Download Catalases and PhoB/PhoR system independently contribute to

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Catalases and PhoB/PhoR system independently contribute to oxidative stress resistance in
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Vibrio cholerae O1
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Carolina L. Goulart§*, Livia C. Barbosa§, Paulo M. Bisch and Wanda M.A. von Krüger
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Laboratório de Física Biológica, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal
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do Rio de Janeiro, Rio de Janeiro, Brazil
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§
Both authors contributed equally to this work.
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*Corresponding author. Mailing address: Laboratório de Física Biológica, Universidade Federal do
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Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, G1-043, Rio de Janeiro – RJ, CEP 21941-902,
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Brazil; E-mail: [email protected]; Tel.: +55 21 3938-6531; fax: +55 21 2280-8193
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Keywords: KatB, KatG, phosphate
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Running title: Oxidative stress resistance in Vibrio cholerae
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Subject category: Physiology and metabolism
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Word count: Abstract – 245 words
Main text – 3919 words
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Abstract
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All cells are subjected to oxidative stress, a condition in which reactive oxygen species (ROS)
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production exceeds elimination. Bacterial defenses against ROS include synthesis of antioxidant
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enzymes like peroxidases and catalases. Vibrio cholerae can produce two distinct catalases, KatB
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and KatG, which contribute to ROS homeostasis. In this study, we analyzed the mechanism behind
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katG and katB expression in two V. cholerae O1 pandemic strains, O395 and N16961, respectively
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of Classical and El Tor biotypes. Both strains express these genes, especially at stationary phase.
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However, El Tor N16961 produces higher KatB and KatG levels and is much more resistant to
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peroxide challenge than the Classical strain, confirming a direct relationship between catalase
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activity and oxidative stress resistance. Moreover, we showed that katG and katB expression levels
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depend on inorganic phosphate (Pi) availability, in contrast to other bacterial species. In N16961,
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katB and katG expression is reduced under Pi limitation relative to Pi abundance. Total catalase
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activity in N16961 and its phoB mutant cells was similar, independently of growth conditions,
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indicating that PhoB/PhoR system is not required for katB and katG expression. However, N16961
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cells from Pi-limited cultures were 50-100 fold more resistant to H2O2 challenge and accumulated
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less ROS than phoB mutant ones. Together, these findings suggest that besides KatB and KatG,
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PhoB/PhoR system is an important protective factor against ROS in V. cholerae N16961. They also
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corroborate previous results from our and other groups, suggesting that PhoB/PhoR system is
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fundamental for V. cholerae biology.
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Introduction
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Under aerobic conditions, all cells produce reactive oxygen species (ROS) as aerobic metabolism
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by-products. Bacteria can also be exposed to high levels of ROS generated by the host defense
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system (Vallet-Gely et al., 2008) or in response to unfavorable environmental conditions (Shimizu,
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2013). ROS accumulation can lead to oxidative stress, which can damage macromolecules such as
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nucleic acids, proteins and lipids, and to avoid such harmful effects bacteria have evolved
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mechanisms to sense ROS levels and to counteract their effects (Lushchak, 2011a).
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Bacterial ROS-sensing mechanisms involve a number of transcription factors that regulate
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antioxidant enzyme expression (Dubbs & Mongkolsuk, 2012). These enzymes include superoxide
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dismutases (SOD), which catalyzes superoxide anion (O2-˙) dismutation to H2O2, and
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hydroperoxidases (catalases and peroxidases) that cleave peroxidic bond in H2O2 and also in
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organic peroxides (Lushchak, 2011b). Hydroperoxidase gene expression regulation is well studied
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in Escherichia coli, a Gram-negative bacterium that produces two catalases. HPI, also known as
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KatG, is a bifunctional catalase-peroxidase whose expression is H2O2-induced in an OxyR-
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dependent manner (Storz et al., 1990). E. coli OxyR is a well-characterized bacterial redox-sensing
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transcription factor that activates a regulon of more than 20 antioxidant genes. HPII, also known as
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KatE, is a typical catalase whose gene is controlled by RpoS, the primary regulator of stationary
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phase genes, and is not induced by H2O2 (reviewed in (Schellhorn, 1995). Both KatG and KatE are
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involved in oxidative stress response in E. coli (Jung & Kim, 2003).
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Vibrio cholerae is a Gram-negative bacterium that can be found in aquatic environments, especially
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in brackish water, either free-living or associated with biotic or abiotic surfaces (Colwell, 1996).
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Several strains are facultative pathogens that can colonize human small intestine and cause watery
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diarrheic disease known as cholera (Finkelstein, 1996). Seven cholera pandemics have been
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reported, all caused by V. cholerae O1 serogroup strains (Salim et al., 2005). Some V. cholerae
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strains can produce two different catalases, KatG (VC1560) and KatB (VC1585), both of which
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contribute to their oxidative stress resistance (Wang et al., 2012). However, little is known about
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katG and katB expression regulation in this species. It has been shown that in V. cholerae El Tor
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biotype strain C7258 katB is repressed by the histone-like nucleoid structuring protein (H-NS) by
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transcriptional silencing (Silva et al., 2008). Differently from E. coli, in V. cholerae El Tor strain
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C6706, KatB and KatG production is independent of OxyR (Wang et al., 2012).
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Under natural conditions, aquatic environments are usually poor in nutrients such as phosphorus, an
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element essential for all forms of life. V. cholerae survival in such habitat depends on its ability to
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cope with phosphorous deficiency. Inorganic phosphate (Pi) is the main source of P for bacteria and
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the maintenance of bacterial Pi homeostasis is controlled by the two-component system PhoB/PhoR
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(Wanner, 1993). This system regulates expression of genes involved in Pi uptake and metabolism
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and in many other processes, including pathogenesis (Goulart et al., 2010; Osorio et al., 2004; von
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Kruger et al., 1999; von Kruger et al., 2006). More recently, we demonstrated for the first time that
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V. cholerae O1 PhoB/PhoR system is also required for full growth and RpoS accumulation at
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stationary phase culture under high Pi level, indicating that it is a fundamental system for bacterium
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biology (Lery et al., 2013). In some bacterial species, Pi limitation induces catalase expression in a
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PhoB-dependent manner, increasing their ability to cope with oxidative stress in such environments
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(Yuan et al., 2005). In previous work, we identified many V. cholerae stress-response related gene
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products whose expression is dependent on PhoB/PhoR and/or Pi levels (von Kruger et al., 2006).
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Although catalases were not among the proteins identified in the mentioned study, we cannot
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discard a relationship between kat genes expression and PhoB/PhoR or Pi availability in V.
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cholerae. This was investigated in the present study using two V. cholerae O1 isolates, the 6th
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pandemic classical biotype O395 and the 7th pandemic El Tor biotype N16961 (Salim et al., 2005).
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Our results led us to conclude that KatG and KatB seem to be involved in oxidative stress response
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in both strains, confirming previous results (Wang et al., 2012). However, differences were found
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between strains O395 and N16961 with respect to catalase genes expression profile and total
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catalase activity. Moreover, in contrast to the observed in other bacterial species (Yuan et al.,
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2005), katB and katG expression in V. cholerae N16961 is repressed under Pi limitation in a PhoB-
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independent manner. However, in this condition, an active PhoB/PhoR system is required for wild-
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type resistance to ROS.
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Material and Methods
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Strains and growth conditions: Vibrio cholerae spontaneous streptomycin-resistant O1 strains El
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Tor N16961, classical O395 and WK10, an N16961 isogenic phoB/phoR mutant strain (von Kruger
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et al., 1999; von Kruger et al., 2006), were routinely grown at 37°C in Luria-Bertani broth (LB)
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under aeration or on solid medium (1.5% agar in LB). For growth under defined Pi concentrations,
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MG (165 mM MOPS pH7.4; 80 mM NaCl; 20 mM KCl; 20 mM NH4Cl; 3 mM Na2SO4; 1 mM
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MgCl2; 200 M CaCl2; 3 M FeCl3; 0.4% glucose; 10 M thiamine) was used with KH2PO4 at 65
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mM (high phosphate medium MGHP) or 65 M (low phosphate medium MGLP) (von Kruger et
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al., 1999). For genetic manipulations, E. coli Top10 (Invitrogen) was used. Antibiotics were added
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to media when required at following concentrations: streptomycin 100 g ml-1, ampicillin 100 g
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ml-1 and kanamycin 50 g ml-1. Bacterial growth was spectrophotometrically determined by
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following OD600nm for 8 hours.
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Genetic procedures: Fragments of 1.7 kb and 2.1 kb containing, respectively, V. cholerae N16961
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katB (vc1585) and katG (vc1560) genes were PCR amplified using N16961 strain chromosomal
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DNA as template and primers KatBADFw-KatBADRv and KatGBADFw-KatGBADRv listed in
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Table 1. Fragments were inserted into pBAD TOPO® (Invitrogen), downstream araBAD promoter,
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according to supplier’s recommendations, to generate arabinose-inducible overexpression systems,
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pAB-katB and pBAD-katG. katB and perA genes from V. cholerae N16961 are 99% identical to the
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same genes in O395 strain.
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Fragments 300bp long of katB regulatory region in N16961 and O395 were PCR-amplified from
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the chromosomal DNA from each strain using primers KatBpro1 and KatBpro2 (Table 1). The
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fragments were digested with Nco I and Xho I and ligated to pIC552 (Macian et al., 1994) digested
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with the same enzymes, to create plasmids pkBN16 and pkBO395. These contained katB regulatory
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region from each strain fused to lacZ gene sequence. All constructions were confirmed by digestion
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and sequencing.
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Induction of catalase genes expression. To induce catalase genes expression from pAB-katB and
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pBAD-katG, plasmids were transformed into O395 competent cells prepared as previously
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described (von Kruger et al., 1999). Transformants were grown in LB till mid-exponential phase
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(OD600nm 0.5-0.7) and arabinose at 0.002%, 0.02% or 0.2% was added to three separate cultures.
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Glucose 0.2% was added to another culture to repress kat genes expression from araBAD promoter
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(negative control). All cultures were incubated for a further 4h at 37˚C under aeration before
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catalase activity was assayed.
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Enzyme assays. Catalase activity was determined in solution and in-gel after non-denaturing
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polyacrylamide gel electrophoresis. For both procedures, V. cholerae cells were cultivated in LB to
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mid-exponential (OD600nm 0.5-0.7) or stationary (OD600 > 2.5) phase or in MGHP and MGLP for
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10h. Cells in a volume corresponding to OD600nm 6 were collected by centrifugation (16,000 x g for
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15 min at 4°C), suspended in 500 μL 50 mM potassium phosphate buffer pH 7.0 containing 5 mM
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EDTA, 10% glycerol and 25 μM PMSF and disrupted by sonication. Unbroken cells and large
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aggregates were removed by centrifugation as above. Lysate protein concentration was determined
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with Bio-Rad Protein Assay Dye Reagent.
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To measure catalase activity in solution, a lysate volume containing a minimum of 12.5 μg proteins
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was diluted in 1mL water to which 500 μL 59 mM H2O2 in 50 mM potassium phosphate buffer
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pH7.0 was added. H2O2 decomposition was monitored spectrophotometrically by measuring
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OD240nm for 3 min. The exponential decay of OD240nm per minute (ΔOD240nm/min) was determined
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and catalase units (U) were defined as (ΔOD240nm/min) x 1000 x 43.6-1 (43.6 represents ε value for
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H2O2 in mM-1 cm-1).
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Native PAGE was used to detect protein bands with catalase activity. Briefly, proteins lysate (50-
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100 μg) was resolved by non-denaturing 4-8% polyacrylamide gradient gel electrophoresis after
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which the gel was stained specifically for catalase activity using potassium ferricyanide solution
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(Woodbury et al., 1971).
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β-galactosidase activities were measured in cell lysates, as previously described (Miller, 1972) and
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expressed in U x μg protein-1 (Protein Assay Dye Reagent, Bio-Rad).
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Mass spectrometry protein identification: Whole cell lysates (30 μg of proteins) were analyzed
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by SDS-PAGE (12.5%). Gel was Coomassie Brilliant Blue-stained and protein bands were cut from
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gel, trypsin digested (Shevchenko et al., 1996) and analyzed by MS ESI-Q-Tof Micro (Waters
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Corporation, USA), coupled to a NanoUPLC (Nano Ultra Performance Liquid Chromatography-
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nanoACQUITY Waters), as recommended by MIAPE-Minimum Information About Proteomics
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Experiment (Taylor et al., 2007). Raw data were processed using the ProteinLynx 2x4 and proteins
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were identified as described (von Kruger et al., 2006).
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Bacterial survival assays after H2O2 treatment: Aliquots of 500 μL the cultures used for catalase
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assay were incubated with H2O2 (15 or 30 mM, depending on strain and growth condition) for up to
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20 min, at 37C, without aeration. Cell survival was calculated by determining CFU ml-1 after
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challenge in comparison to non-treated culture (control, 100%).
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ROS detection: Intracellular ROS levels were measured using dihydrodichlorofluorescein diacetate
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(DCFH-DA). N16961 and WK10 were grown for up to 10 hours in MGHP or MGLP. Aliquots of
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10 μL and 100 μL of the cultures in MGHP and MGLP, respectively, were harvested by
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centrifugation (16,000 x g for 4 min at room temperature) and ressuspended in 1 mL 0.9 % NaCl.
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The suspensions were diluted and plated onto LB-agar 1.5% containing streptomycin 100 g ml-1
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for viable CFU determination. DCFH-DA at a final concentration of 10 μM was then added to the
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bacterial suspension, followed by 30 min incubation at 37 °C. Fluorescence was measured at
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485/530 nm excitation/emission using a microplate reader (Victor3; PerkinElmer) and the results
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were expressed in fluorescence intensity x 1000 / CFU ml-1.
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Results
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Expression of katG and katB in V. cholerae O1 N16961 and O395 is growth-phase dependent
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and varies between the strains
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Genome analysis revealed that classical O395 and El Tor N16961 V. cholerae O1 strains have two
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distinct catalase genes, katG (VC1560) and katB (VC1585), which encode, respectively, KatG and
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KatB. Previous study has shown that both proteins contribute to oxidative stress resistance in E1
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Tor strain C6706 (Wang et al., 2012). However, nothing is known about the expression and
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functions of these genes in the pandemic strains O395 and N16961. In order to investigate whether
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O395 and N16961 produce catalases, cells were grown in rich medium (LB) and enzyme total
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activity was measured in exponential and stationary phase cells (Fig. 1(a) and (b)). In N16961, total
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catalase activity was growth-stage dependent, being higher at stationary phase cells. O395 cells, on
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the other hand displayed lower and similar catalase activity in both culture phases.
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N16961 and O395 exponential and stationary phase whole-cell lysates were also subjected to non-
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denaturing polyacrylamide gel electrophoresis followed by in-gel catalase activity staining
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(Woodbury et al., 1971). Two catalase activity regions (I and II) exhibiting distinct migration
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patterns were detected on gel lanes corresponding to N16961 and O395 stationary-phase cells.
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However, both catalase bands from N16961 were stronger than those from O395 (Fig. 1(b)).
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Exponential cells catalase electrophoretic pattern, on the other hand, differed between strains. While
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N16961 presented only the slow-migrating catalase (band I), no activity was detected in O395
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exponential cells, under conditions analyzed (Fig. 1(c)). This can be explained by the fact that the
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gel staining is less sensitive than the catalase quantitative assay, and also because enzyme activity
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in exponential O395 cells seems to be highly variable (Fig. 1(b)).
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To identify the catalases, bands of high enzymatic activity (I and II from N16961 stationary-phase
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cells electrophoretic profile- Fig. 1(c)) were excised, treated with trypsin and the resulting peptides
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were analyzed by mass spectrometry. While no proteins were identified in band I, several V.
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cholerae proteins were identified in band II (Table S1), including catalase/peroxidade KatG
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(VC1560).
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As strains O395 and N16961 have potential to produce two catalases, KatG and KatB, we
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hypothesized that KatB, although it was not identified in the former experiment, could be the slow-
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moving catalase in the band I (Fig. 1(c)). To test this assumption, two plasmids pAB-katB and
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pBAD-katG, carrying, respectively, KatB and KatG-encoding genes upstream an arabinose
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inducible promoter, were constructed and introduced into V. cholerae O395, since it shows low
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endogenous catalase activity (Fig. 1(b) and (c)). Expression of katG and katB in transformed O395
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cells was induced by arabinose, added to mid-exponential phase cultures. Among arabinose
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concentrations tested, 0.02% was the lowest required for higher expression of both, katG and katB
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(Fig. S1). Thus, 0.02% arabinose was used in the subsequent experiments. Expression of katB and
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katG in induced O395 cells carrying either pBAD-katB or pBAD-katG were evaluated by SDS-
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PAGE and catalase activity assay. A protein band of molecular mass between 45 and 66 kDa (Fig.
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2(a)), consistent with theoretical KatB molecular weight of 64 kDa, was only seen in the
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electrophoretic profile of O395/pBAD-katB induced cells, which also presented strong catalase
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activity at band I (Fig. 2(b)). These results indicate that katB was expressed in O395/pBAD-katB
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cells upon arabinose induction and that KatB is the slow-migrating catalase in band I, also seen in
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Fig. 1(c).
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The protein profile of the induced O395/pBAD-katG cells, on the other hand, presented an extra
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band of molecular mass between 66 and 97 kDa (Fig. 2(a)), consistent with the theoretical KatG
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molecular weight of 86 kDa, which is not present in non-induced cells (Fig. 2(a)). This protein
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showed high catalase activity, as seen in the fast-migrating band II in Fig. 2(b) and 1(c), and was
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identified as KatG by mass spectrometry, as mentioned before.
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In summary, V. cholerae O1 strains El Tor N16961 and classical O395 can express katG and katB
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in a growth-phase dependent manner. However, N16961 produces significantly higher catalase
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levels than classical strain O395. This is not surprising since it has been demonstrated that El Tor
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strains have greater ability to survive stressful environmental conditions than classical ones
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(Finkelstein, 1996). Sunlight, for instance, can lead to H2O2 generation in water environments
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(Cooper & Zika, 1983) and bacterial strains with high capability to neutralize peroxide can persist
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for long periods in water ecosystems (Arana et al., 1992).
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KatB and KatG are involved in oxidative stress response in V. cholerae
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In order to investigate whether KatB or KatG play roles in oxidative stress resistance in V. cholerae,
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non-transformed O395 cells and those carrying pBAD-katB or pBAD-katG were cultivated in LB to
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mid-exponential phase, at 37C, under aeration. At this point, arabinose (to induce
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araBAD promoter) or glucose (to avoid araBAD promoter leaking) was added to cultures that were
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incubated for further 4h. Catalase specific activities and cell survival rates were estimated upon 20
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min exposure to 30 mM H2O2 (Fig. 3). O395/pBAD-katB cells treated with arabinose presented the
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highest catalase specific activity detected, followed by those carrying pBAD-katG, compared to
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non-transformed cells or uninduced transformants. The survival rates of cells challenged with H2O2
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were directly related to their catalase activity. Accordingly, O395/pBAD-katB cells were the most
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resistant (near 100% survival), followed by pBAD-katG cells (around 10% survival). Cells showing
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very low catalase levels were very sensitive to H2O2, with survival rates significantly lower (Fig. 3).
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Taken together these findings show that KatG and KatB catalases seem to be important factors for
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V. cholerae O1 N16961 and O395 strains oxidative stress response, corroborating data from
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previous study on El Tor strain C670 (Wang et al., 2012). However, KatB seems to play a critical
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role in the process (Fig. 3), in agreement with the increased expression of katB gene in V. cholerae
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O1 El Tor strain A1552 variants highly resistant H2O2 (Yildiz et al., 2004).
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Expression regulation of katB gene differs between V. cholerae O1 strains O395 and N16961
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Considering that KatB and KatG are differently expressed in O395 and N16961 cells, we compared
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a 300bp region upstream the putative translation initiation site of katB and katG from both strains.
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While katG regulatory regions in both strains are identical, katB regulatory regions share 98%
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identity (four nucleotide modifications and one gap in O395 sequence, Fig. S2). For that reason, we
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decided to investigate katB expression regulation in O395 and N16961 strains. To this end, 300bp
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DNA fragments upstream the putative translation initiation sites of katB from both, N16961 and
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O395, were fused to lacZ in pIC552 plasmid (Macian et al., 1994) to create pkBN16 and pkBO395
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plasmids (Table 1). Plasmids pkBN16 and pkBO395 were then used to transform V. cholerae
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N16961 and O395 and β-galactosidase activity (lacZ gene product) was measured in exponential
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(OD600 ~ 0.5-0.7) and stationary phase (OD600 > 2.5) cells grown in LB, at 37C, under aeration
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(Fig. 4). β-galactosidase activity level was higher in stationary-phase cells of both strains, relative
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to exponential-phase ones, corroborating results presented before (Fig. 1(b) and (c)). Moreover,
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similar activity levels were observed in cells of each strain harboring either pkBN16 or pkBO395,
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indicating that katB regulatory regions from V. cholerae N16961 and O395 can activate lacZ
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transcription similarly (Fig. 4). However, β-galactosidase activity levels in O395 transformed cells,
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from both culture phases, were about 3-fold lower than the correspondent ones in N16961
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transformants (Fig. 4). These results are in perfect agreement with O395 cells low catalase activity
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(Fig. 1(b) and (c)), and are strong evidence that katB expression regulation differs between strains
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O395 and N16961. Nonetheless, this cannot be attributed to the differences in katB regulatory
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regions between these strains (Fig. S2) and it may, in fact, depend on strain-specific regulatory
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elements.
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katB and katG genes expression is repressed under Pi limitation in a PhoB/PhoR-independent
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manner but this system is required for oxidative stress resistance in V. cholerae O1 N16961
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In previous work, we identified many V. cholerae O1 proteins synthesized in response to Pi
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limitation and/or PhoB cellular levels with roles in oxidative and other stresses conditions (von
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Kruger et al., 2006). Although catalases were not among those proteins, we cannot discard an
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interaction between kat genes expression and Pi metabolism. Induction of catalase katA expression
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by Pi limitation was experimentally demonstrated for three different proteobacteria, Sinorhizobium
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meliloti, Pseudomonas aeruginosa and Agrobacterium tumefaciens (Yuan et al., 2005). In these
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species, katA transcription requires PhoB and Pho-box elements were identified upstream of katA
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genes (Yuan et al., 2005; Yuan et al., 2006). Since nothing is known about a possible interaction
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between catalase production and Pi metabolism, we used the high catalase producer strain N16961
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(Fig. 1(b) and (c)) and WK10, a phoB mutant of N16961, to examine the involvement of
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PhoB/PhoR system in the expression regulation of katB and katG. To this end, specific catalase
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activity was measured in N16961 and WK10 cells grown in MGHP and MGLP with appropriate
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antibiotics, at 37C, under aeration, for 10 h (stationary phase). Total catalase activity did not differ
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between the strains, but the cells grown under Pi abundance presented about 4-fold the activity
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detected in cells from Pi-limited cultures (Fig. 5(a)).
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We next investigated whether Pi limitation and PhoB/PhoR system were involved in katB and katG
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genes expression by electrophoresis of cell lysates on non-denaturing polyacrylamide gel, followed
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by in-gel catalase activity staining. The electrophoretic patterns of the catalases produced by
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N16961 and WK10 in MGHP and MGLP were highly similar (Fig. 5(b)), presenting two bands
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corresponding to KatB and KatG, as in Fig. 1(c) and 2(b). Although KatB and KatG specific
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activity levels did not vary between the strains, they were affected by Pi concentration in culture
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media, being lower in cells grown under Pi limitation (Fig. 5(b)), corroborating data presented
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before (Fig. 5(a)). These results contrast with those obtained for other species (Yuan et al., 2005;
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Yuan et al., 2006) and suggest that katB and katG gene expression in V. cholerae is repressed under
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Pi-limiting conditions in a PhoB/PhoR-independent process.
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N16961 and WK10 cells grown in MGHP and MGLP were also exposed to H2O2 for 5, 10 and 20
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min and their survival rates were determined. The high catalase level in N16961 and WK10 cells
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from MGHP cultures correlated well with their survival rates upon challenge with H2O2 compared
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to cells grown in MGLP (Fig. 6(a)). Interestingly, although N16961 and WK10 grown in MGLP
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cultures presented similar specific catalase activity levels, the wild type cells were about 50 to 100-
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fold more resistant to H2O2 challenge than the mutant ones (Fig. 6(a)). These results and those
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presented before (Fig. 3) highlight a close relationship between total catalase activity and oxidative
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stress response in V. cholerae O1 N16961. They also indicate that extracellular Pi levels regulate
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katG and katB expression in a PhoB/PhoR-independent manner in this strain. However, catalases
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are not the only factor contributing to N16961 resistance to H2O2. PhoB/PhoR system directly or
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indirectly contributes to ROS resistance in V. cholerae O1, confirming previous results from our
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group (Lery et al., 2013; von Kruger et al., 2006).
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PhoB/PhoR system is essential to control cytoplasmic ROS accumulation in V. cholerae O1
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strain N16961 under Pi limitation
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In order to investigate how PhoB/PhoR system protects N16961 cells against oxidative stress, we
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determined intracellular ROS levels in wild type N16961 and phoB/phoR mutant WK10 cells grown
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in MGHP or MGLP for 10 h (Fig. 6(b)). Although N16961 and WK10 cells cultivated in Pi-limiting
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conditions presented similar catalase activities (Fig. 5), similar ROS levels were detected in the
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wild-type cells from both cultures and in the mutant WK10 cultivated in MGHP (Fig. 6(b)). In
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contrast, WK10 cells grown in MGLP presented about 7-fold less ROS levels than in MGHP (Fig.
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6b), indicating that PhoB/PhoR system, in an unknown way, protects V. cholerae O1 against ROS
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accumulation. These findings could help us to understand the phosphate-starvation response of the
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phoB mutant WK3, in comparison to its parental strain from the classical biotype 569B of V.
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cholerae O1 described previously (von Kruger et al., 1999; von Kruger et al., 2006). WK3
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differentially expressed many genes involved in stress resistance and survival in comparison to
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569B under Pi limitation. These included genes of detoxification enzymes, such as the
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alkylhydroperoxide reductase subunit C (AhpC) that is a common scavenger of peroxides in
330
bacteria and a spermidine/putrescine-binding protein that has been associated with DNA protection
331
from oxidative damages (von Kruger et al., 2006). Our results indicate PhoB/PhoR system plays a
332
role in orchestrating detoxifying mechanisms other than catalases to protect cells against oxidative
333
stress.
334
335
Concluding remarks
336
In this study, it is shown for the first time a relationship between Pi levels and katG and katB genes
337
expression in pandemic strains of V. cholerae O1. Although PhoB/PhoR system is not required for
338
full expression of catalase genes, it is involved in V. cholerae O1 response against oxidative stress.
339
This conclusion is based on our findings that besides catalases, a functional PhoB/PhoR system is
340
critical for hydrogen peroxide resistance in V. cholerae O1, especially under Pi limitation.
341
Therefore, this study reveals new roles for PhoB/PhoR, a system that has already been related to
342
many physiological processes and pathogenicity in V. cholerae O1 (Lery et al., 2013; Pratt et al.,
343
2009; Pratt et al., 2010; Sultan et al., 2010; von Kruger et al., 2006).
344
345
Acknowledgments
346
347
We thank FAPERJ, CNPq and CAPES for financial support. We are grateful to Mr. E. Camacho for
348
technical support and to Dr. Rodrigo Fortunato for his assistance in intracellular ROS
349
determination.
350
351
352
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Figure legends
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Table 1 – Plasmids and primers used in this work
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Fig. 1- Catalase activity in exponential and stationary-phase cells of the V. cholerae strains El Tor
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N16961 and classical O395. Cells were grown to exponential or stationary growth phase in LB at
471
37 °C, under aeration. (a) N16961 and O395 growth curves showing exponential (black arrow) and
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stationary (white arrow) phases sampling points. (b) Catalase specific activity was determined in
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cell lysates. Columns represent the average of three individual experiments with standard deviation
474
as error bars. (c) Exponential and stationary-phase whole-cell lysates proteins (60 μg) were resolved
475
by electrophoresis on a 4-8% gradient non-denaturing polyacrylamide gel followed by in-gel
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catalase activity staining. The arrows indicate two bands of catalase activity that will be referred as
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bands I and II in the text.
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Fig. 2 – Protein profiles of O395 cells carrying pBAD-katB or pBAD-katG. O395 cells carrying
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pBAD-katB or pBAD-katG were grown in LB for 4h, at 37˚C, under aeration, after arabinose
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addition to mid exponential phase cultures. Cells were then collected and lysates were prepared and
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gel analyzed. (a) Whole cell lysates electrophoretic profiles (30 μg of proteins/lane) after SDS-
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PAGE (12.5%). Arrows indicate protein bands only seen on arabinose treated cells profile. (b)
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Whole cell lysates catalase activity (50 μg of proteins/lane) after non-denaturing 4-8% gradient gel
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electrophoresis and specific staining.
486
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Fig. 3 – Effect of katG and katB overexpression on V. cholerae O395 resistance to H2O2. Cells were
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grown in LB at 37°C, under aeration, till exponential phase. Cultures were divided in two flasks,
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where glucose 0.2% (Glu) or arabinose 0.02% (Ara) were added. Flasks were incubated for 4 hours
490
at 37°C, under aeration, before catalase activity was measured. To determine cell survival upon
491
exposure to peroxide, aliquots of these cultures were taken and H2O2 at final concentration of 30
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mM was added to each one. Samples were collected 5, 10 and 20 min after H2O2 addition, diluted
493
and plated onto LB-agar. Cell survival was calculated by determining CFU number after challenge
494
and expressed as CFU percentage obtained for the control culture (non-exposed to H2O2). Columns
495
represent the average of four independent experiments with standard deviation as error bars. Each
496
value in the upper box represents the average catalase activity in cells from the same cultures used
497
for assessing H2O2 sensitivity.
498
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Fig. 4 –β-galactosidase specific activity in N16961 and O395 cells harbouring pkB-N16 or pkB-
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O395. Cells were grown till exponential (OD600 ~ 0.5) or stationary (OD600 > 2.5) phases in LB,
501
at 37C, under aeration and β-galactosidase specific activity was measured in their lysates. Data
502
represent the average of four experiments with standard deviation as error bars.
503
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Fig. 5 – Specific catalase activity in V. cholerae N16961 and WK10 cells cultivated under Pi-rich
505
and Pi-limiting conditions. Wild-type N16961 and phoB/phoR mutant WK10 cells were grown in
506
MG medium supplemented with Pi at high (HP) or low (LP) concentration, at 37 °C, under
507
aeration, for 10 hours (stationary phase). (a) Total catalase activity was determined in cell lysates.
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Columns represent five individual experiments average with standard deviation as error bars. (b) V.
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cholerae catalase isozymes observed by non-denaturing polyacrylamide gel. Whole cell lysates
510
(100 μg) were resolved on a 4-8% gradient non-denaturing polyacrylamide gel that was specifically
511
stained for catalase activity.
512
513
Fig. 6 – Oxidative stress resistance and ROS accumulation in V. cholerae N16961 and WK10 cells
514
cultivated under Pi-rich and Pi-limiting conditions. Wild-type N16961 and phoB/phoR mutant
515
WK10 cells were grown in high (MGHP) or low (MGLP) Pi concentration, at 37 °C, under
516
aeration, for 10 hours (stationary phase). (a) Survival rates after 5, 10 and 20 min treatment with
517
H2O2, relative to the control (untreated cells, 100%). N16961 and WK10 cells from MGHP were
518
treated with 30 mM H2O2, while those from MGLP cultures, that do not resist such peroxide
519
concentration, 15 mM H2O2 was used. Columns represent four individual experiments average with
520
standard deviation as error bars. (b) Intracellular ROS accumulation in V. cholerae cells was
521
inferred using DCF-DA, a molecule that penetrates the cell and reacts with cytoplasmic ROS,
522
resulting in fluorescence emission. Data were normalized by the number of cells used in the assay
523
and are presented in fluorescence intensities x 1000 / CFU ml-1. Columns represent the average of
524
three individual experiments with standard deviation as error bars.
525
Table 1
526
Name
Description
Application
pBAD TOPO®
Cloning vector, Invitrogen
Cloning vector
pAB-katB
V. cholerae katB (vc1585) fusioned to araBAD promoter
katB expression
pBAD-katG
V. cholerae katG (vc1560) fusioned to araBAD promoter
katG expression
pIC552
Cloning vector (Macian et al., 1994)
Cloning vector
pkBN16
V. cholerae N16961 katB regulatory region fusioned to
Evaluation of
lacZ
N16961 katB
Plasmid
regulatory
region activity
pkBO395
V. cholerae O395 katB regulatory region fusioned to lacZ
Evaluation of
O395 katB
regulatory
region activity
Primers (5’–3’)
KatBADFw
TGATGAGGAATAATAATTGATAAGTAAAAATATGAG
Cloning (katB
gene)
KatBADRv
CATCGCGGCCAGTTTTGCCACC
Cloning (katB
gene)
KatGBADFw
TGAGAGGAATAATAAATGGAGCACAATAAAGCG
Cloning (katG
gene)
KatGBADRv
CACCAGATCAAATCGATCCGCATTCATT
Cloning (katG
gene)
KatBpro1
GTCTACCATGGATAGCCGTATTGAG
Cloning (katB
regulatory
region)
KatBpro2
GCTACTCGAGCCGTCATTATTTTAAG
Cloning (katB
regulatory
region)
527