JOURNAL OFInorganicBiochemistry

Journal of Inorganic Biochemistry 98 (2004) 68–72

www.elsevier.com/locate/jinorgbio

Study on biological effect of La3þ on Escherichia coli by atomicforce microscopy

Liu Peng a,*, Liu Yi a,*, Lu Zhexue a, Zhu Juncheng a, Dong Jiaxin a, Pang Daiwen a,Shen Ping b, Qu Songsheng a

a Department of Chemistry, College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, PR Chinab School of Life Sciences, Wuhan University, Wuhan 430072, PR China

Received 13 May 2003; received in revised form 21 August 2003; accepted 27 August 2003

Abstract

The biological effects of rare-earth metal ions on the organism have been studied using La3þ as a probe ion and Escherichia coli

cell as a target organism. Atomic force microscopy (AFM) studies reveal that La3þ substantially changes the structure of the outer

cell membrane responsible for the cell permeability. Significant damages of the outer cell membrane are observed using scanning

electron microscopy (SEM) after the introduction of La3þ. In result, the cell becomes easily attacked by lysozyme. Moreover, in-

ductively coupled plasma-mass spectrometry (ICP-MS) measurements show considerable amount of Ca2þ and Mg2þ in the su-

pernatant from the La3þ exposed cells. It is proposed that La3þ can replace Ca2þ from the binding sites because of their close ionic

radii and similar ligand specificities. Lipopolysaccharide (LPS), which forms the outer membrane of Gram-negative bacteria, could

not serve as the cellular envelope steadily after Ca2þ and Mg2þ released from their binding sites on the LPS patches.

� 2003 Elsevier Inc. All rights reserved.

Keywords: La3þ; Lipopolysaccharide; Permeability; Biological effect; Lysozyme

1. Introduction

The physiological functions and biological effects of

the rare-earth elements have been well studied [1–3].

Recently some fertilizers with rare-earth elements addi-

tive have been used in China to improve the yields and

qualities of crops. It has been found out that the rare-

earth elements can accumulate in the soil, bioaccumu-

late in crops and finally transfer to the food chain.

Increasing attention has been paid to the influence ofrare-earth elements on the environment and human

health [4]. Although the toxic action of La3þ as a probe

ion on organisms has been reported [5], the mechanism

of its biological effects is still unclear.

Escherichia coli (E. coli) is a kind of Gram-negative

bacteria with a cell membrane and wall model shown

* Corresponding authors. Tel.: +86-27-87218284; fax: +86-27-

87647617.

E-mail addresses: lpp567@sina.com (L. Peng), liuyi@chem.whu.

edu.cn, liuyi@public.wh.hb.cn (L. Yi).

0162-0134/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2003.08.012

in Fig. 1 [6–8]. The inner membrane is composed of

phospholipid chains and proteins, and the outermembrane consists of lipopolysaccharide (LPS), pep-

tidoglycan and periplasm. LPS forms the surface of

Gram-negative bacteria and acts as a selective per-

meability barrier for E. coli and other Gram-negative

bacteria. Molecular structure of LPS is shown in Fig. 1

[8]. It is composed of lipid A, an inner core, an outer

core and the O-antigen units. Loss or damages of LPS

molecules would lead to an increase in the perme-ability of the cell wall. It is presumed that the

biological effects of rare-earth elements on the or-

ganisms contribute to the change in the membrane

permeability [2,3]. However, no molecule-/nano-level

experiment has been performed to confirm this per-

meability alteration caused by the rare-earth elements

owing to the resolution limit of optical instrumentals.

Atomic force microscopy (AFM) techniques can pro-vide nano-level image of cell surface under physio-

logical conditions [9]. It has been used to widely study

the structure details of biological systems since its

Fig. 1. Schematic molecular representation of E. coli envelop. The envelop of Gram-positive bacterium consists of the outer membrane, the inner

membrane and peptidoglycan layer. MDO is membrane-derived oligosaccharides.

L. Peng et al. / Journal of Inorganic Biochemistry 98 (2004) 68–72 69

invention in 1986 [10]. Three-dimensional (3D) high-

resolution topographs can be in situ available.

In this paper, we choose the E. coli (HB101) cell as a

target organism and La3þ as a probe ion to study the

biological effects of rare earth elements using the AFM

technique. Inductively Coupled plasma-mass spectrom-

etry (ICP-MS) and scanning electron microscopy (SEM)

have been used to further explore the biological effectmechanism of La3þ.

2. Materials and methods

2.1. Cells and reagents

The strain of E. coli (HB101), which was wild type incell wall synthesis, was provided by the Chinese Center

for Type Culture Collections of Wuhan University. The

peptone culture contained NaCl 5 g, peptone 5 g, beef

extract 5 g, Yeast extract 6 g per 1000 ml (pH¼ 7.2). The

culture was sterilized in high-pressure steam at 120 �Cfor 30 min.

Analytical grade reagent Lanthanum nitrate was

obtained from China Medicine (Group) ShanghaiChemical Reagent Corporation. Lysozyme (20 000 U/

mg) extracted from chicken egg albumin was provided

by Amresco. The lysozyme solution was diluted to

100 000 U/ml with TE buffer.

2.2. Sample preparation

When E. coli was grown to primary-log phase at37 �C in a peptone culture, two of 4 ml bacteria sus-

pensions were centrifuged at 5000 r/min for 2 min. The

precipitate was re-suspended in 4 ml ultra-pure water

and was then washed and recovered. This procedure was

repeated 5 times to thoroughly remove the culture me-

dium. 400 lg/ml La3þ was then added into one of the

washed suspensions. After the two cell suspensions were

incubated in the culture media at 37 �C for 1 h with

continuous shaking, the solutions were centrifuged at5000 r/min for 2 min. The supernatant and the bacteria

were used for the following analysis.

2.3. Analysis of Ca2þ and Mg2þ

Inductively Coupled Plasma Mass Spectrometer

(ICP-MS) (Agilent 7500a) was used in this experiment to

determine the amount of Mg2þ and Ca2þ. ICP-MS is amethod for rapid and sensitive determination of all

considered elements and the method is sufficiently sen-

sitive to detect 1 ng/l of elements [11,12]. The concen-

trations of Ca2þ and Mg2þ in the supernatant obtained

from the above experiment were analyzed by ICP-MS.

2.4. Imaging cells

AFM measurements (PicoScan system, Molecular

Imaging Inc, Tempe, AZ) were performed in the air

using a Molecular Imaging PicoScan system (Molecular

Imaging Inc, Tempe, AZ) operating in the Magnetic AC

(MAC) mode. Special coated AFM tips (type II MAC

tips, Molecular Imaging, Inc) were employed. These tips

were coated with a magnetically conductive material and

possess a lower spring constant range (1.2–5.5 N/m)than common tapping mode models due to the lower

Fig. 2. AFM images of the native E. coli (HB101) and cells treated by 400 lg/ml La3þ. The first column (A1, A2, A3) shows images of the native

E. coli in the different scanning area. The second column (B1, B2, B3) presents images of cells treated by La3þ in the different scanning area.

The scanning area of the 1st, 2nd, 3rd row are 14� 21 lm2, 4� 4 lm2, 0.5� 0.5 lm2, respectively. The 4th row presents the 3D images of graph A3

and B3.

70 L. Peng et al. / Journal of Inorganic Biochemistry 98 (2004) 68–72

Table 1

The concentration of Ca2þ and Mg2þ in the supernatant from the

native cells and the cells treated by La3þ

The native

cells (lg/l)The cells treated

by La3þ (lg/l)

Concentration of Mg2þ 24� 1.41 150� 2.12

Concentration of Ca2þ 19� 0.70 150� 2.83

L. Peng et al. / Journal of Inorganic Biochemistry 98 (2004) 68–72 71

amplitude vibrations encountered with employing MAC

mode. The advantage of this configuration is the lower

contact forces compared to other non-contact or inter-

mittent contact configuration.

2.5. Determination of alteration of membrane permeabil-

ity by SEM

Two of E. coli suspensions were grown to primary-

log phase at 37 �C in a peptone culture. One is added

with 400 lg/ml La3þ and another is left for control.

After the two cultures were incubated at 37 �C for 1 h

with continuous shaking, lysozyme solution of 2000 Uwas respectively added into the two suspensions. The

mixtures were incubated at 20 �C for 5 min and then

used for SEM study [13].

Fig. 3. SEM images of the native cells and the cells treated by La3þ

after lysozyme treatment.

3. Results and discussions

In this study, the detailed surface features of E. colicells were visualized by AFM. The typical cells we

chose are about 3–4 lm long, and a little wider than the

actual width because of tip convolution [14]. The

structure details of the native E. coli and cells treated

by La3þ were obtained, as shown in Fig. 2. In

Fig. 2(A2), AFM image reveal that the surface struc-

ture of the native E. coli is smooth at nano-level reso-

lution. We couldn�t observe the protrusion even in 3Dimage, as shown in Fig. 2(A4). By AFM, it may be

technically impossible to resolve the individual LPS and

O-antigen at the terminal of the LPS molecules. The

LPS patches of the cell surface provide an effective

permeability barrier for the Gram-negative bacteria.

However, the surface of the E. coli becomes much

rougher after the introduction of La3þ, as shown in

Fig. 2(B3). For data analysis, the surface roughness ofthe two cell populations was analyzed with visual

scanning probe microscopy (SPM) software (Molecular

Imaging Inc.) to quantify the root mean squared

(RMS). The mean values of RMS of the native cells

and the cells treated by La3þ are 9.91 and 82.46 nm,

respectively. Dark and void spaces on the surface of the

bacteria indicate that some LPS patches have broken

up in the presence of La3þ.With the help of ICP-MS technique, the reason why

LPS patches broke apart in the presence of La3þ was

explored. The cells were first washed five times using

ultra-pure water to remove the culture medium and

other elements thoroughly. After La3þ had interacted

with the cells for 1 h, the cells were then deposited. The

upper solution was analyzed for Ca2þ and Mg2þ by

ICP-MS. The data is shown in Table 1.As compared with the control, much higher

amount of Ca2þ and Mg2þ were found in the solution

of the cells treated by La3þ. It has been reported that

the metal ions such as Ca2þ and Mg2þ on the binding

sites can maintain the structure of the LPS patches.

Molecular dynamics simulation also suggests that the

absence of metals in the LPS assembly facilitates

dissociation of LPS from the assembly [15]. It is

proposed that La3þ can replace Ca2þ from the binding

sites because of their close ionic radii and similar li-

gand specificities [16–19]. But the molecular structureof LPS would be changed after the replacement due to

the difference of the electron charge between La3þ and

Ca2þ. The structure change would lead to the rupture

of LPS patches. Similarly Mg2þ is also released to the

solution after the replacement. According to AFM

images of the cells treated by La3þ with dark and void

spaces seen on the bacteria surface, LPS patches

couldn�t serve as the cellular envelope steadily.LPS is the main component of outer membrane of

Gram-negative bacteria. Lysozyme, a natural antibacte-

rial, breaks down the b ð1 ! 4Þ linkage between NAG

(N-acetyl glucosamine) and NAM (N-acetyl muramic

acid) [20]. The peptidoglycan layer locates under LPS,

so Gram-negatives are less sensitive to lysozyme than

72 L. Peng et al. / Journal of Inorganic Biochemistry 98 (2004) 68–72

Gram-positives because LPS prevents peptidoglycan

from being destroyed by lysozyme directly. Fig. 3 shows

the influence of lysozyme on the morphology of the

native E. coli cell and the cell treated with La3þ. Thenormal cells interacted with lysozyme is still rod-likeexcept for slight expansion of two ends probably owing

to the formation of spheroplasts, as shown in Fig. 3(a).

For the La3þ treated cells lysozyme causes considerable

changes in the morphology of the cells. It seems that

the rods are swollen and becomes interconnected, as

shown in Fig. 3(b). This indicates that the cells are

likely to lay in a dilute medium after the cell wall was

removed, and peptidoglycans were more quickly bro-ken down owing to lysozyme. Residual LPS patches on

the cell surface could not effectively protect the cells

after their rupture, and lysozyme could reach peptido-

glycan. These facts indicate that the permeability is

increased to some extent for the La3þ treated cells.

In summary, La3þ leads to the collapse of some LPS

patches of the cell surface and consequently alters the

permeability and functionality of the outer cell mem-brane. These provide the structural basis for the bio-

effects of La3þ on the microorganism. High resolution

AFM allows the direct visualization of the cell mem-

brane structure at the nanoscopic lever. ICP-MS and

SEM are powerful tools to support our assertion about

the bio-effects of La3þ. Further work is in progress to

investigate the interaction of La3þ with inner membrane

and cytoplasm.

Acknowledgements

We gratefully acknowledge the support of the Na-

tional Natural Science Foundation of China, the

Teaching and Research Award Program for Outstand-

ing Young Professors of High Education Institutes,

MOE, P.R. China.

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