Properties of Arsenite Efflux Permeases (Acr3) from Alkaliphilus metalliredigens and Corynebacterium glutamicum

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.Arsenic is a carcinogen that ranks first on the Superfund List of Hazardous Substances (www.atsdr.cdc.gov). As a consequence of its environmental ubiquity, nearly every organism, from bacteria to humans, has genes that confer resistance to arsenic (1). The most common mechanism of arsenite resistance is efflux from cells catalyzed by members of three unrelated families of transporters. Homologues of the Mrp members of the ATP-binding cassette superfamily catalyze ATP-dependent pumping of As(III)-thiol complexes out of the cytosol. These include Mrp1 and Mrp2 in mammals that extrude As(GS)₃ into blood or bile (2), Ycf1p in yeast that extrudes As(GS)₃ into the vacuole (3), and PgpA in Leishmania that extrudes the As(III)-trypanothione complex into intracellular compartments (4). These pumps are generalized resistance pumps and are not specific for arsenite. In contrast, ArsB, the first identified member of the second family of arsenite efflux proteins, has the physiological role of conferring resistance to inorganic As(III) and Sb(III) (5, 6). The best characterized member of the ArsB family is that encoded by the arsRDABC operon of the conjugative R-factor R773 of Escherichia coli. ArsB is widespread in bacteria and archaea. It has 12 membrane-spanning segments (7), which is similar to members of the Major Facilitator Superfamily (8). It transports As(III) but has higher affinity for Sb(III). ArsB is an antiporter that catalyzes the exchange of trivalent metalloid for protons, coupling arsenite efflux to the electrochemical proton gradient (9).The third arsenic resistance transporter is Acr3, which is a member of the BART (bile/arsenite/riboflavin transporter) superfamily and includes members found in bacteria, archaea, and fungi and is more widely distributed than members of the ArsB family (10) (supplemental Fig. 1). Homologues have recently been identified in plant (Pteris vittata, NCBI accession number {"type":"entrez-protein","attrs":{"text":"ACN65413","term_id":"224814384","term_text":"ACN65413"}}ACN65413) and animal genomes (Danio rerio, NCBI accession number {"type":"entrez-protein","attrs":{"text":"XP_001921075","term_id":"189546053","term_text":"XP_001921075"}}XP_001921075). Unfortunately, the literature is confused by the fact that many members of the Acr3 family are annotated as ArsB even though they exhibit no significant sequence similarity to ArsB. The first identified member of this family is encoded by the ars operon of the skin (sigK intervening) element in the chromosome of Bacillus subtilis (11). The membrane topology of the B. subtilis Acr3 was recently investigated using translational fusions, but the results could not distinguish between 8 and 10 transmembrane-spanning segments (TMs)² (12). Fungal members of this family include the Saccharomyces cerevisiae Acr3p metalloid efflux protein (3, 13). Interestingly, yeast Acr3p appears to be selective for As(III) over Sb(III), which is surprising considering the similarity in chemical properties between the two metalloids. The properties of a more distant homologue from Shewanella oneidensis was examined recently (14). The S. oneidensis homologue confers resistance to arsenate but not arsenite. Similarly, the purified protein binds arsenate, not arsenite, indicating that this protein is not an Acr3 orthologue.Here we examined the properties of Acr3 orthologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum (supplemental Fig. 1). A. metalliredigens is a borate-tolerant Gram-positive alkaliphile and strict anaerobe that uses reduction of metals as electron acceptors (15). It is a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms. The genome of A. metalliredigens QYMF (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_009633","term_id":"150387853","term_text":"NC_009633"}}NC_009633) contains two novel ars operons, arsR₁B_acr3–1D₁A_1–1A_1–2 and arsR₂CB_acr3–2D₂A_2–1A_2–2. The two genes for the AmAcr3s were designated ars_acr3 because they are both in ars operons and are controlled by ArsR repressors, even though they are not homologues of ArsB. Interestingly, both ars operons have genes for ArsD and two genes corresponding to the two homologous halves of ArsA, which we designate AmArsA1 and AmArsA2. ArsD is an arsenic chaperone that transfers As(III) to ArsA (16), which then interacts with ArsB to extrude As(III) from the cells in an ATP-dependent manner (6, 17, 18). Whether or how Acr3 can replace ArsB in this process is a question of considerable interest.C. glutamicum is a Gram-positive soil bacterium that is used for commercial production of glutamate, lysine, and other amino acids, nucleotides, and vitamins and from which the genome sequence has been described (NCBI accession number {"type":"entrez-nucleotide","attrs":{"text":"NC_006958","term_id":"62388892","term_text":"NC_006958"}}NC_006958). It is highly arsenic-resistant and has three genes encoding Acr3 homologues (19). Two of the homologues are in ars operons regulated by ArsRs (arsR₁B_acr3–1C₁C_1′ and arsR₂B_acr3–2arsC₂) and a third orphan gene (arsB_acr3–3) that is not in an operon and may not be expressed to the same extent as the other two. (Again, the genes were misnamed arsB even though they encode Acr3 homologues.)The genes for AmAcr3 and CgAcr3 from the ars1 operons of the respective species were cloned and expressed in the arsenite-hypersensitive E. coli strain AW3110, in which the chromosomal arsRBC operon had been deleted (20). Both conferred resistance to arsenite but not arsenate or antimonite. Examination of the sequence of Acr3 homologues from many species indicates that there is conserved cysteine residues, Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 (supplemental Fig. 1). Those and other nonconserved cysteine residues were changed by mutagenesis, and substitution of only Cys¹³⁸ in AmAcr3 and Cys¹²⁹ in CgAcr3 led to loss of function, suggesting that the conserved cysteine residue participates in As(III) transport. A scanning cysteine accessibility method (SCAM) (21) was used to determine the transmembrane topology of AmAcr3. SCAM analysis is preferable to the use of gene fusions because there are minimal structural changes in the membrane protein, and the sidedness of inserted cysteines can be unambiguously determined with maleimide reagents of differing membrane permeability. A series of single cysteine mutants of AmAcr3 was constructed and the reactivity of each cysteine residue assayed. The results unambiguously demonstrate that Acr3 has 10 TMs.     

Haemagglutinating and antibiotic activities of freshwater microalgae

We analysed the haemagglutinating activity of algal extracts from 44 species of freshwater microalgae against native and trypsin/papain-treated cow, pig, sheep, and human A-, B-, and O-type erythrocytes. Algal extracts obtained with aqueous ethanol exhibited higher haemagglutinating activity than those obtained with aqueous acetone. Most of the algal extracts agglutinated at least one of the erythrocyte types analysed. Human erythrocytes were the most sensitive of the cell types analysed. In the other species, the sensitivity of algal haemagglutinating activity for erythrocytes was pig > sheep > cow. Pre-treating erythrocytes with trypsin and papain improved the detection of most algal agglutinins and increased the haemagglutination titre; pre-treatment with papain was most effective for pig erythrocytes. Algal extracts stored at –20 °C for 4 months lost their haemagglutinating activity. Algal extracts also exhibited strong antibiotic activity against food pathogenic bacteria, especially against Bacillus. Our numerical taxonomy data showed that these microalgae might be grouped into several clusters according to their haemagglutinating activity. The detection of haemagglutinating activity may provide an efficient biochemical or physiological character to classify and differentiate microalgae. Our results suggest that freshwater microalgae might provide a potent source of haemagglutinins and antibacterial compounds for biochemical and medical studies and applications.     

DNA homology study of Corynebacterium diphtheriae v. gravis groups I, II and III, Corynebacterium ulcerans and Corynebacterium pseudotuberculosis (ovis)

The homology of genomes within Krylova 's groups I, II and III of C. diphtheriae, including toxigenic C. diphtheriae and their nontoxigenic precursors within the same group, was confirmed by the method of DNA/DNA molecular hybridization; the homology of DNA within the groups was 89-103%, the thermostability of heteroduplexes being high (on the level of homoduplexes ). The heterogeneity of genomes within these 3 groups of cultivar gravis was confirmed, which made it possible to consider C. diphtheriae, groups I, II and III, to belong to different, though closely related species; in intergroup hybridization the homology of DNA varied, as a rule, between 66% and 73%, while the thermostability of heteroduplexes was low: delta T50 was -3 degrees C to -6 degrees C. The differences in genomes (on the level of different species) between 3 groups of C. diptheriae v. gravis on one hand and C. diphtheriae v. mitis C7 (-) tox- and its convertant C7 (beta) tox+ of phage tox+ on the other hand (DNA homology being 56-62%), as well as between C. diphtheriae v. intermedius No. 328 tox+ on one hand and the representatives of 3 groups of C. diphtheriae v. gravis and C. diphtheriae v. mitis, strain C7 (beta) tox+, on the other hand (DNA homology being 42-43%) were revealed. The heterogeneity of genomes (on the level of different genera) was revealed between C. diphtheriae strains, cultivars gravis (groups I, II and III), mitis (C7(-) tox- and C7 (beta) tox+) and intermedius (No. 328 tox+) on one hand and C. ulcerans and C. pseudotuberculosis (ovis) strains on the other hand; DNA homology was 11-17% for C. ulcerans and 22-26% for C. pseudotuberculosis (ovis), the thermostability of heteroduplexes being at the lowest level (delta T50 was -11 degrees C to -13 degrees C). As a result, C. diphtheriae, classified by Bergey as a single species, was found to comprise 5 species detected by means of marking in accordance with their phenotypical features and genome structure, carried out by the method of DNA/DNA molecular hybridization; among these species were group I, II and III strains of cultivar gravis, strain C7 of cultivar mitis and strain No. 328 of cultivar intermedius. C. ulcerans and C. pseudotuberculosis (ovis) strains investigated in this study can possibly be placed outside the genus including 5 C. diphtheriae species.     

Taxonomic studies on Corynebacterium beticola (Abdou)

Morphological, physiological and chemical studies were performed on the type strain of Corynebacterium beticola in an attempt to clarify its taxonomy. The results indicate that Corynebacterium beticola is a Gram negative, facultatively anaerobic bacterium which should be assigned to the genus Erwinia as Erwinia herbicola .     

Evaluation of differential media for the identification of Corynebacterium genitalium and Corynebacterium pseudogenitalium (group JK corynebacteria)

Tween purple agar containing 1% fructose (TFP agar) differentiated Corynebacterium genitalium from C. pseudogenitalium, which respectively formed colorless and yellow colonies after 72 h incubation at 37 degrees C aerobically or in 5-10% CO2 in air. Thus TFP agar is a differential medium. Corynebacteria-like colonies grown on nonspecific urethritis (NSU) chocolate agar from urogenital material were identified as C. genitalium, C. pseudogenitalium, or commensals when subcultured on TPF agar. TFP agar was unsuitable for their primary isolation since the commensals turned the medium yellow with 24 h incubation. Gentamicin cannot be employed as a selective agent in medium for the isolation of these corynebacteria. TFP agar containing 10 micrograms/mL entamicin inhibited most strains of C. pseudogenitalium and C. genitalium isolated from urogenital infections. It did not inhibit isolates of these corynebacteria from cancer patients or suppress the normal bacterial flora of the urogenital tract. Evidence that gentamicin-resistant strains are characteristic of nosocomial infections is presented.     

Calcium-Binding Properties of the Mitochondrial Channel-Forming Hydrophobic Component

A hydrophobic, low-molecular weight component extracted from mitochondria forms aCa²⁺-activated ion channel in black-lipid membranes (Mironova et al., 1997). At pH 8.3–8.5, thecomponent has a high-affinity binding site for Ca²⁺ with a K_d of 8 × 10^–6 M, while at pH7.5 this K_d was decreased to 9 × 10^–5 M. B_max for the Ca²⁺-binding site did not changesignificantly with pH. In the range studied, 0.2 ± 0.06 mmol Ca²⁺/g component were boundor one calcium ion to eight molecules of the component. The Ca²⁺ binding was stronglydecreased by 50–100 mM Na⁺, but not by K⁺. Treatment of mitochondria withCaCl₂ priorto ethanolic extraction resulted in a high level of Ca²⁺-binding capacity of the partially purifiedcomponent. Cyclosporin A, a specific inhibitor of the mitochondrial permeability transition,when added to the mitochondrial suspension, decreased the Ca²⁺-binding activity of thepurified extract severalfold. The calcium-binding capability of the partially purified componentcorrelates with its calcium-channel activity. This indicates that the channel-forming componentmight be involved in the permeability transition that stimulates its formation.     

Some Properties of the Pili of Corynebacterium renale

Some properties of the pili of the gram-positive bacteria Corynebacterium renale were described. A relationship was found between the morphological features of pili and the types of C. renale. Strains of types I and III usually possessed a small number of pili, whereas those of type II possessed numerous pili. Thick and long bundles of pili characteristic of C. renale were frequently observable in type II strains. Piliation of C. renale was stable under various cultural conditions. No ability to agglutinate red blood cells was demonstrated by piliated strains of C. renale. Pili were isolated from the cells of C. renale and studied serologically by immunodiffusion. The pili of a type II strain were serologically identical with the pili of another type II strain but not with those of the strains belonging to types I and III. The pili were serologically distinct from the cell wall. The pili were broken into short pieces by boiling, but their antigenicity was increased after boiling.     

Properties of Crystalline Creatinine Deiminase from Corynebacterium lilium

The properties of creatinine deiminase (EC 3.5.4.21) were characterized with a crystalline preparation from Corynebacterium lilium ATCC 15990. The molecular weight was determined to be 195,000 by the sedimentation equilibrium method, and the isoelectric point was found to be 4.2 by isoelectric focusing. The enzyme was relatively thermostable and had a broad pH optimum of 7.5 to 10.0. It was specific for creatinine and showed a Km value of 1.27 mm. A compound from creatinine was isolated, with the release of ammonia, and identified as N-methylhydantoin. The enzyme activity was inhibited by heavy metal ions and p-chloromercuribenzoate. The enzyme may be useful in determinations of serum and urinary creatinine.     

Regulatory Properties of Chorismate Mutase from Corynebacterium glutamicum

Regulatory properties of chorismate mutase from Corynebacterium glutamicum were studied using the dialyzed cell-free extract. The enzyme activity was strongly feedback inhibited by l-phenylalanine (90% inhibition at 0.1~1 mm) and almost completely by a pair of l-tyrosine and l-phenylalanine (each at 0.1~1 mm). The enzyme from phenylalanine auxotrophs was scarcely inhibited by l-tyrosine alone but the enzyme from a wild-type strain or a tyrosine auxotroph was weakly inhibited by l-tyrosine alone (40~50% inhibition, l-tyrosine at 1 mm). The enzyme activity was stimulated by l-tryptophan and the inhibition by l-phenylalanine alone or in the simultaneous presence of l-tyrosine was reversed by l-tryptophan. The Km value of the reaction for chorismate was 2.9 } 10^?3 m. Formation of chorismate mutase was repressed by l-phenylalanine. A phenylalanine auxotrophic l-tyrosine producer, C. glutamicum 98–Tx–71, which is resistant to 3-amino-tyrosine, p-aminophenylanaine, p-fluorophenylalanine and tyrosine hydroxamate had chorismate mutase derepressed to two-fold level of the parent KY 10233. The enzyme in C. glutamicum seems to have two physiological roles; one is the control of the metabolic flow to l-phenylalanine and l-tyrosine biosynthesis and the other is the balanced partition of chorismate between l-phenylalanine-l-tyrosine biosynthesis and l-tryptophan biosynthesis.     

Properties of an Antigenic Polysaccharide from Corynebacterium parvum

Corynebacterium parvum strain 10390 is an antitumor agent and stimulant of the reticuloendothelial system and produces a soluble antigen towards the end of its growth cycle. This material, which is a cell wall component and can also be released from the organism by acid or alkaline hydrolysis, has been purified. It is an acidic polysaccharide of molecular weight 100,000 to 150,000 and contains galactose, glucose, fucose, N-acetylgalactosamine, N-acetylglucosamine, uronic acids, sialic acids, and a small proportion of amino acids. The antigen gives a precipitin reaction with antisera raised against the whole organism and also binds to animal cells. The antigenic determinants are extremely resistant to oxidation, reduction, and enzymatic and chemical hydrolysis, but the single cell-binding site is destroyed by alkali and also by Helix pomatia digestive juice, alginase, and neuraminidase without substantially affecting the molecular weight. This site is inaccessible until the molecule is released from the cell surface. The possibility that the soluble antigen is the biologically active fraction of C. parvum is discussed.     

Hydrophobic and Electrostatic Cell Surface Properties of Thermophilic Dairy Streptococci

Microbial adhesion to hydrocarbons (MATH) and microelectrophoresis were done in 10 mM potassium phosphate solutions to characterize the surfaces of thermophilic dairy streptococci, isolated from pasteurizers. Regardless of whether they were grown (in M17 broth) with lactose, sucrose, or glucose added, strains were relatively hydrophilic (showing low initial removal rates by hexadecane) and slightly negatively charged. A tendency exists for cells grown with sucrose added to be more hydrophilic than cells grown with glucose or lactose added. Also, the lowest isoelectric points, i.e., the pH values for which the zeta potentials are zero, were measured for strains with glucose added to the growth medium. The isoelectric points for the strains were all rather high, between pH 3 and 5, indicative of protein-rich surfaces, although X-ray photoelectron spectroscopy did not measure excessively large amounts of nitrogen on the cell surfaces. Both MATH and microelectrophoresis were done as a function of pH. Maxima in hydrophobicity were observed at certain pH values. Usually these pH values were in the range of the isoelectric points of the cells. Thus it appears that MATH measures an interplay of hydrophobicity and electrostatic interactions. MATH measures solely hydrophobicity only when electrostatic interactions are absent, i.e., close to the isoelectric points of the cells. Considering that these thermophilic streptococci are all rather hydrophilic, a possible pathway to prevent fouling in the pasteurization process might be to render the heat exchanger plates of the pasteurizer more hydrophobic.     

Cell Wall-Anchored CshA Polypeptide (259 Kilodaltons) in Streptococcus gordonii Forms Surface Fibrils That Confer Hydrophobic and Adhesive Properties

It has been shown previously that inactivation of the cshA gene, encoding a major cell surface polypeptide (259 kDa) in the oral bacterium Streptococcus gordonii, generates mutants that are markedly reduced in hydrophobicity, deficient in binding to oral Actinomyces species and to human fibronectin, and unable to colonize the oral cavities of mice. We now show further that surface fibrils 60.7 +/- 14.5 nm long, which are present on wild-type S. gordonii DL1 (Challis) cells, bind CshA-specific antibodies and are absent from the cell surfaces of cshA mutants. To more precisely determine the structural and functional properties of CshA, already inferred from insertional-mutagenesis experiments, we have cloned the entire cshA gene into the replicative plasmid pAM401 and expressed full-length CshA polypeptide on the cell surface of heterologous Enterococcus faecalis JH2-2. Enterococci expressing CshA exhibited a 30-fold increase in cell surface hydrophobicity over E. faecalis JH2-2 carrying the pAM401 vector alone and 2.4-fold-increased adhesion to human fibronectin. CshA expression in E. faecalis also promoted cell-cell aggregation and increased the ability of enterococci to bind Actinomyces naeslundii cells. Electron micrographs of negatively stained E. faecalis cells expressing CshA showed peritrichous surface fibrils 70.3 +/- 9.1 nm long that were absent from control E. faecalis JH2-2(pAM401) cells. The fibrils bound CshA-specific antibodies, as detected by immunoelectron microscopy, and the antibodies inhibited the adhesion of E. faecalis cells to fibronectin. The results demonstrate that the CshA polypeptide is the structural and functional component of S. gordonii adhesive fibrils, and they provide a molecular basis for past correlations of surface fibril production, cell surface hydrophobicity, and adhesion in species of oral "sanguis-like" streptococci.     

Properties and biodegradation of a bioemulsifier from Corynebacterium hydrocarboclastus.

An extracellular polymer was produced by continuous fermentation of Corynebacterium hydrocarboclastus on kerosene in a 24 liter reactor. This polymer was composed of protein, lipid, and carbohydrates. The polymer possessed surface active properties, and had two critical micelle concentrations. Its effectiveness was quite comparable to the effectiveness of synthetic surface active agents such as Tween 80 and Span 20; however, its efficiency was much lower. The polymer also had emulsifying properties. Maximum emulsification was obtained at pH 6. The emulsifying properties were unaffected by high salt concentration [up to 5% (w/v) in Na+], and tolerated a water hardness up to 5,000 ppm. A 2 hr treatment of the polymer at temperatures higher than 65 degrees C resulted in a loss of its emulsifying properties. Two microorganisms, named SLYS and Y, isolated from soil, were able to grow on the polymer as sole carbon and energy source, thus proving its biodegradability. SLYS was tentatively identified as Flavobacterium breve and Y as Flavobacterium devorans.     

Hydrophobic regions function in calmodulin-enzyme(s) interactions

Certain naturally occurring lipids (phosphatidylinositol, phosphatidylserine, arachidonic acid) and sodium dodecyl sulfate activate at least two calmodulin-dependent enzymes, bovine brain 3':5'-cyclic nucleotide phosphodiesterase and chicken gizzard myosin light chain kinase in the absence of Ca2+. 2-p-Toluidinyl-naphthalene-6-sulfonate (TNS), which is often used as a probe for hydrophobic groups of proteins, inhibits these two calmodulin-dependent enzymes. Kinetic analysis of inhibition of chicken gizzard myosin kinase by TNS revealed a competitive fashion against calmodulin-induced activation. The interaction between TNS and purified bovine brain calmodulin as demonstrated in the appearance of TNS fluorescence in the presence of 3 microM or more of calcium ion was not observed in the presence of 2 mM EGTA. This suggests that TNS is able to bind to calmodulin in the presence of Ca2+. Moreover, a calmodulin-interacting agent N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide suppressed the TNS fluorescence induced by complex formation with calmodulin in the presence of Ca2+. These results suggest that when Ca2+ binds to the high affinity sites of calmodulin, it induces a conformational change which exposes hydrophobic groups, and the calmodulin is then capable of activating calmodulin-dependent enzymes. We propose that hydrophobic properties of Ca2+-calmodulin are important for the activation of Ca2+-calmodulin-dependent enzymes.