The use of rec-bacteria for testing of carcinogenic substances.

A series of rec-Escherichia coli strains were tested for their sensitivity to four known carcinogenic compounds by examination of a zone of inhibited bacterial growth around a central well containing the test chemical. The mutants recA-, recB-, recC-, and recA- recB- recC- were all more sensitive to the mutagens than the parental strain AB1157. The recB- recC- strain was examined with a larger series of compounds and was found to respond to many of the substances in a similar way as the Salmonella typhimurium strains of Ames but with some notable exceptions. Nitrosamines, with rat liver microsomal activation, could be detected at lower levels and a group of aromatic amino compounds failed to react with these rec-E. coli. An unusual feature of these rec-mutants is their sensitivity to mixtures of nitrosamines and 2-acetyl amino-fluorene in the absence of microsomal activation.     

Intra- and intermolecular recombination of test plasmids in K12 Escherichia coli cells carrying an RTF derivative of the R1drd-19 plasmid

The RTF derivative of the plasmid R1drd-19 was found to stimulate recombination of the tester plasmids in a recB mutant of Escherichia coli K12. The frequency of intramolecular recombination is increased 3.5 and 20-fold, as compared to the one in rec+ and rec- strains, respectively. The frequency of interplasmid recombination is enhanced 4 and 9-fold, respectively. Considerable heterogeneity of the recombination products of the tester plasmid intramolecular recombination in recB-/RTFR1-19 strain has been revealed. It is hypothesized that a "recombinase" encoded by Rldrd-19 plasmid determines a new minor pathway in recB- (Rec P) which differs in activity and, perhaps substrate specificity from the main Rec BCD pathway.     

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.     

Dark Recovery Processes in Escherichia coli Irradiated with Ultraviolet Light II. Effect of uvr Genes on Liquid Holding Recovery

The uvr mutations of Escherichia coli K-12 decrease the ability of cells to survive ultraviolet light (UV), to excise pyrimidine dimers from their deoxyribonucleic acid and to reactivate bacteriophage exposed to UV. The rec mutations decrease the ability of the cells to survive UV and to undergo genetic recombination. Certain rec mutations, including recA1, rec-12, recA13, and rec-56, are necessary for the expression of liquid-holding recovery (LHR), observed as an increase in colony-forming ability when irradiated cells are held in buffer in the dark. These rec mutations appear to act indirectly to permit the detection of LHR rather than to affect its occurrence directly. We have tested the effect of uvr markers on LHR in cells containing one of these rec mutations. Recombinants containing rec-56 together with a uvr marker were constructed and tested for LHR. None of the 39 recombinants examined, carrying uvrA6, uvrB5, or uvrC34, showed LHR. Three rec(-)uvr(-) strains were also tested for photoreactivation. In all three, photoreactivation was observed, indicating that they contained detectable amounts of pyrimidine dimers. Our results are consistent with the idea that uvr mutations inactivate LHR, and suggest that LHR reflects excision-dependent repair of pyrimidine dimers.     

Role of the product of recB-gene in the thymine-less death and characteristics of this phenomenon in a thy-recB--mutant of Escherichia coli K-12]

Thymine requiring mutants of rec+ and recB- Escherichia coli strains have been tested for their response to thymine deprivation. Exonuclease V-deficient mutant is less sensitive to thymine deprivation than the wild type strain, because there is no lag period at thymineless death of recB- thy- cells. However, the mechanism of thymineless death of thy- rec+ and thy- recB- cells may be different. Two types of thymineless death are proposed to exist. The first type is due to DNA primary structure damages (single-strand breaks or gaps), accompanied by DNA degradation. The restoration of the balance disturbed by the thymine deprivation between DNA and protein synthesis rates by their balanced inhibition promotes a complete repair of structural damages in DNA and prevents the death of rec+ cells. The second type of thymineless death is not linked with the formation of DNA damages, and this is observed in recB- thy- mutant, defective in exonuclease V.     

Genetic Analysis of Recombination-Deficient Mutants of Escherichia coli K-12 Carrying rec Mutations Cotransducible with thyA

The rec mutations carried by 20 strains of Escherichia coli K-12 which are defective in genetic recombination and sensitive to ultraviolet light and X rays, and whose lambda lysogens show spontaneous phage production, have been mapped near thyA. In 15 of the strains, the rec mutation fails to complement recB21 but complements rec-22. The other five strains carry a rec mutation which complements recB21 but not rec-22. These mutations map closer to thyA than those which fail to complement recB21. They therefore appear to be defective in a different recombination gene, denoted recC. The order of recB and recC on the linkage map of E. coli K-12 is thyA-recC-recB-argA.     

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.     

Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli

Rutberg, Blanka (Karolinska Institutet, Stockholm, Sweden), and Lars Rutberg. Role of superinfecting phage in lysis inhibition with phage T4 in Escherichia coli. J. Bacteriol. 90:891-894. 1965.-The ability of bacteriophage T4 to induce lysis inhibition upon superinfection was investigated after various treatments of the phage. This ability was found not to be a property of the external protein part of the phage, nor was it dependent on the functional and possibly structural integrity of the phage genetic material.     

Deoxyribonucleic Acid Repair in Bacteriophage T4: Observations on the Roles of the x and v Genes and of Host Factors

Studies were carried out to determine the effect of mutation in the host pol I gene on survival of ultraviolet (UV)-irradiated bacteriophage T4. Whereas a slightly reduced survival was observed in Escherichia coli strain P-3478 (pol A(1)) compared to strain W-3110 (pol A(+)), no such difference was observed in two strains isogenic except for the pol A gene. It was also shown that, whereas bacteriophage T4x is sensitive to UV irradiation, X irradiation, and treatment with methyl-methanesulfonate (MMS), phage T4v(1) is sensitive only to UV irradiation. The survival of damaged phage T4x is neither affected by the presence of the rec A, rec B, or pol A mutations in the host, nor is there evidence that phage T4 effects repair of rec A or pol A mutants previously treated with either UV or MMS.     

Susceptibility of Staphylococcus epidermidis planktonic cells and biofilms to the lytic action of staphylococcus bacteriophage K

AIMS: To evaluate differences in biofilm or planktonic bacteria susceptibility to be killed by the polyvalent antistaphylococcus bacteriophage K. METHODS AND RESULTS: In this study, the ability of phage K to infect and kill several clinical isolates of Staphylococcus epidermidis was tested. Strains were grown in suspension or as biofilms to compare the susceptibility of both phenotypes to the phage lytic action. Most strains (10/11) were susceptible to phage K, and phage K was also effective in reducing biofilm biomass after 24 h of challenging. Biofilm cells were killed at a lower rate than the log-phase planktonic bacteria but at similar rate as stationary phase planktonic bacteria. CONCLUSIONS: Staphylococcus epidermidis biofilms and stationary growth phase planktonic bacteria are more resistant to phage K lysis than the exponential phase planktonic bacteria. SIGNIFICANCE OF STUDY: This study shows the differences in Staph. epidermidis susceptibility to be killed by bacteriophage K, when grown in biofilm or planktonic phenotypes.     

Transformation and Transduction in Recombination-defective Mutants of Bacillus subtilis

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.     

Early Abortive Lysis by Phage BF23 in Escherichia coli K-12 Carrying the Colicin Ib Factor

Growth of phage BF23 was restricted in Escherichia coli K-12 strains carrying a colicin I factor (ColIb); most infected cells lysed early without producing progeny phages. Either addition of chloramphenicol before phage infection or ultraviolet irradiation of phage prevented early abortive lysis, an indication that certain phage functions are required for this phenomenon. Very little or no phage-induced lysozyme was synthesized in the infected ColI(+) cells. This result suggests that early abortive lysis was not due to the lysozyme action. A small fraction (0.05) of BF23-infected ColI(+) cells showed normal phage growth. This "escaped growth" may reflect the physiological state of the host bacteria rather than the heterogeneity of the infecting phage. Host-controlled modification was not observed. A phage mutant, BF23hI, able to grow on ColI(+) cells, was isolated and was characterized to be recessive to the wild-type BF23 in its ability to undergo early abortive lysis. Among the T series phages, T5 induced early abortive lysis, and growth of T5 was restricted upon infection to ColI(+) cells. These results and the other observations, including the occurrence of phenotypic mixing between BF23 and T5, suggest that these two phages are related to each other even though the receptor sites for BF23 and T5 are apparently different.     

The effect of various culture media on infection,growth, lysis,and phage production of B. megatherium

B. megatherium cells were grown in various culture media, centrifuged and washed, and suspended in other culture media containing "C" or "T" phage. The per cent of infection, rate of growth, lysis, and phage production were determined. The behavior of the system depends on the culture medium in which the cells were grown and also on the culture medium in which they were mixed with phage. With the T phage it is possible to set up systems which yield the following results: 1. No infection, normal growth, no phage production. 2. Infection, normal growth, no lysis) phage production. 3. Infection, growth for several hours, lysis, and phage production. 4. Infection, no growth, lysis, and phage production. The C phage system is less affected by changes in the culture medium. The change in the behavior of the cells with T phage probably is not due to selection since it occurs without much growth of the culture, and is readily reversible.     

ESCHERICHIA COLI Rho Factor Is Involved in Lysis of Bacteriophage T4-Infected Cells

A Rid (Rho interaction deficient) phenotype of bacteriophage T4 mutants was defined by cold-sensitive restriction (lack of plaque formation) on rho+ hosts carrying additional polar mutations in unrelated genes, coupled to suppression (plaque formation) in otherwise isogenic strains carrying either a polarity-suppressing rho or a multicopy plasmid expressing the rho+ allele. This suggests that the restriction may be due to lower levels of Rho than what is available to T4 in the suppressing strains.--Rid394 X 4 was isolated upon hydroxylamine mutagenesis and mapped in the t gene; other t mutants (and mot, as well as dda dexA double mutants) also showed a Rid phenotype. In liquid culture in strains that restricted plaque formation Rid394 X 4 showed strong lysis inhibition (a known t- phenotype) but no prolonged phage production (another well-known t- phenotype). This implies that when Rho is limiting the t mutant shuts off phage production at the normal time. Lysis inhibition was partially relieved, and phage production prolonged to varying extents depending on growth conditions in strains that allowed plaque formation. No significant effect on early gene expression were found. Apparently, both mutant (polarity-suppressing) and wild-type Rho can function in prolonging phage production and partially relieving lysis inhibition of Rid394 X 4 when present at a sufficiently high level, and Rho may play other role(s) in T4 development than in early gene regulation.     

Lysis of lysis-inhibited bacteriophage T4-infected cells.

T4 bacteriophage (phage)-infected cells show a marked increase in latent-period length, called lysis inhibition, upon adsorption of additional T4 phages (secondary adsorption). Lysis inhibition is a complex phenotype requiring the activity of at least six T4 genes. Two basic mysteries surround our understanding of the expression of lysis inhibition: (i) the mechanism of initiation (i.e., how secondary adsorption leads to the expression of lysis inhibition) and (ii) the mechanism of lysis (i.e., how this signal not to lyse is reversed). This study first covers the basic biology of the expression of lysis inhibition and lysis of T4-infected cells at high culture densities. Then evidence is presented which implies that, as with the initiation of lysis inhibition, sudden, lysis-associated clearing of these cultures is likely caused by T4 secondary adsorption. For example, such clearing is often observed for lysis-inhibited T4-infected cells grown in batch culture during T4 stock preparation. The significance of this secondary adsorption-induced lysis to wild T4 populations is discussed. The study concludes with a logical argument suggesting that the lytic nature of the T4 phage particle evolved as a novel mechanism of phage-induced lysis.     

Mutations in the new gene stIII of bacteriophage T4B suppressing the lysis defect of gene stII and a gene e mutant.

Mutations of bacteriophage T4B were found which suppress the lysis defect of both gene stII mutants and gene e mutants. The suppressor mutations belong to a new gene, stIII, of phage T4B. Gene stIII is located on the genetic map of T4B between genes stI and e. stIII mutants sometimes form star plaques on Escherichia coli B. The latent period on E. coli 594, but not E. coli B, is shorter with stIII mutants than that with wild-type phage. The premature lysis of E. coli 594 infected with stIII phage does not depend on the expression of both stII+ and e+ function. StIII allele is dominant over the stIII+ with respect to both the ability to suppress the stII defect and the early lysis of infected E. coli 594 cultures.     

Interaction (integration and excision) of the pRP19.6 plasmid, a derivative of the RP1 plasmid containing duplicated sequence IS21, with the chromosome of Escherichia coli K12

A pRP19.6 plasmid is the derivative of the temperature sensitive RPlts12 plasmid and contains a duplicated IS21 (IS8) element. Using temperature sensitive pRP19.6 replication, Hfr strains have been obtained by integration of the plasmid into the chromosome of E. coli rec+ and recA- cells and their properties were studied. According to the results obtained, pRP19.6 insertion into the genome of the rec+ bacteria IS reversible, and its integration into the chromosome of the recA- bacteria produced the stable Hfr strains. To elucidate the mechanism of pRP19.6 excision from the bacterial chromosome, plasmids of R+ transconjugates generated with a low frequency in the crosses between the stable Hfr strains and the rec+ recipients were analyzed. It was shown that the stable Hfr clones might produce stable R1 plasmids as well as a family of deletion KmsTra- derivatives of the pRP19.6. The structure of the KmsTra- was investigated and the mechanism of their formation was proposed. In the light of the data obtained, prospects of pRP19.6 practical application are discussed.     

Phage t: a group T plasmid-dependent bacteriophage

Phage t was isolated from sewage from Pretoria. It formed plaques only on Escherichia coli and Salmonella typhimurium strains that carried plasmids belonging to incompatibility group T. Five of six group T plasmids permitted visible lysis of R+ host strains. There was no visible lysis of E. coli J53-2 or S. typhimurium LT2trpA8 carrying the T plasmid Rts1 although the strains supported phage growth as indicated by at least a 10-fold increase in phage titre. The latter strains transferred the plasmid at high frequency to E. coli strain CSH2 and the resulting transconjugants plated the phage. Proteus mirabilis strain PM5006(R402) failed to support phage growth although it transferred the plasmid and concomitant phage sensitivity to E. coli J53-2. The phage was hexagonal in outline, RNA-containing, resistant to chloroform and adsorbed to the shafts of pili determined by T plasmids.     

The superiority of hamster liver microsomal fraction for activating nitrosamines to mutagens in Salmonella typhimurium

46 chemicals of various classes and structures, including 30 known animal carcinogens, were evaluated for genotoxic effects using the Escherichia coli rec assay with strains WP2 (wild-type) and WP100 (uvrA- recA-) in qualitative and quantitative spot tests and in quantitative suspension tests. The rec assay detected 17 of 30 known carcinogens as genotoxic agents, including mitomycin C and diethylnitrosamine, both negative in the Salmonella/Ames test as utilized in these studies. The rec assay in conjunction with the Salmonella/Ames test detected 20 of 30 known carcinogens as genotoxic agents. Azo/aminoazo carcinogens showed little gentoxicity, and the aromatic amine 2-acetylaminofluorene was non-genotoxic in the rec assay. The rec assay was more effective than pol tests with E. coli strains W3110/p3478 and strains WP2/WP67. Effectiveness of the rec assay was related to the DNA repair-defective nature of the uvrA- recA- genotype of strain WP100.     

Escherichia coli detection by GFP-labeled lysozyme-inactivated T4 bacteriophage

Escherichia coli has been used as an indicator of the fecal contamination of water and food, identifying potential health hazards. In this study, an E. coli-specific bacteriophage, T4, was used to detect E. coli bacteria. The T4 phage small outer capsid (SOC) protein was used to present green fluorescent protein (GFP), an easily detectable marker protein, on the phage capsid. To inactivate phage lytic activity, we used the T4e(-) phage, which does not produce the lysozyme responsible for host cell lysis. Infection of E. coli K12 cells with the GFP-labeled T4e(-) phage (T4e(-)/GFP) enabled the visualization and distinction of E. coli K12 cells from T4 phage-insensitive cells, Pseudomonas aeruginosa. Prolonged incubation of E. coli K12 cells with the T4e(-)/GFP phage did not lead to cell lysis. Propagation of T4e(-)/GFP in host cells increased the intensity of green fluorescence, making the distinction of E. coli cells from other cells simple and effective. This method enables the rapid, conclusive quantitation of E. coli cells within an hour.