Characterization of lipopolysaccharide heterogeneity in Salmonella enteritidis by an improved gel electrophoresis method.

Salmonella enteritidis field isolates of different phage types and pathogenicities were assessed for changes in lipopolysaccharide (LPS) structure, using an improved method of polyacrylamide gel electrophoresis (PAGE) that revealed the same degree of structural detail as mass spectroscopy. The method allowed characterization of an LPS chemotype that may be associated, regardless of phage type, with increased virulence of S. enteritidis. The virulent variant SE6-E21, which efficiently contaminates eggs and yields high numbers of organisms from chick spleens, had an O-antigen/core ratio of 2.8, as determined from gels by densitometry, and 1.67 micrograms of mannose per microgram of 2-keto-3-deoxy-octulosonic acid (KDO), while the avirulent variant SE6-E5 had O-antigen/core ratios of 1.2 and 1.00. The association between O antigen and virulence was also seen on analysis of five new field isolates. One of the new field isolates generated a mixed population of smooth and semismooth variants in agreement with its mixed virulence in chicks. When LPS was purified from large-volume cultures, only the most virulent isolate yielded high amounts of O antigen (1.6 micrograms of mannose per microgram of KDO), while the other isolates had ratios characteristic of semismooth variants (< or = 1.0 microgram of mannose per microgram of KDO), including the isolate of mixed virulence. These results indicate that the improved PAGE method might provide a rapid, sensitive, in vitro assessment of field isolate virulence prior to the performance of definitive infectivity trials.     

Clinical and veterinary isolates of Salmonella enterica serovar enteritidis defective in lipopolysaccharide O-chain polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

Clinical and Veterinary Isolates of Salmonella enterica Serovar Enteritidis Defective in Lipopolysaccharide O-Chain Polymerization

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and the O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, we analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.     

Structural studies of lipooligosaccharides from Haemophilus ducreyi ITM 5535, ITM 3147, and a fresh clinical isolate, ACY1: evidence for intrastrain heterogeneity with the production of mutually exclusive sialylated or elongated glycoforms.

The structures of the lipooligosaccharides (LOSs) from Haemophilus ducreyi ITM 5535 and ITM 3147 and a fresh clinical isolate, ACY1, have been investigated. Oligosaccharides were obtained from phenol-water-extracted LOS by mild acid hydrolysis and were studied by methylation analysis, fast atom bombardment and electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The major oligosaccharide obtained from all strains was a nonasaccharide with the structure beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-a lpha-D-Hepp- (1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp - (1-->3)]4)-L-alpha-D-Hepp-Kdo (Kdo stands for 3-deoxy-D-manno-octulosonic acid) and is thus identical to that identified as the major oligosaccharide in H. ducreyi ITM 2665 (E. K. H. Schweda, A. C. Sundström, L. M. Eriksson, J.A. Jonasson, and A. A. Lindberg, J. Biol. Chem. 269:12040-12048, 1994). Electrospray ionization mass spectrometry on O-deacylated LOS from H. ducreyi ITM 5535 obtained after treatment with anhydrous hydrazine gave evidence for the presence of a sialylated major compound, Neu5Ac alpha(2-->3)-beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-D-Gal p- (1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp -(1-->2)-L- alpha-D-Hepp-(1-->3)]4)-L-alpha-D-Hepp-Kdo(P)-O-deacylated lipid A (Neu5Ac stands for N-acetylneuraminic acid). However, an even larger oligosaccharide could be isolated from all strains as a minor component, viz., the undecasaccharide beta-D-Galp-(1-->4)-beta-D-GlcNAcp-(1-->3)-beta-d-Galp-(1-->4)-beta-D-glcNAcp-(1-->3)-beta-D-Galp-(1-->4)-D-alpha-D-Hepp-(1-->6)-beta-D-Glcp-(1-->[L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)]4-L-alpha-D-Hepp-Kdo, which represents an N-acetyl lactosamine disaccharide unit elongation of the LOS outer core. No Sialylation of this latter minor component undecasaccharide was detected.     

Fourier transform infrared spectroscopy characterization of the lamellar and nonlamellar structures of free lipid A and Re lipopolysaccharides from Salmonella minnesota and Escherichia coli.

The structural polymorphism of free lipid A and deep rough mutant lipopolysaccharide (LPS Re) from Salmonella minnesota strain R595 and Escherichia coli strain F515 was characterized by Fourier transform infrared (IR) spectroscopy. For this, the beta <--> alpha phase states and the three-dimensional supramolecular structures, the latter deduced from small-angle synchrotron radiation x-ray diffraction, were investigated at different water contents, Mg2+ concentrations, and temperatures. The analysis of the IR data for vibrations originating from the hydrophobic moiety shows that the beta <--> alpha acyl chain melting is strongly expressed only for the stretching and scissoring modes of the methylene groups. Vibrational groups originating from the interface region sense the acyl chain melting well (ester carbonyl bands) or only weakly (amide bands), and those resulting from the pure polar moiety not at all. From the x-ray data, the existence of lamellar (L), different cubic, and, for lipid A and LPS R595, also inverted hexagonal (HII) structures could be proven in the temperature range 20-80 degrees C with cubic <--> cubic and cubic <--> HII transitions for the Mg(2+)-free and L <--> HII transitions for the Mg(2+)-containing samples. These structural transitions can be characterized most readily by specific changes of the vibrational bands resulting from the interface region: the ester carbonyl and the amide bands. The magnitude of the changes corresponds to that of the structural rearrangement, i.e., is highest for the L <--> HII, lower for the cubic <--> HII, and lowest for the cubic <--> cubic transitions. The structural transitions are only marginally expressed for vibrational bands of the hydrophobic moiety. Similarly, the band contours of vibrations from the hydrophilic region are no indicators of the structural reorientations except for the carboxylate bands of LPS Re. Particularly the stretching vibrations of the phosphate groups are nearly completely invariant; the absolute values of their half bandwidths, however, differ significantly for lipid A and LPS Re, which seems to be of biological relevance. The ability of IR spectroscopy to detect supramolecular changes also beyond the measurability by x-ray diffraction, i.e., at water contents > 95 to 99.5%, is demonstrated.     

Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay.

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.     

Purification and some properties of alpha-L-arabinofuranosidase from Bacillus subtilis 3-6.

alpha-L-Arabinofuranosidase (EC 3.2.1.55) was purified from culture supernatant of Bacillus subtilis 3-6. The enzyme had a molecular weight of 61,000 and displayed maximum activity at pH 7.0 and 60 degrees C. It released arabinose from O-alpha-L-arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-x ylopyranos e (A1X2), O-beta-D-xylopyranosyl-(1-->4)-[O-alpha-L-arabinofuranosyl-(1-->3)]- O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (A1X3), and arabinan, but not from O-beta-D-xylopyranosyl-(1-->2)-O-alpha-L- arabinofuranosyl-(1-->3)-O-beta-D-xylopyranosyl-(1-->4)-O-beta-D-xylopyr anosyl- (1-->4)-D-xylopyranose (A1X4), arabinoxylan, gum arabic, or arabinogalactan.     

Characterization of the lipopolysaccharide from a wbjE mutant of the serogroup O11 Pseudomonas aeruginosa strain, PA103

The lipopolysaccharide (LPS) of a wbjE mutant of Pseudomonas aeruginosa PA103, a serogroup O11 strain consists of both high and low molecular weight (HMW and LMW) LPSs. The HMW LPS consisted exclusively of rhamnan A-band LPS and no B-band LPS was detected in the wbjE mutant. Interestingly, the LMW LPS from the wbjE mutant showed that it contained a variety of oligosaccharides, each with two or three phosphate groups present as mono- or pyrophosphates. These oligosaccharides consisted of the complete core octasaccharide. The GalN residue was present as an N-acetylated residue in all of these oligosaccharides except the tetrasaccharide in which it is present as an N-alanylated residue. None of these oligosaccharides contained either a d- or l-FucpNAc residue. These results are discussed with regard to the role of wbjE in the biosynthesis of P. aeruginosa PA103 B-band LPS.     

SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis.

KRE6 encodes a predicted type II membrane protein which, when disrupted, results in a slowly growing, killer toxin-resistant mutant possessing half the normal level of a structurally wild-type cell wall (1-->6)-beta-glucan (T. Roemer and H. Bussey, Proc. Natl. Acad. Sci. USA 88:11295-11299, 1991). The mutant phenotype and structure of the KRE6 gene product, Kre6p, suggest that it may be a beta-glucan synthase component, implying that (1-->6)-beta-glucan synthesis in Saccharomyces cerevisiae is functionally redundant. To examine this possibility, we screened a multicopy genomic library for suppression of both the slow-growth and killer resistance phenotypes of a kre6 mutant and identified SKN1, which encodes a protein sharing 66% overall identity to Kre6p. SKN1 suppresses kre6 null alleles in a dose-dependent manner, though disruption of the SKN1 locus has no effect on killer sensitivity, growth, or (1-->6)-beta-glucan levels. skn1 kre6 double disruptants, however, showed a dramatic reduction in both (1-->6)-beta-glucan levels and growth rate compared with either single disruptant. Moreover, the residual (1-->6)-beta-glucan polymer in skn1 kre6 double mutants is smaller in size and altered in structure. Since single disruptions of these genes lead to structurally wild-type (1-->6)-beta-glucan polymers, Kre6p and Skn1p appear to function independently, possibly in parallel, in (1-->6)-beta-glucan biosynthesis.     

Salmonella typhimurium pgtB mutants conferring constitutive expression of phosphoglycerate transporter pgtP independent of pgtC.

PgtC is one of the three components of the atypical "two-component" pgt regulatory system. To investigate whether functional PgtC required for the induction of pgtP expression could be bypassed in the signal transduction process, we sought, and succeeded in isolating, intergenic suppressors arising in the low-copy mini-F plasmid, pSJ11, bearing the entire pgt system except for a 168-bp deletion near the end of the pgtC gene. By transport assays, these suppressors were found to confer constitutive pgtP expression. Intriguingly, all five mutations reside near the 5' end of the pgtB gene, at codons 19 and 21. One mutation alters Arg-19 to Gln, two alter Ala-21 to Thr, one alters Ala-21 to Val, and one alters Ala-21 to Ile. Appropriate strains in which the pgtP promoter was fused to lacZ and which bore the pgtB mutations with and without mutations in pgtC and pgtA genes were constructed, and the epistatic relationships of the wild-type pgtC allele, a mutant pgtA allele, and an essentially total deletion of pgtC to the constitutive pgtB mutations were determined. In the mutant strains bearing the Ala-21 --> Ile and Ala-21 --> Val substitutions, the level of constitutive pgtP-lacZ reporter expression was not affected by the presence of the wild-type pgtC allele, nor was it affected by the total absence of PgtC in the case of the Ala-21 --> Val alteration examined; however, in the mutant strains bearing the Ala-21 --> Thr and the Arg-19 --> Gln substitutions, the extent of constitutive pgtP-lacZ reporter expression was markedly enhanced by the presence of wild-type pgtC allele and, in the case of the Arg-19 -->Gln change examined, by the total absence of PgtC as well. These results indicate that PgtC contains no domain necessary for the kinase activity; that PgtB can be activated in the absence of PgtC mutational alterations of the protein itself; and that PgtB and PgtC interact in the signaling process, with PgtC functioning to activate and modulate the kinase activity of Pgtb. In all strains, the replacement of the wild type pgtA allele with a mutant pgtA allele completely abolished expression of the pgtP-lacZ reporter, indicating that functional pgtA is essential for the constitutivity. His-457 of PgtB, a potential site of autophosphorylation, is also required for the constitutivity because its change to Val drastically reduced pgtP-lacZ reporter expression. The structural basis for the activation of the altered PgtB is discussed in terms of putative structure of PgtB in the membrane.     

The role of helix VIII in the lactose permease of Escherichia coli: I. Cys-scanning mutagenesis.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino acid residue in transmembrane domain VIII and flanking hydrophilic loops (from Gln 256 to Lys 289) was replaced individually with Cys. Of the 34 single-Cys mutants, 26 accumulate lactose to > 70% of the steady state observed with C-less permease, and an additional 7 mutants (Gly 262-->Cys, Gly 268-->Cys, Asn 272-->Cys, Pro 280-->Cys, Asn 284-->Cys, Gly 287-->Cys, and Gly 288-->Cys) exhibit lower but significant levels of accumulation (30-50% of C-less). As expected (Ujwal ML, Sahin-Tóth M, Persson B, Kaback HR, 1994, Mol Membr Biol 1:9-16), Cys replacement for Glu 269 abolishes lactose transport. Immunoblot analysis reveals that the mutants are inserted into the membrane at concentrations comparable to C-less permease, with the exceptions of mutants Pro 280-->Cys, Gly 287-->Cys, and Lys 289-->Cys, which are expressed at reduced levels. The transport activity of the mutants is inhibited by N-ethylmaleimide (NEM) in a highly specific manner. Most of the mutants are insensitive, but Cys replacements render the permease sensitive to inactivation by NEM at positions that cluster in manner indicating that they are on one face of an alpha-helix (Gly 262-->Cys, Val 264-->Cys, Thr 265-->Cys, Gly 268-->Cys. Asn 272-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, and Ala 279-->Cys). The results indicate that transmembrane domain VIII is in alpha-helical conformation and demonstrate that, although only a single residue in this region of the permease is essential for activity (Glu 269), one face of the helix plays an important role in the transport mechanism. More direct evidence for the latter conclusion is provided in the companion paper (Frillingos S. Kaback HR, 1997, Protein Sci 6:438-443) by using site-directed sulfhydryl modification of the Cys-replacement mutants in situ.     

Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus.

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.     

Mutagenesis supports water mediated recognition in the trp repressor-operator system.

High resolution crystallographic analysis of the trp repressor-operator complex indicates that the principal determinants of specificity are water mediated hydrogen bonds between the helix-turn-helix and the identity elements of the operator. One such hydration site involves a conserved G-C base pair (designated G6) six nucleotides away from the dyad which, if changed symmetrically to any other pair (e.g. G6-->A) reduces affinity to nonspecific levels. This same water site also contacts the conserved A5 which, if changed to G (mutation A5-->G), also diminishes affinity. The stereochemistry of the water mediated hydrogen bonding system predicts that the severe deterioration of in vitro binding caused by G6-->A should be reverted by a second deleterious mutation A5-->G. This proved to be the case. No other second mutation at conserved operator position 5 or 7 (flanking the G6-->A) reversed the effect of G6-->A.     

The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification.

Cys-scanning mutagenesis of putative transmembrane helix VIII in the lactose permease of Escherichia coli (Frillingos S. Ujwal ML, Sun J, Kaback HR, 1997, Protein Sci 6:431-437) indicates that, although helix VIII contains only one irreplaceable residue (Glu 269), one face is important for active lactose transport. In this study, the rate of inactivation of each N-ethylmaleimide (NEM)-sensitive mutant is examined in the absence or presence of beta, D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG). Remarkably, the analogue affords protection against inactivation with mutants Val 264-->Cys, Gly 268-->Cys, and Asn 272-->Cys, and alkylation of these single-Cys mutants in right-side-out membrane vesicles with [14C]NEM is attenuated by TDG. In contrast, alkylation of Thr 265-->Cys, which borders the three residues that are protected by TDG, is enhanced markedly by the analogue. Furthermore, NEM-labeling in the presence of the impermeant thiol reagent methanethiosulfonate ethylsulfonate demonstrates that ligand enhances the accessibility of position 265 to solvent. Finally, no significant alteration in NEM reactivity is observed for mutant Gly 262-->Cys, Glu 269-->Cys, Ala 273-->Cys, Met 276-->Cys, Phe 277-->Cys, or Ala 279-->Cys. The findings indicate that a portion of one face of helix VIII (Val 264, Gly 268, and Asn 272), which is in close proximity to Cys 148 (helix V), interacts with substrate, whereas another position bordering these residues (Thr 265) is altered by a ligand-induced conformational change.     

大肠埃希菌脂多糖诱导的血小板降解及急性休克:O抗原在LPS诱导的免疫反应中的作用

目的探讨LPS中的0抗原部分与其它部分在血小板反应中的作用。方法给BALB／c小鼠注人大肠埃希菌野生株E．coli O8、O9、K-12（不含有O抗原）及2株重组变异的K-12株（携带编码O8、O9的O抗原rfb基因）。结果K-12的LPS引起血小板反应及急性休克能力较弱,O8及O9引起一定的反应,而这2种重组的LPS,即在K-12的LPS上带有O8或O9的O抗原．显示出极强的活性。静脉注入补体C5的阻止剂后,重组株LPS的作用消失了。而且在缺乏补体C5小鼠DBA／2中,重组的LPS能引起血小板的聚集但不能降解,也不能引起休克症状。结论诱导血小板反应及急性休克的能力依赖于LPS结构;O抗原及R核心抗原是表现活性的必要结构;LPS诱导的血小板反应及急性休克依赖补体系统。     

Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages.

Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation.     

Complement-mediated killing of Vibrio species by the humoral fluids of amphioxus Branchiostoma belcheri: implications for a dual role of O-antigens in the resistance to bactericidal activity

The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 degrees C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg(2+)-dependent and Ca(2+)-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.     

Carbohydrate dynamics at a micellar surface: GD1a headgroup transformations revealed by NMR spectroscopy.

The conformational dynamics of the carbohydrate headgroup of ganglioside GD1a, NeuAc alpha 2-->3Gal beta 1-->3GalNAc beta 1-->4[NeuAc alpha 2-->3]Gal beta 1-->4Glc beta 1-->1Cer, anchored in a perdeuterated dodecylphosphocholine micelle in aqueous solution, were probed by high resolution NMR spectroscopy. The observed 1H/1H NOE interactions revealed conformational averaging of the terminal NeuAc alpha 2-->3Gal and Gal beta 1-->3GalNAc glycosidic linkages. The pronounced flexibility of this trisaccharide moiety was substantiated further by two-dimensional proton-detected 13C T1, T1 rho and 1H/13C NOE measurements. The anchoring effect of the micelle allowed the detection of conformational fluctuations of the headgroup on the time scale of a few hundred picoseconds. NMR experiments performed on the GD1a/DPC micelles in H2O at low temperatures permitted the observation of hydroxyl proton resonances, contributing valuable conformational information.     

Isolation and structural analysis of polysaccharide containing galactofuranose from the cell walls of Bifidobacterium infantis.

We isolated cell wall polysaccharides (PS-1 and PS-2) from Bifidobacterium infantis Reuter ATCC 15697 and found that the backbone of PS-2 is-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp- (1-->in which beta-D-Galf and alpha-D-Galp are partially substituted at O-6 with beta-D-Glcp. This is the first report of the presence of this disaccharide backbone in a gram-positive bacterium; it resembles the O antigen of some bacteria.     

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10.(ABSTRACT TRUNCATED AT 250 WORDS)