Relating the physical properties of Pseudomonas aeruginosa lipopolysaccharides to virulence by atomic force microscopy

Lipopolysaccharides (LPS) are an important class of macromolecules that are components of the outer membrane of Gram-negative bacteria such as Pseudomonas aeruginosa. P. aeruginosa contains two different sugar chains, the homopolymer common antigen (A band) and the heteropolymer O antigen (B band), which impart serospecificity. The characteristics of LPS are generally assessed after isolation rather than in the context of whole bacteria. Here we used atomic force microscopy (AFM) to probe the physical properties of the LPS of P. aeruginosa strain PA103 (serogroup O11) in situ. This strain contains a mixture of long and very long polymers of O antigen, regulated by two different genes. For this analysis, we studied the wild-type strain and four mutants, ΔWzz1 (producing only very long LPS), ΔWzz2 (producing only long LPS), DΔM (with both the wzz1 and wzz2 genes deleted), and Wzy::GM (producing an LPS core oligosaccharide plus one unit of O antigen). Forces of adhesion between the LPS on these strains and the silicon nitride AFM tip were measured, and the Alexander and de Gennes model of steric repulsion between a flat surface and a polymer brush was used to calculate the LPS layer thickness (which we refer to as length), compressibility, and spacing between the individual molecules. LPS chains were longest for the wild-type strain and ΔWzz1, at 170.6 and 212.4 nm, respectively, and these values were not statistically significantly different from one another. Wzy::GM and DΔM have reduced LPS lengths, at 34.6 and 37.7 nm, respectively. Adhesion forces were not correlated with LPS length, but a relationship between adhesion force and bacterial pathogenicity was found in a mouse acute pneumonia model of infection. The adhesion forces with the AFM probe were lower for strains with LPS mutations, suggesting that the wild-type strain is optimized for maximal adhesion. Our research contributes to further understanding of the role of LPS in the adhesion and virulence of P. aeruginosa.     

Biosynthesis of cryptic lipopolysaccharide glycoforms in Haemophilus influenzae involves a mechanism similar to that required for O-antigen synthesis

It is generally thought that mucosal bacterial pathogens of the genera Haemophilus, Neisseria, and Moraxella elaborate lipopolysaccharide (LPS) that is fundamentally different from that of enteric organisms that express O-specific polysaccharide side chains. Haemophilus influenzae elaborates short-chain LPS that has a role in the pathogenesis of H. influenzae infections. We show that the synthesis of LPS in this organism can no longer be as clearly distinguished from that in other gram-negative bacteria that express an O antigen. We provide evidence that a region of the H. influenzae genome, the hmg locus, is involved in the synthesis of glycoforms in which tetrasaccharide units are added en bloc, not stepwise, to the normal core glycoforms, similar to the biosynthesis of an O-antigen.     

Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

Molecular dynamics simulations of the interaction of phospholipid bilayers with polycaprolactone

The molecular interaction between common polymer chains and the cell membrane is unknown. Molecular dynamics simulations offer an emerging tool to characterise the nature of the interaction between common degradable polymer chains used in biomedical applications, such as polycaprolactone, and model cell membranes. Herein we characterise with all-atomistic and coarse-grained molecular dynamics simulations the interaction between single polycaprolactone chains of varying chain lengths with a phospholipid membrane. We find that the length of the polymer chain greatly affects the nature of interaction with the membrane, as well as the membrane properties. Furthermore, we next utilise advanced sampling techniques in molecular dynamics to characterise the two-dimensional free energy surface for the interaction of varying polymer chain lengths (short, intermediate, and long) with model cell membranes. We find that the free energy minimum shifts from the membrane-water interface to the hydrophobic core of the phospholipid membrane as a function of chain length. Finally, we perform coarse-grained molecular dynamics simulations of slightly larger membranes with polymers of the same length and characterise the results as compared with all-atomistic molecular dynamics simulations. These results can be used to design polymer chain lengths and chemistries to optimise their interaction with cell membranes at the molecular level.     

Regulation of Salmonella typhimurium lipopolysaccharide O antigen chain length is required for virulence; identification of FepE as a second Wzz

Wzz proteins regulate the degree of polymerization of the O antigen (Oag) subunits in lipopolysaccharide (LPS) biosynthesis. Although the pathogenic relevance of Oag is well recognized, the significance of Oag chain length regulation is not well defined. In this report, Salmonella typhimurium was shown to possess two functional wzz genes resulting in a bimodal Oag length distribution. In addition to the previously described wzzST that results in long (L) modal length LPS with 16-35 Oag repeat units (RUs), we now report that wzzfepE, a homologue of Escherichia coli fepE, is responsible for the production of very long (VL) modal length LPS Oag, estimated to contain> 100 Oag RUs. Analysis of a series of isogenic S. typhimurium C5 mutants found that the presence of either wzz gene (and hence either modal length) was sufficient for complement resistance and virulence in the mouse model of infection, suggesting a degree of redundancy in the role of these two wzz genes and their respective Oag modal lengths. In contrast, the wzzST/wzzfepE double mutant, with relatively short, random-length Oag, displayed enhanced susceptibility to complement and was highly attenuated in the mouse. This clearly demonstrates the molecular genetic basis for the longer LPS Oag chains previously identified as the basis of complement resistance in Salmonella. The presence of wzzfepE homologues in the genomic sequences of strains of Escherichia coli, Shigella flexneri and multiple serovars of Salmonella suggests that bimodality of LPS Oag is a common phenomenon in the Enterobacteriaceae.     

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.     

Geometrical constraints limiting the poly(ADP‐ribose) conformation investigated by molecular dynamics simulation

Poly(ADP‐ribosylation) is a post‐transductional modification that regulates protein's function. Most of the proteins subjected to this control mechanism belong to machineries involved in DNA damage repair, or DNA interacting proteins. Poly(ADP‐ribose) polymers are long chains of even 100 monomer length that can be branched at several positions but, not withstanding its importance, nothing is known concerning its structure. To understand, which are the geometrical parameters that confer to the polymer the structural constraints that determine its interaction with the target proteins, we have performed molecular dynamics of three chains of different length, made by 5, 25, and 30 units, the last one being branched. Analysis of the simulations allowed us to identify the main intra‐ and inter‐monomer dihedral angles that govern the structure of the polymer that however, does not reach a unique definite conformation. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 78–86, 2014.     

Lipid chain selectivity by outer membrane phospholipase A

Outer membrane phospholipase A (OMPLA) is a unique, integral membrane enzyme found in Gram-negative bacteria and is an important virulence factor for pathogens such as Helicobacter pylori. This broad-specificity lipase degrades a variety of lipid substrates, and it plays a direct role in adjusting the composition and permeability of bacterial membranes under conditions of stress. Interestingly, OMPLA shows little preference for the lipid headgroup and, instead, the length of the hydrophobic acyl chain is the strongest determinant for substrate selection by OMPLA, with the enzyme strongly preferring substrates with chains equal to or longer than 14 carbon atoms. The question remains as to how a hydrophobic protein like OMPLA can achieve this specificity, particularly when the shorter chains can be accommodated in the binding pocket. Using a series of sulfonyl fluoride inhibitors with various lengths of acyl chain, we show here that the thermodynamics of substrate-induced OMPLA dimerization are guided by the acyl chain length, demonstrating that OMPLA uses a unique biophysical mechanism to select its phospholipid substrate.     

Lipopolysaccharide binding to the periplasmic protein LptA

Lipopolysaccharide (LPS) and the periplasmic protein, LptA, are two essential components of Gram‐negative bacteria. LPS, also known as endotoxin, is found asymmetrically distributed in the outer leaflet of the outer membrane of Gram‐negative bacteria such as Escherichia coli and plays a role in the organism's natural defense in adverse environmental conditions. LptA is a member of the lipopolysaccharide transport protein (Lpt) family, which also includes LptC, LptDE, and LptBFG₂, that functions to transport LPS through the periplasm to the outer leaflet of the outer membrane after MsbA flips LPS across the inner membrane. It is hypothesized that LPS binds to LptA to cross the periplasm and that the acyl chains of LPS bind to the central pocket of LptA. The studies described here are the first to comprehensively characterize and quantitate the binding of LPS by LptA. Using site‐directed spin‐labeling electron paramagnetic resonance (EPR) spectroscopy, data were collected for 15 spin‐labeled residues in and around the proposed LPS binding pocket on LptA to observe the mobility changes caused by the presence of exogenous LPS and identify the binding location of LPS to LptA. The EPR data obtained suggest a 1:1 ratio for the LPS:LptA complex and allow the first calculation of dissociation constants for the LptA–LPS interaction. The results indicate that the entire protein is affected by LPS binding, the N‐terminus unfolds in the presence of LPS, and a mutant LptA protein unable to form oligomers has an altered affinity for LPS.     

Lipopolysaccharide O-antigen chain length regulation in Pseudomonas aeruginosa serogroup O11 strain PA103

The Wzz proteins are important for determining the length of the O-antigen side chain attached to lipopolysaccharide (LPS). Several bacteria, including Pseudomonas aeruginosa strain PAO1 (serogroup O5), produce two such proteins responsible for the preference of two different chain lengths on the surface. Our group has previously identified one wzz gene (wzz1) within the O-antigen locus of P. aeruginosa strain PA103 (serogroup O11). In this study we have identified the second wzz gene (wzz2), located in the same region of the genome and with 92% similarity to PAO1's wzz2 gene. Mutations were generated in both wzz genes by interruption with antibiotic resistance cassettes, and the effects of these mutations were characterized. Wild-type PA103 prefers two O-antigen chain lengths, referred to as long and very long. The expression of the long O-antigen chain length was reduced in the wzz1 mutant, indicating the Wzz1 protein is important for this chain length preference. The wzz2 mutant, on the other hand, was missing O-antigens of the very long chain length, indicating the Wzz2 protein is responsible for the production of very long O-antigen. The effects of the wzz mutations on virulence were also investigated. In both serum sensitivity assays and a mouse pneumonia model of infection, the wzz1 mutants exhibited greater defects in virulence compared to either wild-type PA103 or the wzz2 mutant, indicating the long chain length plays a greater role during these infectious processes.     

C3 binds preferentially to long-chain lipopolysaccharide during alternative pathway activation by Salmonella montevideo

We studied the population of LPS molecules on Salmonella montevideo that bind C3 during alternative pathway activation in serum. LPS molecules of Salmonella are composed of lipid A:core oligosaccharide (one copy per molecule), substituted by an O-polysaccharide (O-PS) side chain, which is a linear polymer of 0 to greater than 60 O-antigen repeat units containing mannose. A mutant of S. montevideo called SL5222 that inserts galactose only into core oligosaccharide and mannose only into O-antigen subunits was grown with [3H]mannose and [14C]galactose, so that LPS molecules bearing large numbers of O-antigen subunits have high 3H to 14C ratios, whereas molecules with few O-antigen subunits have lower 3H to 14C ratios. Double-labeled SL5222 was incubated in C8-deficient (C8D) serum or C8D serum with 2 mM Mg++Cl2 and 10 mM ethylene glycoltetraacetic acid (MgEGTA C8D). LPS molecules with covalently attached C3 were identified by binding to anti-C3. LPS molecules that bound C3 under both incubation conditions had O chains seven to eight times longer than the average LPS molecule. SL5222 was then grown in suboptimal concentrations of mannose in order to decrease the number of LPS molecules with long O-PS side chains. C3 attached to progressively shorter chain molecules of LPS as the mannose input was lowered, but still chose the longest available molecules. This finding and recently published observations indicate that C3 can bind to LPS molecules with short O-PS side chains. We postulate that preferential attachment of C3 to long-chain LPS in SL5222 results because long-chain LPS molecules sterically hinder shorter chain LPS molecules from macromolecules. This study provides direct proof that the O-PS of LPS sterically hinders access of large molecules to the outer membrane and indicates that the LPS coat of these bacteria functions as a barrier against large protein molecules.     

High state of order of isolated bacterial lipopolysaccharide and its possible contribution to the permeation barrier property of the outer membrane.

The conformational properties of the isolated S form of Salmonella sp. lipopolysaccharide (LPS), of Re mutant LPS, and of free lipid A were investigated by using X-ray diffraction and conformational energy calculations. The data obtained showed that LPS in a dried, in a hydrated, and probably also in an aqueous dispersion state is capable of forming bilayered lamellar arrangements similar to phospholipids. From the bilayer packing periodicities, a geometrical model of the extensions of the LPS regions lipid A, 2-keto-3-deoxyoctulosonic acid, and O-specific chain along the membrane normal could be calculated. Furthermore, the lipid A component was found to assume a remarkably high ordered conformation: its fatty acid chains were tightly packed in a dense hexagonal lattice with a center-to-center distance of 0.49 nm. The hydrophilic backbone of lipid A showed a strong tendency to form domains in the membrane, resulting in a more or less parallel arrangement of lipid A units. According to model calculations, the hydrophilic backbone of lipid A appears to be oriented approximately 45 degrees to the membrane surface, which would lead to a shed roof-like appearance of the surface structure in the indentations of which the 2-keto-3-deoxyoctulosonic acid moiety would fit. In contrast, the O-specific chains assume a low ordered, heavily coiled conformation. Comparison of these structural properties with those known for natural phospholipids in biological membranes indicates that the high state of order of the lipid A portion of LPS might be an important factor in the structural role and permeation barrier functions of LPS in the outer membrane of gram-negative bacteria.     

Chemical relaxation of co-operative conformational transitions of linear biopolymers

The kinetics and chemical relaxation of co-operative conformational changes of linear (bio)polymers (e.g. helix-coil transitions of polypeptides) are discussed on the basis of the linear ISING model. Chemical relaxation is in general shown to be described by 4N−5 relaxation times if the polymer chain consists of N elementary reaction sites. It is pointed out that nevertheless substantial simplifications of the theory can be achieved in many special cases of practical interest. Sufficiently short chains exhibit first-order kinetics resulting in a single relaxation time whereas for certain medium chain lengths zero-order kinetics plays a principal role in the relaxation process. For the particularly interesting case of very long chains a set of four relaxation equations is derived. The corresponding relaxation times are calculated assuming strong co-operativity and slow nucleation rates. However, it is almost exclusively the largest one of these relaxation times which actually controls the conformational change as turns out by means of a new approach to compute amplitude factors.     

The thermal polymerization of orosomucoid

1. Orosomucoid was prepared from the urine of a nephrotic patient and polymerized by heating it in a range of salt concentrations at pH4·1. 2. Heating at low ionic strengths produced a `chain' polymer of indefinite length but having the same width as the diameter of the monomer (5·0nm.). Similar treatment in high ionic strengths also produced a spherical (`ball') polymer of limited diameter (14·8nm.). 3. The size and shape of both polymers were determined from ultra-centrifuge, gel-filtration and electron-microscope results. The results suggest that eight monomer units condense to form the ball polymer. 4. Heating orosomucoid at pH1·8 hydrolysed the N-acetylneuraminic acid off the molecule; only chains could then be formed, even in high ionic strengths. 5. Both polymers were stable under normal conditions but could be depolymerized in 3m-guanidine hydrochloride. The monomer could be repolymerized on heating: the `chain monomer' only formed chains at all ionic strengths, but the `ball monomer' was indistinguishable from the original monomer in its immunological properties and polymerization reaction.     

Genetics of lipopolysaccharide biosynthesis in enteric bacteria.

From a historical perspective, the study of both the biochemistry and the genetics of lipopolysaccharide (LPS) synthesis began with the enteric bacteria. These organisms have again come to the forefront as the blocks of genes involved in LPS synthesis have been sequenced and analyzed. A number of new and unanticipated genes were found in these clusters, indicating a complexity of the biochemical pathways which was not predicted from the older studies. One of the most dramatic areas of LPS research has been the elucidation of the lipid A biosynthetic pathway. Four of the genes in this pathway have now been identified and sequenced, and three of them are located in a complex operon which also contains genes involved in DNA and phospholipid synthesis. The rfa gene cluster, which contains many of the genes for LPS core synthesis, includes at least 17 genes. One of the remarkable findings in this cluster is a group of several genes which appear to be involved in the synthesis of alternate rough core species which are modified so that they cannot be acceptors for O-specific polysaccharides. The rfb gene clusters which encode O-antigen synthesis have been sequenced from a number of serotypes and exhibit the genetic polymorphism anticipated on the basis of the chemical complexity of the O antigens. These clusters appear to have originated by the exchange of blocks of genes among ancestral organisms. Among the large number of LPS genes which have now been sequenced from these rfa and rfb clusters, there are none which encode proteins that appear to be secreted across the cytoplasmic membrane and surprisingly few which encode integral membrane proteins or proteins with extensive hydrophobic domains. These data, together with sequence comparison and complementation experiments across strain and species lines, suggest that the LPS biosynthetic enzymes may be organized into clusters on the inner surface of the cytoplasmic membrane which are organized around a few key membrane proteins.     

Lipopolysaccharide size and distribution determine serum resistance in Salmonella montevideo.

The survival of Salmonella montevideo during serum treatment depends on the presence of an O antigen (O-Ag) associated with the lipopolysaccharide molecule. In this organism, the O antigen is a polysaccharide composed of 0 to more than 55 subunits, each containing 4 mannose residues together with glucose and n-acetylglucosamine. We used a mutant strain of S. montevideo that requires exogenous mannose for the synthesis of O-Ag. Lipopolysaccharide (LPS) was prepared from these cells grown under three different conditions where the availability of exogenous mannose was regulated such that the average number of O-Ag units per LPS molecule, the percentage of LPS molecules bearing long O-Ag side chains, and the percentage of lipid A cores bearing O-Ag were all varied. These changes in LPS profiles were monitored on sodium dodecyl sulfate-polyacrylamide gels, and cells with different LPS profiles were tested for their ability to survive treatment with pooled normal human serum. Survival in serum was associated with LPS that contained an average of 4 to 5 O-Ag units per LPS molecule, and 20 to 23% of the LPS molecules had more than 14 O-Ag units per LPS molecule. Serum survival was less clearly associated with the percentage of lipid A cores covered with O-Ag. We propose, based on these data and on previous work, that the O-Ag polysaccharide provides the cell protection from serum killing by sterically hindering access of the C5b-9 complex to the outer membrane and that a critical density of long O-Ag polysaccharide is necessary to provide protection.     

Modulation of the surface architecture of Gram-negative bacteria by the action of surface polymer:lipid A–core ligase and by determinants of polymer chain length

Lipopolysaccharides (LPSs) are complex glycolipids found in the outer membrane of Gram-negative bacteria. The lipid A–core component of the LPS molecule provides a versatile anchor to which a surface polymer:lipid A–core ligase enzyme can attach one or more structurally distinct surface polymers in a single bacterial strain. In some cases the same polymer can be found on the cell surface in both lipid A–core-linked and -unlinked forms. Analysis by SDS–PAGE of populations of LPS molecules extracted from bacterial cells indicates that there is extensive heterogeneity in their size distribution. Much of the heterogeneity results from complex modal distributions in the chain length of the polymers which are attached to lipid A–core. This is the result of preferential ligation of polymers with specific degrees of polymerization during the assembly of the LPS molecule. The surface architecture of the Gram-negative bacterial cell is therefore profoundly affected by the activities of the surface polymer:lipid A–core ligase and by molecular determinants of polymer chain length. Because of the involvement of cell-surface polymers in interactions between pathogenic bacteria and their hosts, these enzymatic activities also have an important impact on virulence. In this review, the organization of LPSs and related surface polymers will be described and the current understanding of the molecular mechanisms involved in surface diversity will be discussed. Emphasis is placed on the Enterobacteriaceae, but similarities to other bacteria suggest that aspects of the enterobacterial system will have broader significance.     

The LptD chaperone LptE is not directly involved in lipopolysaccharide transport in Neisseria meningitidis

The biosynthesis of lipopolysaccharide (LPS) in gram-negative bacteria is well understood, in contrast to the transport to its destination, the outer leaflet of the outer membrane. In Escherichia coli, synthesis and transport of LPS are essential processes. Neisseria meningitidis, conversely, can survive without LPS and tolerates inactivation of genes involved in LPS synthesis and transport. Here, we analyzed whether the LptA, LptB, LptC, LptE, LptF, and LptG proteins, recently implicated in LPS transport in E. coli, function similarly in N. meningitidis. None of the analyzed proteins was essential in N. meningitidis, consistent with their expected roles in LPS transport and additionally demonstrating that they are not required for an essential process such as phospholipid transport. As expected, the absence of most of the Lpt proteins resulted in a severe defect in LPS transport. However, the absence of LptE did not disturb transport of LPS to the cell surface. LptE was found to be associated with LptD, and its absence affected total levels of LptD, suggesting a chaperone-like role for LptE in LptD biogenesis. The absence of a direct role of LptE in LPS transport was substantiated by bioinformatic analyses showing a low conservation of LptE in LPS-producing bacteria. Apparently, the role of LptE in N. meningitidis deviates from that in E. coli, suggesting that the Lpt system does not function in a completely conserved manner in all gram-negative bacteria.     

Distribution of the rol gene encoding the regulator of lipopolysaccharide O-chain length in Escherichia coli and its influence on the expression of group I capsular K antigens.

The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K(LPS). By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K(LPS) antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K(LPS), as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K(LPS) with longer polysaccharide chains. Collectively, these results suggested that group IA K(LPS) is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K(LPS) results from the absence of Rol activity in these strains.