Journal of Bacteriology, March 2004, p . 1239-1248, Vol . 186, No . 5

Identification and Functional Characterization of the Lactococcus lactis rfb Operon, Required for dTDP-Rhamnose Biosynthesis

Ingeborg C . Boels,^1,2, Marke M . Beerthuyzen,^1,2 Marit H . W . Kosters,^1,2, Martijn P . W . Van Kaauwen,^1,2, Michiel Kleerebezem,^1,2* and Willem M . de Vos¹

Wageningen Centre for Food Sciences, Wageningen,¹ NIZO Food Research, Flavor, Nutrition and Ingredients Section, Ede, The Netherlands²

Received 10 July 2003/ Accepted 4 November 2003

 
dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides [EPS] in Lactococcus lactis . We cloned the rfbACBD operon from L . lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L . lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenousRfbAC activities control the intracellular dTDP-rhamnose biosynthesisrate . However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels . A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L . lactis. Limitation of RfbBD activities resulted in the production of altered EPS . The monomeric sugar of the altered EPS consistedof glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2,which is clearly different from the ratio in the native sugar.Biophysical analysis revealed a fourfold-greater molecular massand a twofold-smaller radius of gyration for the altered EPS,indicating that these EPS are more flexible polymers with changedviscosifying properties . This is the first indication that enzymeactivity at the level of central carbohydrate metabolism affectsEPS composition.

 
Bacterial polysaccharides can be present in the cell wall as components of the cell envelope . Information about the structureand biosynthesis pathway of these compounds is fragmented . Glucose, galactose, mannose, N-acetylglucosamine, N-acetylmannose, and rhamnose are often found to be constituents of cell wall polysaccharides [8, 41].

Rhamnose is a 6-deoxyhexose sugar which is widely distributedin O antigens of gram-negative bacteria as part of the lipopolysaccharide [LPS] [43] . Furthermore, this compound is often found in capsularpolysaccharides [CPS], which are covalently bound to the cellwall, and in exopolysaccharides [EPS], which are loosely associatedwith the cell wall . L-dTDP-rhamnose is the sugar-nucleotideprecursor of these rhamnose moieties and is formed in a four-stepreaction from glucose 1-phosphate . The reaction involves theenzyme activities of glucose-1-phosphate thymidylyl transferase,dTDP-glucose-4,6-dehydratase, dTDP-4-keto-L-rhamnose-3,5-epimerase, and dTDP-L-rhamnose synthase encoded by the genes that are commonlydesignated rfbABCD, respectively . These genes have been foundin several gram-negative bacteria, including Escherichia coli[23], Salmonella enterica [30], Xanthomonas campestris [25], and Shigella flexneri [35] . Various rfb mutant strains havebeen described, and the mutations have various effects on therhamnose contents of the cell wall polysaccharides produced,including a loss of O antigen production [31], a reduced levelof LPS production [23], or production of LPS with a reducedamount [25] or complete lack [29] of rhamnose.

In gram-positive bacteria rfb homologues, designated rml genes in Streptococcus mutans [49, 50] and cps genes in Streptococcuspneumoniae [15, 20], have been characterized, and these homologues appear to play an essential role in the production of serotype-specific, rhamnose-containing CPS antigens . In S . mutans, rml mutationsresulted in a change in the composition of the cell wall polysaccharide,which lacked rhamnose, and in a complete lack of productionof the serotype-specific O antigen [49, 50] . S . pneumoniae cps19fLand cps19fN mutants exhibited a so-called rough phenotype anddid not have the capacity to produce CPS, indicating that therfb analogues play an essential role in CPS-19F production [38].

Various lactic acid bacteria, including lactobacilli [7, 11,19, 59], streptococci [45], and lactococci [33, 46], characteristicallycontain rhamnose in their cell walls . Lactococcal cell wallpolysaccharides decorate the peptidoglycan network [for a reviewsee reference 8], and rhamnose is one of the major componentsof these sugar polymers [33, 46] and has been suggested to bethe primary binding site for certain bacteriophages [for a recentreview see reference 14] . Moreover, it is also a component of the EPS produced by L . lactis SBT0495 [40], NIZO B40 [53, 56],and NIZO B39 [54] . Since EPS-producing lactic acid bacteriaare used in the food industry, in which the EPS produced insitu determines dairy product properties like texture, EPS couldprovide a potential new source for food-grade biothickeners[12].

Here we describe cloning and functional analysis of the rfb operon involved in dTDP-L-rhamnose biosynthesis in L . lactis,including complementation of an E . coli rfb mutant, the effectsof homologous overexpression of the rfb genes in L . lactis ondTDP-rhamnose synthesis, and the impact of rfb expression modulationon the production of rhamnose-containing lactococcal EPS . Therfb operon is essential for growth of L . lactis, as shown byusing an rfbBD conditional mutant . In addition, the rfbBD conditional mutant produced an altered EPS with novel physical characteristics.

 
Bacterial strains, plasmids, and media. The lactococcal strains and plasmids used in this study arelisted in Table 1. E . coli MC1061 [6], which was used as a host in cloning experiments, was grown with aeration in tryptoneyeast extract broth at 37°C [44] . L . lactis was grown withoutaeration at 30°C in M17 broth [Merck, Darmstadt, Germany] supplemented with 0.5% [wt/vol] glucose or in a chemically defined medium [32] . When appropriate, the media contained chloramphenicol[10 µg ml^-1], erythromycin [10 µg ml^-1], tetracycline[2.5 µg ml^-1], kanamycin [25 µg ml^-1], or ampicillin[100 µg ml^-1] . To analyze the effect of gene overexpression,the nisin-controlled expression system was used [9, 26] . Briefly,for the overexpression studies L . lactis cells were grown untilthe optical density at 600 nm was approximately 0.5 and thensplit into two cultures . One nanogram of nisin per milliliterwas added to one of the two cultures, and both cultures weregrown for an additional 2 h . For the studies in which the nisin-controlledconditional mutant was used, L . lactis cells were grown overnightin medium containing different levels of nisin and were subsequentlysubcultured in medium lacking nisin for 25 h.

 
DNA manipulations and DNA sequence analysis. Small-scale isolation of E . coli plasmid DNA and standard recombinantDNA techniques were performed as described by Sambrook et al.[44] . Large-scale isolation of E . coli plasmid DNA for nucleotide sequence analysis was performed with JetStar columns by followingthe instructions of the manufacturer [Genomed GmbH, Bad Oberhausen, Germany] . Isolation and transformation of L . lactis DNA were performed as previously described [10].

Automated double-stranded DNA sequence analysis of both strands was performed with an ALFred DNA sequencer [Pharmacia Biotech, Roosendaal, The Netherlands] . Sequence reactions were performedwith an Autoread kit [Amersham Biosciences, Roosendaal, TheNetherlands], were initiated by using Cy5-labeled universaland reverse primers, and were continued with synthetic primerspurchased from Pharmacia Biotech in combination with fluorescein15-dATP by following the instructions of the manufacturer [PharmaciaBiotech] . Sequence data were assembled and analyzed by usingthe PC/GENE program, version 6.70 [Intelli-Genetics, Inc., MountainView, Calif.].

Construction of strains and plasmids. Cloning and characterization of the rfb operon from L . lactis MG1363 were performed prior to release of the L . lactis IL-1403 genome sequence [3] . To do this, an internal fragment of thelactococcal rfbA gene was amplified by PCR by using chromosomalDNA of L . lactis MG1363 [16] as a template and the degenerateprimers 5'-TAYGAYAARCCNATGATHTAYTAYCC-3' and 5'-RTGNGTNCCNGTRTCNARCCA-3' [where H is A, C, or T; N is A, C, G, or T; Y is C or T; andR is A or G], which were based on conserved regions in an alignment[PC/GENE package; Intelli-Genetics, Inc.] of amino acid sequencesof the RfbA analogues RmlA and CPS19FL from S . mutans [accessionno. D78182] and S . pneumoniae [U09239.1] . The 0.6-kb PCR product generated was cloned in pGEM-T [Promega, Leiden, The Netherlands], and sequence analysis revealed a continuous open reading frame [ORF] that was predicted to encode a protein exhibiting highsequence homology with RfbA proteins . The resulting plasmidwas used as a probe in Southern analysis . This fragment hybridizedwith a 3.8-kb SacI/EcoRI fragment and a 2.4-kb HindIII fragment of the L . lactis MG1363 chromosomal DNA, which were cloned in similarly digested pUC18 [60], yielding pNZ4104 and pNZ4106, respectively [Fig . 1A] . Sequence analysis of the inserts revealedthe presence of four ORFs . These ORFs were predicted to encodeproteins consisting of 289, 197, 350, and 300 amino acids that exhibited high sequence identity with RfbA, -C, -B, and -D homologues found in other gram-positive bacteria, including L . lactis IL-1403 [level of identity, >96%], S . pneumoniae [>71%], and S. mutans [>70%] . Sequence comparisons were performed by using the BLAST module at the CMBI web site [www.cmbi.kun.nl].

 
The rfbAC overexpression plasmid was constructed by cloninga 2.0-kb PstI-HindIII fragment of pNZ4106 containing rfbAC intosimilarly digested pNZ8048 [26], which yielded pNZ4115 [Fig.1A] . The rfbAC fragment cloned in pNZ4115 contained a putativetermination sequence upstream of the rfbA coding sequence thathampered expression of the rfbAC genes [data not shown] . Therefore,this sequence was deleted by using a PCR strategy . The 5' codingregion of the rfbA gene was amplified by using pNZ4115 as thetemplate DNA and primers 5'-AATGCAGGCTAATTAATTATGATTATGGAGGTCC-3'and 5'-AGCATATTGAAGATTGATACC-3' . The 223-bp PCR product generatedwas cloned into pNZ4115 digested with PstI-SacI by using the PstI restriction site introduced in the forward primer [underlined] and the SacI restriction site present in the PCR product generated, which yielded the functional rfbAC overexpression construct pNZ4116 [Fig . 1A; also see below] . The rfbACBD overexpressionplasmid was constructed by cloning the 3.8-kb SacI-EcoRI fragmentof pNZ4104, containing the 'rfbACBD ORFs, into PstI-HindIII-digested pNZ4116 after blunting of the EcoRI and HindIII sticky ends with the Klenow fragment, which yielded pNZ4117 [Fig . 1A] . TherfbB gene was amplified by PCR by using pNZ4104 as the templateDNA and primers 5'-CATGCCATGGCAACTGAATTTAAAAATATCGTTGTGACAG-3' and 5'-GCGCTCTAGAGCTAGGATTTCATCAGCAAATTTTGG-3' . The 1.1-kb PCR product generated was cloned into pNZ8048 [26] by using theNcoI and XbaI restriction sites that were introduced by theprimers [underlined], which yielded the rfbB overexpressionplasmid pNZ4118 [Fig . 1A] . Plasmid pNZ4116, pNZ4117, or pNZ4118was transformed into L . lactis strain NZ9000 [26] . All PCR productsthat were cloned in vectors were sequenced to verify that nomutations were accidentally introduced during PCRs . The EPS-producingcapacity was introduced into NZ9000 harboring pNZ4116, pNZ4118,or pNZ4117 by electroporation of plasmid pNZ4030 [56], which contained the B40 eps gene cluster.

To ascertain that the L . lactis rfbACBD operon encodes a functional dTDP-rhamnose biosynthesis pathway, a 3.9-kb ScaI-PstI fragmentof pNZ4117 [see below] was cloned into pK194 [21] . The resultingplasmid, pNZ4110, was transformed into E . coli S874 containingpPM2716 [35].

Several strategies were employed to knock out one of the rfb genes . In the first strategy, which was used to knock out the rfbA gene by single-crossover plasmid integration, a 1.2-kb PCR fragment containing the 3' end of RfbA and the 5' end ofRfbC was amplified by using primers 5'-TATCTATGATAAACCAATGATTTATTATC-3'and 5' GCCCAGTAATCATTAACCAG-3' and cloned into pGEMT [Promega].A 1.2-kb SphI-SpeI fragment from the resulting plasmid was cloned into pUC18Ery [56] . The resulting plasmid, pNZ4105, was transformedinto L . lactis NZ9000, but despite several attempts, no erythromycin-resistant[Ery^r] colonies were obtained . This result provided the firstsuggestion that integration of this plasmid into the rfb locuscould be lethal to L . lactis.

In the second strategy, which was used to knock out the rfbB gene by double-crossover gene replacement, an integration plasmid was constructed, which contained an erythromycin resistance gene cassette flanked by the up- and downstream regions of the rfbB gene . To do this, a 1.1-kb AccI-EcoRI fragment of pNZ4104 containing the rfbB downstream region was cloned into similarly digested pUC18Ery [56], which yielded pNZ4107 [Fig. 1A] . Subsequently,a 1.6-kb HindIII fragment of pNZ4104, containing the rfbB upstreamregion, was cloned into similarly digested pNZ4107, yieldingpNZ4108 . To facilitate direct double-crossover transformantselection, an additional selection marker, tetR, which was isolatedas an SmaI-Ecl136 fragment from pGhost8 [37], was cloned intothe SmaI restriction site of pNZ4108 . The resulting plasmid,pNZ4109, was transformed into L . lactis NZ9000 . No double-crossover transformants were obtained, which supported the postulatedessential role of the rfb genes in L . lactis.

In the third strategy, which was used to knock out the rfbB gene by double-crossover gene replacement, plasmid pNZ4109 was transformed into L . lactis NZ9000 harboring pNZ4118 . The latter plasmid contains a copy of a functional rfbB gene fused to the inducible nisA promoter . Ery^r colonies were screened by replicaplating on GM17 plates containing tetracycline or erythromycinand 1 ng of nisin ml^-1 . The addition of nisin was importantto generate expression of the rfbB gene from pNZ4118 . In contrastto the first two strategies, Ery^r Tet^s integrants were obtainedonly in L . lactis NZ9000 harboring pNZ4118 . Southern analysisconfirmed that all of the Ery^r Tet^s integrants contained a disruptedcopy of the rfbB gene on the chromosome, and one colony, designatedNZ4109, was selected for further analysis.

Since the rfbB gene could be disrupted only when another copy of the rfbB gene was present in trans, we used a fourth strategyto construct a nisin-controlled conditional rfbBD mutant . Inthis mutant expression of rfbBD was placed under control ofthe tightly controlled nisA promoter, while the rfbAC geneswere constitutively expressed . This genetic organization allowednisin-controlled modulation of rfbBD expression, including theshutting down of rfbBD expression by removal of nisin from thegrowth medium, which led to development of the rfbBD mutantphenotype in this strain . For construction of this nisin-controlledconditional rfbBD mutant, a 1.4-kb StuI-SacI fragment of pNZ4118,containing a chloramphenicol [cat] gene-derived terminationsequence that originated from cloning vector pNZ8048 [26], the nisA promoter, and the rfbB gene, was cloned in pUC19 [60] digestedwith SmaI and SacI, which yielded pNZ4111 . In pNZ4111 the 1.5-kbHindIII-EcoRI fragment of pNZ4104, containing a 'rfbAC fragment,was cloned, which yielded pNZ4112 . To facilitate direct double-crossoverselection, we cloned two resistance markers in pNZ4112 . An erythromycinresistance gene cassette was isolated as a 1.2-kb HindIII-KpnIfragment from pUC18Ery [56] and cloned in SmaI-KpnI-digested pNZ4112, after the HindII site was filled with the Klenow fragment. In the resulting plasmid, pNZ4113, a second selection marker, tetR, was cloned . Therefore, a SmaI-Ecl136 fragment from pGhost8[37] was cloned [after the cohesive ends were filled by usingthe Klenow fragment] in the HindIII restriction site of pNZ4113.The resulting plasmid, pNZ4114, was transformed into L . lactisNZ9000 . Integrants were primarily selected on plates containingnisin and tetracycline . After this the integrants were screenedfor erythromycin resistance by replica plating . The desiredTet^r Ery^s colonies that were obtained were further analyzedby Southern analysis, and a single colony, designated NZ4114,was selected . This integrant contained the rfbAC coding sequence,followed by the desired integration of the tetracycline resistancegene cassette, the cat gene-derived termination sequence, andthe nisA promoter followed by the rfbBD coding sequence [Fig.1B].

Preparation of CEs and protein analysis. Lactococcal cells [50 ml] were harvested by centrifugation [3,500 x g, 10 min, 4°C], and the cell pellets were suspended in1 ml of 20 mM sodium phosphate buffer [pH 6.5] containing 50mM NaCl, 10 mM MgCl₂, and 1 mM dithiothreitol . The suspensionswere mechanically disrupted by bead beating in the presenceof zirconium beads [55], and cell debris was removed by centrifugation[3,500 x g, 10 min, 4°C] . The protein content of the cellextract [CE] was determined by the method of Bradford [4] byusing bovine serum albumin as the standard.

Each lactococcal CE was mixed with an equal amount of twofold-concentrated Laemmli buffer, and after boiling, 15 µg of each samplewas analyzed by sodium dodecyl sulfate [SDS]-10% polyacrylamidegel electrophoresis [PAGE] [27].

Northern, Southern, and Western blot analyses. Southern blots were hybridized at 65°C with homologous DNAprobes, which were labeled by nick translation by using establishedprocedures [44], and the blots were subsequently washed witha solution containing 0.015 M NaCl and 0.0015 M sodium citrateat 65°C before exposure.

RNA was isolated from L . lactis cultures, and Northern blot analysis was performed as described by Luesink et al . [34]. The blots were probed with internal fragments of the rfbA and rfbD genes . The internal fragment of the lactococcal rfbA genewas isolated as a 0.4-kb EcoRV-AflII fragment from pNZ4105,and the internal fragment of the lactococcal rfbD gene was isolatedas a 0.7-kb EcoRI-HindIII fragment from pNZ4104.

For Western blot analysis of E . coli, protein samples were prepared by harvesting 1 ml of a cell culture and then resuspending it in 100 µl of distilled water . Subsequently, the resuspendedpellet was mixed with and equal volume of Laemmli buffer andboiled for 3 min, and 10 µl of the resulting suspensionwas applied to an SDS-PAGE gel [27] . Proteins were electrophoretically transferred from SDS-PAGE gels onto nitrocellulose filters [Schleicher and Schuell, Dassel, Germany] [48] by using electroblot equipmentaccording to the instructions of the manufacturer [LKB 2051 Midget Multiblot] . The filters were probed with rabbit antiserum raised against S . flexneri O antigen [Sifin, Berlin, Germany], used at a dilution 1:2,500 . Primary, O antigen-bound antibodies were detected by using goat anti-rabbit peroxidase-conjugated antibodies at a dilution of 1:5,000 and a peroxidase-specific reaction performed according to the instructions of the manufacturer [Pierce, Rockford, Ill.].

Enzyme assays. Enzyme reactions were performed at 30°C in 1-ml [total volume]mixtures by using freshly prepared CEs at various concentrations.The formation of NAD[P][H] was determined by measuring the changein absorbance at 340 nm . The values given below are the meansof at least two independent measurements . Each blank containedthe reaction buffer, cofactors, and the substrate but lackedthe CE.

The glucose-1-phosphate thymidylyl transferase [RfbA; EC 2.7.7.24] reverse reaction assay was based on the assay described by Bernstein [1] . The reaction mixture contained 50 mM Tris-HCl buffer [pH 7.8], 8 mM MgCl₂, 0.3 mM NADP⁺, 2.1 U of -phopsphoglucomutase, 4 U of glucose-6-phosphate dehydrogenase, 4 mM inorganic phosphate, and CE . The reaction was started by addition of 0.1 mM dTDP-glucose. One RfbA activity unit [U_RfbA] was defined as 1 nmol of NADP⁺ converted per min per mg of total protein.

The overall activities of dTDP-glucose-4,6-dehydratase [RfbB;EC 4.2.1.46], dTDP-4-keto-6-deoxy-D-glucose-3,5-epimerase [RfbC;EC 5.1.3.13], and dTDP-4-keto-L-rhamnose reductase [RfbD; EC1.1.1.133] were each determined in a reaction mixture containing50 mM Tris-HCl buffer [pH 8.0], 0.5 mM NADH, and CE; 0.3 mMdTDP-glucose was added to start the reaction [adapted from themethod described by Grobben et al . [18]] . One unit for the overallreaction [U_RfbBCD] was defined as 1 nmol of NADH converted permin per mg of total protein.

The dTDP-D-glucose-4,6-dehydratase [RfbB] [EC 4.2.1.46] reactionmixture [final volume, 700 µl] contained 50 mM Tris-HClbuffer [pH 8.0] and CE . The reaction was started by addition of 43 mM dTDP-glucose . At different times 75-µl samplesof the reaction mixture were taken and added to 600 µlof 0.5 M NaOH . After 10 min of incubation, the formation ofdTDP-4-keto-6-deoxy-D-glucose was determined at 320 nm . Themolar absorption coefficient of dTDP-4-keto-6-deoxy-D-glucose[6.5 x 10³ liters mol^-1 cm^-1] [61] was used to calculate theRfbB specific activity . One RfbB activity unit [U_RfbB] was definedas 1 nmol of dTDP-4-keto-6-deoxy-D-glucose converted per min per mg of total protein.

Sugar nucleotide and EPS analysis. Sugar nucleotides were separated from cell extracts, and individualsugar nucleotide contents were determined by high-performanceliquid chromatography as previously described by Looijesteijnet al . [33] . The values reported below are the averages of atleast two independent determinations . EPS were isolated, quantified,and characterized as described by Looijesteijn and Hugenholtz[32] . The molecular mass and the radius of gyration [R_g] were determined by using the program Insight II [Biosym MS I, Cambridge, United Kingdom] . The intrinsic viscosity [] was calculated asdescribed by Tuinier et al . [51] by using the equation = [10[R_g/1.27]³N_AV]/3M, where N_AV is Avogadro's number and M is the molecular mass.

Isolation of cell wall sugars and characterizations of EPS and cell wall sugars. Isolation of cell wall sugars was performed as described byLooijesteijn et al . [33] . Lactococcal cells [50 ml] grown inchemically defined medium were harvested in the stationary phaseby centrifugation [3,500 x g, 10 min, 4°C] and washed twice with 0.85% [wt/vol] NaCl at 4°C . After disruption with aFrench press [twice at 18,000 lb/in²], whole cells were removed by centrifugation [3,500 x g, 10 min, 4°C], and the supernatantwas centrifuged [200,000 x g, 60 min, 4°C] to harvest cell envelopes . The crude cell envelope fraction obtained was resuspended in 50 mM morpholinepropanesulfonic acid [MOPS] buffer [pH 7] containing 140 µg of RNase per ml and 100 µg ofDNase per ml and incubated for 90 min at 37°C . Cell envelopeswere reisolated by centrifugation [200,000 x g, 60 min, 4°C]and then were resuspended in 0.5 mM MOPS buffer [pH 7] containing2% SDS and incubated at 70°C for 1 h . After centrifugation [200,000 x g, 60 min, 4°C], the pellet was washed twicewith distilled water to remove the SDS and subsequently freeze-dried,which resulted in a purified cell wall fraction . Isolated EPSor cell walls were hydrolyzed in 4 M HCl for 30 min at 100°C.Samples were dried under a vacuum and dissolved in distilledwater . The monomeric sugar composition after hydrolysis was determined by high-performance liquid chromatography [58] . Thevalues presented below are averages based on at least two independentexperiments.

Nucleotide sequence accession number. The nucleotide sequences of the rfbACBD genes have been depositedin the GenBank database under accession no. AF458777.

 
Cloning and functional analysis of the rfb gene cluster. We cloned and sequenced the rfbACBD genes from MG1363 . Sequence alignments [see Materials and Methods] indicated that these genes most likely encode the enzymes involved in the conversionof glucose 1-phosphate to dTDP-rhamnose . The lactococcal rfbACBD genes are organized in an operon-like structure [Fig . 1A] and are all preceded by typical Shine-Dalgarno sequences . A putative promoter region, containing possible -10 [5'-TATAAT-3'] and-35 [5-TTGTGT-3'] sequences, was found to precede the rfbA coding sequence . An inverted repeat sequence [5'-TAATGACTTTGTCATTA-3'] followed by an AT-rich region downstream from the rfbD coding sequence could function as a rho-independent transcriptional terminator [Fig . 1A].

To assess the transcriptional organization of the rfb gene cluster, RNA was isolated from strain L . lactis MG1363 and used for Northern analysis . Internal fragments of the rfbA and rfbD genes were generated by PCR, labeled, and used as DNA probes . Both probes hybridized with a transcript that was approximately 3.8-kb long,and no other transcripts were detected . These results confirmedthat the rfb genes are transcribed as a single 3.8-kb polycistronic mRNA, which probably starts at the postulated promoter upstreamof rfbA and terminates at the putative terminator.

To ascertain whether the L . lactis rfbACBD operon encodes a functional dTDP-rhamnose biosynthesis pathway, these genes were cloned into pK194 [21] . The resulting plasmid, pNZ4110, was transformed into E . coli S874 containing pPM2716, which is aderivative of pPM2213 . pPM2213 contains the complete S . flexneri4 rfb region and directs expression of S . flexneri O antigenproduction in E . coli . The difference that is introduced intopPM2716 when it is produced from pPM2213 is that the S . flexnerirfbBDAC genes are deleted [35] . Cells of E . coli S874 harboringpPM2213, pPM2716, pPM2716 and pNZ4110, or pPM2716 and pK194 were subjected to Western blot analysis by using rabbit antiserum raised against S . flexneri O antigen [Fig . 2] . ImmunoreactiveO antigen could be detected only in cells harboring pPM2213or cells harboring pPM2716 and pNZ4110, indicating that the L . lactis rfbACBD operon was functional . Therefore, we concluded that the genes in the rfbACBD operon most likely encode a glucose-1-phosphatethymidylyl transferase, a dTDP-4-keto-L-rhamnose-3,5-epimerase, a dTDP-glucose-4,6-dehydratase, and a dTDP-L-rhamnose synthase,respectively.

 
Effects of rfb overexpression. To evaluate the control of Rfb activity in sugar nucleotideformation and EPS biosynthesis in L . lactis, pNZ8048 derivativescarrying the lactococcal rfb genes under control of the lactococcalnisA promoter were transformed into NZ9000, which allowed useof the nisin-controlled expression system [9, 26] . Strain NZ9000 harboring the pNZ8048 derivatives carrying rfbACBD [pNZ4117], rfbAC [pNZ4116], or rfbB [pNZ4118] under control of the lactococcalnisA promoter [Table 1] were grown in the presence or absenceof the inducer nisin, and CEs of these cultures were analyzedby SDS-PAGE [Fig . 3] . Nisin-mediated induction of NZ9000 harboringpNZ4116 [rfbAC] resulted in the appearance of additional proteinbands at molecular masses of approximately 32 and 22 kDa, whichare the expected sizes of RfbA and RfbC, respectively [Fig.3, lane 2] . By analogy, nisin-mediated induction of strain NZ9000harboring pNZ4118 [rfbB] resulted in the appearance of an additionalprotein band at a molecular mass of approximately 40 kDa, whichis the expected size of RfbB [lane 4] . Finally, nisin-mediatedinduction of NZ9000 harboring pNZ4117 [rfbACBD] resulted inthe appearance of additional protein bands at molecular massesof approximately 40, 32, and 22 kDa [lane 7] . The RfbA and RfbDproteins appeared to comigrate at an apparent molecular massof 32 kDa, as deduced from the increase in the intensity ofthis band relative to the intensity of the RfbC [22-kDa] bandwhen rfbACBD was expressed instead of rfbAC.

 
The RfbA enzyme activity level was determined in CEs of thewild-type strain [21 ± 0.5 U_RfbA] and the induced culturesof strains that overproduced RfbAC [1,340 ± 200 U_RfbA]and RfbACBD [268 ± 28 U_RfbA]; thus, the levels of overexpression of RfbA were 67- and 13-fold, respectively . The lower RfbA activity measured in the strain that overproduced RfbACBD than in the strain that overproduced RfbAC was probably due to the RfbAenzyme activity assay, in which RfbA and RfbB competed for thesame substrate, dTDP-glucose . The level of RfbB enzyme activityincreased 60- and 45-fold compared to the wild-type level [28± 1 U_RfbB] in the induced strains that overproduced RfbB[1,570 ± 150 U_RfbB] and RfbACBD [1,160 ± 100 U_RfbB],respectively . Moreover, the level of RfbBCD enzyme activitywas 28-fold greater for the strain that overproduced RfbACBD[508 ± 70 U_RfbBCD] than for the wild type [18 ±5 U_RfbBCD] . Remarkably, in contrast to other bacteria, the lactococcalRfbD activity could be measured only by using NADH instead ofNADPH in the RfbBCD enzymatic assay [data not shown], indicatingthat the lactococcal RfbD activity requires NADH as a cofactor.The increased enzyme activities demonstrate that controlledand functional overexpression of the rfbACBD genes was achieved.

The effect of Rfb activity on sugar nucleotide concentrationand glucose 1-phosphate pool conversion was evaluated in thestrains that overexpressed the rfb genes . We anticipated aneffect on different sugar nucleotide levels since the substrateof the Rfb pathway, glucose 1-phosphate, is the central intermediatefrom which UDP-glucose and UDP-galactose are also formed, andmodulation of the sugar nucleotide levels could affect growth.However, functional overexpression of the rfb genes resultedin a maximal growth rate that was not significantly differentfrom that of the wild type [data not shown] . Furthermore, overexpressionof the rfb genes did not influence the absolute level of UDP-glucoseor UDP-galactose [data not shown] . In contrast, overexpressionof rfbAC and rfbACBD resulted in a doubling of the intracellulardTDP-rhamnose levels [7.3 ± 0.6 µmol g of protein^-1,compared to 3.3 ± 0.5 µmol g of protein^-1 in wild-typeor noninduced cells], while the dTDP-glucose levels in thesestrains remained the same [data not shown] . These results demonstratethat RfbAC activities exert control over the dTDP-rhamnose levelsin wild-type cells.

To evaluate the effect of rfb overexpression on EPS production, the EPS-producing capacity was introduced into the rfb-overproducing strains since these strains do not natively produce EPS . This was done by transformation of these strains with pNZ4030, which contains the B40 eps gene cluster [56] . Increased levels ofRfb activity had no effect on the level of EPS production [datanot shown] . Apparently, although increased levels of Rfb activityresulted in increased dTDP-rhamnose levels, the sugar nucleotidechanges did not affect the growth rate or EPS production.

Effect of rfb mutation on growth and EPS production. To evaluate the effect of reduced Rfb activity on growth andEPS biosynthesis in L . lactis, we tried to inactivate the rfb genes [see Materials and Methods] . Several attempts to disrupt the rfbA gene by single-crossover plasmid integration with pNZ4105 failed . Moreover, attempts to select mutants in which the rfbB gene was replaced by an erythromycin resistance gene cassette by direct double crossover, by using the nonreplicative plasmid pNZ4109, were unsuccessful . All erythromycin-resistant colonies obtained when the latter strategy was used appeared to be single-crossover integrants . Southern analysis of these strains revealed thatin all cases the single-crossover plasmid integration had takenplace downstream of the rfbB gene, leaving the rfb operon intact. These results strongly suggest that the rfb genes play an essential role in L . lactis . This suggestion was corroborated by the finding that the desired rfbB::ery strain could be obtained by transformationof pNZ4109 into L . lactis cells harboring an additional copyof the rfbB gene in trans in a replicating plasmid that harborsrfbB under control of the nisA promoter [pNZ4118] . However,the strain obtained when this rescue strategy was used was stillable to grow in the absence of nisin, suggesting that the RfbBactivity level under these noninducing conditions was stillsufficient to sustain growth, probably due to leakage of thenisA promoter in a high-copy system . Therefore, a conditionalrfbBD mutant was constructed in which transcription of the chromosomalrfbBD genes was placed under control of the nisA promoter, whilethe rfbAC genes remained under control of the original rfb promoter [Fig . 1B] . For this purpose, the nonreplicative plasmid pNZ4114was transformed into strain NZ9000, and double-crossover mutantswere selected based on tetracycline resistance and erythromycinsensitivity [see Materials and Methods] . Southern blot analysiswas used to confirm the anticipated genetic organization of the rfb locus, and a single mutant strain, designated L . lactis NZ4114, was used for further analysis . This nisin-controlled conditional rfbBD mutant did not grow in medium without nisin, while its growth in medium containing 1 ng of nisin ml^-1 was similar to that observed for parental strain NZ9000, which confirmed that expression of the rfb genes is essential for growth of L . lactis.

To evaluate the effect of controlled limitation of rfbBD expression on growth, strain NZ4114 was grown overnight in media containing different levels of nisin and subsequently subcultured [2%, vol/vol] in medium lacking nisin, and the optical density was monitored over time [Fig . 4] . Although the growth of each overnightculture was similar to the growth of the parent strain, nisinconcentrations of 1.0, 0.5, and 0.3 ng ml^-1 in the overnightcultures resulted in 22, 59, and 73% reductions in the specificgrowth rates of the subsequent cultures grown without nisin, respectively . Moreover, the stepwise reductions in the final optical densities reached by these cultures corresponded tothe levels of nisin induction used in the overnight cultures[Fig. 4] . However, cells were not washed prior to subculturing, which could have resulted in delayed shutoff of dTDP-rhamnose synthesis and therefore a slower ceasing of growth . Finally,the dTDP-rhamnose levels in NZ4114 cells with reduced rfbBD expression appeared to be below the background level of theassay used, implying that the dTDP-rhamnose levels in thesecells were significantly reduced [at least fivefold lower] comparedto the level observed in wild-type cells [data not shown] . Theseresults validated the anticipated limitation of intracellulardTDP-rhamnose levels by controlled reduction of rfbBD expressionand allowed evaluation of the effects of the limitations onthe biosynthesis of rhamnose-containing sugar polymers.

 
The sugar compositions of the cell wall polysaccharides producedby RfbBD-limited cells appeared to be the same as those of wild-type cells [Table 2], suggesting that incorporation of rhamnose intothe lactococcal cell wall is essential for growth . Besides cell wall polysaccharides [Table 2], rhamnose is known to be a constituentof several lactococcal EPS . To investigate the effect of dTDP-rhamnoselimitation on EPS production [Table 3], the EPS-producing capacitywas introduced into strain NZ4114, which is not a native EPS-producingstrain, by transformation with pNZ4030 [56] . No differencesin growth rate and EPS yield between the mutant and the parentalstrain were observed after 24 h of fermentation when cells weregrown in the presence of nisin [Table 3] . However, the EPS productionby the mutant strain was reduced to approximately 5% of theproduction by the wild type when the cells were grown underrfbBD expression-limiting conditions . These results primarilysuggest that rfb expression is essential for both productionof B40-EPS and growth . However, the observation that the finalculture density decreased stepwise as the nisin concentrationdecreased while the levels of EPS production were reduced equallyin all RfbBD-limited cultures indicates that EPS productionand growth are not coupled . Interestingly, uncoupling of EPSproduction and growth has been reported previously for L . lactisstrain NIZO B40 [32] . Moreover, these results suggest that theprecursor dTDP-rhamnose is preferentially used for the formationof cell wall polysaccharides rather than for EPS production,which may be due to differences in the kinetic properties ofthe different enzymes involved in the two pathways.

 
Characterization of EPS produced by the rfb mutant. The EPS produced by strain NZ4114 were analyzed by static light scattering after size exclusion chromatography . Estimation ofthe molecular masses revealed that the EPS produced by strainNZ4114 had an average molecular mass that was fourfold largerthan that of the EPS produced by the parental strain, NZ9000[Table 3; Fig . 5A] . Furthermore, static light scattering measurements generated an average R_g that was twofold smaller than that measuredfor EPS produced by strain NZ9000 harboring pNZ4030 [Fig . 5B].Moreover, the viscosifying properties of the mutant EPS in chemicallydefined medium, which could be calculated from the molecularmass and the R_g [51], was drastically decreased [Table 3] . Inaddition, monomer sugar analysis after hydrolysis of the purifiedEPS revealed that the EPS produced by the RfbBD conditionalmutant strain consisted of the monosaccharides glucose, galactose,and rhamnose at a molar ratio of 1:0.3:0.2, which is clearlydifferent from the ratio in the parental strain [1:0.6:0.4][Table 3] . These results indicate that the RfbBD conditionalmutant produced EPS with an altered structure as a result ofan at least partially altered repeating unit . Nevertheless,the polymerization and export machinery could still recognizeand handle this altered repeating unit . Moreover, since themolecular mass was increased, the polymerization and exportmachinery may even have extended the polymer to a greater length.Finally, the decreased R_g indicated that there was a more compactlyfolded structure that filled less space, which probably wasthe consequence of the altered structure.

 
In this paper we describe identification and functional analysisof the chromosomal L . lactis rfb MG1363 operon that is involved in dTDP-rhamnose biosynthesis . Sequence analysis led to identification of four rfb genes whose predicted gene products exhibit high levels of homology with proteins involved in the biosynthesis of dTDP-rhamnose . Evidence for the rfb functionality of the L . lactis rfb genes was obtained by overproduction of the Rfb proteins, which led to increased Rfb activities . Moreover, expression of the rfb genes in E . coli complemented an O antigen production mutation that deleted the S . flexneri Rfb homologues.

In both gram-negative and gram-positive bacteria the rfb genes are often genetically linked to genes involved in CPS or O antigen production . Remarkably, even in S . pneumoniae serotypes that produce CPS that do not contain rhamnose, the rfb genes are linked to the cps locus [39] . In contrast, analysis of the chromosomallocalization of the rfb genes in the L . lactis IL-1403 genomesequence revealed that the lactococcal rfb gene cluster is notgenetically linked to genes encoding related functions [3].This resembles the situation in S . mutans, although in thisand various other streptococci the rfbD ortholog was found tobe distant from the rfbA, rfbB, and rfbC genes [49, 50].

Functional overexpression of the rfbACBD or rfbAC genes led to increased levels of Rfb proteins and a twofold increase in the dTDP-rhamnose level . However, the increased Rfb enzyme activities did not result in production of more B40-EPS . Similarly, GalU overproduction resulted in increased UDP-glucose and UDP-galactose levels but did not affect the level of B40-EPS production [2]. These results indicate that there is no correlation betweenthe levels of individual sugar nucleotides and the level ofEPS in L . lactis harboring pNZ4000 derivatives . This apparentlycontradicts several reports that showed that there was a correlationbetween the activity level of enzymes involved in sugar nucleotidebiosynthesis and the level of EPS produced [17, 28] . However,this correlation seems to depend on the type of polysaccharideproduced, as was clearly shown for GalU activity in L . lactis[2, 17] . It is very possible that simultaneous increases inUDP-glucose, UDP-galactose, and dTDP-rhamnose levels could positivelyaffect the B40-EPS level, since the repeating unit of this EPScontains two glucose moieties, two galactose moieties, and arhamnose moiety . Alternatively, the level of B40-EPS producedcould also be controlled by the activity of the specific EPSbiosynthesis machinery encoded by the EPS plasmid rather thanby the level of sugar nucleotides . This hypothesis is supportedby the observation that overexpression of the priming glycosyltransferaseepsD gene in L . lactis resulted in increased levels of B40-EPS[56, 57].

A nisin-controlled conditional rfbBD mutant was constructed by introduction of the nisA promoter upstream of the rfbB gene in the chromosome of L . lactis . This mutant, L . lactis NZ4114,was not able to grow in the absence of nisin, indicating that the rfbB and/or rfbD gene is essential for L . lactis growth.This finding explains our lack of success in construction of an L . lactis rfbB mutant by using conventional knockout strategies. In L . lactis NZ4114, lowering the nisin concentration resulted in a reduction in the growth rate and a lower final optical density . However, the sugar composition of the polysaccharide fraction of the cell wall in this strain appeared to be unaffected. Remarkably, although rhamnose is a major component of cell polysaccharides in L . lactis, as well as in S . mutans [45], inactivation ofany of the four S . mutans rml genes led to viable cells lackingrhamnose in the cell wall polysaccharide [49, 50] . In contrastto these findings for S . mutans, our results suggest that therhamnose moieties in the lactococcal cell wall polysaccharidesare essential for cell wall integrity in L . lactis . Therefore,limitation of dTDP-rhamnose precursor levels could interferewith wild-type cell wall polysaccharide production and resultin a decrease in the growth of L . lactis . We used the nisin-controlledexpression system to construct conditional mutations in essentialgenes like the rfbBD genes, which allowed us to study the correspondingmutant phenotypes . A similar strategy has recently been describedfor the [F₀F₁]-H⁺-ATPase complex in L . lactis [24] . However,since this expression system can be implemented in many othergram-positive hosts [13, 22], this approach has potential tobe used in other bacteria.

The level of EPS production by the conditional rfbBD mutant L . lactis NZ4114 was only 5% of the parental level of EPS production when cells were grown under nisin limitation conditions . This low level of production could be complemented by addition ofnisin to the medium . The effect of a lack of rfbBD expressionon both EPS production and growth in L . lactis can probablybe explained by the hypothesis that dTDP-rhamnose plays a crucialrole in cell wall synthesis and an important role in the biosynthesisof the rhamnose-containing EPS . The enzymes in these pathwaysdiffer, and so may their kinetic properties, and we speculatethat different affinities for dTDP-rhamnose may well explainthe different effects on these processes . Data supporting thissuggestion include recent observations reported by Cartee etal . [5] for reduction of capsule synthesis by S . pneumoniaedue to reduction of the sugar nucleotide concentration . Thesugars of the EPS produced by NZ4114 cells grown under RfbBD-limitingconditions were glucose, galactose, and rhamnose at a ratiothat is different from the ratio for the polymer produced bythe parental strain . These results indicate that the RfbBD conditionalmutant produces EPS with an altered composition as the resultof an at least partially altered repeating unit . This impliesthat it might be possible to change the EPS composition by reducingthe availability of EPS precursors via inactivation of specificprecursor-forming enzymes . Although the yield is limited, theEPS polymerization and export machinery is still capable ofrecognizing and processing EPS with an altered repeating unitand is apparently not exclusively specific for a single repeatingunit . This is corroborated by the finding that expression ofthe Streptococcus thermophilus eps gene cluster in L . lactisresulted in production of very small amounts of EPS with a repeatingunit that differs from the native structure due to a lack oflactococcal UDP-N-acetylglucosamine C₄-epimerase activity leadingto incorporation of a galactose moiety instead of a GalNac moietyin the mutant EPS [47] . However, it remains to be establishedwhat determines the low level of production [6 to 10 mg liter^-1]of EPS in these lactococci.

The global sugar of the altered EPS produced by strain NZ4114 includes fewer rhamnose and galactose moieties and was shownto have a fourfold-greater molecular mass than the EPS producedby the parental strain . Since the distributions of the molecularmasses of both the B40-EPS and the altered EPS follow a typicalsymmetric Gaussian curve [Fig . 5A], we concluded that the altered EPS are homogeneous polymers and do not represent a mixtureof altered and native EPS polymers . Besides the fourfold-greater molecular mass, the altered EPS had a twofold-smaller R_g than the native EPS . This finding suggests that there is a drastic decrease in the viscosifying properties of these EPS comparedto the properties of wild-type B40-EPS [52] . Moreover, the decreased R_g indicates that folding of the backbone of the altered EPSis much less hindered by side chains, resulting in greater chain flexibility and a more compactly folded structure . Hence, itis likely that the increased molecular mass of the altered EPSis due to increased chain length rather than an increase inthe number or size of the side chains . Furthermore, these resultssuggest that the repeating unit of the altered EPS is a modificationof that of the native EPS and partially lacks its side chainsconsisting of rhamnose and galactose phosphate . Finally, theproduction by strain NZ4114 of an EPS that is longer and morecompact than the native EPS suggests that the chain length determinationmechanism is dependent on the three-dimensional conformationof the polymer rather than on only the length of the chain itself.

Evaluation of the dTDP-rhamnose biosynthesis pathway described here allowed assessment of the role of the rfbACBD genes in L . lactis by overexpression and disruption studies of these genes . We could significantly influence the level of dTDP-rhamnose, which is a precursor for cell wall polysaccharides, as wellas for EPS biosynthesis in L . lactis . We were also able to influence the levels of EPS production and even the repeating unit sugar composition by using a conditional rfbBD mutant . Various workers have previously established that it is possible to modulate polysaccharide biosynthesis by engineering at the level of specific eps genes [for a review see reference 57] . To our knowledge,this is the first report showing that modulation of the householdenzyme levels can lead to production of EPS with an altered composition . These results enlarge the knowledge base required for efficient targeting of bottlenecks in EPS biosynthesis and provide new opportunities for creating structural diversityby constucting polysaccharides with novel properties.

 
We thank Jan van Riel for assistance with the sugar nucleotideand EPS analysis, Marja Kanning for assistance with the analysisof the EPS biophysical characteristics, and Douwe Molenaar andRoland Siezen for critically reading the manuscript . NestléResearch Center is acknowledged for providing dTDP-rhamnose.

Part of this work was supported by EC grant BIOT-CT96-0498.

 
* Corresponding author . Mailing address: Flavor, Nutrition and Ingredients, NIZO Food Research, P.O . Box 20, 6710 BA, Ede, The Netherlands . Phone: 31-318-659629 . Fax: 31-318-650400 . E-mail: michiel.kleerebezem@nizo.nl.

Present address: Friesland Coberco Dairy Foods, Corporate Research, Deventer, The Netherlands.

Present address: Plant Research International, Wageningen, The Netherlands.

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