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Archives of Microbiology (v.192, #6)
Advances in molecular detection of Aspergillus: an update by M. Z. Abdin; Malik M. Ahmad; Saleem Javed (pp. 409-425).
Filamentous cosmopolitan fungi of the genus Aspergillus can be harmful in two ways, directly they can be opportunistic pathogens causing aspergillosis and indirectly due to aflatoxin production on food products which can lead to aflatoxicosis. Therefore, a number of methods have been proposed so far for detection of the fungi with lowest possible concentration at the earliest. Molecular methods such as PCR and/or in combination with certain techniques have been found to be useful for Aspergillus detection. We discuss here various technologies that have emerged in recent years and can possibly be used for the molecular detection of Aspergillus in an efficient way. These methods like RSIC, C-probe, and inversion probe with pyrosequencing or direct ss/dsDNA detection have been used for the identification of fungal or bacterial pathogens and thus formulate a ‘gold standard’ for Aspergillus detection.
Keywords: Aspergillus ; Molecular detection; PCR; Polymerase chain reaction-enzyme immunoassay; DNA fingerprinting; Microarray technique; Retrotransposon insertion-site context typing; STRAf ; ELISA
Isolation of bacteria from mouse caecal samples and description of Bacteroides sartorii sp. nov by Thomas Clavel; Anja Saalfrank; Cédric Charrier; Dirk Haller (pp. 427-435).
Caecal samples from wild-type and TNFdeltaARE mice were cultured on selective media containing bile salts, amino acids or casein macro-peptides. Twenty-two strains were isolated and identified by 16S rRNA gene sequencing. Twenty-one strains showed >98% similarity to known bacteria (Blautia spp., Clostridium innocuum, Enterococcus spp., Escherichia coli, Lactobacillus murinus, Parabacteroides goldsteinii and Shigella dysenteriae). One additional isolate, strain A-C2-0, was a new bacterium. The closest relatives were Bacteroides massiliensis, Bacteroides dorei and Bacteroides vulgatus (≤94% similarity). Strain A-C2-0 is a Gram-negative rod that does not form spores and has a G + C content of DNA of 41.5%. Its major cellular fatty acid is C15:0 ANTEISO, and its major respiratory quinone is MK-9. Cells are aerotolerant but grow only under strict anoxic conditions. They are resistant to cefotaxime and tobramycin. When compared with related Bacteroides spp., the new bacterium was positive for α-arabinosidase, negative for glutamyl glutamic acid arylamidase and did not metabolise galactose, glucose, fructose, mannose, raffinose and sucrose. Strain A-C2-0 therefore merits recognition as a member of a novel species within the genus Bacteroides, for which the name Bacteroides sartorii is proposed. The type strain is A-C2-0T (= DSM 21941T = CCUG 57211T).
Keywords: Mouse caecum; Bacterial diversity; Bacteroidetes ; Bacteroides sartorii ; TNFdeltaARE mice; Chronic intestinal inflammation
Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z by Emi Miyamoto; Yuri Tanioka; Ayako Nishizawa-Yokoi; Yukinori Yabuta; Kouhei Ohnishi; Haruo Misono; Shigeru Shigeoka; Yoshihisa Nakano; Fumio Watanabe (pp. 437-446).
Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4°C with 10 μM 5′-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa ± 5.0 kDa using Superdex 200 gel filtration. SDS–polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa ± 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5′-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.
Keywords: Cobalamin; Euglena gracilis Z; Methylmalonyl-CoA mutase; Photoassimilation; Propionate metabolism
Oxidative stress protection and the repair response to hydrogen peroxide in the hyperthermophilic archaeon Pyrococcus furiosus and in related species by Kari R. Strand; Chengjun Sun; Ting Li; Francis E. Jenney Jr.; Gerrit J. Schut; Michael W. W. Adams (pp. 447-459).
Pyrococcus furiosus is a shallow marine, anaerobic archaeon that grows optimally at 100°C. Addition of H2O2 (0.5 mM) to a growing culture resulted in the cessation of growth with a 2-h lag before normal growth resumed. Whole genome transcriptional profiling revealed that the main response occurs within 30 min of peroxide addition, with the up-regulation of 62 open reading frames (ORFs), 36 of which are part of 10 potential operons. More than half of the up-regulated ORFs are of unknown function, while some others encode proteins that are involved potentially in sequestering iron and sulfide, in DNA repair and in generating NADPH. This response is thought to involve primarily damage repair rather than protection, since cultures exposed to sub-toxic levels of H2O2 were not more resistant to the subsequent addition of H2O2 (0.5–5.0 mM). Consequently, there is little if any induced protective response to peroxide. The organism maintains a constitutive protective mechanism involving high levels of oxidoreductase-type enzymes such as superoxide reductase, rubrerythrin, and alkyl hydroperoxide reductase. Related hyperthermophiles contain homologs of the proteins involved in the constitutive protective mechanism but these organisms were more sensitive to peroxide than P. furiosus and lack several of its peroxide-responsive ORFs.
Keywords: Hyperthermophile; Anaerobic; Oxidative stress; Peroxide response
CalA, a cyAbrB protein, binds to the upstream region of ftsZ and is down-regulated in heterocysts in Anabaena sp. PCC 7120 by Dongli He; Xudong Xu (pp. 461-469).
A protein of cyAbrB family of regulators, Alr0946 (CalA), was identified by DNA affinity chromatography using DNA fragments from the promoter region of ftsZ in Anabaena/Nostoc sp. PCC 7120. Electrophoretic mobility shift assays showed that the protein could bind to upstream regions of ftsZ (as biotin-labeled DNA fragments) and that the binding was inhibited by the same DNA fragments and a fragment upstream of hetC but not by 3 fragments upstream of glnA, hetR or ntcA. Both transcriptional fusion with gfp (green fluorescence protein) and Western blot analysis indicated that the expression of calA was down-regulated in heterocysts relative to vegetative cells. An insertional mutant of calA could not be completely segregated. Over-expression of calA in Anabaena/Nostoc caused no change in growth, heterocyst differentiation or expression of ftsZ. CalA as a DNA-binding protein is essential for the growth of Anabaena/Nostoc and may be required for the expression of ftsZ in vegetative cells.
Keywords: CalA; ftsZ ; DNA-binding protein; Anabaena
Pseudomonas putida A ATCC 12633 oxidizes trimethylamine aerobically via two different pathways by Andrés S. Liffourrena; Mario A. Salvano; Gloria I. Lucchesi (pp. 471-476).
The present study examined the aerobic metabolism of trimethylamine in Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine. In both conditions, the trimethylamine was used as a nitrogen source and also accumulated in the cell, slowing the bacterial growth. Decreased bacterial growth was counteracted by the addition of AlCl3. Cell-free extracts prepared from cells grown aerobically on tetradecyltrimethylammonium bromide exhibited trimethylamine monooxygenase activity that produced trimethylamine N-oxide and trimethylamine N-oxide demethylase activity that produced dimethylamine. Cell-free extracts from cells grown on trimethylamine exhibited trimethylamine dehydrogenase activity that produced dimethylamine, which was oxidized to methanal and methylamine by dimethylamine dehydrogenase. These results show that this bacterial strain uses two enzymes to initiate the oxidation of trimethylamine in aerobic conditions. The apparent Km for trimethylamine was 0.7 mM for trimethylamine monooxygenase and 4.0 mM for trimethylamine dehydrogenase, but both enzymes maintain similar catalytic efficiency (0.5 and 0.4, respectively). Trimethylamine dehydrogenase was inhibited by trimethylamine from 1 mM. Therefore, the accumulation of trimethylamine inside Pseudomonas putida A ATCC 12633 grown on tetradecyltrimethylammonium bromide or trimethylamine may be due to the low catalytic efficiency of trimethylamine monooxygenase and trimethylamine dehydrogenase.
Keywords: Trimethylamine; Tetradecyltrimethylammonium; Trimethylamine monooxygenase; Trimethylamine dehydrogenase; Pseudomonas putida
Saccharomyces cerevisiae strain UFMG 905 protects against bacterial translocation, preserves gut barrier integrity and stimulates the immune system in a murine intestinal obstruction model by Simone V. Generoso; Mirelle Viana; Rosana Santos; Flaviano S. Martins; José A. N. Machado; Rosa M. E. Arantes; Jacques R. Nicoli; Maria I. T. D. Correia; Valbert N. Cardoso (pp. 477-484).
Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral treatment with viable or heat-killed yeast Saccharomyces cerevisiae strain UFMG 905 prevents bacterial translocation (BT), intestinal barrier integrity, and stimulates the immunity, in a murine intestinal obstruction (IO) model. Four groups of mice were used: mice undergoing only laparotomy (CTL), undergoing intestinal obstruction (IO) and undergoing intestinal obstruction after previous treatment with viable or heat-killed yeast. BT, determined as uptake of 99mTc-E. coli in blood, mesenteric lymph nodes, liver, spleen and lungs, was significantly higher in IO group than in CTL group. Treatments with both yeasts reduced BT in blood and all organs investigated. The treatment with both yeasts also reduced intestinal permeability as determined by blood uptake of 99mTc-DTPA. Immunological data demonstrated that both treatments were able to significantly increase IL-10 levels, but only viable yeast had the same effect on sIgA levels. Intestinal lesions were more severe in IO group when compared to CTL and yeasts groups. Concluding, both viable and heat-killed cells of yeast prevent BT, probably by immunomodulation and by maintaining gut barrier integrity. Only the stimulation of IgA production seems to depend on the yeast viability.
Keywords: Probiotic; Bacterial translocation; Intestinal permeability; Intestinal obstruction; sIgA; IL-10
Caldimonas hydrothermale sp. nov., a novel thermophilic bacterium isolated from roman hot bath in south Tunisia by Hanene Bouraoui; Imen Boukari; Jean Pierre Touzel; Michael O’Donohue; Mohamed Manai (pp. 485-491).
A polyphasic approach was used to characterize a bacterium, HAN-85T, isolated from thermal water in natural thermal spring at Tozeur, an oasis in southwest Tunisia. The novel isolate was thermophilic, strictly aerobic and amylolytic bacterium, which stained Gram negative. Cells were short rods motile by means of a single polar flagellum. Their optimum temperature and pH required for growth were 55°C and pH 7, respectively. Comparative 16S rRNA gene sequence analyses showed that strain HAN-85T belonged to the genus Caldimonas, with highest sequence similarity to the type strains Caldimonas manganoxidans and Caldimonas taiwanensis. DNA–DNA hybridization measurements revealed low DNA relatedness (35.2–44.5%) between the novel isolate and its closest relative, C. manganoxidans. The major cellular fatty acid components were 16:0, 17:0 cyclo and summed feature 3. The DNA G+C content was 68.3 mol%. Taken together, the results of DNA–DNA hybridization, fatty acids profile, physiological tests and biochemical analyses have allowed the genotypic and phenotypic differentiation of the isolate from currently recognized Caldimonas species. Therefore, we suggest that this isolate is a novel species within the genus Caldimonas and propose that it should be named Caldimonas hydrothermale sp. nov. The type strain is HAN-85T (=DSM 18497T =LMG 23755T). The Gen Bank/Embl/DDBJ accession number for the 16S rRNA gene sequence of strain DSM 18497T is AM283038.
Keywords: Caldimonas ; Thermophile; Amylolytic bacterium; Hammam; Tunisian thermal spring
Single-spore elemental analyses indicate that dipicolinic acid-deficient Bacillus subtilis spores fail to accumulate calcium by Paul E. Hintze; Wayne L. Nicholson (pp. 493-497).
Dipicolinic acid (pyridine-2,6-carboxylic acid; DPA) is a major component of bacterial spores and has been shown to be an important determinant of spore resistance. In the core of dormant Bacillus subtilis spores, DPA is associated with divalent calcium in a 1:1 chelate (Ca–DPA). Spores excrete Ca–DPA during germination, but it is unknown whether Ca and DPA are imported separately or together into the developing spore. Elemental analysis by scanning electron microscopy–energy-dispersive X-ray spectroscopy (SEM–EDS) of wild-type spores and mutant spores lacking the ability to synthesize DPA showed that DPA-less spores also lacked calcium, suggesting that the two compounds may be co-imported.
Keywords: Bacillus subtilis ; Spore; Dipicolinic acid; DPA
Analysis of expression and regulatory functions of the ribosome-binding protein TypA in Mycobacterium tuberculosis under stress conditions by Julia C. Micklinghoff; Mascha Schmidt; Robert Geffers; Werner Tegge; Franz-Christoph Bange (pp. 499-504).
In many bacterial species, the translational GTPase TypA acts as a global stress- and virulence regulator and also mediates resistance to the antimicrobial peptide BPI. On the chromosome of M. tuberculosis, typA is located next to narGHJI, which plays a role in adaptation of the pathogen to various environmental conditions. Here, we show that Mycobacterium tuberculosis is sensitive to P2, a derivative of BPI. Using a typA mutant of M. tuberculosis, we found this phenotype to be independent of TypA. We further tested typA expression in M. tuberculosis under defined stress conditions, such as oxygen- and nutrient depletion, low pH, heat shock, antibiotic stress and the presence of P2, and found that typA expression remains unaffected by any of these conditions. Analysis of growth and whole-genome expression revealed similar growth kinetics and gene expression profiles of the wild type and the mutant under normal growth conditions as well as under stress conditions. Our results suggest that in contrast to the findings in other bacteria, TypA does not act as a global stress- and virulence regulator in M. tuberculosis.
Keywords: Mycobacterium tuberculosis ; typA ; bipA ; Rv1165; Regulation; BPI; Antimicrobial peptide (resistance)
Erratum to: Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan
by Bożena Mierzwa; Barbara Łotocka; Sylwia Wdowiak-Wróbel; Michał Kalita; Sebastian Gnat; Wanda Małek (pp. 505-505).