Bacterial Metabolism & Growth Characteristics. Stijn van der Veen

Transcription

1 Bacterial Metabolism & Growth Characteristics Stijn van der Veen

2 Differentiating bacterial species Morphology (shape) Composition (cell envelope and other structures) Metabolism & growth characteristics Genetics

3 Differentiating bacterial species Morphology (shape) Composition (cell envelope and other structures) Metabolism & growth characteristics Genetics

4 Bacterial metabolism The bacterial metabolism is the combination of all (bio)chemical reactions that occur within bacterial cells that allows them to live, replicate, and maintain cellular integrity. Bacteria can be differentiated by the unique combination of different (bio)chemical reactions they are able o perform. These can be identified by specific substrates they are able to convert or metabolites they are able to produce.

5 Metabolic pathways The metabolism is structured into metabolic pathways that consist of series of consecutive (bio)chemical reactions that are connected through their start and end products. Biochemical reactions in the metabolic pathways are either spontaneous reactions or reactions driven by enzymes (proteins). Metabolic pathways are regulated by enzymes that determine the direction and speed of the biochemical reactions.

6 Metabolic maps Metabolic maps reflect the metabolic pathways and display how all the metabolites (dots) and (bio)chemical reactions (lines) are connected.

7 Metabolic flux Metabolic flux is the turnover of metabolites through metabolic pathways. Metabolic flux is regulated by the enzymes that perform the biochemical reactions

8 Catabolism & Anabolism The metabolism is generally divided into two major groups: Catabolism: Biochemical reactions that convert larger molecules into smaller molecules, thereby generating energy. Anabolism: Biochemical reactions that consume energy to construct larger cellular components such as proteins, lipids, and DNA.

9 Catabolism & ATP Catabolic degradation of larger molecules results in the generation of energy in the form of heat (which is lost) and adenosine triphosphate (ATP). ATP is the most important storage molecule for chemical energy. ATP provide the energy for most of the energy consuming metabolic processes.

10 Generation of ATP Reduction-oxidation (redox) reactions Aerobic respiration: Complete conversion of carbohydrates into water, carbon dioxide and ATP, using oxygen as the final electron acceptor in the electron transport chain (ETC). Fermentation: Anaerobic conversion of carbohydrates into acids, gases, and/or alcohols, and ATP. Anaerobic respiration: Similar to aerobic respiration but instead of oxygen, sulfate or nitrate are used as final electron acceptors. Sunlight Photosynthesis: Use of light energy to energize electron donors (photophosphorylation), which results in the spontaneous movement of electrons through the ETC.

11 Oxygen tolerance Bacteria can be classified according to their oxygen tolerance: Obligate aerobes Require oxygen to stay alive Aerobic respiration Obligate anaerobes Die in the presence of oxygen Fermentation or anaerobic respiration Facultative anaerobes Survive with and without oxygen Combination of aerobic respiration and fermentation Microaerophiles Require low levels of oxygen Combination of growth modes Aerotolerant anaerobes Survive with and without oxygen Fermentation

12 Aerobic respiration Conversion of carbohydrates such as glucose into water, carbon dioxide and ATP is a 4-step process: Glycolysis Pyruvate decarboxylation Krebs (TCA or citric acid) cycle Oxidative phosphorylation (in the ETC) C 6 H 12 O O ADP + 38 P i 6 CO H 2 O + 38 ATP

13 Oxidase test Used to determine the presence of cytochrome c oxidase. Cytochrome c oxidase is part of the ETC and uses oxygen as terminal electron acceptor. The oxidase test uses reagents such as N,N,N,N -tetramethylp-phenylenediamine (TMPD) that turn blue when oxidized and stay colorless when reduced.

14 Detoxification of oxygen radicals During aerobic respiration reactive oxygen species (ROS) are generated as side products. ROS such as superoxide anions(o 2- ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (OH ) are very reactive and damaging to cellular structures such as DNA, proteins, and lipids. Aerobic respiring bacteria contain the detoxifying enzymes superoxide dismutase (SOD), catalase (Kat), and peroxidase.

15 Catalase test The catalase test is used to identify bacteria that contain the catalase enzyme. Hydrogen peroxide is added to a small amount of bacteria and observed for bubble formation (oxygen).

16 Fermentation Conversion of carbohydrates such as glucose into acids, ethanol, and ATP. To maintain the redox balance, after glycolysis pyruvate is converted into waste products (acids, ethanol, etc.). There are many different types of fermentation processes and end products, but the most common types are homolactic fermentation (lactate as end product), heterolactic fermentation (mix of lactate and other acids), and alcohol fermentation.

17 Carbohydrate conversion and acidification Carbohydrate conversion Most bacteria are able to convert glucose into energy. The ability to utilize specific more complex carbohydrates as energy source is variable between bacteria, and this ability is useful for identification. Acidification Homolactic and heterolactic fermentation results in the production of acids. Acidification of media can be detected with ph indicators.

18 Carbohydrate fermentation tests Carbohydrate fermentation tests with ph indicator can show production of acids (color shift). Carbohydrate fermentation tests with durham tube can also show production of gases (collected in durham tube).

19 Citrate conversion Simmon s citrate medium is used to test whether bacteria can utilize citrate as the sole carbohydrate. Citrate conversion results in alkalization of the medium which is indicated with bromothymol blue.

20 Catabolism of proteins and amino acids Proteins are degraded by proteases into peptides and peptides are further degraded into amino acids. Amino acids are converted in various pathways to feed into the TCA (Krebs) cycle and further converted into ATP.

21 Tryptophan conversion (indole test) Some bacteria are able to convert the amino acid tryptophan using the tryptophanase enzyme. Cleavage of tryptophan results in the production of indole. Indole reacts with para-dimethylamino benzaldehyde from Kovacs reagent and produces a red-violet color.

22 Cysteine / methionine conversion Some bacteria are able to convert the sulfide containing amino acids methionine and cysteine. Cleavage of these amino acids results in the production of hydrogen sulfide. The hydrogen sulfide combines with ferrous sulfide (Fe 2 S) in the triple sugar iron (TSI) agar to form a black to dark insoluble precipitate.

23 Urea conversion Urea agar slants are used to test whether bacteria can convert urea into ammonia and carbon dioxide. Production of ammonia results in alkalization of the medium which is indicated with phenol red. Urease activity is important for bacteria that pass through the gastro-intestinal tract and need to survive the acid environment

24 Anabolism The anabolism is the general term for the biochemical reactions leading to the synthesis of cell structures. Anabolism can be divided into four steps: Collection and transport of elements and growth factors Synthesis of monomers Synthesis of polymers Structural assembly of polymers

25 Elements and growth factors The most abundant elements that make up 95 % of the dry weight of a bacterial cell are Carbon, Oxygen, Hydrogen, and Nitrogen. The other 5% consists of Phosphorus, Calcium, Sodium, Potassium, Iron, Copper, Magnesium, and Manganese. Other required elements are trace elements. Growth factors are molecules that are required for growth but bacteria are unable synthesize themselves. These are variable and depend on the specific abilities of each species, but may include some vitamins, amino acids, or nucleic acid precursors.

26 Bacterial structures The bacterial anabolism combines the elements into many different metabolites with the majority finally forming large bacterial structures. Proteins: Forming 50-80% of the dry weight. Sugars: Mainly in the cell wall and capsule. Lipids: Mainly in the cell membrane and outer membrane. Nucleic acids: Mainly DNA and RNA.

27 Bacterial growth Bacterial growth is the asexual replication or division of a bacterium into two daughter cells in a process called binary fission.

28 Generation time Generation time (doubling time) is the average time required for a population of bacteria to double in number. The doubling time for bacteria is variable ranging from 10 min to 30 h or more and also depends on the growth conditions. Organism Clostridium perfringens Generation Time min Escherichia coli Bacillus cereus Staphylococcus aureus min min Mycobacterium tuberculosis hrs Treponema pallidum 30 hrs

29 Exponential or logarithmic growth Generation Cell Number Count , ,048, ,073,741,824 So, in 15 hrs a single cell can turn into a billion cells!!!

30 Bacterial growth factors / conditions Nutrient availability Elements and growth factors. Oxygen pressure Growth mode (respiration, fermentation, etc.) Temperature Important for speed of enzymatic reactions and stability of bacterial structures. Acidity / alkalinity (ph) Impact on proton motive force, stability of bacterial structures, etc. Water activity Determines osmotic pressure

31 Temperature Bacteria are divided into four classes for their ability to grow at specific temperature ranges. This ability is particularly determined by their protein (enzyme) and cell membrane stability at these temperatures. All bacterial pathogens are mesophiles. Thermophiles

32 Acidity / alkalinity Bacteria are divided in three groups for their ability to grow at different ph s. Most bacteria and bacterial pathogens are neutrophiles and have optimum growth around ph , which is the ph of most human organs and tissues. Most important acidophile is Helicobacter pylori, which thrives in the human stomach.

33 Water activity Water is important component of bacterial cells and is involved in many metabolic reactions. Most bacteria die in the absence of water (desiccation). Water activity is determined by the presence of salts and solutes. Water activity determines osmotic pressure. Halophiles (not bacterial pathogens) require high salt concentrations.

34 Measuring bacterial numbers Turbidity of liquid cultures Quantify total bacteria (live and dead) by absorption at 600 nm using a spectrophotometer. Colony counting on agar plates Count colony numbers after plating a known volume of liquid (or serial dilutions). Each colony is derived from a single live bacterial cell.

35 Growth in liquid cultures Growth in liquid media can be measured by turbidity or colony counting on agar plates. Plotting the logarithmic values of turbidity or bacterial cell numbers against time results in a plot called a growth curve. Growth curves are generally characterized by four phases: lag phase, log or exponential growth phase, stationary phase, and death phase.

36 Lag Phase Bacteria are becoming "acclimatized" to the new environmental conditions (ph, temperature, nutrients, etc.). Enzymes and intermediates are formed and accumulate until they are present in concentrations that are permit growth.

37 Log or exponential growth phase Bacteria have adapted to the environmental conditions and start the replicate. The bacterial population is growing rapidly at an exponential rate. This is the most homogeneous state of the bacteria and generally bacteria from this phase are used for most of the biochemical tests, including antibiotic sensitivity tests.

38 Stationary phase When nutrients are becoming limited and metabolic waste products accumulate, growth rates decline until the point that growth rate equals death rate. In this phase there is no increase in the population of live bacteria. Generally, in this phase bacteria produce endospores, toxins, and antibiotics.

39 Death phase The population of live bacteria decreases due to the lack of nutrients and accumulation of toxic metabolic waste products. Some bacteria autolyse in this phase, which might also result in decreased turbidity.

40 Static liquid growth Generally, liquid cultures are grown under shaking conditions, allowing uniform turbidity. Static growth of liquid cultures can result in different patterns: Uniform turbidity Facultative or aerotolerant anaerobes Ring or pellicle at the air-liquid interface Aerobes Sediment at the bottom Anaerobes

41 Growth on solid media Used to obtain a large number of bacteria, isolate identical clones of bacteria (colony), and to perform drug sensitivity test. A colony is a cluster of bacterial cells that propagated (multiplied) from a single cell. Colony can be used to determine the original bacterial numbers by counting colonies and to evaluate viability of bacteria (colony forming units, CFU).

42 Differences in colony morphology

43 Streaking bacteria By spreading bacteria over the surface of a plate, the amount of bacteria is diluted and individual cells are able to form a single pure colony. Procedure: 1. Flame the loop and streak a loop containing bacteria as at A in the diagram. 2. Reflame the loop and cool it. 3. Streak as at B to spread the original inoculum over more of the agar. 4. Reflame the loop and cool it. 5. Streak as at C. 6. Reflame the loop and cool it. 7. Streak as at D. 8. Incubate the plate inverted.

44 Growth on semisolid media Used to test the motility of bacteria (flagellum or pili). - +

45 Bacterial cultivation media Basic nutrient media Supplies all the nutritional requirements for growth of most of the common bacteria. Minimal media Supplies the minimal nutritional requirements for growth of specific bacteria. Enrichment media Supplies additional nutrients for the growth of fastidious bacteria that do not grow on the basic nutrient media.

46 Bacterial cultivation media Selective media Supports the growth of desired bacteria while inhibiting the growth of many or most of the unwanted ones. These media contain selective agents that inhibit growth of unwanted bacteria, while allowing growth of desired bacteria (e.g. antibiotics, bile salts, etc.). Or alternatively, specific nutrients are included or omitted to allow selection. Differential medium Supports the growth of two or more bacterial species, but differentiates between them due to the addition of specific components that react differently with these species (e.g. ph indicators, blood, etc.).

47 Blood agar These are the red plates that most of your cultures will be grown on. The media is made of a basic nutrient agar composed mostly of a mixture of amino acids and peptides, combined with defibrinated blood. When the bacteria produce a membrane toxin, this can lyse the red blood cells (haemolysis) and the media can change colour and become clearer around bacteria producing such toxins. This is the most commonly used media because it is so nutrient rich, many bacteria make recognizable colony shapes on it, and you can see haemolysis.

48 Chocolate agar These are the brown plates (which do not contain chocolate) and are very similar to blood agar. After the blood has been added the media has been re-heated to above 56 degrees to damages the cells to releases more heme (also called growth factor X) and NAD (also called growth factor V) into the media where it is accessible to bacteria that cannot lyse the blood cells. This medium is useful for growth of fastidious bacteria such as Neisseria sp. and Haemophilis sp.

49 MacConkey s Agar Combination of selective and differential medium. It is selective because it contains bile salts that inhibit growth of most bacteria, except for the bacteria that colonize the gut and have adapted to bile salts (such as Enteric bacteria that contain long LPS). It is a differential medium because it contains lactose as sole carbohydrate and the ph indicator neutral red. Acid production during lactose fermentation results in pink-red colonies.

50 Mannitol salt agar Combination of selective and differential medium. It contains high salt concentrations (<7.5% NaCl), which inhibits most bacteria except for Staphylococci (and few others). It also contains the carbohydrate mannitol and ph indicator phenol red to detect acid production from mannitol fermentation. Staphylococcus aureus produce yellow colonies, while other Staphylococci produce pink-red colonies.

51 Bacterial identification flowchart Cell morphology + Gram stain - Cell morphology Cocci Rods Cocci Rods Catalase + - Micrococcus # (or) Streptococcus Staphylococcus Coagulase + - Sta. aureus Sta. epidermidis Optichin sensitive + - Str. pneumonia Str. viridans Atmospheres: Anaerobic: Clostridia Aerobic: Bacillus Facultative anaerobes: Corynebacteria Lactobacillus Oxidase + - Neisseria Not a pathogen Indole test + - Grows with bile salts + - Ferments lactose + - Pseudomonas Haemolysis Alpha (green) Beta (clear) Gamma (not) Not a pathogen Oxidase + - Proteus E. coli Klebsiella Lancefield typing (check for capsule) (can confirm D with growth on bile salts) Haemophilus Urease + - H 2 S + - Salmonella Shigella # Micrococcus has larger cells and looks more yellow. This is a simplified version!!!

52 Next lecture Bacterial Genetics