Journal of Microbiology

Volume 49(4); August 2011

Journal Article

Characterization of Nitrogen-Fixing Bacteria Isolated from Field-Grown Barley, Oat, and Wheat: Anastasia Venieraki , Maria Dimou , Eleni Vezyri , Io Kefalogianni , Nikolaos Argyris , Georgia Liara , Panagiotis Pergalis , Iordanis Chatzipavlidis , Panagiotis Katinakis; J. Microbiol. 2011;49(4):525-534. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0457-y

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Abstract: Diazotrophic bacteria were isolated from the rhizosphere of field-grown Triticum aestivum, Hordeum vulgare, and Avena sativa grown in various regions of Greece. One isolate, with the highest nitrogen-fixation ability from each of the eleven rhizospheres, was selected for further characterisation. Diazotrophic strains were assessed for plant-growth-promoting traits such as indoleacetic acid production and phosphate solubilisation. The phylogenies of 16S rRNA gene of the selected isolates were compared with those based on dnaK and nifH genes. The constructed trees indicated that the isolates were members of the species Azospirillum brasilense, Azospirillum zeae, and Pseudomonas stutzeri. Furthermore, the ipdC gene was detected in all A. brasilence and one A. zeae isolates. The work presented here provides the first molecular genetic evidence for the presence of culturable nitrogen-fixing P. stutzeri and A. zeae associated with field-grown A. sativa and H. vulgare in Greece.

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

Bacterial Structure and Characterization of Plant Growth Promoting and Oil Degrading Bacteria from the Rhizospheres of Mangrove Plants: Flávia Lima do Carmo , Henrique Fragoso dos Santos , Edir Ferreira Martins , Jan Dirk van Elsas , Alexandre Soares Rosado , Raquel Silva Peixoto; J. Microbiol. 2011;49(4):535-543. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0528-0

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32 Citations

Abstract: Most oil from oceanic spills converges on coastal ecosystems, such as mangrove forests, which are threatened with worldwide disappearance. Particular bacteria that inhabit the rhizosphere of local plant species can stimulate plant development through various mechanisms; it would be advantageous if these would also be capable of degrading oil. Such bacteria may be important in the preservation or recuperation of mangrove forests impacted by oil spills. This study aimed to compare the bacterial structure, isolate and evaluate bacteria able to degrade oil and stimulate plant growth, from the rhizospheres of three mangrove plant species. These features are particularly important taking into account recent policies for mangrove bioremediation, implying that oil degradation as well as plant maintenance and health are key targets. Fifty-seven morphotypes were isolated from the mangrove rhizospheres on Bushnell-Haas (BH) medium supplemented with oil as the sole carbon source and tested for plant growth promotion. Of this strains, 60% potentially fixed nitrogen, 16% showed antimicrobial activity, 84% produced siderophores, 51% had the capacity to solubilize phosphate, and 33% produced the indole acetic acid hormone. Using gas chromatography, we evaluated the oil-degrading potential of ten selected strains that had different morphologies and showed Plant Growth Promoting Rhizobacteria (PGPR) features. The ten tested strains showed a promising degradation profile for at least one compound present in the oil. Among degrader strains, 46% had promising PGPR potential, having at least three of the above capacities. These strains might be used as a consortium, allowing the concomitant degradation of oil and stimulation of mangrove plant survival and maintenance.

Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis Analysis of Bacterial Community Structure in the Food, Intestines, and Feces of Earthworms: Sung Wook Hong , Ju Sam Lee , Kun Sub Chung; J. Microbiol. 2011;49(4):544-550. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0423-8

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Abstract: The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other strains, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.

Symbiotic Interaction of Endophytic Bacteria with Arbuscular Mycorrhizal Fungi and Its Antagonistic Effect on Ganoderma boninense: Shamala Sundram , Sariah Meon , Idris Abu Seman , Radziah Othman; J. Microbiol. 2011;49(4):551-557. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0489-3

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48 Citations

Abstract: Endophytic bacteria (Pseudomonas aeruginosa UPMP3 and Burkholderia cepacia UPMB3), isolated from within roots of oil palm (Elaeis guineensis Jacq.), were tested for their presymbiotic effects on two arbuscular mycorrhizal fungi, (Glomus intraradices UT126 and Glomus clarum BR152B). These endophytic bacteria were also tested for antagonistic effects on Ganoderma boninense PER 71, a white wood rot fungal pathogen that causes a serious disease in oil palm. Spore germination and hyphal length of each arbuscular mycorrhizal fungal (AMF) pairing with endophytic bacteria was found to be significantly higher than spores plated in the absence of bacteria. Scanning electron microscopy (SEM) showed that the endophytic bacteria were scattered, resting or embedded on the surface hyaline layer or on the degraded walls of AMF spores, possibly feeding on the outer hyaline spore wall. The antagonistic effect of the endophytic bacteria was expressed as severe morphological abnormalities in the hyphal structures of G. boninense PER 71. The effects of the endophytic bacteria on G. boninense PER 71 hyphal structures were observed clearly under SEM. Severe inter-twisting, distortion, lysis and shrivelling of the hyphal structures were observed. This study found that the effect of endophytic bacteria on G. intraradices UT126 and G. clarum BR152B resembled that of a mycorrhiza helper bacteria (MHB) association because the association significantly promoted AMF spore germination and hyphal length. However, the endophytic bacteria were extremely damaging to G. boninense PER 71.

Methyl Coenzyme M Reductase (mcrA) Gene Based Phylogenetic Analysis of Methanogens Population in Murrah Buffaloes (Bubalus bubalis): Prem Prashant Chaudhary , Sunil Kumar Sirohi , Dheer Singh , Jyoti Saxena; J. Microbiol. 2011;49(4):558-561. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1052-y

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Abstract: The aim of the present study was to decipher the diversity of methanogens in rumen of Murrah buffaloes so that effective strategies can be made in order to mitigate methane emission from these methanogens. In the present study diversity of rumen methanogens in Murrah buffaloes (Bubalus bubalis) from North India was evaluated by using mcr-A gene library obtained from the pooled PCR product from four animals and by using MEGA4 software. A total of 104 clones were examined, revealing 26 different mcr-A gene sequences or phylotypes. Of the 26 phylotypes, 16 (64 of 104 clones) were less than 97% similar to any of the cultured strain of methanogens. Seven clone sequences were clustered with Methanomicrobium mobile and three clone sequences were clustered with Methanobrevibacter gottschalkii during the phylogenetic analysis. Uncultured group of methanogens comes out to be the major component of the methanogens community structure in Murrah buffaloes. Methanomicrobium phylotype comes out to be major phylotype among cultured methanogens followed by Methanobrevibacter phylotype. These results help in making effective strategies to check the growth of dominant methanogenic communities in the rumen of this animal which in turn help in the reduction of methane emission in the environment and ultimately helps us in fighting with the problem of global warming.

Isolation and Evaluation of Terrestrial Fungi with Algicidal Ability from Zijin Mountain, Nanjing, China: Guomin Han , Xiaoguang Feng , Yong Jia , Congyan Wang , Xingbing He , Qiyou Zhou , Xingjun Tian; J. Microbiol. 2011;49(4):562-567. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0496-4

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Abstract: Approximately 60 fungal isolates from Zijin Mountain (Nanjing, China) were screened to determine their algicidal ability. The results show that 8 fungi belonging to Ascomycota and 5 belonging to Basidiomycota have algicidal ability. Of these fungi, Irpex lacteus T2b, Trametes hirsuta T24, Trametes versicolor F21a, and Bjerkandera adusta T1 showed strong algicidal ability. The order of fungal chlorophyll-a removal efficiency was as follows: T. versicolor F21a > I. lacteus T2b > B. adusta T1 > T. hirsuta T24. In particular, T. versicolor F21a completely removed algal cells within 30 h, showing the strongest algicidal ability. The results also show that all 4 fungal species degraded algal cells through direct attack. In addition, most of the tested fungi from the order Polyporales of Basidiomycota exhibited strong algicidal activity, suggesting that most fungi that belong to this order have algicidal ability. The findings of this work could direct the search for terrestrial fungi for bloom control.

Isolation and Analyses of Uranium Tolerant Serratia marcescens Strains and Their Utilization for Aerobic Uranium U(VI) Bioadsorption: Rakshak Kumar , Celin Acharya , Santa Ram Joshi; J. Microbiol. 2011;49(4):568-574. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0366-0

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52 Citations

Abstract: Enrichment-based methods targeted at uranium-tolerant populations among the culturable, aerobic, chemoheterotrophic bacteria from the subsurface soils of Domiasiat (India’s largest sandstone-type uranium deposits, containing an average ore grade of 0.1% U3O8), indicated a wide occurrence of Serratia marcescens. Five representative S. marcescens isolates were characterized by a polyphasic taxonomic approach. The phylogenetic analyses of 16S rRNA gene sequences showed their relatedness to S. marcescens ATCC 13880 (≥99.4% similarity). Biochemical characteristics and random amplified polymorphic DNA profiles revealed significant differences among the representative isolates and the type strain as well. The minimum inhibitory concentration for uranium U(VI) exhibited by these natural isolates was found to range from 3.5-4.0 mM. On evaluation for their uranyl adsorption properties, it was found that all these isolates were able to remove nearly 90-92% (21-22 mg/L) and 60-70% (285-335 mg/L) of U(VI) on being challenged with 100 μM (23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solutions, respectively, at pH 3.5 within 10 min of exposure. his capacity was retained by the isolates even after 24 h of incubation. Viability tests confirmed the tolerance of these isolates to toxic concentrations of soluble uranium U(VI) at pH 3.5. This is among the first studies to report uranium-tolerant aerobic chemoheterotrophs obtained from the pristine uranium ore-bearing site of Domiasiat.

Isolation and Characterization of Ethylbenzene Degrading Pseudomonas putida E41: Lan-Hee Kim , Sang-Seob Lee; J. Microbiol. 2011;49(4):575-584. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0399-4

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Abstract: Pseudomonas putida E41 was isolated from oil-contaminated soil and showed its ability to grow on ethylbenzene as the sole carbon and energy source. Moreover, P. putida E41 show the activity of biodegradation of ethylbenzene in the batch culture. E41 showed high efficiency of biodegradation of ethylbenzene with the optimum conditions (a cell concentration of 0.1 g wet cell weight/L, pH 7.0, 25°C, and ethylbenzene concentration of 50 mg/L) from the results of the batch culture. The maximum degradation rate and specific growth rate (μmax) under the optimum conditions were 0.19±0.03 mg/mg-DCW (Dry Cell Weight)/h and 0.87±0.13 h-1, respectively. Benzene, toluene and ethylbenzene were degraded when these compounds were provided together; however, xylene isomers persisted during degradation by P. putida E41. When using a bioreactor batch system with a binary culture with P. putida BJ10, which was isolated previously in our lab, the degradation rate for benzene and toluene was improved in BTE mixed medium (each initial concentration: 50 mg/L). Almost all of the BTE was degraded within 4 h and 70-80% of m-, p-, and o-xylenes within 11 h in a BTEX mixture (initial concentration: 50 mg/L each). In summary, we found a valuable new strain of P. putida, determined the optimal degradation conditions for this isolate and tested a mixed culture of E41 and BJ10 for its ability to degrade a common sample of mixed contaminants containing benzene, toluene, and xylene.

Microbial Community Analysis and Identification of Alternative Host-Specific Fecal Indicators in Fecal and River Water Samples Using Pyrosequencing: Ju-Yong Jeong , Hee-Deung Park , Kyong-Hee Lee , Hang-Yeon Weon , Jong-Ok Ka; J. Microbiol. 2011;49(4):585-594. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0530-6

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34 Citations

Abstract: It is important to know the comprehensive microbial communities of fecal pollution sources and receiving water bodies for microbial source tracking. Pyrosequencing targeting the V1-V3 hypervariable regions of the 16S rRNA gene was used to investigate the characteristics of bacterial and Bacteroidales communities in major fecal sources and river waters. Diversity analysis indicated that cow feces had the highest diversities in the bacterial and Bacteroidales group followed by the pig sample, with human feces having the lowest value. The Bacteroidales, one of the potential fecal indicators, totally dominated in the fecal samples accounting for 31%-52% of bacterial sequences, but much less (0.6%) in the river water. Clustering and Venn diagram analyses showed that the human sample had a greater similarity to the pig sample in the bacterial and Bacteroidales communities than to samples from other hosts. Traditional fecal indicators, i.e., Escherichia coli, were detected in the human and river water samples at very low rates and Clostridium perfringens and enterococci were not detected in any samples. Besides the Bacteroidales group, some microorganisms detected in the specific hosts, i.e., Parasutterella excrementihominis, Veillonella sp., Dialister invisus, Megamonas funiformis, and Ruminococcus lactaris for the human and Lactobacillus amylovorus and Atopostipes sp. for the pig, could be used as potential host-specific fecal indicators. These microorganisms could be used as multiple fecal indicators that are not dependent on the absence or presence of a single indicator. Monitoring for multiple indicators that are highly abundant and host-specific would greatly enhance the effectiveness of fecal pollution source tracking.

Characterization of Antibiotic Resistance Determinants in Oral Biofilms: Seon-Mi Kim , Hyeong C. Kim , Seok-Woo S. Lee; J. Microbiol. 2011;49(4):595-602. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0519-1

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42 Citations

Abstract: Oral biofilms contain numerous antibiotic resistance determinants that can be transferred within or outside of the oral cavity. The aim of this study was to evaluate the prevalence and the relative level of antibiotic resistance determinants from oral biofilms. Oral biofilm samples that were collected from healthy subjects and periodontitis patients were subjected to qualitative and quantitative analyses for selected antibiotic resistance determinants using PCR. The prevalence of tet(Q), tet(M), cfxA, and blaTEM was very high both in the patient and the healthy subject group, with a tendency toward higher values in the patient group, with the exception of erm(F), which was more prevalent in the healthy group. The two extended spectrum β-lactam (ESBL) resistance determinants blaSHV and blaTEM showed a dramatic difference, as blaTEM was present in all of the samples and blaSHV was not found at all. The aacA-aphD, vanA, and mecA genes were rarely detected, suggesting that they are not common in oral bacteria. A quantitative PCR analysis showed that the relative amount of resistance determinants present in oral biofilms of the patient group was much greater than that of the healthy group, exhibiting 17-, 13-, 145-, and 3-fold increases for tet(Q), tet(M), erm(F), and cfxA, respectively. The results of this study suggest that the oral antibiotic resistome is more diverse and abundant in periodontitis patients than in healthy subjects, suggesting that there is a difference in the diversity and distribution of antibiotic resistance in oral biofilms associated with health and disease.

Journal Article

Cyclic Lipopeptide Profile of Three Bacillus subtilis Strains; Antagonists of Fusarium Head Blight: Christopher A. Dunlap , David A. Schisler , Neil P. Price , Steven F. Vaughn; J. Microbiol. 2011;49(4):603-609. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1044-y

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Abstract: The objective of the study was to identify the lipopetides associated with three Bacillus subtilis strains. The strains are antagonists of Gibberella zeae, and have been shown to be effective in reducing Fusarium head blight in wheat. The lipopeptide profile of three B. subtilis strains (AS43.3, AS43.4, and OH131.1) was determined using mass spectroscopy. Strains AS43.3 and AS43.4 produced the anti-fungal lipopeptides from the iturin and fengycin family during the stationary growth phase. All three strains produced the lipopeptide surfactin at different growth times. Strain OH131.1 only produced surfactin under these conditions. The antifungal activity of the culture supernatant and individual lipopeptides was determined by the inhibition of G. zeae. Cell-free supernatant from strains AS43.3 and AS43.4 demonstrated strong antibiosis of G. zeae, while strain OH131.1 had no antibiosis activity. These results suggest a different mechanism of antagonism for strain OH131.1, relative to AS43.3 and AS43.4.

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

Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov., Rosy Colored β-Glucosidase-Producing Bacteria Isolated from Soil: Sathiyaraj Srinivasan , Jae-Jin Lee , Myung Kyum Kim; J. Microbiol. 2011;49(4):610-616. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-1017-1

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11 Citations

Abstract: Two strains PB196T and PB62T of Gram-negative, non-motile, and non-spore-forming bacteria, were isolated from soil in South Korea and characterized to determine their taxonomic positions. 16S rRNA gene sequence analysis showed that the two strains belonged to the genus Sphingomonas. The highest degree of sequence similarity of strain PB196T was found with PB62T (98.9%), Sphingomonas humi PB323T (98.9%), Sphingomonas kaistensis PB56T (98.2%), and Sphingomonas astaxanthinifaciens TDMA-17T (98.0%). The highest degree of sequence similarity of strain PB62T was found with Sphingomonas humi PB323T (98.8%), Sphingomonas astaxanthinifaciens TDMA-17T (98.2%), and Sphingomonas kaistensis PB56T (98.1%). Chemotaxonomic data revealed that they possessed ubiquinone-10 (Q-10) as common in the genus Sphingomonas, that the predominant fatty acids were summed feature 7 (C18:1ω7c/ω9t/ω12t), summed feature 4 (C16:1ω7c/C15:0 iso 2OH), C16:0, and C17:1ω6c, and that they contained sphingoglycolipid, phosphatidylglycerol (PG), and phosphatidylethanolamine (PE) in common but they showed difference for diphosphatidylglycerol (DPG). Based on these data, PB196T (=KCTC 12339T =JCM 16604T) and PB62T (=KCTC 12336T =JCM 16605T =KEMB 9004-005T) should be classified as type strains of two novel species, for which the names Sphingomonas rosea sp. nov. and Sphingomonas swuensis sp. nov. are proposed, respectively.

Paenibacillus telluris sp. nov., a Novel Phosphate-Solubilizing Bacterium Isolated from Soil: Jae-Chan Lee , Chang-Jin Kim , Ki-Hong Yoon; J. Microbiol. 2011;49(4):617-621. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0471-0

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Abstract: A phosphate-solubilizing bacterial strain designated PS38T was isolated from farm soil. The isolate was a Gram-positive, motile, endospore-forming, rod-shaped bacterium. It grew optimally at 37oC and pH 7.5. The predominant cellular fatty acids were anteiso-C15:0, anteiso-C17:0, and iso-C16:0. The DNA G+C content was 49.5 mol% and the predominant menaquinone was MK-7. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain PS38T belonged to the genus Paenibacillus and was most closely related to Paenibacillus chibensis JCM 9905T, P. barengoltzii SAFN-016T, P. timonensis 2301032T, and P. motobuensis MC10T with 96.3%, 96.0%, 95.9%, and 95.5% 16S rRNA gene sequence similarity, respectively. On the basis of morphological, chemotaxonomic, physiological, and phylogenetic properties, strain PS38T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus telluris sp. nov. is proposed. The type strain is PS38T (=KCTC 13946T =CGMCC 1.10695T).

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

Identification of Enriched Conjugated Linoleic Acid Isomers in Cultures of Ruminal Microorganisms after Dosing with 1-13C-Linoleic Acid: Yong-Jae Lee , Thomas C. Jenkins; J. Microbiol. 2011;49(4):622-627. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0415-8

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Abstract: Most studies of linoleic acid biohydrogenation propose that it converts to stearic acid through the production of cis-9 trans-11 CLA and trans-11 C18:1. However, several other CLA have been identified in ruminal contents, suggesting additional pathways may exist. To explore this possibility, this research investigated the linoleic acid biohydrogenation pathway to identify CLA isomers in cultures of ruminal microorganisms after dosing with a 13C stable isotope. The 13C enrichment was calculated as [(M+1/M)×100] in labeled minus unlabeled cultures. After 48 h incubation, significant 13C enrichment was observed in seven CLA isomers, indicating their formation from linoleic acid. All enriched CLA isomers had double bonds in either the 9,11 or 10,12 position except for trans-9 cis-11 CLA. The cis-9 trans-11 CLA exhibited the highest enrichment (30.65%), followed by enrichments from 21.06 to 23.08% for trans-10 cis-12, cis-10 trans-12, trans-9 trans-11, and trans-10 trans-12 CLA. The remaining two CLA (cis-9 cis-11 and cis-10 cis-12 CLA) exhibited enrichments of 18.38 and 19.29%, respectively. The results of this study verified the formation of cis-9 trans-11 and trans-10 cis-12 CLA isomers from linoleic acid biohydrogenation. An additional five CLA isomers also contained carbons originating from linoleic acid, indicating that pathways of linoleic acid biohydrogenation are more complex than previously described.

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

Identification of an Extracellular Thermostable Glycosyl Hydrolase Family 13 α-Amylase from Thermotoga neapolitana: Kyoung-Hwa Choi , Sungmin Hwang , Hee-Seob Lee , Jaeho Cha; J. Microbiol. 2011;49(4):628-634. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0432-7

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Abstract: We cloned the gene for an extracellular α-amylase, AmyE, from the hyperthermophilic bacterium Thermotoga neapolitana and expressed it in Escherichia coli. The molecular mass of the enzyme was 92 kDa as a monomer. Maximum activity was observed at pH 6.5 and temperature 75°C and the enzyme was highly thermostable. AmyE hydrolyzed the typical substrates for α-amylase, including soluble starch, amylopectin, and maltooligosaccharides. The hydrolytic pattern of AmyE was similar to that of a typical α-amylase; however, unlike most of the calcium (Ca2+)-dependent α-amylases, the activity of AmyE was unaffected by Ca2+. The specific activities of AmyE towards various substrates indicated that the enzyme preferred maltooligosaccharides which have more than four glucose residues. AmyE could not hydrolyze maltose and maltotriose. When maltoheptaose was incubated with AmyE at the various time courses, the products consisting of maltose through maltopentaose was evenly formed indicating that the enzyme acts in an endo-fashion. The specific activity of AmyE (7.4 U/mg at 75°C, pH 6.5, with starch as the substrate) was extremely lower than that of other extracellular α-amylases, which indicates that AmyE may cooperate with other highly active extracellular α-amylases for the breakdown of the starch or α-glucans into maltose and maltotriose before transport into the cell in the members of Thermotoga sp.

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