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On the process of cellular division in Escherichia coli. VI. Use of a methocel-autoradiographic method for the study of cellular division in Escherichia coli.

A technique allowing chain formation by Escherichia coli for the analysis of the segregation of certain cellular components by autoradiographic methods has been developed. Deoxyribonucleic acid labeled with (3)H-thymidine does not appear to segregate preferentially with one or another of the bacteria. Membrane lipids labeled with either (3)H-oleate or (3)H-glycerol are evenly distributed among all progeny cells. A similar pattern of distribution of cell wall material is observed when (3)H-diaminopimelate is used as the marker.

Autoradiography↗

Genetic transfer of Salmonella typhimurium and Escherichia coli lipopolysaccharide antigens to Escherichia coli K-12.

Escherichia coli K-12 varkappa971 was crossed with a smooth Salmonella typhimurium donor, HfrK6, which transfers early the ilv-linked rfa region determining lipopolysaccharide (LPS) core structure. Two ilv(+) hybrids differing in their response to the LPS-specific phages FO and C21 were then crossed with S. typhimurium HfrK9, which transfers early the rfb gene cluster determining O repeat unit structure. Most recombinants selected for his(+) (near rfb) were agglutinated by Salmonella factor 4 antiserum. Transfer of an F' factor (FS400) carrying the rfb-his region of S. typhimurium to the same two ilv(+) hybrids gave similar results. LPS extracted from two ilv(+),his(+), factor 4-positive hybrids contained abequose, the immunodominant sugar for factor 4 specificity. By contrast, his(+) hybrids obtained from varkappa971 itself by similar HfrK9 and F'FS400 crosses were not agglutinated by factor 4 antiserum, indicating that the parental E. coli varkappa971 does not have the capacity to attach Salmonella O repeat units to its LPS core. It is concluded that the Salmonella rfb genes are expressed only in E. coli varkappa971 hybrids which have also acquired ilv-linked genes (presumably rfa genes affecting core structure or O-translocase ability, or both) from a S. typhimurium donor. When E. coli varkappa971 was crossed with a smooth E. coli donor, Hfr59, of serotype O8, which transfers his early, most his(+) recombinants were agglutinated by E. coli O8 antiserum and lysed by the O8-specific phage, Omega8. This suggests that, although the parental E. coli K-12 strain varkappa971 cannot attach Salmonella-specific repeat units to its LPS core, it does have the capacity to attach E. coli O8-specific repeat units.

Agglutination Tests↗

Outer membrane proteins of Escherichia coli. 3. Evidence that the major protein of Escherichia coli O111 outer membrane consists of four distinct polypeptide species.

Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.

Bacterial Proteins↗

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: structure of F100, F152, and F8 and mapping of the Escherichia coli chromosomal region fep-supE-gal-attlambda-uvrB.

The genetic and physical structures of commonly used F-prime factors carrying the galactose region of the Escherichia coli chromosome were analyzed. Deletions in the chromosomal DNA sequences in the F-prime factors were found to be frequent events. A genetic method was developed to reconstruct the original F-prime factors from deletion variants. Heteroduplex analysis of the reconstructed F-prime factors confirmed the derivation of the F-prime factors F100 and F152, from the same Hfr, and finally determined the normal E. coli chromosomal sequence in the region between fep and uvrB, containing about 5 min in genetic units and about 246.5 in kilobase units (kb). This sequence could be connected with the DNA sequences of the lac-purE region, which had been physically determined previously. Together they constituted a total of 528.6 kb. From these combined sequences, the distance from lacPO to galK was calculated to be 412.9 kb, which corresponds to 8.8 min in genetic units.

Chromosome Mapping↗

Salmonella typhimurium newD and Escherichia coli leuC genes code for a functional isopropylmalate isomerase in Salmonella typhimurium-Escherichia coli hybrids.

The supQ newD gene substitution system in Salmonella typhimurium restores leucine prototrophy to leuD mutants by providing the newD gene product which is capable of replacing the missing leuD polypeptide in the isopropylmalate isomerase, a complex of the leuC and leuD gene product. Mutations in the supQ gene are required to make the newD protein available. An Escherichia coli F' factor was constructed which carried supQ- newD+ from S. typhimurium on a P22-specialized transducing genome. This F' pro lac (P22dsupQ394newD) episome was transferred into S. typhimurium strains containing th leuD798-ara deletion; the resulting merodiploid strains had a Leu+ phenotype, indicating that supQ- newD+ is dominant over supQ+ newD+, and eliminating the possibility that the supQ gene codes for a repressor of the newD gene. Furthermore, transfer of the F' pro lac (P22dsupQ39newD) into E. coli leuD deletion strains restored leucine prototrophy, showing that the S. typhimurium newD gene can complment the E. coli leuC gene. Growth rates of the S. typhimurium-E coli hybrid strains indicated that the mutant isopropylmalate isomerase in these strains does not induce a leucine limitation, as it does in S. typhimurium leuD supQ mutants. In vitro activity of the mutant isopropylmalate isomerase was demonstrated; the Km values for alpha-isopropylmalate of both the S. typhimurium leuC-newD isomerase and the S. typhimurium-E. coli hybrid isomerase were as much as 100 times higher than the Km values for alpha-isopropylmalate of the wild-type enzyme, which was 3 x 10(-4) M. Mutagenesis of E. coli leuD deletion strains failed to restore leucine prototrophy, indicating that E. coli does not have genes analogous to the S. typhimurium supQ newD genes, of that, if present, activation of a newD is a rare event or is lethal to the cell.

Escherichia coli↗

Replication of M13 oriC bacteriophages in Escherichia coli rep mutant is dependent on the cloned Escherichia coli replication origin.

The involvement of the Escherichia coli rep protein in the replication of M13 chimeric deoxyribonucleic acids (DNAs) carrying the E. coli chromosomal DNA replication origin (oriC) has been examined. Previous studies indicate that the cloning of a 3,550-base-pair sequence of chromosomal DNA containing oriC into an M13 vector allows extensive replication of the M13 oriC chimeric DNA in an E. coli rep-3 mutant. We have extended these studies by preparing a 330-base-pair deletion that specifically deletes the oriC sequence in the M13 oriC DNAs, to demonstrate that the replication observed in the rep-3 host is dependent on the cloned origin. Thus, a DNA-unwinding enzyme other than the rep protein may be involved in the strand separation process accompanying replication which initiates at oriC in the M13 oriC chimeric DNAs and in the E. coli chromosome. The rep assay used for assessing the functionality of the cloned oriC is useful for analysis of any rep-independent origin of replication functional in E. coli. A direct selection for a cloned origin of replication is possible in the rep-3 recA56 host. Since the cloned origin is nonessential for propagation of the M13 chimeric phage in a rep+ host, mutations in the cloned origin may be constructed, and the mutant phage may be examined by a simple transductional analysis of the rep-3 recA56 mutant strain.

Coliphages↗

Physical mapping of repetitive extragenic palindromic sequences in Escherichia coli and phylogenetic distribution among Escherichia coli strains and other enteric bacteria.

Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeat sequences originally discovered in Escherichia coli and Salmonella typhimurium. We have physically mapped these sequences in the E. coli genome by using Southern hybridization of an ordered phage bank of E. coli (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) with generic REP probes derived from the REP consensus sequence. The set of REP probe-hybridizing clones was correlated with a set of clones expected to contain REP sequences on the basis of computer searches. We also show that a generic REP probe can be used in Southern hybridization to analyze genomic DNA digested with restriction enzymes to determine genetic relatedness among natural isolates of E. coli. A search for these sequences in other members of the family Enterobacteriaceae shows a consistent correlation between both the number of occurrences and the hybridization strength and genealogical relationship.

Base Sequence↗

Identification of the hopG gene, a component of Escherichia coli K-12 type II export system, and its conservation among different pathogenic Escherichia coli and Shigella isolates.

The Escherichia coli K-12 gene coding for a component of a type II export system was identified and characterized. The HopG protein contains a typical prepilin peptidase cleavage site and has a high degree of homology with proteins PulG, OutG, and ExeG, which are components of type II secretion systems from Klebsiella pneumoniae, Erwinia carotovora, and Aeromonas hydrophila.

Amino Acid Sequence↗

Iron transport in Escherichia coli: relationship between chromium sensitivity and high iron requirement in mutants of Escherichia coli.

Utilization of iron (Fe(3+)) by Escherichia coli depends upon a system which is determined by at least two genetic loci. Mutants which carry a deletion of the tonB-trp region of the chromosome grow only when very high concentrations of iron are present in the medium. These strains are sensitive to chromic ion (Cr(3+)) and, unlike the parent strain, fail to grow on MnSO(4) when FeSO(4) is not added to the medium. A second type of mutant, Chr2, which was isolated on the basis of its sensitivity to chromic ion, also requires a high concentration of iron for growth. This mutant can be distinguished phenotypically from the deletion mutants since it grows normally on low concentrations of iron, provided citrate is added to the medium. The chromium sensitivity of both types of mutants can be reversed by high concentrations of exogenous iron. The data are interpreted to indicate that the E. coli mutants studied are defective in iron transport and that residual iron transport is in some way inhibited by chromic ion.

Biological Transport↗

Enterotoxigenic Escherichia coli strains belonging to a new serogroup, Escherichia coli O166.

Thirty-two strains of Escherichia coli belonging to a new O group, O166, were examined. Twenty-one strains had the flagella antigen H27, five had the H15 antigen, five had the H7 antigen, and one was nonmotile. All the H27 strains and the nonmotile strain produced heat-stable enterotoxin but not heat-labile enterotoxin. All the H7 strains produced heat-labile enterotoxin but not heat-stable enterotoxin. The remaining strains were nonenterotoxigenic. None of the strains possessed colonization factor antigens CFA/I, CFA/II, or PCF8775.

Adhesiveness↗

Carumonam enhances reactivity of Escherichia coli with mono- and polyclonal antisera to rough Escherichia coli J5.

Escherichia coli O111 reacts only slightly with antiserum to its rough mutant E. coli J5 in an enzyme-linked immunosorbent assay. When E. coli O111 was grown in the presence of sub-MICs of the monocyclic beta-lactam antibiotic carumonam, however, the enzyme-linked immunosorbent assay titer increased from 1,280 to 81,920. When the bacteria were grown in the presence of carumonam, the titer that was obtained with antiserum against E. coli O111 was not affected. This reaction was abolished after this antiserum was absorbed with E. coli J5 in the case of the carumonam-treated strain, whereas this absorption did not affect the reaction with E. coli O111. Thus, the O-antigenic side chain of E. coli O111 seems to be affected if this strain is cultured in the presence of carumonam. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a relative loss of the O polysaccharide in E. coli O111 when this strain was grown in the presence of carumonam. Also, a much stronger reaction of the antibiotic-affected lipopolysaccharide with a monoclonal antibody against E. coli J5 lipopolysaccharide was shown in immunoblots. The results of this study indicate that there is a synergism between certain antibiotics and monoclonal antibodies, something that could have clinical implications.

Anti-Bacterial Agents↗

Transfer and expression of the genetic determinants for O and K antigen synthesis in Escherichia coli O9:K(A)30 and Klebsiella sp. O1:K20, in Escherichia coli K12.

Escherichia coli serotype O9:K(A)30 and Klebsiella O1:K20 produce thermostable capsular polysaccharides or K antigens, which are chemically and serologically indistinguishable. Plasmid pULB113 (RP4::mini-Mu) has been used to mediate chromosomal transfer from E. coli O9:K30 and Klebsiella O1:K20 to a multiply marked, unencapsulated, E. coli K12 recipient. Analysis of the cell surface antigens of the transconjugants confirmed previous reports that the genetic determinants for the E. coli K(A) antigens are located near the his and rfb (O antigen) loci on the E. coli linkage map. The Klebsiella K20 capsule genes were also found to be in close proximity to the his and rfb loci. Electron microscopy revealed significant differences in the structural organization of capsular polysaccharides in these two microorganisms and the morphological differences were also readily apparent in transconjugants expressing the respective K antigens. These results are consistent with the interpretation that at least some of the organizational properties of capsular polysaccharides may be genetically determined, rather than being a function of the outer membrane to which the capsular polysaccharides are ultimately attached.

Antigens, Bacterial↗

A Col E1 hybrid plasmid containing Escherichia coli genes complementing d-xylose negative mutants of Escherichia coli and Salmonella typhimurium.

The Clarke and Carbon bank of Col El - Escherichia coli DNa hybrid plasmids was screened for complementation of d-xylose negative mutants of E. coli. Of several obtained, the smallest, pRM10, was chosen for detailed study. Its size was 16 kilobases (kb) and that of the insert was 9.7 kg. By transformation or F'-mediated conjugation this plasmid complemented mutants of E. coli defective in either D-xylose isomerase or D-xylulose kinase activity, or both. The activity of D-xylulose kinase in E. coli transformants which bear an intact chromosomal gene for this enzyme was greater than that for the host, due to a gene dosage effect. The plasmid also complemented D-xylose negative mutants of Salmonella typhimurium by F'-mediated conjugation between E. coli and S. typhimurium. Salmonella typhimurium mutants complemented were those for D-xylose isomerase and for D-xylulose kinase in addition to pleiotropic D-xylose mutants which were defective in a regulatory gene of the D-xylose operon. In addition, the plasmid complemented the glyS mutation in E. coli and S. typhimurium. The glyS mutant of E. coli was temperature sensitive, indicating that the plasmid carried the structural gene for glycine synthetase. The glyS mutation in E. coli maps at 79 min, as do the xyl genes. The behaviour of the plasmid is consistent with the existence of a d-xylose operon in E. coli. The data also suggest that the plasmid carries three of the genes of this operon, specifically those for D-xylose isomerase, D-xylulose kinase, and a regulatory gene.

Cloning, Molecular↗

Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate.

Ninety commercial broiler chickens were divided into three equal groups; 30 were injected with brain-heart-infusion broth into the cranial thoracic air sacs (controls), 30 were similarly inoculated with a culture of Escherichia coli, and 30 were similarly inoculated with E. coli cell-free culture filtrate. Birds were examined from 0 to 6 hours post-inoculation. E. coli-inoculated and cell-free culture filtrate-inoculated chickens reacted similarly, with exudation of heterophils into the air sac. Microscopically, heterophils were present in low numbers perivascularly 0.5 hour after inoculation and became more numerous by 3 hours post-inoculation. By 6 hours post-inoculation, there was severe swelling of air sac epithelial cells and thickening of the air sac by proteinaceous fluid and heterophils. Ultrastructurally, air sac epithelial cells were swollen and vacuolated, and interdigitating processes were separated. Histologically and ultrastructurally, all features in control chickens were normal, with only rare heterophils in the air sac interstitium. In E. coli-inoculated and cell-free culture filtrate-inoculated chickens, cell counts (predominantly heterophils) in air sac lavage fluids increased markedly at 3 and 6 hours, with only slight increases in counts from lavages of controls. Heteropenia was observed in E. coli-inoculated chickens, whereas heterophilia was observed in cell-free filtrate chickens and controls. Ninety additional chickens were pretreated with cyclophosphamide, subdivided into three equal groups, and inoculated and examined similarly as above. Cyclophosphamide pretreatment reduced inflammatory changes in air sacs, lowered cell numbers in lavage fluids, and abolished hematologic changes; however, it did not prevent epithelial cell changes. These results indicate that cell-free culture filtrate of E. coli induces changes similar to those induced by cultures of E. coli.

Acute Disease↗

Isolation and characterization of a cDNA from soybean and its homolog from Escherichia coli, which both complement the light sensitivity of Escherichia coli hemH mutant strain VS101.

Using Escherichia coli strain VS101, whose hemH gene encoding the ferrochelatase is partially defective, we isolated and analyzed a clone (designated XWH-1) from a X phage library of soybean (Glycine max) cDNA, which exhibited weak complementation activity against the light sensitivity of VS101. In VS101 bacteria lysogenized with lambdaWH-1, a significant decrease in accumulation of protoporphyrin IX (PROTO IX) was detected as compared with that in non-lysogenic bacteria. On the other hand, in the wild-type E. coli strains lysogenized with lambdaWH-1, significant accumulation of delta-aminolevulinic acid (ALA) was observed, although accumulation of other intermediates such as uroporphyrinogen III (UROGEN III) and coproporphyrinogen III (COPROGEN III), was not observed. The growth of the wild-type bacteria in which the insert cDNA from deltaWH-1 had been introduced via a plasmid vector was markedly inhibited. By constructing, testing and sequencing a series of deletion clones of the insert, it was found that the insert encodes two proteins, a trancated LepA and a hypothetical protein ORF296, and that only ORF296 possesses the ability to block the heme biosynthetic pathway. ORF296 showed about 30% identity with the E. coli hypothetical protein YicL. By cloning and examining the gene for YicL in E. coli, we found that YicL shows the same effect as that of the soybean cDNA. From these findings, we concluded that the clone from soybean and yicL from E. coli block a step in an early stage of the heme biosynthetic pathway (probably the step catalyzed by HemB). Consequently, we postulate that the VS101 bacteria harboring these genes became light resistant as a result of a decrease in accumulated PROTO IX, and that the growth of the bacteria harboring these genes was inhibited because of the inhibition of heme biosynthesis at the step catalyzed by HemB.

Aminolevulinic Acid↗

Sensitivity of Escherichia coli to viral nucleic acid. XVI. Temperature conditions for Ca2+-dependent dna uptake in Escherichia coli.

Properties of Ca2+- or Ba2+-dependent transfection and transformation in Escherichia coli were examined, using phi X174 replicative-form (RF) DNA and plasmid DNA. For the transfection and transformation, a heat pulse step was dispensable and the yield of transfectants was, in most E. coli strains, rather reduced by the heat treatment. The heat pulse step was also detrimental for the transformation of certain strains such as lipopolysaccharide mutants, The first stage of the DNA uptake process (formation of DNA . recipient cell complex) was dependent on low temperature and Ca2+ ion. A substantial amount of the complexed RF-DNA was released from the bacteria, by washing with a chilled Tris buffer. Although a RF-DNA . cell complex was formed even at 37 degree C or in chilled 0.05 m MgC12, the complex did not yield transfectants.

Bacteriophage phi X 174↗

UV inactivation, liquid-holding recovery, and photoreactivation of Escherichia coli O157 and other pathogenic Escherichia coli strains in water.

Drinking water, water used in food production and for irrigation, water for fish farming, waste water, surface water, and recreational water have been recently recognized as a vector for the transmission of pathogenic Escherichia coli, especially serotype O157:H7. We investigated the UV (253.7 nm) inactivation behavior and the capability of dark repair (liquid-holding recovery) and photoreactivation of seven pathogenic (including three enterohemorrhagic E. coli) strains and one nonpathogenic strain of E. coli (ATCC 11229) with respect to the use of UV light for water disinfection purposes. Because most bacteria and yeast are known to be able to repair UV damage in their nucleic acids, repair mechanisms have to be considered to ensure safe water disinfection. We found a wide divergence in the UV susceptibility within the strains tested. A 6-log reduction of bacteria that fulfills the requirement for safe water disinfection was reached for the very most susceptible strain O157:H7 (CCUG 29199) at a UV fluence of 12 J/m2, whereas for the most resistant strain, O25:K98:NM, a UV fluence of about 125 J/m2 was needed. Except for one strain (O50:H7) liquid-holding recovery did not play an important role in recovery after UV irradiation. By contrast, all strains, particularly strains O25:K98:NM, O78:K80:H12, and O157:H7 (CCUG 29193), demonstrated photorepair ability. For a 6-log reduction of these strains, a UV fluence (253.7 nm) up to 300 J/m2 is required. The results reveal that the minimum fluence of 400 J/m2 demanded in the Austrian standard for water disinfection is sufficient to inactivate pathogenic E. coli. A fluence of 160 J/m2 (recommendation in Norway) or 250 J/m2 (recommendation in Switzerland) cannot be regarded as safe in that respect.

Animals↗