Post on 14-May-2023
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: m.trotta@ba.ipcf.cnr.it
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.
340 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349
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.
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) 338–349 341
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).
Cobalt effect on R. sphaeroides F. Pisani et al.
342 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349
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.
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) 338–349 343
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
Cobalt effect on R. sphaeroides F. Pisani et al.
344 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349
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
–14
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.
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) 338–349 345
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)
Cobalt effect on R. sphaeroides F. Pisani et al.
346 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 338–349
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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