Chapter 6 Microbial growth: prokaryotic cell cycle and growth curve 1 Growth • Increase in cellular constituents that may result in: – increase in cell number • binary fission – increase in cell size • coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions • Microbiologists usually study population growth rather than growth of individual cells 2 The Procaryotic Cell Cycle • Cell cycle is sequence of events from formation of new cell through the next cell division • Two pathways function during cycle – replication of DNA and partition – cytokinesis (cell separation) 3 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 6.1 4 The Cell Cycle in E. coli • E. coli requires ~40 minutes to replicate its DNA and 20 minutes after termination of replication to prepare for division Figure 6.2 5 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. 6 Figure 6.3 Beside determination of cell shape, MreB is thought to play a role in chromosome partitioning to daughter cells Chromosome Replication and Partitioning • Most procaryotic chromosomes are circular • Origin of replication – site at which replication begins • Terminus – site at which replication is terminated • Replisome – group of proteins needed for DNA synthesis; parent DNA spools through the replisome as replication occurs • MreB – an actin homolog plays role in determination of cell shape 7 Cytoskeletal Proteins - Role in Cytokinesis • Process not well understood • Protein MreB – similar to eucaryotic actin – plays a role in determination of cell shape and movement of chromosomes to opposite cell poles • Protein FtsZ, – similar to eucaryotic tubulin – plays a role in Z ring formation which is essential for septation 8 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 6.4 9 The Fts Z Ring and Cell Division 10 The Fts Z Ring and Cell Division 11 Cell Wall Synthesis Streptococcus hemolyticus 12 DNA Replication in Rapidly Growing Cells • Cell cycle completed in 20 minutes – 40 minutes for DNA replication – 20 minutes for septum formation and cytokinesis • Look at timing-how can this happen? – 2nd, 3rd, and 4th round of replication can begin before first round of replication is completed – Progeny cells are “born” with two or more replication forks, replication already in progress. 13 The Growth Curve • Observed when microorganisms are cultivated in batch culture – culture incubated in a closed vessel with a single batch of medium • Usually has four distinct phases 14 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. lag phase no increase log phase maximal rate of division and population growth stationary phase population growth ceases death phase decline in population size Figure 6.6 15 Lag Phase • Cell synthesizing new components – to replenish spent materials – to adapt to new medium or other conditions • Varies in length – in some cases can be very short or even absent 16 Exponential Phase • Rate of growth is constant • Population is most uniform in terms of chemical and physical properties during this phase • During log phase, cells exhibit balanced growth – cellular constituents manufactured at constant rates relative to each other 17 Stationary Phase • Total number of viable cells remains constant – may occur because metabolically active cells stop reproducing – may occur because reproductive rate is balanced by death rate 18 Possible reasons for entry into stationary phase • • • • 19 Nutrient limitation Limited oxygen availability Toxic waste accumulation Critical population density reached Starvation responses • Morphological changes – endospore formation • Decrease in size, protoplast shrinkage, and nucleoid condensation • Production of starvation proteins • Long-term survival • Increased virulence 20 Death Phase • Two new alternative hypotheses – Cells are Viable But Not Culturable (VBNC) • Cells alive, but dormant • Programmed cell death – Fraction of the population genetically programmed to die (commit suicide) 21 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Loss of Viability Figure 6.8 22 Prolonged Decline in Growth • bacterial population continually evolves • process marked by successive waves of genetically distinct varients • natural selection occurs Figure 6.9 23 The Mathematics of Growth • Generation (doubling) time – time required for the population to double in size – varies depending on species of microorganism and environmental conditions – range is from 10 minutes to several days for some microorganisms 24 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.1 25 Calculation of number of cells, generation times, and growth rates No = initial population number Nt = population at time t n = number of generations at time t g = generation time If in 8 h an exponentially growing cell population increases from 5 × 106 cells/ml to 5 × 108 cells/ml, calculate g and n. 27 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 6.10 28 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. generation time could be determined using a semilogarithmic graph at the log phase Figure 6.11 29 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.2 30