The survival of the pentachlorophenol-degrading Rhodococcus chlorophenolicus PCP-1 and Flavobacterium sp. in natural soil

The survival of two different pentachlorophenol (PCP)-degrading bacteria were studied in natural soil. The PCP-degraders Rhodococcus chlorophenolicus and Flavobacterium sp., both able to mineralize PCP into CO₂ and chloride in axenic culture, were tested for the capacity to survive and degrade PCP in natural soil. These bacteria were immobilized on polyurethane (PUR) foam and introduced into natural peaty soil to give about 10⁹ cells g^-1 of soil (dry weight). R. chlorophenolicus induced PCP-degrading activity in soil remained detectable for 200 days whether or not a carbon source was added (distillery waste or wood chips). Electron microscopic investigation performed almost a year after inoculation, revealed the presence of R. chlorophenolicus-like cells in the PUR foam particles. PCP-degrading activity of Flavobacterium sp. declined within 60 days of burial in the soil without enhancing the PCP removal. R. chlorophenolicus degraded PCP in soil at a mean rate of 3.7 mg of PCP day^-1 kg^-1 of soil, which corresponds to ca. 5×10^-3 pg of PCP degraded per inoculated R. chlorophenolicus cell day^-1. The solvent extractable organic chlorine contents of the soil decreased stoichiometrically (>95%) with that of PCP indicating that PCP was essentially mineralized.Abbreviations ATCC American type culture collection - DSM Deutsche Sammlung für Mikroorganismen - DW distillery waste - EM electron microscopy - EOX extractable organic halogen - GC/ECD gas chromatograph/electron capture detector - GC/MS gas chromatograph/mass spectrometer - PCP pentachlorophenol - WC wood chips - d.wt. dry weight - w.wt. wet weight - d.s. dry soil - d.H₂O distilled water - PCA polychlorinated aromatics     

Ionophoretic properties and mitochondrial effects of cereulide: the emetic toxin of B. cereus.

The emetic toxin of Bacillus cereus, found to cause immobilization of spermatozoa and swelling of their mitochondria, was purified and its structure found to be identical to the earlier known toxin cereulide. It increased the conductance in black-lipid membranes in KCl solutions in an ionophore-like manner. It formed adducts with K+, Na+, and NH4+ but the conductance was highly selective for K+ in relation to Na+ and H+ (three orders of magnitude). The increase in the kinetics of conductance indicated a stoichiometric ratio between the cereulide and K+. Its ionophoretic properties are thus similar to those of valinomycin. In addition, its effects on rat liver mitochondria were similar: it stimulated swelling and respiration in respiring mitochondria in the presence but not in the absence of K+, it reduced the transmembrane potential under these conditions. In nonrespiring mitochondria, swelling was seen in KNO3- but not in NaNO3-containing media, less in acetate. In NaNO3 media addition of the cereulide caused a transient diffusion potential which was reduced by adding K+. It is concluded that the toxic effects of cereulide are due to it being a K+ ionophore.     

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant‐transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N‐methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host–microbe interactions.     

Detecting species-site dependencies in large multiple sequence alignments

Multiple sequence alignments (MSAs) are one of the most important sources of information in sequence analysis. Many methods have been proposed to detect, extract and visualize their most significant properties. To the same extent that site-specific methods like sequence logos successfully visualize site conservations and sequence-based methods like clustering approaches detect relationships between sequences, both types of methods fail at revealing informational elements of MSAs at the level of sequence–site interactions, i.e. finding clusters of sequences and sites responsible for their clustering, which together account for a high fraction of the overall information of the MSA. To fill this gap, we present here a method that combines the Fisher score-based embedding of sequences from a profile hidden Markov model (pHMM) with correspondence analysis. This method is capable of detecting and visualizing group-specific or conflicting signals in an MSA and allows for a detailed explorative investigation of alignments of any size tractable by pHMMs. Applications of our methods are exemplified on an alignment of the Neisseria surface antigen LP2086, where it is used to detect sites of recombinatory horizontal gene transfer and on the vitamin K epoxide reductase family to distinguish between evolutionary and functional signals.     

Crystal Structure and Biophysical Properties of Bacillus subtilis BdbD: AN OXIDIZING THIOL:DISULFIDE OXIDOREDUCTASE CONTAINING A NOVEL METAL SITE*

BdbD is a thiol:disulfide oxidoreductase (TDOR) from Bacillus subtilis that functions to introduce disulfide bonds in substrate proteins/peptides on the outside of the cytoplasmic membrane and, as such, plays a key role in disulfide bond management. Here we demonstrate that the protein is membrane-associated in B. subtilis and present the crystal structure of the soluble part of the protein lacking its membrane anchor. This reveals that BdbD is similar in structure to Escherichia coli DsbA, with a thioredoxin-like domain with an inserted helical domain. A major difference, however, is the presence in BdbD of a metal site, fully occupied by Ca²⁺, at an inter-domain position some 14 Å away from the CXXC active site. The midpoint reduction potential of soluble BdbD was determined as −75 mV versus normal hydrogen electrode, and the active site N-terminal cysteine thiol was shown to have a low pK_a, consistent with BdbD being an oxidizing TDOR. Equilibrium unfolding studies revealed that the oxidizing power of the protein is based on the instability introduced by the disulfide bond in the oxidized form. The crystal structure of Ca²⁺-depleted BdbD showed that the protein remained folded, with only minor conformational changes. However, the reduced form of Ca²⁺-depleted BdbD was significantly less stable than reduced Ca²⁺-containing protein, and the midpoint reduction potential was shifted by approximately −20 mV, suggesting that Ca²⁺ functions to boost the oxidizing power of the protein. Finally, we demonstrate that electron exchange does not occur between BdbD and B. subtilis ResA, a low potential extra-cytoplasmic TDOR.Disulfide bonds, formed upon oxidation of two cysteine residue side chain thiols, are key for the stability and/or function of many secreted and membrane-bound peptides and proteins in bacteria, and the failure to insert these correctly has wide ranging effects (1–4). To regulate the redox state of cysteine residues on the outside of the cytoplasmic membrane, intricate disulfide bond regulatory systems have evolved. These involve enzymes of the thiol:disulfide oxidoreductase (TDOR)³ family, which contain cysteine residues often arranged in a Cys-Xaa-Xaa-Cys thioredoxin motif (5, 6). These enzymes function in pathways that lead to the formation of disulfide bonds, rearrangement of incorrectly positioned disulfide bonds, or the removal of unwanted disulfide bonds, and the redox properties of the enzymes appear to correlate closely with function.The paradigm system for disulfide bond formation is the DsbA-DsbB system of Escherichia coli, which has been characterized in great detail (7–11). DsbA is a soluble periplasmic TDOR, which has a thioredoxin-like fold with an additional helical domain (8). The protein oxidizes the di-thiol motifs of a range of substrates, generating in each a disulfide bond. Reduced DsbA is rapidly re-oxidized by DsbB, a membrane-bound TDOR (12) that channels the resulting electrons into the membrane quinol pool (13, 14). Dsb-like homologues appear to be extremely widespread in Gram-negative bacteria.Gram-positive bacteria, unlike Gram-negatives, do not have an outer membrane and so have no spatially defined periplasmic compartment. This places different functional and structural demands on extra-cytoplasmic proteins, and one consequence of this is that extra-cytoplasmic TDORs are generally membrane-anchored in Gram-positive bacteria. Systems for the introduction of disulfide bonds appear to be variable in these organisms (15, 16). As an example, Mycobacterium tuberculosis does not contain close homologues of DsbA/B but contains another thioredoxin-like TDOR, DsbE, which has been shown to have redox properties similar to those of DsbA (17). Some Gram-positive bacteria, including Staphylococcus aureus, contain a DsbA homologue but no homologue of DsbB (18). The recent structural and biochemical characterization of S. aureus DsbA revealed major similarities with the E. coli protein but was also consistent with a distinct mechanism of re-oxidation (16).Some Gram-positive bacteria, however, contain clear homologues of both DsbA and DsbB. In the model organism Bacillus subtilis, BdbD and BdbC/BdbB have been identified as homologues of DsbA and DsbB, respectively, and demonstrated to be involved in processes such as natural competence development, which requires the insertion of disulfide bonds (19). BdbD and BdbC are also involved in a number of other pathways that do not require the insertion of a disulfide bond; B. subtilis contains several extra-cytoplasmic TDORs, for example ResA and StoA (required for cytochrome c maturation and endospore biogenesis, respectively), which function to specifically reduce disulfide bonds introduced by BdbD (20, 21). To understand disulfide bond management systems in Gram-positive bacteria, detailed information on each of the different systems found is required.Here we present the crystal structure of the catalytic domain of B. subtilis BdbD in both reduced and oxidized states. The structure is broadly similar to that of E. coli DsbA, but it also reveals the presence of a novel Ca²⁺-binding site remote from the CXXC active site. Using two-dimensional NMR methods and fluorescence kinetic studies of thiolate alkylation, we report the reduction potential and pK_a properties of the soluble protein, which are entirely consistent with an oxidizing function for the protein in vivo. Conformational stability studies and NMR studies showed that the occupancy of the metal site by Ca²⁺ ion is not required for folding/stability but leads to a significant increase of the midpoint reduction potential. The possibility that the principal function of the metal site is to boost the oxidizing power of the protein is discussed.     

Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum

Virulence of Plasmodium falciparum , the most lethal parasitic disease in humans, results in part from adhesiveness and increased rigidity of infected erythrocytes. Pf332 is trafficked to the parasite-infected erythrocyte via Maurer's clefts, structures for protein sorting and export in the host erythrocyte. This protein has a domain similar to the Duffy-binding-like (DBL) domain, which functions by binding to receptors for adherence and invasion. To address structure of the Pf332 DBL domain, we expressed this region, and validated its fold on the basis of the disulphide bond pattern, which conformed to the generic pattern for DBL domains. The modelled structure for Pf332 DBL had differences compared with the erythrocyte-binding region of the αDBL domain of Plasmodium knowlesi Duffy-binding protein (Pkα-DBL). We addressed the function of Pf332 by constructing parasites that either lack expression of the protein or express an altered form. We found no evidence that Pf332 is involved in cytoadhesion or merozoite invasion. Truncation of Pf332 had a significant effect on deformability of the P. falciparum -infected erythrocyte, while loss of the full protein deletion did not. Our data suggest that Pf332 may contribute to the overall deformability of the P. falciparum -infected erythrocyte by anchoring and scaffolding.     

Distribution of Several Activating and Inhibitory Receptors on CD3<Superscript>−</Superscript>CD16<Superscript>+</Superscript> NK Cells and Their Correlation with NK Cell Function in Healthy Individuals

The aim of this study was to estimate the distribution and density of a representative set of activating and inhibitory receptors on gated natural killer (NK) cells, as well as on their bright and dim subsets, and to correlate the receptor expression with NK cell activity for healthy individuals on CD3⁻CD16⁺ NK cells. We show that in 43 healthy controls NK cell activity against K562 target cells was 37.34% (E:T, 80:1) by standard chromium release assay. The expression of receptors on NK cells and their subsets was analyzed by flow cytometry. The cytotoxic CD3⁻CD16^bright NK subset constituted 78.97%, while the regulatory CD3⁻CD16^dim NK subset constituted 21.03% of NK cells. We show the distribution of NKG2D, CD161, CD158a, and CD158b receptors on CD3⁻CD16⁺ NK cells in peripheral blood lymphocytes (PBLs), on gated NK cells, and on the CD3⁻CD16^bright and CD3⁻CD16^dim subsets. Contrary to CD158a and CD158b killer immunoglobulin-like receptors (KIRs), there is a significant positive correlation of NKG2D and CD161 expression with NK cytotoxicity. We show the kinetics of change in CD3⁻CD16⁺NK/K562 conjugate composition, together with the stronger target binding capacity of CD16^bright NK cells. Furthermore, we show that after coculture of PBLs with K562 the expression of CD107a, a degranulation marker, on CD3⁻CD16⁺NK cells and subsets is time dependent and significantly higher on the cytotoxic CD3⁻CD16^bright NK subset. The novel data obtained regarding expression of NK cell activating and inhibitory receptors for healthy individuals may aid in detecting changes that are associated with various diseases.     

Evaluation of methods for recognising strains of the Bacillus cereus group with food poisoning potential among industrial and environmental contaminants

Toxin production, biochemical properties and ribotypes of Bacillus cereus group (B. cereus, B. thuringiensis, B. mycoides) strains originating from industrial and environmental sources (n = 64), from food poisoning incidents (n = 22) and from reference sources (n = 7) were analysed. Forty ribotypes were found among the 93 strains. Eleven strains from food poisoning incidents produced emetic (mitochondrio) toxin, as determined by the boar spermatozoa toxicity test. These strains possessed closely similar ribotypes which were rare among strains of other origins. Sperm toxin producing (cereulide positive) strains did not hydrolyse starch and did not produce haemolysin BL, as determined by the reverse passive latex agglutination test. Sixteen different ribotypes were found among B. cereus strains from board machines (n = 16) and from packaging board (n = 16), indicating many different sources of B. cereus contamination in board mills. Strains originating from packaging board had predominantly different ribotypes from those of dairy and dairy product originating strains. Nine (53%) out of 17 strains from a single dairy process shared the same ribotype whereas strains from milk and milk products from different dairies had different ribotypes indicating that B. cereus group populations were dairy specific. Twenty-two percent of strains isolated from the paperboard industry on non-selective medium were lecithinase negative, including enterotoxin producing strains. This stresses the importance of other detection methods not based on a positive lecithinase reaction.