Characterization and determination of holin protein of Streptococcus suis bacteriophage SMP in heterologous host
© Shi et al; licensee BioMed Central Ltd. 2012
Received: 16 October 2011
Accepted: 22 March 2012
Published: 22 March 2012
Holins are a group of phage-encoded membrane proteins that control access of phage-encoded endolysins to the peptidoglycan, and thereby trigger the lysis process at a precise time point as the 'lysis clock'. SMP is an isolated and characterized Streptococcus suis lytic phage. The aims of this study were to determine the holin gene, HolSMP, in the genome of SMP, and characterized the function of holin, HolSMP, in phage infection.
HolSMP was predicted to encode a small membrane protein with three hydrophobic transmembrane helices. During SMP infections, HolSMP was transcribed as a late gene and HolSMP accumulated harmlessly in the cell membrane before host cell lysis. Expression of HolSMP in Escherichia coli induced an increase in cytoplasmic membrane permeability, an inhibition of host cell growth and significant cell lysis in the presence of LySMP, the endolysin of phage SMP. HolSMP was prematurely triggered by the addition of energy poison to the medium. HolSMP complemented the defective λ S allele in a non-suppressing Escherichia coli strain to produce phage plaques.
Our results suggest that HolSMP is the holin protein of phage SMP and a two-step lysis system exists in SMP.
KeywordsStreptococcus suis Bacteriophage Holin Lysin
- E. coli :
- GAPDH :
Glyceraldehydes-3-phosphate dehydrogenase gene
LB supplemented with ampicillin (100 μg/ml)
LB supplemented with ampicillin (100 μg/ml) and chloramphenicol (30 μg/ml)
LB supplemented with chloramphenicol (30 μg/ml)
Open reading frame
- S. suis :
Holin-lysin lysis systems typically exist in the double-stranded DNA bacteriophages for termination of their growth cycle and release of viral progeny through host cell lysis. By accumulating and forming lesions in the cytoplasmic membrane, holins control access of phage-encoded endolysins to the peptidoglycan and thereby trigger lysis of the host cell at a precise time point. This process determines the length of the infection cycle and is known as the 'lysis clock'.
It is known that holins do not share sequence similarity, although they do have some common characteristics. Firstly, most holins are encoded by the gene adjacent to the endolysin gene. Secondly, at least one hydrophobic transmembrane domain (TMD) occurs in all holins. Thirdly, holins have a highly charged, hydrophilic, C-terminal domain. By identifying these characteristics, it is possible to predict putative holins. Holins can be grouped into three classes by topology. Class I holins, such as bacteriophage λ S protein  and Staphylococcus aureus phage P68 hol15 protein , generally have more than 95 residues and form three TMDs. Class II holins, such as the S protein from lambdoid phage 21  and the Hol3626 protein from Clostridium perfringens bacteriophage Ф3626 , are smaller (65 to 95 residues) and form two TMDs. Class III holins, such as the holin of ФCP39O and ФCP26F , just have one TMD in the central region of the molecule. The schedulings of lysis time by some holin genes are specified by the dual-start model. In the dual-start model, the holin gene is an open reading frame that encodes two proteins, holin and antiholin, with opposing functions that are responsible for the accurate timing of the endolysin release [6, 7]. For example, the prototype class I holin gene, the S gene of bacteriophage λ encodes not only the effector holin, S105, but also an inhibitor, S107, with a Met1-Lys2-Met3... extension at the terminus. An sdi (site-directed initiation) structure near the 5' end of the S gene controls translational initiations from the two initiator codons and determines the ratio of holin to antiholin.
Holins from bacteriophages infecting Gram-negative bacteria have been widely studied, especially bacteriophage λ , bacteriophage T4  and bacteriophage PRD [10, 11]. In Gram-positive bacteria, several studies on holins have been conducted in phage infecting host cells such as Staphylococcus aureus [2, 12], Lactococcus lactis [13, 14], Lactobacillus fermentum , Streptococcus thermophilus , Streptococcus pneumoniae [17, 18], Listeria monocytogenes  and Bacillus cereus . However, no studies on holins from bacteriophages infecting Streptococcus suis (S. suis) have been reported
S. suis is an important pathogen of pigs causing arthritis, endocarditis, meningitis, pneumonia and septicemia . Thirty-five serotypes (types 1 to 34 and 1/2) based on capsular antigens are currently known. Serotype 2 is considered the most virulent and prevalent type in diseased pigs in China. SMP, an S. suis serotype 2 lytic phage, was isolated and characterized in our previous work. Analysis of the complete genomic sequence (GenBank: EF116926) revealed the presence of a putative holin-lysin lysis system , thus providing further evidence that this is the universal mechanism to schedule host lysis for dsDNA phages. Phage-encoded lysins which could degrade peptidoglycan of Gram-positive bacteria exogenously have a bright future as potential therapeutic agents . The extracellular lytic activities of LySMP, the putative endolysin of SMP, on S suis and its biofilm have been tested and confirmed [24, 25], and HolSMP, the putative holin of SMP, was also showed synergistic antibacterial activity against S. suis with LySMP in our recent work (data not published). However, the exact structure and function of HolSMP remained to be investigated. The inability to isolate bacteria lysogenic for SMP limits the functional analysis of the holin gene. Fortunately, holin-dependent induction of membrane lesions is nonspecific, and this enables testing of holins encoded by bacteriophages infecting Gram-positive bacteria in Escherichia coli (E. coli) . Therefore, in this study, the putative holin, HolSMP, was functionally analyzed in E. coli.
Results and discussion
Computational predictions and analyses of HolSMP
Expression of HolSMP in S.suis during phage SMP infection
Relative quantitation of HolSMP using the comparative CT method
HolSMP Average CT
GAPDH Average CT
ΔCT HolSMP- GAPDH a
ΔΔCT ΔCT-ΔCT, 0 min b
HolSMP Rel. to 0 minc
30.61 ± 0.09
16.64 ± 0.03
13.97 ± 0.09
0.00 ± 0.09
30.66 ± 0.03
16.83 ± 0.03
13.83 ± 0.04
-0.14 ± 0.04
29.60 ± 0.05
18.40 ± 0.03
11.20 ± 0.06
-2.77 ± 0.06
24.54 ± 0.03
16.30 ± 0.05
8.24 ± 0.06
-5.73 ± 0.06
21.27 ± 0.02
16.38 ± 0.04
4.89 ± 0.04
-9.08 ± 0.04
17.69 ± 0.03
18.49 ± 0.03
-0.80 ± 0.04
-14.77 ± 0.04
16.10 ± 0.02
18.61 ± 0.05
-2.51 ± 0.05
-16.48 ± 0.05
16.42 ± 0.03
18.55 ± 0.02
-2.12 ± 0.04
-16.09 ± 0.04
15.26 ± 0.07
16.56 ± 0.04
-1.30 ± 0.08
-15.27 ± 0.08
17.94 ± 0.03
18.68 ± 0.01
-0.74 ± 0.03
-14.71 ± 0.03
Expression of HolSMP in E. coli
E. coli is a convenient host for the investigation of holin proteins from phage that infect Gram-positive bacteria . Therefore, the functional identification of putative holin protein HolSMP was performed in BL21(DE3)pLysS (pEXH1) strains. The plasmid pEXH1, containing the HolSMP gene sequence, was constructed. BL21(DE3)pLysS (pEXH1) was created and growth of transformants was monitored after induction with β-D-thiogalactopyranoside (IPTG) (Figure 3A). Growth inhibition of induced cells occurred from 25 to 40 min and was followed by cell lysis with the OD600 value reducing from 0.85 to 0.24. Toxicity of HolSMP to BL21(DE3)pLysS cells was further proven by the viability assay of induced cells. It was showed that the number of viable cells began to decrease at 10 min and a three-log-unit drop was observed (Figure 3A). Moreover, as is characteristic of all holins, HolSMP could be triggered prematurely by the addition of energy poison, potassium cyanide (KCN, 10 mM) (Figure 3A). To determine the kinetics of HolSMP expression, total cellular protein samples were prepared every 8 min, and the accumulation of HolSMP was determined by western blotting. The results showed that a 16 kDa species, consistent with the predicted mass of HolSMP, was detected in samples taken after 24 min, but not before (Figure 3B). The level of HolSMP protein increased from 24 min to 40 min and then declined.
HolSMP accumulated in and damaged the membrane of expressing cells. In order to confirm the subcellular location of HolSMP in cells, cultures of BL21(DE3)pLysS (pEXH1) were collected 40 min after induction to isolate total cellular protein, cytoplasmic protein and membrane protein samples. As the negative control, protein fractions were also prepared from BL21(DE3)pLysS (pEX). Western blots revealed that the dark brown bands indicating HolSMP protein appeared in total cellular protein preparations and the membrane fraction of BL21(DE3)pLysS (pEXH1), but not in the cytoplasmic fraction or in the HolSMP-negative subcellular samples. This suggested that HolSMP accumulates in the membrane of E. coli, consistent with S. suis.
Determination of HolSMP as a holin protein
From these results, protein HolSMP has been identified for the first holin of S. suis bacteriophage. The holin gene, HolSMP, located upstream of the endolysin gene is transcribed, expressed in S. suis after infection by phage SMP. HolSMP, a putative member of class I holins, accumulates on membrane of S. suis. At present, an S. suis strain lysogenic for SMP has not been isolated, and it is not easy to obtain HolSMP-defective SMP mutants. However, biological evidence for the holin-like character of HolSMP was obtained in a Gram-negative background. The HolSMP product caused cellular death, and changes in cell morphology could be accounted for by lesions in the membrane. By forming lesions in the cytoplasmic membrane, HolSMP permitted T7 lysozyme in BL21(DE3)pLysS, LySMP in BL21(DE3), and R in phage λ-infected cells to escape through the membrane and damage the cell wall. HolSMP shows the same host non-specificity and membrane energy sensitivity as other holins [17, 18]. Thereby, the holin-lysin lysis system of SMP was also determined in this study. The HolSMP is able to trigger activity of the LySMP and release viral progeny through host cell lysis. Our further biochemical investigations will shed light of the mechanism of HolSMP action and the application of HolSMP in biopharmacy
Bacterial strains, phages, plasmids and growth conditions
Bacterial strains, phages and plasmids used in this study
Strains, phages or plasmids
Genotype and relevant features
E. coli F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA- argF)U169, hsdR17(rK - mK +), λ-
E. coli F - ompT hsdS(r B - m B - ) gal dcm (DE3)
E. coli F- ompT hsdS(r B - m B - ) gal dcm (DE3) pLysS (CmR)
DP50 supF[supE44 supF58 hsd53(rB mB)dap D8lacY1 glnV44 Δ(gal-uvrB)47 tyrT58 gyrA29 tonA53 Δ(thyA57)]
Streptococcus suis, serotype 2
Wild type phage of S. suis
λ cI857 Sam7
cIts857, Sam7,Lac promoter for expression of cloned genes, phage of E. coli
Expression vector containing hybrid T7-lacPO promoter, Φ10 ribosome binding site, and lacI gene (pBR322 derivative), ampicillin resistance
Low copynumber cloning vector; p15A ori; Tc+; Cm+
Derivative of pET-32a(+); whole ORF of HolSMP gene, HolSMP(429), inserted behind RBS of pET-32a(+)
Derivative of pET-32a(+); LySMP fragment inserted behind RBS of pET-32a(+)
Derivative of pET-32a(+); pEXH1 (Δ HolSMP)
Hind III-SphI fragment of pEXL subcloned into Hind III-SphI sites of pACYC184
Hind III-SphI fragment of pEX subcloned into Hind III-SphI sites of pACYC184
DNA and protein sequence homology alignments were performed using BLAST tools on NCBI. TMHMM http://www.cbs.dtu.dk/services/TMHMM/, SOSUI http://bp.nuap.nagoya-u.ac.jp/sosui/ and PredictProtein servers http://www.predictprotein.org were used to predict the transmembrane helices in HolSMP.
DNA manipulation and plasmid construction
Primers used in this studya
To construct plasmid pEXH1, HolSMP was amplified with oligonucleotides EXH1 and EXH3 to generate a PCR fragment (HolSMP-1) containing the RBS of plasmid pET-32a(+). PCR using fragment HolSMP-1 as the template was performed to generate a final 495 bp PCR fragment (HolSMP-2) with primer EXH2 and the common reverse primer EXH3. HolSMP-2 was cloned into the XbaI and BamHI sites of pET-32a(+) to give plasmid pEXH1. Plasmid pEXL, harboring the LySMP gene, was constructed in the same as was plasmid pEXH1, using primer pairs EXH5/EXH4 and EXH2/EXH4.
The negative control plasmid pEX was constructed using the same strategy as for pEXH1, using primer SYB34 to introduce a stop codon at the beginning of HolSMP. The expression vectors pACEXL and pACEX were constructed by subcloning the HindIII-SphI fragment of pEXL (containing the T7 promoter, RBS, Lac operon and lysine gene) or pEX (containing T7 promoter, RBS, Lac operon and MCS) into HindIII-SphI digested pACYC184.
RNA extraction from SMP-infected host cells
To identify the transcription of the HolSMP gene in SMP-infected S. suis, exponentially growing SS2-H cells (about 109) were infected with phage SMP (109-1010 plaque forming units/ml) at a multiplicity of infection of at least 10. A sample, containing 108 host cells, was collected prior to addition of phage and immediately centrifuged at 13,000 × g for 1 min to pellet cells. The rest of the reaction was incubated at 37°C for 15 min and centrifuged at 13,000 × g for 1 min. The cell pellet was re-suspended gently with THB and incubated at 37°C with shaking at 150 rpm. Samples, containing 108 cells, were collected as described above at 5, 10, 15, 20, 40, 60, 80, 100 and 120 min after infection. Cell pellets were snap frozen in liquid nitrogen as soon as the supernatant was discarded, and stored at -20°C until RNA extraction. Total RNA of all samples was extracted simultaneously with the RNA extraction kit (Omega). Contaminating DNA was removed by digestion. Downstream cDNA synthesis was performed when DNA from SMP and host cells could not be detected by PCR.
Reverse transcription PCR
The transcript levels of HolSMP were determined visually by reverse transcription PCR. MMLV reverse transcriptase (25 U) and random primers (TakaRa) were used for cDNA synthesis. The S. suis housekeeping gene GAPDH was employed as a reference for normalization of samples.
Two pairs of primers, EXH1/EXH3 and GAPDH1/GAPDH2 (Table 3), were used to amplify HolSMP and GAPDH, respectively. PCR was carried out in a final volume of 25 μl, containing 2 μl cDNA (1:5 dilution), 0.4 μM of each primer, and 12.5 μl 2 × PCR mix (Dongsheng Biotech). Amplification was performed for 28 cycles with annealing temperatures of 57.5°C and 60°C for HolSMP and GAPDH, respectively. The volume of each HolSMP PCR product loaded for electrophoresis was adjusted based on the corresponding GAPDH fragments (223 bp). Gels were visualized with an image analysis system after electrophoresis. Then, bands corresponding to the HolSMP gene (473 bp) in each lane were compared.
Real-time quantitative PCR
Further relative quantification of transcript levels of HolSMP was performed by real-time quantitative PCR. Primers SYB35 and SYB36 were designed using primer 5.0 software to amplify 116 bp of HolSMP (Table 3). The S. suis housekeeping gene GAPDH was employed as a reference for normalization of samples. PCR was carried out with a PTC-200 PCR instrument (Bio-Red, Hercules, CA) and MJ option Monitor analysis system. PCR was carried out in a final volume of 50 μl, containing 2 μl cDNA (1:5 dilution), 0.4 μM of each primer, and 1 × SYBR premix EX taq II (Takara). Amplification was performed over 40 cycles of 5 s at 95°C, 30 s at the annealing temperature (57.5°C for HolSMP and 60°C for GAPDH), and 10 s at 72°C. The reaction products were then cooled to 50°C and subjected to a post-PCR melting cycle by increasing the temperature by 0.2°C every 10 s, up to 95°C. The comparative CT method was used to analyze the relative transcription levels of HolSMP after infection.
Membrane protein extraction from SMP-infected host cells
To identify the expression of HolSMP in SMP-infected S. suis, exponentially growing SS2-H cells were infected with phage SMP as described above. Samples, containing 1011 host cells, were collected as described above at 20, 40, 60, 80 and 100 min after infection. Cell pellets were frozen in liquid nitrogen immediately until required for extraction of membrane proteins. To prepare membrane fractions, harvested cells were suspended in 5 ml ice-cold lysis buffer (300 mM NaCl, 50 mM sodium phosphate, pH8.0), and sonicated on ice at 200 W for 50 cycles of 3 s on and 20 s off. The cell fragments were collected by centrifugation at 13,000 × g for 1 min. This process was repeated until cells were completely lysed. The collected supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C to pellet membrane fragments. Each pellet was solubilized with 5 ml ME buffer (1%Triton X-100, 10% glycerine, 0.5 M NaCl, 35 mM MgCl2, 220 mM Tris-HCl, pH8.0) and incubated for 12 h on ice with shaking . The insoluble fraction was discarded after ultracentrifugation at 100,000 × g for 1 h at 4°C. Note that addition of lysozyme must be avoided.
Protein expression and viability assays
BL21(DE3)pLysS harboring plasmid pEXH1, designated BL21(DE3)pLysS (pEXH1), was inoculated and cultured to an optical density at 600 nm (OD600) of 0.5 ~ 0.6. Protein expression was induced by addition of IPTG to a final concentration of 1 mM and shaking at 30°C at 150 rpm. The growth of clones after induction was monitored by measuring OD600. For protein expression analysis, cells in 1 ml cultures were suspended with 100 μl 1 × tricine sample buffer (1 × TSB, 50 mM Tris-HCl [pH 6.8] containing 12% [w/v] glycerol, 4% [w/v] SDS, 2.5% [v/v] mercaptoethanol and 0.01% [w/v] bromophenol blue) and boiled for about 5 min to prepare total cellular protein samples. For viability assays, 20 μl cultures of BL21(DE3)pLysS carrying plasmid pEXH1 was placed on ice at different time points after addition of IPTG. Each sample was serially diluted on ice and 100 μl dilutions were plated in triplicate on LB-Ap. Colonies from three separate experiments were counted after 12 to 16 h of incubation at 37°C.
One litre culture of BL21(DE3)pLysS (pEXH1) induced for 40 min by IPTG was harvested by centrifuging at 13,000 × g for 3 min at 4°C. To prepare the cytoplasmic fraction, harvested cells were suspended in 5 ml ice cold lysis buffer, sonicated on ice at 400 W for 20 min, (3 s on/20 s off cycles), and ultracentrifuged at 100,000 × g for 1 h at 4°C to remove the membrane fraction. Membrane protein preparation from membrane pellets was performed with 5 ml ME buffer as described in membrane protein extraction from SMP-infected host cells. Both cytoplasmic fraction and membrane fraction samples were mixed with 2 × tricine sample buffer and boiled.
Tricine-SDS-PAGE and western blots
HolSMP was separated by tricine-SDS-PAGE and examined by western blotting. For tricine-SDS-PAGE, the protein samples were resolved on 20% (w/v) polyacrylamide gels as previously described . The gel was stained with Coomassie blue or directly used to transfer proteins onto a nitrocellulose membrane by electroblotting. The antibody against a recombinant protein corresponding to TMD2-TMD3-C-terminal sequence of HolSMP was raised in mouse in our lab previously. For immunodetection of HolSMP, the antibody against HolSMP (1:1000 dilution) and the goat anti-mouse immunoglobulin conjugated to horseradish peroxidase (1:2500 dilution; Immunology Consultants Laboratory, Inc.) were used as the primary and secondary antibodies, respectively. The western blots were analyzed with DAB colorimetric western blot kit (Rockland).
Transmission electron microscopy
Culture samples were collected every 5 min in the first hour after the addition of IPTG and centrifuged at 1,160 × g for 3 min to pellet cells. The pellets were re-suspended in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4). Cells were fixed at 4°C for 30 min and centrifuged at 1,160 × g for 1 min. Thin sections of the cells were processed and examined at 60,000 × magnification with a Hitachi H-600 transmission electron microscope.
Co-expression of HolSMP and LySMP in E. coli
To explain the physiological role of HolSMP, HolSMP was co-expressed with LySMP. The HindIII-SphI fragment containing LySMP from pEXL and a negative control sequence from pEX were inserted into plasmid pACYC184. The resulting plasmids were designated pACEXL and pACEX. The chloramphenicol-resistant plasmid pACYC184 harboring the p15A origin of replication was compatible with colE1 of vector pET-32a(+) . Therefore, the recombinant plasmids pACEXL and pACEX were compatible with pEXH1. The E. coli BL21(DE3) strains harboring plasmid combinations pEXH1+pACEXL (harboring both HolSMP and LySMP), pEXH1+pACEX (harboring HolSMP only) and pACEXL+pEX (harboring LySMP only) were grown overnight in LB-Ap-Cm. The strains were diluted (1:100) with fresh medium and cultured to an OD600 of 0.6. Expression of the genes was induced by addition of IPTG, and the growth of clones was monitored by measuring OD600.
Complementation of λ Sam 7 lysis function
BL21(DE3)pLysS (pEXH1) was inoculated and cultured to an OD600 of about 0.5 in LB-Ap. A 200-μl BL21(DE3)pLysS culture was infected at 37°C for 15 min with 10 μl bacteriophage λ cI857 Sam7 (105 plaque forming units/ml). The E. coli BL21(DE3)pLysS and phage were mixed with 5 ml of soft agar containing 0.1 mM IPTG and 100 μg/ml ampicillin, and quickly poured onto LB-Ap plates. Addition of 0.1 mM IPTG to the soft agar induced expression of HolSMP at sub-lethal levels for BL21(DE3)pLysS harboring plasmid pEXH1 unless the R lysin of λ cI857 Sam7 was also present. The plates were incubated face up at 37°C to encourage the formation of plaques, and the number of plaques was determined after overnight incubation. BL21(DE3)pLysS with plasmid pET-32a(+) and VCS257 were used as controls. Before infection, freshly cultured VCS257 was gently resuspended and diluted to an OD600 of 0.5 with sterile 10 mM MgSO4 after centrifugation at 500 × g for 10 min. Antibiotic was not added to the soft agar or plates to culture VCS257.
The authors would like to thank Prof. Lu Chengping from Nanjing Agricultural University for providing constructive suggestions. The research was supported by National Natural Science Foundation (31172381) and fund from State Key Laboratory of Veterinary Etiological Biology (SKLVEB2010KFKT004).
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