Synthesis of Pyruvate Dehydrogenase in Staphylococcus aureus Is Stimulated by Osmotic Stress

The pyruvate dehydrogenase multienzyme complex (PDHC) was found to be upregulated by osmotic stress in the osmotolerant pathogen Staphylococcus aureus. Upregulation was detectable in the levels of both activity and protein and was judged to be about fourfold when sodium chloride was used to adjust the water activity (a_w) of the growth medium to 0.94. The upregulation of the PDHC was also found to be humectant dependent and was greatest when impermeant, nonmetabolizable humectants were used to adjust a_w. Further experiments provided evidence that in addition to osmotic upregulation, the PDHC complex is also subject to catabolite repression, thus providing a possible explanation for the observation that high concentrations of carbohydrates are generally more inhibitory to the growth of this bacterial pathogen than are high concentrations of salts.     

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K_m of 15 ± 4 μm, a k_cat of 5.2 ± 0.7 min⁻¹, and a k_cat/K_m of 5.8 × 10³ m⁻¹ s⁻¹. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H₂O₂, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.     

Linezolid Is a Specific Inhibitor of 50S Ribosomal Subunit Formation in Staphylococcus aureus Cells

Linezolid is an oxazolidinone compound that has been shown to have impressive antimicrobial activity against a number of Gram-positive bacteria. It inhibits an initiation step of protein synthesis, and its binding site has been shown to be on the 50S ribosomal subunit. Linezolid was tested to see whether would interfere with the formation of the 50S subunit in Staphylococcus aureus cells, since a number of other 50S-specific antibiotics have this second inhibitory function. Linezolid inhibited protein synthesis in S. aureus cells with an IC₅₀ of 0.3 μg/ml. A concentration-dependent decline in cell number with an increase in generation time was found. Pulse-chase labeling studies revealed a specific inhibitory effect on 50S particle formation, with no effect on 30S subunit assembly. The compound inhibited 50S synthesis with an IC₅₀ of 0.6 μg/ ml, indicating an equivalent effect on translation and particle assembly. A postantibiotic effect of 1 h was found when cells were initially treated with the drug at 2 μg/ ml. 50S particle numbers recovered more rapidly than translational capacity, consistent with the increase in viable cell numbers. The inhibitory activities of this novel antimicrobial agent in cells are discussed. Received: 28 June 2001 / Accepted: 27 August 2001     

Blepharismin produced by a protozoan Blepharisma functions as an antibiotic effective against methicillin-resistant Staphylococcus aureus

A ciliated protozoan, Blepharisma japonicum, produces a photosensitive red pigment, blepharismin (BLR). This study showed that the pigment inhibits the growth of Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) resistant to arbekacin (ABK), which is the most effective aminoglycoside antibiotic against MRSA and used world wide. Although the minimum inhibitory concentration (MIC) of BLR to the ABK-resistant MRSA strain was 6.25 μg/ml in dark, it was decreased to 1.25 μg/ml by irradiation with white light of 65 W/m² for 30 min, suggesting that the antibacterial activity of BLR is photoactivated. Our findings suggested that the antibacterial activity of BLR in dark is due to inhibition of protein synthesis. In addition, we found that BLR is bactericidal and enhances synergistically the antibacterial activity of ABK.     

Evolution of an inducible penicillin-target protein in methicillin-resistant Staphylococcus aureus by gene fusion

A new beta-lactam-inducible penicillin-binding protein (PBP) that has extremely low affinity to penicillin and most other beta-lactam antibiotics has been widely found in highly beta-lactam(methicillin)-resistant Staphylococcus aureus (MRSA). The gene for this protein was sequenced and the nucleotide sequence in its promoter and close upstream area was found to show close similarity with that of staphylococcal penicillinase, while the amino acid sequence over a wide range of the molecule was found to be similar to those of two PBPs of Escherichia coli, the shape-determining protein (PBP 2) and septum-forming one (PBP 3). Probably the MRSA PBP (Mr 76462) evolved by recombination of two genes: an inducible type I penicillinase gene and a PBP gene of a bacterium, causing the formation of a beta-lactam-inducible MRSA PBP.     

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of chlorotetracycline uptake in Staphylococcus aureus by a fluorescence technique

The antibiotic chlorotetracycline (CTC) is used as a fluorescent chelate probe to investigate the kinetics of its uptake into $\text{Staphylococcus aureus}$. CTC binds to divalent cations in an aqueous solution with enhanced fluorescence. This fluorescence is polarity dependent, being higher in apolar solutions. Upon addition of CTC to dispersions of $\text{S. aureus}$, a time dependent fluorescence enhancement is detected demonstrating that the CTC-divalent cation complex migrates into the apolar regions of the membrane. This uptake, which follows saturation kinetics, is energy dependent. A K_m of 162 μ$\text{M}$ was obtained for CTC concentration ranges of 0.2–100 μgm/ml.     

Cateslytin,a Chromogranin A Derived Peptide Is Active against Staphylococcus aureus and Resistant to Degradation by Its Proteases

Innate immunity involving antimicrobial peptides represents an integrated and highly effective system of molecular and cellular mechanisms that protects host against infections. One of the most frequent hospital-acquired pathogens, Staphylococcus aureus, capable of producing proteolytic enzymes, which can degrade the host defence agents and tissue components. Numerous antimicrobial peptides derived from chromogranins, are secreted by nervous, endocrine and immune cells during stress conditions. These kill microorganisms by their lytic effect at micromolar range, using a pore-forming mechanism against Gram-positive bacteria, filamentous fungi and yeasts. In this study, we tested antimicrobial activity of chromogranin A-derived peptides (catestatin and cateslytin) against S. aureus and analysed S. aureus-mediated proteolysis of these peptides using HPLC, sequencing and MALDI-TOF mass spectrometry. Interestingly, this study is the first to demonstrate that cateslytin, the active domain of catestatin, is active against S. aureus and is interestingly resistant to degradation by S. aureus proteases.     

A Step in the Biosynthesis of Gibberellins that Is Controlled by the Mutation in the Semi-Dwarf Rice Cultivar Tan-Ginbozu

Genetic analysis and a comparison of endogenous levels of gibberellinsbetween the semi-dwarf rice cultivar Tan-ginbozu and the correspondingnormal cultivar Ginbozu have confirmed that Tan-ginbozu is agibberellin deficient mutant and that the semi-dwarfism of Tan-ginbozuis controlled by a single recessive gene. A step in the biosynthesisof gibberellins that is blocked by the mutation in Tan-ginbozuhad been considered to be the synthesis of ent-kaurene or anearlier step. However, the rate of production of ent-kaureneby Tan-ginbozu was almost the same as that by Ginbozu. By contrast,accumulation of only a small amount of ent-kaurene was detectedin Tan-ginbozu, and the amount that accumulated was similarto that in Ginbozu that had been treated with 6.9 x 10^-8 M uniconazole-P(an effective inhibitor of three oxidative steps in the pathwayfrom ent-kaurene to ent-kaurenoic acid via entkaurenol and ent-kaurenal).The height of the treated Ginbozu plants was reduced to thesame as that of Tanginbozu plants. Resembling Tan-ginbozu plants,Ginbozu plants that had been treated with uniconazole-P respondedwell to ent-kaurenoic acid and slightly to ent-kaurene and ent-kaurenol.Since the growth-promoting activity of enf-kaurenal in Tan-ginbozuwas similar to that of ent-kaurene, our results suggest thatthe mutation in Tan-ginbozu blocks the three oxidative stepswhereby ent-kaurene is converted to ent-kaurenoic acid. (Received June 9, 1995; Accepted February 15, 1996)     

Outermost-cell-surface changes in an encapsulated strain of Staphylococcus aureus after preservation by freeze-drying.

The effects of drying time during freeze-drying on the outermost cell surface of an encapsulated strain of Staphylococcus aureus S-7 (Smith, diffuse) were investigated, with special attention paid to capsule and slime production. To quantify capsule and slime production, capsule antigen production and cellular characteristics such as growth type in serum-soft agar, cell volume index, and clumping factor reaction were examined. After freeze-drying the colonial morphology of strain S-7 was altered from a diffuse to a compact type in serum-soft agar. In accordance with these changes, the titer of the clumping factor reaction increased while the cell volume index, capsule and slime production, and capsule antigen production were markedly decreased in parallel with the period of freeze-drying. The ability of the strain to adhere to collagen, fibrinogen, and soybean lectin was also compared before and after freeze-drying. Fibrinogen levels slightly increased when 10% skim milk and 2% honey were used as cryoprotective agents and showed a remarkable increase when 0.05 M phosphate buffer was used as a control. Also, the ability of strain S-7 to adhere to soybean lectin declined, whereas no changes were observed for collagen under any conditions. Strain S-7 was phage nontypable before freeze-drying but the number of typable cells increased after freeze-drying; phage-typable cells reacted to phage 52 alone after 5 h of freeze-drying, but additional cells also proved to be phage typable to phage 42E after 10 h. Electron micrographs indicated that strain S-7, an encapsulated strain, was converted to an unencapsulated state after freeze-drying.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Last Step in Cocaine Biosynthesis Is Catalyzed by a BAHD Acyltransferase

The esterification of methylecgonine (2-carbomethoxy-3β-tropine) with benzoic acid is the final step in the biosynthetic pathway leading to the production of cocaine in Erythoxylum coca. Here we report the identification of a member of the BAHD family of plant acyltransferases as cocaine synthase. The enzyme is capable of producing both cocaine and cinnamoylcocaine via the activated benzoyl- or cinnamoyl-Coenzyme A thioesters, respectively. Cocaine synthase activity is highest in young developing leaves, especially in the palisade parenchyma and spongy mesophyll. These data correlate well with the tissue distribution pattern of cocaine as visualized with antibodies. Matrix-assisted laser-desorption ionization mass spectral imaging revealed that cocaine and cinnamoylcocaine are differently distributed on the upper versus lower leaf surfaces. Our findings provide further evidence that tropane alkaloid biosynthesis in the Erythroxylaceae occurs in the above-ground portions of the plant in contrast with the Solanaceae, in which tropane alkaloid biosynthesis occurs in the roots.One of the most widely known plant alkaloids is cocaine, the benzoate ester of 2-carbomethoxy-3β-tropine (methylecgonine). Cocaine belongs to the tropane class of alkaloids defined by a common chemical substructure, the azabicyclo[3.2.1]octane skeleton (Fig. 1). Esterifications and hydroxylations of the tropane skeleton are common in nature, and more than 200 tropane alkaloids (TAs) with drastically different pharmacological activities are known to exist (Pollini et al., 2006). The physiological effects of these compounds have been ascribed to various features of the tropane skeleton and its substituents. The methylated nitrogen atom mimics that of acetylcholine and thereby leads to inhibition of muscarinic acetylcholine receptors (Schmeller et al., 1995). Binding to dopamine receptors is mediated by the stereochemistry of substituents at the C-2 and C-3 positions (Carroll et al., 1992a; Kelkar et al., 1994), with the strongest affinity being found for compounds containing an aromatic ring connected directly or indirectly to the 3β position (Carroll et al., 1992b). This in turn explains why cocaine exhibits both anesthetic and euphorigenic properties. The main source of cocaine is the South American Erythroxylum coca, a shrub or small tree cultivated for religious and medicinal purposes for more than 8,000 years (Dillehay et al., 2010). The result of long-term cultivation and selection for increasing alkaloid content has given rise to several cultivars containing up to 1% dry weight of cocaine in the leaves (Plowman and Rivier, 1983).Open in a separate windowFigure 1.Structures of selected TAs: numbered tropane nucleus (1), cocaine (2), cinnamoylcocaine (3), methylecgonine (4), pseudotropine (5), tropine (6), and nortropine (7).Many years of in vivo biosynthetic studies of cocaine have led to a proposed pathway (Supplemental Fig. S1) beginning first with Orn or Arg, which produces the polyamine N-methylputrescine (Humphrey and O’Hagan, 2001). After oxidation of N-methylputrescine to 4-methylaminobutanal, which undergoes spontaneous cyclization to an N-methyl-Δ¹-pyrrolinium cation, the equivalent of two acetyl units are added. Some debate remains regarding whether these carbons are supplied via acetate, acetoacetate, or malonate (Leete et al., 1991; Robins et al., 1997). The oxobutanoic acid intermediate formed by this condensation then cyclizes to form a tropane intermediate called methylecgonone (Jirschitzka et al., 2013). In the penultimate biosynthetic step to cocaine, methylecgonone is reduced to form methylecgonine. This reaction is catalyzed by the enzyme methylecgonone reductase (Jirschitzka et al., 2012).The last step in cocaine biosynthesis is the esterification of methylecgonine with a benzoyl moiety hypothesized to utilize benzoyl-CoA as the activated acyl donor (Leete et al., 1988). This moiety was found to be derived from cinnamic acid, but it was not determined whether it arises via benzoyl-CoA or benzaldehyde (Leete et al., 1988; Bjorklund and Leete, 1992). Enzyme activities responsible for the acetylation of other TAs were purified, but no structural genes were isolated (Robins et al., 1991; Rabot et al., 1995). In plants, acylation reactions of secondary metabolites are performed by several acyltransferase families, namely the tyramine N-hydroxycinnamoyltransferase/serotonin N-hydroxycinnamoyltransferase, BAHD, and serine carboxypeptidase-like acyltransferases (Kang et al., 2006; Mugford and Osbourn, 2010). However, of these three groups, only the BAHD acyltransferases are known to utilize activated acyl-CoA thioesters (D’Auria, 2006). In this study, we report the identification and characterization of the enzymes responsible for the last biosynthetic step in the formation of cocaine in E. coca. These convert methylecgonine, a molecule with little physiological activity, into the pharmacologically active cocaine (Williams et al., 1977).