Characterization of a cryptic plasmid pSM429 and its application for heterologous expression in psychrophilic Pseudoalteromonas
© Zhao et al; licensee BioMed Central Ltd. 2011
Received: 31 January 2011
Accepted: 5 May 2011
Published: 5 May 2011
Pseudoalteromonas is an important genus widespread in marine environment, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. By now, there are only a few genetic systems for Pseudoalteromonas reported and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains. The aim of this study is to develop a heterologous expression system for psychrophilic Pseudoalteromonas.
A cryptic plasmid pSM429 isolated from psychrophilic Pseudoalteromonas sp. BSi20429 from the Arctic sea ice, was sequenced and characterized. The plasmid pSM429 is 3874 bp in length, with a G+C content of 28%. Four putative open reading frames (ORFs) were identified on pSM429. Based on homology, the ORF4 was predicted to encode a replication initiation (Rep) protein. A shuttle vector (Escherichia coli, Pseudoalteromonas), pWD, was constructed by ligating pSM429 and pUC19 and inserting a chloramphenicol acetyl transferase (CAT) cassette conferring chloramphenicol resistance. To determine the minimal replicon of pSM429 and to check the functionality of identified ORFs, various pWD derivatives were constructed. All derivatives except the two smallest ones were shown to allow replication in Pseudoalteromonas sp. SM20429, a plasmid-cured strain of Pseudoalteromonas sp. BSi20429, suggesting that the orf4 and its flanking intergenic regions are essential for plasmid replication. Although not essential, the sequence including some repeats between orf1 and orf2 plays important roles in segregational stability of the plasmid. With the aid of pWD-derived plasmid pWD2, the erythromycin resistance gene and the cd gene encoding the catalytic domain of a cold-adapted cellulase were successfully expressed in Pseudoalteromonas sp. SM20429.
Plasmid pSM429 was isolated and characterized, and the regions essential for plasmid replication and stability were determined, helping the development of pSM429-based shuttle vectors. The shuttle vectors pWD and its derivatives could be used as cloning vectors for Pseudoalteromonas, offering new perspectives in the genetic manipulation of Pseudoalteromonas strains. With the aid of pWD-derived vector and its host, the erythromycin resistance gene and the cd gene of a cold-adapted protein were successfully expressed, indicating that the potential use of this system for recombinant protein production, especially for cold-adapted proteins.
Cold-adapted bacteria are excellent candidates for investigations of protein evolution and molecular adaptations to extreme conditions. Moreover, cold-adapted enzymes from psychrophiles offer novel opportunities for biotechnological applications [1, 2]. Native plasmids from cold-adapted bacteria are of particular interest, because plasmids contribute directly in the adaptation of bacteria to their natural environments and provide easy model systems for investigation of basic molecular processes . Furthermore, native plasmids can be used in developing genetic systems.
Pseudoalteromonas is a genus of gamma-proteobacteria that is widespread in the world's oceans, and a lot of psychrophilic Pseudoalteromonas strains thrive in deep sea and polar sea. The genus Pseudoalteromonas contains over 30 marine species , which play an important role in marine ecosystem. Pseudoalteromonas has attracted significant interest for two reasons. First, Pseudoalteromonas species are important for investigation of microbe-host interactions in the sea. Second, many species synthesize biologically active molecules, such as extracellular antibiotics, toxins, extracellular enzymes and polysaccarides [4–8]. However, by now there are only a few genetic systems for Pseudoalteromonas reported [9–12] and no commercial Pseudoalteromonas genetic system is available, which impedes the study of Pseudoalteromonas, especially for psychrophilic strains.
In our laboratory, psychrophilic Pseudoalteromonas strains have been studied for over ten years on their cold-adaptation mechanism and the proteases they secreted [5, 13–18]. To further study the cold-adaptation mechanism of psychrophilic Pseudoalteromonas and protease expression, development of genetic systems for gene knockout and cold-adapted protein expression is essential. In this article, a small cryptic plasmid from psychrophilic Pseudoalteromonas sp. BSi20429 (BSi20429 thereafter), designated pSM429, was isolated and characterized. A shuttle vector pWD was constructed based on pSM429. The regions responsible for replication and stability on pWD were determined by studying the replication of pWD-derived plasmids in Pseudoalteromonas sp. SM20429 (SM20429 thereafter), a plasmid-cured strain of BSi20429. A stable pWD-derived plasmid, pWD2, was successfully used as an express vector to express the erythromycin resistance gene and the cd gene of a cold-adapted cellulase in SM20429.
Materials and methods
Bacterial strains and plasmid isolation
Plasmids used in this study
Source or reference
Cloning vector, Apr
Cloning vector, Apr
Erythromycinr gene source for vector construction
CAT cassette source for vector construction
Cloning vector, Apr
Cryptic plasmid from P. sp. BSi20429
E. coli-Pseudoalteromonas shuttle vector
pWD derivative, 1500-bp fragment deleted from pWD
pWD derivative, 250-bp fragment deleted from pWD1
pWD derivative, 150-bp fragment deleted from pWD2
pWD derivative, 320-bp fragment deleted from pWD3
pWD derivative, 280-bp fragment downstream deleted from pWD4
pWD derivative, 140-bp fragment upstream deleted from pWD4
pWD2-derived ermC expression vector, Plac-ermC fusions
pWD2-derived cd expression vector, Plac-cd fusions
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  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/.
The nucleotide sequence of pSM429 was deposited in the GenBank database under the accession number EU627679.
Determination of plasmid copy number
(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
Pimers used in this study
Sequence (5'-3') *
CGCGGATCC ACGCACCACCCCGTCAGTAG (BamHI)
CCGGAATTC GAAGCACACGGTCACACTG (EcoRI)
AACTGCAG CTAATTTTATAAGGAG (PstI)
ACGCGTCGAC GTTAAGGGATGCAG (SalI)
ACGCGTCGAC TTTACCCTTACGCATAC (SalI)
CGGGATCC TTACGCTTCGCTTGTCTG (BamHI)
ACGCGTCGAC AAGACACTGTGAAGGC (SalI)
CGGGATCC TGCCTTTAAGATTTGC (BamHI)
CGGGATCC AGTCATTCTTGATTAAGTAC (BamHI)
ACGCGTCGAC GAATGGTTAGCCCTTAAG (SalI)
CGGAATTC GGAGCTGCATGTGTC (EcoRI)
ACGCGTCGAC TCTTCCGCTTCCTC (SalI)
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.
The protocol to electroporate plasmids into SM20429 was based on the method described by Kurusu et al.  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  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. . 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 . 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
ORF analysis of pSM429 from Pseudoalteromonas sp. BSi20429
Best BLAST Match
Hypothetical protein of
Hypothetical protein of
Hypothetical protein of
Replication protein A of
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 . 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.
Construction of an ermC-expression vector and expression of ErmC in SM20429
Expression of cold-adapted protein with the constructed expression system
As the first step to develop an effective genetic system for Pseudoalteromonas strains, especially the psychrophilic ones, the Pseudoalteromonas strains isolated from the Arctic sea ice were screened for plasmids. Only one small plasmid, pSM429, was obtained out of over one hundred strains, suggesting that small plasmids from the Arctic sea ice isolates of Pseudoalteromonas are less common. This finding is consistent with the fact that only a few reports have been published about the isolation and characterization of plasmids from cold-adapted Pseudoalteromonas strains, including Pseudoalteromonas sp. PS1M3 , Pseudoalteromonas haloplanktis TAC125  and Pseudoalteromonas sp. 643A . pSM429 has much lower G+C content than other Pseudoalteromonas plasmids, implying its cold-adaptation. The small plasmid pSM429 is valuable for expanding the knowledge about the genetics of extrachromosomal elements in cold-adapted Pseudoalteromonas strains and opens the possibility to construct a genetic system for Pseudoalteromonas strains.
Genetic systems require stable cloning and expression vectors that allow efficient cloning and expression of homologous and heterologous genes. To develop a genetic system of psychrophlic Pseudoalteromonas, a plasmid-cured strain of BSi20429 was obtained to be used as an expression host, and a shuttle vector for Pseudoalteromonas-E. coli was constructed based on pSM429. Sequential deletions were used to study the functionality of ORFs in plasmid pSM429 and to analyze their effect on plasmid stability. The results showed that the rep gene is essential for the autonomous replication of pSM429 in SM20429. This is consistent with the finding that plasmids need specific replication protein to recognize and cut the double-stranded origin of replication for the process to begin . Furthermore, deletion of surrounding sequences of rep gene caused the loss of replication ability of the plasmid in SM20429, probably because these regions contain the promoter sequence of rep gene and the replication origin where the Rep protein binds. Although not essential, an intergenic fragment including some repeat sequences on pSM429 markedly affects the segregational stability. Accordingly, the stable construction pWD2 containing the replication region and the intergenic region related to segregational stability was used as an E. coli- Pseudoalteromonas shuttle vector and for construction of expression vectors.
An expression system in cold-adapted bacteria may circumvent the problem of limited stability of the product that is always encountered during heterogonous expression of psychrophilic proteins in mesophilic hosts [15, 38]. A few low-temperature expression systems have been reported [39, 40]. However, there are still many cold-adapted proteins could not be successfully expressed by a recombinant protein expression system. The alternative systems would be important for the study of cold-adapted proteins. In order to check the usefulness of plasmid pWD2 and its host SM20429 as expression system, the promoter-less ermC gene was cloned into the vector pWD2 and introduced into SM20429. The results showed that the ermC gene was expressed in SM20429 and the recombinant strain displayed resistance to erythromycin, suggesting that the Plac promoter was efficient in SM20429. Then, with this system, the cd gene encoding the catalytic domain of a cold-adapted cellulase from a psychrophilic strain was successfully expressed, suggesting the potential use of this system for overexpression of genes from psychrophiles. Indeed, this low-temperature expression system is still in an early stage of development. Further improvement, such as increasing the yield of proteins and controlling the expression of proteins, is being underway.
In this paper, we report the characterization and use of a plasmid pSM429 from the Arctic Pseudoalteromonas strain BSi20429. The plasmid pSM429 is 3874 bp in length, with a very low G+C content (28%). The ORF4 on pSM429 was predicted to encode a Rep protein. Phylogenetic analysis implied that pSM429 is a novel plasmid. The regions essential for plasmid replication and stability were determined. Accordingly, the stable shuttle vectors based on pSM429 for E. coli and Pseudoalteromonas strains, pWD, pWD1 and pWD2, were developed, which can be used as cloning vectors for the Pseudoalteromonas strain we studied. With the aid of pWD2 and its Pseudoalteromonas host, the heterologous expressions of the erythromycin resistance gene and the cold-adapted cellulase gene were successful, indicating the potential use of this system for recombinant protein production, especially for cold-adapted proteins. These results are helpful for Pseudoalteromonas study.
The work was supported by National Natural Science Foundation of China (31025001, 40876072), Hi-Tech Research and Development program of China (2006AA09Z414, SQ2010AA0900521004), Natural Science Foundation of Shandong Province, China (JQ200910, ZR2009DZ002), Special Fund of China for Marine-scientific Research in the Public Interest (201005032-6), Foundation for Young Excellent Scientists in Shandong Province, China (2007BS07007).
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