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The efficiency of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system, was assessed using real-time PCR, flow cytometry and bioluminescence. The ColE7 antimicrobial system consists of the genetically modified Escherichia coli strain Nissle 1917 harbouring a conjugative plasmid (derivative of the F-plasmid) encoding the "kill" gene (ColE7 activity gene) and a chromosomally encoded "anti-kill" gene (ColE7 immunity gene). On the basis of traJ gene expression in the killer donor cells, our results showed that the efficiency of the here studied antimicrobial system against target E. coli was higher at 4 than at 24 h. Flow cytometry was used to indirectly estimate DNase activity of the antimicrobial system, as lysis of target E. coli cells in the conjugative mixture with the killer donor strain led to reduction in cell cytosol fluorescence. According to a lux assay, E. coli TG1 (pXen lux + Ap r ) with constitutive luminescence were killed already after 2 h of treatment. Target sensor E. coli C600 with DNA damage SOS-inducible luminescence showed significantly lower SOS induction 6 and 24 h following treatment with the killer donor strain. Our results thus showed that bioluminescent techniques are quick and suitable for estimation of the ColE7 bacterial conjugation-based antimicrobial system antibacterial activity., Significance and Impact of the Study: Bacterial antimicrobial resistance is worldwide rising and causing deaths of thousands of patients infected with multi-drug resistant bacterial strains. In addition, there is a lack of efficient alternative antimicrobial agents. The significance of our research is the use of a number of methods (real-time PCR, flow cytometry and bioluminescence-based technique) to assess the antibacterial activity of the bacteriocin, colicin ColE7, bacterial conjugation-based "kill" - "anti-kill" antimicrobial system. Bioluminescent techniques proved to be rapid and suitable for estimation of antibacterial activity of ColE7 bacterial conjugation-based antimicrobial system and possibly other related systems., (© 2018 The Society for Applied Microbiology.)

We report on the application of site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to study possible oligomerization of the bacterial toxin colicin A (ColA) upon membrane insertion in vitro and in vivo. We applied SDSL-EPR protocols and optimized experimental conditions to perform continuous wave EPR experiments and double electron-electron resonance distance measurements on intact Escherichia coli cells interacting with nitroxide spin-labeled ColA. Our data suggest that ColA forms dimers upon membrane insertion, thus explaining previously reported pore diameters of about 1 nm, which are unlikely to be formed by a single colicin A monomer.

A population-based study combining (i) antimicrobial, (ii) genetic, and (iii) virulence analyses with molecular evolutionary analyses revealed segregative characteristics distinguishing human clinical and bovine Escherichia coli O157 strains from western Canada. Human (n = 50) and bovine (n = 50) strains of E. coli O157 were collected from Saskatchewan and Manitoba in 2006 and were analyzed by using the six-marker lineage-specific polymorphism assay (LSPA6), antimicrobial susceptibility analysis, the colicin assay, plasmid and virulence profiling including the eae, ehxA, espA, iha, stx1, stx2, stx2c, stx2d, stx2d-activatable, stx2e, and stx2f virulence-associated genes, and structure analyses. Multivariate logistic regression and Fisher's exact test strongly suggested that antimicrobial susceptibility was the most distinctive characteristic (P = 0.00487) associated with human strains. Among all genetic, virulence, and antimicrobial determinants, resistance to tetracycline (P < 0.000) and to sulfisoxazole (P < 0.009) were the most strongly associated segregative characteristics of bovine E. coli O157 strains. Among 11 virulence-associated genes, stx2c showed the strongest association with E. coli O157 strains of bovine origin. LSPA6 genotyping showed the dominance of the lineage I genotype among clinical (90%) and bovine (70%) strains, indicating the importance of lineage I in O157 epidemiology and ecology. Population structure analysis revealed that the more-diverse bovine strains came from a unique group of strains characterized by a high degree of antimicrobial resistance and high frequencies of lineage II genotypes and stx2c variants. These findings imply that antimicrobial resistance generated among bovine strains of E. coli O157 has a large impact on the population of this human pathogen.

Aim: Detection of viable but uncultivable forms among lyophilized cells of commercial probiotics., Materials and Methods: 9 series of probiotic preparations (colibacterin, bificol, bifidumbacterin, bifiform) with expired (up to 30 years of storage) or valid shelf life were objects of the study. Total quantity of the bacteria was calculated under the microscope in Goryaev chamber, the number of viable cells was determined in luminescence microscope after staining by an array of fluorescent dyes, the quantity of CFU/ml was evaluated by method of seeding into the respective solid or semi-liquid cultivation medium., Results: Juxtaposition of the specified parameter values allowed to establish that in lyophilized preparations of colibacterin with expired shelf life the amount of viable but not forming colonies (uncultivable forms) cells varied from 4.13 to 99.73% depending on date of production. For bifidumbacterin with unexpired shelf life live cells constituted 95.45 and 70.73%. The amount of viable bifidobacteria forming colonies was on the level of 100 and 50%, respectively. In bifidopreparations micro colonies that may possibly be formed spontaneously by awakened uncultivable forms were noted. A possibility of growth stimulation of these cells by 1 and 10% aminopeptide was shown., Conclusion: The presence of uncultivable forms in lyophilized preparations of probiotics was proven experimentally.

Burkholderia cenocepacia, a member of the B. cepacia complex, is an opportunistic pathogen that causes serious infections in patients with cystic fibrosis. We identified a six-gene cluster in chromosome 1 encoding a two-component regulatory system (BCAL2831 and BCAL2830) and an HtrA protease (BCAL2829) hypothesized to play a role in the B. cenocepacia stress response. Reverse transcriptase PCR analysis of these six genes confirmed they are cotranscribed and comprise an operon. Genes in this operon, including htrA, were insertionally inactivated by recombination with a newly created suicide plasmid, pGPOmegaTp. Genetic analyses and complementation studies revealed that HtrA(BCAL2829) was required for growth of B. cenocepacia upon exposure to osmotic stress (NaCl or KCl) and thermal stress (44 degrees C). In addition, replacement of the serine residue in the active site with alanine (S245A) and deletion of the HtrA(BCAL2829) PDZ domains demonstrated that these areas are required for protein function. HtrA(BCAL2829) also localizes to the periplasmic compartment, as shown by Western blot analysis and a colicin V reporter assay. Using the rat agar bead model of chronic lung infection, we also demonstrated that inactivation of the htrA gene is associated with a bacterial survival defect in vivo. Together, our data demonstrate that HtrA(BCAL2829) is a virulence factor in B. cenocepacia.

Shigella is an etiological agent of communicable and food-borne disease worldwide, so it is important to develop typing for Shigella in epidemiological studies. We compared amplified fragment length polymorphism (AFLP), molecular epidemiological typing, to pulsed-field gel electrophoresis (PFGE) and colicin typing in typeability, reproducibility, discriminatory power, ease of interpretation, and ease of use for 51 Shigella isolates to determine AFLP applicability to Shigella. AFLP showed less reproducibility and ease of interpretation although it was superior to PFGE and colicin typing in typeability and discriminatory power. Specifying the reproducibility of these typing methods, the intrastrain similarity of AFLP was 81.9%-90.5% in each of three strains tested in triplicate trials, while PFGE showed higher similarity, ranging from 92.3%-100%. AFLP created a phylogenetic tree and classified four Shigella species taxonomically, despite its lower reproducibility. These results suggest that AFLP is inferior to PFGE as molecular typing for Shigella epidemiologically in outbreaks or sporadic cases, although AFLP can create a phylogenetic tree for taxonomical purposes.

While numerous proteomic analyses have been carried out on Escherichia coli, the vast majority have focused on expression of intracellular proteins. Yet, recent literature reports imply that even in laboratory strains, significant proteins may be found outside the cell. Here, we identify extracellular proteins associated with nonpathogenic E. coli strain W3110. Two-dimensional gel electrophoresis (2DE) revealed approximately 66 prominent protein spots during exponential growth (4 and 8 h shake flask culture) in minimal medium. The absence of detectable nucleic acids in the culture supernatant implies these proteins did not result from cell lysis. MALDI-TOF MS was used to identify 44 proteins, most of which have been previously identified as either outer membrane or extracellular proteins. In addition, 2DE protease zymogram analysis was carried out which facilitated identification of three extracellular proteases, one of which was not observed during standard 2DE. Our results are consistent with previous findings which imply outer membrane proteins are shed during growth.

Aims: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo., Methods and Results: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae., Conclusions: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans., Significance and Impact of the Study: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.

A sensitivity-enhanced 1D (1)H spin diffusion experiment, CHH, for determining membrane protein topology is introduced. By transferring the magnetization of the labeled protein (13)C to lipid and water protons for detection, the CHH experiment reduces the time of the original 2D (13)C-detected experiment by two orders of magnitude. The sensitivity enhancement results from (1)H detection and the elimination of the (13)C dimension. Consideration of the spin statistics of the membrane sample indicates that the CHH sensitivity depends on the (13)C labeling level and the number of protein protons relative to the mobile protons. 5-35% of the theoretical sensitivity was achieved on two extensively (13)C labeled proteins. The experimental uncertainties arise from incomplete suppression of the equilibrium (1)H magnetization and the magnetization of lipid protons directly bonded to natural-abundance carbons. The technique, demonstrated on colicin Ia channel domain, confirms the presence of a transmembrane domain and the predominance of surface-bound helices.

Clinical and bacteriological studies have revealed that the production of colicin by Escherichia coli forming a part of intestinal microbiocenosis is related to the clinical manifestations of inflammatory diseases of the gastrointestinal tract. During the exacerbation of chronic inflammatory processes of the digestive system the proportion of colicin producing E. coli increases (more than 45%) in comparison with that of E. coli fecal strains isolated in children and adolescents regarded as healthy (less than 15%). The possibility of using the colicin producing activity of intestinal microflora for the evaluation of the dysbiotic states of gastrointestinal tract is discussed.

Plasmid DNA of six Escherichia fergusonii colicinogenic strains (three producers of colicin E1, two of Ib and one of Ia) was isolated and the colicin-encoding regions of the corresponding Col plasmids were sequenced. Two new variants of colicin E1, one of colicin Ib, and one of colicin Ia were identified as well as new variants of the colicin E1 and colicin Ib immunity proteins and the colicin E1 lysis polypeptide. The recombinant Escherichia coli producer harboring pColE1 from E. fergusonii strain EF36 (pColE1-EF36) was found to be only partially immune to E1 colicins produced by two other E. fergusonii strains suggesting that pColE1-EF36 may represent an ancestor ColE1 plasmid.

The incidence of bacteriocinogeny and lysogeny was followed in bacteria of 3 recently acknowledged species of the genus Escherichia: E. hermanii, E. vulneris and E. fergusonii. Almost all of the strains examined were of human origin. In 30 strains of E. hermanii no one was found bacteriocinogenic while 57% were lysogenic, in 30 strains of E. vulneris none was found to be bacteriocinogenic and only 10% were lysogenic, and in 50 strains E. fergusonii 12% were bacteriocinogenic and 40% lysogenic. From the 6 bacteriocinogenic strains of E. fergusonii, 3 were producers of colicin E1, 2 of colicin Ib and 1 of colicin Ia. In addition, 3 E. fergusonii strains produced aerobactin.

In order to detect phenotypic characteristics associated with pathogenicity, 25 strains of Escherichia coli, isolated from clinical cases of colisepticemia in broiler chickens, were examined to determine the following properties: colicinogenicity, colicin V production, type 1 fimbriae, hemolysin expression and motility. Colicinogenicity occurred in 72% of the strains, 56% of all strains produced colicin V, 84% were positive for type 1 fimbriae and 80% were positive for motility. None of the strains had hemolytic activity; however, all of them, expressed at least one of the other characteristics studied. These results suggest that the diversity of phenotypes detected partially explain the multifactorial nature of avian colisepticemia.

Colicin production was studied among 137 Escherichia coli strains isolated from pregnant patients with symptomatic or asymptomatic, urinary tract infection (IVU). The observed colicinogeny frequency was 72.92% (n = 96), among the symptomatic patients and 29.26% (n = 41) among the asymptomatic group. The most frequently identified colicins from symptomatic patients were: V (23.9%), A (18.7%) and E1 (17.7%) and within the patients with asymptomatic IVU the most frequently observed colicins were A (9.7%), E1 (17.0%) y V (2.4%). The major frequency of colicin V among the E. coli strains isolated from the symptomatic group against the asymptomatic one, was statistical significant (p > or = 0.025). The results on the observed frequencies of colicins E1 and A were not statistical significant. Among the 137 studied E. coli strains, 37.2% were hemolytic. 83.3% of the colicin V producing strains (n = 24) were hemolytic, among the strains producing other colicin different than colicin V 34% (n = 58) were hemolytic and 12.0% of non colicinogenic strains were hemolytic. These results were statistical significant (p > 0.05). The present data, suggest that colicins production is an important pathogenicity factor among the E. coli strains, specially for those strains producing colicin V. The observed association among hemolysin and colicin V production may be an interesting pathogenicity factor which suggests an increasing ability of uropathogenic strains to produce symptomatic urinary tract infections.

Much computational research aimed at understanding ionizable group interactions in proteins has focused on numerical solutions of the Poisson-Boltzmann (PB) equation, incorporating protein exclusion zones for solvent and counterions in a continuum model. Poor agreement with measured pKas and pH-dependent stabilities for a (protein, solvent) relative dielectric boundary of (4,80) has lead to the adoption of an intermediate (20,80) boundary. It is now shown that a simple Debye-Huckel (DH) calculation, removing both the low dielectric and counterion exclusion regions associated with protein, is equally effective in general pKa calculations. However, a broad-based discrepancy to measured pH-dependent stabilities is maintained in the absence of ionizable group interactions in the unfolded state. A simple model is introduced for these interactions, with a significantly improved match to experiment that suggests a potential utility in predicting and analyzing the acid pH-dependence of protein stability. The methods are applied to the relative pH-dependent stabilities of the pore-forming domains of colicins A and N. The results relate generally to the well-known preponderance of surface ionizable groups with solvent-mediated interactions. Although numerical PB solutions do not currently have a significant advantage for overall pKa estimations, development based on consideration of microscopic solvation energetics in tandem with the continuum model could combine the large deltapKas of a subset of ionizable groups with the overall robustness of the DH model.

A simple approach to estimate the number of alpha-helical and beta-strand segments from protein circular dichroism spectra is described. The alpha-helix and beta-sheet conformations in globular protein structures, assigned by DSSP and STRIDE algorithms, were divided into regular and distorted fractions by considering a certain number of terminal residues in a given alpha-helix or beta-strand segment to be distorted. The resulting secondary structure fractions for 29 reference proteins were used in the analyses of circular dichroism spectra by the SELCON method. From the performance indices of the analyses, we determined that, on an average, four residues per alpha-helix and two residues per beta-strand may be considered distorted in proteins. The number of alpha-helical and beta-strand segments and their average length in a given protein were estimated from the fraction of distorted alpha-helix and beta-strand conformations determined from the analysis of circular dichroism spectra. The statistical test for the reference protein set shows the high reliability of such a classification of protein secondary structure. The method was used to analyze the circular dichroism spectra of four additional proteins and the predicted structural characteristics agree with the crystal structure data.

Intracellularly expressed antibodies have been designed to bind and inactivate target molecules inside eukaryotic cells. Here we report that an antibody fragment can be used to probe the periplasmic localization of the colicin A N-terminal domain. Colicins form voltage-gated ion channels in the inner membrane of Escherichia coli. To reach their target, they bind to a receptor located on the outer membrane and then are translocated through the envelope. The N-terminal domain of colicins is involved in the translocation step and therefore is thought to interact with proteins of the translocation system. To compete with this system, a single-chain variable fragment (scFv) directed against the N-terminal domain of the colicin A was synthesized and exported into the periplasmic space of E. coli. The periplasmic scFv inhibited the lethal activity of colicin A and had no effect on the lethal activity of other colicins. Moreover, the scFv was able to specifically inactivate hybrid colicins possessing the colicin A N-terminal domain without affecting their receptor binding. Hence, the periplasmic scFv prevents the translocation of colicin A and probably its interaction with import machinery. This indicates that the N-terminal domain of the toxin is accessible in the periplasm. Moreover, we show that production of antibody fragments to interfere with a biological function can be applied to prokaryotic systems.

Eight Salmonella typhimurium (Copenhagen) and eight Salmonella dublin isolates from cattle were compared by their antibiotic resistance patterns, by their production of colicin, aerobactin, haemolysin and capsule, by their possession of transmissible R plasmids and the spvC gene, and by their ability to invade and replicate within cultured epithelial cells. The two groups differed in their antibiotic resistance profiles, with more of the host-adapted S. dublin isolates resistant to tetracycline than were the non-host-adapted S. typhimurium (Copenhagen) group, but more of the S. typhimurium (Copenhagen) isolates resistant to the other antibiotics tested. None of the isolates produced colicin, but all produced aerobactin. One isolate in each group was encapsulated. All of the S. typhimurium (Copenhagen) and S. dublin isolates contained plasmids, and all of them contained the spvC-homologous sequences. Four of the S. typhimurium (Copenhagen) isolates were able to transfer an R plasmid to a recipient organism by conjugation. One of the five S. dublin isolates, which showed resistance to some of the antibiotics tested, was able to transfer an R plasmid by conjugation. Both groups of isolates invaded cultured epithelial cells to a similar degree after 1 h, but the S. dublin isolates reached significantly higher levels within the cells than did S. typhimurium (Copenhagen) after 9 h. This ability may, in part, explain the association of S. dublin with more severe forms of salmonellosis and prolonged carrier states. Further study of the intracellular growth of these isolates seems warranted.

Colonization of the intestinal tracts of newly hatched chicks with Escherichia coli was attempted by swabbing test organisms onto the air-shell of 19-day-old embryos. Test organisms consisted of two virulent E. coli isolates, one avirulent isolate, and one laboratory-derived mutant of the avirulent isolate carrying a recombinant plasmid coding for Colicin V production. Chicks were cultured weekly for 3 weeks for total E. coli and for the test organisms using selective media. Control chicks were sampled on weeks 1 and 5, and the normal E. coli intestinal microflora were examined for the production of colicins. The two virulent E. coli isolates maintained colonization of the chicks for the 3-week test period, with titers decreasing from 10' to 10'- colony-forming units (CFU)/g of intestine. The avirulent isolate and laboratory mutant did not consistently colonize the intestinal tracts. The majority of intestinal samples taken from the control chicks at 1 and 5 weeks had colicin-producing E. coli that were inhibitory to the test organisms.

A group of complement-resistant, virulent avian Escherichia coli isolates were compared with a group of complement-sensitive, avirulent avian isolates for the presence of K-1 capsule, smooth lipopolysaccharides (LPS), the traT gene, and Colicin V (ColV) production. These parameters were selected because of their reported association with complement resistance and virulence in E. coli. Lethality in chicken embryos has also been shown to be correlated with virulence of avian E. coli for chickens. The complement-resistant, virulent E. coli isolates did not possess a K-1 capsule. Production of ColV and the presence of smooth LPS were significantly correlated with embryo lethality. There was no correlation between the presence of traT and embryo lethality. These results suggest that complement resistance and virulence in avian E. coli are associated with ColV production and smooth LPS but not with K-1 antigen or traT.

Four hundred twenty turkey and 80 chicken Escherichia coli isolates from colisepticemic birds were examined for the following properties: heat-labile toxin (LT), heat-stable enterotoxin, verotoxin, colicinogenicity, hemolysin, and hydroxamate/aerobactin production. Twenty-four (5.7%) of the 420 turkey isolates and six (7.5%) of the 80 chicken isolates produced an LT that was cytotoxic for both Vero and Y-1 cells. In contrast, 48 (11.4%) of the turkey isolates and 18 (22.5%) of the chicken isolates produced a distinct LT that was cytotoxic only for Vero cells. In addition, 64 (80.0%) of the chicken isolates and 309 (74.0%) of the turkey isolates produced aerobactin. Colicinogenicity occurred in 51 (64.0%) of the chicken isolates, with 41 (51.0%) producing colicin V. By contrast, 254 (61.0%) of the turkey isolates produced a colicin, of which 176 (42.0%) produced colicin V. None of the chicken and turkey isolates produced hemolysin or heat-stable enterotoxin.

30 strains isolated from clinical cases and a laboratory induced R-mutant or S typhi were studied. The synthesis of Colicin V, Siderophores, hydroxamate and phenolate (Aerobactin and Enterochelin) were investigated using biological assays. All strains were positive for Enterochelin but only strains 6586 and 4448 were positive for Aerobactin. An 80 kD receptor for enterochelin was found in the outer membrane of strains. Only strains 4448, 635 and 4693 produced Colicin V. Plasmid related antibiotic resistance was demonstrated in 7 strains and was considered "cryptic" in 3 others. The LD50 for mice ranged from 2.7 x 10(4) to 1 x 10(9) bacteria per ml. The presence of pathogenic factors was not related to the LD50.

Approximately 24% of a sample of pathogenic Escherichia coli strains from different serogroups were found to synthesize colicins. Serogroup O55 had an unusually large proportion of such strains (33%). In a sample of 27 O55 isolates, one synthesized a class A colicin (identified as ColE9), five produced class B colicins (three ColIa, two, unidentifiable), and three a class A and a class B together.

To study the most important epidemiologic features of shigellosis and the application of colicinotyping as an epidemiologic marker for Shigella sonnei. A total of 44.818 stoll-cultures were performed. We classify, using colicinotyping, 156 Shigella sonnei strains isolated from different patients. The incidence of Shigellosis in our media is low (1.08% of all stool-cultures). It is more frequent in pediatric population and increases on late summer and fall. We had been able to show an increasing incidence, with an hyperendemic situation during a three-years period (1981-1983). Shigella sonnei is the most prevalent species (86% of cases), followed by S. boydii (7.3%) and S. flexneri (5.9%). All S. sonnei strains epidemiologically related showed the same colicinotype. Only two strains were not typable and we identified 9 different colicinotypes, being type 13 (30.8%), type 8 (18.6%), type 6 (17.3%) and type 12 (11.54%) the more frequent types. Colicinotype 8 was the more prevalent between 1978-1979. Type 13 was predominant between 1981 and 1985. During 1987 and 1989, at the same time that incidence had risen, types 6 and 12 were prevalent. The total number of different colicinotypes identified during a single year is never greater than five. Colicinotyping of S. sonnei is a simple typing method that gives enough useful epidemiologic information, discriminative and reproducible. Although there are changes of circulating types incidence and the prevalen colicinotype colud vary from one year to another, during longer periods of time there is a reduced number of alternating colicinotypes, which sets up a situation that could be further classified as endemic.

The amino acid sequence of an active fragment (T2A) of colicin E3 composed of 97 amino acid residues was determined. The structures of 6 tryptic peptides obtained from citraconylated T2A were analyzed, mainly by Edman degradation. These tryptic peptides were aligned by examining overlapping peptides obtained from T2A with a Staphylococcal protease. These results, together with sequence analyses of whole T2A from both termini, established the complete amino acid sequence of T2A. The structure and function of colicin E3 are discussed based on the determined primary structure.

Out of 1474 Escherichia coli strains belonging to 70 serogroups and 247 serologically not identified ones, colicin V producers were found in serogroups O78, O1, O7, O18 and also among the not identified strains. The molecular weight of Col V1 plasmid from the standard strain was 70 Mdal. The Col V plasmids carried by E. coli O78 strains isolated from a hospital outbreak in Hungary had a molecular weight of 78 Mdal and also 78 Mdal was the molecular weight of the Col V plasmid carried by Rivier's strain (designated 23), which had caused meningitis in Switzerland. The molecular weight of Col V plasmids of O1: K1, O21, O161 and serologically not identified E. coli strains isolated from sporadic cases was of 94 to 119 Mdal. In the case of three strains the increase of LD50 values, which means the decrease of virulence, resulted in the loss of colicin V production and the loss of Col V plasmid. It was demonstrated by introduction of a transposon, inactivating colicin V production, into a wild type E. coli strain that the production of colicin was not essential for the increase of virulence controlled by Col V plasmid. In the case of one strain the loss of both R and Col V plasmid resulted in a decrease of virulence. No plasmid other than R and Col V was carried by this strain. The virulence determining gene could be eliminated together with both plasmids, which means that this gene could be attached equally to Col V and R plasmid.

The complete nucleotide sequence of the structural gene for colicin A has been established. This sequence consists of 1776 base-pairs. According to the predicted amino acid sequence, the colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62,989. The amino-terminal part is rich in proline and glycine and accordingly secondary structure prediction indicates that this region (1 to 185) is beta-structured. The rest of the molecule (residues 186 to 592) is very rich in alpha-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule, which is involved in the membrane depolarization caused by colicin A. A similar region has been found in colicin E1, which has the same mode of action as colicin A. Three peptides of these bacteriocins were found to be homologous, but a comparison of the bacteriocin genes did not reveal any significant homology out of the corresponding regions. The codon usage of both genes, however, exhibits some similarity and is quite different from that of genes coding for highly or weakly expressed proteins of Escherichia coli.

An Mr = 16,000 receptor-binding fragment of colicin E1 has been obtained by cyanogen bromide digestion of colicin E1. The purified 16-kDa fragment shows binding properties similar to those of an Mr = 38,000 colicin E1 receptor-binding fragment generated by thermolysin treatment. Treatment of the 38-kDa fragment with cyanogen bromide also yields the 16-kDa fragment. By comparing the NH2-terminal amino acid sequence of the 16-kDa fragment with the known colicin E1 sequence, the receptor-binding fragment can be shown to occupy the central region of the colicin molecule, extending from residue 231 to 370. It is inferred that the 16-kDa fragment binds efficiently to the colicin receptor because it is able to protect sensitive cells against the lethal effects of colicins E1 and E2 and, when pre-adsorbed to the cell, to physically displace colicin E1. Unlike the 38-kDa receptor-binding fragment, the 16-kDa fragment was found to be devoid of channel-forming ability previously shown to be associated with the COOH-terminal region of the colicin E1 polypeptide.

Organization of genes for colicin, immunity and lysis and regulation of their expression in colicinogenic plasmid ColD-CA23 were studied. The polypeptides synthesised in minicells carrying the ColD plasmid, its Tn5 mutants and the recombinant plasmids constructed by cloning the ColD EcoRV fragments on pBR325 were analysed. The position of the colicin gene promotor is established and direction of the gene transcription determined. The immunity gene was shown to be expressed independently of the gene coding for colicin and believed to have its own SOS-independent promotor. Treatment with mitomycin C of the ColD-carrying cells leads to induction of the synthesis both of colicin and of a 10 KD protein responsible for cell killing and lysis. This protein can be detected in a cultural medium under lysis. Plasmid mutations abolishing synthesis of the lysis protein prevent release of colicin. The colicin and lysis genes are arranged in an operon, the lysis gene being situated next to that of colicin. The ColD genetic map is suggested.

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.

Conformational investigations, using circular dichroism, on the pore-forming protein, colicin A (Mr 60 000), and a C-terminal bromelain fragment (Mr 20 000) were undertaken to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly alpha-helical with an enrichment of the alpha-helical content in the C-terminal domain carrying the ionophoric activity. The non-negligible beta-sheet structure in the C-terminal domain is unstable and is easily transformed into alpha-helix upon decreasing the polarity of the solvent. No evidence of pH-dependent conformational modification, correlated with modification of colicin A activity, could be obtained. The secondary structure estimated on the basis of experimental data favoured a model in which the pore is built of a minimal number of six transmembrane alpha-helical segments. Search for such segments in the amino acid sequence of the C-terminal domain of colicin A was carried out by combining secondary structure prediction methods with hydrophobicity and hydrophobic movement calculations. Similar calculations on the C-terminal domains of colicin E1 and IB indicate a common structure of the pores formed by colicin A, E1 and IB. Only two or three putative transmembrane segments could be selected in the sequences of colicin A, IB or E1. As a result, it is concluded that the channel is probably not built by a single colicin molecule but more likely by an oligomer.

Colicin M was isolated from Escherichia coli K-12 32T 19F/T1. The purified, biologically active protein had a molecular weight of 27,000. It contained phosphatidyl ethanolamine. The molecular weight found for the polypeptide chain by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 18,000. Colicin M was found to be firmly integrated in the membrane of the producing strain. The action of the colicin seems to be on the membrane, since cells of the susceptible strain E. coli K-12 ROW/V/22.1 lyse rapidly. Using the phase contrast microscope, lysis was followed by decrease in turbidity of the cell culture and release of protein into the medium. Lysis started at about 15 min after addition of colicin M and was completed after 40 to 60 min. At this time, one-third of the protein had been released from the cells. The number of viable cells dropped within 10 min to 0.01%. Colicin M induced formation of spheroplasts in the presence of 16% sucrose. The electron microscope examination revealed that at first bulges in the cell envelope appear, most frequently occurring equatorially but also occurring at sites all over the cell. In the process of spheroplast formation, the cytoplasmic membrane often retreats from one-half of the outer membrane so that the cytoplasm is confined to one hemisphere. Sucrose did not prevent cells from dying unless cells were pregrown in a sucrose containing medium for several generations before colicin M was added. With cells pregrown in the presence of sucrose, the number of survivors was 100 times higher than in the absence of sucrose.

The region of the colicin E1 polypeptide that interacts with immunity protein has been localized to a 168-residue COOH-terminal peptide. This is the length of a proteolytically generated peptide fragment of colicin E1 against which imm+ function can be demonstrated in osmotically shocked cells. The role of particular amino acids of the COOH-terminal peptide in the expression of the immune phenotype was studied. Chemical modification showed that the two histidine residues (His 427 and His 440) and the single cysteine residue (Cys 505) present in the COOH-terminal peptide were not necessary for the colicin-immunity protein interaction. The immunity protein was localized in the cytoplasmic membrane fraction, consistent with previous work of others on the colicin Ia immunity protein and the prediction from the immunity protein amino acid sequence that it is a hydrophobic protein. The distribution of hydrophobic residues along the immunity polypeptide was calculated.

Colicinotype 7 of Shigella sonnei, introduced by Abbott and Graham, is one of those most frequently found in epidemiological screenings. It is represented by the production of a single colicin (acidic protein of m. w. 46 kD) endowed with some striking features. It is unstable at pH 8.0 and at the temperature of 70 degrees C. Its inhibitive activity is specific for the species Shigella sonnei, most strains of which seem to be sensitive. It is the only colicin known so far inactive towards Escherichia coli, including the indicator strains of broad-spectrum sensitivity, such as K12 Row. It shows no cross-resistance with any other colicin type. Its adsorption on sensitive bacteria and inhibitive effect on them proceed extremely quickly. As indicated by indirect fluorimetry, its mode of action is extremely quickly. As indicated by indirect fluorimetry, its mode of action is probably different from those known for other colicins: it does not concern the plasma membrane of sensitive bacteria under aerobiosis, but it interferes with it under anaerobic conditions. These data show clearly that it is a unique colicin type. It is proposed to include it into the current system of classified colicins as "colicin Js".

The scheme of colicin typing of Shigella sonnei includes 26 colicin types. Thirty-three indicator strains were used for typing and 22 of them were prepared by conjugational transfer of col factors from shigellae on recipient strain of E. coli K 13 HfrR nalr or E. coli K 12 ROW. Immune strains of Sh. sonnei or resistant mutants of E. coli K 12 ROW were used as indicator strains only sporadically.

Purification of a bacteriocin, colicin, from Escherichia coli HSC10, is described. A 1,800 fold purified colicin was obtained and found to be an acidic polypeptide with a molecular weight of 82,000 daltons. The effect of colicin on bacterial and mammalian cells during the transition from a crude to a pure preparation is given. A turbidimetric assay for bacterial growth inhibition and 3H-thymidine uptake inhibition for measuring the effect on mammalian cells, was used. Colicin HSC10 caused DNA loss from the bacteriocin-sensitive mammalian cells, which increased with dose and time of exposure. Therefore, flow-cytometry, which detects DNA loss from the bacteriocin-affected mammalian cells, was also used to evaluate the bacteriocin potency. Similar quantitative results were obtained to those using 3H-thymidine uptake inhibition, in terms of microgram protein of pure colicin required to affect adversely 50% of the cells. The bacteriocins were found to retain their activity following freezing and thawing, both as crude and pure preparations, against both bacterial and mammalian cells.

Twelve pathogenic and seven nonpathogenic enterotoxigenic Escherichia coli strains which were previously identified as belonging to serogroup O8:KX105 (A. Broes, J. M. Fairbrother, S. Larivière, M. Jacques, and W. M. Johnson, Infect. Immun. 56:241-246, 1988) were further examined for their phenotypic and genotypic properties. Only the 12 pathogenic strains were confirmed to possess the capsular antigen KX105. The seven nonpathogenic strains did not possess this antigen and thus were incorrectly assigned to have capsular antigen KX105. All seven nonpathogenic strains apparently possessed a previously unrecognized capsular antigen which has been designated K"2829". Studies with antisera prepared against F1 (type 1) fimbriae from three E. coli strains suggested that at least three antigenic subtypes of F1 fimbriae were represented among the O8:KX105 strains examined. By using serotyping, biotyping, and outer membrane protein profile analyses, the O8:KX105 strains were divided into at least two distinct clusters, whereas the O8:K"2829" strains were grouped into a single unique cluster. Most of the strains of the same cluster were further differentiated by testing for antibiotic resistance and colicin production and resistance and by analysis of plasmid content. With the exception of one strain which lost its enterotoxicity during storage, all of the O8:KX105 strains hybridized with the gene probes for the heat-labile (LT) and heat-stable (STb) enterotoxins. For each O8:KX105 strain, a single plasmid ranging in size from 61 to 77 megadaltons carried the LT and STb genes. All of the enterotoxigenic O8: KX105 strains fermented sorbose, whereas the nonenterotoxigenic strain did not. All of the O8:K "2829" strains hybridized with the STb probe only. For each O8:K "2829" strain, the STb genes were located on a single plasmid of 61 or 22 megadaltons. None of the strains demonstrated homology with the genes encoding the F4 (K88), F5 (K99), F6 (987P), and F41 fimbrial antigens and STaP and STaH.

The use of complex typing of Sh. sonnei by the colicinogenic and enzymatic properties in epidemiological analysis of dysentery caused by Sh. sonnei, apart from establishment of epidemiological connections in the foci, offered a possibility of introducing significant corrections in the determination of the sizes and duration of existence of the epidemic foci, as well as in detection of the sources and factors of transmission of the infection established in epidemiological examination. Typing of Sh. sonnei also aided in ascertaining that, apart from general regularities in the individual territories, the epidemic process in individual groups of the population and in different seasons of the year could also course autonomously.

We reported elsewhere (Varenne et al., 1982) that, during synthesis of a number of colicins in Escherichia coli, intermediate nascent chains of discrete sizes accumulated, suggesting a variable rate of translation. In this paper, a detailed analysis provides arguments that this phenomenon, at least for the proteins under study, is not related to aspects of messenger RNA such as secondary structure. It is linked to the difference in transfer RNA availability for the various codons. Experimental analysis of translation of other proteins in E. coli confirms that the main origin for the discontinuous translation in the polypeptide elongation cycle is the following. For a given codon, the stochastic search of the cognate ternary complex (aminoacyl-tRNA-EF-Tu-GTP) is the rate-limiting step in the elongation cycle: transpeptidation and translocation steps are much faster. The degree of slackening in ribosome movement is almost proportional to the inverse of tRNA concentrations. The verification of this model and its possible physiological significance are discussed.