Microbiology, Lec 16.

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Viruses

Let's start by reviewing some basic facts about viruses (chapter 16). The history of Virology goes back a little more than 100 years. Scientists in the late 1800s knew of certain diseases for which they could find no microbe to blame. Pasteur thought that Rabies was caused by bacteria that were much smaller than normal bacteria. Beginning in the 1880s Mayer and Iwanowski began studying a "model system" for viral diseases, the tobacco mosaic disease. They showed that it was contagious and could be spread by spraying juice from sick plants onto healthy plants. But they could not find a bacterium or fungus in the sick plants. They therefore thought it was some sort of toxin. Then in 1897 Beijerinck (lec. 2) showed that it was no ordinary toxin. He used clever dilution experiments to show that the "virus" was multiplying inside infected plants, but he still could not see the virus. It wasn't until 1935 that the existence of viruses was proven by W. Stanley who actually crystallized the tobacco mosaic virus (TMV, using methods that had just been developed for crystallizing proteins) and showed that these crystals could cause the disease. So in a sense he fulfilled most of Koch's Postulates (lec. 2). We know now that TMV is an RNA virus with a protein coat (Figure 16.11).

1) obligate intracellular parasites

2) cannot multiply on their own

3) Ultramicroscopic i.e. less than 0.2 micrometers; some as small as 20 nm (see Fig. 16.10)

They all contain some type of nucleic acid (RNA or DNA) surrounded by a protein coat (= capsid) (Figure 16.13).

Many viruses that attack animals also have a membrane outside the capsid (Figure 16.17). We will come back to animal viruses after spring break....

Viruses contain enough genetic information to take over the protein making machinery (via mRNA) of the host cell and turn the host cell into a virus factory.

Bacteriophages

Diversity of phage is large (Fig. 17.1). The morphological diversity of phage that attack a single bacterium can be quite large. One species of bacteria can have over 20 different phages.

Fig. 17.6 shows the genome of bacteriophage T4. This virus has one of the largest genomes known for any virus which is why it so complex (Fig. 16.19). Genes for related functions are located in one area of the genome and are expressed at the same time (Fig. 17.6).

Viral "Life Cycles"

Lytic Cycle

Fig. 17.5.6 shows the time line for the infection process in T4 (note that the whole lytic cycle takes less than 1/2 hour!).

early mRNA

are made via host RNA polymerase (which is modified by a viral protein injected with the viral DNA) and code for proteins that: 1) degrade host DNA 2) make viral DNA

late mRNA

code for 1) structural proteins, 2) assembly enzymes and 3) cell lytic proteins.

The one-step growth curve = graphical depiction of life cycle... (Fig. 17.2)

Viral assembly can be quite complex (see Fig. 17.11)

Lysogenic Cycle ("Temperate" Bacteriophage, read pgs. 365 - 370)

The example of Lambda Phage:

see Figs. 17.14 - 17.20

3 theories:

1. They've always been here...

2. Retrograde evolution (think of Chlamydia, degenerate intracellular bacterial parasites..)

3. Escaped gene theory (think of transposons and plasmids)

Read Box 16.2, pg. 352.

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Genetic Transfer (cont.)

III. Transduction

is the transfer of DNA from one bacterium to another due to infection by a bacteriophage (a virus that infects bacteria). Bacteriophage infection involves a virus attaching to a specific cell and injecting nucleic acids. The viral nucleic acids "reprogram" the host cell and causes it to replicate the virus that is then assembled and released from the host cell. These new viruses can infect other cells and repeat the process.

Generalized transduction - in this situation, newly assembled viruses may contain DNA from the host cell and upon infection of a new cell can transfer the newly acquired DNA (Fig. 14.19). This typically makes the virus defective because it does not have the necessary information to force a cell to produce new viruses. Once this DNA is injected into a new bacterial cell recombination can occur and incorporate this new DNA.

Specialized transduction - Some viruses form prophages (= viral DNA incorporates itself into the host chromosome) and do not immediately reprogram the cell to make new virus particles. When the prophage is later excised from the chromosome it can take some of the cellŐs chromosomal DNA with it. This new viral DNA that contains the bacterial DNA will then be replicated by the cell and produce new viruses. These may then infect new cells and transfer this DNA (Fig 14.20).

E. coli O157:H7 is the virulent strain that has been in the news lately. It contains a shiga-like toxin that was transferred from a Shigella sp. through transduction. This toxin is a protein that causes severe hemorrhaging of epithelial cells that line the intestine.

Genetic mobility within bacteria

Transposition involves small segments of DNA that can move around a chromosome or plasmid ("jumping genes"). For example, F-plasmids have insertion sequences (Fig. 14.7) that allow the plasmid to integrate into the chromosome (this allows the process shown in Fig. 14.8 to occur). The result is the formation of an Hfr cell.

Insertion sequences (see Table 14.2) are very simple and typically contain only the information needed for insertion.

Transposons are slightly more complex and may contain a number of genes that confer antibiotic resistance and/or toxin production (see Table 14.3). Can be used in experiments to disrupt specific genes.

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Food Microbiology.

We have already discussed the early history of how humans have used microbes in food preservation etc. in lecture 2 (beer and wine) and lec. 7 (cheese and yogurt). Remember that beer and wine making date back at least 6000 years and were important ways to preserve food for ancient peoples. Cheeses and yogurts are probably as old a way of preserving food (evidence of cheese making date to about 4500 years ago in Egypt). There are many other fermented food products from around the world such as Tempeh, Sufu, Kimchi, Poi, Miso, Olives etc. etc. (see Table 43.8). In modern times, these ancient practices have been turned into multi-billion dollar industries.

Microbial Foods.

"Single Cell Protein" - Food for the future?

Yeast are being used more and more as a food supplements due to their high protein and vitamin content.
Other microbes are also used directly as food throughout the world. One example is the use of cyanobacteria (Spirulina platensis) by indigenous peoples of the central African republic of Chad. S. platensisis collected from the bottom of seasonally-dry ponds and lakes and then dried and cut into cakes (called Dihe). Dihe is rich in protein (~65%). Spirulinahas also been grown in S. France where it yields 10 tons of protein per acre. Cows yield only 0.016 tons of protein per acre. (show overhead)

Growing mushrooms on Farm wastes

Figure 43.21

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Cheese

we've already discussed yogurt (lecture 7), but I'd like you to understand a few basic concepts about the making of cheeses.

1) The first step of making cheeses is the coagulation of the milk to form the curd. Coagulation is just the denaturation of milk proteins brought about by lowering the pH (via, e.g. lactic acid bacteria) and by enzymes such as rennin which is an acid protease that was originally obtained from calf stomachs (now it comes from genetically engineered bacteria). Many fungal acid proteases (e.g. from Mucor spp.) are often used instead of rennin.

2) The curd is then pressed to remove the "whey", shaped and then allowed to ripen. Ripening is mostly a matter of further metabolism by microbes associated with (or added to) the cheese. The distinctive aromas, textures and tastes of cheeses are determined by the specific bacteria and fungi that ripen each type of cheese.
There are three main ways of ripening cheeses (see overhead):

(1) via the original bacteria in the cheese (e.g. Parmesan, cheddar, Gouda, Swiss)

(2) via microbes that are added to the inside of the cheese (e.g. Penicillium roquefortii in blue cheeses), (show overhead of Penicillium in blue cheese)

(3) microbes that grow on the outside of the cheese (e.g. Penicillium camemberti on Camembert and Brie cheeses).

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Wine

Let's talk about one other type of microbially produced "food", i.e. wine:

Figure 43.16

Note that the difference between white and red wines has mostly to do with how long the juice (= must) is exposed to the skins (where most of the pigments are) and the occurrence of the malo-lactic fermentation. In both white and red wines yeast (Saccharomyces ellipsoides or S. cerevisiae) are usually added to the wine, but many wines (especially in Europe) are produced just with the natural yeast that are on the grapes (or in the wine making cellars).

You already know the ethanol fermentation from lecture 11, so let's look at the "malo-lactic" fermentation. It turns out that many red wine grapes are picked before they are completely ripe and as a result there is an excess of malic acid in the grapes (that would be converted to sugar in ripe grapes). After the wine has set for a while and most of the sugars are fermented, certain lactic acid bacteria (e.g. some Lactobacillius spp.) carry out a fermentation that converts malic acid to lactic acid and CO2. Because malic is a dicarboxilic acid and lactic is a monocarboxilic acid, this reaction can cut the acidity of the wine by up to half. This step is absolutely essential for the production of high quality red wines, especially in Burgundy and Bordeaux, where the growing season is short.

Dessert Wines

Let's look at one other type of wine that involves some extra microbial steps. In the Sauternes region of France during certain moist years, white wine grapes become infected with a fungus, Botrytis cinerea, which can completely cover the outside of the grape. This fungus digests the pectin in the grape skin leaving most of the sugar in the grape. B. cinerea also causes the grape to lose water, thereby concentrating the sugars in the grape. These fungus-covered grapes are carefully picked and then fermented using a so-called glucophilic yeast which ferments only the glucose in the grape leaving the fructose behind. What results is a sweet wine that has a distinctive taste and a golden color, both of which come from B. cinerea. These Sauternes are among the most expensive wines in the world and are produced in very limited quantities.

Vinegar

To illustrate some basic concepts of industrial processes, let's look at something we mentioned in lecture 6, i.e. the acetic acid or vinegar bacteria (strict aerobic Proteobacteria). When you leave wine exposed to oxygen, these bacteria proliferate and make a living by generating NADH from the oxidation of ethanol to acetic acid (see Figure).

Show some commercial systems for generating vinegar from ethanol. Note that the basic principle of both is to aerate the fluid as much as possible. Reactors similar to these two types of reactors are common to many industrial processes.

1) Continuous Flow Bioreactor involving a biofilm on the wood chips (show Figure).

2) Batch process (show Figure)

Fermented Soy Products

Fermented soybean products have been produced in China and other countries for at least 2000 years. The microorganisms used in these fermentations are quite varied and include Fungi (Rhizopus, Aspergillusand various yeasts) in addition to the usual Bacterial fermentors like Lactobacillus. The production of Tempe and Soya Sauce (shoyu) will be discussed below.

Tempe is one of the main sources of protein in many parts of Indonesia (annual production in 1991 was > 200,000,000 kilograms). It is traditionally made by soaking soybeans overnight (the soak water is discarded), removing the skins and then briefly boiling the beans. The beans are then formed into cakes and inoculated with Rhizopus oligosporus or with some Tempe from the last batch (or sometimes with crushed Hibiscusleaves which are a good natural reservoir for this fungus). The cakes are then wrapped in banana leaves or wax paper to keep them moist. They are then incubated at 30 to 37 degrees C for 1 to 3 days, during which time the fungus grows through the mass of beans holding them together in a firm cake of white mycelium. R. oligosporusproduces high levels of lipases and proteases which render the beans much more digestible for humans. The concentration of B-vitamins (riboflavin, niacin, pyridxine and B12) and antioxidants go way up during the fermentation. These fungal by-products therefore not only increase the nutritional value of the soybeans but also make them more digestible and stable for storage.

Soya Sauce (shoyu) is produced via a succession of different microbes (see overhead) that takes about 3 months to complete. The substrate for shoyu production is a mixture of salted soybeans and various grains (usually wheat).
The main stages of the microbial succession are:

1) growth of Aspergillus spp. and various bacteria (e.g. Micrococcus and Bacillusspp.) that digest the protein and polysaccharides in the soy beans and wheat.
2) later growth of Lactobacillus spp. which decrease the pH of the mixture and finally...
3) Growth of acid-tolerant yeasts (e.g. Torulopsis) which carry out alcoholic fermentations.

The final ethanol content of shoyu is 2-3% and the final lactic acid content is about 1%. Most of the flavor, however, is due to many different primary and secondary metabolites produced by the various organisms involved in the process. There are presently over 25,000 different shoyu manufacturing facilities in Japan alone!...