Deep Rough Lipopolysaccharide Research Articles

Rcs Phosphorelay Responses to Truncated Lipopolysaccharide-Induced Cell Envelope Stress in Yersinia enterocolitica.

Lipopolysaccharide (LPS) is the major component of the outer membrane of Gram-negative bacteria, and its integrity is monitored by various stress response systems. Although the Rcs system is involved in the envelope stress response and regulates genes controlling numerous bacterial cell functions of Yersinia enterocolitica, whether it can sense the truncated LPS in Y. enterocolitica remains unclear. In this study, the deletion of the Y. enterocolitica waaF gene truncated the structure of LPS and produced a deep rough LPS. The truncated LPS increased the cell surface hydrophobicity and outer membrane permeability, generating cell envelope stress. The truncated LPS also directly exposed the smooth outer membrane to the external environment and attenuated the resistance to adverse conditions, such as impaired survival under polymyxin B and sodium dodecyl sulfate (SDS) exposure. Further phenotypic experiment and gene expression analysis indicated that the truncated LPS was correlated with the activation of the Rcs phosphorelay, thereby repressing cell motility and biofilm formation. Our findings highlight the importance of LPS integrity in maintaining membrane function and broaden the understanding of Rcs phosphorelay signaling in response to cell envelope stress, thus opening new avenues to develop effective antimicrobial agents for combating Y. enterocolitica infections.

Ionic Environment Affects Bacterial Lipopolysaccharide Packing and Function.

The interaction of lipopolysaccharides (LPS) with metal cations strongly affects the stability and function of the Gram-negative bacterial outer membrane. The sensitivity of deep rough (Re) LPS packing and function to the ionic environment, as affected by cation valency and ionic radius, has been determined using molecular dynamics simulations and Langmuir balance experiments. The degree of LPS aggregation within the LPS models in the presence of different cations is assessed by measuring the effective mean molecular area (Âm) of each LPS molecule projected onto the interfacial plane at the end of the equilibration. These results are compared to the LPS mean molecular area from experimental measurements in which the LPS monolayers are assembled at the air-water interface using a Langmuir film balance. We found that packing of the LPS arrays is sensitive to the ionic radius and ion valency of the cations present in solution during LPS array packing. Using enhanced sampling of the free energy for the intercalation of oligo(allylamine HCl) (OAH) into deep rough Salmonella enterica LPS bilayers, we obtained the affinity of the core section of LPS to OAH as a function of the nature of the metal cations present in solution. We found that packing of the solvated LPS bilayer models is sensitive to ionic radius and ion valency of the neutralizing cations. This further suggests that ion bridging and steric barriers rather than charge shielding are important factors in mitigating ligand intercalation under conditions with low ionic concentrations.

Quantitative Proteome Profiling of Coxiella burnetii Reveals Major Metabolic and Stress Differences Under Axenic and Cell Culture Cultivation.

Coxiella burnetii is the causative agent of the zoonotic disease Q fever. To date, the lipopolysaccharide (LPS) is the only defined and characterized virulence determinant of C. burnetii. In this study, proteome profiles of C. burnetii Nine Mile phase I (RSA 493, NMI) and its isogenic Nine Mile phase II (RSA 439 NMII) isolate with a deep rough LPS were compared on L-929 mouse fibroblasts and in complex (ACCM-2), and defined (ACCM-D) media. Whole proteome extracts were analyzed using a label-free quantification approach. Between 659 and 1,046 C. burnetii proteins of the 2,132 annotated coding sequences (CDS) were identified in any particular experiment. Proteome profiles clustered according to the cultivation conditions used, indicating different regulation patterns. NMI proteome profiles compared to NMII in ACCM-D indicate transition from an exponential to a stationary phase. The levels of regulatory proteins such as RpoS, CsrA2, UspA1, and UspA2 were increased. Comparison of the oxidative stress response of NMI and NMII indicated that ACCM-2 represents a high oxidative stress environment. Expression of peroxidases, superoxide dismutases, as well as thioredoxins was increased for NMI. In contrast, in ACCM-D, only osmoregulation seems to be necessary. Proteome profiles of NMII do not differ and indicate that both axenic media represent similar oxidative stress environments. Deep rough LPS causes changes of the outer membrane stability and fluidity. This might be one reason for the observed differences. Proteins associated with the T4SS and Sec translocon as well as several effector proteins were detectable under all three conditions. Interestingly, none of these putatively secreted proteins are upregulated in ACCM-2 compared to ACCM-D, and L-929 mouse fibroblasts. Curiously, a higher similarity of proteomic patterns (overlapping up- and downregulated proteins) of ACCM-D and bacteria grown in cell culture was observed. Particularly, the proteins involved in a better adaptation or homeostasis in response to the harsh environment of the parasitophorous vacuole were demonstrated for NMI. This semi-quantitative proteomic analysis of C. burnetii compared axenically grown bacteria to those propagated in cell culture.

Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections.

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.

Antimicrobial Activity of CHIR-090, an Inhibitor of Lipopolysaccharide Biosynthesis, against the Burkholderia cepacia Complex

A striking characteristic of cystic fibrosis (CF) is susceptibility to life-limiting bacterial infections of the respiratory tract (13, 26). Members of the Burkholderia cepacia complex are a particular cause of anxiety to CF individuals (8, 14, 23) since they display high resistance to antibiotics and biocides (3, 32), possibly linked to their relatively large (∼8- to 9-Mbp) genomes (16). At present, the B. cepacia complex consists of 17 species, the majority of which have been recovered from CF patients (4, 10, 11, 36, 37, 40). As an aid to B. cepacia complex studies, two panels of B. cepacia complex reference isolates (Table ​(Table1)1) have been assembled that include B. multivorans and B. cenocepacia strains, the two most prevalent species responsible for CF infections (11, 22). A promising target for the development of new antibiotics against multiresistant Gram-negative pathogens are the lipopolysaccharide (LPS) biosynthetic and modification pathways (27, 29, 30, 39). LPS (also known as endotoxin) is the major component of the bacterial outer membrane. Nine enzymes are required to form the basic core Kdo2-lipid A, and the first six of these enzymes are essential in Escherichia coli. Also, we discovered that a putative locus involved in Ara4N synthesis and LPS modification was essential to B. cenocepacia (28). The metal-dependent UDP-[3-O-(R-3-hydroxymyristoyl)]-N-acetylglucosamine deacetylase (LpxC) (Fig. ​(Fig.1)1) that catalyzes the second step in lipid A biosynthesis (18, 41) has been targeted and, like many metalloenzymes, can be inhibited by hydroxamate-containing compounds (7, 9, 17). The synthetic antibiotic CHIR-090 (Fig. ​(Fig.1)1) (N-aroyl-l-threonine hydroxamic acid [international patent WO 2004/062601 A2]) (1) has been shown to be a slow, tight-binding inhibitor of the LpxCs from different species (5, 6, 24) and displayed good antimicrobial activity against several Gram-negative bacteria (6). FIG. 1. Reaction catalyzed by the deacetylase LpxC and chemical structure of CHIR-090. TABLE 1. Inhibition of strains of Burkholderia genomovars I to IX by CHIR-090 We determined the activity of CHIR-090 against the B. cepacia complex (Table ​(Table1)1) initially by disc diffusion growth inhibition assay according to published guidelines (2). Individual isolates displayed remarkable differences in susceptibility to CHIR-090, even within a single species. Interestingly, CHIR-090 was active against all representative strains of B. multivorans, B. vietnamiensis, B. dolosa, and B. ambifaria. We prepared a panel of clinically relevant B. multivorans strains for MIC determination and included E. coli and Pseudomonas aeruginosa (Table ​(Table2).2). The CHIR-090 MICs were strain dependent, and the values obtained ranged from 0.1 to >100 μg/ml. TABLE 2. MICs of CHIR-090 and polymyxin B against a panel of bacterial strains The LPSs from a number of Burkholderia species display unique structural and inflammatory properties (12, 33); however, there appears to be no correlation between CHIR-090 activity and the LPS profiles of individual strains. For example, CHIR-090 is not active against smooth LPS strain B. cenocepacia K56-2 or its deep-rough LPS derivative SAL1 (20). A BLAST sequence analysis of the Burkholderia genomes (Burkholderia Genome Database) revealed that the LpxC genes are highly conserved and display high sequence homology to LpxCs from P. aeruginosa and E. coli; thus, the reason(s) why CHIR-090 is not active against certain members of the B. cepacia complex remains to be clarified. Our study reports the potential of therapeutic agents against Burkholderia targeted at LPS biosynthesis. Such agents may, possibly in combination with nanoemulsions (19), provide a breakthrough in the treatment of CF-related infections.

Structural Analysis of the Deep Rough Lipopolysaccharide from Gram Negative Bacterium Alteromonas macleodii ATCC 27126T: The First Finding of β‐Kdo in the Inner Core of Lipopolysaccharides

AbstractAlteromonas macleodii ATCC 27126T is a Gram‐negative marine bacterium isolated from a sea water sample collected from around the Hawaiian Islands. The structure of the lipooligosaccharide derived from its outer membrane has been fully determined using either alkaline or acid hydrolysis. Alkaline treatment, aimed at recovering the complete carbohydrate backbone, was carried out by mild hydrazinolysis (de‐O‐acylation) followed by de‐N‐acylation using hot KOH and furnished a single core glycoform. Mild acid hydrolysis was employed to obtain the lipid A moiety which was selectively de‐O‐acylated and analysed to determine its primary structure. The structural elucidation of both fractions was carried out by chemical analyses, 2D NMR spectroscopy and MALDI‐TOF mass spectrometry and revealed a novel lipooligosaccharide with an unusual structure. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Down-Regulation of Key Virulence Factors Makes the Salmonella enterica Serovar Typhimurium rfaH Mutant a Promising Live-Attenuated Vaccine Candidate

Mutants of Salmonella enterica serovar Typhimurium that lack the transcriptional regulator RfaH are efficient as live oral vaccines against salmonellosis in mice. We show that the attenuation of the vaccine candidate strain is associated with reduced net growth in epithelial and macrophage cells. In order to identify the relevant RfaH-dependent genes, the RfaH regulon was determined with S. enterica serovars Enteritidis and Typhimurium using whole-genome Salmonella microarrays. As well as impacting the expression of genes involved in lipopolysaccharide (LPS) core and O-antigen synthesis, the loss of RfaH results in a marked down-regulation of SPI-4 genes, the flagellum/chemotaxis system, and type III secretion system 1. However, a proportion of these effects could have been the indirect consequence of the altered expression of genes required for LPS biosynthesis. Direct and indirect effects of the rfaH mutation were dissociated by genome-wide transcriptional profiling of a structural deep-rough LPS mutant (waaG). We show that truncation of LPS itself is responsible for the decreased intracellular yield observed for DeltarfaH strains. LPS mutants do not differ in replication ability; rather, they show increased susceptibility to antimicrobial peptides in the intracellular milieu. On the other hand, evidence that deletion of rfaH, as well as some other genes involved in LPS biosynthesis, results in enhanced invasion of various mammalian cells is shown. Exposure of common minor antigens in the absence of serovar-specific antigens might be responsible for the observed cross-reactive nature of the elicited immune response upon vaccination. Increased invasiveness of the Salmonella rfaH mutant into antigen-presenting cells, combined with increased intracellular killing and the potential for raising a cross-protective immune response, renders the rfaH mutant an ideal vaccine candidate.

Temperature-Dependent Variations and Intraspecies Diversity of the Structure of the Lipopolysaccharide of Yersinia pestis,

Yersinia pestis spread throughout the Americas in the early 20th century, and it occurs predominantly as a single clone within this part of the world. However, within Eurasia and parts of Africa there is significant diversity among Y. pestis strains, which can be classified into different biovars (bv.) and/or subspecies (ssp.), with bv. orientalis/ssp. pestis most closely related to the American clone. To determine one aspect of the relatedness of these different Y. pestis isolates, the structure of the lipopolysaccharide (LPS) of four wild-type and one LPS-mutant Eurasian/African strains of Y. pestis was determined, evaluating effects of growth at mammalian (37 degrees C) or flea (25 degrees C) temperatures on the structure and composition of the core oligosaccharide and lipid A. In the wild-type clones of ssp. pestis, a single major core glycoform was synthesized at 37 degrees C whereas multiple core oligosaccharide glycoforms were produced at 25 degrees C. Structural differences occurred primarily in the terminal monosaccharides. Only tetraacyl lipid A was made at 37 degrees C, whereas at 25 degrees C additional pentaacyl and hexaacyl lipid A structures were produced. 4-Amino-4-deoxyarabinose levels in lipid A increased with lower growth temperatures or when bacteria were cultured in the presence of polymyxin B. In Y. pestis ssp. caucasica, the LPS core lacked D-glycero-D-manno-heptose and the content of 4-amino-4-deoxyarabinose showed no dependence on growth temperature, whereas the degree of acylation of the lipid A and the structure of the oligosaccharide core were temperature dependent. A spontaneous deep-rough LPS mutant strain possessed only a disaccharide core and a slightly variant lipid A. The diversity and differences in the structure of the Y. pestis LPS suggest important contributions of these variations to the pathogenesis of this organism, potentially related to innate and acquired immune recognition of Y. pestis and epidemiologic means to detect, classify, control and respond to Y. pestis infections.

Biophysical characterization of endotoxin inactivation by NK-2, an antimicrobial peptide derived from mammalian NK-lysin.

NK-2, a membrane-acting antimicrobial peptide, was derived from the cationic core region of porcine NK-lysin and consists of 27 amino acid residues. It adopts an amphipathic, alpha-helical secondary structure and has been shown to interact specifically with membranes of negatively charged lipids. We therefore investigated the interaction of NK-2 with lipopolysaccharide (LPS), the main, highly anionic component of the outer leaflet of the outer membrane of gram-negative bacteria, by means of biophysical and biological assays. As model organisms and a source of LPS, we used Salmonella enterica strains with various lengths of the LPS carbohydrate moiety, including smooth LPS, rough LPS, and deep rough LPS (LPS Re) mutant strains. NK-2 binds to LPS Re with a high affinity and induces a change in the endotoxin-lipid A aggregate structure from a cubic or unilamellar structure to a multilamellar one. This structural change, in concert with a significant overcompensation of the negative charges of LPS, is thought to result in the neutralization of the endotoxic LPS activity in a cell culture system. Neutralization of LPS activity by NK-2 as well as its antibacterial activity against the various Salmonella strains strongly depends on the length of the sugar chains of LPS, with LPS Re being the most sensitive. This suggests that a hydrophobic peptide-LPS interaction is necessary for efficient neutralization of the biological activity of LPS and that the long carbohydrate chains, besides their function as a barrier for hydrophobic drugs, also serve as a trap for polycationic substances.

Cell surface interactions of Rhizobium bacteroids and other bacterial strains with symbiosomal and peribacteroid membrane components from pea nodules.

Samples of Rhizobium bacteroids isolated from pea nodule symbiosomes reacted positively with a monoclonal antibody recognizing N-linked glycan epitopes on plant glycoproteins associated with the peribacteroid membrane and peribacteroid fluid. An antiserum recognizing the symbiosomal lectin-like glycoprotein PsNLEC-1 also reacted positively. Samples of isolated bacteroids also reacted with an antibody recognizing a glycolipid component of the peribacteroid membrane and plasma membrane. Bacterial cells derived from free-living cultures then were immobilized on nitrocellulose sheets and tested for their ability to associate with components of plant extracts derived from nodule fractionation. A positive antibody-staining reaction indicated that both PsNLEC-1 and membrane glycolipid had become associated with the bacterial surface. A range of rhizobial strains with mutants affecting cell surface polysaccharides all showed similar interactions with PsNLEC-1 and associated plant membranes, with the exception of strain B659 (a deep-rough lipopolysaccharide mutant of Rhizobium leguminosarum). However, the presence of a capsule of extracellular polysaccharide apparently prevented interactions between rhizobial cells and these plant components. The importance of a close association between peribacteroid membranes, PsNLEC-1, and the bacterial surface is discussed in the context of symbiosome development.

A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides

We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[α-Kdo-(2→4)-α-Kdo-(2→] and a Kdo trisaccharide [α-Kdo-(2→8)-α-Kdo-(2→4)-α-Kdo-(2→] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.

A novel strategy for the synthesis of neoglycoconjugates from deacylated deep rough lipopolysaccharides.

We report a novel strategy for the preparation of neoglycoconjugates of oligosaccharides which are obtained after complete deacylation of bacterial deep rough lipopolysaccharides (LPS) isolated from recombinant Escherichia coli bacteria synthesizing a Kdo di-[alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] and a Kdo trisaccharide [alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo-(2-->] of Re-type and chlamydial LPS, respectively. Unlike acylated LPS, such oligosaccharides can be obtained in pure form and thus lead to well-defined neoglycoconjugates. Cleavage of the 1-phosphate of the lipid A moiety by alkaline phosphatase treatment leads to a free reducing glucosamine which can be further reacted with allylamine. After reductive amination, spacer elongation of the allyl group with cysteamine and activation with thiophosgene, the ligands were reacted with BSA. We have compared the immunological reactivity of such defined neoglycoconjugates obtained from natural sources with those obtained by chemical synthesis and report that such neoglycoconjugates are immunogenic and well suited as antigens for the study of epitope specificities of monoclonal antibodies. In addition, we have compared these conjugates with those in which ligands were coupled by glutardialdehyde to BSA. Our approach proved to be superior since the latter led upon immunization of mice to a relatively high percentage of antibodies that reacted with glutardialdehyde derivatized BSA without the carbohydrate ligand. This was not the case for cysteamine-spacered ligands coupled via their isothiocyanate-derivatives.

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.

The lipopolysaccharide barrier: correlation of antibiotic susceptibility with antibiotic permeability and fluorescent probe binding kinetics.

Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a permeability barrier, making the outer membrane relatively impermeable to hydrophobic antibiotics, detergents, and host proteins. Mutations in the LPS biosynthetic apparatus increase bacterial susceptibility to such agents. To determine how this increased susceptibility is mediated, we have correlated antibiotic susceptibilities of rough (antibiotic resistant) and deep rough (antibiotic susceptible) bacterial strains with antibiotic permeabilities and fluorescent probe binding kinetics for bilayers composed of LPS purified from the same strains. Bilayer permeabilities of two hydrophobic beta-lactam antibiotics were measured by encapsulating the appropriate beta-lactamases in large unilamellar vesicles. In the presence of MgCl(2), permeabilities of LPS bilayers from rough and deep rough bacteria were similar and significantly lower than those of bacterial phospholipids (BPL). Addition of BPL to the LPS bilayers increased their antibiotic permeability to approximately the level of the BPL bilayers. Binding rates of the fluorescent probe bis-aminonaphthylsulfonic acid (BANS) were 2 orders of magnitude slower for both rough and deep rough LPS bilayers compared to that of bilayers composed of BPL or mixtures of LPS and BPL. On the basis of these results and the observation that deep rough bacteria have higher levels of phospholipid on their surface than do rough bacteria (Kamio, Y., and Nikaido, H. (1976) Biochemistry 15, 2561-2569), we argue that the high susceptibility of deep rough bacteria is due to the presence of phospholipids on their surface. Experiments with phospholipid bilayers showed that the addition of PEG-lipids (containing covalently attached hydrophilic polymers) had little effect on permeability and binding rates, whereas the addition of cholesterol reduced permeability and slowed binding to levels approaching those of LPS. Therefore, we argue that the barrier provided by LPS is primarily due to its tight hydrocarbon chain packing (Snyder et al., (1999) Biochemistry 38, 10758-10767) rather than to its polysaccharide headgroup.

Resistance of Escherichia coli and Salmonella against nisin and curvacin A

We have determined the effects of the following factors on the resistance of Gram-negative bacteria against nisin and curvacin A: (i) chemotype of the lipopolysaccharide (LPS), (ii) addition of agents permeabilizing the outer membrane, (iii) the fatty acid supply of the growth medium, and (iv) the adaptation to acid and salt stress. Bacteriocin activity was determined against growing and resting cells as well as protoplasts. All smooth strains of Escherichia coli and Salmonella enterica serovar Typhimurium were highly resistant towards the bacteriocins, whereas mutants that possess the core of the LPS, but not the O antigen, as well as deep rough LPS mutants were sensitive. Antibiotics with outer membrane permeabilizing activity, polymyxin B and polymyxin B nonapeptide, increased the sensitivity of smooth E. coli towards nisin, but not that of deep rough mutants. Incorporation of 1 g l −1 of either oleic acid or linoleic acid to the growth media greatly increased the susceptibility of E. coli LTH1600 and LTH4346 towards bacteriocins. Both strains of E. coli were sensitive to nisin and curvacin A at a pH of less than 5.5 and more than 3% (w/v) NaCl. Adaptation to sublethal pH or higher NaCl concentrations (pH 4.54 and 5.35 or 4.5% (w/v) NaCl) provided only limited protection against the bacteriocidal activity of nisin and curvacin A. Adaptation to 4.5% (w/v) NaCl did not result in cross protection to bacteriocin activity at pH 4.4, but rendered the cells more sensitive towards bacteriocins.

Identification and cloning of waaF (rfaF) from Bordetella pertussis and use to generate mutants of Bordetella spp. with deep rough lipopolysaccharide.

A DNA locus from Bordetella pertussis capable of reconstituting lipopolysaccharide (LPS) O-antigen biosynthesis in Salmonella typhimurium SL3789 (rfaF511) has been isolated, by using selection with the antibiotic novobiocin. DNA within the locus encodes a protein with amino acid sequence similarity to heptosyltransferase II, encoded by waaF (previously rfaF) in other gram-negative bacteria. Mutation of this gene in B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica by allelic exchange generated bacteria with deep rough LPS phenotypes consistent with the proposed function of the gene as an inner core heptosyltransferase. These are the first LPS mutants generated in B. parapertussis and B. bronchiseptica and the first deep rough mutants of any of the bordetellae.

In Vitro Insertion and Assembly of Outer Membrane Protein PhoE of Escherichia coli K-12 into the Outer Membrane: ROLE OF TRITON X-100

The assembly of the in vitro synthesized outer membrane protein PhoE into purified outer membranes was investigated. The assembly appeared to be strongly stimulated by the presence of low amounts of Triton X-100 (optimal 0.08%, w/v). The role of Triton X-100 in the in vitro system was further examined. Pretreating outer membranes with Triton X-100 did not make the membranes competent for correct assembly, indicating that the detergent did not act on the membrane but at the protein level. PhoE became assembly-incompetent with a half-life of approximately 12 min and 90 s at 37 degrees C in the absence and presence, respectively, of 0.08% Triton X-100. Apparently, Triton X-100 induces an assembly-competent state in the PhoE protein with a very short half-life. Furthermore, the efficiency of correct assembly of PhoE was greatly reduced when outer membranes of deep rough lipopolysaccharide mutants were used, indicating an important role of lipopolysaccharides in the assembly of the porin.

Structure of the core oligosaccharide in the serotype O8 lipopolysaccharide from Klebsiella pneumoniae

Two classes of mutants with O-antigen-deficient lipopolysaccharides were isolated from the serotype O8 reference strain, belonging to Klebsiella pneumoniae subspecies ozaenae. These mutants were selected by resistance to bacteriophage KO1-2, which recognizes and lyses strains with lipopolysaccharide molecules containing the D-galactan II O antigen. Strain RFK-11 contains a defect in O-antigen synthesis and has a complete core, including the attachment site for O antigen. This mutation is complemented by a plasmid carrying the rfb (O-antigen biosynthesis) gene cluster from the related K. pneumoniae serotype O1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the lipopolysaccharide from strain RFK-9 has a mobility typical of deep-rough lipopolysaccharide. RFK-9 lipopolysaccharide lacks the attachment site for O antigen. Lipopolysaccharides from strains RFK-9 and RFK-11 were isolated, and their structures were determined by methylation analyses, muclear magnetic resonance spectroscopy, and mass spectroscopy. The deduced O8 core oligosaccharide includes the partial core structure reported for the K. pneumoniae subspecies pneumoniae serotype O1 lipopolysaccharide (M. Süsskind, S. Müller-Leonnies, W. Nimmich, H. Brade, and O. Holst, Carbohydr. Res. 269:C1-7, 1995), consistent with the possibility of a conserved core structure within the species. The core oligosaccharide differs from those of the genera Salmonella and Escherichia by the absence of a hexose-containing outer core, the lack of phosphate residues in the inner core, and the presence of galacturonic acid residues.

Temperature-sensitive, lipopolysaccharide-deficient mutants of Salmonella typhimurium

Two mutants of Salmonella typhimurium LT2, which were temperature-sensitive for lipopolysaccharide (LPS) synthesis, were isolated from a galE (-) strain based on their resistance to phage C21 and sensitivity to sodium deoxycholate at 42°C. They produced LPS of chemotype Rc at 30°C and deep-rough LPS at 42°C. P22-mediated transductional analysis showed that the mutations responsible for temperature sensitivity are located in the rfa cluster where several genes involved in the synthesis of the LPS core are mapped. A plasmid, carrying rfaC, D and F genes of Escherichia coli K-12, complemented these mutations. These genes are responsible for the synthesis of the inner-core region of the LPS molecule. This indicates that genetic defects in these temperature-sensitive mutants affect the inner-core region of LPS.