Bacterial Transformation

Bacterial transformation usually refers to a specific type of mutation taking place in bacteria.

In fact, it results from DNA of a bacterial cell penetrating to the host cell and becoming incorporated right into the genotype of the host.

The era stretching over 1940s witnessed and recognized that the prevailing inheritence in micro-organisms (bacteria) was adequately monitored and regulated basically by the same mechanisms as could be seen in higher eukaryotic organisms.

Interestingly, it was duly realized that bacteria to designate a ‘useful tool’ to decepher the intri-cate mechanism of heredity as well as genetic transfer ; and, therefore, employed extensively in the overall genetic investigative studies.

Griffith’s Experimental Observations* : Griffith (1928), a British Health Officer, carefully injected mice with a mixture comprising of two different kinds of cells, namely :

(a) A few rough (i.e., noncapsulated and nonpathogenic) pneumococcal cells, and (b) A large number of heat-killed smooth (i.e., capsulated and pathogenic cells.)

Living smooth pneumococci cells — usually causes pneumonia is human beings and a host of animals.

‘Rough’ and ‘Smooth’ — invariably refer to the ensuing surface texture of the colonies of the respective cells.

Consequently, the mice ultimately died of pneumonia, and ‘live smooth cells’ were meticu-lously isolated from their blood. Thus, one may observe critically that there could be certain cardinal factor exclusively responsible for the inherent pathogenicity of the smooth bacteria ; and it had eventu-ally transformed these organisms into pathogenic smooth ones.

Griffith also ascertained that the transforming factor might have been sailed from the trans-formed cells right into their progeny (i.e., offspring), and hence the inheritence of the characteristic features of a gene. Fig. 6.4 depicts the experiment of Griffith.

* From the Office of Technology Assessment.

There are two types of pneumococcus, each of which can exist in two forms :

Type II Type III

R_II S_II R_III S_III

where R represents the rough, nonencapsulated, benign form ; and

S represents the smooth, encapsulated, virulent form.

The experiment consists of four steps :

S

Mice injected with the virulent S die._III Mice injected with nonvirulent R do no become infected._II

S

The virulent is heat-killed. Mice injected with it do not die.S_III Living

When mice are injected with the nonvirulent R and the heat-killed , they die. Type II bacteria wrapped in type III capsules are recovered from these mice.

II

SIII

Fig. 6.4. The Griffith Experiment

[Adapted From : The Office of Technology Assessment.]

Avery, Mcleod, and McCarty (1944) adequately ascertained and identified the aforesaid ‘trans-forming principle’ as DNA. It is pertinent to mention here that these noted microbiologists rightly defined DNA as — ‘the critical chemical substance solely responsible for heredity’.

Transformation : Transformation may be referred to as – ‘a type of mutation occurring in bacteria and results from DNA of a bacterial cell penetrating the host cell, and ultimately becom-ing incorporated duly right into the ‘genotype’ of the host’.

In other words, transformation is the process whereby either ‘naked’ or cell-free DNA essen-tially having a rather limited extent of viable genetic information is progressively transformed from one

bacterial cell to another. In accomplishing this type of objective the required DNA is duly obtained from the ‘donor cell’ by two different modes, such as : (a) natural cell lysis ; and (b) chemical extraction.

Methodology : The various steps that are involved and adopted in a sequential manner are as enumerated under :

(1) DNA once being taken up by the recipient cell undergoes recombination.

(2) Organisms (bacteria) duly inherited by specific characteristic features i.e., markers received from the donor cells are invariably regarded to be transformed.

Example : Certain organisms on being grown in the persistent presence of dead cells, culture filtrates, or cell extracts of a strain essentially having a ‘close resemblance (or similarity)’, shall definitely acquire, and in turn would distinctly and predominantly transmit a definite characteristic feature(s) of the related strain (i.e., with close resemblance).

(3) DNA gets inducted via the cell wall as well as the cell membrane of the specific recipient cell.

(4) Molecular size of DNA significantly affects the phenomenon of transformation. There-fore, in order to have an extremely successful transformation of DNA the corresponding mo-lecular weights (DNA) must fall within a range of 300,000 to 8 million daltons.

Extraction of donor DNA fragments, after cell lysis by chemical or mechanical means

One strand of donor DNA degraded on binding

Binding of donor DNA fragments to

competent recipient cell

Competent recipient cell

Integration of single strand of donor DNA

Cell division, replication of DNA strands

Transformed Cell Donor cell

Fig. 6.5. Major Steps Involved in Bacterial Transformation

(5) Importantly, the actual number of ‘transformed cells’ virtually enhanced linearly with defi-nite increasing concentrations of DNA. Nevertheless, each transformation invariably comes into being due to the actual transfer of a single DNA molecule of the double-stranded DNA.

(6) Once the DNA gains its entry into a cell, one of the two strands gets degraded almost in-stantly by means of the available enzymes deoxyribonucleases ; whereas, the second strand particularly subject to base pairing with a homologous segment of the corresponding recipient cell chromosome. Consequently, the latter gets meticulously integrated into the recipient DNA, as illustrated beautifully in Figure 6.5.

(7) Transformation of Closely Related Strains of Bacteria : In reality, the transformation of closely related strains of bacterial could be accomplished by virtue of the fact that comple-mentary base pairing predominantly occurs particularly between one strand of the donor DNA fragment and a highly specific segment of the recipient chromosome.

However, the major steps involved in the bacterial transformation have been clearly shown in Fig. 6.5.

Examples : The bacterial species which have been adequately transformed essentially include : Bacterial species : Streptococcus pneumoniae (Pneumococcus)

Genera : Bacillus ; Haemophilus ; Neisseria ; and Rhizobium 6.2.9. Bacterial Transcription

Bacterial transcription refers to the – ‘synthesis of a complementary strand of RNA particularly from a DNA template’.

In fact, there exists three different types of RNA in the bacterial cells, namely : (a) messenger RNA ; (b) ribosomal RNA, and (c) transfer RNA.

Messenger RNA (mRNA) : It predominantly carries the ‘coded information’ for the produc-tion of particular proteins from DNA to ribosomes, where usually proteins get synthesized.

Ribosomal RNA : It invariably forms an ‘integral segment’ of the ribosomes, that strategically expatiates the cellular mechanism with regard to protein synthesis.

Transfer RNA : It is also intimately and specifically involved in the protein synthesis.

Process of Bacterial Transcription :

Importantly, during the process of bacterial transcription, a strand of messenger RNA (mRNA) gets duly synthesized by the critical usage of a ‘specific gene’ i.e., a vital segment of the cell’s DNA–as a template, as illustrated beautifully in Figure : 6.6. Thus, one may visualize the vital and important

‘genetic information’ adequately stored in the sequence of nitrogenous bases (viz., A, T, C and G) of DNA, that may be rewritten so that the same valuable ‘genetic information’ appears predominantly in the base sequence of mRNA.

Examples :

(1) In the DNA replication phenomenon, it has been duly observed that a G in DNA template usually dictates a C in the mRNA ; and a T in DNA template invariably dictates an A in the mRNA.

(2) An A in DNA template normally dictates a uracil (U) in the mRNA by virtue of the fact that RNA strategically contains U instead of T*.

* U essentially possesses a chemical structure which is slightly different from T ; however, it base-pairs more or less in the same manner.

(3) In an event when the template segment of DNA essentially possess the base sequence ATG-CAT, consequently the strategic newly synthesized mRNA strand shall predominantly would bear the complementary base sequence UAC GUA.

RNA and RNA

Fig. 6.6. Diagramatic Description of Process of Transcription

[Adapted From : Tortora GJ et al. : Microbiology : An Introduction, The Benjamin/

Cummings Pub. Co. Inc., New York, 1995].

Salient Requirements : The salient requirements for the process of bacterial transcription are as enumerated under :

(1) It essentially needs two cardinal components, namely : (a) RNA– polymerase — an ‘enzyme’, and

(b) RNA–nucleotides — a regular and constant supply.