Microbes Environ. Vol. 21, No. 4, 235–239, 2006 http://wwwsoc.nii.ac.jp/jsme2/

Genetic Transformation System for Members of the Genera, Sphingomonas, , and

MIYUKI SAITO1, YOKO IKUNAGA1, HIROYUKI OHTA1 and YASUROU KURUSU1*

1 Ibaraki University College of Agriculture, 3–21–1 Chuo, Ami, Inashiki, Ibaraki 300–0393, Japan

(Received July 12, 2006—Accepted September 26, 2006)

This paper describes a plasmid transformation system that permits genetic manipulation of the genus Sphin- gomonas. A cryptic indigenous plasmid, pAMI-1, of 10 kb from Sphingobium amiense was isolated and charac- terized. Nucleotide sequencing revealed that pAMI-1 contains five open reading frames, which are predicted to encode proteins associated with integration, recombination, conjugation and replication. -S. amiense shuttle vectors were successfully introduced into Sphingomonas, Sphingobium, Novosphingobium and Sphingopyxis strains by electroporation. The copy number of the shuttle vector was estimated to be 1–2 per chro- mosome in the Sphingobium yanoikuyae cell.

Key words: plasmid, Sphingomonas, Sphingobium, Novosphingobium, Sphingopyxis

Recently, the microbial aerobic degradation of various recently reported for the first time in a prokaryote. This sys- aromatic compounds has been reported9,12). The genus Sph- tem appears to be the origin of endocytosis and phagocyto- ingomonas has received particular attention because it sis in eukaryotes. includes various xenobiotic-degrading . Members of Genetic manipulation of the genus Sphingomonas is nec- this genus are able to degrade compounds such as polycy- essary to improve the ability of these bacteria to degrade clic aromatic hydrocarbons, chlorinated and sulfonated aro- xenobiotic compounds and to elucidate the unique mecha- matics, herbicides, aromatic ethers, and polyethylene nisms involved in degradation; however, a plasmid suitable glycol1). There are several reports indicating that large plas- for genetic manipulation of Sphingomonas has not been mids may be important for the degradation of xenobiotic identified. Some vector systems in sphingomonads with a compounds by Sphingomonas strains. In Sphingomonas broad-host-range plasmid or a cryptic plasmid have been aromaticivorans strain F199T and some other sphin- reported1,11). Also, transformation of sphingomonads by gomonads isolated from the same location, the genes encod- conjugation has been described. Here, we report the isola- ing the machinery for degrading biphenyl, naphthalene, m- tion and characterization of a cryptic indigenous plasmid, xylene, and p-cresol were detected on megaplasmids8,14). pAMI-1, from Sphingobium amiense. Moreover, we Strains of the genus Sphingomonas have a unique character- describe the construction of shuttle vectors based on pAMI- istic: they contain glycosphingolipids, which are ubiquitous 1 and the development of a transformation system for mem- in eukaryotic cell membranes7). When Sphingomonas sp. bers of the genera Sphingomonas, Sphingobium, Novosph- strain A1 assimilates a macromolecule (alginate), a mouth- ingobium, and Sphingopyxis. This is the first report on a like pit (0.02–0.1 µm) is formed on the cell surface through genetic transformation system in these bacteria using an reorganization and/or fluidity of the pleats, causing extra- indigenous plasmid. cellular alginate to be concentrated in the pit5). The pit- dependent system for importing macromolecules was Materials and Methods Bacteria, plasmids, and media * Corresponding author; E-mail: [email protected], Tel: +81– 29–888–8646, Fax: +81–29–888–8525 Sphingomonas strains were routinely cultured at 300°C in 236 SAITO et al.

LB medium. The members of the genus Sphingomonas GCCCATGATGTCACTC-3', and 5'-AAGCATGCCG- sensu lato were classified according to the suggestions of GCACCACGATCA-3' (PstI and SphI sites are underlined) Takeuchi et al.15) as members of the newly created genera for pAMI-K1, pAMI-K2, pAMI-K3, and pAMI-K4, respec- Sphingomonas, Sphingobium, Novosphingobium and tively. PCR products were digested with PstI and SphI and Sphingopyxis. Sphingobium amiense strain JCM11777T then inserted into a PstI/SphI-digested E. coli plasmid vec- containing the plasmid pAMI-1 was isolated originally from tor pHSG298 (Takara Bio, Shiga, Japan), which harbors a a river sediment sample18). Sphingomonas cloacae strain Km resistance gene. The 2.9-kb PCR product amplified JCM10874T 4), Sphingobium yanoikuyae strain JCM737121), with the primers 5'-CCCTGCAGCTTCAAGCATT-3' and Sphingobium chlorophenolicum strain JCM1027513), 5'-GAGCATGCGACGGCGATATACG-3' was also cloned Novosphingobium capsulatum strain JCM745215) and into pHSG398 (Takara Bio) containing a Cm resistance Sphingopyxis alaskensis strain DSM13593T 19) were used for gene to construct pAMI-C1. the transformation experiments. Escherichia coli DH5α [(φ80dlacZ∆M15) endA1 recA1 hsdR17(r−m−) supE44 thi-1 Electroporation λ− gyrA relA1 F− ∆(lacZYA-argF) U169] strains were grown Cells from 50-ml cultures of Sphingomonas strains (opti- at 37°C in LB medium and used as a host for the construc- cal density at 660 nm=0.7 to 0.8) were collected by centrifu- tion of plasmids and routine subcloning. E. coli transfor- gation and washed twice with 10 ml of chilled 10% glyc- mants were selected based on resistance to 20 µg/ml of erol. The cells were resuspended in the same buffer to a chloramphenicol (Cm), 50 µg/ml of kanamycin (Km), or 10 final volume of 100 µl, mixed with plasmid DNA (1 µg), µg/ml of tetracycline (Tc). Transformed Sphingomonas and put into 0.2-cm cuvettes. Electroporation was per- strains were selected based on resistance to 15 µg/ml of Cm, formed using a Gene Pulser (Bio-Rad, Hercules, U.S.A) or 30 µg/ml of Km. with a single pulse at 25 µF and 2.5 kV. The cells were allowed to grow in LB medium for 2 h and then were spread DNA manipulation on selective plates containing for selection. Plasmid DNA was prepared from transformants of E. coli or Sphingomonas by the alkaline lysis method2). Approxi- Nucleotide sequence accession number mately 109 cells were harvested by centrifugation, washed The nucleotide sequence data for the plasmid pAMI-1 with 1 ml of G buffer (50 mM Tris-HCl [pH 8.0], 1 mM appears in the DDBJ, EMBL and Genbank nucleotide EDTA [pH 8.0], and 10% [vol/vol] glycerol) and resus- sequence databases with the accession number DD172051. pended in 100 µl of lysozyme solution (25 mM Tris-HCl [pH 8.0], 1 mM EDTA [pH 8.0], 100 mM NaCl, 15% [wt/ Results and Discussion vol] sucrose, and 0.8% [wt/vol] lysozyme) and incubated at 37°C for 15 min. The cell suspensions were gently mixed Isolation and characterization of a cryptic indigenous with 200 µl of Solution II (0.2 M NaOH and 1.0% [wt/vol] plasmid SDS) and incubated at room temperature until the cells were We isolated a 10-kb cryptic indigenous plasmid, desig- lysed. Subsequently, the cell lysates were mixed with 150 µl nated pAMI-1, from S. amiense strain JCM11777T. To iden- of Solution III (3 M potassium acetate and 11.5% [vol/vol] tify the region essential for plasmid replication, we deter- glacial acetic acid) and placed on ice for 5 min. Finally, the mined the complete nucleotide sequence of pAMI-1. plasmid DNA was prepared by phenol/chloroform extrac- Nucleotide sequence analysis revealed the presence of five tion and precipitation in ethanol and dissolved in TE buffer open reading frames (ORFs) (Fig. 1). Two ORFs, ORF1 (10 mM Tris-HCl [pH 8.0] and 1 mM EDTA) containing 10 (nucleotide positions 9304–9893) and ORF5 (nucleotide µg/ml RNase A. positions 116–3082), were predicted to function in DNA integration or recombination. The deduced amino acid Construction of shuttle vectors sequence of ORF1 was 61% identical to that of DNA The region surrounding the rep gene from pAMI-1 was recombinase from pAG1 of Corynebacterium amplified by PCR using KOD-plus polymerase (Toyobo, glutamicum16). ORF5 shows 39% identity to the transposase Tokyo, Japan) and the following primers: the forward encoded by pVS1 of Pseudomonas aeruginosa3). Two primer 5'-CCCTGCAGCTTCAAGCATT-3'; and the homologs of conjugative genes are found on pAMI-1; spe- reverse primers 5'-GAGCATGCGACGGCGATATACG-3', cifically, the predicted amino acid sequences of ORF2 5'-AAGCATGCCTAATTTTCGCGAC-3', 5'-GGGCAT- (nucleotide positions 7753–8751) and ORF3 (nucleotide Sphingomonas Plasmid 237

Transformation with plasmid by electroporation To construct the host strain for transformation, we iso- lated a pAMI-1-cured strain from S. amiense strain JCM11777T according to Kurusu et al.10). Strain JCM11777T was grown in LB medium supplemented with ethidium bromide (30 µg/ml). After 200 generations of growth, the cultures were spread on LB plates. Screening of 2,000 colonies yielded one pAMI-1-cured colony which was used further as a host strain. A 2.9-Kb fragment containing ORF4 and the surrounding regions of pAMI-1 were cloned into pHSG298 (Kmr) and Fig. 1. Genetic organization of the plasmid pAMI-1. Coding regions r deduced from the complete plasmid sequence of pAMI-1 are pHSG398 (Cm ) to obtain pAMI-K1 and pAMI-C1, respec- shown by arrows indicating the direction of transcription. Arrow- tively. These plasmids were used to transform S. cloacae heads represent direct repeats. strain JCM10874T, the S. amiense pAMI-1-cured strain, S. yanoikuyae strain JCM7371, S. chlorophenolicum strain JCM10275, N. capsulatum strain JCM7452, and S. alasken- positions 2778–3100) are 39% and 40% identical to the sis strain DSM13593T by electroporation, and Km- or Cm- conjugation protein TraA and mobilization protein MobS, resistant transformants were obtained (103–104/µg DNA). respectively. TraA is found on the chromosome of Agrobac- Plasmid DNA extracted from the transformants by the alka- terium tumefaciens20) and MobS is found on pMG160 of line lysis method was subjected to Southern blot analysis Rhodobacter blasticus6). The predicted amino acid sequence using pHSG298 or pHSG398 as a probe. Bands correspond- of ORF4 (nucleotide positions 5073–5723) is 55% identical ing to pAMI-K1 or pAMI-C1 were observed in all the trans- to a putative Rep (replication) protein located on pJK21 of formants (Fig. 2). Therefore, these vectors are useful for the Gluconacetobacter europaeus17). We also identified two 25- genetic analysis of Sphingomonas strains including the bp direct repeats upstream of ORF4. We identified several newly defined genera Sphingomoans, Sphingobium, genes associated with integration, recombination, and con- Novosphingobium, and Sphingopyxis. jugation in pAMI-1, suggesting that this plasmid can be integrated with and excised from the chromosomes and/or Properties of the composite plasmids other plasmids. Moreover, these proteins did not have sig- To determine the minimal region necessary for replica- nificant homology with those found on pNL114), which is a tion of the 2.9-kb fragment containing the rep gene, we completely sequenced megaplasmid of S. aromaticivorans transformed S. yanoikuyae strain JCM7371 with pAMI-K1 strain F199T. Therefore, pAMI-1 and pNL1 may have dif- derivatives containing various deletions (Fig. 3). When ferent origins. pAMI-K2 and pAMI-K3 were introduced into S. yanoikuyae strain JCM7371, Kmr transformants were obtained. However, no transformant was obtained with

Fig. 2. Detection of plasmids pAMI-K1 (left) and pAMI-C1 (right) in various strains. Plasmid DNA was extracted from cells transformed with pAMI-K1 or pAMI-C1, fractionated by 0.8% agarose gel electrophoresis, and transferred to a nitrocellulose filter. Plasmid DNA of pHSG298 (left) or pHSG398 (right) was used as a probe. Lanes: M, λ-HindIII digest marker; 1, S. amiense pAMI-1-cured strain; 2, S. yanoikuyae strain JCM7371; 3, S. chlorophenolicum strain JCM10275; 4, S. cloacae strain JCM10874T; 5, N. capsulatum strain JCM7452; C1, pAMI-K1; C2, pAMI-C1. 238 SAITO et al.

Fig. 3. The ability to transform S. yanoikuyae strain JCM7371 of deletion derivatives of pAMI-K1. Thick bars represent the fragments cloned into pHSG298. Arrowheads represent direct repeats. The transformation of S. yanoikuyae strain JCM7371 is described in the Materials and Methods.

Fig. 4. Estimation of the plasmid copy number. A. Construction of the plasmid for determination of the plasmid copy number. A 1.1-kb SalI fragment from S. yanoikuyae strain JCM7371 chromosomal DNA was obtained by shotgun cloning. A physical map of the1.1-kb SalI fragment is shown. Numbers in the 1.1-kb SalI fragment indicate the size of the SalI restriction fragments (1 to 1145 bp). Plasmids pAMI-K1, pAMI-KCH, and pAMI-KCHT are described in the text. S, SalI restriction site; E, EcoRV restriction site; Kmr, Km resistance gene; Tcr, Tc resistance gene. B. Genomic southern hybridization analysis. Cells containing pAMI-KCHT used in the growth experiments were harvested at the early stationary phase, and total DNA was extracted and digested with SalI. Various amounts (1.0, 5.0, and 10.0 µg) of DNA were fractionated by 0.8% agarose gel electrophoresis and trans- ferred to a nitrocellulose filter. The 1.1-kb SalI fragment of the S. yanoikuyae strain JCM7371 chromosomal DNA was used as a probe for simultaneously detecting the pAMI-KCHT fragment (3.1 kb) and the chromosomal DNA fragment (1.1 kb). The density of various DNA bands was normalized with respect to the 1.1-kb DNA band using 1.0 µg of DNA. pAMI-K4. These results suggested that the replication func- To estimate the copy number of the plasmid pAMI-K1, tion in S. yanoikuyae strain JCM7371 is located within a we determined the amount of plasmid relative to the amount 1.4-kb fragment of pAMI-1. of chromosomal DNA by Southern blot hybridization. First, Sphingomonas Plasmid 239 we cloned a 1.1-kb SalI fragment from S. yanoikuyae strain 8) Kim, E., P.J. Aversano, M.F. Romine, R.P. Schneider and G.J. JCM7371 chromosomal DNA into the SalI site of pAMI-K1 Zylstra. 1996. 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