Journal of Microbiology

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Fleagrass (Adenosma buchneroides Bonati) Acts as a Fungicide Against Candida albicans by Damaging Its Cell Wall.: Youwei Wu, Hongxia Zhang, Hongjie Chen, Zhizhi Du, Qin Li, Ruirui Wang; J. Microbiol. 2024;62(8):661-670. Published online July 3, 2024; DOI: https://doi.org/10.1007/s12275-024-00146-9

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Abstract: Fleagrass, a herb known for its pleasant aroma, is widely used as a mosquito repellent, antibacterial agent, and for treating colds, reducing swelling, and alleviating pain. The antifungal effects of the essential oils of fleagrass and carvacrol against Candida albicans were investigated by evaluating the growth and the mycelial and biofilm development of C. albicans. Transmission electron microscopy was used to evaluate the integrity of the cell membrane and cell wall of C. albicans. Fleagrass exhibited high fungicidal activity against C. albicans at concentrations of 0.5% v/v (via the Ras1/cAMP/PKA pathway). Furthermore, transmission electron microscopy revealed damage to the cell wall and membrane after treatment with the essential oil, which was further confirmed by the increased levels of β-1,3-glucan and chitin in the cell wall. This study showed that fleagrass exerts good fungicidal and hyphal growth inhibition activity against C. albicans by disrupting its cell wall, and thus, fleagrass may be a potential antifungal drug.

Analysis of phylogenetic markers for classification of a hydrogen peroxide producing Streptococcus oralis isolated from saliva by a newly devised differential medium: Ha Pham , Thi Dieu Thuy Tran , Youri Yang , Jae-Hyung Ahn , Hor-Gil Hur , Yong-Hak Kim; J. Microbiol. 2022;60(8):795-805. Published online July 14, 2022; DOI: https://doi.org/10.1007/s12275-022-2261-2

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Abstract: Hydrogen peroxide (H2O2) is produced by alpha-hemolytic streptococci in aerobic conditions. However, the suitable method for detection of H2O2-producing streptococci in oral microbiota has not been setup. Here we show that o-dianisidine dye and horseradish peroxidase were useful in tryptic soy agar medium to detect and isolate H2O2-producing bacteria with the detection limit of one target colony in > 106 colony-forming units. As a proof, we isolated the strain HP01 (KCTC 21190) from a saliva sample using the medium and analyzed its characteristics. Further tests showed that the strain HP01 belongs to Streptococcus oralis in the Mitis group and characteristically forms short-chain streptococcal cells with a high capacity of acid tolerance and biofilm formation. The genome analysis revealed divergence of the strain HP01 from the type strains of S. oralis. They showed distinctive phylogenetic distances in their ROS-scavenging proteins, including superoxide dismutase SodA, thioredoxin TrxA, thioredoxin reductase TrxB, thioredoxin-like protein YtpP, and glutaredoxin- like protein NrdH, as well as a large number of antimicrobial resistance genes and horizontally transferred genes. The concatenated ROS-scavenging protein sequence can be used to identify and evaluate Streptococcus species and subspecies based on phylogenetic analysis.

Regulation of iron homeostasis by peroxide-sensitive CatR, a Fur-family regulator in Streptomyces coelicolor: Yeonbum Kim , Jung-Hye Roe , Joo-Hong Park , Yong-Joon Cho , Kang-Lok Lee; J. Microbiol. 2021;59(12):1083-1091. Published online December 4, 2021; DOI: https://doi.org/10.1007/s12275-021-1457-1

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Abstract: CatR, a peroxide-sensing transcriptional repressor of Fur family, can de-repress the transcription of the catA gene encoding catalase upon peroxide stress in Streptomyces coelicolor. Since CatR-regulated genes other than catA and its own gene catR have not been identified in detail, the understanding of the role of CatR regulon is very limited. In this study, we performed transcriptomic analysis to identify genes influenced by both 􀈟􀂊atR mutation and hydrogen peroxide treatment. Through ChIP-qPCR and other analyses, a new consensus sequence was found in CatR-responsive promoter region of catR gene and catA operon for direct regulation. In addition, vtlA (SCO2027) and SCO4983 were identified as new members of the CatR regulon. Expression levels of iron uptake genes were reduced by hydrogen peroxide and a DmdR1 binding sequence was identified in promoters of these genes. The increase in free iron by hydrogen peroxide was thought to suppress the iron import system by DmdR1. A putative exporter protein VtlA regulated by CatR appeared to reduce intracellular iron to prevent oxidative stress. The name vtlA (VIT1-like transporter) was proposed for iron homeostasis related gene SCO2027.

The transcription factor Cas5 suppresses hyphal morphogenesis during yeast-form growth in Candida albicans: Jong-Myeong Kim , Hye Yun Moon , Dong Wook Lee , Hyun Ah Kang , Jeong-Yoon Kim; J. Microbiol. 2021;59(10):911-919. Published online September 7, 2021; DOI: https://doi.org/10.1007/s12275-021-1326-y

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Abstract: Candida albicans is an opportunistic human pathogen that exists as yeast, hyphal or pseudohyphal forms depending on pH, nutrients, and temperature. The morphological transition from yeast to hyphae, which is required for the complete virulence of C. albicans, is controlled by many transcription factors that activate or repress hypha-specific genes. The C. albicans transcriptional factor Cas5, a key regulator of genes involved in cell wall integrity, affects the susceptibility of C. albicans to fluconazole, an inhibitor of ergosterol synthesis. In this study, we found that deletion of CAS5 in C. albicans decreased the expression levels of a set of ergosterol biosynthesis genes, such as ERG2, ERG3, ERG5, ERG6, ERG11, and ERG24,
result
ing in the accumulation of lanosterol and zymosterol, which are intermediate metabolites in the ergosterol biosynthesis pathway. Interestingly, it was observed that the cas5Δ/Δ mutant could not maintain the yeast form under non-hyphainducing conditions, while the CAS5-overexpressing cells could not form hyphae under hypha-inducing conditions. Consistent with these observations, the cas5Δ/Δ mutant highly expressed hypha-specific genes, ALS3, ECE1, and HWP1, under non-hypha-inducing conditions. In addition, CAS5 transcription was significantly downregulated immediately after hyphal initiation in the wild-type strain. Furthermore, the cas5Δ/Δ mutant reduced the transcription of NRG1, which encodes a major repressor of hyphal morphogenesis, while Cas5 overexpression increased the transcription of NRG1 under hyphainducing conditions. Collectively, this study suggests the potential role of Cas5 as a repressor of hypha-specific genes during yeast-form growth of C. albicans.

Biosynthesis of adipic acid in metabolically engineered Saccharomyces cerevisiae: Xi Zhang , Yingli Liu , Jing Wang , Yunying Zhao , Yu Deng; J. Microbiol. 2020;58(12):1065-1075. Published online October 23, 2020; DOI: https://doi.org/10.1007/s12275-020-0261-7

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Abstract: Adipic Acid (AA) is a valued platform chemical compound, which can be used as a precursor of nylon-6,6. Due to the generation of an enormous amount of nitric oxide metabolites and the growing depletion of oil resources as a result of AA production from a mixture of cyclohexanol and cyclohexanone, the microbial methods for synthesizing AA have attracted significant attention. Of the several AA-producing pathways, the reverse adipate degradation pathway in Thermobifida fusca (Tfu RADP) is reported to be the most efficient, which has been confirmed in Escherichia coli. In this study, the heterologous Tfu RADP was constructed for producing AA in S. cerevisiae by co-expressing genes of Tfu_ 0875, Tfu_2399, Tfu_0067, Tfu_1647, Tfu_2576, and Tfu_ 2576. The AA titer combined with biomass, cofactors and other by-products was all determined after fermentation. During batch fermentation in a shake flask, the maximum AA titer was 3.83 mg/L, while the titer increased to 10.09 mg/L during fed-batch fermentation in a 5-L bioreactor after fermentation modification.

Identification and characterization of a novel light-induced promoter for recombinant protein production in Pleurotus ostreatus: Chaomin Yin , Xiuzhi Fan , Kun Ma , Zheya Chen , Defang Shi , Fen Yao , Hong Gao , Aimin Ma; J. Microbiol. 2020;58(1):39-45. Published online November 4, 2019; DOI: https://doi.org/10.1007/s12275-020-9230-4

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Abstract: A lectin gene (plectin) with a high level of expression was previously identified by comparative transcriptome analysis of Pleurotus ostreatus. In this study, we cloned a 733-bp DNA fragment from the start codon of the plectin gene. Sequence analysis showed that the plectin promoter (Plp) region contained several eukaryotic transcription factor binding motifs, such as the TATA-box, four possible CAAT-box, light responsiveness motifs and MeJA-responsiveness motifs. To determine whether the Plp promoter was a light-regulated promoter, we constructed an expression vector with the fused egfp-hph fragment under the control of the Plp promoter and transformed P. ostreatus mycelia via Agrobacterium tumefaciens. PCR and Southern blot analyses confirmed the Plpegfp- hph fragment was integrated into the chromosomal DNA of transformants. qRT-PCR, egfp visualization, and intracellular egfp determination experiments showed the Plp promoter could be a light-induced promoter that may be suitable for P. ostreatus genetic engineering. This study lays the foundation for gene homologous expression in P. ostreatus.

Competition/antagonism associations of biofilm formation among Staphylococcus epidermidis Agr groups I, II, and III: Sergio Martínez-García , César I. Ortiz-García , Marisa Cruz-Aguilar , Juan Carlos Zenteno , José Martin Murrieta-Coxca , Sonia Mayra Pérez-Tapia , Sandra Rodríguez-Martínez , Mario E. Cancino-Diaz , Juan C. Cancino-Diaz; J. Microbiol. 2019;57(2):143-153. Published online January 31, 2019; DOI: https://doi.org/10.1007/s12275-019-8322-5

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Abstract: Staphylococci have quorum-sensing (QS) systems that enable cell-to-cell communication, as well as the regulation of numerous colonization and virulence factors. The accessory gene regulator (Agr) operon is one of the Staphylococcus genus QS systems. Three groups (I, II, and III) are present in Staphylococcus epidermidis Agr operon. To date, it is unknown whether Agr groups can interact symbiotically during biofilm development. This study analyzed a symbiotic association among Agr groups during biofilm formation in clinical and commensal isolates. Different combinations among Agr group isolates was used to study biofilm formation in vitro and in vivo (using a mouse catheter-infection model). The analysis of Agr groups were also performed from samples of human skin (head, armpits, and nostrils). Different predominant coexistence was found within biofilms, suggesting symbiosis type. In vitro, Agr I had a competition with Agr II and Agr III. Agr II had a competition with Agr III, and Agr II was an antagonist to Agr I and III when the three strains were combined. In vivo, Agr II had a competition to Agr I, but Agr I and II were antagonists to Agr III. The associations found in vitro and in vivo were also found in different sites of the skin. Besides, other associations were observed: Agr III antagonized Agr I and II, and Agr III competed with Agr I and Agr II. These results suggest that, in S. epidermidis, a symbiotic association of competition and antagonism occurs among different Agr groups during biofilm formation.

An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus: Guomin Han , Qian Shao , Cuiping Li , Kai Zhao , Li Jiang , Jun Fan , Haiyang Jiang , Fang Tao; J. Microbiol. 2018;56(5):356-364. Published online May 2, 2018; DOI: https://doi.org/10.1007/s12275-018-7349-3

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Abstract: Aspergillus flavus often invade many important corps and produce harmful aflatoxins both in preharvest and during storage stages. The regulation mechanism of aflatoxin biosynthesis in this fungus has not been well explored mainly due to the lack of an efficient transformation method for constructing a genome-wide gene mutant library. This challenge was resolved in this study, where a reliable and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) protocol for A. flavus NRRL 3357 was established. The results showed that removal of multinucleate conidia, to collect a homogenous sample of uninucleate conidia for use as the transformation material, is the key step in this procedure. A. tumefaciens strain AGL-1 harboring the ble gene for zeocin resistance under the control of the gpdA promoter from A. nidulans is suitable for genetic transformation of this fungus. We successfully generated A. flavus transformants with an efficiency of ~ 60 positive transformants per 106 conidia using our protocol. A small-scale insertional mutant library (~ 1,000 mutants) was constructed using this method and the resulting several mutants lacked both production of conidia and aflatoxin biosynthesis capacity. Southern blotting analysis demonstrated that the majority of the transformants contained a single T-DNA insert on the genome. To the best of our knowledge, this is the first report of genetic transformation of A. flavus via ATMT and our protocol provides an effective tool for construction of genome-wide gene mutant libraries for functional analysis of important genes in A. flavus.

Long-term organic-inorganic fertilization ensures great soil productivity and bacterial diversity after natural-to-agricultural ecosystem conversion: Weibing Xun , Zhihui Xu , Wei Li , Yi Ren , Ting Huang , Wei Ran , Boren Wang , Qirong Shen , Ruifu Zhang; J. Microbiol. 2016;54(9):611-617. Published online August 31, 2016; DOI: https://doi.org/10.1007/s12275-016-6143-3

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Abstract: Natural ecosystems comprise the planet’s wild plant and animal resources, but large tracts of land have been converted to agroecosystems to support the demand for agricultural products. This conversion limits the number of plant species and decreases the soil biological diversity. Here we used highthroughput 16S rRNA gene sequencing to evaluate the responses of soil bacterial communities in long-term converted and fertilized red soils (a type of Ferralic Cambisol). We observed that soil bacterial diversity was strongly affected by different types of fertilization management. Oligotrophic bacterial taxa demonstrated large relative abundances in chemically fertilized soil, whereas copiotrophic bacterial taxa were found in large relative abundances in organically fertilized and fallow management soils. Only organic-inorganic fertilization exhibited the same local taxonomic and phylogenetic diversity as that of a natural ecosystem. However, the independent use of organic or inorganic fertilizer reduced local taxonomic and phylogenetic diversity and caused biotic homogenization. This study demonstrated that the homogenization of bacterial communities caused by natural-to-agricultural ecosystem conversion can be mitigated by employing rational organic-inorganic fertilization managemen

Characterization of Cultivated Fungi Isolated from Grape Marc Wastes Through the Use of Amplified rDNA Restriction Analysis and Sequencing: Spyridon Ntougias , Nektarios Kavroulakis , Kalliope K. Papadopoulou , Constantinos Ehaliotis , Georgios I. Zervakis; J. Microbiol. 2010;48(3):297-306. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9193-y

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Abstract: Microbial assessment of grape marc wastes, the residual solid by-product of the wine-industry, was performed by identifying phylogenetically the fungal culturable diversity in order to evaluate environmental and disposal safety issues and to discuss ecological considerations of applications on agricultural land. Fungal spores in grape marc were estimated to 4.7×106 per g dry weight. Fifty six fungal isolates were classified into eight operational taxonomic units (OTUs) following amplified ribosomal DNA restriction analysis (ARDRA) and colony morphology. Based on 18S rRNA gene and 5.8S rRNA gene-ITS sequencing, the isolates representing OTUs #1, #2, #3, and #4, which comprised 44.6%, 26.8%, 12.5%, and 5.3%, respectively, of the number of the total isolates, were identified as Aspergillus fumigatus, Bionectria ochroleuca, Haematonectria haematococca, and Trichosporon mycotoxinivorans. The isolates of OTU#5 demonstrated high phylogenetic affinity with Penicillium spp., while members of OTUs #6 and #7 were closer linked with Geotrichum candidum var. citri-aurantii and Mycocladus corymbifer, respectively (95.4 and 97.9% similarities in respect to their 5.8S rRNA gene-ITS sequences). The OTU#8 with a single isolate was related with Aspergillus strains. It appears that most of the fungal isolates are associated with the initial raw material. Despite the fact that some of the species identified may potentially act as pathogens, measures such as the avoidance of maintaining large and unprocessed quantities of grape marc wastes in premises without adequate aeration, together with its suitable biological treatment (e.g., composting) prior to any agriculture-related application, could eliminate any pertinent health risks.

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

Panacagrimonas perspica gen. nov., sp. nov., a Novel Member of Gammaproteobacteria Isolated from Soil of a Ginseng Field: Wan-Taek Im , Qing-Mei Liu , Jung-Eun Yang , Min-Seok Kim , Se-Young Kim , Sung-Taik Lee , Tae-Hoo Yi; J. Microbiol. 2010;48(2):262-266. Published online May 1, 2010; DOI: https://doi.org/10.1007/s12275-010-0067-0

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Abstract: A taxonomic study was carried out on Gsoil 142T, a bacterial strain isolated from the soil collected in a ginseng field in Pocheon province, South Korea. Comparative 16S rRNA gene sequence studies showed a clear affiliation of this bacterium to the Gammaproteobacteria, and it was most closely related to Hydrocarboniphaga effusa ATCC BAA 332T (94.4%, 16S rRNA gene sequence similarity), Nevskia ramosa DSM 11499T (94.1%) and Alkanibacter difficilis MN154.3T (92.0%). Strain Gsoil 142T was a Gram-negative, strictly aerobic, motile, and rod-shaped bacterium. The G+C content of the genomic DNA was 69.9% and predominant ubiquinone was Q-8. Major fatty acids were summed feature 8 (C18:1 w7c and/or w6c, 36.3%), summed feature 3 (iso-C15:0 2-OH and/or C16:1 w7c, 20.6%) and C16:0 (17.4%). The major polar lipids detected in strain Gsoil 142T were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, and an unknown glycolipid. On the basis of polyphasic evidence, it is proposed that strain Gsoil 142T should be placed in a novel genus and species, for which the name Panacagrimonas perspica gen. nov., sp. nov. is proposed. The type strain is Gsoil 142T (=KCTC 12982T =LMG 23239T).

Purification and Biochemical Properties of a Glucose-Stimulated β-D-Glucosidase Produced by Humicola grisea var. thermoidea Grown on Sugarcane Bagasse: Cesar Vanderlei Nascimento , Flávio Henrique Moreira Souza , Douglas Chodi Masui , Francisco Assis Leone , Rosane Marina Peralta , João Atílio Jorge , Rosa Prazeres Melo Furriel; J. Microbiol. 2010;48(1):53-62. Published online March 11, 2010; DOI: https://doi.org/10.1007/s12275-009-0159-x

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Abstract: The effect of several carbon sources on the production of mycelial-bound β-glucosidase by Humicola grisea var. thermoidea in submerged fermentation was investigated. Maximum production occurred when cellulose was present in the culture medium, but higher specific activities were achieved with cellobiose or sugarcane bagasse. Xylose or glucose (1%) in the reaction medium stimulated β-glucosidase activity by about 2-fold in crude extracts from mycelia grown in sugarcane bagasse. The enzyme was purified by ammonium sulfate precipitation, followed by Sephadex G-200 and DEAE-cellulose chromatography, showing a single band in PAGE and SDS-PAGE. The β-glucosidase had a carbohydrate content of 43% and showed apparent molecular masses of 57 and 60 kDa, as estimated by SDS-PAGE and gel filtration, respectively. The optimal pH and temperature were 6.0 and 50°C, respectively. The purified enzyme was thermostable up to 60 min in water at 55°C and showed half-lives of 7 and 14 min when incubated in the absence or presence of 50 mM glucose, respectively, at 60°C. The enzyme hydrolyzed p-nitrophenyl-β-D-glucopyranoside, p-nitrophenyl-β-galactopyranoside, p-nitrophenyl-β-D-fucopyranoside, p-nitrophenyl-β-D-xylopyranoside, o-nitrophenyl-β-Dgalactopyranoside, lactose, and cellobiose. The best synthetic and natural substrates were p-nitrophenyl-β-Dfucopyranoside and cellobiose, respectively. Purified enzyme activity was stimulated up to 2-fold by glucose or xylose at concentrations from 25 to 200 mM. The addition of purified or crude β-glucosidase to a reaction medium containing Trichoderma reesei cellulases increased the saccharification of sugarcane bagasse by about 50%. These findings suggest that H. grisea var. thermoidea β-glucosidase has a potential for biotechnological applications in the bioconversion of lignocellulosic materials.

Characterization of Plant-Growth Promoting Diazotrophic Bacteria Isolated from Field Grown Chinese Cabbage under Different Fertilization Conditions: Woo-Jong Yim , Selvaraj Poonguzhali , Munusamy Madhaiyan , Pitchai Palaniappan , M. A. Siddikee , Tongmin Sa; J. Microbiol. 2009;47(2):147-155. Published online May 2, 2009; DOI: https://doi.org/10.1007/s12275-008-0201-4

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Abstract: Diazotrophic bacteria isolated from the rhizosphere of Chinese cabbage were assessed for other plant growth promoting characteristics viz., production of IAA, ethylene, ACC deaminase, phosphate solubilization, and gnotobiotic root elongation. Their effect on inoculation to Chinese cabbage was also observed under growth chamber conditions. A total of 19 strains that showed higher nitrogenase activity identified by 16S rRNA gene sequence analysis were found to be the members of the genera Pseudomonas and Agrobacterium belonging to α- and γ-Proteobacteria groups. These strains were also efficient in producing IAA and ACC deaminase though they produced low levels of ethylene and no phosphate solubilization. In addition, inoculation of selected diazotrophic bacterial strains significantly increased seedling length, dry weight, and total nitrogen when compared to uninoculated control. The colonization of crop plants by diazotrophic bacteria can be affected by many biotic and abiotic factors, and further studies are oriented towards investigating the factors that could influence the establishment of a selected bacterial community.

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