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Bacterial Physiology ,Bacteriophages and bacterial genetics part (I)

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Bacterial multiplication      Bacteria reproduce by  binary fission; each cell divide into 2 daughter cells identical to the parent cell    The doubling time (generation time):  The time required for bacteria to double its number. It ranges from 20 minutes for  E. coli  to 24 hours for  M.  tuberclousis

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Chapter 9 Microbial growth - ppt download

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Binary fission It is the process that bacteria (prokaryotes) use to replicate. Bacterial cell increases in size and elongate. DNA replicates by forming 2 sets of chromosomes which attached by  mesosomes  to cytoplasmic membrane. Protoplasm divides by newly formed cell membrane and cell wall which separate the 2 daughter cells.  Cells may remain attached after division giving the organism characteristic arrangement; pairs or chains

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Requirement of bacterial growth For bacteria to grow and multiply certain requirements must be present which are: Sufficient food (nutrients) Moisture. Temperature. Acidity or Alkalinity (pH) Gaseous requirements. Ions and minerals as iron.

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Bacterial nutrition          Bacteria like all cells require nutrients for maintenance of their metabolism & for cell division. Bacteria vary in their nutritional requirements; two nutritional groups can be distinguished:   Autotrophs: Utilize  simple inorganic substances; these are free living of no medical importance. Heterotrophs :  Can break down organic matter as proteins & sugars into simpler chemical substances. All bacteria of medical importance are heterotrophs.  (Parasitic bacteria) .

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Moisture          Water is essential for bacterial growth acts as vehicle for food transmission. Drying affect some types of bacteria while others resist drying for different periods. Spores resist dryness while delicate bacteria as gonococcus resist dryness only for few hours.

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Temperature       Bacteria have different optimum temperature requirements in which they grow best. Bacteria are classified according to their optimal temperature requirements into: Psychrophiles :  Grow best in cold temperatures between 0 — 20 °C.  Mesophiles :   Grow best in temperatures between 20 — 40 °C.  Thermophiles:   Grow best in temperatures between 40 — 90 °C.  Extreme thermophiles :  Grow best in temperatures above 90 °C.        Most bacteria are  mesophiles  especially pathogens that require temperature around 37 °C. Cold temperature or temperature below the minimum requirement will stop or retard the growth of the bacteria; so,  cooling is used for this purpose as a method for preserving bacterial strains . When bacteria later exposed to a favorable temperature for their growth multiplication will start again.

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Fig () temperature requirements of the bacteria

MICROBIOLOGY 130  STUDY GUIDE FOR CHAPTERS 6,7,20

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Acidity or Alkalinity (pH)          Most pathogenic species of bacteria can grow at a narrow range of pH 7.2-7.6. However, few species as V. cholera can grow at alkaline pH 8 and lactobacilli prefer acid pH 4.

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Gaseous requirements Oxygen  is required for aerobic respiration and energy production. Bacteria are classified according to their oxygen requirements into: - Obligate aerobes   grow only in presence of oxygen.  M. tuberculosis .  Microaerophilic  grow in low level of oxygen (5% oxygen). (No growth in absence of oxygen, high concentration of oxygen is toxic also) e.g.  Campylobacter  jejuni Obligate anaerobes   grow only in absence of oxygen (oxygen is toxic). They lack enzymes that destruct h2o2 and o3 (catalase - Superoxide dismutase). Facultative anaerobes   grow in presence or absence of oxygen.  Aerotolerant  anaerobes   grow in absence of oxygen but are not affected if oxygen is present e.g.  cl.  Perfringens . Carbon dioxide (CO2):  The CO2 present in air is sufficient to most bacteria. Some bacteria require higher concentration of CO2 and called  capnophilicbacteriae.g.Niesseriaspp  and  Brucellaabortus .

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obligate
aerobes
obligate
anaerobes
facultative
anaerobes
c
aerotolerant
anaerobes
microaerophiles

Fig ():   Gaseous requirements of bacteria

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If bacteria inoculated into liquid nutrient medium; bacteria are counted at frequent intervals and results are blotted. A characteristic growth curve with four phases results:   1 . Lag phase:  No  cell division; bacteria adapt to new environment. 2.  Logarithmic phase :  Rapid cell division,  increase  number of bacteria by time.  3.  Stationary phase :  Nutrients exhausted, toxic products accumulate, rate of growth decreases. Number of dying cells = newly formed cells→ number of living bacteria remains  constant . 4.  Decline phase:  As nutrients exhausted, toxic products accumulate, death rate exceeds multiplication rate → number of living bacteria  decreases  steadily.

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10
88
4
Few live cens
Many live cens
Dead cells
Stationary phase
Death
Sorne cells
remain
Lag phase
0
35
Hours
Total cells in population, live and dead, at each phase.

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Bacterial metabolism 1- Catabolism :  Bacteria secrete enzymes e.g. lipases, nucleases and proteinases to break down extracellular material into simple molecules that can be transported into the cell. They oxidized to yield energy  2- Anabolism:  The degradation products and energy are used to build up new molecules. 3- Respiration and energy production  Aerobic respiration:   It occurs in aerobic organisms which use oxygen as a terminal electron acceptor. Superoxide and hydrogen peroxide are released as a result. They are highly toxic. Enzymes  superoxide dismutase and catalase  remove them to prevent cell damage.

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Anerobic  respiration : Anaerobic organisms use other electron acceptors as nitrate, sulfate or CO 2 . These inorganic compounds have a lower reduction potential than oxygen, meaning that respiration is less efficient in these organisms and leads to slower growth rates than aerobes.  Fermentation:  Occur in absence of oxygen and in absence of inorganic compounds. Occur in facultative anaerobes and release the least energy with production of acid or alcohol.

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A bacteriophage  also known informally as a phage is a virus that infects and replicates within Bacteria and  Archaea . The term was derived from "bacteria" and the Greek word ( phagein ), "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and have relatively simple structures. Their genomes may encode as few as four genes and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm. Bacteriophages are ubiquitous viruses, found wherever bacteria exist.

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One of the densest natural sources for phages and other viruses is seawater, where up to 9x10 8 virions per  millilitre  have been found, and up to 70% of marine bacteria may be infected by phages. They have been used for over 90 years as an alternative to antibiotics in the former Soviet Union and Central Europe as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of many bacteria (phage therapy)

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History :          In 1896, Ernest  HanburyHankin  reported that something in the waters of the Ganges and Yamuna rivers in India had marked antibacterial action against cholera and could pass through a very fine porcelain filter. In 1915, British bacteriologist Frederick  Twort  discovered a small agent that infected and killed bacteria.          French-Canadian microbiologist Félix  d'Hérelle , working at the Pasteur Institute in Paris, announced on 1917, discovered that it is a virus and named it  a bacteriophage or bacteria-eater (from the Greek  phagein  meaning "to devour").

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Bacteriophage consists of a head and tail the head is composed of a protein coat (capsid) containing the nucleic acid which is DNA and less commonly RNA. The head may take different shapes. It is usually hexagonal.                                                                       The tail is composed of a hollow core surrounded by a contractile sheath and a terminal base plate. To which are attached tail fibers. The phage tail is the organ of attachment to host cell.

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Lytic phage cycle:  Bacteriophages may have a lytic cycle or a lysogenic cycle, and a few viruses are capable of carrying out both. With lytic phages such as the T4 phage, bacterial cells are broken open (lysed) and destroyed after immediate replication of the  virion . As soon as the cell is destroyed, the phage progeny can find new hosts to infect.   Lytic phages are more suitable for phage therapy. Some lytic phages undergo a phenomenon known as  lysis  inhibition, where completed phage progeny will not immediately lyse out of the cell if extracellular phage concentrations are high. This mechanism is not identical to that of temperate phage going dormant and is usually temporary .

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1-Attachment and penetration            To enter a host cell, bacteriophages attach to  specific receptors  on the surface of bacteria, including lipopolysaccharides,  teichoic  acids, proteins, or even flagella. This specificity means a bacteriophage  can infect only certain bacteria  bearing receptors to which they can bind, which in turn determines the phage's host range. Host growth conditions also influence the ability of the phage to attach and invade them.

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Bacteriophages use a hypodermic  syringe-like motion to inject  their genetic material into the cell. After making contact with the appropriate receptor, the tail fibers flex to bring the base plate closer to the surface of the cell; this is known as reversible binding. Once attached completely, irreversible binding is initiated and the tail contracts, possibly with the help of ATP present in the tail, injecting genetic material through the bacterial membrane. The injection is done through a sort of bending motion in the shaft by going to the side, contracting closer to the cell and pushing back up.

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Fig. ():  Attachment and penetration of the phage

DNA
Tail tube
Long tail fibre
Base plate
Lysozyme compl x
Protein needle
Li p o polysacha
Peptodoglycan
Cell membrane
Receptor

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Viral RNA or
"DNA
Bacteriophage
Viral genome
injected into cell
bacterial genome
Bacterial cell

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2- Synthesis of proteins and nucleic acid            Within minutes, bacterial ribosomes start translating viral mRNA into protein. For RNA-based phages, RNA  replicase  is synthesized early in the process. Proteins modify the bacterial RNA polymerase so it preferentially transcribes viral mRNA.  The host’s normal synthesis of proteins and nucleic acids is disrupted, and it is forced to manufacture viral products instead.

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3-  Virion  assembly           The base plates are assembled first, with the tails being built upon them afterward. The head capsids, constructed separately, will spontaneously assemble with the tails. The DNA is packed efficiently within the heads. The whole process takes about 15 minutes

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4- Release of  virions  Phages may be released via cell  lysis , by extrusion, or, in a few cases, by budding.  Lysis , by tailed phages, is achieved by an enzyme called  endolysin , which attacks and breaks down the cell wall peptidoglycan. Released  virions  are described as free, and, unless defective, are capable of infecting a new bacterium.

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Bacteriophage: Structure, Replication and Uses - Learn Microbiology Online

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(2)  Temperate phage cycle:              In contrast, the lysogenic cycle does not result in immediate lysing of the host cell. Those phages able to undergo  lysogeny  are known as temperate phages. Their viral genome will integrate with host DNA and replicate along with it relatively harmlessly, or may even become established as a plasmid. The endogenous phages (known as  prophages ) become active. At this point they initiate the lytic cycle, resulting in  lysis  of the host cell. As the lysogenic cycle allows the host cell to continue to survive and reproduce, the virus is replicated in all of the cell’s offspring. An example of a bacteriophage known to follow the lysogenic cycle and the lytic cycle is the phage lambda of  E. coli .

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The continuous presence of the  prophage  in the lysogenic bacterium gives it certain characters: It will become immune to infection by another phage. It may acquire new properties, such as production of exotoxin in diphtheria bacilli,  Cl. botulinum ,  Strept .  pyogenes 	 erythrogenic  toxin and  Staph.  aureus  enterotoxin. If the organism loses the  prophage , it will become non toxigenic. Acquisition of new character coded for by the  prophage  is called  (lysogenic conversion) .

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Outcome of the temperate cycle: The cell may continue carrying the  prophage  indefinitely passing it to daughter cells  The  prophage  may be induced to separate from the bacterial chromosome by UV, cold, or alkylating agents) and start a lytic cycle. Rarely an excisional error may occur during separation of the  prophage  from the bacterial chromosome. In this error the  prophge  is induced carrying with it part of the bacterial  chromosaome . When the phage infects another bacterium it will transmit to it anew character, this is called  specialized transduction.

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Specialized Transduction
Prophage gal gene
O Prophage exists in
Bacterial
Galactose-
positive
gal gene
Galactose-
negative
Galac rose-positive
recombinant cell
galactose-using host
(containing the gal
Phage excises,
carrying With it the
gal gene from the host.
O Phage matures and C ell
lyses, releasing
curying gal
O infects a cell that
Can not utilize
(lacking gal gene).
Along with the the
gal gene
integrated into the new
hosts DNA.
O cell can now
galactne.
Copyright@ 2010 Pearson Education, Inc.
Figure 13.13

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Practical uses of bacteriophage : 1- Phage therapy:  Therapeutic uses of bacteriophages to treat bacterial infections in animals, plants and humans are under trials. When used to treat humans the term  biocontrol  is used. They are used to treat bacterial infections that don’t respond to conventional antibiotics mainly those in which bacterial biofilms are involved. Phages are more specific in their action on bacteria than many drugs, and are expected to have minimal or no side effects.

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2- Phage typing :  Since bacteria differ in their susceptibility to different phages, phages are used to identify and type bacteria according to the pattern of  lysis . This is very useful in epidemiologic tracing of sources of outbreaks such as wound infections and food poisoning. 3-  Cloning vectors :  Bacteriophages are used as cloning vectors in recombinant DNA technology. A fragment of DNA (foreign gene) is carried on the phage DNA. When the phage infects a bacterial cell its DNA carrying the foreign gene is incorporated into the bacterial chromosome and the gene is replicated in each cell division.

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All properties of the bacterial cell (e.g., growth, virulence, pathogenicity, and antibiotic resistance) are determined by genetic information contained within its genome. This genetic information is inherited from the parents to daughter cells, so they usually have the same properties.

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In bacteria, the genetic information is carried in three forms: In all bacteria, most of genetic information that is essential for bacterial growth is carried on the chromosome which is a single circular double stranded DNA molecule. The bacterial cell may carry one or more of small circular extra-chromosomal DNA sequences (the plasmid or  episome ) and carry genetic information that are not essential for bacterial growth.

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The bacterial cell may be infected with a bacterial virus (bacteriophage). In some cases, the bacteriophage enters in a case of controlled replication within the bacterial cell without causing  lysis . In this case, the genetic information of bacteriophage are available, and may give additional properties to the bacterial cell.

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Genetic Information In Bacteria
Chromosome
Plasmid
Bacteriophage
Carries properties like virulence,
pathogenicity & resistance
Extrachromosomal genetic
material in the cytoplasm
Replicate independently
Virus infecting bacteria
o

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DNA Replication For inheritance of all properties of the bacterial cell to the progeny cells, it is essential that the chromosome be duplicated . 1) The process begins with unwinding of the double helix at a defined point (origin of replication). The process is assisted by  gyrase  enzyme . 2) DNA synthesis occurs in both directions around the circular chromosome. This occurs using the specific  DNA polymerase  and the two parental strands (as templates). The localized area where the DNA is unwind and DNA synthesis is occurring is referred to as Replication fork.

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DNA
DN A DNA
two DNA
products of the
DNA

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3) The DNA polymerase enzyme incorporates the appropriate nucleotides at each position according to the base pairing rule (A-T, & G-C).  4) To maintain high degree of accuracy, the DNA polymerase possesses a proofreading function to correct any error. 5) The process is described as semi-conservative because each of the two progeny DNA molecules consists of one old ( parentral ) strand and newly synthesized one.

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Proofreading
DNA polymerases
contain proofreading
activity to ensure
accurate replication.
Depends on a 3' to
5'exonuclease acitivity.
Major DNA pol is a
multisubunit enzyme:
separate subunit
Repair system further
increase fidelity
Figure 3.13: Proofreading
Enzyme adds base to growing strand
5'
3'
5'
Enzyme moves on if new base is correct
5'
3'
Base is hydrolyzed and expelled if incorrect
5'
3'
0 201 1 Jones and Bartlett Publishers. LLC (www.ibpubcorn)