microbiology at a glance: November 2009

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GENETIC CODE IN MITOCHONDRIA

Genetic code is universal and does not undergo any change. But during 1980s, it was discovered that the genetic code of mitochondria of yeasts, Drosophila and mammals differs from the universal genetic code. The mitochondrial genome is usually circular DNA molecule and contains complete genetic system. Anderson et al for the first time through DNA sequencing technique presented the complete sequence of 16,569 nuclear, of human mitochondrial genome. The human mitochondrial genome differs from that of nuclear, chloroplast and bacterial genome in the following respect:
(i) Unlike others, every nucleotide appears to be a part of coding sequence.
(ii) The normal codon-anticodon pairing rules are relaxed in mitochondria, therefore, many there are 22 t RNAs in mitochondria and about 55 t RNAs in universal code.
(iii) The genetic code is different from those of the same codons in other genomes. However, the genetic code of mitochondria differs from the universal genetic code on the other hand the mitochondrial genetic code in different group of organisms also differ. For example, UGA which is a stop codon elsewhere is read as tryptophan in mitochondria of yeasts, Drosophila, mammals and protozoa, but as stop codon in mitochondria. The codon AGG normally codes for arginine, but it acts as stop codon in mitochondria of mammals, and codes for serine in Drosophila, UGA codon which codes for isoleusine, specifies methionine in yeasts, Drosophila and mammal’s mitochondria.

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GENE MACHINE

Recently, fully automated commercial instrument called automated polynucleotide synthesizer gene machine is available in market which synthesizes predetermined polynucleotide sequence. Therefore, the gene can be synthesized rapidly and in high amount. For example, a gene for t RNA can be synthesized within a few days through gene machine. It automatically synthesizes of single stranded DNA under the control of microprocessor. The working principle of a gene machine includes (i) development of insoluble silica based support in the form of beads which provides support for solid phase synthesis of DNA chain, and (ii) development of stable deoxyribonucleoside phosphoramidites as synthons which are stable to oxidation and hydrolysis, and ideal for DNA synthesis.

The mechanism of a gene machine is four separate reservoirs containing nucleotides (A,T,C and G) are connected with a tube to a cylinder (synthesiser column) packed with small silica beads. These beads provide support for assembly of DNA molecules. Reservoirs for reagent and solvent are also attached. The whole procedure of adding or removing the chemicals from the reagent reservoir in time is controlled by microcomputer control system i.e. microprocessor.

If one desires to synthesize a short polynucleotide with a sequence of nucleotides T,G,C, the cylinder is first filled with beads with a single T attached. Thereafter, it is flooded with G from the reservoir. The right hand side of each G is blocked by using chemicals from the reservoir so that its attachment with any other Gs can be prevented. The remaining Gs which could not join with Ts are flushed from the cylinder. The other chemicals are passed from the reagent and solvent reservoirs so that these can remove the blocks from G which is attached with the T. In the same way this cycle is repeated by flooding with, C from reservoir into the cylinder. Finally the sequence T.G.C is synthesized on the silica beads which is removed chemically later on.

The desired sequence is entered on a key board and the microprocessor automatically opens the valve of nucleotide reservoir, and chemical and solvent reservoir. In the gene machine the nucleotides are added into a polynucleotide chain at the rate of two nucleotides per hour. By feeding the instructions of human insulin gene in gene machine insulin has been synthesized.

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ARTIFICIAL SYNTHESIS OF A GENE FOR BACTERIAL TYROSINE tRNA

In 1975, Khorana and co-workers completed the synthesis of a gene for E.coli tyrosine
t RNA precursor. E.coli t RNA precursors are formed from the larger precursors. The tyrosine t RNA precursor has 126 nucleotides. They sunthyesized the complete sequence of DNA duplex coding for tyrosine –t RNA precursor of E.coli. though these segments are not the proper structural gene yet are the regions involved in its regulation.

Twenty six small oligonucleotide DNA segment giving rise to t RNA precursor was synthesized which were arranged into six double stranded fragments each containing single stranded ends. These six fragments were joined to give rise complete gene of 126 base pairs for tyrosine t RNA precursor of E. coli.

Khorana completely synthesized a biologically functional tyrosine t RNA suppressor gene of E.coli which was 207 base pairs long and contained (i) a 51 base pairs long DNA corresponding to promoter region, (ii) a 126 base pair long DNA corresponding to precursor region of t RNA, (iii) a 25 base pair long DNA including 16 base pairs contained restriction site for Eco RI. This complete synthetic gene was joined in phage lambda vector which in turn was allowed to transfect E.coli cells. After transfection phage containing synthetic gene successfully multiplied in E.coli.

Khorana made the phosphodiester approach for synthesizing the oligonucleotides of the biologically active t RNA. The demerits of this approach are: (i) the completion of reaction in long time, (ii) rapidly decrease in yield with the increase in chain length, and (iii) time taking procedure of purification.

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OVERLAPPING GENES (GENES WITHIN GENES)

In 1940s, Beadle and Tatum proposed one-gene-one protein hypothesis which explains that one gene encodes for one protein. However, if one gene consists of 1,500 base pairs, a protein of 500 amino acids in length would be synthesized. In addition, if the same sequence read in two different ways, two different amino acids would be synthesized by the same sequence of base pairs. It means, the same DNA sequence can synthesize more than one proteins at different time. It was realized for the first time when the total number of proteins synthesized by X174 exceeded from the coding potential of the phage genome. A similar phonemenon is found in the tumour virus SV40 where the total molecular weight of proteins (i.e. VP1, VP2 and VP3) synthesized by SV40 genes is much more than the size of the DNA molecule (5200 base pairs i.e. 1,733 codons). From this observations the concept of overlapping genes has emerged.

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SPLIT GENES

During 1970, in some mammalian viruses (e.g. adenoviruses) it was found that the DNA sequences coding for a polypeptide were not present continuously but were split into several pieces. Therefore, these genes were variously named as split genes or introns interrupted genes or interventing sequences inserts junk DNA. For the discovery of split genes in adenoviruses and higher organisms, Richards J.Roberts and Phillip sharp were awarded Nobel Prize in 1993.

We can see a DNA sequence codes for m RNA but the complete corresponding sequence of DNA is not found in m RNA. Certain sequences of DNA are missing in m RNA. The sequences present in DNA but missing in m RNA are called intervening sequences or introns, and the sequences of DNA found in RNA are known as exons. The exons code for m RNA.

Before the discovery of split genes in 1977, all the genes analysed in detail were the bacterial genes. Bacteria were considered to resemble the simpler cell from which eukaryotes must have been evolved. Now, it is supposed that split genes are the ancient condition and bacteria lost their introns only after evolution of most of their proteins. Evidence for the ancient origin of introns has been obtained by the examination of the gene that encodes the ubiquitous enzyme, triose phosphate isomerase (TPI). The TPI is coded by a gene that contains six introns (in vertebrates), five of these are present at the same position as in maize. The TPI play a key role in cell metabolism that catalyses the interconversion of glycolysis and glucogenesis. By comparing this enzyme in various organisms it appears that the TPI evolved before the divergence of prokaryotes and eukaryotes from a common ancestor cell-i.e. progenote (Nyberg and Cronhjort, 1992).

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GENETIC DIVERSITY

There is genetic diversity among the microorganisms. The total number of genes in one microorganism differ from that in the other. For example, bacteriophage R17 and QB consist of only three genes. SV40 contains 5-10 genes. Ecoli consists about 4000 genes on about 1mm long chromosome. On the other hand the length of each gene varies in base composition. It may vary between 100 and 3000 nucleotides giving the complexity to proteins.

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REPLICATION Of DNA

one of the most important properties of DNA is that it forms its additional identical copies. The process of forming its replica copy is called replication.Replication is the basis of evolution of all morphologically complexed forms of life. Howard and Pelc demonstrated that in eukaryotes replication occurs during interphase between mitotic cycles and also during interphase are seperated into two daughter cells, and thus equal number of chromosomes is maintained. However, replication does not occur during entire anaphase but is confined only to synthesis (s) phase. There is a post-mitotic gap(G1) between the telophase and S phase. A second premitotic gap (G2) is between the S phase and prophase. Only S phase involves replication process. The G1 phase is most variable and in many eukaryotic cells it is completed within 3 to 4 hours or even months depending on physiological conditions. Mostly DNA synthesis is accomplished in 7 to 8hours. In bacteria growing at long phase, DNA synthesis occurs from the time a cell originates to it give rise to daughter cells. It is networthy that bacteria divide only through binary fission.

In general DNA carries out to important functions such as heterocatalytic function and autocatalytic function. The heterocatalytic function is protein synthesis directed by DNA, and autocatalytic function is the synthesis of its own DNA into replica copies. However replication of DNA in prokaryotes differ from that of eukaryotes.

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IMPORTANT FEAUTURES OF GENETIC CODE

Here are some important features of genetic code :

1. sixty one codons correspond to amino acids.
2. Four codons are the signals. There are three stop codons (UAA, UAG, UGA) and one start codon (AUG). Rarely, GUG also acts as start codon.
3. Amino acids with similar structural property consist of related codons, therefore the aspartic acid codons (GAU and GAC) are related to glutamic acid codons (GAA and GAG). Similarly, the codons of phenylalanine (UUU, UUC), tyrosine (UAU, UAC) and tryptophan (UGG) start with uracil. This characteristic of codons facilitates to minimize the effects of mistakes arising during translation or mutagenic base substitution.
4. For many synonym codons specifying the same amino acid the first two bases of the triplet are constant while the third varies. For example, all the codons starting with CC (CCU, CCC, CCG) specify praline, and all codons starting with AC (ACU, ACC, ACA,ACG) specify threonine. The flexibility in third codon may be to minimize errors.

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GENETIC ENGINERING

Moreover, for centuries human being have been altering the genetic make up of organisms by selective breeding of plant and animals. The deliberate modification in genetic material of an organism by changing the nucleic acid directly is called genetic engineering or gene cloning or gene manipulation and is accomplished by several methods which are collectively known as recombinat DNA (rDNA) technology. Recombinant DNA technology begins a new area of research and applied aspects of biology. Therefore, it is a part of biotechnology which is gaining momentum and much boost in recent years.

However, in breeding programmes much work has been done on alteration of nucleotides by several parasexual or conjugation methods indifferent group of organisms. Now –a-day, a large number of mutagenic agents are available that mutate the genes. It is likely that the changed genes may be beneficial, neutral or lethal. Moreover, the conventional breeding programmes are time taking for making sure that the genes have been altered. In contrast, the rDNA technology has solved several problems which hardly or never are possible through the conventional methods.

Gene cloning or genetic engineering can be defined as changing of genes by using in vitro processes. A unified definition of genetic engineering has been given by smith (1996) as the formation of new combinations of heritable material by the insertion of nucleic acid molecules produced by whatever means outside the cell, into any virus, bacterial plasmid or other vector system so as to allow their incorporation into a host organism in which they do not naturally occur but in which they are capper of continued propagation. In brief, gene technology is the modification of the genetic properties of an organism by sing rDNA technology. Genes are like the biological software filled with programme that govern the growth, development and function of organism. By changing in programme of the software it is possible to bring about alteration in the characters of a given organism (smith,1996).

A gene of known function can be isolated from its normal location by biochemical methods in vitro. Moreover, a gene can be synthesized by using gene machine. The isolated genes can be transferred into the microbial cells (that of course do not contain) via a suitable vector. The transferred gene replicates normally and is handed over to the next progeny over generations. After confirmation for its presence through biochemical procedures clone of the same cell is produced.

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ARTIFICIAL SYNTHESIS OF GENE

For the first time in 1995, Michelson chemically synthesized a dinucleotide in laboratory. Later on in 1970, Har Govind Khorana and K.L. A garwal for the first time chemically synthesized gene coding for tyrosine tRNA are the smallest genes containing about 80 nucleotides. In 1965, Robert w. holley and coworkers worked out first molecular structure of yeast alanine tRNA. This structure lent support to Khorana in deduction of structure of the gene. A gene is responsible for encoding mRNA, and mRNA for polypeptide chain. If the structure of a polypeptide chain is known, the structure of mRAN from genetic code dictionary and in turn the structure of gene can easily be worked out. There are two approaches for artificial synthesis of the gene, by using chemicals and through mRNAs.

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GENE REGULATION

The DNA of a microbial cell consists of genes, a few to thousands, which do not express at the same time. At a particular time only a few genes express and synthesize the desired protein. The other genes remain silent at this moment and express when required. Requirement of gene expression is governed by the environment in which they grow. This shows that the genes have a property to switch on and switch off.
20 different amino acids constitute different protein. All are synthesized by
codons. Therefore, synthesis of all the amino acids requires energy which is useless because all the amino acids constituting proteins are not needed at a time. Hence, there is need to control the synthesis of those amino acids which are not required. By doing this the energy of a living cell is conserved and cells become more competent. Therefore, a control system is operative which is known as gene regulation.

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GENE EXPRESSION

The DNA has two important role in the cell, first is replication and the second is expression. Gene expression is accomplished by a series of events. The information contained in DNA is converted into molecules that determine the metabolism of the cell.

During the process of gene expression DNA is the first copied into an RNA molecule which determines the amino acid sequences of a molecule of a protein. The RNA molecules are synthesized by using a portion of base sequences of single strand of double stranded DNA. This single strand is called template. Hence formation of an RNA transcript is facilitated by an enzyme, RNA polymerase. Therefore the process of synthesis of an RNA molecule corresponding to a gene is called transcription. By using base sequences and RNA molecule , proteins are synthesized in a definite order. Production of an amino acid sequence from an RNA base sequence is called translation. After completion of translation proteins are synthesized. Therefore, gene expression refers to protein synthesis through two events, transcription and translation.

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MICROBIAL GENETICS

In prokaryotes the method of cell division differs from that of eukaryotes. No meiosis or mitosis occurs in prokaryotes because of lack of nucleus. In contrast, in prokaryotes and a few eukaryotic microorganisms most of genetical and molecular works, have been carried out because of easy in handling, fast growth rate, immediate observable characters under any change in conditions and least complex life forms.

Like higher plants and animals, bacteria and the other protists transmit characters to their progenies and in onward generations. Apart from inheritance of characters they also show variability in progenies. These changes are reffered to as phenotype (morphological changes) and genotype (genetic changes). The genotype always remains constant however changes occur through mutation which results in morphological changes or phenotypes in progenies.

In addition, the phenotypic expression depends on the environmental conditions as well. Example is phenotypic changes in agrobacterium radiobacter takes place if it is grown on two different growth media. Mucoid colonies are formed on source salt medium and non-mucoid colonies develop on trypticase soy-agar medium. Secondly yeast produces ethanol when grown in a oxygen deficient conditions, and it is increased its bioms and does not produce ethanol when grown in the presence of sufficient oxygen.

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UNITS OF A GENE

After much extensive work done by the molecular biologists the nature of gene became clear. A gene can be defined as a poly nucleotide chain that consists of segments each controlling a particular trait. Now, genes are considered as a unit of function (cistron), a unit of recombination (recon) and a unit of mutation (muton).

Cistron: A single mRNA is transcribed by a single gene. Therefoer, one-gene-one mRNA hypothesis was put forth. Exceptionally, a single mRNA is also transcribed by more than one gene and it is said to be polycistronic. Therefore, the concept has been given as one-gene-one protien hypothesis. The protiens are the polypeptide chains of amino acids translated by mRNA. Therefore, it has been correctly used as one-gene-one polypeptide hypothesis.

Recon: I n a cistron the recombinational units may be more than one. Thus the smallest unit capable of undergoing recombination is called as recon.

Muton: It may be defined as '' the smallest unit of DNA which may be changed is the nucleotide''. Therefore, cistron is the largest unit in size followed by recon and muton. This can be explained that a gene consists of several cistron, a cistron contains many recons, and a recon a number of mutons. However if the size of a recon is equal to muton, there would be no possibility in recon for consisting of several mutons.

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GROWTH OF MICROBES

Growth is defined as an orderly increase in cellular components. Microorganisms grow in a variety of physical and chemical environments. A number of methods are available for microbial growth. The choice depends upon the measurement, objectives and on available techniques usefulness. In some cases of industrial fermentation which contain complex media, indirect methods for estimation need to be used however, no matter what method is used, considerable care is required in interpreting the results. Bacterial growth can be measured either by colony counting or cell counting, weighing cell i.e. cell mass measurement or by cell activity.

Cell mass is directly proportional to cell number. This can be obtained after centrifugation of a known volume of culture and weighing the pellet obtained. This is called fresh weight but dry weight of cells is obtained by drying of the pelleted cells at 90-110 degrees centigrade overnight.
The cell mass and number is also obtained by using optical density method. Turbidity is developed in the liquid medium due to the presense of cells which make cloudy appearence to the eyes. The more the sample, more cells are present, hence more light is scattered. Turbidity can be measured with a photometer or a spectrometer device that detect the amount of unscattered light recorded in photometer unit.

If the concentration of the cell in the sample is high, light scattered away from the detecting unit by one to one cell can be rescattered back by another. Hence, the one to one correspondense between cell number and turbidity does not follow linearity. Secondly, dead cells also interfere during measurement. Hence this method is reasonable accurate only for measurement of microbial growth till early log phase.

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CLASSIFICATION OF BACTERIA

Although the bacterial structures are quite simple but their classification is quite difficult. However, due to readily available devices in the field of molecular biology it make the taxonomy much easy.The use of old staining techniques such as Gram's staining for differentiation of bacteria on the basis of cell wall structures, their morphological, biochemical characters for proper characterization were known earlier. The use of molecular biological techniques such as restriction fragment length polymorphism, DNA-DNA hybridization and 16S rRNA sequencing make the studies more easy as far as the phylogeny of the bacterial strains are concerned. As a result there has been a considerable revival of interest in bacterial taxonomy.

The bacterial taxonomy has a rapid and radical change due to involvement of modern molecular biological techniques. This is why it has now refined. Much care has also been taken in ascertaining the history of species so that the species names reflect the original description, rather than a popularly used name.As a result of such activity, the species names assigned to bacteria may change.

The basic constitution of bacterial species is different from higher organism due to the reason that in later forms the species is defined as those which shares many common features and its members breed to produce fertile offspring. While in case of bacteria, they reproduce only asexually by binary fission, hence the above concept does not suit in bacteriology.

The use of computers in bacteriology and in particular taxonomy gave an advantage to resolve the impact of the techniques of numerical taxonomy. This is based on comparing the strains of bacteria for the large number of characters. The more closely related organisms are to have the more characteristics in common. A similarity matrix can be drawn up depicting the degree of relationship between all the species examined and it is then presented in the form of dendogram.

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MICROBIAL DIVERSITY

The current list of the world's biodiversity is quite incomplete and that of viruses, microorganisms and invertebrates is especially deficient. The fungal diversity indicate the total number of species in a particular taxonomic group. The estimates of 1.5 million fungal species is based principally on a ratio of vascular plants of fungi of about 1:6.

Attempts to estimate total numbers of bacteria, archaea, and viruses even more problematical because of difficulties such as detection in and recovery from the environment,incomplete knowledge of obligate microbial associations. The microbial diversity, therefore appears in large
measure of reflect obligate or facultative associations with higher organisms and to be determined by the spatio-temporal diversity of their hosts or associates.

The perception of microbial diversity is being radically altered by DNA technique such as DNA-DNA hybridisation, nucleic acid fingerprinting and methods of assessing the outcome of DNA probing, and perhaps most important at present is 16S rRNA sequencing.

Biological diversity or biodiversity is actually evolved as a part of the evolution of organisms and the smallest unit of microbial diversity is a species. Bacteria due to lack of sexuality, fossil records etc., are defined as a group of similar strains distinguished sufficiently from other similar groups of strains by genotypic, phenotypic, and ecological characteristics.

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2009 ·microbiology at a glance by TNB