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Archives of Microbiology (v.192, #11)
Molecular and structural modeling of the Phanerochaete flavido-alba extracellular laccase reveals its ferroxidase structure by Francisco Rodríguez-Rincón; Antonio Suarez; Mathias Lucas; Luis Fernando Larrondo; Teresa de la Rubia; Julio Polaina; José Martínez (pp. 883-892).
The fungus Phanerochaete flavido-alba is highly efficient in the oxidation of olive oil wastewater-derived polyphenols. This capability is largely due to the action of a multicopper-oxidase (MCO), encoded by the pfaL gene. We describe the sequence and organization of pfaL gene and the biochemical characterization and predicted 3D structural model of the encoded protein. pfaL gene organization and peptide sequence are highly similar to those of P. chrysosporium MCOs. However, PfaL is the first MCO in the Phanerochaete genus to show evident laccase activity. Phylogenetic analysis places PfaL in a differentiated sub-branch of ferroxidases. Protein structure analysis reveals close similarity of PfaL and ferroxidases and provides clues about the differences of activity between both types of enzymes. To summarize, P. flavido-alba laccase is the first enzyme in the novel and biochemically poorly defined group of “ferroxidases/laccases” that shows efficacious oxidation of laccase substrates, biotechnologically exploitable in bioremediation approaches.
Keywords: Laccase; Phanerochaete flavido-alba ; Olive oil wastewater; Multicopper oxidase
Antibacterial mechanism of soybean isoflavone on Staphylococcus aureus by Qian Wang; Haitao Wang; Mingjie Xie (pp. 893-898).
Effects of different flavonoids on various bacterial strains have been extensively reported; however, the mechanism(s) of their action on bacterial cells remain largely elusive. In this study, the antibacterial mechanism of soybean isoflavone (SI) on Staphylococcus aureus is systematically investigated using 4′6-diamidino-2-phenylindole (DAPI) staining, pBR322DNA decatenation experiment mediated by topoisomerase and agarose gel electrophoresis for direct decatenation. The results of fluorescence microscopy and fluorescence spectrophotometer indicated that DAPI was integrated in Staphylococcus aureus. Additionally, the quantity of both DNA and RNA reduced to 66.47 and 60.18%, respectively, after treated with SI for 28 h. Effects of SI on topoisomerase I and II were also investigated. SI completely inhibited the pBR322DNA unwinding mediated by topoisomerase I and topoisomerase II at the concentration of 6.4 mg/ml and could denature the plasmid DNA at the concentration of 12.8 mg/ml. These results indicate that topoisomerase I and II are the most important targets by SI to restrain bacterial cell division.
Keywords: Soybean isoflavone; Antibacterial mechanism; Nucleic acids’ synthesis inhibition; DNA topoisomerase
Role of Nitrosomonas europaea NitABC iron transporter in the uptake of Fe3+-siderophore complexes by Neeraja Vajrala; Luis A. Sayavedra-Soto; Peter J. Bottomley; Daniel J. Arp (pp. 899-908).
Nitrosomonas europaea has a single three-gene operon (nitABC) encoding an iron ABC transporter system (NitABC). Phylogenetic analysis clustered the subunit NitB with Fe3+-ABC transporter permease components from other organisms. The N. europaea strain deficient in nitB (nitB::kan) grew well in either Fe-replete or Fe-limited media and in Fe-limited medium containing the catecholate-type siderophore, enterobactin or the citrate-based dihydroxamate-type siderophore, aerobactin. However, the nitB::kan mutant strain was unable to grow in Fe-limited media containing either the hydroxamate-type siderophores, ferrioxamine and ferrichrome or the mixed-chelating type siderophore, pyoverdine. Exposure of N. europaea cells to a ferrichrome analog coupled to the fluorescent moiety naphthalic diimide (Fhu-NI) led to increase in fluorescence in the wild type but not in nitB::kan mutant cells. Spheroplasts prepared from N. europaea wild type exposed to Fhu-NI analog retained the fluorescence, while spheroplasts of the nitB::kan mutant were not fluorescent. NitABC transports intact Fe3+-ferrichrome complex into the cytoplasm and is an atypical ABC type iron transporter for Fe3+ bound to ferrioxamine, ferrichrome or pyoverdine siderophores into the cytoplasm. The mechanisms to transport iron in either the Fe3+ or Fe2+ forms or Fe3+ associated with enterobactin or aerobactin siderophores into the cell across the cytoplasmic membrane are as yet undetermined.
Keywords: Nitrosomonas europaea ; Fe; Iron ABC transporter; Siderophores; Ferrioxamine; Ferrichrome; Pyoverdine; Enterobactin; Aerobactin; Fluorescently labeled siderophore
Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic by Tatina T. Todorova; Anna V. Kujumdzieva; Stéphane Vuilleumier (pp. 909-918).
The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2–2.5-fold upon addition of either arsenate (AsV) or arsenite (AsIII). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3′-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation.
Keywords: Ure2; Glutathione S-transferase; GATA regulation; Arsenic detoxification
Chromate reductase activity of the Paracoccus denitrificans ferric reductase B (FerB) protein and its physiological relevance by Vojtěch Sedláček; Igor Kučera (pp. 919-926).
The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O2 absent) or > 2 (O2 present), the apparent K M value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s−1 (mg protein)−1. Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.
Keywords: Metal bioreduction; Flavoenzyme; Flavin radical; Oxidative stress
Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal by A. Brandão; T. Almeida; E. Muñoz-Atienza; C. Torres; G. Igrejas; P. E. Hernández; L. M. Cintas; P. Poeta; C. Herranz (pp. 927-936).
The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.
Keywords: Enterococcus spp.; Antimicrobial activity; Bacteriocins; Enterocin structural genes
The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures by Ying Jiang; Xi-Ming Chen; Ya-Jie Liu; Yuan-Ting Li; Hai-Hong Zhang; Paul Dyson; Hong-Mei Sheng; Li-Zhe An (pp. 937-943).
The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5′-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.
Keywords: OtsA; N-loop; Arthrobacter ; Trehalose; UDP-Glc; Low temperature
Shotgun proteomics of Xanthobacter autotrophicus Py2 reveals proteins specific to growth on propylene by Christopher A. Broberg; Daniel D. Clark (pp. 945-957).
Coenzyme M (CoM, 2-mercaptoethanesulfonate), once thought to be exclusively produced by methanogens, is now known to be the central cofactor in the metabolism of short-chain alkenes by a variety of aerobic bacteria. There is little evidence to suggest how, and under what conditions, CoM is biosynthesized by these organisms. A shotgun proteomics approach was used to investigate CoM-dependent propylene metabolism in the Gram-negative bacterium Xanthobacter autotrophicus Py2. Cells were grown on either glucose or propylene, and the soluble proteomes were analyzed. An average of 395 proteins was identified from glucose-grown replicates, with an average of 419 identified from propylene-grown replicates. A number of linear megaplasmid (pXAUT01)-encoded proteins were found to be specifically produced by growth on propylene. These included all known to be crucial to propylene metabolism, in addition to an aldehyde dehydrogenase, a DNA-binding protein, and five putative CoM biosynthetic enzymes. This work has provided fresh insight into bacterial alkene metabolism and has generated new targets for future studies in X. autotrophicus Py2 and related CoM-dependent alkene-oxidizing bacteria.
Keywords: Alkene; Coenzyme M; LC-MS/MS; Proteomics; Shotgun; Xanthobacter autotrophicus
Assessment of the modulating effects of fructo-oligosaccharides on fecal microbiota using human flora–associated piglets by Jian Shen; Baorang Zhang; Hua Wei; Chuanyan Che; Dezhong Ding; Xiuguo Hua; Peter Bucheli; Linghua Wang; Yixue Li; Xiaoyan Pang; Liping Zhao (pp. 959-968).
We first used human flora–associated (HFA) piglets, a significantly improved model for research on human gut microbiota, to study the effects of short-chain fructo-oligosaccharides (scFOS) on the gut bacterial populations. Ten neonatal HFA piglets were assigned to receive basal diets alone or supplemented with scFOS (0.5 g/kg body weight/day) from 1 to 37 days after birth (DAB). The impact of scFOS on the fecal bacterial populations of the piglets before (12 DAB), during (17 DAB), and after (25 and 37 DAB) weaning were monitored by PCR-denaturing gradient gel electrophoresis and real-time quantitative PCR. The Bifidobacterium genus was stimulated consistently, except during weaning, confirming the bifidogenic property of scFOS. At 12 DAB, the Clostridium leptum subgroup was decreased and two unknown Bacteroides-related species were increased; at 25 DAB, the C. leptum subgroup and Subdoligranulum variabile-like species were elevated, whereas one unknown Faecalibacterium-related species was suppressed; and at 37 DAB, the Bacteroides genus was decreased. The results showed that effects of scFOS on non-bifidobacteria varied at different developmental stages of the animals, warranting further investigation into the host-development-related effects of prebiotics on the gut microbiota and the host physiology using the HFA piglets as a model for humans.
Keywords: Fructo-oligosaccharides; Prebiotics; Intestinal microbiota; Human flora–associated piglet model; Denaturing gradient gel electrophoresis (DGGE); Real-time PCR
Tellurite-induced carbonylation of the Escherichia coli pyruvate dehydrogenase multienzyme complex by Nataly del P. Contreras; Claudio C. Vásquez (pp. 969-973).
The soluble tellurium oxyanion, tellurite, is toxic for most organisms. At least in part, tellurite toxicity involves the generation of oxygen-reactive species which induce an oxidative stress status that damages different macromolecules with DNA, lipids and proteins as oxidation targets. The objective of this work was to determine the effects of tellurite exposure upon the Escherichia coli pyruvate dehydrogenase (PDH) complex. The complex displays two distinct enzymatic activities: pyruvate dehydrogenase that oxidatively decarboxylates pyruvate to acetylCoA and tellurite reductase, which reduces tellurite (Te4+) to elemental tellurium (Teo). PDH complex components (AceE, AceF and Lpd) become oxidized upon tellurite exposure as a consequence of increased carbonyl group formation. When the individual enzymatic activities from each component were analyzed, AceE and Lpd did not show significant changes after tellurite treatment. AceF activity (dihydrolipoil acetyltransferase) decreased ~30% when cells were exposed to the toxicant. Finally, pyruvate dehydrogenase activity decreased >80%, while no evident changes were observed in complex′s tellurite reductase activity.
Keywords: Pyruvate dehydrogenase complex; Tellurite; Oxidative stress; Escherichia coli
Organic acid production and plant growth promotion as a function of phosphate solubilization by Acinetobacter rhizosphaerae strain BIHB 723 isolated from the cold deserts of the trans-Himalayas by Arvind Gulati; Natasha Sharma; Pratibha Vyas; Swati Sood; Praveen Rahi; Vijaylata Pathania; Ramdeen Prasad (pp. 975-983).
An efficient phosphate-solubilizing plant growth–promoting Acinetobacter rhizosphaerae strain BIHB 723 exhibited significantly higher solubilization of tricalcium phosphate (TCP) than Udaipur rock phosphate (URP), Mussoorie rock phosphate (MRP) and North Carolina rock phosphate (NCRP). Qualitative and quantitative differences were discerned in the gluconic, oxalic, 2-keto gluconic, lactic, malic and formic acids during the solubilization of various inorganic phosphates by the strain. Gluconic acid was the main organic acid produced during phosphate solubilization. Formic acid production was restricted to TCP solubilization and oxalic acid production to the solubilization of MRP, URP and NCRP. A significant increase in plant height, shoot fresh weight, shoot dry weight, root length, root dry weight, and root, shoot and soil phosphorus (P) contents was recorded with the inoculated treatments over the uninoculated NP0K or NPTCPK treatments. Plant growth promotion as a function of phosphate solubilization suggested that the use of bacterial strain would be a beneficial addition to the agriculture practices in TCP-rich soils in reducing the application of phosphatic fertilizers.
Keywords: Acinetobacter rhizosphaerae ; Phosphate-solubilizing rhizobacteria; Phosphate substrates; Plant growth promotion; Organic acids; Maize