THE INHIBITORY EFFECT OF CALCIUM ON

Brazilian Journal of Microbiology (2011) 42: 1547-1559 ISSN 1517-8382

THE INHIBITORY EFFECT OF CALCIUM ON Cylindrospermopsis raciborskii (CYANOBACTERIA) METABOLISM Ronaldo Leal Carneiro1,3*, Ana Carla Nascimento Alípio2, Paulo Mascarello Bisch2, Sandra Maria Feliciano de Oliveira e Azevedo1, Ana Beatriz Furlanetto Pacheco2 1

Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 2Unidade Genômica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil; 3Departamento de Análises Clínicas e Toxicológicas, Universidade de São Paulo, São Paulo, SP, Brasil. Submitted: December 20, 2010; Returned to authors for corrections: February 27, 2011; Approved: May 30, 2011.

ABSTRACT Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca2+) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.

Key words: calcium; Cylindrospermopsis; paralitic shellfish toxin, proteome.

(6, 25). Differently from other geographic regions, several

INTRODUCTION

toxic strains isolated from Brazilian water supplies produce Cylindrospermopsis

raciborskii

is

a

filamentous

diazotrophic freshwater cyanobacterium. It is a cosmopolitan

paralitic shellfish toxins (PSTs) (19, 25, 26, 36). C. raciborskii is consistently recorded as a dominant

diverse

phytoplankton component in eutrophic and hypereutrophic

environments (13, 16, 30, 38). The presence of C. raciborskii

reservoirs of the semi-arid North-eastern region of Brazil (4, 8,

in water supplies used by humans or livestock is potentially

9, 10). In this region, soil composition and climate contribute

harmful since several strains can produce the hepatotoxic

to the characteristic high ionic composition of water (Na2+,

alkaloid cylindrospermopsin and other yet unidentified toxins

Ca2+, Mg2+, Cl- and HCO3- in millimolar range) yielding

species,

recognized

as

highly

adaptable

to

*Corresponding Author. Mailing address: Institute of Biophysics Carlos Chagas Filho, Health Sciences Center, Federal University of Rio de Janeiro.; E-mail: [email protected] / [email protected]

1547

Carneiro, R.L. et al.

Effect of calcium on C. raciborskii

conductivities up to 25000 µS.cm-1 (3). These reservoir waters

of 20 µM. In order to simulate a condition where C. raciborskii

are also defined as hard, routinely evaluated by calcium

occurs in Brazilian water bodies, CaCl2 was added to obtain 10

carbonate content, with high CaCO3 concentration (> 100 ppm)

mM of the ion calcium. The pH of the culture media was

(11). Water hardness implies high Ca

2+

concentrations,

reported as up to 40 mM in those environments (12).

adjusted to 8.0 and the turbidity of both media (ASM-1 and ASM-1 10 mM Ca2+) was measured using a turbidimeter

The influence of salinity (represented by high NaCl

(2100P - HACH). It is expected that part of the added Ca2+ will

concentration) on C. raciborskii metabolism has been

precipitate as CaCO3, which can increase turbidity. Thus, light

previously reported and its effect on saxitoxin production has

intensity was measured with a quantum sensor (QST-100 Box

been investigated using a Brazilian strain (T3) (31). However,

– Biospherical Instruments Inc.) immersed in the same volume

2+

there is no report on the effect of Ca on C. raciborskii growth

of culture medium used in the experiments. In order to

and toxin production. Therefore, considering the common

determine the actual initial concentrations of free Ca2+ in ASM-

incidence of C. raciborskii blooms in water supplies presenting

1 and ASM-1 10 mM Ca2+, 10 mL of each medium were

high concentrations of Ca2+ (conductivity average 2000 µS.cm-

analyzed by ion chromatography using a Dionex ISC-1000

1

system

-1

, max. 19000 µS.cm – 3) in this study we verify the effect of

to

separate

cations.

Analysis

was

performed

a continued exposure to this ion on the physiology of C.

suppressing conductivity by use of a CS12A column (250 mm,

raciborskii T3 strain, evaluating cellular growth, morphology,

4 mm internal diameter), preceded by a CG12A column (50

chlorophyll-a, toxin content and protein expression profiles.

mm, 4 mm internal diameter) and a suppressor pump (CSRS 300 – 4 mm). An isocractic elution with 21 mM metasulfonic

METHOD Strain maintenance

acid was performed online using the software Chromeleon 6.80. The sample (ASM-1 or ASM-1 10 mM Ca2+) injection

In this study, the C. raciborskii T3 strain was used. This

volume was set to 500 µL (loop 500 µL) and the total run time

strain was isolated from a toxic cyanobacterial bloom in Brazil

was 15 min. Concentrations were determined considering the

(19) and has been maintained in the culture collection of the

retention time and according to a calibration curve of cation

Laboratory of Cyanobacterial Ecophysiology and Toxicology

standards (Six cation II- Standard, Dionex).

(UFRJ, Brazil). The T3 strain produces STX, neosaxitoxin (NSTX) and decarbamoyl-NSTX and has been used as a type strain to elucidate the biosynthetic pathway of paralytic shellfish toxins (PSTs) (18, 36). Non-axenic batch cultures of this strain were maintained in ASM-1 medium (15) at 24 ± 2 ºC, with a 12 hour L:D cycle and photon flux of 50 µmol photons.m-2.s-1 (provided by common day light fluorescent lamps). Aeration was produced by a compressed air pump. These culture conditions were established according to the environmental conditions from which this strain was isolated.

C. raciborskii growth in medium with added calcium A culture of C. raciborskii (T3) strain was maintained in the conditions described above for 12 days. Aliquots of this culture were used as inoculum to experimental tests, which were initiated with 5.0 x 105 cells.mL-1. Cells were inoculated into 3liter glass vessels (flat bottom) with 2 L of ASM-1 medium (control) or ASM-1 with 10 mM high Ca2+, in triplicates. Sampling of each experimental culture condition was done every 3 days during 15 days, under aseptic conditions. Estimation of cellular concentration during growth was

Preparation of growth medium with added calcium ASM-1 medium composition has a calcium concentration

performed according to Carneiro et al. (7). Briefly, the length of 30 cells was measured by light microscopy using an

1548



ocular rule. Filaments’ lengths were measured on a FuchsRosenthal hemocytometer. The number of cells.mL

-1

dcNSTX (36). Here, we measured PST production as the sum

was

of STX and NSTX only, since we had no dcNSTX standard

obtained from the total length of filaments divided by the

available. Culture samples of 500 mL were harvested on the 6th

average cellular length. Growth rates (rn) were determined

and 12th days and filtered on borosilicate filters (45 mm

according to the following equation:

diameter, Millipore). Cells retained in the filters were analyzed for intracellular PST content while the volume that passed

Nt = N0·ern·t [1]

through the filters was analyzed for extracellular PST content.

Where Nt is the number of cells in time t; N0 is the initial

Samples were stored at -20º C until the moment of HPLC

number of cells; and rn is the growth rate (33).

analysis, which was performed within 24 h from the extraction

To measure trichome length, aliquots of the experimental

procedure. Extraction of toxins was performed using acetic

cultures were harvested every 3 days over the course of fifteen

acid according to Carneiro et al. (7). PSTs were analyzed

days. In each culture triplicate, a minimum of a hundred and

according to Oshima (29) using a Shimadzu HPLC system with

fifty filaments were measured using a graduated ocular in a

a silica-base reversed phase column (125 mm x 4.0 mm, 5 µm;

light microscope.

Lichrospher 100 RP 18). The chromatographic condition was: mobile phase – 2 mM heptanesulfonate in 30 mM ammonium phosphate and 6% acetonitrile, pH 7.1. PSTs were detected

Chlorophyll-a measurements Chlorophyll-a (Chl-a) content was determined from cells

using a fluorometric detector, with excitation at 330 nm and

taken every 3 days over the course of 15 days of culturing.

emission at 390 nm. STX and NSTX were identified and

Aliquots of 10 mL from each culture were filtered using

quantified by comparison with known retention time and

borosilicate filters (13 mm diameter, Millipore). Cells were

integrated areas of STX and NSTX standards. The standards

transferred to test tubes with 5 mL of methanol (100%) and

were purchased from the Institute of Marine Bioscience –

kept in the dark for 30 minutes. Samples were centrifuged

National Research Council of Canada (Halifax, Canada). PST

(10000 g, 20 min, 4o C) and the supernatants were used to

content is presented as cell quota (fg.cell-1).

determine the optical density at 665 nm (with a turbidity correction at 750 nm). Chl-a concentration was calculated using Mackiney’s extinction coefficient (23), according to the formula:

Protein purification Samples of soluble proteins were obtained from stationary phase cultures at the twelfth day of culturing. Cells were harvested from a volume of 500 mL by centrifugation (10000

C = [(OD665-OD750) ·v] /(V·k·d) [2]

g, 15 min, 4o C) and stored at -20º C. For protein extraction,

where C is Chl-a concentration (µg.L-1), v is the volume of

cells were thawed in ice; 0.5 M acetic acid was added and

methanol (mL), V is the volume of the culture (L), k is

samples were incubated at 25o C for 1h. An aliquot of 2 mL of

Mackiney’s extinction coefficient (74.5L·g

–1

·cm

–1

), and d is

the resulting suspension was combined with 18 mL of a

the distance travelled by light (cm). Chl-a is presented as the

solution containing 90% acetone, 10% trichloroacetic acid and

-1

cell quota (pg.cell ).

0.07% β-mercaptoethanol, mixed, and maintained at -20º C overnight. The precipitated proteins were recovered by

PST analysis The PST profile of C. raciborskii T3 is STX, NSTX and

centrifugation (15000 g, 45 min, 4o C) and the resulting pellet was washed three times with 90% acetone. Proteins were

1549



solubilized at 25o C for 1h in a solution of 2 M thiourea, 7 M

consistently present in replicate gels, thus generating one

urea, 4% Chaps, 40 mM DTT, 0.5% Pharmalyte 3-10, 1 mM

control master gel and one calcium master gel.

o

PMSF. After centrifugation (10000 g, 45 min, 4 C) the

In order to identify differently expressed proteins, images

supernatant was collected and proteins were further purified

of the two master gels were matched. Spot intensity was

using the 2D-Clean up kit (GE Healthcare). The resulting

evaluated using the relative volume parameter, expressed as:

sample had its protein content determined (5) and was stored in liquid nitrogen until use. 2D gel eletrophoresis (2D-PAGE) Samples containing 150 µg of soluble proteins (in 200 µL

[3] Where, spots.

of 2 M thiourea, 7 M urea, 4% Chaps, 40 mM DTT, 0.5% Pharmalyte 3-10, 0.002% bromophenol blue) were used to rehydrate strips of 11 cm, pH 4-7 (Immobiline Dry Strip; GE

is the volume of spot S in a gel containing n

This is a normalized value which minimizes experimental variations between replicate gels (Image Master 2D Platinum software version 5.0 User Manual). Spots were considered

Healthcare) for 16 hours at room temperature. Proteins were focused using the Multiphor II equipment (GE Healthcare) according to the manufacturer recommendations. After

differentially expressed (i) if the mean of

differed more

than two fold between the control and calcium conditions (ii) if values showed little dispersion (mean squared

focusing, proteins were reduced and alkylated (strips were

the

equilibrated in DTT and iodoacetamide solutions) and the

deviation) in replicate gels, and if (iii) dispersion of

strips were placed on a 12% SDS-PAGE gel (34). For

values in each condition did not overlap for more than 25%.

separation of proteins on the second dimension, electrophoresis was carried out on the Multiphor II equipment (GE Healthcare)

Protein identification by mass spectrometry

as a flatbed system, following the conditions recommended by

Protein spots were cut from gels, washed twice in 50%

the manufacturer. The gels were stained with Coomassie blue

acetonitrile (ACN) in 25 mM NH4HCO3, placed in 100% ACN,

G-250, digitalized, and images were analyzed using the

and dried under vacuum. Gel fragments were treated with

ImageMaster 2D Platinum v5.0. software (GE Healthcare). For

porcine trypsin (15 ng.µl-) (Promega) and peptides were

each condition tested (control or calcium) three gels were

extracted with 50 µL of 50% ACN, 5% trifluoroacetic acid

produced, two of them loaded with protein samples extracted

(TFA), vacuum dried, and added to 3 µL of 50% ACN, 0.1%

from the same culture and a third one with a protein sample

TFA. An aliquot of 0.5 µL of sample was mixed with an equal

extracted from a different culture. Isoelectric point (pI) values

volume of a saturated solution of α-cyano-4-hydroxycinnamic

of the proteins of interest were determined using a linear 4–7

acid matrix in 50% ACN, 0.1% TFA. The mix was spotted

distribution and the molecular mass (MM) was estimated based

onto a MALDI-TOF sample plate and allowed to crystallize at

on protein low molecular mass markers (GE Healthcare).

room temperature. MALDI-TOF-TOF analysis was performed

Spots on 2D gels were initially detected automatically

in a 4700 Explorer Proteomics Analyzer (Applied Biosystems).

with ImageMaster 2D Platinum v5.0 software and then

Precursor ion fragmentation was obtained using N2 as

manually confirmed. In each gel class (control or calcium)

collision-induced dissociation gas and collision cell pressure

spots intensities were normalized and the three replicate gels

was kept at 2.8 × 10-6 Torr. Trypsin autolysis peptide masses

were matched (14). A set of spots characteristic of each

842.5 and 2211.1 and calibration mixture 1 or 2 (Sequazyme

experimental condition was defined selecting those spots

Peptide Mass Standard kit, PerSeptive Biosystems) were used,

1550



respectively, as internal and external standards. The resulting

(from 20 µM to 17.93 ± 0.01 µM) and in ASM-1 with added

spectra were used to search the NCBI database (version

Ca2+ (from 10 mM to 7.86 ± 0.88 mM). Therefore, cells were

available on 09/06/2010) using the MASCOT program

maintained in the control condition in 18 µM Ca2+ and in high

interface (www.matrixscience.com). The search parameters

calcium condition in 8 mM Ca2+. Addition of CaCl2 to the

allowed for oxidation of methionine, carbamidomethylation of

culture medium increased turbidity from 3.25 ± 0.38 turbidity

cysteine and one mis-cleavage of trypsin. A mass tolerance of

units in ASM-1 to 7.28 ± 0.35 turbidity units in ASM-1 added

100 ppm was used. The criteria for identification were a

Ca2+.

Mascot score above 50 (P