Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter...

ORIGINAL ARTICLE

Soluble proteome investigation of cobalt effect on thecarotenoidless mutant of Rhodobacter sphaeroidesF. Pisani1, F. Italiano2, F. de Leo3, R. Gallerani1,3, S. Rinalducci4, L. Zolla4, A. Agostiano2,5, L.R. Ceci3

and M. Trotta2

1 Dipartimento di Biochimica e Biologia Molecolare, Universita degli Studi di Bari, Bari, Italy

2 Istituto per i Processi Chimico Fisici (CNR), Bari, Italy

3 Istituto di Biomembrane e Bioenergetica (CNR), Trani and Bari, Italy

4 Dipartimento di Scienze Ambientali, Universita della Tuscia, Viterbo, Italy

5 Dipartimento di Chimica, Universita di Bari, Bari, Italy

Introduction

The interactions between metal ions and micro-organisms

have been widely investigated in the last 15 years as their

understanding may contribute to the development of suit-

able biotechnological approaches to tackle environmental

problems associated with heavy metal pollution. Public

awareness towards heavy metal site contamination and

the fate of such pollutants in the environment, and in

particular throughout the food chain, has steadily

increased over the past 10 years and the national and

transnational legislations have imposed very stringent

limits to the tolerable concentrations of these chemicals

in both civil and industrial wastewaters. In time, next to

the traditional physico-chemical techniques, the use of

micro-organisms in metal remediation has gained

momentum taking advantage of the microbial biomass

capability to bind metal ions on their cellular surface as

well as the capability of growing cells to sequester and

accumulate metal ions inside the cell (Malik 2004).

Among micro-organisms, the photosynthetic ones use

the solar radiation as energy source and several authors

have investigated their interactions with heavy metal ions

(Moore and Kaplan 1994; Rai et al. 1994; Singh and

Kumar 1994; Watt and Ludden 1998, 1999; Danilov and

Ekelund 2001; Singh et al. 2001; Shcolnick and Keren

Keywords

cobalt exposure, photosynthesis,

porphobilinogen deaminase, Rhodobacter

sphaeroides, 2DE PAGE analysis.

Correspondence

Massimo Trotta, Istituto per i Processi Chimico

Fisici (CNR) – Bari, via Orabona 4, 70126 Bari,

Italy. E-mail: [email protected]

F. Pisani and F. Italiano contributed equally to

the research realization.

2008 ⁄ 0165: received 29 January 2008,

revised 28 April 2008 and accepted 21 June

2008

doi:10.1111/j.1365-2672.2008.04007.x

Abstract

Aims: To investigate the surviving capability of Rhodobacter sphaeroides under

phototrophic conditions in the presence of high cobalt concentration and its

influence on the photosynthetic apparatus biosynthesis.

Methods and Results: Cells from R. sphaeroides strain R26Æ1 were grown anae-

robically in a medium containing 5Æ0 mmol l)1 cobalt ions and in a control

medium. Metal toxicity was investigated comparing the soluble proteome of

Co2+-exposed cells and cells grown in control medium by two-dimensional gel

electrophoretic analysis. Significant changes in the expression level were

detected for 43 proteins, the majority (35) being up-regulated. The enzyme

porphobilinogen deaminase (PBGD) was found down-regulated and its activity

was investigated.

Conclusions: The up-regulated enzymes mainly belong to the general category

of proteins and DNA degradation enzymes, suggesting that part of the catabolic

reaction products can rescue bacterial growth in photosynthetically impaired

cells. Furthermore, the down-regulation of PBGD strongly indicates that this

key enzyme of the tetrapyrrole and bacteriochlorophyll synthesis is directly

involved in the metabolic response.

Significance and Impact of the Study: Data and experiments show that the

cobalt detrimental effect on the photosynthetic growth of R. sphaeroides is asso-

ciated with an impaired expression and functioning of PBGD.

Journal of Applied Microbiology ISSN 1364-5072

338 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349

ª 2008 The Authors

2006; Tripathi et al. 2006). It emerges from the literature

that the metabolic response often involves the photosyn-

thetic apparatus as well as several other pathways. It is

then interesting to investigate the response of photosyn-

thetic organisms to heavy metal exposure for identifying

the cellular metabolism modifications to this environmen-

tal stress. Among photosynthetic organisms, the purple

nonsulfur phototrophic bacteria occupy a small but rele-

vant ecological niche in which the photosynthetic growth

is associated to the absorption of wavelengths in the near

infrared (NIR) region of the electromagnetic spectra

(from 700 to 1000 nm, depending on the organism),

which poorly overlaps with the photosynthetic active

radiation used by aquatic oxygenic photosynthetic orga-

nisms. Furthermore, these bacteria can grow in environ-

ments with moderate to severe redox reducing power,

where no oxygenic photosynthesis can take place. Anoxy-

genic bacteria hence are good candidates to be used in

biotechnological approaches towards polluted waters

remediation in cooperation with other photosynthetic

organisms (Podda et al. 2000). Rhodobacter (R.) sphae-

roides is a Gram-negative, purple nonsulfur phototrophic

bacterium belonging to the Alphaproteobacteria class. In

the presence of elevated oxygen partial pressure, it grows

chemoheterotrophically, while when exposed to light at

low oxygen partial pressure it grows photosynthetically

developing a network of membrane invaginations, called

the intra-cytoplasmatic membrane, where the photosyn-

thetic apparatus is located (Sistrom 1960). The carote-

noidless strains, R. sphaeroides R26, and their partial

revertants, R26Æ1, are deletion mutants of the wild-type

R. sphaeroides 2Æ4Æ1, which have been long known to be

highly susceptible to photo-oxidative damage owing to

the absence of photoprotective carotenoid pigments

(Sistrom et al. 1956; Dworkin 1960; Sistrom and Clayton

1964). Carotenoids in both the light-harvesting antenna

protein, LH2, and the reaction centre play two fundamen-

tal roles, as antenna for collecting sunlight and as photo-

protectors of the chlorophyll triplet state against

molecular oxygen. Carotenoidless mutants grow photo-

synthetically at very low oxygen partial pressure, where

substantially no chemoheterotrophic growth is present,

allowing to concentrate our study on the sole photo-

trophic metabolism. Furthermore, the use of these

mutants offers the advantage of simplified optical spectra

where the sole absorption at 860 nm associated to the

bacteriochlorophyll contained in the light-harvesting

antenna protein, LH1, are present in the NIR region

(Cogdell et al. 1980; Cogdell and Thomber 1981; David-

son and Cogdell 1981).

Rhodobacter sphaeroides R26Æ1 is known for its capacity

to grow photosynthetically in the presence of relatively

high concentrations of metal ions, with a marked toler-

ance towards cobalt (Giotta et al. 2006). Cobalt is

required as a trace element in prokaryotes and eukaryotes

to fulfil a variety of metabolic functions. It is an impor-

tant cofactor in vitamin B12-dependent enzymes and in

some other enzymes in animals, yeasts, bacteria, archaea

and plants (Kobayashi and Shimizu 1999). However, as

for any other transition metal, high intracellular concen-

trations of cobalt are toxic to both prokaryotes and

eukaryotes. Cobalt increases oxidative stress in cells by

raising the concentration of reactive oxygen species (Kas-

przak 1991; Leonard et al. 1998); it can mimic or replace

ions like magnesium and calcium in various essential

reactions (Jennette 1981) and tends to bind to proteic

–SH groups, inhibiting the activity of sensitive enzymes

(Bruins et al. 2000). However, the molecular mechanisms

of its toxicity are largely unknown and very little has been

performed in identifying the principal targets of elevated

intracellular Co2+concentrations (Stadler and Schweyen

2002; Ranquet et al. 2007).

Genomic or proteomic global analysis allows a system-

atic overview of thousands of genes or proteins at the

same time. Heavy metal toxicity mechanisms have been

investigated using the recently developed DNA micro-

array technology as a high throughput method for global

gene analysis (Hu et al. 2005). However, proteomics can

produce more accurate and comprehensive information

as protein expressions are regulated not only at the tran-

scriptional but also at the translational levels (Dutt and

Lee 2000; Tyers and Mann 2003). In this paper, the

metabolic response of R. sphaeroides was investigated by

comparing the soluble proteome of cells grown in cobalt-

rich environment and in control media. The analysis of

differentially expressed proteins clearly highlights the

involvement of several metabolic pathways as well as of

some specific periplasmic proteins.

Materials and methods

Chemicals

All chemicals for bacterial cell culture, PMSF (phen-

ylmethanesulfonylfluoride), porphobilinogen, DNAse I

(deoxyribonuclease I from bovine pancreas), RNAse A

(ribonuclease A from bovine pancreas type IIA), methanol

(ACS reagent, ‡99Æ8%), acetone (ACS reagent, ‡99Æ5%),

tri-n-butylphosphate (‡99%), Tris [tris(hydroxymeth-

yl)aminomethane], Triton X-100, glycerol (ACS reagent,

‡99Æ5%), p-dimethylaminobenzaldehyde (DMAB), glacial

acetic acid and perchloric acid (ACS reagent, 70%) were

obtained from Sigma-Aldrich. Urea, thiourea, CHAPS

(3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfo-

nate), DTT (dithiothreitol), IPG (immobilized pH gra-

dient) buffer, pH 3–10 and 4–7, SDS (sodium

F. Pisani et al. Cobalt effect on R. sphaeroides

ª 2008 The Authors

Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349 339

dodecylsulfate), iodoacetamide and IPG strips (13 cm, pH

range: 3–10 or 4–7) were purchased from GE Healthcare.

Agarose, acrylamide, N,N¢-methylene-bis-acrylamide and

Coomassie Brilliant Blue G250 were obtained from Bio-

Rad. The protein molecular weight standard (See-Blu

LC5625) was purchased from Invitrogen.

The following buffers are used in this paper: rehydrata-

tion buffer [7 mol l)1 urea, 2 mol l)1 thiourea, 4% (w ⁄ v)

CHAPS, 1% Triton X-100, 100 mmol l)1 DTT, 0Æ05%

(w ⁄ v) IPG buffer, pH 4–7]; buffer I [50 mmol l)1 Tris-

HCl, pH 8Æ8, 6 mol l)1 urea, 30% glycerol, 2% (w ⁄ v)

SDS, 10 mg ml)1 DTT]; buffer II (50 mmol l)1 Tris-HCl,

pH 8Æ8, 6 mol l)1 urea, 30% glycerol, 2% SDS,

25 mg ml)1 iodoacetamide); TGS buffer [25 mmol l)1

Tris, 192 mmol l)1 glycine and 0Æ1% (w ⁄ v) SDS, pH 8Æ3).

Bacterial strain and culture conditions

The blue-green strain, R. sphaeroides R26Æ1, was grown in

light under anaerobic conditions, according to Buccolieri

et al. (2006). The cells were grown in medium 27 of the

German Collection of Microorganisms and Cell Cultures

(http://www.dsmz.de/), which contains Zn2+, Fe2+, Mn2+,

Co2+, Cu2+, Ni2+and MoO42) as trace elements. Specifi-

cally, [Co2+] < 1 lmol l)1. In this paper, such medium

will be termed as ‘plain’.

Cobalt-enriched media were obtained by dissolving

into the plain medium CoCl2Æ6 H2O to a final concen-

tration of 5Æ0 mmol l)1. The actual Co2+concentration

was routinely checked by AES-ICP as described in

Buccolieri et al. (2006) and found within 5% of the

desired one. Rhodobacter sphaeroides cells from plain

and cobalt-rich cultures were routinely checked for con-

sistent values of growth and photosynthetic apparatus

synthesis.

Photosynthetic apparatus and bacteriochlorophyll

content estimation

The optical spectrum of cells grown in plain and Co2+-

rich media was recorded in the interval 400–1000 nm by

a ultraviolet-visible-NIR spectrometer (Cary 5000; Varian

Inc., Mulgrave, Australia). The absorbance at 535 nm,

where no specific absorption is present, is used to infer

the actual bacterial cell concentration in the culture broth

(Giotta et al. 2006). The entire spectrum was corrected

for scattering using a k)4 interpolating function.

Bacteriochlorophyll content was assessed by adding

100 ll of the cell suspension to 900 ll of a mixture con-

taining acetone, water and 14 mol l)1 of NH4OH

(80 : 20 : 1 by vol.). The solution was vigorously shaken

and centrifuged (18 000 g, 4�C) for 5 min and the super-

natant was supplemented with 200 ll of hexane. The

bacteriochlorophyll concentration is determined in the

hexane layer by recording the absorbance at 770 nm and

using the molar extinction coefficient of

9Æ11 · 104 mol l)2 cm)1 (Smith and Benitez 1955). The

actual concentrations were normalized to 108 CFU ml)1.

Water-soluble protein fraction extraction

Cells in the late exponential growing phase were harvested

by centrifugation (14 000 g, 4�C, 15 min) from 1 l of

plain or cobalt-exposed medium kept under the same

temperature and illumination growth conditions. The

intense-coloured cell pellet was carefully washed with

50 ml of 20 mmol l)1 Tris-HCl at pH 8Æ0, centrifuged

and re-suspended in the same buffer to a final absorbance

of 100 a.u. at 865 nm. Cell wall disruption was accom-

plished by high pressure extrusion method, using a

French pressure cell operating at 15 MPa and 4�C in the

presence of 100 lg ml)1 DNAse I and 1 mmol l)1 PMSF.

A 40-ml aliquot was added with 1 mg ml)1 DNAse I and

10 mg ml)1 RNAse A and gently shaken at room temper-

ature for 90 min. Intact cells and debris were removed by

an initial 20 min centrifugation (18 000 g, 4�C) followed

by a 2 h ultracentrifugation step (150 000 g, 4�C). The

red-brown supernatant (roughly 30 ml) containing the

water-soluble proteome was precipitated by adding 14

volumes of a 12 : 1 : 1 by volume mixture of acetone–

methanol–tri-n-butylphosphate (AMTP) incubated

overnight at 4�C (Mastro and Hall 1999). The pellet was

collected by a 20 min centrifugation (2800 g, 4 �C) and

washed with 30 ml of tri-n-butylphosphate, acetone and

methanol, respectively. The pellet was solubilized in 2Æ5%

CHAPS and the total protein content was determined

according to the Bradford method (Bio-Rad Protein

Assay) using bovine serum albumin as standard. The

CHAPS solution was then added with the rehydratation

buffer to a final protein concentration of 1Æ0–

1Æ5 mg ml)1.

Two-dimensional gel electrophoresis (2-DE)

Exactly 350 lg of proteins were loaded onto the suitable

linear IPG strip for electrophoretic separation. Iso-

electrofocussing (IEF) was performed with a total Vh of

50 000 or 70 000 for 3–10 or 4–7 strips, respectively. IPG

strips coming from IEF were initially equilibrated for

15 min in buffer I, then for another 15 min in buffer II,

and finally sealed to a 15 · 15 cm 10% polyacrylamide

gel by 0Æ5% agarose in TGS buffer. Electrophoresis was

performed at 18�C using TGS as running buffer at 25 mA

for 1 h and at 50 mA for 4 h. 2DE gels were stained with

Coomassie Colloidal G250 (Neuhoff et al. 1988) and

destained with Milli Q-water (Millipore).

Cobalt effect on R. sphaeroides F. Pisani et al.


ª 2008 The Authors

2-DE was run on two classes of four independent solu-

ble protein batches obtained from plain (class P) and

Co2+(class C) biomasses, respectively. 2DE gels were

scanned with an image scanner (Amersham Biosciences)

and analysed with the software Image Master 2D 6Æ0 Plat-

inum (GE Healthcare). Spots from the two classes were

compared and those found with a t-value higher than 7

were recovered from the gels and used for protein identi-

fication. The spots of interest were analysed by MS ⁄ MS.

MS ⁄ MS analysis

Protein spots were excised from stained gels and sub-

jected to in-gel trypsin digestion (Shevchenko et al.

1996). The recovered peptide mixtures were then

separated by using a nanoflow-HPLC system (Ultimate,

Switchos, Famos, LC Packings, Amsterdam, The Nether-

lands) coupled with a high-capacity ion trap HCTplus

(Bruker-Daltonik, Germany). Protein identification was

performed by searching for nonredundant databases

(NCBInr) using the Mascot program (http://www.

matrixscience.com) in the National Center for Biotechno-

logy Information. For positive identification, the result

score had to be above the significant threshold (P < 0Æ05).

Porphobilinogen deaminase (PBGD) assay

Preparation of cell-free extracts

Sedimented cells, after washing by 20 mmol l)1 Tris-HCl

at pH 7Æ0, were resuspended in the same buffer supple-

mented with 4 mmol l)1 DTT kept at 0�C and lysed by

French pressure cell. The extract was centrifuged at

150 000 g for 2 h and the supernatant was used for

PBGD assay.

PBGD assay

PBGD was assayed by the disappearance of its substrate

porphobilinogen (PGB; Davies and Neuberger 1973) in

50 mmol l)1 Tris-HCl at pH 7Æ0. PGB was incubated with

and without cell extract (100 ll) in 1 ml total volume at

37�C for 150 min. The reaction was eventually stopped

by precipitating the proteins through the addition of tri-

chloroacetic acid 10% (w ⁄ v). The proteins were centri-

fuged down (14 000 g, 4�C, 2 min) and an aliquot of the

clear supernatant (500 ll) was added with an equal

volume of modified Ehrlich reagent [1 g of p-dimethyla-

minobenzaldehyde (DMAB), 42 ml of glacial acetic acid,

8 ml of 70% (w ⁄ w) perchloric acid; Mauzerall and Gra-

nick 1956]. The magenta colour due to the condensation

product of the reaction between DMAB and PBG was

allowed to develop for 15 min and detected by measuring

the absorbance at 553 nm using the molar absorption

coefficient of 58 000 l mol)1 cm)1 (Bogorad 1958a–c).

The nonenzymatic loss of PBG at 37�C was found negligi-

ble.

PBGD activity was also measured in dialysed cell extracts

obtained from co-exposed biomass. An aliquot was dialy-

sed overnight at 4�C using a slide-A-Lyzer 3Æ5 K MWCO

Dialysis Cassette (Pierce) against 200 volumes of cobalt-

free 50 mmol l)1 Tris-HCl at pH 7Æ0, 4 mmol l)1 DTT.

Results

Cobalt effect on the growth and photosynthetic

apparatus

The detrimental effect of cobalt on the photosynthetic

growth and photosynthetic apparatus biosynthesis of

R. sphaeroides R26Æ1 are shown in Figs 1 and 2 (a),

respectively. The optical spectra present an intense and

symmetric absorption peak centred around 867 nm whose

intensity decreases with increase in the cobalt concentra-

tion. The modest wavelength shift of the major NIR band

can be explained by the increasing number of antenna

complexes that tend to loose a fraction of the constituent

bacteriochlorophylls and cannot form dimeric units (Raff-

erty et al. 1979). The optical spectra reflect then the

appearance of the monomer bacteriochlorophyll peak

ipsochromic shifted with respect to the dimer. Indeed at

the highest possible concentration (10 mmol l)1), where

the cell growth is strongly inhibited, cobalt induces a

peak-shift to roughly 864 nm (data not shown).

From the normalized peak intensity and bacterio-

chlorophyll content, as function of cobalt concen-

tration, values of 3Æ2 ± 0Æ8 mmol l)1 (Fig. 2b) and 5Æ7 ±

0Æ3 mmol l)1 (Fig. 2c), respectively were found for the

Time (h)0 20 40 60 80 100

ln (

N/N

0)

0

1

2

3

4

Figure 1 Photosynthetic growth curves of Rhodobacter sphaeroides

R26Æ1 in plain and 5 mmol l)1 Co2+culture media (•), plain and (e)

[co2+]=5 mM.


ª 2008 The Authors


EC50 parameters. Such values are half way between the

growth rate (0Æ8 ± 0Æ4 mmol l)1) and the population size

(12 ± 2 mmol l)1) EC50 (Giotta et al. 2006), confirming

that the cobalt inhibition towards the photosynthetic

growth does involve the photosynthetic apparatus

biosynthesis but it cannot be exclusively ascribed to such

a metabolic pathway. To understand the complexity of

the mechanisms regulating the R. sphaeroides

resistence ⁄ tolerance to cobalt, a proteomic investigation

was performed.

Proteomic analysis

The 2DE map of R. sphaeroides soluble proteins obtained

using a 3–10 pH interval in the IEF separation showed

350 spots (data not shown), most of which were localized

in the 4–7 pH range. Therefore, further investigations

were performed in the latter pH interval using IPG strips

with a narrower pH interval. In Fig. 3, gels of the soluble

proteins obtained from R. sphaeroides grown in plain and

5 mmol l)1 cobalt-enriched media are shown. Nearly 800

spots could be resolved. Four gels per class (i.e. classes P

and C – see ‘Material and methods’) were run and within

each class the mean value of the per cent volume of each

spot (�X) was determined along with the SD. The differ-

ence between �X of any chosen spot in the plain and in

cobalt class, �XP and �XC respectively, was used to derive

the t parameter of the ‘two-sample t-test’ (Snedecor and

Cochran 1989) and considered potentially interesting in

terms of differentially expressed proteins for any t > 7.

Forty-three spots having a statistically significant differ-

ence between control and cobalt proteomes were found

and analysed by MS ⁄ MS.

MS and MS ⁄ MS data were used for searching the data-

base in the currently available annotated genome

of R. sphaeroides (http://mmg.uth.tmc.edu/sphaeroides/;

Mackenzie et al. 2001). The results are listed in Table 1

according to the functional category of the identified pro-

teins. In the presence of cobalt, 35 out of 43 proteins

were found up-regulated while eight were down-

regulated.

Among the proteins expressed at the lower level, the

largest categories include enzymes belonging to protein

(25%) and tetrapyrrole biosynthesis (25%) pathways. For

the over-expressed proteins, enzymes involved in pro-

tein ⁄ aminoacid degradation are the largest category

(31%), followed by those required in carbohydrate meta-

bolism (20%), in [Fe–S] cluster assembly and sulfur

aminoacid pathway biosynthesis (17%). Two out of

thirty-five over-expressed proteins are hypothetical ones

with unknown function and six are putative proteins with

nonconfirmed functions.

0·14 (a)

(b) (c)

0·12

0·10

0·08

0·06

0·04

0·02

0·00

1·0

0·0 10–7 10–4 10–1 102

0·5

1·0

0·0

0·5

700 750 850

Wavelength (nm)

Wav

elen

gth

(nm

)

0 1 2 3 [Co2+] mmol l–1

[Co2+] mmol l–1 10–7 10–4 10–1 102

[Co2+] mmol l–1

4 5

867

866

865

864

863

Nor

mal

ized

abs

orba

nce

(a·u

·)

Nor

mal

ized

abs

orba

nce

at p

eak

max

imum

Nor

mal

ized

BC

hl

conc

entr

atio

n

950 800 900 1000

Figure 2 Visible-near infrared spectra of cell

suspensions (a), peak intensity (b) and bacte-

riochlorophyll content (c) as functions of

Co2+concentration for Rhodobacter sphae-

roides R26Æ1. Data were elaborated as illus-

trated in ‘Materials and methods’ section.

Spectra in (a) were corrected from scattering

and normalized to a concentration of

108 CFU ml)1. The inset in (a) shows the peak

wavelength vs [Co2+]. Data in (b) and (c) are

also normalized to a concentration of

108 CFU ml)1. ( , plain; ,

0Æ1 mM; , 0Æ2 mM; ,

0Æ7 mM , 2 mM; 5 mM).



ª 2008 The Authors

PBGD enzymatic assays

Cobalt effect on PBGD was checked by the enzyme acti-

vity in cell-free extracts. PBGD-specific activity was found

to be three time larger in plain extracts than in cobalt-

exposed extracts (see Fig. 4). The latter were also dialysed

for removing residual cobalt, a known inhibitor of PGBD,

and the enzymatic activity was partially restored to 70%

of the control value. A double check was performed by

adding cobalt to the dialysed extract, and PBGD activity

was indeed found to be inhibited by more than 50%. This

finding strongly suggests that the decreased activity of

PBGD is only partially because of direct cobalt inhibition

and hence indirect effect of cobalt should be assumed

(see Discussion). It should be noted that data have been

recorded at pH 7, one unit below the optimal value

reported for PBGD for avoiding cobalt hydroxide precipi-

tation.

Discussion

Heavy metals such as copper, cobalt, manganese, moly-

bdenum and so on, are essential nutrients for bacterial

growth being required in several metabolic pathways

including those involved in the phototrophic growth. The

growth response to these essential elements is typically

represented by a bell-shaped curve with a high-level

plateau indicating a healthy growth in correspondence to

the optimal concentration range. When the element con-

centration falls below or rises above the optimal value,

the bacterial growth is hindered owing to either element

starvation (below optimal concentration) or element tox-

icity (above optimal concentration).

This paper investigates the ability of the photosynthetic

bacterium R. sphaeroides to cope with cobalt in its most

common and stable oxidation state, Co2+, present in con-

centrations well above the optimal one. Cobalt is essential

for bacterial growth being required in the vitamin B12

biosynthesis and of its precursors in the porphyrin and

chlorophyll metabolic pathway.

Cobalt moderate toxicity allows obtaining an evident

metabolic response without fully compromising the bio-

mass production. In R. sphaeroides, typical Co2+concen-

tration required in the culture medium is in the order of

1 lmol l)1 and higher cobalt concentrations lead to toxic

effects that reduce the bacterial ability to grow photosyn-

thetically (Giotta et al. 2006). This suggests an involve-

ment of the bacteriochlorophyll biosynthetic pathway

which, though, cannot be considered the sole detrimental

effect on the cell metabolism as indicated by the different

EC50 values obtained from different parameters. The pro-

teomic approach is definitely suitable in gaining a general

picture of Co2+effect on the bacterium and, based on the

spots listed in Table 1, we hypothesize the following

metabolic response. The protein functions described in

this discussion were identified in the Biocyc Database

(http://www.biocyc.org).

Cobalt affects the photosynthetic apparatus of R. sph-

aeroides by decreasing its ability to harvest light arising

from a lower capacity to synthesize bacteriochlorophyll

(Fig. 2). Accordingly, aconitate hydratase and PBGD (spots

42 and 43 in Table 1) are down-expressed in the presence

Figure 3 Representative gels of soluble proteins from Rhodobacter

sphaeroides R26Æ1 grown in plain media (upper gel) and in 5 mmol l)1

cobalt-enriched media (lower gel). Isoelectrofocussing conditions: pH

4–10 linear gradient, 70 000 Vh (see ‘Materials and methods’ for

more details). Approximately 800 distinguishable spots were obtained

in both the gels.


ª 2008 The Authors


Tab

le1

MS

⁄MS

iden

tifica

tion

of

Rhodobac

ter

sphae

roid

espoly

pep

tides

reso

lved

by

two-d

imen

sional

poly

acry

lam

ide

gel

elec

trophore

sis

Spot

num

ber

Prote

innam

ean

dfu

nct

ional

cate

gory

NC

BIac

cess

ion

num

ber

EC num

ber

Num

ber

of

pep

tides

iden

tified

by

MS

⁄MS

Mas

cot

score

*

%se

quen

ce

cove

rage

t-va

lue�

XC

XP

Car

bohyd

rate

met

abolis

m

1A

lpha

amyl

ase,

cata

lytic

dom

ain

⁄subdom

ain

gi|7

7463009

3Æ2

Æ1Æ1

13

787

16

7Æ7

91Æ6

2

2Fr

uct

ose

-bis

phosp

hat

eal

dola

seI

gi|7

7464860

4Æ1

Æ2Æ1

317

960

47

11Æ4

611Æ3

4

31,4

-alp

ha-

glu

can

bra

nch

ing

enzy

me

gi|7

7463005

2Æ4

Æ1Æ1

820

1144

30

8Æ4

95Æ0

0

4Ph

osp

hoen

olp

yruva

teca

rboxy

kinas

egi|7

7462225

4Æ1

Æ1Æ4

925

1865

64

16Æ7

03Æ0

5

5D-3

-phosp

hogly

cera

tedeh

ydro

gen

ase

gi|7

7464929

–8

515

17

6Æ9

93Æ2

6

6G

luco

se-1

-phosp

hat

ead

enyl

yltr

ansf

eras

egi|7

7463444

2Æ7

Æ7Æ2

713

824

38

31Æ9

830Æ1

9

7Pe

nto

se-5

-phosp

hat

e-3-e

pim

eras

egi|7

7463344

5Æ1

Æ3Æ1

4330

21

10Æ6

18Æ3

7

Oxi

dat

ive

stre

ssre

sponse

8C

atal

ase

gi|7

7462935

1Æ1

1Æ1

Æ627

1634

44

8Æ4

86Æ6

9

Prote

in⁄a

min

oac

iddeg

radat

ion

9Pu

tative

pep

tidyl

-dip

eptidas

egi|7

7464167

–27

1557

39

17Æ8

43Æ6

2

10

S-m

ethyl

-5’-

thio

aden

osi

ne

phosp

hory

lase

gi|7

7462383

2Æ4

Æ2Æ2

86

274

20

7Æ3

46Æ4

8

11

Met

hio

nin

eam

inopep

tidas

e,su

bfa

mily

1gi|7

7464502

3Æ4

Æ11Æ1

89

511

23

12Æ8

5A

bse

nt

inpla

in

12

Bra

nch

ed-c

hai

nam

ino

acid

amin

otr

ansf

eras

egi|7

7464721

–4

253

15

11Æ2

0A

bse

nt

inpla

in

13

Aden

osy

lhom

ocy

stei

nas

e[R

ose

obac

ter

sp.

MED

193]

gi|8

6136724

–4

153

7Æ2

7A

bse

nt

inpla

in

14

His

tidin

ol

deh

ydro

gen

ase

gi|7

7464202

–24

1578

56

9Æ3

02Æ9

1

15

Met

hyl

mal

onic

acid

sem

iald

ehyd

edeh

ydro

gen

ase

gi|7

7463521

–7

308

16

12Æ9

2A

bse

nt

inpla

in

16

Form

ate-

tetr

ahyd

rofo

late

ligas

egi|7

7464234

6Æ3

Æ4Æ3

18

1293

37

14Æ7

516Æ2

7

17

Cyt

oso

lam

inopep

tidas

egi|7

7464956

3Æ4

Æ11Æ1

6388

24

9Æ0

411Æ1

3

18

Puta

tive

ther

most

able

carb

oxy

pep

tidas

e1

gi|7

7465014

–19

1115

44

13Æ5

82Æ5

7

19

Puta

tive

amin

otr

ansf

eras

epro

tein

gi|7

7463951

–22

1564

51

7Æ2

51Æ7

8

Nitro

gen

assi

mila

tion

20

Glu

tam

ate

synth

ase

(bet

a-su

bunit)

gi|7

7464730

–15

1030

40

17Æ7

80Æ3

1

21

Glu

tam

ine

synth

etas

ecl

ass-

Igi|7

7463716

6Æ3

Æ1Æ2

7354

13

46Æ7

3190Æ6

9

Hyp

oth

etic

alpro

tein

s

22

Hyp

oth

etic

alpro

tein

RSP

_3736

gi|7

7465742

–5

240

15

11Æ7

52Æ0

7

23

Hyp

oth

etic

alsi

gnal

pep

tide

pro

tein

gi|7

7462083

–17

910

45

9Æ1

536Æ1

4

Prote

inbio

synth

esis

24

Asp

arty

l⁄glu

tam

yl-t

RN

Aam

idotr

ansf

eras

esu

bunit

Bgi|7

7462533

–19

1197

43

16Æ5

64Æ0

3

25

Ala

nyl

-tRN

Asy

nth

etas

egi|7

7464020

–20

1202

25

8Æ9

53Æ7

5

26

Elongat

ion

fact

or

Tugi|7

7462243

3Æ6

Æ5Æ3

12

564

37

10Æ3

00Æ6

3

27

Elongat

ion

fact

or

Pgi|7

7464048

–8

483

44

9Æ0

60Æ5

2

Fe–S

clust

eras

sem

bly

,su

lfur

amin

oac

idpat

hw

aybio

synth

esis

28

Asp

arta

te-s

emia

ldeh

yde

deh

ydro

gen

ase

gi|7

7464953

2Æ6

Æ1Æ5

27

508

20

13Æ8

61Æ8

9

29

SufC

,A

TPas

egi|7

7464008

–17

1183

64

15Æ4

41Æ7

8

30

Cys

tein

esy

nth

ase

gi|7

7464691

–14

1120

58

11Æ7

52Æ0

7

31

FeS

asse

mbly

pro

tein

SufB

[R.

sphae

roid

esA

TCC

17029]

gi|8

3373147

–4

165

12

8Æ5

51Æ 6

1



ª 2008 The Authors

of cobalt. The first enzyme is involved in the citric acid

cycle metabolic pathway as precursor for the succinate

biosynthesis (Voet et al. 2001). Its down-regulation may

reduce the succinyl-CoA synthesis, a 5-aminolevulinate

precursor, the starting substrate for porphyrins and bacte-

riochlorophyll biosynthesis. For this, aconitase is classified

as belonging to the tetrapyrrole biosynthesis category.

PBGD specifically belongs to the porphyrin and

bacteriochlorophyll biosynthetic pathways and its down-

regulation may account for the reduced ability to synthe-

size bacteriochlorophylls required for assembling LH1.

Indeed, PBGD catalyses the condensation of four PBG

molecules to form the linear tetrapyrrole precursor, the

hydroxymethylbilane (Jordan and Berry 1980). Because of

its central role in the bacteriochlorophyll synthesis,

cobalt’s influence on PBGB activity could give some indi-

cations on the bacterium response to high concentration

of the ion. The measured specific activity (see Fig. 4)

shows that cobalt influence on the PBG condensation

reaction has two contributions: a direct inhibitory effect

that can be suppressed by removing the inhibitor from

the environment and a second indirect contribution that

accounts for nearly one-third of the total inhibition.

Direct inhibition of cobalt has been reported for Saccha-

romyces cerevisiae (Correa Garcia et al. 1991), Rattus

norvegicus (Farmer and Hollebone 1984) and for the

photosynthetic eukaryotes, Pisum sativum L. (Spano and

Timko 1991) and Arabidopsis thaliana (Jones and JordanTab

le1

(Continued

)

Spot

num

ber

Prote

innam

ean

dfu

nct

ional

cate

gory

NC

BIac

cess

ion

num

ber

EC num

ber

Num

ber

of

pep

tides

iden

tified

by

MS

⁄MS

Mas

cot

score

*

%se

quen

ce

cove

rage

t-va

lue�

XC

XP

32

Phosp

hose

rine

amin

otr

ansf

eras

egi|7

7464928

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328

19

11Æ7

37Æ7

0

33

sery

l-tR

NA

synth

etas

egi|7

7462339

6Æ1

Æ1Æ1

16

358

17

17Æ9

23Æ5

2

Lipid

met

abolis

m

34

Enoyl

-CoA

hyd

rata

segi|7

7463724

4Æ2

Æ1Æ1

78

510

25

10Æ2

37Æ7

6

Cel

lw

all,

lipopoly

sacc

har

ide

bio

synth

esis

35

Glu

cose

-1-p

hosp

hat

eth

ymid

ylyl

tran

sfer

ase,

long

form

gi|7

7386275

2Æ7

Æ7Æ2

42

80

828Æ3

0A

bse

nt

inpla

in

Nucl

eic

acid

met

abolis

m

36

Puta

tive

rest

rict

ion

endonucl

ease

or

met

hyl

ase

gi|7

7465362

–3

121

915Æ9

6A

bse

nt

inpla

in

37

DN

A-d

irec

ted

RN

Apoly

mer

ase

bet

a-su

bunit

gi|7

7462251

2Æ7

Æ7Æ6

10

549

810Æ4

538Æ5

8

38

Puta

tive

PmbA

⁄Tld

Dgi|7

7464495

–17

1243

56

10Æ3

91Æ6

6

39

DN

Apoly

mer

ase

IIIsu

bunit

bet

agi|7

7464921

2Æ7

Æ7Æ7

21

1115

50

6Æ6

80Æ9

6

Peripla

smic

pro

tein

s40

Puta

tive

tran

smem

bra

ne

pro

tein

(MdoG

)gi|7

7465201

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737

30

11Æ6

30Æ6

1

41

ABC

moly

bdat

etr

ansp

ort

er,

per

ipla

smic

-bin

din

gpro

tein

ModA

gi|7

7386298

3Æ6

Æ3Æ2

97

513

46

10Æ7

20Æ3

8

Tetr

apyr

role

bio

synth

esis

42

Aco

nitat

ehyd

rata

se1

[Rhodobac

ter

sphae

roid

esA

TCC

17029]

gi|8

3371831

4Æ2

Æ1Æ3

15

941

16

7Æ1

80Æ 4

6

43

Porp

hobili

nogen

dea

min

ase

gi|7

7464252

2Æ5

Æ1Æ6

111

665

40

7Æ0

80Æ5

4

*Th

eM

asco

tSc

ore

isdefi

ned

as)

10

·lo

g(P

),w

her

eP

isth

epro

bab

ility

that

the

obse

rved

mat

chbet

wee

nth

eex

per

imen

taldat

aan

dth

edat

abas

ese

quen

ceis

ara

ndom

even

t.

�The

t-te

stst

atis

tic

isbas

edon

the

diffe

rence

bet

wee

nth

e%

volu

me

mea

nva

lues

(X)

of

the

two

clas

ses

(Pan

dC

),norm

aliz

edto

thei

rst

andar

ddev

iations

(s).

1·0

0·8

0·6

0·4

0·2

0·00 20 40 60 80

Time (min)

Nor

mal

ized

num

ber

of P

BG

mol

es

100 120 140

Figure 4 Normalized porphobilinogen (PBG) deaminase activity in

extracts obtained from cells grown in plain medium [d, specific enzy-

matic activity; 22 ± 2 (100%)], in 5 mmol l)1 cobalt-enriched medium

[h, specific enzymatic activity; 6Æ8 ± 0Æ7 (30Æ6%)], in 5 mmol l)1 dialy-

sed cobalt-enriched medium [s, specific enzymatic activity; 15Æ5 ± 1Æ6

(69Æ2%)] and in 5 mmol l)1 of dialysed cobalt-enriched medium

added with 10 mmol l)1 of CoCl2 [ , enzymatic specific activity

6Æ6 ± 0Æ7 (30%)]. Activities are expressed as nmol of substrate con-

sumed (mg protein))1 min)1. Experimental conditions: T = 37�C,

pH = 7 and PBG is 600 lmol l)1.


ª 2008 The Authors


1994). In this latter case, cobalt seems to inhibit up to

95% of PBGD-specific activity. In R. sphaeroides, less is

known on cobalt inhibition, but a series of divalent ions

show enzyme inhibition ranging from 9% of Fe2+to 97%

of Hg2+(Jordan and Shemin 1973). Cobalt’s indirect inhi-

bition of PBGD activity originates from the decreased

R. sphaeroides ability to express the enzyme, as suggested

also from the proteomic analysis. Altogether, the informa-

tion gathered suggest that cobalt affects the gene expres-

sion of PBGD biosynthesis, either at the transcriptional or

translational levels, in agreement with the recent tran-

scriptome investigation on the R. sphaeroides response to

oxidative stress (Zeller et al. 2005).

Given the effect of cobalt on photosynthesis, the fact

that EC50 have different values when obtained from dif-

ferent parameters suggests that more than one metabolic

pathway may interact with the polluting ion. Further-

more, the relatively high EC50 obtained from the

biomass indicates that the micro-organism could retrieve

energy from sources different from light. Among the

Table 2 Cellular function of specific enzymes in response to cobalt exposure

Enzyme Function Hypothesis on cellular response Note

Glutamate synthase ()) Ammonia assimilation Reduced cellular replication Co2+detrimental effect on bacterial

growth (Fig. 1)DNA polymerase III beta

subunit ())

DNA replication

Elongation factor Tu, P ()) Protein synthesis

Catalase (+) H2O2 detoxification Metal-induced oxidative stress

defense mechanism (Avery and

Tobin 1993; Stohs and Bagchi

1995; Rocha et al. 1996;

Cabiscol et al. 2000;

Shanmuganathan et al. 2004;

Zeller and Klug 2004)

Tetrapyrrole biosynthesis and

photosynthetic genes (Zeller et al.

2005) down-regulation is involved

in the transcriptome response to

oxidative stress in Rhodobacter

sphaeroides (Fig. 2)

DNA-directed RNA

polymerase beta subunit

fragment (+)

Gene transcription Reduced cellular gene expression

or increased degradation

General metabolic response to

cobalt-induced stress

Glutamine synthetase

class-I (+)

Ammonia assimilation Passive response Possibly associated to the

a-ketoglutarate increment induced

by glutamate synthase

down-regulation (Magasanik 1989)

SufC (+) and SufB (+) De novo assembly and ⁄ or

repair of iron–sulfur clusters

(Djaman et al. 2004)

[Fe–S] cluster damage response SUF machinery is initiated by stress

conditions (Nachin et al. 2003;

Outten et al. 2004)

Cysteine synthase (+);

aspartate-semialdehyde

dehydrogenase (+);

phosphoserine

aminotransferase (+);

seryl-tRNA synthetase (+)

Cysteine and methionine

biosynthesis

Increased metallothioneins and

GSH biosynthesis

Yeasts, plants and bacteria

upregulate glutathione,

phytochelatins and metallothioneins

to immobilize toxic metal ions

(Bruins et al. 2000; Cobbett and

Goldsbrough 2002)

PmbA ⁄ TldD (+) Modulator of DNA gyrase (Drlica

1984; Murayama et al. 1996;

Champoux 2001)

Possible response for maintaining

high DNA gyrase activity

Co exposure induces DNA gyrase

up-regulation (Bar et al. 2007).

DNA gyrase inhibition induces an

oxidative damage cellular death

pathway (Dwyer et al. 2007)

Putative transmembrane

protein (MdoG)

Biosynthesis of periplasmic

glucans (Bohin 2000; Cogez

et al. 2002)

Alteration of membrane

permeability

A possible active resistance

mechanism consisting in membrane

permeability alteration or in

periplasmic and cell wall metal

binding (Mergeay 1991; Silver and

Ji 1994; Langley and Beveridge

1999; Rodrigue et al. 2005)

ABC molybdate transporter,

periplasmic-binding protein

ModA

Part of a molybdate ABC

transporter (Imperial et al.

1998)

Hypothetical signal peptide

protein

Unknown

Glucose-1-phosphate

thymidylyltransferase,

long form

Lipopolysaccharide biosynthesis

(Reeves et al. 1996; Zuccotti

et al. 2001)



ª 2008 The Authors

over-expressed proteins, more than 30% (12 out of 35)

are enzymes involved in DNA (spot 36) or protein (spots

from 9 to 19) degradation. The degradation products rep-

resent a possible source of energy when used as substrates

for the gluconeogenesis enzymes. This agrees with the

finding that enzymes involved in glycogenosynthesis

(spots 3 and 6), glycogen mobilization (spot 1) and sub-

sequent glycolysis (spots 2, 4, 5 and 7) are up-regulated

upon cobalt exposure.

Several other enzymes were found differentially

expressed in response to cobalt exposure. At the moment,

a comprehensive evaluation of their metabolic meanings

is not possible and is beyond the aim of this paper. In

Table 2, a collection of enzymes with altered expression

levels is reported, along with their hypothetical cellular

function. Among these, cobalt treatment induces the syn-

thesis of the two proteins, SufC and SufB (spots 29 and

31, respectively), whose homologues in Escherichia coli are

involved in de novo assembly and ⁄ or repair of the dam-

aged iron–sulfur ([Fe–S]) clusters (Djaman et al. 2004);

in particular, it is suggested that iron sulphur cluster

repair (SUF) machinery may be triggered under stress

conditions (iron starvation and oxidative stress) resulting

in [Fe–S] cluster degradation (Nachin et al. 2003; Outten

et al. 2004). Aconitase activity is reported as drastically

decreased in extracts of cobalt-treated E. coli cells (Ran-

quet et al. 2007) and in line with this observation, we

found two [Fe–S] enzymes, aconitase and glutamate syn-

thase, down-expressed consistently under cobalt stress.

Further studies on specific enzymes, trascriptomics and

on the membrane proteome will help in clarifying the

metabolic response of R. sphaeroides to cobalt stress. Fur-

thermore, the over-expression of SufC and SufB might

indicate that the SUF protein machinery specifically and

efficiently contributes to Co2+resistance, but further stud-

ies are required to clarify the role of cobalt in the pertur-

bation of the [Fe–S] cluster assembly process.

Acknowledgements

The authors wish to thank Dr Francesco Milano and

Mr Giovanni Lasorella (CNR – IPCF Bari), Dr Livia

Giotta (University of Lecce) and Luca Losurdo e Rosanna

Caliandro (University of Bari) for their help. This work

was partly funded by Regione Puglia (Progetto Esplora-

tivo PE_057 – ‘Foto Bonifica Biologica’) and by Ricerca

Spontanea a Tema Libero of the Italian National Research

Council (CNR), RSTL-191.

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Soluble proteome investigation of cobalt effect on the carotenoidless mutant of Rhodobacter...

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