2 Differentiating bacterial species Morphology (shape) Composition (cell envelope and other structures) Metabolism & growth characteristics Genetics
3 Differentiating bacterial species Morphology (shape) Composition (cell envelope and other structures) Metabolism & growth characteristics Genetics
4 Genetics Genetics Gene Study of what genes are. How genes carry information. How genes / genomes are replicated. How genes are expressed. Segment of DNA that encodes a functional product, usually a protein. Genome All of the genetic material in a cell.
5 Deoxyribonucleic acid (DNA) The genome of bacteria consists of a polymer of deoxyribonucleic acids (DNA). DNA forms a double helix and is made up of sub-units called nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). The backbone" is deoxyribosephosphate. Strands held together by hydrogen bonds between the nucleobases A and T and between C and G Strands have an antiparallel direction.
6 DNA replication DNA is copied by DNA polymerase. DNA replicates in the 5 3 direction. Replication is initiated by an RNA primer. Leading strand is synthesized continuously. Lagging is strand synthesized discontinuously by joining Okazaki fragments.
7 Energy for DNA replication DNA replication cost a lot of energy, which is provided by hydrolysis of the nucleotides
8 Semiconservative DNA replication DNA replication is semiconservative because each new DNA molecule contains one original and one new strand. Replicated genomes move towards opposite poles during cell division to allow bacterial replication without segregation of the DNA. Figure 8.7
9 Access and use of genetic information The central dogma to go from genetic information to function is: DNA => mrna => Proteins
10 Ribonucleic acid (RNA) RNA is very similar to DNA, but it consists of a single strand (that can be folded on itself). It contains the nucleotide Uracil (U) instead of T. The backbone consists of ribose-phosphate instead of deoxyribose-phsphate. There are three main RNA types Messenger RNA (mrna) Ribosomal RNA (rrna) Transfer RNA (trna)
11 Transcription A sequence of DNA is relaxed and opened up. RNA polymerase synthesizes a strand of RNA. Starting point is a promoter sequence. End point is a terminator
13 Translation mrna is translated in proteins using codons. Codon table Condons are nucleotide triplets that are recognized by trna s carrying specific amino acids. Translation of mrna begins at the start codon AUG Translation ends at the nonsense or STOP codon UAA, UAG, or UGA
14 Translation steps
15 Translation steps
16 Transcription and translation combined Transcription and translation happen simultaneously and are not temporally or spatially separated.
17 Regulation of gene expression Genes can be expressed as single units with their own promoter and terminator or as operon in which two or more genes share a promoter and terminator. Constitutive enzymes are expressed at a fixed rate. Other enzymes are expressed only as needed and are controlled by operator sites. Operator sites can function as repressor, activator, or both, depending on the proteins or metabolites that control them.
18 Two examples of gene regulation Inducible operon: Lactose regulation Repressible operon: Tryptophan regulation
19 Standard condition: gene repression Without lactose, the repressor protein is active, binds the operator sequence, and represses gene expression.
20 Gene activation With lactose, the repressor protein becomes inactive, it can not bind the operator sequence, and gene expression is induced.
21 Standard condition: gene expression Without tryptophan, the repressor protein is inactive, is unable to bind to the operator sequence, and genes are expressed
22 Gene repression With tryptophan, the repressor protein become active, binds to the operator sequence, and gene expression is repressed.
23 Genetic adaptation / evolution Gene regulation allows bacteria to adapt to their environment, however, this type of adaptation is still dependent on the information stored in the genome. Bacteria are also able to genetically adapt to their environment as a result of mutations in the DNA. Mutations can be neutral, beneficial, or harmful. In time, beneficial mutations can be selective and passed on in the process of evolution.
24 Causes of mutations Spontaneous mutations Errors during normal DNA replication and maintenance No external mutagens Mutagens Exposure to external mutagenic agent causes mutations, e.g. UV radiations, chemicals, etc. Uptake of exogenous genetic material Transformation Conjugation Transduction
25 Base substitutions (point mutations) Missense mutation: Change in one base results in change in amino acid
26 Base substitutions Nonsense mutation: Change in one base results in a nonsense or stop codon.
27 Base substitutions Frameshift mutation: Insertion or deletion of one base results in a changed amino acid sequence.
28 Frequency of spontaneous mutations Frequency of spontaneous mutations is variable between different bacterial species. Average spontaneous mutation rate is: 1 in 10 9 (billion) replicated base pairs 1 in 10 6 (million) replicated genes
29 Selection Positive (direct) selection detects mutant cells because they grow or appear different. For instance selection of antibiotic resistant mutants Negative (indirect) selection detects mutant cells because they do not grow. For instance selection of auxotrophic mutants
31 Uptake of exogenous genetic material Transformation Direct uptake of exogenous DNA through a state of competence. Competence can be induced or natural for some bacteria. Conjugation Transfer of genetic material between two bacteria through direct contact. Transduction Transfer of genetic material between bacteria through bacteriophages.
33 Transformation & natural competence
34 Conjugation DNA transferred from one bacteria to another by a sex pillus or mating bridge.
38 Transduction DNA is passed from one bacterium to another in a bacteriophage and put into recipients DNA.
39 Alternative DNA elements Plasmids Self replicating circular DNA that can encode virulence factors, antibiotic resistance markers, toxins, etc. Transposons Segments of DNA that can move from one region of DNA to another. Contain insertion sequences for cutting and resealing DNA. Complex transposons carry other genes such as antibiotic resistance genes.
40 Genetic identification of bacteria Genetic identification methods involve examination of genetic material. Genetic identification methods are rapidly becoming the dominant techniques for bacterial identification because of the speed and accuracy. Genetic identification methods Polymerase Chain Reaction (PCR) methods DNA sequence analysis Other methods like RFLP, Ribotyping, PFGE, etc are becoming obsolete!!
41 Polymerase Chain Reaction (PCR) In vitro amplification of unique target DNA Steps: Two nucleic acid primers are hybridized to the target gene Multiple copies of target gene are made by repeated melting of DNA, hybridization of primers, and amplification of DNA Allows for detection of few bacterial cells. Gel detection of PCR product
42 Real-Time PCR Fluorescent real time detection of PCR product. Amplification of PCR products is detected after each cycle and plotted. Quicker than conventional PCR. Quantitative
43 DNA sequence analysis DNA sequence analysis is the process of determining the nucleotide order (sequence) of DNA fragment. Several platforms exist for DNA sequencing, including small scale Sanger sequencing and large scale pyrosequencing. DNA sequences can be unique for bacterial species, depending on the region of the DNA that is sequenced. Deep sequencing methods can be used to identify mixed bacterial samples.
44 16S rdna sequencing 16S rrna DNA encoding 16S rrna is present in all bacterial species. 16S rdna contains fully conserved and variable regions. Conserved regions are used for design of primers that are compatible with 16S rdna of all bacterial species and which allows for PCR amplification. Variable regions are unique for each bacterial species and allows for identification. 16S rdna
45 Multi Locus Sequence Typing (MLST) Method to distinguish diversity within bacterial species. This is useful to investigate relationships between bacterial isolates during outbreaks or to follow spread of antibiotic resistance. The DNA sequence of internal fragments of multiple housekeeping genes is determined. The different sequences for each housekeeping gene are assigned as distinct alleles and, for each isolate, the alleles at each of the loci define the allelic profile or sequence type (ST).
46 Genome sequencing With the reduced costs for sequencing nowadays, it becomes more and more standard to just sequence whole bacterial genomes for bacterial identification and tracking diversity within bacterial species.
47 Next lecture Bacterial Pathogenicity & Infections