Genome analysis of thirteen Colombian clostridial strains by pulsed field gel electrophoresis

Electronic Journal of Biotechnology ISSN: 0717-3458 © 2006 by Pontificia Universidad Católica de Valparaíso -- Chile Vol.9 No.5, Issue of October 15, 2006 Received November 3, 2005 / Accepted April 4, 2006 RESEARCH ARTICLE DOI: 10.2225/vol9-issue5-fulltext-3 Genome analysis of thirteen Colombian clostridial strains by pulsed field gel electrophoresis Diana Milena Quilaguy Ayure Bioprocesses and Bioprospecting Group Instituto de Biotecnología Universidad Nacional Ciudad Universitaria, Edificio Manuel Ancizar Bogotá, Colombia Tel: 571 3165000. Ext.16970/71 Fax: 571 3165415 E-mail: Zulma Rocío Suárez Moreno* Bioprocesses and Bioprospecting Group Instituto de Biotecnología Universidad Nacional Ciudad Universitaria, Edificio Manuel Ancizar Bogotá, Colombia Tel: 571 3165000. Ext.16972/70 Fax: 571 3165415 E-mail: Fabio Ancizar Aristizábal Gutierrez Bioprocesses and Bioprospecting Group Instituto de Biotecnología Universidad Nacional Ciudad Universitaria Bogotá, Colombia Tel: 571 3165000. Ext.14643 Fax: 571 3165415 E-mail: Jose Mauricio Bernal Morales Bioprocesses and Bioprospecting Group Instituto de Biotecnología Universidad Nacional Ciudad Universitaria, Edificio Manuel Ancizar Bogotá, Colombia Tel: 571 3165000. Ext.16970/71 Fax: 571 3165415 E-mail: Dolly Montoya Castaño Bioprocesses and Bioprospecting Group Instituto de Biotecnología Universidad Nacional Ciudad Universitaria, Edificio Manuel Ancizar Bogotá, Colombia Tel: 571 3165000. Ext.16952 Fax: 571 3165415 E-mail: Financial support: Colciencias, Universidad Nacional de Colombia. Keywords: Clostridium, genome size, PFGE, pSOL1. Abbreviations: AFLP: amplified fragment length polymorphism ATCC: American Type Culture Collection bp: base pairs * Corresponding authors This paper is available on line at http://www.ejbiotechnology.info/content/vol9/issue4/full/3/ Quilaguy Ayure, D.M. et al. (CHEF)-PFGE: contour-clamped homogeneous electric field EC: lysis buffer EDTA: ethylenediaminetetracetic acid ES buffer: EDTA–sarcosine buffer ESP: EDTA-sarcosine-proteinase buffer ET buffer: EDTA-Tris Buffer (Tris-HCl 10 mM, EDTA 100 mM) IBUN: Instituto de Biotecnología de la Universidad Nacional Mbp: mega base pairs OD: optical density ORF: open reading frame PCR: polymerase chain reaction PFGE: pulsed field gel electrophoresis PMSF: phenylmethylsulphonyl fluoride RCM: reinforced clostridial medium RPM: Revolutions Per Minute TBE: Tris-borate-EDTA buffer TE: Tris-EDTA buffer (Tris-HCl 10 mM, EDTA 1 mM) Pulsed field gel electrophoresis was used for estimating the size of the genome and evaluating the presence of megaplasmids in 13 native Colombian solventogenic Clostridium strains. DNA preparation and purification were optimised for obtaining differentiated restriction fragments in electrophoresis. Genomic DNA was digested with ApaI, Eco52I, SmaI and XhoI enzymes. Estimated genome size for native strains ranged from 4.0 to 4.2 mega base pairs. Larger sized plasmids were detected and the presence of genes related to megaplasmid pSOL1 was determined by polymerase chain reaction. adc gene region amplification suggested that genes related to solventogenesis in native strains may be located in an extra-chromosomal element. Determining genome size provides useful information aimed at enhancing native strains' solvent production. 2001). The strains' ability to produce 1,3 propanediol was also evaluated. Multivariate data analysis was used for taxonomically correlating phenotyping and genotyping results; it suggested that 10 out of the 13 native strains could be considered as being a new specie (unpublished data). This research was aimed at estimating the size of the genome for native strains, detecting megaplasmids and evaluating the presence of the pSOL1 plasmid containing important genes encoding enzymes involved in solventogenesis. Determining the presence of solventogenic genes is useful for enhancing native strains' solvent production by means of metabolic engineering. Determining genome size would also contribute towards native strains' taxonomy. MATERIALS AND METHODS Research aimed at gaining knowledge about genetic material has often relied on estimating genome size (Fonstein and Haselkorn, 1995). PFGE has been used for estimating the genome size of different Clostridium strains: 3.5-6.5 Mbp for C. acetobutylicum (Wilkinson and Young, 1993), 5.3 Mbp for C. saccharobutylicum NCP 262 (Keis et al. 2001), 4.0 Mbp for C. botulinum type A (Lin and Johnson, 1995) and 3.8 Mbp for group II (Hielm et al. 1998). The C. acetobutylicum ATCC 824 genome was sequenced and its size was determined to be 3.9 Mbp. It was also determined that the pSOL1 megaplasmid size was 192 Kbp (Nölling et al. 2001). This work is related to 13 native bacterial strains from the Clostridium genus, selected from a strain-bank (consisting of 178 isolates from different Colombian soils), based on their greater ability to produce total solvents than the Clostridium acetobutylicum ATCC 824 strain (Montoya et al. 2000). Native strains were molecularly characterised by sequencing the 16S rRNA gene (Montoya et al. 1999), DNA-DNA hybridisation (unpublished data), plasmid profile characterisation (Arévalo et al. 2002), amplified fragment length polymorphism (AFLP) (Jaimes et al. 2005) and pulsed field gel electrophoresis (PFGE) (Montoya et al. Bacterial strains and culture medium Clostridium IBUN 22A, IBUN 125C, IBUN 140B, IBUN 62F, IBUN 95B, IBUN 13A, IBUN 18A, IBUN 18S, IBUN 62B, IBUN 137K, IBUN 158B, IBUN 18Q and IBUN 64A native strains were used in this study (Montoya et al. 2000). The Clostridium acetobutylicum ATCC 824 strain was used as pattern as it is known to be solventogenic. Cells were activated from strains conserved in silica gel. They were grown in RCM medium (OXOID reinforced Clostridia medium) which had been previously gassed with nitrogen to create anaerobic conditions; all cultures were incubated at 37ºC following the methodology described by Montoya et al. 2000. DNA preparation DNA was prepared in agarose plugs, according to a modified protocol described by Montoya et al. 2001. Bacteria were grown to 0.3 to 0.4 OD (680 nm) in 40 ml RCM medium; up to 180 µg/ml thiamphenicol was then added and bacteria were incubated at 37ºC for 1 hr. Bacterial cultures were placed in an ice bath for 30 min. 542 Genome analysis of thirteen Colombian clostridial strains by pulsed field gel electrophoresis a Table 1. Number and sizes of restriction fragments with C. acetobutylicum ATCC 824 genomic DNA. Fragment Nº 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Estimated genome size (Kbp) 706 ± 21 657 ± 25 523 ± 22 485 ± 12 413 ± 18 379 ± 19 352 ± 15 295 ± 22 283 ± 16 212 ± 11 192 ± 12 181 ± 12 136 ± 12 109 ± 9 88 ± 11 71 ± 7 ApaI Standardised b data x x 523 ± 22 485 ± 12 431 ± 18 379 ± 16 352 ± 15 295 ± 18 283 ± 16 c 212 ± 11 192 ± 12 181 ± 12 136 ± 12 109 ± 9 88 ± 11 71 ± 7 5,081 3,931 ApaI 681 592 465 201 184 149 106 74 13 10 Eco52I Standardised d data 1,355 5 (...truncated)