Journal of Microbiology

Volume 51(3); June 2013

Review

MINIREVIEW] Development of Diagnostic and Vaccine Markers Through Cloning, Expression, and Regulation of Putative Virulence-Protein-Encoding Genes of Aeromonas hydrophila: Vijai Singh , Dharmendra Kumar Chaudhary , Indra Mani , Rohan Jain , B.N. Mishra; J. Microbiol. 2013;51(3):275-282. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2437-x

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Abstract: Aeromonas hydrophila is an opportunistic bacterial pathogen that is associated with a number of diseases in fish, amphibians, reptiles, and humans. In fish it causes several disease symptoms including tail and skin rot, and haemorrhagic septicemia; in human it causes soft-tissue wound infection and diarrhoea. The pathogenesis of A. hydrophila is multifactorial, but the mechanism is unknown so far. It is considered to be mediated by expression and secretion of extracellular proteins such as aerolysin, lipase, chitinase, amylase, gelatinase, hemolysins, and enterotoxins. A number of the putative virulence-protein-encoding genes that are present in the genome of A. hydrophila have been targeted by PCR for molecular diagnosis. These significant genes are also targeted for over-production of proteins by cloning and expression methods. In this review, we emphasize recent progress in the cloning, expression, and regulation of putative virulence-protein-encoding genes of A. hydrophila for a better understanding of the pathogenesis and also help to provide effective strategies for control of diseases.

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

Flavobacterium aquaticum sp. nov., a Member of the Bacteroidetes Isolated from a Freshwater Reservoir: Siwon Lee , Jungnam Lee , Tae-Young Ahn; J. Microbiol. 2013;51(3):283-288. Published online April 26, 2013; DOI: https://doi.org/10.1007/s12275-013-2293-8

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Abstract: A novel bacterial strain, designated ARSA-111T, was isolated from a freshwater reservoir in Cheonan, Korea. Phylogenetic analysis based on 16S rRNA gene sequences suggested that the isolate belonged to the genus Flavobacterium of phylum Bacteroidetes. The 16S rRNA gene sequence of strain ARSA-111T showed a high degree of sequence similarity to those of Flavobacteium cheonanense KACC 14972T (97.3%), F. aquatile JCM 20475T (97.1%), and other type strains of the genus Flavobacterium (＜97.0%). The phylogenetic tree and network analysis (i.e. median-joining) based on 16S rRNA gene sequences showed that strain ARSA-111T is most closely related to F. aquatile JCM 20475T. DNA-DNA hybridization experiment revealed ＜70% of genomic relatedness among strain ARSA-111T, F. aquatile JCM 20475T and F. cheonanense KACC 14972T. The isolate had iso-C15:1, iso-C15:0, and iso-C15:0 3-OH as predominant cellular fatty acids and MK-6 as a predominant menaquinone. The genomic DNA G+C content of the isolate was 35.6 mol%. On the basis of these data, strain ARSA-111T is considered to be a novel species of the genus Flavobacterium, for which the name Flavobacterium aquaticum sp. nov. is proposed. The type strain is strain ARSA-111T (=KACC 14973T =KCTC 23185T = JCM 17070T).

Lysinibacillus tabacifolii sp. nov., a Novel Endophytic Bacterium Isolated from Nicotiana tabacum Leaves: Yan-Qing Duan , Song-Tao He , Qing-Qing Li , Ming-Feng Wang , Wen-Yuan Wang , Wei Zhe , Yong-Hong Cao , Ming-He Mo , Yu-Long Zhai , Wen-Jun Li; J. Microbiol. 2013;51(3):289-294. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2338-z

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26 Citations

Abstract: A Gram-positive, catalase- and oxidase-positive, strictly aerobic, endospore-forming rod bacterium, designated K3514T, was isolated from the leaves of Nicotiana tabacum. The strain was able to grow at temperatures of 8–40°C, pH 5.0–10.0 and NaCl concentrations of 0–7%. The predominant quinones (>30%) of this strain were MK-7(H2) and MK-7. Phylogenetic analysis of 16S rRNA gene sequence showed that strain K3514T was affiliated to the genus Lysinibacillus, with its closest relatives being Lysinibacillus mangiferihumi (98.3% sequence similarity), Lysinibacillus sphaericus (97.9% sequence similarity), Lysinibacillus fusiformis (97.4% sequence similarity), and Lysinibacillus xylanilyticus (97.3% sequence similarity). However, low levels of DNA-DNA relatedness values suggested that the isolate was distinct from the other closest Lysinibacillus species. Additionally, based on analysis of morphological, physiological, and biochemical characteristics, the isolate could be differentiated from the closest known relatives. Therefore, based on polyphasic taxonomic data, the novel isolate likely represents a novel species, for which the name Lysinibacillus tabacifolii sp. nov. and the type strain K3514T (=KCTC 33042T =CCTCC AB 2012050T) are proposed.

Changes in Arbuscular Mycorrhizal Fungus Community Along an Exotic Plant Eupatorium adenophorum Invasion in a Chinese Secondary Forest: Xin Sun , Cheng Gao , Liang-Dong Guo; J. Microbiol. 2013;51(3):295-300. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-3169-7

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Abstract: Knowledge of the changes in arbuscular mycorrhizal (AM) fungi is fundamental for understanding the success of exotic plant invasions in natural ecosystems. In this study, AM fungal colonization and spore community were examined along an invasive gradient of the exotic plant Eupatorium adenophorum in a secondary forest in southwestern China. With increasing E. adenophorum invasion, the density of arbuscules in the roots of E. adenophorum significantly increased, but the AM root colonization rate and the densities of vesicles and hyphal coils in roots of E. adenophorum were not significantly different. A total of 29 AM fungi belonging to nine genera were identified based on spore morphology. Claroideoglomus etunicatum, Funneliformis geosporus, and Glomus aggregatum were the most common AM fungal species. The E. adenophorum invasion significantly decreased the AM fungal spore density in the soil. Furthermore, with increasing of E. adenophorum invasion the spore densities of C. etunicatum, G. aggregatum, and G. arenarium significantly decreased, whereas F. geosporus significantly increased. Nonmetric multidimensional scaling demonstrated that the AM fungus community composition was significantly different (P=0.003) in the different invasive levels of E. adenophorum, and significantly correlated with plant species richness, soil total P, and soil NO3--N. The results suggest that the alteration in AM fungus community might be caused by E. adenophorum invasion via changing the local plant community and soil properties in a Chinese secondary forest ecosystem.

Rhodopirellula rosea sp. nov., a Novel Bacterium Isolated from an Ark Clam Scapharca broughtonii: Seong Woon Roh , Hae-Won Lee , Kyung June Yim , Na-Ri Shin , Jina Lee , Tae Woong Whon , Na-Lae Lim , Daekyung Kim , Jin-Woo Bae; J. Microbiol. 2013;51(3):301-304. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-3210-x

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Abstract: A novel Gram-negative, motile, and ovoid-shaped strain, LHWP3T, which belonged to the family Planctomycetaceae in the phylum Planctomycetes, was isolated from a dead ark clam Scapharca broughtonii collected during a mass mortality event on the south coast of Korea. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the isolate was most closely related to the type strain of Rhodopirellula baltica, with a shared 16S rRNA gene sequence similarity of 94.8%. The isolate grew optimally at 30°C in 4–6% (w/v) NaCl, and at pH 7. The major isoprenoid quinone was menaquinone-6 (MK-6). The dominant polar lipids were phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, and unidentified polar lipids. The predominant cellular fatty acids were C16:0, C18:1 ω9c, and C18:0. The genomic DNA G+C content of strain LHWP3T was 53.0 mol%. Based on polyphasic taxonomic analyses, strain LHWP3T should be classified as a novel species in the genus Rhodopirellula in the family Planctomycetaceae, for which the name Rhodopirellula rosea sp. nov. is proposed. The type strain is LHWP3T (=KACC 15560T =JCM 17759T).

Deinococcus swuensis sp. nov., a Gamma-Radiation-Resistant Bacterium Isolated from Soil: Jae-Jin Lee , Hyun Ji Lee , Gi Seon Jang , Ja Myoung Yu , Ji Yoon Cha , Su Jeong Kim , Eun Bit Lee , Myung Kyum Kim; J. Microbiol. 2013;51(3):305-311. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-3023-y

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Abstract: Strain DY59T, a Gram-positive non-motile bacterium, was isolated from soil in South Korea, and was characterized to determine its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence of strain DY59T revealed that the strain DY59T belonged to the family Deinococcaceae in the class Deinococci. The highest degree of sequence similarities of strain DY59T were found with Deinococcus radiopugnans KACC 11999T (99.0%), Deinococcus marmoris KACC 12218T (97.9%), Deinococcus saxicola KACC 12240T (97.0%), Deinococcus aerolatus KACC 12745T (96.2%), and Deinococcus frigens KACC 12220T (96.1%). Chemotaxonomic data revealed that the predominant fatty acids were iso-C15:0 (19.0%), C16:1 ω7c (17.7%), C15:1 ω6c (12.6%), iso-C17:0 (10.3%), and iso-C17:1 ω9c (10.3%). A complex polar lipid profile consisted of a major unknown phosphoglycolipid. The predominant respiratory quinone is MK-8. The cell wall peptidoglycan contained D-alanine, L-glutamic acid, glycine, and L-ornithine (di-amino acid). The novel strain showed resistance to gamma radiation, with a D10 value (i.e. the dose required to reduce the bacterial population by 10-fold) in excess of 5 kGy. Based on the phylogenetic, chemotaxonomic, and phenotypic data, strain DY59T (=KCTC 33033T =JCM 18581T) should be classified as a type strain of a novel species, for which the name Deinococcus swuensis sp. nov. is proposed.

Paenibacillus marinisediminis sp. nov., a Bacterium Isolated from Marine Sediment: Hae-Won Lee , Seong Woon Roh , Kyung June Yim , Na-Ri Shin , Jina Lee , Tae Woong Whon , Joon Yong Kim , Dong-Wook Hyun , Daekyung Kim , Jin-Woo Bae; J. Microbiol. 2013;51(3):312-317. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-3198-2

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Abstract: A Gram-negative, nonmotile, endospore-forming, rod-shaped bacterial strain LHW35T, which belonged to the genus Paenibacillus, was isolated from marine sediment collected from the south coast of the Republic of Korea. A phylogenetic analysis of 16S rRNA gene sequences indicated that strain LHW35T was most closely related to Paenibacillus taiwanensis G-soil-2-3T (97.2% similarity). The optimal growth conditions for strain LHW35T were 37°C, pH 6.0, and 0% (w/v) NaCl. The main isoprenoid quinone was menaquinone-7 (MK-7) and the major polyamine was spermidine. The diamino acid present in the cell-wall peptidoglycan was meso-diaminopimelic acid. The major fatty acids were anteiso-C15:0 and C16:0. The polar lipids were phosphatidylethanolamine, phosphatidylglycerol, unidentified aminohospholipids, unidentified phospholipids, and unidentified polar lipids. A DNA-DNA hybridization experiment using the type strain of P. taiwanensis indicated <40% relatedness. The DNA G+C content was 45.0 mol%. Based on these phylogenetic, genomic, and phenotypic analyses, strain LHW35T should be classified as a novel species within the genus Paenibacillus, for which the name Paenibacillus marinisediminis sp. nov. is proposed. The type strain is LHW35T (=KACC 16317T =JCM 17886T).

Optimized Transformation of Streptomyces sp. ATCC 39366 Producing Leptomycin by Electroporation: Yong-Qiang Fan , Hong-Jian Liu , Li Yan , Yu-Shi Luan , Hai-Meng Zhou , Jun-Mo Yang , Shang-Jun Yin , Yu-Long Wang; J. Microbiol. 2013;51(3):318-322. Published online April 26, 2013; DOI: https://doi.org/10.1007/s12275-013-2428-y

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Abstract: Streptomyces sp. ATCC 39366 produces leptomycin derivatives. Leptomycin B, a potent and specific inhibitor against the export of nuclear proteins, is the main product; however, the introduction of DNA into this strain is almost impossible, which has impeded its further use. We developed a Streptomyces sp. ATCC 39366 transformation protocol to introduce foreign DNA via electroporation. Various conditions were examined, including treatments of the cell wall with weakening agents, electroporation parameters, and DNA content. We found that only plasmid DNA isolated from a dam- ET12567 strain resulted in successful transformation. The mycelium growing in a yeast-peptone-dextrose medium supplemented with 1% glycine at 28°C on a rotary shaker (220 rpm) was more dispersed than those without supplementation and prone to electroporation. The maximum transformation efficiency of 8×102 CFU/μg plasmid DNA was obtained at a field strength of 13 kV/cm with a time constant of 13 ms (25-μF capacitor; parallel resistance, 600 Ω) using 1-mm electrocuvettes. The results of the transformations of two other Streptomyces species indicated that the optimized conditions established in this study might only be applicable to Streptomyces sp. ATCC 39366. However, this is the first report of successful transformation of Streptomyces sp. ATCC 39366, and will facilitate the construction of a gene knockout mutant in Streptomyces sp. ATCC 39366 to produce series of new leptomycin derivatives.

A Novel Retron of Vibrio parahaemolyticus Is Closely Related to Retron-Vc95 of Vibrio cholerae: Toshi Shimamoto , Ashraf M. Ahmed , Tadashi Shimamoto; J. Microbiol. 2013;51(3):323-328. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2715-7

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Abstract: Some bacteria produce a satellite RNA-DNA complex termed msDNA, multicopy single-stranded DNA. In this report, msDNA from Vibrio parahaemolyticus, a cause of acute gastroenteritis, was identified and named msDNA-Vp96. The retron element containing the ret gene, encoding the reverse transcriptase (RT) that is responsible for msDNA production, was cloned and characterized. Comparison of msDNAVp96 and msDNA-Vc95, from Vibrio cholerae, showed a high level of sequence similarity. We exchanged the two ret genes to examine whether msDNA was produced by the RT from different sources. We found that RT-Vp96 of V. parahaemolyticus was able to synthesize msDNA-Vc95 of V. cholerae and vice versa. To the best of our knowledge, this is the first report that RT from different bacterial species can synthesize msDNA.

Cloning and Functional Characterization of Endo-β-1,4-Glucanase Gene from Metagenomic Library of Vermicompost: Muhammad Yasir , Haji Khan , Syed Sikander Azam , Amar Telke , Seon Won Kim , Young Ryun Chung; J. Microbiol. 2013;51(3):329-335. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2697-5

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Abstract: In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25–55°C) and pH (5.5–8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenomederived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.

Saccharomyces cerevisiae Can Secrete Sapp1p Proteinase of Candida parapsilosis But Cannot Use It for Efficient Nitrogen Acquisition: Zuzana Vinterová , Václava Bauerová , Ji&# , Hana Sychrová , Olga Hru&# , Iva Pichová; J. Microbiol. 2013;51(3):336-344. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2422-4

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Abstract: Secreted aspartic proteinase Sapp1p of Candida parapsilosis represents one of the factors contributing to the pathogenicity of the fungus. The proteinase is synthesized as an inactive pre-pro-enzyme, but only processed Sapp1p is secreted into extracellular space. We constructed a plasmid containing the SAPP1 coding sequence under control of the ScGAL1 promoter and used it for proteinase expression in a Saccharomyces cerevisiae kex2Δ mutant. Because Sapp1p maturation depends on cleavage by Kex2p proteinase, the kex2Δ mutant secreted only the pro-form of Sapp1p. Characterization of this secreted proteinase form revealed that the Sapp1p signal peptide consists of 23 amino acids. Additionally, we prepared a plasmid with the SAPP1 coding sequence under control of its authentic CpSAPP1 promoter, which contains two GATAA motifs. While in C. parapsilosis SAPP1 expression is repressed by good low molecular weight nitrogen sources (e.g., ammonium ions), S. cerevisiae cells harboring this plasmid secreted a low concentration of active proteinase regardless of the type of nitrogen source used. Quantitative real-time PCR analysis of a set of genes related to nitrogen metabolism and uptake (GAT1, GLN3, STP2, GAP1, OPT1, and PTR2) obtained from S. cerevisiae cells transformed with either plasmid encoding SAPP1 under control of its own promoter or empty vector and cultivated in media containing various nitrogen sources also suggested that SAPP1 expression can be connected with the S. cerevisiae regulatory network. However, this regulation occurs in a different manner than in C. parapsilosis.

Candida albicans ENO1 Null Mutants Exhibit Altered Drug Susceptibility, Hyphal Formation, and Virulence: Hui-Ching Ko , Ting-Yin Hsiao , Chiung-Tong Chen , Yun-Liang Yang; J. Microbiol. 2013;51(3):345-351. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2577-z

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Abstract: We previously showed that the expression of ENO1 (enolase) in the fungal pathogen Candida albicans is critical for cell growth. In this study, we investigate the contribution of the ENO1 gene to virulence. We conducted our functional study of ENO1 in C. albicans by constructing an eno1/eno1 null mutant strain in which both ENO1 alleles in the genome were knockouted with the SAT1 flipper cassette that contains the nourseothricin-resistance marker. Although the null mutant failed to grow on synthetic media containing glucose, it was capable of growth on media containing yeast extract, peptone, and non-fermentable carbon sources. The null mutant was more susceptible to certain antifungal drugs. It also exhibited defective hyphal formation, and was avirulent in BALB/c mice.

Involvement of Alternative Oxidase in the Regulation of Sensitivity of Sclerotinia sclerotiorum to the Fungicides Azoxystrobin and Procymidone: Ting Xu , Ya-Ting Wang , Wu-Sheng Liang , Fei Yao , Yong-Hong Li , Dian-Rong Li , Hao Wang , Zheng-Yi Wang; J. Microbiol. 2013;51(3):352-358. Published online April 26, 2013; DOI: https://doi.org/10.1007/s12275-013-2534-x

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Abstract: Sclerotinia sclerotiorum is a filamentous fungal pathogen that can infect many economically important crops and vegetables. Alternative oxidase is the terminal oxidase of the alternative respiratory pathway in fungal mitochondria. The function of alternative oxidase was investigated in the regulation of sensitivity of S. sclerotiorum to two commercial fungicides, azoxystrobin and procymidone which have different fungitoxic mechanisms. Two isolates of S. sclerotiorum were sensitive to both fungicides. Application of salicylhydroxamic acid, a specific inhibitor of alternative oxidase, significantly increased the values of effective concentration causing 50% mycelial growth inhibition (EC50) of azoxystrobin to both S. sclerotiorum isolates, whereas notably decreased the EC50 values of procymidone. In mycelial respiration assay azoxystrobin displayed immediate inhibitory effect on cytochrome pathway capacity, but had no immediate effect on alternative pathway capacity. In contrast, procymidone showed no immediate impact on capacities of both cytochrome and alternative pathways in the mycelia. However, alternative oxidase encoding gene (aox) transcript and protein levels, alternative respiration pathway capacity of the mycelia were obviously increased by pre-treatment for 24 h with both azoxystrobin and procymidone. These results indicate that alternative oxidase was involved in the regulation of sensitivity of S. sclerotiorum to the fungicides azoxystrobin and procymidone, and that both fungicides could affect aox gene expression and the alternative respiration pathway capacity development in mycelia of this fungal pathogen.

Identification and Characterization of an Anti-fungi Fusarium oxysporum f. sp. cucumerium Protease from the Bacillus subtilis Strain N7: Yi Luo , Lifei Sun , Zhen Zhu , Wei Ran , Qirong Shen; J. Microbiol. 2013;51(3):359-366. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2627-6

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Abstract: A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30–60% (NH4)2SO4 precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOFTM Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0–10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na+, Fe3+, Cu2+, Mg2+ (5 mmol/L each) and H2O2 (2%, v/v). However, protease activity was activated by Ca2+, K+, Mn2+ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).

Live and Dead GFP-Tagged Bacteria Showed Indistinguishable Fluorescence in Caenorhabditis elegans Gut: Ju-Ya Hsiao , Chun-Yao Chen , Mei-Jun Yang , Han-Chen Ho; J. Microbiol. 2013;51(3):367-372. Published online June 28, 2013; DOI: https://doi.org/10.1007/s12275-013-2589-8

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Abstract: Caenorhabditis elegans has been used for studying hostpathogen interactions since long, and many virulence genes of pathogens have been successfully identified. In several studies, fluorescent pathogens were fed to C. elegans and fluorescence observed in the gut was considered an indicator for bacterial colonization. However, the grinder in the pharynx of these nematodes supposedly crushes the bacterial cells, and the ground material is delivered to the intestine for nutrient absorption. Therefore, it remains unclear whether intact bacteria pass through the grinder and colonize in the intestine. Here we investigated whether the appearance of fluorescence is indicative of intact bacteria in the gut using both fluorescence microscopy and transmission electron microscopy. In wild-type N2 C. elegans, Escherichia coli DH5α, and Vibrio vulnificus 93U204, both of which express the green fluorescence protein, were found intact only proximal to the grinder, while crushed bacterial debris was found in the post-pharyngeal lumen. Nevertheless, the fluorescence was evident throughout the lumen of worm intestines irrespective of whether the bacteria were intact or not. We further investigated the interaction of the bacteria with C. elegans phm-2 mutant, which has a dysfunctional grinder. Both strains of bacteria were found to be intact and accumulated in the pharynx and intestine owing to the defective grinder. The fluorescence intensity of intact bacteria in phm-2 worms was indistinguishable from that of crushed bacterial debris in N2 worms. Therefore, appearance of fluorescence in the C. elegans intestine should not be directly interpreted as successful bacterial colonization in the intestine.

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