Understanding Bacterial Growth: Measurement, Physiology, and Regulation - Prof. Ranjan N. , Study notes of Health sciences

Download Understanding Bacterial Growth: Measurement, Physiology, and Regulation - Prof. Ranjan N. and more Study notes Health sciences in PDF only on Docsity! 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 reflects 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. 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 finally find 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 difficult 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 filled with bacteria suspended in saline solution and in another only the saline solution is filled. 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 specific volume is plated on nutrient media. The colonies developed on the media are counted to find 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. 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. Changes in metabolic activities: The overall metabolic rate slows down when the cells are starved for nutrients. There is a significant 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 50-70 or more types of new proteins when starved for nutrients like carbon , nitrogen , iron or phosphate. This is due to the induction of high affinity 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 (s ): Under starvation in many bacteria including E.coli the rpoS gene expresses special sigma factor (s) 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 s factor is regulated both at transcription as well as translational levels. Other Regulators: Besides s factor there are other global regulator of gene expression present in the bacterial cells. One of the most important is cyclic AMP(cAMP)- CRP complex which stimulates almost 2/3 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 20,000 to 70,000 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. 0.6 1.0 1.5 2.0 2.5 Doubling Time/h Relative amount RNA Cell Mass Protein DNA 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 E.coli 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 (ori 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 B.subtilis, Caulobacter crescentus, and Pseudomonas putida but not in E.coli. Similar proteins although, are located on E.coli plasmid, responsible for plasmid partitioning. In E.coli Muk B 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 identified to play important role in septum formation including, FtsZ, FtsA, FtsI, FtsQ, FtsL, FtsN, FtsW, and ZipA. Fts stands for filamentous temperature sensitive phenotype. FtsA and FtsZ proteins are located in the cytoplasm while rest are membrane associated. 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. Steady-State 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 flow (F) of limiting nutrient from the reservoir to the growth culture. The dilution rate ,(D): The dilution rate D = F/V, where V is the volume of growing culture. For example if the flow rate is 10ml/h and V is 1 liter, then the D = 10/1000 = 0.01/h. Multiplying dilution rate D with the total cells x gives Dx which is number of cells lost/unit time from the culture during dilution.