Protein Sci, 1992 Aug, 1(8), 961 - 9 Overexpression, purification, and characterization of yeast cyclophilins A and B; Zydowsky LD et al.; Two isoforms of yeast cyclophilins, yCyPA and yCyPB, have been subcloned, expressed in Escherichia coli, and purified to homogeneity . The full-length (163-amino acid) yeast CyPA was easily expressed and purified; however, only a genetically truncated, 186-residue form of yCyPB lacking a putative 20-amino acid signal sequence could be purified . Each yeast cyclophilin isoform is a peptidyl-prolyl isomerase, inhibitable by the immunosuppressive drug CsA (IC50's of 40 +/- 8 nM and 101 +/- 14 nM at 18 nM concentrations of yCyPA and yCyPB, respectively) . Polyclonal antibodies raised against recombinant yCyPA detected native yCyPA in yeast cell extracts by both immunoprecipitation and Western blot analysis . However, polyclonal antibodies raised against recombinant yCyPB detected no native yCyPB in yeast cell extracts by Western blot analysis; small amounts of yCyPB were found in the culture broth, suggesting secretion extracellularly of this isoform . Northern analysis indicated that both yCyPA mRNA and yCYPB mRNA (at a much lower level) were detectable in cell-free extracts . Characterization of the yeast cyclophilin proteins demonstrated that their catalytic properties and sensitivity to CsA parallel those of the human cyclophilins. Prikl Biokhim Mikrobiol, 1992 Jul-Aug, 28(4), 623 - 30 {Effect of physico-chemical properties of polyacrylamide gel media on the growth of Escherichia coli colonies}; Rodin VB et al.; We studied the growth of Escherichia coli LE-392 colonies on polyacrylamide gels (PAAG) depending on the physico-chemical properties of the latter, i.e . polymer concentration in the gel, swelling degree, bound water content (fm), spin-lattice relaxation and spin-spin relaxation times of water molecule protons, and modulus of elasticity (G0) . S- or R-type colonies formed depending on gel properties; the diametral growth rate of S colonies was 3 times less compared with that on the control agar medium (Tryptose broth) . The procedure is proposed for preparation of PAAG which rules out syneresis . Functional relations between the polymer concentrations in uniformly swelling gels and concentrations of copolymers in the reaction mixture, fm and G0 were revealed . The fm and G0 parameters can be used for controlling the quality of PAAG. J Vet Med Sci, 1992 Jun, 54(3), 493 - 9 Acid-induced autoagglutination found in chicken pathogenic Escherichia coli strain; Sekizaki T et al.; Chicken pathogenic Escherichia coli strains were found to autoagglutinate in a static culture of trypticase soy broth (TSB) . One strain, designated PDI-386, was further studied for its autoagglutinating property . Acidity in the cultured medium caused by glucose degradation induced the autoagglutination . The bacterial cells grown in a glucose-free L-broth could be aggregated by adding acid, which suggests a potentiality of autoagglutination of the strain grown in the L-broth . The autoagglutinating parent (Agg) formed small colonies with irregular edges like rough colonies on the TS agar, whereas its non-autoagglutinating variant (Nag) formed larger smooth colonies with a perfectly round edge . The Nag colony was easily generated from the Agg colony on the TS agar . The autoagglutinating property was very unstable when the bacteria was passed in the TSB, but rather stable in the L-broth . Under electron microscope, the Agg were found to possess pili of more than 20 microns in length . However, the phenotypic expression of autoagglutination did not correlate with that of mannose-sensitive hemagglutination against guinea pig erythrocytes . Incubation of the Nag in the L-broth at room temperature for more than 10 days provoked the reversion of the autoagglutination . There was no difference between the Agg and the Nag in terms of surface hydrophobicity, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of membrane proteins and LPS, and plasmid profiles . The virulence of the Agg was higher than that of the Nag . The autoagglutination property is, however, so unstable that the pathogenicity of E . coli isolates from chickens should be carefully evaluated. J Bacteriol, 1992 Apr, 174(7), 2367 - 75 Growth conditions mediate differential transcription of fim genes involved in phase variation of type 1 pili; Schwan WR et al.; Type 1 pili in Escherichia coli undergo phase variation in which individual cells in a population reversibly switch between piliated (Pil+) and nonpiliated (Pil-) states . The switching process is mediated by an invertible DNA fragment which contains the promoter for fimA, the gene encoding the major structural subunit of type 1 pili . Although type 1 pili randomly phase vary in broth cultures, many clinical isolates of E . coli do not express type 1 pili when cultured on agar media . We investigated the role of the invertible element and the upstream genes, fimB and fimE, in the agar-mediated suppression of pili in an agar-negative clinical isolate, strain 149 . Southern hybridization and polymerase chain reaction analyses of the fimA promoter region in broth-grown 149 cells indicated that the invertible element was present in orientations corresponding to both Pil+ and Pil- phenotypes . In contrast, only one orientation of the invertible element, corresponding to the Pil- phenotype, was observed in strain 149 cells cultured on agar . A second clinical isolate, strain 2-7, which expresses type 1 pili on agar was also examined; the invertible element was found in both the Pil+ and Pil- orientations during growth of this strain on agar as well as in broth . The introduction of the fim gene cluster from strain J96 on a multicopy plasmid into agar-negative strain 149 resulted in the production of both J96 and 149 pili during growth on agar . Experiments with subclones of the J96 genes indicated that the presence of an intact fimB gene allowed strain 149 pili to be produced on agar . Differences in pilus production between agar and broth cultures appear to be the result of differential transcription of fimB and fimE under the two growth conditions . In contrast, the pattern of expression of these genes in agar phase-variable strain 2-7 did not differ between broth- and agar-grown cells. J Bacteriol, 1992 Feb, 174(3), 970 - 9 Constitutive sensory transduction mutations in the Bordetella pertussis bvgS gene; Miller JF et al.; The products of the bvgAS locus coordinately regulate expression of the Bordetella pertussis virulence regulon in response to environmental signals . Transcription of bvgAS-activated genes is nearly eliminated by several modulating conditions, including the presence of sulfate anion or nicotinic acid and growth at low temperature . We have isolated spontaneous mutations that result in the constitutive synthesis of multiple bvg-regulated loci . Several of these mutations have been analyzed and were found to result from single-nucleotide substitutions within bvgS, in a region encoding a 161-amino-acid segment which links the transmembrane sequence with cytoplasmic domains that appear to be involved in signaling events . The effect of signal transduction mutations in Escherichia coli was determined by measuring the expression of an fhaB-lacZYA transcriptional fusion, and that in B . pertussis was determined by measuring expression of both fhaB-cat and ptxA3201-cat fusions . The constitutive mutations have little effect on fhaB-cat or fhaB-lacZYA expression in the absence of modulating signals but result in a nearly complete insensitivity to MgSO4, nicotinic acid, or growth at low temperature . Furthermore, insertion and deletion mutations in bvgS sequences encoding the periplasmic domain eliminate activity of the wild-type product, whereas constitutive mutants remain active . In B . pertussis cultures grown in Stainer-Scholte broth, expression of ptxA3201-cat differed from that of fhaB-cat in several respects . In combination with a wild-type bvgS allele, ptxA3201-cat expression required the addition of heptakis-(2,6-O-dimethyl)-beta-cyclodextrin, and this requirement was eliminated by the presence of the constitutive mutations. Vet Pathol, 1992 Jan, 29(1), 68 - 78 Acute airsacculitis in untreated and cyclophosphamide-pretreated broiler chickens inoculated with Escherichia coli or Escherichia coli cell-free culture filtrate; DeRosa M et al.; 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. Acta Microbiol Hung, 1992, 39(3-4), 223 - 8 The mouse ligated intestinal loop assay for the studies on enteroinvasive Escherichia coli; Sakaguchi T et al.; Enteroinvasive Escherichia coli exhibited a positive reaction in the mouse intestinal loop assay except for noninvasive mutant strains . These mean values of fluid weight per gut length of mouse loops inoculated with enteroinvasive E . coli were significantly higher than that given by brain heart infusion broth . Oedema and swelling in all positive loops, increased bacterial cell numbers within intestinal loops were observed. Biochim Biophys Acta, 1991 Dec 2, 1129(1), 1 - 12 Genetic regulation of cardiolipin synthase in Escherichia coli; Heber S et al.; Recombinant DNA and genetic techniques were used to construct Escherichia coli strains SOH92 {phi(cls-lacZ+)} and SOH93 {phi(cls-'lacZ)hyb} . beta-Galactosidase (116 kDa) synthesized by strain SOH92 was primarily present in the particulate fraction . Strain SOH92 produced about 20-fold more beta-galactosidase activity than strain SOH93 . Expression of clsphilacZ in both SOH92 and SOH93 was influenced by the terminal electron acceptor (increasing in the order oxygen, nitrate, fumarate) when the cells were cultured in minimal medium with glycerol as the sole carbon source . As strains SOH92 and SOH93 progressed from early to late log growth phase under aerobic conditions in LB broth, clsphilacZ expression increased about 2.5-fold . Fusion strains containing a pss-1 allele had an increased cardiolipin (CL) level, but no corresponding increase in clsphilacZ expression was observed . A cls::Tn10dTet null mutation was introduced into SOH92 and SOH93 . The strains produced less CL, but no corresponding changes in clsphilacZ expression were observed . A high copy number plasmid bearing the cls gene had no effect on clsphilacZ expression . Taken together, these results indicate that cls is not subject to autogenous regulation. J Bacteriol, 1991 Sep, 173(18), 5631 - 8 Regulation of ompF porin expression by salicylate in Escherichia coli; Rosner JL et al.; The expression of ompF, the gene encoding a major outer membrane protein of Escherichia coli, is regulated by various environmental factors . The mechanism by which salicylate (SAL) drastically reduces ompF expression was studied here by means of lacZ fusions to ompF, ompC, and micF, by sodium dodecyl sulfate-gel electrophoresis of outer membrane proteins, and by measurements of outer membrane permeability . Growth of E . coli in LB broth containing SAL strongly reduced ompF-specific translation of an ompF-lacZ fusion . The extent of this reduction varied with the SAL concentration from 64% at 0.5 mM to 95% at 2 mM and greater than 99% at 10 mM . ompF-lacZ transcription was not affected by SAL, whereas ompC-lacZ transcription was elevated by 70% . Since the micF transcript is antisense to a portion of the ompF transcript and is capable of decreasing the translation of ompF, the effect of SAL on micF transcription was measured in a micF-lacZ fusion strain . SAL-grown cells contained three- to fourfold more micF transcript during the logarithmic phase of growth than did the control cultures . However, micF was not absolutely required for the response to SAL . In micF-deleted strains, the effects of SAL on ompF translation, on OmpF in the outer membrane, and on outer membrane permeability were diminished but still evident . The effect of SAL on ompF expression was independent of the osmolarity of the medium and was epistatic to certain ompB regulatory mutations: the high levels of ompF expression found in envZ3 and ompR472 strains were greatly reduced by growth in SAL . Unexpectedly, the OmpC- phenotypes of these mutants were suppressed by SAL . Thus, growth in SAL severely decreases the translation of ompF while enhancing the transcription of micF and ompC . In this respect, SAL-grown cells resemble certain marA and tolC mutants that have high levels of micF and ompC transcripts and low levels of OmpF. J Gen Microbiol, 1991 May, 137 ( Pt 5), 1163 - 9 Determination of effector molecules in L-arabinose-induced bulge formation and lysis of Escherichia coli IFO 3545; Tanaka T et al.; L-Ribulose 5-phosphate (L-Ru5P) was identified as the primary effector molecule of L-arabinose-induced bulge formation in Escherichia coli IFO 3545 observed in nutrient broth with 5% (w/v) sodium chloride . Hyperinduction of L-arabinose isomerase was due to exogenous sodium chloride and the resulting alteration in the balance of the L-arabinose-metabolizing enzymes resulted in accumulation of L-Ru5P . L-Ru5P induced the lysis of an L-arabinose-negative, L-Ru5P 4-epimerase-less mutant, ara-207, even when directly added to the medium but was not active against the wild-type strain . Some L-arabinose-utilizing (L-arabinose-resistant) revertants of ara-207 were still sensitive to L-Ru5P, indicating the involvement of another mutation in L-Ru5P-sensitivity other than genetic lack of L-Ru5P 4-epimerase . Among the various pentose phosphate esters tested, only L-Ru5P could induce lysis of ara-207 . The lytic activity of L-Ru5P was attributed to its effect on bacterial sugar nucleotide metabolism which caused secondary accumulation of uridine 5'-diphosphate galactose (UDPGal), which provoked lysis induction. Appl Microbiol Biotechnol, 1991 May, 35(2), 185 - 8 Structured model for cell growth and enzyme production by recombinant Escherichia coli; Korte G et al.; A structured cell model has been developed to describe the cultivation of recombinant Escherichia coli K12 with multicopy plasmid under the control of a lambda PR-promoter and a temperature-sensitive lambda cI 857 repressor . The model, based on measurements of a batch culture in a stirred tank reactor, allows statements to be made on the time variation of intracellular processes . Based on cell regulation, the substrate transfer into the cell was considered to be the rate-limiting step for substrate utilization . The model describes substrate utilization, cell growth, and product formation by means of a system of time-dependent, coupled, and partly non-linear differential equations . The solution of these equations allows calculation of the time variation of the concentrations of substrates (glucose and amino acids), dissolved oxygen and cells in the broth as a function of time and the process parameters. J Bacteriol, 1991 Apr, 173(8), 2473 - 80 A novel L-serine deaminase activity in Escherichia coli K-12; Su H et al.; We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2 . A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth . We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely . Both mutations lie near 60.5 min on the E . coli genetic map . The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation. Arch Invest Med (Mex), 1991 Apr-Jun, 22(2), 217 - 22 Frequency of identification of cytotoxigenic strains of Escherichia coli in cases of diarrhea from rural and urban communities; Parra-Maldonado NR et al.; It has been suggested that strains of Escherichia coli producing Vero-Toxin (VTEC) may cause diarrhea or hemorrhagic colitis; however, there are not enough studies to support this hypothesis . We studied the frequency of isolation of VTEC strains in patients with acute diarrhea from rural and urban communities . A total of 1430 strains were analyzed, 361 coming from 118 patients from the rural community (Cadereyta, Qro.) and 1069 from the urban district (D.F.); 95 of these patients were asymptomatic, 213 suffered from watery diarrhea and 43 had bloody diarrhea . For production of toxins, strains were grown in tryptic soy broth for 24h and the culture supernatant was inoculated on HeLa cells; strains were considered cytotoxic when they caused lysis in at least 50% of the cells . In the rural community, VTEC strains were isolated in 20% of the asymptomatics, in 45% of the watery diarrhea patients and in 76% of patients with bloody diarrhea . Frequency of isolation was significantly higher in patients with diarrhea than in asymptomatics (P less than 0.05) . The relative risk to present watery diarrhea was 3 and to present bloody diarrhea was 12 . In the urban district, VTEC strains were isolated in 13, 7.9 and 4.5% from asymptomatics, watery diarrhea and bloody diarrhea patients, respectively; the relative risk for diarrhea was 1 . Colonization by VTEC strains is significantly higher in patients from the rural community and these infected patients have an important risk to develop diarrhea. J Biol Chem, 1991 Mar 15, 266(8), 5055 - 61 A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli; Maki H et al.; Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene . To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit . The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures . The mutator effect of dnaE173 mutation is dominant over the wild type allele . These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule . DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio . We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities . However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase . Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme . It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication. Vet Microbiol, 1991 Feb 15, 26(4), 393 - 400 The importance of enterotoxigenic Escherichia coli containing the 987P antigen in causing neonatal colibacillosis in piglets in Indonesia; Supar et al.; A survey was undertaken in piggeries in the Bogor and Jakarta Capital Territory areas to identify the antigens associated with enterotoxigenic Escherichia coli (E . coli) . Rectal swab samples were collected from 65 normal piglets and from 858 with diarrhoea . Fimbrial and O-antigens were determined by agglutination tests . The 987P antigen was only associated with non-haemolygic E . coli in colonies grown for 3-10 days in tryptic soy broth . Organisms which possessed 987P antigen were isolated from 56.4% of piglets with diarrhoea and from 10.8% of normal piglets . Most cases of diarrhoea that were associated with E . coli 987P occurred within the first 3 weeks of life . Multiple infections occurred in 13.4% of cases and were associated with E . coli K88 in eight cases (1.7%) K99 in 26 cases (5.4%) and F41 in 31 cases (6.4%) . Of 959 isolates of E . coli 987P, 80.7% were O-group 20, 13% were O-group 9 and 0.5% were O-group 141 with 5.7% being non-typable . Heat stable toxin was produced by all five E . coli 987P isolates tested. Vet Microbiol, 1991 Feb 15, 26(4), 359 - 66 Superoxide dismutases of virulent and avirulent strains of Brucella abortus; Sriranganathan N et al.; Extracts of Brucella abortus strains 2308,RB51,45/20 and ST 19 had no significant differences in superoxide dismutase (SOD) activity as measured by the epinephrine assay . These B . abortus strains represent smooth, intermediate and rough colony forms . SOD activity was inhibited 60 to 75% by 2 mM KCN and suggests the presence of Cu/Zn SOD . The SOD activities were similar when the strains were grown in trypticase soy broth containing either 0.5% glucose or erythritol . There were two distinct SOD activity bands in native polyacrylamide gel electrophoresis with identical mobilities for each of the strains . When the native gel was stained for SOD activities in the presence of 2 mM KCN, the SOD band that co-migrated with the bovine erythrocyte Cu/Zn SOD activity disappeared . The band of SOD activity that migrated similar to E . coli iron SOD activity was unaffected by KCN . There were no significant differences in either the total SOD or Cu/Zn SOD activities among the strains . As the Brucella strains represent ranges of virulence, it is difficult to associate any primary role for SOD as a virulence factor. Folia Microbiol (Praha), 1991, 36(4), 395 - 400 Partial purification of alpha-hemolysin in Escherichia coli; Nistiar F et al.; The present paper describes isolation and purification of alpha-hemolysin of Escherichia coli . The optimum production medium was found to be the Todd-Hewitt broth . Out of thirteen fractions obtained after separation on Sephadex G-200, two fractions possessed the highest relative specific activity. J Appl Bacteriol, 1991 Jan, 70(1), 59 - 65 Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer; Raja N et al.; Induction of acid resistance (habituation) in Escherichia coli at pH 5.0 took ca 5 min in broth at 37 degrees C and 30-60 min in minimal medium . Induction occurred at a range of pH values from 4.0 to 6.0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid . Acid resistance was long-lasting; organisms grown at pH 5.0 retained most of their resistance after 2 h growth at pH 7.0 . Organisms grown at pH 5.0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7.0 . DNA repair-deficient strains carrying recA, uvrA or polA1 mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5.0 . Organisms grown at pH 5.0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7.0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated . Induction of acid resistance at pH 5.0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure. Antimicrob Agents Chemother, 1990 Dec, 34(12), 2402 - 6 Potentiation by salicylate and salicyl alcohol of cadmium toxicity and accumulation in Escherichia coli; Rosner JL et al.; The toxicity of Cd2+ in Escherichia coli K-12 was potentiated by salicylate and several related compounds . The efficiency of plating on Luria broth plates was reduced by more than 10(5)-fold when 10 mM salicylate and 200 microM CdCl2 were present simultaneously but was unaffected when either compound was present by itself . Synergistic effects were found at pH 7.4 with certain other weak acids (acetyl salicylate {aspirin}, benzoate, and cinnamate) and with a nonacidic salicylate analog, salicyl alcohol, but not with acetate or p-hydroxy benzoate . Thus, the synergism with Cd2+ is determined by the structure of the compounds and not merely by their acidity . The kinetics of 109Cd2+ uptake by cells grown and assayed in broth indicated the presence of two uptake systems with Kms of 1 and 52 microM Cd2+ and Vmaxs of 0.059 and 1.5 mumol of Cd2+ per min per g of cells, respectively . The kinetics of uptake for cells grown and assayed with 20 mM salicyl alcohol showed 2.5-fold increases in the Vmaxs of both systems but no change in the Kms . Salicylate-grown cells also exhibited increased rates of 109Cd2+ uptake by both systems . Thus, enhanced uptake of Cd2+ may be responsible for the potentiation of Cd2+ toxicity by salicylate and salicyl alcohol. J Bacteriol, 1990 Nov, 172(11), 6411 - 8 Isolation and characterization of mutants with lesions affecting pellicle formation and erythrocyte agglutination by type 1 piliated Escherichia coli; Harris SL et al.; The product of the pilE (also called fimH) gene is a minor component of type 1 pili in Escherichia coli . Mutants that have insertions in the pilE gene are fully piliated but unable to bind to and agglutinate guinea pig erythrocytes, a characteristic of wild-type type 1 piliated E . coli . In this paper we describe the isolation of 48 mutants with point lesions that map to the pilE gene . Such mutants were isolated by using mutT mutagenesis and an enrichment procedure devised to favor the growth of individuals that could form a pellicle in static broth containing alpha-methylmannoside, an inhibitor of erythrocyte binding and pellicle formation . Results indicated that the enrichment favored mutants expressing pilE gene products that were defective in mediating erythrocyte binding . Characterization of 12 of the mutants in greater detail revealed that certain lesions affected pilus number and length . In addition, a mutant that was temperature sensitive for erythrocyte binding was isolated and used to provide evidence that pellicle formation relies on the intercellular interaction of pilE gene products . Our results suggest a molecular explanation for the old and paradoxical observations connecting pellicle formation and erythrocyte agglutination by type 1 piliated E . coli. Biochemistry, 1990 Oct 16, 29(41), 9737 - 45 Secretion of active kringle-2-serine protease in Escherichia coli; Obukowicz MG et al.; Active human tissue plasminogen activator variant kringle-2-serine protease (K2 + SP domains; referred to as MB1004) was synthesized as a secreted protein in Escherichia coli, isolated, and characterized . MB1004 is a relatively large and complex protein, approximately 38 kDa in size and containing nine disulfide bonds . MB1004 without a pro region was secreted into the periplasm of E . coli by fusing the protein to the PhoA leader peptide expressed from the tac promoter . Approximately 1% (20 micrograms/L broth) of the secreted MB1004 was purified from E . coli homogenates as a soluble, active enzyme by using a combination of lysine and Erythrina inhibitor affinity chromatography . Purified MB1004 was monomeric and single-chain, and the N-terminus was identical with the predicted amino acid sequence . The specific activity of purified MB1004 from E . coli was compared against the equivalent recombinant material purified from mammalian cells that was naturally glycosylated (MB1004G) or deglycosylated after treatment with N-glycanase (MB1004N) . Results from four different in vitro assays showed that MB1004 and MB1004N had similar activities . Both exhibited 4-12-fold higher specific activity than MB1004G in plasminogen activation assays . These results suggest that an inaccurate picture of specific activity can be obtained if the effects of glycosylation are not considered . By utilization of secretion in E . coli, nonglycosylated MB1004 was purified without in vitro refolding and was shown to be suitable for structure-function studies. Can J Microbiol, 1990 Oct, 36(10), 725 - 7 Optimization of 7 alpha-hydroxysteroid dehydrogenase production by Escherichia coli 080; Prabha V et al.; 7 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.159) production by Escherichia coli strain 080 was highest when the organism was grown in brain heart infusion broth at pH 6.5 for 72-96 h with shaking at 37 degrees C . The oxygen consumption rate had a strong effect on the production of this constitutive enzyme . Glucose and lactose at 0.2-0.4%, detergents, and ethylenediaminetetra-acetic acid were found to increase the enzyme production. J Bacteriol, 1990 Sep, 172(9), 5103 - 11 Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen; Schmoll T et al.; S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules . In order to determine the influence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme beta-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae . By using a plasmid which carries an R6K origin, the sfaA-lac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E . coli strain 536WT . The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain 536WT . With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth . The growth rate had a strong influence on Sfa expression under aerobic but not under anaerobic conditions . Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature. J Bacteriol, 1990 Aug, 172(8), 4143 - 50 micF RNA in ompB mutants of Escherichia coli: different pathways regulate micF RNA levels in response to osmolarity and temperature change; Coyer J et al.; The repressor RNA, micF RNA, is regulated by temperature, osmolarity, and other stress conditions during growth of Escherichia coli . Northern (RNA) blot analyses showed that levels of micF RNA differ widely in various ompB mutant strains when cells are grown at 24 degrees C in LB broth . For example, relative to the parental strain MC4100, the ompR101 mutant strain (which contains no functional OmpR) had about a 10-fold reduction in micF RNA, whereas the envZ11 strain showed about a 5-fold increase . At 37 degrees C, however, micF RNA levels in the ompR101 and envZ11 strains and other ompB mutants differed by less than two-fold compared with the level in strain MC4100, thus indicating that a factor(s) independent of the ompB locus regulates micF RNA expression with temperature increase and that there is an additional control mechanism(s) which maintains the levels of micF RNA in these mutants close to that of the wild type during growth at high temperatures . In a plasmid strain containing the micF gene but without the upstream OmpR-binding site, steady-state levels of micF RNA increased with temperature increase but did not change with osmolarity increase . This showed that osmolal regulation but not temperature regulation of micF depends on these upstream sequences and suggested that while osmolal regulation of the micF gene depends on OmpR, thermal regulation does not. Infect Immun, 1990 Jun, 58(6), 1951 - 8 Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes; Boehm DF et al.; The calcium requirement for hemolytic activity of Escherichia coli hemolysin was investigated by using hemolytic assays and immunoblotting of toxin-treated erythrocytes . The hemolytic activity of cell culture supernatants obtained during growth of E . coli in Luria-Bertani (LB) broth or calcium-free LB broth was calcium dependent . The hemolytic activity of culture supernatants obtained during growth in LB broth supplemented with calcium was calcium independent . Osmotic protection experiments using Dextran 4 to prevent cell lysis indicated that calcium was required for the binding of hemolysin to erythrocytes at both 4 and 37 degrees C . The binding efficiency at 4 degrees C was 50% of that occurring at 37 degrees C . The calcium-dependent binding was confirmed by immunoblotting saline-washed, toxin-treated erythrocytes with a monoclonal antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins . Bound hemolysin increased the calcium permeability of the cell membranes as evidenced by calcium-induced membrane protein alterations . The alterations in membrane proteins did not directly cause lysis of the cells . The results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells. Proc Natl Acad Sci U S A, 1990 May, 87(10), 3700 - 4 Base changes at position 792 of Escherichia coli 16S rRNA affect assembly of 70S ribosomes; Santer M et al.; To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis . Each base change conferred a unique phenotype on the cells . Cells containing plasmid pKK3535 with G792 or T792 showed no difference in generation time in LB broth containing ampicillin, whereas cells with C792 exhibited a 20% increase in generation time in this medium . To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomycin-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin . Cells containing G792 or C792 showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in 35S assimilation into proteins . Cells with T792 did not grow in spectinomycin-containing medium . Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA . It appeared that C792-containing 30S ribosomes had lost the ability to bind initiation factor 3. Gene, 1990 Apr 30, 89(1), 133 - 7 Requirement of integration host factor (IHF) for growth of Escherichia coli deficient in HU protein; Kano Y et al.; Escherichia coli mutants deficient in histone-like protein, HU, and integration host factor (IHF) were constructed and their growth characteristics were examined . Mutants deficient in both HU and IHF grew slowly and were filamentous at both 30 degrees C and 37 degrees C . These mutants scarcely grew in LB broth at 42 degrees C . They formed minute colonies on LB plates at 42 degrees C and no colonies at 46 degrees C, indicating temperature-sensitive (ts) growth . On the contrary, mutants deficient in either HU or IHF were not ts for growth . These results indicate that IHF compensates for the absence of HU and permits normal cell growth; this suggests functional similarity between HU and IHF. Vet Microbiol, 1990 Feb, 21(4), 353 - 62 Protection of weaned rabbits against experimental Escherichia coli O103 intestinal infection by oral formalin-killed vaccine; Camguilhem R et al.; In an attempt to vaccinate young weaned rabbits against life-threatening enterocolitis caused by Escherichia coli of the O103 serogroup, 32 New Zealand male rabbits were divided into three groups . One group remained unvaccinated as a control (Group C), and each of the other groups received one of two types of vaccine prepared with E . coli strain O103/10 cultured either in trypticase-soy broth (Group A) or in Minca agar (Group B) . Bacteria were killed by formalin and administered per os for 10 consecutive days after weaning at a daily dose of 4 X 10(9) organisms . Six days after the last administration, all the animals were challenged with 1 X 10(4) virulent E . coli O103/10 and the experimental infection was monitored for 26 days . All rabbits in Group A were protected from symptoms of disease and remained alive, whereas two rabbits in Group B developed clinical signs and one died . Protection did not correlate with local or general responses to lipopolysaccharide (LPS) O103, as judged by measurement of anti-LPS O103 IgA in faeces or serum, or by serum agglutinating antibodies . Numbers of E . coli and E . coli O103 were significantly lower in vaccinated animals of Group A as compared with animals of the control group . The differences between both vaccine regimens may be partially explained by a different expression of the adhesins of strain O103/10, depending on the medium used to prepare the vaccine. J Bacteriol, 1990 Jan, 172(1), 94 - 101 Cell volume increase in Escherichia coli after shifts to richer media; Kubitschek HE; Synchronous cultures of Escherichia coli 15-THU and WP2s, which were selected by velocity sedimentation from exponential-phase cultures growing in an acetate-minimal salts medium, were shifted to richer media at various times during the cell cycle by the addition of glucose or nutrient broth . Cell numbers and mean cell volumes were measured electronically . The duration of the division cycle of the shifted generation was not altered significantly by the addition of either nutrient . Growth rates, measured as rates of cell volume increase, were constant throughout the cycle in unshifted acetate control cultures . When glucose was added, growth rates also remained unchanged during the remainder of the cell cycle and then increased abruptly at or after cell division . When nutrient broth was added, growth rates remained unchanged from periods of 0.2 to 0.4 generations and then increased abruptly to their final values . In all cases, the cell volume increase was linear both before and after the growth rate transition . The strongest support for a linear cell volume increase during the cell cycle of E . coli in slowly growing acetate cultures, however, was obtained in unshifted cultures, in complete agreement with earlier observations of cell volumes at much more rapid growth rates . Although cell growth and division are under the control of the synthesizing machinery in the cell responsible for RNA and protein synthesis, the results indicate that growth is also regulated by membrane-associated transport systems. J Parasitol, 1989 Oct, 75(5), 735 - 9 Radiolabeling of infective third-stage larvae of Strongyloides stercoralis by feeding {75 Se}selenomethionine-labeled Escherichia coli to first- and second-stage larvae; Aikens LM et al.; A technique is described for radiolabeling Strongyloides stercoralis larvae with {75Se}selenomethionine . Cultures of an auxotrophic methionine-dependent stain of Escherichia coli were grown in a medium containing Dulbecco's modified Eagle's medium supplemented with 5% nutrient broth, amino acids, and {75Se}selenomethionine . When the 75Se-labeled bacterial populations were in the stationary phase of growth, cultures were harvested and the bacteria dispersed on agar plates to serve as food for S . stercoralis larvae . Use of nondividing bacteria is important for successful labeling because the isotope is not diluted by cell division and death of larvae attributable to overgrowth by bacteria is prevented . First-stage S . stercoralis larvae were recovered from feces of infected dogs and reared in humid air at 30 C on agar plates seeded with bacteria . After 7 days, infective third-stage larvae were harvested . The mean specific activity of 6 different batches of larvae ranged from 75 to 330 counts per min/larva with 91.8 +/- 9.5% of the population labeled sufficiently to produce an autoradiographic focus during a practicable, 6-wk period of exposure . Labeled infective larvae penetrated the skin of 10-day-old puppies and migrated to the small intestine, where the developed to adulthood. Zentralbl Hyg Umweltmed, 1989 May, 188(1-2), 35 - 46 {Use of UV rays for the disinfection of water . III . UV sensitivity of Legionella pneumophila of different ages in cold and warm drinking water}; Martiny H et al.; In drinking water the sensitivity of L . pneumophila serogroup 1 type Philadelphia and L . pneumophila serogroup 5 type Dallas were studied with a flowthrough u.v . light treatment apparatus . By washing purified cells from broth cultures were used as inoculum directly ( = young cultures) or kept 2-3 weeks in drinking water in the dark ( = old cultures) . A decrease of 99,9999% was found after u.v . treatment by 19 mWs/cm2 for young cultures and by 15 mWs/cm2 for old cultures . Reductions of 99,9999% were obtained by 16 mWs/cm2 in cold drinking water (13-16 degrees C) and by 13 mWs/cm2 in warm drinking water (45-47 degrees C) . L . pneumophila serogroup 1 and L . pneumophila serogroup 5 show a very similar susceptibility to u.v.-irradiation . Reductions of 99,9999% were obtained by 14 mWs/cm2 and 15 mWs/cm2, respectively . Thus L . pneumophila-suspensions proved to be more sensitive to u.v.-irradiation than E . coli oder E . faecium in earlier experiments. Proc Natl Acad Sci U S A, 1989 Apr, 86(7), 2172 - 5 One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution; Chung CT et al.; We have developed a simple, one-step procedure for the preparation of competent Escherichia coli that uses a transformation and storage solution {TSS; 1 x TSS is LB broth containing 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethyl sulfoxide, and 50 mM Mg2+ at pH 6.5} . Cells are mixed with an equal volume of ice-cold 2 x TSS and are immediately ready for use . Genetic transformation is equally simple: plasmid DNA is added and the cells are incubated for 5-60 min at 4 degrees C . A heat pulse is not necessary and the incubation time at 4 degrees C is not crucial, so there are no critical timing steps in the transformation procedure . Transformed bacteria are grown and selected by standard methods . Thus, this procedure eliminates the centrifugation, washing, and long-term incubation steps of current methods . Although cells taken early in the growth cycle (OD600 0.3-0.4) yield the highest transformation efficiencies (10(7)-10(8) transformants per micrograms of plasmid DNA), cells harvested at other stages in the growth cycle (including stationary phase) are capable of undergoing transformation (10(5)-10(7) transformants per micrograms of DNA) . For long-term storage of competent cells, bacteria can be frozen in TSS without addition of other components . Our procedure represents a simple and convenient method for the preparation, transformation, and storage of competent bacterial cells. J Clin Microbiol, 1989 Feb, 27(2), 339 - 40 Evaluation of a reversed passive latex agglutination test for detection of Escherichia coli heat-labile toxin in culture supernatants; Scotland SM et al.; One hundred strains of Escherichia coli were tested for the production of the heat-labile enterotoxin by the Y1 adrenal cell test and a commercially available reversed passive latex agglutination test . The strains were grown in Casamino Acids-yeast extract broth, and filtered culture supernatants were tested for the presence of heat-labile enterotoxin . There was perfect correlation between the Y1 test and the reversed passive latex agglutination test, and the latter was simple to perform and completed within 48 h. Infect Immun, 1988 Sep, 56(9), 2430 - 6 Iron regulation of the cloned diphtheria toxin promoter in Escherichia coli; Tai SP et al.; Regulation of the diphtheria toxin promoter by iron was studied in Escherichia coli by using a galK transcriptional fusion . A fragment of the toxin (tox) operon containing the regulatory region was cloned from corynephage beta into a galK transcription vector such that expression of galK activity was controlled by the tox promoter . When E . coli N100 (a galK mutant) harboring this tox-galK fusion plasmid was grown in Luria broth, the specific activity of galactokinase remained constant throughout the exponential phase of growth . When bacteria were shifted from such high-iron medium into low-iron Luria broth, the specific activity of galactokinase increased rapidly, but induction of galactokinase was prevented by the addition of iron to the medium . Measurement of tox-specific mRNA by dot blot hybridization showed that this regulation occurred at the level of transcription . When the plasmid containing the tox-galK fusion was introduced into a fur mutant of E . coli, expression of galK was maximal in both high-iron and low-iron media; but repressibility of galK by iron in this strain was restored by complementation with the fur+ allele . The tox promoter has significant homology with the consensus sequence for other iron-regulated promoters of E . coli that are controlled by fur . These data indicate that the product of the fur gene can function in E . coli as an iron-dependent repressor for the tox promoter from corynephage beta. Appl Environ Microbiol, 1988 Aug, 54(8), 1901 - 6 Metabolic processes involved in repair of Escherichia coli cells damaged by exposure to acid mine water; Wortman AT et al.; Escherichia coli was stressed by exposure to filter-sterilized acid mine water . Synthetic processes required for repair of sublethally injured survivors were studied by the addition of specific metabolic inhibitors to a resuscitation broth . Inhibitors of protein, RNA, DNA, lipid, and peptidoglycan synthesis as well as uncouplers and inhibitors of electron transport and ATPase activity were used . Acid mine water injury was severe, causing damage to the outer and cytoplasmic membranes . Repair of sublethally injured cells required protein, RNA, and lipid synthesis as well as a proton motive force. Appl Environ Microbiol, 1988 Jun, 54(6), 1511 - 5 Cloning of the cellulase gene from Ruminococcus albus and its expression in Escherichia coli; Ohmiya K et al.; The gene for cellulase from Ruminococcus albus F-40 was cloned in Escherichia coli HB101 with pBR322 . A 3.4-kilobase-pair HindIII fragment encoding cellulase hybridized with the chromosomal DNA of R . albus . The Ouchterlony double-fusion test gave a single precipitation line between the cloned enzyme and the cellulase from R . albus . The size of the cloned fragment was reduced by using HindIII and EcoRI . The resulting active fragment had a size of 1.9 kilobase pairs; and the restriction sites EcoRI, BamHI, PvuII, EcoRI, PvuII, and HindIII, in that order, were ligated into pUC19 at the EcoRI and HindIII sites (pURA1) . Cellulase production by E . coli JM103(pURA1) in Luria-Bertani broth was remarkably enhanced, up to approximately 80 times, by controlling the pH at 6.5 and by reducing the concentration of NaCl in the broth to 80 mM. J Assoc Off Anal Chem, 1988 May-Jun, 71(3), 589 - 602 Fluorogenic assay for rapid detection of Escherichia coli in chilled and frozen foods: collaborative study; Moberg LJ et al.; A collaborative study was conducted to compare a proposed LST-MUG method with the AOAC official method for Escherichia coli detection . E . coli produces an enzyme, beta-glucuronidase, which cleaves the substrate, 4-methyl-umbelliferyl-beta-D-glucuronide (MUG), to yield a fluorescent end product . Incorporation of the MUG substrate into lauryl tryptose broth (LST) enables a rapid quantitative method for screening E . coli, which is detected by fluorescence of the medium under longwave UV light . In this collaborative study, 5 food samples, 2 frozen (entree sauce/gravy and dairy topping) and 3 chilled (hamburger, pork sausage, and cheese), were tested for E . coli detection by 17 collaborating laboratories . Results indicate that the LST-MUG method is equal to or better than the current AOAC method for detecting E . coli . The LST-MUG method has been adopted official first action. J Bacteriol, 1988 Apr, 170(4), 1533 - 40 Division behavior and shape changes in isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli during temperature shift experiments; Taschner PE et al.; Isogenic ftsZ, ftsQ, ftsA, pbpB, and ftsE cell division mutants of Escherichia coli were compared with their parent strain in temperature shift experiments . To improve detection of phenotypic differences in division behavior and cell shape, the strains were grown in glucose-minimal medium with a decreased osmolality (about 100 mosM) . Already at the premissive temperature, all mutants, particularly the pbpB and ftsQ mutants, showed an increased average cell length and cell mass . The pbpB and ftsQ mutants also exhibited a prolonged duration of the constriction period . All strains, except ftsZ, continued to initiate new constrictions at 42 degrees C, suggesting the involvement of FtsZ in an early step of the constriction process . The new constrictions were blunt in ftsQ and more pronounced in ftsA and pbpB filaments, which also had elongated median constrictions . Whereas the latter strains showed a slow recovery of cell division after a shift back to the permissive temperature, ftsZ and ftsQ filaments recovered quickly . Recovery of filaments occurred in all strains by the separation of newborn cells with an average length of two times LO, the length of newborn cells at the permissive temperature . The increased size of the newborn cells could indicate that the cell division machinery recovers too slowly to create normal-sized cells . Our results indicate a phenotypic resemblance between ftsA and pbpB mutants and suggest that the cell division gene products function in the order FtsZ-FtsQ-FtsA, PBP3 . The ftsE mutant continued to constrict and divide at 42 degrees C, forming short filaments, which recovered quickly after a shift back to the permissive temperature . After prolonged growth at 42 degree C, chains of cells, which eventually swelled up, were formed . Although the ftsE mutant produced filaments in broth medium at the restrictive temperature, it cannot be considered a cell division mutant under the presently applied conditions. J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 983 - 95 Morphological description of surface structures on strain B41 of bovine enterotoxigenic Escherichia coli bearing both K99 and F41 antigens; Duchet-Suchaux M et al.; In order to describe morphologically the structures on the cell surface of bovine enterotoxigenic Escherichia coli, variants of reference strain B41 (K99+F41+) either negative for K99 and positive for F41 antigens (variants B41A, B41*C), or phenotypically negative for both antigens (variants B41B1, B41B2, B41*CB), and a transconjugant harbouring the K99 plasmid and expressing the K99 adhesin {transconjugant B41 x H510a:H510(2)} were examined by transmission electron microscopy using negative staining . Several negative staining procedures were tested for strain B41 and variant B41A: direct harvesting of strains into ammonium molybdate (2%, w/v), with bacitracin (50 micrograms ml-1) as wetting agent, gave the best results . Three morphologically distinct structures on the cell surface could be identified in cultures grown on Minca medium . Firstly, thin, filamentous, flexible fibrillar structures, presenting a helical structure and a mean diameter of approximately 3 nm, were recognized as K99 fimbriae, since they were present on strain B41 and on transconjugant H510(2), but not on K99-negative variants nor on the recipient strain H510a . Secondly, coil-like structures with a diameter of about 17-20 nm were observed on strain B41 and on variants B41A and B41*C . These structures appeared to consist of two or more curled filaments (diameter 3 nm) joined to coil on themselves into dense spirals . They were very rare in variants B41B1 and B41B2 and were absent on variant B41*CB and on a transconjugant B41* x B41*CB, which had re-acquired the K99 plasmid and which again exhibited K99 fimbriae . Strains B41 and variant B41A gown at 37 degrees C for 24 h on sheep-blood agar exhibited coiled structures like those seen on Minca medium . In contrast, after growth at 18 degrees C for 48 h (which inhibits the synthesis of F41 antigen), coiled structures were no longer expressed on the cell surface of strain B41 and variants B41A and B41*C . Thus the presence of coiled structures correlated with the expression of F41 antigen in strains and variants, which suggests that F41 had a coiled morphology . Finally, straight fimbriae (diameter 6.5-7 nm) were observed on the cell surface of every strain and variant . Their expression on the cell surface was enhanced by several subcultures in th e static broth, and it was inhibited by subculture on agar, but not by culture at 18 degrees C after serial subcultures in static broth . These facts indicated that the straight fimbriae could be common fimbriae, and excluded their being F41 structures. Avian Dis, 1988 Jan-Mar, 32(1), 103 - 7 Effect of oral Escherichia coli inoculation on performance of young turkeys; Schmidt GP et al.; A strain of Escherichia coli isolated from the yolk sac of stunted turkey poults was administered orally to day-old large white poults . Poults were inoculated with either 0.1 ml of sterile broth or 0.1 ml of a 10(-2) dilution of a 24-hr E . coli culture containing 3.4 x 10(8) viable bacteria per ml . Two levels of dietary protein (28 or 22%) were fed from 1 day to 3 weeks of age . Following E . coli inoculation of 3.4 x 10(5) viable bacteria at day one, body weight gain and feed consumption from 0 to 3 weeks of age were numerically increased 4.5 and 2.1%, respectively, and feed efficiency was significantly increased 2.4% . E . coli had a greater effect on performance of poults fed the 28% protein diet than on poults fed the 22% protein diet . Metabolism studies, conducted from 7 to 10 and from 17 to 20 days postinoculation, showed no significant changes in the measurements of nutrient utilization due to E . coli other than a 17% increase in nitrogen retention from 17 to 20 days by those poults fed the 28% protein diet. Med Prog Technol, 1988, 14(1), 25 - 33 Impedancimetric bacterial detection: theoretical and experimental aspects; Felice CJ et al.; By means of the bipolar impedance technique, we detected bacterial growth in an inoculated broth as its time course absolute impedance . From it, the impedance change relative to sterile medium was obtained, calculating also its time derivative . The repeatability of the derivative curves (they overlapped within a band better than 3.3%) permitted the identification of a double-hump pattern which, in principle, could be accepted as an indicator of the type of bacteria (Escherichia coli) . After six experimental series, the growth curves appeared as sensitive to the initial concentration of bacteria and to the culture time preceding inoculation; they were also dependent on the temperature and on the average basal impedance . Temperature showed a greater effect (one order of magnitude) on the lag-phase of the growth curve than on the stationary-phase . This effect occurs because the impedance growth curves tend to get away from the reference offered by the sterile medium . The best working conditions were obtained for an average basal impedance of 510 ohms under well controlled temperature conditions (variations smaller than or equal to 0.20 degrees C) with wire stainless steel electrodes vertically immersed in the culture broth . This impedance technique appears as inexpensive and easy to automatizing for large number of samples. Can J Microbiol, 1987 Dec, 33(12), 1091 - 6 Effect of chlorine injury on heat-labile enterotoxin production in enterotoxigenic Escherichia coli; Walsh SM et al.; Heat-labile enterotoxin (LT) production was examined in chlorine-injured and noninjured populations of enterotoxigenic Escherichia coli (ETEC) by passive immune hemolysis and Y-1 mouse adrenal tumor cell assays . Sublethally injured populations showed reduced LT production after 1, 2.5, and 4 h incubation in trypticase soy broth plus 0.25% glucose, pH 8.0 . Reduction was observed during injury, resuscitation, and for at least 1.5 h following repair . LT levels comparable with that present in noninjured cells were found after 24 h incubation in the same medium, indicating delayed toxigenesis rather than permanent damage . Chlorinated populations failed to incorporate {14C}glucose until repair was completed suggesting a possible explanation for delayed toxin production . The results indicate a temporary loss of virulence among sublethally injured ETEC in chlorinated waters. Undersea Biomed Res, 1987 Nov, 14(6), 485 - 501 Free radical reactions and the inhibitory and lethal actions of high-pressure gases; Thom SR et al.; This study was designed to test whether free radicals are involved in the deleterious effects of compressed gases on cells . The actions of xenon, nitrous oxide, argon, nitrogen, helium, and oxygen and their effects on the toxicity of paraquat (methyl viologen) were studied using Escherichia coli . Growth of E . coli in trypticase-soy broth in an atmosphere of 1.36 MPa (13.6 atm) N2O resulted in an induction of superoxide dismutase (SOD) . In addition, when SOD was induced by oxygen, the resulting cells had increased resistance to the killing action of N2O . The toxicity of paraquat was increased in the presence of N2O but not He, N2, or Ar . However, addition of any of the latter three gases to N2O resulted in increased toxicity of paraquat beyond that due to N2O alone . Oxygen is known to increase the reaction of paraquat radicals within cells and to reduce leakage of the radicals out through the cell membrane . N2O and Xe seem to have this same action, and He, N2, or Ar could enhance the actions of N2O, Xe, or O2 . The data indicate that the inhibitory and lethal actions of these gases may be due to enhanced reactivity of radicals with cell components and reduced leakage of the radicals to the environment. J Bacteriol, 1987 Sep, 169(9), 4036 - 40 Involvement of FtsZ protein in shift-up-induced division delay in Escherichia coli; Kepes F et al.; A nutritional shift-up from glucose minimal medium to LB broth was previously shown to cause a division delay of about 20 min in synchronized cultures of Escherichia coli, and a similar delay was observed after a nutritional pulse (a shift-up followed rapidly by a return to glucose minimal medium) . Using synchronized cultures, we show here that the pulse-induced division delay does not require protein synthesis during the period in LB broth, suggesting that a nonprotein signal is generated by the shift-up and transmitted to the cell division machinery . The cell division protein FtsZ, target of the SOS-associated division inhibitor SfiA (or SulA), seems to be involved in the postshift division delay . Mutants in which the FtsZ-SfiA interaction is reduced, either sfiA (loss of SfiA) or ftsZ(SfiB) (modification of FtsZ), have a 50- to 60-min division delay after a shift-up . Furthermore, after a nutritional pulse, the ftsZ(SfiB) mutant had only a 10- to 16-min delay . These results suggest that the FtsZ protein is the target element of the cell division machinery to which the shift-up signal is transmitted. Biochem J, 1987 Sep 1, 246(2), 287 - 94 Uptake of N-acetylneuraminic acid by Escherichia coli K-235 . Biochemical characterization of the transport system; Rodriguez-Aparicio LB et al.; Kinetic measurement of the uptake of N-acetyl{4,5,6,7,8,9-14C}neuraminic acid by Escherichia coli K-235 was carried out in vivo at 37 degrees C in 0.1 M-Tris/maleate buffer, pH 7.0 . Under these conditions uptake was linear for at least 30 min and the Km calculated for sialic acid was 30 microM . The transport system was osmotic-shock-sensitive and was strongly inhibited by uncouplers of oxidative phosphorylation {2,4-dinitrophenol (100%); NaN3 (66%}) and by the metabolic inhibitors KCN (84%) and sodium arsenate (76%) . The thiol-containing compounds mercaptoethanol, glutathione, cysteine, dithiothreitol and cysteine had no significant effect on the sialic acid-transport rate, whereas the thiol-modifying reagents N-ethylmaleimide, iodoacetate and p-chloromercuribenzoate almost completely blocked (greater than 94%) the uptake of this N-acetyl-sugar . N-Acetylglucosamine inhibited non-competitively the transport of N-acetylneuraminic acid, whereas other carbohydrates (hexoses, pentoses, hexitols, hexuronic acids, disaccharides, trisaccharides) and N-acetyl-sugars or amino acid derivatives (N-acetylmannosamine, N-acetylcysteine, N-acetylproline and N-acetylglutamic acid) did not have any effect . Surprisingly, L-methionine and its non-sulphur analogue L-norleucine partially blocked the transport of this sugar (50%), whereas D-methionine, D-norleucine, several L-methionine derivatives (L-methionine methyl ester, L-methionine ethyl ester, L-methionine sulphoxide) and other amino acids did not affect sialic acid uptake . The N-acetylneuraminic acid-transport system is induced by sialic acid and is strictly regulated by the carbon source used for E . coli growth, arabinose, lactose, glucose, fructose and glucosamine being the carbohydrates that cause the greatest repressions in this system . Addition of cyclic AMP to the culture broth reversed the glucose effect, indicating that the N-acetylneuraminic acid-uptake system is under catabolic regulation . Protein synthesis is not needed for sialic acid transport. Epidemiol Infect, 1987 Aug, 99(1), 155 - 8 The assessment of two media for the confirmation of Escherichia coli in water samples in single-tube tests; Papadakis JA et al.; Two single-tube media, lactose-tryptone-lauryl sulphate broth (LTLSB) and lauryl-tryptose-mannitol broth were tested in parallel in a series of 1111 tests on water samples from a variety of sources . LTLSB was found to be superior both in gas and indole production from Escherichia coli when incubated at 44 degrees C and is recommended as the most suitable single-tube confirmatory test for this organism. Arch Biochem Biophys, 1987 Jul, 256(1), 39 - 49 Oxygen radicals are generated by dye-mediated intracellular photooxidations: a role for superoxide in photodynamic effects; Martin JP et al.; Representative thiazines, xanthenes, acridines, and phenazines photosensitized the oxidation of reduced pyridine nucleotides and reduced glutathione when illuminated with low intensity visible light . Photooxidation resulted in oxygen consumption and in superoxide generation, assayed as the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c . The major pathway of electron transfer involved dye reduction rather than singlet oxygen-mediated oxidation of the substrate, as demonstrated by the relative insensitivity of the oxidation to inhibition by sodium azide and by the observable bleaching of the dye . Hydrogen peroxide was a stable end product of photooxidation . Photosensitive dyes were photoreduced intracellularly . These dyes were transported across the membranes of Escherichia coli B and stimulated a light- and concentration-dependent increase in the cyanide-insensitive respiration . Dyes reduced intracellularly subsequently diffused out of the cell where they reduced extracellular cytochrome c . The photosensitive dyes examined in this study exhibited a light-dependent bacteriostatic effect on E . coli B grown in nutrient broth, manifested as an increased lag prior to growth . Restoration of growth coincided with increased levels of SOD, and the intracellular level of SOD correlated with the level of illumination, the dye concentration, and the reactivity of the dye to NADH in vitro . The thiazine dye, toluidine blue o, imposed a light- and oxygen-dependent lethality on E . coli B grown in glucose minimal medium . Toxicity was relieved by hydroxyl radical scavengers, and their ability to protect the cells was proportional to their reactivity with the hydroxyl radical . The results indicate that oxygen radicals and related species mediate photodynamic effects in E . coli B. J Bacteriol, 1987 Jul, 169(7), 3001 - 6 Mu d-directed lacZ fusions regulated by low pH in Escherichia coli; Slonczewski JL et al.; Methods were devised to isolate strains of Escherichia coli containing Mu d (lacZ Kmr) operon fusions regulated by external pH and by internal pH . External acid-inducible fusions (exa) were detected by plating a Mu d fusion pool on Luria broth with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, buffered at pH 7.4, and then replica plating on the same medium buffered at pH 5.5 . Two exa strains showed induction by external acidification, up to 800-fold and 90-fold . Induction of both fusions was maximal at pH 5.6 and minimal over pH 7.0 to 8.3 . There was no induction by membrane-permeable weak acids which depress internal pH at constant external pH . Anaerobiosis increased the steady-state level of transcription of exa-1 5-fold and of exa-2 2.5-fold at low external pH . Internal acid-inducible fusions (ina) were detected by plating a Mu d fusion pool on MacConkey medium, pH 6.8, and then replica plating with 15 mM benzoate . Two ina strains showed 10-fold induction by 20 mM benzoate at external pH 7.0 . Similar results were obtained with other weak acids; their relative potency (salicylate greater than benzoate greater than dimethoxazoledinedione) was consistent with their relative ability to depress internal pH . In the absence of a weak acid, external pH had almost no effect over the pH range 5.5 to 8.0 . Anaerobiosis did not affect ina induction . To our knowledge, this is the first report of E . coli genes induced specifically by internal but not external acidification and the first report of gene fusions induced by external acidification but not by weak acids. J Biochem (Tokyo), 1987 May, 101(5), 1281 - 8 Direct expression of a synthetic somatomedin C gene in Escherichia coli by use of a two-cistron system; Saito Y et al.; Direct expression of a growth-promoting peptide hormone, somatomedin C/insulin-like growth factor I, which is quite difficult due to the instability of somatomedin C itself in Escherichia coli, has been achieved by the use of a two-cistron system . Assuming that basic somatomedin C might be stabilized by forming a complex with an acidic polypeptide, we constructed synthetic genes consisting of two cistrons; an acidic 93-amino-acid polypeptide was coded in the first cistron followed by a synthetic somatomedin C gene in the second cistron . The chain termination codon for the first polypeptide overlapped the initiation codon for the second polypeptide in the intercistronic region, as occurs in the polycistronic E . coli tryptophan operon, whose products are associated in multi-subunit enzyme complexes . In the expression of the resulting genetic system, recombinant somatomedin C associated with the acidic polypeptide was accumulated to high levels in the cells . After treatment of the product with acetic acid to dissociate the two components, the recombinant somatomedin C was isolated in a yield of 4.0 mg from a liter of culture broth at A600 = 1.6 . It was determined to be Met-somatomedin C by chymotryptic mapping as well as amino-terminal analysis. Am J Vet Res, 1987 May, 48(5), 743 - 8 Shiga-like toxin production and attaching effacing activity of Escherichia coli associated with calf diarrhea; Mainil JG et al.; Four hundred twenty-nine isolates of Escherichia coli from calves were tested for the production of HeLa cell cytotoxin(s) . Isolates that produced enough cytotoxin to be detected in culture supernatants of iron-depleted broth were considered to produce increased amounts of cytotoxins . Isolates also were tested for homology with a DNA probe for a gene that encodes localized adherence of human enteropathogenic E coli . Four isolates produced increased amounts of cytotoxin that was neutralized by Shiga antitoxin (toxin designated as Shiga-like toxin-I {SLT-I}) . A 5th isolate produced increased amounts of cytotoxin (SLT+) that was not neutralized by the Shiga antitoxin, but was neutralized by antitoxin against a variant of SLT (toxin designated as SLT-II) . None of the isolates hybridized with the probe for the localized adherence gene . Three of the SLT+ isolates belonged to human enteropathogenic E coli serogroups O26 and O111 . All 5 of the SLT+ isolates were from calves with diarrhea, but none of the 5 SLT+ isolates contained genes for classic heat-labile or heat-stable enterotoxins, for K99 fimbriae, or for invasiveness; neither did any of them adhere to HeLa cells in culture . Three of the 5 SLT+ isolates had attaching and effacing activities when inoculated into ligated intestinal loops of rabbits . One of the isolates with attaching and effacing activity in rabbits was originally isolated from a calf with lesions characteristic of those produced by attaching effacing E coli (AEEC) . Calves inoculated with this SLT+ AEEC isolate developed focal colonic lesions characteristic of those produced by AEEC, but did not develop diarrhea.(ABSTRACT TRUNCATED AT 250 WORDS) Infect Immun, 1987 Feb, 55(2), 293 - 7 Distribution of type 1 and P pili on uropathogenic Escherichia coli O6; Gander RM et al.; The distribution of type 1 and P pili on individual cells of an O6 uropathogenic Escherichia coli strain, 6260, was determined immunologically with pilus-specific monoclonal antibodies by indirect immunofluorescence and immunogold electron microscopy . Variations in pilus expression under different culture conditions were monitored with an indirect immunofluorescence assay; 63% of piliated cells expressed type 1 pili when grown on agar at 37 degrees C versus 14 to 38% when grown in broth at 37 degrees C . In contrast, generally fewer cells with P pili (18 to 44%) were detected on agar than when grown in broth (up to 86%) . Both type 1 and P pili were absent from cells cultured at 20 degrees C . Immunogold and immunofluorescence double labeling techniques with monoclonal antibodies 11-2 and 91-1 were used to study subpopulations of cells with type 1 and P pili; 39 to 41% of the piliated cells demonstrated only type 1 pili, and 12 to 16% of the cells showed only P pili . The immunogold method proved more sensitive than the immunofluorescence technique for detecting subpopulations expressing both pili types simultaneously, 19 versus 7% . We observed variations between type 1 and P pili, both in expression on individual cells and in the distribution of subpopulations of cells. Gene, 1987, 56(2-3), 209 - 17 Glycosylation and secretion of human alpha-1-antitrypsin by yeast; Moir DT et al.; Human alpha-1-antitrypsin (alpha-AT) is a major serum protein with protease inhibitory activity . Three asparagine residues in alpha-AT are glycosylated with the mammalian 'complex' pattern of carbohydrate as the protein is secreted from cells in the liver . To examine the glycosylation and secretion of human alpha-AT by Saccharomyces cerevisiae, the yeast invertase secretion signal codons were substituted for the native secretion signal coding DNA of an alpha-AT cDNA, and the fusion gene was placed on an autonomously replicating yeast--Escherichia coli shuttle vector under control of the yeast triosephosphate isomerase (TPI) promoter . Yeast strains transformed with this plasmid produce human alpha-AT and secrete about one-fifth of it into the culture broth . Approximately 80% of the alpha-AT produced in yeast is not in the culture broth but is inside the cell within the secretory pathway . This internal alpha-AT is heterogeneous, consisting of molecules with core carbohydrate on either two or all three of the asparagine receptors . Human alpha-AT secreted into the culture broth contains, in addition to core carbohydrate, variable numbers of mannose outer chains, typical of secreted yeast proteins such as invertase . All carbohydrate is removed by endoglycosidase H treatment . Examination of alpha-AT secreted from an mnn9 mutant, which blocks addition of variable numbers of outer mannose chains, revealed a homogeneous alpha-AT product which, like alpha-AT isolated from human serum, bears carbohydrate on three asparagine residues per molecule. J Exp Pathol, 1987 Winter, 3(1), 35 - 60 Activation of murine natural autocytolytic cells which phagocytize normal autologous leukocytes; Watanabe H et al.; We previously described that natural autocytolytic cells (NACs) which were detected in the mesenteric lymph nodes (MLNs) of normal mice, did lyse leukocytes contained in the same lymph nodes . Discrete plaques were observed in monolayers of leukocytes prepared in wells of a microculture plate by NACs phagocytizing and lysing other leukocytes . A large NAC which was morphologically similar to macrophage was found in the middle of each plaque . The mechanisms of autocytolysis by NACs were further investigated in the present study . In contrast to various types of autocytotoxic cells reported previously, for which the incubation with fetal calf serum (FCS) was a prerequisite, the NACs showed their cytolytic activity even in serum-free medium . This excluded the possible "sensitization" by FCS proteins in vitro . The presence of the phagocytosis inhibitor cytochalasin B eliminated the autocytolysis . The peroxide scavenger thioglycollate broth had no effect on the formation of autocytolytic plaques . These results further support the previous finding that autocytolytic plaques were formed by NACs phagocytizing other leukocytes . Autologous nonadherent spleen cells and erythrocytes were also phagocytized by MLN-NACs with plastic adherent characteristics . Ia antigen was demonstrated on the surface of an approximately half population of NACs but neither Thy-1.2 antigen nor immunoglobulins . NACs may represent a subpopulation of macrophages, although the majority of adherent, phagocytic macrophages did not show the NAC activity . The endotoxin lipopolysaccharide (LPS) of Escherichia coli was apparently able to activate MLN-NACs of BALB/c mice in vivo when injected intraperitoneally at a dose of 10 micrograms/ml . A peak of the NAC activity was observed 3 days after the injection.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1986 Nov, 205(2), 376 - 9 A naturally occurring large chromosomal inversion in Escherichia coli K12; Xia XM et al.; Strain 1485IN and its derivatives were found to have a large inversion extending to about 35% of the chromosome . Because of this, the question arose as to whether 1485IN had arisen from an Escherichia coli strain other than K12 . However, 1485IN had a flagellar antigen and a restriction-modification system indistinguishable from those of W3110, a major line of K12, and had retained an amber suppressor and lambda sensitivity that are characteristics of W1485 from which this strain seems to have arisen . Strain 1485IN had acquired proline auxotrophy, but showed the same growth rate as W1485 in nutrient broth at 37 degrees C . Interrupted matings with Hfr strains of 1485IN revealed a gene arrangement of nalA-gal-trp-his-lac-proA-thrleu-ilv, in which gal, trp, and his were on the inverted segment . The termini of the inversion were inferred to be situated between tsx (9.5 min) and purE (12 min) and between his (44 min) and cdd (46.5 min). Can J Microbiol, 1986 Jul, 32(7), 610 - 3 R-plasmid transfer in soil and water; Trevors JT et al.; R-plasmid transfer in Escherichia coli was investigated in nutrient broth, sterile soil, and sterile stream water . Plasmid transfer occurred in broth cultures at 30 and 22 degrees C, but not at 15 degrees C . R-plasmid transfer was not observed at 30, 22, and 15 degrees C in nonamended sterile soil and stream water . The addition of nutrients to sterile stream water and soil allowed plasmid transfer to occur at temperatures ranging from 15 to 30 degrees C . R-plasmid transfer was also observed in soil adjusted from 20 to 100% of its water-holding capacity. J Gen Microbiol, 1986 May, 132 ( Pt 5), 1373 - 8 Iron regulated outer membrane proteins of Escherichia coli: variations in expression due to the chelator used to restrict the availability of iron; Chart H et al.; Iron restriction was induced in Escherichia coli O 111, E . coli O 164 and E . coli C by growing the organisms in trypticase soy broth containing ovotransferrin, desferal, EDDA (ethylenediamine-dihydroxyphenylacetic acid) or alpha,alpha'-dipyridyl . There were marked qualitative and quantitative differences in the iron regulated outer membrane proteins expressed in the presence of the various iron chelators . Differences in the kinetics of growth were also noted . E . coli C was devoid of a ferric enterobactin iron uptake system. Appl Environ Microbiol, 1986 Apr, 51(4), 813 - 20 Removal of endotoxin from water by microfiltration through a microporous polyethylene hollow-fiber membrane; Sawada Y et al.; The microporous polyethylene hollow-fiber membrane has a unique microfibrile structure throughout its depth and has been found to possess the functions of filtration and adsorption of endotoxin in water . The membrane has a maximum pore diameter of approximately 0.04 micron, a diameter which is within the range of microfiltration . Approximately 10 and 20% of the endotoxin in tap water and subterranean water, respectively, was smaller than 0.025 micron . Endotoxin in these water sources was efficiently removed by the microporous polyethylene hollow-fiber membrane . Escherichia coli O113 culture broth contained 26.4% of endotoxin smaller than 0.025 micron which was also removed . Endotoxin was leaked into the filtrate only when endotoxin samples were successively passed through the membrane . These results indicate that endotoxin smaller than the pore size of the membrane was adsorbed and then leaked into the filtrate because of a reduction in binding sites . Dissociation of 3H-labeled endotoxin from the membrane was performed, resulting in the removal of endotoxin associated with the membrane by alcoholic alkali at 78% efficiency. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 Apr, 261(2), 240 - 7 Some enteropathogenic strains of Escherichia coli produce haemagglutinins associated with HEp2 epithelial cell adhesion; Tavendale A et al.; Sixteen of 17 strains of Escherichia coli of traditional infantile enteropathogenic (EPEC) serotypes produced mannose-sensitive haemagglutinin (and type-1 fimbriae) after serial growth in nutrient broth . Two strains also produced mannose-resistant haemagglutinins (MRHAs) after serial growth in phosphate-buffered broth but not in nutrient broth or after growth on phosphate-buffered agar . Some of the type-1 fimbriate EPEC strains adhered modestly in a mannose-sensitive manner to HEp2 epithelial cells in a 30-min in vitro assay; two MRHA+ EPEC strains adhered well in a mannose-resistant manner. Antibiot Med Biotekhnol, 1986 Mar, 31(3), 174 - 8 {Preparative synthesis of the antiviral nucleoside 9-beta-D-arabinofuranosyladenine by using bacterial cells}; ERoshevskaia LA et al.; The conditions of synthesis of 9-beta-D-arabinofuranosyladenine, an antiviral nucleoside by intact cells of Escherichia coli capable of adenine transarabinosylation were studied . The cells were grown in meat-peptone broth supplemented with yeast extract . Cytosine arabinoside served as a donor of the arabinose residues . The pH value, temperature of the reaction medium and concentration of phosphate ions in it were shown to be significant factors defining the cell activity . Under optimal reaction conditions: 0.03 M potassium phosphate buffer, pH 6.75; 0.03 M cytosine arabinoside; 0.01 M adenine; 5 per cent of the cells; incubation time 12 hours; incubation temperature 60 degrees C the efficiency of 9-beta-D-arabinofuranosyladenine synthesis reached 90-95 mol % with respect to the initial adenine level. Am J Vet Res, 1986 Mar, 47(3), 615 - 8 Microscopic alterations in jejunal epithelium of 3-week-old pigs induced by pig-specific, mouse-negative, heat-stable Escherichia coli enterotoxin; Whipp SC et al.; The objective of this study was to determine whether exposure of swine jejunum to crude culture filtrates containing Escherichia coli pig-specific, mouse-negative, heat-stable enterotoxin (STb) induces structural alterations in the jejunal mucosa of pigs . Two ligated intestinal loops in each of twelve 3-week-old pigs were exposed for 2 hours to sterile E coli culture filtrates from each of the following strains: 431 (STa-producing), 1261 (STa and STb-producing), and 1790 (STb-producing); recombinant strain HB101-pRAS-1 (STb-producing); the nontoxigenic K-12 variant HB101; or trypticase soy broth . Formalin-fixed sections from these loops were examined for sloughed cells around villi, and a lesion score was determined, indicating a change in villous epithelium from columnar to cuboidal or squamous cell types or to discontinuous epithelium . Villous lengths and crypt depths also were determined . For loops exposed to culture filtrates containing STa and STb or containing only STb, lesion scores and numbers of sloughed cells were greater (P less than 0.05) and villous length was shorter (P less than 0.01) than in loops not exposed to toxin . For loops exposed to culture filtrates containing STa, lesion scores, villus lengths, and numbers of sloughed cells were not different from those of loops not exposed to toxin . Therefore, exposure of swine jejunum to STb induced structural alterations in intestinal mucosa (ie, loss of villous absorptive cells and partial atrophy of villi) that were consistent with those causing compromised absorptive capacity. J Bacteriol, 1986 Mar, 165(3), 1023 - 5 Inviability of dam recA and dam recB cells of Escherichia coli is correlated with their inability to repair DNA double-strand breaks produced by mismatch repair; Wang TC et al.; The molecular basis for the inviability of dam-3 recA200(Ts) and dam-3 recB270(Ts) cells was studied . The dam-3 recA200(Ts) cells were inviable in yeast extract-nutrient broth or in minimal medium at 42 degrees C . Although the dam-3 recB270(Ts) cells were inviable in yeast extract-nutrient broth at 42 degrees C, they were viable at 42 degrees C in minimal medium, in which the high salt content suppresses the mutant phenotype caused by the recB270(Ts) mutation at 42 degrees C . Under the growth conditions rendering dam rec cells inviable, the cells accumulated double-strand breaks in their DNA . Introduction of a mutL or mutS mutation restored the viability of dam-3 recB270(Ts) cells grown in yeast extract-nutrient broth at 42 degrees C and eliminated the formation of DNA double-strand breaks in these cells . We conclude that the inability to repair DNA double-strand breaks produced by the mismatch repair process accounts for the inviability of the dam recA and dam recB cells. Proc Natl Acad Sci U S A, 1986 Jan, 83(1), 120 - 4 Polynucleotide phosphorylase and ribonuclease II are required for cell viability and mRNA turnover in Escherichia coli K-12; Donovan WP et al.; The isolation of a temperature-sensitive allele of RNase II (rnb) by in vitro mutagenesis has permitted the demonstration that RNase II and polynucleotide phosphorylase (PNPase) are required for cell viability and mRNA turnover in Escherichia coli . Double-mutant strains carrying the pnp-7 and rnb-500 alleles (PNPase deficient and RNase II thermolabile) ceased growing in Luria broth within 30 min after shift to the nonpermissive temperature . Cessation of growth was accompanied by an accumulation of mRNA fragments 100-1500 nucleotides long . In contrast, single-mutant and wild-type control strains grew normally at the nonpermissive temperature and did not accumulate mRNA . No significant changes in rRNA patterns were observed in any of the strains. Infect Immun, 1985 Dec, 50(3), 682 - 6 Relation between the hemolytic property and iron metabolism in Escherichia coli; Lebek G et al.; The hemolytic activity of wild-type strains of Escherichia coli was measured by a standardized method in liquid broth . The system also allowed us to investigate the influence of various Fe3+ concentrations in the cultures on the amount of secreted hemolysin . We found that the hemolysin secretion of all strains was clearly reduced after addition of FeCl3 . However, the influence of additional iron chelators showed remarkable differences . The hemolytic activity of Hly plasmid-containing strains isolated in Berne significantly increased . Most of the strains with a chromosomal hemolysin determinant showed a similar effect, but to a lesser degree . Contrary to this, Hly plasmid-containing strains isolated in Essex and some strains with chromosomal hemolysin determinant were either not affected or even showed reduced hemolysin secretion after limitation of free iron ions in the broth . Our results suggest that hemolysin secretion in E . coli is related to the bacterial iron metabolism, and hemolysin secretion is differentially regulated among E . coli strains. Mikrobiologiia, 1985 Sep-Oct, 54(5), 740 - 4 {Effect of glucose metabolic products on the growth of a recombinant Escherichia coli strain--a superproducer of DNA-polymerase}; Soktoev SA et al.; The dynamics of glucose metabolites production by Escherichia coli CM 5199 was studied under the conditions of batch and continuous cultivation . Acetate and ethanol were shown to be accumulated in the cultural broth in considerable amounts, and the rate of their synthesis was directly proportional to the specific growth rate of the culture . Acetate inhibited E . coli growth as was found in experiments conducted in a turbidostat regimen . As a result, the specific growth rate of the strain decreased during both batch and continuous cultivation. Infect Immun, 1985 Sep, 49(3), 797 - 804 Functional heterogeneity of intestinal Escherichia coli strains expressing type 1 somatic pili (fimbriae): assessment of bacterial adherence to intestinal membranes and surface hydrophobicity; Sherman PM et al.; Although the role of host-specific, nonmannose-sensitive pilus adhesins in the intestinal adherence of pathogenic Escherichia coli is well established, a similar role for mannose-sensitive type 1 or common pili is less clear, since these structures can be expressed by most E . coli, even nonpathogens . We first examined whether type 1 pili, expressed by the rabbit-effacing, adherent, enteropathogenic E . coli strain RDEC-1, mediated interactions with intestinal membranes of several species and compared these interactions with those mediated by the nonmannose-sensitive adhesin of RDEC-1 . We next grew a series of E . coli intestinal strains in static broth to promote type 1 pilus expression and determined whether E . coli expressing type 1 pili differed in their affinity for intestinal membranes (as measured by phase-contrast microscopy and aggregometry), hydrophobic surface properties, net negative surface charge (as measured by hydrophobic interaction chromatography and salt aggregation), and hemagglutination patterns . In contrast to the species-specific attachment to rabbit brush borders of RDEC-1 expressing its nonmannose-sensitive adhesin, type 1 pili on RDEC-1 mediated mannose-sensitive attachment to intestinal membranes of all four species tested . Expression of type 1 pili on other E . coli strains resulted in varying degrees of nonspecies-specific, mannose-sensitive attachment to intestinal membranes . This attachment correlated with increasing surface hydrophobicity rather than with hemagglutination patterns . These results indicate that various E . coli strains expressing type 1 pili are functionally heterogeneous and suggest that some type 1 pili might contribute to in vivo enteroadherence. J Bacteriol, 1985 Aug, 163(2), 506 - 14 Molecular cloning of the cls gene responsible for cardiolipin synthesis in Escherichia coli and phenotypic consequences of its amplification; Ohta A et al.; The cls gene responsible for cardiolipin synthesis in Escherichia coli K-12 was cloned in a 5-kilobase-pair DNA fragment inserted in a mini-F vector, pML31, and then subcloned into a 2.0-kilobase-pair fragment inserted in pBR322 . The initial selection of the gene was accomplished in a cls pss-1 double mutant that had lesions in both cardiolipin and phosphatidylserine synthases and required either the cls or the pss gene product for normal growth at 42 degrees C in a broth medium, NBY, supplemented with 200 mM sucrose . The cloned gene was identified as the cls gene by the recovery and amplification of both cardiolipin and cardiolipin synthase in a cls mutant as well as by the integration of a pBR322 derivative into its genetic locus at 27 min on the chromosome of a polA1 mutant . The maxicell analysis indicated that a protein of molecular weight 46,000 is the gene product . The cls gene is thus most likely the structural gene coding for cardiolipin synthase . Hybrid plasmids of high copy numbers containing the cls gene were growth inhibitory to pss-I mutants under the above selective conditions, whereas they inhibited neither the growth of pss-I mutants at 30 degrees C nor that of pss+ strains at any temperature . Amplification of cardiolipin synthase activity was observed, but was not proportional to the probable gene dosage (the enzyme activity was at most 10 times that in wild-type cells), and cardiolipin synthesis in vivo was at the maximum 1.5 times that in wild-type strains, implying the presence in E . coli cells of a mechanism that avoids cardiolipin overproduction, which is possibly disadvantageous to proper membrane functions. Poult Sci, 1985 Jul, 64(7), 1343 - 9 Dietary protein and yolk sac inoculation with Escherichia coli in young turkeys; Schmidt GP et al.; An experiment was conducted to examine the effect of pathogenic Escherichia coli inoculated into the yolk sac of day-old turkeys . Escherichia coli was isolated from the yolk sac of stunted poults and inoculated directly into the yolk sac of day-old birds . Poults were administered either .1 ml of uninoculated sterile Todd-Hewitt broth or .1 ml of a 10(-3) or 10(-2) dilution of a 24-hr E . coli culture containing 3.4 X 10(8) viable bacteria/ml . In addition, poults were fed either 28 or 22% protein diets from 0 to 21 days of age to form a 3 X 2 factorial arrangement . Body weight gain and feed consumption were measured weekly, and dry matter and protein retention and nitrogen-corrected metabolizable energy were measured from 7 to 10 and 17 to 20 days postinoculation . Intestinal mucosal dipeptidase and maltase activities were determined at 21 days of age . Average mortality by 7 days of age was increased from 1 to 36% from the E . coli inoculation of the yolk sac . Escherichia coli significantly depressed body weight gain and feed consumption 27 and 30, 13 and 16, and 6 and 8%, respectively, during the first, second, and third weeks of the experiment but failed to affect feed efficiency . Feeding a 28% protein diet alleviated the depression in feed consumption and body weight gain to some extent compared with a substantial depression at 22% protein . Nitrogen content and gross energy of the excreta were increased by both dilutions of E . coli for the 7 to 10-day period; this was indicative of a malabsorption of nutrients.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1985 Jul, 163(1), 267 - 74 Evidence for specificity at an early step in protein export in Escherichia coli; Kumamoto CA et al.; We previously described mutations in a gene, secB, which have pleiotropic effects on protein export in Escherichia coli . In this paper, we report the isolation of mutants in which the activity of the secB gene was eliminated . Null mutations in secB affected only a subset of exported proteins . Strains carrying these mutations, although unable to grow on L broth plates, were still viable on minimal media . These secB mutations reversed a block in the translation of an exported protein that was caused by the elimination of another component of the secretion machinery, SecA protein . These results suggest that the secB product acts at an early step in the export process and is involved in the export of only a subset of cell envelope proteins. J Vet Pharmacol Ther, 1985 Jun, 8(2), 150 - 6 Effect of lidamidine-HCl on Escherichia coli heat-stable enterotoxin-induced jejunal water and electrolyte secretion in neonatal piglets; Merritt AM et al.; Neonatal piglets were anesthetized, and two jejunal loops, 20 cm in length, were prepared . Then, either water or 0.12, 0.25, 0.5 or 1.0 mg/kg of lidamidine-HCl was injected intraduodenally on a randomized basis, one treatment per pig . Following this, a crude heat-stable enterotoxin (ST) preparation produced from E . coli no . 1261 was injected into the proximal jejunal loop, and trypticase soy broth (TSB) (with osmolality adjusted to equal the enterotoxin preparation) was injected into the distal jejunal loop . Piglets remained anesthetized for 3 h and were then killed . Fluid was collected from the loops for measurement of volume and Na, K and Cl concentration . Empty loop lengths were measured . There was a significant dose-related reduction of volume and Cl content, and a dose-related, but not significant, reduction in Na content in St-treated loops . A comparison of the mean differences in responses between toxin- and TSB-treated loops indicated that the major 'counter-toxic' effect of the lidamidine was a dose-related increase in water and electrolyte absorption. Biochem Biophys Res Commun, 1985 May 16, 128(3), 1268 - 73 Effect of iso-propyl-thio-beta-D-galactoside concentration on the level of lac-operon induction in steady state Escherichia coli; Cho S et al.; In steady state E . coli cells growing at their maximal rate in broth, maximum induction of beta-galactosidase occurs at 0.10 mM isopropyl-thio-beta-D-galactoside (IPTG) . Although induction of lac is near zero in steady state cells that are growing in 0.01 mM IPTG, induction at mildly subdued levels persists down to at least 0.001 mM in post-steady state cultures . Meanwhile, thiogalactoside transacetylase remains uninduced over the full range in which the cells are in steady state. J Bacteriol, 1985 Mar, 161(3), 1086 - 92 Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli; Shibuya I et al.; Escherichia coli K-12 derivatives with a common genetic background carrying, either alone or in combination, the pss-1 allele coding for a temperature-sensitive phosphatidylserine synthase (A . Ohta and I . Shibuya, J . Bacteriol . 132:434-443, 1977) and cls- for a defective cardiolipin synthase (G . Pluschke et al., J . Biol . Chem . 253:5048-5055, 1978) were constructed . The phospholipid polar headgroup compositions of these strains were significantly different from each other depending on their genotypes and growth temperature, whereas other membrane characteristics such as the total phospholipid content, fatty acid composition, membrane protein profile, and lipopolysaccharide content were practically the same, suggesting that the phenotypes of these strains were the direct consequences of abnormalities in membrane phospholipid composition . The cls pss-1 double mutation caused an unusual accumulation of phosphatidylglycerol with an extremely low content of cardiolipin . The cls mutation alone was found to give a growth defect, and its introduction into a pss-1 mutant resulted in an enhanced temperature sensitivity of growth . Addition to a broth medium of a proper concentration of sucrose, NaCl, Mg2+, or Ca2+ allowed the growth of a pss-1 mutant at otherwise nonpermissive temperature, but a pss-1 cls double mutant required the combined addition of sucrose or NaCl and MgCl2 for full growth at 42 degrees C . The possible mechanisms for these physiological consequences of the mutations are discussed on a molecular basis . The remedial effects of culture supplements allowed the pss-1 mutants to grow at 42 degrees C resulting in enhanced abnormalities of membrane phospholipid composition. Plasmid, 1985 Mar, 13(2), 118 - 28 Transfer systems of K88 and K99 plasmids; Bradley DE; The conjugation systems of three K88-mobilizing plasmids were characterized for the morphology of their pili and type of mating system (surface only or surface + liquid) . pREI had a typical IncI1 transfer system with both thick and thin pili . pVIDO determined aggregating thick flexible pili and pPLS nonaggregating thick flexible pili . All three transferred equally well in broth and on plates . pPLS alone was naturally transfer-depressed . pREI and pVIDO were tested for K88 mobilization efficiency, which was greater from their wild-type host strains to Escherichia coli K-12 than between E . coli K-12 strains . The K99 conjugative plasmid from strain B41 was repressed for transfer and determined thick flexible pili that were receptors for the filamentous phage fd. J Biochem (Tokyo), 1985 Feb, 97(2), 399 - 408 The morphology of L-fucose, D-mannose specific lectin (SFL 100-2) produced by Streptomyces No . 100-2; Matsui I et al.; The morphology of an L-fucose specific lectin, SEL 100-2, from a Streptomyces sp . was studied . Electron microscopic observation showed that purified SFL 100-2 preparation consisted of particles homogeneous in size . The diameter was 25 nm . The digitized images of these particles had 2-fold rotation symmetry . The sedimentation coefficient (s020,w) was determined to be 20.6S . The particle weight and the Stokes radius were calculated to be 8.0 X 10(5) daltons and 94 A, respectively, by three independent methods, i.e., gel filtration, sedimentation equilibrium and velocity measurements . The frictional ratio (f/fmin) was estimated to be 1.53 . These values are quite similar to those of human alpha 2-macroglobulin . 125I-Labeled peptide mapping indicated that these particles were built up of about twelve identical subunits (Mr = 68,000) . The size of SFL 100-2 in culture broth was found to be the same as that of the particles in the purified preparations . The shape and other properties of SFL 100-2 are discussed and compared with those of the tail of lambda phage and type 1 pili of Escherichia coli, whose amino acid compositions were quite similar to that of SFL 100-2 and also those of L-fucose specific plant lectins. Proc Natl Acad Sci U S A, 1985 Jan, 82(1), 227 - 31 Molybdate reduction by Escherichia coli K-12 and its chl mutants; Campbell AM et al.; During anaerobic growth, Escherichia coli can reduce phosphomolybdate . The reduction can also be carried out by washed cells suspended in buffer at pH 5.7 . Phosphate, molybdate, glucose, cells, and anaerobic conditions are required . Reduction is inhibited by 200 microM chromate, 290 microM nitrite, 10 mM tungstate, or 20 mM cysteine . Wild-type (chl+) cells are inhibited by addition of 200 microM nitrate, but chlA, chlB, and chlE mutants are not . The inhibition of chl+ cells results from reduction of nitrate to nitrite . This nitrate reduction is not catalyzed by nitrate reductase . Wild-type cells are more sensitive than chl mutants to inhibition by nitrite and cysteine but more resistant to chromate . Pregrowth of chlD cells in 1 mM Na2MoO4 increases their sensitivity to nitrite and cysteine, and pregrowth of chl+ cells in 1 mM Na2MoO4 increases their resistance to these agents . Assays of biotin sulfoxide reductase show that the tightness of the chlD block depends on growth conditions; chlD cells grown aerobically in tryptone broth make about 50% as much active enzyme as chl+ cells, whereas chlD cells grown anaerobically with tryptone plus glucose make less than 10% . The effect of anaerobic pregrowth on the inhibition of molybdate reduction by added nitrate indicates that in vivo nitrate reduction responds to growth conditions in the same manner as biotin sulfoxide reductase does. Mol Gen Genet, 1985, 200(1), 103 - 9 Function of ribonuclease H in initiation of DNA replication in Escherichia coli K-12; Kogoma T et al.; Escherichia coli rnh mutants lacking ribonuclease H (RNase H) activity can tolerate deletion of the origin of DNA Replication (delta oriC) and transposon-insertional inactivation of an initiator gene (dnaA::Tn10) . Introduction of the recA200 allele encoding a thermolabile RecA protein into rnh- dnaA::Tn10 and rnh- delta oriC mutants strains rendered DNA synthesis and colony formation of these mutants temperature sensitive . The temperature sensitivity and the broth sensitivity (Srm-) of the rnh- dnaA::Tn10 recA200 strain was suppressed by the presence of plasmids (pBR322 derivatives) carrying dnaA+ only when the intact oriC site was present on the chromosome . Lack of RNase H activity neither promoted replication of minichromosomes (pOC24 and p lambda asn20) in the absence of required DnaA+ protein nor inhibited dnaA+-dependent minichromosome replication . These results led to the conclusion that RNase H is not directly involved in the events leading to initiation of DNA replication at oriC . Rather, it functions as a specificity factor by eliminating certain forms of RNA-DNA hybrids which could otherwise be used to prime DNA replication at sites other than oriC. J Bacteriol, 1984 Oct, 160(1), 61 - 6 Identification and characterization of a gene product that regulates type 1 piliation in Escherichia coli; Orndorff PE et al.; The recombinant plasmid pSH2 confers type 1 piliation (Pil+) on a nonpiliated (Pil-) strain of Escherichia coli K-12 . At least four plasmid-encoded gene products are involved in pilus biosynthesis and expression . We present evidence which indicates that one gene encodes an inhibitor of piliation . Hyperpiliated (Hyp) mutants were isolated after Tn5 insertion mutagenesis of pSH2 and introduction of the plasmid DNA into a Pil- strain of E . coli as unique small, compact colonies . Also, Hyp mutants clumped during growth in static broth and were piliated under several cultural conditions that normally suppressed piliation . Electron microscopic examination of Hyp mutants associated an observed 40-fold increase in pilin antigen with an increase in the number and length of pili per cell . All Hyp mutants examined failed to produce a 23-kilodalton protein that was encoded by a gene adjacent to the structural (pilin) gene for type 1 pili, and all Tn5 insertion mutations that produced the Hyp phenotype mapped in this region (hyp) . Piliation in Hyp mutants could be reduced to near parental levels by introducing a second plasmid containing a parental hyp gene . Thus the 23-kilodalton (hyp) protein appears to act in trans to regulate the level of piliation. Infect Immun, 1984 Oct, 46(1), 251 - 9 Cloning and expression of an afimbrial adhesin (AFA-I) responsible for P blood group-independent, mannose-resistant hemagglutination from a pyelonephritic Escherichia coli strain; Labigne-Roussel AF et al.; The uropathogenic Escherichia coli KS52 strain expresses a mannose-resistant hemagglutinin involving an erythrocyte recognition site distinct from the alpha-digalactoside glycosphingolipid receptor identified for the uropathogenic E . coli strains specifying a P adhesin . The KS52 strain showed three major properties . (i) It agglutinated human erythrocytes of all tested blood groups . (ii) Hemagglutinin activity was found both in the supernatant fluid L-broth cultures and in cells grown on L-agar plates . (iii) No fimbriae in organisms grown on L-agar plates were detected by electron microscopy . Whole-cell DNA from the KS52 strain was size fractionated and cloned into the pHC79 cosmid vector . Three recombinant cosmids expressing a mannose-resistant hemagglutination (MRHA) phenotype were characterized and used to subclone the smallest DNA fragment able to confer the same MRHA properties as the parent strain . A 6.7-kilobase chromosomal DNA fragment cloned in pBR322 (pIL14) was shown to be necessary for host-cell MRHA expression and uroepithelial cell adherence . The insert encoded the production of a 16,000-dalton hemagglutinin . This polypeptide could be detected in culture supernatant fluids, in E . coli minicells harboring the pIL14 plasmid, and, by immunoblotting, in the KS52 strain and E . coli whole cells harboring the pIL14 plasmid . No homology was detected by Southern hybridization between the cloned insert and the DNA of the operon responsible for MRHA in the P-specifying, fimbriate strains (pap operon). J Assoc Off Anal Chem, 1984 Sep-Oct, 67(5), 946 - 9 Detection of Escherichia coli enterotoxins by using mouse adrenal cell and suckling mouse assays: collaborative study; Lovett J et al.; The ability of 10 Escherichia coli strains to produce heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT) was determined by 8 analysts in a collaborative study . The suckling mouse model and the mouse adrenal cell line (Y-1) tests were used to detect ST and LT, respectively . Cultures for assay were grown 24 h in casamino acid-yeast extract-trace salts broth at 37 degrees C in a shaker incubator at 250 rpm . Cell-free culture broth prepared by centrifugation and filtration was divided into 2 portions: One was heated for 30 min and used both for ST assay and as a heated control for LT assay; the other was used unheated for LT assay . Results were expressed as positive for ST, positive for LT, positive for ST and LT, or negative for both ST and LT; percent of correct estimates was calculated for each culture for each analyst . At the 95% confidence interval, the overall correct results were 96.3 +/- 2.9 and 95.0 +/- 3.4% for ST and LT, respectively . The test performances thus were satisfactory for detecting ST and LT produced in vitro by E . coli . The method has been adopted official first action. Appl Environ Microbiol, 1984 Aug, 48(2), 285 - 8 Evaluation of a fluorogenic assay for detection of Escherichia coli in foods; Robison BJ; A fluorogenic assay procedure with 4-methylumbelliferyl-beta-D-glucuronide incorporated into lauryl sulfate broth was evaluated to detect and confirm the presence of Escherichia coli in foods . Fluorescence is indicative of the presence of E . coli; extensive biochemical confirmation is unnecessary with this assay . The 4-methylumbelliferyl-beta-D-glucuronide assay was tested concurrently with our present methodology for detection of E . coli on 270 samples of raw ingredients and powdered food products . Total agreement between the two methods was 94.8%; there was a false-positive rate of 4.8% and no false-negatives . We found the 4-methylumbelliferyl-beta-D-glucuronide assay to be rapid, accurate, simple to perform, and inexpensive. J Clin Microbiol, 1984 Jun, 19(6), 798 - 803 Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin; Klipstein FA et al.; The sensitivity of an enzyme-linked immunosorbent assay (ELISA) to detect pure native Escherichia coli heat-stable toxin (ST) and to identify ST-producing strains among clinical isolates was determined . Two synthetically produced ST preparations were used to raise hyperimmune antisera in rabbits and goats: ST(S), which has the same antigenicity as native ST; and ST(C), which is 15-fold more immunogenic . These antisera were used in the double-sandwich technique as either crude double-species antisera or pure single-species antibody . The sensitivity of the assay was increased by using either a purer antibody preparation or the antiserum to the more potent immunogen; the assay in which pure antibody to ST(C) was used was 2,857-fold more sensitive in detecting ST than the assay in which crude antiserum to ST(S) was used . The minimum amount of ST detectable by the ST(C) ELISA was 140 pg/ml, which was an amount 285-fold smaller than that detectable by the suckling mouse assay . Among 50 human E . coli isolates examined by both the ST(C) ELISA and an ELISA for heat-labile toxin (LT), which had a sensitivity of 290 pg/ml for LT, the respective toxins were consistently identified in broth cultures of 10 LT+ and ST-, 15 LT+ and ST+, and 10 LT- and ST+ strains, and there were no false-positive responses . The ST(C) ELISA also detected ST in all of seven ST - producing E . coli strains tested of human origin, which had been shown elsewhere by DNA hybridization probes to have ST-coding genes of either human or porcine origin, and in all of three ST-producing E . coi strains tested of porcine origin . These results indicate that the sensitivity of the ST(C) ELISA is the same as that of previously described LT ELISAs . The concomitant use of both ST and LT ELISAs provides a rapid, simple, and sensitive method for identifying among clinical isolates enterotoxigenic strains of E . coli which produce either toxin. J Clin Invest, 1984 Jun, 73(6), 1515 - 23 Interaction of primate alveolar macrophages and Legionella pneumophila; Jacobs RF et al.; We studied the interaction between Legionella pneumophila, which is principally a pulmonary pathogen, with primate alveolar macrophages (AM), which are the primary pulmonary cellular defense mechanism . For these studies we used L . pneumophila, type I, which were grown in albumin-yeast extract broth, were greater than 80% viable, and were comparable in virulence for guinea pigs to organisms from guinea pig spleen homogenates . For comparison, avirulent agar-passed L . pneumophila, type I, and a strain of Escherichia coli were also used . In the absence of detectable antibody, AM phagocytosed similar numbers of virulent and avirulent Legionella and killed the majority of ingested Legionella in 15-30 min, as determined by two different assays . The virulent and avirulent Legionella appeared to be equally susceptible to the cidal systems of the AM and both were killed more readily than were E . coli under both assay conditions . Phagocytosis of Legionella by AM was associated with a localized respiratory burst, as indicated by nitroblue tetrazolium reduction around ingested organisms . Killing of AM-associated Legionella was inhibited by the hydroxyl radical (OH.) scavenger mannitol (but not by an equiosmolar concentration of sodium sulfate), and by a combination of superoxide dismutase and catalase (but not by either enzyme alone) . These findings suggest a contribution by OH., one generated by the metal-catalyzed interaction of superoxide and hydrogen peroxide (Haber-Weiss reaction) in the anti-Legionella activity of AM . The virulent Legionella that survived intracellularly increased in number from 4 X 10(4) at 1 h to 6 X 10(6) at 96 h after infection . In contrast, avirulent Legionella replicated more slowly, increasing in number from 4 X 10(4) to 1 X 10(5) over the same period . Replication of virulent Legionella destroyed the AM monolayers by 120 h, whereas monolayers containing avirulent organisms remained intact . Thus, virulence of Legionella appears not to correlate with its ability to survive early killing by AM, but rather with the ability of the small fraction of surviving organisms to replicate within these cells. J Hosp Infect, 1984 Jun, 5(2), 155 - 63 The potential of Escherichia coli in enteral feeds to cause food poisoning: a study under simulated ward conditions; Anderton A; The growth of Escherichia coli over a period of 8 h under simulated ward conditions was compared in feeding systems containing either Clinifeed ISO or Nutrient Broth . In both, counts increased from 10(2) to 10(7) ml-1 . Other systems containing Clinifeed ISO were inoculated with E . coli and sampled over 24 h . At 8 and 16 h the contaminated reservoir was replaced, refilled or replaced together with the gastric drip line . When the reservoir was replaced or refilled it was always recontaminated by residual organisms . Even when the reservoir and drip line were replaced, although the contents of the reservoir were sterile, E . coli was still detected in feed collected from the end of the fine-bore tube . Experiments with varying numbers of E . coli in the inoculum demonstrated that even one organism in the reservoir could, within 16 h, multiply to a level that might be harmful, especially to compromised patients. Eur J Biochem, 1984 Apr 16, 140(2), 353 - 61 Metabolic changes in ribosomes of Escherichia coli during prolonged culture in different media; Ramagopal S; The metabolism of ribosomes during the exponential growth and post-exponential phase of Escherichia coli cells was investigated . Incubation of E . coli cells in two rich media: L-broth and phosphate medium, up to stationary phase shows no drop in viability or any changes in ribosomes . However, the survival rate during prolonged culture of the post-stationary-phase cells has been found to be a function of the incubation medium . The decline in viability is only slight in phosphate medium but very rapid in L-broth . So long as the viability is maintained, the level of ribosomes and the relative abundance of rRNA and ribosomal proteins in ribosomes of the post-stationary cultures are remarkably stable and are similar to exponentially growing cells . On the other hand, post-stationary cultures undergoing a rapid drop in cell viability lose 95% of the original ribosomes . These cultures accumulate a large pool of 30S and 50S subunits and a few 70S monosomes, all of which show deficiency in the various ribosomal proteins . No differences in rRNA can be detected but the number and the relative stoichiometry of individual ribosomal proteins are drastically altered . Only 13 of the 53 proteins known in the E . coli ribosome appeared in the same relative amounts as in the ribosomes of the exponentially growing cells . Six proteins (S12, S21, L2, L16, L20, L34) are completely lost and all others undergo partial loss . An analysis of the number and relative abund