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BIODIVERSITY

Microbial alliances

Microbial diversity helps to solve environmental problems and aids in the search for new drugs

Bacteria of the genus Pseudomonas: some species cause disease, others help to clean up the environment

SCIENCE PHOTOBacteria of the genus Pseudomonas: some species cause disease, others help to clean up the environmentSCIENCE PHOTO

A team from the University of São Paulo (USP) Center for Nuclear Energy in Agriculture (CENA) isolated three strains of bacteria in black earth [terra preta] samples from the Amazon: the genera Burkholderia, Pseudomonas and Arthrobacter. Laboratory testing of these samples showed them to be capable of decomposing aromatic hydrocarbons such as diesel, which opens up the possibility of using them against environmental or industrial pollution. In late May, Fernanda Mancini Nakamura identified the bacteria living in the coal cavities (carbon blocks) of the black earth samples taken from a depth of 30 cm in the city of Iranduba, near Manaus (Amazonas State) in northern Brazil.

Nakamura had already identified seven species of bacteria capable of living within the coal blocks, two of which, she believed, would be able to decompose cellulose, because they contained genes for this purpose. This discovery offers the possibility of industrial use if confirmed by further testing, since the breakdown of cellulose is still a challenge when producing alcohol fuel from sugarcane. She says it is interesting to note that bacteria living inside coal particles differ from species living in other parts of black earth. In turn, the microorganisms of patches of black earth—resulting from the accumulation of food and other organic material by pre-Colombian indigenous people—differ from those found in yellow soil, Argisoil or Latosoil types, which are more common in the Amazon.

Microbial diversity has proven to be fertile ground for discoveries of new species and adaptation mechanisms in extreme environments. Microorganisms, especially bacteria, have been shown to live in extreme environments such as the hypersaline waters of the Dead Sea, the extremely arid lands of the Atacama Desert, the Antarctic, and coral at the bottom of the sea. The discovery of new environments and new species is facilitating the use of microorganisms to solve environmental problems and identify potential new medicines.

The giant Samba virus (at different stages of development and isolated on the right): from the waters of the Rio Negro

UFMG Virus LaboratoryThe giant Samba virus (at different stages of development and isolated on the right): from the waters of the Rio NegroUFMG Virus Laboratory

To assess the extent of the diversity of microorganisms in Brazil, researchers from a number of its states founded the Brazilian Microbiome Project (BMP), which was described in the February 2014 issue of the journal Microbial Ecology. Its goal is ambitious, because the diversity of bacteria, viruses and fungi could be even larger than the so-called microfauna and mesofauna (small, invertebrate animals found in soil). Brazil is home to 20% of the world’s known biodiversity, when primarily invertebrates and vertebrates are considered. The first estimates of microbial diversity suggest that just one gram of soil could contain one million species of bacteria, and the top layers of soil harbor species of microorganisms that differ from those living in deeper layers. A typical example: in a study released last month, researchers at New York University reported finding DNA from 3,000 types of bacteria present on one-dollar bills.

Siu Mui Tsai, one of the lead BMP researchers and USP-CENA’s director (and coordinator of the laboratory in which Nakamura works), advises caution when extrapolating data on microbial diversity and notes the importance of field work in all regions of Brazil. “Microbial diversity depends on the interaction of microorganisms with plants and the environment,” she says.  One of the studies of her group at CENA, performed on a farm in the state of Rondônia, indicated that diversity, as measured by the richness and abundance of species, may vary within the same environment. In this case it was an area of Amazon forest, insofar as it showed a homogenous grazing area also evaluated at various collection points.

Little is known about microbial diversity, and it has already been heavily reduced as a result of deforestation, fires, the conversion of native forests into pastures and the planting of monocultures such as soybeans. “There is a rebalancing of microbial diversity in agricultural areas, but this can take 25 years in the case of grazing land,” says Tsai. The increase in global temperatures could exacerbate this situation in some environments. Experiments conducted at the Federal University of Rio de Janeiro (UFRJ) indicated that an increase of two to four degrees Celsius proportionally reduces microbial diversity on coral reefs, which cover less than 1% of the sea bed, but account for 25% of the diversity of marine microorganisms.

Found in black earth: Citricoccus, a group of bacteria capable of degrading hydrocarbons such as diesel

EDUARDO CESARFound in black earth: Citricoccus, a group of bacteria capable of degrading hydrocarbons such as dieselEDUARDO CESAR

Northern Brazil, one of the regions most affected by the loss of natural environments, has yielded enormous surprises for microbiologists. A water sample collected in 2011 from the Rio Negro near Manaus by a team led by Jônatas Abrahão, of the Federal University of Minas Gerais (UFMG), isolated a giant virus that was dubbed Samba. On April 29, 2014, at one of the presentations of a meeting on microorganisms organized by the Biota-FAPESP Program, Abrahão presented Samba as the first giant virus identified in Brazil. Samba was described in an article in the May 2014 issue of the Virology Journal. It measures 600 nanometers—the largest giant viruses, up to one micrometer, can be five times larger than flu viruses and larger than bacteria—and has 938 genes, of which nine are unknown. There are larger varieties such as Pandora dulcis, presented in 2013 with a genome two times larger than that of common viruses, 1,500 genes and nearly one micrometer in size.

“We have already isolated dozens of giant viruses,” says Abrahão. In the Pampulha Lagoon his team found a variety that earned the name Niemeyer [Oscar Niemeyer, Brazilian architect], and in the Serra do Cipó National Park, in Minas Gerais State, the Cipó [the Vine]. A giant virus that Abrahão found in a lagoon in the city of Lagoa Santa, also in Minas Gerais State, he named Kroon, as a tribute to his former advisor Erna Kroon, “the best virologist I know in Brazil,” he says. It has a quadruple outer layer, which gives it more resistance to ultraviolet radiation and temperature extremes. The work is going faster now, after months of frustration when they could not grow the viruses, making them reproduce to facilitate identification, until they found a favorable culture medium, with 40 grams of rice per liter of water, kept in a dark room.

From laboratory to field: bacteria selected in the UFRJ laboratory ...

UFRJFrom laboratory to field: bacteria selected in the UFRJ laboratory …UFRJ

They also now know where to look. “Where there are amoebas, there are probably giant viruses too,” says Abrahão. Amoebas, a type of protozoan, could act as bunkers, protecting the virus from ultraviolet light, heat and deadly chemicals, according to an article that appeared in 2013. This year, the Minas Gerais group identified giant viruses in Amazon monkeys and cattle and, in parallel studies, concluded that well developed viruses could be incorporated into the human body’s microflora to activate the production of defense molecules, such as interferons, which help to combat disease-causing organisms. At least one type of giant virus, Acanthamoeba polyphaga mimivirus can cause pneumonia in humans.

Mangroves
Since the late 19th century, with Robert Koch, Louis Pasteur and others who identified the cause of infectious diseases such as tuberculosis, microorganisms have been associated with disease. However, “only a small number of them cause disease, and this generally occurs when the body is in a state of imbalance,” says Alexandre Soares Rosado, director and researcher at UFRJ’s Institute of Microbiology. “Most of the time they are beneficial to human health and the environment.” Colonies of bacteria in the human gut produce vitamins that are important to the functioning of the body and stimulate the production of molecules of the immune communication system known as cytokines. In the lungs, according to a study of mice published in the May 2014 issue of Nature Medicine, organisms favor the production of immune cells and protection against asthma in adults.

... … are encapsulated (under magnification to the right)...

UFRJ… … are encapsulated (under magnification to the right)…UFRJ

Certain microorganisms obviously continue to be a source of concern. A survey by the World Health Organization conducted in 114 countries indicated that bacterial resistance to antibiotics is now a global phenomenon. According to the report, a number of species, including Escherichia coli, which causes diarrhea, Streptococcus pneumonia and Neisseria gonorrhea, have acquired resistance to antibiotics. “We need to develop new weapons to fight bacteria that are becoming resistant to all antibiotics,” said Karl Klose, a microbiologist at the University of Texas at San Antonio, at a TED conference in April 2014. “We need to avoid a return to the pre-antibiotic era.”

... nourish plants in nurseries...

UFRJ… nourish plants in nurseries…UFRJ

Rosado has explored the friendly side of bacteria. In collaboration with a team at the Petrobras Research Center (Cenpes), the UFRJ group assembled a combination of more than 10 species of bacteria—from the genera Pseudomonas, Actinobacteria and others—which have been able to restore mangrove vegetation. The combination, initially in liquid form and then encapsulated in alginate, was tested for a year and a half in the laboratory. When it produced the anticipated results, it was used to clean up an area of four square kilometers of mangrove of the Baía de Todos os Santos (also known as All Saints Bay), along the Bahian coast, State of Bahía, where there had been successive oil spills.

The first surprise was the effectiveness of the strategy. “The capsules swelled and slowly released the bacteria over a period of six months, protecting them from the tides,” said Rosado. The researchers observed that the bacterial capsules, probably by increasing the fixation of nitrogen, an essential nutrient for plants, increased by 35% the survival rate of the seedlings used to restore the environment, compared to the untreated areas. “The bacteria protect the plants and, instead of 30 years, we were able to restore the vegetation in less than three years, with this technique,” he says. “Microorganisms drive biological and geological cycles, as producers and decomposers of organic material.”

... and help to restore mangrove forests

UFRJ… and help to restore mangrove forestsUFRJ

The UFRL team distributed 300 tubes with oil in the Marambaia mangrove forest, along the coast of Rio de Janeiro—each tube contains three containment barriers to prevent leakage—to assess the action of microorganisms on degrading pollutants and to test the feasibility of techniques with a lower cost than those used in Bahia. At the same time, Rosado’s team is working on describing five potential new species of bacteria, identified from among the 350 isolated in recent years in mangroves and in coastal and interior Antarctica, where surveys are also being conducted.

Medicines
Since the 1940s and the introduction of penicillin, made from fungi of the genus Penicillium, microorganisms have been used to produce medicines. Streptomycin, isolated by culturing a soil bacterium, Streptomyces griseus, was one of the highlights of the scientific meeting on new antibiotics held at the New York Academy of Sciences in January 1946. Streptomycin was an attractive alternative for the treatment of tuberculosis, since penicillin, even though it had a high toxicity, was ineffective. Another species, Streptomyces aerofaciens, provided the aureomycin, of lower toxicity, which a group of New York doctors tested on 35 people with lymphogranuloma venereum, a sexually transmitted bacterial disease, with results they thought were excellent.

Alan Bull and his team at the University of Kent, England, also regarded the in-vitro test results of the antibiotic and antitumor action of substances produced by bacteria of the genus Streptomyces as excellent; they were isolated from the extremely arid regions of the Atacama Desert in Chile. On April 28, 2014, at FAPESP, Bull alluded to the possibility of now being able to work in a more integrated way, that is, on the taxonomy (classification), ecology and genomics of microorganisms, and at the same time, on the identification of substances with antibiotic effects, whose chemical structures could lay the foundation for new medicines. He presented several examples of natural drugs, such as a substance produced by bacteria found at the bottom of a Norwegian fjord; it showed in-vitro action against various types of tumors. He noted that it is important to also evaluate the potential for toxic effects on the body’s healthy cells.

“Discoveries of new substances with antibiotic or antitumor properties do not necessarily lead to new medicines,” he stressed, recalling repeated disappointments. “Pharmaceutical companies are not interested in antibiotics. Their priority is medicines that can be used for a lifetime.” Bull said he does not know why microorganisms living in the desert, on ice, or in the deep sea produce substances that eliminate bacteria, or abnormal cells that combine to form tumors.

Project
The microbiome of Amazonian dark earth: structure and function of the microbial communities from rhizosphere and biochar associated to the biogeochemical cycles (nº 11/50914-3); Grant mechanism Regular Research Grant/Biota/FAPESP; Principal investigator Siu Mui Tsai (CENA-USP); Investment R$ 477,191.18 (FAPESP).

Scientific articles
SANTOS, H.F. et alMangrove bacterial diversity and the impact of oil contamination revealed by pyrosequencing: Bacterial proxies for oil pollutionPLoS ONE. v. 6, n. 3, p. e16943. 2011.
PYLRO, V.S. et alBrazilian Microbiome Project: Revealing the unexplored microbial diversity – Challenges and prospectsMicrobial Ecologyv. 67, n. 2, p. 237-41. 2014.
RODRIGUES, J.L.M. et alConversion of the Amazon rainforest to agriculture results in biotic homogenization of soil bacterial communitiesPNAS. v. 110, n. 3, p. 988-93. 2013.
CAMPO, R.K. et alSamba virus: a novel mimivirus from a giant rain forest, the Brazilian AmazonVirology Journal. v. 11, n. 95. 2014.

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