Abstract: The aims of the study were to clone and identify the putative haloacid permease gene in Rhizobium sp. RC1. The putative dehrP gene encoding an uptake protein in Rhizobium sp. RC1 was identified by DNA sequence analysis. An approximately 3.8 kb DNA sequence upstream of dehalogenase D (dehD) in plasmid pSC1 was analyzed and revealed an open reading frame of 1239 kb which encoded for 412 amino acids with calculated subunit molecular weight of 45 kDa and isoelectric point of 9.78. The amino acid sequence of DehrP gave high sequence identity of 86% with putative monochloropropionic acid permease from Agrobacterium sp. NHG3 and 62% with haloacid-specific transporter from Burkholderia cepacia MBA4. Comparison of the predicted amino acid sequence with the CD server (www.ncbi.nlm.nih.gov) NCBI database also revealed the putative DehrP contained signatures of sugar transport proteins of an integral membrane protein. Therefore, a new Rhizobial dehalogenase genetic organization was proposed. However, further characterization of this transporter protein is required to fully comprehend the dehalogenase uptake system of Rhizobium sp. RC1.
INTRODUCTION
Rhizobial species have versatile capability and have been isolated from various soil and water environment. Moreover, they have been involved in the bioremediation of various pollutants (Johnson et al., 2004; Wei et al., 2008). Rhizobium sp. RC1 was originally isolated from soil using 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon and energy source. Rhizobium sp. RC1 was also capable of utilizing other halogenated compounds for growth for example D,L2-chloropropionic acid (D,L2-CP), Monochloroacetic Acid (MCA) and Monobromoacetic Acid (MBA) but not 3-chloropropionic acid (3CP) (Berry et al., 1979; Stringfellow et al., 1997; Cairns, 1994). Rhizobium sp. RC1 produces inducible dehalogenases in batch culture conditions and all three dehalogenases from this organism have been purified and partially characterized (Huyop et al., 2004, 2008a, b).
To date, all dehalogenases identified are intracellular enzymes that may exist as free enzymes in the cell or periplasmic membrane bound (Tsang and Pang, 2001). In order for the cell to utilize haloacid for growth, the availability of specialized transporter proteins to carry the substrate into the cell would be necessary. Dehalogenase-associated permease has been proposed to mediate the uptake of haloacid into the cell. It is apparent that most substances do not passively enter the cell therefore, transport processes are critical to cellular charged at physiological pH, hence may require assistance for a specialized function. According to Van der Ploeg and Janssen (1995), haloalkanoic acids are negatively carrier protein to enable movement of haloacids through the membrane.
The structural genes of all dehalogenases from Rhizobium sp. RC1 have been isolated and characterized in plasmids pJS771 and pSC1 (Stringfellow et al., 1997; Cairns et al., 1996). In the pJS771 construct, only the dehE gene is found without presence of further upstream nor downstream fragment, whereas pSC1 consists of a truncated open reading frame upstream of the dehD gene (Fig. 1a, b). It was hypothesized that the pSC1 plasmid has a third open reading frame associated with haloalkanoic acid permease. Therefore, our main objectives were to clone and identify the putative haloacid permease gene in pSC1 for further study.
MATERIALS AND METHODS
Source of plasmids: Cloned dehalogenase genes-pSC1 and pJS771 with their original hosts pUC19 and pT7-7, respectively were shown in Fig. 1.
| Fig. 1: | Plasmids used in this course of study. (a) pSC1 showing dehD and dehL. The putative haloacid permease dehrP? gene was hypothesized to locate at upstream of dehD and (b) pJS 771 showing dehE insert without upstream nor downstream |
The plasmids were kindly supplied by Dr. R.A. Cooper (University of Leicester, United Kingdom).
Media and growth conditions: Escherichia coli BL21(DE3) were grown at 37°C in LB (Luria Bertani) broth or on LB agar plates supplemented with ampicillin (100 μg mL-1). Escherichia coli was grown at 37°C with agitation for 16 hours for plasmid preparation.
DNA sequencing and oligodeoxyribonucleotide synthesis: Sequencing was performed by the 1st Base Laboratory, Malaysia. Initial sequencing of both strands were carried out using ABI PRISM 377 DNA sequencer by employing primer designed from known sequence. Sequences were extended by designing downstream primers based on the available determined sequence. These oligodeoxyribonucleotide were synthesized by 1st Base Laboratory, Malaysia.
Construction of a plasmid carrying dehrP gene and nucleotide accession number: DNA fragment containing dehrP was amplified from pSC1 by PCR using forward primer with a NdeI site that includes the atg initiation codon (underline) at the start of the gene 5’ GGA ACA CCA TAT GAC TAC GAC TCT AG 3' and reverse primer, with an EcoRI site (underline) after the STOP codon of the dehrP gene 5’ GGG AAT TCA AAT CAA AGG CAT GCG TCA TAT 3’. The 1.3 kb fragment was cloned into NdeI and EcoRI cut pET 4.3Ia to form plasmid pHJ (containing dehrP gene only). This plasmid was transformed into E. coli K12 strain BL21(DE3) by calcium-chloride method and were selected on the basis of their resistance to ampicillin (Maniatis et al., 1982). The PCR product was re-sequenced to confirm that there is no mutation during PCR. The full sequence was obtained and alignment with the initial sequence was also done which shows 100% identity. The full nucleotide sequence of the dehrP Open Reading Frame (ORF) has been deposited with Gene Bank under accession number AM260971.
Computer analysis: DNA sequence identity were searched using the BLASTn program (Altschul et al., 1990). Amino acid sequence analysis was carried out using the Multiple sequence alignment programme (Corpet, 1988). Gene characterization of putative dehrP gene was carried out using AnnHyb 4.0. Nucleotide and deduced protein analysis was done using BLASTn and BLASTp (www.ncbi.nlm.nih.gov/blast). Prediction of protein physio-chemical properties was carried out using ProtParam Tool as described by Gasteiger et al. (2003) (http://kr.expasy.org/cgi-bin/protparam). Hydrophobic character was analyzed using TMHMM maintained by Centre for Biological Analysis (www.cbs.dtu.dk/services/) and finally the putative conserve domain was searched using CD server (www.ncbi.nlm.nih.gov).
RESULTS
Analysis of DNA sequence and gene expression: The third ORF contained 1239 bp designated dehrP, a putative permease gene, was apparently located within the upstream region of dehD gene in pSC1. The ORF started at position +2080 and stopped at position +3315 of the nucleotide (Fig. 2).
| Fig. 2: | Nucleotide sequence of the upstream region of dehD gene showing the presence of putative haloalkanoic acid permease gene (dehrP) (in blue font) |
Expression of dehrP gene in plasmid pHJ showed the size of the protein was approximately 45 kDa by SDS gel electrophoresis. The complete nucleotide sequence of dehrP gene encoded 412 amino acid sequence with calculated subunit molecular weight of 45 kDa and a theoretical pI of 9.78. The putative 412 amino acid DehrP was then compared to the protein sequence in the NCBI database. There were two protein sequences matched with current DehrP haloacid permease, (a) monochloropropionic acid permease (DehP) from Agrobacterium sp. NHG3 (Higgins et al., 2005), (86% identity) and (b) haloacid-specific transporter (Deh4p) from Burkholderia cepacia MBA4 (Yu et al., 2007), (62% identity) as shown in Fig. 3 and 4, respectively.
Analysis of putative conserved domain: A domain is a compact, locally folded region of tertiary structure. Multiple domains are especially common in the larger globular proteins, whereas small proteins tend to be single folded domain. Domains of a protein are identifiable by their scaffold sequence signatures (the motifs in the protein amino-acid texts that remain recognizable despite millions years of divergent evolution). Knowing the domain architecture underlying the putative DehrP is very important because it may give hints about its potential biochemical or cellular function (Marchler-Bauer and Bryant, 2004).
| Fig. 3: | Amino acid sequence comparison between Rhizobium sp. haloacid permease sequence, DehrP (current study) and Agrobacterium sp. NHG3 putative monochloropropionic acid permease sequence DehP (Higgins et al., 2005). The percent identity is 98% for first 354 amino acid and 86% for the whole protein |
| Fig. 4: | Amino acid sequence comparison between Rhizobium sp. haloacid permease sequence, DehrP (current study) and Burkholderia cepacia MBA4 haloacid-specific transporter Deh4P (Yu et al., 2007). The percent identity is 62% |
| Fig. 5: | Results of the conserved domain search. A sugar transporter was found within DehrP protein with high scoring hit when analyzed using CD server (Marchler-Bauer and Bryant, 2004) |
| Fig. 6: | The location of dehD/dehL and dehrP of Rhizobium sp. RC1. Arrows: direction of transcription |
NCBI conserved domain search (CD server) results suggested that a sugar transporters (sugar_tr) domain was found within 22 to 239 amino acid residues of DehrP proteins (Fig. 5). Although only 211 of 448 conserve domains residues were aligned, the findings still agreed well with the hypothesis that DehrP protein was a transporter protein.
Analysis of transmembrane segments: Hydrophobic regions in a protein could be membrane spanning segments in proteins that anchor themselves into a membrane. DehrP protein sequence was subjected to primary structure analysis using TMHMM to find transmembrane helices in that particular protein (Sonnhammer et al., 1998). From the analysis (data not provided) it was suggested that most regions of DehrP protein were labeled as TM helix transmembrane. This coincided with the prediction that DehrP was transmembrane transport protein that facilitated the transport of halogenated compound into bacteria cell.
Rhizobium sp. RC1 dehalogenase gene organization: The 3.8 kb upstream region of dehD gene in pSC1 have been sequenced. Therefore, overall new genetic organization of the Rhizobial 6.5 kb fragment in pSC1 was proposed (Fig. 6).
DISCUSSION
Microbiological research on the degradation of halogenated compounds has mainly focused on the physiological processes responsible for their mineralization and on the enzyme involved in cleavage of the carbon-halogen bond. The transport system involved in degradation of halogenated compounds were always neglected. Therefore, more efforts should be channeled to investigate the transport system involved in dehalogenase system.
In current study, the putative DehrP protein has significant similarities to various membrane-transport proteins in the database strongly suggested that current dehrP encodes a protein that has an uptake function. High sequence identity of DehrP with putative monochloropropionic acid permease from Agrobacterium sp. NHG3 indicated that these two proteins have a similar transmembrane structure. Since Agrobacterium sp. NHG3 discussed by Higgins et al. (2005) belongs to a member of the Rhizobium family, it was not surprise that haloacid permease gene of Agrobacterium sp. NHG3 and Rhizobium sp. shared high similarity.
This was further supported by the finding that the coding region of DehrP contained a sugar (and other) transporters domains. The sugar transporters belong to a superfamily of membrane proteins responsible for the binding and transport of various carbohydrates, organic alcohols and acids in a wide range of prokaryotic and eukaryotic organisms (Mueckler et al., 1985; Fiegler et al., 1999). These integral membrane proteins are predicted to comprise twelve membrane spanning domains. It is likely that the transporters have evolved from an ancient protein present in living organisms before the divergence into prokaryotes and eukaryotes (Maiden et al., 1987). A sugar transporter domain found within DehrP agreed well with the hypothesis that DehrP protein was a haloacid permease.
Studies on the uptake system of haloorganic metabolites are still in its infancy and not much are known about such systems therefore, putative dehrP gene deserves more study. Earlier study by Slater et al. (1985), indicated that an active uptake of halogenated carboxylic acids was observed in Pseudomonas putida PP3. Subsequent studies on halogen degraders were growth rate based rather than genetics and all efforts were primarily channeled on the dehalogenase enzymes characterization.
At present, there were only two transport proteins identified which functions as an uptake of haloorganic compounds (Higgins et al., 2005; Yu et al., 2007). Nevertheless, accuracy of protein analysis results in this study had some limitation because comparison was limited to alignment between amino acid sequences deduced from nucleotide sequences. Similar study by Higgins et al. (2005), the identification of permease was only a hypothesized protein, whereas haloacid-specific transporter gene (deh4P) from Burkholderia cepacia MBA4 was reported to be a novel haloacid permease (Yu et al., 2007).
The haloacid-specific transporter gene designated as deh4p as reported earlier was located downstream of the coding sequence of DehIVa. Deh4p has a putative molecular weight of 59 kDa bigger than that of DehrP in current study (45 kDa). The nucleotide sequence of deh4p did not show high significant homology (62% only) with current dehrP. This was not surprised since both proteins might have different functions. Additionally, the predicted amino acid sequence of Deh4p showed signatures of sugar transport proteins and identified as an integral membrane protein. These two characteristics were similar to the DehrP and may strongly indicate that both DehrP and Deh4P play a vital role to mediate the uptake of haloacid into the cell.
Further study in gene expression of an active dehrP using gel-shift assay and protein structure-solving techniques based on nuclear magnetic resonance (NMR) or X-ray diffraction will be carried out. The information will give idea on the mechanism of the dehalogenase enzyme uptake system to help enzyme engineering or bacteria used in pollutant degradation.
ACKNOWLEDGMENTS
NHJ thanks Ministry of Science, Technology and Innovation of Malaysia (Vot 79073)/(MOHE-FRGS) 78307 for financial support.