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PubMed1989

Selection and evolution of bacteriophages in cellstat.

Objectives of this work were as follows: 1. to establish a laboratory experimental system utilizable in a biophysical approach to molecular evolution; and 2. to provide real world parameters to theories of molecular evolution, especially to Eigen's theory of quasi-species. Secretion type bacteriophage fd of E. coli, closely related phages and artificial chimera phages of fd, and a virulent phage Q beta of E. coli were cultured continuously in a specially designed fermenter called a "cellstat". A phage is cultured in a flow of host bacterial cells. Due to its high dilution rate, the mutant cell could not be selected in the cellstat. It was therefore recognized that the cellstat is suitable for study of the selection and evolution process of a bacteriophage under well-defined environmental conditions without interference from host cell mutations. Population dynamics of bacteriophages of various types in the cellstat were studied theoretically by computer simulation and experimentally. A genetically invariable pure population of phage behaves like an open non-linear chemical reaction system. An invariable mixed population shows a selection process, while a variable population generates an evolution process. Kinetic constants describing the dynamics were determined by curve fitting between the theoretical and the experimental curve obtained from competition experiments and from biological relaxation experiments. One of the most important kinetic parameters thus obtained was the selection coefficient, and its dependence on the base sequence of phage DNA. We drew a local landscape of the selection coefficient near the fd sequence on the base sequence space. From this landscape we were able to confirm the importance of slightly deleterious mutants in molecular evolution. We also confirmed the possibility of developing an evolutionary molecular engineering using a cellstat as an evolution reactor and fd phage as a working replicon. Novelties of this work were as follows: 1. the first stable continuous culture of a bacteriophage was achieved with a cellstat; 2. a local landscape of selection coefficient near the fd sequence on the sequence space was the first experimental drawing of such a map; 3. a biological relaxation method was realized to measure kinetic constants of a biological kinetic process, or molecular evolution; and 4. a practical engineering process of evolutionary molecular engineering was proposed.

Bacteriophages↗
PubMed1985

The receptor specificity of bacteriophages can be determined by a tail fiber modifying protein.

T-Even type bacteriophages recognize their cellular receptors with the distal ends of their long tail fibers. The distal part of these fibers consists of a dimer of gene product (gp) 37. The assembly of this gp to a functional dimer requires the action of two other proteins, gp57 and gp38. Genes (g) 38 have been cloned from five T-even type phages which use the Escherichia coli outer membrane protein OmpA as a receptor. The phages used differ in their ability to infect a series of ompA mutants producing altered OmpA proteins, i.e., each phage has a specific host range for these mutants. The cloned genes 38 complemented g38 amber mutants of phage T2, which uses the outer membrane protein OmpF as a receptor. The complemented phages had become phenotypically OmpA-dependent and, with one exception, OmpF-independent, but regained the host range of T2 upon growth in a host lacking the cloned g38. The host range of the complemented phages, as determined on the ompA mutants, was identical to, similar to, or different from that of the phage, from which the cloned g38 originated. The results presented show that gp38 from one phage can phenotypically 'imprint', in a finely-tuned manner, a host range onto gp37 of another phage with a different host specificity. In view of the extreme diversity of host ranges observed, it is suggested that gp38 of T2 and of the OmpA-specific phages may remain attached to gp37 in the phage particle and in cooperation with gp37 determine the host range.

Bacterial Outer Membrane Proteins↗
PubMed2000

Big-benefit mutations in a bacteriophage inhibited with heat.

High temperature inhibits the growth of the wild-type bacteriophage phiX174. Three different point mutations were identified that each recovered growth at high temperature. Two affected the major capsid protein (residues F188 and F242), and one affected the internal scaffolding protein (B114). One of the major capsid mutations (F242) is located in a beta strand that contacts B114 in the procapsid during viral maturation, whereas the other capsid mutation (F188) is involved in subunit interactions at the threefold axis of symmetry. Selective coefficients of these mutations ranged from 13.9 to 3.8 in the inhibitory, hot environment, but all mutations reduced fitness at normal temperature. The selective effect of one of the mutations (F242) was evaluated at high temperature in four different genetic backgrounds and exhibited epistasis of diminishing returns: as log fitness of the background genotype increased from -0.1 to 4.1, the fitness boost provided by the F242 mutation decreased from 3.9 to 0. 8. These results support a model in which viral fitness is bounded by an upper limit and the benefit of a mutation is scaled according to the remaining opportunity for fitness improvement in the genome.

Bacteriophage phi X 174↗
PubMed1977

Physical structure of the replication origin of bacteriophage lambda.

The nucleotide sequence of part of the replication region of wild-type bacteriophage lambda and of four mutants defective in the origin of DNA replication (ori-) has been determined. Three of the ori- mutations are small deletions, and one is a transversion. The sequence of the origin region, defined by these mutations, contains a number of unusual features.

Base Sequence↗
PubMed1974

Adriamycin and daunorubicin inhibition of mutant T4 DNA polymerases.

The anticancer drugs, adriamycin and daunorubicin, as well as two other DNA reagents, ethidium bromide and 9-aminoacridine, all exert a differential inhibitory effect on nucleotide incorporation for purified DNA polymerases induced by mutant and wild-type bacteriophage T4. When compared with DNA polymerase of wild-type phage, antimutator enzymes are inhibited to a far greater extent and mutator enzymes to a lesser extent. In contrast, the polymerase-associated 3'-exonuclease activities of wild type and mutants are also inhibited by the compounds but nondifferentially.

Acridines↗
PubMed1994

Comparison of traditional and molecular methods of typing isolates of Staphylococcus aureus.

Fifty-nine Staphylococcus aureus isolates and 1 isolate of Staphylococcus intermedius were typed by investigators at eight institutions by using either antibiograms, bacteriophage typing, biotyping, immunoblotting, insertion sequence typing with IS257/431, multilocus enzyme electrophoresis, restriction analysis of plasmid DNA, pulsed-field or field inversion gel electrophoresis, restriction analysis of PCR-amplified coagulase gene sequences, restriction fragment length polymorphism typing by using four staphylococcal genes as probes, or ribotyping. Isolates from four well-characterized outbreaks (n = 29) and a collection of organisms from two nursing homes were mixed with epidemiologically unrelated stock strains from the Centers for Disease Control and Prevention. Several isolates were included multiple times either within or between the sets of isolates to analyze the reproducibilities of the typing systems. Overall, the DNA-based techniques and immunoblotting were most effective in grouping outbreak-related strains, recognizing 27 to 29 of the 29 outbreak-related strains; however, they also tended to include 3 to 8 epidemiologically unrelated isolates in the same strain type. Restriction fragment length polymorphism methods with mec gene-associated loci were less useful than other techniques for typing oxacillin-susceptible isolates. Phage typing, plasmid DNA restriction analysis, and antibiogram analysis, the techniques most readily available to clinical laboratories, identified 23 to 26 of 29 outbreak-related isolates and assigned 0 to 6 unrelated isolates to outbreak strain types. No single technique was clearly superior to the others; however, biotyping, because it produced so many subtypes, did not effectively group outbreak-related strains of S. aureus.

Bacterial Typing Techniques↗
PubMed1971

Mutants in a nonessential gene of bacteriophage T4 which are defective in the degradation of Escherichia coli deoxyribonucleic acid.

Wild-type bacteriophage T4 was enriched for mutants which fail to degrade Escherichia coli deoxyribonucleic acid (DNA) by the following method. E. coli B was labeled in DNA at high specific activity with tritiated thymidine ((3)H-dT) and infected at low multiplicity with unmutagenized T4D. At 25 min after infection, the culture was lysed and stored. Wild-type T4 degrades the host DNA and incorporates the (3)H-dT into the DNA of progeny phage; mutants which fail to degrade the host DNA make unlabeled progeny phage. Wild-type progeny are eventually inactivated by tritium decay; mutants survive. Such mutants were found at a frequency of about 1% in the survivors. Eight mutants are in a single complementation group called denA located near gene 63. Four of these mutants which were examined in detail leave the bulk of the host DNA in large fragments. All eight mutants exhibit much less than normal T4 endonuclease II activity. The mutants produce somewhat fewer phage and less DNA than does wild-type T4.

Centrifugation, Density Gradient↗