Patent Application: US-57446000-A

Abstract:
compositions and methods for making complex carbohydrates in a bacterial production cell are disclosed . the complex carbohydrates that can be made include oligosaccharides and polysaccharides of bacterial or mammalian origin .

Description:
the present invention provides a method by which the terminal heptose of the core structure of any gram bacterial species which contains the gene rfe ( udp - glcnac : undecaprenol glcnac - 1 phosphate transferase ) ( alexander et al . ( 1994 ) j . bacteriol ., 176 - 7079 - 7084 ) can be modified so as to act as an acceptor for oligosaccharide synthesis . the rfe gene encodes for a protein which catalyzes the transfer of n - acetyl glucosamine ( glcnac , an “ acceptor molecule ”) onto the carrier lipid undecaprenol phosphate . the regulation of this gene can be controlled with a regulatory gene , lsgg , identified in haemophilus influenzae . the increase in rfe expression caused by lsgg mediates the deposition of a glcnac residue on the terminal heptose of lps and los from a variety of gram - negative bacterial species including e . coli , salmonella minnesota and h . influenzae . this glcnac has been found to function as an acceptor molecule forming a scaffold for the sequential addition of saccharide monomers , under the direction of glycotransferases . for example , the galactosyltransferase gene , lsgf , results in the addition of a galactose to the glcnac . the gene sequence coding for the lsg glycotransferases of h . influenzae has been inserted into an escherichia coli k - 12 strain production cell , with the resultant production of h . influenzae - specific los epitopes in e . coli . any production cell containing an initiating enzyme similar to rfe can add an appropriate acceptor to form the scaffold . the production will preferably also contain the regulatory gene lsgg . dna coding for the glycotransferases of other species , strains , tissues , hormones , receptors or other cell - surface carbohydrates can then be inserted into such a production cell , with the resultant production of oligosaccharides or polysaccharidea specific to that species , strain , tissue , hormone , receptor or other cell - surface carbohydrate . complex carbohydrates : any polymer of formula ( ch 2 o ) n , where n equals at least three monomer units , including polymers with additional substituents including but not limited to so 4 , po 4 , co 4 , ch 3 , nh 4 , and such polymers linked to lipids , peptides and proteins . production cell : a production cell useful in this invention is defined as any bacterium which contains an lps or los - saccharide inner core terminating in a molecule and containing an enzyme capable of adding an acceptor molecule to the terminal molecule to serve as a scaffold for elongation and which can be transformed with exogenous dna coding for glycosyltransferases . cells that are otherwise suitable but lack the proper acceptor molecule may be used as production cells if they are co - transformed with genes such as rfe and lsgg , to appropriately modify the lps or los to function as an acceptor molecule for the formation of a scaffold . hib production cell : the preferred cell for production of h . influenzae type b - specific os is preferably a gram - negative bacteriuim , most preferably e . coli k - 12 strain jm 109 . synthetic gene ( s ): the dna coding for the enzyme or enzymes that synthesize the desired complex carbohydrate . these genes include those coding for glycotransferases , lyases , acetylases , sulfatases , phosphorylases , kinases , epimerases , methylases and the like . capsular strains of haemophilus influenzae type b ( hib ) are responsible for various invasive and bacteraemic infections in humans , including meningitis and pneumonia . the surface lipooligosaccharides ( los ) of hib are known to be important factors in microbial virulence and pathogenesis . structural studies of hib los from wild - type and mutant strains have shown that the los contains a conserved heptose trisaccharide core which can be extended with additional sugars on each heptose . recently , a revised structure of the e . coli k - 12 core region was reported which also contains a heptose trisaccharide inner core and a fourth heptose present on the terminus of the main oligosaccharide branch : previous work showed that the core region of e . coli transformed with synthetic enzyme genes could be elongated by the addition of saccharide monomers under the direction of h . influenzae genes to produce a modified e . coli lps that was elongated by approximately five monomer units . it was thought that the monomers were added at each of the heptoses . ( kwaik et al ., molecular microbiology , 5 : 2475 - 2480 ( 1990 ).) therefore , efforts were made to transform an e . coli k - 12 strain termed jm 109 with h . influenzae synthesis genes in an attempt to determine whether an los substantially identical to that of h . influenzae could be produced . escherichia coli strains were routinely cultured at 37 ° c . using lb agar or broth with appropriate antibiotics . vectors used in these studies were previously described . ( kwaik et al . ( 1990 )). lps from e . coli jm 109 was prepared by the extraction procedure of darveau and hancock ( darveau et al . j . bacteriol . 155 ( 2 ), 831 - 838 ( 1983 ).) the lps was separated by sds - page in resolving gels containing 15 % acrylamide , and visualized by silver staining . to determine the structure of this chimeric lps , a few milligrams of lps from each sample were treated with anhydrous hydrazinc at 37 ° c . for 20 minutes , and then precipitated with cold acetone . in order to establish the chemical structure of the e . coli core and determine the e . coli acceptor residue , the lps from e . coli strain jm 109 was partially characterized using composition analysis , linkage analysis , and mass spectrometry as described in example 6 below . hib strain a - 2 was originally isolated from the spinal fluid of a child with meningitis . hib a - 2 was grown on chocolate agar supplemented with amino acids and vitamins or brain heart infusion agar supplemented with 4 % fildes reagent ( difco laboratories , detroit ) at 35 ° c . in 5 % co 2 atmosphere . a gene cluster from hib strain a - 2 containing los synthesis genes ( lsg ) was previously cloned . ( kwaik et al , ( 1990 ). the lsg loci are contained within a 7 . 4 kb dna fragment , consisting of seven complete open reading frames ( orfs ). this region is one of several distinct loci also found in the genome sequence of hib strain rd which has been associated with lipopolysaccharide ( lps ) biosynthesis . dna from the lsg region of hib strain a - 2 was used to construct a genomic library in the lambda bacteriophage embl3 ( kwaik et al , 1990 )). twenty six phage clones were prepared which expressed hib los oligosaccharide epitopes in e . coli strain le392 . the phage transformant designated emblos - i produced a chimeric lps with a 1 . 4 kda oligosaccharide added to the 4 . 1 kda lps of e . coli le392 . monoclonal antibody ( mab ) 6e4 , which recognizes two components in the hib a2 los mixture , also recognized the novel 5 . 5 kda component in the chimeric lps , indicating some immunochemical similarity to hib los . restriction fragments of emblos - i were used to make a series of plasmids which modified e . coli strain jm 109 to give clones which produced a proposed chimeric series of higher mass lps species . the transformants termed pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 generated modified or chimeric lps of 5 . 5 , 5 . 1 , and 4 . 5 kda , respectively . all three apparently modified the 4 . 1 kda lps species from e . coli , although only the lps from pgemlos - 4 expressed the 6e4 epitope . the lps from strain pgemlos - 5 was found to react positively with mab 3f 11 , suggesting the presence of terminal n - acetyllactosamine . the epitope recognized by mab 6e4 is also present in the los of h . influenzae nontypable strain 2019 , as well as the lps from salmonella minnesota re mutant . binding of this monoclonal antibody to h . influenzae los can be inhibited by kdo and the kdo trisaccharide from the re mutant of s . minnesota . because the 6e4 epitope has been associated with the core of haemophilus los , it was originally proposed that the chimeric structures expressed in e . coli might arise from the addition of a haemophilus core structure to an acceptor residue of the e . coli 4 . 1 kda lps species . the hib production cell was transformed with the plasmid pgem3zf + into which different dna restriction fragments from h . influenzae strain a - 2 lsg locus had been ligated ( see table 1 and fig1 ). the plasmid pgemlos - 4 contained a 7 . 4 kb bamhl - pstl fragment of dna which contained all seven open reading frames ( a - g ) comprising the lsg locus . the plasmid pgemlos - 5 contained a 5 . 5 kb hind lll - pstl fragment of dna comprising 5 open reading frames ( c - g ) of the lsg . the plasmid pgemlos - 7 contained a 2 . 8 kb sphl - pstl fragment of dna comprising 2 open reading frames ( f - g ) of the lsg locus . the plasmid pgem3zf + without an insert was also transformed into strain jm 109 . this strain and the lps isolated from it were termed pgem . the lps from pgem ( 31 mg ), pgemlos - 4 ( 25 mg ), pgemlos - 5 ( 15 mg ), and pgemlos - 7 ( 4 . 4 mg ) was hydrolyzed in 1 % acetic acid ( 2 mg lps / ml ) for 2 hours at 100 ° c . the hydrolysates were centrifuged at 5000 g for 20 min at 4 ° c . and the supernatants removed . the pellets were washed with 2 ml of h 2 o and centrifuged again ( 5000 g , 20 min , 4 ° c .). the supernatants and washings were pooled and lyophilized to give the oligosaccharide fractions . as a standard , 10 mg of lps from salmonella typhimurium tv 119 ra mutant ( sigma , st . louis ) was treated in the same fashion . to prepare desalted oligosaccharide pools for esi - ms analysis , small aliquots of the crude oligosaccharide fractions (& lt ; 2 mg ) were chromatographed on two bio - select sec 125 - 5 hplc columns ( bio - rad , richmond , calif .) connected in series , using 0 . 05 m pyridinium acetate ( ph 5 . 2 ) at a flow rate of 1 ml / min . a refractive index detector was used to monitor column effluent and chromatograms were recorded and stored with an integrator . for large scale separations , the oligosaccharide fractions from pgem ( 10 . 2 mg ), pgemlos - 4 ( 9 . 3 mg ), and pgemlos - 5 ( 7 . 0 mg ) were dissolved in 0 . 3 ml of 0 . 05 m pyridinium acetate buffer ( ph 5 . 2 ) and centrifuge - filtered through a 0 . 45 gm nylon - 66 membrane . the pgem and pgemlos - 4 samples were applied to a single bio - gel p - 4 column ( 1 . 6 × 84 cm , & lt ; 400 mesh ; bio - rad ), and the pgemlos - 5 sample was applied to two bio - gel p - 4 columns connected in series ( 1 . 6 × 79 cm and 1 . 6 × 76 . 5 cm ). the columns were equipped with water jackets maintained at 30 ° c . upward elution at a flow rate of 10 ml / h was achieved with a p - 1 peristaltic pump ( pharmacia , piscataway ). effluent was monitored with refractive index and fractions were collected at 10 minute intervals and evaporated to dryness in a concentrator . oligosaccharide fractions were placed in 1 . 5 ml polypropylene tubes and treated with cold 48 % aqueous hydrogen fluoride to make 5 - 10 μg / ml solutions . samples were kept for 18 hours at 4 ° c . and then aqueous hf was evaporated the dephosphorylated samples were then rechromatographed on two bio - select sec 125 - 5 hplc columns connected in series using 0 . 05 m pyridinium acetate ( ph 5 . 2 ). the reactivity with monoclonal antibodies raised to the naturally occurring hib los , as shown in example 2 , indicated that the product had the same immunochemcial function as the naturally occurring hib los . the samples were further analyzed by different techniques in order to determine structural identity to the desired complex carbohydrate . dephosphorylated oligosaccharide fractions were dissolved in 400 μl of 2 m trifluoroacetic acid and heated for 4 hours at 100 ° c . the hydrolysates were evaporated to dryness in a speed - vac concentrator , redissolved in 20 μl h 2 o , and dried again . hydrolysates were analyzed by high - performance anion exchange chromatography with pulsed amperometrie detection using a dionex biolc system ( dionex , sunnyvale , calif .) with a carbopac pa1 column . linkage analysis was performed on dephosphorylated oligosaccharide fractions using the microscale method modified for use with powdered naoh . partially methylated alditol acetates were analyzed by gc / ms in the e1 and c1 modes on a mass spectrometer . lsims was performed using a mass spectrometer with a cesium ion source . oligosaccharide samples ( in 1 μl h 2 o ) were added to 1 μl of glycerol / thioglycerol ( 1 : 1 ) on a stainless steel probe tip . a cs + ion primary beam energy of 10 kev was used and the secondary sample ions were accelerated to 8 kev . scans were taken in the negative - ion mode at 300 s / decade and recorded with an electrostatic recorder . the spectra were mass calibrated manually with ultramark 1621 ( pcr research chemicals , inc ., gainesville , fla .) to an accuracy of better than ± 0 . 2 da . oligosaccharides and o - deacylated lps were analyzed on a mass spectrometer with an electrospray ion source operating in the negative - ion mode . oligosaccharide samples were dissolved in h 2 o mixed with running solvent ( 1 μl in 4 μl ), and injected into a stream of h 2 o / acetonitrile ( 1 / 1 , v / v ) containing 1 % acetic acid , at a flow rate of approx . 20 μl / min . mass calibration was carried out with csi in the negative - ion mode . in some cases , selected oligosaccharide fractions were analyzed at higher resolving power ( m / δm = 2000 ) using a sector - orthogonal time of flight ( tof ) instrument with an array detector operating under esi conditions in the negative - ion mode . in this case , the solvent system and flow rate were essentially the same as described above for the quadrupole esi experiments . a scan speed of 5 sec / decade was used for all samples over the m / z range of 500 to 3000 with an accelerating voltage of 4 kv and an esi needle voltage of between 3 . 5 - 4 kv higher . mass calibration was carried out with an external reference consisting of csl taken under liquid secondary ion mass spectrometry conditions , followed by a one point correction of the doubly charged deprotonated molecular ion of the oligosaccharide from the lps of salmonella typhimurium ra mutant (( m − 2h ) 2 − exact = 973 . 2 )) in the negative - ion es - ms mode . o - deacylated lps samples were analyzed on a voyager or an elite maldi - tof instrument ( perseptive biosystems , framingham , mass .) equipped with a nitrogen laser ( 337 nm ). all spectra were recorded in the negative - ion mode using delayed extraction conditions as described in detail elsewhere . ( gibson et al . j . amer . soc . mass spec . 8 : 645 - 658 ( 1997 )). samples were dissolved in h 2 o ( approx . 250 pmol / μl ), and mixed 1 : 1 with the matrix solution ( a saturated solution of 2 , 5 - dihydroxybenzoic acid in acetone ) and allowed to dry at room temperature on a gold - plated maldi plate . approximately 100 laser shots were recorded for each sample , averaged and then mass calibrated using an external mass calibrant consisting of renin substrate tetradecapeptide , insulin chain b , oxidized , and bovine insulin ( all from sigma ). for external calibrations under these conditions , a mass accuracy of 0 . 1 % was obtained for comparison purposes , a single point correction was made to the spectra of the o - deacylated lps from pgem using the expected lipid a fragment ion (( m − h ) average = 952 . 009 ), and then the spectra for the three chimeric strains were recalibrated using this lipid a fragment ion and an additional ion from pgem ( m / z 2835 . 7 ) present in all four samples . dephosphorylated oligosaccharides were analyzed in the positive - ion mode on a mass spectrometer equipped with a nanospray ion source . the analyzer consists of a high pressure rf - only ion guide followed by a quadrupole mass filter . a high pressure quadrupole collision cell follows the first mass filter . the tof mass analyzer is comprised of a reflection with an effective flight path of 2 . 5 meters . samples were dissolved in h 2 o / acetonitrile ( 1 / 1 , v / v ) containing 1 % acetic acid , and 2 μl of each was injected into a nanospray tip . the nanospray needle voltage was typically 800 - 1000 v . one sample loading usually gave an analysis time of 30 - 40 min , which allowed a conventional mass spectrum to be obtained prior to the selection of several individual ions for cid ms / ms . in ms mode the high resolution capability ( 8 , 000 fwhm ) allowed unambiguous determination of the charge state for each ion . for cid - ms / ms operation the quadropole mass analyzer with a mass window of i m / z unit was used to select precursor ions for fragmentation , which in most cases were doubly charged ( m + 2h ) 2 + . the selected ions were fragmented in a collision cell with air as the collision gas and analyzed in the orthogonal tof operating at an accelerating potential of 20 kv . fragment ion spectra were accumulated under computer control for periods of between 10 seconds and 1 minute . mass assignments based on external calibration were generally within 50 ppm of calculated monoisotopic values whereas internal calibration gave masses accurate to + 5 ppm . we have previously reported the transformation of e . coli strain jm 109 ( a k - 12 strain which produces rough lps ( r - lps ) which lack the o - side - chain ) with a series of plasmids containing overlapping restriction fragments of dna from the lsg region of h . influenzae type b strain a - 2 ( kwaik et al , 1990 ). partial los segments were produced . as diagrammed in fig1 , the pgemlos - 4 clone contains all of the complete orfs ( orfs a - g ) in the lsg region , whereas pgemlos - 5 contains orfs c - g , and pgemlos - 7 contains orfs f - g . the clones pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 were shown by sds - page to produce modified lps structures which added 1 . 4 , 1 . 0 , and 0 . 4 kda moieties , respectively , to the 4 . 1 kda e . coli core ( fig2 ). for initial screening of lps molecular weights and heterogeneity by mass spectrometry , small aliquots of lps from pgem , pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 were treated with anhydrous hydrazine to remove o - linked fatty acids from the lipid a moiety . the pgem o - deacylated lps sample contains several species in the range of 2738 - 3172 da , representing the major e . coli core structures . when fit to proposed compositions , the observed species were found to exhibit heterogeneity in heptose ( hep ), hexose ( hex ), 3 - deoxy - d - mannooctulosonic acid ( kdo ), phosphate ( phos ), and phosphoethanolamine ( pea ) ( table 2 ). specifically , two main core types were observed containing either 3 hex and 3 hep ( with 2 or 3 kdos ) or 4 hex and 4 hep ( with 2 kdos ), with variable amounts of phosphate and pea in both . the pgemlos - 7 o - deacylated lps mixture contained many of these same species , in addition to two major new species at ( m − h )- 3334 . 5 and 3456 . 8 . the m / z 3334 . 5 species apparently arises from the addition of hex and n - acetylhexosamine ( hexnac ) to the pgem core structure containing 4 hex , 4 hep , 2 kdo , 2 phos , and 1 o - deacylated diphosphorylated lipid a ( o - dpla ) moiety . a further addition of 1 pea moiety gives the m / z 3456 . 8 species . these data suggest that the transformation producing pgemlos - 7 results in the addition of a hex - hexnac moiety to the e . coli lps . likewise , the main species in the pgemlos - 5 o - deacylated lps ( m / z 3700 . 6 and 3823 . 6 ) were found to arise from the addition of 2 hex plus 2 hexnac to the pgem core structure containing 4 hex , 4 hep , 2 kdo , 2 phos , 1 o - dpla , and 0 or 1 pea ( see table 2 ). these structures are also found in the pgemlos - 4 o - deacylated lps , in addition to new species arising from the further addition of either another hex ( m / z 4083 . 2 and 4206 . 4 ) or hexnac ( m / z 4124 . 5 and 4246 . 8 ) to , in this case , the pgem core structure containing 4 hex , 4 hep , 3 kdo , 2 phos , 1 o - dpla , and 0 or 1 pea ( see table 2 ). of the chimeric lps structures , only these high molecular weight pgemlos - 4 components contained the third kdo moiety . the lps from pgem , pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 were subjected to mild acid hydrolysis to liberate free oligosaccharides . initially , small aliquots of the oligosaccharide fractions were desalted by size exclusion hplc and analyzed as mixtures by negative - ion esi - ms . the esi - ms spectra contained predominantly doubly charged ions , ( m − 2h ) 2 − . in general , the data were consistent with results from the maldi - tof analysis of o - deacylated lps . the pgem sample was found to contain seven major oligosaccharides and several minor species , ranging in molecular weight from 1459 . 3 to 2016 . 7 da . as shown in table 3 , proposed compositions were determined for the various species which indicated that the structures consisted of two main core types ; one containing 3 hex , 3 hep , and 1kdo , and another containing 4 hex , 4 hep , and 1 kdo . variability in the number of phosphate and pea groups was responsible for the large number of species present in the mixture . the pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 samples contained many of the species found in the pgem sample , in addition to larger molecular weight oligosaccharides ( table 3 ). new lps glycoforms of m r 2177 . 7 and 2302 . 5 were observed in the pgemlos - 7 sample , consistent with the addition of a single hex and hexnac residue to the pgem core structure containing 4 hex , 4 hep , 1 kdo , 2 phos , and 0 or 1 pea the high molecular weight components of the pgemlos - 5 sample ( m r 2543 . 9 and 2666 . 5 ) suggested the further addition of yet another hex - hexnac unit , and the pgemlos - 4 sample contained even higher molecular weight materials ( ranging from m r 2706 . 1 to 2870 . 0 ) consistent with the addition of one more hex or hexnac moiety . to aid in the determination of proposed compositions for these species , oligosaccharides from the pgem , pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 samples were separated by size exclusion chromatography and fractions were analyzed by lsims and / or esi - ms . selected fractions representing the two major pgem core types and the various chimeric structures were then pooled , dephosphorylated with aqueous hf , rechromatographed on size - exclusion hplc , and analyzed again by negative - ion lsims or esi - ms . proposed compositions for the molecular ions observed after hf - treatment are listed in table 4 . upon removal of phosphate and pea moieties , the major high mass species present in the pgemlos - 7 sample is an oligosaccharide of m r ( avg .) 2020 . 3 ( 1hexnac , 5 hex , 4 hep , and 1kdo ). the pgemlos - 5 sample contains an oligosaccharide of m r ( avg .) 2386 . 3 , resulting from the further addition of 1 hex and 1 hexnac to the pgemlos - 7 lps ( 2 hexnac , 6 hex , 4 hep , and 1 kdo ). this species is also present in the pgemlos - 4 sample , in addition to higher molecular weight structures containing an additional hex ( m r ( avg .) 2548 . 4 ) or hexnac ( m r ( avg .) 2589 . 5 ). mass spectrometric analyses of the free oligosaccharides from pgem , pgemlos4 , pgemlos - 5 , and pgemlos - 7 indicated that the different chimeric structures arise from additions of stoichiometric amounts of hexose and hexnac residues to a variably phosphorylated pgem core structure containing 4 hex , 4 hep , and 1 kdo . no chimeric structures were observed to contain the 3 hex , 3 hep , and 1 kdo core . for comparison purposes , dephosphorylated oligosaccharide fractions containing the two pgem core types and the main chimeric structures from pgemlos - 4 , pgemlos - 5 , and pgemlos - 7 were hydrolyzed in 2n trifluoroacetic acid to determine their monosaccharide compositions , and therefore the identities of the hex and hexnac residues . when analyzed by high ph anion exchange chromatography with pulsed amperometric detection , the pgem hydrolysates were found to contain only galactose , glucose , and l - glycero - d - manno - heptose ( table 5 ). ( the kdo residue is not recovered under these hydrolysis conditions .) the two core types were identified as galglc 2 hep 3 and galgc 3 hep 4 . the pgemlos - 7 sample contained glcnh 2 gal 2 glc 3 hep 4 ( table 5 ), suggesting that the larger pgem core was being modified by the addition of one gal and one n - acetylglucosamine ( glcnac ) residue . likewise , the composition of the pgemlos - 5 sample suggested that the larger pgem core was being further glycosylated with only gal and glcnac residues . fraction 2 from pgemlos - 4 , which contained the same species as pgemlos - 5 , gave similar results , and fraction 1 from pgemlos - 4 , which contains three main species ( see table 4 ), contained slightly more glcnh2 . aliquots of the same six dephosphorylated oligosaccharide fractions used for monosaccharide composition analysis were taken for methylation analysis to establish sugar linkage positions . the partially methylated alditol acetates observed by gc / ms are listed in table 6 . again , by comparing the two pgem core types , it is relatively straightforward to see that the second terminal heptose of the larger pgem core is converted to a 1 , 7 - linked heptose in all of the chimeric structures and thus must represent the linkage site for the novel glycosylation . since no chimeric structures were observed with the hep 3 core , it is most likely that the nonreducing terminal heptose recently identified on the oligosaccharide branch in the k - 12 core structure is the modified terminal heptose . additionally , no new trilinked saccharides were obtained from the chimeric oligosaccharides , suggesting that the sugars were most likely all added in a straight chain . to confirm the identity of the linkage site between the e . coli lps core and the novel oligosaccharide moieties , and to determine the sequences of the added sugars , the dephosphorylated oligosaccharides were subjected to ms / ms analysis . for these experiments , samples were run in the positive - ion mode and doubly charged molecular ions , ( m + 2h ) 2 + , were selected for collision - induced dissociation ( cid ). various reducing - terminal ( y - type ) and non - reducing terminal ( b - type ) sequence ions are present in the spectra . for the pgem oligosaccharide , the y ion series including the y 6α , ( m / z 732 . 2 ( 2 +)), y α5 , ( m / z 651 . 2 ( 2 +)), and y 4α , ( m / z 1139 . 3 ) fragment ions , and the corresponding b ion series including the b 3α , ( m / z 517 . 2 ), b 4α , ( m / z 841 . 3 ), b 5 ( m / z 1225 . 4 ), and b 6 ( m / z 1417 . 4 ) fragment ions , support the published structure with the fourth heptose on the non - reducing terminus of the largest oligosaccharide branch . in addition to these sequence ions , several ions present in the spectrum apparently arise from internal cleavages , which can occur under high energy cid conditions . in the spectrum of the pgemlos - 5 oligosaccharide , two similar y and b - type ion series clearly define the sequence and linkage site of the added tetrasaccharide . intense b ions at m / z 366 . 1 ( b 2α ′ t ) and 731 . 3 ( b 4α ,) arise from the sequential cleavage of two hex - hexnac moieties . these losses are also represented by the corresponding y 9α ,′ ( m / z 2020 . 6 ) and 7α , ( m / z 1655 . 5 ) fragment ions . fragment ions at m / z 923 . 3 ( b 5α ,) 0 and m / z 1463 . 4 ( y 6α ,) confirm that the hex - hexnac - hex - hexnac moiety is linked to a heptose , and additional cleavages further along the large oligosaccharide branch confirm that the novel tetrasaccharide is attached to the largest branch of the pgem core structure . in the ms / ms spectra of the chimeric oligosaccharides from pgemlos - 7 and pgemlos - 4 , intense b ions also clearly defined the structures of the added sugar moieties . in the pgemlos - 7 oligosaccharide ( m r 2019 . 7 ), a b ion at m / z 366 . 1 corresponds to a single hex - hexnac disaccharide moiety . the pgemlos - 4 oligosaccharide of m r 2587 . 9 ( hexnac 3 hex 6 hep 4 kdo ) lost a hexnac - hex - hexnac fragment ( m / z 569 . 2 ) and a hexnac - hex - hexnac - hex - hexnac fragment ( m / z 934 . 3 ), whereas the pgemlos - 4 oligosaccharide of m r 2546 . 8 ( hexnac 2 hex 7 hep 4 → do ) lost a hex - hex - hexnac ( m / z 528 . 2 ) and a hex - hex - hexnac - hex - hexnac ( m / z 893 . 3 ,) fragment . in addition to those b - type ions , the latter spectrum also contained large ions at m / z 366 . 1 and 731 . 3 , which apparently arise as internal fragments in that case . assuming that the oligosaccharides are built up sequentially , i . e ., from pgemlos - 7 to pgemlos - 5 to pgemlos - 4 , the ms / ms data , in combination with our methylation analysis results , allows the partial structures of the chimeric oligosaccharides to be deduced as shown in fig3 . the structural data support the prediction that e . coli k - 12 transformed with plasmids containing portions of an eight gene segment from h . influenzae involved in los biosynthesis makes chimeric lps which can be modified to produce oligosaccharide essentially identical to that of h . influenzae . moreover , we have shown that the chimeric lps are segregated hybrid - type structures , where the e . coli r - lps core structure is first synthesized and then serves as a scaffold for h . influenzae los biosynthesis enzymes to add a second independent set of sugars not found in the parent e . coli strain . thus , the biosynthetic pathways appear to be sequential ( segregated ) and not intermixed . before this invention was made , the role of the terminal branch heptose in the e . coli r - lps as the acceptor for oligosaccharide elongation or the requirement for a funcitonal initiator enzyme was unknown . the published structure for the complete e . coli k - 12 core did not contain a second terminal heptose , but rather had this fourth heptose as part of the inner core region . the oligosaccharide branch was believed to terminate in glucose , which was proposed to be the acceptor site for o - antigen and other substituents . the role of the initiator enzyme was unknown . it is now apparent that only e . coli r - lps structures containing this fourth heptose ( i . e ., complete core structures ) underwent elongation in the plasmid - transformed chimeric strains and thus , only those e . coli having this composition are useful as production cells for the production of h . influenzae . in the chimeric structures , glcnac is the first sugar added to the seven position of this heptose . there are two possible explanations for this crucial first step in the elongation sequence . one , an n - acetylglucosamine - specific glycosyltransferase from haemophilus encoded in orff or orfg either has this precise specificity or is promiscuous enough to allow this reaction to occur . two , some analogous e . coli glycosyltransferase gene is being activated by a haemophilus regulatory gene . since sequence comparisons of the seven genes contained in this plasmid suggest that both glycosyltransferase and regulatory genes are present , both explanations are possible . however , the fact that terminal glcnac and 1 , 7 - linked heptose have been found in non - stoichiometric amounts in some other strains of e . coli k - 12 suggests that the addition of this sugar to the terminus of the oligosaccharide branch is accomplished by e . coli enzymes . it was recently reported that when mutations causing the rough phenotype in e . coli k - 12 are complemented , the complemented strains produce an o - antigen which has gicnac at the reducing terminus of the repeat unit . regardless of the mechanism of this first key step in the extension of the pgem core , it appears that addition of this glcnac is rate - limiting , since a large percentage of unmodified pgem core r - lps remains in the chimeric mixtures . furthermore , very little or no intermediate structures are observed as one progresses from pgemlos - 7 to pgemlos - 5 and pgemlos - 4 , suggesting that once this first glcnac is added , the addition of the other haemophilus - related sugars proceeds quickly to defined end points . if other steps in the biosynthesis of the chimeric lps were as incomplete as the addition of this first glcnac , one would expect to see these other intermediate structures , yet none were observed . therefore , it is likely that a n - acetylglucosaminyltransferase from e . coli that is regulated through the product of orff or orfg adds this first key sugar in the chimeric structures . preliminary data on a chimeric construct containing orfg alone showed a mass shift of 203 da ( hexnac ), suggesting that orfg encodes this regulatory gene . the second step in the biosynthesis of the chimeric lps is the addition of galactose to the 3 - position of the terminal glcnac . the resulting disaccharide , gal13glcnac , is the structural moiety observed in pgemlos - 7 , which arises when the transforming plasmid contains orfs f - g from haemophilus . examination of the predicted amino acid sequences of the gene products indicates that orff has high homology ( 66 % identity ) to a galactosyltransferase ( asme ) from erwinia amylovora , suggesting that it may encode a galactosyltransferase in haemophilus . orfg does not show any homology to known oligosaccharide biosynthetic genes , but is homologous ( 64 % identity ) to a gene encoding the mode protein in e . coli . this protein is involved in molybdenum transport and regulation of transcription , suggesting that orfg may encode a regulatory protein from haemophilus ( which may be regulating an n - acetylglucosaminyltransferase gene from e . coli in the chimeric strains ). in the pgemlos - 5 strain , an additional three genes are contained in the transforming plasmid ( orfs c - g ) and an additional glcnac and gal are observed in the resulting lps . these sugars now define the tetrasaccharide gall - 4glcnac - 3gal13glcnac . the lps from this transformant is now reactive to the 3f11 mab , suggesting that this new disaccharide is betalinked to form the terminal trisaccharide , gal14glcnacl - 3gal . all of the new orfs contained in this plasmid have some homology with known glycosyltransferase genes : orfc has homology with the asmd ( 26 % identity ) from erwinia amylovora , which encodes a glycosyltransferase for exopolysaccharide synthesis , and trsd ( 38 % identity ) from yersina entercolitica , a gene involved in lps inner core synthesis , orfd has homology with the sialyltransferase gene ( lst ) ( 27 % identity ) from neisseria gonorrhoeae , and orfe has homology with a putative glycosyltransferase gene ( 77 % identity ) from actinobacillus sp . and the galactosyltransferase gene , amsb ( 27 % identity ) from erwinia amylovora . the fact that these three additional orfs in the transforming plasmid apparently result in the addition of only two more sugars to the growing oligosaccharide chain may indicate that the acceptor for one of the glycosyltransferases is absent in the chimeric lps . when two more orfs are added in the transforming plasmid ( orfs a - g ) to form the pgemlos - 4 chimeric strain , we observe that the 3f11 epitope disappears and the terminal gal residue of the epitope is capped by either a second gal or a glcnac moiety , apparently linked to the 6 - position of the gal . these new species present in the pgemlos - 4 lps population were also observed to contain a third kdo moiety , presumably somewhere in their core regions . while some of the incomplete core structures found in the wild - type e . coli k - 12 lps populations also contain a third kdo , of the chimeric structures , only the structures unique to pgemlos - 4 were found to contain a third kdo . this chimeric strain was also recognized by mab 6e4 which recognizes an inner core , kdo - related epitope in h . influenzae , suggesting that this third kdo forms a different epitope than the one found in the core structure of the wild - type e . coli lps . thus , the addition of orfs a and b to the transforming plasmid which fon - ned strain pgemlos - 4 seems to have multiple effects on the chimeric lps structure . orfb is homologous ( 46 % identity ) to the sialyltransferase gene from n . gonnorhea . orfa is homologous to both the rfb x gene product ( 22 % identity ) from e . coli and trsa ( 24 % identity ) of y . entercolitica . these are putative o - antigen transporters ( 36 , 37 ), suggesting that orfa may encode a flippase . while sialyl - n - acetyllactosamine - containing structures are only minor components of the wild - type h . influenzae type b strain a2 los population , we have previously seen that lsg genes are involved in the synthesis of this epitope . transposon mutagenesis of orfd produced mutant strain 281 . 25 , that lost all ability to add galactose to hib los glycoforms . this strain could not make any of the wild - type los structures larger than the major species containing four glucoses and three heptoses . mutation of orfe ( which is downstream of orfd ) produced strain 276 . 4 which had essentially the same defect , except for one important difference : strain 276 . 4 retained the ability to make the sialyl - n - acetyllactosamine epitope . these results suggest that in the transposon mutants , the knockout of orfd has a polar effect on orfe , which would imply that the gene product of orfe is a galactosyltransferase required for synthesis of the higher molecular weight wild - type structures containing terminal galactose ( s ) on their glucose disaccharide branches and the gene product of orfd is likely an n - acetylglucosaminyltransferase required for the synthesis of the sialyl - n - acetyllactosamine epitope . the case for these assignments can be made on the basis of the homologies noted above ( orfe is homologous to a galactosyltransferase gene ) and the los glycoforms observed in the 276 . 4 and 281 . 25 mutant strains . since no truncated versions of the sialyl - n - acetyllactosamine structure were seen in the 276 . 4 los population ( i . e . ; no species lacking either sialic acid or sialic acid plus galactose ), it seems probable that the orfd gene codes for the glycosyltransferase which adds the glcnac to the oligosaccharide branch . this is also consistent with the observation that one of the genes in orfd c - e is apparently responsible for adding glcnac to the 3 - position of the gal which is terminal in the pgemlos - 7 lps structure . this chimeric carbohydrate expression system has provided information that is relevant to unraveling the functions of these lsg genes and has the additional advantage of being carried out in the absence of the normal endogenous genetic background on h . influenzae . indeed , while gene knockouts of some of the lsg genes in h . influenzae have been completed , downstream or regulatory gene effects can often complicate their functional analysis . in this e . coli expression system , structural analysis of the resulting chimeric lps has shown that synthesis proceeded as a serial ( non - parallel ) synthesis , that is , the new elements of the chimeric lps were added after the formation of the e . coli r - lps . the fact that this synthesis was sequential ( rather than interdigitated with the r - lps synthesis , for example ) allowed for the functions of these h . influenzae gene products to be more readily delineated from the chimeric oligosaccharide structures . moreover , screening of the chimeric lps products with monoclonal antibodies enabled us to follow the formation of terminal sugar sequences ( epitopes ) that are unique to the haemophilus strain from which the plasmid dna originated . all publications and patents cited herein are incorporated by reference as though fully set forth . this invention has been described with respect to specific examples and embodiments . however , it is understood that one skilled in the art may make variations or modifications that are within the spirit and scope of the invention . a relative ion abundances ( given in parentheses ) represent the sum of the monoisotopic molecular ion and anhydro peaks for a given species ( see fig4 ). components listed above the dashed line are chimeric structures . a molar ratios were derived from comparison to the hydrolysate of the s . typhimurium ra oligosaccharide of known composition , and then those values were normalized to either 3 . 0 or 4 . 0 heptoses in each fraction . a peak areas were measured from the gc / ms ei total ion chromatograms , and values were normalized to the 1 , 3 , 6 - glc residue . the data for pgemlos - 7 are the average of two runs . a when lsga and lsgb are both present , the pgemlos - 5 tetrasaccharide structure can be extended with either a 6 - linked galactose or glcnac to form the structures seen in pgemlos - 4 lps , which also contain a third kdo moiety which may be the epitope recognized by mab 6e4 . thus , the functions of these two haemophilus orfs remain unclear . b recent studies with a double gene knockout of lst , a gene with high homology to the sialyltransferase in haemophilus ducreyi ( 15 ), and lsgb suggest that lsgb may also encode a sialyltransferase . c the structure of the pgemlos - 5 lps suggests that one of the three orfs c , d , or e encodes a 1 , 3 - n - acetylglucosaminyltransferase and another encodes a 1 , 4 - galactosyltransferase . based on our previous investigations of the los formed when lsgd or lsge are knocked out in mutant strains 281 . 25 and 276 . 4 , respectively ( 16 ), we can conclude that lsgd is likely to encode a 1 , 3 - n - acetylglucosaminyltransferase and lsgc and lsge are both likely to encode galactosyltransferases . d the los formed when lsge is knocked out ( 16 ) suggest that lsge encodes a galactosyltransferase whose acceptor may be absent in the chimeric lps . 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