Bacterial_metabolism_ES_10042024.pdf

Bacterial metabolism Presented by Dr Eby Sim Faculty of Medicine and Health [email protected] Originally developed by: Dr Christina Adler Faculty of Medicine and Health The University of Sydney Page 1  Learning objectives Define the term “metabolism”, “catabolism”...

Bacterial metabolism Presented by Dr Eby Sim Faculty of Medicine and Health [email protected] Originally developed by: Dr Christina Adler Faculty of Medicine and Health The University of Sydney Page 1  Learning objectives Define the term “metabolism”, “catabolism” and “anabolism” Identify the sources of energy for bacterial metabolism Describe how bacteria are grown in the laboratory and counted Know features of a bacterial growth curve and apply to oral environment Describe the impact on oral bacteria growth of oxygen and nutrients The University of Sydney Page 2 Metabolism “From food to energy” The University of Sydney Page 3  How do bacteria uptake nutrients – Passive transport – Involves nutrient gradient – Active transport – Utilises energy to bring in nutrients. – Iron uptake – Siderophores https://bio.libretexts.org/Bookshelves/Microbiology/Micr obiology_(Bruslind)/11%3A_Microbial_Nutrition The University of Sydney Page 4  Even within the same species, the complement of uptake mechanism can vary Sim et al., 2021. Front Microbiol. doi:10.3389/fmicb.2021.713724 The University of Sydney Page 5  Metabolism Metabolism is the total of all biochemical reactions occurring inside the cell for survival and reproduction. It consists of two opposing chemical reactions in cells. Catabolism – break down of foodstuffs into smaller molecules to generate energy. Anabolism – uses the energy from catabolism to drive the synthesis of other molecules. The University of Sydney Page 6  Energy Energy is produced in cells in the form of a molecule called ATP ATP (Adenosine Triphosphate) – Currency and store of energy in cells – Drive variety of chemical reactions e.g. pumping substance actively across membrane, reproducing and moving the cells – Formed from ADP (Adenosine Diphosphate and Phosphate) in energy consuming reaction – Releases energy when hydrolysed back to ADP The University of Sydney Page 7  Metabolic tasks Chubukov et al., 2014. Nat Rev Microbiol. doi: 10.1038/nrmicro3238. The University of Sydney Page 8  Input and output to aerobic/anerobic respiration The University of Sydney Page 9  Aerobic respiration – Not to be confused with breathing!! – Overall reaction: C6H12O6 + 6O2 à 6CO2 + 6H2O + (energy) – Consist of 4 processes – Glycolysis (Pyruvate generation) – Acetyl COA generation (Pyruvate to acetyl-CoA) – Citric acid (Krebs) cycle – Oxidative phosphorylation The University of Sydney Page 10  Anaerobic respiration – Breaking down of glucose when O2 is lacking. – Some organism can utilise different electron acceptors – Like SO42- , NO3- – Fermentation is another pathway for breaking down glucose. – Occurs after glycolysis Lactic acid fermentation Alcohol fermentation The University of Sydney Page 11 Bacterial growth ‘How do we assess this in a laboratory?’ The University of Sydney Page 12  Bacterial growth – Bacteria grow and replicate primarily by binary fission. – Process whereby one bacterium splits into two. – Require energy to grow The University of Sydney Page 13  Conditions that can affect growth of bacteria. Temperature pH Oxygen Most like 37oC Most like pH 7-8 Strict aerobes = require Vary 0 – 121oC Can vary pH 0 – 11 oxygen for growth Facultative aerobes = prefer oxygen for growth Facultative anaerobes = prefer no oxygen for growth Strict anaerobes = no oxygen for growth The University of Sydney Page 14  Growing bacteria in the lab – culture media – Media is a substance that supports the growth of bacteria in the laboratory. – Culture media are either: – defined (exact composition known) or – complex (extracts from plants or animal; exact composition unknown) – Additives can also be incorporated to media to either enrich or inhibit certain bacteria. The University of Sydney Page 15  Growing bacteria in the lab – media consistency Solid Semi-solid Liquid Allows for the For microaerophilic Allows for uniformed isolation of single bacteria growth until either colonies nutrient limitation or build up of inhibitors The University of Sydney Page 16  Bacteria quantification – Cell counting under a microscope – Serial dilution and plating on agar (colony forming units) – qPCR – Relative quantification, or – Absolute quantification The University of Sydney Page 17  Optical density – Measuring the optical density at wavelength 600 nm is typically how we interrogate growth dynamics in the lab. https://biohackspace.org/projects/od600-spectrophotometer/ https://www.implen.de/od600- diluphotometer/od600/ The University of Sydney Page 18  Growth curve The University of Sydney Page 19  Lag phase – Adjustment period – Bacteria adapting to new environment – Replicating chromosomes to build new new proteins/enzymes The University of Sydney Page 20  Exponential phase – Cells growing and dividing – Maximal growth – Continues until nutrients exhausted or inhibitory molecules accumulate The University of Sydney Page 21  Stationary phase – Cell numbers stop increasing – DNA replication is arrested – Cells produce new proteins to enhance survival during starvation – more resistant to antibiotics via cell wall crosslinking – spore formation – DNA/protein protections The University of Sydney Page 22  Death phase – Cells stop growing, even if supplied with new medium – Q. Do these phases happen just in lab or also in real life? The University of Sydney Page 23  Question In the oral environment, which phase would correspond to the following: A. Immediately after a meal B. In between meals The University of Sydney Page 24 Bacterial growth Oral environment The University of Sydney Page 25  Bacterial growth in oral environment Lag phase = immediately after meal, adjusting to new influx of nutrients Exponential phase = after meal, post adjustment. Cells dividing maximally Stationary = period of fasting in between meals, nutrients limited, bacteria susceptible to antimicrobials Death phase = variable between bacteria e.g. Streptococcus gordonii cannot survive for prolonged periods without nutrients, but others can by forming colonies The University of Sydney Page 26  Nutrient acquisition in oral environment – Oral bacteria breakdown complex macromolecules – Diet – Saliva – Gingival crevicular fluid – Enzymes for breakdown – Proteases = protein – Amylases = carbs – Lipases = fats The University of Sydney Page 27  Oxygen in oral environment Oxygen is present at near atmospheric levels in oral environment (~20%) Aerobic bacteria = need oxygen, capable of detoxifying oxygen free radicals Strict aerobic bacteria = need O2, not many in dental plaque Facultative aerobic bacteria = prefer O2 Facultative anaerobes = do not prefer O2, e.g. oral streptococci Strict anaerobes = no O2, many associated with periodontal disease, e.g. P. gingivalis present in subgingival plaque The University of Sydney Page 28  Question Which type of organisms would be most likely to be found in the supragingival plaque? A. Strict anaerobes B. Strict aerobes C. Facultative aerobes D. Facultative anaerobes The University of Sydney Page 29  Summary – Bacteria can use aerobic and anaerobic respiration in metabolism – Bacteria divide by binary fission and are assessed in the lab using subjective and non-subjective methods – Bacteria grow according to a 5 phase pattern that can be seen in the lab and equated to conditions in life e.g. mouth – Oral bacteria are influenced by nutrient availability and oxygen levels The University of Sydney Page 30