Journal of Microbiology

Volume 49(1); February 2011

Review

REVIEW] An Inward Proton Transport Using Anabaena Sensory Rhodopsin: Akira Kawanabe , Yuji Furutani , Kwang-Hwan Jung , Hideki Kandori; J. Microbiol. 2011;49(1):1-6. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0547-x

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ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacteriorhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity. Proton pumps must have a specific mechanism to exclude transport in the reverse direction to maintain a proton gradient, and in the case of BR, a highly hydrophobic cytoplasmic domain may constitute such machinery. Although an inward proton pump has neither been created naturally nor artificially, we recently reported that an inward-directed proton transport can be engineered from a bacterial rhodopsin by a single amino acid replacement. Anabaena sensory rhodopsin (ASR) is a photochromic sensor in freshwater cyanobacteria, possessing little proton transport activity. When we replace Asp217 at the cytoplasmic domain (distance ~15 Å from the retinal chromophore) to Glu, ASR is converted into an inward proton transport, driven by absorption of a single photon. FTIR spectra clearly show an increased proton affinity for Glu217, which presumably controls the unusual directionality opposite to normal proton pumps.

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Ion-transporting mechanism in microbial rhodopsins: Mini-review relating to the session 5 at the 19th International Conference on Retinal Proteins
Yuji Furutani, Chii-Shen Yang
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Bioinformatic analysis of the distribution of inorganic carbon transporters and prospective targets for bioengineering to increase Ci uptake by cyanobacteria
Sandeep B. Gaudana, Jan Zarzycki, Vamsi K. Moparthi, Cheryl A. Kerfeld
Photosynthesis Research.2015; 126(1): 99. CrossRef
Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin
Kengo Sasaki, Takahiro Yamashita, Kazuho Yoshida, Keiichi Inoue, Yoshinori Shichida, Hideki Kandori, Karl-Wilhelm Koch
PLoS ONE.2014; 9(3): e91323. CrossRef
A role of Anabaena sensory rhodopsin transducer (ASRT) in photosensory transduction
So Young Kim, Sa Ryong Yoon, SongI Han, Yuna Yun, Kwang‐Hwan Jung
Molecular Microbiology.2014; 93(3): 403. CrossRef
The role of protein-bound water molecules in microbial rhodopsins
Klaus Gerwert, Erik Freier, Steffen Wolf
Biochimica et Biophysica Acta (BBA) - Bioenergetics.2014; 1837(5): 606. CrossRef
Microcompartments and Protein Machines in Prokaryotes
Milton H. Saier Jr.
Microbial Physiology.2013; 23(4-5): 243. CrossRef
Quantum chemical modeling of rhodopsin mutants displaying switchable colors
Federico Melaccio, Nicolas Ferré, Massimo Olivucci
Physical Chemistry Chemical Physics.2012; 14(36): 12485. CrossRef
Crystal Structure of the Eukaryotic Light-Driven Proton-Pumping Rhodopsin, Acetabularia Rhodopsin II, from Marine Alga
Takashi Wada, Kazumi Shimono, Takashi Kikukawa, Masakatsu Hato, Naoko Shinya, So Young Kim, Tomomi Kimura-Someya, Mikako Shirouzu, Jun Tamogami, Seiji Miyauchi, Kwang-Hwan Jung, Naoki Kamo, Shigeyuki Yokoyama
Journal of Molecular Biology.2011; 411(5): 986. CrossRef

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

Screening and Identification of a Novel Esterase EstPE from a Metagenomic DNA Library: So-Youn Park , Hyun-Jae Shin , Geun-Joong Kim; J. Microbiol. 2011;49(1):7-14. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0201-7

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Abstract: Esterases represent a large family of hydrolases with broad substrate specificity and functional sequence space. Although many attempts to screen new esterases have been conducted, there have been few reports conducted to discriminate unique enzymes from typical ones based on novel structure and function. In this study, we discovered an esterase and a novel family through a successive assay of whole cells and crude lysates (oxidative open condition). The screened putative esterases from the metagenomic DNA of salted shrimp consisted of 753 bp encoding 27 kDa of polypeptide, namely PE esterase. Sequence analyses revealed that an identical gene was reported from whole genome sequencing of Stenotrophomonas maltophilia K279a. However, its biochemical and phylogenetic characteristics have not yet been evaluated. PE esterase was overexpressed only by the MBP fusion state in E. coli and was easily purified using an affinity column. This enzyme showed a typical spectrum of substrate specificity and possessed the consensus motifs, Ser-Asp-His and GXSXG, which are essential for most esterase/lipase superfamilies. Interestingly, the entire organization of the ORF and consensus sequence around the active site were distinct from the related enzymes, and its structure could be affected by a reducing agent, DTT.

Distinctive Endophytic Fungal Assemblage in Stems of Wild Rice (Oryza granulata) in China with Special Reference to Two Species of Muscodor (Xylariaceae): Zhi-lin Yuan , Zhen-zhu Su , Li-juan Mao , Yang-qing Peng , Guan-mei Yang , Fu-cheng Lin , Chu-long Zhang; J. Microbiol. 2011;49(1):15-23. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0213-3

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Abstract

Ecological niches in the rhizosphere and phyllosphere of grasses capable of sustaining endophytes have been extensively studied. In contrast, little information regarding the identity and functions of endophytic fungi in stems is available. In this study, we investigated the taxonomic affinities, diversity, and host specificities of culturable endophytes in stems of wild rice (Oryza granulata) in China. Seventy-four isolates were recovered. Low recovery rate (11.7%) indicated that there were relatively few sites for fungal infection. Identification using morphology, morphospecies sorting, and molecular techniques resulted in classification into 50 taxa, 36 of which were recovered only once. Nucleotide sequence similarity analysis indicated that 30% of the total taxa recovered were highly divergent from known species and thus may represent lineages new to science. Most of the taxa were classified as members of the classes Sordariomycetes or Dothideomycetes (mainly in Pleosporales). The presence of Arthrinium and Magnaporthaceae species, most often associated with poaceous plants, suggested a degree of host specificity. A polyphasic approach was employed to identify two Muscodor taxa based on (i) ITS and RPB2 phylogenies, (ii) volatile compounds produced, and (iii) an in vitro bioassay of antifungal activity. This to our knowledge is only the second report regarding the isolation of Muscodor spp. in China. Therefore, we hypothesize that wild plants represent a huge reservoir of unknown fungi. The prevalence, novelty, and species-specificity of unique isolates necessitate a reevaluation of their contribution to ecosystem function and fungal biodiversity.

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Prospects of fungal endophytes for stress mitigation in rice (Oryza sativa L.)
Boby Vattekkattu Unnikrishnan, Mahesh Mohan
Oryza-An International Journal on Rice.2022; 59(3): 309. CrossRef
Pest categorisation of Colletotrichum aenigma, C. alienum, C. perseae, C. siamense and C. theobromicola
Claude Bragard, Paula Baptista, Elisavet Chatzivassiliou, Francesco Di Serio, Paolo Gonthier, Josep Anton Jaques Miret, Annemarie Fejer Justesen, Alan MacLeod, Christer Sven Magnusson, Panagiotis Milonas, Juan A Navas‐Cortes, Stephen Parnell, Roel Potting
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Applied Microbiology and Biotechnology.2022; 106(3): 931. CrossRef
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Chijioke E. Ezeobiora, Nwamaka H. Igbokwe, Dina H. Amin, Udoma E. Mendie
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Elucidation of the life cycle of the endophytic genus Muscodor and its transfer to Induratia in Induratiaceae fam. nov., based on a polyphasic taxonomic approach
Milan C. Samarakoon, Benjarong Thongbai, Kevin D. Hyde, Mark Brönstrup, Ulrike Beutling, Christopher Lambert, Andrew N. Miller, Jian-Kui (Jack) Liu, Itthayakorn Promputtha, Marc Stadler
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Louise Barberis, Wilfried Chevalier, Marie-Laure Toussaint, Philippe Binet, Florence Piola, Serge Michalet
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Beatriz Andreo-Jimenez, Philippe Vandenkoornhuyse, Amandine Lê Van, Arvid Heutinck, Marie Duhamel, Niteen Kadam, Krishna Jagadish, Carolien Ruyter-Spira, Harro Bouwmeester
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Carlos Vergara, Karla Emanuelle Campos Araujo, Sônia Regina de Souza, Nivaldo Schultz, Orivaldo José Saggin Júnior, Marcus Vinícius Loss Sperandio, Jerri Édson Zilli
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Identification and characterization of new Muscodor endophytes from gramineous plants in Xishuangbanna, China
Li‐Juan Mao, Jia‐Jie Chen, Chen‐Yang Xia, Xiao‐Xiao Feng, De‐Dong Kong, Zhen‐Yu Qi, Feng Liu, Dian Chen, Fu‐Cheng Lin, Chu‐Long Zhang
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Ya Wang, Bo Liang Gao, Xi Xi Li, Zhi Bin Zhang, Ri Ming Yan, Hui Lin Yang, Du Zhu
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The Colletotrichum boninense species complex
U. Damm, P.F. Cannon, J.H.C. Woudenberg, P.R. Johnston, B.S. Weir, Y.P. Tan, R.G. Shivas, P.W. Crous
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Colletotrichum – current status and future directions
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Halomonas alkalitolerans sp. nov., a Novel Moderately Halophilic Bacterium Isolated from Soda Meadow Saline Soil in Daqing, China: Shuang Wang , Qian Yang , Zhi-Hua Liu , Lei Sun , Dan Wei , Jun-Zheng Zhang , Jin-Zhu Song , Yun Wang , Jia Song , Jin-Xia Fan , Xian-Xin Meng , Wei Zhang; J. Microbiol. 2011;49(1):24-28. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0197-z

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Abstract: A moderately halophilic bacterial strain 15-13T, which was isolated from soda meadow saline soil in Daqing City, Heilongjiang Province, China, was subjected to a polyphasic taxonomic study. The cells of strain 15-13T were found to be Gram-negative, rod-shaped, and motile. The required growth conditions for strain 15-13T were: 1-23% NaCl (optimum, 7%), 10-50°C (optimum, 35°C), and pH 7.0-11.0 (optimum, pH 9.5). The predominant cellular fatty acids were C18:1 ω7c (60.48%) and C16:0 (13.96%). The DNA G+C content was 67.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain 15-13T clustered within a branch comprising species of the genus Halomonas. The closest phylogenetic neighbor of strain 15-13T was Halomonas pantelleriensis DSM 9661T (98.9% 16S rRNA gene sequence similarity). The level of DNA-DNA relatedness between the novel isolated strain and H. pantelleriensis DSM 9661T was 33.8%. On the basis of the phenotypic and phylogenetic data, strain 15-13T represents a novel species of the genus Halomonas, for which the name Halomonas alkalitolerans sp. nov. is proposed. The type strain for this novel species is 15-13T (=CGMCC 1.9129T =NBRC 106539T).

Acinetobacter oleivorans sp. nov. Is Capable of Adhering to and Growing on Diesel-Oil: Yoon-Suk Kang , Jaejoon Jung , Che Ok Jeon , Woojun Park; J. Microbiol. 2011;49(1):29-34. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0315-y

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Abstract: A diesel-oil and n-hexadecane-degrading novel bacterial strain, designated DR1T, was isolated from a rice paddy in Deok-So, South Korea. The strain DR1T cells were Gram-negative, aerobic coccobacilli, and grew at 20-37°C with the optimal temperature of 30°C, and an optimal pH of 6-8. Interestingly, strain DR1T was highly motile (swimming and swarming motility) using its fimbriae, and generated N-acyl homoserine lactones as quorum-sensing signals. The predominant respiratory quinone as identified as ubiquinone-9 (Q-9) and DNA G+C content was 41.4 mol%. Comparative 16S rRNA gene sequence-based phylogenetic analysis placed the strain in a clade with the species A. calcoaceticus, A. haemolyticus, A. baumannii, A. baylyi, and A. beijerinckii, with which it evidenced sequence similarities of 98.2%, 97.4%, 97.2%, 97.1%, and 97.0%, respectively. DNA-DNA hybridization values between strain DR1T and other Acinetobacter spp. were all less than 20%. The physiological and taxonomic characteristics with the DNA-DNA hybridization data supported the identification of strain DR1T in the genus Acinetobacter as a novel species, for which the name Acinetobacter oleivorans sp. nov. is proposed. The type strain is DR1T (=KCTC 23045T =JCM 16667T).

Reductive Divergence of Enterobacterial Repetitive Intergenic Consensus Sequences among Gammaproteobacteria Genomes: Young-Gun Zo; J. Microbiol. 2011;49(1):35-45. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1024-2

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Abstract

Enterobacterial repetitive intergenic consensus (ERIC) sequence is a transcription-modulating, nonautonomous, miniature inverted-repeat transposable element. Its origin and the mechanism of highly varying incidences, limited to Enterobacteriaceae and Vibrionaceae, have not been identified. In this study, distribution and divergence of ERICs along bacterial taxonomic units were analyzed. ERICs were found among five families of gammaproteobacteria, with the copy numbers varying with exponential increments. The variability was explained by genus (45%) and species (36%) affiliations, indicating that copy numbers are specific to subfamily taxa. ERICs were interspersed in genomes with considerable divergences. Locations of ERICs in a genome appeared to be strongly conserved in a strain, moderately in a species or a genus, and weakly in a family. ERICs in different species of a genus were from the identical population of sequences while ERICs in different genera of a family were nearly identical. However, ERICs in different families formed distinct monophylectic groups, implying vertical transmission of diverging population of sequences. In spite of large difference in copy numbers, overall intra-genome evolutionary distances among ERICs were similar among different species, except for a few genomes. The exceptions substantiated hypotheses of genetic drifts and horizontal gene transfers of mobility capacity. Therefore, the confined, variable distribution of ERIC could be explained as a two-step evolution: introduction and proliferation of ERIC in one of the progenitors of gammaproteobacteria, followed by vertical transmission under negative selection. Deterioration of sequences and reduction in copy number were concluded to be the predominant patterns in the evolution of ERIC loci.

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Functional Roles and Genomic Impact of Miniature Inverted-Repeat Transposable Elements (MITEs) in Prokaryotes
Michael F. Minnick
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Occurrence and Antimicrobial Drug Susceptibility Patterns of Commensal and Diarrheagenic Escherichia coli in Fecal Microbiota from Children with and without Acute Diarrhea: Patrícia G. Garcia , Vânia L. Silva , Cláudio G. Diniz; J. Microbiol. 2011;49(1):46-52. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0172-8

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Abstract

Acute diarrhea is a public health problem and an important cause of morbidity and mortality, especially in developing countries. The etiology is varied, and the diarrheagenic Escherichia coli pathotypes are among the most important. Our objectives were to determine the occurrence of commensal and diarrheagenic E. coli strains in fecal samples from children under five years old and their drug susceptibility patterns. E. coli were isolated from 141 fresh fecal samples; 84 were obtained from clinically injured donors with acute diarrhea (AD) and 57 from clinically healthy donors without diarrhea (WD). Presumptive phenotypic species identification was carried out and confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize the diarrheagenic E. coli strains. Drug susceptibility patterns were determined by the disc-diffusion method. In total, 220 strains were recovered from the fecal specimens (61.8% from AD and 38.2% from WD). Diarrheagenic E. coli was identified at a rate of 36.8% (n=50) in diarrheic feces and 29.8% (n=25) in non-diarrheic feces. Enteroaggregative E. coli was the most frequently identified pathotype in the AD group (16.2%) and the only pathotype identified in the WD group (30.9%). Enteropathogenic E. coli was the second most isolated pathotype (10.3%), followed by Shiga toxin-producing E. coli (7.4%) and enterotoxigenic E. coli (2.9%). No enteroinvasive E. coli strains were recovered. The isolates showed high resistance rates against ampicillin, tetracycline, and sulfamethoxazole-trimethoprim. The most effective drugs were ceftazidime, ceftriaxone, imipenem and piperacillin-tazobactam, for which no resistance was observed. Differentiation between the diarrheagenic E. coli pathotypes is of great importance since they are involved in acute diarrheal diseases and may require specific antimicrobial chemotherapy. The high antimicrobial resistance observed in our study raises a broad discussion on the indiscriminate or improper use of antimicrobials, besides the risks of self-medication.

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Babatunde W. Odetoyin, Jennifer Hofmann, Aaron O. Aboderin, Iruka N. Okeke
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Zorangel Amézquita-Montes, Maria Tamborski, Usa G. Kopsombut, Chengxian Zhang, Octavio S. Arzuza, Oscar G. Gómez-Duarte
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Deoxycytidine Production by Metabolically Engineered Corynebacterium ammoniagenes: Yun-Bom Lee , Hong Baek , Sang-Kyum Kim , Hyung-Hwan Hyun; J. Microbiol. 2011;49(1):53-57. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0195-1

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Abstract: Corynebacterium ammoniagenes N424 was metabolically modified to isolate overproducers of deoxycytidine. Inosine auxotrophy (ino-) was initially introduced to prevent the flow of PRPP (phosphoribosyl pyrophosphate) into the purine biosynthetic pathway by random mutagenesis using N-methyl-N'-nitro-N-nitrosoguanidine. Following that, mutants possessing hydroxyurea resistance (HUr) were isolated to increase the activity of ribonucleoside diphosphate reductase, which catalyzes the reduction of ribonucleoside diphosphate to deoxyribonucleoside diphosphate. Then, in order to block the flow of dCTP into the TMP biosynthetic pathway via dUTP, thymine auxotrophy (thy-) was introduced into the mutant IH30 with ino- and HUr. The resulting mutant IM7, possessing the characteristics of ino-, HUr, and thy-, was deficient in dCTP deaminase and produced significantly higher amounts of deoxycytidine (81.3 mg/L) compared to its mother strain IH30 (6.2 mg/L). Deoxycytidine productivity was further enhanced by isolating the mutant IU19, which was resistant to 5-fluorouracil, an inhibitor of carbamoyl phosphate synthase. This enzyme catalyzed the synthesis of carbamoyl phosphate from glutamine, HCO3 -, and ATP. 5-Fluorouracil also inhibited aspartate transcarbamoylase, catalyzeing the condensation of carbamoyl phosphate and aspartate. Finally, 5-fluorocytosine resistance (FCr) was introduced into the mutant strain IU19 to relieve the repression caused by accumulation of pyrimidine nucleosides. The mutant strain IC14-C6 possessing all the five characteristics described above produced 226.3 mg/L of deoxycytidine, which was at least 2,000 fold higher compared to the wild type, and accumulated only a negligible amount of other pyrimidines under shake flask fermentation.

Journal Article

Organic Acids Associated with Saccharification of Cellulosic Wastes During Solid-State Fermentation: Noura El-Ahmady El-Naggar , Mohammed Saad El-Hersh; J. Microbiol. 2011;49(1):58-65. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0288-x

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Abstract: Saccharification of five cellulosic wastes, i.e. rice husks, wheat bran, corn cobs, wheat straw and rice straw by three cellulytic fungi, i.e. Aspergillus glaucus MN1, Aspergillus oryzae MN2 and Penicillium purpurogenum MN3, during solid-state fermentation (SSF) was laboratory studied. Rice husks, wheat bran, and corn cobs were selected as inducers of glucose production in the tested fungi. An incubation interval of 10 days was optimal for glucose production. Maximal activities of the cellulases FP-ase, CMC-ase, and β-glucosidase were detected during SSF of rice husks by P. purpurogenum; however, α-amylase activity (7.2 U/g) was comparatively reduced. Meanwhile, the productivities of FP-ase, CMC-ase, and β-glucosidase were high during SSF of rice husks by A. glaucus; however, they decreased during SSF of corn cobs by P. purpurogenum. Addition of rock phosphate (RP) (75 mg P2O5) decreased the pH of SSF media. (NH4)2SO4 was found to be less inducer of cellulytic enzymes, during SSF of rice husks by A. glaucus or A. oryzae; it also induced phytase production and solubilization of RP. The organic acids associated with saccharification of the wastes studied have also been investigated. The highest concentration of levulinic acid was detected (46.15 mg/g) during SSF of corn cobs by P. purpurogenum. Likewise, oxalic acid concentration was 43.20 mg/g during SSF of rice husks by P. purpurogenum.

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

Production of Anti-Helicobacter pylori Metabolite by the Lichen-Forming Fungus Nephromopsis pallescens: Heng Luo , Yoshikazu Yamamoto , Hae-Sook Jeon , Yan Peng Liu , Jae Sung Jung , Young Jin Koh , Jae-Seoun Hur; J. Microbiol. 2011;49(1):66-70. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0289-9

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Abstract: The present study was conducted to evaluate the antibacterial activity of lichen-forming fungi (LFF) against Helicobacter pylori, and to optimize the culture conditions of LFF for maximum production of natural antibiotics against H. pylori. To accomplish this, a screening assay was first conducted among 19 species of LFF. The extract of Nephromopsis pallescens (KOLRI-040516) exhibited the strongest anti-H. pylori activity. Bioautograghic TLC and HPLC analysis identified usnic acid as the main antibacterial substance produced by N. pallescens. The growth of N. pallescens and production of antibacterial substances produced by the fungus were then investigated under several culture conditions including the culture media, initial medium pHs, incubation temperatures, and the degree of aeration. The results indicated that culture in MY medium with an initial pH of 6.0, a temperature of 15°C and a low degree of aeration supported the largest usnic acid production of the fungus (16.4 μg usnic acid/g dry biomass). Especially, aeration was found to be an important factor that affect both growth and usnic acid production of N. pallescens.

Effect of Light and Reductones on Differentiation of Pleurotus ostreatus: Seung-Rock Lee , Woo-Jeong Joo , Yong-Un Baek , Inyoung Kim , Kee-Oh Chay , Seung-Hyun Cho , Seung-Jae Lee , Sa-Ouk Kang; J. Microbiol. 2011;49(1):71-77. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0507-5

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Abstract: Vegetative mycelia of Pleurotus ostreatus were differentiated into primordia and subsequently into fruit bodies in synthetic sucrose-asparagine medium when exposed to light at low temperature. During photomorphogenesis, L-ascorbic acid-like substances called reductones were produced. L-Ascorbic acid, D-erythroascorbic acid, 5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid, 5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid, 5-methyl-5-O-(α-D-glucopyranosyl)-D-erythroascorbic acid and 5-methyl-5-O-(α-D-xylopyranosyl)-D-erythroascorbic acid were accumulated initially in the illuminated mycelia before the initiation of fruiting. The content of glycosides of erythroascorbic acid and their methylated compounds increased again in the primordia and the fruit bodies. Exogenous L-ascorbic acid induced the formation of primordia from the mycelia in the dark in a dose-dependent manner. Thus, this suggests that these reductones might play a role in mediating the light stimulus in photomorphogenesis.

Research Support, N.I.H., Extramural

Phenotypes Associated with Saccharomyces cerevisiae Hug1 Protein, a Putative Negative Regulator of dNTP Levels, Reveal Similarities and Differences with Sequence-Related Dif1: Eunmi Kim# , Wolfram Siede; J. Microbiol. 2011;49(1):78-85. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0200-8

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Abstract: Saccharomyces cerevisiae Hug1 is a small protein of unknown function that is highly inducible following replication stress and DNA damage. Its deletion suppresses the lethality of deletion of checkpoint kinase Mec1. Although DNA damage responses were largely normal in the HUG1 deletion mutant, we found enhanced resistance towards heat in logarithmic phase. In response to simultaneous carbon and replication stress, overall growth delay and less pseudohyphal filament formation were evident. These novel phenotypes are shared with deletion mutants of the negative regulators of ribonucleotide reductase, Dif1 and Sml1. Microarray analysis showed the influence of Hug1 on the expression of a large number of transcripts, including stress-related transcripts. Elevated dNTP levels in hug1Δ cells may result in a stress response reflected by the observed phenotypes and transcript profiles. However, in contrast to a deletion of structurally related Dif1, Rnr2-Rnr4 subcellular localization is not grossly altered in a Hug1 deletion mutant. Thus, although Hug1 appears to be derived from the Rnr2-Rnr4 binding region of Dif1, its mechanism of action must be independent of determining the localization of Rnr2-Rnr4.

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

Characterization, Gene Cloning, and Heterologous Expression of β-Mannanase from a Thermophilic Bacillus subtilis: Pijug Summpunn , Suttidarak Chaijan , Duangnate Isarangkul , Suthep Wiyakrutta , Vithaya Meevootisom; J. Microbiol. 2011;49(1):86-93. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0357-1

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Abstract

Bacillus subtilis BCC41051 producing a thermostable β-mannanase was isolated from soybean meal-enriched soil and was unexpectedly found to be thermophilic in nature. The extracellular β-mannanase (ManA) produced was hydrophilic, as it was not precipitated even with ammonium sulfate at 80% saturation. The estimated molecular weight of ManA was 38.0 kDa by SDS-PAGE with a pI value of 5.3. Optimal pH and temperature for mannan-hydrolyzing activity was 7.0 and 60°C, respectively. The enzyme was stable over a pH range of 5.0-11.5, and at temperatures of up to 60°C for 30 min, with more than 80% of its activity retained. ManA was strongly inhibited by Hg2+ (1 mM), but was sensitive to other divalent ions to a lesser degree. The gene of ManA encoded a protein of 362 amino acid residues, with the first 26 residues identified as a signal peptide. High expression of recombinant ManA was achieved in both Escherichia coli BL21 (DE3) (415.18 U/ml) and B. megaterium UNcat (359 U/ml).

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Fine Mapping of a Foot-and-Mouth Disease Virus Epitope Recognized by Serotype-Independent Monoclonal Antibody 4B2: Yongzhong Yu , Haiwei Wang , Lei Zhao , Chunyuan Zhang , Zhigang Jiang , Li Yu; J. Microbiol. 2011;49(1):94-101. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-0134-1

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Abstract: VP2 is a structural protein of the foot-and-mouth disease virus (FMDV). In this study, a FMDV serotype-independent monoclonal antibody (MAb), 4B2, was generated. By screening a phage-displayed random 12-peptide library, we found positive phages displaying the consensus motif ETTXLE (X is any amino acid (aa)), which is highly homologous to 6ETTLLE11 at the N-terminus of the VP2 protein. Subsequently, a series of GST-fusion proteins expressing a truncated N-terminus of VP2 were examined by western blot analysis using the MAb 4B2. The results indicated that the motif 6ETTLLE11 of VP2 may be the minimal requirement of the epitope recognized by 4B2. Moreover, a 12-aa peptide 2KKTEETTLLEDR13 was shown to be the minimal unit of the epitope with maximal binding activity to 4B2. Alanine-scanning analysis demonstrated thatThr7, Thr8, and Leu10 are the functional residues of the 4B2 epitope Glu6 and Leu9 are required residues, and Glu11 plays a crucial role in the binding of MAb 4B2. The fine mapping of the epitope indicated that MAb 4B2 has the potential to be used in FMDV diagnosis.

In Vitro Antiviral Activity of Red Alga, Polysiphonia morrowii Extract and Its Bromophenols Against Fish Pathogenic Infectious Hematopoietic Necrosis Virus and Infectious Pancreatic Necrosis Virus: Su-Yeun Kim , Seok Ryel Kim# , Myung-Joo Oh , Sung-Ju Jung , So Young Kang; J. Microbiol. 2011;49(1):102-106. Published online March 3, 2011; DOI: https://doi.org/10.1007/s12275-011-1035-z

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Abstract

Our previous investigation revealed that 80% methanolic extract of the red alga Polysiphonia morrowii has significant antiviral activities against fish pathogenic viruses, infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV). The present study was conducted to identify compounds attributed for its antiviral activities and investigate their antiviral activities against IHNV and IPNV. Activityguided fractionation for 80% methanolic extract of Polysiphonia morrowii using a cell-based assay measuring virus-induced cytopathic effect (CPE) on cells yielded a 90% methanolic fraction, which showed the highest antiviral activity against both viruses among fractions yielded from the extract. From the fraction, two bromophenols were isolated and identified as 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) based on spectroscopic analyses. For both compounds, the concentrations to inhibit 50% of flounder spleen cell (FSP cell) proliferation (CC50) and each viral replication (EC50) were measured. In the pretreatment test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) and 3-bromo-4,5-dihydroxybenzaldehyde (2) exhibited significant antiviral activities showing selective index values (SI = CC50/EC50) of 20 to 42 against both IHNV and IPNV. In direct virucidal test, 3-bromo-4,5-dihydroxybenzyl methyl ether (1) showed significant antiviral activites against both viruses while 3-bromo-4,5-dihydroxybenzaldehyde (2) was significantly effective against only IHNV. Although antiviral efficacies of both compounds against IHNV and IPNV were lower than those of ribavirin used as a positive control, our findings suggested that the red alga Polysiphonia morrowii and isolated two bromophenols may have potential as a therapeutic agent against fish viral diseases.

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