SYNTHESIS OF DNA.RNA.AND PROTEIN 9 of the cell,the nascent chromosor es separate from the membrane but tinueto move toward e ce by a sun ism.Chromosoma wall that will eparate the two offspring(seeTermination of DNA Replication and ning in Cha hin dna i d in tuo RNA polym nrst loc nning allowing RNA polymerase to tran ribe RNA from the DNA template.Before the RNA-called n N(mRNA) -is completely transcribed,a ribosome will As already noted.the ribo ome contains of two subunits.30S and 50S eacl acid RNA) translates mRNA into a triplet cod or transfer RNA (tRNA)molecule acid RNA a charged tRNA molecule. ue ca codon will base-pair with the codo Nascent peptide o888 incoming charged tRNA H R 51 RNA polym AUG Nascent mRNA Transcription"Bubble Fig.1-8.Sequence of events involved in transcription and translation
SYNTHESIS OF DNA, RNA, AND PROTEIN 9 (Fig. 1-7). Note that the chromosome appears attached to the cell membrane as the daughter chromosomes begin to separate. At some point about midway to the ends of the cell, the nascent chromosomes separate from the membrane but continue to move toward the cell poles by a still undefined mechanism. Chromosomal segregation into pre-daughter cells must occur before the cell completes construction of the crosswall that will separate the two offspring (see “Termination of DNA Replication and Chromosome Partitioning” in Chapter 2). The genetic information contained within DNA is processed in two steps to produce various proteins. Protein synthesis (translation) is depicted in Figure 1-8. The enzyme RNA polymerase (DNA-dependent RNA polymerase) first locates the beginning of a gene (promoter). This area of the chromosome then undergoes a localized unwinding, allowing RNA polymerase to transcribe RNA from the DNA template. Before the RNA — called messenger RNA (mRNA) — is completely transcribed, a ribosome will attach to the beginning of the message. As already noted, the ribosome contains of two subunits, 30S and 50S, each composed of special ribosomal proteins and ribosomal ribonucleic acids (rRNA). rRNA molecules do not, by themselves, code for any protein but form the architectural scaffolding that directs assembly of the proteins to form a ribosome. The ribosome translates mRNA into protein by reading three nucleotides (known as a triplet codon) as a specific amino acid. Each amino acid used by the ribosome must first be attached to an adaptor or transfer RNA (tRNA) molecule specific for that amino acid. tRNA containing an attached amino acid is referred to as a charged tRNA molecule. A part of the tRNA molecule called the anticodon will base-pair with the codon in mRNA. 5′ AUG C C A O C O CN H R C O CN H R C O CN H H R 5′ C C A 5′ C O CN H H R Nascent mRNA incoming charged tRNA RNA polymerase 3′ Transcription "Bubble" Nascent peptide Fig. 1-8. Sequence of events involved in transcription and translation
10 INTRODUCTION TO MICROBIAL PHYSIOLOGY amino acids. At this point.the tw amin are attache d to on RNA while the her tRN move alo m g the me essage.at which point mple e protein has be with the C mina amin acid.Als note that the riboso me b e at t 5'end of the m strand ing the is rea by RN the5end.this oesn't referto the strand that template fo RNA polymer e is the rep transcription.and translation are discussed in Chapter 2. Metabolic and Genetic Regulation a cell togrow efficienly.all the basic builing blocks and all the mac romolecules the o be correct prop hich omple of metabolic and genetic regulation are 1.Feedback inhibition of enzyme activity (metabolic regulation) 2.Repression or induction of enzyme synthesis(genetic regulation) )e心 on al p oa pathway d in excessive production of intermed te B results in the by feedback inhibition on,an excess intracellu on t en This action s ge repr or this contro rent whe conside if the ripti the biosyn cont such as c 1-).Different organisms may employ quit on,and indu ton to regulate in greater detail
10 INTRODUCTION TO MICROBIAL PHYSIOLOGY When two such charged tRNA molecules simultaneously occupy adjacent sites on the ribosome, the ribosome catalyzes the formation of a peptide bond between the two amino acids. At this point, the two amino acids are attached to one tRNA while the other tRNA is uncharged and eventually released from the ribosome. The ribosome is then free to move along the message to the next codon. The process continues until the ribosome reaches the end of the message, at which point a complete protein has been formed. Notice that synthesis of the protein begins with the N-terminal amino acid and finishes with the C-terminal amino acid. Also note that the ribosome begins translating at the 5 end of the mRNA while the DNA strand encoding the mRNA is read by RNA polymerase starting at the 3 end. Although the beginning of a gene is usually called the 5 end, this doesn’t refer to the strand that is actually serving as a template for RNA polymerase. It refers to the complementary DNA strand whose sequence is the same as the mRNA (except for containing T instead of U). The details of replication, transcription, and translation are discussed in Chapter 2. Metabolic and Genetic Regulation For a cell to grow efficiently, all the basic building blocks and all the macromolecules derived from them have to be produced in the correct proportions. With complex metabolic pathways, it is important to understand the manner by which a microbial cell regulates the production and concentration of each product. Two common mechanisms of metabolic and genetic regulation are 1. Feedback inhibition of enzyme activity (metabolic regulation) 2. Repression or induction of enzyme synthesis (genetic regulation) In feedback inhibition, the activity of an enzyme already present in the cell is inhibited by the end product of the reaction. In genetic repression, the synthesis of an enzyme (see previous discussion of transcription and translation) is inhibited by the end product of the reaction. Induction is similar except the substrate of a pathway stimulates synthesis of the enzyme. Hypothetical pathways illustrating these concepts are presented in Figure 1-9. In Figure 1-9a, excessive production of intermediate B results in the inhibition of enzyme 1 activity, a phenomenon known as feedback or end-product inhibition. Likewise, an excess of end-product C may inhibit the activity of enzyme 1 by feedback inhibition. In contrast to feedback inhibition, an excess intracellular concentration of endproduct C may cause the cell to stop synthesizing enzyme 1, usually by inhibiting transcription of the genes encoding the biosynthetic enzymes (Fig. 1-9b). This action is referred to as genetic repression. The logic of this control is apparent when considering amino acid biosynthesis. If the cell has more than enough of a given amino acid, that amino acid will activate a repressor protein, which then blocks any further transcription of the biosynthesis genes. In contrast, substrates such as carbohydrates can stimulate the transcription of genes whose protein products consume that carbohydrate. This genetic process is called induction (Fig. 1-9c). Different organisms may employ quite different combinations of feedback inhibition, repression, and induction to regulate a metabolic pathway. In Chapter 5, these and other regulatory mechanisms are discussed in greater detail.
MICROBIAL GENETICS 11 Feedback Inhibition SeA EndproductC (a) Gene Repression Transcription SubstrateA Translation EnzI Enzll Endproduct C (b) Induction Gene Substrate A EnzI Translation anscription Intermediate B Endproduct C nd-product me e in h hypothetical pathway.Arrow indicates activation,line with cross. MICROBIAL GENETICS Having just outlined the processes of transcription,translation,and replication,it is now to define several genetic terms.The gene e may be detined saheritable turn deter the hase nce in a the of bases in an RNA molecule specifies the sequence of amino acid in porate is,the structural appearance and physiological properties of an organism-is referred ene can exist in different forms as a result of nucleotide sequence changes.The e mative gene forms are referred to as alleles.Genetic materia is not ab ut ca change or mutal nge own or wild-type alleles.Spontaneous mutations are thought to arise during replication
MICROBIAL GENETICS 11 Substrate A Intermediate B Endproduct C Feedback Inhibition Repression Substrate A Intermediate B Endproduct C Gene Enz I Transcription Translation Enz II Induction Substrate A Intermediate B Endproduct C Enz I Enz II Gene Transcription Translation (a) (b) (c) Fig. 1-9. Diagrammatic presentation of feedback inhibition of enzyme activity and end-product repression of enzyme synthesis. a, b and c are chemical intermediates in the hypothetical pathway. Arrow indicates activation, line with cross indicates inhibition. MICROBIAL GENETICS Having just outlined the processes of transcription, translation, and replication, it is now possible to define several genetic terms. The gene may be defined as a heritable unit of function composed of a specific sequence of purine and pyrimidine bases, which in turn determines the base sequence in an RNA molecule, and, of course, the sequence of bases in an RNA molecule specifies the sequence of amino acids incorporated into a polypeptide chain. The genotype of an organism is the sum total of all of the hereditary units of genes. The observed expression of the genetic determinants — that is, the structural appearance and physiological properties of an organism — is referred to as its phenotype. An individual gene can exist in different forms as a result of nucleotide sequence changes. These alternative gene forms are referred to as alleles. Genetic material is not absolutely stable but can change or mutate. The process of change is known as mutagenesis. Altered genes are referred to as mutant alleles in contrast to the normal or wild-type alleles. Spontaneous mutations are thought to arise during replication
INTRODUCTON TO MICROBIAL PHYSIOLOGY environm ntal influ ces n the form of x rays uraviolet (UV)rays. the ger ma ng the frequency with which mutations occur are referred to as mutagen ing alteration Since bacterial cells are ha d.mutants are becau re li The use of mutants has been a tremendous tool in the study of most,if not all. biochemical proce Genes are s bya th sed followed by an uppercase letter toindicate different arg gene gene is he indi cated by lo c letters (e.g., At this point.we need to expos ea common mistake made by many aspiring microbial genticists conceming the interpretation of mutant pheno Organisms such source.and a carbon source such as glucose or lactose pecause they can use the carbor skeleton of glu ose to synthe size all the buildng blocksn macromolec % and so forth.A mutant defective in one of the genes ynthesize a huildin block will require as a supplement in the minimal m ium (e.g oundsas carbon ource Howev m ion in a carbon source utilizatic gene (e.g.lac) s not m an I grow on lactose). positions period of time required to trans r by conjugation (10 CHEMICAL SYNTHESIS Chemical Composition cell (the ination of all pro hsfeat wil model cell.The total weight of an average cell is 9.5x1 gwi油 of the cell)contrib ng6.7 10 dry weight thus2.8× RNA (16.7%)transfer RNA (messenser RNA (0)DNA (3.1).lpids (91
12 INTRODUCTION TO MICROBIAL PHYSIOLOGY repair, and recombination of DNA as a result of errors made by the enzymes involved in DNA metabolism. Mutations may be increased by the activity of a number of environmental influences. Radiation in the form of X rays, ultraviolet (UV) rays, or cosmic rays may affect the chemical structure of the gene. A variety of chemicals may also give rise to mutations. Physical, chemical, or physicochemical agents capable of increasing the frequency with which mutations occur are referred to as mutagens. The resulting alterations are induced mutations in contrast to spontaneous mutations, which appear to occur at some constant frequency in the absence of intentionally applied external influences. Since bacterial cells are haploid, mutants are usually easier to recognize because the altered character is more likely to be expressed, particularly if the environment is favorable to mutant development. The use of mutants has been a tremendous tool in the study of most, if not all, biochemical processes. Genes are usually designated by a three-letter code based on their function. For example, genes involved in the biosynthesis of the amino acid arginine are called arg followed by an uppercase letter to indicate different arg genes (e.g., argA, argB). A gene is always indicated by lowercase italic letters (e.g., arg), whereas an uppercase letter in the first position (e.g., ArgA) indicates the gene product. At this point, we need to expose a common mistake made by many aspiring microbial geneticists concerning the interpretation of mutant phenotypes. Organisms such as E. coli can grow on basic minimal media containing only salts, ammonia as a nitrogen source, and a carbon source such as glucose or lactose because they can use the carbon skeleton of glucose to synthesize all the building blocks necessary for macromolecular synthesis. The building blocks include amino acids, purines, pyrimidines, cofactors, and so forth. A mutant defective in one of the genes necessary to synthesize a building block will require that building block as a supplement in the minimal medium (e.g., an arg mutant will require arginine in order to grow). Microorganisms also have an amazing capacity to catabolically use many different compounds as carbon sources. However, a mutation in a carbon source utilization gene (e.g., lac) does not mean it requires that carbon source. It means the mutant will not grow on medium containing that carbon source if it is the only carbon source available (e.g., a lac mutant will not grow on lactose). The chromosome of our reference cell, E. coli, is 4,639,221 base pairs long. Gene positions on this map can be given in base pairs starting from the gene thrL, or in minutes based on the period of time required to transfer the chromosome from one cell to another by conjugation (100-minute map with thrL at 0). CHEMICAL SYNTHESIS Chemical Composition Our paradigm cell (the gram-negative cell E. coli) can reproduce in a minimal glucose medium once every 40 minutes. As we proceed through a detailed examination of all the processes involved, the amazing nature of this feat will become increasingly obvious. It is useful to discuss the basic chemical composition of our model cell. The total weight of an average cell is 9.5 × 10−13 g, with water (at 70% of the cell) contributing 6.7 × 10−13 g. The total dry weight is thus 2.8 × 10−13 g. The components that form the dry weight include protein (55%), ribosomal RNA (16.7%), transfer RNA (3%), messenger RNA (0.8%), DNA (3.1%), lipids (9.1%)
CHEMICAL SYNTHESIS 13 volume being approximately this ons of the ighs ely I of yisurndrd plid. chr 2000different varieties.As you might gather from these figures,the bacterial cell is The metabolites come fromucose or some other.The cataboli vell as mov ment.transport.and so on.Figure 1-10 is a composite diagram of majo (seeFi-10).Three are produced bythe pentose phosphate patha Fiure- illustrates how thesecom unds are siphoned off from the c tabolic pathways and Sufor the but this figure presents an integrated picture of cell metabolism. Energy Another mission of carbohydrate metabolism is the production of energy.The most compound phosphoenol pyruvate)toade nosine diphosphate(ADP)duri ide the used to drive a membrane-bound ATP hydrolase complex to produce ATP from ADP iand chemical gradient the protor rom chemical th e-is passed from one member of the cytochrome chain to another,the energy released
CHEMICAL SYNTHESIS 13 lipopolysaccharides (3.4%), peptidoglycans (2.5%), building block metabolites, vitamins (2.9%), and inorganic ions (1.0%). It is interesting to note that the periplasmic space forms a full 30% of the cell volume, with total cell volume being approximately 9 × 10−13 ml (0.9 femptoliters). An appreciation for the dimensions of the cell follows this simple example. One teaspoon of packed E. coli weighs approximately 1 gram (wet weight). This comprises about one trillion cells — more than 100 times the human population of the planet. When calculating the concentration of a compound within the cell, it is useful to remember that there are 3 to 4 microliters of water per 1 milligram of dry weight. Our reference cell, although considered haploid, will contain two copies of the chromosome when growing rapidly. It will also contain 18,700 ribosomes and a little over 2 million total molecules of protein, of which there are between 1000 and 2000 different varieties. As you might gather from these figures, the bacterial cell is extremely complex. However, the cell has developed an elegant strategy for molecular economy that we still struggle to understand. Some of what we have learned is discussed throughout the remaining chapters. In just 40 minutes an E. coli cell can make a perfect copy of itself growing on nothing more than glucose, ammonia, and some salts. How this is accomplished seems almost miraculous! All of the biochemical pathways needed to copy a cell originate from just 13 precursor metabolites. To understand microbial physiology, you must first discover what the 13 metabolites are and where they come from. The metabolites come from glucose or some other carbohydrate. The catabolic dissimilation of glucose not only produces them but also generates the energy needed for all the work carried out by the cell. This work includes biosynthetic reactions as well as movement, transport, and so on. Figure 1-10 is a composite diagram of major pathways for carbohydrate metabolism with the 13 metabolites highlighted. Most of them are produced by the Embden-Meyerhof route and the tricarboxylic acid cycle (see Fig. 1-10). Three are produced by the pentose phosphate pathway. Figure 1-11 illustrates how these compounds are siphoned off from the catabolic pathways and used as starting material for the many amino acids, nucleic acid bases, and cofactors that must be produced. Subsequent chapters deal with the specifics of each pathway, but this figure presents an integrated picture of cell metabolism. Energy Another mission of carbohydrate metabolism is the production of energy. The most universal energy transfer compound found in living cells is adenosine triphosphate (ATP) (Fig. 1-12). The cell can generate ATP in two ways: (1) by substrate-level phosphorylation in which a high-energy phosphate is transferred from a chemical compound (e.g., phosphoenol pyruvate) to adenosine diphosphate (ADP) during the course of carbohydrate catabolism; or (2) by oxidative phosphorylation in which the energy from an electrical and chemical gradient formed across the cell membrane is used to drive a membrane-bound ATP hydrolase complex to produce ATP from ADP and inorganic phosphate. The generation of an electrical and chemical gradient (collectively called the proton motive force) across the cell membrane requires a complex set of reactions in which H+ and e− are transferred from chemical intermediates of the Embden-Meyerhof and TCA cycles to a series of membrane-associated proteins called cytochromes. As the e− is passed from one member of the cytochrome chain to another, the energy released