The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
Volume 62, Issue 4
Displaying 1-7 of 7 articles from this issue
Full Papers
  • Takuto Kumano, Takahisa Suzuki, Sakayu Shimizu, Michihiko Kobayashi
    2016 Volume 62 Issue 4 Pages 167-173
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: June 01, 2016
    JOURNAL FREE ACCESS
    Supplementary material
    Cyanide is known as a toxic compound for almost all living organisms. We have searched for cyanide-resistant bacteria from the soil and stock culture collection of our laboratory, and have found the existence of a lot of microorganisms grown on culture media containing 10 mM potassium cyanide. Almost all of these cyanide-resistant bacteria were found to show β-cyano-L-alanine (β-CNAla) synthetic activity. β-CNAla synthase is known to catalyze nitrile synthesis: the formation of β-CNAla from potassium cyanide and O-acetyl-L-serine or L-cysteine. We found that some microorganisms were able to detoxify cyanide using O-methyl-DL-serine, O-phospho-L-serine and β-chloro-DL-alanine. In addition, we purified β-CNAla synthase from Pseudomonas ovalis No. 111 in nine steps, and characterized the purified enzyme. This enzyme has a molecular mass of 60,000 and appears to consist of two identical subunits. The purified enzyme exhibits a maximum activity at pH 8.5–9.0 at an optimal temperature of 40–50°C. The enzyme is specific for O-acetyl-L-serine and β-chloro-DL-alanine. The Km value for O-acetyl-L-serine is 10.0 mM and Vmax value is 3.57 μmol/min/mg.
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  • Takuto Kumano, Yuko Takizawa, Sakayu Shimizu, Michihiko Kobayashi
    2016 Volume 62 Issue 4 Pages 174-180
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: June 01, 2016
    JOURNAL FREE ACCESS
    Supplementary material
    One of the nitrile-synthesizing enzymes, β-cyano-L-alanine synthase, catalyzes β-cyano-L-alanine (β-CNAla) from potassium cyanide and O-acetyl-L-serine or L-cysteine. We have identified this enzyme from Pseudomonas ovalis No. 111. In this study, we cloned the β-CNAla synthase gene and expressed it in Escherichia coli and Rhodococcus rhodochrous. Furthermore, we carried out co-expression of β-CNAla synthase with nitrilase or nitrile hydratases in order to synthesize aspartic acid and asparagine from KCN and O-acetyl-L-serine. This strategy can be used for the synthesis of labeled amino acids by using a carbon-labeled KCN as a substrate, resulting in an application for positron emission tomography.
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  • Vera Ulyanova, Raihan Shah Mahmud, Elena Dudkina, Valentina Vershinina ...
    2016 Volume 62 Issue 4 Pages 181-188
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: July 04, 2016
    JOURNAL FREE ACCESS
    Supplementary material
    The potential of microbial ribonucleases as promising antitumor and antiviral agents, determines today’s directions of their study. One direction is connected with biodiversity of RNases. We have analyzed completed and drafted Bacillus genomes deposited in GenBank for the presence of coding regions similar to the gene of an extracellular guanyl-preferring RNase of Bacillus amyloliquefaciens (barnase). Orthologues of the barnase gene were detected in 9 species out of 83. All of these belong to “B. subtilis” group within the genus. B. subtilis itself, as well as some other species within this group, lack such types of RNases. RNases similar to barnase were also found in species of “B. cereus” group as a part of plasmid-encoded S-layer toxins. It was also found that taxonomic states of culture collection strains, which were initially described based on a limited set of phenotypic characteristics, can be misleading and need to be confirmed. Using several approaches such as matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), sequencing of genes for 16S ribosomal RNA and RNA polymerase subunit beta followed by reconstruction of phylogenetic trees, we have re-identified two RNase-secreting Bacillus strains: B. thuringiensis B-388 which should be assigned as B. altitudinis B388 and B. intermedius 7P which should be renamed as B. pumilus 7P. Therefore, small secreted guanyl-preferring RNases are the feature of “B. subtilis” group only, which is characterized by distinctive lifestyle and adaptation strategies to environment.
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  • T. Porter, D. Frederick, E. Johnson, P. G. Jones
    2016 Volume 62 Issue 4 Pages 189-198
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: July 29, 2016
    JOURNAL FREE ACCESS

    As similarly observed in nutrient-poor media at 37°C, Escherichia coli forms small rods in nutrient-rich media at temperatures near 8°C, the minimum temperature of growth. A study was initiated to identify proteins required to facilitate the small rod morphology at low temperature. E. coli contains three nonessential SPOR domain proteins (DamX, RlpA, and DedD) that have been demonstrated to bind to the septal ring. In contrast to the normal growth and small rod morphology of damX and rlpA null mutants at 10°C, the dedD null mutant exhibited reduced growth and formed filamentous cells. The presence of plasmid-encoded DedD restored growth and small rods. Plasmid-encoded FtsN, an essential SPOR domain protein that functions to stabilize the septal ring and to initiate septation, in the dedD null mutant resulted in increased growth and the formation of shorter chained cells. However, plasmid-encoded DedD failed to restore growth and cell division of cells lacking FtsN at 10°C. In contrast to cell division protein DedD, RodZ is a cell elongation protein particularly required for growth at 30°C. However, the rodZ null mutant grew similarly as the wild type strain and produced cocci in LB broth at 10°C. Moreover at 10°C, the concerted deletion of dedD and rodZ resulted in severe inhibition of growth accompanied with the formation of swollen prolate ellipsoids due to a block in septal ring assembly and cell elongation. The data indicate the cellular requirement of both FtsN and DedD for septation as well as RodZ for cell elongation to maintain the small rod morphology at temperatures near 8°C. In comparison to the growth and small rods of the wild type in M9-glucose minimal media at 37°C, the dedD null mutant grew at the same rate and produced elongated cells while the rodZ null mutant grew at a slightly slower rate and produced cocci. The data indicate that DedD and RodZ are also required to maintain the small rod morphology in nutrient-poor media, but there is a higher cellular requirement of DedD for growth and cell division in nutrient-rich media at low temperature.

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  • Akira Také, Takuji Nakashima, Yuki Inahashi, Kazuro Shiomi, Yōko Takah ...
    2016 Volume 62 Issue 4 Pages 199-205
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: July 29, 2016
    JOURNAL FREE ACCESS

    It is the major characteristic of the cell-wall peptidoglycan structure in members of the family Micromonosporaceae that N-acetylmuramic acid (MurNAc) of glycan strand is replaced with N-glycolylmuramic acid (MurNGlyc). Consequently, it is difficult to use enzymatic methods for their peptidoglycan analyses. We therefore developed analysis method of peptidoglycan without using cell wall lytic enzymes as example to take the 3 genera, Micromonospora, Catenuloplanes, and Couchioplanes belonging to the family Micromonosporaceae, and their peptidoglycans were partially hydrolyzed with 4 M HCl at 60°C for 16 h followed by derivatization with Nα-(5-fluoro-2,4-dinitrophenyl)-D-leucinamide (FDLA) or 1-phenyl-3-methyl-5-pyrazolone (PMP) and LC/MS analysis. Peptidoglycan of the genus Micromonospora consisted of a MurNGlyc–Gly–D-Glu–meso-diaminopimelyl (DAP)–D-Ala peptide stem and direct linkage between D-Ala and meso-DAP. In contrast, peptidoglycans of the genera Catenuloplanes and Couchioplanes consisted of a MurNGlyc–Gly–D-Glu–L-Lys–D-Ala peptide stem, and cross-linkage between D-Ala and L-Lys was mediated by an L-Ser residue. This method can be used to analyze the cell-wall peptidoglycan structure of other bacteria as well. By derivatization with FDLA or PMP followed by LC/MS analysis, the structure can be determined using only 0.2 mg of purified peptidoglycan.

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  • Chang-Ho Kang, YuJin Shin, Periasamy Anbu, In-Hyun Nam, Jae-Seong So
    2016 Volume 62 Issue 4 Pages 206-212
    Published: 2016
    Released on J-STAGE: September 12, 2016
    Advance online publication: August 04, 2016
    JOURNAL FREE ACCESS

    Abandoned mine sites are frequently polluted with high concentrations of heavy metals. In this study, 25 calcite-forming bacteria were newly isolated from the soil of an abandoned metal mine in Korea. Based on their urease activity, calcite production, and resistance to copper toxicity, four isolates were selected and further identified by 16S rRNA gene sequencing. Among the isolates, Sporosarcina soli B-22 was selected for subsequent copper biosequestration studies, using the sand impermeability test by production of calcite and extracellular polymeric substance. High removal rates (61.8%) of copper were obtained when the sand samples were analyzed using an inductively coupled plasma-optical emission spectrometer following 72 h of incubation. Scanning electron microscopy showed that the copper carbonate precipitates had a diameter of approximately 5–10 μm. X-ray diffraction further confirmed the presence of copper carbonate and calcium carbonate crystals.

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