Journal of Microbiology

Volume 52(11); November 2014

Review

MINIREVIEW] Modern and Simple Construction of Plasmid: Saving Time and Cost: Hideki Nakayama , Nobuo Shimamoto; J. Microbiol. 2014;52(11):891-897. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4501-6

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11 Citations

Abstract: Construction of plasmids has been occupying a significant fraction of laboratory work in most fields of experimental biology. Tremendous effort was made to improve the traditional method for constructing plasmids, in which DNA fragments digested with restriction enzymes were ligated. However, the traditional method remained to be a standard protocol more than 40 years. At last, several recent inventions are rapidly and completely replacing the traditional method, because they are far quicker with less cost, and requiring less material. We here introduce three such methods that cover up most of the cases. Moreover, they are complementary with each other. Our lab protocols are provided for “no strain, no pain” construction of plasmids.

Research Support, Non-U.S. Gov't

Spatial Distribution of Microbial Communities Associated with Dune Landform in the Gurbantunggut Desert, China: Ruyin Liu , Ke Li , Hongxun Zhang , Junge Zhu , DevRaj Joshi; J. Microbiol. 2014;52(11):898-907. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4075-3

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18 Citations

Abstract: The microbial community compositions and potential ammonia oxidation in the topsoil at different positions of sand dune (stoss slope, crest, lee slope, and interdune) from the Gurbantunggut Desert, the largest semi-fixed desert in China, were investigated using several molecular methods. Actinobacteria and Proteobacteria (especially Alphaproteobacteria) were commonly the dominant taxa across all soil samples. Bacterial communities were similar in soils collected from the stoss slopes and interdunes (HC-BSCs, biological soil crusts with a high abundance of cyanobacteria), containing more abundant cyanobacterial populations (16.9–24.5%) than those (0.2–0.7% of Cyanobacteria) in the crests and lee slopes (LC-BSCs, biological soil crusts with a low abundance of cyanobacteria). The Cyanobacteria were mainly composed of Microcoleus spp., and quantitative PCR analysis revealed that 16S rRNA gene copy numbers of Cyanobacteria (especially genus Microcoleus) were at least two orders of magnitude higher in HC-BSCs than in LC-BSCs. Heterotrophic Geodermatophilus spp. frequently occurred in HC-BSCs (2.5–8.0%), whereas genera Arthrobacter, Bacillus, and Segetibacter were significantly abundant in LC-BSC communities. By comparison, the desert archaeal communities were less complex, and were dominated by Nitrososphaera spp. The amoA gene abundance of ammonia-oxidizing archaea (AOA) was higher than that of ammonia-oxidizing bacteria (AOB) in all soil samples, particularly in the interdunal soils (106–108 archaeal amoA gene copies per gram dry soil), indicating that AOA possibly dominate the ammonia oxidation at the interdunes.

Journal Article

Application of Response Surface Methodology for Rapid Chrysene Biodegradation by Newly Isolated Marine-derived Fungus Cochliobolus lunatus Strain CHR4D: Jwalant K. Bhatt , Chirag M. Ghevariya , Dushyant R. Dudhagara , Rahul K. Rajpara , Bharti P. Dave; J. Microbiol. 2014;52(11):908-917. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4137-6

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23 Citations

Abstract: For the first time, Cochliobolus lunatus strain CHR4D, a marine-derived ascomycete fungus isolated from historically contaminated crude oil polluted shoreline of Alang-Sosiya ship-breaking yard, at Bhavnagar coast, Gujarat has been reported showing the rapid and enhanced biodegradation of chrysene, a four ringed high molecular weight (HMW) polycyclic aromatic hydrocarbon (PAH). Mineral Salt Broth (MSB) components such as ammonium tartrate and glucose along with chrysene, pH and trace metal solution have been successfully optimized by Response Surface Methodology (RSM) using central composite design (CCD). A validated, two-step optimization protocol has yielded a substantial 93.10% chrysene degradation on the 4th day, against unoptimized 56.37% degradation on the 14th day. The results depict 1.65 fold increase in chrysene degradation and 1.40 fold increase in biomass with a considerable decrement in time. Based on the successful laboratory experiments, C. lunatus strain CHR4D can thus be predicted as a potential candidate for mycoremediation of HMW PAHs impacted environments.

Research Support, Non-U.S. Gov'ts

Deodorization of Pig Slurry and Characterization of Bacterial Diversity Using 16S rDNA Sequence Analysis: Ok-Hwa Hwang , Sebastian Raveendar , Young-Ju Kim , Ji-Hun Kim , Tae-Hun Kim , Dong-Yoon Choi , Che Ok Jeon , Sung-Back Cho , Kyung-Tai Lee; J. Microbiol. 2014;52(11):918-929. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4251-5

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7 Citations

Abstract: The concentration of major odor-causing compounds including phenols, indoles, short-chain fatty acids (SCFAs) and branched chain fatty acids (BCFAs) in response to the addition of powdered horse radish (PHR) and spent mushroom compost (SMC) was compared with control nontreated slurry (CNS) samples. A total of 97,465 rDNAs sequence reads were generated from three different samples (CNS, n = 2; PHR, n = 3; SMC, n = 3) using bar-coded pyrosequencing. The number of operational taxonomic units (OTUs) was lower in the PHR slurry compared with the other samples. A total of 11 phyla were observed in the slurry samples, while the phylogenetic analysis revealed that the slurry microbiome predominantly comprised members of the Bacteroidetes, Firmicutes, and Proteobacteria phyla. The rarefaction analysis showed the bacterial species richness varied among the treated samples. Overall, at the OTU level, 2,558 individual genera were classified, 276 genera were found among the three samples, and 1,832 additional genera were identified in the individual samples. A principal component analysis revealed the differences in microbial communities among the CNS, PHR, and SMC pig slurries. Correlation of the bacterial community structure with the Kyoto Encyclopedia of Genes and Genomes (KEGG) predicted pathways showed that the treatments altered the metabolic capabilities of the slurry microbiota. Overall, these results demonstrated that the PHR and SMC treatments significantly reduced the malodor compounds in pig slurry (P < 0.05).

FgFlbD Regulates Hyphal Differentiation Required for Sexual and Asexual Reproduction in the Ascomycete Fungus Fusarium graminearum: Hokyoung Son , Myung-Gu Kim , Suhn-Kee Chae , Yin-Won Lee; J. Microbiol. 2014;52(11):930-939. Published online October 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4384-6

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18 Citations

Abstract: Fusarium graminearum is a filamentous fungal plant pathogen that infects major cereal crops. The fungus produces both sexual and asexual spores in order to endure unfavorable environmental conditions and increase their numbers and distribution across plants. In a model filamentous fungus, Aspergillus nidulans, early induction of conidiogenesis is orchestrated by the fluffy genes. The objectives of this study were to characterize fluffy gene homologs involved in conidiogenesis and their mechanism of action in F. graminearum. We characterized five fluffy gene homologs in F. graminearum and found that FlbD is the only conserved regulator for conidiogenesis in A. nidulans and F. graminearum. Deletion of fgflbD prevented hyphal differentiation and the formation of perithecia. Successful interspecies complementation using A. nidulans flbD demonstrated that the molecular mechanisms responsible for FlbD functions are conserved in F. graminearum. Moreover, abaA-wetA pathway is positively regulated by FgFlbD during conidiogenesis in F. graminearum. Deleting fgflbD abolished morphological effects of abaA overexpression, which suggests that additional factors for FgFlbD or an AbaA-independent pathway for conidiogenesis are required for F. graminearum conidiation. Importantly, this study led to the construction of a genetic pathway of F. graminearum conidiogenesis and provides new insights into the genetics of conidiogenesis in fungi.

Transcriptional Regulation of fksA, a β-1,3-Glucan Synthase Gene, by the APSES Protein StuA during Aspergillus nidulans Development: Bum-Chan Park , Yun-Hee Park , Soohyun Yi , Yu Kyung Choi , Eun-Hye Kang , Hee-Moon Park; J. Microbiol. 2014;52(11):940-947. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4517-y

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12 Citations

Abstract: The temporal and spatial regulation of β-1,3-glucan synthesis plays an important role in morphogenesis during fungal growth and development. Northern blot analysis showed that the transcription of fksA, the gene encoding β-1,3-glucan synthase in Aspergillus nidulans, was cell-cycle-dependent and increased steadily over the duration of the vegetative period, but its overall expression during the asexual and sexual stages was fairly constant up until the time of transcription cessation. In an A. nidulans strain mutated in the eukaryotic bHLH-like APSES transcription factor stuA1, the transcriptional level of fksA, and consequently the content of alkali-insoluble cell wall β-glucan, significantly increased at the conidial chain formation and maturation stage. Electrophoretic mobility shift assays revealed that StuA was bound to StREs (StuA Response Elements) on the fksA promoter region. Promoter analysis with sGFP-fusion constructs also indicated the negative regulation of fksA expression by StuA, especially during asexual development. Taken together, these data suggest that StuA plays an important role in cell wall biogenesis during the development of A. nidulans, by controlling the transcription level of fksA.

Research Support, U.S. Gov't, Non-P.H.S.

Salinity as a Regulator of DMSP Degradation in Ruegeria pomeroyi DSS-3: Paula Salgado , Ronald Kiene , William Wiebe , Catarina Magalhães; J. Microbiol. 2014;52(11):948-954. Published online October 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4409-1

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18 Citations

Abstract: Dimethylsulfoniopropionate (DMSP) is an important carbon and sulfur source to marine bacterial communities and the main precursor of dimethylsulfide (DMS), a gas that influences atmospheric chemistry and potentially the global climate. In nature, bacterial DMSP catabolism can yield different proportions of DMS and methanethiol (MeSH), but relatively little is known about the factors controlling the pathways of bacterial degradation that select between their formation (cleavage vs. demethiolation). In this study, we carried out experiments to evaluate the influence of salinity on the routes of DMSP catabolism in Ruegeria pomeroyi DSS-3. We monitored DMS and MeSH accumulation in cell suspensions grown in a range of salinities (10, 20, 30 ppt) and with different DMSP amendments (0, 50, 500 μM). Significantly higher concentrations of DMS accumulated in low salinity treatments (10 ppt; P < 0.001), in both Marine Basal Medium (MBM) and half-strength Yeast Tryptone Sea Salts (½ YTSS) media. Results showed a 47.1% and 87.5% decrease of DMS accumulation, from salinity 10 to 20 ppt, in MBM and ½ YTSS media, respectively. On the other hand, MeSH showed enhanced accumulations at higher salinities (20, 30 ppt), with a 90.6% increase of MeSH accumulation from the 20 ppt to the 30 ppt salinity treatments. Our results with R. pomeroyi DSS-3 in culture are in agreement with previous results from estuarine sediments and demonstrate that salinity can modulate selection of the DMSP enzymatic degradation routes, with a consequent potential impact on DMS and MeSH liberation into the atmosphere.

Journal Article

Use of Selected Lactic Acid Bacteria in the Eradication of Helicobacter pylori Infection: Jin-Eung Kim , Min-Soo Kim , Yeo-Sang Yoon , Myung-Jun Chung , Do-Young Yum; J. Microbiol. 2014;52(11):955-962. Published online October 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4355-y

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26 Citations

Abstract: Helicobacter pylori is among the major pathogenic bacteria that cause chronic gastritis and peptic ulcer disease and is related to the development of gastric cancer. Several chemicals, including antibiotics, have been used to eradicate H. pylori; however, they do not always curb the infection. Ten representative type strains of lactic acid bacteria (LAB) were screened for antagonism toward H. pylori via inhibition of urease activity. Strains inhibiting the binding of H. pylori to human gastric cell line cells and suppressing H. pylori-induced interleukin-8 (IL-8) production were also screened. Of these, Pediococcus pentosaseus (SL4), which inhibited the adhesion of H. pylori to MKN-45 gastric cancer cells, Bifidobacterium longum (BG7), with urease inhibiting activity, and Lactococcus lactis (SL3), and Enterococcus faecalis (SL5), which suppressed H. pylori-induced IL-8 production within MKN-45 and AGS cells, were selected. In mouse model, these LAB stains in combination significantly suppressed IL-8 levels in serum. Gastric pH also recovered to normal values after the administration of these LAB. These stains effectively suppressed H. pylori viability, although not to the extent of antibiotic treatment. When used as probiotics, LAB may help decrease the occurrence of gastritis and reduce the risk of H. pylori infection without, inducing side effects.

Research Support, Non-U.S. Gov'ts

The Identification of Six Novel Proteins with Fibronectin or Collagen Type І Binding Activity from Streptococcus suis Serotype 2: Hui Zhang , Junxi Zheng , Li Yi , Yue Li , Zhe Ma , Hongjie Fan , Chengping Lu; J. Microbiol. 2014;52(11):963-969. Published online October 31, 2014; DOI: https://doi.org/10.1007/s12275-014-4311-x

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16 Citations

Abstract: Streptococcus suis, a major swine pathogen, is an emerging zoonotic agent that causes meningitis and septic shock. Bacterial cell wall and secreted proteins are often involved in interactions with extracellular matrix proteins (ECMs), which play important roles in the initial steps of pathogenesis. In this study, 2D SDS-PAGE, western blotting-based binding affinity measurements, and microtiter plate binding assays were used to identify cell wall and secreted proteins from S. suis that interact with fibronectin and collagen type І. We identified six proteins from S. suis, including three proteins (translation elongation factor G, oligopeptide-binding protein OppA precursor, and phosphoglycerate mutase) that show both fibronectin and collagen type І binding activity. To the best of our knowledge, these three newly identified proteins had no previously reported fibronectin or collagen type І binding activity. Overall, the aim in this study was to identify proteins with ECM binding activity from S. suis and it represents the first report of six new proteins from S. suis that interact with fibronectin or collagen type І.

Function of VP2 Protein in the Stability of the Secondary Structure of Virus-like Particles of Genogroup II Norovirus at Different pH Levels: Function of VP2 Protein in the Stability of NoV VLPs: Yao Lin , Li Fengling , Wang Lianzhu , Zhai Yuxiu , Jiang Yanhua; J. Microbiol. 2014;52(11):970-975. Published online October 3, 2014; DOI: https://doi.org/10.1007/s12275-014-4323-6

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35 Citations

Abstract: VP2 is the minor structural protein of noroviruses (NoV) and may function in NoV particle stability. To determine the function of VP2 in the stability of the NoV particle, we constructed and purified two kinds of virus-like particles (VLPs), namely, VLPs (VP1) and VLPs (VP1+VP2), from Sf9 cells infected with recombinant baculoviruses by using a Bac-to-Bac? baculovirus expression system. The two kinds of VLPs were treated with different phosphate buffers (pH 2 to pH 8); the secondary structure was then analyzed by far UV circular dichroism (CD) spectroscopy. Results showed that significant disruptions of the secondary structure of proteins were not observed at pH 2 to pH 7. At pH 8, the percentages of α-helix, β-sheet, and β-turn in VLPs (VP1) were decreased from 11% to 8%, from 37% to 32%, and from 20% to 16%, respectively. The percentage of coil was increased from 32% to 44%. By contrast, the percentages of α-helix, β-sheet, and β-turn in VLPs (VP1+VP2) were decreased from 11% to 10%, from 37% to 35%, and from 20% to 19%, respectively. The percentage of coil was increased from 32% to 36%. VLPs (VP1+VP2) was likely more stable than VLPs (VP1), as indicated by the percentage of the secondary structures analyzed by CD. These results suggested that VP2 could stabilize the secondary structure of VLPs under alkaline pH conditions. This study provided novel insights into the molecular mechanism of the function of VP2 in the stability of NoV particles.

NOTE] A Protective Role of Methionine-R-Sulfoxide Reductase against Cadmium in Schizosaccharomyces pombe: Chang-Jin Lim , Hannah Jo , Kyunghoon Kim; J. Microbiol. 2014;52(11):976-981. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3512-7

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5 Citations

Abstract: The Schizosaccharomyces pombe cells harboring the methionine- R-sulfoxide reductase (MsrB)-overexpressing recombinant plasmid pFMetSO exhibited better growth than vector control cells, when shifted into fresh medium containing cadmium chloride (abbreviated as Cd). Although both groups of cells contained enhanced reactive oxygen species (ROS) and nitric oxide (NO) levels in the presence of Cd, ROS and NO levels were significantly lower in the S. pombe cells harboring pFMetSO than in vector control cells. Conversely, the S. pombe cells harboring pFMetSO possessed higher total glutathione (GSH) levels and a greater reduced/oxidized GSH ratio than vector control cells under the same conditions.

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