Which growth phase is an organism most sensitive

In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat Figure 7. A controlled amount of air is mixed in for aerobic processes.

Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment. Biofilms In nature, microorganisms grow mainly in biofilms , complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds. Biofilms are not restricted to solid surface substrates, however.

Almost any surface in a liquid environment containing some minimal nutrients will eventually develop a biofilm. Microbial mats that float on water, for example, are biofilms that contain large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species. Regardless of the environment where they occur, biofilms are not random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.

Observations using confocal microscopy have shown that environmental conditions influence the overall structure of biofilms. Filamentous biofilms called streamers form in rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids.

In still or slow-moving water, biofilms mainly assume a mushroom-like shape. The structure of biofilms may also change with other environmental conditions such as nutrient availability. Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal clusters of microorganisms embedded in a matrix interspersed with open water channels.

The extracellular matrix consists of extracellular polymeric substances EPS secreted by the organisms in the biofilm. The properties of the EPS vary according to the resident organisms and environmental conditions but is composed primarily of polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm. Channels in the EPS allow movement of nutrients, waste, and gases throughout the biofilm.

This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells e. Free-floating microbial cells that live in an aquatic environment are called planktonic cells. The formation of a biofilm essentially involves the attachment of planktonic cells to a substrate, where they become sessile attached to a surface. This occurs in stages, as depicted in Figure 7.

The first stage involves the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels.

Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites.

This stage is referred to as dispersal. Within a biofilm, different species of microorganisms establish metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes.

This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens. The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called quorum sensing.

Quorum sensing—which can occur between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers.

In the lab, we have to continually replace the media. A spectrophotometer can measure how much light a solution of microbial cell transmits; the greater the mass of cells in the culture, the greater its turbidity cloudiness and the less light that will be transmitted.

Metabolic Activity - 3 ways:. The rate of formation of metabolic products, such as gases or acids, that a culture produces. The rate of utilization of a substrate, such as oxygen or glucose.

The rate of reduction of certain dyes. Direct Measurements - Give more accurate measurements of numbers of microbes. Filtration - A known volume of liquid or air is drawn through a membrane filter by vacuum.

The pores in the filter are too small for microbial cells to pass through. Then the filter is placed on an appropriate solid medium and incubated. The number of colonies that develop is the number of viable microbial cell in the volume of liquid that was filtered.

This technique is great for concentrating a sample, ex. Growth Factors - Microbes can exist in a great many environments because they are small, easily dispersed, need only small quantities of nutrients, are diverse in their nutritional requirements.

Physical Factors. Lactobacilllus ferments milk. Vibrio cholerae causes cholera. Halophiles salt lovers inhabit the oceans. Some bacteria have enzyme systems that can repair some mutations. Studies have demonstrated that starved cells exhibit more protective resistance to different stresses as compared to resistance induced during growing stage by non-lethal exposure of stresses Kolter et al. The genes expressed in stationary phase are controlled by promoters, which result in induction of stationary phase.

The promoters, which are turned on, are called SPPs. Promoter mcbA -LacZ fusion showed the induction of transcription in nitrogen, phosphate, and carbon starvation conditions. Similarly, bolA-lacZ fusion demonstrated an increase in expression of approximately to fold during transition to stationary phase.

Since then many SPPs have been isolated and characterized in both Gram-positive and Gram-negative bacteria Tables 2 , 3. Particularly regarding E. Figures 4A,B shows the —10, —35 and spacer region of few SPPs from Gram-positive and Gram-negative bacteria and the consensus sequence at the —10 region is shown as a logo designed using WebLogo software available online Crooks et al.

Sequence alignment of A Gram-positive and B Gram-negative stationary phase promoters. The conserved bases are shown below. Among the SPPs exist a special class of promoters known as the gearbox promoters which include mcbAp, bolAp1, ftsQp to name a few. This class of promoters has been studied in several Gram-negative bacteria including E. Two different highly conserved consensus —10 and —35 sequence have been proposed by Aldea et al.

During unfavorable conditions of growth, reprogramming the cellular machinery for sustaining viability is a natural process of adaptation. In case of bacteria that do not accumulate these polymers, cellular RNA is rapidly degraded for energy generation Matin, What is surprising is that, when in stationary phase, these ribosomes are fairly stable and so degradation occurs only in between the stages.

This raises a very important question: What makes protein synthesis possible at stationary phase? Balaban and coworkers devised a microfluidic device and followed the production of fluorescent proteins at stationary phase. They found that cells after entering stationary phase continue to produce proteins for several days Gefen et al. It has been suggested that cells continue to produce proteins at stationary phase by reusing amino acids from degraded proteins.

In addition, it is shown that each condition resulting in starvation results in induction of specific set of proteins Kolter et al. A strong promoter is the key for developing efficient gene expression systems.

For recombinant protein production, several bacterial hosts have been used as cell factories, with features such as easy purification, improved protein folding and secretion, high production of membrane proteins, etc. Ferrer-Miralles and Villaverde, To develop more such expression systems in bacteria, it is necessary to ensure proper selection of a promoter that would drive the expression of genes at the right time and with maximum amount.

Promoters could be classified as constitutive or inducible, growth-stage limited, tissue specific, etc. Inducible promoters can further be classified into inducer-specific and auto-inducible promoters. Constitutive promoters are not useful for toxic proteins. Inducer-specific promoters involve the cost of inducer. Further, the addition of external inducers often requires growth monitoring which is vital for productivity and hence lead to difficulty in fermentation.

Auto-inducible promoters are ideal for large-scale protein production as they are induced at late log phase or stationary phase. Such promoters induce expression of the recombinant gene without any inducer and thus are economical. However, most of them have low activity Yu et al. The authors have proposed that such a promoter will be useful for protein production.

A SPP-based auto-inducible gene expression system has been constructed using cry3Aa promoter. The Pcry drives the expression of crystal proteins in B. The promoter cry3Aa was tested in B. Similarly, in another Gram-positive bacteria, Gordonia sp. In future, a study of such promoters based on the number of transcripts would be useful to compare the strength.

In Corynebacterium glutamicum , promoter of cg gene coding for flavohemoprotein was found to show higher inducibility in the stationary phase. Then, a synthetic promoter library was prepared to change the spacer and flanking regions in the promoter, to obtain a range of promoter strengths Kim et al. At the end, one of the synthetic promoters that showed up to fold higher strength compared to the original cg promoter was obtained and demonstrated for fed-batch cultivation of glutathione S-transferase in a 5L reactor.

Table 4 depicts the list of SPP-based expression vectors constructed till date. Studies like these indicate that the potential of SPPs is phenomenal. TABLE 4. Stationary phase promoter—based gene expression systems reported from Gram-negative and Gram-positive bacteria. Recombinant production of toxins whose overproduction is detrimental to the growth of cells needs controlled conditions for expression. In such cases, the use of SPP is advantageous as the overproduction will not affect the growth of the host cells.

Many bacteria have been used to demonstrate the utility of cell-density-dependent expression systems for heterologous protein production. Metabolic engineering of bacteria for enhanced production of industrially important chemicals has been carried out since a long time. The fic promoter of E. Using B. It is a well-known fact that the non-growing phase of lactic acid bacteria accounts for a major proportion of flavor production in lactic acid bacteria van de Bunt et al.

Therefore, engineering bacterial cells in such a way that they are expressed at high levels, during the ripening process, by using SPPs would enhance their applicability in food industry. In the bioremediation industry, microorganisms have routinely been employed for removing pollutants.

Due to low nutrient availability in polluted sites, genetic engineering of cells resulting in higher enzymatic activities at lower growth rates have been shown to be highly efficient for bioremediation process. On studying the phenol degradation capability of two non-growing recombinant E. As suggested by Tunner et al. This could save the cost of induction thereby increasing the efficiency of the process. In a very interesting experiment, Rhodospirillum rubrum cells grown photoheterotrophically, evolved hydrogen for about 70 h after growth ceased Melnicki et al.

Stationary phase survival is a means of bacterial adaptation by which bacteria survive under conditions of stress or starvation. The ugly aspect of this is that such a mechanism results in the persistence of pathogenic bacteria which can cause relapsing of infections. However, the good side is represented by the various biotechnological applications that have come up recently based on the promoters of the genes which are upregulated at stationary phase. In the present review, we have discussed not only the changes at the cellular and molecular levels at stationary phase, but also the various promoters characterized, their regulation and the gene expression systems developed.

There are still many unknowns. For example, very little is known about the proteins which are involved in chromosome organization and their interaction with DNA at stationary phase. Such proteins could be important players in regulating gene expression at stationary phase. Further very few SPPs have been experimentally characterized till date.

Such promoters should be highly useful for protein production as the growth and protein production phase can be uncoupled. This will pave way toward constructing improved gene expression systems for recombinant protein production. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors would like to thank Department of Biotechnology, Government of India for the financial support.

Aldea, M. Induction of a growth-phase-dependent promoter triggers transcription of bolA, an Escherichia coli morphogene. EMBO J. PubMed Abstract Google Scholar. Gearbox gene expression and growth rate. World J. Alexander, D. John, A. Characterization of the carbon starvation-inducible and stationary phase-inducible gene slp encoding an outer membrane lipoprotein in Escherichia coli.

Barker, M. Mechanism of regulation of transcription initiation by ppGpp. Effects of ppGpp on transcription initiation in vivo and in vitro. Becker, G. Bohannon, D. Bruno, J. Listeria monocytogenes adapts to long-term stationary phase survival without compromising bacterial virulence.

FEMS Microbiol. Busuioc, M. The pdh operon is expressed in a subpopulation of stationary-phase bacteria and is important for survival of sugar-starved Streptococcus mutans. Cao, Y. Production of phloroglucinol by Escherichia coli using a stationary-phase promoter. The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm cytokinesis. This process of cytokinesis and cell division is directed by a protein called FtsZ.

FtsZ assembles into a Z ring on the cytoplasmic membrane Figure 2. The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Figure 2. FtsZ proteins assemble to form a Z ring that is anchored to the plasma membrane. The Z ring pinches the cell envelope to separate the cytoplasm of the new cells. In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years.

In prokaryotes Bacteria and Archaea , the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments.

Most pathogens grow rapidly, like E. For example, Mycobacterium tuberculosis , the causative agent of tuberculosis, has a generation time of between 15 and 20 hours.

On the other hand, M. It is possible to predict the number of cells in a population when they divide by binary fission at a constant rate. As an example, consider what happens if a single cell divides every 30 minutes for 24 hours. The diagram in Figure 3 shows the increase in cell numbers for the first three generations. The number of cells increases exponentially and can be expressed as 2 n , where n is the number of generations. If cells divide every 30 minutes, after 24 hours, 48 divisions would have taken place.

If we apply the formula 2 n , where n is equal to 48, the single cell would give rise to 2 48 or ,,,, cells at 48 generations 24 hours. When dealing with such huge numbers, it is more practical to use scientific notation. Therefore, we express the number of cells as 2. In our example, we used one cell as the initial number of cells.

For any number of starting cells, the formula is adapted as follows:. N n is the number of cells at any generation n , N 0 is the initial number of cells, and n is the number of generations. Figure 3. The parental cell divides and gives rise to two daughter cells. Each of the daughter cells, in turn, divides, giving a total of four cells in the second generation and eight cells in the third generation.

Each division doubles the number of cells. Microorganisms grown in closed culture also known as a batch culture , in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve.

An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume. In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve Figure 4.

Figure 4. The growth curve of a bacterial culture is represented by the logarithm of the number of live cells plotted as a function of time.

The graph can be divided into four phases according to the slope, each of which matches events in the cell. The four phases are lag, log, stationary, and death. The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth.

The initial phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium.

If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase.

The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum. In the logarithmic log growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions nutrients, temperature, pH, and so forth is genetically determined, and this generation time is called the intrinsic growth rate.

During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is often plotted on a semilogarithmic graph, as shown in Figure 5, which gives the appearance of a linear relationship.

Figure 5. Both graphs illustrate population growth during the log phase for a bacterial sample with an initial population of one cell and a doubling time of 1 hour. Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.

As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth.

The total number of live cells reaches a plateau referred to as the stationary phase Figure 4. In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant.

The culture density in a stationary culture is constant. During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase.

Secondary metabolites, including antibiotics, are synthesized in the stationary phase. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful. As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers.

Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells Figure 4. This is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.

The growth pattern shown in Figure 4 takes place in a closed environment; nutrients are not added and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products.

A chemostat Figure 6 is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.

Figure 6. A chemostat is a culture vessel fitted with an opening to add nutrients feed and an outlet to remove contents effluent , effectively diluting toxic wastes and dead cells.

The addition and removal of fluids is adjusted to maintain the culture in the logarithmic phase of growth. If aerobic bacteria are grown, suitable oxygen levels are maintained. Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists. The number of bacteria in a clinical sample serves as an indication of the extent of an infection.

Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells.

Both direct and indirect methods have advantages and disadvantages for specific applications. Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope.

The calibrated slide is called a Petroff-Hausser chamber Figure 7 and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is etched into squares of various sizes.