Water molecules mediate zinc mobility in the bacterial zinc diffusion channel ZIPB.

Regulated ion diffusion across biological membranes is vital for cell function. In a nanoscale ion channel, the active role of discrete water molecules in modulating hydrodynamic behaviors of individual ions is poorly understood because of the technical challenge of tracking water molecules through the channel. Here we report the results of a hydroxyl radical footprinting analysis of the zinc-selective channel ZIPB from the Gram-negative bacterium, Bordetella bronchiseptica Irradiating ZIPB by microsecond X-ray pulses activated water molecules to form covalent hydroxyl radical adducts at nearby residues, which were identified by bottom-up proteomics to detect residues that interact either with zinc or water in response to zinc binding. We found a series of residues exhibiting reciprocal changes in water accessibility attributed to alternating zinc and water binding. Mapping these residues to the previously reported crystal structure of ZIPB, we identified a water-reactive pathway that superimposed on a zinc translocation pathway consisting of two binuclear metal centers and an interim zinc-binding site. The cotranslocation of zinc and water suggested that pore-lining residues undergo a mode switch between zinc coordination and water binding to confer zinc mobility. The unprecedented details of water-mediated zinc transport identified here highlight an essential role of solvated waters in driving zinc coordination dynamics and transmembrane crossing.

Bordetella bronchiseptica Glycosyltransferase Core Mutants Trigger Changes in Lipid A Structure.

Bordetella bronchiseptica, known to infect animals and rarely humans, expresses a lipopolysaccharide that plays an essential role in host interactions, being critical for early clearance of the bacteria. On a B. bronchiseptica 9.73 isolate, mutants defective in the expression of genes involved in the biosynthesis of the core region were previously constructed. Herein, a comparative detailed structural analysis of the expressed lipids A by MALDI-TOF mass spectrometry was performed. The Bb3394 LPS defective in a 2-amino-2-deoxy-D-galacturonic acid lateral residue of the core presented a penta-acylated diglucosamine backbone modified with two glucosamine phosphates, similar to the wild-type lipid A. In contrast, BbLP39, resulting in the interruption of the LPS core oligosaccharide synthesis, presented lipid A species consisting in a diglucosamine backbone N-substituted with C14:0(3-O-C12:0) in C-2 and C14:0(3-O-C14:0) in C-2', O-acylated with C14:0(3-O-C10:0(3-OH) in C-3' and with a pyrophosphate in C-1. Regarding Bb3398 also presenting a rough LPS, the lipid A is formed by a hexa-acylated diglucosamine backbone carrying one pyrophosphate group in C-1 and one phosphate in C-4', both substituted with ethanolamine groups. As far as we know, this is the first description of a phosphoethanolamine modification in B. bronchiseptica lipid A. Our results demonstrate that although gene deletions were not directed to the lipid A moiety, each mutant presented different modifications. MALDI-TOF mass spectrometry was an excellent tool to highlight the structural diversity of the lipid A structures biosynthesized during its transit through the periplasm to the final localization in the outer surface of the outer membrane. Graphical Abstract.

Regulated, sequential processing by multiple proteases is required for proper maturation and release of Bordetella filamentous hemagglutinin.

Filamentous hemagglutinin (FHA) is a critically important virulence factor produced by Bordetella species that cause respiratory infections in humans and other animals. It is also a prototypical member of the widespread two partner secretion (TPS) pathway family of proteins. First synthesized as a ~370 kDa protein called FhaB, its C-terminal ~1,200 amino acid 'prodomain' is removed during translocation to the cell surface via the outer membrane channel FhaC. Here, we identify CtpA as a periplasmic protease that is responsible for the regulated degradation of the prodomain and for creation of an intermediate polypeptide that is cleaved by the autotransporter protease SphB1 to generate FHA. We show that the central prodomain region is required to initiate degradation of the prodomain and that CtpA degrades the prodomain after a third, unidentified protease (P3) first removes the extreme C-terminus of the prodomain. Stepwise proteolysis by P3, CtpA and SphB1 is required for maturation of FhaB, release of FHA into the extracellular milieu, and full function in vivo. These data support a substantially updated model for the mechanism of secretion, maturation and function of this model TPS protein.

Membrane Vesicles Derived from Bordetella bronchiseptica: Active Constituent of a New Vaccine against Infections Caused by This Pathogen.

Bordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). We recently designed Bordetella pertussis and Bordetella parapertussis experimental vaccines based on outer membrane vesicles (OMVs) derived from each pathogen, and we obtained protection against the respective infections in mice. Here, we demonstrated that OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) protected mice against sublethal infections with different B. bronchiseptica strains, two isolated from farm animals and one isolated from a human patient. In all infections, we observed that the B. bronchiseptica loads were significantly reduced in the lungs of vaccinated animals; the lung-recovered CFU were decreased by ≥4 log units, compared with those detected in the lungs of nonimmunized animals (P < 0.001). In the OMVBbvir+-immunized mice, we detected IgG antibody titers against B. bronchiseptica whole-cell lysates, along with an immune serum having bacterial killing activity that both recognized B. bronchiseptica lipopolysaccharides and polypeptides such as GroEL and outer membrane protein C (OMPc) and demonstrated an essential protective capacity against B. bronchiseptica infection, as detected by passive in vivo transfer experiments. Stimulation of cultured splenocytes from immunized mice with OMVBbvir+ resulted in interleukin 5 (IL-5), gamma interferon (IFN-γ), and IL-17 production, indicating that the vesicles induced mixed Th2, Th1, and Th17 T-cell immune responses. We detected, by adoptive transfer assays, that spleen cells from OMVBbvir+-immunized mice also contributed to the observed protection against B. bronchiseptica infection. OMVs from avirulent-phase B. bronchiseptica and the resulting induced immune sera were also able to protect mice against B. bronchiseptica infection.IMPORTANCEBordetella bronchiseptica, a Gram-negative bacterium, causes chronic respiratory tract infections in a wide variety of mammalian hosts, including humans (albeit rarely). Several vaccines aimed at preventing B. bronchiseptica infection have been developed and used, but a safe effective vaccine is still needed. The significance and relevance of our research lie in the characterization of the OMVs derived from B. bronchiseptica as the source of a new experimental vaccine. We demonstrated here that our formulation based on OMVs derived from virulent-phase B. bronchiseptica (OMVBbvir+) was effective against infections caused by B. bronchiseptica isolates obtained from different hosts (farm animals and a human patient). In vitro and in vivo characterization of humoral and cellular immune responses induced by the OMVBbvir+ vaccine enabled a better understanding of the mechanism of protection necessary to control B. bronchiseptica infection. Here we also demonstrated that OMVs derived from B. bronchiseptica in the avirulent phase and the corresponding induced humoral immune response were able to protect mice from B. bronchiseptica infection. This realization provides the basis for the development of novel vaccines not only against the acute stages of the disease but also against stages of the disease or the infectious cycle in which avirulence factors could play a role.

The Bordetella Bps Polysaccharide Is Required for Biofilm Formation and Enhances Survival in the Lower Respiratory Tract of Swine.

Bordetella bronchiseptica is pervasive in swine populations and plays multiple roles in respiratory disease. Additionally, B. bronchiseptica is capable of establishing long-term or chronic infections in swine. Bacterial biofilms are increasingly recognized as important contributors to chronic bacterial infections. Recently the polysaccharide locus bpsABCD has been demonstrated to serve a critical role in the development of mature biofilms formed by the sequenced laboratory strain of B. bronchiseptica We hypothesized that swine isolates would also have the ability to form mature biofilms and the bpsABCD locus would serve a key role in this process. A mutant containing an in-frame deletion of the bpsABCD structural genes was constructed in a wild-type swine isolate and found to be negative for poly-N-acetylglucosamine (PNAG)-like material by immunoblot assay. Further, the bpsABCD locus was found to be required for the development and maintenance of the three-dimensional structures under continuous-flow conditions. To investigate the contribution of the bpsABCD locus to the pathogenesis of B. bronchiseptica in swine, the KM22Δbps mutant was compared to the wild-type swine isolate for the ability to colonize and cause disease in pigs. The bpsABCD locus was found to not be required for persistence in the upper respiratory tract of swine. Additionally, the bpsABCD locus did not affect the development of anti-Bordetella humoral immunity, did not contribute to disease severity, and did not mediate protection from complement-mediated killing. However, the bpsABCD locus was found to enhance survival in the lower respiratory tract of swine.

Toward a new vaccine for pertussis.

To overcome the limitations of the current pertussis vaccines, those of limited duration of action and failure to induce direct killing of Bordetella pertussis, a synthetic scheme was devised for preparing a conjugate vaccine composed of the Bordetella bronchiseptica core oligosaccharide with one terminal trisaccharide to aminooxylated BSA via their terminal ketodeoxyoctanate residues. Conjugate-induced antibodies, by a fraction of an estimated human dose injected into young outbred mice as a saline solution, were bactericidal against B. pertussis, and their titers correlated with their ELISA values. The carrier protein is planned to be genetically altered pertussis toxoid. Such conjugates are easy to prepare, stable, and should add both to the level and duration of immunity induced by current vaccine-induced pertussis antibodies and reduce the circulation of B. pertussis.

Cyclic-di-GMP signalling regulates motility and biofilm formation in Bordetella bronchiseptica.

The signalling molecule bis-(3'-5')-cyclic-dimeric guanosine monophosphate (c-di-GMP) is a central regulator of diverse cellular functions, including motility, biofilm formation, cell cycle progression and virulence, in bacteria. Multiple diguanylate cyclase and phosphodiesterase-domain-containing proteins (GGDEF and EAL/HD-GYP, respectively) modulate the levels of the second messenger c-di-GMP to transmit signals and obtain such specific cellular responses. In the genus Bordetella this c-di-GMP network is poorly studied. In this work, we evaluated the expression of two phenotypes in Bordetella bronchiseptica regulated by c-di-GMP, biofilm formation and motility, under the influence of ectopic expression of Pseudomonas aeruginosa proteins with EAL or GGDEF domains that regulates the c-di-GMP level. In agreement with previous reports for other bacteria, we observed that B. bronchiseptica is able to form biofilm and reduce its motility only when GGDEF domain protein is expressed. Moreover we identify a GGDEF domain protein (BB3576) with diguanylate cyclase activity that participates in motility and biofilm regulation in B. bronchiseptica. These results demonstrate for the first time, to our knowledge, the presence of c-di-GMP regulatory signalling in B. bronchiseptica.

A deep rough type structure in Bordetella bronchiseptica lipopolysaccharide modulates host immune responses.

The present authors have previously obtained the Bordetella bronchiseptica mutant BbLP39, which contains a deep-rough lipopolysaccharide (LPS) instead the wild type smooth LPS with O antigen. This mutant was found to be altered in the expression of some proteins and in its ability to colonize mouse lungs. Particularly, in BbLP39 the expression of pertactin is decreased. To differentiate the contribution of each bacterial component to the observed phenotype, here mice defective in the LPS sensing receptor TLR4 (TLR4-defective mice) were used. In contrast to wild-type mice, infection of TLR4-defective mice with BbLP39 resulted in lung infection, which persisted for more than 10 days post-challenge. Comparative analysis of the immune responses induced by purified mutant and wild type LPSs showed that the mutant LPS induced significantly higher degrees of expression of TNF-α and IL-10 mRNA than did the wild type. UV matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry analysis revealed that both LPSs had the same penta-acylated lipid A structure. However, the lipid A from BbLP39 contained pyrophosphate instead of phosphate at position 1. This structural difference, in addition to the lack of O-antigen in BbLP39, may explain the functional differences between BbLP39 and wild type strains.

Structural modifications occurring in lipid A of Bordetella bronchiseptica clinical isolates as demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Bordetella bronchiseptica is a respiratory pathogen in mammal species and its cell surface lipopolysaccharide-endotoxin is a potent virulence factor. In order to better characterize the endotoxin structure to virulence relationships, we studied the lipid A structures of B. bronchiseptica isolates from human and rabbit origins as a function of their virulence phases. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has been widely used for the structural characterization of bacterial endotoxins and their lipid A moieties. This method combined with chemical analytical methods proved to be essential for the characterization of small samples and discrete but essential structural modifications. The occurrence of palmitate (C(16)) in the B. bronchiseptica lipid A structures is shown for the first time at two sites. Their presence was also demonstrated for the first time in correlation with the virulence phase of B. bronchiseptica clinical isolates. The recently identified glucosamine modifications of Bordetella lipids A are also reported in these isolates.

NMR structure of the Bordetella bronchiseptica protein NP_888769.1 establishes a new phage-related protein family PF13554.

The solution structure of the hypothetical phage-related protein NP_888769.1 from the Gram-negative bacterium Bordetella bronchoseptica contains a well-structured core comprising a five-stranded, antiparallel ß-sheet packed on one side against two α-helices and a short ß-hairpin with three flexibly disordered loops extending from the central ß-sheet. A homology search with the software DALI identified two Protein Data Bank deposits with Z-scores > 8, where both of these proteins have less than 8% sequence identity relative to NP_888769.1, and one has been functionally annotated as a lambda phage tail terminator protein. A sequence-homology analysis then confirmed that NP_888769.1 represents the first three-dimensional structural representative of a new protein family that was previously predicted by the Joint Center for Structural Genomics, which includes so far about 20 prophage proteins encoded in bacterial genomes.

Antigenic Variation among Bordetella: Bordetella bronchiseptica strain MO149 expresses a novel o chain that is poorly immunogenic.

The O chain polysaccharide (O PS) of Bordetella bronchiseptica and Bordetella parapertussis lipopolysaccharide is a homopolymer of 2,3-diacetamido-2,3-dideoxygalacturonic acid (GalNAc3NAcA) in which some of the sugars are present as uronamides. The terminal residue contains several unusual modifications. To date, two types of modification have been characterized, and a survey of numerous strains demonstrated that each contained one of these two modification types. Host antibody responses against the O PS are directed against the terminal residue modifications, and there is little cross-reactivity between the two types. This suggests that Bordetella O PS modifications represent a means of antigenic variation. Here we report the characterization of the O PS of B. bronchiseptica strain MO149. It consists of a novel two-sugar repeating unit and a novel terminal residue modification, with the structure Me-4-alpha-L-GalNAc3NAcA-(4-beta-D-GlcNAc3NAcA-4-alpha-L-GalNAc3NAcA-)(5-6)-, which we propose be defined as the B. bronchiseptica O3 PS. We show that the O3 PS is very poorly immunogenic and that the MO149 strain contains a novel wbm (O PS biosynthesis) locus. Thus, there is greater diversity among Bordetella O PSs than previously recognized, which is likely to be a result of selection pressure from host immunity. We also determine experimentally, for the first time, the absolute configuration of the diacetimido-uronic acid sugars in Bordetella O PS.

A rapid, small-scale procedure for the structural characterization of lipid A applied to Citrobacter and Bordetella strains: discovery of a new structural element.

Endotoxins [lipopolysaccharides (LPSs)] are part of the outer cell membrane of Gram-negative bacteria. Their biological activities are associated mainly with the lipid component (lipid A) and even more specifically with discrete aspects of their fine structure. The need for a rapid and small-scale analysis of lipid A motivated us to develop a procedure that combines direct isolation of lipids A from bacterial cells with sequential release of their ester-linked fatty acids by a mild alkali treatment followed by MALDI-MS analysis. This method avoids the multiple-step LPS extraction procedure and lipid A isolation. The whole process can be performed in a working day and applied to lyophilized bacterial samples as small as 1 mg. We illustrate the method by applying it to the analysis of lipids A of three species of Citrobacter that were found to be identical. On the other hand, when applied to two batches of Bordetella bronchiseptica strain 4650, it highlighted the presence, in one of them, of hitherto unreported hexosamine residues substituting the lipid A phosphate groups, possibly a new camouflage opportunity to escape a host defense system.

Respiratory disease in kennelled dogs: serological responses to Bordetella bronchiseptica lipopolysaccharide do not correlate with bacterial isolation or clinical respiratory symptoms.

The role of Bordetella bronchiseptica in a natural outbreak of canine infectious respiratory disease was investigated both by culture and serological analysis. B. bronchiseptica was found in the lungs of a large proportion of clinically healthy dogs and in a greater proportion of dogs with respiratory disease. Using a lipopolysaccharide (LPS) antigen-based enzyme-linked immunosorbent assay, we analyzed the serological responses of a large number of dogs. Dogs with high antibody levels showed no protection from disease, and there was no correlation between the development of disease and rising antibody titer. Similarly, there was no difference in antibody levels in dogs with and without B. bronchiseptica in the lungs. Antibodies to LPS have no predictive value in determining which animals will contract respiratory disease, how severe the disease will be, or which dogs will have B. bronchiseptica colonizing the lungs.

Bordetella bronchiseptica PagP is a Bvg-regulated lipid A palmitoyl transferase that is required for persistent colonization of the mouse respiratory tract.

Bordetella bronchiseptica lipopolysaccharide (LPS) expression varies depending on growth conditions, regulated by the Bvg system. A B. bronchiseptica pagP homologue was identified that is required for Bvg-mediated modification of the lipid A core region of LPS that occurs on switching from the Bvg- to the Bvg+ phase. Structural analysis demonstrated that the lipid A of a B. bronchiseptica pagP mutant differed from wild-type lipid A by the absence of a palmitate group in secondary acylation at the C3' position. The putative pagP promoter drove the expression of a green fluorescent protein (GFP) reporter gene in a Bvg-regulated fashion. These data suggest that B. bronchiseptica pagP encodes a Bvg-regulated lipid A palmitoyl transferase that mediates modification of the lipid A as part of the overall Bvg-mediated adaptation of this organism to changing environmental conditions. We also show that pagP is not required for the initial colonization of the mouse respiratory tract by B. bronchiseptica, but is required for persistence of the organism within this organ.

In vitro and in vivo characterization of a Bordetella bronchiseptica mutant strain with a deep rough lipopolysaccharide structure.

Bordetella bronchiseptica is closely related to Bordetella pertussis, which produces respiratory disease primarily in mammals other than humans. However, its importance as a human pathogen is being increasingly recognized. Although a large amount of research on Bordetella has been generated regarding protein virulence factors, the participation of the surface lipopolysaccharide (LPS) during B. bronchiseptica infection is less understood. To get a better insight into this matter, we constructed and characterized the behavior of an LPS mutant with the deepest possible rough phenotype. We generated the defective mutant B. bronchiseptica LP39 on the waaC gene, which codes for a heptosyl transferase involved in the biosynthesis of the core region of the LPS molecule. Although in B. bronchiseptica LP39 the production of the principal virulence determinants adenylate cyclase-hemolysin, filamentous hemagglutinin, and pertactin persisted, the quantity of the two latter factors was diminished, with the levels of pertactin being the most greatly affected. Furthermore, the LPS of B. bronchiseptica LP39 did not react with sera obtained from mice that had been infected with the parental strain, indicating that this defective LPS is immunologically different from the wild-type LPS. In vivo experiments demonstrated that the ability to colonize the respiratory tract is reduced in the mutant, being effectively cleared from lungs within 5 days, whereas the parental strain survived at least for 30 days. In vitro experiments have demonstrated that, although B. bronchiseptica LP39 was impaired for adhesion to human epithelial cells, it is still able to survive within the host cells as efficiently as the parental strain. These results seem to indicate that the deep rough form of B. bronchiseptica LPS cannot represent a dominant phenotype at the first stage of colonization. Since isolates with deep rough LPS phenotype have already been obtained from human B. bronchiseptica chronic infections, the possibility that this phenotype arises as a consequence of selection pressure within the host at a late stage of the infection process is discussed.

Expression of the putative siderophore receptor gene bfrZ is controlled by the extracytoplasmic-function sigma factor BupI in Bordetella bronchiseptica.

A new gene from Bordetella bronchiseptica, bfrZ encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay. A bfrZ null mutant was constructed by allelic exchange. The protein profile of this mutant is similar to that of the wild-type parent strain. The BfrZ(-)-BfrZ(+) isogenic pair was tested for utilization of 132 different siderophores as iron sources. None of these iron sources acted as a ligand for BfrZ. Translational bfrZ::phoA and transcriptional bfrZ::lacZ fusions were introduced into the B. bronchiseptica bfrZ locus. No alkaline phosphatase or beta-galactosidase activity was detected. Sequence analysis of the bfrZ upstream region revealed the presence of two tightly linked genes, bupI and bupR. Both of these genes are located downstream from a Fur-binding sequence. BupI is homologous to Escherichia coli FecI and Pseudomonas putida PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions. BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane. Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension. We found that bfrZ is not transcribed when bupI and bupR are expressed at the same level. However, overexpression of bupI from a multicopy plasmid triggers bfrZ transcription, and under these conditions BfrZ was detected in membrane fractions. By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that bfrZ expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope.

The structure of the carbohydrate backbone of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica.

The structure of the core-lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion-exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results: where for the product from Bordetella hinzii N = H, R = H, beta-FucN4N- or partially N-acetylated Sug-(1-3)-beta-FucN4N and for the product from Bordetella bronchiseptica N = alpha-Hep, R = H, beta-FucN4N, beta-FucN4NMe or partially N-acetylated Sug-(1-3)-beta-FucN4N or Sug-(1-3)-beta-FucN4NMe; Sug = 2,3-diamino-2,3, 4-trideoxy-hex-4-enuronopyranosyl.

Molecular characterization of Bordetella bronchiseptica filamentous haemagglutinin and its secretion machinery.

Two closely related pathogens, Bordetella pertussis and Bordetella bronchiseptica, share a number of virulence factors. Filamentous haemagglutinin (FHA) is widely regarded as the dominant adhesin of B. pertussis, and its multiple binding activities have been well characterized. This large protein is produced and secreted at high levels by B. pertussis and significantly lower levels by B. bronchiseptica strains. FHA secretion is mediated by a single outer-membrane accessory protein, FhaC. The genes encoding FHA and FhaC in B. bronchiseptica were characterized by sequencing and functional analyses and are highly similar to those of B. pertussis. The most distinctive feature of B. bronchiseptica FHA is additional repeats in the N-terminal portion of the predicted protein. Interestingly, a point mutation in the fhaB promoter region of the B. bronchiseptica GP1 isolate, relative to other isolates, was found to be detrimental to promoter activity and to FHA production. FhaC and the N-terminal secretion domain of FHA of B. bronchiseptica were fully functional for secretion in B. pertussis. Thus, the different levels of FHA secretion by these Bordetella species might reflect differences in physiology, composition and structure of cell envelope, or differential protein degradation. Characterization of FHA expression and function may provide clues as to the basis of host species tropism, tissue localization and receptor recognition.

Structure of the lipid A of Bordetella hinzii ATCC 51730.

Bordetella hinzii has recently been isolated from immunocompromised human hosts. The structure of the lipid A of its endotoxin was investigated using chemical analyses, nuclear magnetic resonnance (NMR), gas liquid chromatography/mass spectrometry (GC/MS), plasma desorption mass spectrometry (PDMS) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The lipid A contains the classical bisphosphorylated beta-(1-->6)-linked D-glucosamine disaccharide with hydroxytetradecanoic acid (C14OH) in amide linkages. The lipid A components of B. pertussis, B. bronchiseptica, and B. parapertussis all differ in their acylation pattern but share a residue of tetradecanoyl-3-hydroxytetradecanoic acid in amide linkage at the C-2' position. However, in the B. hinzii species, the tetradecanoic acid (C14) is stoichiometrically replaced by a 2-hydroxytetradecanoic acid (2-C14OH). In the few reported examples of a hydroxylated fatty acid in this position, the substitutions were only partial. The B. hinzii lipid A differs from that of B. pertussis also by replacement of the hydroxydecanoic acid (C10OH) by hydroxydodecanoic acid (C12OH) and by the presence of a hexadecanoic acid (C16) to give a sixth fatty acid. The lipid A was heterogeneous, being composed of three major molecular species: tetra-, penta- and hexaacylated. The fatty acids in ester linkage were localized by PDMS of the native and alkali-treated lipid A. The lipid A components isolated from the O-chain-linked lipopolysaccharides (LPSs) were shown to be more acylated than those from the O-chain-free LPSs.