Introduction

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

Growth curves

DNA Analysis

Protein Analysis

Results

Isolation and Characterization

Growth curves

DNA Analysis

Protein Analysis

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 Bacillus cereus. The sequence has been submitted to UniProt Knowledgebase, accession number is P85307.

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 Bacillus cereus flagellin protein.

Peptide Mass spectrometry fingerprinting of ~36 kDa band isolated from CMG2K4 (Bacillus cereus) was used to search the Swiss-Prot and NCBInr databases using the Mascot search engine (Matrix Science) in order to identify the parent protein (Figure 3). Original, minimized and re-optimized sets of monoisotopic peptide masses were used to perform the searches but no exact match was found. 16 major peptide masses peaks (Figure 4) which were obtained as a result of digestion with trypsin were compared in SWISSPROT database http://c.expasy.org/sprot with peptide mass data of flagellin reported in other strains of Bacillus cereus. Maximum similarity found in exact match of 4 major peaks in Q45XJ8_BACCE ; Q4MU98_BACCE and Q4MU99_BACCE showed exact match of 3 major peaks; while Q4MU96_BACCE , Q4MU97_BACCE and Q45XK1_BACCE showed exact match with two major peaks of peptide masses of ~36 kDa protein of Bacillus cereus strain CMG2K4. There were several potentially modified peptides in all the six strains but minimum unmatched peptide masses were found to be 5 in Q4MU99_BACCE, Q45XJ8_BACCE, and Q45XK1_BACCE; while there were 6 in Q4MU97_BACCE, Q4MU98_BACCE; and 7 in Q4MU96_BACCE.

Figure 1

Figure 1a: Growth curve of CMG2K4 with and without NiCl to check induction through turbidity measurement.

Figure 2

Figure 1b: Growth Curve of CMG2K4 with and without NiCl to check induction through viable count.

Figure 3

Figure 2: SDS-PAGE for CMG2K4 Protein in Presence Of Nickel (Stationary Phase Culture)

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

Figure 4

Figure 3: Mass Spectrum Of ~36 kDa Protein Band of CMG2K4

Figure 5

Figure 4:Partial data set (major peaks only).

Discussion

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 Bacillus cereus. CMG2K4 showed couple of contrasting characters with respect to already known mechanisms of tolerance; firstly CMG2K4 does not contain any plasmid, contrary to the reported mechanisms of nickel tolerance (Liesegang et al., 1993, Grass et al., 2000); secondly, constitutive nickel tolerance mechanism was observed which was evident through optimal density and viable count of growth curves with and without nickel in contrast to the reported mechanisms of tolerance in nickel (Hong et al., 1996).

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 Bacillus cereus and CMG2K4 was also identified as Bacillus cereus, which supports that the homology is not coincidental. Furthermore, length of amino acid sequence of the protein is expected to be 327 and the reported length of some of the Flagellin protein sequence came out to be very close to this number i.e., 284-397.

Several strains of Bacillus cereus have been completely sequenced (Ivanova et al., 2003, Rasko et al., 2004), but the flagellin protein sequences which matched with the 10 amino acid long sequence of CMG2K4 protein were mostly Bacillus cereus G9241 (Hoffmaster et al., 2004). Multiple sequence alignment through CLUSTALW. (1.83) in EBI, Uni-Prot database (Lopez 1997, Higgins et al. 1994) at http://www.ebi.ac.uk/clustalw, of amino acid sequence of flagellin protein of six selected strains of Bacillus cereus on the basis of having amino acid sequence length close to the expected length of the studied protein i.e., 327 amino acids, showed N-terminal and C-terminal homology of the sequences but variability was found in the middle C-terminal half of the sequence.

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 Bacillus cereus strains were compared. All the exactly matched peptides were located at the conserved terminal sequences.

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.

Conclusions

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.

Acknowledgement

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.