Microbial Mythology

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Various misconceptions and oversimplifications commonly appear in textbooks. Here we provide a place for microbiology educators to set the record straight.
Authors: Please provide primary references, in open-access sources whenever possible.


Aerobic or anaerobic: Is a shaken flask truly aerobic?

For decades, researchers have cultured E. coli and other bacteria in a flask shaken or rotated to aerate, providing access to oxygen for respiration. But is the culture truely "aerobic;" that is, does it actually get enough oxygen to respire? It turns out that only during the early part of the growth curve do the bacteria receive enough oxygen to be fully "aerobic" (that is, respiring as fast as they can.) An oxygen electrode shows that up to about optical density of 0.2-0.3 (measured at 600nm), the oxygen concentration declines to zero. Above OD of 0.3, the cells are using oxygen faster than it can be replaced; thus, their respiration is underutilized. The culture is called "microanaerobic," meaning that it uses partly anaerobic metabolism. Later in stationary phase, when metabolism slows down, the culture becomes "aerobic" again. For discussion, see for example Svetlana Alexeeva et al., 2001, J. Bacteriol. 184:1402.
Actually, the situation is even more complicated, depending on the growth medium. Enteric bacteria grown on glucose convert everything to acetate first, no matter how much oxygen they get. Then later, they they take back the acetate and send it to the TCA cycle. For very full discussion, see Alan Wolfe, 2005, MMBR 69:12. (Note: This very long review takes a while to download.)

Centrifugation: Is it a harmless way to "collect" bacteria?

The time-honored way of collecting bacteria from a suspended culture is to centrifuge the suspension; that is, subject the cells to thousands of times the force of gravity (g) so that they sediment in a pellet at the bottom of a tube. But what effect does centrifugation have on cells? They experience g force, loss of oxygen (if previously aerated), nutrient depletion, and cold shock (if previously warmed to 37 deg. C). And the cells experience surface changes that alter their adhesive properties. See for example this article by Caitlin H. Bell, 2005, J. Young Investigators 12:6.

Colonies: Is a bacterial "colony" really a uniform population?

A colony is defined as a population of cells arising through reproduction from a single ancestor, generally a bacterium lying in one spot on agar medium. But the progeny cells soon experience extremely different local conditions; those near the middle of the colony starve for nutrients, and accumulate wastes such as fermentation acids, while those at the growing edge of the colony experience a chemotactic gradient of nutrients. The central cells are in stationary phase, whereas the leading edge cells may be in log phase. Moreover, mutations may occur, resulting in a population that is genetically diverse. Then too, the cells are adjacent to each other, interacting to form a miniature biofilm. "Colony biofilms" are now a major subject of study for antibiotic resistance and other properties. See for example Merritt et al., 2005 and Zuroff et al. 2010.

Conjugation in bacteria: Do pili mediate DNA transfer?

During bacterial conjugation, the donor and recipient cells are brought together by protein filaments called pili. Since the pili are composed of hollow tubes of protein subunits, like a turret, it was thought for a while that DNA might travel down the hollow tube. The hollow tube theory is still taught; see example.

Actually, DNA is transferred across the bacterial envelope by a protein complex embedded in the membranes. For review of classic experiments, see Brigette Dreiseikelmann, 1994. For a more current review, see Inês Chen et al, 2005.

Flaming the Flask: Is it necessary?

A rite of passage in the microbiology lab is learning to "flame the mouth of the flask" when transferring cultures, or when pouring autoclaved media into petri plates. But does flaming the flask actually decrease the incidence of contamination? Not so, according to this ancient reference: Brunker & Fernandez, 1972
In fact, most everyday microbiology techniques don't need flaming. Methanol treatment fixes bacteria to the slide better than heat fixation. In our lab, we recently shut down the gas to the whole floor, to avoid accidental use and require alternative methods.
What does help avoid contamination is (1) rapid transfer, and (2) holding the flask or tube horizontal to avoid dust settling.

Fossil microbes: Do they exist?

The oldest fossil organisms are actually microbes. The earliest convincing microfossils are of colonial cyanobacteria dated to 2 Gya (two billion years ago). More recent fossils known as stromatolites are also the products of microbes affecting the deposition of sediment clasts and the precipitation of chemical sediments.
But microbial fossils are hard to define. Some formations claimed to be fossils have actually turned out to be non-biological. There are no objective criteria for what constitutes a "true" microbial fossil; only a consensus that certain formations have never been seen to result from non-biological chemical or physical processes. For a good general discussion of microbial fossils, see Life on a Young Planet: The First Three Billion Years of Evolution on Earth by Andrew H. Knoll.

Free-Energy Change ΔG: Is Bigger Better?

Textbooks often state that the larger the magnitude of free-energy change ΔG (with negative sign), the better for microbial metabolism. Carbohydrates and lipids are presented as the best energy sources, because their oxidation yields (in theory) the largest amounts of energy for ATP. However, natural environments support vast numbers of bacteria and archaea that reproduce using metabolic reactions with ΔG magnitude less than required to produce one ATP. This is possible because in natural environments such as soil, bacteria and archaea coexist in "syntrophy," a relationship in which the products of one metabolism are immediately consumed as substrates for the next organism. Thus, in nature, most free-living microbes are extremely efficient in their energy usage, and glucose catabolism is rare. Read "Anaerobic microbial metabolism can proceed close to thermodynamic limits," by Jackson and McInerney.

Immune System: Innate Versus Adaptive?

One of the first things we learn about the vertebrate immune system is that it includes "innate nonspecific" components such as macrophages that phagocytose invading bacteria, and "adaptive specific" components such as B cells that make antibodies to specific pathogens. In fact, however, both "innate" and "adaptive" cell types include responses with varying degrees of specificity. Macrophages express cell-surface receptor proteins (Toll-like receptors, TLRs) that recognize pathogen associated molecular patterns (PAMPS) such as LPS or flagellin made by certain classes of bacteria. On the "adaptive" side, B cells start out making antibodies of low specificity; their specificity increases only with exposure to the pathogen. Both innate and adaptive branches of the immune system communicate with each other; for example, the macrophages digest bacteria and viruses to present antigens of optimal size to the T cells for stimulating B cell development and antibody production. Overall, a web of interactions of many types interweaves all components of the immune system.

Glucose Oxidation: Does it Really Generate 36 ATP?

The textbook presentation of oxidative phosphorylation on glucose generally shows pyruvate breakdown to acetyl-CoA with immediate entry to the TCA cycle, leading to electron-transport system and ATPase to produce six CO2 and 36-38 ATP. This picture may come close to what mitochondria do, but bacteria never do this. Escherichia coli, for example, first converts all its glucose to acetate, no matter how much oxygen is provided. Later, as glucose disappears, the acetate is taken back into the cell and metabolized more slowly to formate, ethanol, and eventually CO2. For a detailed picture of acetate metabolism, see Alan Wolfe, 2005, MMBR 69:12.

Nuclear membrane: Do bacteria have one?

Introductory texts often give the impression that bacterial DNA lacks organization, basically distributed in the cytoplasm. In fact, however, bacterial DNA is tightly organized in looped domains of the nucleoid. See for example "The bacterial nucleoid: a highly organized and dynamic structure," Thanbichler et al 2005. Furthermore, some bacteria actually contain their DNA within a membrane very much like a "nuclear membrane." Such bacteria include the Planctomycetes, studied by John Fuerst and colleagues. Planctomycetes are commonly found in soil and water, where they have important ecological functions such as association with invertebrates, and the conduct of anaerobic ammonia oxidation (anammox metabolism). Some archaea associated with sponges also appear to possess something like "nuclear" membranes.

TCA cycle: Does succinyl-CoA synthetase use ATP or GTP?

Succinyl-CoA synthetase, also known as succinate thiokinase, is the enzyme of the TCA cycle that interconverts succinyl-CoA with succinate, coupled to formation of a nucleotide triphosphate. Many textbooks and web sites state that succinyl-CoA synthetase phosphorylates only GDP to GTP. See example.

According to the primary literature, however, ADP phosphorylation predominates in E. coli (Margaret Birney et al, 1996) whereas in Pseudomonas sp., various nucleotide diphosphates are phosphorylated (Vinayak Kapatral et al, 2000). Human mitochondria have two forms of the enzyme, which phosphorylate ATP and GTP respectively (David Lambeth et al, 2004).

Viruses: Can a virus be a cell?

Viruses are traditionally defined as non-cellular infective particles. Yet some giant viruses, such as Mimivirus (which infects amebas) have a genome comparable in size to that of bacteria (1.2 million base pairs) and carry numerous cellular enzymes. ASM News 71:278, 2005 Bioinformatic analysis shows that Mimivirus probably evolved from bacteria by reductive evolution. Similar reductive evolution is likely for other large DNA viruses such as herpesviruses. Mimivirus can even be parasitized by smaller viruses called "virophages." NatureNews 6 Aug. 2008

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