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Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.

Nonsynonymous single nucleotide polymorphisms (SNPs) in fibronectin binding protein A (fnbA) of Staphylococcus aureus are associated with cardiac device infections. However, the role of fnbA SNPs in S. aureus arthroplasty infection is unknown. Bloodstream S. aureus isolates from a derivation cohort of patients at a single U.S. medical center with S. aureus bacteremia (SAB) and prosthetic hip or knee arthroplasties that were infected (PJI, n = 27) or uninfected (PJU, n = 43) underwent sequencing of fnbA and fnbB. A validation cohort of S. aureus bloodstream PJI (n = 12) and PJU (n = 58) isolates from Germany also underwent fnbA and fnbB sequencing. Overall, none of the individual fnbA or fnbB SNPs were significantly associated with the PJI or PJU clinical groups within the derivation cohort. Similarly, none of the individual fnbA or fnbB SNPs were associated with PJI or PJU when the analysis was restricted to patients with either early SAB (i.e., bacteremia occurring <1 year after placement or manipulation of prostheses) or late SAB (i.e., bacteremia >1 year after placement or manipulation of prostheses). In contrast to cardiac device infections, there is no association between nonsynonymous SNPs in fnbA or fnbB of bloodstream S. aureus isolates and arthroplasty infection. These results suggest that initial steps leading to S. aureus infection of cardiovascular and orthopedic prostheses may arise by distinct processes.

Magnetotactic bacteria are a diverse group of prokaryotes that biomineralize intracellular magnetosomes, composed of magnetic (Fe 3 O 4 ) crystals each enveloped by a lipid bilayer membrane that contains proteins not found in other parts of the cell. Although partial roles of some of these magnetosome proteins have been determined, the roles of most have not been completely elucidated, particularly in how they regulate the biomineralization process. While studies on the localization of these proteins have been focused solely on Magnetospirillum species, the goal of the present study was to determine, for the first time, the localization of the most abundant putative magnetosome membrane protein, MamC, in Magnetococcus marinus strain MC-1. MamC was expressed in Escherichia coli and purified. Monoclonal antibodies were produced against MamC and immunogold labeling TEM was used to localize MamC in thin sections of cells of M. marinus . Results show that MamC is located only in the magnetosome membrane of Mc. marinus . Based on our findings and the abundance of this protein, it seems likely that it is important in magnetosome biomineralization and might be used in controlling the characteristics of synthetic nanomagnetite.

Minerals are more complex than previously thought because of the discovery that their chemical properties vary as a function of particle size when smaller, in at least one dimension, than a few nanometers, to perhaps as much as several tens of nanometers. These variations are most likely due, at least in part, to differences in surface and near-surface atomic structure, as well as crystal shape and surface topography as a function of size in this smallest of size regimes. It has now been established that these variations may make a difference in important geochemical and biogeochemical reactions and kinetics. This recognition is broadening and enriching our view of how minerals influence the hydrosphere, pedosphere, biosphere, and atmosphere.

Fibronectin-binding protein A (FnBPA), a protein displayed on the outer surface of Staphylococcus aureus , has a structured A-domain that binds fibrinogen (Fg) and a disordered repeat-region that binds fibronectin (Fn). Amino acid substitutions in Fn-binding repeats (FnBRs) have previously been linked to cardiovascular infection in humans. Here we used microtiter and atomic force microscopy (AFM) to investigate adhesion by variants of full-length FnBPA covalently anchored in the outer cell wall of Lactococcus lactis , a Gram-positive surrogate that otherwise lacks adhesins to mammalian ligands. Fn adhesion increased in five of seven FnBPA variants under static conditions. The bond targeting Fn increased its strength with load under mechanical dissociation. Substitutions extended bond lifetime (1/ k off ) up to 2.1 times for FnBPA-Fn. Weaker adhesion was observed for Fg in all FnBPA variants tested with microtiter. However, mechanical dissociation with AFM showed significantly increased tensile strength for Fg interacting with the E652D/H782Q variant. This is consistent with a force-induced mechanism and suggests that the dock, lock, and latch (DLL) mechanism is favored for Fg-binding under mechanical stress. Collectively, these experiments reveal that FnBPA exhibits bimodal, ligand-dependent adhesive behavior. Amino acid substitutions in the repeat-region of FnBPA impact binding to both ligands. This was unexpected for Fg since all variants have the same A-domain sequence, and the Fg-binding site is distant from the repeat region. This indicates that FnBRs may fold back on the A-domain in a way that impacts the DLL binding mechanism for Fg.

Magnetotactic bacteria mineralize nanometer-size crystals of magnetite (Fe3O4) through a series of protein-mediated reactions that occur inside of organelles called magnetosomes. Mms6 is a transmembrane protein thought to play a key role in magnetite mineralization. We used both electron and fluorescent microscopy to examine the subcellular location of Mms6 protein within single cells of Magnetospirillum magneticum AMB-1 using Mms6-specific antibodies. We also purified magnetosomes from M. magneticum to determine if Mms6 was physically attached to magnetite crystals. Our results show that Mms6 proteins are present during crystal growth, and Mms6 is found in direct contact with the magnetite crystals or within the lipid/protein membrane surrounding the magnetite crystals. Mms6 was not detected at other subcellular locations within the bacteria or isolated fractions. Because Mms6 was found to completely surround the magnetosomes rather than being localized to one specific area of the magnetosome, it appears that this protein could act on the entire magnetite crystal during the biomineralization process. This supports a model in which Mms6 functions to regulate Fe3O4 crystal morphology. This knowledge is important for future in vitro experiments utilizing Mms6 to synthesize tailored nanomagnets with specific physical or magnetic properties.

Force microscopy has been used to quantitatively measure the infinitesimal forces that characterize interactions between Shewanella oneidensis (a dissimilatory metal-reducing bacterium) and goethite (α-FeOOH), both commonly found in Earth near-surface environments. Force measurements with subnanonewton resolution were made in real time with living cells under aerobic and anaerobic solutions as a function of the distance, in nanometers, between a cell and the mineral surface. Energy values [in attojoules (10-18joules)] derived from these measurements show that the affinity between S. oneidensis and goethite rapidly increases by two to five times under anaerobic conditions in which electron transfer from bacterium to mineral is expected. Specific signatures in the force curves suggest that a 150-kilodalton putative iron reductase is mobilized within the outer membrane of S. oneidensis and specifically interacts with the goethite surface to facilitate the electron transfer process.

Magnetotactic bacteria mineralize nanometer-size crystals of magnetite (Fe sub(3)O sub(4)) through a series of protein-mediated reactions that occur inside of organelles called magnetosomes. Mms6 is a transmembrane protein thought to play a key role in magnetite mineralization. We used both electron and fluorescent microscopy to examine the subcellular location of Mms6 protein within single cells of Magnetospirillum magneticum AMB-1 using Mms6-specific antibodies. We also purified magnetosomes from M. magneticum to determine if Mms6 was physically attached to magnetite crystals. Our results show that Mms6 proteins are present during crystal growth, and Mms6 is found in direct contact with the magnetite crystals or within the lipid/protein membrane surrounding the magnetite crystals. Mms6 was not detected at other subcellular locations within the bacteria or isolated fractions. Because Mms6 was found to completely surround the magnetosomes rather than being localized to one specific area of the magnetosome, it appears that this protein could act on the entire magnetite crystal during the biomineralization process. This supports a model in which Mms6 functions to regulate Fe sub(3)O sub(4) crystal morphology. This knowledge is important for future in vitro experiments utilizing Mms6 to synthesize tailored nanomagnets with specific physical or magnetic properties.