Indian Journal of Biotechnology Vol 14, October 2015, pp 489-494 Isolation of moderately halophilic bacteria from Tunisian soil samples and transformation by electroporation using Escherichia coli-Halomonas sp. shuttle vector Toru Matsui School of Bioscience and Biotechnology, Tokyo University of Technology, Katakura-cho, Hachioji, Tokyo, 192-0982, Japan Received 19 December 2014; revised 17 July 2015; accepted 3 August 2015 Halophilic and salt-resistant bacteria were isolated from soil samples collected from Tunisia. Among them, strain 21a grew at NaCl concentration ranging 1-15%, optimum at 3%, showing that it is moderately halophilic bacteria. Phylogenetic analysis based on the 16S rRNA gene sequence showed that strain 21a was closely related to Halomonas aquamarina and H. venusta with sequence similarities of 99.7, and 97.1%, respectively, suggesting that the strain belongs to the genus Halomonas. It was transformed by electroporation either with a plasmid vector pCCMR1, constructed with a replication region fragment from a cryptic plasmid of Chromohalobacter marismortui, or a transposon-complex. Segregational plasmid stability of strain 21a harboring pCCMR1 was higher when cultivated in low NaCl concentrations under non-selective pressure. Keywords: Electroporation, Halomonas aquamarina, halophytic, salt-resistant Introduction conjugation6,7 and transformation by electroporation Halophiles are organisms that require salt for growth has not been reported to date, thus limiting genetic and survival. Among halophilic microorganisms, studies of these species. The use of electroporation halobacteria (extremely halophilic aerobic Archaea) procedures has recently gained popularity, mainly to and moderately halophilic bacteria (which grow best in introduce exogenous DNA into cells. This technique media with 0.5-2.5 M NaCl) predominate and have an facilitates the transformation of various organisms, important ecological role in hypersaline environments1. which otherwise failed using other methods like those Although moderate halophiles have recently gained a employed calcium chloride for Escherichia coli and considerable biotechnological interest2, biochemical/ polyethylene glycol for Bacillus subtilis. genetic characterization has been limited to specific Electroporation can increase the efficiency of enzymes and compatible solutes, such as, ectoine and transformation (it is possible to obtain a higher glycine betaine3,4. number of transformants per microgram of DNA), The isolation and physiological and biochemical and the technique is simple and rapid. Transposon- processes of halophilic bacteria have been extensively directed mutagenesis has emerged as a powerful tool studied5, but our knowledge of cellular properties at for the localization of genes in specific chromosomal the molecular level lags far behind those of other locations. Recently, EZ::Tn transposome systems proteobacteria. Moreover, in view of the widespread were constructed to create random transposon industrial and medical applications of halophiles, libraries for both Gram-positive8 and Gram-negative there has been considerable interest in strain bacteria9. However, the genetic analyses of halophiles improvement and genetic transfer systems for have so far been hindered by the lack of an efficient halophilic bacteria, especially for Halomonas or gene transfer system for generating defined mutants. Chromohalobacter2. One of the reasons for this Very recently, application of electroporation for situation is the lack of rapid gene transformation Halomonas sp. was reported but the detailed procedures. The members of the family information was not available10. Halomonadeceae, such as, Halomonas or Here, we report the successful transformation of Chromohalobacter species, mainly undergo moderately halophilic bacteria, Halomonas sp. strain 21a by electroporation with either a plasmid or transposon __________ Tel/Fax: +81-42-637-2302/2129 vector and characterization of the recombinant strain [email protected] harboring a plasmid-type shuttle vector. 490 INDIAN J BIOTECHNOL, OCTOBER 2015 Materials and Methods samples in Tunisia (30 samples), aliquots of samples Bacterial Strains and Growth Conditions spread on 0.1 times concentration of LB medium The bacterial strains and plasmids employed in the containing 1.5% agar were incubated at 30°C for present study are listed in Table 1. C. marismortui 1 to 2 wk, followed by isolation of the colony for NBRC 103155T was used for isolation of its cryptic growing under the same conditions. Growth of plasmid, pCM1. E. coli JM109 was grown in bacterial cells was estimated spectrophotometrically Luria-Bertani (LB) medium at 37°C11. The medium by measuring the optical density (OD) at 660 nm. was supplemented with antibiotics (kanamycin or Maximum specific growth rate (1/h) was estimated by ampicillin) to a final concentration of 100 µg/L plotting the cell concentration against time in a whenever found necessary. Minimal medium (MM) log-linear plot, as described elsewhere13. was prepared as previously described12 and supplemented with 1% glucose as the sole carbon and DNA Manipulations and Molecular Genetic Protocols energy source and designated as MMG. For the PCRs of 16S rRNA, ectB and repA in pCM1 were performed using Ex-Taq (Takara-bio, Shiga, Japan) screening of isolates from soils and pond-water under the following conditions: initial denaturation at Table 1—Bacterial strains and plasmids used in this study 95°C for 5 min, followed by 35 cycles of denaturation Strain or plasmid Description Source of reference at 95°C for 30 sec, annealing at 53°C for 1 min, extension at 72°C for 1 min and a final extension at Bacterial strains E. coli JM109 recA1, endA1, gyrA96, Takara-bio Co. 72°C for 5 min using a thermal cycler type-PC818 - + (Astec Co., Fukuoka, Japan). Primers used for the thi-1, hsdR17(r m ), K K - - PCR are listed in Table 2. Transformation of e14 (mcrA ), supE44, pCCMR1 or EZ::TN Tnp <KAN-2> into halophilic relA1, ∆ (lac-proAB)/F‘ + bacteria was carried out by electroporation as 〔 traD36, proAB , lac I, described by Choi et al15. Competent cells (100 µL) lacZ∆M15〕 and 1 µL of DNA were mixed and electroporated at C. beijerinckii Type strain NBRC* NBRC 103041 2.5 kV, 25 µF, 200 Ω and 0.2-cm cuvette using a C. marismortui Type strain NBRC MicroPulser electroporator (Bio-Rad, Hercules, CA, NBRC 103155T USA). After electroporation, aliquots mixed with Halomonas sp. 21a Isolate from soil in Present study 1 mL LB broth were incubated at 30°C for 3 h, Chebika, Tunisia followed by spreading the broth on LB plates H. pacifica Type strain NBRC NBRC102220 containing 100 mg/L of kanamycin (Km). H. halodenitrificans Type strain NBRC Transposition efficiency was expressed as NBRC14912 Km-resistant colony forming units (CFU) H. halodurans Type strain NBRC per microliter of the transposome used. Other NBRC15607 techniques for DNA manipulations were performed as H. halophila Type strain NBRC 11 NBRC 102604 described by Sambrook and Russell . H. meridiana Type strain NBRC NBRC 15608 Construction of E. coli-Halomonas sp. Shuttle Vector Plasmids The E. coli-Halomonas sp. shuttle vector was pCM1 Cryptic plasmid from C. Mellado et al7 constructed to examine the transformation efficiency of marismortui the pCM1 replicon as previously described7. Plasmid pHSG298 r Takara-bio Co. E. coli cloning vector; Km pCM1 was isolated from C. marismortui NBRC pCCMR1 E. coli-Halomonas sp. Present study 103155T. The replicon was amplified using the primer shuttle vector harboring 1.6 kb fragment from set MR239 and MR240, based on the sequence of the plasmid pCM1 repA gene (X86092), and ligated with pCR2.1 r pT7 (blue) E. coli cloning vector; Ap Takara-bio Co. (Invitrogen Co., CA) resulting in the construction of pCR2.1 E. coli cloning vector; Life Technologies the E. coli-Halomonas sp. shuttle vector, pCCMR1. r r Co. Ap , Km DNA Sequencing EZ-Tnp<KAN-2> Transposon complex Epicentre Co. r Genomic DNA of bacterial strains was isolated vector; Km using ISOPLANT (Nippon-gene, Tokyo, Japan) and *National Bioresource Research Center, Japan used for PCR of the 16S rRNA, and ectB gene locus. MATSUI: HALOPHILIC BACTERIA FROM TUNISIA 491 The BLAST program (http://ncbi.nlm.nih.gov/ 15% NaCl. As shown in Table 3, the isolates BLAST/; NCBI, Bethesda, MD) was used for gene consisted of not only Gram-positive but also Gram- homology search using the standard program default. negative bacteria, based on the 16S rRNA sequence A phylogenetic tree based on the 16S rRNA gene analyses. Growth characteristics of these isolates in sequence was constructed using the neighbor-joining high NaCl concentration revealed that 7 of the 11 method16 with the Kimura two-parameter model as a isolates were halophilic, whereas the other isolates distance corrector17 after alignment of sequences with were salt-resistant. Strain 21a, belonging to family the CLUSTALX multiple sequence alignment Halomonadaceae, class γ-proteobacteria based on the program18. 16S rRNA sequence was selected for further examinations using a plasmid from Nucleotide Sequences Accession Numbers Chromohalobacter sp. (Fig. 1a). It grew in the Sequences of a fragment of the 16S rRNA gene, presence of NaCl, ranging 1-15%, with a maximum from the isolates examined in the present study, had specific growth rate at 5% NaCl in MMG medium been assigned DDBJ/EMBL/GenBank accession (Fig. 2). PCR analysis using degenerate primers numbers as shown in Table 3. Sequence of the DNA showed the existence of homologue ectB, a gene