Journal of Microbiology

Volume 53(4); April 2015

Review

Replicating poxviruses for human cancer therapy: Manbok Kim; J. Microbiol. 2015;53(4):209-218. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-5041-4

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26 Citations

Abstract: Naturally occurring oncolytic viruses are live, replicationproficient viruses that specifically infect human cancer cells while sparing normal cell counterparts. Since the eradication of smallpox in the 1970s with the aid of vaccinia viruses, the vaccinia viruses and other genera of poxviruses have shown various degrees of safety and efficacy in pre-clinical or clinical application for human anti-cancer therapeutics. Furthermore, we have recently discovered that cellular tumor suppressor genes are important in determining poxviral oncolytic tropism. Since carcinogenesis is a multi-step process involving accumulation of both oncogene and tumor suppressor gene abnormalities, it is interesting that poxvirus can exploit abnormal cellular tumor suppressor signaling for its oncolytic specificity and efficacy. Many tumor suppressor genes such as p53, ATM, and RB are known to play important roles in genomic fidelity/maintenance. Thus, tumor suppressor gene abnormality could affect host genomic integrity and likely disrupt intact antiviral networks due to accumulation of genetic defects, which would in turn result in oncolytic virus susceptibility. This review outlines the characteristics of oncolytic poxvirus strains, including vaccinia, myxoma, and squirrelpox virus, recent progress in elucidating the molecular connection between oncogene/tumor suppressor gene abnormalities and poxviral oncolytic tropism, and the associated preclinical/clinical implications. I would also like to propose future directions in the utility of poxviruses for oncolytic virotherapy.

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

New record and enzyme activity of four species in Penicillium section Citrina from marine environments in Korea: Myung Soo Park , Ji Eun Eom , Jonathan J. Fong , Young Woon Lim; J. Microbiol. 2015;53(4):219-225. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-4700-9

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15 Citations

Abstract: Several strains of Penicillium section Citrina were isolated during a survey of fungi from marine environments along the southern coast of Korea. Based on multigene phylogenetic analyses (?tubulin and calmodulin) and morphological characteristics, the 11 strains were identified as P. citrinum, P. hetheringtonii, P. paxilli, P. sumatrense, P. terrigenum, and P. westlingii. To understand the ecological role of these species, we tested all strains for extracellular enzyme activity; six strains representing four species showed ?glucosidase activity. Four of the identified species ?P. hetheringtonii, P. paxilli, P. terrigenum, and P. westlingii ?are new records for Korea. For these new species records, we describe morphological characteristics of the strains and compare results to published data of type strains.

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

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance§: Ananya Barman , Ranjan Tamuli; J. Microbiol. 2015;53(4):226-235. Published online January 31, 2015; DOI: https://doi.org/10.1007/s12275-015-4465-1

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21 Citations

Abstract: Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca2+/H+ exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca2+/H+ exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca2+ concentration ([Ca2+]c) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)- survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca2+]c, carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

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

Trichoderma reesei Sch9 and Yak1 regulate vegetative growth, conidiation, and stress response and induced cellulase production: Xinxing Lv† , Weixin Zhang† , Guanjun Chen , Weifeng Liu; J. Microbiol. 2015;53(4):236-242. Published online January 31, 2015; DOI: https://doi.org/10.1007/s12275-015-4639-x

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18 Citations

Abstract: Protein kinases are key players in controlling many basic cellular processes in almost all the organisms via mediating signal transduction processes. In the present study, we characterized the cellulolytic Trichoderma reesei orthologs of Saccharomyces cerevisiae Sch9 and Yak1 by sequence alignment and functional analysis. The T. reesei Trsch9Δ and Tryak1Δ mutant strains displayed a decreased growth rate on different carbon sources and produced less conidia. The absence of these two kinases also resulted in different but abnormal polarized apical growth as well as sensitivity to various stresses. In addition, disruption of the genes Trsch9 or Tryak1 resulted in perturbation of cell wall integrity. Interestingly, while the induced production of cellulases was slightly compromised in the Trsch9Δ strain, the extracellular production of cellulases was significantly improved in the absence of Yak1. The results indicate that TrSch9 and TrYak1 play an important role in filamentous growth, stress response and induced production of cellulases in T. reesei.

Multiple roles of a putative vacuolar protein sorting associated protein 74, FgVPS74, in the cereal pathogen Fusarium graminearum: Hee-Kyoung Kim , Ki Woo Kim , Sung-Hwan Yun; J. Microbiol. 2015;53(4):243-249. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-5067-7

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8 Citations

Abstract: Fusarium graminearum, a member of the F. graminearum species complex, is a filamentous ascomycetous group that causes serious diseases in cereal crops. A screen of insertional mutants of F. graminearum, generated using a restriction enzyme-mediated integration method, identified a mutant designated R7048 showing pleiotropic phenotypes in several mycological traits. The vector insertion site in the R7048 genome was identified as the KpnI site within an ORF annotated as FGSG_06346 (designated FgVPS74), which showed similarity to vacuolar protein sorting-associated protein 74 in the baker yeast. Both targeted gene deletion and complementation analyses confirmed that FgVPS74 was involved in hyphal growth, conidiation, sexual development, mycotoxin production, and virulence towards host plants in F. graminearum. Electron microscopy analysis revealed no significant changes in morphology of the vacuole or other organelles, but a greater number of mitochondria were produced in the ΔFgVPS74 strain compared to the wild-type progenitor. Expression of a GFP-tagged FgVPS74 construct under its native promoter in the ΔFgVPS74 strain exhibited localization of GFP signal to putative vesicle structures, but not to the vacuolar membrane. Taken together, these findings demonstrated that a functional vacuolar protein-sorting pathway mediated by FgVPS74 is crucial for fungal growth and development in F. graminearum.

Effect of promoter-upstream sequence on σ38-dependent stationary phase gene transcription: Hyung-Ju Lim , Kwangsoo Kim , Minsang Shin , Jae-Ho Jeong , Phil Youl Ryu , Hyon E. Choy; J. Microbiol. 2015;53(4):250-255. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-4681-8

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3 Citations

Abstract: σ38 in Escherichia coli is required for expression of a subset of stationary phase genes. However, the promoter elements for σ38-dependent genes are virtually indistinguishable from that for σ70-dependent house-keeping genes. hdeABp is a σ38-dependent promoter and LEE5p is a σ70-dependent promoter, but both are repressed by H-NS, a bacterial histone- like protein, which acts at promoter upstream sequence. We swapped the promoter upstream sequences of the two promoters and found that the σ dependency was switched. This was further verified using lacUV5 core promoter. The
results
suggested that the determinant for σ38-dependent promoter lies in the promoter upstream sequence.

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB: Seunghwa Lee , Ji-Hyun Yeom , Sojin Seo , Minho Lee , Sarang Kim , Jeehyeon Bae , Kangseok Lee , Jihwan Hwang; J. Microbiol. 2015;53(4):256-261. Published online March 4, 2015; DOI: https://doi.org/10.1007/s12275-015-5037-0

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9 Citations

Abstract: Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

Journal Article

Spectral characterization of a pteridine derivative from cyanide-utilizing bacterium Bacillus subtilis - JN989651: S. Durairaju Nisshanthini , Antony K. Teresa Infanta S. , Duraisamy Senthil Raja , Karuppannan Natarajan , M. Palaniswamy , Jayaraman Angayarkanni; J. Microbiol. 2015;53(4):262-271. Published online March 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4138-0

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9 Citations

Abstract: Soil and water samples were collected from various regions of SIPCOT and nearby Vanappadi Lake, Ranipet, Tamilnadu, India. Based on their colony morphology and their stability during subculturing, 72 bacteria were isolated, of which 14 isolates were actinomycetes. Preliminary selection was carried out to exploit the ability of the microorganisms to utilize sodium cyanate as nitrogen source. Those organisms that were able to utilize cyanate were subjected to secondary screening viz., utilization of sodium cyanide as the nitrogen source. The oxygenolytic cleavage of cyanide is dependent on cyanide monooxygenase which obligately requires pterin cofactor for its activity. Based on this, the organisms capable of utilizing sodium cyanide were tested for the presence of pterin. Thin layer chromatography (TLC) of the cell extracts using n-butanol: 5 N glacial acetic acid (4:1) revealed that 10 out of 12 organisms that were able to utilize cyanide had the pterin-related blue fluorescent compound in the cell extract. The cell extracts of these 10 organisms were subjected to high performance thin layer chromatography (HPTLC) for further confirmation using a pterin standard. Based on the incubation period, cell biomass yield, peak height and area, strain VPW3 was selected and was identified as Bacillus subtilis. The Rf value of the cell extract was 0.73 which was consistent with the 0.74 Rf value of the pterin standard when scanned at 254 nm. The compound was extracted and purified by preparative High Performance Liquid Chromatography (HPLC). Characterization of the compound was performed by ultraviolet spectrum, fluorescence spectrum, Electrospray Ionization-Mass Spectrometry (ESI-MS), and Nuclear Magnetic Resonance spectroscopy (NMR). The compound is proposed to be 6-propionyl pterin (2-amino-6- propionyl-3H-pteridin-4-one).

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

Statistical experimental design optimization of rhamsan gum production by Sphingomonas sp. CGMCC 6833: Xiao-Ying Xu , Shu-Hao Dong , Sha Li , Xiao-Ye Chen , Ding Wu , Hong Xu; J. Microbiol. 2015;53(4):272-278. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-3662-2

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11 Citations

Abstract: Rhamsan gum is a type of water-soluble exopolysaccharide produced by species of Sphingomonas bacteria. The optimal fermentation medium for rhamsan gum production by Sphingomonas sp. CGMCC 6833 was explored definition. Single-factor experiments indicate that glucose, soybean meal, K2HPO4 and MnSO4 compose the optimal medium along with and initial pH 7.5. To discover ideal cultural conditions for rhamsan gum production in a shake flask culture, response surface methodology was employed, from which the following optimal ratio was derived: 5.38 g/L soybean meal, 5.71 g/L K2HPO4 and 0.32 g/L MnSO4. Under ideal fermentation rhamsan gum yield reached 19.58 g/L ?1.23 g/L, 42.09% higher than that of the initial medium (13.78 g/L ? 1.38 g/L). Optimizing the fermentation medium results in enhanced rhamsan gum production.

Directed analysis of cyanobacterial membrane phosphoproteome using stained phosphoproteins and titanium-enriched phosphopeptides§: Dong-Gi Lee , Joseph Kwon , Chi-Yong Eom , Young-Moon Kang , Seong Woon Roh , Kyung-Bok Lee , Jong-Soon Choi; J. Microbiol. 2015;53(4):279-287. Published online April 8, 2015; DOI: https://doi.org/10.1007/s12275-015-5021-8

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13 Citations

Abstract: Gel-free shotgun phosphoproteomics of unicellular cyanobacterium Synechocystis sp. PCC 6803 has not been reported up to now. The purpose of this study is to develop directed membrane phosphoproteomic method in Synechocystis sp. Total Synechocystis membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and phosphoprotein-stained gel bands were selectively subjected to in-gel trypsin digestion. The phosphorylation sites of the resulting peptides were determined by assigning the neutral loss of [M-H3PO4] to Ser, Thr, and Tyr residues using nano-liquid chromatography 7 Tesla Fourier transform mass spectrometry. As an initial application, 111 proteins and 33 phosphoproteins were identified containing 11 integral membrane proteins. Identified four unknown phosphoproteins with transmembrane helices were suggested to be involved in membrane migration or transporters based on BLASTP search annotations. The overall distribution of hydrophobic amino acids in pTyr was lower in frequency than that of pSer or pThr. Positively charged amino acids were abundantly revealed in the surrounding amino acids centered on pTyr. A directed shotgun membrane phosphoproteomic strategy provided insight into understanding the fundamental regulatory processes underlying Ser, Thr, and Tyr phosphorylation in multi-layered membranous cyanobacteria.

From the traditional Chinese medicine plant Schisandra chinensis new scaffolds effective on HIV-1 reverse transcriptase resistant to non-nucleoside inhibitors: Lijia Xu , Nicole Grandi , Claudia Del Vecchio , Daniela Mandas , Angela Corona , Dario Piano , Francesca Esposito , Cristina Parolin , Enzo Tramontano; J. Microbiol. 2015;53(4):288-293. Published online March 4, 2015; DOI: https://doi.org/10.1007/s12275-015-4652-0

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62 Citations

Abstract: HIV-1 reverse transcriptase (RT) is still an extremely attractive pharmaceutical target for the identification of new inhibitors possibly active on drug resistant strains. Medicinal plants are a rich source of chemical diversity and can be used to identify novel scaffolds to be further developed by chemical modifications. We investigated the ability of the main lignans from Schisandra chinensis (Turcz.) Baill. fruits, commonly used in Traditional Chinese Medicine, to affect HIV-1 RT functions. We purified 6 lignans from Schisandra chinensis fruits and assayed their effects on HIV-1 RT and viral replication. Among the S. chinensis fruit lignans, Schisandrin B and Deoxyschizandrin selectively inhibited the HIV-1 RTassociated DNA polymerase activity. Structure activity relationship revealed the importance of cyclooctadiene ring substituents for efficacy. In addition, Schisandrin B was also able to impair HIV-1 RT drug resistant mutants and the early phases of viral replication. We identified Schisandrin B and Deoxyschizandrin as new scaffold for the further development of novel HIV-1 RT inhibitors.

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