Bacteriophage for ARD

UBC-CERM3 - Hunting for Natural Viruses to Attack the Bacteria that Accelerates the Production of ARD

They descend on the surface of their host like a Lunar Lander. In fact they resemble a Lunar Lander. They drill through the cell wall of their host and inject their DNA deep into the cell to reassemble the cell contents into their offspring. Bacteriophage are "Natural-Born Killers" in the true sense in that they require a specific host cell to perform procreation and in so doing, they kill their host. Not exactly how a guest should act when the host serves dinner!

Electron micrograph of T4 and a variety of other phage types from the ICTV database.

As research continues to study these interesting "critters" and our knowledge grows about the microbiology of the bacteria that enhances the generation of Acid-Rock-Drainage, the twinning of these two fields presents some tantalizing opportunities to develop new approaches to the mitigation of ARD pollution.

Acid-Rock-Drainage (or ARD for short) is generated from almost all hard-rock mining operations that deal with sulfide mineralization - pyrite being one of the most ubiquitous of all sulfides. When pyrite is ground into fine particles, the newly exposed surfaces will react with water and dissolved oxygen to generate ferrous sulfate, ferric sulfate and eventually, sulfuric acid. This causes the pH of waters in contact with waste rock and tailings to slowly fall to about 4.0 to 5.0 from its natural level around 7.0. There are natural bacteria in the environment that use sulfur for food and energy. As these "bugs" proliferate on the surfaces of the sulfide rocks and particles, they contribute to a rapid increase in the speed at which the acid is formed. The pH may decline further into the range of 2.5 to 3.0. Under these conditions, heavy metals such as copper, lead, zinc, cobalt, nickel, and iron are readily leached into solution leading to serious metallic ion pollution which can kill fish and other biota.

ARD prediction and treatment is difficult since the processes that generate ARD are a complex set of interactions between mineralogy, microbial ecology, and hydrogeological features of the waste dumps. The geology, topography and climate of the surrounding region are also important issues. Microbial ecology is perhaps the least understood of these elements. It is known that lowering the pH from neutral to acidic is accompanied by a progression of dominant bacterial populations, ending with the sulfur-oxidizing species Thiobacillus ferrooxidans. The presence of this particular bacterium is associated with an exponential increase in ARD.

What triggers this succession of bacterial populations? One aspect of the microbial environment that has been overlooked todate is the presence of viruses. Viruses (bacteriophage) that attack bacteria are present in all natural systems. Bacteriophage have been isolated from marine ecosystems, soils and other natural environments. Ward et al. (1993) have isolated a bacteriophage for Acidiphilium from coal mine waste dumps and more recently a phage has been found for T. ferrooxidans. Evidence from studies of soil bacteriophage indicates that at acidic pH, these viruses become unstable, (Sykes et al., 1981; Ward et al., 1993). In the context of ARD, the presence or absence of bacteriophage may play an important role in determining the type and quantity of bacteria present which may control the acid-generating micro-environment. Clearly the role of bacteriophage in the microbial ecology of ARD wastes needs to be investigated.

Nathanson (1902) was the first microbiologist to isolate a member of the bacterial genus Thiobacillus, a bacteria noted for its ability to oxidize sulfur. It was not until the late 1940s however that Thiobacillus ferrooxidans was isolated by Colmer and Hinkle (1947). It was subsequently found to oxidize both sulfur and iron. The bacteria has been characterized by Colmer and Hinkle (1947), Colmer et al. (1950) and Temple and Colmer (1951) found the bacteria to be gram-negative, acidophilic and rod-shaped. Conditions for its growth are listed in Table 1.

Other sulfur-oxidizing bacterial species include T. thiooxidans and Leptospirillum ferrooxidans. The latter was identified by Schrenk et al. (1998) as being the dominant species in acid-forming environments at temperatures above 30�C.

Environmental ConditionS	Range/Requirement
pH	1-5
Temperature	10 to 37 �C
Energy Source	Reduced Sulfur and Ferric Ion
Oxygen	Obligate aerobe
Nitrogen source	Ammonium salts, nitrate

T. ferrooxidans catalyzes sulfur oxidation by two mechanisms, one by the direct oxidation of sulfur, and the second indirectly through the oxidation of ferrous ion to ferric ion. The following reactions are catalyzed by the bacteria:

Reaction products from the bio-catalyzed reactions (1) & (2) contribute to abiotic oxidation of sulfides, reaction (3):

Evangelou & Zhang (1995) document that the rate of oxidation of sulfur minerals is increased by six orders of magnitude in the presence of bacteria. Without a bacterial catalyst, oxidation is kinetically slow and generally has only a small effect on the environment.

There are numerous methods to deal with ARD ranging from active treatment using lime and other pH modifiers to passive systems that rely on wetland biology to adsorb metals and prevent them from contaminating the environment.

Attempts have been made to control bacteria populations, especially that of Thiobacillus ferrooxidans, using chemicals and bactericides but most amendments are either too expensive or require frequent replenishment of the poison. There is also the potential for such reagents to be dangerous to the health of other organisms.

A novel alternative to chemicals is to isolate a virus (or bacteriophage) that is specific for the bacteria causing the problem.

Research on certain mine tailings shows a progression of dominant microbial populations as the bulk pH of the tailings decreases (Blowes et al., 1995). In a recent review of the geomicrobiology of sulfide mineral oxidation, Nordstrum & Southam (1998) state that favorable conditions for Thiobacillus species growth occurs in nano-scale environments of low pH that occur on sulfide mineral surfaces. Hence, bulk pH is not an accurate indicator of viable populations of thiobacilli. These nano-scale environments evolve into microenvironments through progressive oxidation and eventually, the bulk pH is affected. The presence and quantity of bacteriophage may be an absolute indication of the bacterial species present and the likelihood or not of future decline in bulk pH conditions.

Bacteriophage were discovered independently by Frederick W. Twort a British microbiologist in 1915 and two years later by a Canadian, Felix d�Herelle (1873-1949) while working as a laboratory technician at the Pasteur Institute in France (Goyal et al., 1987). It was Felix d�Herelle who coined the term "bacteriophage", meaning bacteria-eater, and it was he who recognized the potential of these phage to be antibacterial agents. it was considered that bacteriophage had the potential to kill the bacteria that cause many infectious diseases in humans, as well as in plants and animals. This idea formed the basis for much research as well as for the Pulitzer Prize-winning 1924 novel Arrowsmith by Sinclair Lewis, Scientists of the day embraced the new discovery as a cure-all for the many deadly bacterial diseases rampant at the time (d'Herelle, 1917, 1918, 1921, 1922, 1949), yet research into the efficiency of phage therapy proved inconclusive and eventually the technique was unaccepted by the West. As a result, d'Herelle eventually emigrated to Georgia in the USSR in 1933 where he founded an Institute in Tbilisi, Georgia to study bacteriophage together with Georgian microbiologist George Eliava. Although Eliava was killed in one of Stalin�s purges in 1937, and D�H�relle never returned, the Georgia Bacteriophage Institute survived and continued supplying phage for therapeutic uses to the entire Soviet Union. Almost simultaneously, Max Delbruck and his famous "Phage Group" at Vanderbilt University used bacteriophage to make discoveries that led to the origins of molecular biology (Summers, 1999). Delbr�ck shared the Nobel Prize in 1969 with Salvador Luria and Alfred Hershey, "for their discoveries concerning the replication mechanism and the genetic structure of viruses. Luria then worked at the Massachusetts Institute of Technology (MIT), Cambridge MA, while Max Delbr�ck had become affiliated with the California Institute of Technology, Pasadena, California. Hershey was Director of the Genetics Research Unit of the Carnegie Institution, Cold Springs Harbor on Long Island.

- characterization of insertion sequences, transposons, and invertible DNA segments

When penicillin was discovered in the 1930s, phage research was essentially discontinued for a very long time. In the 1950s a phage research group was formed to investigate the life cycle of phage. With modern scientific techniques, the basic nature and physiology of phage were determined. When �superbugs� began to emerge in the early 1980s resistant to all known antibiotics, there was a resurgence of interest in phage therapy that continues to this day. With more solid scientific information of phage available to researchers today, these therapies have a greater chance of success and indeed successful testing of some phage therapies has been done on animals in several countries.

In the USA, a company called Phage Therapeutics Inc. provided an experimental phage treatment that cured a woman of a bacterial heart infection, although the therapy will not be available in mainstream medicine for several years. The history of phage research and applications in the field of medicine is well documented in several sources (Kutter, 1997, Goyal 1987). However what about phage applications in other industries -- can this technology be transferred?

In April 1996, Nobel laureate Joshua Lederberg, an leading authority on infectious diseases, helped revive interest in phages with an upbeat commentary in the Proceedings of the National Academy of Sciences, which declared that in light of the shortcomings of antibiotics, there "should be a renascence of study of bacteriophages. When people in the scientific community, hear 'phage therapy,' it is not often taken seriously. But today, there's a large body of evidence that suggests that this therapy is a viable approach.

Phage, which are about 1/40th the size of most bacteria, are perhaps the simplest, most abundant organisms on Earth, thriving wherever bacteria grow�in raw sewage, in our bodies, in the oceans, and virtually everywhere. Using their legs to grip the surface of a bacterium, phages bore through the cell membrane with their tail and inject genetic material into the cell. These genes force the host to produce clones of the phage and eventually so many "daughters" are produced�more than 100 in 30 minutes�that they burst the cell wall, destroying the bacterium. The newborn phages then travel forth to adjacent bacteria, repeating their invasion until there are no hosts left to slaughter. Then they simply go "dormant" until other bacteria host cells come along.

Problems with Early Phage Therapy Work

Early research into the use of phage for anti-bacterial applications was inconclusive. The following reasons contributed to this failure;

- Failure to understand the heterogeneity and ecology of the phage and bacteria involved.

- Failure to characterize phage preparation, i.e., to determine the virulence to the target.

- Emergence of resistant bacterial strains likely because of the use of temperate phages.

- Phage are rendered "dormant" by adverse environmental conditions. (gastric fluids).

Advances in research tools such as the Electron Microscope and other techniques, have led to a basic understanding of phage structure and reproduction. Researchers today use this knowledge to avoid the above problems. Of particular importance is determining the host range of the phage. Some phage can infect a number of bacteria strains, while some are more specific and will only infect a particular sub-strain. The ability of the host to mutate may also be an important factor. It is not uncommon for a phage to be isolated and proven virulent on a strain of bacteria, only to find that subsequent generations of the host have mutated and the phage can no longer infect them. Isolation of several phages that have overlapping host ranges is therefore necessary if the phage is to be used at a bactericide.

It is estimated that there are over 10³⁰ different types of phage in the world. Each phage consists of a genetic piece of information, either RNA or DNA, which determines its properties. The genome is encapsulated and protected by a protein coating. Phage are classified according to the type of viral genome they possess. Simple RNA phage are the smallest group and are icosohedral in shape and approximately 26 nm in size. On the other end of the scale are large double-strand DNA phage. These have more complex structures consisting of a head and a tail. They can measure over 200 nm in length and 100 nm wide at the head. Other classes of phage include single-strand DNA phage and small double-strand DNA phage.

The most extensively studied phage is T4 which infects E.Coli. T4 has given the phage the popular image of a lunar landing module. The head is a protein capsule protecting the viral genome while the tail is involved in injecting nucleic acid into the host bacterium cell. The tail fibres are designed for cell recognition and adsorption.

Ward et al., 1993 have discussed the use of phage in the mitigation of ARD, after their discovery of a bacteriophage for the species Acidiphilium. Biological control of ARD was also the subject of a CANMET report (Christison et al., 1986), which looked at bacterial predators for T. ferrooxidans. This report focused on finding a protozoa predator for T. ferrooxidans. Although some species were able to reduce the T. ferrooxidans population in lab samples, the rate of reduction was too low to be a viable control method.

The failure of protozoa as a microbial control agent stems from the predator-prey relationship, without a viable population of T. ferrooxidans, the protozoa can�t survive therefore the two species must coexist in nature. It was concluded that a desirable predator of T. ferrooxidans would be one that can co-exist at low levels of T. ferrooxidans so that acid generation is minimal, but which can then increase rapidly if the population of T. ferrooxidans begins to increase. Bacteriophage meet such criteria.

Isolating and characterizing phage specific for T. ferrooxidans was one of the key recommendations of the CANMET research. Subsequent studies (Johnson & Rang, 1993) have shown that protozoa significantly reduce the number of acidophilic bacteria oxidizing pyrite-rich coal in laboratory cultures. In a review of acidophilic microbial communities, Johnson (1995) states that there is considerable potential either to prevent or mitigate ARD through the use of anti-bacterial agents and other microorganisms indigenous to acidic environments. The question is � can a phage be found that can tolerate such low pH regimes?

Viruses are at the boundary conditions of life. They fall between supra-molecular complexes and very-simple biological entities. Viruses possess some of the structures and exhibit some of the activities common to organic life, but they do not have them all. Viruses are composed of a single strand of genetic information inside a protein capsule. Viruses lack most of the internal structure and mechanics that characterize 'life', including the biosynthesis necessary to reproduce. So in order for a virus to replicate, it must infect a suitable host cell.

Viruses exist in two distinct states. When not in contact with a host cell, the virus is essentially dormant. In this state, there is no biological activity within the virus. The virus is simply an organic particle. In this non-living state viruses are referred to as 'virions'. Virions can remain dormant for long periods of time, simply waiting to come into contact with an appropriate host. When the virion does contact an appropriate host, it becomes active. It is then referred to as a virus. In this state, it displays properties of living organisms and reacts to its environment as it directs its efforts toward self-replication.

The term bacteriophage derives from the Greek word "phagein" which means "to eat". A bacteriophage is a virus which infects bacteria. In particular, the bacteriophage T4 is a virus which infects E. Coli, a bacteria used extensively for molecular biology research. T4 "phage" exemplifies the life cycle of viruses. It exists as an inactive virion until one of its extended 'legs' comes into contact with the surface of an E. Coli cell. Sensors on the ends of its 'legs' recognize binding sites on the surface of the host cell, and triggers the virus into action. First, the bacteriophage binds to the surface of the host. Then it punctures the cell wall with its injection tube or "drill". Once through the wall, it injects its own genetic blueprint into the cell membrane. This genetic information subverts the host cell's normal operation and sets the biosynthetic machinery of the cell to create exact replicas of the virus. These newly created viruses escape from the cell by bursting through the cell wall, effectively killing the host cell. These progeny then float about the solution in a dormant state until they come into contact with a new host cell. The T4 phage contains about 168,800 base pairs of double stranded DNA. This genetic blueprint contains the necessary information to create new T4 viruses.

Phage reproduction takes place via two different methods; a temperate phage reproduces using a lysogenic cycle while a virulent phage employs a lytic cycle. In the latter case, death of the host cell occurs in about half an hour. In the lysogenic case, the DNA of the bacteria is genetically modified and the progeny do not repopulate the environment. Both cycles begin by adsorption of the phage onto the surface of the bacteria followed by insertion of the phage genome into the cell. For virulent phage, a period of growth follows the genome insertion during which time, the genome and other components are replicated using material from within the bacterium cell. The progeny of the phage are then released by "lysing" (or breaking through) the bacterial cell wall.

A temperate phage may reproduce through a lytic cycle, but it can also incorporate its genome into the bacterial DNA in which case the new genome is termed a "prophage". The phage genome is replicated along with the bacterial genome and is inherited by each daughter cell or "lysogen". At some point, induction takes place and the prophage will enter the lytic cycle or else the phage genome is lost from the bacterium preventing further reproduction.

The distinction between the two methods is very important when considering application as an anti-bacterial agent. A virulent phage is required to lyse bacterial cells. Temperate phages through "prophage" formation promote mutations that may be resistant to the phage. They may also prevent adsorption by lytic forms of the phage. So it is important to understand the reproductive character of a phage in order to predict its ability to kill bacteria.

Phage can attack hosts in a wide range of environments. Phage have been isolated for thermophilic bacteria that exist at elevated temperatures (Farrell and Campbell, 1969, Saunders and Campbell, 1966, Sakaki and Oshima, 1976). Phage have also been detected in high salt environments (Zachary, 1974, Torsvik and Dundas, 1978). Phage are capable of infecting acid-tolerant organisms, however the infection always takes place at neutral pH values.

Sykes et al., 1981 studied the effect of pH on phage of streptomycetes, a bacterium commonly found in soils. They found that low pH conditions had variable effects on several stages of phage replication, including adsorption, cell penetration, and the length of the latent period. They were unable to isolate phage from soils with a pH below 6.0, even though acidophilic streptomycetes were present. This apparent instability of phages at low pH has strong implications for detecting phage in ARD-generating environments which typically have a pH of 4.0 or below.

Phage Therapy and Natural Environments

Phage therapy has been used in Russia and Poland for many years as part of normal clinical treatment. Eastern Europe is the only place where clinical trials have been conducted on humans. Today, there are companies in Moscow making phage preparations for therapeutic purposes. In the West, phage therapy is a growing medical field, but has been limited so far to study on animals. With increased understanding of phage properties and controlled studies, the research will likely be more successful than that done early in the 20th Century.

As simple techniques to detect phage have evolved, researchers are beginning to understand the role of these viruses in natural environments. Phage have been isolated from soils (Marsh and Wellingron, 1994; Sykes et al., 1981) and aquatic environments (Noble and Fuhrman, 1998; Armon and Kott, 1993). Practical uses for phage are emerging - as tracers to model sewage flow and other groundwater contaminants through aquifers (Pieper et al., 1997; Paul et al., 1995). Phage have also been applied to protect beef from spoiling (Greer, 1986).

Using biological organisms offers several advantages over traditional chemical bactericides. Biocides are very specific -- they affect only the target organism. They are biodegradable and do not persist or accumulate in the soil after use. They are nontoxic unlike most of their chemical counterparts. They are a natural part of the ecology and so, they are harmless to plants, animals and other non-target organisms, (Greer, 1986).

Biological control of pests is not a new phenomenon. The concept has been used extensively in agriculture as a tool to eradicate crop-eating insects. In the early 1800s, chinch bugs were controlled experimentally using a fungus and early in the twentieth century, grasshoppers in Mexico were controlled using a bacterium. The first commercial biological pesticide was developed in the 1940s, (Greer, 1986).

The first viral pesticide to receive FDA approval was Heliothis Zea nuclear polyhedrosis which targeted bollworms responsible for decimating cotton crops. This virus has seen large-scale application on cotton crops in the United States beginning in the 1970s. Since then, a number of viral pesticides have become commercially available, to target Velvetbean caterpillar (Richter & Fuxa, 1984), western tent caterpillar (Rothman & Myers, 1994), redheaded pine sawfly (Podgwaite et al., 1986), gypsy moth (Webb et al., 1993) and other insects.

Bacteriophage have also been used as biological control agents. Their use has been investigated to mitigate problems with bacteria in beef spoilage (Greer, 1986). Recently there has been much discussion to use phage as an alternative to antibiotics to which certain bacteria have become resistant, (Merril et al., 1995; Kutter 1997). Padival et al., (1995) conducted experiments to control Thiobacillus thiooxidans, which is responsible for corrosion problems in concrete sewers, by using microbial competition. In laboratory tests, acid-tolerant yeast was used as a heterotrophic competitor which succeeded in displacing the T. thiooxidans populations and reducing concrete degradation from acid.

Techniques to isolate phage can be found in several sources, including most microbiology text books. A good outline of the various methods is given by Austin (1988) and in Collins and Lyne's Microbiological Methods (1989), among others. By far the most common method is the plaque assay technique. A lawn of bacterial growth is formed on a solid surface, a sample suspected of containing the phage is added. Virulent phage will lyse the bacteria on the plate forming clear zones called plaques. An aseptic transfer of material from the plaque into a running liquid culture of the host bacteria (i.e. the bacteria are in the exponential phase of their growth curve) will propagate and amplify the phage. The phage is collected by centrifugation and filtration. The result is a concentrated phage solution which can be used for further studies.

Growing bacteria on a solid medium is critical to using the above method which is the easiest way to isolate phage. T. ferrooxidans has also been grown on solid media by several researchers. A good review of the various techniques has been prepared by Johnson (1995).

In the case of a dominant lytic cycle, the ability of this technique to eliminate the bacteria "T. ferrooxidans" is a possible goal. Unfortunately, both modes of infection occur together, resulting in genetically-modified host cells which eventually develop an immunity to the phage. One way to overcome this problem is to use a "cocktail" of several varieties of phage that work together to stave off the onset of this adaptation process.

A second possibility is to deliberately use the lysogenic cycle to genetically modify the bacteria into species that are less harmful. This requires a detailed examination of the bacteria's genetic code and is likely to be a much longer-term research study to find those elements of the DNA that provide the mechanisms to oxidize sulfur and iron so well.

The third possible application, as mentioned above, is to use phage as a monitoring tool in which a long-term study on changes in the presence and intensity of bacteriophage in a particular environment can be a precursor to the eventual onset of ARD. The pH conditions in nano-environments can be monitored using nano-sized particles such as phage. Eventually these changes intensify to affect the overall conditions in the environment and cause the bulk pH to decline.

The presence or absence of bacteriophage is an important microbiological characteristic of an environment. Isolation of the phage that infects T. ferrooxidans could prove useful in both minimizing the impact and/or monitoring the onset of ARD. Another possibility is to use the phage deliberately to modify the genetic code of the host cells and render them less effective as sulfur oxidizers.

B. Austin, 1988. Methods in Aquatic Bacteriology (Modern Microbiological Methods). John Wiley & Sons; New York, 495 pages.

R. Armon and Y. Kott, 1993. A simple, rapid and sensitive presence/absence detection test for bacteriophage in drinking water. J. Appl. Bacteriol. 74:490-496.

D.W. Blowes, C.N. Alpers, L. Lortie, W.D. Gould, J. Jambor, 1995, Microbiological, chemical, and mineralogical characterization of the Kidd Creek mine-tailings impoundment, Timmins Area, Ontario: Geomicrobiology Journal, v. 13, pp. 13-31

C.H. Collins, P.M. Lyne, J.M. Grange, 1989. Collins and Lyne's Microbiological Methods. Oxford University Press; ISBN: 0750606533, 493 pages.

A.R. Colmer, K.L. Temple, and M.E. Hinkle, 1950. An iron-oxidizing bacterium from the drainage of some bituminous coal mines. Journal of Bacteriology, 59, 317-328.

A.R. Colmer, and M.E. Hinkle, 1947, The role of microorganisms in acid mine drainage - a preliminary report: Science, 106(2747) 253-256.

F. d'Herelle, 1917. Sur un microbe invisible antagoniste des bacilles dysent�riques. ). C. R. Acad. Sci. Ser. D 165:373

F. d'Herelle, 1918. Technique de la recerche du microbe filtrant bact�riophage (Bacteriophagum intestinale). . C. R. Soc. Biol. 81:1160

F. d'Herelle, 1921. Le bact�riophage: Son r�le dans l'immunit�. . Presse M�d. 29:463

F. d'Herelle, 1922. The Bacteriophage: Its Role in Immunity. Williams and Wilkins Co., Waverly Press, Baltimore.

J Farrell, L.L. Campbell, 1969. Thermophilic bacteria and bacteriophages. Adv. Microbial Physiol. 3:83-109.

S.M. Goyal, G.P. Gerba, G. and Bitton (eds.), 1987. Phage Ecology. J. Wiley & Sons, NY.

D.B. Johnson, 1995. Acidophilic microbial communities: candidates for bioremediation of acidic mine effluents. International Biodeterioration & Biodegradation, 35, 41-58.

P. Marsh and E.M.H. Wellingron, 1994. Phage-host interactions in soil. FEMS Microbiol. Ecol. 15:99-108.

C.R. Merril, B. Biswas, R. Carlton, N.C. Jensen, G.J. Creed, S. Zullo, S. Adhya, 1996. Long-circulating bacteriophage as antibacterial agents. Proc. Natl. Acad. Sci. USA 93:3188-3192.

R.T. Noble and J.A. Fuhrman, 1998. Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria. Aquat. Microb. Ecol. 14:113-118.

J.H. Paul, J.B. Rose, J. Brown, E.A. Shinn, S. Miller, S.R. Farrah, 1995. Viral tracer studies indicate contamination of marine waters by sewage disposal practices in Key Largo, Florida. Appl. Environ. Microbiol. 61:2230-2234.

A.P. Pieper, J.H. Ryan, R.W. Harvey, G.L. Amy, T.H. Illangasekare, D.W. Metge, 1997. Transport and recovery of bacteriophage PRD1 in a sand and gravel aquifer: Effect of sewage-derived organic matter. Environmental Science & Technology 31:1163-1170.

Y. Sakaki and T. Oshima, 1976. A lipid-containing phage infecting acidophilic thermophilic bacteria. Virology 75:256.

G.F. Saunders and L.L. Campbell, 1966. Characterization of a thermophilic bacteriophage for Bacillus stearothermophilus. J. Bacteriol. 91:340-348.

I. Summers, 1999. Felix D'Herelle and the Origins of Molecular Biology. Yale University Press; Princeton, NJ, ISBN: 0300071272.

I.K. Sykes, S. Lanning, S.T. Williams, 1981. The effect of pH on soil actinophage. J. Gen. Microbiol. 122:271-280.

K.L. Temple and A.R. Colmer, 1951. The autotrophic oxidation of iron by a new bacterium, Thiobacillus ferrooxidans. Journal of Bacteriology, 62, 605-611.

T. Torsvik and I.D. Dundas, 1978. Halophilic phage specific for Halobacterium salinarium str.1. S.R. Caplan and M. Ginzburg (eds.), Energetics and Structure of Halophilic Microorganisms. Elsevier/North Holland, p. 609.

T.E. Ward, D.F. Bruhn, M.L. Shean, C.S. Watkins, D. Bulmer, V. Winston, 1993. Characterization of a new bacteriophage which infects bacteria of the genus Acidiphilium. J. Gen. Virol. 74 ( Pt 11):2419-2425.

A. Zachary, 1974. Isolation of bacteriophages of the marine bacterium Beneckea natriegens from coastal salt marshes. Appl. Microbiol. 27:980-982.

Natural Born Killers:
Bacteriophage and Thiobacillus ferrooxidans

Range/Requirement

Problems with Early Phage Therapy Work

Bacteriophage T4

Phage Therapy and Natural Environments

	Natural Born Killers: *Bacteriophage and Thiobacillus ferrooxidans*
September, 2002.

Natural Born Killers: Bacteriophage and Thiobacillus ferrooxidans

Range/Requirement

Problems with Early Phage Therapy Work

Bacteriophage T4

Phage Therapy and Natural Environments

Natural Born Killers:
Bacteriophage and Thiobacillus ferrooxidans