When an F+ bacteria conjugates with an F-, it makes the other bactaria F+ too. So on the long run, all bactaria should be F+.
Is there any mechanism that converts F- to F+? Could it be degeneration of F plasmid?
So an easy way to convert a cell from F+ to F- is to divide it without correctly replicating the plasmid and transferring it to a daughter cell, leaving you with one F+ and one F-. The cells that are F- now have a selective pressure advantage over the F+ cells, as they now don't require the energy to produce a plasmid when they divide. If that advantage is enough to overcome the frequency of conjugation, you'll end up having a population of F- cells.
Biological immortality (sometimes referred to as bio-indefinite mortality) is a state in which the rate of mortality from senescence is stable or decreasing, thus decoupling it from chronological age. Various unicellular and multicellular species, including some vertebrates, achieve this state either throughout their existence or after living long enough. A biologically immortal living being can still die from means other than senescence, such as through injury, poison, disease, lack of available resources, or changes to environment.
This definition of immortality has been challenged in the Handbook of the Biology of Aging,  because the increase in rate of mortality as a function of chronological age may be negligible at extremely old ages, an idea referred to as the late-life mortality plateau. The rate of mortality may cease to increase in old age, but in most cases that rate is typically very high. 
The term is also used by biologists to describe cells that are not subject to the Hayflick limit on how many times they can divide.
Bacteria: Fossil Record
It may seem surprising that bacteria can leave fossils at all. However, one particular group of bacteria, the cyanobacteria or "blue-green algae," have left a fossil record that extends far back into the Precambrian - the oldest cyanobacteria-like fossils known are nearly 3.5 billion years old, among the oldest fossils currently known. Cyanobacteria are larger than most bacteria, and may secrete a thick cell wall. More importantly, cyanobacteria may form large layered structures, called stromatolites (if more or less dome-shaped) or oncolites (if round). These structures form as a mat of cyanobacteria grows in an aquatic environment, trapping sediment and sometimes secreting calcium carbonate. When sectioned very thinly, fossil stromatolites may be found to contain exquisitely preserved fossil cyanobacteria and algae.
The picture above is a short chain of cyanobacterial cells, from the Bitter Springs Chert of northern Australia (about 1 billion years old). Very similar cyanobacteria are alive today in fact, most fossil cyanobacteria can almost be referred to living genera. Compare this fossil cyanobacterium with this picture of the living cyanobacterium Oscillatoria:
The group shows what is probably the most extreme conservatism of morphology of any organisms.
Aside from cyanobacteria, identifiable fossil bacteria are not particularly widespread. However, under certain chemical conditions, bacterial cells can be replaced with minerals, notably pyrite or siderite (iron carbonate), forming replicas of the once-living cells, or pseudomorphs. Some bacteria secrete iron-coated sheaths that sometimes fossilize. Others may bore into shells or rocks and form microscopic canals within the shell such bacteria are referred to as endolithic, and their borings can be recognized all through the Phanerozoic. Bacteria have also been found in amber -- fossilized tree resin -- and in mummified tissues. It is also sometimes possible to infer the presence of disease-causing bacteria from fossil bones that show signs of having been infected when the animal was alive. Perhaps most amazing are the fossils left by magnetobacteria -- a group of bacteria which form tiny, nanometer-sized crystals of magnetite (iron oxide) inside their cells. Magnetite crystals identifiable as bacterial products have been found in rocks as old as two billion years -- at a size of a few hundred millionths of a meter, these hold the record for the smallest fossils.
NEWS FLASH!One of the hottest science news stories of the decade is the discovery of possible remains of bacteria-like organisms on a meteorite from Mars. But are they really fossils? How would we be able to find out whether or not they are real? And what could they tell us about the history of Mars -- and of life on our own planet? Paleontologists are working together with space scientists to try and answer some basic questions about the possible "Martian bacteria." There will eventually be an exhibit on this server dealing with the "Martian microbes." Until it's ready, you can view photographs and news articles about the find, or learn more about Mars meteorites courtesy of the NASA Jet Propulsion Laboratory. --> Read a UCMP Research Report: "Bacteria and protozoa from middle Cretaceous amber of Ellsworth County, Kansas." Find out more about fossilized filamentous bacteria and other microbes, found in Cretaceous amber -- a unique mode of preservation. This report was originally published in PaleoBios 17(1): 20-26. Dr. Raul Cano, at California Polytechnic State University at San Luis Obispo, has succeeded in isolating and reviving bacteria taken from inside fossilized insects trapped in amber. Read all about it!
Bitter Springs chert fossil image provided by J. William Schopf. The image of Oscillatoria was provided by Alejandro Lopez-Cortes (CIBNOR, Mexico), Mark Schneegurt (Wichita State University), and Cyanosite.
Where Can Bacteria Be Found?
Bacteria are among the most numerous organisms on earth, explains Microbe World. Bacteria exists virtually everywhere on earth. Bacteria can be found in the air, soil, water, on plants, on animals and even on the skin of human beings.
Part of what makes bacteria so plentiful is their ability to inhabit a variety of different types of environments. Some bacteria thrive in extremely hot environments, such as those found in boiling-hot sulfur springs and geysers. Other types of bacteria are capable of surviving sub thermal freezing temperatures buried under many sheets of ice, such as those conditions found in the super-cold Antarctic lakes. Microbes that live in these extreme conditions are called extremophiles.
One single teaspoon of top soil contains as many as one billion bacteria cells. The human mouth alone harbors more than 500 different species of bacteria. Each square centimeter of skin on the human body contains around 100,000 bacteria. Bacteria can survive not only in extreme conditions, they can also survive on surfaces for long periods of time. While removing bacteria from day-to-day life is not feasible, washing hands regularly and disinfecting surfaces you come in contact with on a regular basis helps reduce the amount of bacteria that enters your body.
The Institute for Creation Research
Archaeans are amazing microbes that run on completely different metabolic processes than other microbes. Discovering the first of them must have been like finding a car that runs on hydrogen fuel cells amidst a landscape of gasoline-powered vehicles. This was the privilege of evolutionary biologist Carl Woese, who died on December 30, 2012. 1 How did he interpret these findings, and what should we remember about his contributions?
Woese was famous for adding a whole new major classification of microbes, called archaea, that biology textbooks published about well within his lifetime. But the name assigned to this unique domain of life reflects evolutionary concepts, not science.
The biochemistry of these tiny survivors is so fundamentally different from most oxygen-burning creatures that evolutionists like Woese believed it must have evolved way back when the first normally-functioning bacteria were also inventing themselves. The name "archaea" derives from the Greek word "arkhaios," meaning ancient or primitive.
But though these bacteria defy so many norms of microbial life, they don't appear ancient at all. Scientists observe them alive today, albeit in hostile places like deep sea toxic vents. Why call them ancient if scientists did not actually observe them billions of supposed years ago?
In fact, Woese was familiar with at least two reasons why archaea could never have evolved. First, their fundamentally different biochemistry is fully formed and well designed. 2 It consists of interdependent arrays of molecular machines with form-fitted protein parts. Nature alone couldn't generate all of the miraculous biochemistry on which familiar oxygen-burning cells depend, just like nature alone couldn't generate gasoline burning engines in cars. 3 Therefore the discovery of bacteria that live on sulfur, for example, doubles both the biochemical barriers that evolution cannot hurdle and the credit that the Creator deserves for constructing them. 4
Second, without microbes and other organisms with extreme diets, like those that eat oil 5 or survive radiation 6 and other extreme living conditions like saturated salt, the life-giving properties of earth's atmosphere would not exist. Woese told The New York Times in 1996, "If microbial life were to disappear, that would be it -- instant death for the planet." 7 That means Woese was familiar with the appearance of purpose in bacteria on a planetary level&mdashthey appear to have been created to maintain the grand earth systems that support plants and animals. 8
Revealing the amazing designs of archaeans' unique ways of life is commendable, but naming them "archaean" merely reinforces unscientific evolutionary ideas. Unfortunately, Woese's legacy includes twisting the facts of God's creation to fit the falsehood of evolution.
- Zielinska, E. Evolutionary Biologist Dies. The Scientist. Posted on the-scientist.com January 2, 2013, accessed January 8, 2013.
- Thomas, B. Exploring Earth's Extremes in a Futile Quest for Life in Space. Creation Science Update. May 11, 2010, accessed January 8, 2013.
- Morton, J. S. 1980. Glycolysis and Alcoholic Fermentation. Acts & Facts. 9 (12).
- Sherwin, F. Reheating the Prebiotic Soup. Institute for Creation Research. Posted on icr.org September 1, 2003, accessed January 8, 2013.
- Thomas, B. Oil-eating Bacteria Are Cleaning Up Gulf. Creation Science Update. Posted on icr.org August 27, 2010, accessed January 8, 2013.
- Thomas, B. Life Thrives amid Chernobyl's Leftover Radiation. Creation Science Update. Posted on icr.org February 8, 2011, accessed January 8, 2013.
- Blakeslee, S. Microbial Life's Steadfast Champion. The New York Times. Posted on nytimes.com October 15, 1996, accessed January 8, 2013.
- Thomas, B. New Insights into Earth's Nitrogen-Balancing System. Creation Science Update. Posted on icr.org November 21, 2011, accessed January 8, 2013.
Image credit: National Oceanic and Atmospheric Administration
* Mr. Thomas is Science Writer at the Institute for Creation Research.
An important breakthrough came when Luca Cavalli-Sforza discovered a derivative of an F + strain. On crossing with F − strains this new strain produced 1000 times as many recombinants for genetic markers as did a normal F + strain. Cavalli-Sforza designated this derivative an Hfr strain to indicate a high frequency of recombination. In Hfr ×𠁟 − crosses, virtually none of the F − parents were converted into F + or into Hfr. This result is in contrast with F + ×𠁟 − crosses, where infectious transfer of F results in a large proportion of the F − parents being converted into F + . Figure 7-6 portrays this concept. It became apparent that an Hfr strain results from the integration of the F factor into the chromosome, as pictured in Figure 7-6a.
The transfer of E. coli chromosomal markers mediated by F. (a) Occasionally, the independent F factor combines with the E. coli chromosome. (b) When the integrated F transfers to another E. coli cell during conjugation, it carries along any E. coli DNA (more. )
Now, during conjugation between an Hfr cell and a F − cell a part of the chromosome is transferred with F. Random breakage interrupts the transfer before the entire chromosome is transferred. The chromosomal fragment can then recombine with the recipient chromosome. Clearly, the low level of chromosomal marker transfer observed by Lederberg and Tatum (see Figure 7-2) in an F + ×𠁟 − cross can be explained by the presence of rare Hfr cells in the population. When these cells are isolated and purified, as first done by Cavalli, they now transfer chromosomal markers at a high frequency, because every cell is an Hfr.
The space under your fingernails is completely impervious to the best, most simple means we have of preventing the spread of diseases
The researchers reasoned that could be because the space between the skin and nail creates a perfect environment for the growth and proliferation of these minute lifeforms, thanks to both the physical protection provided by the nail and all that moisture. The prior findings that persistent scrubbing doesn’t sterilise the hand, combined with the finding from their study “that there are significant numbers of bacteria in the subungual compartment suggest[s] that this hand region may be relatively inaccessible to antimicrobial agents during normal hand-washing procedures,” they wrote.
Think about it: the space under your fingernails is completely impervious to the best, and simplest, means we have of preventing the spread of diseases.
Indeed, a small but thriving area of research continues to probe the very nature of the microbial life living on the fingernails of nurses. And not just natural nails, but also artificial ones, or ones covered in polish.
Each fingertip can provide a home to hundreds of thousands of bacteria (Credit: Getty Images)
In 1989, just one year following the University of Pennsylvania study, a group of nurses wrote, “although unanswered questions concerning the safety and practicality of artificial nails remain, many health care workers have succumbed to fashion trends and are now wearing artificial nails”.
The researchers wanted to see whether 56 nurses with artificial nails, which tend to be longer than natural nails and are almost always covered in nail polish, had more bacteria on their fingertips than 56 nurses with natural nails. They also wanted to see whether handwashing was more or less effective for those with artificial nails.
They discovered that nurses with artificial nails had more bacteria on their fingertips than did those with natural nails, both before and after handwashing. That’s not to say that they were actually transferring more bacteria to their patients, necessarily, only that the bacteria living on their fingertips were more numerous. Still, the assumption is that more bacteria at least increases the potential for pathogen transmission.
Triclosan: the good, the bad, and the unknown
A Swiss company called Ciba-Geigy was the first to synthesize and patent triclosan in 1964, and, by 1970, it was in use around the world as a surgical scrub in hospitals. Today, it is estimated that 3 of every 4 antibacterial liquid soaps sold to the typical consumer contains triclosan as the active ingredient.
While it is a useful part of many consumer products such as toothpastes, there are some concerns regarding the use of triclosan. Studies done on cells and animals in labs suggest the chemical can impact hormone signaling and other biological processes. There is also evidence that accumulation of triclosan in the environment negatively impacts organisms like algae in aquatic ecosystems. However, it is also important to point out that, to date, triclosan has not been directly linked to negative health effects in humans. On the other hand, some of the other additives recently banned by the FDA, like hexachlorophene, have been directly shown to be harmful to humans, especially with high or repeated exposure. Fortunately, for chemicals like these, the FDA has had limitations in place for years to ensure over-the-counter exposure to consumers is within safe limits.
Lastly, there are concerns that triclosan use may increase the risk of generating drug-resistant bacteria. It is well documented that bacteria normally found on your skin can become resistant to triclosan itself. Specifically, triclosan-resistant bacteria typically have mutations in proteins called enoyl-acyl carrier protein reductases (ENRs), which are important for the biosynthesis of cell membranes and are also targets for other clinically used antibiotic drugs like Isoniazid. Thus, when bacteria populations are continually exposed to triclosan, especially from environmental accumulation, they develop mutations in their ENRs to survive the exposure. The major public health concern is that these ENR mutations can also make these bacteria resistant to other antibiotics prescribed by doctors (Figure 2). If this is the case, limiting the use of triclosan to only products where it is most effective could be very important.
Figure 2:Environmental exposure to triclosan helps bacterial populations develop resistance mutations to triclosan and other important antibiotics
Why do F- bacteria still exist? - Biology
Bacteria are tiny little organisms that are everywhere around us. We can't see them without a microscope because they are so small, but they are in the air, on our skin, in our bodies, in the ground, and all throughout nature.
Bacteria are single-celled microorganisms. Their cell structure is unique in that they don't have a nucleus and most bacteria have cell walls similar to plant cells. They come in all sorts of shapes including rods, spirals, and spheres. Some bacteria can "swim" around using long tails called flagella. Others just hang out or glide along.
Are bacteria dangerous?
Most bacteria aren't dangerous, but some are and can make us sick. These bacteria are called pathogens. Pathogens can cause diseases in animals and plants. Some examples of pathogens are leprosy, food poisoning, pneumonia, tetanus, and typhoid fever.
Fortunately, we have antibiotics we can take which help to fight off the bad pathogens. We also have antiseptics to help us keep wounds clean of bacteria and antibiotic soap we use to wash to help keep off bad pathogens. Remember to wash your hands!
Not at all. Actually most bacteria are very helpful to us. They play an important role in the planet's ecosystem as well as in human survival.
Bacteria work hard in the soil for us. One type of bacteria, called decomposers, break down material from dead plants and animals. This might sound kind of gross, but it's an important function that helps to create soil and get rid of dead tissue. Another type of bacteria in the soil is Rhizobium bacteria. Rhizobium bacteria helps to fertilize the soil with nitrogen for plants to use when growing.
Yep, there's bacteria in our food. Yuck! Well, they aren't really that bad and bacteria is used when making foods like yogurt, cheese, pickles, and soy sauce.
Bacteria in our bodies
There are many good bacteria in our bodies. A primary use of bacteria is to help us digest and breakdown our food. Some bacteria can also help assist our immune system in protecting us from certain organisms that can make us sick.
Why do F- bacteria still exist? - Biology
Methane fermentation is a versatile biotechnology capable of converting almost all types of polymeric materials to methane and carbon dioxide under anaerobic conditions. This is achieved as a result of the consecutive biochemical breakdown of polymers to methane and carbon dioxide in an environment in which a variety of microorganisms which include fermentative microbes (acidogens) hydrogen-producing, acetate-forming microbes (acetogens) and methane-producing microbes (methanogens) harmoniously grow and produce reduced end-products. Anaerobes play important roles in establishing a stable environment at various stages of methane fermentation.
Methane fermentation offers an effective means of pollution reduction, superior to that achieved via conventional aerobic processes. Although practiced for decades, interest in anaerobic fermentation has only recently focused on its use in the economic recovery of fuel gas from industrial and agricultural surpluses.
The biochemistry and microbiology of the anaerobic breakdown of polymeric materials to methane and the roles of the various microorganisms involved, are discussed here. Recent progress in the molecular biology of methanogens is reviewed, new digesters are described and improvements in the operation of various types of bioreactors are also discussed.
Methane fermentation is the consequence of a series of metabolic interactions among various groups of microorganisms. A description of microorganisms involved in methane fermentation, based on an analysis of bacteria isolated from sewage sludge digesters and from the rumen of some animals, is summarized in Fig. 4-1. The first group of microorganisms secrete enzymes which hydrolyze polymeric materials to monomers such as glucose and amino acids, which are subsequently converted to higher volatile fatty acids, H 2 and acetic acid (Fig. 4-1 stage 1). In the second stage, hydrogen-producing acetogenic bacteria convert the higher volatile fatty acids e.g., propionic and butyric acids, produced, to H 2 , CO 2 , and acetic acid. Finally, the third group, methanogenic bacteria convert H 2 , CO 2 , and acetate, to CH 4 and CO 2 .
Polymeric materials such as lipids, proteins, and carbohydrates are primarily hydrolyzed by extracellular, hydrolases, excreted by microbes present in Stage 1 (Fig. 4-1). Hydrolytic enzymes, (lipases, proteases, cellulases, amylases, etc.) hydrolyze their respective polymers into smaller molecules, primarily monomeric units, which are then consumed by microbes. In methane fermentation of waste waters containing high concentrations of organic polymers, the hydrolytic activity relevant to each polymer is of paramount significance, in that polymer hydrolysis may become a rate-limiting step for the production of simpler bacterial substrates to be used in subsequent degradation steps.
Lipases convert lipids to long-chain fatty acids. A population density of 10 4 - 10 5 lipolytic bacteria per ml of digester fluid has been reported. Clostridia and the micrococci appear to be responsible for most of the extracellular lipase producers. The long-chain fatty acids produced are further degraded by p-oxidation to produce acetyl CoA.
Proteins are generally hydrolyzed to amino acids by proteases, secreted by Bacteroides, Butyrivibrio, Clostridium, Fusobacterium, Selenomonas, and Streptococcus. The amino acids produced are then degraded to fatty acids such as acetate, propionate, and butyrate, and to ammonia as found in Clostridium, Peptococcus, Selenomonas, Campylobacter, and Bacteroides.
Polysaccharides such as cellulose, starch, and pectin are hydrolyzed by cellulases, amylases, and pectinases. The majority of microbial cellulases are composed of three species: (a) endo-(3-l,4-glucanases (b) exo-p-l,4-glucanases (c) cellobiase or p-glucosidase. These three enzymes act synergistically on cellulose effectively hydrolyzing its crystal structure, to produce glucose. Microbial hydrolysis of raw starch to glucose requires amylolytic activity, which consist of 5 amylase species: (a) a-amylases that endocleave a ±1-4 bonds (b) p-amylases that exocleave a ±1-4 bonds (c) amyloglucosidases that exocleave a ±l-4 and a ±l-6 bonds (d) debranching enzymes that act on a ±l-6 bonds (e) maltase that acts on maltose liberating glucose. Pectins are degraded by pectinases, including pectinesterases and depolymerases. Xylans are degraded with a ²-endo-xylanase and a ²-xylosidase to produce xylose.
Hexoses and pentoses are generally converted to C 2 and C 3 intermediates and to reduced electron carriers (e.g., NADH) via common pathways. Most anaerobic bacteria undergo hexose metabolism via the Emden-Meyerhof-Parnas pathway (EMP) which produces pyruvate as an intermediate along with NADH. The pyruvate and NADH thus generated, are transformed into fermentation endo-products such as lactate, propionate, acetate, and ethanol by other enzymatic activities which vary tremendously with microbial species.
Thus, in hydrolysis and acidogenesis (Fig. 4-1 Stage 1), sugars, amino acids, and fatty acids produced by microbial degradation of biopolymers are successively metabolised by fermentation endo-products such as lactate, propionate, acetate, and ethanol by other enzymatic activities which vary tremendously with microbial species.
Thus, in hydrolysis and acidogenesis (Fig. 4-1 Stage 1), sugars, ammo acids, and fatty acids produced by microbial degradation of biopolymers are successively metabolised by groups of bacteria and are primarily fermented to acetate, propionate, butyrate, lactate, ethanol, carbon dioxide, and hydrogen (2).
Although some acetate (20%) and H 2 (4%) are directly produced by acidogenic fermentation of sugars, and amino acids, both products are primarily derived from the acetogenesis and dehydrogenation of higher volatile fatty acids (Fig. 4-1 Stage 2).
Obligate H 2 -producing acetogenic bacteria are capable of producing acetate and H 2 from higher fatty acids. Only Syntrophobacter wolinii, a propionate decomposer (3) and Sytrophomonos wolfei, a butyrate decomposer (4) have thus far been isolated due to technical difficulties involved in the isolation of pure strains, since H 2 produced, severely inhibits the growth of these strains. The use of co-culture techniques incorporating H 2 consumers such as methanogens and sulfate-reducing bacteria may therefore facilitate elucidation of the biochemical breakdown of fatty acids.
Overall breakdown reactions for long-chain fatty acids are presented in Tables 4-1 and 4-2. H 2 production by acetogens is generally energetically unfavorable due to high free energy requirements ( a G o, > 0 Table 4-1 and 4-2). However, with a combination of H 2 -consuming bacteria (Table 4-2, 4-3), co-culture systems provide favorable conditions for the decomposition of fatty acids to acetate and CH 4 or H 2 S ( a G o, < 0). In addition to the decomposition of long-chain fatty acids, ethanol and lactate are also converted to acetate and H 2 by an acetogen and Clostridium formicoaceticum, respectively.
The effect of the partial pressure of H 2 on the free energy associated with the conversion of ethanol, propionate, acetate, and H 2 /CO 2 during methane fermentation is shown in Fig. 4-2. An extremely low partial pressure of H 2 (10 -5 atm) appears to be a significant factor in propionate degradation to CH 4 . Such a low partial pressure can be achieved in a co-culture with H 2 -consuming bacteria as previously described (Table 4-2,4-3).
Methanogens are physiologically united as methane producers in anaerobic digestion (Fig. 4-1 Stage 3). Although acetate and H 2 /CO 2 are the main substrates available in the natural environment, formate, methanol, methylamines, and CO are also converted to CH 4 (Table 4-3).
Table 4-1 Proposed Reactions Involved in Fatty Acid Catabolism by Syntrophomonas wolfei
+ 2 H 2 O 2 CH 3 COO - + 2H 2 + H +
CH 3 CH 2 CH 2 CH 2 CH 2 COO -
+ 4 H 2 O 3 CH 3 COO - + 4H 2 + 2H +
CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 COO -
+ 6 H 2 O 4 CH 3 COO - + 6H 2 + 3H +
+1 H 2 O CH 3 CH 2 COO - + CH 3 COO - +2 H 2 + H +
CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 COO -
+ 4 H 2 O CH 3 CH 2 COO - + 2 CH 3 COO - +4 H 2 + 2H +
CH 3 CHCH 2 CH 2 CH 2 COO -
+ 2 H 2 O CH 3 CHCH 2 COO - + CH 3 COO - + 2H 2 + H +
Table 4-2 Free-Energy Changes for Reactions Involving Anaerobic Oxidation in Pure Cultures or in Co-Cultures with H 2 -Utilizing Methanogens or Desulfovibrio spp.
1. Proton-reducing (H 2 -producing) acetogenic bacteria
A. CH 3 CH 2 CH 2 COO - + 2H 2 O 2 CH 3 COO - + 2H 2 + H +
B. CH 3 CH 2 COO - + 3H 2 O CH 3 COO - + HCO 3 - + H + + 3H 2
2. H 2 -using methanogens and desulfovibrios
C. 4H 2 + HCO 3 - + H + CH 4 + 3 H 2 O
D. 4H 2 + S0 4 2- + H + HS - + 4 H 2 O
A + C 2 CH 3 CH 2 CH 2 COO - + HCO 3 - + H 2 O 4 CH 3 COO - + H + + CH 4
A + D 2 CH 3 CH 2 CH 2 COO - + S0 4 2- 4 CH 3 COO - + H + + HS -
B + C 4 CH 3 CH 2 COO - + 12H 2 4 CH 3 COO - + HCO 3 - + H + + 3 CH 4
B + D 4 CH 3 CH 2 COO - + 3 S0 4 2 " 4 CH 3 COO - + 4 HCO 3 - + H + + 3 HS -
Table 4-3 Energy-Yielding Reactions of Methanogens
CO 2 + 4 H 2 ® CH 4 + 2H 2 O
HCO 3 - + 4 H 2 + H + ® CH 4 + 3 H 2 O
CH 3 COO - + H 2 O ® CH 4 + HCO 3 -
HCOO - + H + ® 0.25 CH 4 + 0.75 CO 2 + 0.5 H 2 O
CO + 0.5 H 2 O ® 0.25 CH 4 + 0.75 CO 2
CH 3 OH ® 0.75 CH 4 + 0.25 CO 2 + 0.5 H 2 O
CH 3 NH 3 + + 0.5 H 2 O ® 0.75 CH 4 + 0.25 CO 2 + NH 4 +
(CH 3 ) 2 NH 2 + + H 2 O ® 1.5 CH 4 + 0.5 CO 2 + NH 4 +
(CH 3 ) 2 NCH 2 CH 3 H + + H 2 O ® 1.5 CH 4 + 0.5 CO 2 + + H 3 NCH 2 CH 3
(CH 3 ) 3 NH+ 1.5H 2 O ® 2.25 CH 4 + 0.75 CO 2 + NH 4 +
Since methanogens, as obligate anaerobes, require a redox potential of less than -300 mV for growth, their isolation and cultivation was somewhat elusive due to technical difficulties encountered in handling them under completely O 2 -free conditions. However, as a result of a greatly improved methanogen isolation techniques developed by Hungate (6), more than 40 strains of pure methanogens have now been isolated. Methanogens can be divided into two groups: H 2 /CO 2 - and acetate-consumers. Although some of the H 2 /CO 2 -consumers are capable of utilizing formate, acetate is consumed by a limited number of strains, such as Methanosarcina spp. and Methanothrix spp. (now, Methanosaeta), which are incapable of using formate. Since a large quantity of acetate is produced in the natural environment (Fig. 4-1), Methanosarcina and Methanothrix play an important role in completion of anaerobic digestion and in accumulating H 2 , which inhibits acetogens and methanogens. H 2 -consuming methanogens are also important in maintaining low levels of atmospheric H 2 .
H 2 /CO 2 -consuming methanogens reduce CO 2 as an electron acceptor via the formyl, methenyl, and methyl levels through association with unusual coenzymes, to finally produce CH 4 (7) (Fig. 4-3). The overall acetoclastic reaction can be expressed as:
Since a small part of the CO 2 is also formed from carbon derived from the methyl group, it is suspected that the reduced potential produced from the methyl group may reduce CO 2 to CH 4 (8).
On the basis of homologous sequence analysis of 16S rRNAs, methanogens have been classified into one of the three primary kingdoms of living organisms: the Archaea (Archaebacteria). The Archaea also include major groups of organisms such as thermophiles and halophiles. Although Archaea possess a prokaryotic cell structure and organization, they share common feature with eukaryotes: homologous sequences in rRNA and tRNA, the presence of inn-ones in their genomes, similar RNA polymerase subunit organization, immunological homologies, and translation systems.
Recombinant DNA technology is one of the most powerful techniques for characterizing the biochemical and genetic regulation of methanogenesis. This necessitates the selection of genetic markers, an efficient genetic transformation system, and a vector system for genetic recombination as prerequisites.
Genetically marked strains are prerequisites for genetic studies: these strains can be employed to develop a genetic-exchange system in methanogens based on an efficient selection system. Since growth of M. thermoautotrophicum is inhibited by fluorouracil, analogue-resistant strains were isolated by spontaneous mutation. Other mutants resistant to DL-ethionine or 2-bromoethane sulfonate (coenzyme M analogue), in addition to autotrophic mutants, were obtained by mutagenic treatment. Several autotrophic strains were also obtained for the acetoclastic methanogen, M. voltae. These mutant strains are listed in Table 4-4.
Although some methanogen genes such as amino acid and purine biosysnthetic genes, transcription and translation machinery genes, and structural protein genes, have been cloned, genes encoding enzymes involved in methanogenesis were chosen as "methane genes" here.
Methyl CoM reductase (MR Fig. 4-3) constitutes approximately 10% of the total protein in methanogenic cultures. The importance and abundance of MR inevitably focused initial attention on elucidating its structure and the mechanisms directing its synthesis and regulation. MR- encoding genes have been cloned and sequenced from Methanococcus vanielli, M. voltae, Methanosarcina barkeri, Methanobacterium thermoautotrophicum and M. fervidus.
Formylmethanofuran transferase (FTR) catalyzes the transfer of a formyl group from formylmethanofuran (MFR) to tetrahydromethanopterin (H 4 MPT) (Fig. 4-3, 4-2). The FTR-encoding gene from M. thermoautotrophicum has been cloned, sequenced, and functionally expressed in E. coli. Formate dehydrogenase (FDH) may sometimes account for 2 to 3% of the total soluble proteins in methanogenic cultures. The two genes encoding the a ± and a ² subunits of FDH have been cloned and sequenced from M formicicum. In addition, the genes encoding F 420 -reducing hydrogenase (Fig. 4-3), ferredoxin, and ATPase have also been cloned.
Table 4-4 Auxotrophic and Drug-Resistant Mutants Applicable To Gene Transfer Experiments