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Archives of Microbiology (v.192, #5)

Bacteriophages and cancer by Paulina Budynek; Krystyna Dąbrowska; Grzegorz Skaradziński; Andrzej Górski (pp. 315-320).

Bacteriophages can be used effectively to cure bacterial infections. They are known to be active against bacteria but inactive against eukaryotic cells. Nevertheless, novel observations suggest that phages are not neutral for higher organisms. They can affect physiological and immunological processes which may be crucial to their expected positive effects in therapies. Bacteriophages are a very differentiated group of viruses and at least some of them can influence cancer processes. Phages may also affect the immunological system. In general, they activate the immunological response, for example cytokine secretion. They can also switch the tumor microenvironment to one advantageous for anticancer treatment. On the other hand, bacteriophages are used as a platform for foreign peptides that may induce anticancer effects. As bacterial debris can interfere with bacteriophage activity, phage purification is significant for the final effect of a phage preparation. In this review, results of the influence of bacteriophages on cancer processes are presented which have implications for the perspective application of phage therapy in patients with cancer and the general understanding of the role of bacteriophages in the human organism.

Keywords: Bacteriophages; Cancer; Phage-display

Bacterial melanin interacts with double-stranded DNA with high affinity and may inhibit cell metabolism in vivo by Jing Geng; Peng Yuan; Chun Shao; Sheng-Bing Yu; Bo Zhou; Ping Zhou; Xiang-Dong Chen (pp. 321-329).

Melanin has been found to interact with a number of molecules including metal ions, antibiotics and proteins. In this study, we showed how melanin from bacteria can interact with double-stranded DNA. Investigation using capillary electrophoresis, various spectroscopic techniques and circular dichroism found that melanin interacts with DNA by intercalating between the base pairs of DNA. And this was further supported by simulating different forms of melanin docking to oligonucleotides. Transmission electron microscopy of recombinant Escherichia coli producing melanin suggested the interaction in vivo. Furthermore, we showed how the cytoplasmic localization of melanin may provide a novel function in inhibiting cellular metabolism using microcalorimetry. The implications of the interaction in prokaryotes and eukaryotes were discussed.

Keywords: Bacterial melanin; Double-stranded DNA; Fluorescence quenching; Dock; Intercalation; Transmission election microscopy; Microcalorimetry

Mutations of ferric uptake regulator (fur) impair iron homeostasis, growth, oxidative stress survival, and virulence of Xanthomonas campestris pv. campestris by Thichakorn Jittawuttipoka; Ratiboot Sallabhan; Paiboon Vattanaviboon; Mayuree Fuangthong; Skorn Mongkolsuk (pp. 331-339).

Iron is essential in numerous cellular functions. Intracellular iron homeostasis must be maintained for cell survival and protection against iron’s toxic effects. Here, we characterize the roles of Xanthomonas campestris pv. campestris (Xcc) fur, which encodes an iron sensor and a transcriptional regulator that acts in iron homeostasis, oxidative stress, and virulence. Herein, we isolated spontaneous Xcc fur mutants that had high intracellular iron concentrations due to constitutively high siderophore levels and increased expression of iron transport genes. These mutants also had reduced aerobic plating efficiency and resistance to peroxide killing. Moreover, one fur mutant was attenuated on a host plant, thus indicating that fur has important roles in the virulence of X. campestris pv. campestris.

Keywords: Fur; Iron; Xanthomonas ; Plant pathogen; Ferric uptake regulator

Insight into the evolutionary history of symbiotic genes of Robinia pseudoacacia rhizobia deriving from Poland and Japan by Bożena Mierzwa; Sylwia Wdowiak-Wróbel; Michał Kalita; Sebastian Gnat; Wanda Małek (pp. 341-350).

The phylogeny of symbiotic genes of Robinia pseudoacacia (black locust) rhizobia derived from Poland and Japan was studied by comparative sequence analysis of nodA, nodC, nodH, and nifH loci. In phylogenetic trees, black locust symbionts formed a branch of their own suggesting that the spread and maintenance of symbiotic genes within Robinia pseudoacacia rhizobia occurred through vertical transmission. There was 99–100% sequence similarity for nodA genes of Robinia pseudoacacia nodulators, 97–98% for nodC, and 97–100% for nodH and nifH loci. A considerable sequence conservation of sym genes shows that the symbiotic apparatus of Robinia pseudoacacia rhizobia might have evolved under strong host plant constraints. In the nodA and nodC gene phylograms, Robinia pseudoacacia rhizobia grouped with Phaseolus sp. symbionts, although they were not closely related to our isolates based on 16S rRNA genes, and with Mesorhizobium amorphae. nifH gene phylogeny of our isolates followed the evolutionary history of 16S rDNA and Robinia pseudoacacia rhizobia grouped with Mesorhizobium genus species. Nodulation assays revealed that Robinia pseudoacacia rhizobia effectively nodulated their native host and also Amorpha fruticosa and Amorpha californica resulting in a significant enhancement of plant growth. The black locust root nodules are shown to be of indeterminate type.

Keywords: Robinia pseudoacacia rhizobia; Phylogeny of sym genes; Nodule structure

The DraC usher in Dr fimbriae biogenesis of uropathogenic E. coli Dr⁺ strains by Beata Zalewska-Piątek; Marta Kur; Sabina Wilkanowicz; Rafał Piątek; Józef Kur (pp. 351-363).

Biogenesis of Dr fimbriae encoded by the dra gene cluster of uropathogenic Escherichia coli strains requires the chaperone-usher pathway. This secretion system is based on two non-structural assembly components, the DraB periplasmic chaperone and DraC outer-membrane usher. The DraB controls the folding of DraE subunits, and DraC forms the assembly and secretion platform for polymerization of subunits in linear fibers. In this study, mutagenesis of the DraC N-terminus was undertaken to select residues critical for Dr fimbriae bioassembly. The DraC-F4A, DraC-C64, DraC-C100A and DraC-W142A significantly reduced the adhesive ability of E. coli strains. The biological activity of the DraC mutants as a assembly platform for Dr fimbriae polymerization was verified by agglutination of human erythrocytes and adhesion to DAF localized at the surface of CHO-DAF⁺ and HeLa cells. The residue F4 of the DraC usher conserved among FGL and FGS chaperone-assembled adhesive organelles can be used to design pillicides blocking the biogenesis of Dr fimbriae. Because the draC and afaC-III genes share 100% identity the range of the virulence determinant inhibitors could also be extended to E. coli strains encoding afa-3 gene cluster. The investigations performed showed that the usher N-terminus plays an important role in biogenesis of complete fiber.

Keywords: Uropathogenic Escherichia coli ; dra gene cluster; DraC usher; Dr fimbriae

Isolation of a novel bacterium, Blautia glucerasei sp. nov., hydrolyzing plant glucosylceramide to ceramide by Hideki Furuya; Yukari Ide; Makiko Hamamoto; Narito Asanuma; Tsuneo Hino (pp. 365-372).

A bacterial strain that is capable of hydrolyzing plant glucosylceramide (GluCer) was newly isolated from dog feces. The novel strain, designated as strain HFTH-1^T, hydrolyzed plant GluCer with a variety of chemical structures, but did not hydrolyze glucosylsphingosine, lactosylceramide, or monosialoganglioside GM₃, indicating that strain HFTH-1^T produces GluCer-specific glucosylceramidase. Strain HFTH-1^T was Gram-positive, anaerobic, oval-spore-forming, rod-shaped, lecithinase-negative, and lipase-negative. It fermented a wide variety of carbohydrates and produced mainly acetate, formate, and lactate from glucose. The G + C content of its DNA was 40.7 mol%. The phylogenetic analysis of 16S rRNA sequence revealed that strain HFTH-1^T is placed in the clostridial rRNA cluster XIVa, with Ruminococcus obeum as the nearest relative. Pairwise comparison revealed approximately 5.0% sequence divergence between strain HFTH-1^T and the type strain of R. obeum. On the basis of its phenotypic characteristics and phylogenetic divergence, it is proposed that the hitherto unknown rod-shaped bacterial strain HFTH-1^T (= DSM 22028^T = NBRC 104932^T) should be placed in the genus Blautia as a novel species, Blautia glucerasei sp. nov, the only currently known isolate of the species.

Keywords: Blautia glucerasei sp. nov.; Glucosylceramide; Ceramide; Glucosylceramidase; Intestinal bacteria

Aspergillus nidulans contains six possible fatty acyl-CoA synthetases with FaaB being the major synthetase for fatty acid degradation by Kathrin Reiser; Meryl A. Davis; Michael J. Hynes (pp. 373-382).

Aspergillus nidulans can use a variety of fatty acids as sole carbon and energy sources via its peroxisomal and mitochondrial β-oxidation pathways. Prior to channelling the fatty acids into β-oxidation, they need to be activated to their acyl-CoA derivates. Analysis of the genome sequence identified a number of possible fatty acyl-CoA synthetases (FatA, FatB, FatC, FatD, FaaA and FaaB). FaaB was found to be the major long-chain synthetase for fatty acid degradation. FaaB was shown to localise to the peroxisomes, and the corresponding gene was induced in the presence of short and long chain fatty acids. Deletion of the faaB gene leads to a reduced/abolished growth on a variety of fatty acids. However, at least one additional fatty acyl-CoA synthetase with a preference for short chain fatty acids and a potential mitochondrial candidate (AN4659.3) has been identified via genome analysis.

Keywords: β-oxidation; Fatty acyl-CoA synthetase; Aspergillus nidulans ; Peroxisome

Extracellular monoenzyme deglycosylation system of 7-O-linked flavonoid β-rutinosides and its disaccharide transglycosylation activity from Stilbella fimetaria by Laura Mazzaferro; Lucrecia Piñuel; Marisol Minig; Javier D. Breccia (pp. 383-393).

We screened for microorganisms able to use flavonoids as a carbon source; and one isolate, nominated Stilbella fimetaria SES201, was found to possess a disaccharide-specific hydrolase. It was a cell-bound ectoenzyme that was released to the medium during conidiogenesis. The enzyme was shown to cleave the flavonoid hesperidin (hesperetin 7-O-α-rhamnopyranosyl-β-glucopyranoside) into rutinose (α-rhamnopyranosyl-β-glucopyranose) and hesperetin. Since only intracellular traces of monoglycosidase activities (β-glucosidase, α-rhamnosidase) were produced, the disaccharidase α-rhamnosyl-β-glucosidase was the main system utilized by the microorganism for hesperidin hydrolysis. The enzyme was a glycoprotein with a molecular weight of 42224 Da and isoelectric point of 5.7. Even when maximum activity was found at 70°C, it was active at temperatures as low as 5°C, consistent with the psychrotolerant character of S. fimetaria. Substrate preference studies indicated that the enzyme exhibits high specificity toward 7-O-linked flavonoid β-rutinosides. It did not act on flavonoid 3-O-β-rutinoside and 7-O-β-neohesperidosides, neither monoglycosylated substrates. In an aqueous medium, the α-rhamnosyl-β-glucosidase was also able to transfer rutinose to other acceptors besides water, indicating its potential as biocatalyst for organic synthesis. The monoenzyme strategy of S. fimetaria SES201, as well as the enzyme substrate preference for 7-O-β-flavonoid rutinosides, is unique characteristics among the microbial flavonoid deglycosylation systems reported.

Keywords: Glycoside hydrolase; Diglycosidase; Hesperidin; α-Rhamnosyl-β-glucosidase

Molecular characterization and isolation of cytochrome P450 genes from the filamentous fungus Aspergillus oryzae by K. H. M. Nazmul Hussain Nazir; Hirofumi Ichinose; Hiroyuki Wariishi (pp. 395-408).

We explored the molecular diversity of cytochrome P450 genes in the filamentous fungus Aspergillus oryzae using bioinformatic and experimental approaches. Based on bioinformatic annotation, we found 155 putative genes of cytochromes P450 in the whole genome sequence; however, 13 of 155 appeared to be pseudogenes due to sequence deletions and/or inframe stop codon(s). There are 87 families of A. oryzae cytochromes P450 (AoCYPs), indicating considerable phylogenetic diversity. To characterize A. oryzae AoCYPs, we attempted to isolate cDNAs using RT-PCR and determined their transcriptional capabilities. To date, we have confirmed gene expression of 133 AoCYPs and cloned 121 AoCYPs as full-length cDNAs encoding a mature open reading frame. Using experimentally deduced sequences and intron–exon organization, we analyzed AoCYPs phylogenetically. We also identified intronic consensus sequences in AoCYPs genes. The experimentally validated exonic and intronic sequences will be a powerful advantage in identification and characterization of novel P450s from various ascomycetous fungi.

Keywords: Cytochrome P450; Aspergillus oryzae ; Molecular diversity; cDNA cloning

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Archives of Microbiology (v.192, #5)

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Archives of Microbiology (v.192, #5)