Methylation analysis of the heptose/3-deoxy-D-manno-2-octulosonic acid region (inner core) of the lipopolysaccharide from Salmonella minnesota rough mutants

A modified methylation analysis is described which allows the elucidation of the structure of the inner core region [heptose/3-deoxy-D-manno-2-octulosonic acid (KDO)] of enterobacterial lipopolysaccharides (LPS) of Salmonella minnesota rough mutants (Re, strain R595; and Rd2P-, strain R4). Methylation, carboxyl-reduction, remethylation, hydrolysis, carbonyl-reduction, and acetylation of the Re-mutant LPS yielded the 2,6-di-O-acetyl and 2,4,6-tri-O-acetyl derivatives of partially methylated 3-deoxyoctitol in equimolar amounts, indicating the presence of a terminal and a 4-linked pyranosidic KDO residue. For Rd2P- LPS, the hydrolysis step involved 0.1M trifluoroacetic acid at 100 degrees for 1 h which cleaved ketosidic linkages, and the final products included the foregoing acetyl derivatives in the molar ratio of 1:02 and a partially methylated and acetylated 3-deoxyoctitol derivative which was substituted at O-5 by a methylated heptopyranosyl residue. Trideuteriomethylation of the latter product followed by methanolysis and acetylation gave 5-O-acetyl-3-deoxy-1,7,8-tri-O-methyl-2,4,6-tri-O-trideuteriomethyl++ +-D- glycero-D-talo/galacto-octitol and 1,5-di-O-acetyl-2,3,4,6,7-penta-O-methyl-L-glycero-D-manno-heptitol++ +. These results prove the presence of a (2----4)-linked KDO disaccharide in Re LPS and show that the core region of Rd2P- LPS contains a terminal alpha-L-glycero-D-manno-heptopyranosyl group and a non-substituted, a 4-O-, and a 4,5-di-O-substituted pyranosidic KDO residue in the molar ratios 1:1:0.2:1.     

Immunochemical studies on R mutants of Yersinia enterocolitica O:3.

Three mutants of Yersinia enterocolitica O:3, namely: YeO3-R1, YeO3-RfbR7 and YeO3-c-trs8-R were classified on the basis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE) profile of isolated lipopolysaccharides (LPS) as belonging to the Ra- (the first) and the Rc-type (the other two mutants). Methylation analysis, in addition to 13C and 1H NMR studies of purified core oligosaccharides revealed structures similar to those established previously for the full core of Y. enterocolitica O:3 in the case of the Ra mutant, and identical to that reported for the Rc mutant Ye75R, in the case of the two other mutants. The O-specific sugar, 6d-L-altrose, which forms a homopolymeric O-chain, was present in small amounts in all three LPS preparations, as well as in the core oligosaccha ride preparations along with the Ra and the Rc sugars, characteristic of the Y. enterocolitica O:3 core. This result is in line with genetic data, indicating that it is the inner core region which is the receptor for the O-specific chain in Y. enterocolitica O:3. This region seems likewise to be the anchoring region for the enterobacterial common antigen (ECA), as shown by SDS/PAGE/Western blot analysis with monoclonal antibodies against ECA. In addition, we also demonstrated that the Ye75R mutant Rc and its parental strain Ye75S, both were ECA-immunogenic strains. So far, ECA-immunogenic strains, i.e. those with LPS-linked ECA, were only identified in E. coli mutants of the R1, R4 and K-12 serotype.     

Selective detection of 3-deoxymannooctulosonic acid in intact lipopolysaccharides by spin-echo 13C NMR

The 3-deoxy-D-mannooctulosonic acid (KDO) region of lipopolysaccharides (LPS) from the heptoseless mutant Salmonella minnesota R595 and inner core and heptoseless mutants derived from Escherichia coli K12 was studied by 13C NMR spectroscopy. A spin-echo spectral editing technique was employed for the selective detection of the quaternary anomeric carbon of ketosidically linked KDO. Only two quaternary carbon resonances attributable to KDO were detected in the anomeric carbon spectral region of each LPS from heptoseless mutants E. coli D31m4 (99.7 and 100.8 ppm) and S. minnesota R595 (100.0 and 100.9 ppm). Integrated signal intensities from fully relaxed normal 13C spectra showed that equivalent molar quantities of KDO and glucosamine (i.e. 2 mol of each) were present in each of these samples. Similarly, only two KDO anomeric carbon resonances were detected in the LPS from the inner core mutants E. coli D21f1 (100.8 and 101.2 ppm) and E. coli D21e7 (100.8 and 101.2 ppm). These data confirm the presence of a KDO disaccharide structure rather than a trisaccharide as determined by others using thiobarbituric acid-based assays. The LPS of E. coli D21 (complete inner core oligosaccharide) exhibited four quaternary anomeric carbon resonances (99.4, 100.7, 101.8, and 102.7 ppm). The unequal intensities of these resonances, however, demonstrated that significant heterogeneity exists with respect to KDO substitution in this LPS. A third KDO moiety present in substoichiometric amounts could be consistent with this observation. However, this possibility could not be distinguished from other modes of substitutional heterogeneity involving only 2 KDO residues.     

Staining of O-specific polysaccharide chains of lipopolysaccharides with ruthenium red

S-form lipopolysaccharides (LPS) from Klebsiella strain LEN-1 (O3: K1-) and from Salmonella minnesota strain 1114 were positively stained with ruthenium red, whereas R-form LPS from Klebsiella strain LEN-111 (O3-: K1-) and Ra, Rb1, RcP+, Rd1P-, and Re LPS from the respective mutant strains of S. minnesota were not or only faintly stained by such treatment. From these results it was concluded that ruthenium red stains the O-specific polysaccharide chains of LPS. The appearance of stained preparations of S-form LPS suggested that the material responsible for this positive staining corresponded to the surface projections which were seen by the negative staining technique as attached to the ribbon-like structures and spherules of the LPS.     

Structure of supported bilayers composed of lipopolysaccharides and bacterial phospholipids: raft formation and implications for bacterial resistance

Lipopolysaccharide (LPS), the major lipid on the surface of Gram-negative bacteria, plays a key role in bacterial resistance to hydrophobic antibiotics and antimicrobial peptides. Using atomic force microscopy (AFM) we characterized supported bilayers composed of LPSs from two bacterial chemotypes with different sensitivities to such antibiotics and peptides. Rd LPS, from more sensitive "deep rough" mutants, contains only an inner saccharide core, whereas Ra LPS, from "rough" mutants, contains a longer polysaccharide region. A vesicle fusion technique was used to deposit LPS onto either freshly cleaved mica or polyethylenimine-coated mica substrates. The thickness of the supported bilayers measured with contact-mode AFM was 7 nm for Rd LPS and 9 nm for Ra LPS, consistent with previous x-ray diffraction measurements. In water the Ra LPS bilayer surface was more disordered than Rd LPS bilayers, likely due to the greater volume occupied by the longer Ra LPS polysaccharide region. Since deep rough mutants contain bacterial phospholipid (BPL) as well as LPS on their surfaces, we also investigated the organization of Rd LPS/BPL bilayers. Differential scanning calorimetry and x-ray diffraction indicated that incorporation of BPL reduced the phase transition temperature, enthalpy, and average bilayer thickness of Rd LPS. For Rd LPS/BPL mixtures, AFM showed irregularly shaped regions thinner than Rd LPS bilayers by 2 nm (the difference in thickness between Rd LPS and BPL bilayers), whose area increased with increasing BPL concentration. We argue that the increased permeability of deep rough mutants is due to structural modifications caused by BPL to the LPS membrane, in LPS hydrocarbon chain packing and in the formation of BPL-enriched microdomains.     

Structure of the hexose region of Shigella sonnei phase II lipopolysaccharide with 3-deoxy-D-manno-octulosonic acid as possible immunodeterminant and its relation to Escherichia coli R1 core.

Comparative chemical analysis (methylation, gas chromatography/mass spectrometry, periodate oxidation, etc.) of the lipopolysaccharides and degraded polysaccharides derived from Shigella sonnei phase I, phase II and galactose-deficient R mutants revealed a structure as shown: (formula: see text) 3-Deoxy-D-manno-octulosonic acid (dOclA) as an immunodeterminant was observed in the passive hemolysis inhibition test by (a) selective inhibition of the phase II system by dOclA; (b) the kinetics of the change of serological activity during mild acid treatment: 1% acetic acid abolished serological activity; (c) a lack of activity in galactose-less R mutants and reactivity with Re mutants including Salmonella minnesota Re. An enhanced sensitivity of phase II lipopolysaccharide to galactose oxidase after prolonged treatment with 1% acetic acid suggests that dOclA is linked to C-6 of the terminal or subterminal galactose. dOclA as immunodeterminant could explain some different polysaccharide structures described for Escherichia coli R1 core.     

Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small-angle X-ray diffraction

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (greater than 90%) at different temperatures (20, 37, and 55 degrees C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20 degrees C, i.e. below the beta----alpha acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 degrees C), 4.60 nm for Re lipopolysaccharide (at 37 degrees C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37 degrees C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20 degrees C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1: square root of 2, 1: square root of 3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.     

On the primary structure of the Escherichia coli R4 cell wall lipopolysaccharide core.

From $\text{Escherichia coli}$ R4, and from some deeper rough mutants of it, the cell wall lipopolysaccharides were isolated and subjected to mild acid hydrolysis. By methylation/g.l.c./m.s. and other analyses of the core oligosaccharides thus obtained, the primary structure of the $\text{E. coli}$ R4 core (hexose and heptose region) was elucidated:

     

Effect of miconazole on the structure and function of plasma membrane of Candida albicans

Abstract Fructose, a rarely occurring sugar constituent of Gram-negative bacterial lipopolysaccharides (LPS), is distributed ubiquitously in LPS of 01 Vibrio cholerae so far examined, but its location in LPS has not hitherto been elucidated. It was found that hydrazinolysis of LPS successfully affords a derivative retaining virtually all the fructose of intact LPS, but no ester-bound phosphate. Structural analysis carried out on the LPS derivative prepared by the hydrazinolysis of R-type LPS isolated from a rough mutant strain (NIH 41R) of 01 V. cholerae NIH 41 (Ogawa) revealed that the fructose is present as a non-reducing terminal residue bound to position C-6 of a glucose residue in the core region. This finding is considered to exclude the possibility that, in the LPS of 01 V. cholerae , the fructose is present in the region of the inner core in place of 2-keto-3-deoxyoctonate.     

Analysis of unmodified endotoxin preparations by 252Cf plasma desorption mass spectrometry. Determination of molecular masses of the constituent native lipopolysaccharides

Nine unmodified endotoxin preparations constituted of Re-, Rd-, and Rc-type lipopolysaccharides (2 to 5 glycoses), representing four species of enterobacteria were analyzed by 252Cf plasma desorption mass spectrometry. The constituent lipopolysaccharides were characterized by the ion pair: (M-H)- and its corresponding lipid fragment ion. The lipid fragment ion is produced by cleavage of the glycosidic bond of the 3-deoxy-D-manno-oct-2-ulosonic acid unit that substitutes O-6' of the glucosamin beta 1'-6glucosamine ("lipid A backbone") disaccharide of the lipid A moiety. These lipid fragment ions were identical to the (M-H)- ions seen in the spectra of homologous isolated lipid A preparations that were obtained by hydrolysis (pH 4.5, 100 degrees C) promoted by sodium dodecyl sulfate. Since the molecular components present in the endotoxin preparations analyzed are known, the ion pair (M-H)(-)-lipid fragment ion defines the molecular compositions of each individual lipopolysaccharide. Heterogeneity of the R-type endotoxin preparations analyzed was due almost exclusively to differing lipid A moieties. In three Salmonella minnesota 595 Re endotoxin preparations 10 different lipopolysaccharides were identified, only two of which were common to all three preparations. Of the nine lipopolysaccharides identified in two S. minnesota R7 endotoxin preparations, only two were present in both.     

rfaP mutants of Salmonella typhimurium

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O-specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmonella minnesota.     

Interaction of lipopolysaccharide with detergents and its possible role in the detergent resistance of the outer membrane of Gram-negative bacteria.

In the presence of MgCl2, amounts of detergents which disrupted phospholipid vesicles caused lipopolysaccharide I from Proteus mirabilis to aggregate and form vesicular, membrane-like structures. Vesicle formation with P. mirabilis lipopolysaccharide II containing longer O-polysaccharide chains was extremely poor. Lipopolysaccharides of Salmonella minnesota R mutants (chemotypes Ra, Rc and Re) displayed a growing tendency for vesicle formation with increasing deficiency of the R core polysaccharide. Lipopolysaccharides of chemotypes Rc and Re produced vesicles even in the absence of MgCl2 and detergent. Spherical aggregates consisting of P. mirabilis lipopolysaccharide I MgCl2 and detergent were unable to either entrap or retain [14C]-sucrose, [3H=inulin or [3H]dextran. On the other hand, S. minnesota R mutant lipopolysaccharides of chemotypes Rc and Re could entrap all three saccharides and retain them for at least short periods of time. Leakage of [3H]-inulin out of re-lipopolysaccharide vesicles was greatly retarded by addition of MgCl2 to the vesicle system. Incorporation of P. mirabilis lipopolysaccharide I or S. minnesota Rc lipopolysaccharide into phospholipid vesicles protected these model membranes from disruption by detergent. This suggested a similar protective function of lipopolysaccharide in the outer membrane of enteric bacteria against the action of surfactants occurring in their normal intestinal habitat.     

Identification of D-galactose-(1 → 7)-3-deoxy-d-manno-2-octulo-pyranosonate as component of the inner core region of escherichia coli EH 100 lipopolysaccharide

The dissaccharide D-galactosyl-3-deoxy-D-manno-2-octulosonic acid (Gal-KDO) from lipopolysaccharides (LPS) of the inner core region of the rough mutant EH100 of E. coli 0100 was isolated after mild acid hydrolysis of LPS by means of dialysis, ion-exchange chromatography, gelfiltration and high-voltage paper electrophresis. By chemical analysis galactose and KDO were identified in a molar ratio of approximately 1 : 1. ¹³C-NMR and ¹H-NMR studies, methylation analysis and GLC of the acetylated R.( – )-2-butyl-galactoside identified the structure .     

Participation of Lipopolysaccharide Genes in the Determination of the Enterobacterial Common Antigen: Analysis of R Mutants of Salmonella minnesota

A series of R (rough) Salmonella minnesota mutants with rfb, rfe, and rfa mutations leading to various defects in the biosynthesis of cell wall lipopolysaccharide was analyzed as to their enterobacterial common antigen (CA) content. All mutants that had functional rfe genes were CA(+) as is the wild-type parent. This includes mutants with the most defective lipopolysaccharide core types, demonstrating that core structures are not a necessary part of CA. All rfe(-) mutants (complete lipopolysaccharide core, defective synthesis of O side chains) were defective in the synthesis of CA. A smooth strain was accidentally found to be CA(-); the mutation responsible for this defect was also located, like rfe, very close to ilv.     

Bindings of axial ligands to cytochrome P-450d mutants: a difference absorption spectral study

By site-directed mutagenesis, we made several cytochrome P-450d (P-450d) mutants as follows: Asn310Phe (D13), Ile312Leu (D14), Glu318Asp (D15), Val320Ile (D16), Phe325Thr (D19), Asn310Phe,Ile312Leu (M6), Glu318Asp,Val320Ile (M7), Phe325Thr, Glu318Asp (M3). This region (Asn-310-Phe-325) is supposed to be located in the distal helix above the heme plane in P-450d, being conjectured from the structure of P-450cam. We studied Soret spectral changes of those mutants by adding several axial ligands such as aniline, pyridine, metyrapone, 2-phenylimidazole and 4-phenylimidazole. Binding constants (Kb) of aniline and pyridine to the single and double mutants were higher than those to the wild type by 2-10-times. The double mutations did not additively increase the Kb values compared with those to the single mutants. In contrast, Kb value (1.0.10(5) M-1) of metyrapone to the double mutant M3 was much higher than that (2.0.10(3) M-1) of the wild type and those of the single mutants, D15 (4.5.10(4) M-1) and D19 (1.6.10(4) M-1). The increased affinity of metyrapone to the mutant M3 may be attributed to an interaction of the hydrophobic group of metyrapone with nearby hydrophobic group(s) produced cooperatively by the double mutation of P-450d. Kb values of 2-phenylimidazole and 4-phenylimidazole to the mutant M3 were also the highest among those of the mutants and the wild type. Therefore, it was suggested that this region (from Asn-310 to Phe-325) must be located at the distal region of the heme moiety and form, at least, a substrate-binding region of membrane-bound P-450d.     

Recognition of heptoses and the inner core of bacterial lipopolysaccharides by surfactant protein d

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-alpha-d-manno-heptose (l,d-heptose), but not by 3-deoxy-alpha-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic alpha1-->3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-alpha-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.     

Mapping of the binding specificity for five monoclonal antibodies recognizing 3-deoxy-D-manno-octulosonic acid in bacterial lipopolysaccharides

Mouse mAb were produced against the deep rough strains Salmonella minnesota R 595, Salmonella typhimurium SL 1102, and Escherichia coli D21f2 and screened by enzyme immunoassay against LPS of several chemotypes. Five antibodies were selected for their ability to bind to chemotype deep rough (Re) LPS which has two 3-deoxy-D-manno-octulosonic acid (Kdo) residues in its nonreducing end. Structurally verified oligosaccharides isolated from rough LPS and synthetic analogues of Kdo were used in an enzyme immunoassay inhibition test to determine the binding epitopes for the antibodies. According to their specificities, the antibodies could be divided into three groups. For two of the groups, the recognized structure was the alpha-Kdo (2----4) Kdo disaccharide and for one group the alpha-Kdo (2----4) alpha-Kdo beta-D-GlcN (1----6) alpha-D-GlcN tetrasaccharide, representing a partial structure of the Re LPS. Inhibition studies with synthetic analogues of Kdo showed that the anomeric configuration and the free carboxyl group of the Kdo residue are important features for antibody binding. Changes in the C-1 to C-6 region of the Kdo molecule do influence the antibody recognition considerably whereas changes in the exocyclic C-7 to C-8 region are of secondary importance. Calculation of the conformation of the inner core region showed that the alpha-Kdo (2----4) alpha-Kdo (2---- disaccharide was free and accessible in chemotype Re LPS, but that linkage of a L-glycero-D-manno-heptose to O-5 of the subterminal Kdo both changes the conformation of the Kdo-disaccharide and covers it thereby making it less accessible.     

Non-typical lipopolysaccharide core regions of some Hafnia alvei strains: structural and serological studies

The lipopolysaccharides of Hafnia alvei strains 23, 1222 and 39 were found to have non-typical core region. On the basis of sugar and methylation analyses, 1H-nuclear magnetic resonance spectra and matrix-assisted laser-desorption ionization-time of flight mass spectrometry, it was concluded that the core oligosaccharide of strains 23 and 1222 has the same structure as Escherichia coli R4 core region, and the core oligosaccharide of strain 39 has the structure of Salmonella Ra core. Using the serological methods (passive hemagglutination, enzyme-linked immunosorbent assay and immunoblotting) and the anti-conjugate sera directed against E. coli R4 and Salmonella Ra core oligosaccharides we have confirmed the structural results presented above.     

Role of a lipopolysaccharide gene for immunogenicity of the enterobacterial common antigen.

It is known that only certain strains of the family of Enterobacteriaceae, notably rough (R) mutants with the type R1 or R4 core, evoked antibodies in high titers against the common enterobacterial antigen (CA) after immunization of rabbits with heated cell suspensions. The present investigation deals with genetic and immunochemical aspects of certain R1 and R4 mutants isolated from Escherichia coli 08 and various Shigella serotypes which, unexpectedly, do not induce CA antibody formation. Immunochemical and genetical (transduction and conjugation) experiments revealed that the rough phenotype of these special mutants was evoked by a mutation of pyrE-linked rfa gene, called rfaL, which is involved in translocation of O-specific polysaccharides onto the lipopolysaccharide core. The transduction of the defective rfaL, allele into appropriate rough recipients results in transductants which have simultaneously lost the ability to evoke CA antibodies. This finding suggests that a close connection exists between the function of the rfaL gene and the expression of CA immunogenicity in R1 and R4 mutants. One of the strains synthesized neither O-hapten nor CA, suggesting a mutation in a region equivalent to the rfe genes of Salmonella.