Biodegradation of Amphipathic Fluorinated Peptides Reveals a New Bacterial Defluorinating Activity and a New Source of Natural Organofluorine Compounds

Three peptides comprising mono-, di-, and tri-fluoroethylglycine (MfeGly, DfeGly, and TfeGly) residues alternating with lysine were digested by readily available proteases (elastase, bromelain, trypsin, and proteinase K). The degree of degradation depended on the enzyme employed and the extent of fluorination. Incubation of the peptides with a microbial consortium from garden soil resulted in degradation, yielding fluoride ions. Further biodegradation studies conducted with the individual fluorinated amino acids demonstrated that the degree of defluorination followed the sequence MfeGly > DfeGly > TfeGly. Enrichment of the soil bacteria employing MfeGly as a sole carbon and energy source resulted in the isolation of a bacterium, which was identified as Serratia liquefaciens. Cell-free extracts of this bacterium enzymatically defluorinated MfeGly, yielding fluoride ion and homoserine. In silico analysis of the genome revealed the presence of a gene that putatively codes for a dehalogenase. However, the low overall homology to known enzymes suggests a potentially new hydrolase that can degrade monofluorinated compounds. 19F NMR analysis of aqueous soil extracts revealed the unexpected presence of trifluoroacetate, fluoride ion, and fluoroacetate. Growth of the soil consortium in tryptone soya broth supplemented with fluoride ions resulted in fluoroacetate production; thus, bacteria in the soil produce and degrade organofluorine compounds

Biotransformation of fluorophenyl pyridine carboxylic acids by the model fungus Cunninghamella elegans

1. Fluorine plays a key role in the design of new drugs and recent FDA approvals included two fluorinated drugs, tedizolid phosphate and vorapaxar, both of which contain the fluorophenyl pyridyl moiety. 2. To investigate the likely phase-I (oxidative) metabolic fate of this group, various fluorinated phenyl pyridine carboxylic acids were incubated with the fungus Cunninghamella elegans, which is an established model of mammalian drug metabolism. 3. 19F NMR spectroscopy established the degree of biotransformation, which varied depending on the position of fluorine substitution, and gas chromatography–mass spectrometry (GC–MS) identified alcohols and hydroxylated carboxylic acids as metabolites. The hydroxylated metabolites were further structurally characterised by nuclear magnetic resonance spectroscopy (NMR), which demonstrated that hydroxylation occurred on the 4′ position; fluorine in that position blocked the hydroxylation. 4. The fluorophenyl pyridine carboxylic acids were not biotransformed by rat liver microsomes and this was a consequence of inhibitory action, and thus, the fungal model was crucial in obtaining metabolites to establish the mechanism of catabolism

Isolation and characterisation of actinomycin D producing Streptomyces spp. from Sudanese soil

Sudanese soil is an unexplored source of antibiotic-producing microorganisms. Here, we reported the screening of soil samples from Sudan for actinomycetes that have antibacterial activity. Two isolates, AH11.4 and AH47, displaying a broad antimicrobial spectrum were selected for further study. Morphological, physiological and biochemical studies indicated that the two isolates are Streptomyces spp. 16S rDNA sequencing confirmed that the closest matches were to other Streptomyces, but phylogenetic analysis demonstrated that the Sudanese strains were on a different node to previously identified strains. The antibiotic activity was isolated by preparative High-performance liquid chromatography (HPLC) and determined to be primarily actinomycin D on the basis of UV, 1H- and 13CNMR, and MS analyses. One strain also produced actinomycin X2 and aB. These strains are distinct from the known producers of actinomycin, thus are new sources of these antibiotics.Keywords: Screening, antibiotic, antitumour, identificationAfrican Journal of Biotechnology Vol. 12(19), pp. 2624-263

Simultaneous removal of malachite green and hexavalent chromium by Cunninghamella elegans biofilm in a semi-continuous system

The present study was conducted to evaluate the potential of the fungus Cunninghamella elegans for simultaneous decolourisation of a triphenylmethane dye malachite green (MG) and hexavalent chromium [Cr(VI)] in the same media. This fungus can degrade MG through its reduction into leucomalachite green and then demethylation followed by oxidative cleavage. Along with MG degradation, C. elegans biofilm could effectively and repeatedly remove Cr(VI) from the liquid cultures even in the presence of high concentrations (40 g L−1) of NaCl and various other metal ions. C. elegans biofilm was also found to adsorb different dyes (reactive black-5, acid orange 7, direct red 81 and brilliant blue G) concurrently with Cr(VI). Based on its potential for simultaneous removal of dyes and Cr(VI) as well as reusability, C. elegans biofilm is envisaged as an efficient bioresource to devise strategies for treatment of wastewaters loaded with multiple pollutants.Environmental Protection AgencyIrish Research Counci

VAST: An ASKAP Survey for Variables and Slow Transients

The Australian Square Kilometre Array Pathfinder (ASKAP) will give us an unprecedented opportunity to investigate the transient sky at radio wavelengths. In this paper we present VAST, an ASKAP survey for Variables and Slow Transients. VAST will exploit the wide-field survey capabilities of ASKAP to enable the discovery and investigation of variable and transient phenomena from the local to the cosmological, including flare stars, intermittent pulsars, X-ray binaries, magnetars, extreme scattering events, interstellar scintillation, radio supernovae and orphan afterglows of gamma ray bursts. In addition, it will allow us to probe unexplored regions of parameter space where new classes of transient sources may be detected. In this paper we review the known radio transient and variable populations and the current results from blind radio surveys. We outline a comprehensive program based on a multi-tiered survey strategy to characterise the radio transient sky through detection and monitoring of transient and variable sources on the ASKAP imaging timescales of five seconds and greater. We also present an analysis of the expected source populations that we will be able to detect with VAST.Comment: 29 pages, 8 figures. Submitted for publication in Pub. Astron. Soc. Australi

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

Drug metabolism in microorganisms

Several wild type and recombinant microorganisms can transform drugs to the equivalent human metabolites. Fungi, such as Cunninghamella spp., and Streptomyces bacteria express cytochrome P450 (CYP) enzymes that enable analogous phase I (oxidative) reactions with a wide range of drugs. The gene encoding the bifunctional CYP102A1 in Bacillus megaterium can be expressed easily in E. coli, and extensive mutagenesis experiments have generated numerous variants that can produce human drug metabolites. Additionally, human CYP isoforms have been expressed in various hosts. The application of microbial CYPs to the production of human drug metabolites is reviewed, and additional applications in the field of drug development are considered

Microbial degradation of fluorinated drugs: biochemical pathways, impacts on the environment and potential applications

Since the discovery over 60 years ago of fluorocortisone's biological properties (9-α-Fluoro derivatives of cortisone and hydrocortisone; Fried J and Sabo EF, J Am Chem Soc 76: 1455–1456, 1954), the number of fluorinated drugs has steadily increased. With the improvement in synthetic methodologies, this trend is likely to continue and will lead to the introduction of new fluorinated substituents into pharmaceutical compounds. Although the biotransformation of organofluorine compounds by microorganisms has been well studied, specific investigations on fluorinated drugs are relatively few, despite the increase in the number and variety of fluorinated drugs that are available. The strength of the carbon-fluorine bond conveys stability to fluorinated drugs; thus, they are likely to be recalcitrant in the environment or may be partially metabolized to a more toxic metabolite. This review examines the research done on microbial biotransformation and biodegradation of fluorinated drugs and highlights the importance of understanding how microorganisms interact with this class of compound from environmental, clinical and biotechnological perspectives

Biotransformation of fluorobiphenyl by Cunninghamella elegans

The fungus Cunninghamella elegans is a useful model of human catabolism of xenobiotics. In this paper, the biotransformation of fluorinated biphenyls by C. elegans was investigated by analysis of the culture supernatants with a variety of analytical techniques. 4-Fluorobiphenyl was principally transformed to 4-fluoro-4′-hydroxybiphenyl, but other mono- and dihydroxylated compounds were detected in organic extracts by gas chromatography–mass spectrometry. Additionally, fluorinated water-soluble products were detected by 19F NMR and were identified as sulphate and β-glucuronide conjugates. Other fluorobiphenyls (2-fluoro-, 4,4′-difluoro- and 2,3,4,5,6-pentafluoro-biphenyl) were catabolised by C. elegans, yielding mono- and dihydroxylated products, but phase II metabolites were detected from 4,4′-difluorobiphenyl only.Other funderEP