Virulence Phenotyping and Molecular Characterization of a Low-pathogenicity Isolate of Listeria monocytogenes from Cow＇s Milk

A low-pathogenicity isolate of Listeria monocytogenes from cow＇s milk, as screened in mouse and chicken embryonated egg models, was examined for virulence-related phenotypic traits. Corresponding virulence genes （iap, prfA, picA, hly, mpl, actA, plcB, InlA and lnlB） were compared with L. monocytogenes reference strains 10403S and EGD to elucidate the possible molecular mechanisms of low virulence. Although L. monocytogenes H4 exhibited similar patterns to strain 10403S in terms of hemolytic activity, in vitro growth and invasiveness and even had higher adhesiveness, faster intracellular growth and higher phospholipase activity in vitro, it was substantially less virulent than the strain 10403S in mouse and chicken embryo models （50% lethal dose： 10^8.14 VS. 10^5.49 and 10^6.73 VS. 10^1.9, respectively）. The genes prfA, picA and mpl were homologous among L. monocytogenes strains H4, 10403S and EGD （〉98%）. Genes iap, hly, plcB, lnlA and lnlB of L. monocytogenes 10403S had higher homology to those of strain EGD （〉98%） than isolate H4. The homology of the gene hly between strain 10403S and isolate H4 was 96.9% at the nucleotide level, but 98.7% at the amino acid level. The actA gene of isolate H4 had deletions of 105 nucleotides corresponding to 35 amino acid deletions falling within the proline-rich region. Taken together, this study presents some clues as to reduced virulence to mice and chicken embryos of the isolate H4 probably as a result of deletion mutations of actA.     

Effect of glucose on Listeria monocytogenes biofilm formation,and assessment of the biofilm’s sanitation tolerance

Listeria monocytogenes is an important cause of human foodborne infections and its ability to form biofilms is a serious concern to the food industry. To reveal the effect of glucose conditions on biofilm formation of L. monocytogenes, 20 strains were investigated under three glucose conditions (0.1, 1.0, and 2.0% w v^–1) by quantifying the number of cells in the biofilm and observing the biofilm structure after incubation for 24, 72, and 168 h. In addition, the biofilms were examined for their sensitivity to sodium hypochlorite. It was found that high concentrations of glucose reduced the number of viable cells in the biofilms and increased extracellular polymeric substance production. Moreover, biofilms formed at a glucose concentration of 1.0 or 2.0% were more resistant to sodium hypochlorite than those formed at a glucose concentration of 0.1%. This knowledge can be used to help design the most appropriate sanitation strategy.     

Metabolism of Poly-β-Hydroxybutyric Acid in Yersinia pseudotuberculosis and Listeria monocytogenes Cultivated at Different Temperatures

A comparative investigation of the intracellular content of poly--hydroxybutyric acid showed that Yersinia pseudotuberculosis strains accumulated, on the average, lower amounts of this reserve substance than Listeria monocytogenes strains. The intracellular pool of poly--hydroxybutyric acid was responsible for the growth of the bacteria at low temperatures (4–6°C) in the absence of any exogenous carbon and energy source.     

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.     

Listeria monocytogenes’ Step-Like Response to Sub-Lethal Concentrations of Nisin

Microbial safety of food products is often accomplished by the formulation of food-grade preservatives into the product. Because of the growing consumer demand for natural substances (including preservatives) in the composition of consumed foods, there is also a growing interest in the natural antimicrobial nisin, which has generally recognized as safe (GRAS) status for certain applications. During the products storage time, concentrations of preservative(s) are decreasing, which may eventually cause a serious problem in the food’s microbial safety. Here, for the first time we report on the non-linear response of a foodborne pathogen, Listeria monocytogenes, to sub-lethal concentrations of nisin.     

Effect of experimental infection with Listeria monocytogenes on the development of pregnancy and on concentrations of progesterone,oestrone sulphate and 15-ketodihydro-PGF2α in the goat

The effect of Listeria monocytogenes infection on hormone levels in pregnant goats was studied. Four goats (Group I) received an intravenous inoculation of a bacterial culture (Type 1) on Days 69–77 and another four goats (Group II) received a similar inoculation on Days 105–106 of gestation. Five non-inoculated goats were used as controls. Plasma was analysed for progesterone, oestrone sulphate and 15-ketodihydro-PGF_2α. The status of the foetus was followed using real-time ultrasonography.Three of the four goats in Group I aborted 8–10 days after inoculation. The fourth goat gave birth to a normal live kid at term. The three goats which aborted showed clinical signs of disease in connection with abortion. In Group II, all goats aborted after 9–11 days. All the goats showed clinical symptoms of disease from a few days after inoculation and the symptoms continued until abortion. The clinical symptoms of disease were more pronounced in Group II than in Group I. L. monocytogenes was isolated from all aborted foetuses. None of the control goats aborted.Ultrasound examination revealed foetal death either immediately before or up to 2 days before abortion. Mummification had begun in the foetus that had been dead for 2 days before expulsion.In comparison with pre-inoculation plasma levels in Group I, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels were observed from Days 4 and 6 after inoculation, respectively. In Group II, a significant decrease (P < 0.05) in progesterone levels and an increase in 15-ketodihydro-PGF_2α levels in plasma were observed from Days 8 and 6, respectively. The oestrone sulphate levels decreased slightly in the inoculated goats a few days before abortion.The pattern of changes in levels around abortion was similar to the pattern present in the control animals around parturition. However, oestrone sulphate levels did not increase in the inoculated groups before abortion in contrast to goats which delivered healthy kids. The changes in levels of 15-ketodihydro-PGF_2α in goats that aborted indicated that the endocrine foetal-placental function was disturbed, which was most likely due to the establishment and development of L. monocytogenes in the placenta and foetus.     

The Timing of IFNβ Production Affects Early Innate Responses to Listeria monocytogenes and Determines the Overall Outcome of Lethal Infection

Dendritic cells (DCs) and natural killer (NK) cells are essential components of the innate immunity and play a crucial role in the first phase of host defense against infections and tumors. Listeria monocytogenes (Lm) is an intracellular pathogen that colonizes the cytosol of eukaryotic cells. Recent findings have shown Lm specifically in splenic CD8a(+) DCs shortly after intravenous infection. We examined gene expression profiles of mouse DCs exposed to Lm to elucidate the molecular mechanisms underlying DCs interaction with Lm. Using a functional genomics approach, we found that Lm infection induced a cluster of late response genes including type I IFNs and interferon responsive genes (IRGs) in DCs. Type I INFs were produced at the maximal level only at 24 h post infection indicating that the regulation of IFNs in the context of Lm infection is delayed compared to the rapid response observed with viral pathogens. We showed that during Lm infection, IFNγ production and cytotoxic activity were severely impaired in NK cells compared to E. coli infection. These defects were restored by providing an exogenous source of IFNβ during the initial phase of bacterial challenge. Moreover, when treated with IFNβ during early infection, NK cells were able to reduce bacterial titer in the spleen and significantly improve survival of infected mice. These findings show that the timing of IFNβ production is fundamental to the efficient control of the bacterium during the early innate phase of Lm infection.     

Critical Role of a Ferritin-Like Protein in the Control of Listeria monocytogenes Cell Envelope Structure and Stability under β-lactam Pressure

The human pathogen Listeria monocytogenes is susceptible to the β-lactam antibiotics penicillin G and ampicillin, and these are the drugs of choice for the treatment of listerial infections. However, these antibiotics exert only a bacteriostatic effect on this bacterium and consequently, L. monocytogenes is regarded as β-lactam tolerant. It is widely accepted that the phenomenon of bacterial tolerance to β-lactams is due to the lack of adequate autolysin activity, but the mechanisms of L. monocytogenes tolerance to this class of antibiotics are poorly characterized. A ferritin-like protein (Fri) was recently identified as a mediator of β-lactam tolerance in L. monocytogenes, but its function in this process remains unknown. The present study was undertaken to improve our understanding of L. monocytogenes tolerance to β-lactams and to characterize the role of Fri in this phenomenon. A comparative physiological analysis of wild-type L. monocytogenes and a fri deletion mutant provided evidence of a multilevel mechanism controlling autolysin activity in cells grown under β-lactam pressure, which leads to a reduction in the level and/or activity of cell wall-associated autolysins. This is accompanied by increases in the amount of teichoic acids, cell wall thickness and cell envelope integrity of L. monocytogenes grown in the presence of penicillin G, and provides the basis for the innate β-lactam tolerance of this bacterium. Furthermore, this study revealed the inability of the L. monocytogenes Δ fri mutant to deplete autolysins from the cell wall, to adjust the content of teichoic acids and to maintain their D-alanylation at the correct level when treated with penicillin G, thus providing further evidence that Fri is involved in the control of L. monocytogenes cell envelope structure and stability under β-lactam pressure.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Influence of Fatty Acid Precursors,Including Food Preservatives,on the Growth and Fatty Acid Composition of Listeria monocytogenes at 37 and 10°C

Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures and increases its content of anteiso-C_15:0 fatty acid, which is believed to be a homeoviscous adaptation to ensure membrane fluidity, at these temperatures. As a possible novel approach for control of the growth of the organism, the influences of various fatty acid precursors, including branched-chain amino acids and branched- and straight-chain carboxylic acids, some of which are also well-established food preservatives, on the growth and fatty acid composition of the organism at 37°C and 10°C were studied in order to investigate whether the organism could be made to synthesize fatty acids that would result in impaired growth at low temperatures. The results indicate that the fatty acid composition of L. monocytogenes could be modulated by the feeding of branched-chain amino acid, C₄, C₅, and C₆ branched-chain carboxylic acid, and C₃ and C₄ straight-chain carboxylic acid fatty acid precursors, but the growth-inhibitory effects of several preservatives were independent of effects on fatty acid composition, which were minor in the case of preservatives metabolized via acetyl coenzyme A. The ability of a precursor to modify fatty acid composition was probably a reflection of the substrate specificities of the first enzyme, FabH, in the condensation of primers of fatty acid biosynthesis with malonyl acyl carrier protein.Listeriosis is a severe and life-threatening human infection encompassing meningoencephalitis, meningitis, focal infections in the immunocompromised, and stillbirths and neonatal sepsis due to infection of pregnant women (2). The disease is caused by the Gram-positive food-borne pathogen Listeria monocytogenes, which is responsible for common-source and sporadic disease involving a variety of different foods (27). Listeriosis has a high fatality rate (24). The U.S. Department of Agriculture has a zero tolerance policy for L. monocytogenes in ready-to-eat products, and high costs are associated with product recalls.L. monocytogenes has a remarkably low minimum growth temperature, e.g., −0.1°C (34), and thus the organism can multiply to dangerous levels when food is kept at refrigeration temperatures. We are interested in the molecular mechanisms of L. monocytogenes psychrotolerance, with a view to applying this knowledge to improve the control of the growth of the organism. Although the adaptations involved in low-temperature tolerance are global in scope, we have focused on changes in fatty acid composition that result in homeoviscous adjustments of membrane fluidity (31, 36). L. monocytogenes has a fatty acid composition that is dominated to an unusual extent (90% or more) by branched-chain fatty acids (BCFAs); the major fatty acids are anteiso-C_15:0, anteiso-C_17:0, and iso-C_15:0. Numerous studies have shown that the major change in fatty acid composition when L. monocytogenes is grown at low temperatures is an increase in the content of anteiso-C_15:0 fatty acid to 65% or more of the total (1, 12, 23, 25, 26, 28). Two cold-sensitive mutants with Tn917 insertions in the branched-chain α-keto acid dehydrogenase gene complex (bkd) were deficient in BCFAs, grew poorly at low temperatures, and had decreased membrane fluidity; all of these defects could be restored by growth in the presence of 2-methylbutyrate (2-MB), a precursor of odd-numbered anteiso fatty acids, including anteiso-C_15:0 fatty acid (1, 7, 13, 37). We believe that anteiso-C_15:0 fatty acid imparts fluidity to the cytoplasmic membrane, as revealed by its low phase transition temperature in model phospholipids (18) and disruption of the close packing of fatty acyl chains (21, 35).The amino acids isoleucine, leucine, and valine are the starting points for the biosynthesis of odd-numbered anteiso, odd-numbered iso, and even-numbered iso fatty acids, respectively (18, 37). The amino acids are converted to their corresponding α-keto acid derivatives through the activity of branched-chain amino acid transaminase. Branched-chain α-keto acid dehydrogenase (Bkd) then converts these α-keto compounds to branched-chain acyl coenzyme A (acyl-CoA) primers of fatty acid biosynthesis (18). These primers are then used to initiate fatty acid biosynthesis through the activity of β-ketoacyl-acyl carrier protein synthase III (FabH), which prefers branched-chain acyl-CoAs to acetyl-CoA as substrates (4, 22, 32). β-Keto-acyl carrier protein synthase II (FabF) is responsible for subsequent rounds of elongation until the acyl chain reaches 14 to 17 carbon atoms (36).We wished to ascertain whether we could manipulate the fatty acid composition of L. monocytogenes by feeding precursors that favored the production of fatty acids other than anteiso-C_15:0 and thereby inhibit the growth of the organism, especially at low temperatures. Kaneda (15, 16) has grouped Bacillus subtilis fatty acids into four pairs based on the precursors from which they are generated, i.e., anteiso-C_15:0 and C_17:0 from isoleucine, iso-C_15:0 and C_17:0 from leucine, iso-C_14:0 and C_16:0 from valine, and n-C_14:0 and n-C_16:0 from acetate or butyrate. The proportions of the fatty acids could be modulated by precursor feeding. We have studied the effects of feeding the potential fatty acid precursors branched-chain amino acids, branched-chain α-keto acids, short branched-chain carboxylic acids, short straight-chain carboxylic acids, medium-length straight-chain carboxylic acids, branched-chain C₆ carboxylic acids, and sodium diacetate (Fig. ​(Fig.1)1) on the growth and fatty acid composition of L. monocytogenes. Various short-chain carboxylic acids are used as food preservatives (5, 8, 29), and it was of interest to see whether any of them had an effect on the fatty acid composition of L. monocytogenes. Precursors giving rise to C₅ and C₆ branched-chain acyl-CoA derivatives, propionate, and butyrate had significant impacts on growth and fatty acid composition. Acetate and precursors that were metabolized to acetyl-CoA had minor effects on fatty acid composition, indicating that their preservative action is not due to effects on fatty acid composition.Open in a separate windowFIG. 1.Structures of potential fatty acid precursors.     

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes–Escherichia coli dual-species biofilms

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes–Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r² = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.     

Rapid, transient, and proportional activation of σ(B) in response to osmotic stress in Listeria monocytogenes

The osmotic activation of sigma B (σ(B)) in Listeria monocytogenes was studied by monitoring expression of four known σ(B)-dependent genes, opuCA, lmo2230, lmo2085, and sigB. Activation was found to be rapid, transient, and proportional to the magnitude of the osmotic stress applied, features that underpin the adaptability of this pathogen.     

Iron Translocation into and out of Listeria innocua Dps and Size Distribution of the Protein-enclosed Nanomineral Are Modulated by the Electrostatic Gradient at the 3-fold ��Ferritin-like�� Pores

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called “ferritin-like” because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.The widely distributed bacterial Dps proteins (1, 2) belong to the ferritin superfamily and are characterized by strong similarities (3) but also distinctive differences with respect to “canonical” ferritins, the ubiquitous iron storage, and detoxification proteins found in biological systems. The structural resemblance is apparent in the overall molecular assemblage because both Dps proteins and ferritins are shell-like oligomers constructed from four-helix bundle monomers. However, Dps proteins are 12-mers of identical subunits that assemble with 23 symmetry, whereas ferritins are built by 24 highly similar or identical subunits related by 432 symmetry. The functional similarities consist in the common capacity to remove Fe(II) from cytoplasm, catalyze its oxidation, and store Fe(III) thus produced in the protein cavity, wherefrom the metal can be mobilized when required by the organism. However, ferritins use molecular oxygen as iron oxidant with the production of hydrogen peroxide, whereas Dps proteins prefer hydrogen peroxide, which is typically about 100-fold more efficient than molecular oxygen (1). This difference is of major importance because it renders Dps proteins capable of removing concomitantly Fe(II) and H₂O₂ whose combination leads to the production of reactive oxygen species via Fenton chemistry (4). This capacity confers H₂O₂ resistance and hence may be a virulence factor in certain pathogens (e.g. Campylobacter jejuni, Streptococcus mutans, and Porphyromonas gingivalis) because the H₂O₂ burst represents one of the first defense lines of the host during infection (5–7).Key to a full understanding of the iron uptake and release processes at a molecular level is the route by which iron enters and exits the protein shell. In both 24-subunit ferritins and 12-subunit Dps proteins, the subunit assemblage creates pores across the protein shell that put the internal cavity in communication with the external medium. In ferritins there are two types of pores: largely hydrophobic ones along the axes with 4-fold symmetry and hydrophilic ones along the axes with 3-fold symmetry. The latter channels are funnel-shaped, with the smaller opening toward the protein cavity, and are lined with conserved glutamic and aspartic residues located in the narrow region of the funnel (8). These 3-fold pores were recognized to provide the route for iron entry into the protein cavity soon after resolution of the horse ferritin x-ray crystal structure (9). Later site-directed mutagenesis studies defined the role of specific residues (Asp¹³¹ and Glu¹³⁴) that line the pore (10, 11), whereas electrostatic calculations related the passage of iron to the existence of a gradient that drives metal ions toward the protein interior cavity (12, 13). More recently, the 3-fold symmetry pores were shown to be involved also in the exit process of iron from the protein cavity. Thus, in H-frog ferritin used as model system, iron exit is affected by local protein unfolding promoted by site-specific mutagenesis of individual amino acid residues (14, 15), by the use of chaotropes (16), and by means of selected peptides designed to bind at these channels (17).In Dps proteins, the protein shell is breached by two types of pores along the 3-fold axes, one type is formed by the N-terminal portion of the monomers and bears a strong similarity to the typical 3-fold channels of 24-subunit ferritins in that it is funnel-shaped, hydrophilic, and lined by conserved, negatively charged residues. It was therefore named “ferritin-like” and assumed to be involved in iron entry into the protein cavity upon resolution of the Listeria innocua x-ray crystal structure (18). The other type of pore is formed by the C-terminal ends of the monomers and was called “Dps type” because it is created at a subunit interface that is unique to Dps proteins. Although somewhat variable in length and in the size of the openings, the Dps type pore is mainly hydrophobic in nature (19).The present paper investigates the role of the ferritin-like pores in the iron uptake and release processes in Dps proteins using the well characterized L. innocua Dps (LiDps) as a model system (18, 20–22). In LiDps, the ferritin-like pores contain a set of three aspartate residues, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ that would be encountered in succession by a metal ion that is attracted by the electrostatic gradient they create and moves down the funnel-shaped pore toward the protein cavity (Fig. 1). Asp¹³⁰, which is located in the narrowest part of the funnel, is conserved significantly among Dps proteins (∼ 80%), whereas Asp¹²¹ and Asp¹²⁶ are less conserved (Fig. 1). Such considerations were used in the design of site-specific variants D121N, D126N, D130N, and D121N/D126N/D130N to elucidate the function of the ferritin-like pores in Dps proteins.Open in a separate windowFIGURE 1.Dps proteins sequences and conservation of the aspartate residues that line the 3-fold ferritin-like pores in L. innocua Dps. A, alignment of multiple Dps sequences from different bacteria: LiDps, non-heme iron-binding ferritin (L. innocua Clip11262]; EcDps, DNA-binding protein Dps (E. coli); HpDps, neutrophil-activating protein (Helicobacter pylori); YpDps, ferritin family protein (Yersinia pestis Angola); GtDps, DNA-protecting protein (Geobacillus thermodenitrificans NG80–2); RmDps, ferritin, and Dps (Ralstonia metallidurans CH34); AtDps, DNA protection during starvation conditions (Agrobacterium tumefaciens str. C58); TeDps Dps family DNA-binding stress response protein (Thermosynechococcus elongatus BP-1); PaDps, putative DNA-binding protein, Dps (Psychrobacter arcticus 273-4); TfDps, hypothetical protein Tfu_0799 (Thermobifida fusca YX); PhDps, DNA-binding DPS protein (Pseudoalteromonas haloplanktis TAC125); SoDps, Dps family protein (Shewanella oneidensis MR-1); BaDps1, general stress protein 20U (Bacillus anthracis str. Ames); BaDps2 general stress protein (B. anthracis str. Ames); LlDps, non-heme iron-binding ferritin (Lactococcus lactis subsp. lactis Il1403); VcDps, DPS family protein (Vibrio cholerae MZO-3); and StDps, DNA-binding ferritin-like protein (oxidative damage protectant) (Streptococcus thermophilus LMD-9). Residues forming the Dps catalytic center are highlighted in pale blue (His³¹, His⁴³, Asp⁵⁸, and Glu⁶² in LiDps); 3-fold pores aspartate residues are highlighted in yellow and marked in bold type. Alignment has been created with ClustalW2 (34). B, view of the junction of three monomers forming the 3-fold ferritin-like pore. C, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ aspartate residues comprised the pore area. D, three-dimensional view of the pore colored by charge. Red, negatively charged residues; blue, positively charged residues; white, uncharged residues. E, schematic representation of the vertical section of the pore. The images were created with PyMol (35).The results demonstrate that iron uses the LiDps ferritin-like pores to enter and leave the protein shell and hence that these pores have the same role as the structurally similar 3-fold channels in 24-subunit ferritins. LiDps residue Asp¹³⁰ is the most important determinant of the negative electrostatic gradient because of its location in the narrow part of the pores. Importantly, the data show for the first time that the electrostatic gradient at the pores modulates cooperativity in the iron uptake process and influences the size distribution of the iron core (23). The effect of the electrostatic gradient can be explained in terms of the electrostatic interaction effects between the fixed negative charges of the aspartate residues at the pores and the mobile positive charges of iron ions.