Structural and functional characterization of three Type B and C chloramphenicol acetyltransferases from Vibrio species

Chloramphenicol acetyltransferases (CATs) were among the first antibiotic resistance enzymes identified and have long been studied as model enzymes for examining plasmid‐mediated antibiotic resistance. These enzymes acetylate the antibiotic chloramphenicol, which renders it incapable of inhibiting bacterial protein synthesis. CATs can be classified into different types: Type A CATs are known to be important for antibiotic resistance to chloramphenicol and fusidic acid. Type B CATs are often called xenobiotic acetyltransferases and adopt a similar structural fold to streptogramin acetyltransferases, which are known to be critical for streptogramin antibiotic resistance. Type C CATs have recently been identified and can also acetylate chloramphenicol, but their roles in antibiotic resistance are largely unknown. Here, we structurally and kinetically characterized three Vibrio CAT proteins from a nonpathogenic species (Aliivibrio fisheri) and two important human pathogens (Vibrio cholerae and Vibrio vulnificus). We found all three proteins, including one in a superintegron (V. cholerae), acetylated chloramphenicol, but did not acetylate aminoglycosides or dalfopristin. We also determined the 3D crystal structures of these CATs alone and in complex with crystal violet and taurocholate. These compounds are known inhibitors of Type A CATs, but have not been explored in Type B and Type C CATs. Based on sequence, structure, and kinetic analysis, we concluded that the V. cholerae and V. vulnificus CATs belong to the Type B class and the A. fisheri CAT belongs to the Type C class. Ultimately, our results provide a framework for studying the evolution of antibiotic resistance gene acquisition and chloramphenicol acetylation in Vibrio and other species.     

Impact of prescribed burning on the nutrient balance of heathlands with particular reference to nitrogen and phosphorus

Question: Can prescribed winter burning compensate atmospheric nutrient loads for dry heathlands? What effects does prescribed burning have on nutrient balances, particularly as regards the limiting nutrients N and P? Location: Lueneburg Heath, NW Germany. Methods: In two burning experiments (in 10/15 year old Calluna‐stands) nutrient balances (for N, Ca, K, Mg, P) were calculated by analysing nutrient inputs (atmospheric deposition, ash deposition), nutrient stores (above‐ground biomass, organic horizon) and nutrient outputs (biomass combustion, leaching). Results: Atmospheric nutrient deposition amounted to 22.8 kg.ha^‐1.a^‐1 for N and < 0.5 kg.ha^‐1.a^‐1 for P. Nutrient stores in the above‐ground biomass were 95/197 kg.ha^‐1 for N and 5/13 kg.ha^‐1 for P (first/second experiment, respectively). From these stores 90/53% (for N) and 25/14% (for P) were removed by burning. Effects of leaching on nutrient balances were low. In the first two years after burning, leaching rates of N increased by about 4/6 kg.ha^‐1, whereas leaching rates of P did not change significantly. Input/output‐ratios showed that prescribed burning leads to positive nutrient balances for N, Ca and Mg in the long term. For example, the amounts of N removed by prescribed burning are equivalent to ca. five years of atmospheric inputs. Applied in ten‐year cycles, this measure alone cannot prevent N accumulation in the long term. Conclusion: Regarding 10/15 year old Calluna‐heaths, we assume that prescribed burning cannot compensate for atmospheric N inputs, thus making long‐term changes in the nutritional state inevitable. Therefore, prescribed burning should be applied in combination with high‐intensity management measures.     

Cyclic condensed metaphosphates and linear polyphosphates in brown and red algae

The occurrence of linear condensed polyphosphates and cyclic condensed metaphosphates was studied by means of pulse-labeling with ³²P-orthophosphate (3–5 h) in a number of Phaeophyceae species: Pylaiella litoralis, Ilea fascia, Ectocarpus siliculosus and also Rhodophyceae species: Ceramium deslongchampsii, C. rubrum, Rhodomela confervoides, Porphyridium purpureum and P. aerugineum. Twodimensional cellulose thin layer chromatography revealed that in all species studied ³²P-radioactivity was generally present in all oligopolyphosphates containing 2 to 7 phosphate residues, in cyclic metaphosphates (tri-, tetra-, penta- and hexametaphosphates) and in high-molecular-weight condensed phosphates which remained at the starting point. Among the low-molecular-weight condensed inorganic phosphates the trimetaphosphate had a significantly higher specific activity than the other oligophosphates which were separated on the chromatography plates as measured by the direct scanning with a Geiger-Muller counter.The phosphate uptake strongly depends on the internal pool of reserve phosphates of the algae cells. The ³²P-orthophosphate incorporation of the cells is low and sluggish when growning in a synthetic medium or in sea water. Accordingly ³²P appeared preferentially in the low-molecular-weight fractions of condensed phosphates since the storage phosphates were not yet used. After previous incubation in a P-free culture medium of the algae the ³²P was rather rapidly incorporated and was found mostly in the highmolecular-weight condensed phosphates.During MAK-chromatography the high-molecular-weight fractions were eluted together with the nucleic acids (tRNA and DNA) while most of the low-molecular-weight fractions left the column immediately on elution.     

Identification and quantification of metabolite conjugates of activated cyclophosphamide and ifosfamide with mesna in urine by ion-pair extraction and fast atom bombardment mass spectrometry

The high bladder toxicity of the alkylating oxazaphosphorine anticancer drugs, cyclophosphamide and ifosfamide is effectively reduced by the concomitant administration of mesna (sodium 2-mercaptoethane sulphonate). The formation and rapid urinary excretion of conjugates of the activated (4-hydroxylated) oxazaphosphorine metabolites with mesna has been suggested as the pharmacological basis for the selective detoxification, but separation and identification of such metabolites in vivo have been extremely difficult due to their high polarity and chemical lability. In this study an ion-pair extraction procedure in combination with positive and negative ion fast atom bombardment mass spectrometry has been developed which enabled the identification and quantification of the conjugation products of activated oxazaphosphorine metabolites with mesna in urine. The conjugates extracted as the tetra-n-butylammonium salts are directly identified by their characteristic positive molecular ion adducts and fragment ions, and the corresponding abundant molecular anions. The pattern of molecular and fragment ion formation was established by comparison of the fast atom bombardment mass spectra of synthetic cyclophosphamide-mesna conjugates with various organic and inorganic counter ions. The ifosfamide-4-(2-thioethylsulphonate) (ifosfamide-mesna) conjugate was identified as a metabolite in the urine of rats, and in patients after administration of the combination, ifosfamide + mesna. By means of a two-step extraction and with the use of suitable analogues as internal standards, procedures for the quantification of parent oxazaphosphorine and of oxazaphosphorine-mesna conjugates by negative ion fast atom bombardment mass spectrometry have been developed, and first examples for the determination of excretion kinetics are described.     

Sexually and Developmentally Dimorphic Grooming: A Comparative Survey of the Ungulata

Grooming is effective in removing fitness‐compromising ectoparasites, such as ticks. Tick‐removal grooming is regulated both by a central control mechanism (programmed grooming model) and by cutaneous stimulation from tick bites (stimulus‐driven model). The programmed grooming model predicts that organismic factors that impact the cost‐benefit ratio of grooming (e.g., sex and body size) will influence the rate of grooming. The ‘vigilance principle’ predicts that breeding males of sexually dimorphic species will groom less than females to maintain high levels of vigilance for rival males and oestrus females. The intraspecific body size principle predicts that juveniles will engage in more frequent grooming than larger‐bodied adults to compensate for the higher cost of tick infestation for smaller animals. To examine the generalizability of these predictions, we surveyed the grooming rate of 53 species/subspecies of ungulates (primarily Bovidae and Cervidae) in tick‐free zoological parks in which stimulus‐driven grooming was controlled for. Matched‐pair analysis supported both predictions. Males delivered fewer oral grooming episodes per hour than females in 85% of sexually dimorphic species (n = 40), but only 56% of monomorphic species (n = 11) exhibited sexually dimorphic grooming, which was not different from random. Juvenile oral episodes per hour exceeded that of adult females in 74% of surveyed species (n = 36). As predicted by the body size principle, the grooming rate of juveniles declined as juveniles grew larger and less vulnerable to tick infestation. In separate analyses of Bovidae and Cervidae to control for common ancestry, both families supported sexually dimorphic grooming, but developmentally dimorphic grooming was supported only for Bovidae. Our results indicate that sexually dimorphic grooming is widespread in the ungulate species surveyed, suggesting that programmed grooming evolved at least as early as the common ancestor to the Artiodactyla.     

Two crystalline pentofuranosyl bromides: tri-O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide and tri-O-(p-nitrobenzoyl)-α,β-D-xylofuranosyl bromide

Methyl α,β-D-ribofuranoside was p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-ribofuranoside (2),and this was treated with HBr in acetic acid to give tri- O-(p-nitrobenzoyl)-β-D-ribofuranosyl bromide (3). Bromide 3 could be converted into 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-allononitrile (4) with Hg(CN)₂, or hydrolyzed to 2,3,5-tri-O-(p-nitrobenzoyl)-D-ribose (5). On p-nitro- benzoylation, 5 gave tetra-O-(p-nitrobenzoyl)-β-D-ribofuranose (6). The synthesis of tri-O-(p-nitrobenzoyl)-α-β-D-xylofuranosyl bromide (11) started with methyl 3,5-O-isopropylidene-β-D-xyldfuranoside (7), which was p-nitrobenzoylated to give ester 8; this was then hydrolyzed, and the product p-nitrobenzoylated to give methyl tri-O-(p-nitrobenzoyl)-β-D-xylofuranoside (10) which, on treatment with HBr in CH₂Cl₂, afforded the desired bromide (11). Nucleophilic replacement with Hg(CN)₂ afforded 2,5-anhydro-3,4,6-tri-O-(p-nitrobenzoyl)-D-gulononitrile (12).