Journal of Microbiology

Volume 51(4); August 2013

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

Molecular Detection and Genotyping of Fusarium oxysporum f. sp. psidii Isolates from Different Agro-Ecological Regions of India: Rupesh Kumar Mishra , Brajesh Kumar Pandey , Vijai Singh , Amita John Mathew , Neelam Pathak , Mohammad Zeeshan; J. Microbiol. 2013;51(4):405-412. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2638-3

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11 Citations

Abstract: Twenty one isolates of Fusarium oxysporum f. sp. psidii (Fop), causing a vascular wilt in guava (Psidium guajava L.), were collected from different agro-ecological regions of India. The pathogenicity test was performed in guava seedlings, where the Fop isolates were found to be highly pathogenic. All 21 isolates were confirmed as F. oxysporum f. sp. psidii by a newly developed, species-specific primer against the conserved regions of 28S rDNA and the intergenic spacer region. RAPD and PCR-RFLP were used for genotyping the isolates to determine their genetic relationships. Fifteen RAPD primers were tested, of which five primers produced prominent, polymorphic, and reproducible bands. RAPD yielded an average of 6.5 polymorphic bands per primer, with the amplified DNA fragments ranging from 200–2,000 bp in size. A dendrogram constructed from these data indicated a 22–74% level of homology. In RFLP analysis, two major bands (350 and 220 bp) were commonly present in all isolates of F. oxysporum. These findings provide new insight for rapid, specific, and sensitive disease diagnosis. However, genotyping could be useful in strain-level discrimination of isolates from different agro-ecological regions of India.

Journal Article

Microbial Community Analysis of a Coastal Hot Spring in Kagoshima, Japan, Using Molecular- and Culture-based Approaches: Minako Nishiyama , Shuichi Yamamoto , Norio Kurosawa; J. Microbiol. 2013;51(4):413-422. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2419-z

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19 Citations

Abstract: Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.

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

Multiple Gene Genealogical Analyses of a Nematophagous Fungus Paecilomyces lilacinus from China: Juan Li , Heng Li , Xiaoxu Bi , Ke-Qin Zhang; J. Microbiol. 2013;51(4):423-429. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2599-6

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Abstract: Paecilomyces lilacinus is a geographically widespread nematophagous fungus and a promising biological control agent against plant parasitic nematodes. However, relatively little is known about its patterns of genetic variation through its broad geographic and ecological contexts. In this study, we analyzed the genetic variation of 2 virulence-associated genes (PLS and PLC) and 4 housekeeping gene fragments (ITS, RPB1, RPB2, and β-tubulin) among 80 P. lilacinus specimens collected from 7 locations in China. Various degrees of polymorphism and haplotype diversity were observed among the six gene fragments. However, no genetic differentiation was observed among the geographic populations, consistent with extensive gene flow among these geographic populations of P. lilacinus in China. Our analysis also suggested that clonal reproduction was the predominant mode of reproduction in natural populations of P. lilacinus.

Bacterial and Fungal Diversity in the Starter Production Process of Fen Liquor, a Traditional Chinese Liquor: Xiao-Ran Li , En-Bo Ma , Liang-Zhen Yan , Han Meng , Xiao-Wei Du , Zhe-Xue Quan; J. Microbiol. 2013;51(4):430-438. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2640-9

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55 Citations

Abstract: Fermented foods and beverages are important parts of human diet. Fen liquor, a Chinese liquor is a fermented beverage that uses a traditional fermentation process. Starters are the main microbial source and also provide nutrients for microorganisms during fermentation. In this study, starters of Fen liquor were produced through a complex traditional fermentation process. To investigate the community structure and the composition of microorganisms in the starter production process, bacterial 16S rRNA and fungal internal transcribed spacer (ITS) regions were sequenced using clone libraries and pyrosequencing, respectively. There was much higher diversity among the bacteria than among the fungi in the starter production process. Bacteria on the surface of the starters belonged mostly to the Lactobacillaceae family, while members of the Bacillacae family were dominant in the interior of the samples that lacked access to air and water. In the fungi population, diversity was high only in the raw material. In all other samples, nearly all of the fungal sequences were from Pichia kudriavzevii, a member of the Saccharomycetaceae family. Nearly all samples showed similar fungal community structures, indicating that there was little change in the fungal community. To the best of our knowledge, this is the first report to reveal the whole process of the starter production of Chinese traditional liquor. The findings obtained in this study provide new insights into understanding the composition of the microbial community during the traditional Chinese liquor starter production process and information about the production process control and monitoring.

Bacterial Diversity in the Mountains of South-West China: Climate Dominates Over Soil Parameters: Dharmesh Singh , Lingling Shi , Jonathan M. Adams; J. Microbiol. 2013;51(4):439-447. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2446-9

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43 Citations

Abstract: Certain patterns in soil bacterial diversity and community composition have become evident from metagenomics studies on a range of scales, from various parts of the world. For example, soil pH has generally been seen as dominating variation in bacterial diversity, above all other soil and climate parameters. It is important however to test the generality of these relationships by studying previously unsampled areas. We compared soil bacterial diversity and community composition under a wide range of climatic and edaphic conditions in mountainous Yunnan Province, SW China. Soil samples were taken from a range of primary forest types and altitudes, reflecting the great variation of forest environments in this region. From each soil sample, DNA was extracted and pyrosequenced for bacterial 16S rRNA gene identification. In contrast to other recent studies from other parts of the world, pH was a weaker predictor of bacterial community composition and diversity than exchangeable Ca2+ concentration, and also the more poorly defined >environmental parameter of elevation. Samples from within each forest type clustered strongly, showing the distinctive pattern of their microbial communities on a regional scale. It is clear that on a regional scale in a very heterogeneous environment, additional factors beyond pH can emerge as more important in determining bacterial diversity.

Phenotypic and Genotypic Analysis of Clarithromycin-Resistant Helicobacter pylori from Bogotá D.C., Colombia: Alba A. Trespalacios , William Otero , Jorge E. Caminos , Marcela M. Mercado , Jenny Ávila , Liliana E. Rosero , Azucena Arévalo , Raúl A. Poutou-Piñales , David Y. Graham; J. Microbiol. 2013;51(4):448-452. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2465-6

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22 Citations

Abstract: Resistance of Helicobacter pylori to clarithromycin is the most common cause of treatment failure in patients with H. pylori infections. This study describes the MICs and the presence of 23S rRNA mutations of H. pylori isolates from Bogotá, D.C., Colombia. H. pylori were isolated from gastric biopsies from patients with functional dyspepsia. Clarithromycin susceptibility was investigated by agar dilution and strains were considered resistant if the MIC was ≥1 μg/ml. DNA sequences of the 23S rRNA gene of strains resistant and sensitive to clarithromycin were determined to identify specific point mutations. Clarithromycin resistance was present in 13.6% of patients by agar dilution. The A2143G, A2142G and A2142C mutations were found in 90.5, 7.1, and 2.4% of H. pylori strains with resistance genotype.The resistant phenotype was associated with 23S rRNA resistance genotype in 85.7% of isolates. The point mutations in 23S rRNA were well correlated with MICs values for clarithromycin.

Screening and Identification of ClpE Interaction Proteins in Streptococcus pneumoniae by a Bacterial Two-Hybrid System and Co-immunoprecipitation: WenJuan Yan , YingYing Cai , Qun Zhang , YuSi Liu , WenChun Xu , YiBing Yin , YuJuan He , Hong Wang , XueMei Zhang; J. Microbiol. 2013;51(4):453-460. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3001-4

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Abstract: Hsp100/Clp proteins have crucial functions in the protein quality control, stress tolerance, and virulence of many pathogenic bacteria. ClpE is an important virulence factor involved in adherence and invasion in Streptococcus pneumoniae. To explore the underlying mechanism, we screened ClpE interaction proteins using a bacterial two-hybrid system and co-immunoprecipitation. We used ClpE as bait and constructed the pBT-ClpE bait plasmid for two-hybrid screening. Then, we constructed ClpE::GFP fusion for co-immunoprecipitation screening using anti-GFP monoclonal antibody. We obtained eight potential ClpE interaction proteins, including carbamoyl-phosphate synthase, pyruvate oxidase (SpxB), phosphoenolpyruvate-protein phosphotransferase, aminopeptidase N (pepN), L-lactate dehydrogenase, ribosomal protein S4, sensor histidine kinase (SPD_2019), and FtsW (a cell division protein). FtsW, SpxB, pepN, and SPD_2019 were confirmed to interact with ClpE using Bacterial Two-hybrid or Co-immunoprecipitation. Morphologic observations found that ΔclpE strain existed in abnormal division. β-Galactosidase activity assay suggested that ClpE contributed to the degradation of FtsW. Furthermore, FtsW could be induced by heat shock. The results suggested that ClpE might affect cell division by regulating the level of FtsW. These data may provide new insights in studying the role of ClpE in S. pneumoniae.

The Pectate Lyase Encoded by the pecCl1 Gene Is an Important Determinant for the Aggressiveness of Colletotrichum lindemuthianum: Andréia Cnossen-Fassoni , Denise Mara Soares Bazzolli , Sérgio Hermínio Brommonschenkel , Elza Fernandes de Araújo , Marisa Vieira de Queiroz; J. Microbiol. 2013;51(4):461-470. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3078-9

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14 Citations

Abstract: Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

Journal Articles

Effect of pH on Conjugated Linoleic Acid (CLA) Formation of Linolenic Acid Biohydrogenation by Ruminal Microorganisms: Yongjae Lee; J. Microbiol. 2013;51(4):471-476. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-1070-z

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11 Citations

Abstract: Conventional beliefs surrounding the linolenic acid (LNA; cis-9 cis-12 cis-15 C18:3) biohydrogenation (BH) pathway propose that it converts to stearic acid (SA) without the formation of conjugated linoleic acid (CLA) as intermediate isomers. However, an advanced study (Lee and Jenkins, 2011) verified that LNA BH yields multiple CLAs. This study utilized the stable isotope tracer to investigate the BH intermediates of 13C-LNA with different pH conditions (5.5 and 6.5). The 13C enrichment was calculated as a 13C/12C ratio of labeled minus unlabeled. After 24 h, eight CLA isomers were significantly enriched on both pH treatment, this result verifies that these CLAs originated from 13C-LNA BH which supports the results of Lee and Jenkins (2011). The enrichment of cis-cis double bond CLAs (cis-9 cis-11 and cis-10 cis-12 CLA) were significantly higher at low pH conditions. Furthermore, the concentration of cis-10 cis-12 CLA at low pH was four times higher than at high pH conditions after a 3 h incubation. These differences support the LNA BH pathways partial switch under different pH conditions, with a strong influence on the cis-cis CLA at low pH. Several mono-, di-, and tri-enoic fatty acid isomers were enriched during 24 h of incubation, but the enrichment was decreased or restricted at low pH treatment. Based on these results, it is proposed that low pH conditions may cause a changed or limited capacity of the isomerization and reduction steps in BH.

Fumigant Activity of Volatiles from Streptomyces alboflavus TD-1 against Fusarium moniliforme Sheldon: Zhifang Wang , Changlu Wang , Fengjuan Li , Zhenjing Li , Mianhua Chen , Yurong Wang , Xi Qiao , Hong Zhang; J. Microbiol. 2013;51(4):477-483. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-2586-y

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35 Citations

Abstract: The fumigant activity of volatiles generated by Streptomyces alboflavus TD-1 against Fusarium moniliforme Sheldon was investigated. The results showed that the mycelial growth, sporulation, and spore germination of F. moniliforme were significantly suppressed, and that membrane permeability was disrupted in the presence of the volatiles. Gas chromatography-mass Spectrometry analysis revealed 31 kinds of volatile organic compound from the volatiles. Among them, two earthy-smelling substances, namely, 2-methylisoborneol (50.97%) and trans-1,10-dimethyl-trans-9-decalinol (3.10%) were found. The most abundant compound, 2-methylisoborneol, exhibited inhibitory activity against F. moniliforme by fumigation. All these results suggested that S. alboflavus TD-1 can be a promising starter for the inhibition of F. moniliforme through fumigant action.

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

Antimicrobial Effects of Herbal Extracts on Streptococcus mutans and Normal Oral Streptococci: Sung-Hoon Lee; J. Microbiol. 2013;51(4):484-489. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3312-5

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18 Citations

Abstract: Streptococcus mutans is associated with dental caries. A cariogenic biofilm, in particular, has been studied extensively for its role in the formation of dental caries. Herbal extracts such as Cudrania tricuspidata, Sophora flavescens, Ginkgo biloba, and Betula Schmidtii have been used as a folk remedy for treating diseases. The purpose of this study was to evaluate and compare the antibacterial activity of herbal extracts against normal oral streptococci, planktonic and biofilm of S. mutans. Streptococcus gordonii, Streptococcus oralis, Streptococcus salivarius, Streptococcus sanguinis, and S. mutans were cultivated with brain heart infusion broth and susceptibility assay for the herbal extracts was performed according to the protocol of Clinical and Laboratory Standard Institute. Also, S. mutans biofilm was formed on a polystyrene 12-well plate and 8-well chamber glass slip using BHI broth containing 2% sucrose and 1% mannose after conditioning the plate and the glass slip with unstimulated saliva. The biofilm was treated with the herbal extracts in various concentrations and inoculated on Mitis-Salivarius bacitracin agar plate for enumeration of viable S. mutans by counting colony forming units. Planktonic S. mutans showed susceptibility to all of the extracts and S. mutans biofilm exhibited the highest level of sensitivity for the extracts of S. flavescens. The normal oral streptococci exhibited a weak susceptibility in comparison to S. mutans. S. oralis, however, was resistant to all of the extracts. In conclusion, the extract of S. flavescens may be a potential candidate for prevention and management of dental caries.

Dysregulation of KSHV Replication by Extracts from Carthamus tinctorius L.: Han Lee , Hyosun Cho , Myoungki Son , Gi-Ho Sung , Taeho Lee , Sang-Won Lee , Yong Woo Jung , Yu Su Shin , Hyojeung Kang; J. Microbiol. 2013;51(4):490-498. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3282-7

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Abstract: Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.

The N3 Subdomain in A Domain of Fibronectin-Binding Protein B Isotype I Is an Independent Risk Determinant Predictive for Biofilm Formation of Staphylococcus aureus Clinical Isolates: An Sung Kwon , Dong Hoon Lim , Hyo Jung Shin , Geon Park , Jong H. Reu , Hyo Jin Park , Jungmin Kim , Yong Lim; J. Microbiol. 2013;51(4):499-505. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3319-y

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8 Citations

Abstract: Fibronectin-binding proteins (FnBP), FnBPA and FnBPB, are purported to be involved in biofilm formation of Staphylococcus aureus. This study was performed to find which of three consecutive N subdomains of the A domain in the FnBP is the key domain in FnBP. A total of 465 clinical isolates of S. aureus were examined for the biofilm forming capacity and the presence of N subdomains of FnBP. In the biofilm-positive strains, N2 and N3 subdomains of FnBPA, and N1 and N3 subdomains of FnBPB were significantly more prevalent. Multivariate logistic regression analysis of 246 biofilm-positive and 123 biofilm-negative strains identified only the FnBPB-N3 subdomain as an independent risk determinant predictive for biofilm-positive strains of S. aureus (Odds ratio [OR], 13.174; P<0.001). We also attempted to delete each of the fnbA-N2 and -N3 and fnbB-N1 and -N3 from S. aureus strain 8325-4 and examined the biofilm forming capacity in the derivative mutants. In agreement with the results of the multivariate regression analysis, deletion of either the fnbA-N2 or -N3, or fnbB-N1 did not significantly diminish the capacity of strain 8325-4 to develop a biofilm, while deletion of the fnbB-N3 did. Therefore, it is suggested that the FnBPB-N3 subdomain of isotype I may be a key domain in FnBP which is responsible for the causing biofilm formation in S. aureus clinical isolates.

Cell-Surface Expression of Aspergillus saitoi-Derived Functional α-1,2-Mannosidase on Yarrowia lipolytica for Glycan Remodeling: Hye Yun Moon , Trinh Luu Van , Seon Ah Cheon , Jinho Choo , Jeong-Yoon Kim , Hyun Ah Kang; J. Microbiol. 2013;51(4):506-514. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3344-x

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16 Citations

Abstract: Expression of proteins on the surface of yeast has a wide range of applications, such as development of live vaccines, screening of antibody libraries, and use as whole-cell biocatalysts. The hemiascomycetes yeast Yarrowia lipolytica has been raised as a potential host for heterologous expression of recombinant proteins. In this study, we report the expression of Aspergillus saitoi α-1,2-mannosidase, encoded by the msdS gene, on the cell surface of Y. lipolytica. As the first step to achieve the secretory expression of msdS protein, four different signal sequences-derived from the endogenous Y. lipolytica Lip2 and Xpr2 prepro regions and the heterologous A. niger α-amylase and rice α-amylase signal sequences-were analyzed for their secretion efficiency. It was shown that the YlLip2 prepro sequence was most efficient in directing the secretory expression of msdS in fully N-glycosylated forms. The surface display of msdS was subsequently directed by fusing GPI anchoring motifs derived from Y. lipolytica cell wall proteins, YlCwp1p and YlYwp1p, respectively, to the C-terminus of the Lip2 prepro-msdS protein. The expression of actively functional msdS protein on the cell surface was confirmed by western blot, flow cytometry analysis, along with the α-1,2-mannosidase activity assay using intact Y. lipolytica cells as the enzyme source. Furthermore, the glycoengineered Y. lipolytica Δoch1Δmpo1 strains displaying α-1,2-mannosidase were able to convert Man8GlcNAc2 to Man5GlcNAc2 efficiently on their cell-wall mannoproteins, demonstrating its potential used for glycoengineering in vitro or in vivo.

Functional Characterization of Autographa californica Multiple Nucleopolyhedrovirus ORF43 and Phenotypic Changes of ORF43-Knockout Mutant: Xue Ying Tao , Jae Young Choi , Yong Wang , Jong Yul Roh , Joo Hyun Lee , Qin Liu , Jong Bin Park , Jae Su Kim , Woojin Kim , Yeon Ho Je; J. Microbiol. 2013;51(4):515-521. Published online August 30, 2013; DOI: https://doi.org/10.1007/s12275-013-3058-0

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7 Citations

Abstract: ORF43 (ac43) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a highly conserved baculovirus gene of unknown function. To investigate the role of ac43 in the baculovirus lifecycle, we constructed an ac43-deleted mutant AcMNPV, Ac43KO. After transfection into Spodoptera frugiperda cells, Ac43KO produced polyhedra much larger in size than those of wild-type AcMNPV. Interestingly, some of the nucleocapsids were singly enveloped in the polyhedrin matrix while the nucleocapsids of AcMNPV are known to be multiply enveloped. Furthermore, Ac43KO led to a defect in the transcription and expression of polyhedrin, which resulted in reduced occlusion body production. However, Ac43KO did not affect production of budded virus as there was no remarkable difference in budded virus titer. These results suggest that ac43 plays an important role in the expression of polyhedrin, the morphogenesis of occlusion body, and the assembly of virions occluded in occlusion bodies.

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