Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Ni²⁺-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni²⁺-binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni²⁺-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.     

Staphylococcal surface display of metal-binding polyhistidyl peptides

Recombinant Staphylococcus xylosus and Staphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni(2+)- and Cd(2+)-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.     

Enhanced Mercury Biosorption by Bacterial Cells with Surface-Displayed MerR

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg²⁺ biosorption than that found in the wild-type JM109 cells. Hg²⁺ binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd²⁺ and Zn²⁺. The Hg²⁺ binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.     

Biochemical characterization of I-CmoeI reveals that this H-N-H homing endonuclease shares functional similarities with H-N-H colicins

Endonuclease assays of the H-N-H proteins encoded by two group I introns in the Chlamydomonas moewusii chloroplast psbA gene revealed that the CmpsbA·1 intron specifies a site-specific DNA endonuclease, designated I-CmoeI. Like most previously reported intron-encoded endonucleases, I-CmoeI generates a double-strand break near the insertion site of its encoding intron, leaving 3′ extensions of 4 nt. This enzyme was purified from Escherichia coli as a fusion protein with a His tag at its N-terminus. The recombinant protein (rI-CmoeI) requires a divalent alkaline earth cation for DNA cleavage (Mg²⁺ > Ca²⁺ > Sr²⁺ > Ba²⁺). It also requires a metal cofactor for DNA binding, a property shared with H-N-H colicins but not with the homing endonucleases characterized to date. rI-CmoeI binds its recognition sequence as a monomer, as revealed by gel retardation assays. K_m and k_cat values of 100 ± 40 pM and 0.26 ± 0.04 min^–1, respectively, were determined. Replacement of the first histidine of the H-N-H motif by an alanine residue abolishes both rI-CmoeI activity and binding to its substrate. We propose that this conserved histidine residue plays a role in binding the metal cofactor and that such binding induces a structural modification of the enzyme which is required for DNA recognition.     

Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif

A gene coding for a de novo peptide sequence containing a metal binding motif was chemically synthesized and expressed in Escherichia coli as a fusion with the maltose binding protein. Bacterial cells expressing the metal binding peptide fusion demonstrated enhanced binding of Cd²⁺ and Hg²⁺ compared to bacterial cells lacking the metal binding peptide. The potential use of genetically engineered bacteria as biosorbents for the removal of heavy metals from wastewaters is discussed.     

Functional analysis of the homeodomain protein SIX5

SIX5 (previously known as myotonic dystrophy associated homeodomain protein-DMAHP) is a member of the SIX [sine oculis homeobox (Drosophila) homologue] gene family which encodes proteins containing a SIX domain adjacent to a homeodomain. To investigate the DNA binding specificities of these two domains in SIX5, they were expressed as GST fusion proteins, both separately and together. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with double stranded oligonucleotides representing putative DNA binding sites. The putative sites included two in the promoter region of DMPK (dystrophia myotonica protein kinase) and the previously characterised murine Six4 DNA binding site in the Na⁺/K⁺ ATPase α1 subunit gene (ATP1A1) regulatory element (ARE). None of the recombinant proteins showed any affinity for the two putative sites in DMPK. However, the two recombinant proteins containing the homeodomain both formed at least one specific complex with the ARE. The recombinant protein containing both domains formed a second specific complex with the ARE, assumed to be a dimer complex. Finally, a whole genome PCR-based screen was used to identify genomic DNA sequences to which SIX5 binds, as an initial stage in the identification of genes regulated by SIX5.     

Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A

An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from a Staphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinant S. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.     

Characterization of Native and Recombinant Forms of an Unusual Cobalt-Dependent Proline Dipeptidase (Prolidase) from the Hyperthermophilic Archaeon Pyrococcus furiosus

Proline dipeptidase (prolidase) was purified from cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme is a homodimer (39.4 kDa per subunit) and as purified contains one cobalt atom per subunit. Its catalytic activity also required the addition of Co²⁺ ions (K_d, 0.24 mM), indicating that the enzyme has a second metal ion binding site. Co²⁺ could be replaced by Mn²⁺ (resulting in a 25% decrease in activity) but not by Mg²⁺, Ca²⁺, Fe²⁺, Zn²⁺, Cu²⁺, or Ni²⁺. The prolidase exhibited a narrow substrate specificity and hydrolyzed only dipeptides with proline at the C terminus and a nonpolar amino acid (Met, Leu, Val, Phe, or Ala) at the N terminus. Optimal prolidase activity with Met-Pro as the substrate occurred at a pH of 7.0 and a temperature of 100°C. The N-terminal amino acid sequence of the purified prolidase was used to identify in the P. furiosus genome database a putative prolidase-encoding gene with a product corresponding to 349 amino acids. This gene was expressed in Escherichia coli and the recombinant protein was purified. Its properties, including molecular mass, metal ion dependence, pH and temperature optima, substrate specificity, and thermostability, were indistinguishable from those of the native prolidase from P. furiosus. Furthermore, the K_m values for the substrate Met-Pro were comparable for the native and recombinant forms, although the recombinant enzyme exhibited a twofold greater V_max value than the native protein. The amino acid sequence of P. furiosus prolidase has significant similarity with those of prolidases from mesophilic organisms, but the enzyme differs from them in its substrate specificity, thermostability, metal dependency, and response to inhibitors. The P. furiosus enzyme appears to be the second Co-containing member (after methionine aminopeptidase) of the binuclear N-terminal exopeptidase family.     

In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA-cleaving deoxyribozyme

A group of highly efficient Zn(II)-dependent RNA-cleaving deoxyribozymes has been obtained through in vitro selection. They share a common motif with the ‘8–17’ deoxyribozyme isolated under different conditions, including different design of the random pool and metal ion cofactor. We found that this commonly selected motif can efficiently cleave both RNA and DNA/RNA chimeric substrates. It can cleave any substrate containing rNG (where rN is any ribonucleotide base and G can be either ribo- or deoxyribo-G). The pH profile and reaction products of this deoxyribozyme are similar to those reported for hammerhead ribozyme. This deoxyribozyme has higher activity in the presence of transition metal ions compared to alkaline earth metal ions. At saturating concentrations of Zn²⁺, the cleavage rate is 1.35 min^–1 at pH 6.0; based on pH profile this rate is estimated to be at least ~30 times faster at pH 7.5, where most assays of Mg²⁺-dependent DNA and RNA enzymes are carried out. This work represents a comprehensive characterization of a nucleic acid-based endonuclease that prefers transition metal ions to alkaline earth metal ions. The results demonstrate that nucleic acid enzymes are capable of binding transition metal ions such as Zn²⁺ with high affinity, and the resulting enzymes are more efficient at RNA cleavage than most Mg²⁺-dependent nucleic acid enzymes under similar conditions.     

Induction of broad CD4+ and CD8+ T-cell responses and cross-neutralizing antibodies against hepatitis C virus by vaccination with Th1-adjuvanted polypeptides followed by defective alphaviral particles expressing envelope glycoproteins gpE1 and gpE2 and nonstructural proteins 3, 4, and 5

Broad, multispecific CD4⁺ and CD8⁺ T-cell responses to the hepatitis C virus (HCV), as well as virus-cross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. In order to recapitulate all of these responses in an ideal vaccine regimen, we have explored the use of recombinant HCV polypeptides combined with various Th1-type adjuvants and replication-defective alphaviral particles encoding HCV proteins in various prime/boost modalities in BALB/c mice. Defective chimeric alphaviral particles derived from the Sindbis and Venezuelan equine encephalitis viruses encoding either the HCV envelope glycoprotein gpE1/gpE2 heterodimer (E1E2) or nonstructural proteins 3, 4, and 5 (NS345) elicited strong CD8⁺ T-cell responses but low CD4⁺ T helper responses to these HCV gene products. In contrast, recombinant E1E2 glycoproteins adjuvanted with MF59 containing a CpG oligonucleotide elicited strong CD4⁺ T helper responses but no CD8⁺ T-cell responses. A recombinant NS345 polyprotein also stimulated strong CD4⁺ T helper responses but no CD8⁺ T-cell responses when adjuvanted with Iscomatrix containing CpG. Optimal elicitation of broad CD4⁺ and CD8⁺ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.     

Fluorescence measurement of intracellular sodium concentration in single Escherichia coli cells

The energy-transducing cytoplasmic membrane of bacteria contains pumps and antiports maintaining the membrane potential and ion gradients. We have developed a method for rapid, single-cell measurement of the internal sodium concentration ([Na⁺]_in) in Escherichia coli using the sodium ion fluorescence indicator, Sodium Green. The bacterial flagellar motor is a molecular machine that couples the transmembrane flow of ions, either protons (H⁺) or sodium ions (Na⁺), to flagellar rotation. We used an E. coli strain containing a chimeric flagellar motor with H⁺- and Na⁺-driven components that functions as a sodium motor. Changing external sodium concentration ([Na⁺]_ex) in the range 1–85 mM resulted in changes in [Na⁺]_in between 5–14 mM, indicating a partial homeostasis of internal sodium concentration. There were significant intercell variations in the relationship between [Na⁺]_in and [Na⁺]_ex, and the internal sodium concentration in cells not expressing chimeric flagellar motors was 2–3 times lower, indicating that the sodium flux through these motors is a significant fraction of the total sodium flux into the cell.     

Lactococcus lactis Expressing either Staphylococcus aureus Fibronectin-Binding Protein A or Listeria monocytogenes Internalin A Can Efficiently Internalize and Deliver DNA in Human Epithelial Cells

Lactococci are noninvasive bacteria frequently used as protein delivery vectors and, more recently, as in vitro and in vivo DNA delivery vehicles. We previously showed that a functional eukaryotic enhanced green fluorescent protein (eGFP) expression plasmid vector was delivered in epithelial cells by Lactococcus lactis producing Listeria monocytogenes internalin A (L. lactis InlA⁺), but this strategy is limited in vivo to transgenic mice and guinea pigs. In this study, we compare the internalization ability of L. lactis InlA⁺ and L. lactis producing either the fibronectin-binding protein A of Staphylococcus aureus (L. lactis FnBPA⁺) or its fibronectin binding domains C and D (L. lactis CD⁺). L. lactis FnBPA⁺ and L. lactis InlA⁺ showed comparable internalization rates in Caco-2 cells, while the internalization rate observed with L. lactis CD⁺ was lower. As visualized by conventional and confocal fluorescence microscopy, large clusters of L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were present in the cytoplasm of Caco-2 cells after internalization. Moreover, the internalization rates of Lactobacillus acidophilus NCFM and of an NCFM mutant strain with the gene coding for the fibronectin-binding protein (fbpA) inactivated were also evaluated in Caco-2 cells. Similar low internalization rates were observed for both wild-type L. acidophilus NCFM and the fbpA mutant, suggesting that commensal fibronectin binding proteins have a role in adhesion but not in invasion. L. lactis FnBPA⁺, L. lactis CD⁺, and L. lactis InlA⁺ were then used to deliver a eukaryotic eGFP expression plasmid in Caco-2 cells: flow cytometry analysis showed that the highest percentage of green fluorescent Caco-2 cells was observed after coculture with either L. lactis FnBPA⁺ or L. lactis InlA⁺. Analysis of the in vivo efficiency of these invasive recombinant strains is currently in progress to validate their potential as DNA vaccine delivery vehicles.The mucosal administration of bacterial carriers to deliver antigens and plasmid DNA constitutes a promising vaccination strategy. Pathogenic bacteria that have the capacity to invade cells, such as Listeria, Salmonella, and Shigella strains, have been used to deliver DNA constructs into mammalian cells (23). Nevertheless, the risk associated with possible reversion to a virulent phenotype of these pathogens is a major concern (5). Lactococcus lactis, the food-grade, gram-positive, noninvasive model bacterium, has been intensively used to deliver antigens and cytokines at the mucosal level (30). We previously showed (i) that native L. lactis can deliver a eukaryotic expression cassette coding for the bovine β-lactoglobulin (BLG), one of the major cow''s milk allergens, into mammalian epithelial Caco-2 cells, and (ii) that these cells were able to express and secrete BLG protein in its native conformation (10). Recently, we demonstrated the ability of native noninvasive L. lactis to deliver a fully functional plasmid to murine intestinal cells in vivo (2).The internalization of the bacterial carrier is a fundamental step to achieve efficient DNA delivery in eukaryotic cells (7). In order to increase DNA delivery by lactic acid bacteria (LAB), invasin genes were expressed in L. lactis. Due to the safety profile of LAB, recombinant lactococci expressing invasin genes from intracellular bacteria are attractive as potential DNA delivery vectors compared to the attenuated pathogens presently used.In this field, we previously demonstrated that L. lactis bacteria expressing the main Listeria monocytogenes invasin, internalin A (L. lactis InlA⁺), were able to invade eukaryotic cells and efficiently deliver a functional green fluorescent protein (GFP) expression plasmid into epithelial/endothelial cells (9). Even though attractive, the experimental use of lactococci expressing InlA in a mouse model has a major bottleneck: InlA, which binds to human E-cadherin (15), does not interact with murine E-cadherin. Consequently, in vivo experimental studies using lactococci expressing InlA as DNA delivery vehicles are limited to transgenic mice expressing human E-cadherin or to guinea pigs (13).Fibronectin binding protein A (FnBPA) of Staphylococcus aureus is another bacterial invasin that is involved in intracellular spreading of S. aureus in the host (27). It is a multifunctional adhesion protein having both fibrinogen and fibronectin binding capacities (24). Its N-terminal part, also called domain A, is responsible for fibrinogen (29) and elastin (20) binding, whereas its C-terminal part, including domains B, C, and D, binds to fibronectin (25). FnBPA is known to mediate adhesion to host tissue and bacterial uptake into nonphagocytic host cells (27). Its expression by L. lactis was previously shown to be sufficient to confer the ability to invade nonphagocytic cells in vitro and in vivo, while the expression of domains C and D confers invasivity only in vitro (19).In this study, we show that L. lactis bacteria expressing full-length FnBPA of S. aureus (L. lactis FnBPA⁺) or a truncated form encompassing only its C and D domains (L. lactis CD⁺) are internalized as efficiently as L. lactis InlA⁺ in the human intestinal cell line Caco-2. We also provide, for the first time, direct microscopic evidence of the intracellular location of the internalized lactococci, showing that the bacteria are heterogeneously distributed in the cell monolayer and that their number per cell can reach a surprisingly high level. However, we demonstrate that FbpA, a fibronectin binding protein from the commensal Lactobacillus acidophilus NCFM, does not mediate bacterial internalization: no difference in invasivity was observed between the wild-type (wt) strain and the mutant with fbpA inactivated. This result indicates that, although widely distributed among bacteria, fibronectin binding proteins are not universal mediators of bacterial internalization, even at low levels. Finally, we also demonstrate that, similarly to L. lactis InlA⁺, L. lactis FnBPA⁺ and L. lactis CD⁺ can efficiently deliver a eukaryotic GFP expression plasmid in Caco-2 cells and trigger GFP expression in these cells. Consequently, L. lactis FnBPA⁺ can be used for further DNA delivery experiments in vivo.     

Effect of O-Side-Chain-Lipopolysaccharide Chemistry on Metal Binding

Pseudomonas aeruginosa PAO1 produces two chemically distinct types of lipopolysaccharides (LPSs), termed A-band LPS and B-band LPS. The A-band O-side chain is electroneutral at physiological pH, while the B-band O-side chain contains numerous negatively charged sites due to the presence of uronic acid residues in the repeat unit structure. Strain PAO1 (A⁺ B⁺) and three isogenic LPS mutants (A⁺ B⁻, A⁻ B⁺, and A⁻ B⁻) were studied to determine the contribution of the O-side-chain portion of LPS to metal binding by the surfaces of gram-negative cells. Transmission electron microscopy with energy-dispersive X-ray spectroscopy was used to locate and analyze sites of metal deposition, while atomic absorption spectrophotometry and inductively coupled plasma-mass spectrometry were used to perform bulk quantitation of bound metal. The results indicated that cells of all of the strains caused the precipitation of gold as intracellular, elemental crystals with a d-spacing of 2.43 Å. This type of precipitation has not been reported previously for gram-negative cells and suggests that in the organisms studied gold binding is not a surface-mediated event. All four strains bound similar amounts of copper (0.213 to 0.222 μmol/mg [dry weight] of cells) at the cell surface, suggesting that the major surface metal-binding sites reside in portions of the LPS which are common to all strains (perhaps the phosphoryl groups in the core-lipid A region). However, significant differences were observed in the abilities of strains dps89 (A⁻ B⁺) and AK1401 (A⁺ B⁻) to bind iron and lanthanum, respectively. Strain dps89 caused the precipitation of iron (1.623 μmol/mg [dry weight] of cells) as an amorphous mineral phase (possibly iron hydroxide) on the cell surface, while strain AK1401 nucleated precipitation of lanthanum (0.229 μmol/mg [dry weight] of cells) as apiculate, surface-associated crystals. Neither iron nor lanthanum precipitates were observed on the cells of other strains, which suggests that the combination of A-band LPS and B-band LPS produced by a cell may result in a cell surface which promotes the formation of metal-rich precipitates. We therefore propose that the negatively charged sites located in the O-side chains are not directly responsible for the binding of metallic ions; however, the B-band LPS molecule as a whole may contribute to overall cell surface properties which favor the precipitation of distinct metal-rich mineral phases.     

Cadmium Ion Biosorption by the Thermophilic Bacteria Geobacillus stearothermophilus and G. thermocatenulatus

This study reports surface complexation models (SCMs) for quantifying metal ion adsorption by thermophilic microorganisms. In initial cadmium ion toxicity tests, members of the genus Geobacillus displayed the highest tolerance to CdCl₂ (as high as 400 to 3,200 μM). The thermophilic, gram-positive bacteria Geobacillus stearothermophilus and G. thermocatenulatus were selected for further electrophoretic mobility, potentiometric titration, and Cd²⁺ adsorption experiments to characterize Cd²⁺ complexation by functional groups within and on the cell wall. Distinct one-site SCMs described the extent of cadmium ion adsorption by both studied Geobacillus sp. strains over a range of pH values and metal/bacteria concentration ratios. The results indicate that a functional group with a deprotonation constant pK value of approximately 3.8 accounts for 66% and 80% of all titratable sites for G. thermocatenulatus and G. stearothermophilus, respectively, and is dominant in Cd²⁺ adsorption reactions. The results suggest a different type of functional group may be involved in cadmium biosorption for both thermophilic strains investigated here, compared to previous reports for mesophilic bacteria.     

Production of Active Chimeric Pediocin AcH in Escherichia coli in the Absence of Processing and Secretion Genes from the Pediococcus pap Operon

Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys⁺¹ to Cys⁺⁴⁴) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH₂ terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys⁺¹-Tyr-Tyr-Gly-Asn-Gly-Val⁺⁷-sequence at the NH₂ terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.     

Contribution of the myosin binding protein C motif to functional effects in permeabilized rat trabeculae

Myosin binding protein C (MyBP-C) is a thick-filament protein that limits cross-bridge cycling rates and reduces myocyte power output. To investigate mechanisms by which MyBP-C affects contraction, we assessed effects of recombinant N-terminal domains of cardiac MyBP-C (cMyBP-C) on contractile properties of permeabilized rat cardiac trabeculae. Here, we show that N-terminal fragments of cMyBP-C that contained the first three immunoglobulin domains of cMyBP-C (i.e., C0, C1, and C2) plus the unique linker sequence termed the MyBP-C “motif” or “m-domain” increased Ca²⁺ sensitivity of tension and increased rates of tension redevelopment (i.e., k_tr) at submaximal levels of Ca²⁺. At concentrations ≥20 μM, recombinant proteins also activated force in the absence of Ca²⁺ and inhibited maximum Ca²⁺-activated force. Recombinant proteins that lacked the combination of C1 and the motif did not affect contractile properties. These results suggest that the C1 domain plus the motif constitute a functional unit of MyBP-C that can activate the thin filament.     

Divalent metal-dependent catalysis and cleavage specificity of CSP41, a chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase superfamily

CSP41 is a ubiquitous chloroplast endoribonuclease belonging to the short chain dehydrogenase/reductase (SDR) superfamily. To help elucidate the role of CSP41 in chloroplast gene regulation, the mechanisms that determine its substrate recognition and catalytic activity were investigated. A divalent metal is required for catalysis, most probably to provide a nucleophile for cleavage 5′ to the phosphodiester bond, and may also participate in cleavage site selection. This requirement distinguishes CSP41 from other Rossman fold-containing proteins from the SDR superfamily, including several RNA-binding proteins and endonucleases. CSP41 is active only in the presence of MgCl₂ and CaCl₂. Although Mg²⁺- and Ca²⁺-activated CSP41 cleave at identical sites in the single-stranded regions of a stem–loop-containing substrate, Mg²⁺-activated CSP41 was also able to cleave within the double-stranded region of the stem–loop. Mixed metal experiments with Mg²⁺ and Ca²⁺ suggest that CSP41 contains a single divalent metal-binding site which is non-selective, since Mn²⁺, Co²⁺ and Zn²⁺ compete with Mg²⁺ for binding, although there is no activity in their presence. Using site-directed mutagenesis, we identified three residues, Asn71, Asp89 and Asp103, which may form the divalent metal-binding pocket. The activation constant for Mg²⁺ (K_A,Mg = 2.1 ± 0.4 mM) is of the same order of magnitude as the stromal Mg²⁺ concentrations, which fluctuate between 0.5 and 10 mM as a function of light and of leaf development. These changes in stromal Mg²⁺ concentration may regulate CSP41 activity, and thus cpRNA stability, during plant development.     

A Putative Multisubunit Na+/H+ Antiporter from Staphylococcus aureus

We cloned several genes encoding an Na⁺/H⁺ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na⁺/H⁺ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na⁺/H⁺ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na⁺/H⁺ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na⁺/H⁺ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na⁺/H⁺ antiporter. We observed a large Na⁺ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na⁺ extrusion was sensitive to an H⁺ conductor, supporting the idea that the system is not a respiratory Na⁺ pump but an Na⁺/H⁺ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.     

The bulge region of HIV-1 TAR RNA binds metal ions in solution

Binding of Mg²⁺, Ca²⁺ and Co(NH₃)₆³⁺ ions to the HIV-1 TAR RNA in solution was analysed by ¹⁹F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for ¹⁹F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg²⁺ and Ca²⁺ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K_d) of 0.9 ± 0.6 and 2.7 ± 1.7 mM, respectively. Argininamide, inducing largest ¹⁹F chemical shifts changes at FU-23, was used as a reference ligand (K_d = 0.3 ± 0.1 mM). In the Pb²⁺-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg²⁺ and Ca²⁺ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb²⁺-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH₃)₆³⁺ > Mg²⁺ > Ca²⁺] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.