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7 Dps: DNA Binding Protein from Starved Cells

7 Dps: DNA Binding Protein from Starved Cells

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et al. 1992). The formation of this tightly packed highly stable crystalline structure

protects the DNA in starved E. coli cells during stationary phase. The tight packing

strongly restricts the accessibility of DNA to damaging agents and protects the

nucleoid against oxidative damage, nuclease cleavage, UV light, thermal shock and

acidic stress (Almirón et  al. 1992; Martinez and Kolter 1997; Choi et  al. 2000;

Frenkiel-Krispin et al. 2004; Nair and Finkel 2004).

The Dps monomer is a structural homolog of ferritin, a family of iron storage

proteins (Grant et al. 1998), consisting of a four helix bundle core (Fig. 8.6a). This

homology explains some of the protective functions of Dps against reactive oxygen

species (ROS) as outlined below. The monomers associate into a Dps dodecamer

with a diameter of approximately 90 Å and a hollow core of ~45 Å in diameter. Dps

contains no classical DNA binding motif and the actual DNA binding mechanism

of Dps still remains to be elucidated. It is clear, however, that Dps makes nonspecific contacts with the DNA. Because the surface of the Dps dodecamer is

negatively charged, a simple electrostatic interaction with likewise negatively

charged DNA must be excluded (Grant et al. 1998). Rather the lysine-containing

disordered N-terminus has been proposed to play an important role for DNA binding.

According to this, three dodecamers within the hexagonal Dps crystal lattice form

holes, in which the lysine-residues of the N-termini are arranged in lines. It is

assumed that the DNA passes through those holes and that the bound DNA becomes

stabilized by the basic residues of the disordered N-termini (Grant et al. 1998; Ceci

et  al. 2004). Actually, Dps variants without positively charged N-termini are

impeded in DNA binding. Moreover, studies with E. coli Dps deletion mutants

lacking the lysine-rich N-terminus confirmed the essential role of Dps in selfaggregation and DNA compaction (Ceci et al. 2004). Figure 8.6c shows a model of

a lattice of Dps dodecamer in which the disordered N-termini of each Dps monomeric subunits are indicated in red.

Dps not only protects from DNA damage by formation of a crystalline structure

with DNA, thereby dramatically reducing the accessibility of vulnerable DNA positions. It also actively protects against reactive oxygen radicals. In accordance with

its structural similarity to ferritin, Dps is able to inhibit Fe2+-dependent generation

of free radicals by the Fenton reaction through binding, sequestering and oxidation

of Fe2+. Protection can take place, whether or not Dps is bound to DNA (Ceci et al.

2003, 2004). These studies suggest that Dps in its non-DNA-bound form is still

involved in the defence of bacterial pathogens by protection against H2O2, which is

produced by the host defence system. Binding of Dps to DNA seems to be responsible for nucleoid condensation and protection against other damaging agents or

factors, such as low pH, nucleases or UV radiation. In line with the notion of being

involved in acid resistance it has been shown that E. coli Dps binds with higher

affinity to DNA at low pH conditions (Ceci et al. 2004). In summary, Dps plays an

important role in protecting bacteria against different types of long-time stress. Its

major function consists in avoiding DNA damage under the harmful conditions,

which the cells encounter during starvation.

In contrast to the other nucleoid-associated proteins no specific function of Dps

as transcription factor with regulatory properties has as yet been described. There



166



Fig. 8.7  DNA-binding and regulatory properties of NAPs. (a) Different modes of DNA structuring by NAPs are schematically illustrated. For each NAP a schematic drawing and a corresponding AFM image is shown. Different effects of NAP binding on the target DNA are displayed:

plectonemic loop (FIS), sharp DNA bends (IHF), DNA-coating (HU), DNA-wrapping (LRP) and

DNA-bending and bridging (H-NS) (Taken with permission from Pul and Wagner (2007)). The

original scanning force microscopy images of FIS and H-NS are courtesy of G. Muskhelishvili

and R.T. Dame, respectively. The IHF, H-NS and LRP pictures were originally published by

Luijsterburg et al. (2006), the HU picture by van Noort et al. (2004). (b) The regulatory network

of genes known to be under the control of the NAPs FIS, LRP, H-NS, IHF and HU, is presented.

Only those genes are indicated which are controlled by at least two NAP members. The data have

been taken from Regulon DB (2008). In addition, coloured arrows indicate autoregulation and

dashed arrows cross-regulation of the NAPs. In case of HU, FIS activates the expression of HUa

(hupA) and represses transcription of HUb (hupB) (Claret and Rouvière-Yaniv 1996)



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are indications from recent studies, however, that Dps may affect replication

through interaction with the DnaA protein, interfering with DnaA-dependent

unwinding of oriC (Chodavarapu et al. 2008).



8.8 Conclusions

All the members of the family of NAPs, especially those exemplified in this

review, share the unique properties of compacting DNA and dynamically modulating the structure of bacterial nucleoids. Many of the NAPs affect DNA supercoiling, which contributes to DNA compaction, but also is essential for different kinds

of DNA transactions, such as replication, recombination or transcription. As such,

NAPs play an important role in the maintenance of a dynamic genome and directly

or indirectly affect gene expression. Their expression levels or activities in the cell

often depend on environmental stimuli or changing growth conditions, which is

consistent with their task as environmental sensors. Their mode of interaction with

DNA is quite variable. The specificity ranges from highly sequence-specific to

completely non-specific. Binding often involves oligomeric forms of the NAPs

and interaction of different NAPs with DNA generally results in defined threedimensional structures. According to their effects on DNA structure NAPs can be

generally categorized in ‘benders’, ‘bridgers’ or ‘wrappers’ (Luijsterburg et  al.

2006, 2008). This is illustrated in Fig. 8.7a. Another characteristic property of all

NAPs consists in their capability to act in concert and to form distinct hetero- or

homomeric protein complexes. Often NAPs share overlapping DNA binding sites,

indicative of synergistic or antagonistic functions. They affect expression of each

other and together coordinate the regulation of a large number of different genes

(Fig. 8.7b).

Together, the importance of NAPs as global regulators of the bacterial cell has

become more and more evident. They are involved in transcription regulation

through many different mechanisms and often in cooperation with each other or

with different gene-specific regulatory factors. This combinatorial regulation leads

to integration of different external or internal signals and contributes to the efficient

adaptation of the cell to environmental changes.



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Chapter 9



Dps and Bacterial Chromatin

Hanne Ingmer



Abstract  When Dps was first discovered in Escherichia coli it was soon realized

that it is an unusual protein. The purified Dps bound efficiently, but non-specifically,

to DNA forming highly ordered complexes and mutants lacking Dps were sensitive

to oxidative stress. Since then Dps proteins have been found in many bacteria.

Structural studies revealed that the Dps monomer forms a bundle structure resembling the iron binding ferritins and bacterioferritins and upon oligomerization it

assembles into a dodecamer with a hollow core. Like ferritins Dps proteins are also

able to sequester and detoxify iron through a ferroxidase center that uniquely to the

Dps family is shared between two monomers. Thus, the protective capabilities of

Dps rely both on its ability to bind and physically protect DNA and its ability to

detoxify iron that otherwise may catalyze the production of toxic free radicals. On

the other hand Dps can also be used for iron storage under iron limiting conditions.

Iron restriction and oxidative stress characterizes the environment that bacterial

pathogens encounter in the human host and Dps proteins are required for full virulence of several pathogens. Thus, Dps is a versatile protein that at multiple levels

protects bacterial cells against stress.

Keywords  Dps • oxidative stress • crystallization • ferroxidase



9.1 Introduction

Oxidative stress is a universal phenomenon that can be experienced by both aerobic

and anaerobic micro-organisms. Particularly exposed are the aerobic bacteria that

generate reactive oxygen species (ROS) as part of respiration. ROS may damage cellular macromolecules including proteins, lipids and DNA and bacterial pathogens



H. Ingmer (*)

Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen,

Stigbøjlen 4, DK-1870, Frederiksberg C, Denmark

e-mail: hi@life.ku.dk

R.T. Dame and C.J. Dorman (eds.), Bacterial Chromatin,

DOI 10.1007/978-90-481-3473-1_9, © Springer Science+Business Media B.V. 2010



175



176



H. Ingmer



encounter ROS when they come into contact with the human host (Farr and Kogoma

1991; Storz et al. 1990; Miller and Britigan 1997). While the toxicity of some reactive

oxygen molecules, like H2O2, is relatively weak they can be transformed into highly

active hydroxyl radicals (OH•) in the presence of transition metals such as Fe2+

(Fe(II)) according to the Fenton reaction: H2O2+Fe2+ →OH• + OH− + Fe3+ (Henle and

Linn 1997; Luo et al. 1994). Thus, the severity of oxidative stress is highly dependent

on the availability of free iron. On the other hand iron is an essential co-factor in many

proteins participating in a variety of cellular processes such as respiration, the TCA

cycle, oxygen transport and DNA biosynthesis (Crosa 1997). In order to cope with

this dilemma, eukaryotic and prokaryotic cells have adopted proteins capable of

sequestering, storing and detoxifying free extracellular iron such as ferritins, transferrin and lactoferrin (Hartford et al. 1993; Wooldridge and Williams 1993). In bacterial

cells two major families of proteins were originally recognized as being involved in

sequestering iron, namely the heme-containing bacterioferritins and the non-heme

ferritins (Andrews 1998). However since the early 1990s, a third class of proteins has

been recognized as possessing a ferritin-like function providing both iron and hydrogen peroxide detoxification properties. This class of proteins is termed Dps for DNA

protection during starvation. Subsequently these proteins have attracted particular

interest as they structurally resemble the ferritins but provide iron detoxification

through a uniquely located ferroxidase center shared by two Dps monomers and

because many members have been shown to bind directly to DNA and physically

compact and protect the DNA from oxidative damage. In this chapter the focus will

be on the biological role of eubacterial Dps proteins in bacterial stress protection and

from the conditions that control the expression of Dps the reader will get some insight

into the environmental niches where Dps is important.



9.2 Dps Structure

Early in the study of the Escherichia coli Dps protein it was realized that an unusual

protein had been discovered. The purified protein bound efficiently to DNA and

formed stable complexes that resisted heat treatment and was visualized by electron

microscopy as large, highly ordered two-dimensional arrays described as honeycombs (Almirón et al. 1992). Six years later the crystal structure was solved at 1.6Å

resolution by X-ray crystallography revealing that the E. coli Dps monomer displays

the same four-helix bundle structure as ferritin and bacterioferritin but upon oligomerization it assembles into a ball-like dodecamer with a hollow core and pores

at three-fold axes of symmetry in contrast to the 24-mer of ferritins (Grant et  al.

1998). In other bacterial species the spherical shape of the dodecamer has consistently been reproduced for a number of Dps family member proteins (Tonello et al.

1999; Ilari et al. 2000; Zanotti et al. 2002; Stillman et al. 2005; Kim et al. 2006).

Little is known about the oligomerization process of the Dps monomers into the

dodecamer. For one of the Dps paralogues present in Mycobacterium smegmatis,

MsDps1, a C-terminal extension of 26 C-terminal amino acids is required for

oligomerization (Roy et  al. 2007) whereas in the other paralogue, MsDps2,



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