Gyrase-dependent initiation of bacteriophage T4 DNA replication: interactions of Escherichia coli gyrase with novobiocin, coumermycin and phage DNA-delay gene products.

Wild-type bacteriophage T4 and DNA-delay am mutants defective in genes 39, 52, 60 and 58–61 were tested for intracellular sensitivity to the antibiotics coumermycin and novobiocin, drugs which inhibit the DNA gyrase of Escherichia coli. Treatment with these antibiotics drastically reduced the characteristic growth of gene 39, 52 and 60 DNA-delay am mutants in E. coli lacking an amber suppressor (su^?). Wild-type phage-infected cells were unaffected by the drugs while the burst size of a gene 58–61 mutant was affected to an intermediate extent. A su^?E. coli strain which is resistant to coumermycin due to an altered gyrase permitted growth of the DNA-delay am mutants in the presence of the drug. Thus, the characteristic growth of the DNA-delay am mutants in an su^? host apparently depends on the host gyrase. An E. coli himB mutant is defective in the coumermycin-sensitive subunit of gyrase (H. I. Miller, personal communication). Growth of the gene 39, 52 and 60 am mutants was inhibited in the himB mutant while the gene 58–61 mutant and wild-type T4 showed small reductions in burst size in this host. Experiments with nalidixic acid-sensitive and resistant strains of E. coli show that wild-type phage T4 requires a functional nalA protein for growth.Novobiocin and coumermycin inhibit phage DNA synthesis in DNA-delay mutant-infected su^?E. coli if added during the early logarithmic phase of phage DNA synthesis. The gene 58–61 mutant showed the smallest inhibition of DNA synthesis in the presence of the drugs. Addition of the drugs during the late linear phase of phage DNA synthesis had no effect on further synthesis in DNA-delay mutant-infected cells. Coumermycin and novobiocin had no effect on DNA synthesis in wild-type-infected cells regardless of the time of addition of the antibiotics. Models are considered in which the DNA-delay gene products either form an autonomous phage gyrase or interact with the host gyrase and adapt it for proper initiation of phage DNA replication.     

Ambivalent bacteriophages of different species active on Escherichia coli K12 and Salmonella sp. strains

A study was made of several bacteriophages (including phages U2 and LB related to T-even phages of Escherichia coli) that grow both on E. coli K12 and on some Salmonella strains. Such phages were termed ambivalent. T-even ambivalent phages (U2 and LB) are rare and have a limited number of hosts among Salmonella strains. U2 and LB are similar to canonical E. coli-specific T-even phages in morphological type and size of the phage particle and in reaction with specific anti-T4 serum. Phages U2 and LB have identical sets of structural proteins, some of which are similar in size to structural proteins of phages T2 and T4. DNA restriction patterns of phages U2 and LB differ from each other and from those of T2 and T4. Still, DNAs of all four phages have considerable homology. Unexpectedly, phages U2 and LB grown on Salmonella bongori were unstable during centrifugation in a CsCl gradient. Ambivalent bacteriophages were found in species other than T-even phages and were similar in morphotype to lambdoid and other E. coli phages. One of the ambivalent phages was highly similar to well-known Felix01, which is specific for Salmonella. Ambivalent phages can be used to develop a new set for phage typing in Salmonella. An obvious advantage is that ambivalent phages can be reproduced in the E. coli K12 laboratory strain, which does not produce active temperature phages. Consequently, the resulting typing phage preparation is devoid of an admixture of temperate phages, which are common in Salmonella. The presence of temperate phages in phage-typing preparations may cause false-positive results in identifying specific Salmonella strains isolated from the environment or salmonellosis patients. Ambivalent phages are potentially useful for phage therapy and prevention of salmonellosis in humans and animals.     

Morphological, Host Range, and Genetic Characterization of Two Coliphages

Two coliphages, AR1 and LG1, were characterized based on their morphological, host range, and genetic properties. Transmission electron microscopy showed that both phages belonged to the Myoviridae; phage particles of LG1 were smaller than those of AR1 and had an isometric head 68 nm in diameter and a complex contractile tail 111 nm in length. Transmission electron micrographs of AR1 showed phage particles consisting of an elongated isometric head of 103 by 74 nm and a complex contractile tail 116 nm in length. Both phages were extensively tested on many strains of Escherichia coli and other enterobacteria. The results showed that both phages could infect many serotypes of E. coli. Among the enterobacteria, Proteus mirabilis, Shigella dysenteriae, and two Salmonella strains were lysed by the phages. The genetic material of AR1 and LG1 was characterized. Phage LG1 had a genome size of 49.5 kb compared to 150 kb for AR1. Restriction endonuclease analysis showed that several restriction enzymes could degrade DNA from both phages. The morphological, genome size, and restriction endonuclease similarities between AR1 and phage T4 were striking. Southern hybridizations showed that AR1 and T4 are genetically related. The wide host ranges of phages AR1 and LG1 suggest that they may be useful as biocontrol, therapeutic, or diagnostic agents to control and detect the prevalence of E. coli in animals and food.     

Cloned genes for bacteriophage T4 late functions are expressed in Escherichia coli

We have constructed derivatives of plasmid pMB9 carrying EcoRI digestion fragments of bacteriophage T4 DNA that code for late gene functions. When Escherichia coli strains carrying these plasmids are infected with T4 amber mutants, burst sizes up to 30% of the wild-type level are obtained. Single burst experiments imply that the phage progeny result from complementation and do not depend on marker rescue. By electrophoretic and immunological techniques, we have established that the cloned T4 late genes are transcribed and translated in uninfected cells. A serum blocking assay has been used to quantitate the levels of one of the T4 gene products, gp11, before and after T4 infection. Uninfected cells containing the cloned T4 gene 11 DNA have 0.1% and mini cells have 1% of the gp11 levels per unit protein found in cells late after T4 wild-type infection. There is little or no additional gp10 and gp11 formed from the cloned genes after T4 infection.     

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

Bacteriophage T4 mutants deficient in alteration and modification of the Escherichia coli RNA polymerase

An extensive screening of coliphage T4 mutants has revealed two distinct classes defective, respectively, in the two sequential phage-induced phosphorylations of the host RNA polymerase, alteration and modification. The existence of these mutants proves that T4-specified functions are involved in both processes. The viabilities of these mutants demonstrate that neither alteration nor modification is essential for growth in Escherichia coli B/r. Physiological studies after infection of E. coli B/r have failed to reveal any abnormalities of phage deficient in alteration or modification. Both mutants normally inhibit host protein and stable RNA synthesis and normally express all classes of T4 genes. Thus, these specific phage-induced structural changes in the host RNA polymerase are not fundamental to the control of gene expression during T4 development. Alteration and modification may be required for growth in some strains of E. coli and hence be selectively advantageous because they extend the normal host range of the phage.Alteration appears to be catalyzed by a T4 function injected with the DNA. A polypeptide of molecular weight 61,000, which is probably cleaved during morphogenesis from a precursor of molecular weight 79,000, is missing in phage particles of alteration-deficient strains and may be the phage activity so injected. The T4 gene involved in alteration is named alt.Modification is controlled by a T4-replicative gene that has been mapped into a region of about 500 base-pairs between genes 39 and 56. These mapping data show that the defect in α modification defines a new T4 gene, named mod.     

Replication and Recombination of Gene 59 Mutant of Bacteriophage T4D

After infection of Escherichia coli B with phage T4D carrying an amber mutation in gene 59, recombination between two rII markers is reduced two- to three-fold. This level of recombination deficiency persists even when burst size similar to wild type is induced by the suppression of the mutant DNA-arrest phenotype. In the background of two other DNA-arrest mutants in genes 46 and 47, a 10- to 11-fold reduction in recombination is observed. The cumulative effect of gene 59 mutation on gene 46-47 mutant suggests that complicated interactions must occur in the production of genetic recombinants. The DNA-arrest phenotype of gene 59 mutant can be suppressed by inhibiting the synthesis of late phage proteins. Under these conditions, DNA replicative intermediates similar to those associated with wild-type infection are induced. Synthesis of late phage proteins, however, results in the degradation of mutant 200S replicative intermediate into 63S DNA molecules even in the absence of capsid assembly. Although these 63S molecules are associated with membrane, they do not replicate. These results suggest a role for gene 59 product, in addition to a possible requirement of concatemeric DNA in late replication of phage T4 DNA.     

Apparent non-involvement of transfer RNA nucleotidyltransferase in the biosynthesis of Escherichia coli suppressor transfer RNAs

Transfer RNA nucleotidyltransferase has previously been shown to be required for the normal growth of Escherichia coli and for the biosynthesis of some bacteriophage T4 tRNAs. In order to obtain information about the involvement of this enzyme in E. coli tRNA biosynthesis we have measured the level of activity of suppressors 1 to 6 in strains carrying either a cca⁺ or cca allele. We found that cca strains, deficient in tRNA nucleotidyltransferase, contained the same amount of suppressor activities as the wild-type cca⁺ strains as determined by suppression of nonsense mutations in both E. coli alkaline phosphatase and in genes of bacteriophage T4. The results suggest that tRNA nucleotidyltransferase is not required for the biosynthesis of tRNAs specified by suppressors 1 to 6.     

Polynucleotide Sequence Divergence Among Strains of Escherichia coli and Closely Related Organisms

Polynucleotide sequence similarity tests were carried out to determine the extent of divergence present in a number of Escherichia coli strains, obtained from diverse human, animal, and laboratory sources, and closely related strains of Shigella, Salmonella, and the Alkalescens-Dispar group. At 60 C, relative reassociation of deoxyribonucleic acid (DNA) from the various strains with E. coli K-12 DNA ranged from 100 to 36%, with the highest level of reassociation found for three strains derived from K-12, and the lowest levels for two “atypical” E. coli strains and S. typhimurium. The change in thermal elution midpoint, which indicates the stability of DNA duplexes, ranged from 0.1 to 14.5 C, with thermal stability closely following the reassociation data. Reassociation experiments performed at 75 C, at which temperature only the more closely related DNA species form stable duplexes, gave similar indications of relatedness. At both temperatures, Alkalescens-Dispar strains showed close relatedness to E. coli, supporting the idea that they should be included in the genus Escherichia. Reciprocal binding experiments with E. coli BB, 02A, and K-12 yielded different reassociation values, suggesting that the genomes of these strains are of different size. The BB genome was calculated to be 9% larger than that of K-12, and that of 02A 9% larger than that of BB. Calculation of genome size for a series of E. coli strains yielded values ranging from 2.29 × 10⁹ to 2.97 × 10⁹ daltons. E. coli strains and closely related organisms were compared by Adansonian analysis for their relatedness to a hypothetical median strain. E. coli 0128a was the most closely related to this median organism. In general, these data compared well with the data from reassociation experiments among E. coli strains. However, anomalous results were obtained in the cases of Shigella flexneri, S. typhimurium, and “atypical” E. coli strains.     

Marker rescue of the adsorption properties of bacteriophage T 4 by bacteriophage T 2

Rescue of adsorption properties from UV-irradiated T4 by T2 as a helper phage, revealed progeny phage with intermediate properties. Fourteen independent progeny phages, plating onE. coli B/2, were plated on several indicator strains and their adsorption properties were also studied with specific T4 antibodies. Two of these, plating onE. coli KS/4, were not inactivated by the T4 antiserum, and were T2h without apparent T4 properties. The other 12 progeny phages did not plate on KS/4, and were inactivated, but at a slower rate than the parental T4. Their mean efficiency of plating onE. coli B/2 (0.83) was significantly lower than that of the parental T4. The efficiency of plating was positively correlated with the velocity of inactivation by T4 antiserum. The observations were explained by assuming that the progeny phages were recombinants of T4 and T2 loci for adsorption sites. Plating of these 12 progeny phages on several indicator strains showed that they were allrII mutants and all, except one, wererI mutants too. In addition, two weretu andh ⁴, respectively. The condition for the appearance of multiple mutants might be a complementation by T2 of UV-damaged functions, which otherwise fail to induce the completion of the lytic cycle in monocomplexes of extracellularly irradiated T4.     

Wide-Host-Range Plasmids Function in the Genus Thiobacillus

Plasmids S-a, RP4, R388, and several RP4 derivatives (pMD101, pDT387, and pDT566) were transmissible by conjugation to Thiobacillus novellus from Escherichia coli. Genetic markers were expressed in T. novellus, with the exception of chloramphenicol resistance and ampicillin resistance. Plasmids were not transmissible by conjugation from E. coli donors to Thiobacillus intermedius, T. perometabolis, T. neapolitanus, or T. acidophilus recipients, although they could be mated into these strains from T. novellus. All Thiobacillus species tested could transfer plasmids back to E. coli, with the exception of T. acidophilus. The donor-specific bacteriophages PRR1 and PRD1 were incapable of initiating the lytic cycle in RP4-bearing strains of T. novellus. The cosmid cloning vehicle pVK100 could be mobilized from E. coli to T. novellus with the aid of the “helper” plasmid pRK2013. pVK100 is stable in T. novellus, but pRK2013 is not maintained in this species. pRK2013 was also used to mobilize another cloning vector, R300B, to T. novellus. A previously unreported cryptic plasmid of approximately 24 megadaltons was observed in T. intermedius. No native plasmids were demonstrated in the other Thiobacillus species except in T. acidophilus, which contained cryptic plasmids ranging in size from 7.6 to 56 megadaltons (molecular mass).     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates I. Identification of T4D Gene 28 Product in the Tail Plug

The T4D bacteriophage gene 28 product is a component of the central plug of the tail baseplate, as shown by the following two independent lines of evidence. (i) A highly sensitive method for radioactive labeling of only tail baseplate plug components was developed. These labeled plug components were incorporated by a complementation procedure into new phage particles and were analyzed by radioautography after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three new structural proteins were found in addition to the three known tail plug proteins (i.e., gP29, gP27, and gP5). One of the three newly identified components had a molecular weight of 24,000 to 25,000 and appeared to be a product of T4D gene 28. (ii) Characterization of mutants of Escherichia coli bacteriophage T4D which produced altered gene 28 products also indicated that the gene 28 product was a viral tail component. T4D 28^ts phage particles produced at the permissive temperature had altered heat labilities compared with parent T4D particles. We isolated a single-step temperature revertant of T4D 28^ts and found that it produced phage particles which phenotypically resembled the original T4D particles. Since the properties of the phage baseplate components usually determine heat lability, these two changes in physical stability after two sequential single mutations in gene 28 supported the other evidence that the gene 28 product was a viral baseplate component. Also, compared with parent T4D particles, T4D 28^ts and T4D 28am viral particles adsorbed at different rates to various types of host cells. In addition, T4D 28^ts particles exhibited a different host range than parent T4D particles. This T4D mutant formed plaques with an extremely low efficiency on all E. coli K-12 strains tested. We found that although T4D 28^ts particles adsorbed rapidly and irreversibly to the E. coli K-12 strains, as judged by gene rescue experiments, these particles were not able to inject their DNA into the E. coli K-12 strains. On the other hand, the T4D 28^ts revertant had a plating efficiency on E. coli K-12 strains that was quite similar to the plating efficiency of the original parent, T4D. These properties of phage particles containing an altered gene 28 product supported the analytical finding that the gene 28 product is a structural component of the central plug of the T4D tail baseplate. They also indicated that this component plays a role in both host cell recognition and viral DNA injection.     

Cloning of linear DNAs in vivo by overexpressed T4 DNA ligase: construction of a T4 phage hoc gene display vector.

A method was developed to clone linear DNAs by overexpressing T4 phage DNA ligase in vivo, based upon recombination deficient E. coli derivatives that carry a plasmid containing an inducible T4 DNA ligase gene. Integration of this ligase-plasmid into the chromosome of such E. coli allows standard plasmid isolation following linear DNA transformation of the strains containing high levels of T4 DNA ligase. Intramolecular ligation allows high efficiency recircularization of cohesive and blunt-end terminated linear plasmid DNAs following transformation. Recombinant plasmids could be constructed in vivo by co-transformation with linearized vector plus insert DNAs, followed by intermolecular ligation in the T4 ligase strains to yield clones without deletions or rearrangements. Thus, in vitro packaged lox-site terminated plasmid DNAs injected from phage T4 were recircularized by T4 ligase in vivo with an efficiency comparable to CRE recombinase. Clones that expressed a capsid-binding 14-aa N-terminal peptide extension derivative of the HOC (highly antigenic outer capsid) protein for T4 phage hoc gene display were constructed by co-transformation with a linearized vector and a PCR-synthesized hoc gene. Therefore, the T4 DNA ligase strains are useful for cloning linear DNAs in vivo by transformation or transduction of DNAs with nonsequence-specific but compatible DNA ends.     

A mutation of the wobble nucleotide of a bacteriophage T4 transfer RNA.

Wild-type bacteriophage T7 is not subject to restriction by the Escherichia coli B and K restriction systems, but T7 mutants that are susceptible to such restriction have been isolated. These mutants are all defective in gene 0.3, the first T7 gene to be expressed after infection. The gene 0.3 protein apparently acts to prevent modification as well as restriction, suggesting that it may interact with a component of the host restriction-modification system that is required for both processes. Mutants in which gene 0.3 is completely deleted are only partially modified by growth on hosts with an active restriction-modification system, presumably because the conditions of T7 infection overload the modifying capacity of the cells. This is in contrast to phages such as lambda that are completely modified during growth. Since gene 0.3 is not essential for growth in non-restricting hosts, it has been possible to isolate deletions which extend to the left of gene 0.3 into the region where E. coli RNA polymerase initiates the synthesis of T7 early RNA. Two of the three strong initiators from which E. coli RNA polymerase transcribes the early region can be deleted.In the course of searching for T7 mutants that are unable to overcome restriction, it was discovered that mutants defective in gene 2 are able to plate on E. coli C with essentially normal efficiency, and most gene 7 mutants are able to plate on both C and K strains. It has not been determined why genes 2 and 7 seem to be needed for growth in some E. coli strains but not in others.     

Effect of T4 infection on initiation of protein synthesis and messenger specificity of initiation factor 3

No alteration in the messenger specificity of initiation factor 3 (IF-3) is observed upon T4 phage infection of several strains of Escherichia coli. IF-3 present in the 1.0 m NH₄Cl washes of ribosomes from T4-infected cells supports the translation of f2 RNA and T4 late mRNA with the same degree of efficiency as the IF-3 in the ribosomal washes obtained from uninfected cells. At high concentrations the ribosomal washes obtained from T4-infected cells are more inhibitory for both f2 RNA- and T4 late mRNA-directed protein synthesis than the ribosomal washes from uninfected cells. Furthermore, this increased inhibition is also observed in the poly(U)-directed synthesis of polyphenylalanine. These data suggest that translational controls exerted at the level of IF-3 probably do not account for the alterations in protein synthesis observed upon T4 infection.     

Insulin action on Escherichia coli Regulation of the adenylate cyclase and phosphotransferase enzymes

Insulin action on Escherichia coli was studied using wild type E. coli B/r and K12 strains and a number of phosphoenolpyruvate phosphotranferase mutants. In vivo, the effects of insulin on the differential rate of tryptophanase synthesis, The rate of α-methyglucoside uptake and the rate of growth on glucose were determined in E. coli B/r. in vitro, the effect of insulin on the adenylate cyclase and the phosphotransferase activities was determined using toluenized cell preparations of E. coli B/r, E. coli K12 and phosphotransferase mutant strains. The specificity of insulin action on E. coli was determined using glucagon, vasopressin and somatropin as well as insulin antisera. Results show the specific action of insulin n E. coli, inhibiting tryptophanase induction and adenylate cyclase activity, while stimulating growth on glucose and uptake and phosphorylation of α-methylglucosode     

Nitrous acid induced damage in T7 DNA and phage

T7 phage was exposed to 56 mM nitrous acid at pH 4.6 causing a 90% decrease in survival for each 10 min duration of exposure. The survival of phage made by encapsulating nitrous acid treated DNA into empty phage heads was nearly the same as the survival of phage exposed to nitrous acid in vivo. In contrast to previous reports, growth of SOS-induced wild-type E. coli showed no increase in survival. The survival of nitrous acid treated phage was not lowered when grown on E. coli strains deficient in DNA polymerase I, exonuclease III, and the uvrA component of the nucleotide excision-repair endonuclease. Therefore, these enzymes are not vital for repair of nitrous acid induced damage in bacteriophage T7.     

Dual Functions of Bacteriophage T4D Gene 28 Product: Structural Component of the Viral Tail Baseplate Central Plug and Cleavage Enzyme for Folyl Polyglutamates II. Folate Metabolism and Polyglutamate Cleavage Activity of Uninfected and Infected Escherichia coli Cells and Bacteriophage Particles

We investigated the role of the T4D bacteriophage gene 28 product in folate metabolism in infected Escherichia coli cells by using antifolate drugs and a newly devised assay for folyl polyglutamate cleavage activity. Preincubation of host E. coli cells with various sulfa drugs inhibited phage production by decreasing the burst size when the phage particles produced an altered gene 28 product (i.e., after infection under permissive conditions with T4D 28^ts or T4D am28). In addition, we found that another folate analog, pyrimethamine, also inhibited T4D 28^ts production and T4D 28am production, but this analog did not inhibit wild-type T4D production. A temperature-resistant revertant of T4D 28^ts was not sensitive to either sulfa drugs or pyrimethamine. We developed an assay to measure the enzymatic cleavage of folyl polyglutamates. The high-molecular-weight folyl polyglutamate substrate was isolated from E. coli B cells infected with T4D am28 in the presence of labeled glutamic acid and was characterized as a folate compound containing 12 to 14 labeled glutamate residues. Extracts of uninfected bacteria liberated glutamate residues from this substrate with a pH optimum of 8.4 to 8.5. Extracts of bacteriophage T4D-infected E. coli B cells exhibited an additional new folyl polyglutamate cleavage activity with a pH optimum of about 6.4 to 6.5, which was clearly distinguished from the preexisting activity in the uninfected host cells. This new activity was induced in E. coli B cells by infection with wild-type T4D and T4D amber mutants 29⁻, 26⁻, 27⁻, 51⁻, and 10⁻, but it was not induced under nonpermissive conditions by T4D am28 or by T4D 28^ts. Mutations in gene 28 affected the properties of the induced cleavage enzyme. Wild-type T4D-induced cleavage activity was not inhibited by pyrimethamine, whereas the T4D 28^ts activity induced at a permissive temperature was inhibited by this folate analog. Folyl polyglutamate cleavage activity characteristic of the activity induced in host cells by wild-type T4D or by T4D gene 28 mutants was also found in highly purified preparations of these phage ghost particles. The T4D-induced cleavage activity could be inhibited by antiserum prepared against highly purified phage baseplates. We concluded that T4D infection induced the formation of a new folyl polyglutamate cleavage enzyme and that this enzyme was coded for by T4D gene 28. Furthermore, since this gene product was a baseplate tail plug component which had both its antigenic sites and its catalytic sites exposed on the phage particle, it was apparent that this enzyme formed part of the distal surface of the phage baseplate central tail plug.     

Evaluation of Lactobacillus strains for selected probiotic properties

Eleven strains of Lactobacillus collected in the Culture Collection of Dairy Microorganisms (CCDM) were evaluated for selected probiotic properties such as survival in gastrointestinal fluids, antimicrobial activity, and competition with non-toxigenic Escherichia coli O157:H7 for adhesion on Caco-2 cells. The viable count of lactobacilli was reduced during 3-h incubation in gastric fluid followed by 3-h incubation in intestinal fluid. All strains showed antimicrobial activity and the three most effective strains inhibited the growth of at least 16 indicator strains. Antimicrobial metabolites of seven strains active against Lactobacillus and Clostridium indicator strains were found to be sensitive to proteinase K and trypsin, which indicates their proteinaceous nature. The degree of competitive inhibition of non-toxigenic E. coli O157:H7 adhesion on the surface of Caco-2 cells was strain-dependent. A significant decrease (P?<?0.05) in the number of non-toxigenic E. coli O157:H7 adhering to Caco-2 cells was observed with all lactobacilli. Three strains were selected for additional studies of antimicrobial activity, i.e., Lactobacillus gasseri CCDM 215, Lactobacillus acidophilus CCDM 149, and Lactobacillus helveticus CCDM 82.     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption. I. Isolation and genetic characterization

Nuclear disruption in T4 phage-infected Escherichia coli as well as the morphology of the nuclear regions in uninfected E. coli can be observed by phase microscopy of cells spread on a thin layer of 17.5% gelatin. We have used this procedure to identify for the first time mutants of phage T4 which fail to induce nuclear disruption. The mutant phenotypes have been further characterized by thin-section electron microscopy.Nuclear disruption is not essential for phage growth. Burst-size and growth-rate experiments indicate that the nuclear disruption-deficient (ndd) mutants grow as well as wild-type T4D under the conditions and in the E. coli strains commonly used in our laboratory.Mapping experiments using multiple amber mutants and rII mutants with deletions extending into the D region adjacent to the rIIB gene indicate that the ndd mutations are located in gene D2b.