Biosynthesis of the variable core domain of lipooligosaccharide[LOS] in Neisseria gonorrhoeae is mediated by glycosyl transferases encoded by lgtABCDE . Changes within homopolymeric runs within lgtA, lgtC, and lgtD affect the expression state of these genes,with the nature of the LOS expressed determined by the functionalityof these genes . However, the mechanism for modulating the amountof multiple LOS chemotypes expressed in a single cell is notunderstood . Using mutants containing polar disruptions within the lgtABCDE locus, we determined that the expression of this locus is mediated by multiple promoters and that disruptionof transcription from these promoters alters the relative levelsof simultaneously expressed LOS chemotypes . Expression of the lgtABCDE locus was quantified by using xylE transcriptional fusions, and the data indicate that this locus is transcribed in trace amounts and that subtle changes in transcription resultin phenotypic changes . By using rapid amplification of 5' cDNAends, transcriptional start sites and promoter sequences wereidentified within lgtABCDE . Most of these promoters possessed50 to 67% homology with the consensus gearbox promoter sequenceof Escherichia coli.

 
Neisseria gonorrhoeae is an obligate human pathogen that causes diseases of mucosal surfaces [see reference 32 for a review].Because the gonococcus is capable of proliferating in differentphysiological milieus, it has developed a variety of mechanismsfor adapting to these environments . Lipooligosaccharide [LOS]is indispensable for disease pathogenesis . It is immunogenic, highly pyrogenic, and responsible for the localized inflammation and scarring characteristic of gonococcal infections and for the septic shock that results from disseminated disease [21-23, 31, 39, 45; H . M . Harper, A . L . Padmore, W . D . Smith, M . K.Taylor, and R . Demarco de Hormaeche, unpublished results [presentedat the Tenth International Pathogenic Neisseria Conference,Baltimore, Md.]] . In addition, specific chemotypes of LOS confercomplement resistance [34] and facilitate intracellular invasion[47] . LOS molecules are heterogeneous, with a single cell oftensimultaneously expressing two or more chemotypes in variousproportions [2, 3, 11, 46] . LOS also undergoes phase variation,and distinct LOS chemotypes are favored for survival of thegonococcus within different regions of the human body and atvarious points during the pathogenic process [15, 32, 49, 53, 55] . We believe that the expression of the correct LOS chemotype[s]by the gonococcus at the correct time during infection is essentialfor establishing and maintaining infection.

Our understanding of the regulatory mechanisms that affect LOS biosynthesis and expression remains incomplete . Biosynthesisof the variable oligosaccharide portion of LOS is mediated byseven LOS glycosyl transferase [lgt] genes [7, 13, 20] . Strandslippage in homopolymeric tracts within the coding sequenceof lgtA, lgtC, lgtD, and lgtG during DNA replication leads toreading frameshifts in these genes . These changes result inthe production of inactivate glycosyl transferases, thus alteringthe LOS chemotype[s] that is expressed by a given cell [7, 11, 13, 56] . Gonococcal LOS expression can be further modified byspecific environmental stimuli, suggesting that regulated geneexpression occurs . Gonococci grown under anaerobic conditionsor in the presence of lactate, which is normally present inthe female genital tract, produce an altered LOS profile [16, 30] . Gonococci grown under acidic and alkaline conditions alsoexpress different LOS profiles [35] . Finally, gonococci grownat different growth rates express different LOS epitopes andpossess different serum sensitivities [33] . The present studyinvestigates the transcriptional organization of the lgtABCDEgenes.

 
Bacterial strains, plasmids, oligonucleotides, and culture conditions. All bacterial strains, plasmids, and oligonucleotide primersused in the present study are listed in Tables 1, 2, and 3.N . gonorrhoeae strains were grown in phosphate-buffered gonococcalmedium [Difco] supplemented with 20 mM D-glucose and growthsupplements [54] either in broth with the addition of 0.042%sodium bicarbonate or on agar where they were incubated in a37° CO₂ incubator . All Escherichia coli strains were grownin Luria-Bertani medium [43] . Kanamycin was used in growth mediaat a concentration of 30 µg/ml, ampicillin was used at60 µg/ml, spectinomycin was used at 50 µg/ml, andX-Gal [5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside] was used at 35 µg/ml . The optical densities of gonococcaland E . coli cultures were determined by using a Klett-Summerson colorimeter fitted with a green filter . One Klett unit correspondsto a culture density of ca . 10⁷ CFU/ml . We used the DNA sequence numbering for the F62 lgtABCDE region, as described in the National Center for Biotechnology Information database under accession number U14554 to orient our constructs, promoter mapping, etc.,to the literature.

 
Chemicals, reagents, and enzymes. Restriction enzymes and T4 DNA ligase were purchased from NewEngland Biolabs [Beverly, Mass.] . PCR buffers and enzymes andbuffers for 5' rapid amplification of cDNA ends [5'-RACE] werepurchased from Invitrogen [Carlsbad, Calif.] and Roche MolecularBiochemicals [St . Louis, Mo.] . All chemicals used for the presentstudy were reagent grade or better and purchased from SigmaChemical Co . [St . Louis, Mo.] unless otherwise specified . The monoclonal antibodies [MAbs] 2-1-L8 and 17-1-L1 were generously provided by Wendell Zollinger of the Walter Reed Army Instituteof Research, Washington, D.C . MAb 1B2 was a gift from J . McLeod Griffiss, University of California, San Francisco.

DNA and RNA isolation procedures. Chromosomal DNA was isolated as described by Rodriguez and Tait[41] . Plasmid DNA was isolated by the method of Birnboim andDoly [8] . RNA was purified chromatographically from gonococcigrown to a Klett reading of 100, by using the High-Pure RNAisolation kit [Roche].

PCR. The PCR was generally performed by using Platinum PCR Supermix[Invitrogen] or the Expand Long-Template PCR kit [Roche] accordingto the manufacturers' directions . Primers were purchased fromBioserve Biotechnologies [Laurel, Md.] or from Integrated DNA Technologies [Coralville, Iowa] . PCRs were resolved on agarose gels containing 500 µg of ethidium bromide/ml in Tris-borate-EDTA running buffer [43].

Transformation. Competent cells of E . coli DH5-MCR were prepared by the methodof Inoue et al . [27] . Recombinant DNA transformation of E . coliwas done according to the standard heat shock protocol [43]. Transformants were verified by digestion of recovered plasmidDNA with the appropriate restriction enzymes . Recombinant DNA transformation into N . gonorrhoeae F62 was done by either the tube or the spot transformation method of Gunn and Stein [24].

Purification of LOS and SDS-PAGE. Gonococcal LOS was prepared from plated cultures as describedby Hitchcock and Brown [25] and diluted 1:25 in lysing buffer.The suspension was boiled for 10 min immediately before 5 µlwas loaded onto a sodium dodecyl sulfate-polyacrylamide gelelectrophoresis [SDS-PAGE] gel . After electrophoresis, the gelwas fixed overnight in a solution of 40% ethanol-5% acetic acidand then oxidized in 0.83% periodic acid for 5 min . The gelwas washed for 2 h in multiple changes of H₂O every 20 min,stained for 5 min in silver staining solution [22.5 mM NaOH,0.42% NH₄OH, 47 mM AgNO₃], and rewashed for 2 h in multiplechanges of H₂O every 20 min . The gel was developed [100 ml of0.005% citric acid-0.007% formaldehyde] until the bands becamesufficiently visible and then photographed.

Immunological methods. Colony blots were performed by transferring cells grown overnighton GCK agar onto nitrocellulose filters . The filters were airdried for 10 min and blocked with filler solution [20 mM Tris,150 mM NaCl, 2% nonfat dry milk, 0.2% NaN₃, 0.002% phenol red[pH 7.4]] for 30 min . Filters were blotted with Whatman 3MMpaper to remove cellular debris prior to incubation in primaryantibody [MAb 2-1-L8 or 17-1-L1] with gentle shaking for 2 hor longer . Filters were washed with Tris-buffered saline [TBS;pH 7.4] three times for 10 min each time . Membranes were incubatedin filler solution containing goat anti-mouse immunoglobulinG conjugated with horseradish peroxidase for 2 h . Filters werewashed three times for 10 min each time in TBS, and the membraneswere developed by incubating them in 50 ml of 50 mM Tris-HCl-1%4-chloro-1-naphthol-0.86% H₂O₂ [pH 8.0].

XylE assay. Qualitative screening for XylE⁺ colonies was performed by sprayingplates with 50 mM catechol, followed by incubation at 37°Cfor as short as 1 min to as long as 1 h to allow for color development.The presence of a yellow color indicated XylE activity . Quantitativeassays were performed by inoculating cells into 20 ml of gonococcalbroth plus supplements, with growth to a Klett reading of 100[indicating an approximate density of 10⁹ CFU/ml] . Cells wereharvested by centrifugation, washed once in 10 ml of 50 mM potassiumphosphate [pH 7.5], and resuspended in 2.5 ml of 100 mM potassiumphosphate-20 mM EDTA-10% [vol/vol] acetone [pH 7.2] . Cells werethen disintegrated with six 10-s pulses from a sonicator [HeatSystems, Inc.] set at 40% output; during this process, the tubecontaining the sample was kept submerged in ice water . The resultingcrude lysate was clarified of cell debris with two rounds ofcentrifugation, first in a swinging bucket centrifuge at 4,000x g for 5 min and then in a microfuge at 10,000 x g for 10 min.The clarified lysate was stored at -80°C . Assays were performedby diluting cell extracts in assay buffer [100 mM potassiumphosphate, 0.2 mM catechol] such that a linear change in absorbanceat 375 nm was seen over time . XylE activity was calculated bylinear regression of the slope over six time points . One microunit of XylE activity corresponds to the formation of 1 nmol of 2-hydroxymuconicsemialdehyde per min at 22°C . XylE activity was normalizedagainst total protein concentration, as determined by the methodof Bradford et al . [10], with bovine serum albumin [New EnglandBiolabs, Beverly, Mass.] as the standard.

5'-RACE. RACE analyses were performed essentially as described by Frohmanet al . [18] . Synthesis of cDNA was performed with 2 µgof total RNA template, 12 pmol of antisense primer, and 1 Uof avian myeloblastosis virus reverse transcriptase [Roche]in reaction buffer supplied by the manufacturer . The reactionwas carried out for 1 h at 55°C, followed by 10 min of incubationat 65°C . cDNA was purified on a Qiagen spin column accordingto the manufacturer's directions, eluted in 50 µl of 10mM Tris-Cl [pH 8.0], and polyadenylated with 0.5 U of terminal transferase [Roche]/µl using the manufacturer's reactionbuffer supplemented with 1.5 mM CoCl₂ and 6.25 µM dATP.Three microliters of polyadenylated cDNA was used in a PCR withthe d[T] forward primer [see Table 3] and a gene-specific antisense primer . The resulting products were subjected to a second round of PCR with the anchor primer and a nested reverse primer andthen resolved on 3% low-melting-temperature agarose gels . The transcriptional start site was determined by DNA sequence analysisof the RACE products.

 
Analysis of transcriptional linkage by interposon mutagenesis. The organization of the genes found in the lgt gene cluster suggested that they would be transcribed as an operon . Analysisof the DNA sequence [up to 500 bp] upstream of the putativelgtA start codon failed to identify any sequences with homologyto known F 70-like promoters . This analysis did identify a DNAsequence that might form a rho-dependent termination sequencein the intergenic region between glyS stop codon and the putativelgtA start codon [see Fig . 1] . However, if this stem-loop structure [26 bp upstream of the putative lgtA start codon] was functioning as a transcriptional stop signal, it was unclear what DNA sequence could be promoting the expression of the lgt gene cluster.

 
In order to determine whether the stem-loop was functioningas a transcription termination signal, we introduced a strong transcriptional stop site 6 bp upstream from the start of the stem-loop structure by inserting the interposon into a BsrGIsite and analyzed the effect of its insertion on LOS expression.The location of the insertion event in each transformant wasverified by PCR amplification of the appropriate region andrestriction digestion of the PCR products . The data indicatethat all transformants analyzed incorporated the interposonsequence at the appropriate chromosomal location [data not shown].LOS was purified from 10 individual transformants and analyzedby SDS-PAGE, and the bands visualized by silver staining the gels . Each of the interposon-derived mutants exhibited an alteredLOS phenotype that possessed the same SDS-PAGE profile . A representativeof the SDS-PAGE profile of one of these mutants [F62BsrGI] isshown in Fig . 2, lane 3.

 
The lgtE gene is the most downstream gene in the lgtABCDE locus, and yet it mediates the first biochemical step in the assembly of a growing LOS molecule . If the lgtABCDE locus is an operon driven by a single promoter, then an interposon insertion atthe BsrGI site should produce a strain whose LOS phenotype resemblesthe LOS of strains deleted in lgtE [produces LOS that will bereferred to as LgtE LOS] . The data presented in Fig . 2 indicatethat F62BsrGI produces an LOS that is larger than the LgtE LOSchemotype, suggesting that this strain possesses LgtE activity.

Western blot analysis of LOS expressed by F62BsrGI indicatedthat the single band seen in Fig . 2, lane 3, reacted with MAbL1-1-17 [data not shown], an MAb that binds to an alternateLOS chemotype in neisserial LOS, one that is produced when LgtAis nonfunctional and LgtC is functional . From the data obtained in the analysis of LOS expressed by F62BsrGI, we concluded thattranscription of lgtA must initiate 5' of the BsrGI . These dataalso indicate that the potential rho-dependent termination sequencestill allows for the transcription of sufficient message suchthat the amount of LgtA present in in vitro-grown cells is notlimiting . Because F62BsrGI was able to produce an LOS that requirestranscription of lgtC, we concluded that a promoter must exist3' of the BsrGI site . These data indicate that the overall transcriptionalorganization of the genes contained within this gene clustermust be driven from multiple promoters.

In order to localize promoter containing regions, we createda family of isogenic strains of N . gonorrhoeae F62 containing the interposon inserted at different points within the lgtABCDE locus and observed the resulting LOS phenotypes . The plasmidsused to make the various gonococcal constructs are describedin Table 2 . LOS was purified from 10 to 12 individual transformants for each construct and visualized after separation on SDS-PAGE gels [data not shown] . Each of the interposon mutants exhibitedan altered LOS phenotype that was consistent across all of theindividual transformants of each mutant that was analyzed, indicatingthat the change in LOS phenotype did not occur due to phasevariation . We verified that the interposon had inserted intothe correct region by PCR amplification of the lgtABCDE locusof each of these mutants, with subsequent restriction digestionanalysis of the amplicons . The expected restriction patternwas generated on an agarose gel from the amplicons generatedfrom each of the mutants, indicating that the interposon hadbeen incorporated into the correct sites [data not shown].

The data presented in Fig . 2 indicate that all strains containing interposon insertions within lgtABCDE continued to produceLOS chemotypes that require LgtE activity, except when the Sinterposon was inserted into an engineered EcoRI site immediately upstream of the lgtE ribosome-binding site [F62EcoRI; Fig . 2,lane 9] . Analysis of these results in detail allowed us to mapthe location of potential promoters . Strain F62AscI [lane 4]produced two LOS components: one that possesses a mobility consistentwith a LgtB LOS [which corresponds to an LOS structure madewhen LgtB is nonfunctional] and one that possesses a mobilitythat corresponded to LgtE LOS . The LOSs expressed by this strainfailed to bind MAbs 1B2, 1-17-L1, and 2-1-L8 . That this straindoes not produce L1 LOS suggests that lgtC transcription hasbeen inhibited . By combining these two results [data shown inFig . 2, lanes 2 to 4], we could map a promoter region to betweenthe BsrGI and AscI sites.

Simultaneous expression of at least two LOS components is seenin four mutant strains [see Fig . 2, lanes 5 and 7 to 9] . In these strains, a mixture of the LgtE LOS and a second LOS chemotypethat binds MAb 1B2 [Western blot data are not shown] is produced.Since the interposon is placed more proximal to the start codonof lgtE, a proportionally greater amount of LgtE LOS is produced.In fact, the shift in the proportions between these two LOScomponents occurs in a stepwise fashion, suggesting the presenceof multiple promoters.

Additional promoter regions were mapped by analyzing changesin the proportion of the two chemotypes that are produced . F62ClaI [Fig . 2, lane 5] produces a roughly 60:40 proportion of 1B2LOS [LOS chemotype made when strain expresses a functional LgtA, and a nonfunctional LgtC and LgtD] and LgtE LOS chemotypes,indicating inhibition of lgtD transcription and decreased transcriptionof lgtE versus that of the parental strain . Strain F62NruI [Fig.2, lane 7] produces a roughly 40:60 proportion of 1B2 LOS and LgtE LOS . Strain F62EcoRV [Fig . 2, lane 8] expresses less 1B2LOS and more LgtE LOS than strain F62NruI . Strain F62EcoRI mutant[Fig . 2, lane 9] only expresses LgtE LOS . Altogether, the introductionof the interposon in successive sites between ClaI and EcoRI results in stepwise increases in the amount of LgtE LOS thatis produced, suggesting a corresponding decrease in LgtE production.These data suggest the existence of promoters between each ofthe interposon insertion sites, except between AscI and ClaI, and support a model of multiple, weak promoters existing throughout the lgtABCDE locus . This further suggests that the cumulative contribution of each of these promoters defines the nature of the LOS that is expressed.

Quantitation of gene expression. In order to quantitate the level of gene expression within thelgtABCDE locus, we constructed three xylE transcriptional fusions.The xylE gene was inserted immediately upstream of lgtA [F62LgtA[xylE]], at the beginning of lgtD [F62LgtC[xylE]], and immediately downstream of lgtE [F62LgtE[xylE]] . Analysis of the LOS profiles of these strains by SDS-PAGE and colony blotting [data not shown] showed that the transformants expressed the expected wild-type LOSprofile when the xylE gene was inserted before or after the lgtABCDE locus and the lgtD LOS when the xylE gene was insertedinto lgtD [Fig. 3].

 
We assayed these xylE fusion strains for catechol-2,3-dioxygenase [XylE] activity [Table 4] . The assays showed a difference inXylE activity among the three strains . XylE activity was sevenfoldhigher in F62LgtA[xylE] than in F62LgtE[xylE] and F62LgtC[xylE].These data indicate that the different genes within the lgtABCDEgene locus can be expressed at different levels due to differentmRNA concentrations . They further indicate that most transcriptionthrough this region terminates before it extends through theentire region.

 
Positional effects of the interposon insertion on XylE activity in F62LgtE[xylE]. The data presented above suggest the presence of multiple promotersequences located within the lgtABCDE locus . In order to quantifythe relative contribution that these sequences might play inoverall gene expression, we introduced the interposon intovarious chromosomal locations in F62LgtE[xylE] . Each transformantwas analyzed by PCR amplification of the region of interest,with subsequent restriction digestion analysis to verify thatthe insertion had incorporated into the correct chromosomal location . In addition, the SDS-PAGE profiles of each isogenic pair [i.e., F62LgtE[xylE]::BsrGI compared to F62BsrGI] wereidentical, indicating that the interposon incorporation was exerting the same phenotypic modulation in each pair of strains. The data presented in Fig . 4 indicate that insertion of the interposon at the BsrGI site did not result in a change in XylE expression . This further supports the data presented abovethat indicated that transcription terminates within the lgtABCDE region . Insertions that occurred within the coding sequencereduced but did not eliminate XylE expression compared to theisogenic parent strain lacking the interposon insertion [F62LgtE[xylE]].As the insertion site of the interposon neared the lgtE codingsequence, the level of transcription decreased . Insertion ofthe interposon in the engineered EcoRI site allowed for XylE activity, suggesting that weak promoter sequences were locatedwithin the lgtE coding sequence . Overall, these data suggestthat multiple promoter sequences must occur within the lgtABCDE coding sequence.

 
Identification of transcriptional start sites. Initial attempts to identify the size of transcripts producedfrom the lgtABCDE region by Northern hybridization experimentswere unsuccessful . However, the XylE expression data describedabove suggest that the reason for this failure is due to thelow level of mRNA that would correspond to this region . Therefore,we used RACE, a more sensitive approach, to identify putativetranscriptional start sites.

RACE products were analyzed on an agarose gel [Fig . 5] . In mostof the lanes, multiple DNA fragments were generated by the RACE reactions . These fragments represent transcriptional start sites [TS] within the lgtDE region or RNA polymerase pause sites. The sequence of the RACE products was determined, and the upstream DNA sequences were analyzed for homology to known promoter sequences. Six of the seven sites identified by RACE corresponded to upstream sequences that showed between 50 and 79% homology with the consensus gearbox promoter sequence of E . coli [1, 9, 29] . The sequenceupstream of the seventh fragment [TS-3626] showed overlappingsequences with 58 and 75% homology to the ⁷⁰ consensus of Neisseriaspp . and the ²⁸ consensus of E . coli, respectively [Fig . 6] [4, 6] . In light of the fact that promoters with less homologyhave frequently been shown to have high expression levels, theseresults indicate that the identification of these sites as promotersis realistic.

 
Due to the extensive homology between lgtA and lgtD and between lgtB and lgtE, we hypothesized that promoters found in lgtDEmay have homologous counterparts in lgtAB . We examined the lgtABsequence for homology within the identified regions . Of theseven promoter sequences identified within lgtDE, only TS-3626had an identical counterpart within lgtAB . Two other sequencesshowed homology with a two-base mismatch [corresponding to TS-4267and TS-4493] . The remaining regions had counterparts in whichthree or more bases mismatched in a manner which gave them lesshomology to the consensus promoter sequence.

 
The lgtABCDE gene cluster encodes most of the genes needed to make the gonococcal -oligosaccharide [20] . Our data indicatethat a complex transcriptional control mechanism is responsiblefor regulating the expression of lgtABCDE . First, we demonstratethat multiple promoter regions exist throughout this cluster.Second, we show that the lgtABCDE genes are transcribed at minutelevels relative to the transcriptional expression of lsi-1 [rfaF] glycosyl transferase [see Table 4] . Subtle differences in the level of transcription appear to modulate the LOS phenotypein terms of the identity and relative amounts of each of thechemotypes that are simultaneously surface expressed.

The interposon is a spectinomycin cassette [aadA] flanked by rho-independent transcriptional terminators derived from bacteriophage T4 . The introduction of the interposon in either orientationresults in at least a 1,000-fold reduction in transcription,causing polar mutations [17, 18] . This interposon cassette hasbeen used to determine linkage relationships and to map thelocation of promoters in a number of organisms, including N.gonorrhoeae [12, 17, 37, 42] . Using interposon insertions withinthe lgtABCDE region, we were able to generate evidence thatsupports the presence of multiple promoters.

As the interposon is placed more proximal to lgtE, a greaternumber of transcripts are terminated at the interposon insertionsite and overall transcription of lgtE is reduced, hence alsothe lower amounts of LgtE . When lgtE is transcribed in limitingamounts, not enough LgtE is produced to process all of the LOSprecursors before they are surface expressed . Using xylE asa reporter gene for measuring lgtE transcription indicated thatthe overall level of transcription of this gene cluster is quitelow . The level of XylE expression at all insertion points withinthis region is very low . This can be seen when we compared thelevel of expression of XylE to that seen when this gene waslinked to another LOS biosynthetic gene, rfaF [14] . Expressionof this gene is almost 1,000 times greater than what is seenfor the two lgt [xylE] insertions . Our data show that lgtE transcription[Fig . 4] and LgtE activity [Fig . 2] decreases if the interposonis inserted within lgtABCDE . The amount of the decrease is moreif the interposon is inserted closer to the start codon oflgtE . This decrease in LgtE activity is seen as an increasein the proportion of LgtE LOS between any two interposon insertionmutants, which occurs between five of the six mutants examined.Therefore, promoter activity originates between each pair ofadjacent interposon insertion sites [see Fig . 2].

Seven potential transcriptional start sites were identified between the ClaI and EcoRI interposon insertion sites by usingRACE analysis . Each of these transcriptional start sites contained-10 and -35 upstream regions that possessed between 50 and 79%homology to consensus E . coli and Neisseria promoter sequences[see Fig . 5 and 6] . Three of these TS had homologous promoter sequences within the lgtAB region . The location of these putative promoters identified by RACE to be within lgtDE is consistent with the phenotypes and reporter gene activities of the F62 lgtE [xylE] interposon insertion mutants . The identificationof a transcriptional start site within the lgtE coding sequence[TS-4493] indicates a promoter which, without further rationalization,appears to serve no biological function in this strain . However,the existence of this promoter within lgtE is logical in termsof evolutionary descent because extensive homology exists foundbetween lgtB and lgtE . Any existing promoters are likely tobe shared between these two genes . It has been demonstratedthat intergenic recombination occurs between lgt genes, givingrise to recombinant lgt loci [5, 28] . Therefore, the putativepromoter upstream of TS-4493 may have had biological functionin the context of lgtB transcription.

The identification of six out of seven putative promoter regions as gearbox promoters is consistent with recent studies and withdata from the present study . In N . gonorrhoeae, the gearbox promoter has been implicated in the expression of two virulence factors, AniA [26] and Tpc [19] . Gearbox promoters generallyexpress at a strength that is inversely proportional to growthrate [52], a finding consistent with our observations that transcriptionof lgtE [xylE] increases fourfold between the mid-logarithmicand early stationary phase [data not shown].

Most significantly, we demonstrated here that limiting the transcription of lgtE leads to simultaneous production of two LOS chemotypes in a balance that is contingent upon the level of transcription. Assays of the xylE fusion strains allowed us to correlate visible proportions of LgtE⁺ and LgtE LOSs with measured levels of transcriptionalexpression [Fig. 2 and 4] . In addition, these results show thatthe lgtABCDE genes are transcribed at very low levels, demonstratingthat subtle changes in transcription are likely to incur significantphenotypic changes.

The transcriptional fusion data also support an earlier studythat demonstrated that a strain of N . gonorrhoeae FA19 withlgtA frameshifted to the "off" position, continued to producetrace amounts of LgtA⁺ LOS [11] . Burch et al . speculated thattranscriptional and/or translational strand slippage was occurringat a frequency just high enough to allow for visible amountsof LgtA⁺ LOS . Transcriptional strand slippage by the RNA polymeraseholoenzyme would result in a small amount of in-frame lgtA transcript,whereas translational strand slippage of out-of-frame mRNA bythe ribosomes would allow for the production of in-frame LgtAprotein . Since the present study demonstrates that small changesin the amount of LgtE dramatically impact the nature of theexpressed LOS, it is feasible that a low level of transcriptionalor translational strand slippage of lgtA mRNA could result insufficient LgtA to account for the LgtA⁺-LgtA^- mixed phenotypeof these strains.

From our data, we are able to formulate a logical explanationfor why N . gonorrhoeae F62 normally produces a mixture of the 1-1-M and 1B2 LOS chemotypes . The LgtD glycosyl transferase facilitates the addition of GalNAc to the terminal Gal of the chain, converting the chain from the 1B2 chemotype to the1-1-M chemotype . Our data show that the lgtD gene is transcribedat a level 1,000-fold less than that of lsi-1 [rfaF] [14] . The low level of lgtD transcriptional expression is such that a distribution of both LgtD⁺ [1-1-M] and LgtD^- [1B2] LOS chemotypesare produced . If transcriptional expression of lgtD were tobe increased, then we would predict a phenotypic shift toward producing a greater proportion of the 1-1-M chemotype.

Control of LOS phenotype by limiting transcription is the likely mechanism in spontaneous L1-reactive phase variants of N . gonorrhoeae F62 . These variants typically express a mixture of immunotype L1 [LgtC⁺] and other [LgtC^-] LOS chemotypes [D . C . Stein, unpublisheddata] . The data from the present study suggests that in thisstrain, lgtC is transcribed in amounts such that insufficientLgtC is being produced to catalyze all its available substrate.It is also possible that lgtC is transcribed and translatedin strains that fail to make the L1 LOS but that the enzymeis outcompeted for substrate by LgtA.

Sequence analysis of the lgtABCDE region using the Neural Network algorithm [40] revealed 29 sequences that share significanthomology with the ⁷⁰ promoter consensus of E . coli and Neisseriaspp . Remarkably, the polyguanine regions of the lgtA, lgtC, and lgtD genes were among the highest scorers, suggesting that the polyguanine tracts may affect promoter functions . Theseregions all had clearly identifiable -35 and -10 regions, butthe spacing between these regions was too long to suggest thatthese sequences were functional in these genes . There are otherexamples of promoter regions spanning homopolymeric runs inthe pathogenic neisseriae . The porA and opc genes of N . meningitidis, respectively, contain a polyguanine and polycytosine run betweenthe -35 and -10 consensus s⁷⁰ sequence [44, 51] . Frameshiftingin these runs alters the spacing between the -35 and -10 sequencesand leads to phase shifting between high, low, and intermediatetranscriptional levels . It is feasible that this same mechanismmay be occurring within the lgtABCDE locus and that these promotersare up- or downregulated based upon heritable changes to theirsequence.

Our model is consistent with observations made by researcherswho have shown that LOS expression is modulated by these environmental stimuli: growth rate, pH, aerobic versus anaerobic conditions,and carbon source [glucose versus lactate versus pyruvate] [16, 30, 33, 35] . At present, the genetic mechanism that mediatesthis variation in expression is unknown . Most of these promotersidentified by the present study are gearbox promoters, whichexhibit expression at levels inversely proportional to growthrate . Since environmental changes can either increase or decreasethe growth rate, changes in the environment would influencethe level of expression from these promoters . Alternatively,additional epistatic factors may interact with these promotersand contribute to LOS expression in response to environmentalconditions.

Our cumulative understanding of LOS phase variation in N . gonorrhoeae can be summarized as follows: N . gonorrhoeae regulates LOS biosynthesisand phase variation via a variety of disparate mechanisms . Thespecific LOS components produced by a particular strain aredefined by on-off strand slippage of homopolymeric tracts withinthe lgtA, lgtC, lgtD, and lgtG genes [7, 13] . Simultaneous production of multiple LOS epitopes is mediated by production of limiting amounts of the LgtA, LgtD, or LgtE . This occurs via transcriptional or translational strand slippage [11], via regulation of transcriptionalexpression [the present study], and via the low kinetic efficienciesof specific glycosyl transferases [36] . In addition, recombinationbetween glycosyl transferases is seen in some strains of Neisseriaand these recombinant loci may consequently invoke one or moreof the above mechanisms of phase variation [5, 36].

 
We thank Peijun He and Anne Corriveau for excellent technical assistance.

This study was supported by a grant from the National Institutes of Health to D.C.S . [AI24452] and an F31 fellowship to D.C.B. [AI09636].

 
* Corresponding author . Mailing address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742 . Phone: [301] 405-5448 . Fax: [301] 314-9489 . E-mail: dcstein@umd.edu .

Present address: Department of Biology, Gallaudet University, Washington, DC 20002.

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