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Archives of Microbiology (v.178, #5)

Characterization of three spiral-shaped purple nonsulfur bacteria isolated from coastal lagoon sediments, saline sulfur springs, and microbial mats: emended description of the genus Roseospira and description of Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov. by Rémy Guyoneaud; Sophie Mouné; Claire Eatock; Virginie Bothorel; Agnès Hirschler-Réa; John Willison; Robert Duran; Werner Liesack; Rodney Herbert; Robert Matheron; Pierre Caumette (pp. 315-324).

Three new spirilloid phototrophic purple nonsulfur bacteria were isolated in pure culture from three different environments: strain CE2105 from a brackish lagoon in the Arcachon Bay (Atlantic coast, France), strain SE3104 from a saline sulfur spring in the Pyrenees (Navarra, Spain) , and strain AT2115 a microbial mat (Tetiaroa Atoll, Society Islands). Single cells of the three strains were spiral-shaped and highly motile. Their intracellular photosynthetic membranes were of the vesicular type. Bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series were present as photosynthetic pigments. Optimal growth occurred under photoheterotrophic conditions and in the presence of 0.5–4% w/v NaCl. These features are similar to those described for Roseospira mediosalina. Comparative sequence analysis of their 16S rRNA genes placed these strains within the α-subclass of Proteobacteria, in a cluster together with Roseospira mediosalina and Rhodospira trueperi. They form a closely related group of slightly to moderately halophilic spiral-shaped purple nonsulfur bacteria. However, the three new isolates exhibited some differences in their physiology and genetic characteristics. Consequently, we propose that they are members of three new species within the genus Roseospira, Roseospira marina sp. nov., Roseospira navarrensis sp. nov., and Roseospira thiosulfatophila sp. nov., with strains CE2105, SE3104, and AT2115 as the type strains, respectively. As a consequence, an emended description of the genus Roseospira is also given.

Keywords: Phototrophic purple nonsulfur bacteria Roseospira Coastal lagoon Saline sulfur spring Microbial mat Moderate halophilic bacteria

Purification and characterization of an ε-poly-L-lysine-degrading enzyme from an ε-poly-L-lysine-producing strain of Streptomyces albulus by Mitsuaki Kito; Rika Takimoto; Toyokazu Yoshida; Toru Nagasawa (pp. 325-330).

An ε-poly-L-lysine-degrading enzyme of an ε-poly-L-lysine-producing strain of Streptomyces albulus was purified and characterized. The enzyme was tightly bound to the cell membrane. After solubilization with NaSCN, the enzyme was purified to homogeneity by phenyl-Sepharose CL-4B column chromatography. The subunit molecular mass of the purified enzyme was 54 kDa. Enzyme activity was inhibited by o-phenanthroline, and could be restored in the presence of 1 mM Mg²⁺, Ca²⁺, Fe³⁺ or Zn²⁺. The mode of ε-poly-L-lysine degradation was of the exo-type, and the enzyme released N-terminal L-lysines one by one. The enzyme acted on various peptides possessing L-lysine residues at the N-terminus and was classified as an aminopeptidase. ε-Poly-L-lysine-degrading activity was found in the membrane fraction of some other Streptomyces strains as well as that of Streptomyces albulus. Streptomyces virginiae IFO 12827 and Streptomyces norsei IFO 15452 exhibited high ε-poly-L-lysine-degrading activity, and both strains could produce ε-poly-L-lysine, indicating a correlation between the distribution of membrane-bound ε-poly-L-lysine-degrading enzyme and ε-poly-L-lysine-producing activity.

Keywords: ε-Poly-L-lysine Streptomyces albulus Membrane-bound ε-poly-L-lysine-degrading enzyme

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes by Teresa Koledin; Gerald L. Newton; Robert C. Fahey (pp. 331-337).

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1D-myo-inosityl 2-L-cysteinylamido-2-deoxy-α-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.

Keywords: mshD Mycothiol synthase Mycothiol Actinomycetes Mycobacteria

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR – implications for GM screening procedures by Simon A. Weller; Sean A. Simpkins; David E. Stead; Andrew Kurdziel; Heather Hird; Rebecca J. Weekes (pp. 338-343).

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.

Keywords: Agrobacterium TaqMan PCR Oil seed rape Canola

The veA gene is necessary for the inducible expression by fructosyl amines of the Aspergillus nidulans faoA gene encoding fructosyl amino acid oxidase (amadoriase, EC 1.5.3) by Hyo-Young Jeong; Myung Song; Jung Back; Dong-Min Han; Xinle Wu; Vincent Monnier; Kwang-Yeop Jahng; Keon-Sang Chae (pp. 344-350).

The faoA gene encoding fructosyl amino acid oxidase (FAOD, EC 1.5.3) was isolated from Aspergillus nidulans and characterized. The complete nucleotide sequence of the faoA (fructosyl amino acid oxidase) gene and its cDNA revealed that the faoA gene encodes a 441-amino-acid polypeptide interrupted by five introns. Expression of the A. nidulans faoA gene was inducible by fructosyl propylamine and fructosyl lysine, as is the case for the gene encoding FAOD in other organisms. The faoA gene was not induced by these fructosyl amines in a null mutant of the veA gene, which has been identified as an activator of sexual development and as an inhibitor of asexual development; the faoA gene was induced greatly in a veA ⁺ wild-type. However, veA gene expression was not affected by fructosyl amines. Even in the absence of fructosyl propylamine, synthesis of the faoA transcript was higher in the veA ⁺ background than in a veA-null mutation background. These results indicated that faoA gene expression is inducible by fructosyl amines and by the veA gene, and that the veA gene is necessary for full induction of faoA gene expression by fructosyl amines. Thus, the faoA gene is the first gene whose expression is dependent on the veA gene. Furthermore, the faoA gene, present in a single copy, seems to be dispensable for development and growth, since the faoA-null mutant grew normally and developed as many conidia and sexual structures as the wild-type.

Keywords: Aspergillus nidulans faoA Fructosyl amino acid oxidase (FAOD) Fructosyl propyl amine veA

The cbs mutant strain of Rhodococcus erythropolis KA2-5-1 expresses high levels of Dsz enzymes in the presence of sulfate by Yasuhiro Tanaka; Osamu Yoshikawa; Kenji Maruhashi; Ryuichiro Kurane (pp. 351-357).

Two mutants of the dibenzothiophene-desulfurizing Rhodococcus erythropolis KA2-5-1, strains MS51 and MS316, which express a high level of desulfurizing activity in the presence of sulfate, were isolated using the transposome technique. The level of dibenzothiophene-desulfurization by cell-free extracts prepared from mutants MS51 and MS316 grown on sulfate was about five-fold higher than that by cell-free extracts of the wild-type. This result was consistent with results of Western-blot analysis using antisera specific for DszA, DszB and DszC, the enzymes involved in the desulfurization of dibenzothiophene. Gene analysis of the mutants revealed that the same gene was disrupted in mutants MS51 and MS316 and that the transposon-inserted gene in these strains was the gene for cystathionine β-synthase, cbs. The cbs mutants also expressed high levels of Dsz enzymes when methionine was used as the sole source of sulfur.

Keywords: Cysteine biosynthesis Desulfurization Rhodococcus erythropolis Transposome Dibenzothiophene

A novel haem compound accumulated in Escherichia coli overexpressing the cydDC operon, encoding an ABC-type transporter required for cytochrome assembly by Gregory M. Cook; Hugo Cruz-Ramos; Arthur J. Moir; Robert K. Poole (pp. 358-369).

cydDC genes encode a heterodimeric ABC transporter required for assembly of the membrane-bound cytochrome bd quinol oxidase and periplasmic cytochromes. Here, we demonstrate that overexpression of functional cydDC genes on a multicopy plasmid results in elevated levels of cytochromes b and d, but most notably formation in anaerobically grown cells of a novel haem-containing component P-574. The pigment has a distinctive absorbance at 574–579 nm and 448 nm in reduced minus oxidised spectra and renders over-producing cells reddish in colour. The highest levels of P-574 were observed in mutants (cydAB) in the structural genes for the polypeptides of cytochrome bd. P-574 is labile; its spectral signal is reduced in cells that are frozen-thawed or subjected to mechanical disruption. P-574 was not detected in cytoplasmic or periplasmic fractions and was predominantly associated with the cell membrane. P-574 did not bind CO or cyanide. Production of P-574 was dependent on haem biosynthesis indicating that it is a haem-containing molecule or derived from haem biosynthesis. These findings suggest that P-574 may result from association of a haem compound with overexpressed transporter subunits, but not with oxidase subunits, and are consistent with an intimate link between the transporter and haem processing during oxidase assembly.

Keywords: Oxidase ABC-type transporter Escherichia coli Haemprotein Cytochrome bd

Impact of the Mg²⁺-citrate transporter CitM on heavy metal toxicity in Bacillus subtilis by Bastiaan P. Krom; Henry Huttinga; Jessica B. Warner; Juke S. Lolkema (pp. 370-375).

Bacillus subtilis possesses a secondary transporter, CitM, that is specific for the complex of citrate and Mg²⁺ but is also capable of transporting citrate in complex with the heavy metal ions Zn²⁺, Ni²⁺ and Co²⁺. We report on the impact of CitM activity on the toxicity of Zn²⁺, Ni²⁺ and Co²⁺ in B. subtilis. In a citM deletion mutant or under conditions in which CitM is not expressed, the toxic effects of the metals were reduced by the presence of citrate in the medium. In contrast, the presence of citrate dramatically enhanced toxicity when the Mg²⁺-citrate transporter was present in the membrane. It is demonstrated that the complex of Ni²⁺ and citrate is transported into the cell and that the uptake is responsible for the enhanced toxicity. At toxic concentrations of the metal ions, the cultures adapted by developing tolerance against these ions. Tolerant cells isolated by exposure to one of the metal ions remained tolerant after growth in the absence of toxic metal ions and were cross-tolerant against the other two toxic ions. Tolerant strains were shown to contain point mutations in the citM gene, which resulted in premature termination of translation.

Keywords: Bacillus subtilis Citrate transporter CitM Heavy metal toxicity

Molecular and biochemical characterization of a novel two-component signal transduction system, amrA-amkA, involved in rifamycin SV production in Amycolatopsis mediterranei U32 by Weiwu Wang; Weiwen Zhang; Hui Chen; Juishen Chiao; Guoping Zhao; Weihong Jiang (pp. 376-386).

Two-component and phosphorelay signal transduction systems are the major means by which bacteria recognize and respond to a variety of environmental stimuli. Although several model systems, including sporulation in Bacillus subtilis and chemotaxis in Escherichia coli, have been extensively studied, the two-component signal transduction systems in industrially important actinomycetes are not well studied. We report the molecular and biochemical characterization of a novel two-component signal system, amrA-amkA, from the rifamycin-SV-producing Amycolatopsis mediterranei U32. The deduced sequences of amkA and amrA contain all the structural features that are highly conserved in the typical bacterial histidine kinases and response regulators, respectively. BLAST analyses showed that AmrA and AmkA displayed high similarities to AfsQ1/AfsQ2 of Streptomyces coelicolor and MtrA/MtrB of Mycobacterium tuberculosis. The amrA and amkA genes were over-expressed and the gene products were purified from E. coli. Biochemical studies showed that AmkA is able to autophosphorylate, supporting its functional assignment as a histidine kinase. That AmrA functions as the cognate response regulator for histidine kinase AmkA was demonstrated by in vitro phosphotransfer from [γ-³²P]ATP-labeled AmkA to AmrA. Rifamycin SV production was also decreased by 10–20% in amrA or amkA gene disruption mutants under the tested condition. Although the detailed regulatory mechanism is still unknown, this is the first report regarding the involvement of two-component signal systems in rifamycin biosynthesis in the genus Amycolatopsis.

Keywords: Amycolatopsis mediterranei U32 Two-component regulatory system Signal transduction Rifamycin SV production

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Archives of Microbiology (v.178, #5)