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1.  Effect of Seven-Valent Pneumococcal Conjugate Vaccine on Staphylococcus aureus Colonisation in a Randomised Controlled Trial 
PLoS ONE  2011;6(6):e20229.
Background
Heptavalent pneumococcal conjugate vaccine (PCV7) shifts nasopharyngeal colonisation with vaccine serotype pneumococci towards nonvaccine serotypes. Because of the reported negative association of vaccine serotype pneumococci and Staphylococcus aureus in the nasopharynx, we explored the effect of PCV7 on nasopharyngeal colonisation with S. aureus in children and parents.
Methodology/Principal Findings
This study was part of a randomised controlled trial on the effect of PCV7 on pneumococcal carriage, enrolling healthy newborns who were randomly assigned (1∶1∶1) to receive PCV7 (1) at 2 and 4 months of age (2) at 2, 4 and 11 months or (3) no PCV7 (controls). Nasopharyngeal colonisation of S. aureus was a planned secondary outcome. Nasopharyngeal swabs were obtained from all children over a 2-year period with 6-months interval and from one parent at the child's age of 12 and 24 months and cultured for Streptococcus pneumoniae and S. aureus. Between July 2005 and February 2006, 1005 children were enrolled and received either 2-doses of PCV7 (n = 336), 2+1-doses (336) or no dose (n = 333) before PCV7 implementation in the Dutch national immunization program. S. aureus colonisation had doubled in children in the 2+1-dose group at 12 months of age compared with unvaccinated controls (10.1% versus 5.0%; p = 0.019). A negative association for co-colonisation of S. pneumoniae and S. aureus was observed for both vaccine serotype (adjusted odds ratio (aOR) 0.53, 95% confidence interval (CI) 0.38–0.74) and nonvaccine serotype pneumococci (aOR 0.67, 95% CI 0.52–0.88).
Conclusions/Significance
PCV7 induces a temporary increase in S. aureus colonisation in children around 12 months of age after a 2+1-dose PCV7 schedule. The potential clinical consequences are unknown and monitoring is warranted.
Trial Registration
ClinicalTrials.gov NCT00189020
doi:10.1371/journal.pone.0020229
PMCID: PMC3112202  PMID: 21695210
2.  Characterization of Genetic and Phenotypic Diversity of Invasive Nontypeable Haemophilus influenzae  
Infection and Immunity  2005;73(9):5853-5863.
The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.
doi:10.1128/IAI.73.9.5853-5863.2005
PMCID: PMC1231076  PMID: 16113304
3.  Relative Immunogenicity of PorA Subtypes in a Multivalent Neisseria meningitidis Vaccine Is Not Dependent on Presentation Form  
Infection and Immunity  2003;71(11):6367-6371.
The hexavalent meningococcal vaccine HexaMen, containing six PorAs on two vesicles, was tested in clinical studies. Although fourfold increases in serum bactericidal activity (SBA) titers against all of the PorAs were observed, there were significant differences between PorA-specific SBA titers. SBA titers were mainly directed against one PorA from each vesicle, P1.5-2,10 and P1.5-1,2-2, and were lower against the other PorAs, especially P1.7-2,4 and P1.19,15-1. We investigated whether these differences were due to immunological interference that resulted in competition between the three PorAs on the same vesicle or whether they were caused by a difference in the immunogenicities of the separate PorAs. Therefore, mice were immunized either with HexaMen, with six monovalent outer membrane vesicles (OMVs) representing the same six PorAs simultaneously (HexaMix), or with only one of the monovalent OMVs. The immunoglobulin G and SBA titers after HexaMen immunization in mice resembled the results obtained in clinical studies. Although immunization with HexaMix gave higher titers than immunization with HexaMen for some PorAs, the pattern of high and low titers was the same. Similar differences in immunogenicity between subtypes were seen after monovalent immunization when interference was eliminated as a cause of the differences. Monovalent immunization resulted in higher titers for P1.5-1,2-2 and P1.7,16 than immunization with HexaMen. However, no significant differences were found for the weakly immunogenic PorAs, P1.7-2,4 and P1.19,15-1. Since immunization with the six PorAs in the trivalent presentation form (HexaMen) and in the mixture of monovalent vesicles (HexaMix) resulted in the same pattern of high and low titers, we concluded that the differences between the PorA-specific responses are due to differences in the immunogenicities of the various PorAs and not due to interference that results in competition between different PorAs.
doi:10.1128/IAI.71.11.6367-6371.2003
PMCID: PMC219571  PMID: 14573657
4.  Distribution of Environmentally Regulated Genes of Streptococcus suis Serotype 2 among S. suis Serotypes and Other Organisms 
Journal of Clinical Microbiology  2002;40(9):3261-3268.
The occurrence of 36 environmentally regulated genes of Streptococcus suis strain 10 among all 35 S. suis serotypes was determined by using hybridization with the amplified genes as probes. In addition, the distribution of these genes among the virulence phenotypes of serotypes 1 and 2 was assessed. Hybridization was also performed with various other streptococcal species and nonstreptococcal bacterial species which may be present in pigs. Interestingly, probe ivs-25/iri-1, similar to agrA and sapR, hybridized only with S. suis serotype 1 and 2 strains with virulent phenotypes and is therefore suitable as a diagnostic parameter. Only one probe was specific for S. suis. This probe's sequence was identical to the epf gene, a putative virulence factor of S. suis. Probe ivs-31 was similar to a virulence factor of S. suis, namely, a gene encoding a fibronectin- and fibrinogen-binding protein. This probe hybridized only with oral streptococci. Nearly half of the probes (45%) hybridized with the oral streptococci (S. oralis, S. milleri, S. sanguis, S. gordonii, and S. mitis) and with Streptococcus pneumoniae. This indicates a close relationship between S. suis, the oral streptococci, and S. pneumoniae with respect to the selected environmentally regulated genes. One probe only hybridized with gram-negative species and therefore seems to be obtained by S. suis from a gram-negative organism by horizontal transfer.
doi:10.1128/JCM.40.9.3261-3268.2002
PMCID: PMC130683  PMID: 12202563
5.  Contribution of Fibronectin-Binding Protein to Pathogenesis of Streptococcus suis Serotype 2 
Infection and Immunity  2002;70(3):1319-1325.
In the present study we investigated the role of the fibronectin (FN)- and fibrinogen (FGN)-binding protein (FBPS) in the pathogenesis of Streptococcus suis serotype 2 in piglets. The complete gene encoding FBPS from S. suis serotype 2 was cloned in Escherichia coli and sequenced. The occurrence of the gene in various serotypes was analyzed by hybridization studies. The FBPS protein was expressed in E. coli and purified, and binding to human FN and FGN was demonstrated. The induction of antibodies in piglets was studied upon infection. An isogenic mutant unable to produce FBPS was constructed, and the levels of virulence of the wild-type and mutant strains were compared in a competitive infection model in young piglets. Organ cultures showed that FBPS was not required for colonization of the tonsils but that FBPS played a role in the colonization of the specific organs involved in an S. suis infection. Therefore, the FBPS mutant was considered as an attenuated mutant.
doi:10.1128/IAI.70.3.1319-1325.2002
PMCID: PMC127759  PMID: 11854216
6.  Modification of Lipid A Biosynthesis in Neisseria meningitidis lpxL Mutants: Influence on Lipopolysaccharide Structure, Toxicity, and Adjuvant Activity 
Infection and Immunity  2001;69(10):5981-5990.
Two genes homologous to lpxL and lpxM from Escherichia coli and other gram-negative bacteria, which are involved in lipid A acyloxyacylation, were identified in Neisseria meningitidis strain H44/76 and insertionally inactivated. Analysis by tandem mass spectrometry showed that one of the resulting mutants, termed lpxL1, makes lipopolysaccharide (LPS) with penta- instead of hexa-acylated lipid A, in which the secondary lauroyl chain is specifically missing from the nonreducing end of the GlcN disaccharide. Insertional inactivation of the other (lpxL2) gene was not possible in wild-type strain H44/76 expressing full-length immunotype L3 lipopolysaccharide (LPS) but could be readily achieved in a galE mutant expressing a truncated oligosaccharide chain. Structural analysis of lpxL2 mutant lipid A showed a major tetra-acylated species lacking both secondary lauroyl chains and a minor penta-acylated species. The lpxL1 mutant LPS has retained adjuvant activity similar to wild-type meningococcal LPS when used for immunization of mice in combination with LPS-deficient outer membrane complexes from N. meningitidis but has reduced toxicity as measured in a tumor necrosis factor alpha induction assay with whole bacteria. In contrast, both adjuvant activity and toxicity of the lpxL2 mutant LPS are strongly reduced. As the combination of reduced toxicity and retained adjuvant activity has not been reported before for either lpxL or lpxM mutants from other bacterial species, our results demonstrate that modification of meningococcal lipid A biosynthesis can lead to novel LPS species more suitable for inclusion in human vaccines.
doi:10.1128/IAI.69.10.5981-5990.2001
PMCID: PMC98725  PMID: 11553534
7.  Interaction of Fimbriae of Haemophilus influenzae Type B with Heparin-Binding Extracellular Matrix Proteins 
Infection and Immunity  2000;68(10):5696-5701.
The interaction of the fimbriae of Haemophilus influenzae type b (Hib) with two heparin-binding extracellular matrix proteins, human fibronectin (Fn) and heparin-binding growth-associated molecule (HB-GAM) from mouse, were studied. The fimbriated Hib strain 770235 fim+, as well as the recombinant strain E. coli HB101(pMH140), which expressed Hib fimbriae, adhered strongly to Fn and HB-GAM immobilized on glass. Purified Hib fimbriae bound to Fn and HB-GAM, and within the Fn molecule, the binding was localized to the N-terminal 30,000-molecular-weight (30K) and 40K fragments, which contain heparin-binding domains I and II, respectively. Fimbrial binding to Fn, HB-GAM, and the 30K and the 40K fragments was inhibited by high concentrations of heparin. The results show that fimbriae of Hib interact with heparin-binding extracellular matrix proteins. The nonfimbriated Hib strain 770235 fim− exhibited a low level of adherence to Fn but did not react with HB-GAM, indicating that Hib strains also possess a fimbria-independent mechanism to interact with Fn.
PMCID: PMC101525  PMID: 10992473
8.  Characterization of Adherence of Nontypeable Haemophilus influenzae to Human Epithelial Cells 
Infection and Immunity  2000;68(8):4658-4665.
The adherence of 58 nontypeable Haemophilus influenzae isolates obtained from patients with otitis media or chronic obstructive pulmonary disease (COPD) and obtained from the throats of healthy individuals to Chang and NCI-H292 epithelial cells was compared. Otitis media isolates, but not COPD isolates, adhered significantly more to both cell lines than did throat isolates. Since high-molecular-weight (HMW) proteins are major adhesins of nontypeable H. influenzae, the isolates were screened for HMW protein expression by Western blotting with two polyclonal sera and PCR with hmw-specific primers. Twenty-three of the 32 adhering isolates (72%) and only 1 of the 26 nonadherent strains were HMW protein or hmw gene positive. Among the 32 isolates adhering to either cell line, 5 different adherence patterns were distinguished based on the inhibiting effect of dextran sulfate. Using H. influenzae strain 12 expressing two well-defined HMW proteins (HMW1 and HMW2) and its isogenic mutants as a reference, we observed HMW1-like adherence to both cell lines for 16 of the 32 adherent isolates. Four others showed HMW2-like adherence to NCI-H292. Of the three other patterns of adherence, one probably also involved HMW protein. Screening of the isolates with six HMW-specific monoclonal antibodies in a whole-cell enzyme-linked immunosorbent assay showed that the HMW proteins of COPD isolates and carrier isolates were more distinct from the HMW proteins from H. influenzae strain 12 than those from otitis media isolates. Characterization of the HMW protein of a COPD isolate by adherence and DNA sequence analysis showed that despite large sequence diversity in the hmwA gene, probably resulting in the antigenic differences, the HMW protein mediated the HMW2-like adherence of this strain.
PMCID: PMC98403  PMID: 10899870
9.  Cloning of Genes of Nontypeable Haemophilus influenzae Involved in Penetration between Human Lung Epithelial Cells 
Infection and Immunity  2000;68(8):4616-4623.
Haemophilus influenzae penetrates between epithelial cells via an unknown mechanism. A chromosomal library of nonencapsulated H. influenzae strain A960053 DNA was constructed in Escherichia coli DH5α to identify bacterial genes contributing to this paracytosis. Two E. coli clones that contained open reading frames (ORFs) homologous to HI0636 to HI0641 of H. influenzae strain Rd and that showed an increased penetration in epithelial cell layers of the human bronchial epithelial cell line NCI-H292 were identified. ORFs HI0636 and HI0638, encoding two small proteins of unknown functions, were further investigated. The clone containing ORFs HI0636 and HI0637 as well as the clone containing ORF HI0638 showed a significant increase in penetration. Disruption of HI0638 by kanamycin box insertion in H. influenzae strain A960053 resulted in loss of penetration into the epithelial cell layers. Disruption of HI0636 had no effect on penetration in this model system. Since a role for HI0637 in the paracytosis of H. influenzae is very unlikely because it encodes TrpS, we conclude that the protein encoded by ORF HI0638 may function as a paracytin, while that encoded by HI0636 may have an auxiliary function.
PMCID: PMC98391  PMID: 10899864
10.  Selection of Recombinant Antibodies Specific for Pathogenic Streptococcus suis by Subtractive Phage Display 
Infection and Immunity  2000;68(7):3949-3955.
A semisynthetic antibody phage display library was used to select recombinant antibodies directed against surface components of a pathogenic strain of Streptococcus suis serotype 2 and against extracellular factor (EF), a protein known to be exclusively associated with pathogenic S. suis serotype 2 strains. Three distinct monoclonal phage antibodies directed against conformational epitopes of surface protein components of S. suis were selected. In addition, three different monoclonal phage antibodies were isolated that recognized EF. To isolate antibody fragments that recognize epitopes specific for a pathogenic S. suis serotype 2 strain, compared to a nonpathogenic serotype 2 strain, we applied a subtractive selection procedure. With this procedure, only one distinct phage antibody was found, and it was shown to be directed against EF. This demonstrates the selectivity of the applied procedure and confirms that EF is indeed differentially expressed by pathogenic and nonpathogenic strains. It also shows that EF is a very dominant antigen in phage antibody selections.
PMCID: PMC101672  PMID: 10858208
11.  Immunogenicity of Outer Membrane Proteins in a Lipopolysaccharide-Deficient Mutant of Neisseria meningitidis: Influence of Adjuvants on the Immune Response 
Infection and Immunity  1999;67(10):4988-4993.
The immunogenicity of outer membrane complexes (OMCs) or heat-inactivated bacteria of a lipopolysaccharide (LPS)-deficient mutant derived from meningococcal strain H44/76 was studied. The immune response in BALB/c mice to the major outer membrane proteins was poor compared to the immune response elicited by wild-type immunogens. However, addition of external H44/76 LPS to mutant OMCs entirely restored the immune response. By using an LPS-deficient mutant, it may be possible to substitute a less toxic compound as adjuvant in meningococcal outer membrane vaccines. Therefore, a broad panel of adjuvants were tested for their potential to enhance the immunogenicity of LPS-deficient OMCs. AlPO4, Rhodobacter sphaeroides LPS, monophosphoryl lipid A and alkali-hydrolyzed meningococcal LPS showed significantly lower adjuvant activity than did H44/76 LPS. Adjuvant activity similar to H44/76 LPS was found for Escherichia coli LPS, meningococcal icsB and rfaC LPS, QuilA, subfractions of QuilA, and MF59. Good adjuvant activity was also found with meningococcal htrB1 LPS, containing penta-acylated lipid A. Antisera elicited with the less active adjuvants showed relatively high immunoglobulin G1 (IgG1) titers, whereas strong adjuvants also induced high IgG2a and IgG2b responses in addition to IgG1. Antisera with the IgG2a and IgG2b isotypes showed high bactericidal activity, indicating that adjuvants promoting the IgG2a and IgG2b response contribute most to the protective mechanism. Thus, this study demonstrates that the immunogenicity of meningococcal LPS-deficient OMCs can be restored by using less toxic adjuvants, which opens up new avenues for development of vaccines against meningococcal disease.
PMCID: PMC96843  PMID: 10496868
12.  Variation in the Composition and Pore Function of Major Outer Membrane Pore Protein P2 of Haemophilus influenzae from Cystic Fibrosis Patients 
We investigated the relationship between susceptibility to β-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulated Haemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinct H. influenzae strains from two patients extensively treated with β-lactam antibiotics. The variants differed in their susceptibilities to at least two β-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to β-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences of ompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.
PMCID: PMC89055  PMID: 9925510
13.  Short-Sequence DNA Repeats in Prokaryotic Genomes 
Short-sequence DNA repeat (SSR) loci can be identified in all eukaryotic and many prokaryotic genomes. These loci harbor short or long stretches of repeated nucleotide sequence motifs. DNA sequence motifs in a single locus can be identical and/or heterogeneous. SSRs are encountered in many different branches of the prokaryote kingdom. They are found in genes encoding products as diverse as microbial surface components recognizing adhesive matrix molecules and specific bacterial virulence factors such as lipopolysaccharide-modifying enzymes or adhesins. SSRs enable genetic and consequently phenotypic flexibility. SSRs function at various levels of gene expression regulation. Variations in the number of repeat units per locus or changes in the nature of the individual repeat sequences may result from recombination processes or polymerase inadequacy such as slipped-strand mispairing (SSM), either alone or in combination with DNA repair deficiencies. These rather complex phenomena can occur with relative ease, with SSM approaching a frequency of 10−4 per bacterial cell division and allowing high-frequency genetic switching. Bacteria use this random strategy to adapt their genetic repertoire in response to selective environmental pressure. SSR-mediated variation has important implications for bacterial pathogenesis and evolutionary fitness. Molecular analysis of changes in SSRs allows epidemiological studies on the spread of pathogenic bacteria. The occurrence, evolution and function of SSRs, and the molecular methods used to analyze them are discussed in the context of responsiveness to environmental factors, bacterial pathogenicity, epidemiology, and the availability of full-genome sequences for increasing numbers of microorganisms, especially those that are medically relevant.
PMCID: PMC98915  PMID: 9618442
14.  The Fimbria Gene Cluster of Nonencapsulated Haemophilus influenzae 
Infection and Immunity  1998;66(2):406-417.
The occurrence of fimbria gene clusters in nonencapsulated Haemophilus influenzae strains from chronic bronchitis patients (n = 58), patients with acute otitis media (n = 13), and healthy carriers (n = 12) was determined by DNA hybridization and PCR, based on sequences of fimbriate H. influenzae type b. Although an average of 18% of all nonencapsulated strains had a fimbria gene cluster consisting of hifA to hifE inserted in the chromosome between purE and pepN, differences in the frequency of fimbria cluster-positive strains were observed, depending on the source of isolates. The compositions of the fimbria gene clusters of seven strains from chronic bronchitis patients and one strain from an otitis media patient were analyzed in more detail. After enrichment for fimbria expression, the promoter of the gene cluster contained 10 TA repeats (n = 2), leading to optimal positioning between the −10 and −35 promoter regions. The promoter regions of five fimbria-negative strains were sequenced; four were found to have nine TA repeats, and one had only four TA repeats. The protein sequence of three ganglioside GM1-specific HifA adhesins consisted of conserved regions intermingled with regions of sequence diversity. hifA appeared to be flanked by intergenic regions that varied between strains and contained both direct and inverted DNA repeats. Since noncoding DNA between hifA and purE has not been found in H. influenzae type b, these DNA sequences are probably not essential for fimbria expression. An analysis of strains lacking the gene cluster revealed the presence of similar sequences in 13 of 15 strains from chronic bronchitis patients, 5 of 5 strains from otitis media patients, and 3 of 5 strains from healthy carriers. The lengths of these intergenic regions were the same for multiple isolates of strains obtained during persistent infections. The presence or absence and the composition of the fimbria gene cluster and other sequences between the flanking genes purE and pepN suggest that the fimbria gene cluster was originally contained on a mobile element.
PMCID: PMC107920  PMID: 9453588
15.  Antimicrobial Susceptibility of Haemophilus influenzae in the Respiratory Tracts of Patients with Cystic Fibrosis 
We analyzed the antimicrobial susceptibilities of Haemophilus influenzae isolates from 157 sputum specimens prospectively collected from 39 cystic fibrosis (CF) patients during a 2-year study. These isolates were characterized by random amplified polymorphic DNA analysis and major outer membrane protein (MOMP) analysis to identify H. influenzae strains and MOMP variants and to assess their persistence in the respiratory tract. Among the 247 H. influenzae isolates, 16 (6.5%) produced β-lactamase. The 231 β-lactamase-negative isolates represented 85 H. influenzae strains, 61 MOMP variants derived from 27 of these strains, and 85 persistent isolates identical to strains or MOMP variants. All β-lactamase-negative isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, cefuroxime, cefotaxime, cefaclor, imipenem, tetracycline, and trimethoprim-sulfamethoxazole by disk diffusion testing. Eleven (13%) H. influenzae strains, 18 (30%) MOMP variants, and 30 (35%) persistent isolates were resistant to one or more of the antibiotics tested. Antimicrobial susceptibility was decreased among MOMP variants and persistent isolates compared to nonpersistent H. influenzae strains, and changes in susceptibility occurred irrespective of MOMP variation. We conclude that the decreased antimicrobial susceptibility of H. influenzae during persistence contributes to the poor eradication of H. influenzae from the respiratory tracts of CF patients.
PMCID: PMC105407  PMID: 9527779
16.  Architecture of the Outer Membrane of Escherichia coli III. Protein-Lipopolysaccharide Complexes in Intramembraneous Particles 
Journal of Bacteriology  1978;134(3):1089-1098.
In a previous paper (A. Verkleij, L. van Alphen, J. Bijvelt, and B. Lugtenberg, Biochim. Biophys. Acta 466:269-282, 1977) we have hypothesized that particles on the outer fracture face of the outer membrane ([Formula: see text]), with corresponding pits on the inner fracture face of the outer membrane ([Formula: see text]), consist of lipopolysaccharide (LPS) aggregates stabilized by divalent cations and that they might contain protein and/or phospholipid. In the present paper the roles of LPS, cations, and proteins in these [Formula: see text] particles are described more extensively, using a strain that lacks the major outer membrane proteins, b, c, and d (b− c− d−), and has a reduction in the number of [Formula: see text] particles of 75%. To study the role of divalent cations in the formation of [Formula: see text] particles, these b− c− d− cells were grown or incubated with Ca2+, Mg2+, or putrescine. The presence of Ca2+ resulted in the appearance of many [Formula: see text] particles and [Formula: see text] pits. Mg2+ and putrescine were less effective than Ca2+. Introduction of these particles was not accompanied by alterations in the relative amounts of LPS and cell envelope proteins. Ca2+ treatment of a heptoseless derivative of a b− c− d− strain did not result in morphological changes. Incubation of Ca2+-treated cells with ethylenediaminetetraacetate caused the disappearance of the introduced particles as well as the release of more than 60% of the cellular LPS. These results strongly support the hypothesis that LPS is involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The roles of various outer membrane proteins in the formation of [Formula: see text] particles were studied by comparing the freeze-fracture morphology of b− c− d− cells with that of cells which contain one of the outer membrane proteins b, c, d, and e or the receptor protein for bacteriophage lambda. The results showed that the presence of any of these five proteins in a b− c− d− background resulted in a large increase in the number of [Formula: see text] particles and [Formula: see text] pits, indicating that these proteins are, independent of each other, involved in the formation of [Formula: see text] particles and [Formula: see text] pits. The simplest explanation for the results is that in wild-type cells each particle consists of LPS complexed with some molecules of a single protein species, stabilized by either divalent cations or polyamines. It is hypothesized that the outer membrane of the wild-type cell contains a heterogeneous population of particles, of which 75% consists of protein b-LPS, protein c-LPS, and protein d-LPS particles. A function of these particles as aqueous pores is proposed.
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PMCID: PMC222359  PMID: 350838

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