Diauxic Culture and Synchronous Culture

 

Diauxic culture

The diphasic response of a culture of microorganisms based on a phenotypic adaptation to the addition of a second substrate; characterized by a growth phase followed by a lag after which growth is resumed.  In batch culture of microorganisms that have inducible enzymes, there will be two growth phases, one on glucose followed by one on the less common sugar. There is a brief delay while the needed enzymes are synthesized. Such a two phase growth is known as diauxic growth.

Catabolite repression is a type of positive control of transcription, since a regulatory protein affects an increase (upregulation) in the rate of transcription of an operon. The process was discovered in E. coli and was originally referred to as the glucose effect because it was found that glucose repressed the synthesis of certain inducible enzymes.  When bacterium was grown in limiting amounts of glucose and lactose the plot of the bacterial growth resulted in a diauxic growth curve which showed two distinct phases of active growth. During the first phase of exponential growth, the bacteria utilize glucose as a source of energy until all the glucose is exhausted. Then, after a secondary lag phase, the lactose is utilized during a second stage of exponential growth.

The Diauxic Growth Curve of E. coli grown in limiting concentrations of a mixture of glucose and lactose

During the period of glucose utilization, lactose is not utilized because the cells are unable to transport and cleave the disaccharide lactose. Glucose is always metabolized first in preference to other sugars. Only after glucose is completely utilized is lactose degraded. The lactose operon is repressed even though lactose (the inducer) is present. The ecological rationale is that glucose is a better source of energy than lactose since its utilization requires two less enzymes.

Only after glucose is exhausted, the enzymes for lactose utilization synthesized. The secondary lag during diauxic growth represents the time required for the complete induction of the lac operon and synthesis of the enzymes necessary for lactose utilization (lactose permease and beta-galactosidase). Only then does bacterial growth occur at the expense of lactose. Since the availability of glucose represses the enzymes for lactose utilization, this type of repression became known as catabolite repression or the glucose effect.

In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP.  So Glucose inhibits the synthesis of cyclic AMP (cAMP), which is required for the initiation of transcription of the lac operon. cAMP is required to activate an allosteric protein called CAP (catabolite activator protein) and stimulates the binding of RNAp polymerase to the promoter for the initiation of transcription.

Thus, to efficiently promote transcription of the lac operon, lactose must be present to inactivate the lac repressor and cAMP must be available to bind to CAP that further bind to DNA and facilitate transcription. 

In the presence of glucose, adenylate cyclase (AC) activity is blocked. AC is required to synthesize cAMP from ATP. Therefore, if cAMP levels are low, CAP is inactive and transcription does not occur. In the absence of glucose, cAMP levels are high, CAP is activated by cAMP, and transcription occurs (in the presence of lactose).

Synchronous or synchronized culture

Synchronous or synchronized culture is a microbiological culture that contains cells that are all in the same growth stage. Non-synchronous cultures have cells in all stages of the cell cycle. Obtaining a culture with a unified cell-cycle stage is very useful for biological research. Since cells are too small for certain research techniques, a synchronous culture can be treated as a single cell.  Synchronous cultures have been extensively used to address questions regarding cell cycle and growth, and the effects of various factors on these.

Synchronous cultures can be obtained in several ways:

External conditions can be changed, so as to arrest growth of all cells in the culture, and then changed again to resume growth. The newly growing cells are now all starting to grow at the same stage, and they are synchronized.

Providing Limiting conditions for some time

For example, for photosynthetic cells light can be eliminated for several hours and then re-introduced. Another method is starvation of cells by eliminating an essential nutrient such as phosphate from the growth medium and later to re-introduce it. One the limiting factor is re-introduced, all the cells will start growing together and will grow in same phase.

Adding growth inhibitors for some time

Cell growth can also be arrested using chemical growth inhibitors. After growth has completely stopped for all cells, the inhibitor can be removed from the culture and the cells then begin to grow synchronously. Nocodazole, for example, is often used in biological research for this purpose.  This method has the disadvantage that the chemical molecule has to be completely removed to initiate synchronous growth.

Size dependent sorting to obtain inoculum

Cells in different growth stages have different physical properties. Cells in a culture can thus be physically separated based on their density or size. This can be achieved using centrifugation (for density) or filtration (for size).

Population of cells is fractio­nated on the basis of size. The cells are filtered so that smallest cells pass through the filter. These small cells are the youngest, and must go through their whole life cycle before dividing. Alternatively, the largest cells, which are ready to divide, may be retained or retarded by a filter. These are then collected separately and used to obtain a synchronous culture.

In the Helmstetter-Cummings technique, a bacterial culture is filtered through a membrane. Most bacteria pass through, but some remain bound to the membrane. Fresh medium is then applied to the membrane and the bound bacteria start to grow. Newborn bacteria that detach from the membrane are now all at the same stage of growth; they are collected in a flask that now harbors a synchronous culture.

The most widely used method for obtaining synchronous cultures is the Helmstetter-Cummings technique. A population of cells is passed through a membrane filter of pore size small enough to trap bacteria in the filter. The filter is then inverted, and fresh nutrient medium-is allowed to flow through it.  Loosely associated bacteria are washed from the filter.  Most bacteria pass through, but some remain bound to the membrane. Fresh medium is then applied to the membrane and the bound bacteria start to grow. Newborn bacteria that detach from the membrane are at the same stage of growth and will divide synchronously.   The method has one disadvantage, that the population size will be very small.

Helmstetter - Cummings filter pad technique

Instead of filtra­tion, density gradient centrifugation is also used to separate the cells. A population of unsynchronised cells is separated into fractions, each composed of the cells of the same density and at the same stage in their life cycle.