defined medium or synthetic medium.
 Media that contain some ingredients of unknown chemical composition are complex media.
 complex media often are needed because the nutritional requirements of a particular microorganism are unknown, and thus a defined medium cannot be constructed.
 This is the situation with many fastidious bacteria, some of which may even require a medium containing blood or serum.
 Complex media contain undefined components like peptones, meat extract, and yeast extract.

• Peptones are protein hydrolysates prepared by partial proteolytic digestion of meat, casein, soya meal, gelatin, and other protein sources.
o They serve as sources of carbon, energy, and nitrogen.
• Beef extract and yeast extract are aqueous extracts of lean beef and brewer’s yeast, respectively.
o Beef extract contains amino acids, peptides, nucleotides, organic acids, vitamins, and minerals.
• Yeast extract is an excellent sourceof B vitamins as well as nitrogen and carbon compounds.



Three commonly used complex media are :
(1) nutrient broth
(2) tryptic soy broth
(3) MacConkey agar.


 Agar is a sulfated polymer composed mainly of D-galactose, 3,6-anhydro-L-galactose, and D-glucuronic acid. It usually is extracted from red algae.
 Agar is well suited as a solidifying agent because after it has been melted in boiling water, it can be cooled to about 40 to 42°C before hardening and will not melt again until the temperature rises to about 80 to 90°C.
 Agar is also an excellent hardening agent because most microorganisms cannot degrade it..
 Other solidifying agents are sometimes employed. For example, silica gel is used to grow autotrophic bacteria

 Types of Media

 Media such as tryptic soy broth and tryptic soy agar are called general purpose media because they support the growth of many microorganisms.

 Blood and other special nutrients may be added to general purpose media to encourage the growth of fastidious heterotrophs. These specially fortified media (e.g., blood agar) are called enriched media.

 Selective media favor the growth of particular microorganisms.

 Bile salts or dyes like basic fuchsin and crystal violet favor the growth of gram-negative bacteria by inhibiting the growth of gram-positive bacteria without affecting gram-negative organisms.
 Endo agar, eosin methylene blue agar, and MacConkey agarthree media widely used for the detection of E. coli and related bacteria in water supplies and elsewhere, contain dyes that suppress gram-positive bacterial growth.
 MacConkey agar also contains bile salts.

 Differential media are media that distinguish between differentgroups of bacteria and even permit tentative identification of microorganisms based on their biological characteristics.


 Blood agar is both a differential medium and an enriched one. It distinguishes between hemolytic and nonhemolytic bacteria.
 Hemolytic bacteria (e.g., many streptococci and staphylococci isolated from throats) produce clear zones around their colonies because of red blood cell destruction.

 MacConkey agar is both differential and selective. Since it contains lactose and neutral red dye, lactose-fermenting colonies appear pink to red in color and are easily distinguished from colonies of nonfermenters.

Isolation of Pure Cultures

 pure culture, a population of cells arising from a single cell, to characterize an individual species(development of pure culture techniques by the German bacteriologist Robert Koch).
1. The Spread Plate
2. Streak Plate
3. The Pour Plate----------Extensively used with bacteria and fungi, a pour plate also can yield isolated colonies. The original sample is diluted several times to reduce the microbial population sufficiently to obtain separate colonies when plating


 Generally the most rapid cell growth occurs at the colony edge. Growth is much slower in the center, and cell autolysis takes place in the older central portions of some colonies. These differences in growth appear due to gradients of oxygen, nutrients, and toxic products within the colony

 At the colony edge, oxygen and nutrients are plentiful. The colony center, of course, is much thicker than the edge. Consequently oxygen and nutrients do not diffuse readily into the center, toxic metabolic products cannot be quickly eliminated, and growth in the colony center is slowed or stopped. Because of these environmental variations within a colony, cells on the periphery can be growing at maximum rates while cells in the center are dying.

SUMMARY


• Microorganisms require nutrients, materials that are used in biosynthesis and energy production.

• Macronutrients or macroelements (C, O, H, N, S, P, K, Ca, Mg, and Fe) are needed in relatively large quantities; micronutrients or trace elements (e.g., Mn, Zn, Co, Mo, Ni, and Cu) are used in very small amounts.
• Autotrophs use CO2 as their primary or sole carbon source; heterotrophs employ organic molecules.
• Microorganisms can be classified based on their energy and electron sources.
• Phototrophs use light energy, and chemotrophs obtain energy from the oxidation of chemical compounds. Electrons are extracted from reduced inorganic substances by lithotrophs and from organic compounds by organotrophs.
• Nitrogen, phosphorus, and sulfur may be obtained from the same organic molecules that supply carbon, from the direct incorporation of ammonia and phosphate, and by the reduction and assimilation of oxidized inorganic molecules.
• Probably most microorganisms need growth factors. Growth factor requirements make microbiological assays possible.
• Although some nutrients can enter cells by passive diffusion, a membrane carrier protein is usually required.
• In facilitated diffusion the transport protein simply carries a molecule across the membrane in the direction of decreasing concentration, and no metabolic energy is required .
• Active transport systems use metabolic energy and membrane carrier proteins to concentrate substances actively by transporting them against a gradient. ATP is used as an energy source by ABC transporters. Gradients of protons and sodium ions also drive solute uptake across membranes .
• Bacteria also transport organic molecules while modifying them, a process known as group translocation. For example, many sugars are transported and phosphorylated simultaneously .
• Iron is accumulated by the secretion of siderophores, small molecules able to complex with ferric iron . When the ironsiderophore complex reaches the cell surface, it is taken inside and the iron is reduced to the ferrous form.
• Culture media can be constructed completely from chemically defined components (defined media or synthetic media) or may contain constituents like peptones and yeast extract whose precise composition is unknown (complex media).
• Culture media can be solidified by the addition of agar, a complex polysaccharide from red algae.
• Culture media are classified based on function and composition as general purpose media, enriched media, selective media, and differential media.
• Pure cultures usually are obtained by isolating individual cells with any of three plating techniques: the spread-plate, streak-plate, and pour-plate methods.
• Microorganisms growing on solid surfaces tend to form colonies with distinctive morphology. Colonies usually grow most rapidly at the edge where larger amounts of required resources are available.























JSR

Microbial Growth

Microbial Growth



Concepts:


• Growth is defined as an increase in cellular constituents and may result in an increase in a microorganism’s size, population number, or both.
• When microorganisms are grown in a closed system, population growth remains exponential for only a few generations and then enters a stationary phase due to factors such as nutrient limitation and waste accumulation. In an open system with continual nutrient addition and waste removal, the exponential phase can be maintained for long periods.
• Water availability, pH, temperature, oxygen concentration, pressure, radiation, and a number of other environmental factors influence microbial growth. Yet many microorganisms, and particularly bacteria, have managed to adapt and flourish under environmental extremes that would destroy higher organisms.
• In the natural environment, growth is often severely limited by available nutrient supplies and many other environmental factors.
• Bacteria can communicate with each other and behave cooperatively using population density–dependent signals.

 Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce by processes like budding or binary fission.

 If the microorganism is coenocytic—that is, a multinucleate organism in which nuclear divisions are not accompanied by cell divisions— growth results in an increase in cell size but not cell number.


The Growth Curve:

A. Lag Phase----When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase.
 The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin.
B. Exponential Phase---- During the exponential or log phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the conditions under which they are growing.

 Rate of growth is constant during the exponential phase; that is, the microorganisms are dividing and doubling in number at regular intervals.
 The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures are usually used in biochemical and physiological studies.
 Exponential growth is balanced growth. That is, all cellular constituents are manufactured at constant rates relative to each other.

C. Stationary Phase

 Eventually population growth ceases and the growth curve becomes horizontal.
 This stationary phase usually is attained by bacteria at a population level of around 109 cells per ml.
 In the stationary phase the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death
 bacteria in a batch culture may enter stationary phase in response to starvation------------------

 Starving bacteria frequently produce a variety of starvation proteins, which make the cell much more resistant to damage in a variety of ways.

 They increase peptidoglycan cross-linking and cell wall strength.


 The Dps (DNA-binding protein from starved cells) protein protects DNA.

 Chaperones prevent protein denaturation and renature damaged proteins.

 As a result of these and many other mechanisms, the starved cells become harder to kill and more resistant to starvation itself, damaging temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine.

 Salmonella typhimurium and some other bacterial pathogens become more virulent when starved.

D. Death Phase
 The death of a microbial population, like its growth during the exponential phase, is usually logarithmic (that is, a constant proportion of cells dies every hour).

The Mathematics of Growth:
 population will double in number during a specific length of time called the generation time or doubling time.

 These observations can be expressed as equations for the generation time.
Let N0 = the initial population number

Nt =the population at time t

n = the number of generations in time t

Then inspection of the results in table 6.1 will show that
Nt = N0 * 2n.

Solving for n, the number of generations, where all logarithms
are to the base 10,

Log Nt = log N0 + n • log 2, and

n = log Nt _ log N0/ log 2= log Nt _ log N0/0.301

 The rate of growth during the exponential phase in a batch culture can be expressed in terms of the mean growth rate constant (k).
 This is the number of generations per unit time, often expressed as the generations per hour.

k = n/ t= log Nt _ log N0/0.301t

 The time it takes a population to double in size—that is, the mean generation time or mean doubling time (g), can now be calculated. If the population doubles (t = g), then

Nt = 2 N0
.
Substitute 2N0 into the mean growth rate equation and solve
for k.
k= log (2NO) _ log NO / 0.301g= log 2 + log N0 - log N0/0.301g

k =1/g

 The mean generation time is the reciprocal of the mean growth rate constant.

g =1/k

 Generation times in nature are usually much longer than in culture.


Measurement of Microbial Growth

a) Measurement of Cell Numbers:
 Petroff-Hausser counting chambers can be used for counting prokaryotes; hemocytometers can be used for both prokaryotes and eukaryotes.

 Larger microorganisms such as protozoa, algae, and nonfilamentous yeasts can be directly counted with electronic counters such as the Coulter Counter.
 It is not as useful in counting bacteria because of interference by small debris particles, the formation of filaments, and other problems.

The Petroff-Hausser Counting Chamber.Formulla:

Bacteria/mm3 = (bacteria/square) (no of squares) (depth of chamber)


 The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates.

b) Measurement of Cell Mass:

 Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of fungi. It is time consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity.




The Continuous Culture of Microorganisms:

 A microbial population can be maintained in the exponential growth phase and at a constant biomass concentration for extended periods in a continuous culture system.

Two major types of continuous culture systems commonly are used:
(1) chemostats
(2) turbidostats.
 A chemostat is constructed so that sterile medium is fed into the culture vessel at the same rate as the media containing microorganisms is removed.
 The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr) and V is the vessel volume (ml).

D = f/V
For example, if f is 30 ml/hr and V is 100 ml, the dilution rate is
0.30 hr_1.
 Both the microbial population level and the generation time are related to the dilution rate .
 The generation time decreases (i.e., the growth rate rises) as the dilution rate increases.
 The microbial population density remains unchanged over a wide range of dilution rates.
 If the dilution rate rises too high, the microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate.
 The limiting nutrient concentration rises at higher dilution rates because fewer microorganisms are present to use it.
 At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate. This is because of the effect of nutrient concentration on the growth rate, sometimes called the Monod relationship.
 The growth rate increases when the total available energy exceeds the maintenance energy.


 The Turbidostat
 Turbidostat, has a photocell that measures the absorbance or turbidity of the culture in the growth vessel.

 The turbidostat differs from the chemostat in several ways.—

 The dilution rate in a turbidostat varies rather than remaining constant, and its culture medium lacks a limiting nutrient.
 The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates.

The Influence of Environmental Factors on Growth:
 Procaryotes such as Bacillus infernus even seem able to live over 1.5 miles below the Earth’s surface, without oxygen and at temperatures above 60°C.

 Microorganisms that grow in such harsh conditions are often called extremophiles.


I. Solutes and Water Activity:

 Because a selectively permeable plasma membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration of their surroundings.

 If a microorganism is placed in a hypotonic solution (one with a lower osmotic concentration), water will enter the cell and cause it to burst
 When microorganisms with rigid cell walls are placed in a hypertonic environment, water leaves and the plasma membrane shrinks away from the wall, a process known as plasmolysis.
 This dehydrates the cell and may damage the plasma membrane; the cell usually becomes metabolically inactive and ceases to grow.




 Many microorganisms keep the osmotic concentration of their protoplasm somewhat above that of the habitat by the use of compatible solutes,( that are compatible with metabolism and growth when at high intracellular concentrations.) so that the plasma membrane is always pressed firmly against their cell wall.

 Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions are also involved to some extent.
 Algae and fungi employ sucrose and polyols—for example, arabitol, glycerol, and mannitol— for the same purpose.
 Polyols and amino acids are ideal solutes for this function because they normally do not disrupt enzyme structure and function.

 A few procaryotes like Halobacterium salinarium raise their osmotic concentration with potassium ions (sodium ions are also elevated but not as much as potassium).
 Halobacterium’s enzymes have been altered so that they actually require high salt concentrations for normal activity
 Since protozoa do not have a cell wall, they must use contractile vacuoles to eliminate excess water when living in hypotonic environments.

 The amount of water available to microorganisms can be reduced by interaction with solute molecules (the osmotic effect) or by adsorption to the surfaces of solids (the matric effect).

 Microbiologists generally use water activity (aw) to express quantitatively the degree of water availability
 It is also equivalent to the ratio of the solution’s vapor pressure (Psoln) to that of pure water (Pwater).

aw = P soln / Pwate

 Water activity is inversely related to osmotic pressure; if a solution has high osmotic pressure, its aw(water potential) is low.
 A microorganism must expend extra effort to grow in a habitat with a low aw value because it must maintain a high internal solute concentration to retain water.
 Some microorganisms can do this and are osmotolerant

• For example, Staphylococcus aureus can be cultured in media containing any sodium chloride concentration up to about 3 M. It is well adapted for growth on the skin.
• The yeast Saccharomyces rouxii will grow in sugar solutions with aw values as low as 0.6.

 The alga Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution.
 Halophiles have adapted so completely to hypertonic, saline conditions that they require high levels of sodium chloride to grow, concentrations between about 2.8 M
 The archaeon Halobacterium can be isolated from the Dead Sea (a salt lake between Israel and Jordan and the lowest lake in the world), the Great Salt Lake in Utah, and other aquatic habitats with salt concentrations.

 Halobacterium and other extremely halophilic bacteria have significantly modified the structure of their proteins and membranes rather than simply increasing the intracellular concentrations of solutes, the approach used by most osmotolerant microorganisms.


• The enzymes, ribosomes, and transport proteins of these bacteria require high levels of potassium for stability and activity. In addition, the plasma membrane and cell wall of Halobacterium are stabilized by high concentrations of sodium ion. If the sodium concentration decreases too much, the wall and plasma membrane literally disintegrate

II. pH

 Acidophiles have their growth optimum between pH 0 and 5.5; Most fungi prefer slightly acid surroundings, about pH 4 to 6; algae also seem to favor slight acidity.e.g-----
 The Archaea Ferroplasma acidarmanus and Picrophilus oshimae can actually grow at pH 0, or very close to it. the alga Cyanidium caldarium and the archaeon Sulfolobus acidocaldarius are common inhabitants of acidic hot springs; both grow well around pH 1 to 3 and at high temperatures.

 neutrophiles, between pH 5.5 and 8.0;
• Most bacteria and protozoa are neutrophiles.

 alkalophiles prefer the pH range of 8.5 to 11.5.
• Extreme alkalophiles like Bacillus alcalophilus maintain their internal pH closer to neutrality by exchanging internal sodium ions for external protons.

 In bacteria, potassium/proton and sodium/proton antiport systems probably correct small variations in pH.
 When the pH drops below about 5.5 to 6.0, Salmonella typhimurium and E. coli synthesize an array of new proteins as part of what has been called their acidic tolerance response.

 If the external pH decreases to 4.5 or lower, chaperones such as acid shock proteins and heat shock proteins are synthesized.

Buffers often are included in media to prevent growth inhibition by large pH changes. Phosphate is a commonly used buffer and a good example of buffering by a weak acid (H2PO4 _) and its conjugate base (HPO4 2–).




III. Temperature
 microbial growth occurs at temperatures extending from _20°C to over 100°C.
 Some species (e.g., Neisseria gonorrhoeae) have a small range; others, like Enterococcus faecalis, will grow over a wide range of temperatures.


1. Psychrophiles------ grow well at 0°C and have an optimum growth temperature of 15°C or lower; the maximum is around 20°C.

 They are readily isolated from Arctic and Antarctic habitats; because 90% of the ocean is 5°C or colder.
 The psychrophilic archaeon Methanogenium has recently been isolated from Ace Lake in Antarctica.
 The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold.that is the reason fo their surviving.




2. psychrotrophs or facultative psychrophiles.

 Many species can grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. These are called psychrotrophs or facultative psychrophiles.

 Psychrotrophic bacteria and fungi are major factors in the spoilage of refrigerated foods e.g--------Pseudomonas fluorescens


3.Mesophiles are microorganisms with growth optima around 20 to 45°C;

 Almost all human pathogens are mesophiles, as might be expected since their environment is a fairly constant 37°C.
e.g Escherichia coli, Neisseria gonorrhoeae,

4. Thermophiles; they can grow at temperatures of 55°C or higher

 The vast majority are procaryotes although a few algae and fungi are thermophilic


 Thermophiles differ from mesophiles in having much more heat-stable enzymes and protein synthesis systems able to function at high temperatures. Their membrane lipids are also more saturated than those of mesophiles and have higher melting points; therefore thermophile membranes remain intact at higher temperatures.



5. Hyperthermophiles. Procaryotes that have growth optima between 80°C and about 113°C are called hyperthermophiles.
 They usually do not grow well below 55°C. Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor.




Oxygen Concentration
 Almost all multicellular organisms are completely dependent on atmospheric O2 for growth—that is, they are obligate aerobes.

 Oxygen serves as the terminal electron acceptor for the electron- transport chain in aerobic respiration. In addition, aerobic eukaryotes employ O2 in the synthesis of sterols and unsaturated fatty acids.

 Facultative anaerobes do not require O2 for growth but do grow better in its presence. In the presence of oxygen they will use aerobic respiration.
 Aerotolerant anaerobes such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or not.
 In contrast, strict or obligate anaerobes (e.g., Bacteroides, Fusobacterium, Clostridium pasteurianum, Methanococcus) do not tolerate O2 at all and die in its presence.

 Aerotolerant and strict anaerobes cannot generate energy through respiration and must employ fermentation or anaerobic respiration pathways for this purpose.

 Finally, there are aerobes such as Campylobacter, called microaerophiles, that are damaged by the normal atmospheric level of O2 (20%) and require O2 levels below the range of 2 to 10% for growth.

The nature of bacterial O2 responses can be readily determined by growing the bacteria in culture tubes filled with a solid culture medium or a special medium like thioglycollate broth, which contains a reducing agent to lower O2 levels

Fungi are normally aerobic, but a number of species— particularly among the yeasts—are facultative anaerobes


Algae are almost always obligate aerobes.
Many microorganisms possess enzymes that afford protection against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD) and catalase, which catalyze the destruction of superoxide radical and hydrogen peroxide, respectively. Peroxidase also can be used to destroy hydrogen peroxide.

Aerotolerant microorganisms may lack catalase but almost always have superoxide dismutase. The aerotolerant Lactobacillus
plantarum uses manganous ions instead of superoxide dismutase to destroy the superoxide radical

All strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O2.

 How to grow anaerobes???????/

1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used.
2) Often CO2 as well as nitrogen is added to the chamber since many anaerobes require a small amount of CO2 for best growth.

(3) One of the most popular ways of culturing small numbers of anaerobes is by use of a Gas- Pak jar . In this procedure the environment is made anaerobic by using hydrogen and a palladium catalyst to remove O2 through the formation of water

Pressure:
 Some bacteria in the gut of deep-sea invertebrates such as amphipods and holothurians are truly barophilic—they grow more rapidly at high pressures.
 These gut bacteria may play an important role in nutrient recycling in the deep sea
 One barophile has been recovered from the Mariana trench near the Philippines (depth about 10,500 m) that is actually unable to grow at pressures below about 400 to 500 atm when incubated at 2°C.

 Thus far, barophiles have been found among several bacterial genera (e.g., Photobacterium, Shewanella, Colwellia). Some members of the Archaea are thermobarophiles (e.g., Pyrococcus spp., Methanococcus jannaschii)

Radiation

 Many forms of electromagnetic radiation are very harmful to microorganisms.
 ionizing radiation, radiation of very short wavelength or high energy, which can cause atoms to lose electrons or ionize. Two major forms of ionizing radiation are
(1) X rays, which are artificially produced, and
(2) Gamma rays, which are emitted during radioisotope decay.


 Low levels of ionizing radiation will produce mutations and may indirectly result in death, whereas higher levels are directly lethal.

 Ionizing radiation can be used to sterilize items. Some procaryotes (e.g., Deinococcus radiodurans) and bacterial endospores can survive large doses of ionizing radiation.



 Ultraviolet (UV) radiation, mentioned earlier, kills all kinds of microorganisms due to its short wavelength (approximately from 10 to 400 nm) and high energy. The most lethal UV radiation has a wavelength of 260 nm, the wavelength most effectively absorbed by DNA.

• The primary mechanism of UV damage is the formation of thymine dimers in DNA . Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and function.
• This damage is repaired in several ways. In photoreactivation, blue light is used by a photoreactivating enzyme to split thymine dimers.
• A short sequence containing the thymine dimer can also be excised and replaced. This process occurs in the absence of light and is called dark reactivation.
• Damage also can be repaired by the recA protein in recombination repair and SOS repair.


Microbial Growth in Natural Environments

Growth Limitation by Environmental Factors

 Liebig’s law of the minimum states that the total biomass of an organism will be determined by the nutrient present in the lowest concentration relative to the organism’s requirements. This law applies in both the laboratory and in terrestrial and aquatic environments.

 Shelford’s law of tolerance states that there are limits to environmental factors below and above which a microorganism cannot survive and grow, regardless of the nutrient supply.


Quorum Sensing and Microbial Populations
 More recently it has become clear that many bacteria can communicate with one another and behave cooperatively.
 A major way in which this cooperation is accomplished is by a process known as quorum sensing or autoinduction.
 Quorum sensing was first discovered in gram-negative bacteria and is best understood in these microorganisms.

 The most common signals in gram-negative bacteria are acyl homoserine lactones (HSLs). These are small molecules composed of a 4- to 14-carbon acyl chain attached by an amide bond to homoserine Lactone.
 Some well-studied examples are-----
(1) bioluminescence production by Vibrio fischeri,
(2) Pseudomonas aeruginosa synthesis and release of virulence factors
(3) conjugal transfer of genetic material by Agrobacterium tumefaciens
(4) antibiotic production by Erwinia carotovora and Pseudomonas aureofaciens.

• Gram-positive bacteria also regulate activities by quorum sensing, often using an oligopeptide signal.


 An interesting and important function of quorum sensing is to promote the formation of mature biofilms by the pathogen Pseudomonas aeruginosa, and it may play a role in cystic fibrosis.


SUMMARY:

1. Growth is an increase in cellular constituents and results in an increase in cell size, cell number, or both.
2. When microorganisms are grown in a closed system or batch culture, the resulting growth curve usually has four phases: the lag, exponential or log, stationary, and death phases .
3. In the exponential phase, the population number doubles at a constant interval called the doubling or generation time . The mean growth rate constant (k) is the reciprocal of the generation time.
4. Exponential growth is balanced growth, cell components are synthesized at constant rates relative to one another. Changes in culture conditions (e.g., in shift-up and shift-down experiments) lead to unbalanced growth. A portion of the available nutrients is used to supply maintenance energy.
5. Microbial populations can be counted directly with counting chambers, electronic counters, or fluorescence microscopy. Viable counting techniques such as the spread plate, the pour plate, or the membrane filter can be employed.
6. Population changes also can be followed by determining variations in microbial mass through the measurement of dry weight, turbidity, or the amount of a cell component.
7. Microorganisms can be grown in an open system in which nutrients are constantly provided and wastes removed.
8. A continuous culture system is an open system that can maintain a microbial population in the log phase. There are two types of these systems: chemostats and turbidostats.
9. Most bacteria, algae, and fungi have rigid cell walls and are hypertonic to the habitat because of solutes such as amino acids, polyols, and potassium ions. The amount of water actually available to microorganisms is expressed in terms of the water activity (aw).
10. Although most microorganisms will not grow well at water activities below 0.98 due to plasmolysis and associated effects, osmotolerant organisms survive and even flourish at low aw values. Halophiles actually require high sodium chloride concentrations for growth .
11. Each species of microorganism has an optimum pH for growth and can be classified as an acidophile, neutrophile, or alkalophile.
12. Microorganisms can alter the pH of their surroundings, and most culture media must be buffered to stabilize the pH.
13. Microorganisms have distinct temperature ranges for growth with minima, maxima, and optima—the cardinal temperatures. These ranges are determined by the effects of temperature on the rates of catalysis, protein denaturation, and membrane disruption.
14. There are five major classes of microorganisms with respect to temperature preferences: (1) psychrophiles, (2) facultative psychrophiles or psychrotrophs, (3) mesophiles, (4) thermophiles and (5) hyperthermophiles.

15. Microorganisms can be placed into at least five different categories based on their response to the presence of O2: obligate aerobes, facultative anaerobes, aerotolerant anaerobes, strict or obligate anaerobes, and microaerophiles .
16. Oxygen can become toxic because of the production of hydrogen peroxide, superoxide radical, and hydroxyl radical. These are destroyed by the enzymes superoxide dismutase, catalase, and peroxidase.
17. Most deep-sea microorganisms are barotolerant, but some are barophilic and require high pressure for optimal growth.
18. High-energy or short-wavelength radiation harms organisms in several ways. Ionizing radiation—X rays and gamma rays—ionizes molecules and destroys DNA and other cell components.
19. Ultraviolet (UV) radiation induces the formation of thymine dimers and strand breaks in DNA. Such damage can be repaired by photoreactivation or dark reactivation mechanisms.
20. Visible light can provide energy for the formation of reactive singlet oxygen, which will destroy cells.
21. Microbial growth in natural environments is profoundly affected by nutrient limitations and other adverse factors. Some microorganisms can be viable but unculturable and must be studied with special techniques.
22. Often, bacteria will communicate with one another in a density-dependent way and carry out a particular activity only when a certain population density is reached. This phenomenon is called quorum sensing.

JSR

Microbial genetics(plasmid,conjugation,transduction,transformation)

 Microbial genetics(plasmid,conjugation,transduction,transformation)


Concepts
1. Recombination is a one-way process in procaryotes:a piece of genetic material (the exogenote) is donated to the chromosome of a recipient cell (the endogenote) and integrated
into it.

2. The actual transfer of genetic material between bacteria usually takes place in one of three ways: direct transfer between two bacteria temporarily in physical contact (conjugation), transfer of a naked DNA fragment (transformation), or transport of bacterial DNA by bacteriophages (transduction).

3. Plasmids and transposable elements can move genetic material between bacterial chromosomes and within chromosomes to cause rapid changes in genomes and drastically alter phenotypes.

4. The bacterial chromosome can be mapped with great precision, using Hfr conjugation in combination with transformational and transductional mapping techniques.

5. Recombination of virus genomes occurs when two viruses with homologous chromosomes infect a host cell at the same time.

General recombination
 The most common form, usually involves a reciprocal exchange between a pair of homologous DNA sequences.
 It can occur anyplace on the chromosome, and it results from DNA strand breakage and reunion leading to crossing-over.

 The Holliday Model for Reciprocal General Recombination.


General recombination is carried out by the prod-
ucts of rec genes such as the recA protein so important for DNA
repair

 In general, a piece of donor DNA, the exogenote, must enter the recipient cell and become a stable part of the recipient cell’s genome, the endogenote.
 During replacement of host genetic material, the recipient cell becomes temporarily diploid for a portion of the genome and is called a merozygote



 Movement of DNA from a donor bacterium to the recipient can take place in three ways: direct transfer between two bacteria temporarily in physical contact (conjugation), transfer of a naked DNA fragment (transformation), and transport of bacterial DNA by bacteriophages (transduction).


 Whatever the mode of transfer, the exogenote has only four possible fates in the recipient (figure 13.4). First, when the exogenote has a sequence homologous to that of the endogenote, integration may occur; that is, it may pair with the recipient DNA and be incorporated to yield a recombinant genome. Second, the foreign DNA sometimes persists outside the endogenote and replicates to produce a clone of partially diploid cells. Third, the exogenote may survive, but not replicate, so that only one cell is a partial diploid. Finally, host cell nucleases may degrade the exogenote, a process called host restriction.

I. Bacterial plasmid:
 Plasmids are small double-stranded DNA molecules, usually circular, that can exist independently of host chromosomes and are present in many bacteria (they are also present in some yeasts and other fungi).

 They have their own replication origins and are autonomously replicating and stably inherited.


 Plasmids have relatively few genes, generally less than 30.

 Their genetic information is not essential to the host, and bacteria that lack them usually function normally.

 Single-copy plasmids produce only one copy per host cell. Multicopy plasmids may be present at concentrations of 40 or more per cell plasmids can be eliminated from host cells in a process known as curing
 Some commonly used curing treatments are acridine mutagens, UV and ionizing radiation, thymine starvation, and growth above optimal temperatures.

Classification of plasmid:

 An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

 conjugative plasmids, have genes for pili and can transfer copies of themselves to other bacteria during conjugation.

 Fertility Factors
 A plasmid called the fertility or F factor plays a major role in conjugation in E. coli and was the first to be described.

 Most of the information required for plasmid transfer is located in the tra operon, which contains at least 28 genes.

 Many of these direct the formation of sex pili that attach the F+ cell (the donor cell containing an F plasmid) to an F_ cell (figure 13.6). Other gene products aid DNA transfer.

 The plasmid contains several transposable elements. IS2 and IS3 are insertion sequences; __ is also called transposon Tn1000. The tra genes code for proteins needed in pilus synthesis and conjugation. The rep genes code for proteins involved in DNA replication. OriV is the initiation site for circular DNA replication and oriT, the site for initiation of rolling circle replication and gene transfer during conjugation.



 Resistance Factors
 Plasmids often confer antibiotic resistance on the bacteria that contain them. R factors or plasmids typically have genes that code for enzymes capable of destroying or modifying antibiotics.

 They are not usually integrated into the host chromosome. Genes coding for resistance to antibiotics such as ampicillin, chloramphenicol, and kanamycin have been found in plasmids.



Col Plasmids

 Bacteria also harbor plasmids with genes that may give them a competitive advantage in the microbial world. Bacteriocins are bacterial proteins that destroy other bacteria.

 Bacteriocins often kill cells by forming channels in the plasma membrane, thus increasing its
permeability. They also may degrade DNA and RNA or attack peptidoglycan and weaken the cell wall.

 Col plasmids contain genes for the synthesis of bacteriocins known as colicins.

I. Other Types of Plasmids

 virulence plasmids, make their hosts more pathogenic because the bacterium is better able to resist
host defense or to produce toxins.
For example, enterotoxigenic strains of E. coli cause traveler’s diarrhea because of a plasmid
that codes for an enterotoxin.
Other plasmid-borne toxins are the tetanus toxin of Clostridium tetani and the anthrax toxin of Bacillus anthracis.

 Metabolic plasmids
carry genes for enzymes that degrade substances such as aromatic compounds (toluene), pesticides (2,4-dichlorophenoxyacetic acid), and sugars (lactose). Metabolic plasmids even carry the genes required for some strains of Rhizobium to induce legume nodulation and carry out nitrogen fixation.




Transposable Elements

• The chromosomes of bacteria, viruses, and eucaryotic cells contain pieces of DNA that move around the genome. Such movement is called transposition. DNA segments that carry the genes required for this process and consequently move about chromosomes are transposable elements or transposons.

• The simplest transposable elements are insertion sequences or IS elements

• They were first discovered in the 1940s by Barbara McClintock during her studies on maize genetics (a discovery that won her the Nobel Prize in 1983).

 A well-studied example of such a Conjugative transposon is Tn916 from Enterococcus faecalis.
 Although Tn916 cannot replicate autonomously, it will transfer itself from E. faecalis to a variety of recipients and integrate into their chromosomes. Because it carries a gene for tetracycline resistance, this conjugative transposon also spreads drug resistance

Bacterial Conjugation
 bacterial conjugation, the transfer of genetic information by direct cell to cell contact, came from an elegant experiment performed by Joshua Lederberg and Edward L. Tatum in 1946.


 F+ * F_ Mating

 In 1952 William Hayes demonstrated that the gene transfer observed by Lederberg and Tatum was polar

 That is, there were definite donor (F+) and recipient (F_) strains, and gene transfer was nonreciprocal. He also found that in F+ * F_ mating the progeny were only rarely changed with regard to auxotrophy (that is, bacterial genes were not often transferred), but F_ strains frequently became F+.



 The F+ strain contains an extrachromosomal F factor carrying the genes for pilus formation and plasmid transfer. During F+* F_ mating or conjugation, the F factor replicates by the rolling-circle mechanism, and a copy moves to the recipient.

 The sex pilus or F pilus joins the donor and recipient and may contract to draw them together.

 Selftransmissible plasmids are present in gram-positive bacterial genera such as Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Streptomyces.

 Hfr Conjugation



 Because certain donor strains transfer bacterial genes with great efficiency and do not usually change recipient bacteria to donors, a second type of conjugation must exist.

 When integrated, the F plasmid’s tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling-circle replication, and transfer genetic material to an F_ recipient cell. Such a donor is called an Hfr strain (for high frequency of recombination) because it exhibits a very high efficiency of chromosomal gene transfer in comparison with F+cells.


 Because only part of the F factor is transferred at the start (the initial break is within the F plasmid), the F_ recipient does not become F+ unless the whole chromosome is transferred.


 The connection usually breaks before this process is finished. Thus a complete F factor usually is not transferred, and the recipient remains F_

 FConjugation
 Because the F plasmid is an episome, it can leave the bacterial chromosome. Sometimes during this process the plasmid makes an error in excision and picks up a portion of the chromosomal material to form an F′plasmid

 The recipient becomes F′and is a partially diploid merozygote since it has two sets of the genes carried by the plasmid. In this way specific bacterial genes may spread rapidly throughout a bacterial population. Such transfer of bacterial genes is often called sexduction


 F′conjugation is very important to the microbial geneticist. A partial diploid’s behavior shows whether the allele carried by an F′plasmid is dominant or recessive to the chromosomal gene.
 The formation of F′plasmids also is useful in mapping the chromosome since if two genes are picked up by an F factor they must be neighbors.

 DNA Transformation
 Discovered by Fred Griffith in 1928.
 Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule
into the recipient chromosome in a heritable form.



 When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes. If a fragment contacts a competent cell, one able to take up DNA and be transformed


 Competency is a complex phenomenon and is dependent on several conditions. Bacteria need to be in a certain stage of growth; for example, S. pneumonia becomes competent during the exponential phase when the population reaches about 107 to 108 cells per ml.


 When a population becomes competent, bacteria such as pneumococci secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins required for transformation



 The Mechanism of Transformation. (1) A long
double-stranded DNA molecule binds to the surface with the aid of a
DNA-binding protein (•) and is nicked by a nuclease ( ). (2) One
strand is degraded by the nuclease. (3) The undegraded strand
associates with a competence-specific protein ( ). (4) The single
strand enters the cell and is integrated into the host chromosome in
place of the homologous region of the host DNA


 Artificial transformation is carried out in the laboratory by a variety of techniques, including treatment of the cells with calcium chloride, which renders their membranes more permeable to DNA.


 When linear DNA fragments are to be used in transformation, E. coli usually is rendered deficient in one or more exonuclease activities to protect the transforming fragments. It is even easier to transform bacteria with plasmid DNA since plasmids are not as easily degraded as linear fragments and can replicate within the host .


 Transformation in Haemophilus influenzae, a gram-negative bacterium, differs from that in S. pneumoniae in several respects. Haemophilus does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species (S. pneumoniae is less particular about the source of its DNA).
 Transduction


 Bacterial viruses or bacteriophages participate in the third mode of bacterial gene transfer


 After infecting the host cell, a bacteriophage (phage for short) often takes control and forces the host to make many copies of the virus. Eventually the host bacterium bursts or lyses and releases new phages. This reproductive cycle is called a lytic cycle because it ends in lysis of the host.



 Bacterial viruses that reproduce using a lytic cycle often are called virulent bacteriophages because they destroy the host cell.Many DNA phages, such as the lambda phage


 The genome remains within the host cell and is reproduced along with the bacterial chromosome. A clone of infected cells arises and may grow for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyse under appropriate environmental conditions. This relationship between the phage and its host is called lysogeny





 Bacteria that can produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to establish this relationship are temperate phages.


 The latent form of the virus genome that remains within the host without destroying it is called the prophage. The prophage usually is integrated into the bacterial genome .

• Sometimes phage reproduction is triggered in a lysogenized culture by exposure to UV radiation or other factors. The lysogens are then destroyed and new phages released. This phenomenon is called induction.



• Generalized Transduction
Generalized transduction occurs during the lytic cycle of virulent and temperate phages and can transfer any part of the bacterial genome.

 The resulting virus particle often injects the DNA into another bacterial cell but does not initiate a lytic cycle. This phage is known as a generalized transducing particle or phage and is simply a carrier of genetic information from the original bacterium to another cell



• Abortive transductants are bacteria that contain this nonintegrated, transduced DNA and are partial diploids.

• Generalized transduction was discovered in 1951 by Joshua Lederberg and Norton Zinder(experiments with Salmonella typhimurium)


• Specialized Transduction
In specialized or restricted transduction, the transducing particle carries only specific portions of the bacterial genome. Specialized transduction is made possible by an error in the lysogenic life cycle.

 The best-studied example of specialized transduction is the lambda phage


 lysates (product of cell lysis) contain only a few transducing particles,they often are called low-frequency transduction lysates (LFT lysates).

 SUMMARY


 In recombination, genetic material from two different chromosomes is combined to form a new, hybrid chromosome. There are three types of recombination:general recombination, site-specific recombination, and replicative recombination.

 Bacterial recombination is a one-way process in which the exogenote is transferred from the donor to a recipient and integrated into the endogenote.

 Plasmids are small, circular, autonomously replicating DNA molecules that can exist independent of the host chromosome. Their genes are not required for host survival.


 Episomes are plasmids that can be reversibly integrated with the host chromosome.

 Many important types of plasmids have been discovered:F factors, R factors, Col plasmids, virulence plasmids, and metabolic plasmids.


 Transposons or transposable elements are DNA segments that move about the genome in a process known as transposition.

 There are two types of transposable elements: insertion sequences and composite transposons.


 Transposable elements cause mutations, block translation and transcription, turn genes on and off, aid F plasmid insertion, and carry antibiotic resistance genes.

 Conjugation is the transfer of genes between bacteria that depends upon direct cell-cell contact mediated by the F pilus.


 In F+ * F_ mating the F factor remains independent of the chromosome and a copy is transferred to the F_ recipient; donor genes are not usually transferred .

 Hfr strains transfer bacterial genes to recipients because the F factor is integrated into the host chromosome. A complete copy of the F factor is not often transferred .


 When the F factor leaves an Hfr chromosome, it occasionally picks up some bacterial genes to become an F′plasmid, which readily transfers these genes to other bacteria .


 Transformation is the uptake of a naked DNA molecule by a competent cell and its incorporation into the genome .

 Bacterial viruses or bacteriophages can reproduce and destroy the host cell (lytic cycle) or become a latent prophage that remains within the host (lysogenic cycle).


 Transduction is the transfer of bacterial genes by viruses.

 In generalized transduction any host DNA fragment can be packaged in a virus capsid and transferred to a recipient.


 Temperate phages carry out specialized transduction by incorporating bacterial genes during prophage induction and then donating those genes to another bacterium.


 The bacterial genome can be mapped by following the order of gene transfer during Hfr conjugation; transformational and transductional mapping techniques also may be used.
 When two viruses simultaneously enter a host cell, their chromosomes can undergo recombination.
 Virus genomes are mapped by recombination and hetero (duplex mapping techniques.








JSR

microbial genetics

 Microbial genetics(plasmid,conjugation,transduction,transformation)


Concepts
1. Recombination is a one-way process in procaryotes:a piece of genetic material (the exogenote) is donated to the chromosome of a recipient cell (the endogenote) and integrated into it.

2. The actual transfer of genetic material between bacteria usually takes place in one of three ways: direct transfer between two bacteria temporarily in physical contact (conjugation), transfer of a naked DNA fragment (transformation), or transport of bacterial DNA by bacteriophages (transduction).

3. Plasmids and transposable elements can move genetic material between bacterial chromosomes and within chromosomes to cause rapid changes in genomes and drastically alter phenotypes.

4. The bacterial chromosome can be mapped with great precision, using Hfr conjugation in combination with transformational and transductional mapping techniques.

5. Recombination of virus genomes occurs when two viruses with homologous chromosomes infect a host cell at the same time.

General recombination
 The most common form, usually involves a reciprocal exchange between a pair of homologous DNA sequences.
 It can occur anyplace on the chromosome, and it results from DNA strand breakage and reunion leading to crossing-over.

 The Holliday Model for Reciprocal General Recombination.


General recombination is carried out by the products of rec genes such as the recA protein so important for DNA repair

 In general, a piece of donor DNA, the exogenote, must enter the recipient cell and become a stable part of the recipient cell’s genome, the endogenote.
 During replacement of host genetic material, the recipient cell becomes temporarily diploid for a portion of the genome and called a merozygote.



 Movement of DNA from a donor bacterium to the recipient can take place in three ways:
1. Direct transfer between two bacteria temporarily in physical contact (conjugation)
2. Transfer of a naked DNA fragment (transformation)
3. Transport of bacterial DNA by bacteriophages (transduction).


 Whatever the mode of transfer, the exogenote has only four possible fates in the recipient (figure 13.4). First, when the exogenote has a sequence homologous to that of the endogenote, integration may occur; that is, it may pair with the recipient DNA and be incorporated to yield a recombinant genome. Second, the foreign DNA sometimes persists outside the endogenote and replicates to produce a clone of partially diploid cells. Third, the exogenote may survive, but not replicate, so that only one cell is a partial diploid. Finally, host cell nucleases may degrade the exogenote, a process called host restriction.






I. Bacterial plasmid:
 Plasmids are small double-stranded DNA molecules, usually circular, that can exist independently of host chromosomes and are present in many bacteria (they are also present in some yeasts and other fungi).

 They have their own replication origins and are autonomously replicating and stably inherited.

 Plasmids have relatively few genes, generally less than 30.

 Their genetic information is not essential to the host, and bacteria that lack them usually function normally.

 Single-copy plasmids produce only one copy per host cell. Multicopy plasmids may be present at concentrations of 40 or more per cell.

 Plasmids can be eliminated from host cells in a process known as curing.
 Some commonly used curing treatments are acridine mutagens, UV and ionizing radiation, thymine starvation, and growth above optimal temperatures.

Classification of plasmid:

 An episome is a plasmid that can exist either with or without being integrated into the host’s chromosome.

 Conjugative plasmids, have genes for pili and can transfer copies of themselves to other bacteria during conjugation.

 Fertility Factors
 A plasmid called the fertility or F factor plays a major role in conjugation in E. coli and was the first to be described.

 Most of the information required for plasmid transfer is located in the tra operon, which contains at least 28 genes.

 Many of these direct the formation of sex pili that attach the F+ cell (the donor cell containing an F plasmid) to an F- cell (figure 13.6). Other gene products aid DNA transfer.

 The F factor also has several segments called insertion sequences that assist plasmid integration into the host cell chromosome. Thus the F factor is an episome that can exist outside the bacterial chromosome or be integrated into it.

 The plasmid contains several transposable elements. IS2 and IS3 are insertion sequences; γ∂ is also called transposon Tn1000.

 The tra genes code for proteins needed in pilus synthesis and conjugation.

 The rep genes code for proteins involved in DNA replication.

 OriV is the initiation site for circular DNA replication and oriT, the site for initiation of rolling circle replication and gene transfer during conjugation.



 Resistance Factors
 Plasmids often confer antibiotic resistance on the bacteria that contain them. R factors or plasmids typically have genes that code for enzymes capable of destroying or modifying antibiotics.

 They are not usually integrated into the host chromosome. Genes coding for resistance to antibiotics such as ampicillin, chloramphenicol, and kanamycin have been found in plasmids.



Col Plasmids

 Bacteria also harbor plasmids with genes that may give them a competitive advantage in the microbial world. Bacteriocins are bacterial proteins that destroy other bacteria.

 Bacteriocins often kill cells by forming channels in the plasma membrane, thus increasing its permeability. They also may degrade DNA and RNA or attack peptidoglycan and weaken the cell wall.

 Col plasmids contain genes for the synthesis of bacteriocins known as colicins.

I. Other Types of Plasmids

 virulence plasmids, make their hosts more pathogenic because the bacterium is better able to resist host defense or to produce toxins.
 For example, enterotoxigenic strains of E. coli cause traveler’s diarrhea because of a plasmid that codes for an enterotoxin.
 Other plasmid-borne toxins are the tetanus toxin of Clostridium tetani and the anthrax toxin of Bacillus anthracis.

 Metabolic plasmids
Carry genes for enzymes that degrade substances such as aromatic compounds (toluene), pesticides (2,4-dichlorophenoxyacetic acid), and sugars (lactose).
 Metabolic plasmids even carry the genes required for some strains of Rhizobium to induce legume nodulation and carry out nitrogen fixation.


Transposable Elements

• The chromosomes of bacteria, viruses, and eucaryotic cells contain pieces of DNA that move around the genome. Such movement is called transposition.
• DNA segments that carry the genes required for this process and consequently move about chromosomes are transposable elements or transposons.

• The simplest transposable elements are insertion sequences or IS elements

• They were first discovered in the 1940s by Barbara McClintock during her studies on maize genetics (a discovery that won her the Nobel Prize in 1983).

 A well-studied example of such a Conjugative transposon is Tn916 from Enterococcus faecalis.


 Although Tn916 cannot replicate autonomously, it will transfer itself from E. faecalis to a variety of recipients and integrate into their chromosomes. Because it carries a gene for tetracycline resistance, this conjugative transposon also spreads drug resistance

Bacterial Conjugation
 Bacterial conjugation, the transfer of genetic information by direct cell to cell contact, came from an elegant experiment performed by Joshua Lederberg and Edward L. Tatum in 1946.


 F+ * F- Mating

 In 1952 William Hayes demonstrated that the gene transfer observed by Lederberg and Tatum was polar

 That is, there were definite donor (F+) and recipient (F-) strains, and gene transfer was nonreciprocal. He also found that in F+ * F- mating the progeny were only rarely changed with regard to auxotrophy (that is, bacterial genes were not often transferred), but F- strains frequently became F+.



 The F+ strain contains an extrachromosomal F factor carrying the genes for pilus formation and plasmid transfer. During F+* F- mating or conjugation, the F factor replicates by the rolling-circle mechanism, and a copy moves to the recipient.

 The sex pilus or F pilus joins the donor and recipient and may contract to draw them together.

 Self transmissible plasmids are present in gram-positive bacterial genera such as Bacillus, Streptococcus, Enterococcus, Staphylococcus, and Streptomyces.




 Hfr Conjugation



 Because certain donor strains transfer bacterial genes with great efficiency and do not usually change recipient bacteria to donors, a second type of conjugation must exist.

 When integrated, the F plasmid’s tra operon is still functional; the plasmid can direct the synthesis of pili, carry out rolling-circle replication, and transfer genetic material to an F- recipient cell. Such a donor is called an Hfr strain (for high frequency of recombination) because it exhibits a very high efficiency of chromosomal gene transfer in comparison with F+cells.


 Because only part of the F factor is transferred at the start (the initial break is within the F plasmid), the F- recipient does not become F+ unless the whole chromosome is transferred.


 The connection usually breaks before this process is finished. Thus a complete F factor usually is not transferred, and the recipient remains F-.

 F'Conjugation
 Because the F plasmid is an episome, it can leave the bacterial chromosome. Sometimes during this process the plasmid makes an error in excision and picks up a portion of the chromosomal material to form an F'plasmid.
 It is not unusual to observe the inclusion of one or more genes in excised F plasmids. The F'cell retains all of its genes, although some of them are on the plasmid, and still mates only with an F- recipient.
 F' * F- conjugation is virtually identical with F+ * F- mating.
 Once again, the plasmid is transferred, but usually bacterial genes on the chromosome are not (figure 13.15b). Bacterial genes on the F'plasmid are transferred with it and need not be incorporated into the recipient chromosome to be expressed.


Figure 13.15 F'Conjugation. (a) Due to an error in excision, the A gene of an Hfr cell is picked up by the F factor. (b) The A gene is then transferred to a recipient during conjugation.

 The recipient becomes F'and is a partially diploid merozygote since it has two sets of the genes carried by the plasmid. In this way specific bacterial genes may spread rapidly throughout a bacterial population. Such transfer of bacterial genes is often called sexduction


 F'conjugation is very important to the microbial geneticist. A partial diploid’s behavior shows whether the allele carried by an F'plasmid is dominant or recessive to the chromosomal gene.
 The formation of F'plasmids also is useful in mapping the chromosome since if two genes are picked up by an F factor they must be neighbors.

 DNA Transformation
 Discovered by Fred Griffith in 1928.
 Transformation is the uptake by a cell of a naked DNA molecule or fragment from the medium and the incorporation of this molecule
into the recipient chromosome in a heritable form.



 When bacteria lyse, they release considerable amounts of DNA into the surrounding environment. These fragments may be relatively large and contain several genes. If a fragment contacts a competent cell, one able to take up DNA and be transformed


 Competency is a complex phenomenon and is dependent on several conditions. Bacteria need to be in a certain stage of growth; for example, S. pneumonia becomes competent during the exponential phase when the population reaches about 107 to 108 cells per ml.


 When a population becomes competent, bacteria such as pneumococci secrete a small protein called the competence factor that stimulates the production of 8 to 10 new proteins required for transformation



 The Mechanism of Transformation. (1) A long double-stranded DNA molecule binds to the surface with the aid of a DNA-binding protein (•) and is nicked by a nuclease .
(2) One strand is degraded by the nuclease. (3) The undegraded strand associates with a competence-specific protein ( ). (4) The single strand enters the cell and is integrated into the host chromosome in place of the homologous region of the host DNA


 Artificial transformation is carried out in the laboratory by a variety of techniques, including treatment of the cells with calcium chloride, which renders their membranes more permeable to DNA.


 When linear DNA fragments are to be used in transformation, E. coli usually is rendered deficient in one or more exonuclease activities to protect the transforming fragments. It is even easier to transform bacteria with plasmid DNA since plasmids are not as easily degraded as linear fragments and can replicate within the host .


 Transformation in Haemophilus influenzae, a gram-negative bacterium, differs from that in S. pneumoniae in several respects. Haemophilus does not produce a competence factor to stimulate the development of competence, and it takes up DNA from only closely related species (S. pneumoniae is less particular about the source of its DNA).
 Transduction


 Bacterial viruses or bacteriophages participate in the third mode of bacterial gene transfer


 After infecting the host cell, a bacteriophage (phage for short) often takes control and forces the host to make many copies of the virus. Eventually the host bacterium bursts or lyses and releases new phages. This reproductive cycle is called a lytic cycle because it ends in lysis of the host.



 Bacterial viruses that reproduce using a lytic cycle often are called virulent bacteriophages because they destroy the host cell.Many DNA phages, such as the lambda phage


 The genome remains within the host cell and is reproduced along with the bacterial chromosome. A clone of infected cells arises and may grow for long periods while appearing perfectly normal. Each of these infected bacteria can produce phages and lyse under appropriate environmental conditions. This relationship between the phage and its host is called lysogeny

 Bacteria that can produce phage particles under some conditions are said to be lysogens or lysogenic, and phages able to establish this relationship are temperate phages.


 The latent form of the virus genome that remains within the host without destroying it is called the prophage. The prophage usually is integrated into the bacterial genome .

• Sometimes phage reproduction is triggered in a lysogenized culture by exposure to UV radiation or other factors. The lysogens are then destroyed and new phages released. This phenomenon is called induction.



• Generalized Transduction
Generalized transduction occurs during the lytic cycle of virulent and temperate phages and can transfer any part of the bacterial genome.

 The resulting virus particle often injects the DNA into another bacterial cell but does not initiate a lytic cycle. This phage is known as a generalized transducing particle or phage and is simply a carrier of genetic information from the original bacterium to another cell



• Abortive transductants are bacteria that contain this nonintegrated, transduced DNA and are partial diploids.

• Generalized transduction was discovered in 1951 by Joshua Lederberg and Norton Zinder(experiments with Salmonella typhimurium)


• Specialized Transduction
In specialized or restricted transduction, the transducing particle carries only specific portions of the bacterial genome. Specialized transduction is made possible by an error in the lysogenic life cycle.

 The best-studied example of specialized transduction is the lambda phage


 lysates (product of cell lysis) contain only a few transducing particles,they often are called low-frequency transduction lysates (LFT lysates).

 SUMMARY


 In recombination, genetic material from two different chromosomes is combined to form a new, hybrid chromosome. There are three types of recombination:general recombination, site-specific recombination, and replicative recombination.

 Bacterial recombination is a one-way process in which the exogenote is transferred from the donor to a recipient and integrated into the endogenote.

 Plasmids are small, circular, autonomously replicating DNA molecules that can exist independent of the host chromosome. Their genes are not required for host survival.


 Episomes are plasmids that can be reversibly integrated with the host chromosome.

 Many important types of plasmids have been discovered:F factors, R factors, Col plasmids, virulence plasmids, and metabolic plasmids.


 Transposons or transposable elements are DNA segments that move about the genome in a process known as transposition.

 There are two types of transposable elements: insertion sequences and composite transposons.


 Transposable elements cause mutations, block translation and transcription, turn genes on and off, aid F plasmid insertion, and carry antibiotic resistance genes.

 Conjugation is the transfer of genes between bacteria that depends upon direct cell-cell contact mediated by the F pilus.


 In F+ * F- mating the F factor remains independent of the chromosome and a copy is transferred to the F- recipient; donor genes are not usually transferred .

 Hfr strains transfer bacterial genes to recipients because the F factor is integrated into the host chromosome. A complete copy of the F factor is not often transferred .


 When the F factor leaves an Hfr chromosome, it occasionally picks up some bacterial genes to become an F'plasmid, which readily transfers these genes to other bacteria .


 Transformation is the uptake of a naked DNA molecule by a competent cell and its incorporation into the genome .

 Bacterial viruses or bacteriophages can reproduce and destroy the host cell (lytic cycle) or become a latent prophage that remains within the host (lysogenic cycle).


 Transduction is the transfer of bacterial genes by viruses.

 In generalized transduction any host DNA fragment can be packaged in a virus capsid and transferred to a recipient.


 Temperate phages carry out specialized transduction by incorporating bacterial genes during prophage induction and then donating those genes to another bacterium.


 The bacterial genome can be mapped by following the order of gene transfer during Hfr conjugation; transformational and transductional mapping techniques also may be used.
 When two viruses simultaneously enter a host cell, their chromosomes can undergo recombination.
 Virus genomes are mapped by recombination and hetero (duplex mapping techniques.








JSR