A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola

Molecular Microbiology (2004)

51

(2) 471ndash481 doi101046j1365-2958200303839x

copy 2003 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd 200351

2471481

Original Article

T denticola phosphatidylcholine pathwayC Kent

et al

Accepted 22 September 2003 For correspondence E-mailfennoumichedu Tel (+1) 734 763 3331 Fax (+1) 734 764 2425

A CDP-choline pathway for phosphatidylcholine biosynthesis in

Treponema denticola

Claudia Kent

1

Patricia Gee

1

Si Young Lee

23

Xuelin Bian

2

and J Christopher Fenno

2

1

Department of Biological Chemistry Medical School University of Michigan Ann Arbor MI 48109ndash1078 USA

2

Department of Biologic and Materials Sciences School of Dentistry University of Michigan Ann Arbor MI 48109ndash1078 USA

3

Department of Oral Microbiology College of Dentistry Kangnung National University Kangnung Korea

Summary

The genomes of

Treponema denticola

and

Treponemapallidum

contain a gene

licCA

which is predicted toencode a fusion protein containing choline kinaseand CTPphosphocholine cytidylyltransferase activi-ties Because both organisms have been reported tocontain phosphatidylcholine this raises the possibil-ity that they use a CDP-choline pathway for the bio-synthesis of phosphatidylcholine This report showsthat phosphatidylcholine is a major phospholipid in

Tdenticola

accounting for 35ndash40 of total phospho-lipid This organism readily incorporated [

14

C]cholineinto phosphatidylcholine indicating the presence ofa choline-dependent biosynthetic pathway The

licCA

gene was cloned and recombinant LicCA had cholinekinase and CTPphosphocholine cytidylyltransferaseactivity The

licCA

gene was disrupted in

T denticola

by erythromycin cassette mutagenesis resulting in aviable mutant This disruption completely blockedincorporation of either [

14

C]choline or

32

Pi into phos-phatidylcholine The rate of production of anotherphospholipid in

T denticola

phosphatidylethanola-mine was elevated considerably in the

licCA

mutantsuggesting that the elevated level of this lipid com-pensated for the loss of phosphatidylcholine in themembranes Thus it appears that

T denticola

doescontain a

licCA

-dependent CDP-choline pathway forphosphatidylcholine biosynthesis

Introduction

Phosphatidylcholine (PC) is a major lipid in eukaryoticmembranes in which it accounts for 40ndash60 of the phos-

pholipids Phosphatidylcholine is a major structuralcomponent of membrane bilayers and lipoproteins andparticipates in several signal transduction pathwaysPhosphatidylcholine is also found in a wide variety ofbacteria notably symbionts and pathogens (reviewed inSohlenkamp

et al

2003) where it may constitute only afew per cent of total lipids as in

Pseudomonas aeruginosa

(Albelo and Domenech 1997) or may be a major lipidcomponent as in

Acetobacter aceti

(Hanada

et al

2001)In eukaryotes PC is made by either of two pathways

the CDP-choline or Kennedy pathway (overview shown inFig 1) consists of three enzymatic steps catalysed bycholine kinase CTPphosphocholine cytidylyltransferase(CCT) and a CDP-choline12-diacylglycerol cholinephosphotransferase The phospholipid N-methyltrans-ferase pathway consists of the stepwise methylation ofphosphatidylethanolamine (PE) with S-adenosylmethion-ine as methyl donor It has been long assumed that bac-teria do not possess the CDP-choline pathway for PCbiosynthesis (Lopez-Lara and Geiger 2001) To dateprokaryotes have been shown to make PC by either thephospholipid N-methyltransferase pathway (Kaneshiroand Law 1964) or the phosphatidylcholine synthase path-way a pathway unique to bacteria in which choline reactswith CDP-diacylglycerol to form PC (de Rudder

et al

1999) Most bacteria having PC as a membrane lipidprobably possess both currently known bacterial path-ways for PC biosynthesis though some including

Borrelia

Pseudomonas

and

Burkholderia

spp possess only onePC biosynthesis pathway (reviewed in Sohlenkamp

et al

2003)

Some bacteria do have a CDP-choline pathway forattaching phosphocholine to complex cell-surface oli-gosaccharides (Fig 1) The

lic1

operon in

H influenzae

and

lic

operon in

S pneumoniae

have been identified asbeing involved in this process (Weiser

et al

1997 Zhang

et al

1999) Gene

licA

was proposed to be a cholinekinase (Weiser

et al

1997) and this identity has recentlybeen confirmed by cloning expressing and characteriz-ing the gene product (H A Campbell and C Kent unpubldata) The

licC

gene has been identified as the CCT ofthis pathway although its primary structure is not similarto that of the eukaryotic CCTs (Campbell and Kent 2001Rock

et al

2001) The gene encoding the choline phos-photransferase that donates phosphocholine to the oli-gosaccharide has not been definitively identified but it has

472

C Kent

et al


Molecular Microbiology

51

471ndash481

been proposed to be

licD

(Zhang

et al

1999 Lysenko

et al

2000)Genomic analysis has revealed that several bacteria

appear to encode a fusion protein in which a

licC

-encodedCCT is the amino terminal half and a

licA

-encoded cholinekinase is the carboxyl terminal half (Fig 2) We refer tothis fusion gene as

licCA

The

licCA

-containing organismsinclude

Treponema pallidum

which causes syphilis

Fuso-bacterium nucleatum

which is involved in periodontaldisease and

Clostridium perfringens

which causes gan-grene Although little information is available on phospho-lipid synthesis and composition in fusobacteria andclostridia it is of interest that the treponemes have beenreported to contain PC as a major membrane phospho-lipid (Livermore and Johnson 1974 Matthews

et al

1979 Barbieri

et al

1981 Belisle

et al

1994) The pres-ence of the

licCA

fusion gene in treponemes suggests thatthey may utilize a CDP-choline pathway for biosynthesisof PC In this report we show that

T denticola

possessesa CDP-choline pathway for PC biosynthesis and that a

licCA

gene similar to that of

T pallidum

encodes proteinsin that pathway This is the first report of a CDP-cholinepathway for PC biosynthesis in bacteria

Results

Phosphatidylcholine in

T denticola

Several species of

Treponema

including

T denticola

have been reported to contain PC and several other

phospholipids including PE and PG (Livermore andJohnson 1974 Smibert 1976 Barbieri

et al

1981) butthe amount of each phospholipid in

T denticola

has notpreviously been reported quantitatively In order to con-firm the presence of and measure the amount of PC in

Tdenticola

total lipids were extracted and chromato-graphed by thin-layer chromatography in several differentsolvent systems The major phospholipids PC PEand phosphatidylglycerol (PG) were identified by co-chro-matography with known standards Phosphatidylcholineand PE were the most abundant phospholipids eachaccounting for about a third of total phospholipids whilePG was about 10 (Fig 3) Cardiolipin was a minor lipid(not shown)

Although the choline-containing sphingolipid sphingo-myelin has been reported to be a constituent of

Tpallidum

(Matthews

et al

1979) we found no evidencefor this lipid in

T denticola

either by measuring lipidphosphate or by incorporation of [

14

C]-choline or

32

Piinto a lipid that co-chromatographed with authenticsphingomyelin

If

T denticola

were using a CDP-choline pathway forPC biosynthesis one would expect that this lipid would bespecifically labelled after incubating the bacteria with radi-olabelled choline Indeed

T denticola

had a robust sys-tem for uptake and incorporation of either [

3

H]choline (notshown) or [

14

C]choline (Fig 4) into lipids Separation bythin-layer chromatography and quantification of the indi-vidual lipids from such an experiment revealed that atleast 96 (

3

H) or 99 (

14

C) of the radioactivity in the totallipid extract was in PC

Cloning and expression of

licCA

and enzymatic activity of the recombinant protein

The

licCA

gene was identified in preliminary

T denticola

genome sequence contigs based on similarity to the pre-dicted

licCA

gene of

T pallidum

(Fraser

et al

1998) The

T denticola licCA

DNA sequence was found to be identi-cal to that shown in the preliminary genomic contigs (datanot shown) The original cloned fragment in pSY107 wasdesigned to contain about 1300 base pairs upstream ofthe translation start site predicted from the

T pallidumlicCA

sequence (

79

MAAGFGShellip Fig 2) Recombinantexpression of a

T denticola

construct that initiated at thistranslation start site however resulted in a protein thatwas insoluble and inactive Inspection of the sequencesupstream of this predicted start site revealed another pos-sible translation initiation site which added 78 residues tothe protein sequence The

T pallidum

and

T denticola

sequences were 45 identical within this segmentExpression of a construct designed to initiate at this newsite (

1

MKRRYFhellip Fig 2) resulted in a protein that wassoluble and active The protein was purified to near homo-

Fig 1

CDP-choline pathways for biosynthesis of phosphatidylcholine and glycoconjugate-linked phosphocholine The CDP-choline path-way for phosphatidylcholine biosynthesis is found in animals plants yeasts and other eukaryotes The CDP-choline pathway for addition of phosphocholine to glycoconjugates has been demonstrated in

H influenzae

and

S pneumoniae

and is presumably present in other bacteria containing the

lic

genes In organisms containing the

lic

genes choline kinase is the product of

licA

cholinephosphate cyti-dylyltransferase is the product of

licC

and the cholinephosphotrans-ferase that transfers phosphocholine to the complex oligosaccharide is the presumed product of

licD

T denticola

phosphatidylcholine pathway

473



51

471ndash481

geneity (Fig 5) and was active in assays for both cholinekinase and CCT (Table 1)

Because the enzyme activities of the LicCA proteinwere 50ndash200 times lower than the activities of the individ-ual enzymes from

S pneumoniae

(Campbell and Kent

2001 Rock

et al

2001 H A Campbell and C Kentunpubl data) we also constructed expression vectors thatwould encode the individual enzymes These were termedC308 for the CCT from the

licC

portion and A317 for thecholine kinase from the

licA

portion Residue 308 was the

Fig 2

Alignment of LicC LicA and LicCA gene products The alignment was according to the

CLUSTALW

algorithm in the

MEGALIGN

program of the Lasergene package Shaded residues are those that are conserved (identical or Y = F =W I = L = V K = R E = D and S = T) in all sequences Boxed residues are the critical Arg and Lys active site residues (see

Discussion

) Sequences abbreviations and accession numbers are Td

Treponema denticola

(AY322155) Tp

Treponema pallidum

(NP_218547) Fn

Fusobacterium nucleatum

(NP_602486 [FnA] and NP_604134 [FnB]) Cp


(NP_561543) Sp

Streptococcus pneumoniae

(AAK94072 [LicC] and AAK94073 [LicA]) Hi

Haemophilus influenzae (NP_439688 [LicC] and P14181 [LicA]) The putative initiator methionine in the T pallidum LicCA is encoded by GTG The last six residues of the T pallidum LicCA are not shown Streptococcus pneumoniae LicA and H influenzae LicA are aligned with the C-terminal regions of LicCA such that the conserved Gly-Met-Thr-Asn motif near the N-terminus of the LicA region (330ndash333 in T denticola) is aligned in all sequences A series of 4-residue tandem repeats at the amino terminus of H influenzae LicA is not shown and is indicated by lsquorsquo

474 C Kent et al

copy 2003 Blackwell Publishing Ltd Molecular Microbiology 51 471ndash481

C-terminus of C308 and residue 317 preceded by aninitiator Met was the N-terminus of A317 These proteinswere also purified to near homogeneity (Fig 5) andassayed for their activities The A317 choline kinase activ-

ity was about fivefold greater than the choline kinase ofthe fusion protein but the C308 CCT activity was negligi-ble (Table 1)

Allelic replacement mutagenesis of licCA

To determine whether LicCA was essential for phosphati-dylcholine biosynthesis in T denticola licCA was dis-rupted by allelic replacement mutagenesis The strategyfor licCA mutagenesis is shown in Fig 6 Briefly a 693 bpBstZ17I-BclI fragment including the 5cent end of licCA inpSY107 was replaced with a 21 kb ermFermB cassetteThe vector sequence was released by restriction enzymedigestion and T denticola was transformed by electropo-ration with the linearized disrupted licCA Six erythromy-cin-resistant isolates were recovered of which two hadthe desired allelic replacement Construction of theisogenic mutant designated T denticola LBE3 was con-

Fig 3 Phospholipid composition of parent and mutant T denticola Levels of major phospholipids were determined as indicated in Exper-imental procedures Data for T denticola 35405 (open bars) and isogenic mutant LBE3 (shaded bars) are the averages and standard deviations from five samples Phospholipids are phosphatidylcholine (PC) phosphatidylethanolamine (PE) phosphatidylglycerol (PG) and total phospholipids (TL)

Fig 4 Incorporation of [14C]choline into lipids in parent and mutant T denticola Parent (35405 solid circles) and two clonal isolates of mutant (LBE Xcents and solid triangles) cells were grown to log phase then 2 mCi ml-1 [14C]choline were added to the culture medium Cells were harvested at the indicated times and processed as described in Experimental procedures Total radioactivity in the lipid extract normalized for the total cellular protein is reported

Fig 5 Analysis of purified recombinant proteins Purified LicCA (lane 2) C308 (lane 3) and A317 (lane 4) were subjected to SDS-PAGE Lane 1 contained molecular weight standards

Table 1 Enzymatic activities of LicCA and fragments

Recombinantprotein

Cytidylyltransferaseactivity (nmol min-1

mg protein-1)Choline kinase Activity (nmol min-1 mg protein-1)

LicCA 813 plusmn 9 228 plusmn 6C308 lt1 NAA317 NA 1100 plusmn 96

NA not applicableValues are the averages and ranges of determinations from twoseparate protein preparations

T denticola phosphatidylcholine pathway 475


firmed by PCR analysis using oligonucleotide primersCX226 and CX227 As expected the amplicon from LBE3was approximately 14 kb larger than that of the parentstrain confirming the double crossover homologousrecombination event Quantitative RT-PCR using a primerset within licCA but downstream of the insertion site dem-onstrated that licCA mRNA was present in 35405 and wasabsent in LBE3 Similarly QRT-PCR using a primer setwithin the open reading frame directly downstream oflicCA in the T denticola genome sequence showed thatthis gene was transcribed in 35405 but was not tran-scribed in LBE3 The polar effect of ermFermB insertionin T denticola is consistent with previous mutagenesisstudies (Lee et al 2002) (data not shown)

The parent and mutant strains displayed no obviousphenotypic differences Growth rates and final densitiesof broth cultures of the parent 35405 and mutant LBE3were not significantly different Cell morphology and motil-ity under phase-contrast microscopy were similar Expres-sion of membrane-associated proteins including MspPrcA and OppA was not altered in LBE3 compared with35405 Peptidase activities of T denticola parent andmutant strains as tested by hydrolysis of chromogenicsubstrates SAAPFNA and BApNA were indistinguishable(data not shown)

Lipid metabolism in the licCA mutant

Targeted disruption of the licCA gene completely elimi-nated incorporation of [14C]choline into lipids and 32Pi intophosphatidylcholine (Figs 4 and 7A) In addition incorpo-ration of [14C]choline into phosphocholine and CDP-choline were also eliminated (Fig 7B) as would beexpected for a licCA disruption In the mutant levels ofsoluble [14C]choline were reduced by about 40 comparedto the parent strain This may be due to absence of tran-scription in LBE3 of the open reading frame downstreamof licCA which has homology with predicted carnitinecholine or glycine betaine transporters (data not shown)

Because T denticola was grown in the presence ofserum it was possible that the mutant was using serumlipoproteins as a source for either PC or lysoPC whichthen might be acylated to form PC Separation and quan-tification of lipid mass however revealed that the mutantcontained little if any PC (Fig 3)

To determine if the levels and biosynthetic rates ofother phospholipids were altered in the mutant a timecourse of 32Pi incorporation was carried out (Fig 8) Asexpected essentially no 32Pi was incorporated into PC inthe mutant The rate of incorporation of 32Pi into PG wasincreased by about 50 although there was not a signif-

Fig 6 Strategy for mutagenesis of licCA Plas-mid pSY107 which contained the original licCA product plus 5cent and 3cent flanking sequences was digested with BclI and BstZ17I to remove a 693 bp fragment of licCA including the 5cent end of the gene The 21 kb ermFermB cassette was isolated from BamHI-PmlI digested pSY118 and ligated to the previously digested pSY107 In the resulting plasmid pSY131 the ermFermB cassette is in opposite transcrip-tional orientation to the disrupted licCA gene pSY131 was digested to completion with SphI and PvuII to release the vector sequence Tre-ponema denticola 35405 was electroporated with the resulting linear DNA resulting in allelic replacement of native licCA Arrows show tran-scriptional orientation of licCA (black) ermFermB (hatched) and predicted open reading frames adjacent to licCA in T denticola (grey)

476 C Kent et al


icant increase in the mass of PG (Fig 3) The rate ofincorporation of 32Pi into PE in the mutant was increasedby 27-fold (Fig 8) and the level of PE mass wasincreased by twofold (Fig 3) Thus it appears that themutant compensates for the lack of PC primarily byincreasing PE levels

Discussion

The results presented here show that T denticola con-tains phosphatidylcholine as a major phospholipid andhas a choline-dependent mechanism for phosphatidyl-choline biosynthesis This mechanism is dependent onan intact licCA gene implying that the two enzymaticfunctions of this gene choline kinase and CCT partici-pate in the T denticola pathway for phosphatidylcholinebiosynthesis Thus it appears that this bacterium uses aCDP-choline pathway for PC biosynthesis which has notpreviously been reported for bacteria Although we can-not definitively rule out the possibility that the LicCAprotein only indirectly governs PC biosynthesis in T den-ticola it is much more likely that the LicCA choline kinaseand CCT activities directly participate in the CDP-cholinepathway Strong evidence for these conclusions comesfrom the disruption of licCA which prevents conversion oflabelled precursors to phosphocholine and PC and elim-inates all detectable PC from this organism Thus thereis no evidence for PC biosynthesis by either sequential

methylation of PE or by a choline-dependent PCsynthase activity in the licCA mutant and the preliminaryT denticola genome sequence does not appear to con-tain genes encoding key enzymes required for thesepathways

The licC and licA genes were originally characterizedin H influenza (Weiser et al 1997) and S pneumonia(Zhang et al 1999) where they are contained in anoperon as separate non-contiguous genes The twogenes are fused however in several organisms (Fig 2)The advantage of such a fusion is not clear It is possiblethat the proximity of the two active sites affords a moreefficient means of catalysis The low activity of the fusionprotein as expressed heterogeneously was surprisingconsidering the much higher activity of the recombinantlicC and licA gene products from S pneumonia (Camp-bell and Kent 2001 Rock et al 2001 H A Campbelland C Kent unpubl data) and eukaryotic cholinekinases (Porter and Kent 1990 Kim et al 1998 Geeand Kent 2003) Possibly the T denticola licCA geneproduct is post-translationally modified or cleaved invivo leading to a higher level of activity in the nativeform Our attempts to produce lsquocleavedrsquo recombinantproteins using molecular tools had mixed results TheA317 fragment had higher choline kinase activity thanrecombinant LicCA but the CCT activity of the C308fragment was much lower than that of recombinantLicCA Further studies are needed to quantify activity ofnative LicCA

Fig 7 Chromatography of radiolabelled precursors and lipidsA Cells were labelled in 5 mCi ml-1 32Pi for 4 h then 32P-labelled lipids from T denticola LBE3 (M) and 35405 parent (P) were prepared and separated by TLC in solvent system I and visualized by autoradiog-raphy Phospholipids are phosphatidylcholine (PC) phosphatidyleth-anolamine (PE) and phosphatidylglycerol (PG)B Cells were labelled in 25 mCi ml-1 [14C]choline for 6 h then soluble metabolites were prepared and separated by TLC and visualized by autoradiography Metabolites are choline (C) phosphocholine (P) and CDP-choline (Cc)

Fig 8 Incorporation of 32Pi into lipids in parent and mutant T denti-cola Parent and LBE3 were incubated with 5 mCi ml-1 32Pi for the indicated times in hours (h) Total lipids were prepared as described in Experimental procedures and separated by TLC in solvent system I before scraping and counting Closed symbols and solid lines par-ent open symbols and dashed lines LBE3 Circles are phosphatidyl-choline triangles are phosphatidylglycerol and squares are phosphatidylethanolamine



In addition to the licCA genes in T denticola and Tpallidum three other putative licCA genes have beenidentified by genomic sequencing one from Clostridiumperfringens and two from Fusobacterium nucleatum(Fig 2) LicCA from C perfringens and one of the LicCArsquosfrom F nucleatum are predicted to have the extendedamino terminal segment that we found necessary for sol-ubility and activity of recombinant LicCA in T denticolaThe other LicCA from F nucleatum however does notappear to have this extension Also of interest is that thetwo longer genes from C perfringens and F nucleatumare altered in a region known to be important for activityin the related GlmURmlA superfamily of nucleotidyltrans-ferases (Brown et al 1999 Blankenfeldt et al 2000)(Fig 2) The alterations leave these two proteins withoutthe critical residues Arg-86 and Lys-96 (numbering for Tdenticola) In the crystal structures of these nucleotidyl-transferases from E coli and S pneumoniae (Brown et al1999 Kwak et al 2002) these residues are found in aloop which might be flexible enough to allow other resi-dues in the altered shorter loop to carry out their functionAlternatively the proteins with the altered catalytic loopmay not have active CCT activity and there may be analternative function of this portion of the LicCA proteinsuch as binding phosphocholine Additional studies inthese organisms will be necessary to determine if thisregion is part of the translated C perfringens and Fnucleatum LicCA proteins and whether they have thesepotential activities

A CDP-choline pathway for PC biosynthesis would alsonecessitate the presence of a gene encoding a cholinephosphotransferase which catalyses the last step inwhich phosphocholine is transferred from CDP-choline todiacylglycerol (Fig 1) The TP0671 gene in T pallidum isquite similar to the choline- and ethanolaminephospho-transferases of yeast and humans with the highest simi-larity in the CDP-alcohol phosphotransferase motif knownto be part of the active site (Williams and McMaster1998) A sequence similar to TP0671 can be found in Tdenticola the Treponema homologues are 49 identicalto each other in the amino terminal half which containsthe active site (not shown) Thus it is reasonable to pro-pose that both of these treponemes encode a cholinephosphotransferase and would have a complete CDP-choline pathway for PC biosynthesis

Of the relatively small number of prokaryotes that syn-thesize PC most have specific symbiotic or pathogenicassociations with eukaryotic hosts In at least some ofthese microbes PC synthesized from host-derived cho-line appears to be important in either bacterial growth orfor specific microbendashhost interactions (Lopez-Lara andGeiger 2001) In T denticola PC does not appear to beessential under in vitro culture conditions and PC incor-poration was not a result of the uptake of exogenous PC

from serum in the medium In the absence of a functionallicCA gene this organism appears to compensate for thelack of PC by increasing its content of PE It is possiblethat this may be due a bias in nutrient availability in thecomplex culture medium required for growth of this organ-ism Some other PC-producing bacteria have the abilityto modulate membrane phospholipid expression inresponse to environmental conditions (Tang and Holling-sworth 1998 Hanada et al 2001 Russell et al 2002)This phenomenon is distinct from the phase-variableexpression of phosphocholine in H influenzae which isproduced from the LicC and LicA activity of this organism(Weiser et al 1997) but is consistent with the hypothesisthat production of PC or phosphocholine in variouseukaryote-associated bacteria is important for bacterialsurvival in these environments Further studies arerequired to understand the unique membrane physiologyof treponemes as well as the potential role of cholinemetabolites in the interaction between these organismsand host cells

Experimental procedures

Chemicals

Unless otherwise noted chemicals were purchased at thehighest available purity from Sigma Chemical Co (StLouis MO) or Fisher Scientific (Chicago IL) [14C-methyl]-choline [3H-methyl]-choline [14C-methyl]-phosphocholine[14C-methyl]-CDP-choline and 32Pi were from Amersham

Bacterial strains plasmids and growth conditions

Treponema denticola ATCC 35405 and isogenic mutantswere grown and maintained under anaerobic conditions inNOS broth medium as previously described (Haapasaloet al 1991) with erythromycin (40 mg ml-1) added as appro-priate For radiolabelling experiments anaerobic pouches(Mitsubishi Gas Chemical Company) were used For allelicreplacement mutagenesis mutants were selected on NOSGN plates (Chan et al 1997) containing erythromycin(40 mg ml-1) as described previously (Li et al 1996 Fennoet al 1998) Cultures were examined by phase-contrastmicroscopy for purity and typical strain morphology beforeuse

Escherichia coli strains JM109 and JM110 (Yanisch-Perronet al 1985) were used for routine subcloning and plasmidpreparations and E coli BL21(DE3) was used for re-combinant expression studies Plasmid vector pSTBlue-1(Novagen) was used for cloning of PCR fragments Plasmidvector pET21b (Novagen) was used for expression studiesThe ermFermB cassette from pVA2198 (Fletcher et al1995) was PCR-amplified using oligonucleotide primersCX247 and CX249 (Table 1) and ligated into the TA cloningsite of pSTBlue-1 to yield pSY118 which was used as thesource for ermFermB in constructing plasmids for allelicreplacement mutagenesis E coli strains were grown inLB broth or agar medium supplemented with ampicillin

478 C Kent et al


(50 mg ml-1) kanamycin (50 mg ml-1) chloramphenicol(34 mg ml-1) or erythromycin (200 mg ml-1) as appropriate

Labelling and analysis of phospholipids

For radiolabelling experiments the label was added at 1ndash5 mCi ml-1 to log phase T denticola cells which were thenaliquoted incubated in anaerobic pouches at 37infinC andharvested at the indicated times At harvest cells were cen-trifuged at 4000 gmax for 15 min at 4infinC The medium wasremoved and the pellet was washed by addition of 1 ml ofphosphate-buffered saline followed by gentle resuspensionand the centrifugation was repeated The final cell pelletwas resuspended in 10 ml H2O and aliquots were taken forlipid extraction and protein determination (Lowry et al1951)

Lipids were extracted by the Bligh-Dyer method (Bligh andDyer 1959) The aqueous fraction was evaporated to drynesswith air and the aqueous metabolites were separated bythin-layer chromatography (TLC) on silica gel G plates inmethanol05 NaClNH4OH 50501 14C-Labelled stan-dards were also chromatographed and standards as well asexperimental samples were visualized by autoradiographyRadioactive TLC spots were scraped into scintillation vialsthen suspended in 05 ml of H2O and 45 ml Ecolite (ICN)scintillation fluid Levels of radioactivity were determined in aBeckman liquid scintillation counter

The chloroform phase from the lipid extraction was washedby re-extracting with methanol and saline then evaporatedto dryness under N2 Lipids were dissolved in chloroformmethanol 21 and the lipid classes separated by TLC in threesystems I silica gel H plates developed in chloroform2-propanolmethyl acetatemethanol025 KClacetic acid2525251054 II silica gel H plates developed in chloro-formmethanolNH4OH 45454 III silica gel G plates in chlo-roformmethanolacetic acidH2O 503084 Non-radioactivestandards were visualized by exposure to iodine vapour andradioactive lipids were visualized by autoradiography

For determination of phospholipid mass organic phospho-rus was assayed as described (Ames 1967) Silica gel con-taining lipids was scraped from TLC plates and the lipids wereeluted from the silica gel in chloroformmethanolacetic acidH2O 503084 and methanol The solvents were then evap-orated before organic phosphorus determination

Enzymatic activity assays

Choline kinase (Gee and Kent 2003) and CCT (Morand andKent 1989) were assayed as described Peptidase activitiesof T denticola parent and mutant strains were tested byhydrolysis of chromogenic substrates succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-p-nitroanilide (SAAPFNA)and N-a-benzoyl-L-arginine-p-nitroanilide (BApNA) asdescribed previously (Fenno et al 2001)

Recombinant DNA methods

Unless stated otherwise standard methods found in Ausubelet al (1995) or Sambrook et al (1989) were followed DNAfragments were eluted from agarose gels using the GeneClean II kit (QBiogene La Jolla CA) Genomic DNAcents andplasmid DNAcents were isolated using the Wizard Genomic DNAPurification Kit and Wizard Plus SV Minipreps Kit (PromegaMadison WI) respectively Oligonucleotide primers (Invitro-gen Carlsbad CA) were designed using the GeneFisheralgorithm (Giegerich et al 1996) or Primer Express software(Perkin-Elmer Applied Biosystems)

Cloning of licCA

The licCA region identified in preliminary unannotated con-tigs of the T denticola genome sequence (httpwwwtigrblasttigrorgufmg) was amplified from T denticolagenomic DNA with oligonucleotide primers CX226 andCX227 (Table 2) The 44 kb PCR product including approx-imately 13 kb upstream of licCA and 15 kb downstream ofthe licCA stop codon was cloned in pSTBlue-1 yieldingpSY107 For expression studies DNA fragments of interestwere cloned by PCR with Vent polymerase with pSY107 astemplate Fragments were cloned for optimal expression fromthe T7 promoter in pET21b Full-length licCA was cloned withforward primer EX10 and reverse primer EX11 (Table 2) ThelicC portion C308 was cloned as the first 308 residues ofthe licCA gene with EX10 as forward primer and EX12 asreverse primer The licA portion A317 was cloned with for-ward primer EX13 which added an ATG codon in front ofcodon 317 and was extended to the end of the licCA genewith reverse primer EX11 For expression plasmid-contain-ing cells were grown in LB media to an optical density of 08

Table 2 Oligonucleotide primers used in this study

Primer Targeta Sequence

CX226 5cent to licCA (F) 5cent dACC CAT ACC TGC TTC ATT C 3centCX227 Beyond licCA 3centend (R) 5cent dCTA CCT ATA CCC TCC GTT ATG 3centCX247 ermFermB 5centend (F) 5cent dGGC ATA TGC GAT AGC TTC CGC TAT TG 3centCX249 ermFermB 3centend (R) 5cent dGGC ATA TGA GCT GTC AGT AGT ATA CC 3centCX329 licCA (F) 5cent dGGA TGC CCG TAA TCC TGA AGA 3centCX330 licCA (R) 5cent dCGT TCA CGT AGA TCA AAA GAA TGC 3centCX323 16S rRNA (F) 5cent dAGG GAT ATG GCA GCG TAG CA 3centCX324 16S rRNA (R) 5cent dTTG CGG GAC TTA ACC CAA CA 3centEX10 licCA (F) 5cent dCG CGG ATC CAT ATG AAA AGA AGA TAT TTT CAA ATT ATA AAA CTT ATGEX11 licCA (R) 5cent dCCG CTC GAG TCA TAG ATT ACC TCC TAG TTC TTT TAT TTT TTT ATA ATA ATC cEX12 C308 (R) 5cent dCCG CTC GAG TCA CTC AAG CCA TTT ATC ATG GGA GGEX13 A317 (F) 5cent dcgc GGA TCC CAT ATG CCT GAG CAC AGC ATC

a Orientation of the primer (F forward R reverse) with respect to gene of interest



then induced with 1 mM isopropylthiogalactopyranosideLicCA was induced for 3 h at 37infinC and C308 and A317 wereinduced for 20 h at 25infinC Cell pellets were stored at -20infinC

Quantitative RT-PCR

Total RNA was extracted from cultures harvested duringactive growth (optical density at 600 nm of 02) using theRNeasy Mini Kit (Qiagen) RNA samples were reversed tran-scribed from random primers using SuperScriptTM First-Strand Synthesis System (Invitrogen) One microlitre of theresulting cDNA was amplified using QuantiTectTM SYBRGreen PCR (Qiagen) in a 25 ml reaction using licCA primersCX329 and 330 Amplification of 16SrRNA with primersCX323 and 324 served as an internal control (Table 2) Ther-mal cycling was performed in an iCycler iQTM Multi-Color RealTime PCR Detection System (Bio-Rad) at 95infinC for 15 minand followed by 40 cycles of 94infinC for 30 s 50infinC for 30 s and72infinC for 30 s

DNA sequence analysis

DNA sequencing of expression plasmids was performed bythe Molecular Biology Core University of Michigan MedicalCenter Analysis of DNA sequence data was performed usingSeqEd 10 (Applied Biosystems) and DNA Strider 13(Service de Biochimie Department de Biologie Institut deRecherche Fondamentale Commissariat a lrsquoEnergieAtomique Saclay France) The non-redundant SWISS-PROT PIR EMBL and GenBank databases were searchedfor homologous peptide and nucleotide sequences using theBLAST (Altschul et al 1990) network service at the NationalCenter for Biotechnology Information National Institutes ofHealth USA

Allelic replacement mutagenesis

Isogenic defined mutants were constructed as described pre-viously (Lee et al 2002) Briefly T denticola was electropo-rated with linear DNA consisting of the selectable ermFermBgene cassette (Fletcher et al 1995) cloned between frag-ments of the target sequence DNA fragments to be intro-duced into T denticola were UV-irradiated at 25 mJcm asdescribed by Picardeau et al (2001) before electroporationFollowing 24 h recovery in NOS broth mutants were selectedby growth in NOSGN agar containing erythromycin(40 mg ml-1)

Protein production and purification

All procedures were performed at 4infinC Cell pellets from250 ml cultures were resuspended in 20 ml of lysis buffer(10 mM Tris-HCl pH 75 30 mM NaCl 2 mM dithiothreitol02 mM EDTA 25 mg ml-1 leupeptin 2 mg ml-1 chymostatin2 mg ml-1 pepstatin 1 mg ml-1 antipain 10 mg ml-1 p-aminobenzamidine 10 mg ml-1 benzamidine and 02 mMphenylmethylsulphonylfluoride) The cells were disrupted bythree 30 s intervals of sonication The cell lysate was centri-fuged at 330 000 g for 25 min to produce a cleared lysate

The LicCA cleared lysate was passed over a 2-mlCM-Sepharose column (Pharmacia) equilibrated with bufferD (20 mM Tris-HCl pH 75 2 mM EDTA and 1 mM 2-mercaptoethanol) and eluted with 05 M KCl in buffer D Theprotein was diluted 200-fold with buffer D then passed overa 1-ml Blue-Sepharose column equilibrated with buffer D andeluted with 1 M KCl in buffer D The C308 cleared lysate waspassed over a 05-ml TMAE column (EM Science) were equil-ibrated with buffer D The flow-through was collected andsaturated ammonium sulphate was added to 60 saturationThe precipitate was removed by centrifugation for 10 min at18 000 g then the supernatant was brought to 75 satura-tion and the precipitation repeated The 75 pellet was dis-solved in buffer D and the C308 protein was further purifiedby gel filtration on Sephacryl-S100 The A317 cleared lysatewas passed over a 1-ml Decyl-Sepharose column (Pharma-cia) equilibrated with buffer D containing 1 M KCl and elutedin 100 ml buffer D The eluate was passed over a 05-mlTMAE column and eluted with 05 M KCl in buffer D In initialpurification of each protein fractions containing significantprotein peaks were assayed by SDS-PAGE and enzymaticactivity to identify the active enzyme peak In subsequentpurifications the corresponding protein peaks were identifiedwithout assaying enzyme activity

Nucleotide sequence accession number

The nucleotide sequence of T denticola licCA has beenassigned GenBank accession number AY322155

Acknowledgements

This work was supported by Public Health Service grantsGM60510 (CK) from the National Institute of General Med-ical Sciences and DE13565 from the National Institute ofDental and Craniofacial Research (JCF) Partial support forSYL was provided by Kangnung National University KoreaThe authors thank Yu Ning for technical assistance

References

Albelo ST and Domenech CE (1997) Carbons from cho-line present in the phospholipids of Pseudomonas aerugi-nosa FEMS Microbiol Lett 156 271ndash274

Altschul SF Gish W Miller W Myers EW and LipmanDJ (1990) Basic local alignment search tool J Mol Biol215 403ndash410

Ames BN (1967) Assay of inorganic phosphate total phos-phate and phosphatases Methods Enz 10 115ndash118

Ausubel FM Brent R Kingston RE Moore DDSeidman JG Smith JA and Struhl K (eds) (1995)Current Protocols in Molecular Biology New York Wiley-Interscience

Barbieri JT Austin FE and Cox CD (1981) Distributionof glucose incorporated into macromolecular material byTreponema pallidum Infect Immun 31 1071ndash1077

Belisle JT Brandt ME Radolf JD and NorgardMV (1994) Fatty acids of Treponema pallidum andBorrelia burgdorferi lipoproteins J Bacteriol 176 2151ndash2157

480 C Kent et al


Blankenfeldt W Asuncion M Lam JS and Naismith JH(2000) The structural basis of the catalytic mechanism andregulation of glucose-1-phosphate thymidylyltransferase(RmlA) EMBO J 19 6652ndash6663

Bligh EG and Dyer WJ (1959) A rapid method of totallipd extraction and purification Can J Biochem Physiol 37911ndash917

Brown K Pompeo F Dixon S Mengin-Lecreulx DCambillau C and Bourne Y (1999) Crystal structure ofthe bifunctional N-acetylglucosamine 1-phosphate uridyl-transferase from Escherichia coli a paradigm for therelated pyrophosphorylase superfamily EMBO J 184096ndash4107

Campbell HA and Kent C (2001) The CTP phosphocho-line cytidylyltransferase encoded by the licC gene of Strep-tococcus pneumoniae cloning expression purificationand characterization (1) Biochim Biophys Acta 1534 85ndash95

Chan ECS DeCiccio A McLaughlin R Klitorinos Aand Siboo R (1997) An inexpensive solid medium forobtaining colony-forming units of oral spirochetes OralMicrobiol Immunol 12 372ndash376

Fenno JC Wong GW Hannam PM and McBride BC(1998) Mutagenesis of outer membrane virulence determi-nants of the oral spirochete Treponema denticola FEMSMicrobiol Lett 163 209ndash215

Fenno JC Lee SY Bayer CH and Ning Y (2001) TheopdB locus encodes the trypsin-like peptidase activity ofTreponema denticola Infect Immun 69 6193ndash6200

Fletcher HM Schenkein HA Morgan RM Bailey KABerry CR and Macrina FL (1995) Virulence of a Por-phyromonas gingivalis W83 mutant defective in the prtHgene Infection Immunity 63 1521ndash1528

Fraser CM Norris SJ Weinstock GM White O Sut-ton GG Dodson R et al (1998) Complete genomesequence of Treponema pallidum the syphilis spirocheteScience 281 375ndash388

Gee P and Kent C (2003) Multiple isoforms of cholinekinase from Caenorhabditis elegans cloning expressionpurification and characterization Biochim Biophys Acta1648 33ndash42

Giegerich R Meyer F and Schleiermacher C (1996)GeneFisher ndash software support for the detection of postu-lated genes Proc Int Conf Intell Syst Mol Biol 4 68ndash77

Haapasalo M Singh U McBride BC and Uitto V-J(1991) Sulfhydryl-dependent attachment of Treponemadenticola to laminin and other proteins Infect Immun 594230ndash4237

Hanada T Kashima Y Kosugi A Koizumi Y YanagidaF and Udaka S (2001) A gene encoding phosphatidyle-thanolamine N-methyltransferase from Acetobacter acetiand some properties of its disruptant Biosci BiotechnolBiochem 65 2741ndash2748

Kaneshiro T and Law JH (1964) Phosphatidylcholine syn-thesis in Agrobacterium tumefaciens I Purification andproperties of a phosphatidylethanolamine N-methyltrans-ferase J Biol Chem 239 1705ndash1713

Kim KH Voelker DR Flocco MT and Carman GM(1998) Expression purification and characterization ofcholine kinase product of the CKI gene from Saccharomy-ces cerevisiae J Biol Chem 273 6844ndash6852

Kwak BY Zhang YM Yun M Heath RJ Rock COJackowski S and Park HW (2002) Structure and mech-anism of CTP phosphocholine cytidylyltransferase (LicC)from Streptococcus pneumoniae J Biol Chem 277 4343ndash4350

Lee SY Bian XL Wong GW Hannam PM McBrideBC and Fenno JC (2002) Cleavage of Treponemadenticola PrcA polypeptide to yield protease complex-associated proteins Prca1 and Prca2 is dependent onPrtP J Bacteriol 184 3864ndash3870

Li H Ruby J Charon N and Kuramitsu H (1996) Geneinactivation in the oral spirochete Treponema denticolaconstruction of an flgE mutant J Bacteriol 178 3664ndash3667

Livermore BP and Johnson RC (1974) Lipids of theSpirochaetales comparison of the lipids of several mem-bers of the genera Spirochaeta Treponema and Lep-tospira J Bacteriol 120 1268ndash1273

Lopez-Lara IM and Geiger O (2001) Novel pathway forphosphatidylcholine biosynthesis in bacteria associatedwith eukaryotes J Biotechnol 91 211ndash221

Lowry OH Rosebrough NJ Farr AL and Randall RJ(1951) Protein measurement with the folin phenol reagentJ Biol Chem 193 265ndash275

Lysenko E Richards JC Cox AD Stewart A MartinA Kapoor M and Weiser JN (2000) The position ofphosphorylcholine on the lipopolysaccharide of Haemophi-lus influenzae affects binding and sensitivity to C-reactiveprotein-mediated killing Mol Microbiol 35 234ndash245

Matthews HM Yang TK and Jenkin HM (1979) Uniquelipid composition of Treponema pallidum (Nichols virulentstrain) Infect Immun 24 713ndash719

Morand JN and Kent C (1989) Localization of the mem-brane-associated CTP phosphocholine cytidylyltrans-ferase in Chinese hamster ovary cells with an alteredmembrane composition J Biol Chem 264 13785ndash13792

Picardeau M Brenot A and Saint Girons I (2001) Firstevidence for gene replacement in Leptospira spp Inactiva-tion of L biflexa flaB results in non-motile mutants deficientin endoflagella Mol Microbiol 40 189ndash199

Porter TJ and Kent C (1990) Purification and character-ization of cholineethanolamine kinase from rat liver J BiolChem 265 414ndash422

Rock CO Heath RJ Park HW and Jackowski S(2001) The licC gene of Streptococcus pneumoniaeencodes a CTP phosphocholine cytidylyltransferase JBacteriol 183 4927ndash4931

de Rudder KE Sohlenkamp C and Geiger O (1999)Plant-exuded choline is used for rhizobial membrane lipidbiosynthesis by phosphatidylcholine synthase J Biol Chem274 20011ndash20016

Russell NJ Coleman JK Howard TD Johnston E andCogdell RJ (2002) Rhodopseudomonas acidophila strain10050 contains photosynthetic LH2 antenna complexesthat are not enriched with phosphatidylglycerol and thephospholipids have a fatty acyl composition that is unusualfor purple non-sulfur bacteria Biochim Biophys Acta 1556247ndash253

Sambrook J Fritsch EF and Maniatis T (1989) Molecu-lar Cloning a Laboratory Manual 2nd edn Cold SpringHarbor New York Cold Spring Harbor Laboratory

Smibert RM (1976) Cultivation composition and physiol-



ogy of avirulent treponemes In The Biology of ParasiticSpirochetes Johnson RC (ed) New York AcademicPress pp 49ndash56

Sohlenkamp C Lopez-Lara IM and Geiger O (2003)Biosynthesis of phosphatidylcholine in bacteria Prog LipidRes 42 115ndash162

Tang Y and Hollingsworth RI (1998) Regulation of lipidsynthesis in Bradyrhizobium japonicum low oxygen con-centrations trigger phosphatidylinositol biosynthesis ApplEnviron Microbiol 64 1963ndash1966

Weiser JN Shchepetov M and Chong ST (1997) Dec-oration of lipopolysaccharide with phosphorylcholine aphase- variable characteristic of Haemophilus influenzaeInfect Immun 65 943ndash950

Williams JG and McMaster CR (1998) Scanning alaninemutagenesis of the CDP-alcohol phosphotransferase motifof Saccharomyces cerevisiae cholinephosphotransferaseJ Biol Chem 273 13482ndash13487

Yanisch-Perron C Vieira J and Messing J (1985)Improved M13 phage cloning vectors and host strainsnucleotide sequences of the M13mp18 and pUC19 vec-tors Gene 33 103ndash119

Zhang JR Idanpaan-Heikkila I Fischer W andTuomanen EI (1999) Pneumococcal licD2 gene isinvolved in phosphorylcholine metabolism Mol Microbiol31 1477ndash1488

472

C Kent

et al



51

471ndash481

been proposed to be

licD

(Zhang

et al

1999 Lysenko

et al

2000)Genomic analysis has revealed that several bacteria

appear to encode a fusion protein in which a

licC

-encodedCCT is the amino terminal half and a

licA

-encoded cholinekinase is the carboxyl terminal half (Fig 2) We refer tothis fusion gene as

licCA

The

licCA

-containing organismsinclude

Treponema pallidum

which causes syphilis

Fuso-bacterium nucleatum

which is involved in periodontaldisease and


which causes gan-grene Although little information is available on phospho-lipid synthesis and composition in fusobacteria andclostridia it is of interest that the treponemes have beenreported to contain PC as a major membrane phospho-lipid (Livermore and Johnson 1974 Matthews

et al

1979 Barbieri

et al

1981 Belisle

et al

1994) The pres-ence of the

licCA

fusion gene in treponemes suggests thatthey may utilize a CDP-choline pathway for biosynthesisof PC In this report we show that

T denticola

possessesa CDP-choline pathway for PC biosynthesis and that a

licCA

gene similar to that of

T pallidum

encodes proteinsin that pathway This is the first report of a CDP-cholinepathway for PC biosynthesis in bacteria

Results

Phosphatidylcholine in

T denticola

Several species of

Treponema

including

T denticola

have been reported to contain PC and several other

phospholipids including PE and PG (Livermore andJohnson 1974 Smibert 1976 Barbieri

et al

1981) butthe amount of each phospholipid in

T denticola

has notpreviously been reported quantitatively In order to con-firm the presence of and measure the amount of PC in

Tdenticola

total lipids were extracted and chromato-graphed by thin-layer chromatography in several differentsolvent systems The major phospholipids PC PEand phosphatidylglycerol (PG) were identified by co-chro-matography with known standards Phosphatidylcholineand PE were the most abundant phospholipids eachaccounting for about a third of total phospholipids whilePG was about 10 (Fig 3) Cardiolipin was a minor lipid(not shown)

Although the choline-containing sphingolipid sphingo-myelin has been reported to be a constituent of

Tpallidum

(Matthews

et al

1979) we found no evidencefor this lipid in

T denticola

either by measuring lipidphosphate or by incorporation of [

14

C]-choline or

32

Piinto a lipid that co-chromatographed with authenticsphingomyelin

If

T denticola

were using a CDP-choline pathway forPC biosynthesis one would expect that this lipid would bespecifically labelled after incubating the bacteria with radi-olabelled choline Indeed

T denticola

had a robust sys-tem for uptake and incorporation of either [

3

H]choline (notshown) or [

14

C]choline (Fig 4) into lipids Separation bythin-layer chromatography and quantification of the indi-vidual lipids from such an experiment revealed that atleast 96 (

3

H) or 99 (

14

C) of the radioactivity in the totallipid extract was in PC

Cloning and expression of

licCA

and enzymatic activity of the recombinant protein

The

licCA

gene was identified in preliminary

T denticola

genome sequence contigs based on similarity to the pre-dicted

licCA

gene of

T pallidum

(Fraser

et al

1998) The

T denticola licCA

DNA sequence was found to be identi-cal to that shown in the preliminary genomic contigs (datanot shown) The original cloned fragment in pSY107 wasdesigned to contain about 1300 base pairs upstream ofthe translation start site predicted from the

T pallidumlicCA

sequence (

79

MAAGFGShellip Fig 2) Recombinantexpression of a

T denticola

construct that initiated at thistranslation start site however resulted in a protein thatwas insoluble and inactive Inspection of the sequencesupstream of this predicted start site revealed another pos-sible translation initiation site which added 78 residues tothe protein sequence The

T pallidum

and

T denticola

sequences were 45 identical within this segmentExpression of a construct designed to initiate at this newsite (

1

MKRRYFhellip Fig 2) resulted in a protein that wassoluble and active The protein was purified to near homo-

Fig 1

CDP-choline pathways for biosynthesis of phosphatidylcholine and glycoconjugate-linked phosphocholine The CDP-choline path-way for phosphatidylcholine biosynthesis is found in animals plants yeasts and other eukaryotes The CDP-choline pathway for addition of phosphocholine to glycoconjugates has been demonstrated in

H influenzae

and

S pneumoniae

and is presumably present in other bacteria containing the

lic

genes In organisms containing the

lic

genes choline kinase is the product of

licA

cholinephosphate cyti-dylyltransferase is the product of

licC

and the cholinephosphotrans-ferase that transfers phosphocholine to the complex oligosaccharide is the presumed product of

licD

T denticola


473



51

471ndash481



S pneumoniae

(Campbell and Kent

2001 Rock

et al


licC


licA


Fig 2


CLUSTALW

algorithm in the

MEGALIGN


Discussion


Treponema denticola

(AY322155) Tp

Treponema pallidum

(NP_218547) Fn




(NP_561543) Sp




474 C Kent et al










Recombinantprotein














476 C Kent et al



Discussion













Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al










































T denticola


473



51

471ndash481



S pneumoniae

(Campbell and Kent

2001 Rock

et al


licC


licA


Fig 2


CLUSTALW

algorithm in the

MEGALIGN


Discussion


Treponema denticola

(AY322155) Tp

Treponema pallidum

(NP_218547) Fn




(NP_561543) Sp




474 C Kent et al










Recombinantprotein














476 C Kent et al



Discussion













Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al










































474 C Kent et al










Recombinantprotein














476 C Kent et al



Discussion













Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al



















































476 C Kent et al



Discussion













Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al










































476 C Kent et al



Discussion













Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al

















































Chemicals





478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al










































478 C Kent et al












Cloning of licCA









Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al













































Quantitative RT-PCR











Acknowledgements


References







480 C Kent et al










































480 C Kent et al



















































Documents

A CDP-choline pathway for phosphatidylcholine biosynthesis in Treponema denticola