Bacterial Physio HSCI 4607
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Bacterial Phys HSCI 46075707 Ch 17 Protein Export and Secretion Introduction Many proteins are exported after their synthesis in cytoplasm to the cell membrane periplasm outer envelope cell wall or secreted outside into the medium Cell membrane alone contains about 300 proteins while the periplasm in gramnegative bacteria may contain more than 100 different types of protein The outer membrane in gram negative bacteria contain 50 different proteins In addition in both gramnegative and positive bacteria there are several proteins associated with the surface layers of capsule appendages like mbriae pili and agella There are several extracellular hydrolytic enzymes like proteases lipases nucleases and amylases that are secreted out Pathogenic bacteria secrete proteinic toxins To understand the protein transport one should be able to answer the following questions 1 What is about the structure that helps these proteins to be transported to their location through the semipermeable membranes 2 What is the mechanism of translocation 3 What is the source of energy The Sec System Most of the research in this eld has been done using E coli All the proteins involved in the protein translocation have been puri ed and the genes have been cloned The system is known as Sec System or General secretory pathway GSP The Components of the Sec system The components of the Sec system include 1 a leader peptide 2 a chaperone protein SecB and 3 a membrane bound translocase SecAYEG Other proteins involved in the translocation are SecD SecF and yajC The leader peptide Most of the protein to be translocated are synthesized with leader peptide or known as signal sequence or leader sequence at the amino terminal end It is necessary for the attachment and insertion of the protein into the membrane and is removed during or after protein translocation Leader pept1de The protein with its leader peptide is called either preprotein or presecretory protein if it is to be secreted outside Mutation or deletion of leader peptide leads to impaired or no translocation of the protein The structure of the leader peptide can be divided into three regions 1 A basic region at the Nterminal which is positively charged at neutral pH that attaches to the membrane negatively charged phospholipids 2 Central hydrophobic region that inserts into the membrane and 3 ACterminal region that contains recognition site for a peptidase that removes the leader peptide during or after translocation The primary sequence of leader peptides vary considerably It is required for the translocation but does not play any role in deciding the destination since exchange of leader peptides between two proteins does not change their nal destination Moreover there are no differences sometimes in the amino acid sequences of leader peptides of two different proteins Chaperone Proteins After their synthesis in the cytoplasm the preproteins leave ribosome and bind to a speci c soluble protein called Chaperone Chaperone proteins carry them to the membrane and deliver them to the translocating protein in the membrane Chaperones also prevent their tight folding and aggregation it recognizes the unfolded topology of the protein and binds to the mature domain not the leader sequence of the preprotein The major chaperone protein in E coli is SecB which binds to the unfolded protein and delivers it to SecA which is a peripheral component of the membrane bound translocase Many proteins do not use SecB but use different chaperone proteins for their translocation The Membrane bound TranslocaseSecA amp SecYEG The preprotein is transferred from SecB to SecA which is attached to the membranebound translocase SecYEG It is not known how translocase moves the proteins through the membrane but it is assumed that it forms hydrophilic channels for the transport since many secreted proteins are not hydrophobic The translocase is a integral membrane protein complex consist of three different polypeptides SecY SecE and SecG The SecY spans the membrane several times and may form channel SecG is not absolutely necessary for the transport but is found to stimulate the transport using proteoliposomes along With SecY and SecE A Model For Protein Secretion The translocation of protein through the membrane is a multistep process utilizing ATP as an energy source Step 1 The preproteinSecB protein complex binds to the SecAtranslocase complex in the membrane Step 2 The preprotein binds to SecA The SecB protein may be released at this step Step 3 SecA binds ATP According to a different model SecA rst binds to ATP which brings about conformational changes in SecA to facilitate its binding to the preprotein Step 4 The positively charged amino terminal of the leader peptide binds to the SecYEG complex and remains attached to the cytoplasmic side of the membrane The hydrophobic region of the leader peptide spontaneously inserts into the membrane forming a loop Step 5 The Nterminus of the leader peptide remains on the cytoplasmic side of the membrane while the Cterminal ips into the lipid bilayer as the preprotein enter into the translocase channel It is believed that the unhydrolyzed ATP provides energy for the conformational changes required for the translocation Step 6 ATP is hydrolyzed with the release of SecA from the preprotein and the membrane Step 7 The rest of the protein is translocated with the help of membrane potential since this step is inhibited by the uncouplers even if the cellular ATP level is unaffected Step 8 The leader sequence is cleaved by a leader peptidase with the release of the translocated protein into the periplasm Recycling of SecA Experimentally it has been shown in vitro that in the absence of Ap ATP can drive the translocation to completion by recycling SecA with ATP hydrolysis This might also occur in vivo a SecA ATP dependent translocation Protein Export independent of Sec Proteins Although most of the proteins are translocated Via Sec proteins there are proteins that do not require these systems These include certain integral membrane proteins as well as phage M13 procoat protein These proteins may have leader sequence but do not require Sec proteins The Translocation of Membranebound proteins The transport of several innermembrane proteins is also Sec dependent These proteins are not transported through the membrane to the periplasm but remain anchored into the membrane The internal hydrophobic regions spanning 10 to 15 amino acids stop transfer signal help these protein to get anchored to the hydrophobic membranes Sometimes in these proteins even the signal sequence is not susceptible to peptidase therefore it is retained and it helps the protein to get anchored Inner membrane protein translocation The E coli Signal Recognition Particle SRP The process of insertion of proteins into the innermembrane in E coli is not well understood as compare to their Sec dependent translocation into the periplasm In E coli the placement of some inner membrane proteins involve signal recognizing particle or SRP In E coli it is ribonucleoprotein particle consist of 45 S RNA along with 48 kD protein Ecoli also has membrane receptor for SRP known as FtsY The mutants lacking SRP and FtsY are not viable but transport of the proteins to the periplasm or outer membrane is not affected profoundly This indicates that they are primarily involved in the placement of the inner membrane proteins E coli SRP and FtsY are homologous to the eukaryotic SRP and the receptor It is speculated that E coli SRP and the receptor may function in the same manner as their eukaryotic counter part SRP Dependent protein translocation across the endoplasmic reticulum ER membrane in Eukaryotes Eukaryotes have a 168 ribonucleoprotein particle called SRP that binds to the signal sequence as the protein being translated on the cytosolic ribosome Binding of SRP stops the translation The SRPribosome nascent polypeptide complex then diffuses to the ER SRP binds to the receptor known as docking protein and delivers the ribosomenascent protein complex to the ER bound translocase With the release of SRP the translation resumes and the polypeptide is translocated by ERtranslocase as being synthesized The eukaryotic translocase is heteromeric complex called Sec61 which is similar in some respect to the prokaryotic SecYEG The released SRP then guides another ribosomenascent polypeptide complex to the ER Extracellualr Protein Secretion The extracellular proteins include proteases lipases carbohydrases toxins and other virulent factors are secreted out in the medium both by gramnegative and grampositive bacteria In certain cases a particular sequence of amino acids at carboxy terminal is suf cient for the secretion of the product across the outer membranes However in most of the cases special machinery is required for the secretion of proteins There are four such machinery 1 Two secindependent known as type I and III and 2 two Secdependent type II and IV There is a fth system which is called type V which is not well characterized but consists of 10 proteins which form a pore through which DNA or proteins can be transferred across the membrane from cell to cell The type V system is responsible for conjugal transfer as well as transfer of oncogenic TDNA by Agrobacterium tumefaciens into plant cells and the secretion of Bordetella pertussis toxin SecIndependent protein secretion Type I These proteins do not have leader peptides and do not require Sec system for secretion The proteins are secreted directly from the cytoplasm to out side the cell The machinery consists of two inner membrane proteins and an outer membrane protein The carboxy terminus of the secreted protein is required for secretion The secretory apparatus recognizes this region The inner membrane complex is presumably the ABC type transporter which has ATP binding domain The mechanism is not very well understood but it is believed that secretory proteins form channel through the inner and outer membrane allowing proteins to move directly from the cytoplasm to the external medium without entering the periplasm The Secdependent Protein secretion The type II secdependent pathway appears to be the most common route through which proteins are secreted in gramnegative bacteria Type II pathway consists of two steps Step 1 Uses the GSP general secretory pathway to export the protein to the periplasm and the leader sequence is removed Step 2 This step is not very well understood It is speculated that the protein from the periplasm move in the vesicles to the outer membrane and fuse with the channel formed by type II secretory apparatus There are several examples of type 11 systems each secreting more than one type of proteins For example in Erwinia carotovora and E chrysanthemi the system secretes pectate lyase exopolyocD galacturonosidase pectin methylesterase and cellulase Secdependent type IV pathway This pathway also uses GSP for exporting the proteins to the periplasm but does not require any pathway to export it through the outer membrane These proteins are known as autotransporters The proteins themselves can form a pore into the outer membrane and get diffused into the medium The example of such protein is secretion of immunoglobulin A protease by Neisseria gonorrhoeae the causative agent of gonorrhoeae The preprotein in this case is synthesized in the cytoplasm and secreted into the periplasm by GSP system Then the carboxy terminus of the protein with its helper domain gets attached to the outer membrane The helper domain helps the protein to traverse the outer membrane and get released in to the medium by autoproteolytic cleavage leaving the Cterminus helper peptide embedded in the membrane Proteolytic step may be catalyzed by separate enzyme Secindependent Type 111 protein secretion Type 111 protein secretion apparatus does not require GSP system and are commonly used by gramnegative pathogenic bacteria such as Yersinia Salmonella Shigella and several plant pathogens Type 111 system consists of approximately 20 different proteins mostly inner membrane These proteins are quite similar in unrelated bacteria but they secrete different proteins The outer membrane protein involved in type III secretion is homologous to the type II outer membrane protein component The typical characteristics of type 111 system is 1 There is no leader sequence typical of this pathway There is no processing of Nterminal end during secretion 2 They all require accessory proteins for secretion 3 Proteins are secreted through both the inner as well as outer membranes generally into the target cell cytoplasm 4 Secretion is initiated upon reception of an active signal for an example adhesion to the target cell Bacterial Physiology HSCI 5607 Ch 2 Growth And Cell Division Introduction The end result of thousands of metabolic activities that are carried out in an individual cell is growth It is important to understand the growth related changes brought about by the physiological activities to accurately measure the population growth Understanding the changes taking place during the growth process is also very important to manipulate population growth in either batch or continuous culture The growth of population actually re ects the physiological state of individual cells in the population The study of growth includes the study of methods for measurement of growth effect of nutritional conditions cell division and growth kinetics Measurement of Growth Growth can be de ned as increase in the mass and can be measured by measuring any component which increases proportionately with the mass of the culture The most common methods include the measurement of turbidity total and viable cell count measuring the dry weight and protein estimation Turbidity This is the routinely used method in most of the laboratories Bacterial cells scatter light and within certain limit the scattered light is linearly proportionate to the mass of the cells If the average mass per cell is constant then one can also use the turbidity measurement for cell count Usually the light coming out of the cells that is the transmitting light is measured The amount of transmitted light is inversely proportionate to the mass But this can be easily converted mathematically to the amount of scattered light which is called turbidity or absorbance or optical density OD Turbidity Turbidity is measured with a colorimeter or spectrophotometer Turbidity is directly proportionate to the mass as well as length of the path light has to travel Width of the quvette according to the Lambert Beer law In practice the standard curve is plotted by measuring the optical density of different cell suspension with varying densities and also counting the cells per ml from each of these suspensions The curve is linear up to certain cell density but beyond that it does not remain linear since the increase in the cell density leads to rescattering of some light towards the phototube lowering the turbidity reading Thus turbidometric growth measurements are the simplest and most rapid way to measure growth Total Cell Counts If the average mass per cell is constant then sampling and cell counting over the period of growth can be a method of choice There are two ways in which one can count the number of cells 1 Total counting and 2 Viable count 1 Total Count Total cell counting is done using counting chamber Counting chamber is a glass slide with tiny square wells of known area and depth marked on them A drop of culture is placed on these tiny chambers and are covered with cover slip Each square holds known volume of culture in which the cells are counted microscopically to nally nd out the total numbers of organisms present per ml of culture suspension Limitations One of the limitation of this method is that it does not distinguish between the dead and the living cells Secondly it is dif cult to count the cells when the count is too low less than 106 cells per ml Total Electronic Cell counting The equipment used for electronic counting consist of two chambers connected by a microscopic pore and a electrode in each chamber One chamber is lled with bacteria suspended in saline solution and in another only the saline solution is lled Bacterial solution is pumped through the microscopic pore to the second chamber Whenever a bacterium passes through the pore it decreases the electrical conductivity since the conductivity of cell is less than that of the saline This results in a voltage pulse which is counted electronically The dual advantage of this method is that the size of the cell can also be measured since the size of he pulse is proportionate to the size of the cell Viable Cell Counts For Viable cell count the bacterial culture suspensions are serially diluted and speci c volume is plated on nutrient media The colonies developed on the media are counted to nd a Viable count of cells per ml There are some limitations of this method For example clumps of cells Will represent single colony Secondly the nutritional requirements supplied may not support growth of all kinds of bacteria lowering the frequency of colony formation Dry Weight and Protein Measurement of the dry weight of the cells is one of the most direct way of measuring the growth The cells in measured volume of sample are harvested by centrifugation or ltration then dried to a constant weight and weighed Protein Estimation Estimation of protein in the sample is another method for measuring growth since concentration of protein increases proportionately with growth For protein estimation cells are harvested by centrifugation or ltration or rst chemically precipitated with acid or alcohol and recovered by centrifugation or ltration Protein estimation is then carried out by suitable method Physiology of Growth Growth physiology includes the regulation of rates of synthesis of macromolecules the regulation of timing of DNA synthesis and cell division and adaptive physiological responses to nutrient availability Adaptive changes include changes in gene expression homeostasis adaptation to the external environment and regulation of metabolism Growth Phases When one measures the growth of bacteria in a population as a function of time and plots a graph one can observe four distinct phases of bacterial growth as shown in the graph Stationary Phase cheap quot 39 39 quot 6a ase Growth 10g Viable cell number Exponential phase Time h Lag Phase This is the initial phase of growth which starts once the inoculum is transferred to the fresh medium Usually lag phase is observed when the cells from the stationary phase are transferred to the fresh medium This is because the cells from the stationary phase of growth take time to prepare physiologically for the growth Duration of lag phase depends on several factors like 1 The duration of time they are kept in stationary phase 2 Composition of the fresh medium 3 The growth phase of the inoculum The graph shows that the cell mass increases but the numbers take time to rise This is because in the initial phase the several enzymes and proteins are synthesized as a preparation of growth increasing the size of the cells but they only start dividing at the later stage Exponential or Log Phase This phase follows the lag phase and is metabolically the most active phase of growth The cells grow exponentially during this phase This phase is followed by stationary phase Stationary Phase There are several reasons why the cells stop growing at the end of exponential phase These include exhaustion of nutrients limitation of oxygen or the accumulation of toxic products Accumulation of toxic product is often a problem during fermentation processes where the substrates are not completely converted into cell components but to toxic waste which is secreted out in the medium Death Phase Depletion of cellular energy or activity of autolytic enzymes result in death of the cells in the stationary phase In some cases the bacteria die Within short period of entering into the stationary phase While some bacteria can survive for longer period of time in stationary phase Some bacteria can form spores or cyst like structure Which are Viable for longer period of time and germinate When transferred to the fresh medium Adaptive responses to Nutrient Limitations Bacteria do face periods of starvation under natural environment and undergo span of either no growth or slow growth Their generation time may extend from days to even months due to very low level of nutrient availability Under such circumstances several bacteria either induce high af nity system for scavenging nutrients from the surrounding environment or may start synthesizing new enzymes for the use of alternative source of nutrients Once cells can not bring nutrients inside the cells they stop growing and enter into stationary phase or some bacteria can form spores or cysts which can survive for longer period of time under the condition of starvation Bacterial cells undergo several morphological biochemical and metabolical changes as they enter stationary phase Changes in the cell size Cells entering into stationary phase stop growing but keep on multiplying for 1 to 2 hours This results in the decrease in the size of the cells Many times due to this the bacilli appear as coccobacilli or cocci Morphological changes As noted above the shape of the cells also change from rodshaped to coccoid cells eg E coli Klebsiella Vibrio and Pseudomonas Changes in surface properties Certain marine bacterial surface becomes hydrophobic and more adhesive under starvation while other bacteria like Vibrio produce mbriae which help them to stick to the surface in order to absorb nutrients Changes in membrane phospholipids Certain bacteria like E coli undergo chemical changes in their phospholipid composition under starvation Signi cance of these changes is still unknown Changes in metabolic activities The overall metabolic rate slows down when the cells are starved for nutrients There is a signi cant increase in the turnover rate of breakdown and resynthesis of proteins and RNA presumably used as a energy source Changes in Protein Composition Bacteria synthesize almost 5070 or more types of new proteins when starved for nutrients like carbon nitrogen iron or phosphate This is due to the induction of high af nity systems required to acquire the nutrients available in very low concentration Changes in resistance to environmental stress Cells entering stationary phase also become more resistant to environmental stresses such as heat osmotic pressure and toxic chemicals such as hydrogen peroxide The rpoS gene encoding special sigma factor 039s Under starvation in many bacteria including Ecoli the rpoS gene expresses special sigma factor 039s which is responsible for the synthesis of several proteins required for starvation condition The rpoS gene product The rpoS gene product seems to be very important and globally involved in the transcription of many proteins induced under wide variety of stress caused by slow growth high temperature and high osmolarity Increased level of rpoS have been observed under these conditions The level of 039s factor is regulated both at transcription as well as translational levels Other Regulators Besides 039s factor there are other global regulator of gene expression present in the bacterial cells One of the most important is cyclic AMPcAMP CRP complex which stimulates almost 23 of the genes expressed during carbon starvation The control of rRNA synthesis The synthesis of ribosome is coupled to the growth rate of cells The number of ribosomes per cell in E coli can vary from 20000 to 70000 depending upon the growth rate There are DNA binding proteins like Fis and H NS and regulators like guanosine tetra phosphate ppGpp which play important role in controlling the expression of rRNA and tRNA Macromolecular composition as a function of Growth Rate It has been observed that the amount of macromolecules like proteins DNA and RNA in the cells increase with the increase of the doubling rate of the cells This is because in rapidly growing cells contain huge number of ribosomes polymerizing aminoacids at the constant rate Thus when the growth rate of the cells increases there is a need to make more proteins and cell regulates this by synthesizing more ribosomes rather than making ribosome to work faster Ribosome consists of 65 RNA and 35 protein by weight and thus there is a increase in the amount of RNA and proteins RNA 11 M Relatlve Ce ass amount Protem DNA 0 51014 9 n 7 g Doubling Timeh Diauxic Growth Many bacteria including E coli prefer to use glucose as a carbon source When such an organism is grown in the presence of glucose and other sugar eg lactose the cells utilize rst glucose as a carbon source then enter temporarily into a stationary phase followed by an exponential growth on lactose The preferential growth on one carbon source before utilizing the other is known as DiauXic growth Growth on lactose Lactose gt Glucose Time Catabolite repression by Glucose The preferential use of glucose is regulated by phenomenon known as catabolite repression under which glucose inhibits the expression of genes involved in the metabolism of other carbon sources It also interferes with the uptake of the alternative sugars the phenomenon known as Inducer exclusion In natural environment preferential use of glucose is justi ed because of its easy availability as well as constitutive production of the enzymes required for glucose metabolism Not all bacteria utilize glucose as a preferential carbon source there are organisms that utilize organic acids as a preferable carbon source eg Rhizobium and Pseudomonas aeruginosa Cell Division The growth cycle of bacteria involve two important events replication of DNA and cell division Cell division is an event through which mother cell is divided into two daughter cells separated by a septum Most of the research is done using gram negative Ecoli or Salmonella or gram positive Bacillus subtilis DNA replication takes place just before the cell division through septum formation DnaA protein initiates the replication by binding at the origin 0ri C which opens up the duplex allowing the series of DNA binding proteins to bind in order to replicate the DNA At the end of replication sister molecules separate and move towards the opposite poles so that when cell divides with septum formation each cell receives single copy of chromosome Movement of sister chromosome is known as partitioning In many bacteria earlier stage of partitioning involves the action of Par proteins which anchor the site of origin and direct them towards the opposite poles Cell division Proteins homologous to Par proteins have been found in Bsubtilis Caulobacter crescentus and Pseudomonas putida but not in E coli Similar proteins although are located on E coli plasmid responsible for plasmid partitioning In Ecoli MukB protein is postulated to be responsible for chromosomal partitioning The Septum formation The movement of chromosome to the opposite pole is followed by the formation of a septum in the center of the cell The site of the septum formation in E coli is somehow governed by three linked genes minC minD and minE all part of minB operon The septum formation begins with the centripetal synthesis of a septum with inward growth of the innermembrane and the peptidoglycan layers In gram negative bacteria there is also a invagination of the outer membrane Septum Several genes have been identi ed to play important role in septum formation including F tsZ FtsA FtsI FtsQ F tsL FtsN FtsW and ZipA Fts stands for lamentous temperature sensitive phenotype FtsA and F tsZ proteins are located in the cytoplasm while rest are membrane associated Growth Yields When the growth of bacteria is limited by a single source of nutrient eg glucose as a carbon source it is possible to calculate the Growth yield constant Y The growth yield constant Y is the amount of the dry weight of cells produced per the amount of limiting nutrient used Ie Y wt of cells produced wt of nutrient used For example if Yglucose 05 it means that 50 of glucose is converted to cell material while 50 is oxidized to C02 Growth Kinetics The equation for exponential growth During exponential growth the mass in the culture doubles after each generation The equation for exponential growth will be x x0 2quot Where x is the number of cells x0 is the starting number of cells and Y is the number of generation If you take log10 of both the sides log10 x log10 x0 0301Y The generation time which is the average time cell takes to double can be de ned as g tY where g is generation time and t is the total time of growth Rearranging this equation for Y tg thus replacing the value of Y in the above equation log10 x log10 x0 0301gt When x is plotted against t on semilog paper the slope is 0301g and the intercept is x0 log K Slope 0301g log xo Time The Generation time g Generation time is the time the population takes to double It is an important parameter of growth It is usually determined from the plot of x versus t on a semi log paper SteadyState growth and continuous Growth When all the cells in a population double at each division grow exponentially without entering stationary phase and maintain the constant ratio to one another the population is said to be in steady state In case of continuous culture also the population is at steady state The chemostat The chemostat is the device which is used for continuous culture This is accomplished by continuous regulated ow F of limiting nutrient from the reservoir to the growth culture medium resevoir 39 ow rate regulator siphon over ow growth chamber A The dilution rate D The dilution rate D FV where V is the volume of growing culture For example if the ow rate is 10mlh and V is 1 liter then the D 101000 001h Multiplying dilution rate D with the total cells x gives Dx which is number of cells lostunit time from the culture during dilution Bacterial Physiology HSCI 5607 Chapter 1 Structure and Function Introduction Prokaryotes do not contain organelles such as nuclei mitochondria chloroplast Golgi apparatus etc Nevertheless their metabolic activities are still compartmentalized Compartmentallization is found in multicomponent enzymes within periplasm in intracellular membranes and the cell membranes and within the inclusion bodies which store various enzymes pigments and storage products Prokaryotic cells do contain well developed cell walls and surface appendages such as fimbriae pili and agella Prokaryotes are divided into two distinct groups 1 Bacteria 2 Archaea Archaea differ from bacteria in ribosomal RNA sequences in cell chemistry as well as certain physiological aspects Archaea commonly belong to one of the three phylogenetic groups 1Methanogenic 2Extremely halophilic 3Extremely thermophilic Phylogenetic relationships based on rRNA sequences Bacteria Archaea Eucarya Animal G reen Ennmoebu Sllrne nonsulfur molds mm Euryarchaeota Fungi Mathnourcml Cram Halopnnes Gram unmann MIMIquots archaeofa bacterium Purple blend Himm Themlaprotcus ococcun I can Cyanohlcterl I Flagelllles Fuvoblclerln Trlchomnnlds Thtmotoglles Mlcrosporidla D I p I omonldl Table 12 Comparison between Bacteria Archae and Eucarya Characteristic Bacteria Archaea Eucarya Peptidoglycan Yes No No Lipids Ester linked Ether linked Ester linked Ribosomes 705 705 805 Initiator tRNA Formylmethionine Methionine Methionine Introns in rRNA No Yes Yes Ribosome sensitive to diphtheria toxin No Yes Yes RNA polymerase One 4 subunits Severai 8 12 Three 12 14 Ribosome sensitive to chlorarnphenicol streptomycin kanamycin subunits each No subunits each No Comparison of Archaea Bacteria and Eukarya 1 In Archaea the membrane lipids are long hydrocarbon chains are linked by etherlinkage to glycerol while in bacteria as well as in eukarya the fatty acids are ester linked to glycerol 2 Archaea lack peptidoglycan in their cell wall but some archaeal cell wall contain compound known as Pseudomurein which is not found in bacterial cell wall 3 Archaea contain histones similar to that is found in eukarya Histones bind archaeal DNA into compact structures like nucleosomes 4 The Archaea contain RNA polymerase 810 subunits different than bacterial RNA polymerase 4 subunits but is similar to that of eukaryotic RNA polymerase 1012 subunits 5 Archaeal protein synthesis differs from bacterial protein synthesis The archaeal ribosomes are not sensitive to the antibiotics affecting bacterial ribosomes indicating the structural differences in their ribosomes 6 The halophilic archaea have light driven ion pumps not found in bacteria 7 The methanogenic archaea have several unique coenzymes that are not found in bacteria These are used for the reduction of C02 to methane and formation of Acetyl CoA from H2 and C02 Cell Appendages Cell appendages include 1 Flagella which is used for motility 2 Fimbrae pili used for adhesion on different surfaces 3 Sex pili used for mating by some bacteria Flagella Motile bacteria contain one or more agella on the cell surface Some bacteria can move without agella by gliding The agellum is a semirigid long helicalright or left handed lament which rotates like a propeller The number and arrangement of agella vary in different species Bacterial agella is different than eukaryotic agella The most studied bacterial agella are those of E coli and S typhimurium Functions 1 Flagella helps the motile bacteria to drive towards nutrients light and electron acceptor or drive away from toxic environments 2 Plays important role in bacterial virulence eg movement of Treponema pallidum through connective tissues Flagella General Structure The agellum consists of mainly three parts A Basal body B A hook and C A lament and additional proteins required for motor function The agellum is made up of approximately 20 different types of polypeptides and requires almost 40 different genes for its assembly and function The motor rotates either clockwise or counterclockwise determining the direction of swimming and it also responds to chemotactic signals Exceptionally in Rhodobacter sphaeroides and Rhizobium meliloti the agella rotate only in one direction The Basal Body Basal body is at the base of the agellum embedded in the membrane It consists of two stacked rings in gram positive bacteria while four rings in gram negative bacteria through which a central rod attached to the lament passes The innermost M ring is attached to the inner membrane next to which is a S ring Both are made up of same type of protein called FliF protein Both rings appear to be single ring under electron microscopy In gram negative bacteria L and P rings serve as bushings and are attached to outer membrane and peptidoglycan layer respectively Recently it has been shown that the L ring in Styphimurium is made up of lipoprotein The lipid component probably helps its attachment to the lipids of an outer membrane Proteins required for Motor Function Mutations in MotA and MotB genes result in paralyzed agella The products of these genes MotA and MotB proteins are located in the membrane and it is suggested that they surround MS rings in a ring of 812 proteins These proteins are believed to transduce the proton potential into the rotation of motor by some unknown mechanism The agella in bacteria such as E coli and S typhimurium change direction of rotation periodically This function is supposed to be regulated by the products of iG iM and iN genes namely switch proteins FliG F liM and FliN respectively FliG seems to be bound to the cytoplasmic side of the M ring while F liM and F liN proteins are believed to be a part of cytoplasmic cylindrical attachment to the M ring called the C ring The Hook The central rod which originates from the base is attached to a curved hook that is made of multiple copies of protein known as Hook protein a product of gE gene There are additional hookassociated proteins HAPl and HAP3 products of gK and gL genes respectively These proteins are necessary to form junction between the hook and the lament The HAP2 protein which is a product of gD gene caps the the agellar lament Mutants lacking these proteins secrete agellin into the medium The Filament Filament is semirigid and helical structure attached to the hook It is made up of protein agellin Thousands of copies of agellin are present The type of agellin varies from species to species Their molecular weight vary from 20 to 65 kD in different species They show homology at the N and C terminal regions while the central part may vary considerably The agellin subunits are arranged in such a manner that there is a central 60 angstrom unit hole This hole may play important role in transporting agellin during agellar growth at the tip 2 Mechanism of Motor Function The rotational force which originates in the MS ring with the help of Mot proteins rotates the central rod and eventually the lament The mechanism through which the Mot proteins generate rotational force using proton ef uX is not yet understood One of the set of rings must act as stator in order to hold the motor in place and allowing the torque to rotate the lament 3Growth of Flagella The agellum grows at the tip This has been demonstrated by the use of uorescent amino acids or radioisotopes The agellin monomers are supposed to be transported through the central hole to the site of growthassembly at the tip of the lament 4Differences in the agellar structure Some bacteria like Vch01eraelipoprotein and Spirochaetes protein have sheathed agella There are differences in the type and numbers of agellin present Some bacteria have agella having smooth surface called Plain agella while some have helical patterns of ridges and groves on surface and these are compleX agella Plain agella can rotate in both the direction while complex agella can rotate only clockwise with intermittent stops Additional rings or particles around basal bodies of unknown function are also found in certain bacteria Flagella in spirochaetes do not protrude out but wrapped around the length of the helical cell between the cell membrane and the outer membrane 5 Archaeal Flagella Archaeal agella are not studied very well They appear to be similar but there are some differences Sequence of protein agellin show no homology with bacterial agellin Within archaeal agellins there is some homology at the Nterminus The basal body investigated in one archaea Methanospirillum hungatei shows much simpler structure in which there are no rings but simple knob like structure is present Fimbriae Pili Filaments and F ibrils The lamentous structures called mbriae or pili or laments or brils are commonly observed in gram negative bacteria but also found in gram positive Corynebacterium renale and Actinomyces viscosus Their length vary from short 02 pm to long 20 um While their thickness ranges from 3 to 14 nm or greater They are made up of protein Pilin Some of them originate from the basal body in the cell membrane but in most of the cases it is not known how are they attached to the cell surface They are commonly found in freshly isolated culture but tend to be lost during subculturing and handling Usually they are classi ed into two groups 1Fimbriae and 2 Sex Pili Fig 13 Electron micrograph of a metal shadowed preparation of Salmonella typhi showing agella and mbriae The cell is about 09 gm in diameter Source Reprinted with permission of P Dugnid 1 Fimbriae The lamentous structures which mediate attachment of bacterial cells on various cell surfaces including that of other bacteria animal plant or fungi are called mbriae Thus they play important role in colonization of bacteria They are also referred to as Adhesive pili They posses adhesins on their tips which help them to stick to other surfaces Adhesins are the proteins at the tip that recognize speci c receptors on the cell surface The receptors on animal cell surface include glycolipids and glycoproteins embedded in the cell membranes in such a way that their oligosaccharide moieties are presented on the surface Their cell adhesive properties are very useful in medical diagnostics eg hemagglutination Where mbriae is used to attach to the surface of red blood cells 2 Sex Pili Sex pili is the type of pili which is used for the attachment to other bacterial cell for the purpose of transmitting DNA from the pili containing cell to the one which doesn t contain pili recipient The requirement of pili for mating is not universal but certainly found in enteric bacteria like Ecoli and Pseudomonads The seX pilus grows on male bacteria and used to donate DNA to the female recipient bacteria The genes responsible for coding seX pili are located on conjugative F plasmid in male E coli The tip of Fpilus contains adhesin which helps the male cells to get attached to the recipient cells This is followed by retraction by depolymerization of pilin This brings the surface of two cells together until their surface are in contact DNA is then transferred at the site of contact Many bacteria do not need pili for gene transfer eg gram positive bacterium Enterococcusfaecalis In this case mating is induced by seX pheromones secreted in the medium by recipient cells Fig 14 Fpilus mediated conjugation Transfer of a sex plasmid The donor cell has a plasmid and an F pilus encoded by plasmid genes 1 The F pilus binds to the recipient cell 2 The pilus retracts bringing the two cells together This is due to a depolymerization of the pilus subunits 3 The plasmid is transferred as it replicates so that when the cells separate each has a copy of the plasmid and is a potential donor The Glycocalyx Glycocalyx is the term used to describe the outer most layer surrounding the cell wall The glycocalyX may be in the form of S layers capsules or slime S layers S layers are the array of protein or glycoprotein subunits on the cell wall surface They are found in wide range of gram positive and gram negative eubacteria They are also found in archaea where the S layer sometimes covers the cell membrane and serves as the cell wall itself Capsules Capsule is a extracellular brous material which is either loosely or tightly attached to the surface of bacteria When it is loosely attached to the cell surface it is also referred to as slime layer or slime capsule or extracellular polysaccharide Capsule is covalently attached to either phospholipid or lipid A embedded in the cell surface Chemical composition of Capsule Most of the glycocalyces are made up of polysaccharides but some are made up of protein The example of polypeptide capsule is the polymer of Dglutamate found in B anthracis Polysaccharide capsules are diverse in chemical composition and structure Some are homopolymers While others are hetropolymers The monosaccharides involved are linked to each other by glycosidic linkage to form straight or branched chains The chemical composition of polysaccharide may vary in different strains of the same organism For example there are more than 80 different types of polysaccharides found in E coli strains Sometimes same types of polysaccharides may be found in two different species Functions One of the most important function of capsule is adhesion to the other cell surfaces or on inanimate surfaces to form bio lm Protection from phagocytosis and thereby increasing the virulence is another important function of the capsules Other functions include prevention of dehydration of cell Cell Wall In most of the bacteria cell membrane is surrounded by a rigid wall like structure known as cell wall Cell wall is responsible for speci c shape and protects the cell from bursting Most of the bacteria can be divided into two groups on the basis of the type of cell wall they contain They can be distinguished on the basis of gram stain reaction The one which retain gram stain are gram positive and the one which do not retain the stain are known as gram negative The Gram Stain Christian Gram invented gram staining procedure in 1884 According to the procedure the cells are divided into two groups on the basis of whether they can retain the Crystal violetiodine complex or not The one which retain are gram positive while the one which loose and are stained with counter stain safranin are gram negative Mechanism of Gram Staining The mechanism of gram staining is based on the differences the two groups have in their cell wall composition and thickness In case of gram positive bacteria because of their thick peptidoglycan cell wall the stain complex is trapped In case of gram negative bacteria their cell wall is thin containing less peptidoglycan and phospholipid rich outer membrane The outer membrane is made leaky by solvent treatment due to which it looses the stain complex Archaea can stain either positive or negative but they contain different cell wall composition and structure Thus peptidoglycan and lipid content plays very important role in gram reaction ulcr membrane ampcplidoglycm cell lt r membrane gt gram gram negative posmvc Fig 17 Bacterial cell walls Schematic illustra tion of a gram negative wall and a grampositive wall Note the prcsencc of an outer membrane also called outer cnvclope in the gramrncgativc wall and the much thicker peptidoglycan layer in the gram positive wall Peptidoglycan The peptidoglycan is responsible for the strength and the rigidity of the bacterial cell wall It is a glycoprotein made up of two types of sugar N acetyleglucosamine GlcNAc or G Nacetylmuramic acid MurNAc or Mlinked with 314 linkage and a tetrapeptide attached to M Two glycan chains are cross linked with peptide bonds between the tetrapeptides of two chains Tetrapeptide usually consists of Lalanine Dglutamate a diamino acid and Dalanine Peptidoglycan forms three dimensional structure surrounding the cell membrane with covalent glycosidic and peptide bonds The strength is due to the presence of many covalent bonds Peptidoglycan In gram negative bacteria peptidoglycan layer is free and only noncovalently attached to the outer envelope which can be isolated as murein sac In gram positive bacteria the peptidoglycan is bonded to various polysaccharides and teichoic acids and can not be isolated as pure peptidoglycan sac In gram negative bacteria the diamino acid is generally the diaminopimelic acid DAP while it is present sometimes in gram positive bacteria In gram negative bacteria the peptides are linked directly Whereas in gram positive bacteria usually peptide bridge eg pentaglycine bridge in Staphylococcus aureus is present Gram Positive Cell Walls Relatively thick about 1530 nm Wide and consist of several polymers and mainly peptidoglycan PG Nonpeptidoglycan polymers usually can consist of almost 50 of the dry weight of the gram positive bacterial cell wall 39 They are bound to the glycan chain of PG quot The nonpeptidoglycan polymers include Teichoic acids polymers of ribitol or glycerol phosphates Teichuronic acid Acidic polysaccharides containing uronic acids Neutral polysaccharides common in Streptococci Lactobacilli lipoteichoic acids Phosphodiester linked a glycerol phosphate bound to lipid glycolipids and Mycolic acid common in genus Mycobacterium Gram negative cell wall The gram negative cell wall is more complex chemically as well as structurally than gram positive cell wall It consists of an outer membrane made up of LPS phospholipid and protein and an underlying PG layer The compartment between the inner amp outer membrane is called periplasm where the PG layer is located 1LPS is made up of three regions Lipid A Core and repeating oligosaccharide 0 antigen They are arranged in such a way that core and oligosaccharides are exposed out side in the medium while the lipid A Phospholipid is embedded inside the membrane as a part of lipid bilayer Gram negative cell wall Besides being important in increasing the virulence LPS also acts as a permeability barrier to hydrophobic compounds and also bile salts Therefore enteric bacteria survive in presence of bile in the intestine Bile salts are also used for selective growth of gram negative organisms eg EMB agar 2 Lipoproteins Lipoproteins in gram negative bacteria form a connection between the outer membraneOM and PG layer The lipid portion of lipoprotein is attached hydrophobically to the OM while the protein end is bound to PG layer Thus it helps OM to remain attached to the cell surface 3 Porins and other proteins Porins are the major outer membrane proteins They are the hydrophilic channels which allow the nonspeci c entry of molecules smaller than 600 Da E coli has three major types of porins namely OmpF OmpC and PhoE OmpC is smaller than OmpF making membrane less permeable PhoE is responsible for inorganic phosphate and other anions transport and is only produced under phosphate limitations Porins Since porins exclude molecules larger than 600 Da gram negative outer membrane translocates larger molecules with the help of other proteins For example LamB protein transports maltose and maltodextrins BtuB transports Vitamin B12 FepA transports ferricenterobactin a siderophore produced by E coli Tsx transports nucleosides and there are many more Archaeal Cell Wall The main difference between the bacterial cell wall and archaeal cell wall is that the archaeal cell wall do not contain Peptidoglycan The cell walls have different structures and they are made of either pseudopeptidoglycan polysaccharide or protein S layer Pseud0peptid0glycan is made up of Nacetylglucosamine same as PG and Nacetyltalosaminuronic acid instead of N acetylmuramic acid The sugars are linked by 313 linkage instead of B14 as in PG The tetrapeptide consists of Laminoacid instead of Damino acids as in PG CHZOH NH 0 HO G 0T O 0 Ifquot glu K Cl0 4393 z CH3 ly glu E Periplasm In gram negative bacteria the space between the inner membrane and the outer membrane is termed as Periplasm It is lled with aqueous solutions containing proteins oligosaccharides salts and also the PG layer Many specialized functions like oxidationreduction osmoregulation nutrient transport protein secretion and enzymatic hydrolyses are carried out in this space Periplasmic space contains many solute binding proteins involved in transport component of electron transport eg cytochrome C hydrolytic enzymes like phosphatases and nucleases and detoxifying enzymes like Blactamase Another important protein partially located in periplasm is TonB This protein is anchored to inner membrane but extends to the periplasm It is involved in energy transduction to many transport processes including iron and vitamin B12 transport It is also involved in energizing drug ef ux systems in bacteria Recently it has been reported that even gram positive bacterial cell perhaps possesses compact periplasmic space Cell Membrane CM Bacterial cell membranes are made up of phospholipid bilayer and protein arranged in uid mosaic structure Phospholipids are formed by esteri cation of phospho glycerides with fatty acids Since these molecules have both polar hydrophilic phosphate and nonpolar hydrophobic fatty acids they are known as amphipathic molecules These molecules spontaneously aggregate in such a way that the hydrophobic ends interact with each other and hydrophilic phosphate face aqueous periplasm and cytoplasm Proteins are embedded in the lipid bilayer These proteins are classi ed into two groups Integral and Peripheral Integral proteins are completely embedded and are attached with the hydrophobic fatty acids while the peripheral proteins are located on the surface and are attached with phospholipids with ionic interactions Functions of CM There are more than 100 proteins located in CM involved in diversi ed functions such as proton pump in ATPase ATPsynthesis movement of agella electron transport nutrient transport photosynthesis biosynthesis of lipids cell wall polymers secretion of proteins and signal transduction The phospholipid bilayer also acts as a permeability barrier to most of the water soluble solutes Archaeal Cell membrane The lipids found in archaeal membranes are different than the ones found in bacteria They consist of isopranoid alcohols either 20 or 40 carbon long The isopranoid alcohols are either etherlinked to glycerol to form monoglycerol diethers or to two glycerols to form diglycerol tetraethers Not many archaeal membrane proteins have been studied except Bacteriorhodopsin and Halorhodopsin both involved in light driven functions The thermoacidophilic and some methanogenic archaea have tetraether glycerolipid having polar end at both the ends These lipids form lipid monolayer which are resistant to high temperature This is the only example of membrane having lipid monolayer Isopranoid alcohols in Archaeal Cell membranes A B H0 EH OW HO A Monoglycerol Diether B Diglycerol tetraether Cytoplasm The Viscous material containing high concentration of proteins DNA RNA salts and thousands of metabolites surrounded by cell membrane is called cytoplasm It also contains many aggregates of proteins as well as different types of inclusion bodies Intracytoplasmic membranes are also found in some bacterial cytoplasm The soluble part of cytoplasm is termed as Cytosol Intracytoplasmic membranes ICM When present the intracytoplasmic membranes have specialized functions It is not very clear that Whether they are formed by the invagination of cell membrane or are form independently in the cytoplasm But in some cases they are attached to cell membrane There are many examples 1 In methanotrophs the ICM functions as site for methane oxidation 2 In nitrogen fixing bacteria like Azotobacter vinelandii the ICM surrounds and provides anaerobic condition to the Nitrogenase enzyme 3 In nitrifying bacteria like Nitrosomonas and Nitrobacter the ammonia and nitrite oxidizing enzymes are located in ICM 4 In phototrophs the photosynthetic machinery is located in the ICM Inclusion bodies Multienzyme aggregates and Granules Some bacteria do contain organelle like structures Which are surrounded by membrane or coat different from the lipid bilayer membrane Which surrounds typical eukaryotic organelles They are called inclusion bodies Bacteria also contain numerous aggregates and multienzyme complexes of large size The examples are 1 Gas vesicles found in aquatic cyanobacteria photosynthetic bacteria and some nonphotosynthetic bacteria The gas vesicles allow them to float in the lake at the depth that favors their growth by providing optimum light temperature or nutrients 2 Carboxysomes They are the large polyhedral inclusions observed in many bacteria that obligately utilize C02 as a sole source of carbon These inclusion bodies contain the enzyme Ribulose biphosphate carboxylase the enzyme Which incorporates C02 into organic compounds 3 Chlorosomes are ellipsoid inclusions found in many photosynthetic bacteria They act as light harvesting bodies since they contain light harvesting photopigments 4 Different bacteria contain many types of granules made up of either lipoidal substance called polyBhydroxybutyric acid PHB or glycogen or polyphosphate or elemental sulfur 5 Ribosomes They are the site of protein synthesis They are made up of three different types of RNA 238 168 and SS and 50 different types of proteins Their approximate size is 22nm by about 30nm On the basis of sedimentation rate both bacterial and archaeal ribosomes sediment at 708 but archaeal ribosomes are still different structurally than bacterial ribosomes 6 Nucleoid It is an area where an amorphous mass of DNA is located almost in the center of the cytoplasm bound to the membrane Sometimes in rapidly growing bacterial cells you can observe two such area containing single copy of chromosome The DNA present in the nucleoid is tightly coiled DNA string if stretched would be 500 times longer than the length of the cell DNA is also bound to RNA which is freshly transcribed and many proteins including enzyme RNA polymerase several DNA binding proteins 7 Multienzyme complexes There are many large multienzyme complexes located Within the cytoplasm The examples are 1 Pyruvate dehydrogenase from Ecoli is a complex of three enzyme consist of total 50 proteins having total size of 4648 x 106 Da The enzyme complex oxidizes pyruvate to acetyl CoA and C02 2 ocKetoglutarate dehydrogenase also consists of three enzymes and made up of 48 proteins having total size of 25 x 106 Da This enzyme oxidizes ocKetoglutarate to succinyl CoA and C02 Cytosol The cytosol is a liquid portion of the cytoplasm Which can be isolated by centrifuging the broken cells at 10500 x g for 1 to 2 hours Centrifugal force separates soluble fraction of the cytoplasm the Cytosol as supernatant from the membranes protein aggregates and DNA as a sediment Cytosol contains all the soluble enzymes responsible for catalyzing thousands of biochemical reactions of carbohydrate lipid protein and nucleotide metabolism Therefore the protein concentration of cytosol is very high making it very viscous Analogues to tubulin and actomyosin There are increasing evidence that there is a presence of Cytoskeleton like structures made up of proteins like tubulin are present in bacteria and 1 Electron TranSport k gt 51 y V y 91 9 I F535 gt JL 39 quot r j v 39 Iquot 39 I 1 i 39 I 1 quotn 39ng 9 v 39LJT 3991 ngr 39LLFF g gt l A v i 1 A A v i 39v v 4quot 39 quoth w v 4 1 1 V i 7 j p gt v v 39 VP 1 A 1 1 7 5 V V 1 x A l 1 e t fun A P 39IV e f i t n A If I quota r v i v D 5 V 93 v 7 v i3 V 13 V A no 39 ww 39 7 H 7 kV V V l P39 l rquot t L p 3 v 3 I 39 i quotv 539 39 quot 139 u I 739 LP 39 err It a if 3 air I Air 1 Eff 3 ir A r 39A V i Vv 7 A r cterial Physiology HSCIS607 Chapter 34 39Mes mbr ane Bioenergetics A ri 39 rw 4 0 Au Chemiosmotic Theory Introduction of chemiosmotic theory by Peter Mitchell has revolutionized the eld of membrane bioenergetics According to the chemiosmotic theory membranes pump protons across the membrane generating electrochemical gradient spanning outside and inside the membrane The gradient drives the ow of protons from outside to inside generating proton motive force Which is used for many functions besides ATP generation This mechanism operates not only in bacterial membranes but also mitochondrial as well as chloroplast membranes In order to generate energy protons must return through specialized conductors which may be transport membrane proteins or ATP synthesizing unit or motor that drives agellar rotation Chemiosmotic mechanism is central to bacterial physiology and bacteria commonly use ion gradients to couple energy yielding exergonic and energy requiring endergonic reactions Mechanism of Chemiosmotic theory According to this theory the protons are translocated across the membrane by exergonic reactions egrespiration photosynthesis or ATP hydrolysis The translocation of protons often leave behind negatively charged counterion inside producing positive potential outside and negative inside When protons return inside towards the negative pole of potential it provides energy for the work to be done This is very similar to a battery which a maintains a potential difference between the poles and the ow of electron generates energy for the work to be done The only difference here is that instead of electrons there is a ow of proton It is important that membrane has a low permeability to protons and are not leaky so that majority of the protons return through the proton conducting channels generating energy It is known that lipid bilayer is relatively impermeable to protons Membrane potential can also induce in ux of other ions like Potassium or Sodium Electrochemical energy of Chemiosmotic system The energy in proton gradient is both electrical and chemical Electrical energy is generated due to the movement of positively charged proton to one side creating a charge separation Actually when protons move outside against the charge it requires the energy which it conserves in the form of electrical eld When proton moves inside with the electric eld the energy which is released is Electrical energy and can be used to perform work Similarly when protons move outside they move against the concentration gradient The energy stored in the concentration gradient is released as Chemical energy which is used for work The sum of the changes in both the types of energy is known as Electrochemical Energy Electron Transport The energy required for the growth related biosynthetic as well as nutrient transport activities is obtained by coupling these activities to the ow of electrons in membrane and creation of electrochemical proton gradient The electrons ow from primary electron donors to terminal electron acceptors through a series of electron carriers proteins and lipids called quinones These ow of electron through the carrier is referred to as respiration If the nal electron acceptor is oxygen it is termed as aerobic respiration while if it is not oxygen it is known as anaerobic respiration nitrate or sulfate or organic compound as Fumerate Proton translocation takes place during respiration and electrochemical potential is created at the coupling site The proton potential is then used to drive solute transport ATP synthesis agella rotation and other membrane activities Electron transport pathways are almost same in bacteria and in mitochondria Within prokaryotes the types of primary donor and terminal acceptor may vary The Electron Carriers There are different types of electron carrier through which electrons ow 1 Flavoproteins Hydrogen and electron carriers 2 Quinones Hydrogen and electron carrier 3 IronSulfur proteins electron carrier 4 Cytochromes Electron carrier Amongst these carriers quinones are lipids while others are proteins and exist as multienzyme complexes known as OXidoreductases The electrons are carried by nonproteinic part known as Prosthetic group such as F eS in ironsulfur proteins and avin in avoprotein Flavoproteins Flavoproteins have organic molecule avin as a prosthetic group Its yellow in color and is synthesized by cells from ribo avin vitamin B2 Two types of avins found are avin mononucleotide FMN and avin adenine dinucleotide FAD When they are reduced they carry two hydrogenselectrons They are involved in many cytoplasmic oxidationreduction reactions besides the electron transport Quinones Quinones are lipid electron carriers Due to their hydrophobicity they are mobile in lipid bilayer and carry hydrogens and electrons to and from the immobile protein carriers Isoprenoid side chains contribute to their hydrophobicity Bacteria make two types of quinones a Ubiquinone UQ and a Menaquinone MK Menaquinones are derived from Vitamin K have lower electrode potential than UQ and are used predominantly during anaerobic respiration A third type of quinone known as Plastoquinone is used in photosynthetic electron transport Ironsulfur Proteins They contain nonheme iron and acid labile sulfur Ironsulfur ratio is usually 11 There may be more than one cluster of ironsulfur present per protein In that case electron may travel from one cluster to another These proteins also contain Cysteine sulfur Which binds the iron to the protein There are several different types of ironsulfur proteins Which catalyze oxidationreduction reaction in cytoplasm amp in membranes Cytochromes Cytochromes are electron carriers that have heme as the prosthetic group Heme consists of four pyrrole rings attached With each other by methane bridges therefore also called tetrapyrrole Each pyrrole ring has side chain and tetrapyrrole With side chain is called Porphyrin and Without side chains Porphin Hemes are classified on the basis of the types of side chains attached to them into five classes abcd and o Hemes d and o are only found in prokaryotes Cytochrome bdd and boo are examples of bacterial cytochromes In the center of each heme the iron is attached to the nitrogen of the pyrrole rings Iron acts here as electron carrier which is oxidized to ferric and reduced to ferrous during electron transport Bacterial Electron Transport Chain The type and sequence of electron transport components vary amongst bacteria and also Within the same bacteria depending upon the growth conditions Similar to mitochondrial electron transport chains bacterial chains are also organized into dehydrogenase and oxidase complexes connected with quinones Mitochondrial I 5390 L H W En 320mV T 39 397 quot quotquot 1 139 815mV NADH gtrp gt Fes gt UQ gtb gtc l Dc gtLaa3J gt02 L 4 L V J Tia 11 LfEJ summit Bacterial Elquot02 AH2 gt gtquinone gt gtc gt 02 AH gt dehydrogenase gt quinon gt b gt c gt gt 02 El 02 B AH gt dehydrogenase quinnnc gt mduclase Y Bacterial ETC The quinones accept electrons from dehydrogenases and transfer these to oxidase complexes that reduce the terminal electron acceptor In bacteria terminal electron acceptor may be molecular oxygen or inorganic compound like nitrate or organic compound like fumerate under anaerobic respiration Similarly bacteria also contain variety of terminal oxidases eg quinol oxidases cytochrome c oxidases Sometimes 2 or 3 different oxidases may be found in the same bacteria The oxidases differ in their af nities for oxygen as well as the type of metal and heme they contain Some of them are proton pumps while some are not Two major differences between the bacterial and mitochondrial ETC is that 1 The routes to oxygen in the bacteria are branched specially under aerobic conditions the branched point being at the quinone or cytochrome 2 Many bacteria can alter their ETC depending upon their growth conditions Branching gives exibility under different growth conditions Coupling sites The sites in electron transport chain where redox reactions are coupled to proton extrusion creating a electrochemical potential Ap are called Coupling sites Each of these sites is also a site for ATP synthesis since the protons extruded reenter via ATP synthase to make ATP The number of protons extruded per two electrons vary depending upon the complex A consensus value of 10 protons travel per the transfer of two electrons from NADH to oxygen while the bcl complex translocates four protons per two electrons 39 z E I n v L p V 3 p h gt J The MLGrawvH Fmpnnias hm Miaan Iun fm repmduanm m dianl v M H 39 W 39 Cc w W 0 mm M WW 039 W zW w y E d uMMMW W Ui obql a J v Number of ATPs generated According to chemiosmotic theory the ATP made per the coupling site equals to the ratio of protons extruded at the coupling site to protons that reenter Via ATP synthase This number may not be a Whole number i K I i H ATP synthase y 32 E Fig 410 The ratio of protons extruded to pro tons translocated through the ATPase determines the amount of ATP made The ratio of protons entering Via ATP synthase to ATP made is called HATP Values of two to four have been reported but consensus value of three is used for calculation Q Loops Q Cycles And Proton Pumps The redox reaction is thought to be coupled to proton extruSion in two Ways 1 Q lOop or Q Cycle 2 ProtOn pump 39 IQ Loop or Cycle In the Q loopCycle reduced quinone site 1 proton pump Z H sin 010013 2m CYTOPLASM r OzlZHquot 320 m4 Carries hydrogen across the membrane and become oxidized releasing protons on the external surface as the electrons return electrogeni cally Via electron carrier to the inner membrane surface On the cytoplasmic side at the same time same number of protons are taken up as oxygen is reduced to water Thus net result is translocation of protons from inside tooutside39 Although not the same protOns traverse the membrane Proton pumps Proton pumps are electron carrier proteins that couple electron transfer to the electrogenic translocation of protons through the membrane For example in mitochondria and some bacteria proton extrusion accompanies cytochrome aa3 oxidase reaction When cytochrome C is oxidized by oxygen Bacterial PhysiologyHSCI 46075607 CH 9 Metabolism of Lipids Nucleotides Aminoacids and Hydrocarbons LIPIDS Introduction Structurally lipids are heterogeneous group of substances Their distinguished properties include They are made up of fatty acids Highly soluble in nonpolar solvents such as ethanol methanol acetone chloroform and so on Relatively insoluble in water Lipids are important components of bacterial and eukaryotic cell membranes The major lipid present in these membrane is phospholipid which is also called phophoglycerides Phosphoglycerides are made up of fatty acids esteri ed to glycerol phosphates Archaea also have phospholipids but they have different chemical structure and mode of synthesis Fatty Acids Fatty acids are chain of methylene carbons with a carboxyl group at one end The carbon chain can be branched saturated no double bonds unsaturated one or more double bonds or hydroxylated Different fatty acids differ in the numbers of carbon atoms they contain the number and position of double bonds that they may have and Whether it is branched or having ring structure They are usually 16 to 18 carbon long and either saturated or have one double bond Grampositive bacteria are richer in containing branched fatty acid than gramnegative bacteria In bacteria fatty acid do not occur freely but are covalently attached to either glycerol phosphate membranes or carbohydrates eg lipid A or to protein lipoprotein which is covalently attached to peptidoglycan Table 91 Some fatty acids Number of carbon atoms Fatty Acid CH3CH214COOH CH3CHZ15COOH CH3CH2J7CHCHCH27COOH CH3CH24CHCHCH2CHCHCH27COOH CH3CH25CH CHCH29COOH CH2 CH3CH27CHCH23COOH CH3 Palmitic Stearic Oleic Linoleic Lactobacillic Tuberculostearic Fatty Acid Degradation BOxidationzMany bacteria when they grow on long chain fatty acids they oxidize fatty acids to acetylCoA Via pathway called BOxidation Reaction 1 In the rst step the fatty acid is converted to AcylCoA derrivative in a reaction catalyzed by AcylCoA synthetase This takes place in two steps First the ATP molecule loses PP and gets attached to the fatty acid at carboxyl end to form Acyl adenylate AcylAMP In a second step the AMP molecule is replaced by CoASH to form Fatty AcylCoA Reaction 2 In this step AcylCoA dehydrogenase oxidizes and forms double bond between the C2 and C3 Reaction 3 The double bond is hydrated by 3hydroxyacyl CoA hydrolase during this step Reaction 4 The hydroxyl group is oxidized in this step to form keto group by L3hydroxyacylCoA dehydrogenase 0 II CH3 CHzn CH24CH2 c 0H l PPi COASH AIVIP 391 CH3 CH CH2 CH CSC0A 2 FAD FADHZ ll CH CH2n CH CH CSCOA H20 3 ll CH3 CHn FH I2 CsCoA OH 4 NAD NADH H o Q II CH3 c11n ltII Hz CSCOA o 39 Colts 5 3 CH3 u H CH3 0 Fig 91 oxidacion of fatty acids Enzyme 1 acyl COA synthetase 2 fatty acyl CoA dehy J 3 2 J poA z z a 4 L 39 39 0A 39 5 3 ketothiolase Reaction 5 In the nal step the carbonyl group of keto acyl CoA is attacked by CoASH displacing Acetyl CoA as a product This reaction is catalyzed by 3 ketothiolase Fate of AcetylCoA AcetylCoA thus released is metabolyzed to C02 in aerobic bacteria Via citric acid cycle or it can be utilized Via glyoxylate cycle The other product Fatty acyl CoA enters into another cycle of Boxidation to generate another molecule of AcetylCoA Thus if the fatty acid is made up of even number of carbons it is completely metabolized to AcetylCoA While if it is odd number chain then the last fragment of propionylCoA is metabolized by variety of oxidative path ways One example is PropionylCoA gtgt AcrylylCoA gtgt LactylCoA gtgt Pyruvate Fatty Acid Synthesis Fatty acid biosynthetic pathways differ from Boxidation pathway in several ways 1 The reductant is NADPH instead of NADH in Boxidation 2 It requires C02 and proceeds via carboxylated derivative of AcetylCoA called MalonylCoA 3 The acyl carrier carrier is ACP Acyl carrier protein instead of CoA in Boxidation ACP is a small protein having MW of 10000 in E coli In eukaryotes the synthesis takes place in the cytosol While the degradation takes place in matrix of mitochondria In bacteria both the reactions take place in the cytoplasm The steps in synthesis Reaction 1 The synthesis begins with the carboxylation of AcetylCoA The reaction is catalyzed by AcetylCoA carboxylase and it requires ATPand biotin The product is MalonylCoA Reaction 2 In this step the CoA is replaced by ACP by an enzyme Malonyl CoAzACP transacetylase Simultaneously similar enzyme displaces CoA from the molecule of AcetylCoA to generate AcetylACP Reaction 3 Reaction 4 During this reaction the malonylACP replaces ACP from AcetylACP with the removal of C02 from MalonylACP and ACPSH from AcetylACP to form 3ketoacylACP The reaction is catalyzed by Bketoacyl ACP synthase Reaction 5 BketoacylACP is then reduced to the hydroxy derivative by an NADPHdependent BketoacylACP reductase to 3 hydroxyl acylACP which is in reaction 6 dehydrated by dehydrase to yield unsaturated acylACP derivative Reaction 7 The unsaturated acylACP is then reduced by enoylACP reductase to saturated AcylACP The ACP chain is elongated by repetition of series of identical reactions initiated by attack of MalonylACP on acylACP chain to remove ACP The biosynthetic pathway is possibly regulated through feed back inhibition When acylACP chain is completed the acyl portion is immediately transferred to membrane phospholipids by the enzyme Glycerol phosphate acyltransferase reactions For the synthesis of unsaturated fatty acids the AcylACP or AcylCoA is dehydrated or desaturated by speci c enzymes depending upon the type of bacteria and aerobic or anaerobic conditions A ersm sssa 4 0 chicwg tez c cu cncsc ou yI nur u 0 4pcquot 4L mam l u m We um malnnyl nunucetyhns uetnse s Shydxaxyncyl mum 1 vmnha a cumuuxnwuiuc n c 91 mam 3 mm aydmxydm gt442 curbo ylnxe 2V s 3krunyl ACP quota ayl ACP dehyd s 9 Elngy m of In C d yz useKyl lxnninrylnl A P 5y ACP admin 7 enuyI ACP uducrnse s Phospholipid Synthesis Phospholipids are the fatty acids covalently attached to glycerol phosphates They are important constituents of cell membranes Bacterial phospholipids also contain other molecules like amino acids an amine or sugar covalently attached to them eg Phosphatidyl serine contains amino acid serine Synthesis Their synthesis starts with glycolysis intermediate Dihydroxy acetone phosphate DHAP which is reduced to Glycerol 3Phosphate by enzyme glycerol phosphate dehydrogenase In step 2 the fatty acids from newly synthesized AcylACP are transferred to the C1 and C2 of the glycerol phosphate to yield the rst Phospholipid called Phosphatidic acid The enzyme catalyzing this reaction is G3P acyl transferase Other phospholipids are synthesized from Phophatidic acid Phospholipid Biosynthesuls HC OH quotff 0H may I o H c R NADHHP Ho lc H R R Hlt o r o l H1c o z 039 R C O CH 0 H mm 63 AH o f a 39 o e a n c o lrn o II II 04 R C D T H D R g quot2 II II ch o r o cnk fu NH HocHZHNHh R C 0 C H o no u 0 39 Hc o Po CMP mm corducyluymnl 5 co 6 KlymolF 5 MP nc o cn R R C O H R o TH 39139 1i R c o c H Mc o o cu cu Nuk H o mc o fo curtlsu cmo 0 PE 0 on 0 Par 7 up o o u R C OCH Ski 04 1 I 3 n c o c n PC I E u I quot3 I R H O gOCHZIIHC ior O C z Hc o r o cn ltlH cuou on n 3960 on 0 CI Archaeal Lipids There are notable differences between archaeal and bacterial lipids as follows 1 Archaeal lipids contain longchain alcohols called isopranyl alcohols instead of fatty acids 2 The linkage to glycerol is Via an ether bond instead of an ester bond NUCLEOTIDES There are three components of nucletides 1 Nitrogeneous base Purine Adenine or Guanine or Pyrimidine Thymine Cytosine or Uracil 2 Pentose SugarzRibose RNA or Deoxyribose DNA 3 Phosphate group The nucleotide Without phosphate is called nucleosides that is nucleotides are nucleoside phosphates The pyrimidine nucleosides are called as Cytidine thymidine uridine while purine nucleoside are called adenosine and guanosine The corresponding nucleotides are referred as eg adenosine monoor di or tri phosphates AMP ADP or ATP Sometimes nucleoside monophosphates are also referred to as adenylic acid guanylic acid cytidylic acid thymidylic acid or uridylic acid In DNA the nucleoside phosphates contain deoxyribose Where on C2 the hydroxyl group is replaced by hydrogen STRUCTURES OF NUCLEOTIDES N HO O H0 H0 H0 Nil6 5F 78gt N34 5 Q3 4 EN 6 N l H 0H OH OH purine pyrimidine ribuse deoxyribose HOWE 11on la OW OH OH OH OH nucleoslde deoxynucleuside nucleoside monophosphate NMP Le a nucleoti 17132 0 0 NH 0 N N HN N HNJj CHJ NJ HN lt1 MAI DANHI 0 021i adenine guanine thymine cytosine uracil The Synthesis of Pyrimidine Nucleotides The pyrimidine ring of nucleotide is made from Aspartate Ammonia and Carbon dioxide The phosphoribosyl pyrophosphate donates the ribose phosphate moiety to the nucleotides This pathway leads to the synthesis of Uridine triphosphate which serves as a precursor for the formation of cytidine triphosphate through ammoni cation Methylation of UTP at 5th C forms thymidine triphosphate Thus UTP serves as precursor for the synthesis of the other pyrimidines Biosynthesis of prymidine nucleotides M11 mam p WEEK 1 39 I 39 NHv u if 7 i jclm HNN mu milquotIquot I u rbnimgh p iniuo 4quot l l mm 2 I l L NH E IOH mm 3 Iumnlm outmun quot 0 Amwn uN u I HN H HN HI a 39 7 e i 04 N W W ova wooquot NAme quot 9 9 W H a mum dlhydrnurnlII o Sadquot magigmmm I I I I gurglingplum OH 0quot pyronnlpnm in Fig 99 Origins of atoms in the pyrimidine ring M C2 is derived from C02 and N3 comes from an m quot quotquot ammonia via carbamoyl phosphate The rest of mm 9 the atoms are derived from aspartate quotiiiquot m M quotEl n m 0 T H 7 0 l Am fibula94 ribmaS PP lrMyIn up mam mumpm um j MN 3quot NM 4 04 H PI NH ifMaw o N I ribonS39FPP cylldln Irlphowhlu CTP Fig 910 Th hiasynlheai of pyrimidth nuclculidu Enzymes 1 curbnmoyl yhmphnt nynthmm 2 Mparum rmnlcarbamoylum 3 dihydmommm 4 dlhydrooromu dchydmgcnnle 5 mom phosphoribuuyl transfemsc 6 urolidineSphmphnle decnrboxylnse 7 nuclcosidc manophos in IIquot E nuclcosid diphoiphule kimae 9 CTP iymhcme IO PRI P synthmu 11V pyrophosphamc Biosynthesis of Purine Nucleotides The precursors for purine synthesis are glutamine aspartate glycine CO2 and a C1 unit at the oxidation state of formic acid The amino groups are donated by glutamine and aspartate while glycine C02 and formic acid donate carbon atoms Enzymatic reactions Step1 The synthesis of purine nucleotides begins with ribose phosphate which is derived from PRPP In the rst step the amino group donated by glutamine is attached to PRPP to form 5phospho ribosylamine Thus amino group displaces PP group at C1 and the reaction is driven by the energy released by the hydrolysis of PP Step 2 In this step glycine is added to the amino group to form 5phosphoribosylglycinamide at the cost of ATP hydrolysis The formyl group is added from formylTHF which is followed by addition of another amino group from glutamine This step also consumes one ATP The resulting 5phosphoribosylNformylglycineamidine cyclizes to form imidazole ring with the hydrolysis of ATP molecule Purine synthesis The imidazole ring serves as precursor and additional carbon atoms are contributed by C02 aspartate and formylTHF to ultimately form Inosinic acid Inosinic acid is the precursor to all the purine nucleotides Adenine nucleotide Adenine nucleotide is formed by substitution of the carbonyl oxygen at C6 of IMP with an amino group from aspartateThis reaction requires GTP Guanidine nucleotidezThis is formed by oxidation of IMP at C2 followed by an amination of C2 Glutamine is the donor of amino group The THFzThe tetra hydrofolic acid THF derivatives which are synthesized from vitamin B folic acid are important single carbon donor during purine biosynthesis The deoxyribonucleotides They are synthesized by reductive dehydration of ribonucleoside diphosphates catalyzed by Ribonucleoside diphosphate reductase which uses protein thioredoxin as electron donor coupled with NADPH Purine Biosynthetic Pathway Fig 911 Metabolic origins of atoms in purines 0 WHwca 7 man Lu39nhorllu IIA nylonIII y I I I munm ll Fig 912 Biosynthcnil of purine nutleolidel Enzymu l l Rl l nmidotnnlfemae Z L i v L v v A v luminolmidnzulc curbaxylm 7 Iym 9 39 A39l39PonF ADP o r y Iynthetue quot quot quot 39 L a g um m K o rquot quot mu r l I h n l quot Pquot um quot a1 quot lnnnyl r 1 39mr N N A 7 mo If m gt f ac Hm Dim I 1quot N H v ADP J 1 9K sphapurlnmIN mmyIIyzlmmldlm nun P lllullulluyl hrmylllyuuum tmymr s m it 913 HN N K in F mum um um rlbnlyIl in munon phmphnrl olphnribmyl nynrhmxc 6 pholpharihoxy L J up 39 39 10 Ml cyclohydrolm Abbreviations gin gluumlna glu glutlmue up puma fum fumnme THF G E 2 o g zvz ADI P Fig 913 Synthesis of AMP and GW from LMP Enzymes 1I adenylosuocinate synthemse Ind i u 1 AMP adenylic acid XMP xanthylic acid GMP guanylic acid 15me I 1120 0 ch base I ll R SH XI H nuleoslde l RES deoxynlclmide dlphoaphnte 5 s dlplloaphte NADP HAD 1 Hquot Fig 915 Formation of deoxynucleotides by ribonucleoside diphosphate reductase The deoxynu cleotides are made from the nucleoside diphosphates The OH on C2 of the ribose leaves with its bonding electrons to form water The OH is replaced by a hydride ion HF donated by reduced thiore doxin a dithioprotein Thioredoxin is rereduced by thioredoxin reductase a avoprotein Thioredoxin reductase in turn accepts electrons from NADPH Enzymes 1 ribonucleotide diphosphate reductase 2 thioredoxin reductase Abbreviations R SH thioredoxin Amino acids Proteins are made up of 20 different amino acids Most of them are derived from the intermediates of glycolysis PPP and TCA While histidine is derived from PRPP and ATP Amino acid Synthesis Glutamate and glutamine synthesis Synthesis of glutamate and glutamine in the cell is extremely important as this is the only way in which inorganic nitrogen is incorporated into organic cell materials The inorganic nitrogen is rst converted to ammonia then the ammonia is incorporated as amino group into either glutamate or glutamine The amino group from these amino acid is then transferred to all the nitrogen containing compounds in the cell For example glutamate donates amino group to most of the amino acids While glutamine is the donor of amino group to purines pyrimidines aminosugars histidine tryptophan aspargine NAD and paraaminobenzoic acid GlutamateGlutamine synthesis There are two different ways in which glutamate is synthesized in cell 1 This is catalyzed by enzyme Glutamate dehydrogenase which carries out reductive amination of aketoglutarate The K M of this enzyme for ammonia is high gt1mM therefore this pathway is operative only under high concentration of ammonia 2 Under low concentration bacteria use combination of two enzymes for the incorporation of ammonia One is Lglutamine synthetase which incorporates ammonia into glutamate to form glutamine using ATP as a energy source The other enzyme is glutamate synthase which transfers newly incorporated ammonia into glutamine to ocketoglutarate to form glutamate This enzyme is also known as Glutamine ocoxoglutarate aminotransferase GOGAT enzyme NADPH is the electron donor in this reaction Glutamate synthesis pathways COOH 90 c0 qu c H EH NADPH H NH3 1sz NADPquot H10 sz EH2 ac quot M 533quot e a Fig 916 The glutamate dehzdrggenase radian GlutamateGlutamine Synthesis I Ii H Kl o c g 1 2 w m Baal Luaum mumu 1h 9J7 The GS and GOGAT reactions Enzymes 1 L glumine syntheuu 2 glummim a J mm L Transamination for the synthesis of other amino acids The glutamate donates amino group for the synthesis of other amino acids to aketo carboxylic acid Transaminase is the enzyme which catalyzes the transfer of amino group A IZOOH lt20 HzN CII H R R IZOOH OOH CH CO 142 1332 COOH COOH glutamate aketoulntante GOGAT NADPH IJOOH POOH lann2 IIHNHZ 112 93912 C OH quot2 0 alumni glutnmlne Synthesis ofAspartate and Alanine Aspartate is synthesized from oxaloacetate Via transamination through glutamate While alanine is synthesized Via similar transamination reaction from pyruvate B 00 IEOOH o HgN mZ ll CH CH 500 glutamate uketoghname oxalonceute Wm c coon coon Io lt HallFH CH mam Jimlmmm CH pyruinle 8 a Lnlanilie Fig 918 The transamination reaction A Glutamate donates an amino group to an aketo carboxylic acid to form an aamino acid The glutamate becomes aketoglutarate which is aminated either via the glutamate dehydrogenase or the GS and GOGAT reactions B Formation of Laspartate from oxaloacetate C Formation of Lalanine from pyruvate NADH 1r goon W goon H II OH lhln ll rll m EAL 39 I lto z HNIl39 H CHZO 010 cup sphuphallyomu phalphulydmxy phuphurlu quotmm H10 3 Pl 3 Com CH CSD3A H E H n s HlN H o c cn ou llu lt Lll lll H18 THF I 1 mama mum 00quot Im HzN H CHINE c q llnlm mum Fig 919 Th synthesis of serine glycine and cysteine from 3PGA Enzymes 1 phasphoglycence dehydrogenm 2 phosphoserinc minouansferase 3 phosphoserine phosphatase 4 s 39 I n 4 A Catabolism of amino acids The catabolism of amino acid always starts with removal of amino group to generate the ocketo acid which eventually enters into citric acid cycle All of the 20 amino acids are degraded to seven intermediates that enter citric acid cycle These intermediates are Pyruvate acetylCOA acet0acetyl COA ocketoglutarate succinyl COA fumerate and oxaloacetate EgEEE lacing m mlt MM mm W mum m 92 L L uA 394 L J k u J l l H at acetyl CoA cannot be used for net glucogenesis except in some strict anaerobic bacteria that can carboxyhte acetyl CoA to pyruvate Chapter 13 Removal of the amino group Removal of amino group is carried out oxidatively to generate corresponding ocketo acid Usually this reaction is catalyzed by L or Damino oxidases which use avoproteins as electron acceptors and supply electrons directly to the ETC D amino oxidases are important in bacteria since several bacterial components like peptidoglycans contain Damino acids NH D II R IJ COOH 7f RC m39l 121 R LlCOOH NlI3 H fp f9quot vquot a R II Cm vNAD Hio DRC ml NADHH39NH3 H 39I39HI u R 139quot R Ii COOH CHECm gtR Cm l CH Clm39l H PMquot 1 Lllnlu 11P um H Nmu 1r D quotquot2 1quot NH Hp quotH o Hocm l oouI 1gt cm cmn AL cn 39lcoon H 8 r 1 J p M t 1 u avoproteins that are speci c for L at D amino acids but generally not for the particular amino acid Electrons are transferred m the avoprotein to the electron transport in amino acid dehydrogenase These are NADP linked and more speci c for the amino acid than are the I i u m J 4 L 4 u r L A i 1 u AL I I I J L Di The deamination of serine by scrim dchydruasc Aliphatic Hydrocarbon Degradation Many bacteria can grow on long chain hydrocarbons for example alkanes CmC18 while some grow on short chain hydrocarbons C2C8 The droplets of hydrocarbons either get dissolved into membrane lipids to move further in the membrane or some bacteria Acinetobacter produce droplets of lipid which dissolve hydrocarbon and transfer them to the membrane Once in the membrane they are metabolized rst aerobically in the presence of oxygen by hydroxylation to longchain alcohol This reaction is catalyzed by monooxygenases Longchain alcohol is then oxidized to carboxylic acid by membrane bound enzymes The carboxylic acid is then derivatized with CoASH using ATP as energy source which is eventually oxidized via Boxidation to acetyl CoA in the cytoplasm Bacterial Physiology HSCI 46075707 Ch 16 Solute Transport Introduction Bacterial cell membranes are semipermeable and made up of phospholipid matrix which blocks the diffusion of water soluble molecules into and out of cells It separates the cell s internal environment from the external environment facilitating metabolic activities and growth by maintaining higher concentration of metabolites inside This promotes faster enzymatic reactions and retention of metabolic intermediates within the cells Lipid barrier also prevents diffusion of ions including protons which help in the maintenance of proton and Sodium gradient across the membrane for the production of ATP solute transport etc All water soluble molecules enter or come out of cell through integral membrane proteins including transporters carriers porters and permeases Studies on membrane transport can be carried out using arti cial lipid membrane system known as Proteoliposomes Proteoliposomes Proteoliposomes are arti cial membrane vesicles of protein and phospholipid that are widely used to study the solute transport There are several methods by which proteoliposomes are made but all of them are based on the fact that the membrane proteins are soluble in detergent and can get integrated into the phospholipid bilayers when detergents are removed One of the method is to disperse phospholipids in water where they spontaneously form spherical vesicles called liposomes consisting of concentric layers of phospholipid These are then subjected to highfrequency ultrasonic waves which breaks them to smaller single phospholipid bilayer vesicles which resemble membrane bilayer structures The protein transporter to be studied solubilized in detergent is mixed with the liposomes and is eventually diluted with buffer to dilute the detergent Thus protein leaves the detergent and gets integrated into the liposomes to form proteoliposomes SOLUTE WHORT lipnsomes prutenlipmumes phosphulipids mumquot buffer 0 quot 39 detergent coated proiein Fig 161 Preparation of proteoliposomes Phosphnljpids are dispersed in water and sonicated Small vesicles each surrounded by a lipid bilayer form The vesicles are mixed with detergent39solubilized protein and diluted into buffer Pmteoiiposomes form with the protein incorporated into the hilayer The proteoliposomes can be loanied with substrate or ATP by including these in the dilution buffer Proteoliposomes Proteoliposomes can be supplied with ATPs or any other substrates along with the dilution buffer When proteoliposomes are incubated with the substrate they catalyze transport of the solute which can be measured by separating the proteoliposomes by either centrifugation 0r ltration Kinetics of Solute Uptake 1 Transportermediated uptake One can prove the existence of the transporter by studying the kinetics of the solute transport The kinetic studies are carried out by measuring solute transport at various concentration of solute and plotting the graph of transported solute versus different solute concentrations The graph appears to be a hyperbolic in case of the presence of speci c transporter Vmax Vm aX2 Km S Transportermediated uptake The explanation for the hyperbolic curve is that the entry through the transporters is always limited by the numbers of transporters present in the membranes Thus initially the numbers of the transporters available are more and as the concentration of solute in the medium progressively increases all the transporters get occupied the transport reaches to a maximum velocity Vmax The solute concentration at half the maximum velocity is called as Km Km a af nity constant is frequently used to express the af nity of the solute towards the transporter The value of Km for different transporter vary from less than micromolar to several hundred micromolar 2 Uptake in absence of transporter When the same concentration dependent experiment is carried out for transporterindependent solute transport following type of graph is generated S As seen in the graph the rate of transport is much slower and is proportionate to the concentration of solute The transport does not get saturated even at higher concentration of solute Energy dependent transport Usually a transporter facilitates entry and exit of the solute across the membrane and at equilibrium it does not favor either entry or exit thus does not accumulate solute in the cell against concentration gradient Many transport mechanism can accumulate solute against the concentration gradient and at steady state the concentration inside the cell reaches several times higher than the external concentration This transport against the concentration gradient requires energy and the source of energy can be chemical light or electrochemical eg proton gradient Bacterial Transport Systems Bacterial transport systems can be divided into two categories 1 Primary transport These are driven by an energyproducing metabolic event The examples are protontranslocationdriven by ATP light or oxidationreduction reactions lightdriven chloride transport sodium ion transporting decarboxylases the uptake of organic or inorganic solute driven by ATP hydrolysis etc 2Secondary transport During secondary transport the solute moves down an electrochemical gradient usually of protons or sodium ions Thus secondary transport is coupled to the primary transport Primary transport Hill Energy gt HJ out Secondary transport Hout Sout gt Hin Sin Active transport is referred to as Primary transport which requires energy and solute remains unmodified Secondary transport is catalyzed by uniporters symporters and antiporters that use electrochemical gradients to accumulate solutes For example the uptake of K down its electrochemical gradient is uniport Symport refers to the uptake of two solute in same direction while antiport is movement of two solutes in opposite directionNaHexchange Secondary Transport Generally the secondary solute transporter functions in such a way that either both solute and ion are transported in one direction Symport or the solute in one direction and the ion in the other antiport But there are transporters that transport just an ion along its electrochemical gradient called uniport In all the cases these transporters are the part of an electrical circuit Most of the bacteria use both the proton as well as sodium symporters but mainly the former But certain bacteria like halophiles alkaliphiles and marine bacteria depend heavily on sodium symporters The examples of transports coupled with the electrochemical gradient are 1 Symport of an uncharged solute with protons 2 Symport of a monovalent anion with protons 3Antiport of a monovalent cation with H and 4 Electrogenic uniport of a cation Evidence for soluteProton or solutesodium symport One way to demonstrate coupling of transport to proton or sodium ion in ux is to measure the alkalinization of the medium decrease in protons or a decrease in the Na ion concentration when bacteria or membrane vesicles are incubated with the appropriate solutes During NaH symport the Na or H can be measured by speci c ion sensitive electrodes ATPdriven Primary transport There are many transport systems that are driven by ATP or some phosphorylated derivatives The examples of such transport are 1 H transport ATP synthase 2 K transport in E coli 3 Some transport systems in gram positive bacteria 4 Transport systems in gramnegative bacteria that uses periplasmic binding proteins Shocksensitive transport systems These systems are the characteristics of gramnegative bacteria and are responsible for the transport of Wide range of solutes including sugars amino acids and ions Usually these systems consist of a transportera inner membrane protein compleX consisting of four subunits two of which are identical and a periplasmic solute binding protein Such systems are more complex than Ap driven single transporter transport The transport process consist of several steps They are known as shock sensitive transport system since osmotic shock leads to the loss of these systems due to the loss of the periplasmic proteins Common Steps of Transport 1 The solute enters the periplasm through an outer membrane pore eg Porin 2 The solute binds to a specific periplasmic binding protein to form a complex The binding protein undergoes conformational change that allows it to deliver the ligand to the membrane transporter 3 The liganded binding protein binds to the transporter and delivers the solute to the membrane bound transporter 4 The transporter complex translocates the solute across the cell membrane perhaps through the channel that opens transiently ATP hydrolysis catalyzed by the transporter complex occurs at this step The ATP binding domains are exposed to the cytoplasm 5 The transporter returns to its original conformation after delivering the solute into the cell Functional Model of Ferricsiderophore Transport CYTOPLASM Examples of ATP driven systems 1 Histidine transport Studies using proteoliposomes proved the presence of ATP requiring histidine transport system in E coli Histidine binding protein HisJ in proteoliposome only transported histidine when supplied with ATP 2 ATP driven K in ux Potassium ion is very important for bacteria due to its role not only in the pH and osmotic homeostasis but also it acts as a cofactor for several enzymes and ribosomes Bacteria tend to accumulate K several fold higher concentration than out side There are two transport systems The major uptake is via constitutive TrK system which is relatively low af nity system dependent on both Ap and ATP The second system is expressed at low K concentration called KDP system the induction of which depends upon osmolarity The phosphotransferase system This system differs from the above systems in that it catalyzes the accumulation of carbohydrates as phosphorylated derivatives Phosphoenolepyruvate is the phosphate group donor Bacterial Physiology HSCI 5607 Chapter 12 Inorganic Metabolism Introduction Most of the prokaryotes can utilize various derivatives of sulfur nitrogen and iron as nutrient as well as energy source These nutrients are metabolized using varieties of metabolic pathways 1 Through assimilatory pathways microorganisms incorporate inorganic nitrogen and sulfur into organic cellular components Some bacteria can also utilize chemically inert nitrogen gas as a nitrogen source through a process called Nitrogen fixation 2 In dissimilatory pathways the inorganic compounds are used as electron acceptor instead of molecular oxygen through a process called Anaerobic respiration A1 is created in the same way in which it is created during aerobic respiration There are many organisms which can use nitrate or sulfate as final electron acceptor but there are also bacteria which can use Fe3 or Mn3 as final electron acceptor 3 There are oxidative pathway which oxidize inorganic compounds like H2 NH3 NOZ39 S H28 and Fe2 instead of organic compounds to obtain electrons and energy These organisms are known as chemolithotrophs If they use C02 as carbon source then they are called Chemolithoautotrophs Assimilation of Nitrate Many bacteria can utilize inorganic form of nitrogen like nitrate as a sole source of nitrogen and reduce them to NH3 The ammonia is then incorporated in to glutamate and glutamine using the GSGOGAT system Glutamate and glutamine then act as source of amino groups for the other nitrogen containing compounds The enzymes involved in the assimilatory reduction of nitrate to ammonia are 1 cytoplasmic nitrate reductase which reduces nitrate to nitrite and 2 Cytoplasmic nitrite reductase which reduces nitrite to ammonia which is incorporated into glutamine via GS enzyme The electron donors involved can be NADH ferredoxin or avodoxin depending upon the the bacterium In E coli it is NADH 1 z I 2 quot03 262w K39 quot02 253w K39 mom 2e 2H who 2e392H 3 lEil up H20 H20 Gs Ig39lll 1 Gk m r quotm glu Fig 121 Assimilatory nitrate reduction This pathway is present in all bacteria that reduce nitrate to ammonia which is then incorporated into cell material The enzymes are found in T the cytosol and are not coupled to ATP formation Nitrate is reduced via twoelectron steps to nitrite nitroxyl hydoxylamine and ammonia The ammonia is incorporated into organic carbon f viii glutamine synthetase GS and the GOGAT enzyme or via glutamate dehydrogenase See Section 931 Enzymes 1 nitrate reductase 2 nitrite reductase Abbreviations glu glutamate akg a ketoglutarate Sulfate Assimilation Many bacteria can utilize inorganic sulfate as their sole source of sulfur The sulfate 804239 is rst reduced to sul de H28 and HS and then incorporated into cysteine In the rst step there is a formation of adenosine5 phospho sulfate APS a reaction catalyzed by the enzyme ATP sulfurylase In the second step APS is phosphorylated by APS kinase to form adenosine3 phosphate 5 phophosulfate PAPS The PAPS is then reduced by thioredoxin a sulfahydryl reductant to release AMP3 Phosphate and sul te SO3 Sul te is then reduced by NADPH to hydrogen sul de HZS is very toxic and doesn t accumulate in cell but is immediately incorporated into cysteine The sul de enzymatically displaces acetate in Oacetylserine to form cysteine f R u R mine llao fo Iro 50339 m lrO O39 Q 0 O O 0 PF Idenonlne 39 Pughulna 2P 20 Aquot A 3 2 H10 ADP mltmaur Pi I E amine II II Wm 4 R R Sl39lz O OH 52 so in 3quot 3w pimpledame shfmhte Omylaaine an Lcysuine Fig 122 Assimilatory sulfate reduction Enzymes 1 ATP sulfurylase 2 APS phosphokinase 3 PAPS 2 reductase 4 sul te reductase 5 Oacetylserine sulfhydrylase RSH2 is reduced thioredoxin Dissimilation of Nitrate and Sulfate Dissimilatory pathways use nitrate and sulfate as nal electron acceptor but do not incorporate these elements in cellular materials rather excrete them out Many facultative anaerobes utilize nitrate reduction pathway in the absence of oxygen while obligately anaerobic sulfate reducers use sulfate as nal electron acceptors Nitrate Dissimilation Generally this takes place in facultative anaerobic bacteria under low oxygen level It takes place in the membranes and the products can be nitrite ammonia or nitrogen gas When nitrate or nitrite is reduced to nitrogen gas or nitric or nitrous oxide the process is known as Denitri cation Denitri cation occurs in soil under anaerobic conditions for example in waterlogged soil Examples of denitrifying bacteria are Alcaligenes and Pseudomonas species Dissimilatory Sulfate Reduction Organisms like Desulfovibrio carry out anaerobic respiration during which electrons ow in the cell membrane to sulfate as a terminal electron acceptor reducing it to HZS The process also generates Ap Which is used for ATP synthesis via respiratory phosphorylation The ow of electron in this process is linked With utilization of lactate which is converted to pyruvate generating 4e that are used nally to reduce sulfate to sul de Several components are involved and the process spans across the membrane to the periplasm and back to the cytoplasm It also generates Ap Which is used for ATP synthesis Sulfate Dissimilation cylansm mm periphsm 2 mm 2mm 8quot S 2 co m ZuclylrSCnA 7 1 3 2 3 PP H30 9 2 may 2 ADP 1 ATP 2 mm 4 Fig 123 Pathway for dissimilamry sulfate rL ducnon in Dcsulowbno Enzymes 1 lacuna dehydmgcmse2 39 quot I 39 39 accmtekmnsc 5 Cytoplasmic hydmgcnnsc a pcrlplnsmi hydrogenase 7 ATP sulfurylase s pyruphnsphamse 9 APS reducmse l0 sul tc reducmsc Nitrogen Fixation Nitrogen xation is ecologically one of the most important process carried out by prokaryotes So far it has not been reported in eukaryotes The ammonia produced Via nitrogen xation is incorporated into cellular components using glutamine synthetase and glutamate synthase system Nitrogen xation takes place when N2 is the only source of nitrogen since the genes required for nitrogen xation are repressed by exogenous nitrogen supply other than nitrogen gas Industrial reduction of nitrogen is carried out by Haber process which requires 200 atm pressure and 800 C While prokaryotes carry out same process at atmospheric pressure and temperatures Nitrogen Fixing Systems Nitrogen xing prokaryotes can be divided into three distinct groups 1 Symbiotic legumes These includes organisms belonging to Rhizobium Bradyrhizobium and Azorhizobium which form symbiotic relationship with leguminous plants like soybeans clover alfalfa string beans and peas These bacteria infect the roots of the plants and stimulate nodule formation within which they x nitrogen Plants provide organic nutrients to the organism 2 Symbiotic nonlegumes There are nitrogen xing bacteria in symbiotic relationship with non legume plants The best example is that of water fern Azolla and nitrogen xing Cyanobacterium Anaebaena azollae 3 Non symbiotic nitrogen xation There are many free living soil and aquatic prokaryotes that can x atmospheric nitrogen The examples are Azotobacter Clostridium and certain species of Desulfovibrio the photosynthetic bacteria and cyanobacteria Certain archaea are also reported to x nitrogen The Mechanism of Nitrogen Fixation The enzyme that catalyzes conversion of nitrogen gas to ammonia is Nitrogenase Nitrogenase activity is strongly inhibited by the presence of oxygen therefore different prokaryotes employ different means to protect it from oxygen In most of the nitrogen xing bacteria nitrogenase is a Molybdenum containing enzyme that consists of two multimeric proteins 1 Molybdenumiron protein MoFe Protein or Dinitrogenase or Component 1 2 The second protein is called the Feprotein or Dinitrogenase reductase or Component 11 Both proteins contain FeS centers MoFe protein is a tetramer made up of four polypeptides XZBZ while Fe protein is a dimer yz made up of two polypeptides The Feprotein dimer contains single Fe4S4 cluster which is responsible for the reduction of MoFeprotein during nitrogen xation In Klebsiellapneumoniae 21 genes have been found to be responsible for the expression and regulation of nitrogenase enzyme They are called nif genes The Nitrogenase reaction The nitrogenase reaction is a series of reductions during which 05 mole of N2 and 4 moles of H are reduced to 1 mole of NH3 and 05 mole of H2 The overall reaction is 4e39 05N2 4H 8ATP gt NH3 05H2 8ADP 8Pi Since the oxidation state of N2 is 0 and the oxidation state of NH3 is 3 there is a need for three electrons per nitrogen atom A fourth electron is transferred to a proton to produce hydrogen gas The electrons are transferred one at a time in ATPdependent reaction from the Fe4S4 cluster in Feprotein to MoFeprotein to N2 The details of the electron pathway and the role of ATP is not clearly understood but it is known that two molecules of ATP are required per electron transferred Thus 16 moles of ATP are needed to convert one mole of nitrogen to two moles of ammonia During the reduction of nitrogen protons are used to make hydrogen gas and thus electrons and ATP are apparently wasted but in few organisms like Azotobacter the hydrogen gas is used to generate electrons for the nitrgenase The Nitrogen Fixation SAD 8Pi 4N melamiqu More maul Nz4 II I 4M Fem More protein NH3H2 SATP Fig 124 The nitrogenase reaction The enzyme system consists of two components Component I is called the molybdenum iron protein MoFe protein or dinitrogenase Component 1 is called the iron protein Fe protein or dinittogenase reductase Both of the proteins contain FeS centers A low potential reductant either fertodoxin or avodoxin reduces component II which transfers the electrons to component I Component I reduces N2 There is always some H2 produced ATP is required despite the fact that the overall reduction of N by ferredoxin or avodoxin is an exothermic reaction Lithotrophy Many prokaryotes derive energy from oxidizing inorganic compounds like H2 CO NH3 NOZ39 HZS S 8203239 or Fe This type of metabolism is known as Lithotrophy Table 121 Chemoautotrophs Bacterial group Typical species Electron Electron Carbon 3 donor acceptor source Product Hydrogenoxidizing Alcaiigenes eutropbus H1 0 C02 H20 Carbonmonoxide Pseudomonas C0 03 C0 C0 4 oxidizing aarboxydovorans carboxydobacteria Amoniumoxidizing Nitrosomonas empaea NH 01 C02 No Nitrite oxidizing Nitrobacter winogradskyi N0 02 0 N0 4 Sulfuroxidizing Tbiobaciuus tbiooxidans s szog 01 co2 501 4 Ironoxidizing Tbiobaci us ferrooxidans 39 Fe 02 C0 Fe3 Methanogenic Metbmobacterbtm H C0 C02 CH4 tbennautotropbiam Aoetogenic Acetobacterimn woodii H2 C02 C01 CHJCOO H39 Ammonia oxidizers The bacteria that oxidize ammonia are called nitrifiers They include Nitrosomonas Nitrococcus Nitrosospira Nitrosolobus and Nitrosovibrio They are all chemolithoautotrophs that acquire carbon by assimilating C02 Via calVin cycle Nitrosomonas oxidizes ammonia to nitrite while Nitrobacter oxidize nitrite to nitrate Both together are responsible for major portion of conversion of ammonia to nitrate a process known as Nitri cation Sulfur oxidizers The sulfur oxidizers are of two typesthe photosynthetic sulfur oxidizers and the nonphotosynthetic sulfur oxidizers The acidophilic sulfur oxidizing bacteria can be isolated from sulfur and coal mines that produce sulfuric acid The example is T hiobacillus thiooxidans which can grow at pH of 10 with optimum of 2 0r 3 The sulfur compound commonly used by these bacteria include hydrogen sul de elemental sulfur and thiosulfate 8203239 All of them are oxidized to sulfate Iron oxidizing bacteria Few bacteria derive energy from oxidizing ferrous ion to ferric ion Most of these are acidophilic sulfur oxidizers which oxidize sul de to sulfuric acid The example is T hiobacillus ferroxidans which can obtain energy either from ferrous ion or from sulfur compounds Bacterial Physiology HSCI 5607 Chapter 11 Cell Wall and Capsule Biosynthesis Introduction The cell wall and capsule are important external cell structures which are biosynthesized in the cytoplasm and assembled at the site where they are located The subunits of the cell wall and capsule are synthesized as a watersoluble precursors in the cytoplasm and are transported across the cell membrane to the site of their assembly There is no source of energy available for their assembly outside the cell membrane but this energy is obtained by means of speci c biochemical reactions taking place outside the membrane The major constituent of grampositive bacterial cell wall is peptidoglycan while in gramnegative bacterial cell wall also has an outer membrane which is made up of lipopolysaccharides Bacterial capsules are usually made up of polysaccharides with few exceptions where they are made up of polypeptides cell outer mgmbrane peptidoglycan membrane Peptidoglycan Structure Layers of peptidoglycan surround the bacterial cells and confer rigidity and shape to the cell wall Chemically peptidoglycan is made up of heteropolymer of glycan crosslinked by amino acids The heteropolymer of glycan is made up of alternating residues of Nacetylmuramic acid and NacetylGlucosamine linked by glycosidic B14 linkage between the carbon 1 of MurNAc and carbon 4 of GlcNAc MurNAc is chemically synthesized by modi cation of GlcNAc at 3rd carbon with the addition of lactyl group Moreover a tetra peptide is also attached to MurNAc The tetrapeptide consists of LalanylyDglutamylLR3D alanine R3 varies from species to species Gramnegative bacteria generally have meso diaminopimelic acid DAP while in gram positive bacteria there is much more variability CHZOH CHIOH o 0 0H6 0 o M 1739 CH CO Ifquot I CH3 Tim CH3 Lallnine CHCHJ 390 1 Dglunmate EHCOOH Clmzh CO Wquot LR CIH X C0 15H Dallnlne ElCH3 oou CH3 Lalanine CHZ CHz OH Lhomourine CH2 CH1 NH2 Ldlnmlnobutyric acid CHECHz COOH Lgluumic acid Cth CHI NHZ Lornithine CH23 CH2 NH2 LIysine coon CH2 CHNH LLDAP and mesaDA 2 Cross links The tetrapeptide chains are crosslinked to each other by peptide bonds between the carboxyl group of the terminal Dalanine of one tetrapeptide and amino group of R3 of another tetrapeptide Sometime this linkage is direct peptide While in some cases they are linked with one or more amino acid bridge For example in Staphylococcus aureus ve glycine bridge and three Lalanine and one Lthreonine in Micrococcus roseus link the tetrapeptides Synthesis There are several stages of peptidoglycan synthesis 1 Synthesis of UDPaminosugar derivatives in the cytoplasm 2 The transfer of amino sugars to the lipid carrier in the membrane which carries the precursors across the membrane 3 Polymerization of the peptidoglycan 4 Transpeptidation reaction to crosslink the tetrapeptides of the peptidoglycan GM CDGH GM coon NH ammo and COOH hndsr Synthesis of UDP Derivatives Both the precursor aminosugars are made from fructose6P Formation of UDPNAcetylGlucosamine In the rst step glutamine donates amino group to C2 and forms glucosamine6P In the second reaction acetylCoA donates acetyl group to the amino group to form Nacetylglucosamine6P which is isomerized in the next step to form NAcGlclP NAcGlclP attacks UTP to displace PP to form UDP NAcGlc This reaction is driven to completion by the hydrolysis of pyrophosphate Some of the UDPNAcGlc is used as a precursor for the synthesis of UDPNAcMuramic acid UDPNAcMur and some serve as a precursor to the peptidoglycan Formation of UDPNacetylmuramic acid UDPNAcMur is formed from UDPNAcGlc by addition of lactyl group to the 3rd carbon The OH group of UDPNAcGlc at 3rd carbon attacks the alpha carbon of the phosphoenol pyruvate to displace P and forms UDPNAcGlc3enoylpyruvylether This product is reduced by NADH dependent next reaction to UDPNAcMur The UDPNAcMur is converted into UDPNAcMur penta peptide by sequential addition of ve amino acids Lalanine D glutamate LR3 and the dipeptide LalanylLalanine catalyzed by different ATP dependent enzymes UDPNAcMur is then transferred to the membrane located lipid carrier known as bactoprenol P or Undecaprenyl phosphate Synthesis of UDP derivatives EH10 fl10 CH2 CFO I HNHZ CoASH o gumMl CHCSC0A f HO C H HO C H l 01 H e 0H glummm H I OH quot0 0H 1 4 an 2 TH H li OH I C CH10 CH20 EH Iructose 6P 39 quotquotinquotquot NIcetylglucoum ine P 3 CHZDH CH10H CHZOH o o m7 DH 0 P 01 H0 039 UMY E PEP no O UMP S UTP Ho NH 4 NH H2CC C00H I I 9 0 90 o CH3 CH CH 39 nun N u 1 n 3moylpyruvymhu NADPHH s NADP cup 0 680quot Lzluun 0 0 D1an H0 O UMP 3 N o Dunnyl Dalinmc CH caco I HO 0 UMP MT I I C quotH LIflnnmc ulcrcmman I cu co Dglumm I CH3 111 um elylmurulnlc neld D c I den 1 in UDPmurlmyhpenlipeplld Fig IA Synthesis of N acerylglucosaminc and Nacetylmuramylvpenlapeptide Enzymes glutzminez frucmscSphosphalc aminouansferase 2 glucosaminc phosphate rransacctylase 33M 4m M 39 39 39 39 Qt n The UDP N 1 mm Dgluramate LRg and Dzlanine Dalaninc by separate enzymes Transfer of UDP derivative to the membrane carrier The lipid carrier bactoprenol not only serve as a carrier for peptidoglycan precursors but also serve as carrier for the other cell wall components eg lipopolysaccharide and teichoic acids The UDP derivative diffuses to the membrane where the bactoprenolP attacks the UDPNAcMur Pentapep and displaces UMP to form Lipid PPNAcMur Pentapep Next NAcGlc is transferred from UDPNAcGlc to NAcMur on the lipid carrier displacing the UDP The lipid disaccharide derivative moves to the other side of the membrane facilitated by some unidenti ed proteins On the other side of the membrane the lipiddisaccharide is transferred to the growing end of the glycan chain with the displacement of the lipidPP of the growing end This reaction is catalyzed by membrane bound enzyme transglycosylase The lipidPP released is hydrolyzed to form lipidP which is recycled for the continuation of the growth while the energy thus liberated is utilized to drive glycosylation reaction Extension of Glycan Chain cytoplasm UDP M L UMP UDPG M 2 l UDP G hld cell membrane Plipid PPlipid Pi PPnpid 39 39 H20 lip PP I lipPP 397 lipPP d G LIA G 1gtIipPP IYI G ISIA G IYI G 1H Cross linking of the Peptides As there is no energy available for the formation of the peptide bond outside the membrane the problem of energy is solved by the transpeptidation reaction During this reaction the amino group of the R3 attacks the carbonyl carbon of the peptide bond which is holding the two alanine residues and displaces the terminal alanine with a formation of a new peptide bond between the two peptides This reaction is the target of antibiotic penicillin Bacterial membranes have several penicillin binding proteins PBPs in their membrane These proteins are bifunctional and perform both the transpeptidase and transglycosylation reaction
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