Lipopolysaccharide Profiles Research Articles

Temperature-dependent protein and lipopolysaccharide expression in clinical Aeromonas isolates

Clinical isolates of Aeromonas were grown at eight different temperatures from 10 degrees C to 40 degrees C. Whole cell lysates were examined by SDS-PAGE and major temperature-dependent changes to both protein and lipopolysaccharide (LPS) profiles were identified. Cells grown at the higher temperatures (37 degrees C and 40 degrees C) produced abundantly a protein of c. 60 kDa which was not detected at the lower temperatures. Temperature-dependent expressions of other proteins were also noted but these were more variable among the isolates. An effect of temperature on expression of lipopolysaccharides was also noted in that some strains produced significantly less O-polysaccharides at the higher temperatures. After fractionation of cells, major differences in the expression of cell envelope and outer-membrane proteins between cells grown at low and high temperatures were noted although no unifying patterns could be discerned. Such growth temperature-induced changes in the cell envelope constituents have not been described previously for Aeromonas isolates from man.

Reduced virulence of rifampicin-resistant mutants of Francisella tularensis.

Rifampicin-resistant mutants of a live vaccine strain (LVS) of Francisella tularensis were produced and screened for virulence in mice; 4 avirulent clones with intraperitoneal (ip) LD50s > 10(6) cfu, compared with 10(2) cfu for LVS, were characterized. Growth characteristics at 37 degrees C, surface envelope proteins, and lipopolysaccharide profiles of resistant mutants were identical to those of LVS. Polymerase activity of the mutants was more resistant than the enzyme from LVS to the inhibitory action of rifampicin. Growth rates for mutants and LVS were similar during the first 5 h at 42 degrees C, but viability of the mutants decreased to < 0.01% at 24 h. LVS and mutants differed in their ability to grow in vitro in host macrophages: LVS increased 580-fold over 72 h; mutants increased 33-fold. After ip inoculation of the organisms into mice, increasing numbers of LVS from peritoneal cells were isolated; mutants decreased over 4 days.

The influence of growth medium on serum sensitivity of Bacteroides species

The susceptibility of 12 different Bacteroides strains (representing nine species) to the bactericidal effect of human serum complement was investigated. When grown in nutrient-rich proteose peptone-yeast extract medium, all 12 strains were, to varying degrees, sensitive to serum. However, when grown in Van Tassell and Wilkins's minimal medium, six of the 12 strains became markedly more serum resistant. Five of these six strains became totally resistant to serum when grown in heat-inactivated (56 degrees C, 30 min) sheep serum. By Percoll discontinuous density centrifugation and light microscopy, the ratio of bacteria with large and small capsules was found to vary with the growth medium used. Lipopolysaccharide (LPS) was extracted with aqueous phenol after growth in the three media. Polyacrylamide gel electrophoresis (PAGE) and silver staining of the LPS showed some differences in LPS profiles in all strains tested. Therefore, variation of growth conditions results in alterations of both the expression of surface structures and, in some cases, sensitivity to serum. The biochemical basis for these changes requires further investigation.

Consecutive mutations leading to the emergence in vivo of imipenem resistance in a clinical strain of Enterobacter aerogenes

Three consecutive isolates of Enterobacter aerogenes were obtained from the blood cultures of a hospitalised patient who was receiving antibiotic therapy. The initial isolate possessed an inducible cephalosporinase and was susceptible to third-generation cephalosporins. After ceftazidime treatment, a second isolate resistant to this antibiotic and characterised by stable overproduction of the chromosomal beta-lactamase was obtained, and therapy was altered to a new combination which included imipenem. During this course of treatment, a strain of E. aerogenes was isolated that was resistant to virtually all beta-lactam agents including imipenem. Comparison of biotypes and ribotyping profiles indicated that the three isolates were probably derived from a single strain which had undergone several mutations during antibiotic exposure. Examination of outer-membrane protein (OMP) preparations and lipopolysaccharide (LPS) profiles showed that the imipenem-resistant isolate lacked a major OMP and high molecular mass LPS. Furthermore, this isolate displayed reduced permeability to cephaloridine compared with the initial isolate. The introduction of a plasmid carrying a wild-type ampD allele prevented cephalosporinase production and restored beta-lactam susceptibility in the imipenem-resistant isolate. It was concluded that stable derepression of class-I beta-lactamase production and reduced permeability are both required for expression of imipenem resistance in E. aerogenes, and that previous exposure to cephalosporins may encourage the emergence of such strains.

Genetic relationships and virulence factors among classical enteropathogenic Escherichia coli serogroup O126 strains.

Thirty-nine Escherichia coli strains of the enteropathogenic (EPEC) serogroup O126 isolated from sporadic and outbreak cases of infantile diarrhoea between 1982 and 1988 were studied. These strains consisted of four serotypes showing close genetic relationships between their virulence markers, outer-membrane protein and lipopolysaccharide profiles, and electrophoretic types by multilocus enzyme electrophoresis. None of these strains exhibited localised adherence to HEp-2 cells or the attaching-effacing properties of classical type I EPEC. Of the 39 strains, 31 were of serotype O126:H12 and enterotoxigenic; one strain was serotype O126:H10 and enteroaggregative. The remaining six strains of serotype O126:H21 and one strain of serotype O126:H8 harboured no known virulence factors for diarrhoeagenic E. coli.

Chemical and physicochemical properties of Escherichia coli: variations among three strains and influence of culture conditions

The surface properties of three Escherichia coli strains (AL52, 382 and HB101) were investigated: the surface chemical composition by X-ray photoelectron spectroscopy (XPS), the surface electrical properties by microelectrophoresis and the surface hydrophobicity by partitioning between two aqueous phases. The surface structures were also analyzed: fimbriae by the hemagglutination test and lipopolysaccharide (LPS) profiles by electrophoresis on polyacrylamide gel. The surface chemical composition depended on the composition of the culture medium and on the mode of culture, in liquid nutritive medium or on nutritive agar. Variations between the surface chemical composition of the three strains were also observed. It seemed that the chemical composition could be related to the surface structures. The LPS profile of AL52 was the only one that indicated the presence of long polysaccharide chains; this was consistent with XPS analysis which showed that the surface of this strain was richer in hydroxide functions than the two other strains. There were more type 1 fimbriae (proteinic appendages) on the 382 than on the HB101 strain, and the surface nitrogen concentration detected by XPS was indeed higher for 382 in comparison with the HB101 strain. The chemical composition could be related to the electrophoretic mobility which increased as the phosphate surface concentration increased. However, there was no coherent relation between any parameter of the surface chemical composition and the surface hydrophobicity.

Effect of sub-MIC antibiotics on the cell surface and extracellular virulence determinants of Pseudomonas cepacia.

The effects of sub-MICs of ciprofloxacin and tobramycin on the cell surface characteristics and extracellular virulence factors of Pseudomonas cepacia were evaluated. Cells were grown in batch culture under iron-deficient and iron-replete conditions. At sub-MIC levels that did not affect bacterial growth cell surface hydrophobicity decreased under both iron-replete and iron-depleted conditions with ciprofloxacin, but increased with tobramycin under iron-sufficient conditions. Exopolysaccharide synthesis, lipase production and siderophore production were all significantly increased by the presence of ciprofloxacin under both growth conditions. Outer membrane protein and lipopolysaccharide profiles were not affected by exposure to the two antibiotics.

Profiles of outer membrane proteins and lipopolysaccharide of Pseudomonas aeruginosa grown in the presence of sub-MICs of macrolide antibiotics and their relation to enhanced serum sensitivity.

We have previously reported that erythromycin at sub-inhibitory concentrations enhanced the serum sensitivity of some Pseudomonas aeruginosa strains. To explore the mechanism of this effect, we have now examined the influence of macrolide antibiotics on outer membrane proteins and lipopolysaccharide (LPS) of P. aeruginosa. The strains S-6 and PAO-1 of P. aeruginosa were used as susceptible and resistant strains respectively to assess enhancement of serum sensitivity by erythromycin. The strain S-6 became more serum-sensitive when grown on agar with sub-MICs of erythromycin or azithromycin, but not of josamycin, whereas no change was observed in the serum sensitivity of the strain PAO-1 after growth with any of these antibiotics. The analysis of outer membrane proteins showed that erythromycin treatment resulted in a reduction in the amount of the 38 kDa protein (OprF) and in a prominent increase of 41 kDa protein band in the strain S-6, but not in the strain PAO-1. By an immunoblotting assay, this 41 kDa protein was shown to be highly reactive to the immune serum against untreated P. aeruginosa. LPS of the strain S-6 were examined by SDS acrylamide gel electrophoresis. The treatment with erythromycin or azithromycin, but not with josamycin, reduced the amounts of LPS species with lower molecular weights although the levels of LPS species with high molecular weights were similar to those of untreated bacteria. These results suggest that the enhanced serum sensitivity of P. aeruginosa by erythromycin is associated with changes in bacterial surface components, such as outer membrane proteins and LPS.

Alteration of lipopolysaccharide and protein profiles in SDS-PAGE of rhizobia by osmotic and heat stress

The effects of osmotic and heat stress on lipopolysaccharides and proteins of rhizobia isolated from the root nodules of leguminous trees grown in semi-arid soils of the Sudan, and of agricultural legumes grown in salt-affected soils of Egypt, were determined by SDS-PAGE. The rhizobia were of three types: (1) sensitive strains, unable to grow in 3% (w/v) NaCl in yeast mannitol medium; (2) tolerant strains which could grow in 3% (w/v) NaCl; and (3) halophytic strains which grew with 3 to 10% (w/v) NaCl. The sensitive strains changed their gel pattern or the amount of lipopolysaccharide they synthesized when grown in 1% (w/v) NaCl. The tolerant and halophytic strains often modified their lipopolysaccharides in 3% NaCl, which was evident by a shift in the banding patterns towards longer chain length. Similar effects were observed in cells incubated with sucrose and, to a lesser extent, in cells incubated at growth temperatures near the recorded maximum temperature for growth. The stress-induced changes in lipopolysaccharides were not associated with specific banding patterns of the lipopolysaccharides. During incubation in medium containing elevated concentrations of NaCl or sucrose, the protein patterns of the rhizobia were also changed. A protein with relative mobility of 65 kDa appeared during temperature stress. The maximum growth temperature of the Sudanese rhizobia were up to 44.2°C.

Electrophoretic analysis of the lipopolysaccharides ofBacteroides spp.

Lipopolysaccharide (LPS) extracts of reference strains and isolates of Bacteroides spp. prepared by the proteinase K method were resolved by tricine-sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis and located by silver staining. A considerable diversity of LPS profiles was evident within the Bacteroides genus although profiles were essentially species-specific, with some minor interstrain variations apparent among isolates of B. uniformis, B. ovatus, B. eggerthii and B. thetaiotaomicron. The LPS of most species consisted of a major rough LPS component of 2-5 kDa and a series of higher molecular weight bands which varied with species. B. vulgatus LPS was distinctive in showing an extensive ladder of multiple repeating oligosaccharide units with molecular weights ranging from 4 to > 17 kDa B. stercoris LPS included a high molecular weight (> 17 kDa) ladder of repeating oligosaccharide units. B. fragilis and B. thetaiotaomicron differed from most other species in producing a short ladder of repeating oligosaccharide units interspersing the rough LPS and a 5.6 kDa (B. fragilis) or 9 kDa (B. thetaiotaomicron) yellow-staining component. The heterogeneity of LPS profiles within the Bacteroides genus may reflect the differences in pathogenicity among the species and prove useful for typing.

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.

The importance of secreted virulence factors in Actinobacillus pleuropneumoniae bacterin preparation: a comparison

Current bacterins provide only partial protection against morbidity and mortality in swine following infection by Actinobacillus pleuropneumoniae. We compared the efficacy of a cell-free concentrate from mid-log phase growth cultures of Actinobacillus pleuropneumoniae (APP) serotype 1 to four commercial bacterins. This cell-free preparation contained carbohydrate, endotoxin, and protein, and had hemolytic and cytotoxic activity. Sodium dodecyl-sulfate polyacrylamide gel electrophoresis analysis indicated the presence of one major 110 000-molecular-weight protein. This protein band also stained by the periodic acid Schiff method, indicating the presence of carbohydrate. Cell-free concentrates of APP serotypes 5 and 7 had identical profiles following electrophoresis and staining with either Coomassie blue for protein or Schiff reagent for carbohydrate. Lipopolysaccharide profiles for the cell-free concentrates of serotypes 1 and 5 were semi-rough while the LPS profile for serotype 7 was smooth. Five A. pleuropneumoniae-free SPF pigs per group were vaccinated on days 0 and 21 with cell-free concentrate of serotype 1 plus adjuvant, or one of four commercial bacterins according to the manufacturer's directions. Control pigs were vaccinated with PBS mixed with adjuvant. All pigs were challenged intranasally on day 35 with serotype 1 and necropsied on day 50. Protection was greatest in the cell-free concentrate group, as compared with all other groups, in that no deaths occurred, clinical scores were less severe, and percent lung affected was significantly reduced ( P < 0.05). In addition, whole-cell ELISA titers were significantly increased ( P < 0.05) postvaccination in the cell-free concentrate group, and postvaccination and postchallenge sera neutralized the hemolytic activity of the cell-free concentrate from serotypes 1 and 5 ( P < 0.05), as compared with all other groups. No serum neutralization to the hemolysin of serotype 7 was observed. Immunoblot analysis using antisera derived from gnotobiotic pigs indicated that the cell-free vaccine generated a response that was identical to the response observed following live challenge. Similar, but not identical, responses were observed when antisera generated against the bacterins was used. This study indicates that an acellular vaccine containing multiple virulence factors can provide complete protection from mortality and significantly reduced morbidity to homologous challenge.

A 20-kDa pilus protein with haemagglutination and intestinal adherence properties expressed by a clinical isolate of non-01 Vibrio cholerae.

A clinical isolate of non-01 V. cholerae (10325) was shown to exhibit higher haemagglutination and intestinal adherence activities in vitro when grown in enriched media, such as trypticase soy broth (TSB) as compared to those of cells grown in a synthetic Tris-buffered or 'T'-medium. A comparison of their cell-surface protein and lipopolysaccharide profiles suggested the involvement of a 20-kDa protein in the cellular adherence process. An antiserum, raised specifically against the 20-kDa protein, recognised pilus structures on the surface of TSB grown cells. Further studies showed that the pilus was morphologically as well as antigenically distinct from toxin coregulated pilus (TCP) or other types of pili expressed by both 01 and non-01 organisms. Inhibition data established the involvement of the 20-kDa protein in haemagglutination as well as intestinal tissue adherence activities of the parent organism.

Neisseria gonorrhoeae strain MS11 harbouring a mutation in gene aroA is attenuated and immunogenic

An aroA mutant of gonococcal strain MS11 was constructed (JKD298) and compared with the wild-type (JKD288). The requirement of JKD298 for aromatic compounds, typical of an aroA mutant, was demonstrated using defined media. Other than the expected auxotrophy, no further differences could be demonstrated between the parent and the aroA mutant. SDS-PAGE analysis of protein and lipopolysaccharide (LPS) profiles showed no differences between the strains. Bactericidal assays using human and guinea-pig normal sera showed that both strains were serum sensitive and were similarly converted to serum resistance by in vitro sialylation using CMP-NANA. Infectivity experiments in guinea-pig subcutaneous chambers showed considerably reduced virulence of the aroA JKD298, which could only infect chambers at very high doses. Established infections by either strain elicited a strong PMN response in which similar proportions of each strain were seen intracellularly. Infections by JKD298 provoked a strong antibody response as detected by ELISA using whole sonicated gonococci. This is the first demonstration of attenuation of Neisseria gonorrhoeae by introduction of a defined mutation in a metabolic gene.

Lipopolysaccharide profile typing as a technique for comparative typing of gram-negative bacteria

We have applied the technique of lipopolysaccharide (LPS) profiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis to the typing of 124 isolates of 12 gram-negative species from suspected outbreaks of infection. LPS was prepared by proteinase K digestion or micro-phenol-water extraction. A total of 11 of the 12 species gave clear ladder band profiles, the exception being Acinetobacter baumannii. When compared with conventional typing for Enterobacter cloacae, Pseudomonas aeruginosa, and Serratia marcescens, LPS profile type alone was sufficient to allow relatedness or distinguishability of isolates to be established, and this was corroborated by serotype and phage type data. Serologically nontypeable isolates invariably lacked O repeating units and thus could not be classified by their silver stain profile. We conclude that LPS profiling is useful for the epidemiological investigation of small clusters of isolates in order to determine whether or not cross-infection between patients has occurred.

Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines.

Clonal, noniridescent mutants of Actinobacillus pleuropneumoniae serotypes 1 and 5 were isolated following chemical mutagenesis with ethyl methanesulfonate. The absence of any detectable capsule was confirmed by inhibition radioimmunoassay. There were no differences between the parent and mutant strains in lipopolysaccharide or protein electrophoretic profiles or in hemolytic activity. There was no detectable reversion to the encapsulated phenotype in vitro after passage in mice or pigs or in microporous capsules that were implanted subcutaneously in pigs for 6 weeks. The mutants were able to survive for more than 1 week in pigs following subcutaneous inoculation, which resulted in a strong immune response to whole cells and Apx toxins I and II. Intratracheal challenge of pigs with the serotype 5 mutant at a dose 1 log greater than the 50% lethal dose for the parent resulted in no clinical disease or lesions except in one pig that had slight pneumonia and pleuritis. Twenty-four hours after challenge, A. pleuropneumoniae could not be recovered from the respiratory tracts of any of the challenged pigs except for the one infected pig; this isolate remained noncapsulated. Immunization of pigs with one or both serotypes of noncapsulated mutants protected all pigs against clinical disease following intratracheal challenge with the virulent homologous or heterologous serotype. Nonimmunized control pigs and pigs immunized with a commercial bacterin died or had to be euthanized within 24 h of challenge. Thus, live noncapsulated mutants of A. pleuropneumoniae may provide safe and cost-effective protection against swine pleuropneumonia. These observations support the possibility that noncapsulated mutants of other encapsulated, toxin-producing bacteria may also prove to be efficacious live-vaccine candidates.

Examination of Lipopolysaccharide (O-Antigen) Populations of Thiobacillus ferrooxidans from Two Mine Tailings

Net acid-generating capacities of 39.74 kg of H(2)SO(4) per ton (ca. 0.05 kg/kg) (pH 2.68) for the Lemoine copper mine tailings (closed ca. 8 years ago; located 40 km west of Chibougamau, Quebec, Canada) and 16.07 kg of H(2)SO(4) per ton (ca. 0.02 kg/kg) (pH 3.01) for the Copper Rand tailings (in current use and 50 km distant [east] from those of Lemoine) demonstrate that these sulfide tailings can support populations of acidophilic thiobacilli. Oxidized regions in both tailings environments were readily visible, were extremely acidic (Lemoine, pH 2.36; Copper Rand, pH 3.07), and provided natural isolates for our study. A 10% (wt/vol) oxalic acid treatment, which solubilizes both ferric sulfate and ferric hydroxide precipitates (B. Ramsay, J. Ramsay, M. deTremblay, and C. Chavarie, Geomicrobiol. J. 6:171-177, 1988), enabled the recovery of intact bacterial cells from the tailings material and from liquid synthetic medium for lipopolysaccharide analysis. No viable cells could be cultured after this oxalic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electro-phoretic profiles of lipopolysaccharides extracted from the Lemoine tailings were complex, indicating a heterogeneous population of Thiobacillus ferrooxidans. Six T. ferrooxidans subspecies as identified by lipopolysaccharide analysis (i.e., lipopolysaccharide chemotypes) were eventually isolated from a total of 112 cultures from the Lemoine tailings. Using the same isolate and lipopolysaccharide typing techniques, we identified only a single lipopolysaccharide chemotype from 20 cultures of T. ferrooxidans isolated from the Copper Rand tailings. This homogeneity of lipopolysaccharide chemotype was much different from what was found for the older Lemoine tailings and may reflect a progressive lipopolysaccharide heterogeneity of Thiobacillus isolates as tailings leach and age.

TnphoA Salmonella abortusovis mutants unable to adhere to epithelial cells and with reduced virulence in mice.

Salmonella abortusovis is a pathogenic bacterium highly specific to sheep, causing spontaneous abortion. In order to understand the role of genes involved in pathogenicity, we investigated S. abortusovis with the random mutagenic TnphoA transposon. A total of 95 S. abortusovis TnphoA mutants yielding alkaline phosphatase active fusion protein were obtained. In this way we created a bank of strains in order to identify any phenotypic modification which could affect the periplasmic and/or exported proteins involved in virulence. The TnphoA mutants were screened for the ability to adhere to epithelial cells: a total of 23 mutant strains lost this phenotypic feature. To detect the chromosomal TnphoA insertions, DNA was restricted by the enzyme EcoRV, which does not cleave the TnphoA sequence. Southern blotting analysis revealed the existence of four classes of integration. Colonies of adhesiveless mutants appear to be as smooth as the S. abortusovis wild type, and electrophoretic analysis indicates a normal lipopolysaccharide profile. To identify mutations affecting genes encoding for outer membrane proteins (OMPs), the alkaline phosphatase portion of the fusion proteins was revealed in TnphoA mutants by immunoblotting with specific antibodies. A mutation in OMPs was detected in seven mutants. Restriction analysis identified in four mutants a common region of 2 kb where alterations in genes coding for OMPs occur. We suggested that this region is involved in pathogenicity in mice, since a group of mutant strains has shown reduced virulence in mice and one mutant is completely avirulent. Furthermore, after mice were exposed orally to these mutants, significant protection against oral challenge with the parental virulent strain resulted.

Chromosomal mapping, expression and synthesis of lipopolysaccharide in Pseudomonas aeruginosa: a role for guanosine diphospho (GDP)‐D‐mannose

Pseudomonas aeruginosa can express two distinct forms of lipopolysaccharide (LPS), called A-band and B-band. As an attempt to understand the molecular biology of the synthesis and regulation of these LPS antigens, a recombinant plasmid, pFV3, containing genes for A-band expression was isolated previously. In the present study, P. aeruginosa strain PAO1 was mutagenized with transposon Tn5-751 and yielded a B-band-deficient mutant, called ge6. This mutant was mated with a PAO1 genomic library, and transconjugants were screened for complementation of B-band using B-band-specific monoclonal antibody MF15-4. Recombinant plasmid pFV100 was subsequently isolated by its ability to complement B-band expression in ge6. SDS-PAGE analysis of LPS from ge6 and ge6(pFV100) revealed that ge6 was deficient in expression of B-band, while ge6(pFV100) had an LPS profile similar to that of the parent strain PAO1. With A-band and B-band genes cloned in separate plasmids, pFV3 and pFV100 respectively, we were able to determine the map location of these LPS genes on the P. aeruginosa PAO1 chromosome using pulsed-field gel electrophoresis. A-band genes mapped at 5.75 to 5.89 Mbp (SpeI fragment SpK; DpnI fragment DpF2), while genes involved with expression of B-band LPS mapped at 1.9 Mbp (SpeI fragments SpC, SpI and SpAI; DpnI fragment DpD) on the 5.9 Mbp chromosome. We also performed initial characterization of a gene involved with synthesis of A-band present on pFV3. We previously reported that recombinant plasmid pFV3 and subcloned plasmid pFV36 complemented A-band synthesis in rd7513, an A- mutant derived from A+ strain AK1401. pFV36 was mutagenized with transposon Tn1000 to reveal a one-kilobase region capable of complementing the expression of A-band in the A- strain rd7513. This region was subcloned as a 1.6 kb KpnI fragment into plasmid vector pAK1900 and the resulting clone named pFV39. Labelling of proteins encoded by pAK1900 and pFV39 in Escherichia coli maxicells revealed a single unique polypeptide of approximately 37 kDa expressed by pFV39. Supernatants from disrupted cells of rd7513(pFV39) and AK1401 converted 14C-labelled-guanosine diphospho (GDP)-D-mannose to GDP-rhamnose, while supernatants from rd7513 did not show synthesis of GDP-rhamnose. The data therefore suggest that conversion of GDP-D-mannose to GDP-rhamnose is required for synthesis of A-band LPS, and that a 37 kDa protein is involved in this conversion.

Porins and lipopolysaccharide ofEscherichia coliATCC 25922 and isogenic rough mutants

The lipopolysaccharide and porin profile of Escherichia coli ATCC 25922, a smooth strain commonly used in antibiotic susceptibility testing, and five isogenic rough mutants was examined. The lipopolysaccharide of the parent strain had the characteristic ladder pattern on polyacrylamide gels, while that of the mutants appeared similar to chemotypes Ra and Rc of Salmonella typhimurium with some changes in chemical composition. Of the porins, OmpC appeared markedly reduced in the parent strain while OmpF appeared markedly reduced in the mutants. In addition, a new outer-membrane protein of size intermediate to that of OmpC and OmpF was detected in all mutants. Neither parent nor mutants were susceptible to the LPS core-specific P1 phage or the porin-specific PA2 and K20 phages.