Microbiology,

Microbial Physiology.

My goal for this part of the course is to help you understand the basic ways that microbes "make a living" out there in the world. Because microbes are so small, it is through their physiologies that they affect the world around them. They don't use physical force to change the world but rather a more subtle form force - the forces of enzymatic reactions and metabolic byproducts including toxins etc.

I. The Basics.

Let's start by looking at the basics of how microbes make their livings.

First, as with all living things, microbes are made up C, N, P, O, S, H, K, Fe, Mg, and Ca. In addition, microbes require some micronutrients, e.g. Mn, Zn, Co, Mo....... We'll see where most of these elements fit into metabolism over the next week or so (but you should already know where N, P, C, O, S, Fe are used by all organisms - if not read chapter 5 and pp. 98-101). Interestingly, many microbes don't require Na, which is required by most plants and animals.

Modes of microbial nutrition.

Microbes and all other living things can be classified based on how they obtain their carbon and energy. We have already touched upon some of these groups in lecture 5 and below is an expanded vocabulary of these terms

Heterotrophs

Chemoheterotrophs use organic compounds for energy and carbon (we are chemoheterotrophs as are the Fungi and many Bacteria and some Archaea).

Photoheterotrophs organisms that can use light for energy ("photo") and organic compounds ("hetero") for carbon (only some prokaryotes can do this)

Autotrophs can fix carbon dioxide and turn it into organic molecules.

Photoautotrophs use light energy to do this (plants, cyanobacteria and many other bacteria are photoautotrophs).

Chemoautotrophs use chemical energy to fix CO2 (e.g. sulfur oxidizing and nitrifiers, there are no eukaryotic chemoautotrophs)

"Chemolithoautotrophs" is a term sometimes used for chemoautotrophs that use inorganic compounds for energy, but this is true of most chemoautotrophs so I will use the term chemoautotroph only.

How do microbes feed?

Bacteria, Archaea and Fungi feed by absorbing chemicals from their environment. Everything that gets into the cell must enter via diffusion (e.g. O2, H2O, CO2) or via some sort of transport protein or channel. In contrast, most protozoa can phagocytize chunks of material (including whole bacterial cells) and digest them in vacuoles that act like little stomachs.

Most of what we will discuss below applies to Bacteria, Archaea and Fungi.

Given that Bacteria, Archaea and Fungi take up all their food (and get rid of all their waste molecules) across their plasma membranes as individual molecules, it is not too surprising that they can NOT be any bigger than they are. To better understand the size limit for absorptive feeders we can do a little simple geometry.

Consider a coccoid cell of with a diameter of 1 µm.......

show formula to illustrate that surface area grows much more slowly than does volume as a sphere or cube gets bigger.

Microbes can partially overcome these constraints by being long and thin (fungi, spirochetes, actinomycetes etc.), by having prosthecae (e.g. Hyphomicrobium and Caulobacter, lec. 6) or by having internal membrane-bound vesicles (true of most fungi and some bacteria) in which they can store nutrients or waste products. An example of a Bacterium that has large internal vacuoles is the giant (15 - 40 µm wide) chemoautotroph (H2S = e- donor; NO3- = e- acceptor) Thioploca (lec. 6) that forms huge mats off the coast of Chile (see Fossing et al., 1995). These large Bacteria migrate up and down in the sediments, taking up nitrate (which is stored in their vacuoles) from seawater at the top of the sediments and then migrating down to zones rich in sulfides (the S can then be oxidized using the stored nitrate). These amazing creatures can also store elemental sulfur granuoles in their cytoplasm as inclusion bodies. Thus, they can carry around extra e- donor (S) and extra e- acceptor (nitrate) wherever they go......

Fossing, H., et al. 1995. Concentration and transport of nitrate by the mat-forming sulphur bacterium Thioploca. Nature, vol. 374: 713 - 715

Uptake of nutrients.

But regardless of their shapes they are taking up nutrients across their membranes.

Please read pp. 101 - 105.

Diffusion - small or uncharged molecules can diffuse across the plasma membrane.

Facilitated diffusion is just diffusion that utilizes a specific trans-membrane protein (a permease)

see Figure 5.2

Active Transport - See Figure 5.3 and 5.4

Group Translocation - see Figure 5.4 (occurs only in some Bacteria)

How Microbes eat polymers and insoluble substrates:

Extracellular enzymes

Bacillus and extracellular amylase (show overhead)

Cytophaga and extracellular cellulases (show Sporocytophaga on plant fiber

Siderophores (See lec. 6, pg. 105 and Fig. 5.5)

Emulsifying agents or "biosurfactants" are extracellular compounds produced by microbes that eat oils.
Read pg. 952 and Fig. 44.18.

The above section (Uptake of nutrients) is all about how microbes accumulate molecules from their environment - now let's look at what happens to these nutrients once they are inside the cell....

Let's do non-photosynthetic reactions first..... Catabolic and anabolic reactions

Energy Transductions

(catabolic reactions, Ch. 9)

Oxidation/Reduction (redox) reactions

Catabolic reactions are Redox reactions. All biological oxidations are coupled to reductions.

oxidation is the loss of electrons (Ole-) Let's start with the simplest reaction to understand - Respiration

Remember from lec. 2 that Kluyver and van Niel proposed that all respiratory reactions (aerobic and anaerobic) could be summarized using the simple formula:

AH2 + B -----> A + H2B

Let's apply this to the type of respiration that goes on in mitochondria and many other chemoheterotrophic microbes:

CH2O + O2 -----> CO2 + H2O

or for the specific case of glucose respiration

C6H12O6 + 6 O2 -----> 6 CO2 + 6 H2O

In this case we say that glucose (C6H12O6) is being oxidized to CO2 and O2 is being reduced to H2O. Glucose is the energy source and the electron donor and oxygen is the terminal electron acceptor.

Respiration in More Detail:

Let's look at e- and H+ flow in a typical respiring microbe.....

Aerobic Respiration.

Figure 9.1 = first step - shows the initial steps in the breakdown of a hexose (after it is released from a polysaccharide) by a respiring bacterium or fungus (or human for that matter).

---> two pyruvate molecules
---> e- flow from sugar to NAD+ ---> NADH.
---> ATPs / substrate-level phosphorylation

Pyruvate is further oxidized to CO2 via the Krebs (TCA) cycle or other pathways in microbes (not all microbes have the Krebs cycle).
Fig. 9.1

---> Products are CO2, ATPs and many NADHs

Fate of e- carried by NADH Figures 9.9 - 9.12 = e- transport chain, ATPases, etc....
.

O2 is the terminal electron acceptor
ATPs / electron transport phosphorylation

Contrast electron transport phosphorylation with substrate-level phosphorylation.

Draw picture on board to explain overall process and stress understanding the process and not all of the individual reactions.......

Anaerobic Respiration.

An overview of anaerobic respiration would look just the same as aerobic respiration except that the terminal e- acceptor would be something like nitrate or sulfate.

Compare anaerobic and aerobic respiration in organisms that can do both.....

p. 176 show these processes in Paracoccus denitrificans (an Alpha Proteobacterium). This organism can completely denitrify nitrate all the way to N2.

Let's look at a slightly simpler example, i.e. E. coli with and without oxygen. Note that in the absence of oxygen fewer protons are pumped (therefore fewer ATPs are ultimately made) and that the end products are nitrite and water.

Fermentations are also a series of redox reactions except that there is no terminal e- acceptor, but more about that next time.......