Agrobacterium

Agrobacterium tumefaciens (A.t.) is a soil-inhabiting bacterium that causes a disease known as crown gall in many plant species. Roots naturally exude chemicals into the rhizosphere that can be detected by microbes in the soil. A wound can increase the flow of exudates from a plant and specific compounds, such as precursor molecules for lignin (produced by the plant as wound tissue) can stimulate the process of pathogenesis. An organic matrix is created by the bacteria as they attach to the surface of the plant. This matrix facilitates the chance of successful colonization. The cytoplasm of A.t. contains two types of DNA: (1.) a chromosome form and (2.) a smaller, circular piece of DNA called a plasmid. Basically, the plasmid has a short segment (T-DNA) that has genes for hormone production and opine synthesis. Opines are a carbon compound that the bacteria can utilize. This T-DNA is transferred from the bacterial cell through the cell walls of the plant and into the plant cell nucleus. In the nucleus, the T-DNA integrates into the plant chromosome. The cellular processes of the plant treat the T-DNA as it's own and production of the hormones indoleacetic acid and cytokinin begins. Plant cells proliferate undifferentiated tissue, forming a gall. Opines are also produced that are metabolized only by the bacteria. The bacterium may colonize the roots, crown, and other parts of the plant. A.t. has evolved to genetically colonize its host. This is an amazing feat of cross-kingdom genetic engineering by a common soil dwelling bacterium. A computer animation (21 MB mov) illustrates the natural pathogenesis. A second computer animation (16 MB mov) illustrates how this phenomenon is utilized in the lab for Agrobacterium mediated transformation.

Agrobacterium tumefaciens has Gram-negative cell walls.

Flagellar movement of bacteria. A wounded plant cell will beginto synthesize lignin in order to heal the wound.Specific plant lignin precursors are chemotacticallysensed by the pathogen. Flagellarmovement is a series of tumbles and runs counterclockwise upthe gradient and cells have been reported to move as fast as60 um/second.

A. tumefaciens exhibits polar attachment to the plant cell.The production of cellulose fibrils serve to anchor the bacteria to the plantas well as trap other bacteria. Once the concentration of lignin precursorsreaches approximately 10-5 M, the virulence genes of the Ti plasmid are inducedand the T-DNA is processed.

A Ti-plasmid modelis under construction here (Flash)

The model at left shows in an approximate manner how the T-DNA moves into the plant cell and is passedthrough the nuclear pores. The bacterium forms an external pilus (type IV secretion system) for the transfer of T-DNA into the plant cell. The assembly of this secretion system has been studied. A draft animation of this process is available. Type IV Secretion System

Part II

Metabolism refers to all the biochemical reactions that occur in a cell or organism. The study of bacterial metabolism focuses on the chemical diversity of substrate oxidations and dissimilation reactions (reactions by which substrate molecules are broken down), which normally function in bacteria to generate energy. Also within the scope of bacterial metabolism is the study of the uptake and utilization of the inorganic or organic compounds required for growth and maintenance of a cellular steady state (assimilation reactions). These respective exergonic (energy-yielding) and endergonic (energy-requiring) reactions are catalyzed within the living bacterial cell by integrated enzyme systems, the end result being self-replication of the cell. The capability of microbial cells to live, function, and replicate in an appropriate chemical milieu (such as a bacterial culture medium) and the chemical changes that result during this transformation constitute the scope of bacterial metabolism.
The bacterial cell is a highly specialized energy transformer. Chemical energy generated by substrate oxidations is conserved by formation of high-energy compounds such as adenosine diphosphate (ADP) and adenosine triphosphate (ATP) or compounds containing the thioester bond

(acetyl ~ SCoA) or succinyl ~ SCoA. ADP and ATP represent adenosine monophosphate (AMP) plus one and two high-energy phosphates (AMP ~ P and AMP ~ P~ P, respectively); the energy is stored in these compounds as high-energy phosphate bonds. In the presence of proper enzyme systems, these compounds can be used as energy sources to synthesize the new complex organic compounds needed by the cell. All living cells must maintain steady-state biochemical reactions for the formation and use of such high-energy compounds.
Kluyver and Donker (1924 to 1926) recognized that bacterial cells, regardless of species, were in many respects similar chemically to all other living cells. For example, these investigators recognized that hydrogen transfer is a common and fundamental feature of all metabolic processes. Bacteria, like mammalian and plant cells, use ATP or the high-energy phosphate bond (~ P) as the primary chemical energy source. Bacteria also require the B-complex vitamins as functional coenzymes for many oxidation-reduction reactions needed for growth and energy transformation. An organism such as Thiobacillus thiooxidans, grown in a medium containing only sulfur and inorganic salts, synthesizes large amounts of thiamine, riboflavine, nicotinic acid, pantothenic acid, pyridoxine, and biotin. Therefore, Kluyver proposed the unity theory of biochemistry (Die Einheit in der Biochemie), which states that all basic enzymatic reactions which support and maintain life processes within cells of organisms, had more similarities than differences. This concept of biochemical unity stimulated many investigators to use bacteria as model systems for studying related eukaryotic, plant and animal biochemical reactions that are essentially "identical" at the molecular level.
From a nutritional, or metabolic, viewpoint, three major physiologic types of bacteria exist: the heterotrophs (or chemoorganotrophs), the autotrophs (or chemolithotrophs), and the photosynthetic bacteria (or phototrophs) (Table 4-1). These are discussed below.
Heterotrophic Metabolism
Heterotrophic bacteria, which include all pathogens, obtain energy from oxidation of organic compounds. Carbohydrates (particularly glucose), lipids, and protein are the most commonly oxidized compounds. Biologic oxidation of these organic compounds by bacteria results in synthesis of ATP as the chemical energy source. This process also permits generation of simpler organic compounds (precursor molecules) needed by the bacteria cell for biosynthetic or assimilatory reactions.

The Krebs cycle intermediate compounds serve as precursor molecules (building blocks) for the energy-requiring biosynthesis of complex organic compounds in bacteria. Degradation reactions that simultaneously produce energy and generate precursor molecules for the biosynthesis of new cellular constituents are called amphibolic.
All heterotrophic bacteria require preformed organic compounds. These carbon- and nitrogen-containing compounds are growth substrates, which are used aerobically or anaerobically to generate reducing equivalents (e.g., reduced nicotinamide adenine dinucleotide; NADH + H+); these reducing equivalents in turn are chemical energy sources for all biologic oxidative and fermentative systems. Heterotrophs are the most commonly studied bacteria; they grow readily in media containing carbohydrates, proteins, or other complex nutrients such as blood. Also, growth media may be enriched by the addition of other naturally occurring compounds such as milk (to study lactic acid bacteria) or hydrocarbons (to study hydrocarbon-oxidizing organisms).

THE DIVERSITY OF METABOLISM IN PROCARYOTES

Introduction
A lot of hoopla is made about microbial diversity.  Based on superficial inspection, the bacteria and archea hardly seem diversified. There are but a few basic morphologies, the possibilities of motility and resting cells (spores), and a major differential stain (the Gram stain) to distinguish the procaryotes microscopically. In the eukaryotes, there may be more structural diversity within a single genus of organisms. So what is all the hoopla about? It is about biochemical or metabolic diversity, especially as it relates to energy-generating metabolism and biosynthesis of secondary metabolites. The procaryotes, as a group, conduct all the same types of basic metabolism as eukaryotes, but, in addition, there are several types of energy-generating metabolism among the procaryotes that are non existent in eukaryotic cells or organisms. The diversity of procaryotes is expressed by their great variation in modes of energy production and metabolism.
Even within a procaryotic species, there may be great versatility in metabolism. Consider Escherichia coli. The bacterium can produce energy for growth by fermentation or respiration. It can respire aerobically using O2 as a final electron acceptor, or it can respire under anaerobic conditions, using NO3 or fumarate as a terminal electron acceptor. E. coli can use glucose or lactose as a sole carbon source for growth, with the metabolic ability to transform the sugar into all the necessary amino acids, vitamins and nucleotides that make up cells. A relative of E. coli, Rhodospirillum rubrum, has all the heterotrophic capabilities as E. coli,plus the ability to grow by photoautotrophic, photoheterotrophic or lithotrophic means. It does require one growth factor, however; biotin must be added to its growth media.
Fundamentally, most eukaryotes produce energy (ATP) through alcohol fermentation (e.g. yeast), lactic acid fermentation (e.g. muscle cells, neutrophils), aerobic respiration (e.g. molds, protozoa, animals) or oxygenic photosynthesis (e.g. algae, plants). These modes of energy-generating metabolism exist among procaryotes, in addition to all following types of energy production which are virtually non existent in eukaryotes.
Unique fermentations proceeding through the Embden-Meyerhof pathway
Other fermentation pathways such as the phosphoketolase (heterolactic) and Entner-Doudoroff pathways
Anaerobic respiration: respiration that uses substances other than 02 as a final electron acceptor
Lithotrophy: use of inorganic substances as sources of energy
Photoheterotrophy: use of organic compounds as a carbon source during bacterial photosynthesis
Anoxygenic photosynthesis: photophosphorylation in the absence of O2
Methanogenesis: an ancient type of archaeon metabolism that uses H2 as an energy source and produces methane
Light-driven nonphotosynthetic photophosphorylation: unique archaeon metabolism that converts light energy into chemical energy
In addition, among autotrophic procaryotes, there are three ways to fix CO2, two of which are unknown among eukaryotes, the CODH (acetyl CoA pathway) and the reverse TCA cycle.

Bacterial Metabolism

Heterotrophic Metabolism
Heterotrophic metabolism is the biologic oxidation of organic compounds, such as glucose, to yield ATP and simpler organic (or inorganic) compounds, which are needed by the bacterial cell for biosynthetic or assimilatory reactions.
Respiration
Respiration is a type of heterotrophic metabolism that uses oxygen and in which 38 moles of ATP are derived from the oxidation of 1 mole of glucose, yielding 380,000 cal. (An additional 308,000 cal is lost as heat.)
Fermentation
In fermentation, another type of heterotrophic metabolism, an organic compound rather than oxygen is the terminal electron (or hydrogen) acceptor. Less energy is generated from this incomplete form of glucose oxidation, but the process supports anaerobic growth.
Krebs Cycle
The Krebs cycle is the oxidative process in respiration by which pyruvate (via acetyl coenzyme A) is completely decarboxylated to C02. The pathway yields 15 moles of ATP (150,000 calories).
Glyoxylate Cycle
The glyoxylate cycle, which occurs in some bacteria, is a modification of the Krebs cycle. Acetyl coenzyme A is generated directly from oxidation of fatty acids or other lipid compounds.
Electron Transport and Oxidative Phosphorylation
In the final stage of respiration, ATP is formed through a series of electron transfer reactions within the cytoplasmic membrane that drive the oxidative phosphorylation of ADP to ATP. Bacteria use various flavins, cytochrome, and non-heme iron components as well as multiple cytochrome oxidases for this process.
Mitchell or Proton Extrusion Hypothesis
The Mitchell hypothesis explains the energy conservation in all cells on the basis of the selective extrusion of H+ ions across a proton-impermeable membrane, which generates a proton motive force. This energy allows for ATP synthesis both in respiration and photosynthesis.
Bacterial Photosynthesis
Bacterial photosynthesis is a light-dependent, anaerobic mode of metabolism. Carbon dioxide is reduced to glucose, which is used for both biosynthesis and energy production. Depending on the hydrogen source used to reduce CO2, both photolithotrophic and photoorganotrophic reactions exist in bacteria.
Autotrophy
Autotrophy is a unique form of metabolism found only in bacteria. Inorganic compounds are oxidized directly (without using sunlight) to yield energy (e.g., NH3, NO2-, S2, and Fe2+). This metabolic mode also requires energy for CO2 reduction, like photosynthesis, but no lipid-mediated processes are involved. This metabolic mode has also been called chemotrophy, chemoautotrophy, or chemolithotrophy.

Anaerobic Respiration
Anaerobic respiration is another heterotrophic mode of metabolism in which a specific compound other than 02 serves as a terminal electron acceptor. Such acceptor compounds include NO3-, SO42-, fumarate, and even CO2 for methane-producing bacteria.
The Nitrogen Cycle
The nitrogen cycle consists of a recycling process by which organic and inorganic nitrogen compounds are used metabolically and recycled among bacteria, plants, and animals. Important processes, including ammonification, mineralization, nitrification, denitrification, and nitrogen fixation, are carried out primarily by bacteria.

Bacterial Lysis

Bacterial Killing
Aims and Objectives

• To understand the terms complement, opsonin, opsonisation, phagocytosis.


• To understand the principles of complement activation.


• How complement is involved in the elimination of bacteria.


• To understand the types of cell involved in phagocytosis.


• To understand the process of phagocytosis.


• To understand the biochemical mechanisms of killing and degradation of bacteria by phagocytes.


• To understand the clinical features and management of patient's with antibody, complement or neutrophil deficiences.

Complement, Opsonisation and Phagocytosis
Opsonisation is the process of coating micro-organisms with plasma proteins to increase their adherence to phagocytic cells in preparation for phagocytosis. The two main opsonins are IgG antibody and the third component of complement (C3) which bind to the surfaces of micro-organisms. Phagocytic cells have membrane receptors for IgG (Fc piece) and activated C3 (which is called C3b). IgG antibody binds to micro-organisms because the Fab portion recognises bacterial epitopes. C3b binds because during activation of C3 a thiol bond is exposed which permits the formation of ester or amide bonds (covalent binding) with the bacterial surface structures.
C3 is activated by limited proteolysis by enzymes called C3 convertases. There are two C3 convertases, one produced by activation of the classical pathway which is antibody dependent (IgM or IgG), while the alternative pathway can be activated in the absence of antibody. The classical pathway is inactivated when the C1q subcomponent of first component of complement binds to two adjacent IgG molecules or a single IgM molecule. The C1s subcomponent of C1 becomes an active protease and activates C4 and C2 to form the classical pathway C3 convertase which is a complex of activated C4 (C4b) and C2 (C2a) it is designated C4b2a. The alternative pathway is activated when micro-organisms come into contact with body fluids. The pathway is always being activated slowly to generate small amounts of activated C3 (C3b). When C3b binds to a micro-organism activation of the alternative pathway is amplified so that a large amount of the alternative pathway C3 convertase is formed on the surface, followed by deposition of activated C3 (C3b).
Complement can also kill micro-organisms directly. Once C3b has been formed, same binds to the C3 convertase to change it to a C5 convertase which activates C5 to form C5b. Subsequently C6, C7, C8 and C9 bind together with C5b to form a hollow cylinder which is inserted into cell membranes to produce lysis.

Thus complement kills micro-organisms in two ways:

1. Opsonisation followed by phagocytosis and intracellular killing (indirect).


2. Assembly of the cytolytic C5b-9 membrane attack complex (direct).

Neutrophils and monocytes/macrophages are the two important phagocytic cells. Neutrophils and monocytes circulate in the blood and migrate into the tissues at the post-capillary venule. Initially they adhere to endothelium and then migrate through intercellular junctions. In the tissues they migrate towards bacteria by means of a process called chemotaxis, which is defined as direct movement along a concentration gradients of chemotactic agents (e.g. C5a leukotriene B4, IL-8, bacterial peptides). Phagocytes recognise their targets by specific sugar residues (e.g. mannose or LPS) but binding is greatly enhanced if the organism is opsonised with IgG and/or C3b. Phagocytes possess Fc and C3b receptors and there is co-operation between these receptors. Thus an organism opsonised with IgG and C3b is more effectively phagocytosed.
Ingestion (phagocytosis) is a localised endocytosis process requiring energy. The plasma membrane envelopes the particle and buds off to form an intracellular vesicle, the phagosome. Following fusion of the phagosome with lysosomal granules the phagolysosome is formed and the bacteria are killed by oxygen-dependent and oxygen-independent process.

Intracellular killing mechanisms:

Oxygen dependent:

• Hydrogen peroxide


• Sight oxygen


• Hydroxyl radical


• Hypohalite


• Nitric oxid

Oxygen independent:

• Lysozyme


• Lysosomal contents


• acid hydrolases


• cationic proteins


• Lactoferrin


• Neutral proteases

Facts on Bacteria

What are bacteria?
Bacteria are a successful and ancient form of life, quite different from the eukaryotes (which includes the fungi, plants and animals). They are small cells, found in the environment as either individual cells or aggregated together as clumps, and their intracellular structure is far simpler than eukaryotes. Bacteria have a single circular DNA chromosome that is found within the cytoplasm of the cell as they do not have a nucleus. Indeed they lack any of the intracellular organelles so characteristic of eukaryotic cells, such that they do not have the golgi apparatus, endoplasmic reticulum, lysosomes nor mitochondria. However they are generally capable of `free-living' and therefore they possess all the biosynthetic machinery that is needed for this, including 70S ribosomes (as opposed to the larger 80S forms found in eukaryotes) distributed throughout the cytoplasm. The most complex region of the cell is often the cell surface. The cell wall / outer membrane is described below, but in addition some bacteria may secrete a polysaccharide capsule onto their outer surface, some may have flagella which they require for mobility and some may have external projections such as fimbriae and pili which are useful for adherence in their chosen habitat. Although bacteria are generally far simpler than eukaryotic cells, they are extremely efficient within their own little niche - and this may include the ability to cause human infections. Bacteria multiply by binary fission and there is no sexual interaction.
Gram stain appearances of medically important bacteria
As bacteria are so small, they need to be viewed under a microscope using special stains; the stain that is traditionally used for this is called the "Gram stain". In this process, purple dyes are poured over bacteria that have been spread out thinly on a microscope slide and the cell walls of the bacteria (made out of peptidoglycan) take up the colour. If a solvent is then applied to the slide, bacteria which have only got a cell wall still keep their purple colour, but bacteria which have got an extra cell membrane (made out of phospholipid) outside their cell wall quickly lose the purple stain and become colourless; in order to be able to see these bacteria under the microscope a second red stain is then used.

• Bacteria that manage to keep the original purple dye have only got a cell wall - they are called Gram positive.




• Bacteria that lose the original purple dye and can therefore take up the second red dye have got both a cell wall and a cell membrane - they are called Gram negative.

A table of the staining characteristics for some common bacteria of medical importance is given below. Note that for cocci, it is not just the shape and colour of the individual bacterial cells that is important, but the way that all these cells group together too. Put simply, round purple balls that look like bunches of grapes under the microscope (i.e. Gram-positive cocci in clusters!) suggests that the bacteria are staphylococci. Most of these bacteria are fairly flexible about the conditions they require for their growth - give them roughly the right temperature and a few simple nutrients and they are happy. A few of them are rather more fussy though and bacteria such as Clostridium and Bacteroides are examples of this; they are called anaerobes which means that they can only grow if there is absolutely no oxygen present.




Why should you bother with all this? There are three reasons:

• dividing up new information makes it easier to learn - and unfortunately there is no option but to learn the core material in this table.




• this is the language used by clinicians, especially when they are trying to manage seriously ill patients.




• knowing this information well makes it easier to understand bacteria, to appreciate the different sorts of infections they cause and the antibiotics that can be used to treat them.

Exceptions to the rule: not all bacteria are shown up by the Gram stain, but there are only a couple of important exceptions for you to remember now!

• Mycobacterium tuberculosis has a thick waxy coat that stops the bacterial cells taking up the Gram stain; if these bacteria are suspected, the specific Ziehl Neelson stain must be used.

A small group of organisms, such as Chlamydia and Mycoplasma, do not have conventional cell walls at all and specialised techniques are often required to diagnose infections caused by these bacteria.

How do you get information about bacteria in clinical practice?
A number of different samples can be sent to the diagnostic laboratory for microbiological analysis including fluids (such as blood, urine or cerebrospinal fluid [CSF]), pieces of tissue or swabs taken from infected lesions. For specimens such as CSF that would normally be sterile, microscopy can be very useful as the presence of any bacteria is always abnormal. However, for the great majority of specimens, the sample will have to be spread out onto culture plates to grow the bacteria, to see if there are any in the sample that might be the cause of the infection - this will generally take at least 24 hours. If there are any suspect bacteria there, they will probably need to be identified further and also checked out to ensure that they are not resistant to the effect of antibiotics - at least another 24 hours. In situations where it is not possible to grow bacteria, it may be possible to diagnose infection based upon a person's antibody responses - but this is not usually a rapid method either! So that in many cases, you must make initial decisions about antibiotic treatment based upon a sound knowledge of bacteriology - what is the most likely or most important bacterial cause of this infection and what is the most appropriate antibiotic for treatment? This will usually be backed up by laboratory investigation to confirm your diagnosis (or not!) and help you refine your future management of the patient. To practice clinical medicine effectively you must have a good knowledge of bacteriology, some idea about the service provided by diagnostic laboratories and the ability to interpret the reports that are issued.
In the table of Gram stains above, the bacteria were grouped together and listed by genus name (plural = genera). This is a name given to a collection of bacteria that share many fundamental, major, obvious characteristics. However, by examining bacteria more closely, perhaps by looking at some of their biochemical capabilities for example, it is possible to divide them up further into individual species. This is very often of great clinical significance. An example of how this works in clinical practice is given below. Also, at the end of this chapter there is a table with a list of common bacteria of medical importance, the sites at which they can normally be found and the sorts of infections that they cause when things go wrong. This is intended to be a ready reference as you go through the course and you are not intended to learn all the information straightaway, but hopefully, by the end of the course, you will have become familiar with much of this information.