Biologia 67/6: 1031—1037, 2012 Section Cellular and Molecular Biology DOI: 10.2478/s11756-012-0117-7 Paenibacillus hemolyticus, the first hemolytic Paenibacillus with growth-promoting activities discovered Salmah Ismail1, Teow Chong Teoh1*, Choong Yong Ung2, Saad Musbah Alasil3 &RahmatOmar3 1Institute of Biological Sciences, Faculty of Science,University of Malaya, 50603 Kuala Lumpur, Malaysia; e-mail: [email protected] 2 Department of Mathematics, National University of Singapore, Singapore 3Department of Otorhinolaryngology, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia Abstract: Paenibacillus spp. are Gram-positive, facultatively aerobic, bacilli-shaped endospore-forming bacteria. They have been detected in a variety of environments, such as soil, water, forage, insect larvae, and even clinical samples. The strain 139SI (GenBank accession No.: JF825470.1) from three strains of Paenibacillus isolates investigated here was chosen as the type strain of the proposed novel species. The other two similar strain isolates investigated were 140SI (JF825471.1) and 141SI (JQ734548.1). These strains were identified as members of the genus Paenibacillus on the basis of phenotypic characteristics, phylogenetic analysis and 16S rRNA G+C content. Surprisingly, these strains exhibited a strong hemolytic activity on 5% sheep blood agar. Their crude extracts also showed positive growth-promoting activities in colon cancer and Vero cell lines. To our knowledge, this is the first Paenibacillus with hemolytic and growth-promoting activities reported, and the name Paenibacillus hemolyticus for this novel species is proposed. The capability of this novel species in hemolytic and cell growth activities suggests its potential in both clinical and pharmacological implications. Key words: Paenibacillus hemolyticus; soil bacteria; hemolytic; anticancer and cytotoxic activities; 16S rRNA G+C content. Abbreviations: At, absorbance of test sample; BHI, brain-heart-infusion; CRA, Congo red agar; dNTPs, deoxynucleotide triphosphates; DSMZ, German Collection of Microorganisms and Cell Cultures; %G+C, guanine plus cytosine content in percentage; HCT, hematocrit; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NCBI, National Center for Biotechnology Information; SI, Salmah Ismail. Introduction degradation), nitrogen fixation and proteases (Berge et al. 2002; Hrabák & Martínek 2007). Actual March 2012, A group of anaerobic, Gram-positive and endospore- no hemolytic activity had been reported for Paenibacil- forming bacteria were initially classified under the lus spp. According to DSMZ. genus Bacillus, based on their phenotypic character- Here, we report on the identification and charac- istics. Later, phylogenetic analyses using 16S rRNA terization of a novel Paenibacillus species that is not with 11 identified species isolated from various habi- only capable to show remarkable hemolytic activity, but tats re-classified them as a separate genus, Paenibacil- it also exhibits growth promotion in colon cancer and lus (Ash et al. 1993). To date, there are 137 species Vero cell lines. For the first time, strains of Paenibacil- of Paenibacillus approved and validated according to lus possessing both hemolytic and growth-enhancing ac- the bacterial nomenclature list by Deutsche Sammlung tivities with potential clinical implications are reported. von Mikroorganismen und Zellkulturen GmbH Braun- schweig (DSMZ), Germany (URL: http://www.dsmz. Material and methods de/download/bactnom/bactname.pdf; March 2012). The genus Paenibacillus has been isolated from various Colony isolation Strains Salmah Ismail (SI), 139SI (GenBank accession No.: ecological habitats, including warm springs (Saha et al. JF825470.1; ATCC BAA-2268); 140SI (JF825471.1) and 2005; Chou et al. 2007; Akel et al. 2008), alkaline soils 141SI (JQ734548.1) were originally isolated from an agri- (Yoon et al. 2005), and agricultural soil (Ma & Chen cultural soil obtained from Serdang Agricultural Center, 2008). Various properties were reported among Paeni- Selangor, Malaysia. This soil sample was thoroughly sus- bacillus species, including xylanolytic (polysaccharide- pended in 3 mL of sterile distilled water, the suspension * Corresponding author c 2012 Institute of Molecular Biology, Slovak Academy of Sciences 1032 S. Ismail et al. was streaked on brain-heart infusion (BHI) agar plates sup- form red colonies. Colonies exhibiting a bright black and plemented with 5% sheep blood, and plates were incubated dry opaque black morphology were classified as virulent for 16–24 h at 37 ◦C. Hemolytic colonies obtained from the strains (biofilm producers), whereas red, pink and bordeaux plates were sub-cultured again on BHI blood agar and in- colonies were classified as non-virulent strains (non-biofilm cubated under aerobic conditions. Strains 139SI, 140SI and producers) (Oliveira & Cunha 2010). 141SI were among the isolates that appeared to exhibit a strong hemolytic activity (β-hemolysis) and were routinely Microscopic observation of cellular morphology cultured on BHI blood agar and maintained as a glycerol Cellular morphology was observed under scanning electron suspension (25%, w/v) at −80 ◦C and BHI-slant agar at microscopy using an overnight culture of strains SI grown room temperature. Of 141 isolates of Paenibacilli and Bacilli in BHI broth. To prepare the sample for scanning electron from the soil extract 52% showed negative hemolytic activi- microscope, 1 mL of pure bacterial culture was transferred ties on BHI blood agar and hence were used as negative con- intoa1mm× 1 mm rounded filter paper and air dried for trols against SI strains. To supplement the hemolytic prop- 10 min. The sample was rinsed in 0.1 M phosphate buffer, erty, the biosurfactant activity assay for the crude protein followed by fixation in 2.5% glutaraldehyde in 0.1 M phos- filtrates from strains 139SI, 140SI and 141SI was also con- phate buffer at room temperature for 4 h. The cells were ducted using drop collapsing test (Youssef et al. 2004) and dehydrated by using series of ethanol concentrations: 30%, the positive emulsification activity was observed as milky- 50%, 70%, 85%, 95% and 100%, and then two times with colored smear indicating that the oil and filtrate were mis- 100% acetone. Critical point drying was done by Hitachi cible. HCP-2 critical point dryer. The samples were then mounted on specimen stubs and sputter-coated with gold. The sam- Bacterial Gram identification ples were observed by Zeiss DSM 950 scanning electron mi- A test for bacterial Gram differentiation was performed us- croscope at 15 kV (Ko et al. 2007). ing Gram staining and non-staining methods of an in-house alkaline lysis and aminopeptidase strip test (Merck KGaA, Investigating the cytological effects of the novel Paenibacil- Darmstadt, Germany). For the alkaline lysis method, a so- lus on cell cultures lution of 1% sodium dodecyl sulfate and 0.2 N NaOH was The colon cancer HCT 116 cell line for anticancer experi- introduced to the overnight bacterial culture of strains SI ments and Vero cell line for cytotoxic experiments were pre- prior to two min incubation at 4 ◦C. Aminopeptidase strip pared as described previously (Wilson 2000). Cell suspen- test was carried out as described previously (Farmer 2005). sion of 5,000 cells/well was adjusted to the 96 well microtitre The Gram-negative and Gram-positive control isolates used plate. The filtrate (crude extract) of soil bacterial 139SI and were Escherichia coli ATCC 25922 and Staphylococcus au- 140SI strains grown overnight in BHI agars was obtained by ◦ reus ATCC 25923, respectively. centrifugation at 10,000 rpm for 10 min at 4 C. Filtrate was passed through a 0.22 µm filter (Millipore, USA) to remove Biochemical tests and phenotypic characterizations any existing bacterial pellets or particles. Ten µL of 139SI Biochemical identification tests including catalase, oxidase, and 140SI crude extracts at 56, 112, 224, 448, 896 µgofpro- urease, glucose, sucrose, indole, mannitol and lactose were tein contents, and distilled water as a control, were added performed as described by Cappuccino & Sherman (2011). to the wells separately followed by 48 h incubation. The an- Growth at three temperatures (25 ◦C, 37 ◦Cand50◦C) was ticancer (HCT 116 cell line) and cytotoxic (Vero cell line) assessed after 2–3 days incubation. Salt tolerance was tested activities were then determined by 3-(4,5-dimethylthiazol- on BHI agar medium supplemented with 1–10% (w/v) NaCl 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Mos- after 2–3 days incubation. Caseinase test was performed mann 1983; Wilson 2000). The MTT colored formazan prod- by inoculating one streak of inoculum on BHI agar supple- uct absorbance was measured using a microplate reader at mented with skimmed milk. The plates were inverted and 490 nm. The percentage of cell viability was calculated using incubated at 37 ◦C for 24–48 h for zone inhibition obser- the following formula: vation around and below the colonies. Additionally, plates were inoculated on nutrient agar, trypticase soy agar and % cell viability = (At − Ab)/(Ac − Ab) × 100 MacConkey agar and incubated aerobically for 24–48 h at ◦ 37 C to evaluate their growth properties. where At – absorbance value of test sample, Ab – absorbance value of blank, and Ac – absorbance value of control. The Congo red agar (CRA) method data were expressed as the mean ± standard error (S.E.) To detect the virulence (slime production) of the cultured using Microsoft Excel 2007 software. strains, CRA