Microsoft Word - 82Lambri.doc CCHHEEMMIICCAALL EENNGGIINNEEEERRIINNGG TTRRAANNSSAACCTTIIOONNSS VOL. 38, 2014 A publication of The Italian Association of Chemical Engineering www.aidic.it/cet Guest Editors: Enrico Bardone, Marco Bravi, Taj Keshavarz Copyright © 2014, AIDIC Servizi S.r.l., ISBN 978-88-95608-29-7; ISSN 2283-9216 Transcriptional Analysis of Pha Genes in Pseudomonas Mediterranea CFBP 5447 Grown on Glycerol Grazia Licciardelloa, Giulia Devescovib, Patrizia Bellac, Corinne De Gregorioa, Antonino F. Cataraa, Salvatore P.P. Guglieminod, Vittorio Venturib, Vittoria Catarac. aParco Scientifico e Tecnologico della Sicilia s.c.p.a., z.i. Blocco Palma I, Str.le Lancia 57, 95121 Catania, Italy bBacteriology Group, International Centre for Genetic Engineering and Biotechnology, Area Science Park, Padriciano, Trieste, Italy cDipartimento di Scienze delle Produzioni Agrarie e Alimentari, Università degli Studi di Catania, Via Santa Sofia 100, 95131 Catania, Italy dDipartimento Scienze Biologiche ed Ambientali, Università di Messina glicciardello@pstsicilia.it We analysed the draft genome sequence of Pseudomonas mediterranea CFBP 5447 in order to identify firstly the central metabolic pathways that convert fatty acids or carbohydrate intermediates into mcl-PHA and secondly the genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). Absence of the glpF gene, which codifies for the “glycerol uptake facilitator protein”, was highlighted. In order to understand the expression of the pha gene cluster, we investigated the promoter activity of phaC1, phaC2, phaZ, phaD and phaI genes. When glycerol was present as the carbon source, PI was found to be the most active promoter. Expression analysis of the knock-out mutant of the phaD gene, which is a transcriptional regulator belonging to the TetR family, showed that PhaD acts as an activator of the phaI promoter which, in turn, triggers the transcription of the phaIF operon. The activation of PC1, which controls the phaC1ZC2D, by PhaD, was less efficient than PI. 1. Introduction Polyhydroxyalkanoates (PHAs) are biodegradable polymers naturally produced by bacteria as carbon storage granules, even from renewable resources. Because of they are biodegradable and recyclable, they are considered as a valid green alternative to conventional plastics in order to manufacture frequently used products (Luengo et al., 2003). The large-scale production of PHAs involves high costs due to the fermentation and separation process. Such costs can be reduced by using appropriate low-cost carbon sources and optimized growth conditions (Solaiman et al., 2006). In addition, genome analysis and meta- bolic engineering represent good strategies to increase PHA production, by providing efficient cell facto- ries. Most Pseudomonas species are able to produce medium-chain-length-PHAs (mcl-PHAs) containing mon- omers from 6 to 14 carbon atoms (Timm and Steinbuchel, 1990). The entire pha gene cluster responsible for PHA metabolism, well conserved among Pseudomonas spp., consists of genes encoding two syn- thases (phaC1 and phaC2), a depolymerase (phaZ) responsible for PHA mobilization (type II biosynthetic locus) and the phaD gene encoding a putative transcriptional regulator (Klinke et al., 2000). In addition, the phaF and phaI genes, transcribed divergently to the other pha genes, encode the phasins, thus playing regulatory and functional roles (Prieto et al., 1999). Knowledge of the molecular mechanisms regulating mcl-PHA synthesis and degradation is relatively lim- ited (Prieto et al., 2007, Sandoval et al., 2007). PHA metabolism comprises two central pathways, β- oxidation and fatty acid de novo synthesis, depending on whether the carbon source is related (oleic acid, vegetable oils, fatty acids) or unrelated (glucose, glycerol), respectively. Precursors for mcl-PHA polymer- ases, derived from these pathways, are provided by phaJ or phaG genes, key link enzymes between β- DOI: 10.3303/CET1438049 Please cite this article as: Licciardello G., Devescovi G., Bella P., De Gregorio C., Catara A., Guglielmino S.P.P., Venturi V., Catara V., 2014, Transcriptional analysis of pha genes in pseudomonas mediterranea cfbp 5447 grown on glycerol, Chemical Engineering Transactions, 38, 289-294 DOI: 10.3303/CET1438049 289 oxidation or fatty acid biosynthesis and mcl-PHA biosynthesis, respectively (Rehm and Steinbuchel, 1999). According to P. aeruginosa studies, glycerol uptake and metabolism is mediated first of all by the outer membrane OprB, and then by the glycerol facilitator GlpF, which is involved in glycerol transport, as well as the glycerol kinase (GlpK) and a citoplasmic-membrane-associated G3P dehydrogenase (GlpD) (Schweizer et al., 1997). The glp operon and the key role of GlpR in the optimization of PHA production from glycerol, has been recently demonstrated (Escapa et al., 2013). Within the Pseudomonas fluorescens group (Solaiman et al., 2002), P. mediterranea CFBP 5447 (9.1) is able to bioconvert refined and biodiesel-glycerol into a mcl-PHA (Palmeri et al., 2012), with an approximate molecular weight of 56 KDa, and which produces a transparent odourless film (Pappalardo et al., 2013). P. mediterranea pha locus has only been partially described (Solaiman et al., 2005; Bella et al., 2007). A comparison with the taxonomic related strains P. corrugata 388 and CFBP 5445, demonstrated that this locus lacks a 24 bp sequence in the phaC1–phaZ intergenic region codifying for a putative rho independ- ent terminator responsible for a slight variation in the PHA composition from oleic acid (Solaiman et al., 2008). Our recent draft genome sequence of P. mediterranea CFBP 5447 (submitted in Genbank) helped us to investigate the central metabolic pathways involved in mcl-PHA synthesis, the peripheral pathway encoded by the pha cluster and genes involved in glycerol metabolism (glpF, glpK, glpD, glpR). The activi- ty of the promoter regions of pha genes was monitored 24 hrs and 48 hrs after inoculation. The role of PhaD as an activator of pha cluster was demonstrated by mutagenesis analyses. 2. Materials and Methods 2.1 Bacterial strains, media and growth conditions The strains and plasmids used in this study are listed in Table 1. P. mediterranea CFBP 5447 was routine- ly grown at 28°C in both nutrient agar (Oxoid, Milan, Italy) supplemented with 1% dextrose (NDA) and Lu- ria-Bertani (LB) agar (Sambrook et al., 2001). Antibiotics were added as required, with the following final concentrations: ampicillin, 100 µg mL-1, tetracycline, 15 µg mL-1 (E. coli), or 40 µg mL-1 (Pseudomonas); kanamycin, 50 µg mL-1 (E. coli) or 100 µg mL-1 (Pseudomonas). DNA manipulations, including digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, ligation with T4 ligase, and E. coli transformation were performed as described by Sambrook et al. (2001). Triparental matings from E. coli to P. mediterranea CFBP 5447 were carried out with the helper strain E. coli DH5α (pRK2013) (Figurski and Helinski, 1979). For PHA production, strains were grown in E-medium supplemented with reagent grade glycerol 2% (v/v), as previously reported (Palmeri et al., 2012). Table 1. Strains and plasmids used in this study. 2.2 Construction of P. mediterranea VVD mutant The phaD- genomic mutant was created as follows. An internal part of phaD was amplified by PCR as a 339-bp fragment, using primer PhaDintFw (ATGGCAAGGAACCCCTTGTC) and PhaDintRev (AACAG- CAACGTCAGGGTGAT). It was cloned first in pGEM and then as an EcoRI fragment in the corresponding site in pKNOCK-Km, generating pKMPhaD. This plasmid was then used as a suicide delivery system in order to create a phaD knockout mutant through homologous recombination in strain CFBP 5447, as de- P. mediterranea strains CFBP 5447 Wild type CFBP VVD phaD:: pKnock, Kmr This study Plasmids pMP220 Promoter probe vector, IncP Tcr Spaink et al., 1987 pKnock-Kmr Mobilizable suicide vector, Kmr Alexeyev, 1999 pGEM-T Cloning vector; Ampr Promega pMPPhaC1 phaC1 promoter cloned in pMP220 This study pMPPhaZ phaZ promoter cloned in pMP220 This study pMPPhaC2 phaC2 promoter cloned in pMP220 This study pMPPhaI phaI promoter cloned in pMP220 This study pKMPhaD pKnock containing an internal fragment of phaD gene This study 290 scribed by Alexeyev (1999), thus generating P. mediterranea VVD. The fidelity of the marker exchange events was confirmed by PCR. 2.3 Reporter gene fusion assay Transcriptional fusion plasmids for phaC1, phaC2, phaZ, phaD and phaI promoter regions based on the pMP220 promoter probe vector were constructed. Fragments containing regions upstream the starting co- don of each gene were amplified by PCR using genomic DNA of P. mediterranea CFBP 5447 as the tem- plate and specific primer sets. The DNA fragments were then cloned into pGEM-T (Promega), removed as EcoRI/XbaI, and cloned in pMP220 yielding PC1, PC2, PZ, PD, PI lacZ promoter fusions. β–galactosidase activities were determined during growth in E-medium supplemented with reagent grade glycerol 2% (v/v) following Miller (1972), with the modification of Stachel and associates (1985). All experiments were per- formed in triplicate and the mean value is given. 2.4 RT-PCR analysis P. mediterranea CFBP 5447 and VVD mutant derivative were grown in E-medium supplemented with rea- gent grade glycerol 2% (v/v) at the exponential growth phase (14 hours after inoculation). Total RNA was isolated using a commercial RNA extraction kit (RiboPure-Bacteria Kit, Ambion), as recommended by the manufacturer in triplicate. RNA samples were quantitatively analyzed by Nanodrop. Following a DNAse purification step by Turbo DNA-free kit (Ambion), 200 ng of total RNA was used for the RT reaction using a Transcription System kit (Promega). The reverse transcription reaction was performed at 24°C for 10 min, followed by 15 min at 42°C, and inactivation at 95 °C for 5 min. PCR reactions were performed under the following conditions: an initial 94°C for 3 min, followed by 33 cycles of 94°C for 30 sec, 52°C for 30 sec and 72°C for 1 min, and a final extension of 72°C for 10 min. Eight primers were used to amplify phaC1- phaZ, phaZ-phaC2, phaC2-phaD and phaF-phaI overlapping regions. As a negative control, PCR reac- tions with the same primer sets were performed using RNA samples that had not been reverse tran- scribed. 3. Results 3.1 Genome mining on metabolic pathways involved in PHA biosynthesis from glycerol Using the pFAM search domain and tBlastn applications, genes that codify for enzymes involved in β- oxidation (fad) and fatty acid de novo synthesis (fab), able to convert fatty acids or carbohydrates into pre- cursors for PHA biosynthesis were mapped. These genes were dispersed in different loci along the entire genome. The entire type II PHA locus consisting of six genes in 6725 bp (Figure 1) was identified. Five putative rho independent terminators were mapped within the locus, three downstream of phaD and in the same direc- tion and two downstream of phaF. They represent sites of transcription termination and act as gene regu- lators (Solaiman et al., 2008). We found that there was no putative rho independent terminator in the phaC1-phaZ intergenic region, thus confirming Solaiman et al. (2008). The glp operon responsible for glycerol catabolism was 4240 bp in length and comprised only the genes putatively coding for GlpK, GlpD and GlpR. The “glycerol uptake facilitator protein” coded by glpF gene was not identified in this strain, thus explaining the prolonged lag growth phase in media containing glyc- erol. The same result has been observed in P. corrugata CFBP 5454 genome, recently obtained (Lic- ciardello et al., 2014). Figure 1. The Pha gene cluster of P. mediterranea CFBP 5447 is organized in two operons - phaC1ZC2D and phaIF - as revealed by the presence of rho independent terminators. 291 3.2 Transcriptional analysis of the genes involved in PHA synthesis from glycerol In order to determine the best molecular strategies for the optimization of the PHA biosynthetic process, the transcriptional expression levels of phaC1, phaC2, phaZ, phaD and phaI were investigated. We constructed five lacZ promoter fusions with the upstream region of each gene (named PC1, PC2, PZ, PD, PI promoter regions), which were transferred into P. mediterranea CFBP 5447 by triparental mating. P. mediterranea strains carrying each lacZ fusion were cultured using reagent grade as a carbon source. The β-galactosidase activity was monitored after 24 and 48 hrs (Figure 2). The highest level of reporter expression was detected in the strain carrying the PI::lacZ fusion. It was more than 5-fold higher than that observed in the strain carrying the PC1::lacZ fusion after 24 hrs and 24-fold after 48 hrs. Expression levels of the strain carrying the PC1::lacZ fusion and PC2::lacZ fusion were essentially similar, with a slight decrease after 48 hrs. No reporter activity was detected for Pz and PD promoters. 3.3 PhaD activates transcription of PC1 and PI In order to describe the role of the phaD gene, it was insertionally inactivated thereby creating the VVD genomic mutant of P. mediterranea. The PC1, PC2, PZ, PD, PI lacZ promoter fusions were transferred to the VVD mutant strain and β-galactosidase activity monitored after 24 and 48 hrs. Promoter activity quantifica- tion data showed that PI was strongly reduced in the VVD mutant. A 3-fold lower expression level than the Wt strain was observed for the PC1 in the VVD mutant after 24 hrs, whereas no reduction was detected af- ter 48 hrs (Figure 2). Only a basal expression level was detected in the phaC2 gene in the VVD mutant, suggesting that it is probably driven by an internal promoter PC2. The absence of promoter activity in the upstream regions of phaZ and phaD genes suggests that their expression is controlled by an upstream promoter. The presence of a polycistronic transcription unit (operon) comprised of phaC1, phaZ, phaC2 and phaD was demonstrat- ed by RT-PCR amplification of overlapping phaC1-phaZ, phaZ-phaC2, phaC2-phaD regions. In addition, the presence of three putative transcriptional rho terminators downstream phaD (Figure 1), confirmed this hypothesis. Similarly, the presence of the phaI-F overlapping region and the identification of two putative rho terminators located downstream of phaF highlighted that these two genes are organized as an operon with PI as a promoter directly controlled by PhaD. Preliminary semiquantitative expression of these genes by RT-PCR in the Wt and VVD mutant confirmed the control of phaIF operon by PhaD, whereas no de- tectable difference in phaC1ZC2D operon expression was assessed. These results need to be confirmed by real-time PCR. Figure 2. Promoter activities of phaC1, phaZ, phaC2, phaD and phaI in the parent strain P. mediterranea CFBP 5447 and VVD mutant derivative. The values are averages of at least three independent experi- ments. 4. Conclusions The improvement in PHA bacterial productivity using genetic engineering of our model P. mediterranea strain entails not only defining the genes involved in the bioconversion but also regulating these genes and understanding the links between the metabolic pathways. In this study, the draft genome sequence of P. 292 mediterranea CFBP 5447 was mined enabling us to identify the complete PHA gene cluster and also the glycerol uptake and metabolism genes. We defined the activity of promoter regions of the pha genes in P. mediterranea CFBP 5447. We found that PC1 and PI are the most active and are responsible for the tran- scription of phaC1ZC2D and phaIF operons, respectively. The PI operon was the most active and drives the expression of phaF and phaI genes. Our results are in accordance with findings for P. putida KT2442 (de Eugenio et al., 2010). In the presence of glycerol as a carbon source, the PI promoter is strongly controlled by PhaD which acts as a transcrip- tional activator. In this growth condition, the less efficient activation of phaC1ZC2D operon, in the Wt com- pared to the VVD mutant strain, is similar to findings observed for P. putida KT2442 cultured in glucose. In fact for P. putida KT2442 it has already been demonstrated that PhaD controls the carbon source depend- ence of the transcription profile of this operon by an intermediate of fatty acid β-oxidation which acts as a PhaD inducer (de Eugenio et al., 2010). The discovery that P. mediterranea CFBP 5447 lacks the glpF gene contributes to understanding its pro- longed lag growth phase in the presence of glycerol as a carbon source. This gene is present in other Pseudomonas spp. that produce PHAs, such as P. aeruginosa (Schweizer et al., 1997) and P. putida (Es- capa et al., 2013). However, this is the first time to our knowledge that the absence of glpF has been re- ported in a Pseudomonas strain. Our recent acquisition of the draft genome sequence of the closely relat- ed bacteria P. corrugata CFBP 5454 (Licciardello et al., 2014), similarly revealed the absence of this gene, prompting us to further investigation. We plan new strategies for culture optimization in order to improve the efficiency in P. mediterranea CFBP 5447 of the glycerol bioconversion into PHAs which can then be used as coatings for paper materials and plasticizers thus widening the range of blends. Acknowledgments This work has been funded by MIUR by means of the national Program PON R&C 2007-2013, project “Po- lyBioPlast – Technologies and processes for the production of diversely functionalised sheets based on microbial biopolymers and biosurfactants (PON01_1377)”. 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