Topological analysis of a haloacid permease of a Burkholderi a sp. bacterium with a PhoA-LacZ reporter
© Tse et al; licensee BioMed Central Ltd. 2009
Received: 25 June 2009
Accepted: 31 October 2009
Published: 31 October 2009
2-Haloacids can be found in the natural environment as degradative products of natural and synthetic halogenated compounds. They can also be generated by disinfection of water and have been shown to be mutagenic and to inhibit glyceraldehyde-3-phosphate dehydrogenase activity. We have recently identified a novel haloacid permease Deh4p from a bromoacetate-degrading bacterium Burkholderia sp. MBA4. Comparative analyses suggested that Deh4p is a member of the Major Facilitator Superfamily (MFS), which includes thousands of membrane transporter proteins. Members of the MFS usually possess twelve putative transmembrane segments (TMS). Deh4p was predicted to have twelve TMS. In this study we characterized the topology of Deh4p with a PhoA-LacZ dual reporters system.
Thirty-six Deh4p-reporter recombinants were constructed and expressed in E. coli. Both PhoA and LacZ activities were determined in these cells. Strength indices were calculated to determine the locations of the reporters. The results mainly agree with the predicted model. However, two of the TMS were not verified. This lack of confirmation of the TMS, using a reporter, has been reported previously. Further comparative analysis of Deh4p has assigned it to the Metabolite:H+ Symporter (MHS) 2.A.1.6 family with twelve TMS. Deh4p exhibits many common features of the MHS family proteins. Deh4p is apparently a member of the MFS but with some atypical features.
The PhoA-LacZ reporter system is convenient for analysis of the topology of membrane proteins. However, due to the limitation of the biological system, verification of some of the TMS of the protein was not successful. The present study also makes use of bioinformatic analysis to verify that the haloacid permease Deh4p of Burkholderia sp. MBA4 is a MFS protein but with atypical features.
Haloacids are metabolic products of naturally occurring compounds [1–3] and are also disinfection by-products of sewage and water [4, 5]. It has been shown that some haloacids are toxic and mutagenic [6, 7]. Microorganisms capable of degrading these haloacids can be found in the natural environment. One of these, a soil-borne Burkholderia sp., MBA4, has been isolated for its ability to grow on monobromoacetate (MBA) . This bacterium produces a haloacid dehalogenase that allows the cell to grow on MBA. Since MBA is a more potent mutagen than ethylmethane sulfonate  one would not expect an uptake mechanism for this kind of compound. We have, however, identified a haloacids-transporter protein gene downstream of the dehalogenase gene. This haloacid permease, Deh4p, was expressed, together with the dehalogenase, to enhance the uptake of haloacetates . The gene encoding for Deh4p has been cloned and expressed in E. coli which facilitated the specific uptake of haloacetates . Deh4p is 552 residues long and has a putative molecular weight of 59,414 and an isoelectric point of 9.14.
With the blooming of the sequencing data and the development of bioinformatics, software that predicts the structure of a protein has become more and more readily available [12–21]. Topology prediction programs that use different algorithms are easily accessible from the Internet and their predictions are becoming more and more accurate. Comparative analysis of the primary structure of Deh4p with proteins in the Pfam database  has designated it as a member of the Major Facilitator Superfamily  (MFS, TC 2.A.1). MFS is a major class of membrane transporter with more than a thousand known proteins . It is also described as the uniporter-symporter-antiporter family. Although there are many members in this family, only four of them have well defined structure or topology. These proteins are EmrD , LacY  and GlpT , all from Escherichia coli and OxlT from Oxalobacter formigenes [28, 29]. They have been shown to possess twelve transmembrane segments (TMS) with a 2-fold symmetry roughly dividing the first and the second 6-TMS. The termini of these proteins were found to reside within the cytoplasm. Though MFS transporters with 14 and 24 TMS are known [30, 31], they are relatively few in number . Hence the presence of twelve TMS was believed to be the standard characteristic of the MFS proteins.
Notwithstanding the abundance and improved accuracy of those computer analysis methods, experimental determination is still necessary. The use of reporter fusion analyses is by far the most convenient method and the use of dual-reporters is no doubt a better choice than the use of a single indicator [33, 34]. Here we report the experimental determination of the topology of Deh4p using a PhoA-LacZ dual-reporters system  and the verification using a comparative approach.
Hydropathy analysis of Deh4p
Topological analysis using Deh4p-PhoA-LacZ fusions
Comparative analysis of Deh4p with Metabolite:H+ Symporter (MHS) family proteins
Haloacid permease Deh4p of Burkholderia sp. MBA4 was classified as a member of the MFS based on its sequence similarity . It was predicted to have twelve TMS. In this study dual-reporters - PhoA-LacZ - were used to study the topology of Deh4p. Thirty-six Deh4p-PhoA-LacZ constructs were made and the fusion proteins expressed in E. coli. Analyses of the PhoA and LacZ activities of these constructs verified that the N- and the C-termini were located in the cytoplasm. This is typical for many MFS proteins . The experimentally determined topology of Deh4p was, however, slightly different from typical MFS transporters. Fusion proteins with Deh4p junctions at G52, T62 and S520 were expected to show a higher PhoA than LacZ activity. Cells expressing these fusion proteins actually exhibited higher LacZ activity. This suggested that the presence of the first and the eleventh TMS was not verified. It is possible that these helices have a low average hydrophobicity. Fig. 1 shows that this is indeed the case for TMS 1 and 11.
It can be argued that the presence of a LacZ moiety affected the translocation and correct folding of the PhoA, and thus its activity, in the periplasm. This is rather unlikely as only the LacZα fragment was used. Moreover, if this were true then the shorter the periplasmic loop the more likely that the PhoA activity will be concealed. The second predicted periplasmic loop only has a size of one residue (G114), and cells producing Deh4p1-114-PhoA-LacZ has a positive strength index. This indicated that the dual-reporter registered the location of the periplasmic loop accurately. Another concern arising from using enzymatic reporter assay for topology study is insufficient understanding of the details of membrane protein topogenesis. This concern is very real as current knowledge of topogenesis and membrane insertion mechanisms mainly comes from studies of eukaryotic cell organelles [50–53]. The topology of the transporter may alter if it is truncated and attached to another domain .
Inconclusive illustration of the presence of the TMS by the fusion reporter system has been reported. When -PhoA and -LacZ fusions were constructed near the N-terminal of the Na+/proline transporter PutP of E. coli, similar enzyme activities were detected . Helix I of the E. coli α-ketoglutarate permease KgtP was not detected by a PhoA fusion . In this case the presence of positively charged residues in other TMS was required to neutralize the negatively charged residues (E34 and D37) in helix I in order to place the segment into the membrane correctly. Similar negatively charged amino acids in Deh4p (E31 and D34) were predicted to be situated in the cytoplasm by the SOSUI program but were postulated to be part of helix I by the TOPCON program. It is possible that a similar effect was currently observed.
When the PhoA-LacZ reporter system was first developed, it was tested on the LacY protein. Eight of the LacY-PhoA-LacZα recombinants had the reporters ending in the TMS and seven of them were found to have higher PhoA than LacZ activities regardless of the orientation of the TMS . This is in contrast to the present study where higher LacZ than PhoA activities were detected in the majority of the recombinants with reporters that ended in the middle of a TMS, regardless of the orientation of the TMS (Fig. 2). The inability of the method to mark the boundary of the TMS and the tendency to have higher LacZ activity suggested the risk of having TMS omitted if insufficient number of constructs were made. The use of an E. coli strain, TOP10, with a wildtype phoA gene did not affect the quantification of the PhoA activities. The background enzyme level was negligible in all our experiments. This is similar to cases where a strain, TG1, which has a wildtype phoA gene, was used [33, 56].
The use of a fusion reporter system also failed to characterize membrane protein with atypical features. Helices E-F and P-Q of the E. coli ClcA protein, which has a known 3-D structure, were not detected by PhoA and green fluorescent protein fusions . These helices may have formed helical hairpins  and inserted into the membrane at a later stage of the folding . Further analysis is required to establish whether TMS 1 and 11 of Deh4p have a similar property. Further examination of hydropathy  and amphipathicity  plots by visual inspection also revealed that Deh4p may have less than twelve TMS. High amphipathicity with high hydrophobicity were also observed for the first 90 residues. This is unusual since TMS of structurally known MFS proteins LacY , EmrD , GlpT  and OxlT [28, 29] have high hydrophobicity but not amphipathicity. These analyses suggested that Deh4p may be an atypical MFS.
Comparative analysis of Deh4p with members of TC2.A.1.6 group indicated that it shares a lot of common features with this group of MFS proteins. Not only do they have seven conserved motifs, the organization of these motifs is also similar among the different members. Motif 1, which appeared twice, is the signature region linking TMS 2 and 3, and 8 and 9 of all MFS proteins. These family-specific motifs demonstrated that Deh4p is both a MHS and MFS protein. However, residues spanning 340 to 450 of Deh4p are unique among the MHS. This region is the periplasmic loop of Deh4p. A FASTA  and a BLASTP  search of the protein database UniProt Knowledgebase (UniProtKB) using this loop sequence have identified putative MFS proteins only from the α-, β-, γ- and δ-Proteobacteria. It is likely that this loop region is specific for the transporter proteins found in Proteobacteria except the ε-Class. The role of this loop awaits further study. The presence of such a loop near the C-terminal suggested that Deh4p is not the result of simple tandem duplication and is atypical of MFS proteins. During the preparation of this manuscript Deh4p has been designated as TC2.A.1.6.8 to indicate its difference from the other MHS members.
The use of PhoA-LacZα dual reports is a simple and convenient method to determine the general topology of any membrane protein. Together with bioinformatic analyses it is possible to produce a more reliable model for the protein being examined. Deh4p has been demonstrated to be an atypical MFS protein with an asymmetric organization and a long periplasmic loop. Although high-resolution structural study is ultimately required to elucidate the actual structure of Deh4p with certainty, the current data are sufficient to conclude the major structural features of Deh4p.
Strains and culture conditions
E. coli TOP10 (Invitrogen) was used for gene cloning and expression of the fusion proteins. E. coli cells were grown at 37°C in Luria broth (LB, 1% tryptone, 0.5% yeast extract, 0.5% NaCl) with or without 100 μg/ml ampicillin. Burkholderia sp. MBA4 (previously B. cepacia) was isolated from soil using monobromoacetate as the growth enrichment substrate . MBA4 was grown at 30°C in Luria broth without NaCl.
Construction of PhoA-LacZ reporter plasmids
DNA fragment encoding PhoA and LacZα was PCR amplified from plasmid pMA632  with primers SpeI-reporter-F (5'-ACTAG TGTTC TGGAA AACCG GGCTG CTCA-3') and Reporter-stop-R (5'-GAGCT TCATT CGCCA TTCAG GCTGC GCAAC TG-3'). The amplified fragment was cloned downstream of the lac promoter of vector pCR2.1-TOPO by TOPO-TA cloning (Invitrogen). A plasmid with the reporters in the correct orientation was designated as pHKU1433. Ribosomal promoter S12 of MBA4 (P s 12 ) was amplified from MBA4 total DNA with primers HindIII-S12-Fwd (5'-AAGCT TCGCA AGCCG TTGAC TTAGT TGG-3') and S12-BsiWI-Rev (5'-CGTAC GACCA GTTGG TTGAT GG-3'). The deh4p gene was similarly amplified with primers BsiWI-4p-Fwd (5'-CGTAC GGATG GCGAC TATTG A-3') and 4p552R-speI (5'-ACTAG TGTCC GCGTC ATAGG TAGAA GAACC CTT-3'). Both PCR products were individually cloned into pGEM-T Easy vector (Promega). The PS12-containing fragment was subsequently isolated by digesting the plasmid with Hind III and Bsi WI. The deh4p-bearing fragment was isolated by digesting the plasmid with Bsi WI and Spe I. These DNA fragments were mixed with Hind III and Spe I cut pHKU1433 and ligated with T4 DNA ligase. A plasmid with Ps12-deh4p ligated upstream of phoA-lacZ was assembled and named as pHKU1601-552.
Reverse primers used for the construction of plasmid pHKU1601 series.
Sequence (5' to 3')
ACTAG TGTCA TACCA CTCGA ATACG GTTCC CAA
ACTAG TACCG GAGAA GAACG TTCGG CT
ACTAG TTGTG AACAC AAACC CCGCT GCTG
ACTAG TGCCA AAAGG ACGCA CGGCG
ACTAG TCTTG CGTCC GATCA TGTCT CCAAG
ACTAG TCATC AGCAG GATTG TCGCA AGAA
ACTAG TTCCG TAACC GGGCA ACAAT CCAA
ACTAG TAGCG ATGAA AACAA CCGGC GC
ACTAG TCTCT CCGCC AAGCG CCAG
ACTAG TTGCG TGTTC CGCAA CATAG GTC
ACTAG TCTGG ATCCA TGCGG TCCAT GCG
ACTAG TAATA AACAG GCCAA GCGTA GCCGT
ACTAG TGGCC GCAAA TGTAT CTTCG TTAAG CAA
ACTAG TAACG ATCGA GACAA GGAAA GGAAC G
ACTAG TAACG GGTGA CTCGT GAAGT TGC
ACTAG TCCCG AATGC TTCCG ATAGT GGGG
ACTAG TTAGT GCAAG CAGGA CGATT TTCAG
ACTAG TGCCC GTGTA CCATA CAACC GCC
ACTAG TGCTC GTACC GTCGA CCTTA AGAGT CTG
ACTAG TACCG ATCAG CAACG CGACA G
ACTAG TCTTT CGCCC AATCT TGTCC GACAG
ACTAG TAATC AGGCA GCCTG CCATG ATA
ACTAG TGTAG TGGGC GAGAG CCTTG AAC
ACTAG TGCTC GGATC AGCGA TCATC G
ACTAG TTGCG ACGTC ACACG AACTC G
ACTAG TGACA GTCCC GGCAG GGGC
ACTAG TTTTT GCGTC CGCCG CTTTC
ACTAG TGGCG GGGTA GCCAG CAGTC T
ACTAG TCGAC ATCGG CCAGT TGATC AGCG
ACTAG TGGTG ACGTA GAGCA CGAGT ATCGT CAG
ACTAG TCATC TCCAC CAGCA TTGCT GCG
ACTAG TATAA GGCAG CGACA TTGAG GTGTA TCG
ACTAG TGCCG CCGAA CCAGC CATTG
ACTAG TTGAA TAGAT GTTCC CGCGC GCTG
ACTAG TCGCA ACGGA AGCGA TAACA ATC
ACTAG TGTCC GCGTC ATAGG TAGAA GAACC CTT
Assay of PhoA and LacZ activities
E. coli cells containing pHKU1601- series plasmid were grown in 5 ml Luria Broth with 100 μg/ml ampicillin. The cultures were incubated overnight at 37°C with shaking. One milliliter of overnight culture was saved for β-galactosidase (LacZ) assay and another milliliter for alkaline phosphatase (PhoA) assay. The protocols for PhoA and LacZ activity assay were modified from a previous report that utilized 96-well microtiter plate .
To determine the PhoA activity, 1 ml overnight culture was harvested, washed once in 1 ml Tris-HCl (pH 8.0) and resuspended in 1 ml assay buffer (1 M Tris-HCl, 0.1 mM ZnCl2, pH 8.0). The cells were permeabilized by adding 50 μl of chloroform and 50 μl of 0.1% SDS and gently vortexed for 10 sec. The mixture was incubated at 30°C for 20 minutes. After the chloroform was settled, 200 μl of the upper aqueous phase was transferred to a well of a microtiter plate. The reaction was started by the addition of 25 μl of p-nitrophenylphosphate solution (Sigma, N7653) and kept at 30°C. Formation of p-nitrophenol was measured by absorbance at 405 nm at 2 minutes' interval, followed by 10 seconds of orbital shaking that prevent cell sedimentation, for 1 hour. The cell densities of the samples were measured by absorbance at 600 nm.
Determination of the LacZ activity was also started with a 1 ml culture but this time washed with Z-buffer  and resuspended in 1 ml Z-buffer with 50 mM β-mercaptoethanol. The cells were then permeabilized and transferred to a microtiter plate as in the PhoA activity assay. The reaction was started by the addition of 25 μl of o-nitrophenyl galactopyranoside (Sigma, N1127; 4 mg/ml in Z-buffer). Formation of o-nitrophenol was quantified by absorbance at 420 nm in conditions similar to that of PhoA assay. The cell densities of the samples were also recorded.
The terms R i , PhoA and R i , LacZ represent R i for the PhoA and the LacZ assays, respectively. Di, LacZand Di, PhoArepresent the optical densities at 600 nm for sample i in the LacZ and the PhoA assays, respectively. The term max (Ri, LacZ/Di, LacZ)i = 1...nrepresents the maximum R i /D i value recorded among n samples for the LacZ assays and likewise the term max (Ri, PhoA/Di, PhoA)i = 1...nrepresents the highest R i /D i value registered for the PhoA assays. A natural logarithm (Ln) was taken for the calculated value so that a positive I represents a higher PhoA than LacZ activity, while a negative I indicates that the LacZ activity was higher. Note that R i must be larger than zero to avoid calculation error. If R i was found to be zero or negative, an arbitrary small positive value was assigned.
We thank Herbert Winkler for plasmid pMA632 and Janice Brabyn for reading the manuscript. YMT thanks the University of Hong Kong for a studentship. We gratefully acknowledge the support of the BIOSUPPORT project http://bioinfo.hku.hk for providing bioinformatics resources and computational services from the HKU Computer Centre. This work was supported by the University Seed Funding Programme for Basic Research 2008 and the Research Grants Council of the Hong Kong Special Administrative Region, China (project no. HKU7536/06 M).
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