Unit 2: Metabolism of Prokaryotes PDF

Unit 2 Metabolism of Prokaryotes *Bacteria - Growth curve and kinetics. * Quantification of bacterial growth * Microbial metabolism: Non-biosynthetic pathway, Biosynthetic pathway. * Adaptation mechanism of microbes: Hal...

Unit 2 Metabolism of Prokaryotes *Bacteria - Growth curve and kinetics. * Quantification of bacterial growth * Microbial metabolism: Non-biosynthetic pathway, Biosynthetic pathway. * Adaptation mechanism of microbes: Halophiles, Alkalophiles, Psychrophiles, Piezophiles, Xerophiles. * Bacterial Recombination: Transformation, Transduction, Conjugation  BACTERIAL GROWTH In microbiology, growth is defined as an increase in the number of cells. Microbial cells have a finite life span, and a species is maintained only as a result of continued growth of its population. As macromolecules accumulate in the cytoplasm of a cell, they assemble into major cell structures, such as the cell wall, cytoplasmic membrane, flagella, ribosomes, enzyme complexes, and so on, eventually leading to the process of cell division itself. when one cell eventually separates to form two cells, we say that one generation has occurred, and the time required for this process is called the generation time  Bacterial Cell Cycles Can Be Divided into Three Phases The cell cycle is the complete sequence of events extending from formation of a new cell through the next division. The bacterial cell cycle consists of three phases: (1) a period of growth after the cell is born, which is similar to the G1 phase of the eukaryotic cell cycle; (2) chromosome replication and partitioning period, which functionally corresponds to the S and mitosis events of the M phase of the eukaryotic cycle; and (3) cytokinesis, during which a septum and daughter cells are formed GROWTH IN COCCI AND BACILLI FtsZ is the major cytoskeletal protein in the bacterial cytokinesis machine. It forms a ring (the Z ring) under the membrane at the center of the cell, and this Z ring constricts to initiate division of the cell. MreB is a protein found in bacteria that has been identified as a homologue of actin. MreB controls the width of rod-shaped bacteria.  GROWTH IN VIBRIO The last cell shape we consider is that of comma-shaped cells, as studied in the aquatic bacterium Caulobacter crescentus. In addition to the actin homologue MreB and the tubulin-like protein FtsZ, these cells (and other vibroid-shaped cells) produce a cytoskeletal protein called crescentin, a homologue of eukaryotic intermediate filaments. This protein localizes to one side of the cell, where it slows the insertion of new peptidoglycan units into the peptidoglycan sacculus. The resulting asymmetric cell wall growth gives rise to the inner curvature that characterizes the comma shape.  GROWTH CURVE OF BACTERIA Population growth is often studied by analyzing the growth of microbes in liquid (broth) culture. When microorganisms are cultivated in broth, they usually are grown in a batch culture; that is, they are incubated in a closed culture vessel like a test tube or a flask with a single batch of medium. Fresh medium is not provided during incubation, so as nutrients are consumed, their concentrations decline, and wastes accumulate. Population growth of microbes reproducing by binary fission in a batch culture can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has five distinct phases: Lag Phase Exponential Phase Stationary Phase Death Phase Long-Term Stationary Phase  LAG PHASE When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs. This period is called the lag phase. It is not a time of inactivity; rather cells are synthesizing new components. This can be necessary for a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. The medium may be different from the one the microorganism was growing in previously. In this case, new enzymes are needed to use different nutrients. Eventually, however, the cells begin to replicate their DNA, increase in mass, and divide. As a result, the number of cells in the population begins to increase.  EXPONENTIAL PHASE During the exponential phase, microorganisms grow and divide at the maximal rate possible given their genetic potential, the nature of the medium, and the environmental conditions. Their rate of growth is constant during the exponential phase; that is, they are completing the cell cycle 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. The growth rate during exponential phase depends on several factors, including nutrient availability. When microbial growth is limited by the low concentration of a required nutrient, the final net growth or yield of cells increases with the initial amount of the limiting nutrient present (figure 7.11a). The rate of growth also increases with nutrient concentration (figure 7.11b) but it saturates, much like what is seen with many enzymes. The shape of the curve is thought to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high nutrient levels, the transport systems are saturated, and the growth rate does not rise further with increasing nutrient concentration  STATIONARY PHASE In a closed system such as a batch culture, population growth eventually ceases and the growth curve becomes horizontal. Final population size depends on nutrient availability and other factors, as well as the type of microorganism. In stationary phase, the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death, or the population may simply cease to divide but remain metabolically active. Microbes enter the stationary phase for many reasons. One important reason is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow and eventually stop. Aerobic organisms often are limited by O2 availability. Population growth also may cease due to the accumulation of toxic waste products. DnaA, the protein that binds to the chromosome’s origin to initiate replication, becomes less active in stationary phase.  DEATH PHASE Cells growing in batch culture cannot remain in stationary phase indefinitely. Eventually they enter a phase known as the death phase. During this phase, the number of viable cells declines exponentially, with cells dying at a constant rate. Detrimental environmental changes such as nutrient deprivation and the buildup of toxic wastes cause irreparable harm to the cells.  LONG-TERM STATIONARY PHASE Long-term growth experiments reveal that after a period of exponential death some microbes have a long period where the population size remains more or less constant. This long term stationary phase (also called extended stationary phase) can last months to years. During this time, the bacterial population continually evolves so that actively reproducing cells are those best able to use the nutrients released by their dying brethren and best able to tolerate the accumulated toxins. VBNC - viable but non-culturable state This dynamic process is marked by successive waves of GASP - Growth Advantage in Stationary genetically distinct variants. Phase (GASP) SCDI - stationary phase contact-dependent Thus natural selection can be witnessed within a single inhibition culture vessel. CASP - constant activity stationary phase Ref: Front. Microbiol., 16 October 2017 Sec. Evolutionary and Genomic Microbiology Volume 8 - 2017 | https://doi.org/10.3389/fmicb.2017.02000  The Mathematics of Growth and Growth Expressions The increase in cell number in an exponentially growing bacterial culture can be expressed with simple mathematics based on a geometric progression of the number 2. As one cell divides to become two cells, we express this as 2(0) to 2(1). As two cells become four, we express this as 2(1) to 2(2), and so on. A fixed relationship exists between the initial number of cells in a culture and the number present after a period of exponential growth, and this relationship can be expressed as N = No2n N is the final cell number, N0 is the initial cell number, and n is the number of generations during the period of exponential growth. The generation time (g) of the exponentially growing population is t/n, Where, t is the duration of exponential growth expressed in days, hours, or minutes. From a knowledge of the initial and final cell numbers in an exponentially growing cell population, it is possible to calculate n, and from n and knowledge of t, the generation time, g. Relation equation of N and No to n The equation N = No2n can be expressed in terms of n as follows: N = No2n log N = log No + n log 2 log N – log No = n log 2 n = log N – log No = log N – log No log 2 0.301 = 3.3 (log N – log No)  Yield Coefficient Yield coefficient (Y), is determined on the basis of the quantity of rate- limiting nutrient, normally the substrate converted into the microbial product. In case of biomass production, the yield coefficient relates to the quantity of biomass produced per gram of substrate utilized and is depicted by the equation x = Yx/s(S – Sr) x = biomass concentration (g/L), Yx/s= yield coefficient (g biomass/g substrate utilized), S =initial substrate concentration (g/L), Sr = residual substrate concentration (g/L) Therefore, the higher is the yield coefficient, the greater the percentage of the original substrate converted into microbial biomass. Determination of yield coefficient is important as it will decide how productive and how cost viable is the medium used.  CONTINUOUS CULTURE OF MICROORGANISMS - CHEMOSTATS AND TURBIDOSTATS It is possible to grow microorganisms in a system with constant environmental conditions maintained through continual supply of nutrients and removal of wastes. Such a system is called a continuous culture system. These systems maintain a microbial population in exponential growth, growing at a known rate and at a constant biomass concentration for extended periods. Continuous culture systems make possible the study of microbial growth at very low nutrient levels, concentrations close to those present in natural environments. These systems are essential for research in many areas, particularly microbial ecology. For example, interactions between microbial species in environmental conditions resembling those in a freshwater lake can be modeled. Continuous culture systems also are used in food and industrial microbiology. Two major types of continuous culture systems commonly are used chemostats and turbidostats.  CHEMOSTATS A chemostat is constructed so that the rate at which a sterile medium is fed into a culture vessel is the same as the rate at which the medium containing microorganisms is removed. The culture medium for a chemostat has a limited quantity of an essential nutrient (e.g., a vitamin). Because one nutrient is limiting, growth rate is determined by the rate at which sterile medium is fed into the growth chamber; the final cell density depends on the concentration of the limiting nutrient. 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, D=f⁄V where, f is the flow rate (milliliter/hr) and V is the vessel volume (milliliter).  TURBIDOSTATS The second type of continuous culture system, the turbidostat, has a photocell that measures the turbidity (defined as the amount of light scattered) of the culture in the growth vessel. The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity. Because turbidity is related to cell density, the turbidostat maintains a desired cell density. Turbidostats differ from chemostats in several ways. The dilution rate in a turbidostat varies, rather than remaining constant, and a turbidostat’s culture medium contains all nutrients in excess. That is, none of the nutrients is limiting. A turbidostat operates best at high dilution rates; a chemostat is most stable and effective at lower dilution rates.  Quantification of bacterial growth. Microbial Population Size Can Be Measured Directly or Indirectly There are many ways to measure microbial growth to determine growth rate constants and generation times. Either population number or mass can be followed because growth leads to increases in both. Here the most commonly employed techniques for determining population size are examined and the advantages and disadvantages of each noted. No single technique is always best; the most appropriate approach depends on the experimental situation.  Direct Measurement of Cell Numbers Petroff-Hausser counting chamber The most obvious way to determine microbial numbers is by direct counts using a counting chamber (e.g., Petroff-Hausser counting chamber). This approach is easy, inexpensive, and relatively quick. It also gives information about the size and morphology of microorganisms. Counting chambers consist of specially designed slides and coverslips; the space between the slide and coverslip creates a chamber of known depth. On the bottom of the chamber is an etched grid that facilitates counting the cells. The number of microorganisms in a sample can be calculated from the chamber’s volume and any dilutions made of the sample before counting. One disadvantage of using counting chambers is that to determine population size accurately, the microbial population must be relatively large and evenly dispersed because only a small volume of the population is sampled.  FLOW CYTOMETRY Flow cytometry is increasingly being used to directly count microbes and to gain detailed information about them. A flow cytometer creates a stream of cells so narrow that one cell at a time passes through a beam of laser light. As each cell passes through the beam, the light is scattered. Scattered light is detected by the flow cytometer. Because cells are separated in space, each light scattering event is detected independently. Thus the number of light-scattering events represents the number of cells in the sample. Cells of differing size, internal complexity, and other characteristics within a population can also be counted. This usually involves the use of fluorescent dyes or fluorescently labeled antibodies. These more sophisticated uses of flow cytometry can provide valuable information about characteristics of the population of cells.  COULTER COUNTER Microorganisms also can be directly counted with electronic counters such as the Coulter counter. In the Coulter counter, a microbial suspension is forced through a small hole. Electrical current flows through the hole, and electrodes placed on both sides of the hole measure electrical resistance. Every time a microbial cell passes through the hole, electrical resistance increases (i.e., the conductivity drops), and the cell is counted.  Traditional methods for directly counting microbes in a sample usually yield cell densities that are much higher than the plating methods described next in part because direct counting procedures do not distinguish dead cells from culturable cells. Newer methods for direct counts help alleviate this problem. Fluorescent dyes can differentiate between live and dead cells, making it possible to count the number of live and dead microorganisms in a sample.  Viable Counting Methods Serial Dilution – Pour plate and Spread plate technique Several plating methods can be used to determine the number of viable microbes in a sample. These are referred to as either viable counting methods or standard plate counts(SPC) because they count only those cells able to reproduce when cultured. Two commonly used procedures are the spread-plate and the pour plate technique. The results are often expressed in terms of colony forming units (CFU)  MEMBRANE FILTER The number of bacteria in aquatic samples is frequently determined from direct counts after the bacteria have been trapped on membrane filters. In the membrane filter technique, the sample is first filtered through a black polycarbonate membrane filter. Then the bacteria are stained with nucleic acid fluorescent stains such as acridine orange or DAPI and observed microscopically.  MOST PROBABLE NUMBER (MPN) TEST Sometimes plate counts cannot be used to measure population size. For instance, plate counts are not helpful if the microbe cannot be cultured on solid media or if large colonies overgrow the surface of the plate, making it impossible to get an accurate count. In these cases, another approach is used: most probable number (MPN) determination.  MEASUREMENT OF CELL MASS One approach is the determination of microbial dry weight. 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 filamentous fungi. However, it is time consuming 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. Cell mass can also be estimated by measuring the concentration of some cellular substance, as long as its concentration is constant in each cell. For example, a sample of cells can be analyzed for total protein or nitrogen. An increase in the microbial population will be reflected in higher total protein levels. Similarly, chlorophyll determinations can be used to measure phototrophic protist and cyanobacterial populations, and the quantity of ATP can be used to estimate the amount of living microbial mass.  A more rapid and sensitive method for measuring cell mass is spectrophotometry. Spectrophotometry depends on the fact that microbial cells scatter light. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. The increase in cell concentration result in greater turbidity, and less light is transmitted through the medium. The extent of light scattering (i.e., decrease in transmitted light) can be measured by a spectrophotometer and is called the absorbance (optical density) of the medium. Absorbance is almost linearly related to cell concentration at absorbance levels less than about 0.5. If the sample exceeds this value, it must first be diluted and then absorbance measured. Thus population size can be easily measured as long as the population is high enough to give detectable turbidity.  MICROBIAL METABOLISM NON-BIOSYNTHETIC PATHWAY, BIOSYNTHETIC PATHWAY Microbial growth requires the polymerization of biochemical building blocks into proteins, nucleic acids, polysaccharides, and lipids. The building blocks must come preformed in the growth medium or must be synthesized by the growing cells. Additional biosynthetic demands are placed by the requirement for coenzymes that participate in enzymatic catalysis. Biosynthetic polymerization reactions demand the transfer of anhydride bonds from adenosine triphosphate (ATP). Growth demands a source of metabolic energy for the synthesis of anhydride bonds and for the maintenance of transmembrane gradients of ions and metabolites.  ATP In living organisms, the most commonly used practical form of energy is the nucleotide adenosine 5′-triphosphate(ATP). In a sense, cells carry out certain processes so that they can “earn” ATP, which they “spend” in carrying out other processes. Thus ATP is often referred to as the cell’s energy currency.  NUTRIENTS re growth. ❖Bacteria, like all living cells, require energy and nutrients to build proteins and structural membranes and drive biochemical processes. ❖Nutrients are substances that provides nourishment essential for the maintenance of life and for growth. ❖Nutrients are used in biosynthesis and energy production and therefore are required for microbial growth.  COMMON NUTRIENT REQUIREMENTS Macronutrients * Analysis of microbial cell composition shows that Macronutrients Major Functions over 95% of cell dry weight is made up of a few major elements Potassium (K+) Required for activity by a number of carbon, oxygen, hydrogen, nitrogen, sulfur, enzymes, including some of those phosphorus, potassium, calcium, magnesium, and involved in protein synthesis iron. Calcium (Ca2+) Contributes to heat resistance of bacterial * These are called macroelements or macronutrients spores and many functions because they are required by microorganisms in relatively large amounts. Magnesium(Mg2+) Cofactor for many enzymes, complexes * The first six (C, O, H, N, S, and P) are components with ATP and stabilizes ribosomes and of carbohydrates, lipids, proteins, and nucleic acids. cell membranes * The remaining four macroelements exist in the cell Iron (Fe2+ and Part of cytochromes and cofactor for as cations and play a variety of roles. Fe3+) enzymes and electron carrying proteins MICRONUTRIENTS * All microorganisms require several nutrients in small amounts. * These are called micronutrients or trace elements. * The micronutrients—manganese, zinc, cobalt, molybdenum, nickel, and copper—are needed by most cells. * In nature, micronutrients are ubiquitous and probably do not usually limit growth. * Micronutrients are normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and maintenance of protein structure  NUTRITIONAL TYPES OF MICROBES * Because the need for carbon, energy, and electrons is so important, biologists use specific terms to define how these requirements are fulfilled. * Microorganisms can be classified as either heterotrophs or autotrophs with respect to their preferred source of carbon. * There are only two sources of energy available to organisms: (1) light energy, and (2) the energy derived from oxidizing organic or inorganic molecules. * Phototrophs use light as their energy source; * chemotrophs obtain energy from the oxidation of chemical compounds (either organic or inorganic). * Microorganisms also have only two sources for electrons. * Lithotrophs (i.e., “rock-eaters”) use reduced inorganic substances as their electron source, whereas organotrophs extract electrons from reduced organic compounds. * Despite the great metabolic diversity seen in microorganisms, most may be placed in one of five nutritional classes based on their primary sources of carbon, energy, and electrons  Nutritional Type Carbon source Energy source Electron source Representative microbes Photolithoautotrophy, Purple and green sulfur bacteria, (photolithotrophic CO2 Light Inorganic e -donor cyanobacteria autotrophy) Photoorganoheterotrophy Organic carbon, Purple nonsulfur bacteria, green (photoorganotrophic but CO2 may also Light Organic e donor nonsulfur bacteria heterotrophy) be used Sulfur-oxidizing bacteria, hydrogen-oxidizing bacteria, Chemolithoautotrophy Inorganic chemicals methanogens, nitrifying (chemolithotrophic CO2 Inorganic e donor bacteria, iron-oxidizing autotrophy) bacteria Chemolithoheterotrophy Organic carbon, Some sulfur-oxidizing bacteria or mixotrophy but CO2 may also Inorganic chemicals Inorganic e donor (e.g., Beggiatoa) (chemolithotrophic be used heterotrophy) Most nonphotosynthetic Chemoorganoheterotroph Organic e chemical, microbes, including most Organic e donor, often y (chemoorganotrophic Organic carbon often same as C source pathogens, fungi, many same as C source heterotrophy) protists, and many archaea  GROWTH FACTORS * Organic compounds that are essential cell components or precursors of such components but cannot be synthesized by the organism are called growth factors. * There are three major classes of growth factors: (1) Amino acids, (2) Purines and Pyrimidines, and (3) Vitamins. * Amino acids are needed for protein synthesis. * Purines and pyrimidines for nucleic acid synthesis. * Vitamins are small organic molecules that usually make up all or part of enzyme cofactors and are needed in only very small amounts to sustain growth.  Microbial metabolism: Utilization of energy, nutrient uptake & biosynthesis of important molecules ❖Energy-utilized by the microorganisms ❖Energy-stored in the form of high-energy-transfer compounds (ATP) ❖ Energy-also available in the form of proton motive force (electrochemical proton gradient) ❖In these forms the energy is used to drive many endergonic reactions required for the cell. ❖electrochemical proton gradient – result in the ATP synthesis It can also used for other biological purposes without the synthesis of ATP Eg: used to generate heat rotation of bacterial flagella  Energy utilization in non-biosynthetic processes ❖ ATP formed by the energy producing reactions of the bacterial cell- utilized in various ways ❖ Much energy – ▪ used in the biosynthesis of new cell components - energy-storage inclusion granules such as glycogen and poly-β-hydroxybutyrate ▪ Other metabolic processes – -physical and chemical integrity of the cell -transport of solute across membranes, -activity of locomotor organells (flagella) ❖ maintenance of the physical and chemical integrity of the cell is mainly through reactions that lead to biosynthesis of macromolecules – nucleic acids and proteins that are continuously broken down and need replacement Energy utilization-Bacterial motility: ❖ Bacterial flagella filaments - have no machinery for interconverting chemical and mechanical energy Eg: flagellin – no enzymatic activity, i.e., no detectable ATPase activity (present in cilia and flagella of eucaryotic microorganisms (protozoa)) Thus, the bacterial flagella differ from much larger and more complex cilia and flagella - protozoa Therefore, the ATP is not the immediate source of energy for flagellar rotation ❖ Instead the flagellar motor is driven by the proton motive force (i.e., the force derived from the electric potential and the hydrogen gradient across the cytoplasmic membrane) ❖ M and S ring in the basal body – rotary motor ❖ The rod is fixed rigidly to the M ring – which rotates freely in the cytoplasmic membrane ❖ The S ring – mounted rigidly on the cell wall ❖ The inward flux of Protons drives the flagellar motor ❖ What molecular events cause the conversion of proton motive force – still unknown ❖ It is clear that in the flagellar rotation, proton movements constitute the energy currency and not ATP Nutritional Uptake  Nutritional Uptake (Transport of nutrients by the bacteria ❖ Various processes- by which the ions or molecules cross the cytoplasmic membrane ❖ cytoplasmic membrane-allows the passive passage of certain small molecules and actively concentrates others within the cell ❖Nutrient molecules frequently cannot cross selectively permeable plasma membranes through passive diffusion and must be transported by one of three major mechanisms involving the use of membrane carrier proteins.  Nutrient Uptake Diffusion Simple Passive Facilitated Active Transport ATP H+ (proton motive) Group Translocation Alter molecule PTS (phosphorylate)  Simple diffusion It refers to a process whereby a substance passes through a membrane without the aid of an intermediary such as a integral membrane protein. In bacteria, a small noncharged molecules or lipid soluble molecules pass between the phospholipids to enter or leave the cell, moving from areas of high concentration to areas of low concentration (they move down their concentration gradient). Oxygen and carbon dioxide and most lipids enter and leave cells by simple diffusion Passive diffusion: ❖ Except water and some lipid soluble molecules, few compounds can pass through the cytoplasmic membrane by simple or passive diffusion ❖ In this process solute molecules cross the membrane as a result of difference in concentration of the molecule across the membrane ❖ The difference in concentration (higher outside the membrane than inside) governs the rate of inward flow of the solute molecule ❖ With time, this concentration gradient diminishes until equilibrium is reached ❖ In passive diffusion, no substance in the membrane interacts specifically with the solute molecule Facilitated diffusion: ❖ Similar to passive diffusion ❖ Solute molecule flows from higher to lower conc. ❖ Differs from passive diffusion in that – it involves a specific protein carrier molecule (porter or permease) located in the cytoplasmic membrane Combines reversibly with the solute molecule (Carrier-solute complex) Carrier-solute complex moves between the outer and inner surfaces of the membrane, releasing one solute molecule on the inner surface and returning to bind a new one on the outer surface Eg: the entry of glycerol into bacterial cells A model of facilitated diffusion The membrane carrier can change conformation after binding an external molecule and subsequently release the molecule on the cell interior. It then returns to the outward oriented position and is ready to bind another solute molecule. Because there is no energy input, molecules will continue to enter only as long as their concentration is greater on the outside. Group translocation: ❖ Solute is altered chemically during transport ❖ Eg : phosphoenol pyruvate dependent sugar-phosphotransferase system ▪ Widely distributed in many bacterial genera and mediates the translocation of many sugars and sugar derivatives ❖ These solutes (sugars) enter the cell as sugar phosphates and are accumulated in the cell in phosphoenol pyruvate form Phosphotransferase system sugar uptake and phosphorylation require the participation of several soluble and membrane-bound enzymes These proteins catalyze the transfer of the phosphoryl group of phosphoenolpyruvate to the sugar molecule ❖ The products formed are therefore sugar phosphate and pyruvate; the overall reaction requires Mg2+ ❖ Heat stable carrier protein (HPr) is activated first by transfer of a phosphate group from the high energy compound phosphoenol pyruvate (PEP) inside the cell Enzyme I PEP+HPr pyruvate+phospho-HPr ❖ Enzyme I and HPr are soluble proteins and non specific components of the process ❖ At the same time the sugar combines with enzyme II at the outer membrane surface and is transported to inner membrane surface ❖ Enzyme II is specific for a particular sugar and is an integral component of the cytoplasmic membrane ❖ Here it combines with the phosphate group carried by the activated HPr ❖ The sugar phosphate is released by enzyme II (HPr) and enters the cell enzyme II Sugar+phospho-HPr sugar-phosphate+HPr (Outside cell) (inside cell) 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. ❖ Almost all solutes, including sugars, amino acids, peptides, nucleosides and ions are taken up by cells through active transport ❖ 3 steps: 1. Binding of a solute to a receptor site on a membrane bound carrier protein 2. Translocation of the solute-carrier complex across the membrane 3. Coupling of translocation to an energy yielding reaction to lower the affinity of the carrier protein for the solute at the inner membrane surface so that the carrier protein will release solute to the cell interior ❖ In this, energy released during the flow of electrons through the ETC or the splitting of the phosphate group from ATP drives protons out of the cell This generates a difference in pH value and electric potential between the inside and the outside of the cell or across the membrane This proton gradient gives rise to a proton motive force which can be used to pump the solutes into the cell When proton reenter the cell, the energy released drives the transport mechanism in the cell membrane by including conformational change in the carrier molecule so that its affinity for the solute is decreased and the solute is released into the cell interior  Simple comparison of transport systems Items Passive Facilitated Active Group diffusion diffusion transport translocation carrier Non Yes Yes Yes proteins transport Slow Rapid Rapid Rapid speed against Non Non Yes Yes gradient Specificity Specificity Specificity transport No specificity molecules metabolic No need Need Need Need energy Solutes Not changed Changed Changed Changed molecules  Utilization of energy in biosynthetic processes ❖ Biosynthetic processes in the cell also require energy; energy from ATP is used to convert one chemical substance into another and to synthesize complex substances from simpler ones Synthesis of small molecules: Amino acids ❖ Amino acids-building block of protein ❖ 20 amino acids ❖ The microorganism growing in the medium – have all 20 of the amino acids present in the medium ❖ If they are not available freely in the medium, the microorganism may have to liberate amino acids from proteins by the action of intracellular and extracellular proteolytic enzymes ❖ Sometimes only a few amino acids are present in the medium, in which case the microbes has to convert other amino acids from the available ones into those that are missing ❖ In another instances, the medium may contains only inorganic sources of nitrogen, such as ammonium salts The microorganism then has to synthesize all the required amino acids from these sources of available nitrogen ❖ All these processes, the interconversion and biosynthesis of chemical substances, require the expenditure of energy ❖ Eg.: synthesis of amino acid proline from Glutamic acid by E.coli Synthesis of macromolecules: Structure and biosynthesis of a cell wall peptidoglycan ❖ This particular biosynthesis also serves as an example of how polymers are synthesized outside the membrane ❖ Synthesis of cell wall components is of interest because polymerization takes place outside the cell membrane by enzymes located on the membrane’s outer surface ❖ bacterial cell walls contain a large, complex peptidoglycan molecule consisting of long polysaccharide chains made of alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) residues ❖ Pentapeptide chains are attached to the NAM groups. ❖ The polysaccharide chains are connected through their pentapeptides or by interbridges ❖ Peptidoglycan synthesis complex process ❖ Two carriers involves : -uridine diphosphate (UDP)derivatives - bactroprenol, a lipid carrier, to transport NAG-NAM-pentapeptide units across the cell membrane ❖ cross links are formed by transpeptidation Peptidoglycan Synthesis ❖ Peptidoglycan synthesis is particularly vulnerable to disruption by antimicrobial agents ❖ Inhibition of any stage of synthesis weakens the cell wall and can lead to osmotic lysis ❖ Many antibiotics interfere with peptidoglycan synthesis. ❖ Eg.: penicillin inhibits the transpeptidation reaction, and bacitracin blocks the dephosphorylation of bactoprenol pyrophosphate Synthesis of organic cell material in chemoautotrophic bacteria ❖ chemoautotrophic bacteria-utilize CO2 as the sole source of carbon oxidize inorganic nutrients – hydrogen, ammonia, nitrite and thiosulfate to produce metabolic energy (in the form of ATP) and reducing power (in the form of NADPH2) - to reduce CO2 and convert it to organic cell material Eg. for the CO2 fixation by autotrophic bacteria – Calvin cycle ❖ Cyanobacteria, some nitrifying bacteria, and thiobacilli possess carboxysomes polyhedral inclusion bodies –contain ribulose-1,5-bisphosphate carboxylase site of CO2 fixation or may store the carboxylase and other proteins ❖ Understanding the cycle is easiest if the calvin cycle is divided into three phases: carboxylation, reduction, and regeneration Carboxylation Phase CO2 fixation is accomplished by the enzyme ribulose 1,5-bisphosphate carboxylase or ribulose bisphosphate carboxylase/oxygenase (rubisco), which catalyzes the addition of CO2 to ribulose 1,5-bisphosphate (RuBP), forming two molecules of 3-phosphoglycerate (PGA) Reduction Phase ❖ After PGA is formed by carboxylation, it is reduced to glyceraldehyde 3-phosphate reduction, carried out by two enzymes (phosphoglycerate kinase, is essentially a reversal of a portion of the glycolytic pathway, although the glyceraldehyde 3-phosphate dehydrogenase differs from the glycolytic enzyme in using NADP rather than NAD Regeneration Phase ❖ third phase of the Calvin cycle regenerates RuBP and produces carbohydrates such as glyceraldehyde 3-phosphate, fructose, and glucose ❖ The formation of glucose from CO2 may be summarized by the following equation 6CO2 +18ATP+12NADPH+ 12H+ +12H2O glucose + 18ADP+18Pi + 12NADP+ ❖ ATP and NADPH are provided by photosynthetic light reactions or by oxidation of inorganic molecules in chemoautotrophs ❖ Sugars formed in the Calvin cycle can then be used to synthesize other essential molecules ❖ Not high utilization of reducing power and energy in this cycle Microbial Metabolism: Aerobic respiration – Glycolysis – TCA – Electron Transport Chain (ET) anaerobic bioenergetics -Fermentation Metabolism - all of the chemical reactions within a living organism 1. Catabolism ( Catabolic ) –breakdown of complex organic molecules into simpler compounds –releases ENERGY 2. Anabolism ( Anabolic ) –the building of complex organic molecules from simpler ones –requires ENERGY Basic Concepts Reduction and Oxidation ❖ An atom becomes more reduced when it undergoes a chemical reaction in which it Gains electrons By bonding to a less electronegative atom And often this occurs when the atom becomes bonded to a hydrogen ❖ An atom becomes more oxidized when it undergoes a chemical reaction in which it Loses electrons By bonding to a more electronegative atom And often this occurs when the atom becomes bonded to an oxygen  Basic Concepts Reduction and Oxidation ❖ In metabolic pathways, we are often concerned with the oxidation or reduction of carbon ❖ Reduced forms of carbon (e.g. hydrocarbons, methane, fats, carbohydrates, alcohols) carry a great deal of potential chemical energy stored in their bonds. ❖ Oxidized forms of carbon (e.g. ketones, aldehydes, carboxylic acids, carbon dioxide) carry very little potential chemical energy in their bonds. ❖ Reduction and oxidation always occur together. ❖ In a reduction-oxidation reaction (redox reaction), one substance gets reduced, and another substance gets oxidized. ❖ The thing that gets oxidized is called the electron donor, ❖ the thing that gets reduced is called the electron acceptor.  Basic Concepts Enzymatic Pathways for Metabolism Metabolic reactions take place in a step-wise fashion in which the atoms of the raw materials are rearranged, often one at a time, until the formation of the final product takes place. Each step requires its own enzyme. The sequence of enzymatically-catalyzed steps from a starting raw material to final end products is called an enzymatic pathway (or metabolic pathway)  Basic Concepts Cofactors for Redox Reactions Enzymes that catalyze redox reactions typically require a cofactor to “shuttle” electrons from one part of the metabolic pathway to another part. There are 2 main redox cofactors: NAD and FAD. These are (relatively) small organic molecules in which part of the structure can either be reduced (e.g., accept a pair of electrons) or oxidized (e.g., donate a pair of electrons)  Microorganisms vary not only in their energy sources, but also in the electron acceptors used by chemotrophs This metabolic process is called respiration and may be divided into 2 different types. aerobic respiration: the final electron acceptor is oxygen, anaerobic respiration: different exogenous acceptor. Most often the acceptor in anaerobic respiration is inorganic (e.g., NO3-, SO42-, CO2, Fe3-,SeO42-, and many others), but organic acceptors such as fumarate may be used. Most respiration involves the activity of an electron transport chain. Aerobic Cellular Respiration 4 subpathways 1. Glycolysis 2. Transition Reaction 3. Kreb’s Cycle 4. Electron Transport System 1. Glycolysis (splitting of sugar) 2. Transition Reaction Oxidation of Glucose into 2 molecules of Connects Glycolysis to Krebs Cycle Pyruvic acid Embden-Meyerhof Pathway End Products: End Products of Glycolysis: –2 Acetyl CoEnzyme A 2 Pyruvic acid 2 NADH2 –2 CO2 2 ATP –2 NADH2 3. Krebs Cycle (Citric Acid Cycle) 4. Electron Transport System Series of chemical reactions that begin and end with citric acid Occurs within the cell membrane of Bacteria Products: Chemiosomotic Model of Mitchell 2 ATP 6 NADH2 –34 ATP 2 FADH2 4 CO2 How 34 ATP from E.T.S. ? 3 ATP for each NADH2 2 ATP for each FADH2 NADH2 FADH2 Glycolysis 2 Glycolysis 0 T. R. 2 T.R. 0 Krebs Cycle 6 Krebs Cycle 2 Total 10 Total 2 10 x 3 = 30 ATP 2 x 2 = 4 ATP Total ATP production for the complete oxidation of 1 molecule of glucose in Aerobic Respiration ATP Glycolysis 2 Transition Reaction 0 Krebs Cycle 2 E.T.S. 34 Total 38 ATP ❖ In both aerobic and anaerobic respiration, ATP is formed as a result of electron transport chain activity. ❖ 3 stages of aerobic catabolism ❖ 1st stage : larger nutrient molecules (proteins, polysaccharides, and lipids) are hydrolyzed or otherwise broken down into their constituent parts such as Amino acids, monosaccharides, fatty acids, glycerol, and other products The chemical reactions occurring during this stage do not release much energy ❖ 2nd stage: Amino acids, monosaccharides, fatty acids, glycerol, and other products of the first stage are degraded to a few simpler molecules such as acetyl coenzyme A, pyruvate, and tricarboxylic acid cycle intermediates can operate either aerobically or anaerobically produces some ATP as well as NADH and/or FADH2 ❖ 3rd stage: nutrient carbon is fed into the tricarboxylic acid cycle during the third stage of catabolism, and molecules are oxidized completely to CO2 with the production of ATP, NADH, and FADH2  The cycle operates aerobically and is responsible for the release of much energy. Much of the ATP derived from the tricarboxylic acid cycle (and stage-two reactions) comes from the oxidation of NADH and FADH2 by the electron transport chain. Oxygen, or sometimes another inorganic molecule, is the final electron acceptor. ❖ These metabolic pathways consist of enzyme catalyzed reactions arranged so that the product of one reaction serves as a substrate for the next. Embden-Meyerhof or Glycolytic Pathway ❖ most common pathway for glucose degradation to pyruvate in stage two of catabolism. ❖ It is found in all major groups of microorganisms and functions in the presence or absence of O2 ❖ Glycolysis is located in the cytoplasmic matrix of procaryotes and eucaryotes ❖ The glycolytic pathway degrades one glucose to two pyruvates by the sequence of reactions in 2 stages. ❖ ATP and NADH are also produced. ❖ In the six-carbon stage two ATPs are used to form fructose 1,6-bisphosphate For each glyceraldehyde 3-phosphate transformed into pyruvate, one NADH and two ATPs are formed Because two glyceraldehyde 3-phosphates arise from a single glucose ❖ the three-carbon stage generates four ATPs and two NADHs per glucose ❖ Subtraction of the ATP used in the six-carbon stage from that produced in the three-carbon stage gives a net yield of two ATPs per glucose ❖ Thus the catabolism of glucose to pyruvate in glycolysis can be represented by the following simple equation Glucose+2ADP+2Pi+2NAD+ 2 pyruvate+2ATP+2NADH+2H+  Tricarboxylic Acid Cycle, or citric acid cycle, or Krebs cycle ❖Although some energy is obtained from the breakdown of glucose to pyruvate by the glycolytic pathway, much more is released when pyruvate is degraded aerobically to CO2 in stage three of catabolism ❖The multienzyme system called the pyruvate dehydrogenase complex first oxidizes pyruvate to form CO2 and acetyl coenzyme A (acetyl-CoA), an energy-rich molecule composed of coenzyme A and acetic acid joined by a high energy thiol ester bond ❖Acetyl-CoA further degraded in the tricarboxylic acid cycle ❖The substrate for the tricarboxylic acid (TCA) cycle, citric acid cycle, or Krebs cycle is acetyl-CoA  Electron Transport Chain: ❖The mitochondrial electron transport chain is composed of a series of electron carriers that operate together to transfer electrons from donors, like NADH and FADH2, to acceptors, such as O2 Anaerobic Respiration Electrons released by oxidation are passed down an E.T.S., but oxygen is not the final electron acceptor Nitrate (NO3-) ----> Nitrite (NO2-) Sulfate (SO24-) ----> Hydrogen Sulfide (H2S) Carbonate (CO24-) -----> Methane (CH4) Fermentation Anaerobic process that does not use the E.T.S. Usually involves the incomplete oxidation of a carbohydrate which then becomes the final electron acceptor. Glycolysis - plus an additional step Fermentation Features of fermentation pathways –Pyruvic acid is reduced to form reduced organic acids or alcohols. –The final electron acceptor is a reduced derivative of pyruvic acid –NADH is oxidized to form NAD: Essential for continued operation of the glycolytic pathways. –O2 is not required. –No additional ATP are made. –Gasses (CO2 and/or H2) may be released  Only 2 ATP 1. Lactic Acid Fermenation End Product - Lactic Acid Food Spoilage Food Production Yogurt - Milk Pickles - Cucumbers Sauerkraut - Cabbage 2 Genera: Streptococcus, Lactobacillus  2. Alcohol Fermentation Only 2 ATP End products: alcohol CO2 Alcoholic Beverages Bread dough to rise Saccharomyces cerevisiae (Yeast) 3. Mixed - Acid Fermentation Only 2 ATP Escherichia coli and other enterics 4. Propionic Acid Fermentation Only 2 ATP End Products: Propionic acid CO2 Propionibacterium sp.  Fermentation End Products  Adaptation mechanism of microbes Halophiles, Alkalophiles, Psychrophiles, Piezophiles, Xerophiles.  Extremophiles Extremophiles are organisms which inhabit environments characterized by properties harsh enough to hinder the survival of common cells. They are highly diversified and are classified on the basis of the main extreme property that prevails in the habitat. Six main categories can be distinguished: Thermophiles found in high temperature sites and which can tolerate temperatures sometimes close to that of the boiling point of water; Psychrophiles living in permanently cold habitats with temperatures sometimes well below the freezing point of water; Piezophiles, which tolerate pressure as high as 1000 atm; Halophiles supporting −1 salt concentrations, in some cases, higher than 300 g l ; Acidophiles thriving well at pH sometimes close to zero; and Alkaliphiles, which, on the contrary, tolerate pH largely exceeding neutrality. These organisms are mainly microorganisms and they notably https://doi.org/10.1007/978-3-319-74459-9_1 produce enzymes that are adapted to work in unusual conditions often required in biotechnological processes.  Effect of Temperature on Microbial Growth Temperature affects microorganisms in two opposing ways. As temperatures rise, the rate of enzymatic reactions increases and growth becomes faster. However, above a certain temperature, proteins or other cell components may be denatured or otherwise irreversibly damaged. For every microorganism there is a minimum temperature below which growth is not possible, an optimum temperature at which growth is most rapid, and a maximum temperature above which growth is not possible. These three temperatures, called the cardinal temperatures Psychrotolerants (sometimes called psychrotrophs) grow at 0° C or higher and typically have maxima at about 35°C  MOLECULAR ADAPTATIONS THAT SUPPORT PSYCHROPHILY Psychrophiles produce enzymes that function—often optimally— in the cold and that may be denatured or otherwise inactivated at even very moderate temperatures. Cytoplasmic membranes from psychrophiles tend to contain a higher content of unsaturated and shorter-chain fatty acids, and this helps the membrane remain in a semifluid state at low temperatures to carry out important transport and bioenergetic functions. Other molecular adaptations to cold temperatures include “cold-shock” proteins and cryoprotectants, and these are not limited to psychrophiles. Cold-shock proteins are even present in Escherichia coli and have several functions that include maintaining other proteins in an active form under cold conditions  Effects of pH on Microbial Growth Each species has a definite pH growth range and pH growth optimum. These are extremophilic microorganisms which thrives in roughly alkaline environments (8-11), and have an optimum of pH around 10. Organisms which needs high pH to survive are called as obligate alkaliphiles. There are facultative alkaliphiles and haloalkaliphiles (needs salty environment as well). Two methods for surviving 1. The cell will be having a unique cellular machinery that works best in alkaline range of pH. 2. The cell will have to acidify the cytosol to nullify the effect of the high pH outside the cell.  HALOPHILES Halophiles generally are able to live in their high-salt habitats because they synthesize or obtain from their environment molecules called compatible solutes. Compatible solutes can be kept at high intracellular concentrations without interfering with metabolism and growth. Some compatible solutes are inorganic molecules such as potassium chloride (KCl). Others are organic molecules such as choline, betaines (neutral molecules having both negatively charged and positively charged functional groups), and amino acids such as proline and glutamic acid. Halophiles have adapted to life at high salinity in many different ways. One way is through the modification of their external cell walls. They tend to have negatively charged proteins on the outside of their cell walls that stabilize it by binding to positively charged sodium ions in their external environments. If salt concentrations decline their cell walls may become unstable and break down.  Microorganisms in extreme low humidity/water activity (Xerophiles) Xerophiles are a group of extremophiles that are capable of surviving in environments with low availability of water or low water activity. Generally, xerophilic organisms are capable of growing at aw values lower than xerotolerant organisms (aw below 0.8). Xerophile Mode of adaptation: Dormancy - These organisms under a temporary period of dormancy in the form of spores so that they reduce metabolic activity and resume normal metabolism when appropriate conditions are available. Extracellular polysaccharides and biofilm formation - The extracellular polysaccharides in the biofilms are hydrophilic, which contributes to rapid water absorption rates. Cell membrane - Xerophilic microorganisms adapt to low water activity by increasing the concentration of negatively charged phospholipids that facilitates the preservation of membrane bilayer structural integrity. Eg: Aspergillus penicillioides, Cereus jamacaru, Deinococcus radiodurans, Aphanothece halophytica, Anabaena, Bradyrhizobium japonicum, Saccharomyces bailli Oxygen and Microbial Growth  Piezophile Many microbes found at great ocean depths are barotolerant—that is, increased pressure adversely affects them but not as much as it does nontolerant microbes. Some are truly piezophilic (barophilic). A piezophile is defined as an organism that has a maximal growth rate at pressures greater than 1 atm. An important adaptation observed in piezophiles is that they change their membrane lipids in response to increasing pressure. For instance, bacterial piezophiles increase the amount of unsaturated fatty acids in their membrane lipids as pressure increases. They may also shorten the length of their fatty acids. Eg: Shewanella benthica, Moritella yayanosii, Shewanella violacea, Photobacterium profundum, Moritella japonica, Sporosarcina spp, etc  GENETIC TRANSFER IN BACTERIA (BACTERIAL RECOMBINATION) Genetic transfer is a process whereby genetic material from one bacterium is transferred to another bacterium. Like sexual reproduction in eukaryotes, genetic transfer in bacteria is thought to enhance the genetic diversity of bacterial species. For example, a bacterial cell carrying a gene that provides antibiotic resistance may transfer this gene to another bacterial cell, allowing that bacterial cell to survive exposure to the antibiotic.  TYPES OF BACTERIAL RECOMBINATION Gene transfer in bacteria can be achieved through three naturally occurring ways such as conjugation, transformation and viral transduction. Transformation: In this case, genetic material (extracellular DNA) is released into the environment when a bacterial cell dies and the genetic material then binds to a living bacterial cell, which can take it up. Transduction: In this case, a virus that infects bacteria, a bacteriophage, transfers bacterial genetic material from one bacterium (donor) to another bacterium (recipient). Conjugation: In this situation, the genetic material from a donor bacterium is transferred to a recipient bacterium through a specialized sex pilus or conjugation tube which is formed when two bacteria comes in direct physical interaction.  CONJUGATION Conjugation takes place when genetic material passes directly from one bacterium to another bacterium with the help of conjugation tube. The process was first postulated by Joshua Lederberg and Edward Tatum (1946) in Escherichia coli and awarded the Nobel Prize in 1958 for their work on bacterial genetics The mechanism could be understood following the works of Bernard Davis in 1956. In conjugation two bacteria lie close together and a connection form between them. A plasmid or the part of bacterial chromosome posses from one cell (donor) to another (recipient). In conjugation DNA is transferred from donor to the recipient , with no reciprocal exchange of genetic material. In most bacteria, conjugation depends on a fertility ( F) factor that is present in donor cell and absent in the recipient cell. The cells that contain F are referred to as F + and the cell lacking F are F- The F factor contains an origin of replication and a number of genes required for conjugation. A cell containing F factor produces a sex pili that makes contact with a receptor on F - cell and pulls the two cells together. DNA is then transferred from F+ to F- cells. Conjugation can takes place only between the cell that possess F and a cell that lacks F.  Replication takes place on the nicked strand, proceeding around the circular plasmid and replacing the transferred strand. The plasmid in the F+ cell is always nicked at Ori site, this site always enters the recipient cell first followed by rest of the plasmid. Inside the recipient cell, the single strand replicates and producing a circular double stranded copy of F plasmid. Transfer is initiated when the F plasmid is nicked at the origin. One end of the nicked DNA separates from the circle and passes into the recipient. If the entire F factor is transferred to the recipient F- cells become an F+ cell. When transfer is complete, both cells are F+ double-stranded. The transfer of F factor shown in the diagram.  Conjugation of high-frequency recombinant strains In Hfr (high frequency recombination) strains, the F factor is integrated into bacterial chromosome. The Hfr cell behave as F+ cells, forming sex pili and undergoes conjugation with F- cells. Hfr strains replicate F factor as part of their main chromosome. Conjugation in Hfr strains begins when F+ is nicked at the origin, and F+ and bacteria chromosomal DNA are transferred into the F- cell just as it does in conjugation between F+ and F- cell (using the rolling circle mechanism). In Hfr cell the F factor is linked to bacterial chromosome and so chromosome follows it into the recipient cell. How much of the bacterial chromosome is transferred is depend on the length of time the two cells remain in conjugation. In mating of Hfr x F- , the F- cell almost never become F+ or Hfr, because the F factor is nicked in the middle during the initiation of strand transfer.  To become F+ or Hfr, the recipient cell must receive the entire F factor, requiring that the entire bacterial chromosome is transferred. This events occur rarely because most conjugating cell break apart before the entire chromosome has been transferred. When a F factor is excise from the bacterial chromosome , a small amount of bacterial chromosome may be removed with it, and these chromosomal genes will then be carried with the F plasmid. F plasmid with some bacterial genes are called F prime. They act as donors.  TRANSFORMATION Transformation takes place when a bacterium takes up DNA from the medium in which it is growing. After transformation recombination may occur between the introduced genes and those of the bacterial chromosome. The first demonstration of bacterial transformation was done with Streptococcus pneumoniae in 1928 by an english bacteriologist F. Griffith. It led to discovery that DNA is genetic material. Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) from their surroundings. The cells of S. pneumoniae (also known as the pneumococcus) are usually surrounded by a gummy capsule made of a polysaccharide. When S. pneumoniae grown on the surface of a solid culture medium, the capsule causes the colonies to have a glistening, smooth appearance. These cells are called "S" cells. However, after prolonged cultivation on artificial medium, some cells lose the ability to form the capsule, and the surface of their colonies is wrinkled and rough ("R"). With the loss of their capsule, the bacteria also lose their virulence. Factors affecting Transformation: 1. Size of DNA 2. Competence of the recipient: Some bacteria are able to take DNA naturally. However , these bacteria only take up DNA at particular time in their growth cycle, when they produce a specific protein called a competence factor. At this stage bacteria are said to be competent. Other bacteria are not able to take up DNA naturally. However in these bacteria competence can be induced in vitro by treatment with chemical ( e.g. CaCl2) Steps in Transformation: 1. Uptake of DNA: Uptake of DNA differ in gram positive and gram negative bacteria. In gram positive bacteria the DNA is taken as a single stranded molecule and complementary strand is made in the recipient. In contrast gram negative bacteria take up double stranded DNA. 2. Recombination: After the donor DNA is taken up, a reciprocal recombination events occur between the chromosome and the donar DNA. Recombination require the bacterial recombination genes (Rec A,B and C) and homology between the DNA involved.  TRANSDUCTION Transduction is the transfer of genetic information from a donor to the recipient by way of a bacteriophage. Viruses are unable to multiply autonomously. Instead, they infect and take control of a host cell, forcing it to make many copies of the virus. Viruses that infect bacteria are called bacteriophages, or phages for short. Virulent bacteriophages multiply in their bacterial host immediately after entry. After the progeny phage particles reach a certain number, they cause the host to lyse, so they can be released and infect new host cells. Thus this process is called the lytic cycle. Temperate bacteriophages, on the other hand, do not immediately kill their host. Instead, the phage establishes a relationship with their host called lysogeny, and bacteria that have been lysogenized are called lysogens. Many temperate phages establish lysogeny by inserting their genomes into the bacterial chromosome. The inserted viral genome is called a prophage. Transduction are of two types Generalized transduction: Transduction in which potentially any bacterial gene from donor can be transferred to the recipient. Phages that mediate generalized transduction generally breakdown host DNA into smaller pieces and package their DNA into the phage particle by head full mechanism. Occasionally one of the pieces of host DNA is randomly packed into a phage coat. Thus, any donor gene can be potentially transferred but only enough DNA as can fit into the phase head can be transferred. If a recipient cell is infected by a phage that contains donor DNA, donor DNA enters the recipient. Specialized transduction: Transduction in which only certain donor genes is transferred to the recipient. Different phages may transfer different genes but an individual phage can only transfer certain genes. Specialized transduction is mediated by lysogenic or temperate phage and the genes that get transferred will depend on where the prophage has inserted in the chromosome. During excision of the prophage, occasionally an error occurs where some of the host DNA is excised with the phage DNA Only host DNA on either side of where the prophage has inserted can be transferred (i.e. specialized transduction).