Metabolism of chloroguaiacols by Rhodococcus chlorophenolicus

Summary A polychlorophenol degrader, Rhodococcus chlorophenolicus, was shown to metabolize five different chlorinated guaiacols, namely tetrachloroguaiacol, 3,4,6-trichloroguaiacol, 3,5,6-trichloroguaiacol, 3,5-dichloroguaiacol and 3,6-dichloroguaiacol. Seven different intermediate metabolites, each with three hydroxyl or methoxyl groups, were identified. Four of these metabolites were also dehalogenation products, three carrying one chlorine atom less than the parent compound, and one metabolite from tetrachloroguaiacol where two chlorine atoms had been removed. Tetrachloroguaiacol was shown to undergo reductive dehalogenation. Demethylation of guaiacol to catechol was observed with the dichloroguaiacols, but not with polychloroguaiacols.Abbreviations DCG dichloroguaiacol - TCG trichloroguaiacol - TeCG tetrachloroguaiacol - DCC dichlorocatechol - TCC trichlorocatechol - TeCC tetrachlorocatechol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol. An example of numeration - 346-TCG 3,4,6-trichloroguaiacol - GLC gas liquid chromatography     

Degradation of polychlorinated phenols by Rhodococcus chlorophenolicus

Summary An actinomycete, Rhodococcus chlorophenolicus, isolated from a pentachlorophenol-degrading mixed bacterial culture is a polychlorophenol degrader. It was shown to oxidize pentachlorophenol into carbon dioxide and to metabolize also 2,3,4,5-,2,3,4,6-, and 2,3,5,6-tetrachlorophenol, 2,3,4-, 2,3,5-, 2,3,6-, 2,4,6-, and 2,4,5-trichlorophenol, 2,5-, and 2,6-dichlorophenol and tetrachloro-p-hydroquinone in an inducible manner. Pentachlorophenol set on the synthesis of enzymes required for the metabolism of all these chlorophenols and of tetrachloro-p-hydroquinone. 2,4,5-, and 2,4,6-trichlorophenol and 2,5-, and 2,6-dichlorophenol were degraded by R. chlorophenolicus cells only if these had previous contact to pentachlorophenol. Other chlorophenols mentioned were able to set on the synthesis of enzymes for their own degradation. 2,3,4,5-, and 2,3,4,6-tetrachlorophenol, and 2,3,5-, 2,4,5-, and 3,4,5-trichlorophenol were more toxic to R. chlorophenolicus than the other chlorophenols, but nevertheless 2,3,4,5-, and 2,3,4,6-tetrachlorophenol and 2,3,5-trichlorophenol were readily degraded by the bacteria.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol - PCP pentachlorophenol - TeCH tetrachloro-p-hydroquinone An example of numeration: 2345-TeCP, 2,3,4,5-tetrachlorophenol     

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase^®) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.     

Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 microg ml(-1). The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 microg of cereulide g(-1) (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 microg of cereulide ml(-1) in liquid medium at room temperature (21 +/- 1 degrees C) in 1 to 3 days, during the stationary growth phase when cell density was 2 x 10(8) to 6 x 10(8) CFU ml(-1). Cereulide production at temperatures at and below 8 degrees C or at 40 degrees C was minimal.     

Identification of a gene cluster in Klebsiella pneumoniae which includes citX, a gene required for biosynthesis of the citrate lyase prosthetic group

The biosynthesis of the 2'-(5"-phosphoribosyl)-3'-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2'-(5"-triphosphoribosyl)-3'-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >or=40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.     

Development of an automation system for a tablet coater

An instrumentation and automation system for a side-vented pan coater with a novel air-flow rate measurement system for monitoring the film-coating process of tablets was designed and tested. The instrumented coating system was tested and validated by film-coating over 20 pilot-scale batches of tablets with aqueous-based hydroxypropyl methylcellulose (HPMC). Thirteen different process parameters were continuously measured and monitored, and the most significant ones were logged for analysis. Laser profilometry was used to measure the surface roughness of the coated tablets. The instrumentation system provided comprehensive and quantitative information on the process parameters monitored. The measured process parameters and the responses of the film-coated tablet batches showed that the coating process is reproducible. The inlet air-flow rate influenced the coating process and the subsequent quality of the coated tablets. Increasing the inlet flow rate accelerated the drying of the tablet surface. At high inlet flow rate, obvious film-coating defects (ie, unacceptable surface roughness of the coated tablets) were observed and the loss of coating material increased. The instrumented and automated pancoating system described, including historical data storage capability and a novel air-flow measurement system, is a useful tool for controlling and characterizing the tablet film-coating process. Monitoring of critical process parameters increases the overall coating process efficiency and predictability.     

Analysis of a 2,4,6-trichlorophenol-dehalogenating enrichment culture and isolation of the dehalogenating member Desulfitobacterium frappieri strain TCP-A

An anaerobic, 2,4,6-trichlorophenol ortho-dehalogenating mixed culture was enriched from sediment of the river Saale (Germany). Two isolated dechlorinating colonies (MK1 and MK2) consisted of rods of different lengths and thicknesses, indicating heterogeneity. Following subcultivation with thiosulfate as alternative electron acceptor and cocultivation with Clostridium celerecrescensT, the 2,4,6-trichlorophenol-dehalogenating bacterium Desulfitobacterium frappieri strain TCP-A was isolated and characterized regarding its taxonomic properties and the spectrum of chlorophenols that it dehalogenated. Four other bacterial strains were coenriched and identified as organisms with closest phylogenetic relatedness to the Clostridium type strains C. indolis, C. glycolicum, C. hydroxybenzoicum and C. sporosphaeroides (16S rDNA sequence identities of 99.5, 99.2, 94.4, and 93.5%, respectively). Amplified ribosomal DNA restriction analysis of the original dehalogenating cultures MK1 and MK2 (when not exposed to thiosulfate) confirmed the microbial heterogeneity and revealed the presence of two additional species related to the type strains of C. celerecrescens and Clostridium propionicum. Only one copy of the 16S rRNA genes of Desulfitobacterium frappieri in each of the clone libraries of MK1 and MK2 (containing 136 and 56 clones, respectively) was found by dot-blot hybridization, suggesting a relatively low number of the dehalogenating bacterium within the enrichment culture.     

Two‐dimensional proteome reference map for the radiation‐resistant bacterium Deinococcus geothermalis

2‐DE reference maps for Deinococcus geothermalis cytosolic and cell envelope proteomes were constructed. In total, 403 spots were identified as 299 different proteins. Unique in the proteomes were four subunits of V‐type ATPase and Deinococcus specific proteins constituting one‐fourth of cell envelope proteome. The cytoplasmic proteome included enzymes of the central carbon metabolism, chaperones, enzymes of protein and DNA repair, and oxidative stress. A total of 34 abundant proteins with unknown function may relate to the extreme stress tolerance of D. geothermalis.