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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
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Molecular Research
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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
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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
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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
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M. Hungria et al.
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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
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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
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M. Hungria et al.
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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
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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).
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M. Hungria et al.
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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. Alternative pathways for energy generation, an impressive number
of proteins related to transport, cell motility, and secretion, many proteins related to iron metabolism, osmotically inducible proteins, membrane porins, proteins related to tolerance against temperature, acid and UV stresses, among others, help explain its extraordinary competitiveness
and tolerance to stress.
ACKNOWLEDGMENTS
We thank the Ministério da Ciência e Tecnologia (MCT)/Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support.
REFERENCES
Almiron, M., Link, A.J., Furlong, D. and Kolter, R. (1992). A novel DNA-binding protein with regulatory
and protective roles in starved Escherichia coli. Genes Devel. 6: 2646-2654.
Andrews, S.C. (1998). Iron storage in bacteria. Adv. Microb. Physiol. 40: 281-351.
Armstrong, S.K., Pettis, G.S., Forrester, L.J. and McIntosh, M.A. (1989). The Escherichia coli enterobactin
biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product.
Mol. Microbiol. 3: 757-766.
August, P.R., Grossman, T.H., Minor, C., Draper, M.P., MacNeil, I.A., Pemberton, J.M., Call, K.M., Holt,
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br
M. Hungria et al.
112
D. and Osburne, M.S. (2000). Sequence analysis and functional characterization of the violacein
biosynthetic pathway from Chromobacterium violaceum. J. Mol. Microbiol. Biotechnol. 2: 513519.
Ballantine, J.A., Beer, R.J.S., Crutchley, D.J., Dodd, G.M. and Palmer, D.R. (1958). The synthesis of
violacein and related compounds. Proc. Chem. Soc.: 232-233.
Beckman, K.B. and Ames, B.N. (1998). The free radical theory of aging matures. Physiol Rev. 78: 547-581.
Blattner, F.R., Plunkett 3rd, G., Bloch, C.A., Perna, N.T., Burland, V., Riley, M., Collado-Vides, J.,
Glasner, J.D., Rode, C.K., Mayhew, G.F., Gregor, J., Davis, N.W., Kirkpatrick, H.A., Goeden, M.A.,
Rose, D.J., Mau, B. and Shao, Y. (1997). The complete genome of Escherichia coli K-12. Science
277: 1453-1474.
Boisbaudran, L. (1882). Matière colorante se formant dans la colle de farine. Comp. Rend. Acad. Sci. 94:
562-563.
Bozzi, M., Mignogna, G., Stefanini, S., Barra, D., Longhi, C., Valenti, P. and Chiancone, E. (1997). A novel
non-heme iron-binding ferritin related to the DNA-binding proteins of the Dps family in Listeria
innocua. J. Biol. Chem. 272: 3259-3265.
Brito, F.A., Carvalho, C.B., Santos, F., Gazzinelli, R.T., Oliveira, S.C., Azevedo, V. and Teixeira, S.M.R.
(2004). Chromobacterium violaceum genome: molecular mechanisms associated with pathogenicity. Gen. Mol. Res. 3: 148-161.
Cabiscol, E., Oiulats, E., Echave, P., Herrero, E. and Ros, J. (2000). Oxidative stress promotes specific
protein damage in Saccharomyces cerevisiae. J. Biol. Chem. 275: 27393-27398.
Cai, S. and Inouye, M. (2002). EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J. Biol.
Chem. 277: 24155-24161.
Calamita, G. (2000). The Escherichia coli aquaporin-Z water channel. Mol. Microbiol. 37: 254-262.
Calamita, G., Bishai, W.R., Preston, G.M., Guggino, W.B. and Agre, P. (1995). Molecular cloning and
characterization of AqpZ, a water channel from Escherichia coli. J. Biol. Chem. 270: 29063-29066.
Calamita, G., Kempf, B., Bonhivers, M., Bishai, W.R., Bremer, E. and Agre, P. (1998). Regulation of the
Escherichia coli water channel gene aqpZ. Proc. Natl. Acad. Sci. USA 95: 3627-3631.
Caldas, L.R. (1990). Um pigmento nas águas negras. Cienc. Hoje 11: 55-57.
Caldas, L.R., Leitão, A.A.C., Santos, S.M. and Tyrrell, R.M. (1978). Preliminary experiments on the photobiological properties of violacein. In: Proceedings of the International Symposium on Current
Topics in Radiology and Photobiology (Tyrrell, R.M., ed.). Academia Brasileira de Ciências, Rio de
Janeiro, RJ, Brazil, pp.121-126.
Chen, L., Devries, A.L. and Cheng, C.H. (1997). Convergent evolution of antifreeze glycoproteins in
Antarctic notothenioid fish and Artic cod. Proc. Natl. Acad. Sci. USA 94: 3817-3822.
Chen, S., Rosner, M.H. and Calvo, J.M. (2001). Leucine-regulated self-association of leucine-responsive
regulatory protein (Lrp) from Escherichia coli. J. Mol. Biol. 312: 625-635.
Chiancone, E. (1997). A novel non-heme iron-binding ferritin related to the DNA-binding proteins of the
Dps family in Listeria innocua. J. Biol. Chem. 272: 3259-3265.
Choi, H., Kim, S., Mukhopadhyay, P., Cho, S., Woo, J., Storz, G. and Ryu, S. (2001). Structural basis of the
redox switch in the OxyR transcription factor. Cell 105: 103-113.
Chou, K.C. (1992). Energy-optimized structure of antifreeze protein and its binding mechanism. J. Mol.
Biol. 223: 509-517.
Chrispeels, M.J. and Agre, P. (1994). Aquaporins: water channel proteins of plant and animal cells. Trends
Biochem. Sci. 19: 421-425.
Coderre, P.E. and Earhart, C.F. (1989). The entD gene of the Escherichia coli K12 enterobactin gene
cluster. J. Gen. Microbiol. 135: 3043-3055.
Cole, S.T., Brosch, R., Parkhill, J., Garnier, T., Churcher, C., Harris, D., Gordon, S.V., Eiglmeier, K.,
Gas, S., Barry 3rd, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor,
R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K.A.,
Krogh, A., McLean J., Moule, S., Murphy, L., Oliver, K., Osborne, J., Quail, J.M.A., Rajandream,
M.-A., Rogers, J., Rutter, S., Seeger, K., Skelton, J., Squares, R., Squares, S., Sulston, J.E., Taylor,
K., Whitehead, S. and Barrell, B.G. (1998). Deciphering the biology of Mycobacterium tuberculosis
from the complete genome. Nature 393: 537-544.
Creczynski-Pasa, T.B. and Antônio, R.V. (2004). Energetic metabolism of Chromobacterium violaceum.
Gen. Mol. Res. 3: 162-166.
Crosa, J.H. (1989). Genetics and molecular biology of siderophore-mediated iron transport in bacteria.
Microbiol. Rev. 53: 517-530.
Doyle, D.A., Morais Cabral, J., Pfuetzner, R.A., Quo, A., Gulbis, J.M., Cohen, S.L., Chait B.T. and
MacKinnon, R. (1998). The structure of the potassium channel: Molecular basis of K+ conduction
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br
Stress tolerance and adaptability of Chromobacterium violaceum
113
and selectivity. Science 280: 69-77.
Durán, N. (1990). Violaceína: a descoberta de um antibiótico. Cienc. Hoje 11: 58-60.
Durán, N., Erazo, S. and Campos, V. (1983). Bacterial chemistry-II: antimicrobial photoproduct from pigment of Chromobacterium violaceum. An. Acad. Bras. Cienc. 55: 231-234.
Durán, N., Campos, V., Riveros, R., Joyas, A., Pereira, M.F. and Haun, M. (1989). Bacterial chemistry. III.
Preliminary studies on the trypanosomal activities of Chromobacterium violaceum products. An.
Acad. Bras. Cienc. 61: 31-36.
Durán, N., Antonio, R.V., Haun, M. and Pilli, R.A. (1994). Biosynthesis of a trypanocide by Chromobacterium violaceum. World J. Microbiol. Biotechnol. 10: 686-690.
Ezaki, B., Yamamoto, Y. and Matsumoto, H. (1995). Cloning and sequencing of the cDNAs induced by
aluminum treatment and Pi-starvation in cultured tobacco cells. Physiol. Plant. 93: 11-18.
Ezaki, B., Gardner, R.C., Ezaki, Y., Kondo, H. and Matsumoto, H. (1998). Protective roles of two aluminum
(Al)-induced genes, hsp150 and sed1 of Saccharomyces cerevisiae, in Al and oxidative stress.
FEMS Microbiol. Lett. 159: 99-105.
Fu, J.C., Ding, L. and Clarke, S. (1991). Purification, gene cloning, and sequence analysis of an Lisoaspartyl protein carboxyl methyltransferase from Escherichia coli. J. Biol. Chem. 266: 1456214572 (Erratum in J. Biol. Chem. 267: 11660).
Fuangthong, M. and Helmann, J.D. (2002). The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic acid derivative. Proc. Natl. Acad. Sci. USA 99: 6690-6695.
Fuangthong, M., Atichartpongkul, S., Mongkolsuk, S. and Helmann, J.D. (2001). OhrR of ohrA, a key
organic hydroperoxide resistance determinant in Bacillus subtilis. J. Bacteriol. 183: 4134-4141.
Fuentes, A.M., Diaz-Mejia, J.J., Maldonado-Rodriguez, R. and Amabile-Cuevas, C.F. (2001). Differential
activities of the SoxR protein of Escherichia coli: SoxS is not required for gene activation under iron
deprivation. FEMS Microbiol. Lett. 201: 271-275.
Fushimi, K., Bai, L., Marumo, F. and Sasaki, S. (1997). Isolation of a gene encoding nodulin-like intrinsic
protein of Escherichia coli. Biochem. Mol. Biol. Int. 41: 995-1003.
Goecke, M., Gallant, C., Suntharalingam, P. and Martin, N.L. (2002). Salmonella typhimurium DsbA is
growth-phase regulated. FEMS Microbiol. Lett. 206: 229-234.
Gustavsson, N. and Nystrom, T. (2002). The universal stress protein paralogues of Escherichia coli are
co-ordinately regulated and co-operate in the defense against DNA damage. Mol. Microbiol. 43:
107-117.
Hancock, R.E., Alm, R., Bina, J. and Trust, T. (1998). Helicobacter pylori: a surprisingly conserved
bacterium. Nat. Biotechnol. 16: 216-217.
Hantash, F.M. and Earhart, C.F. (2000). Membrane association of the Escherichia coli enterobactin
synthase proteins EntB/G, EntE, and EntF. J. Bacteriol. 182: 1768-1773.
Heller, K.B., Lin, E.C. and Wilson, T.H. (1980). Substrate specificity and transport properties of glycerol
facilitator of Escherichia coli. J. Bacteriol. 144: 274-278.
Jimenez-Arribas, G., Leautaud, V. and Amabile-Cuevas, C.F. (2001). Regulatory locus soxRS partially
protects Escherichia coli against ozone. FEMS Microbiol. Lett. 195: 175-177.
Jo, J., Jang, Y.S., Kim, K.Y., Kim, I.J. and Chung, W.I. (1997). Isolation of Alu1-P gene encoding a protein
with aluminum tolerance activity from Arthrobacter viscosus. Biochem. Biophys. Res. Commun. 239:
835-839.
Jung, J.U., Gutierrez, C., Martin, F., Ardourel, M. and Villarejo, M. (1990) Transcription of osmB, a gene
encoding an Escherichia coli lipoprotein, is regulated by dual signals. Osmotic stress and stationary phase. J. Biol. Chem. 265: 10574-10581.
Kammler, M., Schon, C. and Hantke, K. (1993). Characterization of the ferrous iron uptake system of
Escherichia coli. J. Bacteriol. 175: 6212-6219.
Klebba, P.E., Rutz, J.M., Liu, J. and Murphy, C.K. (1993). Mechanisms of TonB-catalyzed iron transport
through the enteric bacterial cell envelop. J. Bioenerg. Biomembr. 25: 603-611.
Kriss, A.E., Mitskevich, I.N., Rozanova, E.P. and Osnitskaia, L.K. (1976). Microbiological studies of the
Wanda Lake (Antarctica). Mikrobiologiia 45: 1075-1081.
Kunst, F., Ogasawara, N., Moszer, I., Albertini, A.M., Alloni, G., Azevedo, V., Bertero, M.G., Bessieres, P.,
Bolotin, A., Borchert, S., Borriss, R., Boursier, L., Brans, A., Braun, M., Brignell, S.C., Bron, S.,
Brouillet, S., Bruschi, C.V., Caldwell, B., Capuano, V., Carter, N.M., Choi, S.K., Codani, J.J.,
Connerton, I.F., Cummings, N.J., Daniel, R.A., Denizot, F., Devine, K.M., Düsterhöft, A., Ehrlich,
S.D., Emmerson, P.T., Entian, K.D., Errington, J., Fabret, C., Ferrari, E., Foulger, D., Fritz, C.,
Fujita, M., Fujita, Y., Fuma, S., Galizzi, A., Galleron, N., Ghim, S.-Y., Glaser, P., Goffeau, A., Golightly,
E.J., Grandi, G., Guiseppi, G., Guy, B.J., Haga, K., Haiech, J., Harwood, C.R., Hénaut, A., Hilbert, H.,
Holsappel, S., Hosono, S., Hullo, M.-F., Itaya, M., Jones, L., Joris B., Karamata, D., Kasahara, Y.,
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br
M. Hungria et al.
114
Klaerr-Blanchard, M., Klein, C., Kobayashi, Y., Koetter, P., Koningstein, G., Krogh, S., Kumano, M.,
Kurita, K., Lapidus, A., Lardinois, S., Lauber, J., Lazarevic, V., Lee, S.-M., Levine, A., Liu, H.,
Masuda, S., Mauël, C., Médigue, C., Medina, N., Mellado, R.P., Mizuno, M., Moestl, D., Nakai, S.,
Noback, M., Noone, D., O’Reilly, M., Ogawa, K., Ogiwara, A., Oudega, B., Park, S.-H., Parro, V.T.,
Pohl, M., Portetelle, D., Porwollik, S.A., Prescott, M., Presecan, E., Pujic, P., Purnelle, B., Rapoport,
G., Rey, M., Reynolds, S., Rieger, M., Rivolta, C., Rocha, E., Roche, B., Rose, M., Sadaie, Y., Sato, T.,
Scanlan, E., Schleich, S., Schroeter, R., Scoffone, F., Sekiguchi, J., Sekowska, A., Seror, S.J.,
Serror, P., Shin, B.-S., Soldo, B., Sorokin, A., Tacconi, E., Takagi T., Takahashi, H., Takemaru, K.,
Takeuchi, M., Tamakoshi, A., Tanaka, T., Terpstra, P., Tognoni, A., Tosato, V., Uchiyama, S., Vandenbol,
M., Vannier, F., Vassarotti, A., Viari, A., Wambutt, R., Wedler, E., Wedler, H., Weitzenegger, T.,
Winters, P., Wipat, A., Yamamoto, H., Yamane, K., Yasumoto, K., Yata, K., Yoshida, K., Yoshikawa,
H.-F., Zumstein, E., Yoshikawa, H. and Danchin, A. (1997). The complete genome sequence of the
gram-positive bacterium Bacillus subtilis. Nature 390: 249-256.
Landini, P. and Busby, S.J. (1999). Expression of the Escherichia coli ada regulon in stationary phase:
evidence for rpoS-dependent negative regulation of alkA transcription. J. Bacteriol. 181: 6836-6839.
Landini, P., Hajec, L.I., Nguyen, L.H., Burgess, R.R. and Volkert, M.R. (1996). The leucine-responsive
regulatory protein (Lrp) acts as a specific repressor for sigma s-dependent transcription of the
Escherichia coli aidB gene. Mol. Microbiol. 20: 947-955.
Lankford, C.E. (1973). Bacterial assimilation of iron. Crit. Rev. Microbiol. 2: 273-331.
Leon, L.L., Miranda, C.C., Souza, A.O. de and Durán, N. (2001). Antileishmanial activity of the violacein
extracted from Chromobacterium violaceum. J. Antimicrob. Chemother. 48: 449-450.
Li, C., Ichikawa, J.K., Ravetto, J.J., Kuo, H.C., Fu, J.C. and Clarke, S. (1994). A new gene involved in
stationary-phase survival located at 59 minutes on the Escherichia coli chromosome. J. Bacteriol.
176: 6015-6022. (Erratum in J. Bacteriol 178: 2159).
Liu, J., Duncan, K. and Walsh, C.T. (1989). Nucleotide sequence of a cluster of Escherichia coli
enterobactin biosynthesis genes: identification of entA and purification of its product 2,3-dihydro2,3-dihydroxybenzoate dehydrogenase. J. Bacteriol. 171: 791-798.
Lowe, C.A., Asghar, A.H., Shalom, G., Shaw, J.G. and Thomas, M.S. (2001). The Burkholderia cepacia fur
gene: co-localization with omlA and absence of regulation by iron. Microbiology 147: 1303-1314.
Lu, A.L., Li, X., Gu, Y., Wright, P.M. and Chang, D.Y. (2001). Repair of oxidative DNA damage: mechanisms and functions. Cell Biochem. Biophys. 35: 141-170.
Manchado, M., Michán, C. and Pueyo, C. (2000). Hydrogen peroxide activates the SoxRS regulon in vivo.
J. Bacteriol. 182: 6842-6844.
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.
Michán, C., Manchado, M. and Pueyo, C. (2002). SoxRS down-regulation of rob transcription. J. Bacteriol.
184: 4733-4738.
Moe, P.C., Blount, P. and Kung, C. (1998). Functional and structural conservation in the mechanosensitive
channel MscL implicates elements crucial for mechanosensation. Mol. Microbiol. 28: 583-591.
Nachin, L., El Hassouni, M., Loiseau, L., Expert, D. and Barras, F. (2001). SoxR-dependent response to
oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase.
Mol. Microbiol. 39: 960-972.
Nahlik, M.S., Brickman, T.J., Ozenberger, B.A. and McIntosh, M.A. (1989). Nucleotide sequence and
transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and
entA. J. Bacteriol. 171: 784-790.
Nikaido, H. (1994). Porins and specific diffusion channels in bacterial outer membranes. J. Biol. Chem.
269: 3905-3908.
Nolling, J., Breton, G., Omelchenko, M.V., Markarova, K.S., Zeng, Q., Gibson, R., Lee, H.M., Dubois, J.,
Qiu, D., Hitti, J., Wolf, Y.I., Tatusov, R.L., Sabathe, F., Doucette-Stamm, L., Soucaille, P., Daly, M.J.,
Bennett, G.N., Koonin, E.V. and Smith, D.R. (2001). Genome sequence and comparative analysis of
the solvent-producing bacterium Clostridium acetobutylicum. J. Bacteriol. 183: 4823-4838.
Nunoshiba, T., Hidalgo, E., Amabile Cuevas, C.F. and Demple, B. (1992). Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the
soxS regulatory gene. J. Bacteriol. 174: 6054-6060.
Oschsner, U.A., Vasil, A.I., Hohnson, Z. and Vasil, M.L. (1999). Pseudomonas aeruginosa fur overlaps
with a gene encoding a novel membrane lipoprotein, OmlA. J. Bacteriol. 181: 1099-1109.
Payne, S.M. (1994). Detection, isolation, and characterization of siderophores. Methods Enzymol. 235:
329-344.
Pereira, M., Parente, J.A., Bataus, L.A.M., Cardoso, D.D.P., Soares, R.B.A. and Soares, C.M.A. (2004).
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br
Stress tolerance and adaptability of Chromobacterium violaceum
115
Chemotaxis and flagellar genes of Chromobacterium violaceum. Gen. Mol. Res. 3: 92-101.
Pomposiello, P.J. and Demple, B. (2001). Redox-operated genetic switches: the SoxR and OxyR transcription factors. Trends Biotechnol. 19: 109-114.
Poole, K. and McKay, G.A. (2003). Iron acquisition and its control in Pseudomonas aeruginosa: many
roads lead to Rome. Front Biosci. 8: 661-686.
Postle, K. (1993). TonB protein and energy transduction between membranes. J. Bioenerg. Biomembr. 25:
591-601.
Preston, G.M., Carroll, T.P., Guggino, W.B. and Agre, P. (1992). Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein. Science 256: 385-387.
Qin, L., Cai, S., Zhu, Y. and Inouye, M. (2003). Cysteine-scanning analysis of the dimerization domain of
envZ, an osmosensing histidine kinase. J. Bacteriol. 185: 3429-3435.
Richards, K.D., Schott, E.J., Sharma, Y.K., Davis, K.R. and Gardner, R.C. (1998). Aluminum induces
oxidative stress genes in Arabidopsis thaliana. Plant Physiol. 116: 408-419.
Schultz, J.E. and Matin, A. (1991). Molecular and functional characterization of a carbon starvation gene
of Escherichia coli. J. Mol. Biol. 218: 129-140.
Silakowski, B., Kunze, B., Nordsiek, G., Blocker, H., Hofle, G. and Muller, R. (2000). The myxochelin iron
transport regulon of the myxobacterium Stigmatella aurantiaca Sg a15. Eur. J. Biochem. 267: 64766485.
Silva, R., Araripe, J.R., Edson Rondinelli, E. and Ürményi, T.P. (2004). Gene expression in Chromobacterium violaceum. Genet. Mol. Res. 3: 64-75.
Sneath, P.H.A. (1984). Genus Chromobacterium Bergonzini 1881, 153AL. In: Bergey’s Manual of Systematic Bacteriology (Krieg, N.H. and Holt, J.G., eds.). Vol. I. Williams & Wilkins, Baltimore, MD, USA,
pp. 580-582.
Souza, A.O. de, Aily, D.C.G., Sato, D.N. and Durán, N. (1999). Atividade da violaceína in vitro sobre o
Mycobacterium turbeculosis H37RA. Rev. Inst. Adolfo Lutz 58: 59-62.
Storz, G. and Imlay, J.A. (1999) Oxidative stress. Curr. Opin. Microbiol. 2: 188-194.
Stover, C.K., Pham, X.Q., Erwin, A.L., Mizoguchi, S.D., Warrener, P., Hickey, M.J., Brinkman, F.S.,
Hufnagle, W.O., Kowalik, D.J., Lagrou, M., Garber, R.L., Goltry, L., Tolentino, E., WestbrockWadman, S., Yuan, Y., Brody, L.L., Coulter, S.N., Folger, K.R., Kas, A., Larbig, K., Lim, R., Smith, K.,
Spencer, D., Wong, G.K., Wu, Z., Paulsen, I.T., Reizer, J., Saier, M.H., Hancock, R.E., Lory, S. and
Olson, M.V. (2000). Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406: 959-964.
Strong, F.M. (1944). Isolation of violacein. Science 100: 287.
Sukchawalit, R., Loprasert, S., Atichartpongkul, S. and Mongkolsuk, S. (2001). Complex regulation of
the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves OhrR, a novel organic
peroxide-inducible negative regulator, and posttranscriptional modifications. J. Bacteriol. 183: 44054412.
Takase, H., Nitanai, H., Hoshino, K. and Otani, T. (2000). Requirement of the Pseudomonas aeruginosa
tonB gene for high-affinity iron acquisition and infection. Infect. Immun. 68: 4498-4504.
Touati, D. (2000). Sensing and protecting against superoxide stress in Escherichia coli - how many ways
are there to trigger soxRS response? Redox Rep. 5: 287-293.
Vasconcelos, A.T.R., Almeida, D.F., Hungria, M., Guimarães, C.T., Antônio, R.V., Almeida, F.C., Almeida,
L.G.P., Almeida, R., Alves-Gomes, J.A., Andrade, E.M., 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., Bogo, M.,
Bonatto, S., Bordignon, J., Brigido, M.M., 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., Cunha-Junior, N.C., 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., Hanna, E.S., Jardim, S.N., Laurino, J., Leoi, L.C.T., Lima, L.F.A., Loureiro, M.F.,
Lyra, M.C.C.P., Madeira, H.M.F., Manfio, G.P., Maranhão, A.Q., Martins, W.S., Mauro, S.M.Z.,
Medeiros, S.R.B., Meissner, R.V., Moreira, M.A.M., Nascimento, F.F., Nicolás, 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.P., 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.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). The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability. Proc. Natl. Acad. Sci.
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br
M. Hungria et al.
116
USA 100: 11660-11665.
Visick, J.E., Ichikawa, J.K. and Clarke, S. (1998). Mutations in the Escherichia coli surE gene increase
isoaspartyl accumulation in a strain lacking the pcm repair methyltransferase but suppress stresssurvival phenotypes. FEMS Microbiol. Lett. 167: 19-25.
Vuilleumier, S. (1997). Bacterial glutathione S-transferases: what are they good for? J. Bacteriol. 179:
1431-1441.
Walker, I. (1990). Ecologia e biologia dos igapós e iguarapés. Cienc. Hoje 11: 44-53.
Williams, M.D., Ouyang, T.X. and Flickinger, M.C. (1994). Starvation-induced expression of SspA and
SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival
during growth and prolonged starvation. Mol. Microbiol. 11: 1029-1043.
Wyckoff, E.E., Stoebner, J.A., Reed, K.E. and Payne, S.M. (1997). Cloning of a Vibrio cholera vibriobactin
gene cluster: identification of genes required for early steps in siderophore synthesis. J. Bacteriol.
179: 7055-7062.
Genetics and Molecular Research 3 (1): 102-116 (2004) www.funpecrp.com.br