Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening

RESEARCH LETTER Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening Marcus Schallmey1, Anh Ly1, Chunxia Wang1, Gabriela Meglei1, Sonja Voget2, Wolfgang R. Streit2, Brian T. Driscoll3 & Trevor C. Charles1 1 Department of Biology, University of Waterloo, Waterloo, ON, Canada; 2Institut für Mikrobiologie und Genetik, Universität Göttingen, Göttingen, Germany; and 3Department of Natural Resource Sciences, McGill University, Ste-Anne-de-Bellevue, QC, Canada MICROBIOLOGY LETTERS Present addresses: Marcus Schallmey, Lehrstuhl für Biotechnologie, RWTH Aachen University, D-52074 Aachen, Germany. Chunxia Wang, Virginia Bioinformatics Institute, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061 USA. Wolfgang Streit, Microbiology and Biotechnology, Biozentrum Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany. Abstract We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-hostrange libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages. Received 31 January 2011; revised 26 May 2011; accepted 27 May 2011. Final version published online 23 June 2011. DOI:10.1111/j.1574-6968.2011.02324.x Editor: Alexander Steinbüchel Keywords functional complementation; broad host range cosmid library; bioplastics. Introduction Petrochemically derived plastics are extremely useful materials, and they dominate many sectors of the industrial economy. However, they are inherently costly to the environment. They are produced from nonrenewable fossil fuels, their waste accumulates due to their recalcitrance to biodegradation, and their production cost will likely escalate as oil reserves are depleted. There is much interest in developing viable alternatives to these plastics. Polyhydroxyalkanoates (PHA) are commonly accumulated bacterial intracellular carbon storage polymers (Steinbüchel & Lütke-Eversloh, 2003; Trainer & Charles, c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved 2006; Keshavarz & Roy, 2010). Their function is to guard against stresses at the level of nutritional carbon and energy balance. Genetic studies of polyhydroxyalkanaote synthesis have been carried out in several bacteria. The central enzyme, polyhydroxyalkanaote synthase encoded by phaC, catalyses the polymerization of hydroxyacyl-CoA molecules, driven by the energy released from CoA hydrolysis. These polymers are arranged in the cell as inert granules, complexed with associated proteins. Upon starvation or other stress, they can be depolymerized to provide a source of carbon and energy to sustain the cell. They are thus of central importance to the metabolic functioning of many bacteria. FEMS Microbiol Lett 321 (2011) 150–156 Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 Correspondence: Trevor C. Charles, Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, ON, Canada N2L 3G1. Tel.: 1519 888 4567, ext. 35606; fax: 1519 746 0614; e-mail: tcharles@uwaterloo.ca 151 Polyhydroxyalkanaote synthesis genes from soil metagenome the ability to store carbon as polyhydroxyalkanaote contributes to survival under fluctuating environmental conditions of the soil and rhizosphere (recently reviewed in Castro-Sowinski et al., 2010). Our methods should be useful for the isolation of additional novel polyhydroxyalkanaote synthesis genes from uncultivated bacteria inhabiting environments such as soil. This work continues our development of the Alphaproteobacteria as surrogate hosts for functional metagenomic studies (Wang et al., 2006) (Hao et al., 2010). Materials and methods Bacterial strains, plasmids, and growth conditions Strains and plasmids are listed in Table 1. Luria–Bertani and yeast extract–mannitol (YM) medium supplemented with appropriate antibiotics were used as described previously (Aneja et al., 2004). The metagenomic library was maintained as pooled Escherichia coli HB101 culture, stored longterm at 70 1C in the presence of 7% dimethyl sulphoxide. Nile red (Sigma-Aldrich, N3013, technical grade) added to agar media at a concentration of 0.5 mg mL 1 facilitated the visual identification of stained colonies containing polyhydroxyalkanaote accumulating cells (Spiekermann et al., 1999). Table 1. Bacterial strains and plasmids Strain or plasmid Relevant characteristics Reference or source E. coli DH5a F– F80lacZDM15D (lacZYA-argF)U169 recA1 endA1 hsdR17 phoA supE44 thi-1 gyrA96 relA1 F– thi-1 hsdS20 supE44 recA13 ara-14 leuB6 proA2 lacY1 galK2 rpsL20 xyl-5 mtl-1 pro82 thi-1 endA hsdR17 supE44 recA56 (pRK600) Invitrogen Canada Inc. (Burlington, ON) Promega (Madison, WI) Finan et al. (1986) SU47 str-21 Rm1021 exoY<Tn5 Rm1021 phaC<Tn5 Rm1021 phaC<Tn5-233 Rm1021 phaB<OSmSp Rm1021 exoY<Tn5 phaC<Tn5-233 Meade et al. (1982) Leigh et al. (1985) Charles et al. (1997) Charles et al. (1997) Aneja et al. (2004) This study pRK2013 tra Nmr<Tn9( ! Cmr) IncP oriT cos lacZa (Tcr) oriT lacZ (Gmr) pRK7813 with metagenomic insert complementing S. meliloti Rm11105 on YM medium pRK7813 with metagenomic insert complementing S. meliloti Rm11105 on YM medium pRK7813 with metagenomic insert complementing S. meliloti Rm11105 on YM medium pRK7813 with metagenomic insert complementing S. meliloti Rm11105 on YM medium pRK7813 with metagenomic insert complementing S. meliloti Rm11105 on YM medium pBBR1MCS-5 with BamHI fragment from pCX92 complementing S. meliloti Rm11105 pBBR1MCS-5 with BamHI fragment from pCX9M4 complementing S. meliloti Rm11105 pBBR1MCS-5 with BamHI fragment from pCX9M5 complementing S. meliloti Rm11105 Finan et al. (1986) Jones & Gutterson (1987) Kovach et al. (1995) This study This study This study This study This study This study This study This study HB101 MT616 S. meliloti Rm1021 Rm7055 Rm11105 Rm11144 Rm11347 Rm11476 Plasmids pRK600 pRK7813 pBBR1MCS-5 pCX92 pCX9M1 pCX9M3 pCX9M4 pCX9M5 pMS1 pMS2 pMS3 Antibiotic resistances: Cmr, chloramphenicol; Gmr, gentamycin; Kmr, kanamycin; Nalr, nalidixic acid; Nmr, neomycin; Smr, streptomycin; Tcr, tetracycline; Spr, spectinomycin. FEMS Microbiol Lett 321 (2011) 150–156 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 While the most common polyhydroxyalkanaote is poly-3hydroxybutyrate (PHB), the diversity of polyhydroxyalkanaote is significant, with over 150 different possible monomeric constituents present in different combinations within a given polymer (Steinbüchel & Lütke-Eversloh, 2003). This structural diversity is reflected in the wide range of physical properties demonstrated by these polymers. Polyhydroxyalkanaote polymers are being developed for industrial purposes, as biodegradable replacements for fossil-fuel derived plastics, and as materials with unique properties. Major research efforts are focused on developing the ability to produce these materials in an economically competitive manner so that they will be commercially viable. Polyhydroxyalkanaote’s structure is determined in part by polyhydroxyalkanaote synthase’s substrate specificity, and there is considerable interest in determining the basis for such substrate specificity. We previously reported the development of a phenotypic complementation-based method for the isolation of polyhydroxyalkanaote synthase-encoding genes (Aneja et al., 2004). In the current report, we describe the use of this method for the isolation and characterization of novel polyhydroxyalkanaote synthesis genes from a soil metagenomic library. Many bacteria found in heterogeneous and diverse soil habitats are known to accumulate polyhydroxyalkanaote. In several cases it has been demonstrated that 152 Genetics and molecular biology Polyhydroxyalkanaote analysis For polyhydroxyalkanaote analysis 1-mL precultures were used to inoculate 200 mL YM broth in 500-mL Erlenmeyer flasks. Incubation was carried out at 30 1C on a shaker at 200 r.p.m. for 48 h. Cells were recovered by centrifugation at 5855 g for 15 min in a GSA rotor. Pellets were washed with 50 mL saline and were finally resuspended in 1 mL saline. These suspensions were frozen at 70 1C and lyophilized for 24 h. For derivatization of poly(D-3-oxybutyric acid) to D-3-hydroxybutyric acid methyl ester, 10 mg of dried cell material was mixed with 2 mL of methanol containing 3% (v/v) H2SO4 and 2 mL of chloroform containing 1.5 g L 1 methyl benzoate as the internal standard. The reaction was carried out at 100 1C for 10 h and then cooled on ice. After the reaction mix was cooled down, 1 mL of deionized water was added and vortexed for 1 min. After separation of both phases by gravity the top layer was removed by pipetting and excess water was removed by freezing at 70 1C for 2 h. Finally, residual water was removed from the chloroform phase by drying over 2 g of anhydrous sodium sulphate. A PHB calibration curve was prepared from commercial PHB (Sigma-Aldrich). GC analysis was carried out on a HP Chemstation with a DB-1 column (length, 30 m; diameter, 323 mm; film thickness, 3 mm) with nitrogen as the carrier gas at 2.6 mL min 1 flow rate. Sample injection volumes of 1 mL were analysed by running a temperature profile and subsequent detection by flame ionization. c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Polyhydroxyalkanaote deposits were visualized by transmission electron microscopy. Samples were prepared from 100 mL stationary phase YM cultures. Cells were harvested by centrifugation, washed with phosphate buffer (pH 6), and collected by centrifugation. The cells were then suspended in 1 mL of 2.5% glutaraldehyde in phosphate buffer, and kept at 4 1C for 1 h, followed by three series of centrifugation and resuspension in 1 mL of phosphate buffer. The washed cells were suspended in 1 mL of 0.5% OsO4 in phosphate buffer and kept at room temperature for 16 h, then diluted to 8 mL in phosphate buffer. The cells were collected by centifugation and resuspended in 2% agar, a drop of which was then allowed to harden on a microscope slide. The agar suspended cells were then dehydrated in a series from 50% acetone to 100% acetone, embedded in eponaraldite, sectioned at a thickness of 60–90 nm on a Reichert Ultracut E ultramicrotome, stained with uranyl acetate and lead citrate, and examined on a Philips CM10 transmission electron microscope using an accelerating voltage of 60 kV. DNA sequence data DNA Sequences have been deposited in GenBank and can be accessed via accession numbers EF408057–EF408059. Results and discussion Isolation of PHB synthesis clones from metagenomic library We previously described a novel method for the isolation of PHB synthesis genes by complementation of a dry colony phenotype of S. meliloti PHB synthesis mutants (Aneja et al., 2004). This strategy was applied to one of the soil metagenomic libraries that we had constructed (Wang et al., 2006) and had used to isolate novel genes for the PHB degradation pathway (Wang et al., 2006) and quorum sensing (Hao et al., 2010). The CX9 soil library, consisting of 22 180 cosmid pRK7813 clones, was introduced en masse into the phaC<Tn5 mutant Rm11105 by triparental conjugation. Transconjugants were selected on YM agar supplemented with neomycin and tetracycline, and screened visually for mucoid colony phenotype. The clones from mucoid colonies were transferred to E. coli DH5a by triparental conjugation, and then reintroduced into strain Rm11105 to confirm the associated mucoid colony phenotype on YM agar. Five of these clones, designated pCX92, pCX9M1, pCX9M3, pCX9M4, and pCX9M5, were found to exhibit unique BamH1 restriction patterns. PHB accumulation was confirmed in the transconjugants of all clones by PHB assay (Table 2) and by transmission electron microscopy for the first clone isolated, pCX92 (Fig. 1). FEMS Microbiol Lett 321 (2011) 150–156 Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 Sinorhizobium meliloti genetics (Glazebrook & Walker, 1991) and standard techniques for molecular biology were used. The metagenomic library was transferred to recipient S. meliloti cells by triparental conjugation, and screens for complementation of polyhydroxyalkanaote synthesis were performed by examination of transconjugant colonies for restoration of mucoid phenotype or Nile Red staining on YM agar (Aneja et al., 2004). Along with end sequencing of subcloned cosmid insert fragments, the EZ<TN hKAN-2i insertion kit (Epicentre) was used to generate transposon mutations that facilitated sequencing using the recommended transposon-sequencing primers. Primer walking was used to close gaps when necessary, and trimming and assembly were performed manually. The sequence was obtained at MOBIX (McMaster University) using an ABI 3100 Gene Analyzer instrument, and at the Institut für Mikrobiologie und Genetik, Universität Göttingen. Potential protein-coding sequences were identified using GENEMARK.HMM (Lukashin & Borodovsky, 1998), and supported by BLASTX analysis (Altschul et al., 1997). The predicted ORFs were further analysed by BLASTP and BLASTN. M. Schallmey et al. 153 Polyhydroxyalkanaote synthesis genes from soil metagenome Improvement of the screening protocol Table 2. Intracellular PHB accumulation Sample % PHB per dry weight Relative PHB accumulation compared with wild type Rm1021 (pRK7813) Rm11105 (pRK7813) Rm11105 (pCX92) Rm11105 (pCX9M1) Rm11105 (pCX9M3) Rm11105 (pCX9M4) Rm11105 (pCX9M5) 5.97 0 6.3 10.7 15.3 12.3 7.9 100 0 106 180 256 205 131 Subcloning and sequence analysis of the complementing clones BamH1 fragments were subcloned from the cosmid clones pCX92, pCXM4, and pCXM5 individually into pBBR1MCS5. Complementing subclones were identified after en masse conjugation of transformants from E. coli DH5a into strain Rm11105 or Rm11476, screening transconjugants on YM-NR as described above. These subclones were subjected to in vitro mutagenesis with EZ<TN hKAN-2i transposon to localize the complementing regions. Complete DNA sequences of the complementing BamH1 fragments were determined, facilitated by sequencing from the EZ<TN hKAN-2i transposon insertions using transposon-specific primers, and from the ends of subcloned fragments using vector-specific primers. Thus, pMS1 carries a 16 456-bp fragment from pCX92, pMS2 carries a 5255-bp fragment from pCX9M4, and pMS3 carries a 5015-bp fragment from pCX9M5. In each case, analysis of the sequence confirmed the presence of phaC genes. The complete 33 810-bp sequence of pCX92 insert DNA was determined from a shotgun library prepared by cloning a partial Sau3A1 digest into vector pTZ19R. The identities of the nearest orthologs from a cultured organism and the predicted functions are presented in Table 3, with the relative gene orientations illustrated in Fig. 2. Fig. 1. Transmission electron microscopy showing the complementation of the Sinorhizobium meliloti phaC mutant Rm11105 by metagenomic clone pCX92. (a) The wild type strain Rm1021 cells are packed with PHB granules. (b) The phaC mutant strain Rm11105 cells are devoid of PHB granules. (c) The phaC mutant strain Rm11105 pCX92 transconjugant contains PHB granules. FEMS Microbiol Lett 321 (2011) 150–156 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 The differentiation of mucoid from dry colony phenotype on YM agar required close inspection, and the possibility of missing complemented colonies was a concern. We found that incorporation of 0.5 mg mL 1 Nile red into the YM agar (YM-NR) resulted in bright pink staining of PHB-producing colonies, with no staining of the colonies that did not produce PHB. Examination under long-wave UV light enhanced the fluorescence, but it was not necessary to differentiate between the PHB mutant and the wild-type colonies. The exoY<Tn5 mutant Rm7055, in which the extracellular polysaccharide succinoglycan is not produced, formed colonies that were not mucoid on YM-NR. These dry colonies fluoresced brightly under UV illumination. Strain Rm11476, containing both exoY<Tn5 and phaC<Tn5-233 mutations, was constructed by transduction. On YM-NR, this strain formed dry colonies that did not stain or fluoresce. This was found to be the best genetic background for the detection of PHB-accumulating clones, especially on densely populated plates, and was used to screen for complementing subclones of the originally isolated cosmid clones. 154 Predicted ORF E-value % amino acid sequence identity EED32118; metE, methionine synthase, gamma proteobacterium NOR5-3 ABR89889; phaB, Janthinobacterium sp. Marseille ACR01721; phaC, Thauera sp. MZ1T ABE49865; Zinc/iron permease, COG0428, Methylobacillus flagellatus KT 5e-123 5e-114 3e-172 4e-67 67 80 56 59 CBJ51083; rluD, Ribosomal large subunit pseudouridine synthase D Ralstonia solanacearum EET31017; Protein of unknown function DUF152 Nitromonas sp. AL212 AAA21975; phaC Cupriavidus necator H16 ABE44067; phaA Polaromonas sp. JS666 8e-91 5e-63 0.0 1e-173 58 52 61 76 6e-29 2e-12 43 66 1e-13 3e-150 31 64 3e-129 67 1e-791 3e-28 4e-76 3e-113 3e-147 1e-121 4e-45 1e-133 2e-134 2e-70 2e-132 2e-155 1e-52 1e-39 0.0 0.0 62 46 48 70 51 41 68 67 72 47 67 68 81 64 66 89 2e-10 1e-86 1e-54 4e-50 4e-152 9e-38 50 60 71 75 66 51 No significant match No significant match ABS12985; hypothetical protein Oant_0254 Ochrobactrum anthropi ATCC 49188 EFL15324; conserved hypothetical protein Streptomyces sp. C No significant match EEF58526; Immunoglobulin I-set domain protein, bacterium Ellin514 ABF52205; anion transporter Sphingopyxis alaskensis RB2256 No significant match No significant match EDX78930; transposase IS116/IS110/IS902 family protein Brevundimonas sp. BAL3 No significant match No significant match ABD27707; protein of unknown function DUF152 Novosphingobium aromaticivorans DSM 12444 ZP_06861057; acetyltransferase Citromicrobium bathyomarinum JL354 ABF2208; protein of unknown function DUF185 Sphingopyxis alaskensis RB2256 BAI96396; prolipoprotein diacylglyceryltransferase Sphingobium japonicum UT26S ABD25128; AMP-dependent synthetase and ligase Novosphingobium aromaticivorans DSM 12444 ABQ70935; isoquinoline 1-oxidoreductase Sphingomonas wittichii RW1 EAT07821; Ferrochelatase Sphingomonas sp. SKA58 EAT10179; hypothetical protein Sphingomonas sp. SKA58 ABD24941; fatty acid desaturase Novosphingobium aromaticivoran DSM 12444 ADP82064; esterase/lipase/thioesterase Frankia sp. EuI1c ABD24943; permease YjgP/YjgQ Novosphingobium aromaticivorans DSM 12444 ZP_06861028; permease Citromicrobium bathyomarinum JL 354 ABD24946; ATP-dependent Clp protease adaptor protein ClpS Novosphingobium aromaticivorans DSM 12444 ABD24947; phasin Novosphingobium aromaticivorans DSM 12444 ABD24948; phaC PHA synthase class I Novosphingobium aromaticivorans DSM 12444 ABD24949; aminotransferase Novosphingobium aromaticivorans DSM 12444 No significant match EAQ28048; two-component response regulator Erythrobacter sp. NAP1 ABD24950; enoyl-CoA hydratase Novosphingobium aromaticivorans DSM 12444 ABD25181; molybdenum cofactor guanylyltransferase Novosphingobium aromaticivorans DSM 12444 ABF53548; glyoxalase/bleomycin resistance protein/dioxygenase Sphingopyxis alaskensis RB2256 BAB47713; RNase Mesorhizobium loti MAFF303099 EEF57824; hypothetical protein Cflav_PD0806 bacterium Ellin514 M. Schallmey et al. FEMS Microbiol Lett 321 (2011) 150–156 pCX9M4 (pMS2) (GenBank EF408058) ABN71569/Complement 968..1996 ABN 71570/Complement 1999..2742 ABN71571/Complement 2772..4499 ABN71572/Complement 4576-5255 pCX9M5 (pMS3) (GenBank EF408059) ABN71573/151..1212 ABN71574/1209..1970 ABN71575/2015..3763 ABN71576/3776..4954 pCX92 (GenBank EF408057) ABN71533/Complement o 1..770 ABN71534/Complement 896..1297 ABN71535/Complement 1578..2093 ABN71536/Complement 2069..2641 ABN71537/Complement 2738..3163 ABN71538/Complement 3160..4008 ABN71539/Complement 4270..5670 ABN71540/5807..6223 ABN71541/6322..7560 ABN71542/Complement 7851..8861 ABN71543/Complement 8990..9469 ABN71544/Complement 9730..10278 ABN71545/Complement 10370..11143 ABN71546/Complement 11140..11649 ABN71547/Complement 11646..12629 ABN71548/Complement 12632..13531 ABN71549/13599..15260 ABN71550/15257..17356 ABN71551/17353..17775 ABN71552/Complement 17685..18821 ABN71553/18917..20017 ABN71554/Complement 20009..20986 ABN71555/Complement 21011..22108 ABN71556/Complement 22108..23316 ABN71557/Complement 23484..23879 ABN71558/Complement 24066..24935 ABN71559/Complement 25058..26929 ABN71560/27170..28369 ABN71561/Complement 28599..29078 ABN71562/28746..29171 ABN71563/29203..30015/ ABN71564/Complement 30012..30572 ABN71565/Complement 30565..30945 ABN71566/31044..32813 ABN71567/Complement 32857.. 4 33763 Best match Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Table 3. Nearest orthologs and predicted functions of ORFs 155 Polyhydroxyalkanaote synthesis genes from soil metagenome As expected, each of the clones contains a gene corresponding to phaC (Fig. 2). CDD analysis (Marchler-Bauer et al., 2011) (data not shown) revealed that the predicted gene product of each contains the conserved PhaC Nterminus domain (pfam07167) and the expected a/b hydrolase fold (pfam00561) (Rehm, 2003). Phylogenetic analysis, presented in the Supporting Information (Figs S1 and S2), reinforced that these genes are homologous to, but substantially different from, known PHA synthesis genes. In clone pCX92, phaC is within a cluster of genes with an organization similar to a segment of the genome of Novosphingobium aromaticivorans, a member of the Alphaproteobacteria. The %GC of the pCX92 sequence, at 65.7, is very similar to the %GC of the corresponding region of the N. aromaticivorans genome, at 64.8. For each of the genes, the corresponding N. aromaticivorans gene is the highest match, ranging from 51% to 89% amino acid sequence identity, with the phaC exhibiting 66% amino acid sequence identity. In an arrangement similar to that found in the N. aromaticivorans genome, this clone also contains a putative phasinencoding gene immediately adjacent to the phaC gene. The clone does not contain any other polyhydroxyalkanaote cycle genes, but this is not unusual, as a broad diversity in genomic organization of polyhydroxyalkanaote synthesis genes has been long recognized (Rehm & Steinbüchel, 1999). The sequence of the pCX9M4 subclone pMS2 revealed the phaC gene to share 56% amino acid sequence identity with a phaC gene from Thauera sp. MZ1T, a member of the Betaproteobacteria, and to be adjacent to a phaB gene. The %GC of the pMS2 sequence, at 66.5, is very similar to the FEMS Microbiol Lett 321 (2011) 150–156 Conclusions We have demonstrated the use of phenotypic complementation to isolate novel polyhydroxyalkanaote synthesis genes from metagenomic libraries. The complementation is dependent on having a suitable phenotype to screen, and we have made use of the complex phenotype of S. meliloti phaC mutants that includes lack of mucoidy on high carbon ratio media such as YM, absence of fluorescence on Nile redcontaining media, and reduced growth on polyhydroxyalkanaote cycle intermediates (Aneja et al., 2004). We should also be able to use this strategy to isolate other polyhydroxyalkanaote synthesis genes such as phaA and phaB from metagenomic libraries. We anticipate the use of this method for the construction of diverse collections of genes encoding polyhydroxyalkanaote synthesis enzymes that might be useful for the optimization and improvement of industrial polyhydroxyalkanaote production through pathway engineering. As more polyhydroxyalkanaote synthase genes are isolated from metagenomic libraries using these methods, it will be interesting to see the full range of genes that can be captured. Acknowledgements This work was supported by a Natural Sciences and Engineering Research Council of Canada Strategic Projects grant (T.C.C). 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved c Downloaded from https://academic.oup.com/femsle/article/321/2/150/626948 by guest on 28 June 2022 Fig. 2. Graphical representation of the predicted polyhydroxyalkanaote synthesis genes (dark arrows), and surrounding genes (light arrows), in each of the three sequenced clones. The arrowheads indicate direction of transcription. MS2 and MS3 represent the complete BamH1 fragment that was sequenced, while CX92 includes a similar sized region of the much larger complementing BamH1 fragment in this clone. %GC of the corresponding region of the Thauera sp. MZ1T genome, at 66.0%. Curiously, maximum-likelihood phylogenetic analysis (Figs S1 and S2) clusters the pMS2 phaC sequence with the MZ1T phaC sequence at the amino acid level only, not at the DNA level, despite the very similar %GC. Because the complete sequence of pCX9M4 has not yet been determined, we do not know whether additional polyhydroxyalkanaote cycle genes are present on the clone, but the MZ1T genome has a phaR repressor gene further downstream of phaC-phaB. The sequence of the pCX9M5 subclone pMS3 indicated a phaC gene with 61% amino acid sequence identity to the well-studied phaC gene of Cupriavidus necator H16, also from the Betaproteobacteria. The best-matching genomic fragment, however, was with another member of the Betaproteobacteria, Burkholderia sp. 383, despite differences in %GC, 59.4 for pMS3 compared with 66.9 for Burkholderia sp. 383. The phaC gene is located adjacent to a phaA. Like pCX9M4, the complete sequence of pCX9M5 has not yet been completed, and so we do not know whether other polyhydroxyalkanaote cycle genes are present on this clone. However, Burkholderia sp. 383 has the typical genomic organization of a class I operon (phaCABR). 156 References c 2011 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved auxotrophic mutants of Rhizobium meliloti induced by transposon mutagenesis. J Bacteriol 149: 114–122. Rehm BHA (2003) Polyester synthases: natural catalysts for plastics. Biochem J 376: 15–33. Rehm BHA & Steinbüchel A (1999) Biochemical and genetic analysis of PHA synthases and other proteins required for PHA synthesis. 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