M. Hungria et al. 102 Tolerance to stress and environmental adaptability of Chromobacterium violaceum Mariangela Hungria1, Marisa Fabiana Nicolás1, Claudia Teixeira Guimarães2, Sílvia Neto Jardim2, Eliane Aparecida Gomes2 and Ana Tereza Ribeiro de Vasconcelos3 1 Embrapa Soja, Caixa Postal 231, 86001-970 Londrina, PR, Brasil Embrapa Milho e Sorgo, Caixa Postal 151, 35701-970 Sete Lagoas, MG, Brasil 3 Laboratório Nacional de Computação Científica, Rua Getúlio Vargas 333, 25651-071 Petrópolis, RJ, Brasil Corresponding author: M. Hungria E-mail: hungria@cnpso.embrapa.br 2 Genet. Mol. Res. 3 (1): 102-116 (2004) Received October 13, 2003 Accepted January 12, 2004 Published March 31, 2004 ABSTRACT. Chromobacterium violaceum is a Gram-negative bacterium, abundant in a variety of ecosystems in tropical and subtropical regions, including the water and borders of the Negro River, a major component of the Amazon Basin. As a free-living microorganism, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature and pH, toxic compounds and UV rays. These variations, and the wide range of environments, require great adaptability and strong protective systems. The complete genome sequencing of this bacterium has revealed an enormous number and variety of ORFs associated with alternative pathways for energy generation, transport-related proteins, signal transduction, cell motility, secretion, and secondary metabolism. Additionally, the limited availability of iron in most environments can be overcome by iron-chelating compounds, iron-storage proteins, and by several proteins related to iron metabolism in the C. violaceum genome. Osmotically inducible proteins, transmembrane water-channel, and other membrane porins may be regulating the movement of water and maintaining the cell turgor, activities which play an important role in the adaptation to Genetics and Molecular FUNPEC-RP www.funpecrp.com.br Molecular Research Research 33 (1): (1): 102-116 102-116 (2004) (2004) www.funpecrp.com.br Stress tolerance and adaptability of Chromobacterium violaceum 103 variations in osmotic pressure. Several proteins related to tolerance against antimicrobial compounds, heavy metals, temperature, acid and UV light stresses, others that promote survival under starvation conditions, and enzymes capable of detoxifying reactive oxygen species were also detected in C. violaceum. All these features together help explain its remarkable competitiveness and ability to survive under different types of environmental stresse. Key words: Adaptability, Secondary metabolism, Stress tolerance, Chromobacterium INTRODUCTION Chromobacterium violaceum was first described at the end of the 19th century (Boisbaudran, 1882), after which several reports confirmed its presence in the soil and water of tropical and subtropical regions. Its most notable characteristic is the production of violacein, a purple pigment first isolated in 1944 (Strong, 1944), and chemically characterized a few years later (Ballantine et al., 1958). In Brazil, the abundance of this bacterium in the water and on the borders of the Negro River, in the Amazon Region, and the therapeutic properties of the violacein have been reported since the end of the 1970s (Caldas et al., 1978; Durán et al., 1983, 1989, 1994; Caldas, 1990; Durán, 1990; Souza et al., 1999; Melo et al., 2000; Leon et al., 2001). The competitive success of C. violaceum in several microbial communities, as well as the wide range of its habitats, is certainly indicative of a remarkable ability to survive under different environmental stresses. Indeed, evidence of a wide range of genes related to adaptability was revealed with the complete genome sequencing of free-living C. violaceum (Vasconcelos et al., 2003). GENERAL MECHANISMS RELATED TO STRESS TOLERANCE AND ADAPTABILITY As with other free-living microorganisms, C. violaceum is exposed to a series of variable conditions, such as different sources and abundance of nutrients, changes in temperature, toxic compounds, and UV rays. These variations require fast adaptive responses, usually triggered by transcriptional activation of specific genes. A set of genes controlling basal transcriptional mechanisms, such as RNA polymerase and common sigma factors, e.g., σ70 (rpoD, CV3762), σ54 (rpoN, CV3332), σ32 (rpoH, CV4206) and σ38 (rpoS, CV3682) are present in C. violaceum. Furthermore, a large number of transcriptional activators and repressors that interact with alternative sigma factors involved in bacterial cell response to stress, such as those belonging to LysR (CV0061, CV0270, CV0509, etc.), AraC (CV1314, CV1825, CV2101, etc.), TetR (CV0385, CV0711, CV1077, etc.), among others are also present. For additional information about transcriptional factors and regulation in C. violaceum, see Silva et al. (2004). The C. violaceum genome has a high proportion (6.4%) of ORFs associated with signal transduction mechanisms (COG category T), when compared with other free-living sequenced microorganisms (Vasconcelos et al., 2003), which reflect an enhanced adaptability to Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br M. Hungria et al. 104 diverse environmental conditions. Additionally, 5.8% (257 of 4,431 valid open reading frames, ORFs) of the genes of C. violaceum encode transcriptional regulators, similar to the proportions found in other versatile microorganisms capable of colonizing soil, water, plant, and animal tissues, such as Pseudomonas aeruginosa, E. coli and Bacillus subtilis, which contain 8.4, 5.8 and 5.3% of the ORFs involving regulatory proteins, respectively. In contrast, such genes constitute only 3.0% and 1.1% of the genome in the specialized pathogens Mycobacterium tuberculosis and Helicobacter pylori, respectively (Blattner et al., 1997; Kunst et al., 1997; Hancock et al., 1998; Cole et al., 1998; Stover et al., 2000). The ecological versatility of C. violaceum is also confirmed by the identification of 204 ORFs related to energy metabolism, including fermentation, aerobic and anaerobic respiration (Vasconcelos et al., 2003; Creczynski-Pasa and Antônio, 2004), allowing this bacterium to obtain energy in various types of conditions. Another principal attribute of C. violaceum, when compared with other sequenced microorganisms, is its high percentage of ORFs (5.8%) related to cell motility and secretion (COG category N) described until now (Vasconcelos et al., 2003; Pereira et al., 2004). Genes related to chemotaxis and flagella are typical adaptations of soil and water dwelling bacteria. A large number of transport-related proteins (496 ORFs, 11.2% of the valid ORFs) are associated with metabolic transport, multidrug-resistance proteins, membrane receptors and porins, suggesting intensive interactions with the different habitats (Vasconcelos et al., 2003). Additionally, C. violaceum has several ORFs related to tolerance against antimicrobial compounds (CV0700), solvents and heavy metals, which results in greater competitiveness compared to other microorganisms under the same conditions. All these features, taken together, are strong attributes to enhance its ability to survive in a wide range of soil and water environments, most of them depleted of nutrients and energy sources (Walker, 1990). IRON METABOLISM Synthesis of iron-chelating compounds All microorganisms need iron, the availability of which is limited in most aerobic environments, since it is present only in insoluble mineral complexes or bound to mammalian or plant host’s iron-binding proteins. Therefore, many microorganisms have developed efficient ways to obtain iron under deprived conditions. One important mechanism is gene clusters encoding enzymes or receptors that recognize low molecular weight iron-chelating compounds, called siderophores. These siderophores are secreted into the environment, where they bind ferric iron with high affinity in a mechanism recognized as crucial to increase competitiveness and adaptation to different environmental conditions (Lankford, 1973; Crosa, 1989; Payne, 1994). Enterobactins are classic examples of iron-chelating compounds involved in iron transportation from the environment into the bacterium cytoplasm. In the biosynthetic pathway of enterobactin, EntD is a key protein in the final conversion of 2,3-dihydroxybenzoic acid and Lserine into enterobactin. Studies involving EntD– and EntB– mutants of E. coli have shown that these genes are essential for bacterial growth in iron-deficient environments (Armstrong et al., 1989; Coderre and Earhart, 1989; Liu et al., 1989; Nahlik et al., 1989; Hantash and Earhart, 2000). ORFs related to the synthesis of enterobactin; entA (CV1482), entB (CV1483), entC (CV1485), entE (CV1484), entD (CV2650), entF (CV1486, CV2233) have been identified in Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br Stress tolerance and adaptability of Chromobacterium violaceum 105 C. violaceum. Similar iron-chelating compounds have been described in other microorganisms, e.g., vibriobactin in Vibrio cholerae (Wyckoff et al., 1997), pyochelin and pyoverdine in Pseudomonas spp. (Poole and McKay, 2003), and myxochelin in myxobacteria (Silakowski et al., 2000). In Gram-negative bacteria, outer membrane receptors, such as FepA in E. coli and ViuA in V. cholerae bind the ferrisiderophore complex across the inner membrane (Klebba et al., 1993; Wyckoff et al., 1997). A second receptor complex is needed, the major component of which is TonB, a 27-kDa protein that facilitates energy transfer from the proton motive force to outer receptors. B-12 and colicin receptors also make use of the TonB system to drive active transport in the outer membrane. In E. coli, the TonB protein interacts with outer membrane receptor proteins that carry out high-affinity binding and energy-dependent uptake of specific substrates into the periplasmic space. In the absence of TonB, these receptors bind to their substrates but do not carry out active transport (Klebba et al., 1993; Postle, 1993). The proteins that are currently known to interact with TonB include the ones encoded by the genes btuB, cirA, fatA, fcuT, fecA, fhuE, fptA, hemR, irgA, iutA, pfeA, pupA, and tbp1. Outer membrane receptors fepA (CV2230), fepB (CV2239), fepC (CV2234), fepG (CV2235), fepD (CV2236), and components of a second receptor complex that drive active transport in the outer membrane, with TonB-dependent receptors (CV0077, CV1019, CV1699, CV1970, CV1982, CV3188, CV3896, CV4254) were found in the C. violaceum genome. Furthermore, colicins exbB (CV0399), exbB1 (CV1972), exbB2 (CV1984), exbD1 (CV1985), exbD2 (CV0398), exbD3 (CV1974), exbD4 (CV1986), and exbD5 (CV1973) were detected. Genes related to enterochelin esterase (fes, CV2231), iron-chelator protein (mxcB, CV2466) and ferrisiderophore reductase C (ubiB, CV3784) were also found in this genome. Besides being crucial for the adaptability and competitiveness of C. violaceum, the iron-related genes may also play an important role in the virulence and infectious ability of the microorganisms. Some pathogenic bacteria express surface receptors to capture eukaryotic iron-binding compounds, while others have evolved siderophores to scavenge iron from ironbinding host proteins (Takase et al., 2000). Classic examples are the enterobactin/enterochelin gene clusters found in E. coli and Salmonella spp., with similar moieties in other pathogens (Liu et al., 1989; Nahlik et al., 1989; Poole and McKay, 2003). Further details about genes of C. violaceum related to pathogenicity can be found in Brito et al. (2004). Synthesis of bacterioferritin, and other mechanisms related to iron uptake Iron-depletion can be ameliorated through the use of storage proteins. Bacteria have two types of iron-storage proteins, the haem-containing bacterioferritins (BFR) and the haemfree ferritins. In E. coli, BFR (also known as cytochrome b1 or cytochrome b557) consists of 24 identical subunits that pack together to form a highly symmetrical, nearly spherical shell surrounding a central cavity. Studies using X-ray crystallography have revealed a close structural similarity between BFR and the ferritins, a family of iron-storage proteins found in both eukaryota and prokaryota. Some bacteria contain two BFR subunits, or two ferritin subunits that in most cases co-assemble. Others possess both BFR and ferritin, while some appear to lack any type of iron-storage protein; the reasons for these differences are unknown. Studies with mutants have shown that ferritin enhances growth during iron starvation and accumulates iron in the stationary growth phase (Andrews, 1998). Two ORFs related to BFR (CV3552 and Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br M. Hungria et al. 106 CV3399) and one probable BFR co-migratory oxidoreductase protein (CV2815) have been detected in C. violaceum. Another mechanism of iron uptake detected in C. violaceum is related to ferric citrate transport system permease protein (fecC CV1909, fecD CV1488, CV1794, fecE CV3899), which facilitates iron diffusion in high concentration of this nutrient. Escherichia coli also has an iron (II) transport system (feo), which may make an important contribution to the iron supply of the cell under anaerobic conditions; consequently the transport of ferrous iron may be ATP dependent (Kammler et al., 1993). feoB was detected (CV3553) in C. violaceum, as well as other ORFs, such as fhuA (CV2251) and fhuC (CV1560, 1487), both involved in an active transport against a concentration gradient with the use of metabolic energy inputs. Fifty ORFs are related to iron metabolism in C. violaceum, a considerable number in comparison to other bacterial genomes that have been sequenced, probably indicating a major role for survival under various environmental conditions. CONTROL OF CELL OSMOTIC PRESSURE AND PH Osmotic movement of water across bacterial cell membranes is an extremely important mechanism to maintain cell turgor. Proteins related to adaptation to differences in osmotic pressure in the environment are therefore crucial for cell survival and affect their adaptability. Osmotically inducible proteins were detected in C. violaceum, with high similarity to organic hydroperoxide resistance protein (CV0209, CV2493). However, an ORF showing similarity (6e-16) with the osmotically inducible lipoprotein OsmB (CV3209) was found, but none was found similar to OsmC. In E. coli, the expression of the osmB gene is regulated at the transcriptional level by two promoters that respond to the growth phase and to hyperosmolarity conditions, osmBp1 and osmBp2. The transcription of osmB from osmBp2 is induced by elevated osmolarity, or upon reaching the stationary phase, and transcription from osmBp1 occurs when both osmotic and growth phase signals are present (Jung et al., 1990). Transport of K+ ions, as well as increases in the intracellular concentration of osmoprotectans, such as glutamate, proline, threhalose and glycine, are also important for bacterial survival under osmotic pressure stress (Jung et al., 1990; Doyle et al., 1998). Examples in C. violaceum include the kup1 (CV2731) and kup2 (CV0573) genes (low-affinity potassium transport system), kdpABC (CV1599, 1598, 1597), the kdpDE (CV1596, 1595) operons (highaffinity potassium transport system), the kefB (CV3326) gene (glutathione-regulated potassium-efflux system K+/H+ antiporter transmembrane protein), the inward rectifier potassium channel transmembrane protein (CV1109), genes related to sodium chloride (CV1670, CV2040, CV2223, CV2225, CV2380, CV3281, CV3650, CV4369), sodium-glutamate symporter gltS (CV1105) and gltP (CV1198, CV3409), and proline transports proP (CV1299, CV2901), proV (CV1197) and proY (CV1138). ORFs with functions similar to a sodium/hydrogen exchanger (CV2903), and to a proton-dependent peptide transporter (CV3755), were found. These genes may act to control internal pH, extruding the H+ generated during metabolism. The porins form water-filled channels, which allow the diffusion of hydrophilic molecules into the periplasm, including large antibiotic molecules (Nikaido, 1994). In E. coli, the transcription of outer membrane porin genes ompC and ompF is regulated in response to medium osmolarity, by a two-component regulatory system, EnvZ, which is a transmembrane sensor, and OmpR, the response regulator (Jung et al., 1990; Cai and Inouye, 2002; Qin et al., 2003). In Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br Stress tolerance and adaptability of Chromobacterium violaceum 107 C. violaceum, the outer membrane protein OmpC (CV3424) and the proteins of the regulatory system, EnvZ (CV0217) and OmpR (CV0216, CV3107), were found. Another interesting gene annotated in C. violaceum was aquaporin Z (aqpZ, CV2864). The aquaporins are transmembrane water-channel proteins present in plants and animals (Chrispeels and Agre, 1994). These channels are water selective and do not allow ions or metabolites to pass through them, regulating osmotically driven movement of water in both directions, maintaining cell turgor during volume expansion in rapidly growing cells. The importance of these channels is due to their mediation of rapid entry or exit of water in response to abrupt changes in environmental osmolarity. In bacteria, the first sequence homologous to aquaporin was glpF, which encodes a glycerol facilitator that displays minimal water permeability, and the aqpZ sequence of E. coli encodes a polypeptide with 28-38% identity to known aquaporins (Heller et al., 1980; Calamita et al., 1995). The C. violaceum aqpZ gene shows 7986% of similarity with that of P. aeruginosa. An operon containing glpF, glpD, glpT, glpK, and glpR (CV0252, 0254, 0253, 0251, 0136) genes was also found in C. violaceum. The expression of aqpZ conferred a 15-fold increase in osmotic water permeability in Xenopus oocytes, but it failed to transport nonionic solutes, such as urea and glycerol. In contrast, Xenopus oocytes expressing glpF transported glycerol, but they had limited osmotic water permeability. A phylogenetic comparison of aquaporins revealed a large difference between aqpZ and glpF, consistent with an ancient gene divergence (Preston et al., 1992). AqpZ-like proteins seem also to be necessary for virulence in some pathogenic bacteria (Calamita et al., 1995, 1998; Fushimi et al., 1997; Calamita, 2000). OXIDATIVE STRESS The advent of oxygen in the atmosphere was one of the first major pollution events on earth. The reaction between ferrous iron, very abundant in the reductive early atmosphere, and oxygen results in the formation of reactive oxygen species (ROS), which can damage proteins, DNA and lipids. The ROS include hydrogen peroxide (H2O2), the superoxide anion (O2–), the hydroxyl radical (OH), and organic hydroperoxides (ROOH) (Beckman and Ames, 1998). Bacteria have numerous enzymes to detoxify ROS, such as catalases, superoxide dismutases, alkyl hydroperoxide reductase, and related peroxidases of the AhpC/thiol-specific antioxidant family (Fuangthong et al., 2001; Fuangthong and Helmann, 2002). In E coli, the common responses to oxidative stress are controlled by two major transcriptional regulators, SoxRS and OxyR, which induce the expression of antioxidant activities in response to O2–/H2O2 and H2O2 stress, respectively (Storz and Imlay, 1999; Manchado et al., 2000; Pomposiello and Demple, 2001). The soxRS regulon is induced in a two-stage process (Nunoshiba et al., 1992). Superoxide-generating compounds, such as paraquat or H2O2, activate the transcription factor SoxR by the univalent oxidation of the [2Fe-2S] clusters of the protein. Oxidized SoxR induces the expression of a second transcription factor, SoxS, which in turn activates the transcription of a set of genes in this regulon. Among the SoxRS-regulated genes are micF (antisense RNA to the porin OmpF), sodA (manganese superoxide dismutase), inaA (pH-inducible protein involved in stress response), fumC (heat-resistant fumarase), and fpr (NADPH-ferredoxin reductase). Evidence for several pathways of SoxR activation, mediated by modifications of [2Fe-2S] centers, has emerged from recent data. The direct oxidation of [2Fe-2S] involves any event that may interfere with the pathway maintaining SoxR in a reduced inactive form; hydrogen peroxGenetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br M. Hungria et al. 108 ide and direct nitrosylation by NO can trigger SoxR activation. Recently, it was reported that although it is not involved in genetic regulation, SoxRS decreases the mortality of cells due to ozone (Jimenez-Arribas et al., 2001). The multiple possibilities for SoxR activation, along with signal amplification via the two-stage process, constitute a unique and sensitive system, enabling cells to rapidly induce a protective response to a broad range of environmental changes, including oxidative stress (Richards et al., 1998; Cabiscol et al., 2000; Manchado et al., 2000; Touati, 2000; Jimenez-Arribas et al., 2001; Michán et al., 2002). In C. violaceum, soxR (CV2793) is present, but not the genes soxS, micF, inaA, and sodA. However, soxS is also lacking in other genomes, including Agrobacterium tumefaciens, Bacillus halodurans, Pseudomonas putida, Xanthomonas axopodis pv. citri, X. campestris pv. campestris, Mesorhizobium loti, Sinorhizobium meliloti, and V. choleare. Recent research indicated that SoxR and SoxS might bind to different regions within the fumC promoter, or an unknown intermediate might be acting in the transcription of fumC. Thus, the regulatory role of SoxR may be more complex than what was previously known from E. coli (Fuentes et al., 2001). Besides playing an important role in adaptability to different environments, resistance to oxidative stress may contribute to the virulence of pathogenic bacteria. Indeed, in Erwinia chrysanthemi, SoxR interacted with suf genes in a virulence response (Nachin et al., 2001). In E. coli, the OxyR transcription factor is activated by hydrogen peroxide through the formation of an intramolecular disulfide bond (Choi et al., 2001), which in turn can activate the expression of hydroperoxidase I (KatG), alkyl hydroperoxide reductase (AhpCF), DNA-binding protein (Dps), and other resistance proteins (Storz and Imlay, 1999). OxyR (CV3378) and the oxidative stress protein Dps (CV4253) were also identified in C. violaceum. Another biochemical mechanism to sense peroxidative stress has been described in B. subtilis; it is triggered by OhrR, a member of a conserved family of organic peroxide-sensing transcription factors. OhrR represses transcription of the ohrA resistance gene by cooperative binding to two inverted repeat elements that overlap the promoter. Induction by peroxides requires a conserved Cys residue that is reversibly oxidized by peroxides to a sulfenic acid (Sukchawalit et al., 2001; Fuangthong et al., 2001). C. violaceum has an operon composed of ohrA-ohrR ORFs (CV0209, 0210). Other ORFs in C. violaceum that might be related to oxidative stress are those coding the hydroperoxide resistance protein OhrB (CV2493) and the MutT/nudix (nucleoside diphosphate linked to some other moeity X) family proteins (CV0032, CV1112, CV1586, CV1767, CV3401). The MutT homologues (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication; they are found in a variety of prokaryotic, viral and eukaryotic organisms (Lu et al., 2001). DsbA is the major disulfide oxidase in the bacterial periplasm (Goecke et al., 2002), and it is also present in C. violaceum (CV3998). ORFs related to aidB (acyl-coA dehydrogenase) were detected in C. violaceum (CV1785, CV2084, CV2723, CV3816, CV4136, CV4139). In E. coli, Ada protein activates σ70-dependent transcription at three different promoters (ada, aidB, and alkA). The aidB gene is not expressed constitutively, but its transcription is induced via distinct mechanisms in response to: i) alkylating agents; ii) acetate at a slightly acidic pH, and iii) anoxia. Induction by alkylating agents is mediated by the transcriptional activator Ada, in its methylated form (meAda); the other forms of induction are Ada-independent and require sigma-S, the alternative sigma factor mainly expressed during the stationary phase of bacterial growth. The leucine-responsive protein (Lrp), a global regulatory protein in E. coli that activates expression of more than a Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br Stress tolerance and adaptability of Chromobacterium violaceum 109 dozen operons and represses expression of another dozen (Chen et al., 2001), is also related to the regulation of those genes (Landini et al., 1996; Landini and Busby, 1999). Several glutathione-S-transferases (CV0194, CV0289, CV0905, CV0972, CV1086, CV1164, CV1775, CV2424, CV2745, CV3024, CV3306, CV4373) and glutathione peroxides (CV1107, CV3555, CV3787) that are related to protection mechanisms are also present in C. violaceum, and they may play a crucial role in adaptability. Bacterial GSTs of known function often have a specific, growth-supporting role in biodegradative metabolism. Some regulatory proteins, such as the stringent starvation proteins, also belong to the GST family (Vuilleumier, 1997). In eukaryotes, GSTs participate in the detoxification of reactive electrophilic compounds by catalyzing their conjugation to glutathione. Among other genes involved in oxidative stress, genes encoding GSTs have been induced by aluminum (Al) stress in Arabidopsis (Richards et al., 1998), and tobacco (Ezaki et al., 1995). PROTEINS RELATED TO TOLERANCE TO TEMPERATURE, ACID AND UV LIGHT STRESSES Optimum temperature for growth of C. violaceum ranges from 30 to 37°C, with a minimum of 1 to 15°C and a maximum of 40°C, but 20% of the strains can grow at 44°C (Sneath, 1984), and it has been isolated in Antarctica (Kriss et al., 1976). Marine teleosts from polar oceans can be protected from freezing in icy seawater by anti-freezing proteins or glycoproteins that act by binding to the ice crystals within the fish, preventing the growth of the crystals. A model has been proposed in which the protein binds to an ice nucleation structure, in a zipper-like fashion, via hydrogen bonding of threonine side chains (with an 11-residue period) to oxygen atoms in the ice lattice. The growth of ice crystals is thus stopped, or retarded, and the freezing point depressed (Chou, 1992; Chen et al., 1997). Thirty-nine ORFs showing anti-freezing domain proteins were detected in C. violaceum; these could play a role in mechanisms that allow the bacteria to survive low temperatures, or other kinds of stress, such as acid conditions or high temperature. Several genes related to heat shock proteins were also found in C. violaceum, including an operon of the DnaJ-DnaK-GrpE system (CV1645, 1643, 1642), the GroEL/GroES system (mopA and mopB, CV3233, 3232, CV4014, 4015), sigma factors (CV0585, CV2058, CV3332), the chaperonin ClpA/B system (CV3965), HscA/B co-chaperone (CV1089, CV1091), HtpG (CV1318), HtpX (CV3109, CV4263), HslO (CV2000), HslU (CV0402), and HslV (CV0401). These proteins may also be involved in adaptability to a wide range of temperatures. Although C. violaceum is described as being unable to grow below pH 5.0 (Sneath, 1984), the pH of waters of the Negro River ranges from 3.8 to 4.9 (Walker, 1990). Thus, genes such as the acid shock (asr) (CV4241) gene, which has a high similarity with the same gene in Enterobacter cloacae, may play an important role in survival under these conditions. Furthermore, as most Brazilian soils are acidic and this is often associated with Al toxicity, an Al resistance protein (CV3507) could have a protective role against the toxic levels of Al. This ORF is similar to the one found in Neisseria meningitidis (4e-54) and Bacillus halodurans (3e-52), to a protein associated with Al tolerance in Arthrobacter viscosus (1e-46) (Jo et al., 1997), and to a PP-loop superfamily ATPase that confers Al resistance in Clostridium acetobutylicum (Nolling et al., 2001). Heat shock proteins may also have protective activity under Al stress (Ezaki et al., 1998). Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br M. Hungria et al. 110 An efficient mechanism against UV light was found in the C. violaceum genome, including two copies of uvrA, uvrB, uvrC and uvrD genes (CV1893, CV3152, CV1305), and one copy of uvrD (CV0205). Additionally, the well-studied violacein operon (CV3274, 3273, 3272, 3271) (August et al., 2000) protects C. violaceum from radiation (Caldas et al., 1978). STARVATION GENES Several genes that promote survival under starvation conditions were detected in C. violaceum, some of them also being related to protection against oxidative damage. A DNAbinding stress protein (Dps, CV4253), already described in item Oxidative stress, is similar to the one found in Listeria innocua (Bozzi et al., 1997; Chiancone, 1997). In E. coli, Dps is synthesized during prolonged starvation in order to protect DNA from oxidative damage and from other stress conditions during the stationary phase. Studies performed in vitro showed that Dps forms extremely stable complexes with DNA, without apparent sequence specificity, while mutant cells lacking Dps develop dramatic changes in the pattern of proteins synthesized during starvation (Almiron et al, 1992). Two carbon starvation genes cstA and cstB were described in E. coli; it was suggested that cstA was involved in peptide utilization (Schultz and Matin, 1991). In C. violaceum only cstA was detected (CV1662, CV0762), as in P. aeruginosa. Stringent starvation proteins SspA (CV4005) and SspB (CV4004) were present in C. violaceum. In E. coli these proteins are synthesized for survival during growth and prolonged starvation conditions, being induced by glucose, nitrogen, phosphate or amino acid starvation (Williams et al., 1994). The stationary-phase survival protein (SurE, CV3679) has an important role in stationary-phase survival; E. coli mutants lacking this protein survived poorly under high temperatures and salt concentrations (Li et al., 1994). In E. coli and P. aeruginosa, surE is co-transcribed with pcm, a gene encoding the L-isoaspartyl protein repair methyltransferase, which probably plays a role in the repair and/or degradation of spontaneously damaged proteins. There is evidence of an interaction between pcm and surE to avoid protein damage (Fu et al., 1991; Li et al., 1994; Visick et al., 1998). Two ORFs similar to pcm (CV3680, CV0234) were detected in C. violaceum. The first one is apparently responsible for the co-transcription with surE, while the second could be related to co-transcription with DNA repair genes, since CV3680 is localized within the glp operon (CV0251, 0252, 0253, 0254), associated with cell protection against damage. Another survival protein precursor, a peptidyl-prolyl cis-trans isomerase (SurA, CV4230) was detected in C. violaceum. These molecular chaperones are required in the folding processes of proteins that are necessary to cross the plasma membrane and to be released into the periplasm in Gram-negative bacteria. The ORF CV2816 is probably related to PhoH, a cytoplasmic protein, and a predicted ATPase that is induced by phosphorus starvation. OTHER CELL PROTECTION STRATEGIES Other proteins involved in cell protection are present in C. violaceum (CV1796), such as OmlA, an outer membrane lipoprotein proposed to have the structural role of maintaining the cell envelope integrity under stress conditions (Oschsner et al., 1999). In P. aeruginosa, fur (ferric uptake regulation protein) overlaps with omlA (Oschner et al., 1999) and in Burkholderia both genes are co-localized, although in the latter bacterium the role of fur in iron uptake is not Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br Stress tolerance and adaptability of Chromobacterium violaceum 111 clear (Lowe et al., 2001). Similarly, in C. violaceum, the fur (CV1797) and omlA (CV1796) ORFs are adjacent. Genes that help protect against cell desiccation were also annotated in the genome of C. violaceum, including manA (mannose-6-phosphate isomerase, CV2312), manC (mannose1-phosphate guanylyltransferase, CV3178), ugd (UDP-glucose-6-dehydrogense, CV3041), galE (UDP-galactose-4-epimerase, CV3884) and galU (glucose-1-phosphate uridylyltransferase, CV3901). Another interesting gene detected in C. violaceum is mscL (CV1360). Although its function is still not well understood, similar proteins are found in several bacteria; in E. coli mscL encodes a channel that is opened by membrane stretch force, probably serving as an osmotic gauge, therefore transducing physical stresses at the cell membrane into an eletromagnetical response (Moe et al., 1998). The universal stress proteins are small cytoplasmic proteins whose expression is enhanced several-fold when cellular viability is challenged with heat shock, nutrient starvation, stress agents that arrest cell growth, or DNA-damaging agents. In E. coli these genes are induced by the stationary phase and by a variety of stresses causing growth arrest of cells, and they are not dependent on rpoS. Also in E. coli, Usp-mutants were found to be sensitive to UV light (Gustavsson and Nystrom, 2002). Genes encoding putative universal stress proteins were detected in C. violaceum (CV3769, CV4160, CV4192, CV4193); they might protect the bacterium from normal stress factors, such as exposure to UV light in aquatic environments. CONCLUSIONS Chromobacterium violaceum occurs abundantly in water environments in the Amazon region, as well as in soils and water of other tropical and subtropical regions. Most of those ecosystems are very poor on nutrients, and organisms are frequently submitted to environmental stresses, such as high temperature, UV exposure, and acidity. Several mechanisms that might be related to the remarkable adaptability of this bacterium were revealed in the complete sequencing of its genome. 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