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1. COMPOSITION AND ORGANIZATION OF THE BACTERIAL CELL
Introduction
Bacteria have evolved over the past two billion years or so uniquely adapted to survive and grow in a bewildering variety of habitats under some of the most inhospitable conditions known--temperatures ranging from the freezing to the boiling point of water, pH values ranging from extreme acidity to extreme alkalinity, intense radiation, or extreme dessication, to name but a few. Bacteria must sense and respond in a dynamic fashion to rapid changes in their environments in order to optimize utilization of available resources and opportunities, while minimizing damage caused by inhospitable hosts, toxic agents, or harmful physical conditions. In this course we will be exploring how the morphology, subcellular structure, metabolism, and regulatory circuitry of bacteria have evolved in concert to enable this group of organisms to become the most successful life forms on Earth.
Bacteria are ideal subjects for the study of how cell growth occurs and is regulated, mainly because they grow rapidly (generation times of 30-40 minutes are common) and they can grow in simple media whose components can be defined precisely. Of all the myriad bacteria exisiting in the world, most of the information we know about bacterial physiology and genetics comes from a single Gram-negative species, called Escherichia coli , which has been studied the most extensively. For this reason alone, most of the material in this course will be derived from studies of E. coli. However, for many aspects of bacterial physiology (cellular differentiation, or nitrogen fixation, for example), E. coli is an inappropriate model system. We will therefore discuss topics such as these using the best-studied model organisms for each particular system.
Table 1.1: Chemical composition of bacterial protoplasm--
Assay of a typical E. coli culture:
Water 70%
Elemental composition of dry mass:
C 50% Ca 0.05%
O 20% Mg 0.05%
N 14% Cl 0.05%
H 8% Fe 0.2%
P 3% Mn,
K 2% Co,
S 1% Cu,
Zn,
Mo 0.3% total
Table 1.2: Macromolecular composition of E. coli strain B/r grown under a standard culture condition (i.e., balanced growth, glucose minimal medium, 37°C, mass doubling time of 40 min.):
Macromolecules:
Protein 55% (of total dry weight)
RNA(23S,16S,
5S rRNA,
tRNA,mRNA) 20.5%
DNA 3.1%
Lipid 9.1%
Lipopolysaccharide 3.4%
Murein 2.5%
Glycogen 2.5%
Soluble pool
(amino acids,
vitamins, etc.) 2.9%
Inorganic ions 1.0%
Looking at the data of Table 1.2 above, we can see that:
1. the vast majority of cellular dry mass (96%) is in the form of macromolecules.
2. proteins constitute over half of the dry weight.
3. bacteria contain two unique macromolecular compounds: murein and lipopolysaccharide.
Remember that the parameters of Table 1.2 are defined for one particular strain (B/r) of one microorganism (E. coli) grown under one type of condition (balanced [i.e., logarithmic] growth in glucose minimal liquid medium at 37°C with a doubling time of 40 min). Changing any of these parameters will yield different results, because the organism will undergo regulatory adaptation to optimize its growth rate in the new environment presented to it.
Dynamics of synthesis of cell components during growth
Imagine a culture of bacteria growing exponentially in liquid medium containing glucose as the sole carbon source. The flow of carbon from glucose into various cellular components can be traced by adding to the culture uniformly-labelled [14-C]-glucose, removing samples from the culture at various times after addition of the label, fractionating the cells into their various components, and determining the radioactivity in each fraction. Depending on the type of molecule analyzed, three basic kinetic patterns of labelling are observed (Fig. 1.1):
Fig. 1.1. Idealized time course of labeling of organic molecules following addition of [14-C] glucose to a culture of bacteria in steady-state growth in glucose minimal medium. Curves A, B, and C are idealized data for different classes of macromolecules described below. cpm, counts per minute. From Neidhardt et al. (1990).
Curve A: components such as the TCA-soluble pool of low molecular weight precursor molecules is rapidly labelled to maximal specific activity within a few minutes, indicating rapid flow of carbon through these compounds.
Curve B: macromolecules are labelled to approximately 50% of maximum by the first doubling, 75% at the second, etc. indicating that the labelled glucose is being converted into stable cellular materials (DNA, cell wall, etc.) in proportions consistent with doubling of cell mass in the culture (think about this...).
Curve C: some compounds (ribonucleotide pools, for example) exhibit a labelling curve intermediate between curves A and B, indicating that flow of carbon through reflects both rapid turnover (A) and residence for a period of time as macromolecules (B) which are also subject to degradation and recycling.
Size and chemical composition of an average SINGLE bacterial cell.
It is necessary to relate the above chemical compositions, which were determined using large populations of cells, to the individual cells which constitute the mass which was assayed. So how can we measure the number of cells in our sample? By using either:
Direct counts: manually using a Petroff-Hauser chamber, or automatically using an electronic particle counter.
Viable counts: Serial dilutions, plating on solid media, incubation, counting colonies.
By these methods, the culture we have been considering (E.coli B/r grown in glucose minimal medium, 37°C) contains 1.05 x 10E12 cells per gram (wet weight) of mass. Therefore, the average total wet mass of one cell = 9.5 x 10E-13 gm, and the average total dry mass is 2.9 x 10E-13 gm (Question: how did we arrive at those wet and dry masses ??).
Exactly what is an "average" cell? In a nonsynchronous population of cells growing exponentially by binary fission, individual cells will be at any stage of the growth cycle, from "new" (i.e., newly-divided) to "old" (i.e., almost ready to divide). It follows from this that for every "old" cell in the population, there are two "new" cells, and the age frequency distribution of the population can be described by the equation
f(x) = 2 E (1-x)
where x is the age of the cell ("new" cell = 0 and "old" cell = 1 generation) (Fig. 1.2).
Fig. 1.2. Idealized frequency distribution of cell age in a steady-state culture increasing by binary fission. From Neidhardt et al. (1990).
VARIETY AND NUMBER OF MOLECULES IN A CELL
It is not enough merely to describe the chemical composition of an average bacterial cell in terms of "percent dry mass devoted to protein", for example--there exist an enormous number of different proteins in a cell, each associated with different parts of the cell. In order to understand such parameters as: the energy costs associated with growth, or the regulation of synthesis of particular cellular components under different growth conditions, it is necessary to further analyze the composition of each class of compounds present in the cell.
Protein
Protein is 55% of cell dry mass (Table 1.2). So (0.55) x (2.9 x 10E-13) = 1.6 x 10E-13 gm protein per cell, or approx. 160 femtograms:
1. How many types of proteins are there in a cell?
2. What are their sizes and relative abundances?
3. How many total protein molecules are there in a cell?
These questions can be addressed by the following experiment. E. coli B/r is grown under our standard conditions in the presence of a radioactive compound such as [35-S] sulfate, which will label all proteins that have sulfur-containing amino acids. Separation of the total cellular proteins can be accomplished by 2-dimensional polyacrylamide gel electrophoresis, in which separation in the 1st dimension is on the basis of each protein's isoelectric point, and in the 2nd dimension on the basis of molecular mass. The gel is then exposed to X-ray film, and each spot on the developed autoradiogram represents (ideally) a single protein species. By assigning each spot a coordinate, each protein species can be identified by its molecular weight and isoelectric point. The intensity of each spot is considered proportional to the number of protein molecules in each spot (Fig. 1.3).
Fig. 1.3. Autoradiogram of 2-dimensional gel of protein extract from E. coli B/r grown under standard conditions and labelled with [35-S] sulfate. The grid overlay provides coordinates for individual spots. Letters A to I are from acidic to basic in the 1st (isoelectric focusing) dimension, and the horizontal position of spots denotes their isoelectric points (pI). Proteins are separated by molecular mass in the 2nd dimension. From Neidhardt et al. (1990).
1. Counting of the spots on the autoradiograms reveals approx. 1,800 different labelled proteins.
2. Analysis of the autoradiogram shows that the sizes of proteins range from approx. 10-155 kDal, with an average of 40 kDal. Each spot can be quantitated either by densitometric measurement or by actual excision and scintillation counting of the radioactivity in the spot. Such analysis reveals that the abundances of proteins in the cell vary over a range of 100,000 or so, from quite rare to quite common.
3. Using the average molecular weight of protein (ca. 40,000 daltons) and the dry mass of protein in the cell one can calculate that there exist approximately 2.4 million protein molecules per cell. (Question: How do you arrive at this number?).
This type of analysis is very useful in getting a picture of the complexity of proteins in E. coli. When all of the spots are assigned coordinates and are cataloged in a database, the power of the technique increases enormously. For example, cultivation of the same strain in two different media results in a dramatic difference in the labelling pattern of proteins; some proteins increase dramatically in abundance, while others decrease. Some spots appear in one film but not in the other. In other words, the cells undergo drastic shifts in the pattern of gene expression to optimize their growth rate in the two different environments.
RNA
RNA constitutes 20% of the cell dry mass (Table 1.2). Of this, more than 80% is ribosomal RNA (rRNA), which has three components: 23S, 16S, and 5S. Each of these is present at 18,700 copies per cell. Each ribosome contains one molecule of each type of rRNA; therefore there are also 18,700 ribosomes in our average cell grown under standard conditions. Most of the remainder is transfer RNA (tRNA; 15%) of which there are approx. 200,000 molecules per cell of about 60 different types. Taken together, rRNA and tRNA are called stable RNA because of their relatively long half-lives within the cell.
Messenger RNA (mRNA) constitutes the remaining 4-5% of the RNA in the cell, although it is synthesized at the same rate as tRNA and rRNA. mRNA is subject to rapid turnover via degradation, however, which accounts both for its low steady-state level and for its kinetcs of accumulation in the cell (mRNA would exhibit a labelling curve similar to curve C in Fig. 1.1. As we will see in later sections, it is the very lability of mRNA which accounts for the cell's ability both grow rapidly and to adapt rapidly to changes in its environment.
DNA
Organized as a single, double-stranded circular DNA chromosome. (In addition, other smaller DNA elements such as plasmids may exist in cells, but the presence of these is variable and will not be considered here.) About 3% of the total dry weight of the cell, or 9 femtograms, is DNA. This works out to be a single circular molecule of 2.5 x 10E9 Dal (4.7 million base pairs) with a circumference of 1 mm!!
LIPID
Mostly found in membranes as phospholipids (26 femtograms per cell, or 9% of dry weight). Four major classes:
Phosphatidylethanolamine (75%)
Phosphatidylglycerol (18%)
Cardiolipin (5%)
Phosphatidylserine (trace in E.coli, but a major constituent of other bacteria)
The fatty acids (R1 and R2 groups) are mainly: palmitic acid (16
; 43%), palmitoleic (16:1; 33%), and cis-vaccenic (18
; 25%).
LIPOPOLYSACCHARIDE
All Gram-negative bacteria possess liposaccharide (LPS), although there is a huge variation in the structure of LPS from one strain or species to another. LPS constitutes 3.4% of the dry weight of E. coli, and is all found on the outer surface of the outer membrane.
MUREIN
Murein is the peptidoglycan found in the cell walls of all eubacteria, imparting rigidity and shape to the cell. It constitutes 2.5% of the dry weight of our gram-negative E. coli cell, but is present in much larger amounts in gram-positive bacteria, where it is associated with teichoic acids.
CARBOHYDRATE
E.coli makes a small amount of intracellular glycogen (2.5% of dry weight) during exponential growth, but MUCH more of this storage compound during stationary phase in media containing excess carbon source. Other bacteria store carbon as poly-beta-hydroxybutyrate.
LOW-MOLECULAR-WEIGHT ORGANIC COMPOUNDS
Constitute 3% of total dry weight and consist of precursors of macromolecules, vitamins, metabolic intermediates, etc.
INORGANIC IONS
1% of dry mass, but are essential to growth, as they serve many functions such as: cofactors for many enzymes, osmotic regulation, sources of nitrogen, sulfur, and phosphrous for macromolecules, etc.
ORGANIZATION OF THE PROKARYOTIC CELL
Examination of a transmission electron micrograph of a thin section of E. coli reveals that the prokaryotic cell is organized quite differently from eukaryotic cells. Here we see surrounded by cell membrane, an amorphous cytosol containing a vague "nuclear region", and completely lacking in all of the various membrane-bound organelles seen in eukaryotic cells. In fact, prokaryotic cell organization appears streamlined for maximum growth rate and physiological adaptation:
small size
no membrane-bound compartments
no physical separation between DNA and translational apparatus
no complex organelles
electron transport at cell surface
highly developed nutrient transport mechanisms
minimal structural complexity
(Question: How do each of these features contribute to rapid growth?)
The important morphological structures of the cell are:
1. Cell envelope: composed of inner membrane, murein cell wall, and outer membrane (in gram-negative cells). The periplasm is the space between the inner and outer membranes.
2. Interior: two regions, the cytosol (electron-dense, densely packed with ribosomes) and the nucleoid (less electron-dense, fibrous appearance, contains bacterial chromosome).
4. Exterior:
A.Two types of appendages: Flagella (helix-shaped locomotor organelles) and Pili (or Fimbriae; rod-shaped adhesion structures)
B. Capsule: loose sheath made of polysaccharide of protein; aids in adhesion, competition, evasion of phagocytosis, etc.
The morphology and composition of the envelope determine whether a cell retains crystal violet-iodine stain upon treatment with alcohol or acetone (the Gram stain). Cells retaining the complex are "Gram-positive" and those losing the complex are "Gram-negative". The vast majority of bacteria (the Eubacteria) fall into these two categories; exceptions are the Mollicutes (which lack a cell wall) and the Archaebacteria, which are distantly related to eubacteria and whose cell walls are composed of protein or a compound called "pseudomurein" which lacks muramic and diaminopimelic acids.
وهنا بعض المواقع للفائدة
http://www.annualreviews.org/catalog/special/geneflow/geabs.asphttp://scidiv.bcc.ctc.edu/rkr/Botany110/lectures/cells.htmlhttp://distance.stcc.edu/AandP/AP/AP1pages/introduction/cells.htmhttp://www.horizonpress.com/hsp/abs/absspi.htmlhttp://www.bioinfo.de/isb/1999/01/0017/main.htmlhttp://tccsa.freeservers.com/articles/max_to_olson_12_17.htmlhttp://genomebiology.com/2002/3/8/research/0040http://www.uis.edu/~lemke/review_web_2002.htmhttp://www.healthtech.com/conference/00pex/pex.htmhttp://www.rae.org/introns.htmlhttp://www.bio.upenn.edu/courses/F99/BIOL121/ويا هلا فيك أخوي nawaf_nat
