Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas

Bacterial strains and plasmid isolation

Plasmid sequencing and analysis

The plasmid isolated from BSi20429 was termed as pSM429. Analysis of restriction digests on agarose gel showed that pSM429 contains singular restriction site for Hin dIII and Nde I, respectively. The plasmid was linearised by these two restriction enzymes, and restriction fragments of different sizes were cloned into pGEM-T Easy and sequenced by dideoxy chain termination method using an Applied Biosystems DNA sequencer. The nucleotide sequence of pSM429 was determined by using a combination of subcloning and primer walking. The whole plasmid sequence was covered at least three times by single read. The nucleotide sequence, amino acid sequence and the distribution of direct and inverted repeated sequences were analyzed using DNASTAR. The predicted protein-coding regions were initially defined by searching for ORFs longer than 80 codons. The potential coding regions were then confirmed with prokaryotic gene finder GeneMark.hmm 2.4 for Prokaryotes [20] at http://opal.biology.gatech.edu/GeneMark/gmhmm2_prok.cgi using Pseudoalteromonas haloplanktis TAC125 chromosome I genome as a model. The functional protein domain analysis of predicted ORFs was performed by the InterProScan program at the European Bioinformatics Institute http://www.ebi.ac.uk/InterProScan/. Similarity searches were carried out using Blast http://www.ncbi.nlm.nih.gov/blast/[21].

The nucleotide sequence of pSM429 was deposited in the GenBank database under the accession number EU627679.

Determination of plasmid copy number

The copy number of plasmid pSM429 in strain BSi20429 was determined using the method described by Qin et al. [22]. Strain BSi20429 was grown in marine LB broth at 20°C and total genome DNA was isolated by using PowerMax™Soil DNA Isolation Kit (MO BIO). The genome DNA was diluted by 10 and 100 times in TE Buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5), and then was separated in equal volumes on a 0.7% agarose gel by electrophoresis at 50 V for 2.5 h. By comparing the intensity of the diluted chromosomal DNA band with the non-diluted plasmid DNA band using the image analysis software Gene Tools (Syngene), the copy number of pSM429 was calculated as follows:

(N _c , copy number of the plasmid; S _c , chromosomal DNA size (4.5 M); S _p , plasmid DNA size (3.78 kb); I _p , intensity of plasmid DNA band; I _c , intensity of chromosomal DNA band).

Curing of pSM429 from BSi20429

Construction of the shuttle vector pWD and its derivatives

Plasmids pSM429 and pUC19 were isolated as described above. The complete DNA sequence of pSM429 was amplified by PCR with primers AF (containing a Sal I site) and AR (containing a Bam HI site). The 3.8-kb linearised pSM429 was digested with Sal I and Bam HI and cloned in pUC19. The chloramphenicol acetyl transferase (CAT) cassette was amplified by PCR from pBT (Stratagene), using primers CB (containing a Bam HI site) and CE (containing an Eco RI site). The 1.3-kb product was double-digested using Bam HI and Eco RI and ligated to the constructed pUC19+pSM429 treated with the same restriction enzymes, and then transformed into E. coli DH5α. The constructed shuttle vector was designated as pWD.

Derivatives of pWD (pWD1, pWD2, pWD3, pWD4, pWD5, pWD6) were constructed as outlined in Figure 1. Purified DNA of pSM429 was used as a template in PCR. All primers were designed with sites for Sal I or Bam HI at their 5' ends to allow direct cloning of the amplicons digested with these two enzymes into the Sal I and Bam HI sites of pWD. In new constructs, the complete fragment of pSM429 was replaced by the amplicons with different length. New constructed derivatives were transformed into E. coli DH5α. Plasmids isolated from E. coli DH5α and verified by sequencing were electroporated into SM20429.

Electroporation

The protocol to electroporate plasmids into SM20429 was based on the method described by Kurusu et al. [23] with some modification. A 1% inoculum from over-night culture of SM20429 in 2216E medium was inoculated into 20 ml of the same culture and grown at 20°C. After incubation for 4 h, the logarithmic-phase cells were harvested and washed twice with chilled ddH₂O. The cells were suspended in a final volume of 100 μl ddH₂O containing 20 mM glucose, and mixed with 1 μg plasmid DNA. Electroporation was performed with a Gene Pulser (Bio-Rad) at a capacity of 50 μF and a voltage of 2.0 kV. After one pulse, the cells were grown in 2216E medium supplemented with glucose (20 mM) for 3 h, and then were spread on selective plates with chloramphenicol.

Analysis of plasmid stability

SM20429 strains with plasmid pWD or other derivative plasmids were grown in 2216E broth for about 24 generations by being serially transferred to fresh broth medium. Samples were removed from each transfer and serial dilutions of the sample were plated on 2216E agar medium with or without antibiotics. The fraction of chloramphenicol-resistant (CmR) cells in the total population was determined to calculate the segregational stability of the constructed plasmid vectors. Finally, presence of plasmid DNA in CmR cells was confirmed by plasmid isolation and transformation to E. coli DH5α.

Construction of expression system in SM20429

The erythromycin resistance gene (ermC) from plasmid pMG36e [24] was amplified by PCR using the primers EMF (containing Pst I restriction site) and EMR (containing Sal I restriction site) to construct an ermC-expression vector. The 735-bp PCR product was cloned into vector pMD19T simple (Takara). The fragment of the promoter-less ermC gene in the pMD19T was separated with Pst I and Sal I and was used in the construction of the ermC-expression vector with the smallest stable plasmid pWD2.

The promoter-less erythromycin resistance gene was cloned into pWD2 downstream of the Plac of pUC19. The constructed ermC-expression vector was designated as pWD-ermC. This expression vector was electroporated into SM20429. The transformed SM20429 cells were cultured at 25°C in 2216E broth for 24 h. To test the gene expression in SM20429, the total cell lysates of recombinant strain SM20429 expressing ermC under the control of Plac were analyzed by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). To test whether the recombinant ErmC was active, the transformed SM20429 cells harboring pWD2 and the transformed SM20429 cells harboring pWD-ermC were inoculated in 2216E medium supplemented with 30 μg/ml or 60 μg/ml erythromycin (EM), respectively, and then cultured at 25°C.

Gene cloning and expression of the catalytic domain of the cold-adapted cellulase from P. sp. BSw20308 and cellulase assay

A gene encoding a cold-adapted cellulase Cel308 (GenBank Accession No. HQ997897) has been cloned from the psychrophilic strain Pseudoalteromonas sp. BSw20308 isolated from the Arctic sea ice in our lab. The DNA fragment encoding the catalytic domain (CD) of the cellulase was amplified by PCR from BSw20308, and then ligated into pWD2 to construct the expression vector pWD-cd. The vector pWD-cd was introduced into SM20429 by electroporation for the production of the CD. The transformed SM20429 cells were cultured at 25-30°C in 2216E broth. To analyze the expression of the CD of the cellulase, the cellulase activity in the transformed SM20429 cells was assayed. After grown at 25-30°C for 48 h, the SM20429 cells (3 ml) were harvested and resuspended in 5 ml PBS buffer (0.02 M, pH 7.5), and then sonicated in an ice-water bath. After centrifugation, the cellulase activity in the supernatant was measured as described by Bhat et al. [25]. A mixture containing 100 μl of the supernatant and 900 μl of 1% sodium carboxyl methyl cellulose was incubated at 35°C for 30 min. After incubation, the released amount of reducing sugar was measured using the dinitrosalicylic acid (DNS) method with glucose as standard [26]. One unit of the enzyme activity was defined as the amount of enzyme to release 1 μg of glucose per min.

Characterization of the cryptic plasmid from BSi20429

Searches in the databases revealed that ORF4 showed moderate similarity (25-26% identity, 50% similarity) with the Rep proteins of pFKN [29] and the other plasmids belonging to pPT23A family from Pseudomonas syringae[30, 31]. It also showed some similarity (with identity lower than 26%) with the Rep proteins of the ColE2-related plasmids, as well as the Rep proteins of plasmids isolated from the bacteria of different genus. As plasmids are generally classified based on the amino acid sequence similarity between their replication initiator proteins [32], a phylogenetic tree was constructed using Rep amino acid sequences of some related plasmids as shown in Figure 2. pFKN and pAV505 isolated from Pseudomonas syringae were selected from pPT23A group and the other plasmids were found by homology search using putative Rep amino acid sequence of pSM429. All of the 12 Rep proteins in the phylogenetic tree contain two conserved domains [33], a replicase domain (Pfam Accession No. PF03090) believed to be involved in DNA binding [34], and an alpha helical domain (Pfam Accession No. PF08708) that was found at the C terminus of primases [35]. This suggests that these Rep proteins might be involved in the replication with the similar mechanism. However, pSM429 did not cluster with other plasmids in the phylogenetic tree, implying that pSM429 is a novel plasmid.

Construction of the shuttle vector pWD and transformation system

The shuttle vector pWD, based on plasmid pSM429, was constructed for replication in E. coli and Pseudoalteromonas strains (Figure 1). The 3.8-kb plasmid pSM429 was linearised and cloned in pUC19. In addition, the 1.3-kb chloramphenicol resistance gene was inserted into the resulted plasmid as selection marker to construct the shuttle vector pWD. The presence of the pUC19 ori and pSM429 ori in pWD enabled pWD to replicate in both E. coli and BSi20429. The constructed plasmid pWD could be purified from E. coli in a large amount.

To construct the host strain for the plasmid transformation, the plasmid pSM429 was cured from BSi20429 using the SDS-treated method. Screening of 40 colonies yielded 4 plasmid-cured colonies, showing an efficiency of 10%, which is much more effective than the method used in Pseudoalteromonas strains previously reported [23]. One plasmid-cured strain showing a similar phenotype to the parental strain was designated as SM20429, which was used for further study. The total DNA of SM20429 was purified and used as template for PCR checking. The results showed that the plasmid did not integrate into the genome of SM20429. As SM20429 was sensitive to erythromycin and chloramphenicol (data not shown), chloramphenicol was used for the direct selection of Pseudoaltermonas transformants. The constructed shuttle vector pWD was introduced into SM20429 by electroporation. Chloramphenicol-resistant transformants of SM20429 were obtained at the frequency of 5.0x 10²/μg DNA. To confirm that the Pseudoalteromonas transformants harbored plasmids, several transformants were subjected to plasmid analysis. Electrophoresis showed that plasmids were prepared from all of the selected transformants, and the prepared plasmids showed the same electroporetic DNA pattern as that of pWD (Additional file 2, Figure S2). Moreover, pWD DNA prepared from the Pseudoalteromonas transformants could be transformed into SM20429 at a higher frequency (10⁴/μg DNA) than that prepared from E. coli transformants, suggesting the presence of DNA restriction and modification system in BSi20429.

Properties of the shuttle vector pWD and its derivatives

The shuttle vector pWD constructed above harbors the complete sequence of plasmid pSM429. To check the functionality of the identified ORFs on pSM429 and delete the unnecessary regions on pWD for an expression vector, six plasmids derived from pWD with different deletions were constructed (Figure 1). Each of the six derivatives was checked in E. coli, and then was introduced into SM20429 by electroporation. Except for the two smallest derivatives, pWD5 and pWD6, transformants of the other four derived plasmids were all obtained, indicating that pWD5 and pWD6 were not capable of replication in SM20429. Since pWD5 and pWD6 lack the upstream or the downstream sequences of the rep gene (orf4) compared to pWD4, the results indicated that the fragment containing rep gene and its flanking sequences are the minimal replication region of pSM429 and all of orf1, orf2 and orf3 are not essential for plasmid replication.

The segregational stability of pWD and its four derivatives were tested twice and counts were done in duplicate. Average results were presented in Figure 3. In the absence of selective pressure, nearly 100% of the cells retain plasmids in the first 40 generations of SM20429. However, after 40 generations, the smaller derivatives, pWD3 and pWD4, exhibited less segregational stability than pWD and the other bigger derivatives, pWD1 and pWD2, in SM20429. Compared to pWD2, pWD3 lacks some repeat sequences located between orf1 and orf2, as shown in Figure 4. The above result suggests that these repeat sequences are essential for the segregational stability of the plasmid and possibly related to plasmid partition.

Construction of an ermC-expression vector and expression of ErmC in SM20429

The erythromycin resistance gene ermC was cloned into pWD2 downstream of the Plac promoter to construct an ermC-expression vector, pWD-ermC. This vector was introduced into SM20429 by electroporation and the ermC gene was expressed under the control of Plac promoter. SDS-PAGE analysis showed that the ErmC protein was expressed in SM20429 cells, and the expression level of ErmC in SM20429 was lower than that in E. coli DH5α (Figure 5A). To test whether the expressed ErmC protein is active, the SM20429 strain harboring pWD2 and the SM20429 strain harboring pWD-ermC were inoculated in 2216E medium supplemented with erythromycin (EM), respectively. The pWD2-harboring strain that is sensitive to EM as the wild strain BSi20429 could not grow in the medium supplemented with 30 μg/ml EM, while the pWD-ermC-harboring strain grew well in the medium supplemented with 60 μg/ml EM (Figure 5B). Therefore, the pWD-ermC-harboring strain obtained the ability resistant to EM, indicating that the ErmC protein expressed in SM20429 was active. These results suggest that Plac promoter is efficient for heterologous gene expression in SM20429.

Expression of cold-adapted protein with the constructed expression system

In our previous work, a cellulase named Cel308 was purified from the Arctic sea ice bacterium Pseudoalteromonas sp. BSw 20308 (Additional file 3, Figure S3), and the gene encoding Cel308 was cloned and sequenced. Character analysis showed that Cel308 is a cold-adapted enzyme (Additional file 3, Figure S4). The cd gene encoding the catalytic domain of Cel308 was cloned into pWD2 to construct a cd-expression vector pWD-cd. The plasmid pWD-cd was transformed into SM20429 by electroporation. After cultivation for 48 h, the intracellular cellulase activity of the transformed strain was detected using a quantitative assay. The results showed that 60 units of cellulase enzyme were expressed in 1 ml culture of SM20429. In comparison, no cellulase activity was detected in the cells of SM20429 harboring pWD2 (Figure 6). The results indicated that the constructed expression system consisting of strain SM20429 and vector pWD2 could be used to express cold-adapted protein.