Mutations affecting gluconate catabolism in Escherichia coli. Genetic mapping of loci for the low affinity transport and the thermoresistant gluconokinase

The isolation and properties of strains of Escherichia coli carrying mutations affecting either the low affinity transport for gluconate (gene gntU) or the thermoresistant gluconokinase (gene gntK) are described. A lesion of each type was genetically characterized by transduction experiments. Both mutations mapped in the asd region, and the order was malA-glpD-asd-gntU-gntK, with the last two markers at about min 75.78 and 75.86 on the map, respectively. Mutations altering specifically gntU have not been previously reported.     

Isolation and characterization of Escherichia coli pili from diverse clinical sources.

Bacteria which attach to different mucous membranes should have differing specificities of adherence in vitro. Human Escherichia coli isolates from blood and urine (pathogens) and from stool and throat (commensals) were characterized as to the patterns of hemagglutination (HA), as well as the structure and function of their pili. Bacterial HA was done in microtiter plates and on slides after bacterial growth in broth or agar. Human erythrocytes were agglutinated by 95% of the pathogens and 65 to 70% of the commensals grown in broth or agar. Mannose-resistant HA was characteristically caused by pathogens, and commensals characteristically caused mannose-sensitive HA of guinea pig cells. Strains often had both mannose-resistant and mannose-sensitive reactions, or even a mannose-paradoxical reaction. Pathogens more often caused HA, but titers were lower than those for commensals. Slide HA was less sensitive than the microtiter method. All isolates were piliated. Commensals also had more pili than pathogens when grown in broth (117.8 versus 38.3 pili per bacterium), but pathogens had more pili after growth on agar (32.1 versus 8.1 pili per bacterium). Isolates causing high-titer HA had large numbers of pili (greater than 85 pili per bacterium), but some well-piliated strains were non-hemagglutinating. Pili were purified from seven E. coli strains from different sites of isolation and with different erythrocyte-binding specificity. Pili usually migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, more than one type of pilus could be copurified from some strains since there were two or more bands after separation in octyl-glucoside and two different amino terminal sequences. Protein sequencing was done on five different pili: four resembled type 1 pili and one was a P fimbria. The type 1-like pili (strains 2239 and 9353) had an initial variable sequence of 1 to 5 residues, followed by a common region of 21 residues. The P fimbria (strain 7714) had different erythrocyte-binding specificity but was still 27% homologous with 2239 and 9353. E. coli strains from different body sites have characteristic attachments to erythrocytes. Pili derived from these different sources may also have different binding specificity, but they are similar in primary structure.     

Isolation and characterization of F12 adhesive fimbrial antigen from uropathogenic Escherichia coli strains

The adhesive fimbrial antigen F12 from a strain of uropathogenic Escherichia coli has been isolated and characterized. The antigen was purified by ammonium sulfate precipitation and gel chromatography. The protein subunit of the F12 fimbria has a molecular weight of 18,200; the N-terminal amino acid sequence of the subunit shows close resemblance to that of the subunits of other F fimbriae and the type 1 fimbriae. We identified in these proteins a pattern of alternating conserved and variable amino acid residues which could indicate a special structural and functional feature.     

Isolation and characterization of the localized adherence factor of enteropathogenic Escherichia coli.

The binding factor of enteropathogenic Escherichia coli O111:H- responsible for localized adherence (LA) on HeLa cells was investigated. Inhibition of LA by carbohydrates and lectins showed that the reactive epitope on HeLa cells contains N-acetylgalactosamine units. Treatment of bacteria with EDTA for extraction of lipopolysaccharides eliminated these polymers as binding factors. Such treatment also caused a marked increase in adhesion suggesting steric hindrance by lipopolysaccharides of the LA factor binding capacity. Immunoblotting with rabbit antibodies showed a strong reaction with two components with approximate molecular sizes of 29 and 32 kilodaltons (kDa) present in the outer membrane preparations of bacteria. Both the absorbed rabbit immune serum and the outer membrane preparation of the bacteria inhibited bacterial adhesion by 100%. Outer membrane components were isolated from an N-acetylgalactosamine-agarose column by elution with KSCN, labeled with 125I, and immunoprecipitated with absorbed rabbit hyperimmune antiserum. The only component precipitated was the protein doublet at 29 to 32 kDa corresponding to the components detected by immunoblotting. The predominant component was always the 32-kDa polypeptide. We conclude that this component of the outer membrane is the best candidate for the LA factor in enteropathogenic E. coli.     

Isolation and characterization of intragenic suppressors of an Escherichia coli ftsA mutation

Mutagenesis of a strain of Escherichia coli carrying a temperature-sensitive (Ts) mutation in the cell division gene ftsA yielded a number of temperature-resistant variants. In certain cases, restoration of viability at the restrictive temperature could not be attributed to suppressor mutations occurring in other genes or to structural gene reversion. DNA sequencing of the variants demonstrated the continuing presence of the original Ts mutation (ftsA13) and revealed secondary mutations within the same gene. These secondary mutations are able to rescue the ftsA13 mutation in cis, but not in trans.     

Isolation and characterization of Escherichia coli mutants exhibiting altered response to thymol

The antimicrobial mode of action of the plant essential oil thymol was studied with Escherichia coli. Random transposon-insertion mutants were screened for altered response to thymol. Of four mutants showing more sensitivity, three were found in rfaQ, whose product is involved in lipopolysaccharide biosynthesis, and the fourth in the quorum-sensing gene qseC. Mutants showing more resistance had mutations in genes whose products are involved in the degradation of short-lived regulatory and abnormal proteins (the lon gene), menaquinone biosynthesis (menA), an unknown function of a putative membrane protein (yagF), synthesis of a small hypothetical protein (an intergene region between the two small genes yiiE and yiiF), and the efflux pump of cadaverine and lysine (cadB). The antibacterial activities of carvacrol, menthol, and cymene, essential oils structurally similar to thymol, were also determined. Although the level of resistance toward thymol was conserved in the respective mutants qseC, menA, and cadB, knockout mutants displayed different levels of tolerance to carvacrol; inconsistencies in resistance levels were also noted in mutants challenged with menthol. Wild-type and mutant E. coli responded to thymol exposure with a massive potassium efflux that generally corresponded to the resistant rate. The verity of the loci accounting for E. coli response suggests a multitarget mode of the antimicrobial activity of thymol and multitolerance mechanisms.     

Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures.

The heat-labile enterotoxin (LT) has been isolated in homogeneous form with high specific activity from three sources: cell-free supernatant, NaCl extract, and whole-cell lysates of an enterotoxigenic Escherichia coli strain. In vitro immunological assays were used in lieu of tedious and highly variable bioassays to recognize fractions with activity. This revealed that the major portion of the LT remained adherent to columns containing agarose, from which it could be eluted quantitatively in practically homogeneous form by galactose. Isolated LT has remarkable similarities to the cholera enterotoxin (choleragen) in both subunit structure and amino acid composition, although there are also notable differences in these two enterotoxins, which are related immunologically and by mode of action. Unlike choleragen, in which the A region is totally nicked, E. coli LT, depending on its source, is activated by proteolytic processing. The activity of LT is equivalent to that of choleragen in bioassays on adrenal cells, in rabbit skin, and in rabbit ileal loops, especially when, depending on the source of material, the LT has been activated by treatment with trypsin. The whole-cell lysate is the richest source of LT.     

Isolation and characterization of the alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) from fimbriated Escherichia coli.

The alpha-galactosyl-1,4-beta-galactosyl-specific adhesin (P adhesin) was isolated from the fimbria-adhesin complex (FAC) of recombinant Escherichia coli strains expressing the F7(1), F8, or F13 fimbrial antigens. Separation into fimbriae and adhesin was achieved by heating the FAC to 80 degrees C in the presence of Zwittergent 3-16. After removal of the fimbriae by precipitation with lithium chloride, the adhesin was purified by anion-exchange fast protein liquid chromatography in the presence of 4 M urea. The purified adhesins from the three strains had pIs of 4.8 to 5.0 and molecular weights of approximately 35,000. The fimbrillins were smaller, their molecular weights being different with different F antigens. The amino-terminal amino acid sequence of the F7(1)- and F13-derived adhesins were different, that of the F13-derived adhesin being identical to that extrapolated from the DNA sequence of the papG gene (B. Lund, G. Lindberg, B.-I. Marklund, and S. Normark, Proc. Natl. Acad. Sci. USA 84:5898-5902). An antiadhesive monoclonal antibody which reacted with the three P adhesins was prepared. The FAC and the purified adhesins but not the fimbriae from which the adhesins had been removed agglutinated erythrocytes and galactose-galactose-coated latex beads. The adhesion of erythrocytes to the surface-fixed adhesins could be specifically inhibited with alpha-galactosyl-1,4-beta-galactosyl-1,4-glucosyl. The results indicate that the P adhesin(s) of uropathogenic E. coli represents a group of related proteins with conserved receptor recognition domains. The F13-derived P adhesin is the PapG protein postulated by Normark and his colleagues (Lund et al., Proc. Natl. Acad. Sci. USA 84:5898-5902; B. Lund, F. Lindberg, and S. Normark, J. Bacteriol. 170:1887-1894).     

Isolation and partial characterization of temperature bacteriophages from enteropathogenic strains of Escherichia coli 0111:K58

We report studies on ten strains of Escherichia coli 0111:K58 isolated from children with acute diarrhea. Our results show that these E. coli strains do not produce the pathogenic factors of enterotoxic E. coli (ETEC) and are lysogenic for phages belonging to two groups that differ for host range, kinetics of thermal inactivation, antigenicity and morphology. These data support the hypothesis that these phages may in vivo contribute to reduction of the number of common E. coli strains by lytic infection favouring the development of the enteropathogenic strain of E. coli.     

Isolation and characterization of a monoclonal antibody directed against type 1 fimbriae organelles from Escherichia coli.

We have isolated a mouse immunoglobulin M (IgM) monoclonal antibody directed against type 1 fimbriae from the Escherichia coli K-12-derived strain CSH50. Antibody specificity was demonstrated by (i) the ability of fimbriate but not nonfimbriate bacteria to compete with solid-phase purified fimbriae for antibody binding in an enzyme-linked immunosorbent assay, (ii) the visualized binding of antibody to fimbriae alone by electron microscopy, and (iii) the appearance in a radioimmunoprecipitation assay of a single electrophoretic band comigrating with pure type 1 fimbriae. The monoclonal antibody was further characterized by immunoblot analysis and compared with previously prepared rabbit anti-fimbrial antisera. Whereas the monospecific but polyvalent antisera recognized both fimbrial monomeric subunits and non-disaggregated fimbriae organelles, the monoclonal antibody recognized only the intact organelles even when the samples were prepared under nondenaturing conditions. The monoclonal antibody, therefore, might be directed against an epitope spanning two (or more) adjacent fimbrial subunits.     

Isolation and characterization of specialized transducing bacteriophages for the recA gene of Escherichia coli.

Mg²⁺ stabilized potato virus Y helper component during partial purification by precipitation using polyethyleneglycol. In solutions containing 0.02 M Mg²⁺ the helper component retained most of its activity for 2 days at 4° and for at least 8 months at ?15°. Activity was destroyed on incubation with pronase or trypsin, or by heating for 5 min at 55°, but not by incubation with ribonuclease. Incubation with its own antiserum strongly inhibited helper component activity, but antisera to potato virus Y coat protein or inclusion protein had no more effect than a control serum showing that the component was unrelated to these two proteins. Filtration through a Sephadex G-200 column resulted in a broad peak of activity which produced many protein-staining bands when electrophoresed on polyacrylamide gel. Gel filtration and ultrafiltration experiments both indicated a molecular weight of 100,000–200,000. Some helper component activity was retained by aphids allowed to probe into a sucrose solution for 20 min showing that the helper component is more firmly bound to the aphid than is the tobacco mosaic viruspoly-l-ornithine complex.     

Isolation and genetic characterization of a coinfection of non-O157 Shiga toxin-producing Escherichia coli

A coinfection of O177:NM and O55:H7 Shiga toxin-producing Escherichia coli (STEC) was identified for a child with acute bloody diarrhea and hemolytic uremic syndrome by using culture and serotype-specific molecular reagents. The profile of O157-related genetic islands revealed that the O55:H7 isolate was highly similar to O157 STEC whereas the O177:NM isolate lacked several fimbrial O islands and non-locus-of-enterocyte-effacement effector determinants. However, both STEC serotypes are known to cause serious disease, and the significant repertoire of virulence determinants in both strains made it impossible to determine their individual contributions to the clinical symptoms.     

Isolation, characterization, and epidemiological assessment of shiga toxin-producing Escherichia coli O84 isolates from New Zealand

Shiga toxin-producing Escherichia coli O84 isolates (n = 22) were examined using culture- and molecularly based methods in order to compare their phenotypic and genotypic characteristics. These analyses directly linked Shiga toxin-producing Escherichia coli O84 isolates from cattle and sheep with human isolates indicating that New Zealand livestock may be a reservoir of infection.     

Isolation and characterisation of Shiga toxigenic Escherichia coli strains from northern Palestine

Shiga toxigenic Escherichia coli (STEC) isolates from symptomatic and asymptomatic patients in northern Palestine in 1999 were screened for serotype O157 and characterised for virulence genes by multiplex PCR assay. Of the 176 STEC isolates, 124 (70.5%) were of serotype O157. All these isolates carried the gene for Shiga toxin type 1 (stx,) and 112 (90.3%) carried stx2. The intimin encoding gene locus eae was detected in 16 isolates (12.9%) and the enterohaemolysin encoding gene, hlyA, in 18 (14.5%). Statistical analysis showed a significant association between the presence of eaeA and hlyA, either alone or combined with stx1 and stx2 genes in O157 isolates from symptomatic infection. ERIC-PCR analysis of DNA from 80 serotype O157 isolates revealed three major clonal populations.     

Detection and characterization of aparmycin-resistant Escherichia coli from humans in Korea

To investigate apramycin resistance in humans in Korea, a total of 138 human Escherichia coli strains confirmed as gentamicin-resistant were collected from Korean Culture Collection Antimicrobial-Resistant Microbes. Apramycin resistance (minimum inhibitory concentrations ≥1,024?μg/ml) was observed in 16 (11.6%) of the 138 gentamicin-resistant E. coli (GREC) strains. Among the seven different kinds of aminoglycoside resistance genes tested, only four kinds were detected in the apramycin-resistant GREC strains: aac (3)-II, aac (3)-III, aac (3)-IV, and armA. The aac (3)-IV gene was found in all apramycin-resistant GREC strains, whereas aac(3)-II, aac(3)-III, and armA genes were detected in 8 (50.0%), 6 (37.5%), and 1 (6.3%) GREC strains resistant to apramycin, respectively. Of 16 apramycin-resistant GREC strains, transfer of apramycin resistance was observed in seven (43.8%), and co-transfer of resistance to other antimicrobials along with apramycin resistance was also found in four strains (25.0%) by broth mating. The results of this study suggest that more prudential use of apramycin in animals is needed.     

Isolation and characterization of cephalothin-susceptible Campylobacter coli from slaughter cattle.

In a recent meat survey, 10 of 13 (77%) Campylobacter coli isolates were susceptible to cephalothin. These organisms were isolated from nine slaughter cattle from eight meat packing establishments. All 10 isolates grew at 43 degrees C but not at 25 degrees C, were catalase and oxidase positive, and were susceptible to nalidixic acid (30 micrograms) and cephalothin (30 micrograms). The cultures were subsequently identified as C. coli serogroup 20, biotype I (Lior scheme). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that protein and lipopolysaccharide profiles of whole cell preparations of the 10 cephalothin-susceptible strains and the reference strain for C. coli serogroup 20 were very similar. The plasmid profiles of these 11 strains were identical.     

Isolation and characterization of the non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6

The non-fimbrial adhesin NFA-4 from uropathogenic Escherichia coli O7:K98:H6 mediates the agglutination of human red cells (RBC), notably of blood group MM. The adhesin can be separated from the bacteria by heat extraction and was purified to homogeneity by ammonium sulphate precipitation and anion exchange chromatography in the presence of 8 M urea. NFA-4 consists of non-covalently linked 28 kDa subunits which tend to form aggregates of an apparent molecular weight in excess of 10(6) Da. The first 23 amino-terminal amino acids were sequenced, and no homology of this region was found with that of the blood group M specific non-fimbrial adhesin of an unrelated uropathogenic E. coli. It has, however, an about 70% homology to the corresponding region of the K88 antigen from animal-pathogenic enterotoxic E. coli. Both polyclonal and monoclonal antibodies against NFA-4 were prepared. One of the monoclonal antibodies strongly inhibits the hemagglutinating activity of both whole bacteria and purified NFA-4.     

Isolation and structural characterization of the equine erythrocyte receptor for enterotoxigenic Escherichia coli K99 fimbrial adhesin

The erythrocyte receptor for Escherichia coli K99 fimbrial adhesin was isolated from equine erythrocytes and characterized as Neu5Gc-alpha(2----3)-Galp-beta(1----4)-GLcp-beta(1----1)-Ceramide. This glycolipid acted as the receptor for K99 by four different experimental approaches: inhibition of equine erythrocyte hemagglutination by preincubation of K99-positive bacteria or purified K99 fimbriae with the isolated glycolipid; inhibition of attachment of K99-positive bacteria to porcine intestinal epithelial cells in the presence of the isolated glycolipid; induction of binding of K99-positive bacteria or purified K99 fimbriae to normally unreactive guinea pig erythrocytes by coating these cells with the isolated glycolipid; and isolation of the receptor by affinity chromatography with K99 coupled to CNBr-activated Sepharose 4B, indicating a strong interaction between K99 and the isolated glycolipid.     

Isolation of Vi Antigen from Escherichia coli by a New Method

Vi antigen was isolated from a trichloroacetic acid-deproteinized saline extract of acetone-killed and -dried cells of Escherichia coli (5396/38) by ethyl alcohol precipitation and absorption on diethylaminoethyl-Sephadex. Stepwise elution with distilled water, 0.15 m NaCl, and 0.5 m NaCl resulted in recovery of Vi antigen and two additional antigenic fractions (designated antigens 1 and 2). Vi antigen was an acidic polymer which flocculated readily with albumin, contained 6.0% nitrogen, and had a sedimentation coefficient of 14.7S. Vi was shown to be antigenic in the rabbit by the indirect hemagglutination test and in mice by tests for protection against challenge with Salmonella typhosa, wherein it was more protective by the intraperitoneal (ip) than by the subcutaneous (sc) route. Vi antigen prepared by this method was much more protective for mice than was the Vi antigen used in previous studies. The other antigens, not fully characterized, were also capable of protecting mice. Antigen 1 was more effective by the sc than the ip route, whereas antigen 2 was more effective by the ip route. The relationship between Vi antigen and antigens 1 and 2, as demonstrated by hemagglutination tests with rabbit antisera, remains unclear.     

Isolation of a thermolabile enterotoxin from Escherichia coli and the study of its biological properties

An enterotoxin was isolated from strainEscherichia coli 015 by salt precipitation and gel chromatography. In the process of isolation and purification the toxic activity of the preparation increased: by 60 times according to the ligated segment of rabbit intestine method and 66–100 times according to the skin test. The plateau and second fraction obtained by gel chromatography were inactive according to the ligated segment of intestine method but possessed permeability factor (PF) activity in the skin test. Two hypotheses were put forward: The vascular permeability factor and the diarrheagenic factor are possibly two different, substances (molecules) and the skin test is more sensitive as a method of determining toxicity than the ligated segment of rabbit intestine method.Laboratory of Protective Antigens and Laboratory of Genetics of Vaccine Strains, I. M. Mechnikov Moscow Institute of Vaccines and Sera. (Presented by Academician of the Academy of Medical sciences of the USSR P. A. Vershilova.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 82, No. 10, pp. 1237–1239, October, 1976.