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     

Bioreclamation of chlorophenol-contaminated soil by composting

Summary Microbiological decontamination of technical chlorophenol-containing soil by composting was studied. In two 50 m³ windrows the concentration of chlorophenols went down from 212 mg kg^-1 to 30 mg kg^-1 in 4 summer months and after the second summer of composting it was only 15 mg kg^-1. All chlorophenol congeners present in the technical chlorophenol were degraded, but the main dimeric impurities, polychlorinated phenoxyphenols were recalcitrant. The contaminated soil was found to contain chlorophenol-degrading microbes, 5x10⁶ cfu g^-1 of dry windrow soil. Laboratory experiments with samples from the windrow compost showed that chlorophenols were truly degraded and that chlorophenol loss by evaporation was less than 1.5% under the circumstances studied. Laboratory experiments also showed that degradation of chlorophenols (120 mg kg^-1) was accelerated when sterilized contaminated soil was inoculated with Rhodococcus chlorophenolicus (mineralizer of several chlorophenols) or naturally occurring microbes of the field composts. Biomethylation of chlorophenols in the composts was insignificant compared to biodegradation.     

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     

Bacterial contaminants in liquid packaging boards: assessment of potential for food spoilage

Liquid packaging boards and blanks were examined for microbial contaminants. A total of 218 strains were identified and representatives of the most frequent species were characterized for their potential for food spoilage. Contaminants found were aerobic spore-forming bacteria, mostly Bacillus megaterium, B. licheniformis, B. cereus group, B. pumilus, Paenibacillus macerans, P. polymyxa, P. pabuli and B. flexus. Production of amylolytic, proteolytic, lipolytic and phospholipolytic enzymes was common. Approximately 50% of the B. cereus group strains were positive in the diarrhoeal enterotoxin immunoassay test or in the enterotoxin reversed passive latex agglutination test. Strains capable of growth at 6°C were found among B. cereus group, P. pabuli, P. validus, B. megaterium and P. polymyxa. All B. licheniformis strains grew at 55°C. The spores of B. licheniformis were most resistant to hydrogen peroxide. The B. cereus group strains were recognizable by fatty acid components not present in any of the other paperboard strains, 11-methyldodecanoic acid (13:0 iso) and trans-9-hexadecenoic acid (16:1 ω 7 trans), each contributing 7% or more to the total cellular fatty acids.     

Differentiation of dairy strains of the Bacillus cereus group by phage typing, minimum growth temperature, and fatty acid analysis

A total of 130 Bacillus strains were isolated from dairy products, the dairy environment and from packaging boards and board-producing machines. Ninety-eight of these were members of the B. cereus group ( B. cereus, B. mycoides and B. thuringiensis ) as determined by whole cell fatty acid composition. Fatty acid composition did not differentiate between the three species. Of the 98 strains, which were indistinguishable by biochemical tests, 87 could be assigned into 21 different phage types (11 strains remained untypable) when tested with 12 B. cereus, B. mycoides and B. thuringiensis phages. The distribution of phage types between strains from different sources showed that the source of contamination of the dairy products was of milk origin and not from the packaging materials. Most strains isolated from the dairy products were able to grow below 10°C, whereas strains from the dairy environment and from board mills had higher minimum growth temperatures.     

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.     

Isolation of toxigenic Nocardiopsis strains from indoor environments and description of two new Nocardiopsis Species, N. exhalans sp. nov. and N. umidischolae sp. nov

Nocardiopsis strains were isolated from water-damaged indoor environments. Two strains (N. alba subsp. alba 704a and a strain representing a novel species, ES10.1) as well as strains of N. prasina, N. lucentensis, and N. tropica produced methanol-soluble toxins that paralyzed the motility of boar spermatozoa at <30 microg of crude extract (dry weight) x ml(-1). N. prasina, N. lucentensis, N. tropica, and strain ES10.1 caused cessation of motility by dissipating the mitochondrial membrane potential, Deltapsi, of the boar spermatozoa. Indoor strain 704a produced a substance that destroyed cell membrane barrier function and depleted the sperm cells of ATP. Indoor strain 64/93 was antagonistic towards Corynebacterium renale. Two indoor Nocardiopsis strains were xerotolerant, and all five utilized a wide range of substrates. This combined with the production of toxic substances suggests good survival and potential hazard to human health in water-damaged indoor environments. Two new species, Nocardiopsis exhalans sp. nov. (ES10.1T) and Nocardiopsis umidischolae sp. nov. (66/93T), are proposed based on morphology, chemotaxonomic and physiological characters, phylogenetic analysis, and DNA-DNA reassociations.     

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.