Pseudomonas strains accumulate mediumchain-length poly(R)-3-hydroxyalkanoate (PHA) as carbon and energy source under conditions of limiting nutrients in the presence of an excess of carbon source (Fidler et al.,

1992)

Abdullah, Amirul AI-Ashraf

Kumar, Sudesh

Najimudin, Nazalan

Samian, Mohd Razip

Yit, Le Yau

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[BIO44] Cloning and characterization of polyhydroxyalkanoate (PHA) genes from Pseudomonas sp. isolated from Antarctica  Yit Le Yau, Amirul Al-Ashraf Abdullah, Mohd Razip Samian, Sudesh Kumar, Nazalan Najimudin  School of Biological Sciences, Universiti Sains Malaysia, 11800 Minden, Penang, Malaysia.   Introduction Pseudomonas strains accumulate medium-chain-length poly(R)-3-hydroxyalkanoate (PHA) as carbon and energy source under conditions of limiting nutrients in the presence of an excess of carbon source (Fidler et al., 1992). This bacterial storage material, mainly formed of monomers of 6 to 14 carbon atoms, has potential as a renewable and biodegradable plastic (Prieto et al., 1999). Most of the research on PHAs was done on well-known PHA-producing soil microorganisms. However, PHA-producing microorganisms from the Arctic or Antarctic regions are still unknown. Psychrophilic bacteria are bacteria normally found in temperate countries. They can survive at low temperature (below 10°C). The psychrophiles are subdivided into obligate and facultative groups. Obligate psychrophiles seldom grow above 22°C and facultative psychrophiles (psychrotrophs) grow very well above 25°C. Several genes such as phaC (PHA synthase), phaA (β-ketothiolase), phaB (NADH-acetoacetyl-CoA reductase), phaG (3-hydroxyacyl-carrier protein-coenzyme A transferase) and phaJ (enoyl-CoA hydratase) are responsible in the accumulation of PHA. An enzyme known as PHA depolymerase coded by phaZ gene will then degrade these PHA compounds (Bernd et al., 1999). The PHA biosynthesis genes are often clustered in the bacterial genome as an operon called phaCAB (Steinbüchel et al., 1992). PHA synthase enzymes can be divided into three types; Type I PHA synthases (R. eutropha) preferentially uses coenzyme A thioesters of various short chain length (3HASCL) comprising three to five carbon atoms, whereas type II PHA synthases (P. aeruginosa) preferentially utilize coenzyme A thioesters of various medium chain length with at least five carbon atoms (3HAMCL). Type III PHA synthases (C. vinosum) prefer coenzyme A thioester of 3HASCL. In this paper, we describe the isolation of a PHA-producing bacterium from Antarctica for pha Synthase gene characterization.  Materials and methods Bacteria were isolated from Antarctica seawater sample using different medium and cultured in 4°C. Isolated bacteria were stained with Nile blue A and Nile red for the detection of PHA (Spiekermann et al., 1999). One of the PHA-producing bacteria was chosen and its PHA composition was tested using gas chromatography. The 16S ribosomal DNA was amplified using universal primers and was cloned into pGEM-T vector (Promega). Sequencing result was aligned with sequences in Genbank database. A fragment of the PHA synthase gene was amplified with a pair of specific primer. Primers with the following sequences were used: 5’ GTAGACCTTCATCRTCAGYTAGGAGTAACC 3’ and 5’ ACAGACCTTCATCRTCAGYTAGAGTAACC 3’. Sequencing result were compared with the sequenses available in Genbank database. Genomic library was constructed, utilizing λGEM as its cloning vector (Promega). Positively hybridized clones were screened and its DNA fragment was subcloned into pGEM-T vector and sequenced. Standard molecular biology protocols used in this study were as described in Sambrook et al., 1989.  Results and Discussion The bacteria of study is a psychrotroph (psychrotolerant). This bacterium grew in a wide range of temperature (4°C to 30°C) but not at 37°C. It grew well in both cold and room temperatures. It required about three to five days to grow at 4°C and two to four days at 30°C. Phase contrast microscopy revealed rod-shaped cells with Gram-negative characteristic.  Bacterial culture grown in limiting nitrogen and high level of carbon was The 4th Annual Seminar of National Science Fellowship 2004229prepared in order to harvest the PHA components. PHA extracted from freeze dried and processed cells were then used for gas chromatography (GC) test. GC results indicated that this bacterium produce medium chain length PHA (PHAMCL) comprising 6, 8, 10, 12 and 14 atom carbons. The total PHA content in the cells was about 17% of the cells dry weight. The 16S ribosomal DNA of this bacterium had been cloned and sequenced. The result of the sequencing showed that this sequence shared 99% identity to Pseudomonas species when compared with available sequences deposited in Genbank database. A PCR fragment corresponding to the expected size of 500bp had been successfully cloned and sequenced. Sequencing results indicated that it shared a 92% identity with PHA synthases from several Pseudomonas species. Southern analysis also indicated that this bacterium contain PHA synthase gene in its genome. Genomic library was constructed utilizing λGEM as its cloning vector. Six positive clones were selected in the primary screening. Southern analysis of phage DNA digested with restriction enzyme SphI demonstrated two positively hybridized DNA fragments (1557 bp and 4kb). These fragments were succesfully cloned into pGEM-T vector.  Fragment 1557 bp possesses a putative truncated phaC1 gene and a putative truncated phaZ gene. Meanwhile, fragment 4 kb possesses four putative genes, which are phaC2, phaD, phaF and phaI genes. Another fragment 890 bp was discovered which bridging the 1557 bp and 4 kb fragments together. The assembly of these 3 fragments turns out to be an operon of pha Synthase gene which is common in Pseudomonad famili (FIGURE 1).                               FIGURE 1 An operon of pha Synthase gene of Pseudomonas sp. was discovered by assembly of 3 subcloned fragments from genomic library.    Acknowledgements The authors wished to thank the Ministry of Science, Technology and Innovation (MOSTI), Malaysia for the National Science Fellowship awarded to Yit Le Yau and for Top Down Structural Biology grant from Malaysian Ministry of Science, Technology (MOSTE) awarded to Nazalan Najimudin.   References Bernd H. A., Rehm, Steinbüchel, A. (1999) Biochemical and genetic analysis of PHA syntheses and other proteins required for PHA synthesis. Int. J. Biol. Macromol. 25: 3-19.  Fidler, S. and Dennis, D. (1992) Polyhydroxyalkanoate production in Escherichia coli. FEMS Microbiol. Rev. 103: 231-236.  Prieto, M. A., Bühler, B., Jung, K., Withlot, B. and Kessler, B. (1999) PhaF, a polyhydroxyalkanoate-granule associated protein of Pseudomonas oleovorans Gpo1 involved in the regulatory expression system for pha genes. J. Bacteriol. 181:858-868.   Sambrook, J., E. F. Fritsch, and T. Maniais. (1989) Molecular cloning: a laboratory manual, 2nd ed. N. Y.: Cold Spring Harbor Laboratory Press.  Spiekermann, P., Bernd H. A. Rehm, Kalsheuer, R., Baumeister, D. and Steinbüchel. A. (1999) A sensitive, viable-colony staning method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds. Arch. Microbiol. 171: 73-80.   Steinbüchel, A., Hustede, E., Liebergesell, M., Pieper, U., Timm, A. and Valentin, H. (1992) Molecular basis for biosynthesis and accumulation of Polyhydroxyalkanoic acids in bacteria. FEMS Microbiol. Rev. 103: 217-230.  phaC2 phaD phaI phaF phaC1 phaZ The 4th Annual Seminar of National Science Fellowship 2004230

Cloning and characterization of polyhydroxyalkanoate (PHA) genes from Pseudomonas sp. isolated from Antarctica

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