BACKGROUND
bacterial enzymes have been known to play a major role in the pathogenesis of burkholderia pseudomallei, the causative agent of melioidosis.
this bacterium is known to secrete a number of enzymes such as protease, catalase, peroxidase, superoxide dismutase, phosphatase and phospholipase c , which are said to contribute to the virulence of the organism  <cit> .
in our previous study  <cit> , high levels of leucine aminopeptidase  enzymatic activity had been detected in both clinical and environmental isolates of b. pseudomallei, by apizym analysis .
lap which belongs to the peptidase m <dig> family, is involved in the processing and regular turnover of intracellular proteins by catalyzing the removal of unsubstituted n-terminal amino acids from various peptides  <cit> .
besides proteolytic activities, this enzyme is also known to play an important role as a dna-binding protein in escherichia coli <cit> , and a repressor or activator in the operon regulation of virulence-associated genes in e. coli, vibrio cholerae and pseudomonas aeruginosa <cit> .
the lap enzyme has been proposed as an immunoantigen for vaccination against fasciola hepatica in sheep  <cit>  and a promising drug target for helicobacter pylori infections  <cit> .
as there has not been any study on lap of b. pseudomallei, the objective of the present study was to characterise the lap activity of b. pseudomallei and to examine the intra- and inter-species variation in the nucleotide and deduced amino acid sequences of the lap encoding gene .
a pepa/pcr-rflp was designed to facilitate the identification of lap sequence types and for possible differentiation of phenotypically identical b. pseudomallei isolates.
methods
extraction of lap
one milliliter of an overnight-culture of b. pseudomallei nctc  <dig>  was inoculated into  <dig> liters of bhi broth and incubated at 37°c for 72 h with constant agitation at 120 rpm in a shaker .
the bacterial cells were removed by centrifugation at  <dig>  rpm for 30 min at 4°c, and the flow-through filtered using a  <dig>  μm polyethersulfone membrane .
one part of the filtrate was mixed with  <dig> parts of cold saturated ammonium sulfate solution for 10 min with stirring, prior to centrifugation at  <dig>  rpm for 45 min at 4°c.
the precipitate was dissolved in cold 50 mm tris-hcl buffer .
desalting was performed using hiprep 26/ <dig> desalting column  coupled to a akta™ explorer  <dig> system .
the eluent was concentrated using a vivaspin 15r column  by centrifugation at  <dig>  g. the protein concentration of the sample was determined by quick start™ bradford protein assay  using bovine serum albumin as the standard.
zymographic analysis
zymographic analysis was performed to detect the presence of lap activity in the crude extract of b. pseudomallei nctc  <dig>  the extract was diluted  <dig> fold  and mixed with nativepage™ buffer   in a ratio of 3: <dig>  the sample was analysed by native polyacrylamide gel electrophoresis  using a one mm-thick gel  in a mini-protean® tetra cell  page apparatus at 90 v for 120 min.
the gel was incubated at 37°c for 10 min in 50 mm tris-hcl buffer  containing  <dig>  mm mgcl <dig> and 200 μm l-leucine-7-amido-4-methylcoumarin•hcl  dissolved in  <dig>  ml acetone  <cit> .
five microliters of  <dig> x aminopeptidase i from streptomyces griseus  was used as positive control for lap.
a fluorescent band similar to the control, representing lap activity was visualised under uv light and photographed.
enzymatic characterisation
lap activity of the crude extract was quantitated as described by wahid et al.
<cit> .
eighty microliters of the extract was added to 20 μl of 10 mm l-leucine-p-nitroanilide substrate solution  and 100 μl of 50 mm tris-hcl buffer  in a microtiter well, followed by incubation at 37°c for 2 h. the reaction was stopped by cooling the mixture on ice for 10 min and the optical density at 405 nm was measured using a microplate reader .
the lap activity was quantitated by using a l-leucine-p-nitroaniline  calibration curve and defined as nanomoles of p-na released per minute per milliliter of sample under the assay conditions.
the optimum ph for lap activity was determined by incubating 80 μl of the concentrated bacterial extract with 100 μl of 50 mm buffer solutions prepared at various phs:  <dig> – <dig>  ,  <dig> – <dig>  ,  <dig> – <dig>   and  <dig> – <dig>  .
eighty microliters of the concentrated crude extract was mixed thoroughly with 100 μl buffer of various ph in a microtiter well at 30°c for 10 min, before addition of 20 μl of substrate solution.
the mixtures were incubated at 37°c for 2 h and the lap activity was determined as described above.
the effect of temperature on lap activity was studied by incubating for 2 h, 80 μl of the concentrated bacterial extract with 100 μl of 50 mm tris-hcl buffer  and 20 μl of 10 mm l-leucine-p-nitroanilide substrate solution at different temperatures .
the effect of metallic ions and other inhibitors on the lap activity was investigated by exposing 80 μl of the extract to 10 μl of solution containing metallic ions , ethylenediaminetetraacetic acid  ,  <dig> -phenanthroline , phenylmethylsulfonyl fluoride  and amastatin   and 90 μl of 50 mm tris-hcl buffer .
each mixture was pre-incubated at 30°c for 30 min before addition of 20 μl of the substrate solution.
following further incubation at 37°c for 2 h, the lap activity of each reaction was determined as described above.
sequence determination and analysis of lap gene
pcr primers  were designed using computer software primer <dig>   and tm calculation for oligos   for amplification of a 930 bp fragment encompassing the central region of the pepa gene, using sequences retrieved from b. pseudomallei reference strains: 1106a , k <dig> ,  <dig> , 1710b  and mshr <dig>  and  <dig> different pulsotypes of b. pseudomallei from a previous study  <cit> .
pure colonies of b. pseudomallei on lb agar were suspended in 500 μl miliq water, heated to 100°c for 30 min and cooled in ice for 10 min before centrifugation at  <dig>  rpm for 10 min.
the clear supernatants were used as dna templates for amplification.
each pcr reaction was performed by preparing a 25 μl reaction mixture containing  <dig>  μm of primers pepa273-f and pepa1202-r,  <dig>  mm of dntp,  <dig>  u/μl of dreamtaq™ dna polymerase ,  <dig> x dreamtaq™ buffer,  <dig>  μl of dh2o and 5 μl of template dna.
pcr conditions were: one cycle at  <dig> °c for 5 min, and 30 cycles at  <dig> °c for 1 min,  <dig> °c for 30 s,  <dig> °c for  <dig>  min, followed by one cycle of final extension at  <dig> °c for 5 min.
the pcr products were purified using geneall® expin™ combo gp  and sequenced using primers pepa273-f, pepa1202-r, pepa442-f  and pepa1037-r  in an applied biosystems abi3730xl automatic sequencer.
the contigs of pepa gene sequences were assembled and edited using geneious pro  <dig> .
<dig>  and aligned using mega  <dig> .
<dig> software.
rflp analysis of lap gene fragments
a pcr-rflp assay was designed based on the pepa sequences.
a total of  <dig> randomly selected clinical isolates of b. pseudomallei from malaysia and  <dig> environmental isolates  and  <dig> b. thailandensis isolates were used.
in additional file 1: table s <dig> shows the origins of the b. pseudomallei isolates.
partial fragments  of pepa gene were amplified from each isolate using primers pepa442-f and pepa1037-r using pcr conditions as described above, except for a higher annealing temperature of  <dig> °c.
the amplified products were purified and subjected to digestion using stui followed by hincii restriction endonucleases .
the rflp products were analysed by non-denaturing page using 12% polyacrylamide gel and stained with  <dig>  μg/ml ethidium bromide.
an o’generuler™ ultra low range dna ladder  was used as molecular weight marker.
RESULTS
the pepa gene of b. pseudomallei consists of  <dig> nucleotides and encodes for  <dig> amino acids.
the predicted molecular mass of the expressed protein was  <dig>  kd .
in the zymographic analysis, a fragment with fluorescent activity was observed in the native gel loaded with the concentrated culture supernatant of b. pseudomallei nctc  <dig> .
the enzyme activity was detected in the culture supernatant, suggesting that lap is a bacterial secretory product, detectable at temperatures ranging from 30°c to 60°c  and ph ranging from  <dig> to  <dig> .
the optimal lap activity was at ph  <dig> and at 50°c.
high optimum temperature has been reported for other laps: i.e.
60°c for tomatoes, e. coli and swine  <cit>  and 70°c for arabidopsis <cit> , whereas the alkaline ph of lap has been reported for organisms such as e. coli and arabidopsis thaliana <cit> .
the alkaline ph is said to facilitate the interaction between unprotonated n-terminus substrate and hydrophobic core of lap in order to hydrolyse the substrate  <cit> .
the optimum activity of lap at high temperature and ph  may be an essential factor for b. pseudomallei to be extremely adaptable in a wide variety of environments and able to survive during nutritional deprivation and exposure to high temperature  <cit> .
the effects of metal ions and inhibitors on lap activity are shown in table  <dig>  there was enhancement of lap activity in the presence of metal ions, in the order of mg2+ > ca2+ > na+ > k+.
this observation is in agreement with previous studies whereby a broad range of metal-ion dependence has been demonstrated by metallo-aminopeptidases: i.e.
mn2+ by laps of e. coli <cit> , mn2+ by human cytosolic aminopeptidase  <cit>  and ca2+ by streptomyces griseus <cit> .
in contrast, edta,  <dig> -phenanthroline and amastatin inhibited lap activity completely whereas mn2+ and zn2+ exhibited partial inhibitory effects .
inhibition by chelating agents  is common in animal, plant and prokaryotic laps  <cit> .
the inhibitory effects exerted by the chelating agents are suggestive that the enzyme is a metalloprotease.
amastatin which mimics the lap catalytic transition state, is a strong inhibitor of animal aminopeptidases  <cit> .
hence, inhibition of lap activity by this specific aminopeptidase inhibitor- amastatin, confirmed the identity of this enzyme as an aminopeptidase, as also described for lap of streptomyces hygroscopicus <cit> .
the lap enzyme is probably not a serine protease as little impact was observed by the addition of serine protease inhibitor pmsf .
comparison of the nucleotide sequences of the central region of the pepa gene  of b. pseudomallei reference strains: 1106a , k <dig> ,  <dig> , 1710b  and mshr <dig>  and  <dig> pulsotypes of malaysian isolates of b. pseudomallei revealed  <dig> lap sequence types .
nucleotide polymorphism was found at  <dig> positions:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig>  of which two at positions  <dig> and  <dig> are being reported for the first time.
examination of the deduced amino acid sequences of the enzyme shows three amino acid differences, i.e.
position  <dig> in b. pseudomallei mshr346; position  <dig> in strain  <dig> and position  <dig> in b. pseudomallei 1710b, strains  <dig> and  <dig>
five sequence types were identified from the  <dig> different pulsotypes representing the genetic diversity of b. pseudomallei isolates in malaysia: the majority  were identical to b. pseudomallei strain 1106a, and  <dig> to b. pseudomallei strain  <dig>  three strains  were new sequence types  suggesting slight differences existed in the conserved pepa gene sequence between isolates from malaysia and those in the genbank database.
shows the comparison of the nucleotide and deduced amino acid sequences of pepa gene of b. pseudomallei  with the closely related species .
between b. pseudomallei k <dig> and b. thailandensis e <dig>  there was only  <dig> % similarity in the nucleotide sequences.
comparison of  <dig> b. pseudomallei strains k <dig>  1710b, mshr <dig> and b. mallei atcc  <dig> showed only one amino acid difference.
however, comparison of b. pseudomallei strain k <dig> with b. thailandensis and b. oklahomensis showed  <dig> amino acid differences.
restriction analysis using stui and hincii of the amplified pepa gene enabled the identification of  <dig> restriction fragment polymorphism patterns  for b. pseudomallei: i.e.
type i with fragments of  <dig>   <dig>   <dig> and 20 bp; type ii with fragments of  <dig> and 233 bp and type iii with fragments of  <dig>   <dig> and 83 bp .
type i  and type ii  pepa/rflp types were predominant amongst our clinical and environmental isolates, respectively .
CONCLUSIONS
to the best of our knowledge there are no published reports on the presence or characterization of lap in b. pseudomallei.
dna sequencing of  <dig> different pulsotypes of b. pseudomallei isolates showed that the partial pepa gene sequence was highly conserved, with the detection of  <dig> extra intraspecific nucleotide divergences .
we describe here the characteristics of b. pseudomallei lap: high optimum temperature , alkaline optimum ph , requirement of divalent metal ions  for activity, and inhibition by lap-specific inhibitors  and some metal ions .
the high lap activity detected in both b. pseudomallei and b. thailandensis in both previous  <cit>  and this study, suggests that lap is probably a housekeeping enzyme rather than a virulence determinant.
however, to verify whether lap is truly a housekeeping gene, the use of a deletion mutant of lap from b. pseudomallei will be needed.
in addition, since iron is often correlated with virulence phenotypes, the effect of iron on the lap activity should be determined.
further work to clone and express lap as a recombinant protein is ongoing.
abbreviations
lap: leucine aminopeptidase; pepa: lap encoding gene; pcr-rflp: polymerase chain reaction restriction fragment length polymorphism; pfge: pulse-field gel electrophoresis; bhi: brain heart infusion; edta: ethylenediaminetetraacetic acid; dna: deoxyribonucleic acid; apizym: analytical profile index ; nctc: national collection of type cultures; atcc: american type culture collection; page: polyacrylamide gel electrophoresis; uv: ultraviolet; p-na: l-leucine-p-nitroaniline; pmsf: phenylmethylsulfonyl fluoride; lb: luria bertani; dntp: deoxynucleotriphosphate; dh2o: distilled water; bp: base pair.
competing interests
the authors declare that there is no conflict of interests.
authors’ contributions
this study was carried out as part of research work for master of medical science degree.
all authors read and approved the final manuscript.
supplementary material
additional file 1: table s1
source and origin of clinical and environmental isolates of b.pseudomallei .
table s <dig>  sequence types of the pepa gene of b. pseudomallei.
table s <dig>  comparison of nucleotide and deduced amino acid sequences of pepa genes of b. pseudomallei and closely related species.
table s <dig>  pcr-rflp of partial pepa gene  of b. pseudomallei.
click here for file
acknowledgments
this research was supported by the grants from the short term research fund   and the postgraduate research fund  from the university of malaya.
we wish to thank prof. surasakdi wongkratanacheewin from melioidosis research centre, department of microbiology, faculty of medicine, khon kaen university, khon kaen  <dig>  thailand, dr. e. h. yap from defense, medical & environmental research institute, dso national laboratories, republic of singapore for providing b. pseudomallei environmental isolates, mr. mah boon geat and mr. b. h. chua from axon scientific sdn.
bhd., mr. chang teck ming and mr. jason lim from interscience sdn.
bhd., who have provided scientific expertise.

 bacterial enzymes have been known to play a major role in the pathogenesis of burkholderia pseudomallei, the causative agent of melioidosis. this bacterium is known to secrete a number of enzymes such as protease, catalase, peroxidase, superoxide dismutase, phosphatase and phospholipase c, which are said to contribute to the virulence of the organism. in our previous study, high levels of leucine aminopeptidase enzymatic activity had been detected in both clinical and environmental isolates of b. pseudomallei, by apizym analysis. lap which belongs to the peptidase m family, is involved in the processing and regular turnover of intracellular proteins by catalyzing the removal of unsubstituted n-terminal amino acids from various peptides. besides proteolytic activities, this enzyme is also known to play an important role as a dna-binding protein in escherichia coli, and a repressor or activator in the operon regulation of virulence-associated genes in e. coli, vibrio cholerae and pseudomonas aeruginosa. the lap enzyme has been proposed as an immunoantigen for vaccination against fasciola hepatica in sheep and a promising drug target for helicobacter pylori infections. as there has not been any study on lap of b. pseudomallei, the objective of the present study was to characterise the lap activity of b. pseudomallei and to examine the intra- and inter-species variation in the nucleotide and deduced amino acid sequences of the lap encoding gene. a pepa/pcr-rflp was designed to facilitate the identification of lap sequence types and for possible differentiation of phenotypically identical b. pseudomallei isolates. methods extraction of lap one milliliter of an overnight-culture of b. pseudomallei nctc was inoculated into liters of bhi broth and incubated at 37°c for 72 h with constant agitation at 120 rpm in a shaker. the bacterial cells were removed by centrifugation at  rpm for 30 min at 4°c, and the flow-through filtered using a  μm polyethersulfone membrane. one part of the filtrate was mixed with parts of cold saturated ammonium sulfate solution for 10 min with stirring, prior to centrifugation at  rpm for 45 min at 4°c. the precipitate was dissolved in cold 50 mm tris-hcl buffer. desalting was performed using hiprep 26/ desalting column coupled to a akta™ explorer system. the eluent was concentrated using a vivaspin 15r column by centrifugation at  g. the protein concentration of the sample was determined by quick start™ bradford protein assay using bovine serum albumin as the standard. zymographic analysis zymographic analysis was performed to detect the presence of lap activity in the crude extract of b. pseudomallei nctc the extract was diluted fold and mixed with nativepage™ buffer in a ratio of 3: the sample was analysed by native polyacrylamide gel electrophoresis using a one mm-thick gel in a mini-protean® tetra cell page apparatus at 90 v for 120 min. the gel was incubated at 37°c for 10 min in 50 mm tris-hcl buffer containing  mm mgcl and 200 μm l-leucine-7-amido-4-methylcoumarin•hcl dissolved in  ml acetone. five microliters of x aminopeptidase i from streptomyces griseus was used as positive control for lap. a fluorescent band similar to the control, representing lap activity was visualised under uv light and photographed. enzymatic characterisation lap activity of the crude extract was quantitated as described by wahid et al.. eighty microliters of the extract was added to 20 μl of 10 mm l-leucine-p-nitroanilide substrate solution and 100 μl of 50 mm tris-hcl buffer in a microtiter well, followed by incubation at 37°c for 2 h. the reaction was stopped by cooling the mixture on ice for 10 min and the optical density at 405 nm was measured using a microplate reader. the lap activity was quantitated by using a l-leucine-p-nitroaniline calibration curve and defined as nanomoles of p-na released per minute per milliliter of sample under the assay conditions. the optimum ph for lap activity was determined by incubating 80 μl of the concentrated bacterial extract with 100 μl of 50 mm buffer solutions prepared at various phs: –, –, – and –. eighty microliters of the concentrated crude extract was mixed thoroughly with 100 μl buffer of various ph in a microtiter well at 30°c for 10 min, before addition of 20 μl of substrate solution. the mixtures were incubated at 37°c for 2 h and the lap activity was determined as described above. the effect of temperature on lap activity was studied by incubating for 2 h, 80 μl of the concentrated bacterial extract with 100 μl of 50 mm tris-hcl buffer and 20 μl of 10 mm l-leucine-p-nitroanilide substrate solution at different temperatures. the effect of metallic ions and other inhibitors on the lap activity was investigated by exposing 80 μl of the extract to 10 μl of solution containing metallic ions, ethylenediaminetetraacetic acid, -phenanthroline, phenylmethylsulfonyl fluoride and amastatin and 90 μl of 50 mm tris-hcl buffer. each mixture was pre-incubated at 30°c for 30 min before addition of 20 μl of the substrate solution. following further incubation at 37°c for 2 h, the lap activity of each reaction was determined as described above. sequence determination and analysis of lap gene pcr primers were designed using computer software primer and tm calculation for oligos for amplification of a 930 bp fragment encompassing the central region of the pepa gene, using sequences retrieved from b. pseudomallei reference strains: 1106a, k,, 1710b and mshr and different pulsotypes of b. pseudomallei from a previous study. pure colonies of b. pseudomallei on lb agar were suspended in 500 μl miliq water, heated to 100°c for 30 min and cooled in ice for 10 min before centrifugation at  rpm for 10 min. the clear supernatants were used as dna templates for amplification. each pcr reaction was performed by preparing a 25 μl reaction mixture containing  μm of primers pepa273-f and pepa1202-r,  mm of dntp, u/μl of dreamtaq™ dna polymerase, x dreamtaq™ buffer,  μl of dh2o and 5 μl of template dna. pcr conditions were: one cycle at °c for 5 min, and 30 cycles at °c for 1 min, °c for 30 s, °c for  min, followed by one cycle of final extension at °c for 5 min. the pcr products were purified using geneall® expin™ combo gp and sequenced using primers pepa273-f, pepa1202-r, pepa442-f and pepa1037-r in an applied biosystems abi3730xl automatic sequencer. the contigs of pepa gene sequences were assembled and edited using geneious pro. and aligned using mega. software. rflp analysis of lap gene fragments a pcr-rflp assay was designed based on the pepa sequences. a total of randomly selected clinical isolates of b. pseudomallei from malaysia and environmental isolates and b. thailandensis isolates were used. in additional file 1: table s shows the origins of the b. pseudomallei isolates. partial fragments of pepa gene were amplified from each isolate using primers pepa442-f and pepa1037-r using pcr conditions as described above, except for a higher annealing temperature of °c. the amplified products were purified and subjected to digestion using stui followed by hincii restriction endonucleases. the rflp products were analysed by non-denaturing page using 12% polyacrylamide gel and stained with  μg/ml ethidium bromide. an o’generuler™ ultra low range dna ladder was used as molecular weight marker.  the pepa gene of b. pseudomallei consists of nucleotides and encodes for amino acids. the predicted molecular mass of the expressed protein was kd. in the zymographic analysis, a fragment with fluorescent activity was observed in the native gel loaded with the concentrated culture supernatant of b. pseudomallei nctc. the enzyme activity was detected in the culture supernatant, suggesting that lap is a bacterial secretory product, detectable at temperatures ranging from 30°c to 60°c and ph ranging from to. the optimal lap activity was at ph  and at 50°c. high optimum temperature has been reported for other laps: i.e. 60°c for tomatoes, e. coli and swine and 70°c for arabidopsis, whereas the alkaline ph of lap has been reported for organisms such as e. coli and arabidopsis thaliana. the alkaline ph is said to facilitate the interaction between unprotonated n-terminus substrate and hydrophobic core of lap in order to hydrolyse the substrate. the optimum activity of lap at high temperature and ph may be an essential factor for b. pseudomallei to be extremely adaptable in a wide variety of environments and able to survive during nutritional deprivation and exposure to high temperature. the effects of metal ions and inhibitors on lap activity are shown in table  there was enhancement of lap activity in the presence of metal ions, in the order of mg2+ > ca2+ > na+ > k+. this observation is in agreement with previous studies whereby a broad range of metal-ion dependence has been demonstrated by metallo-aminopeptidases: i.e. mn2+ by laps of e. coli, mn2+ by human cytosolic aminopeptidase and ca2+ by streptomyces griseus. in contrast, edta, -phenanthroline and amastatin inhibited lap activity completely whereas mn2+ and zn2+ exhibited partial inhibitory effects. inhibition by chelating agents is common in animal, plant and prokaryotic laps. the inhibitory effects exerted by the chelating agents are suggestive that the enzyme is a metalloprotease. amastatin which mimics the lap catalytic transition state, is a strong inhibitor of animal aminopeptidases. hence, inhibition of lap activity by this specific aminopeptidase inhibitor- amastatin, confirmed the identity of this enzyme as an aminopeptidase, as also described for lap of streptomyces hygroscopicus. the lap enzyme is probably not a serine protease as little impact was observed by the addition of serine protease inhibitor pmsf. comparison of the nucleotide sequences of the central region of the pepa gene of b. pseudomallei reference strains: 1106a, k,, 1710b and mshr and pulsotypes of malaysian isolates of b. pseudomallei revealed lap sequence types. nucleotide polymorphism was found at positions: and of which two at positions and are being reported for the first time. examination of the deduced amino acid sequences of the enzyme shows three amino acid differences, i.e. position in b. pseudomallei mshr346; position in strain and position in b. pseudomallei 1710b, strains and five sequence types were identified from the different pulsotypes representing the genetic diversity of b. pseudomallei isolates in malaysia: the majority were identical to b. pseudomallei strain 1106a, and to b. pseudomallei strain three strains were new sequence types suggesting slight differences existed in the conserved pepa gene sequence between isolates from malaysia and those in the genbank database. shows the comparison of the nucleotide and deduced amino acid sequences of pepa gene of b. pseudomallei with the closely related species. between b. pseudomallei k and b. thailandensis e there was only % similarity in the nucleotide sequences. comparison of b. pseudomallei strains k 1710b, mshr and b. mallei atcc showed only one amino acid difference. however, comparison of b. pseudomallei strain k with b. thailandensis and b. oklahomensis showed amino acid differences. restriction analysis using stui and hincii of the amplified pepa gene enabled the identification of restriction fragment polymorphism patterns for b. pseudomallei: i.e. type i with fragments of and 20 bp; type ii with fragments of and 233 bp and type iii with fragments of and 83 bp. type i and type ii pepa/rflp types were predominant amongst our clinical and environmental isolates, respectively.  to the best of our knowledge there are no published reports on the presence or characterization of lap in b. pseudomallei. dna sequencing of different pulsotypes of b. pseudomallei isolates showed that the partial pepa gene sequence was highly conserved, with the detection of extra intraspecific nucleotide divergences. we describe here the characteristics of b. pseudomallei lap: high optimum temperature, alkaline optimum ph, requirement of divalent metal ions for activity, and inhibition by lap-specific inhibitors and some metal ions. the high lap activity detected in both b. pseudomallei and b. thailandensis in both previous and this study, suggests that lap is probably a housekeeping enzyme rather than a virulence determinant. however, to verify whether lap is truly a housekeeping gene, the use of a deletion mutant of lap from b. pseudomallei will be needed. in addition, since iron is often correlated with virulence phenotypes, the effect of iron on the lap activity should be determined. further work to clone and express lap as a recombinant protein is ongoing. abbreviations lap: leucine aminopeptidase; pepa: lap encoding gene; pcr-rflp: polymerase chain reaction restriction fragment length polymorphism; pfge: pulse-field gel electrophoresis; bhi: brain heart infusion; edta: ethylenediaminetetraacetic acid; dna: deoxyribonucleic acid; apizym: analytical profile index; nctc: national collection of type cultures; atcc: american type culture collection; page: polyacrylamide gel electrophoresis; uv: ultraviolet; p-na: l-leucine-p-nitroaniline; pmsf: phenylmethylsulfonyl fluoride; lb: luria bertani; dntp: deoxynucleotriphosphate; dh2o: distilled water; bp: base pair. competing interests the authors declare that there is no conflict of interests. authors’ contributions this study was carried out as part of research work for master of medical science degree. all authors read and approved the final manuscript. supplementary material additional file 1: table s1 source and origin of clinical and environmental isolates of b.pseudomallei. table s sequence types of the pepa gene of b. pseudomallei. table s comparison of nucleotide and deduced amino acid sequences of pepa genes of b. pseudomallei and closely related species. table s pcr-rflp of partial pepa gene of b. pseudomallei. click here for file acknowledgments this research was supported by the grants from the short term research fund and the postgraduate research fund from the university of malaya. we wish to thank prof. surasakdi wongkratanacheewin from melioidosis research centre, department of microbiology, faculty of medicine, khon kaen university, khon kaen thailand, dr. e. h. yap from defense, medical & environmental research institute, dso national laboratories, republic of singapore for providing b. pseudomallei environmental isolates, mr. mah boon geat and mr. b. h. chua from axon scientific sdn. bhd., mr. chang teck ming and mr. jason lim from interscience sdn. bhd., who have provided scientific expertise.
the high lap activity detected in both b. pseudomallei and b. thailandensis, suggests that lap is probably a housekeeping enzyme rather than a virulence determinant. sequence analysis of the complete nucleotide and deduced amino acid sequences of lap-encoding gene showed close genetic relatedness to b. mallei, but not with b. thailandensis. leucine aminopeptidase has been known to be a housekeeping protease, dna-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. lap activity was detected in b. pseudomallei culture supernantants by zymographic analysis. optimum activity was at ph  and stable at 50°c. a pepa/pcr-rflp assay was designed to facilitate the identification of major lap sequence types amongst clinical and environmental isolates of b. pseudomallei. this study showed that lap is a secretory product of b. pseudomallei with features similar to lap of other organisms. identification of major lap sequence types of b. pseudomallei was made possible based on rflp analysis of the pepa gene. lap activity was consistently detected in overnight cultures of burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. based on restriction analysis with stui and hincii enzymes of the amplified pepa gene, clinical and environmental isolates showed different predominant rflp types. type i was the most predominant type amongst % of the clinical isolates, while type ii was predominant in % of the environmental isolates. eight pepa sequence types were identified by comparison with a 596 bp dna fragment encompassing central regions of the pepa gene. lap activity was inhibited by edta, -phenanthroline, amastatin, mn2+ and zn2+.
 leucine aminopeptidase has been known to be a housekeeping protease, dna-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. lap activity was consistently detected in overnight cultures of burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. the intra- and inter-species nucleotide and deduced amino acid sequence variation of lap encoding gene was determined. a pepa/pcr-rflp assay was designed to facilitate the identification of major lap sequence types amongst clinical and environmental isolates of b. pseudomallei.  lap activity was detected in b. pseudomallei culture supernantants by zymographic analysis. optimum activity was at ph  and stable at 50°c. enhanced enzymatic activity was observed in the presence of metallic ions mg2+, ca2+, na+ and k+. lap activity was inhibited by edta, -phenanthroline, amastatin, mn2+ and zn2+. sequence analysis of the complete nucleotide and deduced amino acid sequences of lap-encoding gene showed close genetic relatedness to b. mallei, but not with b. thailandensis. eight pepa sequence types were identified by comparison with a 596 bp dna fragment encompassing central regions of the pepa gene. a pepa/pcr-rflp was designed to differentiate pepa sequence types. based on restriction analysis with stui and hincii enzymes of the amplified pepa gene, clinical and environmental isolates showed different predominant rflp types. type i was the most predominant type amongst % of the clinical isolates, while type ii was predominant in % of the environmental isolates.  this study showed that lap is a secretory product of b. pseudomallei with features similar to lap of other organisms. identification of major lap sequence types of b. pseudomallei was made possible based on rflp analysis of the pepa gene. the high lap activity detected in both b. pseudomallei and b. thailandensis, suggests that lap is probably a housekeeping enzyme rather than a virulence determinant. leucine aminopeptidaseburkholderia pseudomalleipcr-rflp 