ABSTRACT. Chromobacterium violaceum is a free-living microorganism, normally exposed to diverse environmental conditions; it has a versatile energy-generating metabolism. This bacterium is capable of exploiting a wide range of energy resources by using appropriate oxidases and reductases. This allows C. violaceum to live in both aerobic and anaerobic conditions. In aerobic conditions, C. violaceum is able to grow in a minimal medium with simple sugars, such as glucose, fructose, galactose, and ribose; both Embden-Meyerhoff, tricarboxylic acid and glyoxylate cycles are used. The respiratory chain supplies energy, as well as substrates for other metabolic pathways. Under anaerobic conditions, C. violaceum metabolizes glucose, producing acetic and formic acid, but not lactic acid or ethanol. C. violaceum is also able to use amino acids and lipids as an energy supply.

Key words: Chromobacterium violaceum, Energetic metabolism, Aerobic metabolism, Anaerobic metabolism

INTRODUCTION

In all living organisms (bacteria, plants and animals), energy-rich substrates are converted into chemical energy as ATP. ATP is used by the cells to drive movement, for active transport, metabolic synthesis, and other processes requiring energy. Membrane-bound H⁺-ATP synthases are the key enzymes of energy metabolism. They catalyze ATP synthesis from ADP and inorganic phosphate, coupled with transmembrane proton transport. Proton transport is driven by transmembrane pH and electric potential differences (Mitchell, 1974; Creczynski-Pasa and Gräber, 1994; Boyer, 1998). Chromobacterium violaceum has an F₀F₁-type ATP synthase, similar to the enzyme found in E. coli (Fischer and Gräber, 1999; Vasconcelos et al., 2003). However, C. violaceum also has alternative means to obtain energy. It is able to live under aerobic and anaerobic conditions as a free-living organism; this important characteristic enables this bacterium to survive under diverse environmental conditions. Furthermore, C. violaceum has a strong chemotactic capacity, and a strong capability to adapt to stress (Vasconcelos et al., 2003). All these processes require large amounts of energy. We present an overview of the energetic metabolism of C. violaceum, based on the information obtained from its genome analysis (Vasconcelos et al., 2003).

RESULTS AND DISCUSSION

As in all chemoheterotrophic bacteria, C. violaceum is able to grow in minimal medium that includes simple sugars, such as glucose, fructose, galactose, or ribose. However, C. violaceum is not able to synthesize glucose through gluconogenesis, since, based on genome analysis, it lacks the gene that codes for glucose-6-phosphatase (Vasconcelos et al., 2003). The Embden-Meyerhoff pathway, tricarboxylic acid and glyoxylate cycles, and the respiratory chain, supply cellular energy, as well as substrates for anabolic pathways under aerobic conditions. This bacterium has the capability to biosynthesize complex polysaccharides, such as cellulose, but not glycogen. C. violaceum mainly uses monosaccharides; however, it seems to be able to utilize cellulose and chitin, but not starch, as carbon sources, since it contains cellulases and chitinase, but lacks alpha amylase genes. In addition, C. violaceum is not able to utilize sucrose or lactose as energy resources, because these metabolic pathways are absent from its genome (Vasconcelos et al., 2003).

The second key enzyme of the aerobic metabolism is cytochrome oxidase, which is a member of a large superfamily of proteins (Calhoun et al., 1994). Phylogenetic and evolutionary analysis of this family indicated three basic types of cytochrome oxidase, which are: SoxM (or aa3, also found in mitochondria), SoxB and FixN (orcbb3) (Castresana, 2001). Whereas SoxM and SoxB work under normal aerobic conditions, the FixN oxidases are involved in respiration at very low oxygen pressure (microaerobic conditions) (Castresana, 2001). Genes homologous to SoxM and FixN, as well as, the cytochrome bd oxidase, have been found in the C. violaceum genome. This enzyme is a terminal oxidase, with a high affinity for oxygen, but it is completely unrelated to the cytochrome oxidase superfamily. The cytochrome bd seems to play an important role in oxidative stress, although it is also able to create an electrochemical membrane gradient, available for energetic requirements (Castresana, 2001).

There is an operon for HCN synthase (hcnA, hcnB and hcnC) in the C. violaceum genome, encoding a formate dehydrogenase and two amino acid oxidases, respectively, similar to those found in Pseudomonas aeruginosa, which are involved in cianidric acid synthesis. Previous work on cyanide (HCN) synthesis in bacteria show that, in vivo, the four electrons produced by HCN synthase are transferred to oxygen, probably throughout the respiratory chain. These reactions occur at low levels of oxygen. Analysis of enzyme sequences has shown that each subunit is similar to known enzymes involved in electron transport (Laville et al., 1998).

Likewise, lipid metabolism in C. violaceum, with the degradation and biosynthesis of various types of lipids (triacylglycerol, phospholipids, and lipopolysaccharides), provides another energy resource. The beta-oxidation of fatty acids leads to the production of reduced nucleotides, and acetyl-CoA leads to energy production.

Extracellular proteases enable the bacterium to utilize the resultant amino acids as substrates for endogenous protein biosynthesis, as well as for carbon and nitrogen resources. The presence of several deaminases, as well as transaminases, results in the production of keto acids from amino acids, which can be converted to acetyl-CoA, or tricarboxylic cycle intermediates, and eventually to an energy resource.

Chromobacterium violaceum also anaerobically metabolizes glucose, with production of mixed organic acids, such as acetic acid and formic acid. CO₂ and H₂ are produced from formic acid, but not from lactate or ethanol. This alternative way of yielding energy, where organic molecules serve as both electron donors and acceptors, is known as fermentation. In the absence of oxygen, the microorganisms decrease pyruvate dehydrogenase activity, and use the pyruvate or one of its derivatives as an electron and a hydrogen acceptor in the reoxidation of NADH. Also, under anaerobic conditions, C. violaceum is able to obtain large amounts of energy using nitrate or fumarate as final electron acceptors in the respiratory chain. All components of anaerobic fumarate reductase are present, as well as all the necessary elements for reduction of nitrate to nitrite, similar to the situation in E. coli. In C. violaceum, most of the enzymes involved in the N cycle, including nitrate reductase, nitrite reductase, as well as the nitric oxide reductase responsible for N₂O production from nitric oxide, are present (Vascocelos et al., 2003; Moura et al., 2003). However, it lacks nitrous oxide reductase, the enzyme that converts nitrous oxide to N₂ (Vasconcelos et al., 2003).

The genes involved in the fermentation of mixed acids (pyruvate-formate lyase) are also present in C. violaceum. Alcoholic fermentation of glucose can also be performed by the Entner-Doudoroff pathway. Although C. violaceum has alcohol dehydrogenase, it is not able to produce ethanol because the pyruvate produced by this pathway, as well as that produced by the Embden-Meyerhoff pathway, cannot be converted to ethanol, since C. violaceum lacks the pyruvate decarboxylase gene (Vasconcelos et al., 2003).

Chromobacterium violaceum is able to synthesize all the amino acids it needs; however, the most notable characteristic of this bacterium is related to the metabolism of a specific amino acid, tryptophan, which is involved in the production of a chemically well-characterized pigment named violacein (Bromberg and Durán, 2001). Violacein is synthesized only when C. violaceum grows in aerobic conditions; however, the function of this pigment in this bacterium is unknown. There is some speculation that violacein is a kind of storage form of tryptophan, but there is no proof for such a hypothesis. There are some indications that violacein has antibiotic, antichagasic (Momen and Hoshino, 2000), antitumoral (Melo et al., 2000; Duran and Menck, 2001) and antileishmanial (Leon et al., 2001) activity. During the last few years, a new property of violacein has been characterized; it appears that this molecule has antioxidant properties (Azevedo et al., 2000; Konzen et al., 2003). Violacein is involved with oxygen metabolism, but no direct relationship with energy metabolism has been found.

When we compare the versatility of the energy metabolism of C. violaceum with that of five other bacteria from other genera (Table 1), C. violaceum has six enzymes involved with energy metabolism in a general way, while the other bacteria lack one to four of these enzymes. The catabolic activity results in ATP synthesis, or in the production of reduced nucleotides, which should be aerobically or anaerobically reoxidized, through one of the mechanisms present in C. violaceum, depending on environmental conditions.

CONCLUSION

The analysis of C. violaceum DNA allowed us to conclude that this bacterium has an efficient apparatus for energy production, under both aerobic and anaerobic conditions. All the genes that are necessary for the glycolysis and tricarboxylic acid cycle were found in its genome, as well as the main proteins involved in electron transport (dehydrogenases, quinones and cytochromes). This apparatus allows a high production of energy under aerobic conditions, since complete degradation of glucose is possible. Besides the energy required for other functions, C. violaceum seems to spend considerable energy on motility, assuring its survival under variable environmental conditions, such as a lack of food, and other types of stress.

ACKNOWLEDGMENTS

The authors thank MCT/CNPq (Brazilian National Genome Project Consortium) and FUNCITEC for their support.

REFERENCES

Azevedo, M.B.M., Justo, G.Z., Rettori, D., Rodriguez, J.A., Haun, M. and Duran, N. (2000). Antioxidant activity of an inclusion complex of violacein and b-cyclodextrin. Proc. Int’ I. Symp. Control. Rel. Bioact. Mater. 27: 6332.

Boyer, P.D. (1998). Energy, life, and ATP. Biosci. Rep. 18: 97-117.

Bromberg, N. and Durán, N.J. (2001). Violacein transformation by peroxidases and oxidases: implications on its biological properties. Mol. Catal. B: Enzymatic. 11: 463-467.

Calhoun, M.W., Thomas, J.W. and Gennis, R.B. (1994). The cytochrome-oxidase superfamily of redox-driven proton pumps. Trends Biochem. Sci. 19: 325-330.

Castresana, J. (2001). Comparative genomics and bioenergetics. Biochim. Biophys. Acta 1506: 147-162.

Creczynski-Pasa, T.B. and Gräber, P. (1994). ADP binding and ATP synthesis by reconstituted H⁺-ATPase from chloroplasts. FEBS Lett. 350: 195-198.

Duran, N. and Menck, C.F.M. (2001). Chromobacterium violaceum: a review of pharmacological and industrial perspective. Crit. Rev. Microbiol. 27: 201-222.

Fischer, S. and Gräber, P. (1999). Comparison of DpH- and Df-driven ATP synthesis catalyzed by the H⁺-ATPases from Escherichia coli or chloroplasts reconstituted into liposomes. FEBS Lett. 457: 327-332.

Konzen, M., Fernandez, H., Vieira, T.O., Cordova, C.A.S., Antônio, R.V., De Marco, D. and Creczynski-Pasa, T.B. (2003). Antioxidant properties of violacein. XXXII Annual Meeting of SBBq, Caxambu, MG, Brazil, 05-17 to 05-20, pp. 243.

Laville, J., Blumer, C., Von Schroetter, C., Gaia, V., Defago, G., Keel, C. and Haas, D. (1998). Characterization of the hcnABC gene cluster encoding hydrogen cyanide synthase and anaerobic regulation by ANR in the strictly aerobic biocontrol agent Pseudomonas fluorescens CHA0. J. Bacteriol. 180: 3187-3196.

Leon L.L., Miranda, C.C., De Souza, A.O. and Duran, N. (2001). Antileishmanial activity of the violacein extracted form Chromobacterium violaceum. Antimicrob. Agents Chemoth. 48: 449-450.

Melo, P.S., Maria, S.S., Vidal, B.C., Haun, M. and Duran, N. (2000). Violacein cytotoxicity and induction of apoptosis in V79 cells. In Vitro Cell Dev. Biol. Anim. 36: 539-543.

Mitchell, P. (1974). A chemiosmotic molecular mechanism for proton translocating adenosine triphosphatases. FEBS Lett. 43: 189-194.

Momen, A.Z.M.R. and Hoshino, T. (2000). Biosynthesis of violacein: Intact incorporation of the tryptophan molecule on the oxindole side, with intramolecular rearrangement of the indole ring on the 5-hydroxyindole side. Biosci. Biotechnol. Biochem. 64: 539-549.

Moura, I., Cabrito, I., Almeida, G., Cunha, C., Romao, M.J. and Moura, J.J.G. (2003). Molecular aspects of denitrification/nitrate dissimilation. J. Inorg. Biochem. 96: 195-195.

Vasconcelos, A.T.R., Almeida, D.F., Almeida, F.C., Almeida, L.G.P., Almeida, R., Alves-Gomes, J.A., Andrade, E.M., Antônio, R.V., Araripe, J., Araújo, M.F.F., Astolfi-Filho, S., Azevedo, V., Baptista, A.J., Bataus, L.A.M., Batista, J.S., Beló, A., van den Berg, C., Blamey, J., Bogo, M., Bonatto, S., Bordignon, J., Brito, C.A., Brocchi, M., Burity, H.A., Camargo, A.A., Cardoso, D.D.P., Carneiro, N.P., Carraro, D.M., Carvalho, C.M.B., Cascardo, J.C.M., Cavada, B.S., Chueire, L.M.O., Creczynski-Pasa, T.B., Duran, N., Fagundes, N., Falcão, C.L., Fantinatti, F., Farias, I.P., Felipe, M.S.S., Ferrari, L.P., Ferro, J.A., Ferro, M.I.T., Franco, G.R., Freitas, N.S.A., Furlan, L.R., Gazzinelli, R.T., Gomes, E.A., Gonçalves, P.R., Grangeiro, T.B., Grattapaglia, D., Grisard, E.C., Guimarães, C.T., Hanna, E.S., Hungria, M., Jardim, S.N., Laurino, J., Leoi, L.C.T., Lima, L.F.A., Loureiro, M.F., Lyra, M.C.C.P., Macedo, M., Madeira, H.M.F., Manfio, G.P., Maranhão, A.Q., Martins, W.S., di Mauro, S.M.Z., Medeiros, S.R.B., Meissner, R.V., Moreira, M.A.M., Nascimento, F.F., Nicolas, M.F., Oliveira, J.G., Oliveira, S.C., Paixão, R.F.C., Parente, J.A., Pedrosa, F.O., Pena, S.D.J., Pereira, J.O., Pereira, M., Pinto, L.S.R.C., Pinto, L.S., Porto, J.I.R., Potrich, D.P., Ramalho-Neto, C.E., Reis, A.M.M., Rigo, L.U., Rondinelli, E., Santos, E.B.P., Santos, F.R., Schneider, M.P.C., Seuanez, H.N., Silva, A.M.R., Silva, A.L.C., Silva, D.W., Silva, R., Simões, I.D.C., Simon, D., Soares, C.M.A., Soares, R.B.A., Souza, E.M., Souza, K.R.L., Souza, R.C., Steffens, M.B.R., Steindel, M., Teixeira, S.R., Urmenyi, T., Vettore, A., Wassem, R., Zaha, A. and Simpson, A.J.G. (2003). Complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci. USA 100: 11660-11665.