Abstract

An Escherichia coliLaribacter hongkongensis shuttle vector (pPW380) was constructed by ligating the 4701-bp Eco RI digested fragment of pHLHK8 to Eco RI digested pBK-CMV. An E. coliL. hongkongensis inducible expression shuttle vector was further constructed by ligating a 2105-bp fragment that contains the tetracycline repressor and tetracycline-inducible promoter region of pALC2084 to the 8897-bp fragment of pPW380, deletion of the green fluorescent protein gene, and insertion of a multiple cloning site. This inducible expression system was able to express two commonly used reporter genes, the green fluorescent protein gene and the glutathione S-transferase gene, efficiently in E. coli and L. hongkongensis.

Introduction

Laribacter hongkongensis, a novel genus and species, was first discovered in Hong Kong in 2001 from the blood and empyema pus of a 54-year old Chinese man with alcoholic cirrhosis and bacteremic empyema thoracis [1]. Phenotypically, it is a facultative anaerobic, motile, non-sporulating, urease-positive, Gram-negative, S-shaped bacillus. Genotypically, by phylogenetic analysis using 16S rRNA gene sequences, L. hongkongensis belongs to the Neisseriaceae family of the β-subclass of Proteobacteria. Since the patient's underlying liver cirrhosis and ascites suggested that the gastrointestinal tract might be a possible primary site of infection, L. hongkongensis was intensively sought in fecal specimens of patients with gastroenteritis. During a period of two months, L. hongkongensis was discovered, on charcoal cefoperazone deoxycholate agar, in three of our patients with community-acquired gastroenteritis [2]. A similar finding was also observed in three other patients in Switzerland [2]. In a recent multi-centered prospective study using cefoperazone MacConkey agar as the selective medium [3], we confirmed that L. hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea [4]. Furthermore, freshwater fish was also confirmed to be a reservoir of L. hongkongensis[ [, []. By comparing the pulsed-field gel electrophoresis patterns of fish and patient isolates, it was observed that most patient isolates were clustered together, suggesting that some clones could be more virulent than others [5]. The isolation of L. hongkongensis from patients who resided in or have recent travel histories to Asia, Europe, America, and Africa implied that the bacterium is likely to be of global importance. Recently, a class C β-lactamase gene of L. hongkongenis, responsible for its resistance to multiple β-lactam antibiotics, was cloned and characterized [6].

Development of genetic manipulation systems is of paramount importance in the study of pathogenesis and virulence factors in L. hongkongensis. In this study, a small plasmid in one of the isolates was completely sequenced and characterized. Using this plasmid, an Escherichia coliL. hongkongensis inducible expression shuttle vector was constructed.

Materials and methods

Strains and plasmids

The bacterial strains and plasmids used in this study are shown in Table 1. All L. hongkongensis strains used in this study were clinical isolates from patients in Hong Kong [ [, [, []. pCL52.2, a temperature sensitive E. coliStaphylococcus aureus shuttle vector and pALC2084, an E. coliS. aureus shuttle vector with a tetracycline-inducible green fluorescent protein (GFP) gene, were gifts from Cheung [ [, [].

1

Bacterial strains and plasmids

Organism/plasmid Feature Source/reference 
Strains   
Laribacter hongkongensis HKU1 Type strain  [1
Laribacter hongkongensis HLHK8 Human strain isolated from patients with community-acquired gastroenteritis in Hong Kong. Origin of pHLHK8  [4
Laribacter hongkongensis HLHK9 Human strain isolated from patients with community-acquired gastroenteritis in Hong Kong. Used in GFP and GST expression experiments  [4
Laribacter hongkongensis HLHK2-4, 10–24 Human strains isolated from patients with community-acquired gastroenteritis in Hong Kong [ [, [
Escherichia coli DH5α F, Φ80d lac ZΔM15, Δ(lac ZYA-argF)U169, end A1, rec A1, hsd R17(rK-mK+) deo R, thi-1, sup E44, λ, gyr A96(Nalr), rel A1 Invitrogen 
Plasmids   
pHLHK8 Plasmid isolated from HLHK8 This study 
pBSKII(−), Apr Cloning vector Stratagene 
pBK-CMV, Kmr Cloning vector Stratagene 
pCL52.2, Apr Temperature sensitive E. coliS. aureus shuttle vector  [8
pBR322, Apr Tcr Cloning vector Promega 
pUC19, Apr Cloning vector Invitrogen 
pALC2084, Apr E. coliS. aureus shuttle vector with a tetracycline-inducible GFP gene  [7
pPW380, Kmr pHLHK8 replicon + pBK-CMV This study 
pPW632, Kmr pALC2084 +Sph I–Aat II adaptor This study 
pPW576, Kmr Aat II fragment of pPW632 + pPW380 This study 
pPW633, Kmr pPW576–GFP This study 
pPW578, Kmr pPW633+ multiple cloning site This study 
pGEX-2T, Apr GST gene fusion vector Amersham Bioscience 
pPW585, Kmr pPW578 + GST This study 
Organism/plasmid Feature Source/reference 
Strains   
Laribacter hongkongensis HKU1 Type strain  [1
Laribacter hongkongensis HLHK8 Human strain isolated from patients with community-acquired gastroenteritis in Hong Kong. Origin of pHLHK8  [4
Laribacter hongkongensis HLHK9 Human strain isolated from patients with community-acquired gastroenteritis in Hong Kong. Used in GFP and GST expression experiments  [4
Laribacter hongkongensis HLHK2-4, 10–24 Human strains isolated from patients with community-acquired gastroenteritis in Hong Kong [ [, [
Escherichia coli DH5α F, Φ80d lac ZΔM15, Δ(lac ZYA-argF)U169, end A1, rec A1, hsd R17(rK-mK+) deo R, thi-1, sup E44, λ, gyr A96(Nalr), rel A1 Invitrogen 
Plasmids   
pHLHK8 Plasmid isolated from HLHK8 This study 
pBSKII(−), Apr Cloning vector Stratagene 
pBK-CMV, Kmr Cloning vector Stratagene 
pCL52.2, Apr Temperature sensitive E. coliS. aureus shuttle vector  [8
pBR322, Apr Tcr Cloning vector Promega 
pUC19, Apr Cloning vector Invitrogen 
pALC2084, Apr E. coliS. aureus shuttle vector with a tetracycline-inducible GFP gene  [7
pPW380, Kmr pHLHK8 replicon + pBK-CMV This study 
pPW632, Kmr pALC2084 +Sph I–Aat II adaptor This study 
pPW576, Kmr Aat II fragment of pPW632 + pPW380 This study 
pPW633, Kmr pPW576–GFP This study 
pPW578, Kmr pPW633+ multiple cloning site This study 
pGEX-2T, Apr GST gene fusion vector Amersham Bioscience 
pPW585, Kmr pPW578 + GST This study 

Plasmid extraction and sequencing and in silico analysis of pHLHK8

Extraction of plasmids of <20 kb in L. hongkongensis strains was performed using the High Pure Plasmid Isolation kit (Roche Applied Science) according to manufacturer's instructions. The extracted plasmids were electrophoresed in 1.0% (w/v) agarose gel, with molecular size markers, Lambda DNA Eco 471 Digest Marker (Roche Applied Science) and Lambda DNA Hin dIII Digest Marker (Roche Applied Science), in parallel. Electrophoresis in Tris-borate-EDTA (TBE) buffer was performed at 100 V for 45 min. The gel was stained with ethidium bromide (0.5 μg ml−1) for 15 min, rinsed and photographed under ultraviolet light illumination.

pHLHK8, the 8266-bp plasmid of L. hongkongensis strain HLHK8, was digested with Cla I and Eco RI and the respective fragments were cloned into pBSKII(−). Both strands of the fragments were sequenced twice with an ABI 3700 DNA analyzer according to manufacturers' instructions (Applied Biosystems), using primers T7 and T3 and additional primers designed from the first and second rounds of the sequencing reactions. The nucleotide and deduced amino acid sequences of the open reading frames of pHLHK8 were compared with sequences in the GenBank. Direct and inverted repeats were determined using dotmatcher (EMBOSS-GUI).

Determination of copy number of pHLHK8

The copy number of pHLHK8 was determined according to a published protocol [9]. Overnight culture of HLHK8 was inoculated into brain heart infusion (BHI) broth. When the culture reached a turbidity of 0.6–1.0 at OD600, 1 ml of the culture was used for plasmid DNA extraction using the High Pure Plasmid Isolation kit (Roche Applied Science), and the number of bacteria was determined by back titration. The concentration of plasmid DNA was calculated by measuring the absorbance of the plasmid DNA solution at 260 nm. A plasmid of known copy number (pBR322 in E. coli DH5α) was used as the control. The experiment was performed three times and the copy number of pHLHK8 was calculated using the following formula:  

formula

Segregational plasmid stability studies

The plasmid stability of pHLHK8 was determined according to a published protocol [10]. Single colony of HLHK8 containing pHLHK8 was inoculated into BHI broth. Cells in the late exponential growth phase (12 h after inoculation) were diluted 1000-fold and the procedure was repeated four times. After these subcultures, bacterial cells were plated on BHI agar plates. Ten colonies were picked and the presence of pHLHK8 was determined by plasmid DNA extraction.

The plasmid stability of the constructed plasmids pPW380 and pPW578 was determined according to a published protocol [11]. Single colony of HLHK9 carrying pPW380 and that carrying pPW578 were cultivated in BHI broth supplemented with 50 μg ml−1 kanamycin for 12 h. The bacterial cultures were subcultured into BHI broth without kanamycin and were incubated for 12 h. This subculture regimen was repeated four times. After these subcultures, bacterial cells were serially diluted in PBS and were plated on BHI agar. Colonies that appeared were picked and cultured on BHI agar with and without kanamycin.

DNA manipulation

All restriction enzymes were purchased from Roche Applied Science and restriction enzyme digestion was carried out according to manufacturers' instructions. Digested products were gel-purified using the QIAquick Gel Extraction kit (QIAgen) according to manufacturers' instructions. Oligonucleotides LPW814 (5′-CGACGTCGCATG-3′), LPW1376 (5′-CACCGGTGGAGACAGATCTTCCGGACACGTGTTAATTAAGAGCT-3′) and LPW1504 (5′-CTTAATTAACACGTGTCCGGAAGATCTGTCTCCACCGGTGAGCT-3′) were purchased from Invitrogen. Self-annealing of LPW814 and annealing of LPW1376 with LPW1504 were performed as described by Sambrook and Russell [12]. DNA ligation was performed as described by Sambrook and Russell [12] using T4 DNA ligase (Invitrogen).

Transformation

Transformation of pBK-CMV, pBSKII(−), pCL52.2, pBR322, pUC19, pALC2084 and pPW380 (the E. coliL. hongkongensis shuttle vector) was performed by electroporation, using 1 μg of plasmids, according to standard protocol [12]. The presence of plasmids in colonies of L. hongkongensis was determined using the High Pure Plasmid Isolation kit (Roche Applied Science) and agarose gel electrophoresis as described above.

Expression of green fluorescent protein in L. hongkongensis

Analysis of GFP expression was carried out as described by Bateman et al. [7] with slight modifications. E. coli and L. hongkongensis containing pPW576 were grown for 16 h in LB and BHI with kanamycin, respectively. The bacterial cultures were diluted 1:100 in LB or BHI containing kanamycin and further cultured at 37 °C, with shaking at 250 rpm, to an absorbance at 600 nm (OD600) of 0.5. Subinhibitory concentrations of tetracycline (0, 50, 125, 250 and 500 ng ml−1) were added to the bacterial cultures. The bacteria were sampled hourly (100 μl) in triplicate and analyzed for fluorescence and OD600 simultaneously in microtiter wells, using a Fusion Universal Microplate Analyzer (Perkin–Elmer). The experiment was performed in duplicate on separate occasions and results were reported as total fluorescence (FL) units per OD600 unit (mean of the two experiments) to minimize the variation in fluorescence due to cell densities.

Expression of glutathione S-transferase in L. hongkongensis

Analysis of glutathione S-transferase (GST) expression was carried out by Western blot assay using our published protocol [13]. Overnight cultures of E. coli and L. hongkongensis cells with pPW585 were induced with 125 and 500 ng ml−1 tetracycline, respectively for 16 h. The cells were centrifuged at 13000 rpm for 5 min and resuspended in PBS with 1% Tween 20 (v/v) and 0.5 mM phenylmethylsulfonyl fluoride. Thirty microliters of the cell extracts obtained were electrophoresed on an SDS–15% polyacrylamide gel and electroblotted onto a nitrocellulose membrane (Bio-Rad Laboratories). The blot was incubated with a 1:1000 dilution of polyclonal antibody against GST (Amersham Bioscience), followed with a 1:4000 dilution of anti-GST-HRP (Zymed Laboratories). Antigen–antibody interaction was detected with the ECL fluorescence kit (Amersham Bioscience).

Accession numbers

The nucleotide sequence of pHLHK8, pPW380 and pPW578 has been lodged within the GenBank sequence database under Accession Nos. AY858987, DQ164215 and DQ164216, respectively.

Results

Sequencing and characterization of pHLHK8

Agarose gel electrophoresis showed that four of the 21 L. hongkongensis strains screened possessed plasmids of <20 kb. One strain, HLHK8, possessed a plasmid, pHLHK8, of about 8 kb (Fig. 1(a)). Sequencing of pHLHK8 revealed that this plasmid consists of 8266 bp (Fig. 1(b)). The overall G + C content is 53.93%. pHLHK8 has four ORFs with all the genes encoded by the same DNA strand. The predicted amino acid sequences of the four ORFs are homologous to those of DNA invertase, type II restriction enzyme Bsu BI, ATPase involved in chromosome partitioning, and plasmid replication initiator protein (Fig. 1(b) and Table 2). The corresponding genes are named as pin, hsr BI, parA and repA, respectively. In addition, there is a predicted origin of replication that consists of a DnaA box and five 16-bp direct repeats (Fig. 1(b)). The copy number (mean ± SD) of pHLHK8 is 1.1 ± 0.3. The plasmid is stable after four passages (about 240 generations) in the absence of selection.

1

Panel A: Plasmid screening gel of HLHK8. Lane 1 – Lambda DNA Eco 471 Digest Marker. Lane 2 – Lambda DNA Hin dIII Digest Marker. Lane 3 – plasmid of HLHK8. Panel B: Organization and replication region of pHLHK8. The thin arrows indicate the five 16-bp direct repeats of the putative origin of replication.

1

Panel A: Plasmid screening gel of HLHK8. Lane 1 – Lambda DNA Eco 471 Digest Marker. Lane 2 – Lambda DNA Hin dIII Digest Marker. Lane 3 – plasmid of HLHK8. Panel B: Organization and replication region of pHLHK8. The thin arrows indicate the five 16-bp direct repeats of the putative origin of replication.

2

Open reading frames on the L. hongkongensis plasmid pHLHK8 having significant database matches

ORFs Characteristics of ORFs Best match to known sequences in public databases 
 Start-end (base) No. of bases Number of amino acids Frame Gene name Organism Description GenBank Accession Nos. E-value Percentage amino acid identity 
ORF 1 3174–3584 411 136 +3 pin Acetobacter pasteurianus DNA invertase AAB69349 2e−51 83 
ORF 2 3832–4806 975 324 +1 hsr BI Bacillus subtilis Type II restriction enzyme Bsu BI P33562 1e−125 58 
ORF 3 5948–6571 624 207 +2 parA Novosphingobium aromaticivorans ATPase involved in chromosome partitioning ZP_00303510 3e−54 55 
ORF 4 7025–7942 918 305 +2 repA Rhodobacter sphaeroides Plasmid replication initiator protein ZP_00207037 7e−63 48 
ORFs Characteristics of ORFs Best match to known sequences in public databases 
 Start-end (base) No. of bases Number of amino acids Frame Gene name Organism Description GenBank Accession Nos. E-value Percentage amino acid identity 
ORF 1 3174–3584 411 136 +3 pin Acetobacter pasteurianus DNA invertase AAB69349 2e−51 83 
ORF 2 3832–4806 975 324 +1 hsr BI Bacillus subtilis Type II restriction enzyme Bsu BI P33562 1e−125 58 
ORF 3 5948–6571 624 207 +2 parA Novosphingobium aromaticivorans ATPase involved in chromosome partitioning ZP_00303510 3e−54 55 
ORF 4 7025–7942 918 305 +2 repA Rhodobacter sphaeroides Plasmid replication initiator protein ZP_00207037 7e−63 48 

Construction of an E. coliL. hongkongensis shuttle vector

An E. coliL. hongkongensis shuttle vector was constructed by ligating the 4701-bp Eco RI fragment of pHLHK8 that contained the parA and repA genes and origin of replication of pHLHK8 to Eco RI digested pBK-CMV (Fig. 2). This resultant E. coliL. hongkongensis shuttle vector, pPW380, can propagate in both E. coli and L. hongkongensis with good transformation efficiencies (Table 3). pPW380 is stable in L. hongkongensis in an extrachromosomal state after four passages (about 240 generations) in the absence of selection.

2

Construction of E. coliL. hongkongensis shuttle vector and E. coliL. hongkongensis inducible expression shuttle vector.

2

Construction of E. coliL. hongkongensis shuttle vector and E. coliL. hongkongensis inducible expression shuttle vector.

3

Transformation of L. hongkongensis and E. coli DH5α with pCL52.2, pBK-CMV and pPW380

Plasmid Replication origin Size (bp) Number of colonies on selective medium [Results expressed as mean (±SD) number of colonies per μg of plasmid DNA for three separate experiments] 
   L. hongkongensis (HLHK strain number) E. coli DH5α 
   10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 
pCL52.2 pSC101 origin 8000 1.3 (±0.3) × 102 2.6 (±0.7) × 102 4.7 (±1.0) × 102 8.9 (±0.6) × 102 41 (±14) 1.3 (±0.3) × 103 6.9 (±0.8) × 102 9.3 (±1.3) × 102 1.0 (±0.2) × 103 1.7 (±0.4) × 102 52 (±41) 15 (±5) 1.1 (±1.1) × 107 
pBK-CMV pUC origin 4518 23 (±4) 1 (±1) 7 (±9) 2 (±2) 1 (±0) 7 (±4) 6 (±3) 2.4 (±0.3) × 108 
pPW380 pHLHK8 origin and pUC origin 9225 5.4 (±1.6) × 106 3.3 (±0.8) × 106 65 (±33) 3.7 (±1.6) × 106 0a 2.7 (±2.7) × 107 2.4 (±0.4) × 104 1.1 (±0.7) × 102 63 (±18) 4.0 (±4.6) × 102 2.1 (±0.4) × 105 70 (±42) 1.3 (±0.7) × 102 2.7 (±0.01) × 104 2.9 (±1.5) × 105 9.9 (±3.0) × 104 29 (±4) 2.5 (±0.5) × 102 65 (±92) 7.4 (±5.1) × 104 5.7 (±4.3) × 104 1.3 (±1.6) × 107 
Plasmid Replication origin Size (bp) Number of colonies on selective medium [Results expressed as mean (±SD) number of colonies per μg of plasmid DNA for three separate experiments] 
   L. hongkongensis (HLHK strain number) E. coli DH5α 
   10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 
pCL52.2 pSC101 origin 8000 1.3 (±0.3) × 102 2.6 (±0.7) × 102 4.7 (±1.0) × 102 8.9 (±0.6) × 102 41 (±14) 1.3 (±0.3) × 103 6.9 (±0.8) × 102 9.3 (±1.3) × 102 1.0 (±0.2) × 103 1.7 (±0.4) × 102 52 (±41) 15 (±5) 1.1 (±1.1) × 107 
pBK-CMV pUC origin 4518 23 (±4) 1 (±1) 7 (±9) 2 (±2) 1 (±0) 7 (±4) 6 (±3) 2.4 (±0.3) × 108 
pPW380 pHLHK8 origin and pUC origin 9225 5.4 (±1.6) × 106 3.3 (±0.8) × 106 65 (±33) 3.7 (±1.6) × 106 0a 2.7 (±2.7) × 107 2.4 (±0.4) × 104 1.1 (±0.7) × 102 63 (±18) 4.0 (±4.6) × 102 2.1 (±0.4) × 105 70 (±42) 1.3 (±0.7) × 102 2.7 (±0.01) × 104 2.9 (±1.5) × 105 9.9 (±3.0) × 104 29 (±4) 2.5 (±0.5) × 102 65 (±92) 7.4 (±5.1) × 104 5.7 (±4.3) × 104 1.3 (±1.6) × 107 

aIncompatibility of plasmids.

Construction of an E. coliL. hongkongensis inducible expression shuttle vector

The Sph I–Aat II adaptor, formed by self-annealing of LPW814, was ligated to the Sph I digested pALC2084, forming pPW632. The 2105-bp Aat II fragment of pPW632, which contained the tetracycline repressor and tetracycline-inducible promoter region of pALC2084, was ligated to the 8897-bp Aat II digested pPW380 fragment, forming pPW576. The 1564-bp Sac I fragment that contained the GFP gene was deleted from pPW576, forming pPW633. The E. coliL. hongkongensis inducible expression shuttle vector for L. hongkongensis, pPW578, was then constructed by inserting a multiple cloning site that contained sequences for Age I, Bbs I, Bgl II, Bsp EI, Pml I and Pac I digestion, formed by annealing LPW1376 and LPW1504, to the Sac I digested pPW633. pPW578 is stable in L. hongkongensis in an extrachromosomal state after four passages (about 240 generations) in the absence of selection.

Transformation

No colonies were observed from any of the 21 strains of L. hongkongensis transformed with pBR322, pBSKII(−), pUC19 or pALC2084. Colonies [median 3.65 × 102 (range 15–1.3 × 103) per μg plasmid DNA] were observed in 12 (57%) out of 21 L. hongkongensis strains when they were transformed with pCL52.2 (Table 3). Colonies [median 6 (range 1–23) per μg plasmid DNA] were observed in 7 (33%) out of 21 L. hongkongensis strains when they were transformed with pBK-CMV (Table 3). However, no plasmids were recovered from the L. hongkongensis colonies that appeared on selective agar after transformation with pCL52.2 or pBK-CMV, suggesting that the plasmids pCL52.2 and pBK-CMV have integrated into the genomes of the L. hongkongensis strains. Colonies [median 2.55 × 104 (range 29–2.7 × 107) per μg plasmid DNA], with plasmids recoverable, were observed in all 20 L. hongkongensis strains (except HLHK8 as a result of plasmid incompatibility) when they were transformed with pPW380 (Table 3).

Expression of green fluorescent protein in L. hongkongensis

Ligated pPW576 was transformed into E. coli and the resultant pPW576 extracted from E. coli was transformed into L. hongkongensis HLHK9. After induction with various concentrations of tetracycline, increasing degree of fluorescence was observed from both E. coli and L. hongkongensis cells (Fig. 3). For E. coli, the degree of fluorescence was directly related to the concentration of tetracycline used. On the other hand, for L. hongkongensis, the highest degree of fluorescence was observed with the use of 125 ng ml−1 of tetracycline in both experiments, followed with 50, 250 and 500 ng ml−1 of tetracycline used.

3

Analysis of GFP expression in E. coli and L. hongkongensis containing pPW576. Results (means of two experiments) were expressed as total fluorescence (FL) units per OD600 FLOD−1600 measured at different times after induction with 0, 50, 125, 250 and 500 ng ml−1 of tetracycline (tet) (panel A, L. hongkongensis; panel B, E. coli). Maximum fluorescence was observed on L. hongkongensis and E. coli cells after 16 h of induction with 125 and 500 ng ml−1 of tetracycline, respectively. No fluorescence was observed at 0 h or after 16 h of induction with 0 ng ml−1 of tetracycline.

3

Analysis of GFP expression in E. coli and L. hongkongensis containing pPW576. Results (means of two experiments) were expressed as total fluorescence (FL) units per OD600 FLOD−1600 measured at different times after induction with 0, 50, 125, 250 and 500 ng ml−1 of tetracycline (tet) (panel A, L. hongkongensis; panel B, E. coli). Maximum fluorescence was observed on L. hongkongensis and E. coli cells after 16 h of induction with 125 and 500 ng ml−1 of tetracycline, respectively. No fluorescence was observed at 0 h or after 16 h of induction with 0 ng ml−1 of tetracycline.

Expression of glutathione S-transferase in L. hongkongensis

Ligated pPW585 was transformed into E. coli and the resultant pPW585 extracted from E. coli was transformed into L. hongkongensis HLHK9. Western blot analysis showed that a band at 26 kDa, compatible with the size of GST, reacted specifically with polyclonal antibody against GST in both E. coli and L. hongkongensis cells (Fig. 4).

4

Western blot analysis of GST in E. coli and L. hongkongensis. Cell extracts of overnight cultures of E. coli (lanes 1–3) and L. hongkongensis (lanes 4–6) cells with pPW585 (lanes 1 and 6, uninduced at 0 h; lanes 2 and 5, uninduced after 16 h; lanes 3 and 4, after 16 h of induction) electrophoresed on an SDS–15% polyacrylamide gel. A band of about 26 kDa can be detected (lanes 3 and 4). Antigen–antibody interaction was detected with polyclonal antibody against GST.

4

Western blot analysis of GST in E. coli and L. hongkongensis. Cell extracts of overnight cultures of E. coli (lanes 1–3) and L. hongkongensis (lanes 4–6) cells with pPW585 (lanes 1 and 6, uninduced at 0 h; lanes 2 and 5, uninduced after 16 h; lanes 3 and 4, after 16 h of induction) electrophoresed on an SDS–15% polyacrylamide gel. A band of about 26 kDa can be detected (lanes 3 and 4). Antigen–antibody interaction was detected with polyclonal antibody against GST.

Discussion

Development of genetic manipulation systems is crucial in the study of pathogenic mechanisms and virulence factors associated with L. hongkongensis. In the first part of the study, we tried to see if plasmids that are commonly used for expression systems in E. coli propagate satisfactorily in L. hongkongensis. Results revealed that none of the six plasmids tested propagated well in L. hongkongensis. Although transformation of L. hongkongensis with pCL52.2 or pBK-CMV resulted in some successful transformants, no plasmids were recovered from the L. hongkongensis colonies that appeared on the selective agar. This suggested that these plasmids probably had integrated into the genomes of the L. hongkongensis strains. As none of the plasmids in E. coli was useful, native plasmids from strains of L. hongkongensis were looked for. Plasmid profile studies of 21 strains of L. hongkongensis showed that plasmids of <20 kb were present in four of them. Sequencing one of the small plasmids, pHLHK8, revealed no selective markers in it.

As there was no ready-to-use plasmid from E. coli or L. hongkongensis, an E. coliL. hongkongensis shuttle vector that can propagate satisfactorily in both E. coli and L. hongkongensis was constructed. As 100% and 20% of L. hongkongensis strains tested so far were resistant to ampicillin and tetracycline, respectively [ [, [], genes that confer resistance to these two antibiotics were not chosen as the selective marker for the shuttle vector. Therefore, pBK-CMV was chosen as the backbone because it contains a kanamycin resistance gene that can act as the selective marker in both E. coli and L. hongkongensis, as all strains of L. hongkongensis tested so far were sensitive to aminoglycosides [4]. As pHLHK8 was the first small plasmid identified in L. hongkongensis, it was chosen as the source of the replication origin of the shuttle vector in L. hongkongensis. Transformation experiments showed that the resultant shuttle vector, pPW380, can be successfully transformed into all 20 strains of L. hongkongensis tested, with a median transformation efficiency of 2.55 × 104L. hongkongensis colonies per μg of pPW380, about 103 times lower than that in E. coli.

Using the shuttle vector as the backbone, an E. coliL. hongkongensis inducible expression shuttle vector was constructed. Initially, the isopropyl-β-d-thiogalactopyranoside inducible lac promoter and the arabinose inducible ara BAD promoter, two promoters that are very commonly used in bacterial expression systems, were independently cloned into the shuttle vector, pPW380, using lacZ as the reporter gene (unpublished results). However, no β-galactosidase activity was detected in both cases. We speculate that this could be due to a lack of membrane transport system for isopropyl-β-d-thiogalactopyranoside and/or arabinose in L. hongkongensis. Alternatively, it could be due to a lack of the catabolite activator protein, a protein essential for the activation of the two promoters, in L. hongkongensis. This is in line with the observation that L. hongkongensis does not utilize any sugar tested [ [, [, []. Therefore, the tetracycline inducible promoter and the tetracycline repressor gene, used in the E. coliS. aureus shuttle vector system [7], were cloned into pPW380 to generate the E. coliL. hongkongensis inducible expression shuttle vector. A multiple cloning site was also inserted downstream to the tetracycline inducible promoter, generating pPW578. This inducible expression system was able to express two commonly used reporter genes in other expression systems, including GFP and GST, efficiently in both E. coli and L. hongkongensis.

Acknowledgements

This work is partly supported by the Research Grant Council Grant (7357/04M); University Development Fund, The University of Hong Kong; and the Research Fund for the Control of Infectious Diseases of the Health, Welfare and Food Bureau of the Hong Kong SAR Government. We thank Dr. Ambrose Cheung for providing us the pCL52.2 and pALC2084 plasmids.

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