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Archives of Microbiology (v.183, #1)
A novel 2-aminophenol 1,6-dioxygenase involved in the degradation of p-chloronitrobenzene by Comamonas strain CNB-1: purification, properties, genetic cloning and expression in Escherichia coli by Jian-Feng Wu; Cui-Wei Sun; Cheng-Ying Jiang; Zhi-Pei Liu; Shuang-Jiang Liu (pp. 1-8).
Comamonas strain CNB-1 was isolated from a biological reactor treating wastewater from a p-chloronitrobenzene production factory. Strain CNB-1 used p-chloronitrobenzene as sole source of carbon, nitrogen, and energy. A 2-aminophenol 1,6-dioxygenase was purified from cells of strain CNB-1. The purified 2-aminophenol 1,6-dioxygenase had a native molecular mass of 130 kDa and was composed of α- and β-subunits of 33 and 38 kDa, respectively. This enzyme is different from currently known 2-aminophenol 1,6-dioxygenases in that it: (a) has a higher affinity for 2-amino-5-chlorophenol (Km=0.77 μM) than for 2-aminophenol (Km=0.89 μM) and (b) utilized protocatechuate as a substrate. These results suggested that 2-amino-5-chlorophenol, an intermediate during p-chloronitrobenzene degradation, is the natural substrate for this enzyme. N-terminal amino acids of the α- and β-subunits were determined to be T-V-V-S-A-F-L-V and M-Q-G-E-I-I-A-E, respectively. A cosmid library was constructed from the total DNA of strain CNB-1 and three clones (BG-1, BG-2, and CG-13) with 2-aminophenol 1,6-dioxygenase activities were obtained. DNA sequencing of clone BG-2 revealed a 15-kb fragment that contained two ORFs, ORF9 and ORF10, with N-terminal amino acid sequences identical to those of the β- and α-subunits, respectively, from the purified 2-aminophenol 1,6-dioxygenase. The enzyme was actively synthesized when the genes coding for the ORF9 and ORF10 were cloned into Escherichia coli.
Keywords: Comamonas2-Aminophenol 1; 6-Dioxygenase; Chloronitrobenzene degradation
FieF (YiiP) from Escherichia coli mediates decreased cellular accumulation of iron and relieves iron stress by Gregor Grass; Markus Otto; Beate Fricke; Christopher J. Haney; Christopher Rensing; Dietrich H. Nies; Doreen Munkelt (pp. 9-18).
The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of 55Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.
Keywords: Iron; Zinc; Efflux; Cation diffusion facilitatorsE. coliFluorescence quenching
Phylogeny of symbiotic cyanobacteria within the genus Nostoc based on 16S rDNA sequence analyses by Mette M. Svenning; Torsten Eriksson; Ulla Rasmussen (pp. 19-26).
A phylogenetic analysis of selected symbiotic Nostoc strain sequences and available database 16S rDNA sequences of both symbiotic and free-living cyanobacteria was carried out using maximum likelihood and Bayesian inference techniques. Most of the symbiotic strains fell into well separated clades. One clade consisted of a mixture of symbiotic and free-living isolates. This clade includes Nostoc sp. strain PCC 73102, the reference strain proposed for Nostoc punctiforme. A separate symbiotic clade with isolates exclusively from Gunnera species was also obtained, suggesting that not all symbiotic Nostoc species can be assigned to N. punctiforme. Moreover, isolates from Azolla filiculoides and one from Gunnera dentata were well nested within a clade comprising most of the Anabaena sequences. This result supports the affiliation of the Azolla isolates with the genus Anabaena and shows that strains within this genus can form symbioses with additional hosts. Furthermore, these symbiotic strains produced hormogonia, thereby verifying that hormogonia formation is not absent in Anabaena and cannot be used as a criterion to distinguish it from Nostoc.
Keywords: CyanobacteriaNostocAnabaenaSymbioses; 16S rRNA gene; Sequencing; Phylogeny
The genome sequence of an anaerobic aromatic-degrading denitrifying bacterium, strain EbN1 by Ralf Rabus; Michael Kube; Johann Heider; Alfred Beck; Katja Heitmann; Friedrich Widdel; Richard Reinhardt (pp. 27-36).
Recent research on microbial degradation of aromatic and other refractory compounds in anoxic waters and soils has revealed that nitrate-reducing bacteria belonging to the Betaproteobacteria contribute substantially to this process. Here we present the first complete genome of a metabolically versatile representative, strain EbN1, which metabolizes various aromatic compounds, including hydrocarbons. A circular chromosome (4.3 Mb) and two plasmids (0.21 and 0.22 Mb) encode 4603 predicted proteins. Ten anaerobic and four aerobic aromatic degradation pathways were recognized, with the encoding genes mostly forming clusters. The presence of paralogous gene clusters (e.g., for anaerobic phenylacetate oxidation), high sequence similarities to orthologs from other strains (e.g., for anaerobic phenol metabolism) and frequent mobile genetic elements (e.g., more than 200 genes for transposases) suggest high genome plasticity and extensive lateral gene transfer during metabolic evolution of strain EbN1. Metabolic versatility is also reflected by the presence of multiple respiratory complexes. A large number of regulators, including more than 30 two-component and several FNR-type regulators, indicate a finely tuned regulatory network able to respond to the fluctuating availability of organic substrates and electron acceptors in the environment. The absence of genes required for nitrogen fixation and specific interaction with plants separates strain EbN1 ecophysiologically from the closely related nitrogen-fixing plant symbionts of the Azoarcus cluster. Supplementary material on sequence and annotation are provided at the Web page http://www.micro-genomes.mpg.de/ebn1/.
Keywords: Complete genome; Anaerobic degradation; Aromatic compounds; Denitrifying bacterium; Betaproteobacteria
Gene cloning, expression and functional characterization of a phosphopantetheinyl transferase from Vibrio anguillarum serotype O1 by Qin Liu; Yue Ma; Lingyun Zhou; Yuanxing Zhang (pp. 37-44).
Phosphopantetheinyl transferases (PPTases) catalyze the essential post-translational activation of carrier proteins from fatty acid synthetases (FASs) in primary metabolism and polyketide synthetases (PKSs) and non-ribosomal polypeptide synthetases (NRPSs) in secondary metabolism. Bacteria typically harbor one PPTase specific for carrier proteins of primary metabolism (ACPS-type PPTases) and at least one capable of modifying carrier proteins involved in secondary metabolism (Sfp-type PPTases). Anguibactin, an important virulent factor in Vibrio anguillarum serotype O1, has been reported to be synthesized by a nonribosomal peptide synthetases (NRPS) system encoded on a 65-kb virulent plasmid pJM1 from strain 775 of V. anguillarum serotype O1, and the PPTase, necessary for the activation of the anguibactin-NRPS, is therefore expected to lie on the pJM1 plasmid. In this work, a putative PPTase gene, angD, was first identified on pEIB1 plasmid (a pJM1-like plasmid) from a virulent strain MVM425 of V. anguillarum serotype O1. A recombinant clone carrying complete angD was able to complement an Escherichia coli entD mutant deficient in Sfp-type PPTase. angD was overexpressed in E. coli and the resultant protein, AngD, was purified. Simultaneously, two carrier proteins involved in anguibactin-NRPS, ArCP and PCP, were overproduced in E. coli and purified. The purified AngD, PCP and ArCP were used to establish an in vitro enzyme reaction, and the PPTase activity of AngD was proved through HPLC analysis to detect the conversion of inactive carrier proteins to active carrier proteins in the reaction mixture. Co-expression of AngD with PCP or ArCP showed that AngD functioned well as a PPTase in vivo in E. coli, modifying PCP and ArCP completely.
Keywords: Vibrio anguillarrumangD gene; Phosphopantetheinyl transferase; Carrier protein; Post-translational modification
Isolation and characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions by Karel Decroos; Steffi Vanhemmens; Sofie Cattoir; Nico Boon; Willy Verstraete (pp. 45-55).
Only about one third of humans possess a microbiota capable of transforming the dietary isoflavone daidzein into equol. Little is known about the dietary and physiological factors determining this ecological feature. In this study, the in vitro metabolism of daidzein by faecal samples from four human individuals was investigated. One culture produced the metabolites dihydrodaidzein and O-desmethylangolensin, another produced dihydrodaidzein and equol. From the latter, a stable and transferable mixed culture transforming daidzein into equol was obtained. Molecular fingerprinting analysis (denaturing gradient gel electrophoresis) showed the presence of four bacterial species of which only the first three strains could be brought into pure culture. These strains were identified as Lactobacillus mucosae EPI2, Enterococcus faecium EPI1 and Finegoldia magna EPI3, and did not produce equol in pure culture. The fourth species was tentatively identified as Veillonella sp strain EP. It was found that hydrogen gas in particular, but also butyrate and propionate, which are all colonic fermentation products from poorly digestible carbohydrates, stimulated equol production by the mixed culture. However, when fructo-oligosaccharides were added, equol production was inhibited. Furthermore, the equol-producing capacity of the isolated culture was maintained upon its addition to a faecal culture originating from a non-equol-producing individual.
Keywords: Isoflavone; Equol; Hydrogen gas; Bacteria; FOS
Chlorinated aliphatic hydrocarbon-induced degradation of trichloroethylene in Wautersia numadzuensis sp. nov. by Chizuko Kageyama; Toshiya Ohta; Kazuyo Hiraoka; Morimasa Suzuki; Tetsuji Okamoto; Kazuo Ohishi (pp. 56-65).
Two strains of trichloroethylene (TCE)-degrading bacteria were isolated from soils at polluted and unpolluted sites. The isolates, strains TE26T and K6, showed co-substrate-independent TCE-degrading activity. TCE degradation was accelerated by preincubation with tetrachloroethylene, cis-dichloroethylene (DCE) and 1,1-DCE. TCE-degrading activities of strains TE26T and K6 were 0.23, 0.24 μmol min−1 g−1 dry cells, respectively. 16S rDNA sequences of strains TE26T and K6 were almost identical (99.7% similarity), and most closely related to Ralstonia basilensis (ATCC17697T) (98.5% similarity). From the results of DNA–DNA hybridizations, strain TE26T was genetically coherent to strain K6 (94 and 88% hybridization), and exhibited lower relatedness to R. basilensis (DSM11853T) (44% and 15%). In addition, because of the differences in chemotaxonomic properties, strain TE26T and strain K6 appear to be distinct from all established species of the Ralstonia group. Based on these results and the proposal of transferring R. basilensis and related species to Wautersia gen. nov., we propose that these strains should be assigned to the genus Wautersia as Wautersia numadzuensis sp. nov.
Keywords: Trichloroethylene; Dichloroethylene; Chlorinated aliphatic hydrocarbon; Trichloroethylene degradation; BioremediationWautersia numadzuensis sp. nov.
Chlorosis during nitrogen starvation is altered by carbon dioxide and temperature status and is mediated by the ClpP1 protease in Synechococcus elongatus by Kara Barker-Åström; Jenny Schelin; Petter Gustafsson; Adrian K. Clarke; Douglas A. Campbell (pp. 66-69).
The interactive effects of inorganic carbon status, temperature and light on chlorosis induced by nitrogen deficiency, and the roles of Clp proteases in this process were investigated. In wild-type cultures grown in high or ambient CO2, following transfer to media lacking combined nitrogen, phycocyanin per cell dropped primarily through dilution of the pigment through cell division, and also suffered variable degrees of net degradation. When grown at high CO2 (5%), chlorophyll (Chl) suffered net degradation to a greater extent than phycocyanin. In marked contrast, growth at ambient CO2 resulted in Chl per cell dropping through dilution. Conditions that drove net Chl degradation in the wild-type resulted in little or no net Chl degradation in a clpPI inactivation mutant, with Chl content dropping largely through growth dilution in the mutant. The chlorotic response of a clpPII inactivation strain was nearly the same as that of wild-type, although phycocyanin degradation may have been slightly accelerated in the former.
Keywords: Carbon concentrating mechanism; Carbon dioxide; Chlorosis; Clp protease; Cyanobacteria; Photosystem I; Phycobilisome