Journal of Microbiology

Volume 52(7); July 2014

Review

Minireview] The History of Aerobic Ammonia Oxidizers: from the First Discoveries to Today: Maria Monteiro , Joana Séneca , Catarina Magalhães; J. Microbiol. 2014;52(7):537-547. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-4114-0

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63 Citations

Abstract: Nitrification, the oxidation of ammonia to nitrite and nitrate, has long been considered a central biological process in the global nitrogen cycle, with its first description dated 133 years ago. Until 2005, bacteria were considered the only organisms capable of nitrification. However, the recent discovery of a chemoautotrophic ammonia-oxidizing archaeon, Nitrosopumilusmaritimus, changed our concept of the range of organisms involved in nitrification, highlighting the importance of ammonia-oxidizing archaea (AOA) as potential players in global biogeochemical nitrogen transformations. The uniqueness of these archaea justified the creation of a novel archaeal phylum, Thaumarchaeota. These recent discoveries increased the global scientific interest within the microbial ecology society and have triggered an analysis of the importance of bacterial vs archaeal ammonia oxidation in a wide range of natural ecosystems. In thismini review we provide a chronological perspective of the current knowledge on the ammonia oxidation pathway of nitrification, based on the main physiological, ecological and genomic discoveries.

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

Bacillus daqingensis sp. nov., a Halophilic, Alkaliphilic Bacterium Isolated from Saline-Sodic Soil in Daqing, China: Shuang Wang , Lei Sun , Dan Wei , Baoku Zhou , Junzheng Zhang , Xuejia Gu , Lei Zhang , Ying Liu , Yidan Li , Wei Guo , Shuang Jiang , Yaqing Pan , Yufeng Wang; J. Microbiol. 2014;52(7):548-553. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3376-x

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11 Citations

Abstract: An alkaliphilic, moderately halophilic, bacterium, designated strain X10-1T, was isolated from saline-alkaline soil in Daqing, Heilongjiang Province, China. Strain X10-1T was determined to be a Gram-positive aerobe with rod-shaped cells. The isolate was catalase-positive, oxidase-negative, non-motile, and capable of growth at salinities of 0–16% (w/v) NaCl (optimum, 3%). The pH range for growth was 7.5–11.0 (optimum, pH 10.0). The genomic DNA G+C content was 47.7 mol%. Its major isoprenoid quinone was MK-7 and its cellular fatty acid profile mainly consisted of anteiso-C15:0, anteiso-C17:0, iso-C15:0, C16:0, and iso-C16:0. The peptidoglycan contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, and phosphatidylglycerol. Phylogenetic analysis based on 16S rRNA gene sequences showed that X10-1T is a member of the genus Bacillus, being most closely related to B. saliphilus DSM15402T (97.8% similarity) and B. agaradhaerens DSM 8721T (96.2%). DNA-DNA relatedness to the type strains of these species was less than 40%. On the basis of the phylogenetic, physiological, and biochemical data, strain X10-1T represents a novel species of the genus Bacillus, for which the name Bacillus daqingensis sp. nov. is proposed. The type strain is X10-1T (=NBRC 109404T =CGMCC 1.12295T).

Bacillus cheonanensis sp. nov. Isolated from Near Poultry Farm Soil: Hyun-Ju Kim , Cheol-Su Park , Siwon Lee , Tae-Young Ahn; J. Microbiol. 2014;52(7):554-558. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3458-9

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Abstract: A novel bacterial strain, designated PFS-5T, was isolated from the soil environment with feces of a live poultry farm located in Cheonan, Republic of Korea. Strain PFS-5T was Gram-staining-positive, motile, strictly aerobic bacterium, rod-shaped, and endospore-forming. The strain contained meso-diaminopimelic acid in their peptidoglycan and MK-7 menaquinone. The major fatty acids were anteiso-C15:0 (44.2%), C16:0 (22.2%), and iso-C15:0 (16.7%). The DNA G+C content was 40.1 mol%. Comparative 16S rRNA gene sequence analysis identified strain PFS-5T in the genus Bacillus, exhibiting the highest level of sequence similarity with type strain of B. herbersteinensis D-1,5aT (96.9%), B. humi LMG 22167T (96.7%), B. alkalitelluris BA288T (96.1%), B. litoralis SW-211T (96.0%), and B. luteolus YIM93174T (95.5%). The major polar lipids of PFS-5T were diphosphatidylglycerol and phosphatidylglycerol. On the basis of result from poly-phasic data, strain PFS-5T represents a novel species, for which the name Bacillus cheonanensis sp. nov. is proposed (Type strain PFS-5T= KACC 17469T= JCM19333T).

Molecular Characterization of Atoxigenic Aspergillus flavus Isolates Collected in China: Dandan Wei , Lu Zhou , Jonathan Nimal Selvaraj , Chushu Zhang , Fuguo Xing , Yueju Zhao , Yan Wang , Yang Liu; J. Microbiol. 2014;52(7):559-565. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3629-8

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31 Citations

Abstract: Aspergillus flavus strains were isolated from peanut fields of Liaoning, Shandong, Hubei and Guangdong Provinces in China, and identified through phenotypic and molecular approaches. Of the 323 A. flavus strains isolated, 76 strains did not produce aflatoxins detectable by UPLC. The incidence of atoxigenic A. flavus strains decreased with increase in temperature and increased with increase in latitude in different geographical locations. Amplification of all the aflatoxin genes in the aflatoxin gene cluster in the atoxigenic isolates showed that there were 25 deletion patterns (A-Y), with 22 deletion patterns identified for the first time. Most of the atoxigenic A. flavus isolates with gene deletions (97%) had deletions in at least one of the four genes (aflT, nor-1, aflR, and hypB), indicating that these four genes could be targeted for rapid identification of atoxigenic strains. The atoxigenic isolates with gene deletions, especially the isolates with large deletions, are potential candidates for aflatoxin control.

Regional Effects on Chimera Formation in 454 Pyrosequenced Amplicons from a Mock Community: Sunguk Shin , Tae Kwon Lee , Jung Min Han , Joonhong Park; J. Microbiol. 2014;52(7):566-573. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3485-6

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Abstract: Chimeras are a frequent artifact in polymerase chain reaction and could be the underlying causes of erroneous taxonomic identifications, overestimated microbial diversity, and spurious sequences. However, little is known about the regional effects on chimera formation. Therefore, we investigated the chimera formation rates in different regions of phylogenetically important biomarker genes to test the regional effects on chimera formation. An empirical study of chimera formation rates was performed using the Roche GSFLXTM system with sequences of the V1/V2/V3 and V4/V5 regions of the 16S rRNA gene and sequences of the nifH gene from a mock microbial community. The chimera formation rates for the 16S V1/V2/V3 region, V4/V5 region, and nifH gene were 22.1–38.5%, 3.68–3.88%, and 0.31–0.98%, respectively. Some amplicons from the V1/V2/V3 regions were shorter than the typical length (~7–31%), reflecting incomplete extension. In the V1/V2/V3 and V4/V5 regions, conserved and hypervariable regions were identified. Chimeric hot spots were located in parts of conserved regions near the ends of the amplicons. The 16S V1/V2/V3 region had the highest chimera formation rate, likely because of long template lengths and incomplete extension. The amplicons of the nifH gene had the lowest frequency of chimera formation most likely because of variations in their wobble positions in triplet codons. Our results suggest that the main reasons for chimera formation are sequence similarity and premature termination of DNA extension near primer regions. Other housekeeping genes can be a good substitute for 16S rRNA genes inmolecularmicrobial studies to reduce the effects of chimera formation.

Assessment of Microbial Diversity Bias Associated with Soil Heterogeneity and Sequencing Resolution in Pyrosequencing Analyses: Sokhee P. Jung , Hojeong Kang; J. Microbiol. 2014;52(7):574-580. Published online May 13, 2014; DOI: https://doi.org/10.1007/s12275-014-3636-9

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Abstract: It is important to estimate the true microbial diversities accurately for a comparative microbial diversity analysis among various ecological settings in ecological models. Despite drastically increasing amounts of 16S rRNA gene targeting pyrosequencing data, sampling and data interpretation for comparative analysis have not yet been standardized. For more accurate bacterial diversity analyses, the influences of soil heterogeneity and sequence resolution on bacterial diversity estimates were investigated using pyrosequencing data of oak and pine forest soils with focus on the bacterial 16S rRNA gene. Soil bacterial community sets were phylogenetically clustered into two separate groups by forest type. Rarefaction curves showed that bacterial communities sequenced from the DNA mixtures and the DNAs of the soil mixtures had midsize richness compared with other samples. Richness and diversity estimates were highly variable depending on the sequence read numbers. Bacterial richness estimates (ACE, Chao 1 and Jack) of the forest soils had positive linear relationships with the sequence read number. Bacterial diversity estimates NPShannon, Shannon and the inverse Simpson) of the forest soils were also positively correlated with the sequence read number. One-way ANOVA shows that sequence resolution significantly affected the α-diversity indices (P<0.05), but the soil heterogeneity did not (P>0.05). For an unbiased evaluation, richness and diversity estimates should be calculated and compared from subsets of the same size.

Responses of Candida albicans to the Human Antimicrobial Peptide LL-37: Pei-Wen Tsai , Yin-Lien Cheng , Wen-Ping Hsieh , Chung-Yu Lan; J. Microbiol. 2014;52(7):581-589. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3630-2

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52 Citations

Abstract: Candida albicans is a major fungal pathogen in humans. Antimicrobial peptides (AMPs) are critical components of the innate immune response in vertebrates and represent the first line of defense against microbial infection. LL-37 is the only member of the human family of cathelicidin AMPs and is commonly expressed by various tissues and cells, including surfaces of epithelia. The candidacidal effects of LL-37 have been well documented, but the mechanisms by which LL-37 kills C. albicans are not completely understood. In this study, we examined the effects of LL-37 on cell wall and cellular responses in C. albicans. Using transmission electron microscopy, carbohydrate analyses, and staining for β-1,3-glucan, changing of C. albicans cell wall integrity was detected upon LL-37 treatment. In addition, LL-37 also affected cell wall architecture of the pathogen. Finally, DNA microarray analysis and quantitative PCR demonstrated that sub-lethal concentrations of LL-37 modulated the expression of genes with a variety of functions, including transporters, regulators for biological processes, response to stress or chemical stimulus, and pathogenesis. Together, LL-37 induces complex responses in C. albicans, making LL-37 a promising candidate for use as a therapeutic agent against fungal infections.

Journal Articles

The Observation of PlcA Mutation and Localization in Aspergillus nidulans: Chun-Seob Ahn , Young Taek Oh , Jeong-Geun Kim , Kap-Hoon Han , Chang-Won Lee , Jae Won Kim; J. Microbiol. 2014;52(7):590-596. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3651-x

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Abstract: To know the function of the plcA gene, which encodes a putative phosphoinositide-specific phospholipase C, in a model filamentous fungus Aspergillus nidulans, it was disrupted thorough homologous recombination and examined. The germination rate of ΔplcA was reduced by approximately 65% and germination of ΔplcA at a lower temperature (25°C) was much slower than germination under normal conditions (37°C), suggesting the plcA is responsible for cold-sensitivity. The hyphal growth of ΔplcA was slightly reduced at 37°C and conspicuously reduced at 25°C. While germinating ΔplcA formed giant swollen spores, and generated short and thick hyphae. The results of the nuclear examination of ΔplcA showed nuclear division with missegregation, and the rate of nuclear division was lower than that of wild type at both 25°C and 37°C. The results of this study showed that plcA is localized to the nucleus through intracellular calcium signaling in A. nidulans. The abnormal nuclear division, resulting from plcA gene deletion, affects conidiation in asexual development. Taken together, these results suggested that plcA is required for normal vegetative growth, morphogenesis, conidiation, and nuclear division
in A. nidulans.

Aeration Effects on Metabolic Events during Sporulation of Bacillus thuringiensis: Mohammad H. Sarrafzadeh , Sabine Schorr-Galindo , Hyun-Joon La , Hee-Mock Oh; J. Microbiol. 2014;52(7):597-603. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3547-9

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Abstract: The metabolism of Bacillus thuringiensis during its sporulation process was investigated under different concentrations of oxygen. At the beginning of sporulation, the aeration conditions were regulated to obtain different oxygen transfer rates (OTR) in four separate fermentations, representing interrupted, limited, non-limited, and saturated oxygenation, respectively. A higher OTR resulted in a higher pH, up to about 9 in the case of saturated oxygenation, while the interrupted oxygenation resulted in a significantly acidic culture. In contrast, the absence of oxygen resulted in rapid sporangia lysis and caused acidification of the medium, indicating a distinctly different sporangia composition and different metabolism. The bacterium also showed different CO2 production rates during sporulation, although amaximum point was observed in every case.With a higher OTR, the maximal value was observed after a longer time and at a lower value (40, 26, and 13 mmol/L/h for limited, non-limited, and saturated cases, respectively). Despite the exhaustion of glucose prior to the sporulation phase, the interrupted oxygenation resulted in acetate, lactate, and citrate in the medium with a maximum concentration of 4.8, 1.3, and 5.0 g/L, respectively. Notwithstanding, while the metabolic events differed visibly in the absence of oxygen, once sporulation was triggered, it was completed, even in the case of an interrupted oxygen supply.

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

An Easy Way for the Rapid Purification of Recombinant Proteins from Helicobacter pylori Using a Newly Designed Expression Vector: Hyung-Lyun Kang , Jin-Sung Jo , Soon-Uck Kwon , Jae-Young Song , Ji-Hyun Seo , Myung-Je Cho , Seung-Chul Baik , Hee-Shang Youn , Kwang-Ho Rhee , Woo-Kon Lee; J. Microbiol. 2014;52(7):604-608. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3679-y

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Abstract: We constructed a H. pylori expression vector which consisted of both a His-tag and a GST tag as purification tools for recombinant protein and a chloramphenicol resistant cat gene as a reporter. The backbone of the vector pBK contained an ColEI origin of replication and a kanamycin resistant gene. A set of oligos for the His-tag and the PCR product of gst (glutathione S-transferase) gene were inserted sequentially in frame in the multi-cloning site of pBK. The orf of cat was inserted downstream of the gst to generate pBKHGC. The 3' part of H. pylori clpB and flaA were cloned into the vector which was introduced into H. pylori. Recombinant proteins were purified by GSH affinity column, digested with thrombin and were analyzed by western blotting. The final recombinant proteins were successfully purified.

Genotyping, Morphology and Molecular Characteristics of a Lytic Phage of Neisseria Strain Obtained from Infected Human Dental Plaque: Ahmed N Aljarbou , Mohamad Aljofan; J. Microbiol. 2014;52(7):609-618. Published online May 30, 2014; DOI: https://doi.org/10.1007/s12275-014-3380-1

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Abstract: The lytic bacteriaphage (phage) A2 was isolated from human dental plaques along with its bacterial host. The virus was found to have an icosahedron-shaped head (60±3 nm), a sheathed and rigid long tail (~175 nm) and was categorized into the family Siphoviridae of the order Caudovirales, which are dsDNA viral family, characterised by their ability to infect bacteria and are nonenveloped with a noncontractile tail. The isolated phage contained a linear dsDNA genome having 31,703 base pairs of unique sequence, which were sorted into three contigs and 12 single sequences. A latent period of 25 minutes and burst size of 24±2 particles was determined for the virus. Bioinformatics approaches were used to identify ORFs in the genome. A phylogenetic analysis confirmed the species inter-relationship and its placement in the family.

Review

Bovine Viral Diarrhea Virus Infection Induces Autophagy in MDBK Cells: Qiang Fu , Huijun Shi , Yan Ren , Fei Guo , Wei Ni , Jun Qiao , Pengyan Wang , Hui Zhang , Chuangfu Chen; J. Microbiol. 2014;52(7):619-625. Published online June 28, 2014; DOI: https://doi.org/10.1007/s12275-014-3479-4

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27 Citations

Abstract: Bovine viral diarrhea virus (BVDV) is an enveloped, positive-sense, single-stranded RNA virus that belongs to the genus Pestivirus (Flaviviridae). The signaling pathways and levels of signaling molecules are altered in Madin-Darby Bovine Kidney (MDBK) cells infected with BVDV. Autophagy is a conservative biological degradation pathway that mainly eliminates and degrades damaged or superfluous organelles and macromolecular complexes for intracellular recycling in eukaryotic cells. Autophagy can also be induced as an effective response to maintain cellular homeostasis in response to different stresses, such as nutrient or growth factor deprivation, hypoxia, reactive oxygen species exposure and pathogen infection. However, the effects of BVDV infection on autophagy inMDBK cells remain unclear. Therefore, we performed an analysis of autophagic activity after BVDV NADL infection using real-time PCR, electron microscopy, laser confocal microscopy, and Western blotting analysis. The results demonstrated that BVDV NADL infection increased autophagic activity and significantly elevated the expression levels of the autophagy-related genes Beclin1 and ATG14 inMDBK cells. However, the knockdown of Beclin1 and ATG14 by RNA interference (RNAi) did not affect BVDV NADL infection-related autophagic activity. These findings provided a novel perspective to elaborate the effects of viral infection on the host cells.

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

Note] Identification of High-Specificity H-NS Binding Site in LEE5 Promoter of Enteropathogenic Esherichia coli (EPEC): Abhay Prasad Bhat , Minsang Shin , Hyon E. Choy; J. Microbiol. 2014;52(7):626-629. Published online March 7, 2014; DOI: https://doi.org/10.1007/s12275-014-3562-x

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8 Citations

Abstract: Histone-like nucleoid structuring protein (H-NS) is a small but abundant protein present in enteric bacteria and is involved in compaction of the DNA and regulation of the transcription. Recent reports have suggested that H-NS binds to a specific AT rich DNA sequence than to intrinsically curved DNA in sequence independent manner. We detected two high-specificity H-NS binding sites in LEE5 promoter of EPEC centered at -110 and -138, which were close to the proposed consensus H-NS binding motif. To identify H-NS binding sequence in LEE5 promoter, we took a random mutagenesis approach and found the mutations at around -138 were specifically defective in the regulation byH-NS. It was concluded that H-NS exertsmaximumrepression via the specific sequence at around -138 and ubsequently contacts α subunit of RNAP through oligomerization.

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