Identification Of A Unique Mechanism Of Tolerance Against Nickel In Bacillus Cereus Isolated From Heavy Metal Contaminated Sites.
E Shoeb, N Ahmed, P Warner, S Morgan, M Azim
bacillus cereus, metal tolerance, nickel, protein
E Shoeb, N Ahmed, P Warner, S Morgan, M Azim. Identification Of A Unique Mechanism Of Tolerance Against Nickel In Bacillus Cereus Isolated From Heavy Metal Contaminated Sites.. The Internet Journal of Microbiology. 2009 Volume 9 Number 1.
Heavy metals are increasingly found in our environment due to natural and industrial processes (Summers, 1984). These are trace metals with a density at least five times that of water (Morris, 1992). Heavy metals such as copper, nickel, zinc, manganese, cobalt, molybdenum, and vanadium are essential for normal life, although elevated concentrations of both essential and non-essential metals result in growth inhibition by inhibiting the activity of sensitive enzymes (Ahmed et al., 2005).
Most of the essential cell components are potential targets for metal induced damage such as DNA replication, as a result of which cell death can occur (Malakul et al., 1998). Bacteria, being one of the most primitive life forms on earth, naturally developed tolerance to a wide range of toxic heavy metals (Silver and Walderhaug, 1992, Silver and Ji, 1994).
Nickel (Ni) is widely distributed in nature and 8.5% of the earth’s crust is nickel (Carson et al., 1986). Nickel is used in the production of stainless steel, as a chemical catalyst in electroplating, ceramics, pigments, batteries and coins (IARC (International Agency for Research on Cancer), 1990). In many bacteria, nickel is required for the activity of enzymes such as urease, dehydrogenase, and hydrogenase (van Valiet et al., 2001), however excess amount is toxic. Nickel binds to proteins and nucleic acids and inhibits enzymatic activities such as DNA replication, transcription, and translation (Grosse et al., 1999). The best known nickel tolerance in bacteria, in Ralstonia eutrophus strain CH34 and related bacteria, is based on nickel efflux driven by a RND transporter. Two systems have been described, a nickel-cobalt resistance Cnr (Liesegang et al., 1993) and a nickel-cobalt-cadmium resistance Ncc (Schmidt and Schlegel, 1994). These systems are closely related to the cobalt-zinc-cadmium resistance system Czc from strain CH34. Flagellin is a component of flagellar filament known to provide motility to the bacteria. The present study describes the involvement of flagellin in nickel tolerance in an indigenous bacterium; B. cereus, CMG2K4.
Materials & Methods
Isolation and Characterization
The media used were Luria-Bertani (LB) medium (Bopp and Ehrlich, 1988) and Tris minimal media (Mergeay et al., 1985). Soil samples were collected from metal electroplating, auto-batteries and automobile workshops of Gulshan-e-Iqbal, Karachi, Pakistan. Strains were isolated according to the standard procedure in the tris-minimal medium containing 0.2% gluconate and supplemented with 0.1 mM NiCl2. Ralstonia eutropha CH34 (the kind gift of Dr. D.H. Nies) (Nies and Silver, 1989) was also used as standard.
All the isolated strains were checked for maximum tolerable concentration (MTC) against NiCl2. Nickel accumulation of all the strains was calculated by considering metal concentration both in supernatant and in pellet in parts per million (ppm) using Atomic Absorption Spectrophotometer (Perkin Elmer AAnalyst 200).
CMG2K4 was identified using partial 16S ribosomal RNA gene sequence homology analysis by HPA (Collindale, London UK). Sequence was analyzed using BLAST algorithm (Altschul et al., 1990) (Web ref: http://www.ncbi.nlm.nih.gov/cgi-bin/Blast).
Growth curves were studied for determining the tolerance mechanism against nickel both through turbidity measurement (OD600) and viable count using overnight cultures grown with and without NiCl2.
Genomic DNA was isolated by using Wizard Genomic DNA Purification Kit Cat No A1125 from Promega (USA) and Puregene DNA isolation kit of Gentra (cat no. D5500A). Plasmid isolation was carried out by QIAprep Spin Miniprep Kit (Cat No 27104 of QIAGEN).
Oligonucleotide primers were designed by using primer designing programme [Primer 3 Output (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_cgi v 0.2)].
For protein analysis SDS-PAGE was employed. 8% Resolving gel and 4% Stacking gel was prepared. SDS-PAGE was performed as described by Baines (2000). Standard protein used in SDS-PAGE was See Blue® plus 2 pre-stained standard (1X) cat no. LC5925 of Invitrogen, which had 10 pre-stained protein bands of 4-250 kDa range. Vertical electrophoresis system of Hoefer miniVE was used with Hoefer EPS 2A200 power pack.
Protein samples for (SDS)-poly acrylamide gel electrophoresis (PAGE) were prepared as described by Cumming and Iceton (2001). Total protein concentration of the sample was quantified by the method of Bradford (1976) as described by Ausbel (1994). Bradford Reagent (Protein dye reagent) Sigma B-6916 was used with IgG (Biorad) as a standard.
Protein sequencing of band of interest was conducted commercially by amino acid sequencing Facility, School of Biochemistry and Microbiology, LIGHT Laboratories, University of Leeds, Leeds, UK.
Resultant sequence was searched for homology in databases, NCBInr at www.ncbi.nih.gov and Uni-Prot at www.ebi.ac.uk, using Blast (version 2.2.13) and MPsrch (version 1.3) options respectively.
Multiple sequence alignment of amino acid sequences of flagellin protein of different strains of
Peptide Mass Fingerprinting (PMF) was conducted commercially at MALDI-Mass Spectrometry Facility, School of Biochemistry and Microbiology, LIGHT Laboratories, University of Leeds, where the dried plate was transferred to a M@LDI L/R mass spectrometer (Waters) each was analyzed and combined to produce a raw spectrum.
The set of monoisotopic peptide masses produced through trypsin digest was used to search the databases in National Centre for Biotechnology Information (NCBI) at www.ncbi.nih.gov and European Bioinformatics Institute, a part of European Molecular Biology Laboratories (EMBL-EBI) at www.ebi.ac.uk using the Mascot search engine (Matrix Science) in order to identify the parent protein. Peptide Mass Fingerprinting data was characterized and identified in Swiss-Prot database at www.expasy.ch using Proteomics Server through FindMod tool (Wilkins et al., 1999, Gasteiger, 2005).
Isolation and Characterization
43 bacterial strains were isolated and purified initially. Maximum tolerable concentrations (MTC) of all the strains against nickel have shown that few strains such as CMG2K4, CMG2K6, CMG2K7, CMG2K8 tolerated high concentrations of nickel, which was as high as 10, 13, 17, and 9 mM respectively. Highest accumulation was found in CMG2K4 which accumulated 50% of the total amount of NiCl2 added. Other high nickel tolerant strains did not show high rate of accumulation, on the basis of tube results CMG2K4 was selected for further studies.
CMG2K4 was identified through 16S rRNA gene DNA sequencing as
Results of growth of CMG2K4 in presence of nickel, both through turbidity measurement and viable count, have suggested that nickel tolerance is constitutive in CMG2K4 (Figure 1a and 1b).
Plasmid isolation indicated that CMG2K4 harbor no plasmid. PCR results did not indicate presence of ncc and cnr operons in CMG2K4.
As a result of SDS-PAGE of CMG2K4 a single band of protein having molecular weight of ~36 kDa (approximately 327 amino acids) was identified from the crude cell extract of culture grown in the presence of 1mM nickel chloride overnight, whereas rest of the bands found in control were all diminished (Figure 2). As a result of N-terminal protein sequencing 10 amino acid long sequence was obtained. As a result of Blast through MPsrch in EMBL-EBI database (UniProt) this 10 amino acid long sequence showed 100% homology with flagellin protein of
Multiple sequence alignment of selected sequences which had the length close to the expected length of amino acid sequence of our protein conducted through CLUSTALW (1.83) has revealed variability in the amino acid sequences of different strains of
Peptide Mass spectrometry fingerprinting of ~36 kDa band isolated from CMG2K4 (
Lane 1= Standard (See Blue 2 Pre-Stained Invitrogen)
2-5 & 7-10= CMG2K4 Protein in Presence of Nickel (2, 5,10, & 8µl)
Lane 6= CMG2K4 Protein without Nickel
The present studies were initiated with the aim to identify indigenous bacteria having potential for heavy metal tolerance and to ruminate about the genetic mechanisms involved. Metal contaminated sites are the potential source of metal tolerant bacteria (Badar et al., 2001). For this study bacterial strains were collected from the soil contaminated with heavy metals such as automobile workshops, where battery servicing, welding and electroplating is performed commonly.
Among several heavy metal tolerating strains, CMG2K4 was found to tolerate NiCl2 and accumulate it. As CMG2K4 was accumulating NiCl2 as well so it was suggested that in CMG2K4 the mechanism of tolerance is not the active efflux of heavy metal ions from the cytoplasm to the extra cellular medium as generally reported for various heavy metals (Rensing et al., 1997).
Partial 16S-rDNA sequencing technique was used to identify CMG2K4 which was identified as
CMG2K4 did not indicate presence of any reported genes with respect to nickel tolerance in presence of respective standards through PCR. Knowing the fact that bacterial transcriptional activator proteins respond to different toxic environmental compounds such as heavy metals, antibiotics or other drugs (Summers, 1992), a different strategy was adopted to locate genetic basis of tolerance against nickel in CMG2K4. Protein contents were extracted from whole cell extract of CMG2K4 grown with and without NiCl2. Crude protein extract of CMG2K4 showed an intense band of ~36 kDa molecular mass which was found to be the band of only over expressed protein in presence of nickel. All the other bands diminished as compared to control both in log phase culture and stationary phase culture. These results indicate that the ~36 kDa band protein is a constitutive protein of CMG2K4 which might play active role for survival even in the presence of high concentration of nickel.
10 amino acids N-terminal sequence of ~36 kDa protein showed ~100% identity with Flagellin protein from B. cereus using MPsrch program identified in ~36 Kda protein as Flagellin (www.ebi.ac.uk ) (Waterman and Smith, 1981). Most of the sequences of flagellin protein which showed homology with our ~36 kDa protein of CMG2K4 were of
Several strains of
In order to confirm the identity of ~36kDa protein, protein mass fingerprinting was performed by MALDI-Mass Spectrometry which resulted in several peptide peaks, out of which major peaks were analyzed in Swiss-Prot database at http://www.expasy.ch through Proteomics server using FindMod tool. Major peptide peaks of ~36 kDa protein with that of other reported peptides peaks of flagellin protein of
N-terminal sequencing, MPsrch sequence searches, multiple alignment and Peptide mass fingerprinting by MALDI mass spectrometry confirmed that the over expressed ~36 kDa protein from nickel tolerant strain of B. cereus CMG2K4 is in fact flagellar protein, Flagellin.
Flagellin is a component of flagellar filament which provides motility to the bacteria. Potential benefits of motility include increased efficiency of nutrient acquisition, avoidance of toxic substances and to access optimal colonization sites within them (Ottemann and Miller, 1997). It has been reported that synthesis of flagellin gets repressed by various antibiotics belonging to different groups having different mode of actions (Kaldalu et al., 2004). The flagellin protein inspite of being recognized as transcriptionally active against different antibiotics, it has not been reported with respect to its role in heavy metal tolerance. This has been revealed as a result of present finding.
N-terminal sequencing, MPsrch sequence searches and multiple alignment has confirmed that the over expressed ~36 kDa protein from nickel tolerant strain of B. cereus CMG2K4 is in fact flagellar protein, Flagellin. The flagellin protein inspite of being recognized as transcriptionally active against different antibiotics, it has not been reported with respect to its role in heavy metal tolerance. This has been revealed as a result of present finding.
The authors acknowledge the enabling role of the Higher Education Commission, Islamabad, Pakistan and appreciates its financial support through Indigenous Ph.D. Scholarship given to Ms. Erum Shoeb.