Development of an efficient conjugation-based genetic manipulation system for Pseudoalteromonas
© Wang et al.; licensee BioMed Central. 2015
Received: 28 October 2014
Accepted: 10 January 2015
Published: 23 January 2015
Pseudoalteromonas is commonly found throughout the world’s oceans, and has gained increased attention due to the ecological and biological significance. Although over fifty Pseudoalteromonas genomes have been sequenced with an aim to explore the adaptive strategies in different habitats, in vivo studies are hampered by the lack of effective genetic manipulation systems for most strains in this genus. Here, nine Pseudoalteromonas strains isolated from different habitats were selected and used as representative strains to develop a universal genetic manipulation system. Erythromycin and chloramphenicol resistance were chosen as selection markers based on antibiotics resistance test of the nine strains. A conjugation protocol based on the RP4 conjugative machinery in E. coli WM3064 was developed to overcome current limitations of genetic manipulation in Pseudoalteromonas. Two mobilizable gene expression shuttle vectors (pWD2-oriT and pWD2Ery-oriT) were constructed, and conjugation efficiency of pWD2-oriT from E. coli to the nine Pseudoalteromonas strains ranged from 10−6 to 10−3 transconjugants per recipient cells. Two suicide vectors, pK18mobsacB-Cm and pK18mobsacB-Ery (with sacB for counter-selection), were constructed for gene knockout. To verify the feasibility of this system, we selected gene or operon that may lead to phenotypic change once disrupted as targets to facilitate in vivo functional confirmation. Successful deletions of two genes related to prodigiosin biosynthesis (pigMK) in P. rubra DSM 6842, one biofilm related gene (bsmA) in P. sp. SM9913, one gene related to melanin hyperproduction (hmgA) in P. lipolytica SCSIO 04301 and two flagella-related genes (fliF and fliG) in P. sp. SCSIO 11900 were verified, respectively. In addition, complementation of hmgA using shuttle vector pWD2-oriT was rescued the phenotype caused by deletion of chromosomal copy of hmgA in P. lipolytica SCSIO 04301. Taken together, we demonstrate that the vectors and the conjugative protocol developed here have potential to use in various Pseudoalteromonas strains.
Genus Pseudoalteromonas belongs to the Gammaproteobacteria class with thirty-eight recognized species reported so far [1,2]. Pseudoalteromonas is ubiquitous in the marine environment [2-10], and many strains have been isolated from deep sea , polar sea [2,9,10], or other extreme marine habitats, highlighting their important and diverse role in marine ecosystems. Pseudoalteromonas strains also produce a range of bioactive compounds with antimicrobial, antifouling, or algicidal activities that have attracted global attentions from microbiologist, ecologists and chemists . To date, over fifty Pseudoalteromonas genomes have been sequenced, laying a solid foundation for comparative studies on their adaptability to ecological niches as well as for the discovery of novel natural products. Several studies have used ectopic expressing genes in E. coli as a complementary means to interrogate genes and their functions in Pseudoalteromonas [12,13]. However, the lack of an efficient and universal genetic manipulation system has limited the comparative studies of Pseudoalteromonas at the molecular level in vivo.
Shuttle vector pWD2 has been successfully isolated previously and can be used as an expression vector in its original derived strain P. sp. SM20429 . Direct transfer of non-mobilizable pWD2 to other Pseudoalteromonas strains is constrained by the need for electroporation. Electroporation does not seem to work in majority of Pseudoalteromonas strains whose growth are usually salt-dependent. Based on our current knowledge, to date, gene deletion systems have only been described for two Pseudoalteromonas strains, P. haloplanktis TAC125 and P. sp. SM9913 [14,15]. Both protocols were designed for the construction of strain-specific isogenic knockouts, thus developing a widely applicable genetic manipulation system for Pseudoalteromonas now becomes a priority.
A few common features of Pseudoalteromonas make genetic manipulation difficult. Harboring multidrug resistance genes and multiple drug efflux pumps in the genome [8,16] can equip cells to survive antibiotic pressure and also can develop further mutations in genes encoding the target sites of antibiotics . Abundant distribution of restriction-modification systems also reduces transformation efficiency by degrading foreign DNAs . In addition, commonly used conjugation protocol does not offer a condition that allow decent growth of the non-marine originated mesophilic donor strain and the marine recipient Pseudoalteromonas strains. Solving these problems is critical for developing efficient genetic manipulation systems for Pseudoalteromonas.
Bacterial conjugation is a genetic exchange mechanism that requires direct contact between donor and recipient cells. Bacterial conjugation machinery is composed of an oriT sequence and tra genes . The oriT sequence needs to be provided by the plasmid in cis, while the tra genes, which encode a relaxase, a mating pair formation complex, and a type IV coupling protein, can be provided in cis or in trans. The relaxase cleaves the nic site within the oriT sequence and covalently attaches to the 5′ end of the transferred strand to produce a single-strand DNA (ssDNA)-relaxase complex with other auxiliary proteins; this is termed the relaxosome . The type IV coupling protein mediates the connection between the relaxasome and the mating pair formation complex, the latter being the secretion system that transfers ssDNA-relaxase complex into recipient cells . Since the DNA transferred by conjugation is single-stranded instead of double-stranded transferred by electroporation, thus it could reduce the possible degradation by restriction-modification systems which preferably degrading double-stranded DNAs . As a result, conjugation techniques have been widely used for genetic manipulations in Gram-negative bacteria and have also been reported in several Gram-positive bacteria (reviewed in ).
Here, we present an efficient conjugation-based genetic manipulation system for Pseudoalteromonas. Nine Pseudoalteromonas strains from different habitats were selected to represent strains from deep-sea sediment, Arctic sea ice, deep-sea hydrothermal vent, Mediterranean coastal water, Antarctic surface seawater, and sediment or surface water in the South China Sea. Based on antibiotic sensitivity test, two different resistance genes are used for selection to construct new vectors for gene expression and gene knockout. A conjugal transfer system with a modified medium using these vectors is developed, and feasibility of this transferring system is confirmed in nine Pseudoalteromonas strains. We further demonstrate that targeted deletion mutants are successfully constructed in four Pseudoalteromonas strains using this system to facilitate studies of these genes or operons in vivo, including P. rubra DSM 6842, P. sp. SM9913, P. lipolytica SCSIO 04301 and P. sp. SCSIO 11900. In addition, gene complementation using this system is also confirmed in one deletion mutant of P. lipolytica SCSIO 04301.
Results and discussion
Antibiotic resistance in different Pseudoalteromonas strains
Bacterial strains and plasmids used in this study
P. sp. SM9913, deep-sea sediment at a water depth of 1855 meters near the Okinawa Trough, 20°C
P. arctica A37-1-2, Arctic sea ice strain, 20°C
P. spiralis DSM 16099, deep ocean hydrothermal vents of the Juan de Fuca Ridge, 30°C
P. telluritireducens DSM 16098, deep ocean hydrothermal vents of the Juan de Fuca Ridge, 30°C
P. rubra DSM 6842, Mediterranean coastal waters off Nice, 25°C
P. sp. SM20429, plasmid curing mutant of P. sp. BSi20429, Arctic sea ice strain, 20°C
P. haloplanktis TAC125, Antarctic surface seawater, 20°C
P. lipolytica SCSIO 04301, sediment at 63 m deep in the South China Sea (18°0=N, 109°42=E), 25°C
P. sp. SCSIO 11900, surface mucus layer of the coral at 4 m deep in the South China Sea (18°13=N, 109°28=E) , 25°C
bsmA gene deletion mutant of P.sp. SM9913
Deletion mutant of the DNA region containing pigM-K genes related to prodigiosin biosynthesis in P. rubra DSM 6842
hmgA gene deletion mutant of P. lipolytica SCSIO 04301
Deletion mutant of the DNA region containing fliFG genes encoding flagellar motor proteins in P. sp. SCSIO 11900
Escherichia. coli strain
RP4 (tra) in chromosome, DAP-, 37°C
E. coli and Pseudoalteromonas shuttle vector, Ampr, Cmr
E. coli and Bacillus thuringiensis shuttle vector, Ampr, Eryr thuringiensis shuttle vector, Ampr, Eryr
Broad-host-range cloning vector, Kanr
pWD2 containing 1.5 kb BamHI fragment with mobilization region from pBBR1MCS-2, Ampr, Cmr
pWD2-oriT containing a 900bp erythromycin resistant gene replaced the chloramphenicol resistant gene, Ampr, Eryr
Widely used gene knockout vector, Kanr
pK18mobsacB containing the chloramphenicol resistant gene from pWD2, Kanr, Cmr
pK18mobsacB containing the erythromycin resistant gene from pHT304, Kanr, Eryr
pK18mobsacB-Cm containing the homologous arms of bsmA gene of SM9913, Kanr, Cmr
pK18mobsacB-Ery containing the homologous arms of the pigM-pigK DNA region of DSM 6842, Kanr, Eryr
pK18mobsacB-Ery containing the homologous arms of hmgA gene of SCSIO 04301, Kanr, Eryr
pK18mobsacB-Ery containing the homologous arms of the fliF-fliG DNA region of SCSIO 11900, Kanr, Eryr
Construction of mobilizable shuttle vectors for Pseudoalteromonas
Conjugation of pWD2-oriT between E. coli and Pseudoalteromonas
The widely used conjugation donor strain E. coli WM3064 has RP4 Tra function integrated in the chromosome, and is an auxotrophic strain whose growth relies on the supplementation of diaminopimelic acid (DAP) in the medium . First, the pWD2-oriT shuttle vector was transformed into E. coli WM3064 to obtain E. coli WM3064/pWD2-oriT. Using it as a donor strain, pWD2-oriT was then transferred into nine recipient Pseudoalteromonas strains individually by intergeneric conjugation. The conjugation protocol was optimized to allow decent simultaneous growth of the donor and recipient strains. Different conjugation temperatures (15, 25, 30, or 37°C) and media with different salt concentrations (LB+DAP, modified LB+DAP, or 2216E+DAP) were tested, respectively. Since E. coli WM3064 is able to grow at temperatures as low as 15°C, the optimal mating temperature was thus determined by the optimal growth temperature of the recipient strain (shown in Table 1). Growth of E. coli WM3064 and Pseudoalteromonas was also affected by the salt concentration in the medium. E. coli WM3064 grew very slowly in 2216E+DAP medium which has high salt concentration, and most Pseudoalteromonas strains had poor growth in LB medium (Additional file 1: Figure S1). Since viability of the donor strain during mating appeared to be a more important determining factor for conjugation efficiency, modified LB with moderate salt concentration was chosen for E. coli WM3064-Pseudoalteromonas mating to guarantee growth of both donor and recipient strains (Additional file 1: Figure S1).
Transfer efficiencies of pWD2-oriT between E. coli and Pseudoalteromonas strains
4.7 × 107
3.0 × 104
6.4 × 10−4
2.5 × 107
1.8 × 102
7.2 × 10−6
3.2 × 107
1.4 × 104
4.4 × 10−4
3.8 × 107
1.6 × 104
4.2 × 10−4
1.5 × 108
1.6 × 103
1.1 × 10−5
9.5 × 107
1.3 × 102
1.4 × 10−6
8.7 × 107
9.4 × 101
1.1 × 10−6
9.7 × 107
1.6 × 105
1.6 × 10−3
3.0 × 107
9.8 × 101
3.3 × 10−6
Construction of gene knockout vectors for Pseudoalteromonas
In-frame deletion of prodigiosin biosynthesis genes in DSM 6842
In-frame deletion of bsmA in SM9913
In-frame deletion of hmgA in SCSIO 04301
In-frame deletion of flagellar motor protein genes in SCSIO 11900
Different strain-specific genetic manipulation systems have been developed for Pseudoalteromonas strains TAC125 and SM9913 [14,15,29]. Here, we developed an efficient and generalizable genetic manipulation system for different Pseudoalteromonas strains. Incubation at 4°C has previously been used to select transconjugants and avoid overgrowth of the donor cells in the method developed for TAC125 [14,29]. However, conjugation at 4°C is unfeasible for knockout of genes essential for cold tolerance or adaptation , and many Pseudoalteromonas strains grow too slowly at 4°C. The mating temperature used in our approach was determined by the optimal growth temperature of the recipient strains, ranged from 20°C to 30°C, overcoming the limitations of cold temperature conditions. E. coli ET12567 (pUZ8002) containing a suicide plasmid was used as the donor strain for gene deletion in SM9913, however, control experiments showed that the donor cells alone can still grow on the selection plate due to lack of donor cell-specific selection pressure . In this study, the auxotrophic E. coli WM3064 strain was used as donor which cannot grow without the addition of DAP, thus can eliminate interference from donor cells and reduce false positive rates.
Most Pseudoalteromonas strains are sensitive to chloramphenicol and erythromycin, including all the strains tested in this study. When used chloramphenicol in the selection of mutants from the the single crossover event, a few false positive colonies may appear, however, this can be easily avoided by using erythromycin for the same purpose. The precise mechanism remains unclear, but it might be attributed to differences in antibiotic killing mechanisms. Therefore, erythromycin selective marker vector is recommended over chloramphenicol for Pseudoalteromonas genetic manipulations.
Here, we developed an efficient and generalizable genetic manipulation system for Pseudoalteromonas strains. Nine Pseudoalteromonas strains were sensitive to chloramphenicol and erythromycin, and thus both resistance genes were used to construct gene expression and gene knockout vectors. A conjugation transfer system was developed by modifying the culture conditions, which resulted in successful transfer of the pWD2-oriT gene expression vector from E. coli into Pseudoalteromonas strains with relatively high efficiency. By modifying a widely-used suicide plasmid (pK18mobsacB), a gene knockout system was developed and knockout of target DNA regions was confirmed in DSM 6842, SM9913, SCSIO 04301 and SCSIO 11900, covering from high or low conjugation efficiencies for pWD2-oriT transfer. In addition, gene complementation was also confirmed using this system. Taken together, conjugation-based genetic manipulation can be used efficiently for gene expression and gene deletion in Pseudoalteromonas strains, which will facilitate future in vivo studies of Pseudoalteromonas.
Materials and methods
Bacterial strains, plasmids, and growth conditions
The Pseudoalteromonas and E. coli strains and the plasmids used in this study are listed in Table 1. E. coli were cultured at 37°C in Luria-Bertani medium (LB) unless specified, and experiments with Pseudoalteromonas were conducted at 20-30°C in 2216E medium (BD Difco) or in SWLB medium (10 g peptone, and 5 g yeast extract dissolved in 1 l artificial seawater). DAP (diaminopimelic acid) was added at 0.3 mM to culture E. coli WM3064 strain. Conjugation assays were performed in modified LB mating medium (10 g peptone, 5 g yeast extract, 500 ml artificial seawater, and 500 ml distilled water) with 0.3 mM DAP. When needed, antibiotics were added at the following concentrations: 100 μg/ml for ampicillin (Amp), apramycin (Apr) and spectinomycin (Spc); 50 μg/ml for kanamycin (Kan); 30 μg/ml for chloramphenicol (Cm); 25 μg/ml for erythromycin (Ery); and 10 μg/ml for gentamycin (Gm) and tetracycline (Tet).
Antibiotic sensitivity assay
Sensitivities of Pseudoalteromonas strains to eight antibiotics were tested (Additional file 1: Table S1). Strains were grown in 2216E medium at 20-30°C to late exponential phase and then diluted to 10−2-10−7 using 10-fold serial dilutions prior to plating on 2216E containing each antibiotic. The plates were incubated at 20-30°C for 48 h. Assays were performed in triplicate, and plates with no antibiotic were used as controls.
Construction of pWD2-oriT and pWD2Ery-oriT mobilizable shuttle vectors
The mobilization module containing a mob gene and the corresponding oriT region was amplified from pBBR1MCS-2 using the oriT-F/oriT-R primer pair (Additional file 1: Table S2). The 1.5 kb PCR product was digested with BamHI and inserted into the corresponding site of pWD2 , resulting in the mobilizable shuttle vector pWD2-oriT. To generate the pWD2Ery shuttle vector, the Ery resistance gene was amplified from pHT304  using the Ery-F/Ery-R primer pair (Additional file 1: Table S2), and the 914 bp PCR product was digested with BamHI/EcoRI and inserted into the corresponding sites of pWD2 . To generate the pWD2Ery-oriT mobilizable shuttle vector, the 1.5 kb DNA region containing the mobilization module was derived from pWD2-oriT by digesting with BamHI and inserted into the corresponding site of pWD2Ery.
Conjugation experiments were performed as previously described  with some modifications. In brief, donor and recipient strains were grown to an OD600 of 0.8-1, and 2 ml donor cells and 1 ml recipient cells were harvested by centrifugation (4000 rpm for 3 min). Cells were washed twice with mating medium (MLB) and re-suspended in 100 μl MLB containing DAP. The donor and recipient cells were mixed briefly, the mixture dropped on MLB with DAP plates, and the plates were incubated for 8 h or more until a lawn was formed at 20-30°C. Cells were collected from the lawn and re-suspended in 2 ml 2216E medium and spread onto 2216E plates with appropriate antibiotics to select the transconjugants. Transfer efficiency was calculated as the ratio of transconjugants per recipient cells for each condition.
PCR and RAPD-PCR analysis
Genomic DNA for PCR and random amplified polymorphic DNA (RAPD) analyses was isolated using a TIANamp Bacterial DNA Kit (Tiangen, Beijing, China). PCR primers are listed in Additional file 1: Table S2. Primers pWD2-S and pWD2-A were designed to amplify the replication region for detecting the pWD2-oriT plasmid in donor strains and transconjugants. RAPD-PCR analysis was performed using the 272 random primer (5′-AGCGGGCCAA-3′)  to identify donor strains, recipient strains, and transconjugants based on amplification profiles. The RAPD-PCR reaction was: 95°C for 5 min followed by 40 cycles of 95°C for 45 s, 36°C for 1 min, and 72°C for 2 min; and 72°C for an additional 10 min. Routine DNA manipulations were carried out following standard methods .
Construction of the pK18mobsacB-Cm and pK18mobsacB-Ery suicide vectors
The 1.2 kb DNA region containing the Cm resistance gene was recovered from pWD2 with BamHI/EcoRI and cloned into the commonly used pK18mobsacB vector for gene knockout , resulting in the pK18mobsacB-Cm suicide vector. The Ery resistance gene was amplified from pHT304  using the Ery-F/Ery-R primer pair, and the 914 bp PCR product was digested with BamHI/EcoRI and cloned into pK18mobsacB to produce the pK18mobsacB-Ery suicide vector.
Construction of the ΔpigM-K mutant strain in DSM 6842
The suicide plasmid used for deletion of a DNA region containing pigM-K genes was based on pK18mobsacB-Ery. The schematic is shown in Figure 3. Two primer pairs (pigM-up-S/pigM-up-A and pigM-down-S/pigM-down-A) were used to amplify the upstream and downstream DNA sequences of the target region from DSM 6842 genomic DNA. The 996 bp and 815 bp PCR fragments were digested with XbaI/EcoRI and EcoRI/HindIII, respectively, cloned into the XbaI/HindIIII sites of pK18mobsacB-Ery, and were transformed into E. coli WM3064. Suicide plasmid pK18Ery-pigM-K was mobilized from E. coli WM3064 into DSM 6842 by intergeneric conjugation. After mating, cells were spread on 2216E plates containing erythromycin (25 μg/ml) to screen for clones in which the suicide vector pK18Ery-pigM-K had integrated into the DSM 6842 genome via a single crossover event. The mutants were then grown at 25°C with shaking in 2216E medium without any antibiotics for 8 h. To select mutants in which the second recombination had occurred, the culture was diluted and spread on 2216E medium containing 10% sucrose and grown at 25°C for 24–36 h. Single colonies were transferred onto 2216E and 2216E containing 25 μg/ml erythromycin plates simultaneously, and colonies sensitive to erythromycin (25 μg/ml) were collected and confirmed by PCR followed by DNA sequencing.
Three other knockout mutants, ΔbsmA in SM9913, ΔhmgA in SCSIO 04301 and ΔfliFG in SCSIO 11900, were constructed using the similar steps as ΔpigM-K, and detailed procedures can be found in the Additional file 1. The procedure for the complementation of ΔhmgA can also be found in the Additional file 1.
Crystal violet biofilm and motility assays
Biofilm formation was assayed in 96-well polystyrene plates (Corning Costar, Cambridge, MA) in SWLB medium at 20°C after 1 d, 2 d, 3 d, and 4 d with crystal violet staining . To account for growth effects, biofilm formation was normalized by dividing the total biofilm by the maximal bacterial growth as measured by turbidity at 620 nm for each strain. Cell motility was examined with 1% tryptone and 0.3% agar dissolved in seawater medium. Motility halos were quantified after 16 h using at least three plates for each condition and two independent cultures for each strain.
This work was supported by the National Basic Research Program of China (2013CB955701), the National Science Foundation of China (31290233, 41406189 and 41230962) and the Chinese Academy of Sciences (XDA11030402). XW is the 1000-Youth Elite Program recipient in China.
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