英文摘要微生物

C5 CONTROL OF GENE EXPRESSION A1 The microbial world Key Notes What is a microbe? The word microbe (microorganism) is used to describe an organism that is so small that, normally, it cannot be seen without the aid of a microscope. Viruses, bacteria, Archaea, fungi, and protista are all included in this category. Prokaryotes and eukaryotes Microbes are found in all three major kingdoms of life: the bacteria, the Archaea and the Eukarya. The Bacteria and the Archaea are prokaryotes, while all other microbes are eukaryotes. There are many differences between prokaryote and eukaryote cells, the major distinction being the presence of a nucleus and other membrane-bound organelles in eukaryotes. The importance of microbiology Microbes are essential to life. Among their many roles, they are necessary for geochemical cycling and soil fertility. They are used to produce food as well as pharmaceutical and industrial compounds. On the negative side, they are the cause of many diseases of plants and animals and are responsible for the spoilage of food. Finally, microbes are used extensively in research laboratories to investigate cellular process. B1 Heterotrophic Pathways Key Notes High-energy compounds Heterotrophy refers to the breaking down of organic molecules to obtain energy. This energy is generally stored in the from of high-energy compounds, such as APT and NAD+. The formation of such compounds relies on balanced redox reactions that generate organic molecules containing oxygen and phosphate groups. Glycolysis Glycolysis is a cytoplasmic pathway that is used by most microorganisms to break down sugars (such as glucose and fructose ) to pyruvate, yielding two molecules of ATP. Pyruvate then enters the citric acid cycle, and its utilization through this pathway yields energy-rich compounds including APT and NADH. Alternatives to glycolysis There are a number of hexose monophosphate pathways (including the Enter-Douder off pathway ,the phosphoketolase pathway and the pentose phosphate pathway )that can be used as alternatives to glycolysis for the oxidation if glucose. These pathways yield less APT per molecule of glucose than glycolysis, but they generate important metabolic intermediates including NADPH and pentose sugars for nucleic acid synthesis. Citric acid cycle and respiration The citric acid cycle occurs in the cytoplasm if aerobic bacteria and in the mitochondria of aerobic eukaryotes. Respiration is the complete oxidation of an organic substrate to carbon dioxide and water. It requires an external electron acceptor, usually oxygen, and results in the formation of lager amounts of APT. For each glucose molecule oxidized by the citric acids cycle, 12 molecules of ATP are generated. Important intermediates for fatty acid synthesis, nucleotide synthesis and amino acid synthesis are also generated by the citric acid cycle. Fermentation Fermentation is the incomplete oxidation of an organic substrate and it occurs under anaerobic conditions. Energy yields from fermentation are lower than comparative yields from respiration. The products of incomplete oxidation can include pyruvate, lactate, formate and ethanol. B2 Electron transport, oxidative phosphorylation and β-oxidation of fatty acids Key notes Electron transport Electron transport is used to create a proton motive force (PMF) across membranes. This PMF is used by all microorganisms to generate APT via a membrane bound ATPase. The Archaea and Bacteria also use PMF to drive the movement of flagellae, allow transport of charged substrates across membranes and maintain their osmotic potential. In eukaryotic microbes, PMF is established across the inner membrane of the mitochondrion. In the electron transport chain, a series of balanced oxidation and reduction reactions drives the movement of electrons through the carrier series from NADH to oxygen. During this process energy is released and ATP is synthesized. Anapleurotic Pathways Lost intermediates from glycolysis and the citric acid cycle are replenished by anapleurotic reactions, where carbon dioxide is fixed into three-carbon compounds by carboxylation reactions. Glyoxalate cycle Some substrates that microbes can utilize as carbon sources, for example the two-carbon compound acetate, can lead to the depletion of citric acid cycle intermediates. Reactions that result in the loss of CO2 during the cycle can be avoided by using the glyoxalate cycle. Fatty acid oxidation Fatty acids can be used as substrates by microorganisms through the fatty acid or beta oxidation pathway. This is the mitochondria of eukaryotes and the cytoplasm of prokaryotes. Anaerobic respiration Many microbes live in low or no oxygen environments. Alternative electron acceptors, such as nitrate and sulfate, can be utilized instead of oxygen by these organisms to complete the electron transport chain. B3 Autotrophic Reactions Key Notes Chemolithotrophy Autotrophic microorganisms can survive in the absence of organic carbon sources by fixing atmospheric or dissolved CO2 to from carbohydrates. Chemolithotrophs have the ability to fix CO2 using the Calvin cycle, and the energy required to derive the reactions comes from the oxidation of inorganic substrates such as ammonia. Photosynthesis Photosynthesis can be divided into two sets of reactions, those that are light-dependent (light reactions) and those that are light-independent (dark reactions). The light reactions convert light into chemical energy through the synthesis of ATP, which is then used to drive the Calvin cycle (dark reactions). Photosynthesis may be described as oxygenic if oxygen is generated (as in the cyanobacteria and the photosynthetic eukaryotes) or as anoxygenic if it is not (as in the green and purple bacteria). the light reaction can be driven by photosystem I and II in eukaryotes, but may only be driven by photosystem I in some Prokaryotes. Light reactions in bacterial photosynthesis Photosynthetic green and purple bacteria contain chlorophyll A and B, and carry out anoxygenic photosynthesis that utilizes only photosystem I. Light reactions in eukaryotic photosynthesis In eukaryotic, photosynthesis occurs in the chloroplasts, and involves photosystem I. and II. The light-dependent reactions generate NADPH+H+, and the resulting proton gradient is uxed to generate ATP by non-cyclic phosphorylation. Dark reactions in eukaryotic photosynthesis The dark (light-independent) reactions of photosynthesis are called the Calvin cycle and use the energy generated from light-dependent reactions to synthesize carbohydrates from CO2 to H2O. B4 Biosynthesis Pathways Key Notes Carbohydrates Carbohydrates are synthesized from precursors by a process termed gluconeogenesis. This pathway is almost the reverse of glucolysis, except that three irreversible glycolytic enzymes are replaced by three synthetic enzymes specific to this pathway. Amino acids Some bacteria can fix atmospheric nitrogen when fixed nitrogen sources (nitrate, nitrite or ammonia) are not available. All other microorganisms are use fixed nitrogen. Only ammonia can be incorporated directly to form amino acids; nitrate and nitrite must be reduced to ammonia first. There are five amino acid families from which 20 different amino acids are synthesized. Nucleic acids Nucleic acids are made of nucleotides, which are cyclic nitrogen-containing compounds. Purines are bicylic, while pyrimidines have a single ring. When linked to a phosphorylated pentose sugar they are termed nucleosides. They are the building blocks of DNA and RNA. Lipids Lipids are synthesized from fatty acids. They are long chain molecules of around 18 carbon atoms, and they may be saturated (contain no double bonds ) or unsaturated (contain one or more double bonds). C1 STRUCTURE AND ORGANIZATION OF DNA Key Notes DNA structure DNA is made up of deoxyribonucleotides joined by 3’,5’-phosphodiester bonds between the deoxyribose sugars. One of four nitrogenous bases, adenine (A), guanine (G), cytosine(C), or thymine (T), is attached to the sugar. Two strands of DNA are held together by specific hydrogen bonding between A and T and G and C to from a right –handed DNA helix. The specific pairing of A:T and G:C is called complementary base-pairing. DNA conformation Most DNA in the cell is in the B-form, which is a right-handed helix with 10 base pairs per turn. Other conformations include the A-form and the Z-form. DNA measurement and description DNA can be measured by weigh, in Daltons; or by length, in micrometers; or number of base pairs. The sequence of the DNA may be described as a sequence of the bases in a 5’-3’ direction; for example, 5’-AGCTTATTCCG-3’. DNA packaging DNA is negatively supercoiled, which places the molecule under torsion and reduces its volume. In prokaryotes, DNA is packaged with proteins and polyamines to form a nucleoid. In eukaryotes, DNA is wound round histone proteins to form nucleosomes which are further twisted to form 30 nm chromatin fibers. At cell division the chromatin becomes condensely packed to form the metaphase chromosomes. Chromosomes Most Bacteria and Archaea have only one chromosome but there are notable exceptions. A cell with only one copy of each chromosome is called haploid. Eukaryotes are typically diploid, having two copies of each chromosome, one from each parent; however many eukaryotic micro-organisms may exist for most of their life cycle in a haploid state. C2 DNA REPLICATION Key Notes Overview DNA replication is the synthesis of new strands of DNA using the original DNA strands as templates. Complementary base-pairing between the bases on the precursor deoxyribonucleotide triphosphates and the exposed bases on the DNA molecule is responsible for the selection of the correct nucleotide to insert into the growing strand. DNA polymerase is the enzyme that forms the phosphodiester bonds. The replication bubble Initiation of DNA replication occurs at one site, oric on the bacterial chromosome. The helix is opened by a DNA helicase to expose the bases, and DNA synthesis starts on both strands. Replication is bidirectional forming a pair of replication forks that move around the chromosome until they meet at a termination site. Topoisomerases remove the positive supercoiling created by the unwinding of the helix for replication. DNA polymerase DNA polymerases synthesize DNA in a 5→3 direction. They also have exonuclease activity which can remove mismatched bases (proof-reading). Because DNA polymerase can only add a nucleotide to an existing 3’-OH group two problems are created: (i) a primer is required to start DNA synthesis, and (ii) one strand must be synthesized in small fragments rather than continuously. Primers The primers to initiate DNA synthesis are short stretches of RNA synthesized by a RNA polymerase called primase. Leading and lagging strands The strand of DNA synthesized continuously is called the leading strand. The other strand (the lagging strand) is synthesized as a series of short fragments called Okazaki fragments. A complex of proteins called a primosome is responsible for the repeated initiation of DNA synthesis on this strand. Eukaryote replication The main differences between prokaryote and eukaryote replication are that in eukaryotes: (i) there are multiple origins of replication on one chromosome; (ii) different polymerases are used for the leading and lagging strand; (iii) a telomerase is required to replenish the telomers at the ends of the chromosome. Rolling circle replication Replication of DNA by rolling circle mechanism produces concatemers of DNA that are essential for the life-cycle of some bacteriophage and viruses. C3 RNA MOLECULES IN THE CELL Key Notes Structure of RNA RNA is a polymer of ribonucleotides with a similar structure to DNA except that it contains a ribose sugar instead of deoxyribose and uracil instead of thymine. RNA is normally single-stranded but can form secondary structures by base-pairing within the molecule. RNA molecules in the cell RNA molecules in the cell can be split into two groups. Comparatively long-lives stable RNAs have a number of different structural and functional roles in the cell including ribosomeal (rRNAs) and transfer RNAs (tRNAs). Messenger RNA (mRNA) molecules that act as templates for protein synthesis have a much shorter life. Catalytic RNA molecules Some RNA molecules have enzymatic activity, these are known as ribosomes. The most common of these are self-splicing introns found in a range of microbes. C4 TRANSCRIPTION Key Notes Overview Transcription is the synthesis of messenger RNA (mRNA) and stable RNA molecules from a DNA template by RNA polymerase, using ribonucleoside triphosphates as precursors. Each mRNA carries the sequence of one gene or in the case of prokaryote operons, a number of genes. Regulation of gene expression may occur at the level of transcription. DNA-dependent RNA polymerases Bacterial cells contain only one RNA polymerase whereas eukaryotes have at least three. In Bacteria a holoenzyme consisting of five subunits (α2, β, β’, δ) is responsible for accurate initiation of transcription. The sigma (δ) subunit (factor) is released to give the core enzyme (α2, β, β’) which is responsible for elongation. Stages of transcription There are three stages of transcription: initiation, which involves the binding of RNA polymerase to promoter sequences in the DNA that signal the start site for transcription; elongation, which is the synthesis by RNA polymerase of a sequence of ribonucleotides that is complementary to the sequence of one of the strands of the DNA ; and termination of RNA synthesis at the end of the gene or the operon. Promoters Promoters consist of conserved sequences necessary for the initiation of transcription. There are many different types of promoters. Many E.coli promoters contain two conserved regions at-35 and –10 bases before the start site for transcription. The first transcribed base is normally a purine. Termination There are several types of terminator sequence in E.coli. Some require a protein factor, rho (ρ) ,for accurate termination of transcription of transcription; the others rely on the formation of hairpin structures in the transcribed RNA causing termination. Transcription in eukaryotes The promoter sites for RNA polymerases I and II are located before the start site for transcription. RNA polymerases III recognizes sites within the gene itself. Eukaryotic RNA polymerases require the presence of additional transcriptional factors for DNA binding and the initiation of transcription. RNA processing Generally in Bacteria only structural RNA molecules are processed post-transcriptionally to give a functional molecule. In contrast, eukaryotic RNAs undergo a number of processes to produce a mature molecule. Introns, non-coding transcribed sequence, are removed by splicing, a methylated guanine cap is added to the 5’ end of the RNA and a poly(A) tail to the 3’ end. Introns are also found in the Archaea. C5 CONTROL OF GENE EXPRESSION Key Notes Why control gene expression? Not all genes in the cell need be expressed at all times. Cellular regulatory systems allow the control of gene expression so that their products are only synthesized when required. Regulation can occur at all stages in the synthesis of proteins in the cell, but much of it is at the level of transcription which gives rapid and sensitive control. General features of regulation Constitutive genes are expressed at all times, whereas inducible genes are expressed when required. Regulation can be specific, controlling just one gene, or operon, or global, affecting a large number of genes. Regulation requires some way of detecting the current situation and a way of responding by controlling transcription. The control may by either negative, where the gene is normally switched on unless a repressor is present, or positive, in which an activator is required to allow transcription to occur. Genes or operons may be controlled by more than one regulatory system. Lactose operon The lacZ, laxY and lacI gene products are require for the use of lactose as a carbon source. Gene expression is controlled by two regulatory systems. A negative control system, which switches off the genes in the absence of lactose, and a positive control system, called catabolite repression, which switches on the genes when glucose is not present in the surrounding medium. Negative control In the absence of lactose, the lactose repressor protein binds to an operator(o) site and inhibits transcription of the lac genes. If lactose is present, a product of lactose, allolactose, acts as the inducer molecule. It binds to the lac repressor and prevents it binding to operator site, thereby allowing transcription to occur. Positive control A number of operons associated with carbon utilization cannot be transcribed unless a catabolite activator protein (CAP) is bound to the promoter. The CAP requires the presence of cAMP in order to bind to the DNA and activate transcription. cAMP acts as the signal that glucose levels are low in the cell and therefore alternative carbon sources are requires. The tryptophan operon The trp operon is negatively controlled by a repressor protein which in only active if it is complexed with tryptophan (a corepressor). Gene expression is also controlled by attenuation (premature termination of trp RNA synthesis in the presence of tryptophan). The combined effect of the two regulatory systems is to reduce transcription by about 700-fold. Two factor control systems These systems consist of a sensor component, which detects the environment around or within the cell and an effector protein that regulates gene expression in response to the environment. The sensor protein modulates the activity of the effector protein by phosphorylation. Other regulatiry mechanisms in prokaryotes There is a range of other regulatory mechanisms found in prokaryotes. Regulation in eukaryotes Regulation in eukaryotes involves trans-acting transcriptional factors that interact with control sequences near the promoter regions called cis-acting elements. These regulate gene expression in response to nutritional and environmental signals but are also important during development and differentiation. C6 STRUCTURE OF PROTEINS Key Notes Proteins in the cell Proteins in the cell consist of polypeptides of L-amino acids joined by peptide bonds in a sequence dictated by genetic information in the cell. There are 20 amino acids found in proteins, which differ in the nature of their side chains. The sequence of the amino acids in the protein dictates its structure, the nature of any changes made to the protein, its final destination in or outside the cell and its function. Structure of proteins There are four levels of structure in proteins: primary structure which is the sequence of amino acids in the polypeptide; secondary structure which is the result of hydrogen bonding between side chains of amino acids to form α–helices and β–sheets; tertiary structure which results from the spontaneous folding of the protein as a result of interactions between the amino acids side-chains, often controlled by chaperone proteins; and quaternary structure which occurs when more than one polypeptide makes up the functional protein. C7 TRANSLATION Key Notes The generic code A sequence of three bases ,called a codon ,in the mRNA codes for one amino acid .tRNA molecules act as the adapter molecules between the mRNA sequence. They carry an anticodon which base-pairs with the codon sequence and the amino acid sequence and a site at which an amino acid is attached. The process of matching the anticodon and codon and the synthesis of the amino acid chain occurs on the ribosome and is called translation. Ribosomes The role of the ribosomes is to hold the complex of mRNA and the amino-acid loaded tRNAs together ,form the peptide bond between the amino acids and ensure accuracy of protein synthesis .Riboso