Folia Microbiol DOI 10.1007/s12223-015-0375-1

Isolation and characterization of a Siphoviridae phage infecting Bacillus megaterium from a heavily trafficked holy site in Saudi Arabia B. A. Othman & Ahmed Askora & Amel S. M. Abo-Senna

Received: 17 May 2014 / Accepted: 16 January 2015 # Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2015

Abstract In this study, we isolated and characterized a Siphoviridae phage isolated from the vicinity of a religious structure (Kaaba) in Makkah, Saudi Arabia. The phage was designated as φBM and characterized using transmission electron microscopy, restriction digestion of its DNA, and host range. Electron micrograph indicated that φBM phage has an icosahedral head with diameter of about 65±5 nm and long, non-contractile tail with length of about 300±10 nm and width of about 17±2 nm, respectively. On the basis of the φBM phage morphology, we thus propose that φBM represents a member of Siphoviridae phages. The φBM phage was shown to be able to infect Bacillus megaterium and two other Bacillus species and has no effect on other tested bacteria. φBM was stable over the pH range of 5–9, chloroform resistant and stable at 4 °C. A one-step growth experiment showed a latent period of about 40 min and a burst size of approximately 65 per infected cell. The purified bacteriophage appeared to consist of ten proteins. The genome size was estimated to be ∼38 kb. To our knowledge, this is the first report on the isolation of a bacteriophage from Kaaba a heavily trafficked holy site in Saudi Arabia.

B. A. Othman Agricultural Microbiology Department, Faculty of Agriculture, Ain Shams University, Cairo, Egypt A. Askora (*) Department of Botany and Microbiology, Faculty of Science, Zagazig University, Zagazig 44519, Egypt e-mail: [email protected] A. S. M. Abo-Senna Botany Department, Faculty of Science, Al-Azhar University (Girls Branch), Cairo, Egypt

Introduction Bacteriophages are a diverse group of viruses that infect bacteria and are found in a variety of environments, which include soil, drinking water, food, and so on (Kutter et al. 2005; Clokie et al. 2011). It is estimated that the biosphere consists of approximately 1031 bacteriophages and archael viruses (Hendrix et al. 1999). Bacteriophages are known to proliferate wherever their bacterial hosts exist (Hendrix et al. 1999). Virion particles can exist independently outside the host; however, all phages are obligate intracellular parasites and need their host to propagate (Jensen et al. 1998). As new phages are discovered, our understanding of molecular biology and microbial genetics will continue to grow and new technologies will be developed that will continue to help molecular, cellular, and developmental biologists to understand the complexity of life (Canchaya et al. 2003). Generally, phages are stable if the environment is not hostile. Various external physical and chemical factors, such as temperature, UV light, acidity, and salinity, can inactivate a phage through damage of its structural elements (head, tail, envelope), but researchers are known to keep phages in their fridges for over 40 years with no reduction in titer (Clokie et al. 2011). In this study, different Bacillus species and other bacterial strains were collected and used as indicator hosts to screen phages from a heavily trafficked area by people in a holy site [a religious structure (Kaaba)] in Makkah, Saudi Arabia. The Kaaba is a large, stone structure at the heart of the Masjid alHaram in the Holy City of Makkah, Saudi Arabia, and is Islam’s holiest building. Constituting a single room with a marble floor, it now stands some 60 ft high, and each side is approximately 60 ft in length. The Kaaba is the focal point (qibla) towards which Muslims worldwide turn to pray (Peterson 1997). Unlike other Saudi Arabian cities, Makkah retains its warm temperature in winter, which can range from 18 °C at night to 30 °C in the afternoon. Summer temperatures

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are considered very hot and break the 50 °C. Rain usually falls in Makkah in small amounts between November and January. Each year, more than two million pilgrims from over 100 countries converge on the holy city of Makkah to reenact the ritual dramas that Muslims have been performing for centuries. Mass gathering events like the gathering of huge numbers of pilgrims traveling to Saudi Arabia’s holy sites during Ramadan and Hajj may increase the opportunities to spread infections in this place. Little information is known about the microbiological characters of the Kaaba environment of Makkah Province, Saudi Arabia (Peterson 1997). Here, we isolate and characterize a siphovirus from this environment in Saudi Arabia, although the Saudis keep Kabaa meticulously clean. This is the first report indicates to occurrence of bacteriophage in the above-mentioned source of the isolation.

Materials and methods Bacterial strains and culture conditions The Bacillus strains and Gram-negative bacteria used in this study are listed in Table 1. B. megaterium was used as indicator strain for bacteriophage isolation. All bacteria strains listed in Table 1 were used in phage host range analysis. Luria– Table 1

Susceptibility of different bacterial hosts to φBM phage

Strain Gram-positive bacteria Bacillus amyloliquefaciens Brevibacillus parabrevis Bacillus megaterium Bacillus cereus Bacilus subtilis Bacillus circulans Bacillus polymxa Bacillus thuringiensis Staphylococcus aureus Gram-negative bacteria Erwinia carotovora P. aeruginosa 101 P. aeruginosa 62 P. aeruginosa P1

φBM

Bacteriophage isolation Samples for phage isolation were collected from different sites in Kaaba, swabs from the floor of Kaaba yard, and swabs from the surfaces of Kaaba wall, Makkah, Kingdom Saudi Arabia. Sampling was performed in October 2012. The samples were preserved at 4 °C until working. The presence of bacteriophages in the collected samples was determined qualitatively by the spot test technique (Adams 1959). Samples was enriched in the presence of 50 mL of Luria–Bertani (LB) broth containing 1 mL of B. megaterium as a host bacterium (108 cfu/mL) and incubated on a rotary shaker for 48 h at 30 °C; the culture supernatant was harvested by centrifugation at 5500×g for10 min. Chloroform was added to the supernatant at the rate of 1:10 (v/v) with vigorously shaking, and phages were obtained from the upper layer. Quantitative assaying of the phages was carried out as described by the soft-agar overlay (double layer) method as reported by (Othman et al. 2008). Bacteriophage propagation and concentration

Source

– – +(6.6×109) +(4.3×107) +(1.7×105) – – – –

MIRCENa MIRCENa MIRCENa MIRCENa MIRCENa MFSMb MIRCENa MFSZc MIRCENa

– – – –

MIRCENa MFSZc MFSZc MFSZc

(+) indicates that the strain is susceptible to the phage and produces plaques, while (−) indicates that no plaques were observed a

MIRCEN (Microbial Resource Centre, Ain Shams University, Faculty of Agriculture, Cairo, Egypt)

b MFSM (Microbiology Laboratory Department of Microbiology, Minia University, Egypt) c

Bertani broth was used for liquid cultures, and 1.5 % solid agar medium was used for plating bacteria. A double-layer agar technique was used for phage plaque-forming assays, with 1.5 % agar medium at the bottom and 0.7 % soft agar medium on the top. All incubations were carried out at 28 °C.

MFSZ (Microbiology Laboratory Department of Microbiology, Zagazig University, Egypt)

High titer phage stock of the Bacillus phage lysate was obtained using the culture method as following: One Erlenmeyer flask (250 mL) containing 100 mL of LB broth medium was prepared and was inoculated with Bacillus megaterium (108 cfu/mL); phage particles (107pfu/mL) were added at multiplicity of infection (MOI) of 0.1 then incubated at 30 °C for 48 h; after incubation, cultures were centrifuged at 5500×g for 15 min, and then, chloroform was added to the supernatant (1:10, v/v). The mixture was vigorously shaken for 3–5 min and left to clarify for 30 min. The suspension containing phage was then transferred into sterile flasks and stored at 4 °C with traces of chloroform. The purification of Bacillus bacteriophage was performed by ammonium sulfate precipitation according to Johnson et al. (1973), where 47.2 g of ammonium sulfate was added to 100 mL phage lysate to get final concentration of 70 %, followed by dialysis treatment to remove the excess of salt. After mixing, the mixture was allowed to stand at 4 °C overnight, followed by centrifugation at 8500×g for 20 min, and the phage pellets were suspended in 20 mL saline buffer and recentrifugated at 8500×g for 20 min, and the supernatant containing the highly concentrated phage was assayed quantitatively using the double over layer agar technique after concentration, and the titer was 1012 pfu/mL. The phage was stored at 4 °C for further investigation.

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Electron microscopy

Physical properties of the isolated phage

The isolated phage was examined as described by (Bradely 1976). A drop of the phage suspension (1012 pfu/mL) was placed on 200 mesh copper grids with carbon-coat formvar films, and the excess was drawn off with filter paper. A solution of 2 % uranyl acetate was then placed on the grids and the excess drawn off as before. Specimens were examined with an electron microscope (Model Beckman 1010) at Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.

Determination of thermal inactivation point

Host specificity The host specificity to a number of different bacterial strains obtained from Faculty of Agriculture, Cairo MIRCEN, Ain Shams University, Egypt, and other bacterial strains collected through this study were determined by the double over layer agar technique. For this, 3 mL of soft top agar was prewarmed to 50 °C, mixed with 100 μL of with different bacterial strains (108 cfu/mL) and 100 μL of concentrated phage suspensions containing 109 pfu/mL, then poured onto LB agar plates. The plates were then incubated for 24 h at 30 °C for plaque development. The titer was measured on the indicator strain. The plaques were counted, pfu/mL was calculated, and the host specificity was repeated two times.

Two milliliters of phage suspension was incubated at different temperatures of 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 °C for 10 min. Phage infectivity was assayed by the spot test and the double over layer agar technique. Effect of pH on the phage stability The method of Jamalludeen et al. (2007) was used to evaluate the activity of phages to survive at different pH levels. Briefly, phage suspension was exposed to a certain pH value adjusted from 2 to 10 using 0.1 mol/L HCl or NaOH over 16 h of incubation at 30 °C and then checked for survival by the spot test and the double over layer agar technique. Phage longevity in vitro The phage suspension was stored at room temperature and 4 °C, and the stability of φBM phage was recorded at day 0, 10, 20, till 6 months, and the phage infectivity was assayed qualitatively by the spot test and the double over layer agar technique. Extraction of phage genomic DNA and restriction analysis

One-step growth curve of φBM phage The φBM phage was added to the host bacterium B. megaterium culture (108 cfu/mL) at an MOI of 1.0, and the mixture was incubated at 28 °C for 10 min. Cells were collected by centrifugation at 8500×g for 10 min and were resuspended in 1 mL of fresh LB medium. This process was repeated twice to remove unadsorbed phage particles; then, the cell suspension was added to the 100 mL LB broth and incubated with shaking at 28 °C for 3 h. Phage titer in the culture was measured by the double-layer agar technique at a 15-min interval. The relative burst size at different times was plotted against time to determine the latent and rise period. The experiments were repeated three times.

Phage DNA was extracted using the phenol/chloroform method according to Sambrook et al. (2001). DNA was digested with restriction enzymes according to the supplier instructions (Takara Bio Inc., Japan). The DNA digestion mixtures were analyzed by electrophoresis at 100 V in a 1.0 % agarose gel stained with ethidium bromide using a DNA ladder as marker. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) purified phage particles were subjected to SDSPAGE [10–12 % (w/v) polyacrylamide] according to Laemmli (1970). Protein bands were visualized by staining the gel with Coomassie brilliant blue.

Results Isolation of B. megaterium phage φBM

Effect of chloroform on phages High titer phage suspension containing 108 pfu/mL was used. Two milliliter of phage suspension was transferred into sterile test tube and one drop of chloroform added. The solution was mixed gently and left for 30 min at room temperature. After this time, samples were collected and assayed for presence surviving phage.

Different samples from the floor around the Kaaba were tested for the presence of bacteriophages specific for Bacillus species and other tested bacteria such as Brevibacillus parabrevis, Pseudomonas fragi, and Staphylococcus aureus using the spot test technique and the double over layer agar technique (Fig. 1a, b). From these, φBM produced turbid plaques on lawns of B. megaterium as host; one single plaque with 3 mm

Folia Microbiol Fig. 1 Plaques formed on B. megaterium as bacterial lawn and φBM phage morphology. a Spot test. One hundred microliters of φBM phage was spotted onto B. megaterium as a host and incubation over night at 30 °C. b Single plaques resulting from 10−7 dilutions by overlayer agar technique. Arrow indicates the size of the plaque. c Morphology of B. megaterium phage particles under TEM. The virion was negatively stained with uranyl acetate. Scale represents 100 nm

A

B

C

in diameter (Fig. 1b) was isolated for further purification and characterization. Morphology and the host specificity of φBM The purified particles of φBM was negatively stained and examined by transmission electron microscopy to determine the morphotype of the phage. The φBM phage had an icosahedral capsid of 65 ± 5 nm in diameter and long noncontractile tail with length of about 300±10 nm and width of about 17±2 nm. The φBM phage appeared to be a member of the Siphoviridae family based on their structure in electron micrograph (Fig. 1c). The host range of φBM was determined against different species of Bacillus and other genera and species. The results in Table 1 showed that φBM could infect only three of seven different Bacillus species used as host in this study and could not infect other bacterial genera, indicating a quite broad host range of this phage (Table 1).

that φBM phage was extremely heat stable, which still remained active after 10 min exposure at 90 °C; to determine exactly the thermal inactivation point, the heat stability each 2° above 90 °C was detected, the results showed that phage losses its infectivity at 92 °C (Fig. 3). Furthermore, the φBM phage was investigated for its ability to survive at different pHs. The results in Table 2 showed that phage maintained its infectivity when incubated in a pH range between 4 and 9. However, phage could not be detected at less than pH 4 and more than 9.

One-step growth curve of φBM The one-step growth curve of φBM phage on B. megaterium as a host was determined (Fig. 2). The growth characteristics of φBM phage revealed a latent period of 40 min, followed by a rise period of 50–55 min. The average burst size was 65 per infected cell (Fig. 2). Effect of heat and pH on the φBM phage stability The stability of φBM phage was assayed by spot test and plaque assay. Data in Table 2 and Fig. 3 showed

Fig. 2 Single-step growth curve for φBM. The plaque forming units (PFUs) per infected cell in cultures of B. megaterium at different times post-infection are shown. Samples were taken at intervals (every 10 min up to 160 min). Error bars indicate standard deviation. The data of error bars for the standard deviation in Fig. 2 were obtained from three independent replicates

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Effect of different pH on the φBM phage infectivity

pH

PFU/mL×107

4 5 6 7 8 9 10

0 + (2.55) + (4.99) + (2.78) + (1.13) + (0.59) 0

Table 3 Stability during storage and effect of chloroform concentration on φBM phage infectivity Temperature (oC)

Different pH values on the φBM infectivity were determined. The φBM phage (8.5×108 pfu/mL) was incubated at different pH, and the optimal pH for φBM stability was determined. (+) Indicates that phage is infective, with titers in parenthesis, while (0) indicates that no plaques were observed

Time (weeks)

4 °C

28–30 °C (room temperature)

Stability during storage and effect of chloroform on φBM phage The longevity in vitro of φBM was 4 months at room temperature (Table 3) and generally more stable at 4 °C (Table 3). Chloroform had no effect on phage viability for 30 min at room temperature. Titers before and after chloroform treatment were the same, indicating chloroform resistance of the φBM phage. Genetic and SDS-PAGE analysis of φBM The φBM1 genome was a linear double-stranded (ds) DNA because it was susceptible to digestion with different restriction enzymes (Fig. 4). The phage genome was digested with different restriction endonucleases enzymes, including, EcoRI, EcoRV, HindIII, and PstI and subsequently subjected to electrophoretic analyses. As shown in Fig. 4, the phage DNA was sensitive to EcoRV, EcoRI, and HincII and resistant to

φBM (PFU/mL)

1 2 3 4

+ (6.34×108) + (5.52×108) + (4.98×108) + (4.39×108)

8 12 16 1 2 3 4 8 12 16

+ (3.57×107) + (2.64×107) + (1.27×106) + (6.10×108) + (4.76×107) + (3.56×107) + (2.82×106) + (2.12×105) + (1.72×105) + (1.26×103)

The φBM infectivity following different storage period at 4 °C and room temperature was determined. (+) Indicates that phage is infective, with titers in parenthesis

HindIII and PstI restriction endonuclease (lane 3, and PstI lane 5). The genome size was estimated to be ∼38 kb, as determined by combining the fragment sizes obtained with the restriction enzymes used. Using SDS-PAGE, we detected virion proteins of φBM; at least ten proteins, ranging from 13 kDa and about 120 kDa, were separated by SDS-PAGE (Fig. 5).

M

1

2 3

4

5 6

M

(kbp) 19.33 7.74 6.62 4.23 3.47 2.69 1.88 1.49

Fig. 3 Effect of heat on the φBM phage stability. φBM phage was incubated at different temperatures for 10 min and the φBM infectivity was investigated

Fig. 4 Restriction enzyme digestion patterns of φBM after treatment with different restriction enzymes and followed by resolution on 0.9 % agarose gel (lanes 1–6). Lane 1 φBM, undigested; lane 2 φBM digested with HincII; lane 3 φBM digested with HindIII, lane 4 φBM digested with EcoRV, lane 5 φBM digested with PstI, lane 6 φBM digested with EcoRI and (M) lambda DNA marker/StyI

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Fig. 5 SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of φBM structural proteins. The purified φBM phage particles were boiled in loading buffer for 5 min and separated in 12.5 % SDS-PAGE. Protein bands were visualized by staining the gel with Coomassie brilliant blue

Discussion Bacteriophages occur abundantly in the biosphere, with different virions, genomes, and lifestyle (Hendrix et al. 1999). In this study, we isolated and characterized a bacteriophageinfecting B. megaterium from a religious structure (Kaaba) in Makkah, Saudi Arabia. The φBM showed typical siphovirus morphology with long, flexible, and noncontractile tail of about 300±10 nm and width of about 17± 2 nm and an isometric head of about 65±5 nm in diameter (Fig. 2). Interestingly, the morphology of φBM phage showed a similarity to those previously reported for B. megaterium specific phages such as phiT (siphovirus, with head diameter of 68 nm and long, non-contractile tail with length of 240 nm) and MJ-1 (siphovirus, with head diameter of 56.4 nm and tail with length of 300 nm) (Young and Wilson 1972; Yehle and Doi 1967; Cooney et al. 1975; Carvalho and Vary 1977;

Reanney and Teh 1976; Candeli et al. 1978). The φBM phage was specific for B. megaterium, B. cereus, and B. subtilis but did not infect other Bacillus species nor any Gram-negative bacteria tested (Table 1). These results are consistent with the host range data of CS-I (Cooney et al. 1975) and MP-7 (Carvalho and Vary 1977). These phages were also specific for few Bacillus species, although the number of species tested was less extensive. Physical characteristics of φBM phage including effect of heat and stability at different pHs and stability during storage were examined. Interestingly, the results in Fig. 3 showed that φBM phage was thermostable, where it still remained active after 10 min exposure at 90°C, unlike other reported B. megaterium phages, such as the MJ-1 phage, which was sensitive to 60 °C, and phage phiT, which was sensitive to 75 °C. Furthermore, the φBM was able to survive in wide range of pH values. The results in Table 2 showed that the optimum pH for physical stability of φBM was from 5 to 8 for long storage. These results are consistent with B. cereus CP-51, where its pH stability range was from 5.6 to 7.0 (Thorne and Holt 1974). The temperature of bacteriophage storage is the most important factor, which determines phage activity. The data presented in Table 3 showed that the φBM was stable outside the host for 4 months at room temperature. Most of the isolated Bacillus phages have dsDNA, and some of them are quite distinct as they contain unusual bases in their DNA (Lee et al. 2011). The genetic material of the isolated B. megaterium phage was found to be double-stranded DNA, since it shows susceptibility to restriction enzymes (Fig. 4), and the genome size was estimated to be ∼38 kb. There are several Bacillus siphoviruses with small (∼40 kb) genomes existing, including phi 105 (NC_004167), TP21-L (NC_011645), and β (NC_007734). The results suggest that φBM is different from these phages. A priority objective of our future research will be to identify the nucleotide sequence and protein of the isolated φBM. In conclusion, we present φBM, a siphovirus phage isolated from a religious structure (Kaaba) in Makkah, Saudi Arabia, that is specific to different species of Bacillus. We hope that the isolated φBM phage may be used as a source of DNA for transfection studies in B. megaterium, and decipher specific rules regarding phage host range in our further study.

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