A bacteriophage infecting Pseudomonas aeruginosa USM AR2 was successfully isolated from hydrocarbon-contaminated muddy soil. Morphological study revealed a phage with icosahedral capsid head and long non-contractile tail. The genome of the phage was determined as double stranded DNA.

Thus, the phage could be a member of the Siphoviridae family

Mat Arip, Yahya

Repository@USM

International Symposium in Science and Technology at Kansai University 2013 Collaboration between Asian Countries in Materials, Chemistry, Life and Food Science, Information Technology, Electric, Civil Engineering, Architecture and Mechanical Engineering Organized by . Graduate School of Science and Engineering, Kansai University Three Faculties for Science and Technology, Kansai University (Faculty of Engineering Science, Faculty of Environmental and Urban Engineering, Faculty of Chemistry, Materials and Bioengineering) August 21-23, 2013 Venue: Kansai University Centenary Memorial Hall CO-ORGANIZERS - Division of Community and Business Partnerships, Kansai University - Organization for Research and Development of Innovative Science and Technology (ORDIST), Kansai University COSPONSOR 8th International Symposium in Science and Technology at Kansai University 2013 Collaboration between Asian Countries in Science and Technology, Abstract August 21-23, 2013, Osaka, Japan Novel Bacteriophage Capable of Inducing Biosurfactant Rhamnolipid Production in Pseudomonas aeruginosa USM AR2 *Yahya Mat-Arip, Wan Khairunnisa Wan Juhari and Ahmad Ramli Mohd Yahya School of Biological Sciences Universiti Sains Malaysia 11800 Penang, MALAYSIA E-mail: ymarip@usm.my *Corresponding author A bacteriophage infecting Pseudomonas aeruginosa USM AR2 was successfully isolated from hydrocarbon-contaminated muddy soil. Morphological study revealed a phage with icosahedral capsid head and long non-contractile tail. The genome of the phage was determined as double stranded DNA. Thus, the phage could be a member of the Siphoviridae family. Genome profiling using different restriction enzymes showed different digestion patterns when compared to the most common Siphoviridae phage lambda and other Siphoviridae phages infect P. aeruginosa. Hence, the isolated phage could potentially be a new member of the Siphoviridae family. A very peculiar observation was noticed regarding the plaques formation when the phage infected P. aeruginosa USM AR2. The plaques formed were different from the usual where slimy substance was observed in addition to clear zones produced. When P. aeruginosa USM AR2 and the isolated phage were cultured together in broth, the broth became slimy as well. Orcinal assay was carried out on the broth and confirmed the presence ofbiosurfactant rhamnolipid with production could reach more than one hundred percent :of the cell biomass in a shake flask. An extensive literature search using various databases, as well as, different websites of search engines could not find anything on phages that are capable of inducing rhamnolipid production either in Pseudomonas or in any other biosurfactant producing bacteria. Perhaps, this is the first ever phage capable of inducing rhamnolipids production and potentially new and novel phage. 1. INTRODUCTION One of the members in microbial communities is bacteriophage or phage for short refer to viruses that infect bacteria. Phages are among the most common biological entities on earth and are found in all habitats in the world where bacteria and archaea proliferate (Clokie et. al. 2011). Recent estimates suggest that there exist globally -100 million phage species; however, only a small fraction of phages have so far been characterized (Rohwer, 2003 & McNair et. af. 2012). Out of the estimation of more than 1031 phages, only approximately 5100 have been identified and reported towards the end of last century (Ackermann, 2001 & Parisien et. al. 2008). On a global scale, it was estimated that ~ 1025 phages initiate an infection every second (Hendrix et. al. 1999). Therefore, it is not surprising that phages play major roles in the ecological balance of microbial life and indirectly influence the world ecology as well. Biosufactants, produced by varieties of microorganisms, are well known and well documented for their role in enhancement of the emulsification of hydrocarbons, potentially solubilizing the hydrocarbon contaminants and increasing their availability for microbial degradation (Salwa, et. al., 2009; Sekhon et. al., 2012). Biosurfactants are structurally diverse and can have various chemical compositions mainly consisting of fatty acids, glycolipids, lipopeptides, lipopolysaccharides and lipoproteins depending on the producing microorganisms, raw matter and process conditions (Makkar et. al., 2011 and Sekhon et. al., 2012). Biosurfactants have the potential to be applied in various fields such as cleaning industry, phannaceuticals, cosmetics, food industry and medical (Banat et. al., 2000; Kitamoto et. al., 2002; Zeraik & Nitschke, 2010). To date, biosurfactants play an important application in petroleum-related industries included enhanced oil recovery, cleaning oil spills, and bioremediation (Bertrand et. al., 1994 & Daziel, et. al., 1996). 8th International Symposium in Science and Technology at Kansai University 2013 Collaboration between Asian Countries in Science and Technology, August 21-23,2013, Osaka, Japan 2. EXPERIMENTAL PROCEDURE 2.1. Isolation and Purification of Bacteriophage Bacteria host P. aeruginosa USM AR2 was cultured in LB broth and incubated overnight at 37°C with shaking at 180 rpm. Bacteria culture was mixed together with 5 g of hydrocarbon-contaminated muddy soil sample and allowed for incubation overnight at 37°C with shaking at 180 rpm. The next morning, the ' incubated culture was filtered with filter paper, centrifuged at 10,000 rpm and followed by filtration using bacteria filter with pore size of 0.45 Jlm. Then, the filtrate was serially diluted in LB broth. 100 JlL of the dilutions was mixed with 100 JlL of P. aeruginosa USM AR2 culture and plated onto LB agar. 3 mL LB top agar was overlay onto the plates and allowed to harden. The plates were incubated at 37°C overnight. The next morning, formations of clear zones or plaques were observed indicating the infection of P. aeruginosa USM AR2 by phages presence in the filtrate. For purification purposes, well isolated plaques were transferred onto a new LB plate that was previously spread with P. aeruginosa USM AR2 and overlaid with LB top agar. The transferwas carried out using sterile toothpicks by poking through the LB top agar. The plate was incubated at 37°C overnight for plaques formation. The LB top agar was scrapped off the LB plate and eluted in TMS buffer overnight at 4°C. The next day, 0.2 mL chloroform was added and shakes gently. The elution was centrifuged and the supernatant was filtered through 0.45 JlM bacteria filter. A small amount of chloroform (not more than 5% of the filtrate volume) was added into the filtrate for storage purpose at 4°C as stock. Plaque assay was performed to determine the phage titer (pfu/mL) of the filtrate. For bacteriophage enrichment, the stock was allowed to infect P. aeruginosa USM AR2 overnight. The infected P. aeruginosa US M AR2 culture was centrifuged, filtered, stored in chlorofonn as enriched bacteriophage stock. 2.2 Transmission Electron Microscopy (TEM) A drop of phage with titer of 1010 pfu/mL was applied onto the surface of a carbon-coated grid (400 mesh copper grid). The sample was negatively stained with 20/0 methyltungstate. The morphology of the phage was observed using transmission electron microscope (Philips CMl2 equipped with analysis system, Philips Electron Optics). 2.3 Genomic Profiling Prior to genome extraction, the phage stock with was treated with DNase and RNase. The phage genome was extracted using phenol/chloroform and ethanol salt precipitation method (Sambrook and Russel, 2001). The extracted genome was digested with either DNase or RNase. Restriction enzyme digestion of the extracted genome was carried out using EcoRI, EcoRV and Ncol. Virtual restriction enzyme digestion patterns were performed using online programme, NEBcutter V2.0, accessible through website http://tools.neb.co!:l1/NEBcutter2L (Vincze et. al., 2003). Phages D3112 and MP29, Siphoviridae phages infecting P. aeruginosa, were used as comparison against restriction enzyme digestion patterns of the isolated phage. 2.3 Orcinol Assay A culture of P. aeruginosa USM AR2 was mixed with the isolated phage to allow infection took place. The mixture was sampled at 12 hours and 24 hours post infection and centrifuged at 10,000 rpm for 15 minutes. The supernatant was collected and appropriate dilutions were prepared for Orcinal assay. The diluted supernatants were mixed with Orcinal reagent and incubated at 70°C for 40 minutes. After incubation, the mixtures were allowed to cool down to room temperature and spectrophotometer readings at OD421 were taken for each dilutions. 8th International Symposium in Science and Technology at Kansai University 2013 Collaboration between Asian Countries in Science and Technology, August 21-23,2013, Osaka, Japan Table 1: Orcinol assay for rhamnolipid detection. Copious amount of rhamnolipid was produced within 48 hours post infection. Sample Average OD421 Biomass Amount of rhamnolipid % rhamnolipid reading (15X dilution) (gIL) produced (giL) production . Non~infected 0.854 0.77 0.51 66.2 24 hours Infected 1.423 1.28 Non-infected 0.736 0.66 0.69 104.5 48 hours Infected 1.496 1.35 For the isolated phage to act as an inducer for rhanmolipid production in P. aeruginosa USM AR2 was ., unheard before. Supported by an extensive literature search using various databases, as well as, different;'. websites of search engines could not find anything on phages that are capable of inducing rhamnolipid ·· production either in Pseudomonas or in other biosurfactant producing bacteria. 4. CONCLUSIONS Based on these results, the isolated phage may be a potentially new and novel phage infecting P. aeruginosa USM AR2 and capable to act as an inducer for rhamnolipid production. 5. ACKNOWLEDGMENTS The authors would to thank School of Biological Sciences and Division of Research & Innovation of Universiti Sains Malaysia for all the supports. 6. REFERENCES Ackermann, H. W. 200 1. Frequency of morphological phage descriptions in the year 2000. Archive Virology. 146:843-857. Banat, I. M. et. al. 2000. Potential commercial applications ofmicrobial surfactants. Appl. Microbiol. Biotechnol. 53: 495-508. Bertrand, 1. C. et. al. 1994. The potential application ofbiosurfactant in combating hydrocarbon pollution in marine environments. Res. Microbiol. 145: 53-56. Clokie, M R. J. et. al. 2011. Phages in nature. Bacteriophage. 1(1): 31-45. Daziel, E. et. al. 1996. Biosurfactant production by a soil pseudomonas strain growing on PAR. Appl. Environ. Microbiol. 62: 1908-1912. Hendrix, R.W. et. al. 1999. Evolutionary relationships among diverse bacteriophages and prophages: all the world's a phage. Proceeding National Academy of Science USA. 96:2192-2197 . . Kitamoto, D. et. al. 2002. A review: functions and potential applications of glycolipid biosurfactants - from energy-saving materials to gene delivery carriers. J. Biosci. Bioeng. 94: 187-201. Makkar, R. S. et. al. 2011. Advances in utilization of renewable substrates for biosurfactant production. AMB Express. 1 :5. McNair, K. et. al. 2012. PHACTS, a computational approach to classifying the lifestyle of phages. Bioinformatics. 28(5): 614-618. NurAsshifa, M N. et. al. 2012. Rhamnolipid produced by Pseudomonas aeruginosa USM-AR2 facilitates crude oil distillation. J. Gen. Appl. Microbiol. 58: 153-161. 8th International Symposium in Science and Technology at Kansai University 2013 Collaboration between Asian Countries in Science and Technology, August 21-23, 2013, Osaka, Japan Parisien, A. et. al. 2008. Novel alternatives to antibiotics: bacteriophages, bacterial cell wall hydrolases, and antimicrobialpeptides. Journal of Applied Microbiology. 104:1-13. Rohwer, F. 2003. Global Phage Diversity. Cell. 113: 141. Salwa, M. S. et. al. 2009. Different feeding strategy for the production of biosurfactant from Pseudomonas aeruginosa USM AR2 in modified bioreactor. Biotechnol. Bioprocess Eng. 14: 763-768. Sambrook, J and Russel, D. W. 2001. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press. New York. Sekhon, K. K. et. al. 2012. Biosurfadant production and potential correlation with esterase activity. J Pet Environ Biotechnol. 3:133. Vincze, T. et. al. 2003. NEBcutter: a program to cleave DNA with restriction enzymes. Nucleic Acids Research. 31(3): 3688-3691. Zeraik, A. E. and Nitschke, M 2010. Biosurfactants as agents to reduce adhesion 0/ pathogenic bacteria to polystyrene surfaces: effect o/temperature and hydrophobicity. CUff Microbiol. 61: 554-559. 

Collaboration between Asian countries in materials, chemistry, life and food science, information technology, electric, civil engineering, architecture and mechanical engineering.

http://eprints.usm.my/32549/1/Collaboration_between_Asian_Countries_in_Materials.pdf

Collaboration between Asian countries in materials, chemistry, life and food science, information technology, electric, civil engineering, architecture and mechanical engineering.

Abstract

Similar works

Full text

Available Versions

Repository@USM