To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment.
To isolate and characterize bacteriocin, BL8, from the bacteria identified as Bacillus licheniformis from marine environment.
One-hundred and twelve bacterial isolates from sediment and water samples collected off the coast of Cochin, India, were screened for antibacterial activity. Strain BTHT8, identified as Bacillus licheniformis, inhibited the growth of Gram-positive test organisms. The active component labelled as bacteriocin BL8 was partially purified by ammonium sulphate fractionation and was subjected to glycine SDS-PAGE. The band exhibiting antimicrobial activity was electroeluted and analysed using MALDI-TOF mass spectrometry, and the molecular mass was determined as 1·4 kDa. N-terminal amino acid sequencing of BL8 gave a 13 amino acid sequence stretch. Bacteriocin BL8 was stable even after boiling at 100°C for 30 min and over a wide pH range of 1–12.
A novel, pH-tolerant and thermostable bacteriocin BL8, active against the tested Gram-positive bacteria, was isolated from Bacillus licheniformis.
This study reports a stable, low molecular weight bacteriocin from Bacillus licheniformis. This bacteriocin can be used to address two important applications: as a therapeutic agent and as a biopreservative in food processing industry.
Bacteriocins are ribosomally synthesized peptides which kill bacteria that are often closely related to the producer strain (Lisboa et al. 2006). They are heterogeneous compounds that display variable molecular weights, biochemical properties, inhibitory spectra and mechanisms of action (O'Sullivan et al. 2002).
The genus Bacillus is a heterogeneous group of Gram-positive, facultative anaerobic, endospore-forming bacteria widespread in the environment, although soil is generally accepted as its natural reservoir (Galvez et al. 1993). Bacillus spp. produce a large number of bacteriocins, subtilosin by B. amyloliquifaciens (Sutyak et al. 2008), bacillocin 490 by B. licheniformis (Martirani et al. 2002), cerein by B. cereus (Oscariz et al. 1999), haloduracin by B. halodurans (Lawton et al. 2006), thuricin by B. thuringiensis (Gray et al. 2006) and subtilin by B. subtilis (Banerjee and Hansen 1988), all of which are mostly active against Gram-positive organisms.
A single Bacillus strain often has the ability to produce several different molecules partially resistant to enzyme treatments, with stability over a wide range of pH and temperature (Baruzzi et al. 2011). The capability to produce endospores allows Bacillus sp. to withstand extreme environmental conditions such as those encountered in food processing. Like the lactic acid bacteria (LAB), the genus Bacillus includes representatives that are ‘generally recognized as safe’ (GRAS), such as B. subtilis and B. licheniformis (Sharp et al. 1989), and can hence find application in the control of food pathogens and spoilage micro-organisms during food processing. The concept of qualified presumption of safety (QPS) for acceptability of bacteria in foods if the taxonomic group did not raise safety concerns, or if safety concerns did exist could be defined and excluded, was recently introduced by the European Food Safety Authority (EFSA 2007, 2008). The qualification concerning QPS for Bacillus species is modified to ‘absence of food poisoning toxins, absence of surfactant activities, absence of enterotoxic activities’ (EFSA 2008). Nevertheless, bacteriocin-producing strains or their bacteriocin preparations could still be used in food preservation provided they meet the criteria established by EFSA.
This study was undertaken to isolate and characterize the bacteriocin-like substance active against Bacillus sp. and other Gram-positive bacteria produced by strain of B. licheniformis.
Water and sediment samples collected off the coast of Cochin, India, and serially diluted were plated on prepared tryptone soya agar plates (HiMedia, Mumbai, India) to obtain isolated bacterial colonies. Standard test organisms availed from National Collection of Industrial Microorganisms (NCIM, NCL, Pune) were included in this study (Table 1).
|Test organisms||NCIM No.||Zone size in mm|
|Clostridium perfringens||2677||13 ± 0·5|
|Staphylococcus aureus||2127||11 ± 1|
|Bacillus cereus||2155||15 ± 0·6|
|Bacillus circulans||2107||10 ± 1|
|Bacillus coagulans||2030||10 ± 0·5|
|Bacillus pumilus||2189||11 ± 1|
Bacteria were grown in Zobell marine broth 2216 (HiMedia), incubated at 28°C for 24 h in environmental shaker (Scigenics, Chennai, India) at 150 rev min−1. The culture broth was centrifuged at 9400 g for 15 min at 4°C (Sigma 3K30, Osterode, Germany), the supernatant collected and filtered through 0·22-μm membrane (Millipore, Billerica, MA, USA). This filtrate with an alkaline pH (pH 8) was used to evaluate antimicrobial activity without any heat treatment or change in the pH.
Antibacterial activity was determined by agar well diffusion assay (Tagg and McGiven 1971). Mueller-Hinton agar (HiMedia) plates were swab-inoculated with the test organisms (Table 1) grown in nutrient broth (HiMedia) for 12 h and wells were cut. 20 μl of culture filtrate was added into the wells. Plates were incubated for 24 h at 37°C and the inhibition zones were measured. Each time, the antibacterial activity was tested against all six Gram-positive test organisms. The assay was conducted in triplicate.
Bacterial DNA was isolated according to the protocol described by Ausubel et al. (1987). 16S rRNA gene (1·5 kb size) was amplified from the genomic DNA using thermal cycler (Bio-Rad, CA, USA) with universal primers for 16S rDNA (Shivaji et al. 2000) with an initial denaturation at 94°C for 1·5 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 2 min and final extension at 72°C for 10 min. Nucleotide sequence of the PCR amplicon was determined by the ABI Prism 310 genetic analyzer (Applied Biosystems, Carlsbad, CA, USA) using big dye terminator kit. The identity of the sequence was determined by comparing the sequences in the NCBI database using Basic Local Alignment Search Tool (Blast) software (Altschul et al. 1990). The sequence was submitted to GenBank and accession number obtained.
As a first step towards purification of the active compound, the supernatant collected after centrifugation was fractionated using ammonium sulphate (0–30%, 30–60% and 60–90%). The precipitate obtained was dialysed using a 2-kDa benzoylated dialysis tubing (Sigma Aldrich, St Louis, MO, USA) against deionized water at 4°C for 36 h with six changes of water.
The total protein content in the partially purified BL8 was estimated according to the protocol described by Bradford (1976). The resazurin assay utilizing microtitre plate (Sarker et al. 2007) has been modified to determine the minimum inhibitory concentration (MIC) values of the bacteriocin against the test organisms. To 50 μl of deionized water, 2 μl of resazurin (1%) was added. 50 μl of BL8 was added in the first row and double-diluted vertically below. Then 100 μl of double-strength nutrient broth was added in each well followed by 50 μl of test organisms (OD600 = 1) and incubated at 37°C for 18–24 h.
The partially purified protein was subjected to glycine SDS-PAGE using 15% resolving gel (Laemmli 1970). The protein was run in two lanes along with protein molecular weight marker (GeNei, Bangalore, India) in a vertical slab electrophoresis system (Genei) at 80 V, and the gel was cut vertically into two halves. One half containing the sample and molecular weight marker was silver-stained. The other half with sample was washed thrice with 0·1% Tween 80 (30 min each), followed by washing with deionized water to remove SDS. The gel was then placed on Mueller-Hinton agar (HiMedia) base plate, overlaid with soft Mueller-Hinton agar (0·8% agar) seeded with 100 μl (OD600 = 1) of test organisms (Yamamoto et al. 2003) and checked for zone of clearance due to the antibacterial activity after overnight incubation at 37°C.
The protein band with antibacterial activity was excised and electroeluted in a 2-kDa benzoylated dialysis tubing (Sigma Aldrich) against 0·01 mol l−1 phosphate buffer. For this, a voltage of 30 V was applied overnight at 4°C, after which the electrodes were reversed and voltage of 30 V was applied for 30 min (Lei et al. 2007).
Intact mass of the electroeluted fraction of bacteriocin BL8 was determined using mass spectrometer having specification of ABI4800MALDI-TOF/TOF (Applied Biosystems).
The N-terminal amino acid analysis of the electro-eluted protein was resolved by automated Edman degradation (Applied Biosystems 494 Procise Protein Sequencing System). The sequence obtained was compared to bacteriocins from Bacillus sp. retrieved from the protein database of NCBI by multiple sequence alignment using ClustalW (Larkin et al. 2007).
Partially purified bacteriocin was used for the study. To evaluate heat stability, bacteriocin was exposed to temperatures ranging from 40 to 100°C for 30 min and 121°C per 105 kPa for 15 min. To study the effect of different pH, samples of bacteriocin were treated with equal amount of buffers with pH range 1–12 and kept for 18 h at 4°C. The buffer systems used included hydrochloric acid/potassium chloride buffer (pH 1–2), citric acid/sodium citrate buffer (pH 3–5), phosphate buffer (pH 6–7), Tris amino methane/hydrochloric acid buffer (pH 8–9), sodium bicarbonate/sodium hydroxide buffer (pH 10), sodium phosphate dibasic/sodium hydroxide buffer (pH 11–12) (Vincent and John 2009). The samples after the treatment were tested for the antimicrobial activity against the test organisms. All assays were conducted in triplicate.
One-hundred and twelve strains were isolated from sediment and water samples. All strains were screened for antibacterial activity. Of these, strain BTHT8 exhibited broad range of antibacterial activity against the test organisms considered to be the major pathogens in foodborne illnesses and other infections, and also showed consistency and reproducibility. The bacterial strain BTHT8 was identified to be Bacillus licheniformis by 16S rDNA sequence analysis. The sequence obtained was submitted to GenBank (accession no. HMO30819).
Maximum antibacterial activity was exhibited by the 30–60% ammonium sulphate fraction. The total protein concentration was found to be 798 μg ml−1. The MIC of bacteriocin BL8 ammonium sulphate fraction for B. circulans and Staphylococcus aureus was found to be 40 μg ml−1, for B. coagulans it was 80 μg ml−1, for B. cereus it was 160 μg ml−1 and for Clostridium perfringens and B. pumilis it was 250 μg ml−1.
Glycine SDS-PAGE and silver staining of the partially purified bacteriocin showed numerous protein bands on gel. A clearing zone on gel overlaid with Mueller-Hinton soft agar seeded with test organisms indicated antibacterial activity (Fig. 1b) at a region with molecular mass below 3 kDa (Fig. 1a).
The band with antibacterial activity was electroeluted, and the protein analysis by SDS-PAGE showed a single band whose intact mass was determined to be 1·4 kDa by mass spectrometric analysis (Fig. 2).
N-terminal amino acid analysis revealed the 13 amino acid sequence stretch: NH2-Ser-Trp-Ser-Cys-Cys-Gly-Asn-Cys-Ser-Ile-Ser-Gly-Ser-COOH. Comparison with the bacteriocins from protein database by multiple sequence alignment showed no significant similarity indicating its novelty (Fig. 3).
Thermal stability studies revealed that the antibacterial activity of bacteriocin BL8 was lost when exposed to high temperatures of 121°C per 105 kPa for 15 min but was retained when exposed to temperatures ranging from 40 to 100°C for 30 min. The bacteriocin activity was stable over a broad pH range of 1–12.
Bacteriocins and BLIS produced by the genus Bacillus are considered as significant as the bacteriocins produced by the lactic acid bacteria (LAB). A diverse array of antimicrobial peptides with several different basic chemical structures is produced by genus Bacillus (Gebhardt et al. 2002; Stein et al. 2005).
The few bacteriocins reported from B. licheniformis have their antibacterial activity restricted to Gram-positive organisms. Bacillocin 490 produced by thermophilic B. licheniformis 490/5 from a dairy product had antibacterial activity against the closely related species Geobacillus stearothermophilus, B. smithii, B. subtilis, B. anthracis, B. cereus and B. licheniformis (Martirani et al. 2002). A bacteriocin-like inhibitory substance (BLIS) produced by B. licheniformis P40 from the Amazon Basin inhibited Listeria monocytogenes, B. cereus and clinical isolates of Streptococcus sp. (Cladera-Olivera et al. 2004), whereas other uncharacterized antimicrobial substances were produced by B. licheniformis T6-5 from an oil reservoir in Brazil (Korenblum et al. 2005) and by B. lichenifomis AnBa9 from sediments of slaughterhouse sewage (Anthony et al. 2009). The spectrum of antibacterial activity of bacteriocin BL8 from B. licheniformis strain BTHT8 clearly shows that it inhibited not only phylogenetically related species but also other Gram positives.
Bacteriocins active against Gram-negative organisms have been previously reported from other Bacillus spp; B. amyloliquefaciens LBM 5006 from the Brazilian Atlantic forest produces a broad antibacterial spectrum BLIS that included activity against L. monocytogenes, B. cereus, Serratia marcescens and Pasteurella haemolytica (Lisboa et al. 2006). Megacin 19 by B. megaterium 19 from fermented vegetable wastes and megacin 22 by B. megaterium 22 from soil show wide antimicrobial spectra against food spoilage bacteria like Salmonella Typhimurium and Staph. aureus (Khalil et al. 2009a,b). BLIS by B. subtilis LFB112 from Chinese herbs was active against domestic animal disease caused by Gram-positive and Gram-negative bacteria including Escherichia coli, Salmonella Pullorum, Pseudomonas aeruginosa, Pasteurella multocida, Cl. perfringens, Micrococcus luteus, Streptococcus bovis and Staph. aureus (Xie et al. 2009).
The present study is on bacteriocin from B. licheniformis of marine origin. Bacteriocins reported earlier from marine bacteria were extracellular inhibitory substances produced by the marine Alteromonas strain P-31 (Barja et al. 1989) and antimicrobial protein from marine bacterium Pseudoalteromonas sp. strain X153 (Longeon et al. 2004).
BL8 with 1·4 kDa size can be grouped with small molecular peptides. Although some bacteriocins are small peptides with 19–37 amino acids, others have molecular weights up to 90 kDa (Joerger 2003). Bacillocin 490 reported from B. licheniformis 490/5 is a 2-kDa peptide (Martirani et al. 2002). BLIS with 3–5 kDa size from B. subtilis (Alam et al. 2011), 6·3 kDa from B. subtilis (Xie et al. 2009) and 5 kDa from B. amyloliquefaciens LBM 5006 have been reported (Lisboa et al. 2006). Cerein GN105 from B. cereus GN105 has an apparent molecular weight of 9 kDa by SDS-PAGE (Naclerio et al. 1993), while that from B. lentus was 11 kDa (Sharma et al. 2009a,b). Pumilicin 4 from B. pumilus with molecular mass of 1·9 kDa (Aunpad and Na-Bangchang 2007) and tochicin with 10·5 kDa from B. thuringiensis ssp. tochigiensis HD868 (Paik et al. 1997) are other small molecular weight bacteriocins.
BL8 activity was stable at 100°C and over wide pH range. Bacillocin 490 from B. licheniformis 490/5 exhibited high stability at 4 and 100°C over a wide pH range (Martirani et al. 2002). BLIS from B. licheniformis P40 showed stability at pH 3–11 and at higher temperature of 100°C but lost its activity on exposure to 121°C for 15 min (Cladera-Olivera et al. 2004). Heating at 100°C for 60 min had no apparent effect on subtilosin (Sutyak et al. 2008). Bacillocin Bb, a BLIS by B. brevis Bb from soil (Saleem et al. 2009), is stable not only over pH range of 1–9, but also at 100°C for 30 min. Megacins 19 and 22 remained stable up to 100°C for 15 min (Khalil et al. 2009a,b) as did Pumilicin 4 but up to 121°C for 15 min and in the pH range of 3–9 (Aunpad and Na-Bangchang 2007). Some bacteriocins lose their activity at very high temperatures. Cerein 8A from B. cereus 8A had broad pH stability (pH 2–11) and was relatively thermostable, losing activity only at temperatures above 75°C for 30 min (Bizani et al. 2005). BLIS from B. amyloliquefaciens LBM 5006 has stability at 80°C for 30 min, but the residual activity decreased at 100°C and all activity lost at 121°C (Lisboa et al. 2006). On the other hand, tochicin from B. thuringiensis was relatively heat stable at 90°C, but activity undetected after boiling for 30 min (Paik et al. 1997).
Production of antimicrobial substances and sporulating capacity confers a double advantage on Bacillus strains for their survival in diverse habitats. The presence of these bacteria in food does not always imply spoilage or food poisoning, with several species or strains used in human and animal food production. B. subtilis strains are used in the production of Natto, an East Asian fermented food (Hosoi and Kiuchi 2003), as a starter culture for fermentation of soya beans into the traditional West African condiment dawadawa (Terlabie et al. 2006) or African mesquite seeds for the Nigerian food condiment okpehe (Oguntoyinbo et al. 2007). Bacteriocins from Bacillus have a potential preservative application in dairy products like milk and cheeses (Sharma et al. 2009a,b).
Bacteriocin BL8 from B. licheniformis strain BTHT8 with its thermostable and antibacterial activity over a broad pH range can be used to address two important aspects: as a therapeutic agent and as a biopreservative in food processing industry considering its antibacterial activity against pathogenic Gram-positive bacteria that cause foodborne illnesses.
The author acknowledges the financial assistance provided by the University Grants Commission (UGC), India, in the form of junior research and senior research fellowship. This work was also supported by the research grant from Ministry of Earth Sciences, Government of India (grant no. MoES/10-MLR/2/2007).