Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses.

A family of mutants of Salmonella typhimurium with altered lipopolysaccharide (LPS) core chain lengths were assessed for sensitivity to freeze-thaw and other stresses. Deep rough strains with decreased chain length in the LPS core were more susceptible to novobiocin, polymyxin B, bacitracin, and sodium lauryl sulfate during growth, to ethylenediaminetetraacetic acid and sodium lauryl sulfate in resting suspension, and to slow and rapid freeze-thaw in water and saline, and these strains exhibited more outer membrane damage than the wild type or less rough strains. Variations in the LPS chain length did not dramatically affect the sensitivity of the strains to tetracycline, neomycin, or NaCl in growth conditions or the degree of freeze-thaw-induced cytoplasmic membrane damage. The deeper rough isogenic strains incorporated larger quantities of less-stable LPS and less protein into the outer membrane than did the wild type or less rough mutants, indicating that the mutations affected outer membrane synthesis or organization or both. Nikaido's model of the role of LPS and protein in determining the resistance of gram-negative bacteria to low-molecular-weight hydrophobic antibiotics is discussed in relation to the stress of freeze-thaw.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Effect of cultural conditions on the protein and lipopolysaccharide profiles of Haemophilus ducreyi analysed by SDS-PAGE

Abstract A comparative study of the protein and lipopolysaccharide (LPS) profiles of 8 strains of Haemophilus ducreyi revealed that protein patterns remained unaffected by changes in medium composition, atmospheric conditions and temperature. In contrast, the LPS patterns exhibited marked variations under the different cultural conditions.     

Heterogeneity in Expression of Lipopolysaccharide and Major Outer-Membrane Proteins by Strains of Escherichia coli O157 with Different H-Serotypyes

A total of 11 strains of Escherichia coli (E. coli) belonging to serogroup O157 were examined for the expression of long-chain lipopolysaccharide (LPS) and major outer-membrane proteins (OMPs) by means of SDS-PAGE. The strains belonged to either one of four different flagellar (H) types or did not express flagella. Four of the eleven strains carried genes encoding Shiga-like toxins (SLTs). All the strains exhibited one of four LPS profiles, designated A, B, C or D. Electron microscopic analysis with the freeze-substitution technique demonstrated the differences in the cell surface structures of strains with each LPS profile. Strains with LPS profile A, B or C had layers of thin fibers 10, 20 and 20 nm long, respectively, on the outer membrane but strains with LPS profile D had no such structure. An analysis of the OMPs showed that all the strains had one of four OMP profiles, designated I, II, III or IV. Both LPS and OMP profiles were dependent on H-serotypes, and the combination pattern of LPS and OMP profiles of the strains was unique for each H-serotype. These data support the existence of heterogeneous groups of O157 strains.     

Ultrastructural examination of the lipopolysaccharides of Pseudomonas aeruginosa strains and their isogenic rough mutants by freeze-substitution.

The majority of Pseudomonas aeruginosa strains synthesize two antigenically distinct types of lipopolysaccharide (LPS), namely, a serotype-specific B-band LPS and a common antigen A-band LPS. A-band LPS consists of uncharged poly-D-rhamnan, which does not bind uranyl ions and is difficult to stain for electron microscopy; the highly charged B-band LPS is more easily visualized. We selected two wild-type strains, PAO1 (serotype O5) and IATS O6 (serotype O6), generated isogenic mutants from them, and examined the distribution of LPS on the surface of these organisms by freeze-substitution and electron microscopy. On PAO1 cells, which express both A-band and B-band LPSs, a 31- to 36-nm-wide fringe extending perpendicularly from the outer membrane was observed. A fine fibrous material was also observed on the surface of serotype O6 (A+ B+) cells, although this material did not form a uniform layer. When the LPS-deficient mutants, strains AK1401 (A+ B-), AK 1012 (A- B-), rd7513 (A- B-), and R5 (an IATS O6-derived rough mutant; A- B-), were examined, no extraneous material was apparent above the bilayer. However, an asymmetrical staining pattern was observed on the outer leaflet of the outer membrane of each of these mutants, presumably conforming to the anionic charge distribution of the core region of the rough LPS. In all cases, expression of the LPS types was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. When optical densitometry on electron microscopy negatives was used to analyze the outer membrane staining profiles, subtle differences in the degrees of core deficiency among rough mutants were detectable. This is the first time an electron microscopy technique has preserved the infrastructure produced in the outer membrane by its constituent macromolecules. We conclude that freeze-substitution electron microscopy is effective in the visualization of LPS morphotypes.     

Variations in Outer-membrane Characteristics of Two Stem-nodulating Bacteria of <Emphasis Type="Italic">Sesbania rostrata</Emphasis> and its Role in Tolerance Towards Diverse Stress

Outer-membrane characteristics may determine the survivability of rhizobia under diverse abiotic and biotic stresses. Therefore, the role of lipopolysaccharides (LPS) and membrane proteins of two stem-nodulating bacteria of Sesbania rostrata (Azorhizobium caulinodans ORS571 and Rhizobium sp. WE7) in determining tolerance towards abiotic and biotic stresses (hydrophobics and phages) was investigated. Outer-membrane characteristics (LPS and membrane–protein profiles) of ORS571, WE7 and thirteen standard strains were distinct. ORS571 and WE7 also showed susceptibility towards morphologically distinct phages, i.e., ACSR16 (short-tailed) and WESR29 (long-tailed), respectively. ORS571 and WE7 were tolerant to hydrophobic compounds (triton X-100, rifampicin, crystal violet and deoxycholate). To ascertain the role of outer membrane characteristics in stress tolerance, phage-resistant transconjugant mutants of ORS571 (ORS571-M8 and ORS571-M20) and WE7 (WE7-M9) were developed. LPS- and membrane–protein profiles of mutants differed from that of respective wild types (ORS571 and WE7). In in vitro assay, phages got adsorbed onto purified LPS-membrane protein fractions of wild types. Phages did not adsorb onto membrane fraction of mutants and standard strains. Mutant with reduced expression of LPS (ORS571-M20 and WE7-M9) showed reduced tolerance towards hydrophobics. However, the tolerance was unaffected in mutant (ORS571-M8) where expression of LPS was not reduced but pattern was different. The tolerance level of mutants towards hydrophobics varied with the expression of LPS, whereas the specificity towards phages is correlated with the specific LPS pattern.     

Diversity of the outer membrane protein P2 gene from major clones of Haemophilus influenzae type b

In previous studies, it has been demonstrated that outer membrane protein P2 from Haemophilus influenzae type b has porin activity and that antibody directed against P2 is protective in an infant rat bacteraemic model. Outer membrane protein subtyping has been employed to subclassify type b Haemophilus isolates. Strain MinnA has the outer membrane protein subtype 1H and is representative of the dominant clonal group of disease-producing isolates in the United States. In the present study, the P2 gene from strain MinnA was employed to probe EcoRI- and Pvull-digested chromosomal DNA from 24 Haemophilus influenzae type b isolates representative of the common outer membrane protein subtype groups observed throughout the world. Restriction fragment length polymorphisms were identified for the members of the outer membrane protein subtype 3L group, but not for the other subtypes examined. The P2 gene from each of four prototype isolates was then cloned, sequenced and compared to the previously reported sequence of the strain MinnA gene. The P2 gene from each of two isolates with the outer membrane protein subtype 3L was identical to the MinnA P2 sequence. The P2 gene from a subtype 2L isolate differed by a single nucleotide and the gene from a subtype 6U isolate differed by 13 nucleotides. Thus, the P2 protein is highly conserved among type b isolates.     

Molecular composition of the outer membrane of Escherichia coli and the importance of protein-lipopolysaccharide interactions

Whole cells of Escherichia coli strains 0111, K12 and B as well as the ampicillin-resistant mutant K12 D21 and several lipopolysaccharide (LPS) mutants derived from this strain were analyzed for their molar LPS content per mg dry weight. An increase of the LPS concentration in some LPS mutants was substantiated by analyzing isolated cell walls and relating the molar LPS content to the murein subunit as measure of cell surface area. The increase of LPS was paralleled by increasing amounts of phospholipid while the overall protein content in the outer membrane decreased.According to the pattern of major outer membrane proteins in the various strains and the respective LPS structures, protein-LPS interactions are discussed as important requirements for outer membrane assembly and stability.Abbreviations LPS lipopolysaccharide - SDS sodium dodecyl-sulfate Dedicated to Dr. Otto Lüderitz on the occasion of his 60th birthday     

Phenotypic characterization of Vibrio vulnificus biotype 2, a lipopolysaccharide-based homogeneous O serogroup within Vibrio vulnificus.

In this study, we have reevaluated the taxonomic position of biotype 2 of Vibrio vulnificus. For this purpose, we have biochemically and serologically characterized 83 biotype 2 strains from diseased eels, comparing them with 17 biotype 1 strains from different sources. Selected strains were also molecularly analyzed and tested for eel and mouse pathogenicity. Results have shown that biotype 2 (i) is biochemically homogeneous, indole production being the main trait that distinguishes it from biotype 1, (ii) presents small variations in DNA restriction profiles and outer membrane protein patterns, some proteins being immunologically related to outer membrane proteins from biotype 1, (iii) expresses a common lipopolysaccharide (LPS) profile, which is immunologically identical among strains and distinct from that of LPS of tested biotype 1 strains, and (iv) contains at least two high-Mr plasmids. Regarding host range, we have confirmed that both biotypes are pathogenic for mice but only biotype 2 is pathogenic for eels. On the basis of these data, we propose that biotype 2 of V. vulnificus constitutes an LPS-based O serogroup which is phenotypically homogeneous and pathogenic for eels. In this article, the serogroup is designated serogroup E (for eels).     

Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus.

The lipopolysaccharide (LPS) of the outer membrane of Caulobacter crescentus was purified and analyzed. Two distinct strains of the species, NA 1000 and CB2A, were examined; despite differences in other membrane-related polysaccharides, the two gave similar LPS composition profiles. The LPS was the equivalent of the rough LPS described for other bacteria in that it lacked the ladder of polysaccharide-containing species that results from addition of variable amounts of a repeated sequence of sugars, as detected by gel electrophoresis in smooth LPS strains. The purified LPS contained two definable regions: (i) an oligosaccharide region, consisting of an inner core of three residues of 2-keto-3-deoxyoctonate, two residues of alpha-L-glycero-D-mannoheptose, and one alpha-D-glycero-D-mannoheptose unit and an outer core region containing one residue each of alpha-D-mannose, alpha-D-galactose, and alpha-D-glucose, with the glucose likely phosphorylated and (ii) a region equivalent to the lipid A region of the archetype, consisting primarily of an esterified fatty acid, 3-OH-dodecanoate. The lipid A-like region was resistant to conclusive analysis; in particular, although a variety of analytical methods were used, no amino sugars were detected, as is found in the lipid A of the LPS of most bacteria.     

The capsular polysaccharide of Haemophilus influenzae type B prevents killing by complement and antibodies against outer membrane protein a

Abstract The ability of antibodies, raised in rabbits against purified outer membrane protein a ( M _r 47 000) of Haemophilus influenzae type b, to promote complement-dependent killing of these encapsulated organisms was investigated. Killing of encapsulated strains was not induced by these antibodies in conjunction with either human, mouse, rabbit or guinea-pig complement. Acapsular mutants were effectively killed by complement in the presence of antibodies against protein a . Killing was dependent on the presence of the 47-kDa protein a and was not influenced by the outer membrane protein subtype or lipopolysaccharide serotype of the strain. The killing-promoting activity could be absorbed from the sera with cells of strains with the same protein a , purified protein a , but not by purified lipopolysaccharide and capsular polysaccharide. Binding experiments showed that the encapsulated strain and its acapsular mutant bound antibodies against protein a with the same rate and to the same extent, indicating that the capsule probably interferes with complement activation or insertion of the membrane attack complex into the bacterial cell.     

Lipopolysaccharide transport to the bacterial outer membrane in spheroplasts

The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner membrane to the outer membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the outer membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner membrane. The outer membrane localization of the LPS was confirmed by its modification by the outer membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the outer membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the outer membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the outer membrane.     

Serum IgG response to Burkholderia cepacia outer membrane antigens in cystic fibrosis: Assessment of cross-reactivity with Pseudomonas aeruginosa

Abstract Burkholderia cepacia (Pseudomonas cepacia) is now recognised as an important pathogen in cystic fibrosis patients, and several reports have suggested that sputum-culture-proven colonisation occurs despite the presence of specific antibody. In an attempt to establish the use of antibody studies as diagnostic and prognostic indicators of B. cepacia infection, we have examined the IgG response to B. cepacia outer membrane proteins and lipopolysaccharide in patients also colonised with P. aeruginosa . The B. cepacia strains were grown in a modified iron-depleted chemically defined medium and outer membrane components examined by SDS-PAGE and immunoblotting. IgG antibodies were detected against B. cepacia outer membrane antigens, which were not diminished by extensive preadsorption with P. aeruginosa . The response to B. cepacia O-antigen could be readily removed by adsorption of serum either with B. cepacia whole cells or purified LPS, whereas we were unable to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells. The inability to adsorb anti-outer membrane protein antibodies using B. cepacia whole cells maybe due to non-exposed surface epitopes. Several B. cepacia sputum-culture negative patients colonised with P. aeruginosa had antibodies directed against B. cepacia outer membrane protein. This study suggests that there is a specific anti- B. cepacia LPS IgG response, which is not due to antibodies cross-reactive with P. aeruginosa . Our studies indicate that much of the B. cepacia anti-outer membrane protein response is specific and not attributable to reactivity against co-migrating LPS.     

Altered antigenicity is seen in the lipopolysaccharide profile of non-serotypeable Actinobacillus actinomycetemcomitans strains

Non-serotypeable Actinobacillus actinomycetemcomitans strains may be derived from the serotypeable ones. In the present study, we compared the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of serotypeable and non-serotypeable A. actinomycetemcomitans strains (n=24) of the same genotype in the same subject (n=6) to find out if alterations on the cell-surface contribute to the non-serotypeability. Serotypeable and non-serotypeable A. actinomycetemcomitans strains showed great similarity in the OMP patterns both within and between subjects. Using serotype-specific antisera, clear immunoblotting LPS profiles in the O-antigenic region were seen in serotype b and c strains but not in non-serotypeable strains from the same subjects. The results suggest that changes in LPS lead to the altered antigenicity of non-serotypeable A. actinomycetemcomitans strains.     

Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids,outer membrane protein profiles and siderophore production

The virulence of 18 strains of Vibrio anguillarum serogroup O1 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it, produced siderophores as well as OM2 but were non-pathogenic to fish. Likewise, among the virulent strains, considerable variation in LD50 values was recorded. Plasmid profiling and restriction analysis showed that the virulence plasmid existed in various molecular weights from 26 to 80 kbp, with 65-67 kbp being the most common, and that this plasmid displayed various restriction profiles. The presence of other plasmids did not seem to affect the pathogenic properties.     

Daring to be different: colicin N finds another way

The mechanisms by which colicins, protein toxins produced by Escherichia coli, kill other E. coli, have become much better understood in recent years. Most colicins initially bind to an outer membrane protein receptor, and then search for a separate nearby outer membrane protein translocator that serves as a pathway into target cells. Many colicins use the outer membrane porin, OmpF, as that translocator, while using a different primary receptor. Colicin N is unique among known colicins in that only OmpF had been identified as being required for uptake of the colicin and it was presumed to somehow serve as both receptor and translocator. Genetic screens also identified a number of genes required for lipopolysaccharide (LPS) synthesis as uniquely required for killing by colicin N, but not by other colicins. Johnson et al. show that the receptor‐binding domain of colicin N binds to LPS, and does not require OmpF for that binding. LPS of a minimal length is required for binding, explaining the requirement for specific elements of the LPS biosynthetic pathway. For colicin N, the receptor‐binding domain does not recognize a protein, but rather the most abundant component of the outer membrane itself, LPS.     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.     

Phenotypic, antigenic, and molecular characterization of Pasteurella piscicida strains isolated from fish.

We compared Pasteurella piscicida strains isolated from different fish species in several European countries with strains isolated in Japan and the United States. The taxonomic analysis revealed that, regardless of the geographic origin and source of isolation, all the strains exhibited the same biochemical and physiological characteristics. Serological assays with different rabbit antisera demonstrated a high level of antigenic similarity among strains, with cross-agglutination titers of 20,480 to 40,960. This serological homogeneity was supported by the lipopolysaccharide (LPS) and membrane protein profiles. All the P. piscicida strains had the same electrophoretic LPS pattern, showing O side chains with a ladder-like structure, and shared at least four major outer membrane proteins, of 20, 30, 42, and 53 kDa. Western blot (immunoblot) analysis with LPS and protein indicated that all the P. piscicida strains are immunologically related. In addition, the chromosomal DNA fingerprint patterns obtained for the European strains with the enzymes EcoRI and BamHI were practically identical to those of the Japanese and U.S. strains. Although some differences were found in the plasmid profiles of P. piscicida, a large number of strains possessed in common plasmid bands of 20 and 7 MDa. In addition, a plasmid of 50 MDa was present in the majority of the European strains. Restriction endonuclease analysis demonstrated the genetic homology of the plasmid bands shared by most of the European strains. All the P. piscicida strains had the same drug resistance patterns, indicating that a correlation between plasmid carriage and resistance to a specific antimicrobial agent cannot be established. The high levels of phenotypic, serological, and genetic homogeneity found among the P. piscicida strains should facilitate the development of DNA probes with diagnostic purposes as well as the design of effective vaccines.