MICROCOCCUS LUTEUS

Jual Culture Micrococcus luteus

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Micrococcus luteus was originally isolated by Alexander Fleming in 1929 as Micrococcus lysodeikticus. It was the primary experimental microbe used in Fleming’s discovery of lysozyme. The microbe can be found in a variety of environments including soil, water, animals, and some dairy products.Micrococcus is generally thought to be a saprotrophic or commensal organism, though it can be an opportunistic pathogen. This is particularly true in hosts with compromised immune systems. Micrococci, like many other representatives of the Actinobacteria, can be catabolically versatile. It has the ability to utilize a wide range of potentially toxic substrates, such as carbon-based pyridine, pesticides, crude oil and petroleum byproducts. As a species, they are likely involved in detoxification or biodegradation of many other environmental pollutants. Other Micrococcus isolates can synthesize various useful products, such as long-chain (C21-C34) aliphatic hydrocarbons for lubricating oils and the biosynthesis of terpenes1. Thus the full sequencing of Micrococcus luteus has been supported due to its potential as a bioremediator of

contaminated water and soil as well as in current and future biotechnology applications.

Micrococcus luteus is able to survive in the environment for long periods. It is very capable of survival under stress conditions, such as low temperature and starvation. However, M. luteus does not form spores as survival structures, as is common in other bacterium. Instead M. luteus undergoes dormancy without spore formation. Its unique ability to undergo resuscitation from dormancy seems to be connected to the Rpf gene (resuscitation promoting factor). Genes similar to rpf are widely distributed throughout theactinobacteria. Many related bacteria, including M. tuberculosis, contain multiple gene homologs. In this regard, Rpf-like proteins are

essential for bacterial growth. M. luteus is unusual in that it contains a single rpf-like gene. Studies have demonstrated that M. luteus secretes an Rpf protein which has specific signaling functions required for its resuscitation from dormancy. The microbe can adopt a state of low metabolic activity when environmental conditions are not conducive for sustained growth. This would obviously permit survival for prolonged periods until optimal environmental conditions are restored [Mukamolova, et al., 2002]. More recently, a non-spore forming cocci, identified as Micrococcus Luteus, was isolated from a 120 million year old block of amber. Although comparison of rRNA sequences from other isolates is unable to confirm the precise age of the bacteria, it is estimated thatMicrococcus luteus has survived for at least 34,000 to 170,000 years on the basis of 16S rRNA analysis. It seems that M.luteus and other related modern members of the genus have numerous genetic adaptations for survival. This includes extreme, nutrient-poor conditions. These phenotypes have assisted the microbe in persistent and prevalent dispersal within the environment. This species has an ability to utilize succinate and terpinerelated compounds (which themselves are major components of natural amber) to enhance and ensure

its survival in oligotrophic environments.

Micrococcus luteus is an organism that is capable of growth on pyridine. Pyridine is a natural byproduct of coal and oil gasification. It is also mobile in soil and is considered an environmental teratogen. M. luteus contains a gene that codes for the enzyme succinate-semialdehyde dehydrogenase. Although the mechanism is not completely understood, the enzyme is actually induced by pyridine. It permits the oxidation of pyridine as a metabolic carbon source and thereby provides cellular energy. In the process it releases the nitrogen contained in the pyridine ring as ammonium (NH3). M. luteus, like species of Bacillus andCorynebacterium, require the _-amino acids arginine, valine, leucine and methionine for enhanced growth on pyridine [Sims, Sommers, Konopka, 1986].

Miccrococus luteus contains two structural genes (hex-a, hex-b) that encode two essential components of Hexaprenyl disphosphate synthase (HexPS). When these two components are combined, they mechanize prenyltransferase activity. This enzyme complex will produce the precursor of the prenyl side chain of menaquinon-6 (HexPP; C30). Terpenoid-Menaquinon biosynthesis in prokaryotes function as electron carriers within the cytoplasmic membrane, and each is required for respiration using different, although overlapping subsets of terminal electron acceptors. Menaquinone is also known as the essential Vitamin K-2, because it is a nutrient that cannot be synthesized by mammals [Shimizu, Tanetoshi, Ogura 1998].

Biotechnology Applications:

As mentioned in the introduction, Micrococcus luteus has important biotechnology applications, especially in the chemical and pharmaceutical industries. M. luteus may be potentially exploited for its capability in isoprene and terpene synthetic reactions. These reactions are the chemical foundation of many important organic compounds. M. luteus has been the platform for isolation of the cisprenyltransferase gene (a Rer2 gene homolog first found in S. cerevisiae). Cis-Prenyltransferase catalyzes the sequential condensation of isopentenyl diphosphate with allylic diphosphate to synthesize polyprenyl diphosphates that play vital roles in cellular activity. Sequence analysis revealed that the protein is highly homologous in several conserved regions in M. luteus,E. coli, and yeast. The enzyme is able to catalyze the formation of polyprenyl diphosphates ranging in carbon number from 100 to 130. This is an essential step in the biosynthesis of terpenes, major components of a number of commercial, carbon-based organic products [Oh et al., 2000].

Even earlier studies have shown that the membranes of M. luteus are rich in enzymes that catalyze the synthesis of prenyl pyrophosphates (small molecules required to make compounds such as cholesterol, carotene and alkylamines) [Saito and Ogura, 1981]. Prenyl pyrophosphates are currently being looked at as possible nonpeptide antigens that stimulate certain T-cells as vaccines to prevent human infections and to treat cancer. T-cells (V_2V_2) can recognize and kill malignant B-cells and other tumor cells. These T-cells represent the most abundant population of T- lymphocytes (gamma-delta) in human blood. They produce and promote strong cytotoxic activity against many pathogens that are implicated in several human infectious diseases. Their activation requires their exposure to small phosphoruscontaining antigens in the family of prenyl pyrophosphates and their related biosynthetic precursors such as isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are naturally occurring metabolites in Micrococcus luteus as well as several microbial pathogens. Various prenyl pyrophosphate and diphosphonate compounds are being tested to find new vaccine candidates

and thus invites the potential use of isoprenoid-pyrophosphonates as specific immunoregulatory

molecules [Zgani,I.,et al., 2004].

Bioremediation:

The fundamental idea behind biodegradation and/or bioremediation is to find bacteria that are capable of using the pollutant or contaminant as a food source. The toxin might also be a source of some essential compound like an ammonia molecule (nitrogen in the form of NH3) and therefore used up while the microbe grows on it. Most organisms do not have enzymes that can degrade the longer chained molecules, like those found in crude oil, or the aromatic circularized ones. The toxic effects of crude oil and petroleum by-products are well documented. There are a few bacteria that do have those enzymes. In this way, they can degrade the molecules found in crude oil by cutting them and converting them to simpler compounds. These are then broken down again by continued enzymatic action and eventually converted to a sugar (or something similar) and used by the bacteria as an energy source. Usually a number of specialized enzymes (3-10) are required. Numerous studies have demonstrated that Micrococcus luteus is such a bacteria [Isola-Kayode,T.M, et al., 2008].

Tolerance of Bacterial Species to Conc. Of Crude Oil

It is also possible to use a bacterium like M. luteus as a type of sensitivity index for various concentrations of petroleum oils in the environment. Thus a comprehensive assessment of bacterial response to oil pollution would be useful to design baseline oil pollution models. This could potentially provide useful information about the pollutioncarrying capacity of an environment and serve as a tool in rapid environmental impact monitoring and assessment of pollution by oil-based fractions. In addition, there are marine strains of Micrococcus luteus. It has been shown that marine M. luteus K- 3 constitutively produces two salt-tolerant glutaminases. These are designated simply as glutaminase I Table insert is from: Response of Resident Bacteria of a Crude Oil-Polluted River to Diesel Oil. Isola-Kayode,T.M, et al., 2008

and II. Maximum activity of glutaminase I was observed at pH 8.0, 50oC and 8-16% NaCl. The optimal pH and temperature of glutaminase II were 8.5 and 50oC. The activity of glutaminase II was not affected by the presence of 8 to 16% NaCl. The presence of 10% NaCl enhanced thermal stability of glutaminase I. Both enzymes efficiently catalyzed the hydrolysis of the amino acid L-glutamine [Moriguchi M, et al.,1994]. Once again, this demonstrates the versatility of M. luteus to function as a potential bioremediator in fresh and salt water habitats.

It is highly likely that strains of Micrococcus luteus are involved in detoxification and biodegradation of many other environmental pollutants. It has been discovered that most strains of the bacteria contain a plasmid capable of degrading pesticides such as malathione and chlorpyriphos. Chlorpyrifos, in particular, is one of the world's most widely used organo-phosphorus pesticides in agriculture. Exposure to chlorpyrifos and its metabolites have been related to a variety of nerve disorders in humans. Chlorpyrifos, which was previously thought to be immune to enhanced biodegradation, has now been shown to undergo enhanced biodegradation by some bacteria using the organo-phosphorus hydrolase enzyme. Two plasmid-harboring strains of Micrococcus (M-36 and AG-43) degrade both malathion and chlorpyriphos. Agarose gel electrophoresis of cell extracts of M-36 and AG-43 revealed the presence of the plasmids in M. luteus [Guha, A., et al., 1997]. Future studies on the genes responsible for enhanced biodegradation are expected to elucidate the precise degradative pathway involved in such microbial biodegradation. While Micrococcus luteuspossesses unusual abilities to use toxic organic molecules as metabolic carbon sources, it also displays a significant tolerance to metals (i.e. gold, copper, strontium, zinc, nickel, lead). For example, cells of Micrococcus luteus have been shown to remove strontium (Sr) from dilute aqueous solutions of SrCl2 under neutral pH conditions.

Studies have indicated that the rapid removal of Sr++ from solution is consistent with the hypothesis that diffusion is a major determinant of the rate of binding. Experiments done with M. luteus and other bacterium, show that an equilibrium exists between bound and dissolved strontium in batch systems, indicating that binding is reversible. Cells cultured under conditions optimal for growth (using fertilizers and biosurfactants) exhibited optimal binding activity, suggesting that the strontium receptor(s) is a normal component of the growing cells. At least a portion of this binding activity is due to an ion exchange mediated by acidic components of the cell envelope. The kinetics of strontium-binding by M. luteus at various temperatures and pH’s has not yet been determined. However, the fact that cells bound similar amounts of strontium over a temperature range of 4 to 35°C suggests that the binding reaction is not endothermic. The use of immobilized cells of an M. luteus culture for the treatment of nuclear industry waste waters is promising, since strontium is an end product of uranium nuclear decay. The ability of this organism to bind strontium in the absence of cofactors, its high affinity for the metal, and the enhanced stability of strontium-binding in the presence of other metals encourages further study [Faison, B, et at., 1990].