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Archives of Microbiology (v.180, #1)
Mercury pretreatment selects an enhanced cadmium-accumulating phenotype in Euglena gracilis by César Avilés; Herminia Loza-Tavera; Norman Terry; Rafael Moreno-Sánchez (pp. 1-10).
Pre-treatment of heterotrophic cultures of Euglena gracilis with 1.5 μM HgCl2 for at least 60 generations resulted in a cell population that showed both increased resistance to Cd2+ and ability to accumulate it, when compared to non-Hg2+-pretreated Euglena. These Hg2+-enhanced capacities were evident in cells cultured in the dark in a medium with lactate, but not in cells cultured with glutamate plus malate. After culturing with 0.1 mM CdCl2 through three consecutive transfers, the mercury-pretreated cells still grew and maintained high levels of glutathione-related metabolites, while the non-Hg2+-pretreated cells died. Cultures of Hg2+-pretreated cells, after transfer to media with or without cadmium, did not alter either their enhanced Cd2+ accumulation or their increased production of glutathione-related metabolites. These observations suggested that the Hg2+-pretreated population underwent a permanent change that improved its Cd2+ resistance. Several factors that contributed to the improved capacities included: (a) higher cellular malate, cysteine and glutathione levels induced by Hg2+ before and after Cd2+ exposure; and (b) increased storage of Cd2+ in mitochondria along with increased intramitochondrial citrate, cysteine, and glutathione levels. These characteristics suggested that this Cd2+ hyper-accumulating strain of E. gracilis might be a suitable candidate for Cd2+-bioremediation of polluted water systems.
Keywords: Euglena gracilis ; Hg2+ pretreatment; Cd2+ resistance; Cd2+ compartmentation; Cd2+ hyperaccumulation; Phytochelatins; Mitochondria
Variations in metabolism of the soy isoflavonoid daidzein by human intestinal microfloras from different individuals by Fatemeh Rafii; Christy Davis; Miseon Park; Thomas M. Heinze; Richard D. Beger (pp. 11-16).
Isoflavonoids found in legumes, such as soybeans, are converted by intestinal bacteria to metabolites that might have increased or decreased estrogenic activity. Variation in the effects of dietary isoflavonoids among individuals has been attributed to differences in their metabolism by intestinal bacteria. To investigate this variation, the metabolism of the isoflavonoid daidzein by bacteria from ten fecal samples, provided at different times by six individuals on soy-containing diets, was compared. After anaerobic incubation of bacteria with daidzein for 2 weeks, four samples had metabolized daidzein and six samples had not. Three of the positive samples were from individuals whose microflora had not metabolized daidzein in previous samples. Dihydrodaidzein was observed in one sample, dihydrodaidzein and equol in another sample, and equol and O-desmethylangolensin in two other samples. These results corroborate the hypothesis that the microflora of the gastrointestinal tract of an individual influences the particular isoflavone metabolites produced following consumption.
Keywords: Isoflavonoids; Daidzein; Equol; Intestinal bacteria; Soybean
Effect of growth conditions on the motility of Photorhabdus temperata by Meggan M. Hodgson; Brandye Day; Donald J. White; Louis S. Tisa (pp. 17-24).
Photorhabdus temperata is a bioluminescent bacterium that lives in mutualistic association with entomopathogenic nematodes of the genus Heterorhabditis. The bacterium exists in two morphologically distinguishable phases (primary and secondary). The swimming behavior of P. temperata was investigated. Both the primary and secondary variants were able to swim in liquid or semisolid media under appropriate conditions. Variation in the oxygen levels had little affect on the chemotaxis and motility of the primary form, but greatly influenced the behavior of the secondary form. Under oxic conditions the secondary form was nonmotile, but motility was induced under anoxic conditions. Several phenotypic traits of the primary form were not expressed under anoxic conditions. The constituents of the growth media affected the motility of both variants. P. temperata required additional NaCl or KCl for optimum motility and chemotaxis. Optimal chemotactic behavior required the presence of bacto-peptone and yeast extract in the swim-migration medium. A mutant that was isolated from the secondary form was able to swim under oxic conditions and possessed an altered salt requirement for motility.
Keywords: Chemotaxis; Photorhabdus ; Signal transduction; Environmental signals; Nematode; Biocontrol agent; Anoxic conditions
Novobiocin biosynthesis: inactivation of the putative regulatory gene novE and heterologous expression of genes involved in aminocoumarin ring formation by Alessandra S. Eustáquio; Thomas Luft; Zhao-Xin Wang; Bertolt Gust; Keith F. Chater; Shu-Ming Li; Lutz Heide (pp. 25-32).
The left ends of the biosynthetic gene clusters of novobiocin (nov), clorobiocin (clo) and coumermycin A1 (cou) from Streptomyces spheroides (syn. S. caeruleus) NCIMB 11891, S. roseochromogenes var. oscitans DS 12.976 and S. rishiriensis DSM 40489 were cloned and sequenced. Sequence comparison suggested that novE, cloE and couE, respectively, represent the borders of these three clusters. Inactivation of novE proved that novE does not have an essential catalytic role in novobiocin biosynthesis, but is likely to have a regulatory function. The gene products of novF and cloF show sequence similarity to prephenate dehydrogenase and may produce 4-hydroxyphenylpyruvate (4HPP) as a precursor of the substituted benzoate moiety of novobiocin and clorobiocin. Coumermycin A1 does not contain this benzoate moiety, and correspondingly the coumermycin cluster was found not to contain a functional novF homologue. The coumermycin biosynthetic gene cluster apparently evolved from an ancestral cluster similar to those of novobiocin and clorobiocin, and parts of the ancestral novF homologue have been deleted in this process. No homologue to novC was identified in the gene clusters of clorobiocin and coumermycin, questioning the postulated involvement of novC in aminocoumarin biosynthesis. Heterologous expression of novDEFGHIJK in Streptomyces lividans resulted in the formation of 2,4-dihydroxy-α-oxy-phenylacetic acid, suggesting that at least one of the proteins encoded by these genes may participate in a hydroxylation reaction.
Keywords: Streptomyces spheroides ; Novobiocin; Clorobiocin; Coumermycin; Gene inactivation; PCR targeting
Identification and functional analysis of six mycolyltransferase genes of Corynebacterium glutamicum ATCC 13032: the genes cop1, cmt1, and cmt2 can replace each other in the synthesis of trehalose dicorynomycolate, a component of the mycolic acid layer of the cell envelope by Sven Brand; Karsten Niehaus; Alfred Pühler; Jörn Kalinowski (pp. 33-44).
By data mining in the sequence of the Corynebacterium glutamicum ATCC 13032 genome, six putative mycolyltransferase genes were identified that code for proteins with similarity to the N-terminal domain of the mycolic acid transferase PS1 of the related C. glutamicum strain ATCC 17965. The genes identified were designated cop1, cmt1, cmt2, cmt3, cmt4, and cmt5 (cmt from corynebacterium mycolyltransferases). cop1 encodes a protein of 657 amino acids, which is larger than the proteins encoded by the cmt genes with 365, 341, 483, 483, and 411 amino acids. Using bioinformatics tools, it was shown that all six gene products are equipped with signal peptides and esterase domains. Proteome analyses of the cell envelope of C. glutamicum ATCC 13032 resulted in identification of the proteins Cop1, Cmt1, Cmt2, and Cmt4. All six mycolyltransferase genes were used for mutational analysis. cmt4 could not be mutated and is considered to be essential. cop1 was found to play an additional role in cell shape formation. A triple mutant carrying mutations in cop1, cmt1, and cmt2 aggregated when cultivated in MM1 liquid medium. This mutant was also no longer able to synthesize trehalose dicorynomycolate (TDCM). Since single and double mutants of the genes cop1, cmt1, and cmt2 could form TDCM, it is concluded that the three genes, cop1, cmt1, and cmt2, are involved in TDCM biosynthesis. The presence of the putative esterase domain makes it highly possible that cop1, cmt1, and cmt2 encode enzymes synthesizing TDCM from trehalose monocorynomycolate.
Keywords: Mycolic acids; Trehalose dimycolate; Trehalose monomycolate; Mycolyltransferase; Cell envelope; Gene deletion
Genetic diversity of fast-growing rhizobia that nodulate soybean (Glycine max L. Merr) by Gustavo Saldaña; Virginia Martinez-Alcántara; José M. Vinardell; Ramón Bellogín; José E. Ruíz-Sainz; Pedro Alberto Balatti (pp. 45-52).
The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.
Keywords: Sinorhizobium fredii ; Genotype; Genetic diversity; PCR; Symbiosis; Soybean
Improved vectors for transcriptional/translational signal screening in corynebacteria using the melC operon from Streptomyces glaucescens as reporter by Sirin A. I. Adham; Sonia Rodríguez; Angelina Ramos; Ramón I. Santamaría; José A. Gil (pp. 53-59).
The tyrosinase operon (melC) from Streptomyces glaucescens was cloned and functionally expressed in Brevibacterium lactofermentum and Corynebacterium glutamicum under the control of the promoter of the kan gene from Tn5. Recombinant corynebacterial cells containing the tyrosinase operon produced melanin on agar plates and in liquid culture when supplemented with copper and tyrosine. A conjugative bifunctional replacement vector for transcriptional/translational signal screening (pEMel-1) was constructed using expression of the melC operon from S. glaucescens, which can be used for cloning promoter sequences as EcoRI–NdeI fragments. When the DNA fragments with promoter activity such as cspBp or trpp were inserted into pEMel-1, B. lactofermentum harboring the chimeric plasmids produced melanin at different stages of growth, allowing temporal detection of promoter activity. The vector was also used to detect the activity of a Streptomyces promoter (xysAp), which was inactive in B. lactofermentum, after PCR mutagenesis. The melC operon can be used for the visual, inexpensive (compared to the high price of starch azure for amylase detection), and non-selective (in contrast to the kan or cat genes) screening of several thousand clones at high colony density without killing of the transformants due to the presence of iodine (as in the case of amylase assay).
Keywords: Corynebacterium glutamicum ; Brevibacterium lactofermentum ; Promoters; melC ; trp ; cspB ; xysA ; Mutagenic PCR; Streptomyces
Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics by D. Barrie Johnson; Naoko Okibe; Francisco F. Roberto (pp. 60-68).
Moderately thermophilic acidophilic bacteria were isolated from geothermal (30–83 °C) acidic (pH 2.7–3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 °C, and pH 1.0–1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria (Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the α-Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also α-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes (SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.
Keywords: Acidophiles; Biodiversity; Iron oxidation; Iron reduction; Moderate thermophiles; Yellowstone National Park
ADP-dependent glucokinase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 by Antje Labes; Peter Schönheit (pp. 69-75).
The hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324 has been shown to degrade starch via glucose using a modified Embden-Meyerhof pathway. The first enzyme of this pathway, ADP-dependent glucokinase, was purified 600-fold to homogeneity. The enzyme is a monomeric protein with an apparent molecular mass of 50 kDa. It had a temperature optimum at 83 °C and showed a significant thermostability up to 100 °C. The enzyme was highly specific for ADP and glucose as substrates; it did not use ATP, CDP, UDP, or GDP as phosphoryl donors, or mannose, fructose and fructose 6-phosphate as phosphoryl acceptors (at 80 °C). Only glucosamine was phosphorylated at significant rates. The apparent K m values for ADP and glucose (at 50 °C) were 0.07 mM and 0.78 mM, respectively; the apparent V max value was about 50 U/mg at 50 °C and 350 U/mg at 80 °C. Divalent cations were required for maximal activity; Mn2+, Mg2+ and Ca2+, which were most effective, could be replaced partially by Cu2+, Ni2+, Co2+ and Zn2+. The N-terminal amino acid sequence (42 amino acids) of ADP-dependent glucokinase was almost identical to that of ADP-dependent glucokinase from Thermococcus litoralis. In the genome of the closely related Archaeoglobus fulgidus strain VC16 a homologous gene for ADP-dependent glucokinase could not be identified.
Keywords: Hyperthermophilic archaea; Archaeoglobus fulgidus strain 7324; ADP-dependent glucokinase; Modified Embden Meyerhof pathway
Distribution of antigenic oligomannosyl side chains in the cell wall mannans of several strains of Candida tropicalis by Hidemitsu Kobayashi; Hiroko Oyamada; Kyoko Matsuda; Nobuyuki Shibata; Shigeo Suzuki (pp. 76-80).
In order to clarify the distribution of antigenic oligomannosyl side chains in the cell wall mannans of the pathogenic yeast Candida tropicalis, the chemical structure of mannans isolated from four C. tropicalis strains was investigated using nuclear magnetic resonance, two-dimensional homonuclear Hartmann-Hahn (2D-HOHAHA) spectroscopy. Two-dimensional maps of the 2D-HOHAHA clearly showed the distribution of oligomannosyl side chains in the mannans. The linear side chain Manα1–3Manα1–(2Manα1–)n2Man [n≥2] is present in the mannans from C. tropicalis IFO 0589 and IFO 1400, but not in the mannans from IFO 0199 and IFO 1647. The mannan of IFO 0589 is the only mannan with the branched side chains, Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man and Manα1–2Manα1–3[Manα1–6]Manα1–(2Manα1-)n2Man [n≥2]. However, this mannan lacked the phosphate group and the β-1,2-linked oligomannosyl side chain which are features of this group. The mannans of the C. tropicalis strains IFO 0589 and IFO 1400 possessed the side chains containing an α-1,3-linked mannose residue previously observed in Candida albicans.
Keywords: Candida tropicalis ; Cell wall mannan; Antigenic factor; Polysaccharide antigen; Nuclear magnetic resonance