1. GENETIC of BACTERIA
Professor M.Boychenko

2. Bacterial genome
DNA molecules that replicate as discrete genetic units in bacteria are called replicons. In some bacterial strains, the chromosome is the only replicon present in the cell. Other bacterial strains have additional replicons, such as plasmids and bacteriophages.

3. Chromosomal DNA
Bacterial genomes vary in size from about 0.4 x 109 to 8.6 x 109 daltons (Da),
Most bacteria have a haploid genome, a single chromosome consisting of a circular, double stranded DNA molecule.

4. Bacterial genome
However linear chromosomes have been found in Gram-positive Borrelia and Streptomyces spp.,
and one linear and one circular chromosome is present in the Gram-negative bacterium Agrobacterium tumefaciens.
V.cholerae possesses 2 circular chromosomes

5. plasmids
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952
Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major kingdoms, Archea, Bacteria and Eukaryote

6. plasmids
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA,which is capable of replicating independently of the chromosomal DNA. In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organism.

7. PLASMIDS
Plasmid size varies from 1 to over 200 kilobase pairs (kbp). The number of identical plasmids within a single cell can range anywhere from one to even thousands

8. types of plasmids
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance; whereas episome integrate into the host chromosome.

9. types of plasmids
One way of grouping plasmids is by their ability to transfer to other bacteria.

10. Types of plasmids
Conjugative plasmids contain so-called tra-genes, which mediate process of conjugation, the transfer of plasmids to another bacterium

11. F + x F-

12. Types of plasmids
Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids.,

13. Types of plasmids
It is possible for plasmids of different types to coexist in a single cell. Seven different plasmids have been found in E.coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.

14. Types of plasmids .
Another way to classify plasmids is by function. There are five main classes:
Fertility-F-plasmid, which contain tra-genes. They are capable of conjugation (transfer of genetic material between bacteria which are touching).
Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.

15. Types of plasmids
Col-plasmids, which contain genes that code for (determine the production of) bactericins proteins, that can kill other bacteria.
Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid.
Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease).

16. Using of plasmids
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Bacteria can be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.

17. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.

18. Using of plasmids
Major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Bacteria can be induced to produce large amounts of proteins from the inserted gene.

20. Plasmid’s profile
Comparing plasmid profiles is a useful method for assessing possible relatedness of individual clinical isolates of a particular bacterial species for epidemiological studies.

21. Mobile genetics elements
.Transposons are segments of DNA that can move from one site in a DNA molecule to other target sites in the same or a different DNA molecule.
The process is called transposition and occurs by a mechanism that is independent of generalized recombination.

22. Mobile genetics elements
Transposons are not self-replicating genetic elements, however, and they must integrate into other replicons to be maintained stably in bacterial genomes

23. Mobile genetics elements
Insertion of a transposon often interrupts the linear sequence of a gene and inactivates it.
Transposons have a major role in causing deletions, duplications, and inversions of DNA segments as well as fusions between replicons.

24. Mobile genetics elements
Most transposons share a number of common features. Each transposon encodes the functions necessary for its transposition, including a transposase enzyme that interacts with specific sequences at the ends of the transposon.

25. Mobile genetics elements
Complex transposons vary in length from about 2,000 to more than 40,000 nucleotide pairs and contain insertion sequences (or closely related sequences) at each end, usually as inverted repeats.

26. IS-elements
Insertion sequences are simplest in structure and encode only the functions needed for transposition. Inverted repeats are at their endsThe DNA between the inverted terminal repeats contains transposase genes

27. During transposition a short sequence of target DNA is duplicated,
The duplication is presumed to involve asymmetric cleavage of DNA at the target site.
If the transposon at a donor site is replicated and a copy is inserted into the target site, however, the process is called replicative transposition

28. Mobile genetics elements
Resolution of the cointegrate into its component replicons is often accomplished by a transposon-encoded resolvase that catalyzes site-specific recombination between the transposons.
Transposition differs from site-specific recombination by duplicating a segment of the target sequence and by using a variety of different target sequences for a single donor sequence.

29. Role of transposone in bacterial evolution
Some of the multiple antibiotic resistant plasmids have individual transposons with several resistance determinants. The stepwise acquisition of resistance determinants can lead, in some cases, to the formation of composite transposons that encode multiple resistance determinants.

30. Role of transposone in bacterial evolution
After a plasmid carrying a transposon is introduced into a new bacterial host, the transposon and its determinants can jump into the chromosome or indigenous plasmids of the new host. Therefore, stability of the mobilizing plasmid in a new bacterial host is not essential for persistence of genetic determinants located on a transposon.

31. .The acquisition of resistance gene arrays involves genetic mobile elements like :
Plasmids
Transposons
Integrons are a system of gene capture and expression composed of an intI gene encoding an integrase, a recombination site attI, and a promoter.

32. P
attI
5'conserved segment
cassette 1
intI
attC1
attI
attC1
cassette 2
attC2
attI
attC2
attC1
Integrons are a system of gene capture and expression composed of an intI gene encoding an integrase, a recombination site attI, and a promoter.

33. The integrase is able to integrate or excise gene cassettes, by a site-specific system of recombination.
Cassette mobility results in a very efficient system of dissemination of resistance genes.

34. Recombination
Recombination is the rearrangement of donor and recipient genomes to form new, hybrid genomes
Recombination involves breakage and joining of parental DNA molecules to form hybrid, recombinant molecules. .

35. Recombination
Several distinct kinds of recombination have been identified that depend on different features of the participating genomes and require the activities of different gene products. Specific enzymes that act on DNA (for example, exonucleases, endonucleases, polymerases, ligases) participate in recombination

36. Generalized recombination
Generalized recombination involves donor and recipient DNA molecules that have homologous nucleotide sequences.
The product of the recA gene is essential for generalized recombination, but other gene products also participate.

37. Site-specific recombination
Site-specific recombination involves reciprocal exchanges only between specific sites in donor and recipient DNA molecules.
The recA gene product is not required for site-specific recombination.
Integration of the temperate bacteriophage l into the chromosome of E coli is a well-studied example of site-specific recombination .

38. Site-specific recombination
In phage l the product of the int gene (integrase) is required for the site-specific integration event in lysogenization;
The products of the int and xis (excisionase) genes are both needed for the complementary site-specific excision event that occurs during induction of lytic phage development in lysogenic cells.

39. Site-specific recombination
The specific attachment (att) sites on the E coli chromosome and l phage DNA have a common core sequence of 15 nucleotides, within which reciprocal recombination occurs

40. Exchange of Genetic Information
Genetic exchanges among bacteria occur by several mechanisms.
In transformation, the recipient bacterium takes up extracellular donor DNA.
In transduction, donor DNA packaged in a bacteriophage infects the recipient bacterium.
In conjugation, the donor bacterium transfers DNA to the recipient by mating.

41. Transfer of genetic’s information

42. transformation
Historically, characterization of "transforming principle" from S pneumoniae provided the first direct evidence DNA is genetic material.

44. transformation
In transformation, pieces of DNA released from donor bacteria are taken up directly from the extracellular environment by recipient bacteria. To be active in transformation, DNA molecules must be at least 500 nucleotides in length, and transforming activity is destroyed rapidly by treating DNA with deoxyribonuclease.

45. transformation
Molecules of transforming DNA correspond to very small fragments of the bacterial chromosome.
Transformation was discovered in S.pneumoniae and occurs in other bacterial genera including Haemophilus, Neisseria, Bacillus, and Staphylococcus

46. transformation
The ability of bacteria to take up extracellular DNA and to become transformed, called competence, varies with the physiologic state of the bacteria.

47. transformation
Many bacteria that are not usually competent can be made to take up DNA by laboratory manipulations, such as calcium shock or exposure to a high-voltage electrical pulse (electroporation).

48. transformation
Competent bacteria may also take up intact bacteriophage DNA (transfection) or plasmid DNA, which can then replicate as extrachromosomal genetic elements in the recipient bacteria. Recombination occurs between single molecules of transforming DNA and the chromosomes of recipient bacteria.

49. transformation

50. Conjugation

51. F+ x F-
In matings between F+ and F- bacteria, only the F plasmid is transferred with high efficiency to recipients.
In matings between F+ and F- strains, the F plasmid spreads rapidly throughout the bacterial population, and most recombinants are F+.

52. conjugation

53. Conjugation
Donor strains with integrated F factors can transfer chromosomal genes to recipients with high efficiency, they are called Hfr (High frequency recombination) strains.

54. Hfr x F-
In matings between Hfr and F- strains, the segment of the F plasmid containing the tra region is transferred last, after the entire bacterial chromosome has been transferred.

55. Conjugation
Formation of recombinant progeny requires recombination between the transferred donor DNA and the genome of the recipient bacterium.

56. Conjugation
Most recombinants from matings between Hfr and F- are phenotypically F-.
In matings between F+ and F- strains, the F plasmid spreads rapidly throughout the bacterial population, and most recombinants are F+.

57. conjugation

58. General transduction
In transduction, bacteriophages function as vectors to introduce DNA from donor bacteria into recipient bacteria by infection.

59. General transduction
For some phages, called generalized transducing phages, a small fraction of the virions produced during lytic growth contain a random fragment of the bacterial genome instead of phage DNA.

60. General transduction
Each individual transducing phage carries a different set of closely linked genes, representing a small segment of the bacterial genome.
When a generalized transducing phage infects a recipient cell, expression of the transferred donor genes occurs

61. General transduction

62. Specialized transduction
Specialized transduction differs from generalized transduction in several ways.
It is mediated only by specific temperate phages,
and only a few specific donor genes can be transferred to recipient bacteria.

63. Specialized transduction
Specialized transducing phages are formed only when lysogenic donor bacteria enter the lytic cycle and release phage progeny.

64. Specialized transduction
The specialized transducing phages lack part of the normal phage genome and contain part of the bacterial chromosome located adjacent to the prophage attachment site.

65. Specialized transduction
Specialized transduction results from lysogenization of the recipient bacterium by the specialized transducing phage and expression of the donor genes.

67. Specialized transduction
Phage conversion and specialized transduction have many similarities, but the origin of the converting genes in temperate converting phages is unknown.

68. 68
Practical genetic

69. Molecular methods in microbial laboratory
1. PCR
2. Micro array technique
3.Ribotyping
4.Finger printing
5.Plasmid’s profile
8.PCR-RT
Identification of microbes without isolation of pure culture
Intra species identification

70. DNA microarrays are: glass slides with hundreds to thousands of
DNA probes bound μm
DNA microarrays are used for the highly parallell analysis of hundreds to tens of thousands of hybridisation reactions. DNA probes are spotted onto glass slides with modified surfaces (allowing for the stable binding of the spotted DNA). Spots are normally of um in diameter.

71. Methodology Community DNA Specific PCR product Oligonucleotide probes
(i.e., 16S rRNA gene)
Oligonucleotide probes
Length: between 15 and 30 nt
Fluorescently labelled target
(DNA or RNA)
Microarray with the above probes
Hybridisation
Data analysis

72. The principle of detection of specific DNA sequences
Emitted light (fluorescence)
Attached fluorophore
Labelled “target” DNA
Many target molucules
High signal
No target
No signal
A few target molecules
Low signal
DNA probes
On DNA microarrays, DNA probes are chemically bound to a modified glass surface. The target is fluorescently labelled DNA or RNA, free floating in the hybridisation solution. During hybridisation, labelled target molecules bind to their corresponding probe counterparts. After hybridisation, positive spots indicate the presence of their corresponding target DNA or RNA in the target mixture. Signal intensity correlates to the relative amount of target molecules in the hybridisation mixture (note: there are other factors also influencing signal intensity!).
Glass slide
The principle of detection of specific DNA sequences

73. Real time pcr

74. Branch-pcr