The first proof that genetic information is stored in DNA rather than proteins was the 1944 demonstration

by Oswald Avery, Colin MacLeod, and Maclyn McCarty that DNA was responsible for transformation in pneumococci was extension of Griffiths experiment. It established that transforming principle was nothing but DNA. The mechanism of transformation has been studied in considerable detail in S. pneumoniae, Bacillus subtilis, Haemophilus influenzae, The basic process is similar in all four species; however, variations in the mechanism occur in each species. S. pneumoniae and B. subtilis will take up DNA from any source,

TRANSFORMATION

and Neisseria gonorrhoeae.

whereas H. influenzae and N. gonorrhoeae will only take up their own DNA or DNA from closely related species. H. influenzae and N. gonorrhoeae will only take up DNA that contains

a special short nucleotide-pair sequence (11 base pairs in Haemophilus; 10 in Neisseria) that is present in about 600 copies in their respective genomes. Even in the bacterial species that have the ability to take up DNA from their environment, not all cells can do so. Indeed, only cells that are expressing the genes that encode proteins required for the process are capable of taking up DNA. SBT&HS

 These bacteria are said to be competent, and the proteins that mediate the transformation process are called competence (Com) proteins. Bacteria develop competence during the late phase of their growth cycle, when cell density is high but before cell division stops. The process by which cells become competent is understood best in B. subtilis, where small peptides called competence pheromones are secreted by cells and accumulate at high cell density. High concentrations of the pheromones induce the expression of the genes encoding proteins required for transformation to occur. The competence genes are located in clusters, and each cluster is designated by an alphabet e.g., A, B, C. In each cluster first gene is designated A, the second B, and so on. Thus, the protein encoded by the first gene in the fifth cluster is designated ComEA. ComEA and ComG proteins bind double-stranded DNA to the surfaces of competent cells. As the bound DNA is pulled into the cell by the ComFA DNA translocase (an enzyme that moves or translocates DNA), one strand of DNA is degraded by a deoxyribonuclease (an enzyme that degrades DNA), and the other strand is protected from degradation by a coating of single-stranded DNA-binding protein and RecA protein (a protein required for recombination). SBT&HS

 With the aid of RecA and other proteins that mediate recombination, the single strand of transforming DNA invades the chromosome of the recipient cell, pairing with the complementary strand of DNA and replacing the equivalent strand. The replaced recipient strand is then degraded. SBT&HS

 If the donor and recipient cells carry different alleles of a gene, the resulting recombinant double helix will have one allele in one strand and the other allele in the second strand. A DNA double helix of this type is called a heteroduplex (a heterozygous double helix); it will segregate into two homoduplexes when it replicates. The DNA molecules taken up by competent cells during transformation are usually only 0.2 to 0.5 % of the complete chromosome. Unless two genes are quite close together, they will never be present on the same molecule of transforming DNA.

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 P1 is a temperate bacteriophage (phage) that infects Escherichia coli and a some other bacteria. The virion is similar in structure to the T4 phage but simpler. It has an icosahedral head containing the genome attached at one vertex to the tail. The tail has a tube surrounded by a contractile sheath. It ends in a base plate with six tail fibres. The tail fibres are involved in attaching to the host and providing specificity. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacterium unlike other phages (e.g. the phage) that integrate into the host DNA. P1 has an icosahedral "head" containing the DNA attached to a contractile tail with six tail fibers. The P1 phage has gained research interest because it can be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA. Also, P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites (Cre-Lox recombination). The genome of the P1 phage is moderately large, around 93Kbp in length (e.g. T4- 169Kbp, - 48Kbp. SBT&HS

P1 Phage

 In the viral particle it is in the form of a linear double stranded DNA molecule. Once inserted into the host it circularizes and replicates as a plasmid. In the viral particle the DNA molecule is longer (110Kbp) than the actual length of the genome. It is created by cutting an appropriately sized fragment from a concatomeric DNA chain having multiple copies of the genome. Due to this the ends of the DNA molecule are identical. This is referred to as being "terminally redundant and this is important for the DNA to be circularized in the host. The genome contains two origins of replication, oriR which replicates it during the lysogenic cycle and oriL which replicates it during the lytic stage. P1 can exist within a bacterial cell as a circular DNA in that it exists by replicating as if it were a plasmid and does not cause cell death. Alternatively, in its lytic phase, P1 can promote cell lysis during growth resulting in host cell death. During lysogeny new phage particles are not produced. In contrast, during lytic growth many new phage particles are assembled and released from the cell. The P1 plasmid has a separate origin of replication (oriL) that is activated during the lytic cycle. SBT&HS

 Replication begins by a regular bidirectional theta replication at oriL but later in the lytic phase,it switches to a rolling circle method. This results in numerous copies of the genome being present on a single linear DNA molecule called a concatomer. The end of the concatomer is cut a specific site called the pac site or packaging site. This is followed by the packing of the DNA into the heads till they are full. The rest of the concatomer that does not fit into one head is separated and the machinery begins packing this into a new head. The location of the cut is not sequence specific. Each head holds around 110kbp of DNA, so there is a little more than one complete copy of the genome (~90kbp) in each head, with the ends of the strand in each head being identical. After infecting a new cell this "terminal redundancy" is used by the host recombination machinery to cyclize the genome if it lacks two copies of the lox locus. If two lox sites are present (one in each terminally redundant end) the cyclization is carried out by the cre recombinase. Once the complete virions are assembled, the host cell is lysed, releasing the viral particles.

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 Lambda Phage is a dsDNA virus that infects E. coli. The dsDNA is linear while it is in the head of the virus, but becomes circular upon infection of a host E. coli cell. The phage DNA contains ~50 Kilobase pairs that code for about 60 different proteins. The 60 viral proteins include 15 structural proteins that make up the capsid of the virus. Bacteriophage Lambda binds to the target E. coli cell, the J protein in the tail tip interacting with the lamB gene product of E. coli, a porin molecule which is part of the maltose operon. The linear phage genome is injected past the cell outer membrane. The DNA passes through a separate sugar transport protein (ptsG) in the inner membrane, and immediately circularises using the cos sites, 12-base G-C rich cohesive "sticky ends". The single-stranded nicks are ligated by host DNA ligase. Host DNA gyrase puts negative supercoils in the circular chromosome, causing A-T rich regions to unwind and drive transcription. Transcription starts from the constitutive PL, PR and PR' promoters producing the 'immediate early' transcripts. Initially these express the N and cro genes, producing N, Cro and a short inactive protein. SBT&HS

Lambda Phage

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 Cro binds to OR3 preventing access to the PRM promoter preventing expression of the cI gene. N binds to the two Nut (N utilisation) sites, one in the N gene in the PL reading frame, and one in the cro gene in the PR reading frame. The N protein is an antiterminator, and functions to extend the reading frames to which it is bound. When RNA polymerase transcribes these regions, it recruits the N and forms a complex with several host Nus proteins. This complex skips through most termination sequences. The extended transcripts (the 'late early' transcripts) include the N and cro genes along with cII and cIII genes, and xis, int, O, P and Q genes. The cIII protein acts to protect the cII protein from proteolysis by FtsH (a membrane-bound essential E. coli protease) by acting as a competitive inhibitor. This inhibition can induce a bacteriostatic state, which favours lysogeny. cIII also directly stabilises the cII protein. On initial infection, the stability of cII determines the lifestyle of the phage; stable cII will lead to the lysogenic pathway, whereas if cII is degraded the phage will go into the lytic pathway. Low temperature, starvation of the cells and high multiplicity of infection (MOI) are known to favor lysogeny. SBT&HS

 The integration of phage takes place at a special attachment site in the bacterial and phage genomes, called att. The sequence of the bacterial att site is called attB, between the gal and bio operons, and consists of the parts B-O-B', whereas the complementary sequence in the circular phage genome is called attP and consists of the parts P-O-P'. The integration itself is a sequential exchange and requires both the phage protein Int and the bacterial protein IHF (integration host factor). Both Int and IHF bind to attP and form an intasome, a DNA-proteincomplex designed for site-specific recombination of the phage and host DNA. The original B-O-B' sequence is changed by the integration to B-O-P'phage DNA-P-O-B'. The phage DNA is now part of the host's genome.

TRANSDUCTION

Transduction was discovered by Norton Zinder and Joshua Lederberg in 1952. They were studying auxotrophic strains of Salmonella typhimurium that required amino acid supplements to grow. One strain required phenylalanine, tryptophan, and tyrosine; the other required methionine and histidine. SBT&HS

 Neither strain could grow on minimal medium lacking these amino acids. However, when Zinder and Lederberg grew the strains together, rare prototrophs were produced. Moreover, when they grew the strains in medium containing DNase, but separated them in the two arms of a U-tube, prototrophic recombinants were still produced. The insensitivity to DNase ruled out transformation as the underlying mechanism, and the fact that cell contact was not required for the appearance of the prototrophs eliminated conjugation. Subsequent experiments showed that one of the strains was infected with a virus called bacteriophage P22 and that this virus was carrying genes from one cell (the donor) to another (the recipient). The rare prototrophs that Zinder and Lederberg detected were therefore due to recombination between bacterial DNA carried by the virus and DNA in the chromosome of the recipient cell. Later studies revealed that there are two very different types of transduction. In generalized transduction, a random or nearly random fragment of bacterial DNA is packaged in the phage head in place of the phage chromosome. SBT&HS

 In specialized transduction, a recombination event occurs between the host chromosome and the phage chromosome, producing a phage chromosome that contains a piece of bacterial DNA. Phage particles that contain bacterial DNA are called transducing

particles. Generalized transducing particles contain only bacterial DNA. Specialized transducing particles always contain both phage and
bacterial DNA.

Generalized Transduction

Generalized transducing phages can transport any bacterial gene from one cell to another thus, the name generalized transduction. The best known generalized transducing phages are P22 in S. Only about 1 to 2 % of the phage particles produced by bacteria infected with P22 or P1 contain bacterial DNA, and only about 1 to 2 percent of the transferred DNA is incorporated into the chromosome of the recipient cell by recombination. Thus, the process is quite inefficient; the frequency of transduction for any given bacterial gene is about 1 per 106 phage particles.

typhimurium and P1 in E. coli.

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Specialized Transduction

Specialized transduction is characteristic of viruses that transfer only certain genes between bacteria. Bacteriophage is the best-known specialized transducing phage; carries only the gal (required for the utilization of galactose as an energy source) and bio (essential for the synthesis of biotin) genes from one E. coli cell to another. The insertion site is between the gal genes and the bio genes on the E. coli The integrated chromosomethe prophagein a lysogenic cell undergoes rare (about one in 105 cell divisions) spontaneous excision, whereupon it enters the lytic pathway. Prophage excision can also be induced, for example, by irradiating lysogenic cells with ultraviolet light. Normal excision is essentially the reverse of the site-specific integration process and yields intact circular phage and bacterial chromosomes. SBT&HS

chromosome , which explains why only transduces these genes.

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 Occasionally, the excision is anomalous, with the crossover occurring at a site other than the original attachment site. When this happens, a portion of the bacterial chromosome is excised with the phage DNA and a portion of the phage chromosome is left in the host chromosome. These anomalous prophage excisions produce specialized transducing phage carrying either the gal or bio genes of the host. These transducing phage are denoted dgal (for defective phage carrying gal genes) and dbio ( defective phage carrying bio genes), respectively. They are defective phage particles because one or more genes required for lytic or lysogenic reproduction were left in the host chromosome. Because of the small size of the phage head, only bacterial genes located close to the prophage can be excised with the phage DNA and packaged in phage heads. Another specialized transducing phage, 80, integrates near the E. coli trp genes (required for the synthesis of the amino acid tryptophan); this phage transduces trp markers. If specialized transducing particles are formed during prophage excision, they should be produced only when lysogenic cells enter the lytic pathway. Indeed, transducing particles are not present in lysates produced SBT&HS

from primary lytic infections. The frequency of transducing particles in lysates produced by induction of lysogenic cells is about one in 106 progeny particles; therefore, these lysates are called Lft (lowfrequency transduction) The fate of the dgal and dbio DNA molecules after their injection into new host cells will depend on which genes are missing. If genes for lytic growth are missing, but an att site and int

lysates.

able to integrate into the host chromosome. However, they will not be able to reproduce lytically unless a wildtype , acting as a helper phage, is present. If the int gene is missing, the defective phage chromosome will be able to integrate only in the presence of a wild-type helper. If a dgal +phage infects a gal - recipient cell, integration of the dgal +will produce an unstable gal+/gal - partial diploid, whereas rare recombination events between gal +in the transducing DNA and gal- in the recipient chromosome will produce stable gal+ If the ratio of phage to bacteria is high, recipient cells will be infected with both wild-type phage and dgal+ thus, these cells will be double lysogens carrying one wild-type prophage and one dgal prophage. SBT&HS

(integrase) gene are present, the defective chromosomes will be

transductants.

 The resulting transductants will be gal+/gal - partial diploids. If the gal+/gal - transductants are induced with ultraviolet light, the lysates will contain about 50 %dgal particles and 50 % wild

particles.

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 Both prophages will replicate with equal efficiency using the gene products encoded by the wild genome. Such lysates are called Hft (high-frequency transduction) lysates. Hft lysates dramatically increase the frequency of transduction events; therefore, Hft lysates are used preferentially in transduction

experiments.

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