Elsevier

Gene

Volume 296, Issues 1–2, 21 August 2002, Pages 121-128
Gene

The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase

https://doi.org/10.1016/S0378-1119(02)00839-9Get rights and content

Abstract

The Gram-positive bacterium Listeria innocua possesses an authentic ferritin with an unusual dodecameric assemblage that resembles the quaternary structure of the DNA-binding proteins designated Dps (DNA-binding proteins from starved cells). The L. innocua gene encoding the above protein, termed ferritin from Listeria innocua (fri), has been localized on a 3-kb HindIII chromosomal fragment cloned in the Escherichia coli strain DH5αF′. DNA sequence analysis reveals an open reading frame of 468 nucleotides matching perfectly the amino acid sequence of the protein. Primer extension analysis indicates the presence of two transcriptional startpoints located 36 (proximal) and 85 nt (distal) upstream the fri start codon, respectively. Each transcriptional startpoint is preceded by suitably located −10 and −35 elements, which match the σA (proximal) and σB (distal) consensus sequences.

In L. innocua and Liseria monocytogenes, fri expression increases both upon entry into stationary phase and, more markedly, under low-iron growth conditions. The effect of iron is apparent in the exponential and stationary phases of growth. An up-regulation by iron limitation has never been observed in other proven ferritins and bacterioferritins, but has been reported for several members of the Dps family. The unusual regulation by iron of the Listeria ferritin gene provides further support to the evolutionary link with the Dps family and suggests that the iron storage function may not be the unique role of ferritin in the physiology of this bacterium.

Introduction

Iron is essential for nearly all forms of life, but is in short supply due to its low solubility at physiological pH. Living organisms therefore have developed specific molecules that permit iron acquisition, transport and storage. The first two processes are effected by molecules of diverse size and chemical nature, notably siderophores in many bacteria and fungi, and transferrins in higher vertebrates, whereas the iron storage function is performed by ferritin, a ubiquitary, highly conserved protein (Andrews, 1998). Ferritins are polymers of 24 identical or similar subunits forming a spherical protein shell where up to 4500 iron atoms can be sequestered. Iron is thereby solubilized in a readily available form and at the same time its potential toxicity is reduced since the metal is unlikely to participate in free-radical-producing reactions (Harrison and Arosio, 1996).

In plants and animals, the physiological role of ferritin as iron store is in line with the enhanced expression of the ferritin gene(s) in iron-rich conditions (Harrison and Arosio, 1996). In Gram-negative bacteria two kinds of well characterized iron-storage proteins, typical ferritins and heme-containing bacterioferritins, are present. In Escherichia coli the bacterioferritin (bfr) and ferritin (ftnA) genes have been well characterized and their expression has been found to increase under iron-rich growth conditions and during the post-exponential phase of growth (Andrews, 1998). Induction by iron has been observed for Helicobacter pylori ferritin (Bereswill et al., 1998) and for bacterioferritin of Rhodobacter capsulatus (Ringeling et al., 1994). Expression of bacterioferritin from the cyanobacterium Synechocystis PCC 6803 is not influenced by iron availability in the concentration range 3–300 μM suggesting that Synechocystis bacterioferritin plays a role as a carrier in the reverse electron-transport system from cytocrome c-522 to NADP+ (Laulhère et al., 1992).

An unusual ferritin has been isolated from the Gram-positive bacterium Listeria innocua (Bozzi et al., 1997). L. innocua ferritin binds and incorporates iron as an authentic ferritin, although its sequence and structure are related to the DNA-binding proteins designated DNA-binding proteins from starved cells (Dps) that are expressed by bacteria under conditions of oxidative or nutritional stress (Almiron et al., 1992, Peña and Bullerjahn, 1995, Yamamoto et al., 2000). In fact, the apoferritin shell of L. innocua does not contain 24 subunits like all ferritins, but is characterized by the dodecameric assemblage described for the Dps protein from E. coli (Bozzi et al., 1997, Grant et al., 1998). Each subunit contains one high affinity iron-binding site that resembles known ferroxidase sites (Ilari et al., 2000). Interestingly, the iron ligands in Listeria ferritin are conserved in the Dps proteins and belong to the group of amino acids forming the so-called DNA-binding signature, an observation in line with the recently reported iron-binding capacity of a neutrophil-activing dodecameric protein from H. pylori (Tonello et al., 1999). At variance with the Dps proteins, Listeria ferritin does not appear to bind DNA (Bozzi et al., 1997).

As part of the Listeria spp. genome project, the ferritin gene (fri) has been identified at 970,638 bp in the 3,010,209 bp L. innocua chromosome and at 979,044 in the 2,944,528 bp L. monocytogenes chromosome (Glaser et al., 2001). In the present work, the fri gene has been isolated and characterized at the transcriptional level in L. innocua. The influence of iron availability and bacterial growth phase on gene expression has been demonstrated for both L. innocua and L. monocytogenes, the latter being the only species of the genus Listeria pathogenic for humans. fri expression is found to be increased slightly upon entry into stationary phase and markedly under low-iron growth conditions, independently of the growth phase. This behaviour resembles that of the Dps family proteins (Sen et al., 2000). The iron dependence of fri expression is of special interest in L. monocytogenes since iron is known to modulate expression of several virulence-related genes; in particular, the low-iron environment of the host is a signal that influences the infection process (Portnoy et al., 1992, Conte et al., 1996).

Section snippets

Bacterial strains and growth conditions

L. innocua strain LS1 (kindly supplied by Dipartimento di Scienze di Sanità Pubblica, University of Rome ‘La Sapienza’) and L. monocytogenes strain LM1, a clinical wild type isolate, were used in this study. Unless otherwise stated both strains were grown in brain heart infusion (BHI), broth containing about 20 μM Fe+3 (Merck). To achieve iron-limiting growth conditions, bacteria were grown in low-iron medium (LIM), obtained by treating BHI with Chelex-100 (Conte et al., 1996), supplemented

Sequence analysis of the ferritin gene in L. innocua

The complete genome sequence of Listeria spp. has been released in October 2001. At the time the present work was undertaken only the primary sequence of L. innocua ferritin was available (Bozzi et al., 1997). Therefore, to clone the fri gene, genomic DNA from L. innocua LS1 strain was digested with HindIII and a 2.8–3.0 kb size-selected plasmid library was screened with the fri-probe. During purification of the plasmids derived from three positive clones, a different behaviour was observed.

Discussion

The present work relates to factors affecting the expression of the gene coding for ferritin, fri, both in L. innocua and in the facultative pathogen L. monocytogenes.

The L. innocua fri gene was mapped on a 3-kb HindIII chromosomal DNA fragment and the corresponding nucleotide sequence, the first encoding a ferritin from a Gram-positive bacterium, was obtained. The fri open reading frame encodes a protein product matching perfectly the amino acid sequence of the protein (Bozzi et al., 1997). By

Acknowledgements

M. Polidoro and D. De Biase contributed equally to the work. Grants from the Istituto Pasteur-Fondazione Cenci Bolognetti and local grants from the Ministero Istruzione Università Ricerca to E.C. and the Consiglio Nazionale delle Ricerche (9801145.CT14) to S.S. are acknowledged. This paper is dedicated to the memory of Prof. Franco Tatò.

References (32)

  • S. Bereswill et al.

    Structural, functional and mutational analysis of the pfr gene encoding a ferritin from Helicobacter pylori

    Microbiology

    (1998)
  • L. Chen et al.

    Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions

    J. Bacteriol.

    (1993)
  • M.P. Conte et al.

    Iron availability affects entry of Listeria monocytogenes into the enterocyte cell line Caco-2

    Infect. Immun.

    (1996)
  • D. De Biase et al.

    The response to stationary phase stress conditions in Escherichia coli: role and regulation of the glutamic acid decarboxylase system

    Mol. Microbiol.

    (1999)
  • O. Dussurget et al.

    An ideR mutant of Mycobacterium smegmatis has a derepressed siderophore production and an altered oxidative-stress response

    Mol. Microbiol.

    (1996)
  • M. Espinosa-Urgel et al.

    A consensus structure for sigmaS-dependent promoters

    Mol. Microbiol.

    (1996)
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